bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 242: Superoxide and Isoketal Neo-antigen formation in Dendritic Cells from Hypertensive mice activate T cells and promote Hypertension
Authors: Kirabo Annet , Wu Jing , Thabet Salim R , Bikineyeva Alfiya T , Dikalov Sergey , Amarnath Venkataraman , Davies Sean S , Roberts Jackson L , Harrison David G
Published: July 2021

Superoxide and inflammation contribute to the genesis of hypertension but the mechanisms involved are not fully understood. We examined the hypothesis that oxidative stress in dendritic cells (DCs) alters endogenous proteins via Isoketal-modification leading to formation of neo-antigens, T cell activation and blood pressure elevation. DCs isolated from mice with angiotensin II-induced hypertension had a significant increase in NADPH oxidase-dependent superoxide production when compared to sham-treated mice (334.0±49.7 versus 65.8±4.5 pmol/mg protein). This was associated with an exuberant DC accumulation of protein-isoketal adducts and activation of IL-6, IL-1β and IL-23 production. DCs from hypertensive mice but not sham mice promoted survival and proliferation of CD8
+
T cells in culture. Scavenging of isoketals not only prevented activation and immunogenicity of DCs, but also markedly attenuated angiotensin II-induced hypertension (142.59 ± 8.98 mmHg versus 175.53 ± 5.19 mmHg in controls). Moreover, adaptive transfer of DCs from hypertensive mice primed development of hypertension in mice given a sub-pressor dose of angiotensin II (157.45 ± 33.86 mmHg versus 119.90 ± 17.33 mmHg in controls). These studies show that angiotensin II-induced hypertension activates DCs, in large part by causing superoxide production and formation of isoketals. We propose that Isoketal-modified proteins can be presented as neo-antigens by DCs, which in turn trigger T cell activation leading to hypertension.
Abstract 244: Sodium Butyrate Improves In Vivo and In Vitro Inflammation and Increases Plaque Stability in Apoe-Deficient Mice
Authors: Aguilar Edenil C , Silva Analina R , Leonel Alda J , Teixeira Lilian G , Capettine Luciano S , Silva Josie F , Santos Robson A , Alvarez-Leite Jacqueline I
Published: July 2021

Butyrate is a short chain fatty acid, formed by fermentation in the large intestine, with a well-known anti-inflammatory property. As inflammation plays a key role in atherogenesis and insulin resistance, we studied the effects of butyrate supplementation on atherosclerosis and glucose homeostasis. ApoE-deficient mice received control diet (CT, n=8) or 1% butyrate supplemented diet (BUT, n=8) for 10 weeks. Oral glucose tolerance test (OGTT), insulin sensitivity test (IST), lipid profile and fasting glucose were evaluated. Atherosclerosis lesion area was studied in two sites, aorta and aortic root. In the latter, CCL2, VCAM-1, collagen, and matrix metalloproteinase-2 (MMP-2) were also assessed. In order to confirm the in vivo data, culture of human endothelial cells (E.A. Hy926) were pre-incubated with butyrate (0.5 or 1.0mM) and stimulated with oxidized LDL (oxLDL) for 4h for CCL2 and VCAM-1 evaluation by immunofluorescence. Lipid profile, fasting glucose and OGTT were similar between groups. Insulin sensitivity was improved in BUT group mice. Butyrate-treated mice showed reduction in the aorta atherosclerosis lesion (CT=4.1%, BUT=1.8%; percentage of the surface area). Despite of similar lesion area in the BUT- treated mice aortic roots presented reduced inflammation-related molecules, CCL2 (CT=0.25FI, BUT=1.2FI) and VCAM-1(CT=0.42FI, BUT=013FI), and improvement on plaque stability with reduced MMP2 (CT=0.51FI, BUT=0.13FI) and increased collagen (CT=0.19FI, BUT=0.44FI). These results suggest that aortic root lesion areas were similar probably due to a more stable and thicker fibrous cap in the BUT group (beneficial factor). The production of VCAM-1 and CCL2 by butyrate pretreated endothelial cells was reduced compared to control cells, suggesting that butyrate reduces leukocyte migration toward lesion reducing atherogenesis. In conclusion our data show that butyrate supplementation ameliorates atherosclerosis due to the anti-inflammatory effect and increased plaque stability.
Abstract 253: Inhibition of Necrosis Attenuates Blood Pressure and Renal Inflammation in Male SHR with no Effect in Females
Authors: Rafikova Olga , Tipton Ashlee J , Baban Babak , Sullivan Jennifer C
Published: July 2021

Inflammation plays an important role in the pathogenesis of hypertension, and we recently published that male spontaneously hypertensive rats (SHR) have more pro-hypertensive, pro-inflammatory Th17 cells in their kidneys than females. However, the mechanisms initiating the inflammatory response remain unclear. Damage-associated molecular patterns (DAMPs) are released from dying cells and induce immune system activation. High mobility group box 1 (HMGB1) is a DAMP actively secreted by apoptotic cells in an oxidized form, or passively released by necrotic cells in a reduced form. Reduced HMGB1 has prolonged activity and is more pro-inflammatory. We hypothesized that sex differences in cell death contribute to sex differences in the immune cell profile and hypertension. Renal necrosis and apoptosis were measured by flow cytometry in 12 wk old male and female SHR. Male SHR have greater necrotic cell death (% total renal cells: 5.3±0.8 vs.1.0±0.4; p=0.004; N=6), while female SHR have more apoptosis (% total renal cells: 1.6±0.4 vs.4.0±0.4; p=0.004; N=4-5). Male SHR also had greater reduced HMGB1 in renal cortex compared to females (HMGB1 oxidized/reduced ratio: 1.0±0.1 vs. 1.7±0.1; p=0.002; N=6). We next measured blood pressure by telemetry in male and female SHR treated with vehicle or necrosis inhibitor NecroX-5 (1 mg/kg per day; 2 wks). The renal T cell and macrophage levels were measured by flow cytometry. NecroX-5 treatment in male SHR prevented age-related increases in blood pressure compared to vehicle-treated rats (ΔmmHg/week: 0.8±1.1 vs. 4.2±0.3; p=0.05; N=3). Necrox5 also significantly decreased renal necrosis (% total renal cells: 5.3±0.8 vs. 0.8±0.1; p=0.005; N=3-6), macrophages (% total renal cells: 2.8±0.6 vs. 0.8± 0.1; p=0.04; N=3-4), Th17 cells (% T cells: 4.5±0.3 vs. 2.7±0.3; p=0.009; N=3-4) and the pro-inflammatory cytokine IL-17 (% total renal cells: 10.0±0.4 vs. 5.3±0.7; p=0.002; N=3-4) in male SHR. NecroX-5 had no effect on blood pressure or pro-inflammatory markers in females. We conclude that necrosis is an important contributor in progression of hypertension in male SHR. Sex differences in cell death may contribute to sex differences in blood pressure and the immune profile.
Abstract 254: Chronic Ang II Hypertension Differentially Impacts the Renal T Cell Profile in Males and Females
Authors: Zimmerman Margaret A , Tipton Ashlee J , Baban Babak , Sullivan Jennifer C
Published: July 2021

