bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 338: Kidney-specific SPAK Inhibits Activity of the Na-K-2Cl Cotransporter
Authors: McCormick James A
Published: March 2022

The WNK-SPAK/OSR1 pathway has been shown to play a critical role in the regulation of ion homeostasis, and the maintenance of extracellular fluid volume and blood pressure. SPAK and OSR1, members of the MAP kinase family, phosphorylate and activate the cation chloride cotransporters NKCC1, NKCC2 and NCC. SPAK and OSR1 are themselves phosphorylated and activated by members of the WNK kinase family. We recently identified a kidney-specific (KS-) SPAK isoform (KS-SPAK) which inhibits phosphorylation of the N-terminus of NKCC2 by SPAK or OSR1
in vitro
. Since changes in NKCC2 phosphorylation status have been reported to incompletely correlate with changes in NKCC2 activity, we tested the ability of KS-SPAK to inhibit the activity of NKCC2 in
Xenopus
oocytes. First,
86
Rubidium (
86
Rb, equivalent to potassium) uptake experiments were performed under hypertonic (300mOsM) conditions, to maximally activate endogenous SPAK and OSR1 in oocytes. KS-SPAK decreased
86
Rb uptake to 38% of that of oocytes injected with NKCC2 alone, and this inhibition required the conserved carboxy-terminus of SPAK, which interacts with cation cotransporters and WNK kinases. We next tested the ability of KS-SPAK to inhibit the activity of endogenous NKCC1, and observed a 67% reduction in
86
Rb uptake compared with water-injected oocytes, similar to the 73% reduction observed with the NKCC1/NKCC2 inhibitor bumetanide. This inhibition likely involves effects on endogenous SPAK or OSR1, since under hypotonic (160mOSM) conditions, when SPAK is relatively inactive, KS-SPAK did not inhibit endogenous NKCC1 activity, whereas bumetanide still inhibited
86
Rb uptake by water-injected oocytes by 50%. Co-injection of KS-SPAK with SPAK only reversed KS-SPAK inhibition of endogenous NKCC2 activity by 50%, despite a co-injection ratio of SPAK:KS-SPAK of 15:1, suggesting the mechanism of KS-SPAK inhibition may not be simple competition for NKCC2 binding. Another SPAK isoform, SPAK2, which has been proposed to be kinase-inactive, did not inhibit activity of co-injected NKCC2 or endogenous NKCC1 at any tonicity. Together, these data show that KS-SPAK is a potent inhibitor of both NKCC1 and NKCC2 activity, whereas the other putative inhibitory isoform SPAK2 had no effect on their activity.
Abstract 34: Deletion of Nos1 In The Macula Densa Induces Salt-sensitive Hypertension
Authors: Lu Yan , Stec David , Cheng Liang , Ge Ying , Fu Yiling , Wei Jin , Pollock Jennifer S. , Juncos Luis A. , Liu Ruisheng
Published: March 2022

Nitric oxide (NO) released from NO synthase 1 (NOS1) which is highly expressed in the macula densa (MD), blunts tubuloglomerular feedback (TGF) response and dilates the afferent arteriole. However, the role of MD NOS1 and TGF in regulation of chronic salt-water balance and blood pressure is unknown. We developed a strain of tissue-specific NOS1 knockout mice to examine the significance of NOS1 in the MD. We first developed a strain of MD Cre mice using the sodium, potassium, 2 chloride cotransporter (NKCC2) promoter. The NKCC2 promoter was cloned into the plasmid pBS594 to create the pNKCC2-Cre transgene. To characterize this new Cre strain, we crossed the NKCC2-Cre mice with an EGFP reporter strain (Rosa). Cre activity measured by EGFP fluorescence was found in MD and was further confirmed via immunofluorescent staining in isolated perfused MD and found to be limited to the MD and not in the distal tubule, indicating a valid NKCC2-Cre strain. Next, we generated tissue specific NOS1 knockout mice by crossing NKCC2-Cre mice with NOS1
flox/flox
mice, which target the exon-6 of NOS1, therefore deletes all splice variants of NOS1. We confirmed NOS1 KO by immunohistochemistry and found no staining of NOS1 in the MD in KO mice, while NOS1 was abundant in the MD of WT mice. To measure NO generation in the MD, we perfused isolated MD and loaded with fluorescent dye DAF-2. When we switch tubular NaCl from 10 to 80 mM, NO generation in the MD was from 114 ± 12 to 165 ± 11 units/min in WT mice, while in KO mice NO were absent at both 10 and 80 mM NaCl. (n=5; p<0.01) To measure TGF response, we measured maximal change of stop flow pressure using micropuncture. In WT mice, TGF was 4.5 ± 0.3 mmHg; while in KO mice, TGF was 8.4 ± 0.7 mmHg. (p<0.05) To determine the effect of MD NOS1 on the blood pressure response to salt intake , we measured mean arterial pressure (MAP) by telemetry in mice fed a low salt diet (0.01% NaCl) for 10 days, followed by a high salt diet (8%NaCl) for 14 days. MAP of KO mice increased by 10.5 ± 2.4; while WT mice increased 3.1± 2.7 mmHg (p <0.01, n=5). These results indicate that NOS1 in the MD and TGF play important roles in the regulation of blood pressure in response to changes in sodium intake.
Abstract 357: Targeting Vascular Endothelium with HO-1 Gene Promotes Release of Positive Regulators of Adipocyte Function
Authors: Sodhi Komal , Rezzani Rita , Peterson Stephen J , Kim Dong Hyun , Kappas Attalah , Abraham Nader G
Published: March 2022

