bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Mechanisms Involved in Adenosine Receptor‐mediated Suppression of Foam Cell Formation
Authors: Bingham Taiese C , Cronstein Bruce N
Published: June 2021

Atherosclerotic lesions contain many types of cells including immune cells, macrophages and T cells. In addition to ABCA1 mutation and deregulation, several groups have shown an increasing interest in the role of inflammation and the pathogenesis of atherosclerosis. Foam cell formation is one of the earliest indications of atherosclerosis. Pro‐inflammatory cytokines and immune complexes impede cholesterol efflux from macrophages, leading to the formation of foam cells. IFN‐γ a pro‐ inflammatory cytokine has been shown to contribute to the pathogenesis of atherosclerosis. Prior studies have suggested that the occupancy of the anti‐inflammatory adenosine A2A receptor minimizes early atherosclerotic changes in the arterial wall. One study determined that A2A receptor occupancy stimulates ABCA1 message. Previous studies have shown that ABCA1 is the rate‐limiting factor in controlling reverse cholesterol transport. Interest has recently been directed at developing agents that interact with the anti‐inflammatory adenosine A2A receptors to prevent or treat vascular and tissue injury. Ligation of adenosine A2A receptor prevents reperfusion injury and atherosclerosis in animal models, inhibits foam cell formation, and prevents IFN‐γ mediated down‐regulation of transcription for 27‐hydroxylase and ABCA1 by a protein kinase A (PKA) ‐cyclic adenosine monophosphate (cAMP)‐ mediated mechanism. Our focus is to determine the molecular mechanisms involved in adenosine receptor mediated suppression of foam cell formation. It is our hypothesis that adenosine A2A receptors modulate foam cell formation by Gs‐mediated modulation of molecules involved in promoting and preventing foam cell formation. Using siRNA technology and other approaches we indicate that the mechanism by which A2A receptor activation modulates ABCA1 expression functions during reverse cholesterol transport.
Ranolazine Attenuates Palmitoyl‐L‐Carnitine Induced Ventricular Diastolic Dysfunction
Authors: Wu Yuzhi , Belardinelli Luiz , Shryock John
Published: June 2021

Background: Palmitoyl‐L‐carnitine (PC), an ischemic metabolite, increases late Na+ channel current, causing cellular Na+ and Ca2+ overload and contractile dysfunction. Ranolazine (Ran) is a selective blocker of late sodium current. This study was conducted to determine whether Ran attenuates PC‐induced diastolic dysfunction.

Methods: Guinea pig isolated hearts perfused at constant flow (Langendorff method) were used to study left ventricular end‐diastolic pressure (LVEDP), coronary perfusion pressure (CPP), ventricular wall stiffness, and cardiac lactate and adenosine release. Hearts were treated with 4 μM PC for 30 min followed by treatment with Ran or vehicle for 30 min.

Results: PC significantly increased LVEDP, CPP, wall stiffness, and lactate and adenosine release. Ran (10 μM) significantly attenuated the PC‐induced increase in LVEDP by 61 ± 5 % (n=6, p<0.001), the PC‐induced increase of CPP by 27 ± 3% (n=6–7, p<0.05), and the PC‐induced increases of lactate and adenosine release by 50 ± 7 and 75 ± 4 %, respectively (n=6, p ≤0.05 for both). Ran reduced the PC‐induced increase of wall stiffness: LVEDP at all intraventricular volumes was lower in the presence of Ran + PC than in the presence of PC alone.

Conclusion: Ran attenuated PC‐induced ventricular diastolic dysfunction, as indicated by reductions of LVEDP, ischemic metabolites, and CPP.
ATPo‐activated chloride channels play a key role in postconditioning‐induced cardioprotection in mouse heart
Authors: Xiang Sunny (Yang) , Ye Linda , Hatton William J. , Duan Dayue
Published: June 2021

Extracellular ATP (ATPo) activates a Cl− current (ICl.ATP) through a P2‐purinoceptor mediated PKC/PKA phosphorylation. Previous study suggests a role of ICl.ATP in ischemic preconditioning cardioprotection. This study is to further investigate whether ICl.ATP is also important for postconditioning (POC) protection against reperfusion injury of the heart. Since ICl.ATP is encoded by CFTR gene we used the CFTR knockout (KO) mice (FABPCFTR strain) to specifically inactivate ICl.ATP in mouse heart. In the isolated working heart model, 45 min ischemia (I) followed by 120 min reperfusion (R) caused a significant decrease in cardiac function and an infarct size of 37.5±3.4% (n=4) of wild‐type (WT, CFTR+/+) heart. POC with a 6 cycles of 10 sec re‐occlusion/reperfusion in the first minute of reperfusion after the 45 min index ischemia significantly reduced the infarct size (17.0±2.0%, n=4, P<0.01) in WT heart but not in KO heart (Control 40.4±4.1% vs POC 45.0±7.7%, n=4, P>0.05). Similarly, in the in vivo POC model, POC reduced infarct size caused by 30 min I/ 24 h R in WT heart from 46.1±8.5% (n=4) to 14.4±2.4% (n=4, P<0.05) but not in KO heart (Control 46.3±8.3%, n=5, vs POC 40.8±1.2%, n=5, P>0.05). These results suggest that Cl.ATP channels may play a key role in POC cardioprotection in mouse heart and may serve as a novel therapeutic target for ischemic heart diseases. (Sponsored by NIH HL63914 and NCRR P20RR15581)
Sepsis affects the expression of Syntaxin1A and Norepinephrine transporter in the myocardium
Authors: Das Padmalaya , Chopra Mani , Sharma Avadhesh C.
Published: June 2021

