bluuebunny/crossref_metadata_2025 huggingface dataset:
Pressure induced changes in endothelial cell morphology and Ca2+ signaling in ex vivo pressurized resistance arteries revealed using 4D diSPIM myography and AI deep-learning
Authors: Al-Aqtash Rua'a , Collier Daniel
Published: May 2023

The vascular endothelium is critical in promoting vasodilation of resistance-size arteries through Ca2+ dependent pathways. Vascular endothelial cells (ECs) are exposed to mechanical stimulation through changing intravascular pressure and flow, yet it is unclear how these forces impact EC morphology and Ca2+ signaling in pressurized arteries. Visualizing how the endothelium of an intact resistance-size vessel responds to changing physical forces requires isometric, sub-micron, resolution. Conventional confocal fluorescent microscope systems are limited by poor axial resolution. 4D dual inverted selective plane of illumination microscopy (diSPIM; 3i) uniquely provides isometric resolution at ~330 nm with high temporal resolution and minimal photobleaching or phototoxicity. We designed custom pressure myography chambers to allow simultaneous 4D diSPIM fluorescent imaging and vessel diameter tracking measurements via transmitted IR light (diSPIM Myography). We captured fluorescent images of ex vivo pressurized resistance-size mouse mesenteric arteries at 80, 60, 40, and 20 mm Hg before and after the generation of myogenic tone and in the absence of extracellular Ca2+. Representative images were annotated for ECs, nuclei, myoendothelial projections (MEPS), and Ca2+ signals. Annotations were used as ground truth for CNN AI model generation and validation using Apeer (Zeiss). Arivis Vision4D (Zeiss) was used to run the AI model and quantify data. As static intravascular pressure is increased in arteries without tone (with or without Ca2+), ECs increase in width as the vessel dilates. Myogenic tone minimizes pressure dilation and changes in EC width, however, ECs compress by up to 1 micron as pressure increases from 20 to 80 mm Hg. Additionally, increasing intravascular pressure in arteries with tone decreases local EC Ca2+ signaling. The data demonstrate a relationship between intravascular pressure and EC shape and Ca2+ signaling in intact resistance arteries under conditions of physiologic temperature, pressure, pH, and tone. diSPIM myography provides a method of studying the effects of physiologically relevant mechanical stimuli and the roles of various mechanosensitive mechanisms on vascular function. AI image annotation reduces analysis bias and increases the breadth of quantifiable microscopy data.

DMC is supported by NIH R00HL133451

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.
Investigation of the role of ATPase Inhibitory Factor 1 in pulmonary arterial hypertension
Authors: Brown Zachary , Batori Robert , Sellers Hunter , Haigh Stephen , Bordan Zsuzsanna , West Madison , Barman Scott , Fulton David
Published: May 2023

Pulmonary arterial hypertension is a rare and debilitating condition with a five-year survival rate of only 34% without treatment. The origins of pulmonary arterial hypertension are complex and incompletely understood and elevated pulmonary artery pressures result from excessive vascular remodeling due to functional changes in endothelial, vascular smooth muscle cells (VSMCs), and adventitial cells. Recent papers have explored the importance of metabolic switching of VSMCs in pulmonary hypertension in the transition towards a proliferative, apoptotic resistant phenotype which involves a shift from fatty acid oxidation to increased glycolysis. We hypothesized that ATPase Inhibitory Factor 1 (ATPIF1) in VSMCs could function as a metabolic switch that promotes a shift from fatty acid oxidation to glycolysis to increase the severity of pulmonary arterial hypertension. This hypothesis will be investigated using Western blot, Seahorse extracellular flux assays, qRT-PCR, and imaging of GFP-labelled ATPIF1. In preliminary data, we have cloned ATPIF1 and transfected cells to show overexpression of ATPIF1 decreases the maximal respiratory rate while increasing extracellular acidification, a measure of glycolysis. Future goals are to investigate the links between hypoxia, hypoxia-inducible factors and ATPIF1, the role of increased glycolysis in pulmonary smooth muscle cells, and ultimately whether ATPIF1 impacts of the development of pulmonary arterial hypertension in mice.

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 relationship between CFTR expression and cAMP-stimulated bicarbonate secretion diverges for the duodenum and ileum of pig
Authors: Mujica Rebecca , Thornell Ian
Published: May 2023

