bluuebunny/crossref_metadata_2025_split huggingface dataset:
Antibody Targeted Against P2Y12 Receptor Reduces Platelet Activity In vitro and In vivo
Authors: Hensch Nicole R , Karim Zubair A , Khasawneh Fadi T 
Published: November 2023

Platelet hyperactivity is known to give rise to thrombotic disorders, and many antiplatelet therapies are designed to reduce the risk of thrombogenesis. ADP is an important platelet activator and activates platelets by binding to the P2Y1 receptor or P2Y12 receptor, both of which are G‐coupled protein receptors located on the platelets’ surface. To this end, although the involvement of the P2Y12 platelet receptor in thrombosis is well characterized, there are currently no parenteral drugs targeting these receptors in clinical use to complement existing antiplatelet therapies. In this study, we designed a parenteral agent, namely an antibody targeted against the P2Y12 receptor ligand biding domain (abbreviated as P2Y12Ab). In vitro studies revealed that the addition of P2Y12Ab could effectively inhibit aggregation induced by ADP. P2Y12Ab was also found to inhibit aggregation activated by U46619 and thrombin. Using FACS analysis, reduced P selectin and Annexin V exposure and reduced integrin activation was also evident in the presence of P2Y12Ab. As for its in vivo activity, the P2Y12Ab also demonstrated inhibitory effects in a FeCl3‐induced thrombosis model, producing a prolonged occlusion time; however, a lengthened bleeding time was also observed. Overall, our findings suggest that P2Y12Ab functionally blocks platelet activity, further indicating that the P2Y12 platelet receptor plays a central role in thrombogenesis. These studies also validate the use of P2Y12Ab as a purposed parenteral antiplatelet agent to be implemented in conjunction with or as an alternative to current antiplatelet strategies.Support or Funding InformationThis research was supported by funds provided by College of Pharmacy, Western University of Health Sciences (to F.T.K).
Hyperpolarization of Mouse Pulmonary Artery Smooth Muscle and Endothelium by Calcitonin Gene Related Peptide
Authors: Norton Charles , Segal Steven 
Published: November 2023

The goal of these experiments was to develop mouse pulmonary arteries (PAs) as a viable model for investigating the electrophysiological actions of the sensory neurotransmitter calcitonin gene related peptide (CGRP) on membrane potential (Vm) and electrical signal transmission in PA smooth muscle cells (SMCs) and endothelial cells (ECs). Intact PAs were isolated from anesthetized male C57BL/6J mice (3 months old). Immunostaining confirmed that PAs were sparsely innervated by sensory nerves containing CGRP. For intracellular recording, intact PAs (diameter, ~150 μm) were cannulated, pressurized to 16 cm H2O and studied at 37°C. Alternatively, SMCs were gently dissociated to obtain intact endothelial tubes studied at 32°C. We tested the hypothesis that CGRP would hyperpolarize SMCs and ECs in a concentration‐dependent manner. During continuous recording of Vm, increasing [CGRP] from 1 nM to 1 μM hyperpolarized SMCs of intact PAs from −34±2 mV (rest) to −52±3 mV and EC of tubes from −37±4 mV (rest) to −58±4 mV (n ≥ 4 per group); respective EC50 values were 6.2 and 6.8 nM. With dual simultaneous intracellular recording in endothelial tubes, current injection (±0.5 nA) into one EC evoked corresponding Vm responses (±5 mV) in a remote EC (separation distance, 500 μm), confirming electrical signal transmission along PA endothelium. These preliminary studies confirm that CGRP effectively hyperpolarizes SMCs and ECs in mouse PA while validating the utility of this model for investigating the nature of electrical signaling in response to sensory neurotransmission along the PA wall.Support or Funding InformationNIH R37‐HL041026
Aging Impairs Spontaneous Ca2+ Signaling and Regulation by CGRP in Smooth Muscle Cells of Mouse Mesenteric Arteries
Authors: Boerman Erika M , Segal Steven S 
Published: November 2023

