bluuebunny/crossref_metadata_2025 huggingface dataset:
Glucosamine Hydrochloride and Tunicamycin Attenuate Palmitate‐Induced Glycosylation of Cyclooxygenase‐2 in Primary Human Vascular Smooth Muscle Cells
Authors: Raman Puneet , Tat Trinh , Gonzales Rayna J
Published: November 2023

Cyclooxygenase‐2 is a well‐studied mediator of inflammation in the vessel wall, expressed minimally in normal physiological conditions but inducible in pathophysiological conditions like atherosclerosis. Increases in COX‐2 transcription result in prostanoid production, such as prostaglandin E2 (PGE2). PGE2 acts through multiple EP receptors in the vasculature to cause vasoconstriction, vasodilation, change vascular permeability, and promote remodeling. Both COX‐2 expression and PGE2 production contribute to the formation of unstable plaques and increased risk for thrombotic events. Recent studies have shown that free fatty acids such as palmitic acid can act as pro‐inflammatory ligands, but their effect on COX‐2 and regulation of vascular function and health is not well elucidated. Thus, the objective of the study was to investigate changes in post‐translational modification of COX‐2 in response to stimulation via palmitate, as well as pharmacological inhibition and the effect on downstream prostanoid production. Human vascular smooth muscle cells (HVSMC) of brain, aortic and coronary artery origin were treated chronically (12 to 18 hr) with vehicle (5% BSA and 0.1% ethanol), palmitate, tunicamycin, or glucosamine. Protein levels were assessed via immunoblotting and prostanoid production was assessed via ELISA. Palmitate increased the expression of COX‐2 and its glycosylation state. Co‐treatment with the N‐linked glycosylation inhibitors tunicamycin and glucosamine attenuated the increase in glycosylation state induced by palmitate. Additionally, palmitate‐induced increases in PGE2 production were attenuated by co‐treatment with tunicamycin. In conclusion, the results suggest that the increase of vascular COX‐2 glycosylation by saturated fatty acids increases COX‐2 activity, an effect attenuated by inhibition of glycosylation. This regulation of COX‐2 activity via post‐translational modification in the vasculature is a possible novel mechanism that may contribute to the development of cardiovascular disease by diminishing the health of vascular tissue.

Support or Funding Information

Valley Research Partnership (RG, PR) and University of Arizona Sarver Heart Center (PR)
Interaction between Sympathomimetic Amines, Norepinephrine, Tyramine and Dopamine at the Monoamine Transporter in Sympathetic Nerve Terminals and the Role of Nitric Oxide
Authors: Simaan Joseph
Published: November 2023

We previously showed that fresh synthesis of nitric oxide (NO) is involved in the transport of sympathomimetic amines, norepinephrine, tyramine, ephedrine and mephentermine across the monoamine transporter (MAT) in sympathetic nerve terminals and also in the blockade of the transporter by cocaine, imipramine, methylphenidate and atomoxetine. This study explored the effect of NO on the transport of dopamine (DA) and compared it with its effect on the transport of norepinephrine (NE), directly–acting on adrenergic receptors, tyramine (T), indirectly–acting by releasing norepinephrine from vesicles in sympathetic nerve terminals and methoxamine (M), not a substrate of MA transporter. Synthesis of NO was blocked with Nw‐nitro‐L‐arginine (L‐NNA) and the rise in arterial pressure it induces was reduced to starting level by restoring the basal concentration of NO through an infusion of nitroglycerin, an NO donor, thus observing the effect of fresh synthesis of NO. Anesthetized Sprague‐Dawley rats were used in which mean arterial pressure was measured. The pressor effect of single doses of NE (0.05, 0.1, 0.2 μg) was potentiated by 44–23% (P< 0,000), of T (0.025, 0.05. 0.1 mg) was reduced by 31–23% (P<0.001) and of DA (5, 10, 15μg) was reduced by 47–41% (P<0.002). The pressor effect of M was not affected. It is concluded that fresh synthesis of NO is significantly involved in the transport of NE, T and DA across the MA transporter and that in the used doses, the pressor effect of DA is mainly due to its indirect effect by releasing NE from sympathetic nerve terminals. Supported by funds from the American University of Beirut.

