Ang II-induced Blood Pressure Research Articles

Deletion of vascular thromboxane A2 receptors and its impact on angiotensin II-induced hypertension and atherosclerotic lesion formation in the aorta of Ldlr-deficient mice

The thromboxane A2 receptor (TP) has been shown to play a role in angiotensin II (Ang II)-mediated hypertension and pathological vascular remodeling. To assess the impact of vascular TP on Ang II-induced hypertension, atherogenesis, and pathological aortic alterations, i.e. aneurysms, we analysed Western-type diet-fed and Ang II-infused TPVSMC KO/Ldlr KO, TPEC KO/Ldlr KO mice and their respective wild-type littermates (TPWT/Ldlr KO). These analyses showed that neither EC- nor VSMC-specific deletion of the TP significantly affected basal or Ang II-induced blood pressure or aortic atherosclerotic lesion area. In contrast, VSMC-specific TP deletion abolished and EC-specific TP deletion surprisingly reduced the ex vivo reactivity of aortic rings to the TP agonist U-46619, whereas VSMC-specific TP knockout also diminished the ex vivo response of aortic rings to Ang II. Furthermore, despite similar systemic blood pressure, there was a trend towards less atherogenesis in the aortic arch and a trend towards fewer pathological aortic alterations in Ang II-treated female TPVSMC KO/Ldlr KO mice. Survival was impaired in male mice after Ang II infusion and tended to be higher in TPVSMC KO/Ldlr KO mice than in TPWT/Ldlr KO littermates. Thus, our data may suggest a deleterious role of the TP expressed in VSMC in the pathogenesis of Ang II-induced aortic atherosclerosis in female mice, and a surprising role of the endothelial TP in TP-mediated aortic contraction. However, future studies are needed to substantiate and further elucidate the role of the vascular TP in the pathogenesis of Ang II-induced hypertension, aortic atherosclerosis and aneurysm formation.

MyD88 in myeloid cells promotes angiotensin II-induced vascular inflammation and is associated with all-cause mortality and incident heart failure in humans with arterial hypertension

Abstract Background Angiotensin II (AngII) causes hypertension and promotes vascular accumulation of inflammatory cells. We aimed to determine how myeloid factor of differentiation 88 (MyD88) contributes to vascular dysfunction and arterial hypertension in mice and humans. Methods Male C57BL/6 as well as MyD88-/-, LysMCre/wtMyD88LSL/LSL, LysMCre/wt, TLR2-/-, TLR4-/-, TLR7-/- and TLR9-/- were investigated in the AngII-model of hypertension (1 mg/kg/d for 7days). Biodata analyses were performed in the Gutenberg-Health Study (GHS), a population based cohort. In addition, we performed computational analyses of known hypertension-related genes from genome-wide association studies (GWAS) to investigate whether they preferentially interact with MyD88 in the human protein network. Results MyD88 deficiency attenuated AngII-induced blood pressure increase and endothelial dysfunction in conductance and resistance vessels. Vascular mRNA expression levels of vcam-1, nos2, nox2 were decreased in AngII-infused MyD88-/- mice compared to C57BL/6 controls. Flow cytometric analyses revealed that AngII-induced aortic accumulation of CD11b+Ly6Chi inflammatory monocytes was significantly dampened in MyD88-/- mice. AngII increased mRNA expression of cd62L, cd68 and ccl2, which was blocked in MyD88-/- mice, indicating a role of MyD88 for myeloid cell activation and differentiation. Additionally, aortic interferon-g+ NK cells and mRNA levels of interleukin-12 and interleukin-1b were reduced in AngII-treated MyD88-/- mice. Bone marrow transfer experiments demonstrated a vasoprotective effect of MyD88 deficiency in bone marrow-derived cells, and selective expression of MyD88 in LysMCre/wtMyD88LSL/LSL mice restored AngII-induced pathology, revealing that myelomonocytic cells drives vascular dysfunction in a MyD88-dependent manner. Furthermore, data from 1,274 individuals in the GHS indicated, that MyD88 mRNA expression was associated with all cause mortality and incident chronic heart failure after a median follow-up of 11.6 years. Computational analysis of the human protein interaction network demonstrated that MyD88 is significantly connected to proteins encoded by genetic loci associated with blood pressure traits in multiple GWAS. Conclusion We provide evidence, that MyD88 expressed by myelomonocytic cells promotes AngII-induced vascular dysfunction and arterial hypertension. MyD88 might be used as an inflammatory diagnostic marker for the risk of death in hypertensive patients.

