Collagenases and their inhibitors:a review

Hide and skin are complex tissue where the most abundant component is collagen.Matrix metalloproteinases and bacterial collagenases are two kinds of collagenases that can cleave the triple-helical domain of native fibrillar collagens.In this paper,the family members and domain composition of matrix metalloproteinases and bacterial collagenases are summarized.The catalytic mechanism of collagen hydrolysis by collagenases is described,and the methods adopted to date for investigating and regulating collagenases and their inhibitors are reviewed.Furthermore,the applications of collagenases and their inhibitors in biomedicine,food processing and the enzymatic unhairing process in the leather-making industry are presented.

Insight into the spoilage heterogeneity of meat-borne bacteria isolates with high-producing collagenase

Chilled chicken is inevitably contaminated by microorganisms during slaughtering and processing,resulting in spoilage.Cutting parts of chilled chicken,especially wings,feet,and other skin-on products,are abundant in collagen,which may be the primary target for degradation by spoilage microorganisms.In this work,a total of 17 isolates of spoilage bacteria that could secrete both collagenase and lipase were determined by raw-chicken juice agar(RJA)method,and the results showed that 7 strains of Serratia,Aeromonas,and Pseudomonas could significantly decompose the collagen ingredients.The gelatin zymography showed that Serratia liquefaciens(F5)and(G7)had apparent degradation bands around 50 kDa,and Aeromonas veronii(G8)and Aeromonas salmonicida(H8)had a band around.65 and 95 kDa,respectively.The lipase and collagenase activities were detected isolate-by-isolate,with F5 showing the highest collagenase activity.For spoilage ability on meat in situ,F5 performed strongest in spoilage ability,indicated by the total viable counts,total volatile basic nitrogen content,sensory scores,lipase,and collagenase activity.This study provides a theoretical basis for spoilage heterogeneity of strains with high-producing collagenase in meat.

Long-term assessment of collagenase treatment for Dupuytren’s contracture:A 10-year follow-up study

BACKGROUND Enzymatic fasciotomy with collagenase clostridium histolyticum(CCH)has revolutionized the treatment for Dupuytren’s contracture(DC).Despite its benefits,the long-term outcomes remain unclear.This study presented a comprehensive 10-year follow-up assessment of the enduring effects of CCH on patients with DC.AIM To compare the short-term(12 wk)and long-term(10 years)outcomes on CCH treatment in patients with DC.METHODS A cohort of 45 patients was treated with CCH at the metacarpophalangeal(MCP)joint and the proximal interphalangeal(PIP)joint and underwent systematic reevaluation.The study adhered to multicenter trial protocols,and assessments were conducted at 12 wk,7 years,and 10 years post-surgery.RESULTS Thirty-seven patients completed the 10-year follow-up.At 10 years,patients treated at the PIP joint exhibited a 100%recurrence.However,patients treated at the MCP joint only showed a 50%recurrence.Patient satisfaction varied,with a lower satisfaction reported in PIP joint cases.Recurrence exceeding 20 degrees on the total passive extension deficit was observed,indicating a challenge for sustained efficacy.Significant differences were noted between outcomes at the 7-year and 10-year intervals.CONCLUSION CCH demonstrated sustained efficacy when applied to the MCP joint.However,caution is warranted for CCH treatment at the PIP joint due to a high level of recurrence and low patient satisfaction.Re-intervention is needed within a decade of treatment.

A randomized clinical trial assessing the efficacy of single and multiple intralesional collagenase injections for treating contracted scars

Background:Scar contractions caused by trauma or burns can cause secondary physical dysfunction and disfigurement.Many minimally invasive methods for scar contraction have shown limited applicability and efficacy.This study investigated the feasibility and efficacy of intralesional collagenase injections for scar contraction treatment.Methods:Patients with contracted scars who had limited joint movement and physical disfiguration for>1 year were enrolled in this single-blind,randomized clinical trial from July 2017 to February 2018 at Shanghai Ninth People’s Hospital.Collagenase was injected into the firm-contracted scar(15 U/cm^(2))three times at 4-week intervals in the multiple treatment group and once in the single treatment group,and a placebo injection was performed in the control group.Scar length and skin texture were documented at the 4-and 12-week follow-ups.The safety of the collagenase treatment was also evaluated.Results:The contracted scar was significantly elongated after both single and multiple collagenase treatments.The results showed that,compared to a one-time treatment,repeated injections were more effective at 12 weeks,with an average improvement of 26.83(15.79%).At 12 weeks,78.9% of the patients in the multiple group and 52.9%in the single group achieved significant improvement at 12 weeks.No severe adverse events were observed.Conclusion:Intralesional collagenase injection showed promising results in improving scar contraction and provides an alternative treatment for patients.