Support or Funding Information

Supported by funds from the American University of Beirut.
Deactivation of 12‐HETE by Peroxisomal β‐Oxidation by Alternatively Activated Macrophages
Authors: Kriska Tamas , Campbell William B.
Published: November 2023

Polarization of macrophages to pro‐inflammatory M1 and to anti‐inflammatory or alternatively activated M2 state has important physiological implications in development of experimental hypertension and inflammation. 12‐ and 15‐hydroxyeicosatetraenoic acids (12‐ and 15‐HETE), the two major arachidonic acid metabolites of 12/15‐lipoxygenase, are essential in the process since the disruption of the encoding gene, Alox15, renders the mice unsusceptible to variety of types of experimental hypertension. The objective of this study was to test the hypothesis that alternatively activated M2 macrophages catabolize 12‐HETE into products that do not enhance angiotensin II‐mediated arterial constriction.

We used cultures of M1‐ or M2‐polarized macrophages to study the metabolism of externally added [14C]‐12‐HETE. The 12‐HETE metabolites were separated by a reverse phase HPLC and identified by mass spectrometry.

M2 polarized peritoneal macrophages converted most of the 12‐HETE into more polar metabolites within 2 hours of incubation. M1 macrophages, however, had little effect on the 12‐HETE catabolism. The same effect was observed in cultures of M1 or M2 polarized bone marrow‐derived macrophages. The major metabolite peaks were identified by mass‐spectrometry as β‐oxidation products. The conversion of 12‐HETE was inhibited by 50 μM thioridazine, a peroxisomal β‐oxidation inhibitor. Mitochondrial β‐oxidation inhibitors, 4‐pentenoic acid and etomoxir, had no effect on conversion. qPCR analysis of peroxisomal and mitochondrial β‐oxidation markers confirmed that only peroxisomal markers were upregulated upon M2 polarization. The major 12‐HETE metabolite peaks were collected and their activities were tested on abdominal aortic rings. While 12‐HETE (100 nM) pre‐incubation enhanced constrictions to angiotensin II (1–100 nM) from 15 % to 25 %, the metabolites lacked such an activity.

Our results indicate that alternatively activated M2 macrophages are capable of degrading 12‐HETE and eliminating 12‐HETE's enhancement of angiotensin II‐induced vascular constriction, while M1 macrophages are not. These data support an antihypertensive role of M2 macrophages.

Support or Funding Information

This research was supported by the grant: NIH HL103673
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 Information

This 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 Information

This 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 Information

NIH 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

Background

Multiple 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 results

Five 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).

Conclusion

In 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 Information

Funding: 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).

PURPOSE

The 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.

METHODS

To 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.

RESULTS

Critical 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).

CONCLUSIONS

These 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 Information

HL119337, F31HL126377
Impact of early mammalian development on the response of the O2 transport system to muscle contraction
Authors: Dodhy Sami C , Pittman Roland
Published: November 2023

