Thrombin stimulates VSMC proliferation through an EGFR-dependent pathway: involvement of MMP-2.

In this study, the role of epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK1/2), heparin-binding EGF-like growth factor (HB-EGF), general metalloproteinases, matrix metalloproteinases-2 (MMP-2) in mediating the mitogenic action of thrombin in rat vascular smooth muscle cells (VSMC) was investigated. The incubation of rat VSMC with thrombin (1 U/ml) for 5 min resulted in significant (p < 0.001) increase of ERK1/2 phosphorylation by 8.7 ± 0.9-fold, EGFR phosphorylation by 8.5 ± 1.3-fold (p < 0.001) and DNA synthesis by 3.6 ± 0.4-fold (p < 0.001). Separate 30-min pretreatments with EGFR tyrosine kinase irreversible inhibitor, 10 µM PD169540 (PD), and 20 µM anti-HB-EGF antibody significantly reduced thrombin-stimulated EGFR and ERK1/2 phosphorylation by 81, 72 % and by 48 and 61 %, respectively. Furthermore, the same pretreatments with PD or anti-HB-EGF antibody reduced thrombin-induced VSMC proliferation by 44 and 45 %, respectively. In addition, 30-min pretreatments with 10 µM specific MMP-2 inhibitor significantly reduced thrombin-stimulated phosphorylation of both EGFR and ERK1/2 by 25 %. Moreover, the same pretreatment with MMP-2 inhibitor reduced thrombin-induced VSMC proliferation by 45 %. These results show that the thrombin-induced DNA synthesis correlates with the level of ERK1/2 activation rather than EGFR activation. These results further suggest that thrombin acts through EGFR and ERK 1/2 signaling pathways involving MMP-2 to upregulate proliferation of VSMC.

A novel hypothesis regarding the possible involvement of cytosolic phospholipase 2 in insulin-stimulated proliferation of vascular smooth muscle cells.

Insulin (INS) via INS receptor acts as a mitogen in vascular smooth muscle cells (VSMCs) through stimulation of multiple signaling mechanisms, including p42/44 mitogen-activated protein kinase (ERK1/2) and phosphatidyl inositol-3 kinase (PI3K). In addition, cytosolic phospholipase 2 (cPLA2) is linked to VSMCs proliferation. However, the upstream mechanisms responsible for activation of cPLA2 are not well defined. Therefore, this investigation used primary cultured rat VSMCs to examine the role of PI3K and ERK1/2 in the INS-dependent phosphorylation of cPLA2 and proliferation induced by INS. Exposure of VSMCs to INS (100 nM) for 10 min increased the phosphorylation of cPLA2 by 1.5-fold ( p < 0.01), which was blocked by the cPLA2 inhibitor MAFP (10 microM; 15 min). Similarly, the PI3K inhibitor LY294002 (10 microM; 15 min) and ERK1/2 inhibitor PD98059 (20 microM; 15 min) abolished the INS-mediated increase in cPLA2 phosphorylation by 59% (p < 0.001), and by 75% ( p < 0.001), respectively. Further, inhibition of cPLA2 with cPLA2 inhibitor MAFP abolished the INS-stimulated ERK1/2 phosphorylation by 65% ( p < 0.01). Incubation of rat VSMCs with INS resulted in an increase of VSMCs proliferation by 85% ( p < 0.001). The effect of INS on VSMCs proliferation was significantly ( p < 0.01) reduced by pretreatment with MAFP. Thus, we hypothesized that INS stimulates VSMCs proliferation via a mechanism involving the PI3K-dependent activation of cPLA2 and release of arachidonic acid (AA), which activates ERK1/2 and further amplifies cPLA2 activity.

Role of L-type calcium channel blocking in epidermal growth factor receptor-independent activation of extracellular signal regulated kinase 1/2.