Activation of the sympathetic nervous system leads to an increase release of norepinephrine (NE) during sepsis. Re‐uptake of NE by the NET (NE Transporter) protein is the primary mechanism by which the biological effects of NE in the synapse are terminated. A SNARE protein, Syntaxin 1A plays a major role in presynaptic regulatory mechanism linking neurotransmitter secretion to neurotransmitter reuptake activity. However the role of Syntaxin 1A in the regulation of NE in septic heart is not known. Therefore, in the present study the effect of increased duration of sepsis on the expression of syntaxin1A and NET in heart was elucidated. Male Sprague‐Dawley rats (300–350g) were randomized into sham, 1‐, 3‐ and 7‐day sepsis groups. Sepsis and sham‐sepsis was induced using 200mg/kg cecal inoculum and 5% dextrose water i.p., respectively. Hearts were isolated to perform the RT‐PCR and immunoblot analyses for Syntaxin 1A and NET levels. Syntaxin 1A mRNA as well as protein expression increased significantly at 1‐day, while reduced at 3‐ and 7‐day post‐sepsis. NET gene expression was not altered significantly in septic animals but NET protein expression was decreased (61%) in 3‐day post‐sepsis group. We also observed increased NE levels (50% compared to sham) at 1‐day post‐sepsis. The data suggest that the elevation of Syntaxin 1A could be responsible for an increased level of myocardial NE at 1‐day post‐sepsis. Disruption of the transporter function at 3‐day post‐sepsis may lead to an extraneuronal accumulation of the previously released NE in the heart. (This work was supported by NHLBI # 66016).
Cardiac‐specific overexpression of P2X4 receptors enhanced myocyte contractility in the calsequestrin (CSQ) model of cardiomyopathy
Authors: Shen Jian‐Bing , Shutt Robin H , Pappano Achilles J , Liang Bruce T
Published: June 2021

Overexpression of the human P2X4 purinoceptor (P2X4R) rescues the heart failure phenotype in the calsequestrin(CSQ)‐overexpressing transgenic (TG) model of cardiomyopathy (Am J Physiol 287:H1096, 2004). The objective is to study the contractility of binary P2X4R/CSQ (Binary) TG myocyte by comparing contraction shortening (CS) in ventricular myocytes. Cell length (102±1.8 μm vs. 108±2.3 μm, SE) and basal CS (5.3%±0.45% vs. 5.4%±0.38% of cell length, SE) at 0.2Hz pacing (22ºC) were similar in WT (N=41) vs. P2X4R myocytes (N=59). Cell length in Binary (151±1.6μm, N=54) was shorter than that in CSQ (158±2.1μm, N=60, p<0.05), indicating less hypertrophy. Basal CS in Binary (4.1±0.23 %) was greater than that in CSQ (2.6±0.20 %, p<0.02). At 2.0 Hz, basal CS was also greater in Binary (2.0±0.14%, N=30) than in CSQ (0.9±0.11 %, N=23, P<0.05) cells. With 3μM 2me‐SATP, CS increased significantly in myocytes from all four genotypes but the CS increases in P2X4R and Binary TG cells were greater than those in WT and CSQ cells respectively (P<0.05).

Conclusion:

Overexpression of the P2X4R in the CSQ model of cardiomyopathy caused enhanced myocyte contractility to agonist as well as increased basal myocyte contractility; the latter may be due to activation of the overexpressed P2X4R by endogenous extracellular ATP in vivo. Enhanced contractility may mediate the heart failure rescuing effect of P2X4R overexpression.
Inhibitory effect of nicotine on collagen and alpha‐smooth muscle actin expression in cardiac fibroblasts
Authors: Lu David , Yokoyama Utako , Insel Paul A.
Published: June 2021

It is well documented that smoking increases risk of cardiovascular disease and myocardial infarction (MI). Extra‐cellular matrix (ECM) protein deposition by cardiac fibroblasts (CF) is a hallmark of post‐MI scarring and wound healing. In this study, we used primary cultures of rat CF (rCF) to assess the effect of nicotine (Nic) on ECM production by CF. We treated rCF with 1 μM nicotine in the absence and presence of 10 ng/ml transforming growth factor beta (TGF‐β), a cytokine that induces CF to produce ECM proteins. Quantitative real‐time PCR revealed that TGF‐β treatment induces a 2‐fold increase in expression of collagen1a1 (Col1a1) and alpha‐smooth muscle actin (α‐SMA) mRNA. Nic alone had no effect on “basal” Col1a1 or α‐SMA expression but abolished the TGF‐β induced increases in Col1a1 and α‐SMA mRNA. These results suggest that Nic has an inhibitory effect on damage‐induced ECM protein production of rCF. The findings imply that Nic intake through smoking of tobacco or other sources may inhibit wound healing and scar tissue formation in the heart following cardiac damage (e.g., post‐MI) by inhibiting collagen synthesis and myofibroblast transformation. Such inhibition may contribute to smoking‐induced exacerbation of cardiovascular disease and damage. (Supported by grants from NIH and the Ellison Medical Foundation).
Inactivation of HCN channels slows rhythm differently in atria and ventricle and improves conduction in the anoxic‐reoxygenated embryonic chick heart
Authors: Gardier Stephany , Sarre Alexandre , Raddatz Eric
Published: June 2021

Hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels control heart rate and are expressed early during cardiogenesis. Distribution of the predominant HCN4 isoform and the involvement of HCN channels in myocardial dysfunction induced by anoxia‐reoxygenation were investigated in the embryonic heart.

Expression of HCN4 channels was determined by western blotting in atria (A), ventricle (V) and outflow tract (OT) from hearts of 4‐day‐old chick embryos. Hearts or isolated ventricles were exposed to the selective inhibitor of HCN channels ivabradine. Effects of 0.3μM ivabradine on ECG, excitation‐contraction (EC) coupling and contractility were determined in hearts submitted to 30 min anoxia and 60 min reoxygenation.

The distribution of HCN4 was inhomogeneous, i.e. A ≥ V ≫ OT. The intrinsic rate was 164±22 in A and 78±24 bpm in isolated V. Under normoxia, 0.3 μM ivabradine decreased A rate by 20% and V rate by 50% and increased A‐V conduction by 20%. These effects persisted throughout anoxia‐reoxygenation, whereas variations of QT duration, EC coupling, contractility and arrhythmias were not affected.

These data indicate that HCN channels are inhomogeneously distributed and control finely atrial and ventricular pacemakers of the embryonic heart. Their inactivation during anoxia‐reoxygenation ameliorates atrial rhythmicity and conduction.

