bluuebunny/crossref_metadata_2025_split huggingface dataset:
Development of the Ex Vivo Mouse Mesometrium Model to Investigate Multicellular Dynamics During Angiogenesis
Authors: Suarez‐Martinez Ariana Diandra , Kaplan Dana , Huang Katie , Meadows Stryder , Bierschenk Sussane , Sperandio Markus , Murfee Walter Lee 
Published: November 2023

The development of biomimetic in vitro or ex vivo models that can incorporate the complexity of intact microvascular networks would enable the investigation of multicellular dynamics associated with angiogenesis and novel experimental platforms for pre‐clinical therapy testing. Recently, our laboratory has introduced the rat mesentery culture model as such a tool. The applicability of this model would be enhanced with the use of murine tissue, yet unfortunately, the mouse mesentery is avascular. A potential analog to the mesentery is the mesometrium, the connective tissue of the uterine horns. The objective of this study was to demonstrate that the mouse mesometrium contains intact microvascular networks that can be cultured to investigate multicellular dynamics during angiogenesis. Mesometrium windows, defined as the translucent connective tissues attached to the uterine horns, were harvested from adult C57Bl/6 female mice and cultured in minimum essential media with 20% fetal bovine serum for 3 and 5 days. The tissues were immunohistochemically labeled with platelet endothelial cell adhesion molecule (PECAM), neuron‐glial antigen 2 (NG2), and alpha‐smooth muscle actin (α‐SMA) to identify endothelial cells, pericytes, and smooth muscle cells, respectively. The mesometrium proved to have an intact microvascular network with arterioles, venules and capillaries. Arterioles and venules were differentiated by the PECAM‐positive cell morphology, and the α‐SMA‐positive cell morphology that wraps the vessels. α‐SMA and NG2 labeling confirmed the presence of smooth muscle cells along the larger vessels and pericytes along capillaries. After 5 days in culture, tissues remained viable as confirmed with a LIVE/DEAD assay. Compared to day 0, uncultured tissues, capillary sprouts per venule length was dramatically increased by day 3 and day 5 (Day 0: 0.09 ± 0.02; Day 3: 4.10 ± 0.75; Day 5: 4.37 ± 0.60 spouts/mm). For day 3 and day 5, capillary sprouting preferentially occurred of venules versus arterioles. Furthermore, while the number of capillary sprouts were comparable for day 3 and day 5, sprouts appeared to be longer on day 5. Capillary sprouts were also observed to contain NG2‐positive, and SMA‐negative pericytes. These results introduce the mouse mesometrium culture model as a novel ex vivo approach for elucidating pericyte‐endothelial cell dynamics associated with capillary sprouting at specific vessel locations in a real microvascular network scenario.Support or Funding InformationNIH R01AG049821, NIH P20GM103629
Chlorinated Lipids Elicit Neutrophil‐Endothelial Interactions in Mesenteric Microcirculation
Authors: Yu Hong , Wang Meifang , Wang Derek Z. , Kalogeris Ted , Ford David A. , Ronald Korthuis J. 
Published: November 2023

Neutrophils are key early responders to infection, and kill microbes by phagocytosis and oxidant‐mediated mechanisms that involve, in part, myeloperoxidase (MPO). Hypochlorous acid (HOCl) produced by MPO targets plasmalogen phospholipids at vinyl ether bonds to generate 2‐chlorohexadecanal (2‐ClHDA) and 2‐chloropalmitic acid (2‐ClPA). Our previous work showed that levels of 2‐ClPA increased in both plasma and cecum of rats subjected to cecal ligation and puncture (CLP, a model of polymicrobial sepsis) when compared to sham rats. However, it is not clear whether chlorinated lipid species can exert proinflammatory effects including elevated neutrophil‐endothelial interaction, reactive oxygen species (ROS) production and inflammatory markers levels. We have begun to explore this issue by exposing naïve rats to exogenous chlorinated lipids. Mesenteries in four groups of Sprague‐Dawley rats (n=6) were superfused with 10 μM 2‐ClHDA, hexadecanal (HDA), 2‐ClPA and palmitic acid (PA), respectively, and inflammatory responses were measured using intravital microscopy and other assays. Treatment with chlorinated lipids showed significant increases in neutrophil‐endothelial interactions including leukocyte rolling and adhesion as well as ROS production when compared with non‐chlorinated lipids. Tissue MPO levels were also elevated after exposure to chlorinated lipids, while the tissue levels of tumor necrosis factor α (TNFα) were not different when comparing to mesenteries treated with equimolar concentrations of non‐chlorinated lipids. These results show for the first time that chlorinated lipids, which may be formed secondary to the enzymatic action of neutrophilic MPO to produce HOCl in inflammation, elicit neutrophil‐endothelial interactions and other inflammatory response in naïve rats.Support or Funding InformationSupported by NIH grant GM‐115553.
Endothelium protects smooth muscle from oxidative stress in skeletal muscle resistance arteries
Authors: Sinkler Shenghua Y , Segal Steven S 
Published: November 2023

