Remodeling Of Extracellular Matrix Components Research Articles

Higher levels of O-GlcNAcylation induce aortic stiffness by increasing elastase activity in male, but not female rats.

Age-associated structural and functional changes in the aorta, including stiffness, loss of elasticity, and reduced vasodilatation, are known to hamper stable peripheral circulation and lead to organ dysfunction. The extracellular matrix (ECM) is a network of macromolecules produced by resident cells. Remodeling of ECM components, include fragmentation of elastin and excessive deposition and crosslinking of collagens, and is a hallmark of aging which leads to stiffening of the aorta. Recently, O-linked beta-N-acetyl glucosamine (O-GlcNAc) has emerged as an essential regulator of cell stress and survival. Interestingly, higher levels of O-GlcNAc are a biomarker for cardiovascular risks (e.g., hypertension and diabetes). However, it is unknown whether O-GlcNAcylation contributes to age-associated aortic stiffness. The goal of these studies was divided into two parts: 1. To determine how the higher levels of O-GlcNAc impact aortic stiffness, and 2. To investigate any potential sex differences. To address this aims, the thoracic aorta was isolated from male and female Wistar and Fisher 344 rats at 3 (3M) and 16 months of age (16M) and mounted on a tissue puller (DMT) to evaluate vessel strain/stress. Blood pressure was measured directly in the carotid artery under anesthesia, and western blot, immunofluorescence, and histology were performed to measure O-GlcNAc levels, elastase expression, and elastin fragmentation, respectively. To induce O-GlcNAc, aorta from 3M Wistar rats were treated with Thiamet G, a pharmacological inhibitor of OGA (1 uM, 24h). The aorta from 3M males presented with a higher strain than age-matched females, indicating sex difference in stiffness (ß-coefficient: 2.5±0.9 vs. 1.6±0.6, p<0.05). However, this difference was abolished in old male and female rats (5.1±0.9 vs. 4.6±0.6 p<0.05 n= duplicates of 4-5). The systolic blood pressure in 16 MO males was increased by ~ 30 mmHg compared to 3M (87±3 vs. 117±4 p=3-4), while only increasing ~ 16 mmHg when compared to 3M and 16 MO females (83±3 vs. 99±4 p=3-4). We observed elevation in O-GlcNAc protein levels from aortas of 16 MO males and females, modestly higher in females. Counter-intuitively, the Thiamet G increased O-GlcNAc levels and induced aortic stiffness in males (2.1± 0.9 vs. 3.6± 0.9 p<0.05 n= duplicates 10), but not in females (2.6± 0.9 vs. 2.7±0.8 n= duplicates 5). The aortic stiffness in males was associated with increased elastin fragmentation and elastase expression (Control 100±10 vs. Thiamet G 400±70 UA p<0.05 n=duplicate 3), which was not observed in females. Collectively, these findings indicate that higher levels of O-GlcNAc contributed to sex differences in aortic stiffness and blood pressure during aging possibly by inducing elastin fragmentation. NIH (R00HL151889, RO1DK132948), NIA (K01AG061263), and American Heart Association (AHA-916031) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Peptide hormone relaxin: from molecular effects to clinical results

Introduction.The peptide hormone relaxin, which is produced by cells of the corpus luteum during and outside pregnancy, has a huge number of clinically significant effects, mediating many biological mechanisms, including antifibrotic, vasodilatory, angiogenic, anti-inflammatory and antiapoptotic effects.Purpose of the study:based on the study of modern literature data, to analyze the results of scientific research, representing the current system of views on the physiological and pathophysilogical effects of relaxin.Materials and methods.A study f scientific publications for the period from 2005 to 2020 in e PubMed and Elibrary databases was carried out using the keywords: relaxin, pregnancy, relaxin signaling pathway, reproductive system, extracellular matrix.Results and Discussion.The study of modern views on the physiology of relaxin has shown that this hormone mediates its effects by binding to the specific receptor RXFP1, which is localized in a large number of reproductive nonproductive tissues. Relaxin performs many functions related to the remodeling of the extracellular matrix and vasculature. The main uterotropic effects of this peptide include stimulation of growth and vascularization of the uterus, remodeling of extracellular matrix components, and regulation of vascular endothelial growth factor in preparation for implantation.Conclusion.The progress of the last decade in understanding the biochemistry of the hormone relaxin has formed the basis for a deeper penetration into all the variety of its physiological roles. The participation of relaxin in the mechanisms of relaxation of the myometrium during pregnancy, remodeling of the connective tissue of target organs in the antenatal period creases its potential clinical significance. The prospect of a possible therapeutic use of relaxin preparations in stimulating antenatal transformation of the cervix, in vitro fertilization, therapy of preeclampsia, acute heart failure and myocardial ischemia turns it into a potential therapeutic agent for these pathological conditions.