Advancing age is associated with impaired vasomotor function in resistance arteries. Thus, diminished potency of calcitonin gene‐related peptide (CGRP) coupled with loss of sensory nerve inhibition of sympathetic vasoconstriction contribute to impaired vasodilation in mouse mesenteric arteries (MAs; PMC3607869). In light of multiple roles for Ca2+ signaling in governing vasomotor function, we tested the hypothesis that aging impairs both spontaneous and CGRP‐mediated Ca2+ signals in smooth muscle cells (SMCs). MAs (n = 14–18) from Young (~4 months) and Old (~24 months) C57BL/6 mice were isolated, pressurized to 100 cm H2O and studied at 37°C following loading of SMCs with Fluo‐4 (10 μM, 1h, 37 °C ). With ~15 SMCs per field, under control conditions Young MAs had 9 ± 1 sites producing spontaneous local Ca2+ events versus 4 ± 1 sites in Old MAs (P<0.05) while the number of SMCs exhibiting spontaneous Ca2+ waves (7 ± 1) was not different between age groups. Age‐defined EC50 concentrations of norepinephrine (NE; 167 and 600 nM) reduced local Ca2+ event sites in Young and Old MAs (to 3 ± 1 and 1 ± 1, respectively; P<0.05) and increased the number of SMCs with Ca2+ waves in both age groups (to 11 ± 1 and 14 ± 1). In Young but not Old MAs, addition of CGRP (100 nM) restored local Ca2+ event sites and Ca2+ waves to control values. Across age groups the frequency (0.6 ± 0.01 Hz), amplitude (1.3 ± 0.01 F/Fo), rise time (73 ± 8 ms), and duration (FDHM, 164 ± 9 ms) of local Ca2+ events did not differ with age, NE or CGRP treatments. We suggest that the impaired ability of CGRP to restore local Ca2+ signaling (e.g., as associated with BK channel activation) in SMCs of Old MAs during adrenoreceptor activation contributes to age‐related vascular dysfunction underlying impaired vasodilation.Support or Funding InformationNIH F32HL118836 & R37HL041026
Endothelial Conduction of Rapid Onset Vasodilation (ROV) in Mouse Skeletal Muscle
Authors: Sinkler Shenghua Y , Segal Steven S 
Published: November 2023

In response to skeletal muscle contractions, ascending vasodilation (AVD) of feed arteries (FA) increases muscle blood flow and reflects signals initiated downstream from arterioles embedded within active muscle fibers. Muscular activities range from moderate and rhythmic to intense and brief contractions. We tested the hypothesis that the mechanism of AVD differs with the nature of skeletal muscle contraction. Using intravital microscopy, we studied the resistance microvasculature in the gluteus maximus muscle (GM) of anesthetized male C57BL/6J mice (age: 3–4 months; n=6). Under control conditions, a single maximal tetanic contraction (100Hz, 500ms) evoked ROV in primary arterioles (1A) and FA, increasing internal diameter from 30±2μm to 47±2μm in 1A and from 42±2μm to 54±3μm in FA (onset < 1s, peak within 4s). In contrast, during moderate rhythmic contractions at 4Hz, 1A and FA dilations required 10–15s to begin and attained steady‐state diameters of 45±2μm (1A) and 53±4μm (FA) by ~30s. Local stimulation of 1A with acetylcholine (ACh) microiontophoresis (2μm tip, 500ms) increased diameter locally (to 47±3μm) and evoked FA dilation upstream (to 56±3μm). Light‐Dye Treatment (LDT) to disrupt the endothelium midway between 1A and FA eliminated ROV in FA. LDT also abolished FA dilation to ACh microiontophoresis onto 1A downstream while 1A dilations to rhythmic contractions and to ACh remained intact. Remarkably, FA continued to dilate in response to rhythmic contractions following LDT until nitric oxide (NO) synthesis was inhibited (L‐NAME; 10−4M). We conclude that AVD of FA in response to brief intense skeletal muscle contraction (i.e., ascending ROV) reflects intercellular conduction along the endothelium upon initiation in downstream arterioles. In contrast, AVD during rhythmic contractions is mediated by NO release during sustained functional hyperemia.Support or Funding InformationAHA 15PRE22840000; NIH R37‐HL041026
PGC‐1α Activation Confers Plasticity in the Mediator of Dilation in Healthy and Diseased Human Arterioles
Authors: Kadlec Andrew , Hockenberry Joseph C. , Beyer Andreas M. , Gutterman David D. 
Published: November 2023

