Regulation of the platelet-derived growth factor receptor-beta by G protein-coupled receptor kinase-5 in vascular smooth muscle cells involves the phosphatase Shp2

Smooth muscle cell (SMC) proliferation and migration are substantially controlled by the platelet-derived growth factor receptor-beta (PDGFRbeta), which can be regulated by the Ser/Thr kinase G protein-coupled receptor kinase-2 (GRK2). In mouse aortic SMCs, however, we found that prolonged PDGFRbeta activation engendered down-regulation of GRK5, but not GRK2; moreover, GRK5 and PDGFRbeta were coordinately up-regulated in SMCs from atherosclerotic arteries. With SMCs from GRK5 knock-out and cognate wild type mice (five of each), we found that physiologic expression of GRK5 increased PDGF-promoted PDGFRbeta seryl phosphorylation by 3-fold and reduced PDGFRbeta-promoted phosphoinositide hydrolysis, thymidine incorporation, and overall PDGFRbeta tyrosyl phosphorylation by approximately 35%. Physiologic SMC GRK5 activity also increased PDGFRbeta association with the phosphatase Shp2 (8-fold), enhanced phosphorylation of PDGFRbeta Tyr(1009) (the docking site for Shp2), and reduced phosphorylation of PDGFRbeta Tyr(1021). Consistent with having increased PDGFRbeta-associated Shp2 activity, GRK5-expressing SMCs demonstrated greater PDGF-induced Src activation than GRK5-null cells. GRK5-mediated desensitization of PDGFRbeta inositol phosphate signaling was diminished by Shp2 knock-down or impairment of PDGFRbeta/Shp2 association. In contrast to GRK5, physiologic GRK2 activity did not alter PDGFRbeta/Shp2 association. Finally, purified GRK5 effected agonist-dependent seryl phosphorylation of partially purified PDGFRbetas. We conclude that GRK5 mediates the preponderance of PDGF-promoted seryl phosphorylation of the PDGFRbeta in SMCs, and, through mechanisms involving Shp2, desensitizes PDGFRbeta inositol phosphate signaling and enhances PDGFRbeta-triggered Src activation.     

Differential effects of imatinib on PDGF-induced proliferation and PDGF receptor signaling in human arterial and venous smooth muscle cells

Platelet-derived growth factor (PDGF) has been implicated in smooth muscle cell (SMC) proliferation, a key event in the development of myointimal hyperplasia in vascular grafts. Recent evidence suggests that the PDGF receptor (PDGFR) tyrosine kinase inhibitor, imatinib, can prevent arterial proliferative diseases. Because hyperplasia is far more common at the venous anastomosis than the arterial anastomosis in vascular grafts, we investigated whether imatinib also inhibited venous SMC (VSMC) proliferation, and examined possible differences in its mechanism of action between VSMC and arterial SMC (ASMC). Human ASMC and VSMC were stimulated with PDGF-AB, in the presence or absence of imatinib (0.1-10 microM). Proliferation was assayed using the 5-bromo-2'-deoxyuridine (BrdU) incorporation assay, while PDGFR, Akt and ERK1/2-mitogen activated protein kinase (MAPK) signaling pathways were investigated by immunoblotting. The proliferative response to PDGF at 50 and 100 ng/ml was 32 and 43% greater, respectively, in VSMC than in ASMC. Similarly, PDGF-stimulated proliferation was more sensitive to inhibition by imatinib in VSMC than ASMC (IC(50) = 0.05 microM vs. 0.4 microM; P < 0.01). Imatinib also more effectively inhibited PDGF-induced phosphorylation of PDGFRbeta and Akt in VSMC, compared to ASMC. These data highlight inherent pharmacodynamic differences between VSMC and ASMC in receptor and cell signaling functions and suggest that imatinib therapy may be useful for the prevention of venous stenosis in vascular grafts.     

