bluuebunny/crossref_metadata_2025 huggingface dataset:
miR155 Expression is Increased by Inflammation and Modulates the Expression of CD11a in Monocytes
Authors: Syed Safiya , Amin Mohammad , Rabquer Bradley
Published: June 2021

Inflammation is a key process in innate immunity and helps drive the adaptive immune response. In addition, inflammatory mediators are often dysregulated in autoimmune diseases. Monocytes are key players in inflammation that reside in the blood and migrate into tissues to exert their effects. Adhesion molecules are molecules that are located on the surface of many cells and are necessary for monocyte migration. The adhesion molecule CD11a forms a complex with CD18, also known as lymphocyte function‐associated antigen‐1 (LFA‐1), and interacts with intercellular adhesion molecule‐1 (ICAM‐1) to facilitate tight binding of monocytes to the endothelium. miRNAs are known to regulate gene expression in a variety of cell types and in response to various stimuli. We hypothesized that inflammation regulates miRNA expression in monocytes and that these miRNAs play specific roles in regulating adhesion molecule expression and monocyte migration. THP‐1 monocytes were cultured, stimulated with TNF‐α, and miRNA was isolated (n=6 replicates). cDNA was prepared and qPCR was performed using primers specific for miR155, miR181a, miR181c, miR15a, miR9‐1, miR429, and miR466k. TNF‐α stimulation increased the expression of all the miRNAs, except for Mir9‐1. miRNA inhibitors were then used to assess the role of these miRNAs in regulating the expression of CD11a. After confirming inhibitor specificity and efficiency, we found that the inhibitor of miR155 reduced the expression of CD11a mRNA by 93%. In conclusion, we found that miRNAs are inducible by TNF‐α in THP‐1 monocytes and that miR155 regulates the expression of CD11a.
Platelet‐Endothelial Association with Fibrinogen/Fibrin, Coupled with Oxidative Stress, Protein Nitrosylation, and Fibrosis may underlie Pulmonary Endothelial Cell Dysfunction in a Mouse Model of Type 1 Diabetes
Authors: Roberts Andrew , Jagadapillai Rekha , Dixon James , Tan Yi , Cai Lu , Gozal Evelyne
Published: June 2021

Diabetes is characterized by inflammation and abnormal microvascular function. Pulmonary complications, although not well characterized, have been reported in diabetic patients. We hypothesized that oxidative stress, altered nitric oxide synthesis and enhanced fibrinogen‐endothelial interaction in the diabetic lung may underlie pulmonary vascular dysfunction. FVB mice, 8‐10 weeks‐old, were injected with streptozocin (FVB DM; 50mg/kg for 5 days) to induce type 1 diabetes, or with vehicle (FVB Ctr). Lung superoxide formation (DHE) and protein nitrosylation (3‐NT) were assessed after 5 months as indicators of oxidative stress, and type I and type III collagen deposition (Sirius Red) as a marker of pulmonary fibrosis. Superoxide production (152.74 ± 9.11 DM vs. 110.23 ± 3.34 Ctr) and collagen deposition (259.47 ± 8.9 DM vs. 192.89 ± 17.68 Ctr) significantly increased in FVB‐DM (p<0.05; n=3). Protein nitrosylation was elevated, but did not reach significance (102.61 ± 19.18 DM vs. 84.97 ± 10.48 Ctr). Fibrinogen, endothelial cell (CD‐31) and platelet (CD‐61) interactions, assessed by immunochemical staining, showed enhanced expression and co‐localization in FVB DM mice. These data indicate that the lung may be an important target of diabetes‐induced inflammatory injury, inducing decreased nitric oxide bioavailability, significant pulmonary microvascular injury and fibrosis. We conclude that diabetic pulmonary complications could lead to ventilation‐perfusion abnormalities that would exacerbate systemic complications of diabetes.
Ascorbic Acid Supplementation: Influence of Delivery Method on Vitamin C Bioavailability and Capacity to Protect Against Ischemia‐Reperfusion Injury
Authors: Davis J , Paris H , Beals J , Binns S , Giordano G , Scalzo R , Schweder M , Blair E , Bell C
Published: June 2021

Intravenous administration of vitamin C has been shown to decrease oxidative stress and, in some instances, improve physiological function in adult humans. Oral vitamin C administration is typically less effective, due in part to decreased vitamin C bioavailability. The purpose of this study was to determine the efficacy of oral delivery of vitamin C encapsulated in liposomes. On 4 separate occasions, occurring in random order, 11 obese adults (age: 53 ± 2 years; body mass index: 34.1 ± 1.0 kg/m2 (mean ± SE)) were administered an oral placebo, or 4 g of vitamin C via: oral, oral liposomal, or intravenous delivery. The area under the plasma vitamin C concentration curve, determined over 4 hours, was greatest (P < 0.001) following intravenous administration (57.0 ± 6.9 (mg/dL)·h). Liposomal vitamin C delivery (10.3 ± 0.9) evoked a greater (P = 0.002) area under the curve than oral (7.6 ± 0.4) that in turn was greater (P < 0.001) than placebo (3.1 ± 0.4). Forearm ischemia‐reperfusion (20 minutes of occlusion) increased circulating thiobarbituric acid reactive substances, a marker of oxidative stress, 40 minutes post‐reperfusion (P < 0.03); this increase was prevented by all of the vitamin C treatments (P > 0.05). These data suggest that oral delivery of vitamin C encapsulated in liposomes: 1) produces circulating concentrations of vitamin C that are greater than traditional oral administration; and, 2) provides protection from ischemia‐reperfusion that is similar to the protection provided by intravenous administration.

