bluuebunny/crossref_metadata_2025 huggingface dataset:
Anemia prevalence in Honduran children is significantly higher at increased altitudes
Authors: Kemmer Teresa Marie , Gidvani Vinod , Lougee Douglas , Coello Miguel , Amador Wilmer , Aviles Ricardo , Saenz Guillermo , Duron Carlos
Published: June 2021

When determining anemia at altitudes >3,000 ft, hemoglobin (Hb) concentration values are adjusted. Objectives of this study were to determine 1) prevalence of anemia in children ages 6‐60 months living in rural Honduras and 2) if prevalence of anemia varies depending on altitude.

Random sampling was used and data were collected at the household level in three health regions in Honduras. On‐the‐spot Hb levels were obtained and anemia was determined using age specific altitude adjusted Hb concentration values.

Data were obtained from 1024 children (49.6% female). Mean age was 32.45 mo. Mean altitude was 4703 ft. Overall anemia prevalence was 31.1%. Anemia prevalence by region (mean altitude in ft) was Chinacla (4995), 26.9%; Santiago de Purlinga (4278), 28.3%; and Lepaterique (4909), 35.0%. In children living at >5,000 ft, 41.4% were anemic and 27.7% living at <5,000 ft were anemic (P=0.000). By using logistic regression and controlling for age, gender, and health region, anemia was associated with lower age (P=0.000), health region (P=0.02), and higher altitude (P=0.005).

Anemia prevalence is high within the three mountainous health regions evaluated. Anemia prevalence is significantly higher in children living at altitudes > 5,000 ft. Funded by: US Air Force Southern Command, South Dakota State University, and the Center for Disaster and Humanitarian Assistance Medicine.
Effects of food allergy and dietary factors on atopic dermatitis among children
Authors: Lee Kyunghee , Jeong Sunsim , Joung Hyojee
Published: June 2021

The purpose of this study was to investigate the association of dietary factors and atopic dermatitis (AD) among children. Study subjects were 895 children aged 7 to 13 years enrolled in elementary schools in Seoul, Korea. Total of 702 children took a medical examination of AD. Both children and parents completed a structured questionnaire. Dietary intakes were evaluated by the 3‐day food records. AD patients group (n=156) and gender‐age matched healthy control group (n=312) were compared. The risk of AD was significantly higher in the group of not‐been‐breastfed (OR = 1.59, 95% Cl 1.01‐2.52), residents in new‐or‐repaired house within 12 months after birth (OR =2.36, 95% Cl 1.14‐4.89), diagnosed of food allergy (OR = 2.46, 95% Cl 1.11‐5.45) and curry consumers for three days (OR = 2.04, 95% Cl 1.04‐3.99). The risk of AD was lower among those who consumed fish more than 27g for 1 day (OR = 0.41, 95% Cl 1.11‐5.45). These results suggest that food allergy and dietary intakes such as breastfeeding and fish and curry consumption have significant association with atopic dermatitis after controlling for environmental factors. (This work was supported by the Seoul Metropolitan Government and the Brain Korea 21 Project in 2008.)
Ascorbic acid offsets the inhibitory effect of bioactive dietary polyphenolic compounds on basolateral iron transport in Caco‐2 human intestinal cells
Authors: Kim Eun‐Young , Ham Soo‐Kyung , Han Okhee
Published: June 2021

It was previously reported that bioactive dietary polyphenolic components inhibit basolateral iron transport but increase apical uptake of non‐heme iron in intestinal cells suggesting that polyphenols inhibit non‐heme iron absorption by decreasing basolateral iron export rather than apical iron uptake. We determined how the dietary polyphenols, (‐) ‐epigallocatechin‐3‐gallate (EGCG) and grape seed extract (GSE) increase apical iron uptake, tested if the dietary polyphenols inhibit basolateral iron transport in a dose‐dependent manner, and tested whether ascorbic acid reverses the inhibitory effects of EGCG and GSE on iron absorption. The results demonstrate that EGCG and GSE ‐mediated apical iron uptake is partially due to uptake of a polyphenol‐iron complex. Both EGCG and GSE decreased basolateral iron transport but increased apical uptake of non‐heme iron in a dose dependent manner. Addition of ascorbic acid to the solution containing polyphenolic compounds stimulated basolateral iron transport in a dependent upon the ratio of ascorbic acid to polyphenols. The stimulatory influence of ascorbic acid on basolateral iron transport from solutions containing polyphenolic compounds was associated with an increase of apical iron import. These results suggest that the inhibitory effects of bioactive polyphenolic compounds on iron absorption can be reversed by ascorbic acid.
Bacterial Protein Expression is Altered by Treatment with Zinc Nanoparticles
Authors: Grider Arthur , Kabengi Nadine J. , Bertsch Paul M. , Neal Andrew
Published: June 2021

The growing production and utilization of ZnO nanoparticles (ZON) in numerous areas ranging from medicine and manufacturing to microelectronics and personal care products will lead to their introduction to the environment through accidental releases during production, transport, manufacturing and through disposal. The purpose of this research is to determine whether treating bacteria with ZON alters protein expression compared to controls. Cupriavidus necator, a common soil bacterium, was grown in nutrient broth containing either 0.1 or 1.0 umol/L zinc as ZON or zinc acetate. The soluble fraction of the cells was isolated for the separation of cytosolic proteins via two‐dimensional gel electrophoresis. Twelve proteins exhibited differential expression when the nanoparticle treatments were compared to their respective zinc acetate controls. The proteins ranged in molecular weight from 100 kDa to 10 kDa. Bacteria treated with 0.1 umol/L ZON exhibited an increase in the expression of 12 proteins compared to zinc acetate treated cells. Bacteria treated with 1.0 umol/L ZON exhibited four proteins that were increased, and eight proteins that were decreased compared to zinc acetate treated cells. Mass spectrometry will be used to identify the differentially expressed proteins. These results indicate that treatment with ZON alters protein expression in bacteria.
Play foods in the marketplace: What health messages are toy retailers sending to children?
Authors: Flanagan Gerard , Hansen‐Petrik Melissa
Published: June 2021

