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.

Detrimental Effects of ApoE ε4 on Blood-Brain Barrier Integrity and Their Potential Implications on the Pathogenesis of Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease representing the most common type of dementia in older adults. The major risk factors include increased age, genetic predisposition and socioeconomic factors. Among the genetic factors, the apolipoprotein E (ApoE) ε4 allele poses the greatest risk. Growing evidence suggests that cerebrovascular dysfunctions, including blood-brain barrier (BBB) leakage, are also linked to AD pathology. Within the scope of this paper, we, therefore, look upon the relationship between ApoE, BBB integrity and AD. In doing so, both brain-derived and peripheral ApoE will be considered. Despite the considerable evidence for the involvement of brain-derived ApoE ε4 in AD, information about the effect of peripheral ApoE ε4 on the central nervous system is scarce. However, a recent study demonstrated that peripheral ApoE ε4 might be sufficient to impair brain functions and aggravate amyloid-beta pathogenesis independent from brain-based ApoE ε4 expression. Building upon recent literature, we provide an insight into the latest research that has enhanced the understanding of how ApoE ε4, secreted either in the brain or the periphery, influences BBB integrity and consequently affects AD pathogenesis. Subsequently, we propose a pathway model based on current literature and discuss future research perspectives.

Targeting multiple disease hallmarks using a synergistic disease-modifying drug combination ameliorates osteoarthritis via inhibition of senescence and inflammation.

AIMS: Osteoarthritis (OA), is a debilitating disease characterized by progressive cartilage degradation, synovial inflammation, and chondrocyte senescence. Various treatment agents independently targeting these hallmarks have been investigated. However, due to the complex multifaceted nature of OA, no disease-modifying osteoarthritis drugs are clinically available. In an attempt to overcome this, we developed a combinatorial approach and demonstrated the efficacy of TsC [Tissue inhibitor of metalloproteinase-3 (TIMP3) + sulfated carboxymethylcellulose (sCMC)] and piperlongumine (PL) combination for the amelioration of OA in a goat ex vivo OA model. MAIN METHODS: The efficacy of the drug combination was evaluated using the goat ex vivo OA explant model and results were validated in clinically relevant human OA cartilage explants. The chondroprotective effects were evaluated in terms of reduced inflammation and cartilage matrix loss, reduction in chondrosenescence, and reduced oxidative stress. KEY FINDINGS: A combination of TsC and PL (TsC-PL) significantly reduced inflammation, cartilage matrix loss, chondrosenescence, and oxidative stress in the goat ex vivo OA model and showed chondroprotective effects. Further, similar chondroprotective effects were observed in human OA cartilage. Additionally, the coefficient of drug interaction analysis indicated that the combination of TsC and PL had a synergistic effect in reducing matrix degrading proteases and inflammation (goat ex vivo OA model) and Reactive oxygen species (ROS) production (human OA cartilage). SIGNIFICANCE: Combinatorial treatment with TsC and PL demonstrated potential disease-modifying effects for the treatment of osteoarthritis via inhibition of inflammation and senescence and supports the usage of treatment strategies targeting multiple pathological factors of OA simultaneously.

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.

TIMP3 induces gene expression partly through PI3K and their association with vascularization and heart rate.

