**3. Vascularization by ASCs in tissue repair**

Tissue repair and regeneration is reliant upon vascularization. Newly formed tissues must have sufficient blood flow to maintain their health and support their growth. Early in vitro studies with ASCs suggest the capacity to differentiate into endothelial cells and to form vessellike structures. For example, using simple in vitro induction conditions, ASCs express typical markers of endothelial cells, such as von Willebrand Factor (vWF) and function as endothelial cells, taking up acetylated LDL and forming tubular structures on Matrigel substrates [40], [41], [86]. Tubule formation, LDL uptake and CD31 expression by ASCs are also found upon in vitro exposure to shear stress [87], [88]. Such evidence provides strong support for the use of ASCs in the induction of vessel formation and some have attempted to isolate the specific ASC subpopulation that might be responsible for endothelial differentiation. For example, Wos‐ nitza et al. postulate that a population of CD31-ve, S100+ve ASCs are capable of endothelial differentiation [89], while CD34-ve ASCs have been observed to undergo differentiation by others [90].


**Table 2.** Growth factor secretion by ASCs

class I) and HLA-DP, DM and DR (or MHC class II), HLA receptors display proteins on the cell surface for immune surveillance. Of particular interest is the HLA/MHC class II protein, which is found on the surface of antigen-presenting cells and plays critical roles in immuno‐ tolerance and transplantation (for reviews see [83], [84]). The absence of this class of HLA protein may allow the ASC to evade the host's immune surveillance machinery. Of additional interest is a recent study by DelaRosa et al. [85], who note that human ASCs have lower susceptibility to natural killer (NK) cell-mediated lysis in comparison to bone marrow MSCs.

**ASC type Disease Model Inflammatory/Immunosuppressive action**

human murine dystrophy decreased CD3+ve T cells, increased IL-4, IL-10 synthesis

Th17 cells

activity

emphysema decreased inflammatory infiltration

human rheumatoid arthritis inhibition of CD4+ T cell proliferation, increase in

human experimental colitis decrease in Th1-driven inflammation, decrease

increased T cell responses

increased IL10 synthesis

of Treg cell development

Th1-biased immune reponse

increased IL-2 and IL-10 synthesis

increased Treg proliferation

secretion of IL-10, decreased proliferation of Th1,

decreased inflammatory markers, decreased Th1

decreased Th17 production, decrease IL-17 synthesis

IL-10 producing T cells and monocytes, stimulation

inflammatory cytokines, increased IL-10 activity

increased Th2-type shift in cytokine production[80]

demyelination – IL-4 overexpressing ASCs

**Author and Year (Reference)**

Pinheiro et al. 2012

Schweitzer et al. 2011 [64]

Gonzalez-Rey et al. 2010 [74], Gonzalez et al. 2009 [75]

Gonzalez-Rey et al. 2009 [77], Gonzalez et al. 2009 [78]

Constatin et al. 2009 [80]

Payne et al. 2012 [71] human autoimmune

54 Regenerative Medicine and Tissue Engineering

Hyun et al. 2011[67], mouse IgA-induced

human, mouse

Lai 2011 et al. [72] human systemic lupus

Kuo 2011 et al. [73] rat hind limb

Wan et al. 2008 [59] rat orthotopic liver

**Table 1.** Immunosuppressive action of ASCs

Zhou et al. 2011 [65] human autoimmune hearing

loss

nephropathy

erythamatosis

allotransplantion

Zhou 2011 et al. [66] human rheumatoid arthritis decreased Th1, Th17 proliferation/expansion,

Cho et al. 2010 [76] mouse airway allergic disease decreased airway inflammation, shift from a Th2 to a

Kim et al. 2007 [79] human hemorrhagic stroke decreased brain inflammation markers

transplant

encephalolyelitis (multiple sclerosis)

mouse autoimmune

[68]

