bluuebunny/crossref_metadata_2025 huggingface dataset:
Targeting Lactate Efflux Inhibition for the Development of Potent Antiproliferative Agents
Authors: Messeha Samia , Badisa Ramesh , Mazzio Elizabeth , Zarmouh Najla , Gendy Sherif , Soliman Karam
Published: June 2021

In cancer cells, much of the pyruvate from glycolysis is directed away from the mitochondria to form lactate. Monocarboxylate transporters (MCT) mediate the fluxes of lactate and represent an attractive target to inhibit lactate shuttle that maintain tumor growth. In this study, we screened 906 ethanolic plant extracts for their ability to inhibit lactate efflux using Neuroblastoma (N2A) cell line. These extracts were tested at 4 different concentrations (1.0‐0.05 mg/ml) and the obtained results indicate that the plant extracts of Fructus Chebulae (Chinese herb used for cough) and Bupleurum Chinense (an East Asian herb used to treat infections) were very effective inhibitors of lactate efflux at the lowest concentration tested (0.05 mg/ml). Dose‐response curves for Fructus Chebulae and Bupleurum Chinense were established and the IC50 for these 2 plants extracts were 0.003mg/ml and 0.022 mg/ml, respectively. The plant extracts inhibition was more effective than the well‐known MCT inhibitor phloretin which caused inhibition to lactate production with IC50 at 0.048 mg/ml. The data obtained showed that Fructus Chebulae had an inhibitory growth of N2A with EC50 of 0.0038 mg/ml and for Bupleurum Chinense with EC50 of 0.022 mg/ml. Moreover, the effect of these 2 plants on primary rat astrocytes viability was examined and the obtained data indicate that Fructus Chebulae was safer than Bupleurum Chinense (EC50 were 0.018 and 0.021 mg/ml, respectively). In conclusion, the results indicate that the extract of Fructus Chebulae and Bupleurum Chinense were highly selective to inhibit lactate efflux and thus may also inhibit cancer cell proliferation. (Supported by NIH grants from NIMHD G12 MD007582 and P20 MD006738)
ARC Knockout Does Not Alter Apoptotic Enzyme Activities in Skeletal Muscle in Response to Angiotensin II Exposure
Authors: Campbell Troy , Mitchell Andrew , Mandur Talveer , Quadrilatero Joe
Published: June 2021

Although apoptotic signalling does not cause complete skeletal muscle fiber elimination, apoptotic protease activation contributes to contractile protein degradation. The anti‐apoptotic protein apoptosis repressor with caspase recruitment domain (ARC) inhibits both intrinsic and extrinsic apoptotic pathways. Interestingly, although ARC expression levels are relatively high in mature skeletal muscle, its importance in mediating apoptotic signalling is underexplored. Therefore, the purpose of this study was to examine if ARC knockout (KO) would alter apoptotic enzyme activities in response to angiotensin II (AngII) exposure. Wild‐type (WT) and ARC KO mice were implanted with a subcutaneous placebo or AngII‐releasing pellet. No differences in caspase‐3, ‐8, or ‐9 activity were detected between groups in red gastrocnemius; however, a trend (p=0.07) towards an interaction effect was observed for calpain activity. In white gastrocnemius, there was a significant increase in caspase‐3 activity with AngII treatment; however, no differences were seen between genotypes. Although a trend (p=0.06) towards decreased caspase‐8 activity with AngII treatment was observed, no significant differences were detected. A significant decrease in caspase‐9 activity with AngII treatment was also observed. Finally, an interaction effect was detected for calpain activity. In conclusion, apoptotic enzyme activities are not increased in ARC KO compared to WT mice in response to AngII exposure. This may be due to adaptations occurring during development which may counteract the absence of ARC.
The influence of thermal factors on postexercise haemodynamics in trained and untrained men
Authors: Meade Robert , Barrera‐Ramirez Juliana , Paull Gabrielle , Fujii Naoto , Lamarche Dallon , Kenny Glen
Published: June 2021

Previous work suggests that the cardiovascular mechanisms underlying the postexercise reduction in mean arterial pressure (MAP) differ between exercise‐trained and untrained men. However, these studies compared responses based on the same percentage of peak oxygen consumption (VO2peak) resulting in different rates of heat production during exercise. Thus, we evaluated if exercise‐induced differences in the requirement for heat loss, and therefore sweat loss, may influence postexercise haemodynamics. Twelve trained (T60) (VO2peak, 64 ± 4 mL/kg/min) and twelve untrained (UT60) (VO2peak, 49 ± 3 mL/kg/min) males, individually matched for body surface area, cycled for 60 min at 60% of VO2peak­. On a separate day the untrained group performed an additional 60 min of cycling at the same heat production as their trained match in T60 (UTMatch). Sweat loss was assessed by change in body weight (ΔBW). MAP and cardiac output (CO) were measured at baseline and every 10 min during 60 min of postexercise recovery. ΔBW was similar between T60 and UT­Match (T60: ‐1.32 ± 0.36 kg; UTMatch: ‐1.32 ± 0.32 kg) and greater in these conditions relative to UT60 (‐0.95 ± 0.13 kg; both P < 0.05). Further, MAP was ~5 mmHg below baseline following 20 min of recovery in each condition (all P < 0.05). Postexercise CO was ~7% below baseline in T60 (P < 0.05) whereas it was elevated relative to T60 (both P < 0.05) and similar to baseline (both P > 0.05) in UT60 and UTMatch. We show that the differing mechanisms between trained and untrained males underlying the postexercise reduction in MAP are not the result of differences in exercise‐induced heat load and therefore fluid loss. Support: Natural Sciences and Engineering Research Council of Canada.
Best Anesthetics for Assessing Left Ventricular Systolic Function by Echocardiography in Mice
Authors: Pachon Ronald , Scharf Bruce , Yan Lin , Vatner Dorothy , Vatner Stephen
Published: June 2021

