bluuebunny/crossref_metadata_2025 huggingface dataset:
Alpha1‐adrenergic receptor subtype specific activation of ERK in human primary smooth muscle cells (1065.4)
Authors: Brown Kaitlyn , Aungst Timothy , Castro Christian , Bommareddy Ajay , VanWert Adam , McCune Dan
Published: June 2021

Three α1‐adrenergic receptor (AR) subtypes have been cloned and characterized, the α1A‐,α1B‐, and α1D‐AR. Evidence suggests functional effects of receptor activation in various tissues is subtype selective. For example, ERK activation in cardiomyoctyes appears to be α1A‐AR dependent, and may be cardioprotective in heart failure. We examined α1‐AR expression and subtype specific ERK activation in human primary arterial smooth muscle cells. Simultaneous co‐expression of all three subtypes was demonstrated by western blot, and further confirmed by RT‐PCR utilizing subtype specific primers. ERK activation was assessed by western blot. Preliminary data suggests ERK activation is blocked by α1D‐AR selective antagonists and is not induced by α1A‐AR selective agonists. If α1A‐AR selective agents were used in heart failure, our data would suggest that ERK activation in peripheral vasculature would be minimal, therefore mitigating concern of α1A‐AR mediated vascular injury as a result of therapy. However, additional subtype selective effects will need to be better defined by future studies.
Identification of novel signaling regulators of the mu opioid receptor (1066.1)
Authors: Edwards Katie , LaVigne Justin , Moses‐Fynn Emmanuel , Mullen Nate , Streicher John
Published: June 2021

Recent evidence has suggested that specific downstream signaling pathways of the mu opioid receptor (MOR) carry out specific aspects of the behavioral response, such as antinociception or side effects. These findings have led to a drug discovery effort to develop functionally selective MOR ligands which activate the desired cascades (G protein) and not the undesirable cascades (βarrestin2). However, the βarrestin pathway is the only such functionally selective target identified for MOR drug discovery. In order to discover additional novel MOR regulators, we have developed two unbiased screening approaches. First, we have used co‐immunoprecipitation of the drug activated MOR signaling complex in MOR‐expressing CHO cells, followed by proteomic analysis. In addition, we have developed a pooled small‐hairpin RNA screen to find regulators of MOR induced signaling (ERK MAPK readout) in MOR‐expressing HEK293 cells. We present here our current progress using these screens, the outcome of which are lists of candidate MOR signaling regulators. One possible MOR‐regulating scaffold protein has been knocked down in MOR‐expressing CHO cells using small‐interfering RNA and shows evidence of regulating ERK MAPK pathways induced by the MOR. By defining the MOR signaling complex in this way, we hope to identify future targets for functionally selective drug discovery in order to improve the treatment of chronic pain.

Grant Funding Source: Supported by UNE COBRE Seed Grant P20GM103643
Effects of eicosapentaenoic acid and the GPR120 agonist TUG‐891 on lysophosphatidic acid signaling in human prostate cancer cells (1066.10)
Authors: Hopkins Mandi , Liu Ze , Meier Kathryn
Published: June 2021

There is a high incidence of prostate cancer in the Western world, which may be partially attributed to Western diets that are generally deficient in omega‐3 fatty acids (n‐3 FAs). Dietary n‐3 FAs have been shown to inhibit proliferation of prostate cancer cells in culture. The G protein‐coupled receptor GPR120, which is expressed in normal prostate and prostate tumors, serves as a receptor for the n‐3 FA eicosapentaenoic acid (EPA). We therefore hypothesized that the inhibitory effects of n‐3 FAs on prostate cancer cell proliferation are mediated by GPR120. To test this hypothesis, we performed proliferation and signal transduction studies, comparing the effects of lysophosphatidic acid (LPA), EPA, the GPR120 agonist TUG‐891, and the GPR40 agonist GW9508 in DU145 and PC‐3 cells. We found that EPA and TUG‐891 inhibit proliferation of human prostate cancer cells in response to LPA. In addition, brief (15 minute) pre‐incubation of serum‐starved cells with EPA or TUG‐891 suppresses subsequent activation of ERK, p70S6K, and FAK in response to LPA. This rapid effect of EPA is consistent with mediation by a GPCR. Our data suggest that GPR120 shows promise as a therapeutic target in prostate cancer.

Grant Funding Source: Washington State University College of Pharmacy
Brain tissue oxygenation in response to changes in arterial pressure in patients with mild cognitive impairment (1068.6)
Authors: Liu Jie , Tarumi Takashi , Tseng Benjamin , Hill Candace , Armstrong Kyle , Hynan Linda , Hodics Timea , Zhang Rong
Published: June 2021

Purpose: To test the hypothesis that brain oxygenation responses to changes in arterial pressure are altered in patients with amnestic mild cognitive impairment (aMCI).

Methods: Forty‐five aMCI patients (18 males, 65 ± 6 yr) and 30 age and education matched cognitively normal control subjects (NC, 15 males, 66 ± 6 yr) underwent stepwise changes in mean arterial blood pressure (MAP) induced by intravenous infusion of sodium nitroprusside and phenylephrine. Transcranial Doppler and near‐infrared spectroscopy were used to measure the cerebral blood flow velocity (CBFV) at the middle cerebral artery and cortical tissue oxygenation index (TOI) at the prefrontal lobe, simultaneously. All measurements were normalized as percentage changes from baseline (Δ, %). Random coefficient mixed model was used to estimate the regression slopes of ΔCBFV% and ΔTOI% to changes in ΔMAP%.

Results: Drug induced changes in MAP were from ‐26% to 49% (65~143 mmHg) relative to the baseline. The mixed model regression slopes of ∆CBFV% to ∆MAP% showed no differences between aMCI and NC (0.313 ± 0.047 vs. 0.219 ± 0.065, P = 0.238), suggesting the presence of intact cerebral autoregulation in aMCI. However, the slope of ∆TOI% to ∆MAP% was steeper in aMCI than in NC (‐0.253 ± 0.024 vs. ‐0.147 ± 0.032, P = 0.007), indicating an enhanced cortical oxygenation response to changes in blood pressure in aMCI.

