Guanghong Jia

Insulin-like growth factor-1 and TNF-α regulate autophagy through c- jun N-terminal kinase and Akt pathways in human atherosclerotic vascular smooth cells

A balance between programmed cell death and survival of vascular smooth muscle cells (VSMC) in the fibrous cap, which is primarily composed of VSMC and extracellular matrix, appears to best correlate with plaque instability or stability and is controlled by growth factors and cytokines. Autophagy is also involved in programmed cell death. We assessed the effect of TNF‐α and insulin‐like growth factor‐1 (IGF‐1) on the expression of autophagic genes, microtubule‐associated protein 1 light chain 3 (MAPLC‐3) and Beclin‐1 in VSMC isolated from atherosclerotic plaques. Transmission electron microscopy showed a significantly higher number of vacuolated cells in the TNF‐α‐treated VSMC and a markedly lower number in the IGF‐1‐treated VSMC when compared with the untreated control group. TNF‐α‐induced MAPLC‐3 mRNA expression through c‐jun N‐terminal kinase and protein kinase B pathways and induced Beclin‐1 protein expression through the c‐jun N‐terminal kinase pathway. Expression of MAPLC‐3 and Beclin‐1 correlated with autophagic cell death of plaque VSMC. IGF‐1 inhibited MAPLC‐3 mRNA transcripts through the Akt pathway. These findings suggest that the expression of autophagy genes can be influenced by IGF‐1 and TNF‐α through c‐jun N‐terminal kinase or Akt pathways and autophagy might be involved in the regulation of plaque stability.

Involvement of connexin 43 in angiotensin II-induced migration and proliferation of saphenous vein smooth muscle cells via the MAPK-AP-1 signaling pathway

Proliferation and migration of vascular smooth muscle cells (VSMCs) lead to intimal thickening and influence the long-term patency of venous graft post coronary arterial bypass graft. There is increasing evidence that connexins are involved in the development of intimal hyperplasia and restenosis. We assessed connexin 43 (Cx43) expression and its role in angiotensin II-induced proliferation and migration of smooth muscle cells and the signal pathways involved in human saphenous vein bypass conduits. Angiotensin II significantly increased gap junctional intercellular communication and induced the expression of Cx43 in human saphenous vein SMCs in a dose- and time-dependent manner through angiotensin II type 1 receptor. The effect of angiotensin II was blocked by siRNA of ERK 1/2, p38 MAPK and JNK, respectively. Overexpression of Cx43 markedly increased the proliferation of saphenous vein SMCs. However, siRNA for Cx43 inhibited angiotensin II-induced proliferation, cyclin E expression and migration of human saphenous vein SMCs. In dual-luciferase reporter assay, angiotensin II markedly activated AP-1 transcription factor, which was significantly attenuated by a dominant-negative AP-1 (A-Fos) with subsequent inhibition of angiotensin II-induced transcriptional expression of Cx43. These data demonstrate the role of Cx43 in the proliferation and migration of human saphenous vein SMCs and angiotensin II-induced Cx43 expression via mitogen-activated protein kinases (MAPK)-AP-1 signaling pathway.

Autophagy of vascular smooth muscle cells in atherosclerotic lesions.

Autophagy genes were first identified in the yeast system and some of their mammalian orthologues have also been characterized. Increasing lines of evidence indicate that various intracellular proteins, including G proteins, mammalian target of rapamycin (mTor) and PI3K/Akt/PKB, of transmembrane signaling pathways are involved in the regulation of autophagy genes. We have recently discovered autophagy as a mechanism of cell death in atherosclerotic vascular smooth muscle cells (VSMCs). Tumor necrosis factor-alpha (TNF-alpha), insulin-like growth factor-1 (IGF-1), and 7-ketocholesterol can regulate the expression of autophagic genes, including microtubule-associated protein 1 light chain-3 (MAP1LC3) and Beclin 1, through Akt/PKB and c-jun N-terminal signal pathways in VSMCs. However, the balance between cell death and survival of VSMCs in the fibrous cap of atherosclerotic plaques appears to best correlate with plaque instability. Understanding the underlying cellular and molecular mechanisms of autophagy can provide key insights into the cell death machinery of atherosclerotic diseases. Addendum to: Insulin-Like Growth Factor-1 and TNF-Alpha Regulate Autophagy through c-jun N-Terminal Kinase and Akt Pathways in Human Atherosclerotic Vascular Smooth Cells G. Jia, G. Cheng, D.M. Gangahar and D.K. Agrawal Immunol Cell Biol 2006; 84:448-54

