bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 391: Enhanced Coronary Vasoconstriction in Western Diet-Induced Obesity is Associated with Alterations in NHE1, SERCA2a and 3
Authors: Habibi Javad , Bender Shawn B , Garro Monna , Mugerfeld Irina , Rehmer Nathan , Manrique Camila , Nistala Ravi , Whaley-Connell Adam , Sowers James R
Published: July 2021

Cardiovascular disease (CVD) remains a major risk factor and cause of mortality in type 2 diabetes but, the effect of western diet (WD) on coronary artery vasoconstriction has not been well characterized. Therefore, the purpose of this study was to identify potential calcium (Ca
2+
) molecular mechanism(s) underlying alterations in coronary constriction in WD-induced obesity. C57Bl/6J mice were fed a WD or control diet for 16wks. Coronary vascular function was assessed with wire myography and human coronary vascular smooth muscle cells (VSMC) were cultured. WD enhanced coronary constriction to the thromboxane analog
U46619.
This was associated with increased coronary VSMC expression of sodium-hydrogen exchanger 1 (NHE1) and SERCA3 with decreased expression of SERCA2a. Oxidation of SERCA2a cysteine-674, a signal for SERCA2a degradation, was increased in WD coronary VSMC. Exposure of cultured human coronary VSMC to hyperinsulinemic conditions elicited similar changes in SERCA2a, 3 and NHE1. This study suggests that enhanced thromboxane-mediated coronary constriction in WD and insulin resistance/hyperinsulinemia may result from NHE1-dependent changes in intracellular pH and/or impaired Ca
2+
handling at the level of the sarcoplasmic reticulum.
Abstract 392: Blood Pressure Variability: An Independent Marker to Morbimortality
Authors: Paulini Janaina , Nishi Fernanda , Jorge Luciana , Pires Romulo , Krieger Eduardo Moacir , Irigoyen Maria Claudia
Published: July 2021

Introdution:
The purpose of this study was to investigate the mechanisms enrolled in the relationship between increased BP variability, heart damage and mortality in different pathophysiological conditions as hypertension, heart failure and myocardial infarction.

Method:
Wistar rats were divided into control and S (n=10, 8weeks). Sinoaortic denervation (S) was performed using the method previously described. BP and HR were analysed using a data acquisition system. BP variability was analyzed by spectral analysis. Left ventricle systolic ejection and diastolic isovolumetric relaxation time were evaluated by echocardiography. Cardiac fibrosis and cardiomyocytes diameter were quantified by histological analysis. CO, cardiac blood flow and cardiac index were evaluated using colored microspheres.

Result:
No difference was observed in BP and HR (C:104±2 vs 106±2 mmHg; C:313±7 vs 329±3 bpm), while BP variability was increased after S (C:29±2 vs 46*±5 mmHg). Left ventricle systolic ejection was decreased (C:72±0.9 vs 64*±1.5%) and diastolic isovolumetric relaxation time (C:21±1 vs 35*±1 ms) was increased in S group. Moreover, it was observed a reduction in CO (C:111±5 vs 81*±5 mL/min), an increase in cardiac resistance (C:35±1.8 vs 66*±2.6 mL/min/mmHg) and a decreased in cardiac index (C:320±9.7 vs 270*±8.1 l/min/m2) in S group. Those alterations resulted in decreased myocardial blood flow (C:3.47±0.21 vs 1.6*±0.3 mL/min/g). An increased cardiomyocytes diameter (C:14.9±0.3 vs 18.9*±0.3μm),fibrosis (C:0.85±0.08 vs 3.28*±0.2%) and cardiac hypertrophy index (C:3.07±0.06 vs 3.97*±0.1) were observed in S group.The association of all those changes lead to a 72% higher mortality in animals with increased blood pressure variability.(*p<0.03)

Conclusion:
An increased BP variability resulted in a reduced cardiac blood flow, CO and cardiac index and an increased in myocardial resistance this adaptations occours as a attempt to homeostasis maintenance. Howerer, the morphofunctional alterations showed a diastolic dysfunction, increased cardiac hypertrophy index and fibrosis as a resulted. These adjustements could be a determinant factor in cardiac damage and in mortality in different pathological conditions, independently of BP levels.
Abstract 395: Heme Oxygenase-1-mediated PPARd Improves Cardiac Fibrosis and Inflammation in SCID Mice via Induction of T Reg Cells
Authors: Touchon Robert , Thompson Ellen , Sodhi Komal , Puri Nitin , Favero Gaia , Rezzani Rita , Fabrizio Rodella Luigi , Dial Larry , Shapiro Joseph , Abraham Nader G
Published: July 2021

Introduction:
Post-infarcted cardiac failure is the consequence of pathological remodeling after myocardial infarction (MI). A subtype of T lymphocytes, regulatory T (Treg) cells play an important role in improving cardiac function after MI via inhibition of inflammation and direct protection of cardiomyocytes. Heme oxygenase (HO)-1 induction improves heart function after ischemic damage by its anti-inflammatory, anti-oxidative and anti-fibrotic effects. To assess the role of Treg cells in post infarcted myocardium and on renal arteriolar vasoconstriction in immunosuppressed mice via the interplay of HO-1-PPAR δ axis.

Methods:
We examined the effect of HO-1 induction on post-ischemic heart failure induced by left anterior descending coronary artery ligation, in T-lymphocyte immunosuppressive (BALB SCID) mice. Mice were divided into 4 groups: sham, myocardial infarction (MI), MI treated with the HO-1 inducer cobalt protoporphyrin (CoPP) with and without the HO activity inhibitor stannous mesoporphyrin (SnMP). Thirty days after surgery all mice underwent cardiac and renal Doppler sonography. Cardiac fibrosis and necrosis were analyzed and densitometry analysis of HO-1, PPAR δ and thromboxane synthase (TxA2) in cardiac tissues was done.