The objective of this study was to establish baseline data for examining the healthfulness represented by play foods available through major U.S. toy retailers. Play foods have been little studied although they may influence dietary patterns and preferences in early childhood and thereby influence lifelong dietary patterns. Websites for the three largest U.S. toy retailers (Wal‐Mart, Target, and Toys 'R Us) were methodically searched for play foods. Three researchers independently grouped the 125 identified play food sets without predetermined categories and reconciled differences in categorization until all came to agreement. This resulted in identification of eight primary categories. The largest of these was mixed foods including general mixed foods, restaurant sets, kitchen sets, and supermarket sets (n=65, 52%). Dominant foods in this category included fruit (54%), vegetables (46%), hamburgers (45%), cakes and cookies (35%), pizza (31%), milk (31%), hot dogs (29%), fried chicken/turkey (25%), ice cream (22%), and French fries (22%). Of the 60 non‐mixed food sets, the four largest groups were sweets (37%), fruits and vegetables (23%), breads (15%), and pizza (10%). Findings suggest that fruits and vegetables are well represented among play foods, but the high prevalence of fast foods and sweets has potential to adversely impact children's food choices. Supported by the CEHHS Mary Helen Byers Award.
Isoflavone aglycone supplementation affects bone turnover in relation to estrogen receptor polymorphism
Authors: Katsuyama Hironobu , Arii Masayuki , Fushimi Shigeko , Tomita Masafumi , Okuyama Toshiko , Hidaka Kazuo , Watanabe Yoko , Tamechika Yoshie , Li LiHua , Saijoh Kiyofumi
Published: June 2021

Soybean isoflavones have structural similarity to estrogen and have been paid much attention for prevention of postmenopausal symptoms. While isoflavones exist naturally occurring glycosidic forms, they need to be hydrolyzed to aglycones by intestinal flora. In the present study, bone turnover parameters were examined among workers at a local hospital whose isoflavone aglycone supplementation was different. Eighty‐one subjects were recruited with informed consent and all of them were premenopausal women. They took placebo or isoflavone aglycone supplement for 3 month and the changes of bone turnover parameters were investigated. No statistical differences in nutrients intake were observed among the group. There were also no significant differences in bone turnover parameters such as carboxylated osteocalcin (Gla), undercarboxylated osteocalcin (uc‐OC), deoxypridinoline (DPD), and bone mineral parameter (OSI) measured by ultrasound method. Since univariate analysis was insufficient to clarify the association between bone turnover parameters and isoflavone aglycone supplementation, multiple regression analysis was performed. This analysis showed that OSI was correlated with isoflavone aglycone supplementation, Gla and estrogen receptor polymorphism. Since this study was performed only 3 month, long‐term administration of isoflavone aglycone should be further performed. This study was supported in part by a Research Project Grant (No. 17‐501) from Kawasaki Medical School.
Augmentation of the Anticancer Effect of Proteasome Inhibitors by Butyrate in Human Colorectal Carcinoma
Authors: Abaza Mohamed Salah I.
Published: June 2021

The ubiquitin‐proteasome pathway plays a critical role in the degradation of cellular proteins and cell cycle control. Dysregulating the degradation of such proteins should have profound effects on tumor growth and causes cells to undergo apoptosis. This study deals with the apoptotic effect of both proteasome inhibitors; MG115, MG132, proteasome inibitor‐1 (PSI‐1), proteasome inhibitor‐2 (PSI‐2) and epoxomicin; and sodium butyrate (NaB), their synergistic interaction and whether NaB , a natural product capable of inducing apoptosis in a number of tumor cells and exhibit a low degree of clinical toxicity, can enhance the anticancer effect of the proteasome inhibitors in colorectal cancer. The results showed that proteasome inhibitors and NaB inhibited the proliferation cancer cells in a dose and time dependent manner. The combinations of MG115, MG132, PSI‐1, PSI‐2 or epoxomicin and NaB produced prominent, additive and/or synergistic dose and time dependent growth inhibitory effects as well as a marked induction of apoptosis compared to single treatment with proteasome inhibitors or NaB. Cancer cells treated with NaB, proteasome inhibitors and their combinations were growth arrested at G1, G2‐M/S and G1 or S/G2‐M phases of cell cycle, respectively. p53, p21Waf1 , p27Kip1 and Bax were over‐expressed. Meanwhile, Bcl2 was down‐regulated after treatment with proteasome inhibitors and their combinations with NaB. Taken together, these results indicated a synergistic interaction between proteasome inhibitors and sodium butyrate that could represent a new tool in tumor therapy.

This study was supported by Kuwait University, Research Grant No. SL04‐02
Dysregulation of MicroRNAs that Regulate DNMT3b Expression in Primary Breast Cancers
Authors: Sandhu Rupninder , Coleman William B
Published: June 2021

A subset of breast cancer cell lines express a hypermethylation defect related to DNMT3b overexpression resulting from loss of expression of regulatory microRNAs (miRs). Most primary breast cancers overexpress DNMT3b compared to normal breast epithelia, and certain subsets of breast cancer (basal‐type) display gene expression signatures that reflect methylation‐dependent silencing of multiple genes, consistent with the expression of a hypermethylation defect in vivo. Several miRs have been implicated in the regulation of DNMT3b. We examined the expression of miRs‐29a, 29b, 29c, 148a, and 148b, in a group of 15 primary breast cancers (n=6 luminal A, n=2 luminal B, n=2 Her2+, n=5 basal) and two normal breast tissues. miR expression levels <50% of normal were found in 9/15 (60%) tumors for 29a/29c, 10/15 (67%) tumors for 29b, 12/15 (80%) tumors for 148a, and 8/15 (53%) for 148b. Further, 12/15 (80%) tumors lost expression =2 miRs, 9/15 (60%) lost =3 miRs, and 8/15 (53%) lost =4 miRs. The aggregate miR expression score did not differ for luminal A versus basal breast cancers, suggesting that loss of additional factors may govern the hypermethylation defect. These results suggest that loss of expression of miRs‐29a, 29b, 29c, 148a, and 148b occurs commonly among primary breast cancers, but may contribute to the expression of a hypermethylation defect in a subset of these neoplasms.

