bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of adiponectin in high‐fat mediated cardioprotective programs (837.5)
Authors: Wani Mashhood , Haar Lauren , Amratia Avni , Gonzalez Christopher , Jones W.K.
Published: June 2021

The heart protects itself from injury through protective intrinsic mechanisms. Understanding the mechanisms that elicit this cardioprotective effect is vital to the prevention of myocardial infarction. It has been established in the Jones Lab that an acute high fat diet elicits a cardioprotective effect in a murine model. The adipose‐derived circulating adipokine adiponectin may play a crucial role in the activation of cardioprotective gene programs in this high fat model as it has been previously shown to be cardioprotective. This study will address the hypothesis that high fat diet mediates cardioprotection through circulating adiponectin acting through receptors in the heart that activate autophagic and anti‐apoptotic programs necessary for preconditioning.

To address this hypothesis, an in vitro model was established by simulating I/R in HL‐1 cells, and a surgical model that utilizes 30 minute occlusion of the LAD to create infarct. Beclin‐1 (a marker of autophagy) expression was assessed in mice after a 24hr high fat diet through western blot analysis.

From these results, we are able to establish that an acute high fat diet elicits a cardioprotective effect in a murine model. Furthermore, the cardioprotective effect of the high fat diet is lost in adiponectin knockout mice, meaning that adiponectin is a key player in high fat mediated cardioprotection. The upregulation of Beclin‐1 after a high fat diet suggests that adiponectin and the high fat diet is priming the cells for an autophagic state. Beclin‐1 is a downstream target of NF‐κB, a key player in cardioprotective mechanism, and this suggests that adiponectin is interacting with NF‐κB and eliciting a cardioprotective effect.

Grant Funding Source: Provost Pilot Grant‐University of Cincinnati(COM)
The role of dopaminergic signaling in Daphnia magna swimming (839.5)
Authors: Barrozo Enrico , Fowler David , Ingebretson Justin , Beckman Matthew
Published: June 2021

Daphnia are freshwater crustaceans that have been used for decades in ecotoxicology research. In order to determine the role of dopamine in Daphnia movement we have carried out a screen of 10 dopamine receptor drugs; three drugs affected movement. To screen for drug effect on behavior in Daphnia we have developed a two‐dimensional video imaging system to measure the movement of Daphnia magna swimming in shallow circular‐well plates. At one, two, and six hours of treatment with 10µM drug, the D1‐like receptor agonist (1R,3S)‐1‐(Aminomethyl)‐3‐phenyl‐3,4‐dihydro‐1H‐isochromene‐5,6‐diol (A68930), the D2‐like agonist (5'a)‐2‐Bromo‐12'‐hydroxy‐2'‐(1‐methyl舃ethyl)‐5'‐(2‐methylpropyl)ergotaman‐3',6',18‐trione mesylate (bromocriptine), and the D4‐like receptor antagonist 3‐(4‐[4‐Chlorophenyl]piperazin‐1‐yl舃)‐methyl‐1H‐pyrrolo[2,3‐b]pyridine trihydrochloride (L‐745, 870) all decreased movement with different time‐courses of inhibition. For two of the drugs which exhibited significant effects on daphnid behavior at one hour of treatment, A68930 and bromocriptine, the IC50 values were determined to be in the low micromolar range. Preliminary results from experiments with the dopamine neurotoxin 1‐Methyl‐4‐phenylpyridinium iodide (MPP+) are consistent with a previous study showing 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) treatment decreased Daphnia swimming behavior. Last, we have performed genome mining studies which have identified a suite of dopaminergic genes in the Daphnia magna genome. Taken together, the data provide evidence that specific dopamine signaling pathways are involved in Daphnia swimming behavior. We propose that Daphnia is a useful model to use in studies of dopaminergic signaling in healthy and neurotoxin‐induced disease states.

Grant Funding Source: Supported by The Augsburg URGO, McNair, and NorthStar STEM Programs and the College Dean's Office
Targeting glial activation for treating chronic pain and improving opioid efficacy (84.3)
Authors: Watkins Linda
Published: June 2021

Research spanning 20 years into the mechanisms of neuropathic pain has defined a new therapeutic target: glial cell activation. Glial cells surround nerve cells throughout the central nervous system, interacting with the nerve cells and responding to their signaling. In neuropathic pain, glial cells become activated, causing them to release substances that pathologically amplify pain. This new understanding has also provided an explanation for the clinical experience that opioids often have pain‐promoting side effects. Opioid administration causes glial activation, which paradoxically opposes the pain‐modulating effect of opioids. Reversal of the pro‐inflammatory activation of glia would thus not only reverse neuropathic pain in general but should also enhance the clinical efficacy of morphine and other opioids.
GI‐safe NSAIDs and the suppression of colon cancer (840.5)
Authors: Lichtenberger Lenard , Waters David , Phan Tri , Dial Elizabeth
Published: June 2021

The consumption of NSAIDs is linked with a reduction in cancer incidence, metastasis, and mortality. Our lab has developed a family of phosphatidylcholine (PC)‐associated NSAIDs which have reduced GI toxicity both in animals and humans, while possessing full COX‐inhibitory activity. This study was designed to test whether PC‐NSAIDs may also possess significant chemopreventive action. Methods: 1) MC‐26 colon cancer cells were incubated for 24 hours with (0.1‐2mM) of: Aspirin‐PC, or Ibuprofen‐PC; and 2) Sprague‐Dawley rats were inoculated with the carcinogen, azoxymethane (AOM), followed by daily oral NSAID dosing (20 mg NSAID/kg) and after 4 weeks the animals were evaluated for GI toxicity and colonic aberrant crypt formation (ACF). Results: Each of the PC‐NSAIDs showed direct inhibitory effects on growth of MC‐26 cells at 0.5‐1 mM. In vivo, both Aspirin‐PC and Ibuprofen‐PC significantly reduced colonic ACF in rats pre‐treated with AOM without evidence of GI toxicity. Conclusion: Both Aspirin‐PC and Ibuprofen‐PC demonstrated a direct inhibitory effect on the growth of colon cancer cells in vitro and in carcinogen/AOM‐treated rats, significantly reduced the development of colonic ACF, which is a precursor of colonic dysplasia and cancer, suggesting the potential utility of PC‐NSAIDs in patients at risk for colon cancer/recurrence. Supported by R03 CA 171613 and R41 CA171408.

