bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of Web‐logs (Blogs) to Promote Student Ownership
Authors: Oliver Kendra H , Picard Danielle , Wikswo John , Brame Cynthia
Published: November 2023

Blended or hybrid learning is a teaching approach in which some course elements—e.g., content delivery or discussions—occur online while other course elements occur face‐to‐face. Current best practices suggest that the digital environment can be used to guide instruction and to enhance student‐student exchange of ideas. We explore the benefits of online student interactions within an introductory science course as a means of enhancing face‐to‐face interaction and personalization of the learning experience. Weblogs (blogs) have the potential to increase contemplation, community, and collaboration within the classroom through an intimate online framework. In particular, we are interested in building student project ownership that is grounded in student interaction and builds natural learning development through “doing.” However, the effectiveness of blogs to promote ownership has not been fully examined.

We investigated the potential of blogs to promote project ownership. Ownership was tested after the addition of two elements to the blog design: instructor curation of the blog and awarding a grade percentage based on student participation. We explored how these modifications alter student educational experience and project ownership. This was measured using de‐identified student data to quantitatively examined the amount of online student‐student interactions. Additionally, we qualitatively analyzed blog content, the quality of students’ final writing samples, and student perception of project ownership via a survey at the end of the semester. Our overall study goal is to develop student fluency in scientific discourse and science project ownership by constructing an online, student‐driven learning blog community. These findings will elucidate the potential for blogs to build student project ownership, driving learner engagement, and academic success.

Support or Funding Information

Blended Online Learning Design Fellowship, Center for the Integration of Research, Teaching, and Learning, Center for Teaching
Evaluation of Smooth Muscle Cell Function in Cultured Microvascular Networks
Authors: Motherwell Jessica M. , Azimi Mohammad S. , Katakam Prasad V.G. , Murfee Walter L.
Published: November 2023

Ex vivo biomimetic models are valuable for investigating the underlying mechanisms of angiogenesis. Common models currently in use include 2D or 3D culture systems and microfluidic devices. However, these systems are limited by their lack of complexity compared to intact‐tissue scenarios. Based on an alternative top‐down approach, our laboratory has recently introduced the rat mesentery culture model as a tool to investigate cell‐cell interactions during angiogenesis in viable, intact microvascular networks. The objective of this study was to determine if vascular smooth muscle cells within the rat mesenteric microvascular networks maintain functionality during culture. Adult Wistar rat mesenteric tissues were harvested and cultured in Modified Eagle's Medium (MEM) + 10% serum. At 0 or 24 hours, tissues were exposed to vasoconstrictors (20nM Endothelin‐1 and 50mM KCl) for 5 minutes. Diameters of arterioles (22μm–27μm) were compared before and after treatment. Vessel constriction measured at the point of maximum diameter change along a vessel was observed at 0‐ and 24‐hours for both vasoconstrictor treatments. The percentage of constriction for arterioles in networks treated with endothelin‐1 at 0‐ and 24‐hour culture were 26 ± 6.2% (n=5 tissues) and 25 ± 3.9% (n=4 tissues) respectively (mean ± SEM). The percentage of constriction for arterioles treated with KCl at 0‐ and 24‐hour culture were 38 ± 4.6% (n=5 tissues) and 25 ± 4.4% (n=4 tissues) respectively (mean ± SEM). We also observed vasoconstriction of venules for both treatment groups at 0‐ and 24‐hour culture. After washing of 50mM KCl vessels were observed to return to baseline measurements supporting the vasoconstriction effect is readily reversible. These results suggest that the vascular smooth muscle cells maintain their functionality in cultured microvascular networks and supports the physiological relevance of the rat mesenteric culture model.

Support or Funding Information

NIH 5‐P20GM103629‐04
Effect of Betulinic acid on diabetic atherosclerosis in apolipoprotein‐E gene knockout mice
Authors: Yoon Jung Joo , Lee Yun Jung , Han Byung Hyuk , Choi Eun Sik , Namgung Seung , Tan Rui , Kho Min Chol , Park Ji Hun , Kang Dae Gill , Lee Ho Sub
Published: November 2023

Atherosclerosis, a chronic and progressive disease, is a leading cause of endothelial dysfunction, diabetes mellitus, hypertension, and hypercholesterolemia. Betulinic acid (BA), a pentacyclic triterpene, has been reported to have a variety of biological effects, such as anti‐inflammatory and immunomodulatory properties. Diabetic apolipoprotein‐E gene knockout (ApoE KO) mice were treated with BA for 12 weeks to examine the beneficial effects on atherosclerosis. As a results that BA‐treated ApoE KO mice significantly lowered systolic blood pressure. Metabolic parameter showed that BA markedly decreased BUN, triglyceride and total‐cholesterol levels. Blood glucose, insulin, glucose tolerance test, and HOMA‐IR index were significantly better in BA treated ApopE KO mice. In addition, consistent with the change in lipid profile, Oil Red O and H&E staining revealed that treatment with BA significantly reduced atherosclerotic lesion such as roughened endothelial layers and fibrous cap formation. Furthermore, BA restored the reduction of endothelial nitric oxide synthase (eNOS) expression, leading to the inhibition of intracellular adhesion molecule‐1 (ICAM‐1) and endothelin‐1 (ET‐1) expression. These results suggest that BA may be useful in the treatment and prevention of early atherosclerosis via attenuation of the endothelial dysfunction in ApoE KO mice.
Attenuated Rapid‐Onset Vasodilation with Greater Force Production in Skeletal Muscle of Caveolin‐2−/− Mice
Authors: Fernando Charmain , Liu Yajun , Sowa Greg , Segal Steven
Published: November 2023

