bluuebunny/crossref_metadata_2025 huggingface dataset:
Cardiovascular risk factors are related to vitamin D status in midlife and older African Americans
Authors: Lemacks Jennifer Lynn , Ernst Jasminka Ilich , Ralston Penny A. , Young-Clark Iris , Coccia Catherine , Wickrama Kandauda , Harris Cynthia M.
Published: June 2021

Some epidemiological studies suggest that CVD risk factors are negatively related to vitamin D status. The evidence is not clear and is sparse in African Americans (AAs) who are already at higher risk for CVD and have lower vitamin D status. Our objective was to examine the relationship between CVD risk factors and vitamin D status in midlife/older AAs enrolled in a church‐based health intervention project. 71 participants (73% women) had following mean±SD values: age 59.5±10.1y; BMI 34.9±9.2kg/m2; serum vitamin D (25OHD) 56.6±31nmol/L. 50% participants were vitamin D deficient/inadequate based on IOM's classification (serum 25OHD≤50nmol/L). Participants were divided into inadequate (IND) or adequate (ADD) vitamin D status groups and independent t‐tests revealed ADD had significantly lower BMI (31.5 vs 36.3kg/m2) and LDL (111.6 vs 126.1mg/dL) compared to IND. There were no significant differences in triglycerides, TotalChol, VLDL, HDL and blood pressure between groups. Separate regression models, controlled for age, gender, season, BMI, PTH and calcium/protein intake, showed negative influence of LDL, TotalChol, and ApoB/ApoA and positive of HDL on 25OHD. Based on these results, some CVD risk factors and markers of obesity are negatively related to vitamin D, strengthening the importance of higher vitamin D status in this population. NIH Institute on Minority Health & Health Disparities (R24MD002807‐02)

Grant Funding Source : NIH
Antihyperglycemic and antioxidant activities of Nopal (Opuntia ficus indica) in patients with type 2 diabetes
Authors: Lopez Patricia , Pichardo-Ontiveros Edgar , Avila-Nava Azalia , Pedraza-Chaverri Jose , Tovar Armando R , Torres Nimbe
Published: June 2021

Nopal (Opuntia ficus indica), a Mexican variety of cactus is widely used as a food and as a popular treatment for type 2 diabetes (T2D). Thus, the aim of this work was to evaluate whether the consumption of 300 g of steam nopal in a high carbohydrate breakfast (HCB) or in a high soy protein breakfast (HSPB) modified blood glucose, serum insulin, plasma GIP, GLP‐1 and antioxidant activity in plasma of patients with T2D. Fourteen patients with T2D, aged 48.7 ± 2.1 y, with a BMI of 28.99 ± 1.0 kg/m2 were recruited. Blood samples were drawn before and 15, 30, 45, 60, 90, 120,150 min following breakfast tests. HPB was significantly more effective to regulate the postprandial glucose regardless of the presence of nopal, however the combination of HPB+nopal was more effective to control GIP and showed more postprandial antioxidant activity than just the HPB. These results suggest that nopal in combination with soy protein is an effective antioxidant to reduce reactive oxygen species (ROS) and also is an antihyperglycemic food to control postprandial glucose in patients with T2D. Supported by Grupo Produce México (to NT, 2010)
Bioavailability and Brain Deposition of Proanthocyanidin (PAC), Anthocyanin and Flavonoid in Combi‐Phenol Treated Rats on High Fat (HF) or Low Fat (LF) Diet
Authors: Chen Tzu-Ying , Janle Elsa , Ferruzzi Mario , Weaver Connie , Simon James , Wu Qingli , Talcott Stephen , Marambaud Philippe , Wang Jun , Ho Lap , Pasinetti Giulio M.
Published: June 2021

Consumption of grape derived polyphenols is associated with the prevention of neurodegenerative diseases such as Alzheimer's disease (AD). HF diet, on the other hand, has been shown to be a risk factor on AD onset and progression. To further understand the physiological impact of these compounds ingested as a grape polyphenolic preparation termed ‘Combi‐Phenol’ and the influence of dietary fat on absorption and metabolism of PAC monomer, flavonoid, and anthocyanin, studies were conducted with Sprague‐Dawley rat fed on HF or LF diet. Rats were divided into four groups: LF‐control, HF‐control, LF‐Combi‐Phenol, HF‐Combi‐Phenol and fed on individual diet for 2 weeks prior to Combi‐Phenol treatment. Plasma pharmacokinetics was determined after dosing of Combi‐Phenol or water for 10 days. LC‐MS/MS analysis showed that glucuronide and methylated glucuronide are the main metabolites for (epi)catechin, quercetin, and resveratrol. Glucosides were the major derivatives for anthocyanins present in plasma and brains following Combi‐Phenol consumption regardless fat content in the diet. These data illustrate that dietary fat content did not affect the bioavailability of Combi‐Phenol and their access to the brain was also not influenced.

Supported by NIH Grant: P01AT004511

Grant Funding Source: NIH
The effects of the interaction between adiponectin genotype and dietary folate, vitamin B6 and vitamin E on serum TNF‐α level in the patients with type 2 diabetes
Authors: Kim Ki Nam - , Kim Soyeon , Kim Hyesook , Choi Young Ju , Huh Kap Bum , Chang Namsoo
Published: June 2021

