Sex-specific effects of NLRP6/AVR and ADM loci on susceptibility to essential hypertension in a Sardinian population.

Coronary artery disease, heart failure, fatal arrhythmias, stroke, and renal disease are the most common causes of mortality for humans, and essential hypertension remains a major risk factor. Elucidation of susceptibility loci for essential hypertension has been difficult because of its complex, multifactorial nature involving genetic, environmental, and sex- and age-dependent nature. We investigated whether the 11p15.5 region syntenic to rat chromosome 1 region containing multiple blood pressure quantitative trait loci (QTL) detected in Dahl rat intercrosses harbors polymorphisms that contribute to susceptibility/resistance to essential hypertension in a Sardinian population. Initial testing performed using microsatellite markers spanning 18 Mb of 11p15.5 detected a strong association between D11S1318 (at 2.1 Mb, P = 0.004) and D11S1346 (at 10.6 Mb, P = 0.00000004), suggesting that loci in close proximity to these markers may contribute to susceptibility in our Sardinian cohort. NLR family, pyrin domain containing 6/angiotensin-vasopressin receptor (NLRP6/AVR), and adrenomedullin (ADM) are in close proximity to D11S1318 and D11S1346, respectively; thus we tested single nucleotide polymorphisms (SNPs) within NLRP6/AVR and ADM for their association with hypertension in our Sardinian cohort. Upon sex stratification, we detected one NLRP6/AVR SNP associated with decreased susceptibility to hypertension in males (rs7948797G, P = 0.029; OR = 0.73 [0.57-0.94]). For ADM, sex-specific analysis showed a significant association between rs4444073C, with increased susceptibility to essential hypertension only in the male population (P = 0.006; OR = 1.44 [1.13-1.84]). Our results revealed an association between NLRP6/AVR and ADM loci with male essential hypertension, suggesting the existence of sex-specific NLRP6/AVR and ADM variants affecting male susceptibility to essential hypertension.

DEspR T/CATAAAA-box promoter variant decreases DEspR transcription and is associated with increased BP in Sardinian males.

Essential hypertension is highly prevalent in the elderly population, exceeding 70% in people older than 60 yr of age, and remains a leading risk factor for heart disease, stroke, and chronic renal disease. Elucidation of genetic determinants is critical but remains a challenge due to its complex, multifactorial pathogenesis. We investigated the role DEspR promoter variants, previously associated with male essential hypertension susceptibility, in blood pressure (BP) regulation. We detected a single nucleotide polymorphism within the DEspR 5'-regulatory region associated with increased BP in a male Sardinian cohort accounting for 11.0 mmHg of systolic BP (P<10(-15)) and 9.3 mmHg of diastolic BP (P<10(-15)). Sequence analysis of three normotensive subjects homozygous for the rs6535847 "normotension-associated T-allele" identified a canonical TATAAAA-box in contrast to a CATAAAA-motif in three hypertensive subjects homozygous for the rs6535847 "hypertension-associated C-allele." In vitro analysis detected decreased transcription activity with the CATAAAA-motif promoter-construct compared with the canonical TATAAAA-box promoter-construct. Although BP did not differ between DEspR+/- knockout male mice and wild-type littermates at 6 mo of age, radiotelemetric BP measurements in 18 mo old inbred DEspR+/- knockout male mice known to have decreased DEspR RNA and protein detected higher systolic, mean, and diastolic BPs in DEspR+/- mice compared with littermate wild-type controls (P<0.05). Our results demonstrate that promoter variants in DEspR associated with hypertension susceptibility and increased BP in Sardinian males affect transcription levels, which then affect BP in an age-dependent and male-specific manner. This finding is concordant with the late-onset and sex-specific characteristics of essential hypertension, thus reiterating the mandate for sex-specific analyses and treatment approaches for essential hypertension.

Overlapping genes in Nalp6/PYPAF5 locus encode two V2-type vasopressin isoreceptors: angiotensin-vasopressin receptor (AVR) and non-AVR.

