Days Of Angiotensin II Infusion Research Articles

Potential therapeutic efficacy via avB3-integrin dependent interaction of developmental endothelial locus-1 (Del-1) after established angiotensin-II dependent hypertension

Abstract Introduction Cardiovascular diseases (CVDs) contribute to about 31% of global death. Hypertension is a major risk factor and also increases the risk of CVDs. An important regulator of systemic blood pressure in the body, the Renin Angiotensin Aldosterone system becomes overactivated during hypertension releasing a critical product, angiotensin-II (ANG-II), a known vasoconstrictor, activating the immune system leading to inflammation and as such mediating the effects of hypertension and subsequent end-organ damage due to the critical role of the innate and adaptive cells in producing pro-inflammatory cytokines such interleukin-17. Del-1, an extracellular matrix protein expressed mostly by endothelial cells is a known potent inhibitor of leukocyte recruitment. Aim We tested the hypothesis that, Del-1 via αvβ3-integrin dependent interaction protects against ANG-II induced cardiovascular remodeling after established hypertension. Methods Wild type (WT) mice were infused with 2 mg/kg/day of ANG-II for 14 days. Soluble Del-1 (Del-1-Fc) and a point mutant unable to bind to αvβ3-integrin (Del-1-RGE-Fc) was injected (50 μg/per injection) daily after 6 days of ANG-II infusion. SBP was measured using tail-cuff. Endothelial function was assessed using Mulvany myograph and quantification of inflammatory cells and IL-17 by flow cytometry. Histological staining for fibrosis, myocardial/medial hypertrophy and elastin degradation was used to evaluate both aortic and cardiac remodeling. Results After 6 days, mice infused with ANG-II showed significantly higher SBP of 140 mmHg as compared to control mice (110 mmHg). However, further progression of SBP after 6 days was blunted in infused mice treated with soluble Del-1 (ANGII+Del-1-Fc) as compared to similar increase in SBP in both infused mice treated with the point mutant (ANGII+Del-1-RGE-Fc) and non-treated infused mice (ANGII+Fc) leading to a difference of ∼20 mmHg. This effect was due to, the well protected endothelium dependent relaxation and reduced wall tension of the aorta in ANGII+Del-1-Fc mice. Further infiltration of CD45+ leukocytes, CD45+/IL-17A+ and TCRβ+T cells was significantly reduced in the aorta and heart of ANGII+Del-1-Fc mice as compared to both ANGII+Del-1-RGE-Fc and ANGII+Fc mice. However, the aorta and heart of ANGII+Del-1-Fc mice showed increased levels of anti-inflammatory regulatory T-cells (CD25+FOXP3+Treg). Also, aortic medial thickness, adventitial collagen and elastin degradation was decreased significantly in ANGII+Del-1-Fc mice. Additionally, in comparison to both ANGII+Del-1-RGE-Fc and ANGII+Fc mice, the hearts of ANGII+Del-1-Fc mice had significantly less interstitial and pericoronary collagen, smaller cardiomyocyte area and subsequently higher lumen-to-wall ratio. Conclusion Our study reveals the cardiovascular protective effects of Del-1 mediated via its association with αvβ3-integrin after established hypertension which could serve as potential therapeutic target. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): German Research Foundation (DFG)National Institutes of Health (NIH)

Abstract P101: Suppression Of Serum Amyloid A Limits Progression Of Obesity Associated Abdominal Aortic Aneurysms

Obesity increases the risk for abdominal aortic aneurysms (AAA) in humans, and enhances angiotensin II (AngII)-induced AAA formation in C57BL/6 mice. Obesity is also associated with increases in serum amyloid A (SAA). We previously reported that deficiency of SAA significantly reduces AngII-induced inflammation and AAA in apoE-deficient mice. In this study we investigated whether SAA plays a role in progression of an established AAA in obese C57BL/6 mice. Approach and Results: Male C57BL/6 mice were fed a high fat diet (60% kcal as fat) throughout the study. After 4 months of diet the mice were infused with angiotensin II (AngII) at 1000ng/kg/min until the end of the study. Ultrasound (US) was performed in all mice before and after 28 days of AngII infusion, and mice that had at least a 25% increase in the luminal diameter of the abdominal aorta were stratified by luminal diameter into 3 groups. Group 1 was killed to establish baseline AAA. Groups 2 and 3 continued to receive AngII for a further 8 weeks along with an antisense oligonucleotide (ASO) that suppresses all 3 acute phase SAA isoforms (SAA-ASO), or a control ASO (5 mg/kg/wk). US was repeated at study end to assess AAA progression. Plasma SAA at the end of the experiment was 89.2±83.2 mg/L in the control ASO group, and 18.6±0.7 mg/L in the SAA-ASO group (mean±SD , p=0.008). There was no impact of SAA suppression on body weight, body fat, or blood pressure. After the first 4 weeks of AngII infusion, the average luminal diameter in all mice was 1.81±0.40 mm (mean±SD). Mice that received the control ASO had continued aortic dilation (average luminal aortic diameter 2.06±0.42 mm), whereas the mice that received the SAA-ASO had a significant reduction in progression of aortic dilation (average luminal diameter 1.64±0.43 mm, p=0.0015 for interaction between time and group). Conclusions: We demonstrate for the first time that suppression of SAA protects obese C57BL/6 mice from progression of AngII-induced AAA. Suppression of SAA may be a therapeutic approach to limit AAA progression.