However, the efficacy of ASCs in tissue repair may not be entirely due to their direct differ‐ entiation into endothelial lineages, but also to their secretion of paracrine factors capable of increasing vascularization. In support of this, co-culture of ASCs with postnatal cardiomyo‐ cytes results in the formation of stable, branching CD31+ve vessel-like structures that disas‐ semble in the absence of ASCs [99]. Similarly, ASC-conditioned media can induce the formation of vessel-like tubules within Matrigel [105]. More recently, while rat ASCs express Flt-1, CD31 and vascular endothelial cadherin, when injected into a wire injury model in the rat femoral artery, induction of endothelial repair occurs without any observable differentia‐ tion of these ASCs into endothelial cells [106]– a finding that can be explained if repair is driven through the production of soluble factors. In the hopes of identifying what angiogenic factors improve a tissue's vasculature, numerous studies have characterized the secretion of growth factors by ASCs (Table 2). Of all of these factors, perhaps the most commonly reported is VEGF, with secretion of this factor being reported under normal culture conditions [98], hypoxic conditions [104] in models of wound healing [96], [107] and cell-assisted lipotransfer [97]. The ability of VEGF to stimulate neoangiogenesis is well known [108]-[110]. Consistent with this, conditioned medium from ASCs, maintained under hypoxic culture conditions in order to increase production of HGF, VEGF and TGFβ, has been found to increase endothelial cell (EC) growth and reduce their apoptosis [104]. In addition, VEGF secretion by ASCs is significantly upregulated in vitro upon metabolic induction of ischemia [111]. However, the role of other secreted factors cannot be ruled out as suppression of HGF production by ASCs through RNA interference significantly impairs ischemic tissue revascularization [112] and SDF-1α from ASCs has been identified as being involved in myocardial vascularization [92]

#### **3.1. Ischemia/ischemia-reperfusion injury**

Today, there are several model systems that study the paracrine-mediated vascularization potential of ASCs but some of the most common are: ischemia and ischemia-reperfusion (IR) injuries, wound healing and cardiac infarct treatment. Enhanced angiogenesis within ischemic limbs has been reported following treatment with freshly isolated ASCs (i.e. the stromal vascular fraction) and vessels derived from these cells confirmed [113]. However, the use of such a heterogenous population makes it difficult to confirm direct ASC involvement. Fortunately, there have been numerous studies describing the beneficial use of cultured/ purified ASCs in the treatment of ischemia [86], [90], [93], [114]-[117]. Consistent with paracrine action, improved vascularization within ischemic limbs has been associated with increased levels of plasma VEGF [93]. In addition, human ASCs cultured in vitro as spheroids improve neovascularization and limb survival when compared to the implantation of dissociated ASCs – a finding thought to be due to the induction of vascular factors, like HGF, VEGF and bFGF, by the hypoxic conditions of the spheroid [118]. In support of this, decreases in the ability of ASCs to induce reperfusion in ischemic hindlimbs are observed if secretion of HGF by the ASC is inhibited [112]. However, the role of the ASC in angiogenesis may not be restricted to their secretion of established angiogenic factors. Transplantation of ASCs transfected with siRNA to either MMP3 or MMP9 to ischemic hind-limbs results in lower blood flow recovery and higher tissue injury [119], suggesting that ASCs may also promote angiogenesis through their secretion of matrix-remodelling enzymes.

Whereas prolonged ischemia can cause significant tissue damage, there is evidence now that the reperfusion period is also associated with injury, amplified by the production of reactive oxygen species and inflammatory cascades [120]. Events such as these are a major obstacle to successful tissue transplantation. However, the ASC may ameliorate IR injury through its secretion of pro-angiogenic factors, thus increasing the density of developing capillaries within the reperfused tissue. Consistent with this, a significant increase in pro-angiogenic factors can be confirmed in IR skin flap models treated with ASCs [121]. Long-lasting improvement in cardiac function with increased angiogenesis and vasculogenesis can also be observed in IR in minipigs treated with a trans-endocardial injection of ASCs [122] and a higher number of CD31+ve and vWF+ve cells have been found in models of lung IR followed by ASC injection [123]. While the finding that ASCs can form vessel-like structures in Matrigel in vitro and reendothelialize carotid injuries in vivo [87], [124] may suggest that the observed angiogenesis is due to differentiation by ASCs, the failure to observe significant ASC engraftment in IR models [122] again suggests that the role of ASCs may be paracrine in nature.

In addition to stimulating angiogenesis, the ASC may also lessen the damaging effects of IR through paracrine secretion of a combination of anti-inflammatory and anti-oxidant factors. The production of oxidative toxins such as free radicals and reactive oxygen species in ischemia and IR is well-established [125]-[128]. The synthesis of enzymatic anti-oxidants, such as superoxide dismutase and glutathione peroxidase, not only can be detected by proteomic analysis in ASC-conditioned media, but this media is able to protect dermal fibroblasts from oxidative damage [129]. Therefore, the ASC may be an excellent candidate for protection against oxidative damage. In support of this, Chen and co-workers, using a model of kidney IR treated with either conditioned medium from ASCs or direct injection of ASCs during reperfusion, find increased clearance of creatinine and urea from blood plasma in ASC/IR groups together with higher levels of the anti-oxidant markers NAD(P)H quinine oxidore‐ ductase, heme-oxygenase 1/HO-1, glutathione peroxidase and glutathione reductase [130]. Increased anti-oxidant marker levels (i.e. NAD(P)H quinine oxidoreductase and HO-1) have also been reported, together with increased eNOS expression and decreased hepatic oxidative stress versus controls upon multiple injections of ASCs in hepatic IR models [131]. These antioxidant actions by ASCs are not only likely to protect the reperfused tissue from oxidative damage but may also protect the ASC itself. A recent study by Suga and colleagues suggests that resident ASCs are resistant to ischemia-mediated damage, surviving within ischemic adipose grafts [132]. Moreover, this work specifically postulates that the actions of these resident ASCs may be responsible for the observed increases in vascular density and the number of new adipocytes over time. Therefore, ASCs may be resistant to the toxic environ‐ ment of ischemic tissues and may retain their functional capacities, thus being able to either differentiate or secrete paracrine factors for critical for angiogenesis.

