bluuebunny/crossref_metadata_2025 huggingface dataset:
Angiotensin ll Type 2 (AT2) Receptors: Novel Target in Asthma
Authors: Kurade Mangesh , Patel Mehaben , Rajalingam Sahith , Siddiquee Armaan , Narke Deven , Ponnoth Dovenia S.
Published: June 2021

Angiotensin ll (Ang ll) is produced locally in the lungs and may potentiate airway responsiveness in asthmatic patients. Ang ll induces its effects by activating two GPCR subtypes: AT1 and AT2. This lab has previously found that AT1 antagonist losartan reduces airway inflammation and reactivity. The AT2 receptors counterbalance the effects of AT1 and are known to be anti‐inflammatory. However, it is not known if AT2 receptors have any effects in asthma. We studied the effects of AT2 agonist novokinin and antagonist PD 12339 administered in vivo in a mouse model of asthma. Mice were divided into control (non‐allergic; CON) and allergen sensitized‐challenged (SEN) groups, and were sensitized i.p. on days 1, 6 with 0.2μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11–13. Whole body plethysmography (measuring airway responsiveness as enhanced pause, Penh) and bronchoalveolar lavage (BAL) studies were performed. Methacholine (MCh; 48mg/ml) produced significantly higher airway responsiveness in allergic mice compared to control (563.71±40% in SEN vs. 208.79±20.84% in CON, p<0.05). However, novokinin (i.p. 0.3mg/kg) treated SEN mice had significantly attenuated MCh‐indcued airway responsiveness (563.71±40% in SEN vs. 252±11% in SEN+Novokinin, p<0.001). The AT2 antagonist PD 12339 (i.p.5mg/kg) significantly increased MCh airway responsiveness (563.71±40% in SEN vs. 757±30% in SEN+PD 12239, p<0.05). Differential BAL cell analysis showed novokinin also decreased eosinophils (221±21 in SEN vs. 122±18 in SEN+PD 12239, p<0.001), while PD 12239 had no effect on eosinophils (245± 16 cells in SEN+PD 12339). Our data suggests that activation of AT2 receptors may attenuate airway reactivity and inflammation in our model of asthma.

Support or Funding Information

Long Island University start‐up funds (DSP)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Theoretical Considerations of Novel Nanocarbon Onion‐like Fullerene Material (NOLF) in Mitochondrial Quantum Processes, Mediation of Disruptive Perturbation, and Maintenance of Intracellular Quantum Electrodynamics
Authors: Bourassa Daniel J. , Tulp Orien L. , Einstein George P.
Published: June 2021

Medical research into fullerenes, though still currently in its early phases of discovery, has revealed novel applications due to their unique characteristics and compatibility with living cells. This has produced some exciting and potentially promising medical applications. Research supports evidence of enhanced ROS protection, anti‐inflammatory action, HIV protease inhibition, mitochondrial SOD mimetic, and possible longevity benefits among other uses. Fullerenes are increasingly being modified or functionalized as transporter molecules, phototherapy agents, and even considered for primary treatment options in a wide array of diseases, including cancer. However, there is much we don't know. The six‐carbon aromatic ring is among the most electrodynamically stable carbon structures and essential to the known biochemistry of life due to dense pi cloud properties. All fullerenes are composed largely of aromatic six carbon rings. Nanocarbon onion‐like fullerene material (NOLF) all have the basic structure C60n2 and derive most of their electrodynamic properties from their outer two shells. This creates a very large and diffuse but dense pi cloud that seems to share C60s biological compatibility and benefits but potentially offers more due to multi‐shell electrodynamics, diamagnetism, and increased capacitance characteristics. These mechanisms help explain how NOLF assists mitochondria to resist disruptive perturbations and restore homeostasis in disease states as well as maintain and regulate it in health. With interest increasing in the cellular electrodynamics, ultra‐weak photonic emission, electromagnetic resonance, and the role of the quantum cell in health and disease; the electrodynamic properties of NOLF and observed biological effects merit greater attention. We present a theoretical basis, derived from current research, for the influence of NOLF in the enhancement of mitochondrial quantum processes, mediation of disruptive perturbation, and assistance in maintenance of intracellular quantum electrodynamics in chronic degenerative disease and cancer, and suggest future research to investigate these proposed quantum effects in mitochondria and cells.

Support or Funding Information

Support from Institutional Resolurces

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
BPC 157 Rescues NO‐System in Perforated Stomach and Caecum
Authors: Drmic Domagoj , Vidovic Tinka , Vlainic Josipa , Samara Mariam , Gojkovic Slaven , Krezic Ivan , Jeyakumar Jerusha , Bilic Zdenko , Seiwerth Sven , Sikiric Predrag
Published: June 2021

Aim

We focused on perforated defect in stomach and caecum, and NO‐tissue levels in rats and the therapy with the stable gastric pentadecapeptide BPC 157. Effective in perforated stomach lesion and perforated caecum lesion as well as ischemic colitis, ischemia and reperfusion, BPC 157 rapidly activates vessels “running” toward the perforated defect (stomach perforation (Gastroenterology 2017; 152, Issue 5, Supplement 1, S889–S890; Gastroenterology 2017; 152, Issue 5, Supplement 1, S890–S891) or to bypass vessel obstruction (ischemic colitis)(World Journal of Gastroenterology, in press, 2017) and largely attenuates lesions course.

