bluuebunny/crossref_metadata_2025 huggingface dataset:
The role of PECAM‐1 mediated signaling in neutrophil polarization.
Authors: Khan Mohammed Azizuddin , Shin Jae‐Won , Komarova Yulia , Malik Asrar
Published: November 2023

PECAM‐1, also known as CD31, is a 150kDA transmembrane glycoprotein comprised of six extracellular Ig repeats that form trans‐interactions with PECAM‐1 on the cell surface of endothelial cells. Neutrophil trans‐endothelial migration (TEM) is critically dependent on these interactions. While the role of PECAM‐1‐mediated signaling in endothelial cells is well‐studied, and linked to activation of calcium flux, and re‐organization of actin cytoskeleton, the role of PECAM‐1 in neutrophils remains unclear. Hence, we investigated this question using PECAM‐1 coated biomimetic surfaces. We report here that PECAM‐1 homophillic ligation induces prompt nucleation of actin cytoskeleton and neutrophil polarization without the presence of a chemoattactant (fMLF). This was not seen when neutrophils were seated on biomimetic glass coated with fibronectin. We also observed an increase in SHP2 and RhoGTPase activity at the sites of PECAM‐1 homophilic interaction. We propose that PECAM‐1 induces SHP2/RhoA signaling and the resultant polymerization of actin cytoskeleton through the mDia2 pathway. Hence, our data suggests a novel role of PECAM‐1 in neutrophil TEM.
Development and Characterization of a Fluorescent Protein‐Based Biosensor for the Detection of Extracellular ATP
Authors: Conley Jason M , Radhakrishnan Saranya , Valentino Stephen A , Tantama Mathew C
Published: November 2023

Extracellular adenosine 5′‐triphosphate (ATP) is a fundamental component of purinergic signaling that contributes to wide ranging physiological processes and pathophysiological conditions. New methods that enable direct measurement of extracellular ATP dynamics with high spatial and temporal resolution are expected to advance our understanding of purinergic signaling mechanisms in healthy and pathological states. Several distinct classes of fluorescent protein‐based biosensors have emerged as powerful molecular tools that report real‐time intracellular ATP dynamics with single‐cell and subcellular resolution, but there are no reports of leveraging such technology for the detection of extracellular ATP. Therefore, the focus of this study was to develop and characterize a series of fluorescent protein‐based biosensors for the detection of extracellular ATP. Our strategy was to anchor several candidate ATP biosensors to the plasma membrane and to position the biosensor in the extracellular space, allowing for the detection of extracellular ATP. We present the extensive characterization of one such cell surface‐tethered candidate biosensor. Live‐cell fluorescence microscopy was used to verify biosensor expression and localization at the cell surface and to measure sensor response to exogenous application of ATP when expressed by cell types including Neuro2A and HEK293 cells. Furthermore, we present progress using this sensor to measure extracellular ATP dynamics that are modulated by cellular processes, including ligand‐stimulated cellular release of ATP and degradation of ATP by ectonucleotidase activity. In conclusion, we are developing a genetically‐encoded fluorescent protein‐based biosensor that currently demonstrates the ability to measure dynamic changes in extracellular ATP levels in live‐cell proof‐of‐concept studies.

Support or Funding Information

We acknowledge funding support from National Institutes of Health grant NS092010
Alternative Therapeutic Nanoparticle Approach For Treatment Of Invasive Streptococcal Infections
Authors: Escajadillo Tamara , Olson Joshua , Luk Bryan , Zhang Liangfang , Nizet Victor
Published: November 2023

Bacterial infections continue to be a significant cause of morbidity and mortality worldwide. Streptococcus pyogenes, also known as group A Streptococcus (GAS), is an important human‐specific Gram‐positive bacterial pathogen, capable of causing a broad spectrum of disease from mild superficial infections such as pharyngitis and impetigo to serious invasive infections including necrotizing fasciitis and streptococcal toxic shock syndrome. Pathogenic bacteria like GAS express a multitude of virulence factors with diverse properties and modes of action that act in concert to overcome the host's innate immune defense mechanisms. Pore forming toxins (PFTs) are the most common bacterial cytotoxic proteins (25–30% of all cytotoxic proteins), which make them the single largest category of virulence factors. The PFT streptolysin O (SLO) is a well characterized virulence factor produced by nearly all GAS clinical isolates, and is necessary and sufficient to promote dermal necrosis, blood vessel contraction, increased capillary permeability, intravascular thrombosis, cardiotoxicity, and death during severe invasive infection. In the present work, we describe how the pharmacological presentation of red blood cell (RBC) derived biomimetic nanoparticles (“nanosponges”) can sequester SLO and block the ability of GAS to damage host cells, thereby preserving innate immune function and increasing bacterial clearance in vitro and in vivo. Nanosponge administration protected human neutrophils, macrophages and keratinocytes against SLO‐mediated cytoxicity. This therapeutic intervention prevented SLO‐induced macrophage apoptosis and increased neutrophil extracellular trap formation, allowing increased GAS killing by the respective phagocytic cell types. In a murine model of GAS necrotizing skin infection, local administration of the biomimetic nanosponges was associated with decreased lesion size and reduced bacterial colony‐forming unit recovery. Utilization of a toxin decoy and capture platform that inactivates the secreted SLO before it comes into contact with the host cell membrane presents a novel virulence factor targeted strategy that could be a powerful adjunctive therapy in severe GAS infections where morbidity and mortality are high despite antibiotic treatment.

