bluuebunny/crossref_metadata_2025 huggingface dataset:
Cardiometabolic conflux at the carotid body
Authors: Thakkar Pratik , Pauza Audrys , Murphy David , Paton Julian
Published: May 2023

Background: Diabetes is the fastest-growing disease in New Zealand, and when combined with high blood pressure (hypertension), so-called sweet pressure (SP) is the leading cause of mortality. However, controlling SP remains challenging clinically. One target that remains unexploited is the sympathetic nervous system (SNA). As the carotid body (CB) has a pivotal role in regulating SNA, blood pressure and blood glucose, we searched for novel signalling pathways linking the CB with metabolism. Using RNA sequencing, we discovered melanocortin type-4 receptor (MC4R) expression was upregulated in the CB of spontaneously hypertensive rats (SHR) versus Wistar rats; these receptors have an essential role in regulating cardiovascular and energy homeostasis. Hypothesis: We hypothesised that blockade of MC4R within the CB would reduce SNA and alleviate SP. Methods: The SHR was used as a hypertensive-diabetic model as it has glucose intolerance. Using a novel in situ preparation, we recorded SNA activity while injecting an MC4R antagonist (HS014) into the CB via the internal carotid artery. Results: The upregulated MC4R expression in the CB of SHR versus Wistar rat was validated using qPCR and immunocytochemistry. Antagonising MC4R within the CB lowered SNA and prevented the SNA hyperactivation triggered by hyperglycaemia (high glucose). No such responses were found in control Wistar rats. Conclusion: MC4R signalling is a novel mechanism within the CB that depresses ongoing SNA and, importantly, the elevated SNA caused by hyperglycaemia; whether this reduces blood glucose awaits testing in vivo. These data provide a potential mechanism for controlling co-comorbidity of hypertension and blood glucose.

Funding: HRC, Royal Society Te Apārangi and Sidney Taylor Trust funded this research.

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
The GP.Mur red cell phenotype (higher band 3 expression) affects respiratory frequency and the inspiratory/expiratory ratio in a breath cycle during exhaustive exercise
Authors: Hsu Kate , Hsu Kate , Tseng Wei-Chin , Chen Li-Yang , Chen Pin-Lung , Lu Yu-Xian , Chen Yung-Sheng , Tseng Kuo-Wei
Published: May 2023

The GP.Mur red cell phenotype is characterized by the unique presence of glycophorin B-A-B hybrid protein on the erythrocyte membrane which promotes band 3 expression. Band 3, or anion exchanger-1, transports HCO3 - across the erythrocyte membrane by exchange of Cl-; this function of band 3 works in concert with intraerythrocytic carbonic anhydrase II (CAII) to facilitate blood CO2 transport. People with the GP.Mur blood type express 10-20% more band 3 protein than those without the special blood type. We thus hypothesized that respiration in people with the GP.Mur blood type might be distinctly different when being challenged. In this study, we recruited 36 age-matched male elite athletes (13 GP.Mur and 23 without GP.Mur [control]) for an incremental running test to exhaustion. While running on a treadmill, the subjects were continuously monitored using a Quark CPET (cardiopulmonary exercise testing) unit (COSMED, Italy). Two statistical methods were employed for data analysis: (1) unpaired t-test for comparison at individual timepoints; and (2) linear mixed modeling for serial cardiopulmonary measurements collected during the running test. We found that their running abilities were similar. However, the subjects with GP.Mur showed higher respiratory rates and smaller inspiratory/expiratory (I/E) ratios than the control subjects throughout the entire exhaustive running test. That is, GP.Mur+ subjects spent a larger fraction of the respiratory cycle time on CO2 excretion than inspiration; their respiration also appeared to respond faster to the same level of workload. These resulted in lower end-tidal CO2 pressure in the running GP.Mur subjects compared to the controls. In conclusion, we presented direct evidence that band 3-mediated HCO3 -/CO2 exchange at the erythrocyte level could impact the respiratory pattern of CO2 excretion.

Taiwan National Science and Technology Council; MacKay Memorial Hospital

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Mechanotransduction meets thermosensation: Coupling of TRP channels in ventilator-induced lung injury
Authors: Michalick Laura , Zergane Meryam , Pickerodt Philipp , Kuebler Wolfgang
Published: May 2023