Type I collagen synergistically enhances PDGF-induced smooth muscle cell proliferation through pp60src-dependent crosstalk between the alpha2beta1 integrin and PDGFbeta receptor

Smooth muscle cells (SMCs) are exposed to both platelet-derived growth factor (PDGF) and type I collagen (CNI) at the time of arterial injury. In these studies we explore the individual and combined effects of these agonists on human saphenous vein SMC proliferation. PDGF-BB produced a 5.5-fold increase in SMC DNA synthesis whereas CNI stimulated DNA synthesis to a much lesser extent (1.6-fold increase). Alternatively, we observed an 8.3-fold increase in DNA synthesis when SMCs were co-incubated with CNI and PDGF-BB. Furthermore, stimulation of SMCs with PDGF-BB produced a significant increase in ERK-2 activity whereas CNI alone had no effect. Co-incubation of SMCs with PDGF-BB and CNI resulted in ERK-2 activity that was markedly greater than that produced by PDGF-BB alone. In a similar fashion, PDGF-BB induced phosphorylation of the PDGF receptor beta (PDGFRbeta) and CNI did not, whereas concurrent agonist stimulation produced a synergistic increase in receptor activity. Blocking antibodies to the alpha2 and beta1 subunits eliminated this synergistic interaction, implicating the alpha2beta1 integrin as the mediator of this effect. Immunoprecipitation of the alpha2beta1 integrin in unstimulated SMCs followed by immunoblotting for the PDGFRbeta as well as Src family members, pp60(src), Fyn, Lyn, and Yes demonstrated coassociation of alpha2beta1 and the PDGFRbeta as well as pp60(src). Incubation of cells with CNI and/or PDGF-BB did not change the degree of association. Finally, inhibition of Src activity with SU6656 eliminated the synergistic effect of CNI on PDGF-induced PDGFRbeta phosphorylation suggesting an important role for pp60(src) in the observed receptor crosstalk. Together, these data demonstrate that CNI synergistically enhances PDGF-induced SMC proliferation through Src-dependent crosstalk between the alpha2beta1 integrin and the PDGFRbeta.     

TGF-β and Smad3 modulate PI3K/Akt signaling pathway in vascular smooth muscle cells

Transforming growth factor-β (TGF-β) is upregulated at the time of arterial injury; however, the mechanism through which TGF-β enhances the development of intimal hyperplasia is not clear. Recent studies from our laboratory suggest that in the presence of elevated levels of Smad3, TGF-β stimulates smooth muscle cell (SMC) proliferation. This is a novel phenomenon in that TGF-β has traditionally been known as a potent inhibitor of cellular proliferation. In these studies we explore the signaling pathways through which TGF-β mediates its proliferative effect in vascular SMCs. We found that TGF-β phosphorylates and activates Akt in a time-dependent manner, and this effect is significantly enhanced by overexpression of Smad3. Furthermore, both chemical and molecular inhibition of Smad3 can reverse the effect of TGF-β on Akt. Although we found numerous signaling pathways that might function as intermediates between Smad3 and Akt, p38 appeared the most promising. Overexpression of Smad3 enhanced p38 phosphorylation and inhibition of p38 with a chemical inhibitor or a small interfering RNA blocked TGF-β-induced Akt phosphorylation. Moreover, TGF-β/Smad3 enhancement of SMC proliferation was blocked by inhibition of p38. Phosphorylation of Akt by TGF-β/Smad3 was not dependent on gene expression or protein synthesis, and immunoprecipitation studies revealed a physical association among p38, Akt, and Smad3 suggesting that activation requires a direct protein-protein interaction. Our findings were confirmed in vivo where overexpression of Smad3 in a rat carotid injury model led to enhancement of p-p38, p-Akt, as well as SMC proliferation. Furthermore, inhibition of p38 in vivo led to decreased Akt phosphorylation and SMC proliferation. In summary, our studies reveal a novel pathway whereby TGF-β/Smad3 stimulates SMC proliferation through p38 and Akt. These findings provide a potential mechanism for the substantial effect of TGF-β on intimal hyperplasia and suggest new targets for chemical or molecular prevention of vascular restenosis.     