The angiotensin-vasopressin receptor (AVR) responds with equivalent affinities to angiotensin II (ANG II) and vasopressin and is coupled to adenylate cyclase and hence a V2-type vasopressin receptor. AVR maps to the Nalp6 locus and overlaps with the larger Nalp6/PYPAF5 reported to be a T cell/granulocyte-specific, cytoplasmic-specific proapoptotic protein, thus questioning the existence of AVR. Here we confirm, through different experimental modalities, that AVR is distinct from Nalp6/PYPAF5 based on different mRNA and protein sizes, subcellular localization, and tissue-specific expression patterns. Binding studies of PYPAF5-specific Cos1 transfectants detect high-affinity binding to vasopressin but not ANG II, thus assigning PYPAF5 as a non-AVR (NAVR). Signaling array analysis reveals that AVP stimulation of AVR- and NAVR-specific Cos1 transfectants results in diametrical activation as well as coactivation of signaling pathways known to mediate renal sodium and water balance. Likewise, ANG II stimulation of Cos1-AVR transfectants reveals a signaling profile distinct from that of AVP-stimulated Cos1-AVR transfectants. Analysis of genomic organization of the AVR/NAVR locus shows an overlapping gene arrangement with alternative promoter usage resulting in different NH(2) termini for NAVR and AVR. In addition to core promoter elements, androgen and estrogen response elements are detected. Promoter analysis of NAVR/AVR 5'-regulatory region detects transcriptional upregulation by testosterone and synergistic upregulation by testosterone and estrogen, thus suggesting that AVR and/or NAVR contribute to sex-specific V2-type vasopressin-mediated effects. Altogether, confirmation of AVR and identification of NAVR as vasopressin receptors are concordant with emerging vasopressin functions not attributable to V1a, V1b, or V2 receptors and add molecular bases for the multifunctional complexity of vasopressin-mediated functions and regulation.

Embryonic lethality in Dear gene-deficient mice: new player in angiogenesis.

The dual endothelin-1/angiotensin II receptor (Dear) binds endothelin-1 (ET-1) and angiotensin II (ANG II) with equal affinities in the Dahl S/JRHS rat strain. To elucidate its physiological significance within the context of multiple receptor isoforms and diverse ET-1 and ANG II functions spanning blood pressure regulation, tumor proliferation, and angiogenesis, we characterized mouse Dear and Dear-deficient mice. Unlike null mutant models of ET-1, ANG II, and all other ET-1 and ANG II receptors, Dear(-/-) deficiency results in impaired angiogenesis, dysregulated neuroepithelial development, and embryonic lethality by embryonic day 12.5. Interestingly, mouse Dear does not bind ANG II, similar to Dahl R/JRHS rat Dear, but binds ET-1 and vascular endothelial growth factor (VEGF) signal peptide (VEGFsp) with equal affinities, suggesting a putative novel multifunction for VEGFsp and a parsimonious mechanism for coordination of VEGF-induced and Dear-mediated pathways. Consistent with its developmental angiogenic role, Dear inhibition results in decreased tumor growth in B16-F10 melanoma cell-induced subcutaneous tumor in female Dear(+/-)/C57BL6BC10 mice, but not in males (age 3.5 mo), and in 127Cs radiation-induced orthotopic mammary tumors in Sprague-Dawley female rats (age range 3-6.5 mo). Altogether, the data identify Dear as a new player in angiogenesis during development downstream to, and nonredundant with, VEGF-mediated pathways, as well as a putative modulator of tumor angiogenesis acting within a gender-specific paradigm.

Sex-specific effects of dual ET-1/ANG II receptor (Dear) variants in Dahl salt-sensitive/resistant hypertension rat model.

