bluuebunny/crossref_metadata_2025 huggingface dataset:
GPER‐1 Agonist Reduces Angiotensin‐II Induced Oxidative Stress in Rat Aortic Smooth Muscle Cells
Authors: Duong Jennifer Lee , Baker Tyler Pierce , Katakam Prasad , Lindsey Sarah Hoffman
Published: November 2023

We previously showed that the G protein‐coupled estrogen (GPER) reduces oxidative stress in the kidneys and aorta of female mRen2 rats, a model of angiotensin (Ang) II‐dependent hypertension. The goal of the current study was to assess the mechanisms by which GPER reduces oxidative stress. We hypothesized that GPER activation reduces Ang II‐mediated oxidative stress in rat aortic smooth muscle cells (ASMC) by inhibiting phosphorylation and translocation of Rac1, a small GTPase that recruits subunits to the NADPH oxidase complex. A7r5 cells were treated overnight with either 100 nM of Ang II in the presence of vehicle or 100 nM G‐1. Superoxide levels were measured via electron spin resonance spectroscopy, and RAC and phospho‐RAC were measured via Western blot. Activation of GPER by the agonist G‐1 significantly reduced Ang II‐induced superoxide levels (P<0.05) in AMSC. While there were no significant changes in RAC expression, phospho‐RAC was reduced with G‐1 treatment (P<0.05). We conclude that GPER exerts vascular protection against Ang II‐mediated superoxide production by reducing the phosphorylation of RAC. This anti‐oxidant action of the novel GPER may be one mechanism by which estrogen protects against cardiovascular disease.

Support or Funding Information

This study was funded by National Institutes of Health grant 4R00HL103974 to S.H.L
Chronic Paroxetine Treatment in Mice Leads to Adiposity and Glucose Intolerance
Authors: Zha Weibin , Wang Joanne
Published: November 2023

Selective serotonin reuptake inhibitors (SSRIs) are the most widely prescribed class of antidepressants for the treatment of depression and anxiety disorders. Recent epidemiologic results have suggested an association between long term use of SSRIs and an increased risk for type 2 diabetes. However, depression itself has been associated with diabetes, and it therefore is unclear whether the association is solely due to SSRI use, depression, or an interaction of both circumstances. The goal of this study is to determine the effects of chronic paroxetine (a common SSRI) treatment on adiposity and glucose tolerance in wild‐type (WT) mice and mice with targeted disruption of serotonin reuptake transporter (SERT−/−). Three ‐to‐four week old male C57BL/6 mice (n=5–6 per group) were treated with paroxetine (10 mg/kg/day) or control vehicle for 12 weeks. This dose is known to achieve serum concentrations in mice comparable to the therapeutic levels in humans. Compared to vehicle control, paroxetine‐treated WT mice exhibited significantly increased weight gain (+20.4±0.7 vs +17.1±0.4 g, P<0.05 vs. WT‐control), adipose tissue expansion, and pro‐lipogenic gene expression. Glucose and insulin tolerance tests further revealed that paroxetine‐treated WT mice exhibited glucose intolerance [glucoseAUC0–120 min(2 g/kg IP following fasting):30.6±0.7 vs. 26.6±0.7g*min/dl, P<0.05 vs. WT‐control] and insulin resistance [glucose AUC0–120 min(following fasting and insulin0.75 units/kg IP): 24.9±1.2 vs. 20.1±1.2g*min/dl, P<0.05 vs. WT‐control]. In parallel, three to‐four week old male SERT−/− mice (n=5–6 per group) were also treated with paroxetine or control vehicle for 12 weeks. Compared to non‐treated SERT−/− mice, paroxetine‐treated SERT−/− mice showed minimal difference in weight gain and a significant attenuation of fat mass expansion. Collectively, these findings demonstrate that chronic paroxetine treatment at a clinically relevant dose leads to adiposity and glucose intolerance in mice, and this effect is largely SERT‐dependent.

Support or Funding Information

This study is supported by NIH grant R01 GM066233 and the Elmer M. Plein Endowment Research Fund.
A Nutraceutical Formulation Improves Cardiometabolic Parameters in Obese/Overweight Subjects
Authors: Nair Sreejayan , Hua Yinan , Ren Dylan Y , Wang Qiurong , Rasmussen Tyler Pat , Smith Derek T , Robinson Timothy J , Nair Rama P , Muhammed Majeed , Kalyanam Nagabhushanam
Published: November 2023

Obesity is a growing epidemic worldwide and is an independent risk factor for diabetes and cardiovascular disease. GlycaCare™ ‐Plus is a novel proprietary dietary formulation comprising Pterocarpus masupium extract, cinnamon extract, biotin, piperine, magnesium fumarate, zinc monomethionine, copper lysine, chromium polynicotinate and bimaltoxovanadium.

The objective of this study was to evaluate the effect of GlycaCare™‐Plus on glucose tolerance and cardiometabolic parameters in a randomized, double blind, placebo‐controlled study.

Methods

Overweight or obese subjects (n=21) were randomly assigned to receive either GlycaCareTM Plus oral (500 mg/day) or placebo (microcrystalline cellulose), twice daily, for 90 days. Multiple endpoints including blood pressure, body weight, oral glucose‐tolerance test, serum glucose, insulin, lipid profile, body‐fat distribution, serum markers of inflammation (C‐reactive peptide, interleukin‐6), and serum oxidative stress (8‐isoprostane) were assessed at baseline and after 90‐days following treatment. Subjects receiving GlyacaCare™‐Plus supplement exhibited attenuated insulin resistance as and attenuation of serum oxidative stress compared to those that received placebo, as evidenced by a reduction in HOMA‐IR and serum 8‐isprostane levels respectively in subjects. The supplement did not alter body mass, fat distribution, inflammatory parameters, serum lipids or glucose disposal.

