bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 037: A Role for IL-17 to Activate Cytolytic Natural Killer Cells in Response to Placental Ischemia
Authors: Cornelius Denise C , Thomas D'Andrea S , Elfarra Jamil , McAfee Taia R , LaMarca Babbette
Published: July 2021

Women with preeclampsia (PE), newly developed hypertension and renal dysfunction during pregnancy, have small-for-gestational-age babies and demonstrate an increase in the inflammatory cytokine IL-17, placental oxidative stress, and cytolytic natural killer (NK) cell activation. The stimulus of the cytolytic NK cell phenotype during PE is currently unknown. Moreover, the specific role of cytolytic NK cells in the pathophysiology of preeclampsia has not been clearly defined. The reduced uterine perfusion pressure (RUPP) model of placental ischemia exhibits many of the characteristics of preeclampsia including hypertension, renal dysfunction, chronic inflammation and intrauterine growth restriction (IUGR). In this study, we tested the hypothesis that placental ischemia results in cytolytic activation of NK cells, and examined a role for the increased IL-17, in response to placental ischemia, to activate cytolytic NK cells. In this study, blood pressure (MAP) and pup weight were measured, and PBMCs and placental lymphocytes were examined via flow cytometry for surface makers of cytolytic NK cell activation. MAP significantly increased in response to placental ischemia from 103±4.1 mmHg in NP (n=6) to 129.1±3.1 mmHg (n=8) in RUPP rats (p<0.001). Neutralization of IL-17 with a soluble receptor attenuated the blood pressure response to 106.3±2.3 mmHg in RUPP+IL-17RC rats (n=3). Pup weight is significantly decreased in RUPP rats (2.52±0.18g in NP vs 2.03±0.05g in RUPP (p<0.05)), which increased to 2.54±0.36g in RUPP+IL-17RC. Cytolytic activation of circulating NK cells was not significantly changed among any of the groups (NP: 2.49±1.1%; RUPP: 7.74±3.2%; RUPP+IL-17RC:5.50±2.8%). However, cytolytic activation of placental NK cells increased in response to placental ischemia (NP: 3.4±1.1% vs RUPP 10.0±3.4%), and was completely attenuated after treatment with the soluble IL-17 receptor (RUPP+IL-17RC: 0.33±0.17%). These results suggest a role for placental ischemia and increased IL-17 to stimulate cytolytic NK cells. Furthermore, this study links the IL-17 pathway with cytolytic NK cell activation and IUGR in response to placental ischemia, potentially identifying new therapeutic targets to improve maternal and fetal outcomes of PE.
Abstract 038: Placental Growth Factor Supplementation Abolishes Placental Ischemia-induced Hypertension
Authors: Spradley Frank T , Tan Adelene Y , Joo Woo S , Daniels Garrett , Kussie Paul , Karumanchi S A , Granger Joey P
Published: July 2021

Preeclampsia (PE) is a pregnancy-specific disorder of new-onset hypertension. Unfortunately, the most effective treatment is early delivery of the fetus and placenta. Progress toward potential therapeutic targets has found that placental ischemia/hypoxia induced in animals by reduced uterine perfusion pressure (RUPP) or in human patients stimulates the release of hypertensive placental factors into the maternal circulation. For example, the antiangiogenic factor sFlt-1, which antagonizes and reduces bioavailable vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), is significantly elevated in PE patients and RUPP rats. It is clear that reductions in VEGF promote hypertension in RUPP rats as supplementation with recombinant VEGF at 180 ug/kg/day abolished the hypertension. However, it is unknown if reductions in PlGF levels also contribute to the hypertensive response. Thus, we tested the hypothesis that PlGF treatment would reduce placental ischemia-induced hypertension. On gestational day 14, Sprague Dawley rats were randomized to three groups: normal pregnant (NP, N=6), RUPP (N=5) and RUPP + 180 ug/kg/day PlGF (N=7). The rPlGF (AG31, a purified-recombinant human PlGF) was infused via i.p. osmotic minipump. Mean arterial blood pressure (MAP, carotid catheter) and pregnancy weights were assessed on day 19. MAP was significantly higher in RUPP than NP rats (123±4 vs. 104±1, P<0.05). PlGF reversed these levels to NP values (105±3, P<0.05 vs. RUPP). Placental weights (NP: 0.5±0.02; RUPP: 0.51±0.04; and RUPP+rPlGF: 0.5±0.05) and fetal weights (NP: 2.30±0.07; RUPP: 2.00±0.15; and RUPP+rPlGF: 2.09±0.07) were similar among all groups. The number of live fetuses was reduced in RUPP than NP rats (5±2 vs. 12±1, P<0.05) with a slight increase in the RUPP+rPlGF group (8±1). The number of fetal absorptions was increased in RUPP than NP rats (9±2 vs. 2±1, P<0.05) with a slight increase in the RUPP+rPlGF group (5±1). In conclusion, these data indicate that the reductions in PlGF that occur as a result of placental ischemia contribute to the development of maternal hypertension. Our novel finding that rPlGF abolishes placental ischemia-induced hypertension suggests a strong role and therapeutic potential for this growth factor in PE.
Abstract 042: Cd74-dysregulation of Macrophage-trophoblastic Interactions in the Preeclamptic Placenta
Authors: Przybyl Lukasz , Haase Nadine , Golic Michaela , Rugor Julianna , Solano Maria E , Arck Petra C , Gauster Martin , Huppertz Berthold , Bernhagen Jürgen , Bucala Richard , Luft Friedrich C , Staff Anne C , Mueller Dominik N , Dechend Ralf , Herse Florian
Published: July 2021

Objectives:
Preeclamptic pregnancies feature placental anomalies. Villous trophoblast differentiation during placental development is regulated by feto-placental macrophages and disturbance of this well-balanced regulation can lead to pathological pregnancies. We hypothesized that Cluster of differentiation 74 (CD74) dysregulation of placental macrophages, leading to altered macrophage-trophoblast interaction, is involved in preeclampsia.