Supported by Servier and SNSF n°3100A0‐105901
The Early Phase of Preconditioning is Abrogated by COX‐2 Inhibition, but not by a Specific Prostaglandin Receptor Antagonist
Authors: Booth Erin A , Lucchesi Benedict R
Published: June 2021

COX‐2 is an important mediator of cardiovascular protection after ischemia and reperfusion and is essential for ischemic preconditioning (PC), mediated by the synthesis of PGE2 and/or PGI2. Recently, the importance of the PGI2 in the late phase of ischemic preconditioning has been established. The present study was designed to determine the contribution of PGI2 to the early phase of ischemic preconditioning. Male New Zealand white rabbits were administered the prostaglandin receptor (IP) antagonist RO3244794 (3mg/kg), nimesulide (5mg/kg), or vehicle thirty minutes prior to PC, 30 minutes of ischemia and 4 hours of reperfusion. PC reduced myocardial infarct size as a percent of area at risk (12.0 +/− 2.2 vs. control 42.3 +/− 2.9; P < 0.001) and treatment with the COX‐2 inhibitor nimesulide abolished the protective effect (41.0 +/− 2.7). Surprisingly, treatment with the IP receptor antagonist had no effect on PC (17.0 +/− 3.1; P < 0.001 vs. control). The data suggest that COX‐2 does not protect the myocardium from the early phase of preconditioning through the production of PGI2. These results differ from those seen in the late phase of PC, where RO3244794 abrogated the protective effects of PC, suggesting that the upregulation of COX‐2 differentially modulates protection in the early and late phases of ischemic preconditioning.
Alpha 1A‐Adrenergic Receptor Signaling Protects the Heart From Ischemic Injury Through an ERK‐Dependent Mechanism
Authors: Rorabaugh Boyd R , Handel Evelyn M , Waterson Rachael E , Talbot Jeffery N , Perez Dianne M
Published: June 2021

Brief periods of ischemia stimulate an endogenous mechanism in the heart that protects the myocardium from subsequent episodes of prolonged ischemia. This process, called “ischemic preconditioning”, is mimicked by signaling through α1A‐adrenergic receptors (ARs). Extracellular regulated kinases (ERK‐1 and ERK‐2) have been implicated in ischemic preconditioning. Therefore, we used mice expressing constitutively active mutant α1A‐adrenergic receptors (CAM α1A‐ARs) to determine whether α1A‐ARs protect the heart from ischemic injury through an ERK‐dependent mechanism. Western blots demonstrated that phosphorylation of ERK‐1 and ERK‐2 is significantly increased in CAM α1A‐AR hearts relative to nontransgenic hearts. Isolated hearts were perfused using the Langendorf method, and contractile function was monitored by an intraventricular balloon. Hearts isolated from CAM α1A‐AR or nontransgenic mice were subjected to 30 min ischemia and 1 hour reperfusion. Contractile function of CAM α1A‐AR hearts, but not nontransgenic hearts, completely recovered from 30 min ischemia. Perfusion with U0126 (MEK inhibitor) prior to the onset of ischemia blocked ERK phosphorylation and abolished the ability of CAM α1A‐AR hearts to recover from ischemia. These data support the conclusion that α1A‐ARs protect the heart from ischemic injury through an ERK‐dependent signaling pathway.

(This study was supported by a Bower, Bennet, & Bennet Endowed Research Chair Award from Ohio Northern University).
Post‐transcriptional regulation of calpastatin by Protein Interacting with Never in Mitosis Gene A‐1 (PIN1)
Authors: Liu Tongzheng , Likhotvorik Rostislav , Hoyt Dale G
Published: June 2021

The peptidyl‐proline isomerase, PIN1, regulates protein expression at many levels. PIN1 is composed of WW docking and catalytic domains that each interact with p(S/T)‐P motifs in substrates. Previously, we found that depletion of PIN1 with short hairpin RNA increased the amount of the endogenous calpain inhibitor, calpastatin, in endothelial cells. Here we investigated the mechanism and roles of the 2 functional domains of PIN1 in this effect. Murine PIN1 and calpastatin were cloned from mRNA by RT‐PCR. A WW domain mutant, W33A, and a catalytic mutant, K64A, were then generated by site directed PCR mutagenesis. Calpastatin was also fused to a V5 epitope tag in pcDNA3.1. HEK293T cells were co‐transfected with calpastatin‐V5 and PIN1 constructs, extracted after 48 h, and western blotted with anti‐V5 antibody. Wildtype PIN1 prevented expression of calpastatin‐V5. Expression of a control fusion protein, poly(ADP‐ribose)polymerase‐V5, was not affected, suggesting that transcription from the vector CMV promoter was not generally repressed. Mutation of either the WW or the catalytic domains prevented the suppressive effect of PIN1 on calpastatin‐V5. These results suggest that PIN1 acts by a post‐transcriptional mechanism to limit calpastatin expression. Furthermore, both the WW and catalytic domains are necessary. Repression of calpastatin by PIN1 may generally affect calpain‐dependent processes.
Cinnamaldehyde inhibits the tumor necrosis factor‐£\‐induced expression of adhesion molecules in endothelial cells by suppressing NF‐£eB activation
Authors: wung being‐sun
Published: June 2021

The production of adhesion molecules and subsequent attachment of leukocytes to endothelial cells (ECs) are critical early events in atherogenesis. In this study, we have examined the effect of cinnamaldehyde on cytokine‐induced monocyte/ECs interactions. We find that these compounds inhibit the adhesion of TNFα‐induced monocytes to endothelial cells and suppress the expression of the adhesion molecules. In TNFα‐treated ECs, NF‐κB was also abolished. Interestingly, cinnamaldehyde exerts its anti‐inflammatory effects by blocking the degradation of the inhibitory protein IκB‐α, but only in short term pretreatments, whereas it does so via the induction of Nrf2‐related genes, including heme‐oxygenase‐1 (HO‐1), over long term pretreatments. Treating ECs with zinc protoporphyrin, HO‐1 inhibitor, partially blocks the anti‐inflammatory effects of cinnamaldehyde. We also found that cinnamaldehyde can upregulate Nrf2 in nuclear extracts, and can increase ARE‐luciferase activity. Moreover, cinnamaldehyde increases cellular GSH levels after 9 hours exposure. Hence, our present findings indicate that cinnamaldehyde suppresses TNF‐induced singling pathways via distinct mechanisms that are activated by different pretreatment periods.
Regulation of expression of Suppressors of Cytokine Signalling (SOCS) genes in hypothyroid male rats by Growth hormone, Thyroid Hormones and Estradiol
Authors: Santana‐Farré Ruymán , Flores‐Morales Amilcar , Valtueña Natalia Alvarez , Cabrera‐Galván Juan J. , Nóvoa‐Mogollón Javier , Norstedt Gunnar , Fernández‐Pérez Leandro
Published: June 2021

