bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of soluble epoxide hydrolase by t‐AUCB modulates coronary reactive hyperemia in isolated mouse hearts (1071.3)
Authors: Hanif Ahmad , Morisseau Christophe , Nayeem Mohammed
Published: June 2021

Coronary reactive hyperemia (RH) is impaired in hypertrophic hearts and in hearts with metabolic syndrome. Epoxyeicosatrienoic acids (EETs) exert cardioprotective effects against ischemia / reperfusion injury. Soluble epoxide hydrolase (sEH) is the main enzyme responsible for EETs breakdown. We hypothesized that the inhibition of sEH results in increased RH in C57BL/6J isolated mouse hearts through increased generation of EETs compared to non‐treated mouse hearts in response to no‐flow ischemia. Coronary flow (CF) in isolated C57BL/6J wild type mouse hearts was measured before and after sEH‐inhibition using a well characterized and selective sEH inhibitor, trans‐4‐[4‐(3‐adamantan‐1‐yl‐ureido)‐cyclohexyloxy]‐benzoic acid (t‐AUCB) in Langendorff system. In each experiment we used the same isolated heart as its own control. Perfused isolated hearts were exposed to 15‐second ischemia and RH parameters were assessed before and after 15‐minute infusion of t‐AUCB (10‐5 M). Following ischemia, flow repayment (calculated as the area under the curve and normalized to heart weight (ml/g)) was significantly increased in response to t‐AUCB (5.84 ± 0.80 in vehicle vs. 9.52 ± 1.68 in t‐AUCB, p<0.05). Moreover, t‐AUCB significantly increased baseline coronary flow (16.62 ± 0.85 in vehicle vs. 14.59 ± 0.63 in t‐AUCB, p<0.05). Also, we observed that t‐AUCB tends to increase repayment to debt ratio (1.53 ± 0.22 in vehicle vs. 2.32 ± 0.43 in t‐AUCB). No significant differences were observed in debt area, peak hyperemic flow and repayment duration between the two groups. Our results demonstrate that sEH inhibition by t‐AUCB significantly increases RH. These data suggest that sEH inhibition by t‐AUCB could have a beneficial effect on myocardial recovery from ischemic insult. Supported by HL‐114559 to MAN

Grant Funding Source: Supported by HL‐114559 to MAN
ADAM17 mediates TNF release from pericardial adipose tissue arteries to remotely impair coronary dilation in patients with type 2 diabetes (1071.5)
Authors: Dou Huijuan , Feher Attila , Romero Maritza , Bagi Zsolt
Published: June 2021

Pericardial adipose tissue (PAT) is a major source of pro‐inflammatory cytokine TNF, which contributes to the development coronary microvascular dysfunction in type 2 diabetes (T2DM). We hypothesized that TNF is actively cleaved from arteries of PAT by the sheddase, ADAM17 to alter the function of coronary resistance arteries (CRA) in T2DM. Arterioles were dissected from PAT (PATA) and also from the right atrial appendages obtained at the time of cardiac surgery of patients. PATAs and CRA were cannulated and serially connected and changes in CRA diameter were measured with video microscopy. The endothelium‐dependent vasodilator, bradykinin elicited substantial dilation in T2DM CRA, which was significantly reduced when the CRA connected to PATA from T2DM but not from nonDM PATA. Incubation of PATA with soluble TNF receptor, TNF receptor functional blocking antibody or inhibitor of ADAM17, all restored bradykinin‐induced dilation of CRA to the control level. We found an abundant expression of ADAM17 in the endothelium of PATA in both T2DM and nonDM patients. Despite no differences in the level of ADAM17 protein expression, a significantly increased ADAM17 activity was measured in PATA of T2DM patients. We conclude that PATA from T2DM patients are an important source of circulating TNF that can remotely impair dilation of CRA and may contribute to the pathogenesis of coronary microvascular disease in DM.

Grant Funding Source: NIH HL104126
Diabetes complications in congenic leptin receptor deficient BBDR.cg‐lepr.cp rats (1072.6)
Authors: Berglund Lisa , Åkesson Lina , Garcia Vaz Eliana , Zetterqvist Anna , Kotova Olga , Dutius Andersson Anna‐Maria , Johansson Martin , Wierup Nils , Jönsson‐Rylander Ann‐Cathrine , Lernmark Åke , Gomez Maria
Published: June 2021

Diabetes is a chronic disease that is associated with devastating complications. Here, we characterize micro‐ and macrovascular changes in a novel congenic BBDR.cg‐lepr.cp rat line, generated by introgression of the Koletsky leptin receptor mutation onto BioBreeding Diabetes Resistant rats. Interestingly, two subgroups of male BBDR.cg‐lepr.cp rats could be distinguished based on their blood glucose (BG), one severely diabetic (D) and one with moderately (M) elevated BG. Both groups were obese and showed significant enlargement of liver, heart and kidneys when compared to control littermates. Serum insulin, leptin, triglycerides and cholesterol were increased in all BBDR.cg‐lepr.cp rats when compared to controls, but with significant differences between groups; triglycerides and cholesterol were higher in D rats, leptin and insulin were higher in M rats. Albumin/creatinine ratio was dramatically increased and histological examination of kidneys revealed dilated and filled tubuli and loss of glomerular podocin expression, this was more pronounced in D rats when compared to M rats. All BBDR.cg‐lepr.cp rats had significantly higher systolic blood pressure, but surprisingly only M rats had increased aortic mRNA expression of endothelial activation and inflammatory markers. Collectively, our results point to different underlying factors for micro‐ and macrovascular disease in diabetes.

