Disease-Causing TIMP3 Variants and Deep Phenotyping of Two Czech Families with Sorsby Fundus Dystrophy Associated with Novel p.(Tyr152Cys) Mutation.

We aim to report the ocular phenotype and molecular genetic findings in two Czech families with Sorsby fundus dystrophy and to review all the reported TIMP3 pathogenic variants. Two probands with Sorsby fundus dystrophy and three first-degree relatives underwent ocular examination and retinal imaging, including optical coherence tomography angiography. The DNA of the first proband was screened using a targeted ocular gene panel, while, in the second proband, direct sequencing of the TIMP3 coding region was performed. Sanger sequencing was also used for segregation analysis within the families. All the previously reported TIMP3 variants were reviewed using the American College of Medical Genetics and the Association for Molecular Pathology interpretation framework. A novel heterozygous variant, c.455A>G p.(Tyr152Cys), in TIMP3 was identified in both families and potentially de novo in one. Optical coherence tomography angiography documented in one patient the development of a choroidal neovascular membrane at 54 years. Including this study, 23 heterozygous variants in TIMP3 have been reported as disease-causing. Application of gene-specific criteria denoted eleven variants as pathogenic, eleven as likely pathogenic, and one as a variant of unknown significance. Our study expands the spectrum of TIMP3 pathogenic variants and highlights the importance of optical coherence tomography angiography for early detection of choroidal neovascular membranes.

TIMP-3 Alleviates White Matter Injury After Subarachnoid Hemorrhage in Mice by Promoting Oligodendrocyte Precursor Cell Maturation.

Subarachnoid hemorrhage (SAH) is associated with high mortality and disability rates, and secondary white matter injury is an important cause of poor prognosis. However, whether brain capillary pericytes can directly affect the differentiation and maturation of oligodendrocyte precursor cells (OPCs) and subsequently affect white matter injury repair has still been revealed. This study was designed to investigate the effect of tissue inhibitor of metalloproteinase-3 (TIMP-3) for OPC differentiation and maturation. PDGFRßret/ret and wild-type C57B6J male mice were used to construct a mouse model of SAH via endovascular perforation in this study. Mice were also treated with vehicle, TIMP-3 RNAi or TIMP-3 RNAi + TIMP-3 after SAH. The effect of TIMP-3 on the differentiation and maturation of OPCs was determined using behavioral score, ELISA, transmission electron microscopy, immunofluorescence staining and cell culture. We found that TIMP-3 was secreted mainly by pericytes and that SAH and TIMP-3 RNAi caused a significant decrease in the TIMP-3 content, reaching a nadir at 24 h, followed by gradual recovery. In vitro, the myelin basic protein content of oligodendrocytes after oxyhemoglobin treatment was increased by TIMP-3 overexpression. The data indicates TIMP-3 could promote the differentiation and maturation of OPCs and subsequently improve neurological outcomes after SAH. Therefore, TIMP-3 could be beneficial for repair after white matter injury and could be a potential therapeutic target in SAH.

Review: Mechanisms of TIMP-3 accumulation and pathogenesis in Sorsby fundus dystrophy.

Sorsby fundus dystrophy (SFD) is a rare, inherited form of macular degeneration caused by mutations in the gene encoding tissue inhibitor of metalloproteinases 3 (TIMP-3). There are 21 mutations currently associated with SFD, with some variants (e.g., Ser179Cys, Tyr191Cys, and Ser204Cys) having been studied much more than others. We review what is currently known about the identified SFD variants in terms of their dimerization, metalloproteinase inhibition, and impact on angiogenesis, with a focus on disparities between reports and areas requiring further study. We also explore the potential molecular mechanisms leading to the accumulation of extracellular TIMP-3 in SFD and consider how accumulated TIMP-3 causes macular damage. Recent reports have identified extraocular pathologies in a small number of SFD patients. We discuss these intriguing findings and consider the apparent discrepancy between the widespread expression of TIMP-3 and the primarily retinal manifestations of SFD. The potential benefits of novel experimental approaches (e.g., metabolomics and stem cell models) in terms of investigating SFD pathology are presented. The review thus highlights gaps in our current molecular understanding of SFD and suggests ways to support the development of novel therapies.

Matrix metalloproteinases are associated with severity of disease among COVID-19 patients: A possible pharmacological target.

