bluuebunny/crossref_metadata_2025 huggingface dataset:
Dried plum polyphenols decreased markers of inflammation and lipid peroxidation in RAW264.7 macrophage cells
Authors: Kumar Ajay , Hooshmand Shirin , Arjmandi Bahram H.
Published: June 2021

Chronic inflammation is characterized by accumulation of macrophages at injury site and macrophages start the inflammatory cascade. Polyphenols have been found to possess strong anti‐inflammatory and anti‐oxidant properties. To substantiate this assumption, we tested the chlorogenic acid rich dried plum polyphenols using RAW264.7 macrophage cells. Macrophages express nitric oxide (NO) and cyclooxygense‐2 (COX‐2) dependent prostaglandins under chronic inflammatory conditions. RAW264.7 macrophage were stimulated with 1 µg/ml of bacterial lipopolysaccharide (LPS) to induce inflammation and treated with different doses of polyphenols (0, 0.1,1,10,100 and 1000 µg/ml), for 12 hrs. Polyphenols at a dose of 1000 µg/ml was able to significantly reduce the NO production by 43%. Moreover, LPS (10 ng/ml) induced expression of COX‐2 was significantly reduced by 1000 µg/ml polyphenols. To investigate the anti‐oxidant activity of polyphenols, macrophage cells were stimulated with 100 µg/ml of FeSO4 + 1mM/ml of H2O2 to induce lipid peroxidation. Polyphenols at a dose of 1000 µg/ml showed 32% reduction in melondialdehyde production. Conclusively, it can be stated that dried plum polyphenols are potent anti‐inflammatory and anti‐oxidative agents and can have implications in prevention of diseases of chronic inflammatory origin including bone loss.
China's Hypertension Increase Linked Significantly to Its Rapid Rise in Overweight: A 16‐year Longitudinal Analysis: China Health and Nutrition Survey, 1991‐2006
Authors: Du Shufa , Wang Huijun , Popkin Barry M
Published: June 2021

While the rapid increases in stroke in China and hypertension across the developing world are understood, the underlying nutrition causes of this increase in hypertension are unexplored. Using longitudinal data on 8806 subjects aged 18 and older followed over six waves of the China Health and Nutrition Survey, we studied the effect of changes in weight on blood pressure and hypertension. Trained nutritionists obtained weighed dietary data over 3 days combined with individual 24‐h recalls. Trained physicians collected 3 measures of blood pressure over an hour, weight and height data with standardized methods. Multivariate longitudinal logistic regression models were used to estimate the relative risk, controlled for age, gender, education, and urban residence. Prevalence of overweight increased rapidly from 12.8% in 1991 to 27.2% in 2006 (faster among the poorest income tertile: 8% to 23.3%); prevalence of hypertension increased at the same pace from 15% to 27.8% (again fastest among the poorest tertile: 11.3% to 28.1%). Each additional kg of body weight was associated with a 9% increase in risk of being hypertension (RR=1.09, p<0.001). Sodium intake did not modify this relationship. Increased weight accounted for 3.7 per 100 individuals or 11% of the close to 13 percentage point increase in the prevalence of hypertension in China over this period.

This research was supported by NIH R01‐HD30880.
Correlates of ambulatory blood pressure in healthy young women: nutrition, physical activity and stress
Authors: Bedford Jennifer L. , Barr Susan I.
Published: June 2021

Young adult blood pressure predicts cardiovascular risk in later life. This study explored relationships between ambulatory blood pressure (ABP) and dietary intake, physical activity and stress in 120 healthy, non‐obese, 19‐35 yr old women. Participants completed a food frequency questionnaire, the Baecke physical activity questionnaire and the Perceived Stress Scale (PSS). Height, weight and 24‐hr urinary free cortisol (UFC) were measured. An ABP monitor was worn for 12 hr with measurements every 30 min to determine average systolic (SBP) and diastolic (DBP) blood pressure, mean arterial pressure (MAP) and pulse pressure (PP). Height, weight and physical activity correlated positively with PP. Age correlated negatively with PP and positively with DBP. Partial correlations, controlling for age, height, weight, physical activity and energy, revealed few significant associations (p<0.05) between nutrients and ABP: SBP correlated positively with saturated fat; SBP and MAP correlated negatively with thiamin; and DBP correlated positively with sodium. Several nutrients correlated negatively with PP: protein as % of energy, dietary folate equivalents, sodium, and selenium. SBP, DBP and MAP were positively associated with UFC (r=0.26, 0.24 & 0.26, p<0.01). ABP was not related to PSS score, though UFC and PSS score correlated positively. In this study, relationships between ABP and dietary intake or physical activity were weak, and most were with PP, an indirect index of arterial stiffness, rather than with SBP, DBP or MAP. In contrast, stress (as reflected by UFC) appeared to be a stronger correlate of ABP in these young women. Project funded by CIHR MOP 79563.

Grant Funding Source

Canadian Insitutes of Health Research
Dietary & health predictors associated with overweight & obesity in young adults: the Bogalusa Heart Study
Authors: Deshmukh‐Taskar Priya R. , Mendoza Jason A. , Nicklas Theresa A. , Liu Yan , Berenson Gerald S.
Published: June 2021

