bluuebunny/crossref_metadata_2025 huggingface dataset:
ATP‐Sensitive K+ Channels in Smooth Muscle Cells from Rat Mesenteric Lymph Vessels VESSELS
Authors: Garner Brittney R. , Stolarz Amanda J. , Fletcher Terry W. , Rusch Nancy J.
Published: November 2023

The ATP‐sensitive potassium (KATP) channel is expressed in plasma membranes of many types of smooth muscle cells (SMCs). It is disinhibited when intracellular ATP concentrations are reduced during conditions of metabolic stress, and the resulting K+ efflux through KATP channels mediates membrane hyperpolarization and SMC relaxation. However, in the lymphatic smooth muscle cells (LSMCs) of rhythmically contracting lymphatic vessels, KATP channels reportedly play an important role in regulating the basal rhythmic contractions that establish lymph flow even in the absence of overt metabolic challenge. The KATP channels in LSMCs also may participate in the dilator responses of lymphatic vessels (LVs) to nitric oxide, inflammatory mediators, and compromise lymph flow in other pathologies associated with oxidative stress1,2,3. Although it is recognized that the properties and the subunit composition of KATP channels show tissue‐specificity, the identity and properties of KATP channels in LSMCs are unknown. The goal of this study was to begin to define the function and properties of KATP channels in rat mesenteric LVs. Isolated rat mesenteric LVs were cannulated on glass pipettes, pressurized at 4.5–6 mmHg, and edge detection was used to continuously monitor changes in vessel diameter. Addition of the KATP channel agonist cromakalim (0.01 μM‐100 μM; half‐log units) progressively increased LV diameter and abolished rhythmic contractions (n=3). In contrast, addition of the KATP channel antagonist, glibenclamide (0.01 μM‐100 μM; half‐log units) resulted in no change in LV diameter but suppressed rhythmic contractions. The latter finding confirmed the important role of KATP channels as regulators of LV contractile function under basal conditions. Subsequently, we have begun to characterize KATP channel currents in SMCs freshly isolated from rat mesenteric LVs. Patch‐clamp studies in the cell‐attached mode reveal unitary K+ currents in symmetrical 145 K+ recording solutions at negative patch potentials near the resting membrane potential of LSMCs. Unitary amplitudes calculated over a range of negative patch potentials from −80 mV to −30 mV indicate a single‐channel conductance of ~ 37 pS. Thus, we have tentatively identified the KATP channel in rat LSMCs as a first step toward defining it's regulation by endogenous substances and pharmacological interventions that may alter lymph flow.

Support or Funding Information

Supported by T32 GM106999 (BRG) and R21 CA187325‐01A1 (NJR) from the National Institutes of Health.
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 Information

NIH 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 Information

Supported 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 Information

NIH 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 Information

This 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

Rationale

Lymph 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.

Objective

We aimed to understand how lymph flows from the peri‐nodal adipose tissue to the LNs.

Methods and Results

Using 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.

Conclusions

The 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 Information

Dianne & 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

Introduction

QT 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.

Methods

Optical 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.

Results

We 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.

Conclusion

We 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 Information

NIH 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 Information

NIH/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 objectives

The 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 results

Male 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.

Conclusion

In 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.
Preeclampsia alters androgen metabolite‐induced vasorelaxation of rat uterine artery
Authors: Hanson Andrea E. , Stallone John N.
Published: November 2023

