Julio C. Fraulob-Aquino

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.

γδ T Cells Mediate Angiotensin II-Induced Hypertension and Vascular InjuryClinical Perspective

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 &ggr;&dgr; T cell receptor (TCR) rather than the &agr;&bgr; 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 &ggr;&dgr; T cells; deficiency in &ggr;&dgr; T cells blunted Ang II-induced hypertension, vascular injury, and T-cell activation; and &ggr;&dgr; T cells are associated with human hypertension. Methods: Male C57BL/6 wild-type and Tcr&dgr;−/− mice, which are devoid of &ggr;&dgr; T cells, or wild-type mice injected IP with control isotype IgG or &ggr;&dgr; 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 &ggr; 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 &ggr;&dgr; T cells and SBP. Results: Seven- and 14-day Ang II infusion increased &ggr;&dgr; T-cell numbers and activation in the spleen of wild-type mice (P<0.05). Fourteen days of Ang II infusion increased SBP (P<0.01) and decreased mesenteric artery endothelial function (P<0.01) in wild-type mice, both of which were abrogated in Tcr&dgr;−/− mice (P<0.01). Anti-TCR&ggr;&dgr; antibody-induced &ggr;&dgr; T-cell depletion blunted Ang II-induced SBP rise and endothelial dysfunction (P<0.05), compared with isotype antibody-treated Ang II-infused mice. Ang II-induced T-cell activation in the spleen and perivascular adipose tissue was blunted in Tcr&dgr;−/− mice (P<0.01). In humans, the association between SBP and &ggr;&dgr; T cells was demonstrated by a multiple linear regression model integrating whole blood TCR &ggr; constant region gene expression levels and age and sex (R2=0.12, P<1×10-6). Conclusions: &ggr;&dgr; T cells mediate Ang II-induced SBP elevation, vascular injury, and T-cell activation in mice. &ggr;&dgr; T cells might contribute to the development of hypertension in humans.

Gamma Delta T Cells Mediate Angiotensin II-Induced Hypertension and Vascular Injury

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 &ggr;&dgr; T cell receptor (TCR) rather than the &agr;&bgr; 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 &ggr;&dgr; T cells; deficiency in &ggr;&dgr; T cells blunted Ang II-induced hypertension, vascular injury, and T-cell activation; and &ggr;&dgr; T cells are associated with human hypertension. Methods: Male C57BL/6 wild-type and Tcr&dgr;−/− mice, which are devoid of &ggr;&dgr; T cells, or wild-type mice injected IP with control isotype IgG or &ggr;&dgr; 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 &ggr; 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 &ggr;&dgr; T cells and SBP. Results: Seven- and 14-day Ang II infusion increased &ggr;&dgr; T-cell numbers and activation in the spleen of wild-type mice (P<0.05). Fourteen days of Ang II infusion increased SBP (P<0.01) and decreased mesenteric artery endothelial function (P<0.01) in wild-type mice, both of which were abrogated in Tcr&dgr;−/− mice (P<0.01). Anti-TCR&ggr;&dgr; antibody-induced &ggr;&dgr; T-cell depletion blunted Ang II-induced SBP rise and endothelial dysfunction (P<0.05), compared with isotype antibody-treated Ang II-infused mice. Ang II-induced T-cell activation in the spleen and perivascular adipose tissue was blunted in Tcr&dgr;−/− mice (P<0.01). In humans, the association between SBP and &ggr;&dgr; T cells was demonstrated by a multiple linear regression model integrating whole blood TCR &ggr; constant region gene expression levels and age and sex (R2=0.12, P<1×10-6). Conclusions: &ggr;&dgr; T cells mediate Ang II-induced SBP elevation, vascular injury, and T-cell activation in mice. &ggr;&dgr; T cells might contribute to the development of hypertension in humans.

Erythropoietin-induced hypertension and vascular injury in mice overexpressing human endothelin-1: exercise attenuated hypertension, oxidative stress, inflammation and immune response.

