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Background ACE2 has been identified as the entry receptor for coronaviruses into human cells, including SARS-COV-2 that causes COVID-19. Since hypertension is a leading comorbidity in non-survivors of COVID-19, we tested for association between ACE2 gene and hypertension in interaction with specific pre-existing conditions known to be associated with COVID-19 severity. Methods Genetic analysis of ACE2 gene was conducted in French-Canadian and British populations. Results In French-Canadian individuals, the T allele of the single nucleotide polymorphism (SNP) rs2074192 of ACE2 gene was a risk factor for hypertension in adult obese males [odds ratio (OR)=1.39, 95% confidence interval (CI) 1.06-1.83)] and even more so in obese males who smoked (OR=1.67, CI: 1.24-2.55), but not in lean males, non-smoker males or females. The T allele was significantly associated with severity of hypertension and with earlier penetrance of hypertension in obese smoking males. Significant interaction between the T allele and obesity was present in both sexes. The association of ACE2 (rs233575) genotype with blood pressure was also seen in adolescents but the interaction with obesity was present only in females. Several variants in ACE2 gene were found to be associated with hypertension in obese, smoking males in British individuals of the UK Biobank. In addition, we observed more severe outcomes to COVID-19 in association with ACE2 risk alleles in obese, smoking males. Conclusion This is the first report that ACE2 variants are associated with earlier penetrance and more severe hypertension and with more severe outcomes of COVID-19 in obese smoking males., Graphical Abstract Graphical Abstract

Aims/hypothesis Type 2 diabetes increases the risk of cardiovascular and renal complications, but early risk prediction could lead to timely intervention and better outcomes. Genetic information can be used to enable early detection of risk. Methods We developed a multi-polygenic risk score (multiPRS) that combines ten weighted PRSs (10 wPRS) composed of 598 SNPs associated with main risk factors and outcomes of type 2 diabetes, derived from summary statistics data of genome-wide association studies. The 10 wPRS, first principal component of ethnicity, sex, age at onset and diabetes duration were included into one logistic regression model to predict micro- and macrovascular outcomes in 4098 participants in the ADVANCE study and 17,604 individuals with type 2 diabetes in the UK Biobank study. Results The model showed a similar predictive performance for cardiovascular and renal complications in different cohorts. It identified the top 30% of ADVANCE participants with a mean of 3.1-fold increased risk of major micro- and macrovascular events (p = 6.3 × 10−21 and p = 9.6 × 10−31, respectively) and a 4.4-fold (p = 6.8 × 10−33) higher risk of cardiovascular death. While in ADVANCE overall, combined intensive blood pressure and glucose control decreased cardiovascular death by 24%, the model identified a high-risk group in whom it decreased the mortality rate by 47%, and a low-risk group in whom it had no discernible effect. High-risk individuals had the greatest absolute risk reduction with a number needed to treat of 12 to prevent one cardiovascular death over 5 years. Conclusions/interpretation This novel multiPRS model stratified individuals with type 2 diabetes according to risk of complications and helped to target earlier those who would receive greater benefit from intensive therapy. Graphical abstract

Introduction: We assessed the performance of our newly developed polygenic risk score (PRS) to predict microvascular and macrovascular complications of type 2 diabetes (T2D) in men and women. The PRS is composed of 600 common genomic variants associated to diabetes, cardiovascular and renal diseases and their key risk factors selected from summary statistics of meta-analyses of published genome-wide association studies performed in over 1.2 million of individuals. The performance of the polygenic model was assessed by c-statistics in 4098 genotyped participants of European descent of the ADVANCE trial (46.4% women) followed during a period of five years. Methods: The logistic regression model that included the PRS adjusted for the principal component (PC1) of genetically determined ancestry, age at diagnosis and T2D duration, and treatment assignment, did not include any clinical or outcome data. Results: The discrimination between cases (having a specific complication) from controls (free of this complication) at entry in ADVANCE had AUCs for microvascular complications of 0.63 (0.60-0.65) in men and 0.66 (0.63-0.69) in women, sex differences p = 0.07. AUCs for macrovascular complications were 0.56 (0.54-0.58) in men and 0.57 (0.54-0.61) in women; p = 0.41. The AUCs for prediction of incident cases, defined as having an outcome during the ADVANCE trial (free of outcome at baseline) compared to controls that did not have a specific outcome at any time during the study, were for microvascular events 0.66 (0.63-0.70) in men and 0.71 (0.66-0.77) in women; p = 0.15. AUCs for macrovascular events were 0.65 (0.62-0.68) and 0.72 (0.68-0.76) respectively, p = 0.01. AUC for prediction of cardiovascular death occurring during the trial was 0.71 (0.67-0.75) in men and 0.77 (0.72-0.82) in women; p = 0.04. Conclusion: Our polygenic model demonstrated an overall better performance in women than in men and a better prediction capacity in individuals free of previous events in both sexes. Disclosure J. Tremblay: Research Support; Self; Servier. Stock/Shareholder; Self; OPTITHERA. R. Attaoua: None. M. Haloui: None. R. Tahir: None. C. Long: None. C. Hizel: None. J. Chalmers: None. S. Harrap: None. M. Woodward: Consultant; Self; Amgen, Kyowa Hakko Kirin Co., Ltd. P. Hamet: Research Support; Self; Servier. Stock/Shareholder; Self; OPTITHERA. Funding Genome Quebec; Canadian Institutes of Health Research; MEIE; CQDM; Opti Thera; Servier, Canada Research Chair in Predictive Genomics