A continuous supply of O2 to the cells in the body is necessary to maintain normal physiological function and the microcirculation is especially important in this matter as it is the site of O2 exchange. Under conditions of active/functional hyperemia following muscle contractions, the O2 transport system in older subjects does not appear to respond as rapidly and to the same degree as in younger subjects. With aging, elements of the O2 transport and regulatory systems are changed in such a way that matching O2 supply to O2 demand does not work as well in older as in younger subjects. An intravital microscopic approach is being used to show that developmental changes in the O2 transport system begin earlier than previously thought. Significant and rapid changes in the microvascular network of skeletal muscle have been observed during the first few weeks of postnatal development. Four different developmental groups of male Sprague‐Dawley rats (8, 16, 24, and 52 weeks) are used to investigate changes in the O2 demand component of the transport system by measuring oxygen consumption (VO2) and the PISFO2 dependence of VO2 under conditions of rest and contraction. VO2 was measured with a quasi‐continuous, flash‐synchronized, rapidly pressurizing airbag system to briefly arrest flow and determine the rate of change in oxygen tension (dPO2/dt). Changes in the O2 supply component are investigated by measuring capillary and interstitial fluid PO2 (PcapO2 and PISFO2 respectively, using phosphorescence quenching microscopy) and surface area available for O2 exchange (i.e., capillary density). Preliminary experiments were carried out on six animals that ranged from 8 w to 20 w in age with an average weight of 331 ± 35 g. Average baseline VO2 for the 8 w group was 307 ± 26 nL O2/cm3·s (n = 6), average baseline VO2 for the 16 w group was 287 ± 31 nL O2/cm3·s (n = 6), and average baseline VO2 for those in the 24 w group was 264 ± 12 nL O2/cm3·s (n = 6). Average baseline PISFO2 for the 8 w group was 68 ± 3 mmHg (n = 6), average baseline PISFO2 for the 16 w group was 74 ± 4 mmHg (n = 6), and average baseline PISFO2 for the 24 w group was 68 ± 3 mmHg (n = 6). The observed decreases in tissue VO2 are consistent with literature showing that whole body VO2 decreases from 499, 396, to 345 nL O2/cm3·s at 8 w, 16 w, and 24 w, respectively. However, it is still unclear how well matched O2 supply and demand are. As O2 demand increases by increasing muscle contraction, O2 supply should also increase to match it and PISFO2 is at least one element that links the two components of the transport system which requires further investigation.
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 Information

This 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 Information

Supported 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 Information

Supported 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 Information

Missouri Sate University Faculty Research Grant
Ligustilide activates Nrf2/HO‐1 induction, NO synthesis and ameliorates vascular inflammation in HUVECs
Authors: Choi Eun Sik , Yoon Jung Joo , Namgung Seung , Jeong Da Hye , Kim Hyun Ju , Lee Yun Jung , Kang Dae Gill , Lee Ho Sub
Published: November 2023

(Z)‐ligustilide is a bioactive phthalide derivative isolated from Cnidii Rhizoma (Cnidium officinale, rhizome) and Angelicae Gigantis Radix (Angelica gigas Nakai, root). Vascular endothelium is a central spot in developing cardiovascular diseases and chronic vascular inflammation results in atherosclerosis development. We investigated anti‐inflammatory effects of ligustilide against TNF‐α, and whether ligustilide can activates Nrf2/HO‐1 induction, intracellular NO synthesis in endothelial cells. Ligustilide significantly suppressed adhesion of HL‐60 monocyte to HUVECs via suppressing expression of VCAM‐1, ICAM‐1, and E‐selectin against TNF‐α. Also, ligustilide significantly inhibited ROS production and IκB‐α phosphorylation, and it leaded to suppression of NF‐κB activation. Furthermore, ligustilide up‐regulated induction of HO‐1 via Nrf2 nuclear translocation and synthesis of intracellular NO in HUVECs. Present study demonstrates that ligustilde has vascular protective potential and it might prevents developing cardiovascular complications such as thrombosis or atherosclerosis.

Support or Funding Information

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (2008‐0062484).
Mechanisms of Vasodilatory Effects of Total Flavonoids from Euphorbia humifusa in Rat Aorta
Authors: Ji Xiao Wei , Wu Bo , Zhou Zi Qi , Kang Dae Gill , Lee Ho Sub , Cho Kyung Woo , Jin Song Nan , Wen Jin Fu
Published: November 2023