Reviewing the literature of the major cardiovascular journals for the past three years, revealed that of all the studies using anesthesia for mouse echocardiography, the predominant anesthetic was isoflurane, which was used in 76% of the articles. The goal of this investigation was to determine if isoflurane is indeed the best anesthetic. Accordingly, we compared this anesthetic with tribromoethanol, ketamine/xylazine, and ketamine on different days in the same mice also studied in the conscious state. A randomized crossover study design was employed to compare the effects on left ventricular (LV) systolic function and heart rate of the 4 different anesthetic agents assessed by echocardiography and compared with values obtained in the conscious state. Fourteen mice (C57BL/6J, 129SVJ, FVB/N) underwent high resolution echocardiography. As expected each anesthetic depressed LV ejection fraction and heart rate compared with values in conscious mice. Surprisingly, isoflurane was not the best, but actually second to last in maintaining normal LV function and heart rate. The anesthetic with the least effect was ketamine alone at a dose of 150 mg/kg, which induced a surgical level of anesthesia, followed by tribromoethanol, 290 mg/kg, isoflurane, 3% induction and 1‐2% maintenance, and lastly ketamine/xylazine, 100/10 mg/kg.In summary, these results indicate that ketamine alone exerts the least depressant effects on LV function and heart rate, with avertin second; suggesting that these anesthetics should be used, when it is not feasible to study the animals in the conscious state, as opposed to the most commonly used anesthetic, isoflurane.
Intermittent Hypoxia Conditioning (IHC) and Delta Opioid Receptor (DOR) Expression in Canine Atrium
Authors: Estrada Juan , Mallet Robert , Downey H , Caffrey James
Published: June 2021

Extended hypoxia exposures make hearts vulnerable to damage and reportedly suppress DOR expression in cell models resulting in cell damage. However, 20 days of limited daily IHC produced a robust resistance to ischemia reperfusion injury in canine hearts (Exp Biol Med 2004;229:806‐812). DOR blockade with naltrindole during daily IHC abrogates this cardioprotection. Since DOR stimulation enhances cardioprotective cholinergic systems this study evaluated whether controlled IHC alters DOR, cholinergic and/or adrenergic systems to augment cholinergic control of the heart during ischemia reperfusion. Dogs underwent normoxic sham conditioning, IHC [5‐8 cycles of 5‐10 min hypoxia (9.5‐10% FIO2) + 4 min normoxia], or IHC + daily sc naltrindole before IHC, for 20 d. On day 21, atrial tissue was biopsied for immunoblot analysis of proteins. Relative to sham values, after IHC DORs trended lower but after IHC + naltrindole were higher relative to sham (p<0.05) and IHC (p<0.01). Since DOR blockade abrogates IHC mediated protection, these data suggest that DOR stimulation during IHC down‐regulates DOR with a compensatory increase after DOR blockade. Atrial tyrosine hydroxylase was largely unchanged following IHC, while multiple markers of cholinergic function were mixed, resulting in a potential increase in cholinergic influence.
Role of Toll‐Like Receptor 4 Activation in Mitochondrial Respiration in Mouse Liver and Heart
Authors: Pickering Brian , Sharda Daniel
Published: June 2021

Ischemia reperfusion (I/R) injury results in activation of Toll‐like receptor 4 (TLR4) in cardiomyocytes, which contributes to tissue loss. Inhibiting TLR4 upon I/R results in improved recovery, as, paradoxically, does pretreatment with lipopolysaccharide (LPS), an activator of TLR4. However, it is unclear how TLR4 affects cardiomyoctes during LPS pretreatment or I/R injury. One clue may come from the recent discovery that TLR4 activation in macrophages enhances production of mitochondrial reactive oxygen species (mROS) due to perturbations in complex I of the electron transport chain (ETC). Therefore, we hypothesized that TLR4 activation in cardiomyocytes would decrease complex I respiration. Furthermore, we hypothesized that TLR4 activation would lead to mitochondrial uncoupling suggestive of mROS. In this study, mitochondria were isolated from hearts and livers of CD1 mice that had been pretreated for 1 hour i.p. with 1mg/kg LPS or vehicle control (PBS), and states 1‐4 of complexes I, II, and IV were analyzed using a Clarke‐type electrode for respiratory affects. Here we report that LPS did not affect complexes I (p=0.38 for state 3), II (p=0.37), and IV (p=0.41) in the heart, or complex II in the liver (p=0.36), indicating that LPS pretreatment does not modify ETC activity. Moreover, mitochondrial coupling displayed similar values for the respiratory control index (RCI) and respiratory control ratio (RCR) suggesting ROS production is unlikely to be affected by LPS pretreatment. Together, these data suggest that any pretreatment affects conferred by LPS at one hour are separate from the ETC, and remain a topic for future investigation.

This work was funded by the Elbert Pence and Fanny Boyce Undergraduate Summer Research Grant.
LRRC8A (IClswell) Anion Channels Modulate TNFα‐Dependent Activation of NF‐κB
Authors: Ettinger Nicholas , Choi Hyehun , Nguyen Hong‐Ngan , Lamb Fred
Published: June 2021

Leucine‐Rich Repeat‐Containing 8 (LRRC8) family members are essential components of IClswell, the evolutionarily‐conserved anion conductance that modulates cytoplasmic volume in response to cell swelling. Our previous work has demonstrated that the 2Cl‐/H+ anti‐porter ClC‐3 is required for TNFα‐induced endosomal superoxide (O2•) production by Nox1, as well as for subsequent activation of IClswell and NF‐κB in both HEK293 and vascular smooth muscle cells (VSMC). We hypothesized that IClswell activation by TNFα is a required step in NF‐κB signaling. Immunostaining of expressed LRRC8A and Nox1 demonstrated co‐localization in cytoplasmic vesicles of HEK293 cells. An NF‐κB‐luciferase reporter construct demonstrated a dose‐dependent decrease in NF‐κB activation by TNFα (10ng/ml) in the presence of the IClswell inhibitors tamoxifen (85±8% at 50μM) or DCPIB (64±6% at 50μM). Targeting LRRC8A expression with siRNA reduced NF‐κB activation by 64±2%. Similar effects of drugs and siRNA were observed in murine aortic VSMCs. N2‐cavitated HEK293 cell lysates were cleared of nuclei and mitochondria (10,000g, 15 min). These lysates made O2• in the presence of NADPH (0.5mM) and lucigenin (5μM). O2• production was inhibited by DPI (75±10%, 10μM), but enhanced by DCPIB (40±17%). These data suggest that endosomal IClswell activity is required for TNFα signaling. We hypothesize that IClswell is present in the endosomal membrane and acts as a pathway by which O2• release mediates local negative feedback to regulate Nox1 activity and O2•‐dependent inflammatory cell signaling.

