Estrogen Signaling and Portopulmonary Hypertension: The Pulmonary Vascular Complications of Liver Disease Study (PVCLD2).

BACKGROUND AND AIMS: Portopulmonary hypertension (POPH) was previously associated with a single-nucleotide polymorphism (SNP) rs7175922 in aromatase (cytochrome P450 family 19 subfamily A member 1 [CYP19A1]). We sought to determine whether genetic variants and metabolites in the estrogen signaling pathway are associated with POPH. APPROACH AND RESULTS: We performed a multicenter case-control study. POPH patients had mean pulmonary artery pressure >25 mm Hg, pulmonary vascular resistance >240 dyn-sec/cm-5 , and pulmonary artery wedge pressure ≤15 mm Hg without another cause of pulmonary hypertension. Controls had advanced liver disease, right ventricular (RV) systolic pressure <40 mm Hg, and normal RV function by echocardiography. We genotyped three SNPs in CYP19A1 and CYP1B1 using TaqMan and imputed SNPs in estrogen receptor 1 using genome-wide markers. Estrogen metabolites were measured in blood and urine samples. There were 37 patients with POPH and 290 controls. Mean age was 57 years, and 36% were female. The risk allele A in rs7175922 (CYP19A1) was significantly associated with higher levels of estradiol (P = 0.02) and an increased risk of POPH (odds ratio [OR], 2.36; 95% confidence interval [CI], 1.12-4.91; P = 0.02) whereas other SNPs were not. Lower urinary 2-hydroxyestrogen/16-α-hydroxyestrone (OR per 1-ln decrease = 2.04; 95% CI, 1.16-3.57; P = 0.01), lower plasma levels of dehydroepiandrosterone-sulfate (OR per 1-ln decrease = 2.38; 95% CI, 1.56-3.85; P < 0.001), and higher plasma levels of 16-α-hydroxyestradiol (OR per 1-ln increase = 2.16; 95% CI, 1.61-2.98; P < 0.001) were associated with POPH. CONCLUSIONS: Genetic variation in aromatase and changes in estrogen metabolites were associated with POPH.

Activation of angiotensin type 2 receptor attenuates testosterone-induced hypertension and uterine vascular resistance in pregnant rats†.

Preeclampsia is a pregnancy-related hypertensive disorder with unclear mechanisms. While hypersensitivity to angiotensin II via vasoconstrictive angiotensin type-1 receptor (AT1R) is observed in preeclampsia, the importance of vasodilatory angiotensin type-2 receptor (AT2R) in the control of vascular dysfunction is less clear. We assessed whether AT1R, AT2R, and endothelial nitric oxide synthase (eNOS) expression are altered in placental vessels of preeclamptic women and tested if ex vivo incubation with AT2R agonist Compound 21 (C21; 1 µM) could restore AT1R, AT2R, and eNOS balance. Further, using a rat model of gestational hypertension induced by elevated testosterone, we examined whether C21 (1 µg/kg/day, oral) could preserve AT1R and AT2R balance and improve blood pressure, uterine artery blood flow, and vascular function. Western blots revealed that AT1R protein level was higher while AT2R and eNOS protein were reduced in preeclamptic placental vessels, and AT2R agonist C21 decreased AT1R and increased AT2R and eNOS protein levels in preeclamptic vessels. In testosterone dams, blood pressure was higher, and uterine artery blood flow was reduced, and C21 treatment reversed these levels similar to those in controls dams. C21 attenuated the exaggerated Ang II contraction and improved endothelium-dependent vasorelaxation in uterine arteries of testosterone dams. These C21-mediated vascular effects were associated with decreased AT1R and increased AT2R and eNOS protein levels. C21 also increased serum nitrate/nitrite and bradykinin production in testosterone dams and attenuated the fetoplacental growth restriction. Thus, AT1R upregulation and AT2R downregulation are observed in preeclampsia and testosterone model, and increasing AT2R activity could help restore AT1R and AT2R balance and improve gestational vascular function.

Genetic aetiology of blood pressure relates to aortic stiffness with bi-directional causality: evidence from heritability, blood pressure polymorphisms, and Mendelian randomization.

