bluuebunny/crossref_metadata_2025_split huggingface dataset:
Macrophage 12‐HETE Enhances Angiotensin II Vasoconstriction Through Thromboxane B2 Release in Murine Aortas
Authors: Kriska Tamas , Pfister Sandra L. , Campbell William B. 
Published: November 2023

12/15‐Lipoxygenase (12/15‐LO), encoded by Alox15 gene, metabolizes arachidonic acid to 12‐ and 15‐hydroxyeicosatetraenoic acid (12‐ and 15‐HETE). We have shown that macrophages are the exclusive source of 12/15‐LO among immune cells that play crucial role in development of hypertension. The objective of this study was to test the hypothesis that macrophage 12‐HETE contributes to angiotensin II‐mediated arterial constriction and thus to development of angiotensin II‐induced experimental hypertension.We used the Alox15−/− mouse model that is resistant towards angiotensin II‐induced hypertension and immune cell accumulation in arteries and kidney. Chronic treatment of WT mice with angiotensin II (1000_ng/kg/day) increased mean arterial pressure from 100.6±0.9 to 136.2±4.3 mmHg. However, angiotensin II did not change the blood pressure significantly in Alox15−/− mice (98.9±0.7 vs. 103.0±2.3 mmHg). Angiotensin II hypertension increased macrophage and T‐lymphocyte accumulation in aortas of WT mice by 2.6‐fold and 2.2‐fold, respectively. These increases were blunted in Alox15−/− mice. Angiotensin II (100 nM) caused 15±3.4% constriction in isolated mouse aortic rings whereas 12‐HETE alone was without effect. In the presence of 12‐HETE (100 nM), angiotensin II‐induced constriction was further enhanced by 10±4.1%. 15‐HETE (100 nM) did not alter the angiotensin II constriction. The presence of WT macrophages, the major source of 12‐HETE, also enhanced the angiotensin II‐induced constriction, while the effect of Alox15−/− macrophages on constriction was blunted. Pre‐treatment of aortic rings with superoxide inhibitors, thromboxane receptor antagonist, or thromboxane synthase inhibitor or removal of the endothelium eliminated the enhancing effect of 12‐HETE on angiotensin constriction, while externally added thromboxane mimetic U46619 enhanced the constriction in a similar manner to 12‐HETE. In mouse coronary artery endothelial cell cultures, angiotensin II (100 nM) increased the thromboxane B2 production by 1.8 fold. In the presence of 12‐HETE (100 nM), the angiotensin II‐induced increase in thromboxane B2 was enhanced by 3.4 fold.These results indicate that macrophage 12/15‐LO pathway mediates the hypertensive actions of angiotensin II by promoting the synthesis of vasoactive thromboxane B2 through superoxide formation.Support or Funding InformationThis study was supported by the grant: NIH HL103673
Characterization of GPR40 as a Low Affinity Epoxyeicosatrienoic Acid (EET) Receptor in Vascular Cells
Authors: Park Sang‐Kyu , Herrnreiter Anja , Pfister Sandra L. , Falck John R. , Campbell William B. 