S-40-4: ANGIOTENSIN II-INDUCED BLOOD PRESSURE ELEVATION WAS POTENTIATED TOGETHER WITH INCREASES IN INTERFERON-GAMMA PRODUCING T CELLS IN INTERLEUKIN-23 RECEPTOR-DEFICIENT MICE

Objectives: We previously demonstrated a role for γδ T cells in hypertension and vascular injury. In inflammatory conditions γδT17 are a prominent producer of IL-17A, which has been shown to contribute to hypertension. The development and expansion of γδT17 cells is regulated in part through IL-23 receptors (IL-23R). We hypothesized that angiotensin (Ang) II-induced blood pressure (BP) elevation and vascular injury would be blunted in mice with dysfunctional IL-23R. Design and method: Wild-type (WT) and Il23r knock-in (Il23r gfp/gfp) mice were infused or not with Ang II (490ng/kg/min, SC) for 7 or 14 days. BP was monitored via telemetry, mesenteric artery function and remodeling using pressurized myography, and T cell profiling in mesenteric artery perivascular adipose tissue (PVAT) by flow cytometry. Results: Il23r gfp/gfp mice exhibited a greater BP elevation in response to Ang II than WT by day 3 (152 ± 5 vs 144 ± 7 mm Hg, P < 0.05), which was sustained through day 9 (169 ± 2 vs 155 ± 5 mm Hg, P < 0.05), but eventually similar to that of WT mice by the end of the Ang II infusion (167 ± 2 vs 167 ± 4 mm Hg). Il23r gfp/gfp mice were not protected from vascular dysfunction and remodeling after 14 days of Ang II. Il23r gfp/gfp mice had less γδT17 cells in PVAT than WT mice (40 ± 8 vs 108 ± 15 cells/PVAT, P < 0.05). Ang II increased interferon-γ producing γδ T cells in WT (13 ± 3 vs 5 ± 1 cells/PVAT, P < 0.05) and Il23r gfp/gfp mice (14 ± 3 vs 3 ± 1 cells/PVAT, P < 0.05), and interferon-γ producing CD4+ (125 ± 27 vs 38 ± 13 cells/PVA, P < 0.05) and CD8+ T cells (76 ± 13 vs 33 ± 8 cells/PVA, P < 0.05) only in Il23r gfp/gfp mice. Conclusion: Functional IL-23R deficiency exaggerated BP elevation during the initiation of Ang II-induced hypertension, potentially due to the increased number of interferon-γ producing T cells.

Angiotensin II-induced a steeper blood pressure elevation in IL-23 receptor-deficient mice: Role of interferon-γ-producing T cells.

A subset of interleukin (IL)-17A-producing γδ T cells called γδT17 cells may contribute to progression of hypertension. γδT17 cell development is in part dependent upon IL-23 receptor (IL-23R) stimulation. We hypothesized that angiotensin (Ang) II-induced blood pressure (BP) elevation and vascular injury would be blunted in Il23r knock-in (Il23rgfp/gfp) mice deficient in functional IL-23R. To test this hypothesis, we infused wild-type (WT) and Il23rgfp/gfp mice with Ang II (490 ng/kg/min, SC) for 7 or 14 days. We recorded BP by telemetry, assessed vascular function and remodeling using pressurized myography, and profiled T cell populations and cytokine production by flow cytometry. An additional set of Il23rgfp/gfp mice was infused with Ang II for 7 days and injected with interferon (IFN)-γ-neutralizing or control antibodies. Il23rgfp/gfp mice had smaller and stiffer mesenteric arteries and were not protected against Ang II-induced BP elevation. BP was higher in Il23rgfp/gfp mice than WT mice from day 3 until day 9 of Ang II infusion. Il23rgfp/gfp mice had less γδT17 cells and more IFN-γ-producing γδ, CD4+, and CD8+ T cells than WT mice. Seven days of Ang II infusion led to increased IFN-γ-producing γδ, CD4+, and CD8+ T cells in Il23rgfp/gfp mice, whereas only IFN-γ-producing γδ T cells were increased in WT mice. Blocking IFN-γ with a neutralizing antibody reduced the pressor response to 7 days of Ang II infusion in Il23rgfp/gfp mice. Functional IL-23R deficiency was associated with increased IFN-γ-producing T cells and exaggerated initial development of Ang II-induced hypertension, which was in part mediated by IFN-γ.

Abstract 131: Interleukin-23 Receptor Deficiency Increased Interferon-gamma Producing T Cells And Potentiated Blood Pressure Elevation In Angiotensin II-infused Mice

Introduction: We previously demonstrated a role for γδ T cells in hypertension and vascular injury. In inflammatory conditions γδT17 are a prominent producer of IL-17A, which has been shown to contribute to hypertension. The development and expansion of γδT17 cells is regulated in part through IL-23 receptors (IL-23R). Hypothesis: We hypothesized that angiotensin (Ang) II-induced blood pressure (BP) elevation and vascular injury would be blunted in mice with dysfunctional IL-23R. Methods: Wild-type (WT) and Il23r knock-in ( Il23r gfp/gfp ) mice were infused or not with Ang II (490ng/kg/min, SC) for 7 or 14 days. BP was monitored via telemetry, mesenteric artery function and remodeling using pressurized myography, and T cell profiling in mesenteric artery perivascular adipose tissue (PVAT) by flow cytometry. Results: Il-23r gfp/gfp mice exhibited a greater BP elevation in response to Ang II than WT by day 3 (152±5 vs 144±7 mm Hg, P <0.05), which was sustained through day 9 (169±2 vs 155±5 mm Hg, P <0.05), but eventually similar to that of WT mice by the end of the Ang II infusion (167±2 vs 167±4 mm Hg). Il-23r gfp/gfp mice were not protected from vascular dysfunction and remodeling after 14 days of Ang II. Il23r gfp/gfp mice had less γδT17 cells in PVAT than WT mice (40±8 vs 108±15 cells/PVAT, P <0.05). Ang II increased interferon-γ producing γδ T cells in WT (13±3 vs 5±1 cells/PVAT, P <0.05) and Il23r gfp/gfp mice (14±3 vs 3±1 cells/PVAT, P <0.05), and interferon-γ producing CD4 + (125±27 vs 38±13 cells/PVA, P <0.05) and CD8 + T cells (76±13 vs 33±8 cells/PVA, P <0.05) only in Il23r gfp/gfp mice. Conclusion: Functional IL-23R deficiency exaggerated BP elevation during the initiation of Ang II-induced hypertension, potentially due to the increased number of interferon-γ producing T cells.

