bluuebunny/crossref_metadata_2025 huggingface dataset:
The Structure‐function Remodeling and Therapeutic Effect of Tongxinluo Capsule in Rabbit Hearts of Myocardial Infarction
Authors: LI LI , Yunlong Huo
Published: June 2021

Objective

Animal models are important to study basic mechanism of ischemic heart failure (HF). The objective of the study was to create rabbit models of myocardial infarction to investigate the structure‐function remodeling of the left ventricle (LV) and coronary arterial trees and the therapeutic effects of Tongxinluo Capsule.

Method

New Zealand white rabbits were randomly divided into sham group, model group and therapeutic group. The rabbit models were created through multiple coronary artery ligations. Echocardiographic, Micro‐CT and histological measurements were performed at postoperative 6 weeks and postoperative 12 weeks, respectively.

Result

Echocardiographic measurements of the model group showed an approximately monotonic slope of ejection fraction and fractional shortening from 73% and 40% to 58% and 28% as well as persistent enlargement of LV cavity and slight mitral regurgitation at postoperative 12 weeks. Measurements of micro‐CT and histology revealed that coronary vascular sparseness and cardiac fibrosis related to inflammation occurred concurrently in adjacent zone of MI at postoperative 12 weeks although there was compensatory vascular growth at postoperative 6 weeks. Compared with the model group, the therapeutic group presented significant improvements in ejection fraction, fractional shortening, LV end‐systolic dimension and volume of cardiac output. The number of capillary vessels increased distinctly, while the infarct area decreased correspondingly.

Conclusions

In conclusion, these findings validate the proposed rabbit model and prove that the interaction of the degenerated coronary vasculature and increased ventricle wall stress relevant to cardiac fibrosis in vicinity of myocardial infarction (MI) facilitates the occurrence and development of ischemic HF. Furthermore, Tongxinluo Capsule has been proved in our study to play an essential role in protecting myocardium, improving blood supply of myocardium and relieving myocardial injury. To summarize, the post‐MI rabbit model can serve as a reference to test various drugs for treatment of ischemic HF, and contributes to the clinical application of Tongxinluo Capsule in the future.

Support or Funding Information

This research is supported in part by the National Natural Science Foundation of China Grant 11372010, 11672006 (Y Huo); Shenzhen Science and Technology Innovation Institution (China) Grant JCYJ20160427170536358 (Y Huo); and Hebei University (China) Grant 2015A1002 (Y Huo).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enhanced NLRP3 Inflammasome Activation in the Arterial Endothelium with Acid Sphingomyelinase Transgene in Mice
Authors: Yuan Xinxu , Bhat Owais M. , Lohner Hannah , Li Pin‐Lan
Published: June 2021

Endothelial NLPR3 inflammasome activation during hypercholesterolemia triggers arterial inflammation and neointima formation leading to atherosclerosis. However, the mechanisms activating and regulating NLRP3 inflammasomes remain poorly understood. The present study investigated whether endothelial acid sphingomyelinase (ASM) and its product, ceramide are required for endothelial NLRP3 inflammasome activation and atherosclerotic lesions during hypercholesterolemia. We fed Smpd1trg/ECcre mice, a mouse strain with endothelial (EC)‐specific ASM transgene (Smpd1 is the code for mouse ASM gene) and their control littermates (Smpd1trg/WT and WT/WT mice) with the Western diet (WD) and then performed partial left carotid ligation (PLCA). With such animal model, we found that the neointima formation in the carotid artery with ligation was more remarkably increased by 2.6 folds in Smpd1trg/ECcre mice than their control littermates. Using confocal microscopy, enhanced colocalization of NLRP3 with ASC or with caspase‐1 was found in the endothelium of the carotid arteries in WD‐treated Smpd1trg/ECcre mice, which was much less in their control littermates. IL‐1β levels in the carotid intima also more significantly increased by 2.2 folds in Smpd1trg/ECcre mice, which was accompanied by overexpression of ASM and elevated ceramide level. In addition, flotilin‐1 as a membrane raft (MR) marker was found to be aggregated with ASM, ceramide, gp91phox and p47phox more in the intima of Smpd1trg/ECcre mice than their control littermates. Carotid intimal O2.− levels as measured by in situ dihydroethidium (DHE) staining was much higher in WD‐treated Smpd1trg/ECcre mice than their controls. These results suggest that ASM or ceramide‐mediated MR redox signaling platform formation and NADPH oxidase activation are essential for endothelial NLRP3 inflammasome activation and consequent atherogenic changes in the arterial wall during hypercholesterolemia.

