bluuebunny/crossref_metadata_2025 huggingface dataset:
Drug Discovery from Peruvian Medicinal Plants for the Treatment of Psychosis
Authors: Tume Luis , Gallo Carla , Málaga‐Trillo Edward , Poletti Giovanni , Rojas Rosario , Vaisberg Abraham Jaime
Published: November 2023

Background

Schizophrenia is a severe mental disorder that affects approximately 1% of the population. The main symptom is psychosis in which thought and emotions are impaired. Current pharmacological treatments for schizophrenia are partially effective and its long term intake causes side effects. In Peru, native communities since ancient times have considered the psychosis like “madness” and they have treated it using preparations from natural products. The Peruvian traditional medicine is a potential source of drugs because its effectiveness, possible low side effects and they could be acting on different molecular mechanisms compared to the current drugs on the market.

Aim

In this work, as a part of drug discovery process, we tested compounds‐containing fractions from four ethanolic extracts (EEs) on psychosis animal model, mammalian cells and receptors altered in mental diseases.

Methods

We carried out the separation of compounds‐containing fractions from EEs 1, 2, 3 and 4 based on solubility using medium pressure liquid chromatography. The selection of each fraction was based on thin layer chromatography profile. These fractions were tested in mice treated with MK‐801 (N‐methyl D‐aspartate receptor antagonist) to induce psychosis‐like symptoms. We used the open field test (OFT) to measure the hyperactivity over a period of time and prepulse inhibition (PPI) test to measure the startle response. Cytotoxicity assays were performed in mammalian cells to determine the IC50 using the sulforhodamine B method. Moreover, we determined for the EEs, functional and binding assays on a panel of receptors altered in mental diseases.

Results

Four compounds‐containing fractions (A, B, C and D) were obtained for each EE. In behavioural tests, the fraction A, C and D of EE1, the fraction A and B of EE2 suppressed psychosis‐like symptoms in OFT (p <0.001) and PPI test. Fraction C and D of EE3 only showed an effect on PPI test and all fractions of EE4 were not active neither in the OFT nor in the PPI test. The IC50 for each EE was >0. 01563mg/ml. Finally, functional and binding assays indicates that the four EEs have an effect on serotonin, dopamine, glutamate, adenosine receptors.

Conclusion

We obtained potential antipsychotic compounds‐containing fractions as initial stage of drug discovery from four EE of medicinal plants used for the treatment psychotic‐like symptoms. The four EE has an effect on receptors altered in this disease suggesting an effect on signalling pathways altered in psychosis. Therefore, purification and structure elucidation of the active(s) molecule(s) will help us to better understanding of the molecular mechanism by which they are doing their antipsychotic effect.

Support or Funding Information

National Council of science of technology (CONCYTEC) through CIENCIACTIVA and Universidad Peruana Cayetano Heredia.
Sestrin2 Modulates Substrate Metabolism and Prevents Age‐related Intolerance to Ischemia and Reperfusion Injury
Authors: Li Ji , Quan Nanhu , Cates Courtney
Published: November 2023

A novel stress‐inducible protein, Sestrin2, declines in the heart with aging. AMP‐activated Protein Kinase (AMPK) has emerged as a pertinent stress‐activated kinase shown to have cardioprotective capabilities against myocardial ischemic injury. We identified the interaction between Sestrin2 and AMPK in the ischemic heart. It is hypothesized that the ischemic AMPK activation modulated by Sestrin2‐AMPK complex in the heart is impaired in aging that sensitizes heart to ischemic insults.

Methods

C57BL/6 young mice (3–4 months), middle age mice (10–12 months) and aged mice (24–26 months) were subjected to left anterior descending coronary artery occlusion for in vivo regional ischemia. The ex vivo working heart system was used for measuring substrate metabolism. The protein level of Sestrin2 in the hearts were gradually decreased with aging. Intriguingly, ischemic AMPK activation was blunted in the aged hearts as compared with young hearts (p<0.05), the AMPK downstream glucose uptake and the rate of glucose oxidation were significantly impaired in the aged hearts during ischemia and reperfusion (I/R) (p<0.05 vs. young hearts). The myocardial infarction size was larger in the aged hearts (p<0.05 vs. young hearts). The immunoprecipitation with Sestrin2 antibody revealed that cardiac Sestrin2 forms a complex with AMPK and upstream LKB1 during ischemia, intriguingly, the binding affinity between Sestrin2 and AMPK upstream LKB1 is impaired in the aged hearts during ischemia (p<0.05 vs. young hearts). Furthermore, Sestrin2 knock out hearts demonstrate a similar cardiac phenotype and response to ischemic stress as the wild type aged hearts, i.e. impaired ischemic AMPK activation and higher sensitivity to I/R‐induced injury. Adeno‐associated virus (AAV)‐Sestrin2 were delivered into the aged hearts via a coronary delivery approach significantly rescued the protein level of Sestrin2 and the ischemic tolerance of aged hearts. Therefore, Sestrin2 is a scaffold protein that mediates AMPK activation in the ischemic myocardium via an interaction with AMPK upstream LKB1. The decreased Sestrin2 levels in aging lead to a blunted ischemic AMPK activation, alterations in substrate metabolism and an increased sensitivity to ischemic insults.

Support or Funding Information

ADA 1‐14‐BS‐131, NIHR21AG044820
PET Imaging of Isoproterenol‐Induced Myocardial Injury
Authors: Houson Hailey , Hedrick Andria , Awasthi Vibhudutta
Published: November 2023

Background

Clinical assessment of myocardial injury is primarily based on the presence of serum biomarkers and electrocardiogram, with a significant input from perfusion imaging by single photon emission computed tomography (SPECT). Positron emission tomography (PET) is another imaging modality which has the potential to improve diagnostic accuracy by virtue of its high resolution and sensitivity. The objective of this study was to assess isoproteronol‐induced cardiomyopathy by PET using 18F‐fluorodeoxyglucose (FDG) and compare it with conventional SPECT using 99mTc‐Sestamibi (MIBI).