Methods and Results:
We performed microarray analysis of placental tissue. CD74 was one of the most down-regulated (-2.5 fold) genes in placentas from preeclamptic women. We confirmed this finding in early onset (<34 gestational week, n=26) and late onset (≥34 gestational week, n=24) samples from preeclamptic women, compared to healthy pregnant controls (n=28) by real-time RT-PCR and on protein level by Western blot and flow cytometry. We localized CD74 expression in placental macrophages by immunofluorescence, flow cytometry, and real-time RT-PCR. Number and mean fluorescence intensity (MFI) of CD74-positive macrophages were significantly lower in preeclamptic placentas (7692 MFI ± 4402), as compared to controls (16283 MFI ± 3047). In CD74-silenced macrophages, expression of adhesion molecules ALCAM (-2 fold), ICAM4 (-2.1 fold), and Syndecan-2 (-1.9 fold) was lower compared to control. Macrophage adhesion to a trophoblast layer were diminished (-1.3 fold). Naïve and activated macrophages lacking CD74 showed a shift towards a pro-inflammatory signature with an increased secretion of TNFalpha(21.8 pg/ml ± 13.2 vs. 8 pg/ml ± 4.3), CCL5 (1.9 ng/ml ± 0.4 vs. 0.8 ng/ml ± 0.2) and MCP-1 (3 ng/ml ± 2.6 vs. 1 ng/ml ± 0.5), when co-cultured with trophoblasts compared to control macrophages. CD74-knockout mice showed disturbed placental morphology, reduced junctional zone (1.6 mm2 ± 0.3 vs. 2.3 mm2 ± 0.5) and smaller placentas (0.09 g ± 0.01 vs. 0.11 g ± 0.02) with fetal growth restriction (0.7 g ± 0.1 vs. 0.9 g ± 0.2) when compared to WT mice.

Conclusions:
We found that CD74 downregulation in placental macrophages is present in preeclampsia. CD74 downregulation led to altered macrophage activation towards a pro-inflammatory signature, a disturbed crosstalk with trophoblasts and an abnormal placental morphology.
Abstract 063: Impaired Myogenic Response of the Af-art Contributes to the Increased Susceptibility to Hypertension and Diabetic Induced Renal Disease in Milan Normotensive Rats
Authors: Ge Ying , Fan Fan , Roman Richard J
Published: July 2021

Previous studies have indicated that Milan normotensive (MNS) rats are more susceptible to the development of hypertension and diabetic induced renal injury than Milan hypertensive (MHS) rats, but the genes and pathways involved are unknown. MNS also develop proteinuria and chronic kidney disease (CKD) as they age, whereas hypertensive MHS do not. We compared the myogenic response of isolated perfused Af-Art and autoregulation of RBF and glomerular capillary pressure in 6-9 week old MNS and MHS rats. The diameter of Af-Art of MNS rats increased from 14.0 ± 0.5 to 14.2 ± 0.6 μm (n=6) when elevation in perfusion pressure from 60 to 120 mmHg. In contrast, the diameter of the Af-Art decreased significantly from 14.3 ± 0.5 to 11.5 ± 0.6 μm (n=6) in MHS rats. In vivo, RBF increased by 26% when RPP was increased from 100 to 140 mmHg in MNS rats but it remained unchanged in MHS rats. Glomerular capillary pressure rose by 11 mmHg in MNS following the elevation in RPP from 100 to 140 mm Hg but not in MHS rats. Protein excretion increased from 8.9 ± 0.7 to 158.2 ± 23.1 mg/day in MNS rats as the increased in age from 3 to 9 months of age but it did not increase in MHS rats. In com-parison to other strains susceptible and resistant to CKD, we noticed that both MNS and Fawn Hooded hypertensive (FHH) rats that do not autoregulate RBF also share the same sequence variant in the Adducin 3 gene. We performed a genetic complementation study to test whether this mutation might be responsible for the impaired myogenic response in MNS. The diameter of the Af-Art isolated from an F1 cross of MNS &FHH rats increased from 17.2 ± 0.9 to 18.5 ± 0.9 μM (n=5) in response to increase in perfusion pressure and RBF was not efficiently autoregulated in these animals. These data indicate a mutation in Adducin 3 which impairs myogenic response of the Af-Art and increased transmission of pressure to the glomerular capillaries may contribute to the development of CKD in MNS rats similar to what is seen in FHH rats.
Abstract 070: AntagomiR-762 Prevents Angiotensin II Induced Aortic Fibrosis and Stiffening
Authors: Montaniel Kim Ramil C , Wu Jing , Bersi Matthew R , Xiao Liang , Itani Hana A , Vickers Kasey C , Humphrey Jay D , Harrison David G
Published: July 2021

We and others have shown that hypertension (HTN) is associated with a striking deposition of collagen in the vascular adventitia. This causes vascular stiffening, which increases pulse wave velocity and contributes to end-organ damage. Through a screen of vascular microRNAs (miRNAs), we found that miR-762 is the most upregulated miRNA in mice with angiotensin II (Ang II)-induced HTN. qRT-PCR confirmed that miR-762 is upregulated 6.35±1.22 (p=0.03) fold in aortas of Ang II-infused mice compared with controls. This was a direct effect of Ang II, as miR-762 upregulation was not eliminated by lowering blood pressure with hydralazine and hydrochlorothiazide and was increased only 2-fold in DOCA salt HTN. To study the role of miR-762 in HTN, we administered a locked nucleic acid inhibitor of miR-762 (antagomiR-762). AntagomiR-762 administration did not alter the hypertensive response to Ang II, yet it normalized stress-strain relationships and aortic energy storage that occurs in systole (Table). Further studies showed that antagomiR-762 dramatically affected vascular matrix proteins, reducing mRNA for several collagens and fibronectin and dramatically upregulating collagenases MMP1a, 8 and 13 (Table). Thus, miR-762 has a major role in modulating vascular stiffening and its inhibition dramatically inhibits pathological fibrosis, enhances matrix degradation and normalizes aortic stiffness. AntagomiR-762 might represent a new approach to prevent aortic stiffening and its consequent end-organ damage.
Abstract 072: TRIF-Pathway of Innate Immune Responses Mediates Angiotensin II Hypertension
Authors: Singh Madhu V , Cicha Michael Z , Chapleau Mark W , Abboud François M
Published: July 2021