COVID-19 is a devastating disease and imbalanced matrix metalloproteinase (MMP) activity may contribute to its pathophysiology. This exploratory study examined whether increased circulating concentrations of MMP-2 and MMP-9, and their endogenous inhibitors, the tissue inhibitors of MMP (TIMP)-1, TIMP-2, TIMP-3 and TIMP-4 are persistently found in patients 2 weeks after their recovery from severe or critical COVID-19 as compared with those in healthy controls. Subjects who had severe (n = 26) or critical (n = 25) PCR-confirmed COVID-19 and healthy controls (n = 21) had blood samples drawn 2 weeks after recovery and serum MMP-2, MMP-9, TIMP-1, TIMP-2, TIMP-3 and TIMP-4 were determined using two Human Luminex® Discovery Assays. Circulating MMP activity was also determined by gel zymography. Patients who had severe or critical COVID-19 had increased circulating MMP-9 and MMP-2 concentrations, with increased MMP-9/TIMP-1 and MMP-2/TIMP-2 ratios indicating increased MMP activity, confirmed by gel zymography (all p < 0.05). Higher circulating MMP-9 (but not MMP-2) concentrations were found in critical versus severe COVID-19 (p < 0.05). We found increased circulating MMP-9 and MMP-2 concentrations and activity many days after recovery from the acute disease, with MMP-9 levels associated with disease severity. These biochemical alterations suggest that MMP-2 and MMP-9 may be important pharmacological targets in COVID-19.

Tissue Inhibitor of Metalloproteinase 3: Unravelling Its Biological Function and Significance in Oncology.

Tissue inhibitor of metalloproteinases-3 (TIMP3) is vital in regulating several biological processes. TIMP3 exerts antitumour effects via matrix metalloproteinase (MMP)-dependent and MMP-independent pathways. Due to promoter methylation and miRNA binding, TIMP3 expression has been observed to decrease in various cancers. Consequently, the migration and invasion of cancer cells increases. Conflicting results have reported that expression levels of TIMP3 in primary and advanced cancers are higher than those in healthy tissues. Therefore, the role of TIMP3 in cancer biology and progression needs to be elucidated. This review provides an overview of TIMP3, from its biological function to its effects on various cancers. Moreover, gynaecological cancers are discussed in detail. TIMP3 has been associated with cervical adenocarcinoma as well as cancer development in serous ovarian cancer and breast cancer metastasis. However, the relationship between TIMP3 and endometrial cancers remains unclear. TIMP3 may be a useful biomarker for gynaecological cancers and is a potential target for future cancer therapy.

TIMP3/Wnt axis regulates gliosis of Müller glia.

BACKGROUND: Previous studies have confirmed the expression of tissue inhibitor of metalloproteinase-3 (TIMP3) in Müller glia (MG). However, the role of TIMP3 in MG remains unknown. METHODS: A mouse model of laser-induced retinal damage and gliosis was generated using wild-type C57BL/6 mice. TIMP3 and associated proteins were detected using Western blotting and immunofluorescence microscopy. RNA sequencing (GSE132140) of mouse laser-induced gliosis was utilized for pathway analysis. TIMP3 overexpression was induced in human MG. Human vitreous samples were obtained from patients with proliferative diabetic retinopathy (PDR) and healthy controls for protein analysis. RESULTS: TIMP3 levels increased in mouse eyes after laser damage. Morphology and spatial location of TIMP3 indicated its presence in MG. TIMP3-overexpressing MG showed increased cellular proliferation, migration, and cell nuclei size, suggesting TIMP3-induced gliosis for retinal repair. Glial fibrillary acidic protein (GFAP) and vimentin levels were elevated in TIMP3-overexpressing MG and laser-damaged mouse retinas. RNA sequencing and Western blotting suggested a role for ß-catenin in mediating TIMP3 effects on the retina. Human vitreous samples from patients with PDR showed a positive correlation between TIMP3 and GFAP levels, both of which were elevated in patients with PDR. CONCLUSIONS: TIMP3 is associated with MG gliosis to enhance the repair ability of damaged retinas and is mediated by the canonical Wnt/ß-catenin. Changes in TIMP3 could potentially be used to control gliosis in a range of retinal diseases However, given the multifaceted nature of TIMP3, care must be taken when developing treatments that aim solely to boost the function of TIMP3. FUNDING: National Cheng Kung University Hospital, Taiwan (NCKUH-10604009 and NCKUH-11202007); the Ministry of Science and Technology (MOST 110-2314-B-006-086-MY3).