Background: Tissue inhibitor of metalloproteinase 3 (TIMP3) was recently demonstrated capable to regulate some gene expression in a myocardial infarction model. Here we aim to explore the gene expression profile in TIMP3-treated cardiomyocytes and related potential cardiovascular functions. Methods: Total RNA extracted from cultured neonatal rat ventricular myocytes (NRVMs) were used for RNA sequencing analysis and real-time PCR. KEGG pathway enrichment assay and Ingenuity Pathway Analysis (IPA) were performed to study the signaling pathways and downstream effects. Western blot was used to detect phosphorylation of protein kinase B (Akt). A Cell Counting Kit-8 assay was employed to evaluate the proliferation of human umbilical vein endothelial cells (HUVECs). Contraction rate of NRVMs was measured with microscopy. Results: RNA sequencing data showed that expression of 2,526 genes were significantly modulated by recombinant TIMP3 (rTIMP3, 100 ng/ml) in NRVMs. Some differentially expressed genes (DEGs) were validated with real-time PCR. Several KEGG pathways including the phosphoinositide-3-kinase (PI3K)-Akt pathway were significantly regulated by rTIMP3. Phosphorylation of Akt was increased by rTIMP3 and a PI3K inhibitor LY294002 suppressed rTIMP3-induced up-regulation of some genes. Some DEGs were predicted by IPA to increase vascularization, and some to decrease heart rate. RTIMP3 could reduce the contraction rate of NRVMs and its conditioned media increased the proliferation of HUVECs. Conclusion: TIMP3 can regulate expression of multiple genes partly through PI3K. Some DEGs were associated with activation of vascularization and some with heart rate reduction. This study suggests that TIMP3 can potentially modulate cardiovascular functions via DEGs.

Chronic Kidney Disease: Combined Effects of Gene Polymorphisms of Tissue Inhibitors of Metalloproteinase 3, Total Urinary Arsenic, and Blood Lead Concentration.

The tissue inhibitor of metalloproteinase 3 (TIMP3) is known to be an anti-fibrotic factor. Arsenic, lead, and cadmium exposure and selenium intake may affect TIMP3 expression. The downregulation of TIMP3 expression is related to kidney fibrosis. Genotypes of TIMP3 are related to hypertension and cardiovascular diseases. Therefore, this study explored whether TIMP3 polymorphism is associated with hypertension-related chronic kidney disease (CKD). In addition, the combined effects of TIMP3 polymorphism and total urinary arsenic, blood lead and cadmium, and plasma selenium concentrations on CKD, were investigated. This was a case-control study, with 213 CKD patients and 423 age- and sex-matched controls recruited. Polymerase chain reaction-restriction fragment length polymorphism was used to determine TIMP3 gene polymorphisms. The concentrations of urinary arsenic species, plasma selenium, and blood lead and cadmium were measured. The odds ratio (OR) of CKD in the TIMP3rs9609643 GA/AA genotype was higher than that of the GG genotype at high levels of total urinary arsenic and blood lead; the OR and 95% confidence interval (CI) were 0.57 (0.31-1.05) and 0.52 (0.30-0.93), respectively, after multivariate adjustment. High blood lead levels tended to interact with the TIMP3rs9609643 GG genotype to increase the OR of CKD, and gave the highest OR (95% CI) for CKD of 5.97 (2.60-13.67). Our study supports a possible role for the TIMP3rs9609643 risk genotype combined with high total urinary arsenic or with high blood lead concentration to increase the OR of CKD.

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.

Effects of LncRNA MEG3 on the invasion and migration of prostate cancer cells by regulating microRNA-181b-5p/TIMP3 / 现代泌尿外科杂志

