bluuebunny/crossref_metadata_2025 huggingface dataset:
Genetic Inhibition of Chac1 Leads to Dysregulation of Body Composition
Authors: Crawford Rebecca R , Prescott Eugenia T , Mungrue Imran N
Published: November 2023

CHAC1 is a gamma‐glutamylcyclotransferase downstream of ATF4 in the Unfolded Protein Response. CHAC1 degrades glutathione, the primary, intracellular antioxidant, and decreases Notch signaling. Notch is the receptor for a well‐conserved signaling pathway that is essential for development and proper maintenance of adult tissues, such as mucle and bone. Due to the importance of glutathione and Notch, it is likely that CHAC1 is critical for cell function and development. Chac1−/− mice were generated to elucidate the effects of Chac1 in vivo; however, knockout of Chac1 is lethal. Interestingly, Chac1+/− mice have decreased body weight, suggesting Chac1 is important in body composition regulation.

To dissect the underlying cause for reduced body mass in Chac1+/− mice, four metrics were analyzed: food intake, fat mass, muscle mass, and bone function. Analysis of food intake illustrates that there is no change in food intake; thus, food intake does not account for the decrease in body weight, but does suggest a misregulation of energy balance. Abdominal fat pad weight is not consistently changed at any 6, 13, or 33 weeks of age ages, suggesting no changes in fat mass. Chac1+/− mice of both sexes have decreased gastrocnemius weights at 6 weeks and 33 weeks of age, suggesting decreased muscle mass as the cause of the reduced body weight. Finally, markers of osteoblasts were analyzed in the femur of male and female Chac1+/− mice versus control. Markers for osteoblasts, such as Runx2 and Col1a1, are decreased in Chac1+/− mice at 6 weeks. These data suggest that there is a decrease in osteoblast differentiation and decreased bone density.

Overall, these data illustrate that genetic inhibition of Chac1 in mice leads to multiple changes in body composition, including lower muscle mass and changes in bone development, demonstrating the importance of Chac1 in the proper regulation of muscle and bone. Furthermore, Chac1 may play an important role in the progression of muscle and bone pathology in conditions such as osteoporosis, aging, and muscle wasting.

Support or Funding Information

NIH #HL09470; COBRE P30GM106392; Louisiana Board of Regents Fellowship #LESQSF(2012‐17)‐GF‐08 to RRC
2‐Methoxyestradiol protect against isoproterenol‐induced cellular hypertrophy in the human ventricular cardiomyocytes, RL‐14 cells, through MAPK‐ and NF‐κB‐dependent mechanisms
Authors: Almaayah Zaid Hussein , El‐Kadi Ayman O.S.
Published: November 2023

Accumulating data supported the evidence that 2‐methoxyestradiol (2ME) is a biologically active metabolite and mediates multiple effects on the cardiovascular systems that are largely independent of estrogen receptor. 2ME is a major cytochrome P450 1B1 metabolite and has been reported to have a vasoprotective and anti‐inflammatory actions. However, whether 2ME would inhibit isoproterenol (ISO)‐induced cellular hypertrophy has not been investigated yet. Therefore, the overall objectives of the present study are to elucidate the potential anti‐hypertrophic effect of 2ME in the human ventricular cardiomyocyte, RL‐14 cells, and to explore the mechanism(s) involved. For this purpose, RL‐14 cells were treated with 100 μM ISO in the presence and absence of 200 nM 2ME. Thereafter, the cellular hypertrophy markers and cell volume were determined using real‐time polymerase chain reaction and phase contrast imaging, respectively. Phosphorylated mitogen activated protein kinases (MAPK) levels and nuclear factor kappa B (NF‐κB) binding activity were determined using a commercially available kits. Our results showed that 2ME significantly inhibited the ISO‐induced cellular hypertrophy in RL‐14 cells as evidenced by a decrease in the induction of β‐myocin heavy chain/α‐myocin heavy chain and cell volume. Mechanistically, the protective effect of 2ME against ISO‐induced cellular hypertrophy was mediated through a significant inhibition of the phosphorylated p38 and ‐Jun NH2‐terminal kinases (JNK)‐induced by ISO, whereas it normalizes the ISO‐mediated inhibition of extracellular‐regulated kinases1/2 (ERK1/2) signaling pathway. Furthermore, 2ME inhibited the ISO‐induced NF‐κB binding activity. In conclusion, our study provides the first evidence that 2ME attenuate ISO‐induced cellular hypertrophy through MAPK‐ and NF‐κB‐dependent mechanisms.

Support or Funding Information

This work was supported by a grant from the CIHR to A.O.S.E. Z.H.M. is the recipient Izaak Walton Killam Scholarship and Alberta Innovates‐Health solution Scholarship.
Fibroblast Growth Factor 21 Deficiency Attenuates DSS‐induced Acute Colitis Through Activation Of Intestinal Epithelial Cell Stat3‐mediated Pathway
Authors: Liu Liming , Zhao Cuiqing , Dryden Gerald , McClain Craig , Feng Wenke
Published: November 2023

Background

Fibroblast growth factor 21 (FGF21) is a novel metabolic regulator in glucose and lipid metabolism. Recent studies showed that FGF21 is also induced by inflammatory stimuli. However, its role in inflammatory bowel disease (IBD) has not been examined.

Methods

Experimental IBD model was induced by adding 2.5% (wt/vol) dextran sulfate sodium (DSS) into drinking water for 3, 5 and 7 days, respectively, in FGF21 knockout (KO) and wide type (WT) mice. Mouse colon tissues were analyzed for colitis severity and inflammatory cell infiltration. FGF21 and inflammatory makers were determined in serum and tissues samples. The T84 intestinal epithelial cell line was used for additional in vitro studies.