Methods

At 15 minutes post‐injury, we determined nitric oxide (NO) levels in stomach caecum tissue samples using the Griess reaction (Griess Reagent System, Promega, USA). Sulfanilamide was added to the homogenized tissue, incubated and then N‐1‐naphthylethylenediamine dihydrochloride was added. The Griess reaction is based on diazotization reaction in which acidified nitrite reacts with diazonium ions and in a further step are coupled to N‐1‐naphthylethylenediamine dihydrochloride forming chromophoric azo derivate. Absorbance was measured at 540 nm, using sodium nitrite solution as standard. NO levels are reported in μmol/mg protein. Proteins were determined using the commercial kit (BioRad Protein DR Assay Reagent Kit, USA). Medication was bath (BPC 157 10 ug, 10 ng/kg/1ml bath/rat; equivolume saline (controls)) at the ligated perforated injury, in stomach or caecum, at 1 min after perforated injury induction.

Results

Stomach. In the early term, we noted fewer NO‐levels in rats underwent stomach perforation, but a reversal with BPC 157 administration, and thereby, NO levels like in the healthy appear in rats with perforated stomach underwent BPC 157 medication (Fig. 1). Caecum. Likewise, NO level assessment demonstrated a drop in level in rats which underwent caecum perforation, but the NO values in rats with perforated caecum which underwent BPC 157 medication were similar to the NO‐levels in healthy rats (Fig. 2).

Conclusions

Rapid activation of vessels “running” toward the perforated defect (perforated defect healing and attenuated bleeding, counteraction of free radicals formation previously described by BPC 157 application, occurs with the parallel rescue of the NO‐system in the both of the perforated tissues (stomach perforation (Gastroenterology 2017; 152, Issue 5, Supplement 1, S889–S890; caecum perforation (Gastroenterology 2017; 152, Issue 5, Supplement 1, S890–S891)).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Caustic Lesions of Esophagus in Rats and Therapy with Stable Gastric Pentadecapeptide BPC 157
Authors: Peklic Marina , Drmic Domagoj , Pavlov Katarina Horvat , Blagaic Alenka Boban , Seiwerth Sven , Sikiric Predrag
Published: June 2021

Aim

We focused on caustic lesions of the esophagus in rat and therapy with the stable gastric pentadecapeptide BPC 157. Caustic lesions of the esophagus are one of the life‐threatening morbidities after ingestion of acid or alkali, as an accidental ingestion affecting mostly children. BPC 157 is an anti‐ulcer peptide, stable in human gastric juice, and shown as a novel mediator of Robert's cytoprotection with the beneficial effect in the whole gastrointestinal tract (Curr Pharm Des. 2017;23(27):4012–4028; Curr Pharm Des. 2014;20(7):1126–35; Curr Pharm Des. 2010;16(10):1224–34). Providing a direct cytoprotective effect of BPC 157 therapy on esophagus as well (Curr Pharm Des. 2010;16(10):1224–34), we assume its beneficial effect when given after caustic substance ingestion.

Methods

Deeply anesthetized Albino Wistar rats, 200 g b.w., received directly 0.5 mL of the caustic substance (10% NaOH or 20% HCl respectively) at the proximal part of the esophagus with the free passage to the stomach, and immediately thereafter, BPC 157 10 μg/kg or saline (5 mL/kg) intraperitoneally. Assessment (macro/microscopy), was at the sacrifice, at 1, 2, 5 and 10 minutes. At 1 and 10 minutes, in proximal, middle and distal esophagus tissue samples, oxidative stress assessment was by thiobarbituric acid (TBA) reactivity as malondialdehyde equivalents (MDA). Likewise, at 1 and 10 minutes post‐injury, we determined nitric oxide (NO) levels in proximal, middle and distal esophagus tissue samples using the Griess reaction (Griess Reagent System, Promega, USA).

Results

Gross assessment at all time intervals revealed a marked reduction of the lesions induced by both caustics in rats that received BPC 157 therapy. Microscopic examination was along with macroscopic evaluation. Thus, BPC 157‐rats exhibited markedly less damage in the proximal, middle and distal esophagus when challenged with caustics. Of note, in addition to the extensive esophageal caustic lesions, controls exhibited a huge increase of the oxidative stress and NO‐levels following caustic application. By contrast, rats damaged by caustic that subsequently received BPC 157 therapy had markedly less MDA and NO‐tissue level, even close to those values noted in the healthy tissue samples.

Conclusion

BPC 157 has a cytoprotective effect on the esophagus. Likewise, of particular importance is the evidence that this beneficial effect is obtained when the BPC 157 therapy is given after caustic ingestion. Thus, a practical importance of these findings should be emphasized.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
D‐Ribose Induced NLRP3 Inflammasome Activation in Podocytes by Increase in Advanced Glycation End‐Products
Authors: Hong Jinni , Zhang Qinghua , Li Guangbi , Ritter Joseph , Li Weiwei , Wang Xuemei , Li Pinlan
Published: June 2021