Support or Funding Information

UCSD Graduate Training Program in Cellular and Molecular Pharmacology: T32 GM007752
Computational approaches to understanding ARB resistance: a route to personalized ARB therapy
Authors: Anderson Shane D. , Nies Isaac , Santos Priscilla , Soong Tik Hang , Thu Yin Win , Botello‐Smith Wesley M. , Luo Yun Lyna , Andresen Bradley T.
Published: November 2023

The binding of Angiotensin II to the Angiotensin II type 1 receptor (AT1Rs) plays a pivotal role in the regulation of blood pressure and other physiological processes. AT1R blockers (ARBs) are commonly prescribed anti‐hypertensive drugs; however, not all patients respond equally to ARBs and some patients are ARB resistant. One documented reason behind these clinical observations is non‐synonymous polymorphisms within the AT1R that alter ARB affinity. There are 103 known polymorphisms within the crystallized AT1R structure that we utilized for computational approaches to predict which polymorphisms contribute to ARB resistance. To accomplish this, a 150 ns molecular dynamic (MD) simulation was performed to create a stable‐empty AT1R in an 87% POPC and 13% cholesterol membrane. Each polymorphism was made in the MD generated AT1R via MOE and minimized prior to docking performed with autodock4. Each ARB, including the active metabolite of Losartan, was docked using an experimentally derived grid that produced affinities in the wild‐type AT1R comparable to published reports. In total, 93,600 drug‐receptor complexes were analyzed and the data indicates that polymorphisms fall into two general categories: ARB specific resistance and total ARB resistance. Polymorphic AT1Rs displaying ARB specific resistance display wild‐type affinities for a subset of ARBs and in some cases, such as C101Y, G196V, and F313C, not the common ARBs valsartan and losartan; whereas, total ARB resistance, such as W84C, V108I, H132Y, and C289W, indicates that all ARBs are likely ineffective. In conclusion, patients that appear ARB resistant should have their AT1R sequenced and the sequence could be used to determine which ARB may be effective or suggest discontinuing ARB therapy.

Support or Funding Information

This project is funded by internal WesternU funds.
IGF‐1 deficiency alters hypertension induced cerebrovascular remodeling in mice
Authors: Fulop Gabor Aron , Ramirez‐Perez Francisco I , Tarantini Stefano , Sjoelund Virginie , Martinez‐Lemus Luis A. , Sonntag William E. , Csiszar Anna , Ungvari Zoltan
Published: November 2023

Hypertension induced cerebral microhemorrhages (CMHs) may contribute to cognitive decline and predict larger strokes. Previous studies of our group found evidence that IGF‐1 deficiency exacerbates hypertension‐induced cerebral microhemorrhages (CMHs) mimicing an age‐related phenotype, but the underlying mechanisms remain elusive. We hypothesized that increased cerebrovascular stiffness and altered hypertension induced remodeling of the vessels may contribute to higher number of CMHS in IGF‐1 deficiency. Liver‐specific knockdown of IGF‐1 (Igf1f/f + TBG‐iCre‐AAV8) and control mice were chronically treated with angiotensin‐IIleading to similar hypertension in both groups (CTRL syst.: 151.375±2.08, diast.:109.65±3.27; IGF syst.: 150.10±2.00, diast.: 106.48±2.53). Middle cerebral (MCA) arteries of both groups were examined, by fixing them to a canulated isobaric vascular system and loaded with calcein. Smooth muscle cell related characteristics likepressure induced alteration in wall thickness and vessel diameter were measured using confocal microscopy. We found impaired cellular hypertrophic response in the IGF‐1 deficient hypertensive (HT) group, leading to smaller increase in wall thickness (1.43±0.04 μm in CTRL HT vs. 1.25±0.04 μm in IGF‐1 deficient HT). Based on the wall thickness and vessel diameter data circumferential stress and circumferential strain were calculated at different pressure levels (5–160 mmHg). Circumferential stress was higher in the IGF‐1 deficient group at any given pressure comparing to wild type group, while there was no change in the circumferential strain. Young's elastic modulus (Emod) was calculated by linear fitting of the stress‐strain curve. The Emod at lower pressures (describes elastin based elasticity) was higher in IGF‐1 HT group (468.20±73.56 vs. 306.77±36.22) while there was no change in the Emod at higher pressures (describes collagen based elasticity). Based on these data we can conclude that age‐related IGF‐1 deficiency alters hypertension‐induced cellular and extracellular matrix remodeling, which may lead to increased stiffnening of microvessels, possibly leading to incrased risk of pressure‐induced rupture.

Support or Funding Information

This work was supported by grants from the American Heart Association, the National Center for Complementary and Alternative Medicine, and the National Institute on Aging
Comprehensive Geometric and Hemodynamic Analysis of Complete Microvascular Networks in Rat Gluteus Maximus Muscle: An Integrated Model Derived from Experimental Data
Authors: Farid Zahra , Lemaster Kent , Al Tarhuni Mohammed , Frisbee Jefferson C. , Jackson Dwayne N. , Goldman Daniel
Published: November 2023