Support: Empirical Labs
Black currant Suppresses Metabolic Syndrome induced by High–Fructose Diet
Authors: Park Ji Hun , Kho Min Chul , Kim Hye Yoom , Tan Ruy , Lee Yun Jung , Kang Dae Gil , Lee Ho Sub
Published: June 2021

Metabolic syndrome is a medical disorder characterized by obesity, hyperglycemia, dyslipidemia, and hypertension. Black currant (Ribes nigrum L, BC) has been used for horticultural crop in europe nations. The present study was designed to investigate the beneficial effect of BC on metabolic syndrome. To induce metabolic syndrome, rats were fed the 65% high fructose diet. Treatment with BC (100 or 300 mg/kg/day BC for 8 weeks) significantly suppressed the increments of epididymal fat weight, blood pressure, plasma total cholesterol (T‐cho), triacylglycerol (TG), LDL‐cholesterol, and insulin levels and ameliorated oral glucose tolerance, respectively, in the metabolic syndrome rat. In addition, BC markedly prevented increases of adipocyte size and hepatic accumulation of TG. BC ameliorated endothelial dysfunction by down regulation of endothelin‐1 (ET‐1) and adhesion molecules in the aorta. Moreover, vascular relaxation of thoracic aortic ring by acetylcholine was improvement by BC. These findings suggest that BC ameliorates dyslipidemia, hypertension, insulin resistance in rats with Metabolic syndrome. Taken together, BC may be used for therapeutic approach for metabolic syndrome.
Assessment of Endoplasmic Reticulum Stress by Graded Sarcoplasmic Reticulum Ca2+ Store Depletion in Coronary Smooth Muscle
Authors: Dineen Stacey , Sturek Michael
Published: June 2021

This study examined the threshold of sarcoplasmic reticulum (SR) Ca2+ store depletion necessary for induction of endoplasmic reticulum (ER) stress in coronary smooth muscle cells (CSM). Coronary arteries from Ossabaw swine with and without coronary artery disease (CAD) were sectioned into arterial rings and denuded of endothelium for assessment of ER stress. Phosphorylation of eukaryotic initiation factor 2α was examined by immunoblot as an index of ER stress and was elevated in CAD arteries, compared to healthy. CSM were enzymatically isolated and loaded with the fluorescent intracellular Ca2+ indicator, fura‐2. In arteries with overt CAD, CSM from atherosclerotic plaques were isolated separately from the medial layer. The SR Ca2+ store was released with caffeine in Ca2+‐free physiologic salt solution (0Ca‐PSS) and the resultant transient rise in cytosolic Ca2+ was examined as an index of SR Ca2+ store. The SR Ca2+ store was increased ~1.5‐fold in CSM from arteries with mild CAD compared to healthy, but was decreased in overt CAD. Sarco/endoplasmic reticulum Ca2+ ATPase activity as assessed by recovery of cytosolic Ca2+ below baseline followed the same pattern. Following release with 1, 5, or 10 mM caffeine, the SR Ca2+ store was clamped in 0Ca‐PSS for 30 minutes, after which the remainder of the SR Ca2+ was released. This SR Ca2+ store release was assessed as a percentage of maximum. The SR Ca2+ store was depleted in a dose‐dependent manner, indicating the feasibility of clamping SR Ca2+ independent of cytosolic Ca2+. Together, these results indicate that CSM display ER stress in CAD and that SR Ca2+ store is decreased in overt CAD, providing clues to the mechanisms behind CSM changes underlying CAD progression.

(Support: NIH HL062552)
Resveratrol Attenuates Thromboxane‐Prostanoid Receptor‐Mediated Vasocontraction via NO‐ and Endothelium‐ Independent Mechanisms
Authors: Holder Peter , Denniss Steven , Rush James
Published: June 2021

The purpose of this study was to investigate the effect of resveratrol (RSV) on thromboxane‐prostanoid receptor (TPr)‐mediated vasocontraction. Dose‐response to the TPr agonist U46619 (‐9.0 ‐ ‐6.0 LogM) was examined in vitro in tissue baths using isometrically mounted common carotid arterial rings isolated from 20‐30 week old Wistar Kyoto rats (WKY; n=8) and Spontaneously Hypertensive rats (SHR; n=8). Endothelium‐intact (E+) rings were pre‐incubated (for 30 minutes) with either no drug (ND); the nitric oxide synthase inhibitor L‐NAME (LN; 100 µM); RSV at either 20, 50, or 100 µM; or, LN+RSV‐20µM. Endothelium‐denuded (E‐) rings were pre‐incubated with either ND or RSV‐20µM. The EC50 for U46619 in E+ was decreased in LN vs. ND WKY (LN: ‐8.12±0.05 Log M vs ND: ‐7.72±0.06, p<0.05), but not SHR. The combination effect of E+ LN+RSV‐20 µM significantly decreased the maximum amplitude in WKY but not SHR. Increasing doses of RSV at 50 µM and 100 µM resulted in further increases in EC50 for both WKY (‐7.16±0.04 and ‐6.83±0.08, respectively) and SHR (‐7.17±0.06 and ‐6.80±0.04,respectively), while only WKY exhibited a significant (p<0.05) decrease in the maximum amplitude. In E‐, WKY (RSV‐20µM: ‐7.46±0.08 LogM vs. ND) and SHR (RSV‐20µM: ‐7.63±0.06 LogM vs. ND) had significantly increased EC50 with RSV‐20 µM incubation (p<0.05), while the maximum amplitude remained unaffected. These results suggest that acute pre‐incubation with RSV dose‐dependently attenuates TPr‐stimulated vasocontractile activity by a cell‐signalling mechanism that does not appear to require nitric oxide synthase or the endothelium.

Funded by NSERC Canada
Myocardial Calcium Handling is Up‐Regulated During the Spontaneous Resolution of Endotoxemic Cardiomyopathy in Mice
Authors: Hobai Ion , Morse Justin , Siwik Deborah , Colucci Wilson
Published: June 2021

Sepsis and systemic inflammation induce a specific cardiomyopathy that resolves spontaneously in surviving patients, through mechanisms that are currently unknown. Here we studied cardiomyocyte calcium (Ca2+) handling during endotoxemic cardiomyopathy (EC) resolution in mice.

Male C57Bl6 mice were administered lipopolysaccharide (LPS, 7 μg/g, ip). LPS induced an inflammatory shock syndrome, cardiomyopathy and 59% mortality. We measured sarcomere shortening (SS) and Ca2+ transients (ΔCai) in externally paced cardiomyocytes, at 37o C.

