bluuebunny/crossref_metadata_2025 huggingface dataset:
Sodium salicylate rescues inflammation‐associated decrease in phosphorylated‐eNOSser1177 in human aortic endothelial cells through an AMPK dependent mechanism (LB706)
Authors: Siefers Kyle , Wegman‐Points Lauren , Witmer Jordan , Pierce Gary
Published: June 2021

Obesity is associated with impaired nitric oxide (NO)‐mediated vascular endothelial function (VEF). Pro‐inflammatory cytokines, such as TNF, may contribute to reduced VEF in part through a direct reduction of endothelial NO synthase (eNOS) expression and/or phosphorylation. Sodium salicylate (NaSal), a non‐acetylated aspirin that inhibits nuclear factor B and activates AMP kinase (AMPK), improves VEF in obese humans but the molecular mechanisms remain unclear. We tested the hypothesis that NaSal increases eNOS expression/phosphorylation in TNF‐stimulated endothelial cells through an AMPK‐dependent mechanism. Human aortic endothelial cells (HAECs) incubated in vitro with TNFα (10 ng/ml, 2 hrs) demonstrated decreased (vs. control) expression (Western blotting) of eNOS serine 1177 phosphorylation (p‐eNOSser1177, n=6; P<0.01), eNOS (n=6, P=.078) and phosphorylated AMPK (p‐AMPKThr172, n=2, P<0.05). Pretreatment of HAECs with NaSal (5 mM, 30 min) prohibited the TNF‐stimulated decrease in eNOS (n=4; P=0.77), p‐eNOSser117 (n=4; P=0.1) and p‐AMPKThr172 (n=2; P=0.22). Co‐treatment with the AMPK inhibitor compound C (10 μM, 30 min) abolished the TNF‐induced rescue of p‐eNOSser1177 (n=2; P<0.05) but not eNOS (n=2, P=0.77) by NaSal. These preliminary data suggest that NaSal contributes to increased phosphorylated eNOSser1177 in TNF‐stimulated endothelial cells in part through inhibiting AMPK.

Grant Funding Source: Supported by the University of Iowa Fraternal Order of Eagles Diabetes Research Center pilot grant.
Contribution of Ang (1‐7) to Sex Differences in the Renal T cell Profile Following Chronic Ang II Infusion (LB714)
Authors: Zimmerman Margaret , Tipton Ashlee , Baban Babak , Sullivan Jennifer
Published: June 2021

Angiotensin (Ang) II is pro‐inflammatory, and T cells contribute to Ang II hypertension in male animal models; little is known in females. Initial studies treated 12 wk old male and female Sprague Dawley rats (SD) with Ang II (200 ng/kg/min) and renal T cell profiles were measured via flow cytometry. Ang II increased renal T cell infiltration in both sexes; as well as immuno‐suppressive T regulatory cells (Tregs) only in kidneys of female SD (p=0.004), and male SD had greater increases in pro‐inflammatory Th17 cells (p<0.001). We have previously shown that females also have greater renal Ang (1‐7) levels than males, and Ang (1‐7) has been suggested to be anti‐inflammatory. Therefore, the goal of the current study was to test the hypothesis that chronic Ang II infusion increases renal T cells in both sexes; however, greater Ang (1‐7) results in more Tregs in females than males. Additional male and female SD were treated with Ang II, or Ang II plus the Ang (1‐7) mas receptor antagonist A‐779 (48 µg/kg/hr). CD4+ T cell counts were greater in males compared to females following Ang II infusion (p<0.01), inhibition of Ang (1‐7) did not alter counts in either sex. Ang II infusion increased Tregs in females and Th17 in males, inhibition of Ang (1‐7) did not alter these counts. Renal CD8+ T cell counts were reduced in males relative to females following Ang II infusion (p<0.01), and inhibition of Ang (1‐7) caused a significant but comparable decrease in renal CD8+ T cell counts in both sexes (p=0.0003). In conclusion, our data implicates a role for Ang (1‐7) to modulate Ang II‐induced changes in CD8+ T cell counts; however, our results show that Ang (1‐7) does not mediate the sex difference in Tregs and Th17 in SD during Ang II infusion.

Grant Funding Source: Supported by NIH grant 1R01 HL093271‐01A1 to J.C.S, and AHA Predoctoral Fellowship 12PRE11470003 to M.A.Z.
New β‐ENaC Polymorphisms among Salt Sensitive and Salt Resistant Nigerian Subjects (LB718)
Authors: Elias Simiat , Sofola Olusoga , Jaja Smith
Published: June 2021

The relationship between the β‐T594M polymorphism of the epithelial sodium channel (ENaC) and hypertension remains equivocal. This study was designed to determine the occurence of the β‐ENaC polymorphism among Nigerians as a first step towards determining its importance as a determinant of hypertension in this populace. Forty‐seven normotensive (NT) and fifty‐three age‐matched hypertensive (HT) subjects earlier divided into salt sensitive (SS) and salt resistant (SR) subgroups were recruited for this study. Ethical approval (CM/COM/8/Vol.XXI) was granted by University of Lagos. Blood samples were dried on Whitman filter paper and stored at room temperature until analysed. DNA was isolated from the blood spots followed by ampification by polymerase chain reaction (PCR). The amplicons were thereafter sequenced for β‐ENaC polymorphisms. Polymorphisms were recorded in 13% of the subjects. Five (5%) of the subjects were positive for the β‐T594M polymorphism: 2 NT (both SS) and 3 HT (2 SS and 1 SR).Three new non‐synonymous mutations were recorded among HT subjects: β‐E632V and β‐E636V respectively among two SS HT subjects while β‐D638Y was recorded in an SR HT subject. A fourth new mutation β‐L628Q was recorded in an SS NT subject. A synonymous polymorphysm, β‐T577t was observed in three SS HT subjects and one SS NT subject. The non‐synonymous mutations are likely to be important in the development of salt sensitive hypertension among this populace considering the prevalence (9/100) in such a modest number of subjects. It is of interest that the β‐T594M and the β‐D638Y were also recorded in SR subjects as this might question the relationship between ENaC and salt sensitivity.