Grant Funding Source: NIH R03CA171613; R41 CA171408
Glucocorticoid signaling mediates stress‐induced impairment of neurovascular coupling (841.4)
Authors: Longden Thomas , Dabertrand Fabrice , Hammack Sayamwong , Nelson Mark
Published: June 2021

Stress influences the progression and severity of many diseases. Here we examined the effects of stress on neurovascular coupling (NVC), which matches cerebral blood flow increases to local neuronal activity. NVC was studied in brain slices from the amygdala, where dilation of parenchymal arterioles (PAs) was measured in response to neuronal stimulation.

We administered a 7‐day stress paradigm to male Sprague‐Dawley rats, resulting in anxiety‐like behavior and attenuated weight gain. After stress, PA vasodilation evoked by neuronal stimulation was reduced by 66%. This reduction was similar to that previously reported for the effect of blocking inward rectifier K+ (Kir) channels during NVC1. Indeed, blocking Kir channels with barium inhibited NVC in control but not stressed slices. We thus hypothesized that the impairment of NVC by stress reflects a loss of PA Kir channels. Consistent with this, we found that Kir channel current density in smooth muscle cells from PAs was reduced by 90%. Moreover, elevation of external K+ from 3 to 8 mM, which normally causes profound dilation of PAs through Kir activation, was almost without effect on arterioles from stressed rats. In vivo delivery of corticosterone (without stressors) or the glucocorticoid receptor blocker RU486 (prior to stressors) mimicked or prevented NVC impairment by stress, respectively.

We conclude that stress causes a glucocorticoid‐mediated decrease in functional Kir channels in PA myocytes in the amygdala. This renders arterioles less responsive to K+ released from endfeet during NVC, thus impairing vasorelaxation. These studies may pave the way for the development of novel treatment options for brain disorders with a stress component.

1. Filosa et al. (2006) Nat Neurosci 9(11): 1397‐1403

Grant Funding Source: Supported by National Institutes of Health and the American Heart Association.
Does cigarette smoke exposure contributes to the progression of ovarian cancer? (844.7)
Authors: Sumanasekera Wasana , Conzelman Robert , Beckmann Taralein , Nguyen Lena , Petty Kayla , Kakar Sham
Published: June 2021

Rationale and Objective: Investigating risk factors for ovarian cancer is at extreme importance due to the asymptomatic nature of this disease during early stages. Although a correlation between history of cigarette smoking in women and risk of ovarian cancer occurrence has been reported, there is no primary literature linking cigarette smoke (CS) as a causative factor in ovarian cancer development. Here we conducted an in‐vitro approach to detect effects of CS exposure on ovarian cancer progress. We hypothesized that cigarette smoke exposure cause promotion of ovarian cancer via attenuating apoptosis and increasing proliferation of cancer cells. Methods: A2780 ovarian cancer cells were used as the experimental model. Cigarette smoke extract (CSE) was created using a modification of a published method. Following serum starvation, A2780 cells were treated with either control (serum free media) or different concentrations of CSE for 24 hours. To detect effects of CSE on ovarian cancer cell apoptosis, apostain and tryphan blue exclusion assays were performed. To detect CSE effects on cell proliferation, BrdU cell proliferation assay was conducted. Results: 24 hour Exposure of CSE caused increase in cancer cell proliferation, and decrease in cancer cell apoptosis. Conclusions: CS promotes ovarian cancer progression via accelerating cancer cell proliferation and attenuating apoptotic cell death.

Grant Funding Source: Sullivan University Faculty Development Grant
Cholesterol accumulation in skeletal muscle: a potential novel targetable pathway in insulin resistance (854.11)
Authors: Llanos Paola , Contreras‐Ferrat Ariel , Osorio César , Espinosa Alejandra , Hidalgo Jorge , Hidalgo Cecilia , Jaimovich Enrique
Published: June 2021

Cholesterol is enriched in the skeletal muscle transverse tubule system, where translocation of GLUT4 glucose transporters takes place in response to insulin. We tested whether feeding mice a high fat diet (HFD), besides producing insulin resistance, increased TT cholesterol content and modified insulin‐induced glucose uptake. Male C57BL/6J mice were fed a normal or a HFD (60% fat) for 8 weeks. HFD‐fed mice displayed higher body weight and insulin resistance. Insulin‐stimulated glucose uptake in fibers from HFD‐fed mice was lower than in controls whereas cholesterol levels in triads from HFD‐fed mice were 30% higher. Pre‐incubation with Methyl‐β‐cyclodextrin (MβCD) reduced Akt phosphorylation and increased both basal and insulin‐induced glucose uptake in muscle fibers from controls or HFD‐fed mice. MβCD treatment restored normal glycemia and insulin sensitivity in HFD mice. Indinavir, a GLUT4 antagonist, inhibited the effects of MβCD. MβCD increased membrane GLUT4 content and elicited intracellular calcium signals inhibited by Dantrolene. Dantrolene also reduced MβCD‐mediated glucose uptake. These results provide a rationale for the clinical evaluation of cyclodextrins, as an approach to the treatment of insulin resistance and the control of diabetes.