12h after LPS administration, SS and ΔCai were decreased to 53 ± 10% and 78 ± 5% of baseline, respectively (n >30 cells from 3 mice, 30/3, for each group; p<0.05 for this and the following) in association with a decrease in sarcoplasmic reticulum Ca2+ pump (SERCA) reuptake (to 88 ± 5% of control, n > 19/3). In surviving mice, ΔCai was fully recovered 3 days after LPS administration (Day 3, D3) and reached levels exceeding normal at D6 (124 ± 7 % of baseline, n > 18/3). ΔCai recovery was associated with SERCA supra‐normal activation (138 ± 4% of control, n > 28/3) at D3, due to phospholamban (PLB) down‐regulation (to 35 ± 9% of control, n = 6 hearts each group). At D6, membrane Na+/Ca2+ exchange (NCX) function was 51 ± 4 % of baseline (n > 12/3), contributing to the supra‐normal ΔCai levels.

In conclusion, myocardial recovery during the spontaneous resolution of EC is the result of active cellular mechanisms that include down‐regulation of PLB and NCX and lead to supra‐normal ΔCai.
C‐Type Natriuretic Peptide as Mediator of an Endothelial Cell to Mast Cell Communication
Authors: Kuhn Michaela , Gassner Birgit , Rabenhorst Anja , Hartmann Karin , Chen Wen
Published: June 2021

Mast cells (MC) play a critical role in leukocyte recruitment to sites of inflammation after ischemia/reperfusion (I/R). They reside in the perivascular tissue and release proteases and chemokines having detrimental effects on their immediate surroundings, such as endothelial cells. The triggers and counterregulators of MC activation are still enigmatic. We investigated whether endothelial C‐type natriuretic peptide (CNP) via its cGMP forming guanylyl cyclase B (GC‐B) receptor modulates the activity of MC and inflammation after I/R.

Intravital microscopy was used to visualize the microcirculation within the cremaster muscle of anesthetized mice. Blood flow was temporally blocked for 30 min of ischemia. FITC‐Dextran was used to quantify macromolecule leakage during reperfusion. Ruthenium Red was used to stain perivascular degranulated MC. I/R provoked marked degranulation of perivascular resident MC (~7‐fold vs controls) and prominent leakage of FITC‐Dextran from postcapillary venules to the adjacent interstitial space. Local superfusion of CNP during reperfusion, significantly reduced the number of degranulated MC (to ~1.8‐fold vs controls) and concomitantly prevented vascular leakage. These effects were mimicked by the membrane‐permeable cGMP analog, 8‐bromo cGMP. In cultured murine bone marrow and human MC CNP activated GC‐B/cGMP signaling and cGMP protein kinase I‐dependent phosphorylation of the cytoskeleton‐associated vasoactive stimulated phosphoprotein (VASP) at Ser237.

Our study indicates that the endothelial peptide CNP inhibits I/R‐induced degranulation of perivascular MC and thereby attenuates microvascular inflammation.

Supported by SFB 688 and CHFC Würzburg
Ammonia Stimulates Heme Oxygenase‐1 Gene Expression in Human Endothelial Cells
Authors: Durante William , Liu Xiao‐ming , Peyton Kelly
Published: June 2021

Ammonia is continuously generated during cell metabolism via the catabolism of purines, pyrimidines and polyamines, and deamination of amino acids. Although endothelial cells (ECs) produce substantial amounts of ammonia, the role of this gas in ECs is not known. Since heme oxygenase‐1 (HO‐1) is an important regulator of EC function, we investigated whether ammonia modulates the expression of HO‐1 in cultured human umbilical vein ECs. Treatment of ECs with ammonia, given as ammonium chloride or ammonium hydroxide, stimulated a concentration‐ and time‐dependent increase in HO‐1 protein. A significant rise in HO‐1 protein was first detected after 4 hours and levels progressively increased over 24 hours of ammonia exposure. Ammonia‐mediated increases in HO‐1 protein expression were paralleled by significant elevations in HO‐1 mRNA. The induction of HO‐1 by ammonia was dependent on de novo RNA synthesis since it was blocked by the transcriptional inhibitor actinomycin D. Ammonia also stimulated a rapid and sustained increase in the production of reactive oxygen species (ROS) beginning one hour after ammonia administration. However, incubation of ECs with the antioxidant N‐acetyl‐L‐cysteine prevented the formation of ROS and blocked the induction of HO‐1 by ammonia. In conclusion, this study found that ammonia stimulates HO‐1 gene expression in vascular endothelium via a transcriptional‐ and redox‐sensitive pathway. The ability of ammonia to induce HO‐1 may provide a potential mechanism by which this gas regulates EC function. Supported by NIH grant HL59976.
Signaling Magnitude of the G‐Protein Coupled Bradykinin Receptor Depends on Flow: Role of the Glycocalyx
Authors: Jiménez Azucena , Espinosa Ricardo , Rubio Rafael
Published: June 2021

Flow is a stimulus whose mechanism is still under discussion and may act on the luminal endothelial glycocalyx (LEG), generating paracrine messengers targeting specific functions and metabolisms of the parenchymal cells. We have shown that LEG's lectinic transmembrane proteins such as GPCR (L) and diverse oligosaccharides (O) are required for flow detection. Since L and O have affinity toward each other, then, flow may disturb the endothelial paracrine signaling. This hypothesis could explain the molecular mechanism of action of flow and also why the flow is a multifunctional stimulus. Methods: Isolated carotid arteries from guinea pigs (500 g) were cannulated on its proximal end with a catheter, perfused with Krebs‐Henseleit solution at 11 or 16 ml/min and GPCR (L) identified and challenged by its specific agonist Bradykinin (Bk). In addition, artery distal ends were cannulated to have a resistance to flow. Perfusion pressure an indicator of smooth muscle tone was continuously recorded. At each level of flow dose‐response curves (signaling of GPCR) for Bk was determined before and after either enzymatic removal of specific O or addition of exogenous synthetic O.