Vascular endothelium is a secretory organ which not only regulates vascular function but also secretes bioactive compounds to modulate tissue homeostasis. We aimed to characterize the physiological effects of up regulating endothelial heme oxygenase-1 (HO-1) on adipogenesis in mesenchymal stem cells (hMSCs). Human microvessel endothelial cells (hMEC-1) were cultured along with a lentiviral construct expressing human HO-1 under the control of endothelium specific promoter VE-cadherin (VECAD-HO1) with VECAD-GFP used as a negative control. In complementary experiments hMEC-1 cells were cultured in the presence or absence of siRNA-HO-1 with scrambled siRNA as a negative control. Three days post-transfection, the conditioned media (CM) from hMEC-1 cells was used, along with adipogenic growth factors, to promote adipogenesis in hMSCs. Supporting in vivo experiments were conducted in SD rats injected with either VECAD-HO-1 or VECAD-GFP construct via the tail vein. Transduction efficiency was confirmed in hMEC-1 cells by demonstrating over expression (p<0.05) of HO-1 or GFP in respectively treated cells. hMSCs exposed to CM from VECAD-HO-1 transfected cells demonstrated reduced (p<0.05) adipogenesis (0.16± 0.008 vs 0.12±0.006 ) as compared to hMSCs exposed to CM from VECAD-GFP cells. On the contrary, CM from hMEC-1 cells treated with siRNA for HO-1 promoted higher adipogenesis (p<0.05) with increased adipocyte hypertrophy as opposed to hMSCs treated with CM from hMEC-1 cells transfected with the control siRNA (p<0.05) (0.17±0.008 vs 0.14±0.04). These effects on hMSCs were corroborated by immunoblot analysis demonstrating reduced expression (p<0.05) of positive regulators of adipogenesis, PPARγ, and increased levels (p<0.05) of negative regulators, β-catenin, Wnt 5b and hedgehog in cells treated with CM from hMEC-1 transfected with VECAD-HO1. In SD rats transfected with VECAD-HO-1, plasma adiponectin levels were enhanced (p<0.05) along with increased (p<0.05) adipose tissue levels of pAKT and pAMPK. In conclusion, we show here that a crosstalk between the vascular endothelium and its microenvironment has the potential to regulate adipogenesis and improve adipocyte function via HO-1-depndent pathways.
Abstract 360: Genetic Variants Associated With ACE Inhibitor-Associated Angioedema
Authors: Pare Guillaume , Kubo Michiaki , Byrd James B , McCarty Catherine A , Woodard-Grice Alencia , Zurich Rebecca L , Bradford Yuki , Ross Stephanie , Ritchie Marylyn , Brown Nancy J
Published: March 2022

We conducted a genomewide association study in 179 individuals with ACE inhibitor-associated angioedema and 489 ACE inhibitor-exposed controls from Nashville, TN and Marshfield, WI. We performed a replication study in 19 cases and 57 age-, sex-, and ancestry-matched controls from the Ongoing Telmisartan Alone and in Combination with Ramipril Global Endpoint Trial (ONTARGET). There were no genomewide significant associations (p<5x10
-8
) of any SNP with angioedema in either African or European Americans. Sixteen SNPs in African Americans and 41 SNPs in European Americans that were moderately associated with angioedema (p<10
-4
) were evaluated for replication in ONTARGET. The T allele of rs500766, a SNP in the gene encoding for protein kinase C θ, was associated with a reduced risk of angioedema in both the Nashville/Marshfield sample (OR 0.42, 95% CI 0.28-0.63, p=2.97x10
-5
in the additive model and OR 0.42, 95% CI 0.26-0.67, p=3.04x10
-4
in the dominant model) and in ONTARGET (OR 0.28, 95% CI 0.09-0.89, p=0.030 in the dominant model). The G allele of rs2724635, a SNP in ETS variant gene 6, was associated with increased risk of ACE inhibitor-associated angioedema in both the Nashville/Marshfield sample (OR 2.78, 95%CI 1.67-4.00, p=2.73x10
-5
in the additive model; OR 3.23, 95%CI 1.75-6.25, p=2.11x10
-4
dominant model; OR 5.56, 95%CI 1.85-16.6, p=2.01x10
-3
recessive mode) and in the ONTARGET sample (OR 3.27, 95%CI 1.03-10.35, p=0.044 in the recessive model). We also conducted a candidate-gene study, using the smaller ONTARGET sample for discovery, and the Nashville/Marshfield study for replication. In the candidate gene study, rs989692 in the gene encoding for neprilysin (
MME
), was significantly associated with angioedema in ONTARGET and in African Americans in the Nashville/Marshfield sample. Variants in genes involved in immune regulation and in neprilysin, an enzyme that degrades bradykinin and substance P, were associated with ACE inhibitor-associated angioedema.
Abstract 363: Urinary Free Cortisol Variations in Essential Hypertension are Associated with Altered Glucocorticoid Receptor Binding in ex vivo Human Neutrophils
Authors: Chamarthi Bindu , Pulido Patricia , Nieva Adriana , Prado Gregory N , Maldonado Enrique R , Torres-Grajales Manuel , Williams Gordon H , Rivera Alicia , Romero Jose R
Published: March 2022

The role of cortisol in essential hypertension (HTN) is yet unclear. Urinary free cortisol (UFC) levels have been shown to be bimodally distributed among hypertensives with about 30% of individuals with HTN showing high levels of UFC (high mode) and significant familial aggregation of this trait. These data suggest that UFC is a genetically determined intermediate phenotype of HTN. The mechanisms underlying this UFC variation in HTN are unknown. Prior studies have shown that despite higher UFC, the high mode group has less sodium sensitivity of blood pressure and no clinical manifestations of high cortisol, suggesting an element of glucocorticoid resistance. We evaluated the glucocorticoid receptor (GR) binding characteristics in neutrophils obtained from hypertensives with high mode versus normal/low mode UFC by immunomagnetic isolation of untouched circulating human blood cells following density gradient sedimentation with Polymorphprep. Flow cytometric analyses showed >97% of these cells were positive for CD45, CD16 and CD66B. These results were consistent with manual and automated white blood cell differential counts. Our isolation procedure produced >96% live cells as estimated by Live/Dead fluorescent viability assays. Dexamethasone (DEXA), a synthetic cortisol that binds and activates GR, showed increases in mitochondrial membrane potential but no effect on superoxide production or degranulation in these cells. In contrast, increases in superoxide production and degranulation (
P
<0.02 vs vehicle; n=6) were clearly observed when
ex vivo
cells were incubated with either 100nM fMLP or 10nM PMA, two well-described neutrophil activators. We determined DEXA binding parameters in neutrophils isolated from hypertensives with high mode and low mode UFC using
3
H-DEXA. Both the K
d
(85±16 vs 199±51 nM, n=5,
P
<0.030) and the B
max
(20.3±5 vs 61.2±20 fmol/10
6
cells, n=5,
P
<0.040) for DEXA were lower in cells from high mode subjects compared to low mode. We confirmed binding to GR as
3
H-DEXA was displaced by >90% with RU-486, a GR antagonist. Thus our results suggest that hypertensives with high mode UFC have altered GR binding, which may drive the higher UFC levels
in vivo
and may have important clinical implications that warrant further investigation.
Abstract 377: Expression of Soluble and Full-length (pro)renin Receptor by Dehydration
Authors: Tamura Yuma , Mori Nobuyoshi , Ito Osamu , Rong Rong , Xu Bin , Kim Jong Woo , Nakamura Takahiro , Hirose Takuo , Takahashi Kazuhiro , Totsune Kazuhito , Kohzuki Masahiro
Published: March 2022