BackgroundCoronary artery disease (CAD) and increased intraluminal pressure (IILP) shift the mediator of flow‐induced dilation (FID) from nitric oxide (NO) to hydrogen peroxide (H2O2) in the human microcirculation. PGC‐1α, a master regulator of mitochondrial biogenesis, regulates reactive oxygen species (ROS) production and blood pressure. PGC‐1α is decreased in vessels from CAD patients. It is not known whether this important regulator of cell function is critically involved in endothelial‐mediated changes in vascular tone.ObjectiveTo determine whether a stimulated increase in PGC‐1α levels can restore NO as the mediator of dilation in healthy vessels exposed to IILP, and in CAD vessels.MethodsHuman adipose arterioles were incubated overnight with ZLN005, a novel transcriptional activator of PGC‐1α, and subsequently cannulated on glass micropipettes. Healthy (non‐CAD) vessels were exposed to physiological pressure conditions (60mmHg) or IILP (150mmHg for 30 min). Diseased (CAD) vessels were only exposed to 60mmHg pressure. Dilation was elicited by flow in the absence and presence of a nitric oxide synthase inhibitor, L‐NAME (10−4 M), a H2O2 inhibitor, PEG‐catalase (500 units/mL), or both to determine the mediator of dilation. Fluorescence microscopy using intraluminal MitoPY1 probed for H2O2 production in response to IILP.ResultsIn healthy vessels treated with ZLN, a trend toward a reduction in FID from control was observed after addition of either L‐NAME or PEG‐catalase under physiological pressure conditions (n=6–8; NS). PEG‐catalase alone reduced FID after IILP (n=7; P < 0.05), with a similar trend in unstressed vessels from patients with CAD (n=9; NS). Complete inhibition of FID was observed in PGC‐1α‐activated vessels after addition of both L‐NAME and PEG‐catalase and in vessels from subjects with CAD (n=8; P < 0.05) or without CAD under physiological pressure conditions (n=6; P < 0.05). Fluorescence data confirmed that H2O2 production persisted after IILP in healthy treated vessels.Conclusion and SignificanceIn contrast to the exclusively NO‐or H2O2‐mediated dilation seen in healthy or diseased conditions, respectively, activation of PGC‐1α with ZLN005 allows both NO and H2O2 to simultaneously contribute to dilation under basal pressure conditions and in the presence of CAD, but not after acute high pressure stress. These data provide support for the notion that dilation may be potentiated by more than one factor in the human microcirculation and that PGC‐1α may regulate plasticity in the mechanism of dilation in health and disease.Support or Funding InformationFunding support provided by R01‐HL‐113612 (to D. D. Gutterman) and T32‐GM‐08020206 [Medical Scientist Training Program Grant] (to J. T. Barbieri)
Effect of pulmonary flow heterogeneity and impaired diffusing capacity on oxygen uptake
Authors: Roy Tuhin K , Secomb Timothy W 
Published: November 2023

Severe impairment of oxygenation in critically ill patients is a major cause of morbidity and mortality, and can occur even with normal alveolar ventilation and cardiac output under conditions of decreased diffusing capacity or increased flow heterogeneity. A theoretical model of pulmonary oxygen uptake under resting conditions is used to simulate decreased diffusing capacity (DL) as well as increased heterogeneity (characterized by an increase in the coefficient of variation [CV] of pulmonary capillary blood flow), with the assumed normal value of CV based on observations of microvascular networks. Under resting conditions with nominal values of DL and CV, the model predicts an oxygen saturation (SO2) of 97% (PO2 = 94 mmHg). Simulating a 10‐fold reduction in DL leads to an SO2 of 85% (PO2 = 51 mmHg), whereas doubling the CV from its normal value leads to an SO2 of 95% (PO2 = 78 mmHg); both of these predictions suggest a high degree of reserve in the healthy resting lung. However, simulating these conditions simultaneously leads to a large reduction in SO2 to 71% (PO2 = 37 mmHg), exhausting the reserve capacity of the lung and demonstrating the detrimental synergistic effect of heterogeneity in the context of an attenuated diffusing capacity. These results imply that a combination of increased heterogeneity and decreased diffusing capacity can explain the severe impairment in pulmonary oxygen uptake and decreased saturations seen in critical illness.Support or Funding InformationSupported by NIH grant HL070657.
Geometric and Topological Analysis of Arteriolar Networks in the Rat Gluteus Maximus Muscle: One Network to Rule Them All?
Authors: Al Tarhuni Mohammed , Goldman Daniel , Jackson Dwayne N 
Published: November 2023