Support: NIH CA 78343
Autocrine serotonin signaling mediates spontaneous canine myxomatous mitral valve disease
Authors: Disatian Sirilak , Orton Christopher
Published: June 2021

Serotonin and serotonergic drugs are known to cause myxomatous‐like valvulopathy, but the role of serotonin in spontaneous myxomatous valve disease (MVD) is unknown. The study objective was to test the hypothesis that autocrine serotonin signaling mediates spontaneous MVD.

METHODS

Normal and myxomatous mitral valves from dogs were studied. Expression of serotonin 2B receptor (5HT2BR), tryptophan hydroxylase 1 (TPH1), serotonin transmembrane transporter (SERT), total and phosphorylated extracellular signaling‐regulated kinase (ERK)1/2 were studied by immunohistochemistry and immunoblot analysis.

RESULTS

Both 5HT2BR and TPH1, the key serotonin synthetic enzyme, were increased in myxomatous valves; whereas SERT, a key protein in serotonin metabolism, was decreased in myxomatous valves. Phosphorylated, but not total, ERK1/2 was increased in myxomatous valves consistent with enhanced active serotonin signaling.

CONCLUSION

Expression patterns for key serotonin signaling proteins suggest that autocrine serotonin signaling mediates spontaneous canine MVD. Research supported by the Heart to Heart foundation.
Skeletal muscle‐specific overexpression of insulin‐like growth factor I (IGF‐1) decreases oxidative stress and prevents angiotensin II‐induced skeletal muscle wasting: novel potential therapy to treat cachexia in congestive heart failure
Authors: Sukhanov Sergiy , Semprun‐Prieto Laura , Yoshida Tadashi , Vaughn Charlotte , Delafontaine Patrick
Published: June 2021

Congestive heart failure is associated with elevated levels of angiotensin II (ANG) and skeletal muscle wasting (cachexia). We have shown that ANG induces cachexia and increases oxidative stress in FVB mice. Since we also found that IGF‐1 exerts anti‐oxidant effect in a murine model we hypothesized that IGF‐1 overexpression would decrease ANG‐induced cachexia and/or oxidative stress.

Mice overexpressing IGF‐1 under control of the skeletal muscle‐specific type II myosin light chain promoter (MLC) and control FVB mice were infused with 1 ug/kg ANG or vehicle (SHM) for 7d. ANG decreased skeletal muscle weight in FVB (ANG/SHM, gastrocnemius: 99.8±3.6/122.3±4.2mg; tibialis anterior: 32.0±1.3/41.3±1.3mg; quadriceps:117.6±6.1/160.6±6.7mg), however no significant cachexia was found in ANG‐infused MLC. ANG induced leftward shift in myofibers number/size distribution and decreased cross‐sectional area (CSA) of type I and II myofibers in FVB (type I, 443±22/528±28 um2; type II, 620±40/835±44 um2) without effect on myofibers distribution and CSA in MLC (immunostaining with isoform‐specific a/b). ANG increased superoxides in FVB by 67±8% (p<0.05) but did not significantly change superoxides in MLC (assay with superoxide‐sensitive dye L‐012). ANG‐induced superoxides were suppressed by apocynin (a specific NADPH oxidase inhibitor) and also ANG increased NOX4 (a NADPH oxidase subunit) expression (RT‐PCR) in both mouse models, however NOX4 upregulation was greater in FVB compared to MLC (3.6‐ vs. 2.1‐fold increase). Thus, IGF‐1 overexpression suppresses oxidative stress and efficiently blocks skeletal muscle wasting, possibly via inhibition of NADPH oxidase‐mediated superoxide formation.
PI3K‐R1 expression in hepatic tissue of horses with laminitis
Authors: Carlisle Katherine , McGeachy Matthew , Garza Frank , Stokes Ashley Michelle
Published: June 2021

Phosphatidylinositol‐3‐Kinase (PI3K) is an inhibitor of pro‐inflammatory processes regulated by two converging pathways. It is a cytoplasmic component of the Toll‐Like Receptor Signaling Pathway and is also affected by insulin receptor activation. The objective was to examine gene expression of the regulatory subunit p85 (R1) of PI3K in equine liver samples from normal (n=13), naturally acquired laminitis (n=11), experimentally induced laminitis (n=7), and metabolic disorder (n=3) cases.

Total cellular RNA was extracted, analyzed by agarose gel electrophoresis, quantified by spectrophotometry, reverse transcribed to cDNA, and quantified by TaqMan RT‐PCR.

The most significant finding of this study was a difference between PI3K expression and laminitis activity (p=0.0441). Horses with active cases of laminitis, Obel grade ¡Ý 1, demonstrated significantly less expression of PI3K than horses with inactive cases (fold decrease=10.26). We also found a marked increase of PI3K in horses with metabolic disorders compared to those with laminitis (fold increase=14.949).

Our study suggests a relationship may exist between PI3K down‐regulation and the pathogenesis of laminitis. Therefore, PI3K‐R1 deserves further investigation as an inflammatory mediator that can be targeted in prevention and treatment of laminitis.