We tested the role of two distinct adaptors of toll-like receptor (TLR) signaling on Ang II-induced hypertension and cardiac hypertrophy. These TLR adaptors, myeloid differentiation protein 88 (MyD88) and TIR domain-containing adaptor inducing interferon β (TRIF) facilitate distinct inflammatory signaling pathways. In an earlier study, we reported that MyD88-/- mice are protected from cardiac hypertrophy and pro-inflammatory gene expression after myocardial infarction. Our current results with 3 weeks infusion of Ang II (3000 ng/kg/min) vs. saline indicate that in MyD88-/- mice, the pressor response to Ang II and cardiac hypertrophy were increased more than in wild type (WT) mice. In Ang II-infused WT, systolic blood pressure (SBP) peaked at 147 ± 4 mmHg whereas in Ang II-infused MyD88-/- mice SBP reached a peak value of 163 ± 6 mmHg. However, in mice with non-functional TRIF adaptor mutant (Trifmut), SBP did not increase during Ang II infusion and remained similar to the SBP in saline-infused mice (115 ± 3 mmHg). Baseline SBP was not different among WT, MyD88-/- and Trifmut mice. The increase in heart weight to body weight ratio (HW/BW) between saline and Ang II-infused mice was greater in MyD88-/- mice than WT mice (60% increase in MyD88-/- vs. 40% increase in WT), whereas it was less in Trifmut mice (22% increase). Accordingly, expression of several inflammatory genes (Tnfa, Nox4 and Agtr1a) was significantly greater (P< 0.05) in the heart and kidney of Ang II-infused MyD88-/- mice compared with Ang II-infused WT mice, whereas expression of these genes in Trifmut mice was either unchanged or reduced. We conclude that- (1) Ang II-induced hypertension, cardiac hypertrophy and inflammatory gene expression are mediated by activation of a TRIF-dependent pathway, but not by the MyD88-dependent pathways, and (2) Enhanced Ang II effects on SBP and hypertrophy in MyD88-/- mice suggest that MyD88 may serve as a negative regulator of the TRIF pathway in Ang II-induced hypertension. Selective targeting of these adaptor proteins may have significant therapeutic implications.
Abstract 078: Functional Significance of the Chymase/Ang-(1-12) Pathway on Cardiomyocytes
Authors: Zhang Xiaowei , Li Tiankai , Ahmad Sarfaraz , Varagic Jasmina , Groban Leanne , Dell'Italia Louis J , Cheng Che Ping , Ferrario Carlos M
Published: July 2021

Angiotensin-(1-12) [Ang-(1-12)] functions in rodents and humans as a tissue substrate for the direct generation of Ang II via chymase. Since its direct cardiac effect have not been studied, the importance of this renin-independent mechanism for Ang II paracrine/intracrine actions in modulating cardiac contractility were determined in freshly isolated myocytes from 11 normal SD rats. Systolic amplitude (SA), peak velocity of shortening (dL/dtmax), the peak velocity of relengthening (dR/dtmax), and changes in the peak calcium transient ([Ca2+]i) were evaluated before and following exposure to Ang II (10-6 M), Ang-(1-12) delivered alone (range: 2x10-6 to 4x10-6 M) or after 1 h incubation with human recombinant chymase (10 μg protein/mL at 37°C). Both Ang II and the mixture of Ang-(1-12) with chymase elicited positive inotropic responses in freshly isolated cardiac myocytes associated with significant increases in peak systolic [Ca2+]i (Figure) while superfusion of Ang-(1-12) alone elicited an increase in dL/dtmax without significant changes in [Ca2+]i. The increases in contractility elicited by Ang II or the Ang-(1-12)/chymase mixture were abolished by prior exposure of the myocytes to losartan (10-5 M) or the chymase inhibitor chymostatin (8x10-5 M). We conclude that in single adult rat myocytes Ang-(1-12) stimulates contractile function through a chymase mediated action and by mechanisms that implicate a paracrine/intracrine activation of intracellular calcium.
Abstract 079: G Protein-Coupled Estrogen Receptor Deletion Exacerbates Pulse Pressure in Female but not Male Hypertensive Mice
Authors: Lindsey Sarah H , Trimmer Emma H , Zimmerman Margaret A
Published: July 2021

The protective cardiovascular effects of estrogen are mediated by both the classic steroid receptors (ERα/β) and the novel G protein-coupled estrogen receptor (GPER). Our previous work demonstrates a role for GPER in the beneficial effects of estrogen on blood pressure, vascular tone, end organ damage, and aortic remodeling in hypertensive females, due to both direct vasodilatory effects and local regulation of the renin-angiotensin system. Therefore, we hypothesized that genetic deletion of GPER in female mice exacerbates Ang II-induced hypertension and associated tissue damage. Male (M) and female (F) wt and GPER ko mice were implanted with radiotelemetry probes at 11-13 weeks of age to measure baseline blood pressure before infusing Ang II (700 ng/kg/min) for two weeks. MAP (in mmHg) was similar at baseline (F-wt: 108 ± 2, n=4, F-ko: 105 ± 2, n=3, M-wt: 106 ± 4, n=3, and M-ko: 105 ± 0.5, n=3; P = 0.62), and after Ang II (F-wt: 136 ± 11, F-ko: 131 ± 3, M-wt: 148 ± 7, M-ko: 135 ± 5; P=0.51). Pulse pressure was significantly higher in response to Ang II in both sexes (*P<0.0001), and GPER deletion exacerbated this increase in females (30 ± 1 vs 43 ± 4 mmHg, *P<0.05) but not males (37 ± 3 vs. 39 ± 4, P=0.61). Further evaluation of the circadian pattern revealed that pulse pressure was particularly higher in F-ko during the day (28 ± 1 vs. 40 ± 4 mmHg, *P<0.05). Moreover, F-ko mesenteric arteries exhibited enhanced contractility to both receptor-dependent (PGF2α, *P<0.05) and receptor-independent (KCl, *P<0.05) stimulation. Our findings indicate that in females with intact ERα/β signaling, GPER deletion does not alter the initial pressor response to Ang II but exacerbates pulse pressure and resistance artery contractility during hypertension. Pulse pressure is an indirect measure of arterial stiffness and an independent risk factor for adverse cardiovascular events. While pulse pressure increases with aging in both sexes, this increase is markedly exacerbated in women. Our results indicate that loss of endogenous GPER activation due to low circulating estrogen may underlie increased pulse pressure in aging postmenopausal women.
Abstract 127: Agonistic Autoantibodies to Angiotensin II Type I Receptor Contributes Partly to Placental Ischemia-induced Cerebrovascular Abnormalities
Authors: Warrington Junie P , Fan Fan , LaMarca Babbette B , Dechend Ralf , Wallukat Gerd , Roman Richard J , Drummond Heather A , Granger Joey P , Ryan Michael J
Published: July 2021

