Time-course and muscle-specific gene expression of matrix metalloproteinases and inflammatory cytokines in response to acute treadmill exercise in rats.

BACKGROUND: The extracellular matrix (ECM) of skeletal muscle plays a pivotal role in tissue repair and growth, and its remodeling tightly regulated by matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), and inflammatory cytokines. This study aimed to investigate changes in the mRNA expression of MMPs (Mmp-2 and Mmp-14), TIMPs (Timp-1 and Timp-2), and inflammatory cytokines (Il-1ß, Tnf-α, and Tgfß1) in the soleus (SOL) and extensor digitorum longus (EDL) muscles of rats following acute treadmill exercise. Additionally, muscle morphology was examined using hematoxylin and eosin (H&E) staining. METHODS AND RESULTS: Male rats were subjected to acute treadmill exercise at 25 m/min for 60 min with a %0 slope. The mRNA expression of ECM components and muscle morphology in the SOL and EDL were assessed in both sedentary and exercise groups at various time points (immediately (0) and 1, 3, 6, 12, and 24 h post-exercise). Our results revealed a muscle-specific response, with early upregulation of the mRNA expression of Mmp-2, Mmp-14, Timp-1, Timp-2, Il-1ß, and Tnf-α observed in the SOL compared to the EDL. A decrease in Tgfß1 mRNA expression was evident in the SOL at all post-exercise time points. Conversely, Tgfß1 mRNA expression increased at 0 and 3 h post-exercise in the EDL. Histological analysis also revealed earlier cell infiltration in the SOL than in the EDL following acute exercise. CONCLUSIONS: Our results highlight how acute exercise modulates ECM components and muscle structure differently in the SOL and EDL muscles, leading to distinct muscle-specific responses.

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.

Association of TIMP2 418 G/C and MMP Gene Polymorphism with Risk of Urinary Cancers: Systematic Review and Meta-analysis.

Aim: The matrix metalloproteinases (MMPs) inhibit tissue inhibitors of metalloproteinases (TIMPs), playing a notable role in various biological processes, and mutations in TIMP2 genes impact a variety of urinary cancers. In this study, we analyze and evaluate the potential involvement of the TIMP2 418 G/C and MMP gene polymorphism in the etiology of urinary cancer. Methodology: For suitable case-control studies, a literature search was undertaken from various database sources such as PubMed, EMBASE, and Google Scholar. Incorporated into the analysis were case-control or cohort studies that documented the correlation between TIMP2 418 G/C and urological cancers. MetaGenyo served as the tool for conducting the meta-analysis, employing a fixed-effects model. The collective odds ratios, along with their corresponding 95% confidence intervals, were calculated and presented to assess the robustness of the observed associations. Results: A total of seven studies involving controls and cases out of recorded 1265 controls and 1154 cases were analyzed to ascertain the significant association of the TIMP2 gene with urologic cancer. No statistically significant correlation was observed between allelic, recessive, dominant, and overdominant models for the genetic variant under investigation. A 95% confidence interval (CI) and odds ratio (OR) were computed for each model, considering p-values <0.05. The OR and 95% CI for the allelic model were 0.99 and 0.77-1.27, respectively, whereas the respective values were 1.00 and 0.76-1.32 for the recessive model. In the dominant contrast model, OR and 95% CI were 1.09 and 0.62-1.90, while the same were 0.93 and 0.77-1.12 for the overdominant model. A funnel plot was used to reanalyze and detect the results as statically satisfactory. Conclusions: As a result of the data obtained, the TIMP2 gene polymorphism does not correlate statistically with cancer risk. The significance of this finding can only be confirmed using a large population, extensive epidemiological research, a comprehensive survey, and a better understanding of the molecular pathways associated.

TIMP2 protects against sepsis-associated acute kidney injury by cAMP/NLRP3 axis-mediated pyroptosis.

