bluuebunny/crossref_metadata_2025_split huggingface dataset:
Oncostatin M Induces Barrier Dysfunction in Human Brain Microvascular Endothelium via Snail‐Dependent Repression of TAMPs
Authors: Wertz Travis S , Hoettels Brian A , Tawara Ken , Oxford Julia T , Jorcyk Cheryl L , Beard Richard S 
Published: November 2023

Homeostasis of barrier integrity in brain microvessels is maintained by dynamic regulation of endothelial cell‐cell adhesive proteins. During aberrant inflammatory responses, a host of factors act upon the endothelium to modify these proteins and diminish barrier integrity, leading to leakage of plasma components, neurotoxic substances, and potentially edema. The pleiotropic cytokine oncostatin M (OSM) is one such factor, which has been shown to be elevated in several inflammatory conditions, such as multiple sclerosis, that are associated with increased microvascular leakage. Indeed, recent evidence suggests that OSM may directly induce brain endothelial cell barrier dysfunction, but the mechanisms remain unclear. The objective of this study was to determine the effect of OSM on the barrier integrity of human brain microvascular endothelial cells (BMVECs). First, we confirmed that BMVECs were responsive to OSM by measuring activation of STAT3, which is known to be downstream of OSM receptor signaling in other cell types. Indeed, OSM triggered phosphorylation of STAT3 at Y705 in BMVECs as early as 15 minutes following stimulation. Next, we identified that treating BMVEC monolayers with OSM promoted barrier dysfunction, as demonstrated by time‐and‐dosage‐dependent decreases of transendothelial electrical resistance (TER). Concomitant with TER changes, OSM induced a significant decrease in the tight‐junction‐associated MARVEL proteins (TAMPs) occludin and tricellulin. Interestingly, OSMs effect on downregulation of endothelial cell‐cell junctional proteins appears to be exclusive to TAMPs, as there were no noticeable changes in claudin‐ 1, 3, 5, 12 or the adherens junction protein VE‐cadherin. Since it has previously been identified in epithelial cells that occludin can be negatively regulated by the transcription factor SNAI1, or Snail, we evaluated a potential role of Snail in OSM‐mediated barrier dysfunction of BMVECs. At baseline, there was minimal detection of Snail; however, OSM induced a robust upregulation of Snail protein preceding the onset of barrier dysfunction. Finally, siRNA‐mediated knockdown of SNAI1 both increased baseline levels of TAMPs and attenuated OSM‐induced TAMP downregulation and BMVEC barrier dysfunction. In conclusion, elevated OSM induces human brain endothelial barrier dysfunction in a Snail‐dependent manner, and further exploration of this signaling pathway in brain endothelium holds multiple facets of therapeutic potential.Support or Funding InformationSupported by The Birt Family and NIH grants: P20 GM109095 and 2P20GM103408.
Ginsenoside Rb1 protects against ischemia/reperfusion‐induced myocardial injury via energy metabolism regulation mediated by RhoA signaling pathway
Authors: Han Jing‐Yan , Cui Yuan‐Chen , Pan Chun‐Shui , Yan Li , Hu Bai‐He , Chang Xin , Liu Yu‐Ying , Sun Kai , Li Quan , Fan Jing‐Yu 
Published: November 2023

Cardiac ischemia and reperfusion (I/R) injury remains a challenge for clinicians. Ginsenoside Rb1 (Rb1) has been reported to have the ability to attenuate I/R injury, but its effect on energy metabolism during cardiac I/R and the underlying mechanism remain unknown. In this study, we detected the effect of Rb1 on rat myocardial blood flow, myocardial infarct size, cardiac function, velocity of venule red blood cell, myocardial structure and apoptosis, energy metabolism and change in RhoA signaling pathway during cardiac I/R injury. In addition, the binding affinity of RhoA to Rb1 was detected using surface plasmon resonance (SPR). Results showed that Rb1 treatment at 5 mg/kg/h protected all the cardiac injuries induced by I/R, including damaged myocardial structure, decrease in myocardial blood flow, impaired heart function and microcirculation, cardiomyocyte apoptosis, myocardial infarction and release of myocardial cTnI. Rb1 also inhibited the activation of RhoA signaling pathway and restored the production of ATP during cardiac I/R. Moreover, SPR assay showed that Rb1 was able to bind to RhoA in a dose‐dependent manner. These results indicate that Rb1 may prevent I/R‐induced cardiac injury by regulation of RhoA signaling pathway, and may serve as a potential regime to improve percutaneous coronary intervention outcome.Support or Funding InformationAPS
Adipose Tissue – Skeletal Muscle Crosstalk: Obese Human Subcutaneous and Visceral Adipose Tissues Both Supress Muscle Insulin Signalling in Men and Women
Authors: Sarr Ousseynou , Strohm Rachel J. , MacDonald Tara L. , Reed John K. , Foute‐Nelong Jules , Dyck David J. , Mutch David M. 
Published: November 2023

