bluuebunny/crossref_metadata_2025 huggingface dataset:
Direct inhibition by angiotensin II of insulin‐dependent glucose transport activity in mammalian skeletal muscle involves a ROS‐dependent mechanism
Authors: Diamond‐Stanic Maggie Keck , Henriksen Erik J.
Published: June 2021

Angiotensin II (Ang II) is elevated in hypertension and impairs insulin signaling and insulin‐stimulated glucose transport activity in muscle cell lines. No previous study has directly investigated how Ang II impacts insulin action in isolated mammalian skeletal muscle. We investigated the molecular actions of Ang II on insulin signaling and glucose transport activity in isolated skeletal muscle from female lean Zucker rats. Soleus strips were incubated with insulin (5 mU/ml) and/or Ang II (500 nM) for 2 hr. Ang II alone did not affect glucose transport or distal insulin signaling (Akt and glycogen synthase kinase‐3β (GSK‐3β)). Ang II inhibited insulin‐stimulated glucose transport (39%) and decreased phosphorylation of Akt Ser473 (37%) and GSK‐3β Ser9 (42%). Ang II did not affect phosphorylation of IRS‐1 Ser307 or p38 MAPK. We used the superoxide dismutase mimetic, tempol (1 mM), to determine if reactive oxygen species (ROS) contribute to Ang II‐mediated insulin resistance. Tempol partially reversed (42%) the Ang II‐induced inhibition of insulin‐stimulated glucose transport. These results indicate that Ang II inhibits distal insulin signaling and insulin‐stimulated glucose transport activity in isolated mammalian skeletal muscle, and this effect is partially mediated by ROS. Ang II may represent a factor that contributes to the etiology of both hypertension and insulin resistance in skeletal muscle.
Impact of diabetes on ceramides in skeletal muscle subcellular fractions
Authors: Zabielski Piotr , Blachnio‐Zabielski Agnieszka Urszula , Persson Xuan‐Mai , Lanza Ian R , Jensen Michael D , Nair K. Sreekumaran
Published: June 2021

Insulin has been shown to have major impact on muscle mitochondrial function. Recent data show that ceramide can decrease oxygen consumption and ATP production in isolated mitochondria. The aim of the present study was to determine whether insulin deficiency alters ceramide (CER) and its metabolites ‐ sphinganine (SPA), sphingosine (SPH) and sphingosine‐1‐phosphate (S1P) ‐ in mitochondrial (mt) and sarcoplasmic (src) fractions from quadriceps muscle of streptozotocin‐diabetic C57BL/6 male mice. We studied non‐diabetic controls (C); diabetic, insulin‐treated (D+); and diabetic, insulin‐deprived (D−). D− and D+ group received LinBit insulin implants, whereas C group received blank implants. Implants from D− group were removed 7 days prior to sacrifice. CER and its metabolites were measured in a tandem mass spectrometer connected to a UPLC system. Insulin deprivation increased CER in muscle src although decreased in mt. Insulin treatment normalized CER content in src, but further lowered CER content in mt, which was accompanied by elevation in mitochondrial SPH content. Changes in SPA and S1P levels in both the src and mt were non‐significant. In conclusion, diabetes and insulin have profound effects on CER concentrations in muscle subcellular fractions.

C
D−
D+

src
mt
src
mt
src
mt

SPA

Mean
0.34
nd
0.32
nd
0.34
nd

SEM
±0.04
±0.02
±0.04

SPH

Mean
0.44
1.63
0.61*
2.15
0.47
2.55*

SEM
±0.07
±0.23
±0.10
±0.14
±0.03
±0.21

S1P

Mean
0.32
1.14
0.27
1.06
0.23
0.91

SEM
±0.03
±0.2
±0.05
±0.14
±0.01
±0.1

CER

Mean
85.7
326.0
162.5*
267.3*
93.0
199.7*

SEM
±17.4
±38.4
±19.8
±24.0
±4.4
±11.2
Regulation of novel sites on AS160 by insulin and AICAR in human skeletal muscle
Authors: Sharoff Carrie Grace , Langlais Paul , Yi ZhengPing , Mandarino Lawrence J.
Published: June 2021

AS160 is an important mediator of both insulin and contraction‐stimulated glucose uptake in skeletal muscle. Phosphorylation by upstream kinases at several serine and threonine residues directly affects the function of this protein; however, the knowledge of this is limited to sites where phospho‐specific antibodies are available. To more effectively study the effects of Akt and AMPK on AS160 phosphorylation, we employed HPLC‐electrospray ionization hybrid (LTQ‐FT/ICR) tandem mass spectrometry techniques to identify additional serine and threonine residues that are phosphorylated in AS160. CHO/IR cells were transiently transfected with cDNA encoding full length wild‐type human AS160. 24 hours after transfection, serum starved cells were treated with insulin (n=2, 100nM, t=15min) or AICAR (n=3, 2mM, t=60 min). Top 10 data‐dependent HPLC‐ESI‐MS/MS analysis confirmed the identity of 18 phosphorylation sites, 13 of which are previously unidentified. AICAR increased pSer725 by 33±0.3% while insulin increased pSer787 by 46±0.2% (p<0.05) in these novel sites. Next, we assessed the effect of insulin on pSer787 in vivo in human skeletal muscle. Basal and 2‐hr insulin stimulated muscle biopsy samples from lean individuals were analyzed. Insulin increased pSer787 by 60%. These data show that insulin and AICAR independently regulate phosphorylation at Ser787 and Ser725, respectively.
Effect of elevated nutrient availability on intracellular balance redox and insulin resistance in skeletal muscle
Authors: Silveira Leonardo Reis , Pinheiro Carlos Hermano , Alberici Luciane Carla , Fiamoncini Jarlei , Hirabara Sandro , Carneiro Everardo Magalhaes , Barbosa Marina Rodrigues , Curi Rui
Published: June 2021

