The inhibition of matrix metalloproteinase activity in chronic wounds by a polyacrylate superabsorber

Excessive matrix metalloproteinase (MMP) levels have been observed in wound fluid of impaired healing wounds. This is thought to interfere with granulation tissue formation as newly formed extracellular matrix and cytokines are degraded and the wound becomes deadlocked, unable to progress to the next healing stages. In the cleansing phase, associated with high MMP activity levels, hydroactive wound dressings containing polyacrylate superabsorber particles are particularly effective. We tested whether these particles can block MMP activity in wound fluid obtained from chronic venous leg ulcers. Polyacrylate superabsorber particles inhibited MMP activity by more than 87% in a fluorogenic peptide substrate assay. Further analysis revealed two underlying molecular mechanisms. First, experiments showed direct binding of MMPs to the particles. Secondly, polyacrylate superabsorber particles can bind Ca2+ and Zn2+ ions competing with MMPs for divalent ions required for enzymatic activity. Furthermore, we provide the first evidence in vivo that MMPs bind effectively to polyacrylate superabsorber particles within the hostile environment of chronic wounds. We conclude that polyacrylate superabsorber particles can rescue the highly proteolytic microenvironment of non-healing wounds from MMP activity so that more conductive conditions allow healing to proceed.     

A peptidomimetic inhibitor of matrix metalloproteinases containing a tetherable linker group

Successful wound repair and normal turnover of the extracellular matrix relies on a balance between matrix metalloproteinases (MMPs) and their natural tissue inhibitor of metalloproteinases (TIMPs). When overexpression of MMPs and abnormally high levels of activation or low expression of TIMPs are encountered, excessive degradation of connective tissue and the formation of chronic ulcers can occur. One strategy to rebalance MMPs and TIMPs is to use inhibitors. We have designed a synthetic pseudopeptide inhibitor with an amine linker group based on a known high-affinity peptidomimetic MMP inhibitor and have demonstrated inhibition of MMP-1, -2, -3, and -9 activity in standard solutions. The inhibitor was also tethered to a polyethylene glycol hydrogel using a facile reaction between the linker unit on the inhibitor and the hydrogel precursors. After tethering, we observed inhibition of the MMPs although there was an increase in the IC??s that was attributed to poor diffusion of the MMPs into the hydrogels, reduced activity of the tethered inhibitor, or incomplete incorporation of the inhibitor into the hydrogels. When the tethered inhibitors were tested against chronic wound fluid, we observed partial inhibition in proteolytic activity suggesting this approach may prove useful in rebalancing MMPs within chronic wounds.     

Matrix metalloproteinases in wound repair (review)

Wound repair is initiated with the aggregation of platelets, formation of a fibrin clot, and release of growth factors from the activated coagulation pathways, injured cells, platelets, and extracellular matrix (ECM), followed by migration of inflammatory cells to the wound site. Thereafter, keratinocytes migrate over the wound, angiogenesis is initiated, and fibroblasts deposit and remodel the granulation tissue. Cell migration, angiogenesis, degradation of provisional matrix, and remodeling of newly formed granulation tissue, all require controlled degradation of the ECM. Disturbance in the balance between ECM production and degradation leads to formation of chronic ulcers with excessive ECM degradation, or to fibrosis, for example hypertrophic scars or keloids characterized by excessive accumulation of ECM components. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases, which as a group can degrade essentially all ECM components. So far, 20 members of the human MMP family have been identified. Based on their structure and substrate specificity, they can be divided into subgroups of collagenases, stromelysins, stromelysin-like MMPs, gelatinases, membrane-type MMPs (MT-MMPs), and other MMPs. In this review, the role of MMPs in normal wound repair as well as in chronic ulcers is discussed. In addition, the role of signaling pathways, in particular, mitogen-activated protein kinases (MAPKs) in regulating MMP expression is discussed as possible therapeutical targets for wound healing disorders.     