Conclusion

Preliminary studies demonstrate that GlyacaCare™‐Plus supplement alleviates insulin resistance and reduces plasma oxidative stress in overweight/obese subjects.

Support or Funding Information

Funded by a research grant from Sabinsa Corporation, New Jersey.
The Role of CaMKIId in Angiotensin II‐Induced Cardiac Inflammation and Fibrosis
Authors: Willeford Andrew
Published: November 2023

The Ca2+/calmodulin‐dependent kinase, CaMKIId, is an established mediator of the development of heart failure and myocardial injury. Cardiac inflammation has been increasingly recognized as an important player in these cardiac pathophysiological changes. We previously demonstrated that CaMKIId contributes to cardiac inflammation induced by ischemia/reperfusion through activation of cardiomyocyte nuclear factor kappa B (NF‐kB). In the current study we ask whether angiotensin II (Ang II) promotes cardiac inflammation through CaMKIId and its effects on NF‐kB activation. In addition, Ang II treatment promotes cardiac fibrosis and we hypothesize that this response is initiated through activation of CaMKIId in the cardiomyocyte and subsequent inflammatory responses. Here, we report on preliminary data demonstrating attenuation of inflammatory gene expression (e.g. MCP‐1, MIP1a, IL‐6, TNFa) in cardiomyocyte‐specific CaMKIId KO (CKO) mice following 1 and 7 days of Ang II infusion. In addition, the expression of fibrotic markers (e.g. col1a1, col3a1, TGFβ, and CTGF) in response to Ang II infusion is decreased in CKO mice following 7 days of infusion. This is associated with attenuated fibrosis as evident in histological analysis of CKO vs control heart sections. To note, we do not see increased expression of fibrotic markers after 1 day Ang II infusion, suggesting that inflammation precedes fibrosis. Furthermore, we have demonstrated that inhibition of CCR2, the receptor for MCP‐1, attenuates inflammatory gene expression induced by 1 day of Ang II infusion. Currently, we are determining whether Ang II acts through CaMKIId in the cardiomyocyte to activate NF‐kB‐mediated MCP‐1 gene transcription and whether blockade of CCR2 is sufficient to attenuate Ang II‐induced fibrosis. Findings from these studies may implicate CaMKIId as a promising therapeutic target for attenuating cardiac fibrosis.

Support or Funding Information

Pharmacology Training Grant (2T32‐GM007752) and UCSD Skaggs School of Pharmacy and Pharmaceutical Sciences
Kruppel‐Like Factor 15: A Critical Transcriptional Regulator Of Hypoxia Induced Endothelial Arginase 2
Authors: Pandey Deepesh , Hori Daijiro , Santhanam Lakshmi , Berkowitz Dan , Romer Lewis
Published: November 2023

Objective

Kruppel Like Factor 15 has recently been shown to be critical for activation of proinflammatory processes in vascular smooth muscle and atherogenesis. Although KLF15 is abundantly expressed in vascular endothelium there is a significant lack of knowledge regarding the role of KLF15 in the regulation of vascular endothelial function. Here we tested the hypothesis that a KLF15 is a critical regulator of Arg2 transcription in response to hypoxia.

Approach and Results

Adenovirally mediated overexpression of KLF15 in human pulmonary microvascular endothelial cells (HPMEC) and in rat pulmonary arteries decreased Arg2 mRNA, protein, and activity. HPMECs exposed to 48 hours of hypoxia exhibited a robust increase in Arg2 protein and mRNA expression and a reciprocal decrease in KLF15 protein levels that remain sustained after 24 hours of reoxygenation. Further, decreased KLF15 protein levels observed in hypoxia exposed HPMEC occurs through augmented conjugation of Ubiquitin to KLF15 that results in enhanced clearance of KLF15 via proteasomal degradation. Chromatin immunoprecipitation indicated direct binding of KLF15 to the Arg2 promoter, which was relieved when HPMEC were exposed to hypoxia, Finally, overexpression of KLF15 reversed hypoxia‐induced augmentation of arginase activity and decrements in Nitric Oxide production in HPMEC, and also reversed hypoxia‐induced endothelial dysfunction in isolated mice aortic and rat pulmonary artery rings were.

Conclusions

KLF15 is a critical regulator and repressor of endothelial Arg2 expression, and thereby, NO and pulmonary endothelial function. Overexpression or activation of KLF15 may represent novel therapeutic strategies for pulmonary hypertension.

Support or Funding Information

NIH R01‐HL089668 NIH R01‐HL124213 American Heart Association Postdoctoral Fellow Award from American Heart Association (13POST16810011) to D. Pandey
Characterization Of β2‐containing Receptors in Rat Brainstem Using [125I]A‐85380
Authors: Davila‐Garcia Martha Isabel , Kamendi Harriet , Ogunlade Blessing Ogunlade , Robinson Anastasia
Published: November 2023

Nicotinic receptor diversity in brainstem has prompted us to determine the distribution and pharmacological characteristics of the β2‐containing. We used [125I]A‐85380 radioligand which is selective for receptors containing the β2 subunit. Our hypothesis was that β2‐containing nicotinic receptors would be present particularly in cardiorespiratory centers in the brainstem. First, we characterized the binding of [125I]A‐85380 in rat cerebral cortex (CTX). [125I]A‐85380 reached equilibrium in two hours and was found to have a Kd value of ~180pM and a Bmax of ~ 110 fmol/mg protein. In competition assays the rank order of potency for nicotinic receptor agonists was epibatidine > unlabeled A‐85380 > cytisine > nicotine > DMPP and for the antagonists the rank was DHbE > mecamylamine while upto 1mM k‐bungarotoxin did not displace any [125I]A‐85380 binding. In receptor binding and autoradiography studies where cytisine, DMPP, DHβE, epibatidine and nicotine were used to displace [125I]A‐85380, no additional binding sites were found, in all cases, binding was completely blocked by these ligands. Binding of [125I]A‐85380 was found at moderate to high levels in medial raphe areas some of which were insensitive to DHβE, and in most lateral regions of both caudal and rostral medulla.

