**Laser Doppler Flowmetry Evaluation of the Microcirculation in Dentistry Provisional chapterLaser Doppler Flowmetry Evaluation of the Microcirculation in Dentistry**

Carmen Todea, Silvana Canjau, Mariana Miron, Bogdan Vitez and Gheorghe Noditi Carmen Todea, Silvana Canjau, Mariana Miron, Bogdan Vitez and Gheorghe Noditi

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64926

#### **Abstract**

This chapter presents the most important features of laser Doppler (LD) techniques: LD flowmetry (LDF) and LD imaging (LDI), together with examples of their clinical applications in dentistry. LDF gives a constant estimation of blood flow at a specified point, whereas LDI gives a 'snapshot' of perfusion at a given point. These methods are non-invasive laser-based techniques for monitoring gingival and pulpal blood flow and could be used as a diagnostic tool. In paediatric dentistry and odontology, LDF proved to be an atraumatic real-time method used for determining the tooth vitality by monitoring the pulp microcirculation in traumatized teeth, fractured teeth and teeth undergoing different conservative treatments (e.g. bleaching, dental preparation for prosthetic restorations, etc.). In periodontology, recent studies showed the ability of LDF to evaluate the health of gingival tissue in different types of periodontal diseases. By using LDF, it is also possible to evaluate the outcome after different periodontal treatments. The laser Doppler line scanning can be used for recording the gingival healing process after a surgical procedure in the anterior area of the oral cavity.

**Keywords:** microcirculation, dental pulp, gingiva, laser Doppler flowmetry, laser Doppler imaging

## **1. Introduction**

The microcirculation consists of vessels with the diameter less than 100 μm. The structure and topological organization of the microcirculation located within organs differ from the larger conduit vessels that distribute blood flow to the organs. The rheological properties of blood

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

in the microcirculation differ from those in the large vessels due to the Fahraeus-Lindqvist effect, which lead to diameter-dependentreduction in hematocrit and effective blood viscosity in microcirculatory vessels [1]. The main function of the microcirculation is to deliver nutrients to and remove waste products from the various tissues as well as support the exchange of respiratory gasses. It also plays an essential role in fluid exchange between blood and tissue, delivery of hormones from endocrine glands to target organs, and bulk delivery between organs for storage or synthesis and provides a line of defence against pathogens [2]. An ideal technique for measurement of tissue oxygenation should provide quantitative, accurate and reproducible real-time information about oxygen supply and utilization in specific tissue beds. For clinical applications, such a device should be safe, non-invasive and easy to use.

Laser Doppler flowmetry (LDF) and laser Doppler imaging (LDI) have been widely used to assess tissue micro-vascular function. These techniques have functioned as clinical surrogate markers. However, the lack of standardization in data expression limits the use of these tests in routine practice. Nowadays, LDF is commonly used to assess tissue blood flow; yet, data exhibit great spatial variability. Another way of getting around spatial variability could be to evaluate tissue blood flow over wider areas by using LDI. Successful wound healing following periodontal surgery is strongly influenced by revascularization rate as well as by preservation and reconstruction of the micro-vascularity of the gingival tissues [3, 4]. Regular post-operative assessment of flap perfusion by members of the microsurgery team trained in the use of laser Doppler line scanning might, therefore, represent a practical alternative to more complex and invasive monitoring techniques.

There are numerous applications where LDF was used to non-invasively monitor changes in blood flow in living tissues. LDF has been used to assess blood flow for intact microvascular systems such as the skin, the retina, gut mesentery, renal cortex and mucous membranes [5, 6]. Dental applications include LDF readings (LDFRs) of periodontal ligament [7], pulpal blood vessels [8–12], gingival or sulcular blood flow in health and disease [13–18], evaluation of the degree in healing and revascularization of surgical wounds [19], the effect of orthodontic treatment [20] or the injection of vasoconstrictive anaesthetics on blood flow [21]. Single-point LDF, the technique mentioned above, shows good temporal resolution, poor spatial resolution and poor reproducibility in low capillary density tissue areas [22, 23]. This latter issue can be overcome by using either integrated probes with several transmitting and/or receiving fibres or full field techniques such as LDI. This technique shows excellent spatial resolution but poor temporal resolution for most devices (especially when scanning large areas) [24], but it provides a more valid measure of tissue blood flow [25].

## **2. Methods and results**

The pulpal and gingival blood flows (GBFs) in the clinical situations described in this chapter were monitored using a MoorLab Laser Doppler (LD) equipment (Moor Instruments Ltd., Axminster, UK) with a straight optical probe, MP3b, 10 mm. A double silicone impression fixed perpendicularly on the buccal cervical surface of the tooth was used for stabilizing the probe. The Moor Instruments MoorLab LD monitor uses laser radiation generated by a semiconductor laser diode operating at a wavelength of 780 + 10 nm and a maximum accessible power of 1.6 mW. The programmed bandwidth of the recorded LD signal was 20 Hz–20 kHz while sampling frequency was 40 Hz. Calibration was performed according to the manufacturer instructions. LDF was recorded and analysed using MoorSoft MoorLab V2.01 software. The physical parameters assessed were flux, expressed in perfusion units (PU) and perfusion measurement (DC). The term used to estimate blood flow is flux—a quantity proportional to the average speed of the blood cells and their concentration. This is expressed in arbitrary perfusion units (AU) that are linearly related to flux. DC gives an indication of the backscattered laser light intensity. The DC signal indicates a correct positioning of the optical probe, showing the reflected laser radiation level from the level of concerned area. The DC signal is the one that indicates the mechanic stability of the optical probe placed at the level of acquisition area. The data were processed using statistical analysis software SPSS v16.0.1.

## **2.1. Microcirculation of the dental pulp**

in the microcirculation differ from those in the large vessels due to the Fahraeus-Lindqvist effect, which lead to diameter-dependentreduction in hematocrit and effective blood viscosity in microcirculatory vessels [1]. The main function of the microcirculation is to deliver nutrients to and remove waste products from the various tissues as well as support the exchange of respiratory gasses. It also plays an essential role in fluid exchange between blood and tissue, delivery of hormones from endocrine glands to target organs, and bulk delivery between organs for storage or synthesis and provides a line of defence against pathogens [2]. An ideal technique for measurement of tissue oxygenation should provide quantitative, accurate and reproducible real-time information about oxygen supply and utilization in specific tissue beds.

For clinical applications, such a device should be safe, non-invasive and easy to use.

invasive monitoring techniques.

204 Microcirculation Revisited - From Molecules to Clinical Practice

**2. Methods and results**

provides a more valid measure of tissue blood flow [25].

Laser Doppler flowmetry (LDF) and laser Doppler imaging (LDI) have been widely used to assess tissue micro-vascular function. These techniques have functioned as clinical surrogate markers. However, the lack of standardization in data expression limits the use of these tests in routine practice. Nowadays, LDF is commonly used to assess tissue blood flow; yet, data exhibit great spatial variability. Another way of getting around spatial variability could be to evaluate tissue blood flow over wider areas by using LDI. Successful wound healing following periodontal surgery is strongly influenced by revascularization rate as well as by preservation and reconstruction of the micro-vascularity of the gingival tissues [3, 4]. Regular post-operative assessment of flap perfusion by members of the microsurgery team trained in the use of laser Doppler line scanning might, therefore, represent a practical alternative to more complex and

There are numerous applications where LDF was used to non-invasively monitor changes in blood flow in living tissues. LDF has been used to assess blood flow for intact microvascular systems such as the skin, the retina, gut mesentery, renal cortex and mucous membranes [5, 6]. Dental applications include LDF readings (LDFRs) of periodontal ligament [7], pulpal blood vessels [8–12], gingival or sulcular blood flow in health and disease [13–18], evaluation of the degree in healing and revascularization of surgical wounds [19], the effect of orthodontic treatment [20] or the injection of vasoconstrictive anaesthetics on blood flow [21]. Single-point LDF, the technique mentioned above, shows good temporal resolution, poor spatial resolution and poor reproducibility in low capillary density tissue areas [22, 23]. This latter issue can be overcome by using either integrated probes with several transmitting and/or receiving fibres or full field techniques such as LDI. This technique shows excellent spatial resolution but poor temporal resolution for most devices (especially when scanning large areas) [24], but it

The pulpal and gingival blood flows (GBFs) in the clinical situations described in this chapter were monitored using a MoorLab Laser Doppler (LD) equipment (Moor Instruments Ltd., Axminster, UK) with a straight optical probe, MP3b, 10 mm. A double silicone impression fixed perpendicularly on the buccal cervical surface of the tooth was used for stabilizing the The tooth vitality preservation is one of the most important aims in conservative dentistry. This is why the reliable vitality assessment of the dental pulp has always been problematic and therefore, many methods have been suggested to test pulp vitality [26]. Pulp vitality tests should attempt to examine the presence of pulp blood flow, offering a precise, objective and quantitative assessment as opposed to the conventional tests that rely on the patient's subjective sensitivity [27, 28].

It is reported in the literature that the LDF technique is reliable for measuring human pulpal blood flow (PBF) to determine pulp vitality [29, 30]. The technique can measure perfusion quantitatively in real time [31]. However, it has also been claimed that signals from human teeth do not necessarily indicate pulpal blood flow and could be confused with a signal obtained from nearby gingival tissues, suggesting that periodontium and other neighbouring tissues can contribute to the signal [32–34]. Polat et al. [34] examined the scattering and penetration properties of the laser used in LDF by using a camera with slow speed shutters. They demonstrated that the laser can densely penetrate up to 4 mm in depth and less densely for up to 13 mm. This also suggests that even with proper isolation of the tooth, some signal contamination from the periodontium is inevitable. They also demonstrated that without isolation, the laser light could scatter from the source tooth to the whole oral cavity, which can also potentially contribute to signal contamination. Karayilmaz and Kirzioglu [35] indicated that LDF could reliably discriminate the vitality of the teeth with a sensitivity and specificity of 1.0 for studied sample. LDF was found to be a more reliable and effective method than pulse oximetry (PO) and electric pulp tester (EPT) in assessing the pulpal status of human teeth.

The isolation method before LDF measurements is crucial for obtaining an accurate signal. Therefore, many authors have used different isolation techniques, thus the different results. This is why many studies suggest that 45–82% of the blood flow recorded with LDF from human teeth may not be from the pulp [36–39]. Soo-Ampon et al. [33] found that up to 80% of the LDF output signal in human incisors may be non-pulp in origin if attempts at tooth isolation are not made. Polat et al. [34] compared teeth that had undergone a pulpectomy with contralateral healthy pulps as controls. They also found that approximately 70% of the LDF readings from teeth with the pulps removed were non-pulp in origin. The results obtained by our group [40] show that about 69% of the acquired LD signal is of non-pulp origin, consistent with the existing literature [38, 39, 41, 42].

For this reason, in our studies investigating dental pulp blood flow, a silicone impression combined with light cure periodontal liquid dam was used in order to reduce the signal contamination. This method offered an excellent isolation certified by the DC values obtained during the measurements. It has been shown that the light from a LDF probe placed at 2 mm above the buccal cement-enamel junction is transmitted apically towards the radicular pulp [41]. There are several studies that have reported the placement of the LDF probe at 1–1.5 [43], 2 [44], 2–3 [45, 46], ~3 [47] and 4–5 mm [48] coronal to the gingival margin. In our studies, the probe was placed on the cervical third of the tooth, at 3 mm away from the gingival margin (**Figure 1**).

**Figure 1.** The acquisition technique of laser Doppler signals. (a) Silicone holder with the optical fibre inserted in the canal previously created and (b) intra-oral positioning of the silicone holder together with the stabilized optical fibre.

#### *2.1.1. Bleaching and pulp microcirculation*

The treatment of teeth whitening can be performed in the dental office, by the dentist, or at patient's home, and uses whitening agents, such as hydrogen peroxide gel (3–38%), carbamide peroxide (10–30%) or a mixture of hydrogen peroxide and sodium carbonate. Tooth bleaching, as one of the most required dental cosmetic procedures, must imply a consequent tooth vitality assessment. Sensitivity is strongly related to concentration, time and rate of usage of the bleaching gel [39, 42, 49–51]. In general, the activation systems have a role in increasing the temperature of the whitening agent, which penetrates rapidly the dental hard tissues, an aspect that favours the obtaining of an optimal result in a short interval of time but with the risk of increasing the inner pulp temperature. Therefore, this procedure can cause a local irritation to the dental pulp, which affects its micro-vascularization. In one of our studies, we chose the 1064 nm laser instrument for activating the bleaching gel and we compared it with the conventional 'in office' bleaching procedure, using LDF measurements.

In the Nd:YAG (1064 nm) laser-assisted bleaching, the pulp had a much better recovery (**Figure 2**), suggesting that LDF is a suitable method for a continuous monitoring of the dental pulp microcirculation [52].

**Figure 2.** Interval plot of mean recorded before, immediately after and 1 week after treatment, indicating the evolution of the pulp blood flow over time for laser-assisted bleaching procedure.

#### *2.1.2. Prepared teeth and pulp microcirculation*

are not made. Polat et al. [34] compared teeth that had undergone a pulpectomy with contralateral healthy pulps as controls. They also found that approximately 70% of the LDF readings from teeth with the pulps removed were non-pulp in origin. The results obtained by our group [40] show that about 69% of the acquired LD signal is of non-pulp origin, consistent

For this reason, in our studies investigating dental pulp blood flow, a silicone impression combined with light cure periodontal liquid dam was used in order to reduce the signal contamination. This method offered an excellent isolation certified by the DC values obtained during the measurements. It has been shown that the light from a LDF probe placed at 2 mm above the buccal cement-enamel junction is transmitted apically towards the radicular pulp [41]. There are several studies that have reported the placement of the LDF probe at 1–1.5 [43], 2 [44], 2–3 [45, 46], ~3 [47] and 4–5 mm [48] coronal to the gingival margin. In our studies, the probe was placed on the cervical third of the tooth, at 3 mm away from the gingival margin

**Figure 1.** The acquisition technique of laser Doppler signals. (a) Silicone holder with the optical fibre inserted in the canal previously created and (b) intra-oral positioning of the silicone holder together with the stabilized optical fibre.

The treatment of teeth whitening can be performed in the dental office, by the dentist, or at patient's home, and uses whitening agents, such as hydrogen peroxide gel (3–38%), carbamide peroxide (10–30%) or a mixture of hydrogen peroxide and sodium carbonate. Tooth bleaching, as one of the most required dental cosmetic procedures, must imply a consequent tooth vitality assessment. Sensitivity is strongly related to concentration, time and rate of usage of the bleaching gel [39, 42, 49–51]. In general, the activation systems have a role in increasing the temperature of the whitening agent, which penetrates rapidly the dental hard tissues, an aspect that favours the obtaining of an optimal result in a short interval of time but with the risk of increasing the inner pulp temperature. Therefore, this procedure can cause a local irritation to the dental pulp, which affects its micro-vascularization. In one of our studies, we chose the 1064 nm laser instrument for activating the bleaching gel and we compared it with the

conventional 'in office' bleaching procedure, using LDF measurements.

with the existing literature [38, 39, 41, 42].

206 Microcirculation Revisited - From Molecules to Clinical Practice

*2.1.1. Bleaching and pulp microcirculation*

(**Figure 1**).

Determination of pulpal health represents an objective of endodontic diagnosis. It is important to assess pulp vitality prior to undertaking extensive tooth preparation in order to improve the prognosis of the restoration. It is also desirable to confirm periodically the pulp vitality in teeth that have undergone pulp preservation procedures or have had extensive restorations [53].

Full crown preparation procedures are probably the greatest restorative injury to which the dental pulp is subjected [54, 55]. The extensive cutting during crown preparation, desiccation, thermal injury and bacterial contamination has been implicated in the injury associated with tooth preparation [56]. Crown preparation without water spray causes about 95% reduction in the pulpal blood flow by 1 h after preparation. In contrast, the use of water spray virtually eradicates any alteration in pulpal blood flow. The reduction in coronal pulp blood flow is the result of an increased blood flow through the apically positioned arteriovenous (AVA) shunts and a redistribution of blood flow from the drilled side to the opposite side of the pulp [28, 57].

However, few reports were found in the literature regarding the use of LDF in assessing the pulpal blood flow in teeth that underwent prosthetic preparations [58, 59]. That is why the aim of our study was to evaluate how teeth preparation for full crown coverage may affect the pulpal blood flow.

The results obtained in our study show a linear increase in pulpal blood flow (PBF) values for all samples after dental prosthetic preparation. The values recorded 7 days after the preparation were higher than those recorded at 24 h after the preparation (**Figure 3**), which suggests that the increase in values does not relate only to optical changes due to the reduction of dental hard tissue but rather to the establishment of proper PBF. As a consequence, it may be assumed that a phenomenon of micro-irritation has appeared in the investigated area.

