Stem Cells and Angiogenesis: Implications and Limitations in Enhancing Chronic Diabetic Foot Ulcer Healing

Nonhealing diabetic foot ulcers (DFUs) are a continuing clinical issue despite the improved treatment with wound debridement, off-loading the ulcer, medication, wound dressings, and preventing infection by keeping the ulcer clean. Wound healing is associated with granulation tissue formation and angiogenesis favoring the wound to enter the resolution phase of healing followed by healing. However, chronic inflammation and reduced angiogenesis in a hyperglycemic environment impair the normal healing cascade and result in chronically non-healing diabetic foot ulcers. Promoting angiogenesis is associated with enhanced wound healing and using vascular endothelial growth factors has been proven beneficial to promote neo-angiogenesis. However, still, nonhealing DFUs persist with increased risks of amputation. Regenerative medicine is an evolving branch applicable in wound healing with the use of stem cells to promote angiogenesis. Various studies have reported promising results, but the associated limitations need in-depth research. This article focuses on summarizing and critically reviewing the published literature since 2021 on the use of stem cells to promote angiogenesis and enhance wound healing in chronic non-healing DFUs.


Introduction
The diabetic foot ulcer (DFU) is a vascular complication of diabetes mellitus (DM) affecting nearly 6.3% of the global population and a prevalence of 13% in the USA [1,2]. Wound debridement, off-loading the ulcer, medication, wound dressings, and preventing infection by keeping the ulcer clean are the gold standard in the treatment of diabetic foot ulcers (DFU). Despite the advancement in methods of wound healing, chronic DFU remains a critical clinical problem associated with costly and prolonged treatment, risk of amputation, and a high degree of morbidity and mortality. Diabetic vasculopathy, decreased angiogenesis, ischemia, chronic inflammation, and the wound environment interfering with the effect of endogenous factors regulating the healing response render the wound in a chronic inflammatory state without progressing to the resolution phase [3][4][5][6]. Wound healing comprises four phases including homeostasis/coagulation, inflammatory cell recruitment, the proliferative phase, and the maturation phase [6,7]. Angiogenesis and granulation tissue formation plays a critical role in wound healing by providing nutrition, oxygen, and matrix. Angiogenesis is increased in the early phase of healing while the density of blood vessels decreases with healing and scar formation. Granulation tissue acts as a matrix for proliferating vessels, fibroblast migration, and collagen formation. Impaired granulation tissue formation and angiogenesis are distinctive features of non-healing DFUs [8,9].