OBJECTIVE: Vascular smooth muscle cell (VSMC) differentiation, growth and survival, key events in the development of cardiovascular diseases, are under the control of signaling enzymes including extracellular signal regulated kinase 1/2 (ERK 1/2), Akt and epidermal growth factor receptor (EGFR) activation. EGFR trans-activation is known to mediate thrombin- or angiotensin II (AII)-stimulated ERK 1/2 activation. However, our laboratory has demonstrated, in thrombin-stimulated VSMC, that the prevention of intracellular Ca2+ elevation ([Ca2+]i) by BAPTA-AM pretreatment unveiled EGFR-independent ERK 1/2 activation. Since calcium channel blockers (CCBs) also impair agonist-induced [Ca2+]i elevation, we investigated whether EGFR-independent ERK 1/2 activation could occur in VSMCs treated by CCBs such as amlodipine, isradipine and verapamil, and examined the possible role of Akt. METHODS: Cultured VSMCs were pretreated or not with CCBs and with various inhibitors of the signaling pathways under study, prior to stimulation by thrombin or AII, and the phosphorylation/activation status of EGFR, Akt and ERK 1/2 was determined by Western blotting using phospho-specific antibodies. RESULTS AND CONCLUSION: Unlike BAPTA, CCBs did not impair stimulus-induced EGFR trans-activation, hence ERK1/2 phosphorylation. However, when EGFR kinase was inhibited, CCBs and BAPTA dose-dependently protected stimulus-induced ERK1/2 phosphorylation. The effect of amlodipine could not be mimicked by its R+ enantiomer, which is devoid of CCB activity, suggesting that the effects of CCBs were accounted for by their L-type Ca2+ channel-blocking property. Altogether, our results indicated that in G-protein-coupled receptor (GPCR)-stimulated VSMCs, the prevention of [Ca2+]i elevation by CCBs unmasked an EGFR kinase-independent phosphorylation of ERK 1/2. Since EGFR kinase inhibitors are supposed to be efficient in the treatment of some cancers, such a mechanism might be clinically relevant in hypertensive patients with cancer.

Dual mechanism of action of amlodipine in human vascular smooth muscle cells.

OBJECTIVES: It has been recently shown that calcium channel blockers (CCBs) could also control smooth muscle cell (SMC) growth/reactivity through mechanisms that were unrelated to their CCB property. Here, we investigated the effects of amlodipine and isradipine on Ca2+ movements and p42/p44 mitogen-activated protein kinase (ERK 1/2) activities, which are two early signalling events triggered by growth factors such as thrombin and basic fibroblast growth factor (bFGF). METHODS: In cultured human SMCs isolated from internal mammary arteries, Ca2+ movements and ERK 1/2 activation were studied by measurement of the intracellular Ca2+ concentration in Fura 2-labelled SMCs and by Western blots, respectively. RESULTS: In thrombin- and thapsigargin-stimulated SMCs, amlodipine and not isradipine dose-dependently reduced Ca2+ mobilization (i.e. Ca2+ release from internal stores); these dihydropyridines did not affect either Ca2+ influx or ERK 1/2 activation. In bFGF-stimulated SMCs, amlodipine and isradipine reduced both Ca2+ influx and ERK 1/2 activation without affecting Ca2+ mobilization. ERK 1/2 activation could also be directly stimulated by the l-type channel agonist Bay K 8644, demonstrating the involvement of voltage-gated Ca2+ influx in this process. Most of the observed effects described were obtained with approximately 10 nmol/l amlodipine/isradipine (i.e. concentrations close to the peak plasma level in treated patients). CONCLUSIONS: In human SMCs, amlodipine can (i) specifically alter Ca2+ mobilization, likely by interacting with the sarcoplasmic reticulum and (ii) inhibit voltage-dependent Ca2+ influx and the resulting ERK 1/2 activation. It is likely that amlodipine exerts its growth-inhibitory potency by interfering with multiple branches of mitogenic signalling pathways.

Regulation of Endothelial Nitric Oxide Synthase in Pathophysiological Conditions.

The nitric oxide (NO) cascade and endothelial NO synthase (eNOS) are best known for their role in endothelium-mediated relaxation of vascular smooth muscle (VSM). NO generated by eNOS has been established as a key regulatory signaling molecule in the vasculature. The activities of eNOS are controlled by intracellular calcium/calmodulin (CaM) and by binding of the molecular chaperone heat-shock protein 90 (Hsp90). A number of studies have demonstrated a close association between insulin resistance (IR) and NO bioactivity. Some recent studies demonstrate that insulin signaling is essential for normal cardiovascular (CV) function and lack of it such as IR result in CV dysfunction and disease. A key step in the initiation and progression of atherosclerosis is a reduction in the bioactivity of endothelial cell-derived NO. Multiple changes in endothelial function and eNOS activity accompany the onset and development of Type 2 diabetes mellitus (T2DM) and contribute to the development of cardiovascular disease (CVD). This review focuses on recent findings about regulation of eNOS in pathophysiological conditions such are: IR, T2DM and CVD.