Grant Funding Source: Supported by International Junior Research Grant of The Physiological Society
Inhibition of endoplasmic reticulum stress attenuates aorta coarctation induced hypertension and kidney injury in diabetic Goto Kakizaki rats (LB724)
Authors: Wang Zhen , do Carmo Jussara , da Silva Alexandre , Hall John
Published: June 2021

Hypertension (HT) is common in diabetes and substantially increases the risk for diabetic nephropathy. To investigate the mechanisms by which HT and diabetes interact to promote nephropathy, 6 month‐old male Goto‐Kakizaki (GK) rats, a model of spontaneous type 2 diabetes, were used in this study. HT was induced by aorta coarctation (AC) between the renal arteries to produce HT plus diabetes in the right kidney (above AC) and normal blood pressure (BP) plus diabetes in the left kidney (below AC). BP above AC increased to 151±8 mmHg 8 wks after AC (n=5, p<0.05 compared to baseline of 108±5 mmHg). To examine the role of endoplasmic reticulum (ER) stress in the development of hypertensive‐diabetic nephropathy, GK‐AC rats were treated with the ER stress inhibitor tauroursodeoxycholic acid (TUDCA, 200 mg/kg/day, IP) from 2 to 8 wks after AC. After 6 wks of treatment, ER stress marker, GRP‐78, was significantly reduced in the right HT kidney compared to the right HT kidney from untreated rats. TUDCA treatment also significantly lowered the BP above the AC (137±7 vs 151±8 mmHg, n=4, p<0.05 compared to untreated group). Glomerular filtration rate in the right HT kidney was increased to 0.99 ml/min/g of kidney weight in the TUDCA treated group compared to 0.67 ml/min/g in the untreated group (n=4, p<0.05). TUDCA treatment for 6 weeks also attenuated renal glomerular injury including thickness of glomerular basement membrane and expansion of mesangial matrix on the right HT kidney. These results suggest that ER stress contributes to kidney injury when HT is imposed on the diabetic kidney. Pharmacological inhibition of ER stress may attenuate increases in BP and kidney injury in hypertensive‐diabetic nephropathy.

Grant Funding Source: Supported by NHLBI PO1 HL51971, NIGMS P20GM104357, AHA 14POST18160019
Cyclooxygenases (COX) play a key role in the regulation of renal arterial tone, but not in glomerular function,of mouse with 5/6 nephrectomy (LB725)
Authors: Zhu Bijun , Jin Jianjun , Zhao Zhonghua , Zhang Zhigang , Lu Limin , Zhu Tongyu , Vanhoutte Paul , Shi Yi
Published: June 2021

Objectives. Hyperfusion induces shear stress, leading to macrovascular endothelial dysfunction. However, effects of hyperfusion on glomerular endothelial cells (microvascular) remain unclear.

Methods. C57/BL6 mice were randomly grouped into sham‐operated (2K), 1 kidney removal (1K) and 5/6 nephroectomy groups (5/6K‐off). Renal artery and glomerulus were collected for Western blotting, polymerase chain reaction and immunohistochemistry (IHC) assay.

Results. Four weeks after the surgery, arterial blood pressure was comparable among the three groups. Compared with 2K and 1K groups, 5/6K‐off mice had significantly higher levels of renal blood flow. Consistently, relaxations to acetylcholine were blunted in renal arteries, but not in aortae, of 5/6K‐off mice. Indomethacin, a non‐selective cyclooxygenase inhibitor, reduced acetylcholine‐induced relaxation in 2K group, suggesting that prostacyclin is a vasodilator in renal arteries. Indomethacin did not affect acetylcholine‐induced relaxation in 1K mice, but improved it in 5/6K‐off mice. These results indicated that prostanoids, other than prostacyclin, are involved in vasoconstriction. Western blot and IHC experiments confirmed the upregulation of cyclooxygenase‐2 expression in the renal artery of 5/6K‐off mice. Urine protein levels were similar in the three groups, when normalized with remaining glomeruli. Glomerular filtration experiments also indicated that there were no significant increase in levels of macromolecules in 5/6K‐off group. This suggested that mouse glomeruli in 5/6K‐off group have normal function. In addition, Western blotting and IHC experiments did not detect any significant changes in cyclooxygenases expressions in glomeruli.

Conclusion. This study presents some intriguing effects of cyclooxygenases in a mouse kidney hyperfusion model. Prostanoids acts as a vasoconstrictor in renal artery of 5/6K‐off mice, but produces a protective effect in 2K group. However, cyclooxygenases are not involved in glomerular function. Further experiments will focus on examining the underlying mechanisms.

Grant Funding Source: National Natural Science Foundation of China 81373412
Acetazolamide: an improved treatment for lithium‐induced NDI? (LB726)
Authors: de Groot Theun , Sinke Anne , Kortenoeven Marleen , Baumgarten Ruben , Devuyst Olivier , Loffing Jan , Wetzels Jack , Deen Peter
Published: June 2021

Lithium causes nephrogenic diabetes insipidus (Li‐NDI) and hydrochlorothiazide (HCTZ) forms, together with amiloride, the mainstay treatment for Li‐NDI patients. Earlier, we found that HCTZ, primarily known for inhibiting the NaCl‐co‐transporter (NCC), also reduces Li‐NDI in NCC knockout mice, coinciding with alkalinized urine, suggesting that inhibition of carbonic anhydrase (CA) by HCTZ plays a role. To test whether CA inhibition is a useful alternative therapy in Li‐NDI, the effect of acetazolamide (ACZ) was tested.

Polarized mouse cortical collecting duct (mpkCCD) cells and mice were used to study the effect of ACZ on Li‐NDI.