T cells contribute to angiotensin (Ang) II hypertension in male animal models yet less is known in females. We previously showed that hypertensive female rats have more immuno-suppressive T regulatory cells (Tregs) and males have more pro-inflammatory Th17 cells in their kidneys. This study tested the hypothesis that Ang II results in greater expression of renal Tregs in females and Th17 cells in males. 12 wk old male and female Sprague Dawley rats (SD) were treated with Ang II (200 ng/kg/min) or vehicle for 2 wks; blood pressure (BP) was measured via tail cuff and renal T cell profiles were measured via flow cytometry. Ang II infusion increased BP in both sexes (mmHg: males: 130±2 to 152±2; females: 123±1 to 146±1; p<0.05). Females had lower CD3
+
T cell counts at baseline vs. males (p<0.0001). Ang II significantly increased CD3
+
T cells in both sexes (p<0.0001). Baseline CD4
+
T cell counts were comparable between male and female SD. Ang II infusion increased CD4
+
T cells in both sexes (p<0.0001), however, expression remained lower in females (p=0.04). Females had greater expression of Tregs (p<0.0001) at baseline. Ang II infusion increased Treg expression in females (p=0.004), with no effect in males. Males expressed more baseline Th17 cells than females (p<0.001). Though Ang II increased levels in both sexes (p<0.001), females maintained significantly reduced Th17 cell expression vs. males (p<0.001). Therefore, Ang II resulted in female SD having greater renal expression of Tregs and males having greater expression of Th17 cells. In conclusion, sex differences in the T cell profile may contribute to observed sex differences in BP control.
Abstract 258: Azilsartan Ameliorates Hypertension and Kidney Damage in Zucker Diabetic Fatty (ZDF) Rat
Authors: Khan Md Abdul H , Haines Jasmine , Imig John D
Published: July 2021

Azilsartan, an angiotensin II receptor blocker, demonstrated anti-hypertensive and organ protective effects in hypertension. We investigated if azilsartan can be utilized in ameliorating kidney damage associated with cardiometabolic syndrome using Zucker Diabetic Fatty (ZDF) rats. Two groups of 6-8 week-old male ZDF rats were treated with vehicle or azilsartan (10 mg/kg/d, p.o.) for 8 weeks. A set of male Zucker Diabetic Lean (ZDL) rats were used as the control group. At the end of the treatment period, urine and plasma samples were collected for biochemical analysis while kidney tissues were collected for histopathological examination (n=6/group). ZDF rats were diabetic with fasting blood glucose levels averaging 287±45 mg/dL compared to 107±6 mg/dL in ZDL rats. Azilsartan treatment ameliorated hyperglycemia in ZDF rats (167±46 mg/dL). ZDF rats were also hypertensive compared to ZDL rats (181±4 vs 126±4 mmHg), and azilsartan treatment attenuated blood pressure in ZDF rats (109±4 mmHg). ZDF rats demonstrated marked renal damage compared to ZDL with elevated urinary excretion of albumin (ZDF vs ZDL, 92±47 vs 4±2 mg/d) and nephrin (842±364 vs 16±4 μg/d). ZDF rats also had 2-4-fold higher tubular cast formation and glomerular injury compared to ZDL rats. In ZDF rats, azilsartan treatment markedly reduced renal injury by attenuating elevated urinary albumin (7±2 mg/d) and nephrin (73±28 ug/d) along with a reduction in renal tubular cast formation and glomerular injury index. ZDF rats demonstrated increased inflammation and oxidative stress with elevated urinary monocyte chemoattaractant protein-1 (MCP-1) excretion (ZDF vs ZDL, 645±373 vs 319±76 ng/d) and kidney malondialdehyde (MDA) content (5±1 vs 10±1 μmol/mg). Azilsartan treatment reduced inflammation and oxidative stress in ZDF rats by reducing MCP-1 (335±80 ng/d) and kidney MDA content (6±1 ug/d). In summary, we demonstrate marked kidney protective, anti-inflammatory, and anti-oxidative effects of azilsartan in cardiometabolic syndrome ZDF rats.

Support provided by Takeda Pharmaceuticals Inc., USA
Abstract 266: Neural Circulatory Responses to Isolated Muscle Metaboreflex Activation in Postmenopausal Women
Authors: Greaney Jody L , Matthews Evan L , Fadel Paul J , Farquhar William B , Wenner Megan M
Published: July 2021

Understanding the neural circulatory responses to exercise in postmenopausal women (PMW) is important given their greater risk for developing hypertension. During exercise, blood pressure is controlled, in part, by the exercise pressor reflex, which is a feedback mechanism originating in skeletal muscle and compromised of mechanically and metabolically sensitive afferents. A recent study reported an enhanced blood pressure response during exercise in normotensive PMW due to greater muscle metaboreflex activation, but the mechanism(s) underlying these responses are unknown. Herein, we tested the hypothesis that metaboreflex activation elicits exaggerated sympathetic nervous system responses in PMW compared to young women, contributing to the enhanced blood pressure response during exercise.

Methods:
Blood pressure (BP, Finometer) and muscle sympathetic nerve activity (MSNA, peroneal microneurography) were continuously measured in 7 PMW (age 59±2 years; BMI 24±1 kg/m
2
) and 7 young women (age 23±2 years; BMI 22±2 kg/m
2
) during 2-minutes of isometric handgrip exercise performed at 30% of maximal voluntary contraction followed by 3-minutes of forearm ischemia (post-exercise ischemia, PEI) to isolate muscle metaboreflex activation.

