AngII Hypertension Research Articles

Collagen receptor- and metalloproteinase-dependent hypertensive stress response in mesangial and glomerular endothelial cells.

Progressive alteration of the extracellular matrix (ECM) is the characteristic of hypertensive nephropathy (HN). Both mesangial and endothelial cells have the ability to synthesize and degrade ECM components, including collagens through the activation of matrix metalloproteinases (MMPs) in stress conditions, such as in hypertension. On the other hand, hydrogen sulfide (H2S) has been shown to mitigate hypertensive renal matrix remodeling. Surprisingly, whether H2S ameliorates receptor-mediated (urokinase plasminogen activator receptor-associated protein, uPARAP/Endo180) collagen dysregulation in Ang-II hypertension is not clear. The purpose of this study was to determine whether Ang-II alters the expression of Endo180, tissue plasminogen activator (tPA), MMPs, and their tissue inhibitors (TIMPs) leading to the dysregulation of cellular collagen homeostasis and whether H2S mitigates the collagen turnover. Mouse mesangial cells (MCs) and glomerular endothelial cells (MGECs) were treated without or with Ang-II and H2S donor GYY (GYY4137) for 48h. Cell lysates were analyzed by Western blot and RT-PCR, and cells were analyzed by immunocytochemistry. The results indicated that, while Ang-II differentially expressed MMP-13 and TIMP-1 in MCs and in MGECs, it predominantly decreased tPA, Endo 180, and increased plasminogen activator inhibitor-1 (PAI-1), MMP-14, and collagen IIIA and IV in both the cell types. Interestingly, H2S donor GYY treatment normalized the above changes in both the cell types. We conclude that Ang-II treatment causes ECM remodeling in MCs and MGECs through PAI-1/tPA/Endo180 and MMP/TIMP-dependent collagen remodeling, and H2S treatment mitigates remodeling, in part, by modulating these pathways.

Abstract 488: Overexpression of the Prostaglandin E2 EP3 Receptor Reduces Cardiac Function in an Angiotensin II Model of Hypertension

Prostaglandin E2 (PGE2) EP receptor subtypes EP3 and EP4 are present in the heart and signal via decreased and increased cAMP production, respectively. Previously we reported that cardiomyocyte-specific EP4 KO mice develop a phenotype of dilated cardiomyopathy with reduced ejection fraction, that PGE2 decreases contractility via EP3 in vivo, in vitro and ex vivo; and that EP3 expression in the heart increases when mice are subjected to Angiotensin (Ang) II-hypertension (HTN). We therefore hypothesized that overexpression of EP3 in the heart would exacerbate Ang II-HTN. To test this hypothesis, we used 10-12 week old male EP3 transgenic (Tg) mice with EP3 overexpression in cardiomyocytes and their wild type (WT) controls infused with either vehicle or 1.4 mg/kg/d Ang II for 2 weeks. Echocardiography was performed on conscious mice at the end of the experiment and systolic blood pressure (SBP) was measured every 2 days by tail cuff. Results: Table 1. All data are reported as means ± SEM. N= 3-6 mice per group. Statistical significance: * and *** p < 0.05 and p < 0.005 vs WT + vehicle, ++ p < 0.01 vs WT + Ang II, §§§ p < 0.005 vs Tg + vehicle. As shown in the table, cardiac function was unaffected in WT mice after Ang II infusion. Baseline cardiac function was reduced in EP3 Tg mice receiving vehicle and in contrast to WT mice, was further diminished after Ang II infusion. These differences were not due to altered systolic blood pressure. In conclusion, EP3 Tg mice present with reduced cardiac function at baseline and in support of our hypothesis, exhibit an enhanced response to Ang II. Antagonism of the EP3 receptor may be a novel treatment for heart failure associated with hypertension.

Abstract P158: Sulforaphane Rich Broccoli Powder Attenuates the Augmented Angiotensin II Induced Renal Inflammation and Injury in GSTM1 Deficient Mice

Glutathione- S -transferase μ-1 ( GSTM1 ) enzyme belongs to the superfamily of phase II antioxidant glutathione- S -transferases (GSTs) that are downstream targets of the Nrf2 antioxidant transcription factor. In humans, a common GSTM1 gene deletion variant, the null allele GSTM1(0) , results in decreased or complete absence of GSTM1 enzymatic activity and is associated with higher levels of oxidative stress. We reported that GSTM1(0) is associated with accelerated kidney disease progression in the African Americans Study of Kidney Disease (AASK) participants. To understand the direct impact of GSTM1 deficiency on renal inflammation and oxidative stress, we generated Gstm1 deficient mice (KO) to determine their response to angiotensin II, delivered @ 1000 ng/kg/min for 4 weeks via mini-osmotic pump. Blood pressure (BP) was measured by radiotelemetery. Kidney histopathology was assessed by a renal pathologist blinded to genotype and experimental conditions. Renal leukocyte populations were analyzed quantitatively by flow cytometry. Gstm1 KO mice had significantly higher levels of baseline systolic BP (SBP) and ~ 17 mmHg higher SBP after 4 weeks of Ang II-HTN, compared to WT mice. Gstm1 KO mice have increased renal superoxide levels by nearly 3 folds - independent of activation of Nox2 and Nox4 NADPH oxidases or alteration in superoxide dismutase - and worse kidney injury. These changes were associated with significantly increased renal expression of genes involved in inflammation - chemokine ligand 1 (CXCL-1), monocyte chemotactic protein 1 (MCP-1), Interleukin-1β (IL-1β) and IL-6 ( P <0.05). By flow cytometry, Gstm1 KO mice displayed ~ two fold increases in all leukocyte populations in the kidney, with the exception of the numbers of B cells that were not significantly different between groups. This increased renal inflammation was attenuated by dietary administration of broccoli powder that is rich in sulforaphane, a phytochemical that increases Nrf2 expression, independent of BP. In Ang II-hypertension, loss of GSTM1 enzyme may be deleterious by augmenting oxidative stress and inflammation in the kidney. Stimulation of the Nrf2 pathway in hypertension may be a novel therapeutic approach to prevent kidney disease progression in GSTM1 deficiency.