Complex role of matrix metalloproteinases in angiogenesis

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMP) play a significant role in regulating angiogenesis, the process of new blood vessel formation. Interstitial collagenase (MMP-1), 72 kDa gelatinase A/type IV collagenase (MMP-2), and 92 kDa gelatinase B/type IV collagenase (MMP-9) dissolve extracellular matrix (ECM) and may initiate and Promote angiogenesis. TIMP-1, TIMP-2, TIMP-3, and possibly,TIMP-4 inhibit neovascularisation. A new paradigm is emerging that matrilysin (MMP-7), MMP-9, and metalloelastase (MMP-12) may block angiogehesis by converting plasndnogen to angiostatin, which is one of the most potent angiogenesis antagonists. MMPs and TIMPs play a complex role in regulating angiogenesis. An understanding of the biochemical and cellular pathways and mechanisms of angiogenesis will provide importal information to allow the control of angiogenesis, e.g. the stimulation of angiogenesis for coronary collateral circulation formation; while the inhibition for treating arthritis and cancer.

溶藻弧菌实时荧光定量PCR快速检测方法的建立

为建立溶藻弧菌(VA)的快速检测方法,本研究以VA Collagenase基因为靶基因设计合成引物及Taq Man探针,建立了实时荧光定量PCR快速检测VA的方法。结果显示,对15株实验菌株进行荧光定量PCR检测,只有VA菌株检测为阳性,表明该检测方法特异性强;该方法的灵敏度为18 cfu/m L;稳定性和重复性实验结果表明,同一样品重复检测4次Ct值的变异系数均小于2%;利用该检测方法对采集的165份样品进行检测,共计检出2份VA阳性样品,与SN/T2564-2010行标法检测结果一致,显示了良好的实用性。该检测方法灵敏度高、特异性强,具有良好的实用性。

Effects of glycyrrhetinic acid on collagen metabolism of hepatic stellate cells at different stages of liver fibrosis in rats

INTRODUCTIONLiver fibrosis is a dynamic course leading tocirrhosis from a various chronic liver diseases. Thepathological basis of fibrosis is the disturbance ofproduction and degradation of the extracellularmatrix (ECM), which causes accumulation of ECMin the liver[1,2].

具胶原蛋白酶活性铜绿假单胞菌的筛选

从成都皮革厂等堆积废弃毛皮、皮革的场所采集土样 ,通过以明胶为主要基质培养基进行富集和初筛 ,获得 95株有明胶酶活性菌株。挑选其中 2 8株明胶酶活性较高的菌株进行牛皮消化试验 ,有 1 2株菌能在 48h内完全消化小牛皮。以III型酸溶性胶原为底物 ,测定了 1 2株菌发酵培养液中胶原蛋白酶活性 ,确认这 1 2株菌都具有胶原蛋白酶活性 ,酶活力基本相同 ,约 1 0～ 1 6U mL。经形态观察、生理生化特征分析及BIOLOG微生物鉴定仪鉴定 ,这 1 2株菌分为两类 ,分别是铜绿假单胞菌 (Pseudomonasaeraginosa)和火神发光杆菌 (Photobacteriumlogei) (结果另文发表 )。对铜绿假单胞菌产生胶原蛋白酶粗酶性质进行了研究 ,其酶活最适温度为 32℃ ,最适pH为 7 5 ,可以被金属蛋白酶抑制剂EDTA和EGTA部分抑制 ,不能被PMSF抑制。对铜绿假单胞菌产胶原蛋白酶发酵条件的研究表明 ,不仅培养基中氮源、碳源和金属离子影响产酶量 ,而且发酵工艺对胶原蛋白酶的产生也有较大影响。