Adipose tissue is an important endocrine organ that communicates with other peripheral tissues via the secretion of proteins, termed adipokines. In particular, altered secretion of specific adipokines (e.g., TNF‐α and adiponectin) has previously been shown to regulate skeletal muscle insulin sensitivity. For the current study, we established a human adipose tissue – muscle crosstalk model to investigate the impact of different adipose tissue depots on skeletal myocyte insulin signaling, and if this differs between men and women. Visceral (VAT) and subcutaneous (SAT) adipose tissue samples were collected from obese men and women during laparoscopic bariatric surgery. Adipose tissue samples were subsequently cultured for 48h and secretion media was collected. Secretion media was then transferred on to human skeletal myocytes derived from healthy, non‐diabetic male and female donors for 24h. Content of insulin signaling proteins (phosphorylated and total AKT) in mature myocytes incubated with either control M199 media or secretion media from VAT or SAT was assessed using Western Blotting in basal and insulin‐stimulated conditions (100uU/well). Adipokines in secretion media were measured with a multiplex immunoassay. In men, VAT had higher interleukin‐6 gene expression compared to SAT (p=0.004), while no differences were seen in women (p=0.271). Adiponectin and TNF‐α gene expression were not different between VAT or SAT for men or women. Secretion media collected from male SAT and VAT reduced p‐AKTThr308 activation in insulin‐stimulated myocytes compared to controls (p <0.01). Interestingly, myocytes incubated with VAT secretion media, compared to SAT secretion media, showed a significant reduction in p‐AKTSer473 in men after insulin stimulation (p<0.01). Secretion media collected from female VAT and SAT caused significant reductions in p‐AKTThr308 and p‐AKTSer473 activation following insulin stimulation, compared to controls (p<0.01). Secretion media from VAT or SAT for men or women did not affect total‐AKT levels. Independent of sex, 14 out of 18 detected adipokines (i.e., a panel consisting of cytokines, chemokines and growth factors) were more abundant in VAT secretion media compared to SAT secretion media (p<0.01). Only interleukin‐21 levels showed a sex difference, with higher levels detected in secretion media from female adipose tissue (p=0.003). In conclusion, SAT and VAT secretion media from obese men and women suppress insulin signaling in myocytes to a similar extent. These results reveal the power of this human adipose tissue – muscle model to investigate crosstalk between these two tissues.Support or Funding InformationThis work was supported by the Natural Sciences and Engineering Research Council of Canada.
Changes in Plasma β‐Adrenergic Receptor Kinase‐1 Following Left Ventricular Assist Device Placement
Authors: Denfeld Quin , Lee Christopher S. , Woodward William R. , Habecker Beth A. 
Published: November 2023

As a result of chronic sympathetic stimulation, β‐adrenergic receptor kinase‐1 (βARK1) is upregulated in human heart failure (HF). It is unknown, however, how plasma βARK1 changes following unloading of the heart through left ventricular assist device (LVAD) therapy, and moreover, the relationship between plasma βARK1 and patient‐oriented outcomes (i.e. health‐related quality of life (HRQOL)). The aim of this study was to quantify changes in plasma βARK1 expression following LVAD placement. This was a secondary analysis of plasma samples collected from a subset of patients with HF pre‐ and post‐ (one‐, three‐, and six‐months) LVAD placement. βARK1 was measured using a commercially‐available ELISA kit. In a sample of patients (n = 10) with known improvements in HRQOL over 6 months, average βARK1 was 11.8±5.5 pg/mL (picograms/milliliter, ±SEM) at baseline (pre‐LVAD). βARK1 was increased at 1 month (40.4±16.3 pg/mL) and 3 months (38.4±11.4 pg/mL) but decreased closer to baseline at 6 months (20.9±6.2 pg/mL). In a small subset of patients with known improvements in HRQOL, these results indicate βARK1 significantly increases following LVAD placement but returns close to baseline six months post‐LVAD placement.Support or Funding InformationNational Institutes of Health/National Heart, Lung, & Blood Institute 5T32HL094294 (Denfeld); National Institutes of Health/National Institute of Nursing Research R01NR013492 (Lee); National Institutes of Health/National Heart, Lung, & Blood Institute HL093056 (Habecker)
The superposition of work on a pathologic microenvironmental stiffness may regulate actin assembly via phosphorylation and sarcomeric redistribution of PKCɛ
Authors: Mkrtschjan Michael , Wondmegegn Admasu , Arena Anthony , Li Jieli , Russell Brenda 
Published: November 2023