【Objective】 To explore the effects of long non-coding RNA maternally expressed gene 3 (lncRNA MEG3) on the invasion and migration of prostate cancer cells (PC3 cells) by regulating microRNA-181b-5p (miR-181b-5p)/tissue inhibitor of metalloproteinase 3 (TIMP3). 【Methods】 The prostate cancer tissues and adjacent tissues were collected from 20 prostate cancer patients treated in our hospital during Dec.2020 and Dec.2021. The expressions of MEG3 and miR-181b-5p in tissues were detected with quantitative real-time PCR (qRT-PCR). P3 cells were randomly divided into control group (untreated), pcDNA3.1-NC (transfected with pcDNA3.1-NC), pcDNA3.1-MEG3 group (transfected with pcDNA3.1-MEG), pcDNA3.1-MEG3+miR-NC group (pcDNA3.1-MEG3 co-transfected with miR-NC), pcDNA3.1-MEG3+miR-181b-5p mimic group (pcDNA3.1-MEG3 co-transfected with miR-181b-5p mimic). The expressions of MEG3 and miR-181b-5p in PC3 cells were detected with qRT-PCR. The cell viability, invasion and migration ability were determined with MTT assay, Transwell assay and scratch assay. The protein expressions of TIMP3, matrix metalloproteinase (MMP)9 and MMP2 in PC3 cells were detected with Western blot. The targeting relationship of MEG3, miR-181b-5p and TIMP3 was analyzed with dual luciferase assay. 【Results】 The expressions of MEG3 in prostate cancer tissues ( 0.37±0.05 vs. 1.00±0.04) and cells (0.31±0.06 vs. 1.00±0.01) were significantly decreased (P<0.05). Compared with the control group, the pcDNA3.1-MEG3 group had significantly decreased expression of miR-181b-5p (0.26±0.04 vs.1.00±0.02 ), cell survival rate (53.60±5.22 vs.100.00±0.00), number of invasive cells (62.33±9.85 vs.162.34±21.30), cell migration rate (32.85±3.80 vs.75.22±5.96), expressions of MMP9 (0.61±0.08 vs.1.62±0.23) and MMP2 (0.73±0.10 vs.1.20±0.16), but significantly higher expressions of MEG3 (2.31±0.36 vs. 1.00±0.01) and TIMP3 (1.32±0.24 vs. 0.53±0.08) (P<0.05). Overexpression of miR-181b-5p reversed the above changes (P<0.05). MiR-181b-5p had a targeting relationship with MEG3 and TIMP3. 【Conclusion】 Overexpression of lncRNA MEG3 can inhibit miR-181b-5p to promote the expression of TIMP3, thereby inhibiting invasion and migration of PC3 cells.

TIMP3 Gene Polymorphisms of -1296 T > C and -915 A > G Increase the Susceptibility to Arsenic-Induced Skin Cancer: A Cohort Study and In Silico Analysis of Mutation Impacts

Long-term exposure to arsenic may induce several human cancers, including non-melanoma skin cancer. The tissue inhibitor of metalloproteinase (TIMP)-3, encoded by the TIMP3 gene, may inhibit tumor growth, invasion, and metastasis of several cancer types. In this study, we aimed to investigate effects of the TIMP3 -1296 T > C (rs9619311) and -915 A > G (rs2234921) single-nucleotide polymorphisms (SNPs) on skin cancer risk in an arsenic-exposed population, and to evaluate the influence of allele-specific changes by an in silico analysis. In total, 1078 study participants were followed up for a median of 15 years for newly diagnosed skin cancer. New cases were identified through linkage to the National Cancer Registry of Taiwan. A Cox regression analysis was used to evaluate the effects of TIMP3 variants. Transcription factor (TF) profiling of binding sites of allele-specific changes in SNPs was conducted using the JASPAR scan tool. We observed borderline associations between TIMP3 genotypes and skin cancer risk. However, when combined with high arsenic exposure levels, the rs9619311 C allele, rs2234921 G allele, or C-G haplotype groups exhibited a greater risk of developing skin cancer compared to the respective common homozygous genotype group. The in silico analysis revealed several TF motifs located at or flanking the two SNP sites. We validated that the C allele of rs9619311 attenuated the binding affinity of BACH2, MEIS2, NFE2L2, and PBX2 to the TIMP3 promoter, and that the G allele of rs2234921 reduced the affinity of E2F8 and RUNX1 to bind to the promoter. Our findings suggest significant modifications of the effect of the association between arsenic exposure and skin cancer risk by the TIMP3 rs9619311 and rs2234921 variants. The predicted TFs and their differential binding affinities to the TIMP3 promoter provide insights into how TIMP3 interacts with arsenic through TFs in skin cancer formation.

Nanoparticles design considerations to co-deliver nucleic acids and anti-cancer drugs for chemoresistance reversal.