Temporal Profiling of Molecular Alterations in the Hypothalamic Paraventricular Nucleus During Angiotensin‐II Hypertension

The hypothalamic paraventricular nucleus (PVN) plays a central role in cardiovascular regulation including hypertension development in response to elevations in angiotensin-II (Ang-II). Upstream circumventricular nuclei located outside of the blood-brain-barrier sense circulating Ang-II and influence PVN endocrine and autonomic regulation via efferent synaptic projections. Despite progress in unraveling the function of circumventricular-PVN networks in neurogenic hypertension development, the underlying PVN mechanisms responsible for hypertension development remain unclear. In particular, most investigations have examined PVN alterations during established hypertension, which limits insight into molecular changes that may occur prior to hypertension development. Based on this, we performed a detailed temporal profiling of key molecular targets in the PVN during Ang-II hypertension progression. Male C57Bl/6J mice were implanted with subcutaneous osmotic minipumps for chronic delivery of low dose Ang-II (600 ng/kg/min). Brains were harvested for immunohistochemistry analysis of target molecules across rostral to caudal subregions of the PVN (n=3-4/group) at baseline (day 0), pre-hypertensive (days 3 and 7), and established-hypertension (day 14) timepoints. Previous work has suggested a role for arginine vasopressin (AVP) in Ang-II-induced hypertension. In line with this, AVP immunoreactivity in the PVN was elevated within 3 days of Ang-II infusion; this was evident only in the caudal PVN (day 3 fold change: 2.5±0.2, density/cell, p<0.05), whereas AVP expression in the rostral and medial portions was unchanged. Interestingly, caudal AVP immunoreactivity returned to basal levels by day 7 and remained low for the remainder of the protocol. In contrast to AVP, oxytocin expression was unchanged across the rostral to caudal PVN during 2-week Ang-II hypertension development. We also measured preproenkephalin (PPE) immunoreactivity, and found a progressive increase throughout the entire PVN with elevations initiated at pre-hypertensive times (day 7 fold change: 2.0±0.1, density/area, p=0.08) that were sustained during established hypertension (day 14 fold change: 2.6±0.3, density/area, p<0.05). To test possible involvement of the arcuate nucleus, which transmits circulating information from the median eminence to the PVN, in Ang-II hypertension, we measured alpha-melanocortin stimulating hormone (alpha-MSH) levels. Histological analysis for alpha-MSH immunoreactivity in the PVN, as an indicator of synaptic innervation strength from arcuate nucleus proopiomelanocortin neurons, revealed no changes during Ang-II infusion. Collectively, these findings indicate elevations in PVN AVP and PPE, but not oxytocin and alpha-MSH, in response to elevations in circulating Ang-II. Furthermore, the temporal responses suggest that different PVN molecules may be responsible for the initiation (e.g. AVP) versus maintenance (e.g. PPE) of Ang-II hypertension.

IMMUNE CELLS MEDIATED EFFECTS OF SODIUM CHLORIDE ON THE DEVELOPMENT OF VASCULAR INFLAMMATION AND ABDOMINAL AORTIC ANEURYSM IN ANGIOTENSIN II-TREATED APOE-KO MICE

Objective: Chronic high salt (NaCl) intake is a major risk factor for cardiovascular diseases. Recent studies showed that excessive chronic NaCl intake affects immune response and thereby contributes to the pathogenesis of vascular injury. However, it remains unclear whether NaCl intake initially primes immune cell response by promoting memory T cells and sensitize the development of vascular injury in response to a subsequent stimulus. Design and method: Therefore, we investigate whether 14 days of NaCl treatment (1% NaCl) followed by a 7 day washout period affects the incidence and outcome of abdominal aortic aneurysms (AAA) in apolipoprotein E deficient (ApoE-KO) mice infused chronically with angiotensinII (AngII) (1000ng/kg/min) via osmotic minipumps for 10 days. Assessment of AAA was done by magnetic resonance imaging (MRI). To visualize vascular inflammation in vivo, perfluorocarbon nano-emulsions (PFC) injected intravenously before and 2, 4, 7 and 10 days after chronic AngII infusion were detected by combining 1H/19F MRI using a 19F RARE sequence. After 10 days of AngII infusion, aortas with perivascular fat were dissected. Aortic inflammation and immune cells infiltration were analyzed by flow cytometery, immunohistology and realtime PCR. Results: The survival rate of ApoE-KO mice pretreated with NaCl was significantly lower and the AAA incidence rate was significantly higher compared to sham-treated apoE-KO mice. The amount of 19F signal detected in areas of AAA was significantly increased in apoE-KO mice pretreated with NaCl. Histological analysis confirmed these results and showed that most of the immune cells were in the PVAT rather than dwelling in the vascular tissues. At day 10, proinflammatory cytokines like TNF-alpha, iNOS and TGF-ß as well as numbers of neutrophils, monocytes, effector memory CD4 and CD8 T cells were significantly higher in aortas of NaCl pretreated apoE-KO mice compared to sham-treated mice. Moreover, NaCl augmented the development of effector Th1 CD4 and effector CD8 cells from naïve T cells in vitro, respectively. Conclusions: In conclusion, these results suggest a significant role of NaCl in priming immune cell phenotype and thereby mediating vascular inflammation and damage in AngII infused apoE-KO mice.

Depletion of CD11c+ dendritic cells in apolipoprotein E-deficient mice limits angiotensin II-induced abdominal aortic aneurysm formation and growth.

