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Chromosome 2 introgression from normotensive Brown Norway (BN) rats into hypertensive Dahl salt-sensitive (SS) background (SS-chromosome 2 BN /Mcwi; consomic S2 B ) reduced blood pressure and vascular inflammation under a normal-salt diet (NSD). We hypothesized that BN chromosome 2 contains anti-inflammatory genes that could reduce blood pressure and vascular inflammation in rats fed NSD or high-salt diet (HSD). Four- to 6-week old male SS and congenic rats containing the BN chromosome 2 distal portion (SS.BN-[ rs13453786-rs66377062 ]/Aek; S2 B a) and middle segment (SS.BN-[ rs106982173-rs65057186 ]/Aek; S2 B b) were fed NSD or HSD (4% NaCl) up to age 12 to 13 weeks. Systolic blood pressure determined by telemetry was higher in SS rats fed HSD versus NSD. Systolic blood pressure was lower in both congenic rats than in SS under NSD, but similar under HSD versus SS. Reactive oxygen species generation using dihydroethidium staining, expression of vascular cell adhesion molecule-1 and monocyte chemoattractant protein-1, and immune cell infiltration by immunofluorescence demonstrated that S2 B a rats present less inflammation under NSD and more under HSD versus SS rats. RNA sequencing and reverse transcription-quantitative PCR identified 2 differentially expressed genes encoded within BN chromosome 2 distal portion that could act as regulators of vascular inflammation. These were downregulated glutamyl aminopeptidase ( Enpep ) that was anti-inflammatory under NSD and upregulated heparan sulfate 2-O-sulfotransferase 1 ( Hs2st1 ) that was proinflammatory under HSD. In conclusion, 2 differentially expressed genes encoded within introgressed BN chromosome 2 distal fragment were identified: Enpep associated with reduced vascular inflammation under NSD, and Hs2st1 , associated with increased vascular inflammation under HSD.

Aims NADPH oxidase (NOX) 1 but not NOX4-dependent oxidative stress plays a role in diabetic vascular disease, including atherosclerosis. Endothelin (ET)-1 has been implicated in diabetes-induced vascular complications. We showed that crossing mice overexpressing human ET-1 selectively in endothelium (eET-1) with apolipoprotein E knockout (Apoe-/-) mice enhanced high-fat diet-induced atherosclerosis in part by increasing oxidative stress. We tested the hypothesis that ET-1 overexpression in the endothelium would worsen atherosclerosis in type-1 diabetes through a mechanism involving NOX1 but not NOX4. Methods and results Six-week-old male Apoe-/- and eET-1/Apoe-/- mice with or without Nox1 (Nox1-/y) or Nox4 knockout (Nox4-/-) were injected intraperitoneally with either vehicle or streptozotocin (55 mg/kg/day) for 5 days to induce type-1 diabetes and were studied 14 weeks later. ET-1 overexpression increased 2.5-fold and 5-fold the atherosclerotic lesion area in the aortic sinus and arch of diabetic Apoe-/- mice, respectively. Deletion of Nox1 reduced aortic arch plaque size by 60%; by contrast, Nox4 knockout increased lesion size by 1.5-fold. ET-1 overexpression decreased aortic sinus and arch plaque alpha smooth muscle cell content by ∼35% and ∼50%, respectively, which was blunted by Nox1 but not Nox4 knockout. Reactive oxygen species production was increased 2-fold in aortic arch perivascular fat of diabetic eET-1/Apoe-/- and eET-1/Apoe-/- / Nox4-/- mice but not eET-1/Apoe-/- / Nox1y/- mice. ET-1 overexpression enhanced monocyte/macrophage and CD3+ T cell infiltration ∼2.7-fold in the aortic arch perivascular fat of diabetic Apoe-/- mice. Both Nox1 and Nox4 knockout blunted CD3+ T cell infiltration whereas only Nox1 knockout prevented the monocyte/macrophage infiltration in diabetic eET-1/Apoe-/- mice. Conclusions Endothelium ET-1 overexpression enhances the progression of atherosclerosis in type-1 diabetes, perivascular oxidative stress and inflammation through NOX1. Translational perspective We demonstrate that endothelial cell-restricted human ET-1 overexpression worsens atherosclerosis in diabetes and causes perivascular oxidative stress and inflammation, extending our previous findings to a model of type-1 diabetes mellitus. We also show that these effects are mediated through NOX1 and that atherosclerosis is worsened by loss of NOX4, providing evidence that NADPH oxidase isoforms play differential roles in plaque progression. These results offer new approaches to prevent the progression of atherosclerosis in diabetes, which is associated with significantly increased risk of cardiovascular events.

Vascular injury is an early manifestation in hypertension and a cause of end-organ damage. MicroRNAs play an important role in cardiovascular disease, but their implication in vascular injury in hypertension remains unclear. This study revealed using an unbiased approach, microRNA and mRNA sequencing with molecular interaction analysis, a microRNA-transcription factor coregulatory network involved in vascular injury in mice made hypertensive by 14-day Ang II (angiotensin II) infusion. A candidate gene approach identified upregulated miR-431-5p encoded in the conserved 12qF1 (14q32 in humans) microRNA cluster, whose expression correlated with blood pressure, and which has been shown to be upregulated in human atherosclerosis, as a potential key regulator in Ang II–induced vascular injury. Gain- and loss-of-function in human vascular smooth muscle cells demonstrated that miR-431-5p regulates in part gene expression by targeting ETS homologous factor. In vivo miR-431-5p knockdown delayed Ang II–induced blood pressure elevation and reduced vascular injury in mice, which demonstrated its potential as a target for treatment of hypertension and vascular injury.