Study objective: Mechanical ventilation can damage the lung by causing endothelial barrier failure, edema formation and lung injury. Yet, the exact mechanisms underlying the mechanotransduction at the microvascular barrier are still unclear. In previous work, we had identified a critical role for the endothelial Ca2+ channel transient receptor potential vanilloid 4 (TRPV4) in lung microvascular barrier failure subsequent to mechanical overventilation. Our preliminary data reveal an involvement of thermosensitive TRPV1 in this scenario, which prompted us to test for TRPV1’s contribution to TRPV4-induced Ca2+ signaling and lung injury induction in vivo, ex vivo and in vitro. Hypothesis: TRPV1 acts as amplifier of mechano- and thermosensitive TRPV4-mediated pulmonary vascular barrier failure during mechanical ventilation. Methods: Anesthetized wild type or Trpv1-/- mice were ventilated for 2 h with low (7 mL/kg) and high (20 mL/kg) tidal volumes (LVT/HVT) at different inspired gas temperatures (20-40°C) in vivo, and parameters such as edema formation, vascular permeability and inflammation was assessed . Endothelial [Ca2+]i in response to pressure elevation from 5 to 15 cmH2O was quantified in isolated-perfused lungs ex vivo, and in vitro in human pulmonary microvascular endothelial cells (HPMECs) after activation of TRPV1 and/or TRPV4 by capsaicin and/or GSK1016790A with or without pharmacological inhibition of TRPV1 and TRPV4. Additionally, we measured barrier function by ECIS ® and examined the induction of apoptosis after TRPV1/4 activation. Results: TRPV1 deficiency or inhibition, as well as reduced temperatures (< 25°C) of inspired gas attenuated VILI in vivo, and reduced the endothelial [Ca2+]i response to high pressure inflation ex vivo. In vitro HPMECs showed a characteristic increase in endothelial [Ca2+]i in response to agonism of TRPV4, yet not TRPV1 alone. In line with these findings, the dual activation of both TRPV channels amplified and prolonged the TRPV4-mediated Ca2+ response, induction of apoptosis and loss of barrier integrity. Conclusions: Here, we demonstrate a critical role for TRPV1 as amplifier of the TRPV4-mediated [Ca2+]i response and vascular barrier failure in VILI as a function of inspired gas temperature. Our findings unveil a mechano- and temperature-sensitive increase in TRPV1/4 activation, which contributes to endothelial barrier dysfunction and edema formation during mechanical ventilation and could be targeted by lower inspired gas temperatures.

This study is supported by the German Centre for Cardiovascular Research (DZHK), partner site Berlin, Germany.

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Significance of Cell Membrane Repair in Parkinson's Disease
Authors: Goncalves Isabella , Goncalves Isabella
Published: May 2023

Objective: Parkinson’s disease (PD) is the second most common neurodegenerative disorder and affects an estimated 10 million people worldwide. PD pathogenesis involves the death of neuronal cells due to the breakdown of their cell membranes. Membrane repair is a mechanism that appears in nearly every eukaryotic cell that repairs disruptions in the plasma membrane to maintain cell health and function. The aim of the present study was to determine the if compromised membrane repair can contribute to the progression of PD. Materials and Methods: Using a glass bead mechanical damage assay, we injured N2A neuroblastoma cells with three different conditions: GFP transfected control cells, cells expressing alpha synuclein, and cells expressing A53T, an alpha synuclein mutant linked to PD. We then measured the amount of an intracellular enzyme, lactase dehydrogenase (LDH), that was released from the cells after injury with glass beads to determine how well the membrane repaired. (n=16) We also injured the membrane of these cells with a laser injury heat damage assay. In this mechanism, a laser is used to injure the cells, and influx of FM4-64 dye into the cell is measured to determine the rate of membrane repair. (n=60) Results: In the mechanical damage assay, cells expressing alpha synuclein released more LDH, which suggests that membrane repair may be compromised in PD. In the laser damage assay, cells expressing alpha synuclein showed significantly more FM4-64 dye influx. We observed increased dye influx in WT Alpha Synuclein cells. This shows that Alpha Synuclein transfected cells do not repair as effectively as control cells when exposed to the same conditions. These findings support that membrane repair may be compromised in PD. Elucidating that membrane repair is impaired in PD leads us to investigate if existing membrane repair enhancing agents could be potential therapeutics for the disease.

American Physiological Society Summer Undergraduate Research Fellowship (APS SURF)

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
CD8+ T cells stimulate renal sodium transporters contributing to salt-sensitive hypertension
Authors: Deck Katherine , Guo Yunping , Benson Lance , Liu Yunmeng , Mora Christoph , Mu Shengyu
Published: May 2023