Support or Funding Information

American Heart Association (MDG) 9930049N
Unexpected Negative Role of the AT1R Adaptor Protein βarrestin1 in the ERK1/2 Activation of SHR‐Derived Primary Astrocytes
Authors: Negussie Shmuel , Lymperopoulos Anastasios , Clark Michelle Alexa
Published: November 2023

Objective

To investigate the role of βarrestin1 in angiotensin II (AngII) type 1 receptor (AT1R)‐mediated extracellular signal‐regulated kinase (ERK1/2) activation in spontaneously hypertensive rat (SHR)‐derived primary astrocytes.

Background

The βarrestins, consisting of two isoforms, βarrestin1 and ‐2, originally discovered as terminators of G protein signaling by G protein‐coupled receptors (GPCRs), are now known to also mediate their own signaling to ERK1/2 activation independently of G proteins. The AT1R is a GPCR that can also signal to ERK1/2 via βarrestins in various cell types. However, whether it can do so in the brain has not been investigated yet. Thus herein, we sought to examine the involvement of βarrestins in the AT1R‐mediated ERK1/2 activation in SHR‐derived primary astrocytes.

Methods

Primary cultures of rat astrocytes from brainstem were isolated from the brains of 2–3 days old pups obtained from pregnant SHR. 100 nM AngII and 10 μM SII (an AngII peptide analog which is βarrestin‐biased agonist) were used to study the AT1R mediated ERK1/2 activation. The results were compared to normotensive Wistar rats. Protein analysis was done using Western blotting and mRNA measurement was done using real time PCR.

Results

Our results indicate that βarrestin1 is the major arrestin protein in astrocytes from both SHR and normotensive rats. At the mRNA level however, both βarrestin1 and βarrestin2 display comparable expression. AngII activates ERK1/2 in SHR astrocytes much more robustly than in normotensive rat ones. On the other hand, SII strongly activates ERK1/2 in normotensive rat astrocytes and only minimally in SHR ones. Importantly, AngII also upregulates βarrestin1 protein in normotensive rat astrocytes, whereas in SHR astrocytes it has no significant effect. Finally, AngII downregulates both βarrestins at the mRNA level in both SHR and normotensive rat astrocytes.

Conclusion

The AngII‐AT1R‐induced ERK1/2 activation of SHR astrocytes, known to induce hypertension, appears to be almost exclusively G protein‐dependent, contrary to normotensive rat astrocytes. Since βarrestin1 protein is also upregulated by AngII in normotensive rat astrocytes and appears downregulated in SHR astrocytes, it is quite plausible that βarrestin1 may actually inhibit ERK1/2 activation in astrocytes. This might be a beneficial effect of βarrestin1 in preventing development of hypertension in the SHR condition.

Support or Funding Information

Funding for this research was provided through a Nova Southeastern University Health Professions Division Grant
Calpain‐2 Activates Akt via TGFβ1‐mTORC2 Pathway in Pulmonary Artery Smooth Muscle Cells
Authors: Abeyrathna Prasanna Kumara , Kovacs Laszlo , Su Yunchao
Published: November 2023

Calpain, a calcium‐dependent nonlysosomal neutral cysteine endopeptidase, activates an intracrine TGFβ pathway in pulmonary artery smooth muscle cells (PASMCs) and plays an important role in smooth muscle cell hyperproliferation and collagen synthesis in pulmonary vascular remodeling of pulmonary arterial hypertension (PAH). Akt is a serine‐threonine kinase involved in many cellular processes including cell proliferation and collagen synthesis. In this study, we investigated the connection between calpain activation and Akt in the collagen synthesis and cell proliferation of PASMCs caused by PDGF. We found that inhibition of calpain‐2 by calpain inhibitor, MDL28170 and calpain‐2 siRNA attenuated Akt phosphorylation at serine‐473 (S473) and threonine‐308 (T308) as well as collagen synthesis and cell proliferation of PASMCs induced by PDGF. Overexpression of calpain‐2 in PASMCs induced dramatic increases in Akt phosphorylation at S473 and T308. The cell‐permeable specific TGFβ receptor inhibitor SB431542 attenuated Akt phosphorylation at both S473 and T308 induced by PDGF and by overexpressed calpain‐2 in PASMCs. Moreover, SB‐431452 and knocking down ALK5 significantly reduced PDGF‐induced collagen synthesis and cell proliferation of PASMCs. Nevertheless, neutralizing extracellular TGFβ1 using a cell‐impermeable TGFβ1 neutralizing antibody did not affect PDGF‐induced Akt phosphorylation at S473 and T308. Further, inhibition of mTORC2 by knocking down its component protein Rictor prevented PDGF‐induced Akt phosphorylation at S473 and T308. Additionally, calpain inhibitor MDL28170 inhibited PDGF‐induced increase in mTORC2 activity. These data provide first evidence supporting that calpain‐2 up‐regulates PDGF‐induced Akt phosphorylation via an intracrine TGFβ1/mTORC2 mechanism.