Treatment of mpkCCD cells with ACZ attenuated AQP2 downregulation by lithium. ACZ did, in contrast to amiloride, not affect transcellular voltage. Treatment of Li‐NDI mice with ACZ revealed a significant antidiuresis and increased urine osmolality, which was indifferent from Li‐NDI mice treated with HCTZ/amiloride. However, unlike HCTZ/amiloride, ACZ treatment did not result in hyponatremia, hyperkalemia, hypercalcaemia and metabolic acidosis. Moreover, ACZ rescued AQP2 expression over the entire length of the collecting duct, whereas HCTZ/amiloride only increased AQP2 levels in the renal papilla.

In conclusion, our data reveal that ACZ attenuates lithium‐induced downregulation of AQP2 and NDI development. Moreover, as ACZ caused fewer side effects than the conventional treatment, ACZ may represent a better therapeutic agent than HCTZ/amiloride to treat Li‐NDI.

Grant Funding Source: Supported by a VICI grant, a RUNMC grant and a grant from the Society of Experimental Laboratory Medicine.
Effect of Deep Brain Stimulation on Sympathetic outflow in humans (LB733)
Authors: Sverrisdottir Yrsa , Green Alexander , Aziz Tipu , Bahuri Nor Faizal , Hyam Jonathan , Basnayake Shanika , Paterson David
Published: June 2021

Electric deep brain stimulation (DBS) in midbrain nuclei in humans alters cardiovascularparameters, presumably by modulating autonomic and baroreflex function. Baroreflex modulation of sympathetic outflow is crucial for cardiovascular regulation and is hypothesized to occur at two distinct brain locations. This study aimed to evaluate sympathetic outflow in humans with DBS electrodes during ON and OFF stimulation of specific midbrain nuclei known to regulate cardiovascular function.

Muscle sympathetic nerve activity (MSNA) was recorded during DBS ON and OFF in patients with chronic neuropathic pain (n=7) and Parkinson’s disease (n=10). Arterial blood pressure (ABP), heart rate and respiration were monitored during the recording session and spontaneous vasomotor and cardiac baroreflex sensitivity (BRS) were assessed. Head‐up tilt testing was performed separately in the Parkinson’s patients post‐operatively.

Stimulation of the dorsal most part of the subthalamic nucleus (STN) and ventrolateral periaqueductal gray (PAG) resulted in improved vasomotor BRS, decreased MSNA burst frequency and ABP, unchanged burst amplitude distribution and a reduced fall in ABP after tilt. Stimulation of the dorsolateral PAG resulted in a shift in burst amplitude distribution towards larger amplitudes, decreased spontaneous BP variability and unchanged burst frequency, BRS and ABP.

Baroreflex regulation of sympathetic outflow occurs in the STN and PAG. Our results may have implications in understanding abnormal sympathetic discharge in cardiovascular disease and provide an opportunity for therapeutic targeting.

Grant Funding Source: supported by, BHF, NIHR BRC
The effect of maternal high fat diet during gestation on cardiovascular responses to dehydration and air jet stress (LB736)
Authors: Dissanayake Hasthi , Prestipino Louise , Hegedus Elizabeth , Polson Jaimie
Published: June 2021

Maternal high fat diet (MHFD) is associated with programmed hypertension in offspring. In this study we tested the effect of MHFD on cardiovascular and autonomic responses to dehydration (DS) and air jet (AJ) stress. Pregnant rats were fed a MHFD (59% fat) or control (5% fat) diet. At 6 months, offspring were implanted with telemetry blood pressure (BP) transmitters. BP, spontaneous baroreflex sensitivity (sBRS), heart rate variability (HRV) and BP variability (BPV) were measured at rest, and during DS and AJ. BP was higher in MHFD offspring than controls throughout the study (baseline systolic BP 131±6 vs 112±3 mmHg, n=4, P<0.03). This was associated with increased LF:HF HRV (0.58±0.09 vs 0.34±0.02, P<0.01) and sBRS (2.4±0.4 vs 1.5±0.3 ms/mmHg, P<0.01) in females. DS increased BP, accompanied by increased low frequency HRV (37±7%, P<0.05) and BPV (56±16%, P<0.05). During AJ systolic BP was higher in MHFD (156±3 vs 142±3 mmHg, P<0.05), however the size of the increase was similar (38±3 mmHg) in both groups. Increased BP during AJ was accompanied by increased HRV in all frequencies (total power, 1.1±0.2 vs 4.3±1.1 mmHg2, P<0.05), however we found no differences between groups. Our data show that MHFD caused hypertension associated with altered autonomic function. Although BP remained higher in MHFD offspring throughout DS and AJ we found no significant differences in the cardiovascular and autonomic responses to DS and AJ.
The traditional herbal medicine, Ge‐Gen‐Tang, inhibits pacemaker potentials by nitric oxide/cGMP dependent ATP‐sensitive K+ channels in cultured interstitial cells of Cajal from the mouse small intestine (LB746)
Authors: Kim Byung Joo
Published: June 2021

Interstitial cells of Cajal (ICCs) are pacemaker cells that activate the periodic spontaneous depolarization (pacemaker potentials) responsible for the production of slow waves in gastrointestinal (GI) smooth muscle. The effects of Ge‐Gen‐Tang (GGT), a traditional herbal medicine, on pacemaker potentials in cultured ICCs from the mouse small intestine were investigated. The patch‐clamp technique for whole‐cell recording was used in cultured ICCs. Under the current clamping mode, ICC generated pacemaker potentials. The resting membrane potential was ‐ 52 4 mV and the amplitude was 18.3 1.6 mV. GGT (10‐50 mg/ml) decreased the frequency and amplitude of pacemaker potentials in a concentration‐dependent manner, but had no effects on membrane potentials. In the presence of 50 mg/ml GGT, the amplitude was 1.2 0.3 mV and the frequency was 0.3 0.6 cycles/min at 50 mg/ml. However, these effects were blocked by intracellular GDPβS, a G‐protein inhibitor, and glibenclamide, a specific ATP‐sensitive K+ channels blocker. Pretreatment with SQ‐22536, an inhibitor of adenylate cyclase, before the application of GGT had no effect on pacemaker potentials. Similarly, pretreatment with ODQ, an inhibitor of guanylate cyclase, did not inhibit the pacemaker potentials. However, GGT inhibited the pacemaker potentials in the presence of SQ‐22536, not in ODQ. The amplitude was 1.7 0.6 mV in the presence of SQ‐22536 and 17.1 2.6 mV in ODQ at GGT 50 mg/ml. Also, pretreatment with L‐NAME (an inhibitor of nitric oxide (NO) synthase) before the application of GGT abolished its effects on pacemaker potentials, but L‐NAME itself had no effect. In the presence of L‐NAME, the amplitude was 18.7 0.6 mV at GGT 50 mg/ml. Taken together, GGT inhibits pacemaker potentials in ICC in a G protein‐, cGMP‐ and NO‐dependent manner. Our data suggest that the gastroprokinetic effects of GGT are mediated by the induction of pacemaker potentials in ICCs.

