bluuebunny/crossref_metadata_2025 huggingface dataset:
In Utero Exposure to Alcohol Alters Reactivity of Cerebral Arterioles
Authors: Cananzi Sergio Giuseppe , Mayhan William Gerard
Published: November 2023

Background

Fetal alcohol spectrum disorders (FASD) have long been linked with developmental abnormalities that often result in brain damage and mental disabilities. It is conceivable that these abnormalities may be related alterations in the regulation of cerebral blood flow (CBF) by in utero exposure to alcohol. Our goal was to examine whether in utero alcohol impaired reactivity of cerebral arterioles during development.

Methods

Sprague‐Dawley dams were fed a liquid diet with or without alcohol (3% and 6.4%) for the duration of pregnancy (about 21 days). The pups were weaned at three weeks of age. At four weeks of age, we measured CBF (Laser Doppler) in pups in response to hypercapnia. In addition, we made a craniotomy over the parietal cortex and measured responses of cerebral arterioles to agonists that activate endothelial Nitric Oxide Synthase (ADP), neuronal Nitric Oxide Synthase (NMDA), and an agonist that dilates independent of NOS (nitroglycerin). We also used an inhibitor of NOS (L‐NMMA) and tested responses to agonists of eNOS and nNOS. We also measured the expression of NOX‐2 in the brain cortex.

Results

In utero exposure to alcohol (3 and 6.4%) attenuated rCBF responses to hypercapnia. Responses to agonists that activate eNOS were reduced in cerebral arterioles of alcohol pups. Nitroglycerin produced similar responses in all groups. Protein expression of NOX‐2 from cortex tissue was increased in both 3% and 6.4% alcohol groups.

Conclusions

Our findings suggest that in utero alcohol exposure negatively affects the cerebral arterioles and that NOX‐2 may play an important role in the observed impairments. Vascular dysfunction and upregulated expression of NOX‐2 may increase infarct damage from ischemic stroke during FASD.

Support or Funding Information

LSU Health Sciences Center‐Shreveport and the Center for Cardiovascular Diseases and Sciences.
Maternal separation stress causes mis‐programming of arterial smooth muscle maturation
Authors: Reho John , Fisher Steven A
Published: November 2023

Early life stress (ELS) has been implicated in the developmental programming of hypertension however there is little understanding of the programming/mis‐programming of the smaller arteries where vascular resistance and blood pressure are determined. We have previously demonstrated a critical postnatal window for the developmental maturation of the vascular smooth muscle contractile gene program and that this program is under the control of the sympathetic nervous system (SNS). We tested the hypothesis that stress induced hyper‐activation of the SNS during this critical postnatal maturational window would accelerate vascular maturation and potentially lead to adverse cardiovascular effects during adulthood. We used the maternal separation (MS) stress model as a model of ELS where pups were separated from their mothers for 3hrs/day from PND2‐14 and correlated changes in contractile mRNA and resistance mesenteric artery (MA) function throughout postnatal maturation to adulthood. ELS increased expression of CPI‐17, the inhibitory subunit of myosin phosphatase, smMHC, and the a1‐adrenergic receptor, key components of the SNS‐mediated contractile pathway in resistance mesenteric arteries (MA) at PND21 with some effects still present at PND35. This was commensurate with 2–5 fold increases in phenylephrine, KCl, and calcium induced force generation by MAs at PND21. This effect hadt had abated by PND35. Blockade of SNS signaling via the α‐adrenergic blocker Terazosin suppressed this stress induced increases in contractile gene expression and function. No differences were found in adult male rats subjected to ELS under basal or in response to a second stressor. Interestingly, adult females subjected to 1hr restraint stress subsequently demonstrated decreased PE induced constriction of MAs. This was not observed in females subjected to ELS. Thus, ELS results in mis‐programming in the maturation of MA smooth muscle, with evidence for disparate response in males and females. α‐adrenergic receptor blockers administered during ELS may be particularly effective in suppressing the accelerated trajectory of maturation of the constrictor function of these arteries.

Support or Funding Information

NIH R01‐HL066171, T32‐HL072751
Exposure to PM2.5 during the First Trimester Contributes to Adult Cardiac Dysfunction
Authors: Tanwar Vineeta , Gorr Matthew , Eichenseer Clayton , Wold Loren
Published: November 2023

Objective

The present study was designed to investigate the effects of PM2.5 exposure during the first trimester of the in utero period on adult cardiac function.

Methods

Female FVB mice were exposed either to filtered air (FA) or particulate matter (PM2.5) at an average concentration of 51.69 μg/m3 from the Columbus, OH region for 6 h/day, 7 days/wk for the length of the first trimester of pregnancy. To examine cardiac function, male offspring at 12 weeks of age underwent baseline echocardiographic assessment. Following sacrifice, cardiomyocytes were isolated to assess cardiac dysfunction. Protein isolated from LV tissue from separate groups of mice was used to study underlying mechanisms.