Modulation of Matrix Metalloproteinases by Plant-derived Products.

Matrix metalloproteinases (MMPs) are a group of zinc-dependent metalloendopeptidases that are responsible for the degradation, repair, and remodeling of extracellular matrix components. MMPs play an important role in maintaining a normal physiological function and preventing diseases, such as cancer and cardiovascular diseases. Natural products derived from plants have been used as traditional medicine for centuries. Its active compounds, such as catechin, resveratrol and quercetin, are suggested to play an important role as MMPs inhibitors, thereby opening new insights into their applications in many fields, such as pharmaceutical, cosmetic, and food industries. This review summarises the current knowledge of plant-derived natural products with MMP-modulating activities. Most of the reviewed plant-derived products exhibit an inhibitory activity on MMPs. Amongst MMPs, MMP-2 and MMP-9 are the most studied. The expression of MMPs is inhibited through respective signaling pathways, such as MAPK, NF-κB and PI3 kinase pathways, which contribute to the reduction in cancer cell behaviors, such as proliferation and migration. Most studies have employed in vitro models, but a limited number of animal studies and clinical trials have been conducted. Even though plant-derived products show promising results in modulating MMPs, more in vivo studies and clinical trials are needed to support their therapeutic applications in the future.

Role of thrombospondin‑1 and thrombospondin‑2 in cardiovascular diseases (Review).

Thrombospondin (TSP)-1 and TSP-2 are matricellular proteins in the extracellular matrix (ECM), which serve a significant role in the pathological processes of various cardiovascular diseases (CVDs). The multiple effects of TSP-1 and TSP-2 are due to their ability to interact with various ligands, such as structural components of the ECM, cytokines, cellular receptors, growth factors, proteases and other stromal cell proteins. TSP-1 and TSP-2 regulate the structure and activity of the aforementioned ligands by interacting directly or indirectly with them, thereby regulating the activity of different types of cells in response to environmental stimuli. The pathological processes of numerous CVDs are associated with the degradation and remodeling of ECM components, and with cell migration, dysfunction and apoptosis, which may be regulated by TSP-1 and TSP-2 through different mechanisms. Therefore, investigating the role of TSP-1 and TSP-2 in different CVDs and the potential signaling pathways they are associated with may provide a new perspective on potential therapies for the treatment of CVDs. In the present review, the current understanding of the roles TSP-1 and TSP-2 serve in various CVDs were summarized. In addition, the interacting ligands and the potential pathways associated with these thrombospondins in CVDs are also discussed.

Systems Chemo-Biology and Transcriptomic Meta-Analysis Reveal the Molecular Roles of Bioactive Lipids in Cardiomyocyte Differentiation.

Lipids, which are essential constituents of biological membranes, play structural and functional roles in the cell. In recent years, certain lipids have been identified as regulatory signaling molecules and have been termed "bioactive lipids". Subsequently, the importance of bioactive lipids in stem cell differentiation and cardiogenesis has gained increasing recognition. Therefore, the aim of this study was to identify the biological processes underlying murine cardiac differentiation and the mechanisms by which bioactive lipids affect these processes. For this purpose, a transcriptomic meta-analysis of microarray and RNA-seq data from murine stem cells undergoing cardiogenic differentiation was performed. The differentially expressed genes identified via this meta-analysis, as well as bioactive lipids, were evaluated using systems chemo-biology tools. These data indicated that bioactive lipids are associated with the regulation of cell motility, cell adhesion, cytoskeletal rearrangement, and gene expression. Moreover, bioactive lipids integrate the signaling pathways involved in cell migration, the secretion and remodeling of extracellular matrix components, and the establishment of the cardiac phenotype. In conclusion, this study provides new insights into the contribution of bioactive lipids to the induction of cellular responses to various stimuli, which may originate from the extracellular environment and morphogens, and the manner in which this contribution directly affects murine heart morphogenesis.