Ectopic localization of matrix metalloproteinase-9 in chronic cutaneous wounds

It has been hypothesized that excessive activity of matrix metalloproteinases (MMPs), in particular the gelatinases MMP-9 and MMP-2, contributes to poor healing of chronic skin ulcers. We compared MMP-9 and MMP-2 in wound margin biopsies of standardized acute partial-thickness wounds in healthy volunteers (n = 6) and in venous leg ulcer patients (n = 12) with those of chronic wounds of different etiologies (n = 34) by a combination of specific analyses of activity and protein localization. We also studied MMP-14 by immunohistochemistry and in situ hybridization in parallel. Neither MMP-9 (P =.814) nor MMP-2 (P =.742) endogenous activities differed significantly between acute and chronic wound tissues. Acute wound healing was characterized by induction of MMP-9 in the advancing epithelium. In chronic wounds, prominent MMP-9 immunostaining was seen in neutrophils and macrophages in the ulcer bed, but virtually no MMP-9 was detected in wound edge keratinocytes. MMP-2 was increased and activated with acute wound age. MMP-2 was found abundantly in dermal fibroblasts and endothelial cells beneath, but not in new epithelium of acute and chronic wounds. MMP-14 mRNA or protein was detected solely in the stroma of both acute and chronic wounds. In conclusion, the overall activity of gelatinases MMP-9 and MMP-2 was not increased in chronic wounds compared to normally healing wound tissues. Chronic nonhealing wounds may not be caused by excessive gelatinase activity, but are distinguished from healing wounds by an unfavorable distribution and persistance of MMP-9.     

Development of fungal mycelia as a skin substitute: characterization of keratinocyte proliferation and matrix metalloproteinase expression during improvement in the wound-healing process

SACCHACHITIN membranes, prepared from the waste residue of the fruiting body of Ganoderma taugae, were used in our previous study to enhance skin wound healing in animal models. In the present study, the effects of the membrane on the growth of keratinocytes and the activity of matrix metalloproteinases (MMPs), as well as on the healing of skin wounds in humans, were estimated. Fresh human foreskin was employed as the source of the keratinocyte culture, and a modified keratinocyte-SFM medium supplemented with 0.2 ng/mL of recombinant epidermal growth factor and 30 microg/mL bovine pituitary extract was used to enhance the successful growth of keratinocytes under an atmosphere of 5% CO2, at 37 degrees C. The results indicated that 0.01% SACCHACHITIN enhanced the proliferation of keratinocytes in the culture on the fourth and fifth days, and cells showed neither morphological alteration nor disordered proliferation. This evidence clearly indicated that SACCHACHITIN was not cytotoxic to and was safe for the growth of keratinocytes. Thus, SACCHACHITIN might play a positive role in the proliferation and differentiation of keratinocytes around wounds and in accelerated wound healing of epidermal tissue. In addition, microscopic observations during the growth of keratinocytes showed that normal proliferation and differentiation took place along the margin of the SACCHACHITIN membrane. This indicates that SACCHACHITIN is possibly cytocompatible with keratinocytes. Electrophoretic analysis and inhibition tests for the binding effect of SACCHACHITIN on MMPs showed that SACCHACHITIN reduced MMPs in extracellular matrix degradation and facilitated establishment of an extracellular matrix around wounds; these effects resulted in rapid wound healing. SACCHACHITIN was used as a skin dressing for patients who had skin chronicle ulcer, which had not healed for over 7 months. Preliminary clinical observations showed that the wound improved and began to heal. An analysis of MMPs by ELISA in tissue of the wound indicated a significant decrease in MMP levels.     

Matrix metalloproteinases and atherosclerosis. Therapeutic aspects

Matrix metalloproteinases (MMPs) play a key role in the physiology of connective tissue development, morphogenesis and wound healing, but their unregulated activity has been implicated in numerous disease processes including arthritis, tumor cell metastasis and atherosclerosis. MMP family consists of at least 20 members; MMPs are produced by the different cell types (vascular smooth muscle cells, monocytes, endothelial cells) involved in the atheromatous plaque formation and participate to extracellular matrix remodelling and cell infiltration or migration. Since excessive tissue remodelling and increased matrix metalloproteinase activity have been demonstrated during atherosclerotic lesion progression (including plaque disruption), MMPs represent a potential target for therapeutic intervention to modify vascular pathology, by restoring the MMP/TIMP physiological equilibrium. This review highlights the structures of MMPs and their physiological inhibitors, the Tissue Inhibitors of MMPs (TIMPs), and describes the current developments in pharmacological MMP inhibition.     