Results:
Resting mean arterial pressure (MAP) was similar between PMW (85±3 mmHg) and young women (82±2 mmHg; P>0.05). During exercise, the increase in MAP was greater in PMW (Δ18±2mmHg) compared to young women (Δ 12±2 mmHg; P<0.05), and this was maintained during PEI (Δ13±1 mmHg PMW vs. Δ 6±1 mmHg young women; P<0.05). Resting MSNA was higher in PMW (24±4 bursts/min) compared to young women (9±3 bursts/min; P<0.05). Interestingly, the increase in MSNA during exercise was comparable between groups (P>0.05), whereas during PEI, the increase in MSNA was approximately 50% greater in PMW compared to young women (Δ13±2 burst/min PMW vs. 7±2 bursts/min young women; P<0.05).

Conclusions:
These preliminary data suggest that compared to young women, PMW exhibit an exaggerated MSNA response to isolated muscle metaboreflex activation.
Abstract 277: Elevated C-Reactive Protein Contributes to Preeclampsia via Kinin Signaling Pathways
Authors: Parchim Nicholas , Wang Wei , Iriyama Takayuki , Liu Chen , Siddiqui Athar H , Blackwell Sean , Sibai Baha , Kellems Rodney E , Xia Yang
Published: July 2021

Preeclampsia (PE) is a serious pregnancy disease characterized by hypertension and proteinuria. Despite intensive research efforts, the underlying cause of PE remains a mystery. PE is, however, associated with abnormalities of the immune system. Here we report that the levels of C-reactive protein (CRP), an important acute phase reactant, were significantly elevated in the plasma of human with PE at the third trimester. Next, we found that CRP protein levels in the placentas of PE patients were also significantly increased compared to controls. In an effort to determine the exact role of elevated CRP in PE, we infused CRP into pregnant mice. We found that injection of CRP into pregnant mice induced hypertension (170 mmHg mean systolic vs. 125 mmHg mean systolic control; p<0.05) and proteinuria (25 mg/ug vs 12 mg/ug vehicle; p<0.05), indicating the direct role of CRP in PE. CRP is known to bind with phosphocholine on damaged cell membranes. Recent studies identified that neurokinin B (NKB), a placental enriched neuropeptide and known pathogenic molecule for PE, is phosphocholinated. This posttranslational modification increases its stability and enhances NKB-mediated receptor activation. These findings raise an intriguing hypothesis that CRP may bind with NKB coupled to NK3R activation and contribute to PE. To test this hypothesis, we conducted a pulldown assay, and we found that CRP bound with NKB. Next, using a cellular invasion assay, we revealed that CRP decreased invasion of human trophoblast cells (0.7 to 0.07 invasion index, p<0.05), while treatment with an NK3R selective antagonist, SB222200, ameliorated this shallow invasion. Finally, we provided
in vivo
evidence that inhibition of NK3R by SB222200 or knockdown of NK3R by specific siRNA in a potent nanoparticle delivery system significantly reduced CRP-induced hypertension and proteinuria in pregnant mice (170 mmHg mean systolic CRP-injected vs. 130 mmHg mean systolic siRNA NK3R; p<0.05 and proteinuria 25 mg/ug vs. 15 mg/ug; p<0.05). Overall, our findings demonstrate that elevated CRP contributes to PE and NKB/NK3R is a novel mechanism underlying CRP-mediated shallow invasion and disease development. These studies suggest novel pathogenic biomarkers and innovative therapeutic targets for PE.
Abstract 279: Hypertension in Hemolysis, Elevated Liver Enzyme, Low Platelet Syndrome is Associated with Increased Endothelin-1
Authors: Wallace Kedra , Morris Rachael , Chatman Krystal , Moseley Janae , LaMarca B. Babbette
Published: July 2021

INTRODUCTION.
HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets) is a severe form of preeclampsia, a hypertensive disorder that occurs during pregnancy. We have shown that HELLP syndrome in both women and in an animal model causes hypertension and elevated TNF-α, IL-6, and agonistic autoantibodies to the angiotensin II receptor, all of which have been shown to activate the endothelin-1 system (ET-1). Therefore we tested the hypothesis that hypertension in response to HELLP syndrome is associated with ET-1 activation.

METHODS.
Plasma was collected from women with HELLP syndrome or pregnant women without maternal/fetal complications at the time of delivery. Endothelin (ET-1) was extracted from the plasma and measured with a Quantikine ET-1 enzyme linked immunosorbent assay kit.

On gestational day (GD) 12 miniosmotic pumps infusing sEndoglin (7ug/kg/day) and sFlt-1 (4.7ug/kg/day) were implanted into normal pregnant rats to induce HELLP syndrome. On GD18 carotid catheters were inserted into HELLP and normal pregnant (NP) rats and mean arterial pressure (MAP) was recorded on GD19; serum, urine and tissues were collected for molecular analysis.

RESULTS.
Women with HELLP syndrome (n=4) had significantly more circulating ET-1 (3.35pg/mL) compared to normal pregnant women (0.75pg/mL; n=3; p=0.006) and increased MAP (120.8±6.2 mmHg HELLP vs. 87.33±6.67mmHg NP; p=0.015). In rats MAP increased from 91±2mmHg in NP rats (n=4) to 129.8±4.5mmHg in HELLP rats (n=4; p=0.0002). Preproendothelin mRNA expression was 3 fold higher in placentas and 1.3 fold higher in livers from HELLP rats compared to NP rats (p=0.02; p=0.05). In addition we measured ET-1 secretion from endothelial cells exposed to serum from NP and HELLP rats. ET-1 in response to NP serum was 4.03±.68pg/mg/mL and increased to 9.69±3.5pg/mg/mL with HELLP serum (p=0.04) after 24hrs.

CONCLUSION.
These data support the hypothesis that hypertension in both women and animals with HELLP syndrome is accompanied by increased ET-1 expression.
Abstract 287: Radiofrequency Renal Nerve Ablation Attenuates Hypertension in Spontaneously Hypertensive Rats (SHR)
Authors: Gao Juan , Smart Frank W , Kapusta Daniel R
Published: July 2021

Radiofrequency catheter ablation (RF-ABL) of the renal arteries decreases blood pressure in patients with drug-resistant hypertension. This study investigated whether RF-ABL of the renal arteries alters blood pressure (BP) in an animal model of essential hypertension, the spontaneously hypertensive rat (SHR).