We examined independent associations between diet & lifestyle behaviors; differences in markers of cardiovascular disease (CVD), type 2 diabetes mellitus (T2DM), & self‐reported health problems among normal weight (NW), overweight (OW), & obese (OB) young adults. Cross‐sectional data on participants (n =1214, 19‐39 years, 74.1% whites, 60.8% women) from a semi‐rural community of Louisiana were analyzed using covariate‐adjusted multinomial logistic regression, ANOVA & Cochran‐Armitage trend test. OB participants had lower odds of consuming dairy products (OR=0.77; CI=0.62‐0.96); fruits/100% juices/vegetables (OR=0.83; CI=0.75‐0.93); & alcohol (OR=0.16; CI=0.06‐0.37) than NW. OB participants had higher odds of consuming burgers/sandwiches (OR=2.81; CI=1.52‐5.20); processed meats (OR=6.95; CI=2.20‐21.96); high‐fat‐miscellaneous foods (OR=1.60; CI=1.02 ‐2.51); sweetened beverages (OR=1.20; CI=1.01‐1.43); & diet beverages (OR=1.27; CI=1.02‐1.58) than NW. Compared to NW, OB individuals had higher odds of being physically inactive (OR=2.65; CI=1.64‐4.29) & lower odds of being smokers (OR=0.83; CI=0.71‐0.97). Diet explained 4‐9% & smoking & physical activity together explained 3‐7% of the variance in OW & OB. Risk markers for CVD/T2DM (p<.05) & obesity related health problems (p<.05) increased with increasing OW. OW/OB young adults may benefit from modifying dietary & lifestyle behaviors. Supported by NHLBI.
Population reference values for methylmalonic acid (MMA) and its relation with age, race, supplements use, serum creatinine and serum vitamin B‐12 in the post‐folic acid fortification era
Authors: Ganji Vijay , Kafai Mohammad R
Published: June 2021

Serum MMA concentrations indicate tissue vitamin B‐12 deficiency in individuals with normal renal function. The aim of this study was to investigate the relation of serum MMA concentrations with sex, age, race, supplement use, serum creatinine, and serum vitamin B‐12 in the US population using the data from three National Health and Nutrition Examination Survey cycles, 1999‐2000, 2001‐2002, and 2003‐2004 (NHANES 1999‐2004) conducted in the post‐folic acid fortification period (n=18,569). Adjusted mean serum MMA concentrations were significantly higher in women than in men (P<0.033), in Whites than in Blacks and Hispanics (P<0.001), in older persons than in younger persons (P<0.001), and in non‐supplement users than in supplement users (P<0.001). The likelihood of having high serum MMA concentrations (>350 nmol/L) was significantly higher in older persons (=60 y) than in younger persons (<60 y) (OR: 3.82; P<0.0001), in non‐supplement users than in supplement users (OR: 1.81; P<0.0001), in vitamin B‐12 deficient persons (<148 pmol/L) than in vitamin B‐12 sufficient persons (=148 pmol/L) (OR: 13.8; P<0.0001), and in person with high serum creatinine (=115 µmol/L) than in persons with low serum creatinine (<115 µmol/L) (OR: 10.63; P<0.0001). Vitamin B‐12 status, kidney function, age, race, and supplement use were significant determinants of serum MMA concentrations in the post‐folic acid fortification period.
Zinc modulation of osterix expression in osteoblastic MC3T3‐E1 cells
Authors: Alam Md. Jahangir , Cho Young‐Eun , Alcantara Ethel H , Seo Hyun‐Ju , Kwun In‐Sook
Published: June 2021

Osterix is zinc finger transcription factor and downstream of Runx2, which is essential for bone formation and mineralization. In our previous study, Runx2 protein expression decreased by Zn deficiency during osteoblast differentiation phases and at 24 h, which suggests a negative regulation of Runx2. In the present study, we investigated whether Zn deficiency decreases osterix expression, and this modulation will be due to the modulation of Runx2 expression in osteoblasts. In Exp.1, osteoblastic MC3T3‐E1 cells were cultured in Zn‐dependent level (0, 0.25, 0.5, 1, 5, 15 µM ZnCl2) with the cellular Zn depletion by the addition of 5 µM TPEN for 24 h. Osterix gene and protein expression was increased in a Zn concentration‐dependent manner, and Runx2 gene and protein expression showed the similar with the transition pattern of the highest Runx2 expression at 1‐5 µM Zn, which suggests Runx2 expression shifts from 15 µM Zn to the lower Zn level at 24 h. Again, to evaluate Zn effect, in Exp 2, the cells were cultured with 0‐20 µ Zn, Osterix and Runx2 gene expression showed the same pattern of Zn‐dependent. The study results suggest that osterix expression is modulating by cellular Zn level and this modulation would be due to the modulation of Runx2, which also affected by Zn in osteoblasts. (Supported by KOSEF, grant No. R01‐2006‐000‐10640‐0 and KRF grant, No. KRF‐2007‐313‐C00810)
Genistein reduces plasma homocysteine concentrations in a folate deficient rat model
Authors: Nonnecke Eric Brian , Schalinske Kevin L.
Published: June 2021

Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Homocysteine (Hcy) is an intermediate in methyl group metabolism, as S‐adenosylmethionine (SAM), the methyl group donor for most biological methylations, results in the formation of S‐adenosylhomocysteine and ultimately Hcy. Genistein, a 17β‐estradiol‐like isoflavone, has been shown to maintain normal methyl group metabolism in the agouti mouse model fed a methyl‐deficient diet. Although dietary genistein does not provide a methyl group, it may modulate the one‐carbon pool and influence DNA methylation in estrogen‐responsive tissues. To date, it is not known to what extent genistein directly influences Hcy and methyl group metabolism. In the present study, the effects of genistein supplementation on elevated Hcy during moderate folate deficiency were examined. Male Sprague Dawley rats (n=24) were fed ad‐lib control and folate deficient (FD) diets for 14 d, followed by supplementation of each diet with 300mg/kg diet genistein for an additional 24 d. Hcy concentrations in the genistein supplemented FD animals (18.2 + 0.9μM) were reduced (24%; P = 0.01) relative to concentration in unsupplemented, FD rats (24.0 + 2.2μM). Analysis of other hepatic enzymes (i.e. GNMT, PEMT) indicate that the Hcy‐lowering effect of genistein does not reside in reduced production, but rather in increased metabolism.
Consumption of Cranberry Reduces Weight Gain in Mice Fed High Fat Diets
Authors: Boswall Ian Alexander , Shaughnessy Kevin , Slemmer Jennifer , Neto Catherine , Sweeney‐Nixon Marva
Published: June 2021