This research was supported in part by an NICHD training grant to the Department of Pediatrics at Vanderbilt University Medical Center (T32 HD068256‐03).
Shear‐Induced Extracellular Regulated Kinase Signaling to eNOS is Increased When Autophagy is Compromised in Endothelial Cells
Authors: Ruan Ting , Bharath Leena , Mueller Robert , Goodrich Rebekah , Graham Timothy , Symons J David
Published: June 2021

Frictional forces (i.e., “shear stress”) exerted by flowing blood along the surface of the endothelium are potent stimuli for endothelial nitric oxide (NO) synthase (eNOS) phosphorylation and NO production. Recently we reported that shear‐induced increases in p‐eNOSS1177 (a positive eNOS regulatory site) and NO generation are prevented when the process of autophagy is disrupted in ECs via knockdown of AuTophaGy‐related protein 3 (Atg3) using siRNA. We sought to determine whether shear‐induced kinase stimulation / inhibition of eNOS is altered when autophagy is compromised in ECs. In ECs transfected with scrambled siRNA, shear stress (3 h x 20 dyn/cm2) increased (p<0.05) autophagy (e.g., ↑LC3‐II:LC3‐1, ↓p62, ↑LC3‐GFP puncta formation), p‐eNOSS1177, and NO production (assessed via DAF, EPR), but p‐eNOST495 (a negative eNOS regulatory site) was unchanged. In ECs with >85% knockdown of Atg3 protein expression via siRNA, shear–induced increases in autophagy, p‐eNOSS1177, and NO generation were prevented, but p‐eNOST495 increased (p<0.05). As such, shear‐induced increases (p<0.05) in p‐eNOSS1177 : p‐eNOST495 occur in ECs with intact but not compromised autophagy. Extracellular regulated kinase 1/2 (ERK1/2), which phosphorylates p‐eNOST495 in ECs, increased (p<0.05) in response to shear stress only after Atg3 siRNA. Interestingly, when ERK1/2 activity was inhibited (FR180204) after Atg3 siRNA, shear‐induced increases p‐eNOSS1177 : p‐eNOST495 were restored. Work in progress is exploring mechanisms whereby autophagy suppression might alter kinase signaling to eNOS.
Dyslipidemia Induces Endothelial Cell Stiffening and Alterations in Angiogenic Potential
Authors: Bogachkov Yedida , Oh Myung‐Jin , Baruah Jugajyoti , LeMaster Elizabeth , Adamos Crystal , Wary Kishore , Levitan Irena
Published: June 2021

Dyslipidemia is a modification in lipid composition that potentially causes adverse cardiovascular outcomes. An increase in oxidized low density lipoprotein (oxLDL), a common dyslipidemic condition, is considered to be detrimental. We show here that pathological levels of oxLDL induces endothelial cell (EC) stiffening and this stiffening is RhoA dependent. Furthermore, we tested the immediate downstream effector of RhoA, Rho kinase (ROCK). ROCK inhibition caused a complete abrogation of oxLDL induced EC stiffening. 7‐ketocholesterol (7KC), an important oxysterol component of oxLDL, has previously been shown to induce EC stiffening. We now extend our study to the inhibition of RhoA, which abolished the stiffening effect of 7KC. Previous studies have observed that oxLDL increases network formation in vitro. Here we now demonstrate that oxLDL increases migration and proliferation in vitro, and capillary formation in vivo. Similarly to oxLDL induced EC stiffening, the effects of oxLDL on angiogenesis were also inhibited by blocking RhoA and ROCK signaling. Additionally, PGPC and POVPC, components of oxLDL, were tested on capillary formation. We propose that oxLDL induced EC stiffening may play a potential role in angiogenesis under dyslipidemic conditions.
Association Between NADPH Oxidase Activity and NO Bioavailability in Human Blood Arteries of Hyperhomocysteinemic Patients
Authors: KALINOWSKI LESZEK , PLOSKA Agata , SIEKIERZYCKA Anna , DOBRUCKI LAWRENCE , WOJCIECHOWSKI JACEK , WOZNIAK MARCIN , ROGOWSKI JAN , DOBRUCKI IWONA
Published: June 2021

Nitric oxide (NO) deficiency is a hallmark of the vascular disorders induced by risk factors, including hyperhomocysteinemia (Hhcy). NADPH oxidase and eNOS are sources of reactive oxygen and nitrogen species (NROS). Hhcy can promote NROS stress leading to the endothelial dysfunction. Here we investigated the mechanism of NROS stress in the endothelium of patients with induced Hhcy.

Patients qualified for the carotid endarterectomy received 100 mg/kg p.o. methionine or placebo 6 h before surgery. Fragments of isolated carotid arteries were used in the study. NADPH oxidase activity was measured with lucigenin‐based assay. NO, O2‐ and peroxynitrite (ONOO‐) release from endothelial cells (ECs) were measured using electrochemical nanosensors. The expressions of eNOS and NADPH oxidase subunits were analyzed by Western blotting.

Hhcy group revealed significant increase of NADPH oxidase activity and the expression of total and membrane fractions of p47phox subunit. eNOS stimulation resulted in lower NO levels and increased production of both O2‐ and ONOO‐ in group with Hhcy. The presence of apocynin improved NO release in ECs with Hhcy. eNOS expression was significantly higher in the Hhcy ECs. The ratio of eNOS dimers to the total eNOS was significantly decreased in patients with Hhcy.