Results:
Mice with MI had increased levels of cardiac fibrosis and necrosis (p<0.05) compared to sham operated controls. Echocardiography showed that fractional area shortening (FAS) was increased in the CoPP−MI group compared to the MI group (p<0.01). Renal pulsatiltiy index was increased in the MI group compared to the sham operated group (sham 0.72 ±0.08, MI 1.37±0.37, p<0.05). CoPP improved renal vasoconstriction as compared to the MI group (p<0.05). Increased expression of HO-1, PPAR-δ, pAMPK and pAKT and decreased expression of TxA2 was detected in cardiac tissues in CoPP-treated animals (p<0.02) as compared to the MI group. All these beneficial effects were reversed by SnMP.

Conclusion:
Upregulation of HO-1 attenuates post myocardial fibrosis and inflammation and renal vasoconstriction in immunosuppressed mice via recruitment of Treg lymphocytes and PPAR delta activation.
Abstract 397: HMOX1 Ameliorates Fatty Liver and Metabolic Syndrome by Reduction of Hepatic Heme and PGC1a
Authors: Sodhi Komal , Douglas Wade G , Peterson Stephen J , Dial Larry , Khawaja Imran T , Shapiro Joseph , Abraham Nader G
Published: July 2021

Introduction:
Nonalcoholic fatty liver (NAFLD) occurs in a setting of high fat diets, insulin resistance, obesity and dyslipidemia. Individuals with NAFLD have an increased risk of developing metabolic syndrome. Heme oxygenase-1 (HMOX1), a major cytoprotective enzyme, attenuates oxidative stress and obesity and increases insulin sensitivity. The antioxidant effect of HMOX1 is due to an increase in ferritin, and bilirubin and a decrease in heme, a pro-oxidant. The aim of this study was to examine the role of increased hepatic HMOX activity in decreasing steatosis, adiposity and vascular dysfunction and to determine the mechanism underlying these metabolic changes in obese mice.

Methods:
Obese mice were administered cobalt protoporphyrin (CoPP) and HMOX activity inhibitor stannous mesoporphyrin (SnMP) for 6 weeks. Blood pressure, body weight and blood glucose levels were measured in all the groups. Glycogen content, hepatic fibrosis, heme levels, fatty acid synthase (FAS) and lipid droplet size in liver were also assessed.

Results:
CoPP administration increased hepatic HMOX1 protein levels and HMOX activity, decreased blood pressure, body weight, blood glucose levels, hepatic heme content (p<0.05) as compared to obese mice. Our results also showed that HMOX1 induction causes a significant decrease in lipid steatosis ( lipid droplet size and FAS levels; p<0.01) as compared to obese mice. Densitometry analysis showed increased expression of PPARα and Glut 1 along with decreased expression of PGC1α in hepatic tissue. These beneficial effects were reversed by administration of SnMP.

Conclusion:
This novel study demonstrates the role of hepatic HMOX1 in attenuating the fatty liver and metabolic homeostasis by decreasing PGC1α and heme content and enhancing glycogen levels. Pharmacological agents that increase HMOX1 levels or gene targeting of HMOX1 offer a promising therapeutic target for NAFLD and suggest the existence of a significant link between the heme-HMOX system and the extent and severity of heme-dependent fatty liver.
Abstract 399: Whole-Body Deletion of PPAR? in Mice Increases Sympathetic Nerve Activity
Authors: Yang Tianxin , Aoyagi Toshinori , Dowtown Maicy , Liu Mi , Whitehead Kevin , Jia Zhanjun
Published: July 2021

PPARγ and sympathetic nerve activity (SNA) antagonistically regulate energy metabolism and cardiovascular function with the former promoting anabolism and vasorelaxation and the later favoring catabolism and vasoconstriction. However, the precise relationship between these two factors is not known. The present study examined the influence of PPARγ deletion on SNA. Systemic inactivation of PPARγ was

generated constitutively by using Mox2-Cre mice (MoxCre/flox) or inducibly by using the tamoxifen system (EsrCre/flox/TM). Radiotelemetry demonstrates consistent increases in resting daytime heart rate (HR) in both strains of null mice; this was associated a more than 10-fold increase in urinary epinephrine and norepinephrine levels. Subsequently, more detailed analysis of SNA was performed in Esr/flox/TM mice. In response to a bolus ip injection of the gangalion blocker pentolinium at 7.5 mg/kg, the decreases in HR and MAP were greater in the null mice than in the flox controls (delta HR:-254.2 vs. 92.3 beat/min; delta MAP: -67.8 vs. 33.4 mmHg). The HR decrease in response to the beta-blocker metaprolol (12 mg/kg, ip) was greater in the null mice than in the flox controls so was the MAP decease in response to the alpha1-blocker prazosin (1 mg/kg, ip). Baroreflex sensitivity (BRS) was assessed by the measurement of the HR responses to bolus injections of sodium nitroprusside (NP-BRS) or phenylephrine (PE-BRS). EsrCre/flox/TM mice displayed normal PE-BRS but blunted NP-BRS. Echocardiography under anesthesia demonstrated that the ejection fraction (EF) was 11% lower after administration of metaprolol contrasting to a drop of only 1.5% for the flox controls, in parallel with a greater reduction of HR in the KO mice. In contrast, the increase in HR following administration of the beta1 agonist dobutamine (0.8 mg/kg, ip) was less in the KO mice than in the flox controls. Together, we conclude: 1) PPARγ deletion causes the activation of SNA, 2) the parasympathetic nervous system may be unaffected, 3) the blunted response to dobutamine suggest suppressed beta1 receptor activity possibly as a result of compensatory response to sympathoactivation.
Abstract 404: 20-HETE Regulates Angiogenic Functions of Human Endothelial Progenitor Cells and Contributes to Ischemia-induced Compensatory Neovascularization
Authors: Chen Li , Zhang Frank F , Gotlinger Katherine H , Falck John R , Arbab Ali S , Scicli A G , Schwartzman Michal L , Guo Austin M
Published: July 2021

Objective:
Circulating EPC contribute to postnatal neovascularization, which is an important adaptation for recovery from critical ischemia. It is thus essential to understand the factors that regulate EPC involvement in the neovascularization process. We have identified the CYP4A-20-HETE system as a novel regulator of EPC angiogenic functions
in vitro
. Here, we explored cellular mechanisms by which 20-HETE regulates EPC angiogenic functions and assessed its contribution to EPC-mediated angiogenesis and ischemia-induced compensatory neovascularization
in vivo
.

