bluuebunny/crossref_metadata_2025 huggingface dataset:
Homozygosity for an Alpha-Adducin Polymorphism (460TRP) Is Associated with Salt-Sensitivity and Low-Renin Hypertension in Humans
Authors: Grant Frederick D , Romero Jose R , Jeunemaitre Xavier , Hunt Steven C , Hopkins Paul N , Hollenberg Norman K , Williams Gordon H
Published: July 2021

P133

Single nucleotide polymorphisms of the adducin genes have been associated with the development of elevated blood pressure in the rat. However, previous studies of the 460Trp polymorphism of human alpha-adducin have not clearly identified an association with the development of human hypertension. In this study, 281 hypertensive subjects were subgrouped by the intermediate phenotypes of plasma renin status and sensitivity of systolic blood pressure to dietary sodium. The frequency of the 460Trp allele was 19% and 9 of 281 subjects (3.2%) were homozygous (TT) for the 460Trp allele. The systolic blood pressure response to changes in dietary sodium (>150 meq/d vs. ≤30 meq/d) was significantly (p < 0.05) greater in TT subjects (25 ± 2 mmHg) compared to those heterozygous (GT) for the 460Trp allele (12 ± 2 mmHg) or homozygous (GG) for the 460Gly allele (14 ± 1 mmHg). There was a significant (p < 0.005) association between homozygosity for the 460Trp allele and low-renin hypertension. Six of 67 (9%) low-renin subjects, but only 3 of 214 (1.4%) normal- renin subjects had the TT genotype. Heterozygous (GT) subjects did not have increased salt-sensitivity of systolic blood pressure and did not have an increased frequency of low-renin hypertension. Erythrocyte sodium content was significantly (p < 0.01) less in subjects with the TT genotype (24.5 ± 1.0 mmol/kgHgb) compared to all subjects (33.2 ± 0.4 mmol/kgHgb). Subjects with the TT genotype had a significantly (p < 0.05) lower sodium-lithium countertransport (SLC) (0.16 ± 0.04 mmol/l/h) than other subjects (0.28 ± 0.01 mmol/l/h). However, the mean SLC of all low-renin hypertensive subjects (0.25 ± 0.02 mmol/l/h) was not significantly different than the mean SLC of normal-renin hypertensive subjects (0.26 ± 0.01 mmol/l/h). Thus, a decreased SLC is associated with the TT genotype and is not a characteristic of all individuals with low-renin hypertension. These findings suggest that homozygous expression of the 460Trp polymorphism of the alpha-adducin gene alters sodium handling in humans and may be an important contributor to the development of low-renin hypertension in some individuals.
Single Nucleotide Polymorphisms in Promoter Region of Tgf-β
1
Gene Distinguish African Americans from Caucasians: A Possible Genetic Basis for Racial Differences in Epidemiology of Hypertension and End Stage Renal Disease.
Authors: Ding Ruchuang , Li Baogui , Gulhati Mohini , Zhang Yun , Helseth Geraldine , Ward Latonia , Sharma Vijay , August Phyllis , Suthanthiran Manikkam
Published: July 2021

P135

Transforming growth factor-beta 1 (TGF-β
1
) excess is a candidate risk factor for hypertension and hypertensive complications including LVH, vascular remodeling and end stage renal disease (ESRD). We reported that hyperexpression of TGF-β
1
protein is more prevalent in African Americans (AA) compared with Caucasians (C), particularly AA with hypertension and/or ESRD. Single nucleotide polymorphisms (SNPs) have been reported in the TGF-β
1
gene, and there is evidence for heritable control of TGF-β
1
protein levels. In this study we tested the hypothesis that TGF-β
1
SNPs distinguish AA from C. We determined the frequencies of all 6 known biallelic TGF-β
1
DNA polymorphisms in 793 subjects (AA=342 [normal: 77, hypertension: 66, ESRD: 199], C=451 [normal: 142, hypertension: 81, ESRD: 228]). SNPs as well as cis/trans combination of alleles (haplotypes) were identified by designing and using allele specific primers in an amplification refractory mutation system PCR . Our data demonstrate that SNPs at -800bp and -509bp and the haplotype frequencies (GC, GT, AC, AT) are significantly different between AA and C (Table). In conclusion, genetically determined differences in TGF-β
1
production may explain TGF-β
1
excess in AA as well as provide a molecular basis for the excess burden of hypertension and hypertensive complications in AA.
Blood Pressure and Insulin Resistance Cosegregate at Two Distinct Regions on Chromosome 7
Authors: Cheng Li S-C , Davis Richard C , Raffel Leslie J , Xiang Anny H , Quinones M , Hsu Grace , Wen P-Z , Toscano E , Azen Stan P , Hsueh Willa A , Buchanan Thomas A , Rotter Jerome I
Published: July 2021

P137

Purpose: Insulin resistance (IR) and hyperinsulinemia precede hypertension in Mexican Americans and may be a preclinical phenotype for hypertension. We tested the hypothesis that IR and blood pressure (BP) share genetic regulation by conducting a genome scan in healthy adult offspring of hypertensive probands, chosen so that the traits would not have been influenced by either the disease process or treatment. Methods: A total of 390 Mexican-Americans in 77 nuclear hypertension families were genotyped with 386 microsatellite markers. We performed two-point (by SIBPAL) and multipoint linkage analysis (by SOLAR) for BP, IR, and related traits. Results: In direct support of our hypothesis, we observed evidence for two loci on chromosome 7 that influenced both IR and BP. Two-point analysis showed evidence for linkage of a locus on 7p (∼18cM) with diastolic BP (DBP), systolic BP (SBP), mean arterial pressure (MAP), and two-hour insulin (p=0.01-0.05), with a LOD of 1.44 for SBP. More impressively, the locus on 7q (∼133cM) showed lod scores of 1.78 for SBP, and 2.97 for fasting insulin. Additional quantitative traits mapping to the 7q locus are plasma levels for leptin (LOD=1.5) and apoAII (LOD=2.0). This region overlaps with the diabesity locus reported in the Pima (Hanson et al. 1998). Two candidate genes exist in the region: PPP1R3 (∼135cM), which regulates skeletal muscle glycogenesis and has a role in type 2 diabetes (Xia et al. 1998), and leptin (∼126cM). Of particular interest, it appears that the two loci interact. Thus, the 7p locus lod score for MAP increases from 0.90 to 2.37 when an interaction with 7q is introduced Conclusions: 1) The coincident mapping of loci for BP and insulin supports the role of IR in the pathogenesis of hypertension in this population. 2) There appear to be at least two different genes on chromosome 7 responsible for BP and IR. 3) There is an interaction of these two genes, suggesting they are part of a common pathway for the IR-BP syndrome.
Persistent Activation of Both Extracellular Signal-Regulated Protein Kinases and Nuclear Factor-κB Is Required for Induction of Nitric Oxide Synthase by Interleukin-1β
Authors: Jiang Bingbing , Brecher Peter
Published: July 2021