Published: November 2023

Endothelium‐derived EETs have a number of vascular actions including dilating arteries, promoting angiogenesis and reducing inflammation. These actions may be mediated by high and low affinity G protein‐coupled receptors. GPR40 is activated by long chain free fatty acids and EETs. We investigated the role of GPR40 and its signaling mechanisms in some vascular actions of EETs. 14,15‐EET, 11,12‐EET and the GPR40 agonist GW9508 increased intracellular calcium concentration in HEK293 cells overexpressing human GPR40 (EC50= 0.66 ± 0.13 μM, 0.98 ± 0.09 μM and 19 ± 0.37 nM, respectively) but were without effect in non‐transfected HEK293 cells. 8,9‐EET, 11,12‐DHET and 14,15‐DHET were less active. GW1100 (10 μM), the GPR40 antagonist, blocked these increases in calcium by EETs and GW9508. Using PCR and immunoblotting, GPR40 expression was detected in human and bovine endothelial cells (ECs), smooth muscle cells and arteries. 11,12‐EET caused concentration‐related relaxations of U46619‐preconstricted bovine coronary arteries; however, these relaxations were not altered by GW1100 (EC50 = 0.46 ± 0.14 μM vs 0.63 ± 0.37 μM; control vs GW1100). 11,12‐EET increased intracellular calcium in human umbilical vein ECs. In these ECs, 11,12‐EET (1 μM) increased MAP kinase phosphorylation of ERK and increased the expression of connexin 43 (Cx43) and cyclooxygenase‐2 (COX‐2). GW1100 and the MAP kinase inhibitor U0126 (1 μM) both inhibited 11,12‐EET‐mediated ERK phosphorylation and Cx43 and COX‐2 increases. In addition, the NFκB inhibitor andrographolide (40 μM) also inhibited the 11,12‐EET‐mediated COX‐2 increase in ECs. Our results indicate that GPR40 is a low affinity EET receptor that is expressed in vascular cells and arteries. GPR40 does not mediate EET‐induced vasorelaxation;, however, 11,12‐EET through GPR40 increases Cx43 and COX‐2 expression in ECs via ERK phosphorylation. Thus, Cx43 and COX‐2 play important roles in cell to cell gap junctional communication and prostaglandin production to mediate some of vascular actions of EETs.Support or Funding InformationThis work was funded by grants from the National Institutes of Health (HL‐83297), Regulation of adrenal tone by steroidogenesis cells.
Dynamics of Skeletal Muscle Microvascular and Interstitial PO2 from Rest to Contractions
Authors: Hirai Daniel M. , Colburn Trenton D. , Craig Jesse C. , Musch Timothy I. , Poole David C. 
Published: November 2023

Oxygen pressure (PO2) gradients within the microcirculation are required for diffusive O2 transport and, thus, to support oxidative metabolism. The greatest resistance to O2 flux into the skeletal muscle is considered to reside in the short distance between the red blood cell and the adjacent sarcolemma. Given the high flux density and absence of an O2 carrier within this diffusion path, we tested the hypothesis that PO2 gradients between muscle microvascular and interstitial spaces (PO2mv and PO2is; respectively) would be present at rest and maintained or increased during contractions. Oxyphor probes G2 (Pd‐meso‐tetra‐(4‐carboxyphenyl)‐porphyrin; i.a.) and G4 (Pd‐meso‐tetra‐(3,5‐dicarboxyphenyl)‐tetrabenzoporphyrin; tissue microinjection) were used to determine PO2mv and PO2is, respectively, via phosphorescence quenching in the exposed rat spinotrapezius muscle at rest and during submaximal twitch contractions (1 Hz, 6 V, 3 min). Due to overlapping spectral features of G2 and G4, separate measurements of PO2mv (right spinotrapezius; second protocol) and PO2is (left spinotrapezius; first protocol) were performed in the same animals (Sprague‐Dawley; n=7). There were no differences in mean arterial pressure or heart rate during the rest‐contraction transient between protocols (P>0.05 for all). PO2mv was higher than PO2is in all instances from rest to contractions (baseline: 34.3±2.3 vs. 13.8±1.4; steady‐state: 27.4±1.8 vs. 9.3±1.6 mmHg, respectively; P<0.05 for both) such that the mean PO2 gradient throughout the entire protocol was 18.3±2.2 mmHg (z‐test P<0.05). No differences in the magnitude of the PO2 fall induced by contractions were observed between the microvascular and interstitial spaces (amplitude: 9.3±1.4 vs. 11.1±0.9 mmHg, respectively; P>0.05). The speed of the PO2is fall during contractions was slower than that of PO2mv (time constant: 13.0±1.9 vs. 9.6±2.0 s, respectively; P<0.05). Consistent with our hypothesis, a significant gradient between PO2mv and PO2is was sustained (but not increased) from rest to contractions in skeletal muscle. These data support that the carrier‐free region is the site of a substantial PO2 gradient driving blood‐myocyte O2 flux. Therefore, Oxyphor G2 and G4 probes allow discrete investigation of O2 exchange in the microvascular and interstitial spaces. Differences in PO2mv and PO2is kinetics illustrate the potential for muscle microcirculatory O2 exchange dynamics at sites closer to the mitochondria to provide novel insights into the control of muscle oxidative function in health and dysfunction in disease.Support or Funding InformationNIH HL‐2‐108328
Increased NO Sensitivity is Associated with Maintained Coronary Microvascular cGMP‐PDE5 Signaling in Exercising Swine with Multiple Co‐Morbidities
Authors: van Drie Ruben W.A. , Sorop Oana , van de Wouw Jens , Merkus Daphne , Duncker Dirk J. 
