bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 427: Mammalian Target of Rapamycin (mTOR) Inhibition Attenuates Angiotensin II-Induced Hypertension and Endothelial Dysfunction
Authors: Didion Sean P , Sarmiento Joel A , Mezzetti Erin M
Published: July 2021

Angiotensin (Ang) II produces hypertension, endothelial dysfunction as well as vascular hypertrophy. mTOR is a key regulator of cell growth and protein synthesis, however the contribution of mTOR to hypertension and endothelial dysfunction produced by Ang II has not been previously elucidated. The goal of this study was to test the hypothesis that mTOR contributes to the hypertension and endothelial dysfunction produced by Ang II. C57Bl/6 mice were infused (via osmotic minipump) with vehicle or rapamycin (an inhibitor of mTOR; 2 mg/kg/d) for 10 days following which mice were then infused with Ang II (1000 ng/kg/min) plus vehicle or rapamycin for 14 subsequent days. Systolic blood pressure (SBP) was measured on Days -10, 0, and 14 using tail-cuff plethysmography and vascular function was examined in carotid arteries (in vitro) on Day 14. Ang II infusion produced a significant degree (P<0.05) of hypertension in wild-type mice (eg, SBP: 114±4 vs. 145±3 mmHg on Day 0 and Day 14 of Ang II infusion, respectively). In rapamycin-treated mice, Ang II infusion had no effect (P>0.05) on SBP (eg, SBP: 110±6 vs. 116±9 mmHg, on Day 0 and Day 14 of co-infusion of rapamycin plus Ang II, respectively). Rapamycin alone or vehicle infusion had no effect (P>0.05) on blood pressure during Day -10 through Day 0. In terms of endothelial responses, acetylcholine-induced relaxation was impaired (P<0.05) in Ang II-infused mice by approximately 50%. In contrast, rapamycin was associated with a significant degree of protection against Ang II-induced endothelial dysfunction (eg, 100 μM acetylcholine produced 47±10 vs. 90±4% relaxation in carotid arteries from Ang II- and Ang II plus rapamycin-infused mice, respectively). Tempol, a scavenger of superoxide was very effective in improving endothelial responses in Ang II-infused mice. Molecular analysis revealed that Ang II-infusion was associated with increased mTOR expression as well as mTOR phosphorylation. Taken together, these data provide strong pharmacologic and molecular evidence that mTOR activity contributes to the hypertension and endothelial dysfunction produced by Ang II.
Abstract 435: Adiposity Increases Blood Pressure in Blacks More Than in Whites
Authors: Tu Wanzhu , Eckert George , Yu Zhangsheng , Li Zhuokai , DiMeglio Linda , Pratt J Howard
Published: July 2021

Strong associations between adiposity and blood pressure (BP) have been observed consistently in most human populations. While obesity and hypertension are more prevalent in blacks, it is unclear whether adiposity affects BP differentially in blacks and whites and whether mechanisms by which adiposity exerts its influence are common across populations. The current study examined the effects of body mass index (BMI) on BP using longitudinally collected data from healthy black and white children. Using nonparametric regression techniques, we assessed the concurrent influences of age and BMI on BP. The analysis was based on 9113 observations from 1112 subjects (463 blacks and 649 whites; enrollment ages 4-17 years). To accommodate age and sex-related variations in BP and to compare BP in subjects of different ages, we modeled age and sex adjusted BP percentile values (BP%). The mean systolic BP% were 43% and 38% for blacks and whites respectively (p<0.0001); mean BMI% values were 71% and 60% for blacks and whites (p<0.0001). Model fitting results were presented in colored contour plots, which showed that at the same age and BMI level, black subjects tended to have higher BP%. (See Fig 1) The results were consistently observed for male and female subjects (data not shown). Blacks had significantly lower plasma renin activity than whites (PRA; 2.8 vs 3.4 ng/mL/h, p<0.0001), and in addition BMI was negatively associated with PRA in blacks (p=0.001). In conclusion, adiposity increases BP in blacks more than in whites. The lower PRA levels and negative BMI-PRA association observed in blacks suggest that the increase of adiposity effect may be related to expanded extracellular fluid volume.
Abstract 447: Activation of IL-1β-Producing Inflammasomes Triggered by RNA Receptor RIG-I in Mouse Endothelial Cells
Authors: Zhang Yang , Li Xiang , Li Xiao-Xue , Pitzer Ashley L , Li Pin-Lan
Published: July 2021

Retinoic acid-inducible gene-I (RIG-I) is a putative RNA helicase and recently identified as a cytosolic RNA receptor in mammalian cells. The role of RIG-I in the regulation of vascular function under physiological and pathological conditions is unknown. The present study tested whether RIG-I activation triggers inflammasome formation, turning on inflammation in mouse endothelial cells (EOMA cell line). By real time RT-PCR and Western blot analysis, transfection of mouse ECs with RIG-I specific agonist, 5’-triphosphate double-stranded RNA (3pRNA, 0.5 mg/L) increased RIG-I mRNA level by 106% and protein level by 81% compared to those in control double-stranded RNA (dsRNA) transfected ECs. ELISA analyses showed that 3pRNA significantly increased release of type I IFN alpha by 31 folds and IL-1 beta (a prototype cytokine from inflammasome activation) by 8 folds in these ECs. Proatherogenic stimulation of mouse ECs with cholesterol crystals or 7-ketocholesterol also markedly increased protein expression of RIG-I, but had no effect on RIG-I mRNA levels. Measurements of active caspase-1, an inflammasome activation marker using FLICA fluorescent probe that specifically binds to cleaved caspase-1, demonstrated that 3pRNA doubled FLICA positive cells compared to that in control dsRNA transfected ECs. Interestingly, cholesterol crystals significantly increased FLICA positive cells by 3 folds. This activation of caspase-1 in ECs by cholesterol crystals was further confirmed by increase in cleaved caspase-1 (p10) using Western blot analysis and by enhanced IL-1 beta release as detected by ELISA. In the presence of 3pRNA, cholesterol crystal-induced inflammasome activation was not further augmented. These data indicate that increased expression and activity of RIG-I activate IL-1 beta producing inflammasomes in ECs, which may represent an early molecular mechanism mediating vascular inflammation or injury upon atherogenic stimulations.
Abstract 448: Cyclosporine A-Induced Increases in Th17 Cells, Endothelial Dysfunction, and Blood Pressure are Prevented by IL-17 Inhibition
Authors: Chiasson Valorie L , Chatterjee Piyali , Young Kristina J , Kopriva Shelley E , Mitchell Brett M
Published: July 2021