P143

A role of extracellular signal-regulated kinase (ERK) in the signaling pathway by which interleukin-1β(IL-1) increases the expression of inducible nitric oxide synthase (iNOS) in rat vascular smooth muscle cells was studied. IL-1 clearly induced iNOS expression in cultured vascular smooth muscle cells, as demonstrated by nitrite determination, immunodetection of iNOS protein and Northern blot analysis of iNOS mRNA. Western blot analysis showed that IL-1 induced a transient phosphorylation of ERK within 1 h followed by a more prolonged phosphorylation persisting for 24 h. Pretreatment of the cells with PD98059, a selective inhibitor of ERK activation, inhibited IL-1-induced ERK phosphorylation and also inhibited iNOS induction at the level of transcription. Addition of PD98059 at various time points before or after IL-1 treatment showed that the sustained ERK activation was required for IL-1 to induce iNOS gene transcription. Electrophoretic mobility shift assays revealed that NF-κB DNA-binding also was a persistent event following IL-1 addition, increasing progressively over a 16 h incubation period. Pretreatment of the cells with PD98059 did not influence the early activation of NF-κB, but effectively reduced the subsequent increase in DNA binding, suggesting a relationship between ERK activity and the prolonged NF-κB activation. Further analysis by Western blotting showed that I-κBβ, but not I-κBα, degradation induced by IL-1 was clearly attenuated by PD98059 treatment. These data suggest that persistent activation of ERK and NF-κB is required for IL-1 induction of iNOS gene expression. Upon IL-1 stimulation, ERK activity is not required for the early initiation of NF-κB nuclear translocation and DNA-binding but is required for prolonged NF-κB activation, an event possibly mediated by I-κBβdegradation.
Aldosterone Antagonism and Epidermal Growth Factor in Cerebral Ischemia.
Authors: Dorrance Anne M , Osborn Heather L , Grekin Roger , Webb R. Clinton
Published: July 2021

P166

Two factors that may contribute to the vascular remodeling involved in the pathogenesis of ischemic cerebrovascular disease are aldosterone (aldo) and epidermal growth factor (EGF). Spironolactone (spir) is known to reduce the frequency of hemorrhagic infarcts, its effects on ischemic infarcts are unknown. We have previously shown that the mRNA for EGF and its receptor (EGFR) are increased in the cerebral blood vessels from stroke-prone spontaneously hypertensive rats (SHRSP) compared to Wistar Kyoto (WKY) rats. We hypothesized that this, along with an altered response to aldo would contribute to a larger experimentally induced ischemic cerebral infarct. Six week old male SHRSP and WKY rats were treated with spir (200mg) or placebo for six weeks. Cerebral ischemia was induced by middle cerebral artery occulsion, infarct size was quantified and expressed as percentage of the hemisphere infarcted. RNA was extracted from aorta and RT-PCR was performed for EGF and EGFR. mRNA was quantified by phosphorimage analysis and corrected for GAPDH. Treatment did not affect blood pressure in either group. Aldo antagonism reduced the size of experimentally induced cerebral infarct in SHRSP (51.7±3.6 vs. 22±6.7 % SHRSP vs. SHRSP + spir p<0.05) and WKY rats (14.2±2.1 vs. 3.2±1.5 % WKY vs. WKY + spir p<0.01). Expression of mRNA for EGFR was higher in the aorta of SHRSP compared to WKY (1.09±0.25 vs. 0.17±0.03 p<0.05 SHRSP vs WKY). The same was true for EGF mRNA expression (13.61 ± 3.73 vs 3.80 ± 1.30 p<0.05 SHRSP vs. WKY). Spir treatment reduced the expression of EGFR mRNA in the aorta of SHRSP (1.09±0.25 vs. 0.56±0.11 SHRSP vs. SHRSP + spir p<0.05) but not WKY rats. Spir treatment had no effect on the EGF mRNA expression in either group. Plasma aldo levels were similar for SHRSP and WKY. The reduction in ischemic infarct size is a novel observation. The mechanism by which spir reduces infarcts in SHRSP may be by its reduction in the expression of the EGFR mRNA leading to reduced vascular remodeling. It is possible that locally produced aldo is responsible for the inceased EGFR mRNA as aldo levels were the same for SHRSP and WKY. Interestingly, the mechanism for the reduction in infarct size appears to be different in the WKY rat.
Dietary Estrogen Protects Against NaCl-Sensitive Hypertension in Estrogen-Depleted Female Spontaneously Hypertensive Rats.
Authors: Fang Zhiwu , Carlson Scott H. , Wyss J. Michael
Published: July 2021

