Physiological adaptation of normal blood vessels to acute or chronic changes in blood flow is endothelium dependent. In vitro studies have shown that, among other genes, NO synthase (NOS) 3 mRNA and protein expression is enhanced by acute elevation of shear stress in endothelial cells. We have investigated the effect of chronic high blood flow on NOS3 mRNA and protein expression in rat aorta. NOS3 mRNA levels were measured by quantitative polymerase chain reaction (PCR) in the aortas of 12 rats with arteriovenous fistulas and 9 sham-operated control rats. The PCR assay indicated that NOS3 mRNA levels were significantly enhanced (twofold) during high blood flow. Western blots showed that immunoreactive NOS3 levels were also increased to a similar extent. Furthermore, the Ca(2+)-dependent NOS activity, measured by the L-arginine to L-citrulline conversion assay, and the cGMP content were also significantly increased in the proximal aortic wall submitted to the arteriovenous shunt. These results indicate that NOS3 mRNA and protein expression is enhanced in vivo during chronic high blood flow.

Mammalian rRNA genes are preceded by a terminator element that is recognized by the transcription termination factor TTF‐I. In exploring the functional significance of the promoter‐proximal terminator, we found that TTF‐I associates with the p300/CBP‐associated factor PCAF, suggesting that TTF‐I may target histone acetyltransferase to the rDNA promoter. We demonstrate that PCAF acetylates TAFI68, the second largest subunit of the TATA box‐binding protein (TBP)‐containing factor TIF‐IB/SL1, and acetylation enhances binding of TAFI68 to the rDNA promoter. Moreover, PCAF stimulates RNA polymerase I (Pol I) transcription in a reconstituted in vitro system. Consistent with acetylation of TIF‐IB/SL1 being required for rDNA transcription, the NAD+‐dependent histone deacetylase mSir2a deacetyl ates TAFI68 and represses Pol I transcription. The results demonstrate that acetylation of the basal Pol I transcription machinery has functional consequences and suggest that reversible acetylation of TIF‐IB/SL1 may be an effective means to regulate rDNA transcription in response to external signals.

ERSpublicationsGene panel sequencing unravels the genetic architecture of pulmonary hypertension in adult and paediatric cases, emphasises the importance of BMPR2, EIF2AK4, BMP9 and TBX4 mutations, and suggests BMP10 as a new gene for the disease http://ow.ly/Oxes30mXnrI

The human endothelial nitric-oxide synthase gene (heNOS) is constitutively expressed in endothelial cells, and its expression is induced under hypoxia. The goal of this study was to search for regulatory elements of the endothelial nitric-oxide synthase (eNOS) gene responsive to hypoxia. Levels of eNOS mRNA, measured by real time reverse transcriptase-PCR analysis, were increased, and heNOS promoter activity was enhanced by hypoxia as compared with normoxia control experiments. Promoter truncation followed by footprint analysis allowed the mapping and identification of the hypoxia-responsive elements at position ؊5375 to ؊5366, closely related to hypoxia-inducible factor (HIF)-responsive element (HRE). To test whether known HIF-1 and HIF-2 are involved in hypoxia-induced heNOS promoter activation, HMEC-1 and HUVEC were transiently transfected with HIF-1␣ and HIF-1␤ or HIF-2␣ and HIF-1␤ expression vectors. Exogenous HIF-2 markedly increased luciferase reporter activity driven by the he-NOS promoter in its native location. The induction of luciferase was conserved with the antisense construct and was increased in cotransfection experiments when this fragment was cloned 5 to the proximal 785-bp fragment of the eNOS promoter. Deletion analysis and sitedirected mutagenesis demonstrated that the two contiguous HIF consensus binding sites spanning bp ؊5375 to ؊5366 relative to the transcription start site were both functional for heNOS promoter activity induction by hypoxia and by HIF-2 overexpression. In conclusion, we demonstrate that heNOS is a hypoxia-inducible gene, whose transcription is stimulated through HIF-2 interaction with two contiguous HRE sites located at ؊5375 to ؊5366 of the heNOS promoter.The endothelial isoform of nitric-oxide synthase (eNOS) 1 produces nitric oxide (NO) in the endothelium by converting arginine and oxygen into citrulline and NO (1-3). NO rapidly diffuses to vascular smooth muscle cells and stimulates guanosine 3Ј,5Ј-cyclic monophosphate formation, which leads to vasodilation. The enzymatic production of NO in endothelial cells relies on the constitutively expressed eNOS whose activity is regulated by two major pathways, calcium/calmodulin binding or phosphorylation by AKT (4, 5). In addition, different physiological and pathological conditions can modulate the level of eNOS expression in endothelial cells (6) by acting at both the transcriptional (7, 8) and post-transcriptional levels (9 -11).In response to hypoxia, systemic arteries vasodilate, allowing more blood to be delivered to peripheral tissues. This vasodilation occurs within seconds after hypoxia and is maintained for hours (12). The molecular basis of hypoxic vasodilation is not fully understood. Possible mechanisms include direct relaxation of vascular smooth muscle cells induced by changes in pH and ion channel conductance or a decrease in ATP levels (13). Endothelial cells also reduce the amount of released vasoconstrictors such as endothelin or thromboxane and release increased amounts of vasodilators such as adenosin...

