An Overview on Current Issues and Challenges of Endothelial Progenitor Cell-Based Neovascularization in Patients with Diabetic Foot Ulcer

Abstract

Diabetic foot ulcer's impaired wound healing, which leads to the development of chronic non-healing wounds and ultimately amputation, is a major problem worldwide. Although recently endothelial progenitor cell-derived cell therapy has been used as a therapeutic intervention to treat diabetic wounds, thereby promoting neovascularization, the results, however, are not satisfactory. In this article, we have discussed the several steps that are involved in the neovascularization process, which might be impaired during diabetes. In addition, we have also discussed the reported possible interventions to correct these impairments. Thus, we have summarized neovascularization as a process with a coordinated sequence of multiple steps and thus, there is the need of a combined therapeutic approach to achieve better treatment outcomes.

Introduction

Diabetic foot ulcer, a major end-stage complication of diabetes mellitus (DM), imposes a considerable morbidity and mortality along with heavy financial burden for healthcare services (Elsharawy et al., 2012; Ramsey et al., 1999). The progressive development of a sensory, vasomotor, and autonomic neuropathy in diabetic patients leads to loss of protective sensation, joint and bone deformities that increase plantar foot pressure, and alterations in auto-regulation of dermal blood flow (Falanga, 2005; Liu and Velazquez, 2008; Tecilazich et al., 2013). Although all of these factors, in turn, lead to the development of a diabetic foot ulcer, its impaired wound healing leads to the development of chronic, non-healing wounds and, ultimately, amputation (Falanga, 2005). Several physiological factors, such as angiogenic growth factors, collagen accumulation, epidermal barrier functions, keratinocytes and fibroblast migration, and number of epidermal nerves and bone healing, that are required for normal wound healing have been reported to decrease in diabetes (Falanga, 2005; Galkowska et al., 2006).

In recent years, studies have demonstrated the efficacy and associated healing mechanisms of endothelial progenitor cell (EPC)-based cell therapy in a diabetic ischemic foot ulcer (Falanga, 2005; Gu et al., 2007; Kirana et al., 2007; Yang et al., 2005). However, neovascularization by EPC is a coordinated sequence of multiple steps, of which each step is controlled by a specific mechanism(s) (Brem and Tomic-Canic, 2007; Liu and Velazquez, 2008). Several of these mechanisms are impaired in diabetes. Thus, a combination of therapeutic approaches to target individual impaired steps in neovascularization and wound healing is essential to synergize cell-based therapy for a more successful treatment outcome in treating diabetic wounds.

Furthermore, the low quantity and quality of EPCs isolated from bone marrow (BM) and peripheral blood of certain patient populations such as elderly, diabetic, cardiovascular disease (CVD) patients, and so forth are among the challenges faced by EPC-based cell therapy (Sukmawati and Tanaka, 2015). In addition to this, which cell type is appropriate for which particular complication of diabetes is still a difficult question to be answered (Jarajapu and Grant, 2010). To overcome these hurdle strategies to develop different culture mediums and methods as well as an ex vivo manipulation of EPCs (e.g., genetic modification, ex vivo treatment with small molecules), an in vivo manipulation of EPCs as well as a manipulation of the diabetic host environment are being explored (Jarajapu and Grant, 2010). It is beyond the scope of the present article to discuss these methods. Rather here, in the present article, we have discussed different impairments occurring at various steps of EPC-induced neovascularization in diabetes and likely major to correct it, thereby proposing the importance of a combined therapeutic approach toward diabetic foot ulcers.

EPC-Induced Neovascularization During Wound Healing

An essential part of normal healing is new blood vessel formation, which according to recent studies, in addition to being derived from pre-existing vessels (angiogenesis), can also be achieved by BM-derived EPC cells and is known as vasculogenesis (Patenaude et al., 2010; Rafii et al., 2002; Schatteman et al., 2007). In other words, it is now well accepted that both angiogenesis and vasculogenesis participate in tissue vascularization. The recruitment of EPCs from BM to circulation and then homing, that is, their migration to the site of injury, is a step in the EPC-based wound repair process that is regulated by many factors, including chemokines and growth factors (Liu and Velazquez, 2008).

In addition to being an angiogenesis stimulator, growth factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), transforming growth factor beta 1 (TGF-β1), and platelet-derived growth factor (PDGF), are also mitogenic and chemoattractive for fibroblasts. Kulwas et al. (2015) showed decreased levels of VEGF-A in patients with DM complicated diabetic foot syndrome (DFS) compared with diabetic patients without DFS. Furthermore, Galkowska et al. (2006) demonstrated the lack of upregulation of bFGF, IGF-1, TGF-β1, and interleukin-15 (IL-15) expression in the vascular endothelium in diabetic foot ulcer patients. Similarly, different angiogenic chemokines such as C-X-C motif chemokine 12 [(CXCL12), also known as stromal cell-derived factor 1 (SDF-1)], C-X-C motif chemokine 1 (CXCL1), and so on act as not only chemoattractants but also adhesive compounds, thereby promoting EPC adhesion, as demonstrated in a study by Kanzler et al. (2013).