Local blockage of EMMPRIN impedes pressure ulcers healing in a rat model

Excessive extracellular matrix degradation caused by the hyperfunction of matrix metalloproteinases (MMPs) has been implicated in the failure of pressure ulcers healing. EMMPRIN, as a widely expressed protein, has emerged as an important regulator of MMP activity. We hypothesize that EMMPRIN affects the process of pressure ulcer healing by modulating MMP activity. In the rat pressure ulcer model, the expression of EMMPRIN in ulcers detected by Western blot was elevated compared with that observed in normal tissue. To investigate the role of EMMPRIN in regulating ulcer healing, specific antibodies against EMMPRIN were used via direct administration on the pressure ulcer. Local blockage of EMMPRIN resulted in a poor ulcer healing process compared with control ulcers, which was the opposite of our expectation. Furthermore, inhibiting EMMPRIN minimally impacted MMP activity. However, the collagen content in the pressure ulcer was reduced in the EMMPRIN treated group. Angiogenesis and the expression of angiogenic factors in pressure ulcers were also reduced by EMMPRIN local blockage. The results in the present study indicate a novel effect of EMMPRIN in the regulation of pressure ulcer healing by controlling the collagen contents and angiogenesis rather than MMPs activity.     

Differential expression of extracellular matrix metalloproteinase inducer (CD147) in normal and ulcerated corneas: role in epithelio-stromal interactions and matrix metalloproteinase induction

Extracellular matrix metalloproteinase inducer (EMMPRIN) was originally identified on the tumor cell surface as an inducer of matrix metalloproteinase (MMP) production in neighboring fibroblasts. Here we demonstrate a role for EMMPRIN in MMP induction during corneal wound healing. MMP and EMMPRIN expression was analyzed in normal and ulcerated human corneas, as well as in corneal epithelial and stromal cells in culture using confocal microscopy, zymography, immunoblots, and real-time polymerase chain reaction. In normal cornea EMMPRIN was predominantly expressed in the epithelium but was markedly induced in the anterior stroma of ulcerated corneas. This coincided with MMP-2 induction that co-localized with EMMPRIN at the epithelio-stromal boundary. The role of epithelial-stromal interaction in MMP induction was investigated in an in vitro co-culture system and demonstrated an induction and co-localization of EMMPRIN and MMP-2 in the fibroblasts at the interface with epithelial cells. Direct contact of fibroblasts with EMMPRIN-containing purified epithelial cell membranes also induced MMP-1, MMP-2, and EMMPRIN and this was inhibited by a blocking anti-EMMPRIN antibody, suggesting that EMMPRIN was primarily responsible for this induction. These findings, and the up-regulation of EMMPRIN by epidermal growth factor and transforming growth factor-beta, demonstrate a role for EMMPRIN in wound healing and suggest that sustained local up-regulation of EMMPRIN and MMPs in chronic situations in which healing is delayed may lead to excessive matrix degradation and corneal melts.     

The effect of a hydroxamic acid-containing polymer on active matrix metalloproteinases

Matrix metalloproteinase (MMP) sequestering polymer microspheres were prepared by a post-polymerization hydroxamic acid derivatization of poly(methyl methacrylate-co-methacrylic acid). The microspheres were designed to selectively bind MMPs from solutions on contact through a direct interaction between the polymer-bound hydroxamic acid groups and the characteristic catalytic site zinc atom common to all MMPs. MMP activity assays showed that the hydroxamic acid microspheres reduce MMP activity on contact in a time and concentration-dependent fashion. This effect was observed for several MMP subclasses (MMP-2, -3, -8 and -13) suggesting that the microspheres possess a broad-spectrum MMP binding capacity. However, inactive pro-forms of MMPs showed little binding affinity for the microspheres indicating that the interaction was dependent on MMP activation. The preferential binding of active MMPs was confirmed by MMP-3 and MMP-8 activation studies, which demonstrated significant increases in microsphere binding on activation. The MMP sequestering effect of the microspheres was also demonstrated in a physiologically relevant solution (human chronic wound fluid extract) indicating that the binding interaction was effective in a multi-component, competitive adsorption environment. Thus, the hydroxamic acid-containing microspheres may find use as localized, broad-spectrum MMP inhibitors for the treatment of a number of disease conditions characterized by elevated MMP activity.     