Grant Funding Source: ACT 1111, Fondecyt 3110105, 11130267, 11090301
Quantum dots reveal endothelial NOX2 induces VCAM expression in LPS‐challenged inflamed whole lung (855.2)
Authors: Orndorff Rebecca , Hong Nan Kang , Yu Kevin , Zern Blaine , Feinstein Sheldon , Fisher Aron , Muzykantov Vladimir , Chatterjee Shampa
Published: June 2021

Quantum Dot (QD) technology coupled with fluorescence imaging is a powerful tool to detect cellular proteins in vivo. Here we employ QDs as an imaging tool to detect vascular cell adhesion molecule‐1 (VCAM) expression in a model of lung inflammation. A key event in inflammation is the expression of VCAM. VCAM induction is reportedly responsible for the accumulation of neutrophils in lungs and for subsequent production of reactive oxygen species (ROS). NADPH oxidase 2 (NOX2), expressed by both endothelial cells and neutrophils, is a major contributor to ROS production post inflammation in the lungs. To date, the cellular sources of NOX2 (neutrophils or endothelium) that regulate VCAM expression in inflamed lungs is unknown. Using QD probes specific for VCAM, we found increased VCAM expression post LPS‐challenge in vitro, which was abrogated by the NOX2 inhibitor, apocynin. Ex vivo and in vivo lungs exhibited an 8‐10 fold higher incidence of VCAM expression in LPS‐challenged lungs over NOX2 null lungs. We next employed an endothelial specific NOX2 expressing transgenic mouse in a NOX2 null background (endoNOX2Tg) to reveal that endothelial NOX2 induces VCAM expression at levels that are intermediate between those expressed in wild‐type (WT) and NOX2 null mice. This result indicates that endothelial NOX2 is sufficient to induce VCAM expression due to an inflammatory stimulus.

Grant Funding Source: Supported by T32‐HL07748.
Discordant neurohumoral responsiveness to orthostatic stress in amenorrheic physically active premenopausal women (858.4)
Authors: O'Donnell Emma , Goodman Jack , Mak Susanna , Floras John , Morris Beverley , Harvey Paula
Published: June 2021

In light of the known modulatory effects of estrogen on neurohumoral control of blood pressure (BP), we tested the hypothesis that muscle sympathetic nerve activity (MSNA) and serum renin angiotensin aldosterone system (RAAS) levels would differ between estrogen deficient physically active women with functional hypothalamic amenorrhea (ExFHA) and eumenorrheic estrogen replete women. MSNA, serum RAAS components (renin, angiotensin II and aldosterone), and brachial artery BP (mmHg) were recorded at baseline and during graded lower body negative pressure (LBNP; ‐10, ‐20, and ‐40mmHg) in 17 recreationally active eumenorrheic women (ExOv; 24±1 years; body mass index 20.9±0.5kg/m2; mean±SEM) and 12 ExFHA women (25±1 years; 20.7±0.7 kg/m2). At baseline, heart rate (HR; b/min) and systolic BP (SBP) were lower (p<0.05) in ExFHA (47±2 and 94±2, respectively) compared with ExOv (56±2 and 105±2) women. Baseline MSNA, RAAS components, and mean arterial BP (MAP) were similar (p>0.05) between the groups. In response to graded LBNP, HR was increased (p<0.05) and SBP decreased (p<0.05) in both groups, but responses were lower (p<0.05) in ExFHA women. MSNA (bursts/100 heart beats) increased in both groups, but responses were greater (p<0.05) in ExFHA than ExOv women at ‐20 and ‐40 mmHg (37±4 vs. 22±4; 45±4 vs. 33±4, respectively). LBNP elicited increases (p<0.05) in all RAAS components in ExOv, but not ExFHA, women (p>0.05). In conclusion, these findings suggest that in hypoestrogenic ExFHA women, sympathetic nerve activity, but not the RAAS, defends arterial BP during orthostatic stress.

Grant Funding Source: Supported by a Cardiovascular Independent Research Award (NRA 3840028) awarded from Pfizer
Specific inhibition of AT1‐AAs by direct binding improves maternal and fetal outcome in response to placental ischemia during pregnancy: emphasizing the importance of novel drug development in the treatment of preeclampsia (860.15)
Authors: Moseley Janae , Castillo Javier , Amaral Lorena , Cornelius Denise , LaMarca Babbette
Published: June 2021

Preeclampsia, (PE) hypertension with proteinuria during pregnancy, is associated with antibodies that activate the angiotensin II receptor (AT1‐AA). Infusion of AT1‐AA increases blood pressure (MAP) and causes many characteristics of PE in pregnant rodents. The objective of the study was to determine if specific inhibition of AT1‐AA improves pregnancy outcomes in RUPP pregnant rat model of PE.

Specifically AT1‐AA inhibitory peptide (2ug/ml saline) was infused via miniosmotic pumps into RUPP rats beginning day 14 of gestation, immediately following the RUPP procedure. On day 19 blood pressure, MAP, blood and tissue were collected.

MAP was 99+/‐2 in NP(n=18), 121+/‐2 in control RUPP rats (n=19;P<0.001) and was 109 +/‐6 mmHg (n=9 P<0.02) in RUPPs + AT1‐AA inhibitory peptide. Pup weight in NP was 2.4 +/‐ 0.04 in NP; 1.9 +/‐ 0.06 in RUPP, improved significantly to 2.2 +/‐ 0.14 g (P<0.05) in RUPPs + AT1‐AA inhibitory peptide.These studies indicate that specific AT1‐AA inhibition improves maternal blood pressure and fetal outcome in response to placental ischemia, thereby emphasizing the importance of drug discovery for AT1‐AA inhibition to improve pregnancy outcomes in preeclamptic patients.

Grant Funding Source: Ferring Pharmaceuticals, NIH NICHD
Is preeclampsia all in the head? First‐trimester prediction of preeclampsia via maternal plasma levels of the vasopressin pro‐segment copeptin (860.20)
Authors: Santillan Mark , Santillan Donna , Scroggins Sabrina , Min James , Leslie Kimberly , Hunter Stephen , Zamba Gideon , Gibson‐Corley Katherine , Grobe Justin
Published: June 2021