Published: November 2023

BackgroundMultiple comorbidities, including diabetes mellitus (DM), hypercholesterolemia (HC) and chronic kidney disease (CKD) are thought to cause coronary microvascular dysfunction through a reduction in microvascular NO signaling, leading to impaired myocardial perfusion. Here we investigated whether NO‐cGMP‐PDE5 coronary microvascular signaling is modified in swine with DM+HC+CKD, by studying the vasodilator responses to PDE5 inhibition and to exogenous NO in chronically instrumented swine.Methods and resultsFive female swine (DM+HC+CKD) with Diabetes Mellitus (streptozotocin 3×50mg/kg), chronic kidney disease (renal embolization) were fed high fat diet for 6 months, while six healthy female swine served as controls (CON). Hyperglycemia (21±1.4 vs 8.9±1.5mmol/l in in DM+HC+CKD vs CON, P=0.0002), hypercholesterolemia (7.8±1.0 vs 2.8±1.2mmol/l, P=0.013) and chronic kidney disease (GFR: 123±12 vs 191±9ml/min, P=0.001) were present. Myocardial oxygen delivery was impaired in DM+HC+CKD swine, forcing the myocardium to increase its oxygen extraction both at rest and during exercise (Figure), and resulting in reduced coronary venous oxygen content compared to CON (both P<0.05). PDE5 inhibition with sildenafil (8mg i.v.) decreased myocardial oxygen extraction in both DM+HC+CKD (P=0.016) and CON (P=0.005, Figure), to a similar extent (P=0.34), indicating maintained NO‐cGMP vasodilator influence. As myocardial NO production was decreased (0.19±0.03 vs 0.34±0.13μM NO2−+NO3−/mg protein, P=0.02), we investigated whether an increase in NO sensitivity contributed to maintain NO‐cGMP vasodilator influence, by infusion of the NO donor sodium nitroprusside (SNP). Coronary vascular conductance (CVC) decreased more in DM+HC+CKD compared to CON, indicating an increased NO‐sensitivity (P=0.043, Figure).ConclusionIn swine with multiple co‐morbidities reduced bioavailability of NO appears to be compensated by increased sensitivity to NO, which is associated with maintained cGMP‐mediated vasodilator influence.Support or Funding InformationFunding: This study was supported by grants from the European Commission FP7‐Health‐2010 grant MEDIA‐261409, the Netherlands CardioVascular Research Initiative: an initiative with support of the Dutch Heart Foundation [CVON2014‐11 (RECONNECT)].
Oxygenation Characteristics of the Vastus Lateralis During Cycling Exercise Performed Above Critical Power
Authors: Hammer Shane M. , Alexander Andrew M. , Didier Kaylin D. , Baumfalk Dryden R. , Barstow Thomas J. 