Placental ischemia, a characteristic feature of preeclampsia, leads to impaired cerebral blood flow (CBF) autoregulation, cerebral edema, and increased blood-brain barrier (BBB) permeability; however, the placental factors that contribute to these cerebral abnormalities are not clear. Agonistic autoantibodies to the angiotensin II type 1 receptor (AT1-AA) are increased in preeclamptic patients as well as in a rat model of preeclampsia induced by placental ischemia. In this study, we tested the hypothesis that AT1-AA mediates placental ischemia-induced cerebrovascular abnormalities. To determine whether the AT1-AA contributes to impaired CBF autoregulation, we infused purified rat AT1-AA into normal pregnant rats from gestational day (GD) 12 to 19 via mini-osmotic pumps and measured CBF using laser Doppler flowmetry on GD 19. Autoregulatory index increased from 0.7 to 1.0±0.2 in the AT1-AA infused group over the range of 120 to 160 mmHg compared to pregnant controls (0.3 to 0.5±0.1 over the same range of pressures, p<0.05) suggesting impaired CBF autoregulation. However, AT1-AA infusion did not affect brain water content at baseline blood pressures (104±2 mmHg in normal pregnant rats vs. 113±2 mmHg in AT1-AA infused rats, p<0.01). To determine the role of endogenous AT1-AA in mediating placental ischemia-induced cerebrovascular abnormalities, losartan (5 mg/kg/day), an AT1 receptor antagonist, was administered in the drinking water from GD 14 to 19. Losartan reduced anterior brain water content from 79.6±0.2% in placental ischemic rats to 79.2±0.1% (compared to 79.1±0.1% in normal pregnant untreated rats) and BBB permeability from 0.06±0.01 in placental ischemic rats to 0.03±0.03 (compared to 0.03±0.004 in normal pregnant untreated rats). These results indicate that impaired CBF autoregulation in response to placental ischemia is due, at least in part, to increases in circulating AT1-AA. While AT1-AA infusion, by itself, did not alter brain water content at baseline blood pressures, the beneficial effects of losartan in placental ischemic rats suggests that the renin-angiotensin system may interact with other placental factors to promote cerebral vascular changes common to preeclampsia.
Abstract 130: Sphingosine-1-Phosphate Signaling Plays a Role in High Blood Pressure Programmed by Intrauterine Growth Restriction in Mouse
Authors: Intapad Suttira
Published: July 2021

Intrauterine growth restriction (IUGR) is a risk factor for hypertension and cardiovascular (CV) disease in later life, but the underlying mechanisms remain unclear. The bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) is critically involved in CV development in the fetus, and plays a significant role in the regulation of CV health in adulthood. S1P receptor (S1PR) type 1, 2 and 3 are widely expressed in CV system which S1PR1 has a protective role against kidney injury, while S1PR3 is involved in controlling BP. Yet, the contribution of S1P on BP in IUGR is unknown. In the present studies, we tested the hypothesis that IUGR alters renal S1P receptors expression during- and post-nephrogenesis, which contributes to high BP in male IUGR mouse. C57bl/6J mice underwent sham or reduced uterine perfusion (RUP) at day 13 of gestation with delivery at full term. IUGR offspring (from RUP dam) had a lower birth weight than control (p<0.05). Kidneys were isolated from 2 day old male pups or adult 24 week old male control and IUGR. S1PR3 protein expression was increased in 2 day old IUGR kidneys (2.4 fold vs control, N=3, p< 0.01). At 24 weeks of age, S1PR3 mRNA levels were increased (1.2 fold vs control, N=4, p< 0.05) whereas S1PR3 protein levels were decreased (0.75 fold vs control, N=4, p< 0.05) in IUGR kidneys. mRNA and protein expression levels of S1PR1 and S1PR2 were not different between control and IUGR kidneys. Finally, we assessed the role of S1PRs agonist on BP of IUGR. Male IUGR offspring had a significantly higher BP compared to male control via carotid catheter in the conscious state (control: 112.1±2.1, IUGR: 125.0±3.7 mmHg; N=7,
P
<0.05). Acute administration of FTY720 (1 mg/kgBW i.p, Fingomod), a S1P receptor type 1, 3 agonist did not significantly alter BP in control (106.0 ± 5.7 mmHg) but significantly decreased BP in IUGR (105.7±2.3 mmHg, p< 0.05). A dose response to FTY720 (10 mg/kgBW) decreased BP in both control (94.0±2.0 mmHg, p< 0.05) and IUGR (99.3±2.3 mmHg, p< 0.05). Together our data suggest that IUGR programs an alteration of renal S1PR3 expression in both during- and post-nephrogenesis thereby contributing to an increase in sensitivity to S1PRs agonist. Thus, S1P signaling is a putative mechanism underlying the hypertension of IUGR offspring.
Abstract 142: A Fructose-but Not a Glucose-enriched Diet, Induces a Salt-dependent Increase in Blood Pressure and Enhances NKCC2 Phosphorylation in Normal Rats
Authors: King-Medina Keyona N , Henson Emily , Ortiz Pablo
Published: July 2021