Objective: Erythropoietin used to correct anaemia in chronic kidney disease (CKD) has been shown to increase blood pressure (BP) in CKD patients and experimental animals. Endothelin (ET)-1 expression is increased in CKD animals and patients, and enhanced by erythropoietin. Erythropoietin-induced BP rise was blunted by ETA receptor blockers. This study was designed to determine whether preexisting endothelin (ET)-1 overexpression is required for erythropoietin to cause adverse vascular effects and whether this could be prevented by exercise training. Methods: Eight to 10-week old male wild-type mice and mice with endothelial-specific ET-1 overexpression (eET-1) were treated or not with EPO (100 IU/kg, SC, 3 times/week). eET-1 was subjected or not to swimming exercise training (1 h/day, 6 days/week) for 8 weeks. SBP, mesenteric artery endothelial function and remodelling, NADPH oxidase activity, reactive oxygen species (ROS) generation, vascular cell adhesion protein (VCAM)-1, monocyte/macrophage infiltration, T regulatory cells (Tregs) and tissue ET-1 and plasma endothelin were determined. Results: Erythropoietin increased SBP by 24 mmHg (P < 0.05) and decreased by 25% vasodilatory responses to acetylcholine (P < 0.01) in eET-1 mice. Erythropoietin enhanced ET-1 induced increase in resistance artery media/lumen ratio (31%, P < 0.05), aortic NADPH oxidase activity (50%, P < 0.05), ROS generation (93%, P < 0.001), VCAM-1 (80%, P < 0.01) and monocyte/macrophage infiltration (159%, P < 0.001), and raised plasma and aortic ET-1 levels (≥130%, P < 0.05). EPO had no effect in wild-type mice. Exercise training prevented all of the above (P < 0.05). Conclusion: Erythropoietin-induced adverse vascular effects are dependent on preexisting elevated ET-1 expression. Exercise training prevented erythropoietin-induced adverse vascular effects in part by inhibiting ET-1 overexpression-induced oxidative stress, inflammation and immune activation.

Matrix metalloproteinase-2 knockout prevents angiotensin II-induced vascular injury

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.

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 21-06 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.

MPS 01-01 A CONSERVED microRNA CLUSTER AS A POTENTIAL MASTER GENE EXPRESSION REGULATOR IN ANGIOTENSIN II-INDUCED VASCULAR DAMAGE

Objective: Non-coding RNAs (ncRNAs), including long ncRNAs (lncRNAs) and microRNAs (miRNAs), account for ∼98% of the transcribed RNAs. They have been shown to play a role in cardiovascular disease. Vascular damage is an early manifestation and a cause of end-organ damage in hypertension. However, it is unknown whether ncRNAs are involved in the development of vascular injury in hypertension. We hypothesize that ncRNA regulation plays a role in vascular remodeling in hypertension. Design and Method: Ten week-old male C57BL/6 mice underwent sham surgery or angiotensin (Ang) II infusion for 7 or 14 days. Blood pressure (BP) was measured by telemetry. Total RNA was extracted from mesenteric arteries and used to construct libraries for total RNA and small RNA deep sequencing using Illumina HiSeq-2500. Differential expression analysis and heat maps were made in R. Targetscan was used to predict interactions between differentially expressed miRNAs (DEmiRs) and genes (DEGs). MEME Suite was used to predict differentially expressed transcription factor targets in the DEGs. Cytoscape was used to perform functional enrichment analysis and construct molecular networks integrating the above interactions and the gene expression profile. Results: Differentially expressed mRNAs, lncRNAs, miRNAs and other small ncRNAs were identified in the Ang II-treated groups. Functional enrichment analysis showed enrichment of extracellular matrix in both 7-day and 14-day Ang II-induced DEGs, but developmental process in only the 14-day Ang II-induced DEGs. We identified 10 DEmiRs whose expression levels were correlated with BP, 9 of which are located in a single miRNA cluster that is highly conserved in humans. Conclusions: We have identified a conserved miRNA cluster that may play a pivotal role in the regulation of vascular damage. A sub-network of genes presenting the interaction between the miRNA cluster and other BP-correlated ncRNAs has been selected for future investigation to identify therapeutic targets.

OS 15-02 MATRIX METALLOPROTEINASE 2 KNOCKOUT PROTECTS FROM ANGIOTENSIN II-INDUCED VASCULAR INJURY IN PART VIA INHIBITION OF EGFR AND ERK1/2 ACTIVATION.