The luminal pH of the small intestine influences the digestion and absorption of nutrients. HCO3– secretion from Brunner’s glands and the ampulla of Vater initially alkalinize the acidic chyme upon entering the small intestine, and less-frequent Brunner’s glands and the surface epithelia of the small intestine further alkalinize chyme as it moves away from the ampulla of Vater. People with cystic fibrosis alkalinize chyme more slowly than their peers, suggesting that the anion channel CFTR (cystic fibrosis transmembrane conductance regulator) participates in the alkalinization. To further understand the relationship between CFTR expression and HCO3– secretion across duodenal and ileal epithelia, we studied newborn pigs with disrupted CFTR alleles ( CFTR–/–) and their littermates ( CFTR+/– and CFTR+/+). We hypothesized that CFTR–/– pigs would lack HCO3– secretion and that CFTR+/– pigs would have decreased HCO3– secretion because of reduced CFTR expression. To measure the electrogenic HCO3– secretion rate, we recorded short-circuit current ( Isc) across resected intestinal tissue bathed in solutions containing gluconate and HCO3–. Forskolin/IBMX-stimulated Isc across the duodenum of CFTR+/– pigs was half the rate observed for CFTR+/+ pigs, and forskolin/IBMX-stimulated Isc was absent across CFTR–/– duodenum. However, Isc across the ileum was similar between CFTR+/+ and CFTR+/– pigs and absent across CFTR–/– ileum. CFTR–/– pigs expressed CFTR mRNA transcripts at nominal levels for both intestinal segments, whereas CFTR+/– pigs expressed half the CFTR mRNA transcripts of CFTR+/+ littermates for each intestinal segment. These data are consistent with CFTR involvement in neutralizing luminal contents in the duodenum and ileum. Furthermore, these data suggest that the relationship between CFTR expression and the electrogenic HCO3– secretion rate diverges for these two intestinal segments. Electrogenic HCO3– secretion in the duodenum directly relates to CFTR expression, whereas electrogenic HCO3– secretion across the ileum appears to saturate at a CFTR expression level between CFTR–/– and CFTR+/– pigs.

Cystic Fibrosis Foundation Research Development Program

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.
Timed Release Ion Matrix Enhances Muscle Force in Dystrophic Mice
Authors: Kendra Jacob , Brow Richard , Segal Steven , Morton Aaron
Published: May 2023

Background: Duchenne Muscular Dystrophy (DMD) is an x-linked, recessive chromosomal mutation of the gene dystrophin, affecting 1 in 3,500 males. DMD is associated with limb muscle dysfunction from repetitive damage to myofibers beginning at 2 years of age. In healthy muscle, dystrophin is responsible for maintaining the sarcolemma through the dystrophin-glycoprotein complex, while muscle with dystrophin deficiencies seen in DMD patients experience severe sarcolemmal disruption following muscle contraction. Despite major advances in genetic engineering, the functional outcomes of FDA-approved therapeutic strategies have failed to enhance the function of dystrophin-deficient myofibers. We have taken an alternative approach to apply a novel biomaterial, Time Release Ion Matrix (TRIM), that has the potential to release ions favorable to regeneration, restoring muscle structure and function in DMD mice. Hypothesis: We hypothesized that muscle force and myofiber cross-sectional area (CSA) of the tibialis anterior muscle (TA) in an established mouse model of muscle dystrophy [D2.B10 mdx (DBA. mdx)] would be enhanced 14 days post-injection of TRIM. Methods: The left TA of adult male DBA. mdx mice (n=3-9, age 6-7 months) was injected with 250 μg of TRIM suspended in saline or saline alone (vehicle control). At 14 days post injection, the TA muscle was isolated for in situ muscle force measurements using direct electrical stimulation. The TA was then removed and frozen in optimal cutting temperature compound (OCT) for histological analysis. Muscle cross-sections (thickness, 10 μm) were labeled with laminin to identify myofiber borders and myosin heavy chain as a marker of myofibers able to generate force. Images were analyzed using Nikon NIS-Elements software. To simulate body fluid, 300 mg of TRIM was suspended in physiological salt solution (PSS) for 120 hours. Results: The dissolution of TRIM in PSS over 120 hours was 40% (179 mg remaining). Peak isometric force of TAs injected with TRIM (60.0 ± 2.3 g) was 11% greater compared to saline alone (52.4 ± 2.4 g) (means ± SE, n=6-9 per group; P=0.048). There were fewer small diameter myofibers (< 400 μm2) in TAs injected with TRIM (10 ± 7%) compared to saline controls (24 ± 5 %) (n=3/group; P=0.03). Conclusion: 14 days following injection of TRIM into the TA of dystrophic mice, muscle force and myofiber CSA were enhanced by 11 and 14% respectively. We suggest that TRIM may be an effective treatment to restore muscle function in DMD and serve as a powerful tool for discovering therapeutic targets against DMD.

University of Missouri Coulter Biomedical Accelerator (SSS, ABM), Texas A&M Department of Kinesiology and Sport Management (JAK, ABM)

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.
Using activators of NF-KB to model inflammation
Authors: Shivers Mitch , Sellers Hunter , Brown Zachary , Haigh Stephen , Bordan Zsuzsanna , West Madison , Batori Robert , Stepp David , Fulton David
Published: May 2023

Chronic inflammation underlies many chronic diseases such as obesity and diabetes and can lead to vascular and organ dysfunction, disability and death. Inflammation has been widely studied due to its broad involvement in numerous disease states, but current models of inflammation suffer from limitations. Models such as LPS treatment are non-specific and require multiple doses over extended periods of time which can lead to septic shock. The goal of our study was to develop an alternative model offering cell and organ specificity using an AAV-based approach. We hypothesize that delivering Ikk2, a potent activator of NF-KB will lead to chronic inflammation in the targeted cells and tissues. This approach has the benefit of cell and tissue specificity using specialized promoters and modified AAV capsids. Additionally, it will allow for consistent and regulatable expression over time. In preliminary studies I compared two know activators of NFKB, IKBKE and IKK2 to investigate differences in expression of inflammatory markers. Western blot analysis revealed ICAM-1 and IRF-1 protein levels were increased with IKK2 expression. In comparison, the inhibitior of apoptosis, CIAP-2, was more highly increased under IKBKE expression. We conclude that IKK2 would be more suitable to induce inflammation in target cells. Future studies will employ qPCR, Western blot, cytokine analysis and vascular function studies to characterize the effects in vivo.