Results

Mice exposed to PM2.5 during the first trimester demonstrated increased LVESd (2.597 ± 0.082 FA, 2.844 ± 0.077 PM2.5, P=0.04) and LVEDd (3.925 ± 0.062 FA, 4.235 ± 0.084 PM2.5, P=0.01) dimensions and reduced PWTs (1.292 ± 0.039 FA, 1.151 ± 0.039 PM2.5, P=0.02) at 3 months of age. Morphological alterations were associated with lower systolic function as indicated by reduced %FS (34.73 ± 1.741 FA, 29.19 ± 0.817 PM2.5, P=0.007) in PM2.5 exposed mice compared to FA controls. Cardiomyocytes isolated from PM2.5 exposed hearts showed reduced %PS (13.60 ± 0.8970 FA, 10.91 ± 0.7080 PM, P=0.02) compared to FA exposed hearts. −dL/dt was significantly reduced (−4.672 ± 0.3680 FA, 3.581 ± 0.2636 PM2.5, P=0.01) and TR90 (0.1868 ± 0.007295 FA, 0.2158 ± 0.006476 PM, P=0.004) and tau FURA (0.1125 ± 0.008169 FA, 0.1865 ± 0.01068 PM, P=0.0001) were significantly increased between the groups. Western blot analyses demonstrated decreased SERCA‐2 ATPase, p‐PLN and Cav1.2 expression in PM2.5 exposed hearts compared to FA control.

Conclusion

Exposure to PM2.5 during the first trimester of the in utero period triggers adverse developmental changes resulting in global adult cardiac dysfunction similar to exposure during the entire developmental period, suggesting critical windows of priming for adult cardiovascular disease.

Support or Funding Information

Project was supported by a grant from the National Institutes of Health (RO1ES019923) to LEW
Nitric oxide donor [Ru(terpy)(bdq)NO]3+ (TERPY) promotes eNOS activation
Authors: Potje Simone Regina , Oliveira Suellen D'Arc , Chen Zhenlong , Bonini Marcelo G , da Silva Roberto S. , Bendhack Lusiane M , Antoniali Cristina , Minshall Richard D
Published: November 2023

Introduction

TERPY ([Ru(terpy)(bdq)NO]3+) is a ruthenium complex nitric oxide (NO) donor that promotes relaxation of the mesenteric artery and aorta to a greater extent in hypertensive rats as compared to normotensive control. To better understand the mechanism of TERPY action, we sought to investigate whether it acts as both an NO donor and endothelial NO synthase (eNOS/NOS3) activator, as shown previously for nitroglycerin.

Objective

The aim of this study was to determine whether TERPY directly or indirectly (i.e., via NOS3 activation) increases the intracellular concentration of NO.

Methods

Human umbilical vein endothelial cells (HUVEC) and human embryonic kidney 293 cells transfected with empty vector (HEK) or eNOS cDNA (HEK‐eNOS) were treated with TERPY (1 μM) for different time lengths (5–60 min). NOS3 expression and Ser1179 phosphorylation were evaluated by Western Blot, and NO accumulation in HUVEC, HEK, and HEK‐eNOS cells was measured using a copper‐based fluorescent probe (1 μM). It was used to quantify the contribution of NO released from the donor source vs NO produced by eNOS (i.e, L‐NAME sensitive NO production).

Results

First, we demonstrated that in HEK, TERPY has the ability to release NO without additional stimulus, indicating that this compound is an NO donor. Moreover, in HEK‐eNOS cells, TERPY‐induced NO production was even greater with maximum values observed after 20 min. At this level of NO production, 26% could be blocked by co‐treatment with L‐NAME (non‐selective inhibitor of NOS, 0.1 mM). When cells were stimulated with TERPY for 20 min treatment, TERPY increased NOS3 Ser1179 phosphorylation in HUVEC and HEK‐eNOS further suggesting that TERPY induces NOS3 activation. Consistent with the hypothesis that activated eNOS could be inactivated by Src‐phosphorylated caveolin‐1 as part of a negative feedback mechanism, we have observed caveolin‐1 Tyr14 phosphorylation after 20 min.

Conclusion

Taken together, these data suggest that TERPY acts as a long‐acting NO donor and also as a NOS3 activator, thereby increasing the NO bioavailability. The potential benefits and mechanisms of eNOS activation by TERPY, as compared to nitroglycerin, requires further characterization in cell cultures and rodent models of hypertension.

Support or Funding Information

Financial support: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) [Grant 232217/2014‐9], Fundação Lemann, and NIH HL‐60678.
N‐myc downstream regulated gene‐1 promotes vascular inflammation through activating both NF‐κB and c‐Jun/AP‐1 pathways in endothelial cells
Authors: Zhang Guanxin , Qin Qing , Yi Bing , Guo Zhifu , Wei Xin , Yin Litian , Yin Yongqiang , Cheng Fang , Sun Jianxin
Published: November 2023

Objective

N‐myc downstream regulated gene‐1(NDRG‐1) is a member of NDRG family that has been shown to play essential roles not only in the regulation of cancer metastasis but also in the development of cardiovascular diseases, such as atherosclerosis and thrombus. The objective of this study is to determine whether NDRG1 is involved in vascular endothelial cell (EC) activation, and if so, to determine the molecular mechanism (s) involved.

Methods and Results

In response to the stimulation of inflammatory cytokines, such as interleukin‐1β (IL‐1β) and tumor necrosis factor‐alpha (TNF‐α), we show that the expression of NDRG1 in vascular ECs was markedly increased in a dose and time‐dependent manner, as determined by both quantitative real‐time PCR (qRT‐PCR) and western blot analysis. To determine the role of NDRG1 in endothelial activation, we performed a loss‐of‐function study by using NDRG1 specific small interference RNA (siRNA). Our results demonstrate that knockdown of NDRG1 in vascular ECs by siRNA substantially attenuates both IL‐1β and TNF‐α‐induced inflammatory responses in human umbilical vein endothelial cells (HUVECs). The expression of inflammatory cytokines and chemokines, such as IL‐6, IL‐8, and MCP‐1, was markedly inhibited in NDRG1 knockdown ECs. Intriguingly, the IL‐1β‐induced expression of thrombotic markers, such as plasminogen activator inhibitor type 1 (PAI‐1) and tissue factor (TF), was also attenuated in NDRG1 knockdown cells. To further elucidate the functional significance of NDRG1 in vivo, we assessed the expression of NDRG1 in the vessels subjected to the carotid artery ligation‐induced injury. Our results demonstrate that the expression of NDRG1 was markedly increased in the injured vessels as compared with their un‐injured controls. Furthermore, we found that in a mouse model of carotid artery ligation induced injury, knockdown of NDRG1 expression substantially attenuates vascular remodeling, as manifested by decreased neointimal formation, microphage infiltration, and expression of adhesion molecules in injured arteries. Mechanistically, we found that inhibition of NDRG1 markedly inhibited the transcriptional activities of both NF‐κB and AP‐1 pathways in vascular ECs, as determined by both promoter‐driven luciferase and electrophoretic mobility shift assays.

