绿脓杆菌对角膜基质细胞介导的胶原降解作用的实验研究

目的 探讨绿脓杆菌性角膜炎组织破坏机制,为临床治疗提供理论依据。方法 Ⅰ型胶原凝胶,混悬及不混悬角膜基质细胞,与绿脓杆菌培养液共同培养２４ｈ,超滤去除天然胶原纤维,超滤液经过水解,分光光度计测定其羟脯氨酸含量。同时检测基质金属蛋白酶抑制剂Ｇａｌａｒｏｄｉｎ对胶原降解作用的影响。结果 绿脓杆菌培养液可直接降解Ⅰ型胶原,如同时存在角膜基质细胞此作用增强。无论 角膜基质细胞存在与否,Ｇａｌａｒｄｉｎ可明     

Galardin inhibits collagen degradation by rabbit keratocytes by inhibiting the activation of pro-matrix metalloproteinases.

We investigated the inhibitory action of a synthetic peptidyl hydroxamate inhibitor of matrix metalloproteinase (MMP), Galardin (GM6001), on collagen degradation by rabbit corneal stromal fibroblasts (keratocytes) cultured three-dimensionally in the type I collagen gel with medium containing interleukin 1alpha (IL-1alpha) and/or plasminogen. Degradation of collagen fibrils during culture was measured by the release of hydroxyproline, and activation of MMPs was also analyzed by gelatin zymography and Western blotting. Plasmin activity was measured using a synthetic substrate. In the absence of plasminogen, treatment of the cells with IL-1alpha in collagen gel greatly enhanced the production of proMMP-1, -3 and -9, but no significant degradation of collagen was detected. In the presence of plasminogen, IL-1alpha stimulated collagen degradation by keratocytes in a dose-dependent manner. This resulted from the plasminogen activator-plasmin system-dependent activation of proMMP-1, -3 and -9. Galardin inhibited the collagen degradation in a dose-dependent fashion in the presence of plasminogen, whether IL-1alpha was present or not. Galardin inhibited the activation of proMMP-3, and also prevented the activation of proMMP-9 and the conversion of MMP-1 intermediates to the fully active MMP-1. Galardin did not affect plasmin activity. The present results suggest that Galardin inhibits IL-1alpha-stimulated collagen degradation in the presence of plasminogen, resulting from not only inhibiting active MMPs but also preventing the conversion of proMMPs to active MMPs.     

Stimulatory effect of pseudomonal elastase on collagen degradation by cultured keratocytes

PURPOSE. The pathobiology of corneal ulceration induced by Pseudomonas aeruginosa was investigated by characterization of the pseudomonal pathogenic factors responsible for degradation of the collagen matrix. METHODS. Three-dimensional gels of type I collagen containing (or not) rabbit keratocytes were incubated in the presence of either culture supernatant of P. aeruginosa strain PAO1 or pseudomonal pathogenic factors (elastase, lipopolysaccharide, or exotoxin A), and the extent of collagen degradation was assessed after 24 hours by measurement of released hydroxyproline. Activation of matrix metalloproteinases (MMPs) produced by keratocytes was also examined by gelatin zymography and immunoblot analysis. RESULTS. In the absence of keratocytes, the PAO1-conditioned medium increased the extent of collagen degradation. The conditioned medium also promoted keratocyte-mediated collagen degradation. Of the pseudomonal pathogenic factors examined, only elastase degraded collagen directly as well as stimulated keratocyte-mediated collagen degradation. Culture supernatant of elastase-deficient P. aeruginosa (lasR or lasB) mutants had no effect on collagen degradation in the absence or presence of keratocytes. Elastase also induced the conversion of the inactive precursors of MMP-1, -2, -3, and -9 produced by keratocytes to the active forms of the enzymes. CONCLUSIONS. These results suggest that pseudomonal elastase both degrades type I collagen directly and promotes collagen degradation mediated by keratocytes, the latter effect being likely attributable, at least in part, to the activation of proMMPS:     

Differential regulation of collagen degradation by rabbit keratocytes and polymorphonuclear leukocytes