Skeletal muscle microcirculatory networks consist of sets of branching arterioles, which terminate at capillary beds, which subsequently give rise to a branching array of collecting venules. Generally, microcirculationists study each component of the microcirculation in isolation and provide hemodynamic analyses based on their discrete component of study. However, the interconnectivity of arterioles, capillaries, and venules suggests that measuring and predicting accurate hemodynamic outcomes requires collecting data from complete microvascular networks. Having studied blood flow in arteriolar networks both experimentally and computationally, we are now beginning to consider the roles of capillary resistance and venular network geometry. In particular, we are considering how capillaries and venules alter both overall microvascular network resistance and the flow pattern in terminal arterioles (TA's) supplying capillary beds. The venular networks used were reconstructed from intravital videomicroscopy (IVVM) data used previously to reconstruct complete arteriolar geometries, hence there was an exact correspondence between the arteriolar and venular networks. Preliminary work used an arteriolar network consisting of 1657 nodes, ~200 unbranched vessels, and 89 terminal arterioles, as well as an established steady‐state model of plasma and red blood cell (RBC) flow distribution. When resistive elements were added to the TA's to represent downstream capillary beds, overall flow resistance increased by 32% and the coefficient of variation (CVTA, standard deviation/mean) of TA RBC flow decreased by 9%. When the corresponding reconstructed venular network was added to form a complete arteriolar‐venular unit (2589 nodes), flow resistance increased by 69% and CVTA decreased by 10% compared to the arteriolar network alone. Not only was heterogeneity of TA RBC flow altered by adding capillaries and venules, but the ordering of TA's in terms of amount of RBC flow was also affected. Thus, these results imply the importance of considering complete microvascular networks when seeking to understand blood flow resistance and distribution. We believe entire networks will also be key in understanding regulation of local blood flow and its dysregulation in diseases such as type 2 diabetes and the metabolic syndrome.

Support or Funding Information

Natural Sciences and Engineering Research Council of Canada (NSERC) grants R4218A03 (DNJ) and R4081A03 (DG)
Dependence of Erythrocyte Deformability on Mechanical Stress and Oxygenation
Authors: Yalcin Ozlem , Ugurel Elif , Saglam Gokay , Erten Ahmet , Aksu Ali Cenk
Published: November 2023

Mechanical properties of erythrocytes are known to be affected by their oxygenation status. Several studies suggested that cytoskeletal rearrangements are carried out in an oxygen dependent manner. The structure of the cytoskeleton determines the mechanical properties of erythrocyte membrane. However, oxygen‐dependent mechanical characteristics of erythrocyte are poorly studied whether oxygenated state could alter erythrocyte deformability. In this study, we investigated shear stress induced improvements in erythrocyte deformability through their oxygenation status. Venous blood was collected from male, healthy volunteers (n=10) between 25–50 ages. An informed written consent was obtained from each subject participated in the study according to Declaration of Helsinki. The hematocrit of blood samples adjusted to 0.4 l/l with autologous plasma. Whole blood samples were diluted with polyvinylpyrrolidone (PVP) solution (Mechatronics, Hoorn, Netherlands) with a dilution ratio of 1/200. Blood samples were equilibrated with either ambient air or nitrogen gas for at least 10 minutes at room temperature. Erythrocyte deformability was measured by a laser‐assisted optical rotational cell analyzer (LORRCA MaxSis, Mechatronics, Netherlands) applying shear stresses (SS) ranging between 0.3 to 50 Pa. Then, a constant SS of 5, 10 and 20 Pa were applied continuously for 300 seconds and erythrocyte deformability was measured immediately afterwards. Maximal erythrocyte elongation index (EImax) and the SS required for one‐half of this maximal deformation (SS1/2) were calculated by using the linear Lineweaver‐Burke (LB) model. Deoxygenation of blood samples significantly decreased SS1/2 values both before and after SS applications (p < 0.001). EImax was significantly increased in deoxygenated blood before applying 5 Pa SS (p < 0.05). However, there were no significant differences after continuous SS in oxygenated and deoxygenated blood. Deoxygenation significantly decreased SS1/2/EImax values both before and after SS applications (p < 0.01). SS1/2/EImax values in both oxygenated and deoxygenated blood were significantly decreased after 5 and 10 Pa continuous SS applications although they were not significantly decreased after applying 20 Pa SS. Our study showed for the first time that erythrocyte deformability is improved in deoxygenated conditions in contrast to results presented in previous studies. This deformability improvement may control blood flow and consequently erythrocyte distribution within hypoxic tissues. Our study also demonstrated the relationship of oxygenation‐deoxygenation shifts and magnitude of shear stress on erythrocyte deformability.

Support or Funding Information

This study was supported by The Scientific and Technological Research Council of Turkey Grant 214S186.
Protease Inhibition to Optimize the Intestinal Mucosal Barrier to Digestive Enzymes during Hemorrhagic Shock
Authors: DeLano Frank A , Zhang Hongyu , Schmid‐Schönbein Geert W
Published: November 2023

Our recent evidence indicates that escape of pancreatic digestive enzymes from the lumen into the wall of the intestine after breakdown of the mucosal barrier causes autodigestion and multiorgan failure in hemorrhagic shock. We hypothesize that initial opening of the mucosal barrier in the small intestine is mediated by matrix‐metallo proteinases (MMPs). We determined the level of pancreatic trypsin leakage into the intestine by immunohistochemistry of intestinal sections after hemorrhagic shock (35 mmHg mean arterial pressure for 2 hours). Two forms of protease inhibition were studied, single enteral inhibition with a MMP inhibitor (doxycycline, 5 mg/kg, i.v.) without and with enteral serine protease inhibition (127 mM tranexamic acid in 17 ml distributed uniformly over the small intestine of a 300 gm rat by local injections). As shown previously, the enteral inhibition of protease activity with tranexamic acid served to reduce the entry of pancreatic trypsin (detected with a mAb) across the mucosal barrier into the villi and mucosal tissue. Dual blockade with the MMP inhibitor (i.v.) and enteral serine protease inhibitor reduced the level of pancreatic trypsin in the intestinal wall to a level that was not significantly different from non‐shock controls. We detected a similar level of protection against entry of pancreatic amylase into the wall of the intestine by the combined enteral and i.v. protease inhibition. These results indicate that the irreversible opening of the mucosal barrier in hemorrhagic shock involves multiple proteases and a dual inhibition serves to minimize the leakage of digestive enzymes out of the lumen of the small intestine. The dual blockade of proteases may have clinical utility in shock patients.