Preeclampsia (PE), a multi‐system disorder characterized by pregnancy‐induced hypertension and proteinuria, is a major cause of both fetal and maternal morbidity and mortality, yet the pathophysiology remains poorly understood. In pregnancy, the placenta produces abundant androgens. These rapid, nongenomic vasodilators may serve to maintain uterine blood flow during normal pregnancy, and may be impaired in PE. Of the known placental androgens, 5β‐dihydrotestosterone (5β‐DHT), the genomically inactive metabolite of testosterone (TES), often exhibits the greatest vasorelaxation efficacy in systemic blood vessels. This study aimed to examine the acute vascular effects of TES and its metabolites, 5α‐dihydrotestosterone (5α‐DHT) and 5β‐DHT on the rat uterine artery (UA), using the DOCA‐salt model of PE. Endothelium intact UA rings (200 μm, dia.) from normal pregnant (NP) and DOCA‐treated pregnant (DSP) female (F) Sprague‐Dawley rats (250–300g) were prepared for measurements of isometric contractile force (DMT myograph). UA were precontracted with U‐46619 (thromboxane A2 agonist, 1μM) and concentration‐dependent relaxation to TES, 5α‐DHT, and 5β‐DHT was assessed (0.1–300 nM). Data are means ± SE (n = 7–9 rats). In NP, maximal relaxation was similar for 5β‐DHT (21.7±3.4%), TES (26.4 ± 4.6%), and 5α‐DHT (29.2 ± 4.1%), with a rank potency of 5α‐DHT >TES > 5β‐DHT. In DSP, maximal response to 5β‐DHT increased notably by 45% (31.5 ± 6.1), while maxima to TES (33.1 ± 6.1) and 5α‐DHT (34.1 ± 6.1) were unchanged. Potency of 5β‐DHT also increased, with a rank of 5β‐DHT > TES = 5α‐DHT. These data suggest that: 1) there is a selective increase in efficacy and potency of vasorelaxation to 5β‐DHT in UA of DSP rats; and 2) this may reflect upregulation of reactivity to 5β‐DHT resulting from PE‐induced deficiencies in 5β‐DHT biosynthesis by the placenta, which may contribute to the impaired placental blood flow and maternal hypertension, characteristic of PE. (State of Texas)
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 Information

NRF‐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.

Methods

Mice 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.

Results

There 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 Information

NIH R01 HL092933‐01
Mitochondrial Transcription Factor A & Cardiac Stability
Authors: Kunkel George H. , Theilen Nicholas , Jeremic Nevena , Weber Gregory , Tyagi Suresh C.
Published: November 2023

Heart failure is a functional lack of myocardial performance due to a loss of molecular control over increases in calcium and ROS, resulting in proteolytic degradative advances and cardiac remodeling. Mitochondria are the molecular powerhouse of cells, shifting the sphere of cardiomyocyte stability and performance. Functional mitochondria rely on the molecular abilities of safety factors such as TFAM to maintain physiological parameters. Mitochondrial transcription factor A (TFAM) creates a mitochondrial nucleoid structure around mtDNA, protecting it from mutation, inhibiting NFAT (ROS activator/hypertrophic stimulator), and transcriptionally activating Serca2a to decrease calcium mishandling. Current literature depicts major decreases in TFAM as HF progresses. We aim to assess TFAM function against Calpain 1 and MMP 9 proteolytic activity and its role in cardiac remodeling. To this date, no publication has surfaced describing the effects of aortic banding (AB) as a surgical heart failure model in TFAM‐TG mice. HF models were created via AB in TFAM Transgenic (TFAM‐TG) and C57BLJ‐6 (control) mice. Eight weeks post‐AB, functional and histological analysis revealed a successful banding procedure, resulting in cardiac hypertrophy as observed via echocardiography. Pulse wave and color doppler show increased aortic flow rates as well as turbulent flow at the banding site. Preliminary results of cardiac tissue immuno‐histochemistry of HF‐control mice show decreased TFAM and compensatory increases in Serca2a fluorescent expression, along with increased calpain 1 and MMP9 expression. Protein, RNA, and IHC analysis will further assess TFAM‐TG results post‐banding. Echocardiography show more cardiac stability and functionality in HF induced TFAM‐TG mice than the control counterpart. These preliminary findings, along with our invitro results, suggest that TFAM has molecular therapeutic potential to reduce protease activity.

Support or Funding Information

The study is supported by NIH Grants HL‐74185 and HL‐108621 to SCT and NIH F31 Grant 1F31HL132527‐01 to GHK
Unraveling the Identity of Active Serine Proteases in Inflammatory Bowel Diseases
Authors: Vergnolle Nathalie , Deraison Celine , Chrystelle Bonnard , Edir Anissa , Gilmore Brendan , Quaranta Muriel , Sebert Morgane , Alric Laurent , Bonnet Delphine , Bunnett Nigel , Souza Alexandre Denadai
Published: November 2023

Introduction

Our team recently revealed that an upregulation in proteolytic activity is a central mechanism in the pathophysiology of Inflammatory Bowel Disease (IBD), such as Crohn's disease (CD) and ulcerative colitis (UC). Otherwise, the identity of proteases underlying this process remains elusive.