P169

In male spontaneously hypertensive rats (SHR) a high NaCl diet produces a rapid and sustained increase in hypertension. In contrast, in female SHR a high NaCl diet causes only a small, transient increase in arterial pressure. Elimination of most endogenous estrogen (via ovariectomy) produces mild NaCl-sensitive hypertension in female SHR, but simultaneous ovariectomy and elimination of exogenous dietary plant estrogens (phytoestrogens) nearly quadruples the hypertensive effect of the high NaCl diet, although phytoestrogen removal has a negligible effect on blood pressure in SHR fed a basal NaCl diet. This suggests that phytoestrogens (particularly genistein, the primary dietary phytoestrogen) protect ovariectomized female SHR from NaCl-sensitive hypertension. However, other reports suggest that the beneficial effects of phytoestrogens may only be present when genistein is combined with a high soy diet. The present study tests the hypothesis that genistein can independently reduce NaCl-sensitive hypertension in estrogen-depleted female SHR. Female SHR were ovariectomized at 3 weeks of age and placed on one of four casein-based, non-soy containing, diets (AIN-76A), i.e., a high (8%) or basal (0.6%) NaCl diet with or without genistein (0.06%; the approximate phytoestrogen concentration in the diet) added to the diet. After 5 weeks, femoral arterial catheters were implanted and 3 days later arterial pressures were recorded from all rats. The high NaCl diet increased arterial pressure by 68 ± 6 mm Hg in the phytoestrogen-free SHR, but produced only a 17 ± 5 mm Hg rise in arterial pressure in the genistein-supplemented group (similar to the rise previously observed in ovariectomized SHR fed a normal high NaCl diet). These results demonstrate that dietary genistein reduces NaCl-sensitive hypertension in ovariectomized female SHR. This work was supported by NHLBI Grant HL-37722.
Differential Regulation of the Epithelial Sodium Channel (ENaC) in Rat Kidney, Lung and Distal Colon in Response to Aldosterone.
Authors: Masilamani Shyama M E , Kim Gheun-Ho , Knepper Mark A
Published: July 2021

P170

The mineralocorticoid hormone, aldosterone increases renal tubule Na absorption via increases in the protein abundances of the α-subunit of the epithelial sodium channel (ENaC) and the 70 kDa form of the γ- subunit of ENaC (JCI 104:R19-R23). This study assesses the affect of dietary salt restriction on the regulation of the epithelial sodium channel (ENaC) in the lung and distal colon, in addition to kidney, using semiquantitative immunoblotting. Rats were placed initially on either a control Na intake (0.02 meq/day), or a low Na intake (0.2 meq/day) for 10 days. The low salt treated rats demonstrated an increase in plasma aldosterone levels at day 10 (control = 0.78 + 0.32 nM; Na restricted = 3.50 + 1.30 nM). In kidney homogenates, there were marked increases in the band density of the α-subunit of ENaC (286 % of control) and the 70 kDa form of γ-subunit of ENaC (262 % of control), but no increase in the abundance of the β-subunit of ENaC. In lung homogenates, there was no significant change in the band densities of the α, β, or γ subunits of ENaC. In distal colon, there was an increase in the band density of the β-subunit of ENaC (311 % of control) and an increase in both the 85 kDa (2355% of control) and 70 kDa (843 % of control) form of the γ subunit of ENaC in response to dietary Na restriction. However, there was no significant difference in the band density of the α-subunit of ENaC. These findings demonstrate tissue specific regulation of the three subunits of ENaC in response to dietary salt restriction.
Obesity But not Hypertension Is the Major Determinant of Endothelial Activation in Type II Diabetes.
Authors: Braatvedt Geoff D , Bagg Warwick , Desideri Giovambattista , Gamble G , Ockleford Paul , Santucci Anna , Ferri Claudio
Published: July 2021

P174

In order to evaluate the independent role of hypertension and other risk factors in promoting endothelial activation in type II diabetes, several markers of endothelial damage were studied in 43 type II diabetic patients (24 F,30-65 ys), before and after the random assignment to a 20-week period of either aggressive (AC, n=20) or usual (UC, n=22) metabolic control. Compared to matched controls (n=15) type II diabetic patients showed a significant (p<0.05 or less) elevation of circulating P-selectin, E-selectin, ICAM-1, VCAM-1 and Endothelin-1 (ET-1) levels and a simultaneos decrement of total nitric oxide(NO). Multivariate analysis showed that only BMI not dependently influenced (p=0.01 or less) E-selectin, ICAM-1 and total NO, with a trend for P-selectin levels (p=0.06). Accordingly, basal BMI correlated with ICAM-1 (r=0.45, p=0.003), E-selectin (r=0.41, p=0.007) and P-selectin (r=0.34, p=0.02). In addition, P-selectin and E-selectin resulted higher(p<0.03), while total NO lower (p=0.0001), in the 26 patients with a BMI >28 kg/m
2
(33.4±0.7 kg/m
2
) than in the non-obese ones (BMI=24.8±0.6 kg/m
2
). These latter showed similar endothelial adhesin, ET-1 and NO levels than controls. Age, race, gender, blood pressure, albumin excretion and all metabolic variables with the exclusion of BMI did not predict endothelial factor levels. Hypertensive (i.e. blood pressure >140/90 mmHg) and normotensive diabetic patients had similar levels of circulating adhesins, ET-1 and NO. AC was followed by a significant reduction of plasma E-selectin (p=0.04) and HbA
1c
levels (-2.2%, 95% CI 1.5 to 2.9%, p=0.0001). E-selectin decrements correlated with HbA
1c
changes after 20 weeks in AC (r=0.42, p=0.01). In conclusion,E-selectin reduction after AC suggests metabolic control may influence endothelial activation in type II diabetics. However, multivariate analysis showed that only obesity influenced the whole endothelium. Surprisingly, neither blood pressure nor other well-recognized cardiovascular risk factors influenced endothelial adhesins, ET-1 and NO production. Thus, central obesity is the main determinant of endothelial activation in type II diabetics.
Leptin Causes Vasodilation in Humans
Authors: Nakagawa Keigo , Higashi Yukihito , Sasaki Shota , Ohshima Tetsuya , Matsuura Hideo , Kajiyama Goro
Published: July 2021