Biomechanical regulation of hedgehog signaling in vascular smooth muscle cells in vitro and in vivo

Hedgehog (Hh) signaling has recently been shown to be both responsive to mechanical loading in vitro and to control vascular development in vivo. We investigated the role of cyclic strain and pulsatile flow in modulating Hh signaling and growth of adult rat vascular smooth muscle cells (SMC) in culture. Exposure of SMC to defined equibiaxial cyclic strain (0% and 10% stretch, 60 cycles/min, for 24 h) significantly decreased sonic hedgehog (Shh) and patched 1 (Ptc1) expression while concurrently inhibiting Gli₂-dependent promoter activity and mRNA expression, respectively. Cyclic strain significantly decreased SMC proliferation (cell counts and proliferating cell nuclear antigen expression) concomitant with a marked increase in SMC apoptosis (fluorescence-activated cell sorter analysis, acridine orange staining of apoptotic nuclei and Bax/Bcl-x_L ratio). These strain-induced changes in proliferation and apoptosis were significantly attenuated following addition of either recombinant Shh (3.5 µg/ml) or overexpression of the Notch 3 intracellular domain (Notch IC). Further studies using a perfused transcapillary culture system demonstrated a significant decrease in Hh signaling in SMC following exposure of cells to increased pulsatile flow concomitant with a decrease in proliferation and an increase in apoptosis. Finally, the pulsatile flow-induced decreases in Hh signaling were validated in vivo following flow-induced rat carotid arterial remodeling after 28 days. These data suggest that Hh expression is diminished by biomechanical stimulation in vitro and in vivo and thus may play a fundamental role in arterial remodeling and atherogenesis in vivo. cyclic strain; apoptosis; proliferation     

NDUFA4L2 in smooth muscle promotes vascular remodeling in hypoxic pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is characterized by a progressive increase in pulmonary vascular resistance and obliterative pulmonary vascular remodelling (PVR). The imbalance between the proliferation and apoptosis of pulmonary artery smooth muscle cells (PASMCs) is an important cause of PVR leading to PAH. Mitochondria play a key role in the production of hypoxia-induced pulmonary hypertension (HPH). However, there are still many issues worth studying in depth. In this study, we demonstrated that NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4 like 2 (NDUFA4L2) was a proliferation factor and increased in vivo and in vitro through various molecular biology experiments. HIF-1α was an upstream target of NDUFA4L2. The plasma levels of 4-hydroxynonene (4-HNE) were increased both in PAH patients and hypoxic PAH model rats. Knockdown of NDUFA4L2 decreased the levels of malondialdehyde (MDA) and 4-HNE in human PASMCs in hypoxia. Elevated MDA and 4-HNE levels might be associated with excessive ROS generation and increased expression of 5-lipoxygenase (5-LO) in hypoxia, but this effect was blocked by siNDUFA4L2. Further research found that p38-5-LO was a downstream signalling pathway of PASMCs proliferation induced by NDUFA4L2. Up-regulated NDUFA4L2 plays a critical role in the development of HPH, which mediates ROS production and proliferation of PASMCs, suggesting NDUFA4L2 as a potential new therapeutic target for PAH.     

Modification of Akt2 by 4-hydroxynonenal inhibits insulin-dependent Akt signaling in HepG2 cells

The production of reactive aldehydes such as 4-hydroxy-2-nonenal (4-HNE) is a key component of the pathogenesis in a spectrum of hepatic diseases involving oxidative stress such as alcoholic liver disease (ALD). One consequence of ALD is increased insulin resistance in hepatocytes. To understand the effects of 4-HNE on insulin signaling in liver cells, we employed a model using hepatocellular carcinoma cell line HepG2. Previously, we have demonstrated an increase in the level of Akt phosphorylation is mediated by 4-HNE inhibition of PTEN, a direct regulator of Akt. In this work, we evaluated the effects of 4-HNE on insulin-dependent stimulation of the Akt2 pathway. We demonstrate that 4-HNE selectively leads to phosphorylation of Akt2. Although Akt2 is phosphorylated following 4-HNE treatment, the level of downstream phosphorylation of Akt substrates such as GSK3β and MDM2 is significantly decreased. Pretreatment with 4-HNE prevented insulin-dependent Akt signaling and decreased intracellular Akt activity by 87%. Using biotin hydrazide capture, it was confirmed that 4-HNE treatment of cells resulted in carbonylation of Akt2, which was not observed in untreated control cells. Using a synthetic GSK3α/β peptide as a substrate, treatment of recombinant human myristoylated Akt2 (rAkt2) with 20 or 40 μM 4-HNE inhibited rAkt2 activity by 30 or 85%, respectively. Matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF) identified Michael addition adducts of 4-HNE with His196, His267, and Cys311 of rAkt2. Computation-based molecular modeling analysis of 4-HNE adducted to His196 and Cys311 of Akt2 suggests inhibition of GSK3β peptide binding by 4-HNE in the Akt2 substrate binding pocket. The inhibition of Akt by 4-HNE provides a novel mechanism for increased insulin resistance in ALD. These data provide a potential mechanism of dysregulation of Akt2 during events associated with sustained hepatocellular oxidative stress.     