Methods

Cardiomyopathy was induced in Sprague Dawley rats by injecting high dose of isoproteronol (100 mg/kg, i.p.) on two consecutive days. FDG/PET and MIBI/SPECT were performed before and on the fourth day of isoproteronol administration. FDG (0.1 mCi, 0.2–0.4 mL) and MIBI (2 mCi, 0.5–1 mL) were injected via the tail vein and the images were acquired 1 h post‐injection. The images were analyzed by drawing region of interest (ROI) around heart tissue. Isoproteronol‐induced myocardial injury was verified at necropsy by tissue staining with tetrazolium chloride (TTC) and plasma cardiac troponin.

Results

Isoproteronol administration resulted in a significant increase in circulating troponin I (p < 0.05) as compared to the basal levels. FDG/PET images showed detectable alterations in cardiac accumulation of FDG and ROI analysis revealed a 75% drop of myocardial FDG uptake after isoproteronol treatment. The uptake of MIBI decreased by 45%, but the images did not show any discernible visual change in perfusion. At necropsy, TTC staining showed remarkable changes in all isoproteronol ‐treated hearts.

Conclusion

The results suggest that high‐dose isoproteronol significantly impacts cardiac glucose metabolism without commensurate change in myocardial perfusion. PET imaging of glucose metabolism can be employed as a diagnostic criterion in myocardial injury.

Support or Funding Information

Funding: Sandra K and David L Gilliland Chair in Nuclear Pharmacy
Angiotensin (1‐7) Attenuates Angiotensin II‐Induced Cardiac Hypertrophy via a Sirt3‐Dependent Mechanism
Authors: Guo Lirong , Zhong Tiecheng , O'Rourke Stephen T. , Sun Chengwen
Published: November 2023

The objectives of the current study were to investigate the effect of Ang‐(1‐7) on the development of cardiac hypertrophy, and to identify the intracellular mechanism underlying this action of Ang‐(1‐7). Blood pressure and heart rate were recorded using radiotelemetry before and after chronic subcutaneous infusion of control (PBS), Ang II, Ang‐(1‐7), or Ang II+Ang‐(1‐7) for 4 weeks in normotensive rats. Chronic administration of Ang‐(1‐7) did not affect either basal blood pressure or the Ang II‐induced elevation in blood pressure. However, Ang‐(1‐7) significantly attenuated Ang II‐induced cardiac hypertrophy and perivascular fibrosis in these rats. These effects of Ang‐(1‐7) were confirmed in cultured cardiomyocytes, in which Ang‐(1‐7) significantly attenuated Ang II‐induced increases in cell size. This protective effect of Ang‐(1‐7) was significantly attenuated by pretreatment with A779, a Mas receptor antagonist; Mito‐TEMPO, mitochondria‐targeting superoxide scavenger, as well as blockade of Sirt3, a deacetylation‐acting protein, by viral vector‐mediated overexpression of Sirt3‐shRNA. Western blot analysis demonstrated that treatment with Ang‐(1‐7) dramatically increased Sirt3 expression. In addition, Ang (1‐7) attenuated the Ang II‐induced increase in mitochondrial ROS generation, an effect that was abolished by A779 or Sirt3‐shRNA. Moreover, Ang‐(1‐7) increased FoxO3a deacetylation and SOD2 expression, and these effects were blocked by Sirt3‐shRNA. In summary, the protective effects of Ang‐(1‐7) on Ang II‐induced cardiac hypertrophy and increased ROS production are mediated by elevated SOD2 expression via stimulation of Sirt3‐dependent deacetylation of FoxO3a in cardiomyocytes. Thus, activation of the Ang‐(1‐7)/Sirt3 signaling pathway could be a novel therapeutic strategy in the management of cardiac hypertrophy and associated complications.

Support or Funding Information

This work was supported by Grants from the National Institute of Neurological Disorders and Stroke, National Institutes of Health (NIH, NS55008), American Heart Association (10GRNT3170012), Norman Bethune Program of Jilin University (2015221), and Education Department of Jilin Province (2015498).
Mouse c‐kit+ Cardiac Progenitor Cells Do not Age Gracefully
Authors: Castaldi Alessandra , Dodia Ramsinh Mansinh , Orogo Amabel M. , Najor Rita H. , Gustafsson Asa B. , Brown Joan Heller , Purcell Nicole H.
Published: November 2023

Although c‐kit+ cardiac progenitor cells (CPCs) are currently used in clinical trials there remain considerable gaps in our understanding of the molecular mechanisms underlying their proliferation and differentiation. Despite the fact that cardiac injury affects mainly the older human population, studies aimed at understanding CPC biology and their ability to contribute to cardiac regeneration have been performed on young animal models. In the present study we asked whether CPCs isolated from young (3 month) and aged (24 month) mice present different characteristics that could account for different outcomes in future CPC‐based therapeutic treatments for cardiac injury. We observed morphological differences and increased senescence as measure by the presence of senescence associated β‐galactosidase and increased p16 mRNA. The aged CPCs proliferated slower than their young counterpart and the expression of the stemness marker LIN28 was extremely low in the lines. We subsequently treated the cells with Dexamethasone (Dex), routinely used to induce cardiac commitment in CPCs, for 7 days and analyzed the expression of cardiac lineage marker genes. While MEF2C, GATA4, GATA6 and PECAM mRNAs were significantly upregulated in response to Dex treatment in young CPCs, their expression was not increased in aged CPCs. Interestingly, Dex treatment of aged CPCs failed to increase mitochondrial biogenesis and expression of the mitochondrial protein Complex III and IV, consistent with a defect in mitochondria complex assembly in the aged CPCs. This study should have an impact on the design of future CPC‐based therapeutic approaches for the treatment of older patients suffering from cardiac injury.

