**2. Altered levels of oxygen in cell cultures**

### **2.1. Methods of altered oxygen levels in cell culture**

Hyperbaric oxygen treatment of cell cultures can be performed in hyperbaric cell culture chamber *in vitro*. Hyperbaric chambers are available commercially and offer sterile cell culture conditions for short- or long-term maintenance. Cells are usually exposed to 100% oxygen in these chambers; however, some studies comprise 98% oxygen and 2% CO2 [11]. The level of hyperbaric pressure varies between 1.5 and 3 atmospheres absolute. Compression and decompression times may be applied according to focus of interest and study protocol. Standardization of basic research protocols is key to move the latest investigations with HBOT to clinical translation. Besides HBOT, oxygen levels may be modified for normal or low oxygen (hypoxic) conditions. For normoxic treatment (21% O<sup>2</sup> ), general cell culture conditions are suitable (5% CO<sup>2</sup> , 95% normal air). Hypoxia can be induced by replacing oxygen with nitrogen in cell culture incubators. Mostly, 5 or 10% oxygen levels are investigated in cell culture studies. The same cell culture media and culturing surface can be used in altered oxygen levels, HBOT and in normal conditions [12, 13].

More detailed studies comprise direct quantification of oxygen consumption levels in cellular cultures. These data provide information also on metabolomics status, indirectly on cellular energy homeostasis and metabolic activity of the investigated cultures [14]. Planning studies with direct measurement of oxygen consumption levels enable investigation of cellular function keep with oxygen consumption.

## **2.2. Endothelial cells, angiogenesis**

HBOT may be used by pulmonologists, internal medicine specialists, surgeons and obstetrics as well. Evidence-based medicine recommends its use in decompression sickness to protect severe lung injury and to enhance recompression [1]. Carbon monoxide intoxication is another severe, life-threatening emergency scenario, where HBOT enhances CO discard and saves lives [2]. HBOT is recommended in severe carbon monoxide intoxication when conservative ventilation techniques are not efficient to eliminate CO, linked with hemoglobin. These time-sensitive conditions shout for widely available HBOT; however, in low-income

68 Hyperbaric Oxygen Treatment in Research and Clinical Practice - Mechanisms of Action in Focus

Interestingly, HBOT proved to be effective in wound healing applications, for example, ulcers, scar formation after burn injury or plastic surgery operations [3]. Cardiovascular diseases are the leading cause of death in industrialized countries. Peripheral atherosclerotic diseases and diabetes often go side-by-side. Additionally, venous circulation may also be impaired in these patients. Considering the high burden of cardiovascular diseases, number of patients suffering from not-healing ulcers is constantly increasing. Furthermore, retinal arterial stenosis severely impairs vision, in which condition HBOT is on the palette of treatment applications. Wound healing and scar formation in plastic surgery have a huge esthetic impact and because

Next argument for HBOT is that recent publications suggest its beneficial role in neurodegenerative diseases, such as multiple sclerosis [6]. Latest treatment options, for instance mesenchymal stem cell (MSC) implantation, also comprise hyperbaric treatment or preconditioning.

Other clinical applications of HBOT are severe anemia, crush injury and gas embolism, necrotizing fasciitis, osteomyelitis, brain abscesses and delayed radiation injury. Evidence is lack-

*In vitro* models of HBOT utilize wide range of cell lines and tissue cultures [9]. HBOT can be combined with modification of cell culture circumstances, for example, adding active drugs, small molecules, growth factors or signaling drives, according to the focus of interest of the study protocol. Mostly, hyperbaric treatments are applied in parallel with normoxic and hypoxic conditions to implicate useful comparative data. Importantly, *in vitro* models have severe limitations as they are not capable to model the whole pathology and tissue characteristics treated with HBOT. *In vitro* models usually follow the clinical protocols of HBOT, regarding timing and incubation periods [10]. In this chapter, altered oxygen levels of human endothelial cell cultures, fibroblasts cultures, human MSC and pluripotent stem cell (PSC) cultures will be discussed, mirroring the effects of HBOT on angiogenesis, blood clotting,

Hyperbaric oxygen treatment of cell cultures can be performed in hyperbaric cell culture chamber *in vitro*. Hyperbaric chambers are available commercially and offer sterile

of this, HBOT draws significant attention from cosmetic companies as well [5, 6].

Therapeutic potency of MSC improves after hyperbaric modification [8–10].

ing in application for Parkinson's disease and autism.

wound healing and future cell therapy/tissue engineering issues.

**2. Altered levels of oxygen in cell cultures**

**2.1. Methods of altered oxygen levels in cell culture**

countries, its use is still optional.