SOCS proteins act as negative regulators of GH signalling. Recently, E2 was demonstrated to induce SOCS‐2 synthesis and inhibit GH signalling in liver and kidney cells. To evaluate the role of E2 on GH‐regulated SOCS in vivo, this work used orchidectomized‐hypothyroid (TX‐OX) male rats to minimize the influence of internal hormones on treatment. OX‐TX rats were treated 3 weeks with vehicle or E2 before hormonal replacement with GH, T3, or T3 plus GH during 7 days. We analyzed body growth, serum lipids and steady‐state levels of SOCS‐2, ‐3, and CIS mRNA expression in liver in relation to cited hormones. Hypothyroidism resulted in an over 2‐fold increase in serum cholesterol (Cho) levels and 2‐fold reduction in serum triglyceride (Tg) levels. T3 treatment decreased Cho and GH treatment resulted in 3‐4‐fold reduction in serum FFAs levels. Hypothyroidism reduced the levels of SOCS‐2 and CIS mRNA and induced SOCS‐3. E2 reversed the effects of hypothyroidism on SOCS2 and CIS but not on SOCS3 mRNA levels. Unlike T3, the effects of hypothyroidism on SOCS2 and CIS mRNA levels were reversed by GH. T3 reversed the effects of hypothyroidism on SOCS3. Whereas E2 enhanced the effect of T3 on SOCS2 and CIS, T3 effect on SOCS3 was antagonized. These results indicate that SOCS are differently regulated in vivo and an important physiological role for E2, GH and T3 in maintaining levels of SOCS in male liver [MEC‐SAF2006‐07824‐ and Pfizer Spain].
β3‐Adrenergic receptor gene polymorphisms: comparison of human with chimpanzee and Rhesus macaque
Authors: Michel Martin Christian , Teitsma Christine A , Vrydag Wim , Bontrop Ronald , de la Rosette Jean JMCH , Baas Frank
Published: June 2021

The Trp64Arg single nucleotide polymorphism (SNP) of the human β3‐adrenergic receptor is potentially associated with several disease states including obesity and type 2 diabetes. However, in vitro data with site‐directed mutagenesis have not consistently shown this SNP to yield an altered function. This raises the possibility that the SNP may be an indicator in linkage disequilibrium with functionally more relevant SNPs. To explore this and gain evolutionary insight, we have sequenced the β3‐adrenergic receptor gene from 91 humans, 12 chimpanzees and 12 Rhesus macaques. Four additional SNPs (G1219T, A2135G, G2502C, A3558T) and a tandem repeat polymorphism ΔTG3273/4) were detected in the intron and 3′‐UTR of the human gene. Their consistent association indicates the presence of a haploblock. Within the limited monkey and ape populations no polymorphisms were detected, but for the one coding and two 3′‐UTR human SNPs the human wild‐type differed from the monkey and ape wild‐type. We conclude that the intron polymorphisms of the β3‐adrenergic receptor gene have developed evolutionarily later than the coding and 3′‐UTR SNPs. Source of funding: none
Liver X Receptor alpha Gene Polymorphisms and Simvastatin Therapy
Authors: Alzahrani Sarah A. , Dzimiri Nduna
Published: June 2021

Liver X receptor alpha subtype gene (LXRα) is involved in the regulation of cholesterol (Chol) metabolism and lipid biosynthesis, and may be downregulated by statins. The objective of this study was to investigate the prevalence and effect of two single nucleotide polymorphisms (SNPs), rs326220 and rs7934351, of the LXRα gene on patient responses to simvastatin therapy. Compared to 192 angiographic controls, the heterozygote C/T of the rs326220 was slight greater in patients (n =159) than in controls (5% vs 2.6%). Carriers of the homozygous C/C genotype in patients (151) exhibited greater reduction in total Chol (5.03 ± 0.11 pre vs 4.50 ± 0.10 post; p < 0.001) compared to the heterozygote C/T carriers (5.56 ± 0.37 vs 4.95 ± 0.44; p < 0.3) following 6 months treatment with 20mg/day simvastatin. Similar results were observed for low density lipoprotein (LDL) responses (p < 0.001 for C/C vs p < 0.07 for C/T). Interestingly, for the rs7934351, the homozygote A/A (2.5%) was present only in patients. The reduction in Chol (p < 0.001 vs p < 0.8) and LDL (p < 0.001 vs p < 0.9) was significantly greater in the heterozygote A/G (155) than in the A/A (4) carriers. Our results suggest that the heterozygote C/T of the rs326220 and homozygote A/A of the rs7934351are associated with lack of response to simvastatin therapy.

Funding: The King Abdulaziz City for Science and Technology and King Faisal Specialist Hospital & Research Centre
Role of 14‐3‐3zeta in Anoikis Resistance and Anchorage‐independent Growth of Lung Cancer Cells
Authors: Zhao Jing , Li Zenggang , Du Yuhong , Park Hae Ryoun , Sun Shi‐yong , Khuri Fadlo , Fu Haian
Published: June 2021

14‐3‐3 proteins have emerged as critical regulators of diverse cellular responses ranging from cell proliferation, to cell cycle control and apoptosis. Here we report an important role of 14‐3‐3ζ in tumorigenesis through a mechanism that involves anoikis resistance. 14‐3‐3ζ is upregulated in a number of cancer types, including lung cancer. Through an RNAi approach using human lung adenocarcinoma‐derived A549 cells as a model system, we have found that knockdown of a single ζ isoform of 14‐3‐3 is sufficient to restore the sensitivity of cancer cells to anoikis and to impair their anchorage‐independent growth. Enhanced anoikis appears to be mediated in part by upregulated BH3‐only proteins, Bad and Bim, coupled with decreased Mcl‐1, resulting in the subsequent activation of Bax. Our study suggests a model in which anchorage‐independent growth of lung cancer cells requires the presence of 14‐3‐3ζ. This work not only reveals a critical role of 14‐3‐3ζ in anoikis suppression in lung cancer cells, but also identifies and validates 14‐3‐3ζ as a potential molecular target for anticancer therapeutic development.
Overexpression of CYP4A1‐20‐HETE in U251 Glioma Cell Induces Hyperproliferative Phenotypes in vitro and in vivo
Authors: guo austin meng , Roman Richard J , Falck John R , Jafari Kourosh , Edwards Paul A , Scicli Alfonso Guillermo
Published: June 2021