Human consumption of fructose as a sweetener has increased in the past 30 years. High fructose intake has been implicated in the development of hypertension, diabetes, and obesity. In the US, the upper 10th percentile of the population consumes up to 40% of their caloric intake from added sugars, in which fructose represents half of these. Fructose metabolism is strikingly different from that of glucose. Yet, the effect of a fructose or glucose-enriched diet in salt handling by the kidney, affecting blood pressure, and its interaction with high salt intake has been poorly studied. In genetic models of salt-sensitive hypertension, the activity of the Na
+
/K
+
/2Cl
-
cotransporter (NKCC2) in the thick ascending limb (TAL) is abnormally enhanced. We hypothesized that chronic fructose in drinking water induces a salt-dependent increase in blood pressure and stimulates NKCC2 during high salt intake in normal rats. Sprague-Dawley rats were given 20% fructose or 20% glucose in drinking water for 1 week after which a high salt (HS) diet (4% Na
+
in chow) was started for 3 weeks. When we measured systolic blood pressure (SBP) by tail cuff plethysmography in fructose-fed and glucose-fed rats on a HS diet, only the fructose-fed rats had an increased SBP from 120±10 to 132±6 mmHg on day 7 of HS (p<0.01). SBP continued to increase up to 144±18 mmHg after 3 weeks (p<0.01 vs glucose). Fructose or glucose alone did not increase SBP after 4 weeks. We then repeated the protocol using radiotelemetry to monitor the blood pressure (BP). In rats fed fructose, by day 5 of HS the SBP increased by 12±3 mmHg (p<0.02) and SBP remained elevated for 3 weeks (delta: 10±2.5 mmHg, n=3). In rats fed glucose, a HS diet did not significantly change SBP for 3 weeks (n=5). Moreover, NKCC2 activity in the TAL is enhanced by phosphorylation at Thr96, 101. We found that NKCC2 phosphorylation was higher in rats fed fructose plus HS (p<0.02) but not in rats fed glucose plus HS for 3 weeks (HS: 100, fructose+HS: 250±40%, glucose+HS: 95±10%). Therefore, we conclude that a high fructose (but not a glucose) diet in normal rats induces a salt-dependent increase in BP independently from caloric intake. Thus, the increase in BP may in part be due to the stimulation of NKCC2 phosphorylation in the TAL by fructose.
Abstract MP04: Cardiac ACE2/Angiotensin-(1-7) in Rats Expressing Human Angiotensinogen Gene
Authors: Varagic Jasmina , VonCannon Jessica , Ahmad Sarfaraz , Bader Michael , Ferrario Carlos M
Published: July 2021

When compared to Sprague Dawley (SD) control rats, transgenic rats expressing the human angiotensinogen (AGT) gene [TGR(hAGT)L1623] exhibit hypertension associated with cardiac hypertrophy and higher cardiac tissue angiotensin (Ang) II. Whether the hypertension and cardiac hypertrophy in these rats expressing the human AGT are related to a non-canonical pathway for Ang II formation or suppression of the counter regulatory mechanism mediated by ACE2 and Ang-(1-7) has not been established. Consequently, cardiac peptides were determined by RIA in 9 [TGR(hAGT)L1623] and 11 SD male rats (17 weeks of age). ACE2 activities in homogenized heart tissues were determined by HPLC. Cardiac Ang II content was four times higher (37.05 ± 5.04 vs. 9.62 ± 0.93 fmol/mg protein; p <0.0001) while the Ang-(1-7) level increased only 1.3 times (18.02 ± 1.62 vs 13.37 ± 1.74 fmol/mg protein; p=0.06) in TGR(hAGT)L1623 rats when compared with SD rats. Although, the Ang II/Ang-(1-7) ratio was higher in transgenic rats harboring the human AGT gene (2.10 ± 0.27 vs 0.90 ± 0.19; p <0.005), ACE2 activities between these two strains of animals were not different (12.21 ± 0.76 vs. 10.80 ± 0.91 fmol/min/mg; p >0.05). Since human AGT protein is not cleaved by rat renin, our data continues to support the view that hypertension and cardiac hypertrophy in this transgenic strain are induced by activation of a non-renin mechanism rather than a primary suppression of the compensatory Ang II degrading pathway mediated by ACE2. Further studies are necessary to determine the role of enzymes affecting Ang-(1-7) metabolism in the observed inadequate balance between Ang II and Ang-(1-7).
Abstract MP07: Endogenous Chemerin Amplifies Nerve-Dependent Arterial Contraction
Authors: Darios Emma , Watts Stephanie W
Published: July 2021

The adipokine chemerin causes contraction of isolated arteries and is implicated in blood pressure regulation, especially in the obese population that have elevated levels of circulating chemerin. Because chemerin is expressed in the perivascular adipose tissue (PVAT) that facilitates the sympathetic innervation of the blood vessel, we tested the hypothesis that chemerin (endogenous and exogenous) would amplify the effects of the sympathetic nervous system in mediating electrical field stimulated (EFS) contraction. The model was the superior mesenteric artery with PVAT, mounted into tissue baths for isometric contraction. Immunohistochemistry validated a robust expression of chemerin in the PVAT surrounding the superior mesenteric artery. EFS (0.3-20 Hz) caused a frequency-dependent, prazosin-sensitive contraction that was reduced (~40%) by the chemerin receptor ChemR23 antagonist CCX832 (100 nM; figure) but not by the inactive congener CCX826 (100 nM). Exogenous chemerin (1 μM) amplified EFS-induced contraction in a manner that was also blocked by CCX832. Chemerin did not directly modify contraction of the superior mesenteric artery (-PVAT) to cumulative concentrations of norepinephrine (1 nM - 10 μM), supporting that contractile amplification by chemerin was not at the level of smooth muscle. These studies raise the interesting possibilities that endogenous chemerin and/or ChemR23 modifies nerve-mediated contraction. This is significant because of the well appreciated role of the sympathetic nervous system in blood pressure control.
Abstract MP10: Nobelty and Significance of the Rapid Measurement of Plasma Aldosterone Concentration and Active Renin Concentration
Authors: Satoh Fumitoshi , Ono Yoshikiyo , Morimoto Ryo , Iwakura Yoshitsugu , Nezu Masahiro , Omata Kei , Tezuka Yuta , Kudo Masataka , Seiji Kazumasa , Takase Kei , Nakamura Yasuhiro , Gomez-Sanchez Celso E , Sasano Hironobu , Ito Sadayoshi
Published: July 2021