Figure 1.
Angiogenesis in wound healing. Vascular endothelial growth factor (VEGF) secreted from fibroblasts in the wound environment activates endothelial cells (ECs) leading to increased secretion of proteolytic proteins. Increased matrix metalloproteinases (MMPs) from macrophages and proteolytic enzymes facilitate the disruption of the basement membrane, migration of ECs, and sprouting of the new vessels into the wound. This process is facilitated by increased expression of adhesion proteins (VCAM-1) and integrin receptors (αvβ1, αvβ 3, and αvβ 5) and mediators of angiogenesis such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblasts growth factor (FGF), transforming growth factor (TGF)-α and β, prostaglandin E2 (PGE2), angiotensin (Ang)-1, interleukin (IL)-8, and tumor necrosis factor (TNF)-α. Increased recruitment of vascular smooth muscle cells (VSMCs) and pericytes facilitate neo-angiogenesis and vasculogenesis. Once vessels are formed and wound healing enters the later phase of healing angiogenesis is suppressed by the inhibitory form of TGF-β and increased secretion of endostatin (collagen XVIII). Angiogenesis in wound healing. Vascular endothelial growth factor (VEGF) secreted from fibroblasts in the wound environment activates endothelial cells (ECs) leading to increased secretion of proteolytic proteins. Increased matrix metalloproteinases (MMPs) from macrophages and proteolytic enzymes facilitate the disruption of the basement membrane, migration of ECs, and sprouting of the new vessels into the wound. This process is facilitated by increased expression of adhesion proteins (VCAM-1) and integrin receptors (αvβ 1, αvβ 3, and αvβ 5) and mediators of angiogenesis such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblasts growth factor (FGF), transforming growth factor (TGF)-α and β, prostaglandin E2 (PGE2), angiotensin (Ang)-1, interleukin (IL)-8, and tumor necrosis factor (TNF)-α. Increased recruitment of vascular smooth muscle cells (VSMCs) and pericytes facilitate neo-angiogenesis and vasculogenesis. Once vessels are formed and wound healing enters the later phase of healing angiogenesis is suppressed by the inhibitory form of TGF-β and increased secretion of endostatin (collagen XVIII).  Therapeutic strategies involving growth factors (PDGF, EGF, FGF, and VEGF), nongrowth factor proteins (insulin, erythropoietin, stromal-cell derived factor-1, spidroin, and  thymosin beta 4), peptides (anti-microbial peptides, cathelicidins, LL-37 derivatives, and  vasoactive intestinal peptide), blood-derived factors (PDGF-BB, TGF-β1, FGF-2, VEGF-A, EGF, and platelet factor-4), microRNAs, drugs and small molecules (adenosine triphosphate, statins, deferoxamine, natural compounds, and hyaluronan), nanomaterials in the scaffold, and stem cells (adipose-derived stem cells, bone marrow-derived mesenchymal stem cells, induced pluripotent stem cells, and placenta-derived mesenchymal stem cells) have been discussed to enhance angiogenesis and wound healing [13,14]. Becaplermin, a recombinant platelet-derived growth factor, is an FDA-approved drug to treat a neuropathic ulcer in diabetes, systemic bioavailability is a limitation [15]. Thus, exploring new treatment strategies is warranted for the treatment of ischemic DFUs. Using stem cells for wound healing is another promising approach. Multiple articles in the literature have reviewed the mechanism and the role of stem cells in enhancing wound healing [16][17][18][19][20][21][22][23]. Hence, this review focuses on the role of stem cells in promoting angiogenesis to enhance wound healing.

Angiogenesis and Wound Healing
The process of angiogenesis is well regulated by angiogenic (TGF-β, TNF-α, VEGF, PDGF, FGF, angiogenin, and angiopoietin-1) and anti-angiogenic (angiostatin, TIMP-2, TSP-1, endostatin, sprouty 2, PEDF, PF-4, IFNα/β) factors [8,12,24] involving oxidative stress (HIF-1α), inflammation, and Sonic Hedgehog (Shh) signaling. Reduction of angiogenesis in the last phase of wound healing is essential for proper wound healing without scarring and selective reduction of angiogenesis and inflammation has been suggested for proper wound healing [12]. This suggests that increased angiogenesis plays a critical role in normal wound healing during the early phase, but attenuation of angiogenesis is needed for proper healing. However, this dynamic regulation of angiogenesis may alter during diabetes due to changes in the expression levels of pro-and anti-angiogenesis proteins ( Figure 2), however, the available literature is not conclusive [25]. The inconsistencies between the results of the expression of pro-and anti-angiogenic factors may be due to loss of tissue, presence of necrosis and gangrene, the differential response of fibroblasts to hypoxia, different levels of inflammation, tissue fibrosis, vascular thrombosis, arteriolar sclerosis, differential expression of these mediators in different cells in healthy versus wound tissues and healthy versus diabetic tissue as well as different levels of hyperglycemia. Another issue may be a differential expression between different types of diabetic ulcers. Difficulties in translating the lab findings to therapy in clinics to improve angiogenesis and wound healing are also due to the differential expression of these mediators in human versus murine models [25]. Temporary hypoxia is needed for angiogenesis as increased expression of HIF-1α dimerizes and activates hypoxia response elements causing increased expression of VEGF, but the presence of hyperglycemia affects the stability and activation of HIF-1α and attenuates angiogenesis through suppression of PDGF, VEGF, and TGF-β [26].