Support or Funding Information

NIH grants HL057244, HL075316 and HL122937).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Gut Microbial Metabolite TMAO Induces Endothelial Dysfunction by Activating the HMGB1/TLR‐4 Signalling Pathway
Authors: Singh Gurinder Bir , Mohammad Razia S. , Kshirasagar Naresh , Zhang Yang , McConnell Bradley K. , Li Pin‐Lan , Boini Krishna M. , Koka Saisudha
Published: June 2021

The intestinal microbe derived metabolite trimethylamine N‐oxide (TMAO) is implicated in the pathogenesis of cardiovascular diseases. However, the molecular mechanisms of how TMAO induces atherosclerosis and CVD progression are still unclear. In this regard, high‐mobility group box protein 1 (HMGB1), an inflammatory mediator, has been reported to destroy cell‐cell junctions, resulting in vascular endothelial hyper permeability leading to endothelial dysfunction. The present study tested whether TMAO associated endothelial dysfunction results via HMGB1 activation in in vitro and in vivo models. Biochemical and RT‐PCR analysis showed that TMAO increased the HMGB1 expression in dose dependent manner in endothelial cells compared to control cells. However, prior treatment with glycyrrhizin, an HMGB1 binder abolished the TMAO‐induced HMGB1 production in endothelial cells. Furthermore, western blot and immunofluorescent analysis showed significant decrease in the expression of cell‐cell junction proteins ZO‐1, ZO‐2, Occludin and V‐cadherin in TMAO treated endothelial cells compared to control cells. However, prior treatment with glycyrrhizin attenuated the TMAO‐induced cell‐cell junction proteins disruption. In addition, TMAO induced the HMGB1 expression through TLR‐4 signalling pathway in endothelial cells. In in vivo studies, C57BL/6J wild mice were treated with 3 % choline chloride in drinking water to produce enhanced TMAO levels in plasma. Real time PCR analysis showed that HMGB1, TLR‐4 and inflammatory markers (ICAM & VCAM) were significantly increased in carotid arteries of choline treated mice compared to control mice. In addition, it was also found that collagen I and III expression (fibrotic markers) were significantly increased in carotid arteries of choline treated mice. In conclusion, our results suggested that TMAO modulated endothelial cells phenotype via activating HMGB1 associated TLR‐4 signalling pathway.

Support or Funding Information

supported by NIH grant, DK104031

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
AMPK as a metabolic sensor regulates inflammatory response during ischemic insults
Authors: Li Ji , Chen Xu , Cates Courtney , Rousselle Thomas , Li Xuan
Published: June 2021

Introduction

AMP‐activated Protein Kinase (AMPK) is a stress‐activated kinase that protects against cardiomyocyte injury via modulating substrate metabolic shift between glucose and fatty acid during ischemia and reperfusion. c‐Jun N‐terminal kinase (JNK), a mitogen activated protein kinase, is activated by ischemia and reperfusion for modulating inflammatory response. NF‐κB is an important transcription factor involved in inflammation cascade during ischemia and reperfusion injury.

Methods and Results

The intrinsic activation of AMPK attenuates inflammation occurred during ischemia/reperfusion through modulating the JNK mediated NF‐κB signaling pathway. Rat cardiac myoblast H9c2 cells were subjected to hypoxia and/or reoxygenation to investigate the signal transduction that occurred during myocardial ischemia/reperfusion. Mitochondrial function was measured by the Seahorse XF24 V7 PS system. Hypoxia treatment triggered AMPK activation in H9c2 cells in a time dependent manner. Inhibition of hypoxic AMPK activation by pharmacological approach (Compound C) or siRNA knockdown of AMPK α catalytic subunits caused dramatic augments in JNK activation, inflammatory NF‐κB phosphorylation, and apoptosis during hypoxia and reoxygenation. Inhibition of AMPK activation significantly impaired mitochondrial function and increased the generation of reactive oxygen species (ROS) during hypoxia and reoxygenation. In contrast, pharmacological activation of AMPK by a diabetes drug metformin significantly inhibited mitochondrial permeability transition pore (mPTP) opening and ROS generation. Moreover, AMPK activation significantly attenuated JNK‐NF‐κB signaling cascade and inhibited mRNA levels of pro‐inflammatory cytokines such as TNF‐α and IL‐6 during hyopoxia/reoxygenation in H9c2 cells. Intriguingly, both pharmacologic inhibition of JNK by JNK‐IN‐8 and siRNA knockdown of JNK signaling pathway attenuated NF‐κB phosphorylation and apoptosis but did not affect AMPK activation in response to hypoxia and reoxygenation. The results indicate that AMPK as a metabolic sensor regulates substrate metabolism and maintains the integrity of mitochondria in order to limit the inflammatory response during ischemia and reperfusion stress conditions.

Conclusions

AMPK activation regulates substrate metabolism and modulates JNK‐NF‐kB signaling cascade during hypoxia and reoxygenation stress conditions. Cardiac AMPK activation plays a critical role in maintaining mitochondrial function and inhibiting the inflammatory response caused by ischemic insults.

Support or Funding Information

American Diabetes Association 1‐17‐IBS‐296, NIH R01AG049835, R01HL12587701, P01HL051971, and P20GM104357

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Age‐related dysfunction in the satellite cell niche
Authors: Thomas Aaron , Nederveen Joshua , Snijders Tim , Joanisse Sophie , Bell Kirsten , Phillips Stuart , Kumbhare Dinesh , Parise Gianni
Published: June 2021

