Expression profiles of collagens,HSP47, TGF-beta1, MMPs and TIMPs in epidermolysis bullosa acquisita

Epidermolysis bullosa acquisita (EBA) is a chronic, uncommon, sub-epidermal blistering disease involving the skin and mucous membranes that heals with scar formation and milia. Collagens, matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are important components that play an essential role(s) in matrix remodeling during scar formation. However, the possible involvement of these components in EBA-induced scarring is not yet known. In the present study, we examined the expression profile of collagens, collagen-binding heat shock protein 47 (HSP47), MMPs and their inhibitory enzymes, TIMPs, in matrix remodeling during conjunctival scarring. The involvement of TGF-beta1, a fibrogenic factor, was also studied. Compared to the controls, an increased expression of type I collagen, type III collagen and HSP47 was detected in conjunctival biopsy sections of patient with EBA using immunohistochemistry. Similar increase in the expression of type I collagen, type III collagen and HSP47 was noted in conjunctival fibroblasts obtained from the patient with EBA. Up-regulation in the expression of MMP-1 and MMP-14 was also noted in conjunctival fibroblasts isolated from the patient with EBA, while no significant changes in the expression of MMP-3, MMP-8, MMP-9 and MMP-13 were seen. As for TIMPs, conjunctival fibroblasts isolated from the patient with EBA, grown in vitro, exhibited increased expression of TIMP-1, TIMP-2 and TIMP-3, when compared with fibroblasts grown from control conjunctival tissues, although the expression level varies with different molecules of the same family. Additionally, compared to the control conjunctival fibroblasts, an increased expression of TGF-beta1 was detected in fibroblasts isolated from the conjunctival tissues of patient with EBA.This study suggests that there is up-regulation in the production of collagens (type I and III), collagen-binding protein (HSP47), matrix degrading collagenases (MMP-1 and 14), and their inhibitory enzymes (TIMP-1, 2 and 3) during the process of conjunctival matrix remodeling in the patient with EBA. The presented data is preliminary and could serve as a basis for further studies to enhance our understanding about the molecular mechanisms of conjunctival scarring in patients with EBA.     

Role of extracellular matrix and its regulators in human airway smooth muscle biology

Altered extracellular matrix (ECM) deposition contributing to airway wall remodeling is an important feature of asthma and chronic obstructive pulmonary disease (COPD). The molecular mechanisms of this process are poorly understood. One of the key pathological features of these diseases is thickening of airway walls. This thickening is largely to the result of airway smooth muscle (ASM) cell hyperplasia and hypertrophy as well as increased deposition of ECM proteins such as collagens, elastin, laminin, and proteoglycans around the smooth muscle. Many growth factors and cytokines, including fibroblast growth factor (FGF)-1, FGF-2, and transforming growth factor (TGF)-α₁, that are released from the airway wall have the potential to contribute to airway remodeling, revealed by enhanced ASM proliferation and increased ECM protein deposition. TGF-α₁ and FGF-1 stimulate mRNA expression of collagen I and III in ASM cells, suggesting their role in the deposition of extracellular matrix proteins by ASM cells in the airways of patients with chronic lung diseases. Focus is now on the bidirectional relationship between ASM cells and the ECM. In addition to increased synthesis of ECM proteins, ASM cells can be involved in downregulation of matrix metalloproteinases (MMPs) and upregulation of tissue inhibitors of metalloproteinases (TIMPs), thus eventually contributing to the alteration in ECM. In turn, ECM proteins promote the survival, proliferation, cytokine synthesis, migration, and contraction of human airway smooth muscle cells. Thus, the intertwined relationship of ASM and ECM and their response to stimuli such as chronic inflammation in diseases such as asthma and COPD contribute to the remodeling seen in airways of patients with these diseases.     