Impaired salt handling by the kidneys acts as one source of hypertension. However, the exact identity of this kidney defect remains unknown. As established recently, the adaptive immunity, T cells in particular, acts as an important contributor to the pathogenesis of hypertension. In this regard, our laboratory has reported that CD8+ T cells (CD8Ts), when adoptively transferred from salt-sensitive hypertensive mice to normotensive mice, stimulate Sodium Chloride Cotransporter (NCC). Through this stimulation, sodium retention occurs, and salt-sensitive hypertension develops. However, whether other sodium transporters within the nephron are affected by hypertensive CD8Ts and contribute to this pathogenesis of hypertension remains uncharacterized. We hypothesize hypertensive CD8Ts will also have an impact on other sodium channels. Here, we demonstrate the impact of hypertensive CD8Ts on sodium-potassium-co-transporter (NKCC) and epithelial sodium channel (ENaCs) in the kidney and the potential of these regulations contributing to salt-sensitive hypertension.Through in vivo studies, we employed a CD8T-adoptive transfer model as described in our previous publications. The kidneys from mice receiving adoptive transfer of hypertensive CD8Ts were harvested and protein levels of NKCC, αENaC, βENaC, and γENaC were analyzed by western blot. With in vitro studies, we co-cultured mouse CD8Ts with Collecting Duct epithelial cells (CDs, M1 cells) to examine the direct interaction between CD8Ts and CDs in media containing various levels of corticosteroids. Similar western blots were performed after this co-culture, as well as measurement of sodium retention through an intracellular sodium indicator and flow cytometry, as reported previously.In the results of in vivo studies, we found that the kidneys of mice receiving adoptive transfer of hypertensive CD8Ts demonstrated higher expression of NKCC, βENaC and γENaC, but no change in αENaC. In the results from in vitro studies, we further found that in charcoal-stripped co-culture conditions, pre-activated CD8Ts stimulate higher expression of ENaC subunits and ENaC-mediated sodium retention in M1 cells. However, these effects are masked in culture conditions with physiological hormone levels or with additional corticosteroids.Overall, our results suggest that, as a potential kidney defect, hypertensive CD8Ts strongly stimulate NCC and NKCC in the kidney to mediate excessive salt retention. Although CD8Ts themselves can increase ENaC, this effect might be masked in physiologic conditions.

NIH R01-HL146713 (Mu), AHA 15BGIA25730047 (Mu), and SPaT T32-GM10699 (Deck)

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Microvascular Function in Skeletal Muscle of Acute Adiponectin Knockout Mice
Authors: Medarev Steven , Faal Madikay , Asher Andy , Smith Cole , Qasim Muhammad , Belenko Jasen , Hendrickson Dylan , Mitchell Logan , Ojah-Maharaj Jayden , Faynberg Victor , Cannon Nicole , Muller-Delp Judy
Published: May 2023

Adiponectin contributes to the regulation of vascular and metabolic functions in skeletal muscle. Although circulating adiponectin is known to increase with aerobic exercise training, a direct role for adiponectin in adaptation of microvasculature of skeletal muscle to aerobic exercise training has not been documented. We exercise trained young adult mice with normal circulating adiponectin (wild type, WT) and mice in which adiponectin was acutely deleted (Cre-Lox conditional knockout system induced with Tamoxifen chow, AdipoKO) before undergoing 8 weeks of exercise training. Sedentary WT and AdipoKO mice were housed under similar cage conditions for 8 weeks. We determined skeletal muscle oxidative capacity, exercise tolerance, lean and fat mass, and we assessed reactivity of 1A arterioles from the gastrocnemius muscle. Exercise training increased exercise tolerance in both WT and AdipoKO mice. Exercise training increased lean mass and reduced fat mass in both WT and AdipoKO mice. Myogenic constriction to intraluminal pressure changes was increased by exercise training in gastrocnemius muscle 1A arterioles from both WT and AdipoKO mice. In contrast, phenylephrine-induced contractile responses were increased by exercise training in WT, but not AdipoKO mice. These data suggest that adiponectin is an important contributor to adaptations of contractile function that occur in skeletal muscle in response to exercise training; however, the training-induced increase in exercise tolerance and muscle mass that occurred in both WT and AdipoKO mice suggests that loss of adiponectin impacts smooth muscle function independently of metabolic adaptations in skeletal muscle.

NIH R56AG068156

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Spinal microglial morphology in a rat model of Pompe Disease
Authors: Deegan Kyle , Martinez Robert , Rana Sabhya , Byrne Barry , Fuller David
Published: May 2023

Pompe disease is a monogenic neuromuscular disorder caused by a mutation in the gene encoding the lysosomal enzyme acid alpha-glucosidase. This leads to widespread glycogen accumulation, cardiorespiratory failure, and early mortality. There is clear evidence for neurodegeneration and neuroinflammation in animal models of Pompe disease (Turner et al., Resp. Phys. Neurobio., 2016) as well as human Pompe tissues (Fuller et al., J. Neurophys., 2021). However, the appearance of microglia has not been comprehensively evaluated in Pompe disease. Here we examined this question using a rat model of Pompe disease. Mid-cervical spinal cord tissue sections of 10-13 mo old male Pompe ( Gaa null, n=3) and Sprague Dawley rats (n=3) were histologically evaluated. We focused on the mid-cervical spinal cord because this region contains the phrenic motoneurons which innervate the diaphragm muscle. Immunohistochemical processing was performed on coronal spinal cord sections using a neuronal nuclear protein (NeuN) antibody to label neurons, and an ionized calcium-binding adaptor molecule 1 (IBA-1) antibody to label microglia. Robust and reliable staining of neurons and microglia was observed in white and grey matter regions of the spinal cord section using fluorescent imaging. Putative motoneurons in the Pompe mid-cervical ventral grey matter had the “prototypical appearance” of motoneurons in lysosomal storage diseases. This included a considerably enlarged (“swollen”) soma and extensive vacuolization. Spinal cord microglia in Pompe tissues had a fundamentally different appearance as compared to Sprague-Dawley. Specifically, microglia appeared with increased density in Pompe spinal cord sections and were frequently observed to be in immediate proximity (e.g., “wrapping around”) to ventral spinal neurons which displayed a swollen and vacuolated morphology. Pompe microglia also showed a rounded appearance that is consistent with the “reactive” state known to occur during neuroinflammation. These histological data are consistent with neurodegeneration and ongoing microglia-driven phagocytosis of neurons in the mid-cervical spinal cord of adult Pompe disease rats.