Moderate exercise benefits the heart, but chronic demand leads to failure. Thus, acute and chronic loading may drive cell contractility and cell hypertrophy differentially. We test the hypothesis that, in response to changes in demand, the actin assembly is regulated through PKCɛ signaling to alter cell hypertrophy. Cultured neonatal rat ventricular myocytes (NRVMs) were chronically stressed by increased stiffness of the microenvironment, resembling tissue fibrosis in diseased hearts. Physiologic or pathologic work load was mimicked by one‐hour or 24‐hour treatment with 2.5 μM isoproterenol, respectively. NRVMs were plated on polyacrylamide (PAA) or polydimethylsiloxane (PDMS) substrata mimicking the stiffness of normal (10 kPa) or diseased (100 kPa) hearts, in addition to plating directly on glass (>1 GPa). Cell area increased with increasing stiffness following three‐day culture but not with 24‐hour isoproterenol treatment. Contractility was assessed by beat frequency and time to peak tension by line‐scan kymographs, increasing with substrata stiffness and also with one or 24‐hour isoproterenol treatment. Actin dynamics, as assessed by fluorescence recovery after photobleaching (FRAP) using actin‐GFP, were altered by changes in work load. Both chronic stiffness and one‐hour isoproterenol treatment increased kFRAP to much higher values of ≈0.00075 s−1 on glass or on 100 kPa with isoproterenol treatment compared to untreated cells on 100 kPa, ≈0.0004 s−1 (P<0.05, n≥10 cells). To seek a mechanistic basis for regulation of actin assembly and cell hypertrophy, GFP‐PKCɛ positive NRVMs were observed using time lapse imaging over 1 hour. PKCɛ translocated to the sarcomere within minutes, was sustained for approximately 30 minutes, and declined by one hour. Immunostaining confirmed that translocated PKCɛ is localized with alpha‐actinin at the Z‐band. In order to study whether PKCɛ phosphorylation was necessary, we used a phospho‐specific PKCɛ antibody on fixed cells, which showed an increased Z‐disk localization between alpha‐actinin and phospho‐PKCɛ at 15 min as quantified by Pearson's coefficient (P<0.05), but this difference dissipated by 1 hour. Since the PKCɛ translocation was transient and actin assembly was sustained, hypertrophy may be triggered by the arrival of phospho‐PKCɛ at the Z‐band. The superposition of work on a pathologic microenvironmental stiffness may regulate actin assembly and maladaptive hypertrophy by localization of signaling molecules. This basic research may help understanding of hypertrophy found in heart failure in many cardiac disease states.Support or Funding InformationHL 62426.
Endothelial specific epithelial sodium channel activation prompts vascular stiffness in female mice
Authors: Jia Guanghong , Aroor Annayya R , Barron Brady J , Manrique Camila , Lastra Guido , Whaley‐Connell Adam , Meininger Gerald A , Jaisser Frederic , Sowers James R 
Published: November 2023