Support or Funding Information

Supported by NIH, FAMRI and Department of Veterans Affairs
Lubiprostone Stimulates HCO3− Secretion in Rat Stomach and Duodenum mediated by Different EP receptor Subtypes
Authors: Takeuchi Koji
Published: November 2023

A recent study showed that lubiproston, a bicyclic fatty acid derived from prostaglandin (PG) E1, stimulated CFTR‐dependent duodenal HCO3− secretion in rat, and this action was mediated by the activation of EP4 receptors. In the present study, we examined the stimulatory effect of lubiprostone on HCO3− secretion in the rat stomach and duodenum, focusing on the EP receptor subtypes involved in this action. Male SD rats were used after 18 h fasting. Under urethane anesthesia, an ex‐vivo chambered stomach or a duodenal loop was perfused with saline, and HCO3− secretion was measured using a pH stat‐method. lubiprostone significantly increased HCO3− secretion in both the stomach (>30 μM) and duodenum (>1 μM). The stimulatory action in the stomach was significantly abrogated by prior administration of the EP1 antagonist but not by the EP3/EP4 antagonists or the CFTR inhibitor, while that in the duodenum was significantly attenuated by the EP3 antagonist and the EP4 antagonist as well as the CFTR inhibitor. These results suggest that lubiprostone stimulates HCO3− secretion in the stomach and duodenum mediated by different EP receptor subtypes; the former mediated by EP1 receptors and the latter mediated by both EP3 and EP4 receptors. It is also assumed that CFTR is involved in modulating HCO3− secretion in the duodenum but not the stomach

Support or Funding Information

None
Anti‐inflammatory and Antioxidant Effects of Flavonoid‐Rich Fraction of Bergamot Juice (BJe) in a Mouse Model of Intestinal Ischemia/Reperfusion Injury
Authors: Impellizzeri Daniela , Cordaro Marika , Campolo Michela , Esposito Emanuela , Cuzzocrea Salvatore , Navarra Michele
Published: November 2023

The flavonoid fraction of bergamot juice (BJe) has demonstrated radical‐scavenging and anti‐inflammatory activities. The aim of this study was to investigate the effects of BJe on the modulation of the ileum inflammation caused by intestinal ischemia/reperfusion (I/R) injury. This was induced in mice by clamping the superior mesenteric artery and the celiac trunk for 30 min, followed by release of the clamp, allowing reperfusion for 1 h. After 1 h of reperfusion, animals were killed for histological examination of the ileum tissue, the expression of proinflammatory cytokines (tumor necrosis factor TNF‐α and interleukin IL‐1β) and adhesion molecules (intercellular adhesion molecule 1 ICAM‐1 and P‐selectin); for biochemical assays such as myeloperoxidase activity (MPO) and malondialdehyde (MDA) levels. Moreover, by Western blot analysis, we also investigated the expression of nuclear factor NF‐κB, IκB‐α, inducible nitric oxide synthase (iNOS) and the expression of antioxidant such as MnSOD. The results obtained from histological and molecular examinations showed that BJe was able to reduce histological damage, cytokines production, adhesion molecules expression, neutrophil infiltration and oxidative stress. In addition, NF‐κB and MAP kinases pathways were also modulated by BJe treatment. In conclusion, in this study we demonstrated that BJe could represent a possible treatment against secondary events of intestinal I/R injury.
Use of a Non‐Native Chloride Channel as a Treatment for Interstitial Cystitis
Authors: Beckel Jonathan M , Tillman Tommy S , Tang Pei , de Groat William C , Xu Yan
Published: November 2023

Interstitial cystitis (IC) is a disease of the urinary bladder that causes increased urgency to void and pelvic pain. It is commonly thought that increased bladder sensations in IC are due to sensitization of bladder afferent nerves by inflammatory mediators. Current therapies aimed at known targets in the inflammatory or nociceptive pathways have, to date, been ineffective at reducing the symptoms in IC patients. To find potential new treatments for IC, we expressed a non‐native chloride channel (EG3RF) in bladder afferent neurons. This family of channels can be activated by either inflammatory conditions or by an exogenous pharmaceutical compound, but has no activity under normal physiological conditions. We hypothesize that successful transfection of the EG3RF chloride channel into rat bladder afferent nerves will diminish bladder hyperexcitability in the cyclophosphamide model of bladder inflammation. To transfect bladder afferent nerves, an expression plasmid containing the sequence for EG3RF was encapsulated in liposomes and instilled into the urinary bladder of Sprague Dawley rats using a transurethral catheter. Bladder activity was measured 1–2 weeks later using metabolic cages or bladder cystometry. Bladder inflammation was induced by intraperitoneal injection of cyclophosphamide (CYP, 150 mg/kg) 1 hour prior to measurement of bladder activity. Successful expression of the EG3RF channel up to 2 weeks following transfections was confirmed in the bladder smooth muscle, urothelium and dorsal root ganglia (T11–12, L1–L2 and L6‐S1 levels) using RT‐PCR, Western blot, and immunofluorescence analyses. Metabolic cage and cystometry experiments indicated no differences in voiding function between the control and non‐inflamed EG3RF‐transfected animals. However, EG3RF‐transfected rats were resistant to the excitatory effects of cyclophosphamide treatment. Treatment with cysteamine, which has been shown to activate EG3RF, decreased CYP‐induced bladder activity in both metabolic cage and cystometry experiments (31 mg/kg ip or iv, respectively) in EG3RF‐transfected rats but was ineffective in control rats. Intravesical instillation of cysteamine (5 mM) during cystometry in EG3RF‐transfected rats excited bladder reflexes while iv administration (31 mg/kg) decreased peak micturition pressure. These data suggest that expression of a non‐native chloride channel in bladder afferent nerves may be an effective treatment for painful bladder disorders such as interstitial cystitis. Further development of the technique may lead to significant advances in efficacy over existing treatments.