Approach and Results:
20-HETE induced EPC adhesion to fibronectin and EC monolayer by 40 ± 5.6 and 67 ± 10%, respectively, which were accompanied by a rapid induction of VLA-4 and CXCR4 mRNA expression. Basal and 20-HETE-stimulated increases in adhesion were negated by inhibition of the CYP4A-20-HETE system. Real-time PCR analysis and EPC proliferation assay indicated that a positive feedback regulation exists between 20-HETE and HIF-1α/VEGF pathways. Furthermore,
in vivo
addition of EPC increased angiogenesis by 3.6±0.2 fold and these effects were markedly reduced by inhibition of 20-HETE system. Systemic inhibition of the CYP4A-20-HETE system reduced the compensatory neovascularization in response to ischemia in a mouse ischemic hindlimb model. Importantly, ischemic hindlimb muscles produce markedly elevated amounts of 20-HETE compared to non-ischemic controls (124 ± 27 versus 17 ± 17 pg/mg of protein).

Conclusions:
These results strengthen the notion that 20-HETE regulates the angiogenic functions of EPC
in vitro
and
in vivo
in autocrine and paracrine manner via up-regulation of key pro-angiogenic factors. Increases in 20-HETE at ischemic sites promote and contribute to

compensatory neovascularization.
Abstract 410: Combination Therapy With Lercanidipine and Enalapril Improves Wave Reflection in Hypertensive Patients With Metabolic Sindrome
Authors: Ghiadoni Lorenzo , Bruno Rosa Maria , Stea Francesco , Cartoni Giulia , Virdis Agostino , Grassi Davide , Ferri Claudio , Taddei Stefano
Published: July 2021

Arterial stiffness and wave reflection are independent predictors of cardiovascular events. This study compared the effect on arterial stiffness and wave reflection of a combination therapy with an ACE-inhibitor plus calcium channel blocker or thiazide diuretic in essential hypertensive patients with metabolic syndrome uncontrolled by ACE-inhibitor monotherapy. In a multicenter randomized, open, parallel group study, 76 hypertensive patients with metabolic syndrome, after 4 weeks run-in with Enalapril 20 mg, were randomized to a combination therapy with Lercanidipine (LER, 10-20 mg) or Hydrochlorothiazide (HCT, 12,5-25 mg) for 6 months. Applanation tonometry was used to measure aortic stiffness (carotid to femoral pulse wave velocity, PWV), central blood pressure (BP) and augmentation index (AI), a marker of wave reflection. At screening, office BP was 153±4/95±2 mmHg in both groups. After run in, BP was 139±18/84±12 and 142±17/84±10 mmHg, in the LER and HCT groups, respectively, and after 6-month it was 134±15/79±9 mmHg in the LER group and 134±14/79±10 mmHg in the HCT group. No significant difference between the two groups was observed. Central BP values had a similar behavior (6 months: LER 120±13/80±9 mmHg; HCT 122±13/79±9 mmHg). PWV was similar at baseline and was equally reduced by the two treatments (LER from 8.6±1.5 to 8.1±1.3 m/s; HCT from 8.5±1.2 to 8.2±1.0 m/s). Finally, both drugs reduced AI, but this reduction resulted significantly greater in LER than in HCT arm (LER from 26.8±10.9 to 20.6±9.1%; HCT from 28.2±9.0 to 24.7±8.7%). In conclusion, the addition of LER caused the same PWV reduction as compared to HCT but greater reduction in AI in hypertensive patients with metabolic syndrome not controlled with Enalapril alone. These results indicate a positive effect of the combination with LER on wave reflection, suggesting a potential role for cardiovascular protection.
Abstract 417: AT2R Stimulation Prevents TNFa-Induced Vascular Inflammation In Vitro and Ex Vivo
Authors: Sampson Amanda K , Irvine Jennifer C , Huet Olivier , Barnes Tyrone A , Widdop Robert E , Chin-Dusting Jaye P
Published: July 2021

Vascular inflammation, involving the recruitment, adhesion and infiltration of monocytes to the sub-endothelial space, is a critical early event in the development of atherosclerosis. The renin angiotensin system plays an important role in inflammation via activation of the angiotensin type I receptor (AT1R), which induces pro-inflammatory effects. The angiotensin II type 2 receptor (AT2R) counter-regulates the effects of the AT1R, including AT1R-mediated pro-inflammatory cytokine expression. We investigated the anti-inflammatory effects of AT2R stimulation in vascular inflammation by examining leukocyte to endothelial adhesion. We quantified the effect of AT2R stimulation (Compound 21: C21, 100μM) on TNFα (10ng/mL)-induced monocyte adhesion to cultured human umbilical vascular endothelial cells
in vitro
. AT2R stimulation attenuated TNFα-induced monocyte adhesion (unstimulated: 8±4% of TNFα: 100%, C21+TNFα: 59±12% of TNFα-induced adhesion). Adhesion of monocytes to the endothelial monolayer following incubation with TNFα+C21+AT2R antagonism (PD 123319, 10μM) was not different to TNFα-induced monocyte adhesion (93±5% of TNFα); demonstrating that the anti-inflammatory effects of C21 are mediated by the AT2R. Furthermore, C21 treatment attenuated TNFα-induced upregulation of adhesion molecules ICAM-1 and E-selectin and abolished TNFα-induced ROS production (unstimulated: 2±2, TNFα: 55±16, TNFα+C21: -3±5 dihydroethidium fluorescence intensity units). We quantified TNFα-induced leukocyte adhesion in intact mouse thoracic aorta
ex vivo
in real time in the presence and absence of AT2R activation. Consistent with our
in vitro
findings, we observed that direct AT2R activation (C21, 10μM) abolished TNFα-induced leukocyte adhesion (TNFα: 30±4, vs TNFα+C21: 11±4 adhered leukocytes/field of view (FOV), P<0.01) an effect which was abolished by co-incubation with PD 123319 (10μM: 31±5 adhered leukocytes/FOV). This study provides the first functional evidence that direct AT2R stimulation prevents TNFα-induced leukocyte adhesion, ICAM-1 and E-selectin expression and ROS production revealing the anti-inflammatory and therapeutic potential of the AT2R in the treatment of inflammation-induced cardiovascular disease.
Abstract 429: Ambulatory Blood Pressure Dipping is Significantly Related to Night/Day Urinary Norepinephrine Ratio
Authors: Bursztyn Michael , Därr Roland , Pamporaki Christina , Peitzsch Mirko , Eisenhofer Graeme
Published: July 2021