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.
Relationship of body surface area (BSA) and BSA·mass-1 ratio on exertional heat stroke risk in healthy young men and women
Authors: Giersch Gabrielle , Taylor Kathryn , Caldwell Aaron , Epstein Yoram , Charkoudian Nisha
Published: May 2023

Exertional heat stroke (EHS) is an ongoing challenge for military personnel, for athletes and for individuals with occupational stressors that involve prolonged activity in hot environments. We recently showed that body mass index (BMI) was significantly associated with increasing risk for EHS in a cohort of activity duty U.S. Soldiers between 2016-2021. During exercise, heat is generated primarily by contracting skeletal muscle (and other metabolically active body mass) and dissipated based on body surface area (BSA). Thus, in compensable environments, a higher BSA·mass-1 ratio would be a benefit to heat dissipation & decrease risk of EHS. The purpose of the present analysis was to test the hypothesis decreasing BSA·mass-1 ratio is more strongly associated with increased EHS risk compared to increasing BMI in a cohort of healthy young men and women using the USARIEM Soldier Performance, Health and Readiness (SPHERE) database. We employed a matched case-control approach, where each individual with a diagnosis of EHS was matched to 5 controls based on calendar time, unit ID, and job category, for a total of 744 EHS and 3720 controls). As such, we captured control individuals who were matched to EHS events by location, time, and activity. We used a multivariate logistic regression model including variables of BSA·mass-1, age, military rank, and race. Results show that BSA·mass-1 was a significant predictor of EHS risk (P < 0.02), such that people with higher BSA·mass-1 were at lower risk of developing EHS when controlling for other potential factors such as age and race. These data suggest biophysical factors play an important role in EHS risk, with more research needed to better elucidate the role of age and race in this population.

Funded by USAMRDC; author views not representative of the Army/ DoD.

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.
Daunorubicin-induced arterial stiffness in Down syndrome
Authors: Buckman Michelle , Vasileva Anastasiia , Vasilyev Mikhail , Jedlicka Charles , Kalra Hardik , DeRuisseau Lara , Tomasson Michael , Bates Melissa
Published: May 2023

Children with Down syndrome have a high risk of leukemia and are at a higher risk for delayed early onset heart failure after treatment. Cardiac toxicity is more severe in female patients. Traditional risk factors for cardiovascular diseases in the general population may not be predictive of chemotherapy-induced heart failure in the Down Syndrome population. We used the Ts65Dn Down syndrome mouse model to investigate the impact of the chemotherapy agent daunorubicin on survival and arterial stiffness, a predictor of heart failure risk. We hypothesized that arterial stiffness will be increased in Ts65Dn mice treated with daunorubicin. Four-month-old Ts65Dn (n=12) and control wild-type Ts65Dn (WT Ts65Dn, n=8) mice were treated with low (2mg/kg), high (4mg/kg) dose of daunorubicin, or saline. Mice were injected twice over two weeks. Following treatment, we measured pulse wave velocity (PWV) weekly for 8 weeks using the Indus mouse Doppler flow velocity system. There was a significant decrease in PWV in mice treated with high-dose daunorubicin (p<0.001). Additionally, high-dose daunorubicin treatment resulted in a decreased survival in Ts65Dn mice (p<0.01). Overall, high-dose daunorubicin reduced PWV and survival in Ts65Dn and WT Ts65Dn mice. Daunorubicin has a dose-dependent impact on PWV in Ts65Dn and WT Ts65Dn mice. High-dose daunorubicin decreased aortic stiffness and increased mortality in Ts65Dn and WT Ts65Dn mice. This is the first demonstration that daunorubicin, the well known cardiotoxic agent, significantly impacts the conducting vasculature in Down syndrome as well.

NIH R21 HD099573

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.
Loss of Nr4a3 results in increased adipose mass and impaired adipocyte respiration
Authors: Carter Jared , Ellsworth Peter , Herring Jacob , Tessem Jeffery
Published: May 2023

Diabetes is a chronic disease that affects millions of people and will affect millions more in the coming generation. Symptoms of type 2 diabetes (T2D) include weight gain, impaired glucose tolerance, and impaired insulin secretion. Nuclear receptor Nr4a3 controls gene expression of targets essential for fuel metabolism and respiration. In patients with type 2 diabetes Nr4a3 promoter is hypermethylated, and Nr4a3 expression is downregulated. Here we demonstrate the effect of systemic Nr4a3 loss. Full body Nr4a3 KO animals were fed a standard diet. Male, but not female, Nr4a3 KO mice demonstrate impaired glucose and insulin tolerance. Male Nr4a3 KO mice have normal muscle, liver, and kidney respiration rates, while adipose tissue respiration is impaired. The impaired adipose respiration corresponds with increased mass of all adipose depots, increased adipocyte size, and increased lipid droplet size. Our data demonstrate the critical role of Nr4a3 in adipose tissue.