Results

In WT mice treated with DSS induced an elevation in serum FGF21 and a significant body weight loss in a time dependent manner. Gene expression studies showed that liver and epididymal white adipose tissues were the major sources of circulation FGF21. Surprisingly, the body weight loss and colitis severity were significantly attenuated in FGF21 KO mice. Further analysis showed that FGF21 KO mice had less colon shortening, histological damage, less neutrophil infiltration compared to WT mice. FGF21 deficiency decreased DSS treatment‐increased serum concentrations of IL‐6, TNF‐α and IL‐1β and their mRNA levels in colon tissues. Colonic epithelial cell proliferation, examined by BrdU staining, was increased in the KO mice. DSS treatment caused a decrease in epithelial cell proliferation and Paneth and Goblet cell numbers in WT mice, which were attenuated in the KO mice. Importantly, FGF21 depletion dramatically increased signal transducer and activator of transcription‐3 (STAT3) activation in intestinal epithelial cells, along with a decreased suppressor of cytokine signaling‐3 (Socs3) expression. FGF21 pretreatment inhibited IL6‐induced STAT3 phosphorylation in human intestine epithelial T84 cells.

Conclusion

Circulating levels of FGF21 were increased in acute colitis in mice. FGF21 deficiency attenuated DSS induced acute colitis, which is likely mediated by the inhibitory effect of FGF21 on IL‐6‐induced‐STAT3 activation in intestinal epithelial cells.

Support or Funding Information

This research was supported by the NIH and Veterans Administration.
Adherens Junction Proteins in Cochlear Lateral Wall Capillaries are Altered by Loud Sound‐induced Stress to the Ear
Authors: Yu Qing , Foster Sarah , Dziennis Suzan , Wilson Teresa , Nuttall Alfred L
Published: November 2023

Background

The capillaries of the cochlear lateral wall are part of the blood‐labyrinth‐barrier (BLB) that separates the inner ear fluid from systemic circulation. The BLB controls the exchange of solutes, protein, and water, and plays an essential role in maintaining inner ear homeostasis and the endocochlear potential. Compromise of the BLB's integrity is a contributing factor in various hearing disorders including presbycusis, noise‐induced hearing loss, and autoimmune inner ear disease. This crucial barrier's function relies on endothelial cell‐cell junctions including those formed by the adherens junction (AJ) proteins. However, the mechanisms which regulate the function of AJs in the cochlear lateral wall capillaries remain largely unknown.

Objective

To characterize the role of AJ protein complexes and the associated signaling pathways in modulating the integrity of lateral wall capillaries under normal and loud sound‐induced stress conditions.

Methods and Results

The spiral ligament and stria vascularis were isolated from the cochlea of 10 week old CBA/CaJ mice and immunolabeled for VE‐cadherin, β‐catenin, and FAK. Confocal imaging revealed that the fluorescent signals for VE‐cadherin and β‐catenin overlapped at endothelial cell to cell contacts whereas FAK revealed nuclear, cytoplasmic, and focal adhesion distribution without overlap with VE‐cadherin. To evaluate the dynamic changes of VE‐cadherin and its associated signaling pathway components under stress conditions, we exposed the CBA/CaJ mice to loud sound (106 dB SPL, 8–16 kHz, 2 hours). The following immunofluorescence analysis of the capillaries revealed an increase of specific phosphorylation sites for β‐catenin and FAK in the endothelial cells of noise exposed mice. Airyscan imaging showed alterations of the AJ proteins at the cell membrane as well as increased translocation of FAK to the endothelial cell‐cell junctions.

Conclusions

Adherens junctions are dynamic structures that allow for rapid and specific responses to changes in the perivascular microenvironment. Loud sound‐induced alterations of AJ components and intracellular partners may reduce the integrity of the BLB. Unraveling the molecular mechanisms and signaling pathways that modulate the function of AJ complexes will contribute to the future discovery of breakthrough therapeutic treatments that maintain inner ear barrier integrity.

Support or Funding Information

Supported by grants 5R01DC000105 (ALN) and P30DC005983.
Ex Vivo Validation of Intravascular Ultrasound/Photoacoustic Tissue Characterization
Authors: Kole Ayeeshik , Hui Jie , Cao Yingchun , Wang Pu , Cheng Ji‐Xin , Sturek Michael
Published: November 2023

Coronary heart disease is the leading cause of death in the United States and incidence is projected to increase by 18% by 2030. Yet, there remains a pressing clinical need for tools to detect vulnerable atherosclerotic plaques that can rupture and lead to major adverse cardiac events. Plaques that are considered most vulnerable for rupture are thin‐capped fibroatheromas, which are grossly defined by hallmarks of a thin fibrous cap, a large lipid‐rich necrotic core, inflammatory infiltrate, and positive remodeling. These plaques are often structurally non‐obstructive to moderately obstructive, thus asymptomatic and clinically unidentifiable with routine angiography and stress testing. Rather, their vulnerability is a product of their chemical composition. Here, we present a dual‐mode intravascular catheter which is capable of producing co‐registered cross‐sectional images of vessel wall morphology and lipid content, via ultrasound and photoacoustic modes, respectively. A prototype design of the catheter was used to image a formalin‐fixed carotid artery harvested from a miniature Ossabaw swine with metabolic syndrome. Preliminary results suggest the catheter was able to detect a moderately obstructive fibrocalcific plaque identified by gross inspection. A dense pattern of superficial calcium was demonstrated by intense echogenic signal with acoustic shadowing of the ultrasound signal peripheral to the deposit and a lipid‐rich core underlying this was shown by strong photoacoustic signal. In addition, a fresh carotid artery harvested from a healthy domestic swine served as a negative control in which we observed a clean lumen with no detectable photoacoustic signal. These findings will be compared to gold standard histopathology and future studies will be extended to include interrogation of fresh, post‐mortem atherosclerotic coronary arteries from humans obtained at autopsy.

