Tissue Inhibitor Of Metalloproteinase-1 Expression Research Articles

HIV Protease Inhibitors Block HPV16-Induced Murine Cervical Carcinoma and Promote Vessel Normalization in Association with MMP-9 Inhibition and TIMP-3 Induction.

Antiretrovirals belonging to the human immunodeficiency virus (HIV) protease inhibitor (HIV-PI) class exert inhibitory effects across several cancer types by targeting tumor cells and its microenvironment. Cervical carcinoma represents a leading cause of morbidity and mortality, particularly in women doubly infected with high-risk human papillomaviruses (HR-HPV) and HIV; of note, combined antiretroviral therapy has reduced cervical carcinoma onset and progression in HIV-infected women. We evaluated the effectiveness and mechanism(s) of action of HIV-PI against cervical carcinoma using a transgenic model of HR-HPV-induced estrogen-promoted cervical carcinoma (HPV16/E2) and found that treatment of mice with ritonavir-boosted HIV-PI, including indinavir, saquinavir, and lopinavir, blocked the growth and promoted the regression of murine cervical carcinoma. This was associated with inhibition of tumor angiogenesis, coupled to downregulation of matrix metalloproteinase (MMP)-9, reduction of VEGF/VEGFR2 complex, and concomitant upregulation of tissue inhibitor of metalloproteinase-3 (TIMP-3). HIV-PI also promoted deposition of collagen IV at the epithelial and vascular basement membrane and normalization of both vessel architecture and functionality. In agreement with this, HIV-PI reduced tumor hypoxia and enhanced the delivery and antitumor activity of conventional chemotherapy. Remarkably, TIMP-3 expression gradually decreased during progression of human dysplastic lesions into cervical carcinoma. This study identified the MMP-9/VEGF proangiogenic axis and its modulation by TIMP-3 as novel HIV-PI targets for the blockade of cervical intraepithelial neoplasia/cervical carcinoma development and invasiveness and the normalization of tumor vessel functions. These findings may lead to new therapeutic indications of HIV-PI to treat cervical carcinoma and other tumors in either HIV-infected or uninfected patients.

Extracellular matrix collagen biomarkers levels in patients with chronic thromboembolic pulmonary hypertension.

Limited data exist on changes in the extracellular matrix (ECM) collagen biomarkers levels during chronic thromboembolic pulmonary hypertension (CTEPH) development. This study aimed to investigate ECM collagen biomarkers levels in stable patients with CTEPH. Patients with CTEPH and healthy persons were enrolled. Serum levels of procollagen III N-terminal peptide (PIIINP), carboxyterminal propeptide of type I procollagen (PICP), matrix metalloproteinases (MMP2), MMP9, and tissue inhibitor of metalloproteinases 1(TIMP1) were measured by ELISA. Clinical data coincident with samples were collected. The pulmonary endarterectomy (PEA) and control pulmonary artery tissue samples were analyzed for genetic and immunohistochemical differences. The serum concentrations of PIIINP, PICP, MMP2, and MMP9 decreased significantly in CTEPH patients compared to healthy controls (P < 0.001 for each). CTEPH patients had higher serum concentrations of TIMP1 (median, 111.97 [interquartile range, 84.35-139.93]) compared to healthy controls (74.97 [44.03-108.45] ng/mL, P < 0.001). The MMP2 to TIMP1 ratio was lower in patients than in the controls (P < 0.001). After adjusting for the body mass index (BMI), the MMP2 to TIMP1 ratio correlated negatively with pulmonary vascular resistance (PVR) (r = - 0.327, P = 0.025). Increased TIMP1 (P = 0.04) gene expression was identified in tissues of CTEPH patients. Immunohistochemistry results of vascular walls substantiated qRT-PCR results. This study indicates that ECM collagen biomarkers levels were significantly different in stable patients with CTEPH and healthy controls with significantly increased TIMP1 and decreased MMP2 and MMP9. Differences in TIMP1 expression should be expected not only among healthy controls and patients serum, but also across pathological tissue regions. These findings suggest that the state of vascular remodeling in pulmonary vascular bed in stable patients may be represented by ECM collagen biomarkers levels. We conclude that TIMP1 may play an important role in pulmonary vascular reconstruction in stable CTEPH patients.