Euphorbia humifusa (EH) is, rich in flavonoids, used for treating bacillary dysentery and enteritis in many Asian countries. It is noteworthy that the beneficial actions of EH, such as inhibiting inflammation, antioxidation, enhancing immunity and lowering blood pressure, might be mainly attributed to its abundant flavonoids. However, the effects of total flavonoids of EH (TFEH) on the vascular function and its pharmacological mechanisms of action have not been studied. The aim of the present study was to evaluate the vascular effects of TFEH and its action mechanisms in rats. The vasoactive effect of TFEH and its underlying mechanisms in rat thoracic aortas were investigated using the organ bath system. TFEH induced a concentration‐dependent vasorelaxation in endothelium‐intact (+E) aortic rings, which was abolished by the removal of endothelium. The nitric oxide synthase inhibitor L‐NAME and the soluble guanylate cyclase inhibitor ODQ significantly attenuated the endothelium‐dependent vasorelaxation of TFEH. However, inhibition of cyclooxygenases with indomethacin had no effects on the TFEH‐induced vasorelaxation. Extracellular Ca2+ depletion, inhibition of L‐type Ca2+ channels with diltiazem and modulators of the store‐operated Ca2+ entry (SOCE), Gd3+ and 2‐aminoethyl diphenylborinate markedly attenuated the TFEH‐induced vasorelaxation in +E vessels. Wortmannin, an inhibitor of Akt, significantly attenuated the TFEH‐induced vasorelaxation in +E vessels. Furthermore, K+ channel blockers, such as a non‐selective KCa channels blocker tetraethylammonium and KATP channel blocker glibenclamide, and inhibitors of muscarinic and adrenergic receptors had no significant effects on the vasorelaxation. These data demonstrate that TFEH induces vasorelaxation through activation of endothelium‐dependent two‐step signaling: first is an activation of the Akt‐ and Ca2+‐eNOS‐NO signaling in the endothelial cells and then subsequent stimulation of the NO‐soluble guanylyl cyclase‐cGMP signaling in the vascular smooth muscle cells.

Support or Funding Information

This work was supported by research grants from the National Natural Science Foundation of China (Nos. 81270920, 31571175), the Natural Science Foundation of Shandong Province (No. ZR2015HM037), and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (2008‐0062484).
The Functional Contribution Of Myoendothelial Feedback To Arterial Tone Is Determined By The Nature Of The Vasoconstricion
Authors: Kerr Paul M , Wei Ran , Lunn Stephanie E , Gust Stephen L , Classen Boyd , Murphy Timothy V , Sandow Shaun L , Plane Frances
Published: November 2023

Constriction of resistance arteries in response to global application of agonists is modulated by activation of the endothelium via myoendothelial feedback. Recent evidence supports a model of myoendothelial feedback in which generation of localized InsP3‐dependent Ca2+ transients leads to activation of intermediate conductance Ca2+‐activated K+ (IKCa) channels to hyperpolarize the endothelium. We have now investigated the functional contribution of this pathway to limiting responses to two physiologically important vasoconstrictor stimuli, activation of sympathetic nerves and increases in intravascular pressure. In isolated segments of rat mesenteric resistance artery mounted in a wire or pressure myograph, pharmacological inhibition of IKCa channels significantly potentiated increases in tone elicited by global application agonists such as noradrenaline, but did not significantly alter increases in tone elicited by stimulation of sympathetic perivascular nerves, or myogenic reactivity elicited by step‐wise increases in intravascular pressure. Similarly, in the endothelium‐intact perfused mesenteric bed, blockers of IKCa channels enhanced vasoconstriction to exogenous noradrenaline but had no effect on nerve‐evoked vasoconstriction. However, the influence of the endothelium on responses to sympathetic nerve stimulation was inhibited by blockers of nitric oxide signaling and by inhibition of small conductance Ca2+‐activated K+ (SKCa) channels. These data demonstrate that the contribution of myoendothelial feedback to endothelium‐dependent modulation of tone is determined by the stimulus for smooth muscle contraction. Although activated by global application of contractile agonists, the myoendothelial feedback pathway does not appear to play a significant role in modulating myogenic reactivity or nerve‐evoked responses in resistance arteries. Furthermore, in the intact mesenteric vascular bed, under conditions of constant flow, increases in shear stress rather than myoendothelial feedback, may be the predominant stimulus for engagement of the endothelium to oppose nerve‐evoked vasoconstriction.