Conclusion

Taken together, our results for the first time identify NDRG1 as a critical regulator implicated in vascular inflammation and remodeling, and suggest that targeted inhibition of NDRG1 in vasculature may represent a novel therapeutic strategy for preventing certain cardiovascular diseases, such as atherosclerosis and thrombosis.

Support or Funding Information

NIH:RO1 HL103869‐01, NSCF: 81541003, SHSCF:15ZR1413400
Enteroendocrine Cell is a Source of VEGF‐A in the Neonatal Intestine
Authors: Yan Xiaocai , Liu Xueli , Managlia Elizabeth , De Plaen Isabelle
Published: November 2023

Necrotizing enterocolitis (NEC) is the most common gastrointestinal disease affecting premature newborns, characterized with a high mortality and chronic morbidity. We have previously found that VEGF‐A is decreased in human and experimental NEC and may play a role in NEC. However, which cell type(s) produce(s) VEGF‐A in the neonatal intestine remains unknown. Here, to determine the cellular origin of VEGF expression in normal mouse and human intestines, fixed small intestinal tissues of newborn mice and a human neonate with intestinal atresia were examined by immunofluorescence staining for VEGF‐A together with CD31, chromogranin A. To determine whether VEGF‐A was expressed in monocytes, intestines from CX3CR1‐GFP mice were used. In both intestinal tissues of mouse and human neonates, we found that VEGF‐A is strongly present in epithelial cells expressing chromogranin A, in myenteric neural plexus cells and in CD31+ cells. However, VEGF‐A was not detected in CX3CR1+ cells in the mouse intestine or in CD45+ cells in the intestine of humans. In conclusion, VEGF‐A is strongly expressed in enteroendocrine cells, myenteric neural plexus cells and endothelial cells, but not in myeloid‐derived cells. Therefore, we speculate that enteroendocrine cells may be critical for supporting the development of the intestinal villi microvasculature.

Support or Funding Information

Memorial Foundation (Friends of Prentice) and NIH grant R01HD060876 (IDP).
Heme Oxygenase‐1 Counteracts HIV Protease Inhibitor‐Mediated Endothelial Cell Dysfunction
Authors: Durante William , Liu Xiao‐ming , Durante Zane E. , Peyton Kelly J.
Published: November 2023

The use of human immunodeficiency virus (HIV) protease inhibitors has extended the duration and quality of life for HIV‐positive individuals. However, there is increasing concern that this antiviral therapy may promote premature cardiovascular disease by impairing endothelial cell (EC) function. In the present study, we evaluated the effect of HIV protease inhibitors on EC function and determined if the vasoprotective enzyme heme oxygenase‐1 (HO‐1) influences the biological action of these drugs. Treatment of human umbilical vein ECs with three distinct protease inhibitors, including ritonavir, atazanavir, and lopinavir, stimulated the expression of HO‐1 protein in a time‐ and concentration‐dependent manner. The induction of HO‐1 protein was preceded by a rise in HO‐1 mRNA and resulted in a significant increase in HO activity. Furthermore, protease inhibitors evoked a marked increase in Nrf2 activity and an elevation in the production of reactive oxygen species (ROS). Protease inhibitors also stimulated HO‐1 promoter activity, and this was prevented by mutating the antioxidant responsive elements of the promoter or by overexpressing a dominant‐negative mutant of Nrf2 that lacked the transactivation domain. In addition, the protease inhibitor‐mediated induction of HO‐1 and activation of Nrf2 was abolished by the antioxidant N‐acetyl‐L‐cysteine. Finally, protease inhibitors blocked EC proliferation and DNA synthesis in a concentration‐dependent fashion that was potentiated by the HO‐1 inhibitor tin protoporphyrin‐IX or by silencing the expression of HO‐1. In conclusion, this study found that HIV protease inhibitors stimulate HO‐1 gene expression in ECs via the ROS‐Nrf2 pathway, and that the induction of HO‐1 counteracts the anti‐proliferative action of protease inhibitors. Therapeutic strategies targeting HO‐1 represent a novel approach in alleviating protease inhibitor‐mediated EC dysfunction and vascular disease in HIV‐positive patients.

