bluuebunny/crossref_metadata_2025 huggingface dataset:
C.pneumoniae infection increases NADPH oxidase activity in vascular smooth muscle cells
Authors: Rivera Jennifer , Drummond Grant , Strugnell Richard , Sobey Christopher , Walduck Anna
Published: June 2021

Up‐regulation of NADPH oxidases, leading to increased reactive oxygen species (ROS) formation and oxidative stress is a key feature of atherogenesis. Chlamydia pneumoniae has been detected in human atheromas including vascular smooth muscle cells (VSMCs), suggesting a link between the bacteria and atherosclerosis. We examined whether C.pneumoniae induces NADPH oxidase activity in VSMCs. The mouse VSMC cell line, MOVAS, was infected with C.pneumoniae (104‐106 inclusion forming units) and superoxide production (L‐012 chemiluminescence), expression of NADPH oxidase subunit p47phox (immunoblotting), and C.pneumoniae inclusion counts (immunostaining) was assessed. C.pneumoniae caused a dose‐ and time‐dependent increase in superoxide production over 72 hours (n=6, p<0.001). This response was significantly reduced by superoxide scavengers SOD, PEG SOD and tempol (p<0.001, n=6), as well as the NADPH oxidase inhibitor, apocynin (p<0.05, n=6). In preliminary studies (n=2), infection appeared to have no effect on expression of p47phox, suggesting it is not essential for C.pneumoniae‐induced superoxide production. PEG SOD, SOD and the hydrogen peroxide scavenger, catalase, reduced Chlamydia inclusion counts in VSMCs (by 19%, 25%, and 35% respectively). These data suggest that C.pneumoniae increases NADPH oxidase activity in VSMCs and that ROS may promote bacterial growth in these cells. (NHMRC 436825)
The lectin‐like domain of TNF, but not cAMP, protects from Listeriolysin O‐induced endothelial hyperpermeability
Authors: Xiong Chenling , Yang Guang , Kumar Sanjiv , Black Stephen , Verin Alexander , Catravas John , Fischer Bernhard , Chakraborty Trinad , Lucas Rudolf
Published: June 2021

Listeriosis can lead to potentially lethal pulmonary permeability edema in newborns and immune‐compromised patients. Listeriolysin‐O (LLO), the main virulence factor of L. monocytogenes, induces a dose‐dependent myosin light chain (MLC) phosphorylation and hyperpermeability in human lung microvascular endothelial cells (HL‐MVEC), which can promote edema formation. The lectin‐like domain of TNF is spatially distinct from the TNF receptor binding sites and can be mimicked by the 17 amino acid TIP peptide. This peptide was shown to reduce pulmonary edema formation in in vivo and in situ murine and rabbit models. The objective of this study was to investigate, using electrical cell‐substrate impedance sensing (ECIS) and Western Blotting techniques, whether the TIP peptide can protect from LLO‐induced endothelial hyperpermeability in HL‐MVEC. In contrast to a cell‐permeant cAMP analogue, the TIP peptide blunts LLO‐induced hyperpermeability in monolayers of HL‐MVEC, as measured in ECIS. The peptide moreover inhibits LLO‐mediated MLC phosphorylation (Western Blotting) and blocks LLO‐induced generation of reactive oxygen species (EPR measurements), which increase endothelial permeability. These results indicate that the lectin‐like domain of TNF has a potential therapeutic value in protecting from LLO‐induced endothelial hyperpermeability and subsequent pulmonary permeability edema.
Macrolide antibiotics improve phagocytic capacity and reduce inflammation in sulfur mustard‐exposed monocytes
Authors: Gao Xiugong , Ray Radharaman , Xiao Yan , Ishida Keiko , Ray Prabhati
Published: June 2021

Sulfur mustard (SM) inhalation causes apoptosis and death of airway epithelial cells as well as inflammation in the airway. Efficient clearance of the cell debris by alveolar macrophages is necessitated to reduce the inflammation. The present study investigated the effects of four FDA‐approved macrolide antibiotics, namely azithromycin, clarithromycin, erythromycin, and roxithromycin, on macrophage chemotactic and phagocytotic function and on inflammatory cytokines/mediators production in vitro using SM‐exposed monocytes. It has been found that chemotaxis and phagocytosis of the monocytes reduced upon exposure to 10 µM SM (8.1% and 17.5%, respectively) were restored by treatment with 10 µM of any of the four macrolides. Overexpression of inflammatory cytokines following SM exposure was decreased by 50% ‐ 70% with macrolide treatment. Similarly, exaggerated expression of iNOS induced by SM exposure was largely inhibited by treatment with macrolides. These data suggest that treatment by macrolide antibiotics following SM inhalation may lead to improved clearance of apoptotic material in the airway and ultimately result in reduced airway inflammation and injury, further suggesting that macrolide antibiotics may serve as potential vesicant respiratory therapeutics. This work was supported by JSTO‐CBD/DTRA Project No. 3.F0003_05_WR_C.
G219A polymorphism in CRP gene and occurrence of atrial fibrilation after cardiac surgery
Authors: Lavandero Sergio , Roldan Juan , del Campo Andrea , Verdejo Hugo , Garcia Lorena , Becerra Elia , Pinto Melissa , Navarro Mario , Solis Margarita , Corbalan Ramon , Mellado Rosemarie , Prat Hernán
Published: June 2021

Atrial fibrillation (AF) is prevalent arrhythmia in adult population. Inflammation is a risk factor for AF and might have important in its occurrence. Increased C‐reactive protein plasma levels (CRP) have been found in AF and they could be determined by polymorphisms in the CRP gene. We investigate here if the G219A polymorphism in CRP gene is associated to AF occurrence. In this study, 116 patients with elective CABG were included. Systemic inflammatory markers were measured at baseline and 72 h after surgery. G219A polymorphism in the CRP gene, located 219‐bp downstream of exon 2 in the 3′ flanking sequence, was determined by PCR and we correlated with plasma CPR levels and AF occurrence. This study was approved by the Ethics Committee of each participating health center. All patient gave signed an informed consent. Mean age of patients was 61±11 years and 85% of them were male and 48% had a history of hypertension. Allelic frequencies for allele G and A were 0.57 and 0.43, respectively. There were no differences between genotypes with regard to diagnosis, risk factors, smoking, medical history and medications at entry. Postoperative FA incidence was 13%. Mean preoperative and postoperative PCR levels were 3.35 and 75.6 mg/L, respectively. We concluded that no association between G219A polymorphism in the CRP gene, CRP levels and AF were found.