Support or Funding Information

Supported by a grant‐in‐aid from the Heart and Stroke Foundation of Alberta, Nunavut and the North West Territories to FP (G130003076)
Vasoactivity of the Vasculature Directly Supplying the Rat Sciatic Nerve
Authors: Williams Elise , Klein Amanda Taylor , Jackson Dwayne N , Twynstra Jasna
Published: November 2023

Objectives

The purpose of this study was to determine the vasoactivity of the vasculature directly supplying the sciatic nerve in response to phenylephrine (PE) and acetylcholine (ACh) using plasma fluorescence and intravital microscopy.

Methods

PE (10−4M, n=6) and ACh (10−4M, n=9) were applied to an exposed sciatic nerve in an anesthetized Sprague Dawley rat model (PE: 321.6±11.7g, ACh: 307.56±41.57g). A fluorescent dye, FITC (0.3 ml at 5%), was infused via a jugular catheter to fluoresce the plasma stream, while intravital microscopy was used to visualize the plasma stream through the vessels running directly over the sciatic nerve. Each vasoactive agent was topically applied over the sciatic nerve for 6 minutes. Measurements were taken at baseline (immediately prior to drug application), 2 minutes, 4 minutes and 6 minutes after the start of drug application, and finally at 4 minutes after PE or ACh application was stopped (recovery). Maximal diameter was achieved via application of 10−4M of Sodium Nitroprusside (SNP). Data are expressed as mean ± standard error.

Results

Following PE and ACh application, mean arterial pressure (baseline PE: 105.6±11.1mmHg; PE: 102.0±9.9mmHg, baseline ACh: 100.8±4.1mmHg, ACh: 100.2±4.6mmHg) was not affected. PE application resulted in a decreased diameter of the plasma stream (maximal change in diameter: −5.8±1.9um), while ACh application did not result in changes in diameter (maximal change in diameter: 1.5±0.7um). Maximal diameter in response to SNP was not significantly different from the baseline diameter (Baseline: 19.36±6.62um, SNP: 19.80±7.01um)

Conclusion

PE application resulted in a vasoconstriction of the vessels directly supplying the sciatic nerve, while application of ACh or SNP failed to produce significantly vasodilation. Overall, the vessels supplying the sciatic nerve appeared to be maximally dilated at baseline, which may explain the lack of dilation in response to ACh or SNP.

Support or Funding Information

This study was supported by Brescia University College Research Grant.
Acid‐Sensing Ion Channels: Mediator of Cerebrovascular Responses to CO2
Authors: Faraci Frank , Taugher Rebecca , Fan Rong , Wemmie John
Published: November 2023

Because normal brain function is critically dependent on cerebral blood flow (CBF), insufficient CBF contributes to neurological dysfunction and degeneration. Carbon dioxide (CO2), via effects on local acidosis, is one of the most potent regulators of CBF. Although a role for nitric oxide (NO) in intermediate signaling has been implicated, mechanisms that initiate CO2 induced‐vasodilation remain unclear. Acid‐sensing ion channel‐1A (ASIC1A) is a proton‐gated cation channel that is activated by extracellular acidosis. Based on work that implicated ASIC1A in the amygdala and stria terminalis in CO2‐ and acid‐evoked behavior, we hypothesized that ASIC1A might also mediate microvascular response to CO2. To test this hypothesis, we genetically and pharmacologically manipulated ASIC1A and assessed effects on CO2‐induced dilation of small cerebral arterioles in vivo. Genetic deficiency in ASIC1A (Asic1a−/−) or local treatment with psalmotoxin (an ASIC inhibitor) in Asic1a+/+ mice eliminated the majority of the effect of 5 or 10% CO2 on arteriolar diameter (P<0.05). Vasodilator effects of acetylcholine, which acts via endothelial NO synthase were unaffected, suggesting a non‐vascular source of NO was key for CO2 responses. We then hypothesized that neurons may be the cell type through which ASIC1A influences microvessels. In mice in which Asic1a was specifically disrupted in neurons, effects of CO2 on arteriolar diameter were also greatly attenuated. To further explore a role for ASIC1A in the regulation of NO production, we measured NO concentrations in whole brain lysates immediately after exposure to CO2. We found that 10% CO2 induced NO production in wild‐type mice, but not in mice lacking Asic1a globally or specifically in neurons. Together, these data are consistent with a model wherein activation of neuronal ASIC1A is critical for CO2‐induced NO production and vasodilation. With these findings, ASIC1A emerges as major regulator of microvascular tone in brain.