R01HD052682 (DDF and BJB)

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Circadian timing of grape intake and the oxidative stress response
Authors: Blanton Cynthia , Aghazadeh-Habashi Ali , Ghimire Biwash , Khajeh Pour Sana
Published: May 2023

Background: The immune system displays circadian rhythm and timing of the ingestion of foods with immunoactive properties may modify their impact. Objective: The purpose of this investigation was to evaluate the effect of time of day of grape consumption on oxidative stress. Hypothesis: We hypothesized that the oxidative stress response to a high-fat meal would be 1) greater in the a.m. vs. p.m. and that 2) this response would be blunted by ingestion of a grape vs. placebo drink. Methods: The study followed a 2 x 2 factorial randomized, controlled, double-blind design wherein healthy men and women (n=32) consumed either a grape (equivalent to 1.5 servings of fresh grapes) or placebo drink with a high-fat meal (an oxidative challenge) in either the morning (7:00 a.m.) or evening (6:00 p.m.). Urine was collected for a measure of oxidative stress (F2-isoprostane) at baseline and post-meal at hour 1 and batched over hours 1-6. F2-isoprostane concentration was determined using Liquid Chromatography-Tandem Mass Spectrometry and values were normalized to creatinine levels determined by enzyme-linked immunoassay. Results: ANOVA showed a significant increase above baseline in urine F2-isoprostane levels (ng/mg creatinine) at hour 1 and hours 1-6 (P<0.0001) that were significantly greater in the a.m. vs. p.m. (P=0.04) but were not significantly different between grape and placebo. Expressed as % baseline, F2-isoprostane levels at hour 1 and hours 1-6 tended to be lower in the grape vs. placebo group (P=0.058) and were significantly higher in the a.m. vs. p.m. (P=0.01). Total isoprostane excretion expressed as % baseline showed a significant increase in the a.m. placebo group (P=0.03) that was prevented by grape ingestion. Conclusions: 1) Grape may suppress the oxidative stress response to a high-fat meal and 2) The oxidative stress response to a high-fat meal appears greater in the morning vs. evening. Thus, timing the consumption of immunoactive food may alter the immune response.

Funding and grape/placebo powders were provided by the California Table Grape Commission.

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Aortic baroreceptor afferent activity during systemic inflammation
Authors: Brognara Fernanda , Castania Jaci , Dias Daniel , Salgado Helio , Paton Julian , Carnio Evelin
Published: May 2023

Objective: Neuroimmune interactions have been extensively explored to find new therapeutic targets for inflammatory diseases. We postulated that baroreceptors (essential for arterial pressure control) might be susceptible to systemic inflammation. Thus, the present study investigated the effect of early-stage systemic inflammation on aortic baroreceptor afferent activity. Hypothesis: Aortic depressor nerve activity is increased during systemic inflammation. Methods: Anesthetized (urethane; 1 g/kg; i.p.) male Sprague-Dawley rats (7-8 weeks old) were used, and lipopolysaccharide (LPS; 1.5 mg/kg; i.v.) injected to induce systemic inflammation. The femoral artery and vein were cannulated for pulsatile arterial pressure recording and LPS administration, respectively. Following 30 min since injections of either saline (control; n = 10) or LPS (n = 16), pulsatile arterial pressure and aortic depressor nerve activity were recorded. Nerve activity recordings were rectified and integrated on a time basis. Body temperature was measured via rectal thermometer. The results are presented as mean ± standard error of the mean. Results: Compared to saline-treated rats, there was an increase in aortic depressor nerve activity (LPS: 1.04 ± 0.02 vs saline: 0.98 ± 0.01 a.u.; p = 0.007); this was accompanied by increases in diastolic arterial pressure (LPS: 83 ± 2 vs saline: 73 ± 4 mmHg; p = 0.031), mean arterial pressure (LPS: 102 ± 3 vs saline: 91 ± 4 mmHg; p = 0.035) but not systolic pressure (LPS: 140 ± 6 vs saline: 122 ± 6 mmHg; p = 0.057; strong trend) or heart rate (LPS: 425 ± 11 bpm vs saline: 393 ± 14; p = 0.085). Importantly, in the LPS-injected rats, a reduction in body temperature was noted compared to saline-treated rats (LPS: 36.7 ± 0.1 vs saline: 37.2 ± 0.2°C; p = 0.005), an indicative of systemic inflammation. Conclusions: Since the diastolic and mean arterial pressures increased after LPS administration, it remains unclear whether the elevated aortic depressor nerve activity reflects this increased pressure or results from the inflammatory mediated sensitization. However, our preliminary data support the notion that aortic baroreceptor afferents participate in the neuroimmune communication response in the early stages of inflammation.