Dynamics of extracellular matrix production and turnover in tissue engineered cardiovascular structures

Appropriate matrix formation, turnover and remodeling in tissue-engineered small diameter vascular conduits are crucial requirements for their long-term patency and function. This complex process requires the deposition and accumulation of extracellular matrix molecules as well as the remodeling of this extracellular matrix (ECM) by matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs). In this study, we have investigated the dynamics of ECM production and the activity of MMPs and TIMPs in long-term tissue-engineered vascular conduits using quantitative ECM analysis, substrate gel electrophoresis, radiometric enzyme assays and Western blot analyses. Over a time period of 169 days in vivo, levels of elastin and proteoglycans/glycosaminoglycans in tissue-engineered constructs came to approximate those of their native tissue counter parts. The kinetics of collagen deposition and remodeling, however, apparently require a much longer time period. Through the use of substrate gel electrophoresis, proteolytic bands whose molecular weight was consistent with their identification as the active form of MMP-2 (approximately 64--66 kDa) were detected in all native and tissue-engineered samples. Additional proteolytic bands migrating at approximately 72 kDa representing the latent form of MMP-2 were detected in tissue-engineered samples at time points from 5 throughout 55 days. Radiometric assays of MMP-1 activity demonstrated no significant differences between the native and tissue-engineered samples. This study determines the dynamics of ECM production and turnover in a long-term tissue-engineered vascular tissue and highlights the importance of ECM remodeling in the development of successful tissue-engineered vascular structures.     

Global gene expression profiles during acute pathogen-induced pulmonary inflammation reveal divergent roles for Th1 and Th2 responses in tissue repair

T helper 1 responses are typically proinflammatory, while Th2 responses have been considered regulatory. Interestingly, Th2 responses characterize a number of pulmonary diseases, many of which terminate in tissue remodeling and fibrosis. We developed a mouse model using Schistosoma mansoni eggs and cytokine-deficient mice to induce highly polarized Th1- or Th2-type inflammation in the lung. In this study, we examined the pathology and cytokine profiles in Th1- and Th2-polarized environments and used oligonucleotide microarray analysis to decipher the genes responsible for these effects. We further elaborated on the results using IL-10- and IL-13-deficient mice because these cytokines are believed to be the central regulators of Th2-associated pathology. We found that the Th1-polarized mice developed small granulomas with less fibrosis while expressing genes characteristic of tissue damage. Th2-polarized mice, in contrast, formed large granulomas with massive collagen deposition and up-regulated genes associated with wound healing, specifically, arginase, collagens, matrix metalloproteinases (MMPs), and tissue inhibitors of MMP. In addition, several members of the chitinase-like family were up-regulated in the lung following egg challenge. We also developed a method of defining the net collagen deposition using the expression profiles of several collagen, MMP, and tissue inhibitors of MMP genes. We found that Th1-polarized mice did not elaborate collagens or MMPs and therefore did not have a significant capacity for repair in this model. Thus, Th1-mediated inflammation is characterized by tissue damage, while Th2 directs wound healing and fibrosis.     

Expression of genes for gelatinases and tissue inhibitors of metalloproteinases in periodontal tissues during orthodontic tooth movement

Orthodontic tooth movement progresses by a combination of periodontal ligament (PDL) tissue and alveolar bone remodeling processes. Besides the remodeling of alveolar bone around the moving teeth, the major extracellular matrix (ECM) components of PDLs, collagens, are degenerated, degraded, and restructured. Matrix metalloproteinases (MMPs) and their specific inhibitors, tissue inhibitors of metalloproteinases (TIMPs), act in a co-ordinated fashion to regulate the remodeling of periodontal tissues. We hypothesized that the expression levels of the genes for MMP-2, MMP-9, and TIMPs 1–3 are increased transiently in the periodontal tissue during orthodontic tooth movement. To test this hypothesis, we employed an animal model of tooth movement using rats, as well as in situ hybridization to analyze the expression levels of Mmp-2, Mmp-9, and Timps 1-3. The expression levels of these genes increased transiently in cells of periodontal tissues, which include cementoblasts, fibroblasts, osteoblasts, and osteoclasts, at the compression side of the moving teeth. The transient increases in gene expression at the tension side were mainly limited to osteoblasts and cementoblasts. In conclusion, the expression levels of Mmp-2, Mmp-9, and Timps 1-3 increase transiently during orthodontic tooth movement at both the tension and compression sides. The expression of these genes is regulated differentially in the periodontal tissue of the tension side and compression side. This altered pattern of gene expression may determine the rate and extent of remodeling of the collagenous ECM in periodontal tissues during orthodontic tooth movement.     