Chemoresistance and hence the consequent treatment failure is considerably challenging in clinical cancer therapeutics. The understanding of the genetic variations in chemoresistance acquisition encouraged the use of gene modulatory approaches to restore anti-cancer drug efficacy. Many smart nanoparticles are designed and optimized to mediate combinational therapy between nucleic acid and anti-cancer drugs. This review aims to define a rational design of such co-loaded nanocarriers with the aim of chemoresistance reversal at various cellular levels to improve the therapeutic outcome of anticancer treatment. Going through the principles of therapeutics loading, physicochemical characteristics tuning, and different nanocarrier modifications, also looking at combination effectiveness on chemosensitivity restoration. Up to now, these emerging nanocarriers are in development status but are expected to introduce outstanding outcomes.

Comprehensive analyses of DNA methylation of the TIMP3 promoter in placentas from early-onset and late-onset preeclampsia.

Preeclampsia (PE) is classified into late-onset (LOPE) or early-onset (EOPE) according to gestational age of onset (≥34 or <34 weeks, respectively), and into preterm and term (delivery at <37 or ≥37 weeks, respectively). An imbalanced expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) impairs proper placentation in PE, and DNA methylation (DNAm) may affect their expression. We performed comprehensive analyses of DNAm and TIMP3 expression in placentas from PE reclassified into EOPE, LOPE, and term PE. We identified significant differentially methylated probes at the TIMP3 promoter in PE (28), EOPE (38), LOPE (20), and term PE (4) compared to controls, and in EOPE vs. LOPE (8). Moreover, we found a hypomethylation >70% in all groups (except EOPE vs. LOPE) and an increased TIMP3 expression in corresponding placental samples from PE, EOPE and LOPE compared to controls (p<0.05). Our findings highlight the role of DNAm of the TIMP3 promoter region regarding an epigenetic mechanism in PE.

Increased expression of secreted protein acidic and rich in cysteine and tissue inhibitor of metalloproteinase-3 in epidermotropic melanoma metastasis.

Primary cutaneous melanoma generally arises in the epidermis, followed by invasion into the dermis. Although infrequent, invasive melanoma cells can, alternatively, migrate to the intraepidermal area and form epidermotropic melanoma metastasis (EMM). In this study, we focused on this unique manner of metastasis. To identify the key molecules which affect EMM, gene expression in EMM was compared with that in common skin metastasis (CSM). Polymerase chain reaction (PCR) analysis was performed for genes affecting the extracellular matrix, cellular adhesion, and tumor metastasis on three EMM and three CSM samples as an initial screening. For molecules showing altered expression in the EMM, expression levels were further verified using real-time quantitative PCR (qPCR) and immunohistochemistry. Five molecules showed an expression difference in the initial screening. Among these, secreted protein acidic and rich in cysteine (SPARC) was preferentially expressed in EMM (p = 0.01) by real-time qPCR. Another candidate molecule, tissue inhibitor of metalloproteinase-3 (TIMP3), was not statistically significant (p = 0.07), but showed the tendency of higher expression. These results correlated negatively to expression of N-cadherin and ß-catenin. The upregulation of SPARC and TIMP3 may disrupt the continuity of the canonical Wnt pathway. This pathway regulates adhesion activity of melanoma cells to localize within the dermis, which consequently promotes EMM. Our study highlights the potential role of SPARC and TIMP3 as key molecules in EMM, and analysis of EMM may contribute for understanding melanoma invasion between the epidermis and the dermis.

Inhibited microRNA-494-5p promotes proliferation and suppresses senescence of nucleus pulposus cells in mice with intervertebral disc degeneration by elevating TIMP3.

