Protein tyrosine phosphatase 1B inhibition as a potential therapeutic target for chronic wounds in diabetes.

Non-healing diabetic foot ulcers (DFUs) are a serious complication in diabetic patients. Their incidence has increased in recent years. Although there are several treatments for DFUs, they are often not effective enough to avoid amputation. Protein tyrosine phosphatase 1B (PTP1B) is expressed in most tissues and is a negative regulator of important metabolic pathways. PTP1B is overexpressed in tissues under diabetic conditions. Recently, PTP1B inhibition has been found to enhance wound healing. PTP1B inhibition decreases inflammation and bacterial infection at the wound site and promotes angiogenesis and tissue regeneration, thereby facilitating diabetic wound healing. In summary, the pharmacological modulation of PTP1B activity may help treat DFUs, suggesting that PTP1B inhibition is an outstanding therapeutic target.

The role of circulating soluble fms-like tyrosine kinase-1 in patients with diabetic foot ulcer: A possible mechanism of pathogenesis via a novel link between oxidative stress, inflammation and angiogenesis.

BACKGROUND: Diabetic foot ulcer (DFU) is one of the most devastating diabetic consequences leading to amputations. Oxidative stress, inflammation, vascular insufficiency and neuropathy have been linked to DFU development. Since soluble fms-like tyrosine kinase-1 (sFlt-1) is one of the anti-angiogenic factors regulating vascular endothelial growth factor (VEGF) biological activity. So, we aimed to evaluate its role in pathogenesis of DFU and its correlation with oxidative stress and inflammatory markers. METHODS: 60 type 2 diabetic patients: 30 without DFU and 30 with DFU in addition to 20 healthy controls were enrolled in the study. sFlt-1 and VEGF mRNA relative gene expressions and levels and sFlt-1/VEGF ratio were assessed. Also, Advanced oxidation protein products (AOPPs), malondialdhyde (MDA), Total thiol and, tumor necrosis factor alpha (TNF-α) levels were measured. RESULTS: sFlt-1 expression and level, AOPPs, MDA and TNF-α were significantly higher in diabetic patients as compared with the control group with highest levels in DFU patients. However, there were significant decrease in total thiol level and VEGF expression and level in diabetic patients with DFU. CONCLUSION: This study revealed that sFlt-1 is a major player in DFU pathogenesis and may be considered as a novel diagnostic biomarker for early detection of DFU.

Matrix metalloproteinase 9 induces keratinocyte apoptosis through FasL/Fas pathway in diabetic wound.

Apoptosis is a mechanism to remove unwanted cells in the tissue. In diabetic wound, which is characterized by delayed healing process, excessive apoptosis is documented and plays a crucial role. Matrix metalloproteinase 9 (MMP9), which is elevated in non-healed diabetic wound, is necessary for healing process but its abnormality resulted in a delayed healing. The classical function of MMP9 is the degradation of extracellular matrix (ECM). However, there is some literature evidence that MMP9 triggers cell apoptosis. Whether the excessive MMP9 contributes to epidermis cell apoptosis in delayed healing diabetic wound and the underlying mechanisms is not clear. In this study, we aimed to explore whether MMP9 induced keratinocyte apoptosis and investigate the plausible mechanisms. Our in vitro study showed that advanced glycation end products (AGEs) induced keratinocyte apoptosis and enhanced MMP9 level. Besides, MMP9, both intra-cellular expressions and extra-cellular supplement, promoted cell apoptosis. Further, MMP9 resulted in an increased expression of FasL, other than Fas and p53. These findings identified a novel effect that MMP9 exerted in delayed diabetic wound healing, owing to a pro-apoptotic effect on keratinocyte, which was mediated by an increase of FasL expression. This study increases understanding of elevated MMP9 which is involved in diabetic wound repair and offers some insights into novel future therapies.

Impact of lysyl oxidase (G473A) polymorphism on diabetic foot ulcers.

Lysyl oxidase (LOX) is an extra-cellular matrix-modifying enzyme that has been linked to cell proliferation, metastasis, angiogenesis and wound healing. This study was designed to examine the association of LOX gene polymorphism G473A, G>A, (rs1800449) located in exon 1 of the LOX gene in diabetic subjects with and without diabetic foot ulcers (DFU) and its impact of expression on DFU. Genotypic analysis of 906 samples showed a significant increase in the presence of 'A' allele in type 2 diabetes mellitus (T2DM) and DFU when compared to that of control subjects. Allele wise analysis showed a higher frequency of 'A' allele in the T2DM (36.23%, OR 1.069, P value 0.29) and DFU (41.69%, OR 1.195, P value 0.003) when compared to that of control subjects (33.17%). Interestingly, real time RT-PCR results showed significant increased transcript level of the LOX gene on the AA genotype of DFU when compared to that of the AA genotype of T2DM and control subjects. Our finding predicts that there is an association of LOX gene polymorphism (G473A) on diabetes and DFU patients when compared to that of healthy controls. Thus, this study merits further evaluation on a mechanistic approach of this gene.