DI‐06‐09‐2, FONDECYT 1070641
Effect of altered lipid homeostasis on cytochrome P450 2A5 expression
Authors: Nichols Kathleen , Kirby Gordon
Published: June 2021

Unlike most cytochrome P450 (CYP) enzymes, murine hepatic CYP2A5 and human CYP2A6 are elevated during hepatitis, fasting, and chemically‐induced hepatotoxicity. Since these enzymes metabolize nicotine and can activate pro‐carcinogens, their upregulation could affect tobacco addiction and carcinogenesis. However, the stimulus responsible for their induction remains unclear. Since CYP2A5 upregulation is usually associated with liver dysfunction, we hypothesized that a pathophysiological change associated with hepatotoxicity regulates CYP2A5 expression. This study examined the role of altered lipid homeostasis in CYP2A5 regulation. C57Bl6 mice were treated with saline or the hepatotoxin pyrazole for 24 hours, and hepatic gene transcripts were analyzed by Affymetrix microarray. Differential expression was observed for 109 genes associated with lipid synthesis, metabolism, and transport, confirming pyrazole alters lipid homeostasis. Furthermore, treatment of primary mouse hepatocytes with hepatic lipid extracts significantly increased CYP2A5 mRNA levels three‐fold. Analysis of hepatic fatty acid profiles indicated a significant elevation of linolenic acid in pyrazole‐treated mice that was absent in tetracycline‐treated animals which had no increase in CYP2A5 level. Ongoing work is investigating a role for linolenic acid in the regulation of CYP2A5. Funded by NSERC and CIHR.
Using team based learning (TBL) to teach pharmacology to second year medical students
Authors: Sabra Ramzi , Simaan Joseph A , Zgheib Nathalie K
Published: June 2021

A modified TBL method was incorporated in 2 case based discussion sessions of the pharmacology course that is provided to second year medical students at AUB: (1) a relatively easy drug metabolism and pharmacogenetics session and (2) a more challenging pharmacokinetics session. Individual and group answers for all cases were recorded and an evaluation form was collected. Students provided consistently more positive feedback for the drug metabolism session than for the pharmacokinetics session (e.g. 95% vs. 73% respectively, indicated they benefited considerably or more). The teaching and learning processes in both TBL sessions were considerably more interactive than in regular case based discussions. TBL appeared to be a less successful approach in the pharmacokinetics session compared with the drug metabolism session in that the group deliberations did not consistently improve outcome over individual performance. Overall, it appeared that the difficulty of the questions determined the degree of success, with a range of 30‐70% difficulty level being the most appropriate. This study describes the first application of TBL in a pharmacology course at AUB. The greater difficulty of the pharmacokinetics session and the lack of familiarity with the topic rendered TBL not as successful an experience as anticipated. The results provide clues to optimal design of cases and problems for TBL, so that the experience is beneficial and well received by students.
Supplementation of breads containing Soybean and rice retarded incorporation of trans fatty acids into plasma lipid in Healthy Korean Adults
Authors: Noh Kyunghee , Heo Young , Cho Mikyung , Jang Jihyun , Lee Seunghwan , Kim Dohoon , Cho Kyunghwan , Song Youngsun
Published: June 2021

Human can't synthesize trans fat(tFA), therefore the presence of tFA in human tissues and fluids is related to dietary intake, intestinal absorption, metabolism and storage, exchanges among compartments. This study investigated the effect of soybean and rice on the incorporation tFA into plasma lipid through short term (postprandial trial) and long term studies (6weeks trial). 33 healthy volunteers were divided into 3 groups and fed soybean bread, rice bread or wheat bread containing equivalent amounts of tFA (elaidic acid rich, 3.75 g/day), respectively. In a short term trial, postprandial lipid profiles at 0, 1, 2, 3, 4 hours after a respective meal were studied. In a randomized 6‐week study, blood samples were collected at 0. 2. 4. 6 weeks of consumption. Postprandial data indicated that consumption of soybean and rice breads with 3.75g tFA retarded the appearance of C18:1 and C18:2 tFA in plasma lipid compared to wheat bread. Consumption of soybean bread with tFA for 6 weeks also affected plasma fatty acid composition including tFA. TFA levels were lowered in the following order, soybean bread > rice bread > wheat bread. These results imply that soybean bread with high dietary fiber content may lowered the absorption of tFA and thus the level of plasma tFA.
S‐allylcysteine modulates the expression of E‐cadherin and inhibits the malignant progression of human oral cancer
Authors: Tang Feng‐Yao , Lin Meng‐Qiu , Cho Hsin‐Ju
Published: June 2021

Oral cancer is a prevalent type of cancer in the Asian countries. Several studies indicated that garlic extracts such as diallyl disulfide (DADS) and diallyl trisulfide (DATS) have anti‐cancer effects. However, the inhibitory effects of water soluble garlic extracts, S‐allylcysteine (SAC), on the tumor malignancy of oral cancer have not been studied well yet. Thus, the purpose of this study was to investigate the inhibitory effects of SAC on the proliferation and progression of human oral squamous cancer CAL‐27 cells.