Support or Funding Information

Supported by NIH grant R01 HL59976.
Reciprocal Regulation of eNOS and Caveolin‐1 Function in Endothelial Cells
Authors: Chen Zhenlong , Minshall Rihcard
Published: November 2023

Endothelial nitric oxide (eNOS)‐mediated NO production plays a critical role in the regulation of vascular tone, adhesivity, and maintenance of endothelial barrier function. Caveolin‐1 (Cav‐1), which is found highly expressed in endothelia, is an important regulator of vascular tone via modulation of eNOS activity. eNOS‐derived NO may also affect Cav‐1 functions directly and indirectly through nitrosation and phosphorylation‐dependent modifications, although the exact molecular mechanisms and effects on eNOS expression and activity in endothelial cells are not clear. Here, reciprocal regulation by the two proteins was investigated. First, we determined by semi‐quantitative Western blotting that the ratio Cav‐1 to eNOS expression in primary culture endothelial cells was approximately 200:1. Secondly, upon stimulation of ECs with Ca2+ ionophore A23187 to activate Ca2+‐dependent signaling, Cav‐1 nitrosation, phosphorylation, and monomerization occurred within minutes which could be blocked by L‐NAME (eNOS inhibitor) and PP2 (Src kinase inhibitor). Furthermore, oligmerization of Cav‐1 was enhanced when eNOS expression was reduced by siRNA. These data suggest that eNOS, through a NO‐Src signaling pathway, induces a conformational change to the functional oligomeric form of Cav‐1. Thirdly, Cav‐1 knockdown by siRNA reduced eNOS expression level, although NO production and eNOS phosphorylation per molecule were elevated 2‐fold. Finally, in HEK cells overexpressing eNOS, transfection of Cav‐1 resulted in a concentration‐dependent increase followed by a decrease in eNOS expression and activity. Thus, eNOS‐derived NO production promotes Cav‐1 nitrosylation, phosphorylation and monomerization, while lower levels of Cav‐1 expression stabilize eNOS expression and higher levels inhibit eNOS activity via phosphorylation‐dependent binding as part of a negative feedback regulatory mechanism.

Support or Funding Information

Financial Support: P01 HL60678 and Department of Anesthesiology
Endothelium‐Derived Microvesicles Alter Permeability of Human Brain Microvascular Endothelial Monolayers
Authors: Hunter Larry W. , Jayachandran Muthuvel , Miller Virginia M.
Published: November 2023

The disruption of the cerebral microvascular endothelium and increases in capillary permeability are characteristic of many brain pathologies, including Alzheimer's disease, small vessel disease, ischemia/hypoxia, and hemorrhagic stroke. This study was designed to investigate whether microvesicles (MVs) released from activated human brain microvascular endothelial cells (HBMECs) alter permeability of endothelial monolayers. Male donor HBMECs were cultured to confluence in flasks, then treated for 24 hrs with medium alone (control) or with medium plus either tumor necrosis factor (TNFa, 20 ng/ml), thrombin (THR, 2 U/ml), angiotensin II (AngII, 10 nM), or hydrogen peroxide (H2O2, 0.8 mM). MVs released into the medium were isolated by differential centrifugation, labeled with annex in V plus antibodies against endothelial adhesion molecules which have modified expression during inflammation (PECAM‐1, ICAM‐1, E‐select in, integrin aV, or MCAM); and then analyzed by FACS. Total number of MVs released (MVs/ml) by each of the agonists (TNFa, 615.8±13.9 MV/μL; THR, 113.8±4.8 MV/μL; and H2O2, 152.8±23.5 MV/μL) were significantly greater (P<0.05) compared the number released from cells treated medium alone (82.2±5.4 MV/μL). Each agonist treatment released distinct types of MV identified by FACS. Permeability (% control) of the HBMEC monolayer assessed using FITC‐dextran transport across the monolayers increased after 90 minute exposure to all agonist treatments: 199.5%, 234.1%, 126.8%, and 588.3% for TNFa, THR, AngII, and H2O2, respectively. MV derived from the treated cells were labeled with PKH67 and applied to monolayers of (control) HBMECs cultures. Within 30 minutes of exposure punctate structures with PKH67 labeling were observed within the cytoplasm, and after 60 min, similar structures also associated with nuclei and some of which were co‐labeled with the lysosome marker lysotracker red. Co‐localization of labeled MVs with early endosomes, (labeled with anti‐EEA‐1 Ab/Alexa Fluor 647) was absent at these time points. Within these parameters, no differences in vesicle‐trafficking were observed between MVs generated from cells exposed to either medium, TNFa, or H2O2. These results suggest that treatment of HBMECs with agonists associated with inflammation and thrombosis increase release of distinct MVs which in turn increase paracellular permeability. Capillary endothelial permeability may be disturbed by populations of MVs released from proximal activated cells or MV derived from activated cells and carried in the blood.

Support or Funding Information

Supported by the Aurora Fnd, NIH HL88988 and Mayo Clinic
How the activity of PKCα is increased and maintained in Heart Failure despite the up‐regulation of molecular braking mechanism?
Authors: Aslam Naveed , ZAHEER IRUM
Published: November 2023

Heart failure (HF) disease is marked by dampened cardiac contractility. A milder target which could improve the contractile function without desensitization of β‐Adregenric system may help during HF condition and potentially improve survival. It seems that inhibition of PKCα activity might fit this criterion of a milder therapeutic target to boost contractility for HF patients. Observations show that PKCα activity is increased during HF disease. This seems to be bizarre as the increase in PKCα activity is also accompanied by up‐regulation of a molecular braking mechanism. Here, we explore the question that how PKCα activity can be increased and maintained during HF despite the presence of molecular brakes? More specifically, we investigate the possibility of a positive feedback loop through which PKCα might sustain its activity in heart disease condition. Through a computational approach we show that PKCα activity in cardiomyocytes is regulated through a local DAG signaling system. Our results also show that local DAG cascade is regulated through a two compartment signaling system. Our results imply that after large MI local homeostasis of DAG is disrupted and the loss of this balance leads to prolonged activation of PKCα, a key molecular target linked with LV remodeling and dysfunctional filling and ejection characteristics in mammalian heart. This study also proposes an explanation for how the DAG homeostasis is regulated during normal systolic and diastolic functions?