PURPOSE: Both activated keratocytes and infiltrated polymorphonuclear leukocytes (PMNs) contribute to corneal ulceration by degrading stromal collagen. The regulation of such collagen degradation by inflammatory cytokines was investigated with rabbit keratocytes and PMNs cultured in three-dimensional collagen gels. METHODS: Rabbit keratocytes or PMNs were cultured for 24 h in three-dimensional gels of type I collagen in the presence of plasminogen and various concentrations of either interleukin (IL)-1alpha, IL-6, IL-8, or tumor necrosis factor-alpha (TNF-alpha). Degradation of collagen during culture was assessed by measurement of released hydroxyproline. RESULTS: IL-1alpha increased the amount of collagen degraded by keratocytes or PMNs in a dose-dependent manner, whereas IL-6 had no effect on collagen degradation by either cell type. IL-8 increased the extent of collagen degradation by PMNs but not that by keratocytes, and TNF-alpha promoted collagen degradation by keratocytes but not that by PMNs. CONCLUSION: Inflammatory cytokines regulate collagen degradation by rabbit keratocytes and PMNs in culture in a differential manner, and therefore may contribute to the roles of these cells in corneal ulceration.     

Collagenolytic Activity of Keratocytes Cultured in a Collagen Matrix

To study the mechanism of collagen degradation by keratocytes, we developed the new in vitro model in which keratocytes were cultured three-dimensionally in a collagen matrix. Subcultured rabbit keratocytes were embedded in a type I collagen matrix and cultured in serum-free medium. Collagenolytic activity of the cells was determined by measuring the amount of hydroxyproline released into the medium from degraded collagen. Activities of collagenase in the medium were also measured, using collagen labeled with fluorescein isothiocyanate as a substrate. The presence of plasminogen was required for collagen degradation by keratocytes. In the presence of plasminogen, the amount of collagen degradation depended on both the cultivation period and the number of cells. The addition of interleukin-1 (IL-1) stimulated the collagen degradation in a dose-dependent manner. This stimulatory effect of IL-1 was completely inhibited by the addition of IL-1 receptor antagonist (IL-1ra). Collagenase activity in the medium was stimulated by the addition of IL-1, and IL-1ra antagonized this stimulatory effect. These findings indicate that our present model may be useful for investigating the mechanism of collagen degradation by keratocytes.     

Role of protein kinase C signaling in collagen degradation by rabbit corneal fibroblasts cultured in three-dimensional collagen gels

PURPOSE: To understand the mechanism of corneal ulceration by characterizing the intracellular signaling pathways that regulate collagen degradation by corneal fibroblasts cultured in three-dimensional type I collagen gels. Specifically, the potential roles of protein kinase C (PKC) and protein kinase A (PKA) in collagen degradation were investigated. METHODS: Rabbit corneal fibroblasts were cultured in three-dimensional type I collagen gels for 24 hours in the presence of plasminogen and in the absence or presence of activators or inhibitors of PKC or PKA. Degradation of collagen fibrils was then evaluated by measurement of released hydroxyproline, and the production of matrix metalloproteinases (MMPs) was assessed by gelatin zymography and immunoblot analysis. RESULTS: The PKC activator phorbol 12-myristate 13-acetate (PMA) increased the extent of collagen degradation by corneal fibroblasts in a dose-dependent manner, with the maximal effect apparent at a concentration of 0.1 microM. The inactive analog 4alpha-PMA had no effect on collagen degradation. The PKC inhibitor H-7 reduced the extent of collagen degradation by corneal fibroblasts in the absence or presence of PMA. Phorbol 12-myristate 13-acetate also increased the production of proMMP-1, -3, and -9 by corneal fibroblasts, whereas H-7 inhibited this effect. Neither the PKA activators 8-bromo-cAMP, isobutylmethylxanthine, and forskolin nor the PKA inhibitor HA1004 affected collagen degradation by corneal fibroblasts. CONCLUSION: These results demonstrate that PKC plays an important role in collagen degradation by corneal fibroblasts in three-dimensional type I collagen gels, whereas PKA does not appear to participate in this process.     

Pseudomonas protease. Purification, partial characterization, and its effect on collagen, proteoglycan, and rabbit corneas.