In the present study, we demonstrated that SAC dose dependently inhibited the growth of human oral squamous cancer cells. Our results showed that SAC induced the expression of E‐cadherin adhesion molecule. Immunocytochemical staining result also revealed that SAC could restore the distribution of E‐cadherin molecule on cell membrane. We further demonstrated that SAC stabilized the adherent junction complex of E‐cadherin/β‐catenin in oral cancer cells. Treatment of MAPK/MEK specific inhibitor, PD098059, could upregulate the expression of E‐cadherin molecule. Furthermore, SAC significantly inhibited the activation of MAPK/ERK signaling pathway. These findings were associated with the down regulation of Slug repressor protein.

In conclusion, our results indicated that SAC effectively inhibited the proliferation, upregulated the expression of E‐cadherin molecule, and stabilized the E‐cadherin/β‐catenin adherent junction complex in human oral squamous cancer cells. The mechanism of action was in part through the suppression of MAPK/ERK signaling pathway and downregulation of Slug repressor protein.
Carbon Isotope Ratio Analysis of Bovine Endogenous Ouabain and Commercial Sources of Plant Ouabain by High Resolution Mass Spectrometry
Authors: Hamlyn John , Tashko Gerti , Tilva Varun , Liu Yong , Hamilton Bruce , Rogowski Robert
Published: June 2021

Endogenous ouabain (EO) and digoxin‐like materials (DILFs) have been identified in the circulation, brain, adrenal glands and urine of mammals. Although EO and DILF appear to be natural products of the mammalian adrenal cortex, their biosynthetic pathway(s) are unknown. Despite overwhelming evidence for EO, some have speculated that EO is a laboratory contaminant (Nicholls et al, J Hypertens 2009;27:3‐8). Arguments for the former view have been rebutted (Manunta et al, J Hypertens 2009;27:9‐18) and additional proof is provided by the present work. We isolated ~6 µg of EO from 6 Kg of bovine adrenal glands. The bioactivity of the bovine EO was followed through serial chromatography by a radioreceptor assay. Using high resolution mass spectrometry, we compared the exact mass and carbon isotope ratios in bovine EO with commercially available plant sources of ouabain (Sigma Chemical Co. and Calbiochem) that were the only other sources of ouabain in the laboratory environment. As expected, high resolution mass spectrometry showed identical exact mass to charge ratios for protonated molecular ions of EO and the two plant ouabains (M+H+ = 585.292 m/z). Crucially, the abundance of 13C in the bovine material (δ‐53.7‰) was significantly lower (p<0.001) than that in the plant ouabains (δ‐22.4‰, ‐16.6‰). We conclude that carbon isotope ratios distinguish bovine EO from that in ouabain synthesizing plants and exclude the possibility that bovine EO is a laboratory contaminant. As the depletion of 13C in bovine EO extends beyond that expected for plant metabolism (‐10 to ‐30‰), EO biosynthesis is likely to involve one or more enzymes with heightened preference for bonds with adjacent 12C atoms. Supported by NHLBI 078870/75584
Novel expression and modification of the cytoskeletal protein Myozenin during hypertrophic growth
Authors: Paulsson Anna Kristina , Warburton Sarah , Mitchell‐Jordan Scherise A , Ren Shuxun , Wang Yibin , Vondriska Thomas M
Published: June 2021

Cardiovascular disease is the leading cause of death in the developed world and includes growth of the heart (hypertrophy) resulting from altered mechanical and neural/hormonal factors. As a major clinical problem resultant from atherosclerosis and heart attacks, novel insights into hypertrophy are needed, in particular regarding changes in the diseased nuclear and cytoskeletal proteomes. To this end, transverse aortic constriction (TAC) was performed to induce pressure overload hypertrophy in mice. Hearts were excised between 1‐8 weeks post‐TAC, nuclei isolated and proteins separated by 2D or 1D PAGE and subjected to LC/MS/MS. Of ~1000 nuclear proteins exhibiting reproducible changes in abundance (p>0.05) after pressure overload, myozenin‐a molecular link between the phosphatase calcineurin and the structural protein α‐actinin‐was found to be upregulated. Further analysis of myozenin via immunoblotting and MS/MS revealed a truncated form that appears during hypertrophy. In addition, novel phosphorylation of residues S106, S116, T107 and T111 on myozenin‐2 was observed. Comparison of apparent mass using electrophoresis, immunoblotting and MS peptide maps revealed regions of modification on myozenin following injury. These studies add a new dimension to the role of myozenin in the heart and provide insights into nuclear‐cytoskeletal communication during cardiac hypertrophy.

Support: NIH, AHA
Intragenic suppressor mutations of a constitutively active allele of Gs alpha
Authors: Sultan Dahlia , Tobar‐Rubin Raquel , Rylaarsdam Robin
Published: June 2021

Heterotrimeric G‐proteins cycle between an active/GTP‐bound conformation and an inactive/GDP‐bound conformation. Reduction in GTP hydrolysis by the G‐protein alpha subunit results in constitutive activity of the protein. McCune‐Albright Syndrome (MAS) is a human genetic disorder caused by a mutation that constitutively activates the Gs alpha subunit by inhibiting GTP hydrolysis. Arginine 201 substitutions with histidine, serine, or cysteine have been reported in biopsies from MAS patients. Previous work in this laboratory modeled the MAS mutation in a yeast system, and identified an intragenic suppressor of the MAS mutation, which substituted two residues in the GTP‐binding site. The homologous mutations (F222P, D223V) in the human Gs alpha subunit also suppressed the constitutive activity of the MAS mutation when the protein was expressed in human cells. To extend these studies, F222 and D223 were mutated to a variety of other amino acids to determine the structural requirements for suppression of the MAS mutation. Basal and receptor‐stimulated cAMP levels were measured in HEK293 cells transfected with the different alleles of Gs alpha. While some single mutations at either F222 or D223 were able to suppress the MAS R201H mutation, a range of levels of suppression were observed.
Anchored PKA and regulation of calcium handling in cardiomyocytes
Authors: Jones Brian William , McKnight G Stanley
Published: June 2021