The immunosuppressive drug cyclosporine A (CsA) decreases anti-inflammatory regulatory T cells (Tregs); however its effect on pro-inflammatory Th17 cells and whether IL-17 secreted by Th17 cells plays a role in the development of CsA-induced endothelial dysfunction and hypertension are unknown. We hypothesized that CsA causes endothelial dysfunction and hypertension in part by increasing Th17 cells and these effects can be prevented by IL-17 inhibition. Daily treatment of male C57BL6/J mice for 1 week with CsA (50 mg/kg/day, ip) significantly increased SBP compared to vehicle-treated controls (Con: 99±3 mmHg vs. CsA: 138±3 mmHg; p<0.05) and significantly decreased maximal aortic endothelium-dependent relaxation responses (Con: 69±3% vs. CsA: 25±2%; p<0.05). These effects were associated with a significant increase in both splenic and circulating levels of Th17 cells and a significant decrease in both splenic and circulating Tregs. Injections of an IL-17 neutralizing antibody (nAb) on days 0, 3, and 6 of CsA treatment normalized SBP (108±9 mmHg) and aortic endothelium-dependent relaxation responses (68±2%). IL-17 nAb treatment also decreased both splenic and circulating levels of Th17 cells and increased both splenic and circulating levels of Tregs. Injections of an isotype nAb on days 0, 3, and 6 had no effects on SBP in either CsA-treated (141±3 mmHg) or vehicle-treated (98±6 mmHg) mice. The isotype nAb also had no effects on aortic relaxation responses, splenic or circulating levels of Th17 cells, or splenic or circulating levels of Tregs in either CsA-treated or vehicle-treated mice. Together, these data suggest that the hypertension and endothelial dysfunction caused by the immunosuppressive drug CsA is mediated in part by increased Th17 cells and IL-17. Inhibition of IL-17 may have beneficial cardiovascular effects in CsA-treated patients.
Abstract 456: High Salt Induces Pathogenic Th17 Cells and Exacerbates Autoimmune Diseases
Authors: Kleinewietfeld Markus , Manzel Arndt , Titze Jens , Kvakan Heda , Yosef Nir , Linker Ralf A. , Hafler David A. , Muller Dominik N
Published: July 2021

There has been a marked increase in the incidence of autoimmune diseases in the last half-century. While the underlying genetic basis of this class of diseases has recently been elucidated implicating predominantly immune response genes1, changes in environmental factors must ultimate be driving this increase. The recently identified population of interleukin (IL)-17 producing CD4+ helper T cells (Th17 cells) play a pivotal role in autoimmune diseases2. Pathogenic IL-23 dependent Th17 cells have been shown to be critical for the development of experimental autoimmune encephalomyelitis (EAE) an animal model for multiple sclerosis (MS) and genetic risk factors associated with MS are related to the IL23/Th17 pathway1, 2. However, little is known regarding the environmental factors that directly influence Th17 cells. Here we show that increased salt (sodium chloride; NaCl) concentrations found under physiological conditions in vivo dramatically boost the induction of murine and human Th17 cells. High salt conditions activates the p38/MAPK pathway involving the tonicity-responsive enhancer binding protein (TonEBP/NFAT5) and the serum- and glucocorticoid-inducible kinase 1 (SGK1) during cytokine-induced Th17 polarization. Gene silencing or chemical inhibition of p38/MAPK, NFAT5 or SGK1 abrogates the high salt induced Th17 cell development. The Th17 cells generated under high salt display a highly pathogenic and stable phenotype as compared to cells differentiated under normal conditions characterized by the up-regulation of the pro-inflammatory cytokines GM-CSF, TNFα and IL-2. Moreover, mice fed with a high salt diet develop a very severe form of EAE in line with increased central nervous system infiltrating and peripherally induced antigen specific Th17 cells. Thus, increased dietary salt intake might represent an environmental risk factor for the development of autoimmune diseases through the induction of pathogenic Th17 cells.
Abstract 461: Role of Physical Training Intensity in Metabolic and Cardiovascular Dysfunctions in a Fructose Overload Model
Authors: Machi Jacqueline F , Bernardes Nathalia , Dias Danielle S , Mostarda Cristiano , Moreira Edson , De Angelis Kátia , Krieger Eduardo M , Irigoyen Maria Cláudia C , Wichi Rogéo B
Published: July 2021

