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.     

Attenuation of protease activity in chronic wound fluid with bisphosphonate-functionalised hydrogels

Chronic ulcers are an important and costly medical issue, imposing considerable pain, reduced mobility and decreased quality of life. The common pathology in these chronic wounds is excessive proteolytic activity, resulting in degradation of key factors critical to the ulcer's ability to heal. Matrix metalloproteinases (MMPs), a large family of zinc-dependent endopeptidases, have been shown to have increased activity in chronic wound fluid (CWF), with many authors suggesting that they need to be inhibited for the ulcer to heal. The studies we report here show that the excessive MMP activity in CWF can be inhibited with the bisphosphonate alendronate, in the form of a sodium salt, a functionalised analogue, and tethered to a poly(2-hydroxy methacrylate) (PHEMA) hydrogel. Furthermore, these functionalised alendronate hydrogels appear to be biologically inert as assessed in a three-dimensional ex vivo human skin equivalent model. Together, these results highlight the potential use of a tethered MMP inhibitor to inhibit protease activity in wound fluid. This approach may improve wound healing as it still allows MMPs to remain active in the upper cellular layers of the ulcer bed where they perform vital roles in wound healing; thus may offer an attractive new device-orientated wound therapy.     

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.     

Androgens influence expression of matrix proteins and proteolytic factors during cutaneous wound healing

Excessive proteolytic activity is a feature of chronic wounds such as venous ulcers, in which resolution of the inflammatory response fails and restorative matrix accumulation is delayed as a consequence. The inflammatory actions of native androgens during the healing of acute skin wounds have lately been characterized. We have now investigated the hypothesis that such activities may impact upon the balance between anabolic and catabolic processes during wound healing. We report that wound deposition of both type I collagen and fibronectin is increased in castrated rats compared with control animals. This response is accompanied by early increases and later decreases in overall wound levels of the key collagenolytic enzymes, matrix metalloproteinase (MMP)-1 and MMP-13. Moreover, the activities of MMP-2 and MMP-9, two further enzymes that contribute to collagen digestion during venous ulceration, were significantly decreased in the wounds of castrated rats. Additional analyses provide evidence that androgens directly stimulate dermal fibroblast collagen production, which supports the suggestion that increased wound collagen deposition in androgen-deprived rats results from reduced matrix degradation (as opposed to enhanced matrix protein biosynthesis). Androgen-mediated dysregulation of the parallel processes of collagen deposition and turnover may underscore the delayed healing of cutaneous wounds in elderly male patients and further contribute to the increased incidence of non-healing wounds in this population.     

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 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.     

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.     

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.     

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.     

Electrospun hyaluronate–collagen nanofibrous matrix and the effects of varying the concentration of hyaluronate on the characteristics of foreskin fibroblast cells

In this study we propose a novel electrospinning fabrication process for the production of a nanofibrous matrix composed of collagen and hyaluronate. This procedure utilized 1,1,1,3,3,3-hexafluoro-2-propanol and formic acid as a mixed solvent. Fluorescence microscopy photographs revealed that the resulting electrospun nanofibers contained both collagen and hyaluronate. The mean diameter of the composite nanofibrous matrix (as observed using scanning electron micrographs) was approximately 200 nm; this dimension is similar to that of native fibrous protein within the extracellular matrix. The expression of proteinases (e.g. matrix metalloproteinases, MMPs) and tissue inhibitors of metalloproteinases (TIMPs) have been implicated in epidermal repair during wound healing. Moreover, the characteristics of scarless wounds are known to be related to a decreased ratio of TIMP to MMP expression. In the present study the ratio of expression of TIMP1 to MMP1 was lower in foreskin fibroblast cells that were cultured on a hyaluronate–collagen composite nanofibrous matrix than in those cultured on an exclusively collagen nanofibrous matrix. This indicates that the hyaluronate–collagen composite nanofibrous matrix could potentially be used as a wound dressing for the regeneration of scarless skin.     

Differential expression of tissue inhibitors of metalloproteinases (TIMP-1, -2, -3, and -4) in normal and aberrant wound healing.

Wound healing is characterized by hemostasis, re-epithelialization, granulation tissue formation, and remodeling of the extracellular matrix. Matrix metalloproteinases and their specific inhibitors, TIMPs, contribute to these events. We investigated a total of 47 samples of normally healing wounds, chronic venous ulcers, ulcerative vasculitis, and suction blisters using immunohistochemistry and in situ hybridization, to clarify the role of TIMPs in normal and aberrant wound repair. Expression of TIMP-1 and -3 mRNAs was found in proliferating keratinocytes in 3- to 5-day-old normally healing wounds, whereas no epidermal expression was detected in chronic ulcers. However, TIMP-3 protein was found in the proliferating epidermis in 20 of 24 samples representing both full-thickness acute and chronic wounds. TIMP-1 and TIMP-3 also were abundantly expressed by spindle-shaped, fibroblast-like, and plump, macrophage-like stromal cells, as well as by endothelial cells. In normally healing wounds, TIMP-2 protein localized under the migrating epithelial tip and to the stromal tissue under the eschar more frequently than in chronic ulcers. Occasional staining for TIMP-4 protein was detected in stromal cells of chronic ulcers near blood vessels. Our results indicate that TIMP-1 and TIMP-3 may be involved both in the regeneration of the epidermis by stabilizing the basement membrane zone and in the regulation of stromal remodeling and angiogenesis of the wound bed. Lack of TIMP-2 near the migrating epithelial wound edges might contribute to uncontrolled activity of MMP-2 in chronic ulcers. We conclude also that TIMPs are temporally and spatially tightly regulated and that the imbalance between metalloproteinases and TIMPs-1, -2, and -3 may lead to delayed wound healing.     

