bluuebunny/crossref_metadata_2025 huggingface dataset:
Higher Dietary Niacin Intake is Related to Greater Vascular Endothelial Function Associated with Lower Oxidative Stress Among Healthy Middle‐Aged and Older Adults
Authors: Kaplon Rachelle E , Seals Douglas R , Gano Lindsey B
Published: June 2021

Endothelium‐dependent dilation (EDD) is impaired with age due in part to decreased activity of the nicotinamide adenine dinucleotide (NAD)‐dependent histone deacetylase sirtuin‐1. We hypothesized that EDD would be related to dietary intake of the NAD precursor niacin among middle‐aged and older (MA/O) adults. In 156 healthy adults aged 45–78, EDD (brachial artery flow‐mediated dilation, FMD) was positively related to self‐reported dietary niacin intake (%Δ: r=0.19, P=0.02). This difference persisted after accounting for clinical characteristics using multiple regression analysis (r=0.16, P=0.04). In a subset of subjects, inhibition of superoxide (infusion of ascorbic acid) improved FMD in niacin insufficient (dietary intake ≤16mg, n=8, 3.7±1.1 vs. 4.7±0.9%, P<0.01), but not sufficient (>16mg, n=19, 5.4±0.5 vs. 5.9±0.6%, P=0.10) adults. Moreover, niacin intake was inversely related to arterial endothelial cell expression of the superoxide‐producing enzyme NADPH oxidase (n=37, r=−0.44, P=0.01). Endothelium‐independent dilation (sublingual nitroglycerin), a measure of vascular smooth muscle sensitivity to nitric oxide, did not differ between groups (P>0.05). These findings suggest that higher dietary niacin intake is associated with greater vascular endothelial function linked to lower oxidative stress among healthy MA/O adults.

NIH AG013038, AG006537, AG000279, UL1 RR025780
Polyamine supplementation enhances autophagy and reverses age‐related arterial stiffening
Authors: LaRocca Thomas J , Gioscia-Ryan Rachel A , Fleenor Bradley S , Seals Douglas R
Published: June 2021

Aging leads to stiffening of large elastic arteries as a result of changes in structural proteins and increased macromolecular damage. Polyamines (PA) play a causal role in survival/longevity and have been reported to induce autophagy, the cellular process of recycling damaged biomolecules. However, the potential for PA to reduce age‐related arterial stiffness is unknown. We studied young (Y: 5 mo, n=7) and old (O: 28–29 mo, n=7) male C57/BL6 mice, and old mice treated with the PA spermidine (OPA: 29 mo, n=7). Aortic pulse wave velocity (aPWV), an index of arterial stiffness, was higher in O vs. Y mice (465 ± 25 vs. 383 ± 21 cm/s, p<0.05). This was associated with ~50% greater aortic levels of both protein carbonyls, an index of oxidative damage, and advanced glycation end products (AGEs), a marker of protein cross‐linking (dot and western blotting, both p<0.05, O vs. Y). PA supplementation (3 mM drinking water × 4w) ameliorated aortic stiffening in old mice (OPA aPWV pre: 482 ± 17 vs. OPA post: 343 ± 14 cm/s, P<0.05) and reversed age‐related increases in protein carbonyls and AGEs. The effects of PA treatment were associated with enhanced autophagy as indicated by an ~20% reduction in acetylation (inhibition) of histone H3, an ~25% increase in expression of the transcription factors AMPK and FOXO3a, and a corresponding increase in LC3‐II and decrease in p62, markers of autophagy and autophagic protein aggregation/damage, respectively (western blotting, all p<0.05, O vs. OPA). These results suggest that PA supplementation may be a novel strategy for reversing age‐related arterial stiffening, perhaps by enhancing autophagic clearance of damaged/aggregated proteins.

NIH AG013038, AG039210, HL007822
Impaired vasomotor function in uremic rats is directly related to NO‐resistance of smooth muscle cells
Authors: Kolk Felix , Molin D.G.M , De Mey J.G.R , Post M.J.
Published: June 2021

The number of patients with renal failure is 240 million worldwide, with 20% being dependent on dialysis. Renal failure increases the risk to develop cardiovascular diseases and could be related to vessel wall dysfunction.

Gaining a better understanding of arterial dysfunction under uremic conditions and especially its effect on NO‐soluble guanylate cyclase (sGC) mediated vasorelaxation.

For uremic rats, 5/6 nephrectomy (n=21) was performed in Wistar rats. After six weeks, both carotid arteries were removed and analyzed in a wire‐myograph. Different compounds were used to investigate NO‐dependent and ‐independent vasomotor pathways.

SNP and Deta NONOate showed significant differences in vasorelaxation between sham and 5/6 nephrectomy group with a 0.5 log IC50 difference in NO‐induced relaxation (P<0.0001). This difference was endothelial independent as acetylcholine induced effects were blocked with L‐NAME. Also NO independent, but sGC heam dependent and independent, (Bay 41‐2272 and Bay 60‐2770, respectively) vasorelaxation showed significant differences (p<0.01 and p<0.0001). When sGC was oxidized with ODQ, sham group was more potent in relaxation for both compounds as compared to uremic rats.

Reduced vasorelaxation in uremic rats is related to NO‐resistance of smooth muscle cells. Our results point toward an oxidized state of sGC as the main cause of vasomotor dysfunction in uremic rats.
Impaired L‐arginine Uptake Contributes to Aortic Endothelial Dysfunction in Chronic Kidney Disease
Authors: Martens Christopher R , Kuczmarski James M , Lennon-Edwards Shannon , Edwards David G
Published: June 2021

Reduced nitric oxide (NO) bioavailability contributes to endothelial dysfunction in chronic kidney disease (CKD) and is related to an increased cardiovascular disease risk. Vascular uptake of the NO substrate L‐arginine is attenuated under uremic conditions and may be an important mechanism of endothelial dysfunction in CKD. We sought to determine if impaired L‐arginine transport contributed to endothelial dysfunction in an animal model of CKD. 12 week old, male Sprague‐Dawley rats underwent 5/6 ablation/infarction (AI) surgery to induce CKD and were compared to a Sham control group. Endothelial‐dependent relaxation (EDR) of aortic rings to acetylcholine (Ach; 10−9–10−5M) was significantly impaired in AI vs. Sham after 8 weeks, demonstrated by an attenuated maximal response (65.02±5.02 vs. 102.10±4.94%, p < 0.0001) and rightward shifted LogEC50 (−6.659±0.121 vs. −7.707±0.111, p<0.0001) and was not improved by L‐arginine treatment. In support of this finding, expression of the L‐arginine transporter CAT‐1 was attenuated in CKD (relative to β‐actin, p=0.002) and appears to have resulted in reduced aortic uptake of L‐[3H]arginine (49.01±15.48 vs. 100.08±16.33 dpm/mg, p = 0.06). These preliminary data suggest that uremic inhibition of L‐arginine uptake may act as a novel mechanism by which endothelial dysfunction occurs in CKD and may be a potential therapeutic target to treat vascular dysfunction in CKD.