Endothelium-derived endothelin (ET)-1 has been implicated in the development of hypertension and end-organ damage, but its exact role remains unclear. We have shown that tamoxifen-inducible endothelium-restricted human ET-1 overexpressing (ieET-1) mice exhibited blood pressure rise after a 3-week induction in an ET type A (ET A ) receptor-dependent manner, in absence of vascular and renal injury. It is unknown whether long-term ET-1 overexpression results in sustained blood pressure elevation and vascular and renal injury. Adult male ieET-1 and control tamoxifen-inducible endothelium-restricted Cre recombinase (ieCre) mice were induced with tamoxifen and 2.5 months later, were treated with or without the ET A receptor blocker atrasentan for 2 weeks. Three-month induction of endothelial human ET-1 overexpression increased blood pressure ( P P P P Col1A1 and Col3A1 expression, and perivascular adipose tissue oxidative stress ( P P P + ) and myeloid-derived suppressive cell (CD11b + Gr-1 + ) renal infiltration ( P P P A receptors.

Aims Matrix metalloproteinases (MMPs) have been implicated in the development of hypertension in animal models and humans. Mmp2 deletion did not change Ang II-induced blood pressure (BP) rise. However, whether Mmp2 knockout affects angiotensin (Ang) II-induced vascular injury has not been tested. We sought to determine whether Mmp2 knockout will prevent Ang II-induced vascular injury. Methods and results A fourteen-day Ang II infusion (1000 ng/kg/min, SC) increased systolic BP, decreased vasodilatory responses to acetylcholine, induced mesenteric artery (MA) hypertrophic remodelling, and enhanced MA stiffness in wild-type (WT) mice. Ang II enhanced aortic media and perivascular reactive oxygen species generation, aortic vascular cell adhesion molecule-1 and monocyte chemotactic protein-1 expression, perivascular monocyte/macrophage and T cell infiltration, and the fraction of spleen activated CD4+CD69+ and CD8+CD69+ T cells, and Ly-6Chi monocytes. Study of intracellular signalling showed that Ang II increased phosphorylation of epidermal growth factor receptor and extracellular-signal-regulated kinase 1/2 in vascular smooth muscle cells isolated from WT mice. All these effects were reduced or prevented by Mmp2 knockout, except for systolic BP elevation. Ang II increased Mmp2 expression in immune cells infiltrating the aorta and perivascular fat. Bone marrow (BM) transplantation experiments revealed that in absence of MMP2 in immune cells, Ang II-induced BP elevation was decreased, and that when MMP2 was deficient in either immune or vascular cells, Ang II-induced endothelial dysfunction was blunted. Conclusions Mmp2 knockout impaired Ang II-induced vascular injury but not BP elevation. BM transplantation revealed a role for immune cells in Ang II-induced BP elevation, and for both vascular and immune cell MMP2 in Ang II-induced endothelial dysfunction.

Background: Innate antigen-presenting cells and adaptive immune T cells have been implicated in the development of hypertension. However, the T-lymphocyte subsets involved in the pathophysiology of hypertension remain unclear. A small subset of innate-like T cells expressing the γδ T cell receptor (TCR) rather than the αβ TCR could play a role in the initiation of the immune response in hypertension. We aimed to determine whether angiotensin (Ang) II caused kinetic changes in γδ T cells; deficiency in γδ T cells blunted Ang II-induced hypertension, vascular injury, and T-cell activation; and γδ T cells are associated with human hypertension. Methods: Male C57BL/6 wild-type and Tcrδ −/− mice, which are devoid of γδ T cells, or wild-type mice injected IP with control isotype IgG or γδ T cell-depleting antibodies, were infused or not with Ang II for 3, 7, or 14 days. T-cell profiling was determined by flow cytometry, systolic blood pressure (SBP) by telemetry, and mesentery artery endothelial function by pressurized myography. TCR γ constant region gene expression levels and clinical data of a whole blood gene expression microarray study, including normotensive and hypertensive subjects, were used to demonstrate an association between γδ T cells and SBP. Results: Seven- and 14-day Ang II infusion increased γδ T-cell numbers and activation in the spleen of wild-type mice ( P P P Tcrδ −/− mice ( P P Tcrδ −/− mice ( P R 2 =0.12, P -6 ). Conclusions: γδ T cells mediate Ang II-induced SBP elevation, vascular injury, and T-cell activation in mice. γδ T cells might contribute to the development of hypertension in humans.

Background: Chromosome 2 (Chr2) introgression from normotensive Brown Norway (BN) rats into hypertensive Dahl salt-sensitive (SS) background (consomic S B 2) reduced blood pressure (BP) and vascular inflammation under normal-salt diet (NSD). We hypothesized that BN Chr2 contains anti-inflammatory genes that could reduce BP elevation and vascular inflammation in rats fed NSD and high-salt diet (HSD). Method: Four- to 6-week old male SS and congenic rats containing the BN Chr2 distal portion (S B 2a) and middle segment (S B 2b) were fed NSD or HSD (4% NaCl) for 8 weeks. We determined systolic BP (SBP) by telemetry, reactive oxygen species (ROS) generation using dihydroethidium staining, and vascular cell adhesion molecule 1 (VCAM-1), monocyte chemoattractant protein-1 (MCP-1) expression and monocyte/macrophage (MoMϕ) infiltration by immunofluorescence in aorta or perivascular fat (PVAT). RNA was extracted from aorta and used for small and total RNA sequencing and data were analyzed using a systems biology approach. Differentially expressed genes (DEGs) were identified with fold change >1.3 and fold discovery rate Results: SS SBP was 145±2 mm Hg under NSD and 168±1 mm Hg under HSD, which was lower in S B 2a and S B 2b SBP (125±3 and 127±6, P B 2a present less and more inflammation under NSD and HSD, respectively, compared to SS. DEGs were identified in S B 2a vs SS uniquely under NSD (15↑ and 8↓) and HSD (318↑ and 221↓) and under both diet (3↑ and 3↓), and in S B 2b vs SS uniquely under NSD (43↑ and 65↓) and HSD (4↑ and 7 ↓) and under both diet (3↑ and 4 ↓). DEGs encoded within BN Chr2a were uniquely identified under NSD (1↑ and 1↓) and HSD (6↑ and 1↓), and in both diets (1↑ and 1↓). Gene enrichment analysis revealed that under NSD, 2 BN Chr2a DEGs are involved in regulation of BP, and under HSD, 7 BN Chr2a DEGs in cell proliferation, cell differentiation, signal transduction and the immune system. RT-qPCR validated DEGs encoded within BN Chr2a under NSD (2) and HSD (4). Conclusions: DEGs encoded within BN Chr2a fragment are associated with anti- and pro-inflammatory effects under NSD and HSD, respectively.