Recently, (pro)renin receptor ((P)RR), a specific receptor for renin and prorenin, was newly identified as a member of the renin angiotensin system (RAS). (P)RR binding to the prorenin, causes the generation of angiotensin II via the increased enzymatic activity. (P)RR exists in two forms, one is full-length form (f(P)RR), and the other is soluble form (s(P)RR). RAS as well as vasopressin system contributes to the regulation of body fluid balance in a state of dehydration, but roles of (P)RR are unidentified. The purpose of this study was to examine the effects of dehydration on (P)RR expression in the plasma, the urine and kidney. Twelve week-old, male Wister Kyoto rats (n=10) were randomly divided into two groups, and the dehydration was induced by water restriction for 72 h in metabolic cage. Food intake and urinary output were measured. After 72h, rats were killed, and the protein expression of (P)RR in the plasma, the urine and kidney was investigated by using Western blotting. Compared with control rats, dehydration significantly increased osmotic pressure in the plasma and urine (286.8±2.9 vs 305.6±16.8 mOsm/L, p<0.05, 409.0±450.6 vs 1566.0±381.5 mOsm/L, p<0.01, respectively). Similarly, dehydration significantly increased in plasma renin activity and angiotensin II(8.5±3.8 vs 13.5±2.8 ng/ml/h, 409.6±175.6 vs 3595.6±1086.9 pg/ml, p<0.01, respectively). Dehydration decreased the s(P)RR expression in the plasma, but increased in the urine (0.3-fold, 1.5-fold respectively) by direct Western blotting. Dehydration increased the f(P)RR expression levels in the renal homogenates, microsome fraction, and cytosol fraction (2.3-fold, 3.0-fold, 22.9-fold respectively). Dehydration tended to decrease s(P)RR in renal cytosol, but it had no significant difference. These results indicated that dehydration increased the f(P)RR expression in the kidney with the downregulation of s(P)RR levels in the plasma, suggesting a specific stimulation of renal RAS by (P)RR.
Abstract 389: Angiotensin-(1-7) Modulates Renal Vascular Resistance Through Inhibition of Mitogen- Activated Protein Kinase (MAPK) P38 in Apolipoproteine (apoE) Deficient Mice
Authors: Potthoff Sebastian A , Sivritas Sema , Stamer Stefanie , Mori Yuri , Clasen Tilman , Fäing Michael , Patzak Andreas , Rump Lars C , Stegbauer Johannes
Published: March 2022

ApoE-KO mice are characterized by endothelial dysfunction and increased vasoconstrictor response. Previously, we have shown that chronic Ang-(1-7) treatment ameliorates endothelial dysfunction in apoE-KO. However, the impact of Ang-(1-7) on vasoconstrictor response to AngII is unknown. To examine the role of Ang-(1-7) in vascular resistance in atherosclerosis, apoE-KO and wild type (WT) mice fed on western diet were treated via osmotic minipumps either with Ang-(1-7) (82μg/kg/hr) or saline for 6 weeks. Vascular resistance was tested in isolated perfused kidneys. AngII induced renal pressor response was significantly increased in apoE-KO compared to WT mice. Remarkably, chronic Ang-(1-7) treatment attenuated AngII induced pressor response in apoE-KO mice. Accordingly, in apoE-KO, phospho-MLC20, a marker of enhanced contractility was increased in preglomerular vessels compared to WT and reduced after Ang-(1-7) treatment. To examine the underlying cause, we measured reactive oxygen species (ROS) production and changes in NADP(H) expression levels. In apoE-KO mice, p47phox was increased (1.5-fold;
P
<0.05) compared to WT and reduced after chronic Ang-(1-7) treatment. Concordantly, urinary isoprostane-8 excretion and H2O2 generation were significantly decreased in Ang-(1-7) treated apoE-KO mice. ROS is known to activate the MAPK p38. Moreover, p38 has shown to modulate pressor response. To test the role of p38 in our model, we measured p38 activity and renal pressor response to AngII in the presence of SB203580, a specific p38 inhibitor. Thus, in preglomerular vessels of apoE-KO, the phospho-p38 / p38 ratio was increased compared to WT. Chronic Ang-(1-7) treatment restored this ratio almost to WT levels (2.8±0.5 vs. 1.0±0.1 vs. 1.3±0.2
P
<0.05). Administration of SB203580 (5μmol/L) reduced renal pressor response to AngII in apoE-KO but not in Ang-(1-7) treated mice. Hence, chronic inhibition of p38 with BIRB796 (30mg/kg) attenuated the increased pressor response to AngII in apoE-KO mice. In summary, p38 plays a crucial role in regulating renal vascular resistance in apoE-KO mice. Moreover, Ang-(1-7) attenuates pressor response to AngII by reducing p38 activity. Thus, this study revealed a new pathway how Ang-(1-7) modulates vascular function.
Abstract 393: Chymase: Augmented Renal Tubular Protein Expression in Diabetes
Authors: Harrison-Bernard Lisa M , Berner Paul M , Zhang Xun , Husain Ahsan , Naqvi Nawazish
Published: March 2022