Supported by: P20 RR016472
Obesity impairs alpha 1 adrenergic receptor mediated contraction in carotid arteries
Authors: Dorrance Anne McLaren , McClain Jonathon L
Published: June 2021

Hypertension is generally thought to cause a hyper contractile state in arteries. This does not appear to be the case in aorta from rats with diet‐induced obesity associated hypertension. We tested the hypothesis that contractile responses would also be impaired in carotid arteries from rats fed a high fat (HF) diet. Male Sprague Dawley rats received a HF diet from 3–20 weeks of age; control rats were fed normal chow (n=4). Carotid arteries were removed and isometric force generation was measured in response to phenylephrine (PE) and the thromboxane A2 receptor agonist U46619. The HF rats were heavier than the controls (401±9 vs 465±10g, control vs HF p<0.05) and had more abdominal fat (5.6±0.3 vs 16.5±1.2g, control vs HF p<0.05). The PE mediated contraction was impaired in carotids from the HF rats as evidenced by an increase in the EC50 and a reduction in the maximum contraction. This was not the case for U46619 where the responses were similar in the control and HF rats. These studies suggest that alpha 1 adrenergic receptor mediated contraction is impaired by a HF diet; this may be linked to the increased sympathetic drive observed with obesity.

PE
U46619

Max contraction (mN)
EC50
Max contraction (mN)
EC50

Control
8.3±0.4
−7.0±0.1
20.4±0.6
−8.0±0.3

HF
6.8±0.6 *
−6.7±0.1*
20.0±0.7
−7.8±0.4

Different from control
A kinetic model for sodium transport via a non‐gastric H+(Na+)/K+ ATPase
Authors: Nadal-Quiros Monica , Nieves-Gonzalez Aniel , Moore Leon C. , Marcano Mariano
Published: June 2021

Experimental results have suggested that macula densa (MD) cells have a unique way of regulating cellular sodium concentration via an apical H+(Na+)/K+‐ATPase. We developed a kinetic model to represent the sodium transport via this transporter. The model assumes a 2:2 stoichiometry for both the hydrogen and sodium cycles. Rate constant values for the hydrogen cycle were obtained from a renal H+/K+‐ATPase model (A.M. Weinstein, Am J Physiol Renal Physiol 274:F856‐F867, 1998) which were, in turn, based on values obtained from a gastric H+/K+‐ATPase model (P. Brzezinski et al., Biochim. Biophys. Acta 942:215–219, 1988). Rate constants for the sodium cycle are assumed to be equal to those in the hydrogen cycle. Results showed that sodium extrusion is very sensitive to changes in luminal potassium concentration from 0 to 20 mM. In addition, sodium extrusion as a function of internal sodium concentration saturates at ~70 mM. This ATPase pump model is essential in the development of larger scale models of ion transport by the MD cells.

Research supported by MBRS‐RISE grant 2R25GM061151‐09 and by NIH‐NIGMS grant SC1GM084744.
Role of the epithelial sodium channel (ENaC) in the development of salt‐sensitive hypertension
Authors: Pavlov Tengis S. , Levchenko Vladislav , Ilatovskaya Daria V. , O'Connor Paul M. , Cowley Allen W. , Staruschenko Alexander
Published: June 2021

Dahl salt‐sensitive (SS) rats fed a high salt diet exhibit increased blood pressure and renal damage compared with SS rats fed a low salt diet and consomic, salt‐resistant SS‐13BN rats. Amiloride‐sensitive sodium entry, via ENaC, is the rate‐limiting step for Na+ absorption in the aldosterone sensitive distal nephron (ASDN). Previous studies reported that the handling of salt and water in the kidney of SS rats is defective and these defects are mediated by enhanced Na+ reabsorption in the TAL of the loop of Henle. The contribution of ENaC‐mediated pathways in the ASDN in the development of salt‐sensitive hypertension is poorly understood. Initially we studied an effect of ENaC inhibitors on MAP in vivo. Benzamil, given through drinking water (15 mg/L), and amiloride given i/v (6 mg/kg/day) preclude the development of hypertension in SS rats. Western blotting and immunohistochemistry demonstrate that expression of ENaC subunits is increased in SS rats fed a high salt diet compared to SS rats fed a low salt diet and SS‐13BN rats. Patch clamp analysis of ENaC activity in split opened tubules also demonstrated upregulation of ENaC activity in SS rats fed a high salt diet compared to control SS and consomic SS‐13BN rats. Thus, we hypothesize that ENaC is upregulated in SS rats fed a high salt diet and that ENaC‐mediated sodium absorption in the ASDN is of critical importance in the development of salt‐sensitive hypertension.
A carbohydrate restricted – high fat diet reduces blood pressure in spontaneously hypertensive rats without causing insulin resistance
Authors: Jalili Thunder , Bosse John , Dolinsky Vernon , Zhang Quan-Jiang , Abel E. Dale , Symons J. David
Published: June 2021