Vascular dysfunction associated with oxidative stress is characteristic of cardiovascular disease. Advanced age can protect microvascular endothelium from oxidative stress (PMC4422569) however it is unknown whether endothelial cells (ECs) or smooth muscle cells (SMCs) of intact microvessels are more susceptible to damage during oxidative stress. Using the superior epigastric artery freshly dissected from anesthetized male C57BL/6 mice (3–4 months old), we tested the hypothesis that, compared to SMCs, ECs are more susceptible to damage during acute oxidative stress. Individual vessels (length, ~2 mm; internal diameter, ~150 μm) were cannulated at both ends and pressurized to 100 cmH2O with physiological salt solution (PSS), then superfused (2 ml/min) with PSS containing 200 μM H2O2 at 37°C (pH=7.36). At defined durations, the lumen was perfused (0.1 ml/min) with PSS containing Hoechst 33342 (turquoise, 1 μM) and propidium iodide (red, 2 μM) to stain nuclei of all cells and nuclei of cells with damaged membranes, respectively. Staining was quantified in the vessel mid‐region (~500 μm × 150 μm; n=5) which contained nuclei of 93±1 ECs (parallel to vessel axis) and 114±2 SMCs (perpendicular to vessel axis). Within 20–30 min of H2O2 superfusion, membrane disruption (red nuclei) occurred in ECs but not SMCs. Following 50 min of H2O2, 60±2% of SMC and 37±2% of EC nuclei stained red (n=5; P<0.05). Neither EC nor SMC nuclei stained red following equivalent superfusion with control PSS. In vessels denuded of endothelium (50 μm diameter tungsten wire), 50 min superfusion with H2O2 stained 90±2% of SMC nuclei red (n=5; P<0.05, denuded vs intact endothelium). In contrast, SMC nuclei of denuded vessels did not stain red following equivalent superfusion with control PSS. Thus, with H2O2 delivered abluminally, membrane damage occurred sooner for ECs than for SMCs and denuding the endothelium exacerbated SMC damage. We suggest that the endothelium of resistance arteries is more vulnerable to oxidative stress while protecting its surrounding smooth muscle layer.Support or Funding InformationNIH R37HL041026
Neurovascular uncoupling predicts cognitive decline and gait abnormalities in a clinically relevant mouse model of whole brain irradiation
Authors: Yabluchanskiy Andriy , Tarantini Stefano , Hertelendy Peter , Valcarcel‐Ares Marta Noa , Gautam Tripti , Farkas Eszter , Sonntag William E. , Csiszar Anna , Ungvari Zoltan 
Published: November 2023

Whole brain irradiation (WBI) is known to promote accelerated brain senescence and leads to progressive cognitive dysfunction in up to 50% of tumor patients surviving long‐term after treatment, due to, at least in part, gamma‐irradiation induced cerebromicrovascular injury. Moment‐to‐moment adjustment of cerebral blood flow (CBF) via neurovascular coupling (NVC) has a critical role in maintenance of healthy cognitive function. To determine whether cognitive decline induced by WBI associate with impaired NVC, C56BL/6 mice (3 mo) subjected to a clinically relevant protocol of fractionated WBI (5 Gy twice weekly for 4 weeks) and control mice were compared. Mice were tested for spatial memory performance (radial arm water maze), sensorimotor coordination (computerized gait analysis, CatWalk) and NVC (laser Doppler flowmetry) at 3 and 6 months post‐irradiation. We found that mice with WBI exhibited impaired NVC at 3 months post‐irradiation, which was associated with impaired performance in the radial arm water maze, while gait parameters remained unaltered. At 6 months post‐irradiation persisting neurovascular dysfunction and cognitive impairment were evident, which were associated with significant impairment in gait coordination (including changes in the regularity index and phase dispersion). Collectively, our findings provide evidence for early and persisting neurovascular un‐coupling after a clinically relevant protocol of fractionated WBI, which predict early manifestations of cognitive impairment.Support or Funding InformationThis work was supported by grants from the American Heart Association, the National Center for Complementary and Alternative Medicine, and the National Institute on Aging
Lymphatic vessel‐independent lymph flow pattern from the peri‐nodal adipose tissue to the lymph node
Authors: Lin Yujia , Liao Shan 
Published: November 2023