Chymase is a serine protease that is predominantly found stored in granules of mast cells. Chymase expression is increased in mesangial cells, podocytes, vascular smooth muscle cells, and cardiomyocytes under hyperglycemic culture conditions. We have presented evidence for enhanced intrarenal chymase-dependent Ang II formation on afferent arteriole vasoconstriction in the diabetic kidney (
Hypertension.
56: e55, 2010). We tested the hypothesis that renal chymase protein expression is enhanced in the diabetic kidney. Kidneys were harvested from littermate control (
db/m
) and type II diabetic (
db/db
) mice at 36-wk-old. Body weight was significantly lower (33 ± 3 vs 40 ± 2 g), while plasma glucose was significantly higher (552 ± 40 vs 220 ± 21 mg/dl) in diabetic (n=6) compared to control (n=7) mice. However, plasma insulin levels were not different from controls (0.77 ± 0.10 vs 0.43 ± 0.11 mg/L), indicating that the diabetic mice are transitioning from type II to type I diabetic disease. Immunohistochemistry was performed using primary rabbit polyclonal antibodies [mMCP-4 (chymase, A Husain), anti-malignant T cell amplified sequence 1 (chymase, Abcam), H
+
-ATPase (D Brown), and aquaporin 2 (AQP2, J Sands)]. Renal subcapsular and adipose tissue mast cell granules stained positively for toluidine blue and chymase. Chymase protein expression was localized to the apical membrane of connecting tubules and cortical collecting ducts of both control and diabetic kidneys. Chymase was co-localized to AQP2 positive and H
+
-ATPase negative cells indicating that chymase is specifically expressed in the principal cells of the distal nephron. Prominent chymase immunostaining was observed in the renal papilla and endothelial cells of renal arteries and arterioles in all diabetic, but none of the control kidneys. Chymase was not localized to renal vascular smooth muscle cells. The renal localization of chymase was identical using custom and commercially available polyclonal antibodies. These are the first studies to identify chymase protein expression in the principal cells of the distal nephron. These novel data suggest that the serine protease actions of chymase may be linked to altered epithelial transport mechanisms in the distal nephron in diabetic kidney disease.
Abstract 395: Regulation of G Protein-coupled Receptor Kinase 4 on AT1 Receptor Expression and Function in Artery
Authors: Zeng Chunyu , Liu Li , Cheng Ken , Chen Caiyu , Jose Pedro A
Published: March 2022

G protein-coupled receptor kinases (GRKs) constitute a family of seven mammalian serine/threonine protein kinases that phosphorylate/modify agonist-bound or activated G protein-coupled receptors (GPCRs) as their primary substrates. GRK4 is distinguished from other members of the GPCR family by its constitutive activity and limited tissue expression. Our previous study found that GRK4 variants (A142V, A486V and R65L) increases GRK4 activity; A142V transgenic mice develop hypertension with impaired sodium excretion by impairment of renal D
1
dopamine receptor and augmented renal AT
1
receptor expression and function. Whether there is GRK4 in the artery is not known. Our preliminary study found GRK4 expression in the artery, determined by immunoblotting and histochemistry. We hypothesize that GRK4 might also regulate AT
1
receptor expression and function in vascular smooth muscle cells (VSMCs). A10 cells, an embryonic thoracic aortic smooth muscle cell lines, were transfected with A142V in this study. It resulted A142V increased AT
1
receptor protein and mRNA expressions in A10 cells, meanwhile, decreased AT
1
receptor phosphorylation. The regulation of A142V on AT
1
receptor is physiological significance, because the angiotensin II-mediated VSMC proliferation was greater in the A142V transfected cells than in the control cells. Moreover, the vasoconstrictive effect of angiotensin II was greater in A142 transgenic mice than control mice. The above-mentioned phenomenon is confirmed by siRNA, GRK4 siRNA decreased AT
1
receptor expression, increased AT
1
receptor phosphorylation in primary cultured VSMCs from spontaneously hypertensive rats. Our further study found that there was co-localization between GRK4 and AT
1
receptor in A10 cells, transfected with A142V increased the linkage of GRK4/AT
1
receptor. It indicated that arterial GRK4 regulation of AT
1
receptor expression and function, which might be engaged in the pathogenesis of hypertension.
Abstract 398: Angiotensin-[1-7] Infusion Reduces Skeletal Muscle Fibrosis and Restores Locomotor Activity and Sympathovagal Balance in a Mouse Model of Muscular Dystrophy
Authors: Sabharwal Rasna , Chapleau Mark W
Published: March 2022

Muscular dystrophy is a catastrophic, fatal neuromuscular disease in need of new therapies. Angiotensin II binding to AT
1
receptors (AT
1
R) contributes to fibrosis in dystrophic skeletal muscle and AT
1
R blockers are currently being tested in clinical trials. Recently, we reported that autonomic dysregulation precedes and predicts development of cardiomyopathy in sarcoglycan delta deficient (Sgcd-/-) mice with muscular dystrophy
(Clin Auton Res, 2010
). We hypothesized that infusion of the angiotensin peptide Ang-[1-7] will rescue skeletal muscle, locomotor, and autonomic nervous system phenotypes in young Sgcd-/- mice. Control and Sgcd-/- mice were infused with Ang-[1-7] (300 ng/kg/min) for 8 wks beginning at 3 wks of age. Blood pressure (BP), heart rate (HR) and activity were recorded by telemetry in treated and untreated mice. Baroreflex sensitivity (BRS, sequence technique) and resting cardiac vagal and sympathetic tone (HR responses to atropine and propranolol) were measured. BP, activity, BRS and vagal tone were lower in Sgcd-/-
vs.
control mice, whereas sympathetic tone was higher (Table). Ang-[1-7] normalized activity, BRS, and sympathovagal balance in Sgcd-/- mice without affecting BP, and did not influence any variable in control mice. Skeletal muscle fibrosis present in Sgcd-/- mice (18±1%, n=9) was markedly reduced by Ang-[1-7] (3±1%, n=5). We conclude that Ang-[1-7] reduces skeletal muscle fibrosis and restores locomotor activity and sympathovagal balance in Sgcd-/- mice, without lowering BP. Ang-[1-7] and/or enhancement of its endogenous production may provide a novel therapeutic approach to muscular dystrophy.
Abstract 410: Hyptertension and Baroreflex Dysfunction After Loss of Glia in Rat NTS
Authors: Talman William T , Jones Susan , Nitschke Dragon Deidre , Lin Li-Hsien
Published: March 2022