Sympathetic stimulation of the heart elevates cAMP levels leading to the rapid phosphorylation of several protein targets by protein kinase A (PKA). Immediate consequences of this phosphorylation include increased Ca2+ entry through voltage‐gated Ca2+ channels (CaV1.2), Ca2+ release from sarcoplasmic reticulum (SR) ryanodine receptors, and Ca2+ reuptake into the SR by the calcium ATPase (SERCA). Enhanced Ca2+ cycling amplifies the force of contraction and rate of relaxation, substantially improving cardiac performance. Timely and specific phosphorylation of PKA substrates is made possible by scaffolding molecules called A Kinase Anchoring Proteins (AKAPs) which bind directly to PKA, phosphatases, ion channels, and other proteins and membranes to coordinate discrete molecular signaling complexes. Several AKAPs are expressed in the heart. Disruption of all AKAP‐PKA binding abolishes the effect of sympathetic stimulation on Ca2+ cycling. Our work utilizes AKAP knockout/mutant mice to identify specific AKAP(s) that are essential for CaV1.2 and PLB/SERCA regulation. Genetically targeted AKAPs include AKAP7 (15/18), AKAP5 (79/150), and AKAP1.
Acute and long‐term effects of aldosterone on vascular remodeling
Authors: Eatman Danita , Peagler Katie , Watson Jana , Bayorh Mohamed A
Published: June 2021

We have previously demonstrated that aldosterone‐induced upregulation of cyclooxygenase (COX) expression in the kidney is blocked by eplerenone. The objective of this study was to determine whether ALDO causes biochemical and molecular remodeling of the vasculature. The effects of aldosterone on arachidonic acid metabolism and on the expression of thromboxane (TXA2) receptor (TP) were evaluated in the Dahl salt sensitive (DS) rat aorta, renal, femoral and carotid arteries. DS rats on a low salt (0.3% NaCl) diet were treated with or without ALDO for four weeks. Indirect blood pressure (BP), the release of TXA2, prostacyclin (PGI2) and prostaglandin E2, and the expression of COX‐2 and TP were measured. ALDO increased BP (17± 1%) and the basal release of PGE2 but reduced the basal release of TXA2. ALDO enhanced vascular reactivity to phenylephrine and up regulated the expression of COX‐2 in both aorta and renal vessels but reduced COX‐2 expression in the femoral artery. The TP expression was down regulated in the aorta but up regulated in the carotid by ALDO. These data reveal that the effect of ALDO in the vasculature is tissue specific and involves the release of vasoconstrictor prostanoids in the rat aorta by up regulating the expression of COX‐2 and TP in the vasculature. Therefore, it can be stated that the prostanoids play a pivotal role in the vasculopathic effect of aldosterone. Supported by NIDDK grant 1SC1DK082385.
Moderate increases in plasma cholesterol blunt the inflammatory response to salt‐induced hypertension
Authors: Rodrigues Stephen , Granger D. Neil
Published: June 2021

Although an increased leukocyte and platelet adhesion is observed in cerebral venules of mice with either hypertension (HTN) or hypercholesterolemia (HCh), it remains unclear whether the combination of HTN and HCh exerts a comparable effect on leukocyte and platelet recruitment in the cerebral microvasculature. We examined whether HCh alters platelet and leukocyte adhesion in cerebral venules of hypertensive mice. A 50 mg DOCA pellet was implanted s.c. in uninephrectomized C57Bl/6 mice placed on 1 % saline for a period of 3 weeks. A 3‐week HCh diet did not alter blood pressure either alone or in combination with HTN. Donor platelets labeled with CSFE were administered i.v., along with rhodamine, for in vivo observation of platelets and leukocytes using intravital microscopy. The HTN‐induced recruitment of adherent leukocytes in cerebral venules was blunted by the presence of HCh. Platelet adhesion was unaffected by either HTN, HCh or a combination thereof. The role of HDL cholesterol in the leukocyte adhesion response was assessed using human apolipoprotein A‐I transgenic mice (TGApoA1). No significant differences in leukocyte adhesion were observed between TGApoA1 mice ± HTN. Our findings indicate that moderate increases in total plasma cholesterol blunt the pro‐inflammatory phenotype that is induced in brain by DOCA‐salt HTN.

Group
BP (mmHg)
Plasma Total Cholest. (mg/dL)
HDL (mg/dL)
Leukocyte Adhesion (per mm2)

Sham
104±2
70±3
40±4
29±12

HCh
104±1
111±11*
64±6
67±24

HTN
138±2*
85±5
55±2
129±15*

HTN/HCh
140±3*
120±7*
77±6*
72±9

TGApoA1
100±2
175±9*
148±1*
60±15

Sham

HTN
118±5*#
258±9*#
212±9*#
110±16*

TGApoA1

P<0.05 vs Sham,

P < 0.05 vs TGApoA1 Sham. (Supported by HL26441).
Signs of aging‐related inflammation and adaptive reserves of aged mesenteric lymphatic vessels
Authors: Gashev Anatoliy A. , Nagai Takashi , Akl Tony , Thangaswamy Sangeetha , Chatterjee Victor , Cote Gerard , Bridenbaugh Eric A.
Published: June 2021

Adequate adaptation of mesenteric lymphatic vessels (MLV) to various volumetric loads is vital for whole‐body fluid/macromolecule homeostasis, lipid adsorption, and immunity. Such adaptive reserves are greatly diminished in aged MLV due to severe alterations in their stretch‐ and shear‐induced contractile responses. We recently found signs of shear‐independent, NO‐dependent depletion of contractility in MLV isolated from adult (9 mo old) and aged (24 mo old) F‐344 rats. Subsequent experiments with high‐speed imaging of aged MLV in vivo provided additional evidence of significant aging‐induced contractile depression in this lymphatic network. Results of experiments with non‐selective nitric oxide synthase (NOS) inhibition by L‐NAME, performed both in and ex vivo, support the idea of constant activation of inducible NOS (iNOS) in aged MLV. These findings correlate with elevated immunohistochemical labeling of iNOS in aged MLV and with the biochemical signs of a chronic inflammatory environment in and around of aged lymphatic wall. Cumulatively, these findings demonstrate a link between age‐related inflammation and functional disturbances in aged MLV, which leads to their diminished ability to adapt effectively to normal and potential abnormal volumetric challenges in elderly. NIH RO1 AG030578, HL094269.
The Role of Potassium Voltage Gated Kv 1.2 Channels in Coronary Metabolic Dilatation
Authors: Ohanyan Vahagn A , Bratz Ian N , Kolz Christopher L , Guarini Giacinta , Luther Daniel J. , Yin Liya , Pung Yuh Fen , Chilian William M.
Published: June 2021