Recent studies have demonstrated that D‐ribose levels are increased in patients with type II diabetes mellitus and that increased blood D‐ribose levels are involved in the development of diabetic complications such as diabetic encephalopathy and nephropathy. However, the mechanisms mediating the pathogenic role of D‐ribose remain poorly understood. In the present study, we treated podocytes with different concentrations of D‐ribose and examined its potential effects on NLRP3 inflammasome activation and podocyte function. Using confocal microscopy, D‐ribose was found to markedly increase the co‐localization of caspase‐1 and NLRP3 by 1.8 folds compared to control, which suggests enhanced NLRP3 inflammasome formation. By ELISA, we demonstrated that in podocytes treated with D‐ribose, a major product of NLRP3 inflammasome‐IL‐1β was significantly increased by 2.05 folds, indicating the activation of NLRP3 inflammasomes. Western blot analysis further confirmed that D‐ribose increased the level of active caspase‐1 in podocytes by 2.09 folds, another parameter for activation of NLRP3 inflammasomes, which was substantially attenuated by caspase‐1 inhibitor, YvAD and ROS scavenger, NAC, respectively. In addition, D‐ribose‐induced NLRP3 inflammasome activation could be blocked by inhibitor of advanced glycation end‐products (AGEs) formation and their chemical breaker or receptor antagonist, aminoguanidine, aylthiazolium chloride and RAGE siRAGE by 43%, 50% and 73%, respectively. These results suggest that D‐ribose induces NLRP3 inflammasome activation in podocytes in an AGE‐dependent manner, which may be a triggering mechanism of diabetic nephropathy.

Support or Funding Information

Supported by NIH grants DK54927 and HL075316 to Pinlan Li, NSFC grants 81573763 to Weiwei Li, and NSFC grants 81530099 to Xuemei Wang

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential inactivation mechanism and covalent adduct formation of ALDH1A1 and ALDH1A2 by WIN18,446
Authors: Zhong Guo , Zelter Alex , Davis Trisha N. , Hoopmann Michael R. , Mortiz Robert L. , MacCoss Michael , Nelson Wendel L. , Isoherranen Nina
Published: June 2021

WIN18,446 [N,N′‐1,8‐octamethylenebis‐(2,2‐dichloroacetamide)] was discovered as a reversible male contraceptive, which inhibits spermatogenesis in many species, including humans. The contraceptive activity of WIN18,446 is believed to be due to inhibition of the biosynthesis of the critical regulator of spermatogenesis, intratesticular all‐trans‐retinoic acid (atRA), via inhibiting aldehyde dehydrogenase 1A1 (ALDH1A1) and ALDH1A2. However, the usefulness of WIN18,446 is limited due to its off‐target inhibition of ALDH2, the key enzyme eliminating acetaldehyde. Previous studies have shown that in the presence of NAD+, WIN18,446 is a reversible inhibitor of human ALDH1A1, and a time‐dependent, irreversible inhibitor of ALDH1A2. However, neither the mechanism of ALDH1A2 inactivation by WIN18,446 nor the discrepancy between ALDH1A1 and 1A2 inhibition is understood. Using Q‐TOF MS, we identified an aldehyde metabolite of WIN18,446 formed by ALDH1A2, in the presence of cofactor NAD+. In contrast, the aldehyde metabolite was formed by ALDH1A1 only in the absence of NAD+. Similarly, the inactivation of ALDH1A2 by WIN18,446 was NAD+ dependent while ALDH1A1 was only inactivated in the absence of NAD+. Adducts of WIN18,446 with ALDH1A2 were identified with shotgun proteomics and a modified version of Kojak software that allows searching of any mass modified peptides. Using the proteomic MS data, a novel WIN18,446 derived adduct of Cys320 and Cys319 of ALDH1A2 was identified with mass change of 295Da. Based on the identity of this adduct, a mechanism of ALDH1A2 inactivation by WIN18,446 and the pathway to the aldehyde metabolite was proposed (Fig. 1). Interestingly, the proposed mechanism of inactivation does not require NAD+ suggesting that NAD+ acts as an allosteric modulator affecting WIN18,446 binding orientation within the ALDH1A active site instead of contributing to the catalysis. This interpretation is consistent with existing knowledge of ALDH family enzyme structure‐function. In conclusion, using novel MS proteomics methods we have identified the inactivation mechanism of ALDH1A by WIN18,446, which provides important information to guide development of novel male contraceptive inhibitors of ALDH1A enzymes and to assess structure‐function of ALDH1A inhibitors.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pharmacological Evaluation of Novel Chloroquine Analogues in Breast Cancer Cells
Authors: Cao Peter , Patel Dhvani , Ventura Dominic , Heakal Yasser
Published: June 2021

Targeting autophagy, a conserved cellular process required for the degradation and recycling of proteins and cellular organelles, has emerged as a potential therapeutic approach for cancer treatment. Although, its role in cancer progression remains controversial, autophagy inhibition has been shown to be potentially beneficial in pre‐clinical studies. Constitutively active autophagy due to cellular stress has been implicated in tumor progression and therapeutic resistance. Pharmacological inhibition of autophagy using chloroquine, an approved antimalarial agent, has been investigated in preclinical cancer models and is currently being investigated in several ongoing clinical trials. However, several problems may prevent the translation of preclinical findings into the clinic such as the potential adverse events associated with chloroquine. Additionally, most preclinical studies utilize high chloroquine concentrations to inhibit autophagy which may not be achieved clinically. Therefore, we hypothesized that the design and synthesis of novel chloroquine polyamine conjugates may result in compounds that inhibit autophagy in breast cancer cells at lower concentrations due to enhanced cellular uptake through the polyamine transporter. Assessment of the biological activity of the chloroquine analogs using MTT assay demonstrates the chloroquine polyamine conjugates induce cell death at lower concentrations relative to chloroquine. Additionally assessment of the ability of these compounds to inhibits autophagy indicate that they inhibit autophagy at lower concentrations as indicated by the accumulation of LC3B‐II and p62, a protein known to undergo autophagy‐dependent degradation. Future studies will aim at investigating the mechanism of the enhanced cytotoxic activity of these compounds.