Support or Funding Information

Privately funded
Spilanthol from Acmella oleracea lowers Na+‐K+‐2Cl− ‐cotransporter activity and water channel aquaporin 2 membrane expression in mouse renal cells
Authors: Gerbino Andrea , Schena Giorgia , Milella Luigi , Miglionico Rocchina , Armentano Mariafrancesca , Barbosa Alan Franco , Procino Giuseppe , Svelto Maria , Carmosino Monica
Published: November 2023

Objective

Acmella oleracea is well recognized in Brazilian traditional medicine as diuretic, although few scientific data have been published to support this effect. Aim of this study was to determine the molecular effect of Acmella oleracea extract and its main alkylamide spilanthol on two major processes involved in the urine concentrating mechanism: Na‐K‐2Cl symporter (NKCC2) activity in the thick ascending limb and water channel aquaporin 2 accumulation at the apical plasma membrane of collecting duct cells.

Methods

Phosphorylation of NKCC2 was evaluated as index of its activation by Western blotting. Rate of aquaporin 2 apical expression was analysed by confocal laser microscopy. Spilanthol‐induced intracellular signalling events were dissected by video‐imaging experiments.

Results

Exposure to spilanthol reduced the basal phosphorylation level of NKCC2 both in freshly isolated mouse kidney slices and in NKCC2‐expresing HEK293 cells. Spilanthol‐induced effect on NKCC2 activity was significantly larger than that exerted by the methanol plant extract. In addition, exposure to spilanthol strongly reduced both desmopressin and low Cl−‐dependent increase in NKCC2 phosphorylation in mouse kidney slices and NKCC2‐expressing HEK293 cells, respectively. Similarly, exposure to spilanthol reduced both desmopressin‐ and forskolin‐stimulated aquaporin 2 accumulation at the apical plasma membrane of collecting duct in mouse kidney slice and MCD4 cells. Finally, at cellular level, spilanthol rapidly reduced or reversed basal and agonist‐increased cAMP levels through a mechanism involving increases in intracellular [Ca2+].

Conclusions

Spilanthol‐induced inhibition of cAMP production negatively modulates urine‐concentrating mechanisms thus holding great promise for its use as both diuretic and aquaretic.

Support or Funding Information

This work was supported by the Ricerca di interesse locale RIL to M. Carmosino, University of Basilicata and by Fondo per gli Investimenti della Ricerca di Base‐Rete Nazionale di Proteomica to M. Svelto (grant number RBRN07BMCT_009).
Postconditioning Reduces Early Reactive Oxygen Species Production in the Brain in a Porcine Model of Cardiac Arrest and Resuscitation
Authors: Matsuura Timothy R , Yannopoulos Demetris , Bartos Jason A , Olson Matthew D , Rees Jennifer N , Tsangaris Adamantios , Shekar Kadambari Chandra , McKnite Scott H , Aufderheide Tom P , Bienengraeber Martin P , Neumar Robert W , Dikalova Anna E , Dikalov Sergey I , Douglas Hunter F , Salzman Michele M , Riess Matthias L
Published: November 2023

Introduction

Postconditioning during cardiopulmonary resuscitation (CPR) is an effective intervention to improve neurological function and survival in a porcine model of prolonged cardiac arrest (CA). We tested if ischemic (IPoC) and anesthetic postconditioning (APoC) decrease reactive oxygen species (ROS) production in the brain upon reperfusion with CPR.

Methods

After 15 min of untreated ventricular fibrillation, 10 pigs were randomized to receive active compression/decompression CPR with IPoC (three 20‐sec pauses in CPR), APoC with 4 Vol% sevoflurane for 3 min, or without postconditioning (control) before organ harvest at 4 min. Cortical brain tissue was immediately frozen in liquid nitrogen and stored at −80°C until ROS measurements were performed by Electroparamagnetic Resonance (EPR). Data are mean±SD. Statistics: ANOVA with SNK‐post hoc testing, alpha = 0.05.

Results

EPR spectral changes showed that APoC (7.0±0.3 arbitrary units) more so than IPoC (7.9±0.3) reduced ROS compared to controls (9.1±0.2).

Conclusions

Both IPoC and APoC result in decreased ROS production in the brain during early CPR. Either strategy has been shown to improve mitochondrial coupling of oxidative phosphorylation and ATP synthesis and delay mPTP opening post CA. Together, our findings provide further evidence that preservation of mitochondrial function is a key element of improving neurological outcome after CA and CPR.

Support or Funding Information

R01HL123227
Malignant Hyperthermia‐associated Mutation of RyR1 Induces Mitochondrial Damages and Cellular Oxidation in the Heart
Authors: O‐Uchi Jin , Mishra Jyotsna , Jhun Bong Sook , Hurst Stephen , Fu Deming , Gomez Ludovic , Sheu Shey‐Shing
Published: November 2023