Caveolin‐2 (Cav2) is a major protein component in caveolae, minute invaginations in plasma membranes of endothelial cells. While absence of Cav2 increases pulmonary endothelial cell proliferation and inhibits tumor growth (PMC133690; PMC4233177), the role of Cav2 in the microcirculation of skeletal muscle is unknown. We tested the hypothesis that genetic deletion of Cav2 would alter the reactivity of microvascular resistance networks controlling muscle blood flow. Internal diameter (ID) responses to physiological agonists and to contractile activity were studied at 35°C in the left gluteus maximus muscle (GM) of anesthetized Cav2−/− and littermate wild‐type (WT) mice (n=5 males/group; 6 months old). For feed arteries (FA), first‐ (1A), second‐ (2A) and third‐order (3A) arterioles, resting IDs (means: 45, 35, 25, 12 μm) and maximal IDs (65, 55, 45, 30 μm) were not different between groups, nor were cumulative endothelium‐dependent dilations to acetylcholine (ACh, 10−9–10−5 M); all right‐shifted ~1 log unit during nitric oxide synthase inhibition (L‐NAME, 10−4M). Constriction‐response curves to norepinephrine (NE, 10−9–10−5 M) overlapped for respective vessel branches. During twitch contractions (2 & 4 Hz; 30 s), steady‐state dilations (range, 5–20 μm) were again similar between groups. However, for single maximal tetanic contractions (100 Hz; 100, 250 & 500 ms), rapid onset vasodilation (ROV) increased with contraction duration but was reduced by 20–50% across vessel branches of Cav2−/− vs. WT (P<0.05). Remarkably, maximal force production (mN/mm2) was ~40% greater in Cav2−/− (152 ± 14) vs. WT (110 ± 3). Thus, while microvascular reactivity is maintained to ACh, NE and moderate contractile activity, diminished ROV with enhanced force production reveals a novel phenotype for Cav2 deletion in mouse skeletal muscle

Support or Funding Information

NIH R37‐HL041026
Role Of Gamma‐Adducin Gene In Cerebral Vascular Myogenic Response
Authors: Pabbidi Mallikarjuna , Ge Ying , Harder David R , Roman Richard J , Fan Fan
Published: November 2023

Previous studies suggest that middle cerebral arteries (MCA) isolated from FHH rat exhibit impaired myogenic response (MR) that is associated with elevation in large conductance potassium (BK) channel function. Impaired MR is restored in FHH.1BN congenic strain in which 2.4‐Mbp region containing 15 genes including Gamma‐adducin (Add3) in chromosome 1 from BN rats is transferred onto FHH rat. The present study tested the hypothesis that a mutation in Add3 gene, is responsible for the elevation of BK channel function that contribute to impaired vascular MR in FHH rats. We used 27‐mer Dicer‐Substrate RNAi (DsiRNA) to silence Add3 gene in cerebral vessels and HEK293 cells that are permanently transfected with BKα subunit to measure vascular function and BK channel function respectively using arteriomyography, patch clamp and western blot methods. MCA transfected with Add3 DsiRNA and cultured for 36hrs reduced mRNA expression in a dose‐dependent manner (50±0.2% reduction; n=3). MCA transfected with Add3 DsiRNA exhibited impaired MR when luminal pressure is increased from 50 to 150 mmHg (112±1 to 121±2μm; n=3). In contrast, the MR is intact in vessels treated with scrambled DsiRNA (50mmHg: 131±16 to 150mmHg: 91±5 μm; n=3). Total outward K channel currents were ~3 fold higher in smooth muscle cells freshly isolated from MCA that were transfected with Add3 DsiRNA compared with non‐transfected cells (DsiRNA:141±18 pA/pF; non‐ transfected: 44±10 pA/pF; n=10–11). Similarly, BK channel current was ~3 fold higher in HEK293 cells expressing BKα that were transfected with Add3 DsiRNA (+120mV; DsiRNA: 334±8 pA/pF; non‐ transfected: 109±17 pA/pF; n=3–4). Finally, expression of BKα protein is ~2 fold greater in the membrane of cerebral vessels isolated from FHH compared to FHH.1BN rats (FHH: 2.6±0.5; FHH.1BN: 1.3±0.1; n=3). In conclusion, these results indicate that Add3 is a viable candidate gene that may play a causal role in impaired MR by elevation of BK channel activity in FHH rats.

Support or Funding Information

This research has received funding support from the American Heart Association, Scientist Development Grant (13SDG14000006) to Pabbidi and NIH 1R01DK104184‐01 to Richard J. Roman.
Effects of Muscle Stretching on Skeletal Muscle Microcirculation in Old Rats
Authors: Hotta Kazuki , Behnke Bradley J , Ghosh Payal , Chen Bei , Churchill Alexandra , Elam Marcus L , Pourafshar Shirin , Arjmandi Bahram H , Maher Patrick , Kurien Daniel , Verma Rahul , Christou Demetra , Muller‐Delp Judy M
Published: November 2023