Zingerone Attenuates Pi-induced Vascular Calcification via AMPK-mediated TIMP4 Expression.

ObjectiveVascular calcification requires the differentiation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells. This phenomenon can be enhanced by inflammation and oxidative stress. Zingerone is one of the active ingredients present in the ginger plant that has anti-inflammatory and antioxidant effects. Other functions include anti-obesity, anti-nausea effects. However, the functions of zingerone on vascular calcification has not yet been elucidated. This study investigated the effect of zingerone on vascular calcification and its molecular mechanism.MethodsReverse transcription-polymerase chain reaction (PCR), real-time PCR and Western blot analysis was used to measure expression levels of osteogenic marker genes and to investigate whether calcification was regulated by the expression of AMP-activated protein kinase (AMPK) and tissue inhibitor of metalloproteinase 4 (TIMP4). Alizarin red S staining was used to measure calcium deposition. Studies were carried out in VSMCs.ResultsZingerone induced the expression of 2 markers of VSMCs differentiation (α-smooth muscle actin (α-SMA) and smooth muscle 22α (SM22α)) and decreased the expression of core-binding factor α-1 (CBFA1). Additionally, zingerone decreased inorganic phosphate (Pi)-induced expression of distal-less homeobox 5 and CBFA1. AMPK phosphorylation and TIMP4 expression were increased by zingerone. Importantly, zingerone protected VSMCs from calcification, and this protective effect was confirmed by increased TIMP4 via overexpression of AMPK, and inhibition of TIMP4 by Compound C. Zingerone upregulated AMPK/TIMP4 expression and recovered Pi-induced inhibition of TIMP4.ConclusionsTaken together, our results show that zingerone inhibits Pi-induced vascular calcification by regulating the AMPK/TIMP4 signaling cascade in VSMCs. These results suggest that the natural product zingerone could be useful for treating vascular and metabolic diseases.

Long non-coding RNA HAND2-AS1 delays cervical cancer progression via its regulation on the microRNA-21-5p/TIMP3/VEGFA axis.

Cervical cancer is a common cause of cancer-related mortality in women. Mounting evidence suggests that long non-coding RNAs (lncRNAs) function vitally in many cancers. In this study, we discovered that the regulation of the heart and neural crest derivatives expressed 2-antisense RNA 1 (HAND2-AS1) in cervical cancer. RT-qPCR was conducted to detect the expression of HAND2-AS1 and microRNA-21-5p (miR-21-5p). The relationship of HAND2-AS1 and miR-21-5p was identified by dual-luciferase reporter gene assay. The roles of HAND2-AS1, miR-21-5p and tissue inhibitor of metalloproteinases-3 (TIMP3) in cervical cancer were accessed via gain- and loss-of-function approaches. The expression of related proteins in the vascular endothelial growth factor A (VEGFA) signaling pathway was detected through Western blot analysis. Finally, xenografts of cervical cancer in nude mice were established to assess the effect of HAND2-AS1 on tumorigenesis in vivo. HAND2-AS1 and TIMP3 were downregulated in cervical cancer, which were identified to be associated with a poor prognosis of patients with cervical cancer. Moreover, HAND2-AS1 was upregulated the expression of TIMP3 through competitively binding to miR-21-5p. Overexpressed HAND2-AS1 or downregulated miR-21-5p inhibited cell proliferation, migration, and invasion while promoting cell apoptosis, in association with increased expression of proteins in VEGFA signaling pathway. These changes were reversed by silencing of TIMP3. Overexpressed HAND2-AS1 reduced the tumor formation ability in nude mice. In summary, HAND2-AS1 may exert inhibitory effects on cervical cancer cell growth and cervical cancer development through its regulation on the miR-21-5p/TIMP3/VEGFA axis. This highlights that HAND2-AS1 may serve as a potential target for cervical cancer diagnosis and treatment.

Myeloid-Derived TSP1 (Thrombospondin-1) Contributes to Abdominal Aortic Aneurysm Through Suppressing Tissue Inhibitor of Metalloproteinases-1.