Funding provided by the LSU Equine Health Studies Program.
Risks of nutritional deficiencies in young adult type 1 diabetic (T1D) women
Authors: Masse Priscille G , Elisha Belinda , Tranchant Carole C , Ericson Karen , Donovan Sharon , Ouellet Rodney
Published: June 2021

Although nutrition plays a crucial role in the treatment of T1D, there are few studies on the nutritional status. This study aimed to evaluate a target group at risk of nutritional deficiencies. Women with well‐controlled T1D and without renal complications (n=22) and healthy controls (n=27), aged 30‐40 yr‐old, were rigorously selected, having normal body weight and being not pregnant nor vegetarian, among other selection criteria. The two groups were comparable in many aspects. T1D, diagnosed since 21 yrs in average, was monitored by HbA1c level. Serum transferrin (P<0.02) and IGF‐1 concentrations (P<0.001) were reduced in the T1D group. There was no significant difference for albumin. Serum iron concentration was also reduced (P<0.03), although body stores (ferritin) were not affected. Contrarily to other studies, serum Mg and plasma pyridoxal phosphate (PLP, vitamin B6 coenzyme) were not reduced, although alkaline phosphatase, which regulates the concentration of this vitamin, was increased (P<0.01). Plasma PLP was below reference value. Erythrocyte folate was greater (P<0.03) in TID group due to a greater number of subjects taking supplements and more control subjects with values in the low range. This study evidenced T1D‐induced metabolic changes in regard to protein and iron status. It also showed a risk of deficiency for vitamin B6.
Sympathetic α3β2‐nAChRs mediate cerebral neurogenic nitrergic vasodilation
Authors: Lee Hui‐Chao , Chen Po‐Yi , Chen Mei‐Fang , Kuo Jon‐Son , Lindstrom Jon , Lee Tony Jer‐Fu
Published: June 2021

The nicotinic acetylcholine receptor (nAChR) on the sympathetic nerves innervating cerebral arteries of the pig crossbreed between Landrace and Yorkshire (LY) in Illinois is α7‐nAChR. Nicotine‐induced cerebral neurogenic vasodilation in pigs crossbreed among Landrace, Yorkshire and Duroc (LYD) in Hualien (Taiwan), however, is not blocked by α‐bungarotoxin (α‐BTGX, a highly selective α7‐nAChR antagonist). The cerebral perivascular sympathetic nAChR subtype of LYD pigs, therefore, was examined. The nicotine‐induced dilatation of isolated basilar arteries was not affected by α‐conotoxin IMI (an α7‐nAChR antagonist) or α‐conotoxin AuIB (an α3β4‐nAChR antagonist). The vasodilation was inhibited by preferential α3‐containing nAChR antagonists (tropinone and tropane) and α‐conotoxin MII (a selective α3β2‐nAChR antagonist). Using reverse transcription PCR, α3‐, α7‐, β2‐ and β4‐subunits of nAChRs were expressed in fresh superior cervical ganglia. The mRNA levels of α3‐, β2‐ and β4‐subunits were significantly higher than that of α7‐subunit. Furthermore, nicotine‐induced inward currents in α3β2‐nAChR‐expresssing oocytes were blocked by α‐conotoxin MII but not by α‐BTGX or α‐conotoxin AuIB. In conclusion, the predominant nAChR on cerebral perivascular sympathetic nerves mediating cerebral nitrergic neurogenic vasodilation in LYD pigs is α3β2‐subtype (supported by TCU & NSC).
ACTIVATION OF ACID SPHINGOMYELINASE DRIVES LYSOSOMAL TRIFFICKING AND FUSION TO MEMBRANE IN CORONARY ENDOTHELIAL CELLS
Authors: Bao JunXiang , Jin Si , Brimson Christopher , Poklis Justin L , Li PinLan
Published: June 2021

Recent studies have demonstrated that activation of death receptor results in lysosomal fusion to cell membrane to form a lipid rafts (LRs) signaling platform. However, the driving forces are still unknown. The present study was designed to test a hypothesis that acid sphingomyelinase (ASMase) is firstly activated to produce ceramide, then drives lysosomal fusion and LRs clustering. We first performed confocal microscopy and fluorescence resonance energy transfer (FRET) detection in bovine coronary arterial endothelial cells (BCAECs) and found that FasL significantly led to colocalization of Lamp‐1 and CTX‐B, and their FRET efficiency increased from 5 to 19%. We also found a lysosome‐membrane fusion pattern of fluorescence changes induced by FasL which was blocked by siRNA of ASMase. Both activator and inducer of ASMase markedly increased colocalization of Lamp‐1 with CTX‐B and resulted in lysosome‐membrane fusion. However, the de novo synthesis of ceramide had no such effects. In isolated bovine coronary arteries, endothelium‐dependent vasodilation was impaired after ASMase had been activated, which could be reversed by lysosome functional inhibitor. It is concluded that ASMase activation precedes lysosomal fusion and LRs clustering and may serve as a driving force for lysosome trafficking and fusion to cell membrane in BCAECs. (Supported by NIH Grants HL‐57244, HL‐75316, and DK54927).
sEH‐deficiency attenuates neointima formation in the femoral artery cuff model of hyperlipidemic mice
Authors: Revermann Marc , Schloss Manuel , Barbosa‐Sicard Eduardo , Fleming Ingrid , Morriseau Christophe , Hammock Bruce , Brandes Ralf Peter
Published: June 2021

Epoxyeicosatrienoic acids (EETs) have anti‐inflammatory effects and promote normal endothelial function. The soluble epoxide hydrolase (sEH) metabolizes EETs to their less active diols. We hypothesized that knockdown and inhibition of sEH prevents neointima formation in hyperlipidemic ApoE‐/‐ mice induced by the femoral artery cuff model. Inhibition of sEH by 12‐(3‐adamantan‐1‐yl‐ureido) dodecanoic acid (AUDA) or knockout of the enzyme significantly increased the plasma EET concentration. sEH activity was detectable in femoral arteries. sEH‐knockout and inhibition results in a significant reduction of neointima formation (50±6,3% and 42±4,7% reduction in intima/media‐ratio, p<0.05). The proinflammatory gene expression at the site of cuff placement was significantly reduced in sEH‐/‐‐mice. Moreover, an in vivo‐BrdU‐assay revealed significantly attenuated SMC‐proliferation in sEH‐/‐. Accordingly, SMC‐migration activity in cultured cells was attenuated by EETs and this effect was abolished by protein kinase A inhibition. These observations suggest that inhibition of sEH prevents vascular remodelling.