The Lipoxin A4 receptor is coupled to SHP-2 activation: implications for regulation of receptor tyrosine kinases

Mesangial cell proliferation is pivotal to the pathology of glomerular injury in inflammation. We have previously reported that lipoxins, endogenously produced eicosanoids with anti-inflammatory and pro-resolution bioactions, can inhibit mesangial cell proliferation in response to several agents. This process is associated with elaborate receptor cross-talk involving modification receptor tyrosine kinase phosphorylation (McMahon, B., Mitchell, D., Shattock, R., Martin, F., Brady, H. R., and Godson, C. (2002) FASEB J. 16, 1817-1819). Here we demonstrate that the lipoxin A(4) (LXA(4)) receptor is coupled to activation and recruitment of the SHP-2 (SH2 domain-containing tyrosine phosphatase-2) within a lipid raft microdomain. Using site-directed mutagenesis of the cytosolic domain of the platelet-derived growth factor receptor beta (PDGFRbeta), we report that mutation of the sites for phosphatidylinositol 3-kinase (Tyr(740) and Tyr(751)) and SHP-2 (Tyr(763) and Tyr(1009)) recruitment specifically inhibit the effect of LXA(4) on the PDGFRbeta signaling; furthermore inhibition of SHP-2 expression with short interfering RNA constructs blocked the effect of LXA(4) on PDGFRbeta phosphorylation. We demonstrate that association of the PDGFRbeta with lipid raft microdomains renders it susceptible to LXA(4)-mediated dephosphorylation by possible reactivation of oxidatively inactivated SHP-2. These data further elaborate on the potential mechanisms underlying the anti-inflammatory, proresolution, and anti-fibrotic bioactions of lipoxins.     

Lipid peroxidation and cell cycle signaling: 4-hydroxynonenal,a key molecule in stress mediated signaling

Role of lipid peroxidation products, particularly 4-hydroxynonenal (4-HNE) in cell cycle signaling is becoming increasingly clear. In this article, recent studies suggesting an important role of 4-HNE in stress mediated signaling for apoptosis are critically evaluated. Evidence demonstrating the modulation of UV, oxidative stress, and chemical stress mediated apoptosis by blocking lipid peroxidation by the alpha-class glutathione S-transferases (GSTs) is presented which suggest an important role of these enzymes in protection against oxidative stress and a role of lipid peroxidation products in stress mediated signaling. Overexpression of 4-HNE metabolizing GSTs (mGSTA4-4, hGSTA4-4, or hGST5.8) protects cells against 4-HNE, oxidative stress (H(2)O(2) or xanthine/xanthine oxidase), and UV-A mediated apoptosis by blocking JNK and caspase activation suggesting a role of 4-HNE in the mechanisms of apoptosis caused by these stress factors. The intracellular concentration of 4-HNE appears to be crucial for the nature of cell cycle signaling and may be a determinant for the signaling for differentiation, proliferation, transformation, or apoptosis. The intracellular concentrations of 4-HNE are regulated through a coordinated action of GSTs (GSTA4-4 and hGST5.8) which conjugate 4-HNE to GSH to form the conjugate (GS-HNE) and the transporter 76 kDa Ral-binding GTPase activating protein (RLIP76), which catalyze ATP-dependent transport of GS-HNE. A mild stress caused by heat, UV-A, or H(2)O(2)with no apparent effect on the cells in culture causes a rapid, transient induction of hGST5.8 and RLIP76. These stress preconditioned cells acquire ability to metabolize and exclude 4-HNE at an accelerated pace and acquire relative resistance to apoptosis by UV and oxidative stress as compared to unconditioned control cells. This resistance of stress preconditioned cells can be abrogated by coating the cells with anti-RLIP76 antibodies which block the transport of GS-HNE. These studies and previous reports discussed in this article strongly suggest a key role of 4-HNE in stress mediated signaling.     