P177

Background: Recently, it was reported that the acute infusion of the leptin markedly increased sympathetic nerve activity, but did not change arterial pressure, and it was also reported that the leptin receptor was expressed in human endothelium. Therefore, we suggested that the leptin might induce the vasodilation that counterregulate vasoconstricting response which is induced by sympathetic activation. Methods: To evaluate the effect of the leptin on forearm circulation, we measured the forearm blood flow(FBF)response to intra-arterial infusion of the human recombinant leptin(1, 10, 100 ng/kg/min, for 5 minutes, respectively) in seven healthy male subjects (age 23.3±1.7 years). FBF was measured using venous occlusion plethysmography. Results: The leptin infusion significantly increased the FBF in the dose-dependent manner (5.9±2.2%, 13.9±3.0%, 14.4±3.6%, at each doses, p<0.05). The intraarterial infusion of NG-monomethyl-L-arginine (L-NMMA), a nitric oxide synthese inhibitor, did not alter FBF response to the leptin (3.3±4.2%, 21.2±9.7%, 15.5±3.7%, at each doses, p<0.05). No significant changes in blood pressure or heart rate were detected during the infusion of leptin, and leptin in conbination with L-NMMA in all subjects. Conclusion: These findings suggest that the leptin per se directly causes the vasodilatation, and that leptin-induced vasodilatation is not due to the increase in nitric oxide production in healthy subjects.
Pressor Responses to Central Sodium and Ouabain Are Attenuated in Transgenic Rats Deficient in Brain Angiotensinogen
Authors: Huang Bing S , Ganten Detlev , Leenen Frans Hh
Published: July 2021

P195

Studies using AT
1
-blockers suggest that the brain renin-angiotensin system (RAS) contributes to sympatho-excitation and hypertension by high dietary salt or central sodium loading. To more specifically examine this role of the brain RAS, [TGR (AsrAOGEN)] transgenic rats were used. These rats express an antisense RNA against angiotensinogen mRNA specifically in the brain, and the brain angiotensinogen level is reduced by more than 90%. In freely moving TGR and SD controls, BP and HR responses to intracerebroventricular (icv) infusion (3.8 μl/min for 10 min) of artificial CSF (aCSF) and Na
+
-rich aCSF (containing 0.2, 0.3 and 0.45 M Na
+
) as well as icv injection of ouabain (0.3 and 0.6 μg/2 μl) were assessed. The vasopressin antagonist [d(CH
2
)
5
Tyr(Me)]AVP (30 μg/kg) was given iv before each icv infusion. Data are means±SEM (n=6, for each).* p<0.05, vs SD rats. Somewhat surprisingly, angiotensin I and II levels of not only hypothalamus but also plasma were lower in TGR vs. SD rats. The clearly attenuated sympatho-excitatory and pressor effects to ouabain and Na
+
-rich aCSF support the concept that the brain RAS plays an important role in the sympatho-excitatory effects of ouabain and CSF sodium.
Novel Therapeutic Strategy for Restenosis Using Ribozyme Oligonucleotides Against Transforming Growth Factor-β
Authors: Yamamoto Kei , Morishita Ryuichi , Tomita Naruya , Nakagami Hironori , Aoki Motokuni , Higaki Jitsuo , Kaneda Yasufumi , Ogihara Toshio
Published: July 2021

P197

Background Since the mechanisms of atherosclerosis or restenosis after angioplasty have been postulated to involve an increase in TGF-β, a selective decrease in TGF-βmay have therapeutic value. Thus, we employed the ribozyme a unique class of RNA molecules that not only store information but also process catalytic activity, to selectively inhibit TGF-βexpression. Methods & Results We constructed ribozyme oligonucleotides targeted to the sequence of TGF-βgene which shows 100 % homology among human, rat and mouse. The specificity of ribozyme against TGF-βgene was confirmed by selective inhibition of TGF-βmRNA in cultured human VSMC as well as balloon-injured blood vessels in vivo. In vitro study Transfection of TGF-βribozyme ON into human VSMC by cationic liposome significantly decreased mRNA expression and protein of TGF-βinduced by Ang II as compared to control In vivo study Transfection of FITC-labeled ribozyme ON resulted in fluorescence in the balloon-injured vessels at 1 day after transfection. The fluorescence was localized primarily in cell nuclei , and persisted for up to 2 weeks after transfection.And then,transfection of TGF-βribozyme ON into rat cartid balloon injry model significantly decreased mRNA expression of TGF-βinduced by balloon injry ,and have significant inhibitory effects on neointimal formation after vascular injury(P<0.01), whereas DNA-based control oligonucleotides and mismatched ribozyme oligonucleotides did not have any inhibitory effect on neointimal formation. Inhibition of neointimal formation was accompanied by 1) a reduction in collagen synthesis and mRNA expression of collagen I and III, and 2) a significant decrease in DNA synthesis as assessed by PCNA staining. Conclusion Overall, this study provides the first evidence that selective blockade of TGF-βby ribozyme resulted in inhibition of neointimal formation, accompanied by a reduction in collagen synthesis and DNA synthesis in a rat model. We anticipate that ribozyme ON pharmacokinetics will facilitate the potential clinical utility of the ribozyme strategy.
Abstract 100: Electrocardiographic Changes in Patients ≥ 100 Years Of Age
Authors: Thakar Saurabh , Shetty Vijay , Sadiq Adnan , Lichstein Edgar , Hollander Gerald , Shani Jacob
Published: July 2021

Background:
Centenarian population in the United States is increasing, and cardiovascular disease is the most common cause of death in this group of patients. We report electrocardiographic changes in a group of 124 centenarians.

METHODS:
We included ECGs of 124 patients aged 100 to 112 years (mean age 102 years), who were seen either on office visits or on recent admission to the hospital. ECGs were analyzed by 2 different investigators.

Results:
Women were at least 2 times men in number (31% vs 69%, 39 vs 85 of 124); thus women outlived men in the centenarian group. 58% (72 of 124) patients had normal sinus rhythm and only 7 of 124 patients (6%, all women) had normal ECGs. The most common electrocardiographic abnormalities were: left-axis deviation (29%, 36 of 124), atrial fibrillation (AF) (27%, 34 of 124), right bundle branch block (15%, 18 of 124), left anterior fascicular block (14%, 17 of 124), left ventricular hypertrophy (14%, 17 of 124), first-degree atrioventricular (AV) block (13%, 16 of 124), nonspecific ST-T changes (13%, 16 of 124), premature atrial complexes (13%, 16 of 124), premature ventricular complexes (10%, 13 of 124) and pacemaker rhythm (10%, 13 of 124). Almost all Q waves were suggestive of old inferior wall infarcts and were present in 8% (10 of 124) of patients. Only 3 centenarians had QTc prolongation.