Abdominal aortic aneurysm is characterized by the progressive loss of aortic integrity and accumulation of inflammatory cells primarily macrophages. We previously reported that global deletion of matricellular protein TSP1 (thrombospondin-1) protects mice from aneurysm formation. The objective of the current study is to investigate the cellular and molecular mechanisms underlying TSP1's action in aneurysm. Approach and Results: Using RNA fluorescent in situ hybridization, we identified macrophages being the major source of TSP1 in human and mouse aneurysmal tissues, accounting for over 70% of cells that actively expressed Thbs1 mRNA. Lack of TSP1 in macrophages decreased solution-based gelatinase activities by elevating TIMP1 (tissue inhibitor of metalloproteinases-1) without affecting the major MMPs (matrix metalloproteinases). Knocking down Timp1 restored the ability of Thbs1-/- macrophages to invade matrix. Finally, we generated Thbs1flox/flox mice and crossed them with Lyz2-cre mice. In the CaCl2-induced model of abdominal aortic aneurysm, lacking TSP1 in myeloid cells was sufficient to protect mice from aneurysm by reducing macrophage accumulation and preserving aortic integrity. TSP1 contributes to aneurysm pathogenesis, at least in part, by suppressing TIMP1 expression, which subsequently enables inflammatory macrophages to infiltrate vascular tissues.

The fatty-acid amide hydrolase inhibitor URB597 inhibits MICA/B shedding

MICA/B proteins are expressed on the surface of various types of stressed cells, including cancer cells. Cytotoxic lymphocytes expressing natural killer group 2D (NKG2D) receptor recognize MICA/B and eliminate the cells. However, cancer cells evade such immune recognition by inducing proteolytic shedding of MICA/B proteins. Therefore, preventing the shedding of MICA/B proteins could enhance antitumor immunity. Here, by screening a protease inhibitor library, we found that the fatty-acid amide hydrolase (FAAH) inhibitor, URB597, suppresses the shedding of MICA/B. URB597 significantly reduced the soluble MICA level in culture medium and increased the MICA level on the surface of cancer cells. The effect was indirect, being mediated by increased expression of tissue inhibitor of metalloproteinases 3 (TIMP3). Knockdown of TIMP3 expression reversed the effect of URB597, confirming that TIMP3 is required for the MICA shedding inhibition by URB597. In contrast, FAAH overexpression reduced TIMP3 expression and the cell-surface MICA level and increased the soluble MICA level. These results suggest that inhibition of FAAH could prevent human cancer cell evasion of immune-mediated clearance.

Matrix Metalloproteinases and Tissue Inhibitors of Metalloproteinases in Extracellular Matrix Remodeling during Left Ventricular Diastolic Dysfunction and Heart Failure with Preserved Ejection Fraction: A Systematic Review and Meta-Analysis.

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are pivotal regulators of extracellular matrix (ECM) composition and could, due to their dynamic activity, function as prognostic tools for fibrosis and cardiac function in left ventricular diastolic dysfunction (LVDD) and heart failure with preserved ejection fraction (HFpEF). We conducted a systematic review on experimental animal models of LVDD and HFpEF published in MEDLINE or Embase. Twenty-three studies were included with a total of 36 comparisons that reported established LVDD, quantification of cardiac fibrosis and cardiac MMP or TIMP expression or activity. LVDD/HFpEF models were divided based on underlying pathology: hemodynamic overload (17 comparisons), metabolic alteration (16 comparisons) or ageing (3 comparisons). Meta-analysis showed that echocardiographic parameters were not consistently altered in LVDD/HFpEF with invasive hemodynamic measurements better representing LVDD. Increased myocardial fibrotic area indicated comparable characteristics between hemodynamic and metabolic models. Regarding MMPs and TIMPs; MMP2 and MMP9 activity and protein and TIMP1 protein levels were mainly enhanced in hemodynamic models. In most cases only mRNA was assessed and there were no correlations between cardiac tissue and plasma levels. Female gender, a known risk factor for LVDD and HFpEF, was underrepresented. Novel studies should detail relevant model characteristics and focus on MMP and TIMP protein expression and activity to identify predictive circulating markers in cardiac ECM remodeling.