Extracellular matrix remodeling of the testes through the male reproductive cycle in Teleostei fish.

During the fish reproductive cycle, testes undergo morphological changes related to germinal epithelium and remodeling of extracellular matrix components (ECM). ECM is degraded mainly by action of matrix metalloproteinases (MMPs). Due to the natural renewal of ECM in fish testes, we choose Pimelodus maculatus to study remodeling of ECM throughout reproductive cycle, using picrosirius (to identify type I, II, III collagen) and reticulin (type III collagen), and to immunolocalize MT1-MMP (membrane type 1-matrix metalloproteinase) and MMP-2 in testis cells. Testes were classified in four reproductive phases: regenerating, development, spawning capable and regressing. Picrosirius and reticulin demonstrated a differential distribution of total collagen fibers during the reproductive cycle. Immunohistochemistry showed MT1-MMP only in acidophilic granulocyte cells mainly inside blood vessels, in connective tissue of capsule close to the germinal compartment, and also infiltrated in interstitial connective tissue. MMP-2 was detected in fibroblast and endothelial cells of interstitial and capsule blood vessels, in epithelial cells of capsule, and in acidophilic granulocyte cells at same description for MT1-MMP. The fish testes ECM were remodeled throughout reproductive cycle in according to morphophysiological alterations. During reproductive season (spawning capable), the interstitium increased in total collagen fibers (type I, II, III). After spermiation period (regression and regenerating), the amount of collagen fibers decreased in response to action of MMPs on collagen degradation and other interstitial components (not assessed in this study). MMPs seem to be indispensable components for natural cyclic events of ECM remodeling of fish testes and for guarantee tissue homeostasis throughout reproductive cycle.

PDGF/PDGFR Signaling and Targeting in Cancer Growth and Progression: Focus on Tumor Microenvironment and Cancer-associated Fibroblasts

Traditionally, the studies on cancer growth and progression have been focused on the transformed, malignant cells. However, it is now well recognized that the tumour stroma represents a crucial parameter in tumour development, growth and progression. Indeed, several cancers are characterized by a prominent stromal compartment and it is the interactions between cancer cells, stromal cells and extracellular matrix (ECM) components that control the overall tumour growth. Among stromal cells, fibroblasts represent the most important type. They are responsible for deposition and remodeling of ECM components, as well as for the release of cytokines and growth factors, including platelet-derived growth factor (PDGF), acting in a paracrine manner on cancer cells. In this review we elucidate the role of tumor stroma interactions, the roles of PDGF receptor signaling in cancer-associated fibroblasts via alteration of stromal matrix composition and the mitogenic effects of cancer-derived PDGFs. Focus on the targeting of tumor microenvironment at the level of PDGF/PDGF receptor (PDGFR) is also presented as to stimulate further studies for designing and development of novel pharmaceutical agents and combined pharmaceutical interventions. Conclusively, PDGF/PDGFR axis is of paramount importance in the tumour microenvironment context and the inhibition of PDGF receptors' activation represents a major target for future anticancer therapies.

The ‐1562C/T MMP‐9 and the ‐511C/T IL‐1β gene polymorphisms in primary open‐angle glaucoma patients

