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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.

Objective Mechanisms of blood pressure (BP) regulation by endothelin (ET)-1 produced by endothelial cells are complex and remain unclear. Long-term exposure to human ET-1 (hET-1) in mice inducibly overexpressing hET-1 in the endothelium (ieET-1) caused sustained BP elevation. ET-1 has been shown to stimulate the release of aldosterone. Whether aldosterone plays a role in hET-1 overexpression-induced BP elevation and vessel injury is unknown. Method Nine- to 12-week-old male ieET-1 mice and control mice expressing a tamoxifen-inducible Cre recombinase (CreERT2) in the endothelial cells (ieCre) were treated with tamoxifen for 5 days and studied 3 months later. Results Endothelial hET-1 overexpression increased plasma aldosterone levels, which was reversed by 2-week treatment with atrasentan, an endothelin type A receptor blocker. Aldosterone synthase and cryptochrome 2 adrenal cortex mRNA expression was decreased in ieET-1 mice. Two-week treatment with eplerenone, a mineralocorticoid receptor antagonist, reduced systolic BP by 10 mmHg in ieET-1 mice during rest time. Saline challenge-induced sodium excretion and renal cortex thiazide-sensitive sodium-chloride cotransporter mRNA expression were decreased in ieET-1 mice. The sensitivity of mesenteric arteries to contraction by norepinephrine was increased in ieET-1 mice, and was abrogated by eplerenone treatment, whereas sensitivity of endothelium-independent relaxation responses to sodium nitroprusside was enhanced. Resistance artery remodeling was reduced in eplerenone-treated ieET-1 vs. ieET-1 and ieCre mice. Conclusion These results demonstrate that aldosterone contributes to BP elevation and vascular norepinephrine sensitivity and remodeling caused by hET-1 overexpression in endothelium in mice.

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.

Background: The mechanisms of blood pressure (BP) regulation by endothelin (ET)-1 produced by endothelial cells (ECs) are complex and remain unclear. Transgenic mice with tamoxifen-inducible EC-restricted human ET-1 overexpression (ieET-1) exhibited BP elevation 3 months after induction. ET-1 has been shown to stimulate the release of aldosterone from adrenal cortex (AC). Whether aldosterone plays a role in EC ET-1 overexpression-induced BP elevation and vascular injury is still unknown. Methods and results: Nine to 12-week-old male ieET-1 mice and control ieCre mice expressing a tamoxifen-inducible Cre recombinase under the control of EC-specific Tie2 promoter were treated with tamoxifen (1 mg/kg/day, SC) for 5 days and studied 3 months later. Plasma aldosterone levels measured by ELISA was increased in ieET-1 vs. ieCre mice (1.21±0.14 vs. 0.70±0.09 ng/mL, P Cyp11b2 ) was decreased in AC of ieET-1 vs. ieCre mice (fold change: 0.52±0.06 vs. 1.00±0.16, P P -7 ±6x10 -8 vs. 4x10 -6 ±7x10 -7 , P -6 ±5x10 -6 , P -8 ±1x10 -8 vs. 1x10 -7 ±2x10 -8 , P Conclusions: Increased aldosterone production contributes to EC human ET-1 overexpression-induced daytime SBP elevation, enhanced contractile response to norepinephrine and endothelial dysfunction.