Support or Funding Information

T32 GM075770 (Xu); K01 DK106115 (Beckel); R01 DK091253 (de Groat)
VEGF knockdown in muscle improves recovery of blood flow and arteriolargenesis after ischaemia
Authors: Machado Maria J C , Riu Federica , Benest Andrew V , Bates David O
Published: November 2023

Vascular Endothelial Growth Factor (VEGF) signalling is essential for endothelial cell homeostasis and growth. It has thus been assumed that VEGF would be important during the revascularization process that occurs in ischaemic limbs after arterial occlusion. We used a transgenic mouse line in which VEGF is inducibly knocked out in skeletal muscle cells (ACTA1‐rtTA, tetO‐Cre, VEGF‐LoxP mice), to test the hypothesis that lack of VEGF‐A hinders the recovery of blood flow after ischaemia. In adult triple transgenic mice, homozygous for VEGF‐LoxP (ACLL mice), skeletal muscle specific VEGF‐A knockout was achieved by dosing with doxycycline (+Dox) for 10 weeks. They then underwent unilateral hindlimb ischaemia surgery under isoflurane anesthesia. Blood flow to the hindpaws was monitored by laser speckle imaging. The ratio of blood flow in the ischaemic to contralateral limb was significantly improved when compared to ACLL mice dosed with vehicle alone (0.81±0.09 +Dox vs 0.47±0.05 ‐Dox at day 21; p<0.001). Although there was no difference in capillary density (mean±SEM, mm−2) between these groups of mice (334±24 +Dox vs 261±21 ‐Dox, p>0.05), the arteriole density was significantly higher in ACLL +Dox mice (41±6 +Dox vs 20±2 ‐Dox, p<0.01). Exogenous administration of VEGF by local injection of adenovirus over‐expressing VEGF‐A165a to ACLL +Dox mice, inhibited the increase in blood flow recovery (p<0.01). Injection of a control virus (Ad.eGFP) did not affect the rate of recovery after ischaemia in ACLL +Dox mice. These results suggest that VEGF of skeletal muscle origin is detrimental, not beneficial, to arteriolargenesis (the growth of new arterioles).

Support or Funding Information

Medical Research Council and British Heart Foundation
Serine 275 and serine 328 in the carboxyl‐terminus regulates phosphorylation‐dependent anti‐proliferative properties of connexin 37
Authors: Jacobsen Nicole Lynne , Taylor Samantha , Pontifex Tasha , Burt Janis
Published: November 2023

Connexin 37 (Cx37), but not Cx40 or Cx43, suppresses the proliferation of rat insulinoma (Rin) cells, an effect requiring both its channel function and regulation mediated by the carboxyl‐terminus (CT). Additionally, data from cells expressing alanine or aspartate substituted forms of Cx37 suggest that phosphorylation at specific serine residues (275, 285, 302, 319, 321, 325, 328) within the CT directs cell survival and proliferation; “dephosphorylated” Cx37‐CT (use of alanine substitution) results in cell cycle arrest while “phosphorylation” of the Cx37‐CT (aspartate substitution) induces cell death. Interestingly, aspartate substitution at only serines 275, 319, 328 (S3D3) neither limits Rin cell growth nor induces cell death. Rather, there is no effect on the rapid Rin cell proliferation despite its expression. As the first identified full‐length form of Cx37 that retains channel functionality while supporting cell proliferation, we investigated whether phosphorylation at a single site would be sufficient to control cell cycle progression. Single point mutations with alanine and aspartate were made at each of the three sites and subsequently expressed in Rin cells. Aspartate substitution at serine 275, a putative MAPK site, induces cell death while alanine substitution does not have a significant impact on the anti‐proliferative effects of Cx37‐WT. Mutation of serine 319 to either an alanine or aspartate no longer limits Rin cell proliferation, suggesting that this site may not be critical to phenotype determination. Finally, expression of Cx37‐S328A (alanine substitution at a putative PKC site) results in cell death but Cx37‐S328D supports proliferation. Together, these data may suggest that serine 275 and 328 cooperate in modulating cell proliferation in a phosphorylation‐dependent manner.

Support or Funding Information

R01HL058732 and T32HL007249
Therapeutic Transplant Efficacy of Endothelial Progenitor Cells is Impaired by NK Cells
Authors: Exner Eric C , Abel Alex C , Malarkannan Subramaniam C , Greene Andrew C
Published: November 2023

Adult stem cells, including endothelial progenitor cells (EPCs), have long been proposed as a source of cells for therapeutic transplantation in cardiovascular (CV) disease. Studies investigating the therapeutic potential of EPCs have shown mixed results, likely due to the negative impact of chronic disease and aging on EPC function in the high CV risk patients enrolled in such studies. Using genetically controlled rats, we have established a model of EPC dysfunction in the setting of hypertension. In the SS/MCWi rat, a genetic model of salt‐sensitive hypertension, EPC function is compromised. Introgression of the 13th chromosome of the Brown Norway rat onto the SS background results in the SS‐13BN/MCWi (BN13) rat, which is protected from both hypertension and EPC dysfunction. Previously, we found increased expression of the natural killer cell receptor/ligand 2B4 (CD244) on the cell membrane of EPCs isolated from SS rats. This increase in CD244 expression was correlated with increased destruction by NK cells from both SS and BN13 rats. Here, we hypothesize that increased membrane expression of CD244 by SS EPCs leads to destruction by NK cells in vivo, which compromises their therapeutic efficacy. We first found that siRNA mediated CD244 knockdown rescued EPCs from NK cell mediated killing in vitro using an LDH release assay. We next used an NK cell depleting antibody to assess whether NK cells were responsible for EPC dysfunction in animals receiving EPCs as therapy. Finally, we transplanted EPCs treated with CD244 siRNA into naïve recipient animals to confirm that CD244 impacts EPC function in vivo. Our results suggest that increased CD244 on the SS EPC membrane leads to increased NK cell mediated killing of SS EPCs in vivo, contributing to the impaired therapeutic efficacy in our model.