It is widely accepted that endothelial cells play a key role in a number of important physiological conditions and in pathological steps as well. Endothelial functions comprise regulation of blood flow via regulating vascular tone, vasodilation or vasocontraction. Furthermore, endothelial cells and their expressed factors are cornerstones in initiating or inhibiting platelet activation and blood clotting. Next role is inflammatory mechanisms, white blood cell rolling and diapedesis. Furthermore, special sites of endothelial barriers are the blood–brain barrier, the renal glomeruli and the portal endothelial cells. All these sites have complex barrier and gating functions. All endothelial functions can be modeled *in vitro* and may be investigated and modified via changing oxygen levels or by application of HBOT for cultures.

Additionally, endothelial cells regulate and are involved in embryonic vasculogenesis and somatic angiogenesis as well. Neo-angiogenesis is a key pathological step in tumorous proliferation and metastases development as well. To fulfill these tasks, endothelial cells produce and secrete wide range of angiogenesis-related proteins and small molecules. These may be investigated on gene expression or on the translational (protein) level.

Endothelial cells are keen to proliferate *in vitro*, wide range of cell lines and primary cultures are also available commercially. Widely used endothelial lines *in vitro* are the human umbilical vein endothelial cells (HUVEC), the human coronary arterial endothelial cell (HCAEC), capillary endothelial cells and others from human and animal sources as well. Arterial and venous endothelial cells can be divided via cell surface markers and genotype properties. Arterial and venous endothelial phenotypes differ also *in vitro* because the arterial and venous vessels have largely different functional tasks *in vivo*. As an example, arterial endothelial cells are the major regulators of peripheral vascular resistance, while venous capillary endothelium is the localization for white blood cells' rolling and diapedesis [15]. Furthermore, venous endothelial junctions are thinner, and vessels have greater compliance. Interestingly, arterial and venous plasticity exists *in vitro*, for example, HUVEC surprisingly express arterial markers *in vitro* [16].

Endothelial cells may be cultured in universal cell culturing dishes, on various surfaces, for example, gelatin, fibronectin, collagen and laminin. Common endothelial cell culture media are DMEM and endothelial growth media. To enhance proliferation of mature cells or differentiation from stem cells, a range of growth factors and cytokines can be applied to culture. Important characteristic of mature endothelial cells *in vitro* is the contact inhibition of proliferation [17]. This means that endothelial cells are only capable to proliferate in monolayer trend and grow onto free surfaces. Once the monolayer surface is full-grown, endothelial cell refuses to proliferate *in vitro*.

When investigating endothelial culture, most important *in vitro* characteristics of endothelial cells are the following: phenotype appearance (cobblestone pattern), expression of endothelial specific cell surface markers (CD31, CD144, vascular-endothelial cadherin), acetylated low-density-lipoprotein uptake, tube formation on Matrigel surface and wound healing assay [18]. During passage mechanisms, usually trypsin-based enzymatic digestion is utilized.

Interestingly, these endothelial characteristics were studied in HBOT circumstances as well (**Figure 1**). The morphology of adult somatic endothelial cells in response to HBOT did not change. They retained their cobblestone pattern after HBOT [19]. Importantly, viability of endothelial cells improved after 24 h of HBOT. Increase in viability was related to increase in proliferative capacity as well. Nitric oxide synthase (NOS) has pivotal role in endothelium-dependent vasoactive actions. Role of HBOT treatment was investigated on gene expression levels and on protein levels of primary microvascular capillary endothelial cell cultures. The mechanisms of actions needed further investigations, briefly NOS levels were increased in genomic and protein levels as well [20]. In-depth micro-array analyses of microvascular endothelial cells' genome proved huge impact of HBOT on angiogenesis-related gene expressions [21]. In these studies, HBOT dramatically increased tube formation capacity of endothelial cells on Matrigel [22]. Other studies also proved that HBOT had significant effects on endothelial cells tube formation and migration capacity. Short-term (6–8 h) HBOT treatment resulted in increased migration capacity and enhanced tube formation also by length and density of the network [20]. Ingenuity pathway analyses of the microarray expression data proved top responder's genes for HBOT. These top responder genes were all related to cell-matrix adhesion and matrix degradation processes. The analyses further provided quantitative data on the absolute percentage of endothelial cells that have a specific modulation, such as cellular growth and proliferation 41%, cell death 39%, gene expression 34%, cell morphology 16% and cell cycle 13% [23].

**2.3. Endothelial cells, blood clotting**

at 6 h follow-up of treatment [28].

understanding is warranted.