RationaleLymph nodes (LNs) anatomically are distributed along the lymphatic vessels to survey materials carried by the lymph flow. Adipose tissue is a reservoir of hormones, cytokines, metabolites that can regulate immune responses and cell homeostasis in the LNs. Although lymphatic vessel and LNs are always surrounded by adipose tissue, lymph or cell communication between the peri‐nonal adipose tissue and the LNs remains unclear.ObjectiveWe aimed to understand how lymph flows from the peri‐nodal adipose tissue to the LNs.Methods and ResultsUsing high‐resolution, super‐resolution and 3D reconstruction images, we studied the lymph distribution pattern from the adipose tissue to and through the LN. We found that a subset of LN conduits that are initiated at the LN capsule (LNC conduits), connect the afferent subcapsular sinus (SCS) and efferent medullary sinus (MS) and facilitate rapid lymph flow through the LN parenchyma. Super‐resolution images showed the unprecedented heterogeneous flow patterns inside LNC conduits. Surprisingly, we found that the LN capsule and its associated LNC conduits connect to the collagen channels in the fat tissue and provide an additional route supports lymph flow from the peri‐nodal adipose tissue to the LN (Fat‐LNC conduits). The type of materials entering the Fat‐LN conduits is dictated by molecular size. Finally, small molecules can enter LN after the afferent lymphatic vessels are sutured.ConclusionsThe lymph distribution pattern suggests that Fat‐LNC conduit support lymphatic vessel‐independent lymph flow from peri‐nodal adipose tissue to the LN, which provide a “highway” for fat‐derived factors to regulate LN microenvironment.Support or Funding InformationDianne & Irving Kipnes Foundation, Natural Sciences and Engineering Research Council of Canada (NSERC RGPIN‐2015‐03641), Canadian Institute of Health Research (CIHR) to SL and Canada Foundation for Innovation to SL.
LITAF, A Novel Regulator of Cardiac Excitation
Authors: MOSHAL KARNI S , Roder Karim , Werdich Andreas A , Dural Nilufer Turan , Kim Tae Yun , Cooper Leroy L , Lu Yi Chun , Choi Bum‐Rak , Terentyev Dmitry , MacRae Calum , Koren Gideon 
Published: November 2023

IntroductionQT interval is a predictor of arrhythmia and sudden death. Genome‐wide association studies identified LITAF (lipopolysaccharide‐induced TNF factor) as one of the novel loci associated with a significant QT prolongation. Our goal is to investigate and understand the role of LITAF in regulating cardiac excitation.MethodsOptical mapping was performed in zebrafish hearts and rabbit cardiomyocytes to determine the effect of LITAF on action potential duration (APD) and Ca2+ transients. Effect of LITAF on cardiomyocyte Ca2+ handling was determined by live cell confocal Ca2+ imaging. To determine the effect of LITAF on cardiac L‐type calcium channel (LTCC) current (ICa,L), we use whole cell patch clamping. We use heterologous expression system to study the mechanism by which LITAF regulates expression of LTCC.ResultsWe observed that LITAF loss‐of‐function in zebrafish increases the Ca2+ transient amplitude. Overexpressed LITAF in rabbit cardiomyocytes (CM) downregulates LTCC, decreases Ca2+ transients and calcium inward current through ICa,L. In neonatal rabbit CM (NRbCM), overexpressed LITAF shortens APD, likely due to suppression of LTCC. The knockdown of LITAF in NRbCM prolongs the APD and causes an increase in spontaneous cytosolic Ca2+ under isoproterenol. In HEK cells, overexpressed LITAF downregulates the total and surface pools of LTCC via increased Cava1c ubiquitination. A physical interaction between LITAF and the ubiquitin ligase NEDD4‐1 is required to increase Cava1c ubiquitination and its degradation in HEK cells.ConclusionWe show that LITAF acts as molecular adaptor to link NEDD4‐1 ubiquitin ligases to the cardiac LTCC and control its membrane expression and function and thus cardiac excitation.Support or Funding InformationNIH R01 Grant (4R01HL110791‐04) to GK
Connexin 40 Plays a Crucial Role in the Myocardial Ischemia Reperfusion Injury of Type 2 Diabetic Mice
Authors: Si Rui , Guo Rui , Truong Aaron H , Makino Ayako 
Published: November 2023