Injection into the rat NTS of anti-dopamine-beta-hydroxylase-saporin (anti-DBH-SAP), which causes local loss of neuronal DBH immunoreactivity (IR), or of stabilized substance P- SAP (SSP-SAP), which causes local loss of neuronal neurokinin-1 receptor (NK1R) IR, leads to lability of arterial pressure and diminished arterial baroreflex function. We have found that anti-DBH-SAP and SSP-SAP also lead to loss of IR for glial fibrillary acidic protein (GFAP) in the NTS. To further characterize cellular changes produced by introducing into the NTS conjugates containing SAP, we sought to compare and contrast cardiovascular effects of anti-DBH-SAP, SSP-SAP, SAP (unconjugated), blank-SAP (non-targeted peptide conjugate), 6-hydoxydopamine (6-OHDA, a neurotoxin without SAP) and PBS injected into NTS. We assessed effects of injected agents both on cellular markers [NMDAR1 (NMDA receptor subunit 1), GluR2 (AMPA receptor subunit 2), neuronal nitric oxide synthase (nNOS), tyrosine hydroxylase (TH), vesicular glutamate transporters (VGluTs), choline acetyl transferase (ChAT), GFAP, connexin 43 (Cx43), DBH, NK1R and protein gene product 9.5 (PGP 9.5)] and on arterial pressure and baroreflex function. We found that each compound containing SAP (including blank-SAP and unconjugated SAP) led to loss of GFAP and Cx43 immunofluorescent labeling in the NTS as well as lability of arterial pressure. Those outcomes occurred despite neuronal specificity for each of the SAP conjugates. For example, anti-DBH-SAP decreased TH/ DBH IR but not NK1R IR in NTS while SSP-SAP led to loss of NK1R IR as well as glutamate receptors but not DBH IR. SSP-SAP also caused PGP9.5 loss, not seen with other agents. SAP and blank-SAP, on the other hand, led to loss of GFAP and Cx43 but no loss of neuronal markers. Injection of SAP alone in NTS led to significantly attenuated baroreflex responses and to significant hypertension. In contrast, 6-OHDA led to loss of TH and DBH IR and decreased Cx-43, but it increased GFAP. No baroreflex or cardiovascular effects were seen after injection of 6-OHDA, which spared glia. This study supports a critical role for NTS glia in central cardiovascular control and in maintenance of normal blood pressure.
Abstract 417: Central PPAR-γ Activation Attenuates Angiotensin II-Induced Hypertension by Inhibiting Brain Inflammation and Renin-Angiotensin System Activity in Rats.
Authors: Yu Yang , Zhang Zhi-Hua , Wei Shun-Guang , Xue Bao-Jian , Beltz Terry G , Guo Fang , Johnson Alan K , Felder Robert B
Published: March 2022

Upregulation of inflammation and renin-angiotensin system (RAS) activity in the brain contributes to hypertension through effects on fluid intake, vasopressin release, and sympathetic nerve activity. Peroxisome proliferator-activated receptor (PPAR)-γ, a nuclear transcription factor, is expressed in key brain areas involved in drinking, vasopressin release and cardiovascular regulation, including the paraventricular nucleus of hypothalamus (PVN). We recently reported that activation of brain PPAR-γ reduced inflammation and RAS activity in the PVN and ameliorated the peripheral manifestations of heart failure. Here we hypothesized that activation of central PPAR-γ might have beneficial effects on angiotensin II (ANG II)-induced hypertension. Spague-Dawley rats received a continuous 2 week subcutaneous infusion of ANG II (120 ng/kg/min), combined with intracerebroventricular infusion of vehicle (VEH) or the PPAR-γ agonist pioglitazone (3 nmoL/h). At 2 weeks, blood pressure (BP) had significantly (*P<0.05) increased (131±3* vs 98±3 mmHg) and the BP response to ganglionic blockade (70±4* vs 43±3 mmHg) was greater in conscious VEH-treated ANG II rats compared with normal (control) rats. Water intake (34±2* vs 25±2 mL) and plasma vasopressin following 4 hours of water restriction (20.79±2.62* vs 10.62±1.27 pg/mL) were increased. PPAR-γ mRNA in the PVN was unchanged in VEH-treated ANG II rats, compared with control rats, but PPAR-γ DNA binding activity (0.05±0.01* vs 0.09±0.01 OD
450nm
) was reduced and mRNA for interleukin-1β (2.76±0.22* vs 1.09±0.21 fold change), tumor necrosis factor-α (1.58±0.09* vs 1.04±0.12 fold change) and ANG II type-1 receptor (1.71±0.15* vs 1.03±0.10 fold change) was augmented. All of these findings were ameliorated (by 24-36%*) in ANG II rats treated with pioglitazone, which increased PPAR-γ mRNA and PPAR-γ DNA binding activity in the PVN (by 73-77%*). The results suggest that activating brain PPAR-γ to reduce central inflammation and RAS activity, thereby suppressing fluid intake, vasopressin release and sympathetic drive, may be a novel approach to the treatment of ANG II-dependent hypertension.
Abstract 419: Central Angiotensin II Increases Circulating Endogenous Ouabain, Arterial Myocyte Na/Ca Exchanger Expression and Blood Pressure via Brain Aldosterone and Mineralocorticoid Receptors
Authors: Hamlyn John M , Leenen Frans H , Golovina Vera A , Linde Cristina I , Gao Junjie , Huang Bing , Ahmad Monir , Chen Ling , Li Meng , Blaustein Mordecai P
Published: March 2022