Tissue inhibitor of metalloproteinase 1 regulates matrix metalloproteinase activity during newt limb regeneration.

Matrix metalloproteinase (MMP) activity is important for newt limb regeneration. In most biological processes that require MMP function, MMP activity is tightly controlled by a variety of mechanisms, including the coexpression of natural inhibitors. Here, we show that gene expression of one such inhibitor, tissue inhibitor of metalloproteinase 1 (NvTIMP1), is upregulated during the wound healing and dedifferentiation stages of regeneration when several MMPs are at their maximal expression levels. Newt MMPs and NvTIMP1 also exhibit similar spatial expression patterns during the early stages of limb regeneration. NvTIMP1 inhibits the proteolytic activity of regeneration-related newt MMPs and, like human TIMP1, can induce a weak mitogenic response in certain cell types. These results suggest that NvTIMP1 may be functioning primarily to maintain optimal levels of MMP activity during the early stages of limb regeneration, while possibly serving a secondary role as a mitogen.     

Calprotectin inhibits matrix metalloproteinases by sequestration of zinc.

BACKGROUND/AIMS: Calprotectin, a 36 kDa protein present in neutrophil cytoplasm, has antimicrobial and apoptosis inducing activities, which are reversed by the addition of zinc. Matrix metalloproteinases (MMPs), a family of zinc dependent enzymes, are important in many normal biological processes including embryonic development, angiogenesis, and wound healing, but also pathological processes such as inflammation, cancer, and tissue destruction. The aim of this study was to investigate whether calprotectin can inhibit MMP activity, and whether such inhibition could be overcome by the addition of zinc. METHODS: MMP activity was measured by the degradation of substrates precoated on to microwells, and visualised by Coomassie blue staining of residual substrate. Seven metalloproteinases (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, and MMP-13) were tested against two substrates: gelatin and alpha-casein. RESULTS: All MMPs except MMP-1 were active against gelatin, whereas MMP-7 was the only enzyme active against alpha-casein. The addition of calprotectin inhibited the activity of all the MMPs, but different concentrations of the protein, from 0.3 microM to > 11microM, were necessary to produce a 50% inhibition of the MMPs. Inhibition by calprotectin was largely overcome by the addition of zinc. CONCLUSIONS: The findings suggest that calprotectin inhibits MMPs by sequestration of zinc. The data also suggest that MMPs have different affinities for zinc and that calprotectin has a lower zinc affinity than the MMPs.     

TGF-beta antisense oligonucleotides reduce mRNA expression of matrix metalloproteinases in cultured wound-healing-related cells

The pathology of chronic dermal ulcers is characterized by excessive proteolytic activity which degrades extracellular matrix. The transforming growth factor-beta (TGF-beta) has been identified as an important component of wound healing. Recent developments in molecular therapy offer exciting prospects for the modulation of wound healing, specifically those targeting TGF-beta. We investigated the effect of TGF-beta antisense oligonucleotides on the mRNA expression of matrix metalloproteinases in cultured human keratinocytes, fibroblasts and endothelial cells using multiplex RT-PCR. The treatment of keratinocytes and fibroblasts with TGF-beta antisense oligonucleotides resulted in a significant decrease of expression of mRNA of MMP-1 and MMP-9 compared to controls. Accordingly, a decreased expression of MMP-1 mRNA in endothelial cells was detectable. Other MMPs were not affected. Affecting all dermal wound-healing-related cell types, TGF-beta antisense oligonucleotide technology may be a potential therapeutic option for the inhibition of proteolytic tissue destruction in chronic wounds. Pharmaceutical intervention in this area ultimately may help clinicians to proactively intervene in an effort to prevent normal wounds from becoming chronic.     