Only in one sample, the PBF recorded at 24 h was tremendously different from the initial moment and even from the PBF recorded at day 7. The patient did not report clinical symptoms of pulpal inflammation, such as pain and tenderness to percussion.

Yanpiset et al. [43] found LDF measurements to be extremely accurate in differentiating a revascularized (vital) tooth from a necrotic tooth pulp. An exciting finding of their study was that an accurate LDF reading of pulpal revascularization could be established at the fourth week after treatment, which is much earlier than it would be expected from standard sensitivity tests. This finding corresponds to those from the study by Skoglund et al. [60]. The LDF is extremely accurate in non-vital teeth, with almost 100% accuracy, but not as good in vital teeth. The blood vessels, fibroblasts and fibrous connective tissue that occupy the central portion of the pulp chamber can be affected without having a significant inflammatory reaction. While this tissue is vital and would give a radiographic picture of continued root development, the amount of moving blood cells creating a Doppler shift would be minimal. Another reason is that the revascularized teeth containing predominantly osteoid tissue may have a different optical property and the flux value reading from a revascularized tooth may be different from a normal tooth pulp. These teeth might give a false negative result [44]. Clinically, it has to be assumed that one may not rely solely on the LDF, but an estimation of signs of the pulpal or periapical pathology would still be necessary, before initiating endodontic treatment.

## *2.1.3. Pulp capping and pulp microcirculation*

The results obtained in our study show a linear increase in pulpal blood flow (PBF) values for all samples after dental prosthetic preparation. The values recorded 7 days after the preparation were higher than those recorded at 24 h after the preparation (**Figure 3**), which suggests that the increase in values does not relate only to optical changes due to the reduction of dental hard tissue but rather to the establishment of proper PBF. As a consequence, it may be assumed

Only in one sample, the PBF recorded at 24 h was tremendously different from the initial moment and even from the PBF recorded at day 7. The patient did not report clinical symptoms

Yanpiset et al. [43] found LDF measurements to be extremely accurate in differentiating a revascularized (vital) tooth from a necrotic tooth pulp. An exciting finding of their study was that an accurate LDF reading of pulpal revascularization could be established at the fourth week after treatment, which is much earlier than it would be expected from standard sensitivity tests. This finding corresponds to those from the study by Skoglund et al. [60]. The LDF is extremely accurate in non-vital teeth, with almost 100% accuracy, but not as good in vital teeth. The blood vessels, fibroblasts and fibrous connective tissue that occupy the central portion of the pulp chamber can be affected without having a significant inflammatory reaction. While this tissue is vital and would give a radiographic picture of continued root development, the amount of moving blood cells creating a Doppler shift would be minimal. Another reason is that the revascularized teeth containing predominantly osteoid tissue may have a different optical property and the flux value reading from a revascularized tooth may be different from a normal tooth pulp. These teeth might give a false negative result [44]. Clinically, it has to be

of pulpal inflammation, such as pain and tenderness to percussion.

that a phenomenon of micro-irritation has appeared in the investigated area.

**Figure 3.** The PBF in time for the prepared teeth.

208 Microcirculation Revisited - From Molecules to Clinical Practice

Injuries to permanent anterior teeth account for the most frequent form of orofacial trauma at a young age. According to various epidemiological studies, the permanent central incisors are mostly involved in traumatic events, sustaining nearly 80% of all registered injuries [61, 62].

Crown fractures may be uncomplicated involving enamel and dentin, without pulp exposure, or complicated, with pulp involvement. Therefore, an efficient clinical evaluation of an injured tooth requires symptomatic, visual and radiographic assessment. This is where LDF steps in, allowing a more accurate assessment of vascularization status in injured teeth whenever required, meaning immediately after the traumatic episode, as well as during and after treatment, justified by the method's safeness and non-invasiveness.

In one of our studies, we aimed to investigate the use of LDF, the pulpal healing process in complicated and uncomplicated crown fractures—with and without pulpal exposure when laser-assisted therapy combined with calcium hydroxide was used. After rubber dam placement, indirect pulp capping and preparation for resin composite restoration were performed for the upper right (#1.1) and left (#2.1) central incisors using Er:YAG laser irradiation (wavelength of 2940 nm; energy 240–80 mJ, SSP). Immediately after the treatment, the LDFRs were analysed and the results showed an increase in PBF on both teeth especially for tooth 2.1. After 7 days, the LDFRs evaluation was performed, and it showed a decrease in PBF in both teeth. The decrease was more notable in tooth 2.1 where the indirect pulp capping was performed. The last LDFRs evaluation was performed after 6 weeks, which revealed the recovery of PBF to a normal value, demonstrating that the pulp reached normal healthy status (**Figures 4** and **5**).

After 7 days, the pulp tissue was not restored to a healthy condition, with normal blood flow as shown by LDFRs, but after 6 weeks, the PBF recorded by LDF and the clinical assessment also showed almost a complete restoration of PBF. Vascular changes are essential to the initiation of acute as well as chronic inflammation, and blood flow is essential to its resolution. The inflammation process involves vasodilatation, thus increased circulation and perfusion. Therefore, a successful pulp capping is obtained when the following clinical conditions are met: uninflamed pulp, good antibacterial seal and the use of a capping material tolerated by the pulp tissue; better outcome is mainly registered in young teeth. Consequently, the clinical signs of inflammation correlated with the changes in PBF. LDF may therefore play a key role in clarifying the importance of PBF dynamics in the treatment of young traumatized teeth. Moreover, the recovery of PBF after laser indirect pulp capping was spectacular. This fact has been attributed to laser treatment for preparing the area for a hermetic sealing of the pulp. The practician must pay attention to the cavity preparation as well as to optimal placement of the capping material, which is in the benefit of the formation of tertiary dentine. Laser-assisted pulp capping represents a new treatment opportunity that improves the working conditions and the biological quality of the irradiated surface, thus increasing the effectiveness of the interaction between pulp tissue and capping agent.

**Figure 4.** The descriptive graphic for LDFRs in traumatized tooth 1.1.

**Figure 5.** The descriptive graphic for LDFRs in traumatized tooth 2.1.

#### *2.1.4. Traumatology and pulp microcirculation*

LDF has been shown to be valuable in monitoring revascularization of teeth following severe dental trauma. During follow-up examinations the traumatized tooth can be unresponsive to traditional vitality testing during the first 6 months; however, LDF indicated that revascularization had occurred much sooner. Until recently, CO2 ice has been the most effective method for sensitivity testing in trauma cases but LDF is able to give the assurance that we could defer invasive care during critical time period when the root canal therapy might have been initiated for the patient [63]. The information obtained by LDF is of additional importance for the treatment planning. Since the clinical examination of traumatized teeth is sometimes inconclusive, LDF could be regarded as a further diagnostic tool but it cannot replace the radiological or clinical examination [64].

A prospective, cohort study conducted by Emshoff et al. [65] on patients with dental injuries developed prediction rules for the treatment response related to the management of dental injuries. Treatment response (success or failure) was categorized based on findings of clinical and radiographic evaluation after 9 months. The most important variables were sub-luxation, root fracture, baseline PBF level and a change in PBF level at 3-month follow-up. The results show that the outcome following the management of dental injuries may be predicted from variables collected with LDF and physical examination. Predictive modelling may provide clinicians with the opportunity to identify 'at-risk' patients early and initiate specific treatment approaches.

## **2.2. Microcirculation of the gingiva**

**Figure 4.** The descriptive graphic for LDFRs in traumatized tooth 1.1.

210 Microcirculation Revisited - From Molecules to Clinical Practice

**Figure 5.** The descriptive graphic for LDFRs in traumatized tooth 2.1.

LDF has been shown to be valuable in monitoring revascularization of teeth following severe dental trauma. During follow-up examinations the traumatized tooth can be unresponsive to traditional vitality testing during the first 6 months; however, LDF indicated that revascularization had occurred much sooner. Until recently, CO2 ice has been the most effective method

*2.1.4. Traumatology and pulp microcirculation*

There is quite little information in the literature about the vascular dynamics of the gingival circulation in healthy and diseased sites. LDF emerged more than 30 years ago as a noninvasive and real-time method for perfusion measurements [66]. The LD technique made it possible to demonstrate that blood flow wave patterns differ consistently among gingival tissue types [67, 68] and that there are no within-subject differences over time in LDFRs [16].

One of the earliest signs of any inflammatory process is the change in the vascular architecture and microvasculature. This is also true for gingivitis [69]. The healthy gingiva is characterizes by a sub-epithelial vascular plexus consisting of a capillary network with loops arching towards the epithelium [70]. Gingival inflammation presents an increased vascularity with larger vessel size, more capillary loops, [71] slowed blood flow [72] and a restriction of the afferent blood vessels [73]. The capillary units are among the first vessels affected by inflammation in the crestal gingiva [74]. If changes of the vascular morphology in inflammation are related to blood flow changes, they may be the first sign to predict the onset of pathological events in the gingiva [75]. Thus, gingival blood flow (GBF) may serve as a prognostic marker. Gingival microcirculation (GM) has lacked exact evaluation for a long time. This was mainly due to methodological difficulties. Different methods, such as impedance plethysmography or the implantation of microspheres, have been employed to study GBF [76–82]. Unfortunately, most of them were invasive or inapplicable to humans. Other studies on dogs have shown that predictable morphologic changes occur in the blood vessels at the gingival margin with the onset of inflammation. These vascular changes precede recognizable histopathological alterations, starting as early as 2 days after the induction of gingivitis [36, 37, 83].

In our studies, in order to obtain a correct LDF measurement of the gingival blood flow, the probe was positioned 4 mm above the cervical line of the upper incisors and was also distanced using a gingival dam (LC Block-Out Resin, Ultradent Products, Inc.) before creating the silicone holder. This distance was necessary in order to avoid pressure on the gingival tissue when applying and removing the silicone holder during measurements phases. A silicone rubber holder was used in order to secure the gingival LDF probe in position at the studied site. A small hole for the laser probe was placed in the holder at 4 mm away from the gingival margin, using a high-speed handpiece and a 1.5 mm diameter fissure bur. After calibration and disinfection, the laser probe was inserted into a rigid opaque plastic tube with a 1.5 mm diameter and 0.1–0.2 mm longer than the fibre. The plastic tube was used to reduce the movement artefacts of the fibre inside the impression, by increasing adherence and protection of the active optic surface. The plastic tube was forcefully inserted in the canal carved in the impression and positioned afterwards according to study protocol. With the purpose of insuring the reproducibility of LD signal acquisition, a guiding mark was set on the fibre in order to allow its placement in the same position for each testing.

#### *2.2.1. Healthy and inflamed gingiva*

Previous researchers have shown that an interaction between GBF and gingival health exists [84]. One of our studies [18] aimed at evaluating the microcirculation in subjects with gingivitis compared to healthy gingiva by using LDF. The subjects of the present study were young adults in whom oral hygiene and dietary habits were well established. Ramsay et al. [85] indicated that the reliability of blood flow measurements required accurate repositioning of the measurement probe; that is why the technique used in the study aimed at achieving a correct reproducibility of the LDF measurements.

The results showed that LDF could be a useful non-invasive, sensitive, reproducible and harmless method for measuring GM in humans. LDF may therefore be an important element in clarifying the role of GBF dynamics in clinical gingivitis as well as in understanding the blood flow dynamics in the gingiva. At the seventh day, the gingiva was not restored to a healthy condition, with normal blood flow as shown by LDFRs but after 14 days, the GM recorded by LDF and the clinical assessment also showed almost a complete restoration of the gingivitis group. Consequently, the clinical signs of inflammation correlated with the changes in GBF (**Figure 6**).

**Figure 6.** The mean values of the gingival blood flow (GBF) recorded at various moments of time; interval plot of the four moments of time in which the LDF measurements were carried out (*SD* = 74.9411); A. (a) sites with gingivitis; (b) healthy gingival site; B. restored gingival health after 14 days.

The results showed significant statistical differences between the four recordings in time. At 24 h after the initiation of therapy, the GBF was significantly increased compared to the baseline values suggesting local inflammation of the tissues after the initial therapy. No significant differences were noticed between initial moment and 7 days after the treatment and also between initial moment and 14 days after. The GBF values at 14 days were not significantly different compared to the control group (**Figure 7**).

**Figure 7.** Fisher individual 95% CIs. Comparison of GBF values of the gingivitis group among the four moments of time recorded in the study. Showing that there are no statistical significant differences between the initial and the 7-day groups as well as between the initial and the 14-day groups.

### *2.2.2. Laser periodontal surgery and gingival recovery*

applying and removing the silicone holder during measurements phases. A silicone rubber holder was used in order to secure the gingival LDF probe in position at the studied site. A small hole for the laser probe was placed in the holder at 4 mm away from the gingival margin, using a high-speed handpiece and a 1.5 mm diameter fissure bur. After calibration and disinfection, the laser probe was inserted into a rigid opaque plastic tube with a 1.5 mm diameter and 0.1–0.2 mm longer than the fibre. The plastic tube was used to reduce the movement artefacts of the fibre inside the impression, by increasing adherence and protection of the active optic surface. The plastic tube was forcefully inserted in the canal carved in the impression and positioned afterwards according to study protocol. With the purpose of insuring the reproducibility of LD signal acquisition, a guiding mark was set on the fibre in

Previous researchers have shown that an interaction between GBF and gingival health exists [84]. One of our studies [18] aimed at evaluating the microcirculation in subjects with gingivitis compared to healthy gingiva by using LDF. The subjects of the present study were young adults in whom oral hygiene and dietary habits were well established. Ramsay et al. [85] indicated that the reliability of blood flow measurements required accurate repositioning of the measurement probe; that is why the technique used in the study aimed at achieving a correct

The results showed that LDF could be a useful non-invasive, sensitive, reproducible and harmless method for measuring GM in humans. LDF may therefore be an important element in clarifying the role of GBF dynamics in clinical gingivitis as well as in understanding the blood flow dynamics in the gingiva. At the seventh day, the gingiva was not restored to a healthy condition, with normal blood flow as shown by LDFRs but after 14 days, the GM recorded by LDF and the clinical assessment also showed almost a complete restoration of the gingivitis group. Consequently, the clinical signs of inflammation correlated with the changes

**Figure 6.** The mean values of the gingival blood flow (GBF) recorded at various moments of time; interval plot of the four moments of time in which the LDF measurements were carried out (*SD* = 74.9411); A. (a) sites with gingivitis; (b)

order to allow its placement in the same position for each testing.

*2.2.1. Healthy and inflamed gingiva*

212 Microcirculation Revisited - From Molecules to Clinical Practice

reproducibility of the LDF measurements.

healthy gingival site; B. restored gingival health after 14 days.

in GBF (**Figure 6**).

When performing gingivoplasty by conventional methods, there are limitations regarding healing by secondary intention, post-operative bleeding, loss of keratinized gingiva and inability to treat the underlying osseous deformities, which leads to the inability to complete the treatment [87]. Performing surgical procedure using laser technology can solve most of these limitations.

LDF found an excellent utility in the evaluation of the gingival recovery after surgery performed with the high-end methods available today.

When using lasers, the depth and amount of soft tissue ablation are more precisely established than with mechanical instruments [88, 89]. In particular, Er:YAG laser is very adequate and useful for aesthetic periodontal soft tissue management because this laser is capable of accurately ablating soft tissues using various handpiece tips, and therefore, the healing process is faster and favourable due to the minimal thermal alteration of the treated surface [90].

Diode lasers act as a useful tool for cutting gingival tissue, producing good haemostasis and reducing bacterial growth in periodontal surgery. There is evidence that this wavelength can reduce gingival inflammation and also the need for local anaesthesia during surgical procedures.

In order to establish the efficiency of one laser in comparison with other, we decided to perform a study where LDF was used to compare GBF after Er:YAG (Fotona Fidelis Plus II) and 980 nm diode laser (Diode Laser Smile Pro 980 Biolitec) gingivectomy.

The evaluation was carried out on 20 anterior teeth that underwent reshaping of gingiva in five female patients (four anterior teeth/patient), aged between 20 and 35, capable of adequate compliance. The Er:YAG laser was used in Long Pulse: 600 μsec (LP) and Very Long Pulse: 1000 μsec (VLP) modes, 140–250 mJ, 10–20 Hz frequency, contact mode and using cylindrical sapphire tips. The parameters were established according to previous research [26] and were found suitable for soft tissue without causing visible major thermal damage to root dentin or bone. The 980 nm diode laser was used in continuous wave mode, 4 W, contact mode and cooling with saline solution using a 360 μm diameter quartz fibre as delivery system (**Figure 8**).

**Figure 8.** (a). Initial intra-oral status, (b) immediately after laser surgery, (c) 24 h after the laser surgery with indirect provisional restorations, and (d) clinical intra-oral aspect 2 months after treatment with the final ceramic restorations.