Support or Funding Information

IUPUI Graduate Imaging Research Fellowship, NIH T32 GM077229, HL125385
Electronic Cigarettes Modulate Platelet Inflammatory, Adhesion, Activation and Aggregation Responses
Authors: Hom Sarah , Wang Tony , Chen Li , Yin Wei , Rubenstein David Alan
Published: November 2023

Electronic cigarettes (e‐cig) have recently seen a surge in popularity as nicotine‐based vaporizers and as a “healthy” alternative to traditional tobacco cigarettes. However, the health effects, the potential risks and the amount of chemicals consumed during inhalation of electronic vapor has not been fully characterized. Additionally, some of the constituents of e‐vapor are similar to those found in tobacco smoke and it has been well‐established that the tobacco smoke constituents promote disease development, including cardiovascular diseases through the actions of these constituents on platelets. Thus, the goal of this work was to determine the effects of e‐vapor on platelet activities that have been associated with cardiovascular disease progression. We hypothesized that with a longer duration exposure to e‐vapor, platelets would exhibit a pro‐thrombotic and pro‐inflammatory phenotype consistent with those observed during cardiovascular disease progression. Additionally, with increasing nicotine concentration in the e‐juice, we hypothesized that platelet responses would be enhanced as compared with lower nicotine doses. To test this, platelets were incubated with various extracted e‐vapor or tobacco smoke (from different commercially available products) for up to 2 hours. Complement deposition and complement receptor expression was assessed with an ELISA approach. Platelet adhesion potential was assessed with flow cytometry. The ability for platelets to aggregate or activate was quantified with optical platelet aggregometry and a modified prothrombinase assay. We observed an increased deposition of C3b after exposure to e‐vapor, but not C1q or C5b‐9, suggesting that these exposure enhanced the alternative complement pathway but complement could not proceed to completion. The expression of gC1qR and cC1qR (the receptors for C1q) was also enhanced after exposure to e‐vapor suggesting that the potential for complement deposition was enhanced. Additionally, platelet adhesion potential, platelet activation (as quantified via thrombin generation) and platelet aggregation were all enhanced after the exposure to e‐vapor. These enhancements were further perpetuated with longer duration exposures, but interestingly, they were largely independent on the nicotine concentration within the e‐cigarette. Overall, these data suggest that e‐cigarettes may be detrimental to cardiovascular health through the actions of e‐vapor constitutes on platelet activities that have been associated with cardiovascular disease progression.
Platelet adhesion to vascular endothelial cells through PECAM‐1 regulates endothelial cell inflammatory responses
Authors: Yin Wei , Meza Daphne , Rubenstein David Alan
Published: November 2023

Both vascular endothelial cells and platelets are sensitive to blood flow induced shear stress. We have recently reported that platelet‐endothelial cell interaction could greatly affect platelet activation and platelet surface adhesion protein expression under shear flow. In the present study, we aim to investigate how platelet‐endothelial cell interaction affects endothelial cell inflammatory responses. Human coronary artery endothelial cells were exposed to normal or low pulsatile shear stress with or without the presence of platelets in a cone and plate shearing device. Following shear exposure, endothelial cell ICAM‐1 expression was measured using a solid phase ELISA approach, Western blot and real‐time PCR. The results demonstrated that pathological low shear stress induced significant increase in endothelial cell ICAM‐1 expression, at both the protein and RNA levels, compared to normal shear stress. However, when endothelial cells were exposed to shear stress with platelets, the presence of platelets inhibited low shear stress induced endothelial cell activation by reducing ICAM‐1 expression, at both protein and RNA levels. Platelet adhesion to endothelial cell monolayer was observed, with enhanced adhesion occurring under low pathological shear stress, localized to endothelial cell junctions. To determine the mechanisms that are involved, platelet surface adhesion protein expression (GPIb and GPIIb) was measured, and endothelial cell surface von Willebrand factor expression was measured, using flow cytometry and ELISA respectively. However, with the presence of platelets, low pulsatile shear stress did not cause any changes in these protein expression. Interestingly, significant changes in endothelial cell surface PECAM‐1 phosphorylation was observed. When endothelial cells were exposed to shear stress alone, pathological low shear stress significantly enhanced endothelial cell PECAM‐1 phosphorylation (and ICAM‐1 expression). With the presence of platelets, endothelial cell PECAM‐1 phosphorylation was reduced significantly under low pulsatile shear stress (so did ICAM‐1). When endothelial cell PECAM‐1 expression was suppressed using siRNA, platelet adhesion to endothelial cells under low pulsatile shear stress decreased greatly. Therefore, PECAM‐1 may be a mediator to enhance platelet adhesion to endothelial cells under pathological low shear stress. Platelets binding to endothelial cell surface PECAM‐1 could have interfered with endothelial cell mechano‐sensing through PECAM‐1, an important endothelial cell surface mechanotransducer, therefore affecting endothelial cell inflammatory responses towards pathological shear flow.
Molecular Mechanism of Tethers and Slings in CD4+ T cells
Authors: Abadier Michael , Ley Klaus
Published: November 2023