The tissue inhibitor of metalloproteinases 2 (TIMP2) has emerged as a promising biomarker for predicting the risk of sepsis-associated acute kidney injury (SA-AKI). However, its exact role in SA-AKI and the underlying mechanism remains unclear. In this study, we investigated the impact of kidney tubule-specific Timp2 knockout mice on kidney injury and inflammation. Our findings demonstrated that Timp2-knockout mice exhibited more severe kidney injury than wild-type mice, along with elevated levels of pyroptosis markers NOD-like receptor protein 3 (NLRP3), Caspase1, and gasdermin D (GSDMD) in the early stage of SA-AKI. Conversely, the expression of exogenous TIMP2 in TIMP2-knockout mice still protected against kidney damage and inflammation. In in vitro experiments, using recombinant TIMP2 protein, TIMP2 knockdown demonstrated that exogenous TIMP2 inhibited pyroptosis of renal tubular cells stimulated by lipopolysaccharide (LPS). Mechanistically, TIMP2 promoted the ubiquitination and autophagy-dependent degradation of NLRP3 by increasing intracellular cyclic adenosine monophosphate (cAMP), which mediated NLRP3 degradation through recruiting the E3 ligase MARCH7, attenuating downstream pyroptosis, and thus alleviating primary tubular cell damage. These results revealed the renoprotective role of extracellular TIMP2 in SA-AKI by attenuating tubular pyroptosis, and suggested that exogenous administration of TIMP2 could be a promising therapeutic intervention for SA-AKI treatment.NEW & NOTEWORTHY Tissue inhibitor of metalloproteinase 2 (TIMP-2) has been found to be the best biomarker for predicting the risk of sepsis-associated acute kidney injury (SA-AKI). However, its role and the underlying mechanism in SA-AKI remain elusive. The authors demonstrated in this study using kidney tubule-specific knockout mice model of SA-AKI and primary renal tubule cells stimulated with lipopolysaccharide (LPS) that extracellular TIMP-2 promoted NOD-like receptor protein 3 (NLRP3) ubiquitination and autophagy-dependent degradation by increasing intracellular cyclic adenosine monophosphate (cAMP), thus attenuated pyroptosis and alleviated renal damage.

The association between activation of the ERK1/2-NF-κB signaling pathway by TIMP2 expression and chronic renal allograft dysfunction in the CRAD rat model.

PURPOSE: The tissue inhibitor of metalloproteinase 2 (TIMP2), a natural inhibitor of matrix metalloproteinase (MMP), regulates inflammation, fibrosis, and cell proliferation. Chronic renal allograft dysfunction (CRAD) is a primary factor affecting the long-term survival of renal allografts. We assessed whether up-regulation of TIMP2 expression may affect the ERK1/2-NF-κB signaling pathway and CRAD development. METHODS: Lewis rats received orthotopic F344 kidney allografts to establish the classical CRAD model. The treatment group was injected with a lentivirus encoding a TIMP2-targeting small hairpin (sh)RNA (LTS) at 5 × 108 TU/ml monthly after kidney transplantation. A second CRAD group was injected with a lentivirus TIMP2-control vector (LTC). After 12 weeks, blood, urine, and kidney tissue were harvested to evaluate renal function and pathological examinations. Hematoxylin and eosin staining, Masson staining, and Periodic acid-Schiff staining were performed for renal histopathological evaluation according to the Banff criteria. TIMP2, phospho (p)-ERK1/2, p-p65 (NF-κB) expression levels were measured via immunohistochemical and Western blot analyses. RESULTS: Compared to the F344 and Lewis control groups, the expression of TIMP2, p-ERK1/2, and p-p65 were significantly higher in the CRAD and CRAD+LTC renal tissues (p < 0.05). There were also increased levels of serum creatinine, nitrogen, and 24 h urinary protein in these two groups (p < 0.05). Typical histopathological changes of CRAD were observed in the CRAD and CRAD+LTC groups. Administration of LTS effectively decreased the expression of TIMP2, p-ERK1/2, and p-P65, and reduced interstitial fibrosis and macrophage infiltration in the treatment group (p < 0.05). Additionally, MCP1 and ICAM-1, which are downstream cytokines of the NF-κB pathway, were also inhibited in the renal rat kidney from the LTS group (p < 0.05). Furthermore, renal function was well preserved in the LTS group compared to the CRAD group and CRAD+LTC group. CONCLUSION: A decrease of TIMP2 can alleviate the progression of inflammation in CRAD via inhibition of the ERK1/2-NF-κB signaling pathway.

Pharmacological inhibition of EZH2 using a covalent inhibitor suppresses human ovarian cancer cell migration and invasion.