Results

We show that level of flow alters the sensitivity to the hormone (D‐R curve). In addition, the D‐R curves are altered by removal of LEG hyaluronidate, but not by heparinate or by addition of exogenous synthetic O that likely compete with the endogenous O for lectinic binding. Clearly, D‐R curve (GPCR signaling) depends 1) on the level of flow, 2) at a given flow on the O composition of LEG. Supported by Conacyt to RET (223350‐M).
Intermittent Fasting with a High Fat Diet Reveals the Contribution of Mitochondrial Free Radicals to the Endothelium‐Dependent Relaxation of Mouse Aorta
Authors: MARTEL Cécile , BELANGER Alexandre , LUO Xiaoyan , TRESCASES‐THORIN Nathalie , THORIN Eric
Published: June 2021

Obesity leads to endothelial dysfunction, promoting age‐related cardiovascular diseases. In mice, a high fat diet (HFD) impairs endothelial function, while caloric restriction slows age‐dependent endothelial dysfunction and increases lifespan. Whether intermittent fasting (IF) influences endothelial function is unknown. Three‐month old C57BL6 mice were fed ad libitum (AL) or every other day (IF) either a regular diet (RD) or a HFD for 16 weeks. Caloric intake was similar in mice fed a RD either AL or in IF; however, RD‐IF prevented body weight gain (p<.05), improved glucose tolerance and insulin sensitivity (p<.05). As expected, HFD‐AL increased caloric intake (+27 %), body weight (+7 g), adipose tissue and liver weight (2 fold), glucose intolerance and insulin resistance (p<.05). Combining HFD and IF reduced by 50 % the rise in caloric intake compared to HFD‐AL (p<.05), fully prevented HFD‐induced gain in body weight, kept optimal (equal to RD‐IF) glucose tolerance, and normalized insulin sensitivity (p<.05). Acetylcholine (Ach)‐induced relaxation was measured in isolated aortic rings. Ach‐induced maximal relaxation (Emax, see Table) was increased in HDF‐AL mice (p<.05) due to a compensatory increase in eNOS activity. IF tended to ameliorate Emax (p=.06), irrespectively of the diet and without changing eNOS activity. In the 4 groups, TEMPOL (SOD mimetic) led to higher (p<.05) vascular sensitivity and Emax to Ach. Interestingly, mitoTEMPO targeting mitochondria, reduced (p<.05) endothelium‐dependent relaxation in HFD‐IF fed mice only, revealing the contribution of mitochondrial free radicals to the endothelium‐dependent relaxation of mouse aorta.

Emax (%)
RD‐AL
HFD‐AL
RD‐IF
HFD‐IF

Control
52 ± 6
78 ± 4 *
68 ± 5
68 ± 7

L‐NNA (eNOS inhibitor)
22 ± 7 $
41 ± 10 $
33 ± 10 $
31 ± 13 $

TEMPOL (SOD mimetic)
95 ± 2 $
90 ± 3
90 ± 4 $
85 ± 5

mitoTEMPO (mitochondrial SOD2 mimetic)
66 ± 12
70 ± 6
66 ± 8
46 ± 8 $

data are presented as mean ± SEM; * p<0.05 vs. RD‐AL; $ p<0.05 vs. Control.
Shear Stress Increases Sirtuin‐1 Expression to Mediate Shear‐Induced Autophagy in Endothelial Cells
Authors: Wang Ling
Published: June 2021

Blood flow‐induced shear stress acted on endothelial cells (ECs) is essential for normal vascular function. Autophagy represents a homeostatic mechanism crucial for cell survival. The conversion of non‐lipidated form LC3‐I to autophagosome‐membrane‐associated lipidated form LC3‐II is an indicator of autophagic activity. Sirtuins (SIRT1‐7), a family of NAD+‐dependent deacetylase, mediate adaptive responses to wide spectrum of stresses. Earlier studies showed that Sirtuin 1 (SIRT1) participated in the process of autophagy. In our current study, we demonstrate that shear stress to ECs increases autophagy and SIRT1 participates in this shear stress‐induced autophagy. Human endothelial cells (ECs) were subjected to shear stress (5 or 12 dynes/cm2) in a well‐defined parallel plate flow chamber system. Shear stress increased SIRT1 expression. Shear flow also enhanced NAD+/NADH ratio that is essential for Sirtuins activities. Shear stress stimulated autophagy as demonstrated by 1) an increase of LC3‐II/LC3‐I ratio 2) a decrease of P62 level and 3) an increase of RFP‐LC3 puncta numbers in sheared ECs. Shear‐induced autophagy is shown as time‐dependent but shear force‐independent manner. Interestingly, ECs treated with Sirtuins activator (SRT1720) also increased autophagy. Furthermore, shear stress to ECs with SIRT1 knockdown by SiRNA attenuated autophagy indicating that shear stress‐induced autophagy is mediated at least partially through SIRT1. Furthermore, ECs treated with ROS scavenger (NAC) decreased shear‐induced autophagy indicating ROS play a role in shear‐induced autophagy. In conclusion, ECs under shear stress increase SIRT1 expression to promote autophagy and thus enhances endothelial homeostasis.
The Cephalic Phase Responses of Celiac Artery Blood Flow and Gastric Motility to Oral Sweet Taste
Authors: Eguchi Kohei , Kashima Hideaki , Endo Masako , Fukuba Yoshiyuki
Published: June 2021

In humans little is known as to whether oral sensation of sweetness elicits any changes of splanchnic blood flow and gastrointestinal motility. The aim of this study, therefore, was to examine whether any effect of different oral sweet stimuli on celiac artery blood flow (CABF, which predominantly perfuses the stomach) and gastric motility occur. The oral stimulation was performed as follows: Subjects sipped a taste solution without swallowing. The fluid was then spat out, and subjects remained for post‐stimulation recording. During the protocol, CABF and gastric motility were continuously measured using pulsed Doppler ultrasonography and electrogastrography (EGG), respectively. In the experiment 1, 18% glucose (Glu), 18% maltodextrin (Mal) and mineral water (MW) were used for orosensory stimulation. Whereas all solutions had no effect on gastric motility, the stimulus by Glu (i.e. sweet taste solution) increased CABF acutely (p<0.05), but those by Mal and MW (i.e. non‐sweet taste solutions) did not. Accordingly, as the experiment 2, different concentrations of glucose (0, 4, 16, and 48%) were adopted to examine the dose‐response relationship of oral sensation of sweetness on CABF. While three glucose solutions, excepting 0%, increased the CABF (16, 21, and 23% from baseline, respectively; p<0.05), there were no significant differences among them. These results suggest that oral sensation of sweetness acutely increases CABF without augmentation of gastric motility, while it is not certain that the CABF is increased proportionally with the sweet taste intensity.
Different Roles for P2Y2 and P2Y6 Receptors in Large and Smaller Coronary Arteries
Authors: Haanes Kristian , Larsen Stine , Syberg Susanne , Jorgensen Niklas , Robaye Bernard , Boeynaems Jean‐Marie , Edvinsson Lars
Published: June 2021