Conclusion: The enhanced cortical oxygenation responses to changes in blood pressure in patients with aMCI suggest that abnormalities of the cerebral microvasculature in regulation of brain tissue oxygenation may occur early in Alzheimer disease.

Grant Funding Source: NIH grant R01AG033106‐01 and R01HL102457
Age‐related differences in carotid and cerebral blood flow regulation (1069.4)
Authors: Kruse Sarah , Ranadive Sushant , Taylor Jennifer , Joyner Michael , Barnes Jill
Published: June 2021

Common carotid artery (CA) and middle cerebral artery (MCA) blood flow are controlled by multiple factors to ensure that the brain receives adequate blood supply. Additionally, CA arterial stiffness may affect blood flow regulation. The purpose of this study was to investigate the effect of cyclooxygenase (COX) inhibition on arterial stiffness and the relationship between CA and MCA blood flow in young (26±5y) and older (66±7y) adults to determine if aging modifies this relationship. While baseline CA flow and flow velocity were similar between groups, baseline MCA flow velocity was greater in young (59±6 cm/s) compared to older (40±4 cm/s) adults (p<0.05). During COX inhibition, CA flow, CA flow velocity and MCA flow velocity decreased from baseline in young (∆: ‐116±35 mL/min, ‐6±1 cm/s, ‐23±5 cm/s; respectively) and older adults (∆: ‐153±40 mL/min, ‐5±1 cm/s, ‐11±2 cm/s; respectively). The association between CA flow velocity and MCA flow velocity was altered by COX inhibition (r=0.60 vs. r=0.15). β‐stiffness was higher in older compared to young adults (13±2 vs. 5±1 AU; p<0.05). Among older adults, there was a trend for COX inhibition to increase β‐stiffness. Our results suggest that CA and MCA blood flow are regulated differently and that COX inhibition alters the correlation between CA and MCA blood flow. Therefore, it may be incorrect to assume that CA flow is a surrogate measurement for cerebral blood flow.
Effects of aging and high fat diet on endothelial function in middle cerebral arteries is not additive (1070.11)
Authors: Reihl Kelly , Walker Ashley , Henson Grant , Donato Anthony , Lesniewski Lisa
Published: June 2021

Vascular dysfunction in cerebral arteries has been associated with cognitive impairments with advancing age and disease. It is not known, however, how aging and consumption of a high fat (HF) obesigenic diet interact to impact cerebrovascular function. We examined endothelium dependent dilation (EDD) to acetylcholine in the middle cerebral arteries of young (6‐8 mo) and old (29‐31 mo) normal chow (NC) and HF diet fed mice. Maximal EDD was reduced with aging (68 ± 6 vs. 40 ± 2%, P<0.01) in NC mice. HF reduced EDD in young (41 ± 3%, P<0.01) but was without effect in old (34 ± 5%, P=0.3) mice. Differences in maximal EDD with aging and after HF diet were eliminated after inhibition of nitric oxide (NO) synthase by L‐NAME, indicating that the impairments resulted from reduced NO bioavailability. There were no differences in endothelium independent dilation to sodium nitroprusside between groups (P’s>0.5). Advancing age and consumption of a HF diet independently reduce cerebrovascular endothelial function through reductions in NO, but there is no further decrement in old mice fed HF diet.

Grant Funding Source: Supported by NIH grants: R21 AG033755, K01 AG033196, R01 AG040297
Antihypertensive therapy reduces myogenic tone and increases the resting diameter of cerebral penetrating arterioles in hypertensive rats (1070.3)
Authors: Pires Paulo , Jackson William , Dorrance Anne
Published: June 2021

Hypertension impairs neurovascular coupling, possibly by altering the structure and function of cerebral penetrating arterioles (PenA). We hypothesized that an antihypertensive therapy (AhT) would reduce myogenic tone and increase the lumen diameter of PenA in stroke‐prone spontaneously hypertensive rats (SHRSP). Twelve‐week‐old male SHRSP received hydralazine (150mg/L)+hydrochlorothiazide (50mg/L)+reserpine (4mg/L) in the drinking water for 6 weeks (SHRSP+AhT). Controls received regular drinking water (SHRSP). PenA function and structure were studied by pressure myography. Data are shown as means±SEM, SHRSP vs SHRSP+AhT, n=8 for each group. AhT significantly reduced blood pressure (systolic blood pressure: 214±4 vs 130±6, p<0.05). AhT reduced PenA myogenic tone (%tone: 47.9±4.4 vs 29.1±3.3, p<0.05), and increased PenA resting diameter (34.6±1.9 vs 43.8±4.5 μm, p<0.05). PenA from SHRSP+AhT dilated less to 0.1 μM nifedipine (change in diameter from resting: 24.3±2.5 vs 12.5±1.8 μm, p<0.05). AhT did not increase PenA passive lumen diameter (63.7±3.3 vs 60.2±3.7 μm), but reduced wall thickness (11.7±1.0 vs 9.2±0.8 μm, p<0.05). These data support our hypothesis and indicate that AhT may improve local perfusion and prevent the consequences of hypertension on the cerebral microcirculation.

Grant Funding Source: Supported by AHA (12PRE8960019:PWP, 13GRNT17210000:AMD) and NIH (P01‐HL07687)
Mild deoxycorticosterone (DOCA)/salt‐induced hypertension causes cerebral microvascular dysfunction via a rho kinase dependent mechanism (1070.4)
Authors: De Silva T. Michael , Lynch Cynthia , Grobe Justin , Faraci Frank
Published: June 2021

Hypertension is a major cause of cerebrovascular disease. Microvascular dysfunction in brain is a key feature of small vessel disease that increases the risk of stroke and cognitive impairment. We tested whether mild DOCA/salt‐induced hypertension alters cerebral microvascular function. We treated male C57Bl6 mice with DOCA and 0.9% salt in their drinking water for 3 weeks and measured microvascular reactivity in anesthetized mice using a cranial window preparation. Blood pressure was elevated after DOCA/salt treatment (79±2 vs 95±3 mmHg; P<0.01). Endothelium‐dependent dilation of small cerebral arterioles was selectively impaired in mice treated with DOCA/salt compared with sham (eg, 10 μM acetylcholine: 29±5% vs 11±2%; P<0.01). Endothelium‐independent vasodilation to nitroprusside (an NO donor) or nifedipine (a Ca2+ channel blocker) was similar between groups. Rho kinase is a potential mediator of cerebrovascular disease. Local inhibition of Rho kinase with Y27632 did not affect endothelium‐dependent dilation in sham. In contrast, Y27632 restored endothelium‐dependent dilation to normal in DOCA/salt mice (3 µM) (10 μM acetylcholine: 10±1% vs 22±4%; P<0.01). These findings demonstrate that mild DOCA/salt‐induced hypertension profoundly impacts cerebral microvascular function. Our findings identify Rho kinase as a key contributor to DOCA/salt‐induced endothelial dysfunction in brain.