循环纤维细胞与慢性创面愈合

背景：循环纤维细胞是近些年来在外周血液发现的具有成纤维细胞特性的一种白细胞亚群,由于具有合成多种细胞外基质蛋白、细胞因子以及递呈抗原、收缩创面、促进新生血管形成的能力,因此被认为可以促进创伤的修复。但其促进慢性创面修复的潜在作用研究尚少。 目的：通过文献检索,对循环纤维细胞的生物学特性及其在慢性创面修复中的潜在作用进行文献综述。 方法：分别以“循环纤维细胞、慢性创面、糖尿病足、创面愈合、细胞治疗”和“circulating fibrocytes、An-healing wounds、diabetic foot ulcer、wound healing、cell therapy”为关键词进行检索,CNKI数据库的检索时限为2000至2014年,PubMed数据库的检索时限为1994至2015年,西文生物医学期刊文献数据的检索时限为2000至2015年,检索内容为循环纤维细胞、慢性创面的难愈机制以及细胞治疗在慢性创面愈合中的应用。保留符合纳入标准的54篇文献进行总结分析。 结果与结论：循环纤维细胞因其安全、有效并能较好的发挥促进创面愈合的作用,细胞治疗已开始应用于创面修复。循环纤维细胞是在外周血发现的具有成纤维细胞特性的一个新型白细胞亚群,具有合成多种细胞外基质蛋白、细胞因子以及递呈抗原、收缩创面、促进新生血管形成的能力并在伤后早期进入损伤部位,在创伤修复过程中发挥着积极作用。动物研究证实,应用循环纤维细胞可改善慢性创面尤其是糖尿病慢性创面的修复。   中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

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.     

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.     

Human neutrophil elastase and collagenase sequestration with phosphorylated cotton wound dressings

The design and preparation of wound dressings that redress the protease imbalance in chronic wounds is an important goal of wound healing and medical materials science. Chronic wounds contain high levels of tissue and cytokine-destroying proteases including matrix metalloprotease and neutrophil elastase. Thus, the lowering of excessive protease levels in the wound environment by wound dressing sequestration prevents the breakdown of extracellular matrix proteins and growth factors necessary for wound healing. Phosphorylated cotton wound dressings were prepared to target sequestration of proteases from chronic wound exudate through a cationic uptake binding mechanism involving salt bridge formation of the positively charged amino acid side chains of proteases with the phosphate counterions of the wound dressing fiber. Dressings were prepared by applying sodium hexametaphosphate and diammonium phosphate in separate formulations to cotton gauze by pad/dry/cure methods. Phosphorylated cotton dressings were assessed for their ability to lower elastase and collagenase activity. The phosphorylated cotton dressings lowered elastase and collagenase activity 40-80% more effectively than the untreated cotton wound dressings under conditions that mimic chronic wound exudate. Efficacy of the phosphorylated cotton was found to be related to the level of phosphorylation and a lower pH due to protonated phosphate at the surface of the dressing. The capacity of the modified gauze to sequester continued elastase secretions similar to that found in a chronic wound over a 24-h period was retained within a 80% retention of elastase sequestration and was dose-dependent.     

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.     

Specific protease activity indicates the degree of Pseudomonas aeruginosa infection in chronic infected wounds

Chronic non-healing wounds are a major health problem with resident bacteria strongly implicated in their impaired healing. A rapid-screen to provide detailed knowledge of wound bacterial populations would therefore be of value and help prevent unnecessary and indiscriminate use of antibiotics-a process associated with promoting antibiotic resistance. We analysed chronic wound fluid samples, which had been assessed for microbial content, using 20 different fluorescent labelled peptide substrates to determine whether protease activity correlated with the bacterial load. Eight of the peptide substrates showed significant release of fluorescence after reaction with some of the wound samples. Comparison of wound fluid protease activities with the microbiological data indicated that there was no correlation between bacterial counts and enzyme activity for most of the substrates tested. However, two of the peptide substrates produced a signal corresponding with the microbial data revealing a strong positive correlation with Pseudomonas aeruginosa numbers. This demonstrated that short fluorescent labelled peptides can be used to detect protease activity in chronic wound fluid samples. The finding that two peptides were specific indicators for the presence of P. aeruginosa may be the basis for a diagnostic test to determine wound colonisation by this organism.