Grant Funding Source: Supported by the Traditonal Korean Medicine R&D Project, Ministry of Health & Welfare, Republic of Korea (HI12C1886, B120008)
Evaluation of Cathepsins (K, L and S) expression during 3T3‐L1 differentiation (LB757)
Authors: Prado‐Palma Renata , Christoffolete Marcelo
Published: June 2021

Objectives: Obesity is a crescent global disease that can trigger type II diabetes, cardiovascular disease and many others health problems. Obesity is characterized by white adipose tissue (WAT) accumulation. Adipocytes proliferate via pre‐adipocytes differentiation, which comprehends a well orchestraded sequence of events. Therefore, adipocyte differentiation is key step in development of obesity. Cathepsins play an important role on WAT differentiation, evidenced by the reduction of WAT in Cathepsin K or L KO mice. The objective of this study is to evaluate the expression of Cathepsins K, L and S (CatK, CatL and CatS) during the course of adipocyte differentiation. Methods: 3T3‐L1 cell culture was differentiated according to standard protocol, after 2 days of confluence cells were tretated with differentiation cocktail (isobutilmetylxantine, dexamethazone and rosiglitazone), and differentiation followed for additional 8 days. Cells were harvested at 0, 2, 4, 6, 8 and 10 days time points of differentiation. Total RNA was obtained by Trizol and submitted to RT‐real time PCR. Expression of differentiation marker (ap2/FABP4), later regulators (C/EBPα and PPARγ2) and CatK, CatL and S were evaluated. Results: Cathepsin L is expressed at very high levels in 3T3‐L1 cells, followed by a high level expression of CatK. CatS is expressed at very low levels in 3T3‐L1 cells, when compared to the other Cathepsins. CatK has a steady expression troughout differentiation except for a 2.5 fold increased expression at Day 2. The CatL was not modulated by differentiation. CatS is very poorly expressed in 3T3‐L1, however Day 2 and Day 10 showed 10 fold higher levels when compared to Day 0. Conclusion: Cathepsins are expressed in 3T3‐L1 cells similarly to WAT. The peak expression of CatK and CatS on Day 2 suggests a possible role in the early stages of adipocytes differentiation.

Financial Support: FAPESP (2008/10700‐1); UFABC.

Grant Funding Source: FAPESP, UFABC
Vagal nerve stimulation reduces metabolic rate and uncouples the relationship between heart rate and oxygen consumption in conscious mice (LB775)
Authors: Dias Daniel , Morgan Donald , Rahmouni Kamal , Stauss Harald
Published: June 2021

We tested the hypothesis that an increase in metabolic rate contributes to the weight loss associated with chronic vagal nerve stimulation (VNS) that has been observed in patients with epilepsy or depression and in animal studies. Blood pressure (BP) sensors (DSI) and cervical vagal nerve stimulators were implanted in C57Bl6 mice (n=5). On the following day, arterial BP, heart rate (HR), oxygen consumption (VO2), and respiratory exchange rate (RER) were recorded in conscious mice during application of different VNS intensities (3V, max. current 1mA, 1ms pulses at frequencies ranging from 1 Hz to 10 Hz in steps of 1 Hz, randomized order). Each VNS intensity was applied for 20 min and preceded by a baseline (20 min). In average (all VNS intensities pooled), VNS reduced HR (374±37 vs. 504±14 bpm, P<0.05), mean BP (95±5 vs. 107±8 mmHg, P<0.05), and VO2 (1,556±231 vs. 2,201±178 mL/kg/h, P<0.05) but did not alter RER (0.85±0.09 vs. 0.77±0.03). The significant linear correlation between HR and VO2 during baseline without VNS (R2: 0.34±0.08) was lost during VNS (R2: 0.06±0.03). These data suggest that an increase in metabolic rate does not contribute to weight loss associated with chronic VNS. Furthermore, VNS uncouples the linear relationship between HR and VO2. We speculate that VNS may shift energy homeostasis to a “starvation‐like” mode that is associated with reduced food intake and, subsequently decreased metabolic rate.
MiR‐17~92 promotes hypoxia‐induced pulmonary hypertension by regulating proliferation and contraction of pulmonary arterial smooth muscle cells (LB779)
Authors: Zhou Guofei , Chen Tianji , Zhou Qiyuan , Tang Haiyang , Yuan Jason , Raj J Usha
Published: June 2021

Introduction: Recent studies suggest that microRNAs (miRNAs) play important roles in development and pathogenesis of human diseases. We sought to identify novel miRNAs that may be involved in the pathogenesis of pulmonary arterial hypertension (PAH) and to investigate underlying mechanisms.