It has been unraveled that microRNAs (miRNAs) played crucial roles in processes of human diseases, while the role of miR-494-5p in intervertebral disc degeneration (IDD) remains scarcely studied. We aimed to investigate the mechanisms of miR-494-5p in IDD with the involvement of tissue inhibitor of metalloproteinase 3 (TIMP3). Expression of miR-494-5p and TIMP3 in IDD clinical specimens was assessed. The IDD models were established by needle punching, which were then injected with low expression of miR-494-5p or TIMP3 overexpression lentivirus to observe their effects on pathology and apoptosis in IDD mice. The nucleus pulposus cells were isolated and, respectively, treated with miR-494-5p inhibitor or TIMP3 overexpression plasmid to determine the viability, apoptosis, and senescence in vitro. Furthermore, the expression of Aggrecan, Col-2, Caveolin-1, and SA-ß-gal in nucleus pulposus cells in vitro were measured. The target relation between miR-494-5p and TIMP3 was determined. An increased expression of miR-494-5p and a decreased expression of TIMP3 were found in IDD. Downregulation of miR-494-5p or overexpression of TIMP3 could relieve pathology and suppress nucleus pulposus cell apoptosis in IDD mice, as well as promote the viability and attenuate the apoptosis and senescence of nucleus pulposus cells from IDD mice. Moreover, inhibition of miR-494-5p or overexpression of TIMP3 upregulated Aggrecan and Col-2 expression while downregulated Caveolin-1 and SA-ß-gal expression, and TIMP3 was the target gene of miR-494-5p. Results of this study indicated that the degradation of miR-494-5p ameliorates the development of IDD by elevating TIPM3, which may provide new targets for IDD treatment.

Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation.

Purpose: Endogenous tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) has powerful regulatory effects on inflammation and angiogenesis. In this study, we investigated the role of TIMP-3 in regulating inflammation in the diabetic retina. Methods: Vitreous samples from patients with proliferative diabetic retinopathy (PDR) and non-diabetic patients were subjected to Western blot analysis. Streptozotocin-treated rats were used as a preclinical diabetic retinopathy (DR) model. Blood-retinal barrier (BRB) breakdown was assessed with fluorescein isothiocyanate (FITC)-conjugated dextran. Rat retinas, human retinal microvascular endothelial cells (HRMECs) and human retinal Müller glial cells were studied by Western blot analysis and ELISA. Adherence of human monocytes to HRMECs was assessed and in vitro angiogenesis assays were performed. Results: Tissue inhibitor of matrix metalloproteinase-3 in vitreous samples was largely glycosylated. Intravitreal injection of TIMP-3 attenuated diabetes-induced BRB breakdown. This effect was associated with downregulation of diabetes-induced upregulation of the p65 subunit of NF-κB, intercellular adhesion molecule-1 (ICAM-1), and vascular endothelial growth factor (VEGF), whereas phospho-ERK1/2 levels were not altered. In Müller cell cultures, TIMP-3 significantly attenuated VEGF upregulation induced by high-glucose (HG), the hypoxia mimetic agent cobalt chloride (CoCl2) and TNF-α and attenuated MCP-1 upregulation induced by CoCl2 and TNF-α, but not by HG. TIMP-3 attenuated HG-induced upregulation of phospho-ERK1/2, caspase-3 and the mature form of ADAM17, but not the levels of the p65 subunit of NF-κB and the proform of ADAM17 in Müller cells. TIMP-3 significantly downregulated TNF-α-induced upregulation of ICAM-1 and VCAM-1 in HRMECs. Accordingly, TIMP-3 significantly decreased spontaneous and TNF-α- and VEGF-induced adherence of monocytes to HRMECs. Finally, TIMP-3 significantly attenuated VEGF-induced migration, chemotaxis and proliferation of HRMECs. Conclusion: In vitro and in vivo data point to anti-inflammatory and anti-angiogenic effects of TIMP-3 and support further studies for its applications in the treatment of DR.

MAb NJ001 inhibits lung adenocarcinoma invasiveness by directly regulating TIMP-3 promoter activity via FOXP1 binding sites.