Type 2 diabetes increases the risk of cardiovascular and renal complications, but early risk prediction can lead to timely intervention and better outcomes. Through summary statistics of meta-analyses of published genome-wide association studies performed in over 1.2 million of individuals, we combined 9 PRS gathering genomic variants associated to cardiovascular and renal diseases and their key risk factors into one logistic regression model, to predict micro- and macrovascular endpoints of diabetes. Its clinical utility in predicting complications of diabetes was tested in 4098 participants with diabetes of the ADVANCE trial followed during a period of 10 years and replicated it in three independent non-trial cohorts. The prediction model adjusted for ethnicity, sex, age at onset and diabetes duration, identified the top 30% of ADVANCE participants at 3.1-fold increased risk of major micro- and macrovascular events (p=6.3×10−21 and p=9.6×10−31, respectively) and at 4.4-fold (p=6.8×10−33) increased risk of cardiovascular death compared to the remainder of T2D subjects. While in ADVANCE overall, combined intensive therapy of blood pressure and glycaemia decreased cardiovascular mortality by 24%, the prediction model identified a high-risk group in whom this therapy decreased mortality by 47%, and a low risk group in whom the therapy had no discernable effect. Patients with high PRS had the greatest absolute risk reduction with a number needed to treat of 12 to prevent one cardiovascular death over 5 years. This novel polygenic prediction model identified people with diabetes at low and high risk of complications and improved targeting those at greater benefit from intensive therapy while avoiding unnecessary intensification in low-risk subjects.

Genome-wide association studies or GWAS yielded an impressive amount of data that improved the understanding of complex diseases. However, the initial results were deceiving, when the early GWAS of Wellcome Trust, that was successful in reporting many significant associations for several diseases, claimed that no association could be observed for hypertension. It is only later, using carefully collected phenotypes on over 120,000 subjects that 29 SNPs were found to be significantly associated with hypertension. To explain a meaningful proportion of variability in a complex trait and, more importantly, to use this knowledge in risk assessment at an individual level, one needs to construct a genetic (polygenic) risk score (GRS) based on a large number of genotyped variants. From initial 5 to 10 allele-containing GRS reports, more recent publications combine hundreds or even thousands of alleles into so-called polyGRS (or PRS). As the number of SNPs included in the GRS increases, the accuracy of PolyGRS-based risk estimate improves. Any PolyGRS that is predictive in the long term (from birth on) and that has a predictive performance as good as “traditional risk factors,” would significantly improve the clinical utility of existing risk assessment algorithms for hypertension, encouraging its implementation in the practice of personalized medicine.