We hypothesized that a low carbohydrate ‐ high fat (LC) diet will attenuate blood pressure (BP) and cardiac hypertrophy in spontaneously hypertensive rats (SHR). SHR (6 wk old, n=54) and Wistar Kyoto rats (WKY, 6 wk old, n=53; normotensive control) were fed control (C, 70% carbohydrate, 10% fat, 20% protein) or LC diet (20% carbohydrate, 60% fat, 20% protein). After 10 wk mean arterial BP was 11 mmHg lower (p<0.05) in SHR‐LC vs. SHR‐C, however, cardiac S6K phosphorylation was augmented (p<0.05) and heart:tibia length was unchanged. Compared to SHR‐C, mesenteric arteries of SHR‐LC had improved (p<0.05) endothelium dependent (acetylcholine) & independent (sodium nitroprusside) relaxation, reduced (p<0.05) contraction (potassium chloride, phenylephrine), and increased (p<0.05) eNOSSer1177 phosphorylation. Plasma glucose was similar among all groups. Insulin was lower (p<0.05) in rats fed LC vs. C. SHR‐LC had greater (p<0.05) peripheral insulin sensitivity (insulin tolerance test) vs. WKY‐C. Insulin stimulated (10h fast, 2U/kg IP) phosphorylation of AktSer473 and S6 in heart and gastrocnemius was not altered SHR‐ LC vs. C. In conclusion, an LC diet reduced blood pressure and improved arterial function without producing insulin resistance or altering insulin mediated signaling in heart and skeletal muscle.
Dietary isoflavones and supplemental selenium show interactive effects on blood‐glucose homeostasis in male FVB mice
Authors: Cardon Brandon R. , Stallings Michael T. , Brunson Scott E. , Hart Chris M. , Swiss Maria D. , Hepworth Squire D. , Christensen Merrill J. , Hancock Chad R.
Published: June 2021

Isoflavones and supplemental selenium have shown interactive effects on body weight, body fat, and blood‐glucose homeostasis. To further elucidate the effects of the interaction, FVB mice were fed either high or low‐isoflavone (HIF or LIF) diets, with or without supplemental selenium. Custom diets were designed to be equivalent in vitamin, mineral and amino acid profiles, but to differ in soy protein and specific isoflavone content. The HIF diet was tested to ensure 500mg/kg aglycone equivalents of genistein + daidzein. Selenium was administered by gavage daily (3mg/kg body wt/day) as Se‐methylselenocysteine, either from conception (Sel‐C), or weaning (Sel‐W). All groups were compared against a LIF‐no‐Sel control group. After 5 months HIF‐Sel‐W mice had elevated fasting glucose levels (HIF‐no sel 106.2 ± 2.76, HIF‐Sel‐W 121.6 ± 2.79 mg/dL, p=.008). LIF‐Sel‐W mice followed the same trend, but values were not significant (p=.109) Homeostatic Model Assessment of Insulin Resistance showed a 274% ± 36% increase of insulin resistance in the LIF‐Sel‐C group (p=.034), but not in HIF groups. Although blood‐glucose and insulin levels were affected by treatments, body weights and abdominal fat pads were not different, contrary to findings in other models. These results demonstrate an interactive effect of dietary isoflavone content and supplemental selenium on glucose regulation in the absence of changes in body fat.
Renal extracellular matrix in three rat‐models of hypertensive kidney damage: A microarray study of SHR, SHRSP and 2K1C
Authors: Skogstrand Trude , McBride Martin , McClure John , Hultström Michael
Published: June 2021

Introduction

The aim of this work was to identify common genetic pathways in kidneys of three hypertensive rat models during development of kidney damage.

Methods

Kidney microarray expression profiles were determined from 60‐week‐old Spontaneously Hypertensive Rats (SHR) versus Wistar Kyoto (WKY); 21‐week‐old salt‐loaded Stroke‐Prone SHR (SHRSP), and 30‐week‐old Wistar rats with two kidney, one clip (2K1C) hypertension for 24 weeks versus sham‐operated controls.

Results

Differentially expressed genes (FDR < 0.05: 2033 in 2K1C, 3721 in SHR, and 519 in SHRSP) identified 93 common between models. Top genes by rank‐sum were: SPP1, TIMP1, LCN2, COL3A1, WTAP, FN1, KIFC1, SLPI, MGP, TNFRSF12A. Analysis of gene ontology showed enrichment in extracellular matrix (p<2e‐8), cell‐substrate adhesion (p<0.001), defence response (p<1e‐4), and protein binding (p<0.005). Ingenuity Pathway Analysis identified cardiovascular inflammation (p<1e‐8) and renal injury (p<1e‐5) as two major activated pathways.

Conclusion

Comparing three models of hypertensive kidney damage identify a common core of extracellular matrix and cell‐matrix binding genes activated during the development of kidney damage in hypertension.

Funding from the Health Authority of Western Norway, Haukeland University Hospital Strategic Research, Lars Hiertas Minne, Magn. Bergvall, British Heart Founation.
Febuxostat inhibition of xanthine oxidase does not impact blood pressure in the established DOCA‐salt hypertension
Authors: Szasz Theodora , Davis Robert Patrick , Garver Hannah S , Burnett Robert J , Fink Gregory D , Watts Stephanie W
Published: June 2021

Xanthine oxidase (XO) has been implicated in cardiovascular disease such as hypertension. We have previously shown that allopurinol inhibition of XO does not attenuate hypertension in the rat DOCA‐salt model. However, this treatment raised questions of efficacy and specificity. We now hypothesized that febuxostat (FBX), a newer and more specific XO inhibitor, would decrease blood pressure (BP) of DOCA‐salt hypertensive rats through a vascular mechanism. FBX (5 mg/kg/day) or vehicle was administered orally for 2 weeks to male DOCA rats after 3 weeks of DOCA‐salt treatment. BP was measured via radiotelemetry. XO inhibition was assessed by HPLC measurement of XO product UA and substrates hypoxanthine and xanthine in serum, urine and tissues. End‐organ damage was grossly evaluated by measuring heart and kidney weight, as well as aorta wall/lumen ratio. FBX administration resulted in significantly reduced product and increased substrate levels in serum and tissues (serum UA ug/mg protein: vehicle=3.43±0.68, FBX=0.63±0.13). However, this treatment did not alter BP of DOCA rats or improve end‐organ damage. Additionally, contractile responses to norepinephrine, endothelin‐1 and angiotensin II of aorta and vena cava were unmodified by FBX incubation. We conclude that XO inhibition does not impact established hypertension in the DOCA‐salt rat model. Support provided by Takeda Pharmaceuticals North America, Inc.
Epigenetically‐induced systemic vascular dysfunction and hypertension in offspring of restrictive diet pregnancy in mice
Authors: Rexhaj Emrush , Rimoldi Stefano , Giacobino Ariane , Sartori Claudio , Allemann Yves , Scherrer Urs
Published: June 2021

Children born from mothers with preeclampsia display generalized vascular dysfunction. In mice, hypoxia induces pulmonary vascular dysfunction by an epigenetic mechanism in offspring of restrictive diet pregnancy (RDP) (a model mimicking preeclampsia in humans). However, little is known regarding systemic vascular function in this model.

