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The use of human pluripotent stem cells for in vitro disease modelling and clinical applications requires protocols that convert these cells into relevant adult cell types. Here, we report the rapid and efficient differentiation of human pluripotent stem cells into vascular endothelial and smooth muscle cells. We found that GSK3 inhibition and BMP4 treatment rapidly committed pluripotent cells to a mesodermal fate and subsequent exposure to VEGF-A or PDGF-BB resulted in the differentiation of either endothelial or vascular smooth muscle cells, respectively. Both protocols produced mature cells with efficiencies exceeding 80% within six days. On purification to 99% via surface markers, endothelial cells maintained their identity, as assessed by marker gene expression, and showed relevant in vitro and in vivo functionality. Global transcriptional and metabolomic analyses confirmed that the cells closely resembled their in vivo counterparts. Our results suggest that these cells could be used to faithfully model human disease. Cowan and colleagues report a method to generate mature endothelial or vascular smooth muscle cells from human pluripotent stem cells with high efficiency and purity.

Elevated blood glucose (hyperglycemia) is a hallmark metabolic abnormality in diabetes. Hyperglycemia is associated with protein kinase A (PKA)-mediated stimulation of L-type Ca2+ channels in arterial myocytes resulting in increased vasoconstriction. However, the mechanisms by which glucose activates PKA remain unclear. Here, we showed that elevating extracellular glucose stimulates cAMP production in arterial myocytes, and that this was specifically dependent on adenylyl cyclase 5 (AC5) activity. Super-resolution imaging suggested nanometer proximity between subpopulations of AC5 and the L-type Ca2+ channel pore-forming subunit CaV1.2. In vitro, in silico, ex vivo and in vivo experiments revealed that this close association is critical for stimulation of L-type Ca2+ channels in arterial myocytes and increased myogenic tone upon acute hyperglycemia. This pathway supported the increase in L-type Ca2+ channel activity and myogenic tone in two animal models of diabetes. Our collective findings demonstrate a unique role for AC5 in PKA-dependent modulation of L-type Ca2+ channel activity and vascular reactivity during acute hyperglycemia and diabetes.

Abstract Chronic obstructive pulmonary disease (COPD) is a multisystemic respiratory disease that is associated with progressive airway and pulmonary vascular remodeling due to the increased proliferation of bronchial smooth muscles cells (BSMCs) and pulmonary arterial smooth muscle cells (PASMCs) and the overproduction of extracellular matrix (e.g., collagen). Cigarette smoke (CS) and several mediators, such as PDGF (platelet-derived growth factor) and IL-6, play critical roles in COPD pathogenesis. HDAC6 has been shown to be implicated in vascular remodeling. However, the role of airway HDAC6 signaling in pulmonary vascular remodeling in COPD and the underlying mechanisms remain undetermined. Here, we show that HDAC6 expression is upregulated in the lungs of patients with COPD and a COPD animal model. We also found that CS extract (CSE), PDGF, and IL-6 increase the protein levels and activation of HDAC6 in BSMCs and PASMCs. Furthermore, CSE and these stimulants induced deacetylation and phosphorylation of ERK1/2 and increased collagen synthesis and BSMC and PASMC proliferation, which were outcomes that were prevented by HDAC6 inhibition. Inhibition of ERK1/2 also diminished the CSE-, PDGF-, and IL-6–caused elevation in collagen levels and cell proliferation. Pharmacologic HDAC6 inhibition with tubastatin A prevented the CS-stimulated increases in the thickness of the bronchial and pulmonary arterial wall, airway resistance, emphysema, and right ventricular systolic pressure and right ventricular hypertrophy in a rat model of COPD. These data demonstrate that the upregulated HDAC6 governs the collagen synthesis and BSMC and PASMC proliferation that lead to airway and vascular remodeling in COPD.