Background: Preeclampsia, a devastating gestational hypertensive disorder, is characterized by low circulating renin relative to normal pregnancies. Vasopressin plays an important role in selected low‐renin hypertensive states. Therefore, we hypothesized a role for vasopressin in preeclampsia. Methods: Copeptin, a biologically inert, 4 kDa pro‐segment of vasopressin with a stable and slow plasma elimination rate, is secreted in a 1:1 molar ratio with vasopressin. In this case‐control study, copeptin was measured in banked maternal plasma throughout pregnancy from women who did or did not develop preeclampsia. Results: Maternal plasma copeptin was significantly elevated in all trimesters in pregnancies that did (n=51) or did not (n=30) develop preeclampsia (first trimester: 903 vs. 2045 pg/mL, p=0.008; second trimester: 706 vs. 1806, p=0.02; third trimester: 822 vs. 1890, p=0.0006). While controlling for covariates such as age, BMI, chronic hypertension, twin gestation, diabetes, and history of preeclampsia, the association of elevated copeptin and the development of preeclampsia remained significant. ROC analyses reveal that copeptin is significantly predictive of the development of preeclampsia in all trimesters. Further, vasopressin infusion was sufficient to generate preeclampsia phenotypes in pregnant C57Bl/6J mice. Conclusions: These data establish copeptin as a potent predictor of preeclampsia throughout pregnancy, as early as the first trimester (AUC=0.80, p=0.005, at a cutoff of 1018 pg/mL). The temporal order of changes in copeptin (in early first‐trimester) versus other known mechanisms of preeclampsia (mid‐ or late‐pregnancy) supports our working hypothesis that vasopressin secretion may precipitate or induce other known humoral, vascular, immune, and morphological mechanisms of preeclampsia.

Grant Funding Source: HD000849, RR024980, HL098276, HL084207
Aging‐related loss of skeletal muscle strength: the role of muscle quality (863.8)
Authors: Barbat‐Artigas Sebastien , Pion Charlotte , Gouspillou Gilles , Belanger Marc , Hepple Russell , Morais Jose , Aubertin‐Leheudre Mylene
Published: June 2021

Objective: Skeletal muscle aging is associated with a progressive loss of mass and strength. In this process, the loss of muscle quality has been suggested to cause a disproportionate loss in muscle strength compared to the loss in mass. This study aimed at investigating the role of changes in muscle quality in the loss of muscle strength with aging.

Methods: Ten young (24±3 yo) and 9 old (72±4 yo) active men were recruited and matched for physical activity levels. Body composition (DXA and MRI) and knee extension strength (KES) were assessed. Muscle quality indexes (quadriceps mass/KES, volume/KES and CSA/KES) were then calculated. Muscle biopsies were performed in the vastus Lateralis to assess fiber type proportion and size, and intracellular lipid content (Oil Red O).

Results: KES, quadriceps mass, volume and CSA were lower in old vs. young adults (p<0.05). Fat mass percentage was higher in old vs. young adults (p<0.05), while total body weight was similar. No differences were observed between young and old men in muscle quality indexes, intracellular lipid content, fiber size and type proportion. However, type IIa fibers tended to be smaller in old than in young adults (p=0.052).

Discussion: Our results suggest that, in active old men, the loss in muscle strength is mainly attributable to a decrease in muscle mass. Physical activity may contribute to the preservation of the quality of contraction per unit of muscle.
Effect of rosiglitazone and 4‐PBA on the capacitive calcium entry‐induced contraction of mesenteric artery from diabetic rats (865.16)
Authors: Espinosa‐Tanguma Ricardo , Ortiz‐Mesina Mónica
Published: June 2021

The intracellular calcium handling in diabetes mellitus (DM) is altered due, in part, to a decrease in the function/expression of SERCA pump. Objective: to evaluate the Rosiglitazone (Rgl) and 4‐PBA effects on the capacitive calcium‐entry induced contraction. Methods: DM was induced with Streptozotocin (65 mg/Kg iv) in male wistar rats. Six weeks after inducing DM the treatments were administered: Group 1 Rgl (5 mg/kg/day) and group 2 with 4‐PBA (40 mg/Kg/day) for two weeks. Under deep anesthesia mesenteric arteries were isolated for isometric tension experiments. Results: in the control group the tension evoked by addition of calcium (1.1 mM) was 67.3 ± 1.6% (n = 8); compared to DM which was 77.9 ± 1.6% (n = 8, p < 0.001). The increase in tension observed on rings of diabetic rats was reversed with the Rgl application to 70.2 ± 1.8% (n = 6, p < 0.05) and with 4‐PBA to 65.8 ± 1.7% (n = 7, p < 0.05). Conclusions: Treatment with Rgl and 4‐PBA inhibits the increase in tension observed in DM. The results suggest that both drugs could modulate changes in tension observed in DM by regulating the SERCA activity and/or expression.
Lower soluble RAGE and higher aortic stiffness in African American adolescents: possible protective effect of higher sRAGE in white youth (867.10)
Authors: Tank Ericka , Wegman‐Points Lauren , Siefers Kyle , Guinter Mark , Pajaniappan Mohana , Darracott Katherine , Pierce Gary
Published: June 2021

Advanced glycation end‐products (AGEs) are associated with increased aortic stiffness and cardiovascular disease (CVD) risk in middle‐aged/older adults. AGEs increase cross‐linking of extracellular matrix (ECM) proteins and binding to the Receptor for AGE (RAGE) activates vascular pro‐inflammatory/oxidant signaling. Matrix metalloproteinases (MMPs) degrade vascular ECM proteins such as elastin and cleave cell surface receptors such as RAGE. The cleaved soluble isoform of RAGE (sRAGE) serves as a ‘sink’ for circulating AGEs, so that low sRAGE is associated with increased CVD risk and high sRAGE may be vascular protective. Interestingly, sRAGE is lower in African‐American (AA) compared with white middle‐aged/older adults and is hypothesized to contribute in part to higher CVD risk in AAs. However, it is unknown if lower sRAGE is present in AA youth and associated with increased aortic stiffness. Circulating sRAGE was significantly lower in AA (n=20, age 16.8±1.4 yrs; BMI 24±3 kg/m2) compared with white (n=24; age 16.5±1.5 yrs; BMI 23±4 kg/m2) healthy adolescents (930 ± 432 vs. 1567 ±338 pg/ml, P<0.01), in the absence of differences in blood pressure (BP), AGEs, MMP9 and MMP2 (P>0.05). Aortic stiffness (carotid‐femoral artery pulse wave velocity, CFPWV) was higher in AA compared with whites (5.9±0.9 vs. 5.3±0.8 m/sec, P<0.05). After adjusting for age, BMI and mean BP, sRAGE was negatively correlated with CFPWV only in females (r=‐0.60, P<0.01) mostly from the relation among white (r=‐0.86, P<0.01) and not AA (r=‐0.51, P=0.16) females. In males, MMP2 was positively correlated with CFPWV (r=0.53, P<0.05) and sRAGE (r=‐0.54, P<0.05). Young AAs demonstrate lower circulating sRAGE and higher aortic stiffness compared with their white age‐ and BMI‐ matched peers. Whether lower sRAGE contributes to aortic stiffness in AAs or higher sRAGE has a vascular protective effect on large elastic arteries in whites deserves further investigation.