We therefore assessed vascular function in vitro (mesenteric artery) and in vivo (blood pressure, carotid artery) in offspring of RDP (12 wks) and control mice.

In offspring of RDP, endothelium‐dependent mesenteric‐artery vasodilation in vitro was markedly impaired (P<.0001 vs. control) and was associated with arterial hypertension in vivo (119±7 vs. 112±5 mmHg, X±SD, P=.04 vs. ctrl). To test for an epigenetic mechanism, we examined DNA methylation of imprinted genes and the promoter of the eNOS gene in the aorta of male offspring of RDP and control mice. In offspring of RDP the DNA methylation of imprinted genes Peg3 (P<.03), Snrpn (P<.03), Gtl2 (P<.01) and the promoter of the eNOS gene (P= .01) were altered compared to control mice. This vascular dysfunction was transmitted to the next generation further suggesting an epigenetic mechanism.

We demonstrate for the first time that RDP induces systemic vascular dysfunction by an epigenetic mechanism. We speculate that in humans, preeclampsia induces vascular dysfunction by a similar mechanism.
Dexamethasone Differentially Regulates Blood Pressure in Transgenic Mice Containing ‐217A/G Polymorphism of Human Angiotensinogen Gene
Authors: Pandey Varunkumar G. , Jain Sudhir , Maharjan Shreekrishna , Mopidevi Brahmaraju , Arudra Sri Krishna Chaitanya , Kumar Ashok
Published: June 2021

The human angiotensinogen (hAGT) gene locus is associated with hypertension. Earlier studies have shown that hAGT gene has an A/G polymorphism at ‐6 position and ‐6A allele is associated with increased blood pressure. We have shown that this gene also contains an A/G polymorphism at ‐217, and frequency of ‐217A allele is increased in African‐American hypertensive patients. Nucleotide sequence of hAGT gene promoter containing ‐217A has homology with the consensus CCAAT/enhancer binding protein (C/EBP) and GR binding site. Our in vitro studies show that dexamethasone (Dex) differentially increases the promoter activity of plasmid containing haplotype ‐217A as compared to the plasmid with haplotype ‐217G. Therefore, we have generated transgenic mice containing either haplotype ‐217A or ‐217G of the hAGT gene. These transgenic mice were treated with Dex (25ug/mL in drinking water) for 1 week. We found that mice containing haplotype ‐217A has 2.3 fold and 3.5 fold dex induced expressioin of hAGT in liver and kidney respectively whereas no significant changes were observed in haplotype ‐217G. Additionally, the SBP of mice containing haplotype ‐217A increases by 15mmHg after Dex treatment as compared to 6mmHg in the mice containing haplotype ‐217G. Results from these experiments suggest that glucocorticoids may cause differential increase in blood pressure in individuals with ‐217A haplotype as compared to those with ‐217G haplotype.

Supported by NIH grants to AK
Epigenetic mechanism of atherosclerosis and hypertension in Hyperhomocysteinemia
Authors: Narayanan Nithya , Tyagi Neetu , Pagni Sebastian , Tseng Michael T , Tyagi Suresh C
Published: June 2021

Although elevated levels of homocysteine (Hcy), known as Hyperhomocysteinemia (HHcy) is associated with aortic stenosis, dissection and hypertension, the mechanisms are not known. Aortic dissection tissue specimen from patients suffering from aortic aneurysm and stenosis were obtained and the mRNA levels of CBS, MTHFR, SAHH, PRX, NOX, Collagens, Elastin, Tissue Inhibitor of Metalloproteinases (TIMPs) and Matrix Metalloproteinases (MMPs) were measured. The levels of CBS, MTHFR, TIMP1, TIMP4, MMP1 and SAHH were high in aortic dissection samples compared to the controls. Since Hcy plays an important role in regulating the DNA methylation and to study the epigenetic control behind Hcy‐mediated atherosclerosis and hypertension, the methylation levels of CBS and MBD2 were measured. Bisulfite treated DNA was amplified using CBS and MBD2 primers and the level of methylation was measured in cardiac tissue of wild type (WT), CBS (−/+), TIMP2 (−/−) and MMP9 (−/−) knockout mice. CBS was shown to be hypermethylated in CBS (−/+), TIMP2 (−/−) and MMP9 (−/−) when compared to WT samples. Also elevated mRNA levels of DNMT3a was observed in CBS (−/+) mouse. These findings suggest that differential expression and methylation levels of genes associated with Hcy metabolism are observed in human aortic stenosis and hypertension.
Progressive increase in renal sympathetic nerve activity in cold‐induced hypertension in rats
Authors: Miki Kenju , Yagi Kana , Yoshimoto Misa
Published: June 2021

The study investigated how renal sympathetic nerve activity was involved in the development of cold‐induced hypertension in rats. Male Wistar rats were chronically instrumented to allow recording of renal (RSNA), lumbar (LSNA) sympathetic nerve activity, measurement of systemic arterial pressure and infusion of vasoactive drugs. At least three days after the surgery, rats were placed in an environmental temperature of 10 °C for 96 hours. The cold exposure caused an immediate increase in arterial pressure from 103 }2 to 118 }1 mmHg (P<0.05) associated with simultaneous increases in RSNA, of 55%, LSNA, of 211% and heart rate, of 86beats/min (all P<0.05). Subsequently, RSNA increased further in a progressive manner reaching 152% at the end of the cold exposure while LSNA and heart rate remained at the initial increased levels. These data suggest that cold exposure activates RSNA in a cumulative manner, which may contribute to the development of cold‐induced hypertension in rats. JSPS Grant.
Role of Substance P in Renal Injury during DOCA‐salt Hypertension
Authors: Wang Youping , Wang Donna H
Published: June 2021