NLRP3 inflammasome contributes to endothelial dysfunction in angiotensin II-induced hypertension in mice

AimsInflammation is a key feature of endothelial dysfunction induced by angiotensin (Ang) II. The purpose of this study was to explore the role of Nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome in endothelial dysfunction in Ang II-induced hypertension. Materials and methodsWe analyzed blood pressure and vascular function of wild-type (WT) and Nlrp3 knockout (Nlrp3−/−) mice, treated with Ang II. In vitro, we mainly tested the endothelial nitric oxide synthase (eNOS) phosphorylation expression of human umbilical vein endothelial cells (HUVECs). Key findingsHere we showed that 14-day Ang II infusion into mice resulted in the elevation of blood pressure, NLRP3 expression, serum interleukin (IL)-1β level, and the decline of endothelium-dependent relaxation function, p-eNOS-Ser1177 expression in aortas. Nlrp3 deficiency reduced Ang II-induced blood pressure elevation and endothelial dysfunction. In vitro, NLRP3 was involved in the effect of Ang II on reducing p-eNOS-Ser1177 expression. Moreover, the direct effect of IL-1β on vascular endothelial injury could be observed in both vivo and vitro. SignificanceOur result demonstrates that the NLRP3 inflammasome is critically involved in the detrimental effects of Ang II on vascular endothelium in hypertension via the activation of IL-1β, placing NLRP3 as a potential target for therapeutic interventions in conditions with endothelial dysfunction in hypertension.

MFAP4 Deficiency Attenuates Angiotensin II-Induced Abdominal Aortic Aneurysm Formation Through Regulation of Macrophage Infiltration and Activity.

Objective: Abdominal aortic aneurysm (AAA) is a common age-related vascular disease characterized by progressive weakening and dilatation of the aortic wall. Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix (ECM) protein involved in the induction of vascular remodeling. This study aimed to investigate if MFAP4 facilitates the development of AAA and characterize the underlying MFAP4-mediated mechanisms.Approach and Results: Double apolipoprotein E- and Mfap4-deficient (ApoE−/−Mfap4−/−) and control apolipoprotein E-deficient (ApoE−/−) mice were infused subcutaneously with angiotensin II (Ang II) for 28 days. Mfap4 expression was localized within the adventitial and medial layers and was upregulated after Ang II treatment. While Ang II-induced blood pressure increase was independent of Mfap4 genotype, ApoE−/−Mfap4−/− mice exhibited significantly lower AAA incidence and reduced maximal aortic diameter compared to ApoE−/− littermates. The ApoE−/−Mfap4−/− AAAs were further characterized by reduced macrophage infiltration, matrix metalloproteinase (MMP)-2 and MMP-9 activity, proliferative activity, collagen content, and elastic membrane disruption. MFAP4 deficiency also attenuated activation of integrin- and TGF-β-related signaling within the adventitial layer of AAA tissues. Finally, MFAP4 stimulation promoted human monocyte migration and significantly upregulated MMP-9 activity in macrophage-like THP-1 cells.Conclusion: This study demonstrates that MFAP4 induces macrophage-rich inflammation, MMP activity, and maladaptive remodeling of the ECM within the vessel wall, leading to an acceleration of AAA development and progression. Collectively, our findings suggest that MFAP4 is an essential aggravator of AAA pathology that acts through regulation of monocyte influx and MMP production.

Loss of myeloid Bmal1 exacerbates hypertensive vascular remodelling through interaction with STAT6 in mice.

In addition to its involvement of inflammatory responses, limited information is available on the phenotype and behaviour of vascular macrophages during hypertensive vascular remodelling. Here, we aim at studying the contribution of BMAL1 to the pro-fibrotic macrophage phenotype in the vasculature during hypertension, which leads to enhanced vascular remodelling and promoted blood pressure increase. Wild type Bmal1f/f and myeloid cell selective Bmal1 knockout Bmal1f/f; LysMCre/+ mice were infused with AngII for 4 weeks to induce hypertension. AngII-induced blood pressure increase, vascular media thickness and vascular dysfunction were enhanced in Bmal1f/f; LysMCre/+ mice, accompanied with a pro-fibrotic M2 phenotype of the vascular macrophages. Bmal1f/f; LysMCre/+ mice also have more up-regulations of MMP9 and MMP13 expression in the vascular wall, accompanied by enhanced collagen deposition after AngII infusion. Loss of Bmal1 in bone marrow-derived macrophages enhanced STAT6 activation induced by IL4, and the subsequent MMP13 up-regulation and activity. In macrophages, loss of Bmal1 enhanced the phosphorylation and nuclear translocation of STAT6 triggered by IL4, through possibly a direct interaction between BMAL1 and STAT6. To further determine whether IL4-induced signalling in macrophage contributes to enhanced vascular remodelling in hypertensive mice, we showed that deletion of myeloid IL4Rα in Il4raf/f; LysMCre/+ mice attenuated blood pressure increase and hypertensive vascular remodelling after AngII infusion. Our results suggested a tonic effect of BMAL1 deletion on hypertensive vascular remodelling. BMAL1 might inhibit IL4-STAT6 signalling in macrophages through the interaction with STAT6 to reduce STAT6 activation and target gene transcription, especially MMP9 and MMP13, contributing to vascular remodelling.