Support or Funding Information

Supported by GM 85072
Age‐related activation of ADAM17/TNF in adipose tissue leads to remote coronary microvascular dysfunction in obese patients
Authors: Dou Huijuan
Published: November 2023

A disintegrin and metalloproteinase ADAM17 (TNF‐α converting enzyme) regulates soluble TNF levels. Here, we examined whether age‐related changes in adipose tissue (AT)‐expressed ADAM17 contributes to the development of coronary microvascular dysfunction (CMD) in obesity. Human coronary arterioles (CA,~100 μm) and mediastinal AT were examined in patients who underwent open‐heart surgery. CA and AT were also studied in young (6‐month) and aged (24‐month) mice fed a 12‐week high‐fat‐diet (HFD). We found that bradykinin‐induced dilation in isolated human CA significantly declines with age in obese, but not lean patients. Similarly, CA dilation was reduced in aged HFD mice when compared to young. The impaired CA dilation was accompanied by increased ADAM17 activity in AT and elevated serum TNF levels. Transplantation of AT from aged HFD to young mice increased plasma TNF levels and impaired CA dilation in the young recipient mice. In patients we found an age‐related increase in AT ADAM17 activity, which was attributed to expression of ADAM17 in the vascular endothelium of AT, but not adipocytes. Excess release of TNF from AT arteries in older patients was sufficient to impair vasodilator function in a bioassay in which the AT artery was serially connected to a CA. The present study demonstrates age‐related increases in vascular endothelial ADAM17 activity and soluble TNF release in the human and rodent AT, which may contribute to the development of CMD in older obese patients.

Support or Funding Information

AHA16PRE27550006
Direct Current Stimulation of Endothelial Monolayers Induces a Transient and Reversible Increase in Transport Due to the Electroosmotic Effect
Authors: Cancel Limary Melissa , Arias Katherin , Bikson Marom , Tarbell John
Published: November 2023

Transcranial direct current stimulation (tDCS) is currently investigated for treatment of neurological disorders, as an aid in rehabilitation, and to facilitate cognitive performance in healthy individuals. Validating the efficacy of tDCS and optimizing therapy dose requires an understanding of the underlying mechanisms. We present, for the first time, a study of the potential impact of tDCS on endothelial cells (EC) layers that form the blood‐brain‐barrier (BBB). To begin to address the possible role of the BBB in tDCS efficacy and safety, we applied 10 minutes of direct current stimulation (1mA) to monolayers of cultured endothelial cells and characterized their transport barrier response by measuring fluid and solute flux across the monolayers. We found that an applied electric field induced fluid movement across the EC layer that persisted only as long as the current was applied. Solute flux increased for 70kDa Dextran, whose transport is convectively dominated, but not for the small molecule TAMRA, which easily diffuses across the monolayer. In addition we found that the direction of transport could be reversed by changing the polarity of the electric field – a hallmark of the electroosmotic effect. A mathematical model based on a two pore description of the endothelial transport barrier describes the experimental data accurately and predicts enhanced significance of this mechanism in tighter (less permeable) monolayers. This study shows that tDCS relevant currents transiently alter the transport function of the BBB suggesting a new mechanism that may play a role in the efficacy of tDCS.
Water extract of Imperatae Rhizom induces vascular relaxation via NO‐cGMP‐PKG signaling
Authors: Kim Hye Yoom , Ahn You Mee , Jin Xian Jun , Hong Mi Hyeon , Kim Hyun Ju , Yang Sun Nye , Lee Yun Jung , Lee Ho Sub , Kang Dae Gill
Published: November 2023

The purpose of the present study was to investigate the underlying cellular mechanisms of the nitric oxide (NO)‐mediated property of the aqueous extract of Imperatae Rhizoma (AIR), pronounced anti‐inflammatory herb, in vascular endothelium. The roles of the NO signaling in the AIR‐induced effects were tested in human umbilical vein endothelial cells (HUVECs) and aortic ring. HUVEC treated with AIR produced higher amount of NO compared to control. AIR increased in the expression and phosphorylation levels of eNOS and Akt in HUVECs, which were attenuated by L‐NAME, a NOS inhibitor, and wortmannin, a PI3K inhibitor. mRNA or protein levels of guanosine triphosphate cyclohydrolase I (GTPCH) was upregulated in HUVECs treated with AIR, suggesting a role of eNOS coupling. In addition, AIR‐induced dose‐dependent relaxation of phenylephrine‐precontracted aorta was abolished by removal of functional endothelium. Pretreatment with L‐NAME, and ODQ inhibited the AIR‐induced vasorelaxation. Wortmannin and LY‐294002 an upstream signaling molecule of eNOS, attenuated the AIR‐induced vasorelaxation. Incubation of endothelium‐intact aortic rings with AIR increased the production of cGMP, however, which were attenuated by L‐NAME and ODQ. Two‐kidney‐one‐clip (2K1C) hypertensive rats were established to investigate the hypotensive effect of AIR in renovascular hypertension. 2K1C rats were treated with AIR at dose of 100 mg/kg/day orally for 3 weeks. AIR significantly lowered blood pressure. Interestingly, AIR ameliorated both endothelium‐dependent and independent vascular relaxation in the phenylephrine‐precontracted thoracic aorta. 2K1C‐induced hypertension model increased plasma renin activity, however, AIR attenuated those activities. Taken together, the present study suggests that AIR ameliorates vascular dysfunction via the regulation of renin‐angiotensin system and endothelium‐dependent activation of through the PI3K/Akt‐mediated NO‐cGMP‐PKG signaling.

