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.
KeywordsDiabetic foot ulcer Tissue inhibitor of metalloproteinase 3 (TIMP3) Vascular insufficiency
The diabetic foot ulcer is a common complication of diabetes mellitus due to its high frequency and difficulty of complete healing. Peripheral neuropathy, vascular insufficiency, and diminished immune response are major factors in the development of skin ulceration and infection. With the increasing incidence of diabetes, management of diabetic foot ulcers remains a growing challenging task for health care as well as for vascular services [1, 2].
Elevated levels of matrix metalloproteinases (MMPs) and reduced levels of their endogenous tissue inhibitors (TIMPs) have been shown in chronic wounds, including diabetic foot lesions, and can result in excessive proteolysis of tissue, as well as of growth factors and their receptors [3, 4].
Tissue inhibitor of metalloproteinase 3 (TIMP3) is a natural inhibitor of MMPs, such as MMP2, MMP9, and A Disintegrin and A Metalloprotease Domain 17 (ADAM17) . The TIMP3/ADAM17 dyad has been shown to exert different biological properties independent of MMP inhibitory activity, including inhibition of angiogenesis, induction of apoptosis, and regulation of inflammatory signals [6, 7]. TIMP3 and ADAM17 have been involved in the pathogenesis of diabetes and its complications in different models and tissues including peripheral atherosclerotic disease and diabetic microangiopathy nephropathy in humans and experimental models [8, 9, 10, 11, 12, 13, 14]; here, we assessed the role of TIMP3 and its targets in neuropathic and ischemic diabetic foot ulcers by analyzing biopsy samples from patients with type 2 diabetes mellitus.
Patients and biopsies
General patient characteristics at enrollment
Ischemic ulcers (n = 21)
Neuropathic ulcers (n = 14)
Age (year, aa)
68.3 ± 12.0
67.8 ± 8.8
Sex (male, %)
Total cholesterol (mg/dl)
181.9 ± 52.1
166.3 ± 48.1
HDL cholesterol (mg/dl)
44.9 ± 11.1
44.2 ± 10.8
LDL cholesterol (mg/dl)
109.0 ± 46.6
96.3 ± 37.5
165.8 ± 89.6
140.2 ± 49.7
Diabetes duration (aa)
24.7 ± 9.2
17.6 ± 9.9
7.5 ± 1.2
7.7 ± 1.2
MMP-2 (gelatinase A) and MMP-9 (gelatinase B) were measured in biopsy homogenates by quantitative gelatin zymography as previously described .
Real-time quantitative RT-PCR analysis
Total RNA and single-strand cDNA were obtained as described . Real-time PCR RNA expression analysis was performed with ABI PRISM 7000 System (Applied Biosystems, Foster City, CA) and normalized to 18S rRNA. Each reaction was carried out in duplicate and analysis performed by 2ΔΔCt method as described previously .
A Disintegrin and A Metalloprotease Domain 17/TNF-Alpha Converting Enzyme (ADAM17/TACE) activity
Proteins were extracted as previously described . TACE activity was determined by the SensoLyte 520 TACE Activity Assay Fluorimetric Kit (AnaSpec, San Jose, CA), based on a peptide sensitive to cleavage mostly by ADAM17/TACE and in minor part by other ADAMs metalloprotease, according to the manufacturer's instructions.
Results were expressed as mean ± SD, and a Student’s t test was used for statistical analyses. A p value <0.05 was considered statistically significant. All analyses were performed using GraphPad Prism 5.0.
To focus on disease-specific factors in patients with diabetes, we examined whether foot ischemia or neuropathy promotes different activation in MMP enzymes in subjects with diabetic foot ulcer. Clinical characteristics are reported in Table 1.
To evaluate the mechanism of increased MMP9 activity in ischemic samples, we performed RT-qPCR analysis from diabetic foot ulcer biopsies. Obtained results revealed that, among metalloproteinases such as ADAM17/TACE, ADAM15, MMP9, and their inhibitors, such as TIMP1, TIMP2, TIMP3, and TIMP4, only TIMP3 expression was lower in ischemic compared to neuropathic samples (p < 0.05) (Fig. 1c), suggesting that in a context of a vascular insufficiency, a reduction in TIMP3 expression may reflect the increase in MMP9 and ADAM17/TACE activities, contributing to skin vulnerability and refractory foot ulcers.
Management of diabetic foot ulcers remains a rather challenging task. Enhancement of the inflammatory response and decreased function of vascular endothelium play a crucial role in the mechanism of severe ischemia of the lower extremities. Moreover, increased inflammation and expression of MMPs and serum growth factors are the main factors associated with failure to heal diabetic foot ulcers, and this represents a major interest in the treatment for diabetic ulceration [3, 15]. In the current study, we examined whether ischemia or neuropathy promote a different activation of MMPs in diabetic foot ulcer analyzing skin biopsies taken directly from the diabetic foot. Results indicate that MMP9 activity was higher and TIMP3 abundance was lower in ischemic samples. The reduction in TIMP3 expression may reflect the persistence of increased concentrations of MMP9 during the wound healing process contributing to the failure of the acute wound to heal.
Reduction in TIMP3 expression directly reflects the modulation of other multiple systems such as ADAM17/TACE, that in turn, through the ectodomain shedding of various transmembrane proteins, including EGFR, cytokine, adhesion molecules, may impact on crucial signaling for inflammation, development, and regeneration. TIMP3 is also implicated in the control of VEGF/VEGFR2 interaction therefore involved in the modulation of the angiogenesis process and capillary network stabilization. These data suggest that the TIMP3 reduction specifically in ischemic ulcers may represent a causative factor in the ulcer progression and a target in the treatment for chronic wounds. Given the availability of animal models overexpressing TIMP3 from inflammatory cells that reconvene at sites of chronic inflammation [16, 17], it would be possible to explore the therapeutic value of TIMP3 in ischemic peripheral artery disease.
This manuscript was funded by JDRF RRG 1-2007-665, Fondazione Roma Sostegno alla Ricerca Biomedica 2008, Telethon GGP08065 2008, PRIN 2009FATXW3_002 and Ministero della Salute, Ricerca Finalizzata 2007 and 2008 (all to MF).
Conflict of interest
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