bluuebunny/crossref_metadata_2025 huggingface dataset:
Oxidative Damage during a 10‐ to 12‐d Saturation Dive
Authors: Zwart Sara R. , Smith Scott M.
Published: June 2021

The NASA Extreme Environment Mission Operations (NEEMO) underwater habitat is a useful spaceflight analog. The increased air pressure in the habitat exposes crew members to higher oxygen pressures, which increase their risk for oxidative damage to DNA, proteins, and lipids. DNA oxidation occurs at an increased level, similar to that observed in smokers and astronauts returning from space. Astronauts in space and NEEMO crew members also have changes in iron metabolism. Newly formed red blood cells are destroyed and body iron stores are elevated. Excess iron can act as an oxidant and cause tissue damage. In this study we investigated aspects of oxidative damage and tested whether toxic forms of iron were present when iron stores increased during NEEMO missions. Subjects (n = 12) participated in 10‐ to 12‐d saturation dives, and blood and 24‐h urine samples were collected twice before, twice during, and twice after the dive. During the dive, ferritin was significantly (P < 0.001) higher, transferrin tended to be lower (P = 0.053), and transferrin receptors were lower (P < 0.001). Total homocysteine was significantly (P < 0.001) elevated and superoxide dismutase activity was decreased (P < 0.05). Labile plasma iron was measurable during the dive but not before or after it. These data indicate that the NEEMO environment increases iron stores, which may cause oxidative damage. This study was supported by the NASA Human Research Program.
Micellarization of beta‐carotene during in vitro digestion of maize and uptake by Caco‐2 intestinal cells is minimally affected by xanthophylls
Authors: Thakkar Sagar K , Failla Mark L
Published: June 2021

Currently consumed varieties of maize contain limited pro‐vitamin A (VA) carotenoids despite relatively high levels of lutein (LUT) and zeaxanthin (ZEA). Here we determined the relative bioaccessibility of pr‐VA carotenoids and their interactions with xanthophylls. First, we examined four accessions of maize containing 0.4–11.3 μg/g β‐carotene (BC) + β‐cryptoxanthin (BCX) with ratios of xanthophylls to pro‐VA carotenoids ranging from 1.9 – 7.0. Recovery of endogenous carotenoids exceeded 80% after simulated digestion of cooked maize. Mean efficiencies of micellarization of BC, BCX, LUT and ZEA were 16.7, 27.7, 30.3 and 27.9%, respectively, and was independent of the ratio of xanthophylls to pro‐VA carotenoids. We also digested white maize to which 3.3 μg/g BC and increasing amounts of LUT (0–33.3 μg/g) were added. Efficiency of micellarization of BC was 21.7% in absence of LUT but was significantly higher when LUT content was 7x that of BC. LUT also significantly increased incorporation of BC into synthetic micelles when LUT to BC ratio ≥10. Caco‐2 cells accumulated 270, 240 and 180 pmol/mg cell protein (p < 0.05) in absence of LUT and at LUT to BC ratios of 7 and 13, respectively. These results suggest that the potential bioavailability of pro‐VA carotenoids in maize does not appear to be markedly affected by the relative levels of xanthophylls found in cultivars of maize. Supported in part by HarvestPlus & OARDC.
Presence of apo‐lycopenals in food products and human blood plasma
Authors: Kopec Rachel Elizabeth , Riedl Kenneth M , Curley Robert W , Harrison Earl H , Schwartz Steven J
Published: June 2021

Recent research has suggested that lycopene, like β‐carotene, may be metabolized in mammals by eccentric cleavage to aldehydes, catalyzed by β‐carotene oxygenase 2. Since apo‐6′‐ and apo‐8′‐lycopenal have been reported in raw tomato, we hypothesized that several lycopenals may be present in raw and processed foods as well as blood plasma. Lycopenal standards were prepared by in vitro oxidation of lycopene, and an atmospheric pressure chemical ionization negative HPLC‐MSMS method was developed to separate and detect the apo‐6′‐, 8′‐, 10′‐, 12′‐, and 14′‐lycopenal products formed in the reaction. Hexane/acetone extracts of raw tomato, processed tomato products, grapefruit, and watermelon were analyzed, as well as blood plasma of individuals consuming tomato juice for 8 weeks. Apo‐6′‐, 8′‐, 10′‐, 12′‐, and 14′‐lycopenals were detected in all food products tested, and apo‐6′‐, 8′‐, 10′‐, 12′‐ were detected in blood plasma. We conclude that several lycopenals, in addition to apo‐6′‐ and 8′‐, are present in lycopene containing food products and that the presence of apo‐lycopenals in plasma may partly derive from the absorption of lycopenals directly from food.
A single nucleotide polymorphism in the calcitonin receptor gene (CALCR) is associated with bone integrity in response to dietary phosphate deficiency
Authors: Alexander Lindsey Sharone , Cutler Sara Anne , Qu Ani , Mahajan Avanika , Rothschild Max , Stahl Chad H
Published: June 2021