Role of matrix Gla protein in angiotensin II-induced exacerbation of vascular calcification

Vascular calcification predicts an increased risk for cardiovascular events in atherosclerosis, diabetes, and end-stage kidney diseases. Matrix Gla protein (MGP), an inhibitor of calcification, limits calcium phosphate deposition in the vessel wall. There are many factors contributing to the progression of atherosclerosis, including hypertension, hyperlipidemia, the renin-angiotensin system, and inflammation. Angiotensin II (ANG II) plays a crucial role in the atherogenic process through not only its pressor responses but also its growth-promoting and inflammatory effects. In this study, we investigated the role of MGP in ANG II-induced exacerbation of vascular calcification in human vascular smooth muscle cells (VSMCs). The expression of MGP, calcification, and apoptosis in human VSMCs were examined by Western blot analysis, real-time PCR, in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, and enzyme-linked immunosorbent assay, respectively. Increase in VSMC calcification in human atherosclerotic plaques upregulates MGP expression and apoptosis in a negative feedback manner. ANG II inhibited MGP expression in VSMCs via and in vitro in a dose- and time-dependent manner through ANG II type 1 receptor and NF-κB signaling pathway. Meanwhile, MGP inhibited the calcification, caspase-3 activity, activation of runt-related transcription factor 2, and release of inflammatory cytokines by VSMCs induced by calcification medium (2.5 mM P(i)) and ANG II in vitro. These observations provide evidence that ANG II exacerbates vascular calcification through activation of the transcription factors, runt-related transcription factor 2 and NF-κB, and regulation of MGP, inflammatory cytokines expression in human VSMCs.

Differential effects of insulin-like growth factor-1 and atheroma-associated cytokines on cell proliferation and apoptosis in plaque smooth muscle cells of symptomatic and asymptomatic patients with carotid stenosis

Morbidity and mortality from atherosclerosis are associated with complicated atherosclerotic lesions due to plaque rupture, which is regulated by a balance between proliferation and apoptosis of vascular smooth muscle cells (VSMC). We examined insulin‐like growth factor‐1 (IGF‐1)‐induced survival of plaque VSMC from carotid endarterectomy specimens and investigated the underlying cellular mechanisms in the presence and absence of IL‐12 and IFN‐γ. Both IL‐12 and IFN‐γ were strongly expressed in symptomatic atherosclerotic plaques as compared with asymptomatic plaques. In asymptomatic plaque VSMC, IGF‐1 induced the survival and proliferation of VSMC and accelerated VSMC into S‐phase. IL‐12 or IFN‐γ inhibited proliferation and VSMC were arrested in the G0–G1 phase. IGF‐1 markedly inhibited the expression of p27kip and p21cip and significantly induced cyclin E and cyclin D. Both cytokines by themselves increased the expression of p27kip and p21cip and inhibited cyclin E and cyclin D. On the contrary, in symptomatic VSMC there was already increased apoptosis of VSMC and there was no significant effect of IGF‐1 or inflammatory cytokines on proliferation, apoptosis or the expression of p27kip and p21cip and cyclin D and E. These data suggest that IGF‐1 is more potent in inducing the survival of VSMC from the endarterectomy specimens of asymptomatic patients as compared to that of symptomatic subjects and cytokines associated with atheroma lesions decrease the activity of IGF‐1‐induced survival in the VSMC of asymptomatic plaques. The different expression and activity of cell cycle regulatory proteins could be responsible for apoptosis of VSMC and destabilization of atherosclerotic plaques.