Support or Funding Information

D'Youville College and D'Youville College School of Pharmacy

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PAR1 and p38 MAPK Regulation of Endothelial Pro‐Inflammatory Responses
Authors: Rada Cara , Grimsey Neil , Canto Isabel , Mejia‐pena Hilda , Trejo JoAnn
Published: June 2021

Endothelial cells line the lumen of blood vessels and create a semi‐permeable barrier that is disrupted during vascular inflammation resulting in increased permeability, tissue edema, and subsequent organ failure. Endothelial dysfunction also results in cytokine production and leukocyte recruitment. However, the molecular mechanisms behind this pro‐inflammatory state remains to be elucidated. Protease‐activated receptor‐1 (PAR1) is a G protein‐coupled receptor for the coagulant protease thrombin and mediates hemostasis, thrombosis, and inflammatory response to vascular injury. However, the distinct mechanism of how endothelial PAR1 promotes vascular pro‐inflammatory responses represents a major gap in knowledge. We recently discovered that thrombin activation of PAR1 stimulates p38 MAPK activation through a non‐canonical pathway mediated by TAB1 to promote endothelial permeability. PAR1‐induced p38 activation has been linked to vascular barrier disruption; however, the mechanism as to how p38 may be integrated into major signaling pathways that promote pro‐inflammatory responses is not known. We found that thrombin‐stimulated p38 signals independent of the classic g‐protein pathways of RhoA activation and MLC phosphorylation to induce endothelial barrier permeability. Instead, thrombin‐stimulated p38 appears to be signaling through phosphorylation of the MK2 and HSP27. We further show MK2 activity is PAR1 and p38 dependent and regulates differential phosphorylation of its direct substrate, HSP27. Additionally we show HSP27 activity is p38 and PAR1‐dependent. The p38‐MK2‐HSP27 signaling pathway also appears to modulate HUVEC monolayer permeability. These studies show thrombin‐induced p38 signals through MK2 and HSP27 cascade to regulate endothelial barrier permeability.

Support or Funding Information

T32 Pharmocological Sciences Training Grant (NIGMS T32 GM007752) (C. Rada) NIH NIGM R01 GM116597 (J. Trejo)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Novel PRKAG2 Mutation (K475E): Early‐Onset Cardiac Phenotype and Targeted Therapy
Authors: XU YANCHUN , Liu Hong , Phornphutkul Chanika , Hardie Grahame , Dudley Samuel C. , Padbury James F. , Tseng Yi‐Tang
Published: June 2021

Background

Mutations in PRKAG2 have been implicated in hypertrophic cardiomyopathy (HCM). We identified a novel PRKAG2 mutation, K475E, in a neonate with prenatal onset of HCM noted on a 27‐week prenatal ultrasound. We have demonstrated in vitro that K475E mutation led to increases in cardiomyocytes size and area which can be reversed by rapamycin.

Objective

To characterize K475E‐induced cardiac phenotype in vivo, we developed two new transgenic mouse lines with cardiac‐specific expression of the human wild type (TgWT) or K475E mutant (TgK475E) PRKAG2. The goal of this study was to explore the effectiveness of rapamycin for targeted therapy for PRKAG2 mutation‐induced HCM.

Methods

Human WT and K475E DNA constructs were inserted into a pBluescript vector with the mouse α‐myosin heavy chain promoter. Transgenic mice were established by pronucleus microinjection. Rapamycin was treated from two‐week to six‐aged mice. Cardiac hypertrophy was examined and cardiac function changes were monitored by echocardiography.

Results

Hypertrophy in TgK475E mice was confirmed by their significant increases in heart weight/body weight and heart weight/tibial length ratios versus the non‐transgenic control and TgWT mice. Histology revealed large vacuoles and high glycogen content in TgK475E myocytes throughout the ventricles. Echocardiography confirmed that LV mass dramatically increased in TgK475E mice starting from 5 weeks of age. Rapamycin treatment dramatically decreased 1) the ratios of heart weight/body weight and heart weight/tibia length and 2) glycogen overload in TgK475E mice. Moreover, K475E mutation caused inhibition of AMPK which was rectified by rapamycin treatment.

Conclusion

These results indicate the transgenic mouse model reflects the early onset phenotype of the K475E mutation‐induced HCM. The mTOR signaling pathway is involved in K475E induced HCM. Our study suggests that mTOR inhibition strategy as targeted therapy for PRKAG2 induced HCM is effective and should be studied further.

Support or Funding Information

1. NIH P30GM114750 2. Oh‐Zopfi Research Award, Women & Infant's Hospital, RI.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anti‐inflammatory effect of a new formulation: Palmitoylethanolamide/Baicalein in a murine model of Myocardial Ischaemia/Reperfusion Injury
Authors: Cuzzocrea Salvatore , D'Amico Ramona , Fusco Roberta , Gugliandolo Enrico , Crupi Rosalia , Impellizzeri Daniela , Di Paola Rosanna
Published: June 2021

Myocardial ischemia/reperfusion (I/R) injury is the principal cause of death, happens after prolonged occlusion of the coronary arteries. The first intervention to limit myocardial damage is directed toward restoration of perfusion as soon as possible, however infarction triggers a powerful inflammatory response and a significant oxidative stress.