German Research Foundation & Goethe‐University
Statins Block the Formation of Lipid Raft Redox Signaling Platforms in Coronary Endothelial Cells
Authors: Hu Jun‐Jun , Jia Su‐Jie , Li Pin‐Lan
Published: June 2021

Recent studies have reported that lipid raft (LR) redox signaling platforms associated with NADPH oxidase are involved in coronary endothelial dysfunction upon different injury factors. The present study was designed to test whether statins could interfere with the formation of LR redox signaling platforms to protect the coronary arterial endothelium from injury. By confocal microscopy, we first detected the formation of LRs clustering in human coronary arterial endothelial cells (HCAECs) exposed to FasL, a death receptor ligand. In these LR clusters, NADPH oxidase subunits, gp91phox and p47phox were aggregated with LR, which was almost completely blocked by statins (lovastatin, provastatin and simvastatin). To further explore the functional relevance of this action, we performed detergent resistant membrane floatation to isolate LRs. Electron spin resonance spectrometry showed that superoxide (O2.−) production was 5‐fold higher in the LR fractions from FasL‐treated HCAECs than that from untreated cells. This FasL‐induced enhancement of O2.− production in LR fractions was substantially blocked by pretreatment with statins. Our results indicate that blockade of LR redox signaling platform formation in endothelial cell membrane may be another important therapeutic mechanism of statins in preventing endothelial injury and atherosclerosis.(Supported by NIH grants HL057244, HL075316, and DK054927)
NADPH oxidase is critically involved in CCL2‐induced endothelial cell activation
Authors: Zhang Wenbo , Rojas Modesto A. , Wiseman Dean A. , Behzadian M.A. , Bartoli Manuela , Black Stephen M. , Caldwell Robert W. , Caldwell Ruth B.
Published: June 2021

CCL2 plays an important role in vascular diseases via recruitment of leukocytes and by altering integrity of the vascular wall. Molecular mechanisms of direct CCL2‐induced activation of vascular endothelial cells (ECs) are largely unknown. Here we assessed the hypothesis that NADPH oxidase is critically involved in regulating EC responses to CCL2. Studies using EPR spin trap CMH and DCF imaging showed that CCL2 induces a significant and time‐dependent increase in the reactive oxygen species (ROS) which was blocked by pretreating ECs with NADPH oxidase inhibitors or anti‐oxidants. Measurement of directed EC migration showed that CCL2 induced a dose‐dependent increase in EC migration. The increase in migration was blocked by inhibiting NADPH oxidase activity. Immunoblot analysis revealed that CCL2 rapidly activates Akt, ERK, and p38 MAPK. Pretreatment of ECs with NADPH oxidase inhibitors, antioxidants, or the superoxide scavenger, significantly attenuated CCL2‐induced activation of Akt, but did not alter the activation of ERK or p38 MAPK. Inhibiting activity of mitochondrial complex II did not prevent the CCL2‐induced Akt activation. These studies indicate that CCL2‐mediated activation of ECs and induction of EC migration are mediated by NADPH oxidase‐derived ROS and that the process involves the redox dependent activation of Akt.

Supported by EY04618, EY11766, and VA (R.B.C.); HL70215 (R.W.C); AHA (W. Z.)
Structure activity relationships of caffeic acid phenethyl ester (CAPE) and its amide derivative caffeic acid phenethyl amide (CAPA) against oxidant stress in human endothelial cells
Authors: Yang John , Kerwin Sean Michael , Wang Xinyu , Stavchansky Salomon , Bynum James , Bowman Phillip
Published: June 2021

Numerous reports have described the amelioration of ischemia/reperfusion injury by CAPE post‐injury, and we have recently shown that induction of heme oxygenase‐1 (HO‐1) in vitro is highly correlated with this cytoprotection. In vivo, the presence of esterases which are abundant in blood and tissues, would severely limit the effectiveness of CAPE by degrading it to caffeic acid and phenethyl alcohol, neither of which is cytoprotective in vitro. Therefore an amide derivative of CAPE, CAPA was synthesized and screened for cytoprotection by examining its ability to induce HO‐1 mRNA in human endothelial cells. CAPA was produced by a Wittig reaction between 3,4 dihydroxy‐benzaldehyde and the corresponding amide phosphonium salt and was 95% pure by nuclear magnetic resonance spectroscopy. CAPA was as effective as CAPE in inducing HO‐1 mRNA (9‐fold over vehicle control) as determined by RT‐PCR. It remains to be determined if it exhibits greater stability than CAPE in vivo.
Central rimonabant administration prevents endotoxic hypotension by inhibiting norepinephrine release in the preoptic anterior hypothalamic area
Authors: Yilmaz Mustafa S , Villanueva Alex , Millington William R , Cutrera Rodolfo A , Cheer Joseph F , Feleder Carlos
Published: June 2021

Lipopolysaccharide (LPS) is thought to lower arterial pressure by stimulating release of tumor necrosis factor‐α (TNF) and other mediators. However, recent evidence indicates that endotoxic hypotension is initiated through a central mechanism that involves noradrenergic neurotransmission in the preoptic anterior hypothalamic area (POA). In this study, we tested whether cannabinoid‐1 (CB1) receptors participate in the control of arterial pressure during endotoxemia, based on evidence that hypothalamic neurons express CB1 receptors. We found that central administration of the CB1 receptor antagonist rimonabant (250 ng icv) inhibited the initial fall in arterial pressure evoked by LPS (1 mg/kg iv) significantly in both conscious and anesthetized rats. Interestingly, rimonabant also attenuated the second, delayed hypotensive phase that ultimately leads to tissue ischemia and death. Rimonabant also prevented the LPS‐induced rise in extracellular fluid norepinephrine (NE) concentrations in the POA which suggests that rimonabant prevents initiation of LPS hypotension by inhibiting NE release in the POA. The characteristic increase in plasma TNF concentrations was also inhibited significantly. These data indicate that CB1 receptors in the brain mediate endotoxic hypotension. This study supported by American Foundation for Pharmaceutical Education.
Effects of a diet rich in omega‐3 essential fatty acids on behavior in an attentional task and monoamine transmitter levels in the medial prefrontal cortex of adolescent rats
Authors: Mokler David J. , McGaughy Jill A. , Staiti Amanda M. , Morgane Peter J. , Galler Janina R. , Brym Francine J. , Bass Donna C.
Published: June 2021

