Lovastatin Induces Relaxation and Inhibits L-Type Ca2+ Current in the Rat Basilar Artery.

Statins inhibit cholesterol biosynthesis and protect against ischaemic stroke. It has become increasingly apparent that the beneficial effects of statin therapy may extend beyond lowering of serum cholesterol. The present study was done to explore possible pleiotropic statin effects at the level of the cerebral vascular smooth muscle. Lovastatin, lovastatin acid, simvastatin and pravastatin, were added to segments of the rat basilar artery and effects on contraction and Ca2+ handling were examined. Pravastatin had no effect on contraction. Simvastatin, lovastatin, and, to a lesser degree, lovastatin acid, caused relaxation (IC50=0.8, 1.9 and 22 μmol/l) of both intact and denuded arteries precontracted with 5-HT or high-K+. This effect was not reversed by mevalonate, suggesting that it was not related to cholesterol or isoprenoid metabolism. Relaxation was associated with a reduction of the intracellular Ca2+ concentration measured with Fura 2 and with a reduced Mn2+ quench rate, suggesting a direct effect on ion channels in the smooth muscle cell membrane. Current measurements in isolated and voltage clamped basilar artery muscle cells demonstrated that both lovastatin and lovastatin acid inhibit L-type Ca2+ current. We propose that lipophilicity is an important factor behind the effects of statins on vascular tone and that Ca2+ current inhibition is the likely mechanism of action

Regulation of Ca2+ channel and phosphatase activities by polyamines in intestinal and vascular smooth muscle - implications for cellular growth and contractility.

Polyamines added extracellularly to intestinal and vascular smooth muscle cells cause relaxation through inhibition of Ca2+ channel activity. Intracellularly applied polyamines also affect Ca2+ channel properties. Polyamines do not readily pass over the plasma membrane because of their positive charges but in permeabilized smooth muscle preparations they have free access to the cytoplasm. In this system they increase sensitivity of the contractile machinery to Ca2+ through inhibition of myosin phosphatase activity. The magnitude of Ca2+ channel and phosphatase inhibition depends on the number of positive charges on the polyamine molecule. Polyamines have an obligatory, but yet undefined, role in regulation of cell growth and proliferation. Several groups of protein kinases, such as tyrosine and mitogen activated protein (MAP)-kinases transmit the growth signal from the plasma membrane to the cell nucleus where mitosis and protein synthesis are initiated. The data reviewed here show that polyamines may affect such signal transmission via inhibition of phosphatase activity

Expression of microRNAs is essential for arterial myogenic tone and pressure-induced activation of the PI3-kinase/Akt pathway.

The myogenic response is the intrinsic ability of small arteries to constrict in response to increased intraluminal pressure. Although microRNAs have been shown to play a role in vascular smooth muscle function, their importance in the regulation of the myogenic response is not known. In this study, we investigate the role of microRNAs in the regulation of myogenic tone by using smooth muscle-specific and tamoxifen-inducible deletion of the endonuclease Dicer in mice

The Polyamine Pathway as a Potential Target for Vascular Diseases: Focus on Restenosis

Polyamines are organic polycations expressed by all living organisms, which are known to play an essential role in cell proliferation and differentiation. Recent studies revealed their involvement also in cell contractility and migration and in programmed cell death. These processes are known to contribute to restenosis, a pathophysiological process occurring in 10-20% of patients submitted to revascularization procedures. The advent of bare metal stents and of drug-eluting stents has significantly reduced but not eliminated the incidence of restenosis, which thus remains a clinically relevant problem. Despite the potential role of the polyamine pathway as a therapeutic target due to its involvement in proliferation, apoptosis and migration of vascular cells, experimental inhibition of polyamine synthesis and/or uptake has been poorly investigated in animal models of vascular disease. Here we review the current knowledge about molecular mechanisms related to polyamine functions, with particular reference to the role played by polyamines in vascular cell pathophysiology, together with experimental evidence obtained so far in animal models of (re) stenosis. We also evaluate the advantages of different routes of administration of polyamine synthesis/transport inhibitors and polyamine analogue molecules. Increasing knowledge about the molecular mechanisms and functions of polyamines is expected to shed new light on their potential role as a therapeutic target for restenosis reduction

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

The G Protein-Coupled Estrogen Receptor 1 (GPER1/GPR30) Agonist G-1 Regulates Vascular Smooth Muscle Cell Ca Handling.