Previous work has demonstrated that genetic background may impact P utilization in young pigs. In order to examine the impact of a polymorphism in the CALCR (represented by three genotypes: 11, 12, and 22) on P utilization in growing pigs, we utilized 42 young female pigs fed either a P adequate diet or a 20% P deficient diet for 14 wk. Plasma P concentrations were higher in P adequate animals at 8 wk and all subsequent samplings (P < 0.05). While plasma concentrations of 1, 25 (OH)2 vitamin D were elevated (P < 0.05) among P adequate pigs of the 11 and 12 genotypes at 8 wk, PTH concentrations, regardless of genotype, were decreased among P adequate animals at the same time point (P < 0.05). For animals of the 11 or 12 genotypes, P adequacy resulted in higher bone strength and ash % when compared to P deficient animals within the same genotype (P < 0.05). Similar differences were not observed within the 22 genotype, suggesting that animals of this genotype are more resistant to dietary P deficiency. These data are suggestive of an association between this polymorphism and the response to dietary P restriction. Understanding the underlying genetic mechanisms that regulate P utilization may lead to novel strategies to maximize bone integrity as well as minimize the environmental impact of pig production. This work funded in part by BRDC and the NC Agriculture Research Service.
Bone microarchitecture and strength are not enhanced by onion in an ovariectomized rat model
Authors: Lanfair Deborah , Deyhim Farzad , Patil Bhimanagouda S. , Stoecker Barbara J.
Published: June 2021

Onion contains an abundance of flavones and was previously found to have beneficial effects on bone quality. Our study examined the potential of four varieties of onion to prevent bone loss in an ovariectomized (OVX) rat model for osteoporosis. Female Sprague‐Dawley rats were fed the AIN‐G diet and were sham‐operated or ovariectomized at 90 days of age. One of the OVX groups received the same dietary treatment as the SHAM group and the other four groups had diets supplemented with 5% of one of four different varieties of freeze‐dried onion. The experimental period continued for 60 days. At necropsy, the tibia and 4th lumbar vertebra (L4) were removed. Bone mineral density (BMD) of the tibia was assessed using Hologic dual energy X‐ray absorptiometry (DEXA) with high resolution software. Tibia and L4 were scanned each 20 microns using microcomputed tomography (μCT) (Scanco Medical, Ltd). Microarchitecture of trabecular bone was evaluated on a contoured volume of interest (VOI) from L4. Cortical bone was evaluated at the tibia midshaft. OVX significantly reduced BMD, trabecular bone quality and force for compression of the VOI from L4, but supplementation with onion did not improve any parameters of bone quality. Cortical bone at the tibia midshaft was not affected by treatment. Consuming some fruits and vegetables inhibits bone resorption, which may be because of their antioxidant content. However, under our experimental conditions, onion did not inhibit bone loss due to ovariectomy.
Development of functional tofu fortified with garlic and calcium carbonate
Authors: Chang Kyung Ja , Cheong Sun Hee
Published: June 2021