Na+ transporters, electrolyte excretion and renal injury in female and male C57Bl/6 and IL17A−/− mice during AngII hypertension

Experimental AngII hypertension is reported to be blunted in female (F) vs. male (M) mice and in mice lacking the inflammatory cytokine IL17A (time point dependent). This study aimed to determine how sex impacts renal Na+ transporters, Na+ and K+ excretion, and markers of renal injury during AngII hypertension (A) in both C57Bl/6 (WT) and IL17A−/− (IL17−/−) mice.11–16 wk old F and M mice were infused with AngII (490 ng/kg/min) or sham treated for 3 wk (n=6/group). Rate of excretion of a saline load (10% b.wt. subcutaneous) was slowed in all AngII infused groups, indicating Na+ reabsorption activation. Blood pressure (BP), by tail cuff, increased similarly across groups (in Δ mmHg): WTMA: 40 ± 1.6, WTFA: 37 ± 10, IL17−/−MA: 37 ± 3.8, IL17−/−FA: 46.4 ± 4.7. Thus, no blunting of hypertension was detected at 3 wk time point by tail cuff in F or in IL17−/−.In both M and F WT and IL17−/−, AngII provoked activation (‐phosphorylation) of NKCC2 and NCC, and activation of ENaC (more ‐cleaved (Cl), less full length (FL)). These distal anti‐natriuretic responses were balanced by natriuretic responses including: decreased abundance of proximal NHE3, NaPi2, and decreased medullary thick ascending limb NHE3, NKCC2 and NaK‐ATPase (see Figure). We previously proposed that increased ‐NCCp is a secondary compensation, driven by AngII activation of ENaC, to minimize K+ secretion (PMID27600183). Providing further support for this idea, urinary Na/K ratio (UNa/K), collected overnight (during fasting +water), was significantly reduced by AngII (WTM: from 0.71 ± 0.1 at baseline to 0.41 ± 0.9 with AngII, WTF: 0.80 ± 0.1 to 0.28 ±0.05, IL17−/−M: 0.99 ±0.13 to 0.41 ± 0.041, IL17−/−F: 1.05 ± 0.07 to 0.46 ± 0.46). UNa/K fell due to decreased o/n UNa as o/n UK was unaltered by AngII. Urine volume (V), unaltered by AngII treatment, was 2‐fold higher in M vs. F of both genotypes, and 2‐fold higher in IL17−/− vs. WT within each sex.During AngII, the injury marker U albumin increased across groups. Renal cortical angiotensinogen was lower in IL17−/− vs. WT (by 50% in F and 25% in M). KIM1 also tended to be lower in IL17−/− vs. WT in both sexes during AngII.From these results, we propose: AngII stimulation of ENaC drives UK secretion which activates NCCp (more in WT than IL17−/− males), thus reducing UNa to limit K secretion and lowering UNa/K which may contribute to BP elevation. IL17−/− mice exhibit higher UV and lower intrarenal angiotensinogen and KIM1 which suggests less tissue injury.Support or Funding InformationAHA GIA (15GRNT23160003) and an NIH NIDDK R01 (DK083785) to A.A. McDonough, NIH NHLBI K08 award (HL121671), Gilead Cardiovascular Scholars Grant, and NIH DP2 award (HL137166) to M.S. MadhurThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 060: Role of Mir-214 in the Regulation of Perivascular Fibrosis in Angiotensin II Induced Hypertension

Objective: Hypertension (HT) is associated with perivascular inflammation and increased vascular fibrosis. MicroRNAs (miR) are a novel gene expression regulation mechanism and play a pivotal role in a range of pathological processes. The role and mechanism of miR214 in vascular fibrosis is unknown. Methods: 3-month-old C57BL/6, miR214KO and wild-type littermates were treated with angiotensin II (AngII, 490ng/kg/min; n=6-10) or control buffer for 14 days. PVATs from C57BL/6 animals were analysed using TaqMan_Rodent_microRNA_Arrays. Histological studies, wire myography, lucigenin-enhanced luminometry and cytometrical analysis was conducted, followed by statistical analysis with ANOVA or t-test. Data are expressed as a mean±SEM. Results: Out of 381 miRs, 16 were significantly overexpressed in C57BL/6 AngII animals, with only miR214 showing 8-fold induction (p<0.01) after Bonferroni correction. Also, 3-fold elevation of pri-miR-214 was observed. Interestingly, hydralazine treatment prevented both these changes (p<0.01). AngII infusion in miR214 KO animals did not alter blood pressure when compared to WT mice. Mir214 KOs exhibited diminished peri-aortic fibrosis (44779±2491 vs 78805±8696μm, p<0.01), upon AngII hypertension. This was associated with a significantly reduced induction of COL1A1, COL3A1 and TGFβ1 mRNA expression in PVAT and aortas (p<0.05). Vascular studies revealed improved endothelial function (69±10 vs. 22±4%, p<0.01), protection against oxidative stress (66±7 vs 118±19 RLU/sec/mg, p<0.001) and NOX2 mRNA expression (1.9±0.2 vs1.1±0.1, p<0.05) in AngII miR-214-KO aortas, while these parameters were not altered in mesenteric arteries. Recruitment of T cells into aortic PVAT was abolished in KO HT animals in comparison to control group (192±65 vs. 603±164 cell/mg; p<0.05). AngII HT was associated with 4-fold increase of miR-214 expression in the circulating peripheral blood T cells and 2-fold in the spleen. Moreover, AngII infusion increased TNFα mRNA expression in WT T cells (1±0.1 vs 1.6±0, p<0.01) whereas this effect was not seen in miR214 KO T cells (0.9±0.3 vs 0.9±0.1). Conclusions: MiR-214 plays a major role in modulation of aortic fibrosis, vascular function, oxidative stress and perivascular inflammation.