Conclusion:
Fewer men live long enough to reach 100 years of age, consistent with higher incidence of ischemic heart disease in men. Centenarians had higher incidence of AF indicative of increased incidence of AF with age. Increased incidence of left-axis deviation, left anterior fascicular block and left ventricular hypertrophy in centenarians can be attributed to age related cardiac hypertrophy and prevalence of hypertension. Also higher prevalence of conduction disturbances including AV block and bundle branch block indicates that a degenerative conduction system disorder may progress with advancing age. The finding of pacemaker rhythm in a few subjects confirms that pacemaker implantation may allow some patients to reach extreme longevity. Overall centenarians represent a model of successful aging and have satisfactory electrocardiographic framework that reflects a good cardiac function, contributing to attainment of extreme longevity.
Abstract 105: 20-HETE: An Inducer of ACE in Human Microvascular Endothelial Cells (HMVEC).
Authors: Garcia Victor , Cheng Jennifer , Ding Yan , Wu Cheng-Chia , Falck John R , Errabelli Ramu , Schwartzman Michal L
Published: July 2021

20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450 derived arachidonic acid metabolite, is manufactured in the microcirculation and shown to sensitize the smooth muscle to constrictor stimuli and cause endothelial activation and dysfunction. Increased vascular production of 20-HETE promotes hypertension that is dependent, in part, on the renin angiotensin system. Microarray analysis of endothelial cells treated with 20-HETE revealed a five-fold upregulation of angiotensin converting enzyme (ACE). In cultured human microvascular endothelial cells (HMVEC), 20-HETE (5 nM) induced ACE mRNA by 3.1-fold (± 0.16 p<0.05), increased ACE protein levels by 4.2-fold (±0.99 p<0.05) and ACE activity by 2-fold (±0.23 p<0.05). These effects were abrogated by co-treatment with 20-HEDE, a 20-HETE antagonist. 20-HETE induction of ACE mRNA was abolished by inhibitors of EGFR-tyrosine kinase, MAPK and IKKβ activation but was not affected by PKC inhibition. Moreover, transfection of cells with IKKβ siRNA prevented 20-HETE from inducing ACE mRNA. Collectively, theses results suggest a role for 20-HETE as a regulator of endothelial ACE. This regulatory activity impacts vascular function since downregulation of ACE in endothelial cells treated with siRNA exogenous 20-HETE from inhibiting NO synthesis and stimulating O
2
-
production. This study demonstrates that induction of ACE expression and activity is critical to the actions of 20-HETE on endothelial cell function, further implicating the induction of ACE and activation of RAS as a key mechanism by which excessive production of 20-HETE within the vasculature leads to hypertension.
Abstract 128: Six Months of Lifestyle Intervention Reduces Carotid Intima-media Thickness in African Merican Adults
Authors: Veerabhadrappa Praveen , Kashem Abul , Kothapalli Priya , Diaz Keith , Ling Chenyi , Feairheller Deboah , Kretzschmar Jan , Sturgeon Kathleen , Williamson Sheara , Crabbe Deborah , Ahrensfield Deborah , Brown Michael
Published: July 2021

Background:
African Americans tend to have greater carotid intima-media thickness (IMT), and are 60% more likely to have a stroke than Caucasians. Data on the effects of lifestyle intervention on carotid IMT in African Americans does not exist. The aim of our study was to assess the efficacy of 6 months of diet and exercise intervention on carotid IMT in sedentary African American adults.

Methods:
Nineteen African American subjects (15F/4M, 52 ± 6yrs) who were sedentary, non smoking, non diabetic, free of cardiovascular disease, not on antihypertensive or cholesterol medications underwent 6 weeks of dietary counseling under the supervision of a registered dietician and were required to follow an AHA low fat, low salt diet for the duration of the study. Compliance was assessed by a diet log (3 days/week). Subjects completed 6 months of supervised aerobic exercise training (3 days/week, 40 minutes/session at 65% of their estimated VO
2max
). Common carotid artery IMT was assessed using high resolution ultrasonography.

Results:
At baseline, IMT significantly correlated with age (r = 0.58,
P
= 0.003) and total cholesterol (r = 0.41,
P
= 0.04). At 6 months, there were significant changes in basal metabolic index (ΔBMI, -0.83 kg/m
2
,
P
= 0.01); Δtriglycerides (-13.7 mg/dL,
P
= 0.03) and Δfasting glucose (-7.2 mg/dL,
P
= 0.01). On repeated measures ANCOVA, the mean IMT were statistically significantly different (F (1, 15) = 7.999,
P
= 0.013) between before vs. after training (Mean ± SD; 0.617 ± 0.092mm vs. 0.572 ± 0.068mm), after adjusting for changed variables (ΔBMI, Δtotal cholesterol and Δmean arterial pressure) as covariates.

Conclusion:
Our preliminary study showed that 6 months of diet and exercise intervention reduced carotid IMT by ∼7.3% and improved the cardio-metabolic profile in sedentary African Americans. Reduction in IMT from early and effective lifestyle modification strategies may help to improve cerebrovascular health and prevent stroke in this at-risk stroke population.
Abstract 129: Effects of Pulse Pressure and Antihypertensive Therapy on Short and Long Term Outcomes in Stroke Patients
Authors: Murthy Avinash , Arora Jaspreet , Shah Amar , Khawaja Hussain , Torosoff Mikhail
Published: July 2021

Background:
Effects of pulse pressure and benefits of blood pressure lowering with intravenous anti-hypertensive medications and beta-blockers in CVA patients have not been well investigated.

Material and Methods:
Demographic, clinical, and echocardiographic data were collected and long-term outcomes (55+/-21 months) were ascertained in 356 consecutive cerebro-vascular accident (CVA) patients. ANOVA, chi-square, Kaplan-Meier, and logistic regression tests were employed. Study was approved by the institutional IRB.

