Pyk2 inhibition promotes contractile differentiation in arterial smooth muscle.

Modulation from contractile to synthetic phenotype of vascular smooth muscle cells is a central process in disorders involving compromised integrity of the vascular wall. Phenotype modulation has been shown to include transition from voltage-dependent toward voltage-independent regulation of the intracellular calcium level, and inhibition of non-voltage dependent calcium influx contributes to maintenance of the contractile phenotype. One possible mediator of calcium-dependent signaling is the FAK-family non-receptor protein kinase Pyk2, which is activated by a number of stimuli in a calcium-dependent manner. We used the Pyk2 inhibitor PF-4594755 and Pyk2 siRNA to investigate the role of Pyk2 in phenotype modulation in rat carotid artery smooth muscle cells and in cultured intact arteries. Pyk2 inhibition promoted the expression of smooth muscle markers at the mRNA and protein levels under stimulation by FBS or PDGF-BB and counteracted phenotype shift in cultured intact carotid arteries and balloon injury ex vivo. During long-term (24-96 hr) treatment with PF-4594755, smooth muscle markers increased before cell proliferation was inhibited, correlating with decreased KLF4 expression and differing from effects of MEK inhibition. The Pyk2 inhibitor reduced Orai1 and preserved SERCA2a expression in carotid artery segments in organ culture, and eliminated the inhibitory effect of PDGF stimulation on L-type calcium channel and large-conductance calcium-activated potassium channel expression in carotid cells. Basal intracellular calcium level, calcium wave activity, and store-operated calcium influx were reduced after Pyk2 inhibition of growth-stimulated cells. Pyk2 inhibition may provide an interesting approach for preserving vascular smooth muscle differentiation under pathophysiological conditions.

Injury to rat carotid arteries causes time-dependent changes in gene expression in contralateral uninjured arteries.

Vascular surgery aimed at stenosis removal induces local reactions often leading to restenosis. Although extensive analysis has been focused on pathways activated in injured arteries, little attention has been devoted to associated systemic vascular reactions. The aim of the present study was to analyse changes occurring in contralateral uninjured rat carotid arteries in the acute phase following unilateral injury. WKY (Wistar-Kyoto) rats were subjected to unilateral carotid arteriotomy. Contralateral uninjured carotid arteries were harvested from 4 h to 7 days after injury. Carotid arteries were also harvested from sham-operated rats and uninjured rats. Carotid morphology and morphometry were examined. Affymetrix microarrays were used for differential analysis of gene expression. A subset of data was validated by real-time RT-PCR (reverse transcription-PCR) and verified at the protein level by Western blotting. A total of 1011 genes were differentially regulated in contralateral uninjured carotid arteries from 4 h to 7 days after arteriotomy (P<0.0001; fold change, >or=2) and were classified into 19 gene ontology functional categories. To a lesser extent, mRNA variations also occurred in carotid arteries of sham-operated rats. Among the changes, up-regulation of members of the RAS (renin-angiotensin system) was detected, with possible implications for vasocompensative mechanisms induced by arteriotomy. In particular, a selective increase in the 69 kDa isoform of the N-domain of ACE (angiotensin-converting enzyme), and not the classical somatic 195 kDa isoform, was observed in contralateral uninjured carotid arteries, suggesting that this 69 kDa isoenzyme could influence local AngII (angiotensin II) production. In conclusion, systemic reactions to injury occur in the vasculature, with potential clinical relevance, and suggest that caution is needed in the choice of controls during experimental design in vivo.

Ablation of SM22alpha decreases contractility and actin contents of mouse vascular smooth muscle.

The actin-binding protein SM22alpha marks contractile differentiation in smooth muscle, but its function is unknown. We tested its role in arterial contractility and stretch-sensitive vascular protein synthesis. Active stress in depolarised mesenteric resistance arteries and portal veins was reduced by 40% in SM22alpha(-/-) mice. Passive and active arterial circumference-force relationships were shifted leftwards, whereas alpha(1)-adrenergic responses were increased. Actin contents were 10-25% lower in vessels from SM22alpha(-/-) mice, but protein composition was otherwise similar. Synthesis of SM22alpha, calponin and alpha-actin, but not beta-actin, was sensitive to stretch. Ablation of SM22alpha did not affect stretch sensitivity of any of these proteins. Thus, SM22alpha plays a role in contractility, possibly by affecting actin filament organisation.

PYK2 selectively mediates signals for growth versus differentiation in response to stretch of spontaneously active vascular smooth muscle.