In conclusion, the disruption of dimeric structure of overexpressed eNOS contributes to overproduction of O2‐ and induction of eNOS‐dependent NROS stress in patients with Hhcy.
Role Of Caveolin‐1 In Signaling Pathways Involved In Angiotensin II‐Induced Activation Of Cerebral Vascular Hypertrophy And Remodeling
Authors: Umesalma Shaikamjad , Gerhold Thomas , Baumbach Gary
Published: June 2021

Angiotensin II (Ang II) is a major determinant of vascular remodeling and hypertrophy in the cerebral circulation during chronic hypertension, which presumably plays an important role in production of stroke. We previously examined the effects of Caveolin‐1 (Cav1) on Ang II‐induced structural alterations in cerebral arterioles and found that deficiency of Cav1 prevented cerebral arteriolar remodeling and hypertrophy. The goal of this study was to examine the signaling pathways initiated by Cav1 during Ang II‐mediated cerebrovascular changes. Cav1 deficient (Cav1‐/‐) and B6129SF2/J wild type (WT) mice were infused with either Ang II (1000 ng/kg/day) or saline via osmotic minipumps for 28 days. Systolic arterial pressure (SAP) was measured by a tail‐cuff method. Expression of Cav1, p‐EGFR and p‐Akt was determined in cerebral arteries by western blotting and in cerebral arterioles by immunohistochemistry. Infusion of Ang II significantly increased SAP in both WT (149±3 vs 119±4 mmHg in saline infused, P<0.05) and Cav1‐/‐ mice (147±2 vs 108±5 mmHg, P<0.05), thus deficiency of Cav1 did not affect Ang II‐induced hypertension. Ang II increased expression of Cav1 in cerebral arteries and arterioles in WT mice, confirming that Ang II stimulated activation of Cav1. Further more Ang II stimulated EGFR transactivation and phosphorylation of Akt expression significantly (P<0.05) in cerebral arterioles in WT, but not in Cav1‐/‐ mice. These results suggest that Cav1 participates in Ang II stimulated transactivation of EGFR and Akt, both of which may play important roles in Ang II‐induced vascular hypertrophy and inward remodeling.
Knockdown of Exocyst Sec10 in Mouse Skeletal Muscle Cells and Analysis of Insulin‐Stimulated GLUT4 Exocytosis
Authors: Lee Amanda , Ines Eden , Lui Vanessa , Fogelgren Ben
Published: June 2021

Type II diabetes is a metabolic disorder characterized by insulin resistance and high glucose in the blood. The GLUT4 receptor is the major glucose transporter that allows insulin‐stimulated glucose uptake into metabolic tissues such as adipose and skeletal muscle. In cultured adipocyte 3T3‐L1 cells, the eight‐protein exocyst complex has been shown to exocytose GLUT4 vesicles to the plasma membrane when stimulated by insulin. Our lab has extensively studied the activity of the exocyst trafficking complex in renal epithelial cells, and we have successfully generated a conditional knockout mouse for the exocyst subunit Sec10. Previous studies have been limited to cultured adipocytes, but skeletal muscles are responsible for taking in a higher percentage of glucose, and it has been shown that exercise helps maintain glucose levels to prevent the onset of type II diabetes.

To evaluate whether the exocyst also plays a role in GLUT4 regulation in skeletal muscle cells, we used shRNA to generate stable Sec10 knockdowns in C2C12 mouse skeletal muscle cells. After confirming the knockdown, we measured glucose uptake using two different methods. First, we measured the amount of glucose in the media of Sec10 knockdown and control cells over the course of 2 weeks. Second, we compared insulin‐stimulated glucose uptake using a fluorescent glucose analog, 2‐NBDG. Based on preliminary findings, knockdown of Sec10 in mouse skeletal muscle cells results in decreased glucose uptake. Ongoing work with skeletal muscle‐specific Sec10 knockout with our new mouse model will help with better characterization of the role of the exocyst in insulin signaling and GLUT4 exocytosis that may reveal new therapeutic targets for human type II diabetes and pre‐diabetic insulin resistance.
Mesenchymal Stem Cells (MSC) Improve Both Stenotic and Contralateral Kidneys in the Renovascular Hypertension
Authors: Oliveira‐Sales Elizabeth , Maquigussa Edgar , Varela Vanessa , Shimoura Caroline , Gomes Guiomar , Bergasmachi Cassia , Campos Ruy , Boim Mirian
Published: June 2021

Previous data showed that mesenchymal stem cells (MSC) improved the renal morphology and attenuated inflammation, microvascular rarefaction, fibrosis and proteinuria in the stenotic kidney induced by 2 Kidney‐1 clip (2K‐1C) model. Here we evaluated the effect of MSC on the contralateral kidney. Three weeks after left renal artery occlusion, fluorescently tagged MSC (2x105 cells/animal) were injected weekly into the tail vein of the 2K‐1C rats. Flow cytometry showed labeled MSC in the cortex and medulla of both kidneys. MSC significantly reduced blood pressure (BP) and proteinuria in 2K‐1C rats. Glomerular filtration rate (GFR) was reduced in the stenotic kidney and renal plasma flow (RPF) was diminishedin both kidneys. MSC treatment improved these parameters in both kidneys. Contralateral kidney presented altered medullary morphology, fibrosis and vascular loss and MSC improved the morphology and decreased the fibrotic areas. In contrast, the efficiency of MSC improving vascular rarefaction was not observed in the contralateral kidney. Thus, MSC therapy in the 2K‐1C model prevented the progressive increase of BP, improved renal function, morphology and reduced fibrosis in both stenotic and contralateral kidneys but it was more efficient improving vascular rarefaction in stenotic than in contralateral kidney. This therapy may be a promising strategy to treat renovascular hypertension and its renal consequences in the near future. Supported by CAPES.
P2X7R Antagonism Improves Renal Medullary Blood Flow and Oxygenation in Angiotensin II‐dependent Hypertension
Authors: Bailey Matthew , Menzies Robert , Mullins John
Published: June 2021