Grant Funding Source: Supported by VR 2011‐3900; the SUMMIT consortium (IMI‐2008/115006), Påhlsson and Diabetes Found.
The G protein‐coupled receptor (GPER/GPR30) activates endothelial nitric oxide synthase (1075.5)
Authors: Fredette Natalie , Meyer Matthias , Prossnitz Eric
Published: June 2021

Endogenous estrogens mediate protective effects in the cardiovascular system in part due to rapid activation of endothelial nitric oxide synthase (eNOS), which involves the classical estrogen receptor (ER) α. In intact blood vessels, nitric oxide (NO) bioactivity is also regulated by the G protein‐coupled estrogen receptor (GPER/GPR30), suggesting an alternative pathway of estrogen‐dependent eNOS activation. We have further investigated this mechanism by analyzing GPER signaling in telomerase‐immortalized human umbilical vein endothelial (TIVE) cells. Using an antibody recognizing the second extracellular loop of GPER, immunofluorescence microscopy revealed that GPER is expressed predominantly in intracellular membranes as determined by permeabilizing vs. non‐permeabilizing conditions. Similar to the non‐selective ER agonist 17β‐estradiol, the GPER‐selective agonist G‐1 stimulated phosphorylation of Akt (ser437) and eNOS (ser1171) in a dose‐dependent manner. Additional studies have examined the roles of EGFR and ERK in G‐1‐mediated eNOS activation. Pharmacological inhibition of GPER was used to assess its contribution to estrogen‐mediated NO production. Our results further characterize the signaling pathway of GPER‐mediated eNOS activation, suggesting that the beneficial NO‐dependent effects of endogenous estrogens in the cardiovascular system may partly be mediated by GPER.
Changes in vasomotor function following mitochondria‐targeted antioxidant treatment depend on baseline antioxidant capacity in hypercholesterolemic mice (1076.3)
Authors: Roos Carolyn , Zhang Bin , Thalji Nassir , Miller Jordan
Published: June 2021

Reduced antioxidant enzyme activity and subsequent impairments in endothelial function are major contributors to progression of atherosclerosis. We previously reported that endothelium‐dependent relaxation is well‐preserved in manganese superoxide dismutase‐deficient mice (MnSOD+/‐) due to production of a compensatory, H2O2‐derived endothelial hyperpolarizing factor (EDHF). It is unclear, however, whether similar phenomena occur in hypercholesterolemic mice, and whether reconstitution of mitochondrial antioxidant capacity with mitochondria‐targeted antioxidants (mito‐Tempo) can reverse this phenomena. We used Ldlr‐/‐ApoB100/100 (LA) mice that were either wild‐type (LA‐MnSOD+/+) or heterozygous (LA‐MnSOD+/‐) for MnSOD. After 6 months of Western diet, mice were treated with either saline (SAL) or MT (0.7mg/kg/day) using osmotic minipumps for 2 weeks. Endothelial function in aorta was assessed using isolated organ chamber baths in the presence or absence of apocynin (an NAD(P)H oxidase inhibitor, APO). Osmotic minipump implantation significantly worsened baseline endothelial function compared to non‐implanted mice. Overall, relaxation to acetylcholine (MRACH) was unchanged between LA‐MnSOD+/+ and LA‐MnSOD+/‐ treated with SAL (3.8±4.5% and 13.6±14.5%, respectively). Treatment with MT significant improved endothelial function in LA‐MnSOD+/+ mice (21.6±7.1%), and MRACH was further improved after acute incubation with APO (25±6.7%). In LA‐MnSOD+/‐, MT greatly improved MRACH (38.6±3.4%). In contrast to LA‐MnSOD+/+, however, acute incubation of vessels with APO impaired MRACH (27.3±10%). Collectively, our data suggest that increasing mitochondrial antioxidant capacity substantially improves endothelial function in hypercholesterolemic mice, but does not completely reverse compensatory molecular mechanisms that preserve endothelial function during prolonged mitochondrial antioxidant deficiency.

NHLBI

Grant Funding Source: Supported by NHLBI
Hyal‐1 deficiency may protect the endothelium in a mouse diabetic model (1076.4)
Authors: Dogne Sophie , Rath Geraldine , Dessy Chantal , Caron Nathalie , Flamion Bruno
Published: June 2021

Background. Hyaluronic acid (HA) is a major component of the glycocalyx and thereby participates in vascular wall permeability. In diabetes, the size and permeability of the glycocalyx are altered. In addition, type 1 diabetic patients have increased plasma levels of both HA and its somatic hyaluronidase Hyal‐1, which is endocytosed by different cell types including endothelial cells.

Objective. To investigate the potential implication of Hyal‐1 in the development of endothelium dysfunction linked to diabetes.

Methods. Plasma HA and ICAM‐1/VCAM‐1, myocardial capillary glycocalyx (using transmission electron microscopy) and mesenteric artery endothelial dysfunction were measured in Hyal‐1 KO mice and wild‐type (WT) mice made diabetic by daily injections of streptozotocin during 5 days.

Results. KO mice had higher plasma HA concentrations than WT mice. Plasma HA increased with diabetes in the WT mice but did not increase further in KO mice. Basal plasma levels of ICAM‐1 and VCAM‐1 were lower in Hyal‐1 KO than in WT mice. ICAM‐1 but not VCAM‐1 was significantly up‐regulated by diabetes. The glycocalyx had a similar baseline thickness in KO vs WT mice but reacted in opposite ways to 4 weeks of hyperglycemia, almost disappearing in WT mice while increasing in KO mice. Endothelium‐dependent vasodilation did not differ between healthy WT and KO mice. However, in WT diabetic mice, EDHF‐mediated vasorelaxation vanished whereas KO diabetic mice had a preserved EDHF pathway.