Human pedigrees carrying the ryanodine receptor type 1 (RyR1) mutations frequently show skeletal muscle (SM) disorders including malignant hyperthermia (MH). There are also case reports describing sudden cardiac death (SCD) in MH patients in the conscious condition without anesthesia. These observations cannot be explained as a secondary effect of their SM dysfunction; however, the molecular mechanism underlying cardiac phenotypes in MH is still unknown. We previously reported that a low level of RyR1 is expressed in the mitochondria (termed as mitochondrial RyR1: mRyR1), but not in the sarcoplasmic reticulum (SR) in the cardiomyocytes and serves as an important mitochondrial Ca2+ influx pathway in addition to the mitochondrial Ca2+ uniporter in cardiomyocytes. We also reported, using knock‐in mice carrying a MH‐related RyR1 mutation Y522S (YS) that YS hearts exhibit disrupted mitochondrial morphology as well as compromised mitochondrial functions with a high cellular oxidative state, which can account for cardiac phenotypes in MH. In addition, YS heart developed multiple ventricular extrasystoles by β‐adrenergic stimulation. Therefore, we hypothesize that YS‐RyR1s form “leaky channel” at mitochondria and induce mitochondrial Ca2+ overload, which alters the cellular Ca2+ handling in cardiomyocyte. Using isolated mitochondria or isolated cardiomyocytes under confocal microscope, we found that YS cardiomyocytes have higher basal mitochondrial Ca2+ concentration, depolarized mitochondrial membrane potential and slower cytosolic Ca2+ clearance as compared to WT. Moreover, pretreatment of RyR1 blocker dantrolene prevented these changes in YS cardiomyocytes and normalized their Ca2+ handling profiles similar to those in WT. In conclusion, these results indicate that chronic mitochondrial Ca2+ overload via leaky mutant mRyR1 damages cardiac mitochondrial functions/structures, reduces cytosolic Ca2+ buffering capacity and induces cellular oxidation, which may increase arrhythmogenic events in MH.

Support or Funding Information

This work was partly supported by American Heart Association (AHA) grant (14BGIA18830032 to J.O.‐U.), Medical Research Grant from W.W. Smith Charitable Trust (H1403 to J.O.‐U.) and NIH grants (2R01HL093671 and 1R01HL122124 to SSS).
Angiotensin II‐mediated Mitochondrial Ca2+ Uptake and Superoxide Generation Activate Proliferative Pathway in Neonatal Cardiac Fibroblasts
Authors: O‐Uchi Jin , Fu Deming , Mishra Jyotsna , Jhun Bong Sook , Hurst Stephen , Sheu Shey‐Shing
Published: November 2023

Cardiac fibroblasts (CFs) are the most prevalent cell type in the heart in addition to cardiomyocytes and play key roles in regulating myocardial physiological function and pathophysiological remodeling. Clinical observations and basic research data strongly suggest that CFs can respond to various stimuli including, angiotensin II (AT‐II), the levels of which are increased in the remodeling heart and participates in remodeling of the failing heart by AT‐II‐mediated pathological CF proliferation. It has been shown that CF proliferation may occur via enhanced production of reactive oxygen species (ROS), but the detailed signal transduction remains unclear. We previously reported that the enhancement of mitochondrial Ca2+ uptake by mitochondrial Ca2+ uniporter (MCU) induces mitochondrial superoxide (mtSO) generation in cardiomyocytes. Therefore, we hypothesize that Ang‐II stimulation enhances mitochondrial Ca2+‐ induced mtSO generation in CFs, which can activate ROS‐dependent proliferation signaling in CFs. First, using a mitochondria‐targeted Ca2+ biosensor, we found that AT‐II (≥1 μM) stimulation induces significant mitochondrial Ca2+ uptake in response to the Ca2+ release from the endoplasmic reticulum in neonatal rat CFs (NCF). In addition, AT‐II stimulation increases the mtSO levels detected by a mtSO indicator MitoSOX Red. These effects were significantly blocked by the pretreatment of an AT‐receptor antagonist losartan. Next, we confirmed that AT‐II application activates proliferative pathway, including ERK1/2, p38 and JNK1/2 in time‐dependent manner, which was abolished by losartan pretreatment. Lastly, pretreatment of a mitochondria‐targeted antioxidant, Mito‐tempo also significantly inhibited AT‐II‐mediated activation of the proliferative pathway without changing the AT‐II‐induced the mitochondrial Ca2+ uptake profile. Our results indicate that mtSO generation induced by mitochondrial Ca2+ accumulation works as a stimulator of the Ang II–induced proliferative pathway in cardiac fibroblasts.

Support or Funding Information

This work was partly supported by American Heart Association (AHA) grant (14BGIA18830032 to J.O.‐U.), Medical Research Grant from W.W. Smith Charitable Trust (H1403 to J.O.‐U.) and NIH grants (2R01HL093671 and 1R01HL122124 to SSS).
High fat‐diet induced obesity does not necessarily promote vascular remodeling/fibrosis in rats
Authors: Fernandes Roxanne , Carter‐Taylor Evan , Galligan James , Xu Hui
Published: November 2023

Inflammation and hyperglycemia are responsible for obesity associated vascular remodeling/fibrosis. Our lab has previously observed that high fat (HF) diet‐induced obesity and hyperglycemia promote vascular remodeling/fibrosis, but HF diet does not necessarily promote hypertension in male mice. However, it is unclear if vascular remodeling/fibrosis and inflammation are unique changes in all HF diet‐induced obese rodent models. In current studies, we compared the blood pressure, vascular and tissue histological structures of heart, mesentery, and kidney in male and female Sprague Dawley rats fed with HF (60 kcal% fat) or control (10 kcal% fat) diet up to 20 or 48 weeks. Vascular responsibilities to norepinephrine and wall thickness/distensibility were evaluated in pressurized mesenteric arteries (MA) and veins (MV) in vitro. The plasma levels of TNA‐α, monocyte chemoattractant protein‐1 (MCP‐1), insulin, glucose and leptin were also evaluated in these rats. HF fed female (20 weeks) and male (48 weeks) rats were obese and hyperleptinemia, but normotensive and with unchanged vascular contractility. These rats did not exhibit vascular remodeling/fibrosis, hyperglycemia, and systemic inflammation, when compared with control diet fed rats. After 48 weeks of exposure to HF diet, female rats were obese, hyperleptinemia, hypertensive and with reduced vascular reactivity to norepinephrine, however, these rats did not exhibit vascular remodeling/fibrosis, hyperglycemia, or systemic inflammation either. Our findings suggested that obesity is not the determinant in the development of vascular fibrosis/remodeling. HF diet induced hyperglycemia and tissue inflammation may be the predominant mediators in obesity associated vascular remodeling/fibrosis.

