272 research outputs found
Endothelium-derived microparticles from chronically thromboembolic pulmonary hypertensive patients facilitate endothelial angiogenesis.
11 p.-4 fig.-1 tab.Background: Increased circulating levels of endoglin+ endothelial microparticles (EMPs) have been identified in several cardiovascular disorders, related to severity. Endoglin is an auxilary receptor for transforming growth factor Ī² (TGF-Ī²) important in the regulation of vascular structure.Results: We quantified the number of microparticles in plasma of six patients with chronic thromboembolic pulmonary hypertension (CTEPH) and age- and sex-matched pulmonary embolic (PE) and healthy controls and investigated the role of microparticle endoglin in the regulation of pulmonary endothelial function in vitro. Results show significantly increased levels of endoglin+ EMPs in CTEPH plasma, compared to healthy and disease controls. Co-culture of human pulmonary endothelial cells with CTEPH microparticles increased intracellular levels of endoglin and enhanced
TGF-Ī²-induced angiogenesis and Smad1,5,8 phosphorylation in cells, without affecting BMPRII expression. In an in vitro model, we generated endothelium-derived MPs with enforced membrane localization of endoglin. Co-culture of these MPs with endothelial cells increased cellular endoglin content, improved cell survival and stimulated
angiogenesis in a manner similar to the effects induced by overexpressed protein.Conclusions: Increased generation of endoglin+ EMPs in CTEPH is likely to represent a protective mechanism supporting endothelial cell survival and angiogenesis, set to counteract the effects of vascular occlusion and endothelial damage.This research was supported by a project grant (PG 11/13/28765) from the British Heart Foundation and by grants from Ministerio de Economia y Competitividad of Spain (SAF2013-43421-R to CB)Peer reviewe
The ADMA/DDAH Pathway Regulates VEGF-Mediated Angiogenesis
Objectivesā Asymmetrical dimethylarginine (ADMA) is a nitric oxide synthase (NOS) inhibitor and cardiovascular risk factor associated with angiogenic disorders. Enzymes metabolising ADMA, dimethylarginine dimethylaminohydrolases (DDAH) promote angiogenesis, but the mechanisms are not clear. We hypothesized that ADMA/DDAH modifies endothelial responses to vascular endothelial growth factor (VEGF) by affecting activity of Rho GTPases, regulators of actin polymerization, and focal adhesion dynamics.
Methods and Resultsā The effects of ADMA on VEGF-induced endothelial cell motility, focal adhesion turnover, and angiogenesis were studied in human umbilical vein endothelial cells (HUVECs) and DDAH I heterozygous knockout mice. ADMA inhibited VEGF-induced chemotaxis in vitro and angiogenesis in vitro and in vivo in an NO-dependent way. ADMA effects were prevented by overexpression of DDAH but were not associated with decreased proliferation, increased apoptosis, or changes in VEGFR-2 activity or expression. ADMA inhibited endothelial cell polarization, protrusion formation, and decreased focal adhesion dynamics, resulting from Rac1 inhibition after decrease in phosphorylation of vasodilator stimulated phosphoprotein (VASP). Constitutively active Rac1, and to a lesser extent dominant negative RhoA, abrogated ADMA effects in vitro and in vivo.
Conclusionā The ADMA/DDAH pathway regulates VEGF-induced angiogenesis in an NO- and Rac1-dependent manner
miR-150-PTPMT1-cardiolipin signaling in pulmonary arterial hypertension.
