Synergistic role of protein phosphatase inhibitor 1 and sarco/endoplasmic reticulum Ca2+ -ATPase in the acquisition of the contractile phenotype of arterial smooth muscle cells.

BACKGROUND: Phenotypic modulation or switching of vascular smooth muscle cells from a contractile/quiescent to a proliferative/synthetic phenotype plays a key role in vascular proliferative disorders such as atherosclerosis and restenosis. Although several calcium handling proteins that control differentiation of smooth muscle cells have been identified, the role of protein phosphatase inhibitor 1 (I-1) in the acquisition or maintenance of the contractile phenotype modulation remains unknown. METHODS AND RESULTS: In human coronary arteries, I-1 and sarco/endoplasmic reticulum Ca2+ -ATPase expression is specific to contractile vascular smooth muscle cells. In synthetic cultured human coronary artery smooth muscle cells, protein phosphatase inhibitor 1 (I-1 target) is highly expressed, leading to a decrease in phospholamban phosphorylation, sarco/endoplasmic reticulum Ca2+ -ATPase, and cAMP-responsive element binding activity. I-1 knockout mice lack phospholamban phosphorylation and exhibit vascular smooth muscle cell arrest in the synthetic state with excessive neointimal proliferation after carotid injury, as well as significant modifications of contractile properties and relaxant response to acetylcholine of femoral artery in vivo. Constitutively active I-1 gene transfer decreased neointimal formation in an angioplasty rat model by preventing vascular smooth muscle cell contractile to synthetic phenotype change. CONCLUSIONS: I-1 and sarco/endoplasmic reticulum Ca2+ -ATPase synergistically induce the vascular smooth muscle cell contractile phenotype. Gene transfer of constitutively active I-1 is a promising therapeutic strategy for preventing vascular proliferative disorders.

The Notch pathway attenuates interleukin 1ß (IL1ß)-mediated induction of adenylyl cyclase 8 (AC8) expression during vascular smooth muscle cell (VSMC) trans-differentiation.

Vascular smooth muscle cell (VSMC) trans-differentiation, or their switch from a contractile/quiescent to a secretory/inflammatory/migratory state, is known to play an important role in pathological vascular remodeling including atherosclerosis and postangioplasty restenosis. Several reports have established the Notch pathway as tightly regulating VSMC response to various stress factors through growth, migration, apoptosis, and de-differentiation. More recently, we showed that alterations of the Notch pathway also govern VSMC acquisition of the inflammatory state, one of the major events accelerating atherosclerosis. We also evidenced that the inflammatory context of atherosclerosis triggers a de novo expression of adenylyl cyclase isoform 8 (AC8), associated with the properties developed by trans-differentiated VSMCs. As an initial approach to understanding the regulation of AC8 expression, we examined the role of the Notch pathway. Here we show that inhibiting the Notch pathway enhances the effect of IL1ß on AC8 expression, amplifies its deleterious effects on the VSMC trans-differentiated phenotype, and decreases Notch target genes Hrt1 and Hrt3. Conversely, Notch activation resulted in blocking AC8 expression and up-regulated Hrt1 and Hrt3 expression. Furthermore, overexpressing Hrt1 and Hrt3 significantly decreased IL1ß-induced AC8 expression. In agreement with these in vitro findings, the in vivo rat carotid balloon-injury model of restenosis evidenced that AC8 de novo expression coincided with down-regulation of the Notch3 pathway. These results, demonstrating that the Notch pathway attenuates IL1ß-mediated AC8 up-regulation in trans-differentiated VSMCs, suggest that AC8 expression, besides being induced by the proinflammatory cytokine IL1ß, is also dependent on down-regulation of the Notch pathway occurring in an inflammatory context.

Calcium signaling in vascular smooth muscle cells: from physiology to pathology.

Cyclic variations in calcium (Ca(2+)) concentrations, through a process called excitation-contraction coupling, allow regulation of vascular smooth muscle cells contractility and thus modulation of vascular tone and blood pressure. As a second messenger, Ca(2+) also activates signaling cascades leading to transcription factors activation in a process called excitation-transcription coupling. Furthermore, recent evidences indicate an interaction between post-transcriptional regulation by microRNAs (miRNAs) and Ca(2+) signaling. All these actors, which are frequently altered in vascular diseases, will be reviewed here.

miR-424/322 regulates vascular smooth muscle cell phenotype and neointimal formation in the rat.

AIMS: Our aim was to identify new microRNAs (miRNAs) implicated in pathological vascular smooth muscle cells (VSMCs) proliferation and characterize their mechanism of action. METHODS AND RESULTS: MicroRNAs microarray and qRT-PCR results lead us to focus on miR-424 or its rat ortholog miR-322 (miR-424/322). In vitro mir-424/322 level was decreased shortly after the induction of proliferation and increased in a time-dependent manner later on. In vivo its expression increased in the rat carotid artery from Day 4 up to Day 30 after injury. miR-424/322 overexpression in vitro inhibited proliferation and migration without affecting apoptosis and prevented VSMC dedifferentiation. Furthermore, miR-424/322 overexpression resulted in decreased expression of its predicted targets: cyclin D1 and Ca(2+)-regulating proteins calumenin and stromal-interacting molecule 1 (STIM1). Using reporter luciferase assays, we confirmed that cyclin D1 and calumenin mRNAs were direct targets of miR-322, whereas miR-322 effect on STIM1 was indirect. Nevertheless, consistent with the decreased STIM1 level, the store-operated Ca(2+) entry was reduced. We hypothesized that miR-424/322 could be a negative regulator of proliferation overridden in pathological situations. Thus, we overexpressed miR-424/322 in injured rat carotid arteries using an adenovirus, and demonstrated a protective effect against restenosis. CONCLUSION: Our results demonstrate that miR-424/322 is up-regulated after vascular injury. This is likely an adaptive response to counteract proliferation, although this mechanism is overwhelmed in pathological situations such as injury-induced restenosis.