Abstract 13: P2X7 Receptor Knockout Attenuates Angiotensin II-Induced Hypertension, Vascular Injury And CD8 + T Cell Infiltration

Background: The P2X7 receptor (P2RX7) recognizes damage associated molecule patterns such as adenosine triphosphate (ATP), and triggers the activation of immune cells. Elevated plasma ATP levels have been observed in hypertensive patients, providing a potential mechanism for P2RX7 activation. Additionally, a hypomorphic polymorphism for P2X7 is correlated with a decreased risk for essential hypertension in Chinese post-menopausal women. However, it is unknown whether P2RX7 activation contributes to angiotensin (Ang) II-induced blood pressure (BP) elevation and vascular damage. We hypothesized that P2rx7 knockout would blunt Ang II-induced BP elevation, vascular injury, and infiltration of activated immune T cells into perivascular adipose tissue (PVAT). Methods: Ten-to-12-week-old male C57BL/6J male wild-type (WT) and P2rx7 -/- mice were infused or not with Ang II (1000ng/kg/min) for 14 days. BP was determined by telemetry, mesenteric artery function and remodeling using pressurized myography, aortic stiffening by ultrasound and infiltration of activated immune T cells in aortic PVAT by flow cytometry. Results: Ang II-infused P2rx7 -/- mice display a reduced systolic BP (164±3 vs. 176±2 mm Hg, P <0.05) and pulse pressure (37±4 vs. 53±3 mm Hg, P <0.001) in comparison to WT mice. Aortic stiffening occurred in WT mice treated with Ang II, demonstrated by an increased pulse wave velocity (7.7±0.7 vs. 5.9±0.3 m/s, P <0.05), accompanied by a 3.8-fold increased infiltration of activated CD8 + T cells in aortic PVAT (60±16 vs 16±3 cells/aortic PVAT, P <0.001), which were both absent in P2rx7 -/- mice (6.4±1.4 vs 5.5±1.1 m/s and 27±7 vs 16±3 cells/aortic PVAT). In addition, the frequency of IFN-γ producing CD8 + T cells in the spleen of Ang II-treated WT mice increased (2.6±0.2% vs 1.2±0.2%), which did not occur in P2rx7 -/- mice (1.7±0.3% vs 1.7±0.2%). Ang II-infusion induced mesenteric artery endothelial dysfunction in WT mice (61±7 vs 83±4% relaxation response to acetylcholine, P <0.05), which was absent in P2rx7 -/- mice (89±3 vs 90±3%). Conclusion: P2rx7 knockout attenuates Ang II-induced hypertension, vascular injury, and infiltration of activated CD8 + T cells into aortic PVAT.

Mitochondrial activity regulates the differentiation of skin-derived mesenchymal stem cells into brown adipocytes to contribute to hypertension

BackgroundBrown adipocytes (BAs) are major components of brown adipose tissue (BAT), which is involved in blood pressure regulation. BAs are derived from multiple progenitors, including PDGFRα+ adipose-derived stem cells (ASCs). Skin-derived mesenchymal stem cells (S-MSCs) have the capacity to differentiate into adipocytes; however, their ability to differentiate into BAs remains unexplored. We aim to study the ability and regulatory mechanism of the differentiation of S-MSCs into BAs and the direct role of BAT in blood pressure regulation.MethodsProtein expression was measured by flow cytometry or Western blotting, and gene mRNA levels were quantified by real-time quantitative PCR (RT-PCR). To induce the differentiation of S-MSCs into BAs, S-MSCs were stimulated with a brown adipogenic cocktail comprising insulin, IBMX, dexamethasone, triiodothyronine (T3), and rosiglitazone for the indicated periods. The oxygen consumption rate (OCR) was measured with an XF24 Extracellular Flux Analyzer. Mitochondrial mass was determined by flow cytometry and fluorescence staining. Hypertension was induced in WT mice by infusion of angiotensin II (Ang II), and systolic blood pressure (SBP) was measured using a tail cuff. Interscapular brown adipose tissue (iBAT)-deficient mice were generated by surgical removal of the iBAT depot, after which the animals were allowed to recover for 6 days. Aortic, iBAT, and heart tissue sections were analyzed by hematoxylin and eosin (HE) staining.ResultsWe found that in vitro, S-MSCs isolated from the mouse dermis expressed the stem cell markers CD90/105 and PDGFRα and readily differentiated into BAs. Mitochondrial biogenesis and oxygen consumption were markedly increased during differentiation of S-MSCs into BAs. In vivo, iBAT was converted to white adipose tissue (WAT) in Ang II-induced hypertensive mice. We assessed the direct role of BAT in blood pressure (BP) regulation by using iBAT-deficient mice (generated by surgical removal of iBAT) and C57BL/6 (wild-type (WT)) mice and found that Ang II-induced BP elevation and vascular damage were markedly aggravated in iBAT-deficient mice compared with WT mice.ConclusionsThis study demonstrates that PDGFRα+ S-MSCs are able to differentiate into BAs and that this differentiation is regulated by mitochondrial activity. We also show that BAT plays a direct role in ameliorating Ang II-induced hypertension. The therapeutic potential of BAT for the prevention of hypertension-induced organ remodeling thus warrants further investigation.Graphical abstract. Schematic of the in vitro differentiation of PDGFRα+ S-MSCs into BAs via a process regulated by mitochondrial activity. BAT plays a direct role in Ang II-induced hypertension and target organ remodeling