The objective of this study was to investigate the effects of calcium lactate as a coagulant and garlic on the quality including color, textural properties and shelf‐life of tofu. Statistical analysis of data was carried out using the SPSS 12.0 program. The yield of garlic tofu was of the order: 0.75% addition of garlic juice〉 1.00% addition of garlic juice〉 0.00% addition of garlic juice〉 0.50% addition of garlic juice〉 0.25% addition of garlic juice. The lightness (L value) was significantly decreased with increasing the concentration of garlic juice addition with increasing the concentration of garlic juice addition (p<0.05). The hardness and cohesiveness were significantly decreased with increasing the concentration of garlic juice addition (p<0.05). However, the hardness of tofu with calcium lactate was significantly lower compared to the other samples using different coagulants (p<0.05). All tofu had initial bacterial concentrations of 9.6×103 CFU/g at 0 day of storage. Tofu prepared with 0.75% and 1.0% addition of garlic juice did not show any changes of viable counts by the second day and the fourth day of storage, respectively. However, viable microbial counts of tofu prepared with 0.0%, 0.25%, 0.50% addition of garlic juice increased more rapidly than those of tofu prepared with 0.75% and 1.0% addition of garlic juice during longer storage periods. These results can further help in process optimization that becomes essential in retaining maximum nutritive value in the tofu especially when the choices of coagulants are plenty.
Structure‐Activity Relationships of Components from the Roots of Pueraria thunbergiana Having Aldose Reductase Inhibitory and Antioxidative Activity
Authors: Jeon Young Eun , Kang Il‐Jun , Park Chang‐Hun , Lim Soon Sung , Lee Dong‐Ung
Published: June 2021

Aldose reductase (AR), the key enzyme of the polyol pathway, has been demonstrated to play an important role in the etiology of diabetic complications; therefore, AR inhibitors would be expected to be effective in preventing cataract formation in diabetes mellitus. The 80% methanol extract and fractions of the roots of Pueraria thunbergiana were tested for their inhibitory activities on rat lens AR in order to find the most active fraction. For activity‐guided separation, we isolated four isoflavonoids, daidzin, daidzein, puerarin, and ononin, and identified their structures by comparison of their spectral data with previously reported data. The effects of the constituents of Pueraria root, including the above compounds 3–6, compound 7 (formononetin) prepared from compound 3 and the commercial isoflavonoids, genistin and genistein, and flavonoids, baicalin and baicalein, were estimated with rat lens AR using DL‐glyceraldehyde as a substrate. And also, antioxidative activities of the fractions and the constituents were investigated. Considering the close association between AR inhibitor and antioxidant, isoflavonoid 4 and flavonoid 9 would be acceptable compounds for which further trials including in vivo testing should be conducted. Supported by Brain Korea 21.
Effect of Pycnogenol on Glucose Transporter4 expression and glucose uptake stimulatory activity in 3T3‐L1 adipocytes
Authors: Lee Hee‐Hyun , Kim Kui‐Jin , Yoon Kye‐Yoon , Lee Boo‐Yong
Published: June 2021

Pycnogenol, a standardized extract from the bark of the French maritime pine, consists of phenolic compounds including catechin, taxifolin, procyanidins, and phenolic acids. Procyanidins of pycnogenol are flavonoids with an oligomeric structure, and it has been shown that they can improve the pathological oxidative state of a diabetic situation. To evaluate whether the effects of pycnogenol were due to their action on insulin‐sensitive tissues, we used the insulin‐sensitive cell line 3T3‐L1 adipocytes. And then we used the glucose uptake assay and western blotting to see Gult4 movement. Pycnogenol stimulated glucose uptake in 3T3‐L1 adipocytes by a dose‐dependent manner. Pycnogenol significantly stimulated glucose uptake in 3T3‐L1 adipocytes at the concentration of 250ug/mL. To determine whether PI3K and p38 MAPK were involved in the signaling pathways by Pycongenol, we performed assays using specific inhibitors of these kinases. As a result, the effect of Pycnogenol on glucose uptake was sensitive to wortmannin, an inhibitor of phosphoinositol 3‐kinase and to SB203580, an inhibitor of p38MAPK. Conclusively Pycnogenol have insulin‐like effects in insulin sensitive cells that could help to explain their anti‐hyperglycemic effect in vivo. These results suggest that Pycnogenol may be useful for the prevention and treatment of diabetes.
Thrombosis and platelet aggregation is enhanced in mice deficient in alpha(1,3)‐fucosyltransferases‐IV and ‐VII
Authors: Homeister Jonathon W. , Bodary Peter F. , Machlus Kellie R.
Published: June 2021