Insulin resistance is focused on disordered lipid metabolism. This study evaluated the effects of elevated nutrient availability on intracellular balance redox and insulin resistance. The rat soleus muscles were isolated and pre‐incubated in krebs medium during 4h containing glucose 5,6 mM at presence of either palmitate (750 uM) plus insulin (100 μU/mL) or insulin (100 μU/mL). The muscles were also treated with N‐Acetylcysteine (NAC) (1 mM) or electrically submitted to muscle contraction (60 min). The medium was wash‐out and the muscle incubated in krebs ringer containing glucose 5,6 mM at presence of insulin (100 μU/mL) during 60 min. The oxygen consumption; ROS; uptake of 2‐deoxyglucose (2‐DG); NAD+/NADH ratio; mRNA of PGC1α and SIRT1 and glycogen synthesis were evaluated. The O2 consumption and glucose uptake were significantly reduced at presence of palmitate plus insulin (39.9±1U vs 57.1±0.1, p<0.05) and (195±3 vs 100±2%, p<0.05). The glycogen content was also reduced in this conditions (100±1 vs 65±1%, p<0.05). The NAD+/NADH ratio as well as mRNA of PGC1α and SIRT1 were reduced at presence of palmitate plus insulin (p< 0.05). The nitrated protein level at presence of palmitate plus insulin (p< 0.05) was increased (p<0.05). The antioxidant NAC partially improved glucose uptake compared with control (200±2 vs 100±3%, p<0.05). Whereas, muscle contractions increased glucose uptake compared with control (305±5 vs 100±3%, p<0.05). Our results showed that elevated nutrient availability reduce mitochondrial oxygen consumption favoring ROS production and consequently insulin resistance.
Effect of arginase on NO‐mediated protein modifications in endothelial cells
Authors: Krotova K. , Zharikov S. , Block E. R. , Patel J. M.
Published: June 2021

It is known that arginase is able to modify Nitric Oxide (NO) production through competition with NO synthase for the common substrate L‐arginine. We hypothesize that by modifying NO production arginase may affect NO‐mediated protein modifications – S‐nitrosylation and nitration. We studied NO production and arginase activity in human endothelial cells (EC) exposed to hypoxia. We found that the exposure of EC to 1% O2 for 24h induces an increase in NO production and an elevation in the expression of arginase II (Arg II) isoform which localizes in mitochondria. To study the effect of Arg II on S‐nitrosylation and nitration we used the arginase‐specific inhibitor BEC. After exposure of EC to hypoxia with or without BEC, nitrated proteins were precipitated with nitro‐tyrosine antibody, and then nitration of individual proteins was analyzed by Western blotting. In another set of experiments S‐nitrosylated proteins in cell lysates were analyzed by biotin‐switch assay. We found that the treatment of EC with BEC induced remarkable nitration of the mitochondrial protein cytochrome c oxidase (COX) both in normoxia and hypoxia. For non‐mitochondrial protein GAPDH treatment with BEC upregulated both S‐nitrosylation and nitration only in hypoxic conditions. Hence, we report the ability of Arg II to modify NO‐mediated protein modifications in EC.

This work was supported by Florida DOH (KK), VA and NIH (JMP).
Novel role for Caveolin‐1 in the regulation of eNOS‐derived Superoxide
Authors: Karuppiah Kanchana M , Cardounel Arturo
Published: June 2021

Under conditions of tetrahydrobiopterin (BH4) depletion, recombinant eNOS has been shown to generate superoxide, ·O2‐, a phenomenon termed “eNOS uncoupling.” In contrast, studies from hyperglycemic endothelial cells have demonstrated that increased BH2 rather than loss of BH4 is the molecular trigger for eNOS uncoupling. These results suggest that the endothelium imposes regulatory process that prevents oxidase activity from BH4‐free eNOS. Therefore, studies were carried out to identify the endothelial mechanisms for suppression of eNOS uncoupling from BH4 depleted cells. Endothelial cells were depleted of BH4 using the GTPCH1 (rate limiting enzyme in BH4 synthesis) inhibitor DAHP. Results demonstrated that DAHP treatment resulted in undetectable levels of BH4 in the absence of any eNOS derived ·O2‐. We hypothesized that cav‐1 may sequester BH4 free eNOS and prevent catalytic activity as cav‐1‐eNOS association was increased 2‐fold. In support, DAHP treatment of cav‐1 null endothelial cells significantly increased eNOS derived ·O2‐ to values near those of hyperglycemic cells. The combination of cav‐1 deletion and Akt inhibition unmasked the cellular inhibitory effects on eNOS uncoupling with rates of eNOS‐derived ·O2‐ reaching those observed in the hyperglycemic group. These studies demonstrate a new role for cav‐1 in the regulation of eNOS‐derived ·O2‐.
Endothelial KCa ion channels: their compartmentation in caveolae and relevance to cardiovascular pathologies
Authors: Kaistha Anuradha , Brähler Sebastian , Kaistha Brajesh Pratap , Wulff Heike , Köhler Ralf , Hoyer Joachim
Published: June 2021

Ca2+‐activated K+‐channels KCa2.3 and KCa3.1 are expressed in caveolae rich endothelium and their activation plays an important role in EDHF‐dilations. Yet, specific localization of these channels and role in EDHF and hypertension are not fully understood. Here we characterized caveolar compartment to study localization of these channels in endothelium. Effect of caveolin‐1 loss on functioning of these channels was studied using Cav‐1 deficient mice, and furthermore, to clarify specific contributions of these channels, we generated mice deficient of both channels. Sucrose density gradient and immunofluorescence revealed differential localization of endothelial KCa2.3 and KCa3.1 channels into caveolar and non‐caveolar compartments, respectively. Pressure myography showed that KCa3.1 deficiency has severe impact on acetylcholine‐induced EDHF‐dilation, whereas KCa2.3 deficiency impairs shear stress dilation. KCa3.1/KCa2.3‐deficient mice exhibited elevated blood pressure, most prominent during physical activity, as determined by telemetry. Cav‐1 deficient mice, exhibited impaired EDHF‐dilations in response to both, Ach and shear stress. We conclude that these endothelial channels are important in the control of vascular tone and overall circulatory regulation. Loss of caveolae results in impaired functioning of these channels and thereby contributes to cardiovascular pathologies.
Endothelial ephrin‐B2 is essential for acute arterial vasodilation and ischemic recovery in adult mice
Authors: Wang Rong A. , Lin Yuankai , Jiang Weiya , Ng Jennifer , Jina Asmita
Published: June 2021

Arterial vasodilation is critical for acute increases in tissue perfusion, yet molecular mechanisms of arterial‐specific dilation are unknown. Here we show that the cell surface protein ephrin‐B2, expressed in arteries but not veins, is a critical regulator of arterial dilation. Mice lacking the ephrin‐B2 gene specifically in endothelial cells were defective in acute arterial vasodilation in response to increased flow and acetylcholine. The protein, not RNA, levels of endothelial nitric oxide (NO) synthase (eNOS) along with its product NO, a vasodilator, were diminished in mutant arteries, but not veins. Ischemic recovery after femoral artery occlusion was also impaired in the mutants. S‐nitrosoglutathione, an NO donor, alleviated both the vasodilatory and ischemic protection defects in mutant mice. Thus, our data suggest that ephrin‐B2, potentially acting through eNOS, is a specific regulator of arterial dilation that is necessary to protect against ischemia.

