MicroRNA-10a regulation of proinflammatory phenotype in athero-susceptible endothelium in vivo and in vitro

Abstract

A chronic proinflammatory state precedes pathological change in arterial endothelial cells located within regions of susceptibility to atherosclerosis. The potential contributions of regulatory microRNAs to this disequilibrium were investigated by artery site-specific profiling in normal adult swine. Expression of endothelial microRNA10a (miR-10a) was lower in the athero-susceptible regions of the inner aortic arch and aorto-renal branches than elsewhere. Expression of Homeobox A1 (HOXA1), a known miR-10a target, was up-regulated in the same locations. Endothelial transcriptome microarray analysis of miR-10a knockdown in cultured human aortic endothelial cells (HAEC) identified IkappaB/NF-kappaB-mediated inflammation as the top category of up-regulated biological processes. Phosphorylation of IkappaBalpha, a prerequisite for IkappaBalpha proteolysis and NF-kappaB activation, was significantly up-regulated in miR-10a knockdown HAEC and was accompanied by increased nuclear expression of NF-kappaB p65. The inflammatory biomarkers monocyte chemotactic protein 1 (MCP-1), IL-6, IL-8, vascular cell adhesion molecule 1 (VCAM-1), and E-selectin were elevated following miR-10a knockdown. Conversely, knockin of miR-10a (a conservative 25-fold increase) inhibited the basal expression of VCAM-1 and E-selectin in HAEC. Two key regulators of IkappaBalpha degradation--mitogen-activated kinase kinase kinase 7 (MAP3K7; TAK1) and beta-transducin repeat-containing gene (betaTRC)--contain a highly conserved miR-10a binding site in the 3' UTR. Both molecules were up-regulated by miR-10a knockdown and suppressed by miR-10a knockin, and evidence of direct miR-10a binding to the 3' UTR was demonstrated by luciferase assay. Comparative expression studies of endothelium located in athero-susceptible aortic arch and athero-protected descending thoracic aorta identified significantly up-regulated MAP3K7, betaTRC, phopho-IkappaBalpha, and nuclear p65 expression suggesting that the differential expression of miR-10a contributes to the regulation of proinflammatory endothelial phenotypes in athero-susceptible regions in vivo.

Fig. 1.

Arterial regions of endothelial isolation. Endothelial cells were scraped gently from the inner curvature of AA, the DT, and the distal renal artery (A and B) as well as from the caudal and cranial regions of the aorto-renal bifurcations (C and D). (Scale bars, 1 cm.)

Fig. 2.

Low expression of endothelial miR-10a/b at athero-susceptible arterial sites. (A and B) Suppression of endothelial miR-10a and miR-10b at athero-susceptible AA compared with DT (n = 10). (C and D) Suppression of endothelial miR-10a and miR-10b at athero-susceptible cranial and caudal walls of the aorto-renal bifurcations compared with distal renal artery (n = 3 paired samples; each paired sample was pooled from the same four animals). Data represent mean ± SEM. *P < 0.05.

Fig. 3.

Endothelial specificity of miR-10a/b. (A) In situ detection of endothelial miR-10a and miR-10b in frozen sections of freshly isolated swine DT by LNA-FISH. (B) Relative expression of endothelial miR-10a/b in freshly isolated swine aortic endothelial cells (n = 22, 11 AA and 11 DT) and in cultured HAEC (n = 8). Data represent mean ± SEM. *P < 0.05.

Fig. 4.

Activation of NF-κB signal transduction in miR-10a knockdown (KD) HAEC. (A) Suppression of total IκBα in miR-10a knockdown HAECs (n = 3). β-actin was used as an internal control (Ctl). (B and C) Increased nuclear expression and decreased cytoplasmic expression of NF-κB p65 in miR-10a knockdown cells. Nuclear (n = 4) and cytoplasmic (n = 3) expression of p65 was normalized to laminB1 and β-actin, respectively. (D) Up-regulation of E-selectin (E-SEL), VCAM-1, IL-6, IL-8, and MCP-1 in miR-10a knockdown HAEC. mRNAs of the inflammatory biomarkers were normalized to ubiquitin expression (n = 6 or 7). (E) Increased phosphorylated IκBα in miR-10a knockdown cells (n = 3). IκBα phosphorylation was stimulated by 5 ng/mL TNF-α for 10 min, and phospho-IκBα expression was normalized to total IκBα. Data represent mean ± SEM. *P < 0.05.

Fig. 5.

Role of miR-10a in canonical NF-κB signaling. MAP3K7 and βTRC contain an evolutionarily conserved miR-10a putative binding site, and both promote proteasomal degradation of IκBα and p65 nuclear translocation with induction of downstream inflammatory biomarkers.

Fig. 6.

miR-10a negatively regulates MAP3K7 and βTRC through their 3′ UTR binding site. (A and B) Evolutionarily conserved putative miR-10a binding site within the 3′ UTR of MAP3K7 and βTRC. (C and D) Increased expression of MAP3K7 in miR-10a knockdown HAEC. Gene (n = 7–10) and protein (n = 3) expressions were normalized to GAPDH and β-actin, respectively. (E) Reduced luciferase activity in HEK293 cells overexpressing miR-10a following insertion of miR-10a binding elements cloned from MAP3K7 and βTRC 3′ UTRs. Data represent mean ± SEM. ns, not significant. *P < 0.05.

Fig. 7.

Endothelial protein expression in swine athero-susceptible AA andathero-protected DT. (A) Differential endothelial expression of MAP3K7, βTRC, IκBα, phosphorylated IκBα (n = 5), and nuclear p65 (n = 4; each sample was pooled from the seven animals) in paired comparisons of AA and DT. Data represent mean ± SEM. *P < 0.05. (B) Representative Western blots.