Lack of LRP8 triggers the formation of aortic dissection in angiotensin II infused mice

Factor XI-thrombin amplification loop depends on platelets and monocytes and augments angiotensin II (AngII) driven vascular injury in experimental hypertension in mice. Low-density lipoprotein receptor-related protein 8 (LRP8) plays an important role in apolipoprotein E homeostasis and was also found to be important in endothelial barrier stabilization. Moreover, LRP8 is able to form a complex with the platelet glycoprotein Ib alpha (GPIbalpha) which is the main receptor for von Willebrand factor. To explore the role of LRP8 and GPIbalpha in the development of hypertension related to platelet-dependent thrombin generation. We infused LRP8 KO mice with AngII for either 1 or 4 weeks using osmotic minipumps. Blood pressure was recorded using tail cuff measurement. Vascular reactivity was assessed in isolated aortic segments. Leukocyte activation and infiltration were assessed by flow cytometry of aortic tissue and intravital videomicroscopy imaging (IVM). Histology of aortas was performed with sirius red staining. Infusion of AngII for 1 week in LRP8 KO mice altered endothelial dependent and independent vascular relaxation to the same extend as in control mice. Vascular rolling and adherence to the carotid endothelium, visualized by IVM was similar between LRP8 KO and control mice infused with AngII. There were no significant differences in AngII-induced blood pressure increase between the LRP8 KO and control mice. Unexpectedly, LRP8 KO mice demonstrated a drastically increased mortality rate in response to AngII infusion. In the 4 weeks AngII infusion experiments, 80% of the LRP8 KO mice died within the first 10 days of AngII infusion due to aortic dissection complicated by hemorrhage. LRP8 receptor dysfunction lead to the formation of aortic dissection in AngII infused mice. Further studies are needed to better characterize the role of immune cells and platelets in the development of these dissections.

Abstract P1113: P2x7 Receptor Plays a Role in Angiotensin II-Induced Hypertension and Vascular Remodeling

Introduction: Inflammasome activation by binding of ATP released from damaged cells to the purinergic receptor P2X7 (P2RX7) could play a role in hypertension and vascular injury through release of interleukin (IL)-1β and immune cell activation. Elevated ATP levels were observed in the renal interstitial fluid of angiotensin (Ang) II-infused rats, and treatment of these rats with a non-selective P2 receptor blocker prevented Ang II-induced inflammation and renal damage. P2RX7 knockout ( P2rx7 –/– ) prevented deoxycorticosterone acetate-salt-induced blood pressure (BP) elevation and renal damage. However, it is unknown whether P2RX7 plays a role in Ang II-induced BP elevation and vascular damage. We hypothesize that Ang II-induced hypertension, vascular injury, and inflammation will be blunted in P2rx7 -/- mice. Methods: Ten to 12-week-old male C57BL/6J male wild-type (WT) and P2rx7 –/– mice were sham-treated or infused with Ang II (490ng/kg/min) for 14 days. BP was determined by telemetry. Mesenteric artery function and remodeling was assessed using pressurized myography. P2RX7 expression in spleen immune cells was determined by flow cytometry. IL-1β secretion from bone marrow-derived macrophages (BMDM) isolated from WT and P2rx7 –/– mice was assessed by ELISA. Results: Stimulation with lipopolysaccharides and ATP caused IL-1β release in WT (339±162 pg/mL) but not P2rx7 –/– BMDMs. In WT mice, Ang II increased P2RX7 expression in dendritic cells (mean fluorescence intensity: 1927±120 vs 3983±983) and macrophages (2541±265 vs 3314±273 respectively). P2rx7 knockout reduced Ang II-induced systolic (159±3 vs 165±7 mm Hg) and diastolic BP elevation (110±4 vs 126 ±7 mm Hg) compared to WT mice. Acetylcholine-induced vascular relaxation was unaffected by Ang II or P2rx7 knockout. Ang II increased the media-to-lumen ratio in WT (4.71±0.18% vs 3.89±0.21%, P <0.01) but not in P2rx7 –/– mice. Conclusion: This study demonstrated that P2RX7 plays a role in Ang II-induced hypertension and vascular remodeling.

MiR-431-5p Knockdown Protects Against Angiotensin II-Induced Hypertension and Vascular Injury.

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.

Xanthine Oxidase Inhibition by Febuxostat in Macrophages Suppresses Angiotensin II-Induced Aortic Fibrosis.