Methods and Results: We performed miRNA qPCR array using pulmonary arterial smooth muscle cells (PASMC) isolated from lungs of normal donor and PAH patients. We found that all of the six members of the miR‐17~92 cluster were down‐regulated in PAH PASMCs. In hypoxia‐induced PAH mouse model and cultured human PASMCs, we found that short term hypoxia induced miR‐17~92 whereas prolonged hypoxia suppressed miR‐17~92. We generated a strain of SMC‐specific miR‐17~92 knockout (sm‐17~92‐/‐) mouse by crossbreeding miR‐17~92 fl/fl mice with sm22α‐Cre mice and subsequent backcrossing. We exposed 8‐10 week old sm‐17~92‐/‐ mice along with their wild type littermates to hypoxia (10% O2) or normoxia for five weeks. We showed that knockout of miR‐17~92 in SMC attenuated hypoxia‐induced increase in measured their right ventricular systolic pressure (RVSP), RV hypertrophy (RV/(LV+S) ratio), and pulmonary arterial wall thickness, suggesting that SMC‐specific miR‐17~92 is essential for the development of PAH. To investigate the underlying mechanism, we over‐expressed the miR‐17~92 cluster in human PASMC by a lenti‐virus encoding this miRNA cluster. Overexpression of mi‐17~92 induced expression levels of SMA, SM22α, Calponin, and PCNA and BrdU incorporation. Human PASMC from idiopathic PAH (IPAH) patients expressed decreased levels of SMA, SM22α, and Calponin, whereas over‐expression of miR‐17~92 restored the expression of SMA, SM22α, and Calponin in PASMCs from IPAH patient.

Summary: We provide evidence that miR‐17~92 is dysregulated in hypertensive PASMC and suppression miR‐17~92 in SMC is sufficient to inhibit hypoxia‐induced PAH in vivo. We also show that miR‐17~92 induces PASMC proliferation and expression of contractile proteins. These results suggest that miR‐17~92 may promote PAH by regulating vascular remodeling and that miR‐17~92 may represent a novel therapeutic target for the treatment of PAH. Support: NIH HL110829 and Gilead Sciences Research Scholars Program.

Grant Funding Source: Support by NIH HL110829 and Gilead Sciences Research Scholars Program.
Connexin 43 mediates LPS‐induced permeability in lung microvascular endothelial cells (LB785)
Authors: Alli Shanta , Manna Jayeeta , Parthasarathi Kaushik
Published: June 2021

Increased microvessel permeability is a major characteristic of lung inflammation. We showed inhibiting connexin 43 (Cx43)‐containing gap junctions blunts permeability increases in microvessels due to an inflammatory insult. However, it is unclear whether Cx43 levels directly influence the lung endothelial barrier. Toward this, we cultured rat lung microvascular endothelial cells (RLMVEC) on transwell inserts and determined permeability via flux of FITC dextran 40 kd (FDx40) across the endothelial monolayer. In vehicle‐treated monolayers, FDx40 fluorescence in the bottom chamber was 92±2 gray levels (mean±SEM; n=3; measurements in duplicate). Treating RLMVEC with LPS (100 ng/ml) for 4 hours increased FDx40 permeability to 99±3 gray levels. To determine responses to reductions in Cx43 levels, we treated RLMVEC monolayers with Cx43 shRNA lentiviral vector for 24 hours. FDx40 permeability in Cx43 shRNA‐treated monolayers was 13% lower than control shRNA‐treated cells. Further, LPS‐treatment of Cx43 shRNA‐treated monolayers induced a more than 27% decrease in permeability compared to control (P<0.05). To determine responses to elevated levels of Cx43, we transfected RLMVEC monolayers with Cx43‐cDNA. FDx40 permeability in Cx43 cDNA‐treated monolayers was 76% higher than control cDNA‐treated monolayers. Further, LPS‐treatment of Cx43 cDNA‐treated monolayers induced a more than 45% increase in permeability compared to control. Together, these data show for the first time that reduction and increase in Cx43 levels,respectively blunts and augments endothelial monolayer permeability. Thus we interpret, endothelial Cx43 levels directly influence endothelial barrier leak.

Grant Funding Source: HL75503
A novel method for detecting striatal hypoperfusion in Parkinson disease ‐Saturated Iron oxide Nanoparticle Contrast (SINC) MRI (LB79)
Authors: Chang Chen , Chen Chiao‐Chi , Wu Chin‐Tien
Published: June 2021

Striatal dopaminergic denervation is a major characteristic of Parkinson’s disease (PD). Such neuronal damage may lead to disrupted perfusion that reflects changes in regional metabolism and neural activity. Current imaging findings regarding striatal perfusion alterations in PD remain inconclusive. The present study proposed a new perfusion weighted MRI technique that takes advantage of saturated iron oxide nanoparticle contrast (SINC) to estimate cerebral blood flow (CBF), cerebral blood volume (CBV) , and the mean transit time (MTT) in a PD rat model induced by 6‐hydroxydopamine. This SINC‐MRI method uses a high dose of iron oxide nanoparticles (IRON) for inducing signal changes on gradient echo T2* images acquired repeatedly over 60 time frames. As soon as IRON is injected, the signal decreases rapidly from a baseline to an undershoot, and then reaches a saturated steady state. CBV is represented directly by ΔR2* between the baseline and the steady state. Moreover, the derivative of the measured signal changes was modeled as the delta function of impulse response by fitting pixelwisely the signal‐to‐time curve using the piecewise Hermite polynomial function. The modeled signal was then deconvoluted through a local arterial input function to obtain the tissue concentration of the contrast agent so as to estimate CBF and MTT. Our results showed that, in the lesioned striatum, there were consistent decreases in the CBV and CBF by approximately 4‐7% as compared to the intact striatum. Importantly, this difference was not found by conventional dynamic susceptibility contrast MRI method. SINC‐MRI appears as a reliable and sensitive approach in detecting subtle perfusion changes.
Regulation of Adrenaline Synthesis by Intermittent Hypoxia (LB793)
Authors: Khurana Sandhya , Venkataraman Krishnan , McDonald Erika , Yao Jiefei , Studer‐Rabeler Karen , Tai T.C.
Published: June 2021