This work was supported by NIH R01 HL075033 to R.A.W., NIH T32 CA009043 to Y.L., and by a generous grant from the Foundation for Accelerated Vascular Research.
Inhibitory effect of vitexicarpin TNF‐α‐induced vascular inflammation in human umbilical vein endothelial cells
Authors: Lee So Min , Lee Yun Jung , Kim Jin Sook , Kang Dae Gill , Lee Ho Sub
Published: June 2021

Vascular inflammation is pivotal event in the pathogenesis of many human diseases, including atherosclerosis. Recent pharmacological studies have demonstrated that vitexicarpin, flavonoid isolated from Vitex rotundifolia, was found to show potent inhibition against lymphocyte proliferation and inflammation. In this study, we investigated whether vitexicarpin prevented tumor necrosis factor‐alpha (TNF‐α)‐induced vascular inflammation in primary cultured human umbilical vein endothelial cells (HUVEC). We found that pretreatment with vitexicarpin decreased TNF‐α (10 ng/ml)‐induced of cell adhesion molecules (CAMs) such as vascular cell adhesion molecule‐1 (VCAM‐1), intracellular adhesion molecule‐1 (ICAM‐1) and endothelial cell selectin (E‐selectin) as well as matrix metalloproteinase‐2, ‐9 (MMP‐2/‐9) expression in HUVEC. Vitexicarpin significantly inhibited the TNF‐α‐induced increase in HL‐60 monocyte adhesion to HUVEC. Vitexicarpin significantly decreased TNF‐α‐induced intracellular ROS production. Furthermore, vitexicarpin inhibited TNF‐α‐induced transcriptional activity of NF‐κB p65 and IκB‐α phosphorylation. In conclusion, vitexicarpin significantly reduced the increased vascular inflammation, through out inhibition of ROS‐NF‐κB pathway in HUVEC.
Genetic Effect of Endothelin and Angiotensin Receptor Blockade on Renal Injury in Salt‐Sensitive and Salt‐Resistant Hypertension
Authors: Lee Tsung‐Ming , Chang Nen‐Chung
Published: June 2021

Proteinuria was inhibited by angiotensin receptor antagonists with a variable response. It remained unclear whether failure of some experimental models of hypertension to respond to treatment with the angiotensin antagonists may be due to genetic background. To investigate genetic effects on renal injuries in response to endothelin‐A blocker (ABT‐627), endothelin‐B blocker (A‐192621), and angiotensin receptor blocker (valsartan), we compared deoxycorticosterone acetate (DOCA)‐salt rats and spontaneously hypertensive rats (SHR) with the genetic control Wistar‐Kyoto rats. Drugs were initiated after hypertension was established. Compared with controls, arterial pressure increased in both strains and was not decreased after 4 weeks’ drug intervention. In both hypertensive strains, renal creatinine clearance decreased, and urinary excretion of protein, interstitial fibrosis, and TGF‐β mRNA levels were significantly increased. In vehicle‐treated DOCA‐salt rats, such damage was overcome by ABT‐627 and substantially blunted by valsartan, but not with A‐192621. The improvement degree of proteinuria in DOCA‐salt rats was significantly greater in rats treated by ABT‐627 than those treated by valsartan. In SHR, valsartan, but not ABT‐627, can significantly attenuate the renal injury. These results strongly support the important role of genetic predisposition in different renal responses to treatments. In DOCA‐salt rats, endothelin‐A receptor– and angiotensin receptor‐mediated action plays an important role in the pathogenesis of renal damage. On the other hand, only angiotensin receptor antagonist is useful for treatment in SHR.
DOCA/Salt‐induced hypertension and associated increase in vascular reactivity and cardiac and vascular hypertrophy are mediated by cytochrome P450 1B1
Authors: Firat Seyhan Sahan , Jennings Brett L , Estes Anne M , Farjana Nasreen , Khan Amir I , Fang Xiao R , Malik Kafait U
Published: June 2021

Arachidonic acid (AA) metabolites derived via lipoxygenase and cytochrome P450 (CYP) 4A1, 12‐ and 20‐hydroxyeicosatetranoic acids (HETEs) respectively, have been implicated in the development of hypertension. CYP1B1 also metabolizes AA into HETEs in vitro. This study was conducted to determine the contribution of CYP1B1 in DOCA/Salt‐induced hypertension. DOCA, 30 mg/kg given sc. per week with 1% NaCl + 0.1% KCl in drinking water to uninephrectomized rats for 6 weeks, increased mean arterial pressure (MAP) (154 ± 1 mmHg) compared to sham operated normotensive rats (95 ± 1 mmHg) (P<0.05). Administration of CYP1B1 inhibitor, 2, 4, 3′, 5′‐tetramethoxystilbene (TMS, 300 μg/kg, i.p. every 3rd day) initiated at 4th week of DOCA/Salt treatment, decreased MAP to levels not different from normotensive levels (96 ± 1 mmHg) (P>0.05). TMS also decreased CYP1B1 activity but not its expression in the aorta, heart and kidney and prevented the increase in mesenteric artery response to phenylephrine, cardiac and vascular hypertrophy, reactive oxygen species (ROS) production and ERK1/2 and p38 MAPK activity in the aorta produced by DOCA/Salt. These data suggest that DOCA/Salt‐induced hypertension is mediated by CYP1B1 as a result of increased ROS production, ERK1/2 and p38 MAPK activity, vascular reactivity and hypertrophy and could serve as a novel target for the development of agents like TMS for the treatment of hypertension.
Urinary Renin Excretion is augmented in Chronic Angiotensin II‐infused Sprague‐Dawley Hypertensive Rats
Authors: Liu Liu , McCormack Michael , Seth Dale M , Kobori Hiroyuki , Navar L. Gabriel , Prieto Minolfa C
Published: June 2021