Support or Funding Information

This work was supported by grants from the National Heart, Lung and Blood Institute Grants: HL085577 (JHB, NHP, ÅBG and AC), HL028143 (JHB), HL114949 (NHP), and HL087023 (ÅBG) and by the National Institutes of Health NRSA Predoctoral Fellowship HL123309 (AMO). ÅBG is supported by an AHA Established Investigator Award.
Helium Influences Caveolae Numbers in Left Ventricular Endothelial Cells and Alters Metabolic Profile in Mice
Authors: Warmbrunn Moritz Valerian , Schilling Jan M , Zemljic‐Harpf Alice E , Hollmann Markus W , Roth David M , Patel Hemal H , Weber Nina C
Published: November 2023

Background

Myocardial ischemia and reperfusion (I/R) injury is a major cause of morbidity and mortality. IR injury leads to calcium overload, increased oxidative stress, mitochondrial dysfunction and ultimately apoptosis. Helium, an inert gas, causes cardioprotection potentially through secretion of cellular factors into serum. This study aimed to elucidate the protective potential of helium conditioned serum, the metabolite content of serum, and the impact of helium on endothelial cell ultrastructure. We hypothesized that helium induces the release of circulating factors to mediate conditioning and impacts endothelial cell ultrastructure.

Materials and methods

Mice were exposed to 70% helium/30% oxygen or 30% oxygen in air. Serum was harvested at 30 min, 4 hrs and 24 hrs after gas exposure (n=7/group). In addition, mice (n=4/group) were prepared and fixed for transmission electron microscopy. High magnification images (11000×) of left ventricular tissue were analyzed for the amount of caveolae in endothelial cells and myocytes. Serum from exposed mice was analyzed for metabolites of primary metabolism and complex lipids by mass spectrometry. Metabolites were categorized by physiologic pathways and multivariate analysis was used to determine interaction between helium and recover time.

Results

Endothelial cell caveolae number was increased at 30 min and 24 hrs post exposure in the helium group by 39% (p < 0.01, C: 3.5 ± 0.2, n=83 images; He: 4.8 ± 0.3 caveolae/mm, n=67 as mean ± SEM) and 18% (p < 0.05, C: 4.3 ± 0.3, n=72; He: 5.3 ± 0.3 caveolae/mm, n=61), respectively. No differences were observed in myocyte caveolae formation. Interaction of time and drug showed a trend for oxidative phosphorylation (p= 0.097) and galactose metabolism pathway (p= 0.0798). An overall drug effect between the different time points was observed for biotin metabolism (p < 0.05). The metabolites of the two pathways, valine/leucine/isoleucine and pantothenate/CoA biosynthesis showed a trend towards a drug effect (p=0.053; p=0.057 respectively). Three pathways were significantly changed in the 4 hrs group and in the 24 hrs group biosynthesis of unsaturated fatty acids was influenced. In addition three cardioprotective ceramides were significantly upregulated by helium (p < 0.05).

Conclusion

Helium exposure altered endothelial cell ultrastructure by inducing the formation of caveolae and induced secretion of factors resulting in different metabolic profiles of primary metabolism and complex lipids. Such changes in cell ultrastructure and metabolite secretion may be key to the conditioning effects induced by helium.
Potent Blood Pressure Lowering Effect of Optogenetic Stimulation of ‘Angiotensin‐sensitive’ Neurons within the Nodose Ganglion
Authors: da Silva Souza Gean Domingos B , Bruce Erin B. , Hiller Helmut , Smeltzer Michael D. , Tan Yalun , Brandi Igor Viana , Sumners Colin , Raizada Mohan K , Krause Eric G. , de Kloet Annette D
Published: November 2023

Neurons within the nodose ganglion (NG) convey baroreceptor status to the brain and their stimulation is known to reduce blood pressure and heart rate; however, whether a distinct population of angiotensin‐sensitive neurons within the NG mediate these effects is not clear. Here, we use neuroanatomical and optogenetic techniques to test the hypothesis that angiotensin type 1‐a receptor (AT1a)‐expressing neurons within the NG influence hemodynamic responses in male mice. Initial neuroanatomical studies revealed that AT1a‐containing neurons are localized to the NG and uncovered a dense localization of terminals arising from AT1a neurons to the intermediate nucleus of the solitary tract, consistent with their localization to the vagal afferent neurons of the NG. To examine the functional relevance of these distinct angiotensin sensitive neurons, the Cre/Lox system was then used to generate male mice expressing channelrhodopsin‐2 and yellow fluorescent protein (eYFP) specifically in AT1a‐expressing neurons. These mice (n=6) were catheterized into the left carotid artery and the left NG was identified and isolated from adjacent tissues in order to optogenetically stimulate AT1a‐containing neurons within the area. Consistent with our hypothesis, photo‐activation (473nm blue light;1, 15 or 30Hz; 1 min) of these AT1a‐expressing neurons led to significant decreases in mean arterial pressure (MAP, Δ= −16±3, −36±4 and −44±7 mmHg) and heart rate (HR, Δ= −13±7, −104±44 and −163±59 bpm) in AT1a‐ChR2‐eYFP mice but not in control mice subjected to the same stimulation parameters. Collectively, our results demonstrate that activation of AT1a‐expressing neurons in NG effectively reduces MAP and HR in mice, suggesting that AT1a in the NG might play an important role in cardiovascular equilibrium.