Grant Funding Source: Supported by APS Undergraduate Research Fellowship; Univ of Iowa ICRU Fellowship; GHSU CHDI
Collagen C‐peptide roles in post‐myocardial infarction remodeling (867.15)
Authors: de Castro Bras Lisandra , DeLeon‐Pennell Kristine , Ma Yonggang , Yabluchanskiy Andriy , Iyer Rugmani , Fields Gregg , Lindsey Merry
Published: June 2021

Collagen type I is the main extracellular protein present in the left ventricle (LV). The aim of this study was to determine the mechanisms whereby collagen peptide (C‐peptide 1158/9), initially generated by several proteases and later degraded by matrix metalloproteinase (MMP)‐9, mediates LV remodeling following myocardial infarction (MI). We performed cleavage assays of recombinant collagen type I by MMP‐2, ‐8, ‐9, and ‐11. MMP‐2, ‐8, and ‐9 all cleaved collagen, but only MMP‐2 and ‐9 formed peptides <24 kDa. Increased doses of MMP‐9 resulted in further degradation of the <24 kDa collagen peptides. The cleavage products were analyzed by mass spectrometry and the MMP‐2/9 cleavage site was identified (aa1158/9). In vitro, C‐peptide 1158/9 stimulated cardiac fibroblast wound healing, suggesting a role in cell migration. In vivo, we found higher levels of the C‐peptide in MMP‐9 null infarcted LV (LVI) at day 5 post‐MI compared to wild type, indicating accumulation of peptide in the absence of MMP‐9. C‐peptide 1158/9 in vivo treatment attenuated LV dysfunction at day 7 post‐MI, evidenced by higher ejection fraction compared to saline MI. In summary, C‐peptide 1158/9 improved LV function by stimulating the cardiac repair response.

Grant Funding Source: Supported by POST14350034, HHSN 268201000036C, R01 HL075360, HL051971, 5I01BX000505
Overfed Burmese pythons undergo physiological remodeling of the aorta (867.5)
Authors: Eng Jason , Dodson R. , Pham Bryant , Kloor Jackson , DeVan Allison , Doptis Ryan , Harrison Brooke , Leinwand Leslie , Fleenor Bradley
Published: June 2021

After feeding, Burmese pythons (Python molurus bivittatus) undergo physiological cardiac remodeling associated with increased cardiac output. Because the left ventricle (LV) of the heart and aorta are anatomically and mechanically coupled, we tested the hypothesis that chronic overfeeding causes aortic remodeling. Nine‐month old snakes consumed 25% of their total body weight every 2 weeks (normal fed, NF, n=5) or once every 3 days (overfed, OF, n=7) for 12 weeks. Compared with NF, OF had a greater aortic lumen diameter (D, 2.42 ± 0.11 vs. 1.47 ± 0.04 mm, p<0.01) and total wall thickness (WT, 353 ± 12 vs. 227 ± 12 μm, p<0.001), with unchanged WT:D ratio (p=0.67). This remodeling was associated with increased intrinsic mechanical stiffening (elastic modulus: 6,457 ± 670 vs. 3,667 ± 501 kPa, p=0.01; stiffness: 18,811 ± 4,318 vs. 7,093 ± 1,187 kPa*m, p<0.05) and expression of collagen I (2.27 ± 0.34 vs. 1.00 ± 0.15 AU, p<0.05) and advanced glycation end‐products (1.84 ± 0.33 vs. 1.00 ± 0.35 AU, p<0.05). However, no difference in the oxidative stress marker nitrotyrosine was observed between groups, and OF actually had greater expression of the antioxidant enzyme CuZnSOD (2.45 ± 0.06 vs. 1.00 ± 0.25 AU, p=0.05). Chronic overfeeding induces a physiological remodeling of the aorta in Burmese pythons that is not mediated by oxidative stress, but may be an adaptive response to increased blood flow and cardiac remodeling.
microRNA‐378 inhibition promotes angiotensin II induced cardiac hypertrophy and fibrosis: role of paracrine signaling from cardiomyocytes to cardiac fibroblasts (868.1)
Authors: Gupta Madhu , Nagalingam Raghu , Noor Mariam , Sundaresan Ravi , Gupta Mahesh , Solaro R
Published: June 2021

MicroRNAs play significant roles in cardiac remodeling during heart failure. We have recently shown that miR‐378 is mainly expressed in cardiac myocytes (JBC 2012;287(16):12913‐26) and its over‐expression counteracts cardiac hypertrophy (JBC 2013;288(16):11216‐32). Intriguingly, whole body knockout of miR‐378 has also shown benefits against high fat diet induced obesity. However, cardiac effects of miR‐378 deficiency remain unknown. We examined cardiac response of miR‐378 depletion in mice 7 days following 3 consecutive injections (70 mg/kg, i.p.) of LNA‐ modified 378‐antimiR. We found induction of hypertrophy markers and fibrotic gene expression, and de‐repression of targets, IGF1R and Grb‐2. Ang‐II‐induced cardiac dysfunction and remodeling was drastically enhanced by 378‐antimiR treatment. Inhibition of miR‐378 alone led to induction of TGFβ1 expression in mouse hearts. It stimulated TGFβ1 promoter activity via inducing AP‐1 transcriptional activity and Ras‐signaling. Conditioned medium (CM) of miR‐378 depleted cardiomyocytes resulted in cardiac fibroblast cell differentiation with enhanced fibronectin (FN) and collagen (Col1) expression, which was blocked by inclusion of TGFβ1‐neutralizing antibody in the CM. MiR‐378 over‐expressing cardiomyocytes were found to secrete miR‐378 in the culture medium. The secreted miR‐378 was effectively taken up by cardiac fibroblasts where it repressed AngII‐stimulated FN and Col1 expression. Our findings disclose a new paracrine role of cardiac myocytes in the control of fibroblast cell differentiation involving miR‐378. Our data demonstrate that miR‐378 inhibition promotes adverse ventricular remodeling. Thus, previously suggested therapeutic targeting of miR‐378 in metabolic disorders should be exercised cautiously as it could have deleterious cardiac consequences particularly in conditions of preexisting cardiac disorders.

