Increased ratio of serum matrix metalloproteinase-9 against TIMP-1 predicts poor wound healing in diabetic foot ulcers

ObjectiveLittle is known about serum concentrations of Matrix Metalloproteinase-9 (MMP-9), MMP-2, TIMP-1 and TIMP-2 in diabetic patients with foot ulcers. This study demonstrates their relationship with wound healing.MethodsNinety-four patients with diabetic foot ulcers were recruited in the study. Serum MMP-9, MMP-2, TIMP-1 and TIMP-2 were measured at the first clinic visit and the end of 4-week treatment and followed up till 12 weeks. According to the decreasing rate of ulcer healing area at the fourth week, we divided those cases into good and poor healers. Through analyses, we explore the possible relationship among those factors and degree of wound healing.ResultsThe median level of serum MMP-9 in good healers was lower than poor healers at first visit (124.2 μg/L vs 374.6 μg/L, p < 0.05), and after 4-week therapy it decreased 5-fold approximately. In contrast, the change in MMP-9 concentration did not reach statistical significance in poor healers. MMP-2, TIMP-1 and TIMP-2 varied slightly in both good healers and poor healers. The MMP-9/TIMP-1 ratio better reflected the healing than MMP-9 alone before therapy and after 4 week treatment (r = ? 0.6475 vs ? 0.3251, r = ? 0.7096 vs ? 0.1231, respectively). Receiver Operator Curve (ROC) showed that the cutoff for MMP-9/TIMP-1 ratio at < 0.395 best predicted a reduction in wound area of 82% at the end of 4-week treatment with a sensitivity of 63.6% and a specificity of 58.6% (area under the curve 0.658, p < 0.001).ConclusionsDetecting serum MMP-9/TIMP-1 ratio on admission might be a predictor of healing and might provide a novel target for the future therapy in diabetic foot ulcers.     

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.     

Expression of tissue inhibitor of metalloprotease 3 is reduced in ischemic but not neuropathic ulcers from patients with type 2 diabetes mellitus

Diabetic foot ulceration remains one of the most common and most serious consequences of diabetes. Persistently high levels of matrix metalloproteases (MMPs) contribute to wound chronicity. Our aim was to assess the concentrations of MMPs and tissue inhibitors of metalloproteinases (TIMPs) in neuropathic and ischemic diabetic foot ulcers by analyzing biopsy samples. In this study, biopsies were taken from 35 diabetic foot ulcers of type 2 diabetes mellitus patients and distinguished in neuropathic (n = 14) or ischemic (n = 21). Zymography assay was utilized for the analysis of MMP-2 and MMP-9 activity. TACE activity was evaluated by a specific fluorimetric assay. mRNA levels of MMPs as well as TIMPs were detected using quantitative real-time polymerase chain reaction. The activity of MMP9 and A Disintegrin and A MetalloProtease Domain 17/TNF-Alpha Converting Enzyme (ADAM17/TACE) was significantly increased in ischemic compared to neuropathic biopsies. No differences were detected between both groups for the mRNA levels of MMPs as well as of ADAMs. However, TIMP3 mRNA expression was decreased in ischemic samples. The combination of increased activity of MMP9 and ADAM17/TACE with decreased concentrations of TIMP-3 mRNA expression in ischemic diabetic foot ulcers compared to neuropathic samples suggests that the increased proteolytic environment may represent a causative factor in the ulcer progression. New treatment strategies for healing diabetic foot ulcers could be directed toward increasing levels of TIMP3.     