Renin is upregulated in principal cells of the collecting ducts of angiotensin II (AngII)‐dependent hypertensive rats; however it remains unclear if increased distal nephron‐derived renin is secreted into the urine during AngII‐dependent hypertension. Urinary renin (uRen), angiotensinogen (uAGT) and AngII (uAngII) excretion was measured in chronic AngII‐infused male Sprague‐Dawley rats [80 ng/min, SC minipumps for 14 d, n=5] and control, sham‐operated rats [n=5]. Systolic blood pressures increased in the AngII rats by Day 5 and continued to increase throughout the study (Day 13; AngII: 175±10 vs. sham: 116±2 mm Hg). Although plasma renin activity was suppressed in the AngII‐infused rats (AngII: 0.3±0 vs. sham: 5.5±2 ng Ang I/ml/h); renin content in renal medullary tissues did not change (AngII: 10,564±1,476 vs. sham: 9,438±1,588 ng Ang I/h/mg; p=ns). Excretion of uAGT and uAngII increased in the AngII rats compared to sham rats, respectively [uAGT (AngII:1107±106 vs. sham:60±26 ng/day); uAngII (AngII: 3813±431 vs. sham: 2080±361 fmol/day). Importantly, uRen excretion rate at Day 13 was markedly increased in the AngII rats (AngII: 9±3 vs. sham: 1±1×10−5 Enzyme Units/day; p<0.05). Augmented uRen and uAGT may contribute to increased intrarenal AngII formation in AngII‐dependent hypertensive rats.

NIH [IdeA (P20‐RR‐017659‐06), Tulane‐BIRCWH (K12HD043451)]; and AHA (09PRE2209079 and 09BGIA2280440).
Arterial Pressure Wave Reflection Site Shifts Periphery with Aging
Authors: Sugawara Jun , Hayashi Koichiro , Tanaka Hirofumi
Published: June 2021

Early return of reflection wave from the periphery to the heart relates to the augmentation of central pulse pressure, a risk for cardiovascular disease. We determined the impact of aging on the location of a major reflection site which would directly affect the timing of the reflected wave. Arterial length (via 3‐D artery tracing of MRI) and carotid‐femoral pulse wave velocity (PWV) were measured in 218 adults. Aortic blood pressure was estimated via the radial pressure waveform analysis. The major reflection site was detected by carotid‐femoral PWV and the time of return of the reflected wave (Δt) (via carotid pressure waveform analysis). The major reflection sites were located at abdominal aorta (e.g., proximally to aortic bifurcation) in most of subjects (82%). The length from heart to the major reflection site (Lref) gradually increased with aging (r=0.53). Multi‐regression analyses revealed that Lref was significant independent determinant of aortic systolic and pulse pressure (β=−0.24 and β=−0.19). These results suggest that the major reflection site shifts periphery with aging, and that such age‐related shift of reflection site may partly attenuate the augmentation of central pulse pressure.
Contribution of Cytochrome (CYP) P450 1B1 in Various Models of Hypertension in Rats and Mice: A Novel Target for the Development of Anti‐Hypertensive Agents
Authors: Jennings Brett Lawrence , Sahan‐Firat Seyhan , Estes Anne M , Malik Kafait U
Published: June 2021

Previously, we reported that inhibition of CYP1B1 with 2, 4, 3′, 5′‐tetramethoxystilbene (TMS) in rats prevented the development and maintenance of angiotensin II (Ang II)‐induced hypertension. We have found that TMS (300 μg/kg, i.p. every 3rd day) also reduces mean arterial pressure (MAP) in DOCA/Salt‐hypertensive rats (154 ± 1 mmHg to 96 ± 1 mmHg, P < 0.05) and in spontaneously hypertensive rats (163 ± 14 mmHg to 116 ± 4 mmHg, P < 0.05). To further elucidate the role of CYP1B1 in hypertension, studies were conducted in CYP1B1 knockout (CYP1B1−/−) and wild‐type (CYP1B1+/+) mice. Ang II infused by mini osmotic pumps (1mg/kg/min sc.) for 13 days produced a significantly smaller increase in MAP (P < 0.05) in CYP1B1−/− mice (112 ± 1 to 128 ± 2 mmHg) than in CYP1B1+/+ mice (113 ± 3 mmHg to 158 ±3 mmHg). In Ang II‐treated CYP1B1+/+ mice, responses of the mesenteric artery to phenylephrine and endothelin‐1, media to lumen ratio and aortic ROS production were significantly increased compared to Ang II‐treated CY1B1−/− mice. Administration of DOCA (35 mg/kg/week im. + 1% NaCl in drinking water) increased MAP from 102 ± 1 to 153 ± 4 mmHg in CYP1B1+/+ but not in CYP1B1−/− mice. These data suggest that CYP1B1 contributes to the development of hypertension, most likely via increased ROS generation and vascular reactivity and hypertrophy, and could serve as a novel target for the development of agents like TMS for the treatment of hypertension.
Transcriptional regulation and expression of guanylyl cyclase/natriuretic peptide receptor‐A gene by δEF1:Role of TGF‐β1 signaling
Authors: Sen Anagha S , Kumar Prerna , Vellaichamy Elangovan , Pandey Kailash N
Published: June 2021

The binding of atrial and brain natriuretic peptides (ANP and BNP) to guanylyl cyclase‐A/natriuretic peptide receptor‐A (GC‐A/NPR‐A) produces second messenger cGMP, which plays a critical role in various intracellular signaling pathways including; hypertension, cardiovascular events, and cancer. Transcription factor δEF1, an E‐2 box repressor has been predicted to have binding sites in the 5′ flanking region of the Npr1(coding for GC‐A/NPRA) gene promoter. The objective of this study was to determine the repressive effect of δEF1 on transcriptional regulation of murine Npr1 gene promoter in mouse mesangial cells (MMCs) and rat thoracic aortic vascular smooth muscle cells (RTASMCs). MMCs were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and ITS (insulin, transferrin, and selenium). RTASMCs were cultured in DMEM containing 10% FBS. Co‐transfection of δEF1 expression plasmid with the Npr1 gene promoter‐luciferase constructs showed 70–75% reduction in the luciferase activity in MMCs and RTASMCs. Also, Npr1 gene promoter‐luciferase constructs embodying δEF1 sites showed up to 90 % reduction in luciferase activity after treatment with TGF‐β1 in a time‐ and dose‐ dependent manner. Deletion of δEF1 sites eliminated the repression of luciferase activity induced by TGF‐β1. Electrophoretic mobility shift assay as well as supershift assay confirmed the binding of δEF1 to the specific sites of Npr1 gene promoter. The findings of this study suggest that δEF1 exerts a repressive effect on Npr1 gene transcription and expression, which plays a critical role in hypertension and cardiovascular regulation. This work is supported by National Institutes of Health Grants (HL57531 and HL 62147).
Chronic high salt intake induces renal inflammation and endothelial dysfunction that are reversed by statin
Authors: Fiore Maria Cecilia , Jimenez Pablo M , Cremonessi David , Juncos Luis I , Garcia Nestor H
Published: June 2021