This study evaluated the chronic effects of the run and walk in the metabolic and cardiovascular parameters of a metabolic syndrome experimental model. Male Wistar rats were divided into 4 groups(n=8): Control (C),Sedentary Fructose (SF), Fructose Run (FR) and Fructose Walk (FW, n= 8). Metabolic syndrome (MS) induction was performed with D-fructose in drinking water for 18 weeks. The exercise training was initiated after the nineth week of treatment with fructose and was held for 8 weeks (60 minutes/day, 5 times / week). The FW and FR were performed on a treadmill (1 h/day; 5 days/wk for 8 wk), with ∼20% and 60% intensities respectively of the maximum speed in a maximal exercise test. Plasma glucose, triglycerides, insulin resistance, adipose tissue, blood pressure, heart rate, baroreceptor sensitivity and sympathetic and parasympathetic tone, were evaluated at the end of protocol. The results showed that run and walking decreased the adipose tissue (FR: 2.97±0.2; FW: 4.26±0.9; SF: 6.49±0.6; C: 3.23±0.2 g). The glycemia values remained within the normal range,(FR: 86.7±2.3; WF: 91.0±1.4; SF: 70.2±1.9; C: 84±2.3 mg/dl), however only the FR group decreased the triglycerides levels (FR: 133±8.8; FW: 159±10.2; SF: 220±6.3; C: 96± 4.2 mg/dl), and the insulin resistance (FR: 4.37±0.1; FW: 3.55±0.2; SF: 2.79±0.3; C: 4.86±0.3 %/min). The FR group showed a reduction in mean arterial pressure (FR: 111±4.5, FW: 125±4.1; SF: 137±2.6, C: 113±1.5 mmHg) and increased of bradycardic (FR 1.76±0.08; FW 1.31±0.10; SF 1.37±0.10; C 1.72±0.14 bpm/mmHg) and tachycardic response to BP changes (FR 4.02±0.32; FW 2.56±0.16; SF 1.97±0.15; C (and C 3.25±0.37 bpm/mmHg). Finally we observed that only the FR group showed an increase of the vagal tone (FR: 72.3±8.1, FW: 47.3±6.7; FS: 40.3±4.6, C: 60.7±6.5 bpm). In conclusion, our results suggest that training walk (FW), a practice widely recommended, is especially effective for the treatment of metabolic disorders, whereas controlled exercise (FR) seems to encompass hemodynamic and metabolic aspects. This application is easy and within reach of the majority of the population, indicating that this practice should be encouraged and may be effective in managing cardiovascular risk in MS as start therapeutic. Sources of Funding:FAPESP.
Abstract 474: Impaired Excitability of Renal Afferent Innervation in an in-vitro Model of Inflammation.
Authors: Freisinger Wolfgang , Lale Nena , Ditting Tilmann , Heinlein Sonja , Karl Annalena , Schmieder Roland , Amann Kerstin , Veelken Roland
Published: July 2021

INTRODUCTION AND AIMS:
Renal denervation may be beneficial in hypertension with inflammatory renal disease: in hypertension renal afferent control of sympathetic nerve activity could be impaired. Recently, we found that afferent renal neurons showed a characteristic excitability, exhibiting predominantly a sustained firing upon current injection. So far, excitability of these neurons under pathological conditions such as renal inflammation in hypertension is unclear. Hence, in an
in vitro
model we wanted to test the hypothesis that a proinflammatory mediator like CXCL1 could alter the firing pattern of neurons with renal afferents and thus decrease their excitability.

METHODS:
Dorsal root ganglion (DRG) neurons (Th11-L2) were incubated with the chemokine CXCL1 (1,5nmol/ml) for 12 hours before patch clamp recordings. Labelling (DiI) allowed the identification of renal afferent neurons. Current clamp was used to characterize neurons as “tonic”, i.e. sustained action potential (AP) firing or “phasic”, i.e. <5 APs according to their firing response to current injections. AP properties were determined in renal and non-renal neurons incubated with CXCL1 and compared to controls.

Results:
Renal afferent DRG neurons exhibited in 57% (23 of 41) a tonic firing pattern vs.11,7% (3 of 27)* in non-renal neurons. However, exposed to the chemokine, renal DRG neurons exhibited significantly to a lesser degree tonic firing (35,6% [35 of 101] vs. 57% [23 of 41], *p<0,05) but instead an increased occurrence of phasic firing. Renal DRG neurons with phasic firing pattern showed a significantly lower threshold for AP-firing (600pA [320-1000] vs.1000pA [400-3200]) and a significantly shorter AP-duration at threshold-level (2,095ms [1,75-4,25] vs. 5,15ms [4,3- 8,7]) after exposure to CXCL1.

Conclusion:
We could show that after exposure to a proinflammatory mediator like CXCL1, renal afferent DRG neurons exhibited a significantly higher proportion of neurons with a phasic pattern (<5 APs) and decreased excitability as compared to control conditions. Significant changes in action potential properties of phasic neurons point to an altered sodium channel expression likely inducing a faster inactivation of these channels with decreased firing activity.
Abstract 486: Artificial miRNA Based Adenoviral Adam17 Silencing Reveals Essential Role of This Metalloprotease For EGF Receptor Transactivation In Primary Cultured Vascular Smooth Muscle Cells
Authors: Elliott Katherine , Bourne Allison , Takayanagi Takehiko , Takaguri Akira , Eguchi Kunie , Eguchi Satoru
Published: July 2021

siRNA mediated gene silencing has been recently utilized as a powerful molecular tool to study the functional significance of a specific protein. However, due to the transient nature of silencing and insufficient transfection efficiency, this approach can be problematic in primary cell culture. To overcome such weakness of the siRNA based silencing and in order to establish reliable gene silencing in vascular cells, we devised an adenoviral-encoded miRNA based gene silencing system. Here we report the results of silencing ADAM17 in cultured rat vascular smooth muscle cells (VSMCs) and its functional consequences in angiotensin II (AngII) signal transduction. Four distinct miRNA sequences targeting rat ADAM17 were chosen based on recommendations from Invitrogen’s Block-iT RNAi Designer algorithm. The miRNA sequences were inserted into a mammalian expression vector, pcDNA 6.2-GW/EmGFP-miR, and the effective silencing by these vectors was confirmed in HEK cells expressing HA-tagged rat ADAM17. The 4 cassettes carrying the miRNAs were inserted into pAd/CMV/V5-DEST and adenoviral solutions were obtained. Greater than 95% silencing of ADAM17 was achieved when VSMC were infected with 100-200 moi of the ADAM17 miRNA encoding adeonvirus for 72 h with enhancement of infection by fugene6. Relatively linear time and concentration dependencies were observed between 1 to 3 days and 10 to 100 moi of the infection. A miR-ADAM17 (100 moi) but not miR-control (100 moi) completely inhibited 100 nM AngII-induced HB-EGF shedding in VSMCs as assessed by a reporter assay. A miR-ADAM17 but not miR-control also inhibited AngII-induced EGF receptor transactivation and subsequent ERK1/2 activation in VSMCs as assessed by immunoblotting with phospho-selective antibodies. In conclusion, ADAM17 was found to be a major sheddase for HB-EGF contributing to the growth promoting signals induced by AngII in VSMCs. An artificial miRNA-base adenoviral approach appears to be a reliable gene-silencing strategy for signal transduction research in primary cultured vascular cells.
Abstract 499: Salt-sensitivity During Aging: Good or Bad?
Authors: Chugh Gaurav , Asghar Mohammad
Published: July 2021

