Inhibition of SOC/Ca2+/NFAT pathway is involved in the anti-proliferative effect of sildenafil on pulmonary artery smooth muscle cells

<p>Abstract</p> <p>Background</p> <p>Sildenafil, a potent phosphodiesterase type 5 (PDE5) inhibitor, has been proposed as a treatment for pulmonary arterial hypertension (PAH). The mechanism of its anti-proliferative effect on pulmonary artery smooth muscle cells (PASMC) is unclear. Nuclear translocation of nuclear factor of activated T-cells (NFAT) is thought to be involved in PASMC proliferation and PAH. Increase in cytosolic free [Ca²⁺] ([Ca²⁺]_i) is a prerequisite for NFAT nuclear translocation. Elevated [Ca²⁺]_iin PASMC of PAH patients has been demonstrated through up-regulation of store-operated Ca²⁺channels (SOC) which is encoded by the transient receptor potential (TRP) channel protein. Thus we investigated if: 1) up-regulation of TRPC1 channel expression which induces enhancement of SOC-mediated Ca²⁺influx and increase in [Ca²⁺]_iis involved in hypoxia-induced PASMC proliferation; 2) hypoxia-induced promotion of [Ca²⁺]_ileads to nuclear translocation of NFAT and regulates PASMC proliferation and TRPC1 expression; 3) the anti-proliferative effect of sildenafil is mediated by inhibition of this SOC/Ca²⁺/NFAT pathway.</p> <p>Methods</p> <p>Human PASMC were cultured under hypoxia (3% O₂) with or without sildenafil treatment for 72 h. Cell number and cell viability were determined with a hemocytometer and MTT assay respectively. [Ca²⁺]_iwas measured with a dynamic digital Ca²⁺imaging system by loading PASMC with fura 2-AM. TRPC1 mRNA and protein level were detected by RT-PCR and Western blotting respectively. Nuclear translocation of NFAT was determined by immunofluoresence microscopy.</p> <p>Results</p> <p>Hypoxia induced PASMC proliferation with increases in basal [Ca²⁺]_iand Ca²⁺entry via SOC (SOCE). These were accompanied by up-regulation of TRPC1 gene and protein expression in PASMC. NFAT nuclear translocation was significantly enhanced by hypoxia, which was dependent on SOCE and sensitive to SOC inhibitor SKF96365 (SKF), as well as cGMP analogue, 8-brom-cGMP. Hypoxia-induced PASMC proliferation and TRPC1 up-regulation were inhibited by SKF and NFAT blocker (VIVIT and Cyclosporin A). Sildenafil treatment ameliorated hypoxia-induced PASMC proliferation and attenuated hypoxia-induced enhancement of basal [Ca²⁺]_i, SOCE, up-regulation of TRPC1 expression, and NFAT nuclear translocation.</p> <p>Conclusion</p> <p>The SOC/Ca²⁺/NFAT pathway is, at least in part, a downstream mediator for the anti-proliferative effect of sildenafil, and may have therapeutic potential for PAH treatment.</p

Diffusion-weighted MR Imaging in the evaluation of normal appearing white matter in patients with tuberous sclerosis

The aim of the present study was to assess the presence of changes in diffusivity in the brains of patients with tuberous sclerosis (TS) in areas free from signal alterations on conventional MR sequences. Ten patients and controls were evaluated. ROI were placed on ADC maps in the white matter (frontal, parietal and centrum semiovale) excluding areas with signal alterations on FLAIR images. ADC values of supratentorial white matter turned out to be higher than controls (P < .0001). Our data indicate that TS is more widely diffused than expected in the brains of affected patients

Lezioni di Neuroradiologia

Corso didattico per studenti di medicina e specializzandi di neurologia e radiologia. Il libro illustra le principali metodiche neuroradiologiche sia diagnostiche che terapeutiche (neuroradiologia interventistica) e le principali applicazioni nelle malattie di interesse neurologico e neurochirurgico

Early angiographic and CT findings in patients with hemorrhagic infarction in the distribution of the middle cerebral artery.

Hemorrhagic infarction subsequent to ischemic brain damage, even if small, slight, or marbled, can be detected by CT. The mechanisms that give rise to this transformation in humans are not well elucidated. Previous reports indicate that hemorrhagic infarction is most common in embolic stroke and large infarcts, and can worsen the clinical state of ischemic patients. We examined 36 patients with supratentorial ischemic signs and symptoms within the first hours after onset. CT was used to judge if hypodensity on early CT studies might predict the development of hemorrhagic infarction. Angiography was used to observe the site of arterial occlusion, the state of collateral circulation, and the mechanisms of late reperfusion. Hemorrhagic infarction was present in 18 of our 36 patients. Angiography revealed occlusion of the middle cerebral artery or internal carotid artery (three cases) in all patients. Hypodensity was present on early CT studies in all of the 18 patients who developed hemorrhagic infarction. The finding of hypodensity on CT studies performed soon after embolic ischemic stroke is strongly predictive of hemorrhagic transformation