AIMS: Haemodynamic determinants of blood pressure (BP) include cardiac output (CO), systemic vascular resistance (SVR), and arterial stiffness. We investigated the heritability of these phenotypes, their association with BP-related single-nucleotide polymorphisms (SNPs), and the causal association between BP and arterial stiffness. METHODS AND RESULTS: We assessed BP, central BP components, and haemodynamic properties (during a single visit) including CO, SVR, and pulse wave velocity (PWV, measure of arterial stiffness) in 3531 (1934 monozygotic, 1586 dizygotic) female TwinsUK participants. Heritability was estimated using structural equation modelling. Association with 984 BP-associated SNP was examined using least absolute shrinkage and selection operator (LASSO) and generalized estimating equation regression. One and two-sample Mendelian randomization (MR) was used to estimate the causal direction between BP and arterial stiffness including data on 436 419 UK Biobank participants. We found high heritability for systolic and pulsatile components of BP (>50%) and PWV (65%) with overlapping genes accounting for >50% of their observed correlation. Environmental factors explained most of the variability of CO and SVR (>80%). Regression identified SNPs (n = 5) known to be associated with BP to also be associated with PWV. One-sample MR showed evidence of bi-directional causal association between BP and PWV in TwinsUK participants. Two-sample MR, confirmed a bi-directional causal effect of PWV on BP (inverse variance weighted (IVW) beta = 0.11, P < 0.02) and BP on arterial stiffness (IVW beta = 0.004, P < 0.0001). CONCLUSION: The genetic basis of BP is mediated not only by genes regulating BP but also by genes that influence arterial stiffness. Mendelian randomization indicates a bi-directional causal association between BP and arterial stiffness.

Intestinal SIRT1 Deficiency-Related Intestinal Inflammation and Dysbiosis Aggravate TNFα-Mediated Renal Dysfunction in Cirrhotic Ascitic Mice.

In advanced cirrhosis, the TNFα-mediated intestinal inflammation and bacteria dysbiosis are involved in the development of inflammation and vasoconstriction-related renal dysfunction. In colitis and acute kidney injury models, activation of SIRT1 attenuates the TNFα-mediated intestinal and renal abnormalities. This study explores the impacts of intestinal SIRT1 deficiency and TNFα-mediated intestinal abnormalities on the development of cirrhosis-related renal dysfunction. Systemic and renal hemodynamics, intestinal dysbiosis [cirrhosis dysbiosis ratio (CDR) as marker of dysbiosis], and direct renal vasoconstrictive response (renal vascular resistance (RVR) and glomerular filtration rate (GFR)) to cumulative doses of TNFα were measured in bile duct ligated (BDL)-cirrhotic ascitic mice. In SIRT1IEC-KO-BDL-ascitic mice, the worsening of intestinal dysbiosis exacerbates intestinal inflammation/barrier dysfunction, the upregulation of the expressions of intestinal/renal TNFα-related pathogenic signals, higher TNFα-induced increase in RVR, and decrease in GFR in perfused kidney. In intestinal SIRT1 knockout groups, the positive correlations were identified between intestinal SIRT1 activity and CDR. Particularly, the negative correlations were identified between CDR and RVR, with the positive correlation between CDR and GFR. In mice with advanced cirrhosis, the expression of intestinal SIRT1 is involved in the linkage between intestinal dysbiosis and vasoconstriction/hypoperfusion-related renal dysfunction through the crosstalk between intestinal/renal TNFα-related pathogenic inflammatory signals.

Haemodynamic phenotypes and survival in patients with systemic sclerosis: the impact of the new definition of pulmonary arterial hypertension.