Methods
Nineteen-week old male SHR were instrumented with radio-telemetry probes for chronic measurement of BP (Systolic, SBP; Diastolic, DBP). After 1-week, control BP was measured in rats for 3 days. The next day SHR were anesthetized and randomly received either bilateral Sham-ABL (n=5) or RF-ABL (n=6) of the renal arteries (Biosense Webster Stockert 70 generator and RF-probe). The tip of the RF-probe was applied externally to a small dissected segment of each renal artery and stimulated circumferentially (10 Watts). BP was then measured three times a week for 8 weeks.

Results
In anesthetized hypertensive SHR, RF-ABL of a single renal artery did not alter BP. In contrast, subsequent RF-ABL of the contralateral renal artery produced an immediate (<15 seconds) decrease in mean arterial blood pressure (MAP, range Δ -60 to -100 mmHg). BP in hypertensive SHR was not altered during or after the Sham-RF procedure. In conscious SHR, BP was significantly decreased the day after RF-ABL (153±8/102±4 mmHg) and throughout the post-ABL study (4-week SBP/DBP, 173±7/120±5; 8-weeks, 171±6/116±4 mmHg) compared to pre-ABL control levels (SBP/DBP, 187±9/130±5 mmHg). At the end of week-1 the peak increase in MAP to cage switch stress was significantly blunted and the time to return to basal BP was shortened in RF-ABL (peak Δ 18±5 mmHg; 30-min to basal) vs. Sham-ABL (peak Δ 32±3 mmHg; 70-min to basal). At the end of week-8, the hypotensive response to i.p. chlorisondamine was blunted in RF-ABL (Δ -39±4 mmHg) vs. Sham-ABL rats (Δ -53±2 mmHg).

Conclusions
These data demonstrate that in hypertensive SHR, bilateral RF-ABL of the renal arteries produces, presumably via blockade of renal sympathetic afferent/efferent neural pathways, a reduction in BP, which by 4-weeks is reset to a lower basal level. These findings indicate that RF-ABL can be used in rodents to investigate the therapeutic mechanisms by which endovascular renal denervation reduces blood pressure in hypertension.
Abstract 289: Effect of Central Sympatholytic Treatment on the Anti-Hypertensive Response of Renal Denervation in the Spontaneously Hypertensive Rat
Authors: Phelps Jeremiah , Fink Gregory
Published: July 2021

Renal nerve ablation has been shown to elicit a chronic, anti-hypertensive effect in drug-resistant hypertensive patients. Precise understanding of the mechanisms underlying the clinical success of renal denervation is currently unknown, and as a consequence, it is predicted the technology will be under-utilized until such information is uncovered. Retrospective multivariate analyses of responders suggest treatment with a central sympatholytic may correlate with a successful response to renal denervation. However, this hypothesis remains untested. This study tested the hypothesis that pretreatment with a central sympatholytic would augment the response to renal denervation (RDX) in the spontaneously hypertensive rat. In rats pre-treated for 1 week with clonidine (125ug/kg/day), MAP was significantly reduced from baseline but there was no difference in response to clonidine between groups (Sham(n=7): -21.3±0.8 vs RDX(n=7): -22.8±1.3mmHg, p>0.05). During clonidine treatment RDX significantly reduced MAP within 48hrs in RDX animals compared to shams (Sham: 145.9±2.5 vs. RDX: 135.0±1.7mmHg, p<0.05). However, this reduction was abolished by day 5 after RDX (Sham: 145.1±2.5 vs RDX: 140.4±3.0 mmHg, p>0.05). Discontinuation of clonidine caused blood pressure to rise, but once pressures stabilized, the average MAP was significantly lower in RDX treated rats (Sham:157.4 ± 0.8 vs. RDX: 146.4 ± 0.8mmHg, p<0.05). Administration of hydralazine, which reflexively increases sympathetic activity, lowered MAP similar to the magnitude observed in clonidine treatment (Sham: -19.6±1.0 vs RDX: -18.2±0.9mmHg). The anti-hypertensive effect of RDX was not augmented by hydralaizine however, it was also not abolished. These findings do not support the idea that patients taking effective doses of centrally acting sympatholytics will enhance the response to renal denervation.
Abstract 295: Sodium Restriction Increases Ophthalmic Artery Systolic Velocity in Healthy Subjects
Authors: Shafiq Qaiser , Fisher Naomi D , Stallings Jennifer E , Boyce Siobhan F , Silwal Sujeeta , Hollenberg Norman K
Published: July 2021

Changes in ocular blood flow occur in systemic diseases. A high peak systolic velocity (PSV) in the ophthalmic artery (OA) has been observed in diabetes mellitus and hypertension, conditions often associated with high tissue renin-angiotensin system (RAS) activity and reduced renal plasma flow (RPF). Since our group has demonstrated that sodium restriction (
a high RAS state
) is associated with a decrease in RPF which is correctable with ACE inhibition, we hypothesized that sodium restriction would result in higher ophthalmic PSV. Sixteen healthy volunteers (age 46.2±13.2, male 69%, white 75%, BMI 26±3.7) were placed on a low sodium (LS) diet (10 mmol/day) for 1 week followed by high sodium (HS) diet (200 mmol/day). Sodium balance was assessed by 24hr urine collection. BP, RPF (PAH clearance) and OA hemodynamic measurements (Multigon, NY) were made after overnight fasting and rest in the supine position. Sodium restriction did not affect BP (systolic/diastolic LS: 121/73 ± 12/7 vs HS: 122/72 ± 15/9 mmHg, mean ± SD, NS) or heart rate (69 ± 16 vs 70 ± 15/min, NS). As expected, RPF was lower on LS compared with HS (599.2 ± 89.8 vs 633.4 ± 93.5ml/min/1.73 m
2
, p=0.006). Sodium restriction was associated with higher OA peak (38.8 ± 6.4 vs 33.2 ± 6.0 cm/sec, p=0.018) and mean (19.7±2.9 vs 17.1±3.5, p=0.003) systolic velocity (
Figure
). There was no change in end-diastolic velocity (10.6±2.5 vs 9.7±2.4, p=0.266) with sodium restriction. In conclusion, sodium restriction was associated with an increase in OA peak and mean systolic velocity in healthy individuals. Further studies are required to investigate the potential relevance of our results to diabetic and hypertensive retinopathy.
Abstract 297: Crataegus Special Extract WS 1442 Retards Replicative Senescence in Coronary Artery Endothelial Cells: Role of eNOS-derived NO
Authors: Idris-Khodja Noureddine , Kheloufi Marouene , Schini-Kerth Valérie B
Published: July 2021