To determine the role of substance P (SP) in salt‐sensitive hypertension‐induced renal damage, we induced hypertension by uninephrectomy and deoxycorticosterone (DOCA)‐salt in mice with or without selective NK1 antagonists. DOCA‐salt treatment for 5 weeks increased mean arterial pressure (MAP) (P<0.01) and urinary albumin excretion (P<0.05), and also induced renal hypertrophy (P<0.01) compared with the control. Periodic acid‐ Schiff and Masson's trichrome staining showed that DOCA‐salt treatment caused obvious glomerulosclrosis and tubulointerstitial injury in the renal cortex (P<0.05), and tubulointerstitial fibrosis compared with the control mice. Consistent with the results, renal collagen level determined using hydroxyproline assay was higher in DOCA‐salt‐treated mice compared with the control mice (P<0.05). Blockade of the NK1 receptor with RP‐67580 (8 mg/kg/day, ip) or L‐733,060 (20 mg/kg/day, ip) had no effect on MAP and renal hypertrophy. However, they partially prevented renal morphological damage and reduced the increase in urinary albumin excretion and renal collagen (P<0.05). Our data showed that blockade of NK1 receptors with RP‐67580 or L‐733,060 prevented renal damage induced by DOCA‐salt hypertension independently of their effects on blood pressure. The results suggest that activation of NK1 receptors by SP may contribute to renal damage during DOCA‐salt hypertension. This work was supported in part by the National Natural Science Foundation of China (No. 81170243).
Decreased expression of Gcm1 and its downstream targets Syncytin A and B is associated with severity of feto‐placental phenotypes in the BPH/5 model of preeclampsia (PE)
Authors: Sones Jenny Liford , Zhou Yi , Woods Ashley K. , Davisson Robin L.
Published: June 2021

BPH/5 mice spontaneously develop the cardinal features of PE during pregnancy, including late gestational hypertension and proteinuria, along with fetoplacental abnormalities. We have utilized ultrasonography to categorize fetoplacental units (FPUs) at e10.5 into three increasingly severe phenotypic classes (I, II, and III). We tested the hypothesis that Gcm1, a key transcription factor in early trophoblast differentiation, and its downstream targets, Syncytin A and B, are dysregulated in BPH/5 FPUs at e10.5, and this is associated with phenotypic severity. Real‐time PCR revealed downregulation of each of these genes in compromised Class II and Class III BPH/5 FPUs vs C57 controls: Gcm1 (II= 0.3±0.16; III= 0.01±0.003 fold vs C57, n=3 p<0.05), Syncytin A (II= 0.6±0.21, III= 0.05±0.008 fold vs C57, n=3 p<0.05), and Syncytin B (II= 0.69±0.02, III= 0.30±0.14 fold vs C57, n=3 p<0.05). In situ hybridization for Gcm1, Syncytin A and B confirmed this and localized downregulation to the developing labyrinth. Finally, Western analysis revealed decreased Gcm1 protein levels in BPH/5 Class I (29.1±14.3%, n=4, p<0.01) and Class II (43.4±5.2%, n=4, p<0.01) placentas vs C57 (n=6). This suggests that dysregulation of Gcm1 and its downstream targets Syncytin A and B may be involved in the abnormal labyrinth formation and fetoplacental demise observed in this model of PE. Funding: QNRF NPRP09‐1099‐279.
High fat feeding promotes sympathetic axon outgrowth and cardiac hyperinnervation in male Sprague‐Dawley rats
Authors: McCully Belinda H. , Streiff Cole T. , Habecker Beth A. , Brooks Virginia L.
Published: June 2021

Obese individuals are susceptible to cardiac arrhythmias and sudden cardiac death. Female rats fed a high fat diet (HFD) also exhibit arrhythmic events, which are attributed to sympathetic hyperinnervation of the heart. Whether this occurs in male rats, and whether the hyperinnervation is due to enhanced sympathetic nerve growth, is unknown. To test this, male Spraque‐Dawley rats (~250g) were fed either normal chow (CON; 13.5% kcal fat) or a HFD (33% kcal fat) for 4–5 weeks. HFD rats were separated into obesity‐resistant (OR) or prone (OP) groups, based on the bottom and top tertile of weight gain (CON: 459±21g, OR: 444±5g, OP: 564±12g; P<0.01, no difference between OR and CON). Sympathetic axon outgrowth was quantified in superior cervical ganglia explants. Growth rate (μm/hr) was higher in OR (n=4; 23.9±0.9) and OP (n=6; 21.1±0.9) versus CON (n=3; 17.7±1.9, P<0.05), with similar growth in OR and OP. Immunohistochemical staining for tyrosine hydroxylase (TH; % area), the rate limiting enzyme in norepinephrine synthesis, revealed sympathetic hyperinnervation in the right ventricle of both OR (n=5; 1.5±0.1%) and OP (n=5; 1.6±0.2%) versus CON (n=6; 1.1±0.1, P<0.05), but no differences between groups in the left ventricle. These data suggest that the consumption of a HFD, independent of obesity, stimulates sympathetic nerve growth and can lead to sympathetic hyperinnervation of the heart. Support: AHA (7500041 BHM, 2060630 VLB), NIH (HL068231 BAH).
Metabolic and appetite responses to fasting and refeeding in mice with Shp2 deletion in forebrain neurons
Authors: do Carmo Jussara M , da Silva Alexandre A , Hall John E
Published: June 2021