Support or Funding Information

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (2008‐0062484).
Myogenic Oscillations during Alpha‐2 agonist Administration in Humans
Authors: Talke Pekka O , Diab Mohamed
Published: November 2023

Dexmedetomidine is a highly selective alpha‐2 adrenergic agonist. While studying peripheral vasomotor effects of alpha‐2 agonists in humans, we observed unique, low frequency, large amplitude oscillations in the infrared photoplethysmograph (PPG) signal recorded from fingertips. We observed these oscillations in both awake and anesthetized subject but only during dexmedetomidine infusions. The purpose of this study was to determine if the origin of these vascular oscillations is central or if they are mediated locally in the fingers (i.e. myogenic oscillations). Infrared PPG data were recorded at 62.5 Hz from six surgical patients during nitrous‐narcotic general anesthesia. All patients received a continuous dexmedetomidine (1.4 ± 0.6 μg/kg/hr) infusion as part of the anesthetic. PPG data were recorded simultaneously from distal phalanxes of right and left index, middle and ring fingers throughout surgery. All sensors were optically isolated to prevent crosstalk. Data were analyzed offline. After visual inspection of the PPG data of each subject, a 5 min artifact free epoch during which PPG from all six sensors displayed low frequency oscillations was selected for analysis. For frequency analysis of these oscillations, PPG data were band pass filtered (0.01–0.5 Hz) followed by Chirp Z‐Transform spanning 0–0.25 Hz with 0.0005 Hz resolution. Perfusion index (PI) defined as PI=AC/DC*100% of the PPG was determined for each cardiac pulse. PI data were used to analyze the amplitude of the low frequency oscillations. Low frequency PPG oscillations were recorded from all six sensors of all six subjects during majority of dexmedetomidine infusion. The Chirp Z‐Transform analysis showed two distinct frequency peaks. One was at the frequency of the mechanical ventilator. The second frequency peak had a significantly larger amplitude. It's minimum and maximum frequencies during the study were 0.021 and 0.037 Hz respectively. In each subject these oscillations had similar but different frequencies in all six fingers, i.e. none of the oscillations were synchronous in any of the subjects. The average frequencies of these oscillations from each subject were 0.028 ± 0.005 Hz. The average amplitude (% modulation) of the oscillations was 19 ± 3 %. In conclusion, we observed low frequency, high amplitude PPG oscillations in all six fingers of every subject during dexmedetomidine infusion. Centrally mediated oscillations would have been expected to be bilaterally synchronous and in phase. Oscillations originating from large arteries (brachial, ulnar, radial) would have been expected to show some synchrony within the three sensors in each hand. Thus, the asynchrony of the oscillation suggests local origin (myogenic oscillations).

Support or Funding Information

Supported by departmental funds
Amyloid Beta Peptides and Inflammatory Cytokines Depress Human Brain Vascular Smooth Muscle Tonic Contractility: Relevance to Alzheimer's Disease Impaired Cerebral Autoregulation
Authors: Yun J. Winny , Washington Caretia , Minagar Alireza , Alexander J. Steve
Published: November 2023

Alzheimer's Disease (AD) is a neurodegenerative disorder that is characterized both by the presence of tau protein neurofibrillary tangles and amyloid beta (Aβ) containing extracellular plaques. AD is also a neuroinflammatory condition in which Th1 cytokines are elevated and appear to contribute to the intensification of AD severity. The cleavage of amyloid precursor protein (APP) can result in the release of several different forms of Aβ. Although Aβ toxicity to neurons has been described extensively, stressful effects of Aβ peptides on components of the neurovasculature such as endothelial and smooth muscle cells have been less well characterized despite AD exhibiting impaired vascular properties. In particular, cerebral autoregulation, the process by which blood flow is controlled despite alterations in feeding pressure, is disturbed in AD. The failure of autoregulation in AD may make the brain susceptible to cerebral microbleeds through a reduced capacity to limit blood flow when pressure is increased. Conversely, reduced vasodilation during low flow could also exacerbate tissue hypoxia. Currently, whether and how Aβ peptides and inflammatory cytokines depress brain smooth muscle cell tonic contraction is not known, but could reveal important targets in the preservation of autoregulation which is disturbed in AD. We used a collagen contractility assay to evaluate the influence of Aβ 25–35, Aβ 1–40 and Aβ 1–42 peptides on the contractility of human brain vascular smooth muscle (HBVSM) as an in vitro model of cerebral autoregulation. We found that Aβ 1–42 significantly depressed HBVSM contractility at 5 and 10μM while 5–20μM Aβ 1–40 did not affect contractility. Conversely Aβ 25–35 increased tonic contractility at 1, 5, 10 and 20μM. Interestingly, the inflammatory cytokines TNFα (20ng/ml), IL‐1β (20ng/ml) and IFNγ (1000U/ml) also depressed HBVSM contractility. These data suggest that both the inflammatory milieu seen in AD as well as the abundance of Aβ peptides may lead to autoregulatory failure and susceptibility to cerebral microbleeds which are an important characteristic of AD.