Kidney disease is a complex disorder with a genetic contribution. We have shown that the Fischer (F344) rat is more susceptible to hypertensive renal injury than the Lewis rat. Interrogation of microarray identified P2X7R as a candidate gene for injury susceptibility in F344 rats. Renal P2X7R were expressed in the endothelium of the preglomerular arteries and in the afferent arteriole, vasa recta and renal tubules. Infusion of angiotensin II for a 14‐day period did not increase the abundance of P2X7R in whole kidney homogenates but induced expression of the receptor in the vascular smooth muscle of the renal, interlobar and arcuate arteries, the site of vascular injury. P2X7R were also expressed in the glomerulus of hypertensive rats, in which increased numbers of macrophages were identified. The specific P2X7R antagonist AZ11657312, administered IV to angiotensin II treated rats, caused a dose‐dependent reduction in arterial blood pressure and increased blood flow in the renal artery and renal medulla, measured by Doppler flow and flux respectively. BOLD‐MRI also identified a transient decrease in renal R2* signal following P2X7R antagonism, consistent with improved oxygenation of the renal medulla. These data support the hypothesis that P2X7R activation exerts adverse vascular effects within the kidney. Acute antagonism of this receptor lowers blood pressure and increases renal perfusion/oxygenation and may offer a multi‐layered approach to treat kidney disease.

We thank the British Heart foundation for funding and AstraZeneca for AZ11657312.
Metabolic Consequences of Experimental Uninephrectomy
Authors: Arsenijevic Denis , Cajot Jean‐Francois , Scerri Isabelle , Montani Jean‐Pierre
Published: June 2021

Metabolic syndrome, dyslipidemia, obesity, diabetes and cardiovascular diseases may lead to reduced kidney function. Here we chose to test the converse: does reduced kidney function impact on lipid metabolism? Six‐week old male Sprague Dawley rats were submitted to either unilateral nephrectomy (UniNX) or sham surgery, and maintained on an isocaloric normal chow diet equivalent to 90% of ad lib feeding. We then compared blood and tissue parameters at 1, 2 and 4 weeks after surgery. Compared to sham‐operated animals, UniNX resulted in decreased body fat (measured by whole carcass analysis) and decreased fat pad weights (by anatomical dissection). This was associated with evidence of increased lipolysis (increased plasma levels of glycerol, increased mRNA expression of adipocyte triglyceride lipase and hormone‐sensitive lipase in white adipose tissues) rather than with decreased fatty acid synthase. The increase in lipolysis was not related to blood hormones insulin, ghrelin, corticosterone, T3 and leptin, which were similar between the two groups. However, it could be related to a low‐grade inflammation as evidenced by increased circulating levels of certain cytokines, such as interferon‐gamma, granulocyte macrophage colony stimulating factor and acylation stimulating protein, which have some lipolytic properties. Our study suggests that UniNX under slight caloric restriction is characterized by a mild phenotype with lipolysis and low‐grade inflammation, whose mechanisms remain to be determined. (funded by the Swiss NCCR‐kidney.ch)
MD2 may be a new target for the treatment of obesity‐induced cardiac injury
Authors: Qian Yuanyuan , Zhong Peng , Wang Yi , Liang Guang
Published: June 2021

Background

Obesity is strongly associated with cardiovascular diseases. Accumulating evidences have demonstrated a vital role of TLR4 signaling in the pathogenesis of obesity‐induced complications. Myeloid differentiation 2 (MD2), an obligate partner for TLR4, was required for LPS‐associated inflammation and TLR4 signaling activation. However, whether MD2 is also involved in the obesity‐associated inflammation is still unknown. The aim of this study is to investigate the involvement of MD2 in the pathogenesis of obesity‐induced cardiac diseases.

Methods

Cardiac H9C2 cells exposed to free fatty acid were pretreated L6H9, a specific MD2 inhibitor or MD2 silencing siRNA. Rats and APOE‐/‐ mice were given high fat diet for eight weeks and administrated with L6H9 by daily gavage.

Results

In H9C2 cells, FFA was found to induce inflammation, oxidative stress, ER stress, mitochondrial dysfunction, and ultimately cardiac hypertrophy, fibrosis, and apoptosis. Interestingly, all these alterations were significantly inhibited by pharmacological inhibition and genetic silence of MD2. In vivo, two obese models markedly induced cardiac inflammation and oxidative stress and ultimately lead to cardiac remodeling including cardiac disorganization, hypertrophy, apoptosis and fibrosis. All these molecular and pathological alterations were remarkably attenuated by the administration with MD2 inhibitors.

Conclusion

MD2 is involved in the pathogenesis of obesity‐associated cardiac remodeling via mediating FFA‐induced inflammation, and MD2 can be a therapeutic target in treating obesity‐induced complications.
Exercise Training Initiated Late After Myocardial Infarction is Related With Better Cardiac Function, Oxidative and Antioxidative Protein Expression
Authors: Almeida Simone , Claudio Erick Roberto , Mengal Vinicius , Oliveira Suelen , Merlo Eduardo , Podratz Priscila , Gouvea Sonia , Graceli Jones , Abreu Glaucia
Published: June 2021

To evaluate the effects of exercise training (ET) initiated early or late after myocardial infarction (MI) on cardiac dysfunction and ventricular remodeling in ovariectomized (OVX) rats. OVX Wistar rats were subjected to MI or Sham surgery and divided in SHAM, MI, MI+ET3d and MI+ET15d groups. ET was performed on a treadmill (5x/wk, 60 min/day, 8 weeks). Cardiac function was assessed by ventricular catheterization. Collagen deposition, myocyte hypertrophy and infarct size were assessed by histology. Catalase, SOD‐2, Gp91phox and AT1 receptor (AT1R) expression were evaluated by western blotting. Left ventricular end‐diastolic pressure increase in MI rats which was reduced only in the group MI+ET15d and +dP/dt decreased after MI and was improved by the training in both times compared with MI‐sedentary rats. AT1R and Gp91phox expression increase after MI. However, the increase in AT1R was prevented only by the training initiated 15 days after MI. Catalase expression was increased only in the group MI+ET15d compared with other groups. Therefore, ET initiated late after MI is related to a better cardiac function and oxidative and antioxidative protein expression in ovariectomized rats.
Circadian clock gene expression in human buccal cells: potential use as a biomarker for circadian rhythm disorders.
Authors: Speed Joshua , Hamrick William , Burch Mariah , Siddiqui Mohammed , Hyndman Kelly , Pollock David , Calhoun David , Young Martin , Pollock Jennifer
Published: June 2021

