Temporal PTEN inactivation causes proliferation of saphenous vein smooth muscle cells of human CABG conduits

Internal mammary artery (IMA) coronary artery bypass grafts (CABG) are remarkably resistant to intimal hyperplasia (IH) as compared to saphenous vein (SV) grafts following aorto-coronary anastomosis. The reason behind this puzzling difference still remains an enigma. In this study, we examined the effects of IGF-1 stimulation on the PI3K-AKT/PKB pathway mediating proliferation of smooth muscle cells (SMCs) of IMA and SV origin and the specific contribution of phosphatase and tensin homologue (PTEN) in regulating the IGF-1-PI3K-AKT/PKB axis under these conditions. Mitogenic activation with IGF-1, time-dependently stimulated the phosphorylation of PI3K and AKT/PKB in the SV SMCs to a much greater extent than the IMA. Conversely, PTEN was found to be significantly more active in IMA SMCs. Transient overexpression of PTEN in SMCs of SV and IMA inhibited AKT/PKB activity and upstream of AKT/PKB, caused a reduction of IGF-1 receptors. Downstream, PTEN overexpression in SV SMCs induced the transactivation of tumour suppressor protein p53 by down-regulating the expression of its inhibitor MDM2. However, PTEN overexpression had no significant effect on MDM2 and p53 expression in IMA SMCs. PTEN overexpression inhibited IGF-1-induced SMC proliferation in both SV and IMA. PTEN suppression, induced by siRNA transfection of IMA SMCs diminished the negative regulation of PI3K-PKB signalling leading to greater proliferative response induced by IGF-1 stimulation. Thus, we show for the first time that early inactivation of PTEN in SV SMCs leads to temporally increased activity of the pro-hyperplasia PI3K-AKT/PKB pathway leading to IH-induced vein graft occlusion. Therefore, modulation of the PI3K-AKT/PKB pathway via PTEN might be a novel and effective strategy in combating SV graft failure following CABG.     

Fibronectin promotes cell cycle entry in smooth muscle cells in primary culture

Smooth muscle cell proliferation after arterial injury is regulated by growth factors and components of the extracellular matrix. We have previously demonstrated that fibronectin promotes a phenotypic modulation of freshly isolated rat smooth muscle cells from a contractile to a synthetic phenotype in primary culture and supports the ability of the cells to respond to growth factors. Here, we analyzed if fibronectin promotes cell cycle entry in freshly isolated rat aortic smooth muscle cells during primary culture. Cell cycle analysis showed that cells seeded on fibronectin remained in the G(0)/G(1) phase of the cell cycle during the first 6 days of culture. During this period, there was an increased expression of cyclin D1 and p27(KIP1) in the absence of exogenous growth factors. Addition of serum was followed by enhanced cyclin D1 expression, decreased p27(KIP1) levels, hyperphosphorylation of Rb protein, induction of cyclin A and cyclin D3 expression, and cell cycle progression into S phase. The results indicate that fibronectin initiates cell cycle entry in freshly isolated smooth muscle cells by promoting the induction of cyclin D1 and thereby facilitates further cell cycle progression together with growth factors.     

The clopidogrel after surgery for coronary artery disease (CASCADE) randomized controlled trial: clopidogrel and aspirin versus aspirin alone after coronary bypass surgery [NCT00228423]

Background

Saphenous vein graft disease remains a major limitation of coronary artery bypass graft surgery. The process of saphenous vein intimal hyperplasia begins just days after surgical revascularization, setting the stage for graft atherosclerotic disease and its sequalae. Clopidogrel improves outcomes in patients with atherosclerotic disease, and is effective at reducing intimal hyperplasia in animal models of thrombosis. Therefore, the goal of this study will be to evaluate the efficacy of clopidogrel and aspirin therapy versus aspirin alone in the prevention of saphenous vein graft intimal hyperplasia following coronary artery bypass surgery.

Methods

Patients undergoing multi-vessel coronary artery bypass grafting and in whom at least two saphenous vein grafts will be used are eligible for the study. Patients will be randomized to receive daily clopidogrel 75 mg or placebo, in addition to daily aspirin 162 mg, for a one year duration starting on the day of surgery (as soon as postoperative bleeding has been excluded). At the end of one year, all patients will undergo coronary angiography and intravascular ultrasound assessment of one saphenous vein graft as selected by randomization. The trial will be powered to test the hypothesis that clopidogrel and aspirin will reduce vein graft intimal hyperplasia by 20% compared to aspirin alone at one year following bypass surgery.

Discussion

This trial is the first prospective human study that will address the question of whether clopidogrel therapy improves outcomes and reduces saphenous vein graft intimal hyperplasia following cardiac surgery. Should the combination of clopidogrel and aspirin reduce the process of vein graft intimal hyperplasia, the results of this study will help redefine modern antiplatelet management of coronary artery bypass patients.     