Skeletal muscle satellite cells (SC) are important for maintenance and repair of myofibers. SC function and responsiveness is regulated, at least in part, through interactions with niche components in which they reside. Recent evidence suggests that structural changes occur in the SC niche as a function of aging. In the present study, we aimed to investigate whether age has an impact on the properties of the SC niche. Muscle biopsies were obtained from the vastus lateralis of previously untrained but otherwise healthy young men (YM: 21±0.4 yrs; n=10) and old men (OM: 66±0.9 yrs; n=11) at rest. In addition, OM (n =10) performed a single bout of resistance exercise and additional muscle biopsies were taken 24 h and 48 h post‐exercise. Skeletal muscle SC niche measurements were assessed using high resolution immunofluorescent and confocal microscopy. We report changes in the muscle SC niche with aging and that many of these differences are primarily associated with type II fibres. Type II SC niche laminin thickness was significantly greater in OM (1.86 ± 0.06 μm) as compared to YM (1.55 ± 0.09 μm, p<0.05). Mean intensity of collagen IV was significantly lower in OM (92 ± 10 a.u) as compared to YM (512 ± 17 a.u, p<0.05). In OM, the fibrotic index as measured by Masson's trichrome staining was greater (3.6 ± 0.5 %) at baseline, as compared to YM (2.0 ± 0.5 %; p<0.05). We also observed the presence of SC fully incarcerated by the basal lamina. At baseline, 100% of OM possessed SC that were incarcerated, as compared to ~40% of YM. The percentage of SC in mixed muscle that were incarcerated by laminin was greater in OM (19.0 ± 1.6%) as compared to YM (7.2 ± 2.1; p<0.05). Although this phenomenon was present in both Type I and Type II fibres, it appeared exaggerated in Type I fibres (OM: 28.4 ± 8.2%, YM: 3.6 ± 2.1%; p<0.05). In all cases the greatest proportion of active SC were found in the non‐incarcerated population, whereas incarcerated cells were less likely to activate (24 h post exercise – Non‐incarcerated:32 ± 6% active, Incarcerated: 8 ± 2% active, p<0.05). Consistent with this, at 24 h, there was a significantly lower number of quiescent SC in the non‐incarcerated condition (62 ± 7 %) as compared to the incarcerated condition (92 ± 4%, p<0.05). Additional evidence of age‐related dysfunction includes larger myonuclear CSA, increased expression of COL4α1 mRNA and Laminin subunits α2 and β1 was significantly greater in OM as compared to YM. Together this data suggests there are age‐related changes in the niche that are potentially detrimental to SC function and skeletal muscle health.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dynamics of blood lactate concentration during recovery period of short duration‐high intensity exercise in acute hypoxia or normoxia
Authors: Takei Naoya , Kakinoki Katsuyuki , Hatta Hideo
Published: June 2021

We studied dynamics of blood lactate concentration during recovery period of short duration‐high intensity exercise in acute hypoxia or normoxia. University track and field athletes were recruited for single‐blind randomized crossover designed study. All subjects completed wingate test in both acute hypoxia (FiO2=0.145) and normoxia (FiO2=0.21). Two wingate tests were separated at least 48 hours. After 10 min cycling warm‐up (100 W), subjects took 5 min rests and then conducted 30 sec wingate tests. After the wingate test, subjects of both conditions took 30 min rests in normoxia. Blood samplings were conducted 1 min before and, 0.5, 1, 2, 3, 4, 5, 7, 10, 20 and 30 min after the wingate tests to examine the blood lactate concentration. Peak power output (PPO), mean power output (MPO) and fatigue index (FI) were not different in both condition (PPO; 984.3±49.3 W and 971.0±32.1 W, MPO; 674.3±37.4 W and 680.0±36.3 W, FI; 49.2±7.1% and 49.6±5.2%, normoxia and hypoxia respectively). The AUC of blood lactate, when examined pre to 10 min values, was significantly higher in acute hypoxia than normoxia (p<0.05). The rates of reduction of blood lactate concentration was higher in hypoxia than normoxia(p<0.05, 0.42±0.02 and 0.28±0.03 mmol/L/min, respectively). These results suggested that muscle glycogen breakdown was higher in acute hypoxia than normoxia in spite of same wingate test performances. It also suggests that lactate oxidation may be higher in acute hypoxic group when resting at normoxia.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparison of Measures of Compensatory Reserve and Tissue Oxygen in Differentiating Tolerance to Simulated Hemorrhage
Authors: Schlotman Taylor E. , Howard Jeffrey T. , Convertino Victor A.
Published: June 2021

Background

We previously reported that average measurements of compensatory reserve (CRM) and muscle oxygen saturation (SmO2) obtained from a large cohort of humans provided earlier indication of reduced central blood volume than standard vital signs (e.g., blood pressure, heart rate, arterial oxygen saturation). Additionally, CRM was more sensitive and specific in earlier prediction of decompensation than SmO2 overall.

Objective

In the present study, we hypothesized that CRM derives its superior predictive qualities from its ability to differentiate between individuals with high (HT) and low (LT) tolerance to central hypovolemia compared with measures of SmO2.

Methods

Continuous noninvasive measures of CRM (calculated from feature changes in the photoplethysmographic arterial waveforms) were collected from 55 healthy volunteer subjects before and during stepwise lower body negative pressure (LBNP) to the point of presyncope. Near infrared spectroscopy was used on the forearm to obtain measures of deep SmO2, hydrogen ion concentration ([H+]), and hemoglobin volume (HbT; decreases reflect vasoconstriction). Using a progressively increasing stepwise protocol, tolerance group was defined by those subjects who did (HT) or did not (LT) complete 60 mmHg LBNP. Data were analyzed using generalized estimating equations (GEE) to account for the repeated measures design.