Obesity and dyslipidemia are major risk factors for cardiovascular disease. To test the health effects of antioxidant‐enriched diets, high‐fat diets (20% by weight) were fed to control (C57BL/6J) and apolipoprotein E‐deficient (ApoE‐/‐) mice, a strain that develops atherosclerotic lesions. Mice were fed either normal AIN‐93 purified diet (CON) or one that contained 5% freeze‐dried cranberry (Vaccinium macrocarpon; CB) for 8 weeks. There was less weight gain in animals fed CB from weeks 3 through 8 (p<0.05; two‐way ANOVA). At week 8, control mice fed CON weighed 39.4 ± 1.95 g, compared to 35.4 ± 1.55 g for control mice fed CB. Similarly, at week 8, ApoE‐/‐ mice fed CON weighed 40.5 ± 1.22 g, compared to 37.5 ± 1.60 g for those fed CB. The amount of abdominal white adipose tissue was lower in CB‐fed mice. Diets were found to be isocaloric, and there were no differences measured in the levels of carbohydrates, fats, cholesterol, and protein in the 2 diets. ApoE‐/‐ mice had elevated plasma cholesterol, HDL, and triglyceride values (p<0.0001), but diet had no effect on any of these endpoints. Plasma pyruvate, lactate, and keto acids will be measured to determine a potential metabolic mechanism for reduced weight gain. These data suggest that CB diet may improve energy balance which would benefit cardiovascular disease and obesity. (Funded by AIF and NSERC).
Effects of supplementation of conjugated linoleic acid with gamma‐oryzanol on body fat mass in healthy overweight Korean women
Authors: Kim Jung Hee , Kim Ok Hyun , Jung Hana
Published: June 2021

This study was done to investigate the enhancement effects of human body fat reduction of CLA with the supplementation of γ‐oryzanol(OZ) on overweight Korean women(n=51, BMI>23). The subjects were divided into 4 groups of control (olive oil), CLA, glyceride form of CLA(GCLA), CLA plus OZ(CLA‐OZ) in randomized, double‐blinded placebo‐controlled study. The soft‐gel capsule(500mg) was used to deliver control(500mg olive oil), CLA(500mg CLA), GCLA(500mg GCLA), and CLA‐OZ(500mg CLA+ 50mg OZ). Three capsules were taken twice a day for 12 weeks. There were no significant differences among 4 groups in energy intake and energy expenditure for the test periods. The CLA‐OZ treatment reduced 1.14kg body fat mass(BFM), whereas CLA, GCLA treatment reduced only 0.36 and 0.71 kg BFM determined by DEXA. Control treatment rather elevated 0.02 kg BFM. As considered subject variations, CLA‐OZ reduced body fat ranged from 7.9% to ‐2.7%, equivalent to 5.6 kg loss to 0.7 kg gain in body fat mass, against CLA. Therefore CLA‐OZ treatment seemed to most effectively reduce body fat, relative to CLA, GCLA and control treatment groups although the reduction by CLA‐OZ were not significantly different from CLA or GCLA group. Also no difference was seen in LBM from all treatments. All biochemical markers analyzed for safety were not significant different within or between groups and were within the normal range. The CLA‐OZ supplement significantly reduced blood pressure, as compared to the supplementation of CLA, GCLA and control. These results that OZ could be a useful ingredient to mix with CLA for the reduction of human body fat. However further studies are needed to confirm the enhancement effect of CLA‐OZ with more subjects and longer treatment periods.
Quercitrin inhibits oxidized LDL‐induced expression of scavenger receptors and vascular endothelial growth factor in cultured murine macrophages
Authors: Choi Jung‐Suk , Kang Young‐Hee
Published: June 2021

Oxidized LDL (oxLDL) has been implicated in the pathogenesis of atherosclerosis accompanying lipid accumulation, pro‐inflammatory responses, and apoptotic endothelial cell death. One pro‐atherogenic mechanism of oxidized LDL involves local up‐regulation of pro‐inflammatory mediators such as vascular endothelial growth factor (VEGF). We examined the ability of polyphenoric quercitrin to inhibit oxLDL‐induced macrophage foam cell formation via class A scavenger receptors (SR‐A). Murine macrophages J774A.1 were cultured with 10 µg/ml oxLDL for 4 h in the presence of 10 µM quercitrin. Submicromolar quercitrin blocked apoptotic cell death induced by cytotoxic oxLDL at 100 µg/ml and considerably lessened the oxLDL‐induced foam cell formation, evidenced by Oil Red O staining. Western blot analysis revealed that oxLDL up‐regulated SR‐A expression and VEGF, which was rapidly abolished by 10 µM quercitrin within 4 h. In addition, 10 µM quercitrin down‐regulated oxLDL‐induced activation of peroxisome proliferator‐activated receptor gamma (PPARγ). These results demonstrate that quercitrin dampens the expression of macrophage SR‐A and VEGF with blocking PPARγ activation. We conclude that quercitrin may be an anti‐atherogenic agent blocking foam cell formation pertaining to VEGF production. Supported by Brain Korea 21 from Korea Research Foundation.
Barley protein supplementation and oxidative damage
Authors: Wong Julia M W , Srichaikul Kristie , Fozdar Nishant , Kendall Cyril W C , Jenkins David J A
Published: June 2021

Objective

To assess the effect of vegetable protein as high protein barley flour, baked into bread, on risk factors for heart disease (i.e. serum lipids and markers of oxidative damage).

Method

In a randomized controlled crossover design, 23 hypercholesterolemic adults (16F, 7M; 57±7y; LDL‐C, 3.90±0.21mmol/L) completed two 4‐week treatments of bread supplementation containing either 30g/d (per 2000 kcal diet) of barley protein (treatment) or dairy protein (calcium caseinate placebo). Outcomes included serum lipids and markers of oxidative damage, measured as serum protein thiols, and MDA and conjugated dienes in the LDL fraction.

Results

At week 4, supplementation with barley protein did not provide evidence of improved LDL‐C and TC:HDL‐C, expressed as the difference from baseline (‐0.05±0.12 mmol/L, P=0.685 and 0.19±0.15 mmol/L, P=0.216, respectively) compared to control (‐0.02±0.12 mmol/L, P=0.858 and 0.19±0.11 mmol/L, P=0.094, respectively). No effect was seen on serum protein thiols, as an overall marker of oxidative status. In addition, neither MDA nor conjugated dienes in the LDL fraction, as markers of oxidized LDL, were affected differently by barley compared to dairy protein.