Results:
Widened pulse pressure on admission was significantly elevated in CVA patients who expired in the hospital or during the long-term follow-up (62+/-21mmHg for long-term survivors vs. 72+/-20mmHg for hospital deaths vs. 69+/-28 mmHg for long-term deaths, p=0.01). There was a trend towards increased hospital mortality (14% in long-term survivors vs. 25% in hospital deaths vs. 22% in long-term deaths, p=0.110) in CVA patients requiring IV anti-hypertensive therapy. Utilization of beta-blockers was lower in patients who suffered hospital death, but more likely in patients experiencing long-term death (42% use in hospital deaths vs. 48% in long-term survivors vs. vs. 66% in long-term deaths, p=0.003). Beta-blocker use was not predictive of hospital outcomes but was strongly predictive of adverse event long-term events (HR 2.1, 95%CI 1.3-3.4, p=0.002). When adjusted for demographic parameters and co-morbidities in multivariate analysis, pulse pressure and IV anti-hypertensive therapy were not predictive of short or long-term outcomes, while beta-blocker treatment was associated with reduced hospital (0.3, 95%CI 0.1-0.9, p=0.029) but not long-term mortality.

Conclusions:
Widened pulse pressure and need for IV anti-hypertensive therapy are not predictive of adverse short- or long-term outcomes when demographics and co-morbidities are accounted for. Effects of beta-blocker therapy on outcomes in CVA patients are complex. Wider beta-blocker use in acute CVA may be associated with better hospital outcomes, while increased long term mortality with beta-blocker therapy may be indicative of poor cardiovascular health leading to adverse outcomes
Abstract 132: Hypoxia-inducible Factor Prolyl-hydroxylase-2 Mediates Transforming Growth Factor Beta 1-induced Epithelial-mesenchymal Transition In Renal Tubular Cells
Authors: Han Weiqing , Hu Jun-Pin , Li Pin-Lan , Li Ningjun
Published: July 2021

Transforming growth factor beta 1 (TGFβ1)-induced epithelial-mesenchymal transition (EMT) in kidney epithelial cells plays a key role in renal tubulointerstitial fibrosis in chronic kidney diseases. As hypoxia-inducible factor (HIF)-1α is found to mediate TGFβ1 signaling pathway, we tested the hypothesis that HIF-1α and its upstream regulator prolyl hydroxylase domain-containing proteins (PHDs) are involved in TGFβ1-induced EMT in renal tubular cells. Our results showed that TGFβ1 treatment for 48 h stimulated EMT in cultured renal tubular cells as indicated by the decrease in epithelial marker P-cadherin from 1.0 ± 0.02 to 0.40 ± 0.05 (
P
< 0.05), and the increase in mesenchymal markers α-smooth muscle actin (2.14 ± 0.32 fold,
P
< 0.05) and fibroblast-specific protein (2.0 ± 0.17 fold,
P
< 0.05) as shown in Western blot assay. Meanwhile, TGFβ1 time-dependently increased HIF-1α, which reached its maximum value (2.36 ± 0.2 fold,
P
< 0.05) at 24 h, and that HIF-1α siRNA significantly inhibited TGFβ1-induced EMT, suggesting that HIF-1α mediated TGFβ1 induced-EMT. Real-time PCR showed that PHD1 and PHD2, rather than PHD3, could be detected, with PHD2 as the predominant form of PHDs (PHD1 : PHD2 = 0.21:1.0). Importantly, TGFβ1 time-dependently decreased PHD2 mRNA and protein level, which reached their maximum value from 1.0 ± 0.15 to 0.45 ± 0.08 (
P
< 0.05) for mRNA at 16 h and from 1.0 ± 0.08 to 0.26 ± 0.08 (
P
< 0.05) for protein at 24 h, respectively. In contrast, TGFβ1 had no effect on PHD1 mRNA and protein levels. Furthermore, over-expression of PHD2 transgene almost fully prevented TGFβ1-induced HIF-1α accumulation and EMT marker changes, indicating that PHD2 is involved in TGFβ1-induced EMT. Finally, Smad2 inhibitor SB431542 prevented TGFβ1-induced PHD2 decrease, suggesting that Smad2 may mediate TGFβ1-induced EMT through PHD2/HIF-1α. It is concluded that TGFβ1 decreased PHD2 expression via a Smad2-dependent signaling pathway, thereby leading to HIF-1α accumulation and EMT in renal tubular cells. The present study suggests that PHD2/HIF-1α is a novel signaling pathway mediating the fibrogenic effect of TGFβ1 and that manipulating PHD2/HIF-1α pathway may be used as a therapeutic strategy in chronic kidney diseases. (support: NIH grant HL89563 and HL106042)
Abstract 135: Alubuminuria Causes Disorder of Cytochrome P450 Metabolism of Arachidonic Acid in the Kidney of Nephrotic Rats
Authors: Muroya Yoshikazu , Ito Osamu , Rong Rong , Sakata Yoshiko , Takashima Kenta , Ito Daisuke , Cao Peng-Yu , Kohzuki Masahiro
Published: July 2021

Alubuminuria is an aggravating factor for chronic kidney disease. Fatty acids bound to albumin are overloaded to the proximal tubules in alubuminuria and contribute to tubulointerstitial damage. It is known not only the β-oxidation ability but the ability of cytochrome P450 (CYP) metabolism of arachidonic acid (AA) is high in the kidney. Epoxyeicosatrienoic acids synthesized primarily by CYP2C and 20-hydroxyeicosatetraenoic acid synthesized by CYP4A affect tubular function and renal circulation. However, it isn’t clear CYP metabolism of AA would change in alubuminuria. The study tested changes of CYP metabolism of AA in the kidney of nephrotic rats. Sprague-Dawley rat (SD) and Nagase Analbuminemic rat (NAR) with inherited hypoalbuminemia were used and nephrotic syndrome was induced by Puromycin Aminonucleoside (PAN; 100 mg/kg, iv). Rats were randomly divided into four groups; (1) SD group; (2) SD treated with PAN group (PAN group); (3) NAR group; (4)NAR treated with PAN group (NAR+PAN group). Rats were killed on the 14th day and urinary 8OHdG, a marker of oxidative stress in the kidney, and urinary NAG, a marker of proximal tubular cell damage, were measured. The protein level of enzymes in the kidney was analyzed by Western blot and immunohistochemistry. Compared with the SD group, albuminuria, 8OHdG and NAG increased respectively to 847%, 154% and 903% in the PAN group, and Western blot showed the protein levels of CYP2C23 and CYP4A decreased to 45% and 49% without change of the protein level of PPARα, a nuclear receptor regulating fatty acid metabolism, in the PAN group. Immunohistochemistry showed the localization of CYP2C23 and CYP4A in the proximal tubule and confirmed the results by Western blot. Otherwise, we could find no difference in albuminuria, tubulointerstitial damage and the protein level of CYP2C23 between the NAR group and the NAR+PAN group. Compared with the NAR group, the protein level of CYP4A decreased to 68% in the NAR+PAN group. Alubuminuria caused tubulointerstitial damage and decreased the expressions of CYP2C23 and CYP4A in the nephrotic kidney. This study suggested the disorder of CYP metabolism of AA would affect renal circulation in alubuminuria and CYP4A would be affected by anything except alubuminuria in PAN rats.
Abstract 141: Histone Deacetylase 1 Regulates Nitric Oxide Production and Nitric Oxide Synthase-3 Acetylation in Endothelial Cells
Authors: Hyndman Kelly A , Ho Dao H , Pollock Jennifer S
Published: July 2021