BACKGROUND Increased endothelin (ET)-1 expression causes endothelial dysfunction and oxidative stress. Plasma ET-1 is increased in patients with diabetes mellitus. Since endothelial dysfunction often precedes vascular complications in diabetes, we hypothesized that overexpression of ET-1 in the endothelium would exaggerate diabetes-induced endothelial dysfunction. METHODS Diabetes was induced by streptozotocin treatment (55mg/kg/day, i.p.) for 5 days in 6-week-old male wild type (WT) mice and in mice overexpressing human ET-1 restricted to the endothelium (eET-1). Mice were studied 14 weeks later. Small mesenteric artery (MA) endothelial function and vascular remodeling by pressurized myography, reactive oxygen species (ROS) production by dihydroethidium staining and mRNA expression by reverse transcription/quantitative PCR were determined. RESULTS Endothelium-dependent vasodilatory responses to acetylcholine of MA were reduced 24% by diabetes in WT ( P < 0.05), and further decreased by 12% in eET-1 ( P < 0.05). Diabetes decreased MA media/lumen in WT and eET-1 ( P < 0.05), whereas ET-1 overexpression increased MA media/lumen similarly in diabetic and nondiabetic WT mice ( P < 0.05). Vascular ROS production was increased 2-fold by diabetes in WT ( P < 0.05) and further augmented 1.7-fold in eET-1 ( P < 0.05). Diabetes reduced endothelial nitric oxide synthase (eNOS, Nos3 ) expression in eET-1 by 31% ( P < 0.05) but not in WT. Induction of diabetes caused a 52% ( P < 0.05) increase in superoxide dismutase 1 ( Sod1 ) and a 32% ( P < 0.05) increase in Sod2 expression in WT but not in eET-1. CONCLUSIONS Increased expression of ET-1 exaggerates diabetes-induced endothelial dysfunction. This may be caused by decrease in eNOS expression, increase in vascular oxidative stress, and decrease in antioxidant capacity.

We investigated the role of angiotensin type 1a receptors (AGTR1a) in vascular injury induced by aldosterone activation of mineralocorticoid receptors in Agtr1a −/− and wild-type (WT) mice infused with aldosterone for 14 days while receiving 1% NaCl in drinking water. Aldosterone increased systolic blood pressure (BP) by ≈30 mm Hg in WT mice and ≈50 mm Hg in Agtr1a −/− mice. Aldosterone induced aortic and small artery remodeling, impaired endothelium-dependent relaxation in WT mice, and enhanced fibronectin and collagen deposition and vascular inflammation. None of these vascular effects were observed in Agtr1a −/− mice. Aldosterone effects were prevented by the AGTR1 antagonist losartan in WT mice. In contrast to aldosterone, norepinephrine caused similar BP increase and mesenteric artery remodeling in WT and Agtr1a −/− mice. Agtr1a −/− mice infused with aldosterone did not increase sodium excretion in response to a sodium chloride challenge, suggesting that sodium retention could contribute to the exaggerated BP rise induced by aldosterone. Agtr1a −/− mice had decreased mesenteric artery expression of the calcium-activated potassium channel Kcnmb1 , which may enhance myogenic tone and together with sodium retention, exacerbate BP responses to aldosterone/salt in Agtr1a −/− mice. We conclude that although aldosterone activation of mineralocorticoid receptors raises BP more in Agtr1a −/− mice, AGTR1a is required for mineralocorticoid receptor stimulation to induce vascular remodeling and inflammation and endothelial dysfunction.

Background: The immune system plays an important role in hypertension. Chromosome 2 (Chr2) introgression from normotensive Brown Norway (BN) rats into hypertensive Dahl salt sensitive (SS) background (consomic SB2) reduced blood pressure (BP), and vascular inflammation under a normal-salt diet (NSD). We hypothesized that BN Chr2 contains anti-inflammatory genes that could prevent BP elevation and vascular inflammation in rats fed NSD and high-salt diet (HSD). These genes will be identified using microRNA (miRNA) and total RNA sequencing in aorta of congenic rats containing different portions of BN Chr2 under NSD and HSD. Methods and Results: Four-to-6 week-old male SS and congenic SB2a and SB2b rats were fed a NSD or HSD (4% NaCl) for 8 weeks. Telemetry systolic BP (SBP) was lower in SB2a and SB2b compared to SS (125±3, 127±6 vs 146±2 mm Hg, P Ddah1 was down-regulated in SB2a vs SS rats fed NSD and HSD, while Acad9 , Agtr1b, ncRNA AABR07012047.1 , Fbxw7, Ptgfrn were down-regulated and ncRNA AABR07012585.3 , Bbs12, Kcnab1 were up-regulated in SB2b vs SS rats fed NSD and HSD. DE RNAs were confirmed by reverse transcription-quantitative PCR (RT-qPCR). Correlation between RNA-sequencing and RT-qPCR data was demonstrated for 6 of 9 tested RNAs: Kcnab1 (r=0.86, P Agtr1b (r=0.85, P Ddah1 (r=0.78 , P , Bbs12 (r=0.72, P Ptgfrn (r=0.54, P AABR07012047.1 (r=0.48, P Conclusions: BN Chr2 encoded DE genes were identified in aorta of congenic SB2a and SB2b rats fed NSD and HSD. Whether these genes play a role in vascular inflammation remains to be determined.