Insulin, thrombine, ERK1/2 kinase and vascular smooth muscle cells proliferation.

Vascular smooth muscle cells (VSMC) respond to arterial wall injury by intimal proliferation and play a key role in atherogenesis by proliferating and migrating excessively in response to repeated injury, such as hypertension and atherosclerosis. In contrast, fully differentiated, quiescent VSMC allow arterial vasodilatation and vasoconstriction. Exaggerated and uncontrolled VSMC proliferation appears therefore to be a common feature of both atherosclerosis and hypertension. Signal transduction pathways in eukaryotic cells integrate diverse extracellular signals, and regulate complex biological responses such as growth, differentiation and death. One group of proline-directed Ser/Thr protein kinases, the mitogen-activated protein kinases (MAPKs), plays a central role in these signalling pathways. Much attention has focused in recent years on subfamilies of MAPKs, the extracellular signal regulated kinases (ERKs). Here we overview the work on ERKs 1 to 2, emphasising when possible their biological activities in VSMC proliferation. It is clear from numerous studies including our own, that ERK1/ERK2 pathway has an important role in VSMC proliferation induced by insulin (INS) and thrombin. Despite the physiological and pathophysiological importance of INS and thrombin, possible signal transduction pathways involved in INS and thrombin regulation of VSMC's proliferation remains poorly understood. Thus, this review examines recent findings in signaling mechanisms involved in INS and thrombin- triggered VSMC's proliferation with particular emphasis on ERK1/2 signaling pathways. Future investigations should now focus on the mechanisms of MAPK activation which might therefore represent a new mechanism involved in the antiproliferative effect revealed in this review.

Involvement of ERK1/2 kinase in insulin-and thrombin-stimulated vascular smooth muscle cell proliferation.

It is well recognized that the proliferation of vascular smooth muscle cells (VSMCs) is a key event in the pathogenesis of various vascular diseases, including atherosclerosis and hypertension. We have previously shown that among extracellular signal-regulated protein kinases (ERKs), the 42- and 44-kDa isoforms (ERK1/2) participate in the cellular mitogenic machinery triggered by several VSMCs activators, including insulin (INS) and thrombin (Thr). However, understanding of the intracellular signal transduction pathways involved is incomplete. This review considers the recent findings in INS and Thr signaling mechanisms that modulate the proliferation of VSMCs with particular emphasis on the ERK1/2 signaling pathway, an important mediator of VSMCs hypertrophy and vascular disease. Moreover, because the ERK1/2 pathway have been acknowledged as an important mediator of VSMCs hypertrophy, ERK1/2 is identified as a key target for novel therapeutic interventions to minimize irreversible tissue damage associated with hypertension and atherosclerosis.

Role of PI3K/AKT, cPLA2 and ERK1/2 signaling pathways in insulin regulation of vascular smooth muscle cells proliferation.

Vascular smooth muscle cells (VSMCs) respond to arterial wall injury by intimal proliferation and play a key role in atherogenesis by proliferating and migrating excessively in response to repeated injury, such as hypertension and atherosclerosis. In contrast, fully differentiated, quiescent VSMCs allow arterial vasodilatation and vasoconstriction. Exaggerated and uncontrolled VSMCs proliferation appears therefore to be a common feature of both atherosclerosis and hypertension. Phosphorylation/dephosphorylation reactions of enzymes belonging to the family of mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) play an important role in the transduction of mitogenic signal. We have previously shown that among extracellular signal-regulated protein kinases (ERKs), the 42 and 44 kDa isoforms (ERK1/2) as well as Akt and cytosolic phospholipase 2 (cPLA2) participate in the cellular mitogenic machinery triggered by several VSMCs activators, including insulin (INS). The ability of INS to significantly increase VSMCs proliferation has been demonstrated in several systems, but understanding of the intracellular signal transduction pathways involved is incomplete. Signal transduction pathways involved in regulation of the VSMCs proliferation by INS remains poorly understood. Thus, this review examines recent findings in signaling mechanisms employed by INS in modulating the regulation of proliferation of VSMCs with particular emphasis on PI3K/Akt, cPLA2 and ERK1/2 signaling pathways that have been identified as important mediators of VSMCs hypertrophy and vascular diseases. These findings are critical for understanding the role of INS in vascular biology and hyperinsulinemia.