BACKGROUND: In this study, we investigated the impact of the new haemodynamic definition of pulmonary arterial hypertension (PAH) as proposed by the 6th PH World Symposium on phenotypes and survival in patients with systemic sclerosis (SSc). METHODS: In SSc patients who were prospectively and consecutively screened for PAH including right heart catheterisation in Heidelberg or Zurich, haemodynamic and clinical variables have been reassessed according to the new PAH definition. Patients have been followed for 3.7±3.7 (median 3.4) years; Kaplan-Meier survival analysis was performed. Patients with significant lung or left heart disease were excluded from comparative analyses. RESULTS: The final dataset included 284 SSc patients, 146 patients (49.2%) had mean pulmonary arterial pressure (mPAP) ≤20 mm Hg, 19.3% had mPAP 21-24 mm Hg and 29.4% had mPAP ≥25 mm Hg. In the group of mildly elevated mPAP, only four patients (1.4% of the whole SSc cohort) had pulmonary vascular resistance (PVR) values ≥3 Wood Units (WU) and could be reclassified as manifest SSc-APAH. Twenty-eight (9.8%) patients with mPAP of 21-24 mm Hg and PVR ≥2 WU already presented with early pulmonary vascular disease with decreased 6 min walking distance (6MWD) (p<0.001), TAPSE (p=0.004) and pulmonary arterial compliance (p<0.001). A PVR ≥2 WU was associated with reduced long-term survival (p=0.002). PVR and 6MWD were independent prognostic predictors in multivariate analysis. CONCLUSION: The data of this study show that a PVR threshold ≥3 WU is too high to enable an early diagnosis of PAH. A PVR threshold ≥2 WU was already associated with pulmonary vascular disease, significantly reduced survival and would be more appropriate in SSc patients with mild PAH.

Gene expression profiles of ion channels and receptors in mouse resistance arteries: Effects of cell type, vascular bed, and age.

OBJECTIVE: Receptors and ion channels of smooth muscle cells (SMCs) and endothelial cells (ECs) are integral to the regulation of vessel diameter and tissue blood flow. Physiological roles of ion channels and receptors in skeletal muscle and mesenteric arteries have been identified; however, their gene expression profiles are undefined. We tested the hypothesis that expression profiles for ion channels and receptors governing vascular reactivity vary with cell type, vascular bed, and age. METHODS: Mesenteric and superior epigastric arteries were dissected from Old (24-26 months) and Young (3-6 months) C57BL/6J mice. ECs and SMCs were collected for analysis with custom qRT-PCR arrays to determine expression profiles of 80 ion channel and receptor genes. Bioinformatics analyses were applied to gain insight into functional interactions. RESULTS: We identified 68 differences in gene expression with respect to cell type, vessel type, and age. Heat maps illustrate differential expression, and distance matrices predict patterns of coexpression. Gene networks based upon protein-protein interaction datasets and KEGG pathways illustrate biological processes affected by specific differences in gene expression. CONCLUSIONS: Differences in gene expression profiles are most pronounced between microvascular ECs and SMCs with subtle variations between vascular beds and age groups.

The role of genetics in pulmonary arterial hypertension.

Group 1 pulmonary hypertension or pulmonary arterial hypertension (PAH) is a rare disease characterized by proliferation and occlusion of small pulmonary arterioles, leading to progressive elevation of pulmonary artery pressure and pulmonary vascular resistance, and right ventricular failure. Historically, it has been associated with a high mortality rate, although, over the last decade, treatment has improved survival. PAH includes idiopathic PAH (IPAH), heritable PAH (HPAH), and PAH associated with certain medical conditions. The aetiology of PAH is heterogeneous, and genetics play an important role in some cases. Mutations in BMPR2, encoding bone morphogenetic protein receptor 2, a member of the transforming growth factor-ß superfamily of receptors, have been identified in 70% of cases of HPAH, and in 10-40% of cases of IPAH. Other genetic causes of PAH include mutations in the genes encoding activin receptor-like type 1, endoglin, SMAD9, caveolin 1, and potassium two-pore-domain channel subfamily K member 3. Mutations in the gene encoding T-box 4 have been identified in 10-30% of paediatric PAH patients, but rarely in adults with PAH. PAH in children is much more heterogeneous than in adults, and can be associated with several genetic syndromes, congenital heart disease, pulmonary disease, and vascular disease. In addition to rare mutations as a monogenic cause of HPAH, common variants in the gene encoding cerebellin 2 increase the risk of PAH by approximately two-fold. A PAH panel of genes is available for clinical testing, and should be considered for use in clinical management, especially for patients with a family history of PAH. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Mutation of Plekha7 attenuates salt-sensitive hypertension in the rat.