Besides angiogenesis, orchestrating of blot clotting is a foremost characteristic of endothelial cells. Importantly, altered oxygen circumstances can change endothelial responsiveness, platelet activation and clotting mechanisms. Tissue plasminogen activator is the most powerful enzyme to catalyze thrombin via activating plasminogen to cleave thrombin. Interesting *in vitro* studies proved that HBOT has the potential to modify tissue plasminogen activator secretion from endothelial cells [27]. The changes observed would be clinically significant and beneficial, even more, considering advantageous effects of HBOT on blood-brain barrier function. Others also measured tissue plasminogen activator in combination with plasminogen activator-inhibitor from endothelial supernatants, immediately after HBOT treatment. Surprisingly, both peptides were significantly increased after short-term HBOT. Increased expression was observed immediately after the HBOT and remained also significantly higher

Increased NOS production

expressions **HBOT**

Increased angiogenic gene

**Figure 1.** Angiogenesis-related effects of HBOT on endothelial cells.

Increased viability

**Endothelial cells**

Increased proliferation

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Increased migration and tube formation

Additionally, beside regulating endothelial cells-related clot cytokines, HBOT also had notable effects on platelet activity and function as well. Interestingly, platelets responded to HBOT in a manner that their NOS secretion increased significantly [29]. This phenomenon can have significant effects on platelet clotting and thrombus formation as well [9]; however detailed

Some human clinical studies investigated platelet count and activity after HBOT and surprisingly found no significant difference before and after HBOT [30]. *In vitro* models often use altered levels and timing of HBOT and most *in vitro* effects are not directly translatable to

In angiogenesis, main initiative steps are orchestrated by VEGF. Both by sprouting and intussusceptive angiogenesis, the main drive brings activation by VEGF isoforms and their receptors. These VEGFs set communication between tip, phalanx, stalk cells and pericytes [24]. Many other endothelial growth factors and small molecules take part in this process, for example, fibroblast growth factor, epidermal growth factor, insulin-like growth factor, Ephrins and Ephrin receptors, angiopoietin-1, angiopoietin-2 and their receptors [25]. Furthermore, the complex regulatory pathway of the renin-angiotensin-aldosteron system also interacts with vascular mechanism. Amount of secreted angiogenesis-related factors can be measured *in vitro* from cell culture supernatants and from cell lysates via proteolysis. Interestingly, angiogenesis-related steps and molecules may also play a role in chronic tinnitus, which tends to be a future disease to be treated with HBOT [26].

Cell Culture Effects of Altered Oxygen Levels and Hyperbaric Treatment *In Vitro* http://dx.doi.org/10.5772/intechopen.75378 71

**Figure 1.** Angiogenesis-related effects of HBOT on endothelial cells.

#### **2.3. Endothelial cells, blood clotting**

Endothelial cells may be cultured in universal cell culturing dishes, on various surfaces, for example, gelatin, fibronectin, collagen and laminin. Common endothelial cell culture media are DMEM and endothelial growth media. To enhance proliferation of mature cells or differentiation from stem cells, a range of growth factors and cytokines can be applied to culture. Important characteristic of mature endothelial cells *in vitro* is the contact inhibition of proliferation [17]. This means that endothelial cells are only capable to proliferate in monolayer trend and grow onto free surfaces. Once the monolayer surface is full-grown, endothelial cell

70 Hyperbaric Oxygen Treatment in Research and Clinical Practice - Mechanisms of Action in Focus

When investigating endothelial culture, most important *in vitro* characteristics of endothelial cells are the following: phenotype appearance (cobblestone pattern), expression of endothelial specific cell surface markers (CD31, CD144, vascular-endothelial cadherin), acetylated low-density-lipoprotein uptake, tube formation on Matrigel surface and wound healing assay [18]. During passage mechanisms, usually trypsin-based enzymatic digestion is utilized.

Interestingly, these endothelial characteristics were studied in HBOT circumstances as well (**Figure 1**). The morphology of adult somatic endothelial cells in response to HBOT did not change. They retained their cobblestone pattern after HBOT [19]. Importantly, viability of endothelial cells improved after 24 h of HBOT. Increase in viability was related to increase in proliferative capacity as well. Nitric oxide synthase (NOS) has pivotal role in endothelium-dependent vasoactive actions. Role of HBOT treatment was investigated on gene expression levels and on protein levels of primary microvascular capillary endothelial cell cultures. The mechanisms of actions needed further investigations, briefly NOS levels were increased in genomic and protein levels as well [20]. In-depth micro-array analyses of microvascular endothelial cells' genome proved huge impact of HBOT on angiogenesis-related gene expressions [21]. In these studies, HBOT dramatically increased tube formation capacity of endothelial cells on Matrigel [22]. Other studies also proved that HBOT had significant effects on endothelial cells tube formation and migration capacity. Short-term (6–8 h) HBOT treatment resulted in increased migration capacity and enhanced tube formation also by length and density of the network [20]. Ingenuity pathway analyses of the microarray expression data proved top responder's genes for HBOT. These top responder genes were all related to cell-matrix adhesion and matrix degradation processes. The analyses further provided quantitative data on the absolute percentage of endothelial cells that have a specific modulation, such as cellular growth and proliferation 41%,