Skeletal muscle blood flow during exercise declines with age, in part, due to impaired microvascular dysfunction of skeletal muscle. Mechanical stretch alters morphology and function of vascular endothelial cells; however, little is known about the effect of muscle stretching on endothelium‐dependent vasodilation and skeletal muscle microcirculation. We tested the hypothesis that daily passive muscle stretching enhances endothelium‐dependent vasodilation of skeletal muscle arterioles and increases skeletal muscle blood flow in old rats. Old (n=45, 20 months old) male Fischer 344 rats underwent daily muscle stretching or remained cage controls. The left hindlimb was passively stretched by placing a splint on the left ankle (30 min/day, 5d/wk for 4 wks). The right hindlimb served as a contralateral control limb. At the end of the muscle stretching, soleus muscle blood flow was measured at rest and during treadmill exercise. The relaxation responses to acetylcholine was assessed in isolated soleus muscle arterioles. Three dimensional images of the microvasculature in soleus muscle were obtained using a high‐resolution microcomputed tomography. Exercising blood flow and relaxation responses to acetylcholine were greater in soleus muscle of the stretched hindlimb as compared with the contralateral hindlimb and cage control rats. Microcomputed tomography imaging of vessel connectivity, which is defined as the number of vessel branches, was greater in soleus muscle of the stretched hindlimb than the contralateral hindlimb. These results suggest that muscle stretching enhances microvascular endothelial function and induces angiogenesis, likely contributing to increased exercising blood flow in skeletal muscle of old rats.

Support or Funding Information

NIH 1R 21AG044858‐01A1 and Japan Heart Foundation
Thermally Induced Erythrocyte Aggregation by Surface Nanobubble Coalescence
Authors: Seidner Harrison S. , Gunter Geoffrey , Weber‐Fishkin Samantha , Frame Mary D.
Published: November 2023

Erythrocyte aggregation is a reversible phenomenon, of which the mechanism is unknown. Pathological conditions, such as thermal burn injury and the presence of plasma proteins are associated with an increase of erythrocyte aggregation, particularly in the temperature range of 42°C to 45°C. Aggregation contributes to two‐phase flow, increasing the throughput of red blood cells. Excessive aggregation occludes vessels, inhibiting blood flow to tissues downstream. Reduced perfusion leads to hypoxia and ischemia in the region of thermal injury, ultimately resulting in thermal burn injury progression. During low Reynolds flow and stasis, fluid shear is less likely to monodisperse the red blood cells. Following thermal injury, aggregation happens within seconds. Aggregates remaining in the presence of fibrin for a four hour period will progress to clots. While the mechanism for aggregation is unknown, several models have been proposed, including the bridging model and depletion layer model; however, neither account for all possible biophysical phenomena. Here we propose a model in which nanobubbles on neighboring red blood cell surfaces have potential to coalesce, effectively increasing red cell aggregation. We hypothesize that during the onset of thermal injury, nanobubbles are formed by rapid blood‐gas dissolution furthered by simultaneous oxy‐hemoglobin dissociation. Nanobubble dynamics are a function of gas concentration, surface tension, and geometric properties of blood constituents. Using MATLAB (MathWorks), a mathematical model of gas content and bubble dynamics was constructed to predict the likelihood of nanobubble formation, coalescence, and contribution to aggregation. The results of the model predict temperature dependent erythrocyte aggregation only at pH, and oxygen partial pressures similar to that of venous blood.
Erythrocyte Flux vs. Flow in Microchannels as a Function of Bifurcation Architecture and Re
Authors: Darvishi Samira , Frame Mary
Published: November 2023

In microvascular systems with arteriolar diameters approximately 5 to 10 times larger than the average red blood cells (RBC) the distribution of RBC at bifurcations is not only influenced by flow properties such as Reynolds number (Re), but also by the geometry of the bifurcation region (bifurcation angle, radius of curvature). Red blood cells, while they do not follow the traditional Poiseuille flow streamlines, they do follow the path with lower resistance, which is a function of both the flow properties and complex regional geometry. Yet, the flux vs. flow relationship remains poorly understood. We study these coupled effects using a fabricated PDMS microchannel system (square, 25μm), and observe RBC distribution at bifurcations with varying angles (45°, 90° and 135°) and radius of curvature (0.5, 1 and 2 tube diameters). The flow rate in the feed, Qf, feed continuation, Qfc, and branch, Qb, were confirmed by the velocities of 0.5μm fluorescently labeled microspheres (particle: tube ratio = 1:50) The main observation, also seen in vivo, is that regardless of the Re number, the relative flux of RBC into the branch is always less than the relative flow rate into the branch. This trend is more significant at higher Re (Re: 0.001 to 0.1). Moreover, 90‐degree bifurcation angles draw fewer RBCs (lower flux) than the other two angles; one cause could be the decreased cross‐sectional area of the branch opening associated with 90° vs. 45° or 135° angles. Although viscous effects are dominant in our system and we cannot use the ‘potential flow theories’ fully, the existing flow paths suggest a stagnation point in each microchannel that affects the particle flow direction, and thus the RBC flux distribution to the branch vs. continuing along the feed. Therefore we can predict the RBC distribution at a bifurcation knowing the flow properties (Re), the geometry that creates this stagnation point (angle, radius of curvature) and radial position of the RBC 2–3 tube diameters upstream of the junction.
Acute Reduction of Ambient Air Pressure Enhances Arterial Vasodilation in Murine Resistance Arteries Independently of Endothelial Factors
Authors: Shahid Anmol , Morton Jude S , Davidge Sandra T , McMurtry Michael Sean
Published: November 2023

Introduction

Epidemiological studies have found populations living at higher elevations are at lower risk for acute myocardial infarction (MI) than those at lower elevations, even after adjustments for confounders. The mechanism for this observation is not known. Since atmospheric air pressure varies inversely with elevation, and air pressure exerts compressive force on vascular structures, reduced compressive force on arteries due to elevation‐related reductions in atmospheric pressure might alter arterial function. This could be a potential mechanism. We hypothesized that acute exposure to lower air pressure will dilate arteries and increase arterial vasodilation to reduce peripheral vascular resistance.