Grant Funding Source: NIH/NHLBI 5R01HL022231‐35
Diaphragm muscle denervation following mid‐cervical contusion injury (871.6)
Authors: Alvarez‐Argote Santiago , Mora Juan , Stowe Jessica , Sieck Gary , Mantilla Carlos
Published: June 2021

Respiratory failure is one of the most important complications of cervical spinal cord injury. The level of injury critically determines the severity of the complications. We hypothesized that higher cervical contusion injuries will compromise the motor neuron pool and the descending bulbospinal projections, causing greater functional compromise than contusions at lower levels. Unilateral contusion was performed on adult rats at either the C3 or C5 levels vs. laminectomy. No deficit was seen in ventilatory and non‐ventilatory behaviors as assessed by whole body plethysmography or chronic diaphragm electromyography up to 2 weeks. Contusion injuries at either level resulted in similarly sized cavity formation. Phrenic motor neuron loss differed, with C3 contusion causing a 50% loss of phrenic motor neurons, and C5 contusion only 20% loss compared to the contralateral side. The percent of NMJs that were denervated followed a segmental pattern. Following C3 contusion, ventral regions of the diaphragm displayed denervation of ~30% of NMJs compared to 1‐3% in the C5 contusion and laminectomy groups. In contrast, following C5 contusion, denervation was evident only in dorsal regions (~10%). These results show minimal ventilatory impairment following contusion injuries to the mid‐cervical spinal cord that result in phrenic motor neuron loss and evidence of diaphragm muscle denervation.

Grant Funding Source: Supported by NIH grant R01 HL096750 and the Mayo Clinic
Interaction of central and peripheral chemoreflexes in neonatal mice: evidence for hypo‐addition (872.11)
Authors: Cummings Kevin
Published: June 2021

The nature of peripheral and central chemoreceptor interaction in the neonatal period is unknown. Given recent findings in adult rodents, the hypothesis addressed here is that mild systemic hypercapnia will blunt the respiratory response to sudden excitation and inhibition of the peripheral chemoreceptors. To test this, postnatal day 12 mouse pups were exposed to three brief (30 sec) episodes of 10% O2/balance N2 (HPX) or 100% O2 (HPRX) using a mask‐pneumotach system that allowed re‐breathing. Challenges were given during normocapnic conditions (inspired CO2= 0‐0.5%; NC (n=6)), or when inspired CO2 was increased ~1.5% (HC; n=6) via re‐breathing. Respiratory frequency (fB), tidal volume (VT) and ventilation (VE) were calculated, on a breath‐to‐breath basis, during room air and during each HPX or HPRX exposure. The hyperventilation induced by HPX reached steady‐state by 50 breaths (~15‐20 sec). The HPX‐induced increase in fB and VE was significantly greater in NC (+45 ± 6 breaths.min‐1 and +560 ± 81 ml.min‐1.kg‐1, respectively) than in HC (+19 ± 9 and +311 ± 109, respectively) (P<0.05 for both). Hypoventilation was induced by HPRX in the first 25 breaths (~5‐10 sec). The HPRX‐induced reduction in VT and VE was greater in NC (‐1.1 ± 0.5 mL/breath and ‐364 ± 141 ml.min‐1.kg‐1, respectively) than in HC (+0.3 ± 0.3 and ‐15 ± 79, respectively) (P<0.01 for both). These data show that when CO2 is slightly elevated, respiration is relatively unresponsive to sudden stimulation or inhibition of the peripheral chemoreceptors. This suggests that in neonatal life, a hypo‐additive interaction exists between peripheral and central chemoreceptor inputs.
Effects of acute intermittent hypoxia on sympathetic and expiratory activities in rats (872.8)
Authors: Lemes Eduardo , Bassi Mirian , Colombari Débora , Colombari Eduardo , Zoccal Daniel
Published: June 2021

The exposure to acute intermittent hypoxia (AIH) produces a long‐lasting increase of phrenic and sympathetic activities in anesthetized rats. In the present study we aimed to evaluate the effects of AIH on baseline expiratory motor activity as well as the role of retrotrapezoid nucleus in the AIH‐induced sympathetic and respiratory changes. In the in situ working heart‐brainstem preparations of juvenile rats (n=4), AIH exposure (10 x 5‐7% O2 for 30 s, every 5 min) increased baseline phrenic (Δ: 20±6%), abdominal (Δ: 65±30%) and sympathetic (Δ: 89±48%) nerve activities (P<0.05) that was maintained for at least 60 min after the last hypoxic episode. In conscious adult rats (n=9), AIH evoked an increase in baseline arterial pressure (Δ: 7±2 mmHg), associated with enhanced sympathetic‐ and respiratory‐related variabilities (P<0.05). In adult urethane‐anesthetized rats with intact vagus (n=5), AIH increased baseline arterial pressure (Δ: 9±1 mmHg) and abdominal muscle contraction amplitude (Δ: 412±117%), which were reverted by pharmacological inhibition of retrotrapezoid nucleus (P<0.05). These data indicate that AIH promotes a sustained increase of expiratory activity, in addition to inspiratory and sympathetic activities. Moreover, AIH‐induced sympathetic and expiratory long‐term facilitation may involve, at least in part, the activation of the retrotrapezoid nucleus.