Grant Funding Source: Supported by NIH (HL‐62984, HL‐113863) VA (BX001399), Fondation Leducq and NHMRC (1053786)
Pulse pressure oscillations regulate cerebrovascular reactivity to flow (1070.5)
Authors: Raignault Adeline , Bolduc Virginie , Lesage Frédéric , Thorin Éric
Published: June 2021

In vivo, arterial blood pressure is oscillatory and pulsated. This pulse pressure (PP) corresponds to the difference between the systolic and diastolic pressure, and its frequency is dictated by heart rate (bpm). Our aim was to determine, in vitro, the unknown impact of PP (30 mm Hg, 550 bpm) on endothelial shear stress sensitivity (flow‐mediated dilatation) of mouse cerebral arteries. Methods/results: We developed an original system that generates a pulse‐wave adjustable in amplitude and frequency, and can be connected to a pressure arteriograph. Middle cerebral arteries (≍160 µm diameter, n=10 per group) were isolated from 3‐month old C57Bl6 mice and pressurized at 60 or 100 mm Hg either in static pressure (SP) or PP conditions. At 60 mm Hg, pre‐constricted arteries similarly dilated to an increase in flow up to a shear stress of 15 dyn/cm2, to 59±6% of the maximal diameter in PP, and 52±10% in SP conditions. In response to a higher shear stress of 20 dyn/cm2, however, arteries further dilated in PP (73±7%; p<0.05) unlike in SP (43±9%) conditions. At 100 mm Hg, arteries dilated in response to low shear stress (8 dyn/cm2) but less at high shear stresses (20 dyn/cm2): dilation was reduced by 50% in SP (from 44±11% to 23±11%; p<0.05) and 75% in PP (from 34±3% to 8±4%; p<0.05) conditions. Conclusion: PP increases the sensitivity of the endothelium to shear stress at physiological pressure (60 mm Hg) while it limits its sensitivity to high shear stress at high pressure (100 mm Hg), suggesting that PP regulates the endothelial function to critically adjust vessel diameter and flow to neuronal metabolic demand.

Grant Funding Source: CIHR
Gestational chronic intermittent hypoxia alters glucose homeostasis in rat male offspring (1072.3)
Authors: Iqbal Waseem , Hardy Daniel , Ciriello John
Published: June 2021

We have shown that exposure to chronic intermittent hypoxia (CIH) during pregnancy results in offspring with increased fat deposition that are hyperglycemic, hyperinsulinemic, and have impaired early‐stage glucose tolerance in adulthood, but the underlying mechanisms are unknown. This study was done to examine changes in the glucoregulatory function of the liver as a result of CIH exposure during gestational development. Female Sprague‐Dawley rats were mated and exposed daily to CIH throughout gestation. Offspring were sacrificed at postnatal day‐1 or 6‐wks of age to determine immediate and long‐term changes in liver function. By 6 wks of age, male CIH offspring had higher plasma corticosterone concomitant with higher liver protein expression of 11β‐hydroxysteroid dehydrogenase type I and glucocorticoid receptor (GR). These were associated with lower liver X receptor (LXR) protein expression. Analysis of GR and LXR‐direct target genes in gluconeogenesis showed that glucose‐6‐phosphatase expression was higher in CIH offspring, while no changes in phosphoenolpyruvate carboxykinase protein levels were observed. Taken together, these data suggest that gestational exposure to CIH results in long‐term alteration of hepatic glucose homeostasis via increased glucocorticoid signaling and decreased regulation of gluconeogenic enzymes by LXR. Supported by HSF of Ontario and CIHR.

Grant Funding Source: Heart and Stroke Foundation of Ontario and Canadian Institutes of Health Research
O‐GlcNacase overexpression restores coronary endothelial dysfunction in type 1 diabetic mice (1076.1)
Authors: Dai Anzhi , Donthamsetty Reshma , Han Ying , Scott Brian , Wang Hong , Dillmann Wolfgang , Makino Ayako
Published: June 2021

Cardiac ischemia is a leading cause of morbidity and mortality in diabetes, and endothelial cell (EC) dysfunction is implicated in coronary arterial disease. O‐linked N‐acetyglucosamine (O‐GlcNAcylation) is a post‐translational modification that is tightly controlled by two enzymes: O‐GlcNAc transferase (OGT, catalyzes the addition of an O‐GlcNAc to proteins) and O‐GlcNAcase (GCA, removes GlcNAc from proteins). In this study, we examine the role of GCA in coronary endothelial dysfunction in type 1 diabetic mice.

We generated EC‐specific, inducible GCA transgenic mice and induced diabetes in these mice with streptozotocin. In diabetic mouse coronary endothelial cells (MCECs), GCA expression was significantly decreased and protein O‐GlcNAcylation was increased. GCA overexpression in diabetic mice decreased O‐GlcNAcylation in MCECs. Capillary density in the left ventricle was decreased in diabetes, while GCA overexpression significantly increased the density to the control level. Endothelium‐dependent relaxation was attenuated in diabetic coronary arteries (CAs) and restored by GCA overexpression. These data suggest that elevated protein O‐GlcNAcylation due to decreased GCA protein expression in MCECs contributes to coronary endothelial dysfunction in diabetes, whereas overexpression of GCA in ECs improves endothelial function and may have a beneficial effect on coronary vascular complications in diabetes.