#### **3.2. Wound healing**

increasing vascularization. In support of this, co-culture of ASCs with postnatal cardiomyo‐ cytes results in the formation of stable, branching CD31+ve vessel-like structures that disas‐ semble in the absence of ASCs [99]. Similarly, ASC-conditioned media can induce the formation of vessel-like tubules within Matrigel [105]. More recently, while rat ASCs express Flt-1, CD31 and vascular endothelial cadherin, when injected into a wire injury model in the rat femoral artery, induction of endothelial repair occurs without any observable differentia‐ tion of these ASCs into endothelial cells [106]– a finding that can be explained if repair is driven through the production of soluble factors. In the hopes of identifying what angiogenic factors improve a tissue's vasculature, numerous studies have characterized the secretion of growth factors by ASCs (Table 2). Of all of these factors, perhaps the most commonly reported is VEGF, with secretion of this factor being reported under normal culture conditions [98], hypoxic conditions [104] in models of wound healing [96], [107] and cell-assisted lipotransfer [97]. The ability of VEGF to stimulate neoangiogenesis is well known [108]-[110]. Consistent with this, conditioned medium from ASCs, maintained under hypoxic culture conditions in order to increase production of HGF, VEGF and TGFβ, has been found to increase endothelial cell (EC) growth and reduce their apoptosis [104]. In addition, VEGF secretion by ASCs is significantly upregulated in vitro upon metabolic induction of ischemia [111]. However, the role of other secreted factors cannot be ruled out as suppression of HGF production by ASCs through RNA interference significantly impairs ischemic tissue revascularization [112] and SDF-1α from

ASCs has been identified as being involved in myocardial vascularization [92]

Today, there are several model systems that study the paracrine-mediated vascularization potential of ASCs but some of the most common are: ischemia and ischemia-reperfusion (IR) injuries, wound healing and cardiac infarct treatment. Enhanced angiogenesis within ischemic limbs has been reported following treatment with freshly isolated ASCs (i.e. the stromal vascular fraction) and vessels derived from these cells confirmed [113]. However, the use of such a heterogenous population makes it difficult to confirm direct ASC involvement. Fortunately, there have been numerous studies describing the beneficial use of cultured/ purified ASCs in the treatment of ischemia [86], [90], [93], [114]-[117]. Consistent with paracrine action, improved vascularization within ischemic limbs has been associated with increased levels of plasma VEGF [93]. In addition, human ASCs cultured in vitro as spheroids improve neovascularization and limb survival when compared to the implantation of dissociated ASCs – a finding thought to be due to the induction of vascular factors, like HGF, VEGF and bFGF, by the hypoxic conditions of the spheroid [118]. In support of this, decreases in the ability of ASCs to induce reperfusion in ischemic hindlimbs are observed if secretion of HGF by the ASC is inhibited [112]. However, the role of the ASC in angiogenesis may not be restricted to their secretion of established angiogenic factors. Transplantation of ASCs transfected with siRNA to either MMP3 or MMP9 to ischemic hind-limbs results in lower blood flow recovery and higher tissue injury [119], suggesting that ASCs may also promote angiogenesis through their

**3.1. Ischemia/ischemia-reperfusion injury**

56 Regenerative Medicine and Tissue Engineering

secretion of matrix-remodelling enzymes.

