SORBS2-Mediated inhibition of malignant behaviors in esophageal squamous cell carcinoma through TIMP3.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is characterized by high invasiveness and poor prognosis. The role of Sorbin and SH3 domain-containing protein 2 (SORBS2) in ESCC remains largely unexplored. METHODS: The expression levels of SORBS2 in ESCC were detected using RNA-seq and proteomics data. The biological functions of SORBS2 in ESCC were investigated through in vivo and in vitro experiments. The mechanism of SORBS2 was explored using RIP-seq technology, which identified the key downstream molecule metalloproteinase-3 (TIMP3). The interaction between SORBS2 and TIMP3, including specific binding sites, was validated through RIP-qPCR and RNA pull-down assays. The impact of altered SORBS2 expression in ESCC on HUVECs was assessed using endothelial tube formation assays. RESULTS: SORBS2 expression was significantly downregulated in ESCC tissues, and its decreased expression was associated with poor prognosis. Overexpression of SORBS2 in ESCC cell lines inhibited cell proliferation, migration, and invasion both in vitro and in vivo. Mechanistically, SORBS2 bound to the 3' UTR of TIMP3 mRNA, enhancing its stability and thereby regulating TIMP3 expression. Rescue experiments demonstrated that increased TIMP3 expression could reverse the promotive effects of SORBS2 knockdown on ESCC, confirming TIMP3 as a critical downstream molecule of SORBS2. Furthermore, downregulation of SORBS2 in ESCC cells was associated with activation of HUVEC functions, whereas upregulation of TIMP3 could reverse this effect. The SORBS2/TIMP3 axis may exert tumor suppressive effects by influencing extracellular matrix degradation. CONCLUSION: This study confirms that SORBS2 inhibits ESCC tumor progression by regulating extracellular matrix degradation through TIMP3, providing a potential therapeutic target for future treatment interventions.

Tissue Inhibitor of Metalloproteinase 3 (TIMP3) mutations increase glycolytic activity and dysregulate glutamine metabolism in RPE cells.

OBJECTIVES: Mutations in Tissue Inhibitor of Metalloproteinases 3 (TIMP3) cause Sorsby's Fundus Dystrophy (SFD), a dominantly inherited, rare form of macular degeneration that results in vision loss. TIMP3 is synthesized primarily by retinal pigment epithelial (RPE) cells, which constitute the outer blood-retinal barrier. One major function of RPE is the synthesis and transport of vital nutrients, such as glucose, to the retina. Recently, metabolic dysfunction in RPE cells has emerged as an important contributing factor in retinal degenerations. We set out to determine if RPE metabolic dysfunction was contributing to SFD pathogenesis. METHODS: Quantitative proteomics was conducted on RPE of mice expressing the S179C variant of TIMP3, known to be causative of SFD in humans. Proteins found to be differentially expressed (P < 0.05) were analyzed using statistical overrepresentation analysis to determine enriched pathways, processes, and protein classes using g:profiler and PANTHER Gene Ontology. We examined the effects of mutant TIMP3 on RPE metabolism using human ARPE-19 cells expressing mutant S179C TIMP3 and patient-derived induced pluripotent stem cell-derived RPE (iRPE) carrying the S204C TIMP3 mutation. RPE metabolism was directly probed using isotopic tracing coupled with GC/MS analysis. Steady state [U-13C6] glucose isotopic tracing was preliminarily conducted on S179C ARPE-19 followed by [U-13C6] glucose and [U-13C5] glutamine isotopic tracing in SFD iRPE cells. RESULTS: Quantitative proteomics and enrichment analysis conducted on RPE of mice expressing mutant S179C TIMP3 identified differentially expressed proteins that were enriched for metabolism-related pathways and processes. Notably these results highlighted dysregulated glycolysis and glucose metabolism. Stable isotope tracing experiments with [U-13C6] glucose demonstrated enhanced glucose utilization and glycolytic activity in S179C TIMP3 APRE-19 cells. Similarly, [U-13C6] glucose tracing in SFD iRPE revealed increased glucose contribution to glycolysis and the TCA cycle. Additionally, [U-13C5] glutamine tracing found evidence of altered malic enzyme activity. CONCLUSIONS: This study provides important information on the dysregulation of RPE glucose metabolism in SFD and implicates a potential commonality with other retinal degenerative diseases, emphasizing RPE cellular metabolism as a therapeutic target.