IntroductionA detailed evaluation of in vivo geometry and topology of skeletal muscle arteriolar networks is essential to understanding microvascular blood flow regulation. Beyond increasing understanding of skeletal muscle hemodynamics, such data provides critical experimental inputs for theoretical blood flow simulation studies. Generally, past studies have relied on data collected from vascular casting experiments or data collected from incomplete microvascular trees or segments. The objective of this study was to comprehensively analyze the geometry and topology of complete arteriolar networks within the rat gluteus maximus (GM) muscle. The data generated were used to generate an index of geometric/topological homology across GM networks and produce essential experimental data to use as inputs for future theoretical studies of hemodynamics.MethodsThe rat GM provides the ideal experimental model to study locomotive skeletal muscle. Its planar geometry and uniform thinness enable access (within a single focal plane) to its entire microcirculation for microscopic imaging and perturbation. Using intravital videomicroscopy, the GM (n=4) was scanned under baseline conditions and photomontages were compiled (~400 images per network). Photomontages were registered to a MATLAB x–y coordinate system and scaled digital networks were generated. Arteriolar diameters and lengths were measured along the arteriolar network and a centrifugal ordering algorithm was applied, resulting in arterioles ranging from 1st (feed) to 9th (terminal) order. Mean diameters, lengths, and number of segments were outputted and grouped with respect to arteriolar order.ResultsThe coefficients of variation at each order were consistently greater for lengths than diameters. The relationships of arteriolar diameter and segment length as a function of vessel order were fitted with an exponential decay function and resulted in relatively stronger goodness of fit values for diameters (R2 = 0.62 to 0.75) than lengths (R2 = 0.32 to 0.53), suggesting greater predictability and homology between arteriolar diameter and vessel order. Topological analyses were extended to plotting the logarithm of diameters and lengths as a function of vessel order in an effort to validate Horton's laws in the GM and derive diameter, length, and bifurcation ratios. Finally, a “mean” arteriolar network that describes all relevant geometric and topological parameters was explored.ConclusionThis study provides the first comprehensive analysis of rat GM topology and geometry. The data presented herein will serve as ideal experimental inputs for future computational studies of skeletal muscle hemodynamics and microvascular blood flow regulation.Support or Funding InformationNSERC
Global Cerebrovascular Dysfunction in the Metabolic Syndrome Following Ischemic Stroke
Authors: Branyan Kayla , Brooks Steve , DeVallance Evan , Asano Shinichi , Frisbee Jefferson , Chantler Paul 
Published: November 2023

Insulin resistance is suggested to impact endothelial function by attenuating nitric oxide (NO) synthase activity, which is important for blood flow control. This reduction in insulin sensitivity and decrease in endothelial function leads to an increased risk for poor cardiovascular outcomes such as stroke. Many studies have examined the ipsilateral middle cerebral artery (MCA) to determine the effect of stroke on the cerebral microvasculature. Neither the contralateral MCA, nor the posterior cerebral arteries (PCA) have been thoroughly scrutinized to determine the more global impact of stroke on cerebrovascular function. Obese Zucker rats (OZRs) are used as a representative model of insulin resistance, where their lean counterparts (LZRs) are considered healthy controls. To look at the possible effect of decreased NO bioavailability on the ipsilateral and contralateral hemispheres of the brain, OZRs and LZRs were induced with stroke by transient middle cerebral artery occlusion (tMCAO) for 60 minutes. The purpose of this study was to compare the reactivity of both the ipsilateral and contralateral MCAs and PCAs at 24 hours post‐stroke to determine if stroke impacted the cerebral hemispheres differently. OZRs and LZRs were separated into sham and tMCAO groups. The ipsilateral and contralateral MCA and PCA were isolated and hung in a pressurized microvessel myobath. To assess endothelial function, the MCAs and PCAs were exposed to increasing doses of acetylcholine. A DAF‐FM diacetate assay was used to detect the amount of NO production in the aorta as a representative measure of NO synthesis within the vasculature. After 24 hours, LZR tMCAOs had lower NO production than the sham animals (p<0.05) but OZR tMCAOs did not have a significant decrease in NO release. LZR and OZR tMCAOs had similar NO production values after 24 hours. Both groups had decreases in their MCA and PCA reactivity 24 hours post‐stroke (p<0.05). In the MCAs, ipsilateral reactivity was lower in OZRs than in LZRs. This effect was not seen in the PCA, where both the contralateral and ipsilateral had similar decreases in reactivity in LZRs. OZR ipsilateral PCA reactivity was further attenuated from the contralateral (p<0.05). OZR MCA reactivity was improved by incubation with TEMPOL but not LZRs. This same effect was not seen in PCAs where TEMPOL did not improve dilation in either group. The results from this study suggest that NO bioavailability is important for endothelial function after an ischemic stroke. It also suggests that both hemispheres of the brain are affected by stroke, although not to the same degree. Likewise, vessels such as the PCA are also impaired by occlusion of the MCA suggesting a more global effect on the cerebral microvasculature.Support or Funding InformationAHANIH
Activation of RhoA, but not Rac1, mediates early stages of S1P‐induced endothelial barrier enhancement
Authors: Zhang Xun , Adderley Shaquria , Breslin Jerome 
Published: November 2023