Grant Funding Source: supported by NIH/NHLBI(HL115578)
Expression and function of BK and Kv1.5 channels in aortic smooth muscle from lean and obese Zucker rats (1077.5)
Authors: Rottgen Trey , Fancher Ibra , Dick Gregory
Published: June 2021

We tested the hypothesis that metabolic syndrome is associated with reduced expression and function of K+ channels in smooth muscle. Aortic smooth muscle from male lean and obese Zucker rats was used as a model. Whole‐cell patch clamp experiments indicated that Ca2+‐activated (BK) and voltage‐dependent (KV) K+ currents dominated membrane conductance. Penitrem A (1 uM) and DPO‐1 (10 uM), selective antagonists of BK and KV1.5 channels were used to determine the expression of these channels. In lean rats, penitrem A inhibited 37 ± 6% of current at +100 mV, whereas subsequent addition of DPO‐1 inhibited 74 ± 4% of the remaining current. In obese rats, penitrem A inhibited 19 ± 6% of current at +100 mV, whereas subsequent addition of DPO‐1 inhibited 78 ± 3% of the remaining current. The penitrem A‐sensitive BK current was 2.7 ± 0.7 fold greater in lean rats. In contrast, and surprisingly, the DPO‐1‐sensitive KV1.5 current was 1.5 ± 0.2 fold greater in obese rats. Western blots supported the patch clamp studies, as BK channel protein was 1.4 ± 0.1 fold greater in lean rats, while KV1.5 protein was 2.5 ± 0.4 fold greater in obese rats. These data indicate that metabolic syndrome decreases the expression of BK channels, but simultaneously increases KV1.5 channel expression. We suggest this opposite change in K+ channel expression may represent a compensatory stage of metabolic disease that serves to preserve cardiovascular function.

Grant Funding Source: Supported by T32HL090610
High‐density lipoprotein modulates glucose metabolism in cardiomyocytes (1078.12)
Authors: Siebel Andrew , Heywood Sarah , Henstridge Darren , Carey Andrew , Delbridge Lea , Kingwell Bronwyn
Published: June 2021

The effects of HDL on the coronary vasculature are well understood, however its direct actions on the myocardium remain to be elucidated. We have previously shown that HDL stimulates glucose uptake into skeletal muscle, but whether HDL‐mediated glucoregulatory mechanisms exist within cardiac muscle is unknown. We sought to determine the effect of HDL on myocardial glucose uptake, glycolysis, glucose oxidation, oxidative metabolism and the associated intracellular signalling mechanisms using a neonatal rat ventricular cardiomyocyte model. HDL (50µg/ml) treatment increased glucose uptake by 47±10% and glycolysis by 43±8% in association with rapid activation of Akt/PKB and its downstream target AS160, a regulator of GLUT4 translocation. HDL also increased basal and maximal mitochondrial respiration by 40±8% and ATP turnover by 48±8%. Glucose oxidation was increased by 30±6% with 50µg/ml HDL and may contribute to observed increases in total mitochondrial respiration. HDL‐mediated increases in both glycolytic and mitochondrial respiratory capacity may limit ATP depletion during ischaemia‐reperfusion and thereby minimise myocardial cell damage.

Grant Funding Source: Supported by the National Heart Foundation of Australia, National Health & Medical Research Council
Coronary arterial wave form relates to compression of intramural vasculature controlled by left ventricular end diastolic pressure (1079.11)
Authors: Spaan Jos , Rolandi Cristina , Wiegerinck Esther , Piek Jan , Baan Jan , Siebes Maria
Published: June 2021

The aim of this study was to test the hypothesis that left ventricular, LV, end diastolic pressure, LVEDP, contributes to coronary arterial flow waveform since it reflects venous pressure determining microvascular intramural blood volume, MIBV.

In 12 patients undergoing coronary catheterization, coronary flow velocity and pressure were measured simultaneously in undiseased vessels with a 0.014‐in dual‐sensor guidewire and LV pressure with an open ended catheter. Measurements were performed under intact autoregulation. The signals were analyzed with wave intensity analysis that allows separating the contribution to the net coronary energy coming from the aorta and from the microcirculation.

Both the microcirculatory‐originating waves, generated during contraction (backward compression wave, BCW) and relaxation (backward expansion wave, BEW), were linearly related to LVEDP (p<0.04 for both waves) while both the aortic‐generating waves during contraction and relaxation were not.

It is known that LVEDP relates well to coronary venous pressure hence, an increase in LVEDP reduces the pressure head over the microcirculation. Its effect on flow can be compensated by autoregulation but also by increasing MIBV due to increasing microvascular pressure pressure. Increased MIBV results in an enhanced BCW by the elastance effect when the myocardium is contracting and consequently it results also in a higher BEW because of restoration forces due to compliance of the microcirculation. By increasing the energy of the BCW and BEW, LVEDP has an influence on the coronary flow waveform.
Peroxynitrite disrupts endothelial caveolae leading to eNOS uncoupling and diminished flow‐mediated dilation in coronary arterioles of diabetic patients (1082.3)
Authors: Bagi Zsolt , Feher Attila , Cassuto James
Published: June 2021

Peroxynitrite (ONOO‐) contributes to coronary microvascular dysfunction in diabetes (DM). We hypothesized that in DM ONOO‐ interferes with the function of coronary endothelial caveolae, which plays an important role in nitric‐oxide (NO)‐dependent vasomotor regulation. Flow‐mediated dilation (FMD) of coronary arterioles was investigated in DM (n=41) and nonDM (n=39) patients undergoing heart surgery. NO‐mediated coronary FMD was significantly reduced in DM patients, which was restored by ONOO‐ scavenger, uric‐acid, whereas exogenous ONOO‐ reduced FMD in nonDM subjects. Immuno‐electronmicroscopy demonstrated an increased 3‐nitrotyrosine formation (ONOO‐ specific protein nitration) in endothelial plasma membrane in DM, which co‐localized with caveolin‐1 (Cav‐1), the key structural protein of caveolae. The membrane‐localized Cav‐1 was significantly reduced in DM and also in high glucose‐exposed coronary endothelial cells. We also found that DM patients exhibited a decreased number of endothelial caveolae, whereas exogenous ONOO‐ reduced caveolae number. Correspondingly, pharmacological (methyl‐β‐cyclodextrin) or genetic disruption of caveolae (Cav‐1‐knockout mice) abolished coronary FMD, which was rescued by sepiapterin, the stable precursor of NO synthase cofactor, tetrahydrobiopterin. Sepiapterin also restored coronary FMD in DM. Thus, we propose that ONOO‐ selectively targets and disrupts endothelial caveolae, which leads to NO synthase uncoupling, hence reduced NO‐mediated coronary vasodilation in DM patients.