Kv channels are important for function of vascular smooth muscle. Coronary arterioles express members of the Kv1 channel family: Kv1.2, 1.3, and 1.5. These channels are redox and oxygen regulated which makes them possible transducers of metabolic dilation in the heart. Total knockout of Kv1.2 channels is lethal, which may reflect their importance coupling metabolism to flow. Thus, we hypothesized that Kv1.2 channels are critical for coronary metabolic dilation. To investigate this, we studied the relationship between cardiac work and myocardial blood flow in genetically modified mice heterozygous null for the Kv 1.2 channel (Kv 1.2 +/−) and their wild type controls (Kv1.2+/+). Cardiac work (stroke volume [SV] X heart rate [HR] X mean arterial pressure [MAP]) was measured during infusion of norepinephrine (NE 0.5, 1.0, 2.5 and 5.0 μg/kg/min, iv.). MAP and HR were measured using a Millar transducer, SV via echocardiography and MBF using contrast echocardiography. At baseline MBF was equivalent in Kv 1.2 +/− mice and WT. At high levels of cardiac work produced by NE, the increase in MBF was 42%±6 less in Kv 1.2+/− mice compared to WT (P<0.05). Interestingly at high doses of NE, cardiac function deteriorated in the Kv 1.2+/− mice suggesting that flow was inadequate to match the metabolic needs of the myocardium. Based on our data, we conclude that Kv 1.2 channels play a key role in coupling myocardial metabolism to MBF.
Receptor for advanced glycation end products is involved in remodeling of diabetic coronary arterioles
Authors: Trask Aaron J , Katz Paige S , Stewart James A , Lucchesi Pamela A
Published: June 2021

The aim of this study was to determine whether the receptor for advanced glycation end products (RAGE) contributes to remodeling of type 2 diabetic coronary arterioles (<150 μm diameter) of leptin‐receptor deficient db/db mice. Coronary arterioles isolated from control Db/db, type 2 diabetic db/db, and RAGE−/− × db/db mice at 16 weeks of age were mounted on a pressure myograph to measure and record wall thickness and diameters over a range of pressures (0–125 mmHg). Coronary arterioles isolated from db/db mice exhibited decreased luminal diameter (Db/db: 118 ± 5 μm vs. db/db: 103 ± 4 μm p < 0.05) with thicker microvascular walls (Db/db: 6.9 ± 0.6 μm vs. db/db: 9.7 ± 0.5 μm p < 0.01), which significantly increased the wall:lumen ratio (Db/db: 6.0 ± 0.8 vs. db/db: 9.5 ± 0.4, p < 0.01). Compared to db/db mice, coronary arterioles isolated from RAGE−/−x db/db mice had similar internal diameter (104 ± 9 μm, p > 0.05), while wall thickness (6.1 ± 0.5 μm, p < 0.001), wall:lumen ratio (6.2 ± 0.8 μm, p < 0.01), and growth index (db/db: 28.1 vs. RAGE−/−x db/db: 22.3, % of control) were reduced. External diameters were not different among groups, although there was a tendency for reduced external diameter in RAGE−/−x db/db. These data suggest that the RAGE receptor is at least partially responsible for inward hypertrophic remodeling observed in coronary arterioles of type 2 diabetic mice.

Support: NIH T32HL098039 to AJT; NIH HL056046 to PAL.
Redox Regulation of Arterial L‐Type Calcium Channels
Authors: Amberg Gregory C , Frey Madeline M
Published: June 2021

Arterial walls are largely composed of smooth muscle cells. By contracting or relaxing, these cells regulate arterial diameter, thus influencing blood flow and blood pressure. The concentration of calcium in arterial smooth muscle largely determines the degree of arterial contraction. The main source of calcium entry into these cells is through voltage‐gated L‐type calcium channels. We recently reported that local NADPH oxidase‐mediated stimulation of PKC alpha‐dependent L‐type calcium channel activity (“calcium sparklets”) results in increased calcium influx and arterial contraction. However, details regarding the molecular mechanisms underlying redox‐dependent regulation of L‐type calcium channels are poorly understood. Using a combination of TIRF microscopy and patch clamp electrophysiology, here we investigate detailed aspects of redox‐dependent regulation of arterial L‐type calcium channels including experiments examining the molecular composition of the NADPH oxidase complex involved as well as determining the identity of the participating reactive oxygen species. Preliminary data suggest that Rac1‐dependent NADPH oxidase activity results in the generation of hydrogen peroxide that in turn stimulates PKC alpha‐dependent L‐type calcium channel function. This work was supported by the Pew Scholars Program (GCA).
The cardiac KATP channel associates with the glycolytic enzyme complex
Authors: Hong Miyoun , Kefalogianni Eirini , Bao Li , Malester Brian , Delaroche Diane , Neubert Thomas A , Coetzee William A
Published: June 2021