The role of chronic inflammation in abdominal aortic aneurysm (AAA) is controversial. CD11c+ antigen-presenting cells (APCs) (dendritic cells (DCs)) have been reported in human AAA samples but their role is unclear. The effect of conditional depletion of CD11c+ cells on experimental AAA was investigated in the angiotensin II (AngII)-infused apolipoprotein E-deficient (ApoE-/-) mouse model. CD11c-diphtheria toxin (DT or D.tox) receptor (DTR), ovalbumin (OVA) fragment aa 140-386, and enhanced green fluorescent protein (eGFP)-ApoE-/- (CD11c.DOG.ApoE-/-) mice were generated and CD11c+ cell depletion achieved with D.tox injections (8 ng/g body weight, i.p., every-other-day). AAA formation and growth were assessed by measurement of supra-renal aortic (SRA) diameter in vivo by serial ultrasound and by morphometry assessment of harvested aortas at the end of the study. Depletion of CD11c+ cells by administration of D.tox on alternative days was shown to reduce the maximum diameter of AAAs induced by 28 days AngII infusion compared with controls (D.tox, 1.58 ± 0.03 mm vs Vehicle control, 1.81 ± 0.06 mm, P<0.001). CD11c+ depletion commencing after AAA establishment by 14 days of AngII infusion, was also shown to lead to smaller AAAs than controls after a further 14 days (D.tox, 1.54 ± 0.04 mm vs Vehicle control, 1.80 ± 0.03 mm, P<0.001). Flow cytometry revealed significantly lower numbers of circulating CD44hi CD62Llo effector CD4 T cells, CD44hi CD62Llo effector CD8 T cells and B220+ B cells in CD11c+ cell-depleted mice versus controls. CD11c+ depletion attenuated SRA matrix degradation indicated by decreased neutrophil elastase activity (P=0.014), lower elastin degradation score (P=0.012) and higher collagen content (P=0.002). CD11c+ cell-depletion inhibited experimental AAA development and growth associated with down-regulation of circulating effector T cells and attenuated matrix degradation. The findings suggest involvement of autoreactive immune cells in AAA pathogenesis.

Abstract 095: Mutation in the Furin Cleavage Site of the Prorenin Receptor Attenuates Angiotensin-II Induced Hypertension and Albuminuria

Cleavage of the extra-cellular domain of the (pro)renin receptor (PRR) yields a soluble fragment (sPRR) which can promote angiotensin-II (Ang-II) formation. Although alterations in plasma sPRR levels have been reported in hypertension, the causal role of sPRR in hypertension is unknown. To investigate this, we mutated the furin cleavage site (R276A, R279A) of the PRR using CRISPR/Cas9. Because the gene encoding PRR is on the X-chromosome and male mutant mice are infertile, only male mice were studied. Mutant mice had markedly lower plasma sPRR levels (control: 16.2 ± 0.3 vs mutant 0.2 ± 0.03 ng/ml). Mutant mice had normal survival and development and no histological renal abnormalities up to 12 months of age. During normal salt intake, no differences in blood pressure, body weight, urinary water or Na + excretion, or acid-base status were observed between control and mutant mice. Compared to controls, mutant mice had an attenuated hypertensive response to 2 weeks of Ang-II infusion (400 ng/kg/min) (Figure 1). Mutant mice also had lower albuminuria (control: 327 ± 114 vs mutant: 58 ± 8 μg/day) at day 7 post Ang-II infusion. No differences in urinary Na + excretion were detected between control and mutant mice after 7 days of Ang-II infusion (control: 33.6 ± 3.1 vs mutant: 35 ± 2.7 μmol/day/gram body weight). These results suggest that sPRR plays a role in Ang-II induced hypertension and renal injury.

Neutrophil Depletion Influences Renal Outcomes in Male Mice with Chronic Angiotensin II Infusion

Hypertension is the leading cause of morbidity and mortality worldwide; yet the exact cause for many cases remain unknown. Recent research has highlighted the significance of the immune system in the development and progression of HTN in animal models. Much of this research has focused on classical immune pathways involving antigen presenting cells and subsequent activation of the adaptive immune response (primarily CD8+ T‐cells). These activated lymphocytes then support the development of HTN by mediating end‐organ damage in organs tasked with regulating blood pressure and extracellular volume (such as the kidneys). In the current study, we set out to determine the role of neutrophils in a murine model of HTN – the chronic angiotensin II (AngII) infusion model. All animal experiments were performed following animal protocols approved by the Liberty University IACUC and conform to the FASEB Statement of Principles for the use of animals in research and education. Baseline blood pressure was recorded in male C57Bl/6 mice (N=10) for 3 weeks using the CODA‐8 tail cuff volume pressure recording system. Neutrophil depletion was accomplished via intraperitoneal injections of a monoclonal anti‐Ly6G antibody (1A8, n=5), or IgG control (2A3, n=5) every 48 hours (250μg/mouse). 18 hours following the first antibody injection, a mini‐osmotic pump was implanted into the rear flank delivering 500ng/kg*min AngII. 3 days after pump implantation, blood pressure readings resumed and continued for 14 days. After 14 days, mice were individually housed in metabolic cages and urine was collected for quantification of albuminuria. Renal blood flow was estimated in anesthetized mice using contrast‐enhanced ultrasonography. Following renal blood flow estimation, mice were euthanized and blood, kidney, and spleens collected for determination of leukocyte content by flow cytometry. All data were analyzed using general linear models procedures in SPSS. Significant differences in blood pressure were observed periodically during the 14 days of AngII infusion (P≤0.05 for days 8, 12, and 14); however, the overall effect of treatment (1A8 vs. 2A3) did not reach statistical significance (P&lt;0.2). Mice receiving the 1A8 neutrophil depleting antibody also tended (P≤0.07) to have a higher urinary output and albuminuria as compared to mice receiving the isotype control (respectively). No difference in estimated renal blood flow was detected between the treatment groups. Tissue analysis by flow cytometry confirmed the depletion and/or significant reduction of circulating and splenic neutrophils in mice receiving 1A8, but not 2A3, injections after 2 weeks. In the kidneys, mice receiving the 1A8 neutrophil depleting antibody had reduced (P=0.03) CD45+ leukocyte content. Further analysis of renal single cell suspensions revealed a significant reduction (P=0.04) in B‐cells and higher (P=0.03) CD8+ T‐cells in 1A8 treated animals. Taken together, our data suggest that neutrophil depletion alters several renal outcomes in the AngII murine model of HTN, with neutropenic mice tending to have higher blood pressure, greater urinary output and albuminuria as compared to control mice. These effects could be mediated by the significant differences in renal leukocyte content between the treatment groups. Further studies are needed to confirm our results and delineate the molecular mechanisms responsible for altered renal function and inflammation due neutrophil depletion in this animal model.Support or Funding InformationThis work was supported by Liberty University, College of Osteopathic Medicine intramural grant #2016‐07This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Temporal Association Between Endoplasmic Reticulum Stress and Reactive Oxygen Species in the Subfornical Organ During Angiotensin‐II‐Mediated Hypertension

