Suellen C. Coelho

Inducible Human Endothelin-1 Overexpression in Endothelium Raises Blood Pressure via Endothelin Type A Receptors

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<0.01) and ≥20 mm Hg elevation in systolic blood pressure (P<0.01), which could be reversed by atrasentan (P<0.05). Endothelin-1 overexpression did not cause vascular or kidney injury or changes in kidney perfusion or function. However, endothelin type A and B receptor expression was differentially regulated in the mesenteric arteries and the kidney. Our results demonstrate using this ieET-1 mouse model that 21 days of induction of endothelin-1 overexpression caused endothelin-1–dependent elevated blood pressure mediated by endothelin type A receptors.

Aldosterone-Induced Vascular Remodeling and Endothelial Dysfunction Require Functional Angiotensin Type 1a Receptors

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.

Three-Month Endothelial Human Endothelin-1 Overexpression Causes Blood Pressure Elevation and Vascular and Kidney InjuryNovelty and Significance

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 (ETA) 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 ETA receptor blocker atrasentan for 2 weeks. Three-month induction of endothelial human ET-1 overexpression increased blood pressure (P<0.01), reduced renal artery flow (P<0.001), and caused mesenteric small artery stiffening (P<0.05) and endothelial dysfunction (P<0.01). These changes were accompanied by enhanced mesenteric small artery Col1A1 and Col3A1 expression, and perivascular adipose tissue oxidative stress (P<0.05) and monocyte/macrophage infiltration (P<0.05). Early renal injury was demonstrated by increased kidney injury molecule-1 expression in renal cortex tubules (P<0.05), with, however, undetectable lesions using histochemistry staining and unchanged urinary albumin. There was associated increased myeloid (CD11b+) and myeloid-derived suppressive cell (CD11b+Gr-1+) renal infiltration (P<0.01) and greater frequency of myeloid and renal cells expressing the proinflammatory marker CD36 (P<0.05). Atrasentan reversed or reduced all of the above changes (P<0.05) except the endothelial dysfunction and collagen expression and reduced renal artery flow. These results demonstrate that long-term exposure to endothelial human ET-1 overexpression causes sustained blood pressure elevation and vascular and renal injury via ETA receptors.

OS 21-01 NOX1 OR NOX4 DELETION PREVENTS TYPE 1 DIABETES-INDUCED ENDOTHELIAL DYSFUNCTION.

Objective : The prognosis of type-1 diabetes remains poor and is primarily related to the increased risk of vascular complications. 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 microangiopathy 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. Design and Method : Diabetes-related vascular injury were studied in atherosclerosis-prone apolipoprotein knockout (Apoe-/-) mice. Six-week-old male Apoe-/- mice, Apoe-/- mice deficient in Nox1 (Apoe-/-/Nox1y/-) and Nox4 (Apoe-/-/Nox4-/-) were rendered diabetic by streptozotocin treatment (STZ, 55 mg/kg/day, ip) for 5 days. 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 to acetylcholine of only 46%, which was further decreased by ∼50% by diabetes. In contrast, endothelium-dependent relaxations to acetylcholine were 1.5-fold higher in diabetic Apoe-/-/Nox1y/- and Apoe-/-/Nox4-/- mice compared to vehicle-treated Apoe-/- mice. Endothelium-independent relaxation responses to the nitric oxide donor, sodium nitroprusside, were similar in all groups. Diabetes decreased MA stiffness in Apoe-/- mice, as indicated by a rightward displacement of the stress-strain curves, which was blunted by Nox1 or Nox4 knockout. MA media/lumen was unaltered by diabetes. Knockout of Nox4 but not Nox1 increased MA media/lumen 1.4-fold in diabetic Apoe-/- mice. 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 also seems to have dual actions on the vasculature, as it is also protective against vascular remodeling of small arteries in type 1 diabetes.

OS 21-06 THREE-MONTH EXPOSURE TO ENDOTHELIN-1 OVEREXPRESSION INCREASES BLOOD PRESSURE AND CAUSES SMALL ARTERY INJURY.

Objective: 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 a BP increase after three weeks of induction in an ET type A receptor-dependent manner, in absence of evident vascular injury. It is unknown whether long-term exposure to ET-1 overexpression results in persistent BP elevation and vascular injury. Design and Method: 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. BP by telemetry, mesenteric artery (MA) endothelial function and vascular remodeling using pressurized myography and reactive oxygen species (ROS) generation using dihydroethidium staining and immune cell infiltration by immunofluorescence in MA or perivascular fat (PVAT) were determined at the end of the study. Results: Systolic BP was increased by 27 mmHg in ieET-1 compared with ieCre mice (P < 0.001). Endothelium-dependent relaxation responses to acetylcholine were decreased by 50% in ieET-1 compared to ieCre mice (P < 0.01), whereas endothelium-independent relaxation to sodium nitroprusside were unchanged. MA media/lumen and media cross-sectional area were similar in both groups, but stiffness was increased in ieET-1 compared to ieCre mice, as indicated by leftward displacement of the stress-strain curves (14% decrease in strain at 140 mmHg, P < 0.05). ROS generation was enhanced 1.5-fold in PVAT of ieET-1 compared to ieCre mice (P < 0.05). Monocyte/macrophage infiltration was similar whereas CD3+ cell infiltration was 1.4-fold higher in MA PVAT of ieET-1. Conclusions: The results demonstrate that long-term exposure to endothelial ET-1 overexpression caused sustained BP elevation, endothelial dysfunction and vascular stiffening, oxidative stress and CD3+ cell infiltration.