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.
Implementation of a 10-meter radius centrifuge model into a simulator of the human cardiovascular system: Effect on heart rate, stroke volume, and aortic pressure
Authors: Holcomb Matthew , Kilburg Jackson , Wellens Zach , Detwiler Trevin , Harycki Katya , Petridis Athanasios , Luttrell Meredith
Published: May 2023

Greater interest in long term space travel is creating demand to develop artificial gravity systems that minimize the negative effects of long-term microgravity exposure. However, due to practical constraints of understanding the physiological effects of artificial gravity systems, we propose a cardiovascular simulation to better understand the physiological impacts of an artificial gravity gradient created through the centrifugal force of a rotating spacecraft. The purpose of these simulations is to understand the effects of artificial gravity systems during space travel. We hypothesized that an artificial gravity system will not have significant effects on the cardiovascular system if it induces the same gravity gradient in an upright human as exists on Earth. For this reason, a 10-meter radius, 1 radian/second centrifuge system was chosen to mimic the effect of the gravity gradient on Earth. We used a modified version of the CVSIM program developed to model cardiovascular responses to orthostatic challenges ( https://physionet.org/content/cvsim/1.0.0/ ). CVSIM is a 21-compartment, lumped parameter model with control systems for the cardiopulmonary and arterial baroreflexes. The existing tilt test simulation within the CVSIM program was modified to create a simulation of the centrifuge. A 500-second-long simulation resulted in an average heart rate of 79 beats/minute, an average stroke index of 46 mL/beat/m2, and average pressures in the ascending aorta of 99 mmHg during systole, and 72 mmHg during diastole. These values were achieved by 150 seconds into the 500-second simulation. In comparison, prior to the onset of the centrifugation, initial heart rate, stroke index, and ascending aortic pressure compliance were set at 70 beats/min, 46 mL/beat/m2, and 0.28 ml/mmHg, respectively, based on published norms in humans. These preliminary data suggests that this artificial gravity system may produce viable physiological conditions for the cardiovascular system. Further research, especially with longer simulations, is necessary to determine if a rotating spacecraft could ease the negative impacts of microgravity over an extended time.

This work is supported by Iowa Space Grant Consortium/NASA BASE grant No.103428-19-20 (AP, MJL)

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 WNK-SPAK/OSR1 kinases and NKCC1 control epithelial barrier function by regulating claudin-2 expression
Authors: Koumangoye Rainelli , Delpire Eric
Published: May 2023

Conditions that cause the loss of epithelial barrier integrity are often accompanied by dysregulation of tight junction protein expression and/or localization. Recently, we have reported that patients with mutations in SLC12A2; the gene encoding the basolateral Na+-K+-2Cl- cotransporter (NKCC1), suffer from severe gastrointestinal deficits, including chronic gastrointestinal inflammation, gastrointestinal hemorrhage, intestinal obstruction, and constipation. Although the intestinal inflammation observed in patients with loss of NKCC1 function may or may not be due to tight junction dysfunction, we investigated whether the loss of NKCC1 function affects paracellular ion transport and epithelial barrier function. Wild-type HT29-MTX-E12 and CRISPR/Cas9-mediated NKCC1 knockout HT29 clones were tested for tight junction protein expression and localization. Tightness of epithelial cell monolayer was assessed by measurement of transepithelial resistance and permeability of molecular tracers in transwell filters. Tight junction protein localization was assessed by immunofluorescence. Loss of NKCC1 expression strongly increases the expression of claudin-2. Genetic deletion of WNK1, WNK4, SPAK and OSR1 resulted in a substantial increase in claudin-2 expression, confirming an essential role of WNK-SPAK/OSR1 pathway in tight junction regulation. Loss of NKCC1 significantly reduces the transepithelial electrical resistance (TER) indicating an increase in paracellular ions flux, consistent with upregulation of the cation-selective and channel-forming claudin-2. In addition, NKCC1-KO monolayers showed a significant increase in the paracellular flux of small molecules like fluorescein (0.33 kDa), while the permeability of higher molecular weight TRITC-Dextran (4 kDa and 70 kDa) remained unchanged. Thus, NKCC1 regulates tight junction protein expression and loss of NKCC1 function affects epithelial barrier integrity.

