bluuebunny/crossref_metadata_2025 huggingface dataset:
Angiotensin II‐induced arterial fibrosis and stiffness is prevented in arginase 1‐deficient mice (1065.8)
Authors: Bhatta Anil , Xu Zhimin , Caldwell Ruth , Caldwell R. William
Published: June 2021

Arterial stiffness (AS) is an independent risk factor for cardiovascular morbidity and mortality. Increases in smooth muscle cell (SMC) proliferation and collagen synthesis are key features in development of AS. Arginase (ARG), an enzyme implicated in many cardiovascular diseases and fibrosis, can compete with nitric oxide (NO) synthase for their common substrate, L‐arginine. Increased ARG, in addition to reducing NO production, can also elevate levels of ornithine for synthesis of polyamines via ornithine decarboxylase (ODC) and proline/collagen via ornithine aminotransferase (OAT), leading to increases in cell proliferation and collagen formation, respectively. In order to investigate the role of ARG1 in AS, wild type (WT) or ARG1 KO mice were infused with angiotensin II (Ang II, 4 wks). Ang II infusion in WT mice increased levels of aortic ARG activity/expression, ODC mRNA, hydroxyproline, type I collagen, fibrosis, and induced AS (as shown by increased pulse wave velocity). These effects did not occur in Ang II infused ARG1 KO mice. Exposure of rat aortic SMCs to Ang II (1 μM, 48 hrs) increased ARG activity (57%), cell proliferation (22%), type 1 collagen protein (44%), ODC mRNA (25%), and hydroxyproline (22%). Treatment with ARG inhibitor ABH prevented these changes. In summary, ARG1 is crucially involved in arterial SMC proliferation, fibrosis and stiffness and represents a promising therapeutic target.
Chronic baclofen treatment influences GABAB activation of G‐proteins and phosphorylation of FAK, GSK3β, and DARPP32 in mesocorticolimbic rat brain regions (1066.3)
Authors: Keegan Bradley , Beveridge Tom , Pezor Jeffrey , Xiao Ruoyu , Sexton Tammy , Childers Steven , Howlett Allyn
Published: June 2021

The GABAB receptor agonist baclofen is being explored as a potential treatment for cocaine addiction, as therapeutic activation of the GABAergic system is theorized to counterbalance over‐stimulation of the dopaminergic system in brain regions involved in reward. Rats acutely administered i.p. baclofen (5 mg/kg, single dose 15 min. prior to sacking) showed no immediate changes in GABAB‐stimulated GTPγS binding in any brain region but displayed significantly greater phosphorylation of focal adhesion kinase (FAK) in key mesocorticolimbic regions (caudate putamen, cortex, hippocampus, thalamus, and prefrontal cortex) compared to controls, as well as elevated levels of glycogen synthase kinase 3‐beta (GSK3β) phosphorylation in these and other regions (nucleus accumbens, septum, and globus pallidus). In contrast, GABAB‐stimulated GTPγS binding in rats administered chronic (5 mg/kg, t.i.d. for five days) baclofen was significantly lower in the prefrontal cortex, septum, amygdala, and parabrachial nucleus than in the controls. While chronically‐treated rats exhibited similar increases in FAK phosphorylation, they displayed attenuated levels of GSK3β phosphorylation and wide‐spread elevations in levels of DARPP32 phosphorylation compared to those treated acutely or with vehicle. These findings indicate a desensitization of GABAB‐mediated signal transduction in rats following chronic administration of baclofen.

Grant Funding Source: Supported by the National Institute on Drug Abuse: R01‐DA03690 and P50‐DA006634.
Structural requirements for FPR2/ALX interaction with formyl peptides (1066.7)
Authors: Ye Richard , He Hui‐Qiong , Troksa Erica , Caltabiano Gianluigi , Pardo Leonardo
Published: June 2021

Unlike formyl peptide receptor 1 (FPR1), FPR2/ALX (FPR2) interacts with peptides of diverse sequences but has low affinity for the Escherichia coli chemottactic peptide fMet‐Leu‐Phe (fMLF). We investigated the structural requirements for FPR2 to interact with formyl peptides of different composition. In calcium flux assays, the N‐formyl group of these peptides is necessary for activation of both receptors while the composition of the C‐terminal amino acids appears more important for FPR2 than FPR1. FPR2 interacts better with pentapeptides (fMLFII, fMLFIK) than tripeptide (fMLF) and tetrapeptides (fMLFK, fMLFW), but not peptides with negatively charged side chains at the C‐terminus (e.g. fMLFE). In contrast, FPR1 does not discriminate against the C‐terminal negative charges. A CXCR4‐based homology model of FPR1 and FPR2 revealed that Asp2817.32 could be crucial for FPR2 interaction with certain formyl peptides as its negative charge might be repulsive with the terminal COO‐ group of fMLF and the negatively charged Glu in fMLFE. Asp2817.32 is a candidate for a stable interaction with the positively charged Lys in fMLFK. Site‐directed mutagenesis was performed to remove the negative charge at position 281 in FPR2. The resulting mutant, D2817.32G, showed a 10‐fold higher affinity for fMLFE and fMLF, but it reduced interaction with fMLFK compared with wild type FPR2. These results show that FPR1 and FPR2 use different structural determinants for interaction with formyl peptides.
Pre‐exposure of the urotensin II receptor to urotensin II or urotensin II‐related peptide differentially reduces the response to subsequent additions (1066.8)
Authors: Warren Taylor , Clark Stewart
Published: June 2021

The Urotensin II receptor (UIIR) is a G protein‐coupled receptor which is activated by urotensin II (UII) and urotensin‐related peptide (URP). Studies show equivalent Kd values and a similar EC50 in Ca2+assays in vitro. The co‐expression of the peptides, spatially and temporally, in vivo leads us to hypothesize that the ligands mediate different post‐activation events. Ca2+ mobilization was used to monitor receptor activation. CHO‐rUIIR cells pre‐treated with 1pM UII have a 40% reduction in response to a second addition of 10nM UII. The same experiment was carried out with equimolar URP; no significant decrease was seen. The same phenomenon was seen when cells were pre‐treated for 6 or 19 minutes, washed, and a second addition added one hour later. UII and URP have identical cyclic regions, but UII has an additional short n‐terminal tail containing a charged residue. The Ca2+ assay was repeated using peptide truncations, and these truncations resulted in differential desensitization. Desensitization mechanisms may lead to proliferative pathways. Therefore, cells were treated with UII or URP overnight. UII dose‐dependently increased MTS reduction (130%; MTS assay) compared to control; URP showed no significant increase. Data suggests low concentrations of UII and URP induce different post‐activation events. A charged residue at the n‐terminal of the cyclic region may alter receptor conformation upon binding.