Essential (polygenic) hypertension is a complex genetic disorder that remains a major risk factor for cardiovascular disease despite clinical advances, reiterating the need to elucidate molecular genetic mechanisms. Elucidation of susceptibility genes remains a challenge, however. Blood pressure (BP) regulatory pathways through angiotensin II (ANG II) and endothelin-1 (ET-1) receptor systems comprise a priori candidate susceptibility pathways. Here we report that the dual ET-1/ANG II receptor gene (Dear) is structurally and functionally distinct between Dahl salt-sensitive, hypertensive (S) and salt-resistant, normotensive (R) rats. The Dahl S S44/M74 variant is identical to the previously reported Dear cDNA with equivalent affinities for both ET-1 and ANG II, in contrast to Dahl R S44P/M74T variant, which exhibits absent ANG II binding but effective ET-1 binding. The S44P substitution localizes to the ANG II-binding domain predicted by the molecular recognition theory, providing compelling support of this theory. The Dear gene maps to rat chromosome 2 and cosegregates with BP in female F2(RxS) intercross rats with highly significant linkage (LOD 3.61) accounting for 14% of BP variance, but not in male F2(RxS) intercross rats. Altogether, the data suggest the hypothesis that modification of the critical balance between ANG II and ET-1 systems through variant Dear contributes to hypertension susceptibility in female F2(RxS) intercross rats. Further investigations are necessary to corroborate genetic linkage through congenic rat studies, to investigate putative gene interactions, and to show causality by transgenesis and/or intervention. More importantly, the data reiterate the importance of sex-specific factors in hypertension susceptibility.

Analysis of gender-specific atherosclerosis susceptibility in transgenic[hCETP]25DS rat model.

Epidemiological and clinical data demonstrate differences in atherosclerotic coronary heart disease prevalence between age-matched men and premenopausal women. Mechanisms underlying relative athero-susceptibility in men and athero-resistance in premenopausal women remain to be elucidated. Lack of informative animal models hinders research. We report here a moderate-expresser line transgenic for human cholesteryl ester transfer protein (CETP) in the Dahl salt-sensitive hypertensive rat strain, Tg25, that recapitulates premenopausal female athero-resistance. Having ascertained identical genetic background, environmental factors, and equivalent CETP hepatic RNA levels, we detect worse hypercholesterolemia, hypertriglyceridemia, coronary plaques and survival outcome in Tg25 male rats compared with Tg25 females. Hepatic transcription profiles of Tg25 males and females normalized to respective gender- and age-matched non-transgenic controls exhibit significant differences. Genes implicated on hierarchical cluster analysis and quantitative real-time RT-PCR pinpoint pathways associated with coronary plaque progression such as inflammation and arachidonic acid epoxygenation, and not just cholesterol metabolism pathways. The data demonstrate gender-specific factors as key modulators of atherosclerosis phenotype and suggest a possible role for the liver in atheroma progression as a large organ source of proatherogenic systemic factors.

Genome-wide scan identifies novel QTLs for cholesterol and LDL levels in F2[Dahl RxS]-intercross rats.

Hypercholesterolemia is a significant risk factor for coronary artery disease development. Genes influencing nonmonogenic hypercholesterolemia susceptibility in humans remain to be identified. Animal models are key investigative systems because major confounding variables such as diet, activity, and genetic background can be controlled. We performed a 121-marker, total genome-analysis of an F2[Dahl RxS]-intercross selected for contrasting parental strain susceptibilities for hyperlipidemia on regular rat diets at 6 months of age. Quantitative traits studied were plasma total cholesterol, triglyceride, HDL, and LDL levels adjusted for obesity. Genome-wide analysis of 200 F2-intercross male rats detects two QTLs with highly significant linkage for total cholesterol (TC) on chromosome (chr) 5-133.3 Mbp (LOD 5.8), and chr5-54.2 Mbp (LOD 4.8), and two QTLs with significant linkage for TC: on chromosome 8, chr8-60.4 Mbp (LOD 3.8), and chromosome 2, chr2-243.5 Mbp (LOD 3.4). A QTL for LDL with significant linkage is detected on chromosome 5, chr5-104 Mbp (LOD 3.7). These QTLs contribute from 7% to 12% of total trait variance, respectively, with Dahl-S allele effects resulting in increased TC and LDL levels consistent with hyperlipidemia susceptibility in the parental Dahl-S rat strain. Predicted QTL-peaks do not coincide with previous genome scans. Human homologues of two TC-QTLs span genes listed in a LocusLink profile for cholesterol. Only suggestive loci were detected for HDL and total triglyceride levels. Altogether, the data demonstrates the contribution of multiple QTLs to hypercholesterolemia making a multipathway pathogenic framework imperative. QTL-peak candidate genes delineated are syntenic between rat and human genomes, increasing clinical relevance and mandating further study.