Support or Funding Information

National Heart, Lung, and Blood Institute Grant HL122494 (EGK), HL125805 (ADdK), HL‐033610 (CS and MKR)
AT1 receptor on glutamatergic neurons regulate cardiac function through modulation of excitability and sympathetic outflow
Authors: Xu Jiaxi , Molinas Adrien , Zsombok Andrea , Lazartigues Eric
Published: November 2023

Previously we generated a mouse model with Angiotensin (Ang)‐II type 1 (AT1) receptor deletion from the central nervous system (CNS) neurons. Compared to their control littermates, those mice exhibited attenuated hypertension and improved autonomic function after DOCA‐salt treatment (DOCA 1mg/g body weight sc + 1% saline po for 3 weeks). Moreover, unlike controls, no increase in left ventricle (LV) mass or decrease in cardiac systolic function was observed, suggesting those mice with neuronal AT1R deficiency were protected from the cardio‐hypertrophic process induced by DOCA‐salt treatment, indicating a possible role of neuronal AT1R in regulation of cardiac function. To further investigate this hypothesis, a new mouse model (AT1G) was generated, in which AT1aR was knocked‐out from glutamatergic neurons. Interestingly, at baseline, AT1G mice (n=7) already exhibit impaired cardiac function, as reflected by reductions in ejection fraction (−11.63 ±3.95%; P=0.0048) and fractional shortening (−6.61 ±2.25%; P=0.0049), despite no significant changes in morphology (such as LV mass, chamber size, and wall thickness). Blood pressure (BP) and heart rate (HR) were also unaltered even though spontaneous baroreflex sensitivity was slightly reduced (2.31 ±0.29 vs. 3.01 ±0.23 msec/mmHg, P=0.04). After 2 weeks of DOCA‐salt treatment (n=5/group), control mice exhibited a significant decrease in cardiac systolic function (P=0.01) and increase in the LV chamber size (P<0.01), which were not observed in DOCA‐salt‐treated AT1G mice. Moreover, when echocardiography was performed (14 day of DOCA‐salt treatment), there was no significant difference in BP and HR between AT1G mice and their control littermates, both of which were hypertensive due to DOCA‐salt treatment (127.3 ±8.89 vs. 131.5 ±1.84, P=0.27), suggesting the change in cardiac function is mediated directly by the CNS and not secondary to a BP rise. To further dissect the mechanism, qRT‐PCR and patch‐clamp recording were performed. In the hypothalamus of AT1G mice, mRNA levels for GABAA receptors and glutamic acid decarboxylase (GAD) 67 were significantly higher, by +52 ±17% and +36 ±14% respectively, compared to controls (n=4/group, P<0.01). Meanwhile, lower action potential (AP) frequency was recorded in kidney‐related PVN neurons of AT1G mice (0.43 ±0.06 vs. 2.04±0.96 Hz, n=3, P=0.01), suggesting that those neurons indeed have lower excitability compared to controls. Altogether, our data provide evidence that central renin‐angiotensin system has an essential role in regulating cardiac function by mediating neuronal excitability and sympathetic outflow.

Support or Funding Information

This work was supported by research grants from the National Institutes to Health (HL093178 and GM106392 to E.L.) and The American Heart Association to E.L. (12EIA8030004), J.X. (15POST25000010).
Blunted respiratory responses in the STZ‐induced rat model of Alzheimer's disease
Authors: Ebel Dalton Louis , Torkilsen Christopher G , Ostrowski Tim Daniel
Published: November 2023

Alzheimer's disease (AD) is a debilitating disease of the central nervous system that results in a drastic decline in cognition and memory. In addition, respiratory dysfunction is frequently observed in AD patients, as shown by altered breathing during sleep and blunted minute ventilation during heightened oxygen demand with peak exercise. The combination of AD symptoms leads to a decrease in day‐to‐day function and increased mortality. At this time, the mechanisms behind the respiratory alterations are widely unknown.

For this study, an AD rat model was induced by injecting 1.5 mg/kg Streptozotocin (STZ) in the lateral ventricle to trigger pathological changes consistent with sporadic AD. We used plethysmography to analyze the peripheral and central chemoreflex in response to hypoxia (14 ‐ 8% O2, equilibrated with N2) or hypercapnia (5% CO2, equilibrated with O2), respectively. We compared respiratory parameters before and after injection of STZ or vehicle (aCSF). Morphological changes in the brainstem and hippocampus were immunohistochemically analyzed using an antibody against S‐100B for astrocytes.

Similar to the inflammation in the CNS of AD patients, microinjection of STZ induced astrogliosis in the CA1 region of the hippocampus. We also found reactive astrocytes in the commissural part of the nucleus tractus solitarii, an important center for the control of respiration and responses to low oxygen conditions. In comparison, astrocytes in the pre‐Bötzinger complex of AD rats did not change. Analyzing the respiratory pattern, we found an expected age‐dependent decrease in minute ventilation in our control group. This age‐dependent decrease was absent in the AD group, indicating elevated minute ventilation following STZ administration. Peripheral chemoreflex activation with hypoxia showed significant alterations in the AD group, as observed by a blunted elevation of respiratory rate and minute ventilation. Central chemoreflex activation with hypercapnia only decreased respiratory rate following STZ.

We show that the STZ‐induced AD rat model displays significant respiratory dysfunction at rest and in response to hypoxia. Thus, this study provides the first model to elucidate the mechanisms behind respiratory dysfunction of sporadic Alzheimer's disease.
An intrinsic tryptophan metabolite acts as pulmonary vasodilator in pulmonary hypertension
Authors: Nagy Bence , Nagaraj Chandran , Meinitzer Andreas , Papp Rita , Foris Vasile , Ghanim Bahil , Kwapiszewska Grazyna , Kovacs Gabor , Klepetko Walter , Pieber Thomas R. , Mangge Harald , Olschewski Horst , Olschewski Andrea
Published: November 2023

The tryptophan metabolite kynurenine is associated with pulmonary hypertension (PH) in dyspnea patients and is increased in PH. In addition, it causes dilation in systemic arteries. Our aim was to investigate the role of kynurenine in the pulmonary circulation.

Serum tryptophan, kynurenine and kynurenic‐acid levels were measured in 20 IPAH patients, 20 healthy controls and 20 patients with chronic lung disease or metabolic syndrome without PH. Laser‐dissected pulmonary arteries from IPAH and control lungs were tested for the expression of IDO, the enzyme responsible for tryptophan‐kynurenine conversion. Acute effects of kynurenine were tested in pulmonary vascular preparations, two different models of chronic PH, and in human pulmonary arterial smooth muscle cells (hPASMC).