PLEKHA7 (pleckstrin homology domain containing family A member 7) has been found in multiple studies as a candidate gene for human hypertension, yet functional data supporting this association are lacking. We investigated the contribution of this gene to the pathogenesis of salt-sensitive hypertension by mutating Plekha7 in the Dahl salt-sensitive (SS/JrHsdMcwi) rat using zinc-finger nuclease technology. After four weeks on an 8% NaCl diet, homozygous mutant rats had lower mean arterial (149 ± 9 mmHg vs. 178 ± 7 mmHg; P < 0.05) and systolic (180 ± 7 mmHg vs. 213 ± 8 mmHg; P < 0.05) blood pressure compared with WT littermates. Albumin and protein excretion rates were also significantly lower in mutant rats, demonstrating a renoprotective effect of the mutation. Total peripheral resistance and perivascular fibrosis in the heart and kidney were significantly reduced in Plekha7 mutant animals, suggesting a potential role of the vasculature in the attenuation of hypertension. Indeed, both flow-mediated dilation and endothelium-dependent vasodilation in response to acetylcholine were improved in isolated mesenteric resistance arteries of Plekha7 mutant rats compared with WT. These vascular improvements were correlated with changes in intracellular calcium handling, resulting in increased nitric oxide bioavailability in mutant vessels. Collectively, these data provide the first functional evidence that Plekha7 may contribute to blood pressure regulation and cardiovascular function through its effects on the vasculature.

Off-Target Vascular Effects of Cholesteryl Ester Transfer Protein Inhibitors Involve Redox-Sensitive and Signal Transducer and Activator of Transcription 3-Dependent Pathways.

Elevated blood pressure was an unexpected outcome in some cholesteryl ester transfer protein (CETP) inhibitor trials, possibly due to vascular effects of these drugs. We investigated whether CETP inhibitors (torcetrapib, dalcetrapib, anacetrapib) influence vascular function and explored the putative underlying molecular mechanisms. Resistance arteries and vascular smooth muscle cells (VSMC) from rats, which lack the CETP gene, were studied. CETP inhibitors increased phenylephrine-stimulated vascular contraction (logEC50 (:) 6.6 ± 0.1; 6.4 ± 0.06, and 6.2 ± 0.09 for torcetrapib, dalcetrapib, and anacetrapib, respectively, versus control 5.9 ± 0.05). Only torcetrapib reduced endothelium-dependent vasorelaxation. The CETP inhibitor effects were ameliorated by N-acetylcysteine (NAC), a reactive oxygen species (ROS) scavenger, and by S3I-201 [2-hydroxy-4-[[2-(4-methylphenyl)sulfonyloxyacetyl]amino]benzoic acid], a signal transducer and activator of transcription 3 (STAT3) inhibitor. CETP inhibitors increased the phosphorylation (2- to 3-fold) of vascular myosin light chain (MLC) and myosin phosphatase target subunit 1 (MYPT1) (procontractile proteins) and stimulated ROS production. CETP inhibitors increased the phosphorylation of STAT3 (by 3- to 4-fold), a transcription factor important in cell activation. Activation of MLC was reduced by NAC, GKT137831 [2-(2-chlorophenyl)-4-[3-(dimethylamino)phenyl]-5-methyl-1H-pyrazolo[4,3-c]pyridine-3,6-dione] (Nox1/4 inhibitor), and S3I-201. The phosphorylation of STAT3 was unaffected by NAC and GKT137831. CETP inhibitors did not influence activation of mitogen-activated proteins kinases (MAPK) or c-Src. Our data demonstrate that CETP inhibitors influence vascular function and contraction through redox-sensitive, STAT3-dependent, and MAPK-independent processes. These phenomena do not involve CETP because the CETP gene is absent in rodents. Findings from our study indicate that CETP inhibitors have vasoactive properties, which may contribute to the adverse cardiovascular effects of these drugs such as hypertension.

Polymorphisms and haplotype of ROCK2 associate with high altitude essential hypertension in native high altitude Ladakhi Indian population: a preliminary study.