Leukocyte recruitment towards the site of injury is a tightly regulated multi‐step process initiated by rolling along the luminal surface of inflamed endothelium in the presence of high shear flow. The interaction of endothelial P‐selectin‐to‐P‐selectin glycoprotein ligand (PSGL)‐1 constitutively expressed in leukocytes mediates leukocyte rolling by rapid formation and breakage of selectin‐to‐ligand bonds to balance the hydrodynamic drag forces exerted by blood shear stress. Once pulling forces due to the high shear exceed a critical threshold, tethers and slings become essential to support leukocyte rolling. Slings, recently discovered by our group, are long cell adhesive structures derived from long tethers. Here, we polarized naïve T cells towards distinct lineages of CD4+ effector T cells (Th1, Th2, Th17 and Treg). Considering the critical role of functional PSGL‐1 for T cell rolling, we tested functional PSGL‐1 by P‐selectin‐Fc binding in flow cytometry. PSGL‐1 in Th1, Th17 and Treg is functional in 70–80% of cells, but only in 10–20% of Th2 cells and 0% of Naïve CD4 T cells. To visualize the dynamic process of T cell rolling, we performed live cell imaging under high shear stress (>5 dyn/cm2). Interestingly, the rolling speed of Th2 cells was significantly higher when compared to the other T cell subsets. This was mirrored by observing tethers and slings in Th1, Th17, Treg but to a lesser extent in Th2. Surprisingly, some of the few Th1 or Th17 cells bearing non‐functional PSGL‐1 were able to form tethers and slings, suggesting that functional PSGL‐1 binding to P‐selectin allows for T cell rolling at reduced speed but might not be the sole requirement for tether and sling formation. To identify the molecular and cellular requirements for tether and sling formation, Naïve CD4+ T cells and neutrophils will be used as negative and positive controls, respectively. By developing gene expression maps we will identify the differentially regulated genes during T cell lineage differentiation. Upregulated genes in Th1, Th17 and Treg, but not in Th2, are excellent candidates for being involved in tether and sling formation.

Support or Funding Information

Swiss National Science Foundation and P01 HL078784/NIH
Delayed β1 Adrenergic Receptor Blockade Attenuates Cardiac Oxidative Stress, Remodeling, and Improves Cardiac Function in Post‐Myocardial Infarction
Authors: Bao Weike , Kuziw Joanne , Zacco Anna , Ringenberg Mike , Schnackenberg Christine
Published: November 2023

Our previous study showed that selective β1 adrenergic receptor blockade prevents cardiac oxidative stress and damage associated with acute myocardial ischemia/reperfusion (I/R) injury. A recent human study demonstrated that early intravenous β1 blockade before primary percutaneous coronary intervention reduced infarct size and increased left ventricular (LV) ejection fraction without adverse events. Another clinical study showed that β1 blocker treatment post‐hospitalization improved patient survival after acute myocardial infarction (MI). However, the mechanisms for the cardiac and survival benefits in post‐MI remain unclear. In the present study, we tested the hypothesis that delayed metoprolol treatment reduces cardiac maladaptive remodeling and improves cardiac function in a rat model of myocardial ischemia followed by a prolonged reperfusion, which mimics the contemporary clinical situations in which patients with heart attacks undergo revascularization. Male Sprague‐Dawley rats were subjected to a 30 min occlusion of the left anterior descending coronary artery followed by four weeks of reperfusion or sham surgery (n=6). In I/R rats, vehicle (n=12) or metoprolol (n=12, 100 mg/kg/day in drinking water) treatment commenced two days after I/R and continued until the end of the study. LV hemodynamics (pressure‐volume loop catheter), heart and LV weights (gravimetric measurements), cardiac histology (H&E and Masson's Trichrome stains), and cardiac oxidative stress (cardiac myosin binding protein C (cMyBPC) carbonylation) were determined at the end of the study. Compared to sham animals, I/R + vehicle significantly reduced stroke volume and ejection fraction by 40% and 21%, respectively, increased heart and LV weights by 19% and 16% (both p<0.01), respectively, increased cMyBPC carbonylation by 596%, resulted in fibroplasia and inflammatory cell infiltrates and mature collagen bundles in the lesion area, and infarcted the LV by 13%. After one week of I/R, metoprolol significantly reduced heart rate (EKG) by 11%, which confirmed the pharmacologically active dose. After four weeks of I/R, metoprolol significantly improved stroke volume and ejection fraction by 52% and 18%, respectively, significantly reduced heart and LV weights by 9% and 9%, respectively, significantly reduced cMyBPC carbonylation by 57%, trended towards less severe histological grades while it had no effect on infarct size. Our data indicate that in a rat model of MI, β1 adrenergic receptor blockade initiated after cardiac injury reduced oxidative stress and improved cardiac remodeling and function independent of infarct size.
Soluble guanylate cyclase activation during ischemic injury in mice protects against post‐ischemic inflammation at the mitochondrial level
Authors: Wang Derek , Jones Allan , Wang Walter , Wang Meifang , Korthuis Ronald
Published: November 2023

Preconditioning with BAY 60‐2770, a selective activator of oxidized soluble guanylate cyclase (sGC), was shown to protect against postischemic reperfusion (I/R) injury in wild type (WT) and heme‐oxygenase‐1 KO (HO‐1−/−) mice. Here a similar effect occurred when administered during ischemia; as measured by reduced leukocyte rolling and adhesion to intestinal venules, as well as prevention of increased TNF‐α and circulating neutrophil levels that accompany I/R. We hypothesized that a component of BAY 60‐2770 protection involves maintenance of mitochondrial membrane integrity during I/R. Measures on isolated mucosal cells of Calcein fluorescence and JC‐1 fluorescence ratio were reduced by I/R, indicating formation of mitochondrial permeability transition pores (mPTP). These indices remained at control levels following treatment with BAY 60‐2770 as well as cyclosporin A and SB 216763. Western blots of mucosal cells from WT and HO‐1−/− indicated that glycogen synthase kinase‐3β (GSK‐3β) phosphorylation on serine9 (inhibitory site) was reduced following I/R alone (increased GSK‐3β activity), and increased (reduced GSK‐3β activity) following treatment with BAY 60‐2770. Studies by others have associated phosphorylation of the GSK‐3β substrate, cyclophilin D (pCyPD) with mPTP formation. We observed an increase in pCyPD after I/R, whereas BAY 60‐2770 treatment of sham and I/R groups reduced pCyPD. In conclusion, selective activation of oxidized sGC of WT and HO‐1−/− during ischemia protects against I/R induced inflammatory processes and preserves mucosal integrity in part by reducing pCyPD production and mPTP formation.