Among all investigated tryptophan metabolites, the strongest association to PH was found with kynurenine. In IPAH vs. control, kynurenine was significantly elevated (3.6±0.2μM vs. 2.6±0.1μM, p<0.0001), and strongly associated with PH (AUC=0.86), but kynurenine levels were not elevated in lung disease‐ and metabolic syndrome‐patients. Kynurenine displayed strongest correlation with mean pulmonary arterial pressure (mPAP) (ρ:0.770, p<0.0001). Tryptophan was significantly decreased in IPAH however, IDO expression was not changed. In hPASMCs, kynurenine increased both cAMP and cGMP; in the rat and mouse it relaxed preconstricted intrapulmonary arteries; and in two models of established PH it acutely decreased mPAP.

Our data suggest that kynurenine elevation might be specifically associated with mPAP; kynurenine increases both cAMP and cGMP in hPASMC and it exerts acute pulmonary vasodilatation. Kynurenine might provide both a new biomarker for PH and a new therapeutic option.
Microparticles from Aged Stored Platelets Impair Microvascular Pulmonary Endothelial Cell Barrier Function
Authors: McVey Mark J , Maishan Mazharul , Srbely Victoria , Lee Warren , Spring Chris , Semple John , Kuebler Wolfgang M
Published: November 2023

BACKGROUND

Transfusion‐related acute lung injury (TRALI) has a mortality of 5–15% and an incidence ranging from 0.02–1%. TRALI causes chest infiltrates and hypoxia in part due to lung endothelial cell (EC) barrier failure and edema formation. Aged platelets which have a high risk of causing TRALI, accumulate microparticles (PMPs) throughout storage. PMPs have been shown to mediate pro‐inflammatory processes such as neutrophil priming.

RESEARCH QUESTION

Considering the role of ECs in TRALI, we set out to determine if aged platelet derived PMPs could mediate EC barrier disruption?

HYPOTHESIS

Specifically, we hypothesized that PMPs from aged but not fresh platelets will lead to EC monolayer disruption.

METHODS

C57BL/6 mouse platelets were stored for 1 (fresh) or 5 (aged) days duration prior to PMP isolation (centrifugation/filtration) and enumeration (flow cytometry). Human microvascular pulmonary ECs (passage 6–8) were cultured with PBS (sham) or 0.1 mg/ml LPS two hours prior to addition of PBS, 5×105 of fresh or aged PMPs, or PMP depleted supernatants from stored platelet pools. Transendothelial electrical resistance (TEER) readings were obtained every 30 minutes for 8 hours in triplicate. Alternatively aged 10 ml/kg PMPs (≈ 5×106) or saline (sham) was transfused into primed (2 mg/kg LPS intraperitoneal) BALB/c mice for assessment of lung tissue wet‐to‐dry (W/D) ratios 6 hours post transfusion.

RESULTS

PMPs were found to be increased more than 2 fold in aged as compared to fresh platelet storage pools. ECs exposed to LPS alone did not reveal decreases in TEER relative to PBS sham. When aged PMPs were added 2 hours after PBS there was a marked drop in TEER within 2 hours which became maximal (nearly 50% decrease relative to PBS alone) by 5 hours. Addition of LPS instead of PBS prior to aged PMPs led to similar decreases in TEER readings but with faster onset. Addition of platelet and PMP depleted supernatants (fresh or aged) or fresh PMPs with LPS did not show signs of decreased TEER readings relative to PBS sham. Beyond being more numerous, aged PMPs appear more injurious than fresh PMPs when delivered in our EC model at identical doses. Aged PMPs are able to reduce EC monolayer integrity within 2 hours and sustain this effect for up to 6 hours +/− LPS. LPS primed mice transfused aged PMPs had higher W/D ratios than LPS primed mice transfused saline (p<0.05).

CONCLUSIONS

PMPs may in part contribute to TRALI when aged platelets which are a rich source of PMPs are transfused. In our model aged PMPs were injurious, impairing EC barrier integrity in vitro and leading to increased W/D ratios in vivo. Targeting aged PMPs may prove a therapeutic option to reduce the risk of aged cellular blood products from causing TRALI.

Support or Funding Information

Canadian Institutes of Health Research (CIHR)

Canadian Blood Services (CBS)
Novel PKA‐mediated mechanisms of microvascular endothelial barrier enhancement
Authors: Batori Robert Karoly , Kumar Sanjiv , Shaw Mary , Bordan Zsuzsanna , Gorshkov Boris , Kovacs‐Kasa Anita , Fulton David , Verin Alexander
Published: November 2023

Acute lung injury (ALI) is devastating disorder with an unacceptably high level of mortality. The pharmacological treatment of ALI relies on supportive care and control of initiating causes. Therefore, novel therapies are urgently needed to improve clinical outcomes. Endothelial cells (EC) form a semi‐permeable barrier between the interior space of blood vessels and the underlying tissues. ALI is characterized by significant pulmonary inflammatory response resulting in a protein‐rich pulmonary edema due to the loss of endothelial barrier integrity. The mechanisms that govern increased EC permeability are under intense investigation, however, the processes determining the preservation/restoration of the EC barrier function remain poorly understood. Our group has previously shown that extracellular purines, ATP and adenosine are able to preserve vascular barrier in murine model of LPS‐induced ALI and enhance EC barrier in human pulmonary artery EC (HPAEC) via activation of myosin light chain (MLC) phosphatase (MLCP). MLCP activation may involve PKA‐dependent phosphorylation of the MLCP targeting subunit 1 (MYPT1) at S695, which may activate MLCP directly or indirectly by preventing its Rho kinase‐mediated phosphorylation of the subsequent T696 residue, however, the direct link between MLCP, PKA and purine‐induced EC barrier enhancement was not established.