Results

Of 55 participants, there were 35 (64%) HT and 20 (36%) LT, 26 (47%) males and 29 females (53%), with a mean age of 28 years (SD=7), height of 172 cm (SD=10) and weight of 72 kg (SD=15). CRM decreased by 97% in a linear fashion across progressive LBNP levels from baseline to −90 mm Hg, while SmO2 decreased by only 39% from baseline to −90 mm Hg. The slope for CRM values across progressive LBNP levels differed significantly between LT and HT individuals (slope difference −0.14; p=0.03). The slope for SmO2 values did not differ between LT and HT individuals across LBNP levels (slope difference=0.06; p=0.18).

Conclusion

Consistent with our hypothesis, CRM exhibited greater reduction across progressive LBNP levels than SmO2. These data support the notions that: 1) CRM can enable more accurate triage of casualties than SmO2 due to superior ability to differentiate those individuals at highest risk for shock; and 2) continuous monitoring of CRM can allow for earlier detection of continued casualty deterioration.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bicarbonate is required for proliferation of pulmonary endothelial cells
Authors: Sayner Sarah L. , Maulucci Marcy , Harvell Tyler
Published: June 2021

Rationale

The second messenger cAMP is widely known to regulate proliferation in numerous cell types. Many of these studies implicate cAMP generated by membrane‐bound cyclases (AC1‐9). While these membrane‐bound ACs generate cAMP in the near membrane compartment, cAMP can also be generated in unique subcellular compartments by the soluble adenylyl cyclase (sAC or AC10). Soluble AC localizes to the cytosol as well as organelles, such as the mitochondria, and is uniquely stimulated by bicarbonate to generate cAMP. Recently, AC10 was observed to be overexpressed in prostate carcinoma, suggesting a role for AC10 in cellular proliferation; however, the independent role of bicarbonate in proliferation has not been investigated. Thus, we tested the hypothesis that bicarbonate is required for proliferation of pulmonary endothelial cells.

Methods

Pulmonary microvascular endothelial cells (PMVECs) and pulmonary artery endothelial cells (PAECs) were seeded at 100, 000 cells per well in media with and without bicarbonate. The cells were counted for seven consecutive days to generate growth curves and blood gas analysis performed to determine pH, pO2, pCO2, and HCO3− concentrations of the media. Additionally, a separate set of PMVECs were seeded at 100, 000 cells per well in bicarbonate free media and bicarbonate added back to the media after 24, 48, 72, 96, 120, 144, and 168 hours and counted for five days following bicarbonate add back.

Results

Growth curves revealed that PMVECs and PAECs seeded in the bicarbonate‐containing media entered into log phase growth followed by a plateau phase at cell confluence; however, PMVECs and PAECs seeded in the bicarbonate free media did not enter log phase growth nor reach confluence. When bicarbonate was added back to PMVECs initially grown in a bicarbonate free environment, the cells proliferated to numbers comparable to PMVECs seeded in bicarbonate containing media throughout the growth curve; however, if PMVECs were incubated in bicarbonate free media for more than 96 hours, these cells did not enter into log phase growth and did not form a confluent monolayer.

Conclusion

These data reveal a requirement for bicarbonate for proliferation of pulmonary endothelial cells, since neither PAECs nor PMVECs formed confluent monolayers in the absence of bicarbonate.

Support or Funding Information

NIH 121513

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mesenchymal Stromal Cell Microparticles Enhance Lung Endothelial Barrier Through CD44 and the S1P/ceramide Rheostat
Authors: Maishan Mazharul , McVey Mark J. , Lee Warren L. , Curley Gerard F. , Kuebler Wolfgang M.
Published: June 2021

Introduction

Acute respiratory distress syndrome (ARDS) is a fatal condition characterized by hyperinflammation and pulmonary microvascular leak. Treatment is only supportive with no pharmacological cure. Recently, mesenchymal stromal/stem cells (MSCs) have emerged as potential therapy, but greater knowledge on their mechanism(s) of action could yield development of cell‐free therapeutics.

Objective

To determine if MSC‐produced microparticles (MPs) ameliorate endothelial barrier disruption and identify their biomolecular contents responsible for such therapeutic effect.

Methods

To re‐capitulate lung capillary leak in ARDS, primary human pulmonary microvascular endothelial cell (HPMEC) monolayers grown on Transwell inserts were injured with thrombin to induce barrier disruption as measured by transendothelial electrical resistance (TEER). MPs were purified out of conditioned medium produced by MSCs either stimulated with Ca2+ ionophore or unstimulated. MPs were administered to treat thrombin‐injured HPMEC monolayers; surface markers were compared by flow cytometry and lipid content analyzed by HPLC‐MS.

Results

MPs from unstimulated MSCs enhanced HPMEC barrier function and TEER recovery after thrombin injury, while MPs from stimulated MSCs were injurious. Stimulated MSCs produced MPs with significantly less surface CD44. Antibody blockade of CD44 on unstimulated MPs eliminated their therapeutic effect on HPMEC monolayers. Moreover, stimulated MPs had significantly more total ceramides and less sphingosine‐1‐phosphate (S1P) than unstimulated MPs. Ceramide degradation by neutral ceramidase blunted the injurious effect of stimulated MPs. The therapeutic function of unstimulated MPs was eliminated with ceramide over‐production by acid sphingomyelinase or S1P degradation by S1P lyase.