The G protein-coupled estrogen receptor GPER1/GPR30 is implicated in blood pressure regulation but the mechanisms are not identified. Here, we hypothesize that GPER1 controls blood pressure by regulating vascular smooth muscle cell Ca(2+) handling. Treatment with the GPER1 agonist G-1 (in the µM concentration range) acutely reduced spontaneous and synchronous Ca(2+) spike activity in A7r5 vascular smooth muscle cells expressing mRNA for GPER1. Furthermore, G-1 (1 µM) attenuated the thromboxane A2 analogue U46619-stimulated Ca(2+) spike activity but had no effect on the U46619-induced increase in the basal level of Ca(2+). The voltage-sensitive L-type Ca(2+) channel blocker nifedipine (100 nM) reduced Ca(2+) spike activity similar to G-1. Pharmacological, but not physiological, concentrations of the estrogen 17β-estradiol reduced Ca(2+) spike activity. The GPER1 antagonist G-15 blocked G-1-induced downregulation of Ca(2+) spike activity, supporting a GPER1-dependent mechanism. G-1 (1 µM) and nifedipine (100 nM) attenuated the 30-mM KCl-evoked rise in intracellular Ca(2+) concentration, suggesting that G-1 blocks inflow of Ca(2+) via voltage-sensitive Ca(2+) channels. In conclusion, we demonstrate that the GPER1 agonist G-1 regulates vascular smooth muscle cell Ca(2+) handling by lowering Ca(2+) spike activity, suggesting a role for this mechanism in GPER1-mediated control of blood pressure. © 2013 S. Karger AG, Basel

Regulation of microRNA expression in vascular smooth muscle by MRTF-A and actin polymerization

Producción CientíficaThe dynamic properties of the actin cytoskeleton in smooth muscle cells play an important role in a number of cardiovascular disease states. The state of actin does not only mediate mechanical stability and contractile function but can also regulate gene expression via myocardin related transcription factors (MRTFs). These transcriptional co-activators regulate genes encoding contractile and cytoskeletal proteins in smooth muscle. Regulation of small non-coding microRNAs (miRNAs) by actin polymerization may mediate some of these effects. MiRNAs are short non-coding RNAs that modulate gene expression by post-transcriptional regulation of target messenger RNA. In this study we aimed to determine a profile of miRNAs that were 1) regulated by actin/MRTF-A, 2) associated with the contractile smooth muscle phenotype and 3) enriched in muscle cells.The Swedish Research Council (grant 2012-2197)The Crafoord Foundation (grant 20150629)Instituto de Salud Carlos III (grant RD12/0042/0006)Ministerio de Economía, Industria y Competitividad (grant BFU2013-45867-R

Local inhibition of ornithine decarboxylase reduces vascular stenosis in a murine model of carotid injury

Objectives: Polyamines are organic polycations playing an essential role in cell proliferation and differentiation, as well as in cell contractility, migration and apoptosis. These processes are known to contribute to restenosis, a pathophysiological process often occurring in patients submitted to revascularization procedures. We aimed to test the effect of alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, on vascular cell pathophysiology in vitro and in a rat model of carotid arteriotomy-induced (re) stenosis. Methods: The effect of DFMO on primary rat smooth muscle cells (SMCs) and mouse microvascular bEnd. 3 endothelial cells (ECs) was evaluated through the analysis of DNA synthesis, polyamine concentration, cell viability, cell cycle phase distribution and by RT-PCR targeting cyclins and genes belonging to the polyamine pathway. The effect of DFMO was then evaluated in arteriotomy-injured rat carotids through the analysis of cell proliferation and apoptosis, RT-PCR and immunohistochemical analysis of differential gene expression. Results: DFMO showed a differential effect on SMCs and on ECs, with a marked, sustained anti-proliferative effect of DFMO at 3 and 8 days of treatment on SMCs and a less pronounced, late effect on bEnd. 3 ECs at 8 days of DFMO treatment. DFMO applied perivascularly in pluronic gel at arteriotomy site reduced subsequent cell proliferation and preserved smooth muscle differentiation without affecting the endothelial coverage. Lumen area in DFMO-treated carotids was 49% greater than in control arteries 4 weeks after injury. Conclusions: Our data support the key role of polyamines in restenosis and suggest a novel therapeutic approach for this pathophysiological process. (C) 2013 Elsevier Ireland Ltd. All rights reserved