P‐selectin is a link between inflammation and venous or microvascular thrombosis. Selectin‐mediated leukocyte/endothelial interactions require ligands that contain sialyl Lewis x, which is absent in mice deficient in alpha(1,3)‐fucosyltransferases (FucT)‐IV and ‐VII. We tested whether mice deficient in the FucTs had the predicted anti‐thrombotic phenotype in a photochemical‐induced injury model of carotid artery thrombosis. Surprisingly, the time to thrombosis was decreased in FucT‐IV(−/−)/FucT‐VII(−/−) mice [16.5±5.1 min, n=4, vs WT=36.2±2.6 min, n=8; p≤0.05]. The prothrombotic phenotype was not accounted for by alterations of the prothrombin time [FucT‐IV(−/−)/FucT‐VII(−/−)=12.2±0.4 sec, n=8 vs WT=12.4±0.3 sec, n=8; p≥0.05], activated partial thromboplastin time [FucT‐IV(−/−)/FucT‐VII(−/−)=28.2±2.6 sec, n=8 vs WT=29.5±1.9 sec, n=8; p≥0.05], or platelet count [FucT‐IV(−/−)/FucT‐VII(−/−)=775±121 × 103/ul, n=9 vs WT=721±107 × 103/ul, n=9; p≥0.05]. In contrast, collagen‐stimulated whole blood platelet aggregation in FucT‐deficient mice had a shorter lag time [FucT‐IV(−/−)/FucT‐VII(−/−)=68±5.8 sec, n=5 vs WT=90±5.7 sec, n=5; p≤0.05], and a faster rate [FucT‐IV(−/−)/FucT‐VII(−/−)=16.2±1.5 sec−1, n=5 vs WT=13.4±1.7 sec−1, n=5; p≤0.05]. FucT‐deficient mice have a prothrombotic phenotype that is associated with enhanced platelet aggregation. Support by AHA FTF Award 0275023N.
In vivo receptor occupancy of N‐desmethylclozapine (ACP‐104) and Clozapine
Authors: Littler Pey‐Lih H , Schmelzer Kara R , McFarland Krista , Bonhaus Douglas W , Lameh Jelveh
Published: June 2021

Acadia is currently developing ACP‐104 (N‐desmethylclozapine, NDMC) as an antipsychotic with potential procognitive benefits. A number of clinical and preclinical lines of evidence suggest that NDMC, a predominant metabolite of clozapine, may contribute to clozapine's superior efficacy. Clozapine is an antagonist at serotonin 5‐HT2A, muscarinic M1, and dopamine D2 receptors, while we have shown that ACP‐104 is an M1 agonist and D2 partial agonist. In vivo studies have shown that ACP‐104 has both antipsychotic and procognitive activity. We utilized ex vivo binding assays to study the in vivo receptor occupancy of clozapine and ACP‐104. In vivo binding to 5‐HT2A and M1 receptors was assessed in rat pre‐frontal cortex by [3H] MDL100, 907 and [3H] pirenzepine, respectively. Our data show that clozapine and ACP‐104 bind to cortical 5‐HT2A receptors. Moreover, both ACP‐104 and clozapine bind to M1 receptors in the prefrontal cortex. These data demonstrate that ACP‐104 interacts with 5‐HT2A and M1 receptors in vivo at doses that are effective in antipsychotic and procognitive animal models. Our findings support the potential utility of ACP‐104 as a procognitive antipsychotic agent in the treatment of schizophrenia.
Adenosine A2A receptor knock‐out mice have impaired vasorelaxation and endothelial function
Authors: Ponnoth Dovenia , Ledent Catherine , Mustafa S Jamal
Published: June 2021

The A2A adenosine receptor (AR) has been implicated in vasorelaxation in many species. The present study was undertaken to investigate the role of the A2A AR on vasoreactivity in A2A AR knock‐out (KO) and wild‐type (WT) mice. AR agonists and antagonists were used in organ bath experiments to produce concentration‐response curves in isolated aorta. NECA (non‐selective adenosine analog; EC50 = 6.78μM) and CGS 21680 (A2A AR selective agonist; EC50 = 0.013μM) produced a concentration‐dependent relaxation (25% and 28% relaxation with NECA and CGS, respectively) in WT aorta. In KO aorta, NECA (EC50 = 0.075μM) induced a concentration‐dependent contraction (47%; p<0.05 compared to WT) while CGS produced no response. SCH 58261 (10−6M; A2A AR selective antagonist) abolished vasorelaxation in WT (p<0.05 compared to controls). When DPCPX (10−5M; A1 AR selective antagonist) was used, higher vasorelaxation was observed in WT (50% vs. 25% in controls; p<0.05) while KO showed less contraction (5% vs. 47% in controls; p<0.05). Aortic endothelial function, determined by the response to acetylcholine, was significantly higher in WT compared to KO (66% in WT vs. 51% in KO; p<0.05). In conclusion, A2A AR KO mice had significantly lower aortic relaxation and endothelial function, suggesting that the A2A AR plays an important role in vasorelaxation, probably, in part, through an endothelium‐dependent mechanism. Supported by HL‐027339
1,4‐Benzoquinone forms an unstable thioether bond with cysteine residues that is eliminated in basic conditions
Authors: Labenski Matthew T. , Fisher Ashley A , Chapman John D. , Monks Terrence J , Lau Serrine S
Published: June 2021