cell death 39%, gene expression 34%, cell morphology 16% and cell cycle 13% [23].

future disease to be treated with HBOT [26].

In angiogenesis, main initiative steps are orchestrated by VEGF. Both by sprouting and intussusceptive angiogenesis, the main drive brings activation by VEGF isoforms and their receptors. These VEGFs set communication between tip, phalanx, stalk cells and pericytes [24]. Many other endothelial growth factors and small molecules take part in this process, for example, fibroblast growth factor, epidermal growth factor, insulin-like growth factor, Ephrins and Ephrin receptors, angiopoietin-1, angiopoietin-2 and their receptors [25]. Furthermore, the complex regulatory pathway of the renin-angiotensin-aldosteron system also interacts with vascular mechanism. Amount of secreted angiogenesis-related factors can be measured *in vitro* from cell culture supernatants and from cell lysates via proteolysis. Interestingly, angiogenesis-related steps and molecules may also play a role in chronic tinnitus, which tends to be a

refuses to proliferate *in vitro*.

Besides angiogenesis, orchestrating of blot clotting is a foremost characteristic of endothelial cells. Importantly, altered oxygen circumstances can change endothelial responsiveness, platelet activation and clotting mechanisms. Tissue plasminogen activator is the most powerful enzyme to catalyze thrombin via activating plasminogen to cleave thrombin. Interesting *in vitro* studies proved that HBOT has the potential to modify tissue plasminogen activator secretion from endothelial cells [27]. The changes observed would be clinically significant and beneficial, even more, considering advantageous effects of HBOT on blood-brain barrier function. Others also measured tissue plasminogen activator in combination with plasminogen activator-inhibitor from endothelial supernatants, immediately after HBOT treatment. Surprisingly, both peptides were significantly increased after short-term HBOT. Increased expression was observed immediately after the HBOT and remained also significantly higher at 6 h follow-up of treatment [28].

Additionally, beside regulating endothelial cells-related clot cytokines, HBOT also had notable effects on platelet activity and function as well. Interestingly, platelets responded to HBOT in a manner that their NOS secretion increased significantly [29]. This phenomenon can have significant effects on platelet clotting and thrombus formation as well [9]; however detailed understanding is warranted.

Some human clinical studies investigated platelet count and activity after HBOT and surprisingly found no significant difference before and after HBOT [30]. *In vitro* models often use altered levels and timing of HBOT and most *in vitro* effects are not directly translatable to *in vivo* human responses. For instance, platelet rich plasma in experimental circumstances improved after HBOT and had more advantageous effects in a pro-inflammatory, pro-thrombotic area *in vivo* [31]. Mostly, these mechanisms act differently in whole bodies *in vivo*.

Interestingly, blood-brain barrier function of endothelial cells can also be modeled and investigated *in vitro*. This very special and crucial endothelial site of the human body is key in pharmacological interventions and critically ill patients. Altered oxygen levels have different effects on blood-brain barrier. It is widely believed that decreased oxygen availability, for example, ischemic attack of the brain, has huge impact on the existence and proper function of blood-brain barrier. In stroke, blood-brain barrier lacks its gating function and medications