Stem Cells and Angiogenesis in DFUs
Angiogenic therapy using stem cells to enhance healing in refractory wounds has shown promising therapeutic efficacy. Stem cells migrate to the site of the wound, differentiate, proliferate, promote granulation tissue formation, collagen deposition, and angiogenesis, and ameliorate neuro-ischemia and inflammation thereby enhancing wound healing. Angiogenesis is promoted by increasing the secretion of angiogenic factors including VEGF and Von Willebrand factor and endothelial cell recruitment through TNF-α. Mesenchymal stem cells (MSCs) have been used to promote angiogenesis in preclinical and clinical studies [16,18,27]. Paracrine signaling and the capability of stem cells to differentiate into specialized cells including fibroblasts, vascular endothelial cells, and epithelial cells contribute to the efficacy of stem cells in stimulating angiogenesis, neovascularization, and re-epithelialization. Stem cells heal the wound by providing a favorable environment by secreting cytokines, chemokines, and growth factors necessary to produce an extracellular matrix and promoting neo-angiogenesis. Thus, stem cells alter the wound microenvironment favorable for healing and promote tissue regeneration at the wound site. Administration of stem cells is associated with the advantage of angiogenesis promotion, attenuated inflammation, and enhanced wound healing but is also associated with side effects (Table 1). Further, stem cells have the advantage of administering them along with other treatments to exploit their role in a better way for the treatment of nonhealing DFUs [16][17][18]28].

Table 1. Advantages and limitations of the stem cells enhancing healing in DFUs.
Stem cells showed improved clinical efficacy in enhancing wound healing in DFUs by increased angiogenesis and re-epithelialization [17]. The common side effects associated with stem cell therapy are whole body urticaria, diarrhea, oral ulceration, the elevation of serum creatinine level, number, and differentiated potential decline with aging, nausea, and vomiting. Bone marrow-mesenchymal stem cells (BM-MSCs), umbilical cord blood-

Stem Cells and Angiogenesis in DFUs
Angiogenic therapy using stem cells to enhance healing in refractory wounds has shown promising therapeutic efficacy. Stem cells migrate to the site of the wound, differentiate, proliferate, promote granulation tissue formation, collagen deposition, and angiogenesis, and ameliorate neuro-ischemia and inflammation thereby enhancing wound healing. Angiogenesis is promoted by increasing the secretion of angiogenic factors including VEGF and Von Willebrand factor and endothelial cell recruitment through TNF-α. Mesenchymal stem cells (MSCs) have been used to promote angiogenesis in pre-clinical and clinical studies [16,18,27]. Paracrine signaling and the capability of stem cells to differentiate into specialized cells including fibroblasts, vascular endothelial cells, and epithelial cells contribute to the efficacy of stem cells in stimulating angiogenesis, neovascularization, and re-epithelialization. Stem cells heal the wound by providing a favorable environment by secreting cytokines, chemokines, and growth factors necessary to produce an extracellular matrix and promoting neo-angiogenesis. Thus, stem cells alter the wound microenvironment favorable for healing and promote tissue regeneration at the wound site. Administration of stem cells