Support or Funding Information

NIH R01‐AA022108 and P01‐HL ‐095486. Bayer Schering Pharma AG provided BAY 60‐2770 as a gift
Mast Cells‐Dependent Compromised Response of Aged Mesenteric Lymphatic Vessels and Surrounding Tissues to the Acute Inflammatory Challenge
Authors: Skoog Hunter , Dusio Giuseppina F. , Tobin Richard , Zawieja David C. , Newell‐Rogers M. Karen , Gashev Anatoliy A. , Nizamutdinova Irina Tsoy
Published: November 2023

Mesenteric lymphatic vessels (MLVs), being located at the border between the biologically aggressive environment of the gut lumen and inner compartments of the abdomen have not been studied in depth as an active component of the anti‐inflammatory defense system of the body. Aging creates several challenges for lymphatic vessels by altering their ultrastructure, contractility and consequently lymph flow as well as their microenvironment. We found an increase in number and degree of basal activation of mast cells (MC) nearby aged mesenteric lymphatic vessels. Such, as we predicted, will delimit ability of aged mesenteric tissue to respond to acute inflammatory challenge. In current study we attempted to establish mechanistic links between the aging‐associated changes in functional status of MCs and the altered responses of mesenteric tissue and MLVs to acute inflammatory insult. We used in vivo model of induction of acute peritoneal inflammation by lipopolysaccharide (LPS) treatment (24 hours after single IP injection of LPS in dose of 10 mg/kg body weight into adult (9‐mo) and aged (24‐mo) F‐344 rats). Analysis of data obtained by studies based on ruthenium red staining of MCs and by flow cytometry‐based measurements demonstrate that LPS‐induced degranulation of MCs in aged mesentery is greatly diminished. Latter co‐exists with basal activation of NF‐κB signaling in aged MLVs and mesenteric tissue therefore additional LPS‐induced NF‐κB activation is also reduced. Furthermore both mast cell stabilization by Cromolyn, or application of mixture of Histamine H1–H4 receptor blockers, are able to prevent the NF‐κB activation in mesentery/MLVs. NF‐κB, as hallmark of aging‐associated low‐grade inflammation, controls the release of pro‐inflammatory cytokines. To identify the corresponding aging‐associated differences, 24 hours after LPS injection we compared blood and mesenteric tissue (including MLVs) samples from adult and aged rats for 27 major pro‐inflammatory cytokines, using a Multiplex cytokine assay system (kit RECMAG65K27PMX, Millipore, US). The obtained results show that there is an elevation of NF‐κB‐dependent pro‐inflammatory cytokines in aged mesentery/MLVs with imbalance of Th1/Th2 cytokine profile and subsequent changes in T and B cells populations. Cumulatively, our data indicate that pre‐existing activation of MCs in aged mesentery by MLVs linked to its altered response to LPS, associated with histamine/NF‐κB‐dependent specific changes in cytokine levels. Latter can contribute to imbalance of immune cells profile and decreased ability of the aged immune system to respond to acute inflammatory challenge in peritoneal compartment of the body.

Support or Funding Information

NIH/NIDDK 1R56DK099161
Mathematical Model of Calcium and Electrical Dynamics in Lymphatic Endothelium
Authors: Jafarnejad Mohammad , Behringer Erik J , Scallan Joshua P , Davis Michael J , Segal Steven S , Moore James E
Published: November 2023

Contraction of collecting lymphatic vessels is essential for immunity and tissue fluid balance. The underlying role of lymphatic endothelium is unknown. In contrast to blood vessels, lymphatic endothelial cell tubes (LECTs; mouse popliteal fossae) do not govern membrane potential (Vm) via small‐ and intermediate‐conductance Ca2+ activated K+ channels (SKCa/IKCa). Consequently, despite similarity in the intracellular Ca2+ ([Ca2+]i) response to acetylcholine (1 μM) (i.e., rapid initial peak followed by secondary “plateau” phase), depolarization of Vm (ΔVm: 29±4 mV, n=11 LECTs) ensues in contrast to classic hyperpolarization in blood vessels (ΔVm: −38±3 mV, n=4 popliteal arteries). To further resolve the contribution of respective ions and channel [e.g. transient receptor potential (TRP)] currents to LECT depolarization, we adapted mathematical models of [Ca2+]i dynamics and ion fluxes (e.g., Hodgkin‐Huxley). Modeling reproduced [Ca2+]i responses and accompanying depolarization observed experimentally. Furthermore, depolarization required a combination of Na+ and Ca2+ influx via cation membrane channels (e.g., TRP canonical & vanilloid 4, Orai) during the plateau phase. Roles of other ions (e.g., K+, Cl−) and respective channels (e.g., inward‐rectifying K+ & volume‐regulated anion) were also apparent. Altogether, the absence of SKCa/IKCa function during robust Na+ and Ca2+ mobilization determines the electrical activity of lymphatic endothelium. Such novel cellular signaling may regulate the efficacy of lymphatic muscle cell activity to control lymph transport during health while disruption of these interactions may contribute to disease.