Absence of the normal 10% reduction of ambulatory blood pressure (ABP) from day to night is associated with morbidity and mortality. The cause of this reduction (or its absence) is not completely understood. We studied excretion of norepinephrine (NE) and epinephrine (EPI) on the same day ABP monitoring was performed in 37 healthy volunteers aged 52±7years, with body mass index (BMI) 25±4 Kg/m
2
, and 24-hour ABP 130±11/82±9 mmHg. Overall SBP decreased from day to night 134±13 to 117±10 mmHg, diastolic blood pressure (DBP) from 86±9 to 72±8 mmHg, and heart rate (HR) from 80±10 to 70±9 beats per minute (bpm). Urinary NE and EPI excretion decreased (p<0.005) from 19±10 to 10±6 μmol/mol creatinine, and from 4±2 to 0.8±0.4 μmol/mol creatinine, respectively. Awake or asleep SBP was not correlated to the corresponding NE excretion (r=-0.2, and 0.19). We defined SBP dippers (n=22) as those with a night/day ratio (NDR) of <0.9. There were no differences in age, gender, BMI, 24-hour ABP, daytime DBP, sleep ABP, daytime or sleep HR, or daytime or overnight NE or EPI excretion. On the other hand awake SBP 129±11 mmHg was lower, p<0.05, and NDR DBP 0.89±0.05, NDR HR 0.90±0.05, and NDR NE 0.66±2.7, were significantly higher p<0.05, in the non-dippers (n=15) , than in dippers 138±17mmHg, 0.81±0.05, 0.86±0.8, 0.49±0.18, respectively. In a stepwise multiple regression analysis model with SBP NDR as the dependent variable, awake SBP, p=0.0008, and NDR NE p=0.03, were significant predictors, whereas age and BMI were excluded from the model. In conclusion although day and night blood pressure and urinary NE are not related, the nocturnal decrease of SBP is closely related to the degree of reduction in sympathetic activity as reflected by urinary NE excretion on the same day.
Abstract 43: Renal Hemodynamic and Excretory Responses to Systemic Administration of Interleukin-10 in Anesthetized Mice
Authors: Castillo Alexander , Majid Dewan S
Published: July 2021

Recent studies have demonstrated that the anti-inflammatory cytokine, interleukin-10 (IL-10) is normally present in plasma and its protein is constitutively expressed in the renal tissue. However, the possible role of IL-10 in the regulation of renal hemodynamics and excretory function is not yet clearly defined. In the present study, we have examined the systemic and renal responses to intravenous administration of incremental doses of recombinant mouse IL-10 (0.015, 0.075 and 0.15 ng/min/g ; 45 min in each dose) in anesthetized mice (n=5). To determine the specificity of IL-10 actions, the responses to the middle dose were also assessed in the presence of its receptor antagonist, (mouse IL-10 Rα antibody; 1.5 ng/min/g; R&D system) in a separate group of mice (n=6). Standard clearance techniques were used to assess renal responses to infusions of IL-10 and its receptor antagonist. Renal blood flow (RBF) was determined by PAH clearances and glomerular filtration rate (GFR) was determined by inulin clearance. Infusion of IL-10 doses resulted in significant decreases in systemic mean arterial pressure (MAP; 98±1.9 to 89±3.9, 88±2.6 and 84±3.3 mmHg) and renal vascular resistance (RVR, 13.5±1.1 to 9.1±0.6, 7.1±0.5 and 7.1±0.5 mmHg/mL/min/g), increases in RBF (7.4±0.5 to 9.7±0.6, 10.6±0.5 and 9.8±0.6 mL/min/g) and GFR (1.06±0.05 to 1.45±0.13, 1.51±0.13 and 1.53±0.28 mL/min/g) without significant changes in urine flow (6.6±0.5 to 5.3±0.5, 5.5±1.1 and 5.3±1.1 μL/min/g ) or sodium excretion (0.58±0.08 to 0.52±0.18, 0.57±0.19 and 0.69±0.26 μmol/min/g). Administration of IL-10 receptor antagonist alone did not cause significant changes in MAP (90±3.7 to 89±5.3 mmHg), RVR (11.5±1.9 to 11.6±3.0 mmHg/mL/min/g), RBF (8.8±1.3 to 10.4±2.5 mL/min/g), GFR (1.06±0.08 to 1.20±0.19 mL/min/g) or other renal parameters. Infusion of the middle dose of IL-10 in the receptor antagonist pretreated mice failed to cause significant changes in RBF or GFR or other parameters confirming the specificity of its receptor activity in the mediation of observed renal responses to IL-10 infusion. These data suggest that the anti-inflammatory cytokine, IL-10 exerts vasodilator and hyper-filtration effects in the kidney but minimally influences the renal excretory function.
Abstract 445: Increased Dietary Salt Intake Enhances Sympathetic and Cardiovascular Responses to Various Stressors
Authors: Stocker Sean D , Simmonds Sarah S
Published: July 2021