Plasma matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 as biomarkers of ulcerative colitis activity

AIM:Overexpression of mucosal metalloproteinases(MMP) has been demonstrated recently in inflammatory bowel disease.Their activity can be counterbalanced by the tissue inhibitor of metalloproteinases(TIMP).The aim of this study was to evaluate the effect of ulcerative colitis(UC)on MMP- 1 and TIMP-1 plasma concentrations,as two possible biomarkers of the disease activity. METHODS:MMP-1 and TIMP-1 plasma concentrations were measured with an enzyme immunoassay in 16 patients with endoscopically confirmed active UC. RESULTS:Plasma concentrations of both MMP-1(13.7±0.2 ng/ml)and TIMP-1(799±140 ng/ml)were significantly elevated in UC patients in comparison to healthy controls (11.9±0.9 ng/ml and 220±7 ng/ml respectively).There was no correlation between TIMP-1 and MMP-1 concentrations (r=0.02).TIMP-1 levels revealed significant positive correlations with scored endoscopic degree of mucosal injury, disease activity index and clinical activity index values as well as C-reactive protein concentration.There was no correlation between MMP-1 and laboratory,clinical or endoscopic indices of the disease activity.CONCLUSION: These results confirm the role of both MMP- 1 and TIMP-1 in the pathogenesis of ulcerative colitis. However only TIMP-1 can be useful as a biomarker of the disease activity, demonstrating association with clinical and endoscopic pictures.

Antitumor activity of anti-type IV collagenase monoclonal antibody and its lidamycin conjugate against colon carcinoma

AIM： Type IV collagenase including MMP-2 and -9 plays an important role in cancer cell invasion and metastasis and is an attractive target for rnAb-directed therapy. The irnrnunoreactivity of rnAb 3G11, a rnAb directed against type Ⅳ collagenase in human colorectal carcinomas, was studied by irnrnuno-histochernical （IHC） staining, rnAb 3G11 was conjugated to an antiturnor antibiotic lidarnycin （LDM）. The antiturnor activity of 3G11-LDM conjugate against colon carcinoma was investigated in mice. METHODS： ELISA, gelatin zyrnography, and Western blot assay were used for the biological characterization of rnAb 3G11. The irnrnunoreactivity of rnAb 3G11 with human colorectal carcinomas was detected by IHC staining. The cytotoxicity of LDM and 3G11-LDM conjugate to human colon carcinoma HT-29 cells was examined by clonogenic assay and MTT assay. The therapeutic effect of conjugate 3G11-LDM was evaluated with colon carcinoma 26 in mice. RESULTS： As shown in ELISA, mAb 3Gll reacted specifically with type IV collagenase, while 3G11-LDM conjugate also recognized specifically its respective antigen. In IHC assay, mAb 3G11 showed positive irnrnunoreactivity in most cases of colorectal carcinoma, and negative irnrnunoreactivity in the adjacent non-malignant tissues. By gelatin zyrnography, the inhibition effect of rnAb 3G11 on the secretion activity of type IV collagenase was proved. In terms of IC50 values in MTT assay, the cytotoxicity of LDM to human colon carcinoma HT-29 cells was 10 000-fold more potent than that of rnitornycin C （MMC） and adriarnycin （ADM）. 3G11- LDM conjugate also displayed extremely potent cytotoxicity to human colon carcinoma HT-29 cells with an IC50 value of 5.6× 10^-19 mol/L. 3G11-LDM conjugate at the doses of 0.05 and 0.1 mg/kg inhibited the growth of colon carcinoma 26 in mice by 70.3 and 81.2%, respectively. CONCLUSION： mAb 3G11 is immunoreactive with human colorectal carcinoma and its conjugate with LDM is highly effective against colon carcinoma in mice.