The burden of oxidative stress is increased in chronic obstructive pulmonary disease (COPD). However, whether the intra-cellular mechanisms controlling the oxidant/anti-oxidant balance in structural airway cells such as airway smooth muscle in COPD is altered is unclear. We sought to determine whether the expression of the NADPH oxidase (NOX)-4 is increased in airway smooth muscle in COPD both in vivo and primary cells in vitro and its role in hydrogen peroxide-induced reactive oxygen species generation. We found that in vivo NOX4 expression was up-regulated in the airway smooth muscle bundle in COPD ( n  = 9) and healthy controls with >20 pack year history ( n  = 4) compared to control subjects without a significant smoking history ( n  = 6). In vitro NOX4 expression was increased in airway smooth muscle cells from subjects with COPD ( n  = 5) compared to asthma ( n  = 7) and upregulated following TNF-α stimulation. Hydrogen peroxide-induced reactive oxygen species generation by airway smooth muscle cells in COPD ( n  = 5) was comparable to healthy controls ( n  = 9) but lower than asthma ( n  = 5); and was markedly attenuated by NOX4 inhibition. Our findings demonstrate that NOX4 expression is increased in vivo and in vitro in COPD and although we did not observe an intrinsic increase in oxidant-induced reactive oxygen species generation in COPD, it was reduced markedly by NOX4 inhibition supporting a potential therapeutic role for NOX4 in COPD.

Background Abdominal aortic aneurysms (AAA) are a critical global health issue with increasing prevalence. Dexmedetomidine (DEX) is a highly selective α2-adrenoceptor agonist that has previously been shown to play a protective role in AAA. Nevertheless, the mechanisms underlying its protection effect remain not fully understood. Methods A rat AAA model was established via intra-aortic porcine pancreatic elastase perfusion with or without DEX administration. The abdominal aortic diameters of rats were measured. Hematoxylin-eosin and Elastica van Gieson staining were conducted for histopathological observation. TUNEL and immunofluorescence staining were utilized to detect cell apoptosis and α-SMA/LC3 expression in the abdominal aortas. Protein levels were determined using western blotting. Results DEX administration repressed the dilation of aortas, alleviated pathological damage and cell apoptosis, and suppressed phenotype switching of vascular smooth muscle cells (VSMCs). Moreover, DEX activated autophagy and regulated the AMP-activated protein kinase/mammalian target of the rapamycin (AMPK/mTOR) signaling pathway in AAA rats. Administration of the AMPK inhibitor attenuated the DEX-mediated ameliorative effects on AAA in rats. Conclusion DEX ameliorates AAA in rat models by activating autophagy via the AMPK/mTOR pathway.

Asthma is associated with airway narrowing in response to bronchoconstricting stimuli and increased airway smooth muscle (ASM) mass. In addition, some studies have suggested impaired β-agonist induced ASM relaxation in asthmatics, but the mechanism is not known. To characterize the potential defect in β-agonist induced cAMP in ASM derived from asthmatic in comparison to non-asthmatic subjects and to investigate its mechanism. We examined β(2)-adrenergic (β(2)AR) receptor expression and basal β-agonist and forskolin (direct activator of adenylyl cyclase) stimulated cAMP production in asthmatic cultured ASM (n = 15) and non-asthmatic ASM (n = 22). Based on these results, PDE activity, PDE4D expression and cell proliferation were determined. In the presence of IBMX, a pan PDE inhibitor, asthmatic ASM had ∼50% lower cAMP production in response to isoproterenol, albuterol, formoterol, and forskolin compared to non-asthmatic ASM. However when PDE4 was specifically inhibited, cAMP production by the agonists and forskolin was normalized in asthmatic ASM. We then measured the amount and activity of PDE4, and found ∼2-fold greater expression and activity in asthmatic ASM compared to non-asthmatic ASM. Furthermore, inhibition of PDE4 reduced asthmatic ASM proliferation but not that of non-asthmatic ASM. Decreased β-agonist induced cAMP in ASM from asthmatics results from enhanced degradation due to increased PDE4D expression. Clinical manifestations of this dysregulation would be suboptimal β-agonist-mediated bronchodilation and possibly reduced control over increasing ASM mass. These phenotypes appear to be \"hard-wired\" into ASM from asthmatics, as they do not require an inflammatory environment in culture to be observed.