Paracrine action is also likely to play a significant role in the beneficial effects of ASCs in wound healing models. ASCs isolated from debrided skin are capable of producing an epithelial layer when seeded into collagen gels, together with a dermis when seeded fibrin gels are co-cultured with ASC/collagen/epithelial constructs, suggesting that the ASC would be an excellent cell source for healing skin wounds [133]. In support of this, increased collagen density has been reported in full-thickness rat skin grafts injected with ASCs [134] and Lim et al. [135] note improved wound healing rates upon implantation of ASCs. These wound healing rates are significantly higher than in controls treated with ASC extracts, suggesting that production of paracrine factors by viable ASCs are necessary in order to direct the formation of new tissue within the wound. In vitro culture of immortalized keratinocytes or dermal fibroblasts with ASC-conditioned medium results in increased proliferation of these cells, in addition to increased transcription and production of collagen type I, suggesting that secreted ASCderived factors may ultimately influence keratinocyte-mediated healing in skin grafts [136], [137]. Finally, Jung and colleagues have reported that conditioned medium from ASCs can increase CNI, CNIII and hyaluronic acid synthesis by human dermal fibroblasts and that neutralizing antibodies to TGFβ1 can abolish this effect [138]. However, it is equally likely that improved wound-healing using ASCs is due to their secretion of angiogenic factors, thus improving healing through augmentation of vascularization. As proponents of this theory, Reichenberger et al. [139] and Gao et al. [107] report higher blood flow and skin flap survival, respectively when the flaps are combined with ASCs. In addition, Gao and colleagues report increased capillary density, together with increased expression of VEGF within the dermis in the ASC-treated groups. In support of this, increased VEGF expression and microvascular density is also measured in ASC-treated rat skin grafts [134]. Interestingly, recent studies suggest that AKT/c-myc signaling pathways may mediate increased VEGF secretion in ASCs as injection of constitutively active AKT/v-myc-expressing ASCs promote better wound healing compared to normal controls [140]. How exactly the ASC promotes wound healing is likely to be a combination of increased tissue healing and vascularization as directed by their secretion of specific paracrine factors. In support of this, GFP-labelled ASCs not only secrete the angiogenic factors VEGF, HGF and bFGF in vivo, but co-stain with keratin and CD31 in excisional wound healing models in normal and diabetic rats, possibly undergoing both epithelial and endothelial differentiation [96]. Similar differentiation by human ASCs, implanted into skin wounds via silk/chitosan scaffolds, has also been reported by Altman and colleagues [141]. Therefore, the successful use of ASCs in wound healing models may be due to their paracrine action in promoting angiogenesis by the host and their autocrine action in promoting differentiation in themselves.

#### **3.3. Infarct treatment**

In a 2007 study by Fotuhi, freshly isolated ASCs injected into porcine transmural infarcts were shown not to cause arrhythmia, bradycardia or conduction block. Moreover, these ASC-treated hearts required extra-stimuli to induce an arrhythmia, suggesting that ASCs could be used in the treatment of cardiac infarcts [142]. With in vitro studies confirming the cardiomyogenic potential of these stem cells, infarct treatment could be mediated through the differentiation of ASCs into cardiomyocytes. However, there is a debate on whether the ASC contributes directly to cardiac muscle regeneration or supports this event through the production of angiogenic growth factors and cytokines. An example of this debate can be seen in the 2007 article by Zhang et al. [143]. Rabbit ASCs injected into transmural infarcts in hearts three wks after occlusion decrease transmural scar and improve left ventricle ejection fraction (LVEF), end-diastolic pressure and myocardial performance relative to saline controls, with ASCs preinduced with 5-azacytidine for 24 hours giving slightly better results versus untreated controls. When the infarct region is examined histologically, the ASCs form islands of cardiac tissue in and around the scar. However, all infarcts treated with ASCs also show greater capillary density, with the ASCs also differentiating into endothelial cells. Increased capillary densities/ angiogenesis have previously been reported using bone marrow mononuclear cells and endothelial progenitors and MSCs are known to cause improvement in cardiac function by incorporating into newly formed capillaries and releasing angiogenic factors [144]. Similar events may also be induced by ASCs. In support of this, mouse ASCs injected into murine infarcts take up residence in the infarct area, with EKGs showing stability of LVEF [145]. Murine ASCs [146] or rat ASCs [147] transplanted into rat infarcts result in significant improvement in heart function and tissue viability. Human ASCs not only increase peri-infarct capillary density in rat infarcts but increase numbers of nerve sprouts [148]. Finally, while Beitnes and co-workers show significant improvement in LVEF, smaller infarct sizes and increased vascularization when human ASCs are injected into infarcts in nude rats, they specifically observe an absence of ASC engraftment [149]. However, it is important to note that ASC engraftment was examined in this study 4 weeks post-transplant. It is possible that the long-term beneficial effects of ASCs on infarct treatment can result from short-term engraft‐ ment. In support of this, while transdifferentiation of human ASCs into cardiomyocytes or endothelial cells is also not observed in rat cardiac infarcts, the expression of VEGF, bFGF and SDF-1α can be confirmed in these hearts within the first few days of transplant and improved heart function and vascular density is ultimately observed [92]. Therefore, long-term survival of ASCs within the myocardium may not be necessary for their beneficial effects on cardiac function to be realized. Such a possibility would be extremely exciting if this treatment modality is translated into the clinic.