Loss of TIMP3, but not TIMP4, exacerbates thoracic and abdominal aortic aneurysm.

AIMS: Aorta exhibits regional heterogeneity (structural and functional), while different etiologies for thoracic and abdominal aortic aneurysm (TAA, AAA) are recognized. Tissue inhibitor of metalloproteinases (TIMPs) regulate vascular remodeling through different mechanisms. Region-dependent functions have been reported for TIMP3 and TIMP4 in vascular pathologies. We investigated the region-specific function of these TIMPs in development of TAA versus AAA. METHODS & RESULTS: TAA or AAA was induced in male and female mice lacking TIMP3 (Timp3-/-), TIMP4 (Timp4-/-) or in wildtype (WT) mice by peri-adventitial elastase application. Loss of TIMP3 exacerbated TAA and AAA severity in males and females, with a greater increase in proteinase activity, smooth muscle cell phenotypic switching post-AAA and -TAA, while increased inflammation was detected in the media post-AAA, but in the adventitia post-TAA. Timp3-/- mice showed impaired intimal barrier integrity post-AAA, but a greater adventitial vasa-vasorum branching post-TAA, which could explain the site of inflammation in AAA versus TAA. Severity of TAA and AAA in Timp4-/- mice was similar to WT mice. In vitro, Timp3 knockdown more severely compromised the permeability of human aortic EC monolayer compared to Timp4 knockdown or the control group. In aneurysmal aorta specimens from patients, TIMP3 expression decreased in the media in AAA, and in adventitial in TAA specimens, consistent with the impact of its loss in AAA versus TAA in mice. CONCLUSION: TIMP3 loss exacerbates inflammation, adverse remodeling and aortic dilation, but triggers different patterns of remodeling in AAA versus TAA, and through different mechanisms.

Sublytic C5b-9 induces TIMP3 expression by glomerular mesangial cells via TRAF6-dependent KLF5 K63-linked ubiquitination in rat Thy-1 nephritis.

Rat Thy-1 nephritis (Thy-1N) is an experimental model for studying human mesangioproliferative glomerulonephritis (MsPGN), and its pathological features are glomerular mesangial cell (GMC) proliferation and extracellular matrix (ECM) accumulation. Although we have confirmed that renal lesions of Thy-1N rats are sublytic C5b-9-dependent, and ECM accumulation is related to tissue inhibitor of matrix metalloproteinase (TIMP) inhibiting matrix metalloproteinase (MMP) activity, whether sublytic C5b-9 can induce TIMP production by GMC in Thy-1N rat and the underlying mechanism remains unclear. In the study, we proved that the expressions of TIMP3, krϋppel-like transcription factor 5 (KLF5) and tumor necrosis factor receptor-associated factor 6 (TRAF6) were simultaneously up-regulated both in the renal tissues of Thy-1N rats (in vivo) and in the GMC exposed to sublytic C5b-9 (in vitro). Further mechanism exploration discovered that KLF5 and TRAF6 as two upstream molecules could induce TIMP3 gene transcription through binding to the same region i.e., -1801nt to -1554nt (GGGGAGGGGC) and -228nt to -46nt (GCCCCGCCCC) of TIMP3 promoter. In the process, TRAF6 mediated KLF5 K63-linked ubiquitination at K99 and K100 enhancing KLF5 nuclear localization and binding to TIMP3 promoter, augmenting its gene activation. Furthermore, the experiments in vivo exhibited that silencing KLF5, TRAF6 or TIMP3 gene could markedly lessen renal KLF5 K63-linked ubiquitination or TIMP3 induction, ECM accumulation and other pathological changes of Thy-1N rats. Besides, the positive expressions of above-mentioned these proteins and ECM accumulation and their correlation in the renal tissues of MsPGN patients were also demonstrated. Overall, our findings implicate that KLF5 and TRAF6 play a promoting role in sublytic C5b-9-triggered TIMP3 gene transcription and expression, which might provide a novel mechanistic insight into rat Thy-1N and human MsPGN.