Therefore, we can conclude that impairment in any of these factors in diabetic patients may affect the EPC-based neovascularization (discussed later in detail in the “Mobilization of EPCs from BM to Peripheral Circulation” section).

Figure 1 shows the normal EPC-based neovascularization process in normal and diabetic patients.

FIG. 1.

Different stages of bone marrow EPC led to neovasculogenesis during wound healing in healthy people versus diabetic patients. Tissue injury or ischemia releases the EPC-mobilizing factors such as angiogenic growth factors, cytokines, hormones, and so on. These mobilizing stimuli might signal through their cognate receptors to activate the eNOS within the BM stromal cells. eNOS induces production of NO, which stimulates and/or maintains MMP-9 activity, resulting in the cleavage soluble kit ligand (sKitL) from the membrane-bound kit ligand (mKitL) to mobilize c-Kit EPCs from the BM niche into the circulation. There is a decrease or an alteration in the release of these mobilizing factors in diabetic patients, and, thus, eNOS phosphorylation in the BM is impaired, limiting the EPC mobilization. Homing and recruitment of EPCs into the active remodeling sites require a coordinated multistep process, including chemoattraction, adhesion, transendothelial migration, and tissue invasion. Locally enhanced levels of cytokines and growth factors provide a chemotaxic trigger for homing and EPC adhesion to the endothelium. The SDF-1 expression is decreased in epithelial cells and myofibroblasts in diabetic wounds along with a decreased release of cytokines and growth factors, which limits wound healing in diabetic patients. BM, bone marrow; eNOS, endothelial nitric oxide synthase; EPC, endothelial progenitor cell; SDF-1, stromal cell-derived factor 1.

Identification of Specific Subpopulation of EPCs

Although most of the studies have reported the importance of EPCs in neovascularization, some reports suggested that EPC administration partially rescued cardiovascular dysfunction after myocardial injury, with some evidence for EPC contribution to new vessel growth (Kuethe et al., 2004, 2005). After searching several available data, we came to the conclusion that the EPCs are organized in a hierarchy of progenitor stages that vary in proliferative potential and these subpopulations of EPCs with distinct phenotypes (as discussed in a subsequent paragraph) give rise to different outcomes (Nagano et al., 2007; Yoder et al., 2007). Thus, the choice of incorrect phenotypic EPCs in aforementioned studies might have led to such conflicting results.

In general, two approaches have been used to isolate EPCs: (1) culture and colony assays and (2) selection of subpopulations based on surface markers. However, all of these diverse culturing methods and identification of EPCs based on various combinations of different surface antigens are challenged by the close relationship and overlapping markers of the endothelial and hematopoietic lineages (Fadini et al., 2012).

Culture and colony assays

EPCs can be established in culture from a number of sources, including embryonic stem cells, umbilical cord blood, BM, and, most recently, from peripheral blood (Fadini et al., 2012).

Depending on the culture method, which mainly differs in culture time, early and late EPC phenotypes have been identified.

The short-term culture method entails to culturing peripheral blood mononuclear cells on fibronectin for 4 days in VEGF containing medium, giving rise to so-called early EPCs or colony-forming unit-endothelial cells (CFU-EC)s with myeloid/hematopoietic characteristics, sharing features with immune cells, particularly monocytes/macrophages. However, in the long-term culture method, peripheral blood mononuclear cells are cultured on collagen for 2–4 weeks. This gives rise to so-called late EPCs or out growth EPCs or endothelial colony-forming cells (ECFCs), which are a more mature EC phenotype and lack hematopoietic and myeloid markers as well as have lower cytokine release and different growth patterns (Balaji et al., 2013; Werling et al., 2013). Early EPCs expresses CD45 and CD14 on their surface, other than CD31, displaying an overlap between endothelial, monocyte, and macrophage cell markers; whereas the late EPCs, which are believed to be a subset of CD14⁻/CD34⁻/KDR⁻ (Kinase insert Domain Receptor) cells, do not express CD45 and CD14 (Hur et al., 2004).

According to the study by Yoder et al. (2007), CFU-ECs are descendants of hemopoietic stem cells (HSCs) that retain some of the myeloid progenitor activity with no ability to form secondary EC colonies or perfused vessels in vivo. Furthermore, CFU-ECs differentiate into phagocytic macrophages and not ECs. In contrast, ECFCs are rare circulating EPCs with a robust proliferative potential and vessel-forming activity in vivo. However, the generation of ECFCs in culture seems to be an on/off phenomenon, implying that ECFCs cannot be efficiently obtained from all donors, especially in relation to age and pre-existing pathological conditions (Meneveau et al., 2011; Wang et al., 2011).