Cardiac ATP‐sensitive potassium (KATP) channels are primarily gated by high‐energy nucleotides, a property that allows them exquisite sensitivity to changes in intracellular energy metabolism. An emerging view is that the KATP channel is a multi‐subunit protein complex, and that the associated proteins provide an additional layer of regulation. Using bioinformatic approaches, we identified putative coiled‐coil (CC) domains in SUR subunits. We utilized these CC‐domains as baits in a two‐hybrid screen against a rat cardiac cDNA library and identified two glycolytic enzymes (GAPDH and aldolase A) as putative interacting proteins. Interaction between aldolase and the SUR CC‐domain was confirmed using GST pull‐down assays. We further demonstrated the interaction with full‐length SUR subunits with co‐immunoprecipitation assays using heterologous expression assays. Mass spectrometry was performed with KATP channel subunit immunoprecipitates obtained from rat membrane fractions, which identified glycolysis as the most enriched biological process. Using co‐immunoprecipitation approaches with rat cardiac membrane preparations, we confirmed interaction for several glycolytic enzymes (aldolase, enolase, fructose 6 phosphate kinase and hexokinase). These glycolytic enzymes were co‐localized with KATP channel subunits in isolated cardiac myocytes, as determined with immunocytochemistry. The catalytic activity of aldolase and pyruvate kinase inhibited KATP channel activity in inside‐out patch clamp experiments, whereas D‐glucose was without effect. Overall, our data demonstrate close physical association and functional interaction of the glycolytic process (particularly the distal ATP generating steps) with cardiac KATP channels.
Enhanced Ca2+‐activated Cl− conductance and TMEM16A expression in pulmonary arterial smooth muscle in a rat model of chronic hypoxic pulmonary hypertension
Authors: Sun Hui , Li Anlong , Paudel Omkar , Lo Alexander K. , Yang Xiao‐Ru , Sham James S.K.
Published: June 2021

Ca2+‐activated Cl− Channel (CaCC) encoded by TMEM16A plays an important role in the regulation of vascular tone. Here we sought to determine whether TMEM16A expression and CaCC currents (ICl.Ca) in pulmonary arterial smooth muscle cells (PASMCs) are altered in a rat model of chronic hypoxic pulmonary hypertension (CHPH). TMEM16A mRNA was quantified by qRT‐PCR and protein by immunoblotting. ICl.Ca was recorded using whole‐cell or perforated patch clamp technique under K+ free conditions. The TMEM16A mRNA was more abundant in pulmonary arteries (PA) compared to cerebral, tail, mesenteric and renal arteries in normoxic rats. In Fluo‐3/AM‐loaded PASMCs, caffeine‐induced Ca2+ release evoked robust outward currents at 0 mV (19.9±2.4 pA/pF) and inward current at −80 mV (−21.7±3.1 pA/pF). The current was inhibited by niflumic acid, and reversed at the predicted ECl following the changes of [Cl−] gradient, as consistent with ICl.Ca. Chronic hypoxia (10% O2, 4 weeks) caused an increase in ICl.Ca density evoked by caffeine (30.0 ± 6.8 pA/pF at 0 mV) or by depolarization with [Ca2+]i clamped at 750 nM (124.2 ± 24.1 vs. 24.4 ± 3.3 pA/pF at +120 mV) in PASMCs, accompanied by ~ 2‐fold increase in TMEM16A mRNA and 5‐fold increase in protein levels in PA. Our results show that TMEM16A and functional CaCC are highly expressed in rat PA. Their upregulation by hypoxia may contribute to the enhanced pulmonary vasoconstriction observed in CHPH.
Protein O‐GlcNAcylation Promotes Post‐hypoxic Survival of Cardiac Progenitor Cells
Authors: Zafir Ayesha , Li Qianhong , Bolli Roberto , Jones Steven P
Published: June 2021

Background

Cardiac progenitor cells (CPCs) represent a viable route to promote post‐infarct cardiac repair. We hypothesized that the auto‐protective stress signal, known as protein O‐GlcNAcylation, would confer a pro‐survival benefit in CPCs.

Methods/Results

We genetically and pharmacologically augmented O‐GlcNAc levels in CPCs (c‐kit+/lin−) with loss‐ and gain‐of‐function approaches, i.e., adenoviral (Ad) overexpression of OGT (adds the O‐GlcNAc modification to proteins) and inhibition of OGA (removes the O‐GlcNAc modification from proteins) by PUGNAc (0.2 mM). We also used DON (0.05 mM) as an inhibitor of the rate‐limiting enzyme of the hexosamine biosynthetic pathway, GFAT. CPCs were subjected to 3 h hypoxia, followed by a 3 h reoxygenation period. Augmenting O‐GlcNAc levels (via AdOGT or PUGNAc) significantly attenuated post‐hypoxic cell death (according to LDH release), whereas reducing O‐GlcNAc levels (via DON) augmented post‐hypoxic cell death.

Conclusions

Enhancing protein O‐GlcNAcylation represents a novel approach to minimize cell death in CPCs. Such approaches could be explored with the goal of improving CPC survival at the time of adoptive transfer to bolster the reparative capacity of CPCs in vivo.

Financial support: NIH, AHA.
Uncoupling effect of bafilomycin A1 on HIF and cell bioenergetics
Authors: Dmitriev Ruslan I , Zhdanov Alexander V , Papkovsky Dmitri B
Published: June 2021

The inhibitors of the V‐ATPase, bafilomycin A1 (Baf) and concanamycin A (CMA) are widely used drugs in cell physiology studies as well as pre‐clinical trials. We showed for the first time that Baf can uncouple mitochondria in vivo (acting as potassium ionophore) thereby decreasing the mitochondrial membrane potential, pH and Ca2+. At the same time, the observed effects of CMA were related to inhibition of V‐ATPase only. Here we demonstrate that such an uncoupling activity of Baf strongly affects cell bioenergetics and hypoxia‐dependent regulatory pathways in PC12 and HCT116 cells. We show that the mechanism of uncoupling by Baf differs from that of other potassium ionophore, valinomycin: cells, treated with Baf remain in state of “mild uncoupling”, having increased basal respiration and partially depolarized mitochondria for hours. The cells treated with Baf (but not with valinomycin) are capable to respond to FCCP treatment by further increase in respiration. Intracellular O2 levels were strongly decreased upon Baf treatment at hypoxia. In these conditions Baf treatment leads to stabilization of HIF1a in HCT116 or HIF2a in PC12 cells independently on V‐ATPase inhibition. Thus, treatment with Baf may trigger hypoxia‐specific gene expression, which leads to cell adaptation to the conditions of low O2 availability, and can have effects on cell bionergetics unrelated to inhibition of V‐ATPase. Supported by SFI.
Acute intermittent hypoxia alters cell fate choice in postnatal neural precursors
Authors: Ross Heather Hamilton , Sandhu Milap S , Sher Warren J , Cheung Tina F , Tiemeier Alexander J , Reynolds Brent A , Fuller David D
Published: June 2021