BACKGROUND: Previously, we developed a monoclonal antibody (mAb) NJ001 that binds to the antigen SP70 in human non-small cell lung cancer (NSCLC) cells and showed it could inhibit lung adenocarcinoma (AD) growth. Here, we investigated the effect and mechanisms of NJ001 in lung AD metastasis. METHODS: Human lung AD cells (SPC-A1 and A549) were treated with different concentrations of mAb NJ001, and the effects of NJ001 on cell migration and invasive activity were investigated using wound-healing and Matrigel assays, respectively. The molecular mechanism of this inhibition was explored by microarrays, qRT-PCR, western blot, luciferase assays and electrophoretic mobility shift assays (EMSA). RESULTS: MAb NJ001 markedly suppressed lung AD cell migration; and the invasiveness of SPC-A1 and A549 cells treated with mAb NJ001 was diminished by 65%. Tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) was highly expressed in SPC-A1 cells treated with mAb NJ001, whereas knockdown of TIMP-3 by shRNA significantly increased SPC-A1 and A549 invasiveness. MAb NJ001 affects lung AD by inhibiting TIMP-3 through direct transcriptional regulation of FOXP1 binding sites in the TIMP-3 promoter region, as shown in luciferase assays and EMSA. CONCLUSIONS: MAb NJ001 inhibits invasiveness and metastasis in lung AD through the FOXP1 binding sites in the TIMP-3 promoter region. It may have clinical applications in preventing and treating metastatic lung AD.

Overexpression of TIMP3 inhibits discogenic pain by suppressing angiogenesis and the expression of substance P in nucleus pulposus.

Approximately 50% of the cases of low back pain (LBP) are attributed to discogenic origin. The causes of discogenic pain are complicated and consist of a complex biochemical cascade. Neovascularization of intervertebral discs (IVDs) is believed to be associated with discogenic pain. The anti­angiogenesis ability of tissue inhibitor of metalloproteinase­3 (TIMP3) has been reported in many tumors, yet whether TIMP3 is associated with neovascularization of IVDs remains unknown. In the present study, both in vitro and in vivo models were used to investigate the association between discogenic pain and TIMP3 expression in nucleus pulposus (NP). PCR results demonstrated that inflammation induced downregulation of TIMP3 expression in NP cells. By using an adenovirus system to upregulate TIMP3 expression, the effect of TIMP3 on angiogenesis was measured by endothelial cell migration and tube formation assays. The results demonstrated that overexpression of TIMP3 suppressed angiogenesis in NP without the regulation of vascular endothelial growth factor (VEGF) expression. TNF­α converting enzyme (TACE) expression was downregulated by TIMP3, thus inhibiting the TACE­induced activation of TNF­α in NP cells. Immunohistochemical staining of IVDs also confirmed that TIMP3 inhibited the expression of substance P in NP. Taken together, the present results indicated the expression of TIMP3 in NP may have a key role in the development of discogenic pain.

Protein expression pattern of tissue inhibitor of metalloproteinase-3 (TIMP3) in endometriosis and normal endometrium.

Given the involvement of different extracellular matrix (ECM) metalloproteinases (MMPs) in endometriosis, the protein expression pattern of tissue inhibitor of metalloproteinase-3 (TIMP3) was analyzed in this study in endometriosis and normal endometrium. Tissue samples were collected prospectively from 64 premenopausal patients undergoing operative laparoscopy. Protein expression of TIMP3 was analyzed immunohistochemically in endometriotic lesions (n = 30) and normal eutopic endometrium from patients with (n = 35) and without (n = 29) endometriosis. Comparison between the three different groups of tissue samples showed that TIMP3 was differentially expressed between the three groups (p = .04). Pair-wise comparisons showed that TIMP3 expression was lower in endometriotic lesions as compared with normal eutopic endometrium from controls (p = .006); the same non-significant trend was found, in the comparison between endometriosis lesions and matched eutopic endometrium. There were no differences in TIMP3 expression in the normal eutopic endometrium between patients with and without endometriosis. In conclusion, TIMP3 seems to be involved in the pathogenesis, pathophysiology, and maintenance of endometriosis and it might be useful as a diagnostic and prognostic marker of endometriosis. Future studies should further investigate this issue, as well as the interplay between TIMPs and different extracellular MMPs in endometriosis.

miR-21-5p inhibits neuropathic pain development via directly targeting C-C motif ligand 1 and tissue inhibitor of metalloproteinase-3.