Conclusion. Decreased levels of Hyal‐1 or increased plasma levels of HA orient the diabetic endothelial response towards a reinforced glycocalyx, less inflammation, and a lesser damage to the EDHF‐dependent vasodilation pathway. The mechanisms of this protection are under current investigation.
Chromium inhibits phenotypic switching of vascular smooth muscle cells in response to hyperglycemic conditions in vitro (1076.8)
Authors: Ganguly Rituparna , Sahu Soumyadip , Boyer Brooke , Raman Priya
Published: June 2021

Hyperglycemia is an independent risk‐factor for macrovascular complications accounting for increased morbidity and mortality in diabetes. Diabetic patients have increased incidence of vascular smooth muscle cell (VSMC) migration and proliferation attributing to accelerated macrovascular lesion development. We recently showed that trivalent chromium (Cr), a beneficial mineral nutrient, reduces high glucose‐induced expression of thrombospondin‐1 (TSP‐1), a potent proatherogenic matricellular protein implicated in atherosclerosis. Here we show that Cr specifically inhibits high glucose induced proliferation of human aortic SMC (HASMC) vs. serum‐stimulated proliferation via attenuated ERK activation. Further in glucose‐sitmulated cells using protein‐DNA array we demonstrate that Cr reduces activation of transcription factors including SP1, ELK1, YY1, known repressors of SMC contractile gene expression. In parallel, Cr increases activation of SRF, a known transcriptional activator of contractile marker, α‐SMA. Finally, Cr’s antiproliferative effect was attenuated in TSP‐1 transgenic (TSP1‐Tg)‐SMC, constitutively overexpressing TSP‐1 in arterial SMC, compared to WT‐SMC suggesting specificity of Cr’s effect for TSP‐1. Together, our data reveal anti‐atherogenic effect of Cr on VSMC function and suggest TSP‐1 as a potential therapeutic target of Cr in diabetic vascular complications.
Defective endothelial connexin40 raises blood pressure in mice through impairment to conducted dilation (1079.14)
Authors: Morton Susan , Howitt Lauren , Baillie Brett , Heisler Jillian , Nicholson Bruce , Lau Katherine , Ashton Anthony , Matthaei Klaus , Hill Caryl
Published: June 2021

Global deletion or functional impairment of Cx40 in mice attenuates conducted dilation and causes hypertension. Although impairment of conducted dilation might be expected to increase peripheral resistance, the hypertension is renal in origin. Endothelial‐specific deletion of Cx40 also impairs conducted dilation, without change in blood pressure. However, these latter studies are complicated by coordinate loss of Cx37 from the endothelium. In order to impair Cx40 function selectively in the endothelium, without coordinate loss of Cx37 expression, we have generated a mouse expressing a mutant Cx40 (Cx40T152A) under control of the endothelial specific promoter Tie‐2, alongside native Cx40. We show that this single point mutation impairs both chemical and electrical conduction in vitro, although paradoxically, heterotypic pairings with Cx37 rescue gap junction function. Endothelial Cx37 expression is normal but Cx40T152A mice are hypertensive (105±1mmHg vs wildtype 97±1mmHg; P<0.05) without change in heart rate. While the EDH‐vasodilatory pathway remains intact in Cx40T152A arteries, conducted vasodilation is markedly attenuated. We conclude that myoendothelial gap junction communication remains intact in Cx40T152ATg mice, but impairment to conducted vasodilation arising from defective endothelial Cx40 signalling raises peripheral resistance and blood pressure.
Structural remodeling of coronary resistance arteries: effects of age and exercise training (1079.24)
Authors: Hanna Mina , Taylor Curtis , Chen Bei , La Hae‐Sun , Maraj Joshua , Kilar Cody , Behnke Bradley , Delp Michael , Muller‐Delp Judy
Published: June 2021

Age induces medial thickening and stiffening of conduit arteries; however, little is known of the effects of age on remodeling and mechanical properties of coronary resistance arteries. We determined 1) whether age increases wall thickness and stiffness of coronary resistance arteries, and 2) whether exercise training reverses age‐induced increases in wall thickness and stiffness. Young and old rats remained sedentary or underwent 10 wks of exercise training. Coronary resistance arteries were isolated for determination of wall/lumen ratio, wall stiffness, and myogenic contractile responses to intraluminal pressure changes. Collagen and elastin content of the vessel wall were assessed histologically. Age increased wall/lumen ratio, but reduced wall stiffness. Collagen/elastin ratio did not change with age. Exercise training reduced wall/lumen ratio and collagen/elastin ratio, but increased stiffness in old arteries. Myogenic responses were impaired with age and restored by exercise training. Thus, age induces inward remodeling and reduces wall stiffness of coronary resistance arteries, leading to loss of myogenic responsiveness. Exercise training induces outward remodeling and increases wall stiffness of aged coronary resistance arteries, thus restoring myogenic function. The restorative effects of exercise training may be due to changes in smooth muscle and extracellular matrix proteins.

Grant Funding Source: NIH R01 HL 90937
Combinational effects of gp91 ds‐tat and SS‐31 in reducing myocardial/ischemia reperfusion injury (1080.8)
Authors: Patel Harsh , Walker Sydney , Chau William , Devine Issachar , Ondrasik Regina , Chen Qian , Barsotti Robert , Young Lindon
Published: June 2021