Omega‐3 essential fatty acids (Ω3‐EFAs) have been suggested as a treatment for depression and ADD. Male adolescent rats were put on a diet balanced 1:1 with Ω3:Ω 6 essential fatty acids (EFAs) or a diet high in Ω6 EFAs acids (25:1 Ω6:Ω 3) from postnatal days 21 to 45. Male adolescent rats were tested in the intradimension/extradimensional attentional set‐shifting (ID/ED) task on days 40‐45 of age. The ID/ED has previously been used only in adult animals. Following completion of the ID/ED, animals were implanted with a 2mm microdialysis probe into the ventral mPFC (vmPFC). After determination of basal levels of norepinephrine (NE), DA and 5‐HT, animals were again run in the ID/ED task. Implantation of a microdialysis probe into the vmPFC did not affect performance in the ID/ED task. Basal levels of NE were higher in the 25:1 dietary group while basal levels of DA and 5‐HT did not differ between groups. Extracellular 5‐HT decreased during the task in the 1:1 dietary group but not in the 25:1 group. These data suggest that a diet rich in omega‐3 EFAs in adolescent rats produces changes in neurotransmitter levels but does not alter attention in an ID/ED task. Supported by EAST and MH074811‐01.
Effects of the novel anti‐inflammatory drugs robenacoxib and AHI‐805 on recovery of ischemic injured equine jejunum ex vivo
Authors: Marshall John Fraser , Bhatnagar Adria , Bowman Susan , Morris Natalie , Skorich Dinah , Blikslager Anthony
Published: June 2021

Flunixin is an NSAID administered to horses to ameliorate shock, but which inhibits recovery of barrier function in ischemic‐injured intestine. We examined the effect of robenacoxib, a COX‐2 inhibitor, and AHI‐805, an aza‐thia‐benzoazulene derivative, on recovery of mucosal barrier function. Jejunum was subjected to ischemia for 2 hours in 6 horses. Control and ischemic‐injured mucosa was treated with Ringer's alone (control) or containing 0.27μM flunixin, robenacoxib, or AHI‐805 in Ussing chambers. Trans‐epithelial electrical resistance (TER) and mucosal‐to‐serosal flux of 3H‐mannitol were measured over a 4‐hour period. TXB2 and PGEM in the serosal bathing solution were measured to assess COX‐1 and COX‐2 function. Expression of COX‐1 and ‐2 was determined by immunoblot and histology was performed. Ischemic injury significantly increased 3H‐mannitol flux, epithelial denudation, and COX‐2 expression (P<0.05). TER of ischemic‐injured tissue treated with flunixin or AHI‐805 was significantly lower than control over the recovery period (P<0.05). Robenacoxib did not inhibit recovery of TER. PGEM and TXB2 were significantly decreased by flunixin and AHI‐805 when compared to Ringer's alone or robenacoxib. Robenacoxib permits recovery of ischemic‐injured jejunum judging by indices of barrier function, and this is linked to its ability to allow enhanced PG production.
PKA PHOSPHORYLATION OF SPINOPHILIN MODULATES ITS INTERACTION WITH THE alpha2AAR AND ALTERS TEMPORAL PROPERTIES OF alpha2AAR INTERNALIZATION
Authors: Chen Yunjia , Xu Jianmin , Cottingham Christopher , Lu Roujian , Jiao Kai , Wang Qin
Published: June 2021

Spinophilin plays critical roles in regulating trafficking and signaling of the alpha2‐adrenergic receptor (AR) both in vitro and in vivo (Wang Q, etc. (2004) Science 304, 1940‐1944). In the present study, we demonstrate that PKA phosphorylation of spinophilin modulates the spinophilin‐alpha2AAR interaction to regulate alpha2AAR internalization. Activation of PKA by forskolin abolishes the agonist‐enhanced interaction between spinophilin and the alpha2AAR, and this event can be blocked by Ser‐>Ala mutations at the PKA phosphorylation sites of spinophilin. Furthermore, a Ser‐>Asp mutation that mimics the phosphorylated state at the PKA phosphorylation site Ser177, which is located within the alpha2AAR‐binding region of spinophilin, is sufficient to block the spinophilin‐alpha2AAR interaction in intact cells. In cells expressing mutant spinophilin carrying the S177D mutation, agonist‐induced internalization of the alpha2AAR is accelerated and enhanced, as revealed by both intact cell ELISA and quantitative immunofluorescent studies. These results support that PKA phosphorylation of spinophilin is functionally relevant in regulating alpha2AAR trafficking. Therefore, modulation of spinophilin‐receptor interaction through phosphorylation of spinophilin may represent a novel mechanism whereby PKA regulates GPCR trafficking.
In vitro system for studying blood, perfluorocarbons (PFC) and Sickle Cell Anemia
Authors: Narayanan Srinivasan Vasudevan , Berger Brian , Torres Filho Ivo
Published: June 2021

PFCs are inert compounds that dissolve more oxygen (O2) than plasma. A mixture of PFC and sickle cell blood could potentially hold and deliver more O2 to tissues, compensating for sickle cell red blood cell's (RBC) decreased affinity for O2. A system was developed for in vitro studies using blood mixed with PFC. The system (priming volume: 10 ml) includes a computer‐controlled pump, gas mixer, oxygenator, deoxygenator and interconnected tubing that allowed the flowing blood to be exposed to various O2 and CO2 tensions, yielding controlled levels of hemoglobin O2 saturation (SO2) and O2 partial pressures (PO2). SO2 and PO2 were measured using a blood gas analyzer and a CO‐oximeter. With the addition of 5, 10, and 20% of PFC by volume of blood, the total O2 content, O2 delivery and consumption increased linearly with the PFC concentration. The data suggest increased O2 transport by the PFC. The SO2 of blood mixed with PFC did not differ significantly from baseline. Changes in blood cell morphology can also be evaluated at each oxygenation level and PFC concentration using video‐microscopy. The percentage of RBC that sickle at a given PO2 with and without PFC will be determined. The system allows several variables to be studied using blood sampled from animals and patients.