Angiotensin II AT1 receptor (AT1R) and dopamine D1 receptor (D1R) are key kidney receptors involved in sodium metabolism and blood pressure regulation. Recently, we reported that aging in Fischer 344 X Brown Norway (FBN) rats is associated with higher renal AT1R and diminished D1R functions, while in Fischer 344 (F344) rats it is associated with only diminished D1R function. We tested the hypothesis that FBN and not F344 rats are salt sensitive during aging. Old (21-24 month) FBN and F344 rat strains were fed with high salt (HS, 8% NaCl) for 4 weeks and different parameters were measured. Both day (in mmHg; Control vs HS: 119+2 vs. 125+2) and night (in mmHg; Control vs HS: 116+2 vs. 123+2) blood pressures (BP) increased with HS in FBNs. BP did not change with HS in F344 rats (in mmHg; Control vs HS: 110+7 vs 111+9). Despite a similar food intake, there were marked decrease in body weight gain (in gm; Control vs HS: 38+7 vs 18+5) as well as fasting levels of blood glucose (in mg/dl; Control vs HS: 127+6 vs 105 + 4) and triglyceride (in mg/dl; Control vs HS: 157+14 vs 90+7) in HS fed compared to control FBNs. Plasma insulin and blood HDL cholesterol levels remained unchanged between HS fed and control FBNs. These results, together with our previous report, suggest that (1) both higher renal AT1R and diminished D1R function contributes to salt-sensitive hypertension in aging. (2) FBN and F344 rats may serve as useful aging models to study molecular targets that leads to and protect from the development of salt-sensitive hypertension, respectively, and (3) HS via changes in metabolic process may produce some beneficial effects.
Abstract 51: Periostin: a Novel Mediator of Renal Disease
Authors: Mael-ainin Mouna , Abed Ahmed , Conway Simon , Dussaule Jean-Claude , Chatziantoniou Christos
Published: July 2021

Introduction:
Chronic Kidney Disease (CKD) is characterized by progressive decrease in renal function related to a progressive accumulation of fibrosis. The lack of efficient treatment makes urgent the finding of novel targets for therapy. In previous studies we have shown that periostin, a protein normally involved in the regulation of osteoblasts’ differentiation, is abnormally and highly expressed in several models of CKD, and is inversely correlated with the decline of renal function.

Objectives:
To investigate whether periostin, in addition to be a biomarker of CKD, is also involved in mechanisms leading to the development of renal disease.

Methods:
Unilateral ureteral obstruction (UUO) was applied in periostin knock-out/beta-galactosidase knock-in and wild type littermate mice. The expression of periostin was evaluated by quantitative RT-PCR, Western Blot and immunocytochemistry in the wt mice. The localization of periostin synthesis within renal cortex was determined by the reporter gene LacZ in the KO mice. The expression of other genes, well-known for their pro-fibrotic and inflammatory action was also studied by RT-PCR, WB and IHC.

Results:
Following UUO, periostin expression (mRNA and protein) was several-fold induced in the tubular interstitium of wt mice. Periostin synthesis occurred within the collecting duct in the beginning of the disease, and then around altered/dilated tubules as the disease progressed. Periostin KO mice were protected compared to wild type as they displayed less tubular dilation and fibrosis (p<0.01), and the activation of genes associated to renal disease like collagen I, TGFbeta, vimentin and MCP-1 was blunted (p<0.01). In addition, periostin KO mice displayed increased rates of proliferation of tubular cells ( p<0.001), and preserved E-cadherin expression.

Conclusion:
Induction of periostin expression is an early and important event of CKD. Genetic inhibition of periostin expression leads to a better preservation of the renal tissue in the UUO model. These results suggest that blocking periostin can be a novel and promising target against the development of CKD
Abstract 517: The CYP4A-20-HETE System Regulation of Endothelial Progenitor Cell Functions that Associated with Angiogenesis
Authors: Chen Li , Braverman Alexander , Zhang Frank , Falck John , Arbab Ali Syed , Schwartzman Michal , Scicli A Guillermo , Guo Austin M
Published: July 2021