Type 2 diabetes serves as an independent risk factor for the development of ischemic heart disease and ischemic heart diseases is a leading cause of high mortality in diabetes. In addition to atherosclerosis, microvascular disease due to endothelial dysfunction is implicated in the development and progression of ischemic heart disease. Connexin 40 (Cx40) predominantly expresses in vascular endothelial cells (ECs) and regulates endothelium‐dependent vascular relaxation and angiogenesis. We previously proved that downregulation of Cx40 is associated with coronary endothelial cell dysfunction in type 1 diabetic mice, however the involvement of Cx40 in cardiac ischemia reperfusion (I/R) injury in diabetes has never been revealed. In this study, we explore the role of Cx40 in cardiac I/R injury using type 2 diabetic mice (T2D mice). T2D mice were generated by a single injection of low‐dose streptozotocin (75 mg/kg) with a high‐fat diet (60 % kcal). T2D mice exhibited a significant decline of cardiac contractile function and larger infarction size after I/R compared to the control mice. T2D mice also showed attenuated endothelium‐dependent vascular relaxation in coronary arteries, decreased capillary density and enhanced endothelial apoptosis in the myocardium. Mouse coronary endothelial cells (MCECs) isolated from T2D mice exhibited significantly lower expression of Cx40 protein than in control MCECs. Therefore, we next examined the myocardial endothelial function using Cx40 knockout (Cx40−/−) mice. Our results demonstrated that a) Cx40 deletion impaired endothelium‐dependent relaxation via diminishing endothelium‐dependent hyperpolarization‐induced relaxation in coronary arteries, b) capillary density was decreased in the myocardium of Cx40−/− mice, c) cardiac contractility was attenuated in Cx40−/− mice, and d) infarction size after I/R was significantly increased in Cx40−/− mice compared to wild type mice. In conclusion, our data suggest that CX40 is an important protein to maintain myocardial endothelial function and decreased CX40 level might be one of crucial mechanisms which alter cardiac functions and lead to more severe damage after cardiac I/R in T2D mice.Support or Funding InformationNIH/HL115578
Sex difference in the patho‐physiological consequences of adipocyte PRR deficiency
Authors: Gatineau Eva , Cohn Dianne , Liu Yongshi , Gong Ming , Yiannikouris Frédérique 
Published: November 2023

Introduction and objectivesThe epidemic of obesity is currently affecting more than 30% of the population in the United States. Obesity increased the expression of the (pro)renin receptor (PRR), a component of the renin‐angiotensin system, in adipose tissue. In male mice, the deletion of adipocyte PRR prevented the development of obesity and the accumulation of fat mass. However, similar to mouse model with lipodystrophy and mouse model with obesity, adipocyte‐PRR deficient (PRRAdi/Y) mice are characterized by a fatty liver and by an elevated systolic blood pressure (SBP). Sex‐related differences in factors associated with obesity exist between male and females, therefore the objective of our study was to determine whether the remarkable phenotype of the male PRRAdi/Y mice was sustained in female.Methods and resultsMale PRRAdi/Y mice expressing adiponectin‐driven Cre recombinase were bred with female mice PRRfl/fl to generate female control (PRRfl/fl) and adipocyte‐PRR deficient mice (PRRAdi). Female mice were fed a standard diet (SD) or a high fat diet (HF) for 23 weeks (n=6–13/groups). Body weight was measured weekly, body composition monthly and blood pressure by radiotelemetry at the end of the study. The deletion of adipocyte PRR in females did not change body weight (BW) curves in SD‐fed mice, but prevented the development of high fat diet‐induced obesity. Interestingly, the resistance to obesity in female mice was less pronounced than in male mice. Similar to males, the deletion of adipocyte PRR in female mice decreased by over 85% adipose tissue mass (SD: PRRfl/fl, 4.4±0.7 g; PRRAdi, 1.7±0.05 g and HF: PRRfl/fl, 5.8±1.2 g; PRRAdi, 0.6± 0.1; P<0.05). Similar to males, female PRRAdi mice developed hepatomegaly (SD: PRRfl/fl, 1.3 ± 0.1 g; PRRAdi, 2.6 ± 0.1 g, P<0.05) associated with an increase of neutral lipid contents in livers of female PRRAdi compared to control mice. At 22 weeks, radiotelemetry devices were implanted in female mice. In contrast to males, the increase in systolic blood pressure was not significantly different in SD‐fed PRRAdi female mice (PRRfl/fl, 129 ± 3 mmHg; PRRAdi, 141 ± 9 mmHg) suggesting a sex difference in PRR mediated‐regulation of blood pressure. Blood pressure measurement in HF‐fed mice are under investigation.ConclusionIn contrast to males, our results demonstrated that, elevation of blood pressure due to adipocyte PPR deficiency is abolished in female mice. Future studies will define whether sex difference in blood pressure regulation mediated by PRR depends on the renin angiotensin system and on hormonal regulation. Similar to male mice, adipocyte PRR deficiency prevents the development of obesity and decreases fat mass in female mice. Future studies will determine the mechanism by which PRR regulates lipid metabolism.
Identification of a molecular mechanism underlying estrogen‐mediated neuroprotection in neuronal cells against oxidative stress
Authors: Park Sookyoung , Won Jinyoung , Hong Yonggeun 
Published: November 2023