Previous studies indicate that increased dietary salt intake enhances sympathetic nerve activity (SNA) and arterial blood pressure (ABP) responses evoked from sympathetic neurons of the rostral ventrolateral medulla. The present study sought to extend these findings and determine whether dietary salt intake enhances SNA and ABP responses various sympathetic reflexes that depend on RVLM neurotransmission. Male Sprague-Dawley rats were fed 0.1% (n=6-8) or 4.0% (n=6-8) NaCl diets for 14-21 days. Then, animals were anesthetized with Inactin. Electrical stimulation (1-20 Hz, 1 ms pulse, 500 uA) of sciatic afferents produced frequency-dependent changes in SNA and ABP in both groups. However, rats ingesting 4% versus 0.1% NaCl displayed significantly larger increases in lumbar SNA (5Hz: 213±25 vs 146±25%, P<0.05), renal SNA (5Hz: 187±24 vs 120±11%), splanchnic SNA (5Hz: 203±21 vs 136±9%), and mean ABP (5Hz: 28±2 vs 12±2 mmHg). Rats ingesting 4% vs 0.1% NaCl also displayed greater increases in lumbar SNA (24±6 vs 13±2%, P<0.05) and mean ABP (12.1±0.9 vs 8.2±1.3mmHg, P<0.05) during increases in cerebrospinal fluid sodium concentration produced by intracerebroventricular infusion of 1M NaCl (5ul/10min). Lastly, hypercapnia (7% CO2, 33% O2, 63% N2, 60s) produced greater increases in lumbar SNA in rats ingesting 4% versus 0.1% NaCl (24±2% versus 9±3%, respectively; P<0.01). These findings suggest increased dietary salt intake enhances several sympathetic and cardiovascular reflexes.
Abstract 449: Assessment of Pharmacokinetic Drug Interaction Between LCZ696 and Digoxin
Authors: Ayalasomayajula Surya , Jordaan Pierre , Pal Parasar , Albrecht Diego , Langenickel Thomas , Sunkara Gangadhar , Chandra Priyamvada
Published: July 2021

Objective:
LCZ696 is a first-in-class angiotensin receptor neprilysin inhibitor (ARNI) being developed for the treatment of cardiovascular diseases including hypertension and heart failure. Ingestion of LCZ696 results in systemic exposure to AHU377 (an inactive prodrug converted to LBQ657, a neprilysin inhibitor) and valsartan (angiotensin receptor inhibitor). Digoxin, a narrow therapeutic index drug, is a commonly administered medication to heart failure patients. Since LCZ696 and digoxin may be co-administered in this patient population, this study was conducted to evaluate the pharmacokinetic drug-drug interaction potential between LCZ696 and digoxin.

Methods:
This study employed an open label, two-period, single sequence study design in 24 healthy subjects. In period 1, subjects received 200 mg LCZ696 b.i.d for 3 days and a single dose of 200 mg LCZ696 on Day 4 morning. Following a 4-7 day washout, in period 2, all subjects received 0.25 mg digoxin q.d. for 14 days and 200 mg LCZ696 b.i.d co-administered from Day 11 to Day 14. Serial PK samples were collected in both treatment periods and analyzed using validated LC/MS/MS bioanalytical methods. The PK parameters including Cmaxss and AUCtau of LCZ696 analytes (LBQ657, valsartan) and digoxin were determined using non-compartmental analysis and the results were statistically evaluated.

Results:
The 90% confidence intervals of the geometric mean ratios (test/reference) for Cmaxss and AUCtau of digoxin were within the 0.8-1.25 range indicating that LCZ696 did not affect the PK of digoxin. Similarly, the 90% confidence intervals of the geometric mean ratios for Cmaxss and AUCtau for both LBQ657 and valsartan were within the 0.8-1.25 range indicating that digoxin did not affect the PK of LCZ696 analytes.

Conclusion:
After co-administration of LCZ696 200 mg b.i.d with digoxin 0.25 mg q.d., exposures of digoxin and the LCZ696 analytes (LBQ657 and valsartan) remained unchanged. LCZ696 200 mg b.i.d was safe and well tolerated in healthy subjects when administered alone and in combination with digoxin 0.25 mg qd.
Abstract 453: Angiogenesis Inhibition-Induced Hypertension and Nephrotoxicity: Not All Antihypertensives are the Same
Authors: Lankhorst Stephanie , Kappers Mariëtte H , Van Esch Joep H , Smedts Frank M , Sleijfer Stefan , Danser Jan A , van den Meiracker Anton H
Published: July 2021

Angiogenesis inhibition with the VEGF inhibitor sunitinib is an established anti-cancer therapy inducing hypertension and nephrotoxicity. Here we compared the effects of the endothelin antagonist (ET-R) macitentan (30 mg/kg/day, p.o; n=8), the calcium channel blocker (CCB) amlodipine (3 mg/kg/day, p.o; n=9), or the ACE-inhibitor captopril (3 or 12 mg/kg/day, s.c.; n=9) vs. no treatment (n=14) in sunitinib (26.7 mg/kg/day, p.o)-exposed hypertensive WKY rats. Treatment lasted 8 days; mean arterial pressure (MAP) was monitored telemetrically. At the end of the treatment period, 24-hour urine samples for protein and endothelin-1 (ET-1) measurements, and blood samples and kidneys for histological evaluation were collected.

With sunitinib, MAP increased from 94.7±0.9 mmHg to 125.8±1.5 mmHg (Δ31.1±0.9 mmHg, p<0.001). Co-administration of macitentan (Δ12.3±1.5 mmHg, p<0.001) or amlodipine (Δ11.4±1.7 mmHg, p<0.001) attenuated the sunitinib-induced MAP rise, whereas low and high captopril doses did not (Δ28.1±2.0 and Δ27.2±1.1 mmHg). With sunitinib, serum creatinine increased from 8.0±2.7 to 29.9±4.6 μmol/l (p<0.01) and proteinuria from 7.5±1.3 to 33.3±4.8 mg/day, p<0.05. Although no agent could prevent the sunitinib-induced rise in creatinine, the induced proteinuria was attenuated by 62% (p<0.01) with macitentan and by 88% with low and 114% (p<0.001) with high dose of captopril, while proteinuria increased by 56% (p=NS) with amlodipine. With sunitinib, urinary ET-1 increased from 3.3±0.5 to 4.8±1.0 pg/day (p<0.05). Macitentan and captopril abolished this increase. Renal histology revealed extensive glomerular ischemia and endothelial cell swelling. Concomitant with the decrease in proteinuria, glomerular intra-epithelial protein deposition decreased with macitentan and captopril.