Hesperetin, a bioflavonoid, inhibits rat aortic vascular smooth muscle cells proliferation by arresting cell cycle

Diet can be one of the most important factors that influence risks for cardiovascular diseases. Hesperetin, a flavonoid present in grapefruits and oranges, is one candidate that may benefit the cardiovascular system. In this study, we have investigated the effect of hesperetin on the platelet-derived growth factor (PDGF)-BB-induced proliferation of primary cultured rat aortic vascular smooth muscle cells (VSMCs). Hesperetin significantly inhibited 50 ng/ml PDGF-BB-induced rat aortic VSMCs proliferation and [(3)H]-thymidine incorporation into DNA at concentrations of 5, 25, 50, and 100 microM. In accordance with these findings, hesperetin revealed blocking of the PDGF-BB-inducible progression through G(0)/G(1) to S phase of the cell cycle in synchronized cells. Western blot showed that hesperetin inhibited not only phosphorylation of retinoblastoma protein (pRb) and expressions of cyclin A, cyclin D, cyclin E, cyclin-dependent kinase 2 (CDK2) as well as proliferating cell nuclear antigen (PCNA) protein, but also downregulation of cyclin-dependent kinase inhibitor (CKI) p27(kip1), while did not affect CKI p21(cip1), p16(INK4), p53, and CDK4 expressions as well as early signaling transductions such as PDGF beta-receptor, extracellular signal-regulated kinase (ERK) 1/2, Akt, p38, and JNK phosphorylation. These results suggest that hesperetin inhibits PDGF-BB-induced rat aortic VSMCs proliferation via G(0)/G(1) arrest in association with modulation of the expression or activation of cell-cycle regulatory proteins, which may contribute to the beneficial effect of grapefruits and oranges on cardiovascular system.     

Circular RNA circENTPD7 suppresses the accumulation of PTEN to promote cell proliferation in non-small cell lung cancer

The oncogenic role of circular RNA ENTPD7 (circENTPD7) in cancer biology has been reported in glioblastoma, while its role in non-small cell lung cancer (NSCLC) is unknown. This study was performed to investigate the involvement of circENTPD7 in NSCLC. NSCLC tissues and paired non-tumor tissues were collected from 64 NSCLC patients and the expression of circENTPD7 and PTEN were determined by RT-qPCR. Expression levels of PTEN protein in these tissue samples were measured by ELISA. The 64 NSCLC patients were subjected to a follow-up study to explore the role of circENTPD7 in predicting the survival of NSCLC. Overexpression of circENTPD7 was achieved in NSCLC cells, and the effects of overexpression of circENTPD7 on the expression of PTEN were measured by RT-qPCR and Western blot at mRNA and protein level, respectively. Cell proliferation was assessed by CCK-8 assay. CircENTPD7 was upregulated in NSCLC and high expression levels of circENTPD7 predicts the poor survival rate of NSCLC cells. In NSCLC tissues, circENTPD7 was inversely correlated with PTEN protein but not mRNA. In NSCLC tissues, overexpression of circENTPD7 resulted in downregulation of PTEN, but did not alter the expression of PTEN mRNA. Cell proliferation analysis showed that overexpression of circENTPD7 promoted the proliferation of NSCLC cells and reduced the inhibitory effects of overexpression of PTEN on cell proliferation. CircENTPD7 may suppress the accumulation of PTEN to promote cell proliferation in NSCLC.     

Cell cycle arrest by the PTEN tumor suppressor is target cell specific and may require protein phosphatase activity

PTEN, a tumor suppressor commonly targeted in human cancer, possesses phosphatase activities toward both protein and lipid substrates. While PTEN suppresses gliomas through cell cycle inhibition which requires its lipid phosphatase activity, PTEN's effects on other tumor types and the role of its protein phosphatase activity are controversial or unknown. Here we show that exogenous wild-type PTEN arrests some, but not all human breast cancer cell lines in G1, in a manner independent of endogenous PTEN. Unexpectedly, the G129E mutant of PTEN selectively deficient in the lipid phosphatase activity still blocked the cell cycle of MCF-7 cells, while the G129R and H123Y mutants lacking both phosphatase activities were ineffective. These results suggest that PTEN's protein phosphatase activity likely contributes to its tumor suppressor function in subsets of tumors and that elucidation of downstream targets which dictate cellular responses to PTEN may have important implications for future cancer treatment strategies.     

USP52 inhibits cell proliferation by stabilizing PTEN protein in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer. Ubiquitination is closely related to the development of lung cancer. However, the biological importance of newly discovered ubiquitin-specific peptidase (USP) 52 (USP52) in NSCLC remained unclear. Here, our findings identify USP52 as a novel tumor suppressor of NSCLC, the low expression of USP52 predicts a poor prognosis for NSCLC patients. The present study demonstrates that USP52 inhibits cancer cell proliferation through down-regulation of cyclin D1 (CCND1) as well as AKT/mTOR signaling pathway inhibition. Meanwhile, USP25 also suppresses NSCLC progression via enhancing phosphatase and tensin homolog (PTEN) stability in cancer cells, which further indicates the significance/importance of USP52 in NSCLC suppression.     