BACKGROUND AND OBJECTIVES: High-altitude essential hypertension (HAEH) is a disease occurring in permanent residents of high-altitude regions. The disease is characterized with SBP ≥140 mmHg and DBP ≥90 mmHg. HAEH is known to run in families, i.e. the disease has genetic component. Rho-associated coiled-coil containing protein kinase 2 (ROCK2) is a stress-activated serine-threonine kinase known to disturb vascular-homeostasis leading to an increase in systemic vascular resistance, hallmark of HAEH. ROCK2 is implicated in sea-level essential hypertension but its role in HAEH is yet to be elucidated. METHODS: The present study deals with genotyping 13 polymorphisms of ROCK2 gene in demographicaly matched human cases (n = 65) and controls (n = 38) by Sequenom MS (TOF)-based MassARRAY platform using iPLEX Gold technology. RESULTS: A significant association was observed for GG genotype (SNP, rs978906), AA genotype (SNP, rs6753921), GG genotype (SNP, rs10495582) and AA genotype (SNP, rs2230774) with HAEH (p < 0.05). The 4 SNPs were tagged to each other and formed a 35 kb LD block (r(2 )> 0.90). Haplotype AGCC, composed of wild-type alleles of the SNPs was over represented in controls. In contrast, haplotype GAGA, composed of variant-alleles was observed to be in higher proportion in cases. Moreover, SBP levels (mmHg) were higher in cases with risk genotype against the ones having protective genotype (p = 0.05). Bioinformatic analysis revealed binding of a critical transcription factor, SRF to variant-allele G of SNP rs10495582. SRF has been reported in previous studies to promote ROCK2 transcriptional expression. INTERPRETATION AND CONCLUSIONS: The data clearly suggests association of ROCK2 polymorphisms and haplotypes with HAEH.

Vasculopathy and pulmonary hypertension in sickle cell disease.

Sickle cell disease (SCD) is an autosomal recessive disorder in the gene encoding the ß-chain of hemoglobin. Deoxygenation causes the mutant hemoglobin S to polymerize, resulting in rigid, adherent red blood cells that are entrapped in the microcirculation and hemolyze. Cardinal features include severe painful crises and episodic acute lung injury, called acute chest syndrome. This population, with age, develops chronic organ injury, such as chronic kidney disease and pulmonary hypertension. A major risk factor for developing chronic organ injury is hemolytic anemia, which releases red blood cell contents into the circulation. Cell free plasma hemoglobin, heme, and arginase 1 disrupt endothelial function, drive oxidative and inflammatory stress, and have recently been referred to as erythrocyte damage-associated molecular pattern molecules (eDAMPs). Studies suggest that in addition to effects of cell free plasma hemoglobin on scavenging nitric oxide (NO) and generating reactive oxygen species (ROS), heme released from plasma hemoglobin can bind to the toll-like receptor 4 to activate the innate immune system. Persistent intravascular hemolysis over decades leads to chronic vasculopathy, with ∼10% of patients developing pulmonary hypertension. Progressive obstruction of small pulmonary arterioles, increase in pulmonary vascular resistance, decreased cardiac output, and eventual right heart failure causes death in many patients with this complication. This review provides an overview of the pathobiology of hemolysis-mediated endothelial dysfunction and eDAMPs and a summary of our present understanding of diagnosis and management of pulmonary hypertension in sickle cell disease, including a review of recent American Thoracic Society (ATS) consensus guidelines for risk stratification and management.

A genome-wide expression quantitative trait loci analysis of proprotein convertase subtilisin/kexin enzymes identifies a novel regulatory gene variant for FURIN expression and blood pressure.

Proprotein convertase subtilisin/kexin (PCSK) enzymes cleave and convert their immature substrates into biologically active forms. Polymorphisms in the PCSK genes have been reported to associate with human diseases and phenotypes, including hypercholesterolemia and blood pressure (BP), and targeting PCSKs is considered a promising future form of drug therapy. PCSK processing is readily induced upon upregulation of the enzyme, but the genetic factors contributing to PCSK expression have not been thoroughly characterized. To gain a comprehensive understanding of the genetic regulation of PCSK expression, we performed, for the first time, a genome-wide expression quantitative trait loci (eQTL) analysis using mRNA expression in >1400 human peripheral blood samples from the Cardiovascular Risk in Young Finns Study and ca. ten million single-nucleotide polymorphisms (SNPs). The expression data showed clear expression for FURIN, PCSK5, PCSK7 and MBTPS1 (membrane-bound transcription factor peptidase, site 1) mRNAs in virtually all tested samples. A discovery analysis demonstrated a genome-wide significant (p < 5 × 10(-8)) association with the selected PCSK probes for 1024 variants, which were located at ten independent loci. Of these loci, 5/10 could be confirmed to regulate PCSK expression in two additional and independent sample sets. Finally, a phenotypic analysis demonstrated that a novel cis-eQTL SNP rs4702 for FURIN is strongly associated with both diastolic (p = 0.012) and systolic (p = 0.035) BP levels, as well as peripheral vascular resistance (p = 0.003). These findings indicate that the expression of the PCSK enzymes is regulated by genetic factors, which have biological roles in health and disease.