A lack of diurnal variation in blood pressure is a risk factor for end‐organ disease. Time‐of‐day‐dependent oscillations in biological processes are mediated by a number of oscillating transcription factors, collectively termed the “molecular clock,” that control transcription of various regulators of physiological function. In particular, the genetic disruption of BMAL1 is known to impair blood pressure rhythms in mice. The goal of this feasibility study was to determine if human buccal cell BMAL1 mRNA is expressed in a time‐of‐day‐dependent manner. Following IRB approval, we recruited 11 healthy, adult volunteers (7 females, 4 males; 23‐50 years old) who were not on any medications, and had normal sleep patterns. Volunteers were instructed to swab for buccal cells at 6 AM, 12 noon, 6 PM, and 10 PM for three consecutive days and to return the swabs after the third day for RNA extraction and subsequent analysis by qRT‐PCR. We consistently obtained greater than 1 μg of total RNA in all buccal samples from all participants. BMAL1 mRNA displayed a reproducible rhythmic pattern in 9 of the 11 volunteers over the 3 day period (e.g. Day 1, 6 AM 2248±413, 12 PM 1149±336, 6 PM 715±211,10 PM 904±385 copy number/μg total RNA). These data suggest that human buccal cells exhibit rhythms in the circadian “molecular clock,” and may prove useful as a biomarker for determining mechanisms of circadian rhythm disorders such as nocturnal hypertension.
S‐glutathionylation of Epithelial Sodium Channels in Neonatal Mouse Lung Regulates Alveolar Fluid Clearance in vivo
Authors: Trac Phi , Helms My
Published: June 2021

Hyperoxia causes oxidative stress and leads to a positive shift in the GSH/GSSG redox potential (Eh) in the lung. We hypothesize that an increase in the concentration of GSSG can post‐translationally modify lung epithelial sodium channels (ENaC) via S‐glutathionylation. We measured the Eh value of bronchoalveolar fluid (BAL) from postnatal day (PN) 10 C57Bl6 mice housed under room air (CTR; 21% O2) or hyperoxia (H85; 85% O2) and found that chronic H85 exposure elevates GSSG concentrations. In support of our hypothesis, single channel patch clamp analysis showed that GSSG (0.4 mM) decreases ENaC activity in alveolar type 2 cells (from 0.39 + 0.06 to 0.13 + 0.05). Real‐time X‐ray imaging illustrated that H85 and GSSG (0.4 mM) treatment significantly impairs the rate of lung fluid clearance compared to PN 10 controls; thus verifying the translational significance of single channel measurements. Predictive software analysis showed that a, b, and g‐ENaC subunits have conserved Cys thiols that can be directly S‐glutathionylated by GSSG. ENaC subunit pull‐down with biotinylated glutathione ethyl esters (BioGEE) confirms post‐translational modification of ENaC via S‐glutathionylation under hyperoxia‐induced oxidative stress. In conclusion, our data indicated that S‐glutathionylation of ENaC Cys thiols attenuates net salt and water reabsorption in the lungs. A better understanding of this novel regulatory pathway could lead to improved therapeutics for newborn infants under respiratory distress reliant on oxygen therapy.

Acknowledgements: We acknowledge support from the APS Short‐Term Research Education Program to Increase Diversity in Health‐Related Research Fellowship.
Molecular Dynamics Modeling of Sodium And Water Transport through a Claudin Pore
Authors: Yu Alan , Laghaei Rozita , Rosenthal Rita , Coalson Rob
Published: June 2021

Background

Claudin‐2 is a paracellular channel. Na and water flow through it driven by an osmotic or Na concentration gradient, suggesting that Na and water pass through a common pore and that their fluxes are frictionally coupled. We investigated the mechanism by molecular dynamics simulations.

Methods

The claudin pore was modeled either as a double cone or a cylindrical nanotube with narrowest diameter of 6.5 Å and 6 negatively charged sites in the middle of the pore (each ‐0.2e). A hydrostatic pressure gradient was applied via an additional constant force in the channel axis direction to a layer of water molecules parallel to the membrane interface. Simulations were performed using the CHARMM27 force field and the TIP3P water model, with full electrostatics was included.

Results

With an osmotic gradient, water molecules oriented within the pore in 2 water wires and exhibited directional flow with Pf= 4.7 x 10‐13 cm3/s and a water:Na flux ratio of 233. Increasing the magnitude of the negative charge increased permeability only when the charges were smeared evenly along the pore surface. Application of a Na concentration gradient, also drove water flow with water:Na flux of 14.6. Increasing the pore diameter to 7Å also increased water flow significantly by allowing 3 water wires to form.

Conclusions

Our findings confirm that water can permeate through claudin‐2 via a water wire mechanism, demonstrate that water permeability is dependent on the magnitude and distribution of intrapore charges and on the pore diameter, and provide a molecular explanation for the frictional coupling of Na and water fluxes within the pore.
Claudin‐2 Mediates Sodium and Water Transport through a Common Pore
Authors: Rosenthal Rita , Günzel Dorothee , Schulzke Joerg , Fromm Michael , Yu Alan
Published: June 2021

Background

The tight junction protein Claudin‐2 is mostly expressed in leaky epithelia like proximal kidney tubule and small intestine. Claudin‐2 protein abundance is increased in inflammatory bowel diseases like Crohn's disease, ulcerative colitis, and celiac disease. Former studies of our group revealed that claudin‐2 forms paracellular channels for small cations like sodium and potassium and also paracellular channels for water. Aim of this study was to analyze whether the diffusive transport of sodium and water occurs through a common pore of the claudin‐2 channel.