Endothelial cell senescence promotes endothelial dysfunction, which has been suggested to have a critical role in the initiation and/or progression of atherosclerosis, and also to contribute to the pathogenesis of age-associated vascular disorders. Endothelial senescence is characterized by an irreversible cell cycle arrest, which involves an increased activity of p53 and its downstream effector p21. Endothelial senescence is also associated with a decreased expression of endothelial nitric oxide synthase (eNOS). The present study has evaluated whether the
Crataegus
special extract WS
®
1442, a rich source of polyphenols and a potent inducer of eNOS activation, prevents replicative senescence of porcine coronary artery endothelial cells, and, if so, to elucidate the underlying mechanism. Replicative senescence was induced by sequential passaging of primary cultures of endothelial cells up to the fourth passage (P4). Changes of endothelial senescence were determined by measuring senescence-associated β-galactosidase (SA-β-gal) activity. Western blot was used to analyze the protein expression of p53, p21 and eNOS. Compared to P1, the SA-β-gal activity was 240% increased in cells at P4 (
P
<0.001), and this effect was associated with 93% (
P
<0.001) and 56% (
P
<0.001) increased expression of p53, p21 and a 87% decreased expression of eNOS (
P
<0.001). Treatment of P3 cells with the p53 inhibitor (pifithrin) reduced 43% SA-β-gal activity indicating a role of p53 activity in replicative senescence (
P
<0.001). Treatment of endothelial cells with the Crataegus extract reduced by 56% the SA-β-gal activity (
P
<0.01), improved by 131 % eNOS expression (
P
<0.01) and reduced by 39% the up-regulation of p21 in cells at P4 without affecting the expression level of p53. The inhibitor of eNOS, L-NAME promoted the induction of endothelial senescence at P1 and reduced the inhibitory effect of the Crataegus extract on SA-β-gal activity at P3. In conclusion, the present findings indicate that the Crataegus extract delays endothelial cell replicative senescence most likely by preventing the downregulation of eNOS expression and activity and the upregulation of the p53/p21 pathway.
Abstract 302: Aerobic Exercise Effects on Endothelial Health Status in Pre- and Post Menopausal African American Women
Authors: Grimm Heather , Jan Kretzschmar Jan , Thakkar Sunny , Ling Chenyi , Diaz Keith M , Sturgeon Kathleen , Feairheller Deborah L , Perkins Amanda , Lee Hojun , Williamson Sheara , Babitt Dianne , Brown Michael D
Published: July 2021

Background:
When compared to other racial groups, African American women (AAW) reach menopause at an earlier age and are at an increase risk for CVD and endothelial health complications. A known intervention for the treatment of both CVD and menopausal changes is aerobic exercise training (AEXT). The purpose of this study was to compare the effects of AEXT on endothelial function between pre- and post- menopausal AAW.

Methods:
Sedentary, non-smoking pre- and post- menopausal AAW not on hormone replacement therapy and free of CVD completed AEXT. Participants exercised at 65% of their VO2max for 40 min, 3 days/wk, for 6 months. Participants completed flow-mediated dilation (FMD) testing and blood sample collection. Endothelial microparticles from plasma samples were analyzed by labeling with late stage apoptotic antibodies CD31+/CD42b- and early phase endothelial dysfunction and endothelial activation CD62E+ and quantified using flow cytometry.

Results:
No difference in FMD was found between groups before (7.11±1.2 vs. 7.73±0.8 %) or after (10.38±0.2 vs. 9.88±0.6 %) AEXT. Both pre- (7.11±1.2 vs. 10.38±0.2 %, p≤0.005) and post- (7.73±0.8 vs. 9.88±0.6 %, p≤0.043) menopausal women significantly increased FMD after AEXT.

CD31+/CD42b- EMPs did not differ between pre- and post-menopausal AAW before (4.52±0.9 vs. 2.9±0.4 events/μL) or after (2.1±0.4 vs. 2.31±0.5e vents/μL) AEXT. CD31+/CD42b- levels significantly decreased in pre-menopausal AAW (4.52±0.9 vs. 2.1±0.4 events/μL, p≤0.015) after AEXT.

CD62E+ EMPS were not different between pre- and post-menopausal groups before (44±8.5 vs. 42.5±9 events/μL) or after (26.64±8.4 vs. 19.79±3.2 events/μL) AEXT. Both pre- (44±8.5 vs. 26.6±8.4 events/μL, p≤0.034) and post-menopausal (42.5±9 vs. 19.8±3.2 events/μL, p≤0.008) groups had lower levels of CD62E+ EMPs after AEXT.

Discussion:
No difference was found in terms of vascular function or health between pre- and post-menopausal AAW. Both groups showed improvements in vascular function and health after AEXT. Our findings suggest AEXT has significant improvements in terms of endothelial function and vascular health in AAW regardless of being a pre- or post-menopausal.
Abstract 309: Sirt3 Regulates Oxygen Sensors and Blood Vessel Formation in the Heart
Authors: He Xiaochen , Zeng Heng , Hou Xuwei , Chen Jian-Xiong
Published: July 2021