Metastasis is the cause of poor prognosis in ovarian cancer (OC). Enhancer of Zeste homolog 2 (EZH2), a histone-lysine N-methyltransferase enzyme, promotes OC cell migration and invasion by regulating the expression of tissue inhibitor of metalloproteinase-2 (TIMP2) and matrix metalloproteinases-9 (MMP9). Hence, we speculated that EZH2-targeting therapy might suppress OC migration and invasion. In this study, the expression of EZH2, TIMP2, and MMP9 in OC tissues and cell lines was analyzed using The Cancer Genome Atlas (TCGA) database and western blotting, respectively. The effects of SKLB-03220, an EZH2 covalent inhibitor, on OC cell migration and invasion were investigated using wound-healing assays, Transwell assays, and immunohistochemistry. TCGA database analysis confirmed that the EZH2 and MMP9 mRNA expression was significantly higher in OC tissues, whereas TIMP2 expression was significantly lower than that in normal ovarian tissues. Moreover, EZH2 negatively correlated with TIMP2 and positively correlated with MMP9 expression. In addition to the anti-tumor activity of SKLB-03220 in a PA-1 xenograft model, immunohistochemistry results showed that SKLB-03220 markedly increased the expression of TIMP2 and decreased the expression of MMP9. Additionally, wound-healing and Transwell assays showed that SKLB-03220 significantly inhibited the migration and invasion of both A2780 and PA-1 cells in a concentration-dependent manner. SKLB-03220 inhibited H3K27me3 and MMP9 expression and increased TIMP2 expression in PA-1 cells. Taken together, these results indicate that the EZH2 covalent inhibitor SKLB-03220 inhibits metastasis of OC cells by upregulating TIMP2 and downregulating MMP9, and could thus serve as a therapeutic agent for OC.

Trophoblast cellular adhesion molecule expression regulated by different genes and their correlation with pregnancy-induced hypertension.

It was to study trophoblast cell (TC) adhesion molecules regulated by different genes in the placental tissue (PT) of patients with pregnancy-induced hypertension (PIH), and the correlation with the severity of PIH. 42 patients with PIH (13 cases in the mild PIH group, 11 cases in the moderate PIH group, and 18 cases in the severe PIH group) and 40 patients with normal pregnancy (NP group) were included. mRNA and protein levels in matrix metalloproteinase (MMP)-9, MMP-2, tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2 of all patients were determined by semi-quantitative polymerase chain reaction (PCR) and Western blotting (WB), respectively. Compared to the NP group, MMP-9 and MMP-2 mRNA levels as well as their proteins in PT significantly decreased in PIH groups (P<0.05). MMP-9 mRNA was greatly lower in the severe PIH group than mild PIH group (P<0.05). MMP-2 mRNA in moderate and severe PIH groups was much lower than NP and mild PIH groups, and that in the severe PIH group was considerably lower than the moderate PIH group (P<0.05). TIMP-1 mRNA and its protein highly increased in PT in PIH groups than NP group (P<0.05). TIMP-2 mRNA was remarkably higher in the severe PIH group than in the NP group (P<0.05). mRNA and proteins of MMP-9 and MMP-2 decreased in PT of PIH patients, while TIMP-1 mRNA and its protein increased, which were correlated with the severity of PIH. MMP-9, MMP-2, and TIMP-1 were involved in the pathogenesis of PIH by regulating the infiltration of TCs.

TIMP2 ameliorates blood-brain barrier disruption in traumatic brain injury by inhibiting Src-dependent VE-cadherin internalization.