17beta-estradiol affects proliferation and apoptosis of rat prostatic smooth muscle cells by modulating cell cycle transition and related proteins

Abundant evidence indicates that estrogens have an important role in the pathology of benign prostatic hyperplasia (BPH). To investigate the effect of 17beta-estradiol (E2) on the proliferation and apoptosis of prostatic smooth muscle cells (PSMCs), rat PSMCs were obtained and exposed to gradient concentrations (0.1-100 nmol/l) of E2 over varying amounts of time. The progression of cell cycle, cellular apoptosis, cyclin D1, Bcl-2 and Bax proteins were detected. The data show that the effect of E2 on rat PSMCs is bilateral: it promotes cell proliferation by enhancing the expression of cyclin D1, which accelerates G1 to S phase transition; on the other hand, it induces apoptosis of the cells by up-regulating the expression of Bax. We thus suggest that an increase in estrogen may exert a launching effect in the pathology of BPH.     

PTEN in smooth muscle cells is essential for colonic immune homeostasis

Colonic immune homeostasis is essential for normal gastrointestinal tract functioning. In this study, we report that specific gene targeting of phosphatase and tensin homolog (PTEN) in smooth muscle cells caused age-related colonic lymphoid hyperplasia followed by global immune activation in mice. Beginning at 5 weeks of age, these mutant mice displayed massive neutrophil infiltration in the colonic lamina propria. The gene expression levels of pro-inflammatory cytokines and chemokines, including those code for monocyte chemotactic protein-1 (Mcp-1), stromal cell-derived factor 1α (Sdf-1α), and chemokine (C-C motif) ligand 28 (Ccl-28), were upregulated in the colon. Accordingly, permeability and proliferation of the colonic epithelium was compromised. These abnormalities were alleviated to a great extent when the mutants were crossed with Akt1-null mice, indicating that the pathogenesis was mediated by Akt1 signaling. Our results suggest that in smooth muscle cells, PTEN is crucial for maintaining colonic immune homeostasis.     

Regulation of store-operated Ca entry in pulmonary artery smooth muscle cells

Store-operated Ca²⁺ entry (SOCE) is an important mechanism for Ca²⁺ influx in smooth muscle cells; however the activation and regulation of this influx pathway are incompletely understood. In the present study we have examined the effect of several protein kinases in regulating SOCE in pulmonary artery smooth muscle cells (PASMCs) of the rat. Inhibition of protein kinase C with chelerythrine (3 μM) potentiated SOCE by 47 ± 2%, while the tyrosine kinase inhibitors genistein (100 μM) and tyrphostin 23 (100 μM) caused a significant reduction in SOCE of 55 ± 9% and 43 ± 7%, respectively. It has been proposed that Ca²⁺-insensitive phospholipase A₂ (iPLA₂) is involved in the activation of SOCE in many different cell types. The iPLA₂ inhibitor, bromoenol lactone had no effect on SOCE, suggesting that this mechanism was not involved in the activation of the pathway. The calmodulin antagonists, calmidazolium (CMZ) (10 μM) and W-7 (10 μM) appeared to potentiate SOCE in PASMCs. Further investigation established that CMZ was actually activating a Ca²⁺ influx pathway that was independent of the filling state of the sarcoplasmic reticulum. The CMZ-activated Ca²⁺ influx was blocked by Gd³⁺ (10 μM), but unaffected by 2-APB (75 μM), indicating a pharmacological profile distinct from the classical SOCE pathway.     

Aspirin inhibits human coronary artery endothelial cell proliferation by upregulation of p53

Aspirin (acetylsalicylic acid, ASA) is effective in the primary and secondary prevention of vascular events. This effect is mediated in large part by platelet inhibition; however, non-platelet-mediated effects may also be relevant in the overall efficacy of ASA. We determined the effect of ASA on the synthesis of DNA and total proteins in cultured human coronary endothelial cells (HCAECs). Fourth generation HCAECs were cultured and treated with ASA and rate of synthesis of DNA and total proteins was determined by incorporation of [3H]thymidine and [3H]proline, respectively. ASA inhibited DNA synthesis by 50% at a concentration of 1mM and protein synthesis by 50% at a concentration of 2mM. The inhibitory effect of ASA was observed as early as 2h after treatment of HCAECs. The inhibition of DNA and protein synthesis could be reversed within 24h after removal of the drug from the culture medium. Indomethacin also inhibited DNA and protein synthesis. Western blot analysis revealed that the expression of p53 protein was increased after treatment of the cells with ASA. These observations indicate that ASA decreases endothelial cell proliferation through cell cycle arrest mediated by enhanced p53 expression. Arrest of endothelial proliferation and activation may be an important mechanism of the beneficial effect of ASA in acute coronary syndromes.     