The brain, together with altered arterial function, plays a crucial role in the pathogenesis of hypertension. We have suggested that endogenous ouabain (EO), and its actions on arterial Na
+
and Ca
2+
transporters, are key in some forms of hypertension; these entities may be controlled by brain mechanisms (review: Blaustein et al.,
Am J Physiol Heart Circ Physiol
302:
H1031-49, 2012). One commonly-used hypertension model involves infusion of angiotensin II (Ang II) in rodents. When Ang II was infused (subcutaneous minipump, 400 ng/kg/min x 15 days) into 8 normal ∼35 g C57Bl/6 mice, 24 hr mean blood pressure (MBP in mm Hg, telemetry), was 119±4, vs 97±2 in 8 vehicle-infused controls,
C
(
P
<0.001). Expression of Na/Ca exchanger-1 (NCX1, immunoblots with R3F1 antibody) was increased by 171±21% (
P
<0.001 vs
C
) in the aortas of the Ang II-treated mice. To test the role of the brain in Ang II-induced hypertension, vehicle (
C
;
n
= 7) or Ang II (
A
;
n
= 8) was administered by intra-cerebroventricular infusion (Ang II, 3.6 μg/day x 12 days) in 200-250 g Wistar rats. In some rats, the Ang II infusion contained the mineralocorticoid receptor (MR) blocker, eplerenone (
E
, 5 μg/day x 12 days;
n
= 7), or the aldosterone synthase inhibitor, FAD-286 (
F
, 25 μg/day x 12 days;
n
= 8). At the end of the treatment, the following MBPs were recorded (mm Hg, telemetry):
C
: 101±2;
A
: 123±4 (
P
<0.05 vs
C, E
&
F
);
E
: 108±2;
F
: 112±2 (
P
<0.05 vs
C
). Upon euthanasia, blood, pituitary and adrenal samples were taken for EO radioimmunoassay (RIA), and aortas were harvested for NCX1 immunoblotting. The following results were obtained: 1) Plasma EO (nM):
C
, 0.34±0.07;
A
, 0.58±0.08 (
P
<0.05 vs
C
;
P
<0.01 vs
F
);
E
, 0.37±0.08;
F
, 0.30±0.05. The plasma EO increase in
A
was confirmed by off-line liquid chromatography-RIA on pooled samples. 2) NCX1 expression (normalized to
C
= 1.0±0.2):
A
, 2.6±0.3 (
P
<0.01 vs
C
;
P
<0.05 vs
E
);
E
, 0.9±0.3;
F
, 1.3±0.2. 3) Adrenal and pituitary EO were not changed. These results provide the first evidence that circulating EO levels and arterial NCX expression are regulated by the brain Ang II-aldosterone-MR system. Thus, in addition to regulating BP by direct, neurally-mediated vasoconstriction, the brain also influences BP by modulating ion transport pathways involved in arterial Na
+
and Ca
2+
homeostasis.
Abstract 420: Role of Ang II and Glutamate in the PVN of Rats with Ang II-induced Hypertension
Authors: Gabor Alexander , Leenen Frans H
Published: March 2022

A chronic increase in circulating Ang II increases neuronal activation in hypothalamic nuclei such as the paraventricular nucleus (PVN) and causes progressive hypertension. These effects can be largely prevented by central infusion of blockers of an aldosterone-ouabain neuromodulatory pathway that enhances the activity of angiotensinergic sympathoexcitatory pathways. A number of models of chronic sympathetic hyperactivity are associated with an increase in glutamate receptor and AT
1
-receptor activation in the PVN. The present study evaluated whether these mechanisms in the PVN also contribute to the elevated BP from a chronic increase in plasma Ang II by chronic subcutaneous (sc) infusion of Ang II for 2 weeks. One week following bilateral stereotaxic cannulation of the PVN, bilateral infusion (300 nL/min/side) of AT
1
and glutamate receptor blockers, candesartan and kynurenate were performed, and BP and HR were recorded in conscious Wistar rats. Data are mean±SE; (n=4-5/group). *p<0.05 vs. baseline

Kynurenate or candesartan in the PVN both fully reversed the increase in BP from sc infusion of Ang II for 2 weeks. Infusion of candesartan after kynurenate or kynurenate after candesartan did not further lower BP. These findings suggest that enhanced AT
1
-receptor and glutamate receptor activation in the PVN both contribute to the elevated BP in Wistar rats with chronically elevated plasma Ang II. The effects of increased AT
1
-receptor activation in the PVN appear to be mediated by local glutamate release.
Abstract 428: Complete Blockade of the Vasorelaxant Effects of Ang-(1-7) and Bradykinin in Murine Microvessels by Antagonists of the Receptor Mas
Authors: Peiró Concepción , Vallejo Susana , Gembardt Florian , Palacios Erika , Azcutia Veróa , Rodríez-Mañas Leocadio , Sánchez-Ferrer Carlos F. , Walther Thomas
Published: March 2022

The heptapeptide angiotensin-(1-7) is a biologically active metabolite of angiotensin II, the predominant peptide of the renin-angiotensin system. Recently, we have shown that the receptor Mas is associated with angiotensin-(1-7)-induced signalling and mediates, at least in part, the vasodilatory properties of angiotensin-(1-7). However, it remained controversial whether an additional receptor can account for angiotensin-(1-7)-induced vasorelaxation. Here, we used two different angiotensin-(1-7) antagonists, A779 or D-Pro-angiotensin-(1-7), to address this question. We also studied the influence of both antagonists on the vasodilatation induced by bradykinin. Isolated mesenteric microvessels from both wild-type and Mas deficient C57Bl/6 mice were pre-contracted with norepinephrine, and vascular reactivity to angiotensin-(1-7) and bradykinin was subsequently studied using a small vessel myograph. Both peptides, angiotensin-(1-7) and bradykinin, triggered a concentration-dependent vasodilation in wild-type microvessels, that was totally absent in the presence of the nitric oxide synthase inhibitor ω-nitro-L-arginine methyl ester (L-NAME). In these vessels, the pre-incubation with the Mas antagonists A779 or D-Pro-angiotensin-(1-7) totally abolished vasodilatatory capacity of both angiotensin-(1-7) and bradykinin, which was nitric oxide-mediated. Accordingly, Mas deficient microvessels lacked the capacity to relax in response to either angiotensin-(1-7) or bradykinin. In murine microvessels, the angiotensin-(1-7) antagonists A779 and D-Pro-angiotensin-(1-7) equally block a receptor, which mediates the complete angiotensin-(1-7)-induced vasodilation in mesenteric microvessels. Importantly, beyond mediating the vascular actions of angiotensin-(1-7), Mas appears to be also a critical player in NO-mediated vasodilation induced by renin-angiotensin system-independent agonists.
Abstract 43: A Functional Angiotensin Type 1a Receptor Is Required for Aldosterone to Induce Small Artery Endothelial Dysfunction, Inflammation and Oxidative Stress
Authors: Briet Marie , Mian Muhammad Oneeb Rehman , Barhoumi Tlili , Coffman Tom M , Paradis Pierre , Schiffrin Ernesto L
Published: March 2022

Background:
Aldosterone induces hypertension, endothelial dysfunction, vascular inflammation and oxidative stress. Recent
in vitro
studies suggest that there may be cross-talk between angiotensin II and aldosterone pathways. We hypothesized that
in vivo
vascular effects of aldosterone require a functional angiotensin II type 1a receptor (Agtr1a).