PKCδ inhibition normalizes the wound-healing capacity of diabetic human fibroblasts.

Abnormal fibroblast function underlies poor wound healing in patients with diabetes; however, the mechanisms that impair wound healing are poorly defined. Here, we evaluated fibroblasts from individuals who had type 1 diabetes (T1D) for 50 years or more (Medalists, n = 26) and from age-matched controls (n = 7). Compared with those from controls, Medalist fibroblasts demonstrated a reduced migration response to insulin, lower VEGF expression, and less phosphorylated AKT (p-AKT), but not p-ERK, activation. Medalist fibroblasts were also functionally less effective at wound closure in nude mice. Activation of the δ isoform of protein kinase C (PKCδ) was increased in postmortem fibroblasts from Medalists, fibroblasts from living T1D subjects, biopsies of active wounds of living T1D subjects, and granulation tissues from mice with streptozotocin-induced diabetes. Diabetes-induced PKCD mRNA expression was related to a 2-fold increase in the mRNA half-life. Pharmacologic inhibition and siRNA-mediated knockdown of PKCδ or expression of a dominant-negative isoform restored insulin signaling of p-AKT and VEGF expression in vitro and improved wound healing in vivo. Additionally, increasing PKCδ expression in control fibroblasts produced the same abnormalities as those seen in Medalist fibroblasts. Our results indicate that persistent PKCδ elevation in fibroblasts from diabetic patients inhibits insulin signaling and function to impair wound healing and suggest PKCδ inhibition as a potential therapy to improve wound healing in diabetic patients.

Expression and vitamin D-mediated regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in healthy skin and in diabetic foot ulcers.

Diabetic foot ulcers (DFUs) are chronic wounds with high matrix metalloproteinase (MMP) activity, and are a frequent complication on diabetics. This work studied the expression of selected MMP and tissue inhibitor of metalloproteinases (TIMP) gene family members in DFU and normal skin biopsies, and in vitamin D-treated keratinocytes cultured from those biopsies. We report for the first time the expression of some of these genes in healthy skin. Our results suggest that vitamin D may modulate the expression of some MMP gene family members in keratinocytes. Gene expression in DFU and in non-diabetic healthy skin (control) biopsies was evaluated by RT-qPCR for MMP-1, MMP-3, MMP-8, MMP-9, MMP-10, MMP-19, TIMP-1 and TIMP-2, and also by immunohistochemistry for MMP-1 and MMP-9. Primary keratinocytes cultured from DFU and healthy skin biopsies were used for gene expression analyses of selected MMPs and TIMPs by RT-qPCR, both in the presence and absence of calcitriol. The expression of MMP-1, MMP-8, MMP-9, MMP-10, and TIMP-2 in healthy skin is reported here for the first time. DFUs showed increased MMP-1, MMP-9 and TIMP-1 expression, compared to healthy skin. Calcitriol down-regulated MMP-1 and MMP-10 expression in DFU-derived keratinocytes but not in those derived from healthy skin. Our data demonstrate the expression of certain MMPs that had not been previously described in healthy skin, and further support previous reports of MMP and TIMP up-regulation in DFUs. Our results point to calcitriol as a potential modulator for the expression of certain MMP members in DFUs.

AMP-activated protein kinase activators in diabetic ulcers: from animal studies to Phase II drugs under investigation.

INTRODUCTION: Diagnosed cases of diabetes have gradually increased year by year, and research on diabetes mellitus (DM) has attracted greater attention from the medical profession. Diabetic ulcers present persistent pain and the risk of bacterial infection. However, no promising treatment methods have been found. As a regulator of cellular energy balance, 5' adenosine monophosphate-activated protein kinase (AMPK) has been suggested as a drug target for DM, including such drugs as metformin. AREAS COVERED: This review summarizes the current research and clinical trials of AMPK activators on diabetic wound healing and diabetic ulcers. Furthermore, it discusses the feasibility of AMPK activators in the treatment of diabetic wounds. EXPERT OPINION: Animal studies have demonstrated that AMPK activators are a potential treatment for diabetic ulcers. AMPK activators alleviate tissue inflammation and promote re-epithelialization in diabetic wounds. However, due to the complicated pathological mechanism of diabetic foot ulcers, AMPK activators should be combined with other approaches. The new strategies for combination therapy with AMPK activator may provide a therapeutic advantage for patients with diabetic ulcers.