The plasma aldosterone/renin ratio (ARR) for the clinical diagnosis of primary aldosteronism (PA) is calculated with plasma aldosterone concentration (PAC) per plasma renin activity (PRA) or active renin concentration (ARC). It takes 3-4 days to get the results. Of late, we developed the novel rapid non-RIA assays of PAC and ARC, which are measurable in 10 minutes. Both were simultaneously measured by chemiluminescent enzyme immunoassay (CLEIA) system machine with their specific monoclonal antibodies and were automatically washed by the immobilized magnetic particles. We retrospectively compared RIA-assayed PAC,PRA and ARC with CLEIA-measured PAC and ARC in 243 patients with aldosterone producing adenoma (APA, n=86), bilateral hyperaldosteronism (BHA, n=78) and essential hypertension (EH, n=79), and investigated whether the calculation of ARR with ARC (ARR-C, ng/dL per pg/mL) instead of PRA (ARR-A, ng/dL per ng/m/hour) affects the diagnosing of the APA patients, which are potentially surgically curable. CLEIA-measured PAC were significantly correlated with RIA-assayed PAC (y=0.9846 x + 2.5708, Spearman's r =0.9072, P<0.0001). CLEIA-measured ARC (the lower detection limit = 0.25 pg/mL) were also significantly correlated with RIA-assayed ARC (y=1.0103 x + 0.9156, Spearman's r =0.8166, P<0.0001). ARR-A and ARR-C of APA patients were 129 ± 4.3 and 25.1±2.7 (Mean ± SEM), respectively. ARR-A and ARR-C of BHA patients were 40.4 ± 2.9 and 6.7 ± 0.5, and those of EH patients 17.2 ± 2.1 and 3.2 ± 0.4, respectively. Our novel rapid CLEIA-assays of PAC and ARC will be able to be clinically more useful for detecting APA or BHA because it takes only ten minutes to get the results.
Abstract P013: Meta-analysis of Poor Outcomes and Permissive Hypertension after Acute Ischemic Stroke
Authors: Ivanov Alexander , Mohamed Ambreen , Korniyenko Alex
Published: July 2021

Background:
Stroke is the leading cause of long term disability and second leading cause of death worldwide. The effectiveness of primary and secondary prevention of stroke by antihypertensive medications is well validated, however, support for permissive hypertension in the early course of acute ischemic stroke (AIS) has been questioned.

Materials and methods:
We searched Pubmed, Embase, and Cochrane databases to identify RCTs comparing different blood pressure reduction regimens with placebo in AIS patients within 48 hours after symptom onset and sample size of 100 or more patients. We excluded studies that do not report mortality or functional outcomes at the end of follow up. The main outcomes were all-cause mortality and death or severe morbidity which was defined as: modified Rankin Score >2 or Bartel ADL index<60, Glasgow outcome scale3, Mathew Impairment Scale <14. Relative risks (RR) and corresponding 95% confidence intervals (CI) were calculated using random-effect model.

Results:
In our analysis we included 20 trials involving 17,209 patients. There was no difference in all-cause mortality RR 1.04 (95% CI 0.95-1.13), p=0.4 nor in mortality or severe disability RR 1.03 (95% CI 0.99-1.08), p=0.16 between active blood pressure reduction and permissive hypertension strategy. There was no evidence of heterogeneity between studies for both outcomes I
2
=2.6% and I
2
=14.1% , p for heterogeneity =0.42 and =0.27, respectively.

Conclusion:
Use of antihypertensive therapy in acute period of ischemic stroke does not have an effect on disability or all-cause mortality.
Abstract P020: Decreased Survival Rate in Female Obese Leptin Receptor Mutant Dahl Salt-Sensitive Rats that Develop Chronic Kidney Disease
Authors: McPherson Kasi , Guillory Devin , Taylor Lateia , Spires Denisha , Johnson Ashley C , Garrett Michael R , Williams Jan M
Published: July 2021

Obesity contributes to sex differences in the risk for chronic kidney disease (CKD) in which males tend to develop CKD earlier in life than females. Therefore, in the current study, we examined whether there were sex differences in the development of CKD in the obese leptin receptor mutant Dahl salt-sensitive (SS
LepR
mutant) strain which was derived from Zinc-finger nucleases. We observed an increase in body weight in both female and male SS
LepR
mutant rats when compared to SS rats throughout the study. Glucose tolerance was impaired significantly in female and male SS
LepR
mutant rats versus SS

rats by 18 weeks of age. The SS
LepR
mutant strain also developed hyperinsulinemia in comparison to their lean SS counterparts (6.86±0.83 vs 0.72±0.04 ng/mL, respectively, n=6). However, blood glucose in the SS
LepR
mutant strain remained within normal range throughout the course of the study regardless of sex. Female and male SS
LepR
mutant rats developed severe systolic hypertension (via tail-cuff) by 18 weeks of age when compared to the values measured in SS rats (199±7 and 201±10 vs. 159±5 and 155±4 mmHg, respectively, n=6). Yet, the rise in arterial pressure occurred earlier in female SS
LepR
mutant rats than males. Protein excretion was significantly higher in the SS
LepR
mutant strain as opposed to the values observed in SS rats at 18 weeks of age regardless of sex (488±61 and 631±86 vs. 50±17 and 149±23 mg/day, respectively, n=6). At the end of study, kidneys from the SS
LepR
mutant strain displayed increased glomerulosclerosis and interstitial fibrosis than SS rats. Female and male SS
LepR
mutant rats had a significant increase in plasma creatinine levels and averaged 2.1±0.4 mg/dL (n=6) compared to the normal value of 0.5±0.1 mg/dL (n=6) observed in the SS strain suggesting the presence of severe CKD. While both, female and male, SS rats survived the length of study, the survival rate of female SS
LepR
mutant rats was markedly reduced compared to their male counterparts (62%, 21 of 34 vs. 25%, 6 of 24, respectively). Overall, these data indicate that the SS
LepR
mutant strain may be a useful model to study sex differences during the development of CKD associated with obesity.
Abstract P027: GCH1 and GULP1 Play a Role in Hypertension Associated With Ovarian Hormone Loss Independently of Body Weight Gain
Authors: Pai Amrita V , Woperrer Samuel , Ji Hong , Wu Xie , Li James , Sandberg Kathryn
Published: July 2021