In conclusion, ET-R antagonism and CCB effectively reduced the sunitinib-induced hypertension, whereas ACE-inhibition did not. Both ET-R antagonism and ACE inhibition diminished the sunitinib-induced proteinuria. Since ET-R antagonists are not yet available as anti-hypertensive agents, CCBs are preferred in angiogenesis inhibition-induced hypertension, while adding an ACE-inhibitor might be considered when proteinuria is also present.
Abstract 454: Hypertension Control by Decrease of Sympathetic Hyperactivity in SHR Rats: Comparison Between Renal Denervation and Dopamine ß-hydroxylase Inhibition
Authors: Pires Nuno , Igreja Bruno , Wright Lyndon , Soares-da-Silva Patrício
Published: July 2021

Activation of the sympathetic nervous system has been described in the development and progression of systemic hypertension. Recently, catheter-based renal nerve ablation for treatment of drug-resistant hypertension has been developed. An alternative strategy for modulation of sympathetic nerve function is to reduce the biosynthesis of norepinephrine (NE) via inhibition of dopamine β-hydroxylase (DβH), the enzyme that catalysis the conversion of dopamine (DA) to NE in sympathetic nerves.

Renal denervation was assessed in the rat by transient (90 sec) ischemia of renal tissues. Bilateral renal artery occlusion (RAO) was assessed in spontaneously hypertensive rats (SHR) to evaluate the effect of renal denervation on NE levels and blood pressure throughout a 28-day period. Oral treatment with the selective peripheral DβH inhibitor etamicastat (30 mg/Kg body weight) was performed in a second cohort of SHR and NE levels and blood pressure was assessed.

Renal denervation resulted in marked depletion of NE levels, selective for the renal sympathetic innervations, 5 days after RAO in renal cortex (16.5±7.7% of control values, P<0.0001). Decreased NE levels persisted up to 28 days after RAO (40.8±19.4% of control values, P<0.0001). No differences in left ventricle NE levels were observed. Renal denervation significantly decreased systolic blood pressure (SBP) 5 days (165±6 vs. 187±2 mmHg, P<0.05) and 14 days post-surgery (194±3 vs. 204±2 mmHg, P<0.05), but had no effect on heart rate. Systolic blood pressure recovered to basal values 28 days post-surgery. Likewise, etamicastat treatment resulted in a depletion of NE levels in both renal cortex (70.9±9.1% of control values, P<0.05) and left ventricle (55.8±3.9% of control values, P<0.01). Etamicastat treatment decreased SBP (228±7 vs. 174±9 mmHg, P<0.001) without effects on heart rate.

In conclusion, these results suggest that renal denervation and DβH inhibition similarly decreases sympathetic hyperactivity and high blood pressure in the SHR. Downregulation of sympathetic drive with DβH inhibitors may be useful in the treatment of hypertension.
Abstract 457: Assessment of Pharmacokinetic Drug-drug Interaction between LCZ696 and Carvedilol
Authors: Hsiao Hsiu-Ling , Greeley Michael , Pal Parasar , Langenickel Thomas , Sunkara Gangadhar , Chandra Priyamvada
Published: July 2021

Objective:
LCZ696 is a first-in-class angiotensin receptor neprlysin inhibitor (ARNI) being developed for the treatment of hypertension and heart failure. Ingestion of LCZ696 results in systemic exposure to AHU377 (inactive prodrug of LBQ657, a neprilysin inhibitor) and valsartan (angiotensin receptor blocker). Carvedilol is a third-generation, non-selective beta-blocker with vasodilating properties and one of three beta-blockers with efficacy in reducing the risk of death in patients with heart failure. Since LCZ696 may be co-administered with carvedilol for optimal blood pressure control, this study was conducted to evaluate the pharmacokinetic (PK) drug-drug interaction potential between LCZ696 and carvedilol.

Methods:
An open-label, three-period, single sequence study in 28 healthy subjects was conducted. In Period 1, subjects received oral LCZ696 400 mg qd x 5 days and were discharged for a 4-10 day washout. In Period 2, subjects received oral carvedilol 12.5 mg bid x first 2 days, then 25 mg bid x 4 days, and in Period 3, LCZ696 400 mg qd + carvedilol 25 mg bid x 5 days. Serial PK samples were collected and analyzed by a validated LC-MS/MS method. PK parameters (AUCtau,ss, Cmax,ss) of LCZ696 analytes(LBQ657, valsartan) and carvedilol (R(+)-and S(-)-carvedilol) in plasma were determined using non-compartmental analysis, and results were statistically evaluated.

Results:
The 90% CIs of the geometric mean ratio for AUCtau,ss and Cmax,ss of LBQ657, and AUCtau,ss of valsartan fell within the range of (0.80-1.25); the lower bound for Cmax,ss of valsartan (0.88, 0.78-0.98) was below the range, indicating PK of LBQ567 was not altered but Cmax,ss of valsartan decreased by 12% when co-administered with carvedilol. Those for AUCtau,ss and Cmax,ss of both R(+)-and S(-)carvedilol fell within the range (0.80-1.25), indicating no change in PK of Carvedilol in combination with LCZ696.

Conclusion:
When LCZ696 400 mg qd and carvedilol 25 mg bid were co-dministered, PK of carvedilol (R(+)-and S(-)-carvedilol) was unchanged. PK of LBQ657 or AUCtau,ss of valsartan was unchanged, while Cmax,ss decreased by 12%. LCZ696 400 mg qd was safe and well tolerated in healthy subjects when administered alone and in combination with carvedilol 25 mg bid.
Abstract 458: Relaxin Did Not Improve Angiotensin II-induced Target Organ Damage
Authors: Haase Nadine , Hupfer Stefan , Golic Michaela , Herse Florian , Qadri Fatimunnisa , Müller Dominik N , Dechend Ralf
Published: July 2021

We investigated whether or not Relaxin ameliorates the hypertension induced damage to heart and kidney. We used double transgenic rats harboring both human renin and angiotensinogen genes (dTGRs). These rats develop moderately severe Hypertension but die of end-organ cardiac and renal damage by week 7. Untreated dTGRs had an around 50% mortality rate at 7 weeks. The heart shows necrosis and fibrosis, whereas the kidneys esemble the hemolytic-uremic syndrome vasculopathy.