Intermittent compressive force regulates matrix metalloproteinases and tissue inhibitors of metalloproteinases expression in human periodontal ligament cells.

OBJECTIVE: This study aims to evaluate the effects of intermittent compressive force (ICF) on the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) by human periodontal ligament cells (hPDLCs). DESIGN: hPDLCs were subjected to ICF with a magnitude of 1.5 g/cm2 and loaded for 24 h. mRNA and protein expression of several MMPs and TIMPs were assessed using RT-PCR and ELISA analyses. An inhibitor of TGF-ß (SB431542) was used to assess a possible role of TGF-ß in the expression of MMPs and TIMPs under ICF. RESULTS: mRNA and protein analyses showed that ICF significantly induced expression of TIMP1 and TIMP3, but decreased expression of MMP1. Incubation with the TGF-ß inhibitor and applied to ICF showed a downregulation of TIMP3, but expression of MMP1 was not affected. CONCLUSION: ICF is likely to affect ECM homeostasis by hPDLCs by regulating the expression of MMP1 and TIMPs. Moreover, TGF-ß1 regulated expression of TIMP3. These findings suggest ICF may decrease the degradation of ECM and may thus be essential for maintaining PDL homeostasis.

Demethylation of TIMP2 and TIMP3 Inhibits Cell Proliferation, Migration, and Invasion in Pituitary Adenomas.

OBJECTIVE: Tissue inhibitors of matrix metalloproteinases (TIMPs) are prognostic markers in cancers. However, the role of TIMPs in DNA methylation during invasive pituitary adenoma (PA) remains unclear. The purpose of this study was to assess the effects of TIMP2 and TIMP3 promoter demethylation on the proliferation, migration, and invasion of invasive PA cells. METHODS: Methylation-specific polymerase chain reaction (PCR), quantitative PCR, and western blots were used to analyze the promoter methylation and expression of TIMP1-3. Cell counting kit-8 (CCK-8), wound healing, and transwell assays were carried out to determine the effects of TIMP2 and TIMP3 demethylation. RESULTS: TIMP1-3 showed downregulated expression in invasive PA tissues and cell lines (p < 0.05). The low expression of TIMP1-3 was due to promoter methylation of these genes (p < 0.05). The results showed that downregulation of TIMP2 and TIMP3 can promote cell proliferation, migration, and invasion (p < 0.05), whereas overexpression of TIMP2 and TIMP3 can inhibit cell proliferation, migration, and invasion (p < 0.05). After treatment with 5-azacytidine (5-AzaC), the cell activity decreased, the proliferation rate decreased, and the invasion ability weakened (p < 0.05). Treatment with 5-AzaC increased TIMP2 and TIMP3 expression and decreased DNA (cytosine-5-)-methyltransferase 1 (DNMT1), DNMT3a, and DNMT3b expression (p < 0.05). CONCLUSIONS: We showed that DNA methylation causes the silencing of TIMP2 and TIMP3 in invasive PA, it can also lead to malignant cell proliferation and cause pathological changes, whereas the use of 5-AzaC can inhibit the methylation process and can inhibit cell proliferation. Our results provide a novel method for clinical diagnosis and prevention of invasive PA.

Tissue Inhibitors of Metalloproteinase 1 (TIMP-1) and 3 (TIMP-3) as New Markers of Acute Kidney Injury After Massive Burns.