A novel arterial pouch model of saccular aneurysm by concomitant elastase and collagenase digestion

Background: An ideal aneurysm model of cerebral aneurysm is of great importance for studying the pathogenesis of the lesion and testing new techniques for diagnosis and treatment. Several models have been created in rabbits and are now widely used in experimental studies; however, every model has certain intrinsic limitations. Here we report the development of a novel saccular aneurysm model in rabbits using an arterial pouch that is subject to in vitro pre-digestion with combined elastase and collagenase. Methods: A segment of right common carotid artery (CCA) was dissected out and treated with elastase (60 U/ml, 20 min) followed by type I collagenase (1 mg/ml, 15 min) in vitro. The graft was anastomosed to an arterial arch built with the left CCA and the remaining right CCA, while the other end of the graft was ligated. The dimension and tissue structure of the pouch were analysed immediately, 2 or 8 weeks after operation. Findings: Ten terminal aneurysms were produced. The gross mor-phology of the aneurysm resembles the human cerebral terminal aneurysms. We have observed the following pathological changes: (1) growth of the aneurysm (mean diameter increased from (2.0±0.1) to (3.2±0.3) mm at 2 weeks, P【0.001, n=7~10); (2) thinning of the aneurysmal wall (the mean wall thickness decreased to 44% at 2 weeks), which was accompanied by significant losses of elastic fibres, collagen and the cellular component; and (3) spontaneous rupture (3 out of 9, one aneurysm ruptured 24 h after operation with the other two at 2 and 4 weeks respectively). Conclusion: This rabbit arterial pouch model mimics human cerebral aneurysms in relation to morphology and histology. In particular, this model exhibited an increased tendency of spontaneous rupture.

Influence of hepatic arterial blockage on blood perfusion and VEGF,MMP-1 expression of implanted lver cancer in rats

AIM: To investigate the influence of hepatic arterial blockage on blood perfusion of transplanted cancer in rat liver and the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-1 (MMP-1), and to explore the mechanisms involved in transarterial embolization (TAE)-induced metastasis of liver cancer preliminarily. METHODS: Walker 256 carcinosarcoma was transplanted into rat liver to establish the liver cancer model. Hepatic arterial ligation (HAL) was used to block the hepatic arterial blood supply and simulate TAE. Blood perfusion of tumor in control, laparotomy control, and HAL group was analyzed by Hoechst 33342 labeling assay, the serum VEGF level was assayed by ELISA, the expression of VEGF and MMP-1 mRNA was detected by in situ hybridization. RESULTS: Two days after HAL, the number of Hoechst 33342 labeled cells which represent the blood perfusion of tumor directly and hypoxia of tumor indirectly in HAL group decreased significantly compared with that in control group (329+/-29 vs 384+/-19, P【0.01). The serum VEGF level in the HAL group increased significantly as against that of the control group (93 ng.L(-1)+/-44 ng.L(-1) vs 55 ng.L(-1)+/-19 ng.L(-1), P【0.05). The expression of VEGF and MMP-1 mRNA in the tumor tissue of the HAL group increased significantly compared with that of the control and the laparotomy control groups (P【0.05). The blood perfusion data of the tumor, represented by the number of Hoechst 33342 labeled cells, showed a good linear inverse correlation with the serum VEGF level (r=-0.606, P【0.05) and the expression of VEGF mRNA in the tumor tissue ( r =-0.338, P【0.01). CONCLUSION: Blockage of hepatic arterial blood supply results in decreased blood perfusion and increased expression of metastasis-associated genes VEGF and MMP-1 of transplanted liver cancer in rats. Decreased blood perfusion and hypoxia may be the major cause of up-regulated expression of VEGF.

Impact of vasculature damage on the outcome of spinal cord injury:a novel collagenase-induced model may give new insights into the mechanisms involved

The deleterious effect of vasculature damage on the outcome of spinal cord injury has long been recognized, and numerous clinical studies have shown that the presence of hemorrhage into the spinal cord is directly associated with a poorer neurological outcome. Vascular damage leads to de- creased blood flow to the cord and the release of potentially toxic blood-borne components. Here we consider the mechanisms that may be contributing to hemorrhage-induced damage and discuss the utility of a new model of spinal cord hemorrhage, which was urgently required as most of our current understanding has been extrapolated from intracerebral hemorrhage studies.