Acceleration of diabetic wound healing using a novel protease–anti-protease combination therapy

Nonhealing chronic wounds are major complications of diabetes resulting in >70,000 annual lower-limb amputations in the United States alone. The reasons the diabetic wound is recalcitrant to healing are not fully understood, and there are limited therapeutic agents that could accelerate or facilitate its repair. We previously identified two active forms of matrix metalloproteinases (MMPs), MMP-8 and MMP-9, in the wounds of db/db mice. We argued that the former might play a role in the body’s response to wound healing and that the latter is the pathological consequence of the disease with detrimental effects. Here we demonstrate that the use of compound ND-336, a novel highly selective inhibitor of gelatinases (MMP-2 and MMP-9) and MMP-14, accelerates diabetic wound healing by lowering inflammation and by enhancing angiogenesis and re-epithelialization of the wound, thereby reversing the pathological condition. The detrimental role of MMP-9 in the pathology of diabetic wounds was confirmed further by the study of diabetic MMP-9–knockout mice, which exhibited wounds more prone to healing. Furthermore, topical administration of active recombinant MMP-8 also accelerated diabetic wound healing as a consequence of complete re-epithelialization, diminished inflammation, and enhanced angiogenesis. The combined topical application of ND-336 (a small molecule) and the active recombinant MMP-8 (an enzyme) enhanced healing even more, in a strategy that holds considerable promise in healing of diabetic wounds.Diabetes affects 340 million people in the world, including 29.1 million individuals in the United States (1). A complication in diabetic patients is the inability of wounds to heal, which resulted in 73,000 lower-limb amputations in the United States in 2010 (1). The standard treatment for diabetic foot ulcers includes debridement of the wound, treatment of infection with antibiotics, and reducing or eliminating weight pressure from the lower extremities (2). There is a paucity of pharmacological therapeutics that accelerate wound healing. Although becaplermin (PDGF) is approved for use in diabetic neuropathic ulcers, malignancies have been reported, and an increased risk of mortality was observed in patients treated with becaplermin (3).In diabetic patients, high blood sugar triggers prolonged chronic inflammation, with concomitant elevated levels of matrix metalloproteinases (MMPs). The detrimental effect of MMPs in the diseased tissue has been attributed to the rapid turnover of potential growth factors, receptors, and the newly formed extracellular matrix, which are essential for wound healing (4). Hence, wound healing is impaired and delayed in diabetic patients. However, this process is not well understood, and the actual instigator MMPs are not known.MMPs are a family of zinc-dependent endopeptidases that are capable of degrading extracellular matrix components and are involved in tissue remodeling and restructuring (5). MMPs are expressed as zymogens or pro-MMPs. Activation by proteolytic removal of the N-terminal prodomain is required for their catalytic functions. Active forms of MMPs are highly regulated by binding of tissue inhibitors of metalloproteinases (TIMPs). MMPs are presumed to play various roles in regulating inflammatory and repair processes (6) as well as in wound healing (7).We recently reported on the identification and quantification of active MMP-8 and MMP-9 in a mouse model of diabetic wound healing by the use of an inhibitor-tethered resin that binds exclusively to active MMPs, in conjunction with proteomics analyses (8). Because MMP-9 was observed to be up-regulated only in diabetic wounds, whereas MMP-8 was found in both diabetic and nondiabetic wounds, we hypothesized that MMP-8 is beneficial and that MMP-9 is detrimental in diabetic wound healing. We now report that the use of either a novel and highly selective MMP-9 inhibitor of our design (ND-336, compound 1) or the application of the active recombinant MMP-8 accelerates wound healing in db/db mice. We further confirm the detrimental effect of MMP-9 on diabetic wound healing by the use of MMP-9–knockout mice. Finally, we document that the combination of a selective MMP-9 inhibitor (a small molecule) plus the active recombinant MMP-8 (an enzyme) accelerated wound healing even further in db/db mice. This combination is a potential pharmacological treatment for diabetic wound healing and holds great promise for recourse in this devastating disease.Open in a separate windowScheme 1.Structures of compounds 1 and 2.     

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

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.     

血清基质金属蛋白酶9、糖基化终产物与糖尿病足的相关研究

比较糖尿病足患者(糖尿病足组)、2型糖尿病但非糖尿病足患者(非糖尿病足组)及健康体检者血清基质金属蛋白酶9(MMP-9)、晚期糖基化终产物(AGE)水平.糖尿病患者MMP-9、AGE水平高于健康者,糖尿病足组MMP-9高于非糖尿病足组.血清MMP-9升高似可预测糖尿病足发生及足溃疡的预后.     

