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.

Comparative Study of Cytokine Levels in Different Respiratory Samples in Mild-to-Moderate AECOPD Patients.

BACKGROUND: Matrix metalloproteinase-12 (MMP-12) and Tissue inhibitor of metalloproteinase-4 (TIMP-4) play important roles in the pathophysiology of chronic obstructive pulmonary disease (COPD). Subjects of many previous studies were patients with severe and very severe COPD. However, there are comparatively few studies on patients with mild-to-moderate COPD. Our aim was to measure MMP-12 and TIMP-4 levels and to compare its levels in various materials in patients with mild-to-moderate acute exacerbation of chronic obstructive pulmonary disease (AECOPD). We also compared which of the two materials of these biomarkers was better correlated with disease severity and DODE index. METHODS: A total of 39 patients with AECOPD and 25 control subjects were enrolled in our study. MMP-12 and TIMP-4 in different respiratory samples were detected by ELISA. RESULTS: Expression levels of MMP-12 in bronchoalveolar lavage fluid (BALF) and exhaled breath condensate (EBC) and TIMP-4 in BALF were significantly higher in AECOPD patients than that in healthy subjects (P < 0.001). However, there was no significant difference in TIMP-4 level in EBC of AECOPD patients compared to healthy subjects (P = 0.0527). The levels of MMP-12 in BALF and EBC and TIMP-4 in BAFL of AECOPD patients were significantly correlated with FEV1% predicted (P < 0.001). However, in AECOPD patients, there was no significant correlation between TIMP-4 levels in EBC and BODE index (r = 0.4175, P = 0.0559). CONCLUSION: During mild-to-moderate AECOPD, the levels of MMP-12 and TIMP-4 in BALF were better correlated with FEV1% predicted and BODE index than that in EBC, indicating that they may be new target interventions for pharmacology to prevent and/or treat AECOPD.

Genome-wide interaction analysis of folate for colorectal cancer risk.

BACKGROUND: Epidemiological and experimental evidence suggests that higher folate intake is associated with decreased colorectal cancer (CRC) risk; however, the mechanisms underlying this relationship are not fully understood. Genetic variation that may have a direct or indirect impact on folate metabolism can provide insights into folate's role in CRC. OBJECTIVES: Our aim was to perform a genome-wide interaction analysis to identify genetic variants that may modify the association of folate on CRC risk. METHODS: We applied traditional case-control logistic regression, joint 3-degree of freedom, and a 2-step weighted hypothesis approach to test the interactions of common variants (allele frequency >1%) across the genome and dietary folate, folic acid supplement use, and total folate in relation to risk of CRC in 30,550 cases and 42,336 controls from 51 studies from 3 genetic consortia (CCFR, CORECT, GECCO). RESULTS: Inverse associations of dietary, total folate, and folic acid supplement with CRC were found (odds ratio [OR]: 0.93; 95% confidence interval [CI]: 0.90, 0.96; and 0.91; 95% CI: 0.89, 0.94 per quartile higher intake, and 0.82 (95% CI: 0.78, 0.88) for users compared with nonusers, respectively). Interactions (P-interaction < 5×10-8) of folic acid supplement and variants in the 3p25.2 locus (in the region of Synapsin II [SYN2]/tissue inhibitor of metalloproteinase 4 [TIMP4]) were found using traditional interaction analysis, with variant rs150924902 (located upstream to SYN2) showing the strongest interaction. In stratified analyses by rs150924902 genotypes, folate supplementation was associated with decreased CRC risk among those carrying the TT genotype (OR: 0.82; 95% CI: 0.79, 0.86) but increased CRC risk among those carrying the TA genotype (OR: 1.63; 95% CI: 1.29, 2.05), suggesting a qualitative interaction (P-interaction = 1.4×10-8). No interactions were observed for dietary and total folate. CONCLUSIONS: Variation in 3p25.2 locus may modify the association of folate supplement with CRC risk. Experimental studies and studies incorporating other relevant omics data are warranted to validate this finding.

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

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

Gene Expression in Intracranial Aneurysms-Comparison Analysis of Aneurysmal Walls and Extracranial Arteries with Real-Time Polymerase Chain Reaction and Immunohistochemistry.

BACKGROUND: This study was aimed at evaluating the gene expression levels of 4 genes in the intracranial aneurysm wall and comparing them with extracranial arteries. The analysis was done using real-time polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC). Also, a correlation of the differential genetic expression was done with various patient clinical and radiologic factors. METHODS: The quantitative assessment of ribonucleic acid levels was done with RT-PCR and was validated with IHC. The genes studied were collagen 1A2 (COL1A2), tissue inhibitor of metalloproteinase 4 (TIMP4), cathepsin B (CTSB), and alpha-1 antitrypsin (α-1 AT). The analysis was done on 24 aneurysm sacs and superficial temporal/occipital artery samples from patients undergoing surgical clipping. RESULTS: The mean fold change of COL1A2 in the aneurysm sample was 8.89, that of TIMP4 was 10.16, that of CTSB was 1.02, and that of α-1 AT was 1.46 when compared with normal control vessel on PCR. On semiquantitative IHC, COL1A2 was 94.44%, α-1 AT was 77.8% overexpressed, CTSB was positive in 50%, and the expression of TIMP4 was 94.4% underexpressed in aneurysmal walls. There was no statistically significant correlation between patient profile and gene expression. CONCLUSIONS: On RT-PCR and IHC analysis, COL1A2 and α-1 AT were overexpressed, CTSB was marginally overexpressed, and TIMP4 had equivocal expression in the aneurysmal sac when compared with the normal extracranial vessel. This is the first study of its kind in the Indian population with the largest sample size on live human patients.