A clear understanding of the physiological regulation of the coronary blood flow is important in order to recognize the changes after a pathological incident, such as a heart attack. Extracellular pyrimidines activate P2Y receptors on both smooth muscle cells and endothelial cells in the vasculature, leading to vasoconstriction and relaxation respectively. The aim of the present study was to examine the receptor profiles along the vasculature, particularly in the coronary circulation. We studied this in arteries utilizing stable pyrimidine analogues and P2Y2 and P2Y6 receptor KO mice in a myograph setup and also performed blood pressure measurements. The results show that P2Y6 mediates most of the UTP and UDP induced contraction in the larger coronary arteries (lumen diameter ~150 µm) and that P2Y2 is an endothelia dependent relaxing receptor. In the smaller coronary arteries (lumen diameter ~ 50 µm) the expression is opposite, with P2Y2 being the contractile receptor and P2Y6 the relaxant receptor, although together with P2Y2. We therefore propose that released UTP can act as a vasodilator downstream of its release, after being degraded to UDP, also without affecting the contractile pyrimidine receptors.The obtained results gives a completely new view of the purinergic regulation in the vascular bed and can be used as an explanation of the many differences in the expression of purinergic receptors both between species and in various arteries.

Funding: Lundbeck foundation (R167‐2013‐15379) and Lundbeck Grant of excellence
Inhibition of Soluble Epoxide Hydrolase Increases Coronary Flow and Improves Cardiac Work in Mice
Authors: Qin Jun , Jiang Houli , Kandhi Sharath , Froogh Ghezal , Thompson Carl , Huang An , Sun Dong
Published: June 2021

We tested the hypothesis that compromising hydrolysis/degradation of epoxyeicosatrienoic acids (EETs) via deletion of the gene encoding soluble epoxide hydrolase (sEH) improves coronary circulatory response to myocardial work in mice. Hearts were isolated from male wild type (WT) and sEH knockout (KO) mice and perfused with constant flow at different pre‐loads. Compared to the WT controls, sEH‐KO hearts required significantly greater basal coronary flow to maintain the perfusion pressure at 100 mmHg suggesting a reduced basal coronary resistance. Increases in cardiac work initiated greater reduction of coronary flow resistance in sEH‐KO than in WT mice. Moreover, an improved cardiac contractile function was observed in response to increases in pre‐load in sEH‐KO mice. Adaptation of the coronary circulation in sEH‐KO hearts in response to increased work loads was prevented with the EET receptor antagonist 14,15‐EEZE, while inhibition of nitric oxide synthesis had no effect. Cardiac expression of CYP2J2 (EET synthase) was comparable in both groups of mice. Levels of EETs were dramatically higher in hearts from sEH‐KO which were accompanied with lower levels of DHETs (hydrolyzed EETs) compared to those of WT hearts. In conclusion, the elevation of cardiac EETs, as a function of deletion of sEH gene, plays significant roles in increasing coronary flow and improving cardiac function.
Chronic Hypoxia Alters the Response of Fetal Ovine Middle Cerebral Arteries to Endothelin‐1 (ET‐1)
Authors: Silpanisong Jinjutha , Kim Dahlim , Williams James , Adeoye Olayemi , Thorpe Richard , Pearce William
Published: June 2021

This study explores the hypothesis that chronic hypoxia alters the expression and intracellular coupling of endothelin receptors to induce phenotypic transformations of smooth muscle cells within the arteries of fetal cerebrovasculature. Endothelium‐denuded middle cerebral arteries (MCAs) harvested from term fetuses of pregnant adult sheep kept at sea level or 3820m for 110 days were used in contractility experiments or cultured with ET‐1 and various kinase inhibitors. Chronic hypoxia had no effect on plasma levels of ET‐1 but altered contractile responses to ET‐1 and increased ETA receptor expression in fetal MCAs. ET‐1 treatment increased arterial thicknesses in both normoxic and hypoxic arteries. Organ culture of MCAs with ET‐1 decreased colocalization of MLCK and α‐actin and this effect was attenuated by chronic hypoxia. ET‐1 also decreased MLC20 and MLCK colocalization, but this effect was similar in normoxic and hypoxic arteries. The effects of ET‐1 on colocalization were reversed by inhibitors of either PKC (7 µM chelerythrine) or p38 (10 µM SB203580) in both normoxic and hypoxic arteries. From these results we conclude that chronic hypoxia modulates phenotypic responses of fetal cerebrovascular smooth muscle to ET‐1 through elevated expression of ET‐1 receptors, which act downstream through PKC and p38 dependent pathways on contractile protein expression.