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In the in vitro model of blood-brain barrier, brain microvascular endothelial cells can be cultured and trans-endothelial electric potential, as a measure of barrier function, can be evaluated in different oxygen circumstances [34]. Mainly cell interactions, tight junctions and endothelial-pericyte interactions are damaged in blood-brain barrier dysfunction. Cell adhesion molecules are often investigated *in vitro* as well. Endothelial and pericyte co-cultures (e.g., insert plate) offer studying communication between these cellular compartments [35]. In co-culture, cellular models of endothelial interactions with white blood cells were also observed. White blood cells' diapedesis, rolling and pooling in microcirculation are the determinants of local inflammatory responses. Attenuating these would have dramatic therapeutic effects, for example, in chronic, not-healing wounds. Neutrophils' adhesion to endothelial cells was reversed and delayed in HBOT circumstances [36]. The underlying molecular mechanism was mainly the reduced expression of neutrophil-endothelial adhesion molecule, ICAM-1. As a result of low neutrophil adhesion, local levels of ROS were also decreased [36]. Further studies proved that HBOT may have direct effects on endothelial gene expression as well. HCAEC modified their angiogenesis-related gene expression, shortly after HBOT. Shortterm HBOT (4–6 h) resulted in increased TNF-α secretion from HCAEC. Related to this, HBOT also modified expression of a range of peptides and small molecular, which have strong role in glucose metabolism and inflammatory reactions as well [20]. Additionally, all of these mechanisms were also linked to altered expressions of certain kinases and altered phosphorylation status. These were related to visceral fat accumulation, atherosclerosis, inflammation and increased cardiovascular risk. Remarkable results proved that HBOT also have metabolomics effects on treated endothelial cells. Short-term HBOT altered glucose uptake in HCEAC. These key results showed that metabolomics disturbances may also be modified under HBOT cir-

cumstances, which has key message to future therapeutic human applications [20].

Interestingly, HBOT had robust effect on inflammation-related cytokine expression, for example, level of anti-inflammatory angiogenin decreased, while the level of pro-inflammatory cytokines (IL-6 and IL-8) significantly decreased in response to HBOT. This *in vitro* model was established from and *in vivo* septic small animal model. Endothelial cells from septic and control rats were cultures and inflammatory cytokines were measured from endothelial supernatants [37]. Others also showed significant decrease in pro-inflammatory cytokines,

Latest *in vitro* studies demonstrated that hypoxic damage of blood-brain barrier may be reversed via HBOT [34]. Hypoxia induced cellular endothelial fragmentation and impair of cell adhesion molecular. On the contrary, HBOT after hypoxia was able to attenuate the effects and improve cellular junctions [34]. These data have very important message to clinical trials, as HBOT may have undistinguishable role in stroke treatment in the acute clinical phase (**Figure 3**) [39].

may have altered neurological side effects as well.

such as TNF-α following HBOT [38].

Some studies concluded that HBOT may also have disadvantageous effects *in vivo*, if the timing and longevity of treatment is not optimal. Interestingly, in experimental setup, HBOT was able to modify renal erythropoietin production. Disadvantageous results came after HBOT was released and rebound effects ameliorated normal erythropoietin levels. Thus, renal tissue failed to cope with sudden and frequent changes in oxygen levels. The observed results were unrelated to circadian rhythm of erythropoietin production [32].

Point-of-care whole blood and platelet clot analyzer systems also brought disappointing data. Some of these ex vivo analyses proved that short-term HBOT may initiate *in vitro* steps which are characteristics of a disseminated intravascular coagulation (DIC) [33]. DIC is a severe, life threating condition, comprising both pro-thrombotic and not-clotting elements, resulting in a severe clinical case, when blood is unable to clot, but small capillaries are impaired by thrombi. In response to HBOT, an increase in the maximum clot firmness and thrombo-elastic component in clot firmness was depicted (**Figure 2**) [33].

#### **2.4. Endothelial cells, barrier and inflammation**

Nitric oxide (NO) is one of the most important factors released by endothelial cells. NO plays a pivotal role in setting vascular tone and regulating blood pressure, via arterioles. On the venous circuit site, NO also has vasodilatory effects, thus is a major vasoactive factor at the site of white blood cells diapedesis and extravasation. Beside these, NO also counteracts with angiogenic activities.

**Figure 2.** Effects of HBOT on blood clotting. PAI: plasminogen activator inhibitor, DIC: disseminated intravasulcar coagulation.

Interestingly, blood-brain barrier function of endothelial cells can also be modeled and investigated *in vitro*. This very special and crucial endothelial site of the human body is key in pharmacological interventions and critically ill patients. Altered oxygen levels have different effects on blood-brain barrier. It is widely believed that decreased oxygen availability, for example, ischemic attack of the brain, has huge impact on the existence and proper function of blood-brain barrier. In stroke, blood-brain barrier lacks its gating function and medications may have altered neurological side effects as well.

*in vivo* human responses. For instance, platelet rich plasma in experimental circumstances improved after HBOT and had more advantageous effects in a pro-inflammatory, pro-thrombotic area *in vivo* [31]. Mostly, these mechanisms act differently in whole bodies *in vivo*.