High salt (HS) intake induces harmful vascular effects that are independent from blood pressure (BP) changes. Thus, we hypothesized that HS intake could also impair renal structure and function independently from BP changes. Four groups of rats were treated for 6 weeks as follows: Normal salt (NS) (0.8%), HS (4%), NS plus Atorvastatin (NSAt) and HS plus Atorvastatin (HSAt). We found no differences between groups in systolic BP, body weight and serum cholesterol at the end of the study. In the HS group creatinine clearance increased (p< 0.02), proteinuria rose (6.0±3.1 to 28.9±6.9 mg/day, p <0.05) while glomerular volume increased by 33% (p<0.001). In this HS group, acetylcholine‐induced vasodilatation decreased by up to 40% (p <0.05). In addition, the HS diet decreased eNOS expression by 62% (p<0.05) and caused mild glomerular sclerosis, interstitial mononuclear cell infiltration and increased cortical expression of TGF‐β1. All of the changes induced by the HS diet were reversed by Atorvastatin (At) except for the increased creatinine clearance and renal TGF‐β1 expression. We conclude that long term (6 weeks) HS intake induces inflammatory and hemodynamic changes in the kidney that are independent from BP. In these earlier stages of salt induced kidney damage, At corrected all but the increased TGF‐β1 expression suggesting a prevailing role of the NO‐oxidative stress balance over TGF‐β1.
BAY 58‐2667, a Novel NO‐Independent Activator of Soluble Guanylate Cyclase, Protects against Ischemia/Reperfusion Injury: Potential Role of Hydrogen Sulfide Signaling
Authors: Salloum Fadi N , Das Anindita , Chau Vinh Q , Hoke Nicholas N , Ockaili Ramzi A , Stasch Johannes‐Peter , Kukreja Rakesh C
Published: June 2021

BAY 58‐2667 is a soluble guanylate cyclase (sGC) activator that acts independent of nitric oxide. It protects the heart against ischemia/reperfusion (I/R) injury through protein kinase G signaling. Hydrogen sulfide (H2S) is a gaseous molecule which plays a critical role in protection against I/R injury. We tested the hypothesis that H2S mediates BAY‐induced cardioprotection. Following baseline echochardiography, adult male ICR mice were treated with either BAY (10 μg/kg, ip) or 10% DMSO (vehicle) 30 min prior to regional ischemia for 30 min and reperfusion for 24 h. The H2S producing enzyme, cystathionine‐γ‐lyase (CSE), specific inhibitor, dl‐propargylglycine (PAG, 50 mg/kg, ip) was given 30 min before ischemia in pretreated mice. At the end of reperfusion, echocardiography was performed prior to sacrifice and infarct size (IS) was measured. IS was reduced with BAY compared to vehicle (80% reduction, P<0.05). PAG blocked the cardioprotective role of BAY as shown by an increase in IS. Moreover, fractional shortening was preserved with BAY treatment. In adult murine cardiomyocytes, BAY treatment increased cGMP levels and also protected against necrosis and apoptosis following simulated ischemia/reoxygenation. These results suggest a potent cardioprotective effect of BAY, which is mediated by H2S. This research was funded in part by NIH (HL51045, 79424 and 93685 to RCK) and Bayer Schering Pharma AG.
Glucose deprivation induced Cyclooxygenase‐2 and Heme oxygenase‐1 expression via P38 pathway in neonatal rat ventricular cardiomyocytes
Authors: Zou Luyun , Chatham John C
Published: June 2021

We have reported that in neonatal rat ventricular cardiomyocytes (NRVM) glucose deprivation (GD) resulted in increased levels of O‐linked‐N‐acetylglucosamine (O‐GlcNAc) on nucleocytoplasmic proteins, which was associated with decreased O‐GlcNAcase (OGA) levels. Others have shown that in cardiac fibroblasts GD increased expression of both COX (Cyclooxygenase)‐2 and HO (Heme oxygenase)‐1, which was mediated in part by p38 MAPK. The goal of this study, therefore, was to determine if in NRVM the GD induced increase in O‐GlcNAc levels was p38 dependent. We found that in NRVM GD significantly increased COX2 and HO1 expression in time dependent manner, which was blocked by the p38 inhibitor, SB203580, in a dose dependent manner. However, treatment with SB203580 had little or no effect on the GD induced changes in O‐GlcNAcylation or OGA levels. In contrast augmenting hexosamine biosynthesis pathway (HBP) flux with glucosamine attenuated the GD induced increase in O‐GlcNAc levels and prevented the decrease in OGA in dose dependent manner; however glucosamine had no effect on the GD‐induced increase in COX2 or HO1 levels. Thus, in NRVM the GD induced increase in COX2 and HO1 is mediated by p38 but not HBP, whereas the increase in O‐GlcNAc levels is mediated by HBP and not p38. These results provide new insights into the mechanisms underlying the stress induced increase in O‐GlcNAcylation. Supported by: NIH HL067464/HL079364.
Inflammatory response underlies Pulmonary Hypertension
Authors: Mathew Rajamma , Huang Jing , Gewitz Michael H
Published: June 2021