This work was supported by National Institutes of Health Grants HL‐54120, HD‐31266, HL‐64867, and NS‐076945 and the Loma Linda University School of Medicine.
L‐2‐Oxothiazolidine‐4‐Carboxylic Acid Mitigates the Thromboembolic Effects and Systemic Toxicityinduced by Sub‐acute Exposure to Cadmiumin Mice
Authors: Fahim Mohamed , Nemmar Abderrahim , Singh Sarabjit , Shafiullah Mohamed , Yasin Javed , Hasan Mohamed
Published: June 2021

Exposure to cadmium (Cd) poses a deleterious health effects and its toxicity has been attributed to oxidative stress. Therefore, we examined the sub‐acute systemic and thrombotic influence of Cd exposure in vivo, and assessed the possible protective effects of the antioxidant L‐2‐Oxothiazolidine‐4‐Carboxylic acid (OTC). Mice received Cd at a dose of 1 mg Cd/kg/day, i.p., for 3 weeks. OTC pre‐treatment (80mg/kg/day, i.p.) was started one hour before Cd administration. Cd compared with control significantly reduced both the time (in seconds) for the first platelet aggregate (48.00+3.74 vs 79.33+20.73) and the time until blood flow stops (152+17.24 vs 179.00+13.62) in pial microvessels. OTC either by itself or concurrently with Cd significantly increased both the time for the first aggregate (156.67+10.98 vs 135.33+13.60) and until flow stops (239.00+20.22 vs 201.17+2.93). Cd induced a significant increase in total WBC and CK. A significant increase was also observed in liver enzymes after Cd exposure. The antioxidants Superoxide dismutaseand catalase activities significantly increased in mice treated with Cd. OTC in combination with Cd alleviated most of these adverse effects induced by Cd. OTC, through its antioxidant actions, appeared to effectively protect Cd induced thrombosis in mice. Hence, OTC represents a potential candidate to protect the cerebral microvessels from the detrimental effect of Cd toxicity.
The Probiotic Kefir Improves Cardiovascular Function in Spontaneously Hypertensive Rats
Authors: Friques Andreia , Klippel Brunella , Leal Marcos , Kalil Ieda , Andrade Tadeu , Pereira Thiago , Meyrelles Silvana , Vasquez Elisardo
Published: June 2021

In the present study, we evaluated the effects of the probiotic kefir on the cardiovascular function in spontaneously hypertensive rats (SHR). SHR were chronically administered with oral 0.3 mL/100 g b.w. of kefir (SHR‐k) or vehicle (SHR‐v) for 60 days and compared with Wistar rats (WR). In comparison with NWR, SHR‐v exhibited higher mean arterial pressure, which was significantly attenuated in SHR‐k (108±2, 194±5 and 142±8 mmHg, respectively, p<0.01). Similarly effects were observed on angiotensin converting enzyme activity (91±7, 119±9 and 78±6%, respectively, p<0.01). The evaluation of the baroreceptor function showed that the attenuated gain (bpm/mmHg) of the baroreflex observed in SHR‐v (3.85±0.03) when compared with WR (6.3±0.05) was significantly improved in SHR‐k (5.8±0.04). Evalulation of vascular endothelium function showed that aortic rings from SHR‐v had impaired maximum relaxing responses (%) and sensitivity (‐log M) to acetylcholine (48±1.7 and 6.7±0.09) when compared with WR (78±1.4 and 8.0±0.07), which were significantly improved in SHR‐k (59±1.3 and 7.4±0.07). These data show evidence of beneficial effects of the probiotic kefir on high blood pressure and endothelial dysfunction in the this genetic model of hypertension. The current study expands the many health benefits have been attributed to kefir. Ongoing experiments are being directed to clear the mechanisms of the beneficial effects of this probiotic on the cardiovascular function. Financial support from: FAPES and CNPq.
Endothelium‐Dependent and ‐Independent Vasodilation in Women at Risk of Hypertension
Authors: Harvey Ronee , Laughlin‐Tommaso Shannon , Stewart Elizabeth , Curry Timothy , Joyner Michael , Barnes Jill
Published: June 2021

Women with uterine fibroids (UF), benign tumors of the myometrium, have a greater prevalence of hypertension than women without UF. The mechanism of this predisposition is unclear. Individuals with borderline hypertension have endothelial dysfunction in the form of decreased endothelium‐dependent vasodilation in response to intra‐arterial acetylcholine infusion. It is possible that women with UF also demonstrate impaired endothelium‐dependent vasodilation prior to overt clinical hypertension. We measured forearm blood flow (FBF) using venous occlusion plethysmography at baseline and during intra‐arterial acetylcholine (ACH; 1.0‐4.0 μg/100 ml tissue/min) and sodium nitroprusside (SNP; 0.25‐2.00 μg/100 ml tissue/min) infusions in 4 normotensive women with UF (41±3 y) and 4 age‐matched normotensive women without UF. These women did not differ in body mass index or mean arterial pressure (97±4 vs. 103±6 mmHg, Control vs. UF; p>0.05). Baseline FBF was not different between the two groups (1.6±0.4 vs. 3.0±1.3 ml/100 ml tissue/min; Control vs. UF; p>0.05). FBF increased significantly from baseline in both groups at the highest doses of ACH and SNP; however, the change in FBF was not significantly different between groups (ACH: Δ8.6±1.6 vs. 9.5±4.1 ml/100 ml tissue/min; SNP: Δ9.8±3.0 vs. 13.1±4.6 ml/100 ml tissue/min; Control vs. UF; p>0.05). Our preliminary results suggest that FBF responses to ACH and SNP do not differ between women with and without UF; thus, impairments in endothelium‐dependent and ‐independent vasodilation do not appear to contribute to hypertension risk in normotensive women with UF.
Funded by AHA and NIH.
Sinoaortic denervation reduces the complexity of heart rate variability in mice
Authors: Silva Luiz Eduardo , Rodrigues Fernanda , Oliveira Mauro , Salgado Helio , Fazan Rubens
Published: June 2021

Heart rate variability (HRV) is recognized as a phenomenon highly complex, presenting nonlinear dynamics with fractal correlations and multiscale properties. Several cardiovascular and systemic diseases are related to loss of complexity in HRV, which has been recognized as an independent risk factor for life‐threatening cardiovascular outcomes. Considering that most cardiovascular diseases are accompanied by autonomic imbalance and baroreflex dysfunction, we hypothesise that the baroreflex may play an important role in the genesis of HRV complexity. Mice were submitted to sinoaortic denervation (SAD) and received catheters into femoral artery and jugular vein 5 days prior to the experiment. Baseline arterial pressure (AP) was recorded and pulse interval (PI) time series were generated from the intervals between consecutive diastolic pressures. The complexity of the HRV was determined using detrended fluctuation analysis (DFA) and multiscale entropy (MSE). DFA α1 scaling exponent (a short‐term index) was remarkably decreased in the SAD mice (0.79±0.06 vs. 1.13±0.04 for the control mice), whereas SAD slightly increased the α2 scaling exponent (a long‐term index: 1.12±0.03 vs. 1.04±0.02 from control). In SAD mice, total MSE was decreased (13.2±1.3) compared to the control counterparts (18.9±1.4). In conclusion, both fractal and regularity structures of HRV are altered by SAD in mice, affecting short‐ and long‐term mechanisms of complexity, suggesting that the baroreceptors play a important role in the complex structure of HRV.