Hypertension affects one in three adults and is directly associated with diseases of the heart, kidney, and brain. While a number of factors may play a role, dysregulation in Angiotensin‐II (Ang‐II) signaling in the brain is well accepted to contribute to hypertension development. In particular, Ang‐II signaling within the subfornical organ (SFO), a forebrain circumventricular region situated outside of the blood‐brain‐barrier, plays a critical role. Ang‐II‐mediated oxidative stress and endoplasmic reticulum (ER) stress have both been implicated in Ang‐II‐dependent hypertension. However, the interplay between these key hypertensive mechanisms in the SFO remains unclear. Therefore, as a first step, we hypothesized that SFO oxidative stress and ER stress occur at the same time during the development of Ang‐II hypertension. Adult male C57B1/6 mice underwent subcutaneous implantation of osmotic mini‐pumps for continuous 2 wk delivery of Ang‐II (600 ng/kg/min) to induce hypertension, or vehicle control (BSA). At 14 days of infusion (peak hypertension), brains were collected and dihydroethidium (DHE) fluorescence staining was performed to evaluate reactive oxygen species in the SFO (n=10/group). In a separate cohort (n=4–5), immunohistochemistry for protein disulfide isomerase (PDI), an ER chaperone protein that is upregulated under conditions of ER stress, was performed. While DHE fluorescence in the SFO was minimal in control animals, 2 wk infusion of Ang‐II resulted in a robust increase in reactive oxygen species in the SFO (1.0±0.1 vs. 1.7±0.1 fold control; control vs. Ang‐II, p&lt;0.05). Interestingly, immunohistochemistry analysis at 14 days also demonstrated marked Ang‐II‐mediated ER stress in the SFO, as indicated by upregulation of PDI (8.8±1.6 vs. 14.9±1.3 PDI density/area; control vs. Ang‐II, p&lt;0.05). To further delineate the temporal relationship between oxidative and ER stress, in another cohort we subsequently performed PDI western blot evaluation in micropunches of the SFO at pre‐hypertensive (7 days) and established hypertension (14 days) timepoints. Importantly, we have previously shown a pre‐hypertensive (day 7) increase in SFO reactive oxygen species during Ang‐II hypertension (Young et al. AJP Cell, 2015). Following 7 day chronic infusion of Ang‐II, an ~85% increase in SFO PDI protein expression was found (1.00±0.24 vs. 1.85±0.7 fold day 0; day 0 vs. 7 day Ang‐II, p&lt;0.05) and this ER chaperone upregulation was maintained at 14 days of Ang‐II infusion (1.58±0.16 fold day 0, p&lt;0.05). Together, these findings indicate a close temporal relationship between Ang‐II‐induced ER stress and reactive oxygen species production in the SFO, which occurs prior to and in concert with the development of Ang‐II‐mediated hypertension.Support or Funding InformationAPS Undergraduate Summer Research Fellowship (M.J.); HL116776 (C.N.Y.)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Angiotensin II infusion into ApoE-/- mice: a model for aortic dissection rather than abdominal aortic aneurysm?

Angiotensin II-infused ApoE-/- mice are a popular mouse model for preclinical aneurysm research. Here, we provide insight in the often-reported but seldom-explained variability in shape of dissecting aneurysms in these mice. N = 45 excised aortas were scanned ex vivo with phase-contrast X-ray tomographic microscopy. Micro-ruptures were detected near the ostium of celiac and mesenteric arteries in 8/11 mice that were sacrificed after 3 days of angiotensin II-infusion. At later time points (after 10, 18, and 28 days) the variability in shape of thoraco-abdominal lesions (occurring in 31/34 mice) was classified into 7 different categories based on the presence or absence of a medial tear (31/31), an intramural hematoma (23/31) or a false channel (11/23). Medial tears were detected both in the thoracic and the abdominal aorta and were most prevalent at the left and ventral aspects of celiac and mesenteric arteries. The axial length of the hematoma strongly correlated to the total number of ruptured branch ostia (r2 = 0.78) and in 22/23 mice with a hematoma the ostium of the left suprarenal artery had ruptured. Supraceliac diameters at baseline were significantly lower for mice that did not develop an intramural hematoma, and the formation of a false channel within that intramural hematoma depended on the location, rather than the length, of the medial tear. Based on our observations we propose an elaborate hypothesis that explains how aortic side branches (i) affect the initiation and propagation of medial tears and the subsequent adventitial dissection and (ii) affect the variability in shape of dissecting aneurysms. This hypothesis was partially validated through the live visualization of a dissecting aneurysm that formed during micro-CT imaging, and led us to the conclusion that angiotensin II-infused mice are more clinically relevant for the study of aortic dissections than for the study of abdominal aortic aneurysms.