The Continuing Saga of Tissue Inhibitor of Metalloproteinase 2: Emerging Roles in Tissue Homeostasis and Cancer Progression.

Tissue inhibitors of metalloproteinases (TIMPs) are a conserved family of proteins that were originally identified as cytokine-like erythroid growth factors. Subsequently, TIMPs were characterized as endogenous inhibitors of matrixin proteinases. These proteinases are the primary mediators of extracellular matrix turnover in pathologic conditions, such as cancer invasion and metastasis. Thus, TIMPs were immediately recognized as important regulators of tissue homeostasis. However, TIMPs also demonstrate unique biological activities that are independent of metalloproteinase regulation. Although often overlooked, these non-protease-mediated TIMP functions demonstrate a variety of direct cellular effects of potential therapeutic value. TIMP2 is the most abundantly expressed TIMP family member, and ongoing studies show that its tumor suppressor activity extends beyond protease inhibition to include direct modulation of tumor, endothelial, and fibroblast cellular responses in the tumor microenvironment. Recent data suggest that TIMP2 can suppress both primary tumor growth and metastatic niche formation. TIMP2 directly interacts with cellular receptors and matrisome elements to modulate cell signaling pathways that result in reduced proliferation and migration of neoplastic, endothelial, and fibroblast cell populations. These effects result in enhanced cell adhesion and focal contact formation while reducing tumor and endothelial proliferation, migration, and epithelial-to-mesenchymal transitions. These findings are consistent with TIMP2 homeostatic functions beyond simple inhibition of metalloprotease activity. This review examines the ongoing evolution of TIMP2 function, future perspectives in TIMP research, and the therapeutic potential of TIMP2.

Tissue Inhibitor Of Metalloproteinase 3 As A Potential Staging Biomarker In Hepatocellular Carcinoma.

Objectives: To assessthe potential role of tissue inhibitor of metalloproteinase 3 as a staging marker of hepatocellular carcinoma. Method: The experimental study was conducted at Faculty of Pharmacy, Kafrelsheikh University, Egypt, from December 2020 to March 2022 after approval from the Faculty of Pharmacy, Kafrelsheikh University, Egypt, and comprised male albino rats. The subjects were divided into 4 groups. The control group was administrated a single intraperitoneal injection of normal saline, while the other groups were generated post-induction of hepatocellular carcinoma. The induction was done by injecting rats intraperitoneally with a single dose of 200mg/kg diethyl nitrosamine, followed by the administration of 0.05% phenobarbital sodium in drinking water daily. Rats were euthanised at 8, 16 and 24 weeks after the injection to obtain three groups related to the 3 stages of hepatocellular carcinoma. Serum was used for measuring the alpha protein level. Liver homogenates were used for the assessment of the hepatic tissue inhibitor of metalloproteinase 3 expression, B-cell lymphoma 2-associated X protein expression, and the hepatic content of matrix metalloproteinase -9 and cyclin D1. Data was analysed using Graph Prism 8. RESULTS: Of the 24 rats with weight range 120-130g, 6(25%) were in each of the 4 groups. The relative protein and messenger ribonucleic acid tissue inhibitor of metalloproteinase 3 expressions were significantly decreased in the intervention groups compared to the control group, with more decline as the hepatocellular carcinoma stage increased. The matrix metalloproteinase -9 and cyclin D1 concentrations and the relative hepatic protein Ki67 expression were significantly raised in the intervention groups compared to the control group (p<0.05). The relative expression of hepatic B-cell lymphoma 2-associated X protein was markedly decreased in the intervention groups compared to the control group (p<0.05). CONCLUSIONS: Tissue inhibitor of metalloproteinase 3 might be a promising diagnostic and staging biomarker in hepatocellular carcinoma.

Suramin analogues protect cartilage against osteoarthritic breakdown by increasing levels of tissue inhibitor of metalloproteinases 3 (TIMP-3) in the tissue.