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.
Filling the Gaps: implementation of two programs to engage highly motivated scholars at crucial gaps in their careers
Authors: Becker Rena , Watts Makayla , Wilson Christopher , Gutierrez Orlando , Pollock Jennifer
Published: May 2023

Providing opportunities to engage highly motivated scholars in kidney, urology, and non-malignant hematology (KUH) research areas is of great need to advance research and practice in these underserved fields. High school, undergraduate, pre-doctoral and post-doctoral programs have helped improve interest and connection with the areas, however, there are gaps in education at crucial career decision points that are often overlooked. We developed and implemented two training programs during this pre-matriculation “gap.” The UAB KUH Gap year INternship (GAIN) program and the PRedOctoral PhD and MD research training in teams (PROmoTE) program engage scholars as pre-matriculates, after their undergraduate or master’s education and prior to their professional education for PhD studies or MD education. Through creating opportunities for in-depth training and exposure to KUH research areas at this crucial space in their careers, highly motivated individuals work with highly engaged mentors to be introduced and engaged in KUH research. The GAIN program, initiated in 2021, connects interested and motivated scholars who have recently completed their undergraduate or master’s degree to commit 1-2 years in a UAB KUH laboratory for scientific and laboratory training opportunities through seminars, laboratory research training, and mentorship. The PROmoTE program provides a summer research experience for pre-matriculating UAB PhD and MD scholars. The NIH R25 short-term experience is team-based with pre-matriculating PhD and MD students paired with PhD and MD mentors in a translational research project. Thus, the pre-matriculants are engaged in KUH research directly with both clinical and basic science researchers for the summer prior to their UAB PhD program or UAB MD education. The PROmoTE program seeks to engage pre-matriculating scholars to continue their careers in KUH research areas, to inspire translational research, as well as to promote the careers of underrepresented individuals. PROmoTE has engaged with 26 pre-matriculating scholars of which 46% are underrepresented individuals, with 9 scholars (3 PhD students, 6 medical students) continuing to engage in KUH research. We will address the opportunities these programs provide to enhance engagement and diversity in KUH research areas, challenges and obstacles overcome when developing and enhancing the programs, and provide preliminary outcomes. In addition, we will address how these programs have enhanced connection to peers, the institution, and ultimately the KUH scientific community.

NIH R25 DK112731

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.
Contribution of smooth muscle-derived vascular progenitor cells to atherosclerosis
Authors: Dubner Allison , Lu Sizhao , Jolly Austin , Noble Tysen , Moulton Karen , Nemenoff Raphael , Weiser-Evans Mary
Published: May 2023

Purpose: Atherosclerosis is a major cause of morbidity and mortality worldwide, but current therapies fail to adequately meet clinical needs. Emerging evidence implicates the outer layer of the blood vessel, the adventitia, in the pathogenesis of atherosclerosis. Specifically, it has been suggested that expansion of adventitial microvessels, the vasa vasorum (VV), drives atherosclerosis by facilitating inflammatory cell infiltration. Our group previously identified a unique population of multipotent resident vascular stem cells (AdvSca1-SM cells) that derive from mature vascular smooth muscle cells (SMCs) and reside in the vessel adventitia, where they are poised to respond to vascular injury. The goal of this project was to determine the contribution of AdvSca1-SM cells to the chronic vascular injury response. We hypothesized that in the setting of atherosclerosis, AdvSca1-SM cells contribute to plaque growth or VV expansion to drive disease progression. Methods: We generated a highly specific lineage tracing mouse model to track AdvSca1-SM cells in vivo over time. Lineage tracing mice were placed on either normal chow or modified Western diet (combined with PCSK9 AAV) for 8 to 30 weeks, then vascular tissue was analyzed using IF microscopy, scRNA-Seq, and flow cytometry. Results: scRNA-Seq and flow cytometry revealed a large reservoir of AdvSca1-SM cells in a stem-like state in the setting of atherosclerosis, but these cells showed significant phenotypic shifts compared to control conditions. Additionally, genetic modulation of the AdvSca1-SM cells influenced the transcriptional profiles of non-AdvSca1-SM-derived cells, indicating a signaling role for AdvSca1-SM cells in the vascular microenvironment. When AdvSca1-SM cells differentiate into other cell types, they primarily become mature SMCs, modulated SMCs, and myofibroblasts. These AdvSca1-SM-derived SMCs and myofibroblasts can be found in the core of the plaque, the fibrous cap, and the media wall. Additionally, these cells can be associated with adventitial microvessels in plaque regions, suggesting a role in plaque neovascularization. Contrary to our preliminary data, AdvSca1-SM cells very rarely differentiate into endothelial cells or macrophages. Conclusions: These studies emphasize the dynamic nature of AdvSca1-SM cells in the vascular injury response, demonstrating the potential of these cells to both differentiate into a variety of cell types as well as signal to other cells in the vascular wall. Ongoing studies will further characterize the distinct contributions of AdvSca1-SM cells to atherosclerosis.