Site-specific increases in utero- and fetoplacental arterial vascular resistance in eNOS-deficient mice due to impaired arterial enlargement.

The sites of elevated vascular resistance that impede placental perfusion in pathological pregnancies are unknown. In the current study, we identified these sites in a knockout mouse model (eNOS(-/-)) with reduced uterine (-55%) and umbilical (-29%) artery blood flows caused by endothelial nitric oxide synthase deficiency. Uteroplacental and fetoplacental arterial vascular trees of pregnant mice near term were imaged using x-ray microcomputed tomography (n = 5-10 placentas from 3-5 dams/group). The resulting three-dimensional images were analyzed to assess vessel geometry and vascular resistance. In control and eNOS(-/-) trees, ∼90% of total uteroplacental vascular resistance was located in the radial arteries. Changes in eNOS(-/-) vessel geometry, including 30% reductions in uterine, radial, and spiral artery diameters, were calculated to increase arterial resistance downstream of the uterine artery by 2.3-fold, predicting a 57% decrease in uterine blood flow. Despite large reductions in eNOS(-/-) spiral arteries (-55% by volume) and maternal canals (-67% by volume), these vessels were relatively minor contributors to resistance. In the eNOS(-/-) fetoplacental tree, the number of arterioles (50-75 µm diameter) increased by 26%. Nevertheless, calculated resistance rose by 19%, predominantly because arteries near the periphery of the tree selectively exhibited a 7%-9% diameter reduction. We conclude that previously observed decreases in uterine and umbilical blood flows in eNOS(-/-) pregnancies are associated with markedly divergent structural changes in the uteroplacental versus fetoplacental circulations. Results showed the radial arteries were critical determinants of uteroplacental resistance in mice and therefore warrant greater attention in future studies in pathological human pregnancies.

Epigenetic regulation of aortic remodeling in hyperhomocysteinemia.

Hyperhomocysteinemia (HHcy) is prevalent in patients with hypertension and is an independent risk factor for aortic pathologies. HHcy is known to cause an imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), leading to the accumulation of collagen in the aorta and resulting in stiffness and development of hypertension. Although the exact mechanism of extracellular matrix (ECM) remodeling is unclear, emerging evidence implicates epigenetic regulation involving DNA methylation. Our purpose was to investigate whether 5-aza-2'-deoxycytidine (Aza), a DNA methyltransferase (DNMT1) inhibitor, reduces high blood pressure (BP) by regulating aortic ECM remodeling in HHcy. Wild-type and cystathionine ß-synthase (CBS)(+/-) HHcy mice were treated with Aza (0.5 mg/kg body weight). In HHcy mice, Aza treatment normalized the plasma homocysteine (Hcy) level and BP. Thoracic and abdominal aorta ultrasound revealed a reduction in the resistive index and wall-to-lumen ratio. Vascular response to phenylephrine, acetylcholine, and sodium nitroprusside improved after Aza in HHcy mice. Histology showed a marked reduction in collagen deposition in the aorta. Aza treatment decreased the expression of DNMT1, MMP9, TIMP1, and S-adenosyl homocysteine hydrolase (SAHH) and upregulated methylene tetrahydrofolate reductase (MTHFR). We conclude that reduction of DNA methylation by Aza in HHcy reduces adverse aortic remodeling to mitigate hypertension.

microRNA and Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a lethal vasculopathy associated with complex etiology that involves remodeling of distal pulmonary arteries leading to elevation of pulmonary vascular resistance. This process results in right ventricular (RV) hypertrophy and ultimately RV failure. In addition, PAH is associated with systemic impairment in the skeletal muscle contributing to exercise intolerance. It has only been a few decades since microRNAs (miRNAs) have been implied in the development and progression of PAH regarding every organ affected by the disease. Indeed, impairment of miRNA's expression has been involved in vascular cell remodeling processes such as adventitial fibroblast (AdvFB) migration; pulmonary arterial smooth muscle cell (PASMC) proliferation and pulmonary arterial endothelial cell (PAEC) dysfunction observed in PAH. At the molecular level miRNAs have been described in the control of ion channels and mitochondrial function as well as the regulation of the BMPR2 signaling pathways contributing to PAH lung impairment. Recently miRNAs have also been specifically implicated in RV dysfunction and systemic angiogenic impairment, observed in PAH. In this chapter, we will summarize the knowledge on miRNA in PAH and highlight their crucial role in the etiology of this disease.