Conclusion

Barley protein appears similar to low fat dairy protein in its effect on blood lipids and markers of oxidative damage.

Research Support: NSERC, Loblaw Companies Limited

Grant Funding Source

Canadian Institutes of Health Research
Oven‐dried Brussels sprouts with undetectable levels of glucosinolates or isothiocyanates increased expression of detoxification enzymes in vitro and in vivo
Authors: Williams Melissa , Barnes David , Hanlon Paul
Published: June 2021

Many individuals use supplementation to increase exposure to crucifers in order to decrease their risk of chronic disease. However supplements require extensive processing including grinding and drying that can affect their ability to induce detoxification enzymes. This study measured the ability of oven‐dried (OD) (without glucosinolates (GLS); without myrosinase activity (MYR)), freeze‐dried (FD) (with GLS; with MYR), and blanched, freeze‐dried (BFD) (with GLS; without MYR) Brussels sprouts to increase detoxification enzyme expression in vitro and in vivo. A 48 hour treatment with OD and FD, but not BFD, Brussels dose‐dependently induced quinone reductase (QR) activity and the expression of RNA for cytochrome P450 (CYP) 1A1, CYP1A2, heme oxygenase, and QR in HepG2 human carcinoma cells. In vivo, mRNA expression of detoxification enzymes was measured in the livers of mice fed diets containing BFD or OD Brussels (20%, 10% or 5%) for 14 days. Mice fed diet containing OD Brussels demonstrated a dose‐dependent induction of CYP1A1, CYP1B1, and QR in the liver, while BFD Brussels had no effect. Despite lacking the phytochemicals typically thought to be responsible, the OD Brussels sprouts induced detoxification enzymes. Additionally BFD Brussels failed to induce detoxification enzymes in vitro and in vivo, suggesting that endogenous myrosinase activity is required for the bioactivity of FD Brussels sprouts.
High protein and resistant starch have a synergistic effect on reducing body fat levels and increasing fermentation in rats
Authors: Vidrine Kirk , McCutcheon Kathleen L , Zhou Jun , Raggio Anne M , Goita Mfamara , Lammi‐Keefe Carol , Tulley Richard T , Williams Cathleen , Martin Roy J , Keenan Michael J
Published: June 2021

Obesity and colon health is a growing medical problem in the U.S. Previous studies have shown independent effectiveness of resistant starch (RS) and high protein (HP) diets in decreasing body fat. HP diets have been shown to reduce food intake by altering brain neuropeptides. RS may produce this effect through mechanisms related to colonic fermentation that also improves colon health. The objective of this study was to determine whether there are additive effects of RS (20%) and high protein (40 %) on the reduction of fat accumulation. Forty female rats were fed control diets for a 4 week free feeding period. Rats were then stratified by weight and placed in 4 diet treatment groups (n=10): HP, RS, RS/HP and control. They were meal fed for a 2 week restricted feeding period followed by an 8 week free feeding rebound period. Analysis was a 2x2 factorial followed by LSD mean comparisons (p<0.05). HP/RS showed a significant reduction in mesenteric and % total dissected fat. There were synergistic interactions between RS and HP as the RS/HP group had the lowest pH and greatest cecal contents which are indicators of fermentation. This combination may reduce body fat levels and benefit the health of the colon. Funded by the LSU AgCenter.
Induction of Phase 2 Detoxifying Enzymes by Dehydroglyasperin C Isolated from Licorice
Authors: KIM Jong‐Sang , Seo Ji‐Yeon , LIM Ji‐Sun , Quintos Lesley , LIM Soon Sung , Lee Choong Hwan , Yoon Park Jung Han
Published: June 2021

This study has been performed to confirm the potential of dehydroglyasperin C isolated from licorice to induce phase 2 detoxifying (antioxidant) enzymes using cultured cell and mouse model systems. Dehydroglyasperin C caused a dose‐dependent induction of QR enzyme activity in hepa1c1c7 mouse hepatoma cells and its mutant BPRc1 cells. The compound increased the luciferase activity of HepG2‐C8 cells, transfectants carrying antioxidant response element (ARE)‐luciferase gene, in a dose‐dependent manner, suggesting ARE‐mediated transcriptional activation of antioxidant enzymes. Dehydroglyasperin C also stimulated nuclear accumulation of Nrf2 as evaluated by immunocytochemistry. Furthermore, the compound resulted in a significant induction of QR activity of kidney and large intestine in mice when injected at the dose of 5 mg/kg b.w. three times for one week, suggesting that dehydroglyasperin C might have the anticarcinogenic potential in whole body system.
Nitric oxide inhibition of chronic Hcy‐induced endothelial cell (EC) activation is cGMP‐independent
Authors: Graham Anne Margaret , Alkhoury Kenan , Naseem Khalid , Parkin Susan Mary
Published: June 2021

Hyperhomocysteinemia (hHcy) upregulates expression of cell adhesion molecules (CAM) on EC which recruits leukocytes and initiates atherosclerotic lesion development. Nitric oxide (NO) is anti‐adhesive for leukocytes.

Objective

to study effects of exogenous NO on hHcy CAM expression in human umbilical vein endothelial cells (HUVEC) and recruitment of leukocytes under flow.

HUVEC were cultured for 5‐9 days in medium containing 1mM Hcy or Cysteine (negative control). Some Hcy‐stimulated cells were treated with NO donor (S‐nitrosoglutathione: GSNO: 20µM, 10min) in absence of presence of a cGMP inhibitor (ODQ 40µM, 30min). Confluent cells were subjected to flowing neutrophils (1x106cells/ml) at 1.1dynes. Tethered, rolled, fixed and transmigrated neutrophils on HUVEC were counted. Immunofluorescence antibodies against ICAM‐1, E‐selectin and P‐selectin quantified adhesion molecule expression.

Hcy induced expression of ICAM‐1, E‐selectin and P‐selectin (p<0.05) and significantly increased interactions between neutrophils and HUVEC while cysteine had no effect. When Hcy‐treated HUVEC were exposed to GSNO with or without ODQ, numbers of all interactions decreased dramatically but significantly decreased only P‐selectin expression (p<0.05).