Previous reports showed that NOS3 is regulated by acetylation through transcriptional mechanisms via histone acetylation or through direct lysine acetylation. Histone deacetylase (HDAC) enzymes and histone acetyltransferases (HATs) modulate acetylation processes. Recent work by our lab, demonstrated increased expression of aortic HDAC1 and HDAC6 while HATs were unchanged in a mouse model of early life stress with endothelial dysfunction. These data suggest a negative correlation between endothelial dysfunction and HDAC expression. The purpose of this study was to test the hypothesis that HDAC1 and 6 regulate endothelial NO production and/or NOS3 acetylation. Initial immunoprecipitation studies with anti-acetyl lysine and anti-NOS3 antibodies demonstrated that NOS3 is basally acetylated in primary bovine aortic endothelial cells (BAECs). Treatment with the HDAC inhibitor, trichostatin A (500 nM) for 1 hr, significantly increased NOS3 acetylation. BAECs were transfected with HDAC1, HDAC6, vector expression plasmids, or untransfected, with nitrite production determined by HPLC and NOS3 acetylation and expression probed by immunoprecipitation and Western blotting. Untransfected and vector transfected control BAECs had similar NO production (357 ± 10 and 344 ± 30 pmol/mg pr/h, respectively, N=6) as well as NOS3 acetylation (7.8 ± 1.6 and 6.8 ±0.3 AU, N=3). HDAC6 transfected BAECs had similar NO production to the control BAECs (272 ± 93 pmol/mg pr/h, N=3) with an increase in NOS3 acetylation (17.4 ± 1.7 AU, N=3). In contrast, HDAC1 overexpression significantly decreased NO production (89 ± 50 pmol/mg pr/h, P< 0.05, N=3) and reduced NOS3 acetylation (3.8 ± 0.5 A.U, N=3), P <0.05). Control transfections, HDAC6, and HDAC1 transfected BAECS all had similar NOS3 expression (10.14 ± 1.8; 9.8 ±1.6; 8.9 ± 1.5; 10.6 ± 1.0 AU, respectively, N=3). Thus, we conclude that HDAC1 regulates NO production via direct lysine deacetylation of NOS3.
Abstract 149: Association Between Hypertension and Chronic Exposure in Bangladesh
Authors: Islam Md Rafiqul , Khan Ismail , Hassan Sheikh Md Nazmul , McEvoy Mark , D'Este Catherine , Attia John , Azim Syed , Akhter Ayesha , Akter Shahnaz , Milton Abul Hasnat
Published: July 2021

Objective:
To determine the association between chronic arsenic exposure via drinking water and prevalence of hypertension among rural Bangladeshi adults.

Design, Setting, Participants:
This is an analytical cross sectional study among 1004 participants from 1682 eligible men and women (Participation rate 60%) aged >30 years living in Bangladesh who had continuously consumed arsenic-contaminated drinking water for at least 6 months.

Main Outcome Measures:
Hypertension was defined as systolic blood pressure > 140 mm of Hg or a diastolic blood pressure > 90 mm of Hg or in combination of the both.

Results:
A total of 1004 individuals participated in the study. The prevalence of hypertension was 6.6% (95% CI 5.1-8.3%). After adjustment for participant’s age, sex, education, religion, marital status, sign of arsenical skin lesions, monthly household income and BMI, no excess risk of hypertension was observed for drinking water arsenic exposure over 50μg/L. Also, there was no increased risk for higher concentration of arsenic in the drinking water. Arsenic concentration >50 μg/L in drinking water showed no association with systolic, diastolic hypertension separately however, it shows a strong relationship with increased pulse pressure when pulse pressure categorized as <55 and ≥55 mmHg (Adjusted OR: 3.06, CI: 1.22-7.65). Also, duration of exposure to arsenic didn’t show any impact on an increased risk to hypertension except pulse pressure when exposed to arsenic for ≥10 years (Adjusted OR: 4.74, CI: 1.25-17.88).

Conclusion:
Unlike other studies conducted in developing countries that reported a significant association, this study suggests no association between higher drinking water arsenic concentration and hypertension except for pulse pressure.
Abstract 154: GWAS Candidate Gene Knockout in Rats Reveals a Role for Erk/mapk Signaling in Hypertension
Authors: Endres Bradley , Sarkis Allison B , Priestley Jessica , Casati Marc , Hoffman Matt , Lazar Jozef , Schilling Rebecca , Dwinell Melinda , Lombard Julian , Cowley Allen W , Mattson David , Moreno Carol , Geurts Aron M , Jacob Howard J
Published: July 2021

The data revealed by GWAS studies provide a wealth of candidate human disease genes now requiring functional validation in animal models. In two years, we disrupted a large set of GWAS candidate genes for human hypertension and chronic kidney disease by Zinc Finger Nuclease (ZFN)-mediated gene targeting in the SS (Dahl salt-sensitive) strain background. Phenotyping male rats from gene-disrupted strains revealed five genes (
Sh2b3
,
Rasgrp3
,
Gpr73
,
Ulk3
, and
Wdr72
) significantly altering the salt-induced hypertension in and renal damage phenotypes in this model strain of human hypertension compared to age matched wild type controls.