BACKGROUNDSeveral reports from basic researches and clinical studies have suggested that xanthine oxidase (XO) inhibitors have suppressive effects on cardiovascular diseases. However, the roles of a XO inhibitor, febuxostat (FEB), in the pathogenesis of vascular remodeling and hypertension independent of the serum uric acid level remain unclear.METHODSTo induce vascular remodeling in mice, angiotensin II (Ang II) was infused for 2 weeks with a subcutaneously implanted osmotic minipump. FEB was administered every day during Ang II infusion. Aortic fibrosis was assessed by elastica van Gieson staining. Mouse macrophage RAW264.7 cells (RAW) and mouse embryonic fibroblasts were used for in vitro studies.RESULTSFEB suppressed Ang II-induced blood pressure elevation and aortic fibrosis. Immunostaining showed that Ang II-induced macrophage infiltration in the aorta tended to be suppressed by FEB, and XO was mainly colocalized in macrophages, not in fibroblasts. Transforming growth factor-β1 (TGF-β1) mRNA expression was induced in the aorta in the Ang II alone group, but not in the Ang II + FEB group. Ang II induced α-smooth muscle actin-positive fibroblasts in the aortic wall, but FEB suppressed them. XO expression and activity were induced by Ang II stimulation alone but not by Ang II + FEB in RAW. FEB suppressed Ang II-induced TGF-β1 mRNA expression in RAW.CONCLUSIONSOur results suggested that FEB ameliorates Ang II-induced aortic fibrosis via suppressing macrophage-derived TGF-β1 expression.

A6321 Complement in Hypertension and Target Organ Damage

Objectives: Immunity and inflammation play pivotal roles in the development of hypertension, including innate and adaptive immunity. Complement system provides a bridge between innate and adaptive immune cells. We aim to elucidate the mechanism of complement activation in hypertension and related target organ damage. Methods: Complement C3, C3aR, C5aR knock out (KO) mice were utilized for performing hypertension model, including deoxycorticosterone acetate (DOCA)/salt and angiotensin II (Ang II)-induced hypertension. Macrophage and T cells were separated for stimulating with complement factors. Results: We firstly found that perivascular adipose tissue (PVAT) expressed abundant C3, which stimulated adventitial fibroblast migration and phenotype transdifferentiation. Subsequently, we showed that C5a regulated M1/M2 macrophage polarization and inhibited adiponectin production in the PVAT, which contributed to vascular inflammation in hypertension. More important, we demonstrated that double C3aR and C5aR KO (DKO) prevented Ang II-induced blood pressure elevation. Mechanically, DKO activated the immunosuppressive functio of regulatory T cells (Tregs). Adoptive transfer of DKO Tregs attenuated Ang II-induced hypertension and target organ damage. Conclusion: Our results demonstrate that complement activation-mediated immune cell infiltration is involved in the development of hypertension and target organ damage. Blockade of complement activation may be an alternative novel approach for hypertension treatment.

Abstract 041: Brain Cytochrome P450 1B1-Testosterone Generated Metabolite 6Beta-Hydroxytestosterone Contributes to Angiotensin II-Induced Increased Sympathetic Outflow and Blood Pressure

Cytochrome P450 1B1 (CYP1B1)-generated testosterone (T) metabolite 6β-hydroxytestosterone (6β-OHT) contributes to angiotensin (Ang) II-induced hypertension in the male mice. However, the site of action of CYP1B1-testosterone metabolite 6β-OHT in Ang II-induced hypertension is unknown. The demonstration that Ang II increases blood pressure by its action in the subfornical organ and the presence of CYP1B1 in the brain led us to hypothesize that T contributes to Ang II-induced hypertension via its metabolism to 6β-OHT in the brain. To test this hypothesis, we examined the effect of intracerebroventricularly (ICV) administered T in castrated (Cas) wild-type ( Cyp1b1 +/+ ) and Cas Cyp1b1 -/- mice on the action of systemic Ang II (700 ng/kg/min) for 14 days. T (3 μg/2μL/injection/every 2nd day) or its vehicle (2-Hydroxypropyl-β-cyclodextrin dissolved in artificial CSF) was injected via a cannula implanted in the brain. Mean arterial blood pressure (MAP, mmHg) was measured by radiotelemetry (n=4-5). Ang II increased MAP in T but not its vehicle-injected Cas Cyp1b1 +/+ mice on Day 12 (159±2 vs. 98±2, P<0.05). In Cas Cyp1b1 -/- mice Ang II increased MAP in ICV injected 6β-OHT (1.5 μg/2μL/injection every 2nd day) but not T (151±3 vs. 123±5, P<0.05). Power spectral analysis of the data on day 12 showed that Ang II increased the low to high-frequency ratio of heart rate variability, index of sympathetic outflow modulation in ICV T injected Cas Cyp1b1 +/+ but not Cas Cyp1b1 -/- mice (3.8±0.0.4 vs. 1.9±0.0); whereas ICV 6β-OHT in the later mice increased this ratio (4.7±0.6). Ganglionic blocker hexamethonium (30 mg/Kg, ip) on day 14 of Ang II infusion resulted in greater reduction in MAP (P<0.05) in centrally 6β-OHT injected Cas Cyp1b1 -/- , and T-injected Cas Cyp1b1 +/+ (Δ81±11 and Δ87±10) than in T injected Cas Cyp1b1 -/- mice (Δ50±8). Furthermore, in intact Cyp1b1 -/- , but not Cas Cyp1b1 -/- mice transduction with adenovirus (Ad) of CYP1B1-DNA (ICV 2μL of 1.0 X 10 12 particles/mL) increased Ang II-induced systolic blood pressure (P<0.05, n=5/group) measured by tail-cuff on Day12 (176±11 vs. 120±2, mm Hg). These data suggest that T contributes to Ang II-induced hypertension most likely via generation by the brain CYP1B1 to 6β-OHT, which increases sympathetic outflow in male mice.