Osteoarthritis is a chronic degenerative joint disease affecting millions of people worldwide, with no disease-modifying drugs currently available to treat the disease. Tissue inhibitor of metalloproteinases 3 (TIMP-3) is a potential therapeutic target in osteoarthritis because of its ability to inhibit the catabolic metalloproteinases that drive joint damage by degrading the cartilage extracellular matrix. We previously found that suramin inhibits cartilage degradation through its ability to block endocytosis and intracellular degradation of TIMP-3 by low-density lipoprotein receptor-related protein 1 (LRP1), and analysis of commercially available suramin analogues indicated the importance of the 1,3,5-trisulfonic acid substitutions on the terminal naphthalene rings for this activity. Here we describe synthesis and structure-activity relationship analysis of additional suramin analogues using ex vivo models of TIMP-3 trafficking and cartilage degradation. This showed that 1,3,6-trisulfonic acid substitution of the terminal naphthalene rings was also effective, and that the protective activity of suramin analogues depended on the presence of a rigid phenyl-containing central region, with para/para substitution of these phenyl rings being most favourable. Truncated analogues lost protective activity. The physicochemical characteristics of suramin and its analogues indicate that approaches such as intra-articular injection would be required to develop them for therapeutic use.

Targeting of HBP1/TIMP3 axis as a novel strategy against breast cancer.

Malignant proliferation and metastasis are the main causes of breast cancer death. The transcription factor high mobility group (HMG) box-containing protein 1 (HBP1) is an important tumor suppressor whose deletion or mutation is closely related to the appearance of tumors. Here, we investigated the role of HBP1 in breast cancer suppression. HBP1 enhances the activity of the tissue inhibitors of metalloproteinases 3 (TIMP3) promoter, thereby increasing protein and mRNA levels of TIMP3. TIMP3 increases the phosphatase and tensin homolog (PTEN) protein level by inhibiting its degradation and acts as a metalloproteinase inhibitor to inhibit the protein levels of MMP2/9. In this study, we demonstrated that the HBP1/TIMP3 axis plays a crucial role in inhibiting the tumorigenesis of breast cancer. HBP1 deletion interferes with the regulation of the axis and induces the occurrence and malignant progression of breast cancer. In addition, the HBP1/TIMP3 axis promotes the sensitivity of breast cancer to radiation therapy and hormone therapy. Our study opens new perspectives on the treatment and prognosis of breast cancer.

The Histone Methyltransferase SETDB2 Modulates Tissue Inhibitors of Metalloproteinase-Matrix Metalloproteinase Activity During Abdominal Aortic Aneurysm Development.

OBJECTIVE: To determine macrophage-specific alterations in epigenetic enzyme function contributing to the development of abdominal aortic aneurysms (AAAs). BACKGROUND: AAA is a life-threatening disease, characterized by pathologic vascular remodeling driven by an imbalance of matrix metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). Identifying mechanisms regulating macrophage-mediated extracellular matrix degradation is of critical importance to developing novel therapies. METHODS: The role of SET Domain Bifurcated Histone Lysine Methyltransferase 2 (SETDB2) in AAA formation was examined in human aortic tissue samples by single-cell RNA sequencing and in a myeloid-specific SETDB2 deficient murine model induced by challenging mice with a combination of a high-fat diet and angiotensin II. RESULTS: Single-cell RNA sequencing of human AAA tissues identified SETDB2 was upregulated in aortic monocyte/macrophages and murine AAA models compared with controls. Mechanistically, interferon-ß regulates SETDB2 expression through Janus kinase/signal transducer and activator of transcription signaling, which trimethylates histone 3 lysine 9 on the TIMP1-3 gene promoters thereby suppressing TIMP1-3 transcription and leading to unregulated matrix metalloproteinase activity. Macrophage-specific knockout of SETDB2 ( Setdb2f/fLyz2Cre+ ) protected mice from AAA formation with suppression of vascular inflammation, macrophage infiltration, and elastin fragmentation. Genetic depletion of SETDB2 prevented AAA development due to the removal of the repressive histone 3 lysine 9 trimethylation mark on the TIMP1-3 gene promoter resulting in increased TIMP expression, decreased protease activity, and preserved aortic architecture. Lastly, inhibition of the Janus kinase/signal transducer and activator of the transcription pathway with an FDA-approved inhibitor, Tofacitinib, limited SETDB2 expression in aortic macrophages. CONCLUSIONS: These findings identify SETDB2 as a critical regulator of macrophage-mediated protease activity in AAAs and identify SETDB2 as a mechanistic target for the management of AAAs.