NO inhibits HUVEC: leukocyte interaction through inhibiting the expression of P‐selectin in a cGMP‐independent manner.

Funding: HRUK, ORSAS
Role of cellular senescence in the age‐associated risk of venous thrombosis
Authors: Cardenas Jessica Caroline , Krishnamurthy Janakiraman , Sharpless Norman , Whinna Herbert , Church Frank
Published: June 2021

During normal aging, expression of the tumor‐suppressor gene, p16ink4a, results in cellular senescence in many tissues, including the vasculature. We hypothesize that p16ink4a‐mediated cellular senescence is associated with impaired hemostasis and contributes to a prothrombotic phenotype. In this study, we have used in vivo murine models of vascular injury to examine the role of p16ink4a and cellular senescence in venous thrombosis. Transgenic mice over‐expressing p16ink4a were developed and real‐time PCR data show a 6.5 fold increase in expression of p16ink4a in the saphenous vein/artery bundle. When challenged with an acute ferric chloride injury to the saphenous vein, the p16ink4a transgenic mice have a significantly shorter occlusion time when compared to wild‐type controls. Wild‐type mice show an average occlusion time of 20 minutes versus 13.5 minutes in the p16ink4a transgenic mice. Additional studies have shown that p16ink4a does not alter blood cell populations or venous flow velocity. Currently, we are examining the role of cellular senescence in the response to injury over time. Our results suggest the p16ink4a transgenic mice have a delayed response to injury and are more sensitive to damage induced by ferric chloride. Our studies with the transgenic p16ink4a mice are the beginning to our understanding the role of cellular senescence in the risk of venous thrombosis.

NIH support 1R21AG031068‐01.
Differential thrombin generation inhibition by branded and generic low molecular weight heparins (LMWHs) as studied using a fluorescence substrate based kinetic method
Authors: Adiguzal Cafer , Litinas Evangelos , Cunanan Josephine , Hoppensteadt Debra , Fareed Jawed
Published: June 2021

Thrombin generation reflects the overall activity of the coagulation cascade. Conventional clot‐based methods are not useful for monitoring LMWHs or fondaparinux. In order to compare the relative inhibitory effects of branded enoxaparin (Lovenox) and several generic LMWHs (Lupenox, Loparin, Dripanina, Dilutol, Cutenox and Clenox), we used a thrombin generation assay (Technothrombin® TGA) based on cleavage of a fluorescent substrate. Each LMWH was supplemented to plasma at concentrations of 2.5 to 10µg/ml. Plasma samples were activated by reagents containing various concentrations of tissue factor (TF) and negatively charged phospholipids and the change in fluorescence was measured. The change in fluorescence is proportional to the amount of thrombin generated. Each LMWH concentration dependently inhibited thrombin generation. With the lower concentration of TF (TGA RC‐high), almost complete inhibition of thrombin generation was noted with 10µg/ml LMWH. The IC50s for the LMWHs ranged from 2.5 to 5µg/ml. Only partial inhibition of the thrombin generation was noted at a LMWH concentration of 10 µg/ml when a higher concentration of TF (TGA RD) was used. Each LMWH showed a distinct inhibitory profile. These studies showed that LMWHs can be differentiated based on their ability to inhibit thrombin generation. Furthermore, the TGA RC‐high is more suitable for the monitoring of LMWHs in contrast to the TGA RD.
Neutralization of Hemorrhagic and Antithrombotic Activities of Heparins by a Novel Salicylamide Derivative
Authors: Kuziej Jacqueline , Litinas Evangelos , Hoppensteadt Debra , Liu Dahui , Walenga Jeanine M. , Fareed Jawed , Jeske Walter
Published: June 2021

We previously showed that salicylamide derivatives neutralize heparin activity in vitro. This study evaluates the ability of one salicylamide derivative (PMX60056; PolyMedix, Radnor, PA) to neutralize hemorrhagic/antithrombotic actions of heparins in defined animal models. Blood was collected to evaluate circulating anticoagulant activity. At 2 mg/kg, UFH and enoxaparin prolonged bleeding time versus vehicle (UFH: 29.1±5.1 min; enoxaparin: 16.7±3.6 vs. vehicle: 7.3±1.4 min; p<0.001). Protamine and PMX60056 dose‐dependently reduced heparin‐induced bleeding; at equigravimetric doses, both agents produced near complete neutralization of heparin‐induced bleeding. PMX60056 was somewhat more effective than protamine (7.4±1.2 vs. 8.7±1.5 min) at neutralizing enoxaparin‐induced bleeding. Residual antithrombotic activity was comparable in the UFH and enoxaparin‐treated rats (7.2±1.1 and 7.0±0.6 clampings, respectively vs. 3.4±0.5 for vehicle). Comparable neutralization of antithrombotic activity by protamine and PMX60056 was observed. Residual anticoagulant/anti‐protease activity correlated poorly with neutralization of bleeding. These results suggest that PMX60056 is as effective as protamine at neutralizing antithrombotic/hemorrhagic actions of UFH and may be slightly better at neutralizing enoxaparin. Further characterization of the salicylamide derivatives is warranted.
Thrombin induces cyclooxygenase‐2 expression through MAPKs and NF‐¿B in osteoblast MC3T3‐E1 cells
Authors: Chiang Yi‐Chen , Hsieh Hsi‐Lung , Yang Chuen‐Mao
Published: June 2021