Support or Funding Information

NIH grants R01 HL094269, U01 HL123420 (JEM), R01 HL120867 (MJD), R37 HL041026 (SSS), K99/R00 AG047198 (EJB), K99 HL124142 (JPS); The Royal Society, The Royal Academy of Engineering, and The Sir Leon Bagrit Trust (JEM).
Endothelial nitric oxide synthase protein distribution in endothelial cells along the coronary vascular tree
Authors: Israel Jessica H , Bray Jeffrey F , McIntosh Avery L , Schroeder Friedhelm , Heaps Cristine L
Published: November 2023

Previous investigations have compared the levels of endothelial nitric oxide synthase (eNOS) protein among different branch orders of the coronary arterial tree. In these studies, eNOS protein levels were determined in whole vessels and thus were likely influenced by variable amounts of non‐endothelial cell types in the lysate as vessel wall thickness varied. For the current study, we isolated endothelial cells from both coronary conduit arteries and microvasculature to test the hypothesis that the content of eNOS protein remains similar in coronary endothelial cells regardless of vessel radius. Porcine hearts were obtained from a local abattoir and transported to the lab in iced Krebs solution. Large (conduit) and small arteries as well as arterioles were dissected free of myocardium and homogenized as whole vessels. Additionally, endothelial cells were isolated from both conduit arteries and left ventricular myocardium by mincing the tissue into small pieces, digesting with collagenase, followed by endothelial cell isolation using biotinylated‐anti‐CD31 and streptavidin‐coated paramagnetic beads. Purity of isolated coronary endothelial cells was confirmed by immunofluorescence and immunoblot. In whole vessel lysate, immunoblot analysis revealed that protein content for eNOS was significantly greater in arterioles compared to small arteries and large arteries. In contrast, eNOS protein levels in endothelial cells isolated from coronary microvasculature were not significantly different from those isolated from large conduit arteries. Taken together, these data indicate that endothelial cell eNOS protein content is comparable regardless of whether endothelial cells are isolated from the coronary microcirculation or conduit arteries. Furthermore, use of whole vessel lysate among different branch orders to compare levels of endothelium‐specific proteins may be unsuitable due to variable contamination by other cell types.

Support or Funding Information

NIH R01‐HL064931
Whole Brain Irradiation Impairs Cerebromicrovascular Function and Promotes Cognitive Decline: Potential Role of Endothelial Senescence
Authors: Yabluchanskiy Andriy , Hertelendy Peter , Ashpole Nicole M , Tarantini Stefano , Logan Sreemathi , Owen Daniel B , Farley Julie A , Ungvari Zoltan , Sonntag William E , Csiszar Anna
Published: November 2023

Whole brain irradiation (WBI) leads to a progressive cognitive dysfunction in up to 50% of tumor patients surviving long‐term after treatment, due to, at least in part, γ‐irradiation induced cerebromicrovascular injury. Moment‐to‐moment adjustment of cerebral blood flow (CBF) via neurovascular coupling has a critical role in maintenance of healthy cognitive function. To determine whether cognitive decline induced by WBI associates with cerebrovascular dysfunction and neurovascular un‐coupling, C56BL/6J mice (3 mo) subjected to fractionated WBI (5Gy twice weekly for 4 weeks) and control mice were compared. Mice were tested for spatial memory performance (radial arm water maze), neurovascular coupling, endothelial function, and were sacrificed for molecular and immunohistochemical (IHC) analyses 90 days post‐irradiation. Mice with WBI showed impaired performance in the radial arm maze (increased path length vs control; p<0.05). WBI significantly decreased endothelium‐mediated CBF responses (laser Doppler flowmetry) in the somatosensory cortex evoked by contralateral whisker stimulation. In cultured cerebromicrovascular endothelial cells, γ‐irradiation induced replicative senescence and acquisition of a senescence‐associated secretory phenotype (SASP). In the mouse brain, WBI resulted in up‐regulation of the senescence‐associated genes, including Cdkn2a (p16INK4a), Serpine1 (PAI‐1), and the SASP genes Mcp2, IL6, IL1b (qPCR). IHC analysis revealed that the DNA damage marker H2AX was co‐localized with CD31 positive cells in sections from WBI mice. Collectively, our findings provide evidence for persistent neurovascular un‐coupling, highlighting the potential role of endothelial senescence in cerebromicrovascular dysfunction and cognitive decline induced by WBI.

Support or Funding Information

This study is supported by the Donald W. Reynolds Foundation and NINDS grant NS056218‐08 to WES.
Orphan Nuclear Receptors Nur77 and Nor1 Are Novel Regulators of Thrombotic Function in Vascular Endothelial Cells
Authors: Wei Xin , Yang Ping , Zhang Jian , Yi Bing , Zhang Guanxin , Yin Litian , Sun Jianxin
Published: November 2023

Objective

Thrombomodulin (TM) is highly expressed on the lumenal surface of vascular endothelial cells (ECs) and possesses potent anticoagulant, anti‐fibrinolytic, and anti‐inflammatory activities in the vessel wall. However, the regulation of TM expression and function in ECs remains largely unknown.