Ethanol-induced modulation of hepatocellular extracellular signal-regulated kinase-1/2 activity via 4-hydroxynonenal

Modulation of the extracellular signal-regulated kinases (ERK-1/2), a signaling pathway directly associated with cell proliferation, survival, and homeostasis, has been implicated in several pathologies, including alcoholic liver disease. However, the underlying mechanism of ethanol-induced ERK-1/2 modulation remains unknown. This investigation explored the effects of ethanol-associated oxidative stress on constitutive hepatic ERK-1/2 activity and assessed the contribution of the lipid peroxidation product 4-hydroxynonenal (4-HNE) to the observations made in vivo. Constitutive ERK-1/2 phosphorylation was suppressed in hepatocytes isolated from rats chronically consuming ethanol for 45 days. This observation was associated with an increase in 4-HNE-ERK monomer adduct concentration and a hepatic cellular and lobular redistribution of ERK-1/2 that correlated with 4-HNE-protein adduct accumulation. Chronic ethanol consumption was also associated with a decrease in hepatocyte nuclear ELK-1 phosphorylation, independent of changes in total nuclear ELK-1 protein. Primary hepatocytes treated with concentrations of 4-HNE consistent with those occurring during oxidative stress displayed a concentration-dependent decrease in constitutive ERK-1/2 phosphorylation, activity, and nuclear localization that negatively correlated with 4-HNE-ERK-1/2 monomer adduct accumulation. These data paralleled the decreased phosphorylation of the downstream kinase ELK-1. Molar ratios of purified ERK-2 to 4-HNE consistent with pathologic ratios found in vivo resulted in protein monomer-adduct formation across a range of concentrations. Collectively, these data demonstrate a novel association between ethanol-induced lipid peroxidation and the inhibition of constitutive ERK-1/2, and suggest an inhibitory mechanism mediated by the lipid peroxidation product 4-hydroxynonenal.     

Transfection with 4-hydroxynonenal-metabolizing glutathione S-transferase isozymes leads to phenotypic transformation and immortalization of adherent cells.

4-Hydroxy-2-trans-nonenal (4-HNE), one of the major end products of lipid peroxidation, has been shown to induce apoptosis in a variety of cell lines. It appears to modulate signaling processes in more than one way because it has been suggested to have a role in signaling for differentiation and proliferation. We show for the first time that incorporation of 4-HNE-metabolizing glutathione S-transferase (GST) isozyme, hGSTA4-4, into adherent cell lines HLE B-3 and CCL-75, by either cDNA transfection or microinjection of active enzyme, leads to their transformation. The dramatic phenotypic changes due to the incorporation of hGSTA4-4 include rounding of cells and anchorage-independent rapid proliferation of immortalized, rounded, and smaller cells. Incorporation of the inactive mutant of hGSTA4-4 (Y212F) in cells by either microinjection or transfection does not cause transformation, suggesting that the activity of hGSTA4-4 toward 4-HNE is required for transformation. This is further confirmed by the fact that mouse and Drosophila GST isozymes (mGSTA4-4 and DmGSTD1-1), which have high activity toward 4-HNE and subsequent depletion of 4-HNE, cause transformation whereas human GST isozymes hGSTP1-1 and hGSTA1-1, with minimal activity toward 4-HNE, do not cause transformation. In cells overexpressing active hGSTA4-4, expression of transforming growth factor beta1, cyclin-dependent kinase 2, protein kinase C betaII and extracellular signal regulated kinase is upregulated, whereas expression of p53 is downregulated. These studies suggest that alterations in 4-HNE homeostasis can profoundly affect cell-cycle signaling events.     