NHLBI F31 (Dubner, HL160149-01); NHLBI R01 (Weiser-Evans, HL121877)

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.
Blockade of mTOR stimulates hyperglycemia but inhibits severe proteinuria in obese SS rats prior to puberty
Authors: Mandal Sautan , Ekperikpe Ubong , Safir Sarah , Johnson Tyler , Cooper Jonita , Cornelius Denise , Williams Jan
Published: May 2023

Prepubertal obesity (PPO) is a public health problem that is now associated with renal injury. Recently, we reported that the obese SSLepRmutant rat develops inflammation and renal injury before puberty. Moreover, the progression of renal disease in the SSLepRmutant rat is associated with the increased renal expression of the mammalian target of rapamycin (mTOR). Previous studies have shown that the mTOR pathway contributes to inflammation and renal disease. Therefore, this study tested the hypothesis that rapamycin (a mTOR inhibitor) will attenuate renal inflammation and early progressive renal injury in SSLepRmutant rats. Four-week-old SS and SSLepRmutant rats were treated with either vehicle (saline) or rapamycin (1.5 mg/kg/day, ip) for 4 weeks. At baseline, body weight was significantly higher in SSLepRmutant vs SS rats and remained elevated throughout the study. Treatment with rapamycin significantly decreased body weight in SSLepRmutant rats without affecting SS rats. While blood glucose levels were in the normal physiological range throughout the study in SSLepRmutant and SS rats (≤120 mg/dL), treatment with rapamycin only increased blood glucose in SSLepRmutant rats (561±54 mg/dL) without affecting SS rats (116±23 mg/dL). At the end of the study, we observed significant elevations in plasma insulin in SSLepRmutant vs SS rats, and treatment with rapamycin significantly decreased insulin levels by >50% in SSLepRmutant rats. At baseline proteinuria was significantly higher in SSLepRmutant vs SS rats (72±52 vs. 6±6 mg/day), and it remained higher throughout the study (500±125 vs. 26±12 mg/day). Interestingly, treatment with rapamycin only decreased proteinuria in SSLepRmutant rats (62±8 mg/day). Glomerular injury and renal fibrosis were markedly greater in SSLepRmutant vs SS rats, and treatment with rapamycin ameliorated these histological changes. At the end of the study, we observed increased GFR (via creatinine clearance) in SSLepRmutant vs SS rats (3.93±0.5 vs. 0.56±0.2 mL/min/gkwt), and treatment with rapamycin reduced GFR in SSLepRmutant rats (0.47±0.1 mL/min/gkwt). While there were no detectable differences in the renal expression of IL-4 in SSLepRmutant vs SS rats, there was a significant decrease in the renal expression of IL-10 in SSLepRmutant vs SS rats. Treatment with rapamycin significantly increased the renal expression of IL-4 and IL-10 in SSLepRmutant rats, while not affecting SS rats. In conclusion, although rapamycin causes hyperglycemia, it prevents early progressive proteinuria by reducing GFR and increasing renal anti-inflammatory cytokines during PPO.

Funding: DK109133 and HL151407

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.
Voltage-gated ion channels regulate ion and fluid transport in Malpighian tubule epithelia of larval lepidopterans
Authors: Dates Jocelyne , Castaneda Alexis , Kolosov Dennis
Published: May 2023

Insects account for ~80% of the world’s species, translating to approximately 200 million insects for every human. Moths and butterflies, from the order Lepidoptera, belong to second most diverse order of insects. The Cabbage Looper, Trichoplusia ni, is a prolific crop pollinator. Unfortunately, its larvae are worldwide agricultural pests because they consume copious quantities of food, eating 3-4 times their body weight in food daily, and growing 1000-fold in just a few weeks. These invasive agricultural crop pests are generalist feeders (eating >160 beneficial host plants) and consume large quantities of food to support their rapid growth. Rapid larval growth requires specialized regulatory mechanisms and an efficient excretory system. The Malpighian tubules (MTs) function as the insects’ primary excretory organ (equivalent to the vertebrate kidney), regulating ion and fluid transport, and xenobiotic and metabolic waste excretion. MT epithelium of larval lepidopterans is of particular interest because it can rapidly (~10 min) switch between ion reabsorption (where ions are transported from tubule lumen to hemolymph) and ion secretion (where ions are transported from hemolymph to tubule lumen). Recent studies have shown that this switch has been shown to be regulated in part by voltage-gated ion channels (VGICs). In larval T. ni, CaV1 channels expressed in the MTs distal ileac plexus region (DIP) have been shown to directly regulate K+ secretion. Pharmacological inhibition of voltage-gated ion channels (Kir1, HCN1, Cav1) independently from each other altered cell-based K+ transport. In the current study, biological assays measured changes in fluid secretion rate and membrane potential of MT epithelia of larval lepidopterans in response to pharmacological inhibition and/or activation of VGICs. In addition, immunohistochemistry assay using custom-made antibodies, revealed cellular and membrane localization of voltage-gated TRPA/ painless in the epithelia of the MT’s. In the current study we demonstrate that activating and inhibiting voltage-gated K+ and Ca2+ channels altered fluid secretion rate and membrane potential in the MTs of larval lepidopterans. VGICs have been extensively studied in excitable tissues. In contrast, their roles in the regulation of ion transport in the non-contractile, non-innervated epithelia of animals remains largely understudied. The novel data obtained in this study provides further insight about the fast-acting response mechanisms of VGICs, and how they play a vital role in the regulation of ion and water transp

California State University startup and seed grant funding

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.
Deletion of myostatin resolves obesity-induced myosteatosis and sarcopenia and improves angiogenesis in obese mice
Authors: Guilfoyle-Speese Andrew , Padgett Caleb , Bridgewater Cody , Corley Zachary , Mintz James , Fulton David , Stepp David
Published: May 2023