MI/R injury results in cardiac contractile dysfunction, and increased cell death principally due to the reperfusion of blood following ischemia. This injury is initiated in part by a decrease in endothelial derived nitric oxide bioavailability and an increase in reactive oxygen species (ROS). Two key sources of ROS are NADPH oxidase and damaged mitochondria. We’ve shown that gp91 ds‐tat, a NADPH oxidase assembly inhibitor peptide and SS‐31, a mitochondrial targeted antioxidant, dose dependently improved post‐reperfused left ventricular developed pressure (LVDP) and reduced infarct size in rat hearts subjected to I (30min)/R (45min). This led us to the question, whether the combinational effects of low dose gp91 ds‐tat and SS‐31 would act synergistically to improve LVDP and reduce infarct size compared to the independent effects of each peptide and untreated control? Early data show that the combination of gp91 ds‐tat (5μM) + SS31 (10μM) (n=3) improved post‐reperfused LVDP by 63 ± 11% of baseline and reduced infarct size (IS) to 32 ± 9% compared to control (44 ± 10% of baseline, n=7; 42 ± 5% IS) and to SS‐31 10μM (25 ± 1% of baseline, n=3; 41 ± 10% IS); gp91 5μM (56 ± 9% of baseline, n=6; 28 ± 5% IS). Although this combination improved post‐reperfused LVDP more so than either drug alone or control, at these concentrations the combinations didn’t act synergistically. Other combinational doses will be tested in the future.
Improving survival with cytoprotective therapeutics administered after a myocardial infarction in the absence of reperfusion (1080.9)
Authors: Platt Mathew , Henry Scott , Huber Jason , Lutchmedial Sohrab , Brunt Kieth , Simpson Jeremy
Published: June 2021

A strong negative correlation between the size of a myocardial infarction (MI) and the long‐term survival of the patient exists in the clinical population. The current gold standard treatment for reducing infarct size in MI patients is acute reperfusion therapy (ART). Due to various contraindications, up to 30 percent of eligible patients do not receive ART. Thus, there is an urgent need for pharmacological alternatives in this substantive population. As patients experiencing a MI can only be treated after the event precipitates symptoms, the purpose of this study was to establish if a cytoprotective agent, hemin, can reduce infarct size and subsequent mortality when administered after a permanent coronary occlusion without reperfusion. Methods. Mice were treated with either hemin or vehicle 30 minutes following the induction of a MI. Results. Four weeks post MI, infarct size was significantly reduced in hemin treated (HT) mice compared to vehicle controls (VC) indicating preserved myocardial tissue (44.0±4.2% and 60.2±0.8% respectively; p<0.05). Kaplan‐Meier curves demonstrate HT mice had significantly higher 4‐week survival rates than VC (78% and 56% respectively; p<0.05). Conclusions. This data demonstrates the potential of a cytoprotective therapeutic to reduced infarct size and improve survival when administered after a permanent occlusion in the absence of reperfusion.

Grant Funding Source: Canadian Institute of Health and Research
Age impacts the developmental programming of blood pressure regulation in the intrauterine growth‐restricted male rat (1085.10)
Authors: Dasinger John , Intapad Sattira , Backstrom Miles , Alexander Barbara
Published: June 2021

Age impacts the developmental programming of blood pressure regulation in the intrauterine growth restricted male rat John Henry Dasinger, Sattira Intapad, Miles A. Backstrom, Barbara T. Alexander. Department of Physiology University of Mississippi Medical Center, Jackson MS, 39216 Intrauterine growth restriction (IUGR) induced by placental insufficiency programs hypertension, enhanced blood pressure (BP) response to acute angiotensin II (Ang II) and a two‐fold increase in circulating testosterone (T) at 4 months of age versus male control. Hypertension and the enhanced pressor response to acute Ang II at 4 months of age were abolished by castration implicating that programming of cardiovascular risk is T‐dependent. T levels decrease with age in men. We recently demonstrated that plasma T also decreases with age in male IUGR resulting in normalizing of circulating T levels relative to male control by one year of age. Thus, we tested the hypothesis that the age‐related reduction in circulating T in male IUGR rats would normalize BP and attenuate hyperresponsiveness to acute Ang II relative to age‐matched male controls. BP and Ang II sensitivity were measured in conscious, chronically catheterized male rats plus or minus Ang II (100ng‐kg‐1‐min‐1 for 20 minutes) at one year of age. BP in untreated animals was not significantly elevated in male IUGR (131±5 mmHg) relative control (123±3 mmHg). The enhanced pressor response to acute Ang II was not observed in male IUGR relative to control (IUGR vs. Control, Δ17 vs. Δ18 mmHg). Thus, these data indicate that developmental programming of hypertension and enhanced Ang II sensitivity is lost with the age‐related reduction in circulating T in male IUGR rats.

Grant Funding Source: Supported by HL074927, HL51971, 12POST11980021
The diverse mechanisms of cardiomyopathy and heart failure in obese and non‐obese insulin‐resistant rat models (1086.2)
Authors: Huang Jiung‐Pang , Hung Li‐Man
Published: June 2021

Currently, studies in cardiomyopathy and heart failure (HF) were mainly chosen an obese related insulin resistance animal model. However, the CVD morbidity in non‐obese insulin resistance (non‐obese IR) patient was still high. Therefore, it is important to elucidate the different mechanisms in obese and non‐obese IR induced cardiomyopathy/HF. In the study, we have designed obese (45% calorie from fat) and non‐obese (60% calorie from fructose) IR animal models (SD rat). The rats were fed with normal chow, high fat and high fructose diet for 12 weeks. Both obese and non‐obese animal were exhibited insulin resistance syndrome compared with control mice. In addition, obese rat myocardium exhibited severe fibrosis and ROS elevation, hyper‐activation of neurohumoral systems, concentric hypertrophy with slightly impairment of cardiac mechanical performance (Pressure‐Volume catheter), and fewer changes in both plasma and myocardial metabolites profiling (LC‐MS metabolites analysis) compared to non‐obese IR rats. In contrast, cardioyocytes shape change (much leaner and longer), elevation of TUNEL positive signal and higher autophage related proteins expression were co‐contributed in the impairment of cardiac contractility with worse mechanical efficiency in non‐obese IR rats. Both total and phosphorylated cardiac troponin I were downregulated; however, it was upregulation in plasma imply the harmful myocardium damage and HF risk were observed in non‐obese IR animals. Plasma and myocardial metabolites identification suggested LysoPC (18:0) may play a crucial role of cardiomyopathy/HF progression in non‐obese IR group, respectively. LysoPC(18:0) treated neonatal cardiomyocyte trigger cardiomyocyte longer and cell death, which confirmed our hypothesis. In conclusion, this study demonstrated the diverse cardiomyopathy/HF mechanisms in obese and non‐obese IR rats, which may provide a new effective CVD therapeutic target in non‐obese IR patient.
ENaC‐independent impaired natriuresis and increased blood pressure in mouse models of diet‐induced hyperinsulinemia (1086.3)
Authors: Nizar Jonathan , Dong Wuxing , McClellan Robert , Labarca Mariana , Zhou Yuehan , Satlin Lisa , Bhalla Vivek
Published: June 2021