Angiotensin II-Induced Hypertension Is Attenuated by Overexpressing Copper/Zinc Superoxide Dismutase in the Brain Organum Vasculosum of the Lamina Terminalis.

Angiotensin II (AngII) can access the brain via circumventricular organs (CVOs), including the subfornical organ (SFO) and organum vasculosum of the lamina terminalis (OVLT), to modulate blood pressure. Previous studies have demonstrated a role for both the SFO and OVLT in the hypertensive response to chronic AngII, yet it is unclear which intracellular signaling pathways are involved in this response. Overexpression of copper/zinc superoxide dismutase (CuZnSOD) in the SFO has been shown to attenuate the chronic hypertensive effects of AngII. Presently, we tested the hypothesis that elevated levels of superoxide (O2 ∙−) in the OVLT contribute to the hypertensive effects of AngII. To facilitate overexpression of superoxide dismutase, adenoviral vectors encoding human CuZnSOD or control adenovirus (AdEmpty) were injected directly into the OVLT of rats. Following 3 days of control saline infusion, rats were intravenously infused with AngII (10 ng/kg/min) for ten days. Blood pressure increased 33 ± 8 mmHg in AdEmpty rats (n = 6), while rats overexpressing CuZnSOD (n = 8) in the OVLT demonstrated a blood pressure increase of only 18 ± 5 mmHg after 10 days of AngII infusion. These results support the hypothesis that overproduction of O2 ∙− in the OVLT plays an important role in the development of chronic AngII-dependent hypertension.

Functional aortic stiffness: role of CD4+ T lymphocytes

The immune system is suggested to be essential in vascular remodeling and stiffening. To study the dependence upon lymphocytes in vascular stiffening, we compared an angiotensin II-model of vascular stiffening in normal C57BL/6J mice with lymphocyte-deficient RAG 1−/− mice and additionally characterized the component of vascular stiffness due to vasoconstriction vs. vascular remodeling. Chronic angiotensin II increased aortic pulse wave velocity, effective wall stiffness, and effective Young's modulus in C57BL/6J mice by three-fold but caused no change in the RAG 1−/− mice. These functional measurements were supported by aortic morphometric analysis. Adoptive transfer of CD4+ T helper lymphocytes restored the angiotensin II-mediated aortic stiffening in the RAG 1−/− mice. In order to account for the hydraulic vs. material effects of angiotensin II on pulse wave velocity, subcutaneous osmotic pumps were removed after 21 days of angiotensin II-infusion in the WT mice to achieve normotensive values. The pulse wave velocity (PWV) decreased from three- to two-fold above baseline values up to 7 days following pump removal. This study supports the pivotal role of the CD4+ T-lymphocytes in angiotensin II-mediated vascular stiffening and that angiotensin II-mediated aortic stiffening is due to the additive effect of active vascular smooth muscle vasoconstriction and vascular remodeling.

Angiotensin II mobilizes spleen monocytes to promote the development of abdominal aortic aneurysm in Apoe-/- mice.

Abdominal aortic aneurysm (AAA) is widespread among elderly people and results in progressive expansion and rupture of the aorta with high mortality. Macrophages, which are the main population observed within the site of aneurysm, are thought to derive from circulating monocytes although no direct evidence has been provided to date. In this study, we were particularly interested in understanding the trafficking behavior of monocyte subsets in AAA and their role in disease pathogenesis. Using bone marrow transplantation in Apoe(-/-) mice, we showed that circulating monocytes give rise to abdominal aortic macrophages in hypercholesterolemic mice submitted to angiotensin II (AngII). Detailed monitoring of monocyte compartmentalization revealed that lymphocyte antigen 6C(high) and lymphocyte antigen 6C(low) monocytes transiently increase in blood early after AngII infusion and differentially infiltrate the abdominal aorta. The splenic reservoir accounted for the mobilization of the 2 monocyte subsets after 3 days of AngII infusion. Spleen removal or lymphocyte deficiency in Apoe(-/-) Rag2(-/-) mice similarly impaired early monocyte increase in blood in response to AngII and protected against AAA development, independently of blood pressure. Reconstitution of Apoe(-/-) Rag2(-/-) mice with total splenocytes but not with B-cell-depleted splenocytes restored monocyte mobilization in response to AngII and enhanced susceptibility to AAA. Taken together, the data show that lymphocyte antigen 6C(high) and lymphocyte antigen 6C(low) monocytes are mobilized from the spleen in response to AngII. Intriguingly, the process is dependent on the presence of B cells and significantly contributes to the development of AAA and the occurrence of aortic rupture.

Over-expression of copper/zinc superoxide dismutase in the median preoptic nucleus attenuates chronic angiotensin II-induced hypertension in the rat.