PS 16-31 ENDOTHELIN-1 EXAGGERATES TYPE-1 DIABETES-ACCELERATED ATHEROSCLEROSIS THROUGH NADPH OXIDASES 1 AND 4

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. Design and method: Six-week-old male Apoe−/− mice, eET-1/Apoe−/− and eET-1/Apoe−/− mice deficient in Nox1 (eET-1/Apoe−/−/Nox1y/−) or Nox4 (eET-1/Apoe−/−/Nox4−/−) were rendered diabetic with 55 mg/kg/day streptozotocin (STZ) IP 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. Results: Diabetic Apoe−/− mice presented an impaired maximal endothelium-dependent vasodilatory response to acetylcholine (21%), which was not observed in diabetic eET-1/Apoe−/−, eET-1/Apoe−/−/Nox1y/− or eET-1/Apoe−/−/Nox4−/− mice (99%). Endothelium-independent relaxation to the nitric oxide (NO) donor sodium nitroprusside was similar between groups. Blockade of NO synthase with L-NAME completely blunted endothelium-dependent relaxation to acetylcholine in diabetic eET-1/Apoe−/− mice, which was prevented by Nox1 but not by Nox4 knockout. ET-1 overexpression caused a 1.8-fold increase in MA media/lumen of diabetic Apoe−/− mice, which was further exaggerated 1.2-fold by Nox4 but not Nox1 knockout. ET-1 overexpression exaggerated > 2-fold the atherosclerotic lesion area in the aortic sinus in diabetic Apoe−/− mice, which was reduced ∼40% by Nox1 and Nox4 knockout. Conclusions: Increased levels of ET-1 exaggerate diabetes-accelerated atherosclerosis through NOX1 and NOX4, despite paradoxically improving endothelium-dependent relaxation in small arteries.

MPS 18-08 MAPPING OF CHROMOSOME 2 REGIONS LINKED TO VASCULAR INFLAMMATION USING CONGENIC RATS

Objective: Chromosome 2 (chr2) introgression from normotensive Brown Norway rats (BN) into hypertensive Dahl salt sensitive (SS) background (consomic SS2BN) reduced blood pressure (BP) and vascular inflammation under a normal-salt diet. Mapping of chr2 using congenic rats revealed that the distal portion of BN chr2 (SS2BNa) but not the middle segment (SS2BNb) on the SS background under a normal-salt diet contains anti-inflammatory genes. However, the role of chr2 in the regulation of vascular inflammation under a high-salt diet (HSD) remains unknown. We hypothesized that SS2BNa but not SS2BNb rats would have reduced vascular inflammation under HSD. Design and Method: Four-to-6 week old male SS, SS2BNa and SS2BNb rats were fed a HSD (4% NaCl) for 8 weeks or until they developed a stroke as manifested by seizures. Vascular remodeling was assessed in mesenteric arteries (MA) and middle cerebral arteries (MCA) using pressurized myography. Reactive oxygen species (ROS) production by dihydroethidium staining, vascular cell adhesion molecule (VCAM)-1 expression and CD3+ T cell infiltration by immunofluorescence were determined in aorta or perivascular adipose tissue (PVAT). BP was measured by telemetry after 6 weeks of HSD. Results: Systolic BP tended to be higher in SS2BNb compared to SS. The incidence of seizures was 2.5-fold higher in SS2BNb. MA media/lumen was 1.3-fold higher in SS2BNa (P < 0.01). MCA stiffness was increased in SS2BNa, as indicated by a leftward displacement of the stress-strain curve. PVAT ROS production was 1.8-fold higher in SS2BNa (P < 0.01) and tended to be lower in SS2BNb. Aortic VCAM-1 was increased 2.1-fold in SS2BNa (P < 0.05). Aortic PVAT CD3+ T cell infiltration was 55% lower in SS2BNb (P < 0.05). Conclusions: Unexpectedly, SS2BNa rats present increased vascular injury under HSD. The absence of vascular inflammation and remodeling in SS2BNb rats despite slightly higher BP seems maladaptive and may explain the increased incidence of stroke.

PS 07-16 CROSSTALK BETWEEN ALDOSTERONE-STIMULATED MINERALOCORTICOID RECEPTOR AND ANGIOTENSIN RECEPTOR PATHWAYS PLAYS A ROLE IN ALDOSTERONE-INDUCED VASCULAR INJURY

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 angiotensin type 1a receptor (AGTR1A). We hypothesized that aldosterone-induced vascular damage require AGTR1A. Design and Method: Wild-type and Agtr1a knockout (Agtr1a−/−) mice were infused with aldosterone (600 &mgr;g/Kg/d) for 14 days while receiving 1% saline in drinking water. Systolic blood pressure (SBP) was measured by telemetry. Endothelial function and vascular remodeling using pressurized myography, reactive oxygen species (ROS) production by dihydroethidium staining, collagen content by Sirius red staining, fibronectin, and monocyte chemotactic protein-1 (MCP-1) expression by immunofluorescence and mRNA expression of mineralocorticoid receptor (MR) target genes regulating vascular tone by reverse transcription-quantitative PCR were assessed in mesenteric arteries. Natriuretic responses to a saline challenge was assessed using metabolic cage. Results: Aldosterone increased SBP by ∼30 mmHg in wild-type mice, and ∼50 mmHg in Agtr1a−/− mice. Aldosterone induced vascular remodeling and impaired endothelium-dependent relaxation in wild-type mice, and enhanced fibronectin and collagen deposition, and vascular inflammation. None of these vascular effects were observed in Agtr1a−/− mice. Agtr1a−/− mice infused with aldosterone did not increase sodium excretion in response to a sodium chloride challenge, suggesting sodium retention that could contribute to the exaggerated blood pressure 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. Conclusions: We conclude that although aldosterone activation of MR raises BP more in Agtr1a−/− mice, AGTR1a is required for MR stimulation to induce endothelial dysfunction and vascular remodeling and inflammation.