Previous studies showed that Src homology‐2 tyrosine phosphatase (Shp2) is an important regulator of body weight homeostasis. Whether Shp2 is involved in normal refeeding response after prolonged fasting is still unknown. In this study we examined whether mice with Shp2 deletion in forebrain neurons (Shp2/CamK2‐Cre mice) exhibit impaired appetite and metabolic responses to refeeding after 24‐hrs of fasting. Shp2/CamK2‐Cre (n=6) and control mice (Shp2flox/flox, n=5) at 22‐weeks of age were placed in a controlled apparatus to measure 24‐hr food intake (FI), oxygen consumption (VO2), and body core temperature (BT) before and after a 24‐hr fasting protocol. Shp2/CamK2‐Cre mice were heavier, hyperphagic (4.7±0.3 vs. 3.0±0.3 g), exhibited lower VO2, but similar BT. Fasting reduced body weight (52.0±3.8 to 48.6±3.6 and 32.9±2.2 to 30.3±2.4 g), VO2 (2154±135 to 1898±138 and 2424±197 to 2093±132 ml/kg/hr), and BT (36.5±0.2 to 35.4 ±0.2 and 36.2±0.0 to 35.5±0.1 oC) in Shp2/CamK2‐Cre and Shp2flox/flox mice. The increases in metabolic rate and appetite in response to refeeding were attenuated in Shp2/CamK2‐ Cre compared to control mice (VO2: +6 vs. +11% and FI: +6 vs. +54%). Our results demonstrate that in addition to promote obesity, Shp2 deletion in forebrain neurons is also involved in the normal FI and metabolic responses to refeeding after prolonged fasting. (NHLBI‐PO1HL51971/AHA SDG5680016)
Differential effects of prenatal stress on metabolic programming in diet‐induced obese and dietary resistant rats
Authors: Balasubramanian Priya , Varde Pratibha , Garcia Fernando , Jeyaraj Ninitha Margret Julfiya Asirvatham , MohanKumar P.S. , MohanKumar Sheba M.J.
Published: June 2021

The prevalence of obesity has reached epidemic proportions in the United States. Recent studies suggest that prenatal influences could contribute to the development of obesity and the metabolic syndrome. In this study, we hypothesized that diet‐induced obese (DIO) rats are more susceptible to prenatal stress‐induced metabolic programming compared to dietary resistant (DR) rats. Pregnant DIO and DR (n=5) moms were subjected to chronic stress (restraint stress‐3 times/day for 45 minutes from day 14–21 of gestation) and controls were undisturbed throughout pregnancy. After weaning, male pups were maintained on chow diet for 9 weeks and were placed on 1 week of high fat (HF) diet during the 10th week. Birth weight, weekly body weight and food intake were recorded. At the end of the 10th week, the animals were sacrificed, and we measured blood glucose levels, visceral adipose mass and serum C‐peptide levels. Chronic stress during pregnancy did not affect any of the morphometric or metabolic parameters measured in DR rats. In contrast, the stress paradigm significantly decreased birth weight in DIO male offspring compared to their non‐stressed counterparts. After weaning, there was no apparent difference in body weight between pups from the stressed and non‐stressed DIO dams. However, when placed on a HF diet for 1 week, there were significant increases in body weight in DIO pups from stressed dams compared to non‐stressed dams. This was also accompanied by increases in serum C‐peptide levels. There was no difference in visceral adipose mass and blood glucose levels between the groups. These results suggest that chronic prenatal stress is capable of metabolically programming DIO rats resulting in higher levels of C‐peptide that is most probably responsible for diet‐induced obesity in this model.
Molecular determinant of KCa3.1 that affects cell proliferation?
Authors: Hamilton Kirk L. , Devor Daniel C.
Published: June 2021

It has been demonstrated that the KCa3.1, the intermediate‐conductance Ca2+‐activated K+ channel KCNN4, increases cell proliferation. It still remains to be determined how this occurs. Recently, we reported that wild‐type KCa3.1, a non‐conducting KCa3.1 or non‐trafficking KCa3.1 channel increased cell proliferation when transiently expressed in HEK cells (Millership et al., Am J. Physiol Cell Physiol 300:C792‐C802, 2011). In the present study, we have used chimeric channels of KCa3.1 (IK) and KCa2.2 (SK; has no affect on cell proliferation) to uncover the potential molecular portion of KCa3.1 that alters cell proliferation. Transient transfection of HEK cells with IK increased cell number after 3 days to 131±7% (n = 7) compared to mock‐transfected cells (n = 7) while SK had no effect on cell number (n = 4). HEK cells transfected with a chimeric channel of SK‐200IK (SK+S5‐pore‐ S6‐C‐terminus of IK) significantly increased cell number to 119±3% (n = 5) when compared with mock‐transfected cells, however, SK‐287IK (SK+C‐terminus of IK) did not increase cell count (n = 3). We are currently exploring other chimeric and mutated channels to further define the portion of KCa3.1 responsible for altered cell proliferation. At present, our data suggest that the S5‐pore‐S6 portion of KCa3.1 is crucial for modulation of cell proliferation. This work was funded by Department of Physiology at UoO (KLH) and NIH (DCD).
Expression and functional significance of Ca2+‐activated Cl‐channels in dendritic cells
Authors: Shumilina Ekaterina , Szteyn Kalina , Schmid Evi , Nurbaeva Meerim K. , Yang Wenting , Kunzelmann Karl , Lang Florian
Published: June 2021

The functions of dendritic cells (DCs), professional antigen presenting cells linking innate and adaptive immunity, are under regulation of Ca2+. Ca2+ entry is sensitive to the membrane potential and may depend on Cl− channel activity. The present study employed whole‐cell patch clamp experiments on mouse bone marrow‐derived DCs to identify expression and regulation of Cl− channels. Outwardly rectifying Cl− currents were activated by elevation of cytosolic Ca2+ triggered either by ionomycin in the presence of extracellular Ca2+ or by inositol‐triphosphate‐induced Ca2+ release from intracellular stores. Most importantly, Ca2+‐activated Cl− channels (CaCCs) were activated by the chemokine CCL21, which induces DC migration to the lymph nodes. The currents were inhibited by Cl− channel and CaCC blockers: tannic, digallic and niflumic acids and AO1. According to Fura2 fluorescence, the CaCC blockers blunted the store operated Ca2+ entry following depletion of intracellular Ca2+ stores with thapsigargin or upon stimulation with CCL21. The CaCC blockers further inhibited spontaneous, and chemokine‐ (CXCL12, CCL21)‐induced migration and secretion of interleukin‐2 (IL‐2) and IL‐6. According to RT‐PCR and Western blot data, Anoctamin 6 is expressed and can contribute to CaCCs in DCs. Our data identify Ca2+‐activated Cl− channels in mouse DCs as major players in Ca2+ homeostasis and Ca2+‐regulated DC functions.
Effect of clinical mutations on functionality of the hRFT‐2 in intestinal epithelial cells
Authors: Nabokina Svetlana , Subramanian Veedamali S , Said Hamid M
Published: June 2021