New blood vessels formation is driven by deprivation of nutrients and oxygen during myocardial ischemia. The interaction between nutrients and oxygen sensors in the regulation of new blood vessel formation still remains incompletely understood. Sirtuins belong to a family of NAD+-dependent deacetylases which are involved in the regulation of angiogenesis and energy hemostasis. In the present study, we investigated the functional role of Sirtuin 3 (Sirt3), a metabolic sensor, in the regulation of blood vessel formation and its interaction with oxygen sensors-prolyl hydroxylases (PHDs)/hypoxia-inducible factors (HIFs) system. The levels of PHD1-3, HIFs, and angiogenic growth factors as well as blood vessel formation were examined in the hearts of Sirt3 knockout (Sirt3 KO) mice and wild type (WT) mice. Our data showed that the expression of angiopoietin-1 and angiopoietin-2 was significantly reduced, whereas the expression of Tie-2 and VEGF was unchanged in the hearts of Sirt3 KO mice compared to WT mice. Intriguingly, loss of Sirt3 had different effects on the PHDs/HIFs system. The expression of PHD1, PHD2, and HIF1α was significantly up-regulated, but PHD3 and HIF2α expression was down-regulated in Sirt3 KO mice. Ex vivo angiogenesis assay revealed that vessel outgrowth was significantly reduced in Sirt3 KO mice compared to WT mice. Our immunohistochemistry study further confirmed that the arteriole formation was significantly reduced in the hearts of Sirt3 KO mice compared to that in WT mice. This was accompanied by a reduction of cardiac ejection fraction (EF %) and fraction shortening (FS %). Moreover, the phosphorylation level of Akt was dramatically decreased together with increased Wnt7b, caspase-3 expression and apoptosis in the hearts of Sirt3 KO mice. Therefore, our data implicates that Sirt3 is important in controlling of blood vessel growth and cell survival by affecting oxygen sensors and angiopoietins expression in the heart.
Abstract 31: Rt-pcr Profiling Reveals the Essential Role of Endothelial Hif-1a in Hypertensive Nephropathy
Authors: Luo Renna , Zhang Weiru , Zhao Cheng , Zhang Yujin , Grenz Almut , Eltzschig Holger K , Tao Lijian , Kellems Rodney E , Xia Yang
Published: July 2021

Chronic kidney disease is a devastating disease featured with kidney injury and renal fibrosis. Hypertension is a major cause of CKD. However, the specific factors underlying hypertensive nephropathy are unclear. Here we sought to identify specific factors that contribute to hypertensive CKD. Using RT-PCR array, we discovered that the hypoxia induced factor-1α (HIF-1α) mRNA was significantly increased in the kidneys of angiotensin II (AngII)-infused mice, a well-accepted animal model of hypertensive CKD. Immunohistochemsitry revealed that HIF-1α was significantly elevated in the endothelial cells of capillary lumen of glomeruli of AngII-infused mice. To determine the role of elevated endothelial HIF-1α in hypertensive CKD, we generated mice with specific endothelial HIF-1α deficiency by mating floxed HIF-1α mice with the V-Cadherine-Cre mice containing transgene expressing Cre recombinase only in the endothelial cells. First, we found that Ang II-induced-hypertension and proteinuria were significantly reduced in endothelial HIF-1α deficient mice compared to the control mice (150.88mmHg vs. 176.33mmHg systolic, p<0.05; 15.49ug/mg vs. 129.16ug/mg, p<0.001). Histological studies showed genetic deletion of endothelia HIF-1α attenuated glomerular damage and renal fibrosis in Ang II-infused mice. Mechanistically, we further revealed that genetic deficiency in endothelial HIF-1α significantly reduced levels of endothelin-1 (ET-1), a potent vasoconstrictor, in both kidneys and circulation of Ang II-infused mice. Intriguingly, using primary mouse kidney organ cultures, we provide genetic evidence that Ang II-mediated ET-1 production was significantly reduced in the cultured kidneys from endothelial HIF-1α-deficient mice, indicating that Ang II is a hypoxia-independent factor capable of inducing HIF-1α production and contributing to ET-1 elevation in the mouse kidneys. Lastly, we revealed that both HIF-1α and ET-1 levels were significantly elevated in the kidneys of CKD patients compared to normal individuals and further elevated in hypertensive CKD patients. Overall, both the mouse and human studies provide strong evidence that elevated endothelial HIF-1α-mediated excess ET-1 is a prominent driver of hypertensive CKD.
Abstract 310: Deoxycorticosterone Acetate (doca)-salt Exacerbates Hypertension and Vascular Dysfunction in Mice Expressing Dominant Negative Peroxisome Proliferator-activated Receptor-gamma (pparg) in Smooth Muscle
Authors: Ketsawatsomkron Pimonrat , Davis Deborah R , Hilzendeger Aline M , Grobe Justin L , Sigmund Curt D
Published: July 2021

PPARG, a ligand-activated transcription factor plays a critical role in the regulation of blood pressure and vascular function. We hypothesized that smooth muscle cell (SMC) PPARG protects against hypertension (HT) and resistance vessel dysfunction. Transgenic mice expressing dominant negative PPARG (S-P467L) in SMC or non-transgenic controls (NT) were implanted with DOCA pellet and allowed ad libitum access to 0.15 M NaCl for 21 days in addition to regular chow and water. Blood pressure was monitored by telemetry and mesenteric arterial (MA) function was assessed by pressurized myograph. At baseline, 24-hour mean arterial pressure (MAP) was similar between NT and S-P467L mice, while the transgenic mice were tachycardic. DOCA-salt increased MAP to a much greater degree in S-P467L mice (Δ MAP; S-P467L: +34.2±6.0, NT: +13.3±5.7, p<0.05 vs NT). Heart rate was similarly decreased in both groups after DOCA-salt. Vasoconstriction to KCl, phenylephrine and endothelin-1 did not differ in MA from DOCA-salt treated NT and S-P467L, while the response to vasopressin was significantly reduced in S-P467L after DOCA-salt (% constriction at 10-8 M, S-P467L: 31.6±5.6, NT: 46.7±3.8, p<0.05 vs NT). Urinary copeptin, a surrogate marker for arginine vasopressin was similar in both groups regardless of treatment. Vasorelaxation to acetylcholine was slightly impaired in S-P467L MA compared to NT at baseline whereas this effect was further exaggerated after DOCA-salt (% relaxation at 10-5 M, S-P467L: 56.1±8.3, NT: 79.4±5.6, p<0.05 vs NT). Vascular morphology at luminal pressure of 75 mmHg showed a significant increase in wall thickness (S-P467L: 18.7±0.8, NT: 16.0±0.4, p<0.05 vs NT) and % media/lumen (S-P467L: 8.4±0.3, NT: 7.1±0.2, p<0.05 vs NT) in S-P467L MA after DOCA-salt. Expression of tissue inhibitor of metalloproteinases (TIMP)-4 and regulator of G-protein signaling (RGS)-5 transcript were 2- and 3.5-fold increased, respectively, in MA of NT with DOCA-salt compared to NT baseline. However, this induction was markedly blunted in S-P467L MA. We conclude that interference with PPARG function in SMC leads to altered gene expression crucial for normal vascular homeostasis, thereby sensitizing the mice to the effects of DOCA-salt induced HT and vascular dysfunction.
Abstract 312: Mechanisms Underlying Impaired KCl-induced Contractility After Long-term Serum-free Organ-culture of Rat Isolated Mesenteric Artery
Authors: Morita Tomoka , Okada Muneyoshi , Yamawaki Hideyuki
Published: July 2021