Finacial Support: FAPESP, CAPES, CNPq
Nonlinear Identification of the Total Baroreflex Arc
Authors: Moslehpour Mohsen , Kawada Toru , Sunagawa Kenji , Sugimachi Masaru , Mukkamala Ramakrishna
Published: June 2021

The total baroreflex arc – the open‐loop system relating carotid sinus pressure (CSP) to arterial pressure (AP) – exhibits nonlinear behaviors such as saturation, thresholding, and steady‐state responses to pulsatile changes. However, few studies have quantitatively characterized the nonlinear system dynamics. The aim was to develop a nonlinear dynamic model of the total baroreflex arc. Five normal rats were studied according to a protocol approved by the Animal Subjects Committee of the National Cerebral and Cardiovascular Center. Under anesthesia, the vagal and aortic arch depressor nerves were sectioned; the carotid sinus regions were isolated and attached to a servo‐controlled piston pump; and AP and sympathetic nerve activity (SNA) were measured. CSP was perturbed using a Gaussian white noise signal. A second‐order Volterra model was established using nonparametric identification. The second‐order kernel was mainly diagonal, but the main diagonal was not the same as the first‐order kernel. A simplified second‐order nonlinear model was accordingly developed using nonparametric identification. This model predicted AP changes 15% better than a linear model in response to Gaussian white noise CSP data not used in model development. Linear and nonlinear models of the neural arc – the system relating CSP to SNA – and peripheral arc – the system relating SNA to AP – were likewise established. These two models indicated that the nonlinearity of the total baroreflex arc mainly arises from the neural arc rather than the peripheral arc.
Baroreflex Sympatho‐activation During Muscle Metaboreflex Activation Improves Blood Flow to the Ischemic Muscle
Authors: Kaur Jasdeep , Spranger Marty , Machado Tiago , Krishnan Abhinav , Alvarez Alberto , O'Leary Donal
Published: June 2021

Muscle metaboreflex and arterial baroreflex regulate arterial pressure through distinct mechanisms. Muscle metaboreflex activation (MMA) elicits a pressor response virtually solely by increasing cardiac output (CO) while baroreceptor unloading increases mean arterial pressure (MAP) primarily through peripheral vasoconstriction. We activated the muscle metaboreflex in chronically instrumented canines during dynamic exercise (via graded reductions in hindlimb blood flow; HLBF) followed by simultaneous baroreceptor unloading (via bilateral carotid occlusion; BCO). We hypothesized that simultaneous activation of both the reflexes would result in an exacerbated pressor response owing to both an increase in CO and vasoconstriction. We observed that MMA caused a significant increase in MAP, heart rate (HR), CO, and non‐ischemic vascular conductance (NIVC; conductance of all vascular beds except the hindlimb vasculature). When followed by BCO, there were further increases in MAP and HR however, NIVC decreased by 25% indicating substantial peripheral vasoconstriction. Although the hindlimb resistance in this setting also increased indicating vasoconstriction of the ischemic muscle, the remaining vasculature vasoconstricted more, thereby redirecting blood flow to the ischemic muscle. We conclude that baroreceptor unloading during MMA induces preferential peripheral vasoconstriction to improve blood flow to the ischemic active skeletal muscle. HL‐55473
Post‐Junctional Adrenergic Neurotransmission is Inhibited by Nitric Oxide (NO) in Humans
Authors: Del Pozzi Andrew , Medow Marvin , Stewart Julian
Published: June 2021

NO, can modulate adrenergic vasoconstriction by central reduction of sympathetic outflow and by peripheral effects in animal models. We investigated whether NO inhibits post–synaptic α‐1‐adrenergic neurotransmission in humans. To accomplish this we performed intravenous phenylephrine dose‐response experiments under three conditions: during a control saline infusion, after NOS inhibition with L‐NG‐monomethyl Arginine (L‐NMMA), and following restoration of NO by adding sodium nitroprusside (SNP) to L‐NMMA. We studied 11 healthy controls (21 ±3 yrs) using repeated measures ANOVA. All subjects received at least 0.5, 1.0, and 2.0 µg/kg/min in sequence. Patients were instrumented with an oscillometric BP cuff, EKG, and beat‐to‐beat BP, cardiac output (CO), and total peripheral resistance using a Finometer with ModelFlow software. We measured cerebral blood flow velocity (CBFv) in the middle cerebral artery with transcranial Doppler ultrasound. Saline and L‐NMMA experiments were performed on different days. L‐NMMA increased pre‐phenylephrine MAP by 7% while reducing HR by 8% and increasing CBFv by 13%. The change in MAP, and TPR were significantly increased with L‐NMMA (P < 0.001) and returned to baseline following administration of SNP (P > 0.05) for all levels of the dose‐response. HR and CO were significantly decreased with L‐NMMA (P < 0.001) and was returned to baseline with SNP (P > 0.05) for all levels of the dose‐response. The change in CBFv was unaffected (curves were parallel). We conclude that post‐junctional adrenergic neurotransmission is inhibited by NO in humans. Inhibition could occur by reduction of α‐1‐adrenergic properties or of neurovascular transduction.
Cyclooxygenase Inhibition Attenuates the Muscle Sympathetic Nerve Activity Responses to Venous Distension in Humans
Authors: Cui Jian , McQuillan Patrick , Blaha Cheryl , Sinoway Lawrence
Published: June 2021