Cytochrome P450 (CYP) omega hydroxylases such as CYP 4A1 and 4F enzymes convert arachidonic acid to 20‐HETE. 20‐HETE is a potent stimulator of the growth of human glioma cells in vitro. Herein, we report that increasing the endogenous generation of 20‐HETE by overexpressing CYP4A1 in U251 results in a ∼ 2 fold higher proliferation rate than control U251. This is accompanied by an increased S phase in the cell. U251 O show a disorganized and disrupted cytoskeleton, and have constitutive activation of phosphorylated ERK1/2 and increased expression of cyclin D1/2. The hyperproliferative and signaling effects in U251 O are abolished by the selective CYP4A inhibitor HET0016 and by the putative 20‐HETE antagonist 20‐HEDE. This demonstrates that the hyperproliferative state is caused by the high endogenous production of 20‐HETE by CYP4A1. Dihydroethidium assay show that the U251 O cells have marked increases in superoxide formation. Treatment with PEG‐SOD abolished the hyperproliferative state, indicating the crucial contribution of superoxide to the hyperproliferative phenotype. In vivo, MRI studies showed that the tumor of the rats implanted with U251 O had a ∼10 fold higher tumor volume compared with U251. High 20‐HETE leads to enhanced proliferation and to a fast‐growing tumor in vivo. These findings suggest that 20‐HETE synthases and 20‐HETE could play an important role in growth regulation of human gliomas.
Poly(ADP‐ribose)polymerase inhibition decreases angiogenesis.
Authors: Rajesh Mohanraj , Mukhopadhyay Partha , Godlewski Grzegorz , Bátkai Sandor , Pacher Pal
Published: June 2021

Inhibitors of poly(ADP‐ribose)polymerase (PARP), a nuclear enzyme involved in regulating cell death and cellular responses to DNA repair, show considerable promise in the treatment of cancer both in monotherapy as well as in combination with chemotherapeutic agents and radiation. Here, we show dose‐dependent reduction of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF)‐induced proliferation, migration, and tube formation of HUVECs in vitro by various PARP inhibitors (3‐aminobenzamide or PJ‐34, 5‐aminoisoquinolinone‐hydrochloride (5‐AIQ) and 1,5‐isoquinolinediol (IQD). Moreover, PARP inhibitors decrease the sprouting of rat aortic ring explants in an ex vivo assay of angiogenesis. These results establish the novel concept that PARP inhibitors have antiangiogenic effects, which may have tremendous clinical implications for the treatment of various cancers, tumor metastases, and certain retinopathies.
Biochemical and structural properties of four CYP1B1 mutations present in primary congenital glaucoma (PCG) patients
Authors: choudhary Dharamainder , Jansson Ingela , Sarfarazi Mansoor , Schenkman John B
Published: June 2021

Methods: The study involves heterologous protein expression of four mutant CYP1B1 forms (Y81N, E229K, A330F, R368H) in E. coli, analyzing their stability and ability to metabolize the endogenous substrates, estradiol, arachidonic acid and retinoids.

Results: PCG is a major cause of childhood blindness and occurs due to an abnormal development of the trabecular meshwork (TM) and anterior chamber region of the eye. Our group was the first to link mutations in the CYP1B1 gene to the PCG phenotype. The role of CYP1B1 in eye development may involve its oxidative metabolic activity, whereby it could either generate or degrade an active signaling molecule, e.g. a morphogen, which affects the downstream transcriptional expression of genes associated with proper eye development. The data showed a high degree of instability for the Y81N and E229K mutant proteins. All the four mutants affected the metabolic activity significantly. The results were correlated with the structural changes in the CYP1B1 protein due to amino acid substitutions, based upon CYP1A2 crystal structure.

Conclusion: The findings suggest that altered enzymatic properties of the CYP1B1 mutants towards endogenous substrates may be the cause of impaired eye development and the PCG phenotype.

(Support: NIH grants R01 EY11095, 5R03 EY014654; American Health Assistance Foundation award AHAF G2007‐036)
EFFECTS OF STRUCTURALLY AND FUNCTIONALLY DIVERSE GROUP OF PHYTOCHEMICALS ON ANILINE HYDROXYLATION (CYP2E1) OF CONTROL AND ACETONE‐INDUCED RAT LIVER MICROSOMES
Authors: Parekh Jigna , Sengupta Amiritaparna , Ray Sidhartha D.
Published: June 2021

Although the last decade focused on exploring disease fighting properties of hundreds of phytochemicals, major gaps remain to be filled in understanding their mechanisms of actions in vivo. Our laboratory pioneered investigating anti‐toxic properties of several phytochemicals, however, information on their bioavailability, impact on drug metabolism, precise role during pathogenesis of a number of diseases, and drug‐drug interactions remains unclear. Interestingly, CYP2E1 has evolved as a major player in this field. This study investigated the effects of structurally & functionally diverse group of phytochemicals on aniline hydroxylation pattern of control and acetone induced (1% in drinking water for 6 days) rat liver microsomes in vitro. Trans cinnamic acid (TCA), Resorcinol (RSL), Rutin (RUT), Quercetin (QTN), Hesperidin (HES) and Curcumin (CUR) were used at 25, 50, 100μM, 1 and 5mM concentrations. Data show that TCA and HES did not impact aniline hydroxylation at all, whereas RSL turned out to be a potent inhibitor. RUT showed 22% inhibition at 25–100 μM, 33% and 53% inhibition at 1mM & 5mM respectively. CUR and QTN showed slight inhibition, 9% and 33% respectively but only at highest dose. This study suggests that different phytochemicals may have different mechanisms of interfering with drug metabolism; therefore, products containing these phytochemicals must be used with caution during drug therapy.
Lacritin, a Novel Tear Glycoprotein, Stimulates Tear Production in Rabbits without Ocular Toxicity
Authors: Samudre Sandeep S , Loose‐Thurman Patricia , McKown Robert , Laurie Gordon , Lattanzio Frank , Williams Patricia
Published: June 2021