Some studies concluded that HBOT may also have disadvantageous effects *in vivo*, if the timing and longevity of treatment is not optimal. Interestingly, in experimental setup, HBOT was able to modify renal erythropoietin production. Disadvantageous results came after HBOT was released and rebound effects ameliorated normal erythropoietin levels. Thus, renal tissue failed to cope with sudden and frequent changes in oxygen levels. The observed results were

Point-of-care whole blood and platelet clot analyzer systems also brought disappointing data. Some of these ex vivo analyses proved that short-term HBOT may initiate *in vitro* steps which are characteristics of a disseminated intravascular coagulation (DIC) [33]. DIC is a severe, life threating condition, comprising both pro-thrombotic and not-clotting elements, resulting in a severe clinical case, when blood is unable to clot, but small capillaries are impaired by thrombi. In response to HBOT, an increase in the maximum clot firmness and thrombo-elastic

Nitric oxide (NO) is one of the most important factors released by endothelial cells. NO plays a pivotal role in setting vascular tone and regulating blood pressure, via arterioles. On the venous circuit site, NO also has vasodilatory effects, thus is a major vasoactive factor at the site of white blood cells diapedesis and extravasation. Beside these, NO also counteracts with angiogenic activities.

> Increased plasminogen activator levels

**HBOT Blood clotting**

**Figure 2.** Effects of HBOT on blood clotting. PAI: plasminogen activator inhibitor, DIC: disseminated intravasulcar coagulation.

Modify renal erythropoietin production

Increased PAI levels

Increased NOS secretion of platelets

unrelated to circadian rhythm of erythropoietin production [32].

72 Hyperbaric Oxygen Treatment in Research and Clinical Practice - Mechanisms of Action in Focus

component in clot firmness was depicted (**Figure 2**) [33].

**2.4. Endothelial cells, barrier and inflammation**

May cause DIC pattern In the in vitro model of blood-brain barrier, brain microvascular endothelial cells can be cultured and trans-endothelial electric potential, as a measure of barrier function, can be evaluated in different oxygen circumstances [34]. Mainly cell interactions, tight junctions and endothelial-pericyte interactions are damaged in blood-brain barrier dysfunction. Cell adhesion molecules are often investigated *in vitro* as well. Endothelial and pericyte co-cultures (e.g., insert plate) offer studying communication between these cellular compartments [35].

In co-culture, cellular models of endothelial interactions with white blood cells were also observed. White blood cells' diapedesis, rolling and pooling in microcirculation are the determinants of local inflammatory responses. Attenuating these would have dramatic therapeutic effects, for example, in chronic, not-healing wounds. Neutrophils' adhesion to endothelial cells was reversed and delayed in HBOT circumstances [36]. The underlying molecular mechanism was mainly the reduced expression of neutrophil-endothelial adhesion molecule, ICAM-1. As a result of low neutrophil adhesion, local levels of ROS were also decreased [36].

Further studies proved that HBOT may have direct effects on endothelial gene expression as well. HCAEC modified their angiogenesis-related gene expression, shortly after HBOT. Shortterm HBOT (4–6 h) resulted in increased TNF-α secretion from HCAEC. Related to this, HBOT also modified expression of a range of peptides and small molecular, which have strong role in glucose metabolism and inflammatory reactions as well [20]. Additionally, all of these mechanisms were also linked to altered expressions of certain kinases and altered phosphorylation status. These were related to visceral fat accumulation, atherosclerosis, inflammation and increased cardiovascular risk. Remarkable results proved that HBOT also have metabolomics effects on treated endothelial cells. Short-term HBOT altered glucose uptake in HCEAC. These key results showed that metabolomics disturbances may also be modified under HBOT circumstances, which has key message to future therapeutic human applications [20].

Interestingly, HBOT had robust effect on inflammation-related cytokine expression, for example, level of anti-inflammatory angiogenin decreased, while the level of pro-inflammatory cytokines (IL-6 and IL-8) significantly decreased in response to HBOT. This *in vitro* model was established from and *in vivo* septic small animal model. Endothelial cells from septic and control rats were cultures and inflammatory cytokines were measured from endothelial supernatants [37]. Others also showed significant decrease in pro-inflammatory cytokines, such as TNF-α following HBOT [38].