Our research suggests that enhanced endothelium mineralocorticoid receptor (ECMR) signaling induces endothelium epithelial sodium channel (EnNaC) activation to promote vascular stiffness. This occurs, in part, by reduction of endothelial nitric oxide (NO) synthase (eNOS) activity and bioavailable NO. This study demonstrated that consumption of a Western Diet (WD) containing excess fat and refined sugar for 16 weeks increases aldosterone level and MR activation resulting in impaired NO mediated vascular relaxation and aortic stiffness in females. These abnormalities were prevented by low doses of the MR antagonist, spironolactone. As ECMR KO also prevented these abnormalities we posited that ECMR/EnNaC activation was critical. Accordingly, we hypothesized that EC‐specific EnNaC activation would mediate vascular stiffness and impair flow‐mediated vasodilation through reduction of bioavailable NO. Four week old C57BL6/J mice were fed a WD containing high fat (46%), sucrose (17.5%), and high fructose corn syrup (17.5%) with or without a very low dose of amiloride (1 mg/kg/day) for 16 weeks. Female EnNaC KO (EnNaC−/−) and wild‐type littermate females were treated with aldosterone (250 μg/kg/day) via osmotic minipumps for 3 weeks. Amiloride, an antagonist for EnNaC for 16 weeks, could significantly attenuate WD‐induced increases in aortic stiffness in vivo as measured by pulse wave velocity and in vitro endothelial stiffness measured by atomic force microscopy. Meanwhile, amiloride further improved flow mediated dilation in mesenteric arteries and endothelium‐dependent relaxation with acetylcholine (10−9–10−4 mol/L) stimulation in wire myograph. Importantly, EnNaC KO mice also prevent aldosterone‐induced endothelium stiffness and impairment of endothelium‐dependent relaxation. These data suggest that the sequence of activated ECMR leading to cell specific EnNaC activation and signaling decreases bioavailable NO. This cascade plays a key role in the development of vascular stiffness and dysfunction.Support or Funding InformationNIH (R01 HL73101‐01A and R01 HL107910‐01) and the Veterans Affairs Merit System (0018) to Dr. Sowers
Shear Stress Induces Apoptosis via Cytochrome C Release from Dynamic Mitochondria in Endothelial Cells
Authors: Hu Ying‐Li , Hur Sung Sik , Lei Lei , Wang Yingxiao , Chien Shu 
Published: November 2023

Mechano‐sensors on endothelial cells (ECs) detect shear stress and transduce it into biochemical signals to trigger vascular adaptive responses. The variations in shear stress patterns can result in different consequences of mechanotransduction and propensity for atherosclerosis. In the regions of disturbed, oscillatory flow with low shear stress, excess cell turnover generates spatial differences in apoptosis in the endothelium. The aims of the present study were to determine whether oscillatory shear stress (OS) and laminar shear stress (LS) could induce EC apoptosis and modulate the cytochrome c (Cyt c) release from mitochondria. ECs were subjected to oscillatory and laminar flows, with shear stresses of 0.4±4 and 12 dynes/cm2, respectively. A significantly increased apoptosis was seen following OS over 24 h, compared with LS.

Apoptosis (%)
4 h
24 h

Oscillatory Flow / Static
1.85±0.06
2.89±0.05*

Laminar Flow / Static
1.73±0.03
1.24±0.04

Values are mean±SEM.
P<0.05 for Oscillatory vs. Laminar Flow.

Endothelial mitochondria are essential to the functional integrity of ECs as they integrate a wide range of cellular processes. The mitochondrial morphology changes dynamically due to their frequent fusion and fission in response to various cellular conditions. Mitochondrial fission is an early event during apoptosis and it occurs very close in time to Cyt c release, and the fragmented mitochondria can readily be seen to retain Cyt c. Cyt c staining of the mitochondria was detected in ECs under both types of flow. In comparison to static control, Cyt c was significantly reduced in the mitochondria of ECs exposed to both flow patterns for 4 h and 24 h. The reduction of Cyt c in endothelial mitochondria was greater under oscillatory flow for 24 h.

Mitochondrial Cyt c
4 h
24 h

Oscillatory Flow / Static
0.16±0.05
0.59±0.05*

Laminar Flow / Static
0.19±0.03
0.90±0.03

Values are mean±SEM.
P<0.05 for Oscillatory vs. Laminar Flow.

We used time‐lapse imaging to monitor the spatial‐temporal dynamics of mitochondria in live ECs transfected with RFP‐mito. During the shearing of live ECs, mitochondria networks are dramatically reorganized from long filamentous, interconnected tubules into small punctate spheres under both flow patterns. Under oscillatory flow, more small fragmentations of endothelial mitochondria were observed than under laminar flow. The results show that mechanical forces affect the mitochondria dynamics in ECs to cause Cyt c release, which was greater under oscillatory than laminar flow. The findings suggest that shear stress induces apoptosis via mitochondrial pathway. Understanding of the endothelial mitochondria responses to different flow patterns will help to elucidate the mechanism of the region‐specific localization of atherosclerosis in the arterial system.Support or Funding InformationThis work was supported by NHLBI Research Grants HL‐104402 and HL‐106579 (S.C.)
Microparticle Delivery of Cyclic AMP to Pulmonary Microvascular Endothelial Cells
Authors: Scruggs April K , Leavesley Silas J , Annamdevula Naga S , Rich Thomas C , Bauer Natalie N 
Published: November 2023