Matrix metalloproteinase inhibition negatively affects muscle stem cell behavior

Skeletal muscle is a large and complex system that is crucial for structural support, movement and function. When injured, the repair of skeletal muscle undergoes three phases: inflammation and degeneration, regeneration and fibrosis formation in severe injuries. During fibrosis formation, muscle healing is impaired because of the accumulation of excess collagen. A group of zinc-dependent endopeptidases that have been found to aid in the repair of skeletal muscle are matrix metalloproteinases (MMPs). MMPs are able to assist in tissue remodeling through the regulation of extracellular matrix (ECM) components, as well as contributing to cell migration, proliferation, differentiation and angiogenesis. In the present study, the effect of GM6001, a broad-spectrum MMP inhibitor, on muscle-derived stem cells (MDSCs) is investigated. We find that MMP inhibition negatively impacts skeletal muscle healing by impairing MDSCs in migratory and multiple differentiation abilities. These results indicate that MMP signaling plays an essential role in the wound healing of muscle tissue because their inhibition is detrimental to stem cells residing in skeletal muscle.     

实时荧光PCR法定量小腿慢性静脉性溃疡表面的细菌分布

背景：小腿慢性静脉性溃疡表面的细菌在溃疡的愈合过程中起重要作用,但细菌的种类、密度与溃疡愈合的关系仍然存在争议。 目的：探讨小腿慢性静脉性溃疡中的常见菌群种属和细菌量与溃疡愈合的关系。 方法：纳入2009-05/2010-09重庆医科大学第一附属医院血管外科诊断为小腿慢性静脉性溃疡患者39例,共42条患肢。所有患者均进行标准的加压疗法治疗,经过6个月的随访以确定溃疡的愈合率,42条患肢中,28条患肢在6个月内完全愈合,为愈合组,14条患肢未完全愈合,为未愈合组。获取治疗前所有溃疡的组织样本,提取样本中的细菌基因组DNA,合成细菌的通用引物及下肢慢性静脉性溃疡中常见细菌种属的特异性引物,运用常规PCR及荧光定量PCR方法比较愈合组与未愈合组溃疡表面的细菌差异。 结果与结论：愈合组和未愈合组标本中都可检测到细菌,最常见的细菌为金黄色葡萄球菌和铜绿假单胞杆菌。荧光定量PCR结果显示未愈合组标本中的细菌总量较愈合组明显增多(P < 0.05),两组中金黄色葡萄球菌、铜绿假单胞杆菌的含量无明显差异(P  > 0.05)。实验结果中未见任何一种溃疡的常见细菌种属与溃疡的愈合具有直接关系,而溃疡中的细菌总量与溃疡的愈合有较大的关联。结果表明溃疡表面细菌总量的增加引起的皮肤慢性感染可能是导致小腿慢性静脉性溃疡长期不愈合的重要原因。     

Fibronectin profiles in normal and chronic wound fluid

Fibronectin, an adhesion protein found in blood and tissues, is an important factor in wound repair. Recently, we began studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting to compare fibronectin in the fluid of normal and chronic wounds. We found similar fibronectin profiles in suction blister fluid (2 normal volunteers) and mastectomy fluid (5 patients). There was primarily an intact 250 kilodalton fibronectin subunit and in addition some higher molecular mass fibronectin-containing complexes. On the other hand, fibronectin in diabetic ulcer wound fluid (2 patients) was partially degraded, and there was no intact fibronectin in stasis ulcer wound fluid (2 patients). The most prominent degradation products were 93 and 125 kilodalton polypeptides. When intact fibronectin was added back to stasis ulcer wound fluid, fragmentation occurred within 15 minutes at 37 degrees C indicating the presence of protease activity in the wound fluid.     

伤口组织与电刺激调控的相关性研究

慢性伤口迁延不愈。外源性电刺激提供促进慢性伤口愈合的电流,其独特优势是使电荷调控伤口,促进伤 口愈合,具有良好的应用前景,并成为目前组织学研究者和临床医生的研究热点之一。现介绍一些常见慢性伤口 的形态学和电生理特性,主要探讨目前电荷调控伤口愈合的形态学改变、可能作用机制及其影响因素。     