Support or Funding Information

Supported by Malcolm Feist Cardiovascular Center Predoctoral Fellowship and Annette Funicello Research Foundation
KATP Channels Mediate CGRP‐Dependent Hyperpolarization of Mouse Pulmonary Artery Smooth Muscle and Endothelium
Authors: Norton Charles E , Segal Steven S
Published: November 2023

Calcitonin gene related peptide (CGRP) hyperpolarizes membrane potential (Vm) of smooth muscle cells (SMCs) and endothelial cells (ECs) in pulmonary arteries (PAs) (FASEB J. 30, 945.18). In the systemic circulation, protein kinase A (PKA) can phosphorylate ATP‐sensitive potassium channels (KATP) as a mechanism of vasodilation to CGRP. We hypothesized that this signaling pathway is effective in the pulmonary circulation. Intact PAs were isolated (internal diameter, ~150 μm) from male C57BL/6J mice (age, 3 months), cannulated, pressurized to 16 cm H2O, and studied at 37°C. Alternatively, intact endothelial tubes were studied at 32°C following dissociation of SMCs. Vm was recorded continuously using intracellular microelectrodes. At rest, Vm was −43±2 mV for SMCs of intact PAs and −38±1 mV for ECs in endothelial tubes. Increasing concentrations (1 nM to 10 μM) of pinacidil (KATP activator) hyperpolarized SMCs by 38±2 mV (to −80±3 mV) and ECs by 36±3 mV (to −73±3 mV); respective EC50 values were 72 and 40 nM (n=4/group). Hyperpolarization of SMCs and ECs with 10 μM pinacidil was reversed by glibenclamide (KATP inhibitor) in a concentration‐dependent manner, with complete reversal at 1 μM. Increasing [CGRP] from 0.1 nM to 1 μM hyperpolarized SMCs of intact PAs by 17±3 mV (to −52±3 mV) and ECs by 20±4 mV (to −57±3 mV). With no effect on resting Vm, 1 μM glibenclamide attenuated (P<0.05) hyperpolarization of both SMCs (4±1 mV; n=5) and ECs (5±3 mV; n=4) to 1 μM CGRP. Inhibiting PKA with protein kinase inhibitor [PKI (14–22) amide, myristoylated; 10 μM] attenuated hyperpolarization to 1 μM CGRP in SMCs (5±1 mV; n=3) and in ECs (4±2 mV; n=4). In PAs preconstricted to ~50% of resting diameter with 5 μM uridine triphosphate, peak dilation to 1 μM CGRP was 87±4% (n=5) and 1 μM glibenclamide attenuated this effect to 14±5% (n=4). We conclude that CGRP‐dependent hyperpolarization of pulmonary arterial SMCs and ECs is mediated by KATP channel activation via PKA, leading to vasodilation. These findings provide mechanistic insight into therapies for vascular diseases associated with impaired pulmonary perfusion.

Support or Funding Information

Support: NIH R37HL041026
PIP2‐Regulation of Endothelial KIR2.1 Channels in Cerebral Arteries
Authors: Sancho Maria , Welsh Donals G.
Published: November 2023

In the cerebral circulation, inwardly rectifying potassium (KIR) channels are robustly expressed in both endothelial and smooth muscle cells. This pattern of expression is unusual for vascular cells that are electrically coupled and it suggests that each pool is differentially regulated driving a distinct subset of physiological responses. In this context, this study characterized the electrical/molecular properties of KIR2.x channels in cerebral arteries and ascertained the importance of phosphatidylinositol‐bis‐phosphate (PIP2), a regulatory phospholipid, in modulating each channel pool. Endothelial and smooth muscle cells were freshly isolated from rat cerebral arteries; patch clamp electrophysiology and Q‐PCR were used to ascertain KIR activity and expression, respectively. Our electrophysiological results highlight that a Ba2+‐sensitive KIR current is present and stable in both cell types. Endothelial current density was ~8 fold greater than smooth muscle (−20 vs −2.5 pA/pF at −100mV) and Q‐PCR revealed that KIR2.x mRNA subunits were present in both cell types, albeit it at variable levels. Intriguingly, KIR channels in endothelial but not smooth muscle cells were PIP2 sensitive; neomycin impaired, and exogenous PIP2 elevated channel activity. In summary, our observations indicate that while endothelial and smooth muscle both expressed KIR channels, they are uniquely regulated by PIP2. A complete picture of the expression/regulation of KIR channels within the vascular wall will provide further advances in our understanding of their role in cerebral blood flow control in health and disease.

Support or Funding Information

Canadian Institute of Health Research (CIHR)
The Role of NADPH Oxidase in Regulating Cerebral Vascular Myogenic Tone
Authors: Mazumdar Neil , Jalali Hanna , Hashad Ahmed Mohamed , Welsh Donald Gordon
Published: November 2023

The myogenic response refers to the intrinsic ability of arteries to constrict to elevated pressure. The underlying mechanism has yet to be elucidated but recent evidence suggests that the angiotensin II type 1 receptor (AT1R) is a key mechanosensor, linking intravascular pressure to tone development. One intriguing aspect of this receptor is its ability to activate NADPH oxidase (Nox), an enzyme responsible for the production of reactive oxygen species. The goal of this study was to ascertain the role of Nox in myogenic tone development. Briefly, rat cerebral arteries were mounted in a myograph, pressurized to 60mmHg and diameter monitored prior to and following the application of apocynin (a general Nox inhibitor) or ML171 (a Nox1 specific inhibitor). Myogenic constriction progressively rose as vessels were pressurized from 20 to 60 mmHg. These pressure‐induced responses were attenuated by apocynin in a concentration dependent manner (37% vs 24% at 60 mmHg) irrespective of whether the endothelium was present or absent. ML171 (30μM) was equally effective at moderating the myogenic response (36% vs 24%); control experiments showed no discernible role for Nox2. Similar attenuating effects were observed in human cerebral arteries. These findings indicate Nox1 is expressed in rat cerebral arterial smooth muscle and may be a regulator of pressure‐induced responses. While the precise downstream mechanism has yet to be resolved, the L‐type Ca2+ channel is a likely target of Nox regulation. This work sheds new light on mechanosensation and how unique signaling proteins impact the ability of resistance arteries to respond to intravascular pressure.