Objective: To test the hypothesis that restored muscle mass achieved through deletion of myostatin improves angiogenesis in obese mice through the resolution of myosteatosis. Methods: db/db mice, a well-described model of obesity, were crossed with mice lacking myostatin (MSTN KO), a myokine that negatively regulates muscle differentiation and growth, or stearoyl Co-A desaturase 1 (SCD1 KO), the rate limiting enzyme for oleate synthesis and subsequent triglyceride production, to generate db +/-_MSTN +/- and db +/-_SCD1 +/- mice. We utilized confocal and nuclear magnetic resonance (NMR) spectroscopy to assess skeletal muscle (SKM) histology and morphology in db +/-_MSTN +/- and db +/-_SCD1 +/- mice. Muscle lipid content was analyzed via mass spectrometry. Femoral artery ligation was used to stimulate in vivo angiogenesis in response to ischemia; whereas, 1mm aortic rings cultured in ECBM (2% FBS) were used to study ex vivo angiogenesis. Gene expression was assessed by RNA-Seq with protein expression follow-up in isolated gastrocnemius SKM via RT-qPCR and western blotting. Results: As previously described, MSTN deletion results in significantly increased muscle mass without altering whole-body mass or fat percentage in obese db/db mice. Additionally, we observed that obesity inhibits and MSTN deletion restores angiogenesis in both hind limb ischemia and aortic ring assay models. Despite persistent plasma lipedema, ectopic lipid deposition in SKM (Myosteatosis) was largely ameliorated in obese mice lacking MSTN, a reduction largely explained by a decrease in tissue triglyceride levels. Confocal microscopy revealed the accumulation of lipid in obese mouse myocytes that was lacking in obese mice with MSTN KO. RNA-Seq analysis of SKM exposed marked upregulation of lipogenesis pathways, notably the expression of SCD1. SCD1 was elevated at both the protein and RNA levels in db -_MSTN + SKM but not in db -_MSTN - SKM. Similar to MSTN KO, confocal imaging reveals that SCD1 deletion reverses lipid accumulation in obese mice. Conclusion: In summary, restored muscle mass in obese mice provides potent protection to vascular and metabolic health. A potential correlate of this improvement is the resolution of myosteatosis in obese MSTN KO mice. These data suggest that resolution of myosteatosis, independent of muscle mass, may afford similar protection, possibly by targeted deletion or blockade of SCD1.

A. C. Guilfoyle-Speese is supported by Kirschstein National Research Service Award (NRSA) Institutional Research Training Grant (T32) T32HL155011. D.W. Stepp and D.J. Fulton are supported by NIH 1R01HL147159.

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.
Transcription factor HNF4A activity is suppressed by microbiota and inhibits IFN beta production
Authors: Tillman Matthew , Lickwar Colin , Soderblom Erik , Horner Stacy , Rawls John
Published: May 2023

Intestinal epithelial cells (IECs) separate the intestinal lumen from lamina propria resident immune cells, positioning IECs as mediators of microbial signals to the immune system. Understanding these microbe-IEC-immune interactions may represent potential therapeutic targets for immunological diseases. We previously discovered that microbiota regulate the activity of the nuclear receptor transcription factor Hepatocyte nuclear factor 4 alpha (HNF4A). Microbiota suppress HNF4A-activated genes in zebrafish and lead to genome-wide reduction of HNF4A DNA occupancy in mouse IECs. HNF4A activates lipid metabolism and epithelial differentiation gene programs, while also suppressing inflammatory and immune response gene programs in the intestine. We therefore hypothesized that microbial suppression of HNF4A contributes to activation of immunological pathways. We conducted an unbiased coimmunoprecipitation-mass-spectrometry analysis comparing HNF4A interacting partners in IECs from germ-free and conventionalized (germ-free mice colonized with SPF feces for 2 weeks) mice. HNF4A interacted with significantly more proteins in germ-free IECs, including several proteins involved in promotion of transcription and chromatin regulation, consistent with HNF4A being more active in germ-free mice. Our HNF4A and H3k27ac ChIP-seq data from IECs derived from germ-free and conventionalized mice identified that HNF4A binds ubiquitously to IEC enhancers, but also revealed a small number of enhancers that were activated by microbiota with no HNF4A binding. Motif enrichment analysis showed that these enhancers were enriched for binding sites for Interferon regulatory factors (IRFs). This raises the interesting possibility that there is a selective disadvantage to colocalizing IRF and HNF4A occupancy at a given enhancer, and suggests that these transcriptional regulatory pathways may otherwise communicate. To test this, we used a luciferase reporter assay in HEK293T cells to probe the impact of HNF4A on the IFNβ promoter, which is controlled by IRF3/7. HNF4A potently suppressed IFNβ promoter activity following viral stimulation. HNF4A DNA-binding activity is necessary for this suppression; however, there is no evidence HNF4A binds the IFNβ promoter, suggesting HNF4A likely induces a repressor of the IFNβ promoter. Together these results indicate that HNF4A is both a target of microbiota suppression and a potent suppressor of IFNβ production. Though IRF bound enhancers appear to be insulated against HNF4A binding in IECs, our results show that these two transcriptional pathways communicate, potentially representing a novel mechanism through which microbiota influence innate immunity.