Support or Funding Information

National Institutes of Health grant HL082798 awarded to A.S. Greene, PhD; T32 HL007852 PI: H.V. Forster
Differential Expansion of the Capillary Bed through Physiological Angiogenesis in Skeletal Muscle
Authors: Kissane Roger , Egginton Stuart
Published: November 2023

A range of well‐established in vivo models of angiogenesis currently exists, involving surgical and pharmacological intervention. These approaches generally improve global measures of microvascular supply, but are rarely interrogated for their capacity to improve tissue oxygen tension. The aim of this research was to characterise a variety of muscle recruitment patterns known to induce angiogenesis, determine their effect on capillary spatial heterogeneity, and subsequently local tissue oxygen delivery.

Male Wistar rats underwent seven days of angiogenic remodelling through indirect electrical stimulation of skeletal muscle (at 4, 10 or 40Hz), muscular overload (through synergist ablation) or pharmacological supplementation (Prazosin, alpha‐adrenergic blocker). Muscle samples were snap frozen and capillary content identified through Griffonia simplicifolia lectin staining, with morphometric analysis of muscle composition used as the basis for oxygen consumption modelling.

We demonstrated that seven days of supramaximal indirect electrical stimulation induced significant angiogenesis (capillary to fibre ratio (C:F) increased by 59, 36 and 45% for 4, 10 and 40Hz, respectively). A similar response for muscular overload and prazosin supplementation were seen with a 42 and 27% increase, respectively. This improvement in capillarity was accompanied by a reduction in mean capillary domain size (tissue supply area), but with varying degrees of spatial heterogeneity.

Skeletal muscle overload and 40Hz electrical stimulation showed a substantially more homogeneous capillary distribution, and subsequently improved tissue oxygenation at high levels of simulated exercise. Conversely, the improved capillarity associated with prazosin and 4Hz electrical stimulation showed no extensive change in capillary distribution. We therefore conclude that mechanical stimulation of angiogenesis may be accentuated by a metabolic component.
Angiogenesis and Expression of IGF2R, IGF2, uPAR, and TGFB in a Rat Obesity Model
Authors: Bayless David S , Sheldon Ryan D , Linden Melissa A. , Rector R. Scott , Laughlin M. Harold
Published: November 2023

Introduction

Our group has previously shown that obesity decreases capillary density in skeletal muscle. Here, we tested the hypothesis that decreased capillary density in obesity is the result of increased activity of the insulin growth factor II receptor (IGF2R) in degrading urokinase‐plasminogen receptor (uPAR), activating transforming growth factor beta (TGFB), and degrading insulin growth factor II (IGF2) using an obese, diabetic rat model.

Methods

At four weeks of age, normophagic Long‐Evans Tokyushima Otsuka (LETO; n=20) and hyperphagic Otsuka Long‐Evans Tokushima Fatty (OLETF; n=20) rats were provided with either a normal, low‐fat diet (LFD, 10% kcal fat) or a high‐fat, high‐cholesterol diet (HFD, 45%kcal fat with 1% cholesterol). At 32 weeks of age, samples from rats were taken, including biceps brachii and vastus lateralis muscle, and arterial vessels. Whole tissue muscle samples were snapfrozen and cold sectioned. Slides were stained for IHC highlighting of capillaries, with manual counting of capillaries and muscle fibers in cross‐section. Vessels were homogenized in buffer and processed by way of Western blot procedure, with membranes probed for IGF2R, IGF2, TGFB, and uPAR.

Results

Capillary density in biceps brachii was reduced in OLETF LFD rats (p<0.07) compared to LETO LFD rats, but HFD increased capillary density significantly relative to LFD counterpart, especially in OLETF rats (19%, p<0.01). In addition, vastus lateralis IHC indicated a significant decrease in capillary density in OLETF LFD/HFD and LETO HFD relative to LETO LFD (p<0.05). Western blot analysis of arterial vessel samples did not indicate any significant difference in IGF2R, IGF2, TGFB, or uPAR between LETO LFD and other groups.

Discussion

These data demonstrate that capillary density is significantly downregulated in obese, type 2 diabetic OLETF rats. However, examination of arterial samples suggest that changes may not be related to changes in expression of IGF2R and associated proposed down‐regulators of vascular growth in the arterial vasculature. To complete the test of our hypothesis we will measure IGF2R, uPAR, IGF2, and TGFB expression in muscle tissue to determine whether changes in expression occur in the muscle that may mediate changes in capillarity in obesity/diabetes.

Support or Funding Information

We Acknowledge Support of this research: NIH HL36088(MHL), VHA CDA2 1BX001299 (RSR), Society of Phi Zeta, and the University of Missouri Department of Medical Pharmacology and Physiology.
Increased Cerebral Capillary Density in Aquaporin‐4 Knockout Mouse
Authors: Xu Kui , Almotah Khalil , Zhang Yifan , Radford Thomas S , Yu Xin , Boron Walter F , LaManna Joseph C
Published: November 2023

In the brain, Aquaporin 4 (AQP4) is highly expressed in the blood–brain barrier and brain–CSF interfaces. AQP4 functions as an efficient water‐selective transporting protein and plays an important role in cerebral water balance. It has been shown that knocking out of AQP4 in mice reduces brain edema after acute water intoxication and ischemic stroke. The specific localization of AQP4 to these anatomical and cellular regions of the central nervous system may also suggest a role for AQP4 as a gas channel to facilitate oxygen transport. In this study we investigated the role of AQP4 on cerebral capillary density and cerebral blood flow using AQP4 knockout (AQP4‐KO) mice in comparison with the wild‐type (WT) mice. Cerebral blood flow was measured using arterial spin labeling (ASL) MRI in WT mice (n = 7) and AQP4‐KO mice (n = 4). Mice were then perfusion fixed and cerebral capillary density was identified by GLUT‐1 immunohistochemistry staining and was quantified from the number of the positive counts per unit area (number/mm2). Results showed that the cerebral blood flows were similar in the WT and the AQP4‐KO groups (Mean ± SD, 182 ± 30 vs. 178 ± 52, ml/min/100g tissue). The AQP4‐KO mice exhibited a 24% increase in capillary density as compared to the WT mice (Mean ± SD, 504 ± 49 vs. 406 ± 32, N/mm2). Our results showed that the deletion of AQP4 increased the capillary density without altered blood flow in mouse brain, suggesting a compensation for decreased gas transport in the KO mice.