Funding sources: FAPESP (protocols #2021/03764-8 and #2021/05554-0).

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Improving White Blood Cell Morphology Education Through Standardized Questions
Authors: Memmott Audrey , Tanner Dylan , Cordner Ryan
Published: May 2023

Medical laboratory scientists routinely evaluate blood smears in the clinical laboratory. A high level of skill in white blood cell morphology is required to do the task quickly and accurately. Educational approaches to teaching white blood cell morphology consist of showing examples of cells to students and allowing them to practice using trial and error to hone their skills. Student progress with this traditional approach is often quite slow. In order to expedite the learning process, we created a series of questions that can guide morphological identification; these questions focus on the shape and description of the nucleus, cytoplasm, granules and cell overall. We hypothesized that white blood cells can be accurately and uniformly identified using the criteria we’ve outlined. We confirmed the accuracy of this approach to white blood cell identification using a random forest classifier algorithm on a data set of 3600 cells that we had previously categorized. Our approach has the potential to standardize white blood cell morphology training in undergraduate medical laboratory scientist programs, and could help improve the consistency of manual differential results between hospital laboratories.

This work is funded by a Teaching Development Grant from the Brigham Young University College of Life Sciences

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Activation of renal vascular smooth muscle TRPV4 channels by 5-hydroxytryptamine impairs kidney function in neonatal pigs
Authors: Michael Olugbenga , Afolabi Jeremiah , Falayi Olufunke , Kanthakumar Praghalathan , Mankuzhy Pratheesh , Soni Hitesh , Adebiyi Adebowale
Published: May 2023

Control of microvascular reactivity by 5-hydroxytryptamine (5-HT; serotonin) is complex and may depend on vascular bed type and 5-HT receptors. 5-HT receptors consist of seven families (5-HT1-5-HT7), with 5-HT2 predominantly mediating renal vasoconstriction. Regulation of cyclooxygenase (COX) and smooth muscle cell (SMC) intracellular Ca2+ concentration ([Ca2+]i) levels have been implicated in 5-HT-induced vascular reactivity. Although 5-HT receptor expression and circulating 5-HT levels are known to be dependent on postnatal age, control of neonatal renal microvascular function by 5-HT is unclear. The objective of this study is to use the translational swine model to examine the effects of 5-HT on newborn renal microcirculation. We demonstrate that 5-HT activates human transient receptor potential cation channel subfamily V member 4 (TRPV4) channels that were transiently expressed in Chinese hamster ovary cells. 5-HT2A is the predominant 5-HT2 receptor subtype in freshly isolated neonatal pig renal microvascular SMC. HC-067047 (HC), a selective TRPV4 blocker, attenuated cation currents induced by 5-HT in neonatal pig renal vascular SMC. HC also inhibited the 5-HT-induced increase in renal microvascular [Ca2+]i and constriction. Intrarenal artery infusion of 5-HT had minimal effects on systemic hemodynamics but reduced renal blood flow (RBF) and increased renal vascular resistance (RVR) in the pigs. Transcutaneous determination of glomerular filtration rate (GFR) indicated that kidney infusion of 5-HT reduced GFR. HC diminished 5-HT-induced alteration of RBF, RVR, and GFR. Moreover, plasma and urinary COX-1 and COX-2 levels in 5-HT-treated pigs were unchanged. These data suggest that activation of renal microvascular SMC TRPV4 channels by 5-HT impairs kidney function in neonatal pigs independently of COX production.

NIH R01DK120595 and R01DK127625

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Identification of MEST as a critical player in mechanotransduction of endothelial cells in response to shear stress in atherosclerosis
Authors: Zeng Ye , Fu Bingmei
Published: May 2023

OBJECTIVE: Atherosclerosis (AS) is a chronic disease characterized by the inflammation and the deposition of cholesterol in arterial intima, which is the common pathological basis of various cardiovascular and cerebrovascular diseases. Fluid shear stress (FSS) is an important regulatory factor of cellular cholesterol metabolism during the occurrence and progression of AS. However, the mechanism by which FSS regulated the deposition of cholesterol in AS remains unclear. METHODS: By using a novel cell model of foaming established by piled vascular endothelial cells, the transcriptome sequencing and the bioinformatics data mining were carried out to screen the key differential genes involved in the foaming of vascular endothelial cells and the FSS mechanotransduction, and then the qRT-PCR validation of them was performed. Also, the levels of the identified molecules in ApoE-/- atherosclerotic mice were detected. Finally, the role of MEST in vascular endothelial cells was explored by BODIPY staining upon MEST knockdown. RESULTS: We identified MEST that regulated by FSS is downregulated in foaming endothelial cells, and is also reduced in the atherosclerotic lesions. MEST might be closely associated with lipid metabolism, vascular endothelial glycocalyx, G protein-coupled receptor signaling, and cell-to-cell communication. Knockdown of MEST induced lipid accumulation in vascular endothelial cells. CONCLUSION: MEST is an antiatherogenic molecule, which might control the endothelial injury and atherogenic initiation.