Patients with obesity develop peripheral insulin resistance, hyperinsulinemia, and hypertension that are associated with cardiovascular disease and death. Hyperinsulinemia increases renal sodium reabsorption, in part through the epithelial sodium channel (ENaC) of the distal nephron, and may increase blood pressure. The molecular determinants of this process are poorly understood, and a mouse model is needed to facilitate discovery.

We systematically characterized five potential diet‐induced models of the metabolic syndrome in C57BL/6J mice. We measured sodium avidity, ENaC‐mediated sodium reabsorption in whole animal and in ex vivo microperfusion experiments as well as blood pressure by radiotelemetry.

High‐fat (45% or 60% kcal) diet‐fed (HFD) mice developed obesity and hyperinsulinemia after 6 weeks while fructose‐fed mice lacked hyperinsulinemia. HFD mice demonstrated increased sodium avidity compared to low‐fat (10% kcal) fed (LFD) mice as measured by urine sodium excretion after transition to a high salt diet. Interestingly, HFD animals did not have increased ENaC activity as measured by metabolic cage studies utilizing the ENaC antagonist, benzamil, or microperfusion of the cortical collecting duct. Blood pressure was significantly different in HFD vs. LFD mice, when active and while awake, but not at rest.

HFD mice display manifestations of the metabolic syndrome including increased sodium avidity, which was not mediated through ENaC as has been previously described. In contrast to insulin infusion studies, HFD mice developed increased blood pressure that was both statistically and clinically significant during selected intervals in a 24‐hour cycle.

Grant Funding Source: Supported by Child Health Research Institute, Stanford CTSA, and National Institutes of Health
Hyperglycemia‐driven glycosylation of the AT1 receptor in the vascular endothelium (1086.8)
Authors: Hoffmann Brian , Prisco Anthony , Greene Andrew
Published: June 2021

Vascular dysfunction, leading to coronary artery and peripheral vascular disease, remains an important problem in type 2 diabetes mellitus (T2DM) patients. In T2DM, hyperglycemia is a major causative factor for vascular endothelial dysfunction, yet there is a large gap in studies of the etiology behind the underlying mechanisms. This study evaluated the hypothesis that hyperglycemia‐induced endothelial glycoproteome changes lead to alterations in homeostatic endothelial signaling pathways. To test this hypothesis, innovative advances in glycoproteome and phosphoproteome enrichment, highly sensitive mass spectrometry, and relevant functional assays were utilized to assess alterations of rat microvascular endothelial cells (RMVECs) cultured in normal (5 mM) or high glucose (25 mM). Differential glycosylation of the type‐1A angiotensin II receptor (AT1; p=0.029) on the RMVEC surface was observed during hyperglycemia, along with glycosylation on other important vascular targets. Additionally, hyperglycemia‐induced changes in the synergistic balance of intracellular O‐GlcNAcylation and phosphorylation, along with inhibition of RMVEC tube formation, revealed alterations in AT1‐receptor signaling processes. Overall, this study identified glycoproteome alterations converging to cause endothelial dysfunction and potentially peripheral vascular disease during hyperglycemia and T2DM.

Grant Funding Source: Supported by HL082798 and T32 HL094273
Misclassification of pulmonary hypertension due to automated pulmonary capillary wedge pressure calculations (1089.1)
Authors: Shterental Sebastian , Longhini Anthony , Alam Mian , Bachman Timothy , Corotto Paul , Gupta Shikha , Nguyen Daniel , Shoushtari Ali , Zaidi Farhan , Simon Marc , Champion Hunter
Published: June 2021

Introduction: Misclassification of patients with pulmonary hypertension may occur due to reliance on automated pulmonary wedge pressure (PWP) measurements instead of end‐expiration measurements. The long‐term outcomes of patients who are misclassified are unclear. We performed a retrospective study to characterize misclassified patients and to assess their outcomes.

Methods: Review of clinical and hemodynamic data was performed for all patients who underwent right heart catheterization at University of Pittsburgh Medical Center with a mean pulmonary arterial pressure>25mmHg and PWP<15mmHg from 9/2005 to 3/2011. “Misclassified” was defined by end expiratory PWP value greater than 15mmHg. Demographic and clinical characteristics were gathered by retrospective medical chart review. Significance was assessed at the 5% level. Logistic regression was used to identify parameters that had a significant correlation with misclassification of patients. Time to death was the primary outcome with misclassification as the predictor of interest. Multiple regression was used to test the full model, backwards selection was used for final model selection.

Results: There were 288 patients diagnosed with pulmonary arterial hypertension for whom hemodynamic and clinical data were reviewed. There were 186 patients who were misclassified (64%). Misclassification had a significant relationship with weight (p<.001), interstitial lung disease (p<.001), left cardiomyopathy (p<.001) and pulmonary vascular resistance (p<.001). Unadjusted linear regression showed non‐significant negative association between misclassification and time to death (R2=.023, p=.097). Multiple linear regression test of our model adjusted for age demonstrated significance (R2=.068, p=.0173).