Blood-brain barrier (BBB) disruption is a serious pathological consequence of traumatic brain injury (TBI), for which there are limited therapeutic strategies. Tissue inhibitor of metalloproteinase-2 (TIMP2), a molecule with dual functions of inhibiting MMP activity and displaying cytokine-like activity through receptor binding, has been reported to inhibit VEGF-induced vascular hyperpermeability. Here, we investigate the ability of TIMP2 to ameliorate BBB disruption in TBI and the underlying molecular mechanisms. Both TIMP2 and AlaTIMP2, a TIMP2 mutant without MMP-inhibiting activity, attenuated neurological deficits and BBB leakage in TBI mice; they also inhibited junctional protein degradation and translocation to reduce paracellular permeability in human brain microvascular endothelial cells (ECs) exposed to hypoxic plus inflammatory insult. Mechanistic studies revealed that TIMP2 interacted with α3ß1 integrin on ECs, inhibiting Src activation-dependent VE-cadherin phosphorylation, VE-cadherin/catenin complex destabilization, and subsequent VE-cadherin internalization. Notably, localization of VE-cadherin on the membrane was critical for TIMP2-mediated EC barrier integrity. Furthermore, TIMP2-mediated increased membrane localization of VE-cadherin enhanced the level of active Rac1, thereby inhibiting stress fiber formation. All together, our studies have identified an MMP-independent mechanism by which TIMP2 regulates EC barrier integrity after TBI. TIMP2 may be a therapeutic agent for TBI and other neurological disorders involving BBB breakdown.

Neuronal TIMP2 regulates hippocampus-dependent plasticity and extracellular matrix complexity.

Functional output of the hippocampus, a brain region subserving memory function, depends on highly orchestrated cellular and molecular processes that regulate synaptic plasticity throughout life. The structural requirements of such plasticity and molecular events involved in this regulation are poorly understood. Specific molecules, including tissue inhibitor of metalloproteinases-2 (TIMP2) have been implicated in plasticity processes in the hippocampus, a role that decreases with brain aging as expression is lost. Here, we report that TIMP2 is highly expressed by neurons within the hippocampus and its loss drives changes in cellular programs related to adult neurogenesis and dendritic spine turnover with corresponding impairments in hippocampus-dependent memory. Consistent with the accumulation of extracellular matrix (ECM) in the hippocampus we observe with aging, we find that TIMP2 acts to reduce accumulation of ECM around synapses in the hippocampus. Moreover, its deletion results in hindrance of newborn neuron migration through a denser ECM network. A novel conditional TIMP2 knockout (KO) model reveals that neuronal TIMP2 regulates adult neurogenesis, accumulation of ECM, and ultimately hippocampus-dependent memory. Our results define a mechanism whereby hippocampus-dependent function is regulated by TIMP2 and its interactions with the ECM to regulate diverse processes associated with synaptic plasticity.

Dznep, a histone modification inhibitor, inhibits HIF1α binding to TIMP2 gene and suppresses TIMP2 expression under hypoxia.

Epidemiological studies have shown that patients who recovered from acute kidney injury (AKI) may subsequently develop chronic kidney disease (CKD). AKI is primarily caused by renal hypoxia, and it causes epigenetic alterations, known as hypoxic memory. 3-Deazaneplanocin A (Dznep), an inhibitor of histone modification, suppresses renal fibrosis and the expression of tissue inhibitor of metalloproteinases-2 (TIMP2), a profibrotic factor, in mouse ischemia-reperfusion models. The current study investigated the epigenetic regulation of TIMP2 in human kidney 2 (HK-2) cells. The expression of TIMP2 was upregulated in HK-2 cells under hypoxic conditions and was suppressed by Dznep. ChIP-qPCR showed that Dznep reduced the amount of H3K4me3 at the promoter region of the TIMP2 gene under hypoxic condition. Formaldehyde-assisted isolation of regulatory elements-qPCR of the TIMP2 gene showed that Dznep reduced open chromatin area. In addition, based on ChIP-qPCR of hypoxia-inducible factor 1 alpha (HIF1α), Dznep inhibited the binding of HIF1α to the TIMP2 gene under hypoxic conditions. The reporter assays for the binding region of HIF1α showed enhanced transcriptional activity by hypoxia. Dznep suppresses the expression of TIMP2 under hypoxic conditions by inhibiting the binding of HIF1α to the TIMP2 gene.

Expression analysis of MMP14: Key enzyme action in modulating visceral adipose tissue plasticity in patients with obesity.