At first appointment, the initial measurements were carried out. Post-operative controls and LDF measurements were accomplished after 24 h, 7 and 14 days to evaluate healing and wound evolution on a total of eight points/patient (two points on each tooth) for each patient.

reduce gingival inflammation and also the need for local anaesthesia during surgical proce-

In order to establish the efficiency of one laser in comparison with other, we decided to perform a study where LDF was used to compare GBF after Er:YAG (Fotona Fidelis Plus II) and 980 nm

The evaluation was carried out on 20 anterior teeth that underwent reshaping of gingiva in five female patients (four anterior teeth/patient), aged between 20 and 35, capable of adequate compliance. The Er:YAG laser was used in Long Pulse: 600 μsec (LP) and Very Long Pulse: 1000 μsec (VLP) modes, 140–250 mJ, 10–20 Hz frequency, contact mode and using cylindrical sapphire tips. The parameters were established according to previous research [26] and were found suitable for soft tissue without causing visible major thermal damage to root dentin or bone. The 980 nm diode laser was used in continuous wave mode, 4 W, contact mode and cooling with saline solution using a 360 μm diameter quartz fibre as delivery system (**Figure 8**).

**Figure 8.** (a). Initial intra-oral status, (b) immediately after laser surgery, (c) 24 h after the laser surgery with indirect provisional restorations, and (d) clinical intra-oral aspect 2 months after treatment with the final ceramic restorations.

diode laser (Diode Laser Smile Pro 980 Biolitec) gingivectomy.

214 Microcirculation Revisited - From Molecules to Clinical Practice

(d)

dures.

As for the gingival surgery with Er:YAG laser, significant differences in LDF recordings over time were established between different times (*p* < 0.001 with a significant level *α* = 0.001, Friedman test). The results showed that after 24 h the differences are significant compared to the initial moment; 7 days after the treatment, with the Er:YAG, LDF was slightly raised compared to the initial moment (*p* = 0.256), and after 14 days, LDF the values were insignificantly lower compared to pre-treatment (*p* = 0.431) (**Figure 9**).

**Figure 9.** The descriptive graphic for 'Laser 1' method applied at the four moments of time.

Regarding gingival surgery with the diode laser, significant differences between the four tracings over different times were found (*p* < 0.001 with a significant level *α* = 0.001, Friedman test). After 24 h, the differences were significantly lower compared to the initial moment; whereas after 7 and 14 days, the recorded LDF values were significantly raised compared to the initial moment (*p* < 0.001) (**Figure 10**).

The Levene's test for equality of variances was used in order to establish the equal variances assumed at the initial moment as well as after 14 days, and afterwards, the independent sample test was used for comparing the values obtained for the Er:YAG area and for the diode area at the initial moment (insignificant differences *p* = 0.897) and after 14 days (significant difference *p* < 0.001). We established that after 14 days, the recorded fluxes for the diode area were significantly higher compared to the values obtained for the Er:YAG area (*p* < 0.001).

**Figure 10.** The descriptive graphic for 'Laser 2' method applied at the four moments of time.

The results obtained after the laser treatment on the free gingival area indicate a modification in the micro-vascular blood flow response. Furthermore, our measurements, which are in accordance with other studies [91], indicate that LDF technique can offer information regarding the micro-vascular changes during healing period. These results showed an evident decrease in perfusion for both areas in comparison with the baseline values 24 h after surgical procedure. The micro-vascular blood flow increased significantly after 7 days in both areas but mostly in the diode area. After 14 days, the blood perfusion returned to the initial value in the Er:YAG-treated area. The results in the diode-treated area remained at a higher level, showing that after 14 days, the healing in this area was not complete. The response after laser treatment in both areas was an obviously hyperaemic one. The difference in haemodynamic changes that occurred after 14 days can be explained by the differences in tissue interaction of the different laser procedures applied in our study.

#### *2.2.3. Mucositis and gingival blood flow*

In a study [92] conducted by our group, we evaluated the immediate effects of radiotherapy, more precisely, the oral and perioral soft tissue changes that appear after the radiotherapy treatment period. Additionally, we measured the gingival blood flow using LDF, in order to objectively determine any changes of the microvascular system of the gingiva.

Even after the first radiotherapy exposure, the blood flow values increased towards the irradiated area and remained increased throughout the entire treatment. This suggests that the periodontal tissue responds immediately to radiotherapy (as expected), and an inflammatory state is established even after the first exposure and it persists during treatment. What we found interesting was that this increase in vascularity preceded the clinical modifications, which means that with the help of LDF, we can diagnose an inflammation and we can predict the setting of the clinical side effects of radiotherapy. On the other hand, we did not find any numerical correlation between blood flow values and the severity of the clinical manifestation of the radiation-induced side effects.

LD was a useful instrument in establishing the kind of dental procedures we can perform during treatment. Based on our results, we recommend to perform, during this time frame, mostly conservative measures, surgical measures should be performed keeping in mind that the tissues are inflamed and that the bleeding would be greater than normal and wound healing difficult. Prosthetic treatments, if performed, should be done with consideration towards the periodontal tissue that should not be additionally irritated. The clinician should carefully wage the advantages of the treatment against the possible complications that it could bring. The goal of any dental treatment should be increasing the patients' quality of life and decreasing the risks of interrupting radiotherapy, due to the onset of the side effects that it causes.

## *2.2.4. Smokers and gingival microcirculation*

**Figure 10.** The descriptive graphic for 'Laser 2' method applied at the four moments of time.

laser procedures applied in our study.

216 Microcirculation Revisited - From Molecules to Clinical Practice

*2.2.3. Mucositis and gingival blood flow*

The results obtained after the laser treatment on the free gingival area indicate a modification in the micro-vascular blood flow response. Furthermore, our measurements, which are in accordance with other studies [91], indicate that LDF technique can offer information regarding the micro-vascular changes during healing period. These results showed an evident decrease in perfusion for both areas in comparison with the baseline values 24 h after surgical procedure. The micro-vascular blood flow increased significantly after 7 days in both areas but mostly in the diode area. After 14 days, the blood perfusion returned to the initial value in the Er:YAG-treated area. The results in the diode-treated area remained at a higher level, showing that after 14 days, the healing in this area was not complete. The response after laser treatment in both areas was an obviously hyperaemic one. The difference in haemodynamic changes that occurred after 14 days can be explained by the differences in tissue interaction of the different

In a study [92] conducted by our group, we evaluated the immediate effects of radiotherapy, more precisely, the oral and perioral soft tissue changes that appear after the radiotherapy treatment period. Additionally, we measured the gingival blood flow using LDF, in order to

Even after the first radiotherapy exposure, the blood flow values increased towards the irradiated area and remained increased throughout the entire treatment. This suggests that the periodontal tissue responds immediately to radiotherapy (as expected), and an inflammatory state is established even after the first exposure and it persists during treatment. What we found interesting was that this increase in vascularity preceded the clinical modifications, which means that with the help of LDF, we can diagnose an inflammation and we can predict the setting of the clinical side effects of radiotherapy. On the other hand, we did not find any

objectively determine any changes of the microvascular system of the gingiva.

One of our studies [93] aimed at investigating microcirculatory alterations of the gingiva occurring after smoking tobacco compared the periodontal status of both smoker and nonsmoker patients and also the registered values between the sexes (**Figure 11**).

**Figure 11.** (a) Example of LDF recording from a non-smoker patient; (b) example of LDF recording from a smoker patient; (c) interval plot of flux values (AU) in smoker male group; and (d) interval plot of flux values (A.U.) in smoker female group.

We found no significant differences (*t*-test) between non-smoker male group (I-M) and nonsmoker female group (I-F). On the other hand, LDF in the smoker female group (Group II-F) was significantly elevated compared to the smoker male group (Group II-M). The Group II-M LDF values were slightly increased compared to the Group I-M. The LDF values in the Group II-F were significantly higher than the LDF values in Group I-F.

#### *2.2.5. Laser Doppler imaging and gingival microcirculation*

Essentially, LDI works by scanning a monochromatic laser across the surface of the tissue. Light, which is backscattered from moving erythrocytes, undergoes a shift in frequency proportional to its velocity, according to the Doppler principle. Most laser Doppler set-ups use a helium-neon laser (RED, 632.8 nm), providing an estimate of perfusion up to a depth of 1– 1.5 mm into the dermis of white skin and thus mainly measure the perfusion in arterioles, venules and capillaries. LDI gives a 'snapshot' of perfusion at a given point.

The objective of one of our studies [19] was to evaluate the applicability of LD line scanning in recording the gingival healing process after a surgical procedure followed by two types of plastic provisional restoration. As a secondary objective, we also aimed at testing two different techniques and materials for performing the plastic temporaries. The results were also validated by clinical examination.

The moorLDI2-IR instrument, infrared diode laser 785 nm nominal, maximum power 2.5 mW with a visible diode laser (target beam for infrared systems) 660 nm nominal, maximum power 0.25 mW, was used in our study. The microcirculation in the investigated areas suffered changes in the analysed period (14 days) and was monitored with the Moor laser Doppler line scanner (**Figure 12**).

**Figure 12.** Laser Doppler line scanning procedure.

LDI recordings were performed in the labial regions of the operated areas at the day of the surgery, prior to local anaesthesia, after 24 h, after 7 days and 14 days following the intervention. The scanner used in this study was placed so that it was directed to record the vessels within the selected area. The differences between the four recordings clearly demonstrated adjustments in the micro-vascularity of the region in the healing period. The initial images of the area (**Figure 13(a)**) showed a certain perfusion map that differed completely from the LDI images at 24 h after the surgical procedure and the cementation of the plastic temporaries. The image at 24 h showed increased microcirculation as a reaction to the surgical procedure (**Figure 13(b)**). This situation is represented by an increase in the red colour of the affected areas in the perfusion map. The LDI images, 7 days after the surgical procedure, showed an improvement in the microcirculation healing in the interested area while the LDI images, 14 days after the surgical procedure, confirmed healing by offering a perfusion map similar to the initial one. The clinical examination asserted the changes observed on the perfusion maps in both cases.

With the aid of LDI, it was possible to obtain information regarding the impact of different materials for aesthetic prosthetics temporary restoration after surgical treatment on GM. The two types of plastic materials had no negative influence on the healing process of investigated area.

1.5 mm into the dermis of white skin and thus mainly measure the perfusion in arterioles,

The objective of one of our studies [19] was to evaluate the applicability of LD line scanning in recording the gingival healing process after a surgical procedure followed by two types of plastic provisional restoration. As a secondary objective, we also aimed at testing two different techniques and materials for performing the plastic temporaries. The results were also

The moorLDI2-IR instrument, infrared diode laser 785 nm nominal, maximum power 2.5 mW with a visible diode laser (target beam for infrared systems) 660 nm nominal, maximum power 0.25 mW, was used in our study. The microcirculation in the investigated areas suffered changes in the analysed period (14 days) and was monitored with the Moor laser Doppler line scanner

LDI recordings were performed in the labial regions of the operated areas at the day of the surgery, prior to local anaesthesia, after 24 h, after 7 days and 14 days following the intervention. The scanner used in this study was placed so that it was directed to record the vessels within the selected area. The differences between the four recordings clearly demonstrated adjustments in the micro-vascularity of the region in the healing period. The initial images of the area (**Figure 13(a)**) showed a certain perfusion map that differed completely from the LDI images at 24 h after the surgical procedure and the cementation of the plastic temporaries. The image at 24 h showed increased microcirculation as a reaction to the surgical procedure (**Figure 13(b)**). This situation is represented by an increase in the red colour of the affected areas in the perfusion map. The LDI images, 7 days after the surgical procedure, showed an improvement in the microcirculation healing in the interested area while the LDI images, 14 days after the surgical procedure, confirmed healing by offering a perfusion map similar to the initial one. The clinical examination asserted the changes observed on the perfusion maps in both cases.

With the aid of LDI, it was possible to obtain information regarding the impact of different materials for aesthetic prosthetics temporary restoration after surgical treatment on GM. The

venules and capillaries. LDI gives a 'snapshot' of perfusion at a given point.

validated by clinical examination.

218 Microcirculation Revisited - From Molecules to Clinical Practice

**Figure 12.** Laser Doppler line scanning procedure.

(**Figure 12**).

**Figure 13.** (a) Initial LDI recording and (b) LDI recording at 24 h with an increase in the red colour of the affected areas in the perfusion map.

The major advantages of LDI over LDF are the fact that there is no need for direct contact with the tissue (max. distance 19 cm), the possibility to accomplish multiple measurements allowing to obtaining many images in the area of interest (120 pixel/cm) and most importantly, it allows a global analysis of blood flow in the area of interest.

This technique has been shown to be easy to learn by surgeons. Regular post-operative assessment of flap perfusion by members of the microsurgery team trained in the use of LD line scanning might, therefore, represent a practical alternative to more complex and invasive monitoring techniques. Issues of inter- and intra-examiner reliability have yet to be examined, and in an area where only a low percentage of flaps undergo vascular compromise, this may prove impractical.

One advantage that LDF has over LDI is that it gives a constant measure of blood flow at the specified point, whereas LDI gives a 'snapshot' of perfusion at a given point.

#### **2.3. Limitations**

Although LDF has proved valuable for a variety of clinical applications, there are some limitations to its use in oral medicine. A major drawback is that LDF can only detect red blood cell movement in a small volume of tissue (1 mm3 ); thus, variables such as the number of vessels with active flow, changes in vessel diameter and flow in individual micro-vessels cannot be analysed. The small measuring area may also influence the reproducibility of the results due to the fact that a minimal displacement of the optical probe would lead to a change in the investigated area [20]. Another source of error in LDF measurements are the artefacts caused by tissue motion in relation to the probe. Additionally, oral LDFRs have demonstrated considerable intra- and inter-individual variability [94, 95]. A part of the limitations is being solved by the fact that the velocity of PBF in humans is very low and that LDF modified for the measurement of slow blood flow is appropriate for PBF measurement in humans [96]. One of the most important limitations of the LDF is that each patient presents variation of blood flow because the measurement is influenced by the thickness of the connective tissue and local distribution of the vessels and also the recording site (free gingivae, inter-dental gingivae, attached gingivae or alveolar mucosae) [35–37, 40]. Other limitation of LDF is that flow readings are not only dependent on the blood flow in the measurement volume but also on the scattering properties of the surrounding tissues. It has been reported that up to 80% of LD blood flow signal recorded from an intact human pulp is of non-pulpal origin [41]. The same could be anticipated for LDF measurements performed on the gingivae.

Originally, iontophoresis was used in conjunction with single-point LDF, as opposed to LDI systems, which measure perfusion over a larger area and produce a detailed perfusion map. Laser Doppler flowmetry typically measures within a small volume (∼1 mm3 ) and, as a result, has often suffered from poor reproducibility, mainly due to the spatial heterogeneity of tissue blood flow and movement artefacts [97, 98], although reproducibility has been improved recently by the use of 'integrated probes'. These uses multiple collecting fibres positioned in a ring around a central light delivery fibre, thus increasing the spatial resolution. However, the use of LDI still provides a larger surface area measurement and should be the preferred choice in areas of tissues with high spatial variability, despite the significant difference in costs. This could be detrimental if one is interested in the dynamics of the dilator response. This problem can be partially solved by altering the time taken for a scan. This can be done in two ways: by reducing the area to be scanned and/or increasing the scanning speed of the laser. The latter has the slight disadvantage of producing a slightly less detailed image, but in most cases, it is a compromise worth making. Many studies are not closely concerned with the dynamics of the cutaneous response and are instead focusing more on the maximum response at a given dose, in which case LDI is adequate. The line-of-sight velocity of the moving scatterers is directly proportional to the frequency of the fluctuations. This would suggest that both techniques are linear with respect to velocity. In the case of Doppler, however, it has been accepted for some 30 years that if you take the first moment of the power spectrum of the fluctuations, then it scales linearly with both velocity and concentration (number of moving scatterers) [99]. In the case of blood flow, this is a measure of perfusion. If a Doppler system uses this algorithm (first moment of the power spectrum), then it should be linear with respect to perfusion [100].