Conclusion

These results show MSC‐produced MPs were therapeutic after lung capillary endothelial barrier disruption. Beneficial effects were associated with surface CD44 and greater ratio of S1P:ceramide content. This knowledge provides a mechanistic basis for development of MSC‐based cell‐free therapeutics.

Support or Funding Information

This work was supported by Ontario Research Fund and Canadian Institutes of Health Research.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Myeloperoxidase‐derived 2‐Chlorofatty Acids: Accumulation in Rat and Human Sepsis, and Association with Acute Respiratory Distress Syndrome Mortality in Human Sepsis
Authors: Pike Daniel , Nuala Meyer , Reilly John , Feng Rui , Christie Jason , Albert Carolyn , Hartman Celine , Franke Jacob , McHowat Jane , Ford David
Published: June 2021

Neutrophilic inflammation is a component of sepsis‐associated acute respiratory distress syndrome (ARDS). Neutrophil myeloperoxidase (MPO) activity produces HOCl, which leads to increased plasma 2‐chlorofatty acid (2‐ClFA) levels due to HOCl targeting plasmalogens. Thus, these studies were designed to investigate the role of 2‐ClFA in ARDS during sepsis. In patients with sepsis (n=198), free 2‐ClFA levels were significantly associated with ARDS (odds ratio (OR) 1.87 (95% CI 1.41, 2.49); p < 0.001), and with 30‐day mortality (OR 1.38 (95% CI 1.11, 1.72); p=0.004), for each log increase in free 2‐chlorostearic acid. Of considerable interest, plasma 2‐ClFA levels correlated with plasma levels of angiopoietin‐2, E‐selectin, and soluble thrombomodulin suggesting 2‐ClFAs may mediate endothelial dysfunction in human sepsis. Further studies with human lung microvascular endothelial cells (HMVEC‐L) showed 2‐ClFA treatment resulted in increased adhesion molecule surface expression; increased angiopoietin‐2 release and dose‐dependent endothelial permeability. In contrast, these changes were not observed in endothelial cells treated with non‐chlorinated palmitic acid. Next, the cecal slurry model of rat sepsis was investigated. Injecting rats with 15ml of cecal slurry/kg resulted in mortality within 26h accompanied by significant increases in 2‐ClFA levels in the plasma and lung in comparison to rats injected with vehicle. The ultimate goal of these studies will be to test therapeutic agents that prevent chlorinated lipid‐caused organ injury including ARDS during sepsis. Collectively, these studies suggest that 2‐ClFAs derived from neutrophil MPO‐catalyzed oxidation contribute to pulmonary endothelial injury and ARDS, and have prognostic utility in sepsis‐associated ARDS.

Support or Funding Information

This study was supported by NIH grants R01GM115553 (to DAF and JM). and R01HL122474 (to NJM).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hypoxia Upregulates ErbB Family Members in Human Pulmonary Microvascular Endothelial Cells
Authors: Regan Christopher S. , Jin Yi , Pool Caitlyn S. , Liu Yusen S. , Chen Bernadette S. , Nelin Leif D.
Published: June 2021

Pulmonary hypertension is associated with vascular remodeling that is driven by proliferation of cells in the vascular wall, which involves both endothelial cells and vascular smooth muscle cells. We have previously shown that human pulmonary microvascular endothelial cells (hPMVEC) proliferate in hypoxia, and that hypoxia results in a significant increase in the expression of both epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR/ErbB1). Furthermore, we have shown that hypoxia‐induced proliferation of hPMVEC depends on activation of EGFR/ErbB1. The literature suggests that EGFR/ErbB1 is activated via dimerization, either homodimerization (between the same ErbB family member such as ErbB1:ErbB1), or heterodimerization (between different ErbB family members such as ErbB1:ErbB2). In other cell types, ErbB1, 2, 3 and 4 have been described and the manner by which the ErbB family members dimerize may determine which down‐stream signaling cascades are activated. At present, it is not known in hPMVEC whether all ErbB family members are expressed. As a first step in understanding how ErbB is activated in hPMVEC, we tested the hypothesis that ErbB2, 3, and 4 are expressed in hPMVEC. To begin to address our hypothesis, we incubated hPMVEC in normoxia (21% O2, 5% CO2) at 37° C for 24 hours. Protein was harvested for Western blotting and probed for ErbB1, ErbB2, ErbB3, and ErbB4. We found that hPMVEC express ErbB1, ErbB2, and ErbB4 with only minimal protein expression of ErbB3. To determine the effect of hypoxia on ErbB2 and/or ErbB4 protein expression, hPMVEC were incubated in normoxia or hypoxia (1% O2, 5% CO2) at 37° C for 24 hours. We found that protein levels of both ErbB2 and ErbB4 were greater in hypoxia than in normoxia. In conclusion, our data demonstrate that under the conditions of these experiments, hPMVEC express ErbB1, ErbB2 and ErbB4 protein, with little expression of ErbB3 protein. In addition, hypoxia enhanced ErbB2 and ErbB4 protein expression. These results have important implications for ErbB‐mediated cell signaling in hPMVEC. We speculate that the ErbB members identified in hPMVEC and the mechanism by which ErbB dimerizes represent potential novel therapeutic targets for pulmonary hypertensive disorders associated with hypoxia.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Loss of BMPR2 Confers Proliferative Advantage on Pulmonary Artery Endothelial Cells by Decreasing Dependence on Growth Suppressor Merlin
Authors: Josephs Caleb A. , Ceballos Rhandy , Vasauskas Audrey A. , Morrow Rebekah L. , Morrow Kyle Adam
Published: June 2021