Topical application of the bee hive protectant propolis is well tolerated and improves human diabetic foot ulcer healing in a prospective feasibility study

AimsPropolis is a naturally occurring anti-inflammatory bee derived protectant resin. We have previously reported that topically applied propolis reduces inflammation and improves cutaneous ulcer healing in diabetic rodents. The aim of this study was to determine if propolis shows efficacy in a pilot study of human diabetic foot ulcer (DFU) healing and if it is well tolerated.MaterialsSerial consenting subjects (n = 24) with DFU ≥ 4 week's duration had topical propolis applied at each clinic review for 6 weeks. Post-debridement wound fluid was analyzed for viable bacterial count and pro-inflammatory MMP-9 activity. Ulcer healing data were compared with a matched control cohort of n = 84 with comparable DFU treated recently at the same center.ResultsUlcer area was reduced by a mean 41% in the propolis group compared with 16% in the control group at week 1 (P < 0.001), and by 63 vs. 44% at week 3, respectively (P < 0.05). In addition, 10 vs. 2% (P < 0.001), then 19 vs. 12% (P < 0.05) of propolis treated vs. control ulcers had fully healed by weeks 3 and 7, respectively. Post-debridement wound fluid active MMP-9 was significantly reduced, by 18.1 vs. 2.8% week 3 from baseline in propolis treated ulcers vs. controls (P < 0.001), as were bacterial counts (P < 0.001). No adverse effects from propolis were reported.ConclusionsTopical propolis is a well-tolerated therapy for wound healing and this pilot in human DFU indicates for the first time that it may enhance wound closure in this setting when applied weekly. A multi-site randomized controlled of topical propolis now appears to be warranted in diabetic foot ulcers.     

Chronic hypoxia inhibits MMP-2 activation and cellular invasion in human cardiac myofibroblasts

Cardiac myofibroblasts are pivotal to adaptive remodelling after myocardial infarction (MI). These normally quiescent cells invade and proliferate as a wound healing response, facilitated by activation of matrix metalloproteinases, particularly MMP-2. Following MI these reparative events occur under chronically hypoxic conditions yet the mechanisms by which hypoxia might modulate MMP-2 activation and cardiac myofibroblast invasion have not been investigated. Human cardiac myofibroblasts cultured in collagen-supplemented medium were exposed to normoxia (20% O₂) or hypoxia (1% O₂) for up to 48 h. Secreted levels of total and active MMP-2 were quantified using gelatin zymography, TIMP-2 and membrane-associated MT1-MMP were quantified with ELISA, whole cell MT1-MMP by immunoblotting and immunocytochemistry and MT1-MMP mRNA with real-time RT-PCR. Cellular invasion was assessed in modified Boyden chambers and migration by scratch wound assay.In the human cardiac myofibroblast, MT1-MMP was central to MMP-2 activation and activated MMP-2 necessary for invasion, confirmed by gene silencing. MMP-2 activation was substantially attenuated by hypoxia (P < 0.001), paralleled by inhibition of myofibroblast invasion (P < 0.05). In contrast, migration was independent of either MT1-MMP or MMP-2. Reduced membrane expression of MT1-MMP (P < 0.05) was responsible for the hypoxic reduction of MMP-2 activation, with no change in either total MMP-2 or TIMP-2. In conclusion, hypoxia reduces MMP-2 activation and subsequent invasion of human cardiac myofibroblasts by reducing membrane expression of MT1-MMP and may delay healing after MI. Regulation of these MMPs remains an attractive target for therapeutic intervention.     