Support or Funding Information

This work is supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) for DG Welsh. DG Welsh is a Rorabeck chair in molecular neuroscience and vascular biology at the University of Western Ontario. AM Hashad is a Vanier scholar (CIHR) and was supported by a salary studentship from Alberta Innovates Health solutions (AIHS).
Altered Distribution of Adrenergic Constrictor Responses Contributes to Skeletal Muscle Perfusion Abnormalities in Metabolic Syndrome
Authors: Lemaster Kent , Jackson Dwayne , Welsh Donald , Brooks Steven , Chantler Paul , Frisbee Jefferson
Published: November 2023

Although studies have suggested elevations in adrenergic traffic, signaling and vascular responses with development of metabolic syndrome in obese Zucker rats (OZR), the moderate hypertension and modest impact on organ blood flow in OZR questions the multi‐scale validity of these data. To better understand the role of adrenergic function in contributing to vascular reactivity in OZR, we utilized a multi‐scale approach to investigate pressure responses, skeletal muscle blood flow and vascular reactivity in OZR following adrenergic challenge. For OZR under in vivo conditions, adrenergic challenge resulted increased pressor responses vs. LZR that were mediated via α1 receptors, with minimal contribution by either ROS or NO bioavailability. In situ gastrocnemius muscle of OZR exhibited blunted hyperemic responses to elevated metabolic demand, partially restored with α1 inhibition, although improved muscle performance and VO2 required combined treatment with TEMPOL as well. Within in situ cremaster muscle, proximal resistance arterioles of OZR exhibited a more heterogeneous constrictor response to adrenergic challenge, biased toward hyperresponsiveness, as compared to that in LZR. This increasingly heterogeneous pattern was mirrored in ex vivo resistance arterioles as well, mediated via α1 receptors, with roles for ROS and NO bioavailability evident in hyperresponsive vessels only. These results support observations of the central role of the α1 adrenoreceptor for augmented pressor responses and elevations in vascular resistance, but identify an increased heterogeneity of constrictor reactivity with development of metabolic syndrome that is presently of unclear purpose.

Support or Funding Information

National Institutes of Health and American Heart Association
Effect of aging on vascular smooth muscle contractility in skeletal muscle resistance arteries
Authors: Woodman Christopher , Seawright John , Shin Song Yi , Trache Andreea
Published: November 2023

Objective

We tested the hypothesis that vascular smooth muscle contractility declines with age in skeletal muscle resistance arteries.

Methods

Soleus muscle feed arteries (SFA) from young (4 mo) and old (24 mo) Fischer 344 rats were cannulated with glass micropipettes and pressurized to 90 cmH2O for 1 h. To isolate the role of vascular smooth muscle, SFA were denuded (endothelium removed) by passing 5 ml of air through the lumen of the artery. At the end of the 1 h equilibration period, contractile responses were assessed using increasing whole log doses of norepinephrine (NE; 10−9–10−4 M), angiotensin II (Ang II; 10−11–10−7 M), and phenylephrine (PE; 10−9–10−4 M).

Results

Vasoconstrictor responses NE, Ang II, and PE were significantly impaired in endothelium intact old SFA compared to young SFA. In contrast, vasoconstrictor responses to NE, Ang II, and PE were similar in young and old denuded SFA.

Conclusion

These results indicate that the contractile properties of vascular smooth muscle cells are preserved with age and that impaired constrictor responses are manifest only when the endothelium is intact.

Support or Funding Information

Research support: AHA grant 0765043Y, AHA grant 4150031, The National Space Biomedical Research Institute through NCC 9–58 and a Sydney and J.L. Huffines Institute of Sports Medicine Graduate Student Research Grant.
Presence of Hysteresis in the Cerebral Pressure‐Flow Relationship During Repeated Squat‐Stand Maneuvers in Humans
Authors: Brassard Patrice , Ferland‐Dutil Hélène , Smirl Jonathan David , Paquette Myriam , Le Blanc Olivier , Malenfant Simon , Ainslie Philip Neil
Published: November 2023

In humans, the cerebrovasculature is more efficient at compensating for transient hypertension compared with transient hypotension – a phenomenon called hysteresis. To date, however, these asymmetrical responses of cerebral blood flow (CBF) in response to changes in blood pressure have been reported using cyclic inflation and deflation of thigh cuffs in patients with head injury or via pharmacological interventions in healthy participants. Whether this phenomenon exists during non‐pharmacologically and physiologically‐induced transient hypertension and hypotension is unknown. In this study, we compared the relative change in CBF, as indexed by mean velocity in the middle cerebral artery (MCAvmean; transcranial Doppler), per relative change in mean arterial pressure (MAP; finger photoplethysmography) (%MCAvmean/%MAP) during transient hypertension and hypotension. These changes in MAP were reliability and repetitively induced during 5 minutes of repeated squat‐stand maneuvers performed at 0.05 Hz (10‐second squat–10‐second stand) and 0.10 Hz (5‐second squat–5‐second stand) in 64 healthy participants (Age [(mean±SD)] 32±14 yrs). These large oscillations in MAP (~25–30 mmHg) are transmitted to the cerebrovasculature when executed at frequencies within the high‐pass filter buffering range (<0.20 Hz). However, the results revealed the %MCAvmean/%MAP was attenuated by 26% ([median (range)]: 0.96 (−3.79–5.22) vs. 1.29 (−1.12–8.55) %/%; p=0.02] and 47% [0.92 (−2.93–3.26) vs. 1.74 (−0.29–9.38) %/%; p=0.0001] during transient hypertension compared to transient hypotension performed at 0.05 and 0.10 Hz, respectively. These findings suggest that the human cerebrovascular function is better adapted to compensate for physiologically relevant transient hypertension than transient hypotension.