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 mechanisms of blood pressure (BP) regulation by endothelin (ET)-1 produced by endothelial cells are complex and remain unclear. Long-term exposure to endothelial human ET-1 overexpression causes sustained blood pressure elevation via ETA receptors. ET-1 has been shown to stimulate the release of aldosterone from the adrenal cortex. Whether aldosterone plays a role in ET-1 endothelium overexpression-induced BP elevation is still unknown. Methods and Results: Nine to 12-week-old male ieET-1 mice and control ieCre mice expressing a tamoxifen-inducible Cre recombinase (CreER T2 ) under the control of EC-specific Tie2 promoter, were treated with tamoxifen (1 mg/kg/day, SC) for 5 days and studied 3 months later. Plasma aldosterone level measured by ELISA was higher in ieET-1 compared with ieCre mice (1.21±0.14 vs. 0.70±0.09 ng/mL, P Scnn1a ), TSC22 domain family member 3 ( Rsc22d3 ), serum- and glucocorticoid-induced kinase 1 ( Sgk1 ), and ET type A and B receptors in the kidney or adrenal glands. The mRNA expression of aldosterone synthase ( Cyp11b2 , fold change: 0.52±0.06 vs 1.00±0.16, P Hsd3b1) and 6 (Hsd3b6) or steroidogenic acute regulatory protein ( Star ), were decreased in the adrenal cortex of ieET-1 compared with ieCre mice. CYP11B2 and HSD3B1 protein levels measured by Western Blotting were unchanged. Treatment of ieET-1 mice with MR antagonist eplerenone (100 mg/kg per day) during the last 2 weeks decreased systolic BP more during the day (128±2 vs. 134±3 mm Hg) than during the night (137±2 vs. 140±2 mm Hg) compared to untreated ieET-1 mice. Conclusions: These results showed that aldosterone contributes at least in part to the BP elevation caused by endothelial human ET-1 overexpression.

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.

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 This study evaluated the involvement of tumour necrosis factor α (TNF-α) in orofacial thermal and mechanical hyperalgesia induced by an inflammatory stimulus or by chronic constriction of the infraorbital nerve (CION) using etanercept (Eta), a TNF-receptor fusion protein that inhibits TNF-α action. Methods Animals were treated with Eta (0.5 and 5.0 mg/kg, s.c.) or dexamethasone (Dex, 0.5, 1.0 and 2.0 mg/kg, s.c.) and orofacial thermal (cold and heat) and mechanical hyperalgesia induced by an inflammatory stimulus (carrageenan, Cg 50 and 100 μg/lip) or by chronic CION, a model of neuropathic pain in the orofacial region was evaluated. Treatments with Dex or Eta were carried out before Cg or before or after CION. Results Eta or Dex abolished inflammatory thermal and mechanical hyperalgesia. Also, each drug, when given at the day of the surgery and the subsequent day, was effective to abolish thermal and mechanical hyperalgesia induced by CION, assessed on day 4 and on day 13 after the surgery, respectively. However, Eta, but not Dex, given after the CION, abolished thermal and mechanical hyperalgesia and reduced TNF-α level in the trigeminal ganglion. Conclusions These results suggest that TNF-α has an important role in cold, heat and mechanical hyperalgesia induced by inflammation or neuropathy in the orofacial region and this may contribute for the establishment of new therapeutic strategies to treat orofacial pain.

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.

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.

The mechanisms of blood pressure regulation by endothelin-1 produced by endothelial cells are complex and still unclear. Transgenic mice with endothelium-restricted human endothelin-1 ( EDN1 ) overexpression presented vascular damage but no significant change in blood pressure, which could be because of adaptation to life-long exposure to elevated endothelin-1 levels. We now generated a tamoxifen-inducible endothelium-restricted EDN1 overexpressing transgenic mouse (ieET-1) using Cre/loxP technology. Sixteen days after tamoxifen treatment, ieET-1 mice presented ≥10-fold increase in plasma endothelin-1 ( P P P

Objective:Aldosterone is known to induce hypertension and vascular injury. A large body of evidence suggests a crosstalk between aldosterone and angiotensin II pathways. We demonstrated in vitro that vascular smooth muscle cell activation of signaling pathways such as ERK1/2 requires functional angi