The extracellular protease of a virulent strain of Pseudomonas aeruginosa was purified by DEAE-cellulose chromatography in two steps. SDS-polyacrylamide gel electrophoresis of the purified enzyme revealed a single band, and the enzyme was shown to be the major component of the bacterial filtrate. The protease was fully inhibited by Na2 EDTA, 1,10-orthophenanthroline, L-cysteine and Zn+2 ions but was insensitive to dissopropylphosphofluoridate. The elastase substrates orcein-elastin and acetyl-L-alanyl-L-alanyl-L-alamine-methyl ester were degraded by the enzyme. The protease activity toward soluble and insoluble collagen was found to be limited to the telopeptide region of the collagen molecule. With soluble collagen, conversion of the beta and gamma chains into monomeric alpha chains was observed. About 60% of the total proteoglycans and 1.5% of the total collagen were solubilized from rabbit corneas following incubation with the enzyme, and the solubilized products were nondialyzable. It was concluded that the purified protease has little or no collagenolytic activity and that dissolution of the cornea by Pseudomonas protease infection results essentially from the degradation of the protein backbone of the corneal proteoglycans.     

Matrix metalloproteinase 2: involvement in keratoconus

PURPOSE: The activation of matrix metalloproteinase-2 (MMP-2) is postulated to be a crucial pathogenic factor behind progressive and chronic diseases in which basement membranes are disrupted. An ocular example is keratoconus. The purpose of the present enquiry was therefore to investigate and compare the activities of the MMP-2 secreted by keratocytes of normal and keratoconic corneas. METHODS: The spectrum of MMP-2 activities secreted by cultures of keratocytes derived from normal and keratoconic corneas was analysed by zymography. Subsequently, selected preparations were assayed for peptidase activity, using Type I, Type III, Type IV and Type V collagen as substrate, under native conditions and after treatment with a variety of putative activating reagents. RESULTS: Although MMP-2 of Mr 65,000 on SDS gelatin polyacrylamide gels is the major protease secreted by keratocytes of normal corneas, the keratocytes of early-phase keratoconic corneas secrete an additional zymographic activity of Mr 61,000. From their N-terminal amino acid sequences, both these proteins were shown to be conformers of proMMP-2. Treatment with SDS followed by protein fractionation was required to achieve in vitro activation of the MMP-2 secreted by normal corneal keratocytes. Treatment with SDS alone partially activated the enzyme produced by early-phase keratoconic corneal keratocytes. This procedure and autocatalysis, yielded an enzyme of Mr 43,000 that selectively hydrolysed Type IV and denatured Type 1 collagen. CONCLUSIONS: The zymographic gelatinase activities of apparent Mr 65,000 and 61,000 are conformers of corneal proMMP-2. Activated enzyme, of Mr 43,000, is more readily generated from protein preparations of the culture media of early phase keratoconic corneal keratocytes than from protein preparations of the culture media of normal corneal keratocytes.     

Pseudomonas keratitis: protease IV gene conservation, distribution, and production relative to virulence and other Pseudomonas proteases

PURPOSE: To determine the distribution of the protease IV gene, the production of this and other proteases by multiple strains of Pseudomonas, and the virulence of a mutant specifically deficient in protease IV. METHODS: The protease IV gene was cloned, its sequence analyzed, and its chromosomal location determined by pulse-field gel electrophoresis. Three PCR reactions were used to detect the protease IV gene in 30 Pseudomonas isolates and protease production was determined by Western blot analysis, colorimetric assay, and zymography. An allelic replacement mutant deficient in the protease IV gene was analyzed for enzyme production, corneal growth, and corneal virulence. RESULTS: The protease IV gene was identified in all P. aeruginosa, but none of the non-aeruginosa strains tested. The protease IV genes of strains PA103-29 and PAO1 were in a common chromosomal site and had 98.5% sequence identity with variations occurring mainly in the promoter region. The protease IV activity of the 23 wild-type P. aeruginosa strains tested varied from 2.3 to 221.5 x 10(-3) U/mg protein in the culture supernatant. Protease IV was produced by all P. aeruginosa wild-type strains. A protease IV-deficient mutant derived from strain PA103-29 had reduced virulence compared with its parent strain and unexpectedly produced alkaline protease. CONCLUSIONS: The protease IV gene and its product are common to P. aeruginosa, but not to other Pseudomonas species. Protease IV activity varies among P. aeruginosa strains, and a mutant specifically deficient in this activity produced alkaline protease and had reduced corneal virulence.     