Approaches and Results

In the present study, we characterized the nuclear receptor 4A (NR4A) family as a novel regulator of TM expression in vascular ECs under both basal and inflammatory conditions. We demonstrate that both NR4A receptor Nur77 and Nor1 robustly increase TM mRNA and protein levels in human vascular ECs and in mouse liver tissues after adenovirus‐mediated transduction of Nur77 and Nor1. Moreover, Nur77 gene deficiency and knockdown of Nur77 and Nor1 expression markedly attenuated the basal and VEGF‐stimulated TM expression in ECs. Mechanistically, we found that Nur77 and Nor1 increase TM expression by acting through two different mechanisms. We show that Nur77 barely affects TM promoter activity, but significantly increases TM mRNA stability, while Nor1 enhances TM expression mainly through induction of Kruppel‐like factor 2 and 4 in vascular ECs. Furthermore, we demonstrate that both Nur77 and Nor1 significantly increase the protein C activity and inhibit tumor necrosis factor alpha (TNF‐α)‐induced prothrombotic effects, at least in part, through an inhibition of the NF‐κB activation in vascular ECs. Moreover, in a model of ferric‐chloride‐induced carotid artery thrombosis, Nur77 deficiency markedly exacerbates the formation of arterial thrombosis, whereas the bleeding time was not affected.

Conclusions

Our observations for the first time identified NR4A receptors Nur77 and Nor1 as novel regulators of TM expression and function in vascular ECs. We postulate that targeted increasing expression of Nur77 and Nor1 in vascular endothelium may represent a novel therapeutic approach for the treatment of vascular disorders, such as thrombosis and inflammation.

Support or Funding Information

This research was supported by the US National Institutes of Health (HL103869).
Process of cell‐free layer formation after arteriolar bifurcation and its physiological effects
Authors: Kim Sangho , Ng Yan Cheng
Published: November 2023

Understanding the formation of cell‐free layer (CFL) in arteriolar networks has been of great interest, particularly for its significance in regulation of blood flow. The heterogeneous distribution of red blood cells (RBCs) modulated by the formation of CFL in arteriolar bifurcations potentially leads to alterations in tissue oxygenation (O2) and vascular tone due to varying nitric oxide (NO) production in the endothelium. Accordingly, we investigated the formation of CFL from 2 to 6 vessel‐diameter (2D–6D) downstream of arteriolar bifurcations in the rat cremaster muscle under reduced flow conditions (pseudoshear rates = 15.6 ± 2.0 s−1). We accounted for the spatiotemporal variations in the wall shear stress, velocity profile and core hematocrit along the downstream vessels by extending our previous image‐based two‐dimensional transient model, which was unprecedented in previous computational models. Our experimental results showed that an asymmetric formation of CFL width along the vessel walls was evident up to 6D from the bifurcating point. The recovery of CFL symmetry appeared to be impeded by RBC aggregation. Our numerical prediction of soluble guanylate cyclase (sGC) activity revealed that vasoactivity might not occur uniformly within an inter‐bifurcation distance, in particular under reduced flow and aggregating conditions.

Support or Funding Information

NUS FRC
p66 Shc Contributes to Susceptibility to Cerebrovascular Injury and Intracerebral Hemorrhages
Authors: Beyer Andreas M. , Ait‐Aissa Karima , Miller Bradley , Sorokin Andrey
Published: November 2023

Background

Breakdown of the blood brain barrier (BBB) and loss of cerebral vessels autoregulation in response to elevated systemic pressure are closely linked to intracerebral hemorrhage and micro bleeds. Presently no established models to study the causes of intracerebral hemorrhage exist that does not rely on surgical interventions and existing models have failed to improve clinical understanding of this condition. Overproduction of reactive oxygen species (ROS) following ischaemia/reperfusion (I/R) is considered a key mechanism leading to BBB damage. It has been shown that the adaptor protein p66(Shc) is crucial regulator for ROS production in several disease states including I/R injury of the brain. p66shc when activated translocates to the mitochondria where it sequesters cytochrome C and generates hydrogen peroxide. The contribution of mitochondria to myogenic tone and autoregulation of the cerebral vasculature is not well defined.

We have found previously that rats expressing constitutively active form of p66 Shc (Shc‐CA) have decreased long term survival and increased incidences of stoke despite no significant difference in systemic blood pressure.

Hypothesis

We hypothesis that p66Shc signaling via mitochondrial translocation contribute to vascular dysfunction of cerebral arteries and intracerebral hemorrhage in hypertensive rats by decreasing mitochondrial ATP while elevating mitochondrial ROS.

Results

Vessels from WT rats, lacking p66 Shc protein (Shc‐KO), or expressing Shc‐CA were used for video microscopy. Endothelial dependent dilation to flow was assessed in vessels from Shc‐KO animals compared to their littermate controls (%Max Dilation 100 cm H2O: WT = 1.3±3 vs. Shc‐KO=39±3*; N≥3 P<0.05 one way ANOVA). Shc‐CA failed to develop tone spontaneously or to pharmacological stimuli (as a result no dilator function could be studied) suggesting a defect in vascular smooth muscle function. No difference in tone were observed in WT vs. Shc‐KO. Further evaluation revealed loss of myogenic tone in MCAs of Shc‐CA (% Tone at 150 cm H2O relative to Ca2+ free: WT = 19±6; Shc‐KO = 28±12; Shc‐CA =3±2, n≥2). In vivo cerebral vascular flow was evaluated using Doppler laser probe and revealed an inability of cerebral vessels to respond to altered systemic pressure in vivo. Shc‐CA showed decreased levels of ATP (pmol/mg Protein WT=1±0.2; Shc‐KO=0.7±0.2; Shc‐CA=0.1±0.1*; N≥3 P<0.05 one way ANOVA) and mitochondrial respiration, confirming decreased mitochondrial function in rats that express Shc‐CA.