Nevertheless, it should be acknowledged that not only culture protocols but also specific culture conditions as well as an in vivo environment may influence cell fate and function, leading to their incapability to improve neovascularization (Werling et al., 2013). Furthermore, intercellular communication among EPCs and their environment, by either direct cell-to-cell contact or cell-contact–independent mechanisms, can transfer epigenetic materials, including proteins and nucleic acids, to EPCs (Fadini et al., 2012). For example, Badorff et al. (2003) co-cultured human peripheral blood EPCs with rat cardiomyocytes and found that EPCs differentiated toward a cardiomyocyte cell fate, suggesting that transdifferentiation potential is influenced by the local cellular environment.

Selection of subpopulation by surface markers

Culture assay as is started with the preparation of total mononuclear cells, the interaction of the different cells in the mixture, may influence the cellular phenotype. Therefore, the direct isolation of cell populations by using surface antigens has been proposed to select the defined populations of cells. The EPCs in BM as well as in peripheral blood are characterized by the expression of both stem cell markers, such as CD133, CD34, c-Kit, and Sca-1, and endothelial markers, such as vascular endothelial growth factor receptor 2 (VEGFR2, also known as KDR or Flk1), tyrosine kinase with immunoglobulin-like and EGF-like domains 2 (Tie-2), E-selectin, and vascular endothelial Cadherin (VE-cadherin).

Under endothelial cell culture conditions, freshly isolated EPCs gradually differentiate toward endothelial cells, losing their stem cell markers while gaining endothelial cell markers in the process (Hristov et al., 2003; Masuda and Asahara, 2003). And therefore, currently, investigators tend to characterize EPCs by including antigenic markers defining the stemness and hematopoietic lineage (humans: CD34 and CD133; mice: CD34, c-kit, or stem cell antigen-1 [Sca-1]) in combination with markers demonstrating endothelial commitment (humans: KDR; mice: Flk-1) along with morphological, functional, and clonal expansion characteristics (Balaji et al., 2013).

Several studies till date advocate CD34⁺KDR⁺ or CD34⁺CD133⁺KDR⁺ cells as a BM (BM cell) marker to identify EPCs (Hagensen et al., 2012; Sieveking et al., 2008; Wickersheim et al., 2009). The parallel analysis of CD45 expression has been also proposed to distinguish EPCs, as Case et al. (2007) showed that cord blood and granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood CD34⁺CD45 population forms endothelial colonies in vitro instead of CD34⁺KDR⁺ and CD34⁺CD133⁺KDR⁺ cells, which, though they developed into hematopoietic cells, failed to form endothelial colonies.

However, the relationship between CD34⁺CD45⁻ cells and mature circulating endothelial cells is still to be established. In addition, more recently, Tian et al. (2009) employed Sca-1 as a BM cell marker and used VE-cadherin and E-selectin as endothelial markers to identify EPCs, with encouraging results. To our understanding, there are lineage and functional heterogeneities within the EPC population, and it has a dynamic phenotype in space and time and a detailed functional characterization of the cells is warranted before its therapeutic application.

Mobilization of EPCs from BM to Peripheral Circulation

Although not entirely elucidated, the nitric oxide (NO)-mediated signaling pathway has been proposed as mechanism that is involved in EPC mobilization. EPCs are embedded in a microenvironment (niche) of BM and they are released into circulation in response to trauma or ischemia (Aicher et al., 2003; Heissig et al., 2002, 2003). Bone marrow stromal cells sense the signal molecules, such as VEGF-A, that are released by injured tissue. Endothelial nitric oxide synthase (eNOS) within the BM stromal cells is then activated by VEGF-A and other mobilizing stimuli. eNOS induces production of NO, which then S-nitrosylates by paracrine mechanisms and activates and/or maintains matrix metalloproteinase 9 (MMP-9) activity, resulting in cleavage of soluble kit ligand (sKitL) from the membrane-bound kit ligand (mKitL) to mobilize c-Kit⁺ EPCs from the BM niche into the circulation (Aicher et al., 2003; Heissig et al., 2002, 2003; Nakamura et al., 2004; Rafii et al., 2003; Shintani et al., 2001).

Hyperoxia and EPC mobilization

The mobilization of BM EPCs is affected in diabetes, although a precise mechanism still remains unclear. Many researchers hypothesized impaired eNOS function in diabetes as one of the causes for decreased EPC mobilization, and they, indeed, succeeded in enhancing BM-derived EPC mobilization by hyperbaric oxygen therapy (HBOT). HBOT has been found to upregulate NO production via stimulation of NOS in several tissues as well as in BM stromal cells (Gallagher et al., 2007). Hypoxia and some cytokines such as granulocyte macrophage-colony stimulating factor (GM-CSF) are also known stimuli that are used to mobilize EPCs; hypoxia, in turn, worsens the wound-healing process and GM-CSFs are associated with many systemic side effects (Li et al., 2002; Lindemann and Rumberger, 1993). One advantage of HBOT is that it specifically stimulates BM EPCs to be released into circulation without any significant impact on the inflammatory cell numbers in circulation.