Abstract 020: Sex Difference in T Regulatory Cells After Adoptive Transfer From Hypertensive Donors Leads to Protection Against T Cell-Mediated Hypertension in Premenopausal Female Mice

Activation of T cell-dependent pro-inflammatory responses are required for Ang II hypertension in male mice. However, females are protected from T cell-mediated hypertension and may suppress hypertension by directly preventing Ang II-induced pro-inflammatory T cell activation. Here we sought to determine whether transferring T cells from hypertensive donor mice eliminates female protection against T cell-mediated hypertension. Splenic CD3 + T cells were transferred from normotensive (NT) or Ang II-hypertensive (HT) C57BL/6J male donors to female Rag-1 -/- (NT T cell female-NTF; HT T cell female-HTF) or male Rag-1 -/- (HT T cell male-HTM) recipient mice. Blood pressure was monitored (tail cuff) for 5 weeks post-transfer. Ang II (490ng/kg/min) was infused into recipient mice for 14 days during weeks 4 and 5 post-transfer (NTFA; HTFA; HTMA). Ang II significantly increased MAP in donor male mice (NT 114 vs HT 157 mmHg, p<0.05). Transfer of T cells from HT donors did not induce HT in female or male recipients. Similarly, T cell donor environment did not affect Ang II-induced blood pressure in female recipients, which remained protected compared to male recipients (MAP: NTF 83 + 4 mmHg*, HTF 88 + 6 mmHg*, NTFA 101 + 5 mmHg*, HTFA 103 + 5 mmHg*, HTMA 138 + 3 mmHg, *p<0.05 vs HTMA). Flow cytometry demonstrated similar splenic T cell frequency across all groups (CD3: NTF 18%, NTFA 16%, HTF 17%, HTFA 14%, HTMA 18%, p>0.05). However, regulatory T cells were significantly reduced in male recipients compared to all female groups (Foxp3: NTF 21.6%*, NTFA 22.2%*, HTF 22.8%*, HTFA 22.6%*, HTMA 15.3%, *p<0.05 vs HTMA) Females had significantly less renal T cell infiltration compared to males and infiltration was not impacted by Ang II infusion or T cell donor status (CD3: NTF 12,083*, NTFA 11,317*, HTF 12,656*, HTFA 8,997*, HTMA 22,405, *p<0.05 vs HTMA; CD4: NTF 6,411*, NTFA 4,702*, HTF 5,831*, HTFA 4,579*, HTMA 9,914, *p<0.05 vs HTMA; CD8: NTF 5,397*, NTFA 6,123*, HTF 6,362*, HTFA 3,792*, HTMA 11,727, *p<0.05 vs HTMA). These results demonstrate that female mice prevent T cell-mediated hypertension and renal T cell infiltration regardless of previous T cell exposure to a hypertensive environment, suggesting a direct preventive mechanism in females against pro-hypertensive T cell responses.

Abstract P234: 2-Methoxyestradiol Attenuates Angiotensin II Induced Hypertension: A Novel Paradigm

Estrogen metabolite 2-methoxyestradiol (2ME2) has therapeutic potential in a number of cardiovascular disorders, including hypertension. However, specific or potential targets remains to be determined. In this study, animals were surgically implanted with PA-C40 telemetry in the intra-aortic vessel and 2ME2 (20 mg/kg/day, i.p. injection) effects in AngII infused Wistar-Koyoto (WKY) rats and spontaneously hypertensive rats (SHRs) were determined. In WKY rats 2ME2 treatment resulted in significant reduction in AngII induced blood pressure (BP) compared to vehicle control (systolic; 143.69 ± 2.68 vs. 169.75 ± 1.93 mm Hg, diastolic; 99.91 ± 2.49 vs. 120.16 ± 1.25 mm Hg, and mean arterial pressure; 118.94 ± 2.52 vs. 142.73 ± 1.47 mm Hg). We observed a decrease in heart rate in 2ME2 exposed animals compared to control group (272.67 ± 9.88 vs. 329.12 ± 6.14 BPM). In addition, 2ME2 mediated reduction in workload of the heart when we calculated the rate pressure product or pressure rate index by 13.46 ± 2.32 indicating that 2ME2 can reduce AngII induced hemodynamic stress of the heart. [ 3 H]AngII binding studies showed that in kidney cortex AT1R expression was inhibited by 2ME2 treatment compared to control (2112 ± 71 vs. 3341 ± 248 DPM/mg protein). In a separate 5-week study, 2ME2-treated SHRs displayed significant reduction in diastolic BP (114.31 ± 0.66 vs. 123.28 ± 0.70 mm Hg) and mean arterial pressure (146.35 ± 0.67 vs. 151.54 ± 0.89 mm Hg) without significant change in systolic BP compare to the vehicle treated animals. Moreover, we found significant decrease in heart rate in 2ME2-treated animals (290.03 ± 2.69 vs. 312.92 ± 1.50 BPM). Analyses of kidney cortical tissue revealed downregulation of AT1R expression after 2ME2 treatments (20227 ± 2211 vs. 9515 ± 2115 DPM/mg protein). Consistent with the reduction in BP, the indicators of cardiac hypertrophy including heart weight (1.28 ± 0.08 vs. 0.99 ± 0.02 gram), cardiac collagen deposit and α-smooth muscle actin (α-SMC) positive myofibroblasts were decreased. The data demonstrate for the first time that 2ME2 control BP through downregulation of AT1R in kidney and heart rate, which may provide an insight to the mechanism(s) of cardioprotective effects of 2ME2 through the blockade of AngII induced hypertension.