In vitro exposure of neural progenitor cells (NPCs) to relative hypoxia (e.g. 3% versus 20% O2) can increase their proliferation, survival and neuronal differentiation both in culture and following transplantation. Recently, chronic intermittent hypoxia (CIH) was shown to increase proliferation in the dentate gyrus and subventricular zone (SVZ)—sites of postnatal neurogenesis. Our objective was to determine if in vivo exposure to acute intermittent hypoxia (AIH) could alter expansion and differentiation of subsequent in vitro cultures of SVZ NPCs. Neonatal C57BL/6 mice (postnatal day 4) were subjected to AIH (20×1 minute; alternating 21% and 10% O2), and sacrificed within 15 minutes of protocol termination. SVZ tissue was isolated and NPCs cultured as neurospheres (NS) or adherent monolayers (MASC). Expression of proliferation and neuronal fate markers was assessed with a range of methods including immunocytochemistry, western blot, fluorescence‐activated cell sorting and PCR. Our preliminary results indicate that in vivo exposure to AIH increases expression of proliferation and neuronal differentiation markers as subsequently assessed in in vitro NPC cultures. These data suggest that AIH protocols may provide a means to “prime” NPCs prior to harvest for subsequent transplantation into the injured central nervous system. This work is supported by 5K12HD055929 (HHR); 1R01HD052682‐01A1 (DDF)
Intermittent hypoxia/hypercapnia alters the transcriptome of mouse coronary artery endothelial cells
Authors: Douglas Robert Mitchell , Zambon Alexander C , Makino Aya , Haddad Gabriel G
Published: June 2021

Intermittent hypoxia/hypercapnia (IHH) occurs in a variety of diseases, especially in sleep‐disordered breathing conditions (e.g. obstructive sleep apnea–OSA). IHH may contribute to the development of hypertension and atherosclerosis. To study the impact of IHH on global transcriptional remodeling, we exposed neonatal (NN) and adult C57BL/6 mice to IHH (8% O2; 8% CO2; 4 min cycles) for 4 wks. A recovery group of IHH‐exposed adult mice were returned to room air for 4 wks. Global mRNA levels of isolated coronary artery endothelial cells (ECs) were determined with microarrays. Clustering of differentially expressed genes revealed very little overlap of altered genes in adult and NN ECs (e.g. Notch 1 and Notch 4 were downregulated in NN ECs while Notch 3 was upregulated in adult ECs). In adult ECs three patterns of gene regulation were observed: 1) genes altered by IHH that returned to basal levels with reoxygenation (e.g. leukocyte migration and blood vessel development); 2) genes with sustained changes in IHH and reoxygenation (e.g. autophagy and antigen binding); 3) genes altered only by reoxygenation (e.g. immune response and DNA repair). In conclusion, IHH differentially affects the transcriptome of NN and adult ECs. IHH mediates distinct signaling pathways related to immune and cell stress responses in adult ECs. These changes may contribute to EC dysfunction and the pathophysiology of OSA.
The airway branch regulator, Sprouty2, represses vasculogenesis in fetal lung by direct interaction with the VEGF promoter
Authors: Land Stephen C , Walker David J , Scott Claire L
Published: June 2021

Lung development requires co‐ordinated growth of airway and vascular structures to form the branched network of tubes which supply the blood/gas barrier. We have shown that the inducer of airway outgrowth, Fibroblast Growth Factor‐10 (FGF‐10), promotes vascular signalling from fetal airway epithelium through Sprouty2 cleavage and consequent mTORC1‐dependent activation of HIF‐1α. However, fetal airway epithelium displays a nuclear sub‐population of Sprouty2 which associates with chromatin. We therefore hypothesised that uncleaved Sprouty2 represses HIFα interaction with the VEGF‐A promoter by competitive interaction with the hypoxia response element (HRE). Chromatin immunoprecipitation (ChIP) revealed that Sprouty2 interacts with the VEGF promoter in fetal airway epithelium by: 1) weak, FGF‐10‐dependent, binding to the HRE promoter region, and, 2) constitutive, FGF‐10‐independent binding to GC‐rich regions near the transcriptional start site. This interaction was inhibitory as modulation of Sprouty2 activity by siRNA knockdown or null (Y55F) mutation augmented HIF‐dependent VEGF‐A production. Moreover, immunoprecipitation revealed an FGF‐10‐dependent interaction between Sprouty2 and the HIF‐competitive inhibitor, HIF‐3α. We conclude that Sprouty2 represses HIF‐mediated VEGF‐A production in fetal lung by direct association with critical sequences within the VEGF‐A promoter.
MicroRNA‐21 plays a Role in Hypoxia‐mediated Pulmonary Artery Smooth Muscle Cell Proliferation and Migration
Authors: Sarkar Joy , Gou Deming , Ramchandran Ramaswamy , Raj Usha
Published: June 2021