Objective

Identify active serine proteases in the colonic mucosa of IBD patients by using Activity‐Based Probes (ABP).

Material and Methods

Samples of colonic mucosa supernatants or tissue extracts were incubated with biotinylated ABPs designed to form covalent complexes with active serine proteases. ABP‐serine proteases complexes were then submitted to SDS‐PAGE to generate ABP proteomic profiles and LC‐MS/MS analysis to identify the active proteases.

Results

ABP proteomic profiles revealed that active proteases clustered in 4 main groups with mean MW of 27.8, 32.6, 35.4 and 71.5 kDa. Compared to control patients, there was a trend of upregulation for proteases with mean MW of 27.8 and 32.6 kDa in CD and UC, whereas a protease with 71.5 kDa was detected exclusively in UC. Preliminary LC‐MS/MS analysis of IBD samples identified up to 13 potential serine proteases (i.e. PRSS1/2, TPSAB1, DLD, PREP, etc) and 8 proteases from other clans (i.e. LTA4H, PARK7, etc).

Conclusion

The present study identified proteases not yet described as expressed in the colonic mucosa, as well as proteases derived from genes associated to IBD risk loci with unknown function in the gastrointestinal tract. This data will direct further studies aiming at establishing the relevance of these proteases in the pathophysiology of IBD.

Support or Funding Information

Agence Nationale de la Recherche (ANR) and European Research Council (ERC).
Bacteria‐neutrophil crosstalk: Genomic approaches identify G protein‐coupled receptors as new, potential targets for the treatment of infections
Authors: Corriden Ross , Sriram Krishna , Insel Paul A
Published: November 2023

Each year in the United States, 2 million people are infected with antibiotic resistant pathogens, such as methicillin‐resistant Staphylococcus aureus (MRSA); 23,000 patients die as a result of such infections. With the growth of antibiotic resistance and the slowing in the development of new antibiotics, the discovery of new, effective approaches to treat infections is an important unmet medical need. G protein‐coupled receptors (GPCRs) in polymorphonuclear neutrophils (PMNs), the first line effectors of the innate immune response, are a novel but unexplored potential class of “immune boosting” targets. Using a combination of qPCR based arrays and RNAseq analysis, we defined and quantified GPCR expression in human PMNs and found 174 GPCRs commonly expressed among 3 healthy donors. To help determine which GPCRs might be involved in response to pathogens, we assessed the GPCR profile of PMNs incubated with MRSA for 3 h. We found numerous GPCRs whose expression increased at least 2‐fold upon pathogen exposure. Analyses of PMN function (e.g., chemotaxis, extracellular trap production, bacterial killing) using selective agonists/antagonists of several such GPCRs indicated that pharmacological agents can “boost” the function of PMNs, including the killing of bacteria. The results indicate that: 1) Human PMNs express a large profile of GPCRs, expression of a number of which is altered by exposure to pathogenic bacteria. 2) Previously unrecognized PMN‐expressed GPCRs can boost PMN function, including the ability to kill bacteria. These data thus suggest a new paradigm for enhancement of innate immunity: targeting of neutrophil‐expressed GPCRs as a means to treat bacterial infections, including ones caused by antibiotic‐resistant bacteria.
Osteocytes orchestrate mechanical signal transduction in bone via WNT
Authors: Robling Alexander
Published: November 2023

Mechanical loading is essential to maintain normal bone metabolism and the balance between bone formation and resorption. The cellular mechanisms that control bone cell mechanotransduction are not fully understood, but several key pathways have been identified in osteocytes. One such mechanism involves Wnt signaling—a pathway involved in many physiologic processes including body axis specification/patterning, and morphogenic signaling. The first clues of WNT's role in bone biology began with several genetic studies that sought to identify the cause of Osteoporosis Pseudoglioma (OPPG), an autosomal recessive disease characterized by early‐onset osteoporosis and blindness. Mutant mouse studies revealed that loss‐of‐function mutation in the WNT co‐receptor LDL‐Receptor‐related Protein 5 (Lrp5) caused OPPG, but perhaps more importantly, that osteocytic Wnt signaling through Lrp5 is crucial for load‐induced bone formation. Moreover, mechanical loading strongly regulates Sost/sclerostin expression—a potent inhibitor of Wnt/Lrp5 signaling. Sost, and its protein product sclerostin, are nearly exclusive to the osteocytes—the cell postulated to be the tissue's sensor apparatus in transducing mechanical loading events. The suppression of sclerostin expression by mechanical loading may provide a mechanism by which Wnt signaling is enhanced and bone formation is enacted. Furthermore, Wnt signaling modulation via Sost/sclerostin also appears to modulate the process of disuse osteoporosis. As the mechanisms of mechanical signal transduction in osteocytes become revealed, a greater range of targetable molecules will be manifest, that might ultimately be exploited to improve bone health among patients with a wide range of skeletal disease.