Background: Transgenic mouse with tamoxifen-inducible endothelium-restricted human ET-1 overexpression (ieET-1) exhibited blood pressure (BP) elevation three weeks (short-term) and three months after induction (long-term). Vascular injury was observed only after long-term exposure to endothelial ET-1 overexpression. It is unknown whether short or long-term exposure to ET-1 overexpression results in gene dysregulation. We aimed to identify differentially expressed (DE) microRNAs (miRs) and genes in mesenteric arteries of ieET-1 mice after short- and long-term induction of human ET-1 overexpression. Methods and Results: Ten to 12-week old male ieET-1 mice and control ieCre mice expressing a tamoxifen-inducible Cre recombinase under the control of endothelium-specific Tie2 promoter, were treated with tamoxifen (1 mg/kg/day, s.c.) for 5 days and sacrificed 16 days or 3 month later. RNA was extracted from mesenteric arteries of ieCre and ieET-1 mice and used for small and total RNA-sequencing using Illumina HiSeq-2500. EdgeR was used for differential expression analysis (false discovery rate Khdrbs3 (KH domain containing, RNA binding, signal transduction associated 3), which was up-regulated after both short- and long-term exposure to endothelial ET-1 overexpression, was validated by reverse transcription-quantitative PCR (RT-qPCR). We demonstrated a correlation between RNA-sequencing and RT-qPCR data for short- and long-term groups (r=0.8, P Khdrbs3 was 2 times more in fibroblasts than in smooth muscle cells and endothelial cells (2.37±0.28 vs. 1.11±0.17 and 1.12±0.12, P Conclusions: The results showed that short- and long-term exposure to endothelial ET-1 overexpression up-regulated Khdrbs3 in mesenteric arteries. In vitro study revealed that this gene was expressed to a greater level in fibroblasts than other vascular cells. However, the role of Khdrbs3 in ET-1-induced vascular injury remains to be determined.

Background: Rat chromosome (RNO) 2 introgression from normotensive Brown Norway (BN) rats into hypertensive Dahl salt sensitive (SS) background (consomic SB2) reduced vascular inflammation. We hypothesized that the BN-RNO2 contains genes that reduce vascular inflammation, which could be identified using microRNA (miRNA) and total RNA expression profiling in aorta of congenic rats containing different portions of BN-RNO2 on the SS background. Methods and Results: Twelve-to-13-week-old male SS rats and congenic rats containing the distal portion of BN-RNO2 (SB2a), the middle segment (SB2b) and the proximal segment (SB2e) on the SS background, fed a normal-salt diet, were studied. Systolic blood pressure (SBP) was measured by telemetry. SBP was lower in SB2a and SB2b but not SB2e compared to SS (125±3, 127±6, 138±4 vs 146±2 mm Hg, P Rattus norvegicus release-86, mirdeep2 for miRNA annotation and counting, Htseq-count for mRNA and long non-coding RNA annotation and counting; R for differential expression analysis. Differentially expressed miRNAs and genes (mRNA and non-coding RNA) were identified in SB2a vs SS (miRNAs: 3 up and 2 down, genes: 1 up and 3 down), SB2b vs SS (miRNAs: 2 up and 3 down, genes: 67 up and 112 down) and SB2e vs SS (miRNAs: 29 up and 25 down, genes: 12 up and 35 down), with FDR Conclusions and Perspectives: Differentially expressed BN-RNO2 encoded genes were identified in aorta of congenic SB2a, SB2b and SB2e rats. Whether these genes play a role in inflammation or vascular injury remains to be determined.

Introduction: Endothelium-derived endothelin (ET)-1 has been implicated in hypertension and renal disease but the mechanisms are complex and remain unclear. We have shown that tamoxifen-inducible endothelium-restricted human ET-1 overexpressing (ieET-1) mice exhibited BP rise after 3 weeks of induction in an ET type A receptor (ET A R)-dependent manner, in absence of renal injury. It is unknown whether long-term exposure to ET-1 overexpression results in sustained BP elevation and renal injury. Methods: Adult male ieET-1 and control tamoxifen-inducible endothelium-restricted Cre recombinase (ieCre) mice were treated with tamoxifen (1 mg/kg/day, SC) for 5 days and 2.5 months later were treated or not with an ET A R blocker, atrasentan (10 mg/kg/day, PO) for 2 weeks. Blood pressure (BP) by telemetry, renal artery flow (RAF) by ultrasonography, immune cell infiltration by flow cytometry, kidney injury molecule (KIM)-1 expression by immunofluorescence, 24h urinary albumin by ELISA and creatinine by alkaline picrate method were determined at the end of the study. Results: Induction of ET-1 overexpression for 3 months resulted in greater systolic BP (135±4 vs 114±2 mmHg, P P + cells/kidney, P + Gr-1 + cells/kidney, P + (23.2±1.8 vs 7.5±1.6 % of CD45 + cells, P - , 5.7±0.8 vs 3.2±0.6 % of CD45 - cells, P P P Conclusions: Long-term exposure to endothelial ET-1 overexpression caused sustained BP elevation and renal injury via ET A R.

Background: Vascular injury is an early manifestation of hypertension. microRNAs (miRNAs) play an important role in cardiovascular disease, but their implication in vascular injury remains unclear. Using small and total RNA sequencing, we identified in murine mesenteric arteries (MAs) a conserved angiotensin (Ang) II-upregulated Dlk1-Dio3 miRNA miR-431 that correlated with blood pressure (BP), and an Ang II-downregulated BP-correlated conserved putative miR-431 target, the transcriptional factor ETS homologous factor ( Ehf ). miR-431 might be involved in vascular remodeling as Ehf regulates expression of extracellular matrix genes including alpha-1 type I collagen ( Col1a1 ) and of other Dlk1-Dio3 miRNAs. In this study, we proposed to validate the miR-431- Ehf - Col1a1 interaction in vitro and in vivo , and determine whether miR-431 inhibition antagonizes angiotensin (Ang) II-induced hypertension and vascular injury. Methods and Results: Transfection of miR-431 mimics into human aortic smooth muscle cells decreased Ehf expression (0.13±0.05 fold, P Col1a1 (1.7±0.5 fold, P Ehf (1.5±0.2 fold, P Col1a1 (0.89±0.11 fold, P Ehf siRNA transfection increased 1.2±0.1 fold Col1a1 ( P EHF 3’ UTR binding site decreased 0.51±0.01 fold ( P P Col1a1 (0.58±0.11 fold, P Ehf (2.9±0.8 fold, P P P P Conclusion and Perspectives: miR-431 and its target Ehf may act as master regulators in the pathophysiology of vascular damage in hypertension. miR-431 inhibition has the potential to serve as a novel therapeutic approach for treatment of vascular damage and hypertension.