Healing of diabetic foot ulcers is independently associated with the use of angiotensin receptor blockers but not with those of diuretics and angiotensin conversion enzyme inhibitors

Aim. Dysregulation of the renin angiotensin system (RAS) has been proven in diabetic animal models, and studies in humans show that diuretic use is associated with lower limb amputation in diabetes. While patients with diabetes are often treated with diuretics and RAS blockers, the association between wound healing and these treatments is still unknown. We aimed to determine whether the use of diuretics and RAS blockers could influence healing of diabetic foot ulcers (DFU).Methods. Two hundred seventy-six patients referred to a specialized diabetes foot care unit for a new foot ulcer were included in this retrospective observational study.Results. Healing rate was significantly higher in patients not treated with diuretics than in those receiving diuretics (75.9 vs. 62.9%, P = 0.026) and in patients treated with angiotensin receptor blockers (ARB) than in those not treated with ARB (79.5 vs 64.4%, P = 0.012). The difference was not significant for angiotensin conversion enzyme inhibitor use. ARB use was independently and positively associated with wound healing in a multivariate adjusted model including several factors affecting wound healing (odds ratio (OR) 2.79 [1.13, 6.86] P = 0.025). Diuretic use was negatively associated with wound healing in univariate analysis (OR 0.54 [0.32, 0.91] P = 0.02) but not in multivariate adjusted analysis (OR 0.53 [0.26, 1.10] P = 0.088).Conclusions. This novel study found that ARB use is independently and positively associated with wound healing in 276 patients with DFU. On the contrary, diuretics were associated with healing rate only at univariate analysis. Further prospective studies are needed to confirm our findings.     

Diabetic foot ulcer and multidrug‐resistant organisms: risk factors and impact

Aims The primary objective was to characterize factors allowing the colonization of diabetic foot wounds by multidrug‐resistant organisms (MDRO), and the secondary objective was to evaluate the influence of MDRO colonization/infection on wound healing. Methods In 180 patients admitted to a specialized diabetic foot unit, microbiological specimens were taken on admission. Potential risk factors for MDRO‐positive specimens were examined using univariate and multivariate analyses. Prospective follow‐up data from 75 patients were used to evaluate the influence of MDRO colonization/infection on time to healing. Results Eighteen per cent of admission specimens were positive for MDRO. MDRO‐positive status was not associated with patient characteristics (age, sex, type of diabetes, complications of diabetes), wound duration, or wound type (neuropathic or ischaemic). In the multivariate analysis, the only factors significantly associated with positive MDRO status on admission were a history of previous hospitalization for the same wound (21/32 compared with 48/148; P = 0.0008) or the presence of osteomyelitis (22/32 compared with 71/148; P = 0.025). In the longitudinal study of 75 wounds, MDRO‐positive status on admission or during follow‐up (6 months at least or until healing, mean 9 ± 7 months) was not associated with time to healing (P = 0.71). Conclusion MDROs are often present in severe diabetic foot wounds. About one‐third of patients with a history of previous hospitalization for the same wound, and 25% of patients with osteomyelitis, had MDRO‐positive specimens. This suggests that hygiene measures, or isolation precautions in the case of admission of patients presenting with these characteristics, should be aggressively implemented to prevent cross‐transmission. Positive MDRO status is not associated with a longer time to healing.     

Effects of a synthetic retinoid on skin structure, matrix metalloproteinases, and procollagen in healthy and high-risk subjects with diabetes

Background

In diabetes, foot ulceration may result from increased skin fragility. Retinoids can reverse some diabetes-induced deficits of skin structure and function, but their clinical utility is limited by skin irritation. The effects of diabetes and MDI 301, a nonirritating synthetic retinoid, and retinoic acid have been evaluated on matrix metalloproteinases (MMPs), procollagen expression, and skin structure in skin biopsies from nondiabetic volunteers and diabetic subjects at risk of foot ulceration using organ culture techniques.

Methods

Zymography and enzyme-linked immunosorbent assay were utilized for analysis of MMP-1, -2, and -9 and tissue inhibitor of metalloproteinase-1 (TIMP-1) and immunohistochemistry for type I procollagen protein abundance. Collagen structure parameters were assessed in formalin-fixed, paraffin-embedded tissue sections.