Evaluation of Matrix Metalloproteases by Artificial Intelligence Techniques in Negative Biopsies as New Diagnostic Strategy in Prostate Cancer.

Usually, after an abnormal level of serum prostate-specific antigen (PSA) or digital rectal exam, men undergo a prostate needle biopsy. However, the traditional sextant technique misses 15-46% of cancers. At present, there are problems regarding disease diagnosis/prognosis, especially in patients' classification, because the information to be handled is complex and challenging to process. Matrix metalloproteases (MMPs) have high expression by prostate cancer (PCa) compared with benign prostate tissues. To assess the possible contribution to the diagnosis of PCa, we evaluated the expression of several MMPs in prostate tissues before and after PCa diagnosis using machine learning, classifiers, and supervised algorithms. A retrospective study was conducted on 29 patients diagnosed with PCa with previous benign needle biopsies, 45 patients with benign prostatic hyperplasia (BHP), and 18 patients with high-grade prostatic intraepithelial neoplasia (HGPIN). An immunohistochemical study was performed on tissue samples from tumor and non-tumor areas using specific antibodies against MMP -2, 9, 11, and 13, and the tissue inhibitor of MMPs -3 (TIMP-3), and the protein expression by different cell types was analyzed to which several automatic learning techniques have been applied. Compared with BHP or HGPIN specimens, epithelial cells (ECs) and fibroblasts from benign prostate biopsies before the diagnosis of PCa showed a significantly higher expression of MMPs and TIMP-3. Machine learning techniques provide a differentiable classification between these patients, with greater than 95% accuracy, considering ECs, being slightly lower when considering fibroblasts. In addition, evolutionary changes were found in paired tissues from benign biopsy to prostatectomy specimens in the same patient. Thus, ECs from the tumor zone from prostatectomy showed higher expressions of MMPs and TIMP-3 compared to ECs of the corresponding zone from the benign biopsy. Similar differences were found for expressions of MMP-9 and TIMP-3, between fibroblasts from these zones. The classifiers have determined that patients with benign prostate biopsies before the diagnosis of PCa showed a high MMPs/TIMP-3 expression by ECs, so in the zone without future cancer development as in the zone with future tumor, compared with biopsy samples from patients with BPH or HGPIN. Expression of MMP -2, 9, 11, and 13, and TIMP-3 phenotypically define ECs associated with future tumor development. Also, the results suggest that MMPs/TIMPs expression in biopsy tissues may reflect evolutionary changes from prostate benign tissues to PCa. Thus, these findings in combination with other parameters might contribute to improving the suspicion of PCa diagnosis.

Generation of a TIMP3 knockout stem cell line via CRISPR/Cas9 system.

The tissue inhibitors of metalloproteinases 3 (TIMP3) play an essential role in the tumorigenesis of human pancreatic endocrine tumors and Sorsby fundus dystrophy. To further investigate the significance of TIMP3 in disease, we used CRISPR/Cas9 to create a TIMP3 knock out human embryonic stem cell line (WAe009-A-89) that can differentiate into any desired cell type. Our results show that the WAe009-A-89 cell line retains the typical colony form and normal karyotype of stem cells. The cells strongly expressed pluripotency markers and could differentiate into tissues of all three germ layers in vivo. This cell line allowed exploring the role of the TIMP3 gene in related diseases.

Differential gene expression of ADAMTS-1, ADAMTS-9 and TIMP-3 in periodontitis.

A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) are metalloproteinases that bind to components of the extracellular matrix (ECM) to regulate tissue remodeling and homeostasis. ADAMTS can be inhibited by tissue inhibitors of metalloproteinases (TIMPs). Expression of ADAMTS increases under inflammatory conditions. We investigated the mRNA expression of ADAMTS-1, ADAMTS-9 and TIMP-3 genes in both healthy gingival tissues and periodontitis. Clinical periodontal measurements were conducted and gingival biopsies were obtained from stage IIIgrade C generalized periodontitis and healthy (control) groups. mRNA expression was evaluated using real-time quantitative polymerase chain reaction (RTqPCR). All clinical periodontal parameters were significantly higher in the periodontitis group than for the control group. ADAMTS-1 levels were significantly higher in the periodontitis group and were significantly correlated with clinical attachment level and probing pocket depth. Differences in ADAMTS-9 and TIMP-3 mRNA in the periodontitis group compared to the control group were not statistically significant. Increased ADAMTS-1 mRNA expression in periodontitis indicates that members of the ADAMTS family of metalloproteinases are associated with pathogenesis and progression of periodontal disease. Maintaining balance between ADAMTS and TIMP is important for limiting ECM catabolism and preventing tissue damage.