Abstract P331: Angiotensin-salt Hypertension Requires Ouabain-sensitive Na + Pumps

Dietary salt is a major factor in the pathogenesis of essential hypertension (EH), but the underlying links are unresolved. Animal models indicate that angiotensin (Ang) II and high dietary salt (HS) are convergent signals that act via the brain to elevate blood pressure (BP). Low-dose sc Ang II+HS is a common model for EH. We tested the Na + pump ouabain binding site’s role in this model because it is crucial in some other hypertension models (e.g., ACTH and Nedd4-2-knockout +HS). Mice that express Na + pumps with a mutant, ouabain-resistant α2 catalytic subunit (α2 R/R ; cation transport is normal), and wild type (WT), ouabain sensitive controls (α2 S/S ) were studied. [80-90% of rodent artery myocyte Na + pumps are ouabain-resistant (α1 R/R ); only 10-20% are α2.] BP was measured by telemetry. First, 3 basal 24 hr BPs were recorded. Osmotic 4-week minipumps were then implanted sc in all mice to deliver vehicle (saline; Expt. #1,3), or 400 (Expt. #1,2) or 800 (Expt. #3) ng/kg/min Ang II; simultaneously, in Expt. #2, the diet was switched from 0.4% (standard) to 2% NaCl (HS). BPs were monitored every 3-4 days for up to 4 weeks. Also, in Expt. #2, on day 21, all mice received 2 ip injections, 4 hrs apart, of 10 mg/kg DigiFab, Fab fragments that immuno-neutralize ouabain, while BP was continuously monitored; on day 23, the mice received 2 ip injections of CroFab, anti-crotalus toxin (‘control’) Fab fragments. Results: 1. Basal mean BP (MBP) was 10±2 mm Hg higher in α2 R/R than in WT mice ( P <0.01; n =21 & 29; ANOVA). 2. In WT mice, 400 ng/kg/min sc Ang II and Ang II+HS raised MBP by 15±1 and 34±1 mm Hg, respectively ( P <0.01; n =7-8; ANOVA). 3. The MPB elevation in Ang II+HS α2 R/R (17±2 mm Hg) was only half that in WT mice ( P <0.01; n =7 each; ANOVA). 4. DigiFab rapidly (<1 hr) reduced MBP by 14±2 mm Hg in Ang II+HS hypertensive WT mice ( P <0.001; n =7; T-test), but not in α2 R/R mice ( P <0.01; n =7 each; ANOVA); CroFab did not lower MBP in either strain. 5. 800 ng/kg/min sc Ang II elevated systolic BP by 55±3 mm Hg in WT mice, but by only 37±3 mm Hg in α2 R/R mice ( P <0.05; n =3-5; ANOVA). Conclusions: Ouabain-sensitive α2 Na + pumps and their endogenous ligand are both required for full expression of low-dose Ang II-salt hypertension. Ouabain-sensitive α2 pumps apparently also contribute to high-dose Ang II-hypertension.

AngII Hypertension Differentially Influences Sodium Transporters, Channels and Claudins in Female vs. Male FVB/N Mice

AngII Hypertension Differentially Influences Sodium Transporters, Channels and Claudins in Female vs. Male FVB/N Mice