Published: November 2023

Near‐infrared spectroscopy has been used to empirically describe the oxygenation status of exercising skeletal muscle. Changes in total‐[Hb+Mb] and deoxy‐[Hb+Mb] concentrations have been thought to reflect changes in microvascular hematocrit (an index of diffusive conductance) and fractional oxygen extraction (an index of perfusive conductance), respectively. Our laboratory has previously shown that, following dynamic forearm exercise, no differences exist in the oxygenation status of the exercising muscle at task failure among exercise intensities performed above critical power (CP).PURPOSEThe purpose of this study was to investigate the oxygenation characteristics of the vastus lateralis muscle during cycling exercise performed at multiple intensities above critical power. We hypothesized that no significant differences would be detected in end‐exercise total‐[Hb+Mb] or deoxy‐[Hb+Mb] concentrations among exercise intensities.METHODSTo date, four healthy men (23.5 ± 2.0 yrs; 92.6 ± 4.2 kg; 175.2 ± 4.4 cm) performed an incremental exercise test on a cycle ergometer to determine peak power (Ppeak). Four constant load exercise tests were then performed to the limit of exercise tolerance (Tlim), from which CP was determined. Exercise intensities of the four constant load exercise tests were selected such that Tlim would occur between 2 and 15 minutes. Total‐[Hb+Mb] and deoxy‐[Hb+Mb] concentrations of the vastus lateralis were assessed using near‐infrared spectroscopy during each exercise bout.RESULTSCritical power (178 ± 17 W) was 59.4% ± 2.3% of Ppeak (300 ± 31 W). There was no significant overall effect of exercise intensity on end‐exercise total‐[Hb+Mb] concentration (p=0.26). While a significant overall effect of exercise intensity on end‐exercise deoxy‐[Hb+Mb] concentration was detected (p<0.05), there were no significant differences among exercise intensities. The end‐exercise total‐[Hb+Mb] (61.3 ± 16.1 μM) and deoxy‐[Hb+Mb] (23.0 ± 6.4 μM) concentrations were significantly correlated (r=0.96; p<.001).CONCLUSIONSThese preliminary data suggest that, following cycling exercise performed above critical power, the oxygenation status of the exercising muscle at task failure is not exercise intensity dependent. Further, these data suggest that perfusive (deoxy‐[Hb+Mb]) and diffusive (total‐[Hb+Mb]) conductances are associated at task failure.
Phosphodiesterase 3 Inhibition Does Not Restore ATP Release from Deoxygenated Red Blood Cells of Older Adult Humans
Authors: Racine Matthew L. , Joyner Michael J. , Dinenno Frank A. 
Published: November 2023

Red blood cells (RBCs) release ATP in direct proportion to the degree of hemoglobin (Hb) deoxygenation, which binds to purinergic receptors on the endothelium and stimulates vasodilation. Accordingly, RBCs act as both a ‘sensor’ for oxygen demand and an ‘effector’ for increasing oxygen delivery to facilitate the matching of tissue oxygen supply and demand. We have recently demonstrated that Hb deoxygenation‐induced ATP release is impaired in RBCs from healthy older vs. young adults, but the underlying mechanisms are not fully understood. Cyclic AMP (cAMP) has been shown to be an important component of the signaling cascade for RBC ATP release, and decreased cAMP signaling contributes to impaired ATP release from RBCs of humans with type 2 diabetes. As such, treatment with cilostazol to inhibit phosphodiesterase 3 (PDE3) hydrolysis of cAMP improves Hb deoxygenation‐induced ATP release from RBCs of these patients. Whether similar alterations in cAMP signaling contribute to impaired RBC ATP release in healthy older adults is unclear. Thus, in the present study we tested the hypothesis that PDE3 inhibition would restore Hb deoxygenation‐induced ATP release from RBCs of healthy older adults. Isolated RBC ATP release in response to normoxic (PO2 ~111 mmHg) and hypoxic (PO2 ~26 mmHg) stimuli was quantified in RBCs from young (25 ± 1 years; n = 4) and older (65 ± 3 years; n = 6) adults using the luciferin‐luciferase technique following RBC incubation with either dimethylformamide (DMF; vehicle control) or 100 μM cilostazol (PDE3 inhibitor). The relative change in ATP release from