AbstractPurpose Matrix metalloproteases (MMPs) play a role in the remodeling of extracellular matrix components (ECM) and the development of primary open‐angle glaucoma (POAG). Interleukin 1 (IL‐1β) is considered as MMP’s transcription upregulating factor. The aim of this study was to evaluate an association of the ‐1562C/T MMP‐9 and the ‐511C/T IL‐1β gene polymorphisms with a risk of POAG in a Polish population.Methods DNA samples obtained from 196 POAG patients (mean age 70 ± 14) and 256 control subjects (mean age 67 ± 16) were analyzed by restriction fragment length polymorphism of polymerase chain reaction (PCR‐RFLP).Results The comparison of genotypes distributions showed that the ‐1562C/T genotype (OR 1.69, 95% CI 1.10 ‐ 2.58; P = 0.015) and the T allele (OR 1.57, 95% CI 1.10 ‐ 2.26; P = 0.014) of MMP‐9 exhibit a significant increase of the frequency in POAG patients as compared to healthy controls. A statistically significant increase of the frequency was also found for the ‐511T/T genotype (OR 2.35, 95% CI 1.23‐4.51; P = 0.009) and the T allele (OR 1.40, 95% CI 1.06‐1.85; P = 0.017) of IL‐1β in POAG patients. The analysis of gene‐gene interactions of MMP‐9 and IL‐1β showed a statistically significant increase of the frequency of the C/T‐C/T (OR 2.27, 95% CI 1.25‐4.10; P = 0.006) and the C/C‐T/T (OR 2.23, 95% CI 1.05‐4.71; P = 0.033) genotypes in POAG patients group.Conclusion In conclusion, we suggest that MMP‐9 and IL‐1β gene polymorphisms may be associated with an increased risk of POAG in a Polish population. This work was supported by grants N N402 375838 and N N402 248936 from Polish Ministry of Science and Higher Education.

Matrix remodeling stimulates stromal autophagy, “fueling” cancer cell mitochondrial metabolism and metastasis

We have previously demonstrated that loss of stromal caveolin-1 (Cav-1) in cancer-associated fibroblasts is a strong and independent predictor of poor clinical outcome in human breast cancer patients. However, the signaling mechanism(s) by which Cav-1 downregulation leads to this tumor-promoting microenvironment are not well understood. To address this issue, we performed an unbiased comparative proteomic analysis of wild-type (WT) and Cav-1-/- null mammary stromal fibroblasts (MSFs). Our results show that plasminogen activator inhibitor type 1 and type 2 (PAI-1 and PAI-2) expression is significantly increased in Cav-1-/- MSFs. To establish a direct cause-effect relationship, we next generated immortalized human fibroblast lines stably overexpressing either PAI-1 or PAI-2. Importantly, PAI-1/2(+) fibroblasts promote the growth of MDA-MB-231 tumors (a human breast cancer cell line) in a murine xenograft model, without any increases in angiogenesis. Similarly, PAI-1/2(+) fibroblasts stimulate experimental metastasis of MDA-MB-231 cells using an in vivo lung colonization assay. Further mechanistic studies revealed that fibroblasts overexpressing PAI-1 or PAI-2 display increased autophagy (“self-eating”) and are sufficient to induce mitochondrial biogenesis/activity in adjacent cancer cells, in co-culture experiments. In xenografts, PAI-1/2(+) fibroblasts significantly reduce the apoptosis of MDA-MB-231 tumor cells. The current study provides further support for the “Autophagic Tumor Stroma Model of Cancer” and identifies a novel “extracellular matrix”-based signaling mechanism, by which a loss of stromal Cav-1 generates a metastatic phenotype. Thus, the secretion and remodeling of extracellular matrix components (such as PAI-1/2) can directly regulate both (1) autophagy in stromal fibroblasts and (2) epithelial tumor cell mitochondrial metabolism.

Increase of MT1-MMP, TIMP-2 and Ki-67 proteins in the odontogenic region of the rat incisor post-shortening procedure

MT1-MMP and TIMP-2 are well known for their roles in remodelling of extracellular matrix components. However, reports are emerging on the involvement of these molecules in cell kinetics. In the rat incisor tooth, a shortening treatment increases the eruption and cell proliferation rates. However, the role of MT1-MMP and TIMP-2 proteins in these processes is still to be evaluated. Male Wistar rats were divided in two groups. In the normofunctional group (NF) the lower teeth of the rats remained in a normal eruption process. In the hypofunctional group (HP) rats their lower left incisor tooth was shortened every 2days during 12days. The eruption rate was estimated during the shortening period and MT1-MMP, TIMP-2 and Ki-67 protein expression from the odontogenic region was measured after the treatment. In HP groups an increase in eruption rate, and in MT1-MMP/TIMP-2 and Ki-67 expression were observed. We conclude that there is a relationship between the increase in eruption rate, and in levels of MT1-MMP, TIMP-2 and Ki-67 in the HP group. This suggests that MT1-MMP and TIMP-2 may have some role in cell proliferation during the eruption of the rat incisor tooth.