To investigate an underling physiological relevance, rats were perfused with methylene blue (MB). A substantial increase in MB staining in the brain of Shc‐CA was observed, suggesting an increase in permeability of the BBB.
Effect of Age and High Fat Diet on the Pro‐Atherogenic Response to Oscillatory Blood Flow
Authors: Walker Ashley E , Breevoort Sarah R , Durrant Jessica R , Machin Daniel R , Dobson Parker S , Nielson Elizabeth I , Donato Anthony J , Lesniewski Lisa A
Published: November 2023

Atherogenesis is more likely to occur in arteries at locations of greater oscillations in blood flow, such as branch points and curvatures. However, it is unknown if advancing age or a high fat diet affect the pro‐atherogenic response to increased oscillatory blood flow. To induce greater oscillatory blood flow, we performed partial carotid ligation (PCL) of the left carotid artery of young normal chow fed (YNC, 6.4±0.3 mo, n=10), young high fat fed (YHF, 6.0±0.2 mo, n=11), old normal chow fed (ONC, 29.0±0.1 mo, n=7), and old high fat fed (OHF, 29.7±0.3 mo, n=11) male B6D2F1 mice. After PCL, blood flow of the ligated (left) and control (right) carotids was measured by ultrasound. The pro‐atherogenic response was assessed in histological sections of carotids collected 3‐weeks post‐PCL by measurement of intima media thickness (IMT) and neointima formation. Additionally, a subset of histological samples were evaluated by a pathologist (n=3–5/group).

After PCL surgery, antegrade blood flow was ~10‐fold less (p<0.001), but retrograde blood flow was not different between the ligated and control carotids (p>0.05). This resulted in an oscillatory index that was ~20‐fold higher in the ligated compared to the control carotids (p<0.001). Oscillatory index was not different between age or diet groups (p>0.05). Histological evaluation identified hypertrophied smooth muscle cells and separation of the mural elastic laminae in ligated carotids from all groups. On average, IMT was ~2 fold greater in the ligated carotid compared to the control carotid (p<0.001). Although IMT of the ligated carotid was not different between YNC and ONC mice (p>0.05), it was 52% greater in YHF compared to YNC (p<0.05) and 38% greater in OHF compared to ONC (p<0.05). Neointima formation in the ligated carotid was variable within groups and was primarily composed of smooth muscle cells and macrophages. Neointima was present in 20% of YNC, 45% of YHF, 14% of ONC, and 33% of OHF ligated carotids, but no neointima was present in control carotids from any group. In the ligated carotids, the percent of area within the internal elastic lamina composed of neointima was greater in YHF vs. YNC (19.5±7.4% vs. 4.3±2.8%, p<0.05) as well as in OHF vs. ONC (11.0±5.3% vs. 0.4±0.4%, p<0.05). The majority of YHF and OHF ligated carotids had adventitial or perivascular inflammation, extending into the media and intima in the most severe cases. However, minimal inflammation was present in YNC and ONC ligated carotids.

In conclusion, we find a high fat diet, but not older age, leads to an exacerbated pro‐atherogenic response to increased oscillatory blood flow, indicated by greater IMT, neointima formation and vascular inflammation.

Support or Funding Information

Supported by NIH AG046326, AG043952, AG040297, Department of Veterans Affairs 1I01BX002151
Differential effects of group III/IV skeletal muscle afferents and hypoxia on blood pressure during lower‐extremity dynamic exercise
Authors: Chantigian Daniel P , Cowl Andrielle L , Johnson Bruce D , Keller‐Ross Manda , Olson Thomas P
Published: November 2023

Activation of group III/IV skeletal muscle afferents (ergoreflex) and hypoxia (chemoreflex) are two potent stimuli of the sympathetic nervous system during exercise. Venous distention by graded sub‐systolic occlusion contributes to the ergoreflex during exercise. Although the ergoreflex causes greater increases in blood pressure (BP) compared with the chemoreflex, the interaction of the ergoreflex and hypoxia during lower extremity dynamic exercise is unknown.

Objective

To determine the effect of hypoxia and graded cuff occlusion on hemodynamic responses during low‐intensity cycling exercise.

Methods

19 healthy adults (9 men, 25±5 yr) completed two sessions. Session 1 included a maximal cycle ergometry exercise test. Session 2 included two randomized bouts (hypoxia: 12.5% FiO2 and normoxia: 21% FiO2) of constant‐load cycling at 30% peak workload for 3 min (control, CTL) followed by randomized inflation of bilateral thigh pressure tourniquets to 20, 40, 60, 80, 100 mmHg for 2 min with 2 min of deflation between occlusions. The bouts were separated by 30 minutes of rest. Blood pressure, heart rate and cardiac output (CO) were measured continuously.

Results

During hypoxia, the average SPO2 was 80%. Venous occlusion and hypoxia had differential effects on mean arterial pressure (MAP, occlusion × hypoxia, p=0.02). Significant increases of MAP from CTL occurred at 60 (Δ9 ± 6mmHg), 80 (Δ15 ± 8mmHg), and 100mmHg (Δ17 ± 9mmHg) under normoxia (p<0.05). MAP only increased at 100mmHg (Δ14 ± 9mmHg) under hypoxia (p<0.05). Under hypoxia, MAP was much greater at CTL and 20mmHg as compared to normoxia (p<0.05). SBP exhibited responses similar to MAP under hypoxia (100mmHg increase, p<0.001, occlusion × hypoxia interaction, p=0.02). CO was greater with venous occlusion as mediated by increases in HR (p<0.001). In addition, CO increased under hypoxia (p=0.007) but had no interaction with venous occlusion.

Conclusion

During low pressure venous occlusion, hypoxia had a greater effect on BP, but as pressure increased (60–100mmHg), there were no significant differences in BP between normoxia and hypoxia. Therefore, sympathetically mediated increases of BP during exercise attributed to hypoxia may negate any responses mediated by the ergoreflex when elicited by venous occlusion at low pressures.