The CYP4A-20-HETE system regulates the neovascularization process via its potent vascular effects mainly on endothelial cells and vascular smooth muscle cells. Endothelial progenitor cells (EPC) also actively participate in physiological and pathological neovascularization. Our group first reported that the CYP4A-20-HETE system is present and functional in EPC derived from human umbilical cord blood (HUCB) and that EPC also respond to exogenous 20-HETE with increased proliferation and migration. We hypothesized that the angiogenic actions of the CYP4A-20-HETE system may involve regulation of EPC functions that associated with angiogenesis. In this study, we identified CYP4A11/22 as the main 20-HETE synthases in EPC derived from HUCB by real time PCR. We also examined the effects of exogenous 20-HETE on EPC adhesion since adhesion of EPC to extracellular matrix is an important aspect of EPC homing to the sites where angiogenesis is occurring. We found that 20-HETE (1 μM) increased EPC adhesion to fibronectin and SDF-1α coating by ∼40% and ∼35%, respectively. These increases in adhesion are completely abolished in the presence of 20-hydroxy-6, 15-eicosadienoic acid (20-HEDE), a 20-HETE antagonist. We further established the mouse ischemic hindlimb model to study the effects of pharmacological inhibition of the CYP4A/F-20-HETE system using the 20-HETE synthase inhibitor Dibromo-dodecenyl-methylsulfimide (DDMS) and 20-HEDE on compensatory angiogenesis in response to ischemia. Systemic treatment of animals with 10 mg/kg/day of either DDMS or 20-HEDE inhibited hindlimb compensatory angiogenesis by more than 50% without significant effects on the blood pressure. Specific targeting of the EPC-derived CYP4A-20-HETE system needs to be performed to further dissecting the role of systemic and EPC-derived 20-HETE on angiogenic processes. These findings implicates the CYP4A-20-HETE system as a novel regulator of EPC functions that are associated with angiogenesis and suggests that it can act as both an autocrine and paracrine regulatory factor.
Abstract 524: Extracellular Guanosine Regulates Extracellular Adenosine Levels
Authors: Jackson Edwin K , Gillespie Delbert G
Published: July 2021

Extracellular adenosine modulates cardiovascular and renal function. While measuring extracellular purines in biological samples, we observed a correlation between levels of adenosine and guanosine. This observation led us to test the hypothesis that extracellular guanosine regulates extracellular adenosine levels in the cardiovascular and renal systems. Rat preglomerular vascular smooth muscle cells in culture were incubated with adenosine and/or guanosine. In the absence of added adenosine, exogenous guanosine (30 μmol/L) had little effect on extracellular adenosine levels, indicating that extracellular guanosine does not trigger the release or production of adenosine. Without added guanosine and 1 hour after adding 3 μmol/L of exogenous adenosine, extracellular adenosine levels were only 0.125 ± 0.020 μmol/L, indicating rapid disposition of extracellular adenosine by a monolayer of cells. In contrast, extracellular adenosine levels 1 hour after adding 3 μmol/L of adenosine plus guanosine (30 μmol/L) were 1.173 ± 0.061 μmol/L (9-fold higher; p<0.0001), indicating slow disposition of extracellular adenosine in the presence of extracellular guanosine. Extracellular guanosine impeded the disposition of extracellular adenosine not only in preglomerular vascular smooth muscle cells, but also in rat preglomerular vascular endothelial cells, mesangial cells, cardiac fibroblasts and kidney epithelial cells, as well as in human aortic vascular smooth muscle cells, coronary artery vascular smooth muscle cells and coronary artery endothelial cells. In rats, infusions of guanosine per se had little effect on cardiovascular/renal variables, yet markedly enhanced the effects of co-infusions of adenosine. For example, in control rats, adenosine (0.3 μmol/kg/min) only modestly decreased mean arterial blood pressure (from 114 ± 4 to 100 ± 4 mm Hg). In contrast, in guanosine-treated rats (10 μmol/kg/min), adenosine profoundly decreased blood pressure (from 109 ± 4 to 79 ± 3 mm Hg; p<0.0001 vs non-guanosine treated group).

Conclusion:
Extracellular guanosine powerfully regulates extracellular adenosine levels by altering adenosine disposition and this occurs in many, perhaps most, cell types in the cardiovascular system and kidneys.
Abstract 56: Endothelial Adam17 Contributes to Angiotensin Ii-induced Abdominal Aortic Aneurysm in Mice
Authors: Kobayashi Tomonori , Takayanagi Takehiko , Hashimoto Tomoki , Blobel Carl P , Eguchi Satoru
Published: July 2021

Enhancement of the renin angiotensin II (AngII) system has been implicated in the development of abdominal aortic aneurysm (AAA) in animal models as well as in clinical studies. However, detailed molecular mechanism(s) by which AngII promotes AAA remain uncertain. We have demonstrated the critical roles of a metalloprotease, ADAM17, in AngII signal transduction in cultured vascular cells. Endothelial AT1 receptor has been recently implicated in oxidative stress. Interestingly, others have reported that ADAM17 expression was enhanced in CaCl2-induced AAA in mice and that systemic ADAM17 silencing attenuated AAA formation. However, the cell type that is mediating the deleterious effect of ADMA17 signaling is not well understood. Here we tested our hypothesis that endothelial ADAM17 activation is required for AngII-promoted AAA formation using ADAM17flox/flox mice bred with Tie2 Cre transgenic mice. 8 week old mice were co-infused with AngII 1000 ng/kg/min (4 weeks) and beta-aminopropionitrile, a lysyl oxidase inhibitor, 150 mg/kg/day (2weeks) or control saline (4 weeks) via osmotic mini-pump, and AAA formation was evaluated by the diameters. AAA formation was attenuated in ADAM17flox/flox Tie2 Cre(+/-) mice compared with ADAM17flox/flox Tie2 Cre(-/-) mice (1.50±0.44 mm vs 3.96±0.61 mm). Cardiac hypertrophy evaluated by heart weight body weight ratio was also attenuated in the Tie2 Cre(+/-) mice (0.0063±0.0005 vs 0.0092±0.0013). However, AngII induced hypertension to a similar degree in both mice groups, as assessed by telemetries. The ADAM17 deletion was associated with less oxidative stress as assessed by anti-nitrotyrosine IHC and less extravascular fibrosis/matrix deposition. Also, the aorta was less stained with MMP2 antibody and phospho-JNK antibody. In conclusion, endothelial ADAM17 appears to be a critical metalloprotease contributing to AAA formation and cardiac remodeling but not hypertension induced by AngII. The mechanism by which endothelial ADAM17 promotes AAA seems to involve induction of oxidative stress, JNK activation and subsequent MMP2 induction.
Abstract 607: Antiarrhythmic Effects Of Omega-3 Fatty Acids And Cyp-eicosanoids In A Murine Atrial Fibrillation Model
Authors: Spallek Bastian , Westphal Christina , Konkel Anne , Wallukat Gerd , Zeldin Darryl C , Falck J. R. , Mueller Dominik , Fischer Robert , Schunck Wolf-Hagen
Published: July 2021