For vascular remodeling in hypertension, it is essential that vascular smooth muscle cells (VSMCs) reshape in order to proliferate and migrate. The extracellular matrix (ECM) needs to be degraded to favor VSMC migration. Many proteases, including matrix metalloproteinases (MMPs), contribute to ECM proteolysis and VSMC migration. Bioactive peptides, hemodynamic forces and reactive oxygen-nitrogen species regulate MMP-2 expression and activity. Increased MMP-2 activity contributes to hypertension-induced maladaptive arterial changes and sustained hypertension. New ECM is synthesized to supply VSMCs with bioactive mediators, which stimulate hypertrophy. MMP-2 stimulates the interaction of VSMCs with newly formed ECM, which triggers intracellular signaling via integrins to induce a phenotypic switch and persistent migration. VSMCs switch from a contractile to a synthetic phenotype in order to migrate and contribute to vascular remodeling in hypertension. MMPs also disrupt growth factors bound to ECM, thus contributing to their capacity to regulate VSMC migration. This review sheds light on the proteolytic effects of MMP-2 on ECM and non-ECM substrates in the vasculature and how these effects contribute to VSMC migration in hypertension. The inhibition of MMP activity as a therapeutic target may make it possible to reduce arterial maladaptation caused by hypertension and prevent the resulting fatal cardiovascular events.

Aortic aneurysm and dissection (AAD) is a cardiovascular disease that poses a severe threat to life and has high morbidity and mortality rates. Clinical and animal-based studies have irrefutably shown that fluoroquinolones, a commonly prescribed antibiotic for treating infections, significantly increase the risk of AAD. Despite this, the precise mechanism by which fluoroquinolones cause AAD remains unclear. Therefore, this study aims to investigate the molecular mechanism and role of Ciprofloxacin definitively—a type of fluoroquinolone antibiotic—in the progression of AAD. Aortic transcriptome data were collected from GEO datasets to detect the genes and pathways expressed differently between healthy donors and AAD patients. Human primary Vascular Smooth Muscle Cells (VSMCs) were isolated from the aorta. After 72 h of exposure to 110ug/ml Ciprofloxacin or 100 nmol/L AngII, either or combined, the senescent cells were identified through SA-β-gal staining. MitoTracker staining was used to examine the morphology of mitochondria in each group. Cellular Reactive Oxygen Species (ROS) levels were measured using MitoSox and DCFH-DA staining. Western blot assay was performed to detect the protein expression level. We conducted an analysis of transcriptome data from both healthy donors and patients with AAD and found that there were significant changes in cellular senescence-related signaling pathways in the latter group. We then isolated and identified human primary VSMCs from healthy donors (control-VSMCs) and patients' (AAD-VSMCs) aortic tissue, respectively. We found that VSMCs from patients exhibited senescent phenotype as compared to control-VSMCs. The higher levels of p21 and p16 and elevated SA-β-gal activity demonstrated this. We also found that pretreatment with Ciprofloxacin promoted angiotensin-II-induced cellular senescence in control-VSMCs. This was evidenced by increased SA-β-gal activity, decreased cell proliferation, and elevation of p21 and p16 protein levels. Additionally, we found that Angiotensin-II (AngII) induced VSMC senescence by promoting ROS generation. We used DCFH-DA and mitoSOX staining to identify that Ciprofloxacin and AngII pretreatment further elevated ROS levels than the vehicle or alone group. Furthermore, JC-1 staining showed that mitochondrial membrane potential significantly declined in the Ciprofloxacin and AngII combination group compared to others. Compared to the other three groups, pretreatment of Ciprofloxacin plus AngII could further induce mitochondrial fission, demonstrated by mitoTracker staining and western blotting assay. Mechanistically, we found that Ciprofloxacin impaired the balance of mitochondrial fission and fusion dynamics in VSMCs by suppressing the phosphorylation of AMPK signaling. This caused mitochondrial dysfunction and ROS generation, thereby elevating AngII-induced cellular senescence. However, treatment with the AMPK activator partially alleviated those effects. Our data indicate that Ciprofloxacin may accelerate AngII-induced VSMC senescence through modulating AMPK/ROS signaling and, subsequently, hasten the progression of AAD.