Support or Funding Information

NIH R01 AR053237VA I01 BX001478
Cardiovascular adaptations to moderate anemia maintain cerebral perfusion but are inadequate to prevent renal and splanchnic tissue hypoxia
Authors: Mistry Nikhil , Solish Max , Cahill Lindsay , Zhou Yu‐Qing , Hare Alexander GM , Doctor Allan , Lazarus Alan H , Sled John G , Mazer C David , Hare Gregory MT
Published: November 2023

Mild to moderate anemia is associated with increased organ injury and mortality in surgical patients by undefined mechanisms. We hypothesize that anemia‐induced tissue hypoxia is a potential unifying mechanism. A transgenic mouse model, with luciferase fused to hypoxia inducible factor (HIF ODD‐Luciferase) was utilized to assess the impact of acute antibody‐mediated anemia on adaptive cardiovascular responses, tissue PO2 (PtO2), and HIF expression. A red blood cell (RBC)‐specific antibody (TER119) induced anemia, reducing hemoglobin concentrations from 144±9 to 94±11 g/L (p<0.006) via intravascular hemolysis and RBC sequestration in the spleen and liver. Anemia‐induced cardiovascular adaptations included: 1) increased peripheral arterial oxygen saturation (p=0.018); 2) increased cardiac output (26%, p=0.011); 3) increased internal carotid blood flow (80%, p<0.001); and 4) augmented cerebrovascular reactivity relative to control mice (p<0.001). All of these mechanisms contributed to the maintenance of brain PtO2 (Control vs. Anemia: 23±10 vs.23±5 mmHg O2, p=0.935). By contrast, no increase in renal blood flow occurred (p=0.239), and kidney PtO2 decreased in anemic mice (21±4 vs. 13±4 mmHg O2, p<0.001). Molecular adaptations to tissue hypoxia included increased HIF‐1α expression within the dorsal renal and hepatic region (30%, p=0.006), and ventral gut region (72%, p=0.017). These findings demonstrate anemia‐induced tissue hypoxia is heterogeneous and organ‐specific and support the hypothesis that renal and splanchnic hypoxia may contribute to increased organ injury and mortality in anemic perioperative patients.

Support or Funding Information

The project described was supported by the St. Michael's Hospital AHSC AFP Innovation Fund Grant Support
Comparison of the Ventilatory Responses Elicited by Hypoxic Gas Challenge in Freely‐Moving C57BL6 and A/J Male Mice
Authors: Coffee Gregory A , Getsy Paulina Marie , Lewis Stephen J
Published: November 2023

Genetic differences in humans determine the propensity for disordered breathing including the incidence and severity of sleep apneic events. We have reported that the C57BL6 mouse strain displays a robust series of responses to hypoxic gas challeges.1 Considering evidence that the A/J strain of mouse may be less prone to non‐eupneic breathing,2 our objective was to compare the ventilatory responses elicited by hypoxic challenge in adult male freely‐moving C57BL6 and A/J mice.

Mice were placed in whole‐body plethysmography chambers and subjected to episodes of hypoxic (10% O2, 90% N2) gas challenges (5 episodes of 5 min in duration separated by 15 min). Ventilatory parameters included, frequency of breathing, tidal volume, minute ventilation (= frequency × tidal volume), inspiratory and expiratory times, end inspiratory and expiratory pauses, peak inspiratory and expiratory flows, EF50 (expiratory flow at 50% expired tidal volume) and central inspiratory drive (tidal volume/inspiratory time).