Support or Funding Information

NIH R01 NS 38632
In vivo imaging of infection‐induced cogulopathy in the microcirculation
Authors: Jenne Craig N , Davis Rachelle P , McDonald Braedon
Published: November 2023

Patients with systemic infection often develop disseminated intravascular coagulation (DIC), a condition leading to clot formation throughout the vasculature. This systemic coagulation can impair blood flow within the tissue microvasculature and has been associated with organ damage and dysfunction. Infection also triggers the release of Neutrophil Extracellular Traps (NETs) within the vasculature. NETs are comprised of extracellular DNA covered in a number of nuclear and antimicrobial proteins. This immune response is designed to catch and kill pathogens, but also has the potential to bind platelets and initiate coagulation.

Currently, most studies are limited to the histological analysis of tissue sections or in vitro biochemical assays, providing only snapshots of these complex and dynamic processes. As such the specific interaction between NETs, platelets and coagulation are poorly understood. Using intravital microscopy (IVM), can directly visualize NET formation and fibrin deposition in real‐time, in the blood vessels of live mice following infection. Additionally, we have developed a novel imaging protocol using an enzyme‐activatable fluorescent probe to track the time and location of thrombin activation within the vasculature of live mice. Importantly, IVM allows us to understand not only the complex interactions between the pathogen, immunity and coagulation, but to also the effect of these interactions on vascular perfusion and tissue damage. Following i.v. challenge with bacteria, we measure significant NET deposition within the liver sinusoids. These NETs in turn activate thrombin leading to fibrin deposition, vascular occlusion and tissue damage. Importantly, treatments that prevent NET formation, break‐down NETs within the vasculature, or block key NET components significantly inhibit thrombin activation, improve perfusion and attenuate tissue damage.

Support or Funding Information

This work is supported by grants from the Critical Care Strategic Clinical Network and the Natural Sciences and Engineering Research Council of Canada (NSERC)
Multicolor Two‐Photon Intravital Microscopy Reveals Monocyte Patrolling and Macrophage Migration in Atherosclerosis
Authors: McArdle Sara , Ray Nilanjan , Ley Klaus
Published: November 2023

Atherosclerosis is an underlying cause of two of the leading causes of death in the world, heart attacks and strokes. It is a chronic inflammatory disease characterized by plaque build‐up in large and medium arteries. During disease progression, monocytes in the blood invade the arterial wall, where they can differentiate into macrophages and dendritic cells. Together, these cells phagocytose lipids, clear apoptotic cell debris, present antigen to T cells, and produce pro‐ and anti‐inflammatory cytokines. Both monocyte recruitment to the plaque and macrophage function within the plaque require cell motion. The purpose of this work is to study monocyte and macrophage movement in the context of atherosclerosis.

We developed a system for imaging fluorescent leukocytes in atherosclerotic arteries in vivo. The major challenge in intravital imaging of large arteries is the motion artifacts due to the expansion of the arterial wall with the heartbeat. This system utilizes cardiac triggering and novel image post‐processing algorithms to remove these motion artifacts and allow for cell motion quantification. Apoe−/− Cx3cr1GFP/+ Cd11cYFP mice were generated to visualize labeled monocytes and macrophages. Monocytes were observed patrolling the endothelium of atherosclerotic arteries and the motion characteristics were quantified. Macrophages were seen actively probing their local environment as well as migrating through the plaque. Finally, these macrophages were extensively phenotyped by cell surface markers to link observed motion to known subsets and function.

Support or Funding Information

This work was funded by NIH R01 (115232) to K.L. and AHA (#11PRE7580009), HHMI (56005681), and NHLBI (T32HL105373‐03) to S.M.
The Effect of Splenectomy on the Recruitment of Inflammatory Cells in the Cerebral Microvasculature
Authors: Seifert Hilary , Russell Janice , Granger Daniel Neil
Published: November 2023

Splenectomy decreases inflammatory cell recruitment to the brain following sterile cerebral inflammation that results from stroke. Similarly it is known that non‐sterile cerebral inflammation like systemic lipopolysaccharide (LPS) causes rapid recruitment of leukocytes in the cerebral venules. It is not known if the protection afforded by splenectomy is specific to stroke or if removal of the spleen can protect the brain against systemic LPS. The objective of this study was to determine if splenectomy alters the recruitment of adherent leukocytes and platelets in the cerebral microvasculature following systemic LPS administration. C57Bl/6 mice underwent either splenectomy or sham splenectomy two weeks prior to an intra‐peritoneal injection of LPS (10 ug/mouse). Four hours after LPS injection, mice were imaged with intravital video microscopy. Adherent platelets, labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), and leukocytes, labeled with rhodamine G6, were quantified. Peripheral blood counts were also measured in all groups. LPS significantly increased the number of adherent leukocytes and platelets in cerebral venules. This response was not altered by splenectomy. LPS also caused a significant decrease in circulating leukocytes, which was unchanged by splenectomy. There was no affect on circulating platelets. These results demonstrate that splenectomy does not protect the brain from non‐sterile cerebral inflammation and the protective effects of splenectomy on the inflamed brain appear to be specific to stroke.