We have shown upregulation of IL‐6 mRNA, IL‐6 bioactivity and activation of PY‐STAT3, a proproliferative transcription factor in MCT‐induced pulmonary hypertension (PH), accompanied by a loss of endothelial caveolin‐1, a potent immunomodulator. To investigate the role of inflammation in hypoxia‐induced PH and compare with the MCT model, rats (¡Ö200 g) were subjected to hypobaric hypoxia or given MCT (sc, 60 mg/kg). Hemodynamic data, expression of caveolin‐1, eNOS, PY‐STAT3 were examined at 48h, 1 and 2 wks. Both models revealed progressive PH and right ventricular hypertrophy. Progressive PY‐STAT3 activation was observed at 48h, i.e. before the onset of PH (MCT, control [C], 100¡À0 vs 48h, 134¡À10*, hypoxia, C, 100¡À vs 48h, 453¡À70 *, *= P<0.05 vs C). Transient increase in eNOS expression at 1 wk was observed in both models. Unlike the MCT model, hypoxia‐induced PH did not exhibit loss of caveolin‐1. Caveolin‐1, known to inhibit PY‐STAT3 activation may be dysfunctional in the hypoxia model. In conclusion, dissimilar models of PH show activation of PY‐STAT3, indicating that inflammatory response may have a significant role in the pathogenesis of PH. (Supported in part by MF children¡ s Hosp. Res. Fund).
Habitual aerobic exercise reverses age‐associated increases in transforming growth factor beta 1 in carotid arteries of mice
Authors: Marshall Kurt D , Seals Douglas R , Geolfos Candace , Lesniewski Lisa A , Fleenor Bradley S
Published: June 2021

Aging increases carotid artery stiffness and adventitial collagen in mice. These events are reversed by voluntary wheel running (VR). Transforming growth factor β1 (TGF‐β1) modulates extracellular matrix proteins with aging, but the effect of habitual exercise on TGF‐β1 expression in large elastic arteries is unknown. Carotid artery stiffness was greater in old (O, 31 mo, n=4) vs. young (Y, 7 mo, n=4) adult cage‐control B6D2F1 mice (8.53 ± 2.00 vs. 3.015 ± 1.19 AU, p < 0.05), but not in O (n=4) given access to running wheels (mean distance = 1 km/d) for 12 weeks (3.61 ± 0.83 AU, p < 0.05). Immunohistochemical analysis of carotid arteries from Y (n=6) and O (n=8) cage control mice revealed increases in adventitial TGF‐β1 with aging (5.7E‐05 ± 5.9E‐06 vs. 3.6E‐05 ± 3.5E‐06 AU, P < 0.05), which were reversed by VR in O (n=4) (3.6E‐05 ± 4.2E‐06 AU). In a primary culture of adventitial fibroblasts obtained from Y adult (6 mo) Sprague‐Dawley rats, 24‐hr treatment with TGF‐β1 increased type I collagen (1.38 ± 0.07 vs. 1.0 ± 0.08, P < 0.05, western blot) and superoxide (O2−) production (4514 ± 317 vs. 2828 ± 185, P < 0.05, electron paramagnetic resonance). These preliminary data are consistent with the working hypothesis that habitual aerobic exercise may reverse large elastic artery stiffening with aging in part by reducing O2− production, which inhibits adventitial TGF‐β1‐induced adventitial collagen accumulation.

AG013038 and HL007822
Role of 17[beta]‐estradiol and estrogen receptor [beta] in the genomic response to hypertrophy
Authors: Kararigas Georgios , Fliegner Daniela , Regitz‐Zagrosek Vera
Published: June 2021

We and others have clearly indicated an important role for 17β‐estradiol (E2) and estrogen receptor β (ERβ) in attenuating pressure overload (PO)‐induced left ventricular hypertrophy (LVH). However, the underlying molecular mechanisms are poorly understood. Here, we investigated the role of E2/ERβ signaling in PO‐induced LVH at a genome‐wide level.

Female wild‐type (WT) and ERβ knockout (ERβ−/−) cycling C57Bl6 mice were subjected to transverse aortic constriction (TAC) or sham surgery. At nine weeks post operation, left ventricles were harvested and RNA was hybridized to the Mouse Genome 430 2.0 Array. The microarray data were analyzed with R and Bioconductor.

Assessment of single gene differential expression with a false discovery rate‐adjusted P < 0.05 identified 197 probe sets regulated in WT, 941 in ERβ−/− and 366 in both WT and ERβ−/− mice. Gene set enrichment analysis based on 1000 permutations revealed 26 pathways modulated in WT, while almost the double were modulated in ERβ−/− mice. Of these, 16 were modulated in both WT and ERβ−/− mice. Among others, the cytochrome P450 pathway was repressed in WT/TAC mice, the apoptosis pathway was induced in ERβ−/−/TAC mice, while the ECM‐receptor interaction pathway was induced in both WT and ERβ−/−/TAC mice.

In conclusion, we provide evidence that the E2/ERβ axis plays an important role in the genomic response to LVH modifying the expression of several genes/pathways.
Superoxide production in the medullary thick ascending limb modulates blood pressure
Authors: Gongora Maria Carolina , Lob Heinrich , Blinder Yelena , Harrison David G
Published: June 2021

Superoxide production in the renal medulla has been suggested to play a major role in modulating renal sodium and water handling and blood pressure control. To further study this, we created mice with loxP sites flanking the extracellular superoxide dismutase (SOD3) coding region and other mice with loxP sites flanking the NADPH oxidase subunit p22phox. We also created transgenic mice with Cre driven by the Tamm Horsfall promoter (tgcreth mice). SOD3 and the NADPH oxidase subunit p22phox were then selectively deleted in the medullar thick ascending limb (mTAL) by crossing the phloxed SOD3 and p22phox mice with tgcreth mice. While baseline blood pressures were similar (114 +/−2.0 and 116 +/− 0.8 mmHg), the hypertensive response to two‐week infusion of angiotensin II (490 ng/kg/min) was markedly augmented in mice with deletion of mTAL SOD3 compared to controls (178 +/− 1.6 vs. 158 +/− 7.9, p = 0.007). In contrast, preliminary data show that mice with mTAL deletion p22phox demonstrate a blunted hypertensive response to angiotensin II. We conclude that oxidative stress within the mTAL, as demonstrated in these unique genetically modified mice, critically regulates the hypertensive response to angiotensin II. Supported by AHA 09POST2100151 and NIH PPG PO1HL058000.
CNS Gαi2 proteins participate in the central neural regulation of sodium and water homeostasis and in countering salt‐sensitive hypertension
Authors: Wainford Richard David , Kapusta Daniel R
Published: June 2021

Hypothesis

Central Gαi2‐protein gated pathways mediate the renal sympathoinhibitory, natriuretic and diuretic responses to acute volume expansion (VE) and counter the development of salt‐sensitive hypertension.