Stretch of vascular smooth muscle stimulates growth and proliferation as well as contraction and expression of contractile/cytoskeletal proteins, all of which are also regulated by calcium-dependent signals. We studied the role of the calcium- and integrin-activated proline-rich tyrosine kinase 2 (PYK2) in stretch-induced responses of the rat portal vein loaded by a hanging weight ex vivo. PYK2 phosphorylation at Tyr-402 was increased both by a 10-min stretch and by organ culture with load over several days. Protein and DNA synthesis were reduced by the novel PYK2 inhibitor PF-4594755 (0.5-1 µmol/L), while still sensitive to stretch. In 3-day organ culture, PF-4594755 caused maintained myogenic spontaneous activity but did not affect contraction in response to high-K(+) (60 mmol/L) or to α1-adrenergic stimulation by cirazoline. Basal and stretch-induced PYK2 phosphorylation in culture were inhibited by PF-4594755, closely mimicking inhibition of non-voltage-dependent calcium influx by 2-APB (30 µmol/L). In contrast, the L-type calcium channel blocker, nifedipine (1 µmol/L) eliminated stretch-induced but not basal PYK2 phosphorylation. Stretch-induced Akt and ERK1/2 phosphorylation was eliminated by PF-4594755. PYK2 inhibition had no effect on mRNA expression of several smooth muscle markers, and stretch-sensitive SM22α synthesis was preserved. Culture of portal vein with the Ang II inhibitor losartan (1 µmol/L) eliminated stretch sensitivity of PYK2 and Akt phosphorylation, but did not affect mRNA expression of smooth muscle markers. The results suggest that PYK2 signaling functionally distinguishes effects of voltage- and non-voltage-dependent calcium influx. A small-molecule inhibitor of PYK2 reduces growth and DNA synthesis but does not affect contractile differentiation of vascular smooth muscle.

Down-regulation of endothelial cell estrogen receptor expression by the inflammation promoter LPS.

Endothelial cells express both estrogen receptor (ER) alpha and beta. The objective of this study was to investigate if and how mediators of inflammation regulate endothelial cell ERalpha and ERbeta expression. ERalpha and ERbeta transcript and protein expression were determined by real-time quantitative PCR and Western blotting, respectively, in endothelial cell line bEnd.3 cells stimulated with the inflammation promoter lipopolysaccharide (E. coli LPS). Stimulation with LPS (500 ng/ml and 10 microg/ml) for 4 days reduced both ERalpha and ERbeta mRNA levels. The glucocorticoid dexamethasone (1 microM) had no effect on LPS-induced attenuation of ERalpha and beta transcript expression. Full-length 66-67 kDa ERalpha protein was unaffected by 4 days stimulation with LPS, while the 46-kDa ERalpha isoform was reduced by about 20%. ERbeta protein was reduced by about 40% by LPS at 4 days. Treatment with 17beta-estradiol (E(2), 100 nM) for 4 days increased ERbeta mRNA by about 8 times but had no effect on ERalpha mRNA level. The E(2)-induced increase in ERbeta transcript was not associated with increased ERbeta protein. E(2) increased ERbeta mRNA expression also in the presence of LPS, suggesting that inflammation-induced impairment of ERbeta signalling is rescued by estrogen.

Differential dependence of stretch and shear stress signaling on caveolin-1 in the vascular wall.

The role of caveolae in stretch- versus flow-induced vascular responses was investigated using caveolin 1-deficient [knockout (KO)] mice. Portal veins were stretched longitudinally for 5 min (acute) or 72 h (organ culture). Basal ERK1/2 and Akt phosphorylation were increased in organ-cultured KO veins, as were protein synthesis and vessel wall cross sections. Stretch stimulated acute phosphorylation of ERK1/2 and long-term phosphorylation of focal adhesion kinase (FAK) and cofilin but did not affect Akt phosphorylation. Protein synthesis, and particularly synthesis of smooth muscle differentiation markers, was increased by stretch. These effects did not differ in portal veins from KO and control mice, which also showed the same contractile response to membrane depolarization and inhibition by the Rho kinase inhibitor Y-27632. KO carotid arteries had increased wall cross sections and responded to pressurization (120 mmHg) for 1 h with increased ERK1/2 but not Akt phosphorylation, similar to control arteries. Shear stress by flow for 15 min, on the other hand, increased phosphorylation of Akt in carotids from control but not KO mice. In conclusion, caveolin 1 contributes to low basal ERK1/2 and Akt activity and is required for Akt-dependent signals in response to shear stress (flow) but is not essential for trophic effects of stretch (pressure) in the vascular wall.

Novel blocker of NFAT activation inhibits IL-6 production in human myometrial arteries and reduces vascular smooth muscle cell proliferation.