normoxia to hypoxia in control conditions was impaired in RBCs from older (56.1 ± 11.0%) vs. young (129.1 ± 16.1%) adults (p < 0.05). Cilostazol resulted in a slight, but non‐significant improvement in RBC ATP release in older adults (74.2 ± 15.0%; p = 0.32 vs. control), whereas there was no effect on RBC ATP release in young adults (123.4 ± 37.5%; p = 0.84 vs. control). RBC intracellular ATP was not different between age groups or treatments (p > 0.05). These preliminary data suggest that greater cAMP hydrolysis via PDE3 in response to Hb deoxygenation is not a primary contributor to impaired RBC ATP release in older adults.Support or Funding InformationHL119337, F31HL126377
Effects of Long‐term Administration of the KCa Channel Agonist SKA‐31 on Cardiovascular Function in the Type 2 Diabetic Goto‐Kakizaki Rat
Authors: Mishra Ramesh C , John Cini M , Kendrick Dylan J , Fedak Paul W , Wulff Heike , Braun Andrew P 
Published: November 2023

It is now evident that small and intermediate‐conductance, Ca2+‐activated K+ channels (KCa 2.3 and KCa 3.1, respectively) expressed in vascular endothelium are intimately involved in agonist‐evoked vasodilation of small resistance arteries throughout the circulation. In myogenically active vasculature exhibiting endothelial dysfunction associated with Type 2 Diabetes (T2D), we have shown that pharmacologic “priming” of KCa 2.3 and KCa 3.1 channels with the KCa channel agonist SKA‐31can acutely improve agonist‐stimulated dilation in isolated, myogenically active resistance arteries and fluid flow in the intact coronary circulation of non‐obese T2D Goto‐Kakizaki (GK) rats. In the present study, we have hypothesized that chronic administration of SKA‐31 will mitigate the progression of cardiovascular complications associated with T2D‐associated endothelial dysfunction (e.g. hypertension, cardiac impairment, atherosclerosis). Experimentally, male GK rats were implanted with radio‐telemeters and treated with 10 mg/kg or 30 mg/kg SKA‐31 or drug vehicle by daily I.P. injection for up to 12 weeks. The main cardiovascular parameters being monitored in our study include: blood pressure and heart rate, in vivo cardiac function (echocardiography), plasma metabolic factors, and immune cell/plasma cytokine profiles. Ex vivo assessment of vascular function is carried out at the end of the treatment protocol. The purpose of this presentation is to describe the preliminary data gathered to date.Support or Funding InformationThis work has been supported by research funding to APB from the Alberta Diabetes Institute and Canadian Institutes of Health Research
Sphingosine‐1‐Phosphate Protects Endothelial Cells and Microvascular Barrier Function Following Hypoxic Injury and Hemorrhagic Shock
Authors: Alves Natascha Guimaraes , Yuan Sarah Yuan , Breslin Jerome William 
Published: November 2023

Hemorrhagic shock can cause local hypoxic injury, activation of apoptotic signaling, shedding of the endothelial surface layer (glycocalyx), and elevated microvascular permeability in the gut. Several studies have suggested that the bioactive lipid sphingosine‐1‐phosphate (S1P) can protect endothelial barrier function. The objective of the current study was to test whether S1P reduces hemorrhagic shock and resuscitation (HSR)‐induced microvascular hyperpermeability, and to investigate its potential to protect endothelial cells from hypoxia‐induced damage and maintain the glycocalyx following HSR. For our in vivo studies, we used a rat model of HSR, in which S1P was administered in the intravenous resuscitation fluid. To assess permeability of the mesenteric microvasculature, we measured FITC‐albumin extravasation by intravital microscopy. As an indicator of glycocalyx health, we used an intravenous injection of FITC‐labeled lectin and measured the amount of lectin bound to the microvascular endothelium. For the in vitro studies, cultured primary rat mesenteric microvascular endothelial cells were placed in a low‐oxygen environment for 12h. S1P was applied during the hypoxic challenge to test its potential to prevent cell death. Cell viability was measured using propidium iodide (PI) staining. We have found that S1P reduces microvascular