Mechanisms of Relaxin Action in the Reproductive Tract: Studies in the Relaxin‐Deficient (Rlx−/−) Mouse

The major functions of relaxin (RLX) are associated with female reproductive tract physiology, namely, the regulation of biochemical processes involved in remodeling of extracellular matrix components in the cervix and vagina at term. Studies in RLX-deficient mice (Rlx-/-) demonstrate that although females give birth to live young without apparent dystocia, the pubic symphysis is not elongated, and they have abnormal cervical and vaginal morphology. The current study examined phenotypic differences in collagen, matrix metalloproteinases (MMP), and estrogen receptors (ERs) in the cervix and vagina of pregnant Rlx+/+ and Rlx-/- mice. Neither collagen nor TGFbeta1 mRNA levels in the cervix and vagina differed significantly between Rlx+/+ and Rlx-/- at any stage of gestation, except on gestation day 18.5, with an increase in alpha(1)-I collagen and TGFbeta1 expression in Rlx-/- mice. MMP gene expression was also increased in Rlx-/- mice, especially at term. Administration of recombinant H2 RLX (0.05 microg/microL/h) to Rlx-/- mice for 6 d from gestation day 12.5 caused a significant decrease in alpha1-I collagen and MMP-13 gene expression in the cervix and vagina, but it had no effect on TGFbeta1. There was also a significant reduction in ERbeta expression in RLX-treated Rlx-/- mice. Interestingly, RLX treatment caused a significant decrease in LGR7 expression in these reproductive tissues. In summary, these data show increases in MMP gene expression in Rlx-/- mice that are not correlated with changes in collagen expression. Furthermore, we report a novel ER phenotype in the cervix and vagina of Rlx-/- mice.

Degradation of extracellular matrix components by defined proteinases from the greenbottle larva Lucilia sericata used for the clinical debridement of non-healing wounds.

Larvae of the greenbottle fly Lucilia sericata are used routinely for the clinical treatment of difficult necrotic and infected wounds. Degradation by proteinases contained in larval excretory/secretory (ES) products is thought to contribute to wound debridement by removal of dead tissue. However, proteinase activity may also affect host tissue remodelling processes. To identify proteolytic enzymes derived from L. sericata ES products with activities against fibrin and extracellular matrix (ECM) components. Larval proteinase activities were assayed in vitro using class-specific substrates and inhibitors. Their action against fibrin and ECM components was examined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Three classes of proteolytic enzyme were detected in the secretions using fluorescein isothiocyanate-labelled casein as a model substrate. The predominant activity belonged to serine proteinases (pH optima 8-9) of two different subclasses (trypsin-like and chymotrypsin-like), with a weaker aspartyl proteinase (pH 5) and a metalloproteinase (pH 9) with exopeptidase characteristics also present. Using skin-relevant ECM components as substrates L. sericata ES products solubilized fibrin clots and degraded fibronectin, laminin and acid-solubilized collagen types I and III. Hydrolysis of ECM macromolecules was inhibited by preincubating ES products with phenylmethylsulphonyl fluoride but not 4-amidinophenylmethylsulphonyl fluoride, indicating that degradation was due to the 'chymotrypsin-like' serine proteinase. These data suggest that a combination of L. sericata ES proteinases involving chymotrypsin-like and trypsin-like activities could potentially influence wound healing events when maggots are introduced into necrotic and infected wounds, with the chymotrypsin-like activity involved in the remodelling of ECM components.