Our new data demonstrated that stable ATP analog, ATPγS, is able to strengthen the EC barrier in human lung microvascular EC (HLMVEC). In contrast to ATP, this effect is not accompanied by an inflammatory response but involved PKA activation and decrease in MLC20 phosphorylation suggesting the involvement of MLCP. Surprisingly, in contrast to adenosine, ATPγS‐induced PKA activation does not acompanied by increase in cAMP level suggesting the existance of an unconventional cAMP‐independent model of PKA activation. Further, ATPγS‐induced EC barrier strengthening is not involved EPAC1, but dependent upon a non‐receptor type 11 tyrosine‐phosphatase (PTPN11, also known as Shp2) and AKAP2 (PKA anchoring protein 2) expression. Furher, we demonstrated that AKAP2 can directly interact with MYPT1 and depletion of AKAP2 restored MLC20 phosphorylation level reduced by ATPγS treatment. Collectively, our data suggest that ATPγS‐, but not adenosine‐induced EC barrier enhancement requires coordinated Shp2‐mediated activation of PKA which is then guided by AKAP2 to activate MLCP and dephosphorylate MLC.

Support or Funding Information

Supported by HL101902
Paternal Hyperandrogenism Promotes Behavioral Changes in Swiss Mice Offspring
Authors: Conceição Rodrigo Rodrigues , Souza Janaina Sena , Laureano‐Melo Roberto , Lau Raphael Silva , da Rocha Natalino Monteiro Livia , Dos‐Santos Raoni Conceição , da Silva Almeida Claudio , Giannocco Gisele , da Silva Cortes Wellington
Published: November 2023

The paternal environmental factors before conception and during sperm development determine the health of the offspring in later life. Thus, the aim of our study was to verify how paternal hyperandrogenism is able to change behavioral parameters in mice's offspring during adulthood. For this purpose, two groups (n=6 each, and ~35g) of male Swiss mice were randomly divided into two groups. The control was treated with peanut oil, while the treated group received testosterone cypionate (7.5mg/kg, s.c.) twice a week for five weeks. After this period, these animals were mated and the offspring obtained, by completing 70 days of life, were underwent to a battery tests consisting of the following tests: open field, dark‐light box, elevated plus maze and tail suspension test. At the end of the experiment, hippocampus was dissected for RNA analysis. Statistical analysis was performed by t‐Student and the means were considered significantly different when p<0.05. According to our results, the treated group had higher immobility time in tail suspension test (28%, p=0.02) and decreased opened arms time in elevated plus maze test (27%, p=0.01). Moreover, in light‐dark box test, the treated group offspring had less time in the light side than control (156%, p=0.002). These behavioral changes were accompanied by reduced GAD 67 (17%, p = 0.01) and BDNF (37%, p = 0.005) expression, as well as a higher GABAA receptor α2 subunit (50%, p = 0.03) expression in hippocampus. These findings indicate that paternal hyperandrogenism alters hippocampal neurotrophin and gabaergic system, promoting anxiety and depression‐like behaviors in adults. Also, these results are of important significance when we consider the abusive use of anabolic steroids for fitness purposes.

Support or Funding Information

FAPERJ & CNPq.
Determining the Contribution of Mast Cell‐specific Neurokinin‐1 Receptors in Pressure Overload‐Induced Cardiac Fibrosis
Authors: Widiapradja Alexander , Manteufel Edward , Pena James , Goldspink Paul , Sharma Amit , Imig John D , Lu Bao , Levick Scott
Published: November 2023

Substance P (SP) and mast cells (MCs) both play a detrimental role in initiating myocardial remodeling, including fibrosis, in response to pressure overload. However, it is not known whether MC activation is an underlying mechanism mediating the effects of SP in this setting. We sought to determine whether SP and its cognate receptor, the neurokinin‐1 receptor (NK‐1R), regulate MC function and whether this is a mechanism underlying the pro‐fibrotic actions of SP. Administration of the NK‐1R antagonist, L732138 to spontaneous hypertensive rats (SHR) reduced cardiac MC density by reducing levels of stem cell factor (SCF), which promotes MC proliferation and maturation. SP treatment of bone marrow derived mast cells (BMMCs), as a model of cardiac MCs, showed no change in c‐kit levels (SCF receptor) or apoptosis, ruling out the possibility of SP/NK‐1R in regulating MC number through increasing the response to SCF or via altering apoptosis. Interestingly, SP activation of the NK‐1R does not activate MCs since conditioned media from SP treated MCs had no effect on collagen synthesis by cultured fibroblasts, and reconstitution of MC‐deficient mice with MCs lacking the NK‐1R did not reduce cardiac fibrosis in angiotensin II‐infused mice. Further, whilst SCF is important in MC proliferation, it also does not activate MCs as there was no increase in cardiac collagen in SCF injected mice. In conclusion, SP activation of the NK‐1R increases MC density in the overloaded heart via production of SCF, however, neither SP nor SCF activate MCs in this setting. Thus, the stimulus activating MCs in the setting of cardiac fibrosis is still unknown.

Support or Funding Information

National Institutes of Health HL093215 (S.P.L.), HL132908 (S.P.L.), Greater Milwaukee Foundation–Elsa Schoeneich Medical Research Fund (S.P.L.), MCW Presidential Postdoctoral Fellow Award (A.W.).
Administration of anti‐inflammatory interleukin‐37 ameliorates age‐related vascular, metabolic and physical dysfunction in mice
Authors: Sapinsley Zachary J , Ballak Dov B , Brunt Vienna E , Zigler Melanie C , Gioscia‐Ryan Rachel A , Richey James J , Dinarello Charles A , Seals Douglas R
Published: November 2023

Chronic, low‐grade inflammation that occurs with age, termed inflammaging, is a key mechanism in the etiology of age‐related disease and dysfunction, which has been linked to the pathophysiology of cardiovascular diseases, metabolic diseases including type 2 diabetes mellitus, and physical decline. Interleukin‐37 (IL‐37), a member of the interleukin‐1 family of cytokines, broadly inhibits inflammation and therefore carries strong potential as an anti‐aging intervention.