Ras-transfection up-regulated HaCaT cell migration: Inhibition by Marimastat

Cell migration is an essential process in physiological and pathological conditions such as wound healing and tumor invasion. This phenomenon involves cell adhesion on the extracellular matrix mediated by integrins, and cell detachment promoted in part by metalloproteinases (MMPs). In the present study, the migration of two HaCaT-ras clones (metastatic or not), was compared with HaCaT cells, and normal human primary cultured keratinocytes. Using colloidal gold migration assay, the migration index on type I and type IV collagen was similar for primary cultured keratinocytes and HaCaT, whereas it was markedly higher for the HaCaT-ras clones. High motility of ras-transfected cells was confirmed from an in vitro wound healing assay. It was not correlated with changes in integrin expression or related to a different adhesion on extracellular matrix. The Marismastat (BB-2516), a MMP inhibitor, inhibited in a dose-dependent effect the migration in both assays, demonstrating the important role of MMPs in the migration process. Under our experimental conditions, MMP-1 activity was not detected in HaCaT and MMP-9 activity was secreted by these cells only after their stimulation by EGF. Here, MMP-2 was the major gelatinolytic activity secreted by all the cells and its secretion was markedly higher for HaCaT-ras clones compared with HaCaT. In addition, Western blotting results confirmed a higher expression of MMP-2 associated with a lower expression of TIMP-2 in HaCaT-ras compared with HaCaT. These results suggest that Ha-ras oncogene could be a stimulating factor of migration and might modified the balance between MMP-2 and TIMP-2 in keratinocyte cell lines. This revised version was published online in July 2006 with corrections to the Cover Date.     

Role of matrix metalloproteinases in models of macrophage-dependent acute lung injury. Evidence for alveolar macrophage as source of proteinases.

Matrix metalloproteinases (MMPs) have been implicated in the tissue injury seen in neutrophil-dependent models of acute lung injury. However, the role of MMPs in macrophage-dependent models of lung injury is unknown. To address this issue, the macrophage-dependent immunoglobulin A immune complex-induced lung injury model and the macrophage-dependent portion of the lipopolysaccharide-induced acute lung injury model in the rat were assessed for MMP involvement and for the source of these activities. In both models, injury was inhibited by the recombinant human tissue inhibitor of metalloproteinases-2. Bronchoalveolar lavage fluids (BALFs) from injured animals in both models showed increased levels of MMPs. Characterization of MMP production by isolated lung fibroblasts, endothelial cells, type II epithelial cells, and alveolar macrophages revealed that only the macrophage had the same spectrum of MMP activity as seen in the BALF. Further, isolated alveolar macrophages from injured lungs showed evidence of in vivo activation with the release of the same spectrum of MMP activities. Together these studies show that MMPs are produced during macrophage-dependent lung injury, that these MMPs play a role in the development of the lung injury, and that the alveolar macrophage is the likely source of these MMPs.     

Cancer is a functional repair tissue

When a wound occurs, growth and repair genes (GR genes, such as oncogenes, proto-oncogenes, etc.) in surrounding cells are activated and secretion of growth and repair factors (GR factors, such as growth, stem cell, and stimulating factors, etc.) is induced to heal the wound. However, if the wound is persistent due to chronic physical (radiation, electromagnetic field, trauma, particles, etc.), chemical (carcinogens, toxic chemicals, heavy metals etc.) or biological (aging, free radicals, inflammation, nutrient deficiency, bacteria and virus infections, stress, etc.) damage, amplification of GR gene activation in surrounding cells may lead to a clinical cancer. Based on the commonalities between cancer and wound healing, a new hypothesis of cancer is presented: malignancies are not passive mutated useless masses; rather, they are functional tissues produced by GR gene activation to secrete GR factors in an effort to heal persistent wounds in the body. Based on the hypothesis, current cancer treatments aimed at killing cancer cells only may be misguided. The logical extension of the hypothesis is that cancer treatment focused on wound healing by limiting causes of persistent wounds, providing repair cells, GR factors, and substrates required by repair cells may yield more fruitful results than treatments focused on killing cancer cells alone. Spontaneous regressions of cancer, although rare, may be successful examples of serendipitous spontaneous wound healing. Standard therapies aimed at killing cancer cells, should be limited to adjuvant status for limiting symptoms or buying time for completion of the wound healing process. Attempts to destroy cancer cells without healing underlying persistent wounds will allow for eventual recurrence.