Human Hepatocellular Carcinoma (HCC) Cells Require Both α3β1 Integrin and Matrix Metalloproteinases Activity for Migration and Invasion

Hepatocellular carcinoma (HCC) is the most frequent malignant tumor of the liver; prognosis depends on the tendency to metastasize. Cancer cell invasion is regulated by proteolytic remodeling of extracellular matrix components and by integrin expression. We have shown that matrix metalloproteinase-2 (MMP-2) and membrane-type–1 matrix metalloproteinase (MT1-MMP) cleave Laminin-5 (Ln-5), stimulating cell migration. Here we report that all HCC cells express MT1-MMP, migrate on Ln-1 and Collagen IV, whereas only HCC cells that express α3β1 integrin secrete detectable levels of gelatinases, migrate on Ln-5, and invade through a reconstituted basement membrane (BM). Migration on Ln-5 is blocked by BB-94, an MMP inhibitor, and by MIG1, a monoclonal antibody that hinders migration on MMP-2–cleaved Ln-5. Invasion through a reconstituted BM is also inhibited by BB-94. HCC α3β1-negative cells migrate on Ln-1 and Collagen IV, but not on Ln-5, and do not invade through a reconstituted BM, although they express MT1-MMP. Anti-α3β1 blocking antibodies inhibit gelatinase activation, cell motility, and cell invasion through Matrigel. In vivo, α3β1 integrin and Ln-5 are expressed in HCC tissue but not in normal liver. In conclusion, our data suggest that both α3β1 integrin and gelatinase activity are required for HCC migration and invasion.

Diminished Matrix Metalloproteinase 2 (MMP-2) in Ectomesenchyme-Derived Tissues of the Patch Mutant Mouse: Regulation of MMP-2 by PDGF and Effects on Mesenchymal Cell Migration

Platelet-derived growth factors (PDGF) regulate cell proliferation, survival, morphology, and migration, as well as deposition and turnover of the extracellular matrix. Important roles for the A form of PDGF (PDGF-A) during connective tissue morphogenesis have been highlighted by the murine Patch mutation, which includes a deletion of the α subunit of the PDGF receptor. Homozygous (Ph/Ph) embryos exhibit multiple connective tissue defects including cleft face (involving the first branchial arch and frontonasal processes), incomplete heart septation, and heart valve abnormalities before they die in utero. Analyses of the cell biology underlying the defects in Ph/Ph embryos have revealed a deficit in a matrix metalloproteinase (MMP-2) and one of its activators (MT-MMP) that are likely to be involved in cell migration and tissue remodeling, two processes necessary for normal cardiac and craniofacial development. Morphogenesis of these structures requires infiltration of ectomesenchymal precursors and their subsequent deposition and remodeling of extracellular matrix components. First branchial arch and heart tissue from E10.5 embryos were examined by gelatin zymography and RT-PCR in order to characterize the expression of MMPs in these tissues. Of the MMPs examined, only MMP-2 and one of its activators, MT-MMP, were expressed in the first arch and heart at this stage of development. Tissues from Ph/Ph embryos exhibited a significant decrease in both MMP-2 and MT-MMP compared to tissues from normal embryos of the same developmental stage. In order to assess whether this decrease affects the motile activity of mesenchymal cells, cell migration from Ph/Ph branchial arch explants was compared to migration from normal arch tissue and found to be significantly less. In addition, the migratory ability of branchial arch cells from normal explants could be reduced in a similar manner using a specific MMP inhibitor. Although it is still unclear whether the MMP-2 reduction is a direct result of the absence of response of Ph/Ph cells to PDGF-A treatment of normal branchial arch cells in vitro with recombinant PDGF-AA significantly upregulated MMP-2 protein. Together, these results suggest that PDGF-A regulates MMP-2 expression and activation during normal development and that faulty proteinase expression may be at least partially responsible for the developmental defects exhibited by Ph/Ph embryos.

Overexpression of alveolar macrophage gelatinase B (MMP-9) in patients with idiopathic pulmonary fibrosis: effects of steroid and immunosuppressive treatment.