Dry eye is extremely common and debilitating disease. Current treatment options like artificial tears and cyclosporin are palliative and do not effectively address the underlying pathology of dry eye. Lacritin is a newly discovered glycoprotein that occurs naturally in human tears, produced solely by meibomian, lacrimal and salivary glands. Human recombinant lacritin stimulates tear secretion in cultured rat lacrimal acinar cells, leading us to hypothesize that topical application of lacritin will increase production of tears of normal composition. Bilateral tear production in New Zealand white rabbits was measured at 60, 120 and 240 min after a single topical administration of 50μl lacritin (1, 10, or 50 μg/ml). Tears were collected and slit lamp examination and ocular surface staining measured before and 240 min after treatment. Tear pH, protein and electrolyte concentrations were then measured. A 50 μg/ml lacritin dose increased tear production by 40% within 60 min and maintained for 240 min (p<0.05). With other doses, a significant increase (55% from baseline, p<0.05) was observed only at 240 min. No local irritant effects were noted by slit‐lamp examination. The composition (pH, Na2+ and K+) of tears was also not different from normal tears. In conclusion, lacritin is a novel, safe and effective candidate for dry eye therapy. Lacritin, being mitogenic, may also help restore the natural tear film.
Clotrimazole inhibits inflammation and pathogenic angiogenesis via modulation of IL‐8 in TNF‐α‐induced intestinal inflammation
Authors: Thapa Dinesh , Kwon Jun Bum , Park Byung Chul , Kim Jung‐Ae
Published: June 2021

In the inflammatory bowel diseases (IBD), tissue level of interleukin (IL)‐8 has been reported to be upregulated. Such increased IL‐8 activates and recruits leukocytes to the inflamed site. Recent evidences have shown that IL‐8 also induces angiogenesis at the inflammation site, which further aggravates the condition of IBD. Since clotrimazole (CLT) has been reported to inhibit angiogenesis, in the present study, we investigated whether CLT inhibits intestinal inflammation via its inhibitory action on IL‐8. In TNF‐α‐stimulated HT29 colon epithelial cells, a well recognized in vitro model for colon inflammation, the treatment with CLT significantly suppressed TNF‐α‐induced increase in IL‐8 level at both mRNA and secreted protein levels. In addition, CLT inhibited both TNF‐α‐ and IL‐8‐induced monocyte adhesion to the cells and MCP‐1 expression in a concentration‐dependent manner. In an in vitro angiogenesis assay, CLT suppressed IL‐8‐induced proliferation, tube formation, and invasion of endothelial cells. In addition, CLT inhibited the IL‐8‐induced angiogenesis in vivo using a chick chorioalantoic membrane assay. Collectively, these results suggest that CLT may prevent the progression of intestinal inflammation by not only down‐regulating IL‐8 expression but also inhibiting the action of IL‐8 in both colon epithelial and endothelial cells during pathogenesis of IBD.
Effects of Antioxidants on Contracting Spinotrapezius Muscle Force Production and Oxygen Consumption in Aged (26–30 Month Old) Rats
Authors: Herspring Kyle F , Ferreira Leorando F , Copp Steven W , Poole David C , Musch Timothy I
Published: June 2021

Aged rats exhibit a decreased muscle microvascular O2 partial pressure (PO2mv) at rest and during contractions compared to young rats. Age‐related reductions in nitric oxide bioavailability due, in part, to elevated reactive O2 species (ROS) constrain muscle blood flow (Q̇ m). Therefore antioxidants may restore NO bioavailability, Q̇ m and ameliorate the reduction in PO2mv.

PURPOSE:

To test the hypothesis that antioxidants would elevate Q̇ m and therefore PO2mv.

METHODS:

PO2mv and Q̇ m were measured in the spinotrapezius of 20 anesthetized male F344xBN hybrid rats at rest and during 1 Hz contractions before and after antioxidant intravenous infusion (76mg/kg vitamin C and 52mg/kg tempol).

RESULTS:

Antioxidants did not change Q̇ m at rest but during contractions Q̇ m was reduced from 157 ± 28 to 91 ± 15 ml/min/100g (P<0.05). In the control condition, contractions invoked a biphasic PO2mv that fell from 30.6 ± 0.9 mmHg to a nadir of 16.8 ± 1.2 mmHg with an ‘undershoot’ of 2.8 ± 0.7 mmHg (P<0.05) below the subsequent steady‐state (19.7 ± 1.2 mmHg). Antioxidants elevated baseline PO2mv to 35.7 ± 0.8 mmHg (P<0.05) and reduced the ‘undershoot’ (P<0.05) without changing the steady‐state contracting PO2mv.

CONCLUSION:

At rest, antioxidants increased PO2mv without changing O2 delivery. During contractions antioxidants transformed the aged PO2mv profile to that of young rats.

Supported by AHA Heartland Affiliate Grant #0750090Z.
Connexin40 is required for NADPH oxidase‐derived oxidant production in microvascular endothelial cells
Authors: Tyml Karel , Yu Guo , Bolon Michael , Shao Qing , Laird Dale
Published: June 2021