Pulmonary artery endothelial cells (PAECs) exhibit an abrogated proliferative phenotype when compared to pulmonary microvascular endothelial cells (PMVECs). Pulmonary arterial hypertension (PAH) is a disease characterized by occlusive lesions in the pulmonary vasculature, potentially due to a heightened proliferative capacity of pulmonary endothelial cells. The cytoskeletal linker protein Merlin functions to suppress proliferation in a contact‐dependent manner in a variety of tissues. Loss of Merlin contributes to certain types of lung cancer. Mutations in the bone morphogenic protein receptor 2 (BMPR2) are frequently seen in patients with PAH, and reduced BMPR2 is implicated in a cancer‐like, highly proliferative phenotype of pulmonary microvascular endothelial cells. However, the role of BMPR2 in pulmonary artery endothelial cell proliferation remains unclear. Here, we test the hypothesis that a reduction of BMPR2 confers a growth advantage on PAECs by altering Merlin status. Wild‐type PAECs and PMVECs were grown to sub‐confluence (~80%) or full confluence, cell lysates were harvested, and Merlin levels were assessed. As Merlin functions to suppress growth in a contact‐dependent manner, as expected, PAECs possessed higher levels of Merlin at full confluence than at sub‐confluence. Interestingly, Merlin protein was not detected at sub‐confluence or full confluence in PMVECs. Wild‐type PAECs and PAECs with reduced BMPR2 (BMPR2 knockdown) were assessed for proliferative capacity by traditional and image‐based growth curve analysis. BMPR2 knockdown cells showed increased proliferative capacity when compared with wild‐type PAECs. BMPR2 knockdown and wild‐type PAECs were grown to sub‐confluence and full confluence, cell lysates were harvested, and Merlin levels were assessed. When compared to wild‐type PAECS, BMPR2 knockdown cells appeared to increase Merlin levels to a lesser extent as confluence increased. Thus, we provide evidence of a link between reduced BMPR2 in PAECs contributing to enhanced proliferative capacity, potentially due to a decreased dependence on the growth suppressor Merlin.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Sympathetic Activation on Aortic Stiffness in Young Healthy and Hypertensive Adults
Authors: Lahti Dana S. , Hayward Shayne D. , Wells Calvin R. , McNair Erick D. , Tomczak Corey R.
Published: June 2021

Aortic stiffness is a major link between hypertension and cardiovascular risk. Anatomic features of the artery including the smooth muscle regulate aortic stiffness, in part. Importantly, vascular smooth muscle is under sympathetic nervous system control, and thus aortic stiffness may be influenced by the sympathetic nervous system. When exposed to physiological stress, older hypertensive adults demonstrate an exaggerated blood pressure response that may correspond with transient arterial stiffness increases, but this response in young hypertensive adults remains unknown.

PURPOSE

We tested the hypothesis that sympathetic activation would increase aortic stiffness in young healthy adults (control, CTL), and that aortic stiffness increases during sympathetic activation would be greater in young hypertensive adults (HTN).

METHODS

Six healthy controls (males=4, females=2; 28.3±5.5 years; BMI: 23.5 ± 3.5 kg/m2) and three young hypertensive participants (males=2, females=1; 28.3±6.0 years; BMI: 30.3±8.2 kg/m2) were studied. Mean arterial pressure (MAP) and carotid‐femoral pulse wave velocity (PWV) were measured at rest and during 5 min of brachial circulatory occlusion (sympathetic activation condition) that followed isometric handgrip exercise.

RESULTS

Resting MAP was higher in HTN (100±6 mmHg) than CTL (90±5 mmHg; P=.029). Occlusion MAP was also higher in HTN (116±6 mmHg) than CTL (102±7 mmHg; P=.016). Resting PWV was similar between CTL (8.6±1.6 m/s) and HTN (8.1±0.1 m/s; P=.47). The increase in PWV from rest to occlusion (1.2±1.4 m/s) in CTL was not significant (P=.09), whereas the increase in PWV from rest to occlusion (2.0±0.4 m/s) in HTN was significant (P=.01).

