flow recovery

Legend: NOD, non-obese diabetic; TK, tissue kallikrein; BM-MNC, bone marrow-derived mononuclear cells; STZ, streptozotocin.

Legend: NOD, non-obese diabetic; TK, tissue kallikrein; BM-MNC, bone marrow-derived mononuclear cells; STZ, streptozotocin.

recovery [116]. Experimental DM does not affect either function or angiogenic potential of EpPC.

3.2. DM-Associated Impaired Angiogenic Signaling in Wound Healing Models

A decreased healing capacity is frequently associated with both clinical and experimental DM. The normal wound healing process proceeds through the following phases: hemostasis, inflammation and debridement, proliferation, epithelialization and remodeling [33, 62]. The non-healing nature of the diabetic wound has been linked to disturbances in both the inflammation/debridement phase and the proliferation phase [33]. This prevents the wound from progressing to later stages of the healing process [63]. The adequate course of the healing process is strictly dependent on the development of new vessels. In broader terms, a generalized microangiopathy could also prevent the adequate transfer of nutrients to the wounded tissue, thereby interfering with the normal healing process [117].

The EC is the most important cell type in the process of angiogenesis. ECs can initiate angiogenesis on their own, but periendothelial cells are required for angiogenesis to complete. Studying the process of wound healing provides the possibility to evaluate the adverse effect of DM on the function of perivascular cells partly represented by fibroblasts. Fibroblasts are central to the processes of wound healing, and they play a role in angiogenesis by being involved in ECM deposition and remodeling. The healing cascade requires angiogenesis together with ECM turnover, i.e. ECM production, ECM deposition and collagen constriction by fibroblasts. Fibroblasts are considered as an important synthetic cell type, being directly involved in producing ECM proteins and MMPs and in depositing ECM proteins. Secondly, fibroblasts are involved in intercellular signaling by secreting growth factors important for cell-cell communication during the angiogenic process and by releasing growth factors from ECM depots [59, 62]. Any impairment of fibroblast function prevents normal wound healing and results in chronic, nonhealing wounds as they occur in DM.

Genetically modified diabetic (db/db) mice show delayed wound repair in the incisional skin-wound model, which is characterized by reduced angiogenesis, delayed formation of granulation tissue, decreased collagen content, and low breaking strength as compared with normal littermates [118]. Characteristics of the microvascular derangements during healing in a mouse model of DM include reduced arteriolar density, loss of vascular tone and a reduction in the cross-sectional area of new vessel walls [118]. Full-thickness skin wounds of STZ-treated diabetic mice showed delayed wound healing due to delayed revascularization and delayed and diminished collagen turnover [119]. Delayed angiogenesis and impaired wound healing in diabetic mice are paralleled by low breaking strength of the wound [120] as a result of an impaired local ECM metabolism.

Versatile mechanisms have been elucidated to lead to the impaired angiogenesis and the delayed wound healing in DM. The diminished expression of several angiogenic factors has been reported to significantly contribute to a delayed wound healing. In the normal course of healing, i.e. in healthy animals, an increase in the expression ofVEGF-A [120, 121], angiopoietins [122], members of sonic hedgehog (Shh) [123] and nerve growth factor (NGF) [124] are observed within the first days following injury. In contrast, diabetic mice showed lower VEGF-A levels than their normal littermates [120]. The impaired expression of VEGF has been linked to a high proteolytic activity of the diabetic environment [125]. In addition, the increased generation of reactive oxygen species (ROS), in particular lipid peroxidation (LPO) negatively affected VEGF-A levels [120]. The administration of raxofelast, an inhibitor of LPO, did not modify the process of wound repair in normal mice, but significantly improved the impaired wound healing in diabetic mice through the stimulation of angiogenesis and synthesis and maturation of ECM [120]. The inhibition of LPO could restore the decreased VEGF-A expression during skin repair in diabetic mice. The increased production of ROS in diabetic wounds can be explained by an altered expression of the protective enzyme superoxide dismutase (SOD) [126]. The impairment of nitric oxide (NO) production in DM has been linked to the increased production of ROS including a decrease in the expression of endothelial NO synthase (eNOS) and a reduced availability of NO, as this is directly inactivated by ROS [126]. Gene therapy using SOD and eNOS could restore the levels of NO and resulted in normalized healing.

The use of VEGF-A as a therapeutic agent resulted in an accelerated healing processes by promoting angiogenesis [127, 128]. This may be associated with the recruitment of bone marrow-derived cells [129]. However, circulating CD34+ cells have an impaired function in DM with regard to their contribution to angiogenesis. Experimentally, this problem could be overcome by external supplementation with CD34+ cells from non-diabetic animals, which significantly improved neovascularization in experimental diabetic wounds [119].