Support or Funding Information

Supported by Undergraduate Research in Biomedical Sciences Program (NIH grant HL103156, for Dr. Ewart to support Carter‐Taylor) and NIH grant 2P01HL070687 (For Drs Galligan and Xu)
Hydrogen Sulfide Suppresses Diabetic Myocardial ER Stress: An Uncanonical Way
Authors: Veeranki Sudhakar , Metreveli Naira , Tyagi Suresh C
Published: November 2023

Diabetes critically inhibits cardiac endogenous production of hydrogen sulfide (H2S), a cardioprotective gaseous signaling molecule, by suppressing expression of CBS, CSE and 3MST (known H2S producers endogenously) in the mouse models of type 1 diabetes (Akita or C57BL/6‐Ins2Akita/J). Diabetes induced ER stress has been previously identified as an initiator of cardiomyocyte apoptosis and cardiomyopathy. However, the mechanisms of ER stress induction are not clearly illustrated. Furthermore, H2S has been implicated as an inducer or suppressor of ER stress in a tissue type specific manner and molecular mechanisms of H2S mediated ER stress regulation are not entirely known. Here, we show that chronic H2S supplementation resulted in enhanced cardiac function concurrent with suppression of ER stress with significant reversal of elevated CHOP levels in the diabetic hearts. Interestingly, there were no significant changes in the sXBP1 levels in the diabetic hearts with or without H2S treatment suggesting differential regulation of different arms (PERK, ATF6 and IRE1) of ER stress pathway. Importantly, the ER stress suppression is accompanied by suppression of GRP78 (AKA Bip) mRNA levels with a dramatic and paradoxical elevation in its protein levels. Differential translational regulation and/or protein degradation are implicated in regulation of GRP78 protein levels which further highlighted that H2S supplementation not only suppresses ER stress, but also raises the threshold for ER stress induction through elevation of GRP78 protein levels. Remarkably, the H2S mediated diabetic ER suppression was also accompanied with reversal of elevated TLR4 receptors, a well‐known inducer of ER stress upon stimulation. TLR4 mutant diabetic mice (Akita background) are currently studied to unravel relative contribution of TLR4 dependent and independent mechanisms for H2S mediated reversal of ER stress during diabetic cardiomyopathy. In conclusion, H2S mediated raise in threshold for ER stress induction involve GRP78 protein preservation and TLR4 level suppression in the diabetic hearts.

Support or Funding Information

NIH grants : HL108621, HL074185 and DK104653.
Physiological and transcriptomic data have equal variability in male and female rats
Authors: Dayton Alex , Skelton Meredith , Kaldunski Mary , Greene Andrew S , Cowley Allen W
Published: November 2023

There are well known physiological differences between male and female animals that have been studied in multiple organ systems. Yet, the ratio of male to female animal studies in physiology is estimated at 3.7:1; a sex bias that may impact the interpretation and translation of the results to human conditions.

It is well established that many phenotypes are significantly affected by estrogen and other circulating hormones. It is reasonable to suggest that the varying levels of these hormones during the estrous cycle would confound the study of these phenotypes in female rats by increasing the variance in female experimental groups. If the variance were greater in female rats than in males, it would require more experimental subjects (N) to detect an equivalent difference in phenotype. Since this would necessitate a change in experimental design to study female rats, it is important to determine whether this proposed increase in variance was supported by available data.

We analyzed the phenotypic data available on the Rat Genome Database, a large, publically available dataset of physiological measurements of cardiovascular, renal, biochemical, vascular and lung phenotypes made in male and female rats. In total, this dataset comprised ~40 phenotypes measured in 6 genetically distinct strains of rat. We found no evidence that the variance or coefficient of variation was consistently greater in female rats than in male rats.

We then wanted to see whether estrous stage impacted the blood pressure response to salt in the SS rat. In a smaller discrete study of the blood pressure response to high salt diet in a group of 16 female SS rats, we determined the phase of estrous by daily vaginal smears and cytology. At the end of the study, the number of estrous cycles determined for each animal over the three weeks of study (2 weeks of 8% salt). Six rats continued to cycle during the study with 3–5 cycles confirmed. The remaining eight rats had two or fewer cycles confirmed. When the blood pressure response to the high salt diet was analyzed, there was no difference between the cycling and non‐cycling female SS rats.

To assess the impact of the estrous cycle on transcriptomic data, we analyzed the dataset of Y. Yu et al. (Nature Communications, 2014), in which genomic RNA‐seq measurements were made in several organs from both male and female Fischer 344 rats. This dataset consisted of RNA‐seq measurements made in 11 organs. Each RNA‐seq dataset contained measurements for ~20000 individual transcripts. We found no evidence that the variance or coefficient of variation of expression of individual genes was consistently greater in female rats than in male rats.