Cardiovascular and renal complications of T2D represent a human and societal burden. Early detection of susceptibility to complications could help their prevention. Clinical scores are useful to predict hard outcomes but genomic score can be applied earlier than a risk factor based score and thus save morbidity. We have constructed a polygenic risk score (PRS) based on genetic variants associated to risk factors and outcomes of T2D available from GWAS catalog and tested it on 5,000 genotyped Caucasian subjects with T2D of ADVANCE trial and its 5 year extension in ADVANCE-ON. We selected 620 SNPs which in combination with geo-ethnic principal component, age of onset and diabetes duration achieved a highly significant prediction of outcomes, including cardiovascular (AUC=0.80) and total mortality (AUC=0.77), equalling and surpassing such clinical scores as Framingham and UKPDS. The highest positive predictive values for micro- and macro-vascular prevalent cases were 81% and 78%, respectively. Individuals within the highest tertile of genetic risk for total prevalence of both microvascular and macrovascular events, had significantly higher cardiovascular death than subjects with the lowest genetic score (p=2.02x10-12). The number needed to treat to prevent one CV death over 5 years by ADVANCE therapies, was 12 in subjects with high PRS (p=0.0045) compared to 45 (p=0.14) in subjects with low PRS. Cox cumulative hazard plots demonstrated persistence of benefits for all cause and cardiovascular mortality in high PRS tertile over 10 years of intensive blood pressure control, while for glucose intensive therapy the benefit was restricted to the high PRS group for end-stage renal disease. We conclude that the use of a PRS that detects the risk of micro- and macrovascular complications at the time of T2D diagnosis could effectively assist in preventive and therapeutic measures to attenuate diabetes complications. Disclosure P. Hamet: Consultant; Self; Servier. F. Harvey: None. M. Haloui: None. M. Woodward: None. R. Tahir: None. F. Marois-Blanchet: None. C. Long: None. M. Marre: Board Member; Self; Abbott. Consultant; Self; AstraZeneca. Board Member; Self; Intarcia Therapeutics, Inc.. Consultant; Self; Eli Lilly and Company. Board Member; Self; Merck Sharp & Dohme Corp., Novo Nordisk A/S. Research Support; Self; Sanofi. Board Member; Self; Servier. S. Harrap: Research Support; Self; Servier. J. Chalmers: Research Support; Self; Servier. Other Relationship; Self; Servier. J. Tremblay: Consultant; Self; Servier.

Background: The prevalence of diabetic nephropathy varies according to ethnicity. Environmental as well as genetic factors contribute to the heterogeneity in the presentation of diabetic nephropathy. Our objective was to evaluate this heterogeneity within the Caucasian population. Methods: The geo-ethnic origin of the 3409 genotyped Caucasian type 2 diabetes (T2D) patients of Action in Diabetes and Vascular Disease: Preterax and Diamicron MR Controlled Evaluation was determined using principal component analysis. Genome-wide association studies analyses of age of onset of T2D were performed for geo-ethnic groups separately and combined. Results: The first principal component separated the Caucasian study participants into Slavic and Celtic ethnic origins. Age of onset of diabetes was significantly lower in Slavic patients (P = 7.3 x 10-20), whereas the prevalence of hypertension (P = 4.9 x 10-31) and albuminuria (5.1 x 10-9) were significantly higher. Age of onset of T2D and albuminuria appear to have an important genetic component as the values of these traits were also different between Slavic and Celtic individuals living in the same countries. Common and geo-ethnic-specific loci were found to be associated to age of onset of diabetes. Among the latter, the PROX1/PROX1-AS1 genes (rs340841) had the highest impact. Single-nucleotide polymorphism rs340841 CC genotype was associated with a 4.4 year earlier onset of T2D in Slavic patients living or not in countries with predominant Slavic populations. Conclusion: These results reveal the presence of distinct genetic architectures between Caucasian ethnic groups that likely have clinical relevance, among them PROX1 gene is a strong candidate of early onset of diabetes with variations depending on ethnicity.

In vascular smooth muscle cells and several other cell types, inhibition of Na+/K+-ATPase leads to the expression of early response genes, including c-Fos. We designed this study to examine whether or not a putative Na+i/K+i-sensitive element is located within the c-Fos 5′-UTR from − 650 to + 103 containing all known response elements activated by ‘classic’ stimuli, such as growth factors and Ca2+i-raising compounds. In HeLa cells, the highest increment of c-Fos mRNA content was noted after 6 h of Na+/K+-ATPase inhibition with ouabain that was abolished by actinomycin D, an inhibitor of RNA synthesis. c-Fos protein accumulation in ouabain-treated cells correlated with a gain of Na+i and loss of K+i. Augmented c-Fos expression was also observed under inhibition of Na+/K+-ATPase in K+-free medium and in the presence of the Na+ ionophore monensin. The effect of ouabain on c-Fos expression was sharply attenuated under dissipation of the transmembrane Na+ gradient, but was preserved in the presence of Ca2+ chelators and the extracellular regulated kinase inhibitor PD98059, thus indicating an Na+i-mediated, Ca2+i- and extracellular regulated kinase-independent mechanism of gene expression. In contrast to massive c-Fos expression, we failed to detect any effect of ouabain on accumulation of luciferase driven by the c-Fos 5′-UTR. Negative results were also obtained in ouabain-treated vascular smooth muscle cells and C11 Madin–Darby canine kidney cells possessing augmented c-Fos expression. Our results reveal that Na+i-induced c-Fos expression is not mediated by the 5′-UTR containing transcriptional elements activated by growth factors and other ‘classic stimuli’.