Effect of xiaotan huayu liqiao traditional Chinese medicine compound on myocardial fibrosis in rats with chronic intermittent hypoxia and its mechanism

Objective: To explore the role of transforming growth factor-β (TGF-β) signaling pathway in xiaotan huayu liqiao traditional Chinese medicine compound (XC)'s anti-myocardial fibrosis in chronic intermittent hypoxia (CIH) rats. Methods: Forty SD rats were randomly divided into normoxia group, oxygen + traditional Chinese medicine compound group ( TCMC), Chronic intermittent hypoxia model group (CIH), TCMC + CIH, 10 in each group. CIH cabin was built by filling with nitrogen and oxygen. Firstly, the volume fraction of oxygen in the cabin reduced from 21% to 9% in 90 s by filling the cabin with nitrogen. And then it gradually rose to 21% by reoxygenating in 90s, as a cycle. CIH and TCMC+CIH group rats were placed in the CIH device, while normoxia and TCMC group rats were placed in the normal oxygen chamber. In addition, rats in TCMC +CIH group and TCMC group were treated with XC crude drug (24 g/kg) daily by gavage, while rats in CIH group and normoxia group were given equal volume normal saline. Using sirius red staining, the collagen in myocardial interstitium was visualized. The protein expressions of collagen I, collagen III and fibronectin were detected by Western blot, p-Smad3, p-Smad2 and TGF-β protein in the TGF-β/Smads signaling pathway were also analyzed by Western blot. The mRNA expressions of matrix metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinase -2(TIMP-2) were measured by real-time quantitative polymerase chain reaction (PCR). Results: Compared with the rats exposed to normoxia, the CIH rats showed obvious collagen deposition, protein expressions of collagen I, collagen III and fibronectin were significantly increased in the myocardial tissue (P＜0.01). The protein expression levels of TGF-β, p-smad2 and p-smad3 in the myocardial tissue of the CIH rats were also significantly increased (P＜0.01). The up-regulation of TIMP-2 mRNA in the myocardial tissues resulted in the decrease of MMP-2 mRNA(P＜0.01). XC reduced myocardial fibrosis of CIH rats and inhibited the expressions of collagen I and collagen III and fibronectin protein (P＜0.05,P＜0.01,P＜0.05, respectively). The further mechanism study showed that XC inhibited the expression of TGF-β (P＜0.01), which down-regulated the expressions of p-smad2, p-smad3 and TIMP-2 (P＜0.05). Conclusion: XC could reduce the expression of TGF-β and smad2/3 phosphorylation, down-regulate the expression of TIMP-2, which would inhibit the formation of myocardial fibrosis in CIH rats, and improve the myocardial function of CIH rats.

Cross-talk between synovial fibroblasts and chondrocytes in condylar hyperplasia: an in vitro pilot study

Increasing evidence indicates an interaction between the synovium and the cartilage in the temporomandibular joint (TMJ) and other joints. We recently demonstrated that the expression of proangiogenic factors was enhanced and that of factors promoting matrix degradation was decreased in synovial fibroblasts in condylar hyperplasia (CH). The aim of this study was to explore whether CH chondrocytes can affect the expression of these factors of synovial fibroblasts in a co-culture system. The expressions of vascular endothelial growth factor (VEGF), cluster of differentiation 34 (CD34), fibroblast growth factor 2 (FGF-2), and tissue inhibitor of metalloproteinase 1 (TIMP1) from CH condylar tissues were observed by using immunohistochemical methods. Synovial fibroblasts of control tissues were co-cultured with the chondrocytes of CH, and protein expressions of VEGF, FGF-2, thrombospondin 1 (TSP1), matrix metalloproteinase 3 (MMP3), and TIMP1 were examined by using Western blotting. Positive staining for VEGF, CD34, FGF-2, and TIMP1 was found in the hypertrophic cartilage layer of CH condylar tissues. Protein expressions of VEGF, FGF-2, and TIMP1 were significantly increased in co-cultured synovial fibroblasts, but TSP1 and MMP3 expressions were decreased. The angiogenic factors and matrix degradation-related factors in synovial fibroblasts co-cultured with CH chondrocytes showed the same trends as those in synovial fibroblasts from CH tissue, suggesting potential cross-talk between synovial fibroblasts and chondrocytes during CH progression.

MMP-2 and TIMP-2 expression, quantitative analysis and biomechanical changes in scar hypertrophy after autologous free transplantation of rabbit oral mucosa and scrotal skin.