A New Least Squares Based Calibration for an Ultrasound Tomography Scanner Using Radial Image Processing.

In the context of the development of an ultrasound scanner where a probe turns around the part of the body to be studied, a new tomographic technique has been developed: the Ultrasound Reflection-mode Tomography Using Radial Image Processing (URTURIP Technique). This technique is used when a unique B-scan image is insufficient to correctly describe a cross-section. It utilises B-scan images obtained under different angles of view in the same plane to reconstruct a better cross-sectional image. Before using the URTURIP Technique, a calibration step is required to accurately determine the centre of rotation of the probe. To solve the calibration problem, a reference method has been developed, but it is very time-consuming. This paper presents a faster method based on a least squares fitting and is compared to the reference method in terms of accuracy and time-consuming.

Evidence for ERK1/2 activation by thrombin that is independent of EGFR transactivation.

Thrombin is involved in abnormal proliferation of vascular smooth muscle cells (VSMCs) associated with pathogenic vascular remodeling. Thrombin stimulation results in extracellular signal-regulated kinase (ERK)1/2 activation through transactivation of the epidermal growth factor receptor (EGFR). Here, using specific antibodies and inhibitors, we investigated the thrombin-induced phosphorylation of Src family kinases, nonreceptor proline-rich tyrosine kinase (Pyk2), EGFR, and ERK1/2. Our results show that Src and Pyk2 are involved upstream of the EGFR transactivation that is required for ERK1/2 phosphorylation. The investigation of the role of intracellular calcium concentration ([Ca2+]i) and calcium mobilization with the Ca2+ chelator BAPTA and thapsigargin, respectively, indicated that thrombin- and thapsigargin-induced phosphorylation of the EGFR but not ERK1/2 is dependent on an increase in [Ca2+]i. Moreover, only after BAPTA-AM pretreatment was thrombin-induced activation of ERK1/2 partially preserved from the effects of EGFR and PKC inhibition but not Src family kinase inhibition. These results suggest that BAPTA, by preventing [Ca2+]i elevation, unmasks a new pathway of Src family kinase-dependent thrombin-stimulated ERK1/2 phosphorylation that is independent of EGFR and PKC activation.

Differentiation of vascular myofibroblasts induced by transforming growth factor-beta1 requires the involvement of protein kinase Calpha.

In response to vascular injury, adventitial fibroblasts can modulate their phenotype to myofibroblasts, cells that participate in arterial remodeling. However, the signaling mechanisms underlying the vascular myofibroblast differentiation remain unknown. Since protein kinase C (PKC) is a key enzyme for cell differentiation, we examined whether PKC isoforms were involved in the vascular myofibroblast differentiation. The association between PKCalpha and myofibroblast differentiation was investigated in cultured rat aortic fibroblasts treated with transforming growth factor-beta1 (TGFbeta1). Confocal immunofluorescence microscopy indicated that fibroblasts expressed alpha-smooth muscle actin (alpha-SM actin) after TGFbeta1 treatment. Moreover, TGFbeta1 stimulation increased both PKCalpha mRNA expression (measured by real-time quantitative RT-PCR) and PKC activity (determined by histone-like pseudosubstrate phosphorylation) in adventitial fibroblasts. Western blot analysis indicated that PKCalpha protein expression was higher in TGFbeta1-treated fibroblasts than in untreated cells. TGFbeta1-induced expression of alpha-SM actin was inhibited in a dose-dependent manner by treating cells with a PKC inhibitor, calphostin C, and was abolished by depleting PKCalpha with antisense PKCalpha oligodeoxynucleotides. Our results demonstrate that TGFbeta1 induces adventitial myofibroblast differentiation via a PKCalpha-dependent process.