We started Relaxin-treatment on four weeks old dTGR in low (26 μg/kg/d) and high dose (240 μg/kg/d) and age-matched SD rats. Blood-pressure- and albuminuria-measurements were monitored during the treatment period (three weeks). Seven weeks old animals were killed, hearts and kidneys were isolated and used for histochemical and quantitative TaqMan RT-PCR analysis. Systolic blood pressure increased progressively in untreated dTGRs from 161.6 ± 3.02 mmHg in week 5 to 225.3 ± 4.48 mmHg in week 7. Relaxin treatment had no significant influence on blood pressure (low dose 208.0 ± 5.51 mmHg and high dose 222.2 ± 5.60 mmHg at week 7 ) whereas SD rats were normotensive (106.3 ± 1.15 mmHg). Untreated (5.38 ± 0.13 mg/g) and Relaxin treated dTGRs (low dose 5.20 ± 0.18 mg/g and high dose 5.33 ± 0.29 mg/g) had similar cardiac hypertrophy indices (heart-to-body weight ratio), which were significantly higher compared with nontransgenic SD rats (2.90 ± 0.08 mg/g). BNP and CTFG mRNA expression in the hearts was significant higher in untreated dTGRs compared to SD rats. There were no differences in BNP and CTFG expression between untreated and Relaxin treated dTGRs. Relaxin treatment (low dose 78502 ± 16848 μg/day and high dose 43642 ± 10852

μg/day at week 7) did not ameliorate albuminuria compared with untreated dTGRs (57937 ± 6122 μg/day at week 7). Furthermore the treatment with Relaxin did not prevent matrix deposition in the heart and kidney of d TGRs. Finally, Relaxin treatment did not reduce mortality. Although survival was 52 %, in untreated dTGRs 36 % in low dose and 54 % high dose Relaxin treated dTGRs at week 7.

These data demonstrate that Relaxin did not improve angiotensin II-induced target organ damage.
Abstract 460: Atrasentan Inhibition of Injury-induced Vascular Smooth Cell Proliferation In Vivo is Associated With Reduction of Cell Division Cycle 25 Homolog A Expression
Authors: Chen Songcang
Published: July 2021

Activation of endothelin (ET) signaling in vascular wall plays an important role in the development of secondary atherosclerosis, including re-stenosis post-angioplasty and transplantation vasculopathy. We have shown previously that ET activates vascular smooth muscle cell (VSMC) proliferation through increased expression of the key cell cycle phosphatase, cell division cycle 25 homolog A
(
Cdc25A). In this study, we asked whether a selective ET A receptor antagonist, atrasentan is able to block injury-induced in vivo VSMC proliferation associated with reduction of Cdc25A expression. After measurement of blood pressure (BP) and 24-hour urine sodium excretion, 18 male Sprague-Dawley rats were randomly divided into 3 groups to receive vehicle or atrasentan (5 mg/kg/d) in the drinking water 7 days prior to sham surgery ( control group) or the right common carotid artery (CCA) ligation ( with or without atrasentan groups). The treatment was continued for 21 days after operation. At that time their BP and urine sodium excretion were measured again before sacrifice. There was no significant difference in BP and 24-hour urine sodium excretion among 3 groups before and 21 days after surgery. CCA ligation led to neointimal hyperplasia. Treatment with atrasentan resulted in about a 75% reduction of the hyperplasia. The neointima dominantly consisted of VSMCs confirmed by anti-smooth muscle (SM)-α-actin staining. To determine whether CCA ligation induces Cdc25A expression in VSMCs and this induction is blocked by atrasentan, we conducted co- immunofluorescence assay with anti-SM-α-actin and anti-Cdc25A antibodies. The ligation markedly increased Cdc25A expression in VSMCs and the expression was much higher in neointimal VSMCs than those in medial layer. Atrasentan reduced ligation-mediated Cdc25A expression in neointimal VAMCs by ~80%, while it seemed not significantly decrease VSMC Cdc25A levels in medial layer. These results demonstrate that atrasentan inhibits VSMC proliferation in vivo possibly through a Cdc25A-dependent mechanism and suggest that this compound may prove useful in the management of disorders characterized by acute aberrant proliferation of VSMCs in the vascular wall.
Abstract 468: The Dopamine D
1
-like Receptors Negatively Regulate the Expression and Function of the a
1
A Adrenergic Receptor
Authors: Ennis Riley C , Villar Van Anthony M , Asico Laureano D , Escano Crisanto S , Feranil Jun B , Jurgens Julie A , Jones John E , Jose Pedro A
Published: July 2021

The homeostatic control of blood pressure hinges upon the delicate balance between pro-hypertensinogenic (e.g., sympathetic nervous system) and anti-hypertensinogenic systems (e.g., dopamine system). The D
1
-like dopamine receptors (D
1
R and D
5
R) and the α
1A
adrenergic receptor (ARα
1A
) are endogenously expressed in the renal proximal tubules and engender opposing effects on sodium transport, i.e., natriuresis (D
1
R and D
5
R) or anti-natriuresis (ARα
1A
). We tested the hypothesis that the D
1
R and D
5
R interact with and regulate the ARα
1A
in human renal proximal tubule cells (hRPTCs) and in mice. We found that the D
1
R and D
5
R colocalized with the ARα
1A
in hRPTCs and in proximal tubules in human kidney sections. Both receptors immunoprecipitated, pulled-down, and co-fractionated with ARα
1A
in lipid rafts. Short-term co-treatment with fenoldopam (1 μM, 15 min) reversed the ARα
1
agonist phenylephrine (10 μM, 15 min)-induced Na
+
,K
+
-ATPase (NKA) translocation from the cytosol to the plasma membrane in hRPTCs (plasma membrane NKA: vehicle=100±5% vs. fenoldopam=65±3% vs. phenylephrine=177±5% vs. co-treatment=115±7%; P<0.05, n=3-4). Long-term fenoldopam (1 μM, 24 hr) treatment resulted in decreased D
1
R (70.0±5.9%, P<0.05, n=3) and D
5
R (50.1±10.7%, P<0.05, n=3), consistent with D
1
-like receptor desensitization, but increased ARα
1A
abundance (142.6±4.3%, P<0.05, n=3) in hRPTCs. RNAi silencing of ARα
1A
(48 hr) increased the expression of D
1
R and D
5
R. To determine the extent of regulation of each D
1
-like receptor on ARα
1A
, we used the subclass-selective ARα
1
agonist phenylephrine (5 μg/kg body weight, i.p.) and the receptor-specific ARα
1A
agonist A610603 (25 ng/kg body weight, i.p.) to inhibit Na
+
excretion in three mouse strains. Phenylephrine treatment resulted in 59.6%, 84.2%, 99.3%, and 99.5% reduction from basal level of 24-hr Na
+
excretion_while A61603 treatment resulted in 42.4%, 67.1%, 99.9%, and 100% reduction_in wild-type controls, D
1
R
-/-
, and D
5
R
-/-
knockout mice, and D
1
R/D
5
R
-/-
double knockout mice, respectively, suggesting a stronger regulatory effect of D
5
R on ARα
1A
. Elucidating the intricacies of the interaction among these receptors is crucial for a better understanding of the crosstalk between anti- and pro-hypertensive systems.
Abstract 47: Caveolin-1 is Critical for Abdominal Aortic Aneurysm Induced by Angiotensin II
Authors: Obama Takashi , Takayanagi Takehiko , Crawford Kevin J , Kobayashi Tomonori , Rizzo Victor , Eguchi Satoru
Published: July 2021