Adiponectin is known to relate with insulin resistance and CVD risk due to its anti‐inflammatory effect. To evaluate the effect of interaction between the adiponectin genotype and nutrient factors on tumor necrosis factor‐α (TNF‐α), SNP 276G→T at adiponectin gene was evaluated by a PCR‐RFLP analysis and TNF‐α was measured by ELISA kit. Dietary intakes were analyzed by using semi‐quantitative food frequency questionnaire. Sex, age, BMI, smoking, and alcohol drinking were used for adjustment and energy intake was used only in the analysis of dietary factors. 569 patients with T2D (287 males and 282 females) were enrolled, whose prevalence of G/G, G/T and T/T genotype was 45.9%, 45.7% and 8.4%, respectively. TNF‐α was correlated with HbA1c (r=0.237, P<0.0001), KITT, an insulin resistance index (r=−0.160, P<0.0001), atherosclerosis index (r=0.104, P<0.05) and log‐transformed intakes of dietary folate (r=−0.156, P<0.0001), vitamin E (r=−0.145, P<0.01) and vitamin B6 (r=−0.123, P<0.01). However negative relationships between above vitamins and TNF‐α were significant only in the G/G genotype (P‐values for trend were <0.0001, <0.001, and <0.01, respectively). In conclusion, dietary vitamins could reduce serum TNF‐α, a risk factor of insulin resistance and CVD, and it could be more effective in the T2D patients with normal adiponectin genotype. This work was supported by the Brain Korea 21 project.
Differential Regulation of Liver Gene Expression by in vitro Digested Soy and Caseinate
Authors: Li Jia , Liu Peter , Cope Mark , Crosby Seth , Napawan Nida , Krul Elaine S.
Published: June 2021

The mechanisms underlying the cholesterol lowering effects of soy protein remain unknown. The current study investigated the gene expression profile of a human liver cell line, HepG2, after 18 hours of exposure to 1 mg/ml soy (S) or caseinate (C) protein. S or C was predigested with pepsin and pancreatin to mimic gastrointestinal digestion. Lovastatin (L) (10 μM) and baseline control (Con) were included. Whole genome expression was profiled using an Illumina® microarray. Results indicated that 268 genes were regulated by either S and/or C versus Con, with the patterns of regulation observed being unique to the protein hydrolysate studied. Real‐time quantitative PCR (RT‐qPCR) experiments were conducted using a subset of genes differentially expressed by microarray with a focus on genes that regulate lipid metabolism. RT‐qPCR confirmed the microarray expression profiles observed for L and showed similar trends for S and C compared to Con. RT‐qPCR results showed that the MYLIP gene, whose protein product is responsible for LDL‐R degradation, decreased in HepG2 cells after S treatment compared to Con (0.45 ± 0.076 fold, p = 0.002) or C (0.55 ± 0.092, p = 0.018), with no significant difference between C and Con. Therefore, the cholesterol lowering effect of soy protein may partially be due to increased liver cholesterol uptake as a result of decreased LDL‐R degradation. Funded by Solae, LLC
Retinaldehyde‐Mediated Lipolysis Underlay Sexual Dimorphism in Visceral Obesity in Mice
Authors: Yasmeen Rumana , Reichert Barbara , Duester Gregg , Ziouzenkova Ouliana
Published: June 2021

Increased risk of mortality in men and women is associated with the development of visceral fat that occurs in a sex‐specific manner by poorly understood mechanisms. Vitamin A metabolites: retinaldehyde (Rald) and retinoic acid (RA) are known to regulate key biological processes. Here, we explored the role of Rald and RA in sex‐specific fat formation, in a mouse model deficient in Aldh1a1, a major RA‐producing enzyme in white adipose tissue. The Aldh1a1−/− mice displayed a lean phenotype compared to wild type (WT), even on a high‐fat (HF) diet (300days). While subcutaneous fat remained similar in WT males and females, it was reduced in the Aldh1a1−/− mice. Strikingly, only Aldh1a1−/− females had significant reduction in visceral fat mass (12.3‐fold lower than WT females), while visceral fat reduction in Aldh1a1−/− males was insignificant (2.3‐fold) compared to WT males. We found markedly higher protein levels (compared to WT) of adipose triglyceride lipase (Atgl) in Aldh1a1−/− females vs. males. Consistant with Atgl's key role in lipolysis, visceral fat in Aldh1a1−/− female, but not male mice has Atgl‐positive staining in clusters of multilocular cells, also co‐expressing thermogenic proteins. The effective induction of Atgl protein levels was proven to be mediated by Rald. Altogether, our data suggest that Aldh1 could be a crucial target for treating female visceral obesity.

Grant Funding Source : The EHE SEED Grant, The Ohio State University
Fetal growth and birth weight are determined by maternal fat mass at mid pregnancy in low‐income Brazilian women
Authors: Toro-Ramos Tatiana , Sichieri Rosely , Hoffman Daniel J.
Published: June 2021

The relationship between maternal body composition and fetal development is unclear. The objective of this study was to determine whether maternal body composition determines fetal growth in women with low, normal, and high energy intakes. Two hundred pregnant women were recruited from the Instituto Fernandes Figueira, Rio de Janeiro, Brazil. Dietary intake was assessed using a food frequency questionnaire validated for Brazilian adults and body composition was estimated using bioimpedance analysis. Fetal growth was assessed using serial ultrasound measure and infants underwent anthropometric measurements at birth. Multiple linear regression analyses were used to determine the association between fetal growth and birth weight with percent fat mass (%FM) and percent fat‐free mass (%FFM), controlling for infant gender, maternal serum glucose, and BMI. %FM was positively associated with birth weight adjusted for gestational age (BWTadj), estimated fetal weight change (ΔEFW), and change in fetal length (ΔFL), yet %FFM was negatively associated. When stratified by energy intake, %FM was a significant predictor of BWTadj in women with normal energy intake, but a significant predictor of ΔEFW and ΔFL in women with low energy intake. FM may be used as a reliable indicator of maternal nutritional status for individual recommendations on weight gain during pregnancy. This study was funded by the American Egg Board.
α(1,3)‐Fucosyltransferases‐IV/‐VII modulate the prevalence of murine IL‐17‐producing cells in vivo
Authors: Mackey Lantz C. , Rose Jason M , Homeister Jonathon W.
Published: June 2021