CONCLUSION

Sympathetic activation may significantly increase aortic stiffness in young hypertensive adults, but not young healthy adults.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of Nitric Oxide Synthase (NOS) by NG‐monomethyl‐L‐arginine (L‐NMMA) Reduces Transient Increase in the Blood‐Brain Barrier Solute Permeability in Rat Brain by Transcranial Direct Current Stimulation
Authors: Fu Bingmei M. , Shin Da Wi , Fan Jie , Khadka Niranjan , Luu Eric , Khalid Wasem , Xia Yifan , Bikson Marom
Published: June 2021

Transcranial Direct Current Stimulation (tDCS) is a non‐invasive electrical stimulation technique investigated for a broad range of medical and performance indications. Our prior study has shown that tDCS with proper dosage can achieve non‐invasive, selective and temporary increase in the blood‐brain barrier (BBB) permeability (P). To further investigate the cellular and structural mechanisms by which tDCS increases P, prior to 20 min 1 mA tDCS, we pretreated the cerebral microvessels for ~30 min with a NOS inhibitor, L‐NMMA, through the tail vein injection, at an initial blood concentration of ~1 mM. We then used laser scanning multiphoton microscopy with 840 nm excitation wavelength to determine P of the cerebral microvessels in the rat brain tissue 100–200 μm below the pia mater. Sodium fluorescein (MW 376), or FITC‐dextran 70k in 1% BSA mammalian Ringer was injected into the rat (SD, 250–300g) cerebral circulation via the ipsilateral carotid artery by a syringe pump at a constant rate of ~3 ml/min. P were determined from the rate of dye spreading images around individual microvessels in the brain tissue. It was found that L‐NMMA pretreatment significantly reduced the increased P to sodium fluorescein from ~13‐fold to ~4‐fold of its control, and P to Dextran‐70k from ~88‐fold to ~8‐fold of its control at 5 min post tDCS treatment. Treatment by L‐NMMA alone for ~55 min significantly decreased the P to both solutes to ~66% of their controls. The predictions from our previously developed mathematical model for the BBB suggested that increasing the cleft width between adjacent endothelial cells and the width of basement membrane between the endothelium and astrocyte foot processes, due to contracting of endothelial cells, should be responsible for the transient increase in P by tDCS. In contrast, pretreatment of L‐NMMA may strengthen the structural components, most likely the junction strands in the cleft between endothelial cells to prevent the permeability increase by tDCS.

Support or Funding Information

NIH RO1 NS101362‐01

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
New Hippocampal Circuit Organization Revealed by Viral‐genetic Tracing and Functional Circuit Mapping
Authors: Xu Xiangmin
Published: June 2021

The hippocampal formation plays essential roles in learning and memory, and spatial navigation. Although the general anatomy and circuit organization of hippocampal CA1 has been well‐studied, most of our understanding of hippocampal circuits comes from the conventional anatomical tracing studies which lack cell‐type specificity and quantitative measurements of connectional strengths. New advances in virology and molecular genetics complement traditional approaches and are powerful tools for mapping cell‐type‐specific circuit connectivity and function. Using monosynaptic rabies tracing, we recently discovered a non‐canonical but significant set of back‐projections comprised of both excitatory and inhibitory elements from the subiculum to CA1 in the mouse. This discovery suggests previously unconsidered functional roles for direct subicular modulation of hippocampal CA1 circuit activity. In this talk, I will first report our quantitative analysis of canonical‐ and non‐canonical inputs to excitatory neurons in dorsal hippocampal CA1. We find that comparable strength of subiculum complex and entorhinal cortex (EC) inputs to CA1, significant inputs from presubiculum and parasubiculum to CA1, and a threefold stronger input to proximal versus distal CA1 from CA3. Non‐canonical subicular complex inputs exhibit opposing topographic connectivity gradients whereby the subiculum‐CA1 input strength systematically increases but the presubiculum‐CA1 input strength decreases along the proximal‐distal axis. These results reveal a novel anatomical framework by which to determine the circuit bases for CA1 physiological representations. Further, to test the hypothesis that CA1‐projecting subiculum neurons integrate strong CA1 and local subicular inputs to exert direct feedback regulation of CA1 circuit activity, we map and compare global circuit input and output connections of CA1‐projecting and other excitatory subicular neurons using retrograde monosynaptic rabies tracing and anterograde directed herpes simplex virus (H129 strain) in intact brains, respectively. In conjunction with behavior analysis and genetically targeted neuronal activation and inactivation, in vivo GCaMP6‐based calcium imaging of CA1 neurons in freely moving animals is used to resolve how CA1‐projecting subicular neurons modulate CA1 place cell activity and how they contribute to location dependent learning and memory. Together, these studies provide new insights into this subicular‐CA1 pathway, and advance our understanding of how the subiculum interacts with CA1 to regulate hippocampal circuit activity and learning and memory behaviors.

Support or Funding Information

This research is supported by the National Institutes of Health grant (R01NS078434).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Who am I? Investigating the impact of anatomy education on healthcare students' professional identity.
Authors: Lazarus Michelle , Paynter Sophie , Stephens Georgina , Rees Charlotte
Published: June 2021