Grant Funding Source: Supported By NIH Grant# R00DA024754‐05S1
Transient receptor potential vanilloid type 4 channel as a putative target molecule for anti‐hypertensive therapy (1067.10)
Authors: Walas Dawid , Paton Julian
Published: June 2021

TRPV4 is a non‐selective cation channel causing vasodilatation (Willette et al. 2007) and cerebral arterial re‐modelling in response to shear stress (Schierling et al., 2011). Our hypothesis is that TRPV4 is an intracranial sensor that maintains cerebral blood flow by sympathetically mediated modulation of arterial pressure.

Using qPCR, we demonstrated that TRPV4 mRNA levels from common carotid arteries of the Spontaneous Hypertensive Rat (SHR) were lower compared to a normotensive Wistar (9 weeks old animals, p=0.002, n=3). mRNA levels in the SHR brain vasculature were age related with older rats showing higher levels (e.g. at 9 weeks old mRNA was 2 times lower than 13 weeks old SHR, p=0.06, N=3). This difference was not detected in Wistar rats (p=0.997, n=3).

We next examined the effect of TRPV4 agonist (GSK1016790A) on arterial pressure in anesthetised SHRs fitted with radio‐telemeters. Bolus intravenous injections showed a dose‐dependent reduction in arterial pressure with maximal response achieved at 166 µg/kg and the lowest effective dose being 42 µg/kg (n=3); the latter dose has been reported to cause circulatory collapse in normotensive rats (Willette et al. 2007). Thus, SHR showed a higher tolerance to the agonist consistent with lower mRNA expression.

Presence of TRPV4 in the cerebral vessels and its vasodilatory effect are both consistent with it being an exploitable target for improving cerebral blood flow and for controlling hypertension.
MCA‐APK(Dnp) is not a selective substrate of angiotensin‐converting enzyme‐2 (1067.6)
Authors: Carrera Eduardo , Linares Andrea , Joachim Arline , Jean‐Baptiste Malaika , Speth Robert
Published: June 2021

MCA‐APK(Dnp) is a fluorogenic substrate used in assays of angiotensin‐converting enzyme‐2 (ACE‐2) metabolism. Cleavage of the Pro‐Lys bond removes the quenching Dnp group and induces fluorescence. To ascertain the specificity of MCA‐APK(Dnp) for ACE‐2, we measured metabolism of the substrate in rat kidney in the presence or absence of the ACE‐2 inhibitor MLN‐4760. Frozen rat tissues were homogenized in 19 volumes of 50 mM sodium phosphate at pH 7 with 0.05% Triton X‐100 detergent and centrifuged at 48,000 x g. The supernatant was then diluted 20‐fold in assay buffer for a final concentration of 50 mM sodium phosphate at pH 6, 6.5, 7, or 7.5 and 100 mM NaCl. 20 µL of diluted supernatant, 10 µl of 50 µM MLN‐4760, and 20 µl of 125 µM substrate were added to each well for a reaction volume of 50 µL. Metabolism of MCA‐APK(Dnp) was recorded at 37oC at a wavelength of 393 nm with excitation at 328 nm. Breakdown of MCA‐APK(Dnp) attributable to ACE‐2 represented only 21%, 28%, 44%, and 56% of total enzymatic activity at pH 6, 6.5, 7, and 7.5, respectively. Total activity also increased with decreasing pH. Our results suggest that MCA‐APK(Dnp) is not selective for ACE‐2. When using MCA‐APK(Dnp) as a surrogate substrate for determination of ACE‐2 activity, it is critical to use a selective inhibitor or ACE‐2 activity such as MLN‐4760 to differentiate ACE‐2 versus non‐ACE‐2 mediated metabolism of MCA‐APK(Dnp).

Grant Funding Source: NIH‐HL113905
Major role of ryanodine type 2 receptors in global and local intracellular calcium release in arterial smooth muscle (1067.7)
Authors: Kassmann Mario , Schleifenbaum Johanna , Szijártó István , Anistan Yoland‐Marie , Gollasch Maik
Published: June 2021

Ryanodine receptors (RyR) are calcium‐sensitive ion channels in the sarcoplasmic reticulum (SR) membrane, and are important effectors in arterial smooth muscle excitation‐contraction (EC) coupling. While three different RyR isoforms are expressed in arterial smooth muscle cells (SMCs), the relative contribution of each RyR isoform to vascular function remains unresolved. We generated tamoxifen‐inducible SMC specific knock‐out mice of RyR2 to test the hypothesis of RyR2 contributing in arterial EC coupling, vasoregulation, and systemic blood pressure. Myography and videomicroscopy of isolated arteries, calcium imaging and patch clamp current recordings on isolated SMCs, and radiotelemetric blood pressure measurements were used. Targeted deletion of the gene for RyR2 resulted in a loss in functional coupling of calcium sparks to associated BK potassium channel activation, and increased arterial tone and systemic blood pressure. Aortas, femoral, cerebral and mesenteric arteries from RyR2‐deficient mice showed no contraction in response to RyR activation by 10 mmol/L caffeine. Our data demonstrate that RyR2 are key elements in Cav1.2 channel‐induced EC coupling in large and small arteries. Furthermore, RyR2, by activating BK channels, are important molecular SR calcium release components in translating local calcium signals (Ca2+‐sparks) to vasoregulation and arterial blood pressure.