Support: Virginia Commonwealth University.
Mechanoregulation of extracellular matrix dynamics by endothelial cell surface heparan sulfate
Authors: Helmke Brian P. , Qian Jessie J. , Mott Rosalind E.
Published: June 2021

Decentralized mechanotransmission through endothelial cells (ECs) induces rapid fibronectin (Fn) fibril remodeling in response to hemodynamic shear stress, but mechanisms regulating adaptation of extracellular matrix structure are poorly understood. Since heparan sulfate in the glycocalyx has been proposed to modulate mechanosignaling at the apical cell surface, we hypothesized that it also contributes to mechanotransmission from the basal surface to Fn. Heparan sulfate in primary cultured post‐confluent EC monolayers was primarily in the subendothelial matrix. Heparinase pretreatment decreased initial displacement rate and eliminated directional displacement of Fn fibrils after flow onset. Competitive inhibition of heparan sulfate binding to Fn by heparin allowed coordinated fibril displacement but in random directions rather than in the flow direction. A C‐terminal Fn fragment containing HepII domains but not integrin binding domains reduced heparan sulfate expression but not colocalization with Fn fibrils. Displacement after flow onset remained in the downstream direction but became more heterogeneous, and displacement rate decreased significantly. Thus, heparan sulfate expression and association with Fn fibrils contribute to mechanoregulation of subendothelial matrix dynamics. Supported by NIH grants HL076499, HL071958, and HL080956.
Frequency dependent myofilament desensitization is impaired in rabbit right ventricular hypertrophy
Authors: Varian Kenneth D. , Kijtawornrat Anusak , Gupta Subash C , Torres Carlos AA , Monasky Michelle M , Hiranandani Nitisha , Periasamy Muthu , Hamlin Robert L , Janssen Paul ML
Published: June 2021

The aim of this study was to test the hypothesis that frequency‐dependent myofilament desensitization, a physiological response of healthy myocardium, is altered in hypertrophied myocardium. New Zealand White rabbits underwent pulmonary artery banding (PAB) and after 10 weeks animals were euthanized, and suitable trabeculae harvested from the right ventricle. Twitch contractions, calibrated bis‐fura‐2 calcium transients, and myofilament calcium sensitivity were measured at various frequencies. TnI and MLC2 phosphorylation status and expression levels of other proteins including titin isoforms were assessed. The force frequency response, relaxation frequency response, and calcium frequency relationships were significantly blunted, and diastolic tension increased with frequency in the PAB rabbits. Myofilament calcium sensitivity was identical at 1 Hz (pCa 6.11±0.03 vs. 6.11±0.06) but the frequency‐dependent desensitization that takes place in the sham group (delta pCa 0.14±0.06, p<0.05) was not observed in PAB animals (delta pCa 0.02±0.05). Frequency‐dependent phosphorylation of TnI occurred normally in the sham but not in PAB trabeculae. We conclude that frequency‐dependent myofilament desensitization is impaired in right ventricular hypertrophy, and may be associated with impaired TnI phosphorylation and contribute and/or be responsible for an elevation of diastolic tension.
Modulation of CFTR activity alters submaximal β‐adrenergic receptor stimulated cardiomyocyte contraction rate
Authors: Sellers Zachary M , Xiang Yang , Best Philip M
Published: June 2021

Previously we found that CFTR regulates the contraction rate of spontaneously beating neonatal ventricular cardiomyocytes. In this study we examined if CFTR is involved in β‐adrenergic receptor (β‐AR) stimulated increases in contraction rate. Spontaneous beating cardiomyocytes were generated by isolation and culture of mouse neonatal ventricular myocytes. Isoproterenol dose‐dependently increased contraction rates with an EC50 of 16.6 nM. Inhibition or knockout of CFTR led to a loss of submaximal β‐AR stimulation, resulting in a right‐shift of the dose response curve with an EC50 of 66.7 nM and 31.2 nM, respectively. This targeted response to submaximal β‐AR stimulated contraction rates was specific to CFTR as inhibition of volume‐ or Ca2+‐activated Cl− channels affected both submaximal and maximal concentrations. Furthermore, pre‐activation of CFTR with genistein or PG‐01 potentiated submaximal isoproterenol stimulated contraction rates, resulting in a left‐shift of the dose response curve with an EC50 of 1.1 nM and 0.6 nM, respectively. Our results indicate that CFTR plays a distinct role in regulating contraction rate responses to submaximal, but not maximal, β‐AR stimulation. Future work will be necessary to determine if individuals with alterations in CFTR activity (i.e. cystic fibrosis patients) may be at increased risk for cardiac abnormalities. Funded by the AHA, NIH, University of Illinois.
The activation of calcineurin pathway contributes to the cardiomyocyte hypertrophy induced by thyroid hormone
Authors: Carneiro‐Ramos Marcela Sorelli , Shibata Caroline Emy Rodrigues , Moriscot Anselmo Sigari , Barreto‐Chaves Maria Luiza Morais
Published: June 2021

Objective

T3 exerts strong influence over cardiac trophism and contractility. However, the possible intracellular pathways modulated by T3 are poorly understood. In this manner, several studies have shown that Calcineurin pathway is closely related to hypertrophic process in heart in different experimental models. Thus, the present was undertaken to evaluate Calcineurin pathway contribution to the cardiac hypertrophy triggered by T3 in vivo and in vitro.