Grant Funding Source: Supported by FAPESP (09/54888‐7; 11/20040‐1) and CNPq
Carotid body induced post‐inspiratory neuron channelopathy for neurogenic hypertension (872.9)
Authors: Moraes Davi , Machado Benedito , Paton Julian
Published: June 2021

Post‐inspiratory (post‐I)‐modulation of sympathetic outflows was increased in the pre‐hypertensive spontaneously hypertensive (SH) rats and this excitation can be reduced by carotid body denervation (CBD).In SH and Wistar rats (4‐6 week old), simultaneous recordings of sympathetic and respiratory nerves together with whole cell patch clamp recordings of Bötzinger Complex post‐I neurons from in situ preparations were made. We evaluated whether enhanced post‐I modulation of sympathetic activity is due to changes in the intrinsic ionic properties of post‐I neurons and whether this can be reversed by CBD. In SH rats, the increased post‐I modulation of lumbar sympathetic nerve (n=22) was associated with a greater post‐I discharge in laryngeal motor outflow (n=15). These changes are associated with an increased intrinsic excitability and decreased spike frequency adaptation of post‐I neurons in SH(n=8) relative to Wistar (n=8) rats; this was attributed to a reduced calcium activated potassium conductance in SH rat post‐I neurons. Five days after CBD in SH rats the calcium activated potassium conductance in post‐I neurons was similar to that measured in Wistar rats (n=10). Our data support the novel concept of “respiratory neuron channelopathy” mediated by carotid body over activity in SH rats that may contribute to their excessive sympathetic activity.

Grant Funding Source: Supported by British Heart Foundation and FAPESP
Angiotensin behaves like a paracrine mediator in guinea pig enteric nervous system (874.1)
Authors: Wood Jackie , Wang Xi‐Yu , Wang Guo‐Du
Published: June 2021

Enteric mast cell hyperplasia occurs in inflammatory bowel disease and the irritable bowel syndrome. Mast cell proteases are catalysts for conversion of angiotensin I to angiotensin II (AT‐II). We applied AT‐II to ENS neurons, while recording neuronal electrical and synaptic behavior. The AT‐II acted to elevate or suppress neuronal excitability. Suppression of was reversed by noradrenergic alpha2 receptor blocking drugs. AT‐II enhanced electrically‐evoked noradrenergic inhibitory postsynaptic potentials and suppressed fast and slow excitatory postsynaptic potentials. Selective AT‐II1 receptor blocking drugs, but not selective AT2 receptor antagonists, suppressed all actions of AT‐II. AT2 receptor protein and the AT2 mRNA transcript were expressed in the ENS. The AT1 receptor was not expressed. Immunoreactivity for the AT1 receptor was expressed by ENS neurons with AH‐type 2 and S‐type electrophysiological behavior. Our results predict that mast cell‐induced AT‐II formation will have paracrine function in the ENS that includes enhanced release of norepinephrine from sympathetic postganglionic axons. Elevation of norepinephrine represses excitatory neurotransmission in the neural networks of the brain‐in‐the‐gut and suppresses neurogenic secretion thru inhibition of excitability of secretomotor neurons in the submucosal division of the ENS.

Grant Funding Source: Supported by National Institutes of Health, R01DK037238
Cardiovascular effects of chronically increasing angiotensin II type 2 receptor expression in the nucleus of the solitary tract (874.2)
Authors: Ruchaya Prashant , Blanch Graziela , Speretta Guilherme , Freiria‐Oliveira André , Sumners Colin , Menani José , Colombari Eduardo , Colombari Débora
Published: June 2021

The aim of this study was to investigate the effects of increased angiotensin II type 2 receptor (AT2R) expression in the nucleus of the solitary tract (NTS) on neurogenic hypertension. Ten week old normotensive (NT) Holtzman and spontaneously hypertensive rats (SHRs) were microinjected in the NTS with either AAV2‐CBA‐AT2R or AAV2‐CBA‐eGFP (n=5‐8/group). Telemetry and baroreflex were performed on four experimental groups: NTeGFP, NTAT2R, SHReGFP and SHRAT2R. At termination brains were harvested for RT‐PCR. No significant change in mean arterial pressure (MAP) was observed in all groups from baseline to 4 weeks post viral transfection, but SHReGFP and SHRAT2R showed a significant elevation in MAP compared to NTeGFP and NTAT2R groups (138 ± 4 and 136 ± 4 vs. 94 ± 2 and 97 ± 6 mmHg, respectively). SHReGFP showed a significantly impaired baroreflex compared to NTeGFP and NTAT2R (‐0.5 ± 0.1 vs. ‐1.1 ± 0.1 and ‐1.4 ± 0.1 bpm/mmHg, respectively). SHRAT2R show significant improvement in baroreflex compared to SHReGFP (‐0.70 ± 0.1 vs. ‐0.5 ± 0.1 bpm/mmHg, respectively). SHRAT2R have significantly higher mRNA levels of MAS and angiotensin converting enzyme 2 (ACE2) compared to SHReGFP (MAS: 1.6 ± 0.1 vs. 0.7 ± 0.3 and ACE2: 8.5 ± 1.6 vs. 2.0 ± 0.4). While AT2R is ineffective at modulating chronic MAP, the baroreflex is significantly improved in SHR possibly via the MAS/ACE2 axis, suggesting a potential beneficial effect of AT2R.

Grant Funding Source: Supported by: CNPq/PRONEX, FAPESP/PNDP‐CAPES, NIH HL‐076803
ICV infusion of (pro)renin receptor antagonist (mPRO20) attenuates prorenin and DOCA‐salt induced hypertension (874.5)
Authors: Li Wencheng , Feng Yumei
Published: June 2021