Support or Funding Information

This work was supported by the NIH, the Department of Veterans Affairs, and the Fondation Leducq.
Multiple Sclerosis Alters Dynamic Cerebral Autoregulation during Heat Stress
Authors: Allen Dustin Ryan , Huang Mu Ryan , Purkayastha Sushmita , Brothers Robert Matthew , Davis Scott L
Published: November 2023

Multiple sclerosis (MS) is a neurological disease marked by demyelination within the central nervous system that results in individuals with MS experiencing a myriad of symptoms, including cognitive dysfunction and orthostatic dizziness. Adding complexity, many individuals with MS (60–80%) experience temporary worsening of their symptoms upon increases in core temperature (Tcore), an occurrence known as Uhthoff's phenomenon. This study tested the hypotheses that individuals with MS: 1) have impaired cerebral autoregulation under normothermic baseline conditions, and 2) have exacerbated impairments in cerebral autoregulation as a result of a passive heat stress compared to healthy controls (CON). Seven subjects with relapsing‐remitting MS (age: 44±7 yrs; height: 174±9 cm; weight: 74±17 kg) and 7 matched healthy controls (CON; age: 44±6 yrs; height: 176±11 cm; weight: 81±14 kg) were outfitted in a tube‐lined water perfusion suit in which they were exposed to a normothermic baseline (NT: 34 °C circulating water) condition for 10 minutes followed by a ~60‐minute whole‐body heat stress (WBH: 48 °C circulating water) until a 0.8 °C increase in Tcore was achieved. Continuous measurements of mean middle cerebral artery blood velocity (MCAv; transcranial Doppler ultrasonography) and mean arterial pressure (MAP; finger photopleythsmography) were taken throughout each thermal condition. Transfer function analysis of beat‐to‐beat fluctuations in MAP and MCAv was used to quantify dynamic cerebral autoregulation (dCA) during NT and WBH. All data from transfer function analyses were examined at very low frequency (VLF: 0.02–0.07 Hz), low frequency (LF: 0.07–0.20 Hz), and high frequency (HF: 0.20–0.30 Hz) ranges. Estimates of coherence exceeded 0.5 at LF and HF during NT, and all frequencies during WBH. Contrary to our hypothesis, LF phase (MS: 0.37±0.20 vs CON: 0.55±0.37 radians, p=0.29) and LF gain (MS: 0.83±0.21 vs CON: 0.89±0.12 cm/s/mmHg, p=0.52) were similar during NT. No differences in LF phase during WBH (MS: 0.73±0.30 vs CON: 0.66±0.30 radians, p=0.66). However, differences in LF gain were observed between groups following WBH (MS: 1.26±0.47 vs CON: 0.79±0.21 cm/s/mmHg, p=0.03). These preliminary data demonstrate a negative impact on estimates of transfer function gain that manifest with increases in Tcore, likely indicating MS impairments in dynamic cerebral autoregulation during heat stress.

Support or Funding Information

Supported by NIH (R15HL11722401) and NMSS (RG4696A3/2) grants.