Low density lipoprotein receptor-related protein 1 (LRP1) controls endocytosis and c-CBL-mediated ubiquitination of the platelet-derived growth factor receptor beta (PDGFR beta)

The low density lipoprotein receptor-related protein 1 (LRP1) has been implicated in intracellular signaling functions as well as in lipid metabolism. Recent in vivo and in vitro studies suggest that LRP1 is a physiological modulator of the platelet-derived growth factor (PDGF) signaling pathway. Here we show that in mouse fibroblasts LRP1 modulates PDGF-BB signaling by controlling endocytosis and ligand-induced down-regulation of the PDGF receptor beta (PDGFRbeta). In LRP1-deficient fibroblasts, basal PDGFRbeta tyrosine kinase activity was derepressed, and PDGF-BB-induced endocytosis and degradation of PDGFRbeta were accelerated as compared with control cells. This was accompanied by rapid uptake of receptor-bound PDGF-BB into the cells and by attenuated ERK activation in response to PDGF-BB stimulation. Pulse-chase analysis indicated that the steady-state turnover rate of PDGFRbeta was also accelerated in LRP-deficient fibroblasts. The rapid degradation of PDGFRbeta in the LRP1-deficient fibroblasts was prevented by MG132 and chloroquine. Furthermore, the association of PDGFRbeta with c-Cbl, a ubiquitin E3-ligase, as well as the ligand-induced ubiquitination of PDGFRbeta were increased in LRP1-deficient fibroblasts. We show that LRP1 can directly interact with c-Cbl, suggesting a Sprouty-like role for LRP1 in regulating the access of the PDGFRbeta to the ubiquitination machinery. Thus, LRP1 modulates PDGF signaling by controlling ubiquitination and endocytosis of the PDGFRbeta.     

Estrogen-induced smooth muscle cell growth is regulated by tuberin and associated with altered activation of platelet-derived growth factor receptor-beta and ERK-1/2

The mechanisms that regulate the diverse responses to estrogen (E2) are unknown. Loss of function of the tuberous sclerosis 2 gene (TSC2), a tumor suppressor gene, has been associated with a growth-promoting effect of E2. We hypothesized that tuberin, the protein product of TSC2, binds to estrogen receptors (ER) and regulates the growth effect of E2. An in vivo association between full-length tuberin and ERalpha was observed in HEK 293 cells and ELT-3 smooth muscle cells. In contrast, poor association was observed between tuberin and ERbeta. Complex formation with ERalpha and the C-terminal end of tuberin was also observed in vivo and in vitro, indicating that binding between ERalpha and tuberin occurs at the C-terminal end of the tuberin molecule. We examined the effect of tuberin expression in ELT-3 smooth muscle cells on the growth response to E2. The growth-promoting effect of E2 in tuberin-null ELT-3 smooth muscle cells was ERalpha-specific, associated with up-regulation and activation of platelet-derived growth factor receptor-beta (PDGFRbeta) and activation of the signaling intermediate, extracellular signal-regulated kinase-1/-2 (ERK-1/2). In contrast, the expression of tuberin in ELT-3 smooth muscle cells resulted in significant abrogation of E2-stimulated growth. In parallel with this observation, the expression of tuberin in ELT-3 cells also resulted in significant inhibition of PDGFRbeta and ERK-1/2 activation in response to E2. These results demonstrate that tuberin binds specifically to ERalpha and inhibits E2-induced proliferation of ELT-3 cells. Furthermore, the opposing effects of tuberin on estrogen-induced activation of PDGFRbeta and ERK-1/-2 suggest a pivotal role for tuberin in directing the signaling events that dictate the growth response to E2.     

Autocrine signaling through Ras prevents apoptosis in vascular smooth muscle cells in vitro

Vascular smooth muscle cell (SMC) apoptosis contributes to physiological and pathological vascular remodeling. Autocrine fibroblast growth factor (FGF) signaling promotes survival in SMC in vitro. Interruption of autocrine FGF signaling results in apoptosis that can be rescued by other growth factors such as PDGF (platelet-derived growth factor) or EGF (epidermal growth factor). Such heterologous growth factor rescue is prevented by pharmacological inhibition of MAPK, implicating signaling through Ras in mediating survival. This study was designed to test the hypothesis that signaling through Ras is both necessary and sufficient to mediate SMC survival in vitro. Recombinant adenoviruses encoding dominant-negative (Ras(N17)) and constitutively active (Ras(L61)) mutants of Ras were used. Ras(N17) blocks growth factor-mediated MAPK activation and can itself induce SMC apoptosis. Ras(N17) is synergistic with inhibition of autocrine FGF signaling in triggering apoptosis and prevents heterologous growth factor rescue. Conversely, Ras(L61) prevents apoptosis resulting from inhibition of autocrine FGF signaling. Rescue by Ras(L61) can be partially prevented by pharmacological inhibition of MEK or phosphatidylinositol 3-kinase, two downstream effectors of Ras. These results suggest that Ras signaling is both necessary and sufficient to mediate survival in SMC in vitro. Further work is required to determine how these signaling events are regulated in the context of vascular remodeling in vivo.     