Latest *in vitro* studies demonstrated that hypoxic damage of blood-brain barrier may be reversed via HBOT [34]. Hypoxia induced cellular endothelial fragmentation and impair of cell adhesion molecular. On the contrary, HBOT after hypoxia was able to attenuate the effects and improve cellular junctions [34]. These data have very important message to clinical trials, as HBOT may have undistinguishable role in stroke treatment in the acute clinical phase (**Figure 3**) [39].

damage. Placing the animals in HBOT and treating them resulted in increased MMP inhibitor

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HBOT has a significant effect on the growth of fibroblast cultures. HBOT in increasing pressure and time interval had advantageous effects on the proliferation of fibroblast cultures, suggesting beneficial effects in wound healing steps as well [45]. Parallel with timing and pressure of HBOT, cell numbers increased as well [45]. Additionally, HBOT increased tube formation of endothelial and fibroblast co-cultures [20]. In a wound healing assay, *in vitro*, significant increase was observed after HBOT treatment [22]. Linking to previous clues, HBOT also increases collagen proliferation [22], parallel with beneficial effects on fibroblast growth, and thus all together, HBOT has a beneficial effect on extracellular matrix proliferation, growth and structure [20]. Furthermore, recent experiments proved the increase of fibroblast proliferation and growth also *in vitro* and *in vivo* in epidural fibrous tissue. Mechanisms of action behind these were downregulation of canonical TGF-β and interleukin pathways, which were responsible for maintaining fibroblasts' viability and proliferation (**Figure 4**) [46].

TGF-β is also involved in uncontrolled scar formation, known as keloid scars [47]. Keloid scars contain highly proliferative fibroblasts and connective tissues, which cause significant biomechanical and esthetic problems on affected skins. HBOT were successful to reduce TFG-β levels in these keloids and interestingly proliferation of keloid scar was postponed in response to HBOT [48]. The regulatory steps are not yet characterized in detail, and further

*In vitro* models for burned skins also exist; however, modeling the complex mechanisms of local and systemic response to severe burn and demonstrating and measuring accurately the cytokine storm *in vitro* are almost impossible. Interesting *in vitro* model for burned skin evolved ex vivo available burned skin tissues [50]. Recently, HBOT has been emerged for the treatment of chronic burn-related wounds as inflammatory cytokine release was decreased and bacterial viability also decreased in wound [34, 35]. Burn models proved hyperemia (improved microcirculation) and reduced size of the burned lesions after HBOT of burn wounds [53]. Additionally, fluid homeostasis of burned wounds was also altered beneficially after HBOT [51, 52]. Intercellular edema decreased after HBOT, resulting in better microcircu-

**2.6. Mesenchymal and pluripotent stem cells, cell therapy and tissue engineering** 

comprise biodegradable matrices combined with cellular building blocks.

MSC and other cell types such as the pluripotent stem cells have huge potential for cell therapy and tissue engineering in various diseases. Recently, most clinical trials in cardiovascular field have been performed with MSC or MSC-derivatives [55]. Furthermore, cardiovascular derivatives of pluripotent stem cells are promising tools to differentiate new cardiovascular cells and to build cardiovascular tissue. Latest tissue engineering methods

MSC and PSC behave and differentiate altered in normal hypoxic or in hyperbaric oxygen conditions PSC studies concluded that altered oxygen levels may mimic in utero conditions

investigation is needed to understand the process [49].

latory responses and increased debris elimination [54].

**aspects**

activity and in parallel the tissue damage, cellular apoptosis and necrosis decreased [44].

**Figure 3.** Effects of HBOT on endothleial barrier and local inflammatory reactions.

#### **2.5. Fibroblasts, wound healing**

Fibroblasts are easy to culture and maintain. They have high proliferative capacity and low maintenance circumstances. They grow in any cell culture media, mostly in fibroblast growth media or DMEM. They adhere to plastic surfaces or to any additional, for example, gelatin or fibronectin. Interestingly, fibroblasts proliferative from skin biopsy samples *in vitro* as well. Fibroblasts have rod-shaped, elongated phenotype in culture. Usually they proliferate in monolayer; however, contact inhibition of growth is not as prominent as it is by endothelial cells [40].

Chronic, not-healing wounds are major challenge in dermatology, surgery and plastic surgery [4, 7]. These wounds have valuable impact on diabetic and cardiovascular patients' quality of life. Furthermore, these wounds often become infected or colonized with resistant species, for example, MRSA [41]. Mechanisms of action in these chronic wounds include reactive oxygen species, chronic inflammation and chronic ischemia [42]. The connective tissue, extracellular matrices are also affected and hyper-oxidant status seems to be the common clue behind nonhealing. Growth and proliferation of fibroblasts are often impaired due to aforementioned pathological mechanisms. Thus, fibroblasts offer platform to monitor cellular events on one important component of these wounds. *In vitro* studies are suitable to monitor effects of altered oxygen levels, especially focusing on cytokine release, apoptosis and leukocyte activation.