Methods

Second order mesenteric arteries (n=7) were isolated from C57BL/6 male mice (n=7, age 3.2±0.9 months) and perfused with Ca2+‐free saline solution to assess passive properties of the vessels in a pressure myograph. This system was placed in a custom‐designed hypobaric chamber and arterial function was studied ex‐vivo at acute exposure to three barometric pressure steps: 754 mmHg (room pressure or control), 714 mmHg (p1) and 674 mmHg (p2). Active vessel responses in the presence of L‐NAME, a nitric oxide inhibitor, and Meclofenamate, a cyclooxygenase inhibitor, were assessed with mechanical manipulation of perfusion pressure (4–140 mmHg) or flow rate (0–70 μL/min). Lumen diameter was measured using a micrometer, with manometers for measurement of pressure drop across the vessel.

Results

Under static conditions, vessel diameters at p1 and p2 increased by 20.9±9.3% and 28.2±8.6% compared to baseline diameter at room pressure (p<0.01 vs. control for p1 and p2). Flow‐mediated vasodilation contributed little additional dilation. At a fixed flow rate of 70 μLmin−1, vessels reached maximum diameters of 108.6 ±13.6μm at p1 and 120.5±13.0 at p2 versus 106.0±4.0 μm for the control condition. This observed vasodilation was not diminished in the presence of L‐NAME and Meclofenamate. Vascular resistance was significantly reduced at p2 compared to control (2.14±0.60 mmHg*min/μL vs. 3.21±0.49 mmHg*min/μL, p < 0.05).

Conclusion

We conclude that acute exposure to reduced barometric pressure increases artery diameter and decreases resistance across vessels ex‐vivo in an endothelium‐independent manner.
Comprehensive Hemodynamic Analysis of Arteriolar Networks in the Rat Gluteus Maximus Muscle
Authors: Al Tarhuni Mohammed , Jackson Dwayne N , Goldman Daniel
Published: November 2023

Introduction

Theoretical blood flow models enable comprehensive analysis of blood flow, pressure, resistance, and hematocrit distribution within microvascular networks. However, the validity of data produced by such models is dependent on the detail of its experimentally derived inputs. Currently, many theoretical models of skeletal muscle hemodynamics are limited to incorporating experimental inputs from small network segments or trees, or from non‐locomotive tissues such as the mesentery. Thus, the objective of this study was to utilize recently collected experimental (geometric and topological) data from complete arteriolar networks of locomotive skeletal muscle as inputs for our computational blood flow model. Using outputs from theoretical simulations, we evaluated inter‐network hemodynamic variability in an effort to describe the level of functional homology among skeletal muscle arteriolar networks.

Methods

The 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. Approximately 1500 discrete nodes were used to mathematically model each network, with an average length of approximately 100 μm between each node, resulting in 179 to 239 vessel segments per network. A previously established two‐phase blood flow model (MATLAB) was used to simulate and assess the hemodynamic properties of each arteriolar network including heterogeneity within each network and variability between networks.

Results

Blood flow rate in each vessel segment decayed exponentially with increasing (1A to 9A) arteriolar order for each network (Exponential Decay fit: R2 0.71 to 0.88). Additionally, the coefficients of variation (a measure of blood flow distribution) at each order ranged from 30.24% to 86.79%. Log‐log plots between blood flow and arteriolar diameter showed a strong correlation for each network (Linear Regression, R2 0.65 to 0.86) with similar slopes of regression lines (3.08 to 3.81).

Conclusion

Simulation results show strong homology in blood flow properties between all the networks considered. Other hemodynamic properties such as RBC flow and hematocrit, as well as overall resistance, are now being analyzed with respect to both arteriolar order and diameter. This analysis allows detailed comparison of the hemodynamic properties of the different arteriolar networks studied, including the extent to which observed topological and geometric homologies result in similar hemodynamic outcomes.

Support or Funding Information

NSERC
Endothelial Dysfunction in Children after Cardiopulmonary Bypass
Authors: Krispinsky Luke , Lamb Fred , Stark Ryan , Parra David
Published: November 2023

Objectives

Cardiopulmonary bypass (CPB) is required for surgical repair of congenital heart defects; however, CBP causes an inflammatory response which profoundly impacts post‐operative management. CBP‐induced endothelial dysfunction has been inferred by changes in pulmonary vascular resistance, changes in biomarkers, and the presence of capillary leak. However, peripheral vascular endothelial dysfunction has never been quantified in children. We used laser Doppler perfusion monitoring (LDPM) coupled with iontophoresis of vasoactive compounds to assess endothelial function in cutaneous arterioles in infants undergoing CPB.

Study Design

Enrollment criteria included age less than 1 year and a congenital heart lesion requiring use of CPB. Timing of assessment of endothelial function included pre‐operatively, 2–4 hours and 24 hours after CPB. Trans‐cutaneous iontophoresis of acetylcholine (ACh, endothelium‐dependent vasodilator) was used to trigger increases in local perfusion and LDPM signals were quantified as peak perfusion and area under the curve (AUC). Sodium nitroprusside (NP) provided an endothelium independent control vasodilator. With each assessment of endothelial function, estimates of extracellular water were obtained using bioelectrical impedance analysis. Clinical markers of the severity of postoperative illness were obtained through chart review. Peak serum lactate, total colloid and crystalloid replacement volume, urine output, and peak vasoactive inotrope score will be correlated with the degree of endothelial impairment. Means and standard error of the mean were calculated for peak perfusion and AUC to determine if there was significant change between pre and post‐CPB values.