This project was supported by the National Natural Science Foundation of China (12272246).

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Cytosolic full-length PTEN-induced putative kinase 1 enhances mitophagic potential and mtDNA integrity in angiotensin II-induced hypertensive mice
Authors: Park Joon-Young , Hong Soon-Gook , Shin Junchul , Saredy Jason , Choi Soo-Young , Wang Hong
Published: May 2023

PTEN-induced putative kinase 1 (PINK1) is an essential molecule in mitophagy process in mammalian cells. The primary purpose of this study was to determine the expression and subcellular localization of PINK1 under physiological fluidic conditions in endothelial cells (ECs) and elucidate the implication these phenomena in the endothelial homeostasis and disease prevention. Unidirectional laminar shear stress (LSS) significantly elevated both mRNA and protein expression levels of the full-length PINK1 (FL-PINK1) in ECs. Extensive mitochondrial fractionation assays and confocal microscopic analyses demonstrated significantly reduced FL-PINK1 accumulation in the mitochondrial compartment yet increased in the cytosolic pool of FL-PINK1 after exposed to prolonged high LSS (15 dyne/cm2 for 48h). We also observed that mitophagy flux was significantly decreased without changes in mitochondrial membrane structures and membrane potential under the LSS condition. Moreover, increased cytosolic PINK1 increased mitophagic sensitivity to the cumulative fragmented mitochondria in the ECs. When treated with angiotensin II (AngII), LSS-preconditioning significantly decreased mtDNA lesions and displayed rapid Parkin recruitment followed by mitophagy induction in response to a mitochondrial uncoupler, carbonyl cyanide m-chlorophenyl hydrazone, -mediated oxidative stress condition. In addition, voluntary running exercise-preconditioning showed elevated PINK1 expression and enhanced mitophagy induction and reduced mtDNA lesions in the AngII-induced hypertensive mice. Taken together, LSS specifically increases a cytosolic pool of FL-PINK1, which elevates mitophagic sensitivity to dysfunctional mitochondria in endothelial cells. Our data suggest that enhanced unidirectional laminar shear stress promotes mitochondrial quality control and cell protection mechanisms via a PINK1-dependent pathway.

This work was supported by funding from the National Institute of Heart, Lung, and Blood Institute at the National Institutes of Health (R01 HL126952 to J.Y.P.); and The American Heart Association/Beatrice F. Nicoletti research grant (19POST34450157 to J.C.S).

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Overexpression of ABCB6 does not protect against doxorubicin cardiotoxicity
Authors: Jacintho Moritz Cesar Eduardo , Montalvo Ryan N. , Doerr Vivian , Nguyen Branden L. , Smuder Ashley J.
Published: May 2023

Doxorubicin (DOX) is a highly effective chemotherapeutic agent used in the treatment of a broad spectrum of human cancers. However, the clinical use of DOX is limited due to dose-dependent cardiotoxicity, which adversely affects quality of life and survival. Therefore, new strategies to mitigate the cardiotoxic effects of DOX are needed to improve the clinical usefulness of DOX and long-term health outcomes. The mitochondria-localized ATP-binding cassette (ABC) proteins (i.e. ABCB6, ABCB7, ABCB8 and ABCB10) are a family of transporters with the potential to mediate xenobiotic efflux and/or may play a role in maintaining mitochondrial redox balance and iron homeostasis. Previous data showed that exercise training mediated an increase in the expression of each mitochondria-localized ABC transporter in the heart. To determine the specific role of increased ABCB6 expression in the heart prior to DOX treatment female Sprague Dawley rats were randomly divided into the following groups: 1) Saline-Saline; Saline-ABCB6; 3) DOX-Saline; and 4) DOX-ABCB6. Four weeks before DOX (20 mg/kg; i.p.) or saline administration the animals received rAAV-ABCB6. Dependent measures were evaluated 48 hours after DOX/saline treatment. Cardiorespiratory fitness assessed via a graded exercise tolerance test performed just prior to endpoint demonstrated a significant effect of DOX on time to fatigue, with no beneficial adaptation of ABCB6 overexpression. Moreover, weight change from treatment to endpoint and the heart weight/tibia length ratio were also decreased after treatment in both DOX groups with no effect of ABCB6. Additionally, cardiac fractional shortening (FS) and posterior wall shortening velocity (PWSV) were reduced in both DOX groups. Similarly, myocardial performance index (MPI) increased in the DOX-Saline and DOX-ABCB6 groups. These data demonstrate that overexpression of the ABCB6 transporter in the heart is not capable to mitigate the deleterious effects of DOX treatment on the heart. Further studies are needed to investigate the potential therapeutic effects of mitochondria-localized ABC transporters as a clinical approach to prevent cardiotoxicity effects triggered by DOX.

This research was funded by the National Institute of Health (NIH), R01HL144858.