Regulation of smooth muscle dystrophin and synaptopodin 2 expression by actin polymerization and vascular injury

Producción CientíficaObjective: Actin dynamics in vascular smooth muscle is known to regulate contractile differentiation and may play a role in the pathogenesis of vascular disease. However, the list of genes regulated by actin polymerization in smooth muscle remains incomprehensive. Thus, the objective of this study was to identify actin-regulated genes in smooth muscle and to demonstrate the role of these genes in the regulation of vascular smooth muscle phenotype. Approach and Results: Mouse aortic smooth muscle cells were treated with an actin-stabilizing agent, jasplakinolide, and analyzed by microarrays. Several transcripts were upregulated including both known and previously unknown actin-regulated genes. Dystrophin and synaptopodin 2 were selected for further analysis in models of phenotypic modulation and vascular disease. These genes were highly expressed in differentiated versus synthetic smooth muscle and their expression was promoted by the transcription factors myocardin and myocardin-related transcription factor A. Furthermore, the expression of both synaptopodin 2 and dystrophin was significantly reduced in balloon-injured human arteries. Finally, using a dystrophin mutant mdx mouse and synaptopodin 2 knockdown, we demonstrate that these genes are involved in the regulation of smooth muscle differentiation and function. Conclusions: This study demonstrates novel genes that are promoted by actin polymerization, that regulate smooth muscle function, and that are deregulated in models of vascular disease. Thus, targeting actin polymerization or the genes controlled in this manner can lead to novel therapeutic options against vascular pathologies that involve phenotypic modulation of smooth muscle cells.Instituto de Salud Carlos III - Fondo Europeo de Desarrollo Regional (grant RD12/0042/0006)Ministerio de Economía, Industria y Competitividad (grants BFU2010-15898 and BFU2013-45867-R

Deficiency of SARS-CoV-2 T-cell responses after vaccination in long-term allo-HSCT survivors translates into abated humoral immunity

Recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for hematological diseases are at risk of severe disease and death from COVID-19. To determine the safety and immunogenicity of BNT162b2 and mRNA-1273 COVID-19 vaccines, samples from 50 infection-naive allo-HSCT recipients (median, 92 months from transplantation, range, 7-340 months) and 39 healthy controls were analyzed for serum immunoglobulin G (IgG) against the receptor binding domain (RBD) within spike 1 (S1) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; anti–RBD-S1 IgG) and for SARS-CoV-2–specific T-cell immunity, reflected by induction of T-cell–derived interferon-g in whole blood stimulated ex vivo with 15-mer SI-spanning peptides with 11 amino acid overlap S1-spanning peptides. The rate of seroconversion was not significantly lower in allo-transplanted patients than in controls with 24% (12/50) and 6% (3/50) of patients remaining seronegative after the first and second vaccination, respectively. However, 58% of transplanted patients lacked T-cell responses against S1 peptides after 1 vaccination compared with 19% of controls (odds ratio [OR] 0.17; P 5 .009, Fisher’s exact test) with a similar trend after the second vaccination where 28% of patients were devoid of detectable specific T-cell immunity, compared with 6% of controls (OR 0.18; P 5 .02, Fisher’s exact test). Importantly, lack of T-cell reactivity to S1 peptides after vaccination heralded substandard levels (,100 BAU/mL) of anti–RBD-S1 IgG 5 to 6 months after the second vaccine dose (OR 8.2; P 5 .007, Fisher’s exact test). We conclude that although allo-HSCT recipients achieve serum anti–RBD-S1 IgG against SARS-CoV-2 after 2 vaccinations, a deficiency of SARS-CoV-2–specific T-cell immunity may subsequently translate into insufficient humoral responses