PURPOSE

We tested the hypothesis that two weeks of recombinant IL‐37 (recIL‐37) administration would improve vascular, metabolic, and motor function in old mice.

METHODS

Twenty old (27 mo) and fourteen young (9–10 mo) male C57BL/6 mice were treated with recIL‐37 or vehicle (phosphate buffered saline) for two weeks, injected intraperitoneally (IP) (200μL of 5μg/L) daily. Vascular, metabolic, and motor functions were measured at baseline and again post‐intervention using blood pressure and large elastic artery stiffness testing with pulse wave velocity, 2‐hr glucose and insulin tolerance tests, and RotaRod endurance run time, respectively. Vascular function was further evaluated through ex vivo carotid artery endothelium‐dependent dilation to increasing doses of acetylcholine (10−9 to 10−4 M) without and with nitric oxide (NO) synthase inhibition via 10−4M L‐NG‐nitroarginine methyl ester (L‐NAME).

RESULTS

Two weeks of recIL‐37 administration in old mice: (1) improved ex vivo endothelium‐dependent dilation of carotid arteries (p < 0.05 for 10−7 to 10−5M ACh, n=7–9 per group; 10−5M ACh: recIL‐37: 86.2±2.7%max vs. vehicle: 64.6±3.0%max, p=0.01) seemingly by improving NO‐dependent dilation (difference between dilation to max ACh dose without and with L‐NAME: recIL‐37: 67.4±7%max vs. vehicle 50.5±5.3%max, p=0.08, n=6 per group); (2) improved insulin sensitivity (2‐hr glucose AUC following IP insulin injection [0.75U/kg body weight], recIL‐37: 232±23 mM·min vs. vehicle: 293±17 mM·min, p=0.05) and tended to improve glucose tolerance in a subset of mice (20‐min peak blood glucose following IP glucose injection [2μg/g body weight], recIL‐37: 10.4±0.8mM vs. vehicle: 13.3±1.2mM, p=0.08), and (3) completely restored RotaRod endurance run time (recIL‐37: 36±7 m vs. vehicle: 17±7, p=0.03) to levels comparable to young vehicle‐treated mice (34±8m, P=0.86 vs. old recIL‐37). No changes were observed in blood pressure or pulse wave velocity or in any variables in young mice.

CONCLUSIONS

Two‐week administration of anti‐inflammatory recIL‐37 improves selective vascular, metabolic, and motor functions in old mice. Anti‐inflammatory treatments such as recIL‐37 present a novel strategy for preserving physiological function with advancing age and preventing age‐related diseases.

Support or Funding Information

Supported by NIA grant #RAG053804A.
RORγt‐Dependent Pro‐inflammatory Cytokine IL‐17F in SHR Impairs Endothelium‐Dependent Vascular Relaxation
Authors: Singh Madhu V. , Cicha Michael Z. , Kumar Santosh , Irani Kaikobad , Chapleau Mark W. , Abboud François M.
Published: November 2023

We have previously shown that SHR (spontaneously hypertensive rat) have an abnormally large population of CD161+ immune cells that express RORγt (retinoic acid receptor related orphan receptor gamma) transcription factor. RORγt is the master regulator of T helper 17 (Th17) cell differentiation of CD4+ naive T cells that express CD161, IL‐17A and IL‐17F. This CD161+ abundance in the SHR spleen increases with age and produces a potent pro inflammatory cytokine IL‐17F in a RORγt‐dependent manner. In this study, we tested whether the enhanced inflammatory response seen in splenocytes is manifested in the vascular tissue of the SHR and contributes to a pathological response. We determined CD161+ cell infiltration of the aorta by flow cytometry and measured changes in RNA expression by real time quantitative PCR. We also performed wire myography on aortic rings to determine the effect of IL‐17F on vasorelaxation. We found significantly greater infiltration of CD161+ cells in age‐matched SHR aorta than in WKY (0.2% in WKY vs. 6.2% in SHR), whereas total CD4+ cell infiltration was not different (15.1% in WKY vs. 14.2% in SHR). Upon ex vivo treatment of aorta with phorbol 12‐myristate 13‐acetate (PMA) and ionomycin to induce cytokine production, both WKY and SHR showed increased IL‐17A RNA expression (1.6 fold in WKY vs. 2.4 fold in SHR), whereas IL‐17F expression was increased to a much greater extent in SHR aorta than in WKY (7.9 fold in WKY vs. 15.3 fold in SHR, P<0.05). These results are consistent with our previous findings of greater IL‐17F induction in SHR immune cells. Furthermore, we found that PMA‐ionomycin induces a modest increase in IL‐17A RNA expression in the peri‐vascular adipose tissue (PVAT) of both WKY and SHR (1.7 fold in WKY vs. 2.9 fold in SHR) with a much larger increase in IL‐17F RNA in SHR PVAT (2.8 fold in WKY vs. 13.9 fold in SHR, P<0.05). Treatment of aortic rings from WKY rats with IL‐17F, but not IL‐17A, impaired endothelium‐dependent relaxation of preconstricted vessels by acetylcholine in a dose‐dependent manner. The treatment did not affect endothelium‐independent, sodium nitroprusside‐induced vasorelaxation. We conclude that in SHR, a genetically predisposed abundance of CD161+ immune cells leads to a greater infiltration of these cells in the vasculature and increased potential for IL‐17F production. Moreover, IL‐17F impairs endothelium‐dependent vascular relaxation, a function previously unknown that would contribute to vascular pathology in SHR.