Grant Funding Source: Supported by the Deutsche Forschungsgemeinschaft (DFG)
Stretch‐activation of angiotensin II type 1a receptors contributes to the myogenic response of mouse mesenteric and renal arteries (1067.8)
Authors: Schleifenbaum Johanna , Kassmann Mario , Szijártó István , Hercule Hantz , Weinert Stefanie , Heidenreich Matthias , Pathan Asif , Anistan Yoland‐Marie , Alenina Natalia , Rusch Nancy , Bader Michael , Jentsch Thomas , Gollasch Maik
Published: June 2021

Wall stretch is a major stimulus for the myogenic response of small arteries to pressure. Recent findings suggest that G protein‐coupled receptors can elicit a stretch response. Our aim was to determine if angiotensin II type 1 receptors (AT1R) in vascular smooth muscle cells (VSMC) exert mechanosensitivity and identify the downstream ion channel mediators of myogenic vasoconstriction. We used mice deficient in AT1R signaling molecules and putative ion channel targets, namely AT1R, angiotensinogen, TRPC6 channels or subtypes of the KCNQ (Kv7) gene family (KCNQ3, 4 or 5). We identified a mechano‐sensing mechanism in mesenteric arteries and the renal circulation that relies on coupling of the AT1R subtype a (AT1aR) to a Gq/11‐protein as a critical event to accomplish the myogenic response. The mechano‐activation occurs after block of AT1R, and in the absence of angiotensinogen or TRPC6. Activation of AT1aR suppresses XE991‐sensitive Kv channel currents in VSMCs, blocking these channels enhances mesenteric and renal myogenic tone. Although KCNQ3, 4 and 5 are expressed in VSMCs, XE991‐sensitive K+ current and myogenic contractions persist in arteries deficient in these channels. Our results provide evidence that myogenic responses of mouse mesenteric and renal arteries rely on ligand‐independent mechano‐activation of AT1aR. This signal relies on an ion channel distinct from TRPC6 or KCNQ3, 4 or 5.

Grant Funding Source: Supported by Deutsche Forschungsgemeinschaft (DFG)
Contribution of voltage‐gated potassium channels in cerebrovascular dysfunction associated with a genetic model of ischemic small vessel disease (1068.1)
Authors: Dabertrand Fabrice , Krøigaard Christel , Bonev Adrian , Brayden Joseph , Joutel Anne , Nelson Mark
Published: June 2021

Dominant mutations in the NOTCH3 gene induce the most common heritable cause of stroke and vascular dementia, referred to as Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL). Using a recently developed mouse model of CADASIL (Joutel, JCI, 2010), we examined the consequences of this mutation on the function of intracerebral arterioles. Elevation of intravascular pressure to 20 mm Hg constricted isolated arterioles from control and CADASIL mice to a similar extent. However, above 30 mm Hg, CADASIL arterioles displayed impaired vascular reactivity. At 40 mm Hg, CADASIL arterioles were 38% less constricted, and their smooth muscle membrane potential was 10 mV more hyperpolarized than control. A pharmacologic approach revealed an unchanged activity of small‐, intermediate‐, large‐conductance calcium‐sensitive potassium channels. However the voltage‐gated potassium (Kv) channel blocker 4‐AP enhanced pressure‐induced constriction to a greater extent in both parenchymal arterioles and pial arteries from CADASIL mice. Conversely, mesenteric resistance arteries displayed similar 4‐AP‐induced constriction and similar myogenic response in both groups. In isolated cerebral myocytes, Kv current density was 30% greater in CADASIL than in control, whereas gating properties were similar. These results support the concept that the CADASIL mutation decreases pressure‐induced vasoconstriction through upregulation of smooth muscle Kv channels in cerebral arteries and arterioles.

Grant Funding Source: NIH PO1HL095488, RO1HL44455, the Totman Trust and the Fondation Leducq
Uncoupling of flow‐pressure relationships following sport concussion in elite athletes (1068.14)
Authors: Bishop Scott , Burnett Tanis , Smirl Jonathan , Ainslie Philip , van Donkelaar Paul , Neary Patrick
Published: June 2021

Introduction: Although sports concussion has the potential to impair the brains ability to protect against changes in blood pressure (BP), this has not been examined in in elite adolescent hockey players. We hypothesised that dynamic cerebral pressure‐flow relationships would be altered following a sports‐related concussion.

Methods and Procedure: Using a within‐subject design, 49 elite adolescent hockey players (age = 17.4 ± 1.4 years) were tested. Middle cerebral artery velocity, beat‐to‐beat blood pressure and end‐tidal CO2 were measured. Testing sessions involved resting and driven (squat‐stand maneuvers at very low (VLF, 0.05Hz) frequencies) measures to maximize BP variability and improve assessment of the pressure‐flow relationship using transfer function analysis (TFA). Players recovering from a concussion (n=4) repeated the protocol at day 3 ± 2, day 8 ± 2, and day 16 ± 5.

Results and Discussion: Preliminary VLF findings for baseline vs. post‐injury indicate that phase parameters (baseline = 1.01 ± .07 radians, day3 = 0.46 ± .1 radians; p <0.05) decreased during the first 3 ± 2 days post injury. By the third measurement, no significant differences were found, however phase parameters were still not completely returned to baseline. This initial change suggests that a concussion causes a reduction in the buffering capacity of the cerebral pressure‐flow response in elite adolescent hockey players.
Dynamic cerebral autoregulation and tissue oxygenation in amnestic mild cognitive impairment (1068.7)
Authors: Tarumi Takashi , Dunsky David , Ayaz Muhammad , Liu Jie , Hill Candace , Armstrong Kyle , Martin‐Cook Kristin , Cullum Munro , Zhang Rong
Published: June 2021