Hypoxia stimulates pulmonary artery smooth muscle cell (PASMC) proliferation. Recent studies have implicated an important role for microRNAs (miRNAs) in hypoxia‐mediated responses in various cellular processes including cell proliferation. In this study, we investigated the role of miR‐21 in hypoxia‐induced PASMC proliferation and migration. We first demonstrated that miR‐21 expression increased by ~ 3‐fold in human PASMC after 6 h of hypoxia (3% O2) and remained high (~2‐fold) after 24 h of hypoxia. Knockdown of miR‐21 with anti‐miR‐21 inhibitors significantly reduced hypoxia‐induced cell proliferation, whereas miR‐21 over‐expression in normoxia enhanced cell proliferation. We also found that miR‐21 is essential for hypoxia‐induced cell migration. Protein expression of miR‐21 target genes, specifically, programmed cell death protein 4 (PDCD4), Sprouty 2 (SPRY2) and Peroxisome‐Proliferator‐Activated‐Receptor Alpha (PPARα) was decreased in hypoxia and in PASMC overexpressing miR‐21 in normoxia and increased in hypoxic cells in which miR‐21 was knocked down. In addition, PPARα‐3′UTR‐luciferase‐based reporter gene assays demonstrated that PPARα is a direct target of miR‐21. Taken together, our findings indicate that miR‐21 plays a significant role in hypoxia‐induced pulmonary vascular smooth muscle cell proliferation and migration by regulating multiple gene targets.
Genetic Variation of the Alpha Subunit of ENaC Influences Lung Diffusion during Peak Exercise
Authors: Baker Sarah Elizabeth , Wheatley Courtney M , Cassuto Nicholas A , Wong Eric C , Foxx‐Lupo William T , Snyder Eric M
Published: June 2021

Epithelial Na+ Channels (ENaC) regulate Na+ reabsorption from the alveolar airspace. Increased expression of ENaC has been shown to increase lung fluid clearance. Previous work has demonstrated that substitution of T→A at amino acid 663 of αENaC results in increased Na+ reabsorption. We sought to determine the influence of the αT663A variant of ENaC on lung diffusion with exercise in healthy subjects. 30 subjects (n=17 vs. 13; age=25±7 vs. 30±10yrs; ht=174±11 vs. 171±10cm; wt=69±12 vs. 73±14kg; VO2peak= 95±30 vs. 100±31%pred; mean±SD, wild‐type, WT (TT) vs. non wild‐type, NWT (AT, AA), respectively) were recruited. The diffusion capacity of the lungs for carbon monoxide (DLCO) and nitric oxide (DLNO), alveolar volume (VA), and alveolar‐capillary membrane conductance (DM) were measured at rest and peak exercise. The WT group showed a significantly greater increase in DLCO and DLNO from rest to peak exercise than the NWT group (DLCO=51±12 vs. 36±17%, DLNO=51±24 vs. 32±25%, p<0.05). The WT group also had a greater % increase in DLCO/VA from rest to peak exercise (49±26 vs. 33±26%, p=0.08). The increase in DM from rest to peak exercise was statistically similar, although higher in the WT group (45±21 vs. 32±26%). These results demonstrate that genetic variation of ENaC at amino acid 663 influences lung diffusion at peak exercise in healthy humans suggesting differences in alveolar Na+ and, therefore, fluid handling.
Exercise ancestry decreases lipogenesis‐related gene expression in skeletal muscle of male offspring
Authors: Guth Lisa M. , Ludlow Andrew T. , Witkowski Sarah , Marshall Mallory R. , Lima Laila , Venezia Andrew C. , Xiao Tao , Lee Mei‐Ling Ting , Spangenburg Espen E. , Roth Stephen M.
Published: June 2021

The study objective was to examine the influence of exercise (EX) ancestry compared to sedentary (SED) ancestry on offspring skeletal muscle gene expression. C57/Bl6 mice (F0) were exposed to EX (wheel running, N=20) or a SED condition (no wheel access, N=20) for 12 weeks prior to breeding. EX males and EX females, and SED males and SED females were bred to produce F1 pups. F0 EX mice had continued access to the wheel during breeding, pregnancy, and weaning. F1 pups were sacrificed at 8 wk without direct exercise exposure. Microarray analysis of gene expression was performed on pooled gastrocnemius RNA from F1 males of each ancestry (5 animals per array; 4 total arrays). After processing the microarray data to minimize false positives, 109 transcripts were differentially expressed between F1 EX and F1 SED. Three targets identified as upregulated in F1 SED offspring have been previously associated with lipogenesis and were selected for real‐time PCR validation (Adipoq, Scd1, and Cidec). Adipoq and Scd1 were verified as significantly upregulated in F1 SED offspring (p < 0.05) and Cidec tended to be upregulated in F1 SED offspring (p = 0.06). These preliminary findings indicate that exercise ancestry (i.e., parental exercise stimulus) can affect lipogenesis‐related skeletal muscle gene expression in male mouse offspring.

Supported by the UMD School of Public Health Research Seed Money Program and NIH AG000268.
The effect of vivo‐Morpholinos on Dopamine Receptor 1 (Drd1) and physical activity in mice
Authors: Ferguson David Paul , Wiggs Michael P , Prabhu Greeshma S , Fluckey James D , Lightfoot J. Timothy
Published: June 2021

It is suggested that mice exhibiting low physical activity levels overexpress Drd1 and this may be related to activity. This study aimed to evaluate if vivo‐morpholino (Gene Tools LLC) would knock down Drd1 expression and increase activity. Animal procedures were conducted in conformance with the FASEB Statement of Principles for the use of animals in research and education. Male C3H/HeJ (n=6 control; n=6 morpholino) mice (Jackson Labs) ran on wheels. After one week, the control group received a saline IV injection while the morpholino group received an IV vivo‐morpholino injection (11mg•kg−1) for three days. Four days following injection half of the cohort was sacrificed. Soleus and nucleus accumbens were harvested and analyzed on a western blot. The remaining mice were sacrificed and analyzed one week later. Four days post injection the morpholino group had a significant (p=0.03) knockdown of Drd1 in the soleus. There was no difference in Drd1 expression in the nucleus accumbens (p=0.33) nor was there a difference between the control and morpholino group twelve days post injection (p=0.53). There was no difference in wheel running between the control and morpholino group (p=0.42). The use of vivo‐morpholinos is effective in knocking down Drd1 in muscle but not in the nucleus accumbens potentially due to the blood brain barrier. Furthermore our data suggest that the vivo‐morpholinos gene knock‐down is transient.