#### **3.4. Other vascularization systems**

with ASC/collagen/epithelial constructs, suggesting that the ASC would be an excellent cell source for healing skin wounds [133]. In support of this, increased collagen density has been reported in full-thickness rat skin grafts injected with ASCs [134] and Lim et al. [135] note improved wound healing rates upon implantation of ASCs. These wound healing rates are significantly higher than in controls treated with ASC extracts, suggesting that production of paracrine factors by viable ASCs are necessary in order to direct the formation of new tissue within the wound. In vitro culture of immortalized keratinocytes or dermal fibroblasts with ASC-conditioned medium results in increased proliferation of these cells, in addition to increased transcription and production of collagen type I, suggesting that secreted ASCderived factors may ultimately influence keratinocyte-mediated healing in skin grafts [136], [137]. Finally, Jung and colleagues have reported that conditioned medium from ASCs can increase CNI, CNIII and hyaluronic acid synthesis by human dermal fibroblasts and that neutralizing antibodies to TGFβ1 can abolish this effect [138]. However, it is equally likely that improved wound-healing using ASCs is due to their secretion of angiogenic factors, thus improving healing through augmentation of vascularization. As proponents of this theory, Reichenberger et al. [139] and Gao et al. [107] report higher blood flow and skin flap survival, respectively when the flaps are combined with ASCs. In addition, Gao and colleagues report increased capillary density, together with increased expression of VEGF within the dermis in the ASC-treated groups. In support of this, increased VEGF expression and microvascular density is also measured in ASC-treated rat skin grafts [134]. Interestingly, recent studies suggest that AKT/c-myc signaling pathways may mediate increased VEGF secretion in ASCs as injection of constitutively active AKT/v-myc-expressing ASCs promote better wound healing compared to normal controls [140]. How exactly the ASC promotes wound healing is likely to be a combination of increased tissue healing and vascularization as directed by their secretion of specific paracrine factors. In support of this, GFP-labelled ASCs not only secrete the angiogenic factors VEGF, HGF and bFGF in vivo, but co-stain with keratin and CD31 in excisional wound healing models in normal and diabetic rats, possibly undergoing both epithelial and endothelial differentiation [96]. Similar differentiation by human ASCs, implanted into skin wounds via silk/chitosan scaffolds, has also been reported by Altman and colleagues [141]. Therefore, the successful use of ASCs in wound healing models may be due to their paracrine action in promoting angiogenesis by the host and their autocrine action in

In a 2007 study by Fotuhi, freshly isolated ASCs injected into porcine transmural infarcts were shown not to cause arrhythmia, bradycardia or conduction block. Moreover, these ASC-treated hearts required extra-stimuli to induce an arrhythmia, suggesting that ASCs could be used in the treatment of cardiac infarcts [142]. With in vitro studies confirming the cardiomyogenic potential of these stem cells, infarct treatment could be mediated through the differentiation of ASCs into cardiomyocytes. However, there is a debate on whether the ASC contributes directly to cardiac muscle regeneration or supports this event through the production of angiogenic growth factors and cytokines. An example of this debate can be seen in the 2007 article by Zhang et al. [143]. Rabbit ASCs injected into transmural infarcts in hearts three wks

promoting differentiation in themselves.

58 Regenerative Medicine and Tissue Engineering

**3.3. Infarct treatment**

In addition to wound healing, infarct treatment and ischemia-reperfusion, there are numerous other vascularization systems that might benefit from the putative angiogenic action of ASCs. Hemodynamic abnormalities may be reversed with the treatment of pulmonary arterial hypertension with ASCs through their augmented expression of HGF for angiogenesis and increased number of small pulmonary arteries [95]. Small-for-size liver injury may be treated through their secretion of VEGF. Inhibition of VEGF secretion by ASCs through RNA interference (RNAi) does not prevent apoptosis of liver sinusoidal endothe‐ lial cells in vitro and when cells are transplanted syngeneically results in significant disturbances to graft microcirculation, serum liver functional parameters and graft surviv‐ al [150]. Finally, at the cosmetic level, cell-assisted lipotransfer fat grafts survive at higher levels, are 35% larger and show increased neoangigogenesis when compared to grafts transplanted without isolated ASCs [151].