Background: Vascular disease and dysfunction elevate the risk of Alzheimer’s disease. The objective of this study was to determine whether dynamic regulation of cerebral blood flow and tissue oxygenation is impaired in patients with amnestic mild cognitive impairment (aMCI). Methods: Twenty‐seven patients with aMCI and 15 control subjects underwent measurements of cerebral hemodynamics, brain MRI, and neurocognitive assessment. Dynamic cerebral autoregulation and tissue oxygenation were assessed by transfer function analysis of mean blood pressure (MBP), normalized cerebral blood flow velocity (CBFV%), and cerebral tissue oxygenation index (TOI) at baseline and during a sit‐stand maneuver. Results: Patients with aMCI demonstrated lower cognitive performance in memory and executive function, accompanied by smaller volumes of entorhinal cortex. At baseline, aMCI patients exhibited lower levels of cerebral TOI. Transfer function gain and phase between MBP and CBFV% and between CBFV% and cerebral TOI did not differ between the groups. Within aMCI patients, greater oscillations and transfer function gain of cerebral TOI were associated with the lower scores on delayed recall. Conclusions: An inverse association between transfer function gain of cerebral TOI and memory performance suggests impairment of oxygen transport from the microcirculation to brain tissue in patients with aMCI.

Grant Funding Source: National Institute on Aging
Cerebral hemodynamics in normal aging: associations with central hemodynamics and cerebral small vessel disease (1069.2)
Authors: Tarumi Takashi , Ayaz Muhammad , Liu Jie , Tseng Benjamin , Parker Rosemary , Jonathan Riley , Tinajero Cynthia , Zaidi Waqar , Zhang Rong
Published: June 2021

Background: Blood ejected from the left ventricle perfuses the brain via central elastic arteries, which stiffen with advancing age and may elevate the risk of end‐organ damage. The current study aimed to determine the impact of central arterial aging on cerebral hemodynamics. Methods: Ninety participants aged 22‐80 years were enrolled. Total and pulsatile cerebral blood flows (CBF) were measured by phase‐contrast MR imaging and transcranial Doppler respectively. Central arterial stiffness, wave reflection, and pressure were recorded using applanation tonometry. White matter hyperintensity, a marker of cerebral small vessel disease, was measured by fluid‐attenuated inversion recovery MR imaging. Results: Total CBF decreased with age while systolic and pulsatile CBF increased and diastolic CBF decreased. In the frequency domain, CBF pulsatility at the fundamental harmonic exhibited a steep curvilinear increase with age. Age‐related differences in CBF pulsatility were independently associated with carotid pressure pulsatility. In middle‐aged and older subjects, the prevalence of white matter hyperintensity increased with age and was independently associated with CBF pulsatility. Conclusions: Central arterial aging plays an important role to determine age‐related differences in cerebral hemodynamics. Cerebral small vessel disease is more closely associated with pulsatile CBF than total CBF.

Grant Funding Source: National Institute on Aging
Age‐ and estrogen‐dependent effects on cerebrovascular reactivity: shifting from beneficial to detrimental (1069.6)
Authors: Deer Rachel , Perkins Lisa , Stallone John
Published: June 2021

Although mechanisms underlying the beneficial effects of estrogen on cerebrovascular (CV) function are well known, the age‐dependent deleterious effects are largely unknown. We hypothesized that age enhances the deleterious effects of estrogen on CV function by altering the role of prostanoids in modulating CV reactivity. Female (F) rats approximating key stages of hormonal aging were studied: pre‐menopausal (mature multigravid, MA), and post‐menopausal (reproductively senescent, RS). Rats underwent bilateral ovariectomy (O); some received estrogen replacement therapy (OE). Reactivity to VP (10‐12‐10‐7M) was measured in pressurized middle cerebral arteries (%vasoconstriction) using COX‐1 (SC560, SC, 1μM) or COX‐2 (NS398, NS, 10μM) selective inhibitors. Maximal VP reactivity in MAO (64±0.7%) was attenuated in MAOE by 21%; in contrast, reactivity in RSO (59±1.1%) was enhanced in RSOE by 27%. In MAO and RSO, SC and NS reduced reactivity similarly. In MAOE, SC reduced reactivity to a greater extent than NS (Δ59% vs Δ34%); however, in RSOE this effect was reversed (Δ65% NS vs Δ36% SC). VP‐stimulated release of PGI2 and TXA2 (radioimmunoassay of 6‐keto‐PGF1α and TxB2, stable metabolites, pg/mg dry wt/45min) in MAO (22,805 ± 3,108) and RSO (18,499±1,563) was increased by estrogen in MAOE (+58%) and RSOE (+56%). In contrast, PGI2 in MAO (22,805±3,108) and MAOE (35,975±2,133) was reduced by age in both RSO (‐19%) and RSOE (‐20%). VP‐stimulated TXA2 in MAO (671±57) and RSO (527±71) was increased by estrogen in MAOE (+62%) and RSOE (+165%). TXA2 in MAOE (1,085±70) was increased by age in RSOE (+21%) with no difference in MAO and RSO. These data reveal that the vascular effects of estrogen are distinctly age‐dependent. In younger F, beneficial effects of estrogen are evident (decreased vasoconstriction, increased dilator function). Conversely, in older F, detrimental effects of estrogen dominate (enhanced vasoconstriction, increased constrictor prostanoid function).

Grant Funding Source: NIH: HL‐080402
Vigorous treadmill exercise improves reactivity of cerebral arterioles and reduces brain injury following transient focal ischemia (1070.2)
Authors: Arrick Denise , Yang Shu , Li Chun , Sun Hong , Mayhan William
Published: June 2021