is associated with the advantage of angiogenesis promotion, attenuated inflammation, and enhanced wound healing but is also associated with side effects (Table 1). Further, stem cells have the advantage of administering them along with other treatments to exploit their role in a better way for the treatment of nonhealing DFUs [16][17][18]28]. Table 1. Advantages and limitations of the stem cells enhancing healing in DFUs. Stem cells showed improved clinical efficacy in enhancing wound healing in DFUs by increased angiogenesis and re-epithelialization [17]. The common side effects associated with stem cell therapy are whole body urticaria, diarrhea, oral ulceration, the elevation of serum creatinine level, number, and differentiated potential decline with aging, nausea, and vomiting. Bone marrow-mesenchymal stem cells (BM-MSCs), umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs), adipose-derived mesenchymal stem cells (AMSCs), umbilical cord-derived mesenchymal stem cells (UC-MSCs), placenta-derived mesenchymal stem cells (PMSCs), and human amniotic fluid-derived stem cells (AF-MSCs), human gingiva-derived mesenchymal stem cells (GMSCs). Mesenchymal stem cells induce the mobilization of various angiogenesis factors including SDF-1, VEGF, EGF, insulin-like growth factor-1 (IGF-1), angiopoietin (Ang)-1, keratinocyte growth factor (KGF), MMP-9, macrophage inflammatory protein (MIP)-1α and β and erythropoietin (EPO) to the wound bed stimulating recruitment, proliferation, and differentiation of endothelial progenitor cells and thereby angiogenesis and wound healing [16,27]. Among the MSCs, bone marrow-derived MSCs are more suitable to enhance healing in DFUs [17]. MSCs have been used in various studies and shown to have a beneficial role in enhancing wound healing in DFUs. The molecular mechanism involved in enhancing wound healing in DFUs using stem cells has been extensively reviewed [17,29]. Adult stem cells including bone marrow-derived mesenchymal stem cells (BM-MSC), peripheral blood-derived mesenchymal stem cells (PB-MSC), human umbilical cord-derived mesenchymal stem cells (hUC-MSC), and adipose-derived mesenchymal stem cells (ADSC) have been used in pre-clinical and clinical studies (Table 2). Additionally, enhanced wound healing in a murine model of DFUs using human amniotic MSCs, the micronized amniotic membrane containing human amniotic epithelial cells, human placental MSC, collagen gels containing embryonic fetal liver MSCs, and collagen hydrogel scaffold to deliver human fetal aortic MSCs has also been reported. Moreover, the use of embryonic stem cells, induced pluripotent stem cells, and granulocyte-colony stimulating factors stimulating bone marrow to mobilize endothelial progenitor cells at the wound site, have also been documented and reviewed [19][20][21][22][23]. Table 2. Stem cells enhance angiogenesis and wound healing in DFUs. Neonatal porcine bone marrow-derived mesenchymal stem cell (npBM-MSC), Adipose-derived mesenchymal stem cells (ADSCs), human induced pluripotent stem cell-derived smooth muscle cells (hiPSCSMC), endothelial differentiated adipose-derived stem cells (EC-ADSCs), human umbilical cord blood-derived CD34 + stem cells (UCB-CD34 + SC), bone marrow mononuclear cells (BMMNCs), endothelial progenitor cells, human fetal aorta-derived CD133 + progenitor cells and their conditioned medium (CD133 + CCM), human umbilical cord-derived MSCs (hucMSCs).