Support or Funding Information

This research was supported via the Ministère de l'Education, du Loisir et du Sport du Quebec (PB), the Foundation of the Institut universitaire de cardiologie et de pneumologie de Quebec (PB), and an NSERC Discovery grant (PNA). PB was a Junior 1 Research Scholar of the Fonds de recherche du Québec – Santé.JDS was supported via an NSERC PGS‐D2 Fellowship and the Killam Pre‐Doctoral Fellowship. MP was supported via a scholarship from CIHR.
High Fat Feeding Reduces Cerebral Artery Transient Receptor Potential Channel Vanilloid 4 (TRPV4) Expression In A Mineralocorticoid Receptor‐Dependent Manner
Authors: Dorrance Anne McLaren , Diaz‐Otero Janice , Pires Paulo W , Jackson William F
Published: November 2023

Overweight/obese patients are at an increased risk of developing cognitive impairment associated with chronic cerebral hypoperfusion. We have developed an overweight rat model that exhibits impaired cerebral artery endothelial function and cognitive decline. The mechanisms responsible for the impaired cerebral artery dilation have not been determined. Recent studies from our laboratory have identified the transient receptor potential channel (TRP) vanilloid 4 (TRPV4) as a key regulator of cerebral artery endothelium‐dependent dilation. We therefore hypothesized that TRPV4 mRNA expression would be reduced in cerebral arteries from overweight rats. Cerebral arteries were collected from overweight rats fed a high fat (HF) diet for 17 weeks from 3 weeks of age; these were compared to arteries from control chow fed rats (n=5 in each group). qRT‐PCR with TAQMAN primers was used to assess TRPV4 expression, TRPV3 and TRPA1 expression were also assessed. HF feeding specifically reduced TRPV4 mRNA expression (fold change from control = 0.58±0.11; p<0.01), while it had no effect on TRPV3 (fold change from control = 1.144±0.28; p=0.6) or TRPA1 (fold change from control = 1.08±0.2; p=0.7) mRNA expression. Circulating levels of aldosterone are increased in the overweight population, to test if this plays a role in the reduced TRPV4 expression observed with HF feeding we treated rats with the mineralocorticoid receptor (MR) antagonist canrenoic acid (CAN 20mg/kg/day n=3) for the duration of the HF feeding, these were compared to placebo treated HF fed rats (n=5). MR antagonism increased the mRNA expression of TRPV4 (fold change from HF placebo treated = 2.012±0.24; p<0.01). CAN treatment also increased the expression of doublecortin, a marker of new and immature neurons (fold change from HF placebo treated = 2.184±0.37; p<0.01) and synaptophysin a marker of synapse formation (fold change from HF placebo treated = 2.90±1.05; p<0.01). Taken together these studies suggest the MR antagonism improves expression of TRPV4, a key regulator of cerebral artery dilation. Augmented expression of TRPV4 may improve cerebral perfusion, and may reduce the cognitive decline observed in the overweight population.

Support or Funding Information

Supported by 5P01HL070687‐12.
Cerebrovascular Reactivity in Atrial Fibrillation
Authors: Fisher James P , Braz Igor Dutra , Lucas Samuel J E , Edwards Louisa , van Lieshout Johannes J , Lip Gregory Y H
Published: November 2023

Atrial fibrillation is associated with substantial risk of severe stroke, cognitive decline and dementia. Elevated plasma biomarkers of vascular damage/dysfunction and reduced conduit (brachial) artery function have been reported in atrial fibrillation. We sought to test the hypothesis that patients with atrial fibrillation have a reduced cerebrovascular reactivity to carbon dioxide (CO2) in comparison with age‐matched, healthy control and hypertensive participants.

Middle cerebral artery flow velocity was measured at rest and during step‐wise changes in partial pressure of end‐tidal CO2 (PETCO2) in 14 patients with atrial fibrillation (median [IQR]; 66.5 [59.5–69.3] yr, 3 women), 8 patients with hypertension (65.5 [63.5–71.5] yr, 4 women) and 10 healthy control participants (67.5 [66–70] yr, 4 women). Incremental hypercapnia was delivered via the open circuit steady‐state method, with stages of 4% and 7% CO2. Incremental hypocapnia was comprised of two 2‐min stages of voluntary hyperventilation to reduce PETCO2 in an equal but opposite direction to that observed during hypercapnia. Cerebrovascular reactivity was calculated as the change in middle cerebral artery mean flow velocity, per mmHg change in PETCO2.

Resting middle cerebral artery mean flow velocity tended to be lower in patients with atrial fibrillation (mean [SD]; 54.4 [14.5] cm/s) and hypertension (54.6 [13.0] cm/s), when compared to healthy controls (65.9 [14.4] cm/s) (P=0.127). Hypercapnic cerebrovascular reactivity was ≈50% lower in patients with atrial fibrillation (median [IQR]; 1.7 [1.5–2.4] cm/s/mmHg) and ≈22% lower in patients with hypertension (2.7 [2.2–3.1] cm/s/mmHg), compared to healthy controls (3.4 [2.8–3.8] cm/s/mmHg) (P<0.05). Hypocapnic cerebrovascular reactivity was not significantly different between groups (P>0.05).

In summary, atrial fibrillation patients exhibit blunted hypercapnic cerebrovascular reactivity when compared to healthy control participants. Such impairments in cerebrovascular reactivity may contribute to the well‐established increased risk of cerebrovascular events in this patient group.

Support or Funding Information

Funded by BHF project grant PG/15/45/31579.