Exercise training (ExT) has been shown to play a significant role in the prevention of cardiovascular‐related diseases. Our goal was to examine whether vigorous exercise training (ExT) could influence nitric oxide synthase (NOS)‐dependent dilation of cerebral arterioles and transient focal ischemia‐induced brain injury in rats. Sprague‐Dawley rats were divided into sedentary (SED) or exercised trained (ExT) groups. Treadmill exercise was carried out 5 days/week for a period of 6‐8 weeks. In the first series of studies, a craniotomy was made over the parietal cortex and we measured responses of pial arterioles to eNOS‐dependent (ADP), nNOS‐dependent (NMDA) and NOS‐independent (nitroglycerin) agonists. In a second series of studies, we measured infarct volume in SED and ExT rats following right middle cerebral artery occlusion (MCAO) for 2 hours followed by 24 hours of reperfusion. In a third series of studies, we measured eNOS, nNOS, SOD1 and SOD2 protein levels in cerebral vessels and brain tissue of SED and ExT rats. We found that eNOS‐ and nNOS‐dependent, but not NOS‐independent vasodilation, was increased in ExT compared to SED rats. In addition, we found that ExT significantly reduced total, cortical and subcortical infarct volumes following ischemia/reperfusion when compared to sedentary rats. Surprisingly, we found that eNOS and nNOS protein levels were similar in ExT and SED rats, but SOD1 and SOD2 protein levels were increased by ExT. We suggest that ExT improves NOS‐dependent vascular function and reduces infarct volume by mechanisms that appear to be related to alterations in oxidative stress. We suggest that ExT may be a viable preventative therapeutic approach to lessen ischemia‐induced brain injury.

Grant Funding Source: NIH HL090657
Effect of cold pressor stress on glycemic response of healthy college aged subjects (1072.10)
Authors: Curtis Taylor , Krause Samantha , Schmit Katelyn , Ragsdale Frances , Wilson Ted
Published: June 2021

Stress is associated with type 2 diabetes and acute stress could alter glycemic response. Physiological effect of cold pressor stress (CPS; n=40) and control (no CPS; n=43) on glycemic and cardiovascular response to a 50 gram oral glucose tolerance test drink (OGTT) was examined in healthy humans (18±10 years). CPS consisted of three 30‐second left arm ice bath immersions 10 min pre‐OGTT (0 min‐OGTT ingestion), then 10 and 20 minutes post‐OGTT. Blood glucose for control and CPS at ‐15, 0, 30, 60, 120 min was 89.7±9.0, 91.0±8.7, 154.2±21.5, 138.8±32.7, 96.4±19.1 mg/dl and 90.2±7.7, 89.0±6.4, 156.2±21.4, 134.7±24.2, 98.3±21.4 mg/dl respectively (P=0.78). Systolic, diastolic, and mean arterial pressure changes were not statistically significant between treatments. Heart rate diminished across time in both treatments (P= 0.02) although no significant difference was observed between groups. CPS was not associated with significant changes in glycemic and cardiovascular function. However, longer ice bath immersions may promote a greater level of stress that could be associated with altered glycemic response and cardiovascular function.
Dysregulation of intracellular Ca2+ in lymphocytes and neutrophils in type 2 diabetic patients (1072.11)
Authors: Singh Jaipaul , Kappala Shanti , Espino Javier , Pariente Jose , Rodriguez Ana , Bidasee Keshore
Published: June 2021

Background: Patients with T2D are immune‐compromised, making them more susceptible to infections and long term complications than non‐diabetic individuals. Neutrophils and lymphocytes play key roles in host‐immune responses to infections, and their phagocytic and chemotatic activities are reduced in diabetes. To date, the underlying causes for impairment in neutrophils and lymphocytes functions in T2D remain poorly defined. Dysregulation of intracellular Ca2+ cycling is a common phenotype in many diabetic cells. This study investigated if intracellular Ca2+ cycling is also defective in neutrophils and lymphocytes isolated from individuals with T2D.

Methods and results: Neutrophils and lymphocytes were obtained from patients with T2D and healthy controls with consent, and their intracellular Ca2+ cycling kinetics when challenged with thapsigargin (TG), formyl‐methionyl‐leucyl‐phenylalanine (fMLP) and hydrogen peroxide (H2O2), were compared using time‐lapsed Ca2+ imaging assays. All cells from T2D and control patients responded to TG, fMLP and H2O2. However, the rate of cytoplasmic Ca2+ rise, peak Ca2+ transient amplitude and Ca2+ transient decay times were significantly compromised in neutrophils and lymphocytes from T2D patients compared to healthy controls.

Conclusion: From these data we concluded that neutrophils and lymphocytes from T2DM patients are less responsive to this wide array of intracellular Ca2+ mobilizing agonists compared to control. Moreover, the decreased [Ca2+]i in immune cells might be one of the possible mechanisms of impaired immunity in diabetic patients.
Hemodynamic responses to recumbent positions in healthy adults (1073.1)
Authors: Sasaki Konosuke , Sato Haruka , Bao Sarina , Kanno Emi , Maruyama Ryoko
Published: June 2021

The effect of lateral decubitus positions on the autonomic nervous system and circulatory regulation has not been well clarified in normal healthy adults. Our aim was to evaluate whether the left and right lateral decubitus positions affect heart rate variability (HRV), including hemodynamics, compared to the supine position. We non‐invasively measured blood pressure (BP), heart rate (HR), stroke index (SI), cardiac index (CI) and total peripheral resistance index (TPRI) in each posture in 10 nonsmoking healthy volunteers. After lying in the supine position, the subjects maintained each lateral decubitus position in random order for 10 min at an angle of about 45 degrees. The postural change was performed manually by two trained researchers. No significant differences were observed in mean BP, low frequency HRV, high frequency HRV, and their ratio. However, in the left lateral decubitus position, HR was significantly lower than in the supine position. In both lateral decubitus positions, SI and CI tended to decrease compared to the supine position, and an upward trend was noted for TPRI. The HRV results suggest that the both recumbent positions have no influence on the autonomic nervous system. The reduction in HR and SI in the left recumbent position may not be related to autonomic nervous activity.
Sex differences in heart rate variability and circulation after postural change (1073.3)
Authors: Sato Haruka , Sasaki Konosuke , Bao Sarina , Kanno Emi , Maruyama Ryoko
Published: June 2021