Mice deficient in α(1,3)‐fucosyltransferase (FucT)‐IV and ‐VII (DKO) lack selectin‐dependent leukocyte trafficking and homing, have an increased prevalence of CD4+ TH17 cells, and elevated levels of circulating IL‐17a compared to wild‐type (WT) mice. We sought to determine if FucT‐IV/‐VII‐mediated fucosylation can regulate the prevalence of all IL‐17a‐producing cells. Flow cytometry was used to assess alterations in IL‐17a‐producing cell populations. Mononuclear leukocytes were isolated from blood (BL), spleen (SP), and submandibular lymph nodes (SMLN) of DKO (n=9) or WT (n=9) mice, stimulated with a PMA/Ionomycin and treated with brefeldin A. CD3+ CD4− NK1.1− CD49b+ (iNKT‐ 17) and CD3−CD4−NK1.1−CD90+ (ILC‐17) cells were analyzed for intracellular staining of IL‐17a. DKO mice had increased numbers of iNKT‐17 and ILC‐17 cells compared to WT: iNKT‐17 cells were increased 23‐fold in the SP, 7‐fold in BL, and 30‐fold in SMLN; ILC‐17 cells were increased 17‐fold in the SP, 5‐fold in BL, and 9‐fold in SMLN. Mice deficient in PSGL‐1, P‐selectin, or E‐selectin had WT levels of IL‐17a‐producing cells. These data show that loss of both FucT‐IV/‐VII causes an expansion of IL‐ 17a‐producing populations in vivo and suggests that α(1,3)‐ fucosylation events may regulate the differentiation, proliferation, and/or maintenance of IL‐17a producing cells in a selectin‐independent manner.
Peritoneal Mechanobiology and Metastatic Success in Epithelial Ovarian Cancer
Authors: Burkhalter Rebecca J , Liu Yueying , Marszow Michael , Sun Zhe , Meininger Gerald , Wagner Diane , Stack M. Sharon
Published: June 2021

Less than 30% of women diagnosed with epithelial ovarian cancer (EOC) survive for 5 years due to metastatic disease. EOC progression is accompanied by accumulation of ascites (up to 4 liters), changing intraperitoneal (i.p.) fluid pressure from subatmospheric (−5 mmHg) to >20 mmHg with tense ascites. The overall consequence of altered peritoneal mechanobiology on EOC metastatic success is currently unknown. Due to altered i.p. fluid dynamics the peritoneal mesothelium, an elastic soft tissue, may be modified by exposure to tensional force (strain). Mechanical strain (stretching) on intact murine mesothelial explants causes increased tissue stiffness, as determined by atomic force microscopy, and enhances ovarian tumor cell adhesion. To approximate the compressive force environment on EOC cells in tense ascites, cells were seeded in suspension in polystyrene bags and cultured under a compressive load (Instron, 25mmHg, 8h). Increased fluid pressure enhances proliferation in multiple cell lines and increases expression of N‐cadherin, a mesenchymal marker, in Dov13 cells. These data suggest that ascites‐induced changes in peritoneal mechanobiology may potentiate EOC metastatic success. Support: NIH‐R01CA109545, NIH R01CA086984, NIH F31 R01CA086984‐12S1.
VEGFR2 activation during the early onset of flow is ligand‐dependent and mediated by the release of autocrine VEGF
Authors: dela Paz Nathaniel G. , Frangos John A.
Published: June 2021

Previous studies have shown that VEGFR2 is part of a mechanosensory complex in endothelial cells (ECs) and is activated in response to fluid shear stress in a ligand‐independent manner. Here, we tested the hypothesis that shear stress induces a rapid release of heparan sulfate (HS)‐sequestered VEGF, which subsequently activates VEGFR2 in a ligand‐dependent manner. Our results showed that VEGFR2 is phosphorylated at Y1175 and Y1214 within 15 sec of flow in human coronary artery ECs. Removal of HS by heparinase III digestion led to a decrease in VEGFR2 phosphorylation. Transfection of siRNA against VEGF and blockade of VEGFR2 with a neutralizing antibody were also performed to demonstrate that early activation of VEGFR2 is ligand‐dependent. To determine whether VEGFR2 activation is regulated via the phospholipase C (PLC) pathway, we sheared ECs after pre‐incubation with two structurally different PLC inhibitors, U73122 and Edelfosine. Phosphorylation of VEGFR2 in response to flow was diminished by both drugs, suggesting that VEGFR2 activation may be downstream of PLC activation. Together, these studies aim to identify the role(s) and mechanism(s) by which HSPGs participate in flow‐induced VEGFR2 activation.
Is mass cytological cancer screening possible worldwide?
Authors: Markovic Olivera , Markovic Nenad
Published: June 2021

Our answer is yes, but with appropriate tools.

Although preventable, cervical cancer is still, second to the breast cancer, major killer of women from malignant diseases worldwide. The outreach for preventive cytological screening is less than 10% in the most of the developing counties. Screening for risk factors such as HPV have contributed, but still not sufficiently.

At EB 2004, we presented for the first time our proprietary, biomarker‐based MarkPap® platform technology. The biomarker is cytoplasmic expression of genetic changes in cervical cells during transformation from normal to pre‐malignant (dysplastic) and malignant. At subsequent EB meetings, we presented further developments of this technology from manual to semi‐automated, to diagnosis at distance‐telecytopathology (both digital and wireless), including home self‐collection. Most recently, we made self‐collection kit. With all these small devices, a low‐trained technician at the POC in remote sites, can process the specimen with customer‐friendly MarkPap® kit, locate suspicious cells by the biomarker and transmit microscopic images of those cells via MarkPap® Digital networking system to expert sites for instant diagnosis. Our recent testing with China and India proved that this concept is feasible.

Consequently, development of low‐cost devices for mass cervical cancer screening for developing countries is warranted. .
Novel treatment of bladder cancer with cell therapy
Authors: Jeong Christina , Luria Ayala , Nolta Jan , Kurzrock Eric
Published: June 2021

Our laboratory is studying a potential treatment for transitional cell carcinoma by developing a safe technique for the de‐epithelialization of murine bladder, evaluating aspects of spontaneous native urothelial regeneration, and determining the efficiency of cell replacement therapy. All techniques were performed on cadaveric mice. The mice were catheterized, and their bladders were distended by injecting 0%, 1%, 2%, or 4% trypsin for different periods of time ranging between 30 – 90 minutes at 37°C. Bladders were fixed and stained with hemotoxylin and eosin for histological analysis. The treatments with 1% trypsin had no apparent effect on the epithelial layer. With 2% trypsin, the epithelial cells start to detach leaving patches of urothelium on the basement membrane. Treating the bladder with 4% trypsin for 90 minutes completely removes the epithelial layer with the basement membrane still intact. Our results confirmed that 2% and 4% trypsin treatments can achieve different levels of de‐epithelialization in cadaveric mice. The results will be used as guidelines as we perform the same techniques in vivo. Subsequently, an analysis of cell regeneration and oncogenic‐free urothelium replacement will be done. By applying these tissue engineering technologies to human cancer models in the future, we can offer an entirely new approach to treat bladder cancer.