Results

15 patients have completed the study protocol. Comparing pre‐bypass LDPM responses to ACh with initial (2–4 hour) post‐bypass measurements, we observed a significant decrease in both the peak perfusion values (75.6 ± 10.7 vs. 32.3 ± 7.7 perfusion units (PU), p < 0.05) as well as the AUC (19,417 ± 3286 vs. 6980 ± 2197 PU, p < 0.05). The percent change from baseline responses to ACh at 2–4 hours post‐CPB was −56% ± 8.8% for peak values and −61% ± 11% for AUC. Responses to ACh 24 hours after CBP remained impaired, but this effect was not statistically significant. In contrast, responses to NP were not significantly altered at 2–4 or 24 hours post‐CPB. Analysis of LDPM correlation to biochemical and clinical outcomes is ongoing.

Conclusion

Profound cutaneous vasomotor endothelial dysfunction is present in infants 2–4 hours after separating from CPB. This effect may persist for at least 24 hours. The clinical significance of this finding remains to be determined.

Support or Funding Information

Dr. Fred Lamb's Laboratory at Vanderbilt Children's Hospital
Effects of Hydrogen Sulfide on Renal and Mesenteric Blood Flow
Authors: Barrera Adelaeda , Naik Jay , Kanagy Nancy L.
Published: November 2023

In sleep apnea, recurrent obstruction of the upper airway leads to sleep fragmentation and intermittent hypoxia (IH) during sleep. In animal studies, IH causes wake‐time hypertension by elevating vascular resistance due in part to decreased production of the vasodilator, hydrogen sulfide (H2S). Inhibiting cystathionine γ‐lyase (CSE) to prevent H2S synthesis augments constriction in isolated mesenteric and renal arteries but H2S regulation of resistance in these vascular beds in vivo is unknown. The goal of this study was to evaluate CSE regulation of flow and resistance in the mesenteric and renal circulations of control rats and in rats made hypertensive by exposure to IH for 14 days. We hypothesized that inhibiting CSE would increase vascular resistance and mean arterial pressure (MAP) more in Sham than in IH exposed rats. Rats were anesthetized with inhaled isofluorane (2%) and instrumented with femoral artery and vein catheters and Doppler flow probes on the main mesenteric and renal arteries. Responses to phenylephrine and CSE inhibition (β‐cyanoalanine, BCA, 50 mg/kg/min infusion for 20 minutes) were evaluated. BCA infusion significantly increased MAP in both Sham and IH compared to saline infusion (Sham: 7.2±1.2 vs. −2.8±1.1; IH: 9.3±1.1 vs. 3.3±0.7, % change from baseline, p<0.05, n=6–8 rats per group) and augmented the press or response to phenylephrine (12 mg/kg, bolus) in Sham, but not IH rats. However, BCA did not alter either renal or mesenteric resistance in the Sham and IH groups. Thus, CSE is an important regulator of blood pressure and reduces sensitivity to adrenergic stimulation. However, since resistance arteries were not sensitive to CSE inhibition in anesthetized rats, future studies in conscious animals will determine if this was due to effects of anesthesia or lack of regulation of vascular tone by H2S in these vascular beds.

Support or Funding Information

This work was supported by the National Institutes of Health National Heart, Lung, and Blood Institute [Grants HL123301 and HL007736‐21] and institutional funding from the University of New Mexico.
Melatonin Preserves Blood‐brain Barrier Integrity and Permeability via Matrix Metalloproteinase‐9 Inhibition
Authors: Shaji Chinchusha Anasooya , Alluri Himakarnika , Wiggins‐Dohlvik Katie , Beeram Madhava R , Davis Matthew L , Tharakan Binu L
Published: November 2023

Microvascular hyperpermeability that occurs at the level of the blood‐brain barrier (BBB) often leads to vasogenic brain edema following traumatic/ischemic brain injury. At a cellular level, tight junction proteins (TJPs) regulate the functions of the blood‐brain barrier. Zonula occludens‐1 (ZO‐1) is an important TJ associated protein that binds to the transmembrane TJPs and actin cytoskeleton intracellularly and plays an important role in maintaining the integrity of the blood‐brain barrier. The pro‐inflammatory cytokine, interleukin‐1β (IL‐1β) as well as the proteolytic enzyme, matrix metalloproteinase‐9 (MMP‐9) play major role in promoting brain edema following trauma/ischemia. Recent studies indicate that melatonin a pineal hormone also acts as an endogenous inhibitor of MMP‐9. Our objective was to test if melatonin will attenuate IL‐1β‐induced BBB hyperpermeability via MMP‐9 inhibition in vitro, and also protect the BBB in a mouse model of mild traumatic brain injury. Rat brain microvascular endothelial cells (RBMEC) grown as monolayers on Transwell inserts or chamber slides were exposed to melatonin prior to IL‐1β treatment. Monolayer permeability was studied using FITC‐dextran permeability assay. Tight junction integrity and cytoskeletal organization were studied utilizing ZO‐1 immunofluorescence and rhodamine phalloidin staining of f‐actin, respectively. Effect of IL‐1β on cell viability was studied using Calcein AM assay. MMP‐9 activity was measured fluorometrically. ZO‐1 protein and gene expressions were studied by western blotting and RT‐PCR respectively. The effect of melatonin against BBB hyperpermeability was studied in a mouse controlled cortical impact model of traumatic brain injury. Acute IL‐1β (10 ng/mL; 2 hours)‐induced BBB hyperpermeability was significantly attenuated by pretreatment with melatonin (10 μg/mL; 1 hour) and MMP‐9 inhibitor‐1 (MMP‐9 specific inhibitor; 5 nM; 1 hour). Melatonin attenuated IL‐1β‐induced MMP‐9 activity, loss of ZO‐1 junctional integrity and f‐actin stress fiber formation. IL‐1β treatment had no effect on ZO‐1 protein or gene expression or on cell viability. Melatonin attenuated TBI‐induced BBB hyperpermeability in a mouse model of TBI. In conclusion, these results suggest that, one of the protective effects of melatonin against BBB hyperpermeability occurs due to enhanced BBB integrity via MMP‐9 inhibition. Melatonin treatment provided protection against BBB hyperpermeability in a mouse model of TBI suggesting its potential as a therapeutic agent against brain edema when established in humans.
Inhibition of TRPC3 Channels Underlies Hypoxia/Reoxygenation‐induced Endothelial KCa Channel Dysfunction
Authors: Wang Xiang‐Chong , Huang Jun‐Hao , Sun Wen‐Tao , Yu Cheuk‐Man , He Guo‐Wei , Yang Qin
Published: November 2023