Reduced cystathionine γ-lyase and increased miR-21 expression are associated with increased vascular resistance in growth-restricted pregnancies: hydrogen sulfide as a placental vasodilator.

Increased vascular impedance in the fetoplacental circulation is associated with fetal hypoxia and growth restriction. We sought to investigate the role of hydrogen sulfide (H2S) in regulating vasomotor tone in the fetoplacental vasculature. H2S is produced endogenously by catalytic activity of cystathionine ß-synthase and cystathionine γ-lyase (CSE). Immunohistochemical analysis localized CSE to smooth muscle cells encircling arteries in stem villi. Immunoreactivity was reduced in placentas from pregnancies with severe early-onset growth-restriction and preeclampsia displaying abnormal umbilical artery Doppler waveforms compared with preeclamptic placentas with normal waveforms and controls. These findings were confirmed at the protein and mRNA levels. MicroRNA-21, which negatively regulates CSE expression, was increased in placentas with abnormal Doppler waveforms. Exposure of villus explants to hypoxia-reoxygenation significantly reduced CSE protein and mRNA and increased microRNA-21 expression. No changes were observed in cystathionine ß-synthase expression, immunolocalized principally to the trophoblast, in pathologic placentas or in vitro. Finally, perfusion of normal placentas with an H2S donor, after preconstriction with a thromboxane mimetic, resulted in dose-dependent vasorelaxation. Glibenclamide and N(G)-nitro-l-arginine methyl ester partially blocked the effect, indicating that H2S acts through ATP-sensitive K(+) channels and nitric oxide synthesis. These results demonstrate that H2S is a powerful vasodilator of the placental vasculature and that expression of CSE is reduced in placentas associated with increased vascular resistance.

Disruption of methylarginine metabolism impairs vascular homeostasis.

Asymmetric dimethylarginine (ADMA) and monomethyl arginine (L-NMMA) are endogenously produced amino acids that inhibit all three isoforms of nitric oxide synthase (NOS). ADMA accumulates in various disease states, including renal failure, diabetes and pulmonary hypertension, and its concentration in plasma is strongly predictive of premature cardiovascular disease and death. Both L-NMMA and ADMA are eliminated largely through active metabolism by dimethylarginine dimethylaminohydrolase (DDAH) and thus DDAH dysfunction may be a crucial unifying feature of increased cardiovascular risk. However, despite considerable interest in this pathway and in the role of ADMA as a cardiovascular risk factor, there is little evidence to support a causal role of ADMA in pathophysiology. Here we reveal the structure of human DDAH-1 and probe the function of DDAH-1 both by deleting the DDAH1 gene in mice and by using DDAH-specific inhibitors which, as we demonstrate by crystallography, bind to the active site of human DDAH-1. We show that loss of DDAH-1 activity leads to accumulation of ADMA and reduction in NO signaling. This in turn causes vascular pathophysiology, including endothelial dysfunction, increased systemic vascular resistance and elevated systemic and pulmonary blood pressure. Our results also suggest that DDAH inhibition could be harnessed therapeutically to reduce the vascular collapse associated with sepsis.

5'UTR repeat polymorphisms of the BMPR2 gene in children with pulmonary hypertension associated with congenital heart disease.