Protective Role for Tissue Inhibitor of Metalloproteinase-4, a Novel Peroxisome Proliferator-Activated Receptor-γ Target Gene, in Smooth Muscle in Deoxycorticosterone Acetate-Salt Hypertension.

Loss of peroxisome proliferator-activated receptor-γ (PPARγ) function causes hypertension, whereas its activation lowers blood pressure. Evidence suggests that these effects may be attributable to PPARγ activity in the vasculature. However, the specific transcriptional targets of PPARγ in vessels remain largely unidentified. In this study, we examined the role of smooth muscle PPARγ during salt-sensitive hypertension and investigated its transcriptional targets and functional effect. Transgenic mice expressing dominant-negative PPARγ (S-P467L) in smooth muscle cells were more prone to deoxycorticosterone acetate-salt-induced hypertension and mesenteric arterial dysfunction compared with nontransgenic controls. Despite similar morphometry at baseline, vascular remodeling in conduit and small arteries was enhanced in S-P467L after deoxycorticosterone acetate-salt treatment. Gene expression profiling in aorta and mesenteric arteries revealed significantly decreased expression of tissue inhibitor of metalloproteinase-4 (TIMP-4) in S-P467L. Expression of TIMP-4 was increased by deoxycorticosterone acetate-salt treatment, but this increase was ablated in S-P467L. Interference with PPARγ activity either by treatment with a PPARγ inhibitor, GW9662, or by expressing P467L PPARγ markedly suppressed TIMP-4 in primary smooth muscle cells. PPARγ binds to a PPAR response element (PPRE) in chromatin close to the TIMP-4 gene in smooth muscle cells, suggesting that TIMP-4 is a novel target of PPARγ. The interference with PPARγ and decrease in TIMP-4 were accompanied by an increase in total matrix metalloproteinase activity. PPARγ-mediated loss of TIMP-4 increased, whereas overexpression of TIMP-4 decreased smooth muscle cell migration in a scratch assay. Our findings highlight a protective mechanism induced by PPARγ in deoxycorticosterone acetate-salt treatment, establishing a novel mechanistic link between PPARγ and TIMP-4.

Osteoprotegerin promotes the proliferation of chondrocytes and affects the expression of ADAMTS-5 and TIMP-4 through MEK/ERK signaling.

The involvement of osteoprotegerin (OPG) in bone metabolism has previously been established; however, whether OPG regulates chondrocytes directly and exerts precise cellular and molecular effects on chondrocytes remains to be determined. Thus, the present study aimed to investigate the direct effect of OPG on the viability, proliferation and functional consequences of chondrocytes. Primary chondrocytes were isolated from the knee of Sprague-Dawley rats. Passage 1 chondrocytes were identified by toluidine blue staining and used in the experiments. The cell proliferation induced by OPG at various concentrations was measured by a Cell Counting kit-8 (CCK-8) assay. Following pretreatment with mitogen-activated/extracellular signal-regulated kinase kinase (MEK) inhibitor U0126, extracellular signal-regulated kinase (ERK) inhibitor PD098059, and P38 mitogen-activated protein kinase (P38MAPK) inhibitor SB203580 for 30 min, chondrocytes were treated with OPG, and CCK-8 was performed. The cellular signals of MAPKs, including ERK, P38MAPK and c-Jun N-terminal protein kinase (JNK), were investigated by western blot analysis following treatment with OPG. The functional consequences following treatment with soluble OPG were analyzed by qPCR and western blot analysis. OPG increased chondrocyte proliferation with maximal effect at 10 ng/ml, and induced the phosphorylation of MEK and ERK but not P38MAPK or JNK. Suppression of ERK activity via PD098095 inhibited OPG-induced chondrocyte proliferation. Administration of OPG significantly downregulated ADAMTS­5 and upregulated tissue inhibitor of metalloproteinase (TIMP)-4 production, but had no effect on the expression of TIMP-1, -2 and -3, insulin-like growth factor I, transforming growth factor-ß, basic fibroblast growth factor, bone morphogenetic protein-2, collagen II, aggrecan and ADAMTS-4. Suppression of ERK activity via PD098095 inhibited the alteration of ADAMTS-5 and TIMP-4 expression induced by OPG. OPG therefore regulated the proliferation of chondrocytes via MEK/ERK signaling, and directly affected chondrocytes by influencing the expression profile of ADAMTS-5 and TIMP-4.