Objectives

Although studies have shown that elevation of [Ca2+]i is essential for the activation of endothelial calcium‐activated potassium (KCa) channels and the importance of Ca2+‐permeable channels, such as canonical transient receptor potential channel (TRPC) in endothelial [Ca2+]i regulation, little information is available regarding the association of TRPC and KCa channels in endothelial function. Furthermore, it remains uninvestigated whether modulation of TRPC channels is involved in the compromised endothelial function related to IKCa and SKCa in ischemia/reperfusion injury.

Methods

Vasorelaxation was studied in porcine small coronary arteries (PCAs) in a myograph. IKCa and SKCa channel currents were recorded by patch‐clamp, and coimmunoprecipitation experiments were performed to examine the association of TRPC3 and KCa proteins in primary cultured porcine coronary arterial endothelial cells (PCAECs).

Results

The IKCa and SKCa‐dependent, endothelium‐derived hyperpolarizing factor (EDHF)‐mediated relaxation in response to bradykinin was inhibited in PCAs pretreated with the TRPC3 specific inhibitor Pyr3. Pharmacological inhibition of TRPC3 with Pyr3 and TRPC3 gene silencing by siRNA both significantly suppressed bradykinin‐induced IKCa (6.71±0.85 in Pyr3‐treated cells, 9.46±1.12 in TRPC3 siRNA transfected cells, vs. 16.89±0.93 pA/pF in control) and SKCa (5.51±1.17 and 6.46±0.84, vs. 10.63±1.69 pA/pF) currents. No physical interactions between either TRPC3 and IKCa channels or TRPC3 and SKCa channels were observed. In hypoxia/reoxygenation (H/R)‐exposed PCAECs, bradykinin‐induced IKCa (10.24±1.96 pA/pF) and SKCa (4.62±0.43 pA/pF) currents were markedly suppressed and Pyr3 exhibited less inhibition on both components when compared with the cells without H/R exposure. Activation of TRPC3 channels with 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) restored H/R‐induced suppression of IKCa (15.08±0.99 pA/pF) and SKCa (9.62±0.89 pA/pF) currents and the current enhancement is associated with an improved EDHF response in PCAs.

Conclusions

TRPC3 channels play an important role in IKCa and SKCa channel activation in coronary arterial endothelial cells. The functional association between TRPC3 and KCa channels does not involve physical interaction. Inhibition of TRPC3 channels contributes to H/R‐induced suppression of IKCa and SKCa channel activity, which serves as a mechanism underlying endothelial dysfunction under H/R condition and renders TRPC3 channels a potential target for endothelial protection in ischemic status.

Support or Funding Information

Supported by RGC/GRF CUHK4774/12M & CUHK14118414; Lui Che Woo Institute of Innovative Medicine ‐ CARE theme 8303303, and CUHK Direct Grant 4054182.
Rapid Increases in Fluid Shear Stress Elicit Local Lamellipodia and Elevate Microvascular Endothelial Barrier Function
Authors: Spampinato Sara G , Breslin Jerome W
Published: November 2023

The microvascular endothelium serves as a barrier that controls exchange of fluid and solutes between the blood and tissues. This semipermeable barrier reacts to changes in frictional shear stress from the plasma as it flows over the luminal surfaces of endothelial cells. We tested the hypothesis that step increases in shear stress strengthen endothelial barrier function, due to spreading of endothelial cell membrane near junctions. We also tested the role of the shear stress‐sensing calcium channel Piezo1 in both baseline and mediating shear‐dependent changes in barrier function. An electrical cell substrate impedance sensor (ECIS) was used to determine the barrier function of cultured human cardiac microvascular endothelial cell (HCMEC) monolayers, with transendothelial electrical resistance (TER) serving as an index of barrier integrity. Shear stress was stepped from either 0 or 1 dynes/cm2 up to 10 dynes/cm2 using a computer‐controlled peristaltic pump system. Time‐lapse microscopic images of confluent HCMEC monolayers were obtained to observe junctions and spreading behavior of cells. The number of local lamellipodia per 100 microns of junctional distance over time, before and after step increases in shear stress, was quantified. The Piezo1 inhibitor, GSMTx4 was applied at 1.25 mM for at least 30 min under no‐flow conditions prior to testing step increases in shear stress. The results show that step increases in shear stress cause an increase in TER, that correlates with a robust increase in local lamellipodia. GSMTx4 caused a significant decrease in TER within the first few minutes of application. Step increases in shear stress applied after GSMTx4 still caused increases in TER, but usually not up to the level observed prior to GSMTx4 treatment. Combined, the data shows that rapid changes in fluid shear stress elicit a quick rise in the number of local lamellipodia that protrude from endothelial cells, that appear to tighten the junctions between cultured endothelial cells. The results also suggest that the Piezo1 channel has a role in maintaining basal barrier function of endothelial cells. However, endothelial cells can still enhance their barrier function when Piezo1 is blocked using GSMTx4. Because a compromised endothelial barrier is common in many diseases and after injury, our future goal will be to identify mechanisms that promote local lamellipodia as potential therapeutic targets.