Compromised endothelial barrier function is a hallmark of inflammation. Rho family GTPases are critical in regulating endothelial barrier function, yet their precise roles, particularly in sphingosine‐1‐phosphate (S1P)‐induced endothelial barrier enhancement, remain elusive. We tested the roles of Rac1 and RhoA in this study. Confluent cultures of human umbilical vein endothelial cells (HUVEC) or human dermal microvascular endothelial cells (HDMEC) were used to model the endothelial barrier. Barrier function was assessed by determining the transendothelial electrical resistance (TER) using an electrical cell‐substrate impedance sensor. The results show that pharmacologic inhibition of Rac1 with 50 μM Rac1 Inhibitor II failed to block barrier enhancement elicited by 2 μM S1P. Likewise, expression of a dominant negative form of Rac1, or knockdown of native Rac1 with siRNA, failed to block S1P‐induced elevations in TER. In contrast, blockade of RhoA with the combination of the inhibitors Rhosin and Y16 (6.25 μM respectively) significantly reduced S1P‐induced increases in TER. Assessment of RhoA activation in real time using a fluorescence resonance energy transfer (FRET) biosensor showed that S1P increased RhoA activation primarily at the edges of cells, near junctions. This was complemented by myosin light chain‐2 phosphorylation at cell edges, which could be blocked with Y16 and Rhosin treatment. The results suggest that S1P causes activation of RhoA at the cell periphery, stimulating local phosphorylation of MLC, and resulting in endothelial barrier enhancement. S1P‐induced Rac1 activation, however, does not appear to have a significant role in this process.Support or Funding InformationSupported by NIH Grant R01HL098215
Nrf2 Deficiency Attenuates Synaptic Plasticity in Mice, Mimicking the Aging Phenotype
Authors: Valcarcel‐Ares Marta Noa , Tucsek Zsuzsanna , Tarantini Stefano , Hertelendy Peter , Gautam Tripti , Sonntag William E , Deak Ferenc , Ungvari Zoltan , Csiszar Anna 
Published: November 2023

There is increasing evidence that Nrf2/ARE signaling regulates lifespan and/or health span both in model organisms and laboratory rodents. Aging is associated with progressive Nrf2 dysfunction, impairing cellular resilience and promoting oxidative stress and inflammation. Age‐dependent impairment in the induction of hippocampal long‐term potentiation (LTP) is well documented, providing a likely neural basis for age‐related decline in learning and memory. While previous studies showed that Nrf2 dysfunction promotes neuronal death and administration of Nrf2 activators improve learning and memory in aged rodents, the effects of age‐related Nrf2 dysfunction on synaptic function are not well understood. To determine the effect of Nrf2 deficiency on cognition and synaptic plasticity, we compared performance of Nrf2−/− and control mice on various behavioral tests including the elevated plus maze, the radial arm water maze (RAWM) and novel object recognition test. To determine whether Nrf2 deficiency compromises neuronal mechanisms of learning and memory, extracellular recordings were performed from acute hippocampal slices using multi‐electrode arrays. Field excitatory post‐synaptic potentials were invoked through stimulation of the Schaffer collateral and obtained from the CA1 area. We found that Nrf2−/− mice have a reduced LTP elicited by high frequency stimulation in hippocampal slices. Reduced LTP in Nrf2−/− mice was associated with a compromised performance in the elevated plus maze learning test and the novel object recognition test. Together, these results provide additional evidence that intact Nrf2 signaling importantly contributes to brain health, preserving hippocampal synaptic plasticity and cognitive function. Further studies are needed to elucidate the neuronal mechanisms impacted by Nrf2 deficiency, determining the potential roles of oxidative stress and inflammatory processes.Support or Funding InformationThis work was supported by grants from the American Heart Association and the National Institute on Aging