Atrial fibrillation (AF) is the most common arrhythmia in man. Small animal models of AF are rare, limiting the opportunities of mechanistic studies and of evaluating novel treatment strategies. We found that mice can be rendered susceptible to AF by chronic ß-adrenergic stimulation and used this model to test the hypothesis that dietary n-3 polyunsaturated fatty acids (n-3 PUFAs) and endogenously produced cytochrome P450 (CYP)-dependent epoxyeicosanoids protect against AF. Our study included male wild type C57BL/6 mice given either normal chow (WT-n6-group) or a diet supplemented with n-3 PUFAs (2.5% EPA/DHA added to normal chow; WT-n3-group), as well as transgenic littermates with cardiomyocyte-specific overexpression of the human epoxygenase CYP2J2 (CYP-n6 group). All animal groups received 40mg/kg/d of isoproterenol (ISO) or vehicle over 14 days via osmotic mini-pumps. AF induction was tested
in vivo
by programmed electrical stimulation (PES) and reached 48% (13 of 27 protocols) in ISO-treated WT-n6, compared to 9% (2 of 22) in the vehicle group. ISO-treated WT-n6 mice also featured increased atrial fibrosis, decreased atrial connexin-40 expression, and significantly reduced atrial refractoriness (AERP: 13.1±0.5 vs. 21.7±0.9 ms in vehicle-infused controls). Ventricular refractoriness remained unchanged and ventricular arrhythmias were not inducible. EPA/DHA-supplementation as well as CYP2J2 overexpression significantly reduced ISO-stimulated AF inducibility to 17% (5 of 30 in WT-n3, and 4 of 24 in CYP-n6), decreased atrial fibrosis, and partially prevented connexin-40 down-regulation and AERP reduction (16,1±0,9 in WT-n3 and 17.5±0.5 ms in CYP-n6). Finally, we tested the antiarrhythmic potential of a synthetic analog of 17,18-epoxyeicosatetraenoic acid (17,18-EEQ), an EPA metabolite predominantly produced by CYP2J2 and other epoxygenases. Acute i.v. injection of the 17,18-EEQ compound decreased AF inducibility in ISO-stimulated WT-n6 mice from 67% (14 of 21 in vehicle control) to 33% (10 of 30) and also the mean duration of AF episodes. These results show that ISO-infusion allows establishing a suitable mouse model of AF and indicate an important role of CYP-dependent n-3 PUFA metabolites in preventing AF.
Abstract 613: Interactive Effect of Ace Inhibition and DPP4 Inhibition on Substance P-Stimulated T-PA Release
Authors: Devin Jessica K , Pretorius Mias , Billings Frederic T , Brown Nancy J
Published: July 2021

Dipeptidyl peptidase 4 (DPP4) inhibitors improve glycemic control in patients with T2DM by preventing the degradation of the incretin hormones. DPP4 inhibition prevents the degradation of other peptides with a penultimate proline or alanine, including the vasodilator substance P and the metabolite of bradykinin formed by its cleavage by aminopeptidase P. Substance P and bradykinin are normally inactivated by ACE. This study tested the hypothesis that during ACE inhibition, DPP4 inhibition would potentiate the effects of substance P. Ten healthy, lean (BMI<30 kg/m
2
) subjects, age 24-60 years of old (2 Black, 8 White, 6 Female) participated in this randomized, double-blinded, placebo-controlled crossover study. On separate study days separated by at least one week, Subjects received sitagliptin 200 mg po or matching placebo. Substance P (2, 4, 8 pmol/min) and bradykinin (25, 50, 100 ng/min) were given via the brachial artery before and after administration of intra-arterial enalaprilat (0.33 mcg/min/100mL forearm volume). Enalaprilat potentiated the FBF and tissue plasminogen activator (t-PA) responses to bradykinin and there was no effect of sitagliptin (Figure). Neither ACE inhibition nor DPP4 inhibition altered the FBF flow response to substance P. In contrast, there was an interactive effect of ACE inhibition and DPP4 inhibition on t-PA release in response to substance P (p=0.026).
Abstract 615: Renal Sympathetic Denervation: A Comparison Of Irrigated And Non-irrigated Radiofrequency Ablation In A Porcine Model
Authors: Koruth Jacob S , Dukkipati Srinivaz , Miller Marc A , Neuzil Petr , D'avila Andre , Reddy Vivek Y
Published: July 2021

Introduction:
Renal artery sympathetic denervation using radiofrequency (RF) energy has recently been shown to reduce blood pressure in patients with hypertension. However arterial damage is required to achieve denervation, which has unknown long term consequences. Alternative techniques of denervation that minimize arterial wall damage are therefore preferable. We compared the ability of irrigated RF vs. non-irrigated RF to ablate the nerve fibers with respect to the degree of arterial wall sparing in an in vivo model.

METHODS:
Percutaneous femoral arterial access was obtained in 14 adult

swine and renal angiography was performed to assess for stenosis at baseline and at time of sacrifice . Irrigated (3.5mm tip/30cc) and non-irrigated RF (3.5, 4mm and 2mm tips) energy was delivered bilaterally using custom catheters. All animals were then recovered and survived

for 30-90 days. Renal cortical tissue was collected from bilateral kidneys for norepinephrine levels prior to sacrifice.

Results:
There was no significant interval renal artery stenosis. Arteries were sequentially sectioned, processed, cut at 4-6 μm and stained with hematoxylin&eosin and movat pentachrome. Arterial medial injury (percentage depth) and nerve injury were then scored on a incremental scale of 0-4. Medial injury was significantly less in the irrigated group vs. non-irrigated group (1.15vs 2.2 p<0.05). Nerve injury on the other hand was similar in both groups (1.75 vs. 1.24,p=0.24). Norepinephrine levels were obtained in 11/14 animals. In the irrigated and non-irrigated arms there was a 67% and 53% reduction of norepinephrine levels compared to that of controls.