Introduction: The mechanisms of blood pressure (BP) regulation by endothelin (ET)-1 produced by endothelial cells (EC) are complex and remain unclear. Transgenic mice with constitutive EC-specific human ET-1 ( EDN1 ) overexpression presented vascular injury but no change in BP, which could be due to adaptation to life-long high ET-1 exposure. We have now generated an inducible EC-restricted EDN1 overexpressing mouse (ieET-1) in order to demonstrate the effects of ET-1 on BP regulation independent of developmental effects. Method: Two transgenic mouse lines (C134 and C170) expressing chloramphenicol acetyltransferase ( cat ) and EDN1 before and after Cre-mediated excision, respectively, were crossed with mice expressing tamoxifen-inducible CreER T2 under the control of Tie2 promoter (ieCre) to generate ieET-1 mice. Mice were treated with tamoxifen (1 mg/kg/day, SC) or vehicle for 5 days and sacrificed 16 days later. Additional mice were treated with 5 or 10 mg/kg/day PO of ET type A receptor blocker, atrasentan, from day 10. BP was determined by telemetry. Plasma ET-1 levels were assessed by ELISA. ET type A and B receptors expression, Ednra and Ednrb , were evaluated in the kidney by quantitative PCR. Results: Tamoxifen increased plasma ET-1 in ieET-1 C134 and C170 (7.1±0.7 and 13.2±2 pg/mL, respectively, P (0.8±0.1 pg/mL). ET-1 overexpression increased night systolic BP in ieET-1 C134 and C170 (137±4 and 132±4 mmHg) compared to tamoxifen-treated ieCre (116±7 mmHg), which was reversed partially or completely with 5 or 10 mg/kg/day of atrasentan, respectively (122±3 and 115±4 mmHg, P Ednra expression in renal cortex and medulla (2.9±0.6 and 8.2±3.0 fold, P Ednrb levels in the renal cortex (2.0±0.3 fold, P Conclusion: Our results demonstrate that this new inducible EC-restricted EDN1 overexpressing mouse exhibits ET-1-dependent elevated BP mediated by ET type A receptors. The increased expression of renal ET receptors could play a role in ET-1-induced BP rise.

Objective: To develop a model permitting the study of the mechanisms of endothelin (ET-1)-induced vascular injury, we generated an inducible and endothelial cell (EC)-restricted human ET-1 ( EDN1 ) cDNA overexpressing transgenic mouse, which would accordingly be devoid of developmental effects. Methods and Results: A transgene was engineered that expresses chloramphenicol acetyltransferase ( cat ) cDNA prior to Cre-mediated excision, and EDN1 cDNA after Cre-mediated excision, under the control of cytomegalovirus immediate early enhancer/chicken β-actin promoter (CAG). Co-transfection of Cre expression vector and pCAG- cat - EDN1 caused a 30-fold increase in ET-1 production evaluated by ELISA. A DNA fragment containing the CAG- cat - EDN1 transgene was used to generate transgenic mice. PCR genotyping demonstrated that 14/132 mice were transgenic. Two of 7 transgenic lines were selected based on cardiac cat expression level, which were twice higher in line C-134 than C-170. Inducible EC-restricted EDN1 mice were generated crossing CAG- cat - EDN1 mice with mice expressing Cre recombinase fused to a tamoxifen (TAM)-inducible modified estrogen receptor ligand binding domain ( Cre ER T2 ) under control of EC-specific receptor tyrosine kinase ( Tie2 ) promoter. To investigate the extent of CreER T2 activation by TAM and tissue specificity, Tie2 -CreER T2 mice were crossed with ROSA mT-mG/mT-mG reporter mice expressing a membrane-targeted tandem dimer tomato (mT) before Cre-mediated excision, and membrane-targeted enhanced green fluorescent protein (mG) after excision. Tie2 -CreER T2 / cat - EDN1 and Tie2 -CreER T2 / ROSA mT-mG/+ mice were treated with 1 mg TAM/d SC for 5 d and sacrificed 14-16 d later. mG expression was detected in 22±3% of mesenteric artery EC of Tie2 -CreER T2 / ROSA mT-mG/+ mice. Plasma ET-1 levels were similar in vehicle-treated Tie2 -CreER T2 / cat - EDN1 (1.2±0.1 pg/mL, n=4) and wild-type mice (1.1±0.1 pg/mL, n=2), demonstrating no leaky expression. TAM induced 8-fold increase in plasma ET-1 levels in Tie2 -CreER T2 / cat - EDN1 mice (9.1 ± 0.3 pg/mL, n = 4). Conclusion: Tie2 -CreER T2 / cat - EDN1 -inducible EC-restricted EDN1 overexpressing mice allow the study of ET-1 vascular effects in adult independently of developmental effects.