Palmitoylethanolamide (PEA), is a well‐known fatty acid amide‐signaling molecule that possess an important anti‐inflammatory and analgesic effects. Nevertheless, PEA does not possess the ability to prevent free radicals formation. Baicalein, a bioactive component isolated from a Chinese herbal medicine, has multiple pharmacological activities, such as a strong anti‐oxidative effects. A combination of PEA and Baicalein could have beneficial effects on oxidative stress produced by inflammatory response. In the present study we investigated the effects of composite containing PEA and Baicalein in a model of myocardial I/R injury. Myocardial ischemia/reperfusion injury was induced by occlusion of the left anterior descending coronary artery for 30 min followed by 2 h of reperfusion. PEA‐Baicalein (10:1), was administered (10 mg/kg) 5 min before the end of ischemia and 1 h after reperfusion. In this study, we clearly demonstrated that PEA‐Baicalein treatment reduces myocardial tissue injury, neutrophils infiltration, markers for mast cell activation expression such as chymase and tryptase and pro‐inflammatory cytokines production (TNF‐α, IL‐1β). Moreover, PEA‐Baicalein treatment reduces nitrotyrosine and PAR formation, inhibits NF‐kB nuclear translocation and modulates apoptosis pathways.

Accordingly, these results support the idea that the association between PEA and Baicalein should be further studied as a potent candidate for the treatment of myocardial I/R injury.

Support or Funding Information

no funding

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
AT1R Dependence of Proangiogenic Ang‐(1–7)/Mas Signaling in Endothelial Cells and Endothelial Progenitors
Authors: Exner Eric Charles , Stodola Timothy J. , Hoffmann Brian R. , Greene Andrew S.
Published: June 2021

The renin‐angiotensin system (RAS) is a critical regulator of microvascular function and cardiovascular risk. Success of clinical interventions that interfere with the signaling of angiotensin II (AngII) at the angiotensin II receptor type 1 (AT1R) suggested signaling via AT1R is predominantly associated with pathology; however, recent studies have reaffirmed that AngII/AT1R signaling has a key role in maintaining vascular homeostasis. Angiotensin‐(1–7) (Ang‐(1–7)) is a RAS effector peptide that counteracts many effects of pathologically high AngII. In a previous study, we observed that at low doses, both AngII/AT1R and Ang‐(1–7) signaling through the receptor Mas restore angiogenesis in a low renin model of microvascular dysfunction. We have also observed beneficial effects of low dose AngII on endothelial progenitor cell (EPC) function. While competitive inhibition of ligand binding at the converse receptor did not abolish the effects of each peptide, AT1R was identified as a component of the Ang‐(1–7)/Mas signaling complex. It has been suggested that AT1R and Mas form heterodimers based on data showing rescued function of mutant AT1R upon coexpression of Mas. Here, we test the hypothesis that Ang‐(1–7) has proangiogenic effects in both endothelial cells (ECs) and EPCs that is dependent upon the presence of AT1R. In the Dahl salt‐sensitive (SS) rat, a low‐renin model of microvascular dysfunction, we confirm the beneficial effects of low‐dose Ang‐(1–7) on angiogenesis and observe Ang‐(1–7) mediated restoration of EPC function. In an AT1R knockout on the SS background, the ability of Ang‐(1–7) to restore angiogenesis and EPC function is lost. In addition, in vitro analogues of angiogenic capability recapitulate this phenotype in both ECs and EPCs. Using qPCR arrays and mass‐spectrometry based proteomics, we find both the transcriptional response and the Ang‐(1–7)/Mas signaling complex to be altered in the absence of AT1R. These results support the hypothesis that AT1R is a key component of the Ang‐(1–7)/Mas signaling pathway and further support the previous hypothesis by others and ourselves that AT1R and Mas heterodimerize. Computational models of AT1R and Mas have been constructed to examine the potential mechanism of AT1R and Mas heterodimerization, with biological confirmation to follow.

Support or Funding Information

This work was supported by NHLBI grant F30HL131153.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Neoadjuvant Chemotherapy Decreases Angiogenesis Potential and Microvascular Function in Human Breast Cancer Patients
Authors: Norwood Toro Laura E. , Linn Jasmine , Hockenberry Joseph C. , Kong Amanda L. , Flister Michael J. , Beyer Andreas M.
Published: June 2021

Rational

Chemotherapy (CT) is a necessary treatment to prevent the growth and survival of cancer cells; however, CT is well established to have adverse impact on the cardiovascular (CV) system even years after cessation of treatment. Much work has been dedicated to studying the effects of CT drugs on the tumor vasculature, but little evidence exists showing the effects on the host microcirculation. Microvascular (MV) dysfunction is a known early indicator of numerous CV disease phenotypes, including heart failure. The goal of this study was to evaluate whether previous CT treatment has any lasting effect on the human MV.

Methods

To study the effect of CT on the MV, flow mediated dilation (FMD) was evaluated in freshly isolated adipose microvessels from cancer patients with and without previous CT. Angiogenic potential was assessed using matrigel plug assays with samples isolated from human adipose. Effect of CT on endothelial cell (EC) and cancer cell survival was determined using MTT; EC permeability was established using FITC‐dextran in transwells with a EC monolayer.

Results

In cultured cells doxorubicin (dox; 100 nM) causes a decrease in MTT in human cancer, but not EC. Ex vivo treatment of the vessels with dox reduced nitric oxide (NO) mediated dilation by 90% compared to untreated controls (A). Similarly, MV FMD was severely decreased in vessels from patients with neoadjuvant CT (cessation of therapy ~ four weeks prior) compared to untreated control subjects (B). Smooth muscle dependent dilation to papaverine was not affected in either study (C). Using an ex vivo matrigel assay, we found a significant decrease in angiogenesis potential in samples treated with dox (D). In line with these findings, dox causes a decrease in EC cell permeability, suggesting a potential contributing mechanism.