The Brown‐Vialetto‐Van Laere syndrome (BVVLS) is a rare neurological disorder believed to be due to mutations in the C20orf54 gene, which encodes the human riboflavin (RF) transporter −2 (hRFT‐2). However, there is nothing known about the effect of these mutations on functionality of hRFT‐2, a protein that is expressed in a variety of tissues with high expression in the intestine. We addressed this issue for a number of the mutants using the Caco‐2 cells. Our results showed significant inhibition in RF uptake in Caco‐2 cells expressing W17R, P28T, E36K, E71K, and R132W hRFT‐2 mutants. The inhibition in RF transport in these mutants were not due to a decrease in transcription and/or translation of the hRFT‐2, since mRNA and protein levels of the carrier were similar in mutants and wild‐type hRFT‐2. Confocal images of live Caco‐2 cells transfected with hRFT‐2 mutants (fused with GFP) showed the P28T, E36K, E71K, and R132W mutants were retained within the ER, while the W17R mutant was expressed at the cell membrane; the latter was further confirmed by cell surface biotinylation assay. These results show for the first time that BVVLS associated mutations in hRFT‐2 affect the transporter functionality and that this effect is mediated via alterations in membrane targeting and/or activity of the transporter. [Supported by grants from the DVA and NIH (DK 58057 and DK 84094)].
Epigenetic control of lung fluid clearance
Authors: Wujak Lukasz Andrzej , Becker Simone , Arnoldt Verena , Morty Rory Edward
Published: June 2021

The Na,K‐ATPase regulates ion transport and alveolar fluid clearance (AFC) in the lung. TGF‐β mediates acute lung injury (ALI), and impacts AFC. We hypothesized that TGF‐β impacts Na,K‐ATPase activity by modifying subunit stoichiometry. Cell surface density of the Na,K‐ATPase is regulated by the ATP1B1 subunit. ATP1B1 expression was reduced both in TGF‐β‐treated lung epithelial cells and in lungs from ALI patients or mice with experimental (bleomycin‐induced) lung injury. Luciferase, surface biotinylation and 86Rb+ uptake assays demonstrated that TGF‐β reduced Na,K‐ATPase gene expression, activity, and cell‐surface density, respectively, in primary lung epithelial cells. RNA interference and chromatin immunoprecipitation studies revealed that ATP1B1 downregulation relied on SMAD4, SNAI1 and E2F5 transcription factors, and was critically dependent on HDAC2. The HDAC inhibitor, Trichostatin A, improved edema resolution in experimental lung injury. Collectively, our data suggest that TGF‐β downregulates ATP1B1 expression leading to decreased Na,K‐ATPase activity and consequently impaired AFC in ALI, and that these processes can be reversed by HDAC inhibition.

German Research Foundation
Identification of proteins regulated by 24‐hour aldosterone treatment in late distal convoluted tubules, connecting tubules and initial cortical collecting ducts
Authors: Jensen Thomas Buus , Pisitkun Trairak , Hoffert Jason D , Fenton Robert A , Praetorius Helle A , Jensen Uffe Birk , Knepper Mark A , Praetorius Jeppe
Published: June 2021

Proteomic studies of late distal convoluted tubule and connecting tubule have so far been hindered by insufficient yield and purity of manually isolated tubular cells. Our aim was to determine the effects of 24‐hours elevated plasma aldosterone on the protein expression profile in these tubules. Mice endogenously expressing enhanced green fluorescent protein in the late distal convoluted tubule, connecting tubule, and initial cortical collecting ducts were treated with a single subcutaneous injection of 2.0 mg/kg aldosterone or vehicle. Before the sacrifice, the plasma aldosterone was 5.6‐fold increased in the aldosterone treated mice. Suspensions of single viable kidney cells were subjected to fluorescence activated cells sorting, yielded 1.4 million cells per mouse with over 69% purity. Three biological replicates of pooled protein samples from 10 mice (100 μg) in each vehicle or aldosterone treated group were digested with trypsin and labelled with 8‐plex iTRAQ reagents. Strong cation exchange yielded 18 fractions for liquid chromatography tandem mass spectrometry. The resulting spectra were matched to peptides by the SEQUEST search algorithm, which identified 3904 peptides corresponding to 506 unique proteins. A change in abundance was observed in several of these proteins after 24‐hour aldosterone treatment. Among the increased proteins, pyruvate dehydrogenase E1 and pyruvate kinase isozyme M2 were confirmed by immunohistochemistry. These enzymes are involved in the carbohydrate metabolism, suggesting an increased capacity for energy production after 24‐hours aldosterone treatment.
Quantification and Analysis of Advanced Glycation End Products in avian plasma
Authors: Gu Mary , Ferguson Donna , Braun Eldon J
Published: June 2021

Comparison of avian plasma glucose concentrations with those of mammals of similar body mass shows that birds have much higher levels of plasma glucose. Hyperglycemia in diabetic patients can lead to the binding of glucose to the lysine residues of serum albumin to form glycated albumin. Through a series of reactions, the last of which is non‐reversible, this glycated albumin is converted to Advanced Glycation End‐products (AGEs). Pentosidine, a well‐characterized AGE, has been quantified in humans and has been related to age and diabetic complications. Does the high level of glucose in avian plasma lead to high levels of AGEs? In our study using HPLC, we quantified pentosidine to determine if there is a relationship between pentosidine and glucose in avian (mourning dove) plasma. The results of our HPLC analysis of avian plasma indicated a pentosidine concentration of 44 × 10−3 pmol/μl (14.9 × 10−3). This can be compared to 100 × 10−3 pmol/ μl (56 × 10−3) in normal human plasma. Thus, our results indicate a pentosidine concentration in avian plasma that is 2.3 times lower than that of humans suggesting that the high plasma glucose concentrations of birds do not lead to high levels of AGEs (pentosidine). Moreover given these data, the ratio of glucose to pentosidine is much higher for birds (0.44 × 10−9) than that for humans (0.05 × 10−9).
Molecular mechanisms regulating cardiac contractility: subfunctionalization of fish‐specific paralogs of troponin C in Danio rerio
Authors: Genge Christine Elizabeth , Tibbits Glen F
Published: June 2021