Conclusions:
Irrigated RF ablation is an efficacious and feasible means to achieve renal denervation. Although medial injury occurred in both groups, irrigated RF demonstrates a lower degree of medial injury for similar levels of nerve injury when compared to non-irrigated RF.
Abstract 620: Chronic Allium Bakeri Intake Alleviated Hypertension and Aortic Media Thickness in 2-Kidney, 1-Clip Rats.
Authors: Kitamura Saori , Hashimoto Hiroko , Segawa Yukiko , Kurihara Nobutaka
Published: July 2021

Objective:
Allium bakeri
(AB) known as “rakkyo” and
Allium sativum
known as “garlic” contain alliin. Alliin easily turns into allicin, which is reported to decrease blood pressure (BP) through activating the excitatory ion channel transient receptor potential ankyrin-1. Thus, it is suggested that these vegetable decrease BP. Actually, garlic is reported to decrease BP and aortic media thickness in hypertensive animal models. However, it is practically difficult that garlic shows the effects, because it can hardly be eaten without heating and allicin is thermolabile. On the other hand, AB is usually eaten without heating. Therefore, in this study, we tested whether AB intake alleviates hypertension and aortic media thickness in hypertensive rats.

Method:
We used 2-kidney, 1-clip (2K1C) renovascular hypertension model. Male Sprague-Dawley rats (6 wo) were treated with sham operation (SHAM) or clipping the left renal artery (2K1C). After surgery, SHAM and 2K1C rats were respectively divided into 2 groups, which received a control diet (CONT) or a diet containing 1.0 % (w/w) freeze-dried AB powder for 7 weeks (AB). The systolic BP (SBP) was measured by a tail-cuff method every week. At the end of the protocol, mean arterial BP (MAP) was measured in each rat under anesthesia. Then, thoracic aorta was removed and the section of aorta was stained with hematoxylin and eosin.

Results:
From the 2nd week after surgery until the end of the protocol, SBP in 2K1C-CONT went up significantly compared with SHAM-CONT (174 ± 7 vs 120 ± 5 mmHg, at the end of the protocol, P < 0.01). However, SBP in 2K1C-AB did not go up through the protocol (127 ± 7 mmHg, P < 0.01 with 2K1C-CONT, at the end). In addition, the AB diet showed no significant effects on SBP in SHAM. These observations in SBP were similar to in MAP at the end of the protocol. The aortic media thickening was observed in 2K1C-CONT compared with SHAM-CONT. In 2K1C-AB, the thickness was significantly decreased compared with 2K1C-CONT, and was not significantly different from that in SHAM-AB. There were not significant differences between SHAM-CONT and SHAM-AB.

Conclusion:
Chronic intake of Allium bakeri, which can be eaten without heating, alleviated hypertension and aortic media thickness in rats with renovascular hypertension.
Abstract 63: Relation between Poor Sleep Quality and Resistant Hypertension
Authors: Bruno Rosa Maria , Palagini Laura , Di Giulio Alessia , Gemignani Angelo , Taddei Stefano , Ghiadoni Lorenzo
Published: July 2021

Purpose:
Insomnia and short sleep duration have been associated with increased incidence of hypertension. However, the relationship between sleep loss and resistant hypertension (RH), has not been ascertained yet.

Methods:
270 patients at first access to our Hypertension Outpatient Unit were enrolled. RH was defined according as office BP values >140/90 mmHg with 3 or more antihypertensive drugs, or controlled BP with 4 or more drugs, including a diuretic. Pittsburgh Sleep Quality Index (PSQI), and Beck Depression Inventory (BDI) were assessed. Poor sleep quality was defined as PSQI>5, mild-to-severe depressive symptoms as BDI score >10. Patients with self-reported sleep apneas or snoring were excluded (n=21).

Results:
Complete data were available for 234 patients (males 51%, mean age 58±13 years, BMI 26±5 kg/mq, antihypertensive treatment 84%, previous CV events 12%, diabetes 8%, smoking 15%, hypercholesterolemia 60%). Mean sleep duration was 6.4±1.6 hours, with a 49% prevalence of short sleep duration (<6 hours) and similar in both sexes. Conversely, women had higher PSQI scores (5.2 vs 3.6, p=0.03) and prevalence of poor sleep quality (46% vs 30% in men, p=0.01). Women showed also higher BDI scores (4.5 vs 1.8, p=0.006) and prevalence of depressive symptoms than men (20% vs 7%, p=0.003). RH patients (15%) had higher PSQI (5.8 vs 4.1, p=0.03), a difference shown in women (6.8 vs 4.8, p=0.04) but not in men (4.7 vs 3.5, p=0.37). The association between BDI score and RH showed a similar behavior (overall 3.6 vs 2.8, p=0.02; women 5.1 vs 3.7, p=0.03; men: 2.0 vs 1.9, p=0.53). In a multiple logistic regression analysis (including age, sex, obesity, diabetes, previous CV events, sleep duration, use of hypnotic drugs) poor sleep quality was independently associated with RH (OR 2.2, CI95% 1.1-5.3). However this relationship lost significance when also depressive symptoms were included in the model (p=0.12).

Conclusions:
Short sleep duration is highly prevalent in hypertensive patients. This condition is accompanied by poor sleep quality and depressive symptoms in women. Poor sleep quality is associated with a 2-fold higher probability of having RH. This association could be mediated by the presence of depressive symptoms.
Abstract 632: Flow-induced TRPV4 Activation Mediates Calcium Influx In The Thick Ascending Limb.
Authors: Cabral Pablo D , Garvin Jeffrey L
Published: July 2021

Background:
The thick ascending limb is critical in the regulation of sodium balance and therefore blood pressure. We previously showed that nitric oxide (NO), which inhibits sodium chloride transport and therefore blood pressure is regulated by luminal flow in this segment. Transient receptor potential vanilloid 4 (TRPV4), a member of the TRPV family of cation channels, is expressed in the thick ascending limb. This calcium permeable channel can be activated by luminal flow in other cell types. Therefore, we hypothesized that in the thick ascending limb luminal flow induces TRPV4 activation thereby increasing calcium influx and NO production.

