Abdominal Aortic Aneurysm-Associated MicroRNA-516a-5p Regulates Expressions of Methylenetetrahydrofolate Reductase, Matrix Metalloproteinase-2, and Tissue Inhibitor of Matrix Metalloproteinase-1 in Human Abdominal Aortic Vascular Smooth Muscle Cells.

BACKGROUND: MicroRNAs (miRNAs or miRs) have been highlighted to be involved in abdominal aortic aneurysm (AAA) with the emergence of recent miRNA microarray profiling studies. miR-516a-5p has been shown to be significantly overexpressed in vascular smooth muscle cells (VSMCs) from human AAA tissues from our previous microarray study, suggesting its crucial association with AAA. In addition, further bioinformatics analysis predicted methylenetetrahydrofolate reductase (MTHFR), which regulates homocysteine (Hcy) metabolism and is proposed to be a risk gene for AAA formation and to be the downregulation target of miR-516a-5p. However, the pathogenic role of miR-516a-5p in VSMCs for AAA formation remains unresolved. This study aims to investigate the role of miR-516a-5p in human VSMCs for AAA pathogenesis. METHODS: miR-516a-5p was stably overexpressed and knocked down in VSMCs explant cultured from human abdominal aortic tissues by means of lentiviral system. The MTHFR protein expression was first examined by Western blotting. In addition, the protein expressions of several key components involved in AAA pathogenic features are as follows: matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of matrix metalloproteinase (TIMP)-1 and TIMP-2 for elastin degradation; collagen type 1 alpha 1 for compensatory collagen synthesis; monocyte chemoattractant protein-1 for inflammation, were also evaluated. Apoptotic level of VSMCs was examined by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. RESULTS: Results showed that protein expression of MTHFR was significantly downregulated on miR-516a-5p overexpression (P < 0.05) in VSMCs, whereas it was significantly upregulated on miR-516a-5p knockdown (P < 0.05). Of all the AAA key components investigated, only MMP-2 and TIMP-1 protein expressions were found altered. A significant increase in MMP-2 (P < 0.05) and decrease in TIMP-1 (P < 0.05) expressions were observed on miR-516a-5p overexpression in VSMCs. Apoptosis was not promoted on miR-516a-5p overexpression or knockdown in VSMCs. CONCLUSIONS: Our findings suggested that miR-516a-5p may regulate MTHFR, MMP-2, and TIMP-1 expressions in human VSMCs, possibly promoting the disruption of Hcy metabolism and proteolytic degradation of elastin for AAA formation.

Clearance of matrix metalloproteinase-9 is dependent on low-density lipoprotein receptor-related protein-1 expression downregulated by microRNA-205 in human abdominal aortic aneurysm.

OBJECTIVE: Low-density lipoprotein receptor-related protein-1 (LRP1) has been suggested to be a crucial regulator in the pathogenesis of abdominal aortic aneurysm (AAA) from previous genome association and animal studies. Our prior study using human aortic samples has further revealed a significant reduction of LRP1 protein expression associated with AAA. However, the downregulation of LRP1 in the pathophysiology of AAA remained unresolved. We hypothesized that LRP1 downregulation may be mediated by microRNA (miR) and that LRP1 may function as a scavenger of matrix metalloproteinase-9 (MMP-9), a well-known protease for degradation of extracellular matrix proteins at the aortic wall for AAA pathogenesis. This study investigated the cause of LRP1 downregulation and its potential effect on AAA pathogenesis. METHODS: An observational study of LRP1 protein, LRP1 messenger RNA (mRNA), and its three predicted miR candidates (miR-205, miR-338-5p, and miR-545-3p) was first performed in AAA compared with nonaneurysmal tissues from humans, followed by a functional study testing the effect on LRP1 expression of miR-205 overexpression and knockdown in human vascular smooth muscle cells (VSMCs) explant cultured from human abdominal aortic tissues. Lastly, another functional study was performed to test for the clearance of exogenous MMP-9 upon silencing of LRP1 in human VSMCs. RESULTS: From the observational study, significantly higher miR-205 (P < .001) and lower LRP1 protein (P < .001) expressions were found in human AAA tissues compared with nonaneurysmal aortic tissues, and no significant difference in LRP1 mRNA expression was observed. Further statistical analysis showed a significant negative correlation between miR-205 and LRP1 protein expressions (r = -0.65; P < .01). For the functional study, a significant downregulation of LRP1 protein expression was shown in miR-205-overexpressing VSMCs (P < .05), without any alteration in LRP1 mRNA expression. Moreover, a significantly reduced clearance of exogenous MMP-9 was observed in LRP1-silenced VSMCs (P < .05), and this difference in MMP-9 clearance was completely abolished with a pretreatment of anti-LRP1 antibody. CONCLUSIONS: Our study revealed the downregulation of LRP1 protein expression may be tightly regulated by miR-205 through translational inhibition in human VSMCs. Also, such LRP1 down-regulation in VSMCs may hinder the removal of pericellular MMP-9, leading to excess MMP-9 remaining in the extracellular matrix. Hence, the integrity of the vascular wall may be disrupted, promoting AAA formation.

Identification and characterization of microRNAs in vascular smooth muscle cells from patients with abdominal aortic aneurysms.

BACKGROUND: The role of microRNAs (miRs) in the development of various cardiovascular diseases was recently highlighted in several studies. However, the biological role of miRs in the pathogenesis of abdominal aortic aneurysms (AAAs) is still not well defined. The present study aims to identify and characterize miR expression in the aortic explant cultures of AAA patients. METHODS: A new microarray platform (miChip) using locked nucleic acid-modified capture probes with increased sensitivity and specificity was employed to detect the miR expression profile in human vascular smooth muscle cell cultures from AAA surgical samples. The array data were further validated by real-time quantitative reverse transcription polymerase chain reaction (qPCR) experiments with additional AAA and control aorta samples. Potential target genes of the differentially expressed miRs were predicted by bioinformatics analysis. Some of the results were confirmed by enzyme immunosorbent assay and Western blot analysis. RESULTS: Total RNA extracted from the explant of six AAAs and six normal aortas exhibited notably different miR profiles. A twofold difference of miR-516a-5p and miR-1260 was expressed in AAAs compared with normal aortic cultures (P < .05). The expression of miR-516a-5p was more than three times higher in AAAs, and miR-1260 expression was almost four times lower, as validated by qPCR. Additional qPCR performing on an extra 10 AAAs and 10 control aortas showed similar results. The expression of three predicted targets--secretory interleukin-3, vascular endothelial growth factor A, and collagen type 1, alpha 1 proteins--was significantly elevated in aneurysmal cultures compared with normal aortic cultures (P < .05). CONCLUSIONS: This miR microarray study displayed an altered expression of miR-516a-5p and miR-1260 in AAAs compared with control aortas. Functional annotations of the two miRs via bioinformatics approaches revealed that both are highly involved in some predefined mechanisms of AAA formation. Three of their target genes were also upregulated in AAAs. The results may be critical to elucidate the functional role of miRs in diseased aorta.