Background and Objective:
Ovarian hormone loss is associated with an increased incidence of hypertension, body weight (BW) gain and inflammation. Since BW gain is also associated with an increased incidence of hypertension, it has been difficult to separate the role of the immune system in hypertension from BW gain. Dahl salt-sensitive (DS) and Dahl salt-resistant (DR) rats gain BW after ovariectomy; however, only the DS rat becomes hypertensive. In this study, we took advantage of the DS/ DR model to determine which T cell genes are associated with hypertension induced by ovarian hormone loss-independently of body weight gain.

Methods and Analysis:
DS and DR rats (1 month) were either ovariectomized (OVX) or subjected to sham surgery (Sham). At 4 months of age, a microarray analysis was conducted on isolated splenic T-cells from these 4 groups and differential expression was confirmed by real-time PCR.

Results:
In contrast to DR rats which remain normotensive, the mean arterial pressure (MAP) significantly increased by 14 mm Hg in DS rats after ovariectomy [MAP (mmHg): DS-OVX, 143 ± 3.4 vs DS-Sham, 129 ± 9.4; n=10-11/group; p=0.0002; DR-OVX, 123 ± 15 vs DR-Sham, 126 ± 4.8; n=11-12/group; ns]. Both rat strains, however, exhibited a similar 17-20% increase in BW after ovariectomy compared to sham treatment [BW (g): DS-OVX, 372 ± 34 vs. DS-Sham, 308 ± 13; n=6-7/group; p<0.05; DR-OVX, 323 ± 22 vs, DR-Sham, 276 ± 49; n=7/group; p<0.05]. Microarray analysis suggested that among many others, the genes GTP Cyclohydrolase 1 (GCH1) and Engulfment Adaptor PTB Domain Containing 1 (GULP1) were specifically associated with resistance or susceptibility to hypertension induced by ovarian hormone loss. Real-time PCR confirmed that GCH1 and GULP1 were selectively elevated by ovariectomy in DS but not DR splenic T cells.

Conclusion:
GCH1 and GULP1 play a role in hypertension associated with ovarian hormone loss independently of BW gain, which has implications for women with ovarian hormone deficiency due to premature ovarian failure or elective oophorectomy.
Abstract P056: MHC CLass II-associated Invariant Peptide (CLIP) Antagonism During Chronic Lipopolysaccharide Treatment Preserves Afferent Arteriolar Autoregulatory Behavior
Authors: Van Beusecum Justin , Cook Anthony K , Zhang Shali , Tobin Richard P , Newell-Rogers M. K , Inscho Edward W
Published: July 2021

Lipopolysaccharide (LPS) is a cell wall component of gram-negative bacteria that can activate toll-like receptor 4, which in turn, activates the innate immune system. Chronic immune system activation is linked to blunted afferent arteriolar autoregulatory behavior and kidney injury. MHC Class II-associated invariant peptide (CLIP) provides a critical step in antigen processing, presentation, and adaptive immune system activation. Accordingly, we postulated that treatment with a competitive CLIP antagonist (CAP) during chronic low-dose LPS exposure would preserve afferent arteriolar autoregulatory behavior. Rats were implanted with osmotic minipumps (day 0) for infusion of LPS (0.01mg/kg/day) or saline (0.9% NaCl; 0.5μl/hr) for 8 days and then the kidneys were harvested for juxtamedullary nephron studies. Four groups (n=6/group) were studied: Control, LPS, LPS + CAP and Sham + CAP. Both LPS + CAP and Sham + CAP groups were treated with CAP (3mg/kg/day; i.p.) on days 1-7. Autoregulatory behavior was assessed in these groups by increasing perfusion pressure in 15 mmHg increments from 65 to 170 mmHg. Starting baseline diameters were similar across the control (15.2 ± 1.2 μm), LPS (14.1 ± 1.0 μm), LPS + CAP (15.1 ± 1.0), and Sham + CAP (15.7 ± 0.6) groups. When perfusion pressure was increased from 65 to 170 mmHg, control and sham + CAP afferent arteriolar diameter decreased significantly by 26 ± 4% and 25 ± 2% (P<0.05), respectively. In contrast, afferent diameters from LPS treated kidneys decreased by just 5 ± 2% over the same pressure range indicating impaired afferent arteriolar autoregulatory behavior. In LPS + CAP treated kidneys, afferent arteriolar diameter decreased by 17 ± 2% (P<0.05) over the same pressure range signifying preservation of autoregulatory behavior. These data support the hypothesis that CLIP antagonism during chronic low dose LPS treatment preserves afferent arteriolar autoregulatory behavior. Inhibiting CLIP may open novel therapeutic targets for inflammatory kidney disease.
Abstract P069: 1H-Magnetic Resonance Spectroscopy Reveals Elevation of MyoInositol and Other Markers of Inflammation in the Dorsal Medulla of Children with Orthostatic Intolerance
Authors: Wagoner Ashley L , Olson John D , Fortunato John E , Diz Debra I , Shaltout Hossam A
Published: July 2021