hyperpermeability following HSR in vivo, as seen as a decrease in albumin extravasation. Furthermore, we found that the amount of lectin bound to the mesenteric microvasculature is higher in HSR animals treated with S1P compared to HSR only. In our in vitro studies, we found that S1P prevents hypoxia‐induced cell death, characterized by a decrease in PI‐stained cells. Taken together, our results suggest that S1P has the potential to protect microvascular barrier dysfunction caused by HSR. In addition, S1P may prevent glycocalyx shedding following injury and reduce hypoxia‐induced activation of the apoptotic cascade. S1P's ability to protect the endothelial glycocalyx and prevent derangements produced by traumatic injury should be further investigated.Support or Funding InformationSupported by NIH grants R01HL098215 and R01GM120774
Sphingosine‐1‐phosphate reduces adhesion of malignant mammary tumor cells MDA‐MB‐231 to microvessel walls by protecting endothelial surface glycocalyx
Authors: Fu Bingmei , Zhang Lin , Zeng Min 
Published: November 2023

To test the hypothesis that the plasma phospholipid S1P can reduce tumor cell adhesion to microvessel walls by protecting the endothelial surface glycocalyx (ESG), we quantified the ESG by fluorescence immunostaining of heparan sulfate (HS), the most abundant glycosaminoglycans in the ESG, the microvessel permeability to albumin, and MDA‐MB‐231 tumor cell adhesion in the presence and absence of S1P in post‐capillary venules of rat mesentery. Rats (SD, 250–300g) were anesthetized with pentobarbital sodium given subcutaneously and kept warm on a heating pad. A midline incision (~2 inch) was made in the abdominal wall and the mesentery was gently taken out from the abdominal cavity and arranged on the surface of a glass coverslip for the measurement. A post‐capillary venule of 35–50μm diameter was cannulated by a glass micropipette; after 20 min perfusing with 1% fatty acid free bovine serum albumin Ringer solution with and without 1 μM S1P, the intensity of FITC‐anti‐HS, permeability to albumin and tumor cell adhesion rates were quantified, respectively, in 3 groups of experiments. In the absence of S1P, FITC‐anti‐HS labeled ESG was ~10% of that in the presence of S1P, whereas the permeability to albumin and adherent tumor cells were ~7‐fold and ~3.5‐fold (after 30 min adhesion) those in the presence of S1P, respectively. Our results conform to the hypothesis that protecting ESG by S1P inhibits tumor cell adhesion to the microvessel wall.Support or Funding InformationSupported by NIH SC1CA153325.
Modulation of P2Y2R‐Dependent Vascular Barrier Function: Focal Adhesion Kinase
Authors: Wang Jianjie , Harvey Joseph , Garrad Richard , Erb Laurie , Weisman Gary , Huxley Virginia 
Published: November 2023

We found that stimulation of P2Y2 receptor (P2Y2R) induced transient increase in venule permeability as 5‐folds greater than baseline in C57BL mouse cremaster skeletal muscle. Given that integrin mediates the P2Y2 receptor signaling, we hypothesized that focal adhesion complex would be involved in signaling pathway to regulate P2Y2R‐induced hyperpermeability. To test the hypothesis, we isolated and cultured microvascular endothelial cells derived from both wild‐type mice and P2Y2R knockout mice as in vitro models to assess the focal adhesion kinase signaling mechanism underlying P2Y2R‐induced hyperpermeability. We found that endothelial permeability was 1.27‐fold (1.27 ± 0.09, n=5, P<0.05) greater vs the baseline by 10−5 M UTP stimulation for 10 min in vitro, supporting the in vivo results. The increased permeability response to 10−5 M UTP was inhibited by focal adhesion kinase inhibitor PF‐228 (10−5 M). Phosphorylation of focal adhesion kinase at tyrosine‐397 increased (1.48 ± 0.11, n=3, P<0.05 compared with the basal level) in response to 10−5 M UTP stimulation for 10 min in the wild type microvascular endothelial cells while there was no change in P2Y2R knockout microvascular endothelial cells. The findings demonstrates that focal adhesion kinase plays an important role in the regulation of P2Y2R‐dependent hyperpermeability.Support or Funding InformationMissouri Sate University Faculty Research Grant