Grant Funding Source: Supported by NIH HL104126
Thermal stress conditions endothelial cells and improves cellular function (1084.10)
Authors: Needham Karen , Macrorie Sarah , Minson Christopher , Gilbert Jeffrey
Published: June 2021

It is well established that temporary exposure to a stressor, such as heat, can induce the production of proteins that confer cellular protection (heat shock proteins ‐ HSP). Our lab has shown that exercise training (a stressor) in pregnant rats increases production of HSP 27, 60, and 90 in the placenta. The aim of this study is to determine whether heat shock, resulting in the up‐regulation of HSPs, improves endothelial function in human umbilical vascular endothelium (HUVEC) treated with normal pregnant rat serum or serum from rats with placental ischemia induced hypertension (RUPP). HUVECs were heat shocked at 42.5° C for 4hrs followed by 2hr recovery at 37° C. Extracts from shocked cells had 4x HSP 70 (p<0.01) and 3x HSP 27 (p<0.01) protein levels over the control. Shocked cells and 37° C control cells were treated with NP or RUPP serum and function was assessed with angiogenesis and wound healing assays. Tubule length counts in heat treated cells were at three times the level of no shock controls (p <0.01), without any effect from serum. In the wound assay RUPP/heat shocked cells tended toward greater mitogenesis and motility. These benefits may involve endothelin 1, which has been shown to promote HUVEC proliferation and migration and was elevated by 20% (p<0.05) in the media of RUPP/heat shock HUVECS in our study. These results suggest that human endothelial cells may improve function with conditioning by low level stressors. Such conditioning, as would be conferred by exercise, may improve the outcome for women with preeclampsia.

Grant Funding Source: Supported by NIH RO1HL114096 and AHA 10SDG2600040
Exercise before and during pregnancy does not lower blood pressure in an sFlt‐1 infusion model of preeclampsia in the rat (1084.9)
Authors: Banek Christopher , Gillham Haley , Needham Karen , Johnson Sarah , Beasley Kara , Gilbert Jeffrey
Published: June 2021

Preeclampsia (PE) is a pervasive, pregnancy‐specific syndrome defined by new onset hypertension and proteinuria. Additionally, PE is often associated with increased soluble VEGF Receptor‐1 (sFlt‐1), decreasing the bioavailability of vascular endothelial growth factor (VEGF) creating angiogenic imbalance leading endothelial dysfunction and hypertension. We recently reported exercise training attenuates placental ischemia‐induced hypertension and restores angiogenic balance but the mechanism remains unclear. Thus, we hypothesized exercise training before and during (EBD) pregnancy would increase plasma free VEGF and lower blood pressure in an sFlt‐1 infusion model of PE. EBD rats voluntarily exercised for six weeks before and during pregnancy on an activity wheel. Age‐matched sedentary (Sed) controls were housed without wheel access. After breeding, the dams regained access to the activity wheels. Osmotic minipumps infused sFlt‐1 (500ng/hr) or vehicle from gestational day 14 to day 19 when blood pressures and tissues were collected. In both Sed and EBD groups, sFlt‐1 infusion increased (p<0.05) blood pressure, decreased (p<0.05) endothelial dependent vascular relaxation of mesenteric vessels via wire myography to acetylcholine (ACh) and decreased (p<0.05) endothelial tube formation by HUVECs on Matrigel. EBD had no effect on sFlt‐1 hypertension but improved (p<0.05) vascular endothelial relaxation to ACh and improved (p<0.05) HUVEC tubule formation. In contrast with our hypothesis, these data suggest sFlt‐1‐induced hypertension in pregnancy is not remediated by EBD despite improved tubule formation in vitro and vascular endothelial function ex vivo. Together these data suggest improvements in vascular endothelial function as a result of exercise before and during pregnancy may be due to the stimulation of alternative pro‐angiogenic and vasorelaxant pathways. Further studies are underway to elucidate these alternative mechanisms.

Grant Funding Source: Supported by NIH RO1HL114096 and AHA 10SDG2600040
High fat feeding induces exaggerated endothelial dysfunction and insulin resistance in rats exposed to maternal separation (1085.7)
Authors: Loria Analia , Fox Brandon , Pollock David , Pollock Jennifer
Published: June 2021

We previously reported that adult male rats exposed to MatSep (3 hrs/day from day 2 to 14 of life) display normal baseline cardiovascular parameters but enhanced responses to angiotensin II (AngII) compared to controls (C, non‐MatSep littermates). We tested the hypothesis that a chronic HF diet (60% fat cal., 0.4 salt; 6 months) exaggerates metabolic and/or cardiovascular indices in MatSep rats. Plasma fasting glucose and insulin were higher in MatSep vs. C rats (p<0.05), showing a greater HOMA index in MatSep than C rats (1.0±0.1 vs. 1.7±0.1, p<0.05). Plasma adiponectin was lower in MatSep vs. C rats (19±2 vs. 28±2 ug/ml, p<0.05), although body weight and leptin were similar between groups. No differences in mean arterial pressure or heart rate were observed between MatSep and C rats (105±2 vs. 102±2mmHg; 326±6 vs. 317±3 bpm; n=6). Wire myography of thoracic aortae showed a greater vasoconstrictive response (%max constriction) to AngII (17±2 vs. 6±1, p<0.05) and phenylephrine (59±4 vs. 29±5, p<0.05) from MatSep rats. L‐NAME pre‐incubation revealed a loss of NO buffering capacity to AngII in MatSep aortae only (p<0.05). Impaired acetylcholine‐induced relaxation was greater in aortae from MatSep rats (49±6 vs. 27±3 % relax, p<0.05). These data reveal that MatSep followed by HF feeding induces a synergistic effect on disrupting vascular function and glucose homeostasis increasing the risk to cardiovascular disease.