Thrombin, a serine protease, has hormone‐like effects on cells from many tissues, including bone. Thrombin can induce expression of COX‐2 catalyzed the synthesis of prostaglandin (PG) in osteoblasts, which plays a crucial role in regulating bone resorption. Here, the mechanisms of MAPKs and NF‐κB pathways involved in thrombin‐induced COX‐2 expression were investigated in MC3T3‐E1 osteoblast cells. In this study, we demonstrated that thrombin‐induced COX‐2 protein and mRNA expression in a time‐ and concentration‐dependent manner, which was attenuated by pharmacological inhibitors of MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125) and NF‐κB (Bay11‐7082), revealed by RT‐PCR and Western blot analyses. Consistently, thrombin‐stimulated phosphorylation of p42/p44 MAPK, p38 MAPK and JNK1/2 was attenuated by pretreatment with U0126, SB202190 or SP600125. The implication of NF‐κB in thrombin‐mediated responses was supported by translocation of NF‐κB into the nucleus and degradation of IκB‐α stimulated by thrombin. Taken together, these results suggest that in MC3T3‐E1 osteoblast cells, activation of MAPKs and NF‐κB translocation are essential for thrombin‐induced COX‐2 gene expression. These results provide new insight into the mechanisms by which thrombin may contribute to bone remodeling.
The KIAA0391 and PSMA6 gene cluster confers a risk factor for coronary artery disease
Authors: Dzimiri Nduna , Muiya Paul , Alsmadi Osma , Al‐Saud Haya , Meyer Brian , Alshahid Maie , Al‐Mohanna Futwan
Published: June 2021

We evaluated by molecular beacon‐based genotyping assays the roles of 5 SNPs in the chromosomal region 14q13.2 harbouring the KIAA0391 and PSMA6 gene cluster in coronary artery disease (CAD) in the Saudi population. In a preliminary study involving 502 patients and 474 controls, we depicted rs4981283 (denoted as 5) (χ2=0.014; p=0.015) as conferring risk for CAD and the rs7157492 (2) (χ2=0.027; p=0.028) as protective, while the rs8008319 (1) (χ2=0.045; p=0.055), rs1048990 (3) and rs1287391 (4) were not associated with CAD. Increasing the study size to 1166 patients and 835 controls resulted in the diminishing of the significance for these associations. In contrast, 3 haplotypes consisting of 1T‐2G‐3C‐4G‐5A (χ2=9.99, p<0.0001), 1T‐2A‐3G‐4A‐5G (χ2 = 7.80, p<0.03) and 1A‐2A‐3G‐4G‐5A (χ2=6.93, p<0.03) with frequency of 0.042, 0.010 and 0.001 respectively in controls consistently conferred risk for CAD in a 5‐SNP locus model in both the preliminary and primary studies. Two other haplotypes [1A‐2G‐3G‐4A‐5A (χ2=6.15, p<0.04) and 1T‐2A‐3G‐4G‐5A (χ2=8.2, p<0.04)] with frequencies of 0.01 and 0.003 respectively in the controls, were protective. Notably, the haplotype 1T‐2G‐2C‐4G‐5A also conferred risk for MI (χ2=4.91, p<0.05). Thus, our study identified a haplotype in the chromosomal region encompassing the KIAA0391 and PSMA6 genes as a genetic link between CAD and MI, and points to haplotypes as more informative markers than individual SNPs for identifying disease risk factors.
Generic versions of argatroban can be differentiated from branded argatroban using a thrombin generation assay (TGA)
Authors: Adiguzal Cafer , Litinas Evangelos , Cunanan Josephine , Hoppensteadt Debra , Fareed Jawed
Published: June 2021

Beside direct inhibition of thrombin, argatroban inhibits tissue factor mediated generation of proteases such as thrombin and Xa and the resultant formation of fibrin. Since generic and branded argatrobans have different thrombin inhibitory potencies, we hypothesized that differences in thrombin generation inhibition may also be observed. To test this hypothesis, we used the TECHNOTHROMBIN TGA assay to measure thrombin generation in platelet poor plasma (PPP) and platelet rich plasma (PRP) supplemented with argatrobans (branded and three generics: slovastan, gartban, and argaron) at concentrations from 0.6 to 5 µg/ml. Activators containing tissue factor and phospholipids at varying concentrations were used to generate thrombin. The order of inhibitory potency in PPP was argatroban> gartban> slovastan. All of the generic products showed stronger thrombin generation inhibition in PPP in comparison to argatroban. In PRP, there was a much wider difference among the generic argatrobans and order of potency was slovastan> gartban> argaron. In the fibrinokinetic assays wider differences were noted between generic and branded products; rank order was gartban> slovastan > argaran> branded argatroban. These observations underscore the differences between the generic and branded products and suggest that beside thrombin inhibitory assays argatroban can be differentiated in other biologic assays.
Reactive Oxygen Species Promote Angiogenesis in the Infarcted Rat Heart
Authors: Zhao Wenyuan , Zhao Tiqiang , Chen Yuanjian , Sun Yao
Published: June 2021

Cardiac reactive oxygen species (ROS) production is significantly increased following infarction (MI), particularly at early stage. Herein we sought to determine whether ROS play a role on angiogenesis in the infarcted heart. Experimental MI rats were treated with combined antioxidants, tempol and apocynin, each 120mg/kg/day. Non‐treated MI rats served as controls. Hearts were collected at day 1, 3, 7, 14, 28 and 42. We examined effect of antioxidants on angiogenesis in the infarcted myocardium. Following MI, NADPH oxidase (gp91phox subunit) was significantly increased in the infarcted myocardium at day 3, reached peak at day 7, and gradually declined thereafter. In the infarcted region, existing vessels were lost at day 1 postMI. Newly formed vessels appeared in the border zone as early as day 3, followed by the infarcted myocardium. Vascular density peaked at day 7 and 14, and progressively reduced thereafter. Antioxidants significantly reduced cardiac vascular density at day 7 and 14, but not in the later time points. Thus, angiogenesis is activated in the infarcted myocardium in the first two weeks and becomes quiescent in the late stage of MI, which is temporally and spatially coincident with increased ROS production. Our study also indicates that ROS promote angiogenesis in the infarcted myocardium. Further studies are required to determine the mechanisms responsible for ROS mediated cardiac angiogenesis.
The Level of Cardiac Specific Overexpression of Adenylyl Cyclase Type 2 Dictates the Response to Chronic Pressure Overload
Authors: Guellich Aziz , Hu Che‐Lin , Yan Lin , Hong Chull , Gao Shumin , Fritzky Luke F. , Wagner Thomas , Ishikawa Yoshihiro , Iwatsubo Kosaku , Hittinger Luc , Ghaleh Bijan , Sadoshima Junichi , Vatner Dorothy E. , Vatner Stephen F.
Published: June 2021