Ingenuity Pathway Analysis revealed a putative connection for four of these genes (
Sh2b3
,
Rasgrp3
,
Gpr73
, and
Ulk3
) to the ERK/MAPK signaling pathway and suggested that decreased ERK signaling would exacerbate disease in the SS model. To test this hypothesis, we measured the response of SS rats to a 4% salt diet with daily IP injections of the MEK inhibitor PD98059 or vehicle control. Wild type SS animals treated with PD98059 show a significant increase in mean arterial pressure compared to vehicle-injected controls (n=10, 142±7 vs. 126±6 mmHg respectively, P<0.05) suggesting that ERK signaling plays an important role in the pathogenesis of this model. In conclusion, we have used gene knockout in a hypertensive rat model to identify genes and associated pathway mechanisms of BP regulation. The gene disruption phenotype and

participation of multiple genes in the ERK/MAPK signaling pathway indicates that regulation of this pathway plays a major role in determining blood pressure.
Abstract 161: Identification of Genetic Variants Associated with Kidney Injury That Leads to Increased Blood Pressure
Authors: Harmon Ashlyn C , Johnson Ashley C , Atanur Santosh , Maratou Klio , Aitman Tim , Garrett Michael R
Published: July 2021

Hypertension, diabetes and obesity, along with genetic predisposition, contribute to the growing number of chronic kidney disease patients. Our novel congenic model [S.SHR(11)] was developed through genetic modification of the Dahl salt-sensitive (S) rat, a model of hypertension related renal disease. The S.SHR(11) strain exhibits accelerated kidney injury compared to the already highly susceptible S rat. On either a low or high-salt diet, the S.SHR(11) model predominately exhibited more tubulointerstitial fibrosis compared to the S rat (17.1±1.29% vs. 12.9±1.22%). Increased α-SMA and macrophage infiltration was also observed. The S and S.SHR(11) had similar blood pressure (week 12), despite an early reduction in renal function in the S.SHR(11); however at an advanced age the S.SHR(11) demonstrated significantly higher blood pressure than the S (215±6.6 mm Hg vs. 183±5.9, respectively). This suggests that increased kidney injury is driving the development of hypertension later in life. Since these two animal models are identical with exception of chromosome 11, the causative genetic variants contributing to decreased renal function must reside within this region. The Dahl S and SHR genomes have been sequenced; this data provides a catalog of all the genetic variants between the two models. The 95% confidence interval of the genomic locus contains 28 non-synonymous SNP, with 15 of these SNP occurring within only three genes:
Retnlg
,
Trat1
and
Myh15.
Two of these genes,
Retnlg
and
Trat1,
are known to play a role in immune response leading to our hypothesis that genetic variants in these genes alter protein function and lead to an increased immune response. Bone marrow transplant studies have been initiated to test our hypothesis and preliminary data shows that S rats who receive S.SHR(11) bone marrow have kidney function measurements similar to the S.SHR(11). The sequencing information has also lead to the development of nine new, more refined congenic strains. Through functional analysis of these new congenic animals, identification of the causative genetic variations will be expedited. In summary, we are employing a model of accelerated kidney disease to identify genes or genetic variants responsible for reduced kidney function and hypertension.
Abstract 171: Eukaryotic Elongation Factor 2 Kinase (eEF2K) Mediates Vascular Inflammation and Development of Hypertension in Spontaneously Hypertensive Rats
Authors: Usui Tatsuya , Okada Muneyoshi , Hara Yukio , Yamawaki Hideyuki
Published: July 2021

Eukaryotic elongation factor 2 kinase (eEF2K) is a Ca
2+
/calmodulin-dependent protein kinase that mainly regulates protein translation. We have recently demonstrated that eEF2K protein increases in mesenteric artery from spontaneously hypertensive rats (SHR) compared with Wistar Kyoto rats. Pathogenesis of hypertension is modulated in part by vascular inflammation. We examined whether eEF2K mediates vascular inflammatory responses and development of hypertension. In human umbilical vein endothelial cells (HUVECs) and rat mesenteric arterial smooth muscle cells (SMCs), eEF2K phosphorylation was increased by TNF-α (48% increase in HUVECs and 46% increase in SMCs, n=5, P<0.01). In HUVECs, small interfering RNA (siRNA) against eEF2K inhibited induction of VCAM-1 (62% inhibition, n=4) and e-selectin (29% inhibition, n=6) as well as monocyte adhesion (43% inhibition, n=4) by TNF-α (P<0.01). eEF2K siRNA inhibited phosphorylation of JNK (76% inhibition) and NF-κB (54% inhibition) as well as reactive oxygen species (ROS) production (69% inhibition) by TNF-α (n=4, P<0.01). In SMCs, eEF2K siRNA inhibited VCAM-1 induction (41% inhibition, n=5, P<0.05) and phosphorylation of JNK (65% inhibition, n=6, P<0.01) and NF-κB (83% inhibition, n=4, P<0.05) by TNF-α. In vivo, increased blood pressure (systolic blood pressure, SHR; 199.5 mmHg vs. SHR+NH125; 159.4 mmHg, n=4, P<0.01) and increased eEF2K phosphorylation (68% inhibition, n=4), induction of VCAM-1 (62% inhibition, n=4, P<0.05) and hypertrophy (70% inhibition, n=4) in SHR mesenteric artery was normalized by long-term treatment with NH125 (500 μg/kg/day for 6 weeks). In SHR mesenteric artery, impairment of acetylcholine (ACh)-induced endothelium-dependent relaxation was normalized by NH125 (relaxation induced by 30 nM ACh, SHR; 60.4% vs. SHR+NH125; 90.0%, n=4, P<0.01). The present results for the first time demonstrated in cultured ECs and SMCs that eEF2K mediates TNF-induced inflammatory responses via ROS-dependent mechanism. It is also suggested that eEF2K may mediate development of hypertension in SHR likely via inflammation, hypertrophy and endothelial dysfunction.