Insulin-like growth factor-1 induces phosphorylation of PI3K-Akt/PKB to potentiate proliferation of smooth muscle cells in human saphenous vein

Coronary revascularization by coronary artery bypass grafting (CABG) is recommended in patients with recurrent myocardial ischemia. However, the long-term results of CABG using saphenous vein (SV) graft, compared to internal mammary artery (IMA) graft, have not been satisfactory. The SV graft failure is due to the development of intimal hyperplasia, a process characterized by abnormal migration and proliferation of smooth muscle cells (SMCs) in the intimal layer of the vein graft. Insulin growth factor 1 (IGF-1) is a major mitogenic growth factor released at the site of the shear stress-induced graft injury. This study, for the first time, compares the extent of IGF-1-PI3K-Akt activation in isolated human bypass graft conduits. Human SV and IMA vessels were collected and SMCs isolated and cultured. In cultured SMCs, effect of IGF-1 was examined on total and phosphorylated PI3K, Akt and IGF-1R by Western blot analysis. Cell proliferation was measured using BrdU ELISA. There was no significant difference in the basal expression of phosphorylated PI3K, Akt and IGF-1R in SV and IMA SMCs from human bypass conduits. However, we observed an upregulation of IGF-1 receptors in the SV SMCs in response to IGF-1 stimulation with no effect in IMA SMCs. Furthermore, the immunoblotting and cellular activation of signaling ELISA (CASE) assay demonstrated a significantly higher activity of both PI3K and Akt in IGF-1-stimulated SV SMCs than IMA. This was inhibited by an IGF-1R blocking antibody. IGF-1 induced proliferation in both SV and IMA SMCs was inhibited by a PI3K inhibitor, wortmannin. These data demonstrate differential activity of IGF-1-induced PI3K-Akt activation, which was quantitatively and temporally greater in SV SMCs than in the IMA. This, at least in part, could explain the greater propensity of the SV conduits than the IMA to undergo intimal hyperplasia following CABG.

Cross-talk between angiotensin II and IGF-1-induced connexin 43 expression in human saphenous vein smooth muscle cells

Vascular restenosis following coronary artery bypass graft can cause major clinical complications due to intimal hyperplasia in venous conduits. However, the precise underlying mechanisms of intimal hyperplasia are still unclear. We have recently reported that increased expression of connexin43 (Cx43) is involved in the proliferation of vascular smooth muscle cells (SMCs) in human saphenous vein (SV). In this study, we investigated the signalling transduction pathway involved in Cx43 expression and SV SMC proliferation. Angiotensin‐II (AT‐II, 100 ng/ml) increased AT‐II receptor 1 (AT‐1R) protein expression and insulin‐like growth factor‐1 (IGF‐1) (100 ng/ml) up‐regulated IGF‐1 receptor (IGF‐1R) protein expression in SV SMCs. Interestingly, AT‐1R expression was also increased by IGF‐1 treatment, and IGF‐1R expression was increased by AT‐II treatment, which was blocked by siRNA‐IGF‐1R and siRNA‐AT‐1R, respectively. Furthermore, the effect of AT‐II and IGF‐1 signal cross‐talk i nducing up‐regulation of their reciprocal receptors was blocked by siRNA against extracellular signal‐regulated kinases 1/2 (Erk 1/2) in SMCs of SV. Moreover, AT‐II and IGF‐1‐induced Cx43 expression via phosphorylation of Erk 1/2 and activation of transcription factor activator protein 1 (AP‐1) through their reciprocal receptors in SV SMCs. These data demonstrate a cross‐talk between IGF‐1R and AT‐1R in AT‐II and IGF‐1‐induced Cx43 expression in SV SMCs involving Erk 1/2 and downstream activation of the AP‐1 transcription factor.