## **3. Conclusions**

This technique has been shown to be easy to learn by surgeons. Regular post-operative assessment of flap perfusion by members of the microsurgery team trained in the use of LD line scanning might, therefore, represent a practical alternative to more complex and invasive monitoring techniques. Issues of inter- and intra-examiner reliability have yet to be examined, and in an area where only a low percentage of flaps undergo vascular compromise, this may

One advantage that LDF has over LDI is that it gives a constant measure of blood flow at the

Although LDF has proved valuable for a variety of clinical applications, there are some limitations to its use in oral medicine. A major drawback is that LDF can only detect red blood

with active flow, changes in vessel diameter and flow in individual micro-vessels cannot be analysed. The small measuring area may also influence the reproducibility of the results due to the fact that a minimal displacement of the optical probe would lead to a change in the investigated area [20]. Another source of error in LDF measurements are the artefacts caused by tissue motion in relation to the probe. Additionally, oral LDFRs have demonstrated considerable intra- and inter-individual variability [94, 95]. A part of the limitations is being solved by the fact that the velocity of PBF in humans is very low and that LDF modified for the measurement of slow blood flow is appropriate for PBF measurement in humans [96]. One of the most important limitations of the LDF is that each patient presents variation of blood flow because the measurement is influenced by the thickness of the connective tissue and local distribution of the vessels and also the recording site (free gingivae, inter-dental gingivae, attached gingivae or alveolar mucosae) [35–37, 40]. Other limitation of LDF is that flow readings are not only dependent on the blood flow in the measurement volume but also on the scattering properties of the surrounding tissues. It has been reported that up to 80% of LD blood flow signal recorded from an intact human pulp is of non-pulpal origin [41]. The same

Originally, iontophoresis was used in conjunction with single-point LDF, as opposed to LDI systems, which measure perfusion over a larger area and produce a detailed perfusion map.

has often suffered from poor reproducibility, mainly due to the spatial heterogeneity of tissue blood flow and movement artefacts [97, 98], although reproducibility has been improved recently by the use of 'integrated probes'. These uses multiple collecting fibres positioned in a ring around a central light delivery fibre, thus increasing the spatial resolution. However, the use of LDI still provides a larger surface area measurement and should be the preferred choice in areas of tissues with high spatial variability, despite the significant difference in costs. This could be detrimental if one is interested in the dynamics of the dilator response. This problem can be partially solved by altering the time taken for a scan. This can be done in two ways: by reducing the area to be scanned and/or increasing the scanning speed of the laser. The latter has the slight disadvantage of producing a slightly less detailed image, but in most

); thus, variables such as the number of vessels

) and, as a result,

specified point, whereas LDI gives a 'snapshot' of perfusion at a given point.

could be anticipated for LDF measurements performed on the gingivae.

Laser Doppler flowmetry typically measures within a small volume (∼1 mm3

cell movement in a small volume of tissue (1 mm3

220 Microcirculation Revisited - From Molecules to Clinical Practice

prove impractical.

**2.3. Limitations**

The major advantage of the laser Doppler techniques in general is their non-invasiveness and their ability to measure the microcirculation flux of the tissue and fast changes of perfusion during provocations. The LDF represents an important instrument to assess gingival and pulpal microcirculation in the oral cavity. In this respect, it enables monitoring of the tooth vitality, establishing the pulp revascularization before these data could be derived from traditional sensitivity tests, which can also add more inflammation to the already irritated pulp. LDF can be used to assess the degree and duration of the pulpal inflammation or ischemic episodes, thereby identifying patients at risk for adverse reactions such as irreversible inflammation, avascular necrosis and tissue loss. Further studies are warranted to assess the validity of pulpal blood flow measurements by comparing them with histological tooth pulp changes, and by determining how well the LDF diagnoses of pulp health may predict the course of pre-prosthetic treatment.

In conclusion, LDF is a suitable technique for determining pulp vitality in most clinical situations and can be used together with other indices to evaluate the marginal gingival health status.

## **Author details**

Carmen Todea1\*, Silvana Canjau1 , Mariana Miron1 , Bogdan Vitez1 and Gheorghe Noditi2

\*Address all correspondence to: carmentodea@gmail.com

1 Department of Oral Rehabilitation and Dental Emergencies, Faculty of Dentistry, 'Victor Babes' University of Medicine and Pharmacy, Timisoara, Romania

2 Department of Plastic and Reconstructive Surgery, 'Victor Babeș' University of Medicine and Pharmacy, Timisoara, Romania

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## **Vascular Regeneration by Endothelial Progenitor Cells in Health and Diseases Vascular Regeneration by Endothelial Progenitor Cells in Health and Diseases**

Estefanía Nova-Lamperti, Felipe Zúñiga, Valeska Ormazábal, Carlos Escudero and Claudio Aguayo Estefanía Nova-Lamperti, Felipe Zúñiga, Valeska Ormazábal, Carlos Escudero and Claudio Aguayo

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64529

#### **Abstract**

Human endothelial progenitor cells (hEPCs) are adult stem cells, located in the bone marrow and peripheral blood. These cells can be differentiated into mature endothelial cells, which are involved in processes of angiogenesis and vessel regeneration. Different phenotypes and subtypes of endothelial progenitor cells (EPCs), such as early and late EPCs, have been described according to their functionality. Thus, it has been shown that early EPCs release cytokines that promote tissue regeneration and neovasculogenesis, whereas late EPC and endothelial colony forming cells (ECFCs) contribute to the formation of blood vessels and stimulate tube formation. It has been demonstrated that the number of circulating hEPC is decreased in individuals with hypercholesterolemia, hypertension, and/or diabetes. In addition, the number and the migratory activity of these cells are inversely correlated with risk factors such as hypertension, hypercholes‐ terolemia, diabetes, and metabolic syndrome. On the other hand, the number of circulating hEPC is increased in hypoxia or acute myocardial infarction (AMI). hEPCs have been used for cell‐based therapies due to their capacity to contribute in the re‐ endothelialization of injured blood vessels and neovascularization in ischemic tissues. This chapter provides an overview of the key role of hEPC in promoting angiogenesis and their potential use for cell therapy.

**Keywords:** stem cell, endothelial progenitor cells, angiogenesis, vascular regenera‐ tion, cell therapy

License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution

## **1. Introduction**

Stem cells are characterized by their ability to proliferate and self‐renew in response to signals or stimuli generatedby the microenvironment.These signals can also induce thedifferentiation of stem cells into diverse cell types with specialized features and functions [1, 2]. According to their differentiation potential, stem cells can be classified as either embryonic or adult. The characteristics of both cell populations are summarized in **Table 1**. In this chapter, we will focus on adult stem cell. This subtype of stem cells is present in several tissues and is thought to be a part of the natural tissue repair system (**Figure 1**). Adult stem cells can be present not only in tissues with high regenerationpotential, such as the skin, intestinal epithelium [3], andvascular tissue [3] but also in tissues with lower cell turnover like the brain [4]. They are responsible for tissue regeneration, andtheycanbe classifiedashematopoietic stemcells (HSCs),mesenchymal stem cells (MSCs), and endothelial progenitor cells (EPCs).


+++: high; ++: medium; +: low.

Adapted with permission from Smart and Riley [127] and Adams et al. [128].

**Table 1.** Main characteristics of human stem cells.

**Figure 1.** Types of stem cell and their potential differentiation.

## **2. Hematopoietic stem cells (HSCs)**

**1. Introduction**

232 Microcirculation Revisited - From Molecules to Clinical Practice

+++: high; ++: medium; +: low.

**Table 1.** Main characteristics of human stem cells.

**Figure 1.** Types of stem cell and their potential differentiation.

Stem cells are characterized by their ability to proliferate and self‐renew in response to signals or stimuli generatedby the microenvironment.These signals can also induce thedifferentiation of stem cells into diverse cell types with specialized features and functions [1, 2]. According to their differentiation potential, stem cells can be classified as either embryonic or adult. The characteristics of both cell populations are summarized in **Table 1**. In this chapter, we will focus on adult stem cell. This subtype of stem cells is present in several tissues and is thought to be a part of the natural tissue repair system (**Figure 1**). Adult stem cells can be present not only in tissues with high regenerationpotential, such as the skin, intestinal epithelium [3], andvascular tissue [3] but also in tissues with lower cell turnover like the brain [4]. They are responsible for tissue regeneration, andtheycanbe classifiedashematopoietic stemcells (HSCs),mesenchymal

**Characteristics Embryonic stem cells Adult stem cells**

Proliferation capacity +++ + Potential differentiation +++ ++ Cellular availability +++ + Immunogenicity allogenic ++ +++ Teratogenicity Yes No Ethical acceptability No Yes Complexity of isolation +++ ++ Clinical practice No Yes

stem cells (MSCs), and endothelial progenitor cells (EPCs).

Adapted with permission from Smart and Riley [127] and Adams et al. [128].

HSCs are multipotent tissue-specific stem cells that give rise and maintain lifelong hematopoiesis [5]. HSCs only comprise approximately 0.001–0.01% of total bone marrow cells in mice and approximately 0.01–0.2% of total bone marrow mononuclear cells in humans [6]. Moreover, HSCs express cytokines receptors, allowing them to respond to signals from immune cells and to sense pathogens during inflammation or infection. This capacity allows them to adapt their cycling and differentiation behavior according to the requirements of the body [7].

## **3. Mesenchymal stem cells (MSCs)**

MSCs are bone marrow–derived stem cells that have the capacity to form plastic-adherent colony forming unit-fibroblasts (CFU-f) [8]. They exhibit a well-known phenotype (CD73+ CD90+ CD105+ CD34− CD45− ), and they have the capacity to differentiate into osteoblasts, adipocytes, and chondroblasts [9]. Furthermore, they can be also differentiated into numerous cell types derived from all three embryonic layers, which include muscle, vascular, nervous, hematopoietic, and bone cells, among others. MSCs can be isolated from bone marrow, adipose tissue, synovium, skeletal muscle, dermis, pericytes, amniotic fluid, umbilical cord, and even human peripheral blood [10–13]. These cells are indeed promising candidates for tissue engineering and cell-based therapies not only because of their multipotent differentiation potential but also due to their low immunogenicity [14].

## **4. Human endothelial progenitor cells (hEPCs)**

hEPCs are adult stem cells characterized by the capacity to proliferate [15], self-renew and repair endothelial tissue [16]. They have been successfully isolated from peripheral blood [16], placenta, and bone marrow [17]. Several cell surface markers have been described to identify hEPC, such as CD34 [18], vascular endothelial growth factor (VEGF) receptor-1 or Flt-1 [19, 20], CD133 or prominine-1 (surface glycoprotein), Tie-2 (endothelial receptor tyrosine kinase), Von Willebrand factor, Nanog, and Oct-4 (Octámer-4) [21].

The original description of hEPCs by Asahara et al. was based on (1) the ability of hEPC to adhere to fibronectin-coated surfaces and (2) the surface expression of both immature stem cells (CD34, CD45, VEGFR2, or Flk-1) and mature endothelial cell (EC) markers (CD31, Eselectin, and angiopoietin receptor Tie-2) [20]. In addition, the expression of endothelial nitric oxide synthase (eNOS), the synthesis of nitric oxide (NO), and the ability to incorporate lowdensity lipoproteins (LDL) have been also associated with differentiation of hEPC toward endothelial cells [22].

## **4.1. Origin of hEPCs**

To date, at least four cell sources of circulating hEPCs have been described: (1) HCSs (hemangioblast and myeloid cell), (2) bone marrow–derived MSCs, (3) hEPC not derived from bone marrow (fat and resident cells in tissues such as heart, liver, intestine, and nervous system), and (4) mature ECs migrating from the vascular wall [16, 23]. The best‐characterized and most abundant hEPC are hematopoietic‐derived hEPC, which can be isolated from peripheral blood mononuclear cells (PBMCs), umbilical cord, and placenta [16, 24]. Despite the fact that hematopoietic‐derived hEPC are identified in different tissues, they have similar features, for example, hEPCs from umbilical cord exhibit the same surface markers (CD34, CD146, vWF, and VEGFR2) as hEPC from peripheral blood [25]. Other similarities between hematopoietic‐ derived hEPC include the ability to uptake modified LDL and the capacity to form capillary type structures in matrigel [26]. It has been shown that circulating monocytes have also the potential to differentiate into a variety of cell types (transdifferentiation), including EPCs [27]. Schmeisser et al. showed that CD14+ CD34− cells, isolated from PBMCs and cultured for 2–4 weeks on fibronectin‐coated plates with VEGF supplemented medium, were able to express markers of ECs, such as von Willebrand factor (vWF) [20], vascular endothelial (VE)‐cadherin, and eNOS [28]. In addition, these CD14+ cells changed their phenotype toward endothelial morphology and were able to form capillary type structures on matrigel [29, 30]. The principal surface markers of hEPC are shown in **Table 2**.


VEGFR2, vascular endothelial growth factor receptor; vWF, von Willebrand factor, eNOS, endothelial nitric oxide synthase; VE‐Cad, vascular endothelial cadherin. Adapted with permission from: Hur et al. [56].

**Table 2.** Surface markers of hEPC.

Hematopoietic‐derived hEPCs are maintained in a particular niche in the bone marrow, and they can be released into circulation by cytokines such as VEGF or stromal‐derived factor 1 (SDF‐1), synthesized by ischemic tissues and hormonal stimuli. Once in circulation, hEPCs are recruited to repair damaged endothelium and/or induce blood vessel formation. In target tissues, they can be differentiated into mature ECs to lead re‐endothelialization processes and neovascularization [16].

Circulating hEPCs can be isolated and cultured from PBMCs by three different methods:

**a.** Cell‐culture on fibronectin matrix in the presence of VEGF [20]. Under these conditions, hEPCs are selected by their ability to bind fibronectin. After removing PBMCs in suspen‐ sion, early hEPC can be identified after 3 days of culture, whereas late hEPCs are observed after 2 weeks of culture.


## **4.2. Quantification of circulating EPCs**

marrow (fat and resident cells in tissues such as heart, liver, intestine, and nervous system), and (4) mature ECs migrating from the vascular wall [16, 23]. The best‐characterized and most abundant hEPC are hematopoietic‐derived hEPC, which can be isolated from peripheral blood mononuclear cells (PBMCs), umbilical cord, and placenta [16, 24]. Despite the fact that hematopoietic‐derived hEPC are identified in different tissues, they have similar features, for example, hEPCs from umbilical cord exhibit the same surface markers (CD34, CD146, vWF, and VEGFR2) as hEPC from peripheral blood [25]. Other similarities between hematopoietic‐ derived hEPC include the ability to uptake modified LDL and the capacity to form capillary type structures in matrigel [26]. It has been shown that circulating monocytes have also the potential to differentiate into a variety of cell types (transdifferentiation), including EPCs [27].

weeks on fibronectin‐coated plates with VEGF supplemented medium, were able to express markers of ECs, such as von Willebrand factor (vWF) [20], vascular endothelial (VE)‐cadherin,

morphology and were able to form capillary type structures on matrigel [29, 30]. The principal

VEGFR2+ VE‐cad+ VE‐cad+

VEGFR2, vascular endothelial growth factor receptor; vWF, von Willebrand factor, eNOS, endothelial nitric oxide

Hematopoietic‐derived hEPCs are maintained in a particular niche in the bone marrow, and they can be released into circulation by cytokines such as VEGF or stromal‐derived factor 1 (SDF‐1), synthesized by ischemic tissues and hormonal stimuli. Once in circulation, hEPCs are recruited to repair damaged endothelium and/or induce blood vessel formation. In target tissues, they can be differentiated into mature ECs to lead re‐endothelialization processes and

Circulating hEPCs can be isolated and cultured from PBMCs by three different methods:

**a.** Cell‐culture on fibronectin matrix in the presence of VEGF [20]. Under these conditions, hEPCs are selected by their ability to bind fibronectin. After removing PBMCs in suspen‐

E‐selectin+ E‐selectin+ e‐NOS+ e‐NOS+ vWF+ vWF+

cells, isolated from PBMCs and cultured for 2–4

cells changed their phenotype toward endothelial

CD34−

Hemangioblast Early hEPC Late hEPC Endothelial cell

CD 34+ CD 34+ CD 34+ CD 34+ CD 133± CD 133+ CD 31+ CD 31+ VEGFR2+ CD 31+ VEGFR2+ VEGFR2+

Schmeisser et al. showed that CD14+

and eNOS [28]. In addition, these CD14+

234 Microcirculation Revisited - From Molecules to Clinical Practice

synthase; VE‐Cad, vascular endothelial cadherin. Adapted with permission from: Hur et al. [56].

**Table 2.** Surface markers of hEPC.

neovascularization [16].

surface markers of hEPC are shown in **Table 2**.

Since EPCs can be identified from peripheral blood samples, their detection, quantification, and characterization may be considered as potential diagnostic and prognostic biomarkers and as a novel therapeutic option for cardiovascular disorders. The main methods to quantify EPCs in human studies can be divided into two approaches: flow cytometry and CFU assays; these are also the two most widely used methods for EPCs quantification. Flow cytometry offers the advantage of a multiparameter approach that allows the identification of both endothelial and stem cell markers. However, the gating strategies used to interpret the flow cytometric events are still highly variable and dependent on the criteria of each research group; therefore, a well‐defined and uniform gating strategy to identify these cells has not been fully established yet.

The quantification of EPCs by flow cytometry requires a combination of antibodies that recognize antigens of both progenitor and endothelial cells. This technique has allowed to identify that *in vitro* cultured CD34+ /KDR+ cells home to sites of neovascularization. Based on a review of studies using EPC phenotypes as biomarker in different diseases, the CD34+ /KDR + /CD45dim phenotype appears to be the best option to identify these cells in terms of sensitivity, specificity, and reliability to quantify EPC in the clinical settings [32].