This study examines the relative impact of canonical hypoxia-inducible factor-1alpha- (HIF-1α and Na+ i/K+ i-mediated signaling on transcriptomic changes evoked by hypoxia and glucose deprivation. Incubation of RASMC in ischemic conditions resulted in ∼3-fold elevation of [Na+]i and 2-fold reduction of [K+]i. Using global gene expression profiling we found that Na+,K+-ATPase inhibition by ouabain or K+-free medium in rat aortic vascular smooth muscle cells (RASMC) led to the differential expression of dozens of genes whose altered expression was previously detected in cells subjected to hypoxia and ischemia/reperfusion. For further investigations, we selected Cyp1a1, Fos, Atf3, Klf10, Ptgs2, Nr4a1, Per2 and Hes1, i.e. genes possessing the highest increments of expression under sustained Na+,K+-ATPase inhibition and whose implication in the pathogenesis of hypoxia was proved in previous studies. In ouabain-treated RASMC, low-Na+, high-K+ medium abolished amplification of the [Na+]i/[K+]i ratio as well as the increased expression of all tested genes. In cells subjected to hypoxia and glucose deprivation, dissipation of the transmembrane gradient of Na+ and K+ completely eliminated increment of Fos, Atf3, Ptgs2 and Per2 mRNAs and sharply diminished augmentation expression of Klf10, Edn1, Nr4a1 and Hes1. In contrast to low-Na+, high-K+ medium, RASMC transfection with Hif-1a siRNA attenuated increments of Vegfa, Edn1, Klf10 and Nr4a1 mRNAs triggered by hypoxia but did not impact Fos, Atf3, Ptgs2 and Per2 expression. Thus, our investigation demonstrates, for the first time, that Na+ i/K+ i-mediated, Hif-1α- -independent excitation-transcription coupling contributes to transcriptomic changes evoked in RASMC by hypoxia and glucose deprivation.

Elevation of blood pressure with age is one of the hallmarks of hypertension in both males and females. This study examined transcriptomic profiles in the kidney of 12-, 40-, and 80-week-old spontaneously hypertensive rats and 4 recombinant inbred strains in search for functional genetic elements supporting temporal dynamics of blood pressure elevation. We found that both in males and females of spontaneously hypertensive rats and hypertensive recombinant inbred strains age-dependent blood pressure increment was accompanied by 50% heightened expression of epithelial sodium channel β- and γ-subunits. Epithelial sodium channel subunit expression correlated positively with blood pressure but correlated negatively with renin expression. Increased epithelial sodium channel activity was observed in cultured epithelial cells isolated from the kidney medulla of 80-week-old spontaneously hypertensive rats but not in age-matched normotensive Wistar Kyoto. This difference remained evident after 24-hour treatment with aldosterone. 22Na uptake in the perfused kidney medulla was increased whereas the urinary Na/K ratio was decreased in old spontaneously hypertensive rats compared with normotensive controls. The difference was eliminated by the administration of epithelial sodium channel inhibitor benzamil. Observations in recombinant inbred strains representing various mixtures of parental hypertensive and normotensive genomes suggest that Scnn1g and Scnn1b genes themselves are not implicated in heightened expression and that the increased expression is neither secondary nor required for a partial elevation of blood pressure in contrast to spontaneously hypertensive rats. We suggest that spontaneously hypertensive rats display an intact negative feed-back between renin-angiotensin-system and epithelial Na channel activity whose upregulated expression is supported by a yet unknown mechanism.