OP.3D.02] MICRORNA-214 IS INVOLVED IN THE REGULATION OF PERIVASCULAR FIBROSIS IN HYPERTENSION

Objective: Hypertension is associated with perivascular inflammation and increased collagen deposition in adventitia. MicroRNAs (miR) are a novel mechanism for gene expression regulation and play a pivotal role in a wide range of pathological processes such as vascular fibrosis.The role and mechanism of miRs in this irreversible process are still unknown. Design and method: 3-months-old C57BL/6, miR-214KO and wild type littermates were treated with angiotensin II (AngII, 490ng/kg/min; n = 6–10) or control buffer for 14 days. Perivascular adipose tissues (PVAT) from C57BL/6 animals were analysed using TaqMan_Rodent_microRNA_Arrays. Histological studies, wire myography, lucigenin enhanced luminometry and cytometrical analysis was conducted. Statistical analysis was performed using ANOVA or t-test. Data are expressed as a meanSEM. Results: Out of 381 miRs, 16 were significantly overexpressed in C57BL/6 AngII animals after global normalisation. Mir-214 was the only significantly overexpressed miR with 8-fold induction (p < 0.001) in hypertensive animals after Bonferroni correction. miR214 was also increased the serum of hypertensive animals (p < 0.05). AngII infusion in miR-214KO animals did not alter blood pressure elevation, when compared to WT mice. Mir-214KOs exhibited diminished peri-aortic fibrosis upon AngII hypertension (44779 ± 2491 vs.78805 ± 8696 μm, p < 0.001). Vascular studies revealed improved endothelial function (69 ± 10 vs. 22 ± 4%, p < 0.001 by repeated measures ANOVA) and protection against oxidative stress in AngII miR-214KO aortas (66 ± 7 vs. 118 ± 19 RLU/sec/mg, p < 0.001), while these parameters were not altered in mesenteric arteries. Moreover, miR-214-KO animals were protected from VSMC hypertrophy observed in WT hypertensive animals. Recruitment of T cells into aortic PVAT was abolished in KO hypertensive animals in comparison to control (192 ± 65 vs. 603 ± 164 cell/mg; p < 0.05). This was observed in relation to both CD4 + and CD8 + T cells (107 ± 32 vs. 360 ± 108; p < 0.05, 69 ± 20 vs. 209 63cell/mg; p < 0.05, respectively). Interestingly AngII hypertension was associated with with 4-fold increase of miR-214 expression in the circulating peripheral blood T cells. Conclusions: AngII infusion changes miR profile in murine PVAT. MiR-214 has a major role in modulation of aortic fibrosis, vascular function and recruitment of T cells into PVAT. In the future, MiR-214 may be used as a biomarker or a potential target in vascular fibrosis prevention.

Abstract P192: Both Mineralocorticoid Receptor and Angiotensin II type 1 Receptors in the Subfornical Organ Mediate Angiotensin II Induced Reactive Oxygen Species (ROS) in Brain Angiotensinergic Pathways

Activation of angiotensinergic pathways and central aldosterone (aldo)-MR-ENaC-endogenous ouabain (EO)-AT 1 R pathway play a critical role in Ang II associated hypertension. The SFO contains both MR and AT 1 R and can relay the signals of circulating Ang II to downstream nuclei such as the PVN, SON and RVLM. We evaluated the effect of knockdown of MR and AT 1 R specific in the SFO on reactive oxygen species (ROS) production in downstream nuclei. Wistar rats were intra SFO infused with AAV-MR- or AT 1a R-siRNA and after 7 days received a sc infusion of Ang II at 500 ng/min/kg for 2 weeks. MR and AT 1 R expression were measured by real-time qPCR and western blotting. ROS was assessed by DHE staining. Ang II increased AT 1 R mRNA expression in the SFO. Both MR- and AT 1 R-siRNA in the SFO prevented this increase. Ang II decreased MR mRNA but increased protein expression in the SFO. Both MR- and AT 1 R-siRNA further decreased MR mRNA expression. Ang II significantly increased ROS in the SFO, magno- and parvocellular parts of the PVN, SON and RVLM. Both MR- and AT 1 R-siRNA in the SFO prevented ROS increases in the PVN and RVLM. In contrast, MR- but not AT 1 R-siRNA in the SFO prevented the Ang II-induced ROS in the SON. Both MR- and AT 1 R-siRNA in the SFO prevented most of the Ang II-induced hypertension. These results suggest that aldo-MR signaling in the SFO is needed for the activation of Ang II-AT 1 R signaling from the SFO to the PVN and RVLM. Considering that only MR-siRNA in the SFO prevents circulating Ang II induced ROS in the SON, activation of aldo-MR signaling from the SFO to the SON may via EO enhance AT 1 R dependent activation of pre-sympathetic neurons in the PVN, and thereby to Ang II-hypertension.

Abstract 19932: A Neuroimmune Drive Activates Plgf to Control an Epigenetic Mechanism in the Spleen That Allows T Cells Activation in Hypertension

Introduction and Hypothesis: In the past several years, there has been mounting interest in the roles played by immunity in hypertension. However, where and how the immune system gets activated under hypertensive stimuli to contribute to hypertension, is still enigmatic. The activation of effector T cells in hypertension suggests that antigen presentation by a specialized subset of innate cells represents an important component that participates in this process. In particular, it has been recently shown that T cell costimulation by B7 ligands is an important mechanism in the development of hypertension. Methods and Results: Here we demonstrate a novel splenic mechanism driven by Placental Growth Factor (PlGF) and regulating T cells costimulation in AngII-induced hypertension. We have found that PlGF is necessary to allow CD86 expression, crucial in AngII hypertension. Moreover, we show that this effect is exerted by a Sirt1-dependent epigenetic modulation of Timp3 expression in splenic macrophages, obtained by controlling p53 repressor activity on its promoter. Since the brain plays a pivotal role in AngII-induced hypertension and it is known that a selective removal of sympathetic innervation to the splanchnic district, obtained by celiac ganglionectomy (CGX), markedly attenuated hypertension, we looked whether the early induction of splenic PlGF upon AngII could be mediated by a nervous drive. Indeed, when we performed CGX in WT mice, splenic PlGF was markedly reduced, thus demonstrating that is is activated by Ang-II induced SNS overactivity. Importantly, we show that this neuroimmune mechanism driven by PlGF is crucial for the onset of hypertension, since PlGF KO mice display a clear-cut protection from the typical AngII-hypertensive response and from T cells infiltration in vessels and kidneys. Finally, chimeric mice generated by spleen transplantation (PlGF KO spleen in WT background and vice versa), allowed us to finally demonstrate that PlGF is indispensable in the spleen for the onset of hypertension. Conclusions: Summarizing, our study brought to light both how important the neuroimmune drive in the spleen is in the genesis of hypertension and, at same time, unveil that PlGF is the molecular pathway recruited in the spleen to control BP raising.