METHODS:
We used the TRPV4 antagonists Ruthenium red and RN 1734 and the TRPV4 agonist GSK1016790A and measured intracellular calcium and NO production in the absence and presence of luminal flow in isolated and perfused thick ascending limbs from Sprague Dawley rats. NO production was measured using the NO-sensitive dye DAF FM da. Intracellular calcium was measured using the ratiometric calcium sensitive dye Fura-2 AM.

Results:
Increasing luminal flow from 0 to 20 nL/min stimulated NO production from 8 ± 3 to 45 ± 12 arbitrary units (AU)/min (
p
<0.05,
n
=5). Increasing luminal flow in the presence of the TRPV4 antagonist Ruthenium red (15 μM) eliminated NO production (from 18 ± 5 to 16 ± 9 AU/min;
n
= 4). Flow-induced NO production was also prevented by the selective TRPV4 antagonist RN 1734 (10 μM) (from 11 ± 7 to 9 ± 2 AU/min;
n
= 4). Increasing luminal flow increased Fura 2 ratio units by 113 ± 20 %. However, in the presence of the TRPV4 antagonist Ruthenium red (15 μM) Fura 2 ratio units increased only by 55 ± 12 % in response to luminal flow (
p
<0.04,
n
=6). In the absence of luminal flow, activating TRPV4 using the TRPV4 selective agonist GSK1016790A (1 ηM) increased Fura 2 ratio units by 204 ± 43 %.

Conclusion:
From these data we conclude that flow-induced TRPV4 activation mediates calcium influx and NO production in the thick ascending limb.
Abstract 653: G-protein-coupled Receptor Kinase 4 Binds Preferentially To The Caveolin-1α Isoform In Rat Renal Proximal Tubule Cells
Authors: Bigler Wang Dora , Gildea John J , Felder Robin A
Published: July 2021

Caveolins, localized in lipid rafts of plasma membranes, tether and regulate signaling complexes into functional units and have been shown to inhibit g-protein-coupled receptor kinases GRK2 and GRK5 via the caveolin scaffolding domain. In human renal proximal tubule cells (RPTC), the dopamine-1-receptor (D1R) is phosphorylated and inhibited by G protein-coupled receptor kinase 4 (GRK4). Defects in D1R coupling leads to sodium-retention and a rise in blood pressure. We showed earlier that chronic disruption of lipid rafts or Caveolin-1 (Cav1) protein expression in the kidney induces hypertension in rats by relieving the steric inhibition of Cav1 on GRK4. Here we extend these studies by examining caveolin expression in renal proximal tubule cells microdissected from Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). We discovered a significant decrease in Cav1 and Cav2 expression in SHR compared to WKY (98 ±1.2% reduction; n=3, P<0.001). In order to determine if the D1R coupling defect found in these SHR renal proximal tubule cells is due to loss of either of these proteins, we made stable SHR cell lines expressing either Cav1α, Cav1β or Cav2. Using co-immunoprecipitiation and western blot analysis, we demonstrate for the first time that GRK4 and Cav1 physically interact in rat cells, and that it is the Cav1α isoform that binds predominantly to GRK4 (Cav1β, Cav2 and vector control show a 90 ±1.5%, 84±0.8% and 87±2.1% reduction, respectively; n=3, P<0.001). Similar to an effect found in mouse Cav1 knockout animals, we find that Cav2 is not stable without the co-expression of Cav1. Our data suggest that Cav1α. but not Cav1β, is important for normal D1R function by binding to and inhibiting GRK4, implicating a domain other than the caveolin scaffolding domain in this interaction. These findings might be used in designing selective GRK4 inhibitors as novel antihypertensive compounds.
Abstract 9: Endothelin B Receptors Protect Against Hypertension Induced by Chronic Angiotensin II in Female Rats
Authors: Kittikulsuth Wararat , Pollock David M
Published: July 2021

Endothelin B (ET
B
) receptors mediate vasodilation, anti-inflammation and natriuresis, which ultimately contribute to blood pressure control. We previously showed that renal medullary ET
B
receptor function is maintained in female angiotensin (Ang) II hypertensive rats, while male Ang II hypertensive rats have blunted ET
B
-induced natriuretic responses. Because female rats are more resistance to blood pressure elevation induced by high salt intake and/or Ang II infusion, we hypothesized that ET
B
receptors protect female rats against the hypertensive response and renal injury induced by a high salt diet and chronic Ang II infusion compared to males. Male and female rats received Ang II infusion (150 ng/kg/min; sc.) with 4% NaCl for 4 weeks; blood pressure was measured by telemetry. After a week of Ang II infusion with a high salt diet, subsets of both male and female rats received the ET
B
antagonist, A-192621, at three doses on consecutive weeks (1, 3, and 10 mg/kg/d in food). Male rats had a significantly higher blood pressure compared to females after 4 weeks of Ang II (178±10 vs. 138±10 mmHg; p<0.05). A-192621 resulted in a dose-dependent increase in blood pressure in female Ang II hypertensive rats (167±8 mmHg at 10 mg/kg/d; p<0.05); the increase produced by A-192621 in male Ang II hypertensive rats was not statistically significant (193±10 mmHg). After 4 weeks of Ang II infusion, the level of proteinuria and nephrinuria was higher in male rats compared to female. A-192621 did not further increase urinary excretion of protein or nephrin in both male and female Ang II hypertensive rats. In conclusion, these results support the hypothesis that ET
B
receptors provide more protection against hypertension during chronic Ang II infusion in female rats compared to male.