Grant Funding Source: HL111354 and PO1 HL69999
Vascular dysfunction, arterial hypertension and insulin‐resistance in offspring of a mouse model of preeclampsia: prevention by sildenafil (1085.8)
Authors: Cerny David , Rexhaj Emrush , Bouillet Elisa , Soria Rodrigo , Sartori Claudio , Allemann Yves , Rimoldi Stefano , Scherrer Urs
Published: June 2021

In humans, offspring of mothers with preeclampsia display generalized vascular dysfunction and premature cardiovascular morbidity, but underpinning mechanisms and possibilities for prevention are unknown. In mice, offspring of restrictive diet pregnancy (RDP), a model mimicking preeclampsia in humans, display insulin resistance and vascular dysfunction offering an opportunity to study interventions aimed at preventing these problems. Sildenafil administration to preeclamptic rats increases birth weight in the offspring.

We assessed vascular function in vitro (mesenteric artery dilation), arterial blood pressure (carotid artery) and insulin sensitivity (clamp studies) in vivo in offspring of RDP (Sildenafil 40 mg/kg/day or vehicle in drinking water, day 7 to end of pregnancy) and control mice. As expected, offspring of RDP displayed impaired acetylcholine‐induced mesenteric artery dilation (P<.0001 vs. ctrl), arterial hypertension (119±7 vs. 112±5 mmHg, X±SD, P=.04 vs. ctrl) and insulin resistance (Glucose Infusion Rate 77.4±10.7 vs. 96.7±12.2 mg/kg*min, P<.001 vs. ctrl). Sildenafil administration during RDP prevented these problems in the offspring (all P>.5 vs. ctrl). We show for the first time that in mice sildenafil administration during RDP prevents arterial hypertension and insulin resistance in the offspring. We speculate that in humans sildenafil has similar beneficial effects.
Endoplasmic reticulum stress contributes coronary artery dysfunction in type 2 diabetic mice (1086.7)
Authors: Choi Soo‐Kyoung , Lee Young‐Ho
Published: June 2021

Background: Vascular dysfunction is a major complication in type 2 diabetes. Although endoplasmic reticulum (ER) stress has been suggested to be a contributing factor in cardiovascular diseases, the relationship between ER stress and type 2 diabetic vascular dysfunction remains unclear. Thus, in the present study, we examined whether ER stress contributes coronary artery dysfunction and ER stress inhibition ameliorates vascular function in type 2 diabetes. Methods and Results: Type 2 diabetic and their control mice were treated with or without ER stress inhibitors (taurine‐conjugated ursodeoxycholic acid, TUDCA, and 4‐phenylbutyric acid, 4‐PBA) for 2 weeks. In type 2 diabetic mice, blood glucose, body weight, and insulin level are elevated compared to control mice. The myogenic response is potentiated and endothelium‐dependent relaxation is impaired in type 2 diabetic mice. Interestingly, treatment of ER stress inhibitors normalized myogenic responses and endothelium dependent relaxation.These data were associated with an increase in ER stress marker expression (CHOP, ATF6, XBP‐1, and phosphorylated‐eIF2α) in heart and mesenteric resistance arteries in type 2 diabetic mice, which were reduced by treatment with ER stress inhibitors. Conclusion: ER stress inhibition normalizes myogenic response and improves vascular function in type 2 diabetes. Therefore, ER stress could be a potential target for cardiovascular diseases in diabetes mellitus.

Grant Funding Source: supported by the Basic Science Research Program of the National Research Foundation of Korea (NRF)
Prenatal taurine supplementation decreases adult renal function by mechanisms other than taurine inhibition of prenatal renin‐angiotensin system (1087.1)
Authors: Roysommuti Sanya , Kritsongsakchai Angkana , Wyss J.
Published: June 2021

Taurine inhibits renin‐angiotensin system (RAS). This study tests the hypothesis that the effect of perinatal taurine supplementation on adult renal function is primarily due to prenatal inhibition of the RAS by taurine. Female Sprague‐Dawley rats were fed normal rat chow and given water alone (control) or water containing captopril (400 mg/ml; captopril) or 3% taurine (taurine) from conception until delivery. Then, the rats received normal rat chow and tap water. At 7‐8 weeks of age, renal function at rest and after acute saline load (5% of body weight) was studied in conscious, restrained male rats. Compared to control, prenatal taurine but not captopril treatment slightly increased mean arterial pressure but not heart rate in adult rats. In taurine compared to control and captopril groups, renal blood flow significantly decreased whereas renal vascular resistance significantly increased. Glomerular filtration, filtration fraction, diuretic and natriuretic responses to acute saline load were significantly increased in captopril compared to the other two groups. These data indicate that the prenatal inhibition of RAS increases renal glomerular and tubular functions, while the prenatal taurine supplementation worsens renal vascular and blood pressure control. Thus, the effect of perinatal taurine supplementation on adult renal function is not related to the ability of prenatal taurine to inhibit the RAS.