In terms of absolute quantification, it has been shown that peripheral blood samples from healthy donors (*n* = 10) have a median value of 1.88 CD45dimCD34+ VEGFR2+ EPCs per micro‐ liter. Similar data reported by Van Craenenbroeck et al. showed that the median value of CD34+ VEGFR‐2+ CD133+ EPCs was 1.95 per microliter [33]. Other authors have reported similar values of peripheral blood EPCs [34–36].

The different absolute numbers obtained for circulating EPC quantification could be explained by the use of different gating strategies and phenotypes to identify EPC subpopulation.

### **4.3. Migration, recruitment, and differentiation toward EPCs**

In healthy individuals, hEPC correspond to the 0.0001–0.01% of the total cells in blood circulation [37]. The majority of these cells are located in the bone marrow as stem cells in a quiescent state. In this tissue, hEPCs are surrounded by stromal cells in a microenvironment characterized by low oxygen tension and high levels of chemoattractant molecules [29, 38]. Different factors such as hypoxia, trauma, physical exercise, estrogen, or cytokines can access to the bone marrow from circulation and induce the release of stem cells with the potential to differentiate toward hEPCs. Once released, stem cells migrate via circulatory system to the injury zone. How these cells reach the site of injury is not totally understood; however, it has been described that cells can be guided by the concentration gradient of different chemoat‐ tractant molecules [39].

**Figure 2.** Recruitment and incorporation of hEPCs into ischemic tissue.

It has been shown that hEPC migration and mobilization is related to the secretion of angio‐ genic growth factors such as VEGF‐A, VEGF‐B, stromal cell‐derived factor 1 (SDF‐1), and insulin‐like growth factor‐I (IGF‐1) that attract cells to the site of injury [40]. SDF‐1 is a potent chemoattractant molecule released by platelets during endothelial damage [41], and its effects are dependent on the activation of the CXCR4 receptor. VEGF exerts its effect via tyrosine kinase receptors, VEGFR1 or VEGFR2, VEGFR3, which are mainly expressed in ECs from blood and lymph vessels. VEGF is produced by different cell types, such as ECs and smooth muscle cells, and is a potent angiogenic agent that regulates key steps in the process of angiogenesis, including proliferation and migration of ECs [42] and hEPC [43]. Cytokines, such as tumor necrosis factor alpha (TNF‐α), granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), granulocyte colony‐stimulating factor (G‐CSF), interleukin (IL)‐6, and IL‐3, trigger the mobilization and recruitment of hEPC. *In vivo* studies by Jin et al. in animal models subjected to ischemia demonstrated that the release of soluble proteins such as thrombopoietin (TPO), sKitL hematopoietic cytokines (soluble ligand kit), erythropoietin (EPO), and GM‐CSF induced the release of SDF‐1 from platelets, enhancing neovascularization via mobilization of CXCR4+ VEGFR1+ hemangiocytes [44]. Another study observed that there is an early vascular response involving platelet adhesion to exposed subendothelium, which represents a critical step in the homing of hEPCs to the site of endothelial disruption [45] (**Figure 2**).

As mentioned, hEPCs migrate and home to specific sites following ischemic via growth factor and cytokine gradients. Some growth factors are unstable under acidic conditions of tissue ischemia; therefore, synthetic analogues stable at low pH may provide a more effective therapeutic approach for inducing hEPC mobilization and cerebral neovascularization after an ischemic stroke [46, 47].

Also, the release of hEPC from the stem cell niche in the bone marrow has been associated with the activation of proteinases such as elastase, cathepsin G, and matrix metalloproteinases (MMP) [48]. It has been shown that stromal cells can maintain precursor stem cells or hEPCs in the bone marrow via the interaction of c‐Kit ligand (cKitL), expressed on stromal cells and their receptors expressed on precursor hEPCs. The mechanism of this interaction is under investigation; however, it is known that stromal cells induce the synthesis of nitric oxide (NO) and MMP‐9 in response to VEGF, SDF‐1, and GM‐CSF. The production of these two proteins has been associated with the cleavage of cKitL in stromal cells, allowing the release of hEPCs toward circulation [49–51].

## **4.4. EPCs and angiogenesis** *in vivo*

The different absolute numbers obtained for circulating EPC quantification could be explained by the use of different gating strategies and phenotypes to identify EPC subpopulation.

In healthy individuals, hEPC correspond to the 0.0001–0.01% of the total cells in blood circulation [37]. The majority of these cells are located in the bone marrow as stem cells in a quiescent state. In this tissue, hEPCs are surrounded by stromal cells in a microenvironment characterized by low oxygen tension and high levels of chemoattractant molecules [29, 38]. Different factors such as hypoxia, trauma, physical exercise, estrogen, or cytokines can access to the bone marrow from circulation and induce the release of stem cells with the potential to differentiate toward hEPCs. Once released, stem cells migrate via circulatory system to the injury zone. How these cells reach the site of injury is not totally understood; however, it has been described that cells can be guided by the concentration gradient of different chemoat‐

It has been shown that hEPC migration and mobilization is related to the secretion of angio‐ genic growth factors such as VEGF‐A, VEGF‐B, stromal cell‐derived factor 1 (SDF‐1), and insulin‐like growth factor‐I (IGF‐1) that attract cells to the site of injury [40]. SDF‐1 is a potent chemoattractant molecule released by platelets during endothelial damage [41], and its effects are dependent on the activation of the CXCR4 receptor. VEGF exerts its effect via tyrosine kinase receptors, VEGFR1 or VEGFR2, VEGFR3, which are mainly expressed in ECs from blood and lymph vessels. VEGF is produced by different cell types, such as ECs and smooth muscle cells, and is a potent angiogenic agent that regulates key steps in the process of angiogenesis, including proliferation and migration of ECs [42] and hEPC [43]. Cytokines, such

**4.3. Migration, recruitment, and differentiation toward EPCs**

236 Microcirculation Revisited - From Molecules to Clinical Practice

**Figure 2.** Recruitment and incorporation of hEPCs into ischemic tissue.

tractant molecules [39].

Angiogenesis and re‐endothelialization are required for the maintenance of vascular homeo‐ stasis. Initially, it was thought that these processes occurred exclusively by the migration and proliferation of mature ECs surrounding the endothelial injury. Nowadays, new vascular repair mechanisms involving precursor cells from bone marrow, such as hEPCs have been proposed [52–54]. *In vitro* studies conducted in matrigel angiogenesis have shown that hEPCs have the ability to form capillary structures, depending on their maturation stage [55, 56]. For example, early hEPCs can migrate into a tubular network already formed and secrete IL‐8 and VEGF, but they cannot form new capillary structures [57]. On the other hand, late hEPC lose their secretory capacity, but they can form capillary structures *in vitro* [56]. The ability of hEPC to form capillary structures *in vitro* and *in vivo* allowed the development of new treatments for vascular diseases. It has been demonstrated that cell therapy performed with *in vitro*‐cultured EPCs, successfully promote neovascularization in ischemic tissues without the coadministra‐ tion of angiogenic growth factors [58]. Several studies have shown that hEPCs from peripheral blood can induce endothelial cells turnover, via differentiation into functional mature ECs [59– 62]. Kalka et al*.* performed this therapeutic strategy of neovascularization for the first time in 2000 [63]. They showed improved neovascularization and functional recovery when hEPCs were injected intravenously in immunodeficient mice suffering from ischemia in the lower limbs. In rat models of myocardial ischemia, the treatment with hEPC improved the migration of cells into the neovascularization area, as well as their ability to differentiate into mature ECs, which in turn was associated with the recovery of ventricular function and reduction of the ischemic area size [64, 65]. In another study, Cui et al. injected green fluorescent protein‐tagged EPCs (GFP‐EPCs) in murine models exhibiting damaged endothelium by ligation of the left carotid artery. In these animals, GFP‐EPCs were detected at the site of injury contributing to the process of re‐endothelialization [59]. The presence of GFP‐EPCs in the injury enhances re‐ endothelialization associated with decreased neointimal formation, demonstrating that EPCs have an active role in tissue repair [59] (**Figure 3**). Other research groups have also shown that EPCs have been associated with improvements in the re‐endothelialization and neointimal formation in animal models [60, 62, 65].

**Figure 3.** Mobilization of hEPCs from the bone marrow.

All these studies have shown that hEPC are crucial for vascular repair, and it has been observed that the number and migratory activity of these cells in blood are inversely correlated with the presence of risk factors for coronary artery disease [66]. Therefore, an adequate number and a correct functional state of hEPCs are required for the maintenance of the endothelium and vascular remodeling.

#### **4.5. Mobilization mechanisms of EPCs in ischemia**

One of the main transcription factors induced during acute and chronic ischemia in response to hypoxia is the hypoxia inducible factor 1 (HIF‐1). In general, the activation of the HIF‐1 pathway has been associated with protective responses during ischemia. The mechanism of activation of HIF‐1 has been extensively described by Agani and Jiang [67]. HIF‐1 is a tran‐ scription complex formed by two subunits, alpha (Hif‐1α) and beta (Hif‐1β). While Hif‐1β is constitutively expressed, Hif‐1α levels are highly regulated by cellular oxygen partial pressure, thus Hif‐1α‐mediated cellular responses depend on oxygen levels [68]. After Hif‐1α induction, in response to low oxygen partial pressure, the ECs undergo prosurvival signals, which include the increased expression of VEGF and angiogenesis. HIF‐1α is the main direct regulator of EC function and its upregulation in EPCs promoted differentiation, proliferation, and migration in a model of hindlimb ischemia [69].

HIF‐1α‐transfected EPCs exhibited higher revascularization potential, as increased capillary density was observed at the site of injury. This study suggests that siRNA‐mediated downre‐ gulation of the HIF‐1 α gene can effectively sensitize EPCs to hypoxic conditions. It can also significantly blunt early EPC growth and differentiation into ECs [70]. The underlying mechanisms of the effect of HIF‐1α in EPC have been well described [69–72].

It has been shown that hypoxia‐induced HIF‐1 is reduced in patients with chronic heart failure (CHF) [73]; however, it has been also observed that exercise transiently increases circulating hEPCs in CHF patients. This transient effect can be sustained for approximately 4 weeks when exercise is combined with statins and/or VEGF treatment [43, 63, 74, 75].

This evidence suggests that EPCs mobilization and recruitment could also be mediated by hypoxic conditions via HIF‐1‐induced expression of VEGF.

## **4.6. Cardiovascular risk factors and hEPC function**

limbs. In rat models of myocardial ischemia, the treatment with hEPC improved the migration of cells into the neovascularization area, as well as their ability to differentiate into mature ECs, which in turn was associated with the recovery of ventricular function and reduction of the ischemic area size [64, 65]. In another study, Cui et al. injected green fluorescent protein‐tagged EPCs (GFP‐EPCs) in murine models exhibiting damaged endothelium by ligation of the left carotid artery. In these animals, GFP‐EPCs were detected at the site of injury contributing to the process of re‐endothelialization [59]. The presence of GFP‐EPCs in the injury enhances re‐ endothelialization associated with decreased neointimal formation, demonstrating that EPCs have an active role in tissue repair [59] (**Figure 3**). Other research groups have also shown that EPCs have been associated with improvements in the re‐endothelialization and neointimal

All these studies have shown that hEPC are crucial for vascular repair, and it has been observed that the number and migratory activity of these cells in blood are inversely correlated with the presence of risk factors for coronary artery disease [66]. Therefore, an adequate number and a correct functional state of hEPCs are required for the maintenance of the endothelium and

One of the main transcription factors induced during acute and chronic ischemia in response to hypoxia is the hypoxia inducible factor 1 (HIF‐1). In general, the activation of the HIF‐1

formation in animal models [60, 62, 65].

238 Microcirculation Revisited - From Molecules to Clinical Practice

**Figure 3.** Mobilization of hEPCs from the bone marrow.

**4.5. Mobilization mechanisms of EPCs in ischemia**

vascular remodeling.

hEPCs number and functional status are important for their repair capacity; however, these parameters are greatly influenced by clinical condition and risk factors. Indeed, several studies have shown that patients with cardiovascular risk factors such as age, gender, smoking habits, hypertension, diabetes mellitus (DM), and dyslipidemia have reduced number and function of hEPCs in peripheral blood. In contrast, some cytokines, hormones, drugs, and physical activity can increase not only the circulating number of hEPC but also their function [30, 49, 74, 76] (**Figure 4**).

Vasa et al. showed that the number of hEPC inversely correlates with cardiovascular risk factors (age and LDL cholesterol levels). According to these results, patients with higher cardiovas‐ cular risk factors have lower number of circulating hEPC compared with the control group [66]. Studies by Hill et al. showed a positive correlation between hEPC colony numbers in culture and endothelium‐dependent vasodilatation and a negative correlation between hEPC colony number and the Framingham index [31]. Moreover, a negative correlation between the severity of atherosclerosis and hEPC levels has been described, showing decreased circulating hEPCs levels as an early risk factor of subclinical atherosclerosis [77]. Furthermore, reduced number of circulating hEPCs has been found in patients with hypercholesterolemia, which correlates with the fact that increased plasma cholesterol levels have been linked with endothelial damage. In the same study, the number of hEPCs was negatively correlated with total cholesterol and low‐density lipoprotein (LDL) cholesterol level [78]. On the other hand, it has been also observed that the number of circulating hEPCs increases significantly after exercise [79] and in response to statins [80], antidiabetic (Pioglitazone, Sitagliptin) [81], and antihypertensive drugs (Ramipril and Enalapril) [82, 83].

**Figure 4.** Mechanism of contributes EPC to the repair of injured vessels.

## **4.7. Correlation of EPC and clinical conditions**

In addition, lower numbers of circulating hEPCs have been observed in individuals with stable and unstable angina [84], erectile dysfunction [85], and atherosclerosis [86] compared with healthy volunteers. Patients with type 1 and 2 diabetes also show lower number and func‐ tionality of hEPC than healthy individuals [87]. For instance, poor glycemic control, deter‐ mined by HbA1c levels, appears to be associated with a reduction in the number of circulating EPCs, whereas an adequate control of glycemia seems to increase their numbers [88]. Several mechanisms seem to be involved in that, including advanced glycation end products forma‐ tion [89], reduced activity of silent information regulator 1 (SIRT1), and increased synthesis of platelet‐activating factor (PAF) [90].

Patients with familial hypercholesterolemia and hypertension [91, 92] also showed lower number and function of circulating hEPC. However, this last effect was reversed when angiotensin‐converting‐enzyme inhibitor (ACE‐inhibitor) was used, a phenomenon associated with reduction in the progress of vascular damage [93]. Imanishi et al. have reported that hEPCs senescence is accelerated in both experimental hypertensive rats and in patients with essential hypertension, which may be related to telomerase inactivation [94, 95]. They also found that the hypertension‐induced EPC senescence decreases vascular remodeling process [95].

Other conditions affecting the functionality of hEPC are ischemic heart disease and nonalco‐ holic fatty liver disease (NAFLD) [96]. Also, in patients with stable coronary artery disease (CAD [66, 97]), heart failure deterioration has been correlated with low number of circulating hEPC.

it has been also observed that the number of circulating hEPCs increases significantly after exercise [79] and in response to statins [80], antidiabetic (Pioglitazone, Sitagliptin) [81], and

In addition, lower numbers of circulating hEPCs have been observed in individuals with stable and unstable angina [84], erectile dysfunction [85], and atherosclerosis [86] compared with healthy volunteers. Patients with type 1 and 2 diabetes also show lower number and func‐ tionality of hEPC than healthy individuals [87]. For instance, poor glycemic control, deter‐ mined by HbA1c levels, appears to be associated with a reduction in the number of circulating EPCs, whereas an adequate control of glycemia seems to increase their numbers [88]. Several mechanisms seem to be involved in that, including advanced glycation end products forma‐ tion [89], reduced activity of silent information regulator 1 (SIRT1), and increased synthesis of

Patients with familial hypercholesterolemia and hypertension [91, 92] also showed lower number and function of circulating hEPC. However, this last effect was reversed when angiotensin‐converting‐enzyme inhibitor (ACE‐inhibitor) was used, a phenomenon associated with reduction in the progress of vascular damage [93]. Imanishi et al. have reported that hEPCs senescence is accelerated in both experimental hypertensive rats and in patients with essential hypertension, which may be related to telomerase inactivation [94, 95]. They also found that the hypertension‐induced EPC senescence decreases vascular remodeling process

antihypertensive drugs (Ramipril and Enalapril) [82, 83].

240 Microcirculation Revisited - From Molecules to Clinical Practice

**Figure 4.** Mechanism of contributes EPC to the repair of injured vessels.

**4.7. Correlation of EPC and clinical conditions**

platelet‐activating factor (PAF) [90].