Support or Funding Information

This work was supported by a fellowship from the Center for Cardiovascular Diseases and Sciences, LSU Health Sciences Center‐Shreveport
The Effect of Media Type on Nerve Presence in Cultured Microvascular Networks
Authors: Hodges Nicholas A. , Barr Ryan W. , Murfee Walter L.
Published: November 2023

Neurovascular patterning represents an emerging area of microvascular research. While overlapping molecular signals highlight links between angiogenesis and neurogenesis, advancing our understanding of their spatial and temporal coordination is limited by a lack of in vitro models that contain both systems. Such a potential model is the rat mesentery culture model, which our laboratory has recently introduced as an ex vivo tool to investigate cellular dynamics during angiogenesis and lympangiogenesis in an intact microvascular network scenario. The objective of this study was to evaluate the effect of media type on the maintenance of nerve presence in cultured networks. Mesenteric tissue windows were harvested from adult male Wistar rats and cultured for 3 days in the following media groups: neural basal media (NBM, n = 4 tissues), NBM + nerve growth factor (NGF, n = 7 tissues), and NBM + NGF + 20% fetal bovine serum (FBS, n = 7 tissues). Tissues were immunohistochemically labeled for PECAM (endothelial cell marker) and class III‐β tubulin (nerve marker). Control tissues (n=7) were immediately immunolabeled after harvesting with no culture. The ratio of network feeding arterioles with observable positive class III‐β tubulin nerve alignment in control tissues was significantly increased compared to each of the three cultured groups (Control = 0.83 ± 0.09; NBM = 0.04 ± 0.04; NBM + NGF = 0.33 ± 0.14; NBM + NGF + 20% FBS = 0.40 ± 0.12). These results suggest that the presence of nerves is reduced in cultured networks. The possibility for media type influences on nerve maintenance supports the potential for the future development of the rat mesentery culture model as a unique ex vivo tool to investigate angiogenesis, neurogenesis, and neurovascular patterning simultaneously.

Support or Funding Information

NIH 5‐P20GM103629‐04
Caveolin‐3 plays a critical role in autophagy after ischemia reperfusion
Authors: Kassan Adam , Pham Uyen N. , Cho Eunbyul , Nguyen Quynh My , Patel Piyush P. , Roth David M. , Head Brian P. , Patel Hemal H.
Published: November 2023

Autophagy is a dynamic recycling process responsible of the breakdown of misfolded proteins and damaged organelles, providing nutrients and energy for cellular renovation and homeostasis. Loss of autophagy is associated with cardiovascular diseases. Caveolin‐3 (Cav‐3), a muscle specific isoform, is a structural protein within caveolae and is critical to stress adaptation in the heart. However, it remains unknown whether Cav‐3 plays a role in regulating autophagy to modulate cardiac stress response. In the present study, we used HL‐1 cells, a cardiac muscle cell line, with stable Cav‐3 knockdown (Cav‐3 KO) to study the impact of Cav‐3 in regulation of autophagy in the setting of simulated ischemia‐reperfusion injury (I/R). Cav‐3 co‐immunoprecipitated with beclin‐1, showing an interaction of caveolin with autophagy related proteins. We found that Cav‐3 KO cells have a decreased expression of autophagy markers (beclin‐1, light chain (LC3)‐II) after ischemia and I/R when compared to WT cells. Moreover, Cav‐3 KO cells showed increased cell death and higher level of apoptotic proteins (cleaved caspase‐3 and cytochrome c) with suppressed mitochondrial function in response to ischemia and I/R. Taken together, these results indicate that Cav‐3 regulates autophagy to modulate stress adaptation.

Support or Funding Information

5 T32 MH 19934‐20
Novel Finding of Mitochondrial Biogenesis During Cardiac Surgery: Marker or Mediator of Ischemia/Reperfusion (I/R) Injury, or an Element of the Homeostatic Intracellular Repair Response (HIR2)
Authors: Andres Allen , Sengstock David , Jahania Salik , Dabir Reza , Tucker Kyle , Gottlieb Roberta , Mentzer Robert
Published: November 2023

BACKGROUND

We recently demonstrated that the homeostatic intracellular repair response (HIR2) is activated during cardiac surgery. Part of HIR2 involves brisk mitochondrial turnover in the heart; a process that comprises both mitophagy and mitochondrial biogenesis. We have previously demonstrated that mitophagy is an important element of ischemic preconditioning. However, it is unknown whether mitochondrial biogenesis occurs in the setting of I/R and how it might contribute to cardioprotection or injury.

STUDY DESIGN

Autophagy and mitochondrial turnover were assessed in 20 patients undergoing coronary artery bypass or valve surgery requiring cardiopulmonary bypass (CPB). Biopsies of right atrial appendage obtained before initiation and after weaning from CPB were processed for whole tissue lysate, mitochondria‐enriched heavy membrane, and cytosolic fractions. Samples were analyzed for autophagy by immunoblotting for LC3, Beclin‐1, ATG5‐12, and p62. Mitochondrial turnover was assessed by monitoring Tom70, Cox4, and Parkin. Cytosolic cytochrome c was examined as a marker of injury.

RESULTS

Heart surgery resulted in increased autophagy indicated by depletion of autophagy proteins after bypass. Parkin increased in the mitochondrial fraction and decreased in the cytosol. We observed a marked increase in mitochondrial proteins Tom70 and Cox4, both of which positively correlated with cross‐clamp and bypass time. Cytosolic cytochrome c consistently increased after CPB and correlated with increased mitochondrial mass.

CONCLUSIONS

These findings provide evidence for the first time in humans that coordinated mitophagy and biogenesis occur in response to I/R. Intriguingly, increased cytosolic cytochrome c after bypass paralleled the increase in mitochondrial mass, raising the concern that mitochondrial biogenesis may contribute to I/R injury. Whether mitochondrial biogenesis is beneficial or a liability in the setting of I/R remains to be clarified. Thus, strategies designed to amplify HIR2 and/or modulate mitochondrial biogenesis during cardiac stress may represent a novel approach to myocardial protection in patients undergoing heart surgery.

Support or Funding Information

NIH: P01 HL112730