Manganese superoxide dismutase (MnSOD) gene (SOD2) therapy

Recent studies have shown that high glucose elevates oxidative stress and decreases EPC survival by inhibiting cell proliferation, NO production, MMP-9 activity, and migration (Balestrieri et al., 2008; Barcelos et al., 2009; Kränkel et al., 2005). Hyperglycemia augments the superoxide anion generation in skin tissue by activating NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) and protein kinase C, thereby resulting in delayed wound healing. Normal EPCs are believed to express intrinsically high levels of the antioxidant enzyme MnSOD, which plays a key role in EPC resistance to oxidative stress via scavenging mitochondrial ROS (mtROS), and decreased MnSOD in diabetic patients might be one of the causes for EPC failure to tolerate excessive oxidative stress (Chen and Chen, 2006; Dernbach et al., 2004; He et al., 2004). Marrotte et al. (2010) reported that transplantation of diabetic EPCs after MnSOD gene (SOD2) therapy restored their ability to mediate angiogenesis and wound repair.

Silencing p66Shc gene (SHC1)

In addition to MnSOD, ShcA protein (p66Shc) also plays a major role in the oxidative stress-mediated EPC dysfunction. The p66 isoform of ShcA protein (p66Shc), which is a fundamental regulator of mtROS production, plays a pivotal role in ROS-induced apoptosis by modulating ROS production in the nucleus, the plasma membrane, and the mitochondria, respectively (Wils et al., 2016). In the nucleus, p66Shc inhibits forkhead box sub-group O (FOXO) transcription factors, thereby leading to the decreased expression of ROS-scavenging enzymes catalase (CAT) and MnSOD (Nemoto and Finkel, 2002). At the plasma membrane, p66Shc promotes Ras-related C3 botulinum toxin substrate 1 (RAC1) activation and triggers NADPH membrane oxidase-ROS production (Khanday et al., 2006). In the mitochondrial intermembrane space, p66Shc binds to cytochrome c, acting as an oxidoreductase and generating ROS (Pinton et al., 2007).

These ROS, in turn, activate the permeability transition pore, triggering organelle dysfunction, massive release of mitochondrial apoptotic factors, and ROS and, eventually, inducing cell apoptosis (Giorgio et al., 2005). p66Shc has been found to be modulated by sirutin1 (SIRT1) either through its direct inhibitory role in p66Shc expression involving epigenetic modifications or through its ability to downregulate p53 activation in diabetic conditions. Hence, both p53 and SIRT1 are responsible for the modulation of p66Shc abundance (de Kreutzenberg et al., 2010; Zhou et al., 2011).

Diabetic conditions (hyperglycemia; non-esterified fatty acid) cause an increase in p53, protein kinase C and downregulation of SIRT1, all of which cause p66Shc overexpression. p66Shc over-expression not only results in ROS-induced apoptosis of EPC but also decreases EPC motility by downregulating the antioxidant enzyme and/or or deregulating VEGF, thereby eventually decreasing NO bioavailability (Camici et al., 2007; de Kreutzenberg et al., 2010; Zhou et al., 2011). Despite the role of the p66Shc gene in dysregulation of EPC in diabetic patients, only few studies have investigated its specific role in pro-vascularizing progenitor cells. Di Stefano et al. (2009) demonstrated that EPCs isolated from the BM of mice that were deficient in p66Shc were resistant to a strong hyperglycemic challenge in culture, leading to apoptosis in wild-type cells.

Moreover, p66Shc deficiency prevented the alteration of the angiogenic ability of streptozotocin-induced hyperglycemic mice. Furthermore, Albiero et al. (2014) suggested that BM autonomic neuropathy alters circulating CD34⁺ EPC mobilization by the dysregulation of the p66Shc/SIRT1 axis in diabetic patients, which can be prevented by SIRT1 overexpression. Nevertheless, either silencing of p66Shc gene (SHC1) or overexpression of SIRT1 might prevent the hyperglycemic condition from affecting the number and function of EPCs.

Integrins and angiogenic growth factors

Other factors such as integrins, angiogenic growth factors (VEGF-A, placenta growth factor, bFGF, angiopoietins), cytokines (SDF-1, G-CSF), drugs (statins, CXCR antagonist), and exercise all have been recognized as EPC mobilizing factors. Regulation of the retention of EPCs in the BM microenvironment is carried out by several integrin subunits. α4 and β3 have a role in EPCs, whereas β1 and β2 promote homing (discussed in “Homing” section). α4-Integrins promote the adherence of EPCs to vascular cell adhesion molecule I (VCAM-I), and adhesive interactions between EPC and BM stromal cells. β3-Integrins play an essential role in angiogenesis and hemostasis (Nagano et al., 2007).

Through his study, Qin et al. (2006) demonstrated that inhibiting the availability of α4- and β3-integrins results in enhanced mobilization of EPCs from the BM niche into circulation. Some studies have also shown the role of β1 and β2-integrins in EPC homing to active angiogenic sites and their adherence to activated ECs, VCAM-I, and cellular fibronectin (Balaji et al., 2013).