Dipeptidyl peptidase 4 inhibition may facilitate healing of chronic foot ulcers in patients with type 2 diabetes.

The pathophysiology of chronic diabetic ulcers is complex and still incompletely understood, both micro- and macroangiopathy strongly contribute to the development and delayed healing of diabetic wounds, through an impaired tissue feeding and response to ischemia. With adequate treatment, some ulcers may last only weeks; however, many ulcers are difficult to treat and may last months, in certain cases years; 19-35% of ulcers are reported as nonhealing. As no efficient therapy is available, it is a high priority to develop new strategies for treatment of this devastating complication. Because experimental and pathological studies suggest that incretin hormone glucagon-like peptide-1 may improves VEGF generation and promote the upregulation of HIF-1α through a reduction of oxidative stress, the study evaluated the effect of the augmentation of GLP-1, by inhibitors of the dipeptidyl peptidase-4, such as vildagliptin, on angiogenesis process and wound healing in diabetic chronic ulcers. Although elucidation of the pathophysiologic importance of these aspects awaits further confirmations, the present study evidences an additional aspect of how DPP-4 inhibition might contribute to improved ulcer outcome.

[Modulating effects of protein kinase A on intracellular signal systems in the course of skin wound healing in patients with diabetes mellitus].

The purpose of this study was to examine the modulating effects of protein kinase A (PKA) on the performance of signal systems triggered while activating alpha2-adrenoceptors and eNOS in the course of a wound process in patients with type 2 diabetes mellitus. Patients with the diabetic foot were shown to have impaired adrenergic responsiveness, accompanied by reduced eNOS activity and PKA inhibition. The positive therapeutic effect was noted with the increased stimulating effect of the PKA system on PKG on days 3 to 5 and on eNOS on days 10-14 of therapy. The preserving eNOS activity deficit was compensated for by the PKA-induced increase in PKG stimulation. The modulating effects of PKA are a compensatory intracellular mechanism of adaptation when NO production is decreased, which may be used to develop new therapeutic corrections of the dysregeneratory syndrome.

Expression of matrix metalloproteinases and growth factors in diabetic foot wounds treated with a protease absorbent dressing.

UNLABELLED: Wound healing in diabetes is impaired, and nonhealing ulceration represent clinically relevant complications. Persistently high levels of matrix metalloproteases (MMPs) contribute to wound chronicity. Thus, the topical use of protease inhibitors might influence wound healing and promote transition from a chronic to an acute wound. METHODS: In this study, 33 patients with chronic diabetic foot lesions (stage 2a of the University of Texas Wound Classification system) were included. Fifteen patients received redundant "good standard wound care." In addition, 18 patients were treated with a protease inhibitory modulating matrix (the OCR/collagen Promogran matrix, Ethicon) with dressings changed on a daily basis. Prior to treatment and at 4 and 8 days after treatment, two 3-mm punch biopsies were taken from the center of the wounds and analyzed using ELISA techniques for MMPs, tissue inhibitor of MMP-2 (TIMP-2), and interleukin-1beta (IL-1beta) levels. In addition, mRNA levels of MMPs as well as IL-1beta and TNF-alpha were detected using quantitative real-time polymerase chain reaction (TaqMan, Applied Biosystems, Weiterstadt, Germany). RESULTS: No differences were detected between both groups and at the three time points for the mRNA levels of MMPs as well as of IL-1beta and TNF-alpha. In addition, MMP levels in wound tissue (analyzed by ELISA) were also not significantly different between both groups. However, IL-1beta was increased on day 8 in the treatment group (P=.01) only. Interestingly, we found a significant reduction of the MMP-9/TIMP-2 ratio in the group being treated with the ORC/collagen. These wounds exhibited a more rapid healing rate when treated with the ORC/collagen matrix. CONCLUSIONS: The local treatment with a protease inhibitor has a beneficial effect on wound healing. In contrast to the data on wound fluid, our study demonstrated for the first time the unaltered mRNA levels of MMPs during treatment with a protease inhibitory modulating matrix. At the cellular level, MMPs were also not statistically different. However, the more relevant ratio of MMP-9/TIMP-2 was decreased in the treatment group. An equally important finding was that we did not detect a compensatory increase in the MMP-RNA expression even though wound size was clearly reduced.

Expression of matrix-metalloproteinases and their inhibitors in the wounds of diabetic and non-diabetic patients.