Stem Cell Strategy Parameters Checked Outcome
npBM-MSC [29] Xenotransplantation in mice model of diabetic wound.
Rate of wound closure and the promotion of neovascularization Regarding the route of administration of stem cells to enhance wound healing, nonvascular administration including intradermal, subcutaneous, and intramuscular injections are the most used routes to enhance DFU wound healing. Additionally, local administration of stem cells in a collagen-based hydrogel and systemic venous and arterial administration of stem cells have also shown promising results [19,20]. Further, Yan et al. [37] reported that both systemic and topical application of BM-MSCs have the potential of enhancing wound healing and promoting neo-angiogenesis and vascularization but systemic administration is associated with ameliorating hyperglycemia and improving blood perfusion of the ischemic hindlimb of the diabetic rats with a positive wound distribution and trans-differentiation to ECs.

Stem-Cell Derived Exosomes and Combinational Strategies to Enhance Diabetic Wound Healing
Stem cells not only enhance angiogenesis but also attenuate the effects of diabetes and protect endothelial cells [36]. Low cell retention and integration of stem cells are issues of using hydrogels while administering stem cells to enhance wound healing in DFUs. Shi et al. [38] reported that gelatin microspheres enhance the delivery and integration of locally delivered adipose-derived stem cells from rats. The use of gelatin microspheres was associated with M2 macrophage polarization, collagen deposition, angiogenesis, peripheral nerve recovery, and hair follicle formation suggesting the efficacy of using microspheres in enhancing healing in DFUs. In another study, Takahashi et al. [39] reported that topical application of hydrogels containing conditioned medium from hypoxically cultured amnion-derived mesenchymal stem cells promotes wound healing in diabetic mice by enhancing angiogenesis, accelerating epithelization, and suppressing inflammation. The result of this study obviates the need for stem cell transplantation at the wound site though future research is warranted.
Along with transplanting stem cells in gels or as nanoparticles, exosomes derived from stem cells have also shown a beneficial effect in enhancing angiogenesis and wound healing in DFUs. A study by Wang et al. reported the effectiveness of exosomes derived from epidermal stem cells in improving diabetic wound healing [40]. The study reported that compared to epidermal stem cells (ASC) alone, exosomes isolated from ASC (ASC-Ex) showed better results in wound healing in db/db mice. Enhanced wound healing with ASC-Ex was associated with decreased inflammation, augmented wound cell proliferation, stimulating angiogenesis, and inducing M2 macrophage polarization. Heras et al. [41] reported increased angiogenesis and human dermal fibroblasts proliferation and migration, in vivo, with exosomes isolated from hair follicle-derived MSCs (HF-MSCs) and adipose tissue-derived MSCs (AT-MSCs). Another study by Gondaliya et al. reported enhanced collagen deposition, angiogenesis, and re-epithelialization in diabetic wounds with MSCderived exosomes loaded with miR-155 inhibitor in a mouse model [42]. Accelerated wound healing was associated with keratinocyte migration, restoration of FGF-7 levels, and decreased inflammation. The promotion of angiogenesis with accelerated diabetic wound healing in diabetic mice was also reported with exosomes derived from human umbilical cord mesenchymal stem cells via oxidative stress amelioration [43]. Decreased angiogenesis or neovascularization in hyperglycemic state and old age may be due to senescent endothelial cells (ECs) or decreased ECs proliferation. Xiao et al. [44] reported that mesenchymal stem cell-derived small extracellular vesicles (MSC-sEV) mitigate oxidative stress-induced ECs senescence and stimulate angiogenesis through miR-146a/Src. Increased angiogenesis to enhance wound healing in diabetic rats by promoting HIF-1α-mediated angiogenesis in the PI3K-AKT-mTOR dependent manner with extracellular vesicles derived from human adipose-derived stem cells (hADSC-EVs) in association with accelerated wound healing was reported by Liu et al. [45]. These findings suggest that mitigating EC senescence may enhance angiogenesis and thus might be of therapeutic significance to enhance healing in chronic non-healing DFUs. Further, stem cells have better therapeutic effects in combina- tions with hydrogels, collagen matrix, and nanoparticles in comparison to stem cells alone and pretreatment will enhance efficacy (Table 3). Table 3. Combinational strategies with stem cells enhancing angiogenesis and wound healing in DFUs: Exendin-4 (Ex-4) is a glucagon-like peptide-1 receptor agonist known to have many beneficial effects on diabetes, human adipose tissue-derive stem cells (hADSCs), long noncoding RNA (Lnc), bone marrow mesenchymal stem cells (BMSCs), self-assembled nano-peptide hydrogels with human umbilical cord mesenchymal stem cell spheroids (hUC-MSCsp), acellular dermal matrix (ADM), extra cellular matrix (ECM), hBM-MSCs/T/H/S embedded in an ECM scaffold (S) and preconditioned with hypoxia (H) and the β-adrenergic receptor antagonist, timolol (T), Wharton's jelly mesenchymal stem cell (WJMSC), sodium ascorbyl phosphate (SAP), platelet rich plasma (PRP), human umbilical cord mesenchymal stem cells (hUC-MSCs), 5-aminolevulinic acid photodynamic therapy (ALA-PDT), EVs secreted by human umbilical cord mesenchymal stem cells (hucMSC-EVs), HOTAIR-MSC EVsextracellular vesicles (EVs) isolated from mesenchymal stem/stromal cells (MSCs) transfected to overexpress long non-coding RNA HOX transcript antisense RNA (HOTAIR), endothelial cells (ECs), catechol-functionalized hyaluronic acid (HA-CA) patch.