The brain senses circulating levels of angiotensin II (AngII) via circumventricular organs, such as the subfornical organ (SFO), and is thought to adjust sympathetic nervous system output accordingly via this neuro-hormonal communication. However, the cellular signaling mechanisms involved in these communications remain to be fully understood. Previous lesion studies of either the SFO, or the downstream median preoptic nucleus (MnPO) have shown a diminution of the hypertensive effects of chronic AngII, without providing a clear explanation as to the intracellular signaling pathway(s) involved. Additional studies have reported that over-expressing copper/zinc superoxide dismutase (CuZnSOD), an intracellular superoxide (O2·−) scavenging enzyme, in the SFO attenuates chronic AngII-induced hypertension. Herein, we tested the hypothesis that overproduction of O2·− in the MnPO is an underlying mechanism in the long-term hypertensive effects of chronic AngII. Adenoviral vectors encoding human CuZnSOD (AdCuZnSOD) or control vector (AdEmpty) were injected directly into the MnPO of rats implanted with aortic telemetric transmitters for recording of arterial pressure. After a 3 day control period of saline infusion, rats were intravenously infused with AngII (10 ng/kg/min) for ten days. Rats over-expressing CuZnSOD (n = 7) in the MnPO had a blood pressure increase of only 6 ± 2 mmHg after ten days of AngII infusion while blood pressure increased 21 ± 4 mmHg in AdEmpty-infected rats (n = 9). These results support the hypothesis that production of O2·− in the MnPO contributes to the development of chronic AngII-dependent hypertension.

Angiotensin II Induces Region-Specific Medial Disruption during Evolution of Ascending Aortic Aneurysms

Angiotensin II (AngII) promotes development of ascending aortic aneurysms (AAs), but progression of this pathology is undefined. We evaluated factors potentially involved in progression, and determined the temporal sequence of tissue changes during development of AngII-induced ascending AAs. AngII infusion into C57BL/6J mice promoted rapid expansion of the ascending aorta, with significant increases within 5 days, as determined by both invivo ultrasonography and exvivo sequential acquisition of tissues. Rates of expansion were not significantly different in LDL receptor-null mice fed a saturated fat-enriched diet, demonstrating a lack of effect of hypercholesterolemia. Augmenting systolic blood pressure with norepinephrine infusion had no significant effect on ascending aortic expansion. Pathological changes observed within 5 days of AngII infusion included increased medial thickness and intramural hemorrhage characterized by erythrocyte extravasation in outer lamellar layers of the media. Intramedial hemorrhage was not observed after prolonged AngII infusion, although partial medial disruption was present. Elastin fragmentation and transmural medial breaks of the ascending aorta were observed with continued AngII infusion, which were restricted to anterior aspects. CD45(+) cells accumulated in adventitia but were minimal in media. Similar pathology was observed in tissues obtained from patients with ascending AAs. In conclusion, AngII promotes ascending AAs through region-specific changes that are independent of hypercholesterolemia or systolic blood pressure.

297Osteoglycin regulates cardiac fibrosis in the pressure-overloaded heart

Hypertension and/or aging impose stress on the heart with consecutive cardiac hypertrophy, inflammation and fibrosis, processes that lead to heart failure. Our group has previously shown that non-structural matrix proteins such as TSP-2 and SPARC are critical during hypertension, fibrosis, and age-related cardiac dysfunction. Osteoglycin (OGN) is a class III member of the small leucine rich proteoglycans, and was recently identified as a marker of increased left ventricular mass in a study using genomics in recombinant inbred hypertensive rat strains. Its precise biological role in cardiac remodeling, however, remains unclear. We hypothesize that OGN will influence cardiac hypertrophy and fibrosis and hence is critical in the cardiac response during hypertension and aging. We show in young adult mice (3 months old) that hypertension-induced pressure overload of the heart causes two phases of cardiac fibrosis, following 28 days of angiotensin-II (AngII) infusion. The first phase develops between days 3 to 7 after the AngII-treatment and consists of diffuse interstitially deposited loose fibers. During the second wave of fibrosis, which starts at 14 days, these loose fibers transition into more localized scars of denser collagens. Interestingly, the expression of OGN mRNA also shows a biphasic response, significantly decreasing prior to each phase of fibrosis, at day 1 (96% decrease, p=0.002) and day 14 (84% decrease, p=0.004) respectively. Moreover, hypertension causes exaggerated cardiac fibrosis (7.54±1.58 vs. 3.65±0.62 %, p=0.04) and increased heart weight to body weight ratios (6.39±0.31 vs. 5.42±0.17 mg/g, p=0.01) in young adult OGN null mice when compared to WT littermates, but no differences in the amount of infiltrating leukocytes (31.48±9.82 vs. 36.32±18.75 cells/mm2), after 28 days of AngII infusion. In addition, aging results in decreased cardiac function, as measured by ejection fraction, in 18 months versus 3 months old OGN null mice (42.65±2.74 vs. 58.05±1.37 %, p=0.0001), which is absent in WT mice (51.01±3.11 vs. 53.01±1.67 %, p=0.62). Furthermore, a follow-up study in WT and OGN null mice showed a decrease in stroke volume over time in the OGN null mice (39.30±2.90 μl at 6 months, 35.54±1.95 μl at 12 months and 31.13±1.29 μl at 18 months, p=0.04 between 6 and 18 months) but not in the WT mice (41.61±2.02 μl at 6 months, 39.04±1.95 μl at 12 months and 41.04±3.05 μl at 18 months). Collectively, these data suggest that OGN regulates cardiac fibrosis during pressure overload and is therefore important in the remodeling response to hypertension and aging.