Effects of the Matrix Metalloproteinase Inhibitor GM6001 on the Destruction and Alteration of Epithelial Basement Membrane During the Healing of Post-alkali Burn in Rabbit Cornea

Purpose

To examine the alteration in structure and matrix composition of epithelial basement membrane (BM) during the healing of alkali-burned rabbit cornea, and the roles of matrix metalloproteinases (MMPs) in these alterations.

Methods

The central cornea of one eye of 78 albino rabbits was exposed to 1?N NaOH for 180?s under general and topical anesthesia and allowed to heal with or without subconjunctival injection of GM6001 (an MMP inhibitor). Cryosections of affected corneas were observed by H&;E staining, immunohistochemistry for type IV collagen subtypes, or in situ zymography for detection of localization of MMP activity.

Results

Uninjured corneal epithelial BM exhibited α5 (IV)-immunoreactivity, but lacked the α1/α2-immunoreactivity of collagen IV. Epithelial BM in healing burned cornea transiently exhibited α1/α2-immunoreactivity. Examination by in situ zymography showed an upregulation of MMP activity in the regenerated central epithelium and anterior stroma of the burned corneas at days 7 and 14. GM6001 suppressed degradation of α5-containing epithelial BM in vivo and also in organ culture.

Conclusions

Epithelial BM was degraded by endogenous MMPs during healing following an alkali burn in rabbit cornea. GM6001 had an inhibitory effect on the degradation of the epithelial basement membrane in burned cornea in vivo.?Jpn J Ophthalmol 2006;50:90–95 © Japanese Ophthalmological Society 2006     

Alkaline protease-deficient mutants of Pseudomonas aeruginosa are virulent in the eye

PURPOSE: Alkaline protease has been associated with virulence in Pseudomonas aeruginosa corneal infections. To define the role of this enzyme in such infections, isogenic mutants of P. aeruginosa deficient in alkaline protease production were constructed. This study examines the ability of these mutants to adhere to scarified corneal tissue in vitro and to establish corneal infections in vivo. METHODS: Mutants were constructed by allelic exchange in two phenotypically different wild type strains, PAO1 (invasive) and ATCC 19660 (cytotoxic). Alkaline protease-deficient mutants were characterized by zymography and western blot analysis of bacterial culture supernatants. Allelic exchange was confirmed by PCR analysis of the disrupted aprA gene of the mutants. Adherence of wild type and mutant strains to scarified corneal epithelium was assessed by an in vitro organ culture assay, while ocular virulence of the strains was determined in vivo using a mouse scarification model of bacterial keratitis. RESULTS: Being isogenic, phenotypes of mutants were identical to their respective parents with the exception of the loss of alkaline protease production. The absence of alkaline protease did not alter corneal adherence or ocular virulence of the organisms when compared to similar wild type strains. CONCLUSIONS: These data provide evidence that alkaline protease produced by P. aeruginosa is not essential in the pathogenesis of P. aeruginosa keratitis.     

Identification of a novel secreted protease from Pseudomonas aeruginosa that causes corneal erosions

PURPOSE: The purpose of this study was to identify a new Pseudomonas protease and determine its possible role in keratitis. METHODS: Concentrated culture supernatants of the Pseudomonas aeruginosa strains PA103 and ATCC 19660 were analyzed by zymography. P. aeruginosa small protease (PASP) was purified from strain PA103, and modified elastase B (LasB) was purified from strain ATCC 19660. SDS-PAGE and Western blot analysis were performed on purified PASP and modified LasB. PASP was further analyzed by mass spectrometry and amino-terminal sequencing. The Pasp gene was cloned and expressed, affinity-purified in denatured form from inclusion bodies, and refolded by removal of the denaturant. Purified recombinant PASP was analyzed by zymography for protease activity. PASP and heat-inactivated PASP were injected into rabbit corneas, and the corneas were monitored for erosions caused by protease activity. RESULTS: Each strain produced a protease with a molecular mass of 80 kDa on zymograms. LasB antiserum identified the ATCC 19660 protease as modified LasB. Mass spectrometry defined the PA103 protease as having a molecular mass of 18.5 kDa. Amino-terminal sequencing and analysis of the P. aeruginosa genome sequence determined that the PA103 Pasp gene sequence was >99% identical with the PA0423 sequence of strain PAO1. Recombinant PASP was proteolytic, with a zymogram mass of 50 kDa. PASP purified from PA103 produced extensive corneal epithelial erosions, whereas heat-inactivated PASP produced no erosions. CONCLUSIONS: PASP is a protease that has not been previously identified. It causes corneal epithelial erosions, indicating its likely activity as a virulence-promoting factor in Pseudomonas keratitis.     