MicroRNAs (miRNA) play important roles in neuroinflammation and neuropathic pain development; however, the underlying mechanism requires further investigation. The expression of miR-21-5p was remarkably upregulated in chronic constrictive injury (CCI) rat model. A significant alleviated neuropathic pain development and reduced the expression of cytokines was observed in CCI rat after exogenous injection of miR-21-5p mimic. The dual-luciferase analysis revealed that tissue inhibitor of metalloproteinase-3 (TIMP3) and chemokines C-C motif ligand 1 (CCL1) was direct downstream target of miR-21-5p. Moreover, silencing of TIMP3 and CCL1 could rescue mechanical allodynia, thermal hyperalgesia and cytokine release in CCI rat, suggesting that TIMP3 and CCL1 exert their function by mediating neuroinflammation in neuropathic pain development. Therefore, we have identified a novel miR-21-5p-CCL1/TIMP3-cytokine axis in regulation of neuropathic pain development in CCI rat model, which is valuable for enhancing our understanding of neuropathic pain and developing miRNAs as potential therapeutic options in the future.

Lung Cancer Therapy Targeting Histone Methylation: Opportunities and Challenges.

Lung cancer is one of the most common malignancies. In spite of the progress made in past decades, further studies to improve current therapy for lung cancer are required. Dynamically controlled by methyltransferases and demethylases, methylation of lysine and arginine residues on histone proteins regulates chromatin organization and thereby gene transcription. Aberrant alterations of histone methylation have been demonstrated to be associated with the progress of multiple cancers including lung cancer. Inhibitors of methyltransferases and demethylases have exhibited anti-tumor activities in lung cancer, and multiple lead candidates are under clinical trials. Here, we summarize how histone methylation functions in lung cancer, highlighting most recent progresses in small molecular inhibitors for lung cancer treatment.

TIMP3 deficiency exacerbates iron overload-mediated cardiomyopathy and liver disease.

Chronic iron overload results in heart and liver diseases and is a common cause of morbidity and mortality in patients with genetic hemochromatosis and secondary iron overload. We investigated the role of tissue inhibitor of metalloproteinase 3 (TIMP3) in iron overload-mediated tissue injury by subjecting male mice lacking Timp3 ( Timp3-/-) and wild-type (WT) mice to 12 wk of chronic iron overload. Whereas WT mice with iron overload developed diastolic dysfunction, iron-overloaded Timp3-/- mice showed worsened cardiac dysfunction coupled with systolic dysfunction. In the heart, loss of Timp3 was associated with increased myocardial fibrosis, greater Timp1, matrix metalloproteinase ( Mmp) 2, and Mmp9 expression, increased active MMP-2 levels, and gelatinase activity. Iron overload in Timp3-/- mice showed twofold higher iron accumulation in the liver compared with WT mice because of constituently lower levels of ferroportin. Loss of Timp3 enhanced the hepatic inflammatory response to iron overload, leading to greater neutrophil and macrophage infiltration and increased hepatic fibrosis. Expression of inflammation-related MMPs (MMP-12 and MMP-13) and inflammatory cytokines (IL-1ß and monocyte chemoattractant protein-1) was elevated to a greater extent in iron-overloaded Timp3-/- livers. Gelatin zymography demonstrated equivalent increases in MMP-2 and MMP-9 levels in WT and Timp3-/- iron-overloaded livers. Loss of Timp3 enhanced the susceptibility to iron overload-mediated heart and liver injury, suggesting that Timp3 is a key protective molecule against iron-mediated pathology. NEW & NOTEWORTHY In mice, loss of tissue inhibitor of metalloproteinase 3 ( Timp3) was associated with systolic and diastolic dysfunctions, twofold higher hepatic iron accumulation (attributable to constituently lower levels of ferroportin), and increased hepatic inflammation. Loss of Timp3 enhanced the susceptibility to iron overload-mediated injury, suggesting that Timp3 plays a key protective role against iron-mediated pathology.