Gene expression profile in human late radiation enteritis obtained by high-density cDNA array hybridization

Late radiation enteritis is a sequela of radiation therapy to the abdomen. The pathogenic process is poorly understood at the molecular level. cDNA array analysis was used to provide new insights into the pathogenesis of this disorder. Gene profiles of six samples of fibrotic bowel tissue from patients with radiation enteritis and six healthy bowel tissue samples from patients without radiation enteritis were compared using membrane-based arrays containing 1314 cDNAs. Results were confirmed with real-time RT-PCR and Western blot analysis. Array analysis identified many differentially expressed genes involved in fibrosis, stress response, inflammation, cell adhesion, intracellular and nuclear signaling, and metabolic pathways. Increased expression of genes coding for proteins involved in the composition and remodeling of the extracellular matrix, along with altered expression of genes involved in cell- to-cell and cell-to-matrix interactions, were observed mainly in radiation enteritis samples. Stress, inflammatory responses, and antioxidant metabolism were altered in radiation enteritis as were genes coding for recruitment of lymphocytes and macrophages. The Rho/HSP27 (HSPB1)/zyxin pathway, involved in tissue contraction and myofibroblast transdifferentiation, was also altered in radiation enteritis, suggesting that this pathway could be related to the fibrogenic process. Our results provide a global and integrated view of the alteration of gene expression associated with radiation enteritis. They suggest that radiation enteritis is a dynamic process involving constant remodeling of each structural component of the intestinal tissue, i.e. the mucosa, the mesenchyme, and blood vessels. Functional studies will be necessary to validate the present results.     

Extracellular matrix dynamics in heart failure: A prospect for gene therapy

In chronic congestive heart failure, an illness affecting more than 4 million Americans, there is impairment of myocardial extracellular matrix (ECM) remodeling. Failing human ventricular myocardium contains activated matrix metalloproteinases (MMPs), which are involved in adverse ECM remodeling. Our studies support the concept that impaired ECM remodeling and MMP activation are, in part, responsible for the cardiac structural deformation and heart failure. There is no known program that has declared its aim the investigation of the role of ECM gene therapy in heart failure. The development of transgenic technology, and emerging techniques for in vivo gene transfer, suggest a strategy for improving cardiac function by overexpressing or downregulation of the ECM components such as MMPs, tissue inhibitor of metalloproteinases (TIMPs), transforming growth factor-β1 (TGF-β), decorin, and collagen in cardiomyopathy and heart failure. J. Cell. Biochem. 68:403–410, 1998. © 1998 Wiley-Liss, Inc.     

Differential spatial distribution and temporal regulation of tissue inhibitor of metalloproteinase mRNA expression during rat central nervous system development

The elucidation of the cellular and molecular mechanisms governing the maturation of the central nervous system (CNS) is rapidly emerging. Cell-cell and cell-matrix interactions play critical roles in all phases of developmental tissue remodeling. Throughout development, an intricate balance between extracellular matrix synthesis and degradation is preserved by the opposing actions of matrix metalloproteinases (MMPs) and their specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Although recent evidence suggests that TIMPs exert diverse cell biological functions distinct from their MMP-inhibitory activities, few studies have investigated MMP or TIMP expression during CNS development. The present report analyzes the mRNA expression of the four known TIMPs throughout the course of embryonic and postnatal rat CNS development. The results clearly demonstrate the unique spatial distribution and temporal regulation of TIMP expression and suggest a distinct role for each TIMP during CNS development.     