Abstract 149: Regulator of G-Protein Signaling-1 Modulates Leukocyte Trafficking in Atherosclerosis and Aortic Aneurysm Formation Through Chemokine Receptor Desensitization

The regulation of macrophage recruitment and retention into the vascular wall is critical in the progression of atherosclerosis and aortic aneurysm formation. This can be mediated by chemokine activation of multiple G-protein coupled receptors. The Regulator of G-Protein Signaling-1 (RGS1) acts to deactivate the intracellular response to sustained chemokine stimulation. We have found that RGS1 is upregulated with atherosclerotic plaque progression and with monocyte-macrophage activation but its role is unknown. Rgs1-/- macrophages have significantly enhanced migratory responses to atherogenic chemokines and have impaired desensitization to chemokine re-stimulation (p&lt;0.001). In vivo, RGS1 has a role in the accumulation of macrophages in atherosclerotic lesions and during Angiotensin II (AngII) aortic aneurysm rupture. In the absence of RGS1, atherosclerosis and macrophage accumulation is attenuated in early lesions in the aortic root and aortas of ApoE-/- mice (p&lt;0.001). Rgs1-/- mice are protected from AngII induced aneurysm rupture compared to ApoE-/- mice with 94% survival vs. 56%. Rgs1-/- mice have significantly fewer CD11b+ myeloid cells and CD14+ macrophages in aortas than ApoE-/- mice (p&lt;0.05) after 5 days of AngII infusion. Following bone marrow transplantation, recipient mice receiving ApoE-/- bone marrow were more susceptible to aortic aneurysm rupture (p=0.0124), indicating bone marrow-derived RGS1 is required for aneurysm rupture. Furthermore, AngII treatment increased systolic blood pressure to a greater extent in Rgs1-/- mice than ApoE-/- mice suggesting aneurysm formation in these mice is independent of AngII induced hypertension and this is mediated by vascular-derived RGS1. To gain insight into the mechanism by which RGS1 regulates trafficking, we selectively labelled inflammatory monocytes in vivo to track their movement into aortas following AngII infusion. We found an accumulation of labelled CD45+ cells in the aortas of ApoE-/- mice from day 3 to day 5 but not in Rgs1-/- mice indicating RGS1 as a regulator of macrophage retention in aortic aneurysms. These findings identify a novel role for RGS1 in leukocyte retention in vascular inflammation, highlighting RGS1 as a potential target in cardiovascular disease.

OVLT lesion decreases basal arterial pressure and the chronic hypertensive response to AngII in rats on a high-salt diet.

We have reported that lesion of the organum vasculosum of the lamina terminalis (OVLT) has no effect on basal levels of mean arterial pressure (MAP) but abolishes the hypertensive effects of angiotensin II (AngII) in rats consuming a normal-salt diet. These results suggest that the OVLT does not contribute to regulation of MAP under conditions of normal salt intake, but it is an important brain site for the hypertensive actions of AngII. The OVLT has been proposed as a major sodium sensor in the brain and the hypertensive effects of AngII are exacerbated by high-salt intake. Therefore, the objective of this study was to investigate the role of the OVLT during AngII-induced hypertension in rats fed a high-salt diet. Male Sprague-Dawley rats underwent sham (Sham; n = 9) or OVLT lesion (OVLTx; n = 8) surgery and were placed on a high-salt (2% NaCl) diet. MAP was measured by radio telemetry during three control days, 10 days of AngII infusion (10 ng/kg/min, i.v.), and a 3-day recovery period. MAP was significantly lower in OVLTx (97 ± 2 mmHg) compared to Sham (106 ± 1 mmHg) rats during the control period (P < 0.05). Moreover, the chronic pressor response to AngII was markedly attenuated in OVLTx rats. MAP increased 58 ± 3 mmHg in Sham rats by Day 10 of AngII compared to a 40 ± 7 mmHg increase in OVLTx rats (P < 0.05). We conclude that (1) the OVLT regulates the basal levels of MAP in rats consuming a high-salt and (2) the OVLT is an important brain site of action for the pathogenesis of AngII-salt hypertension in the rat. Supported by HL076312.

Abstract 399: Deficiency of Angiotensin Type 1a Receptors in Adipocytes Increases Fat Mass in Angiotensin II-Infused LDLR-Deficient Mice but Has No Effect on Vascular Pathologies