Conclusions: Use of automated rather than end expiratory PWP value may lead to misdiagnosis of pulmonary arterial hypertension and such misclassification of patients is negatively associated with time to death.

Grant Funding Source: Supported by NIH TPPG
Adeno‐associated viral delivery of TrkB after upper cervical spinal hemisection enhances recovery (1091.6)
Authors: Zambrano Juan , Martinez‐Galvez Gabriel , Zhan Wen‐Zhi , Gransee Heather , Sieck Gary , Mantilla Carlos
Published: June 2021

Cervical spinal hemisection at C2 (SH) paralyzes the diaphragm muscle by interrupting descending bulbospinal pathways to phrenic motoneurons. Signaling via the neurotrophin receptor TrkB in phrenic motoneurons enhances functional recovery after SH. However, it is not clear if the delay in gene expression resulting from adeno‐associated virus (AAV)‐mediated delivery would blunt the therapeutic efficacy of AAV delivered after SH. The goal of the present study was to examine the efficacy of intrapleurally‐delivered AAV7 encoding the full‐length TrkB receptor at 3 days post‐SH. Chronic diaphragm EMG recordings were conducted in adult male Sprague‐Dawley rats confirming absence of ipsilateral activity 3 days after SH. The proportion of animals displaying recovery of ipsilateral diaphragm EMG activity increased over time post‐SH and was greater in those treated with AAV‐TrkB (by 14 days post‐SH: AAV‐TrkB: 10/15 vs. AAV‐GFP: 2/11; p=0.01). Furthermore, peak root‐mean‐square amplitude of the diaphragm EMG activity increased progressively over time post‐SH. During eupnea, animals displaying recovery of ipsilateral diaphragm EMG activity that were treated with AAV‐TrkB achieved 40% of pre‐injury values (vs. 10% in those treated with AAV‐GFP). Targeting phrenic motoneurons via intrapleural delivery of AAV7‐TrkB promotes recovery of diaphragm activity when administered 3 days post‐injury.

Grant Funding Source: Supported by R01 HL96750, T32 HL105355 and Mayo Clinic Center for Regenerative Medicine
Differential effects of neonatal sustained hypoxia exposure on the acute hypoxic ventilatory response among different rat strains (1092.12)
Authors: Warren Philippa , Bozek Danielle , Mayer Catherine , Alilain Warren , MacFarlane Peter
Published: June 2021

Sustained hypoxia (SH) exposure during a vulnerable period of neonatal development (~11‐15 days of postnatal (P) age) attenuates the acute hypoxic (HVR) ventilatory response in rats. In the current study, we investigated whether the effects of SH exposure on the acute HVR varies among different rat strains. Sprague‐dawley (SD), Fisher (F), and Spontaneously Hypertensive (SHR) rats were exposed to SH (11% O2, 5 days) starting at P11. The acute HVR and Dejours test were assessed using whole‐body plethysmography the day after (P16) SH exposure ended. SH abolished the HVR in SHR, but not SD or F rats compared to normoxia raised animals. The transient ventilatory depression following brief (5 minutes) 100% O2 exposure (Dejours test) was also absent in SHR rats following SH treatment. These data indicate a strain difference in the vulnerability of the respiratory system to neonatal SH exposure; possible genetic determinants for this phenomenon requires further study.
The effects of prior hyperventilation duration on central chemoreflex responses using the “Duffin” hyperoxic rebreathing test (1092.16)
Authors: Boulet Lindsey , Jamieson Alenna , Day Trevor
Published: June 2021

The effects of prior hyperventilation duration on central chemoreflex responses using the “Duffin” hyperoxic rebreathing test The “Duffin” hyperoxic rebreathing test was developed from the “Read” rebreathing test by the addition of a five‐minute hyperventilation (HV) prior to switching to the rebreathing circuit. This HV eliminates CO2 stores, such that when the subject equilibrates with the rebreathing circuit, brainstem CO2 is below the ventilatory recruitment threshold (VRT) of the central respiratory chemoreceptors. This allows investigators to quantify the VRT prior to eliciting a central chemoreflex to increases in CO2 during rebreathing. An assumption of the test is that a full five minutes of prior HV is required. However, prolonged HV is uncomfortable for the subject, and there is no experimental evidence to validate this assumption. We aimed to test this assumption experimentally by comparing various durations of prior HV. We hypothesized that there would be no difference in central chemoreflex responses between randomized prior hyperventilation durations (HVD) of one, three and five minutes. Following instrumentation with a pneumotachometer and CO2 and O2 gas analyzers, 19 subjects underwent three hyperoxic rebreathing trials, each with randomized prior HV of one‐, three‐ and five‐minute durations, each coached to 18‐20 Torr PCO2. Rebreathing was carried out by switching to a circuit comprised of a 6L bag containing 7% CO2 and 93% O2. For all three trials, we quantified the VRT (PCO2; Torr), and respiratory frequency (min‐1/Torr CO2), inspired tidal volume (L/Torr CO2) and inspired ventilation (L/min/Torr CO2) with linear regression. We found no differences between HVD in any respiratory variable, within‐subject. Our data suggest that when using the “Duffin” rebreathing test, a one‐minute HV is sufficient to drive CO2 stores below the VRT, and the resulting central chemoreflex responses are no different than the standard five‐minute test.