Isolation and activation of collagenase from fish processing waste

Collagenase was isolated from fish waste (a mixture of haddock, herring, ground fish and flounder) using a Tris-buffer system. The proteins in the crude extract were precipitated using ammonium sulfate (40% - 80%) and purified with gel-filtration chromatography using Sephadex G-100. The results showed that the collagenase enzyme was produced as a latent enzyme and was activated with bovine trypsin and potassium thiocyanate (KSCN). The enzyme activity was affected by pH and temperature. Optimal enzyme activities were found at 35?C and a pH of 7.5 when insoluble collagene type I was used as substrate and the liberated amino acids were measured in relation to L-leucine in the presence of ninhydrin. The enzyme activity was completely inhibited by the action of ethylenediaminetetraacetic acid (EDTA) suggesting that the collagenase enzyme isolated from the fish waste is a metalloproteinase enzyme requiring metal ions for enzyme activity. Dialysis against KSCN decreased the enzyme total activity and increased its specific activity. Sodium dodecyl sulphate polyacryla-mide gel electrophoresis (SDS-PAGE) indicated that the purified procollagenase enzyme have only one band at molecular weight of 50 kilodaltons (kDa). When the enzyme was cleaved with trypsin, it was found to consist of two subunits: a large unit with a molecular weight of 50 kDa and a small unit with a molecular weight of 10 kDa.

Analysis of isolation of cerebral cortical neurons in rats by different methods

The aim of this study was to find a way to efficiently separate neuronal cells from the cerebral cortex of adult rats,providing a reference method for rapid acquisition of neuronal cells from the adult rat brain.Fifteen SD rats were randomly divided into three groups,with five SD rats in each group.Then,neuron cells were isolated from the adult rat cerebral cortex by the grinding method,the trypsin method,and the collagenase II method,respectively.The expression of anti-NeuN in the neurons of each group was analyzed by flow cytometry.The acquisition rates and morphology of neurons of each group were observed by immunofluorescence staining.The grinding or collagenase II method is more suitable for rapid acquisition of neuronal cells from an adult rat’s cerebral cortex.The number of neuron cells obtained by the trypsin method were very few,so it is not convenient for later experiments.

Modified insulin-like growth factor 1 containing collagen-binding domain for nerve regeneration

Insulin-like growth factor 1 （IGF-I） is a potential nutrient for nerve repair. However, it is impractical as a therapy because of its limited half- life, rapid clearance, and limited target specificity. To achieve targeted and long-lasting treatment, we investigated the addition of a binding structure by fusing a collagen-binding domain to IGF- 1. After confirming its affinity for collagen, the biological activity of this construct was examined by measuring cell proliferation after transfection into PC12 and Schwann cells using a 3-（4,5-dimethyl-2-thiazolyl）-2,5-di- phenyl-2-H-tetrazolium bromide assay. Immunofluorescence staining was conducted to detect neurofilament and microtubule-associated protein 2 expression, while real time-polymerase chain reaction was utilized to determine IGF-1 receptor and nerve growth/actor mRNA expression. Our results demonstrate a significant increase in collagen-binding activity of the recombinant protein compared with IGF-1. Moreover, the recombinant protein promoted proliferation of PC12 and Schwann cells, and increased the expression of neurofilament and microtubule-associated protein 2. Importantly, the recombinant protein also stimulated sustained expression of IGF-1 receptor and nerve growth factor mRNA for days. These results show that the recombinant protein achieved the goal of targeting and long-lasting treatment, and thus could become a clinically used factor for promoting nerve regeneration with a prolonged therapeutic effect.

The Role of Neutrophil Collagenase in Endotoxic Acute Lung Injury

The aim of this study was to determine the role of neutrophil collagenase in the pathogenesis of acute lung injury induced by endotoxin. 28 Sprague-Dawley were randomized into control group and LPS-enduced groups. Samples of left lung were obtained in 2 h (group L 1), 6 h (group L 2), 12 h (group L 3) after intravenous LPS. Immunohistochemsitry was employed for detection of expression of neutrophil collagenase. Pathological scores, lung wet/dry weight ratio and the number of neutrophils were measured. The results showed that the concentration of neutrophil collagenase in LPS-enduced groups (group L 1, L 2, L 3) were significantly higher than that of control group (P<0.01). Pathological scores, lung wet/dry weight ratio and the number of neutrophils in LPS-enduced groups (group L 1, L 2, L 3) were also significantly higher than that of control group (P<0.01). Moreover, among group L 1, L 2 and L 3, there were significant correlations in concentration of neutrophil collagenase and pathological scores, lung wet/dry weight ratio, the number of neutrophils (P<0.05). The present study showed that neutrophil collagenase play an important role in the pathogenesis and progress of endotoxic acute lung injury.