Methods

Sprague‐Dawley rats were pre‐treated (24h) intracerebroventricularly (i.c.v.) with a scrambled (S) or Gαi2 oligodeoxynucleotide (ODN; 25 μg). Changes in renal sympathetic nerve activity (RSNA), natriuresis and diuresis evoked by acute i.v. isotonic saline VE (5% body weight) were examined. In chronic studies rats were fed normal 0.4% (NS) or high 8% (HS) NaCl chow for 21‐days and brain Gαi2 protein levels were determined (N=6/gp). Separate rats were maintained on NS or HS and infused i.c.v. with a Gαi2 or S ODN, after 21‐days MAP and renal function were assessed (N=4/gp).

Results

Central Gαi2‐protein down‐regulation attenuated the renal sympathoinhibitory (peak ΔRSNA [% control] S 43±3 vs. Gαi2 88±8, P<0.05 N=4), natriuretic (peak ΔUNaV [μeq/min] S 40±3 vs. Gαi2 26±3, P<0.05 N=6) and diuretic (peak ΔUV [μl/min] S 309±12 vs. Gαi2 225±26, P<0.05 N=6) responses to i.v. VE. In chronic studies HS intake selectively increased Gαi2 protein levels (5.8 fold, P<0.05) in the PVN. When infused with i.c.v. Gαi2 ODN, rats maintained on HS (but not NS) retained sodium (24h Na balance [meq/day] NS + Gαi2 0.3±.0.1 vs. HS + Gαi2 2.75±0.4, P<0.05), retained water (24h H2O balance [ml/day]; NS + Gαi2 12.6±1.3 vs. HS + Gαi2 29.4±3.6, P<0.05) and developed hypertension (MAP [mmHg]; NS + Gαi2 129±2 vs. HS + Gαi2 147±3, P<0.05).

Conclusion

These data demonstrate that central Gαi2‐protein gated pathways contribute to the renal sympathoinhibitory and natriuretic responses to acute VE and participate in maintaining sodium and water homeostasis and normotension during high salt challenge. AHA 2250585, DK43337, HL071212, AHA 0855293E, P20 RR018766
Interaction Between Endothelin (ET) and Norepinephrine (NE) in the Activation of Renal Sensory Nerves
Authors: Kopp Ulla , Cicha Michael , Smith Lori
Published: June 2021

Stimulation of ETB receptors (ETB‐R) contributes to the natriuretic renorenal reflexes elicited by NE‐mediated activation of the renal sensory nerves. We now examined the mechanisms involved in the ETB‐R mediated activation of these nerves. Renal pelvic administration of the ETB‐R agonist IRL1620 increased ARNA 3800±850 %·sec which was blocked by the ETB‐R antagonist BQ788 or indomethacin. Adding IRL1620 to an isolated renal pelvic preparation resulted in increases in the release of PGE2 from 114±18 to 414±64 pg/min and substance P from 7.8±1.0 to 16.6±1.6 pg/min that were also blocked by BQ788 or indomethacin. The concentration of IRL1620 used was 20 μM in vitro vs 10 nM in vivo suggesting reduced responsiveness of ETB‐R in the denervated in vitro preparation. 2 pM NE + 25 nM IRL1620 increased renal pelvic release of PGE2 from 90±9 to 240±30 pg/min and substance P from 5.3±0.3 to 12.4±0.8 pg/min. No effect was produced by 2 pM NE or 25 nM IRL1620 alone. Renal pelvic administration of prazosin reduced the ARNA responses to IRL1620 by 81±13% in vivo. Thus NE facilitates the responsiveness of ETB‐R. The intracellular mechanisms contributing to the ETB‐R activation of renal sensory nerves were examined by renal pelvic administration of the PLC inhibitor U73122 and PKC inhibitor GF109203X (GFX). U73122 and GFX reduced the ARNA responses to IRL1620 by 48±16% and 100±0% in vivo. Likewise, U73122 and GFX reduced the increases in substance P release produced by 2 pM NE+25 nM IRL1620 by 38±5% by and 46±5% in vitro. The NE+IRL1620 induced increases in PGE2 release was reduced by 48±11% by GFX. Conclusion: There is a reciprocal interaction between α1‐adrenoceptors and ETB‐R in the activation of renal sensory nerves. Activation of the PLC/PKC pathway contributes to the ETB‐R induced increases in renal pelvic release of PGE2 and substance P and ARNA.
Potential Role of NADPH Redox in Regulating Thiol Oxidation‐Elicited Subunit Dimerization Activation of Protein Kinase G in the Relaxation of Bovine Pulmonary Arteries to Pentose Phosphate Pathway Inhibitors
Authors: Neo Boon Hwa , Wolin Michael
Published: June 2021

We have previously provided evidence that inhibition of glucose‐6‐phosphate dehydrogenase (G6PD) in the pentose phosphate pathway promotes oxidation of NADPH, and elicits vascular relaxation through coordinating multiple processes that lower intracellular calcium. Since it was reported that H2O2 induces coronary relaxation associated with a NO/cGMP‐independent thiol oxidation/subunit dimerization activation of protein kinase G (PKG), we investigated if inhibition of G6PD activates PKG through disulfide‐mediated subunit dimerization under conditions where relaxation of isolated endothelium‐removed bovine pulmonary arteries (BPA) is observed. BPA precontracted with 20mM KCl under hypoxic conditions were exposed to either 1mM 6‐aminonicotinamide (6‐AN) or 0.5mM epiandrosterone (Epi) to inhibit G6PD. It was observed that both 6‐AN and Epi increased PKG dimerization under conditions where relaxation was observed. These responses were associated with increased phosphorylation of vasodilator‐stimulated phosphoprotein (VASP) at the serine‐239 site known to be mediated by PKG. Thus, inhibition of G6PD promotes thiol oxidation‐elicited dimerization activation of PKG, suggesting that PKG activation is likely to be a participant in mechanisms through which cytosolic NADPH oxidation promotes vascular relaxation.

