Serum response factor regulates expression of phosphatase and tensin homolog through a microRNA network in vascular smooth muscle cells.

OBJECTIVE: Serum response factor (SRF) is a critical transcription factor in smooth muscle cells (SMCs) controlling differentiation and proliferation. Our previous work demonstrated that depleting SRF in cultured SMCs decreased expression of SMC markers but increased proliferation and inflammatory mediators. A similar phenotype has been observed in SMCs silenced for phosphatase and tensin homolog (PTEN), suggesting that SRF and PTEN may lie on a common pathway. Our goal was to determine the effect of SRF depletion on PTEN levels and define mechanisms mediating this effect. METHODS AND RESULTS: In SRF-silenced SMCs, PTEN protein levels but not mRNA levels were decreased, suggesting posttranscriptional regulation. Reintroduction of PTEN into SRF-depleted SMCs reversed increases in proliferation and cytokine/chemokine production but had no effect on SMC marker expression. SRF-depleted cells showed decreased levels of microRNA (miR)-143 and increased miR-21, which was sufficient to suppress PTEN. Increased miR-21 expression was dependent on induction of Fos related antigen (FRA)-1, which is a direct target of miR-143. Introducing miR-143 into SRF-depleted SMCs reduced FRA-1 expression and miR-21 levels and restored PTEN expression. CONCLUSIONS: SRF regulates PTEN expression in SMCs through a miR network involving miR-143, targeting FRA-1, which regulates miR-21. Cross-talk between SRF and PTEN likely represents a critical axis in phenotypic remodeling of SMCs.

SDF-1α induction in mature smooth muscle cells by inactivation of PTEN is a critical mediator of exacerbated injury-induced neointima formation.

OBJECTIVE: PTEN inactivation selectively in smooth muscle cells (SMC) initiates multiple downstream events driving neointima formation, including SMC cytokine/chemokine production, in particular stromal cell-derived factor-1α (SDF-1α). We investigated the effects of SDF-1α on resident SMC and bone marrow-derived cells and in mediating neointima formation. METHODS AND RESULTS: Inducible, SMC-specific PTEN knockout mice (PTEN iKO) were bred to floxed-stop ROSA26-ß-galactosidase (ßGal) mice to fate-map mature SMC in response to injury; mice received wild-type green fluorescent protein-labeled bone marrow to track recruitment. Following wire-induced femoral artery injury, ßGal(+) SMC accumulated in the intima and adventitia. Compared with wild-type, PTEN iKO mice exhibited massive neointima formation, increased replicating intimal and medial ßGal(+)SMC, and enhanced vascular recruitment of bone marrow cells following injury. Inhibiting SDF-1α blocked these events and reversed enhanced neointima formation observed in PTEN iKO mice. Most recruited green fluorescent protein(+) cells stained positive for macrophage markers but not SMC markers. SMC-macrophage interactions resulted in a persistent SMC inflammatory phenotype that was dependent on SMC PTEN and SDF-1α expression. CONCLUSION: Resident SMC play a multifaceted role in neointima formation by contributing the majority of neointimal cells, regulating recruitment of inflammatory cells, and contributing to adventitial remodeling. The SMC PTEN-SDF-1α axis is a critical regulator of these events.

Defining the serum 99th percentile in a normal reference population measured by a high-sensitivity cardiac troponin I assay.

OBJECTIVES: This study determined the serum 99th percentile reference value for cTnI measured using the high sensitivity Erenna cTnI assay. DESIGN AND METHODS: Serum was obtained from healthy adults (n=348); aged 18-76 years of which 147 were males and 201 were females. Nonparametric analysis was performed to determine the 99th percentiles. RESULTS: For all subjects, the 99th percentile was 10.19 ng/L; mean concentration=1.45 ng/L, range=0.2 to 34.56 ng/L. By gender, the male and female 99th percentile values were as follows: male=16.58 ng/L, mean concentration=1.72 ng/L, and female=9.36 ng/L, mean concentration=1.25 ng/L, respectively (p=0.108). CONCLUSION: cTnI measured by the high-sensitivity Erenna cTnI assay measured 100% of normal subjects, allowing prospective diagnostic and risk assessment studies to be performed, which are essential for the early detection of cardiac disease and for the management of patients presenting with symptoms suggestive of acute coronary syndrome.