The major adenylyl cyclase (AC) isoforms in the heart are AC5 and AC6, which when overexpressed, exert different effects on the response to chronic pressure overload (CPO). Accordingly, the goal of this study was to examine the effects of overexpression (OE) of an AC isoform, which does not normally play a major role in the heart, eg, AC2, on the response to CPO. Two lines were generated: one with moderate AC2 OE of the transgene and the other with a higher level. The AC2 protein was 2 times greater in the line with higher OE. At least 6 WT and 6 AC2 OE from each group were subjected to transverse aortic constriction (TAC) or sham (S) operation for 1 week. With moderate AC2 OE, CPO did not affect the LV ejection fraction (EF) measured with echocardiography (75±1%) vs WT banded (72±1%), or with sham operated animals (76±1%). With higher AC2 OE, 1 week of CPO leads to a significant decrease in LVEF compared with WT (47±4 vs 72±1%) and increase in LVEDP (22.1±7.8 vs 3.5±0.5 mmHg). These were associated with more LV hypertrophy [LV weight/body weight (BW) ratio increased 121% vs 31%] and development of heart failure, as reflected by increased lung weight/BW (278%) vs WT (11%), and 3‐fold more fibrosis on histopathological examination. Thus, cardiac expression of AC2 at a moderate level does not affect the response to CPO adversely, but at a high level, results in more LV hypertrophy and reduced cardiac function leading to heart failure.
Heat Shock Protein 70 in Myocardial Ischemia: Isoform Specific Roles?
Authors: Wilhide Michael E , Ren Xiaoping , Tranter Michael , Jones W. Keith
Published: June 2021

NF‐κB has been shown to contribute to myocardial infarction (MI) after ischemia/reperfusion (I/R) and yet mediates pro‐survival signaling after specific manipulations, including after permanent coronary artery occlusion (PO). The aim of this study was to discover the mechanism by which NF‐κB regulates cardioprotection after PO compared to cell injury seen after I/R. We subjected transgenic mice (DN) that block NF‐κB in a cardiac‐specific manner after I/R or PO and microarrays were used to delineate the genes regulated acutely by NF‐κB and regulated by I/R or PO (n=4 for all groups). These results showed that NF‐κB significantly upregulated HSP70.1 after I/R, while HSP70.3 is significantly upregulated by NF‐κB after PO. An in‐vivo mouse model of I/R (30m/24h) or PO (24hr) was used to assess the impact of HSP70 isoform expression on I/R and PO models. After I/R, HSP70.1/.3 KO mice showed a significant increase in infarct size compared to WT and DN. However, after I/R, the HSP70.1 KO had a smaller infarct size relative to WT. In the PO model, HSP70.1/.3 KO mice had a larger infarct size compared to WT, whereas, HSP70.1 KO exhibited no change in infarct size relative to the DN model. These results suggest that NF‐κB regulation of HSP70.1 plays a cell injury role after I/R, while NF‐κB regulation of HSP70.3 in PO contributes to cardioprotection. This work was supported by NIH grant R01 HL63034 (WKJ) and 5F31 HL081923 (MW).
Elevated Advanced Glycation Endproducts (AGEs) in Rat Models
Authors: Zhao Chunsheng , Yang Jieying , Zhang Yan , Tan Ningzhi , Zhu Jia‐Shi
Published: June 2021

It is reported that plasma AGEs are elevated in patients with diabetes mellitus (DM) and in rats with STZ‐induced DM, where non‐enzymatic formation of AGEs causes crosslink of proteins, lipids and DNA. We examined in this study the plasma AGEs fluorescence (ex. 360nm, em. 450nm) in 3 rat models. (1) Male SD rats were given STZ (i.p. 60mg/kg), and continuously drank water or 5% fructose or sucrose water. Addition of fructose in drinking water caused higher mortality (92%) in rats, compare to controls (p<0.001). Addition of sucrose did not increase plasma AGEs in STZ‐induced DM rats (p>0.05). (2) In alloxan‐induced (i.v. 40mg/kg) DM rats, plasma AGEs were increased by 41%, 108% and 239% at Wks 4, 8 and 12, respectively, compared to normal rats (p<0.05). Carboxymethyl lysine (CML) and HbA1c were also increased at Wk 8 by 673% and 239%, respectively (p<0.01). (3) In a high‐calorie diet‐induced Metabolic Syndrome model in male SD rats, plasma AGEs were increased by 12% at Wk 12, as compared to normal rats (p<0.05). In summary, the alloxan‐induced DM rats and diet‐induced Syndrome X rats may serve as animal models for AGEs research, in addition to the STZ model. Fructose or sucrose drinks are not options of increasing plasma AGEs in the STZ‐induced DM rats. It is to be studied if fructose drinks would accelerate plasma AGEs elevation in diet‐induced Syndrome X rats.
Curcumin protects against glucose‐induced endothelial dysfunction through HO‐1 and GC pathway
Authors: Fang Xiao‐dong , Yang Fan , Zhu Li , Shen Yue‐liang , Chen Ying‐ying
Published: June 2021

Aim

To investigate the effect and mechanism of curcumin on high glucose induced injury in blood vessels.

Methods

The thoracic aortic rings with or without endothelium from male SD rats were mounted in an organ bath. Isometric contraction of aortic rings was measured. Heme oxygenase (HO) activity was also evaluated.

Results

(1)After incubation with 44 mmol/L of high glucose for 2 h, the vascular relaxation response to acetylcholine (Ach) decreased in an endothelium‐dependent manner; (2)Coincubation with curcumin (10−13~10−11 mol/L) and high glucose, the high glucose‐induced vasodilation dysfunction was inhibited in a dose‐dependent manner; (3) Curcumin increased the HO activity of thoracic aorta. ZnPPIX (an inhibitor of heme oxygenase‐1) offset the protective effect of curcumin. ; (4) A non‐selective guanylate cyclase (GC) inhibitor methylene blue also completely abolished the protective effect of curcumin.