Objective: Matrix metalloproteinase 2 (MMP2) is involved in cardiovascular disease. Whether MMP2 plays a role in hypertension and vascular damage is unknown. We hypothesized that Mmp2 knockout will prevent angiotensin (Ang) II-induced hypertension and vascular injury. Design and Method: Mmp2 knockout (Mmp2−/−) and wild-type (WT) mice were infused with Ang II (1000 ng/kg/min, SC) for 14 days. Systolic blood pressure (SBP) was measured by telemetry, and mesenteric artery (MA) endothelial function and vascular remodeling by pressurized myography. Reactive oxygen species (ROS) generation using dihydroethidium staining, and vascular cell adhesion protein 1 (VCAM-1), monocyte chemotactic protein-1 (MCP-1) expression and monocyte/macrophage infiltration by immunofluorescence were determined in the aortic wall or perivascular fat (PVAT). Spleen T cell and monocyte profiles were assessed by flow cytometry. Vascular smooth muscle cells (VSMCs) were isolated from MA of WT and Mmp2−/− mice, stimulated 5 min with Ang II (100 nM) and epidermal growth factor receptor (EGFR) and extracellular-signal-regulated kinase 1/2 (ERK1/2) phosphorylation measured by Western blot. Results: Ang II increased SBP by 50 mmHg (P < 0.01), decreased by 50% MA vasodilator responses to acetylcholine (P < 0.01) and increased 1.7-fold MA media-to-lumen ratio (P < 0.01), 1.4-fold media cross-sectional area (P < 0.05), and stiffness (P < 0.01), as shown by a leftward shift of the stress/strain relationship, in WT. Furthermore, Ang II enhanced 12-fold aortic ROS generation (P < 0.01), 3.4-fold aortic VCAM-1 (P < 0.01), 6.5-fold MCP-1 expression (P < 0.01), 8-fold PVAT monocyte/macrophage infiltration (P < 0.05), and ∼2-fold spleen activated CD4+CD69+ and CD8+CD69+ T cells, and pro-inflammatory Ly-6Chi monocytes (P < 0.05) in WT. Ang II increased phosphorylation of EGFR 1.9-fold and ERK1/2 1.4-fold in VSMCs (P < 0.05). Mmp2 knockout prevented or reduced all of the above (P < 0.05) except SBP elevation. Conclusions: MMP2 plays an important role in Ang II-induced vascular injury, which could be mediated at least in part through EFGR and ERK1/2 activation in VSMCs.

PS 07-28 GAMMA/DELTA T CELLS MEDIATE ANGIOTENSIN II-INDUCED HYPERTENSION AND VASCULAR INJURY

Objective: Both innate antigen-presenting cells and the adaptive immune system have been shown to play a role in the development of hypertension. Nevertheless, the T cell subsets involved in the pathophysiology of hypertension remains unclear. There is a small subset of “innate-like” T cells expressing the g/d T cell receptor (TCR) rather than the a/b TCR that could play a role bridging between the innate and adaptive immune systems. However, it is unknown whether g/d T cells contribute to the development of hypertension. We hypothesized that angiotensin (Ang) II-induced hypertension and vascular injury would be blunted in Tcrd−/− mice, which are devoid of g/d T cells. Design and Method: Thirteen to 15-week old male C57BL/6 wild-type and Tcrd−/− mice were infused or not with Ang II (490 ng/kg/min, SC) for 7 or 14 days. Telemetric blood pressure (BP), mesenteric artery endothelial function and vascular remodeling by pressurized myography, and spleen T cell profile by flow cytometry were evaluated. Results: Fourteen days of Ang II increased systolic BP by 42 mmHg (P < 0.01) in wild-type compared to control mice. The frequency of g/d T cells (2.3-fold, P < 0.05) and activated (CD69+) g/d T cells (1.6-fold) was increased after 7 days of Ang II, and 7 days later remained increased or rose further (2.4-fold) in wild-type compared to control mice. Ang II decreased mesenteric artery relaxation responses to acetylcholine by 42% (P < 0.01) and increased media/lumen by 45% (P < 0.01) in wild-type mice compared to controls. BP rise and all the above mentioned Ang II effects were abrogated in Tcrd−/− mice. Conclusions: These data suggest that g/d T cells mediate Ang II-induced BP elevation and vascular injury. g/d T cells could be key immune cells bridging innate and adaptive immune responses during the development of hypertension in mouse models and by extension in humans.