Although topically applied growth factors have been shown to be ineffective (Smiell et al., 1999), the direct application of angiogenic factors, such as VEGF, bFGF, and PDGF (Ackermann et al., 2011), indeed, has shown improved angiogenesis. Ackermann et al. (2014) found that the application of a mixture of VEGF/bFGF/PDGF subcutaneously induces favorable effects on cutaneous incisional wound healing in diabetic mice.

HMGCoA-reductase inhibitors

Even though the pharmacologic mechanisms by which statins affect the neovasculogenesis of circulating EPCs is still unknown, their role in promoting the EPC function through the regulation of C-X-C chemokine receptor type 4 (CXCR4), a SDF-1 receptor expression, activating the endothelial phosphatidyl inositol-3-kinase (PI3K)/Akt [also known as Protein kinase B]/eNOS/NO pathway, and upregulating eNOS expression has been proposed (Chiang et al., 2015; Kureishi et al., 2000; Urbich et al., 2002). Chiang et al. (2015) found that statin treatment led to significantly more CXCR4-positive EPCs being incorporated into ischemic sites and in the blood compared with control mice.

Homing

The term “homing” relates to the signals that attract and stimulate the EPCs to migrate to sites of injury, adhesion to the endothelial cell monolayers, and incorporation into neovessels (Brem and Tomic-Canic, 2007).

Hypoxia-inducible factor-1 and CD26/dipeptidylpeptidase IV (DPP-IV) as a central regulator of stromal cell-derived factor-1α

Similar to the adhesion molecules engaged by leukocytes for recruitment to sites of inflammation, EPC and progenitor cells also use adhesion molecules for homing to sites of neovascularization. Hypoxia-inducible factor 1 (HIF-1) and DPP-IV have been recognized as central regulators of SDF-1α (a chemokine).

HIF-1 not only promotes angiogenesis, by activating the transcription of multiple angiogenic factors such as VEGF, angiopoietin 2, and FGF-2, but also enhances EPC homing to wound sites, by increasing the expression of the HIF-1 target gene SDF-1α and its cellular receptor, CXC receptor type 4 (CXCR4) (Catrina and Zheng, 2016). These receptors are expressed on the EPC cell surface. A recent study demonstrated that the dicarbonyl metabolite methylglyoxal (MGO) accumulates in cells that are exposed to high glucose concentrations, and it modifies HIF-1α, resulting in reduced expression of HIF-1 target genes such as SDF-1α, CXCR4, eNOS, and VEGF (Bento et al., 2010). Local HIF-1α overexpression via the gene transfer of a stable and active form of HIF-1α (Botusan et al., 2008; Mace et al., 2007) as well as the topical application of prolyl hydroxylase domain protein inhibitors such as dimethyloxalylglycine (DMOG), deferoxamine (DFO) (Botusan et al., 2008; Thangarajah et al., 2009), and Cobalt (II) chloride CoCl₂ (Mace et al., 2007) has been reported to improve wound healing in diabetic mice by enhancing EPC homing (Brem and Tomic-Canic, 2007).

Moreover, Gallagher et al. (2007) demonstrated in his study that SDF-1α expression by epithelial cells and myofibroblasts in the granulation tissue of cutaneous wounds decreases in the diabetic murine model. The exogenous administration of recombinant SDF-1α protein to the wounds of these diabetic murine mice significantly enhances EPC recruitment to the wound site (Gallahger et al., 2007).

More recently, SDF-1α has been established as one of the many substrates of DPP-IV, which is a membrane-bound extracellular peptidase that selectively cleaves N-terminal dipeptides from a variety of substrates, including cytokines, growth factors, neuropeptides, and the incretin hormones. SDF-1α is cleaved and inactivated by DPP-IV (Röhrborn et al., 2015). DPP-IV is expressed on many hematopoietic cell populations, including stimulated B and T lymphocytes, endothelial cells, fibroblasts, epithelial cells, and CD34⁺ stem cells (Klemann et al., 2016).

It has been demonstrated that chronic hyperglycemia induces a significant increase in DPP-IV activity in type 1 and type 2 diabetes, which, in turn, leads to a further reduction in incretin hormones (glucagon-like peptide-1 [GLP-1] and glucose-dependent insulinotropic polypeptide [GIP]), causing consequent postprandial hyperglycemia in type 2 diabetic patients with poor metabolic control (Mannucci et al., 2005).

Inhibition of DPP-IV, as it increases the level of these incretin hormones, which are major regulators of post-prandial insulin secretion, increases the half life of insulin action and, thus, has gained considerable interest for the therapy of type 2 diabetic patients (Pederson et al., 1998; Scheen, 2012). In addition, its inhibition has also been found to mobilize EPCs in patients with type 2 diabetes by protecting SDF-1α from enzymatic degradation, thereby strengthening its clinical value for diabetic foot ulcer patients. Zubra et al. (Zaruba et al., 2009) found that genetic deletion or pharmacologic inhibition of DPP-IV increases the homing of CXCR4⁺ EPC at sites of myocardial damage, resulting in a reduced cardiac remodeling and improved heart function and survival in mice. Furthermore, Fadini et al. (2010) demonstrated that type 2 diabetic patients receiving a 4-week course of therapy with the DPP-IV inhibitor sitagliptin show increased SDF-1α plasma concentrations and circulating EPC levels.