Testing of a New Collagenase Blend for Pancreatic Islet Isolation Produced by Clostridium histolyticum

Clostridium histolyticum is used for production of several proteolytic enzymes such as elastase, neutral proteases, clostripain and in particular collagenase. Besides industrial applications, collagenase has been indispensable for medical purposes including isolation of pancreatic islets for diabetes treatment. The aim of this study was to optimize the method for production and partial purification of a new collagenase blend and to test its suitability for successful pancreatic islets isolation in a rat model. Bacterial strain of C. histolyticum was sequenced for presence of the collagenase genes. Different fermentation conditions were tested and the process of collagenase extraction was modified and optimized. Samples of collagenases were taken for western blot detection, activity assessment, and ability for dissociation of pancreatic tissue. Findings indicate that concentrated trypton growth medium with pepton was the most suitable for Clostridium growth and collagenase production. Whole genome sequencing revealed two genes for collagenase and also gene for clostripain. Western blot specific detection helped to select useful production modifications. Following these modifications was also improved the yield, morphology and in vitro function of intact pancreatic islets which were finally comparable or better than those achieved using standard blends of collagenase. The results support the use of the new collagenase blend for islet isolation giving thus the opportunity to choose an alternative product. Our next steps would lead to further enzyme purification through scaling up of the production method for a wider use.

Epidermal collagenase activity and its induction by 20-hydroxyecdysone in the fiddler crab Uca pugilator

The epidermal collagenase activity and its induction by 20-hydroxyecdysone in Uca pugilator were investigated. Zymographic electrophoresis showed four bands of collagenase activity, 16, 19, 22 and 29 kDa in molecular weight, with the former two accounting for 60% and 36% , respectively, of the total collagenase activity. The collagenase activity varies during the molting cycle. Among the molt stages tested, Premolt Stage Do exhibited the highest epidermal collagenase activity for both the 16 and 19 kDa isoenzymes and, as the molt stage proceeded, the enzymatic activity of these two isoenzymes decreased, with the lowest activity for both found in Premolt Stage D3 -4 - Injection of 20-hydroxyecdysone significantly induced the activity of the 16 kDa eollagenase in the epidermis of Uca pugilator, suggesting that the activity of this isoenzyme is under molting hormone control. Although 20-hydroxyeedysone injection did not result in a statistically significant increase in the activity of the 19 kDa isoenzyme, a tendency of the induction was nonetheless demonstrated. This is the first report on epidermal collagenase activity and its induction by the molting hormone in a crustacean .

Therapeutic applications of collagenase(metalloproteases): A review

Non-invasive therapeutic methods have recently been used in medical sciences. Enzymes have shown high activity at very low concentrations in laboratories and pharmaceutical,enabling them to play crucial roles in different biological phenomena related to living organism, especially human medicine. Recently, using the therapeutic methods based on non-invasive approaches has been emphasized in medical society. Researchers have focused on producing medicines and tools reducing invasive procedures in medical.Collagenases are proteins which catalyze chemical processes and break the peptide bonds in collagen. Collagen may be generated more than the required amount or produced in unsuitable sites or may not degrade after a certain time. In such cases, using an injectable collagenase or its ointment can be helpful in collagen degradation. In both in vitro and in vivo tests, it has been revealed that collagenases have several therapeutic properties in wound healing, burns, nipple pain and some diseases including intervertebral disc herniation, keloid, cellulite, lipoma among others. This review describes the therapeutic application of collagenase in medical sciences and the process for its production using novel methods, paving the way for more effective and safe applications of collagenases.