This study aimed to investigate the long-term scar hypertrophy in the rabbit transplanted oral mucosa and scrotal skin with changed matrix environment, as well as the scar location expression, quantitative analysis of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) and biomechanical changes in the transplanted tissues. The split-thickness skin grafts were collected from the oral mucosas and scrotal skins of 30 male rabbits, and prepared into reelpipes for autologous transplantation into the rabbit back muscular tissues. Samples were collected to carry out elastic tensile mechanical detection and histological observation. The maximum longitudinal tensile displacement of scrotal skin before 8 weeks of transplantation was greater than that after 8 weeks of transplantation (P < 0.05). The expression intensities of MMP-2 and TIMP-2 in the oral mucosa and in scrotal skin at 2 W time point were higher than those at To time point (P < 0.05). The expression quantities of TIMP-2 in oral mucosa and scrotal skin during 8–24 W were higher than those of MMP-2 (P < 0.05). At 8 W time point, the TIMP-2/MMP-2 ratio in scrotal skin was higher than that in oral mucosa (P < 0.05). MMP-2 and TIMP-2 expression in normal oral mucosa and scrotal skin is weak, but their expression is remarkably up-regulated after 2 weeks of transplantation, revealing that scar formation was related to the high expression of MMP-2 and TIMP-2. At the 8th–24th weeks, the AOD values of TIMP-2 in oral mucosa and scrotal skin are apparently higher than those of MMP-2; moreover, the TIMP-2/MMP-2 ratio in scrotal skin at the 8th week was higher than that in oral mucosa, which can well explain the earlier scar formation in scrotal skin than in oral mucosa, and it also suggests that the different expression levels between TIMP-2 and MMP-2 may account for the important cause of scar formation.

Leonurus sibiricus root extracts decrease airway remodeling markers expression in fibroblasts.

Bronchial asthma is believed to be provoked by the interaction between airway inflammation and remodeling. Airway remodeling is a complex and poorly understood process, and controlling it appears key for halting the progression of asthma and other obstructive lung diseases. Plants synthesize a number of valuable compounds as constitutive products and as secondary metabolites, many of which have curative properties. The aim of this study was to evaluate the anti-remodeling properties of extracts from transformed and transgenic Leonurus sibiricus roots with transformed L. sibiricus roots extract with transcriptional factor AtPAP1 overexpression (AtPAP1). Two fibroblast cell lines, Wistar Institute-38 (WI-38) and human fetal lung fibroblast (HFL1), were incubated with extracts from transformed L. sibiricus roots (TR) and roots with transcriptional factor AtPAP1 over-expression (AtPAP1 TR). Additionally, remodeling conditions were induced in the cultures with rhinovirus 16 (HRV16). The expressions of metalloproteinase 9 (MMP)-9, tissue inhibitor of metalloproteinases 1 (TIMP-1), arginase I and transforming growth factor (TGF)-β were determined by quantitative polymerase chain reaction (qPCR) and immunoblotting methods. AtPAP1 TR decreased arginase I and MMP-9 expression with no effect on TIMP-1 or TGF-β mRNA expression. This extract also inhibited HRV16-induced expression of arginase I, MMP-9 and TGF-β in both cell lines (P<0·05) Our study shows for the first time to our knowledge, that transformed AtPAP1 TR extract from L. sibiricus root may affect the remodeling process. Its effect can be attributed an increased amount of phenolic acids such as:chlorogenic acid, caffeic acid or ferulic acid anddemonstrates the value of biotechnology in medicinal research.

Expression of Matrix Metalloproteinase-9 and Tissue Inhibitor of Metalloproteinase-1 in Endometriosis Menstrual Blood.

Endometriosis is a gynecological disease characterized by the presence of endometrial-like tissue outside the uterine cavity. Remodeling of the extracellular matrix (ECM) is a prerequisite for tissue implantation. The presence of matrix metalloproteinase (MMP) and its inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), in shed endometrium cells has a significant role in ECM degradation. A case–control study was performed to find other diagnostic markers using menstrual blood. We examined a sample of 68 women who visited the gynecology clinic in Dr Hasan Sadikin General Hospital, 40% of whom were confirmed to have endometriosis, and the rest tested negative by histopathological examination. All endometriotic cases presented MMP-9 and TIMP-1 expression with different cell distribution. MMP-9 expression in endometriosis patients was increased compared to the controls (p = 0.002). Expression of MMP-9 in >80% of endometrial cells was associated with a higher risk for endometriosis (OR 4.44 95% CI 1.31 to 15.56) compared to MMP-9 expression in 50%–80% of cells. TIMP-1 cell expression in women with endometriosis was lower than in the control group (p = 0.030). Subjects with TIMP-1 expression in 20%–50% of endometrial cells had a higher risk for endometriosis (OR 4.5, 95%CI 1.21–17.42) compared those with TIMP-1 expression in 50%–80% of cells. These expressions levels can be useful to predict endometriosis.