OBJECTIVE: We have previously reported distinct genetic architectures of renal impairment in T2D patients of Slavic and Celtic origins participating in the ADVANCE trial (J Hypertens. 2015 Jun;33 Suppl 1:e3). Further analysis suggests that the major driver of the difference in the prevalence of T2D complications between Slavic and Celtic groups is due to an earlier onset of diabetes in Slavic patients. In an attempt to distinguish between environmental and genetic factors on age of onset of diabetes, we have determined the age of onset of T2D in Slavic subjects living in Celtic countries and confirmed the same earlier onset (-2 years) in these subjects, notwithstanding their living environment. DESIGN AND METHOD: We performed GWAS analyses of age of onset of T2D in 3500 T2D patients from ADVANCE trial. Analyses were done for Celtic and Slavic groups separately and combined. RESULTS: 7 loci are associated to age of onset of T2D in patients of both Celtic and Slavic origins, including HDAC9 gene (rs1128745). 9 loci are selectively associated to this phenotype in patients of Celtic origin, including the Il23R (rs1273974) while 9 different loci are associated to it in Slavic patients only. Among the latter, PROX1/PROX1-AS1 genes (rs340841) has the highest effect size. SNP rs 340841 homozygous CC genotype is associated with 2 years earlier onset of T2D in Slavic patients living in Slavic countries or in Celtic countries. This locus is also associated with eGFR decline in Slavic, with macroalbuminuria and hypertension in all ADVANCE subjects of Caucasian origin and with Interleukin-6 levels at baseline. In recent literature search we found that PROX1 gene has been associated with abnormalities of glucose metabolism and risk of diabetes with variations depending on ethnicity. CONCLUSIONS: We conclude that fine granularity of distinction in geo-ethic background assist in resolution of clinically relevant genetic contribution to cardiovascular complication in T2D.

Stimulus-dependent elevation of intracellular Ca(2+) ([Ca(2+)](i)) affects the expression of numerous genes--a phenomenon known as excitation-transcription coupling. Recently, we found that increases in [Na(+)](i) trigger c-Fos expression via a novel Ca(2+) (i)-independent pathway. In the present study, we identified ubiquitous and tissue-specific [Na(+)](i)/[K(+)](i)-sensitive transcriptomes by comparative analysis of differentially expressed genes in vascular smooth muscle cells from rat aorta (RVSMC), the human adenocarcinoma cell line HeLa, and human umbilical vein endothelial cells (HUVEC). To augment [Na(+)](i) and reduce [K(+)](i), cells were treated for 3 hrs with the Na(+),K(+)-ATPase inhibitor ouabain or placed for the same time in the K(+)-free medium. Employing Affymetrix-based technology, we detected changes in expression levels of 684, 737 and 1839 transcripts in HeLa, HUVEC and RVSMC, respectively, that were highly correlated between two treatments (p0.62). Among these Na(+) (i)/K(+) (i)-sensitive genes, 80 transcripts were common for all three types of cells. To establish if changes in gene expression are dependent on increases in [Ca(2+)](i), we performed identical experiments in Ca(2+)-free media supplemented with extracellular and intracellular Ca(2+) chelators. Surprisingly, this procedure elevated rather than decreased the number of ubiquitous and cell-type specific Na(+) (i)/K(+) (i)-sensitive genes. Among the ubiquitous Na(+) (i)/K(+) (i)-sensitive genes whose expression was regulated independently of the presence of Ca(2+) chelators by more than 3-fold, we discovered several transcription factors (Fos, Jun, Hes1, Nfkbia), interleukin-6, protein phosphatase 1 regulatory subunit, dual specificity phosphatase (Dusp8), prostaglandin-endoperoxide synthase 2, cyclin L1, whereas expression of metallopeptidase Adamts1, adrenomedulin, Dups1, Dusp10 and Dusp16 was detected exclusively in Ca(2+)-depleted cells. Overall, our findings indicate that Ca(2+) (i)-independent mechanisms of excitation-transcription coupling are involved in transcriptomic alterations triggered by elevation of the [Na(+)](i)/[K(+)](i) ratio. There results likely have profound implications for normal and pathological regulation of mammalian cells, including sustained excitation of neuronal cells, intensive exercise and ischemia-triggered disorders.