Compromised adipose tissue plasticity is a hallmark finding of obesity orchestrated by the intricate interplay between various extracellular matrix components. Collagen6 (COL6) is well characterized in obese visceral adipose tissue (VAT), not much is known about MMP14 which is hypothesized to be the key player in matrix reorganization. Subjects with obesity (BMI ≥40; n = 50) aged 18-60 years undergoing bariatric surgery and their age-matched controls (BMI < 25; n = 30) were included. MMP14, Col6A3 and Tissue inhibitor of metalloproteinase 2 (TIMP2) mRNA expression was assessed in VAT and their serum levels along with endotrophin were estimated in both groups preoperatively and post-operatively in the obese group. The results were analysed statistically and correlated with anthropometric and glycaemic parameters, namely fasting glucose and insulin, HbA1c, HOMA-IR, HOMA-ß and QUICKI. Circulating levels as well as mRNA expression profiling revealed significant differences between the individuals with and without obesity (p < .05), more so in individuals with diabetes and obesity (p < .05). Follow-up serum analysis revealed significantly raised MMP14 (p < .001), with decreased Col6A3, endotrophin and TIMP2 levels (p < .01, p < .001 and p < .01, respectively). A rise in serum MMP14 protein, simultaneous with post-surgical weight loss and decreased serum levels of associated extracellular matrix (ECM) remodellers, suggests its crucial role in modulating obesity-associated ECM fibrosis and pliability of VAT.

Evaluation of the Efficiency of TIMP-2 as a Biomarker for Acute Kidney Injury in Sepsis.

The aim of this study was to evaluate the biomarker potential of TIMP-2 in septic-induced acute kidney injury (AKI). Healthy male rats (n=56, age 8-10 weeks, body weight 250-300 g) were randomized into 3 groups: controls (intact rats, n=6), sham-operated (SO, n=24), and sepsis model (cecum ligation and perforation, CLP, n=24). Thirty minutes before and 6, 12, 24, and 48 h after surgery, blood samples were collected to measure serum creatinine, blood urea nitrogen (BUN), and TIMP-2 and the kidneys were isolated for histopathological analysis and Western blotting. The key sepsis-related genes were screened through bioinformatics analysis. In 24 and 48 h after surgery, 2 rats in the SO group reached the diagnostic criteria of AKI (increased levels of serum creatinine and BUN). In the CLP group, serum creatinine in 6 h after the surgery was slightly higher than 30 min before the surgery, but this change did not meet the diagnostic criteria for AKI. In the CLP group, BUN was normal 6 h after the surgery, but increased after 12 h. In more than 50% rats of the CLP group, serum creatinine and BUN significantly increased 12 h after operation, so this can be diagnosed as AKI. In rats of the CLP group, plasma TIMP-2 was elevated 6 h after surgery and increased with time, suggesting that plasma TIMP-2 can be used as an early marker of AKI. Histological examination of the kidneys in this group revealed destruction of the renal tubular structure, swelling of renal tubular epithelium, the disappearance of brush edge and collapse of necrotic epithelial cells, etc., and the degree of damage increased with time. Immunohistochemistry showed that TIMP-2 was expressed in rats of the CLP group at all terms of the experiment. The expression of TIMP-2 and pyroptosis-related proteins (NLRP3, IL-1ß, caspase-1, and GSDMD) in the CLP group was higher than in the SO group (p<0.05) and increased with time, suggesting that pyroptosis is involved in AKI. Thus, plasma TIMP-2 is sensitive indicator for the early detection of kidney injury and can be used as an early biomarker of AKI.

TIMP2 facilitates CIRI through activating NLRP3-mediated pyroptosis.

This study aimed to investigate the underlying mechanisms of cerebral ischemia-reperfusion injury (CIRI) in mice using CIR and hypoxia/reoxygenation (H/R) cell models. The study evaluated brain tissue weight, pathological injury, and changes in the expression levels of TIMP2, p-ERK1/2 and NLRP3-mediated pyroptosis-related proteins in brain tissues and hippocampal neurons of CIR mice using established methods such as dry/wet weight measurement, HE staining, qPCR, TUNEL assay, and Western blotting. The results demonstrated a significant increase in brain water content and neuronal apoptosis rate in the experimental groups compared with those in the control group. In particular, the I/R+TIMP2 group showed the highest increase. Additionally, the control group exhibited a clear brain tissue structure, neatly and densely arranged cells with normal morphology, and evenly stained and clear hippocampal tissues. However, the I/R group showed hippocampal structure disorders, interstitial edema, deep nuclear staining, karyopyknosis, and karyorrhexis in brain tissues. The study results further revealed that TIMP2 could aggravate the pathological damage of brain tissues in the I/R+TIMP2 group compared with the I/R group and significantly reduced it in the TIMP2-KD group. Furthermore, the Western blotting results demonstrated that the protein expression levels of TIMP2, p-ERK1/2, t-ERK1/2, NLRP3, IL-1ß, IL-18, GSDMD, Caspase-1, and ASC in brain tissues and hippocampal neurons were significantly higher in the experimental groups than those in the control group. The I/R+TIMP2 group displaying the highest increase and the TIMP2-KD group showing a significant decrease. In conclusion, TIMP2 can contribute to the occurrence and progression of CIRI by activating NLRP3-mediated pyroptosis.