Conclusion

Curcumin could alleviate the high glucose‐induced acute endothelium‐dependent vasorelexation dysfunction in rat aortic rings. HO‐1 and GC were proposed as a mechanism to account for the protection of vascular responses by curcumin.
Localization and coupling of adenylyl cyclase isoforms 2, 3 and 6 with G protein‐coupled receptors in mouse bronchial smooth muscle cells
Authors: Thangavel Muthusamy , Adris Piyatilake , Ostrom Rennolds
Published: June 2021

Several GPCR stimulate adenylyl cyclase (AC) to increase cAMP and promote relaxation in smooth muscle. β‐adrenergic receptors only couple to certain AC isoforms due to compartmentation in lipid rafts. Previous data show AC2, AC3, AC4 and AC6 are expressed in primary mouse bronchial smooth muscle cells (mBSMC). We overexpressed AC2, AC3 or AC6 in mBSMC using recombinant adenoviruses and assessed their localization by cell fractionation and confocal microscopy then determined the coupling of various GPCR to each AC isoform by examining the arborization response to receptor‐specific agonists. Lipid rafts were isolated using a detergent‐based fractionation method and fractions were analyzed by immunoblotting. AC3 and AC6 were expressed primarily in caveolin‐rich fractions, whereas the AC2 expression was excluded from these domains. Forskolin‐induced arborization of mBSMC, measured by time‐lapse microscopy, was enhanced by overexpression of all three AC isoforms. Isoproterenol‐ or beraprost‐mediated arborization was only accelerated by AC6 overexpression. In contrast, butaprost‐mediated arborization was only accelerated by AC2 overexpression. AC3 overexpression did not significantly alter the response to any of these agonists. Our results imply that particular GPCR couple to discreet AC isoforms, likely based upon their colocalization. Supported by HL079166.
Endocrine and neurochemical effects of S(+) and R(‐)‐MDMA in rhesus macaques
Authors: Murnane Kevin Sean , Fantegrossi William , Godfrey Jodi , Neidert Lisa , Howell Leonard
Published: June 2021

3,4‐Methylenedioxymethamphetamine (MDMA) is an amphetamine derivative that produces complex subjective effects in humans. One plausible mechanism for this phenomenon is that racemic MDMA is composed of two isomers that exhibit qualitatively different effects. In particular, drug discrimination studies in rodents have shown that R(‐)‐MDMA tends to have hallucinogen‐like effects whereas S(+)‐MDMA tends to have stimulant‐like effects. However, relatively little work has shown that these qualitative differences extend across taxa. In the present study, the neurochemical and endocrine effects of each isomer were assessed in rhesus macaques after non‐contingent intravenous delivery of each isomer. Specifically, in vivo microdialysis was utilized to assess extracellular concentrations of dopamine in the dorsal striatum and plasma concentrations of prolactin were assessed to determine the systemic effect of each isomer on dopamine and serotonin overflow. Only S(+)‐MDMA significantly increased dopamine concentration, and it produced a blunted increase in prolactin levels compared to R(‐)‐MDMA. These studies demonstrate that qualitative differences in the effects of the isomers of MDMA extend to primates; strengthening the inference that this may be the mechanism producing the complex subjective effects of MDMA in humans. These studies were supported by USPHS grants (DA 10344, DA 00517, and RR 00165).
Repeated social stress reveals two populations characterized by different behavioral and endocrine stress response profiles: a model of stress vulnerability and resilience
Authors: Wood Susan K , Walker Hayley , Valentino Rita J , Bhatnagar Seema
Published: June 2021

Chronic exposure to social stress results in psychological disorders in vulnerable individuals. Repeated social stress (7 days) using a rat resident‐intruder model resulted in two populations, characterized by distinct behavioral and endocrine profiles. One population exhibited a passive response (e.g., freezing) and assumed a defeat posture within a short latency (SL). The second group exhibited proactive behaviors (e.g., boxing) and a longer latency (LL) to be defeated. The proactive behavior of the LL phenotype emerged after 4 sessions of stress and was temporally correlated with habituation of the adrenocorticotropin (ACTH) response to defeat, not observed in SL rats until day 7. The SL phenotype exhibited a stress axis dysfunction resembling melancholic depression, characterized by adrenal hypertrophy, blunted ACTH release to CRF and novel restraint stress, and decreased CRF1 receptors in pituitary compared with controls. SL rats also exhibited depressive‐like behaviors in the forced swim test. In contrast, the LL phenotype exhibited a blunted ACTH response to a CRF infusion; however the ACTH response to restraint was facilitated. These data suggest the LL phenotype is more resilient to adverse consequences of social stress. Elucidating the neural basis of these two phenotypes may increase our understanding of the pathophysiology of stress‐related depression. MH06751 to SB, MH40008, MH58250.
Overexpression of VEGF165b in mouse ovary results in reduced litter size
Authors: Qiu Yan , Murphy David , Harper Steven J. , Bates David O.
Published: June 2021

Physiological angiogenesis occurs in the ovary, and facilitates oxygen, nutrient, and hormone substrate delivery and transfer of synthesized hormones to targeted cells. VEGF plays a key role in the regulation of ovarian follicle development and corpus luteum formation and maintenance.

VEGF is differentially spliced in the terminal exon to form two families of proteins, termed VEGFxxx and VEGFxxxb, where xxx denotes the amino acid number. VEGF165b inhibits VEGF165 induced endothelial cell proliferation, migration, vasodilatation and in vivo angiogenesis,

To identify the biological function of VEGF165b in the ovary, nine female transgenic mice (TG) overexpressing VEGF165b under the control of the MMTV promoter and 10 wild type (WT) female littermates were mated with WT C57Bl6 mice. RT‐PCR indicated strong expression of VEGF165b in ovaries of TG females. TG mothers produced litters of half the size of WT dams (3.3±0.4 vs. 5.8±0.7 pups/litter), or TG males (6.1±0.7 pups/litter). There was no developmental defect identified in either the WT or TG pups from TG mothers suggesting subfertility due to defects in the female reproductive system. We have therefore shown that over‐expression of VEGF165b reduces fertility, possibly by its anti‐angiogenic actions on follicular development. This project is supported by the Wellcome Trust and British Heart Foundation.