Support or Funding Information

Supported by NIH Grant R01HL098215‐S1
Lymphatic Vascular Integrity is Disrupted in a Mouse Model of Diabetes: Dual Regulation by Nitric Oxide
Authors: Scallan Joshua P , Hill Michael A , Davis Michael J
Published: November 2023

Emerging studies support the idea that metabolic diseases such as hypercholesterolemia and obesity are associated with compromised lymphatic vessel function. Whether, or how, insulin‐resistant diabetes affects the lymphatic vasculature remains unexplored. To test the hypothesis that lymphatic vascular integrity is disrupted in diabetes, we developed an innovative approach to measure the permeability of collecting lymphatics from wild‐type (WT) and leptin receptor‐deficient (db/db) mice. Vessels were isolated and mounted on glass micropipettes that could switch repeatedly between perfusion of fluorescent‐labeled or unlabeled albumin solutions. The accumulation of fluorescent albumin outside the vessel was measured over time with photometry, while diameter was monitored simultaneously on a near infrared image. Since nitric oxide (NO) is crucial for maintaining blood vessel integrity and is impaired in diabetes, we first determined whether NO was capable of regulating lymphatic permeability (×10(−7) cm/s). WT lymphatic permeability (3.3±0.7, n=7) was significantly reduced by pharmacologic inhibition (0.6±0.2, n=7) or genetic deletion (0.6±0.1, n=6) of endothelial nitric oxide synthase (eNOS). Histamine, an agent known to elicit endogenous NO production (21±7, n=7), or sodium nitroprusside, an exogenous NO donor (20±5, n=6), both significantly elevated WT lymphatic permeability. Collecting lymphatics from db/db mice exhibited a dramatic increase in permeability compared to age‐matched 25‐week old controls (WT: 2.2±0.3, n=10; db/db: 299±56, n=23). Interestingly, restoration of NO production by acute exposure to L‐arginine rescued this endothelial barrier dysfunction, and could be reversed by pharmacologic inhibition of eNOS with L‐NAME (db/db: 321±84, L‐arginine: 14±5, L‐arginine+L‐NAME: 297±88; n=9). In summary, this approach enables quantitative measurement of lymphatic integrity in transgenic mice for the first time and identified a lymphatic defect in diabetic mice caused by reduced NO bioavailability.

Support or Funding Information

This work was supported by the National Heart, Lung, and Blood Institute at the National Institutes of Health (K99 HL124142 to J.P.S., R01 HL089784 and R01 HL120867 to M.J.D., P01 HL095486 to M.J.D. and M.A.H., and R01 HL085119 to M.A.H.)
Mechanisms coordinating vascular smooth muscle adhesion to the extracellular matrix during contraction
Authors: Huang Huang , Sun Zhe , Hill Michael A , Meininger Gerlad A.
Published: November 2023

Physical coupling of vascular smooth muscle cells (VMSC) to extracellular matrix proteins (ECM) by integrins is required to efficient mechano‐transduction and vessel function. Previous work in our laboratory demonstrated that increased VSMC stiffness, induced by Angiotensin II (AII), is coordinated with enhanced adhesion to fibronectin (FN). However, the mechanism of coordination is not fully understood. In this study, we hypothesized that enhanced adhesion in response to AII was mediated by integrin activation through an inside‐out signaling pathway linked to increases in Ca2+. We first tested that the increase in FN adhesion was due to integrin activation and not increased membrane expression. We found that the adhesion, studied, using atomic force microscopy (AFM) with anti‐alpha 5 integrin‐coated probes, was not increased by AII in contrast to increased adhesion observed with FN coated probes. This suggested that the enhanced adhesion was occurring through an integrin activation process and not an increase in membrane density of the integrin. To examine a role for Ca2+ in this integrin activation, we used a non‐G‐protein coupled vasoconstrictor KCl (60mM), and assessed the coordination between changes in cell elasticity and adhesion to FN. In all VSMC tested, cell stiffness was increased by KCl, and this enhanced stiffness was transient and rapid. Also, cell adhesion probability (number of adhesion events) was transiently increased by KCl but the average adhesion force was unchanged. These data suggest that Ca2+ may play a role in the activation of integrin adhesion during contraction. However, further studies are necessary to confirm a role for Ca2+ and elucidate the signaling pathway linked to integrin activation. d adhesion to FN. In all VSMC tested, cell stiffness was increased by KCl, and this enhanced stiffness was transient and rapid. Also, cell adhesion probability (number of adhesion events) was transiently increased by KCl but the average adhesion force was unchanged. These data suggest that Ca2+ may play a role in the activation of integrin adhesion during contraction. However, further studies are necessary to confirm a role for Ca2+ and elucidate the signaling pathway linked to integrin activation.