UC Davis Cancer Center Developmental Awards
Cocaine dose and self‐administration history, but not initial cocaine‐induced locomotor responsiveness, affect sensitization to the motivational effects of cocaine in rats
Authors: Mandt Bruce H , Gomez Emily , Johnston Nickie L , Zahniser Nancy R , Allen Richard M
Published: June 2021

Cocaine addiction is a significant and complex disease. Part of this complexity is due to variability of the drug experience early in drug use (e.g., initial responsiveness, amount of use, etc.). In rats, individual differences in initial cocaine responsiveness and cocaine self‐administration history both predict the development of cocaine sensitization, a putative mechanism contributing to addiction. Here we sought to determine the role of these factors and cocaine dose on the development of sensitization to cocaine's motivational effects during the earliest stages of self‐administration. Rats were classified as either low or high cocaine responders (LCRs or HCRs, respectively) based on acute cocaine‐induced locomotor activity (10 mg/kg, IP) before learning to self‐administer cocaine (0.75 mg/kg/IV inf) under a fixed ratio 1 (FR1) schedule. Following acquisition, rats advanced to progressive ratio (PR) testing (0.75 or 1.5 mg/kg/inf cocaine) either immediately or after an additional 5 FR1 sessions (0.75 or 1.5 mg/kg/inf cocaine). No LCR/HCR differences were found. However, exposure to the higher dose of cocaine under both schedules produced sensitization to cocaine's motivational effects (i.e., increased break points). Thus, our results reveal the development of sensitization during the earliest stages of drug self‐administration and suggest this process may occur very early in drug addiction.

This work was supported by NIH Grants R01 DA004216 and K05 DA015050 and an ASPET SURF award.
Role of Central Atypical Cannabinoid Receptor GPR18 in Modulating Cardiovascular Function
Authors: Penumarti Anusha , Abdel-Rahman Abdel A
Published: June 2021

Background and objectives

The abnormal cannabidiol (Abn CBD) receptor GPR18 mediates peripheral vasodilatation. N‐arachidonoyl glycine (NAGly) is the endogenous ligand of GPR18. Despite NAGly's abundance in the CNS and GPR18 mediated peripheral vasodilation, there are no studies on the role of central GPR18 in blood pressure control. Therefore, we investigated if GPR18 is: (i) present in the brainstem cardiovascular regulatory nuclei, particularly in presympathetic tyrosine hydroxylase (TH) expressing neurons; (ii) co‐expressed with Cannabinoid 1 receptors (CB1R) in those neurons and, (iii) the impact of GPR18 activation and blockade on blood pressure (BP) and heart rate.

Key results

Our immunofluorescence studies demonstrated GPR18 expression in the rostral vento‐lateral medulla (RVLM). GPR18 is co‐localized with TH expressing neurons as well as with CB1R in RVLM neurons. Intra‐RVLM microinjection in conscious male Sprague Dawley rats demonstrated dose‐dependent reduction in BP following GPR18 activation with Abn CBD. By contrast, intra‐RVLM blockade of GPR18 with the antagonist O‐1918 caused increase in BP and abrogated the Abn CBD‐evoked reduction in BP.

Conclusions

The present studies are the first to demonstrate the expression of GPR18 in the RVLM and to suggest a role for this receptor in modulating central cardiovascular functions.
Thrombin stimulated glioblastoma cell adhesion is mediated by Rap1 and integrin activation
Authors: Sayyah Jacqueline , Stupack Dwayne , Brown Joan Heller
Published: June 2021

Rap1 is a Ras family GTPase that plays an important role in ERK1/2 activation, cell proliferation and adhesion. Little is known about the mechanisms for activation or the role of Rap1 in signaling through GPCRs. We have previously reported that thrombin coupling to the G protein‐coupled receptor PAR‐1 induces a robust and sustained increase in Rap1 activation that is mediated by RhoA‐phospholipase D (PLD) signaling and that this pathway is necessary for sustained ERK1/2 activation and glioblastoma cell proliferation. Rap1 also mediates cellular signals that regulate integrins from inside the cell thereby affecting integrin‐mediated cell adhesion. Accordingly, we asked whether PLD and Rap1 are critical for thrombin induced glioblastoma cell adhesion. Down‐regulation of PLD1 and Rap1 with siRNA significantly blocked thrombin induced glioblastoma cell adhesion to fibronectin by 50–60 percent. In addition, blocking β1 integrin activation via neutralizing antibody inhibited thrombin stimulated ERK1/2 activation in these cells. Collectively, our results suggest that Rap1 activation by thrombin contributes to integrin activation and downstream signaling that could be important for the aberrant growth or migration of glioblastoma cells. We are currently examining in more detail the integrin signaling pathways that are activated downstream of thrombin/PAR‐1 mediated Rap1 activation.
In‐Vitro and In‐Vivo Studies of Antiasthmatic and Mast Cell Stabilizing Activity of Yemeni Sidr Honey
Authors: Alzubier Aman Ahmad , Okechukwu Patrick Nwabueze
Published: June 2021