HMGB1 protein and homing of EPCs

In addition to SDF-1α, few studies have also provided evidence for other factors such as high-mobility group box 1 protein (HMGB1) protein, which enhances integrin-dependent functions such as adhesion and migration of EPCs, thereby playing an important role in EPC homing (Yu et al., 2015). However, the role of HMGB1 in diabetic foot ulcers can be controversial, provided its ability to enhance production of pro-inflammatory mediators, thereby leading to a sustained chronic inflammatory state during diabetes (Tsao et al., 2015).

HMGB1 protein, a member of the damage-associated-molecular-pattern (DAMP) family of proteins, in addition to its intracellular functions such as neurite outgrowth, platelet activation, and cell adhesion, also contributes to extracellular functions such as cytokine and chemokine activity. These extracellular functions of HMGB1 are mediated by HMGB1 receptors (e.g. receptor for advanced glycation end products [RAGE]) (Hori et al., 1995), members of the Toll-like receptor (TLR) (e.g. TLR2, TLR4, and TLR9) (Park et al., 2004), or endocytic HMGB1 (Kang et al., 2014) uptake to activate the downstream signaling pathway (e.g., nuclear factor-kB [NF-kB], interferon regulatory factor-3 [IRF3], and PIK3). Studies have reported the role of HMGB1 in stimulating EPC homing to ischemic tissue sites and also increased EPC adhesion to endothelial cell monolayers, intercellular Adhesion Molecule-1 (ICAM-1), and fibronectin in a RAGE- and an integrin-dependent manner (Bento et al., 2010).

Moreover, HMGB1 has also been reported to promote autophagy in response to oxidative stress. Cytoplasmic HMGB1 promotes starvation- and oxidative stress-induced autophagy by binding Beclin-1 (Tang et al., 2010b). Extracellular HMGB1 induces autophagy by binding to its receptor RAGE (Tang et al., 2010a). Nuclear HMGB1 regulates autophagy by inducing expression of heat shock protein b-1, which allows membrane dynamic trafficking during autophagy and mitophagy (Tang et al., 2011).

Hayakawa et al. (2013, 2014) and Chavakis et al. (2007), through their respective studies, demonstrated that blocking β2 integrins on EPCs or blocking the RAGE on endothelial cells significantly decreased EPC accumulation and EPC-endothelial adherence. Moreover, suppression of HMGB1 with small interfering RNA (siRNA) in vivo significantly decreases EPC numbers as well as proliferates endothelial cell numbers. Although this evidence suggests the beneficial role of HMGB1 protein induction in treating diabetic foot ulcers, some authors have also reported sustained inflammation and cellular damage brought on by the HMGB1-induced NF-kB signaling pathway, which, in turn, upregulates pro-inflammatory cytokines such as tumor necrosis factor, IL-1, and IL-6, causing impaired tissue repair (Chen et al., 2015b). Moreover, Mudaliar et al. (2014) also demonstrated upregulation of TLR4 expression with increased NF-kB activation, IL-8 and ICAM-1 expression with a high glucose concentration. These studies showed a relationship between a high glucose state in diabetes and upregulation of HMGB1 and its receptors.

The reasons for the discrepancies between studies are unclear, but they may be the result of differences in the purity of HMGB1 redox protein preparations and the used dose. Three redox forms of HMGB1 have been identified: (1) all-cysteine-reduced HMGB1, (2) disulfide HMGB1, and (3) all-cysteine-oxidized HMGB1. All-cysteine-reduced HMGB1 has been shown to induce cell migration and inflammatory cell recruitment but not cytokine production in immune cells, whereas disulfide HMGB1 has been reported to induce cytokine activity. All-cysteine-oxidized HMGB1 does not show cytokine or chemotactic activity (Venereau et al., 2012). However, it still has the ability to activate neutrophils and VE cells and to trigger age-related inflammation. In addition to these findings, dose dependency of HMGB1 has also been reported. When large amounts of HMGB1 were passively released from damaged cells, they worsened neuroinflammation and brain injury; whereas when lower levels of HMGB1 were actively secreted from reactive astrocytes, they were found to be beneficial (Hayakawa et al., 2014).

Autophagy

Autophagy is an adaptive catabolic process that maintains cellular homeostasis when nutrients are scarce (e.g., during starvation, immediately after birth, etc.) by degrading long-lived proteins to produce the amino acids required for adenosine triphosphate (ATP) synthesis. It also eliminates functionally redundant or damaged intracellular structures such as peroxisomes, mitochondria, and the endoplasmic reticulum (Meijer and Codogno, 2008). However, the exact role and the regulation of autophagy in diabetes are still controversial.