Alveolar macrophages (AM) express gelatinase B, a member of the matrix metalloproteinase family involved in the degradation and remodeling of extracellular matrix components. We evaluated the expression of gelatinase B in the course of idiopathic pulmonary fibrosis (IPF) by studying alveolar macrophages in culture AM and bronchoalveolar lavage fluid from 12 untreated patients with IPF, 11 patients with IPF under treatment with steroid and immunosuppressive agents, and 10 control subjects. By using zymography and quantitative image analysis, latent gelatinase B, as well an 88-kD active form, were investigated in culture medium (24 h) of AMs and were found to be significantly increased (P < 0.01) in untreated patients exhibiting severe IPF when compared with control subjects (4.1 +/- 1.7 versus 0.3 +/- 0.2 10(5) arbitrary units [AU]/10(4) AM for the 92-kD form). Concomitant studies of gelatinase B levels associated with cultured AM extracts or freshly harvested AM showed similar results, both at the mRNA and protein levels, respectively. Immunocytochemical studies on freshly harvested AM demonstrated that the enzyme was located mainly at the cell, suggesting some involvement of gelatinase B in AM migration. In contrast, gelatinase B activity secreted by AM tended to be normal in patients with IPF under steroid and immunosuppressive treatment. Simultaneously, level of the gelatinase B activity in epithelial lining fluid was increased in untreated IPF patients, whereas it was normal in treated patients. These results suggest that AM of patients with IPF are primed for gelatinase B expression and that steroid and immunosuppressive treatment induces negative modulation of the gelatinase B overexpression. We conclude that gelatinase B may play a role in lung remodeling in IPF.

Regulatory role of extracellular matrix components in expression of matrix metalloproteinases in cultured hepatic stellate cells.

Hepatic stellate cells (HSCs) were changed in their morphology, proliferative activity, and functions by culturing on type I collagen gel, as compared to the culture on polystyrene surface. HSCs have been found to produce extracellular matrix components and matrix metalloproteinases (MMPs). In this study, we have assessed the effects of several types of substrata on the expression of MMPs in HSC culture. MMP-1 expression was detectable in HSC culture on polystyrene surface and on type I collagen gel by immunofluorescence staining and reverse transcriptase-polymerase chain reaction (RT-PCR). The results from in situ zymography revealed the presence of interstitial collagenase activity around HSCs and along their cellular processes. Although proMMP-2 and proMMP-9 were detectable by gelatin zymography in the conditioned medium from both cultures using type I collagen gel and Matrigel as substratum, an active form of MMP-2 but not of MMP-9 was detected only in the culture using type I collagen as a substratum. Tissue inhibitor of metalloproteinase-2 expression was observed by RT-PCR in HSCs cultured on or in type I collagen gel, suggesting the suppression of MMP-2 activity detected in HSC culture using type I collagen. These results indicate a differential expression of MMP activity, hence the remodeling of extracellular matrix components is dependent on the substratum used for HSC culture. The HSC culture using several types of substrata appears to be a useful in vitro model to study the mechanism of extracellular matrix remodeling.

Expression of transforming growth factor-beta receptors during hyperoxia-induced lung injury and repair.

Lung injury and repair processes involve many cellular activities, including cell growth, differentiation, and remodeling of extracellular matrix components. Transforming growth factor-beta (TGF-beta) is a major class of signaling peptide growth factors regulating these cellular activities. Type I (T beta RI) and type II (T beta RII) receptors for TGF-beta are transmembrane serine/threonine kinases that are essential for TGF-beta signaling. To gain insight into the possible molecular mechanisms of lung injury and repair, we investigated the expression of T beta RI and T beta RII in an acute hyperoxia-induced model of lung injury and repair. Localization of message expression of T beta RI and T beta RII in oxygen-exposed rat lung tissue was analyzed by using in situ hybridization. T beta RI mRNA expression was found in the interstitium, capillaries, and the alveolar septa of rat lungs exposed for 60 h to 100% oxygen. The distribution of T beta RII mRNA in oxygen-exposed rat lung tissue overlapped the localization of T beta RI mRNA. Temporal changes of T beta RI and T beta RII mRNA expressions in rat lung during hyperoxic exposure and repair were examined by Northern analysis. We found that expression of T beta RI was upregulated in adult rats undergoing prolonged exposure to 100% oxygen, and the increase of T beta RI expression persisted during 2 wk of repair of lung injury. The pattern of T beta RII expression during hyperoxic exposure and repair was distinct from that of T beta RI. The expression of T beta RII increased with a peak at 3 days postexposure and then declined after 7 days of repair. Changes of T beta RI and T beta RII protein expressions in rat lung during hyperoxic exposure and repair were examined further by Western blot analysis, which correlated with the mRNA expression. The results suggest that T beta RI and T beta RII may play important roles during the lung injury and repair by mediating signaling activity of TGF-beta and may regulate interactions between the mesenchyme and the epithelium.