Yemeni Sidr honey has been reported to possess anti‐inflammatory, antipyretic and analgesic effect. In this study the in‐vitro and in‐vivo antiasthmatic and mast cell stabilizing effect of Yemeni Sidr honey were investigated. The inhibition of contraction and hind paw oedema induced by inflammatory mediators such as; histamine, acetylcholine, serotonin (5HT), bradykinin, and leukotriene by Yemeni Sidr honey were examined using guinea pig ileum in organ bath and rat induced paw oedema. Mast cell stabilizing effect was tested, using 0.5 ml subcutaneous injection horse serum along with 0.5ml of triple antigen containing 20000 million microorganisms. Yemini Sidr Honey showed a significant concentration ‐ dependent inhibition of contraction and hind paw oedema induced by the inflammatory mediators and inhibited the degranulation of mast cells. This study revealed that Yemeni Sidr honey possesses significant dose‐dependent antiasthmatic activity and markedly protected the rats’ cells against antigen‐induced challenge of mast cells. This result is in agreement with the previous report and confirms the traditional use.
Calcium sensitization underlies endothelium‐dependent hypoxic augmentation in the porcine coronary artery
Authors: Vanhoutte Paul M , Chan Calvin K
Published: June 2021

In isolated porcine coronary arteries hypoxia (PO2:30 mm Hg) causes endothelium‐dependent potentiation (hypoxic augmentation) of contractions to vasoconstrictor agonists. Previous studies show that the presence of nitric oxide (NO), but not cyclic GMP, is a prerequisite for this augmentation to occur. Experiments were designed to determine the calcium‐dependency of the phenomenon. Rings (with or without endothelium) of porcine coronary arteries were suspended in organ chambers for the measurement of isometric tension, contracted with the TP receptor agonist U46619 and exposed to hypoxia. Incubation with a voltage operated calcium channel inhibitor (nifedipine) or calcium free buffer did not significantly affect hypoxic augmentation, while the calcium chelator EGTA abolished it. Hypoxia caused no significant increase in intracellular calcium concentration (detected by confocal fluorescence microscopy) in vascular smooth muscle. However, incubation with the Rho kinase inhibitors HA1077 or Y27632 reduced the response while thapsigargin significantly augmented it. These results suggest that hypoxic augmentation requires calcium, but does not depend on influx of extracellular calcium or an increase in the intracellular concentration of the activator ion. Thus, the endothelium‐dependent, NO‐requiring hypoxic augmentation involves calcium sensitization mediated by Rho kinase.
The Role of Alternative Polyadenylation in Mediating Stress‐ Induced Protein Expression of Hsp70.3
Authors: Tranter Michael , McGuinness Michael , Helsley Robert N , Paulding Waltke R , Brokamp Cole , Haar Lauren , Ren Xiaoping , Jones W. Keith
Published: June 2021

Heat shock protein 70.3 (Hsp70.3) expression is increased in response to cellular stress and plays a cytoprotective role. It has been shown that increased expression of Hsp70.3 expression in the heart is protective against ischemia/reperfusion injury. Recent work from our lab shows that Hsp70.3 expresssion is controlled through coordinated post‐transcriptional regulation by miRNAs and alternative polyadenylation (APA).

Results show an APA‐mediated shortening of the Hsp70.3 3’‐UTR upon a stress‐induced increase in expression that facilitates increased protein expression during stress‐induced upregulation of the gene product. This work investigated the mechanisms through which APA‐mediated shortening of the 3’‐UTR contributes to increased protein expression. Our results describe the role of APA on mRNA stability and longevity, micro‐RNA regulation of Hsp70.3, and the role of APA on polysome loading and translational efficiency of the Hsp70.3 transcript.

Our results provide a mechanistic understanding of the role APA plays in regulation of Hsp70.3 gene expression and have broad implications for the understanding of APA as a global modulator of gene expression.

This work was supported by NIH RO1 HL0901478 (WKJ) and NIH T32 HL007382 (MT).
Hypoxia‐induced lipid accumulation involves a HIF‐α independent, oxidative stress‐mediated regulation of FGF‐21 in HepG2 cells
Authors: Liu Yanlong , Wang Yuhua , McClain Craig J , Feng Wenke
Published: June 2021

Fibroblast growth factor 21 (FGF‐21) has emerged as an important metabolic regulator of glucose and lipid metabolism. Previous studies have indicated that FGF‐21 regulates lipolysis in the adipose tissue, and increases fatty acid oxidation in the liver. Although hypoxia has been attributed to lipid metabolic dysfunction in several tissues, it is not known whether hypoxia plays a role in FGF‐21 regulation. In the present study, HepG2 cells were used to determine the role of FGF‐21 regulation in cellular triglyceride accumulation under physical and chemical hypoxia. Incubation of HepG2 cells with a chemical hypoxia inducer, CoCl2, or exposing the cells to 1% O2, significantly increased hypoxia‐inducible factor 1α protein accumulation and the target gene, glucose transport 1, expression, and dramatically reduced cellular FGF‐21 mRNA and secreted FGF‐21 protein levels along with a significant increase in cellular triglyceride content. Chemical hypoxia also induced a significant reduction in PPARα/γ mRNA levels, which have been shown to regulate FGF‐21 expression in hepatocytes and adipocytes. Knocking down HIF‐1α or HIF‐2α using specific siRNAs neither restored CoCl2‐reduced FGF‐21 expression, nor reduced triglyceride accumulation, indicating that HIF‐α is not responsible for CoCl2‐induced FGF‐21 reduction and fat accumulation. However, addition of N‐acetyl cysteine completely restored CoCl2‐induced reduction in FGF‐21, and attenuated the fat accumulation, indicating the role of oxidative stress in hypoxia mediated FGF21 regulation and lipid metabolism. The results suggest that hypoxia regulates FGF‐21 and cellular fat metabolism through a HIF‐α independent, but oxidative stress‐mediated pathway.
Synergistic Effect of Cysteinyl Leukotriene Receptor Antagonist and H‐1 Receptor Antagonist in Acute and Chronic Guinea Pig Asthma Models
Authors: Alluri Ramesh , Routhu Kasi V. , Nadendla Mukharjee , Emandi Hemalatha
Published: June 2021