Grant Funding Source: Supported by Khon Kaen University
Expression of an angiotensin‐(1‐7) endopeptidase in proximal tubules of the sheep and human kidney (1088.10)
Authors: Wilson Bryan , Marshall Allyson , Pirro Nancy , Su Yixin , Rose James , Chappell Mark
Published: June 2021

Angiotensin‐(1‐7) [Ang 7] is a an active peptide of the renin‐angiotensin system (RAS) that generally antagonizes the actions of Ang II. Within the kidney, Ang 7 is derived from Ang I by endopeptidases including neprilysin (NEP) and thimet oligopeptidase (TOP) or from Ang II by the carboxypeptidase ACE2. Ang 7 is primarily degraded by ACE to the Ang‐(1‐5) in the circulation and tissues; however, we identified an endopeptidase in sheep CSF that efficiently degraded Ang 7 to Ang‐(1‐4) (Ang 4) and accounted for the majority of Ang 7 metabolism in this compartment (Marshall et al AJP 2013). The current study determined whether the Ang 7 peptidase is expressed in the kidney, specifically in isolated proximal tubules of the sheep and human renal epithelial cells. Activity was determined by the conversion of 125I‐Ang 7 to 125I‐Ang 4 in a 100,000 xg supernatant fraction. Both the sheep proximal tubules and the human epithelial cells expressed Ang 7 endopeptidase activity. Kinetic analysis revealed an apparent Km of 2.0 ± 1.0 µM and apparent Vmax’ of 93 ± 37 pmol/min/mg protein [n=3]. Peptidase activity in the isolated tubules was attenuated by the metalloendopeptidase inhibitor JMV‐390 with an IC50 of 0.81 ± 0.12 nM [n=3] that is ~50‐fold more potent than that of NEP and TOP. We conclude that an Ang 7 endopeptidase may be a novel component of the intrarenal RAS to regulate the tubular expression of Ang 7.

Grant Funding Source: Supported by NIH grants HL56973, HL52972, HL112237, and HD047584
Adaptation of chronic renal microdialysis to evaluate the role of interstitial ATP and angiotensin II in accelerating nephropathy and oxidative stress during insulin induced hypertension (1088.3)
Authors: Prathipati Priyanka , Alanazi Wael , Fakhruddin Fnu , Jackson Debra , Jackson Keith
Published: June 2021

Various studies demonstrated the role of ATP in regulating hemodynamic functions and play a key role in maintaining tubuloglomerularfeedback (TGF) and renal vascular resistance. Previous studies also demonstrated the role of Ang II and HO‐1 in regulating blood pressure. We thus hypothesize that insulin induced hypoglycemia augments interstitial ATP levels which in turn increases AngII. This stimulates afferent arteriolar vasoconstriction interrupting normal TGF mechanism resulting in hypertension leading to oxidative stress and ultimately renal damage. Male Sprague Dawley rats (200‐225g) were anesthetized and microdialysis probe was inserted into their renal cortex. Post surgery rats were treated with insulin (7U/kg body weight) or vehicle for 2 weeks. Dialysate was collected daily after insulin dosing and analyzed for ATP by luciferin‐luciferase assay and Ang II by ELISA. ATP levels were elevated from 4.75 ng (day 0) to 36.47 ng (day 14). Heart and kidneys were collected at the end and analyzed for oxidative stress using EPR (Electron Paramagnetic Resonance). Oxidative stress was observed from the spectra obtained by using spin probes CMH and CPH. Thus the present study demonstrates a correlation between interstitial ATP and oxidative stress by real‐time chronic collection of renal interstitial samples, demonstrating the ability of hypoglycemia to promote the development of hypertension in turn accelerating oxidative stress.

Grant Funding Source: Board of Regents Research Competitiveness 2010
Glucagon‐like peptide 1 receptor activation acutely increases systolic blood pressure independent of aldosterone‐mediated sodium retention (1088.8)
Authors: Moreno Meagan , Rodriguez Ruben , Lee Andrew , Vazquez Guillermo , Ruilova Steven , Ortiz Rudy
Published: June 2021

Metabolic syndrome is associated with increased activation of the renin‐angiotensin system, high blood pressure and decreased circulating levels of glucagon‐like peptide‐1 (GLP‐1). GLP‐1 mimetics have been shown to decrease systolic blood pressure (SBP) via unknown mechanisms. To assess the effects of angiotensin receptor type 1 (AT1) and GLP‐1 receptor (GLP‐1r) activation on SBP, urinary Na+ (UNaV), aldosterone (UAldoV), and corticosterone (UBV) excretion were recorded for six weeks in five groups of rats: 1) untreated, lean LETO (n=7), 2) untreated, obese OLETF (n=5), 3) OLETF + AT1 blocker (ARB; 10 mg olmesartan /kg/d; n=4), 4) OLETF + GLP‐1 mimetic (Ex; 10 ug exenatide/kg/d; n=7), and 5) OLETF + ARB + Ex (combo; n=6). Ex UAldoV and UBV increased 207% and 123%, respectively, compared to OLETF on day 2 and were associated with a 62% UNaV decrease and 8% SBP increase. However, the 13% reduction in UAldoV in combo was associated with 94% increase in UBV, and 80% and 16% decrease in UNaV and SBP, respectively, compared to OLETF suggesting that activation of AT1 has a greater contribution to the insulin resistance‐associated increase in SBP than impaired UNaV. By day 7, Ex UAldoV was 48% lower than OLETF, but UBV, UNaV, and SBP were similar to OLETF. While GLP‐1r activation acutely increases SBP independently of aldosterone‐mediated sodium retention, ultimately the effect is rectified SBP and normalization of UNaV.

Grant Funding Source: Amylin Pharmaceuticals (Bristol‐Myers Squibb)
microRNA‐301b reduces PPARγ expression in transgenic sickle mice and in hemin‐treated human pulmonary artery endothelial cells (1089.22)
Authors: Kang Bum‐Yong , Tan Fang , Samit Ghosh , Archer David , Sutliff Roy , Ofori‐Acquah Solomon , Hart C. Michael
Published: June 2021

Sickle cell disease (SCD) is characterized by chronic intravascular hemolysis resulting in the release of hemin into the blood. Our preliminary microarray data indicate that PPARγ expression is significantly reduced in hemin‐treated human pulmonary artery endothelial cells (HPAECs). To examine post‐transcriptional mechanisms of PPARγ regulation in pulmonary hypertension in SCD (SCD‐PH), Townes knockin SCD mice aged 12‐weeks were gavaged daily with vehicle or rosiglitazone (RSG, 10 mg/kg/day) for 10 d. PPARγ was decreased and ET‐1 expression was increased in sickle mice (SS) compared to littermate control mice (AA). Using an in vitro model that mimics acute extracellular hemin release, HPAECs were treated with DMSO or hemin (5 μM) for 72 h ± RSG (10 μM) for the final 24 h. Similarly, hemin reduced PPARγ and increased ET‐1 expression in HPAECs. MicroRNA (miR‐301b), which negatively regulates PPARγ, was increased in SCD mice and in hemin‐treated HPAECs. In contrast, PPARγ activation attenuates increases in miR‐301b levels in SS mice and in hemin‐induced HPAECs. These findings suggest that hemolysis in SCD increases miR‐301b to reduce PPARγ and increase ET‐1 levels. These results suggest novel pathways for SCD‐PH pathogenesis and therapy.