Role of 4-hydroxynonenal in stress-mediated apoptosis signaling

In this mini review we summarize recent studies from our laboratory, which show the involvement of 4-hydroxynonenal (4-HNE) in cell cycle signaling. We demonstrate 4-HNE induced apoptosis in various cell lines is accompanied with c-Jun-N-terminal kinase and caspase-3 activation. Cells exposed to mild, transient, heat or oxidative stress acquire capacity to exclude intracellular 4-HNE at a faster rate by inducing hGST5.8 which conjugate 4-HNE to GSH, and RLIP76 which mediates the ATP-dependent transport of the GSH-conjugate of 4-HNE. The cells preconditioned with mild transient stress acquire resistance to H(2)O(2) and 4-HNE induced apoptosis by excluding intracellular 4-HNE at an accelerated pace. Furthermore, a decrease in intracellular concentration of 4-HNE achieved by transfecting cells with mGSTA4-4 or hGSTA4-4 results in a faster growth rate. These studies strongly suggest a role of 4-HNE in stress mediated signaling.     

4-Hydroxynonenal induces Cx46 hemichannel inhibition through its carbonylation

Hemichannels formed by connexins mediate the exchange of ions and signaling molecules between the cytoplasm and the extracellular milieu. Under physiological conditions hemichannels have a low open probability, but in certain pathologies their open probability increases, which can result in cell damage. Pathological conditions are characterized by the production of a number of proinflammatory molecules, including 4-hydroxynonenal (4-HNE), one of the most common lipid peroxides produced in response to inflammation and oxidative stress. The aim of this work was to evaluate whether 4-HNE modulates the activity of Cx46 hemichannels. We found that 4-HNE (100 μM) reduced the rate of 4′,6-diamino-2-fenilindol (DAPI) uptake through hemichannels formed by recombinant human Cx46 fused to green fluorescent protein, an inhibition that was reversed partially by 10 mM dithiothreitol. Immunoblot analysis showed that the recombinant Cx46 expressed in HeLa cells becomes carbonylated after exposure to 4-HNE, and that 10 mM dithiothreitol reduced its carbonylation. We also found that Cx46 was carbonylated by 4-HNE in the lens of a selenite-induced cataract animal model. The exposure to 100 μM 4-HNE decreased hemichannel currents formed by recombinant rat Cx46 in Xenopus laevis oocytes. This inhibition also occurred in a mutant expressing only the extracellular loop cysteines, suggesting that other Cys are not responsible for the hemichannel inhibition by carbonylation. This work demonstrates for the first time that Cx46 is post-translationally modified by a lipid peroxide and that this modification reduces Cx46 hemichannel activity.     

Additive effects of PDGF receptor beta signaling pathways in vascular smooth muscle cell development

The platelet-derived growth factor beta receptor (PDGFRbeta) is known to activate many molecules involved in signal transduction and has been a paradigm for receptor tyrosine kinase signaling for many years. We have sought to determine the role of individual signaling components downstream of this receptor in vivo by analyzing an allelic series of tyrosine-phenylalanine mutations that prevent binding of specific signal transduction components. Here we show that the incidence of vascular smooth muscle cells/pericytes (v/p), a PDGFRbeta-dependent cell type, can be correlated to the amount of receptor expressed and the number of activated signal transduction pathways. A decrease in either receptor expression levels or disruption of multiple downstream signaling pathways lead to a significant reduction in v/p. Conversely, loss of RasGAP binding leads to an increase in this same cell population, implicating a potential role for this effector in attenuating the PDGFRbeta signal. The combined in vivo and biochemical data suggest that the summation of pathways associated with the PDGFRbeta signal transduction determines the expansion of developing v/p cells.     