With respect to baseline values, frequency was lower whereas tidal volume was higher in the A/J than the C57BL6 mice such that the minute ventilation was similar in A/J and C57BL6 mice. Inspiratory and expiratory times were both longer in A/J mice than in C57BL6 mice. The remaining parameters including, end inspiratory and expiratory pauses, peak inspiratory and expiratory flows, EF50 and central inspiratory drive were similar in the two mouse strains.

With respect to the ventilatory responses elicited by hypoxic challenges, there were relatively minor differences between C57BL6 and A/J mice in most of the studied parameters. The key differences were that expiratory times and end expiratory pauses dramatically increased upon return to room‐air following the hypoxic gas challenges in C57BL6 mice whereas these values simply returned to baseline in A/J mice.

This study demonstrates that key differences exist between C57BL6 and A/J mice in baseline values and responses to hypoxic gas challenges. Longer duration post‐hypoxia expiratory times and end expiratory pauses are a hall‐mark of humans with sleep apnea. As such, the dramatic increase in these parameters in the post‐hypoxic challenge phases in C57BL6 mice and their absence in A/J mice provide an avenue to further understand the genetic make up of sleep disordered breathing.

Support or Funding Information

GlaxoSmithKline (GSK) Funding CWRU Internal Number: RES510692
Glucagon‐like peptide‐1 affects mean arterial pressure, renal blood flow and urinary flow rate in spontaneously hypertensive rats despite no renal expression of GLP‐1 receptors
Authors: Sorensen Charlotte Mehlin , Ronn Jonas , Holst Jens Juul , Jensen Elisa Pauline
Published: November 2023

Glucagon‐like peptide‐1 (GLP‐1) has many extrapancreatic effects and the GLP‐1 receptor is expressed in many tissues. GLP‐1 increases renal blood flow (RBF), glomerular filtration rate and sodium and water excretion. Also, GLP‐1 has been shown to reduce blood pressure in some hypertensive models. The aim of this study was to examine expression of the GLP‐1 receptor in the renal vasculature from spontaneously hypertensive rats (SHR) and to assess the renal and vascular effects of GLP‐1.

Methods

The GLP‐1 receptor was localized using both immunohistochemical staining and in situ hybridization for the GLP‐1 receptor on kidney sections from normotensive Sprague‐Dawley (SD) and hypertensive SHR rats. The vasodilatory effect of GLP‐1 was examined in isolated interlobar arteries. In vivo experiments were performed on rats monitoring mean arterial blood pressure (MAP), RBF, heart rate (HR), urine flow and sodium excretion. GLP‐1 was infused directly into the kidney for 20 min before and after infusion of the GLP‐1 receptor antagonist exendin9–39.

Results

No GLP‐1 receptor positive staining was found in kidneys from SHR whereas there was GLP‐1 receptor positive staining in the vascular smooth muscle cells in the renal vasculature from normotensive rats. No vasodilation was elicited in interlobar arteries from SHR whereas arteries from SD responded to increasing concentrations of GLP‐1. Intrarenal infusion of GLP‐1 increased BP, RBF, urine flow and sodium excretion significantly in both rat strains whereas heart rate was unchanged. The effect on RBF and MAP was abolished by exendin9–39 whereas the increase in urine flow and sodium excretion was still present.

Conclusion

The renal effects of GLP‐1 in SHR do not rely on the presence of renal GLP‐1 receptors. It seems likely that the GLP‐1 mediated effects in SHR are mediated via extrarenal GLP‐1 receptors e.g. GLP‐1 receptors in organs responsible for regulation of insulin or ANP.
The choroid plexus sodium‐bicarbonate cotransporter NBCe2 regulates cerebrospinal fluid pH
Authors: Barbuskaite Dagne , Praetorius Jeppe , Christensen Henriette L , Rojek Alexandra , Nielsen Hans Malte , Fuchtbauer Ernst‐Martin , Wang Tobias , Damkier Helle Hasager
Published: November 2023