Hepatoprotective effect and possible mechanism of phytoestrogen calycosin on carbon tetrachloride-induced liver fibrosis in mice.

The study was to explore the hepatoprotective effect and possible mechanism of calycosin on carbon tetrachloride (CCl4)-induced liver fibrosis in mice. Hepatic fibrosis was induced by intraperitoneal injection of CCl4 in C57BL/6 male mice. Serum alanine aminotransferase (ALT) and aspartate transaminase (AST) activity, superoxide dismutase (SOD) activity, and hydroxyproline (Hyp) and malondialdehyde (MDA) levels were determined by biochemical assays. Liver histopathology was assessed by H&E and Masson trichrome staining. The mRNA expressions of α-smooth muscle actin (α-SMA), collagen-I (Col-I), Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) were determined using qRT-PCR. The protein levels of α-SMA, Col-I, estrogen receptor α (ERα), estrogen receptor β (ERβ), tissue inhibitor of metalloproteinase-1 (TIMP-1), matrix metalloproteinase-1 (MMP-1), JAK2, phospho-JAK2 (p-JAK2), STAT3, and phospho-STAT3 (p-STAT3) were detected by Western blotting. The levels of α-SMA and ERβ were measured by immunohistochemistry. Calycosin significantly reduced liver index, MDA level, and ALT and AST activity and increased SOD activity. The α-SMA, Col-I, and Hyp of the calycosin group were significantly lower than those of the model group. Calycosin increased MMP-1 and inhibited TIMP-1 expression resulting in the improvement of MMP-1/TIMP-1 ratio. Importantly, calycosin improved ERβ protein expression, JAK2 and STAT3 mRNA expressions, p-JAK2/JAK2, and p-STAT3/STAT3 relative protein expressions. However, ERα, JAK2, and STAT3 protein expressions were relatively unchanged. Calycosin significantly inhibits liver fibrosis in mice, and its mechanism may involve the following: calycosin inhibits oxidative stress; calycosin inhibits collagen synthesis and balances MMP-1/TIMP-1 system; calycosin increases ERβ expression and activates JAK2-STAT3 pathway.

Isolation and Characterization of primary human trabecular meshwork cells from segmental flow regions: New tools for understanding segmental flow

Segmental flow in the human trabecular meshwork is a well-documented phenomenon but in depth mechanistic investigations of high flow (HF) and low flow (LF) regions are restricted due to the small amount of tissue available from a single donor. To address this issue we have generated and characterized multiple paired HF and LF cell strains. Here paired HF and LF cell strains were generated from single donors. Cells were characterized for growth and proliferation, as well as gene and protein expression of potential segmental region markers. Cells isolated from HF and LF regions have similar growth and proliferation rates. Gene expression data reveals vascular cell adhesion protein 1 (VCAM1), thrombospondin 2 (THBS2), and tissue inhibitor of metalloproteinase 1 (TIMP1) are potential markers of LF cells in vitro. Protein expression of VCAM1, THBS2 and TIMP1 are complex and may reflect the dynamic nature of the TM. Initial protein expression levels of these genes is either similar between HF and LF cells (VCAM1, THBS2), or higher in HF compared to LF in some strains (TIMP1). However, after long term culture LF cells express higher levels of VCAM1, TIMP1 and THBS2 protein compared to HF cells. HF and LF cell strains are a powerful new tool that enable understanding segmental flow allowing for multiple experiments on the same genetic background.

Changes of Tissue Inhibitors of Metalloproteinase Expression by Transient Receptor Potential Melastatin-Subfamily Member 7 Suppression in Renal Cancer Cells

Changes of Tissue Inhibitors of Metalloproteinase Expression by Transient Receptor Potential Melastatin-Subfamily Member 7 Suppression in Renal Cancer Cells

β-Adrenoceptors regulate matrix metalloproteinase expression in human urothelial cells under hydrostatic pressure.