Hydroquinone (HQ) is an environmental toxicant found in skin creams, cigarette smoke, and it is also a byproduct of combustion. After exposure, HQ is oxidized to benzoquinone (BQ) and conjugated with GSH; HQGSHs target the outer stripe of the outer medulla of the kidney causing necrosis. The adverse effects of HQGSHs are caused by its redox cycling capabilities and the ability to adduct proteins. In vitro and in vivo studies have revealed that Lys, Glu and Arg residues are targets of BQ adduction. Cys residues, which are the more commonly reported sites of electrophilic chemical adduction, have been identified as targets of BQ, but only with in vitro experiments. Following reaction of a Cys containing peptide and apocytochrome c (2 Cys) with BQ and mass spec analysis we identified Cys‐BQ adducts. Then by mimicking a standard protein digestion protocol we observed a time‐dependent elimination of the thioether bond. Other electrophiles with affinity for Cys, such as acrolein and iodoacetamide, retained the Cys thioether bond on the peptide during exposure to identical conditions. In conclusion, we have identified BQ‐Cys adducts in peptide and protein models, where the thioether bond is eliminated at basic pH over time. As a result, quinone post‐adduction chemistry ex vivo plays a critical role in determining our ability to identify amino acid targets of these compounds. (GM007890, ES006694, ES007091).
NF‐κB ORCHESTRATES GENE EXPRESSION ASSOCIATED WITH MYOCARDIAL INFARCTION AND PROTECTION
Authors: Wilhide Michael E. , Tranter Michael , Ren Xiaoping , Jones W. Keith
Published: June 2021

NF‐κB has been shown to contribute to myocardial infarction (MI) after ischemia/reperfusion (I/R) and to cardioprotection after permanent coronary occlusion (PO). The aim is to discover the mechanism by which NF‐κB regulates these opposing effects after MI. The hypothesis is that, after different ischemic stimuli, NF‐κB mediates different sets of genes that are responsible for opposite effects. We employed transgenic mice that block NF‐κB in a cardiac‐specific manner. These mice were subjected either to I/R or PO and microarrays were used to delineate the genes regulated acutely by NF‐κB. Interestingly, after I/R NF‐κB activation leads to at least a two‐fold increase in the expression levels of several heat shock proteins. After PO, NF‐κB leads to a three‐fold higher level of Hsp70 compared to I/R. Cox2 was also found to be differentially expressed. Different heat shock proteins are known to be either anti‐apoptotic or pro‐apoptotic, and HSPs may affect processing and activity of Cox2 via endoplasmic reticulum (ER) stress response/processing pathways. Thus, HSP and Cox2 regulation by NF‐κB may underlie, at least in part, the differential effects of NF‐κB. Understanding the role of NF‐κB in the regulation of these sets of genes will aid in the discovery of novel therapeutic targets for the treatment of heart disease.

This work was supported by NIH grant R01 HL63034 (WKJ) and 5F31 HL081923 (MW).
Effect of Sildenafil on mdx Skeletal Muscle
Authors: Parchen Candace M , Gray Heidi , Percival Justin , Froehner Stanley C , Beavo Joseph A
Published: June 2021

Duchenne muscular dystrophy (DMD) affects 1 in 3,500 boys and is a severe X‐linked disease caused by a complete loss of dystrophin. DMD patients are born healthy but by age five, develop progressive skeletal muscle necrosis, fibrosis and abnormal hypertrophy. Sildenafil Citrate (Viagra®) is a phosphodiesterase 5 (PDE5) inhibitor that induces vasodilation and is currently approved for use in treatment of erectile dysfunction and pulmonary hypertension.