BACKGROUND Acute kidney injury (AKI) is a common and serious complication after massive burn injury. One of the postulated etiologies is destruction of the extracellular matrix of nephrons, caused by a local imbalance between matrix metalloproteinases (MMPs) and specific inhibitors. The aim of this study was to analyze the dynamics of tissue inhibitors of metalloproteinases (TIMPs) during the first 5 days after massive thermal injury and the relationship with the risk of AKI. MATERIAL AND METHODS Thirty-three adults (22 men, 11 women) with severe burns were enrolled in the study. The values of TIMPs 1 to 4 were measured in blood serum and urine using the multiplex Luminex system. The associations between TIMPs and the risk of AKI were analyzed by using the generalized linear mixed models for repeated measurements. RESULTS Significant changes in serum and urine activities of TIMPs were confirmed, especially during the first 2 days after burn injury. Almost half of patients presented renal problems during the study. Significant differences between values of TIMPs in AKI and non-AKI status were also observed. However, a significant relationship between concentration of TIMPs and risk of AKI was confirmed only for urine TIMP-1 and serum TIMP-3. CONCLUSIONS The evaluation of TIMPs in the early stage after burn injury has potential benefits. The important roles of urine TIMP-1 and serum TIMP-3, as novel markers of the risk of AKI development, were confirmed. Other parameters require further analysis.

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.

Extensive Macular Atrophy with Pseudodrusen-like appearance: Progression Kinetics and Late-Stage Findings.

PURPOSE: To describe the clinical outcome and late-stage findings of extensive macular atrophy with pseudodrusen-like appearance (EMAP). DESIGN: Retrospective cohort study. PARTICIPANTS: Seventy-eight patients (156 eyes) affected by EMAP. METHODS: We collected data on best-corrected visual acuity, kinetic perimetry, OCT, short-wavelength autofluorescence, and near-infrared autofluorescence findings. Genetic testing for the TIMP3 and C1QTNF5 genes was performed via Sanger sequencing for 58 patients, with no pathogenic variants identified. MAIN OUTCOME MEASURES: The primary outcomes were best-corrected visual acuity at the last examination, visual field at the last examination, and incidence rates and time-to-event curves for blindness with the United States Social Security Administration and World Health Organization (WHO) criteria, foveal involvement, and atrophy enlargement beyond the 30° and 55° field of view. Imaging findings at the last examination were secondary outcomes. RESULTS: At the most recent visit, mean age was 70.9 ± 5.2 years. Using United States criteria, 58.1% of the patients were blind, and 25.8% were blind according to WHO criteria. All eyes showed large central scotomas, which were associated with visual field constriction in 22.2% of eyes. We detected focal openings or large dehiscences of Bruch's membrane (BM) in 25.4% of eyes. Near-infrared autofluorescence showed increased visibility of the choroidal vessels beyond the atrophy in 87.2% of eyes. The incidence rates for blindness were 3.95 per 100 patient-years with United States criteria and 1.54 per 100 patient-years according to WHO criteria. The incidence rates were 22.8 per 100 eye-years for foveal involvement, 12.0 per 100 eye-years for atrophy enlargement beyond 30°, and 6.6 per 100 eye-years for atrophy enlargement beyond 55°. The estimates were not influenced by the age at onset. CONCLUSIONS: We identified characteristic imaging findings, including BM ruptures, in elder patients with EMAP and calculated incidence rates for different functional and anatomic outcomes. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

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).

Abnormal stress promotes intervertebral disc degeneration through WTAP/YTHDF2-dependent TIMP3 m6A modification.

Mechanical environment worsening is an important predisposing factor that accelerates intervertebral disc degeneration (IDD), but its specific regulatory mechanisms remain unclear. In this study, we reveal the molecular mechanisms of WTAP/YTHDF2-mediated m6A modification in abnormal stress-induced intervertebral disc (IVD) matrix degradation. WTAP expression in human nucleus pulposus cells was elevated under tension. Similarly, high WTAP expression was detected in severe degenerated human and rat nucleus pulposus tissues. Functionally, WTAP was found to increase the TIMP3 transcript methylation level under tension, resulting in YTHDF2 recognition, binding, and induction of its degradation. Reduction in TIMP3 caused increases in active matrix metalloproteinases, ultimately inducing extracellular matrix degradation in nucleus pulposus cells. Macroscopically, this promotes IDD. Additionally, in vitro and in vivo inhibition of WTAP expression or TIMP3 overexpression significantly increased stress resistance in the nucleus pulposus, thereby alleviating IDD. Our results show that abnormal stress disrupts IVD matrix stability through WTAP/YTHDF2-dependent TIMP3 m6A modification.