Conclusions

Dox in clinically relevant doses causes increased lethality in cancer cells versus EC, but induces MV dysfunction, decreased angiogenic potential, and elevated EC permeability. Our studies show for the first time the effect of dox on human MV function and may lead to ways to prevent adverse CV side effects from CT without affecting cancer cell lethality.

Support or Funding Information

This work was supported by NIH R01 HL133029, We Care Foundation Grant; MCW‐Cardiovascular Center Pre‐PPG grant, Advancing a Healthier Wisconsin – Redox Biology 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.
Enhanced epithelial sodium channel signaling in endothelial cells promotes arterial stiffness
Authors: Jia Guanghong , Aroor Annayya R. , Hill Michael A. , Yan Yang , Habibi Javad , Manrique Camila , Lastra Guido , Whaley‐Connell Adam , Jaisser Frederic , Sowers James R.
Published: June 2021

Excessive epithelial sodium channel (ENaC) activation in endothelial cells (EnNaC) has been reported to increase oxidative stress and induce inflammation with associated cardiovascular abnormalities. Our previous research has demonstrated that global inhibition of ENaC signaling with amiloride prevented Western diet (WD)‐induced inflammation, fibrosis, endothelial dysfunction and vascular stiffness in mesenteric and coronary resistance arteries and aorta. However, the role of endothelium specific EnNaC activation in the pathogenesis of vascular stiffness has not been investigated. We posited that EnNaC activation promotes obesity‐ and mineralocorticoid receptor‐mediated vascular stiffness. Accordingly, we sought to determine if endothelium specific EnNaC alpha subunit KO would prevent vascular inflammation, vascular stiffness, and impairment of endothelium dependent relaxation. Female EnNaC and wild‐type littermate mice were fed with WD high in refined carbohydrates and saturated fat or a standard chow diet for 12 weeks. In a separate study, female mice were treated with aldosterone (250 μg/kg/day) via osmotic minipumps for 3 weeks. Selective deletion of EnNaC significantly mitigated WD‐induced ex vivo endothelial stiffness measured by atomic force microscopy and – impaired aortic endothelium dependent relaxation in response to acetylcholine (10−9–10−4 mol/L). In addition to reducing mineralocorticoid receptor mediated endothelial stiffness, EnNaC KO also mitigated aldosterone‐induced inward Na+ currents and EnNaC activity in isolated endothelial cells. Furthermore, EnNaC KO attenuated aldosterone‐induced endothelium permeability, vascular inflammation, endothelial stiffness, as well as aortic endothelium dependent relaxation. These data indicate that endothelial specific EnNaC activation mediates vascular endothelium dysfunction and prompts vascular stiffening.

Support or Funding Information

JRS received funding from NIH (R01 HL73101‐01A and R01 HL107910‐01) and the Veterans Affairs Merit System (0018). Dr. Jia received funding from American Diabetes Association (Innovative Basic Science Award #1‐17‐IBS‐201).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Treating the Network: Application of a Combination of Two microRNA Inhibitors to the Brainstem Prevents Hypertension Development in the Spontaneously Hypertensive Rat
Authors: Gorky Jon , DeCicco Danielle , Achanta Sirisha , Schwaber James , Vadigepalli Rajanikanth
Published: June 2021

We here test the concept that a disturbed function or disease state may result not from a single cause but from small changes in a network that are collectively significant, and that treatment might aim to correct these collective changes. We recently showed that development of hypertension (HTN) in the spontaneously hypertensive rat (SHR) model of human essential hypertension is accompanied by small changes in gene expression within a gene regulatory network, and that these changes potentially result from changes in microRNA expression levels tracking the development of HTN. These results led to the hypothesis that preventing the change in microRNA levels might prevent the development of HTN. We propose that hypertension is an emergent property of a network that has been pushed out of a normotensive equilibrium into a compensatory, yet ultimately pathological, state. In this work, we show that small perturbations in the gene regulatory networks in the brainstem by selectively blocking two microRNAs is sufficient to prevent development of hypertension in the SHR model. Furthermore, this effect appears driven by only modest changes in the expression of rate‐limiting genes, some of which are targets of these miRNAs, suggesting that the combination of genes that are targeted in the network is responsible for the effect rather than just one gene or another. While the use of anti‐sense oligonucleotides to treat hypertension is itself novel, the demonstration that hypertension is an emergent property an underlying network suggests that a new treatment paradigm altogether is needed.