Support or Funding Information

NIH Program Project Grant to FMA (HL 14388) VA Merit Review Award to MWC (1I01BX001414), and AHA Innovative Research Grant to MVS (16IRG27260323).
When undergraduate research becomes the curriculum
Authors: Dolan Erin
Published: November 2023

National calls to improve undergraduate STEM education have emphasized the importance of undergraduate research experiences. Course‐based Undergraduate Research Experiences, or CUREs, involve groups of students in addressing research problems or questions in the context of a class, and have been proposed as scalable ways of involving undergraduates in research. This presentation will offer a definition of CUREs, describe what makes them distinctive from other learning experiences, and highlight results from the Freshman Research Initiative at the University of Texas Austin as a unique and highly impactful CURE model.

Support or Funding Information

This work was supported by a grant from the National Science Foundation (NSF DBI‐1061874) and two grants from the Howard Hughes Medical Institute (HHMI #52005907 and #52006958). The contents of the presentation are solely the responsibility of the author and do not necessarily represent the official views of the NSF or HHMI.
TREATMENT WITH PROLYL HYDROXYLASE INHIBITOR REVERSES PRE‐EXISTING DIASTOLIC DYSFUNCTION IN THE AGED SIRT3 KNOCKOUT MICE
Authors: He Xiaochen , Zeng Heng , Chen Jian‐xiong
Published: November 2023

SIRT3 declines with aging and ablation of SIRT3 accelerates the development of cardiovascular diseases. Diastolic dysfunction is emerging as a leading cause of heart failure in aging population and diabetes. However, there is no effective treatment for diastolic dysfunction in the patients with heart failure. In the present study, we investigated the role of SIRT3 in the development of diastolic dysfunction and explored its potential mechanisms. We found that deletion of SIRT3 resulted in impairment of diastolic function, as evidenced by increased isovolumic relaxation time, myocardial performance index, and E/E′ ratio when compared to WT mice. We further investigated the molecular basis by which ablation of SIRT3 causes diastolic dysfunction in mice. Extracellular flux analysis indicated that SIRT3 deficient endothelial cells (ECs) exhibited higher mitochondrial oxygen consumption rate (OCR) than that of WT ECs. We also found that knockout of SIRT3 led to a significant increase in the expression of prolyl hydroxylase domain‐containing protein (PHD) 1 and 2, but a decrease in the expression of PHD3. We then investigated the involvement of PHD in the development of diastolic dysfunction by treatment of SIRT3 KO mice with PHD inhibitor, dimethyloxalylglycine (DMOG). Treatment with DMOG (25 mg/kg/day, i.p.) for 2 weeks increased the expression of hypoxia‐inducible factor (HIF)‐1α in the endothelium of coronary arteries. This was accompanied by a significant increase in coronary flow reserve, indicating improvement of coronary microvascular function. Intriguingly, treatment with DMOG reduced mitochondrial OCR in SIRT3 KO ECs and completely reversed the pre‐existing diastolic dysfunction in the SIRT3 KO mice. Our data demonstrates that disruption of the SIRT3/PHD/HIF signaling may contribute to the impairment of mitochondrial homeostasis and diastolic dysfunction in the SIRT3 KO mice. The present study provides a novel therapeutic approach for treatment of patients with diastolic dysfunction.

Support or Funding Information

This study was supported by National Institutes of Health RO1 HL102042 (Chen).
MitoQuinone Rescues Aortic Endothelial Function mediated through eNOS/NO pathway
Authors: Pung Yuh‐Fen , Kmetz John , Bratz Ian , Murphy Michael P , Smith Robin AJ , Chilian William M
Published: November 2023

Although mitochondrial oxidative stress is known to occur in many models of cardiovascular disease, whether the mitochondrial stressor alone is sufficient to induce endothelial dysfunction, and the underlying mechanisms are unknown. Accordingly, Wistar Kyoto rats (WKY) were divided into 3 groups: Control; Rotenone (Rot): mitochondrial complex I inhibitor infused through Alzet pumps at 1.5 mg/kg, subcutaneously, to induce mitochondrial oxidative stress; MitoQ+Rot: mitochondrial targeted antioxidant, MitoQ, at 50 μM in drinking water to scavenge mitochondrial oxidative stress with Rot for 12 to 14 days. Aortae were collected from Control (n=7), Rot (n=6) and Rot+MitoQ (n=3) groups for functional and molecular analyses. Aortae from controls showed ~53.3% relaxation (isometric vascular ring preparation) in response to acetylcholine (ACh), Rot prevented ACh‐induced dilation suggesting endothelial dysfunction. MitoQ partially reversed the inhibitory effect of Rot (~35.8% vasodilation to ACh) suggesting that mitochondrial oxidative stress is central to this effect. Interestingly, relaxation to sodium nitroprusside (SNP) was greater in the Rot group (~55.1%), than in Control or MitoQ+Rot groups, suggesting that smooth muscle function is not compromised by mitochondrial oxidative stress. To delineate the underlying mechanism(s), enriched aortic ECs fractions were isolated from the groups. To our surprise, activation of eNOS (phosphorylation at s1177) was higher in ECs from Rot, than in Controls and Rot+MitoQ groups (ratio of p‐eNOS to total eNOS: 0.71±0.21 vs 0.44±0.08 and 0.45±0.17, respectively). Similarly, SOD‐2 expression (normalized to porin) was augmented in Rot group, suggesting a possible compensatory mechanism to scavenge excessive mitochondrial superoxide production (Rot: 4.25±0.78, Control: 2.98±0.62 and Rot+MitoQ: 1.38±0.03). Both functional and molecular data imply that mitochondrial oxidative stress uncouples eNOS (activation of eNOS with endothelial dysfunction) and scavenging mitochondrial oxidative stress rescues endothelial function. Collectively our study raises the possibility for mitochondrial therapies as a treatment for vascular disease.

Support or Funding Information

This work was supported by NIH Grants HL32788, R01 83366, RC1HL100828 (to WMC), American Heart Association Post‐doctoral Fellowship 09POST2290021 and Malaysia Ministry of Higher Education Grant FRGS/1/2015/SKK08/UNIM/03/2 (to YFP).