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.

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.

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.

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.

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.

Distinct Expression Patterns of Genes Coding for Biological Response Modifiers Involved in Inflammatory Responses and Development of Fibrosis in Chronic Hepatitis C: Upregulation of SMAD-6 and MMP-8 and Downregulation of CAV-1, CTGF, CEBPB, PLG, TIMP-3, MMP-1, ITGA-1, ITGA-2 and LOX.

Background and Objectives: The aim of this study was to analyze the expression of genes on transcriptomic levels involved in inflammatory immune responses and the development of fibrosis in patients with chronic hepatitis C. Materials and Methods: Expression patterns of 84 selected genes were analyzed with real-time quantitative RT PCR arrays in the peripheral blood of treatment-naive patients with chronic hepatitis C and healthy controls. The panel included pro- and anti-fibrotic genes, genes coding for extracellular matrix (EMC) structural constituents and remodeling enzymes, cell adhesion molecules, inflammatory cytokines, chemokines and growth factors, signal transduction members of the transforming growth factor- beta (TGF-ß) superfamily, transcription factors, and genes involved in epithelial to mesenchymal transition. Results: The expression of SMAD-6 coding for a signal transduction TGF-beta superfamily member as well as MMP-8 coding for an ECM protein were significantly increased in CHC patients compared with controls. Conclusions: Chronic hepatitis C was also characterized by a significant downregulation of a set of genes including CAV-1, CTGF, TIMP-3, MMP-1, ITGA-1, LOX, ITGA-2, PLG and CEBPB encoding various biological response modifiers and transcription factors. Our results suggest that chronic hepatitis C is associated with distinct patterns of gene expression modulation in pathways associated with the regulation of immune responses and development of fibrosis.

Deglycosylation Increases the Aggregation and Angiogenic Properties of Mutant Tissue Inhibitor of Metalloproteinase 3 Protein: Implications for Sorsby Fundus Dystrophy.

Sorsby fundus dystrophy (SFD) is an autosomal dominant macular disorder caused by mutations in tissue Inhibitor of the metalloproteinase-3 (TIMP3) gene with the onset of symptoms including choroidal neovascularization as early as the second decade of life. We have previously reported that wild-type TIMP3 is an endogenous angiogenesis inhibitor that inhibits Vascular Endothelial Growth Factor (VEGF)-mediated signaling in endothelial cells. In contrast, SFD-related S179C-TIMP3 when expressed in endothelial cells, does not have angiogenesis-inhibitory properties. To evaluate if this is a common feature of TIMP3 mutants associated with SFD, we examined and compared endothelial cells expressing S179C, Y191C and S204C TIMP3 mutants for their angiogenesis-inhibitory function. Western blot analysis, zymography and reverse zymography and migration assays were utilized to evaluate TIMP3 protein, Matrix Metalloproteinase (MMP) and MMP inhibitory activity, VEGF signaling and in vitro migration in endothelial cells expressing (VEGF receptor-2 (VEGFR-2) and wild-type TIMP3 or mutant-TIMP3. We demonstrate that mutant S179C, Y191C- and S204C-TIMP3 all show increased glycosylation and multimerization/aggregation of the TIMP3 protein. In addition, endothelial cells expressing TIMP3 mutations show increased angiogenic activities and elevated VEGFR-2. Removal of N-glycosylation by mutation of Asn184, the only potential N-glycosylation site in mutant TIMP3, resulted in increased aggregation of TIMP3, further upregulation of VEGFR-2, VEGF-induced phosphorylation of VEGFR2 and VEGF-mediated migration concomitant with reduced MMP inhibitory activity. These results suggest that even though mutant TIMP3 proteins are more glycosylated, post-translational deglycosylation may play a critical role in the aggregation of mutant TIMP3 and contribute to the pathogenesis of SFD. The identification of factors that might contribute to changes in the glycome of patients with SFD will be useful. Future studies will evaluate whether variations in the glycosylation of mutant TIMP3 proteins are contributing to the severity of the disease.