Support or Funding Information

Funding Sources: T32 AA007463‐28; U01 HL133360; R01 HL111621; SPARC‐OT2‐OD023848

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Palmitic Acid Induces Extracellular Vesicle Release in Human Renal Proximal Tubule Epithelial Cells
Authors: Cobbs Alyssa , Muthusamy Saravanakumar , Chen Xiaoming , Sims Joya , Chatterjee Radhika , Zhao Xueying
Published: June 2021

Lipotoxicity, an accumulation of intracellular lipid metabolites, has been proposed as an important pathogenic mechanism leading to kidney dysfunction in the context of metabolic disease. Palmitate, a predominant lipid derivative, can cause lipoapoptosis and the release of inflammatory extracellular vesicles (EVs) in hepatocytes. This study was aimed to investigate whether free fatty acid palmitate could increase EV release in renal tubular epithelial cells. A human proximal tubule epithelial cell line, HK‐2, was incubated with 1% bovine serum albumin (BSA), BSA‐conjugated palmitic acid (PA, 250 μM), BSA‐conjugated oleic acid (OA, 250 μM), or diluent (control) for 24 hours. The released EVs were isolated by ultracentrifugation, then characterized and quantified by immunoblot and nanoparticle‐tracking analysis (NTA). To examine the uptake of EVs by tubular cells, isolated EVs were further labelled and applied to HK‐2 cells. According to the NTA, the size distribution of HK‐2‐derived EVs was 30–200 nm with a similar mode size in all four groups. Interestingly, incubation of HK‐2 cells with 1%BSA alone significantly increased the release of EVs compared to the vehicle control. Moreover, BSA‐induced EV release was further increased by approximately two‐fold in the presence of PA, whereas EV production was not significantly changed by the addition of OA. PA (250 μM)‐enhanced EV release in HK‐2 is independent of cell apoptosis since apoptosis‐related proteins were not different between BSA and BSA‐PA groups. Both confocal microscopy and flow cytometry analyses confirmed that labeled lipid‐induced EVs were taken up by HK‐2 cells. Collectively, our results indicate that saturated fatty acid palmitate stimulates EV release from HK‐2 cells. Lipid‐induced EVs may serve as an untapped source to evaluate novel signaling pathways contributing to nephropathy progression.

Support or Funding Information

NIH Diabetes and Digestive Kidney Disease Grant Number: R25DK1136559

MSM Interdisciplinary Sleep/Clinical Cardiovascular Research Training Program (T32)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Reno‐protective effects of everolimus vs. bone marrow‐derived stem cells in rat model of glomerulonephritis
Authors: Zedan Mohamed M. , Mansour Ahmed K. , Bakr Ashraf A. , Sobh Mohamed A. , Baban Babak , Elmarakby Ahmed A.
Published: June 2021

Glomerular endothelial injury and effectiveness of glomerular endothelial repair play a crucial role in the progression of renal disease. Recent studies suggest that progenitor cells could be an effective tool to enhance glomerular endothelial repair. We hypothesize that bone marrow‐derived stem cells (BMDSCs) attenuate experimental glomerulonephritis by restoring glomerular endothelial barrier function through improving the repair process. Anti‐Thy1 glomerulonephritis was induced in male Sprague Dawley rats by a single injection of an antibody against Thy1, which is mainly expressed on glomerular mesangial cells. Rats were then treated with the immunosuppressant everolimus daily or injected with bone marrow‐derived endothelial progenitor cells after one week of injection of anti‐Thy1 (n=6–7). After nine days of injection of anti‐Thy1, glomerular albumin permeability and albuminuria were significantly increased when compared to control group (P< 0.05). Everolimus was superior to BMDSCs in attenuating the increase in glomerular albumin permeability and albuminuria in anti‐Thy1 injected rats. Only everolimus treatment significantly reduced the markers of glomerular injury, nephrinuria and podocalyxin excretion levels, in anti‐Thy1 injected rats (nephrinuria was 0.4± 0.1 vs. 0.8± 0.02 mg/day and podocalyxin excretion was 7± 1 vs. 11± 1 μg/day in everolimus treated vs. untreated anti‐Thy1 injected rats, P< 0.05). Injection of Anti‐Thy1 induced hypercellular glomeruli with loss of Bowman space and variable number of lymphocyte when compared to control. Everolimus treatment partially restored Anti‐Thy1 renal pathophysiological changes whereas BMDSCs had no significant effect. Everolimus treatment also reduced the number of renal T cells and macrophage and lowered IL‐17 and caspase 3 expression levels in the anti‐Thy1 injected rat. These data suggest that everolimus early treatment preserves glomerular endothelial barrier function in anti‐Thy1‐induced glomerulonephritis via anti‐apoptotic and anti‐inflammatory mechanisms.

Support or Funding Information

This work was supported by American Heart Association scientist development grant and intramural grant from Augusta University To Dr. Ahmed A. Elmarakby

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In‐Common And Unique Gene Expression Patterns In Acute Kidney Injury Of Different Aetiology Implicates MYC‐Pathway In Damage Progression
Authors: Hultström Michael , Jönsson Sofia , Agic Mediha Becirovic
Published: June 2021

Acute kidney injury (AKI) is a syndrome that may be caused by a number of different diseases. The existence of renal biomarkers that work for many kinds of AKI indicate that there may be an underlying common molecular pathway by which AKI causes progression of chronic kidney disease (CKD). By comparing six high‐quality microarray studies of renal gene expression after AKI in models of different diseases (gram negative sepsis, gram positive sepsis, ischemia‐reperfusion, malignant hypertension, rhabdomyolysis and cisplatin toxicity) a total of 5254 genes were differentially expressed in at least one of the AKI models. 66% we only found in one of the models showing that there are unique features to AKI depending on the precipitating disease. There were in‐common features in the form of four genes that were differentially expressed in all six models, 49 in at least five, and 215 were in‐common between at least four models. The in‐common genes included many of the known biomarkers and damage associated molecular patterns (DAMPs) indicating that these are an integral part of the molecular response to AKI of any etiology. The most connected gene in the network was MYC, which suggests that it may be a central regulator of renal gene expression in AKI. The outlining of this molecular network may be useful for understanding the progression from AKI to chronic kidney disease, but also for identifying new potential biomarkers.