(Background and aim)
Organ culture of blood vessels is a useful technique to investigate the long-term effects of drugs. Organ culture in a serum-free condition is so far the best way to maintain differentiated cell function. However some functional changes may occur from freshly isolated blood vessel (fresh) such as decreased contractility. Mammalian/mechanical target of rapamycin (mTOR) complex 1 is atypical serine/threonine kinase which integrates various signals induced by growth factors, stress, energy status, oxygen, and amino acids. Here, we investigated the mechanism of decrease in smooth muscle contractility after long-term serum-free organ culture specifically focusing on mTOR.

(Methods and results)
Rat isolated mesenteric arteries were cultured for 5 days without (0% serum) or with rapamycin (Rap). In 0% serum, absolute contraction by KCl significantly decreased from fresh (n=21 for fresh, n=7 for 0% serum,
p<0.01
). In Rap, the decreased contraction was significantly normalized (n=7,
p<0.05
vs. 0% serum). In both 0% serum and Rap, sensitivity to KCl significantly increased from fresh (
p<0.01
vs. fresh). In 0% serum, mTOR expression significantly increased from fresh (n=8,
p<0.01
) which was significantly normalized by rapamycin (n=8,
p<0.01
). Morphological examinations showed degenerative changes in smooth muscle layer of 0% serum which was improved by rapamycin. In 0% serum, expression of myocardin, a master regulator of smooth muscle gene expression significantly decreased from fresh (n=9,
p<0.01
), which was significantly normalized by rapamycin (n=9,
p<0.01
).

(Conclusion)
Addition of rapamycin prevented the decreased contractility in serum-free organ cultured mesenteric artery perhaps by normalizing the mTOR and downstream myocardin expression as well as arterial degenerative morphological damage. Further studies are needed to clarify how mTOR controls myocardin expression and also how decreased myocardin expression leads to the decreased smooth muscle contractility. This experimental system may be useful for the hypertension research.
Abstract 358: Excess Cardio-renal Damage in Aldosterone-producing Adenoma Patients With Somatic Mutations in the KCNJ5 Gene
Authors: Rossi Gian Paolo , Seccia Teresa M , Letizia Claudio , Cicala Maria Verena , Zinnamosca Laura , Kuppusamy Maniselvan , Cesari Maurizio , Sciomer Susanna , Mantero Franco , Pessina Achille C
Published: July 2021

Aldosterone exerts detrimental cardiovascular (CV) effects, and aldosterone producing adenoma (APA) patients carrying somatic mutations in the KCNJ5 K+ channel (mutAPA) have higher plasma aldosterone concentration (PAC) than those wild-type (wtAPA).

We therefore investigated whether mutAPA patients develop a more prominent CV and renal damage than wtAPA patients.

From 250 consecutive PA patients, we identified 170 who had a diagnosis of APA by the 'four corners' criteria and high-quality echocardiographic data. Of them 106 with KCNJ5 sequencing information and long-term follow-up data could be compared for echocardiographic changes and eGFR (by CKD-EPI equation) according to presence (mutAPA, 18.8%) or absence (wtAPA, 81.2%) of the KCNJ5 mutations.

At baseline the mutAPA had lower eGFR (75±29 ml/min vs 84±20, p<0.05), and higher left ventricular mass index (LVMI, 61.9±21.4 mg/h2.7vs 49.5±11.3, p=0.001), PAC (48.9 [37.4-77.1] ng/dl vs 37.0 [24.9-48.5]), <0.0001), aldosterone-renin-ratio (ARR, 173.5 [92.2-229.6] ng/dl/ng/ml/h vs 144.3 [69.9-252.1], <0.0001), than the wtAPA patients. They were similar for blood pressure (BP) and need for antihypertensive medications. After adrenalectomy BP, PAC, ARR, and LVMI normalized in all groups, with no difference between mutAPA vs wtAPA.

Compared to the wild-type APA patients those with KCNJ5 mutations showed more prominent cardiovascular and renal damage, likely because of the higher PAC. However, the presence of these mutations did not compromise the chances of being cured from the hyperaldosteronism and the high blood pressure, or the regression of LVMI after adrenalectomy.
Abstract 367: Insulin Resistance is Associated with Increased Sympathetic Activity and Decreased Baroreflex Function in Obese African American Women
Authors: Shibao Cyndya , Celedonio Jorge E , Mariños Alejandro , Ramirez Claudia E , Arnold Amy C , Okamoto Luis E , Diedrich Andre' , Biaggioni Italo
Published: July 2021

Obesity has a greater detrimental impact on the health of African American (AA) women than any other racial or gender group. Nearly 80% of AA women are overweight or obese and have higher incidence of insulin resistance compared to Caucasian women. The sympathetic nervous system (SNS) is increased in obesity and previous studies have reported that it may contribute to insulin resistance. However, most of these studies were performed in Caucasians. Thus, the contribution of the SNS to insulin resistance in African Americans remains unknown.The aim of this study was to determine if sympathetic activity is increased in obese AA women with insulin resistance. A total of 35 obese AA women with metabolic syndrome were studied (age 43±9 years, body mass index 38.7±5.5 Kg/m
2
, percentage body fat 48±4). Insulin sensitivity (SI) was evaluated by frequently sampled intravenous tolerance test and analyzed with Bergman's minimal model (MINMOD). Measurement of sympathetic regulation of vasomotor tone and baroreflex gain were determined with baroreflex-cross spectral analysis. Our results showed that SI was directly associated with baroreflex sensitivity, Figure. Subjects with insulin resistance (SI<3) had lower baroreflex sensitivity compared with those with normal SI (6.2±2.8 vs 10.4±4.5 ms/mm Hg, P=0.009). Subjects with insulin resistance tended to have elevated sympathetic activity as measured by low frequency variability of systolic blood pressure (LF
SBP
; 5.9±3.4 vs 3.7±2.0 mm Hg
2
, P=0.057). In conclusion, insulin resistance is associated with decreased baroreflex gain and increased sympathetic activity in obese AA women.
Abstract 376: Prolonged Excess of Superoxide in Mouse Afferent Arterioles Causes Remodeling and Enhances Myogenic and Angiotensin II Contractions
Authors: Lai En Yin , Li Lingli , Feng Di , Welch William J , Wilcox Christopher S
Published: July 2021