Results

The % of active MMP-1 and -9 was higher and TIMP-1 abundance was lower in subjects with diabetes. Type 1 procollagen abundance was reduced and skin structural deficits were increased in diabetes. Three μM MDI 301 reduced active MMP-1 and -9 abundance by 29% (P<.05) and 40% (P<.05), respectively, and increased TIMP-1 by 45% (P=.07). MDI 301 increased type 1 procollagen abundance by 40% (P<.01) and completely corrected structural deficit scores. Two μM retinoic acid reduced MMP-1 but did not significantly affect skin structure.

Conclusions

These data indicate that diabetic patients at risk of foot ulceration have deficits of skin structure and function. MDI 301 offers potential for repairing this skin damage complicating diabetes.     

Complexity of factors related to outcome of neuropathic and neuroischaemic/ischaemic diabetic foot ulcers: a cohort study

Aims/hypothesis  We sought to identify factors related to short-term outcome of foot ulcers in patients with diabetes treated in a multidisciplinary system until healing was achieved. Methods  Consecutively presenting patients with diabetes and worst foot ulcer (Wagner grade 1–5, below ankle) (n = 2,511) were prospectively followed and treated according to a standardised protocol until healing was achieved or until death. The number of patients lost to dropout was 31. The characteristics of the remaining 2,480 patients were: 1,465 men, age 68 ± 15 years (range 18–96), type 1 diabetes 18%, type 2 diabetes 82% and insulin-treated 62%. Results  The healing rate without major amputation in surviving patients was 90.6% (n = 1,867). Sixty-five per cent (n = 1,617) were healed primarily, 9% (n = 250) after minor amputation and 8% after major amputation; 17% (n = 420) died unhealed. Out of 2,060 surviving patients, 1,007 were neuroischaemic (48.8%). In a multiple regression analysis, primary healing was related to co-morbidity, duration of diabetes, extent of peripheral vascular disease and type of ulcer. In neuropathic ulcers, deep foot infection, site of ulcer and co-morbidity were related to amputation. Amputation in neuroischaemic ulcers was related to co-morbidity, peripheral vascular disease and type of ulcer. Age, sex, duration of diabetes, neuropathy, deformity and duration of ulcer or site of ulcer did not have an evident influence on probability of amputation. Conclusions/interpretation  Patients with diabetic foot ulcer suffer from multi-organ disease. Factors related to outcome are correspondingly complex.     

Monocyte phenotype as a predictive marker for wound healing in diabetes-related foot ulcers

AimsDelayed healing of diabetes-related foot ulcers (DRFUs) is associated with increased macrophage and matrix metalloproteinases (MMPs) at the wound site. Whether circulating monocyte phenotype and/or MMPs are altered in association with wound healing outcome is unknown, and was investigated in this study.MethodsBlood was obtained from 21 participants with DRFU, at initial visit (V1), week-4 (V2), and week-8 (V3) for measurement of monocyte number (CD14⁺), phenotype (CD16, CD163) and chemokine receptors (CCRs) by flow cytometry, and circulating MMPs and TIMP-1 by ELISA.ResultsSix wounds healed during the study. At V1, non-classical CD16⁺⁺ monocytes and MMP-3 were higher in healed vs unhealed (both p < 0.05). At V3, the increased %CD16⁺⁺ persisted and %CCR2⁺ was decreased in healed, but no other monocyte markers nor MMP/TIMP differed between groups. Increased wound closure rate (WCR) at V3 correlated with increased %CD16⁺⁺ monocytes and decreased MMP-2 at V1 or V1 + V2. Receiver operating characteristic (ROC) curves yielded an area-under-the-curve of %CD16⁺⁺ at V1 of 0.78 to predict ulcer healing at V3.ConclusionsThese results indicate that circulating monocyte phenotype and MMPs alter as DRFUs heal. The relationship of %CD16⁺⁺ monocytes with WCR and ROC curve suggest a predictive role of %CD16⁺⁺ monocytes for ulcer healing.     