Anatomy contact time reductions, in favour of early clinical exposure and professionalism coursework, remain trending in healthcare education. These efforts, have not necessarily improved student classroom to clinic transitions, and may have negative impacts on acquisition of knowledge fit‐for‐practice. In an effort to improve clinical integration and transitions from classroom learning to clinical environments, we evaluated the impact of anatomy education on healthcare students' professional identity and role formation. Using a longitudinal qualitative study approach with Monash University physiotherapy and medicine student participants, we probed students' perceptions regarding the impact of anatomy education on their professional identity development through serial online discussion forums during semester and delved deeper into these discussions by engaging participants in interprofessional focus groups at the end of each semester. Preliminary Framework analysis was undertaken by the research team to identify themes. Preliminary results indicate that there are six themes related to professional identity (PI) and role (PR) implicit in anatomy education: (1) Process, (2) Peer Relationships, (3) Educator Relationships, (4) Ethics, (5) Tolerance of Ambiguity (ToA) and (6) Contexts. Interestingly, some of these themes changed with time and teaching methods (e.g. Ethics), while others appear to be impeded by anatomy education (ToA). Integration of professional development into anatomy education is not only possible, it is an existing (albeit sometimes implicit) component of current teaching practices. Understanding the hidden and informal anatomy curriculum and its impact on PI and PR will not only serve to inform current practices, but it may also help healthcare students improve the ease of transitions (from classroom to clinics to practice). This work will serve as a foundation for builidng a framework which fosters purposeful and explicit integration between anatomy learning and PI/PR development. Together this will foster easier transitions into the clinic by providing students a platform for PI development early in their educational progression.

Support or Funding Information

Medical Education Travel Fellowship American Association of Anatomists Travel Fellowship Monash Education Academy Small GrantMonash Facutly of Medicine, Nursing, and Health Sciences Learning and Teaching Grant

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synergizing Non‐technical Skill Development in Medical Education: From Anatomy Classroom to the In‐Hospital Patient Setting and Back
Authors: Lachman Nirusha , Wittich Christopher
Published: June 2021

Medical professionalism is a multifaceted concept that includes maintenance of knowledge, interpersonal skills, teamwork, privacy and honesty. While non‐technical skills are often under‐emphasized in the medical student learning environment these skills are directly associated with providing professional medical care. There is an increasing trend however, for basic science courses such as Anatomy to introduce medical students early onto the concept of professionalism and highlight the importance of non‐technical skill development to patient care. To reinforce these competencies as well as develop an authentic curriculum for non‐technical skill development, anatomists and internists at Mayo Clinic, College of Medicine aligned objectives within medical school and residency programs. Strategies were designed to evaluate (1) teamwork, (2) communication and (3) self‐reflection. The aim of this presentation is to demonstrate how each of these core competencies can be promoted in both residency and medical student curricula and how trainee and student progress may be measured by educational approaches within their learning environments. Strategies include the use of a team charter, self‐reflection/reflective diaries and communication exercises that target patients and the multidisciplinary health care team. Through educational design (1) trainee and student's ability to acknowledge self‐limitation and develop critical thinking skills appeared to evolve and (2) trainee and student appreciation of the importance of communication skills when interacting with different groups as well as ability to translate medical/anatomical terminology to lay language were evident. In addition, the use of reflection and team charters promoted understanding team dynamics and facilitation of effective teamwork. The current multi‐disciplinary health care environment places strong emphasis on team‐work, confidentiality of protected health information and the role of communication in preventing medical errors and improving patient outcome. Anatomy courses designed on team‐based approaches with use of body donors provide a useful simulated learning environment. Medical students may be assessed through a variety of activities targeting team‐work, communication skills and clinical reasoning at different levels. Use of reflective exercises assessing team interaction, evolution of critical thinking skills and course challenges have found an important place in the evolving anatomy curriculum. Early exposure to these critical skills helps prepare for clinical rotations where milestones for these skills will need to be reached. This presentation will demonstrate the translational approach in synergizing non‐technical skill development between anatomy and residency curricula.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of Novel Scaffold for PIM1 Kinase Inhibition by Ligand Based Virtual Screening
Authors: Ranganna Kasturi , Mathew Omana P , Milton Shirlette , Thilgavathi Ramasamy , Selvam Chelliah
Published: June 2021

Prostate cancer afflicts one in six men during their lifetime making this disease the second leading cause of cancer‐related deaths in men in the United States. Given the high prevalence of prostate cancer and the high failure rate of current prostate cancer therapies, development of new therapies for aggressive prostate cancer is a high priority. PIM1 kinase represent as a potential therapeutic target for prostate cancer drug development because they are overexpressed in prostate cancers. There are several PIM kinase inhibitors that have reached to clinical trials and search for the novel PIM1 kinase inhibitor has dramatically accelerated. In silico ligand based virtual high throughput screening is one of the most successful and efficient methods to screen millions of lead like molecules and discover potent lead molecule with novel scaffolds. The present study aim to identify novel scaffolds for selective inhibition of PIM1 kinase by ligand based screening of lead like 4,591,276 compounds from zinc database. A ligand query model with pharmacophore features using vROCS tool was generated by taking a set of well‐known PIM 1 kinase inhibitors. The model was then utilized to screen 4.6 million lead like molecules of ZINC database. The resulted hits were prioritized and purchased 24 compounds from Enamine and Chembridge screening libraries to screen for PIMI kinase inhibitory activity. The PIMI kinase inhibitory activity was performed for all the purchased compounds by using CycLex PIMI kinase assay. The results are displayed in figure 1. The compounds 7, 22, and 23 showed more than 40% inhibition. The best hit molecule identified by ligand based virtual screening is compound 23.

Support or Funding Information

This study was made possible, in part, by Molecular Biology research infrastructure support from grant number 2G12MD007605 from the NIMHD/NIH. We thank the Openeye Inc. for providing the academic license to use the virtual screening tools.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.