Epidemiological studies show a strong correlation between Obstructive Sleep Apnea (OSA) and cardiovascular disorders. OSA patients experience intermittent hypoxia (IH), characterized by brief, but recurring episodes of cessation in breathing. These patients have higher levels of circulating catecholamines and an increased incidence of hypertension; however the mechanisms defining this association are not clearly established. Genetic linkage studies have associated the phenylethanolamine N‐methyltransferase (PNMT) gene to the development of hypertension. PNMT, the terminal enzyme in the catecholamine biosynthetic pathway, directly responsible for adrenaline synthesis, is elevated in hypertensive animals. Recent studies utilizing PC12 cells show an increase in the expression of PNMT and its regulatory transcription factors when exposed to continuous hypoxia. The current study examined the regulation of PNMT under conditions of intermittent hypoxia. The mRNA of PNMT was analyzed to assess if the regulation of PNMT expression entails alternative splicing. The mRNA and protein levels of the transcription factors HIF1α, Egr‐1, GR, and Sp1, was analyzed to assess the cellular pathways involved in regulating PNMT expression. A PNMT promoter‐driven luciferase assay was performed to evaluate promoter activity under IH. Preliminary results lay an antecedent for the regulation of PNMT by IH conceivably via an altered regulation of its transcription factors and establish a possible role for PNMT in IH mediated hypertension in OSA patients.

Grant Funding Source: Supported by NSERC
Heart rate variability Analyses using wearable sensing system in response to parabolic flight (LB799)
Authors: Fujino Hidemi , Kondo Hiroyo , Hirayama Yusuke , Maezawa Toshiyuki , Tanaka Masayuki , Tsutou Akimitsu , Shinozaki Ryo , Nagatomo Fumiko , Ishihara Akihiko
Published: June 2021

New concept as wearable heart rate and gravity sensing system requires the development of bio‐signal acquisition device to be easily integrated into the health management in space, microgravity and hypergravity. The purpose of this study is to examine the acute effects of microgravity and hypergravity on the frequency domain analysis of heart rate variability (HRV) with the new wearable sensing system for electrocardiogram and gravity acquisition during parabolic flight. Six healthy volunteers underwent a series of experiments in sitting position involving parabolic flight. In this study, a new wearable sensing system “Mybeat” (Union Tool, Co., Tokyo) for Electrocardiogram and gravity acquisition was used. The ratio of low frequency to high frequency (LF/HF) components of HRV in the hypergravity phase before microgravity during parabolic flight was higher than that in horizontal before parabolic flight. In microgravity, the LF/HF components of HRV decreased. In contrast, there were no differences in the HF components of HRV between different gravity phases during parabolic flight. These results suggest that the acute effect of the different gravity phases on autonomic nervous activity might be affected by the modulating the dominance between sympathetic and parasympathetic nervous activity. In addition, the system could simultaneously provide information about the gravity and HRV in different gravity phases during parabolic flight. Supported by Grants‐in‐Aid for Science Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology.
Mitochondrial ROS Amplify Nox2 Signaling and Atrophy in the Rat Soleus with 7 Days of Hindlimb Unloading (LB821)
Authors: Lawler John , Lee Yang , Hord Jeff , Guzzoni Vinicius , Kuczmarski J
Published: June 2021

Removal of mechanical loading during spaceflight, bedrest, or immobilization causes rapid and profound atrophy of skeletal muscle. Emerging data suggest that oxidative stress from mitochondrial sources may contribute to unloading‐induced skeletal muscle atrophy. We recently found that the Nox2 isoform of NADPH oxidase is also upregulated in the unloaded soleus muscle, associated with FoxO3a activation and fiber atrophy. We used a mitochondrial specific antioxidant peptide (SS‐31) to test the hypothesis that mitochondrial ROS are involved in feed‐forward or amplification signaling with Nox2 in response to mechanical unloading of the rat soleus muscle. F344 rats were divided (n=7/grp) into the following groups: ambulatory control (CON), ambulatory + SS‐31 (1.5 mg/kg/d) (AS), hindlimb unloaded for 7 days (HU), and HU + SS‐31 (HUS). SS‐31 significantly attenuated the reduction in soleus mass and soleus mass/body mass ratio elicited by HU. SS‐31 also abrogated an increase in Type II fibers during unloading. In addition, SS‐31 significantly blunted upregulation of Nox2 subunit p67phox in the unloaded soleus, using fluorescent signal intensity and p67phox membrane localization as markers. Our results are consistent with the postulate that mechanical unloading of skeletal muscle causes mitochondrial ROS to upregulate Nox2 and thus drive atrophy and fiber‐type shift from slow to fast. Supported by NASA (NNX13AE45G), NIH (AR054084), and the Huffines Institute.

Grant Funding Source: NASA
Effects of capsaicin on tension in the slow skeletal muscle fibers of the chicken (LB823)
Authors: Uribe Calvillo tannia , Trujillo Xóchitl , Montoya‐Perez Rocío , Sánchez‐Duarte Elizabeth , Huerta Miguel
Published: June 2021

Skeletal muscle possesses two types of muscle fibers: 1) fast and 2) slow or tonic. In skeletal muscle fibers exists evidence of vanilloid receptors presence. It is known that vanilloid receptor TRPV1 is activated by capsaicin. On the other hand, primary sensitive neurons from chicks the vanilloid receptors are capsaicin‐insensitive.

By using isometric recording in bundles of the slow skeletal muscle fibers of one or two‐week age chicken dissected from the anterior latissimus dorsi muscle, the effect of capsaicin (10 μM) on caffeine (6 mM) evoked contractures was measured in the presence and absence of the antagonist capsazepine (1 μM). Capsaicin diminished maximum tension in the slow skeletal muscle fibers until 83.25±11.5%, (n=7, p=0.002) respecting to control, an effect that was partially reverted in the presence of the TRPV1 antagonist capsazepine. However, this reduction of maximum tension was not statistically significative (mean was 91.30±5.04%, n=5, p>0.05). These results suggest capsaicin is capable to activate the vanilloid receptor in avian skeletal muscle fibers.