Myofilament Ca2+ sensitivity is a critical factor in regulating cardiac contractility. The key regulator, cardiac troponin (cTn), is made up of three proteins (cTnC, cTnI, cTnT). Though Ca2+‐activated component of cTn,TnC is highly conserved across phylogenetically diverse groups, subtle variations are seen as interspecies orthologs and intraspecies paralogs between tissues. In mammals, two forms of TnC exist with distinct localization patterns (fast skeletal muscle and slow skeletal/cardiac muscle) and Ca2+ affinities, but in fish a third paralog exists annotated ssTnC in zebrafish. Tissue‐specific mRNA profiles in adult zebrafish revealed a distinct localization of gene expression, with ssTnC preferentially expressed in the atrium and cTnC is almost exclusively expressed in the zebrafish ventricle, which may reflect the differing contractile properties between the cardiac chambers. The functionality of these differences is being compared biochemically through measurement of Ca2+ binding kinetics. This subfunctionalization of TnC isoforms lends insight the consequences of variation to the structure‐function of cTn and variation in cardiac muscle contraction.
Mitophagy causes coronary artery endothelial dysfunction in oxidative stress dose‐dependent (i.e. C57>FVB>C3H mice) manner during right ventricle failure (RVF)
Authors: Qipshidze Natia , Tyagi Suresh C
Published: June 2021

Coronary artery disease remains the leading cause of mortality due to pulmonary artery hypertension. We hypothesized that the coronary vascular reactivity is decreased due to mitophagy during elevated levels of oxidative stress, causing coronary vasospasm during RVF. This leads to fibrosis in the coronary, imbalance in MMP/TIMP ratio and NO production by eNOS. Treatment with Mdivi‐I mitigates mitophagy by diminishing oxidative stress, leading to improvement of the coronary artery function. To verify this, C57BL6/J (less), FVB (intermediate), and C3H (most resistant to oxidative stress) strains of mice underwent pulmonary artery constriction (PAC) for 4 weeks with or without the Mdivi‐I (50 mg/kg/day) treatment. Septal and LAD coronary arteries were isolated and reactivity to ACH was measured. Septal coronary arteries isolated from C57‐PAC mice developed more constriction than FVB, and more than C3H mice. LAD isolated from C57‐PAC mice developed constriction. LAD from FVB‐PAC mice does not have any change in reactivity to ACH, and LAD from C3H‐PAC mice had dilatation as in Sham group of mice. Mdivi‐I treatment mitigates this constriction and abolished mitophagy in C57 mice. These results suggest that, Mdivi‐I treatment reduces oxidative stress and ameliorates mitochondrial dysfunction that results in protection of coronary endothelial function during RVF in oxidative stress dose‐dependent manner.
AMPK activation by AICAR induces mitochondrial biogenesis in the inner ear
Authors: Nuttall Alfred L. , Wilson Teresa , Omelchenko Irina , Shi Xiaorui
Published: June 2021

Rationale

The importance of mitochondrial biogenesis to auditory hair cell physiology or that of the non‐sensory cells in the cochlea is poorly understood. Activation of 5′ AMP activated protein kinase (AMPK) upregulates peroxisome proliferator–activated receptor‐γ coactivator‐1α (PGC‐1α) which, in turn, increases the levels of factors involved in mitochondrial biogenesis and respiration. At the same time, AMPK activation can increase antioxidant expression and reduce reactive oxygen species generation.

Objective

To determine whether the AMPK activator 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside (AICAR) can induce mitochondrial biogenesis in the inner ear and protect against noise induced hearing loss.

Methods and Results

AICAR treatment in mice led to higher levels of phosphorylated AMPKα in the cochlea by immunoblot and immunohistochemistry (IHC) analysis. Quantitative real‐time PCR revealed increased PGC‐1α mRNA levels. Extended AICAR treatment increased mitochondrial fission protein 1 and superoxide dismutase 2 protein levels. Auditory brainstem response analysis demonstrated that AICAR treatment accelerated recovery from acoustic trauma.

Conclusion

These results demonstrate that mitochondria in the inner ear have the capacity to be dynamically regulated with increasing energy demands.

Supported by grants 5R01DC000105 (ALN), 1R01DC010844 (XS), and P30DC005983.
(−)‐Epicatechin attenuates prednisolone induced‐hyperglycemia
Authors: Torres-Gonzalez Moises , Ceballos Guillermo , Villarreal Francisco
Published: June 2021

The chronic use of steroids in humans is known to increase blood glucose levels. The consumption of (−)‐epicatechin (Epi) rich cocoa has been associated with improvements in insulin sensitivity and increases in mitochondrial volume and cristae density. During 15 days, rats were treated with prednisolone (Pred) (10 mg/Kg/day, SC) and received either Epi (1 mg/kg/twice a day) or water by gavage, to evaluate the effects of Epi on Pred‐induced hyperglycemia and glucose control systems. Rats not treated with Pred and not receiving Epi served as controls. Epi treated rats demonstrated lower fasting glucose levels than Pred only rats (p<0.05). Epi treated rats also displayed glucose tolerance comparable to that of controls. Western blot analysis of skeletal muscle demonstrated minor changes in proteins levels involved in the insulin‐signaling pathway by Epi. Pred reduced protein levels of the oxidative phosphorylation (OXPHOS) respiratory complexes. However, Epi treatment was able to maintain OXPHOS levels similar to controls. The protein levels of the catalytic subunit E1α of the pyruvate dehydrogenase were increased by Epi. These findings suggest that Epi protection against Pred‐induced hyperglycemia may be due to an improvement in mitochondrial function and glucose metabolism.