Inactivation of the tumour suppressor, PTEN, in smooth muscle promotes a pro-inflammatory phenotype and enhances neointima formation.

AIMS: Phosphatase and tensin homolog (PTEN) is implicated as a negative regulator of vascular smooth muscle cell (SMC) proliferation and injury-induced vascular remodelling. We tested if selective depletion of PTEN only in SMC is sufficient to promote SMC phenotypic modulation, cytokine production, and enhanced neointima formation. METHODS AND RESULTS: Smooth muscle marker expression and induction of pro-inflammatory cytokines were compared in cultured SMC expressing control or PTEN-specific shRNA. Compared with controls, PTEN-deficient SMC exhibited increased phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signalling and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) activity, reduced expression of SM markers (SM-alpha-actin and calponin), and increased production of stromal cell-derived factor-1alpha (SDF-1alpha), monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), and chemokine (C-X-C motif) ligand 1 (KC/CXCL1) under basal conditions. PI3K/Akt or mTOR inhibition reversed repression of SM marker expression, whereas PI3K/Akt or NF-kappaB inhibition blocked cytokine induction mediated by PTEN depletion. Carotid ligation in mice with genetic reduction of PTEN specifically in SMC (SMC-specific PTEN heterozygotes) resulted in enhanced neointima formation, increased SMC hyperplasia, reduced SM-alpha-actin and calponin expression, and increased NF-kappaB and cytokine expression compared with wild-types. Lesion formation in SMC-specific heterozygotes was similar to lesion formation in global PTEN heterozygotes, indicating that inactivation of PTEN exclusively in SMC is sufficient to induce considerable increases in neointima formation. CONCLUSION: PTEN activation specifically in SMC is a common upstream regulator of multiple downstream events involved in pathological vascular remodelling, including proliferation, de-differentiation, and production of multiple cytokines.

Targeted deletion of PTEN in smooth muscle cells results in vascular remodeling and recruitment of progenitor cells through induction of stromal cell-derived factor-1alpha.

We previously showed that changes in vascular smooth muscle cell (SMC) PTEN/Akt signaling following vascular injury are associated with increased SMC proliferation and neointima formation. In this report, we used a genetic model to deplete PTEN specifically in SMCs by crossing PTEN(LoxP/LoxP) mice to mice expressing Cre recombinase under the control of the SM22alpha promoter. PTEN was downregulated with increases in phosphorylated Akt in major vessels, hearts, and lungs of mutant mice. SMC PTEN depletion promoted widespread medial SMC hyperplasia, vascular remodeling, and histopathology consistent with pulmonary hypertension. Increased vascular deposition of the chemokine stromal cell-derived factor (SDF)-1alpha and medial and intimal cells coexpressing SM-alpha-actin and CXCR4, the SDF-1alpha receptor, was detected in SMC PTEN-depleted mice. PTEN deficiency in cultured aortic SMCs induced autocrine growth through increased production of SDF-1alpha. Blocking SDF-1alpha attenuated autocrine growth and blocked growth of control SMCs induced by conditioned media from PTEN-deficient SMCs. In addition, SMC PTEN deficiency enhanced progenitor cell migration toward SMCs through increased SDF-1alpha production. SDF-1alpha production by other cell types is regulated by the transcription factor hypoxia-inducible factor (HIF)-1alpha. We found SMC nuclear HIF-1alpha expression in PTEN-depleted mice and increased nuclear HIF-1alpha in PTEN-deficient SMCs. Small interfering RNA-mediated downregulation of HIF-1alpha reversed SDF-1alpha induction by PTEN depletion and inhibition of phosphatidylinositol 3-kinase signaling blocked HIF-1alpha and SDF-1alpha upregulation induced by PTEN depletion. Our data show that SMC PTEN inactivation establishes an autocrine growth loop and increases progenitor cell recruitment through a HIF-1alpha-mediated SDF-1alpha/CXCR4 axis, thus identifying PTEN as a target for the inhibition of pathological vascular remodeling.