AIMS/HYPOTHESIS: The molecular factors that cause an acute wound in diabetic patients to become chronic have not yet been established. Wound healing is known to require a balance between the accumulation of collagenous and non-collagenous extracellular matrix components and their remodelling by matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs). Our aim was to assess if the concentrations of MMPs and TIMPs were different between acute and chronic wounds in diabetic patients by analysing biopsy samples. METHODS: A 5 mm punch biopsy was taken from 20 diabetic foot ulcers of patients before initiating treatment and from traumatic wounds of 12 non-diabetic patients 2 days after injury. The concentrations of MMP-1, MMP-2(pro), MMP-2(active), MMP-8, MMP-9 and TIMP-2 were measured in detergent extracts of the biopsy homogenates using ELISAs and gelatin-zymography. RESULTS: The concentration of MMP-1 was increased 65-fold in biopsies of diabetic foot ulcers compared with the concentrations measured in biopsies of traumatic wounds. Similarly, MMP-2(pro) were increased threefold, sixfold for MMP-2(active), twofold for MMP-8 and 14-fold for MMP-9 compared to average concentrations in biopsies of traumatic wounds. Furthermore, the expression of TIMP-2 was reduced twofold in diabetic wounds compared with lesions of non-diabetic patients. CONCLUSION/INTERPRETATION: The combination of increased concentrations of MMPs with decreased concentrations of TIMP-2 in chronic diabetic foot ulcers compared with healing wounds in normal patients suggests that the increased proteolytic environment contributes to the failure of diabetic wounds to heal. New treatment strategies for healing chronic diabetic foot ulcers could be directed towards reducing concentrations of MMPs and increasing levels of TIMPs.

Endothelial dysfunction and the expression of endothelial nitric oxide synthetase in diabetic neuropathy, vascular disease, and foot ulceration.

We studied endothelial-mediated microvascular blood flow in neuropathic diabetic patients to determine the association between endothelial regulation of the microcirculation and the expression of endothelial constitutive nitric oxide synthetase (ecNOS) in the skin. Vasodilation on the dorsal foot in response to heating and iontophoresis of acetylcholine (endothelium-dependent) and sodium nitroprusside (endothelium-independent) were measured using single-point laser Doppler and laser Doppler imaging in diabetic patients with neuropathy (DN), with neuropathy and vascular disease (DI), with Charcot arthropathy (DA), and without complications (D), and in healthy control subjects (C). The response to heat was reduced in the DN (321 [21-629] percentage of increase over the baseline, median [interquartile range]) and DI (225 [122-470]) groups but was preserved in the DA (895 [359-1,229]), D (699 [466-1,029]), and C (810 [440-1,064], P < 0.0001) groups. The endothelial-mediated response to acetylcholine was reduced in the DN (17 [11-25]), DA (22 [2-34]), and DI (13 [2-30]) groups compared with the D (47 [24-58]) and C (44 [31-70], P < 0.001) groups. The non-endothelial-mediated response to sodium nitroprusside was also reduced in the DI (4 [0-18]), DN (17 [9-26]), and DA (21 [11-31]) groups compared with the D (37 [19-41]) and C (44 [26-67], P < 0.0001) groups. There was a significant reduction in vasodilation in the DI group compared with all other groups (P < 0.0001). Full thickness skin biopsies from the dorsum of the foot of 15 DN, 10 DI, and 11 C study subjects were immunostained with antiserum to human ecNOS, the functional endothelial marker GLUT1, and the anatomical endothelial marker von Willebrand factor. The staining intensity of ecNOS was reduced in both diabetic groups. No differences were found among the three groups in the staining intensity of von Willebrand factor and GLUT1. We conclude that the endothelium-dependent and endothelium-independent vasodilations are impaired in diabetic patients predisposed to foot ulceration and that neuropathy is the main factor associated with this abnormality. Reduced expression of ecNOS may be a major contributing factor for endothelial dysfunction. These data provide support for a close association of neuropathy and microcirculation in the pathogenesis of foot ulceration.

[The alpha-glycerolphosphate dehydrogenase and adenosinetriphosphatase activity of the peripheral blood lymphocytes in diabetics (a cytochemical determination)]. / Aktyvnist' al'fa-hlitserofosfatdehidrohenazy i adenozyntryfosfatazy limfotsytiv peryferychnoï krovi khvorykh na tsukrovyi diabet (tsytokhimichne vyznachennia).

Decrease in activity of alpha-GPDG and ATP-ase observed in peripheral lymphocytes of patients with diabetes mellitus correlated well severity of the disease. The lowest indices of enzymatic activity were registered in patients with grave diabetes complicated by angiopathies, foot gangrene, and purulent inflammatory processes. Compensation of carbohydrates metabolism and improvement of clinical picture were accompanied by the trend to normalization of enzymes' activity. Dynamic study of alpha-GPDG and ATP-ase in patients with diabetes mellitus allows to evaluate degree of hypoxia, comprehend the nature of compensation mechanisms and assess effectiveness of the treatment.