The bladder wall is constantly subjected to intravesical pressure during the filling and voiding cycles. An imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) under elevated intravesical pressure contributes to pathological changes in the bladder. To investigate the changes in human urothelial cells (HUCs) under elevated intravesical pressure, this study analyzed the effect of β-adrenoceptor signaling on the expression of MMPs and TIMPs in HUCs exposed to pathological hydrostatic pressure (HP) (70 cm H2 O) for 6 hours. Quantitative polymerase chain reaction, Western blot analysis, and cell fluorescence staining were used to explore the effect of β-adrenoceptor signaling on the expression of MMPs and TIMPs in HUCs after agonist and/or antagonist treatment. The expression levels of β2 - and β3 -adrenoceptor, MMP1, and MMP2 were greatly downregulated, while the expression of TIMP1 was greatly upregulated. Formoterol and BRL 37344, which are agonists of β2 - and β3 -adrenoceptor, respectively, significantly increased MMP1 and MMP2 expression under 70 cm H2 O. As a classic downstream pathway of β2 - and β3 -adrenoceptor, protein kinase A (PKA) signaling inhibited MMP1 and MMP2 expression by regulating cAMP response element binding protein (CREB) activity. MMP1 and MMP2 expression in HUCs under 70 cm H2 O was modified by β2 - and β3 -adrenoceptor via the PKA/CREB pathway. This outcome suggests that MMPs likely participate in the pathological effects of elevated intravesical pressure. The underlying mechanism of β2 - and β3 -adrenoceptor in elevated intravesical pressure was also revealed; this mechanism constitutes a new potential therapeutic target for partial bladder outlet obstruction.

Expression of Collagen (Types I, III, and V), HSP47, MMP-2, and TIMP-1 in Retrobulbar Adipose Tissue of Patients with Thyroid-Associated Orbitopathy.

Objective This study aimed to investigate the expression of collagen (types I, III, and V), heat shock protein 47 (HSP47), matrix metalloproteinase-2 (MMP-2), and tissue inhibitors of metalloproteinase-1 (TIMP-1) in the retrobulbar adipose tissues of patients with thyroid-associated orbitopathy (TAO). Materials and Methods The retrobulbar adipose tissues were collected from 4 TAO patients undergoing orbital decompression and 4 ocular enucleation patients with atrophic eyeball caused by ocular trauma between May 2019 and September 2019. Masson staining was performed to analyze the differences in collagen expression and degree of histologic fibrosis in each sample. The protein expressions of collagen (types I, III, and V), HSP47, MMP-2, and TIMP-1 were determined by western blotting. The data of western blotting were analyzed using SPSS version 17.0, with independent t-tests. Results The results of Masson staining showed that the expression of collagen fibers in the TAO group was significantly higher than that in the control group, and the fibers were diffuse and irregular in distribution. The expression level of collagen (types I, III, and V), HSP47, MMP-2, and TIMP-1 in the TAO group were significantly higher than that in the control group (P < 0.05). Conclusion The proliferation and fibrosis of retrobulbar adipose tissue in TAO patients might be related to the increased expression of collagen (types I, III, and V) and HSP47 and decreased degradation of extracellular matrix.

Gene Suppression of the Chloride Channel 2 Suppressed TGF-β1-Induced Proliferation, Collagen Synthesis, and Collagen Gel Contraction Mediated by Conjunctival Fibroblasts

Background: Excessive scarring of filtering blebs is the main cause of surgical failure in glaucoma. Previous studies have highlighted the role of chloride channels (ClCs) in scar formation, whereas the role of ClCs in scarring of filtering blebs has not been studied. Objectives: The objective of this study was to investigate the effects of the chloride channel 2 (ClC-2) on scar formation of filtering blebs after glaucoma filtering surgery. Methods: ClC-2 siRNA-transfected human conjunctival fibroblasts (HConFs) were cultured in type 1 collagen gels in the presence of transforming growth factor (TGF)-β1. Collagen gel contraction was evaluated based on the gel area. 3D-cultured HConFs were treated with the ClC blocker NPPB in the presence of TGF-β1, and cell proliferation collagen synthesis and contractility were measured. The expression levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in HConFs were assessed by Western blotting and qPCR. Results: TGF-β1 induced cell proliferation, cell cycle progression, collagen synthesis, and collagen gel contraction in HConFs. TGF-β1 increased MMP-2 and MMP-9 levels but inhibited the expression of TIMPs. NPPB and ClC-2 siRNA transfection inhibited TGF-β2-induced cell proliferation, cell cycle progression, collagen synthesis, and collagen gel contraction, mediated by HConFs. TGF-β2-induced increases in MMP-2 and MMP-9 were also inhibited by NPPB and ClC-2 siRNA transfection, but TIMP expression was increased by NPPB and ClC-2 siRNA transfection. Conclusions: These findings demonstrate that ClC-2 ClCs modulate TGF-β1-induced cell proliferation, collagen synthesis, and collagen gel contraction of HConFs by attenuating MMP-2 and MMP-9 production and by stimulating TIMP1 production. NPPB may therefore prove to be of clinical value for the inhibition of scar formation of filtering blebs.