Inhibition of the proliferation of smooth muscle cells from human coronary bypass vessels by vasonatrin peptide

Abnormal proliferation of vascular smooth muscle cells (VSMCs) is known to be a key event in the development of atherosclerosis and restenosis. The present study examined the effect of a novel synthetic natriuretic peptide, vasonatrin peptide (VNP), on norepinephrine (NE)-induced proliferation of VSMCs from coronary bypass vessels. Human VSMCs were isolated from an internal mammary artery (IMA) and saphenous vein (SV) by explant culture and stimulated with NE. MTT assay and [3H] thymidine-incorporation were undertaken to analyze cell proliferation and radioimmunoassay was used to determine the level of intracellular cyclic 3',5'-guanosine monophosphate (cyclic GMP). NE (10(-8) - 10(-7) mol/l) had a mitogenic effect in human VSMCs from both SV and IMA. However, NE-stimulated proliferation of VSMCs from SV was greater than that from IMA. Furthermore, low concentration of NE (10(-10) mol/l) promoted cell growth in SV-derived cells but not in IMA-derived cells. VNP (10(-8) - 10(-6) mol/l) reduced NE-induced cell proliferation and increased intracellular cyclic GMP, which were abrogated by HS-142-1. In addition, the growth inhibition of VNP was mimicked by 8-bromo-cGMP. These results indicate that VNP has a significant inhibitory effect on NE-stimulated proliferation of human VSMCs from both IMA and SV, which is mediated by guanylate cyclase-linked receptors by increasing cyclic GMP.     

Hammerhead ribozymes targeted against cyclin E and E2F1 cooperate to down-regulate coronary smooth muscle cell proliferation

BACKGROUND: Anti-proliferative drugs released from endo-vascular stents have substantially contributed to reduce in-stent restenosis rates in coronary arteries bearing single primary lesions by down-regulating coronary smooth muscle cell (CSMC) growth. However, the considerably lower drug efficacy shown in treatment of more complex coronary lesions suggests that alternative anti-proliferative approaches can be beneficial. Thus, we explored the use of hammerhead ribozymes as tools to knock down cyclin E and E2F1, two potent activators of cell proliferation which cooperate to promote the G1 to S phase transition. METHODS: Two ribozymes, one directed against cyclin E and the other against E2F1 mRNAs, were delivered by liposomes to cultured human CSMCs. The influences on cell proliferation were measured evaluating BrdU incorporation into newly synthesised DNA. The effects on cell cycle phase distribution were determined by BrdU and 7-aminoactinomycin D incorporation into DNA. RESULTS: Both ribozymes exhibited a sequence-specific and dose-dependent reduction in BrdU incorporation, which, at a concentration of 280 nM, persisted up to 4 days after transfection of CSMCs. A combined administration of the two ribozymes (210+210 nM) resulted in a more pronounced decrease in BrdU incorporation compared to the administration of an equimolar amount (420 nM) of each of them. Finally, both ribozymes induced a significant (P<0.05) reduction in S phase cells with a concomitant increase of G1/G0 and G2-M phase cells, compared to controls. CONCLUSIONS: The ribozymes selected represent potent tools to prevent CSMC proliferation, especially when administered together, and thus are ideal candidates for in vivo application.     

Inhibition of PTEN expression and activity by angiotensin II induces proliferation and migration of vascular smooth muscle cells

PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a tumor suppressor and has been suggested recently to be involved in the regulation of cardiovascular diseases. The molecular mechanisms of this regulation are however poorly understood. This study shows that down regulation of PTEN expression and activity by angiotensin II (Ang II) increased proliferation and migration of vascular smooth muscle cells (VSMCs). The presence of Ang II induced rapid PTEN phosphorylation and oxidation in accordance with increased AKT and FAK phosphorylation. The Ang II‐mediated VSMC proliferation and migration was inhibited when cellular PTEN expression was increased by AT1 inhibitor losartan, PPARγ agonist rosiglitazone, NF‐κB inhibitor BAY 11‐7082. Over expression of PTEN in VSMCs by adenovirus transduction also resulted in inhibition of cell proliferation and migration in response to Ang II. These results suggest that PTEN down‐regulation is involved in proliferation and migration of VSMCs induced by Ang II. This provides insight into the molecular regulation of PTEN in vascular smooth muscle cells and suggests that targeting the action of PTEN may represent an effective therapeutic approach for the treatment of cardiovascular diseases. J. Cell. Biochem. 114: 174–182, 2012. © 2012 Wiley Periodicals, Inc.