Abstract 043: Acute K+ Intake Does Not Reduce Renal Na-Cl Cotransporter Phosphorylation during Chronic AngII Hypertension

In Sprague Dawley (SD) rats, a single K + rich meal provokes a 50% decrease in DCT Na-Cl cotransporter phosphorylation (NCCp) which shifts Na + reabsorption downstream for reabsorption by CCD ENaC which drives a homeostatic increase in K + secretion. During Ang II dependent hypertension, DCT NCCp increases 2-4 fold as does NCC mediated Na + reabsorption. Motivated by these two recent findings, we tested the hypothesis that NCCp elevated by AngII hypertension (400 ng AngII/kg/min x 14 d in SD rats) could be reduced by a single K + rich meal (3 hr 2% K meal). Three groups (n=5-6) of male SD rats were compared: 1) Untreated fed 0%K meal, 2) AngII infused fed 0%K meal, and 3) AngII infused fed 2%K meal. By immunoblot of renal cortical homogenates, AngII infusion increased the following 2-5 fold compared to the untreated group: NCC, NCCpS71, NCCpT53, NCCpS89, SPAK, SPAKpS373, and cleaved ENaC-gamma. A single 2%K meal did not reduce abundance or phosphorylation of NCC, SPAK or ENaC cleavage. Interestingly, the AngII infused rats exhibited signs of kaliuresis and K + depletion compared to the untreated group: overnight urine K + (UKV) was 4.3 +/- 0.1 vs. 2.3 +/- 0.3 mM, fasting plasma [K + ] was 3.3 +/- 0.1 vs. 3.9 +/- 0.1 mEq in AngII vs. untreated, and while plasma [K + ] climbed to 4.6 +/- 0.1 after the 2% K meal in AngII infused rats, UKV was not elevated compared to AngII rats fed a 0%K meal. The results indicate that a single K + rich meal does not reduce NCCp in AngII infused rats, likely due to our finding that the AngII hypertension protocol provokes kaliuresis and K + deficiency. Thus, the ingested K + replenishes K + stores and is not excreted. The results also suggest that the rise in NCCp during AngII hypertension may be, at least in part, a response aimed to reduce K + excretion.

Abstract 207: Angiotensin -II (Ang-II) Induces Endoplasmic Reticulum (ER) Stress-mediated Overproduction of Mitochondrial Reactive Oxygen Species (mROS) and Alterations in Mitochondria-associated Membrane (MAM) in Neural Cells

ER stress and mROS are identified as key mediators of Ang-II-induced neurogenic hypertension. Although ER and mitochondria are linked both structurally and functionally, the interplay between these two organelles in neural cells and in the subfornical organ (SFO), a key forebrain region in Ang-II hypertension, is unknown. Previously we reported that Ang-II causes mitochondrial fragmentation in murine SFO neurons. Here we tested the hypothesis that Ang II-induced mitochondrial dysfunction is mediated by ER stress and disruption of the MAM. Neuro-2A cells were incubated with Ang-II (300 nM, 24 h) and real-time PCR was performed to examine expression of key molecular markers of MAM (n=3-5). Dynamin related peptide 1 (drp1), a MAM marker and mitochondrial fission protein, was increased in the presence of Ang-II (1.77±0.17-fold vehicle, p&lt;0.05).This effect was blocked when cells were co-treated with the chemical ER stress inhibitor TUDCA (0.77 ± 0.27 fold vs. Ang-II; p&lt;0.05). Ang-II also increased the expression of phosphofurin acidic cluster sorting protein 2 (pacs2, another MAM marker) and the co-administration of TUDCA was able to block this effect (Ang-II: 1.5 ± 0.02-fold vs. vehicle; TUDCA+Ang-II: 0.9 ± 0.03 fold vs. Ang-II; p&lt;0.05). Ang-II-induced mROS production, measured by MitoSox Red dye (n=11-15), was decreased in the presence of TUDCA (12 ± 4% increase from vehicle compared to a 58 ± 2% increase with Ang-II alone ; p&lt;0.05). BD-1047, a chemical disruptor of MAM, also prevented the Ang-II-induced increase in MitoSox fluorescence (9.6 ± 0.2 % vs. Ang-II alone; p&lt;0.05). Furthermore, when MDivi-1, a selective drp-1 blocker, was added, a similar blockade of mROS production was observed (11.9 ± 0.8 %; p &lt; 0.05) In dissociated SFO cells from adult male C57Bl/6 mice (n=8-23), Ang-II-induced mROS production (12.9 ± 2% increase from vehicle, p&lt; 0.05) was blocked by TUDCA (0.8 ± 0.01%, p&lt;0.05) and by losartan (2.2 ± 1.6%, p&lt;0.05). These data demonstrate that induction of ER stress by Ang-II leads to mitochondrial fragmentation mediated by MAM disruption, which then results in mROS overproduction. Collectively, our data suggest a novel role for ER-MAM interaction in Ang-II-mediated hypertension.