Children with orthostatic intolerance (OI) have exaggerated decreases in heart rate variability (HRV) and suppression of baroreflex sensitivity (BRS) with standing. Inflammation is proposed as a possible factor contributing to impaired HRV in cardiometabolic disorders; whether systemic or brain inflammation better predicts impaired HRV is arguable. We used 1H magnetic resonance spectroscopy (MRS) to quantify markers of neuronal and glial integrity in children with OI compared with asymptomatic controls. Fifteen subjects ages 10-18 years were evaluated for blood pressure, HR, and autonomic function in supine and upright positions and 7 tested positive for OI. An average of 2 weeks following OI testing all subjects underwent 1H-MRS scans of dorsal medulla on a clinical 3T magnet while supine. OI subjects had higher myoinositol (mIns) as a marker of glial inflammation than asymptomatic controls (7.8 ± 0.4 vs 5.6 ± 0.9 mmol/L, p = 0.03). Trends were observed for higher glycerophosphocholine (higher GPC, reduced myelination and axonal integrity) (2.3 ± 0.2 vs 1.8 ± 0.2 mmol/L, p = 0.08) and lower N-acetyl aspartate (lower NAA, reduced neuronal integrity) (2.8 ± 0.3 vs. 3.7 ± 0.4 mmol/L, p = 0.1) in OI subjects vs controls (mean ± SEM). mIns concentrations did not correlate with indices of autonomic function measured in the supine position. However, supine measures of mIns correlated with autonomic measures taken in the upright position: negatively correlated with spontaneous BRS (R = -0.64, p = 0.01), parasympathetic tone measured by high frequency alpha index (HFα, R= -0.547, p=0.04) and HRV measured by root of mean square of successive differences (rMSSD, R = -0.45, p = 0.09); there was a positive correlation with HR (R = 0.53, p < 0.05). In summary, children with OI have higher mIns in dorsal medulla while supine that is predictive of impairment in BRS, HRV and parasympathetic tone upon upright posture. This first report that OI in children is associated with elevated mIns, a marker of glial inflammation in a variety of neuropathies, raises the intriguing possibility that brain inflammation plays a role in the autonomic dysfunction observed while standing in these subjects. Support: Centers for Integrative Medicine and Hypertension & Vascular Research; AHA12CRP9420029
Abstract P076: Suppressor of Cytokine Signaling 3 (SOCS3) in POMC Neurons and Its Role in Regulating Blood Pressure, Body Weight and Glucose in Obesity
Authors: Wang Zhen , do Carmo Jussara M , da Silva Alexandre A , Hall John E
Published: July 2021

Suppressor of cytokine signaling 3 (SOCS3), a negative regulator of leptin signaling, may be involved in development of obesity-induced leptin resistance. Although we previously showed that activation of proopiomelanocortin (POMC) neurons mediates the chronic effects of leptin on blood pressure (BP), the role of SOCS3 in modulating BP in obesity is still unclear. In this study, we investigated the role of SOCS3 specifically in POMC neurons in regulating body weight, glucose handling and BP in mice fed a normal or high fat (45%, HFD) chow. Male and female SOCS3flox/flox-POMC/cre mice in which SOCS3 was selectively deleted in POMC neurons and control SOCS3flox/flox mice were used. Food intake and body weight were measured from 8 to 16 weeks of age, and a glucose tolerance test (GTT) was performed at 20 wks of age. At 22 wks of age, mice were implanted with telemetry probe to measure BP and heart rate (HR) and fed a HFD for 6 weeks. Compared to control mice, both male and female SOCS3flox/flox-POMC/cre mice were lighter at 16 wks (29.1 ± 3.5 vs 31.9 ± 3.6 g in male and 21.5 ± 2.2 vs 26.1 ± 5.7 in female, n=9-11, p<0.05) but food intake was similar in both groups. Only male SOCS3flox/flox-POMC/cre mice exhibited improved glucose handling (AUC: 1059 ± 52 vs 1283 ± 54 mg/dL x 120 min, n=7-10, p<0.05 ) and no differences were observed in female mice. When fed normal chow, BP was similar in SOCS3flox/flox-POMC/cre and control mice (116 ± 7 vs 113 ± 5 mmHg) at 23 wks. After a HFD for 6 weeks, SOCS3flox/flox-POMC/cre mice had a greater BP increase compared to control mice (7.2 ± 1.9 vs 0.9 ± 1.8 mmHg, n=4-9, P<0.05) but no significant differences were observed in food intake or body weight between two groups. These results suggest that SOCS3 deletion specifically in POMC neurons reduced body weight in male and female mice, and improved glucose handling only in male mice. HFD increased BP in SOCS3flox/flox-POMC/cre but not in control mice, suggesting that SOCS3 in POMC neurons may modulate BP response to HFD.
Abstract P099: Angiotensin Type I Receptor (AT1R) and (Pro)renin Receptor (PRR) Functional Heterodimer Mediates ERK Phosphorylation
Authors: Quadri Syed Siraj Ahmed , Li Caixia , Culver Silas , Siragy Helmy M
Published: July 2021

Receptor dimerization was shown to enhance the receptor efficacy, trafficking and signal transduction. Angiotensin II type 1 receptor (AT1R) and (Pro)renin receptor (PRR) are expressed in the kidney. Studies demonstrated that these two receptors have some similarities in their signaling mechanisms and effects. In the present study we hypothesized that the AT1R and PRR form functional heterodimer to enhance cellular ERK phosphorylation. Using Immunoprecipitation technique, confocal immunofluorescence staining (IF), and fluorescence resonance energy transfer (FRET), we evaluated the presence of AT1R and PRR heterodimer in rat renal mesangial (RMC) and PC12W cells transfected with AT1R. These two receptors coimmunoprecipitated at 80kD band. IF demonstrated AT1R and PRR co-localization as shown in figure below and FRET demonstrated the physical distance between them to be less than 10nm (<100 Å), providing strong evidence for these receptors heterodimerization. To evaluate the function of the AT1R/PRR dimer, PC12W cells were treated with scramble siRNA or PRR siRNA . There were no significant differences in phosphorylation of ERK between control and scramble siRNA. PRR siRNA treatment significantly reduced the ERK phosphorylation by 35%, P<0.05. There was 60% (P<0.01) increase in ERK phosphorylation in PC12W cells transfected with AT1R. In these cells, PRR siRNA treatment attenuated the increase in ERK phosphorylation by 33% (P< 0.05). We conclude that AT1R-PRR form a heterodimer that is functionally active to enhance ERK phosphorylation.