Background: Three congenic rat strains (SB2a, SB2b and SB2e) were created by chromosome (Chr) 2 fragment introgression from normotensive Brown Norway (BN) rats into hypertensive Dahl salt sensitive (SS) background. SB2a and SB2b rats fed a normal-salt diet presented reduced blood pressure (BP) and inflammation when compared to SS rats. We hypothesized that BN-Chr2 contains antihypertensive and anti-inflammatory genes that could prevent high-salt diet (HSD)-induced BP elevation and vascular injury in SB2a and SB2b rats. These genes will be identified using microRNA (miRNA) and total RNA expression profiling analysis in aorta of congenic rats fed a HSD. Methods and Results: Four-to-6 week-old male SS, SS, SB2a and SB2b rats were fed a HSD (4% NaCl) for 8 weeks or until they developed a stroke as manifested by seizures. Systolic blood pressure (SBP) was measured by telemetry. Systolic BP was higher in SB2b but not SB2a when compared to SS (185±8, 167±7 vs 168±5 mm Hg). Total RNA was extracted from aorta and used to construct libraries for small and total RNA sequencing using Illumina HiSeq-2500. The bioinformatics pipeline included: FastQC for quality control, STAR for genome (Rattus norvegicus, release-86) alignment, mirdeep2 for miRNA annotation and counting, Htseq-count for mRNA and long non-coding RNA annotation and counting; R for differential expression analysis. Differentially expressed miRNAs and genes (mRNAs and non-coding RNAs) were identified in SB2a vs SS (miRNAs: 11 up and 10 down; genes: 92 up and 91 down) and in SB2b vs SS (miRNAs: 3 up and 2 down; genes: 10 up and 13 down) with FDR Conclusions and Perspectives: Differentially expressed BN-Chr2 encoded genes were identified in aorta of congenic SB2a and SB2b rats fed HSD. Whether these genes play a role in HSD-induced BP elevation and vascular inflammation remains to be determined.

Aims Peroxisome proliferator-activated receptor γ (PPARγ) agonists reduce blood pressure and vascular injury in hypertensive rodents. Pparγ inactivation in vascular smooth muscle cells (VSMC) enhances vascular injury. Transgenic mice overexpressing endothelin (ET)-1 selectively in the endothelium (eET-1) exhibit endothelial dysfunction, increased oxidative stress and inflammation. We hypothesized that inactivation of the Pparγ gene in VSMC (smPparγ-/-) would exaggerate ET-1-induced vascular injury. Methods and results eET-1, smPparγ-/- and eET-1/smPparγ-/- mice were treated with tamoxifen for 5 days and studied 4 weeks later. SBP was higher in eET-1 and unaffected by smPparγ inactivation. Mesenteric artery vasodilatory responses to acetylcholine were impaired only in smPparγ-/-. N(omega)-Nitro-L-arginine methyl ester abrogated relaxation responses, and the Ednra/Ednrb mRNA ratio was decreased in eET-1/smPparγ-/-, which could indicate that nitric oxide production was enhanced by ET-1 stimulation of endothelin type B receptors. Mesenteric artery media/lumen was greater only in eET-1/smPparγ-/-. Mesenteric artery reactive oxygen species increased in smPparγ and were further enhanced in eET-1/smPparγ-/-. Perivascular fat monocyte/macrophage infiltration was higher in eET-1 and smPparγ and increased further in eET-1/smPparγ-/-. Spleen CD11b+ cells were increased in smPparγ-/- and further enhanced in eET-1/smPparγ-/-, whereas Ly-6C(hi) monocytes increased in eET-1 and smPparγ-/- but not in eET-1/smPparγ-/-. Spleen T regulatory lymphocytes increased in smPparγ and decreased in eET-1, and decreased further in eET-1/smPparγ-/-. Conclusion VSMC Pparγ inactivation exaggerates ET-1-induced vascular injury, supporting a protective role for PPARγ in hypertension through modulation of pro-oxidant and proinflammatory pathways. Paradoxically, ET-1 overexpression preserved endothelial function in smPparγ-/- mice, presumably by enhancing nitric oxide through stimulation of endothelin type B receptors.

Objective: Increased endothelin (ET)-1 expression has been shown to cause endothelial dysfunction and oxidative stress. Plasma ET-1 is increased in patients with diabetes mellitus. Since endothelial dysfunction often precedes vascular complications in diabetes, we sought to determine whether ET-1 contributes to diabetes-induced endothelial dysfunction. We hypothesized that overexpression of ET-1 in the endothelium will exaggerate diabetes-induced endothelial dysfunction. Methods: Diabetes was induced by streptozotocin IP injections (STZ, 55 mg/kg/day) for 5 days in 6-week-old male wild-type (WT) mice and in mice overexpressing human ET-1 restricted to the endothelium (eET-1). Mice were studied 14 weeks later. Endothelial function and vascular remodeling using pressurized myography, reactive oxygen species (ROS) production by dihydroethidium staining and mRNA expression by reverse transcription-quantitative PCR were assessed in mesenteric arteries (MA). Results: MA endothelium-dependent vasodilatory responses to acetylcholine were reduced 24% by diabetes in WT (E max : 61±6 vs 84±3%, P max : 49±5, P P P P 2 , P 2 , P Nos3) mRNA expression in eET-1 by 50% (0.7±0.1 vs 1.4±0.2, P Sod1 , 1.5±0.2 vs 1.0±0.0, P Sod2 (1.3±0.1 vs 1.0±0.0, P Conclusions: Increased expression of ET-1 exaggerates diabetes-induced endothelial dysfunction. This may be caused by an increase in vascular oxidative stress, a decrease in eNOS expression and a decrease in antioxidant capacity.