The function of connexins (Cx) may extend beyond gap junctional intercellular communication (GJIC). We hypothesized that Cx40 is required for NADPH oxidase‐dependent ROS production in microvascular endothelial cells subjected to hypoxia/reoxygenation (H/R), independent of GJIC. ROS generation and NADPH oxidase activation was assessed after H/R in confluent monolayers of cells of wild‐type (WT), Cx40−/−, gp91phox−/−, or p47phox−/− mice, as well as in monolayers of GJIC‐deficient SKHep1 cells overexpressing GFP‐tagged Cx40. In WT cells, H/R‐initiated ROS increase was not affected by the GJ blocker 18alpha‐glycyrrhetinic acid, nor by preventing cells from establishing contact with each other, but it was absent in Cx40−/− cells. H/R increased ROS in SKHep1 cells expressing Cx40‐GFP, but not in cells expressing the control vector. We also asked if Cx40‐dependent reduction in inter‐endothelial electrical coupling after H/R requires NADPH oxidase. H/R reduced coupling in WT and gp91phox−/− but not in p47phox−/− cells. We suggest that (1) H/R‐initiated signaling in microvascular endothelial cells involves cross‐talk between NADPH oxidase and Cx40, and (2) Cx40 is required for NADPH oxidase‐derived ROS production, independent of GJIC. (Support: HSFO).
Pathways used to transmit conducted dilations are dependent on the stimulus used to initiate the conducted response
Authors: Junuzovic Jasna , Murrant Coral L
Published: June 2021

We hypothesized that the pathways utilized for conducted dilations along skeletal muscle microvasculature are dependent on the dilator used to initiate the conducted response. We initiated conducted responses by micropipette application of either 10−4M acetylcholine (ACh) or 10−5M pinacidil (PIN, a KATP channel opener) to the capillaries or the transverse arteriole (TA, located three branch orders proximal to the capillaries) in the anaesthetized hamster cremaster muscle model in situ. When the capillaries were stimulated we observed changes in diameter of the arteriole directly supplying the capillaries and when the TA was stimulated we observed the TA approx. 1000um upstream (US) and downstream (DS) from the site of dilator application. We reapplied ACh and PIN after a 30 min micropipette application of 40uM 18‐B‐glycyrrhetinic acid (BGA), a gap junction uncoupler, between sites of initiation and observation. Significant conducted dilations were produced in proximal arterioles when either ACh (3.6+/−0.9um) or PIN (3.1+/−0.5um) was applied to the capillaries. BGA significantly attenuated dilation to ACh (0.6+/−0.2um) but not dilations to PIN (3.4+/−0.8um). Significant conducted dilations were observed on the TA in response to both ACh (US: 9.6+/−2.0um, DS: 7.8+/−2.4um) and PIN (US: 5.2+/−1.4um, DS: 7.0+/−1.7um). BGA attenuated the conducted responses to ACh (US: 4.4+/−1.8um, DS: 2.6+/−1.6um), but not to PIN (US: 5.1+/−1.4um, DS: 10.9+/−4.4um). Therefore, conducted responses may use different transmission pathways that are dependent on the stimulus initiating the conducted response. NSERC and PREA.
Adenosine stimulates eNOS phosphorylation by a p42/44 MAPK‐dependent mechanism in microvascular endothelial cells
Authors: Kalogeris Theodore J. , Yusof Mozow , Gaskin F. Spencer , Korthuis Ronald J.
Published: June 2021

Adenosine (Ado) is a key mediator in the protective effects of ethanol preconditioning on microvascular function. In vivo studies indicate that Ado's protective effect mediated by increased activity of endothelial nitric oxide synthase (eNOS). However, the mechanism for Ado‐induced eNOS activation is not clear. In order to address this issue, we studied the effects of Ado, Ado agonists, and several antagonists on phosphorylation of eNOS Ser 1177 in human microvascular endothelial cells (HMEC‐1). Ado, CPCA (an Ado A2 receptor agonist), and NECA (an Ado A1/A2 receptor agonist) significantly increased eNOS phosphorylation within one minute of exposure, but CPA (A1 agonist) had no effect. The effects of Ado and NECA were partially blocked by the A2 receptor antagonist, DMPX, whereas DMPX completely reversed the effect of CPCA. A1 receptor antagonist, DPCPX, had no effect on Ado‐induced phosphorylation. Sp‐cAMPS, a cAMP agonist, increased phosphorylation; this was blocked by PI3K inhibitors, wortmannin and LY‐294002. Ado‐induced phosphorylation was unaffected by the cAMP antagonist, Rp‐cAMPS, wortmannin, and LY‐294002, but was reversed by p42/44 MAPK inhibitor, PD98,059. These results suggest that in HMEC‐1, Ado A2 receptor‐mediated activation of eNOS is independent of activity of either PKA or PKB, but is dependent upon activation of p42/44 MAPK. Supported by AA14945 and DK43785.
Diverse expression of Kir contributes to different resting membrane potential distribution among the cochlear, brain and mesenteric arterioles
Authors: Yang Yu‐Qin , Ma Ke‐Tao , Karasawa Takatoshi , Shi Xiao‐Rui , Nuttall Alfred L , Jiang Zhi‐Gen
Published: June 2021

The resting membrane potentials (RP) of the spiral modiolar artery (SMA) cells exhibit a prominent bimodal distribution peaked near −75 and −40 mV. The RPs of brain artery (BA) also showed a bimodal pattern but with a dominating population around −77 mV, while the mesenteric arterial (MA) displayed a skewed single peak distribution around −72 mV. Using intracellular and whole‐cell recording, immunocytochemical and molecular biological detection of Kir2.x expression, we tested a hypothesis that a diverse expression of Kir underlies the distinct RP distribution. We found that: 1) bathing of 10 mM K+ caused a −7.1, 6.7 and 9.8 mV membrane potential change, 2) 20 mM K+ usually caused a dilation, biphasic dilation followed by a constriction, and only a constriction in the SMA, BA and MA, respectively. 3) Ba2+ caused a depolarization in high RP cells with an EC50 of 89, 158 and 201 μM for the SMA, BA and MA, respectively. 4) A robust Kir current in 60 mM K+ was recorded more frequently in BA and MA than the SMA. Ba2+ suppressed the Kir current with an IC50 of 561 ± 73, 446 ± 30 and 811 ± 49 nM at −120 mV for the SMA, BA and MA. 5) Kir2.1 immunoreactive signal was detected in smooth muscle not endothelial cells of all the vessels. Western blotting data revealed that the SMA, BA and MA express Kir2.1 protein at a ratio of 1.0:1.1:0.83. 6) RT‐PCR analysis showed that the four Kir2.x isoforms all had mRNA transcripts in the three vessels but Kir2.3 was higher in BA and MA than SMA, while Kir2.4 was higher in the SMA than BA and MA. We conclude that heterogeneous expression of Kir2.x subunits may form different heteromeric channels and channel densities in the three vessels, thus contributing to the distinct RP distributions. Supported by NIH DC 004716 (ZGJ) & DC 00105 (ALN).
Hindlimb collateral growth after superficial femoral artery (SFA) ligation in the Ossabaw pig
Authors: Ziegler Matthew A , Alloosh Mouhamad , Bills Randal G , Miller Steven J , Sturek Michael , Unthank Joseph L
Published: June 2021