Dynamic expression profiles of MMPs/TIMPs and collagen deposition in mechanically unloaded rat heart: implications for left ventricular assist device support-induced cardiac alterations

Left ventricular assist devices (LVADs) ameliorate heart failure by reducing preload and afterload. However, extracellular matrix (ECM) deposition after application of LVADs is not clearly defined. The purpose of the present study was to investigate ECM remodeling after mechanical unloading in a rat heart transplant model. Sixty male Lewis rats were subjected to abdominal heterotopic heart transplantation, and the transplanted hearts were pressure- and volume-unloaded. The age- and weight- matched male Lewis rats who had undergone open thoracic surgeries were used as the control. Left ventricle ECM accumulation and the expression/activity of matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases (TIMPs) were measured on the third, seventh, and fourteenth days after transplantation/sham surgery. Compared with the control group, myocardial ECM deposition significantly increased on the seventh and fourteenth days after heart transplantation (P?<?0.05) and peaked on the 14th day. The gelatinase activity as well as mRNA expression of MMP-2 and MMP-9 significantly increased after transplantation (P?<?0.05). Both mRNA and protein levels of TIMP-1 and TIMP-2 significantly increased compared with those of the control group. Mechanical unloading may lead to adverse remodeling of the ECM of the left ventricle. The underlying mechanism may due to the imbalance of the MMP/TIMP system, especially the remarkable upregulation of TIMPs in the pressure and volume unloaded heart.     

MMP-TIMP interaction depends on residue 2 in TIMP-4.

Extracellular matrix remodeling and degradation are of great importance in both physiological and pathological situations. Matrix metalloproteinases (MMPs) and their natural occurring inhibitors - tissue inhibitors of metalloproteinases (TIMPs) - are involved in matrix turnover. Among the TIMPs there is only little specificity for inhibiting individual MMPs. In this report we describe the mutational analysis of the interaction of human TIMP-4 with several MMPs. The effects of different substitutions of residue 2 (Ser(2)) in the inhibitory domain of TIMP-4 were determined by kinetic measurements. Size, charge and polarity of residue 2 in the TIMP structure are key factors in MMP inhibition.     

The molecular biology of matrix metalloproteinases and tissue inhibitors of metalloproteinases in inflammatory bowel diseases

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are thought to be predominant proteases and protease inhibitors involved in the pathogenesis of inflammatory bowel diseases (IBD) through their ability to remodel the extracellular matrix (ECM) in response to inflammatory stimuli and by their immunomodulating effects. An imbalance between MMPs and TIMPs has been linked with acute and chronic inflammation and aberrant tissue remodeling, as seen in IBD. Moreover, recurrent phases of tissue destruction and subsequent tissue repair can cause serious complications in IBD patients such as fistulas and fibrosis. The aims of this review are (i) to summarize current literature on genetic association, mRNA, and protein expression studies with regard to MMPs and TIMPs in IBD patients and various animal models, including those with transgenic and knockout mice; (ii) to compare biochemical and molecular biological data in humans with those obtained in animal model studies and (iii) to critically evaluate and translate how this knowledge may be used in practical terms to understand better the pathophysiology and mechanisms operating in IBD and to apply this for improvement of clinical outcomes at diagnostic, prognostic and therapeutic levels.     

Role of matrix metalloproteinases in melanoma cell invasion

Cutaneous melanomas are notorious for their tendency to metastasize. Essential steps in this process are the degradation of basement membranes and remodeling of the extracellular matrix (ECM) by proteolytic enzymes such as matrix metalloproteinases (MMPs), which are regulated by their tissue inhibitors (TIMPs). An MMP expression is not restricted to tumor cells but is also found in stromal cells, indicating that stroma-derived proteases may contribute to melanoma progression. The MMPs have been shown to interact with a broad range of non-matrix proteins including adhesion molecules, growth factors and mediators of angiogenesis and apoptosis. In this review, we evaluate new insights into the interplay of MMPs and their molecular partners in melanoma progression.     