Kim et al. (2014) reported that the hyper-glycemic condition induces autophagy and mitochondrial impairment in EPCs, leading to dysfunctional EPCs. According to him, autophagy in endothelial cells was induced by methylglyoxal, a metabolite specific to diabetes. Furthermore, Masini et al. (2009) reported that autophagic cell death was enhanced in type 2 diabetes pancreatic beta cells. However, most studies claimed that autophagy is a protective mechanism during diabetes, which is triggered by hyper-glycemic-induced oxidative stress and is actually impaired in the diabetic state. This impaired autophagy leads to accumulation of dysfunctional or damaged cellular components, contributing to cellular dysfunction in diabetes (Meijer and Codogno, 2008).

Identification of many other regulatory molecules such as AMP-activated protein kinase; the eukaryotic initiation factor 2α; BH3-only proteins that contain a Bcl-2 homology-3 (BH3) domain inhibition of the Beclin 1/class III PI3K and a disrupted Bcl-2/Bcl-XL complex; and the tumor suppressor protein, p53, has led to different pharmacological approaches to induce or inhibit autophagy (Levine and Kroemer, 2008). In fact, more recently, even genetic approaches to regulate autophagy have been introduced. Studies have shown that knockout of autophagy-related gene (ATG) by a homologous recombination or knockdown by siRNA inhibits autophagy; whereas on the other hand, inhibition of microRNA-130a (miR-130a) enhances the autophagy (Kang et al., 2007; Xu et al., 2014). In case of diabetes, to enhance autophagy in EPCs, mTOR inhibitor (rapamycin), Beclin 1, and adenosine have been used and have shown encouraging results so far. Figure 2 summarizes the regulation of autophagy in diabetes.

FIG. 2.

Regulation of autophagy in diabetes: Activation of mTOR requires input from both insulin and amino acids. Activation of mTOR stimulates protein synthesis and simultaneously inhibits autophagy via phosphorylation of the protein kinase Atg1. The mechanism by which FFAs stimulate autophagy remains to be elucidated. ROS may be involved. Overexpression of miR-130a suppresses its target runt-related transcription factor 3 (Runx3) and, subsequently, upregulates Bcl2, which not only inhibits apoptosis but also binds to and inactivates Beclin 1, thereby inhibiting the initiation of autophagy. In addition, miR-130a may reduce Beclin 1 levels by suppressing Runx3 and inhibiting autophagy in a Bcl2-independent manner. , stimulate/upregulate; , inhibit/downregulate. FFAs, free fatty acids; ROS, reactive oxygen species.

mTOR inhibitor (rapamycin) and adenosine

Chen et al. (2015a) demonstrated significantly decreased autophagosomes per cellular cross-sectional area, increased mTORC1 expression, and higher mitochondrial membrane potential (MMP) of peripheral blood EPCs in diabetic patients. The increase in MMP indicates that the cells needed to obtain more raw materials for the ATP-synthesizing machinery and that they degraded more metabolic waste. However, in diabetic patients, the level of autophagy, which could degrade metabolic waste and transform it into materials for the ATP-synthesizing machinery, was reduced. This leads to an increase of oxygen-derived free radicals, which, in turn, could cause mitochondrial dysfunction and could even result in cell death (Chen et al., 2015a). Accordingly, adenosine and mTOR inhibitor rapamycin can be used to increase autophagy. Chen et al. (2015a) demonstrated that high extracellular adenosine decreased the level of cellular energy metabolism and increased EPC autophagy. In his study after the addition of adenosine, the expression of EPC autophagy markers LC3-II/I and Beclin-1 significantly increased.

Adenosine is a common messenger that functions mainly through four G protein-coupled receptors, A1, A2a, A2b, and A3. Adenosine acting at A2A receptor promotes endothelial cell proliferation, migration, and secretion of angiogenic growth factor, VEGF. Recent studies have also shown participation of A2B receptors in angiogenesis (Ryzhov et al., 2008).

Hypoxic culture environment-induced autophagy in EPCs

Moreover, culture environment has been shown to significantly affect EPC function.

The embryonic environment in which these cells are often found is typically under low oxygen tension and therefore, it is believed that hypoxic conditions are beneficial to stem and progenitor cell growth and survival (Basciano et al., 2011; Prado-Lopez et al., 2010). Studies have demonstrated that overexpression of HIF-1-dependent Beclin-1, BNIP3, and ATG5 under hypoxic condition significantly enhances the EPC migration and pseudotubule formation capabilities of EPCs (Bellot et al., 2009; Hu et al., 2015; Zhang et al., 2008).