Histamine and cysteinyl leukotrienes are pivotal mast cell mediators which contribute considerably and likely complementary to the symptoms of asthma. The present study hypothesized to explore the direct actions of histamine and cysteinyl leukotrienes, on acute platelet activating factor and chronic Ovalbumin induced guinea pig asthma models. In our In‐ vivo guinea pig studies, oral levocetrizine (0.1mg/kg) and montelukast (0.1mg/kg) had significantly reduced antigen‐induced total leukocyte count and eosinophil count. Similarly, the dose–response characteristics of montelukast (0.1–10mg/kg) was observed against ovalbumin induced guinea pig asthma model. Our studies support the position that histamine and cysteinyl leukotrienes may act collaboratively to elicit infiltration of leukocytes especially eosinophils are the target to treat asthma. Levocetrizine and Montelukast had reduction in IL‐4 &IL‐5 in bronchoalveolar lavage fluid. Combination of levocetrizine and montelukast had produces synergistic effect in both models. Therefore, this combination may therefore useful for treating airway inflammation in bronchial asthma.
Epac/Rap1‐regulated deactivation of PAF‐induced hyperpermeability is independent of eNOS activation
Authors: Zhang Jihang , Rana Roshiniben J , Duran Ricardo G , Kim David , Duran Walter N
Published: June 2021

Platelet‐activating factor (PAF) increases endothelial permeability via phosphorylation and translocation of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production. PAF‐induced hyperpermeability is partly attenuated through Epac/Rap1 pathway. We tested the hypothesis that activation of Epac/Rap1 inhibits PAF‐induced phosphorylation and localization of eNOS. We activated Epac/Rap1 specifically with 8CPT (8‐(4‐chlorophenylthio)‐2′‐O‐methyladenose‐3′,5′‐cyclic monophosphate) in human dermal microvascular endothelial cells (HMVEC). 8CPT activated eNOS‐S1177 phosphorylation and dephosphorylation of eNOS on T495, starting as early as 30”, achieving peak at 1′, and returned to the basal level at 10′; a time‐course that is similar to PAF stimulation. 8CPT applied after PAF did not attenuate eNOSS1177 phosphorylation or dephosphorylation on T495. Furthermore, 8CPT increased NO production in dose‐dependent manner. Interestingly, our immunofluorescence microscopy studies revealed that 8CPT inhibited PAF‐induced eNOS internalization. Our data suggest that Epac/Rap1 reverses PAF‐induced hyperpermeability by limiting eNOS to the cell plasma membrane while allowing eNOS phosphorylation and NO production. (Supported by NIH grants HL070634 and HL088479).
The Vascular Renin Angiotensin System Contributes to Endothelial Dysfunction Induced by Acute High Pressure in Human Adipose Microvessels
Authors: Durand Matthew J. , Phillips Shane A. , Gutterman David D.
Published: June 2021

We have previously shown that transient elevations in intraluminal pressure reduce endothelium‐dependent vasodilation, but the mechanism of this impairment is unknown. Since the renin‐angiotensin system (RAS) is activated by barostress, we hypothesized that the pressure‐induced endothelial dysfunction is mediated by local generation of angiotensin II (ANG II). Arterioles (100–200 μm) from discarded surgical adipose specimens were cannulated onto glass micropipettes, pressurized to 60 mmHg and constricted with endothelin‐1. Vasodilation to acetylcholine (ACh; 10−9 – 10−4 M) or papaverine (10−9 – 10−4 M) was assessed before and after the vessel had been exposed to an intraluminal pressure of 150 mmHg for 30 minutes. Dilation to ACh was significantly reduced in vessels exposed to 150 mmHg of pressure, while the response to papaverine was unaffected. The pressure‐induced endothelial dysfunction was prevented by incubating the vessels with PEG‐SOD (100 U/mL), the angiotensin type 1 receptor antagonist losartan (1 μM), or the angiotensin converting enzyme inhibitor captopril (1 μM). The acute high pressure did not affect vessel sensitivity to ANG II (10−11 – 10−7M). We conclude that local generation of ANG II by the vascular RAS plays a causative role in pressure‐induced endothelial dysfunction, possibly by increasing vascular superoxide. This study was supported by NHLBI grants HL‐094971 and HL‐080704.
Placental growth factor (PlGF) is a potent dilator of uterine and mesenteric veins isolated from pregnant rats
Authors: Mandala Maurizio , Kostin Shannon , Osol George
Published: June 2021

PlGF, a member of the VEGF family, is produced by the placenta; impairment of its signalling has been reported in preeclampsia. We tested the effect of PlGF on uterine and mesenteric veins to evaluate its vasoactive properties in reproductive vs. non‐reproductive tissues. Uterine (pre‐myometrial, pre‐placental) and third order mesenteric veins isolated from pregnant rats (n=12) were cannulated and pressurized (6 mmHg) in an arteriograph. All vessels were preconstricted 40–50% with U46619 prior to being exposed to PlGF (10pM–3nM). PlGF produced vasodilation in a concentration‐dependent manner in all veins. Similar maximal relaxations were observed in pre‐myometrial (32 ± 5%) and pre‐placental (36 ± 10%) veins, while greater efficacy (52 ± 10%, p<0.05) was noted in mesenteric veins. The kinetics of the vasodilatory response were also different, with half times on the order of 20 vs. 10 minutes in uterine vs. mesenteric vessels. In mesenteric veins, inhibition of NOS + COX reduced vasodilation by 65% on average, to 18 ± 9% (p = 0.03). Thus, PlGF is a potent vasodilator of both reproductive and systemic veins, with significantly more potent and rapid effects in the mesenteric circulation, suggesting an important role for the tissue VEGFR‐1 (Flt‐1) receptor in regulating maternal gestational hemodynamic changes, e.g. venous capacitance and return, and cardiac output.