UNLABELLED: The mutations of bone morphogenetic protein receptor type 2 (BMPR2) in patients with idiopathic pulmonary hypertension has been well defined. We investigated the occurrence of BMPR2 mutation and genetic polymorphisms in children with pulmonary hypertension associated with congenital heart disease (aPH/CHD) and correlated with the pulmonary haemodynamic and vasoreactivity. METHODS: BMPR2 mutation/polymorphisms were determined in 30 aPH/CHD children. All children underwent cardiac catheterisation to obtain baseline haemodynamic data. The 5'UTR containing promoter region and all the exons [1-13] of BMPR2 gene were genotyped for possible genetic variants that may be related to the aPH/CHD. RESULTS: None of our 30 patients (median-age 90 months) with aPH/CHD (mean PAP 48±17mmHg, PVR 6.7±4.2WUm(2)) has had any BMPR2 mutation. Fifteen of them had single nucleotide polymorphism, rs1061157 and/or 5'UTR-polymorphism, specifically GGC repeat variant in seven patients; AGC repeat variant in one patient; and nine base pairs duplication (CTTCTTCGG) in one patient. The GGC repeat ≥13 was found in three out of six of children with aPH/CHD with normal PVR vs. two out of 24 children with aPH/CHD with high PVR. The odd ratio between these two subgroups of aPH/CHD is 0.09 (95% CI 0.02-0.34). CONCLUSIONS: In our cohort, there was no BMPR2 mutation in children with aPH/CHD while nine out of 30 of them have 5'UTR repeat polymorphisms. Our data suggests the occurrence of GGC repeat ≥13 at the 5'UTR region may have some protective effect towards pulmonary vasculopathy in children who have been exposed to high pulmonary blood flow due to CHD.

Impaired myogenic constriction of the renal afferent arteriole in a mouse model of reduced ßENaC expression.

Previous studies demonstrate a role for ß epithelial Na(+) channel (ßENaC) protein as a mediator of myogenic constriction in renal interlobar arteries. However, the importance of ßENaC as a mediator of myogenic constriction in renal afferent arterioles, the primary site of development of renal vascular resistance, has not been determined. We colocalized ßENaC with smooth muscle α-actin in vascular smooth muscle cells in renal arterioles using immunofluorescence. To determine the importance of ßENaC in myogenic constriction in renal afferent arterioles, we used a mouse model of reduced ßENaC (ßENaC m/m) and examined pressure-induced constrictor responses in the isolated afferent arteriole-attached glomerulus preparation. We found that, in response to a step increase in perfusion pressure from 60 to 120 mmHg, the myogenic tone increased from 4.5 ± 3.7 to 27.3 ± 5.2% in +/+ mice. In contrast, myogenic tone failed to increase with the pressure step in m/m mice (3.9 ± 0.8 to 6.9 ± 1.4%). To determine the importance of ßENaC in myogenic renal blood flow (RBF) regulation, we examined the rate of change in renal vascular resistance following a step increase in perfusion pressure in volume-expanded animals. We found that, following a step increase in pressure, the rate of myogenic correction of RBF is inhibited by 75% in ßENaC m/m mice. These findings demonstrate that myogenic constriction in afferent arterioles is dependent on normal expression of ßENaC.

The impact of the catechol-O-methyltransferase genotype on the acute responsiveness of vascular reactivity to a green tea extract.

The beneficial effects of green tea catechins, such as the proposed improvement in endothelial function, may be influenced by phase II metabolism during and after absorption. The methylation enzyme, catechol-O-methyltransferase (COMT), has a missense mutation rs4680 (G to A), proposed to result in a 40 % reduction in enzyme activity. In the present pilot study, twenty subjects (ten of each homozygous COMT genotype) were recruited. Green tea extract capsules (836 mg green tea catechins) were given in a fasted state, and a high-carbohydrate breakfast was given after 60 min. Blood samples and vascular function measurements were taken at regular intervals. The change in digital volume pulse stiffness index (SI) from baseline was shown to be different between genotype groups at 120 and 240 min, with a lower SI in the GG individuals (P ≤ 0·044). The change in blood pressure from baseline also differed between genotype groups, with a greater increase in systolic (P = 0·023) and diastolic (P = 0·034) blood pressure at 120 min in the GG group. The GG [corrected] group was shown to have a greater increase in insulin concentrations at 120 min (P = 0·019) and 180 min (P = 0·008) compared with baseline, despite similar glucose profiles. No genotypic differences were found in vascular reactivity measured using laser Doppler iontophoresis, total nitrite, lipids, plasma total antioxidant capacity or inflammatory markers after ingestion of the green tea extract. In conclusion, SI and insulin response to the glucose load differed between the COMT genotype groups, and this may be suggestive of a green tea extract and genotype interaction.