Modification of Matrix Metalloproteinase Activities from Alveolar Macrophages During Chronic Coal Mine Dust Exposure in Rats

Macrophage derived products have been implicated in pneumoconiosis induced by chronic coal dust inhalation. To assess the role of macrophages during chronic inflammatory processes in relation to pneumoconiosis, we investigated their capacity to secrete matrix metalloproteinase (MMP) activities, which are largely involved in the degradation and the remodelling of extracellular matrix components. Two groups of rats (N = 48 per group) were exposed to 100 or 200 mg m-3,6 h per day, 5 days per week and sacrified at 9,28 and 78 days of exposure, and 6 months following the end of exposure. A total of 92 kDa proform and 88 kDa active form collagenase type IV (gelatinase) were investigated by enzymography in macrophage culture medium (MEM), macrophage extracts (MACs) and bronchoalveolar fluid (BAL). In parallel, net gelatinase and interstitial collagenase activities were evaluated by degradation of radiolabelled specific substrates. Pneumoconiotic lesions progressively developed during the late dust exposure and the recovery phase, and were associated with macrophage alveolitis. Results clearly showed that, at each step of chronic dust exposure as well as during the recovery phase, chronic coal dust inhalation induced the increase of total gelatinase activities secreted into the MEM and the BAL. The net gelatinase and collagenase activities were increased in parallel in MEM whereas, they appeared inhibited when secreted in the BAL whatever the dust exposure time. These results suggest that chronic coal mine dust exposure is capable of inducing chronic alveolar MACs activation in regard to their persistant highly increased capacity to degrade in situ extracellular matrix components, namely collagen types IV or V [HIBBS et al, J. din. Invest. 80,1644-1650 (1987)]. Such a dysregulation associated with the acute inhibition process towards MMPs in the alveolar space, allowed us to propose a role of MMPs during pneumoconiosis.

Hyaluronic acid production and hyaluronidase activity in the newt iris during lens regeneration

The process of lens regeneration in newts involves the dedifferentiation of pigmented iris epithelial cells and their subsequent conversion into lens fibers. In vivo this cell-type conversion is restricted to the dorsal region of the iris. We have examined the patterns of hyaluronate accumulation and endogenous hyaluronidase activity in the newt iris during the course of lens regeneration in vivo. Accumulation of newly synthesized hyaluronate was estimated from the uptake of [ 3H]glucosamine into cetylpyridinium chloride-precipitable material that was sensitive to Streptomyces hyaluronidase. Endogenous hyaluronidase activity was determined from the quantity of reducing N-acetylhexosamine released upon incubation of iris tissue extract with exogenous hyaluronate substrate. We found that incorporation of label into hyaluronate was consistently higher in the regeneration-activated irises of lentectomized eyes than in control irises from sham-operated eyes. Hyaluronate labeling was higher in the dorsal (lens-forming) region of the iris than in ventral (nonlens-forming) iris tissue during the regeneration process. Label accumulation into hyaluronate was maximum between 10 and 15 days after lentectomy, the period of most pronounced dedifferentiation in the dorsal iris epithelium. Both normal and regenerating irises demonstrated a high level of endogenous hyaluronidase activity with a pH optimum of 35–40. Hyaluronidase activity was 1.7 to 2 times higher in dorsal iris tissue than in ventral irises both prior to lentectomy and throughout the regeneration process. We suggest that enhanced hyaluronate accumulation may facilitate the dedifferentiation of iris epithelial cells in the dorsal iris and prevent precocious withdrawal from the cell cycle. The high level of hyaluronidase activity in the dorsal iris may promote the turnover and remodeling of extracellular matrix components required for cell-type conversion.