It is well known that sex differences in body weight, blood pressure (BP) and heart rate (HR) exist. However, little research has been conducted on sex differences in circulatory effects, including autonomic nervous activity, between various recumbent positions. This study aimed to evaluate whether male and female young adults show differences in circulatory effects and autonomic nervous activity between 3 positions (supine, right lateral, and left lateral decubitus). Twenty‐nine male (22.5 ± 0.5 years) and 29 female (23.1 ± 0.7 years) healthy volunteers participated in this study. We measured HR variability (HRV) and BP in each of the 3 positions for 10 min. Compared to females, males had a significantly higher systolic BP and low frequency‐to‐high frequency component ratio (LF/HF) in all 3 positions. In females, HF was significantly higher than that in males in the right and left lateral decubitus positions. In both sexes, HR was significantly decreased in the left lateral decubitus position compared to the supine position. Interestingly, neither sex showed significant changes in HF and LF/HF during the change from the supine position to the recumbent positions. The results suggest that there are differences in baseline autonomic nervous activity between males and females. However, both sexes have same reaction to postural changes in circulatory dynamics.
The contribution of cardiac mechanics to isolated increases in peripheral blood flow (1073.4)
Authors: Stöhr Eric , van Mil Anke , Drane Aimee , Pearson James
Published: June 2021

The human heart responds to whole‐body increases in cardiovascular demand by enhancing heart rate (HR) and intrinsic cardiac muscle function. However, it is not known if enhanced left ventricular (LV) muscle function ('LV mechanics') also contributes to localized increases in peripheral blood flow and, if so, whether enhanced peripheral blood flow per se or other arterial hemodynamic signals are the mechanism for acutely adjusting LV mechanics. We performed cardiac and vascular ultrasonography in eight healthy individuals (4 women) during 1) Baseline, 2) Bilateral arm heating (BAH), and 3) BAH with partial brachial artery occlusion (BAH+OCC) at mean arterial pressure (MAP, 85±15 mmHg). BAH markedly increased skin temperatures (32.71±0.69 to 39.21±0.60 deg. C, p<0.0001) while rectal core temperature was unaltered (p=0.54). Brachial artery blood flow measured in the left arm increased almost threefold with BAH (98±46 to 266±96 mL/min, P<0.0001) and returned to baseline levels with BAH+OCC. Arterial wave reflection, as assessed by applanation tonometry, had a tendency to increase with BAH and remained elevated with BAH+OCC (p=0.07). Increased cardiac output (p=0.02) was entirely explained by increased HR of 3‐4 bpm (P=0.002). In contrast, LV mechanics ‐ evaluated by systolic longitudinal strain and diastolic untwisting velocity ‐ did not change with BAH but they declined significantly with BAH+OCC (both p<0.05), which was accompanied by a rise in MAP (P<0.0001).

In conclusion, the heart contributes to isolated increases in peripheral blood flow by enhancing HR but not its intrinsic LV muscle mechanics. Because LV output was maintained during the period of enhanced blood flow demand and increased afterload (=BAH+OCC), reduced systolic and diastolic LV mechanics during this condition may reflect an intrinsic protection of LV myofibers from pressure stress rather than a decline in LV function.
A potential role of memory T cells in hypertension (1074.1)
Authors: Itani Hana , Zhang Feng , Xiao Liang , Chen Wei , Harrison David
Published: June 2021

Immunological memory is an important component of adaptive immunity. We have previously shown that T cells are important in hypertension. We therefore hypothesized that memory T cells contribute to long‐term and repeated hypertensive challenges. To test this, mice received 2 weeks of angiotensin II, were allowed to recover for 3 weeks and then were received a low dose of ang II (140 ng/kg/min) that minimally raises blood pressure in naïve mice. This low‐dose of ang II increased blood pressure to 137±6 mmHg in mice that had previously received a vehicle infusion, but to 172±12 mmHg in mice that had received ang II. In keeping with a memory T cell response, we found that ang II increased CD8+ central memory cells in the kidney 5 to 10‐fold while reducing these effector memory CD8+ T cells in the spleen. CD8+ memory T cells require co‐stimulatory signaling between CD70 on macrophages and CD27 on T cells. We found that ang II infusion increased CD70 mRNA in isolated kidney vessels by 5 to 10‐fold. Similarly, flow cytometry revealed that ang II increased macrophages expressing CD70 and CD8+ T cells expressing CD27 expression in the kidney. Thus, these studies indicate that repeated exposures to ang II promote an immune memory response of CD8+ T cells and markedly enhance the hypertensive response. The role of CD27 and CD70 signaling requires additional study but might serve as a therapeutic target.
Isoketals in monocyte‐derived dendritic cells activate T cells and promote hypertension (1074.2)
Authors: Loperena Roxana , Kirabo Annet , Roberts L. , Davies Sean , Harrison David
Published: June 2021

Fatty acid oxidation leads to formation of highly reactive γ‐ketoaldehydes termed isoketals that adduct to protein lysines. We have shown that hypertension causes isoketals to accumulate in dendritic cells (DCs) and activate T cells. Others have shown that human monocytes traversing the endothelium differentiate into monocyte‐derived DCs (mo‐DCs) that express CD83, MHC‐II, CD11b, and CD11c. We hypothesized that isoketal formation promotes transformation of monocytes to mo‐DCs and that this is fundamental to the genesis of hypertension. Thus, we co‐cultured human monocytes with aortic endothelial cells exposed to a hypertensive (10%) stretch for 48 hours. Using flow cytometry, we detected markers for maturation of mo‐DCs expressing CD14low and CD83high with increased isoketals and CD86 activation marker. Exposure of murine DCs to the oxidant tert‐Butyl hydroperoxide (t‐BHP) also led to formation of mo‐DCs expressing CD11b and CD11c with a 3‐fold increase in isoketals. Co‐culture of t‐BHP treated DCs with T cells after 7 days promoted survival and proliferation of CD8+ T cells and a modest proliferation of CD4+ T cells. Moreover, adaptive transfer of t‐BHP treated DCs to mice elevated the hypertensive response to a generally subpressor dose of angiotensin‐II (128 ± 0.80 vs. 114 ± 0.48 in controls). We conclude that oxidant stress and isoketal formation in murine monocytes promote T cell activation and hypertension. Increased stretch of endothelial cells might transfer an oxidant signal to monocytes and promote their transformation to mo‐DCs to mediate this response. These findings provide a mechanism as to how T cells are activated in hypertension and provide insight into the inflammatory nature of this disease.