Methods

Wild‐type claudin‐2 and claudin‐2 mutants with substituted amino acids of the first extracellular loop were expressed in the tight epithelial cell line MDCK I, using a TetOff inducible system. Sodium and water permeability were measured in modified Ussing chambers.

Results

Neutralization of the negatively charged cation interaction site in the pore with the mutation D65N caused a reduction of both, sodium and water permeability. Steric blocking of the claudin‐2 pore by thiol‐reactive reagents was performed after substitution of the pore‐lining amino acids, isoleucine‐66 and serine‐68 with cysteine. Thiol‐reactive reagents caused a decrease in both, ion conductance and water permeability in these claudin‐2 cysteine mutants whereas wild‐type claudin‐2 was not affected. Plotting all data yielded a strong correlation of claudin‐2‐mediated sodium and water permeability.

Conclusion

Claudin‐2‐mediated sodium and water transport are loosely coupled and share a common pore lined by the amino acids of the first extracellular loop of claudin‐2.
Functional Significance of Glycosylation of Cl‐/oxalate exchanger Slc26a6
Authors: Thomson R. , Thomson Claire , Aronson Peter
Published: June 2021

The brush border Cl‐/oxalate exchanger, Slc26a6, plays a crucial role in enteric oxalate secretion. Mice devoid of Slc26a6 develop profound hyperoxalemia, hyperoxaluria, and calcium oxalate urolithiasis. Modifications such as glycosylation that alter Slc26a6 trafficking or function may therefore be important determinants of oxalate homeostasis. The study aims are to confirm Slc26a6 glycosylation and to characterize its functional significance. By use of glycosidic enzyme digestions and site‐specific mutagenesis of transfected human Slc26a6 expressed in OKP cells, we demonstrate that Slc26a6 is glycosylated and that carbohydrate residues are added via N‐glycosidic linkage at two distinct sites in the putative second extracellular loop. Biotinylation studies suggest that glycosylation is not absolutely essential for surface delivery but does dramatically affect the efficiency by which Slc26a6 is processed and delivered to the cell surface. Moreover, cell‐surface biotinylation trafficking studies indicate that rates of endocytic retrieval of enzymatically deglycosylated Slc26a6 are 2‐3 fold higher than that observed with fully glycosylated Slc26a6, further supporting a role for glycosylation in Slc26a6 trafficking. Finally, investigation of the intrinsic transport function of Slc26a6, assayed as Cl‐ gradient‐dependent uptake of [14C]oxalate normalized to cell‐surface expression, demonstrate that deglycosylation achieved through either mutation or enzymatic digest consistently reduces transport rates by up to 75%. In conclusion, we confirm that Slc26a6 is glycosylated and demonstrate that the presence of glycan residues at two glycosylation sites is critically important for both protein trafficking and oxalate transport function. NIH DK33793.
Heat shock protein 72 inhibits vascular calcification through a PPAR‐dependent pathway
Authors: Soong Ya‐Hue , Xu Jen , Zhuang Jun‐Cheng , Kong Tianqing , Lu Tzongshi
Published: June 2021

Cardiovascular disease (CVD) is the leading cause of mortality globally and caused approximately 600,000 people decease annually in the United States. Vascular calcification (VC) is a significant contributor to CVD mortality in patients with Chronic Kidney Disease and Coronary Heart Disease. We recently reported that heat shock protein 72 (HSP72) can be induced in human arteries and prevent VC. However, the molecular mechanisms of HSP72 involved in VC have yet to be elucidated.

Our results show that inducible HSP72 is significantly expressed following heat shock treatment and abolished by HSP72 siRNA, as well as HSP72 mediated anti‐calcification in HA‐SMCs. We next show that HSP72 gene is significant down regulated at HA‐SMCs and human arteries. In addition, we found that the peroxisome proliferator‐activated receptors γ (PPARγ) inhibitors (CCL4 (chemokine (C‐C motif) ligand 4) and FABP4 (fatty acid binding protein‐4)) and transcription factor AP‐1 (activator protein‐1) that involved in atherosclerosis, cellular transformation and cell death were up‐regulated in calcified human arteries. And. Our data shows that HSP72 formed a protein‐protein complex with PPARγ by co‐immunoprecipitation. Lastly, induction of HSP72 restored expression of the HSP72‐PPARγ complex and inhibited up‐regulation of FABP4, AP‐1 with reduction in calcification in our in vitro calcification model. These vasculo‐protective effects were abolished by HSP72 siRNA.

In conclusion, HSP72 is a powerful inhibitor of VC and exerts its vasculo‐protective effects by functioning as a molecular chaperon through PPARγ dependent pathway.
CRISPR‐Cas9 Genome Editing Of Human Cullin‐3 In Vivo Enhances The Expression Of Bona Fide Cullin‐3 Substrates
Authors: Agbor Larry , Quelle Frederick , Sigmund Curt
Published: June 2021

Human patients with de novo loss‐of‐function mutations in Cullin‐3 (Cul3) resulting in deletion of exon 9, exhibit severe early onset hypertension mediated in part by impaired ubiquitination of WNK4. The objective of this study was to determine if genome editing by the clustered regularly interspersed short palindromic repeats (CRISPR) and CRISPR‐associated (Cas9) technique could permanently introduce deletion and insertion mutations into Cul3 using human embryonic kidney cell line (HEK293T). We hypothesize that genome editing of Cul3 would result in a loss‐of‐function phenotype leading to accumulation of Cul3 substrates. Using a plasmid vector expressing Cas9 enzyme and a GFP selectable marker, CRISPR guide RNA specifically targeting exon 5 and 7 of endogenous Cul3 were cloned into the vector and confirmed by sequencing. HEK293T cells were transfected and positive cells were sorted for GFP by flow cytometry. Using polymerase chain reaction (PCR), Cul3 exon 5 and 7 loci were amplified, cloned into a pCRII‐TOPO vector and sequenced. We found that genome editing induced both monoallelic and biallelic mutations in Cul3 upstream of the protospacer adjacent motif (PAM) in several HEK293T clones. This resulted in a loss of Cul3 protein expression. Interestingly, loss of Cul3 protein expression correlated with accumulation of RhoA, WNK4, and Cyclin E, all bona fide Cul3 substrates. Further, immunocytochemistry staining using confocal microscopy demonstrated significantly enhanced RhoA expression in Cul3‐edited cells compared to wildtype. These studies show that genome editing of Cul3 results in loss‐of‐function phenotype. Future studies will focus on generating HEK293T cells lacking exon 9 of Cul3.
O‐GlcNAc Plays a Role in Nrf2 Regulation in the Myocardium
Authors: Dieter Brad , Johnson Emily , Medford Heidi , Miller Lindsey , Vella Chantal , Marsh Susan
Published: June 2021