[95].

Furthermore, EPCs play an important role in the development and regulation of vasculariza‐ tion in pregnancy. Luppi et al. reported a progressive increase of circulating CD133+ / VEGFR‐2+ cells from the first trimester onwards, with a significant rise of CD34+ /VEGFR+ cells near‐term [98]. In preeclampsia for example, a pregnancy condition associated with hyper‐ tension, Matsubara et al. reported no difference in the number of circulating EPCs [99]. In contrast, studies from Sugawara et al. and Lin et al. showed lower cell numbers of circulating hEPCs in this condition compared with normal pregnancies [100, 101].


**Table 3.** Physiological and pathological conditions and their effect on hEPC.

Patients with obesity were reported to have reduced numbers of circulating hEPCs, and this was inversely associated with an increased intima‐media thickness [102]. Obesity was a more prominent predictor of the number of hEPC than any other cardiovascular risk factors, and weight loss was associated with an increased hEPC count and an improved brachial artery flow-mediated dilation. Similar evidence suggests that overweight is associated with reduced capacity to produce colony-forming units [103].

Altogether these studies support the idea that hEPCs play an important role in the maintenance of vasculature homeostasis. Thus, new therapeutic strategies should aim to increase their number and functionality in circulation. A summary of the main physiological and pathological conditions associated with functionality of hEPC is shown in **Table 3**.

#### **4.8. Clinical translation of EPC therapy**

Stem cell therapy holds great promise to restore damaged vessels. Researchers have made significant progress in cell transplantation in preclinical and clinical settings. For example, initial preclinical studies have reported favorable improvements in left ventricular function in a rat model of acute myocardial infarction (AMI) after intravenous injection of *ex vivo* expanded human CD34+ cells [104]. In another study, the intramyocardial injection of EPC in a swine model of AMI reduced the scar formation and prevented the left ventricular dysfunction after AMI, providing encouraging outcomes in favoring the application of EPCs as a potential therapy in clinical trials [105, 106] (**Figure 5**).

**Figure 5.** Potential therapeutic features and the sources of their extraction of EPC.

In the human studies performed by Li et al. [108] and Lasala et al. [107] it has been shown that intracoronary infusion of hEPC in patients with AMI were associated with the migration and incorporation of hEPCs in the infarcted tissue, a reduction of infarct size, and secretion of angiogenic growth factors including VEGF, SDF‐1, and IGF‐1, which produced more capillar‐ ity and higher transdifferentiation of cells to cardiac progenitor cardiomyocytes [107, 108]. Moreover, these hEPCs also reduced apoptosis of endothelial cells and increased myocardial viability in the infarcted area [109, 110]. Studies from Dobert et al. described increased myocardial viability in patients receiving intracoronary infusion of peripheral blood bone marrow‐derived hEPCs 4 days after myocardial infarction [111]. In addition, other studies [112, 113] suggest that adhesion and differentiation of hEPC into mature ECs in infarcted tissue is partially modulated by fibrin, which in turn promotes angiogenesis. Similar studies have been conducted in patients with chronic critical limb ischemia of the lower extremities. In a Phase II clinical study, patients who received CD133+ cells, obtained from peripheral blood and mobilized with G‐CSF, experienced limb salvage, symptomatic relief, appearance of blood flow, and significant functional improvement at the site of injury [114–116]. Similarly, treatment with autologous G‐CSF‐mobilized peripheral blood CD34+ cells in nonhealing diabetic foot patients have been promising [117].

Bone marrow‐derived EPCs may be mobilized to stimulate angiogenesis and may attenuate tissue ischemia CAD and peripheral arterial disease (PAD). For instance, intramyocardial transplantation of autologous CD34+ cells improved survival in patients with cardiovascular diseases [118]. In another study, patients with refractory angina who received autologous CD34+ cells showed a reduction of angina frequency and improvement of exercise tolerance [119].

In addition, hEPCs may contribute to liver repair and regeneration by promoting the secretion of supportive factors to induce host's endogenous repair mechanisms [120]. EPC treatment has been shown to halt the progression of liver fibrosis in rats by suppressing hepatic cell activation by increasing the MMP activity and regulating hepatocyte [121].

Similar evidence has suggested that hEPCs are involved in the recovery after deep vein thrombosis (DVT). DVT is characterized by a fibrotic vein injury with loss of venous compli‐ ance and subsequent venous hypertension [122]. In this disease, hEPCs were involved in blood vessel recanalization in organized venous thrombi [123]. Human studies suggest that children with idiopathic pulmonary arterial hypertension (IPAH) had no severe adverse events after hEPCs infusion and improved pulmonary functions [124, 125]. In animal models, Baker et al. (2013) described the use of autologous bone marrow‐derived EPCs in a rat model of pulmonary arterial hypertension (PAH) [126]. They found that EPCs reduced the hemodynamics and ventricular weight, at the same time that they increased connexin, eNOS expression and activity, Bcl‐2 expression, and the number of alveolar sacs and small lung arterioles.

## **5. Concluding remarks**

weight loss was associated with an increased hEPC count and an improved brachial artery flow-mediated dilation. Similar evidence suggests that overweight is associated with reduced

Altogether these studies support the idea that hEPCs play an important role in the maintenance of vasculature homeostasis. Thus, new therapeutic strategies should aim to increase their number and functionality in circulation. A summary of the main physiological and patholog-

Stem cell therapy holds great promise to restore damaged vessels. Researchers have made significant progress in cell transplantation in preclinical and clinical settings. For example, initial preclinical studies have reported favorable improvements in left ventricular function in a rat model of acute myocardial infarction (AMI) after intravenous injection of *ex vivo* expanded

model of AMI reduced the scar formation and prevented the left ventricular dysfunction after AMI, providing encouraging outcomes in favoring the application of EPCs as a potential

cells [104]. In another study, the intramyocardial injection of EPC in a swine

ical conditions associated with functionality of hEPC is shown in **Table 3**.

capacity to produce colony-forming units [103].

242 Microcirculation Revisited - From Molecules to Clinical Practice

**4.8. Clinical translation of EPC therapy**

therapy in clinical trials [105, 106] (**Figure 5**).

**Figure 5.** Potential therapeutic features and the sources of their extraction of EPC.

In the human studies performed by Li et al. [108] and Lasala et al. [107] it has been shown that intracoronary infusion of hEPC in patients with AMI were associated with the migration and incorporation of hEPCs in the infarcted tissue, a reduction of infarct size, and secretion of

human CD34+

Vascular regeneration is a dynamic area of research showing remarkable medical advances, both in basic science and in the clinical application field. The preclinical and clinical studies reviewed here strongly support a therapeutic potential use of EPCs in the treatment of cardiovascular diseases; however, the very low number of these cells limits their use for cell‐ based therapies. The number of EPCs needed for therapy in human adults is relatively large, that is, about 3 × 108 to 6 × 108 cells, which means that 8.5–120 L of peripheral blood are required to isolate an adequate number of EPCs. Therefore, protocols aimed to expand EPCs will be needed for future therapies. However, EPCs can be used in the present as a biomarker to identify the state of diverse diseases.

The mechanisms by which EPCs mediate vessel growth and repair could potentially be ascribed to a variety of angiogenic factors produced by EPCs. However, optimal quality/ quantity of EPCs is essential to set up a successful therapeutic EPC‐based approach. In order to get this, it is important to improve the isolation, characterization, and expansion methods to obtain the optimal numbers and functionality of EPCs. In addition, it is also relevant to improve the administration of these cells and the cellular application techniques such as quantification of EPC. Finally, a positive clinical outcome will be the main indicative to demonstrate whether these are able to repair the disease‐based dysfunction by the different mechanism already mentioned in this chapter.

## **Author details**

Estefanía Nova‐Lamperti1 , Felipe Zúñiga2 , Valeska Ormazábal3 , Carlos Escudero4,5 and Claudio Aguayo2,4\*

\*Address all correspondence to: caguayo@udec.cl

1 MRC Centre for Transplantation, King's College London, London, UK

2 Department of Clinical Biochemistry and Immunology, Faculty of Pharmacy, University of Concepción, Concepción, Chile

3 Department of Physiopathology, Faculty of Biological Sciences, University of Concepción, Concepción, Chile

4 Group of Research and Innovation in Vascular Health (GRIVAS Health), University of Bío‐ Bío, Chillán, Chile

5 Vascular Physiology Laboratory, Group of Investigation in Tumor Angiogenesis (GIANT), Department of Basic Sciences, University of Bío‐Bío, Chillán, Chile

## **References**

[1] Pomerantz J, Blau HM. Nuclear reprogramming: a key to stem cell function in regen‐ erative medicine. Nature Cell Biology. 2004;6(9):810–6.


based therapies. The number of EPCs needed for therapy in human adults is relatively large,

to isolate an adequate number of EPCs. Therefore, protocols aimed to expand EPCs will be needed for future therapies. However, EPCs can be used in the present as a biomarker to

The mechanisms by which EPCs mediate vessel growth and repair could potentially be ascribed to a variety of angiogenic factors produced by EPCs. However, optimal quality/ quantity of EPCs is essential to set up a successful therapeutic EPC‐based approach. In order to get this, it is important to improve the isolation, characterization, and expansion methods to obtain the optimal numbers and functionality of EPCs. In addition, it is also relevant to improve the administration of these cells and the cellular application techniques such as quantification of EPC. Finally, a positive clinical outcome will be the main indicative to demonstrate whether these are able to repair the disease‐based dysfunction by the different

, Valeska Ormazábal3

2 Department of Clinical Biochemistry and Immunology, Faculty of Pharmacy, University of

3 Department of Physiopathology, Faculty of Biological Sciences, University of Concepción,

4 Group of Research and Innovation in Vascular Health (GRIVAS Health), University of Bío‐

5 Vascular Physiology Laboratory, Group of Investigation in Tumor Angiogenesis (GIANT),

[1] Pomerantz J, Blau HM. Nuclear reprogramming: a key to stem cell function in regen‐

, Carlos Escudero4,5 and

cells, which means that 8.5–120 L of peripheral blood are required

that is, about 3 × 108

**Author details**

Claudio Aguayo2,4\*

Concepción, Chile

Bío, Chillán, Chile

**References**

Estefanía Nova‐Lamperti1

Concepción, Concepción, Chile

to 6 × 108

244 Microcirculation Revisited - From Molecules to Clinical Practice

mechanism already mentioned in this chapter.

\*Address all correspondence to: caguayo@udec.cl

, Felipe Zúñiga2

1 MRC Centre for Transplantation, King's College London, London, UK

Department of Basic Sciences, University of Bío‐Bío, Chillán, Chile

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## **Perioperative Inflammation and Microcirculation in Surgery: Clinical Strategies for Improved Surgical Outcomes Perioperative Inflammation and Microcirculation in Surgery: Clinical Strategies for Improved Surgical Outcomes**

Robert Schier, Philipp Zimmer and Bernhard Riedel Additional information is available at the end of the chapter

Robert Schier, Philipp Zimmer and Bernhard Riedel

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/64435

#### **Abstract**

Impaired microcirculation secondary to underlying vascular endothelial dysfunction is increasingly recognized to play a central role in the pathophysiology associated with numerous postoperative complications. Noxious stimuli, including direct injury from surgical trauma and hypoxia (e.g., ischemia‐reperfusion injury), trigger adrenergic‐ inflammatory‐thrombotic‐immune cascades to impair the microcirculation, with consequent perfusion‐related postoperative complications. The endothelium, charac‐ terized by exquisite sensitivity to inflammation and low proliferative potential, has limited self‐repair capacity that is dependent on circulating bone marrow‐derived endothelial progenitor cells for regeneration. As such, the extent to which the endothe‐ lial physical and functional integrity is preserved mirrors not only underlying cardiovascular health but is also an important factor in susceptibility to postoperative morbidity. This review explores the effect of perioperative inflammation on the microcirculation and some of the current protective strategies available to clinicians. "Prehabilitation," with preoperative exercise to improve the underlying endothelial function and bone marrow responsiveness for endogenous endothelial repair mecha‐ nisms, and anti‐inflammatory strategies to limit activation of the endothelial‐throm‐ botic‐inflammatory cascades may provide clinical strategies to preserve the microcirculation to engender optimal surgical outcomes.

**Keywords:** microcirculation, endothelial dysfunction, inflammation, perioperative, surgery

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **1. Introduction**

With an estimated 234 million operations performed annually, surgical care is an integral part of health care throughout the world [1]. Furthermore, the World Health Organization (WHO) estimates that the incidence of trauma, predominantly requiring surgery, accounts for 10% of deaths and 16% of disabilities worldwide—considerably more than malaria, tuberculosis, and HIV/AIDS combined [2].

Confounding the underlying comorbidities that patients present with during surgery, patients also suffer a significant biologic perturbation—the "surgical stress response"—a significant stressor to the human body during the perioperative period. A variety of systems are involved in this stress response, including the sympathetic autonomic nervous system, endocrine system, and immune system [3]. Inflammatory mechanisms are intimately tied to the immune system and contribute to direct defense against infection and promote postoperative wound healing. This physiological reaction of the human body can be exaggerated by a systemic inflammatory response syndrome (SIRS) [4]. SIRS results from the release of endogenous factors such as damage‐associated molecular patterns (DAMPs) or alarmins [4, 5] after surgical tissue injury [6]. DAMPs activate the complement system, leading to a rapid generation of C3a and C5a [7–9] and initiation of the release of a myriad of inflammatory mediators such as adiponectin, leptin, C‐reactive protein, interleukins (IL‐8, IL‐10, etc.), soluble tumor necrosis factor‐receptor 1(sTNF‐R1), and 8‐isoprostane.

**Figure 1.** The surgical pro‐inflammatory and pro‐oxidant milieu may result in both functional and structural altera‐ tions in the endothelium, resulting in hemostatic dysregulation and impaired microcirculation with consequent micro‐ vascular‐related postoperative complications (Illustration courtesy of Dr Marissa Ferguson).

Interestingly, these inflammatory mediators, described as a systemic "inflammome," are increased in obese patients presenting for bariatric surgery [10]. Hence, this suggests that a significant number of patients may present to surgery with an underlying pro‐inflammatory state and is also seen in patients with inflammatory comorbidities, such as rheumatoid disease, inflammatory bowel disease, and diabetes mellitus [11, 12]. This inflammatory burden activates cellular processes at affected sites within tissues, with enhanced capillary permea‐ bility to soluble mediators, particles, and cellular trafficking. These systems are in a delicate balance, which can be easily disrupted to exacerbate disease or organ dysfunction [13].

Impaired microcirculation, largely driven by vascular endothelial dysfunction, is increasingly implicated as a central pathophysiological feature of postoperative morbidity. Microcircula‐ tion is affected by certain noxious stimuli, many of which are common to the perioperative period, including direct injury from surgical manipulation or hemodynamic shear stress, hypoxia (e.g., ischemia‐reperfusion injury), and through exposure to inflammatory cytokines and endotoxins. Perioperative inflammation caused in reaction to surgical trauma causes a pro‐inflammatory and pro‐oxidant milieu that results in both functional and structural alterations in the endothelium. This may lead to microcirculation hemostatic dysregulation with impaired local tissue perfusion and consequent micro‐ and macrovascular‐related postoperative complications (**Figure 1**) [14, 15].

## **2. Physiology of the endothelium**

**1. Introduction**

260 Microcirculation Revisited - From Molecules to Clinical Practice

HIV/AIDS combined [2].

factor‐receptor 1(sTNF‐R1), and 8‐isoprostane.

With an estimated 234 million operations performed annually, surgical care is an integral part of health care throughout the world [1]. Furthermore, the World Health Organization (WHO) estimates that the incidence of trauma, predominantly requiring surgery, accounts for 10% of deaths and 16% of disabilities worldwide—considerably more than malaria, tuberculosis, and

Confounding the underlying comorbidities that patients present with during surgery, patients also suffer a significant biologic perturbation—the "surgical stress response"—a significant stressor to the human body during the perioperative period. A variety of systems are involved in this stress response, including the sympathetic autonomic nervous system, endocrine system, and immune system [3]. Inflammatory mechanisms are intimately tied to the immune system and contribute to direct defense against infection and promote postoperative wound healing. This physiological reaction of the human body can be exaggerated by a systemic inflammatory response syndrome (SIRS) [4]. SIRS results from the release of endogenous factors such as damage‐associated molecular patterns (DAMPs) or alarmins [4, 5] after surgical tissue injury [6]. DAMPs activate the complement system, leading to a rapid generation of C3a and C5a [7–9] and initiation of the release of a myriad of inflammatory mediators such as adiponectin, leptin, C‐reactive protein, interleukins (IL‐8, IL‐10, etc.), soluble tumor necrosis

**Figure 1.** The surgical pro‐inflammatory and pro‐oxidant milieu may result in both functional and structural altera‐ tions in the endothelium, resulting in hemostatic dysregulation and impaired microcirculation with consequent micro‐

Interestingly, these inflammatory mediators, described as a systemic "inflammome," are increased in obese patients presenting for bariatric surgery [10]. Hence, this suggests that a

vascular‐related postoperative complications (Illustration courtesy of Dr Marissa Ferguson).