Grant Funding Source: Supported by NHLBI 5P01 HL0580000‐16
Interleukin 17A induces renal SGK1 expression in hypertension (1074.3)
Authors: Norlander Allison , Saleh Mohamed , Harrison David , Madhur Meena
Published: June 2021

We have previously shown that the pro‐inflammatory cytokine, interleukin 17A (IL17A), is upregulated by angiotensin II and plays a critical role in angiotensin II induced hypertension and vascular dysfunction. However, the mechanism by which IL17A promotes hypertension is not known. IL17A is produced by a subset of CD4+ T helper cells termed Th17 cells that also produce the related isoform, IL17F. We have preliminary data that only IL17A, not IL17F, promotes hypertension. Recently, it has been shown that serum and glucocorticoid kinase 1 (SGK1) in T cells induces the differentiation of naïve CD4+ T cells into IL17A‐producing Th17 cells in a salt‐dependent manner. We tested the hypothesis that IL17A could in turn upregulate renal SGK1 to induce salt and water retention in the kidney in a feed forward fashion. Using cultured human proximal tubule cells (HK‐2 cells), we found that IL17A, but not IL17F, upregulated SGK1 mRNA expression 4‐fold in a dose‐dependent manner. In vivo, angiotensin II infusion upregulated renal SGK1 expression in wild type C57 mice, and this effect was abolished in IL17A deficient mice. These studies are the first to describe a link between IL17A and the induction of renal SGK1 and thus provide a potential mechanism by which IL17A, but not IL17F, modulates salt and water retention and blood pressure.
Oligoclonal CD8+ cells in the kidney mediate experimental hypertension (1074.5)
Authors: Trott Daniel , Thabet Salim , Itani Hana , Wu Jing , Norlander Allison , Chen Wei , Harrison David
Published: June 2021

We have shown that T cells are involved in the genesis of hypertension, but the subset(s) involved have not been defined. T cell activation involves clonal expansion of cells with unique T cell receptors (TCRs). We analyzed TCR usage in mice infused with Ang II for two weeks. Ang II did not affect TCR profiles in the spleen, mesenteric vasculature or kidney CD4+ cells but markedly skewed the TCR profile in 3 V families of CD8+ cells in the kidney. This suggests an oligoclonal T cell response in hypertension. To further examine the role of CD8+ cells in hypertension we examined the hypertensive response to Ang II in CD4‐/‐ and CD8‐/‐ mice. After two weeks of Ang II, wild type and CD4‐/‐ mice systolic blood pressure rose to 172 ± 5.7 mmHg and 176 ± 6.3 mmHg, respectively. This response was blunted in CD8‐/‐ mice (146 ± 5.9 mmHg). To determine whether a full repertoire of CD8+ TCRs are required for hypertension we studied OT1 x RAG‐1‐/‐ mice, which have a TCR on CD8+ cells that is restricted to an ovalbumin peptide. Importantly, these mice cannot generate other TCRs. OT1 x RAG‐1‐/‐ mice demonstrated a blunted hypertensive response to Ang II (141 ± 7.8 mmHg) which was similar to that observed in CD8‐/‐ mice. We conclude that hypertension requires a full repertoire of CD8+ TCR, from which select populations of cells proliferate and accumulate in the kidney. These cells likely release mediators that promote salt and water retention and hypertension.
Targeted glyoxalase‐1 gene transfer to prevent cardiac dysfunction in diabetes (1078.8)
Authors: Bidasee Keshore , Alomar Fadhel , Rozanski George , Singh Jaipaul
Published: June 2021

Targeted glyoxalase‐1 gene transfer to prevent cardiac dysfunction in diabetes Keshore R. Bidasee1,2, Fadhel Alomar1, George Rozanski1,2, and Jaipaul Singh3, 1Pharmacol and Exp’t Neurosci, Univ. of Nebraska Medical, Omaha, NE 68198, 2Nebraska Redox Biology Center, Lincoln, Nebraska and 3Forensic and Investigative Sciences, UCLAN, UK.

Background: Heart failure is a common pathophysiology in individuals with diabetes. We attributed this defect to dysregulation of key excitation‐contraction (E‐C) coupling proteins arising from post‐translational modifications by the di‐carbonyl methylglyoxal (MG). MG increases mitochondrial ROS production which further compromises contractile function. Since MG regulates physiologic functions, globally lowering it will precipitate undesired side effects. In this project, we identified a sub‐pool of MG responsible for causing heart failure in diabetes.

Methods and results: In isolated ventricular myocytes, MG induced Ca2+ waves and impaired contractile kinetics. Daily i.p. injections (21 days) of MGO to control rats to attain serum levels similar to that in diabetes did not impair cardiac ejection fraction or fractional shortening. Immuno‐histochemical analyses of hearts from diabetic animals revealed showed increased expression of the MG‐synthesizing enzyme vascular adhesion protein 1 (VAP‐1) and the MG adduct, argpyrimidine in microvascular smooth muscle cells (SMC). Overexpression the MG‐degrading enzyme glyoxalase‐1 (Glo‐1) in SMC after the onset of diabetes (rodent models of type 1 and type 2 diabetes), blunted cardiac function loss, decreased expression of VAP‐1 and argpyrimidine in SMC, and normalized evoked Ca2+ transient and contraction kinetics in ventricular myocytes without lowering serum MG levels.

Conclusion: These data identified for the first time SMC of the microvasculature as a source of myocyte‐damaging MG in diabetes, and pharmacologically lowering this sub‐pool of MG blunts heart failure development.