Abstract 042: Evidence of Angiotensin II (AngII)-dependent Obesity-induced Hypertension in Female Mice Exposed to Postnatal Neglect

Previously, we have shown that female mice subjected to maternal separation with early weaning (MSEW), a model of postnatal neglect, display exacerbated diet-induced obesity and high blood pressure (BP) compared with control mice. Female MSEW mice show activated renin-angiotensin system components, including increased plasma renin activity and adipose tissue-derived angiotensinogen secretion. The goal of this study was to test whether augmented obesity-induced hypertension in female MSEW mice is AngII-dependent. Mouse MSEW was achieved by repeated, daily separations from the dam and 4-day early weaning. Normally reared controls (C) were weaned at postnatal day 21. Each experimental group of female weanlings was comprised of 6 mice each and derived from 3 different litters, that were placed on high fat diet (HFD, 60% kcal from fat). After 18 weeks, mice were implanted with radiotelemetry for BP measurement. At week 20, average 24-hr systolic blood pressure (SBP) was 134±2 mmHg in MSEW mice and 126±2 in C (P<0.05). No significant changes in mean arterial pressure, diastolic blood pressure or heart rate were observed between groups. Next, we also determined the BP sensitivity to the acute administration of AngII (1, 10 and 50 ug/kg, s.c.). AngII-induced BP changes, assessed by BP area under the curve, were similar between MSEW and C mice at all doses (50 ug/kg dose:145±10 vs. 132±15 mmHgx30 min, respectively). Chronic enalapril treatment (2.5 mg/kg/day, drinking water, 7 days) was conducted to block endogenous AngII synthesis. Enalapril reduced SBP 15±2 mmHg in MSEW mice but only 6±1 mmHg in C mice (p<0.05). The BP response to acute AngII doses increased similarly in MSEW and C enalapril-treated mice, (50 ug/kg dose: 200±13 vs. 207±22 mmHgx30 min, respectively). In addition, BP and HR responses to acute injections (i.p) of mecamylamine (5 mg/kg), propranolol (5 mg/kg) or atropine (1 mg/kg) were similar between untreated MSEW and C mice, suggesting that exacerbated BP in female MSEW mice is independent of sympathetic or parasympathetic dysfunction. Taken together, these data provide evidence that increased BP in female MSEW mice results from elevated circulating AngII rather than enhanced AngII sensitivity or sympathetic nerve activity.

BP.06.01] THE EFFECTS OF FEBUXOSTAT ON ANGIOTENSIN II-INDUCED VASCULAR REMODELING

Objective: A xanthine oxidase (XO) inhibitor, febuxostat (FEB) is often used in patients with hyperuricemia. Several reports have suggested that uric acid contributes to endothelial dysfunction and kidney injury. Because XO produces hydrogen peroxide with uric acid in the nucleic acid metabolism, XO inhibitors can also have the anti-oxidative properties. From these reasons, XO inhibitors have the possibilities to suppress cardiovascular diseases. Actually, the effects of FEB on cardiovascular events have been examined in several clinical trials in Japan and several basic researches have suggested that FEB suppressed cardiac fibrosis and renal inflammation, however, those mechanisms remain unclear. In this study, we investigated the effects of FEB on angiotensin II (Ang II)-induced vascular remodeling. Design and method: To induce vascular remodeling, Ang II (2 mg/kg/day, s.c.) was infused by osmotic minipump into the mice. FEB was suspended in 1% carboxymethyl cellulose and administrated daily for 14 days. The mice were divided into four groups: control, FEB alone, Ang II alone, and Ang II+FEB. Medial thickening and vascular fibrosis were assessed by Elastica van Gieson stain. Blood pressure was measured by tail-cuff method. The macrophage infiltration and XO localization in the aortic tissue were assessed by immunofluorescence. In our in vitro studies, protein phosphorylation and cell proliferation were measured by western blotting and MTT assay, respectively. Results: Serum XO activity was lower in the Ang II+FEB group than in the Ang II alone group. FEB administration suppressed Ang II-induced blood pressure elevation and aortic fibrosis, however it did not affect medial thickening of the aortae. In our in vitro studies, FEB did not change the aortic smooth muscle cell proliferation and ERK1/2 phosphorylation induced by Ang II stimulation. We found that XO was expressed strongly in the macrophages, and Ang II-induced macrophage infiltration in the aortae tended to be suppressed by FEB. Conclusions: Our results suggested that FEB affects the mechanisms of fibrosis in vascular cells more than those of smooth muscle cell proliferation induced by Ang II. We supposed that FEB suppresses macrophage-derived XO activity in the mouse model of Ang II-induced vascular remodeling.