Comparison of MMP-2 and TIMP-2 expressions in yak testes at different ages.

This study aimed to investigate the distribution and expression of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) in yak testes. The testes of healthy yaks at different ages: newborn [3 days], young [1 year], adult [4 years], and old [9 years] were collected for microscopic analyses using hematoxylin and eosin staining, immunohistochemistry and immunofluorescence, as well as western blot to compare the expression of MMP-2 and TIMP-2. Furthermore, the levels of MMP-2mRNA and TIMP-2mRNA was detected by real-time quantitative polymerase chain reaction (qPCR). The results of immunohistochemistry and immunofluorescence demonstrated that MMP-2 and TIMP-2 were mainly located in gonocytes of newborn, Sertoli cells of young, spermatozoa of adult and Leydig cells of old. The protein levels of MMP-2 and TIMP-2 exhibited a downward from newborn to adult, but increased again in old yaks. The analysis of qPCR showed that MMP-2 was higher in young compared with newborn or adult(**p < .01), but a lower expression was detected in adult compared with old yak testicular tissues (*p < .05). Compared with adults, TIMP-2 was significantly higher in newborn and young yaks (**p < .01), and slightly higher in old yaks (*p < .05). Hence, The location of MMP-2 and TIMP-2 in gonocytes were associated with the development of newborn yak testes. The expression of MMP-2 and TIMP-2 in Sertoli cells at young and adult yaks suggested that they provided a clue for the regulation of spermatogenesis. The positive labeling of MMP-2 and TIMP-2 in Leydig cells in old yaks suggested that both may be involved in the interstitial metabolism of the testes during this period. This study revealed the possible role of MMP-2 and TIMP-2 in testicular functionality of yaks at different ages.

Utilizing genetic code expansion to modify N-TIMP2 specificity towards MMP-2, MMP-9, and MMP-14.

Matrix metalloproteinases (MMPs) regulate the degradation of extracellular matrix (ECM) components in biological processes. MMP activity is controlled by natural tissue inhibitors of metalloproteinases (TIMPs) that non-selectively inhibit the function of multiple MMPs via interaction with the MMPs' Zn2+-containing catalytic pocket. Recent studies suggest that TIMPs engineered to confer MMP specificity could be exploited for therapeutic purposes, but obtaining specific TIMP-2 inhibitors has proved to be challenging. Here, in an effort to improve MMP specificity, we incorporated the metal-binding non-canonical amino acids (NCAAs), 3,4-dihydroxyphenylalanine (L-DOPA) and (8-hydroxyquinolin-3-yl)alanine (HqAla), into the MMP-inhibitory N-terminal domain of TIMP2 (N-TIMP2) at selected positions that interact with the catalytic Zn2+ ion (S2, S69, A70, L100) or with a structural Ca2+ ion (Y36). Evaluation of the inhibitory potency of the NCAA-containing variants towards MMP-2, MMP-9 and MMP-14 in vitro revealed that most showed a significant loss of inhibitory activity towards MMP-14, but not towards MMP-2 and MMP-9, resulting in increased specificity towards the latter proteases. Substitutions at S69 conferred the best improvement in selectivity for both L-DOPA and HqAla variants. Molecular modeling provided an indication of how MMP-2 and MMP-9 are better able to accommodate the bulky NCAA substituents at the intermolecular interface with N-TIMP2. The models also showed that, rather than coordinating to Zn2+, the NCAA side chains formed stabilizing polar interactions at the intermolecular interface with MMP-2 and MMP-9. Our findings illustrate how incorporation of NCAAs can be used to probe-and possibly exploit-differential tolerance for substitution within closely related protein-protein complexes as a means to improve specificity.