The role of matrix metalloproteinases in infectious corneal ulcers

During infectious keratitis, the production of collagenolytic and inflammatory substances, along with increased corneal matrix metalloproteinase (MMP) activity, induces the degradation of corneal collagen and may cause postkeratitis complications, such as opacity, thinning, and corneal perforation. MMPs, especially MMP-2 and MMP-9, are overexpressed in infectious keratitis and sustained over time by inflammatory and nonmicrobial mechanisms. The high MMP levels are correlated with excessive corneal destruction in bacterial, herpetic, fungal, and acanthamoeba infections. Nonspecific treatments, such as tetracyclines, particularly doxycycline, or corticosteroids, are used as adjuvants to antimicrobials to alleviate the disproportionate degradation and inflammation of the corneal layers caused by corneal MMPs and decrease the recruitment and infiltration of inflammatory cells. Treatments showing inhibition of specific MMPs (Galardin, ZHAWOC7726), interfering with pro-MMP activation (EDTA, ascorbic acid), or showing anticytokine effect (epigallocatechin-2-gallate, TRAM-34) have been reported. Other treatments show a direct action over corneal collagen structure such as corneal cross-linking or have been associated with reduction of MMP levels such as amniotic membrane grafting. Although the use of these drugs has been shown in studies to be effective in controlling inflammation, especially in experimental ones, robust studies are still needed based on randomized and randomized clinical trials to demonstrate their potential effect as adjuvants in the management of infectious keratitis.     

Expression of metalloproteinases from human retinal pigment epithelial cells and their effects on the hydraulic conductivity of Bruch's membrane

PURPOSE: To evaluate the expression pattern of matrix metalloproteinases (MMPs) from retinal pigment epithelial (RPE) cells in culture, and to determine their ability to alter the transport properties of human Bruch's membrane. METHODS: Human RPE cells from either primary cultures or a cell line were maintained under culture conditions. At different time intervals after subculturing of cells the presence of MMPs in the bathing medium was determined by zymography. Cellular MMP activity was determined in a similar series of experiments where serum was omitted from the culture medium. Cultured cells were introduced onto Bruch's membrane, mounted in a modified Ussing chamber, to assess entry of MMPs into the membrane. Fluid flow across Bruch's membrane was determined by hydraulic conductivity for different ages of donor tissue, before and after 24 hours' incubation with active MMPs from the RPE-conditioned medium or after incubation with purified activated MMPs. Latent (inactive) MMPs from medium containing serum were used in control experiments. RESULTS: Cultured RPE cells expressed both MMP-2 and -9, with active MMP-2 becoming detectable from 4 days after subculture through to confluence and activated MMP-9 becoming abundant up to 24 hours after subculture. Both active MMPs significantly increased hydraulic conductivity of Bruch's membrane, with the increase after MMP-9 incubation being far greater than that for MMP-2. Both enzymes showed a trend in hydraulic conductivity change with age such that, MMP-2 produced a relatively constant change, whereas MMP-9 showed a greater increase in older eyes. CONCLUSIONS: Activation of both MMP-2 and -9 by cultured RPE cells appeared to show a temporal relationship with the growth cycle of the cells. The activated enzymes increased fluid flow of Bruch's membrane, and the marked effect observed with MMP-9 in older eyes suggests a mechanism that may allow debris removal.     