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
The Effect of Exercise on Doxorubicin-Induced Nephrotoxicity in Male and Female Rats
Authors: Patel Jay , Montalvo Ryan , Boeno Franccesco , Doerr Vivian , Smuder Ashley
Published: May 2023

Doxorubicin (DOX) is a first-line chemotherapeutic agent used to treat a wide range of malignancies including breast and lymphatic cancers. However, the clinical use of DOX is limited due to off-target toxicity to healthy organs. DOX-induced nephrotoxicity is established in both cancer patients and experimental models, where it is hypothesized that oxidative damage to renal cells increases glomerular capillary permeability and tubular atrophy. In contrast, endurance exercise preconditioning has the potential to reduce renal edema and inflammation, improving glomerular filtration rate and urine creatinine clearance rate. Therefore, the purpose of this study was to investigate whether exercise preconditioning can protect against DOX-induced nephrotoxicity and elucidate differences between male and female rats. Young-adult male and female Sprague-Dawley rats were randomly separated into 4 groups: i) sedentary saline; ii) sedentary DOX (20 mg/kg IP); iii) exercise-trained saline; and iv) exercise-trained DOX. Exercise trained animals underwent 2 weeks of treadmill preconditioning for (5 days/wk, 60 min/day, 30 m/min @ 0% grade). Animals received saline or DOX 24hrs after the final exercise bout, followed by endpoint 48hrs post-treatment. Evaluation of inflammatory markers within the urine (i.e. IP10, AGP and NGAL) demonstrated elevated levels in DOX-treated males and females, regardless of exercise. Male rats receiving DOX had elevated levels of clusterin which was not found in female rodents suggesting a possible sex-difference. Moreover, urine creatinine levels were significantly increased following DOX treatment and reduced with exercise in males, with no significant differences in females. In summary, animals treated with doxorubicin had higher levels of markers for inflammation and renal disease, with minimal effects of exercise preconditioning to prevent these changes. Additionally, males demonstrated higher levels of renal toxicity suggesting that females may have a greater resistance to DOX-induced nephrotoxicity.R01 HL153140R01 HL144858

R01 HL144858 and R01 HL153140

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Targeted disruption of CTCF binding near the Ren gene alters gene expression and plasma renin activity in response to dietary salt
Authors: Vanden Avond Mark , Grzybowksi Michael , Kurth Theresa , Greene Andrew , Liang Mingyu , Cowley Allen , Geurts Aron
Published: May 2023

Hypertension is the leading risk factor for cardiovascular disease and impacts one in three adults globally. One genetic mechanism that may affect blood pressure regulation and alter hypertension risk in the general population is the disruption of chromatin conformation by common genetic variation. CCCTCF-binding factor (CTCF) has many important functions including regulating the conformation of chromatin by binding to specific DNA sequences and can thereby alter gene expression. The renin locus has multiple CTCF motifs surrounding the renin gene and cellular expression of CTCF is critical for proper renin expression. Despite these important functions, the role that local chromatin conformation plays to regulate normal renin expression including its response to stimuli such as dietary salt intake and in the downstream regulation of blood pressure is unknown. We hypothesize that disruption of local chromatin conformation can alter gene expression, intermediate physiological mechanisms, and blood pressure. A mutant rat model was created using CRISPR-Cas9 in the genetic background of the Dahl salt-sensitive (SS) rat with a disruption to the CTCF binding motif located within an intron of Golt1a near the Ren gene which we have previously demonstrated an interaction with the renin proximal promoter. Here we report the preliminary characterization of this model for renin expression and plasma renin activity (PRA). Chromatin immunoprecipitation-qPCR showed a reduction in CTCF binding at the Golt1a locus in our mutant model compared to wild-type (WT), confirming our mutation resulted in a disruption in CTCF binding in vivo. An expected reduction (p<0.05) in renin expression was found in male and female WT and mutant rats in response to a high salt diet. WT males showed an expected inverse PRA relationship to dietary salt (WT: 0.1% NaCl=17.52, 0.4% NaCl=12.87, 4.0% NaCl=7.13; p<0.05) however, interestingly, mutant males failed to increase PRA in response to very low salt (0.1% NaCl) challenge (0.1% NaCl mutant=13.87; p<0.05 compared to WT 0.1% NaCl). Female mutants showed a similar but non-significant trend. Taken together, these data suggest that the disruption of CTCF binding in a region which interacts with the renin proximal promoter can alter renin expression and the plasma renin activity in response to very low dietary salt in SS rats.