Tumor necrosis factor-α regulates p27kip expression and apoptosis in smooth muscle cells of human carotid plaques via forkhead transcription factor O1

Apoptosis of vascular smooth muscle cells (SMCs) is controlled by a balance between the effect of growth factors and cytokines, and is involved in plaque instability in advanced atherosclerotic lesions. Recently, we reported high levels of atheroma-associated cytokines, including tumor necrosis factor-α (TNF-α), in carotid plaques of symptomatic patients. These cytokines induce apoptosis of vascular SMCs, and thus could be responsible for plaque rupture, a clinically devastating event. In this study, we examined the effect of TNF-α on the cell cycle inhibitor p27(kip) and apoptosis of SMCs in human carotid plaques, and the underlying mechanism. Both Forkhead box subclass o1 (FoxO1) and p27(kip) were more strongly expressed in symptomatic than asymptomatic atherosclerotic plaques. TNF-α significantly induced the expression of FoxO1 in asymptomatic plaque SMCs in a dose- and time-dependent manner via JNK signaling pathway. TNF-α also induced phosphorylation of FoxO1, resulting in its cytoplasmic translocation/nuclear exclusion of transcription factors. The effect of TNF-α was blocked by the PI3K inhibitor, LY294002. Meanwhile, TNF-α not only induced the p27(kip) expression and cell cycle arrest in the G(0)-G(1) phase, but also enhanced caspase-3 activity and induced apoptosis in SMCs of asymptomatic plaques. However, the potential effect of TNF-α on the cell cycle inhibitor p27(kip) and apoptosis of SMCs was inhibited by siRNA against FoxO1 in asymptomatic patients. These data suggest the involvement of FoxO1 transcription factor in TNF-α-induced expression of a cell cycle regulatory protein and apoptosis of SMCs, thus regulating the stability of atherosclerotic plaques with carotid stenosis.

Regulation of cell cycle entry by PTEN in smooth muscle cell proliferation of human coronary artery bypass conduits

Proliferation of smooth muscle cells (SMCs) is the key event in the pathogenesis of intimal hyperplasia (IH) leading to coronary artery bypass graft (CABG) occlusion. The saphenous vein (SV) conduits are often affected by IH, while the internal mammary artery (IMA) conduits remain remarkably patent. SMC proliferation is mediated by the cell cycle, under the control of cyclin‐dependent kinases (cdks), cdk‐inhibitors and the retinoblastoma protein (Rb). Early passage of the SMCs through the cell cycle involves crossing the non‐reversible G1 checkpoint, the restriction (R) point. In this study, we investigated the effect of mitogenic insulin‐like growth factor (IGF)‐1 stimulation on the R‐point and its relationship with the phosphorylation of Rb protein and the cdk inhibitors p21 and p27 in SV and IMA SMCs. We observed no change in the R‐point following IGF‐1 activation in either SV or IMA SMCs. However, Rb‐phosphorylation occurred much earlier and was quantitatively greater in SV SMCs than IMA. Overexpression of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in SV SMCs followed by IGF‐1 activation significantly decreased the expression of cyclin E and pRb and induced p27 expression in SV SMCs, while, pRb levels were markedly decreased and p27 levels were significantly increased in IMA SMCs. Silencing the PTEN gene by siRNA transfection of IMA SMCs significantly induced the expression of pRb and inhibited p27 expression, while, the expression levels of cyclin E, pRb, p21 and p27 were unaffected by the silencing of PTEN in SV SMCs. These results demonstrate that the PTEN plays a critical role in regulating cell cycle entry. Therefore, overexpression of PTEN possibly by means of gene therapy could be a viable option in regulating the cell cycle in SV SMCs in the treatment of vein graft disease.

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.