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

Abstract

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.

Figure 1

SMC PTEN depletion induces constitutive PI3K-Akt-mTOR signalling. Rat aortic SMCs stably expressing control (CTRL) or PTEN-specific shRNA were serum-restricted for 72 h. Whole cell lysates were analysed for total PTEN, phosphoAkt, total Akt, phospho-p70S6-kinase, and total p70S6-kinase. β-Actin was used as a loading control. Shown is a representative western of three independent experiments.

Figure 2

PTEN depletion promotes SMC phenotypic modulation. (A) qRT–PCR for SM-α-actin and calponin mRNAs. β-Actin was used for normalization of cDNA. Shown are fold changes in mRNA copy number ± SE from CTRL SMC from three independent experiments; *P < 0.05. (A, right panel) CTRL and PTEN-deficient SMCs were transiently transfected with an SM-α-actin promoter-Luciferase reporter construct. Luciferase activity normalized to β-galactosidase was determined; shown are percent changes ± SE from control SMC. (B) Whole cell lysates were analysed for SM-α-actin and calponin protein levels. β-Actin was used as a loading control. Shown is a representative western and fold changes in densitometry measurements ± SE from CTRL SMC from three independent experiments; *P < 0.05. (C) CTRL and PTEN-deficient SMCs were serum-restricted in the presence or absence of the PI3-kinase inhibitor, LY294002 (10 µM), or the mTOR inhibitor, rapamycin (10 nM). Whole cell lysates were analysed for phosphoAkt, SM-α-actin, and calponin levels. β-Actin was used as a loading control. Shown is a representative western; fold changes in densitometry measurements from CTRL SMC are shown in the graph. (D, left) CTRL and PTEN-deficient SMCs were transiently transduced with empty vector adenovirus (EV) or adenoviruses encoding wild-type PTEN (WT) or phosphatase inactive PTEN (MT) (MOI = 100). Cells were maintained in 0.1% FCS and whole cell lysates analyszed for total PTEN and SM-α-actin levels. β-Actin was used as a loading control. (Right) CTRL and PTEN-deficient SMCs were transiently transfected with an expression plasmid encoding wild-type PTEN (PTEN WT) or phosphatase inactive PTEN (PTEN C124A), along with an SM-α-actin promoter-Luciferase reporter construct. Luciferase activity normalized to β-galactosidase was determined; shown are percent changes from control SMC.

Figure 3

SMC PTEN depletion induces the upregulation of a family of cytokines. (A) qRT–PCR analysis for the indicated mRNAs in serum-restricted CTRL and PTEN-deficient SMCs. β-Actin was used for normalization of cDNA. Shown are fold changes in mRNA copy number ± SE from CTRL SMC from three independent experiments; *P < 0.05. (B) Conditioned media from SMCs described in (A) were analysed by ELISA for SDF-1α, IL-6, MCP-1, or CXCL1/KC protein levels. Shown are the means ± SE from three independent experiments; *P < 0.05.

Figure 4

Cytokine induction mediated by PTEN-depletion is dependent on NF-κB activity. (A) qRT–PCR analysis of serum-restricted CTRL and PTEN-deficient SMCs maintained in the presence or absence of the PI3K inhibitor, LY294002 (10 µM), the mTOR inhibitor, rapamycin (10 nM), or the IKK inhibitor, BAY11-7082 (5 µM). β-Actin was used for normalization of cDNA. Shown are fold changes in mRNA copy number ± SE from CTRL SMC from a minimum of four independent experiments; *P < 0.05 from CTRL DMSO; **P < 0.05 from PTEN DMSO. (B) SMCs were maintained under basal conditions (SFM) for 72 h or growth-arrested followed by stimulation with 100 ng/mL TNFα for 1 h then immunofluorescently stained for NF-κB p65 (green). Blue, DAPI; nuclei. (C) SMCs were transiently transfected with an NF-κB promoter-Luciferase reporter construct. Luciferase activity normalized to β-galactosidase was determined; shown are percent changes ± SE from CTRL SMC from three independent experiments; *P < 0.05. (D) SMCs were serum-restricted in the presence or absence of LY294002, rapamycin, or BAY11-7082 as in (A) and then immunofluorescently stained for NF-κB p65 (green). Blue, DAPI; nuclei. Shown in the graph are percent cells expressing nuclear p65 ± SE; *P < 0.05 from CTRL DMSO; **P < 0.05 from PTEN DMSO.

Figure 5

PTEN-deficient mutant mice exhibit enhanced neointima formation in response to vascular injury. (A) Western analysis of whole aorta from wild-type, global PTEN heterozygote (PTEN+/−), floxed wild-type (PTEN^fl/fl; +/+), and SMC-specific heterozygote (PTEN^fl/+; SM22α-Cre+/−) mice showing reduced total PTEN levels and increased phosphoAkt in tissues from PTEN heterozygous mice. Two mice per genotype were analysed; graphs show means of densitometry measurements. (B) Carotid artery ligation-induced vascular injuries were performed on mice described in (A) and arteries harvested 14-days post-injury. H&E staining on cross-sections from representative injured left carotid arteries. Arrowheads, internal elastic laminae. (C) Medial and intimal areas were measured using SPOT software. Intima-to-media ratios (left) and percent stenotic areas (right) are presented in the graphs. Percent area stenosis = 100 × [1.00 − (stenotic area/native vessel area)] = 100 × {1.00 − [(π × stenotic major axis × stenotic minor axis/4)/(π × native major axis × native minor axis/4)]}. *Different from global wild-type; **different from floxed wild-type; P < 0.05; n = 6.

Figure 6

SMC-specific PTEN-deficient mutant mice exhibit increased intimal cell proliferation, reduced SM marker expression, and overexpression of NF-κB p65 and cytokines. (A) BrdU immunohistochemistry on injured left carotid arteries from wild-type (PTEN^fl/fl;+/+) and SMC-specific heterozygote (PTEN^fl/+;SM22α-Cre+/−) mice (brown nuclei). Percent replicating cells was determined independently for the arterial media and intima and data presented in the graphs as means ± SE; *different from wild-type; P < 0.05. (B and C) Immunohistochemistry for SM-α-actin (B, upper), calponin (B, lower), NF-κB p65 (C, upper), IL-6 (C, middle), or MCP-1 (C, lower) on injured left carotid arteries from wild-type (PTEN^fl/fl;+/+) and SMC-specific heterozygote (PTEN^fl/+;SM22α-Cre+/−) mice (brown reaction colour). Arrowheads, internal elastic lamina; lines in C, the arterial media; n = 4.