Design and methods:
Eight to 10-week old male
agtr1a
knockout (

agtr1a
-/-

) and wild-type mice were implanted with a dummy pump or infused with aldosterone (600 μg/kg/d, s.c.) for 14 days while receiving 1% saline in the drinking water (n=8-10). Systolic blood pressure (SBP) was measured by telemetry. Endothelial function, and reactive oxygen species (ROS) production were assessed in mesenteric arteries using pressurized myography and dihydroethidium staining, respectively. Levels of collagen by Sirius red staining and fibronectin, VCAM-1, and MCP-1 by immunofluorescence were determined in aortic sections.

Results:
Aldosterone-induced higher levels of SBP in

agtr1a
-/-

compared to wild-type mice (about 15 mmHg,
P
<0.01). Maximal vasodilatory responses to acetylcholine in mesenteric small arteries were similar in

agtr1a
-/-

and wild-type mice. Aldosterone reduced maximal vasodilatory responses to acetylcholine in wild-type mice by 48% (
P
<0.05) whereas there was no effect in

agtr1a
-/-

mice. Aldosterone increased ROS production 2-fold in mesenteric arteries of wild-type mice (
P
<0.05) whereas it had no effect in

agtr1a
-/-

mice. Aldosterone increased VCAM-1 (1.8-fold,
P
<0.05), MCP-1 (2.7-fold,
P
<0.05), fibronectin (3-fold,
P
<0.01) and collagen (3.9-fold,
P
<0.05) in aorta of wild-type but not in

agtr1a
-/-

mice.

Conclusion:
Aldosterone requires functional Agtr1a to induce small artery endothelial dysfunction and vascular oxidative stress, inflammation and fibrosis.
Abstract 436: Association Between Coffee Consumption and Hypertension in Asians
Authors: Finkelstein Joseph , Cha Eunme
Published: March 2022

Background:
A link between coffee consumption and hypertension had been investigated over many years. Studies had shown that coffee-drinking is associated with small immediate increase in blood pressure, however long-term association between coffee consumption and hypertension is not clear. Using the Korean National Health and Nutrition Examination Survey (KNHANES), we evaluated the association of coffee consumption and hypertension in Korean population on a population-based level.

Methods:
Caffeinated coffee is one of the popular beverages in South Korea whereas decaffeinated coffee consumption is limited. We used the 4th Korean National Health and Nutrition Examination Survey (KNHANES 2007-2009), a nationally representative sample of Korean adults. All adult population (>=18 y.o.) was divided into those who drink coffee 2-3 times per month or less (light consumption) and those who drink coffee at least once a week (regular consumption). Hypertension was identified by subject self-report. Hierarchical survey weights were employed to generate population-based prevalence. Odds ratio of having hypertension was calculated from a logistic regression model.

Results:
The final dataset included 14,853 participants from whom 79.6% drank coffee at least once a week. The following groups had higher coffee consumption: males (82.0%), age 46-65 years (81.6%), more than 12 years of education (82.2%), current smokers (87.7%), heavy drinkers (84.5%), and those with BMI ≥ 25 (84.2%). Hypertension prevalence in the regular coffee consumption group (15.3%) was significantly (p<0.001) lower than in the light consumption group (19.3%). After controlling for gender, age, education, current smoking status, physical activity, alcohol consumption and BMI, subjects who drank coffee at least once a week were 10% less likely to report that they have hypertension (OR 0.9 95% CI: 0.8-0.9; p=0.05).

Conclusions:
More frequent coffee drinkers have lower hypertension prevalence in Korea. This association remained significant after adjusting for socio-demographic and health behavior factors.
Abstract 437: Reproductive Timing Related Genetic Variants from GWAS are Associated with Blood Pressure in Healthy Adolescents
Authors: He Chunyan , Eckert George J , Wagner Erin K , Yu Zhangsheng , Pratt J. Howard , Tu Wanzhu
Published: March 2022

Blood pressure (BP) increases in an accelerated fashion during puberty. Previous studies have identified genetic variants that were associated with the timing of reproductive aging, namely menarche and menopause. But the effects of these genetic variations on BP have not been investigated. We examined the BP effects of 46 SNPs identified in genome-wide association studies (GWAS) that were linked to age of menarche or of menopause in women of European ancestry. The current analysis was based on longitudinal data collected from 601 adolescents (338 whites and 263 blacks). Subjects were enrolled between ages 4 and 17 and followed for an average of 6 years. After adjusting for multiple testing, we found 3 of the 16 menarche-related SNPs, rs10423674 in
CRTC1
, rs13187289 near
PHF15
and rs9635759 near
CA10
, were associated with a more rapid increase of BP in black males (all P< 0.0023); and two SNPs (rs17268785 in
CCDC85A
and rs7642134 near
VGLL3
) and one SNP (rs1079866 near
INHBA
) were associated with increased BP in black and white females, respectively (all P<0.0026). Of the 8 menarche and weight-related SNPs, rs7647305 near
ETV5
was associated with increased BP in whites (P=0.0028); and 3 of the 7 menarche and height-related NPs, rs10946808 near
HIST1H1D
, rs6440003 in
ZBTB38
and
rs757608
near
TBX2
, were positively associated with BP in white males, white females, and black males, respectively (all P<0.005). Except for one SNP, none of the 15 menopause-related SNPs were associated with BP. The one menopause-related SNP in gene
SYCP2L
, rs2153157, was associated with an increase of BP in whites (P= 0.0028). Most of the associations (9 of 11) were in the direction predicted by previously observed associations with menarche or menopause ages. Furthermore, these associations did not appear to be attenuated by weight and height. In conclusion, genetic variants from previous GWAS that were associated with a women’s reproductive aging showed significant associations with BP in healthy male and female adolescents. The genetic effects were independent of a subject’s sex, race or growth related factors such as weight, height, and BMI, and thus may represent novel candidates for BP genes.
Abstract 442: Pressure Overload Promotes HMGB1-Inflammasome Signaling in the Ischemic-Reperfused Heart.
Authors: Baban Babak , Grochowska Karolina , Masoumy Mohamad , Liu Jun Yao , Mozaffari Mahmood
Published: March 2022