Circulating levels of endothelial miR-150 are reduced in pulmonary arterial hypertension (PAH) and act as an independent predictor of patient survival, but links between endothelial miR-150 and vascular dysfunction are not well understood. We studied the effects of endothelial miR-150 supplementation and inhibition in PAH mice and cells from patients with idiopathic PAH. The role of selected mediators of miR-150 identified by RNA sequencing was evaluated inĀ vitro and inĀ vivo. Endothelium-targeted miR-150 delivery prevented the disease in Sugen/hypoxia mice, while endothelial knockdown of miR-150 had adverse effects. miR-150 target genes revealed significant associations with PAH pathways, including proliferation, inflammation, and phospholipid signaling, with PTEN-like mitochondrial phosphatase (PTPMT1) most markedly altered. PTPMT1 reduced inflammation and apoptosis and improved mitochondrial function in human pulmonary endothelial cells and blood-derived endothelial colony-forming cells from idiopathic PAH. Beneficial effects of miR-150 inĀ vitro and inĀ vivo were linked with PTPMT1-dependent biosynthesis of mitochondrial phospholipid cardiolipin and reduced expression of pro-apoptotic, pro-inflammatory, and pro-fibrotic genes, including c-MYB, NOTCH3, transforming growth factor Ī² (TGF-Ī²), and Col1a1. In conclusion, we are the first to show that miR-150 supplementation attenuates pulmonary endothelial damage induced by vascular stresses and may be considered as a potential therapeutic strategy in PAH
Nanoscale Study of Calcium Handling Remodeling in Right Ventricular Cardiomyocytes Following Pulmonary Hypertension
Pulmonary hypertension is a complex disorder characterized by pulmonary vascular remodeling and right ventricular hypertrophy, leading to right heart failure. The mechanisms underlying this process are not well understood. We hypothesize that the structural remodeling occurring in the cardiomyocytes of the right ventricle affects the cytosolic Ca2+ handling leading to arrhythmias. After 12 days of monocrotaline-induced pulmonary hypertension in rats, epicardial mapping showed electrical remodeling in both ventricles. In myocytes isolated from the hypertensive rats, a combination of high-speed camera and confocal line-scan documented a prolongation of Ca2+ transients along with a higher local Ca2+-release activity. These Ca2+ transients were less synchronous than in controls, likely due to disorganized transverse-axial tubular system. In fact, following pulmonary hypertension, hypertrophied right ventricular myocytes showed significantly reduced number of transverse tubules and increased number of axial tubules; however, Stimulation Emission Depletion microscopy demonstrated that the colocalization of L-type Ca2+ channels and RyR2 (ryanodine receptor 2) remained unchanged. Finally, Stimulation Emission Depletion microscopy and super-resolution scanning patch-clamp analysis uncovered a decrease in the density of active L-type Ca2+ channels in right ventricular myocytes with an elevated open probability of the T-tubule anchored channels. This may represent a general mechanism of how nanoscale structural changes at the early stage of pulmonary hypertension impact on the development of the end stage failing phenotype in the right ventricle
Effect of short-term food restriction on iron metabolism, relative well-being and depression symptoms in healthy women
Tipifarnib prevents development of hypoxia-induced pulmonary hypertension
Aims. RhoB plays a key role in the pathogenesis of hypoxia - induced pulmonary hypertension. Farne sylated RhoB promotes growth responses in cancer cells and we investigated whether inhibition of protein farnesylation will have a protective effect. Methods and Results. The analysis of l ung tissues from rodent models and pulmonary hypertensive patients showed increased levels of protein farnesylation. Oral farnesyltransferase inhibitor tipifarnib prevented development of hypoxia - induced pulmonary hypertension in mice. Tipifarnib reduced hypoxia - induced vascular cell proliferation, increased endothelium - dependent vasodilatation and reduced vasoconstriction of intrapulmonary arteries without affecting cell viability. Protective effects of tipifarnib were associated with inhibition of Ras and RhoB, actin depolymerisation and increased eNOS expression in vi tro and in vivo . Farnesylated - only RhoB (F - RhoB) increased proliferative responses in cultured pulmonary vascular cells, mimicking the effects of hypoxia, while both geranylgeranylated - only RhoB (GG - RhoB) and tipifarnib had an inhibitory effect. Label - fre e proteomics linked F - RhoB with cell survival, activation of cell cycle and mitochondrial biogenesis. Hypoxia increased and tipifarnib reduced the levels of F - RhoB - regulated proteins in the lung, reinforcing the importance of RhoB as a signalling mediator. Unlike simvastatin, tipifarnib did not increase the expression levels of Rho proteins. Conclusions. Our study demonstrates the importance of protein farnesylation in pulmonary vascular remodeling and provides a rationale for selective targeting of this pa thway in pulmonary hypertension