Objectives: Angiotensin II (AngII) promotes atherosclerosis and abdominal aortic aneurysms (AAA) through the angiotensin type 1a receptor (AT1aR). However, the cell type mediating these effects has not been defined. Adipose tissue surrounding the aorta may influence the inflammatory milieu within the vessel wall and thus contribute to disease progression. The purpose of this study was to determine the effect of adipocyte-AT1aR deficiency on development of AngII and diet-induced vascular pathologies in low density lipoprotein receptor deficient (LDLR-/-) mice. Methods and results: LDLR -/- mice with adipocyte-AT1aR deficiency (AT1aRaP2, n = 21) and littermates (AT1aRfl/fl, n=17) were fed a high-fat diet (HF, 42% kcal fat; 0.15 cholesterol) for 1 week prior to, and throughout, 28 days of AngII infusion (1,000 ng/kg/min). Adipocyte-AT1aR deficiency had no effect on AAA incidence (external diameter of aorta; AT1aRfl/fl 1.4±0.1 vs. AT1aRaP2 1.5±0.1 mm, p=0.41) and atherosclerotic lesion areas in the aortic arch (AT1aRfl/fl 7.1±0.8 vs. AT1aRaP2 8.3+0.8% lesion area, p=0.49). Serum cholesterol (AT1aRfl/fl 1981±109 vs. AT1aRaP2 2044±85 mg/dL, p=0.65) and triglyceride (AT1aRfl/fl 450±31 vs. AT1aRaP2 496±33 mg/dL, p=0.32) concentrations were also unaffected by adipocyte-AT1aR deficiency. Interestingly, body weight was significantly increased in AngII-infused AT1aRaP2 mice (AT1aRfl/fl 26.8±0.5 vs. AT1aRaP2 28.0±0.5 g, p=0.013 for interaction between genotype and time), due to increases in adipose mass (retroperitoneal fat; AT1aRfl/fl 0.26±0.03 vs. AT1aRaP2 0.36±0.02 g, p=0.017). Mice fed the HF diet for 3 months without AngII infusion did not exhibit any changes in atherosclerotic lesion area in the aortic arch (AT1aRfl/fl 14.3±1% vs. AT1aRaP2 11.7±1%, p=0.14). Conclusions: Adipocyte-AT1aR deficiency has no effect on development of diet- or AngII-induced atherosclerosis or AAAs in hypercholesterolemic mice. Interestingly, body weight and fat mass were increased in AngII-infused adipocyte AT1aR deficient mice. Future studies will elucidate the mechanisms through which AngII regulates body weight and fat mass under these conditions.

Abstract 254: Cellular Heterogeneity of the Ascending Aorta Relationship to AngII-Induced Medial Hemorrhage

Introduction and Objective: Infusion of angiotensin II (AngII) into mice generates pronounced aneurysms that are localized to the ascending aorta. Following prolonged infusion of AngII, the resulting ascending aneurysms are characterized by pronounced lumen dilation and concentric medial thickening with progressive expansion between elastin layers from the lumen to the adventitia of the aorta. The presence of medial hemorrhage is a common characteristic within 5 days of initiating AngII infusion. The purpose of this study was to determine whether the location of medial hemorrhage coincided with overt structural or cellular transition within the aorta. Methods and Results: Eight-week-old, male, C57BL/6 mice were fed a normal laboratory diet and infused with either saline (N=5) or AngII (1,000 ng/kg/min, N=20) subcutaneously using osmotic mini pumps for 5 days. Mice were terminated after 5 days of AngII infusion. Thirty percent (6 out of 20) displayed hemorrhage, visible from the ascending aorta to the aortic arch. Serial sections (10 μm) were acquired through the entire aortic root and ascending aorta. Alpha-actin and reticular fibroblast (ER-TR7) antibodies detected the location of smooth muscle cells (SMC) and fibroblasts, respectively. In both groups, there was a transition within the aortic root in which the media no longer immunostained positively for alpha actin. Conversely, this region immunostained positively for fibroblasts. Hemorrhages occurred distal to the cellular phenotypic changes seen in the aortic root. Conclusion: These findings suggest that the development of AngII-induced medial hemorrhage within the ascending aorta did not correspond to the medial cellular transitions present in the aortic root.

The effects of angiotensin II on the coupled microstructural and biomechanical response of C57BL/6 mouse aorta

RationaleAbdominal aortic aneurysm (AAA) is a complex disease that leads to a localized dilation of the infrarenal aorta, the rupture of which is associated with significant morbidity and mortality. Animal models of AAA can be used to study how changes in the microstructural and biomechanical behavior of aortic tissues develop as disease progresses in these animals. We chose here to investigate the effect of angiotensin II (AngII) in C57BL/6 mice as a first step towards understanding how such changes occur in the established ApoE−/− AngII infused mouse model of AAA. ObjectiveThe objective of this study was to utilize a recently developed device in our laboratory to determine how the microstructural and biomechanical properties of AngII-infused C57BL/6 wildtype mouse aorta change following 14 days of AngII infusion. MethodsC57BL/6 wildtype mice were infused with either saline or AngII for 14 day. Aortas were excised and tested using a device capable of simultaneously characterizing the biaxial mechanical response and load-dependent (unfixed, unfrozen) extracellular matrix organization of mouse aorta (using multiphoton microscopy). Peak strains and stiffness values were compared across experimental groups, and both datasets were fit to a Fung-type constitutive model. The mean mode and full width at half maximum (FWHM) of fiber histograms from two photon microscopy were quantified in order to assess the preferred fiber distribution and degree of fiber splay, respectively. ResultsThe axial stiffness of all mouse aorta was found to be an order of magnitude larger than the circumferential stiffness. The aortic diameter was found to be significantly increased for the AngII infused mice as compared to saline infused control (p=0.026). Aneurysm, defined as a percent increase in maximum diameter of 30% (defined with respect to saline control), was found in 3 of the 6 AngII infused mice. These three mice displayed adventitial collagen that lacked characteristic fiber crimp. The biomechanical response in the AngII infused mice showed significantly reduced circumferential compliance. We also noticed that the ability of the adventitial collagen fibers in AngII infused mice to disperse in reaction to circumferential loading was suppressed. ConclusionsCollagen remodeling is present following 14 days of AngII infusion in C57BL/6 mice. Aneurysmal development occurred in 50% of our AngII infused mice, and these dilatations were accompanied with adventitial collagen remodeling and decreased circumferential compliance.