Hypertension Promotes the Proliferation and Migration of ccRCC Cells by Downregulation of TIMP3 in Tumor Endothelial Cells through the miR-21-5p/TGFBR2/P38/EGR1 Axis.

Recent studies have demonstrated that hypertension correlates with tumorigenesis and prognosis of clear-cell renal cell carcinoma (ccRCC); however, the underlying molecular mechanisms remain unclear. By analyzing bulk and single-cell RNA sequencing data and experimental examining of surgical excised ccRCC samples, we found that tissue inhibitors of metalloproteinases 3 (TIMP3), a pivotal paracrine factor in suppressing tumor progression, was significantly reduced in the tumor endothelial cells of patients with hypertensive ccRCC. Besides, in tumor xenograft of NCG mouse model, compared with saline normotensive group the expression of TIMP3 was significantly decreased in the angiotensin II-induced hypertension group. Treating human umbilical vein endothelial cells (HUVEC) with the plasma of patients with hypertensive ccRCC and miR-21-5p, elevated in the plasma of patients with hypertensive ccRCC, reduced the expression of TIMP3 compared with normotensive and control littermates. We also found that the inhibition of TIMP3 expression by miR-21-5p was not through directly targeting at 3'UTR of TIMP3 but through suppressing the expression of TGFß receptor 2 (TGFBR2). In addition, the knockout of TGFBR2 reduced TIMP3 expression in HUVECs through P38/EGR1 (early growth response protein 1) signaling axis. Moreover, via coculture of ccRCC cell lines with HUVECs and mouse tumor xenograft model, we discovered that the TIMP3 could suppress the proliferation and migration of ccRCC. IMPLICATIONS: Overall, our findings shed new light on the role of hypertension in promoting the progression of ccRCC and provide a potential therapeutic target for patients with ccRCC with hypertension.

Association of tissue inhibitor of metalloproteinase-3 (TIMP-3) serum level and its genetic polymorphism with pregnancy outcome of patients undergoing in vitro fertilization and embryo transfer.

OBJECTIVES: Tissue inhibitors of metalloproteinase-3 (TIMP-3) and matrix metalloproteinases (MMPs) play a major role in embryo implantation and placentation. This study aimed to investigate the relationship between TIMP-3 serum level and TIMP-3 genetic polymorphism with pregnancy outcome in patients undergoing in vitro fertilization and embryo transfer (IVF-ET). MATERIAL AND METHODS: This project included 100 infertile women who became pregnant after IVF (IVF+) and 100 infertile women who failed to conceive after IVF (IVF-). Genotyping was performed using Restriction Fragments Length Polymorphism Polymerase Chain Reaction (PCR-RFLP), and the serum level was measured by Enzyme-Linked Immunosorbent Assay (ELISA). RESULTS: The frequencies of TT, TC, and CC in the IVF+ group were 41%, 37% and 22%, respectively, while in the IVF- group were 18%, 43% and 39%, respectively. The C and T allele frequencies were 40.5% and 59.5% in the IVF+ group and 60.5% and 39.5% in IVF- group, respectively. The C allele conferred a 2.25-fold increased risk of IVF failure (OR 2.25; 95% CI 1.5-3.35; p = 0.0001). Also, there was a significant increase in TIMP-3 serum levels in the IVF- group (193.29 ± 29.50 ng/mL), which was higher than the IVF+ group (166.74 ± 17.60 ng/mL; p = 0.00002), was demonstrated. It was shown that the TT genotype is associated with decreased TIMP-3 serum levels in IVF- group (CC, CT, and TT, values were 143.19 ± 88.49 ng/mL, 117.55 ± 15.73 ng/mL, 61.17 ± 44.36 ng/mL, respectively). CONCLUSIONS: It is concluded that there is a relationship between TIMP-3 gene polymorphism and its serum concentration with IVF-ET outcome. We also suggest that the TT genotype might be involved in IVF-ET outcome.