Chlamydia pneumoniae accelerates coronary artery disease progression in transgenic hyperlipidemia-genetic hypertension rat model.

Chlamydia pneumoniae (Cpn) has been associated with human coronary artery disease but causal relevance as a risk factor has not been shown. Several rabbit and mouse model studies demonstrate exacerbation of aortic atherosclerosis by Cpn, however impact of Cpn on coronary artery disease (CAD) and survival outcomes has not been shown. To study this, we used specific pathogen-free, inbred, transgenic-CAD Dahl salt-sensitive (S) hypertensive (Tg53) rats and control inbred, non-transgenic Dahl S (nonTg) rats to analyze the effects of Cpn infection on macrophage foam cell formation, coronary artery disease progression, and effect on survival. Cpn infection induced acceleration of foam cell formation in hyperlipidemic Tg53 recruited peritoneal macrophages. This effect is hyperlipidemia-dependent. The transcription profile of Tg53-Cpn macrophage foam cells is different from control mock-inoculated (Tg53-spg) and heat-inactivated (Tg53-iCpn) macrophages (ANOVA P < 0.0001). Decreased survival was detected in Tg53-Cpn compared with control nonTg-Cpn and mock-infected Tg53-mouse pneumonitic rats (P = 0.009) and was associated with "culprit" coronary plaques and left atrial thrombi. These data demonstrate that in the presence of significant hyperlipidemia and hypertension, one-time Cpn infection at 5 mo of age (associated with early CAD stage) accelerates progression to overt-CAD in the Tg53 rat model. The data support the hypothesis that untreated Cpn infection is a causal risk factor for CAD progression most likely mediated by Cpn-induced accelerated macrophage foam cell formation.

Differential regulation of functional gene clusters in overt coronary artery disease in a transgenic atherosclerosis-hypertensive rat model.

BACKGROUND: Human acute coronary syndrome refers to the spectrum of clinical manifestations of overt coronary artery (CAD) disease characterized by atherosclerotic plaque destabilization and resultant myocardial injury. Typically studied as distinct pathologies, emerging pathogenic paradigms implicate multiple processes beyond thrombosis and ischemic cell injury respectively, with significant pathway overlap involving inflammation, apoptosis, matrix degradation, and oxidative stress. However, all these pathways have also been implicated in still-quiescent coronary plaque progression, thus making it harder to pinpoint the turnkey events leading to overt-CAD. Analysis of transcription profiles could identify a working framework of pathogenesis distinguishing overt-CAD. MATERIALS AND METHODS: We investigated the transcription profile associated with overt-coronary artery disease (CAD), in contrast to quiescent-CAD and attenuated, quiescent-CAD using the Tg 53 transgenic atherosclerosis-hypertensive rat model, which exhibits end-stage coronary heart disease simulating human acute coronary syndromes. Using a rat-specific known-gene oligonucleotide array, twice corroborated transcription profiles from four individual Tg 53 rats exhibiting overt-CAD were analyzed and contrasted to transcription profiles of age-matched Tg 53 rats with quiescent-CAD (pooled n = 4) and attenuated, quiescent-CAD (pooled n = 4). RESULTS: Tg 53 male rats with overt-CAD exhibited distinct transcription profiles compared with both quiescent-CAD control groups. Functional gene cluster analysis detects upregulation of genes involved in inflammation (interleukin-1, interleukin-18, Fc gamma II receptor, thyrotropin releasing hormone), matrix balance (membrane type metalloproteinase, TIMP-1, lysyl oxidase), oxidized LDL entry (endothelial oxLDL receptor), which contrast deinduced gene clusters involved in angiogenesis, proliferation, metabolism, ion transport and adrenergic receptors. CONCLUSION: The data demonstrate that transcriptionally mediated events distinguish the onset of overt-CAD and identify a first list of putative "turnkey" genes. This altered molecular framework implies an altered "hardwiring" which a priori would require multifaceted, targeted intervention- currently not implemented to date. Although more studies are necessary, early concordance with current pathogenic paradigms of human coronary plaque destabilization and post-ischemic myocardial response provides translational significance to observations and hypotheses.