Background:
Superoxide (O
2
.-
) is associated with cardiovascular disease (CVD) and inactivating SNIPs of the EC-SOD gene increased CVD, but the mechanisms are unclear. Superoxide dismutases (SODs) have three isoforms: cytoplasmic Cu/ZnSOD (SOD1), mitochondrial MnSOD (SOD2) and extracellular Cu/ZnSOD (SOD3). Complete SOD2-/- was lethal. We tested the hypothesis that these isoforms of SOD modulate angiotensin II (Ang II), myogenic responses (MRs) and remodeling of afferent arterioles (Affs).

Methods and Results:
MRs were assessed from the slope of the regression of active wall tension on perfusion pressure (PP) and Ang II contractions from incubation with 10
-6
M Ang II. O
2
.-
was assessed from PEG-SOD inhibitable ethidium:dihydroethidium fluorescence and remodeling from media:lumen area ratio (M:L).

Results:
1. Compared to +/+, increasing PP from 40 to 80 mmHg or applying 10
-6
M Ang II in Affs increased O
2
.-
more in SOD1-/- (PP, 8 ± 1% vs 5 ± 1% , P<0.05; Ang II, 29 ± 7 vs 10 ± 2%, P<0.05), in SOD2+/- (PP, 15 ± 2% vs 7 ± 1%, P<0.01; Ang II, 32 ± 5 % vs 7 ± 1%, P<0.01), and in SOD3-/- ( PP, 20 ± 4% vs 7 ± 1%, P<0.05; Ang II, 32 ± 5% vs 12 ± 3%, P<0.05). However, O
2
.-
did not increase in SOD1+/- and SOD3+/-. 2. Compared to +/+, MRs (dynes.cm
-1
.mmHg
-1
) were significantly increased in SOD1-/- (3.49 ± 0.30 vs 2.30 ± 0.27, P<0.05), in SOD2+/- (3.85 ± 0.48 vs 1.80 ± 0.34, P<0.05), and in SOD3-/- (3.51 ± 0.20 vs 2.43 ± 0.15, P<0.01), with no increase in SOD1+/- and SOD3+/-. 3. Compared to +/+, Ang II contractions were stronger in SOD1-/- (-68 ± 2% vs -33 ± 3%, P<0.0001), in SOD2+/- (-66 ± 6% vs -42 ± 3%, P<0.01), and in SOD3-/- (-75 ± 3% vs -34 ± 3%, P<0.0001) 4. Compared to +/+, M:L ratio of Affs were greater in SOD1-/- (4.0 ± 0.2 vs 1.8 ± 0.2, P<0.0001), in SOD2+/- (3.7 ± 0.4 vs 1.8 ± 0.4, P<0.001), and in SOD3-/- (4.5 ± 0.2 vs 1.9 ± 0.1, P<0.0001), but were unchanged in SOD1+/- and SOD3+/-. M:L ratio was strongly correlated to MR (r
2
=0.32, P<0.005) and Ang II contractions (r
2
= 0.56, P<0.005) across genotypes. 5. There were no compensatory changes in expression of other SOD isoforms.

Conclusions:
Lifetime increases in cytosolic, mitochondrial or extracellular O
2
.-
enhanced afferent arteriolar myogenic and Ang II contractions that were closely related to vascular remodeling.
Abstract 385: Tenascin-C Worsens Myocardial Inflammation and Fibrosis by Enhancing Macrophage Activation via NF-kappaB in Mouse Hypertensive Heart
Authors: Shimojo Naoshi , Hashizume Ryotaro , Hara Mari , Suzuki Yuka , Nishioka Tomohiro , Yoshida Toshimichi , Imanaka-Yoshida Kyoko
Published: July 2021

Background and Aim:
Tenascin-C (TN-C) is an extracellular matrix glycoprotein, not detected in normal adult heart but it expresses under various pathological conditions. We have previously reported in the enhanced TN-C production and accumulation of macrophages in the perivascular lesions of angiotensin II (AgII)-induced cardiac fibrosis mouse model. To clarify the role of TN-C in molecular mechanism, we analyzed the effect of TN-C in hypertensive heart utilizing wild-type (WT) and TN-C knock-out (TNKO) mice. Furthermore, to assess whether TN-C is involved in macrophage activation, we investigated in vitro study using macrophages isolated from the peritoneal cavity of WT mice.

Methods and Results:
Balb/c WT and TNKO mice were treated with 560 ng/kg body weight/min AgII subcutaneously by osmotic minipump for 4 weeks (WT/AgII and TNKO/AgII), and analyzed histological and molecular biological approaches. AgII treatment increased blood pressure, heart weight/body weight ratio, atrial and brain natriuretic peptide expression level and sizes of cardiomyocytes, but no significant differences were detected between WT/AgII and TNKO/AgII mice. In WT/AgII mice, interstitial collagen fibers (10.29±5.09% vs. WT: 4.6±1.56%, p<0.001) and accumulation of mac-3 positive macrophages (50±15.13 vs. WT: 6±2.55 cells/section, p<0.001) were observed at perivascular regions, and expression levels of interleukin (IL)-6 (1.85±0.39 fold, p<0.05) and monocyte chemoattractant protein (MCP)-1 (3.20±0.53 fold, p<0.001) were up-regulated. These changes were significantly reduced in TNKO/AgII mice. In vitro, TN-C accelerated macrophage migration in the presence of MCP-1. Western blotting and Immunofluorescence staining indicated that TN-C activated rapidly NF-kappaB. RT-PCR analysis demonstrated that TN-C up-regulated IL-6 mRNA on NF-kappaB dependent manner (39.90±18.10 fold, p<0.01). Integrin αVβ3 antagonist P11 suppressed these changes.

Conclusion:
The present study clearly demonstrated that TN-C aggravates inflammation and fibrosis in hypertensive heart by upregulation of IL-6 productions by activating NF-kappaB via integrin αVβ3 on macrophage.