NIH P01-DK094779

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.
Vagal Afferent Innervation of the Atria and Ventricles of the Rat Heart: Anterograde Tracing
Authors: Chen Jin , Cheng Zixi
Published: May 2023

Vagal afferent innervation of the atria from the nodose ganglion has been shown in previous studies but the innervation of the ventricles remains unknown. In this study, tracer Dextran-Biotin was injected into the left nodose ganglion of SD male rats (n=6; 3 months). The tracer was allowed to transport for 14 days, then the rats were perfused through the left ventricle with 40°C phosphate-buffered saline (PBS, 0.1 M, pH = 7.4). Tissues were then fixed by perfusion with 4% paraformaldehyde in 0.1M PBS (4°C, pH 7.4). Whole rat hearts were harvested, and the atria and ventricles were separated. Then, a cut was made along the interatrial septum to separate the left and right atrium, then either a DAB staining or fluorescent immunohistochemistry staining was performed. Intrinsic cardiac ganglionic (ICG) neurons were stained with Cuprolinic Blue or Fluoro-Gold. Labeled single vagal afferent axons in the flat mounts of the whole heart were traced using the Neurolucida system. Consistent with previous findings using fluorescent tracer 1,1’-dioleyl-3,3,3’,3’ tetramethylindocarbocyaninemethanesulfonate (DiI) injection, afferent axons have been observed in the epi-, myo-, and endocardium and vagal axons formed “flower-spray” and “end-net” terminals in the atria. Also, the afferents displayed contact with cardiac muscles. For the first time, the “flower-spray” and “end-net” vagal afferent terminals have been found in both the left and right ventricles. Neither the “flower-spray” and “end-net” vagal afferent terminals on the heart, nor the vagal afferent terminal structures on the stomach such as intraganglionic laminar endings ( IGLEs) and intramuscular arrays ( IMAs) were found to co-localize with calcitonin gene-related peptide (CGRP) or Substance P (SP) in the three rats with injection followed fluorescent labeling and immunohistochemical analysis. In conclusion, our study provides the foundation for further functional studies of vagal afferent innervation of the whole heart (atria and ventricles) in normal and pathological states.

This study was supported by NIH HEAL/SPARC U01 NS113867-01 (ZJC), NIH R15HL137143-01A1 (ZJC).

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.
Inhibition of Histone 3 Lysine 4 Histone Methyltransferase Attenuates DOCA-Salt-Induced Hypertension
Authors: Cooper Silvana , Earley Yumei
Published: May 2023

Emerging evidence suggests that epigenetic regulatory mechanisms contribute to the development of hypertension. However, whether there is significance of epigenetics on neurogenic hypertension development is not fully understood. We previously reported an elevation of Histone 3 Lysine 4 (H3K4) trimethylation in the paraventricular nucleus of the hypothalamus (PVN). Accordingly, we hypothesize that H3K4 histone methyltransferase (HMT) in the PVN contributes to the development of DOCA-salt hypertension. To test our hypothesis, C57Bl/6J mice were implanted with SHAM or DOCA (50mg) pellets and given free access to water or saline (0.9% NaCl) respectively for 3, 7, or 21 days. H3K4 HMT activity were assessed in the PVN (n = 3-5 mice/group) and expressed as fold change. The HMT activity in the PVN was increased as early as 3 days after DOCA-salt compared with SHAM (6.1 ± 0.69 vs. 1.0 ± 0.73, P < 0.05), remained elevated at day 7 (6.3 ± 1.29 vs. 1.0 ± 0.90, P < 0.05) and day 21 (1.6 ± 0.07 vs. 1.0 ± 0.07, P < 0.05) of DOCA-salt treatment compared with SHAM. To examine the contribution of HMT activity to the development of DOCA-Salt hypertension, another cohort of mice (n = 3-5 mice/group) were implanted with telemetry transmitters to mornitor the blood pressure. After 10 days of recovery, mice treated with DOCA-Salt salt and intracerebroventricularly (ICV) infused with either artificial CSF or 5'-methylthioadenosine (MTA), a H3K4 HMT inhibitor, for 21 days. Importantly, ICV infusion of MTA attenuated mean arterial pressure (123.1 ± 6.3 vs. 138.5 ± 7.4 mmHg, P<0.05) compared with the control following DOCA-salt treatment. In addition, ICV infusion of MTA significantly reduced the H3K4 HMT activity (1.0 ± 0.08 vs 1.7 ± 0.07, P < 0.05) compared with aCSF group following 21 days of DOCA-salt treatment confirming the effectiveness of the inhibitor. In summary, these findings suggest the importance of elevated HMT activity in the PVN in DOCA-salt-induced hypertension. Further studies will be performed to identify the downstream targets of HMT and the mechanisms involved.

R01HL122770, 1P20GM130459, R35HL155008-01S1

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.