Ex Vivo Analysis of an Association of Mechanical Strength of Dilated Ascending Aorta with Tissue Matrix Metalloproteinases and Cytokines.

We analyzed the associations of the mechanical strength of dilated ascending aorta wall (intraoperative samples from 30 patients with non-syndromic aneurysms) with tissue MMPs and the cytokine system. Some samples were stretched to break on an Instron 3343 testing machine and the tensile strength was calculated; others were homogenized and the concentrations of MMP-1, MMP-2, MMP-7, their inhibitors (TIMP-1 and TIMP-2), and pro- and anti-inflammatory cytokines were determined by ELISA. Direct correlations between aortic tensile strength and concentrations of IL-10 (r=0.46), TNFα (r=0.60), and vessel diameter (r=0.67) and an inverse correlation with patient's age (r=-0.59) were revealed. Compensatory mechanisms supporting the strength of the ascending aortic aneurysm are possible. No associations of MMP-1, MMP-7, TIMP-1, and TIMP-2 with tensile strength and aortic diameter were found.

Is the Synovium the First Responder to Posttraumatic Knee Joint Stress? The Molecular Pathogenesis of Traumatic Cartilage Degeneration.

OBJECTIVE: The aim of this study was to evaluate if a similar catabolic and inflammatory gene pattern exists between the synovium, hyaline cartilage, and blood of patients with the knee joint tissues and if one precedes the other. DESIGN: A total of fifty-eight patients (34 females and 24 males) with a mean age of 44.7 years (range, 18-75) underwent elective knee arthroscopy due to previously diagnosed pathology. Full blood samples were collected preoperatively from synovium and cartilage samples intraoperatively. Real time PCR with spectrophotometric analysis was performed. Following genes taking part in ECM (extracellular matrix) remodeling were selected for analysis: MMP-1, MMP-2, MMP-8, MMP-9, MMP-13, MMP-14, ADAMTS-4 (Agg1) and ADAMTS-5 (Agg2) proteases, TIMP-1, and TIMP-2 - their inhibitors - and IL-1 and TNF-α cytokines. RESULTS: Analysis revealed a strong and significant correlation between gene expression in synovial and systemic blood cells (p <0.05 for all studied genes) with ADAMTS-4, ADAMTS-5, IL-1, TNF-α and TIMP-2 expression most positively correlated with an R>0.8 for each. An analysis between chondrocytes and systemic blood gene expression shown no significant correlation for all genes. Bivariate correlation of International Cartilage Repair Society grading and genes expression revealed significant associations with synovial MMP-1, MMP-2, MMP-8, MMP-9, IL-1, TNF-α and TIMP-2. CONCLUSION: We suggest that the synovial tissue is the first responder for knee joint stress factors in correlation with the response of blood cells. The chondrocyte's genetic response must be further investigated to elucidate the genetic program of synovial joints, as an organ, during OA development and progression.

Impact of Matrix Metalloproteinases 2 and 9 and Tissue Inhibitor of Metalloproteinase 2 Gene Polymorphisms on Allograft Rejection in Pediatric Renal Transplant Recipients.

OBJECTIVES: Acute and chronic allograft rejection have been continuously an important obstacle in the follow-up of renal transplant recipients. During clinical management, several factors acting simultaneously result in acute rejection and chronic allograft nephropathy. Matrix metalloproteinases and tissue inhibitors of metalloproteinases are responsible for the organization of the extracellular matrix and play roles in cell proliferation and cellular invasion. Changes in matrix metalloproteinase expression levels have been reported to be associated with renal allograft rejection and interstitial fibrosis. In this study, we aimed to investigate functional polymorphisms of MMP2, MMP9, and TIMP2 genes in pediatric renal transplant recipients. MATERIALS AND METHODS: Our study included 68 kidney transplant recipients and 58 control patients. The kidney transplant recipient group was further divided into 2 subgroups: no graft rejection (n = 47) and graft rejection (n =21). MMP2 -735C >T (rs2285053), MMP2 -1306C >T (rs243865), MMP2 -1575G >A (rs243866), MMP9 c.-1562C >T (rs3918242), TIMP2 -418G >C (rs8179090), and TIMP2 303C > T (rs2277698) polymorphisms were analyzed with the use of polymerase chain reaction and restriction fragment-length polymorphism methods. Allele prevalence was compared with reference values of the control group, and Hardy-Weinberg equilibrium was tested. RESULTS: Mean ages were 16.7 ± 3.9 years for the study group and 14.8 ± 5.6 years for the control group. The mean follow-up time after transplant was 37.7 ± 7.9 months. We compared allele frequencies in the 2 groups and calculated a statistically significant difference in rs2285053, rs243865, rs243866, rs3918242, rs8179090, and rs2277698 polymorphism frequencies between the transplant recipients and control patients. When the transplant recipient group was compared in itself with regard to allograft rejection, all investigated polymorphisms except TIMP2 -418G >C (rs8179090) revealed a statistically significant difference between those with and without rejection (P < .05). CONCLUSIONS: Matrix metalloproteinases and their tissue inhibitors could be important predictive biological markers for the follow-up of kidney transplant recipients.