Grant Funding Source: CIC2014‐RMP
Foot muscle of the sea slug Aplysia californica expresses a 5‐HT2 serotonin receptor (LB83)
Authors: Duclos Laura , Schuman Ben , McPherson Duane , Lovett Janice
Published: June 2021

Foot muscle of the sea slug Aplysia californica expresses a 5‐HT2 serotonin receptor Laura Duclos*, Ben Schuman, Duane McPherson, Janice Lovett: Geneseo, NY 14454, Department of Biology, SUNY Geneseo Serotonin (5−HT) has strong modulatory effects on foot and body‐wall muscle in the opisthobranch slug Aplysia. These effects include increased force of muscle contraction and increased rate of muscle relaxation. At the cellular level, 5−HT causes a dose−dependent increase in the cyclic adenosine monophosphate (cAMP) content of foot muscle. Our laboratory has previously isolated and cloned from Aplysia foot a serotonin receptor belonging to the 5−HT7 family, which stimulates adenylyl cyclase and could account for the increase in cAMP. More recently, a 5−HT2 receptor has been cloned from sensory neurons in the CNS of Aplysia (Nagakura et al., 2010). Using the information from that receptor sequence, we have discovered that foot muscle in Aplysia also expresses a 5−HT2 receptor. We are presently at work to clone the complete mRNA for this receptor, to determine whether it is identical to the one previously described. It is possible that 5−HT2 and 5−HT7 receptors act synergistically in the serotonergic modulation of muscle function.
H4K20me3 and H3K79me3 epigenetic changes in Endometriosis and endometrial tissues (LB844)
Authors: Rodriguez‐Deliz Carla , Monteiro Janice , Cruz‐Cosme Ruth , Flores Idhaliz
Published: June 2021

The significance of epigenetic changes in the etiology and pathogenesis of endometriosis is being progressively recognized. However, the function of lysine‐histone epigenetic modifications and furthermore, their role in the pathology of this disease remains unclear. We lead a study aimed to identify specific epigenetic profiles and evaluate possible associations with gene expression to provide insights about the pathological processes occurring in endometriosis. Objective: Identify the methylation pattern of lysine‐histones H4K20me3 and H3K79me3 and chromatin modulation by gene activation in endometrial tissues. Methods: We used endometrial tissues from 9 women, with and without endometriosis. Macro‐dissection was achieved by following pathology protocols. Tissues were homogenized and disaggregated using a bullet blender. Total histone extraction was carried out using an Epiquick Kit (Epigentek) and then, western blots (WB) were performed. Both, the H4K20me3 and H3K79me3 were detected in all endometrial tissues by WB. Chromatin immuno‐precipitation (ChIP) assay was performed using an Epiquick Kit. The promoter gene region between ‐27024569‐ 27025759, 100027589 ‐ 100028694 and ‐41275101 ‐ 4127670 of the ARID1A (220 pb), LOX‐4 (176 pb) and CTNNB1 (241 pb) genes were amplified with the correspondent primers, designed using ENCODE at UCSC Genome Browser program. PCR products were electrophoresed, stained with SYBR™ Green, and visualized. Results: There were higher levels of methylation of both 17kDa H4K20me3 and H3K79me3 in the endometrial tissue from women with endometriosis when compared to women without endometriosis (p < 0.05). ChIP assay demonstrated that ARID1A was expressed with H4K20me3, whereas with H3K79me3, it was silenced. However, Lox‐4 was silenced by both histones. Conclusion: Our results show that histone H4K20me3 and H3K79me3 modifications may be useful as a biomarker for detection and treatment of endometrial lesions.

Grant Funding Source: NIH‐NICHD Grant # R01‐050559, NIGMS S06‐GM 08239 (IF) ARRA supplement to R01‐050559 (IF), RCMI G12‐MD007579, PRCTRC U54‐MD007587, NIH NIGMS R25GM096955 to UPR‐Ponce.
Lymph transport in rat mesenteric lymphatics experiencing edemagenic stress (LB851)
Authors: Rahbar Elaheh , Akl Tony , Cote Gerard , Moore James , Zawieja David
Published: June 2021

Objective: To assess lymphatic adaptations to edema, we evaluated lymph transport function in rat mesenteric lymphatics under normal and increased fluid volume (edemagenic) conditions in situ.

Methods: Twelve rats were infused with saline (venous infusion, 0.2 ml/min/100g body weight) to induce edema. We intravitally measured mesenteric lymphatic diameter and contraction frequency, as well as immune cell velocity and density before, during and after infusion.

Results: A 10‐ and 6‐fold increase in lymph velocity (0.1‐1 mm/s) and flow rate (0.1‐0.6 µL/min), were observed post‐infusion, respectively (p<0.05). There were also increases in contraction frequency and fractional pump flow 1‐minute post‐infusion (p<0.05). Time‐averaged wall shear stress increased 10 times post‐infusion to nearly 1.5 dynes/cm2 (p<0.05). Similarly, maximum shear stress rose from 5 dynes/cm2 to 40 dynes/cm2.

Conclusions: Lymphatic vessels adapted to edemagenic stress by increasing lymph transport. Specifically, increases in lymphatic contraction frequency, lymph velocity, and shear stress were statistically significant. Although the changes in lymphatic diameter were not statistically significant, lymph pumping increased post‐infusion. These results suggest that edemagenic conditions stimulate lymph transport via increases in lymphatic contraction frequency, lymph velocity and flow. These changes, consequently, resulted in large increases in wall shear stress, which will activate nitric oxide pathways and modulate lymphatic transport function.