MMP-9 in atrial remodeling in patients with atrial fibrillation

IntroductionAtrial fibrillation (AF) is the most common arrhythmia and is associated with significant morbidity and mortality. The impact of matrix metalloproteinases (MMPs) on structural atrial remodeling and sustainment of AF in patients with persistent and permanent AF is unresolved.ObjectivesThe aim was to evaluate MMP-9 and its tissue inhibitor-1 (TIMP-1) as markers of atrial remodeling in patients with persistent AF (PAF) who underwent electrical cardioversion (ECV) and in patients with permanent AF (continuous AF, CAF).Patients and methodsPlasma levels of MMP-9 and TIMP-1, clinical findings, and echocardiographic parameters were evaluated in 39 patients with AF and in 14 controls with sinus rhythm.ResultsThe concentrations of MMP-9 were significantly higher in patients with PAF and CAF compared to controls. There was a significant increase of MMP-9 after ECV in the persistent AF group. The values of TIMP-1 were not significantly different between the groups. In patients with AF, MMP-9 levels were positively related to posterior wall thickness of the LV (r = 0.356, P = 0.049) and body mass index (r = 0.367, P = 0.046).ConclusionElevated levels of MMP-9 were related to the occurrence and maintenance of AF. This suggests that MMP-9 can be a marker of atrial remodeling in patients with AF. Regulation of the extracellular collagen matrix might be a potential therapeutic target in AF.     

Liver fibrosis improvement in chronic hepatitis C after direct acting-antivirals is accompanied by reduced profibrogenic biomarkers–a role for MMP-9/TIMP-1

Background and aimsDisturbances in matrix metalloproteinases (MMPs) and corresponding tissue inhibitors (TIMPs) contribute to hepatitis C virus (HCV)-induced fibrosis. This study aimed to determine MMP-9/TIMP-1 levels in addition to MMP-2 and -9 activities; correlating with the improvement of liver fibrosis in patients under direct-acting antiviral (DAA) therapy.MethodsClinical and laboratory follow-up were performed before treatment and after 12 weeks post-treatment, referred as sustained viral response (SVR). We evaluated liver function including non-invasive fibrosis measurements; MMP activity by zymography; and MMP-9/TIMP-1 complex, inflammatory and pro-fibrogenic mediators by immunoenzymatic assays.ResultsCohort included 33 patients (59.5 ± 9.3 years, 60.6% females) whose reached SVR and 11 control-paired subjects (42.5 ± 15 years, 54.5% females). Before treatment, HCV patients presented higher MMP-9/TIMP-1 levels (P < 0.05) when compared to controls, and the highest values were observed in patients with fibrosis (P < 0.05). In addition, MMP-9/TIMP-1 levels were significantly reduced after DAA therapy (P < 0.0001) and were associated with profibrogenic biomarkers. No differences were observed for MMP-2 and -9 activities; however, these biomarkers were significantly associated with inflammatory mediators.ConclusionOur data suggest that MMP-9/TIMP-1 complex can be a promising biomarker of active fibrogenesis, being able to identify the interruption of fibrosis progression after HCV eradication.     

A case series to describe the clinical characteristics of foot ulceration in patients with rheumatoid arthritis

The aim of this study was to describe the clinical characteristics of foot ulceration in patients with rheumatoid arthritis (RA). Adults with RA and current foot ulceration but without diabetes were recruited. Clinical examination included assessment of RA disease activity, foot deformity, peripheral vascular disease, neuropathy and plantar pressures. Location, wound characteristics and time to healing were recorded for each ulcer. Participants completed the Health Assessment Questionnaire and Leeds Foot Impact Scale. Thirty-two cases with 52 current ulcers were recruited. Thirteen patients (41%) experienced more than one current ulcer: 5 (16%) had bilateral ulceration, 15 (47%) had previous ulceration at a current ulcer site. The majority (n = 33) of open ulcers were located over the dorsal aspect of the interphalangeal joints (n = 12), plantar aspect of the metatarsophalangeal joints (MTPJs) (n = 12) and medial aspect of first MTPJs (n = 9). In ulcerated limbs (n = 37), ankle brachial pressure index (ABPI) was <0.8 in 2 (5%); protective sensation was reduced in 25 (68%) and peak plantar pressures were >6 kg/cm² in 6 (16%). Mean ulcer size was 4.84 by 3.29 mm. Most ulcers (n = 42, 81%) were superficial; five (9.6%) were infected. Time to healing was available for 41 ulcers: mean duration was 28 weeks. Three ulcers remained open. In conclusion, foot ulceration in RA is recurrent and multiple ulcers are common. Whilst ulcers are small and shallow, time to achieve healing is slow, posing infection risk. Reduced protective sensation is common in affected patients. The prevalence of arterial disease is low but may be under estimated due to high intolerance of ABPI.     