NIH 5P01HL149620-03

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Mechanisms of Estrogen Induced Skeletal Muscle Fibrosis in Inguinal Herniation
Authors: Potluri Tanvi , Zhao Hong , Stulberg Jonah , Yin Ping , Bulun Serdar , Lieber Richard
Published: May 2023

Background: Abdominal wall fibrosis weakens these muscles, often resulting in inguinal hernias. Despite its high prevalence and public health burden, the etiology or exploration of non-surgical treatments for muscle fibrosis remains a vastly under-explored area. We developed a mouse model (humanized aromatase mouse – Aromhum ) wherein all male mice develop scrotal hernias at puberty. The lower abdominal muscle (LAM) in these mice produces local estradiol (E2), which acts on stromal fibroblasts to proliferate, leading to muscle fibrosis, atrophy, and hernias. We are using this novel model to uncover mechanisms of muscle fibrosis. Objectives & Hypothesis: The study was to understand the mechanisms of E2-induced muscle fibrosis in scrotal hernias. We hypothesized that manipulating of E2-ERα signaling inhibits or reverses hernia formation by reducing or eliminating LAM fibrosis. Methods: Mice were administered E2-ERα antagonist treatment via Fulvestrant slow-release pellets (0.15mg/kg, 90 days) either prior to or post large hernia development. Hernia sizes were measured weekly, and LAM tissues were harvested for immunohistochemistry analysis.For in vitro experiments, fibroblasts were isolated from herniated LAM tissues and cultured under estrogen-replete (+10nM E2) and deplete (+ 100nM Fulvestrant) conditions. The cells were subsequently processed for ChIP-seq, ATAC-seq, and RNA-seq analysis. Sample size: 10-15 mice/group in 3 technical replicates Results: In vivo: Administration of fulvestrant to mice completely reversed scrotal herniation within 4-5 weeks of treatment (n = 10-15/group, p < 0.0001). In addition, the LAM tissues showed a reversal of fibrosis and a concurrent increase in myofiber cross-sectional area (n = 10-15/group, p < 0.0001) compared to placebo-treated mice. In vitro: Overall, 853 differentially expressed genes were observed between E2 and E2+Fulvestrant treated fibroblasts. Genes associated with the extracellular matrix, WNT signaling, TGFβ, and focal adhesions were enriched in our study. Further, transcription factors like Pbx1, Elk1, and Yy1 were also enriched in ERα cistrome. Integrating the three datasets, we identified 58 core genes that play a crucial role in E2-associated muscle fibrosis. Conclusions: We believe that this is the first skeletal fibrosis model that shows reversibility by inhibiting E2-ERα signaling. We provide mechanistic insight into this process by identifying the genetic, epigenetic, and transcriptomic changes associated with fibrosis in skeletal muscle upon stimulation by E2. These genes and pathways pave the way for potential therapeutic targets for the treatment of hernias and other skeletal muscular disorders.

NIH R01 DK121529

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Galectin 3 secretion in pulmonary arterial hypertension
Authors: Haigh Stephen , Bordan Zsuzsanna , Sellers Hunter , Brown Zach , Meadows Mary Louise , Barman Scott , Fulton David
Published: May 2023

Pulmonary arterial hypertension (PAH) is a complex and fatal disorder characterized by an unrelenting increase in pulmonary arterial pressure. Excessive proliferation of pulmonary artery smooth muscle cells (PASMC) leads to increased vascular resistance and eventually right ventricular failure and death. Our lab has hypothesized that circulating Galectin-3 (Gal-3), a readily secreted b-galactoside-binding lectin protein, previously investigated as a biomarker of PAH, has a key role in the pathology of PAH. Co-culture experiments as well as recombinant protein supplementation show that soluble GAL-3 is biologically active and induces SMC growth and proliferation(P<.05). Inversely, inhibition of GAL-3 using blocking antibodies prevents this SMC proliferation. (p<.05) To test our hypothesis in-vivo we injected GAL-3 KO mice with a liver specific AAV overexpressing GAL-3 or GFP (control) and exposed them to sugen/hypoxia to induce PAH. Surprisingly our initial findings indicate no protection with liver GAL-3 overexpression compared to GFP transduced mice. Contrary to our hypothesis, while liver overexpression of GAL-3 was robustly increased, there was no evidence of GAL3 in the lungs or plasma of GAL3KO mice indicating it may not be robustly secreted from the hepatocyte, unlike what was observed in other cell types. This lack of circulating Gal-3 generated by liver overexpression revealed a knowledge gap in the mechanism of Gal-3 secretion into the circulation. To address this we have generated at a number of mutant forms of GAL3 that alter motifs proposed to be relevant to Gal-3 secretion. R186S prevents binding to the all but 1 of the 176 of GAL-3 binding glycans however this appears to have no impact on its uptake in GAL-3 KO cells. Similarly, L131A/L203A GAL-3 which prevents the oligomerization of GAL-3 by preventing interaction of the carbohydrate recognition domain with its n terminal binding domain (responsible for oligomerization) also maintains its ability to interact with these cells. These changes did not interfere with its secretion into the media. However, these interactions do appear to be altered somewhat, when a cell surface biotinylation assay is performed there is significantly less R185S on the cell surface compared to WT GAL-3. Our findings suggest that ECs and SMCs can secrete GAL-3 and exert pathological actions such as increasing proliferation in vitro. Our blocking antibody further suggests that this secreted GAL-3 is capable of regulating SMC proliferation and provides evidence for possible treatment of PAH.

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.