Treatment Combination/Strategy Assessing Parameters Study Outcome
Exosomes from linc00511overexpressing ADSCs [46] hADSCs-derived exosomes were injected into Sprague-Dawley (SD) rats along with human blood-derived EPC  hUC-MSCs combined with ALA-PDT-hUC-MSCs were injected intradermally to diabetic C57BL/6J mice after exposing the mice to ALA-PDT with 10% ALA gel and 25 J/cm 2 of PDT.
To investigate the efficacy of the combinational approach on wound closure, angiogenesis, and inflammation Combining ALA-PDT with hUC-MSCs possesses a significantly enhanced therapeutic efficacy in enhancing wound healing, promoting angiogenesis, and attenuating inflammation and bacterial load suggesting its efficacy in healing refractory wounds.
ADSCs [59] ADSCs combined with HA-CA ADSCs were injected around the wound in diabetic C57BL/6 mice and a patch was deposited on the wound Angiogenesis Wound healing HA-CA + ADSCs enhanced wound healing and angiogenesis synergistically involving PI3K/AKT pathway.
ADSCs [60] Human ADSCs with SDF-1α gene-activated scaffold were tested in vitro using HUVEC Pro-angiogenic properties SDF-1α gene-activated scaffold overcomes the deficiencies associated with diabetic ADSCs and restores pro-angiogenic features ln ADSCs Many studies have also reported enhanced therapeutic efficacy of adipose stem cell released extracellular vesicles and exosomes are being used to enhance wound healing in DFUs [61][62][63]; it is imperative to further investigate the strategies to enhance the therapeutic efficacy of stem cells and their synergistic or additive effects in a combinational approach to promote angiogenesis and wound healing in chronic non-healing DFUs. In-depth research involving exosomes (EVs) isolated from stem cells is also important because EVs isolated from different types of stem cells may have differential effects on angiogenesis and wound healing as reported by Pomatto et al. [64] that EVs isolated from bone marrow and adipose mesenchymal stem cells have differential therapeutic effects on wound healing and angiogenesis. Although these studies (Tables 2 and 3) suggest the promising role of stem cells in inducing angiogenesis in the presence of hyperglycemia most of the studies conducted on the murine model were done either on streptozotocin-induced diabetes mellitus, type I diabetes, or genetically induced diabetic mice. However, type II diabetes is common in human patients with DFUs. Having said that, it is warranted to design clinical trials to establish the role of stem cells in enhancing angiogenesis in DFU wound healing.

Limitations and Future Perspectives
Limited cell viability, reduced cell number, limited proliferation, and differentiating capacity of the stem cells are limiting factors while using stem cells. Stem cells may be autologous or allogenic. Both autologous and allogeneic stem cells have shown improved wound healing in human and animal models, but the pre-clinical and clinical data is still limited and more well-planned studies with an increased number of subjects are warranted for the feasibility of using stem cells in enhancing healing in DFUs [17]. Decreased viability of stem cells due to late glycosylation end products in a hyperglycemic environment diminishes the efficacy of stem cells in enhancing wound healing in DFUs [65]. However, a study by Assis et al. [66] reported that the MSCs from diabetic patients have the same potential for angiogenesis if they are seeded on decellularized micro fragments. The MSCs were isolated from the bone marrow of distal tibial or femoral shafts from the amputated limb. These results suggest the efficacy of autologous SMCs even in a diabetic patient in enhancing angiogenesis during wound healing.
Administering an increased number of stem cells might be an alternative but is associated with over-proliferation and tumorigenicity [67]. Administering stem cells with a biomaterial carrier may mitigate the limitations associated with the number and viability of stem cells but needs extensive research [19,20]. Administration of preconditioned stem cells with photo-biomodulation is another strategy to enhance the viability and activity of stem cells to enhance angiogenesis and wound healing [68][69][70]. With the background of the complications associated with autologous MSCs, Palma et al. [71] reported that one specific subpopulation derived from the Wharton's jelly of the umbilical cord (ucMSC), the differentiated mesenchymal cells (DMCs), has the potential of improving wound healing with increased angiogenesis without any rejection of the transplanted cells in immunocompetent mice. These results showed successful allogeneic transplantation of MSCs. Treating chronic diabetic foot ulcers with adipose-derived stromal vascular fraction cell injections may be a safe strategy to induce vascular repair, angiogenesis, and wound repair [72]. Although the studies discussed in this article showed promising results favoring angiogenesis and wound healing and the limitations of stem cells, the heterogeneity among the results of various randomized clinical trials in human patients also warrants well-planned large-scale randomized clinical trials to establish the best protocols, doses, transplant type (autologous, allogenic, xenotransplant), cell types, cell source, and administration routes to enhance angiogenesis and wound healing [19,20].

Conclusions
Stem cells have been proven beneficial in promoting angiogenesis and enhancing wound healing both in vitro and in vivo but most of the in vivo studies were conducted using animal models including mice, rats, and porcine. The results of various studies showed heterogeneous results due to different sources of stem cells, route of delivery, and stem cell properties like viability and proliferation after administration. Further, EVs isolated from stem cells and a combination of stem cells with other strategies, as discussed here, has also shown promising results. Based on this background, large-scale clinical trials are warranted to harness the beneficial outcome of the lab findings and translate them to the clinics.