Support or Funding Information

NIH 1PO1HL095486 to GAM
Human pericardial fluid‐derived endothelin elicits arterial constriction
Authors: Nemeth Zoltan , Cziraki Attila , Szabados Sandor , Horvath Ivan , Koller Akos
Published: November 2023

Background

Local regulation of blood flow includes extravascular substances released by the parenchymal tissues. Recently, substances derived from fat cells, pericytes and other cells and tissues were shown to exert local vasomotor effects. We hypothesized that pericardial fluid (PF) also affects vasomotor tone, because it has been shown that PF contains vasoactive substances, such as endothelin‐1 (ET‐1).

Methods

Studies were performed using isometric myograph (DMT‐Multi Myograph System, pH=7.4, T=36.8 °C). Human PF‐induced changes in vasomotor tone were studied in carotid arteries isolated from Wistar rats. Human PFs was obtained from patients undergoing coronary artery bypass graft (CABG) or valve replacement (VR) surgery and its vasomotor effect was examined by adding PF to the vessels chamber. Vasomotor capacity of vessels was tested by adding KCl (60 mM) before and after the adding PF. Also, in CABG PF the role of ET‐1 was assessed by using the selective endothelin A receptor (ETA) antagonist BQ123 (10−6 M/L).

Results

Both CABG PF and VR PF induced significant increases in isometric forces of isolated arteries (CABG PF: 3.1±0.7 mN and VR PF 3.0 ± 0.9 mN). Isometric forces induced by CABG PF were significantly reduced in the presence of BQ123 (0.8±0.1 mN vs. 2.6±0.5 mN). We have also observed that contraction to PF were more potent than that of ET‐1. KCl elicited substantial increases in vasomotor tone, both before and after applications of PF (KCl‐1: 6.1±0.2 mN; KCl‐ 2: 6.5±0.9 mN).

Conclusion

Pericardial fluids of patients undergoing cardiac surgery exert substantial constrictor activity in arteries, which in vivo may include microvessels as well. In the vasomotor activity of PF, ET‐1 has a dominant role, but other vasoactive factors may also be present. Thus non‐parenchymal tissues, such as pericardial tissues and fluid may also be involved in the local regulation of coronary blood flow.

Support or Funding Information

Hungarian Scientific Research Fund (OTKA) K 108444. SROP‐4.2.2.A‐11/1/KONV‐2012‐0024, Hungarian Society of Hypertension 2011/2012, 2013/14.
Stromal Interaction Molecule 1 Disruption Protects Myocardial from Ischemia ‐ Reperfusion Injury
Authors: Mali Vishal R , Ali Maha , Haddox Samuel , Belmadani Souad , Trebak Mohamed , Matrougui Khalid
Published: November 2023

Background

It has been reported that the increase in Stromal Interacting Molecule 1 (STIM1) expression is associated with myocardial complication. However, the role of vascular STIM1 in cardiac ischemia‐reperfusion (I/R) injury is unknown. Therefore, the central hypothesis of the present study is to determine the role of deleted STIM1 in endothelial cells or smooth muscle cells (SMC) in cardiac I/R injury. Thus, we performed cardiac I/R in control mice, mice lacking the STIM1 specifically in endothelial cells (Stim1−/−EC) or specifically in SMC (Stim1−/−SMC).

Methods and Results

All mice were subjected to cardiac ischemia for 40 minutes and followed by reperfusion for 24 hours. After 24 hours reperfusion, hearts were incubated with triphenyl tetrazolium chloride to determine the infarct size. In control mice subjected to I/R, the heart develops infarct, which represents 50% of the heart area. Interestingly, in Stim1−/−EC and Stim1−/−SMC, the infarct size was significantly reduced indicating that STIM1 disruption specifically in endothelial cells or SMC protects the heart from I/R injury. Importantly, the heart protection was greater in Stim1−/−SMC than Stim1−/−EC mice. We found an increase in the endoplasmic reticulum (ER) stress in control mice subjected to I/R than in Stim1−/−SMC and Stim1−/−EC mice subjected to I/R.

Conclusion

Our results indicate that STIM1 disruption in endothelial cells and SMC protects the heart from I/R injury likely through the inhibition of ER stress.

Support or Funding Information

NIH
Early Post‐Ischemic Blood‐Brain Barrier (BBB) Disruption in Obesity
Authors: Li Chun , Jiang Zheng , Arrick Denise M , Lu Wei , McCarter Kimberly D , Sun Hong
Published: November 2023

We determined the effect of high fat diet (HFD)‐induced obesity on early BBB disruption following transient focal cerebral ischemia. Male C57BL/6J mice were fed a HFD or standard chow for 16 weeks. A cranial window was prepared over the left frontal, parietal and temporal cortex. Transient focal cerebral Ischemia was induced by directly ligating the middle cerebral artery (MCA) for two hours. Early BBB disruption was assessed by measuring Evans Blue and sodium fluorescein extravasation at 3 hours of reperfusion. The body weight was significantly increased in obesity group (49.2 ± 0.4g) compared to control group (31.9 ± 0.6g). There was no significant difference in conscious blood pressure and fasting blood glucose between control and obesity groups. Transient focal cerebral ischemia produced an early BBB disruption in both control and obesity groups. However, the magnitude of early BBB disruption was significantly greater in obesity group compared to control group. Topical treatment with L‐NPA, 7‐NI (neuronal nitric oxide synthase inhibitors) or L‐NAME (a nonspecific nitric oxide synthase inhibitor) completely abolished the BBB disruption in control group, but only partially suppressed the BBB disruption in obesity group. Interestingly, matrix metallopeptidase (MMP)‐9 activity of cerebral cortex was reduced in obesity group either under basal conditions or following ischemic stroke. Our findings suggest that obesity exacerbates early post‐ischemic BBB disruption via a mechanism that appears to be unrelated to MMP‐9 or NOS.