Grant Funding Source: NSERC
Aging increases resistance to bacterial infection in the tobacco hornworm, Manduca sexta (1100.4)
Authors: Booth Kimberly , Cambron Lizzette , Fisher Nathan , Greenlee Kendra
Published: June 2021

Manduca sexta larvae (tobacco hornworm) early in their fifth and final larval stage have been reported to have more robust immune responses to Photorabdus luminscens compared to animals later in that stage. We hypothesized that the robust immune response in early stage caterpillars would result in varying hemolymph bacterial loads and survival to other bacterial infections. Early stage M. sexta injected with non‐pathogenic E. coli had 25‐fold higher bacterial loads in hemolymph than those injected later. In addition, early stage M. sexta injected with pathogenic Stenotrophomonas maltohphilia died twice as fast as those injected later. These data demonstrate an ineffective immune response in early stage caterpillars. We also hypothesized that the robust immune response in early stage caterpillars would affect other aspects of physiology, such as development and growth. Early stage M. sexta injected with E. coli or S. maltophilia exhibited developmental and growth rates twice as slow as those injected later. Sickness‐induced anorexia occurs in many taxa and may be the cause of the growth rate and developmental delays in infected caterpillars. While it is clear that development affects immunity, it is unclear why the age‐immunity relationship varies with different bacteria. Because of the importance of insects as pests and pollinators, understanding how immunity varies throughout development is critical.
Eggshell calcium regulates embryonic growth and calcium transport in an oviparous snake (1100.9)
Authors: Frye Hannah , Stewart James , Pyles Rebecca , Ecay Tom
Published: June 2021

One hypothesis to explain the high incidence of independent evolutionary transitions from oviparity to viviparity among squamate (snake and lizard) reptile lineages proposed that embryonic development is independent of eggshell calcium. Recent research on embryonic calcium nutrition does not support this hypothesis as at least 25% of the calcium in hatchling oviparous squamates is extracted from the shell. An alternative hypothesis is that shell calcium supplements calcium from yolk in oviparous squamates, but is not obligatory for embryonic development. We tested this hypothesis by physically peeling the outer layers of the shell, and thus removing the calcium, early in development of Pantherophis guttatus (corn snake) eggs. We found support for the hypothesis because survivorship to hatching did not differ for peeled eggs compared to intact eggs, yet hatchlings from peeled eggs were shorter (273.6 ± 3.4 vs. 261.0 ± 3.7 mm, p=0.0028, n=16), lighter (6.36 ±0.22 vs. 5.75 ± 0.23 g, p=0.0158, n=16), and had reduced calcium (40.8 ± 1.7 vs. 30.5 ± 1.8 mg, p<0.001, n=16), which could impact hatchling fitness in the wild. We also tested the hypothesis that embryos detect and respond to calcium availability by assaying the two tissues involved in embryonic calcium acquisition for changes in calcium transport protein expression (by immunoblotting) following shell calcium removal. The yolk sac, involved in yolk calcium transport, showed no detectable change in the developmental expression of calbindin‐D28K, a marker for calcium transport activity. However, the chorioallantois, involved in shell calcium transport, showed reduced calbindin‐D28K expression relative to samples from intact eggs, suggesting eggshell calcium regulates chorioallantoic calcium transport. Our findings suggest that evolution of viviparity is enhanced by a mechanism for detection and mobilization of calcium in oviparous embryos that also functions in the uterine environment. (Supported by an APS‐IOSP fellowship to HF)
Variations in pancreatic glucoregulatory hormones in birds (1101.1)
Authors: Calhoun Matthew , McGraw Kevin , Sweazea Karen
Published: June 2021

Plasma glucose concentrations (PGlu) in birds are naturally 1.5‐2 times greater on average than mammals of similar body mass. However, mechanisms regulating variations in PGlu across species with similar body mass are poorly understood. One theory is that the relative expression of the pancreatic glucoregulatory hormones insulin (lowers PGlu) and glucagon (increases PGlu) differs across species. However, evidence suggests many avian species are resistant to the glucose‐lowering effects of insulin. Therefore, the purpose of this pilot study was to correlate PGlu with pancreatic insulin and glucagon concentrations across diverse species of birds (pigeon, mourning dove, mallard duck, northern pintail duck, and Gambel’s quail). It was hypothesized that, similar to mammals, PGlu would be positively correlated with glucagon and negatively correlated with insulin expression in the pancreas. PGlu was measured using a commercially available kit. Pancreata were isolated, embedded in OCT compound and 12 micron cryosections were collected onto glass microscope slides. Tissue sections were then immunostained for insulin and glucagon. Commercially available ELISAs were used to verify immunohistochemical observations. Consistent with a negative relationship between PGlu and body mass for birds, mourning doves have the highest PGlu (24.02 mM/L) whereas mallard ducks have the lowest PGlu (11.14 mM/L). Insulin was not significantly correlated with variations in PGlu (Spearman coefficient 0.035; p=0.864), supporting prior observations that many species are resistant to the glucose‐lowering effects of insulin. Moreover, immunohistochemical data from doves and quail show higher glucagon expression as compared to insulin, which may contribute to their naturally high PGlu.
Effects of an acute stimulation of the HPA axis on sexual and stress hormones in male northern elephant seals (1101.5)
Authors: Ensminger David , Somo Derek , Sharick Jeffrey , Houser Dorian , Crocker Daniel
Published: June 2021