Grant Funding Source: Supported by AHA‐SDG 13SDG14150004,CEB F16788‐00, Atlanta VA 1I01BX001910, NIH HL074518, DK102167
Antenatal chronic hypoxia and L‐type Ca2+‐dependent contractility of pulmonary arteries from fetal sheep (1089.6)
Authors: Brunelle Alex , Blood Quintin , Longo Lawrence , Wilson Sean
Published: June 2021

Recently we and others have shown that K+‐depolarization induced pulmonary arterial (PA) contraction is due to the combined activation of L‐type Ca2+ channels (CaL) and Rho kinase (ROK). In fetal sheep we demonstrated that CaL inhibition with nifedipine (NIF) reduced 125 mM K+‐mediated contraction up to about one‐half, with ROK contributing the remainder, and that the relationship between these two pathways was preserved following antenatal chronic hypoxia (CH). To better define the relationship between these pathways and the impact of antenatal CH, we tested the hypothesis that direct activation of CaL would elicit a NIF sensitive contraction and this contraction would be preserved following antenatal CH. This was examined using wire‐myography approaches on small diameter PA isolated from normoxic (~700 m) and antenatal CH (3,801 m, for >100 days) late gestation fetal sheep. Our results show that direct activation of CaL with 10 μM FPL 64176 (FPL) elicited a contraction that was approximately 80‐125% of the maximum K+‐induced depolarization (TKmax) in normoxic and hypoxic PA. In both normoxic and hypoxic PA 10 μM NIF reduced FPL‐induced contraction back to baseline. These data illustrate that NIF‐sensitive CaL‐dependent contraction is preserved following antenatal CH and when compared to our previous work that direct CaL activation causes fetal PA constriction in a distinct way compared to K+‐depolarization.

Grant Funding Source: Supported by NSF MRI 0923559, NIH HD‐069746, P01HD031226, R01HD003807
The structural dynamics of translation initiation (109.1)
Authors: Elvekrog Margaret , Wang Jiangning , Caban Kelvin , MacDougall Daniel , Gonzalez Ruben
Published: June 2021

Accurate initiator tRNA and mRNA start codon selection during translation initiation is essential for ensuring the integrity of gene expression; mis‐initiation using an elongator tRNA or a sense codon can lead to proteins containing aberrant N‐termini or to reading‐frame errors that yield truncated and/or mis‐folded proteins. In bacteria, three essential initiation factors (IFs) bind to the small, 30S, ribosomal subunit and control the accuracy of translation initiation by regulating tRNA and codon selection into the 30S initiation complex (30S IC) and joining of the large, 50S, ribosomal subunit to the 30S IC to form an elongation‐competent 70S initiation complex. Although IFs ensure that 50S subunit joining is dependent on the presence of a correctly selected initiator tRNA and start codon within the 30S IC, the mechanism through which the IFs accomplish this remains elusive. Using fluorescently labeled IFs, tRNAs, and ribosomal subunits, we have recently developed several single‐molecule fluorescence resonance energy transfer signals that report on the conformational dynamics of the 30S IC and on the dynamics of 50S subunit joining to the 30S IC. Here we will present the results of our latest studies, which, when integrated with the available biochemical data, strongly suggest that tRNA‐ and codon‐dependent conformational dynamics of the IFs play a critical role in regulating the 50S subunit joining step of translation initiation.
Mutant BMPR2 expression in cardiomyocytes results in an altered hypertrophic response (1090.2)
Authors: Talati Megha , Funke Mitchell , Bylund Jeffery , Trammell Aaron , Majka Susan , Fessel Joshua , West James , Newman John , Hatzopoulos Antonis , Hemnes Anna
Published: June 2021

Background: End‐stage pulmonary arterial hypertension (PAH) is characterized by right ventricular (RV) dysfunction, failure and death. We have shown BMPR2 mutation is associated with impaired RV hypertrophy and cardiomyocyte lipid deposition in both heritable PAH patients and a murine model of mutant BMPR2 expression.

Hypothesis: BMPR2 mutation alters hypertrophic response and fatty acid transport in RV cardiomyocytes.

Methods: Stable mouse embryonic stem cell lines (ESC) were engineered to express BMPR2 mutation using pCI‐Neo‐BMPR2 plasmids with mutation in either cytoplasmic (CD) or kinase domain (KD) or a linearized pCI‐Neo vector (Control) and gene expression confirmed by RT‐PCR. Directed differentiation of the ESC to cardiac myocytes (ESC CM) performed using noggin. ESC CM treated with phenylephrine for 72 hours to induce hypertrophy. Western analysis performed for proteins: CD36 (a fatty acid transporter molecule), brain natriuretic peptide (BNP) and corin (a serine peptidase which cleaves pro‐BNP). Oil‐red‐O stain performed for intracellular lipid deposition in ESC‐CM.

Results: All ESC‐CM expressed α‐SMA and demonstrated characteristic contraction in vitro. In response to hypertrophy, control ESC CM increased expression of BNP and corin, which remained unchanged in CD and KD BMPR2 mutant cells.

At baseline expression of CD36 protein was higher in CD BMPR2 mutant containing ESC CM and Oil‐red‐O staining suggested increased intracellular lipid accumulation in mutant ESC CM relative to control cells. Hypertrophic stimulus reduced the expression of CD36 in CD and KD BMPR2 ESC CM compared to control cells.

Conclusions: Altered BMPR2 signaling due to CD or KD mutations in ESC CM were associated with an impaired hypertrophic response. This impaired response was associated with decreased expression of BNP, corin and CD36. This may explain reduced ability of RV to adapt to increased RV afterload in PAH. Further characterization of the mechanism of impaired hypertrophy is planned.

Grant Funding Source: P01HL108800