The endothelial "organ" is estimated to weigh approximately 1 kg in adults and covers the entire vasculature with a single layer of cells, covering a surface area of approximately 100– 150 m2 and comprising 10–60 trillion cells in a single layer.

For a long time, the endothelium was considered to be inert, tasked with passive maintenance of a non‐thrombogenic blood‐tissue interface. In 1980, however, Furchgott and Zawadzki [16] discovered the endothelium‐derived relaxing factor (nitric oxide), and since then our under‐ standing of the importance of the vascular endothelium has undergone a dramatic evolution.

The endothelium is now recognized as a complex tissue composed of key immunoreactive cells that respond to environmental conditions. Sandwiched between the blood compartment and the vascular smooth muscle cells, the single layered endothelium is ideally located to act as a dynamic sensor‐effector organ. Most of the endothelial cell mass is found in the endothelial lining of the resistance vessels and capillaries, thereby exposing a relatively large endothelial surface to a small volume of blood (up to 5000 cm2 /ml). This facilitates the exchange of nutrients and metabolic products [17], and thus allows the endothelium to exert significant autocrine, paracrine, and endocrine actions on smooth muscle cells, platelets, and peripheral leukocytes. Endothelial cells, thereby, participate actively and reactively in the regulation of a number of key physiological processes, including vascular tone, vascular permeability, hemostasis (thrombosis, fibrinolysis, and platelet adherence), immune and inflammatory (leukocyte adherence) reactions, angiogenesis, and maintenance of the basement membrane. This dynamic "gate keeping" role of the endothelium, modulated through its metabolic and synthetic functions (such as production of nitric oxide, endothelin, prostaglandins, cytokines, growth factors, and adhesion molecules) and through the expression of endothelial cell receptors and glycoproteins on the abluminal surface, allows the healthy endothelium to maintain a dominant state of vasodilation, anti‐thrombosis/pro‐fibrinolysis by inhibition of platelet and leukocyte adhesion—a state that is indispensable for body homeostasis [18].

## **3. Pathophysiology of the endothelium**

In contrast, endothelial dysfunction, activation, and injury are characterized by inhibition of vasodilation, promotion of a pro‐thrombotic/anti‐fibrinolytic state, and promotion of platelet and leukocyte adhesion. Altered release of endothelium‐derived factors appears to be pivotal in pathophysiological changes that occur in disease states, such as atherosclerosis, thrombosis, hypertension, pulmonary hypertension, eclampsia, hyperglycemia, diabetes, metastatic disease, immune diseases, inflammatory syndromes, infectious processes, and sepsis. Indeed, there is increasing evidence that perturbations in the vascular endothelium are directly or indirectly involved in the pathophysiology of numerous disease processes, including postop‐ erative morbid events.

**Figure 2.** The phenotypic expression of the endothelium can be described as a dynamic "set point" that ranges be‐ tween a quiescent, activated, or dysfunctional state. Endothelial cell (EC) dysfunction caused by perioperative inflam‐ mation in response to an acute stressor (surgery, critical illness) is accompanied by microcirculatory hypoperfusion that can lead to end‐organ dysfunction.

The crucial step in the progression of perioperative endothelial dysfunction is the change of the endothelium from a quiescent into an active state. The endothelium, activated by exposure to inflammatory cytokines, becomes prothrombotic, prone to vasoconstriction instead of vasodilation, and more porous with increased fluid extravasation and increased cellular trafficking to the intercellular space. A systemic response to major trauma, associated with a lowered ability to fight infection and susceptibility to sepsis, will further activate the destruc‐ tive inflammatory response [19].

In those patients presenting with underlying impaired preoperative microcirculatory function now confounded by the pathophysiologic changes to the endothelium that accompanies the surgical stress response will be at higher risk of deterioration of the endothelial reserve below a critical "physiologic threshold" required to sustain microvascular integrity and perfusion (**Figure 2**).

## **4. Endothelial regeneration**

receptors and glycoproteins on the abluminal surface, allows the healthy endothelium to maintain a dominant state of vasodilation, anti‐thrombosis/pro‐fibrinolysis by inhibition of platelet and leukocyte adhesion—a state that is indispensable for body homeostasis [18].

In contrast, endothelial dysfunction, activation, and injury are characterized by inhibition of vasodilation, promotion of a pro‐thrombotic/anti‐fibrinolytic state, and promotion of platelet and leukocyte adhesion. Altered release of endothelium‐derived factors appears to be pivotal in pathophysiological changes that occur in disease states, such as atherosclerosis, thrombosis, hypertension, pulmonary hypertension, eclampsia, hyperglycemia, diabetes, metastatic disease, immune diseases, inflammatory syndromes, infectious processes, and sepsis. Indeed, there is increasing evidence that perturbations in the vascular endothelium are directly or indirectly involved in the pathophysiology of numerous disease processes, including postop‐

**Figure 2.** The phenotypic expression of the endothelium can be described as a dynamic "set point" that ranges be‐ tween a quiescent, activated, or dysfunctional state. Endothelial cell (EC) dysfunction caused by perioperative inflam‐ mation in response to an acute stressor (surgery, critical illness) is accompanied by microcirculatory hypoperfusion

The crucial step in the progression of perioperative endothelial dysfunction is the change of the endothelium from a quiescent into an active state. The endothelium, activated by exposure to inflammatory cytokines, becomes prothrombotic, prone to vasoconstriction instead of vasodilation, and more porous with increased fluid extravasation and increased cellular trafficking to the intercellular space. A systemic response to major trauma, associated with a

**3. Pathophysiology of the endothelium**

262 Microcirculation Revisited - From Molecules to Clinical Practice

erative morbid events.

that can lead to end‐organ dysfunction.

Through reconstitution of the endothelial layer, which generally occurs in the presence of angiogenesis and vasculogenesis, endothelial function can be restored. Neovascularization is mediated through migration and proliferation of endothelial cells within the vasculature. Endothelial colony‐forming cells (CFCs) developing endothelial progeny is the key factor in order for mature endothelial cells to proliferate and restore endothelial function [20–22]. For adult vasculogenesis, endothelial progenitor cells (EPCs) play an important role for the *de novo* formation of blood vessels. Historically, the presence of circulating blood cells with the ability to promote vascular repair and regeneration was first described in 1997 [23]. A variety of seemingly endothelial‐specific cell surface antigens were displayed on the cells identified as EPCs. Subsequently, numerous experimental studies have assessed the mechanism induced by tissue ischemia, vascular trauma, tumor growth, and inflammation by which EPC are released from the bone marrow, travel to the sites of active neovascularization, and initiate the homing process in the endothelial layer. Furthermore, some studies suggest EPCs as a biomarker for clinical disorders, such as cardiovascular disease [24], cerebrovascular disease [25, 26], sepsis [27], and numerous types of cancer [28, 29]. Interestingly, there is an inverse correlation between the number of bonemarrow released, circulating EPC and the postoper‐ ative complication risk. Subsequent experimental data from marrow transplantation demon‐ strated that these stem cells are recruited to sites of active neovascularization and differentiate into vascular cells *in‐situ*. However, the frequency of this occurrence and the identification of the cell type involved need to be fully determined [30].

## **5. Endothelial progenitor cell populations**

A major limitation in this field has been the lack of specific markers and different methods used to identify circulating EPCs. Different methods included flow cytometry, cell culture methods, immunostaining, and consequently render comparison difficult. Three functional populations of EPCs have generally been well defined. A cellular population that expresses the phenotype CD34+ AC133+ KDR+ has gained wide acceptance as a measure of circulating EPC in human subjects [31]. These cells, while being recruited to denuded vessels in ischemic sites, do not become persistent vascular endothelial cells or display de novo in‐vivo vasculo‐ genic potential, but rather exhibit potent paracrine properties to regulate new vessel formation through angiogenesis [32, 33]. These cells are referred to as proangiogenic hematopoietic cells [22, 34, 35]. Colony‐forming assays, in which plated human CD34+ peripheral blood cells form cellular clusters on fibronectin‐coated dishes in‐vitro, have identified other populations of EPC. Asahara et al. [23] described that CD34+ peripheral blood cells form clusters, bind acetylated low‐density protein (acLDL) and differentiate into spindle‐shaped endothelial cells. These cell clusters are referred to as EPC colony‐forming units (CFU). A third population of EPCs, identified as yet another type of cell colony emerging from plated peripheral blood mononuclear cells, form tightly adherent cells with a cobblestone appearance and are referred to as endothelial colony‐forming cells (ECFC), late outgrowth cells (OEC), or blood outgrowth endothelial cells (BOEC). These cells become part of the systemic circulation of the host and have vessel‐forming ability [36] These ECFCs, with *in vivo* human vessel‐forming ability, exhibit the greatest features consistent with human postnatal vasculogenic cells [37].

EPC enumeration correlates with cardiovascular risk factors, extent of coronary disease, and risk of future cardiovascular events [24]. EPC enumeration and functional characterization assess the reparative ability and propensity to cardiovascular injury, and thus greatly improves the risk stratification of patients for postoperative morbidity. Given that peripherally circulat‐ ing EPCs and intrinsic stem cells play an important role in accelerating endothelialization and tissue remodeling following vascular damage from both disease and toxic insults, we antici‐ pate that therapeutic attempts to stimulate mobilization and homing of bone marrow‐derived EPC or exogenous administration of cell‐based (progenitor) therapies will likely emerge in clinical medicine over the next decade [38–40]. Comorbid disease states and aging associate with decreased regenerative ability by EPCs and may underlie the etiology of postoperative complications and delayed recovery following surgery. For example, diabetes is characterized by poor bone marrow mobilization and decreased proliferation and survival of EPCs [41]. Inhibiting oxidative stress has been shown to modulate EPCs and normalize post‐ischemic neovascularization in diabetics. Similarly, EPC mobilization is also reported to improve with insulin therapy in diabetic rats [42]. Whether this effect is mediated by insulin itself or through improved glucose control needs to be clarified.

## **6. Impaired endothelium‐dependent vascular function in the clinical setting**

An intact microcirculation is key for the functional success of the cardiovascular system and end‐organ perfusion. In the perioperative period, a wide range of microcirculatory alterations associated with surgery itself, including factors such as anesthesia type, hypothermia, hemodilution, inflammatory reaction, and microemboli formation [43,44], impair endotheli‐ um‐dependent vascular function to decrease blood flow and oxygen supply to the parenchy‐ mal cells. An improved understanding of the different types of microcirculatory alterations may also contribute to reducing perioperative complications. Variants of impaired microcir‐ culation include impaired microcirculatory perfusion where obstructed capillaries are observed next to capillaries with flow, often seen in clinical conditions such as sepsis or reperfusion injury; microcirculatory alterations characterized by increased diffusion distance between oxygen‐carrying red blood cells and tissue cells, often seen in hemodilution that accompanies cardiopulmonary bypass; microcirculatory tamponade, often associated with excessive use of vasopressors and/or increased venous pressure. This fluid overload causes tissue edema that consequently leads to a damage of endothelial cells and losses of hemody‐ namic coherence, glycocalyx barriers, and/or the compromise of adherence and tight junc‐ tions [45].

## **7. Impaired microcirculation during critical illness**

genic potential, but rather exhibit potent paracrine properties to regulate new vessel formation through angiogenesis [32, 33]. These cells are referred to as proangiogenic hematopoietic cells [22, 34, 35]. Colony‐forming assays, in which plated human CD34+ peripheral blood cells form cellular clusters on fibronectin‐coated dishes in‐vitro, have identified other populations of EPC. Asahara et al. [23] described that CD34+ peripheral blood cells form clusters, bind acetylated low‐density protein (acLDL) and differentiate into spindle‐shaped endothelial cells. These cell clusters are referred to as EPC colony‐forming units (CFU). A third population of EPCs, identified as yet another type of cell colony emerging from plated peripheral blood mononuclear cells, form tightly adherent cells with a cobblestone appearance and are referred to as endothelial colony‐forming cells (ECFC), late outgrowth cells (OEC), or blood outgrowth endothelial cells (BOEC). These cells become part of the systemic circulation of the host and have vessel‐forming ability [36] These ECFCs, with *in vivo* human vessel‐forming ability,

exhibit the greatest features consistent with human postnatal vasculogenic cells [37].

**6. Impaired endothelium‐dependent vascular function in the clinical**

An intact microcirculation is key for the functional success of the cardiovascular system and end‐organ perfusion. In the perioperative period, a wide range of microcirculatory alterations associated with surgery itself, including factors such as anesthesia type, hypothermia, hemodilution, inflammatory reaction, and microemboli formation [43,44], impair endotheli‐ um‐dependent vascular function to decrease blood flow and oxygen supply to the parenchy‐ mal cells. An improved understanding of the different types of microcirculatory alterations may also contribute to reducing perioperative complications. Variants of impaired microcir‐ culation include impaired microcirculatory perfusion where obstructed capillaries are observed next to capillaries with flow, often seen in clinical conditions such as sepsis or

improved glucose control needs to be clarified.

264 Microcirculation Revisited - From Molecules to Clinical Practice

**setting**

EPC enumeration correlates with cardiovascular risk factors, extent of coronary disease, and risk of future cardiovascular events [24]. EPC enumeration and functional characterization assess the reparative ability and propensity to cardiovascular injury, and thus greatly improves the risk stratification of patients for postoperative morbidity. Given that peripherally circulat‐ ing EPCs and intrinsic stem cells play an important role in accelerating endothelialization and tissue remodeling following vascular damage from both disease and toxic insults, we antici‐ pate that therapeutic attempts to stimulate mobilization and homing of bone marrow‐derived EPC or exogenous administration of cell‐based (progenitor) therapies will likely emerge in clinical medicine over the next decade [38–40]. Comorbid disease states and aging associate with decreased regenerative ability by EPCs and may underlie the etiology of postoperative complications and delayed recovery following surgery. For example, diabetes is characterized by poor bone marrow mobilization and decreased proliferation and survival of EPCs [41]. Inhibiting oxidative stress has been shown to modulate EPCs and normalize post‐ischemic neovascularization in diabetics. Similarly, EPC mobilization is also reported to improve with insulin therapy in diabetic rats [42]. Whether this effect is mediated by insulin itself or through Alterations of the cerebral microcirculation may represent a key component for the develop‐ ment of postoperative sepsis‐associated encephalopathy. Cerebral hypoperfusion is a common complication of sepsis and its pathophysiology is complex and related to numerous processes and pathways, while the exact mechanisms producing neurological impairment such as delirium in septic patients is not fully understood. Cerebral hypoperfusion is caused by vasoconstriction that may be induced by inflammation and hypocapnia. The underlying endothelial dysfunction in sepsis leads to impairment of microcirculation and cerebral metabolic uncoupling that may further reduce brain perfusion. The natural autoregulatory mechanisms that protect the brain from reduced/inadequate cerebral perfusion can be impaired in septic patients, especially in those with shock or delirium, and this further contributes to cerebral ischemia if blood pressure drops below critical thresholds [46].

Postoperative brain dysfunction (delirium and coma) may relate to impaired microcirculation following surgical trauma and the associated inflammation seen in the postoperative period. Postoperative neurocognitive dysfunction is very prevalent, especially in the elderly surgical patient population. It has been reported to independently associate with prolonged mechanical ventilation, longer and more costly hospitalizations, delayed cognitive dysfunction that persists for months after hospital discharge, and increased mortality [47–53]. Factors impli‐ cated in the pathogenesis of acute brain dysfunction, such as inflammation, abnormal cerebral blood flow, and increased blood‐brain barrier permeability [54, 55], are known to impact endothelial function. Similarly, critical illnesses, such as sepsis and multiple organ dysfunction syndrome, states that circulating inflammatory cytokines affect endothelial nitric oxide production and expression of adhesion molecules [56, 57]. This results in coagulation system activation, altered perfusion, distorted permeability, and decreased ability for vascular repair [58, 59]. In the brain specifically, structural and functional alterations of blood–brain barrier endothelial cells secondary to inflammatory states have been associated with increased microvascular permeability and impaired microcirculatory blood flow [60–63]. This relation‐ ship between endothelial dysfunction and brain dysfunction during critical illness is increas‐ ingly reported in critically ill patients. The observed impact of endothelial dysfunction and injury on brain function will also likely reflect that seen in other end organs, including acute lung injury following surgery [64] or during critical illness [65].