*In vitro* studies proved that HBOT on ischemic wound tissue increased the activity of superoxidedismutase (SOD) enzyme, which is known to be one of the most potent enzymes acting against ROS species-related harm [43]. Interestingly, *in vivo* studies on small animal models of chronic ulcers also proved significant effects of matrix-metalloproteinases (MMP) in the chronic ongoing damage. Placing the animals in HBOT and treating them resulted in increased MMP inhibitor activity and in parallel the tissue damage, cellular apoptosis and necrosis decreased [44].

HBOT has a significant effect on the growth of fibroblast cultures. HBOT in increasing pressure and time interval had advantageous effects on the proliferation of fibroblast cultures, suggesting beneficial effects in wound healing steps as well [45]. Parallel with timing and pressure of HBOT, cell numbers increased as well [45]. Additionally, HBOT increased tube formation of endothelial and fibroblast co-cultures [20]. In a wound healing assay, *in vitro*, significant increase was observed after HBOT treatment [22]. Linking to previous clues, HBOT also increases collagen proliferation [22], parallel with beneficial effects on fibroblast growth, and thus all together, HBOT has a beneficial effect on extracellular matrix proliferation, growth and structure [20]. Furthermore, recent experiments proved the increase of fibroblast proliferation and growth also *in vitro* and *in vivo* in epidural fibrous tissue. Mechanisms of action behind these were downregulation of canonical TGF-β and interleukin pathways, which were responsible for maintaining fibroblasts' viability and proliferation (**Figure 4**) [46].

TGF-β is also involved in uncontrolled scar formation, known as keloid scars [47]. Keloid scars contain highly proliferative fibroblasts and connective tissues, which cause significant biomechanical and esthetic problems on affected skins. HBOT were successful to reduce TFG-β levels in these keloids and interestingly proliferation of keloid scar was postponed in response to HBOT [48]. The regulatory steps are not yet characterized in detail, and further investigation is needed to understand the process [49].

**2.5. Fibroblasts, wound healing**

Decreased levels of proinflammatory cytokines

> Decreased tissue ROS levels

74 Hyperbaric Oxygen Treatment in Research and Clinical Practice - Mechanisms of Action in Focus

**Figure 3.** Effects of HBOT on endothleial barrier and local inflammatory reactions.

Fibroblasts are easy to culture and maintain. They have high proliferative capacity and low maintenance circumstances. They grow in any cell culture media, mostly in fibroblast growth media or DMEM. They adhere to plastic surfaces or to any additional, for example, gelatin or fibronectin. Interestingly, fibroblasts proliferative from skin biopsy samples *in vitro* as well. Fibroblasts have rod-shaped, elongated phenotype in culture. Usually they proliferate in monolayer; however, contact inhibition of growth is not as prominent as it is by endothelial cells [40]. Chronic, not-healing wounds are major challenge in dermatology, surgery and plastic surgery [4, 7]. These wounds have valuable impact on diabetic and cardiovascular patients' quality of life. Furthermore, these wounds often become infected or colonized with resistant species, for example, MRSA [41]. Mechanisms of action in these chronic wounds include reactive oxygen species, chronic inflammation and chronic ischemia [42]. The connective tissue, extracellular matrices are also affected and hyper-oxidant status seems to be the common clue behind nonhealing. Growth and proliferation of fibroblasts are often impaired due to aforementioned pathological mechanisms. Thus, fibroblasts offer platform to monitor cellular events on one important component of these wounds. *In vitro* studies are suitable to monitor effects of altered oxygen levels, especially focusing on cytokine release, apoptosis and leukocyte activation.

Decreased ICAM-1 expression

**HBOT Endothelial barrier** Decreased neutrophil rolling and diapedesis

Decreased local inflammatory response

*In vitro* studies proved that HBOT on ischemic wound tissue increased the activity of superoxidedismutase (SOD) enzyme, which is known to be one of the most potent enzymes acting against ROS species-related harm [43]. Interestingly, *in vivo* studies on small animal models of chronic ulcers also proved significant effects of matrix-metalloproteinases (MMP) in the chronic ongoing *In vitro* models for burned skins also exist; however, modeling the complex mechanisms of local and systemic response to severe burn and demonstrating and measuring accurately the cytokine storm *in vitro* are almost impossible. Interesting *in vitro* model for burned skin evolved ex vivo available burned skin tissues [50]. Recently, HBOT has been emerged for the treatment of chronic burn-related wounds as inflammatory cytokine release was decreased and bacterial viability also decreased in wound [34, 35]. Burn models proved hyperemia (improved microcirculation) and reduced size of the burned lesions after HBOT of burn wounds [53]. Additionally, fluid homeostasis of burned wounds was also altered beneficially after HBOT [51, 52]. Intercellular edema decreased after HBOT, resulting in better microcirculatory responses and increased debris elimination [54].