The reduced expression of NGF in DM was accompanied by the reduction of NGF receptor levels [124], which apparently contributed to a decreased expression of VEGF-A. In turn, the supplementation of NGF normalized VEGF-A wound levels and accelerated reparative angiogenesis. Similarly, Shh has shown favorable effects on angiogenesis in wound healing [123], which could have been mediated by a Shh-induced increase in VEGF-A (and Ang-1) wound levels. Recombinant human erythropoietin had similar effects on angiogenesis and wound healing as well as on VEGF-A expression [130].

Ang-1 and VEGF-A, both of which are EC-specific growth factors, play distinct but additive roles in the process of angiogenesis. VEGF induces the generation of immature and leaking vessels, whereas Ang-1 leads to their maturation [131]. In the light of impaired reparative angiogenesis in DM, the decreased expression of Ang-1 and VEGF-A is worsened by a prolonged and elevated expression of the Ang-1 antagonist Ang-2 in diabetic wounds [3, 132]. Ang-2 prevents Ang-1-mediated activation of the receptor Tie-2 and thereby prevents vessel stabilization. Likewise, Tie-2 expression is decreased in diabetic wounds [132].

Diabetic conditions are characterized by decreased expression of certain other growth factors such as platelet-derived growth factor (PDGF) and bFGF. Both are acting on SMC and fibroblasts, and their reduced expression is associated with an impaired angiogenesis [133]. Moreover, bFGF exerts a strong mitogenic action on ECs and promote their differentiation [134]. bFGF can act on ECs in both autocrine (produced by ECs themselves) and paracrine (released from ECM after being produced by fibroblasts) ways [59]. bFGF is also a potent motogenic stimulus for ECs involving multiple mechanisms: upregulation of uPA [135]; upregulation of aV^3 integrin, which mediates binding of ECs to ECM [136] and allocation of MMP-2 facilitating ECM degradation in the process of EC migration [137].

The impaired function of fibroblasts in DM can significantly contribute to impaired reparative angiogenesis in wound healing. Fibroblasts from diabetic mice revealed altered baseline expression and ability to up-regulate VEGF-A in response to hypoxic conditions ex vivo [138]. Fibroblasts also exhibited a significantly decreased migratory response and were unable to increase neither migration nor VEGF-A production under hypoxic conditions [138].

Decreased expression of VEGF and growth factors in fibroblasts and ECs was suggested to be due to an impaired regulation of transcription. Expression of the homebox (Hox) D3 transcription factor, a member of a family of transcription factors upregulated during normal wound healing [139], is compromised in a diabetic animal healing model [140]. HoxD3 is induced by bFGF [141] expression of which is negatively affected in DM. HoxD3 is involved in the regulation of transcription of aV^3 integrin and uPA, all blunted in the presence of DM [142]. HoxD3 gene transfer restored expression of the target genes in ECs and fibroblasts and facilitated the improvement in the angiogenic response and collagen deposition during the healing process [142].

Impaired ECM degradation is a critical prerequisite for alteration of angiogenic processes. MMPs are important players in this and evidence was generated in diabetic animal models of wound healing about the affected levels and activity of MMPs. Fibroblasts, a major cell type involved in wound healing, are known to express MMPs [58]. As diabetic mice possess significantly reduced pro-MMP-2 levels during wound healing, this may be contributing to the impaired healing by delaying cell migration and angiogenesis. In support of this notion, it has been shown that fibroblasts from sites that exhibit preferential healing, e.g. the oral mucosa, exhibit increased migration and remodeling in association with increased MMP-2 activity [143]. Additionally, diabetic fibroblasts exhibit impaired expression of major ECM protein collagen I, which appears to be linked to aberrant transcriptional regulation by HoxD3 [144].

The monocyte/macrophage is the main MMP-9-expressing cell type within the wound, with neutrophils being known to store, but not to synthesize it [145]. Diabetic mice show delayed monocyte recruitment to the wound [61] that may strongly contribute to the impaired healing. The reduced level of pro-MMP-9 [61] is probably a reflection of this delayed recruitment; however, the reduced MMP-9 levels may be either cause or effect of this delay. On the other hand, the hypertrophic phenotype of fibroblasts [33] apparently results in an increased production and activity of pro-MMP-9 in the presence of diabetes [138]. The pathophisiological differences between acute and chronic wounds are well established [57]; the differences in MMP expression in animal models of DM may, therefore, be attributed to the different time-points of analysis. Alternatively, interspecies differences in experimental models can be considered to explain these differences.

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Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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