The calcineurin/nuclear factor of activated T cells (NFAT) signaling pathway has been found to play a role in regulating growth and differentiation in several cell types. However, the functional significance of NFAT in the vasculature is largely unclear. Here we show that NFATc1, NFATc3, and NFATc4 are expressed in human myometrial arteries. Confocal immunofluorescence and Western blot analysis revealed that endothelin-1 efficiently increases NFATc3 nuclear accumulation in native arteries. Endothelin-1 also stimulates NFAT-dependent transcriptional activity, as shown by a luciferase reporter assay. Both the agonist-induced NFAT nuclear accumulation and transcriptional activity were prevented by the calcineurin inhibitor CsA and by the novel NFAT blocker A-285222. Chronic inhibition of NFAT significantly reduced IL-6 production in intact myometrial arteries and inhibited cell proliferation in vascular smooth muscle cells cultured from explants from the same arteries. Furthermore, by using small interfering RNA-mediated reduction of NFATc3, we show that this isoform is involved in the regulation of cell proliferation. Protein synthesis in intact arteries was investigated using autoradiography of [(35)S]methionine incorporation in serum-free culture. Inhibition of NFAT signaling did not affect overall protein synthesis or specifically the synthesis rates of major proteins associated with the contractile/cytoskeletal system. An intact contractile phenotype under these conditions was also shown by unchanged force response to depolarization or agonist stimulation. Our results demonstrate NFAT expression and activation in native human vessels and point out A-285222 as a powerful pharmacological blocker of NFAT signaling in the vasculature.

Stretch of the vascular wall induces smooth muscle differentiation by promoting actin polymerization.

Stretch of the vascular wall by the intraluminal blood pressure stimulates protein synthesis and contributes to the maintenance of the smooth muscle contractile phenotype. The expression of most smooth muscle specific genes has been shown to be regulated by serum response factor and stimulated by increased actin polymerization. Hence we hypothesized that stretch-induced differentiation is promoted by actin polymerization. Intact mouse portal veins were cultured under longitudinal stress and compared with unstretched controls. In unstretched veins the rates of synthesis of several proteins associated with the contractile/cytoskeletal system (alpha-actin, calponin, SM22alpha, tropomyosin, and desmin) were dramatically lower than in stretched veins, whereas other proteins (beta-actin and heat shock proteins) were synthesized at similar rates. The cytoskeletal proteins gamma-actin and vimentin were weakly stretch-sensitive. Inhibition of Rho-associated kinase by culture of stretched veins with Y-27632 produced similar but weaker effects compared with the absence of mechanical stress. Induction of actin polymerization by jasplakinolide increased SM22alpha synthesis in unstretched veins to the level in stretched veins. Stretch stimulated Rho activity and phosphorylation of the actin-severing protein cofilin-2, although both effects were slow in onset (Rho-GTP, >15 min; cofilin-P, >1 h). Cofilin-2 phosphorylation of stretched veins was inhibited by Y-27632. The F/G-actin ratio after 24 h of culture was significantly greater in stretched than in unstretched veins, as shown by both ultracentrifugation and confocal imaging with phalloidin/DNase I labeling. The results show that stretch of the vascular wall stimulates increased actin polymerization, activating synthesis of smooth muscle-specific proteins. The effect is partially, but probably not completely, mediated via Rho-associated kinase and cofilin downstream of Rho.

Stretch-induced contractile differentiation of vascular smooth muscle: sensitivity to actin polymerization inhibitors.

Signaling mechanisms for stretch-dependent growth and differentiation of vascular smooth muscle were investigated in mechanically loaded rat portal veins in organ culture. Stretch-dependent protein synthesis was found to depend on endogenous release of angiotensin II. Autoradiography after [(35)S]methionine incorporation revealed stretch-dependent synthesis of several proteins, of which SM22 and actin were particularly prominent. Inhibition of RhoA activity by cell-permeant C3 toxin increased tissue mechanical compliance and reduced stretch-dependent extracellular signal-regulated kinase (ERK)1/2 activation, growth, and synthesis of actin and SM22, suggesting a role of the actin cytoskeleton. In contrast, inhibition of Rho-associated kinase by Y-27632 did not reduce ERK1/2 phosphorylation or actin and SM22 synthesis and did not affect tissue mechanical compliance but still inhibited overall growth. The actin polymerization inhibitors latrunculin B and cytochalasin D both inhibited growth and caused increased tissue compliance. Whereas latrunculin B concentration-dependently reduced actin and SM22 synthesis, cytochalasin D did so at low (10(-8) M) but not at high (10(-6) M) concentration. The results show that stretch stabilizes the contractile smooth muscle phenotype. Stretch-dependent differentiation marker expression requires an intact cytoskeleton for stretch sensing, control of protein expression via the level of unpolymerized G-actin, or both.