NCK Associated Protein 1 Modulated by miRNA‐214 Determines Vascular Smooth Muscle Cell Migration, Proliferation, and Neointima Hyperplasia

Abstract

BackgroundMicroRNA miR‐214 has been implicated in many biological cellular functions, but the impact of miR‐214 and its target genes on vascular smooth muscle cell (VSMC) proliferation, migration, and neointima smooth muscle cell hyperplasia is unknown.Methods and ResultsExpression of miR‐214 was closely regulated by different pathogenic stimuli in VSMCs through a transcriptional mechanism and decreased in response to vascular injury. Overexpression of miR‐214 in serum‐starved VSMCs significantly decreased VSMC proliferation and migration, whereas knockdown of miR‐214 dramatically increased VSMC proliferation and migration. Gene and protein biochemical assays, including proteomic analyses, showed that NCK associated protein 1 (NCKAP1)—a major component of the WAVE complex that regulates lamellipodia formation and cell motility—was negatively regulated by miR‐214 in VSMCs. Luciferase assays showed that miR‐214 substantially repressed wild‐type but not the miR‐214 binding site mutated version of NCKAP1 3′ untranslated region luciferase activity in VSMCs. This result confirmed that NCKAP1 is the functional target of miR‐214 in VSMCs. NCKAP1 knockdown in VSMCs recapitulates the inhibitory effects of miR‐214 overexpression on actin polymerization, cell migration, and proliferation. Data from cotransfection experiments also revealed that inhibition of NCKAP1 is required for miR‐214–mediated lamellipodia formation, cell motility, and growth. Importantly, locally enforced expression of miR‐214 in the injured vessels significantly reduced NCKAP1 expression levels, inhibited VSMC proliferation, and prevented neointima smooth muscle cell hyperplasia after injury.ConclusionsWe uncovered an important role of miR‐214 and its target gene NCKAP1 in modulating VSMC functions and neointima hyperplasia. Our findings suggest that miR‐214 represents a potential therapeutic target for vascular diseases.

Downregulation of Cavin‐1 Expression via Increasing Caveolin‐1 Degradation Prompts the Proliferation and Migration of Vascular Smooth Muscle Cells in Balloon Injury–Induced Neointimal Hyperplasia

“…Compared with sham carotids, Cavin‐1 expression in carotid arteries was markedly downregulated at 2, 7, and 14 days after balloon injury (Figure A). To confirm that the vascular injury model was successful, we performed hematoxylin and eosin staining and found that balloon injury significantly stimulated arterial neointimal formation at 14 days after injury (Figure B), in agreement with previous studies . Only monolayer endothelial cells were seen along the inner lumina of sham carotid artery without neointima, while in the balloon‐injured group, the intima was thickening and became a multilayer compartment, and the elastic membrane was disrupted, indicating arterial intimal hyperplasia.…”

“…Neointimal formation is characterized by medial VSMC proliferation and migration from the media to the neointima, which leads to blood vessel occlusion . Our studies showed that the inhibition of Cavin‐1 protein expression affected both proliferation and migration of VSMCs.…”

“…Neointimal formation is characterized by medial VSMC proliferation and migration from the media to the neointima, which leads to blood vessel occlusion. 27,44 Our studies showed that the inhibition of Cavin-1 protein expression affected both proliferation and migration of VSMCs. Reduction of Cavin-1 expression induced no change in cell numbers or phenotypic markers in normally cultured VSMCs ( Figure 4B and 4C).…”

“…To confirm that the vascular injury model was successful, we performed hematoxylin and eosin staining and found that balloon injury significantly stimulated arterial neointimal formation at 14 days after injury ( Figure 1B), in agreement with previous studies. [26][27][28] Only monolayer endothelial cells were seen along the inner lumina of sham carotid artery without neointima, while in the balloon-injured group, the intima was thickening and became a multilayer compartment, and the elastic membrane was disrupted, indicating arterial intimal hyperplasia. Subsequently, we used immunofluorescent staining to examine the expression of Cavin-1 protein at 14 days after surgery.…”

Genetic and Pharmacologic Inhibition of the Neutrophil Elastase Inhibits Experimental Atherosclerosis

“…Real‐time quantitative polymerase chain reaction was performed as previously described 17, 18, 19, 27, 28, 29, 30, 31. Briefly, total RNA was extracted from murine aortas or cells using Trizol reagent (Sigma) according to the manufacturer's instructions and subjected to DNase I (Sigma) digestion to remove potential DNA contamination.…”