Abdominal aortic aneurysm (AAA) is a significant cause of mortality for adults aged >60 years. Accumulating evidence suggests a role of angiotensin II (Ang II) in abdominal aortic aneurysm (AAA) formation. However, the Ang II-sensitive proximal signaling events primarily responsible for AAA formation remain unclear. We recently reported that caveolin-1 (Cav1) enriched membrane microdomains in vascular smooth muscle cells (VSMC) mediate a metalloprotease ADAM17-dependent EGF receptor (EGFR) transactivation, which is linked to vascular remodeling induced by Ang II. Given that ADAM17 expression is one of the key features in AAA, we have tested our hypothesis that Cav1, a major structural protein of caveolae, plays a critical role for development of AAA by Ang II via regulation of ADAM17. 8 week old male Cav1-/- and the control C57Bl/6 wild-type mice (WT) were co-infused with Ang II (1 μg/kg/min) and β-aminopropionitrile (BAPN: 150mg/kg/day) for 4 weeks to induce AAA. In WT with the co-infusion, 58% (14/24) were dead due to aortic rupture/dissection. All surviving WT with co-infusion had AAA with max diameter (mm) of 2.6±0.18 vs 0.93±0.09 with saline infusion (p<0.01). In contrast, we found that Cav1-/- with co-infusion did not die or develop AAA. The max diameter (mm) of AAA in Cav1-/- with co-infusion was 1.0±0.04 vs 1.1±0.06 with saline infusion (n=7). In contrast, both WT and Cav1-/- with the co-infusion developed hypertension assessed by telemetry (MAP mmHg: 151±5 vs 161±7). We found an increased expression of ADAM17 by IHC and qPCR, and enhanced phosphorylation of EGFR by IHC in WT abdominal aortae with aneurysms. These events were markedly attenuated in Cav1-/- aorta with co-infusion (ADAM17/18S mRNAx10,000 = 3.08±0.71 vs 0.97±0.42 p<0.05, n=4). Furthermore, Cav1-/- aortae showed less ER and oxidative stress compared to WT aortae assessed by IHC. In addition, Cav1 silencing induced by adenovirus encoding Cav1 targeting siRNA embedded miRNA in cultured vascular smooth muscle cells prevented Ang II-induced ADAM17 induction and activation. In conclusion, Cav1 and presumably vascular caveolae microdomains appear to play a critical role in the formation of AAA by Ang II via regulation of the ADAM17/EGFR signaling and subsequent ER/oxidative stress.
Abstract 480: NaCl-Independent Dietary Effects are Associated with Altered DNA Methylation in Dahl Salt-Sensitive (SS) Hypertension
Authors: Mattson David L , Liu Pengyuan , Geurts Aron M , Rudemiller Nathan P , Skelton Meredith M , Liu Yong , Yang Chun , Liang Mingyu , Cowley Allen W
Published: July 2021

Salt-sensitive hypertension and renal damage were compared in colonies of SS rats fed casein-and grain-based diets. When 6 week old rats were fed a low NaCl diet (n=7-11/group), mean arterial pressure (MAP) and urinary albumin (UAlb) excretion were not significantly different in grain-fed rats compared to values in casein-fed rats (107±2 mmHg and 6±2 mg/day). Increasing dietary NaCl intake to 4.0% for three weeks led to a significantly greater increase in MAP (171±7 vs 119±7 mmHg), UAlb (164±19 vs 24±6 mg/day) and renal histological damage in SS rats fed the casein diet (p<.05). To assess mechanisms of this NaCl independent effect, an RNA-Seq analysis of renal outer medullary tissue of rats (n=4/group) fed the grain- or casein-based diets was performed. Over 1500 known genes were differentially expressed between the casein- and grain-fed rats on low salt; over 2100 were different between the groups fed the 4.0% chow (FDR<0.05); and 897 genes were common to these two data sets. The differentially expressed genes were significantly enriched for genes related to hematopoietic cell lineage, the complement and coagulation cascade, B-cell signaling, NK cell-mediated cytotoxicity, and primary immunodeficiency. To understand potential mechanisms of the regulation of gene expression, a genome-wide DNA methylation analysis of the renal outer medulla at single-base resolution was performed. Of nearly 3,000 methylated CpG islands identified, 534 and 650 were differentially methylated between the casein and grain fed groups when comparing effects of low to high salt diets, respectively (FDR<0.05). Approximately 200 differentially methylated CpG islands were located in the proximity of transcriptional start sites. We first focused our attention on several genes which exhibited a reciprocal pattern of DNA methylation and RNA abundance. These include Klotho (an aging-related gene), and Cyp39a1 (a cytochrome P450) which exhibited significantly more 5-methylcytosine modifications in the transcription start site and reduced mRNA expression in rats fed the casein-based diet with low salt. These data provide evidence that epigenetic modifications due to NaCl-independent dietary effects may alter salt-sensitive blood pressure and renal disease phenotypes.