Background: The mechanisms of blood pressure (BP) regulation by endothelin (ET)-1 produced by endothelial cells are complex and remain unclear. Recently, we developed a transgenic mouse with tamoxifen-inducible endothelium-restricted human ET-1 overexpression (ieET-1) using Cre/loxP technology. ieET-1 mice exhibited BP rise after three weeks of induction in an ET type A (ET A ) receptor-dependent manner, in absence of vascular and kidney injury. It is unknown whether long-term exposure to ET-1 overexpression results in sustained BP elevation and vascular injury. Design and methods: Nine to 12-week old male ieET-1 mice and control ieCre mice expressing a tamoxifen-inducible Cre recombinase under the control of endothelium-specific Tie2 promoter, were treated with tamoxifen (1 mg/kg/day, s.c.) for 5 days and studied 3 months later. ieET-1 mice were treated or not with ET A receptor blocker, atrasentan (10 mg/kg/day, PO) in the last 2 weeks of the study. BP by telemetry, endothelial function and vascular remodeling by pressurized myography and reactive oxygen species (ROS) generation using dihydroethidium staining in mesenteric artery (MA) or perivascular fat (PVAT) and renal artery flow (RAF) by ultrasonography were determined. Results: Systolic BP was increased in ieET-1 and normalized by atrasentan compared to ieCre mice (141±0 and 124±4 vs 120±0 mm Hg, P P P 2 , P P Conclusions: Long-term exposure to endothelial human ET-1 overexpression caused sustained BP elevation, endothelial dysfunction and vascular stiffening and oxidative stress.

Objective: The prognosis of type-1 diabetes is in part related to the increased risk of vascular complications such as atherosclerosis. Overproduction of reactive oxygen species by NADPH oxidase (NOX) is believed to play an important role in diabetes-related vascular injury. NOX1 may play a role in the macrovascular disease, whereas NOX4 may have protective actions. Nevertheless, their role in diabetic vascular injury is less well understood. We hypothesized that deletion of Nox1 would prevent diabetes-induced endothelial dysfunction and vascular remodeling of small arteries whereas Nox4 would exaggerate vascular injury in atherosclerosis-prone apolipoprotein knockout ( Apoe -/- ) mice. Methods: Diabetes was induced by streptozotocin IP injections (STZ, 55 mg/kg/day) for 5 days in 6-week-old male Apoe -/- mice, Apoe -/- mice deficient in Nox1 ( Apoe -/- / Nox1 y/- ) and Nox4 ( Apoe -/- / Nox4 -/- ). Mice were studied 14 weeks later. Endothelial function and vascular remodeling were assessed in mesenteric arteries (MA) using pressurized myography. Results: Apoe -/- mice presented a maximal endothelium-dependent vasodilatory response (E max ) to acetylcholine of 48±8%, which was further decreased by diabetes to 20±6%. In contrast, endothelium-dependent relaxations to acetylcholine were 1.5-fold higher in diabetic Apoe -/- / Nox1 y/- and Apoe -/- / Nox4 -/- mice compared to non-diabetic Apoe -/- mice (E max : 72±9 and 70±7 vs 48±8%). Diabetes decreased MA stiffness in Apoe -/- mice, as indicated by a rightward displacement of the stress-strain curves (strain at 140 mm Hg: 1.02±0.04 vs 0.75±0.04), which was blunted by Nox1 or Nox4 knockout (strain at 140 mm Hg: 0.77±0.02 and 0.81±0.02). MA media/lumen was unaltered by diabetes. Knockout of Nox4 but not Nox1 increased MA media/lumen 1.4-fold in diabetic Apoe -/- mice (4.1±0.4 and 3.5±0.2 vs 2.9±0.2%). Conclusions: These results suggest that NOX1 and NOX4 play a pathophysiological role in diabetes-induced endothelial dysfunction and contribute to potentially maladaptive changes in vascular stiffness. NOX4 seems to have dual actions on the vasculature, as it is also protective against vascular remodeling of small arteries in type 1 diabetes.

Objective: NADPH oxidase (NOX) 1 but not NOX4-dependent oxidative stress plays a role in diabetic vascular disease, including atherosclerosis. Endothelin (ET)-1 has been implicated in diabetes-induced vascular complications. We showed that crossing mice overexpressing ET-1 selectively in endothelium (eET-1) with apolipoprotein E knockout ( Apoe -/- ) mice exaggerated high-fat diet-induced atherosclerosis in part by increasing oxidative stress. We hypothesized that ET-1 overexpression in the endothelium would exaggerate diabetes-accelerated atherosclerosis through a mechanism involving NOX1 but not NOX4. Methods: Six-week-old male Apoe -/- mice, eET-1/ Apoe -/- and eET-1/ Apoe -/- mice deficient in Nox1 (eET-1/ Apoe -/- / Nox1 y/- ) or Nox4 (eET-1/ Apoe -/- / Nox4 -/- ) were rendered diabetic with 55 mg/kg/day streptozotocin (STZ) IP injections for 5 days and studied 14 weeks later. Endothelial function and vascular remodeling were assessed in mesenteric arteries (MA) using pressurized myography. Aortic atherosclerotic lesions were quantified using Oil Red O staining. Plasma cholesterol, HDL and triglycerides were measured. Results: Diabetic Apoe -/- mice presented an impaired endothelium-dependent vasodilatory response to acetylcholine, which was not observed in diabetic eET-1/ Apoe -/- , eET-1/ Apoe -/- / Nox1 y/- or eET-1/ Apoe -/- / Nox4 -/- mice (E max : 20±6 vs 99±1, 98±1 and 100±0%). ET-1 overexpression caused a 1.8-fold increase in MA media/lumen of diabetic Apoe -/- mice (5.3±0.3 vs 2.9±0.2%), which was further increased 1.2-fold by Nox4 (6.4±0.3%) but not Nox1 knockout (5.5±0.3%). ET-1 overexpression exaggerated >2-fold the atherosclerotic lesion area in the aortic sinus in diabetic Apoe -/- mice (plaque area [x10 5 μm 2 ]: 5.3±0.5 vs 2.9±0.6), which was reduced ~40% by Nox1 and Nox4 knockout (plaque area [x10 5 μm 2 ]: 3.3±0.6 and 3.6±0.6). Plasma triglycerides were unaffected by ET-1 overexpression but reduced by Nox1 (2.2±0.4 vs 3.4±0.3 mmol/L) and Nox4 knockout (1.8±0.4 mmol/L). Plasma HDL and cholesterol were similar between groups. Conclusions: Increased levels of ET-1 exaggerate diabetes-accelerated atherosclerosis through NOX1 and NOX4, despite paradoxically improving endothelium-dependent relaxation in small arteries.