Statins, 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, protect against deleterious effects of chronic nitric oxide inhibition. We aimed at determining the genes and pathways involved in the protective effect of statin treatment during hypertension. Chronic inhibition of nitric oxide synthesis by Nω-nitro- l -arginine methyl ester (L-NAME) induced accelerated hypertension which was slightly reduced by cotreatment with atorvastatin (100 mg/kg/day). Gene expression profile of aortic media was strongly modified by atorvastatin cotreatment which prevented modulation of many genes regulated by L-NAME administration (described in Dupuis, M., Soubrier, F., Brocheriou, I., Raoux, S., Haloui, M., Louedec, L., Michel, J.B., Nadaud, S., 2004. Profiling of aortic smooth muscle cell gene expression in response to chronic inhibition of nitric oxide synthase in rats. Circulation 110, 867–873). The functional classification of these genes highlighted several major biological pathways modulated in aortic media by atorvastatin: effectors involved in smooth muscle tone; extracellular matrix; intracellular mediators of cell proliferation. Moreover, atorvastatin partially prevented arterial wall thickening, TGF-β pathway activation, MCP-1 induction and smooth muscle cell proliferation induced by L-NAME treatment although blood pressure was only slightly reduced, suggesting mechanisms independent of blood pressure levels. The induction of PPARalpha mRNA expression by atorvastatin in L-NAME treated rats also suggests that this pathway could participate in the protective effect. In conclusion, our data show that atorvastatin specifically antagonizes a set of genes modulated by L-NAME-induced accelerated hypertension.

In vascular smooth muscle cells and several other cell types, inhibition of Na(+)/K(+)-ATPase leads to the expression of early response genes, including c-Fos. We designed this study to examine whether or not a putative Na(+) (i)/K(+) (i)-sensitive element is located within the c-Fos 5'-UTR from - 650 to + 103 containing all known response elements activated by 'classic' stimuli, such as growth factors and Ca(2+) (i)-raising compounds. In HeLa cells, the highest increment of c-Fos mRNA content was noted after 6 h of Na(+)/K(+)-ATPase inhibition with ouabain that was abolished by actinomycin D, an inhibitor of RNA synthesis. c-Fos protein accumulation in ouabain-treated cells correlated with a gain of Na(+) (i) and loss of K(+) (i). Augmented c-Fos expression was also observed under inhibition of Na(+)/K(+)-ATPase in K(+)-free medium and in the presence of the Na(+) ionophore monensin. The effect of ouabain on c-Fos expression was sharply attenuated under dissipation of the transmembrane Na(+) gradient, but was preserved in the presence of Ca(2+) chelators and the extracellular regulated kinase inhibitor PD98059, thus indicating an Na(+) (i)-mediated, Ca(2+) (i)- and extracellular regulated kinase-independent mechanism of gene expression. In contrast to massive c-Fos expression, we failed to detect any effect of ouabain on accumulation of luciferase driven by the c-Fos 5'-UTR. Negative results were also obtained in ouabain-treated vascular smooth muscle cells and C11 Madin-Darby canine kidney cells possessing augmented c-Fos expression. Our results reveal that Na(+) (i)-induced c-Fos expression is not mediated by the 5'-UTR containing transcriptional elements activated by growth factors and other 'classic stimuli'.

Objective:The genetic architecture of type 2 diabetes (T2D) has been reported to be different between Asian and Caucasian populations (BBRC 2014;452:213–220). It is also well recognized that renal complications of T2D start earlier and are more severe in Asian subjects. Our objective was to determin

Background— Chronic inhibition of nitric oxide (NO) synthesis by N ω -nitro- l -arginine methyl ester (L-NAME) induces hypertension associated with remodeling of the arterial wall. In this study, we aimed at identifying genes and pathways involved in this process in aortic smooth muscle cells from Fischer 344 rats, which exhibit an accelerated hypertension after administration of L-NAME. Methods and Results— We studied the transcriptional profile of aortic media after 15 days (moderate hypertension) and 30 days (accelerated hypertension) of L-NAME administration (50 mg · kg −1 · d −1 ) by using rat Affymetrix Genechips, and we present a large-scale validation of the DNA chip results by real-time reverse transcription–polymerase chain reaction (RT-PCR). We observed, in aortic media, a progressive increase in the number of modulated genes during L-NAME administration, with 53 genes significantly modulated after 15 days and 147 genes after 30 days. These expression changes were confirmed at 87% by RT-PCR. We found 28 known genes regulated at both 15 and 30 days (96% confirmation by RT-PCR). The functional classification of the regulated genes highlights 3 major biological pathways modulated in aortic media during L-NAME administration: genes regulating cell proliferation, genes involved in the extracellular matrix remodeling, and genes of the NO/cGMP signaling pathway. Conclusions— As a consequence of the genomic approach, we observed a large increase in modulation of gene expression along the evolution of the model and the progressive implication of compensatory mechanisms, making expression profile analysis more complex.