Poly-L-Lactic acid increases collagen gene expression and synthesis in cultured dermal fibroblast (Hs68) through the TGF-ß/Smad pathway.

OBJECTIVE: Poly-L-Lactic Acid (PLLA) is a synthetic polymer which possesses biocompatible and biodegradable properties, and is widely used in the clinical filler material. This study focuses on the potential role of PLLA on the collagen production of dermal fibroblasts and its mechanism. METHODS: The dermal fibroblast Hs60 was treated with different concentration of PLLA. RT-qPCR was conducted for the determination of mRNA levels of collagen type I (COL1) alpha 1 (COL1A1), COL1 alpha 2 (COL1A2), elastin, matrix metalloproteinase 1 (MMP-1), tissue inhibitor of metalloproteinase 1 (TIMP-1), and TIMP-2. Procollagen Type I C-peptide (PIP) enzyme immunoassay (EIA) Kit assay was carried out to analyze procollagen production. Western Blot was employed to examine the effect of PLLA and transforming frown factor (TGF-ß) receptor-specific inhibitor (SB431542) on protein levels of COL1A1 and TGF-ß/Smad signaling pathway related proteins. RESULTS: With the increase of PLLA concentration, the production of procollagen gradually increased, and both protein and mRNA levels of COL1A1 and COL1A2 gradually increased (p < 0.001). Elevated PLLA concentrations increased elastin, TIMP-1, and TIMP-2 levels and attenuated MMP-1 expression. PLLA increased TGF-ß levels in a dose-dependently manner. p-Smad2 and p-Smad3 protein levels were also increased by PLLA, but the influences were reversed by SB431542 (p < 0.001). Similarly, increased levels of COL1A1, COL1A2, TIMP-1, and TIMP-2 caused by PLLA were significantly inhibited by SB431542, whereas MMP-1 was typically elevated (p < 0.001). CONCLUSION: Poly-L-Lactic Acid promotes the collagen production of dermal fibroblasts by activating the TGF-ß/Smad signaling pathway. The findings may lay a foundation for clinical material applications of PLLA.

Cancer-derived exosomal miR-197-3p confers angiogenesis via targeting TIMP2/3 in lung adenocarcinoma metastasis.

Cancer-derived exosomal miRNAs are implicated in tumorigenesis and development of lung adenocarcinoma (LUAD). The objective of this study is to unravel the biological function of exosomal miR-197-3p in LUAD metastasis. qRT-PCR showed that elevated miR-197-3p in LUAD tissues was positively correlated with LUAD metastasis. CCK-8, tube formation, transwell and wound healing assays revealed that exosomal miR-197-3p from LUAD cells promoted the proliferation, angiogenesis and migration of HUVECs in vitro. LUAD cells-derived exosomal miR-197-3p also facilitated tumor growth and angiogenesis in LUAD cells-derived tumor xenograft model. TIMP2 and TIMP3 were identified as target genes of miR-197-3p in HUVECs by bioinformatics analysis and luciferase reporter assay. Functional studies illustrated that exosomal miR-197-3p promoted angiogenesis and migration via targeting TIMP2 and TIMP3 in HUVECs. In vivo data further supported that exosomal miR-197-3p promoted lung metastasis via TIMP2/3-mediated angiogenesis. In conclusion, LUAD cells-derived exosomal miR-197-3p conferred angiogenesis via targeting TIMP2/3 in LUAD metastasis.