Vasodilator Phosphostimulated Protein (VASP) Protects Endothelial Barrier Function During Hypoxia
The endothelial barrier controls the passage of solutes from the vascular space. This is achieved through active reorganization of the actin cytoskeleton. A central cytoskeletal protein involved into this is vasodilator-stimulated phosphoprotein (VASP). However, the functional role of endothelial VASP during hypoxia has not been thoroughly elucidated. We determined endothelial VASP expression through real-time PCR (Rt-PCR), immunhistochemistry, and Western blot analysis during hypoxia. VASP promoter studies were performed using a PGL3 firefly luciferase containing plasmid. Following approval by the local authorities, VASPā/ā mice and littermate controls were subjected to normobaric hypoxia (8% O2, 92% N2) after intravenous injection of Evans blue dye. In in vitro studies, we found significant VASP repression in human microvascular and human umbilical vein endothelial cells through Rt-PCR, immunhistochemistry, and Western blot analysis. The VASP promoter construct demonstrated significant repression in response to hypoxia, which was abolished when the binding of hypoxia-inducible factor 1 alpha was excluded. Exposure of wild-type (WT) and VASPā/ā animals to normobaric hypoxia for 4Ā h resulted in an increase in Evans blue tissue extravasation that was significantly increased in VASPā/ā animals compared to WT controls. In summary, we demonstrate here that endothelial VASP holds significant importance for endothelial barrier properties during hypoxia
RalA interacts with ZONAB in a cell density-dependent manner and regulates its transcriptional activity
Therapeutic potential of KLF2-induced exosomal microRNAs in pulmonary hypertension
Pulmonary arterial hypertension (PAH) is a severe disorder of lung vasculature that causes right heart failure. Homeostatic effects of flow-activated transcription factor KrĆ¼ppel-like factor 2 (KLF2) are compromised in PAH. Here we show that KLF2-induced exosomal microRNAs, miR-181a-5p and miR-324-5p act together to attenuate pulmonary vascular remodeling and that their actions are mediated by Notch4 and ETS1 and other key regulators of vascular homeostasis. Expressions of KLF2, miR-181a-5p and miR-324-5p are reduced, while levels of their target genes are elevated in pre-clinical PAH, idiopathic PAH and heritable PAH with missense p.H288Y KLF2 mutation. Therapeutic supplementation of miR-181a-5p and miR-324-5p reduces proliferative and angiogenic responses in patient-derived cells and attenuates disease progression in PAH mice. This study shows that reduced KLF2 signaling is a common feature of human PAH and highlights the potential therapeutic role of KLF2-regulated exosomal miRNAs in PAH and other diseases associated with vascular remodelling
Deficiency of Vasodilator-Stimulated Phosphoprotein (VASP) Increases Blood-Brain-Barrier Damage and Edema Formation after Ischemic Stroke in Mice
Background: Stroke-induced brain edema formation is a frequent cause of secondary infarct growth and deterioration of neurological function. The molecular mechanisms underlying edema formation after stroke are largely unknown. Vasodilator-stimulated phosphoprotein (VASP) is an important regulator of actin dynamics and stabilizes endothelial barriers through interaction with cell-cell contacts and focal adhesion sites. Hypoxia has been shown to foster vascular leakage by downregulation of VASP in vitro but the significance of VASP for regulating vascular permeability in the hypoxic brain in vivo awaits clarification. Methodology/Principal Findings: Focal cerebral ischemia was induced in Vasp2/2 mice and wild-type (WT) littermates by transient middle cerebral artery occlusion (tMCAO). Evanās Blue tracer was applied to visualize the extent of blood-brainbarrier (BBB) damage. Brain edema formation and infarct volumes were calculated from 2,3,5-triphenyltetrazolium chloride (TTC)-stained brain slices. Both mouse groups were carefully controlled for anatomical and physiological parameters relevant for edema formation and stroke outcome. BBB damage (p,0.05) and edema volumes (1.7 mm360.5 mm3 versus 0.8 mm360.4 mm3; p,0.0001) were significantly enhanced in Vasp2/2 mice compared to controls on day 1 after tMCAO. This was accompanied by a significant increase in infarct size (56.1 mm3617.3 mm3 versus 39.3 mm3610.7 mm3, respectively; p,0.01) and a non significant trend (p.0.05) towards worse neurological outcomes. Conclusion: Our study identifies VASP as critical regulator of BBB maintenance during acute ischemic stroke. Therapeutic modulation of VASP or VASP-dependent signalling pathways could become a novel strategy to combat excessive edema formation in ischemic brain damage
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