We previously reported that the binding of prorenin to (pro)renin receptor (PRR) plays a major role in brain Ang II formation and development of deoxycorticosterone acetate (DOCA)‐salt hypertension. In this study, we designed and developed an antagonistic peptide (mPRO20) to block the binding of prorenin to PRR. Fluorescence‐labeled mPRO20 bound to both mouse and human brain tissue, and the dissociation constant was 14.5nM and 0.9nM respectively. The binding was blocked by co‐incubation with prorenin, indicating the specificity of mPRO20 to PRR. To test the in vivo effect of mPRO20, C57BL/6 mice were implanted with telemetric probes and intracerebroventricular (ICV) cannula. ICV infusion of mouse prorenin (300 ng in 3μl for 10 minutes) increased BP (ΔMAP: 28±3.4 mmHg); and this effect was attenuated by mPRO20 in a dose‐dependent manner (maximum effect, ΔMAP: 7±2.9 mmHg). Chronic ICV infusion of mPRO20 (40μg/kg/day, 21 days) attenuated the development of hypertension (113±3.3 vs. 134±3.6 mmHg, P<0.05), and the increase in brain Ang II levels (904±47 vs. 1371±88 pg/g) induced by DOCA‐salt. In addition, ICV infusion of mPRO20 improved autonomic function and spontaneous baroreflex sensitivity in mice treated with DOCA‐salt. In summary, mPRO20 binds to both mouse and human PRR, decreases Ang II formation and hypertension induced by either prorenin or DOCA‐salt. We conclude that mPRO20 may be a novel PRR antagonist for the treatment of hypertension.
Circulating Ang II modulates the effects of exercise training on skeletal myopathy in heart failure (874.8)
Authors: Gomes‐Santos Igor , Fernandes Tiago , Couto Gisele , Ferreira‐Filho Julio , Salemi Vera , Fernandes Fernanda , Casarini Dulce , Brum Patricia , Rossoni Luciana , Oliveira Edilamar , Negrao Carlos
Published: June 2021

Previous studies show that angiotensin II (AngII) mediates muscle wasting. In chronic heart failure (CHF), exercise training reduces AngII and improves skeletal myopathy. Unknown is whether the improvement in skeletal myopathy in CHF provoked by exercise training depends on AngII reduction. Thus, we studied the effects of exercise training on muscle ubiquitin‐proteasome pathway in CHF rats submitted to an AngII clamp (osmotic minipump). Male Wistar rats underwent coronary artery ligation for CHF induction. At fourth week of surgery, the CHF rats were divided into three groups: 1) Untrained; 2) exercise‐trained and 3) exercise‐trained with AngII. Exercise training was conducted on a treadmill 60min/day, 5 days/wk at 60% VO2max during 8wk. Exercise training reduced E3a, Atrogin and MuRF mRNA expression (RT‐PCR), carbonylated and ubiquitinated proteins (Western Blot), and 26S proteasomal activity (Fluorimetry) in plantaris muscle of CHF rats. AngII infusion, set at the same levels of untrained rats, restrained the exercise‐mediated changes in the ubiquitin‐proteasome pathway. We concluded that circulating AngII modulates the effects of exercise training on skeletal myopathy in CHF.

Grant Funding Source: Supported by FAPESP (2010/50048‐1), CAPES, CNPq, and Fundação Zerbini
Microglia, a new cellular factor, in the development of hypertension (875.6)
Authors: Li You , Li Liang , Bernstein Kenneth , Shen Xiao , Shi Peng
Published: June 2021

The concept of neuroinflammation in the development of hypertension has been acknowledged recently, as studies showed that anti‐inflammation treatments attenuated Ang II induced blood pressure increase. Microglia are the major immune cells in the central nervous system. To examine the role of microglia in hypertension, we applied a microglial depletion strategy by using a transgenic mouse line CD11b‐DTR. This mouse line has an inserted transgene coding simian diphtheria toxin receptor (DTR) under the control of the CD11b promoter. CD11b‐DTR mice were made hypertensive by subcutaneous Ang II infusion. Two weeks later, the mice received intracerebroventricular infusion of either DT or saline. Blood pressure was monitored by tailcuff. It showed that systolic blood pressure reached ~140 mmHg in both groups by 2 wk. Microglial depletion caused a gradual reduction in blood pressure (20 mmHg) by the end of 4 wk. To validate the importance of microglia in neuroinflammation associated with hypertension, the expression of pro‐inflammatory cytokines in paraventricular nucleus (PVN) were analyzed by quantative RT‐PCR. Microglial depletion reduced the expression of both IL‐1β and TNFα in the PVN of hypertensive mice. Moreover, the norepinephrine levels in the kidney were significantly decreased in the microglia‐depleted mice vs. controls. These data strongly support our hypothesis that in hypertension, microglia play an importance role in initiating neuroinflammation and in sustaining blood pressure.

Grant Funding Source: AHA and UCLA CTSI
Visualizing lymph movement in anuran amphibians with computed tomography (879.9)
Authors: Hedrick Michael , Hansen Kasper , Wang Tobias , Thygesen Jesper , Pedersen Michael
Published: June 2021

Lymph flux rates in anuran amphibians are high relative to other vertebrates owing to ‘leaky’ capillaries and a very high interstitial compliance. Lymph movement is accomplished primarily by specialized lymph muscles and lung ventilation that move lymph through interconnected lymph sacs to the dorsally‐located lymph hearts which are responsible for pumping lymph back into the circulatory system. The highly compartmentalized lymph sacs have been described anatomically, but it is unclear how lymph moves through the lymphatic system to the lymph hearts. We used computed tomography (CT) to visualize an iodinated contrast solution, injected into various lymph sacs, through the lymph system in awake cane toads (Rhinella marina). Toads were anesthetized with MS‐222 and small bore cannulae (PE 50) were placed into a variety of lymph sacs(subvertebral, lateral, interfemoral, brachial, crural) to inject contrast solution (0.25‐1.0 ml; VisipaqueTM 320 mg I/ml). Isotropic 0.216 mm3/voxel CT scans were acquired with a 64‐Slice Siemens Somatom Definition (Siemens Medical Solutions, Germany) at 80 kV, tube current 273 mA. Scanning time was approximately 12 seconds and scans were performed repeatedly for 1 min to 180 min following injection of the contrast solution. CT‐data was analyzed qualitatively in the 3D viewer in Osirix‐software where movement of the contrast was clearly visible over time, although clearance rates differed considerably between lymph sacs. We observed vertical movement of contrast in lateral and interfemoral sacs as predicted from previous studies. A surprising result was the observation of a previously undescribed pathway for lymph movement from the subvertebral sac ventrally between the lungs. These visual results confirm predictions about the movement of lymph, but also provide some novel findings regarding the pathways for lymph movement.

Grant Funding Source: Supported by the National Science Foundation and the Danish Research Council