Supported by NIH grants HL31069, HL43023 and HL66331
Activation of Glucose‐6‐Phosphate Dehydrogenase Promotes Acute Hypoxic Pulmonary Artery Contraction
Authors: Rawat Dhawjbahadur K , Gupte Rakhee S , Chettimada Sukrutha , McMurtry Ivan F , Gupte Sachin A
Published: June 2021

Hypoxic pulmonary vasoconstriction (HPV) is a physiological response to a decrease in airway O2 tension, but the underlying mechanism is incompletely understood. We studied the contribution of glucose‐6‐phosphate dehydrogenase (G6PD), an important regulator of NADPH redox and production of reactive oxygen species, to the development of HPV. We found that hypoxia (95%N2‐5%CO2) increased contraction of bovine pulmonary artery (PA) precontracted with KCl or serotonin. Depletion of extracellular glucose reduced NADPH, NADH and HPV, substantiating the idea that glucose metabolism and G6PD play roles in the response of PA to hypoxia. Our data also show that inhibition of glycolysis and mitochondrial respiration (indicated by an increase in NAD+ and decrease in the ATP‐to‐ADP ratio) by hypoxia, or by inhibitors of pyruvate dehydrogenase or electron transport chain complexes I or III, increased generation of reactive oxygen species, which in turn activated G6PD. Silencing G6PD expression in PA using a targeted siRNA abolished HPV and diminished Ca2+‐independent and Ca2+‐dependent myosin light chain phosphorylation otherwise increased by hypoxia. Similarly, G6PD expression and activity were significantly reduced in lungs from G6PDmut(−/−) mice, and there was a corresponding reduction in HPV. Finally, regression analysis relating G6PD activity and the NADPH‐to‐NADP+ ratio to the HPV response clearly indicated a positive linear relationship between G6PD activity and HPV. Based on these findings, we propose that G6PD and NADPH redox are crucially involved in the mechanism of HPV and, in turn, may play a key role in increasing pulmonary arterial pressure, which is involved in the development of pulmonary hypertension. (Supported by NIH grant RO1HL085352)
Impact of cyclic strain on integrin expression in rat pulmonary microvascular endothelial cells
Authors: Villalta Patricia Caridad , Rocic Petra , Townsley Mary I.
Published: June 2021

Detachment of pulmonary microvascular endothelial cells (PMVEC) is observed in mechanical stress‐induced, TRPV4‐mediated (vanilloid transient receptor potential 4 channel) acute lung injury. Integrin dimers, comprised of α and β subunits, that mediate PMVEC tethering and detachment have not been identified. The goals of this study were to characterize integrin expression and to identify the effect of cyclic strain on integrin expression. Rat PMVEC were cultured on collagen I or pronectin flexwell membranes to 90% confluence and then subjected to 0% or 10% uniaxial strain at 30 cycles/min for 24 h. Using PCR and primers for rat integrins, expression of integrin subunits α1, α2, α3, α5, α6, α7, αv, β1, and β6, but not α4, α8 or β3, was identified in PMVEC, irrespective of strain or matrix composition. However, integrin β4 expression decreased with cyclic strain in PMVEC cultured on either matrix. Based on these results, extracellular ligands for integrin dimers, and matrix composition of the alveolar basement membrane, we predict that α1β1, α2β1, α3β1, α6β1, α7β1, and/or α6β4 could tether PMVEC to matrix in vivo and be a target for detachment in TRPV4‐mediated acute lung injury. Supported by HL066299 and HL076125.
LPS induced changes in zinc homeostasis in cultured sheep pulmonary artery endothelial cells (SPAEC)
Authors: Thambiayya Kalidasan , Wasserloos Karla , Klei Linda , St Croix Claudette M. , Pitt Bruce R.
Published: June 2021

Intracellular zinc (Zni) is maintained in a narrow range (100–500 uM) by zinc importers (SLC39A1‐14) and exporters (SLC30A1‐10). Although virtually all (Zni) is protein‐bound, a chelatable pool of (Zni) exists that is capable of participating in intracellular signaling. We recently showed ( FASEB J. 2009 23:999.5 ) that LPS causes a decrease in labile (Zni) in SPAEC as ascertained by decreases in: a) the intensity of a zinc sensitive fluorophore (Fluo‐Zin3); and b) activity of a genetically encoded zinc sensitive chimera (metal responsive element fused to luciferase). A contributory role of decreases in (Zni) in LPS induced apoptosis in SPAEC was revealed by mimicking the effect of LPS with the (Zni) chelator, TPEN and inhibiting LPS induced apoptosis with exogenous zinc in the presence of zinc ionophore, pyrithione. Using quantitative real time PCR (with primers designed from bovine data base), we surveyed acute changes in mRNA of representative members of zinc transporter family after SPAEC were exposed to proapoptotic dose (100 ng/ml) of LPS. Four hr after LPS, there were minimal changes in expression of zinc exporters (SLC30A1,4,5,6,7). In contrast, SLC39A14 (but not SLC39A6 or SLC39A10) increased dramatically after LPS. In three subcultures, SLC39A14 increased an average of 14X and a portion of this increase could be mimicked with TPEN. These data suggest that SLC39A14 may be responding to LPS induced decreases in (Zni) and this may have implications for longer term changes in zinc homeostasis in response to LPS in pulmonary endothelial cells.
Ventilatory effects of focal spinal demyelination within descending respiratory‐related pathways
Authors: Johnson Rebecca Ann , Mitchell Gordon
Published: June 2021

Although respiratory complications are a major cause of morbidity/mortality in advanced myelin disorders (i.e. MS), little is known concerning mechanisms whereby lack of myelin impairs ventilation, or how patients compensate for such changes (i.e. compensatory plasticity). We tested the hypothesis that respiratory function is diminished in a model of focal spinal demyelination within descending bulbospinal pathways at C2. Spinal ethidium bromide (EtBr) was injected unilaterally at a depth of 1.8 mm below the dorsal root entry zone. Ventilatory measurements were made via whole‐body plethysmography in EtBr and sham‐injected rats at baseline and during maximal ventilatory stimulation (7% hypercapnia/11% hypoxia). EtBr produced dose‐dependent (0.5–2 microliter) demyelination lesions in ventrolateral pathways at C2. Tidal volume and mean inspiratory flow (VT/TI) tended to be reduced in EtBr rats during hypercapnia/hypoxia (p=0.06 and 0.07, respectively). Although additional data are needed, these novel preliminary findings suggest ventilatory capacity may be impaired with unilateral, focal demyelination of descending respiratory‐related spinal pathways. If confirmed, such a model will provide the foundation for future mechanistic studies of respiratory insufficiency and possible therapeutic interventions in myelin disorders. Support: UW ICTR via NIH CTSA 1UL1RR025011