Grant Funding Source: NIH Grant No. R01 HL094269, NSF Graduate Research Fellowship
Virtual Team Rounding: A Cross-Specialty Inpatient Care Staffing Program to Manage COVID-19 Surges
Authors: Becker Nora V. , Bakshi Salina , Martin Kate L. , Bougrine Amina , Andrade Jonathan , Massey Paul R. , Hirner Jesse P. , Eccleston Julie , Choudhry Niteesh K. , Britton Kathryn A. , Landman Adam B. , Licurse Adam M. , Carlile Narath , Mendu Mallika L.
Published: June 2021

Problem

The SARS-CoV-2 (COVID-19) pandemic presented numerous challenges to inpatient care, including overtaxed inpatient medicine services, surges in patient censuses, disrupted patient care and educational activities for trainees, underused providers in certain specialties, and personal protective equipment shortages and new requirements for physical distancing. In March 2020, as the COVID-19 surge began, an interdisciplinary group of administrators, providers, and trainees at Brigham and Women’s Hospital created an inpatient virtual staffing model called the Virtual Team Rounding Program (VTRP).

Approach

The conceptual framework guiding VTRP development was rapid-cycle innovation. The VTRP was designed iteratively using feedback from residents, physician assistants, attendings, and administrators from March to June 2020. The VTRP trained and deployed a diverse set of providers across specialties as “virtual rounders” to support inpatient teams by joining and participating in rounds via videoconference and completing documentation tasks during and after rounds. The program was rapidly scaled up from March to June 2020.

Outcomes

In a survey of inpatient providers at the end of the pilot phase, 10/10 (100%) respondents reported they were getting either “a lot” or “a little” benefit from the VTRP and did not find the addition of the virtual rounder burdensome. During the scaling phase, the program grew to support 24 teams. In a survey at the end of the contraction phase, 117/187 (62.6%) inpatient providers who worked with a virtual rounder felt the rounder saved them time. VTRP leadership collaboratively and iteratively developed best practices for challenges encountered during implementation.

Next Steps

Virtual rounding provides a valuable extension of inpatient teams to manage COVID-19 surges. Future work will quantitatively and qualitatively assess the impact of the VTRP on inpatient provider satisfaction and well-being, virtual rounders’ experiences, and patient care outcomes.
Is Academic Attainment or Situational Judgment Test Performance in Medical School Associated With the Likelihood of Disciplinary Action? A National Retrospective Cohort Study
Authors: Sam Amir H. , Bala Laksha , Westacott Rachel J. , Brown Celia
Published: June 2021

Purpose

Disciplinary action imposed on physicians indicates their fitness to practice medicine is impaired and patient safety is potentially at risk. This national retrospective cohort study sought to examine whether there was an association between academic attainment or performance on a situational judgment test (SJT) in medical school and the risk of receiving disciplinary action within the first 5 years of professional practice in the United Kingdom.

Method

The authors included data from the UK Medical Education Database for 34,865 physicians from 33 U.K. medical schools that started the UK Foundation Programme (similar to internship) between 2014 and 2018. They analyzed data from 2 undergraduate medical assessments used in the United Kingdom: the Educational Performance Measure (EPM), which is based on academic attainment, and SJT, which is an assessment of professional attributes. The authors calculated hazard ratios (HRs) for EPM and SJT scores.

Results

The overall rate of disciplinary action was low (65/34,865, 0.19%) and the mean time to discipline was 810 days (standard deviation [SD] = 440). None of the physicians with fitness to practice concerns identified as students went on to receive disciplinary action after they qualified as physicians. The multivariate survival analysis demonstrated that a score increase of 1 SD (approximately 7.6 percentage points) on the EPM reduced the hazard of disciplinary action by approximately 50% (HR = 0.51; 95% confidence interval [CI]: 0.38, 0.69;P< .001). There was not a statistically significant association between the SJT score and the hazard of disciplinary action (HR = 0.84; 95% CI: 0.62, 1.13;P= .24).

Conclusions

An increase in EPM score was significantly associated with a reduced hazard of disciplinary action, whereas performance on the SJT was not. Early identification of increased risk of disciplinary action may provide an opportunity for remediation and avoidance of patient harm.
Individual mortality risk predictive system of patients with acute-on-chronic liver failure based on a random survival forest model
Authors: Zhang Zhi-Qiao , He Gang , Luo Zhao-Wen , Cheng Can-Chang , Wang Peng , Li Jing , Zhu Ming-Gu , Ming Lang , He Ting-Shan , Ouyang Yan-Ling , Huang Yi-Yan , Wu Xing-Liu , Ye Yi-Nong
Published: June 2021

Background:

The basis of individualized treatment should be individualized mortality risk predictive information. The present study aimed to develop an online individual mortality risk predictive tool for acute-on-chronic liver failure (ACLF) patients based on a random survival forest (RSF) algorithm.

Methods:

The current study retrospectively enrolled ACLF patients from the Department of Infectious Diseases of The First People's Hospital of Foshan, Shunde Hospital of Southern Medical University, and Jiangmen Central Hospital. Two hundred seventy-six consecutive ACLF patients were included in the present study as a model cohort (n = 276). Then the current study constructed a validation cohort by drawing patients from the model dataset based on the resampling method (n = 276). The RSF algorithm was used to develop an individual prognostic model for ACLF patients. The Brier score was used to evaluate the diagnostic accuracy of prognostic models. The weighted mean rank estimation method was used to compare the differences between the areas under the time-dependent ROC curves (AUROCs) of prognostic models.

Results:

Multivariate Cox regression identified hepatic encephalopathy (HE), age, serum sodium level, acute kidney injury (AKI), red cell distribution width (RDW), and international normalization index (INR) as independent risk factors for ACLF patients. A simplified RSF model was developed based on these previous risk factors. The AUROCs for predicting 3-, 6-, and 12-month mortality were 0.916, 0.916, and 0.905 for the RSF model and 0.872, 0.866, and 0.848 for the Cox model in the model cohort, respectively. The Brier scores were 0.119, 0.119, and 0.128 for the RSF model and 0.138, 0.146, and 0.156 for the Cox model, respectively. The nonparametric comparison suggested that the RSF model was superior to the Cox model for predicting the prognosis of ACLF patients.

Conclusions:

The current study developed a novel online individual mortality risk predictive tool that could predict individual mortality risk predictive curves for individual patients. Additionally, the current online individual mortality risk predictive tool could further provide predicted mortality percentages and 95% confidence intervals at user-defined time points.