Together, these analyses suggest that the commonly held view that the estrous cycle necessitates changes in experimental design due to increased variance is not supported by experimental data. We conclude that it is appropriate to use same experimental design for both sexes.
Mycophenolate Mofetil Attenuates Hypertension in an Experimental Model of Systemic Lupus Erythematosus
Authors: Taylor Erin , Strawder Akil , Ryan Michael
Published: November 2023

Mycophenolate mofetil (MMF) is an immunosuppressive agent that selectively depletes proliferating lymphocytes and is commonly used to prevent organ transplant rejection or treat autoimmune disease. Systemic lupus erythematosus (SLE) is a chronic multisystem autoimmune disorder that predominately affects women of childbearing age and is associated with a high rate of hypertension, renal injury, and cardiovascular disease. Recent clinical evidence suggests that treatment with MMF can reduce blood pressure in patients with rheumatoid arthritis or psoriasis; however, whether MMF can be effectively utilized in the control of blood pressure during SLE remains unclear. Because immune dysregulation and inflammation have been linked to the pathophysiology of essential hypertension, we hypothesized that MMF will attenuate hypertension and renal injury in an experimental mouse model of SLE. Female SLE (NZBWF1) and control (NZW) mice, aged 26 weeks, were injected intraperitoneally with 5% dextrose (vehicle) or MMF daily at a rate of 60 mg/kg/day for eight weeks. CD45R+ B cells were lower in the peripheral blood of SLE mice treated with MMF for eight weeks (19.5±1.3% SLE‐Vehicle vs. 5.2±1.5% SLE‐MMF, p< 0.01), but the levels of both CD4+ and CD8+ T cells were unchanged. Circulating levels of anti‐dsDNA IgG auto antibodies, a marker of SLE disease activity, were higher in SLE mice as compared to control mice (789±120 vs. 161±57 units/mL, p<0.0001), and were significantly lower in mice treated for eight weeks with MMF (SLE‐MMF: 321±90; p<0.05 vs. SLE‐Vehicle). Urinary albumin excretion, an indicator of glomerular injury, was increased in SLE mice as compared to controls (5.6±1.9 vs. 0.11±0.02 ng/day, p<0.0001), and was lower in SLE mice treated with MMF (SLE‐MMF: 0.38±0.20 ng/day; p<0.001 vs. SLE‐Vehicle). Mean arterial pressure (MAP; mmHg) measured in conscious mice by carotid catheter was higher in SLE mice than in control mice (141±3 vs. 120±3, p<0.05). MAP was significantly lower in SLE mice treated with MMF when compared to vehicle treated SLE mice (SLE‐MMF: 120±5; p<0.001 vs. SLE‐Vehicle). These data suggest that MMF treatment may have added clinical benefit for patients with SLE by helping to control blood pressure, a major contributor to cardiovascular risk in this population. Moreover, given that MMF selectively depleted CD45R+ B cells, these data provide additional evidence that B cells and the production of auto antibodies mechanistically contribute to the pathogenesis of hypertension during SLE.

Support or Funding Information

Research supported by: VA# I01BX002604‐01A2 (MJR), T32HL105324 (EBT), HL051971 (UMMC‐Physiology)
Improvement in Blood Pressure and Renal Injury Following Vagal Nerve Stimulation
Authors: Maloy Charles I , Fairley Amber S , Pham Grace S , Mathis Keisa W
Published: November 2023

The concept that autoimmunity and its subsequent inflammation could play a role in the pathogenesis of hypertension has recently been supported by many studies. Such inflammatory responses are also associated with end organ damage in hypertension. Recent studies have proposed a novel pathway, the cholinergic anti‐inflammatory pathway, which may combat this inflammation and prevent the subsequent damage. Specifically, stimulation of the vagus nerve has been shown to reduce inflammation via the cholinergic anti‐inflammatory pathway. Systemic Lupus Erythematosus (SLE), an autoimmune disease with profound systemic inflammation and a high prevalence of hypertension, provides an appropriate model to test whether an impaired cholinergic anti‐inflammatory pathway plays a role in development of chronic inflammation and hypertension. We tested the hypothesis that pharmacological stimulation of the vagus nerve reduces blood pressure and improves renal injury and function in SLE mice (NZBWF1). Starting at 30 weeks of age, female SLE and control (NZW) mice were treated twice per week over four weeks with CNI‐1493 (Semapimod hydrochloride; 8 mg/kg, IP), which has been shown to stimulate the vagus nerve. Saline was used as the vehicle treatment. At the end of the study, mean arterial pressure (mmHg) was higher in SLE mice compared to controls (143±11 vs. 116±4; p=0.003; n=3–5/group). Treatment with CNI‐1493 attenuated the development of hypertension in the SLE mice (130 ± 4), but did not alter mean arterial pressure in controls (119 ± 5). Renal injury in the form of albuminuria (albumin excretion rate; μg/day) was increased in SLE mice compared to controls (4409±2519 vs. 4 ± 1; p=0.002; n =3–6/group). Albumin excretion rate was diminished in CNI‐1493‐treated SLE mice (420±410; p = 0.008). Renal blood flow (mL/min*g kidney weight) was measured and renal vascular resistance was calculated (mmHg*min*g kidney weight/mL) as indices of renal function. CNI‐1493‐treated SLE mice had higher renal blood flow (3.0 vs. 2.5±0.1; n=1–2/group) and lower renal vascular resistance (21.9 vs. 23.9±1; n=1–2/group) compared to SLE mice treated with saline. These data suggest that stimulation of the vagus nerve may protect the kidney and prevent SLE hypertension; however, future studies are warranted to determine how this occurs. Taken together, our findings suggest that the vagally‐mediated cholinergic anti‐inflammatory pathway plays a mechanistic role in the development hypertension in the setting of chronic inflammation.

Support or Funding Information

Studies supported by the AHA (#14SDG18320033) and UNTHSC (Faculty Pilot Grant and Research Seed Funding)