Background Nitrogen-containing bisphosphonate(N-BP)had been found to inhibit the osteogenic differentiation and calcification in vascular smooth muscle cells (VSMCs), but the mechanism is not clear. We intend to verify that N-BP induces enhancement of OPG expression and inhibition of RANKL expression via inhibition of farnesyl pyrophosphate synthase(FPPS) to inhibit the osteogenic differentiation and calcification in VSMCs. Methods β-glycerophosphate (β-GP) was used to induce the osteogenic differentiation and calcification in VSMCs. VSMCs were treated with N-BP or pretreated with downstream products of farnesyl pyrophosphate synthase(FPPS) in mevalonate pathway, such as farnesol (FOH) or geranylgeraniol (GGOH). Alizarin red S staining and determination of calcium content were used to detect calcium deposition.Western Blotting were used to detect expressions of proteins(OPG and RANKL ) and osteogenic marker proteins (Runx2 and OPN). Results β-GP induced the osteogenic differentiation and calcification in VSMCs, increased RANKL protein expression and had no significant effect on OPG protein expression. With the treatment of N-BP, the expression of OPG protein was increased and expression of RANKL protein was decreased in VSMCs undergoing osteogenic differentiation and calcification. In addition, N-BP reduced the osteogenic marker proteins (Runx2 and OPN) expression and calcium deposition in VSMCs undergoing osteogenic differentiation and calcification. These effects of N-BP on the osteogenic differentiation and calcification in VSMCs were concentration-dependent, which could be reversed by the downstream products of FPPS, such as FOH or GGOH. Conclusion N-BP increases OPG expression and decreases RANKL expression via inhibition of FPPS to inhibit the osteogenic differentiation and calcification in VSMCs.

Angiotensin II (AngII) induces the development of vascular hypertrophy and hypertension. We have shown previously that overexpression of class III deacetylase SIRT1 inhibits AngII-induced hypertrophy in vascular smooth muscle cells (VSMCs). However, the direct role of SIRT1 in VSMCs in response to AngII infusion in vivo remains unclear. Here, we found that the expression and activity of SIRT1 in mouse aortas was decreased significantly by AngII infusion. VSMC-specific SIRT1 transgene (SV-Tg) prevented the increase in systolic blood pressure (SBP) caused by AngII infusion without affecting heart function in mice. SIRT1 overexpression alleviated vascular remodeling in mouse thoracic and renal aortas induced by AngII infusion, and significantly inhibited reactive oxygen species (ROS) generation, vascular inflammation, and collagen synthesis in arterial walls. Reduced expression of transforming growth factor-β 1 (TGF-β1) was also observed in the aortas of AngII-infused SV-Tg mice. Moreover, SIRT1 overexpression decreased AngII-increased binding of nuclear factor-κB on its specific binding sites on TGF-β1 promoter. Taken together, these data demonstrate that SIRT1 overexpression in VSMCs reduces SBP and inhibits AngII-induced vascular remodeling in mice. The inhibition of vascular remodeling contributes, at least in part, to the antihypertensive effect of SIRT1. Key message SIRT1 is reduced in aortas of AngII-infused hypertensive mice. SIRT1 VSMC transgene alleviates AngII-increased systolic blood pressure. SIRT1 VSMC transgene attenuates AngII-induced vascular remodeling. VSMC SIRT1 overexpression inhibits remodeling-related pathological changes. VSMC SIRT1 overexpression reduces AngII-induced TGF-β1 expression.