This study was supported by NIH HL79253
Lymphatic valve lock in response to modest gravitational loads: a contributing mechanism to peripheral lymphedema?
Authors: Davis Michael John , Moore James E , Zawieja David C , Gahsev Anatoliy A , Scallan Joshua P
Published: June 2021

Lymphedematous limbs often have an uninterrupted column of lymph that implies an underlying contractile dysfunction of collecting lymphatic vessels. To test this idea, single lymphangions were isolated, cannulated and connected to a servo system to control input (Pin) and output (Pout) pressures. Intraluminal pressure was measured with vessel diameter and valve position. As Pout and Pin were slowly raised in parallel, with Pout > Pin to mimic conditions in a dependent extremity during development of edema, the vessel progressively distended and the output valve would appear to suddenly “lock” into the open position. Valve lock appeared to result from the interaction of three factors: tone, pump strength, and valve leaflet stiffness. In multiple lymphangion segments, valve lock occurred in the weakest lymphangion and progressed to its neighbors as pressure rose. Surprisingly, valve lock was reversible if tone was enhanced using a contractile agonist, even at elevated Pout levels. We propose that pump strength, valve leaflet stiffness and tone normally interact to minimize the likelihood of valve lock, which reflects an inherent susceptibility of the valves that can be triggered by modest pressure or volume overload. Once valve lock occurs, it would exacerbate edema by creating an uninterrupted lymph column that transmits a pathological pressure level back to the lymphatic capillaries.
Effects of HBOC‐201 in a porcine model of impending myocardial infarction produced by severe coronary artery stenosis
Authors: Hekkert Maaike te Lintel , Dubé Gregory , Lankhuizen Inge , van Deel Elza , de Beer Vincent , Merkus Daphne , Serruys Patrick , Duncker Dirk
Published: June 2021

Hemoglobin oxygen carrier HBOC‐201 can act as a direct oxygen donor and facilitate diffusive oxygen delivery between red blood cells and tissues to improve tissue oxygenation. Recently we showed that pre‐oxygenated HBOC‐201 infused distal to a coronary occlusion, fully restored aerobic metabolism and myocardial function. Here, we studied the effects of systemic administration of HBOC‐201 on left ventricular (LV) function and infarct size (IS) in pigs with coronary stenosis. A stenosis placed in the left anterior descending coronary artery for 120 min reduced flow by 70±2%. HBOC‐201 (1 g/kg), with or without nitroglycerine (NTG), were infused iv over 30 min, starting 15 min after the onset of ischemia. After 120 min of reperfusion, the area‐at‐ risk (AR), infarct area (IA) and IS=IA/AR•100% were determined. HBOC‐201 did not ameliorate stenosis‐induced loss of regional systolic segment shortening (from 20±1% to −3±1%) in the anterior LV wall and had no effect on IS (33±5%) compared to control (39±5%). To balance the purported scavenging of nitric oxide by HBOC‐201, we co‐infused NTG during HBOC‐201 which prevented systemic and coronary vasoconstriction. However, even in the presence of NTG, HBOC‐201 failed to blunt regional dysfunction or reduce IS (40±6%). Thus, despite the established oxygen transport capacity of HBOC‐201, it failed to afford cardioprotection in the setting of ischemia‐reperfusion in pigs.
Effects of Endogenously and Exogenously Elevated Bilirubin on Myocardial Ischaemia Reperfusion Injury
Authors: Bakrania Bhavisha , Bulmer Andrew
Published: June 2021

Cardiovascular disease is the world's greatest killer; additional therapies for targeted prevention/treatment are clearly required. Whether elevated endogenous or exogenous bilirubin attenuates cardiac ischaemia reperfusion injury is currently unknown. Echocardiography did not reveal significant differences (P<0.05) in cardiac structural measures in hyperbilirubinemic Gunn rats, however, showed reduced stroke volume (W‐T, 132 ± 29.2 μL, Gunn, 101 ± 12.8 μL, P=0.049) and peak aortic blood flow velocity (W‐T, 1225 ± 115 mm/s, Gunn, 712 ± 13.9 mm/s, P<0.01) leaving the left ventricle in Gunn rats, which could indicate that bilirubin reduces contractility of the heart. Langendorff perfused Gunn and bilirubin pretreated wild‐type hearts recovered moreso after an ischemic insult versus control hearts. Recovery was most pronounced at 90 minutes of reperfusion (W‐T, 38.5 ± 9.1 %, Gunn, 58.3 ± 11.3 %, P=0.04; Control, 33.5 ± 12.5, Pretreated, 56.3 ± 12.3 %, P=0.036). Tissue unconjugated bilirubin correlated positively with percent recovery in left ventricular developed pressure after ischaemia (r=0.69, P<0.01). These data suggest that the Gunn phenotype (analogous to Gilbert's syndrome in humans) or bilirubin ditaurate pretreatment induces cardioprotection by 1) reducing cardiac contractility at rest and/or 2) protecting from free radical mediated damage during ischaemia reperfusion.
Effects of NADPH oxidase inhibitor apocynin on real‐time blood hydrogen peroxide release in femoral artery/vein ischemia and reperfusion
Authors: Parker C. Woodworth , Bartol Kyle D , Perkins Kerry-Anne , Chen Qian , Young Lindon H
Published: June 2021

Vascular endothelial dysfunction can initiate oxidative stress during ischemia/reperfusion (I/R). Endothelial dysfunction is characterized by an increase in blood hydrogen peroxide (H2O2) and a decrease in endothelial‐derived nitric oxide (NO) bioavailability. Previous studies using Gö 6983, a broad‐spectrum protein kinase C inhibitor that can inhibit NADPH oxidase activity, has attenuated blood H2O2 levels during femoral I/R in vivo. This study examines the effects of apocynin, a direct NADPH oxidase inhibitor, on real‐time blood H2O2 levels in femoral I/R in vivo. H2O2 microsensors (100 μm) were inserted into both femoral veins in anesthetized rats. One hind limb was subjected to ischemia by clamping the femoral artery and vein for 20 minutes and allowed to reperfuse for 45 minutes, while the other hind limb served as a sham. In the control group, H2O2 was significantly increased by 1.3–2.7 μM during 45 minutes of reperfusion in the I/R vein when compared to the sham vein (n=4, P<0.05). Intravenous apocynin (13.7 mg/kg) given at the beginning of reperfusion significantly decreased blood H2O2 levels compared to the control group (n=6, P<0.05). The data suggests that apocynin attenuates oxidative stress in femoral I/R, which may be due to inhibition of leukocyte/endothelial NADPH oxidase.
Normal albumin inhibits histone‐induced platelet aggregation
Authors: Lam Fong Wilson , Leung Hon-Chiu Eastwood , Smith C Wayne , Rumbaut Rolando
Published: June 2021