Grant Funding Source: HL085061, GRTP#1053
Calcium alternans induced by enhanced dephosphorylation of phospholamban predisposes to inducibility of ventricular tachycardia in the type I diabetic Akita mouse (1078.9)
Authors: Jin Hongwei , Rajab Mohammad , Aronovitz Mark , Wang Bo , Park Ho‐Jin , Link Mark , Noujaim Sami , Galper Jonas
Published: June 2021

Introduction: Diabetes mellitus is associated with increased risk of sudden cardiac death. Little evidence exists to support specific mechanisms to account for this association. We studied the inducibility of ventricular tachycardia (VT) in the type I diabetic Akita mouse and ionic and molecular mechanisms of arrhythmogensis. Methods: Inducibility of VT was studied by programmed ventricular stimulation and optical mapping. Left ventricular myocytes were isolated for electrophysiology and calcium imaging studies. Sarcomere shortening and Ca2+ transients were measured using the Ionoptix system. Results: Programmed ventricular stimulation of Akita mice resulted in VT in 80% of mice compared to 20% of WT. Optical mapping of perfused hearts demonstrated multifocal breakthroughs which occasionally gave rise to short lived rotors consistent with focal initiation and maintenance of VT. Both contractile and Ca2+ transient alternans were observed in single ventricular myocytes of Akita mice at field stimulations above 4 Hz. 100nM Iso induced triggered activity in 90% of myocytes from Akita and 7% in WT mice. Ventricular myocytes from Akita mice demonstrated decreased SR Ca2+ reuptake (τ= 170±10 ms vs.138±7ms, n=20, p<0.01) compared to WT and increased cytosolic Ca2+[[Unsupported Character ‐ Codename ­]][[Unsupported Character ‐ Codename ­]]. Caffeine induced Ca2+ release was significantly decreased in Akita mice vs. WT. Western blot analysis showed that phosphorylated ser16 phospholamban (PLB) and the ratio of SERCA2a/total PLB were significantly decreased in Akita mice. Conclusions: Increased dephosphorylation of PLB caused abnormal SR Ca2+ loading resulting in cardiac alternans predisposing to the development of VT in type 1 diabetic Akita mice.

Grant Funding Source: NIH
TRPA1 mediates NADPH oxidase‐dependent cerebral artery dilation (1079.1)
Authors: Sullivan Michelle , Gonzales Albert , Bruhl Allison , Leo M. , Jaggar Jonathan , Welsh Donald , Earley Scott
Published: June 2021

Activation of the Ca2+‐permeable transient receptor potential (TRP) ankyrin (A) channel TRPA1 in the cerebral artery endothelium with electrophilic compounds induces dilation. Endogenous agonists for endothelial TRPA1 have not been reported. We hypothesized that H2O2 is an endogenous agonist for TRPA1 in this tissue. H2O2 can be generated by NADPH oxidase (NOX) activity via production of a O2‐ intermediate or directly by NOX isoform NOX4. We find that activation of NOX with its substrate, NADPH, increases TRPA1 activity and induces dilation of pressurized cerebral arteries (80 mmHg) that is blocked by TRPA1 inhibitor HC‐030031 (HC) and H2O2 degradation by catalase. In the presence of iron, H2O2 is converted to highly reactive oxygen radicals, such as OH•, by the Fenton reaction. To distinguish between direct effects of H2O2 vs. those of OH• or OH• metabolites we inhibited the Fenton reaction by chelating iron with deferoxamine. Deferoxamine attenuated NADPH‐induced TRPA1 activation and vasodilation, suggesting that OH• formation is required for NOX‐dependent TRPA1 activation. OH• peroxidizes membrane lipids yielding lipid peroxidation products (LPP). The LPP 4‐hydroxynonenal increased TRPA1 activity and induced cerebral artery dilation that was blocked by HC. Together, these data suggest that NOX‐dependent LPP generation activates TRPA1 in cerebral artery endothelial cells to induce vasodilation.

Grant Funding Source: Supported by HL091905
Functional hyperemia requires ATP‐sensitive potassium channels and hydrogen peroxide and not adenosine in isolated mice hearts (1079.17)
Authors: Zhou Xueping , Teng Bunyen , Mustafa S. Jamal
Published: June 2021

We previously demonstrated that adenosine contributes to coronary ischemic vasodilation through A2A AR‐mediated H2O2 production and opening of ATP‐sensitive potassium channels (KATP). Whether the same mechanism applies to functional hyperemia remains unknown. This study aims to investigate the role of adenosine, KATP, and H2O2 in pacing‐induced coronary hyperemia using pharmacological approaches combined with adenosine receptor knockout (KO) mice. Coronary flow was measured in isolated Langendorff perfused hearts paced at a graded rate from 400 to 650 bpm. Coronary flow increased linearly from 17.1 ± 0.7 to 24.1 ± 0.8 ml/min/g with increased HR in WT mice (n=7). Neither non‐selective blockade of adenosine receptor by 8‐(p‐Sulfophenyl) theophylline (8‐SPT, 50 μM, n=6), nor selective A2A AR blockade by SCH58261 (1 μM, n=4) or A2A AR KO (n=9) affected the pacing‐induced coronary hyperemia, though the relationship between coronary flow and rate‐pressure product was shifted downward in a parallel manner. Combined A2A KO with A2B selective blockade (CVT6883, 1 μM, n=3) or vice versa (n=3) showed similar effect as 8‐SPT. Inhibition of nitric oxide synthesis by L‐NAME (100 μM) or combined administration with 8‐SPT did not affect the pacing‐induced coronary hyperemia, but attenuated the resting flow to a similar extent (by ~20%, n=5 per group). However, glibenclamide (KATP channel blocker, 10 μM) significantly decreased both pacing‐induced hyperemia (by ~80%) and baseline flow (by ~45%, n=4). Scavenging of H2O2 by catalase (2500 U/min) also decreased baseline flow (by ~16%) and pacing‐induced coronary hyperemia (by ~24%, n=4), but to a lesser extent than glibenclamide. These findings suggest that adenosine modulates coronary vascular tone under both resting and ischemic conditions but not functional hyperemia. However, H2O2 and KATP channels are important local mechanisms responsible for both coronary ischemic and metabolic vasodilation.

Grant Funding Source: Supported by HL027339 and HL 094447