Objective: Peroxisome proliferator-activated receptor γ (PPARγ) agonists reduce blood pressure (BP) and vascular injury in hypertensive rodents. Pparγ inactivation in vascular smooth muscle cells (VSMC) using a tamoxifen inducible Cre-Lox system enhanced angiotensin II-induced vascular damage. Transgenic mice overexpressing endothelin (ET)-1 in the endothelium (eET-1) exhibit endothelial dysfunction, increased oxidative stress and inflammation. We hypothesized that inactivation of the Ppar gene in VSMC (sm Pparγ -/- ) would exaggerate ET-1-induced vascular damage. Methods and Results: Eleven-week-old male control, eET-1, sm Pparγ -/- and eET-1/sm Pparγ -/- mice were treated with tamoxifen (1 mg/kg/day, s.c.) for 5 days and sacrificed 4 weeks later. Systolic BP determined by telemetry was higher in eET-1 (123±5 vs 109±2 mm Hg, P Pparγ inactivation. Mesenteric artery (MA) vasorelaxation to acetylcholine was impaired only in sm Pparγ -/- (E max : 52.0±6.7 vs 82.2±4.9%, P Pparγ -/- ( P Pparγ -/- (2.5±0.3-fold, P Pparγ -/- (331±34 and 326±49 vs 140±8 cells/mm 2 , P Pparγ -/- (557±77, P + cells was increased in sm Pparγ -/- (1.1±0.1 vs 0.47±0.1%, P Pparγ -/- (1.8±0.2%, P hi monocytes was increased in eET-1 and sm Pparγ -/- (24±3 and 27±4 vs 14±1%, P Pparγ -/- . The spleen fraction of T regulatory cells was increased in sm Pparγ -/- (13±2 vs 9±1%, P P Pparγ -/- (3±1%, P Conclusions: These results suggest that increased ET-1 paradoxically preserves endothelial function in mice with inactivated VSMC Pparγ despite enhanced oxidative stress. Flow cytometry data indicate that infiltrating monocyte/macrophages in these mice might be anti-inflammatory.

Background: The mechanisms of blood pressure (BP) regulation by endothelin (ET)-1 produced by endothelial cells are complex and remain unclear. Recently, we developed a transgenic mouse with tamoxifen-inducible endothelium-restricted human ET-1 overexpression (ieET-1) using Cre/loxP technology. ieET-1 mice exhibited BP rise after three weeks of induction in an ET type A receptor-dependent manner, in absence of vascular and kidney injury. It is unknown whether long-term exposure to ET-1 overexpression results in elevated BP elevation and vascular injury. Methods: Nine to 12-week old male ieET-1 mice and control ieCre mice expressing a tamoxifen-inducible Cre recombinase (CreERT2) under the control of EC-specific Tie2 promoter, were treated with tamoxifen (1 mg/kg/day, s.c.) for 5 days and studied 3 months later. Metabolic cages were used to collect 24-hour urine for sodium, potassium and protein measurements. Renal artery flow (RAF) was assessed by ultrasonography. BP was determined by telemetry, plasma aldosterone by ELISA, and small mesenteric artery (MA) endothelial function and vascular remodeling by pressurized myography. Results: Systolic BP was increased in ieET-1 compared with ieCre mice (144±5 vs 117±3 mmHg, P Conclusions: The results demonstrate that long-term exposure to endothelial ET-1 overexpression caused sustained BP rise and small artery stiffening.

Objective: Peroxisome proliferator-activated receptor γ (PPARγ) agonists reduce blood pressure (BP) and vascular injury in hypertensive rodents and humans. Pparγ inactivation in vascular smooth muscle cells (VSMC)using a tamoxifen inducible Cre-Lox system enhanced angiotensin II-induced vascular injury. Transgenic mice overexpressing endothelin (ET)-1selectively in the endothelium (eET-1) exhibit endothelial dysfunction, increased oxidative stress and inflammation. We hypothesized that inactivation of Pparγ in VSMC(smPparγ-/-)will exaggerate ET-1-induced vascular damage. Methods and Results: Elevenweek-old male control, eET-1, smPparγ-/-and eET-1/smPparγ-/- mice weretreated with tamoxifen (1 mg/kg/day, s.c.) for 5 days and sacrificed 4 weeks later. Systolic BP was higher in eET-1compared to control (123±5 vs 109±2 mmHg,P Conclusion: Increased ET-1 paradoxically preserves endothelial function in mice with smPparγinactivation, despite enhancedoxidative stress and inflammation.