Histones have been implicated as mediators of death and thrombosis in sepsis, a major cause of mortality in the U.S. The purpose of our study was to define plasma factors that modulate histone‐induced platelet aggregation, which we hypothesize to be albumin. Washed platelets from healthy adults were incubated with various concentrations of human serum albumin, charge‐neutralized albumin (via N‐ethyl‐N′‐(3‐dimethylaminopropyl) carbodiimide hydrochloride), and urea‐denatured albumin and studied using platelet aggregometry in the presence of histone H4 and control. Normal albumin inhibited maximal histone‐induced aggregation in a dose‐dependent manner (0, 2.5, 5, & 10 mg/mL albumin; 57, 28, 20, & 4% aggregation, respectively; p<0.005), while neutralized and denatured albumin lost this inhibitory effect. Furthermore, preliminary data suggest that normal, but not neutralized or denatured, albumin prevents binding of histone H4 to platelets under flow cytometry. These data suggest a role for albumin as an inhibitor of histone‐induced platelet aggregation and may lead to alternative treatment strategies in sepsis. Merit Review Grant from Dept. of Veterans Affairs.
Mannose‐binding lectin concentrations correlate with enhanced whole blood aggregation and thrombin‐like activity in humans
Authors: McClure Erin , La Bonte Laura R , Stahl Gregory L
Published: June 2021

Mannose‐binding lectin (MBL) and its associated serine proteases (MASPs) are part of the lectin pathway of complement system activation. Recently, the MBL‐complex was shown to have thrombin‐like activity in cellular and murine models, suggesting the complement and coagulation systems interact. The purpose of this study was to determine if the MBL‐complex displays thrombin‐like activity in human whole blood aggregation. Sera from low, normal, and high MBL‐expressing donors were collected and analyzed for thrombin‐like activity using the chromogenic thrombin substrate, S2238. In addition, blood was collected from 14 healthy human volunteers and whole blood aggregation (Chronolog) was assessed. Thrombin substrate cleavage correlated with increasing MBL levels (p < 0.001), and could be inhibited by GlcNAc, a competitive MBL inhibitor, verifying that the observed cleavage was MBL‐complex dependent. Whole blood aggregation was enhanced in donors with normal MBL levels (501 – 2000 ng/mL) compared to low MBL donors (<500 ng/ml; p < 0.01). However, no significant difference in whole blood aggregation was found between normal and high MBL donors, suggesting a saturation‐type effect. These results indicate that human MBL complex activation in vitro significantly activates the coagulation system in whole blood. Further study is required to determine the role of human MBL in in vivo coagulation.
SM22α Phosphorylation: A Novel Mechanism of Activation of PKCδ‐P47phox Axis and ROS Production in VSMCs
Authors: Pin Lv , Mei Han
Published: June 2021

Smooth muscle 22 alpha (SM22α), an actin‐binding protein abundant in vascular smooth muscle cells (VSMCs), is downregulated in atherosclerosis. The recent study indicates that disruption of SM22 promotes arterial inflammation through activation of reactive oxygen species (ROS). However, the molecular mechanisms by which SM22α controls the production of ROS have not been characterized. We investigated the effect of SM22α disruption on ROS production. ROS was detected using dihydroethidium for superoxide. The expression of proteins was detected by Western Blotting after transfection with small interfering RNA. We showed that down‐regulation and phosphorylation of SM22α were associated with angiotensin (Ang) II‐induced increase in ROS production in VSMCs. The phosphorylation of SM22α at Serine 181 was increased in an AT1R‐PKCδ pathway‐dependent manner in VSMCs upon Ang II. Phosphorylated SM22α facilitated Ang II‐inducted ROS production via activation of PKCδ‐p47phox axis through release of PKCδ and actin dynamics in VSMCs and was associated with intimal thickening. These results indicate that disruption of SM22α plays pivot roles in regulation of ROS production in VSMCs, and may be a potential target for the development of new therapeutics for vascular diseases..

Source of fundings: This study was supported by the National Natural Science Foundation of China (31071003).
12/15‐Lipoxygenase gene knockout severely impairs ischemia‐induced angiogenesis due to lack of Rac1 farnesylation
Authors: Singh Nikhlesh K , Kundumani-Sridharan Venkatesh , Rao Gadiparthi N
Published: June 2021

Previously, we have reported that 15(S)‐hydroxyeicosatetraenoic acid (15(S)‐HETE)‐induced migration and tube formation of endothelial cells require Rac1 activation. To understand the mechanisms by which 15(S)‐HETE activates Rac1, here, we have studied the role of HMG‐CoA reductase and RhoGFP, α‐Pix. 15(S)‐HETE by inducing HMG‐CoA reductase expression caused increased farnesylation and membrane translocation of Rac1, where it became activated by Src‐dependent α‐Pix stimulation in human dermal microvascular endothelial cells (HDMVECs) and these events enhanced the migration and tube formation of these cells. Mevalonate rescued 15(S)‐HETE‐induced Rac1 farnesylation in HDMVECs and the migration and tube formation of these cells from inhibition by simvastatin. Hind limb ischemia induced Rac1 farnesylation and activation leading to increased angiogenesis and these effects were blocked by simvastatin and rescued by mevalonate in C57BL/6 mice. In contrast, hind limb ischemia failed to induce Rac1 farnesylation and activation as well as angiogenic response in 12/15‐Lox−/− mice. Activation of Src and α‐Pix were also compromised at least to some extent in 12/15‐Lox−/− mice. Together, these findings demonstrate that HMG‐CoA reductase‐dependent farnesylation and α‐Pix‐dependent GDP/GTP exchange of Rac1 is essential for 12/15‐Lox‐12/15 (S)‐HETE‐induced angiogenesis.