## **8. Therapeutic options to improve perioperative endothelial dysfunction**

Therapeutic modulation of underlying subclinical microvascular endothelial dysfunction holds promise for a significant reduction in perioperative morbidity and specifically for complications such as impaired wound healing and end‐organ dysfunction related to impaired microcirculation following surgery. Perioperative inflammation can be targeted with non‐ steroidal anti‐inflammatory drugs to limit activation of the endothelial‐thrombotic‐inflamma‐ tory cascades with potential to improve perioperative outcomes [66–68]. Other therapeutic interventions, including preoperative exercise capacity, which aim to improve endothelial‐ dependent vascular function before surgery in order to cope with the inflammatory burden are currently under investigation in clinical studies [69, 70].

## **9. Mobilizing of endothelial progenitor cells with preoperative exercise**

Numerous factors have an important role in the mobilization of EPCs [71, 72]. These include growth factors (e.g., GM‐CSF, GCSF, VEGF, placental growth factor, erythropoietin, and angiopoietin‐1), pro‐inflammatory cytokines, chemokines (e.g., stromal cell‐derived factor‐1), hormones (e.g., estrogens, and lipid lowering and antidiabetic drugs), and physical activity [73]. The stimulatory effect of exercise on EPC has been shown in highly trained athletes [74], healthy subjects [71], and importantly also in patients with cardiovascular disease [75]. However, further research is required to understand the potential benefit of exercise to endothelial health in patients with subclinical cardiovascular disease characterized by endothelial dysfunction secondary to comorbidities, including metabolic syndrome or in patients subjected to the acute inflammatory insult of surgery.

Exercise has been shown to improve exercise capacity, specifically the anaerobic threshold (AT) and the maximum oxygen consumption (pVO2), and underlying endothelial reserve. In healthy subjects, Laufs et al. [76] showed that moderate and intense running for 30 min (80– 100% velocity of individuals' AT) increased circulating EPC levels, but this was not seen with running occurred at short intervals (10 minutes). In elderly patients with coronary artery disease, a 4‐week exercise program achieved significant upregulation of circulating EPCs. More recently, this was achieved after an even shorter (15 days) cardiac rehabilitation program, with an increase in EPCs that correlated with improved exercise capacity [73]. Other markers of improved endothelial function from a cardiac rehabilitation program included: a two‐fold increase in EPCs, a three‐fold increase in CFU, increased blood nitrite concentration, and reduced EPC apoptosis [75]. The duration and the intensity of exercise that are needed to adequately stimulate EPC mobilization and improve endothelial function require further investigation [77]. Surgical injury induces the mobilization of EPCs, with significantly higher circulating EPC and bone marrow EPC levels observed 24 hours after surgery in an animal model [78]. The ability to mount an EPC response is also seen in critical illness, and the response is significantly greater in patients that survive sepsis [27], and recover from illness, for example, without fibrotic changes after pneumonia [40].

Given that "responders" who mount a "cellular" stress response to injury, with increased EPC mobilization, have improved organ recovery [40] and improved survival [27], it is increasingly clear that a bone marrow‐derived cellular component must follow the surgical "stress re‐ sponse" to facilitate repair processes. In a recent pilot study, we were able to demonstrate that patients scheduled for major surgery that exhibited an EPC response to the stressor of preoperative exhaustive exercise with a single cardiopulmonary exercise test up to pVO2 suffered significantly fewer postoperative complications [69]. Whether strategies to improve bone marrow capacity and responsiveness will influence a patient's ability to withstand surgical injury remains to be investigated. Increasing this bone marrow‐derived regenerative response through preoperative exercise training may be one potential therapeutic option to optimize patients' health status prior to surgery.

**8. Therapeutic options to improve perioperative endothelial dysfunction**

Therapeutic modulation of underlying subclinical microvascular endothelial dysfunction holds promise for a significant reduction in perioperative morbidity and specifically for complications such as impaired wound healing and end‐organ dysfunction related to impaired microcirculation following surgery. Perioperative inflammation can be targeted with non‐ steroidal anti‐inflammatory drugs to limit activation of the endothelial‐thrombotic‐inflamma‐ tory cascades with potential to improve perioperative outcomes [66–68]. Other therapeutic interventions, including preoperative exercise capacity, which aim to improve endothelial‐ dependent vascular function before surgery in order to cope with the inflammatory burden

**9. Mobilizing of endothelial progenitor cells with preoperative exercise**

Numerous factors have an important role in the mobilization of EPCs [71, 72]. These include growth factors (e.g., GM‐CSF, GCSF, VEGF, placental growth factor, erythropoietin, and angiopoietin‐1), pro‐inflammatory cytokines, chemokines (e.g., stromal cell‐derived factor‐1), hormones (e.g., estrogens, and lipid lowering and antidiabetic drugs), and physical activity [73]. The stimulatory effect of exercise on EPC has been shown in highly trained athletes [74], healthy subjects [71], and importantly also in patients with cardiovascular disease [75]. However, further research is required to understand the potential benefit of exercise to endothelial health in patients with subclinical cardiovascular disease characterized by endothelial dysfunction secondary to comorbidities, including metabolic syndrome or in

Exercise has been shown to improve exercise capacity, specifically the anaerobic threshold (AT) and the maximum oxygen consumption (pVO2), and underlying endothelial reserve. In healthy subjects, Laufs et al. [76] showed that moderate and intense running for 30 min (80– 100% velocity of individuals' AT) increased circulating EPC levels, but this was not seen with running occurred at short intervals (10 minutes). In elderly patients with coronary artery disease, a 4‐week exercise program achieved significant upregulation of circulating EPCs. More recently, this was achieved after an even shorter (15 days) cardiac rehabilitation program, with an increase in EPCs that correlated with improved exercise capacity [73]. Other markers of improved endothelial function from a cardiac rehabilitation program included: a two‐fold increase in EPCs, a three‐fold increase in CFU, increased blood nitrite concentration, and reduced EPC apoptosis [75]. The duration and the intensity of exercise that are needed to adequately stimulate EPC mobilization and improve endothelial function require further investigation [77]. Surgical injury induces the mobilization of EPCs, with significantly higher circulating EPC and bone marrow EPC levels observed 24 hours after surgery in an animal model [78]. The ability to mount an EPC response is also seen in critical illness, and the response is significantly greater in patients that survive sepsis [27], and recover from illness, for example,

are currently under investigation in clinical studies [69, 70].

266 Microcirculation Revisited - From Molecules to Clinical Practice

patients subjected to the acute inflammatory insult of surgery.

without fibrotic changes after pneumonia [40].

However, discovering an inadequate EPC response during acute illness, such as impaired wound healing, pneumonia, acute lung injury [64], or sepsis [65], is likely too late. Hence, using a surrogate stressor, for example, exercise, to allow for early identification of at‐risk patients prior to surgery will enable timely strategies to improve bone marrow responsiveness to be implemented. Importantly, some of the endothelial dysfunction, particularly that acquired in the perioperative period, may be transient or reversible and may not actually involve structural change in the cells of the vascular endothelium, but more likely potentially reversible altera‐ tions in function—so these would not require new cells, just repair of a damaged process. Importantly, whether this lack of EPC response is an epiphenomenon, a surrogate marker, or indeed causative of increased postoperative complications, requires further study. The causative nature is supported by animal studies that suggest that exogenous EPC administra‐ tion can rescue endotoxin‐induced acute respiratory distress syndrome (ARDS), with reduced inflammation, improved oxygenation, and improved survival [38, 39].

Jeong et al. [79], investigating whether diabetic neuropathy could be reversed by local transplantation of EPCs, reported that motor and sensory nerve conduction velocities, blood flow, and capillary density were reduced in sciatic nerves of streptozotocin‐induced diabetic mice; with recovery after hindlimb injection of bone marrow‐derived EPCs that were shown to engraft in close proximity to the vasa nervorum. This study demonstrated that bone marrow‐ derived EPCs could reverse manifestations of diabetic neuropathy, and that cell‐based translational approaches may provide a novel and valid therapeutic alternative in the future.

Exercise [80] and tissue insult from surgery [78] are known to increase the mobilization of EPC. In this manner, cardiopulmonary exercise testing (CPET) can be used as a catalyst to increase the circulating population of EPCs and as a diagnostic tool of a patient's ability to mount an EPC response preoperatively. Additional gas exchange parameters obtained during a diag‐ nostic CPET (anaerobic threshold and peak VO2) can be used to determine patients' individual physiologic capacity and the amount of exercise needed in order to stimulate the population of EPC. Preoperative exercise training could condition patients' individual functional capacity and to improve endothelial reserve by affecting EPC responsiveness. As such, Cesari et al. [73] reported a significant increase in circulating EPCs in those patients that improved their exercise capacity by more than 23%, as assessed by a six‐minute walk test, after completion of a rehabilitation program.

## **10. Exercise and inflammation**

Regular exercise has been described to be involved in risk reduction of many chronic patho‐ logical alterations such as cancer, cardiovascular, and neurodegenerative diseases. One key mechanism, which is frequently discussed in this context, is that exercise contributes to an anti‐inflammatory environment, thereby counteracting a major risk factor of those diseases [81–83]. This hypothesis is supported by a vast body of literature, indicating that acute exercise induces a short‐term strong increase in the pro‐inflammatory cytokine interleukin‐6, which in turn induces a long‐term depression of TNF‐α and the expression of anti‐inflammatory mediators, such as interleukin‐10 and soluble receptors of interleukin‐1 [84]. Furthermore, recent research suggests that regular exercise suppresses over a life‐span the permanent expression of inflammatory cytokines via epigenetic mechanisms. Nakajima et al. [85] showed that the DNA‐methylation in the promoter region of the ASC gene, the products of which induce inflammation, is decreased in older subjects. An intermediate exercise intervention resulted in a re‐methylation of this region; hence, the methylation pattern of 60‐ to70‐year old was corrected to those of 30‐ to 40‐year‐old study participants.

The anti‐inflammatory effect of exercise is mediated by cells which secrete protective cytokines, such as interleukin‐6, which is expressed by skeletal muscle‐tissue during physical activity. However, little is known about the exact mechanism in which exercise triggers the anti‐ inflammatory component. Evidence rises that regular exercise and higher levels of cardiovas‐ cular fitness are related to an increased number of regulatory T‐cells. Since these cells have strong anti‐inflammatory properties (e.g., by secreting Interleukin‐10), they may contribute to the intermediate anti‐inflammatory effect of exercise [86].

Exercise is involved in multiple processes establishing an anti‐inflammatory environment, which counteracts with perioperative inflammatory stress. Therefore, preoperative exercise, which is feasible over a 1‐month time period, may contribute to a reduction of the inflammatory burden that is present in patients undergoing surgery.

## **11. Other aspects of exercise promoting endothelium‐dependent vascular function**

Besides the mobilization of EPCs and its anti‐inflammatory properties, exercise is known to regulate key factors of vascular functioning. Furthermore, exercise induces the expression of the endothelial nitric oxide synthase (eNOS) and increases the levels of VEGF [87–89]. The first studies revealed that the regulation of these factors is at least partially driven by epigenetic mechanisms. Wu et al. [90] revealed that exercise in rats results in a downregulation of the microRNA155. Interestingly, the messenger RNA of eNOS is known to be inhibited by microRNA155. One essential mediator may be displayed by shear‐stress which is also associated with epigenetic modifications of the chromatin (histone modifications) in the eNOS gene region [91, 92]. Fernandes et al. [93] found reduced levels of microRNA126 and 16 in exercising animals. Both microRNAs were previously described to inhibit the expression of VEGF. Although the previous studies give a premature insight into the underlying mechanism, they display that exercise truly contributes to the improvement of vascular function and regeneration on the molecular level. Further research, especially in humans, is warranted to get more information about the mechanism and dose–response relationship of exercise contributing to endothelial and vascular regeneration.

## **12. Conclusion/Summary**

**10. Exercise and inflammation**

268 Microcirculation Revisited - From Molecules to Clinical Practice

was corrected to those of 30‐ to 40‐year‐old study participants.

the intermediate anti‐inflammatory effect of exercise [86].

burden that is present in patients undergoing surgery.

**function**

Regular exercise has been described to be involved in risk reduction of many chronic patho‐ logical alterations such as cancer, cardiovascular, and neurodegenerative diseases. One key mechanism, which is frequently discussed in this context, is that exercise contributes to an anti‐inflammatory environment, thereby counteracting a major risk factor of those diseases [81–83]. This hypothesis is supported by a vast body of literature, indicating that acute exercise induces a short‐term strong increase in the pro‐inflammatory cytokine interleukin‐6, which in turn induces a long‐term depression of TNF‐α and the expression of anti‐inflammatory mediators, such as interleukin‐10 and soluble receptors of interleukin‐1 [84]. Furthermore, recent research suggests that regular exercise suppresses over a life‐span the permanent expression of inflammatory cytokines via epigenetic mechanisms. Nakajima et al. [85] showed that the DNA‐methylation in the promoter region of the ASC gene, the products of which induce inflammation, is decreased in older subjects. An intermediate exercise intervention resulted in a re‐methylation of this region; hence, the methylation pattern of 60‐ to70‐year old

The anti‐inflammatory effect of exercise is mediated by cells which secrete protective cytokines, such as interleukin‐6, which is expressed by skeletal muscle‐tissue during physical activity. However, little is known about the exact mechanism in which exercise triggers the anti‐ inflammatory component. Evidence rises that regular exercise and higher levels of cardiovas‐ cular fitness are related to an increased number of regulatory T‐cells. Since these cells have strong anti‐inflammatory properties (e.g., by secreting Interleukin‐10), they may contribute to

Exercise is involved in multiple processes establishing an anti‐inflammatory environment, which counteracts with perioperative inflammatory stress. Therefore, preoperative exercise, which is feasible over a 1‐month time period, may contribute to a reduction of the inflammatory

**11. Other aspects of exercise promoting endothelium‐dependent vascular**

Besides the mobilization of EPCs and its anti‐inflammatory properties, exercise is known to regulate key factors of vascular functioning. Furthermore, exercise induces the expression of the endothelial nitric oxide synthase (eNOS) and increases the levels of VEGF [87–89]. The first studies revealed that the regulation of these factors is at least partially driven by epigenetic mechanisms. Wu et al. [90] revealed that exercise in rats results in a downregulation of the microRNA155. Interestingly, the messenger RNA of eNOS is known to be inhibited by microRNA155. One essential mediator may be displayed by shear‐stress which is also associated with epigenetic modifications of the chromatin (histone modifications) in the eNOS gene region [91, 92]. Fernandes et al. [93] found reduced levels of microRNA126 and 16 in exercising animals. Both microRNAs were previously described to inhibit the expression of Impaired microcirculation secondary to underlying vascular endothelial dysfunction is increasingly recognized to play a central role in the pathophysiology associated with numer‐ ous postoperative complications. Noxious stimuli, including direct injury from surgical trauma and hypoxia (e.g., ischemia‐reperfusion injury), trigger adrenergic‐inflammatory‐ thrombotic‐immune cascades to impair the microcirculation, with consequent perfusion‐ related postoperative complications.

The endothelium, characterized by exquisite sensitivity to inflammation and low proliferative potential, has limited self‐repair capacity that is dependent on circulating bone marrow‐ derived endothelial progenitor cells for regeneration. As such, the extent to which the endo‐ thelial physical and functional integrity and bone marrow responsiveness, for the circulating progenitor pool, is preserved mirrors not only underlying cardiovascular health but also as an important factor in susceptibility to postoperative morbidity.

This review explores the effect of perioperative inflammation on the microcirculation and some of the current protective strategies available to clinicians. "Prehabilitation," with preoperative exercise to improve underlying endothelial function and bone marrow responsiveness for endogenous endothelial repair mechanisms, and anti‐inflammatory strategies to limit activa‐ tion of the endothelial‐thrombotic‐inflammatory cascades may provide clinical strategies to preserve the microcirculation to engender optimal surgical outcomes.

## **Author details**

Robert Schier1\*, Philipp Zimmer2 and Bernhard Riedel3

\*Address all correspondence to: robert.schier@gmx.net

1 Department of Anaesthesiology and Intensive Care Medicine, University Hospital of Cologne, Cologne, Germany

2 Department for Molecular and Cellular Sports Medicine, Institute for Cardiovascular Re‐ search and Sports Medicine, German Sport University Cologne, Cologne, Germany

3 Perioperative and Pain Medicine, Peter MacCallum Cancer Center, St Andrews Place, East Melbourne, Victoria, Australia

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## *Edited by Helena Lenasi*

The book provides a comprehensive overview of selected topics in microcirculation, from physiology to pathophysiology including molecular mechanisms and clinical aspects. It contains 10 chapters written by reputed authors, which comprehensively sum up the current knowledge and some interesting new insights in the field of microcirculation. It will be useful to a broad range of audience, from students to highly profiled experts, helping them to expand their knowledge on microcirculation and opening up additional questions for further investigation.

Microcirculation Revisited - From Molecules to Clinical Practice

Microcirculation Revisited

From Molecules to Clinical Practice

*Edited by Helena Lenasi*

Photo by tomertu / iStock