The redox sensitive transcription factor NF‐E2–related factor 2 (Nrf2) protects against oxidative stress and its activity is aberrant in type 2 diabetes.. Acute increases in the protein posttranslational attachment of O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) to serine/threonine residues have been shown to mitigate oxidative stress responses in the heart. This suggests that O‐GlcNAc may play a role in Nrf2 signaling; however, the role of O‐GlcNAc modification on Nrf2 remains to be elucidated. We examined Nrf2‐mediated genes in a tamoxifen‐inducible cardiomyocyte‐specific OGT knockdown (OGTKD) mouse. We also augmented O‐GlcNAc in cardiomyocytes (H9C2 cell line) and examined Nrf2 protein levels and mRNA of Nrf2‐mediated genes. Immunoprecipitation demonstrated that Nrf2 is O‐GlcNAc modified in the heart. Our in vivo model showed that Nrf2 protein was increased (p<0.05) while mRNA of the Nrf2 gene‐product catalase was reduced in the OGTKD mouse(p<0.05). Conversely, our in vitro experiments demonstrated that increasing protein O‐GlcNAcylation in H9C2 cells increased transcription of the Nrf2‐mediate genes catalase, glutamate‐cysteine ligase (GCLM), and heme oxygenase 1 (HMOX1) (p<.05). These data provide evidence for O‐GlcNAc as novel post‐translational modification of Nrf2 and suggest that O‐GlcNAcylation plays a role in regulating Nrf2 signaling.
A Translational Kinetic Analysis of Rodent and Human Hearts Shows Species‐specific Differences and Increases in Monoamine Oxidase Activity with Obesity/Diabetes
Authors: Nelson Margaret , Katunga Lalage , Anderson Ethan , Kypson Alan
Published: June 2021

Cardiac monoamine oxidase (MAO) has been shown to have an etiological role in heart failure due to the mitochondrial toxicity of the reactive oxygen (ROS) and catechol‐aldehyde species (RCS) produced by this enzyme. A causal role for MAO in cardiomyopathy with obesity and type 2 diabetes has been proposed due to the elevated sympathetic tone that occurs in these conditions, although this has never been investigated in either experimental or clinical models. We performed a cross‐species kinetic analysis of MAO isoform‐specific (A & B) oxidation of dopamine (DA), phenylethylamine (PEA), serotonin (5‐HT), norepinephrine (NE), and Tyramine (TYR) in heart tissue from nondiabetic/control (Ctl) and obese/diabetic (ObD) mice (n=12), rats (n=12), and humans (n=46) using MAO‐derived ROS production as index. In mouse hearts, maximal MAO‐B activity >> MAO‐A, in rat hearts MAO‐A >> MAO‐B, while in humans the isoform activity appears to be equivalent. Maximal MAO activity (both A & B) was significantly higher in ObD mouse and rat hearts with TYR and DA substrates compared with Ctl. ObD mouse hearts also had higher rates of MAO activity with PEA. In human myocardium, MAO activity (both A & B) was 1.5‐fold and 3‐fold higher in ObD patients compared to Ctl patients with NE and DA, respectively. Cardiac MAO‐B activity with 5‐HT also was significantly greater in ObD patients. These translational findings suggest that in obese/diabetic hearts, increased MAO activity and the ROS/RCS derived from it may contribute to cardiomyopathy, and imply that targeting cardiac MAO may have therapeutic potential.
Calcineurin Alpha Inhibition Induces Nox2 Up‐regulation via NFκB
Authors: Williams Clintoria , Hoover Robert , Gooch Jennifer
Published: June 2021

Although essential for preventing transplant rejection, prolonged calcineurin inhibitor (CNI) use induces nephrotoxicity. We showed that mice lacking the alpha catalytic isoform of calcineurin (CnAα) have a phenotype that mirrors CNI nephrotoxicity. Other studies implicate NADPH oxidase‐2 (Nox2) in the pathogenesis. This study seeks to determine if CnAα and Nox2 function in a common pathway. To determine the mechanism of CNI inhibition‐mediated increases in renal Nox2, WT mice were treated with cyclosporine A (CsA) for 6 weeks. In addition, Nox2 expression and activity were examined in CnAα‐/‐ and CnAβ‐/‐ mice and renal fibroblasts. In kidneys, co‐IP reveals that CsA treatment reduces CnAα association with its binding partner, calmodulin. Consistent with previous data, CnAβ and calmodulin association is not observed. Additionally, reduced CnAα activation is accompanied by increased Nox2 expression and ROS levels, as assessed by Western blot and Amplex Red. Consistently, renal Nox2 expression and ROS levels are increased in CnAα‐/‐ mice but not CnAβ‐/‐. Surprisingly, NFAT transcriptional activity is unchanged in CnAα‐/‐ mice compared to WT and CnAβ‐/‐, as assessed by luciferase activity. However, increased NFκB phosphorylation is observed. In CnAα‐/‐ cells, NFκB inhibition with CAPE attenuates enhanced Nox2 expression and ROS levels. In conclusion, these data indicate that CsA‐induced CnAα inhibition and subsequent NFκB activation stimulates Nox2 up‐regulation. Therapeutic approaches using CNI that do not target CnAα could ameliorate oxidative damage associated with CNI nephrotoxicity.

NIH‐T32 (CRW), NIH‐R01 (RSH), VA‐MERIT (JLG)