Bortezomib, a Proteasome Inhibitor, Attenuates Angiotensin II-Induced Hypertension and Aortic Remodeling in Rats

BackgroundHypertension is a highly prevalent disorder and a major risk factor for cardiovascular diseases. Hypertensive vascular remodeling is the pathological mal-adaption of blood vessels to the hypertensive condition that contributes to further development of high blood pressure and end-organ damage. Hypertensive remodeling involves, at least in part, changes in protein turnover. The ubiquitin proteasome system (UPS) is a major protein quality and quantity control system. This study tested the hypothesis that the proteasome inhibitor, bortezomib, would attenuate AngII-induced hypertension and its sequelae such as aortic remodeling in rats.Methodology/Principal FindingsMale Sprague Dawley rats were subjected to AngII infusion for two weeks in the absence or presence of bortezomib. Mean arterial pressure was measured in conscious rats. Aortic tissue was collected for estimation of wall area, collagen deposition and expression of tissue inhibitors of matrix metalloproteases (TIMP), Ki67 (a marker of proliferation), reactive oxygen species (ROS) and VCAM-1 (a marker of inflammation). AngII infusion increased arterial pressure significantly (160±4 mmHg vs. vehicle treatment 133±2 mmHg). This hypertensive response was attenuated by bortezomib (138±5 mmHg). AngII hypertension was associated with significant increases in aortic wall to lumen ratio (∼29%), collagen deposition (∼14%) and expression of TIMP1 and TIMP2. AngII also increased MMP2 activity, proteasomal chymotrypsin-like activity, Ki67 staining, ROS generation and VCAM-1 immunoreactivity. Co-treatment of AngII-infused rats with bortezomib attenuated these AngII-induced responses.ConclusionsCollectively, these data support the idea that proteasome activity contributes to AngII-induced hypertension and hypertensive aortic vascular remodeling at least in part by modulating TIMP1/2 and MMP2 function. Preliminary observations are consistent with a role for ROS, inflammatory and proliferative mechanisms in this effect. Further understanding of the mechanisms by which the proteasome is involved in hypertension and vascular structural remodeling may reveal novel targets for pharmacological treatment of hypertension, hypertensive remodeling or both.

Abstract 442: Endoplasmic Reticulum (ER) Stress in the Subfornical Organ (SFO) Induces Peripheral Inflammation in Angiotensin II (Ang-II)-dependent Hypertension

The SFO is implicated in peripheral T cell activation and the genesis of Ang-II-dependent hypertension. Our recent studies show that ER stress in the SFO is also a key mechanism underlying the development of Ang-II hypertension. Because the ER is closely integrated with initiation of the adaptive immune response, we hypothesized that ER stress in the SFO contributes to peripheral inflammation in Ang-II hypertension. First, 5 days of intracerebroventricular (ICV) infusion of thapsigargin (Tg, 1 ug/day), a chemical ER stress inducer, caused a significant increase in CD3 + T cells in aortas (Tg: 11.9 ± 3.5 x 10 3 cells/aorta vs. Vehicle: 2.2 ± 0.7 x 10 3 cells/aorta , n = 6, p&lt;0.05) and blood (Tg: 9.9 ± 1.8 x 10 4 cells/ mL vs. Vehicle: 2.9 ± 0.6 x 10 4 cells/ mL, n = 6, p&lt;0.05). Furthermore, quantitative real-time PCR of SFO micropunches showed a 15-fold increase of TNF-α, a pro-inflammatory cytokine, a 3-fold increase of CCL5, a T cell attracting chemokine and a 3-fold increase in CD3, a T cell marker, (n = 4, p&lt;0.05). To test the functional role of ER stress in the SFO in peripheral T cell activation, we targeted an adenovirus encoding GRP78 (AdGRP78), a molecular ER stress inhibitor, to this brain region during chronic systemic Ang-II infusion (600 ng/kg/min, 14 days). Our results demonstrate a significant reduction in T cell accumulation in aortas compared to control virus (AdLacZ) treatment (AdGRP78: 0.5 ± 0.07 x 10 4 cells/aorta vs. AdLacZ: 8.7 ± 2.1 x 10 4 cells/aorta, n = 6, p&lt;0.05). These data show that 1) brain ER stress induces inflammation in the SFO and peripheral vascular T cell activation, and 2) ER stress in the SFO is linked to peripheral vascular T cell activation in Ang-II-dependent hypertension. These results suggest that ER stress and inflammation in the SFO induce peripheral vascular T cell activation and inflammation in Ang-II hypertension.