C-reactive protein and vein graft disease: evidence for a direct effect on smooth muscle cell phenotype via modulation of PDGF receptor-beta

Plasma C-reactive protein (CRP) concentration is a biomarker of systemic atherosclerosis and may also be associated with vein graft disease. It remains unclear whether CRP is also an important modulator of biological events in the vessel wall. We hypothesized that CRP influences vein graft healing by stimulating smooth muscle cells (SMCs) to undergo a phenotypic switch. Distribution of CRP was examined by immunohistochemistry in prebypass human saphenous veins (HSVs, n = 21) and failing vein grafts (n = 18, 25-4,400 days postoperatively). Quiescent HSV SMCs were stimulated with human CRP (5-50 microg/ml). SMC migration was assessed in modified Boyden chambers with platelet-derived growth factor (PDGF)-BB (5-10 ng/ml) as the chemoattractant. SMC viability and proliferation were assessed by trypan blue exclusion and reduction of Alamar Blue substrate, respectively. Expression of PDGF ligand and receptor (PDGFR) genes was examined at RNA and protein levels after 24-72 h of CRP exposure. CRP staining was present in 13 of 18 diseased vein grafts, where it localized to the deep media and adventitia, but it was minimally detectable in most prebypass veins. SMCs pretreated with CRP demonstrated a dose-dependent increase in migration to PDGF-BB (P = 0.02), which was inhibited by a PDGF-neutralizing antibody. SMCs treated with CRP showed a dose-dependent increase in PDGFRbeta expression and phosphorylation after 24-48 h. Exogenous CRP had no effect on SMC viability or proliferation. These data suggest that CRP is detectable within the wall of most diseased vein grafts, where it may exert local effects. Clinically relevant levels of CRP can stimulate SMC migration by a mechanism that may involve upregulation and activation of PDGFRbeta.     

Strain activation of bovine aortic smooth muscle cell proliferation and alignment: Study of strain dependency and the role of protein kinase A and C signaling pathways

Smooth muscle cell (SMC) phenotype can be altered by physical forces as demonstrated by cyclic strain-induced changes in proliferation, orientation, and secretion of macromolecules. However, the magnitude of strain required and the intracellular coupling pathways remain ill defined. To examine the strain requirements for SMC proliferation, we selectively seeded bovine aortic SMC either on the center or periphery of silastic membranes which were deformed with 150 mm Hg vacuum (0–7% center; 7–24% periphery). SMC located in either the center or peripheral regions showed enhanced proliferation compared to cells grown under the absence of cyclic strain. Moreover, SMC located in the center region demonstrated significantly (P < 0.005) greater proliferation as compared to those in the periphery. In contrast, SMC exposed to high strain (7–24%) demonstrated alignment perpendicular to the strain gradient, whereas SMC in the center (0–7%) remained aligned randomly. To determine the mechanisms of these phenomena, we examined the effect of cyclic strain on bovine aortic SMC signaling pathways. We observed strain-induced stimulation of the cyclic AMP pathway including adenylate cyclase activity and cyclic AMP accumulation. In addition, exposure of SMC to cyclic strain caused a significant increase in protein kinase C (PKC) activity and enzyme translocation from the cytosol to a particulate fraction. Further study was conducted to examine the effect of strain magnitude on signaling, particularly protein kinase A (PKA) activity as well as cAMP response element (CRE) binding protein levels. We observed significantly (P < 0.05) greater PKA activity and CRE binding protein levels in SMC located in the center as compared to the peripheral region. However, inhibition of PKA (with 10 μM Rp-cAMP) or PKC (with 5–20 ng/ml staurosporine) failed to alter either the strain-induced increase in SMC proliferation or alignment. These data characterize the strain determinants for activation of SMC proliferation and alignment. Although strain activated both the AC/cAMP/PKA and the PKC pathways in SMC, singular inhibition of PKA and PKC failed to prevent strain-induced alignment and proliferation, suggesting either their lack of involvement or the multifactorial nature of these responses. J. Cell. Physiol. 170:228–234, 1997. © 1997 Wiley-Liss, Inc.