Treatment of chronic diabetic foot ulcers with bemiparin: a randomized,triple‐blind,placebo‐controlled,clinical trial

Aims To assess the efficacy and safety of bemiparin in the treatment of chronic diabetic foot ulcers. Methods A triple‐blind, parallel, randomized, placebo‐controlled trial. Patients aged > 8 years, with diabetes for at least 3 years, and with a foot ulcer persisting for > 3 months were selected from 39 Spanish centres. Bemiparin 3500 IU/day for 10 days, followed by 2500 IU/day for up to 3 months plus standard care for ulcers, was compared with placebo plus standard care for ulcers for 3 months. The primary efficacy end‐point was ulcer improvement, defined as an objective decrease in ulcer area of ≥ 50%, measured by digital photography and ImageJ software, and/or any decrease in Wagner's ulcer grade at 3 months. Results Ulcer improvement rates were 70.3% (26 of 37 patients) in the bemiparin group and 45.5% (15 of 33 patients) in the placebo group [absolute difference 24.8; 95% confidence interval (CI) 2.3, 47.3; P = 0.035] (number needed to treat 4; 95% CI 2, 43). Complete healing rates at 3 months were similar in both groups (35.1% vs. 33.3%; P = 0.874), as were the number of adverse events. Conclusions Bemiparin is more effective than placebo in the management of diabetic foot ulcers and has few side‐effects.     

Exercise ameliorates serum MMP-9 and TIMP-2 levels in patients with type 2 diabetes

AimThis study assessed the impact of regular exercise on inflammatory markers (high-sensitivity C-reactive protein [hsCRP], fibrinogen), and matrix metalloproteinases (MMPs) and their inhibitors (TIMPs), in patients with type 2 diabetes mellitus (T2DM).PatientsFifty overweight patients with T2DM were randomly assigned to two groups: (A) an exercise group (EXG, n = 25), with self-controlled exercise for at least 150 min/week and one additional supervised exercise session/week; and (B) a control group (COG, n = 25), with no exercise instructions. All participants were taking oral antidiabetic drugs, and none had diabetic complications. Clinical parameters, exercise capacity (VO_2peak), ventilatory threshold (VT), insulinresistance indices (fasting insulin, HOMA-IR, HOMA%S), hsCRP, fibrinogen, MMP-2, MMP-9, TIMP-1 and TIMP-2 were assessed at baseline and after 16 weeks.ResultsNo significant changes were found in body mass index, waist/hip ratio, insulin-resistance indices, MMP-2 and TIMP-1 throughout the study in either group (P > 0.05). Compared with controls, the EXG showed a significant decrease in systolic and mean blood pressure, total and LDL cholesterol, and HbA_1c (P < 0.05). Also, exercise significantly suppressed levels of fibrinogen (P = 0.047), hsCRP (P = 0.041) and MMP-9 (P = 0.028), and the MMP-9-to-TIMP-1 ratio (P = 0.038), whereas VO_2peak (P = 0.011), VT (P = 0.008) and plasma TIMP-2 levels (P = 0.022) were considerably upregulated in the EXG vs. COG. Standard multiple-regression analyses revealed that MMP-9 changes were independently associated with fibrinogen and HbA_1c changes, while fibrinogen changes independently predicted TIMP-2 alterations with exercise.ConclusionMostly self-controlled exercise of moderate intensity ameliorated serum levels of pro- and anti-atherogenic markers in patients with T2DM, with no effects on body weight. These data offer further insight into the cardioprotective mechanisms of exercise in patients with T2DM.     