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Pulmonary arterial hypertension (PAH) is a rare and devastating disease, resulting from progressive obliteration of small caliber pulmonary arteries by proliferating vascular cells, and leading to cardiac failure, with an untreated mean survival of less than three years 1,2. PAH can complicate other pathological conditions, or can occur in the context of genetic mutations causing heritable PAH, or can be considered as idiopathic (iPAH), which represents approximately 40% of all PAH 3,4. Low penetrance dominant BMPR2 mutations are found in ~70% of familial PAH (fPAH), and in ~15% of iPAH which are thereafter considered as heritable PAH 5,6. We conducted a Genome-Wide Association Study (GWAS) based on two independent case-control studies for iPAH and fPAH (without BMPR2 mutations) totaling 625 patients and 1,525 healthy individuals, to identify novel genetic factors associated with iPAH and fPAH (i/fPAH) in the absence of BMPR2 mutations. A genome wide significant association was detected at the CBLN2 locus mapping to 18q22.3, the risk allele being associated with an odds ratio for i/fPAH of 1.97 [1.59 – 2.45] (P = 7.47 x 10−10). CBLN2 is expressed in the lung, particularly in pulmonary vascular endothelial cells, and its expression is increased in explanted lungs from PAH patients and in endothelial cells cultured from explanted PAH lungs.

SummaryAging is the main risk factor for cardiovascular diseases, but the associated molecular mechanisms are poorly understood. The Wnt signaling pathway was shown to be induced during aging in muscle and in the skin, but the regulation and role of Wnt signaling in the aged vessel have not yet been addressed. While screening for age‐related changes in gene expression in the intima/media of human mammary arteries, we observed that the expression of frizzled 4 (Fzd4), a Wnt receptor, and of several targets of the Wnt/β‐catenin/TCF signaling pathway [Wnt‐inducible secreted protein 1 (WISP1), versican, osteopontin (SPP1), insulin‐like growth factor binding protein 2 (IGFBP‐2), and p21] were modified with age, suggesting an activation of the Wnt/β‐catenin pathway. In contrast, we did not observe any regulation of forkhead transcription factor (FoxO) target genes. Beta‐catenin‐activating phosphorylation at position Ser675 was increased in aging mammary arteries, confirming the activation of this pathway. We confirmed in vitro that Wnt3a or Wnt1 treatment of human vascular smooth muscle cells (VSMCs) induced β‐catenin phosphorylation at Ser675 and WISP1, SPP1, and IGFBP‐2 expression. In vitro, Wnt treatment induced proliferation and cyclin D1 expression in VSMC from young (6 weeks old) rats but not in cells from older rats (8 months old), even though low‐density lipoprotein receptor–related protein 6 and β‐catenin phosphorylation, and β‐catenin nuclear translocation demonstrated β‐catenin activation in both cell types. Beta‐catenin silencing demonstrated that Wnt induction of cyclin D1 expression is β‐catenin dependent. Altogether, our data show that the Wnt/β‐catenin/TCF pathway is activated in aging human mammary artery cells, but fails to induce the proliferation of aging vascular cells.

The prevalence of germline mutations in paediatric pulmonary hypertension (PH) is poorly documented. The objective of this study was to determine the mutation frequency in PH genes in a paediatric cohort and describe the clinical characteristics of mutation carriers.The study involved 66 index cases with PH: 35 children with idiopathic pulmonary arterial hypertension (IPAH); five children with familial PAH (FPAH); three children with pulmonary veno-occlusive disease (PVOD); and 23 children with PAH associated with congenital heart disease (APAH-CHD).No mutations were found in the 23 children with APAH-CHD. In the 40 children with IPAH or FPAH, 12 mutations were found: five on BMPR2; four on ACVRL1; and three on TBX4. In the three PVOD cases, two carried the EIF2AK4 mutation. Mutation carriers had a more severe disease at diagnosis and more aggressive first-line therapy was required. The three patients with PVOD had a very severe disease at diagnosis and required a lung transplantation.The genetic architecture of paediatric PAH is enriched in ACVRL1 and TBX4 mutations compared to adult PAH, but further studies are required to confirm these results. Childhood-onset PAH in children carrying a mutation in one of the genes tested has a more severe presentation at diagnosis but a similar outcome to that observed in non-carriers.