Loss of TIMP3 expression induces inflammation, matrix degradation, and vascular ingrowth in nucleus pulposus: A new mechanism of intervertebral disc degeneration.

Low back pain (LBP) is one of the most common complains in orthopedic outpatient department and intervertebral disc degeneration (IDD) is one of the most important reasons of LBP. The mechanisms of IDD contain a complex biochemical cascade which includes inflammation, vascular ingrowth, and results in degradation of matrix. In our study, we used both in vitro and in vivo models to investigate the relation between tissue inhibitor of metalloproteinase-3 (TIMP3) expression and IDD. Loss of TIMP3 expression was found in degenerative intervertebral disc (IVD), this change of expression was closely related with the dephosphorylation of smad2/3. Overexpression of TIMP3 significantly inhibited the release of TNF-α and matrix degradation induced by Lipopolysaccharide. Vascular ingrowth was also suppressed by TIMP3 in the in vitro and in vivo models. Further, animal experiments confirmed that the degeneration of IVD was reduced after overexpression of TIMP3 in nucleus pulposus. Taken together, our results indicated TIMP-3 might play an important role in the pathogenesis of IDD and therefore be a potential therapeutic target in the future.

Anti‑inflammatory and anti‑catabolic effect of non‑animal stabilized hyaluronic acid and mesenchymal stem cell‑conditioned medium in an osteoarthritis coculture model.

Previous clinical studies have reported the clinical effectiveness of non‑animal stabilized hyaluronic acid (NASHA) and adipose‑derived mesenchymal stromal/stem cells (MSC) in the treatment of knee osteoarthritis (OA). Unlike MSC secreted mediators, invitro anti‑inflammatory effects of NASHA have not been evaluated. We aimed to evaluate and compare the anti‑inflammatory effect of NASHA and MSC conditioned medium (stem cell‑conditioned medium; SC‑CM), in an explant‑based coculture model of OA. Cartilage and synovial membrane from seven patients undergoing total knee arthroplasty were used to create a coculture system. Recombinant IL‑1β was added to the cocultures to induce inflammation. Four experimental groups were generated: i)Basal; ii)IL‑1β; iii)NASHA (NASHA+IL‑1β); and iv)SC‑CM (SC‑CM+IL‑1β). Glycosaminoglycans (GAG) released in the culture medium and of nitric oxide (NO) production were quantified. Gene expression in cartilage and synovium of IL‑1β, matrix metallopeptidase13 (MMP13), ADAM metallopeptidase with thrombospondin type1 motif5 (ADAMTS5) and tissue inhibitor of metalloproteinases1 (TIMP1) was measured by reverse transcription‑quantitative PCR. Media GAG concentration was decreased in cocultures with NASHA and SC‑CM (48h, P<0.05; 72h, P<0.01) compared with IL‑1β. Production of NO was significantly lower only in SC‑CM after72h (P<0.01). In cartilage, SC‑CM inhibited the expression of IL‑1β, MMP13 and ADAMTS5, while NASHA had this effect only in MMP13 and ADAMTS5. In synovium, SC‑CM decreased the expression level of MMP13 and ADAMTS5, while NASHA only decreased ADAMTS5 expression. Both NASHA and SC‑CM increased TIMP1 expression in cartilage and synovium. Treatments with NASHA and SC‑CM were shown to be a therapeutic option that may help counteract the catabolism produced by the inflammatory state in knee OA. The anti‑inflammatory mediators produced by MSC promote a lower expression of inflammatory targets in our study model.