Objective: Increased endothelin (ET)-1 expression has been shown to cause endothelial dysfunction.Plasma ET-1 is increased in patients with diabetes.Since endothelial dysfunction often precedes vascular complications in diabetes, we sought to determine whether ET-1 contributes to diabetes-induced endothelial dysfunction. We hypothesized that overexpression of ET-1 in the endotheliumwill exaggerate diabetes-induced endothelial dysfunction. Method: Diabetes was induced by streptozotocin treatment (STZ, 55 mg/kg/day, ip) for 5 days in 6weekold male wild-type (WT) mice and in mice overexpressinghuman ET-1 restricted to the endothelium (eET-1). Mice were studied 14 weeks later. Blood glucose,plasma ET-1 levels, mesenteric artery (MA) reactivity, mitochondrial superoxide production in aorta and endothelial nitric oxide synthase (Nos3), superoxide dismutase 1 (Sod1) and 2 (Sod2) mRNA expression in MA were determined. Results: STZ-induced diabetes was confirmed by increased glycemia in WT(27.6±1.5 vs 10.7±1.1 μM, P Conclusions: Increased levels of ET-1 exaggerate diabetes-induced endothelial dysfunction. This may be caused by a decrease in Nos3 expression, an increase in mitochondrial oxidative stress and a decrease in antioxidant capacity.

Objective: Peroxisome proliferator activated receptor γ (PPARγ) agonists reduce blood pressure (BP) and vascular injury in hypertensive rodents and humans. Pparγ inactivation in vascular smooth muscle cells (VSMC) using a tamoxifen inducible Cre-Lox system enhanced angiotensin II-induced vascular remodeling and endothelial dysfunction. Transgenic mice overexpressing endothelin (ET)-1 selectively in the endothelium (eET-1) exhibit endothelial dysfunction, increased oxidative stress and inflammation. We hypothesized that inactivation of the Ppar gene in VSMC (sm Pparγ -/- ) will exaggerate ET-1-induced vascular damage. Methods: Eleven week-old male control, eET-1, sm Pparγ -/- and eET-1/sm Pparγ -/- mice were used. BP by telemetry, mesenteric artery (MA) reactivity and structure by pressurized myography, reactive oxygen species (ROS) by dihydroethidium (DHE) staining and fibronectin expression by immunofluorescence were determined. Results: Systolic BP was higher in eET-1 and eET-1/sm Pparγ -/- compared to control and sm Pparγ -/- (127±3 and 124±7 vs 109±2 and 114 ± 4 mmHg, respectively, P Pparγ -/- ( P Pparγ -/- /eET-1 compared with control (E max : 52.0±6.7%, 66.2±7.6 and 87.1±4.4% vs 82.2±4.9%). Endothelium-independent relaxation responses to the nitric oxide donor, sodium nitroprusside, were similar in the four groups. Media/lumen at 45 mmHg was higher only in eET-1/sm Pparγ -/- compared with control (5.5±0.21 vs 4.5±0.21, P Pparγ -/- and eET-1/sm Pparγ -/- compared to control, as indicated by a leftward displacement of the stress-strain curves (strain at 140 mmHg: 0.82±0.03, 0.81±0.04, 0.80±0.03 vs 0.97±0.02, P Pparγ -/- and 2.8±0.4 fold in eET-1/sm Pparγ -/- compared with control ( P Conclusion: These results suggest that increased ET-1 paradoxically preserves endothelial function in mice with inactivated Pparγ in vascular smooth muscle, despite presence of enhanced oxidative stress.

Background: Peroxisome proliferator activated receptor γ (PPARγ) agonists improve vascular remodeling and endothelial dysfunction in hypertensive rodents and humans. PPARγ activation in vascular smooth muscle cells (VSMC) may be responsible for the vascular protective effects of PPARγ agonists. We previously observed a protective role of VSMC PPARγ in angiotensin II-induced endothelial dysfunction and vascular remodeling. However, it is unknown whether VSMC PPAR plays a similar protective role in adverse vascular effects of aldosterone. We hypothesized that inactivation of the Ppar gene in VSMC (sm Pparγ -/- ) would exaggerate aldosterone-induced vascular injury. Methods: Using a tamoxifen-inducible Cre/loxP system, Pparγ was ablated in VSMC of adult mice. Thirteen week-old control and sm Pparγ -/- mice were infused or not with aldosterone (400 μg/kg/d, SC) for 14 days while receiving 1% NaCl/0.3% KCl in drinking water. Endothelial function and vascular remodeling were assessed in mesenteric arteries (MA) by pressurized myography. Results: Endothelium-dependent relaxation (EDR) responses to acetylcholine were reduced to a similar extent in sm Pparγ -/- and aldosterone-treated control and sm Pparγ -/- mice compared to control mice (E max : 62.5±8.7%, 50.8±8.6% and 56.8±7.9%, respectively, vs 86.4±3.2%, P Pparγ deletion induced an increase in MA stiffness compared to control, which was not worsened by aldosterone (strain at 140 mmHg, 0.67±0.01, 0.65±0.03, vs 0.89±0.03). Conclusion: These results indicate that either VMSC Pparγ inactivation or aldosterone treatment induce vascular remodeling and endothelial dysfunction, which are not mutually exaggerated. This suggests that PPARγ and aldosterone signal intracellularly through different pathways.

Objective: Vascular disease associated with endothelial dysfunction is a major cause of morbidity in patients with type-1 diabetes. Endothelin (ET)-1 plays a role in diabetes-induced vascular complications, since ET-1 type A receptor blockade reduces diabetes-induced vascular injury. However, whether ET-1 contributes to diabetes-induced endothelial dysfunction remains unproven. We hypothesized that vascular ET-1 overexpression will exaggerate diabetes-induced endothelial dysfunction. Methods: Diabetes was induced by streptozotocin treatment (STZ, 55 mg/kg/day, ip) for 5 days in 6-week-old male wild-type (WT) mice and in mice overexpressing ET-1 restricted to the endothelium (eET-1). Mice were studied 14 weeks later. Blood was collected to determine glucose. Mesenteric artery reactivity and remodeling were evaluated using pressurized myography and aortic fibronectin expression by immnunofluorescence. Results: STZ-induced diabetes was confirmed by a 3-fold increase in glycemia in WT and eET-1 ( P P P P P P P P 2 , P 2 , P Conclusion: ET-1 contributes to alterations in several pathways mediating endothelium-dependent relaxation in type-1 diabetes, leading to exaggerated endothelial dysfunction.