Damage-associated molecular patterns (DAMPs) are released following tissue injury and cell death and can activate pro-inflammatory pathways. Prototypical DAMPs include the high-mobility group box 1 (HMGB1) protein which is released from necrotic cells. We have shown that pressure overload increases infarct size in association with poorer functional recovery of the heart subjected to an ischemia-reperfusion insult. In this study, we tested the hypothesis that pressure overload increases HMGB1 expression in association with increased pro-inflammatory changes and consequent cell death. Accordingly, hearts of male Sprague-Dawley rats were subjected to an ischemia (40 min) reperfusion (15 min) insult with perfusion pressure set at either 80 or 160 cmH
2
O. Thereafter, hearts were processed for flow cytometry-based, Western blotting and immunostaining studies. The ischemic-reperfused hearts subjected to the high pressure displayed greater expression of HMGB-1 in association with increased phosphorylation of c-jun-NH2-terminal kinase (JNK) and production of the inflammasome-associated cytokine, interleukin-1β. These changes were associated with greater disruption of mitochondrial membrane potential and increased apoptotic and necrotic cell death in hearts subjected to the high pressure. Collectively, the results suggest that pressure-related upregulation of HMGB1-inflammasome signaling contributes importantly to exacerbated cell death in the ischemic-reperfused heart.
Abstract 443: Peroxisome Proliferator Activated Receptor-α Gene Deletion Exacerbates the Progression of Hypertension and Inflammation in Chronic Angiotensin II-Hypertensive Mice
Authors: Lee Dexter L , Wilson Justin L , Duan Rong , Alhashim Abdulmohsin , Elmarakby Ahmed
Published: March 2022

The anti-inflammatory properties of peroxisome proliferator activated receptor- (PPAR-α) contribute to the incidence of hypertension and its associated cardiovascular disease. Fenofibrate, a PPAR-α agonist has been demonstrated to reduce angiotensin II (Ang II)-induced hypertension via inducing cytochrome P450 4A (CYP4A) and increasing 20-hydroxyeicosatetraenoic acid (20-HETE) production. The current study determines whether deletion of PPAR-α gene exacerbates the elevation in blood pressure, plasma interleukin-6 (IL-6) levels, and renal inflammatory markers during infusion of a slow-pressor dose of Ang II (400 ng/kg/min). Ten to twelve week old male PPAR-α knockout (KO) mice and their B129s wild-type (WT) controls were implanted with telemetry devices and infused with Ang II for 12 days. There was no difference in basal mean arterial pressure (MAP) between WT and PPAR-α KO mice compared to WT (113 ± 1 vs. 111 ± 2 mmHg). On day 14 of Ang II infusion, MAP was elevated in PPAR-α KO mice compared to WT (160 ± 3 vs. 142 ± 4 mmHg, P < 0.05) and fenofibrate administration significantly reduced the elevation in MAP in WT + Ang II mice (134 ± 7 mmHg, P < 0.05) with no significant effect on PPAR-α KO + Ang II mice (158 ± 8 mmHg). Plasma IL-6 levels were significantly higher in PPAR-α KO mice when compared to WT on day 12 of Ang II infusion (30 ± 4 vs. 8 ± 2 pg/ml, P < 0.05) and fenofibrate significantly reduced plasma IL-6 in Ang II-treated WT mice (10 ± 3 pg/ml, P < 0.05) with no significant effect in Ang II-treated PPAR-α KO mice (28 ± 4 pg/ml). Fenofibrate significantly increased renal expression of CYP4A and restored the decrease in renal CYP2J expression in WT + Ang II treated mice with no effect on PPAR-α KO + Ang II. Fenofibrate also significantly reduced the elevation in renal expression of the inflammatory markers intracellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1) and cyclooxygenase-2 (COX-2) in WT + Ang II mice. Our results demonstrate that PPAR-α KO mice have a greater presser response during Ang II hypertension and activation of PPAR-α attenuates Ang II-induced hypertension possibly via up-regulation of renal CYP4A and CYP2J and anti-inflammatory mechanisms involving reducing the inflammatory markers IL-6, MCP-1, ICAM-1 and COX-2.
Abstract 449: Develoment of Endothelin-1 Induced Aneurysms in High-Fat Diet-Fed Apolipoprotein E Knockout Mice Through a Mechanism Involving Oxidative Stress and Inflammatory Cell Infiltration
Authors: Li Melissa W , Paradis Pierre , Schiffrin Ernesto L
Published: March 2022

Background:
Endothelin (ET)-1 has been implicated in the pathogenesis of atherosclerosis. Plasma and tissue ET-1 are increased in human and animal with atherosclerosis. ET-1 overexpression exacerbates high-fat diet (HFD)-induced atherosclerosis in
apolipoprotein E
knockout (

apoE
-/-

) mouse. Abdominal aorta aneurysms (AAA) occur in association with atherosclerosis. We hypothesized that ET-1-induced ROS and inflammation would increase the occurrence of AAA in HFD fed

apoE
-/-

mice.

Design and methods:
Eight-week-old male transgenic mice overexpressing preproET-1 in the endothelium (eET-1),

apoE
-/-

, eET-1/

apoE
-/-

and wild type mice were fed a HFD for 8 weeks. Suprarenal aortic perimeter was determined using Oil Red O stained-sections. ROS production using dihydroethidium staining and monocyte/macrophage and T cell infiltration using immunofluorescence with MOMA-2 and anti-CD4 antibodies, respectively, were determined in perivascular fat and media in suprarenal aorta sections.

Results:
Aneurysms were observed at a suprarenal level in 6 of 15 eET-1/

apoE
-/-

compared to none of 15

apoE
-/-

(
P
<0.05). The aortic perimeter was increased 2.5-fold in eET-1/

apoE
-/-

with AAA compared to

apoE
-/-

(
P
<0.01). ROS production was increased 2.8- and 3.8-fold in perivascular fat and media of eET-1/

apoE
-/-

compared to

apoE
-/-

, respectively (
P
<0.05). Monocyte/macrophage infiltration was increased 2.6-fold in perivascular fat of eET-1/

apoE
-/-

compared to

apoE
-/-

(
P
<0.01). CD4
+
T cell infiltration was observed in perivascular fat and plaque of 6 eET-1/

apoE
-/-

compared to none of 6

apoE
-/-

, respectively (
P
<0.05).

Conclusions:
These results suggest that ET-1 plays an important role in development of AAA by increasing oxidative stress and monocyte/macrophage and T cell infiltration in the aorta.