Support or Funding Information

The study was supported by grants from the Swedish Heart and Lung Foundation, and the Swedish Society for Medical 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.
Metabolic acidosis inhibits AMPK function in kidney cells
Authors: Pastor‐Soler Nuria M. , Rajani Roshan , Mancino Valeria , Omi Kazuhiro , Hallows Kenneth R.
Published: June 2021

The metabolic sensor AMP‐activated protein kinase (AMPK) is stimulated by cellular metabolic depletion. We have shown that AMPK activation inhibits kidney membrane transport proteins. AMPK‐activated processes protect kidney cells from further metabolic depletion and damage. AMPK is tightly regulated, and its pharmacological activation can protect kidneys from superimposed injury, such as acute kidney injury (AKI). Conversely, AMPK is inhibited in chronic kidney disease (CKD). Therefore, pharmacologic AMPK activators are proposed as therapies to slow CKD progression. However, the mechanisms by which AMPK may promote kidney survival are unclear. CKD results in metabolic acidosis (MA) due to the inability of kidneys to excrete non‐volatile acid. MA, if untreated, has severe health sequelae such as worsening of kidney function. We hypothesized that there is cross‐talk between acid‐base status and AMPK signaling, and that dysregulation of AMPK function by MA disrupts kidney regeneration in the setting of CKD. We used kidney epithelial cell lines in culture and adult male C57BL/6 mice (WT) and AMPKβ1‐KO mice for our experiments. Cells were exposed to media at either pH 7.3–7.4 (control) or 6.9–7.0 (acidic) for 3 d ± an AMPK activator (last 16 h). We found that, as compared to cells grown at pH 7.4, kidney cells grown in acidic media have reduced baseline AMPK activity and blunted pharmacologic AMPK activation. We then evaluated changes in AMPK function in WT mice fed a normal diet vs. WT mice given 1.5% saccharin ± 0.28 M NH4Cl in drinking water for 2 mo. To evaluate the AMPK pathway in kidney tissue, we examined levels of phosphorylation of acetyl CoA carboxylase (pACC) by immunoblot. Mice on the acidogenic diet developed MA without significant weight loss compared to mice on the control diet (blood pH 7.2 vs 7.3 ± 0.01). In mice after UNX, the superimposed MA induced a statistically significant decrease in pACC, compared to mice with UNX on a control diet. We also evaluated differences in BUN in WT vs. AMPKβ1‐KO mice 3 mos. after sham surgery or UNX, a model for reduced kidney mass. Both WT and KO mice post‐UNX had significant worsening of kidney function (higher BUN) compared to sham‐operated mice of the same genotype. However, we did not observe any significant differences in BUN between WT and KO mice following sham or UNX. Our results point to worsening in kidney function post‐UNX and to a direct inhibition of the AMPK pathway in the setting of reduced kidney mass and MA. This AMPK inhibition may be detrimental to kidney regeneration in these settings.

Support or Funding Information

Keck School of Medicine of USC; USC/UKRO Kidney Research Center

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Aerobic exercise training prevents renal lipotoxicity associated with insulin resistance in mice
Authors: Muller Cynthia Rodrigues , Américo Anna Laura Viacava , Pereira Renata Oliveira , Matsumoto Alice Jackeline , Fiorino Patricia , Evangelista Fabiana Sant'Anna
Published: June 2021

Insulin resistance is associated with lipid accumulation in the kidney which leads to lipotoxicity, inflammation and renal damage. We investigated the potential of aerobic exercise training (AET) to prevent renal lipotoxicity in mice with diet‐induced insulin resistance. Adult male C57BL6/J mice were assigned into chow‐fed controls (C, n=5), cafeteria diet (CAF, n=5), chow‐fed trained (T, n=5), and cafeteria diet plus trained (CAFT, n=5). The standard chow diet contained 4% of kilocalories from fat, 55% from carbohydrate and 22% from proteins (Nuvilab®, Paraná, Brazil). The cafeteria diet contained 18.7% of kilocalories from fat, 56% from carbohydrate and 14.8% from proteins. AET was performed simultaneously with diet and consisted of 8‐wk running session of 60 min at 60% of maximal speed, 5 days/wk. Experimental procedures were approved by Ethics Committee from Faculty of Medicine of University of São Paulo (#18/2014). Cafeteria diet induced insulin resistance in CAF group compared to C group during insulin tolerance test, which was counteracted by AET in CAFT group. Histological analysis showed that CAF group increased intra‐renal lipid deposition (4.12 ± 0.48 %/area) compared to C group (1.70 ± 0.55 %/area), and that AET was able to prevent this increase in CAFT group (2.11 ± 0.51 %/area). Immunohistochemistry measurement revealed no difference in the expression of TGF‐β, Collagen IV and fibronectin. In the western blot analysis, CAF (2.12 ± 0.7 % vs. C) and CAFT (2.74 ± 0.5 % vs. C) groups showed a tendency (p=0.07) to increase the expression of IL‐6 compared to C group (1 ± 0.07 % vs. C). Furthermore, there was an increase in TNF‐α expression in the CAF (1.72 ± 0.05 % vs. C) and CAFT (1.46 ± 0.02 % vs. C) compared to C group. In conclusion, our results showed that cafeteria diet was able to induce renal lipotoxicity associated with insulin resistance and AET was efficient to prevent this damage independent of inflammatory profile changes.

Support or Funding Information

Financial Support: FAPESP # 2015/04948‐4.

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