Respiratory centers in the central nervous system (CNS) react to changes in pCO2 and maintain systemic acid/base homeostasis by controlling the rate and depth of respiration. Both blood‐brain barrier and blood‐CSF barrier are highly permeable to CO2 enabling swift transmission of arterial pCO2 fluctuations to be coupled with CSF pH changes, whilst both barriers are much less permeable to H+and HCO3−, hence masking the effects of metabolic acid‐base disturbances. The electrogenic Na+‐HCO3−cotransporter NBCe2 is expressed in the CSF‐facing plasma membrane of the choroid plexus epithelial (CPE) cells and could be a regulator of CSF pH. We hypothesized that NBCe2 is responsible for HCO−extrusion into the ventricle lumen to counteract decreases in CSF pH during acidosis. Intracellular pH (pHi) measurements on CPE cells from NBCe2 knockout (KO) mice showed a statistically significant 84% increase in Na+‐dependent acid extrusion following intracellular acidification by ammonium chloride prepulse and an 81% reduction in DIDS‐sensitive base efflux after intracellular alkalization by trimethylamine. Knockout of NBCe2 abolished CSF pH recovery from hypercapnia‐induced acidosis compared to control mice: wt (n=5): 0.0048 ± 0.003 vs ko (n=6): −0.0001±0.0002 pH units/min, p=0.0043. Choroid plexus targeted NBCe2 knockdown mice generated by intracerebroventricular installation of siRNA revealed a similar effect: Control (n=5): 0.002+/−0.0004 vs. NBCe2 knockdown (n=7): −0.0001±0.0007, p=0.047. Knockout of NBCe2 did not affect respiration rate or tidal volume compared to wildtype, and revealed a similar ventilatory response as controls when exposed to 5% CO2. In conclusion, we show that NBCe2 is important for recovery of CSF pH after CO2 induced acidosis by transporting HCO3−into the CSF. This suggests a significant role for NBCe2 in regulating brain pH when faced with an acid challenge independent of the respiratory response.

Support or Funding Information

Dagne Barbuskaite is funded by a PhD stipend from the Faculty of Health and Medical Sciences and the department of Cellular and Molecular Medicine, Copenhagen University. Henriette L. Christensen is funded by a PhD stipend from the Faculty of Health, Aarhus University.

The project is financed by the Danish Council for Independent Research/Medical sciences and the Laege Sofus Carl Emil Friis og Olga Doris Friis' legat.
H,K‐ATPase type 2 participates in the adaptive proliferation of quiescent renal A‐type intercalated cells in response to K+ depletion
Authors: Crambert Gilles , Cheval Lydie , Doucet Alain , Edwards Aurélie , Walter Christine
Published: November 2023

Quiescent renal epithelial cells proliferate in response to specific stimuli such as K+depletion or metabolic acidosis, leading to hypertrophy of the kidney. The role of ion transporters in cell proliferation is now well established: intracellular pH and intracellular K+ and Cl− concentrations are key determinants of cell volume and thereby contribute to cell division. Interestingly, H,K‐ATPase type 2 (HKA2) has recently been shown to participate in cell proliferation in a leukemia cell model and its expression is dysregulated in different cancers (3, 4). HKA2 could interfere with cell proliferation by transporting H+ and/or K+. Taking into account the fact that this transporter is strongly up‐regulated in A‐type intercalated cells (A‐IC) of the collecting duct (CD) in response to K+ depletion (for review see (1)), we investigated whether it participates in the cell proliferation process that occurs under these conditions.

In contrast to WT mice under a low‐K+ diet, HKA2‐null mice (HKA2‐KO) did not exhibit an increased number of either total or proliferative A‐IC. However, K+ depletion increased the expression of early proliferation markers such as PCNA and cyclin D1 similarly in WT and HKA2‐KO. These results indicate that in the absence of HKA2, the process of A‐IC proliferation is initiated but is promptly aborted. This is confirmed by the fact that the number of apoptotic (TUNEL‐positive) cells in the CD of HKA2‐KO mice is 3 times higher than in that of WT after K+ depletion. According to our recent mathematical model of ion transport in the CD (2), the absence of HKA2 may result in a lower A‐IC cell volume (−20%). Therefore, HKA2 may be relevant during K+ depletion by contributing to the cell swelling process required during cell division. A dysfunctional increase in cell volume during cell proliferation may result in the activation of ER stress mechanisms and indeed, under a low K+ diet, CHOP expression is almost twice as high in HKA2‐HO mice relative to WT mice.

Altogether, these results revealed a new role for the renal H,K‐ATPase type 2 that may participate in the process of cell proliferation in the context of K+ depletion.

Support or Funding Information

This study was supported by recurrent grants from the Institut National de la Santé et de la Recherche Médicale (INSERM) and from the Centre National de la Recherche Scientifique (CNRS)