Microparticles (MPs) are important for the exchange of information between cells. MPs are extracellular vesicles that range in size from 0.1 to 1 μm in diameter. They are constitutively released from many cell types and are thought to play a role in vascular homeostasis. Further, the number and content of MPs are altered in pathologies involving inflammation and vascular cell activation. MPs carry intracellular constituents from parent cells that affect target cell function. However, the mechanisms of delivery of the MP payload to target cells are unknown. Understanding the payload delivery system between MPs and target cells is the focus of this project.We recently discovered that cyclic adenosine monophosphate (cAMP), a ubiquitous second messenger, is found in MPs released from pulmonary microvascular endothelial cells (PMVECs). Additionally, MPs isolated from PMVECs treated with β adrenergic agonists and phosphodiesterase (PDE) inhibitors have increased cAMP levels. This suggests that different levels of cAMP are packaged in MPs from the parent cell. MPs containing maximal cAMP were therefore used to study the delivery of MP payload to target cells.We developed mathematical models of MP payload delivery to subcellular compartments of target PMVECs to better understand how the cAMP signaling pathway may be altered by MP‐mediated cAMP delivery. Simulations indicate that if the cAMP contained in one MP were released into a near‐membrane compartment of a target cell, cAMP levels within that compartment would be sufficient to activate protein kinase A (PKA) and Exchange protein activated by cAMP (Epac) for several minutes. Release of cAMP from a single MP released into the larger volume of the bulk cytoplasm would have little effect on target cell signaling. However, several MPs are often internalized by a target cell and their combined cAMP content would sufficiently elevate bulk cytosolic cAMP levels to activate PKA.To experimentally test the predictions of this model, MPs were collected from PMVECs treated with rolipram (a PDE type 4 inhibitor; 10 μM, 5 min) and isoproterenol (a β adrenergic agonist; 1 μM, 10 min). These MPs were added to cells transfected with a cytosolic cAMP FRET sensor. Time lapse hyperspectral image stacks were acquired using a Nikon A1R confocal microscope. Custom hyperspectral analysis scripts in MatLab (MathWorks) were used to unmix fluorescent spectra and quantify cAMP concentration changes throughout the target cell. Preliminary results indicate that MPs significantly increased cAMP levels in target cells compared to control. Together, these data indicate that MPs can deliver cAMP as a functional payload. Future studies will determine whether the MP‐delivered cAMP is sufficient to trigger sustained responses in target cells and contribute to MP‐mediated signaling events.Support or Funding InformationThis work was supported by NIH T32HL076125, P01HL066299, S10RR027535, and AHA 11SDG7390037.
Calcium Influx Through Cav1 and TRP Channels Induced by the Calcium Release Via RyR Activates BKCa in Mouse Mesenteric Artery
Authors: Garcia Daniela Cibeli Guimarães , Lopes Miguel José , Lemos Virgínia Soares , Cortes Steyner França 
Published: November 2023

The sarcoplasmic reticulum (SR)/plasma membrane (PM) junction plays an important role in the control of the excitation‐contraction coupling of the vascular smooth muscle cells (VSMCs). The present work investigated the PM/SR interaction involved in the modulation of the contraction induced by the activation of ryanodine receptors (RyRs) in mouse mesenteric artery. 2nd branch of endothelium‐denuded small mesenteric arteries from male Swiss mice (10–12 weeks old) were studied using wire myography, epifluorescence microscopy, and electrophysiology methods. All experimental protocols were approved by the local ethics committee (protocol n° 86/2015). Caffeine (10 mM) induced a transient contraction in mesenteric arteries and increased of the intracellular calcium signal. In Ca2+‐free solution, the contraction and the calcium signal induced by caffeine were reduced. Nifedipine (10 μM), a selective Cav1 blocker, increased the area under the curve (AUC) resulting from the reduction of the contraction decay time while reducing the intracellular calcium signal. 2‐APB (10 μM), a TRPC blocker, increased the peak and the AUC of the contraction and reduced the calcium signal. Paxilline (1 μM), a BKCa channel blocker, also increased the AUC of the contraction. The contractile effect of caffeine was not changed by apamin (50 nM) and TRAM‐34 (1 μM), respective blockers of SKCa and IKCa. Caffeine evoked a hyperpolarization, which was blocked by nifedipine, 2‐APB, and paxilline. The present work demonstrated that the release of calcium stores sensitive to the activation of RyRs induces an influx of calcium through Cav1 and TRP channels responsible for a BKca‐dependent hyperpolarization, triggering a negative feedback of the contraction induced by caffeine in mouse mesenteric artery.Support or Funding InformationSupported by the Brazilian Council for Scientific and Technological development (CNPq).