Estrogen (17β‐estradiol; E2) has neuroprotective effects in the setting of CNS injury and neurodegenerative disorders however, the mediator for neuroprotection mechanisms by E2 remains largely unknown. Recent studies have demonstrated that E2 might be potent modulator of both expression and activity of telomerase, which is a ribonucleoprotein complex involved in maintenance of the chromosome end and DNA repair. To examine whether E2 treatment exerts cytoprotective effects in neuronal cells, mouse origin neuroblastoma Neuro‐2a (N2a) cells were differentiated into cholinergic phenotypes, and used as cell culture model following exposure to hydrogen peroxide to induce oxidative stress. Neuronal cell death was induced and morphologic changes were observed following oxidative stress. In addition, apoptotic changes including Bax:Bcl‐2 ratio and apoptosis‐related proteases caspase‐3 were increased in neuronal cells rendered by oxidative stress. E2 treatment attenuated these changes, as well as increased cell viability. Our data demonstrated that E2 treatment significantly increased TERT expression in a dose‐dependent manner in neuronal cells. To confirm whether the estrogen signaling is associated with TERT, selective siRNA targeted against both ERα and ERβ were transfected to neuronal cells. TERT mRNA expression was down‐regulated in both ERα and ERβ‐silenced neuronal cells in particular, this change was more distinctive in knockdown of ERα. Taken together, these findings indicate that the telomerase enzyme may be an important mediator of the neuroprotective effects of E2.Support or Funding InformationNRF‐2013R1A2A2A01067169 to Y.H., NRF‐2014R1A1A3051724 to S.P., Republic of Korea.
Chronic ketosis modulates metabolite profiles and cell‐signaling pathways in mouse brain
Authors: LaManna Joseph , Sharma Katyayini , Ye Dan , Berthiaume Jessica M M , Luo Yu , Xu Kui , Puchowicz Michelle A 
Published: November 2023

The mechanism through which diet‐induced ketosis confers neuroprotection remains unknown. Studies have implicated ketosis in modulating cell‐signaling pathways. The aims of this study was to investigate the effects of ketosis on (i) recovery following transient focal cerebral ischemia (middle cerebral artery occlusion; MCAO) and (ii) signaling regulators of cell growth‐survival and to corroborate these effects with circulating and brain tissue metabolites.MethodsMice were fed with a ketogenic diet (KG; 89.5% kcal fat) or standard chow (STD; 27.5% kcal fat) diets for 4 weeks. For the MCAO group, mice underwent 60 min of MCAO and total brain infarct volumes were evaluated 48 h after reperfusion. Cortical brain tissues were analyzed for hypoxic inducible factor‐1alpha (HIF‐1α), total AKT and pAKT, total 5′ AMP‐activated protein kinase (pAMPK) and FOXO3a, by Western Blot analysis. Metabolic profiles (μmol/g tissue) were determined using mass spectrometry targeted analysis.ResultsThere was a proportional decrease in infarct volumes with increasing blood ketone concentrations (beta‐hydroxybutyrate, BHB; KG vs. STD; 4.2 ± 0.6 vs 7.8 ± 2.2 mm3, mean ± SEM). A positive correlation was also observed with HIF‐1α and pAKT levels relative to blood BHB. KG group exhibited significant downregulation in total FOXO3a levels, as well as levels of tissue metabolites correlated with phosphorylation of AKT protein targets, suggesting a concentration‐dependent effect of ketosis on activity of signaling targets. These modifications infer that the neuroprotective properties of ketosis may be through regulation cell survival pathways that are coupled to cell energetics.Support or Funding InformationNIH R01 HL092933‐01