Vascular compensation to femoral artery occlusion was assessed in the Ossabaw pig. In 6 pigs, the right SFA was ligated and 4 weeks later, angiograms of the proximal hindlimbs were obtained using fluoroscopy. An average of 7.2±1.6 collaterals identified as vessels responsible for reconstitution of distal femoral artery originated from the superficial femoral, profunda (deep femoral) and gluteal arteries. The greatest number originated from the profunda (4.0±0.8), but the most prominent (>1 mm diameter) from the gluteal. Histology of the collaterals showed a tremendous increase in a single layer of intimal cells, an intact internal elastic lamina and no neointima. The inner diameter of the common femoral determined by intravascular ultrasound was similar between limbs, 5.0±0.4 mm. Average peak velocity in the common femoral measured with a ComboWire™XT was similar between limbs (19±2 vs 20±2, p=0.68), but decreased ∼50% with acute compression of the SFA. MAP was 64±5 mm Hg at time of velocity measurement. In conclusion, remarkable collateral growth occurs in the young healthy Ossabaw pig, which is able to restore resting flow in the common femoral artery within 4 weeks post‐occlusion. The Ossabaw pig is an excellent model to assess the impact of vascular disease on collateral growth in vessels of comparable size and hemodynamics as in humans.

Support: HL42898, RR013223, HL062552
Vascular defects in mice with mutation in the Lysosomal Trafficking Regulator gene
Authors: Beyer andreas M. , Trantow Colleen , Anderson Michael G , Rahmouni Kamal
Published: June 2021

Exfoliation (XFS) is commonly associated with disturbance in elasticity, oxidative stress and increased risk for aortic aneurism. We hypothesized that vascular defects may be common in XFS which could contribute to the vascular disorders associated with this condition. To test this, we studied the vascular function and structure of a mouse model that recapitulates many phenotypes of human XFS: mice that carry a mutation in the lysosomal trafficking regulator (Lyst) gene. Using aorta rings, we found an attenuated relaxation to acetylcholine in Lystbg‐J mice (50±16%, n=3) as compared to B6 controls (65±7%, n=3) indicating that mutation in the Lyst gene leads to endothelial dysfunction. Histological analysis of the vessels showed a decrease in vascular cross section area in Lystbg‐J mice (0.1022±0.0104 μm2) as compared to controls (0.1291±0.0009 μm2, P<0.05) demonstrating vascular atrophy in the mutant mice. In addition, Lystbg‐J mice exhibit vascular wall thinning. In conclusion, mutation in the Lyst gene in mice results in vascular defects which may account for the disorders associated with XFS in human. Presence of vascular atrophy is consistent with the attenuated aortic relaxation to acetylcholine because vascular atrophy is known to attenuate the endothelial responses to stimuli. Finally, wall thinning is consistent with the increased risk for aortic aneurisms associated with XFS.
Vascular proteomics for protein‐arginine‐methylation in metabolic syndrome
Authors: Adachi Takeshi , Ishiwata Kyoko , Yamamoto Michiko , Yamamoto Takehiro , Suematsu Makoto
Published: June 2021

Objective: Zucker rat is widely employed as an experimental model of metabolic syndrome, which is characterized with obesity, hyperlipidemia and insulin resistance. Although atherosclerosis is related with the mortality of the syndrome, changes in the vascular post‐translational protein modifications in this pathological state had not been well examined. In this study, we analyzed the protein‐arginine‐methylation in aorta from Zucker rat.

Methods: Aortic constriction with phenyrephrin (PE) was assessed with isometric tension measurement. Some rings were pre‐treated with an iNOS blocker, L‐NIL (10 □M). Plasma homocysteine levels were measured by HPLC with fluorescence detection. Proteome analysis of aortic proteins was performed with the differential display of two‐dimensional electrophoresis using Ettan Dige.

Results: PE‐induced vasoconstriction was decreased in aorta from Zucker rat and pre‐treatment of L‐NIL improved it. Plasma homocysteine concentrations were higher in Zucker rat. The proteomics analysis for the protein‐arginine‐methylation with anti‐dimethyl‐arginine (ADMA) antibody revealed the decreased ADMA modifications on several protein spots in aorta from Zucker rat.

Conclusion: Hypomethylation status can be related with early vascular changes in metabolic syndrome and the elevated plasma homocysteine levels and NO from iNOS can be involved.
Physiological implications of high‐heeled shoes on the venous return for healthy women
Authors: Tedeschi‐Filho Wagner , Dezotti Nei Rodrigues Alves , Moriya Takachi , Piccinato Carlos Eli
Published: June 2021

Walking with high‐heeled shoes is a common cause of venous complains like pain, fatigue and heavy legs feeling. In this study, we tested the hypothesis that women using different high‐heeled shoes present an impaired venous return when compared with themselves barefooted. Women without venous disease were evaluated with Air Plethysmograpy, a test that measures changes in air volume on a cuff placed on the calf, while they perform orthostatic flexion and extension foot movements. The test was repeated 4 times: barefooted, with 3.5 cm heel, 7cm heel, and platform heel. The values for the parameters of Venous Filling Index (VFI), Ejection Fraction (EF), Residual Volume Fraction (RVF) were separated in 4 groups, one for each test situation in order to compare them applying Two Way Analysis of Variance. It was found that, on the 7cm high‐heel group, the EF reduces and the RVF increases in relation to barefooted group (p<0,05). The platform heel group presents an increase for RVF values compared with barefooted group (p<0,05). There is no statistical difference when comparing the 3.5 cm high‐heel with the other groups. VFI values did not show difference among the 4 groups. These results indicate that the 7cm and platform heels impair the muscle pump what changes the venous physiology. This is demonstrated by the increase on FVR values. The absence of variation for VFI indicates that the venous valves competence is preserved. FAEPA.