Effect of Irisin on Human Nucleus Pulposus Cells: New Insights into the Biological Cross-talk Between Muscle and Intervertebral Disk.

STUDY DESIGN: In vitro study. OBJECTIVE: To investigate the effect of irisin on human nucleus pulposus cells (hNPCs) in vitro. SUMMARY OF BACKGROUND DATA: Physical exercise (PE) favours weight loss and ameliorates function in patients with low back pain. Although there is no biological evidence that the intervertebral disk (IVD) can respond to PE, recent studies have shown that running is associated with increased IVD hydration and hypertrophy. Irisin, a myokine released upon muscle contraction, has demonstrated anabolic effects on different cell types, including chondrocytes. MATERIALS AND METHODS: hNPCs were exposed to 5, 10, and 25 ng/mL irisin. Cell proliferation, glycosaminoglycan (GAG) content, metabolic activity, gene expression of collagen type II (COL2), matrix metalloproteinase (MMP)-13, tissue inhibitor of matrix metalloproteinase (TIMP)-1 and TIMP-3, aggrecan (ACAN), interleukin (IL)-1ß, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 were assessed. In addition, MTT assay and ADAMTS-5, COL2, TIMP-1, and IL-1ß gene expression were evaluated following incubation with irisin for 24 hours and subsequent culture with 10 ng/mL IL-1ß and vice versa (incubation for 24 hours with IL-1ß and subsequent culture with irisin). RESULTS: Irisin increased hNPC proliferation, metabolic activity, and GAG content, as well as COL2, ACAN, TIMP-1 and TIMP-3 gene expression, while decreasing MMP-13 and IL-1ß mRNA levels. Irisin pretreatment of hNPCs cultured in proinflammatory conditions resulted in a rescue of metabolic activity and a decrease of IL-1ß levels. Similarly, incubation of hNPCs with IL-1ß and subsequent exposure to irisin led to an increment of metabolic activity, COL2 gene expression, and a reduction of IL-1ß and ADAMTS-5 levels. CONCLUSIONS: Irisin increases hNPC proliferation, GAG content, metabolic activity, and promotes anabolic gene expression while reducing catabolic markers. Irisin may be one of the mediators by which PE and muscle tissues modulate IVD metabolism, suggesting the existence of a biological cross-talk between the muscle and IVD.

Circulating levels of tissue inhibitor of metalloproteinase 3, a protein with inhibitory effects on angiogenesis, are increased in pre-eclampsia.

OBJECTIVE: To assess and compare circulating tissue inhibitor of metalloproteinase 3 (TIMP-3) concentrations between women with pre-eclampsia and healthy pregnant women. We also aimed to determine the relationships between circulating TIMP-3 and matrix metalloproteinase 2 (MMP-2), MMP-9, TIMP-1, and TIMP-2 concentrations in pre-eclampsia. METHODS: A primary case-control study included patients with pre-eclampsia (n = 219) and gestational hypertension (n = 118), healthy pregnant women (n = 214), and non-pregnant women (n = 66), and a replication case-control study included patients with pre-eclampsia (n = 177) and healthy pregnant women (n = 124), all from southeastern Brazil. Plasma TIMP-3, MMP-2, MMP-9, TIMP-1, and TIMP-2 concentrations were assessed using commercially available enzyme-linked immunosorbent assay kits, and the relationships between them were analyzed using Spearman's correlation. RESULTS: In our primary study, patients with pre-eclampsia and gestational hypertension exhibited increased TIMP-3 concentrations compared with healthy pregnant women (both P < 0.0001) and non-pregnant women (both P < 0.001). These findings were confirmed in the replication study, showing elevated TIMP-3 concentrations in women with pre-eclampsia versus healthy pregnant women (P < 0.001). We found no difference in TIMP-3 concentrations between early-onset and late-onset pre-eclampsia. Moreover, TIMP-3 concentrations were significantly correlated with plasma concentrations of TIMP-1 (r = 0.2333; P = 0.0086) and MMP-2 (r = 0.2159; P = 0.0156) in pre-eclampsia. CONCLUSIONS: Circulating TIMP-3 concentration is increased in women with pre-eclampsia compared with healthy pregnant women, and it is positively correlated with plasma MMP-2 and TIMP-1 concentrations in pre-eclampsia.