Abstract 313: Effects of Angiotensin II-Induced Hypertension and HFHS-Induced Obesity on PVAT Function in Young Rats

The perivascular adipose tissue (PVAT) is a metabolically active structure that modulates the contractile, proliferative and migratory functions of underlying smooth muscle. In normal conditions, PVAT decreases contractile responses non-specifically in various vascular beds. Alterations in the secretory properties and consequently function of PVAT occur in various cardiovascular diseases, such as hypertension and metabolic syndrome. We have previously observed that contrary to genetic or long term diet-induced obesity, in a model of short-term high fat high sucrose (HFHS) diet-induced obesity, the anti-contractile effects of PVAT were increased. Animal models of hypertension, including angiotensin II-induced hypertension, are associated with a loss of anti-contractile effects of PVAT. Thus, we hypothesized that angiotensin II-induced hypertension opposes the effects of HFHS-induced obesity on PVAT anti-contractile function. We used young control non-treated rats, HFHS obese rats, angiotensin II hypertensive rats, and AngHFHS obese hypertensive rats and compared contractile function in the absence or presence of PVAT in mesenteric resistance arteries. Systolic blood pressure was significantly increased in AngII and AngHFHS rats compared to control (AngHFHS=153±17, AngII=170±15, Con=97±6 mmHg), while not significantly different from control in HFHS rats. Visceral adipose tissue mass was overall significantly increased in HFHS and AngHFHS rats compared to control rats (e.g. retroperitoneal fat AngHFHS=0.47±0.07, HFHS=0.64±0.14, Con=0.23±0.04 % body weight). In the absence of PVAT, contractile responses to PE, 5HT and ET-1 in mesenteric artery were not altered by HFHS obesity alone, however were increased in AngII and AngHFHS conditions. Anti-contractile effects of PVAT were significantly reduced in AngII hypertension but similarly increased in HFHS and AngHFHS rats (% reduction in max contraction, con: PE=13.6, 5-HT=14.04, ET-1=19.53; HFHS: PE=13.3, 5-HT=26.13, ET-1=30.93; AngHFHS: PE=36.06, 5-HT=18.20, ET-1=39.77). These data suggest that the deleterious effects of augmented BP on PVAT function are insufficient in counteracting the increase in anti-contractile PVAT effects in response to HFHS obesity in young rats.

Role of blood cell-associated angiotensin II type 1 receptors in the cerebral microvascular response to ischemic stroke during angiotensin-induced hypertension

BackgroundAngiotensin II type 1 receptor (AT1R) blockers lower the incidence of ischemic stroke in hypertensive patients and attenuate brain inflammation and injury in animal models. Although AT1R on both blood cells (BC) and vascular endothelial cells (EC) can be activated by angiotensin II (Ang II) to elicit inflammation, little is known about the relative contributions of AT1R expressed on BC and EC to the brain injury responses to ischemia and reperfusion (I/R) in the setting of angiotensin-induced hypertension.MethodsThe contributions of BC- and EC-associated AT1R to I/R-induced brain inflammation and injury were evaluated using wild type (WT), AT1aR-/-, and bone marrow chimera mice with either a BC+/EC+ (WT→WT) or BC-/EC+ (AT1aR-/-→WT) distribution of AT1aR. The adhesion of leukocytes and platelets in venules, blood brain barrier (BBB) permeability and infarct volume were monitored in postischemic brain of normotensive and Ang II-induced hypertensive mice.ResultsThe inflammatory (blood cell adhesion) and injury (BBB permeability, infarct volume) responses were greatly exaggerated in the presence of Ang II-induced hypertension. The Ang II-enhanced responses were significantly blunted in AT1aR-/- mice. A similar level of protection was noted in AT1aR-/- →WT mice for BBB permeability and infarct volume, while less or no protection was evident for leukocyte and platelet adhesion, respectively.ConclusionsBC- and EC-associated AT1aR are both involved in the brain injury responses to ischemic stroke during Ang II-hypertension, with EC AT1aR contributing more to the blood cell recruitment response and BC AT1aR exerting a significant influence on the BBB disruption and tissue necrosis elicited by I/R.

IL‐6 Knockout Blocks Low‐Dose Angiotensin II Hypertension Independent of Renal Vasoconstriction

We have reported that IL‐6 knockout (KO) mice have ~50% inhibition of AngII hypertension with a 90 ng/min, ip AngII dose. Lower doses of 5 ng/min (200 ng/kg/min) and 20 ng/min (800 ng/kg/min) have been shown to be slow‐pressor and physiologic hypertensive doses, respectively, of AngII in mice. We instrumented mice with DSI blood pressure transmitters or Transonic renal flow probes to test whether IL‐6 knockout could ameliorate lower dose AngII hypertension and determine the relationship to chronic renal function. MAP averaged 112±5 and 119±7 mmHg in WT (n=6) and KO (n=5) mice, respectively during 4 control days, and in WT mice increased to 124±4 mmHg on day 1 and rose gradually to 146±5 mmHg by AngII day 7. In the KO mice, however, MAP did not increase on any day of AngII, and averaged 123±7 mmHg by day 7. RBF, measured 5 hrs/d, decreased in both WT mice and both KO mice by approximately 50% of control, and this was sustained during the 7‐day AngII infusion. Our previous report with 90 ng/min showed rapid increases in MAP that were not different between WT and KO mice, and the significant difference became evident only during the sustained phase. That suggested the vasoconstrictor responses were not IL‐6 dependent, and the current RBF data concur. Together with the complete blockade of AngII hypertension at this 20 ng/min dose, this suggests that the dependence of AngII hypertension on IL‐6 is due to renal tubular mechanisms. HL74167.