Methods

Hyperthyroidism was induced in rats by i.p. T3 administration (7ug/100g BW) for 12hs, 24hs and 7 days. At the end of treatment, animals were decapitated, hearts collected for gene expression by Real Time PCR or calcineurin protein levels by Western‐Blotting. To evaluate a direct effect of T3, cardiomyocytes primary cultures treated for 24hs with T3 (10nM) or T3 conjugated with a calcineurin inhibitor (Ciclosporin A 500nM) were used. ANF mRNA levels were used as cardiac hypertrophy marker. The results were expressed as mean±SD and P0.05 (n=3).

Results

As expected, 7d T3 treatment was able to induce significant cardiac mass gain and increase calcineurin levels. Data from in vitro experiments showed that concomitant administration of T3 and Cyclosporin A prevented partially cardiomyocytes hypertrophy when compared to that treated with T3 alone. (1,6 ± 0,21 vs. 2,16 ± 0,35, p<0,05). These results suggest an important role for calcineurin in T3 mediated cardiac hypertrophy.

Financial Support: FAPESP.
AT1 receptor knockdown prevents the T3‐induced cardiomyocyte hypertrophy: modulation of the Akt Signaling Pathway
Authors: Diniz Gabriela Placoná , Carneiro‐Ramos Marcela Sorelli , Barreto‐Chaves Maria Luiza Morais
Published: June 2021

Objective

The Renin Angiotensin System plays an important role in TH‐induced cardiac hypertrophy. We recently demonstrated the in vivo participation of Angiotensin II Type 1 receptor (AT1R) in TH‐induced cardiac hypertrophy. The aim of this study was to analyze the contribution of AT1R to the development of T3‐induced cardiomyocyte hypertrophy, as well as to evaluate the role that AT1R plays in the activation of the Akt/GSK‐3β/mTOR signaling pathway promoted by T3.

Methods

Cardiomyocytes cultures were prepared from ventricles of neonatal Wistar rats by colagenase/pancreatin method. The cells were transfected with a selective AT1R small interfering RNA (AT1R siRNA). The AT1R knockdown was evaluated by Real Time PCR and Western Blot. Protein synthesis was quantified by tritiated leucine incorporation. The Akt/GSK‐3β/mTOR signaling pathway was analyzed by Western Blot. The results were expressed as mean±SD and P0.05 (n=3).

Results

The cells transfected with the AT1R siRNA presented reduced mRNA and protein expression of AT1R (P<0.01). AT1R knockdown prevented the T3‐induced cardiomyocyte hypertrophy, as evidenced by the reduction in protein synthesis (P<0.01). In addition, the AT1R siRNA diminished the T3‐induced activation of the Akt/GSK‐3β/mTOR (P<0.01). These data demonstrate for the first time that the AT1R contributes to the trophic effect that T3 has on cardiomyocytes, as well as in the nongenomic T3 effect on the Akt/GSK‐3β/mTOR signaling pathway in cardiomyocytes.

Financial Support: FAPESP.
PKC‐alpha Potentially Mediates Transcription of the Inducible HSP70 via AP‐1
Authors: Mestril Ruben , Mirza Osman
Published: June 2021

Prior research has established the involvement of both PKC and HSP proteins in conferring cardioprotection. The interaction between these two families has implicated that PKC‐α mediates induction of Hsp70 and subsequent protection against simulated ischemia‐reperfusion in neonatal rat ventricular myocytes. Surprisingly, Hsp70 induction by PKC‐α was found to be both highly specific and independent of the conventional heat shock transcription factor HSF‐1. In an effort to elucidate the underlying mechanism, we linked a luciferase reporter gene to chimeric constructs of the 5' promoter region of the rat Hsp70. Constructs were transfected into myogenic H9c2 cells followed by adenoviral mediated overexpression of PKC‐α. Since regulation by PKC‐α was found to be independent of HSF‐1, the significance of other transcription factors exerting their action on the promoter region of Hsp70 was investigated. Our results show that induction of the Hsp70 promoter by PKC‐α is preserved within the first 175 base pairs from the start of transcription. The sequence of the Hsp70 promoter in this segment contains at least two canonic Ap‐1 signaling elements. Further restrictions of the ‐175 bp promoter region may reveal the possible role of these Ap‐1 sites in the transcriptional activation of Hsp70 by PKC‐α. Insertion of Ap‐1 signals upstream of a basal promoter‐luciferase construct could further confirm their significance.
Arrhythmogenicity of Low‐Flow Reperfusion Following Acute Local Ischemia in Excised Rat Hearts
Authors: Swift Luther Mitchell , Asfour Huda , Bhatt Jay , Forleiter Craig , Sarvazyan Narine , Kay Matthew W
Published: June 2021

Clinical studies show that impaired myocardial reperfusion following thrombolytic therapy for coronary occlusions increases the incidence of ventricular arrhythmias; however the mechanisms are poorly understood. We studied arrhythmias during controlled low‐flow reperfusion (LFRP) in excised Langendorff‐perfused hearts (n=6). The left anterior coronary artery (LAD) was cannulated. An HPLC pump delivered perfusate to the LAD at a rate of 2 mL/min, followed by 20 min of local ischemia (0 mL/min) and then 20 min of LFRP (0.2 mL/min). The electrical response at each flow rate was monitored using an ECG and fluorescence imaging of epicardial potentials. We found that the highest heart rates and lowest heart rate variability occurred during LFRP (p<0.05). LFRP also resulted in the highest incidence of VF (p<0.05). During ischemia heart rate variability was highest and the incidence of VF was much lower than during LFRP (p<0.05). Fluorescence images revealed periodic ectopic beats during ischemia and numerous ectopic beats during LFRP. During LFRP, frequent episodes of monomorphic VT were driven by a single rotor and VF was driven by multiple transient rotors. In summary, LFRP fuels the continuous generation of ectopic beats that precede VT/VF. These findings match with our previous in‐vitro studies where we hypothesized that microreperfusion of ischemic border zones could result in ectopic beats and arrhythmias.