Altered circulating levels of matrix metalloproteinases and inhibitors associated with elevated type 2 cytokines in lymphatic filarial disease

Background

Infection with Wuchereria bancrofti can cause severe disease characterized by subcutaneous fibrosis and extracellular matrix remodeling. Matrix metalloproteinases (MMPs) are a family of enzymes governing extracellular remodeling by regulating cellular homeostasis, inflammation, and tissue reorganization, while tissue-inhibitors of metalloproteinases (TIMPs) are endogenous regulators of MMPs. Homeostatic as well as inflammation-induced balance between MMPs and TIMPs is considered critical in mediating tissue pathology.

Methods

To elucidate the role of MMPs and TIMPs in filarial pathology, we compared the plasma levels of a panel of MMPs, TIMPs, other pro-fibrotic factors, and cytokines in individuals with chronic filarial pathology with (CP Ag+) or without (CP Ag−) active infection to those with clinically asymptomatic infections (INF) and in those without infection (endemic normal [EN]). Markers of pathogenesis were delineated based on comparisons between the two actively infected groups (CP Ag+ compared to INF) and those without active infection (CP Ag− compared to EN).

Results and Conclusion

Our data reveal that an increase in circulating levels of MMPs and TIMPs is characteristic of the filarial disease process per se and not of active infection; however, filarial disease with active infection is specifically associated with increased ratios of MMP1/TIMP4 and MMP8/TIMP4 as well as with pro-fibrotic cytokines (IL-5, IL-13 and TGF-β). Our data therefore suggest that while filarial lymphatic disease is characterized by a non-specific increase in plasma MMPs and TIMPs, the balance between MMPs and TIMPs is an important factor in regulating tissue pathology during active infection.     

uPARAP expression during murine lung development

Lung remodeling requires active collagen deposition and degradation. Urokinase plasminogen activator receptor-associated protein (uPARAP), or Endo 180, is a cell-surface receptor for collagens, which leads to collagen internalization and degradation. Thus, uPARAP-mediated collagen degradation is an additional pathway for matrix remodeling in addition to matrix remodeling mediated by matrix metalloproteinases and cathepsins. Using immunohistochemistry, we demonstrate extensive uPARAP expression in the mesenchyme throughout murine lung development. By immunofluorescence, we demonstrate significant overlap of uPARAP expression with collagen IV expression, but minimal overlap with collagen I expression in the developing murine lung. Finally, we compared lung development between wild-type and uPARAP(-/-) mice, and found no significant histologic differences, indicating the presence of alternative collagen degradation pathways during murine lung development.     

Fibrogenesis II. Metalloproteinases and their inhibitors in liver fibrosis

Liver fibrosis is characterized by activation of hepatic stellate cells, which are then involved in synthesis of matrix proteins and in regulating matrix degradation. In the acute phases of liver injury and as liver fibrosis progresses, there is increased expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). Among the changes described, striking features include increased expression of gelatinase A (MMP-2) and membrane type 1-MMP (MT(1)-MMP; MMP-14) as well as TIMP-1 and TIMP-2. These molecules and other family members are involved in regulating degradation of both normal and fibrotic liver matrix. This article outlines recent progress in this field and discusses the mechanisms by which MMPs and TIMPs may contribute to the progression and regression of liver fibrosis. Recently described properties of MMPs and TIMPs of relevance to the pathogenesis of liver fibrosis are outlined. The proposal that regression of liver fibrosis is mediated by decreased expression of TIMPs and involves degradation of fibrillar collagens by a combination of MT(1)-MMP and gelatinase A, in addition to interstitial collagenase, is explored.     