The role of nitric oxide synthase isoforms and arginase in the pathogenesis of diabetic foot ulcers: possible modulatory effects by transforming growth factor beta 1

Aims/hypothesis. l-arginine, an amino acid involved in wound healing, is metabolised by one of two pathways; nitric oxide synthase and arginase. If metabolised by nitric oxide synthase, this can result in tissue destruction, or matrix deposition if metabolised by arginase. The aim therefore was to investigate the role of these enzymes in the pathogenesis of diabetic foot ulcers. Methods. The activity, proteins by Western blot analysis and cellular distribution (using immunocytochemistry) of these enzymes were measured in diabetic foot ulcers, diabetic skin and normal skin. Results. Total and inducible nitric oxide synthase (p < 0.001) and endothelial nitric oxide synthase were increased in diabetic ulcers compared with diabetic and normal skin and were associated with increased plasma nitrite concentrations in diabetic ulcers (p < 0.05). Inducible nitric oxide synthase was the major isoform, with the macrophage being the predominant cellular source. Similarly arginase activity was increased (p < 0.01) in diabetic ulcers. The protein levels corroborated with the activity data, with the fibroblast being the major cellular source. The spatial and cellular distribution of the two enzyme systems was distinct. Transforming growth factor-beta1 was decreased in diabetic ulcers in comparison with diabetic skin and normal skin. Conclusion/interpretation. Increased nitric oxide synthase activity in diabetic foot ulcers may be responsible for the impaired healing in this disease. Furthermore, the increased activity of arginase could account for the characteristic callus formation around these ulcers. In addition, the lower concentrations of transforming growth factor-beta1 in diabetic ulcers may explain the raised concentrations of nitric oxide in this condition. [Diabetologia (1999) 42: 748–757] Received: 23 September 1998 and in revised form: 10 December 1998     

Evaluation of metalloproteinase 2 and 9 levels and their inhibitors in diabetic and healthy subjects

OBJECTIVES: We hypothesized that molecules active in vascular remodeling (i.e. MMPs and their TIMPs) could be modified in diabetic patients, as indirect markers of the diabetes related generalized abnormality of vascular activity. To test this hypothesis, we measured the plasma levels of MMP-2, MMP-9, TIMP-1, and TIMP-2 in type 2 diabetic patients and in healthy subjects. METHODS: We enrolled 181 diabetic patients and 165 controls. We measured body mass index (BMI), glycosylated hemoglobin (HbA(1c)), fasting plasma glucose (FPG), fasting plasma insulin (FPI), homeostasis model assessment index (HOMA index), systolic blood pressure (SBP), diastolic blood pressure (DBP), total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C), high density lipoprotein-cholesterol (HDL-C), triglycerides (Tg), lipoprotein(a) [Lp(a)], plasminogen activator inhibitor-1 (PAI-1), homocysteine (Hct) fibrinogen (Fg), high sensitivity C-reactive protein (hs-CRP), and plasma levels of MMP-2, MMP-9, TIMP-1, and TIMP-2. RESULTS: A significant increase (P<0.0001) of BMI, HbA(1c), FPG, FPI, HOMA index, SBP, DBP, TC, LDL-C, Tg, Lp(a), PAI-1, Hct, Fg, and hs-CRP was present in the diabetic group, with a significant decrease (P<0.0001) of HDL-C levels compared to healthy subjects. MMP-2 and MMP-9 levels were significantly higher (P<0.0001) in diabetic patients. Significant TIMP-1, and TIMP-2 increase was also observed (P<0.0001) in the diabetic group. CONCLUSION: Plasma levels of MMP-2, MMP-9, TIMP-1, and TIMP-2 are increased in diabetic patients which may reflect abnormal extracellular matrix (ECM) metabolism.