转谷氨酰胺酶交联胶原凝胶构建三维角膜基质

目的检测转谷氨酰胺酶交联胶原凝胶对三维培养的角膜基质细胞的影响,探讨可提高机械性能的组织工程角膜基质层新途径。方法胶原酶消化法获取原代兔角膜基质细胞,以加入转谷氨酰胺酶与胶原凝胶交联为实验组,不加酶交联为对照组。倒置显微镜下每日观察细胞生长情况、Alamar-Blue试剂检测细胞增生、免疫荧光法检测凝胶内细胞波形蛋白、检测透光度、酶消化法检测胶原凝胶抗消化能力。结果实验组细胞胶原凝胶内附着和生长优于对照组,细胞在凝胶内呈树枝状生长。2组细胞均随培养时间延长明显增生（P=0.000）。共焦显微镜下见2组细胞胞浆波形蛋白均阳性表达,实验组细胞伪足更丰富。实验组透光度稍差于对照组。实验组抵抗胶原酶消化的能力显著增强。结论酶交联的胶原凝胶对角膜基质细胞无毒性作用,重构的基质层结构更加稳定,有利于组织工程角膜基质层的构建。     

A Pseudomonas aeruginosa strain isolated from a contact lens-induced acute red eye (CLARE) is protease-deficient

PURPOSE: Pseudomonas aeruginosa proteases are thought to be important virulence factors in the pathogenesis of corneal disease. This study examined protease production from two strains of P. aeruginosa responsible for two very distinct clinical diseases: strain Paer1, isolated from a Contact Lens-induced Acute Red Eye (CLARE), and strain KEI 1025, isolated from a corneal ulcer. Strains were compared to a laboratory strain (ATCC 19660) known to produce severe keratitis in experimentally infected mice for protease production and for ocular virulence. METHODS: Protease production was examined with colorimetric assays, gelatin zymography and western blots. Elastase A activity was quantitated with a staphylolytic assay. Ocular virulence was examined using a mouse scratch model of keratitis. RESULTS: In contrast to strains KEI 1025 or ATCC 19660, Paer1 was unable to produce enzymatically active elastase A, elastase, and protease IV. All three strains produced active alkaline protease. Strains KEI 1025 and ATCC 19660 produced a fulminant keratitis in mice whereas Paer1 produced a mild transient infection. Restoration of elastase activity in Paer1 via genetic complementation did not result in a virulent phenotype. Co-infection of mouse eyes with strains Paer1 and ATCC 19660 resulted in the eventual loss of Paer1 from corneal tissue. CONCLUSIONS: These studies suggest that P. aeruginosa elastase A and/or protease IV, but not alkaline protease or elastase, contribute to the ocular virulence of this organism.     

Expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) and MMP-2 in normal and keratoconus corneas

PURPOSE: To determine whether MT1-MMP and MMP-2 are expressed in normal and keratoconic corneas, and to investigate the ability of MT1-MMP, expressed on cultured keratocytes after stimulation with concanavalin A, to activate pro-gelatinase A (pro-MMP 2). METHODS: Specimens of keratoconus corneas (n = 20), removed at corneal transplantation, were obtained from pathology archives, sections were cut, and were stained with an antibody to MT1-MMP, using peroxidase immunohistochemistry. Eye banked corneas served as controls (n = 14). Normal human keratocyte cultures were initiated from eye bank corneas, and after stimulation with con A, MMPs in the media were examined using gelatin zymography and immunoblotting, and MT1-MMP expression was analysed by flow cytometry and immunoblotting. RESULTS: All corneas showed some expression of MT1-MMP and MMP-2, although the degree of staining varied greatly. The MMPs were present in the epithelium, endothelium and stroma. Expression of MT1-MMP, but not MMP-2, in the epithelium and stroma, was significantly elevated in keratoconus, compared to normal corneas. In vitro, keratocytes stimulated with con A expressed MT1-MMP and produced active MMP-2, detected by zymography. These responses to con A were concentration-dependent and MT1-MMP expression and MMP-2 activation correlated significantly (p = 0.0003) In addition, MMP inhibitors abolished MMP-2 activation, providing further evidence that MT1-MMP activated MMP-2. CONCLUSION: The observation that MT1-MMP expression may be up-regulated in keratoconus corneas, taken together with the demonstration that human corneal cells can express this enzyme, which in turn can activate latent MMP-2, provide evidence for a possible role for MT1-MMP in the pathogenesis of keratoconus.