Matrix metalloproteinases and their expression in mammary gland

The matrix metalloproteinases (MMPs) are a family of zine-dependent endopeptidases that play a key role in both normal and pathological processes involving tissue remodeling events.The expression of these proteolytic enzymes is highly regulated by a balance between extracellular matrix (ECM) deposition and its degradation,and is controlled by growth factors,cytokines,hormones,as well as interactions with the ECM macromolecules.Furthermore,the activity of the MMPs is regulated by their natural endogenous inhibitors,which are members of the tissue inhibitor of metalloproteinases (TIMP) family.In the normal mammary gland,MMPs are expressed during ductal development,lobulo-alveolar development in pregnancy and involution after lactation.Under pathological conditions,such as tumorigenesis,the dysregulated expression of MMPs play a role in tumor initiation,progression and malignant conversion as well as facilitating invasion and metastasis of malignant cells through degradation of the ECM and basement membranes.     

Extracellular matrix molecules, long-term potentiation, memory consolidation and the brain angiotensin system

Considerable evidence now suggests an interrelationship among long-term potentiation (LTP), extracellular matrix (ECM) reconfiguration, synaptogenesis, and memory consolidation within the mammalian central nervous system. Extracellular matrix molecules provide the scaffolding necessary to permit synaptic remodeling and contribute to the regulation of ionic and nutritional homeostasis of surrounding cells. These molecules also facilitate cellular proliferation, movement, differentiation, and apoptosis. The present review initially focuses on characterizing the ECM and the roles of cell adhesion molecules (CAMs), matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), in the maintenance and degradation of the ECM. The induction and maintenance of LTP is described. Debate continues over whether LTP results in some form of synaptic strengthening and in turn promotes memory consolidation. Next, the contribution of CAMs and TIMPs to the facilitation of LTP and memory consolidation is discussed. Finally, possible roles for angiotensins, MMPs, and tissue plasminogen activators in the facilitation of LTP and memory consolidation are described. These enzymatic pathways appear to be very important to an understanding of dysfunctional memory diseases such as Alzheimer's disease, multiple sclerosis, brain tumors, and infections.     

The effect of IL-1alpha on the expression of matrix metalloproteinases, plasminogen activators, and their inhibitors in osteoblastic ROS 17/2.8 cells

Interleukin-1 (IL-1) plays key roles in altering bone matrix turnover. This turnover is regulated by matrix metalloproteinases (MMPs), tissue inhibitor of matrix metalloproteinases (TIMPs), and the plasminogen activation system, including tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA) , and plasminogen activator inhibitor type-1 (PAI-1). In this study, we examined the effect of IL-1alpha on the expression of the MMPs, TIMPs, tPA, uPA, and PAI-1 genes in osteoblasts derived from the rat osteosarcoma cell line ROS 17/2.8. The cells were cultured in alpha-minimum essential medium containing 10% fetal bovine serum with 0 or 100 U/ml of IL-1alpha for up to 14 days. The levels of MMPs, TIMPs, uPA, tPA, and PAI-1 expression were estimated by determining the mRNA levels using real-time RT-PCR and by determining protein levels using ELISA. In IL-1alpha cultures, the expression levels of MMP-1, -2, -3, -13, and -14 exceeded that of the control through day 14 of culture, and the expression of MMPs increased markedly from the proliferative to the later stages of culture. The TIMP-1, -2, and -3 expression levels increased from the initial to the proliferative stages of culture. The expression of tPA increased greatly during the proliferative stage of culture, and uPA expression increased throughout the culture period, increasing markedly from the proliferative to the later stages of culture. In contrast, PAI-1 expression decreased in the presence of IL-1alpha through day 14. These results suggest that IL-1alpha stimulate bone matrix turnover by increasing MMPs, tPA, and uPA production and decreasing PAI-1 production by osteoblasts, and incline the turnover to the resolution.