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.

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-1α and HIF-1β or HIF-2α and HIF-1β expression vectors. Exogenous HIF-2 markedly increased luciferase reporter activity driven by the heNOS 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 site-directed 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 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 onBMPR2; four onACVRL1; and three onTBX4.In the three PVOD cases, two carried theEIF2AK4mutation. 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 inACVRL1andTBX4mutations 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.