We have shown that venous distension induced by saline infusion into veins of an arterially occluded arm evokes reflex increases in muscle sympathetic nerve activity (MSNA) and blood pressure (BP) in humans. Recently, Yamauchi and colleagues examined this venous distension reflex in rats and showed that cyclooxygenase blockade with indomethacin attenuated the reflex pressor response. We hypothesized that blockade of prostaglandin synthesis could attenuate MSNA responses to venous distension in humans. BP (Finapres), heart rate, and MSNA (microneurography) were assessed in 11 young healthy subjects during infusion of 6 mg ketorolac tromethamine in 40 ml saline into veins of an arterially occluded arm. In the second experiment, the same amount of saline was infused (control trial). MSNA significantly rose towards to the end of infusion of saline (18.1±2.1 to 27.0±2.7 bursts/min, P<0.01), while no significant change was observed in the ketorolac trial (19.0±2.2 to 22.7±3.2 bursts/min, P=0.77). BP significantly rose towards to the end of infusion in the both trials, and the BP response in ketorolac trial tended to be less than that in saline trial (P=0.07). These observations indicate that blockade of prostaglandin synthesis may attenuate MSNA and BP responses to distension of veins, and suggest that prostaglandins may contribute to the venous distension reflex.
Ionotropic Glutamate Receptors Partially Mediate Pressor and Sympathoexcitatory Responses to Disinhibition of the RVLM after Sinoaortic Denervation and Contralateral RVLM Inhibition in Sedentary and Physically Active Rats
Authors: Dombrowski M. , Azar T. , Mueller P.
Published: June 2021

The RVLM is a bilateral brainstem region important for sympathetic control of arterial pressure (AP). We have demonstrated that after sinoaortic denervation (SAD) and unilateral blockade of RVLM, GABAA receptor blockade (GABAA‐X) in RVLM dramatically increased splanchnic sympathetic nerve activity (SSNA) and AP in sedentary (SED) and wheel running (WR) rats. Whether this excitation is mediated by glutamate is unknown. We tested the hypotheses that 1) ionotropic glutamate receptors (iGluRs) contribute to increases in AP and SSNA in response to GABAA‐X and 2) SED conditions enhance glutamatergic excitation. Prior to microinjections in Inactin‐anesthetized SED (n=6) and WR (n=5) rats, SAD was performed and one RVLM was inhibited (muscimol 2mM, 90nl). Responses to GABAA‐X using bicuculline (Bic 5mM, 90nl) alone and in the presence of the iGluR antagonist kynurenic acid (Kyn 40mM, 90nl) were tested in the contralateral, intact RVLM. As before GABAA‐X alone increased AP and SSNA in SEDs and WRs. Pretreatment with Kyn attenuated increases in AP in SEDs (Δ49±13 mmHg vs Δ24±4 mmHg) and WRs (Δ30±5 mmHg vs. Δ11±5 mmHg) (p<0.05 main effect). However, effects of pretreatment with kyn on increases in SSNA did not reach significance (SEDs: Δ121±28 % vs Δ81±13 %; WRs: Δ97±46 % vs Δ65±30 %) (p=0.149 main effect). Our preliminary data suggests that blockade of endogenous glutamate limits pressor responses to removal of GABA input in the RVLM of barodenervated rats. The residual sympathoexcitation appears to be due to non‐ionotropic glutamatergic input to RVLM that may or may not be altered in SEDs and WRs. R01‐HL096787; R01‐HL096787‐S1
Renal Denervation by Radiofrequency Ablation Attenuates Startle Responses Selectively in Rats with Exaggerated Stress Responsiveness
Authors: Knuepfer Mark , Willingham Laura , Hoffman‐Schepers Nicole
Published: June 2021

Renal denervation (RDN) is reported to reduce arterial pressure (AP) in some patients with resistant hypertension. Patients prone to develop hypertension have been reported to have greater pressor responses to stress. We hypothesized that RDN would selectively reduce hemodynamic responses to startle in those individuals who have greater responsiveness to stress. We instrumented Sprague‐Dawley rats for determination of ascending aortic blood flow using Doppler flowmetry (cardiac output) and for direct measurement of AP. After recovery, rats were tested with air jet (1 sec) and with cold water stress (60 sec) while recording hemodynamic responses. After characterizing responses to the initial startle using both stressors, RDN was performed by radiofrequency ablation (<1mW, 20 s) applied externally to each renal artery 4 times each using an RF generator (100 KHz). One week later, rats were retested for stress responsiveness. Rats were divided into those with greater or lesser AP responses to acute startle according to their initial response averages. Those rats with greater AP responses had significant reductions in the AP response and the increase in systemic vascular resistance to startle after RDN. Startle responses in rats with smaller pressor responses were not affected by RDN. These results suggest that a subset of hyper‐responders to stress may be “normalized” with RDN. Since many have proposed that some individuals develop essential hypertension due to greater stress responsiveness, these results suggest a means by which RDN might be effective in reducing arterial pressure in a subset of the population with hypertension. Supported by St. Louis University.
c‐Fos expression in rat periaqueductal gray induced by white noise sound exposure
Authors: Koba Satoshi , Inoue Ryo , Watanabe Tatsuo
Published: June 2021

We previously reported that parasympathetically‐mediated bradycardia in response to white noise sound (WNS) exposure in conscious rats was suppressed by muscimol, a GABAA receptor agonist, microinjected into the lateral/ventrolateral periaqueductal gray (l/vlPAG). This finding prompted us to determine if the rat l/vlPAG is activated by the WNS exposure. Rats were exposed to 30‐min WNS at 90 dB, which evoked freezing behavior, an index of fear. We examined the distribution of cells expressing Fos, a marker of neuronal activation, in the WNS‐exposed rats and control rats which had not been exposed to WNS (N=6 for each). The WNS exposure significantly (P<0.05) increased Fos positive cells in l/vlPAG, as well as dorsolateral PAG, as compared to the control. Subsequently, we investigated if Fos positive cells in the PAG after the WNS exposure in rats would be immunolabeled with cholera toxin b‐subunit, a retrograde tracer, previously microinjected into the nucleus ambiguus (NA) (N=2). Our pilot data suggested that there were neuronal projections from l/vlPAG to NA which were activated by the WNS exposure. In conclusion, the l/vlPAG‐NA pathway may play a role in evoking bradycardia in response to exposure of WNS. Supported by JSPS Kaken 26670112.