Little is known about variations in hypothalamic‐pituitary‐axis (HPA ) responsiveness and its impacts on metabolism in wild pinnipeds. To characterize changes due to acute stress, 18 free living adult male northern elephant seals were challenged with an intramuscular injection of slow release adrenocorticotropic hormone (ACTH) over 3 sample periods: early in breeding season, after 70+ days of the breeding fast and during peak molt. Subjects were blood sampled every 30 minutes for 2 hrs post‐injection. Breeding animals were recaptured and sampled at 48 hrs. This was not possible in molting subjects due to animal density and mobility. In response to ACTH, cortisol increased two‐three fold in all groups, and remained elevated at 48 hrs in early breeding subjects. ACTH was a stronger secretagogue for aldosterone, causing a 3‐8 fold increase. Cortisol and aldosterone responses did not vary between groups but were significantly correlated among individuals. Endogenous ACTH was suppressed by the challenge in late breeding males, particularly at 48 hrs. Cortisol increases resulted in elevations in plasma glucose only during molting. ACTH challenge caused suppression of testosterone and thyroid hormone (T3) at 48 hrs in early breeding males. ACTH challenge increased plasma non‐esterified fatty acids and blood urea nitrogen in all study groups. These data suggest that sensitivity of the HPA axis is maintained but the metabolic impacts of cortisol and feedback inhibition of the axis varies with life history stage. Strong impacts of the challenge on testosterone, thyroid hormone and protein sparing suggest the importance of maintaining low cortisol levels during breeding .
Cellular aging of skeletal muscle: evidence that physical inactivity is responsible and not age (1102.14)
Authors: Venturelli Massimo , Morgan Garrett , Donato Anthony , Reese Van , Tarperi Cantor , Milanese Chiara , Schena Federico , Bottura Renato , Reggiani Carlo , Naro Fabio , Cawthon Richard , Richardson Russell
Published: June 2021

Telomeres, not only provide a marker of cellular aging, but also play an essential role in maintaining chromosomal integrity in the face of physiological stressors, such as free radicals, that result in DNA damage. Although, the age‐related shortening of telomere length (TL) in highly proliferative tissue, is thought to be predominantly due to the replication process, the mechanism for telomere shortening in skeletal muscle, which is minimally proliferative, is unclear. By studying TL in both the upper and lower limbs of the young (Y), old mobile (OM), and old immobile (OI) and by virtue of the bipedal nature of human locomotion, which declines with age, it may be possible to elucidate the mechanism responsible for cellular aging of skeletal muscle. With this approach, we revealed that TL (~15 kb) in arm skeletal muscle is unaffected by age and mobility. In contrast TL fell progressively in the legs across the young (~15 kb), the old mobile (~13 kb), and old immobile (~11 kb). Interestingly, there was a reciprocal rise in leg muscle free radical concentration across these groups that was significantly correlated with TL (r=0.7), with no such relationship in the arm (r=0.09). Our results document that chronologic age, per se, does not affect cellular aging of skeletal muscle, but reveals that physical inactivity, likely mediated by free radicals, has a profound effect upon this process.

Grant Funding Source: NIH (PO1 HL, 09830), and VA (Merit Grant E6910R)
Cigarette smoke impairs the in situ fatigue resistance of locomotor skeletal muscle in mice (1102.18)
Authors: Trisko Breanna , Nogueira Leonardo , Wagner Peter , Breen Ellen
Published: June 2021

Muscle dysfunction is thought to contribute to exercise intolerance in smokers with COPD. In this study, cigarette smoke (CS) was hypothesized to impair skeletal muscle fatigue resistance. To test this hypothesis, adult C57Bl6 mice were exposed via a nose‐only system to daily periods of CS (5 cigarettes/period for 2 periods, 5 days/week) for 8 weeks and compared to mice delivered CS‐extract (weekly, i.p) and control, unexposed mice. Body weights were recorded each week. Gastrocnemius complex fatigue resistance was evaluated by measuring the time to reach 50% of maximal force when electrically stimulated to contract in situ. Compared to control mice, body weights were decreased in CS‐exposed mice, but not CS‐extract mice, at 3‐weeks (7.4%, p<0.05), 7‐weeks (7.9%, p<0.05), and 8‐weeks (12.4%, p<0.001). However, gastrocnemius, soleus, and plantaris mass / body weight ratios were not different between groups. A trend to earlier fatigue was seen in CS‐extract mice (Con. 342 ± 72 sec., CS‐exposed 310 ± 111 sec., CS‐extract 177 ± 16 sec., p=0.06). These data suggest the route or amount of CS that circulates in the blood may influence the extent of peripheral skeletal muscle dysfunction.

Grant Funding Source: Supported by NIH 1 PO1 HL091830‐01A1
SPRED1 in quadriceps of patients with mild chronic obstructive pulmonary disease (1102.21)
Authors: Porlier Alexandra , Dubé Annie , Debigaré Richard , Maltais François
Published: June 2021

Limb muscle dysfunction contributes to exercise intolerance in chronic obstructive pulmonary disease (COPD). In turn, quadriceps capillarization is a determinant of muscle function. In our previous study, the number of capillaries and mRNA level of VEGFA were both reduced in mild COPD. MicroRNAs (miRs) regulate biological processes such as angiogenesis. Based on the literature, angiogenesis can be regulated by miR‐126 which blocked the expression of an inhibitor of angiogenesis, SPRED1. In this study, we assessed the expression of miR‐126 in the context of capillarization characterization in muscle tissue obtained from a needle biopsy in 5 patients with mild COPD and 5 healthy age‐matched controls. We hypothesized that miR‐126 will be downregulated and SPRED1 will be enhanced in COPD. Vastus lateralis muscle samples were analyzed for miRs and mRNA content by qPCR and the number of capillaries was obtained by immunochemistry. Levels of miR‐126 were similar between mild COPD and healthy controls. SPRED1 level was enhanced by 2.1 fold (p=0.01) and the number of capillaries tended to be reduced in mild COPD compared to healthy controls. SPRED1 may have a role in limb muscle capillarization in mild COPD. Investigations targeting specifically endothelial cells will better dissect the biochemical process involved in muscle angiogenesis in the context of COPD.

Grant Funding Source: CHIR/IRSC