I009 Angiotensinii induced atrial remodelling is worsened in mice overexpressing aldosterone synthase in cardiomyocyte

The aim of this work was to check the hypothesis that increased cardiac aldosterone level combined with arterial hypertension may be deleterious for the heart. Transgenic mice overexpressing Aldo Synthase (AS) in cardiomyocytes and wild type (WT) littermates were submitted to AngII treatment by osmotic pump (1 mg/kg/day) during 3 and 8 weeks. 1) Same levels of hypertension were observed in all treated groups whatever the genotype. Ventricular remodelling was equivalent in terms of LVH at 3wks and 8 wks in AngII-treated groups (ratio HW/BW +10 % for AS and WT after AngII, p<0.05) and fibrosis (×3 for AngII groups, p<0.05). Eplerenone treatment (50 mg/kg/day) prevented these changes. In contrast, after 3 wks of AngII treatment atrial remodelling was worsened with increased dilatation (0.220±0.003 cm vs 0.200±0.003 cm for WT, p<0.05) and fibrosis (3.13±0.26 % vs 2.64±0.16 % for WT, p<0.05) in AS mice. This phenotype was aggravated in 8 weeks treated mice with an atrial diameter of 0.240±0.004 cm in AS vs 0.220±0.004 cm (p<0.05) in WT mice. Atrial fibrosis was 4.2±0.6 % in AS vs 3.6±0.2 % in WT (p<0.55). We also observed a longer P wave duration in AS mice after AngII than in WT (for 3 wks treatment, 18.78±0.45 ms vs 17.15±0.38 ms for WT, p<0.01 and at 8 wks 22.00±0.42 ms vs 20.98±0.83 ms for WT, p<0.05). 2) This electrical change led us to study the expression of atrial connexin (Cx) 40 and 43. AS mice showed a decrease of about 50 % of C×43 at basal state compared to WT but C×40 levels remained unchanged. AngII treatment decreased about 50 % C×43 and C×40 expression in WT mice. For AS mice, AngII induced an increase of about 50 % of C×40 expression but C×43 expression was not affected. In conclusion, these results suggest that cardiac aldosterone combined with AngII hypertension have deleterious effects on left atria: it increases dilatation and fibrosis and it increases conduction time by regulating expression of C×40 and 43.

Contrasting effect of exercise and angiotensin II hypertension on in vivo and in vitro cardiac angiogenesis in rats

Cardiac vessel density (beta-actin immunolabeling) and angiogenic capacity of coronary artery explants (culture in collagen gel) was determined in hypertrophied heart obtained by exercise training (10 wk) or ANG II infusion for 10 days. A group of rats received ANG II the last 10 days of training. The heart weight index was similarly elevated after exercise, and ANG II-hypertension compared with controls (3.16 +/- 0.09 and 3.11 +/- 0.11 vs. 2.68 +/- 0.08 mg/g, respectively), whereas tail cuff pressure (TCP) increased only in sedentary rats infused with ANG II. Vessel density was increased by 36% in trained rats and reduced by 30% in ANG II-infused rats. The number of sprouts generated by coronary rings was reduced by 50% in ANG II-infused rats and increased by 50% in exercise trained rats compared with controls (35 +/- 4 and 113 +/- 5 vs. 71 +/- 1 sprouts per ring, respectively). Exercise-training partly prevented the hypertensive effect of ANG II (TCP of 141 +/- 5 mmHg), whereas heart weight index (3.66 +/- 0.06 mg/g body wt) was not lowered. Myocardial vessel density was normalized, and sprouting from coronary rings increased by 50% in trained rats infused with ANG II compared with sedentary normotensive rats. Cardiac VEGF (Western blot analysis) was higher in hypertensive rats and not affected by exercise. Facing a similar increase in cardiac mass, intense training, but not ANG II hypertension, is accompanied by an increase in vascular density of the heart. The effect of training is unlikely related to changes in resting VEGF and may represent enhanced angiogenic capacity of the coronary vascular bed.

The effects of angiotensin II and genetic hypertension upon extracellular nucleotide hydrolysis by rat platelet ectoenzymes

The extracellular nucleotides, ATP and ADP, as well as adenosine have been implicated in a great number of physiological functions. ADP is one of the major platelet recruiting factors, whereas ATP is considered to be a competitive inhibitor of ADP-induced platelet aggregation and adenosine is able to induce vasodilatation and to inhibit platelet aggregation. The di- and triphosphate nucleosides can be hydrolyzed by members of several families of ectonucleotidases, including ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) and ecto-nucleotide pyrophosphatase/phosphodiesterases (E-NPPs) that, together with an ecto-5'-nucleotidase, catalyze adenosine formation. The renin–angiotensin system is the most important regulator of renal and cardiovascular functions and angiotensin II induces, physiologically, platelet activation. The aim of this study was to clarify the effects of ANGII and genetic hypertension upon extracellular nucleotide hydrolysis by rat platelet ectoenzymes. ANGII, in all tested doses (5, 50, 500 and 5000 pmol), was able to increase ATP (21, 31, 44 and 27%, respectively), ADP (22, 28, 78 and 37%, respectively) and AMP (40, 64, 60 and 64%, respectively) hydrolysis by rat platelets. Furthermore, losartan, a specific antagonist of the AT1 angiotensin-receptor, prevented the nucleotide hydrolysis effects. Additionally, an increase in AMP (about 144%) hydrolysis and a decrease in p-Nph-5'TMP (about 27%) hydrolysis were observed in platelets from spontaneously hypertensive rats (SHR) when compared to Wistar normotensive rats. We, herein, present data to demonstrate interactions between rat platelet angiotensinergic and adenosinergic systems that could contribute to the understanding and treatment of cardiovascular diseases such as hypertension, thrombosis and arteriosclerosis.