BNIP3, a member of the so-called BH3-only proteins that heterodimerize and antagonize the activity of the prosurvival proteins (B cell lymphoma 2 [Bcl-2] and B cell lymphoma-extra large [Bcl-XL]), are actually autophagy receptors containing LC3-interacting regions, which bind directly to LC3 proteins localized within autophagosomes, thereby engaging mitophagy (Hanna et al., 2012; Johansen and Lamark, 2011; Zhu et al., 2013). Maiuri et al. (2007) suggested that BNIP3 might induce autophagy by disrupting interactions between Beclin-1, a highly conserved protein that is required for the initiation of autophagy, and Bcl2 or Bcl-XL. Wang et al. (2013) demonstrated that short-term, 2 hours, hypoxia increases the expression of Beclin-1, a critical protein for autophagosome formation, and LC3, a specific marker for autophagosome structures, thereby increasing autophagy in EPCs. Quantitative polymerase chain reaction analysis and immunofluorescence staining have shown hypoxia to upregulate messenger RNA (mRNA) and protein expression levels of Beclin-1, which, in turn, enhance Beclin 1-dependent autophagy in EPCs.

MicroRNA and Angiogenesis

MicroRNAs (miRs) are endogenously expressed, noncoding small RNAs that post-transcriptionally regulate gene expression by binding to identical or similar sequences in the 3′-UTRs of target cellular mRNAs, hence resulting in inhibition of translation or cleavage of the mRNA target (Meng et al., 2013; Ye et al., 2015). MicroRNAs are involved in key cellular processes, including cell proliferation, differentiation, migration, and apoptosis (Meng et al., 2013). Several studies suggest that miRs are involved in the process of angiogenesis by modulating new vessel formation (Hermeking, 2010; Suarez and Sessa, 2009). Out of the multiple miRNAs known to be regulated during angiogenesis, overexpression of miR-221 and miR-222 (Poliseno et al., 2006) in VE cells was found to significantly reduce endothelial cell migration, proliferation, and angiogenesis; whereas miR-130a, miR-210, miR-424, miR-27-b, let-7f, and miR-17-92 were found to be proangiogenic miRs (Urbich et al., 2008).

Meng et al. (2013) demonstrated that several of these microRNA such as miR-126b, miR-130a, miR-21, miR-27a, and miR-27b were downregulated in EPCs derived from type 2 DM patients. Although he was able to demonstrate that downregulation of miR-126b and miR-130a impairs EPC function, whether other miRs, which are decreased in DM such as miR-21, miR-27a, and miR-27b, are also involved in EPC dysfunction is still to be elucidated. Furthermore, in their study, Meng et al. (2013) found that overexpression of these two miRNAs, miR-126b and miR-130a, significantly improves angiogenesis by promoting EPC migration, differentiation, colony formation, and tubule formation. Downregulation of miR-126b impairs EPC function via its target, Spred-1, and through Ras/ERK/VEGF and PI3K/Akt/eNOS signal pathways; whereas miR-130a regulates angiogenesis via Runx3 (Chen et al., 2010). Furthermore, miR-130a also downregulates the anti-angiogenic homeobox proteins growth arrest homeobox (GAX) and HoxA5, and it functionally antagonizes their inhibitory effects on endothelial cell proliferation, migration, and tube formation (Chen and Gorski, 2008). Figure 3 shows the role of miRNA in angiogenesis.

FIG. 3.

Role of miRNAs in vascular biology: SPRED1 inhibits activation of the Ras/ERK pathway, whereas PIK3R2 negatively regulates the activity of PI3K, thereby inhibiting EPC proliferation and migration. Overexpression of miR-126 inhibits SPRED1 and PIK3R2, which, in turn, activates VEGF and mediates Ras/ERK and PI3K/Akt pathways. Overexpression of miR-130a decreases Runx3 mRNA and protein levels in EPCs, thereby promoting colony formation, proliferation, migration, and differentiation of EPCs. Moreover, miR-130a downregulates the anti-angiogenic homeobox proteins GAX and HoxA5, and it functionally antagonizes the inhibitory effects of GAX on endothelial cell proliferation, migration, and tube formation and the inhibitory effects of HoxA5 on tube formation vitro. , stimulate/upregulate; , inhibit/downregulate. GAX, growth arrest homeobox; VEGF, vascular endothelial growth factor.

Moreover, although the present study is entirely focused on EPC dysfunction in diabetes, angiogenesis is only one aspect in diabetic foot ulcer (DFU) wound healing. Several other factors such as keratinocyte differentiation, proliferation, and migration; fibroblast migration toward the wound; expression of type I and III collagens in fibroblasts; and so on regulate wound healing. In this regard, these aforementioned miRNA have been shown to play a role in angiogenesis, whereas several other miRNA have also been identified that play an important role during the different stages of wound healing, suggesting their possible application in treating patients with diabetic foot ulcers [for a review, see reference Moura et al. (2014)].

Summary

A new development in molecular and cellular biology has led to a better understanding of the etiology of nonhealing, diabetic wounds. It is now clear that multiple steps, working through specific mechanisms, contribute to neovascularization and several of these mechanisms are impaired in diabetic patients. Therefore, a combination of therapeutic approaches, including all these steps involved in neovascularization, would significantly accelerate diabetic wound healing. Future studies applying combined approaches for correcting EPC activation, mobilization, and homing, as well as promoting autophagy would be interesting to look forward to.

Footnotes

Author Disclosure Statement

The authors declare that no conflicting financial interests exist.

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