Thrombin, a serine protease, plays an important role in the progression of atherosclerosis. How atorvastatin could limit the pro-inflammatory response to thrombin was studied in cultured rat aortic smooth muscle cells. The variations in expression of interleukin-6, heme oxygenase-1, p 22phox and Mox-1 mRNAs were evaluated by reverse transcriptase–polymerase chain reaction (RT-PCR). Interleukin-6 release was determined using the B9 cell assay. Nuclear factor-kappa B (NF-κB) translocation was analysed by electrophoretic mobility shift assay (EMSA) and RhoA protein translocation by Western blot. Thrombin activated interleukin-6 secretion and mRNA expression in smooth muscle cells in a dose-dependent manner. The greatest effect on mRNA expression was obtained after 1 h of stimulation. Preincubation (72 h) of the cells with various concentrations of atorvastatin prevented this effect. Simultaneous addition of mevalonate overcame this statin effect. Thrombin was without effect on p 22phox and heme oxygenase-1 mRNA expression but, after 3 h of stimulation, induced a two-fold increase in that of Mox-1. Preincubation with atorvastatin dose-dependently downregulated this Mox-1 mRNA expression. In addition, thrombin induced NF-κB translocation and membrane translocation of RhoA in smooth muscle cells which were both prevented by pre-treatment of the cells by atorvastatin. These data demonstrate the ability of atorvastatin to prevent the induction by thrombin of a pro-inflammatory phenotype in smooth muscle cells.

Objectives: Neutral endopeptidase (NEP) inhibition potentiated the renal action of Atrial Natriuretic Peptide (ANP) and was associated with appearance of the peptide in the urine, providing evidence of protection of the filtrated peptide along the course of the nephron. The macula densa, composed of epithelial cells, receives ionic information from the urinary compartment via Na–K–2Cl cotransport and influences renin secretion by the myoepithelioid cells in the afferent arteriole. bNOS constitutively expressed in the epithelial cells of the macula densa is involved in this feed-back. NEP inhibition was associated with the absence of any increase in renin secretion. The hypothesis is that potentiation of urinary ANP by NEP inhibition could limit renin secretion by directly or indirectly targeting the macula densa in vivo. Methods and results: We tested the interaction between NEP inhibition (candoxatril) and Na–K–2Cl inhibition (bumetanide) on electrolyte and ANP urinary excretion, renin secretion, macula densa activity (NADPH diaphorase activity and bNOS mRNA) and TSC-1 mRNA expression in the renal cortex and BSC-1 in the renal medulla of rats treated for 5 days. Bumetanide increased urinary electrolyte excretion whereas candoxatril did not. Candoxatril increased urinary ANP and cyclic GMP excretion. Bumetanide increased renin and aldosterone secretion whereas candoxatril decreased renin secretion. This effect on renin release was associated with an increase in macula densa NADPH diaphorase activity in the bumetanide-treated group which was blunted by candoxatril. Lastly, bumetanide increased TSC-1 mRNA expression in the cortex and this effect was blunted by candoxatril. Conclusion: These results suggest that potentiation of ANP by NEP inhibition could interfere with tubular function at different levels and limit renin secretion by a urinary pathway involving macula densa activity.

Hypertension represents one of the main risk factors for vascular diseases. Genetic susceptibility may influence the rate of its development and the associated vascular remodeling. To explore markers of hypertension-related morbidity, we have used surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry to study changes in proteins released by the aorta of two rat strains with different susceptibilities to hypertension. Fischer and Brown Norway (BN) rats were divided into a control group and a group receiving low-dose N(Omega)-nitro-L-arginine methyl ester (L-NAME), a hypertensive drug, interfering with endothelial function. In spite of a significant elevation of blood pressure in both strains in response to L-NAME, BN rats exhibited a lower vascular remodeling in response to hypertension. Proteomic analysis of secreted aortic proteins by SELDI-TOF MS allowed detection of four mass-to-charge ratio (m/z) peaks whose corresponding proteins were identified as ubiquitin, smooth muscle (SM) 22alpha, thymosin beta4, and C-terminal fragment of filamin A, differentially secreted in Fischer rats in response to L-NAME. We have confirmed a strain-dependent difference in susceptibility to L-NAME-induced hypertension between BN and Fischer rats. The greater susceptibility of Fischer rats is associated with aortic wall hypertrophic remodeling, reflected by increased aortic secretion of four identified biomarkers. Similar variations in one of them, SM22alpha, also were observed in plasma, suggesting that this marker could be used to assess vascular damage induced by hypertension.