Previous studies have shown Sildenafil activates PKG‐1, resulting in anti‐hypertrophic and anti‐fibrotic effects on pressure‐induced cardiac hypertrophy (Takimoto et al (2005) Nature 11(2):214–222). Recently, another PDE5 inhibitor, tadalafil, has been reported to be effective in ameliorating some of the dystrophic symptoms in the mdx mouse, a dystrophin knockout model of DMD (Asai et al (2007) PLoS ONE 2(8): e806). We have been exploring the use of non‐invasive techniques such as echocardiography and MRI, to track the progression of Sildenafil treatment throughout the life of the mdx mouse. After one year of chronic Sildenafil treatment, we see a decrease in the MRI T2 relaxation time of the hind‐leg muscle, indicating a decrease in dystrophic tissue. Earlier times also show a trend toward beneficial effect. With eight weeks of treatment, there is a decrease in Tibialis Anterior muscle atrophy and centrinucleation, thus less overall damage and a decrease in the need for muscle regeneration. This data provides preliminary evidence that PDE5 regulation can reduce muscle damage in skeletal dystrophic muscle indicating Sildenafil could be a possible treatment for DMD patients. Supported by Charley's Fund and NIH grant DK21623.
Diammonium Glycyrrhizinate protects against trinitrobenzene sulfonic acid‐induced colitis in rats
Authors: Ding Hong , Liu Ying , Mao Yao , Qi Zheng , Wang Ying , Xiang Jin , Zhou Huiping
Published: June 2021

Glycyrrhizinate is the major active component of licorice root extract, an ancient medicinal plant with various pharmacological activates. Diammonium Glycyrrhizinate (DG) has long been used to treat hepatitis in China. Recently, it has been reported that DG could reduce acetic acid‐induced inflammatory injury in a rat ulcerative colitis (UC). However, whether DG has anti‐inflammatory effect in other UC models remains unknown. The aim of the present study is to examine the protective effects of DG on trinitrobenzene sulfonic acid (TNBS)‐induced UC in rats.

Methods: Sprague‐Dawley rats were randomly assigned into five groups: 1) normal control; 2) TNBS; 3) TNBS+DG (2 mg/kg); 4) TNBS+DG (10 mg/kg); 5) TNBS+DG (50 mg/kg); 5) TNBS+ sulfasalazine (SASP, 225mg/kg). After induction of UC with TNBS, DG or SASP was rectally administrated daily for 8 days, the normal control and TNBS groups were administrated with the same volume of saline. Colonic inflammation was evaluated by disease activity index, histological changes, and myeloperoxidase (MPO) activity. Radioimmunoassay was used to measure TNF‐a and IL‐1b levels in the serum.

Results: DG not only significantly inhibited TNBS‐induced increase of MPO, TNF‐α and IL‐1β, but also prevented TNBS‐induced colonic injury.

Conclusions: TNBS‐induced colitis was reliable rat UC model. Rectal administration of DG could effectively prevent TNBS‐induced inflammatory injury.
Antineuroinflammatory polyketide plakortide N from the Brazilian marine sponge Plakortis angulospiculatus
Authors: Mayer Alejandro M , Kossuga Miriam H , Veloso Katyuscya , Ferreira Antonio G , Hajdu Eduardo , Berlinck Roberto G
Published: June 2021

Involvement of brain microglia (BMΦ) in neuroinflammation has been hypothesized (Mayer AMS, MEDICINA 58:377, 1998; Mayer AMS et al., SHOCK 11: 180, 1999). We have reported marine chemicals as a potential source of antineuroinflammatory compounds (Mayer et al. BioMedCentral Pharmacology 5:6, 2005) using Escherichia coli lipopolysaccharide (LPS)‐activated BMΦ as our in vitro model (Mayer AMS et al., SHOCK 11:180, 1999). The purpose of this investigation was to study the effect of several polyketides isolated from the marine sponge Plakortis angulospiculatus on phorbol ester (PMA)‐stimulated thromboxane B2 (TXB2) and superoxide anion (O2−) generation from E. coli LPS‐activated rat BMΦ O2− was determined by superoxide dismutase‐inhibitable reduction of ferricytochrome C and TXB2 using commercially available immunoassays. Only plakortide N potently inhibited TXB2 (IC50=0.93μM) but not O2− (IC50 >10μM), with low lactic dehydrogenase release (LDH50>10μM). Because LDH release was low, our current data suggests that the significant inhibitory effect of plakortide N on BMΦ TXB2 release was pharmacologic rather than toxic in nature, although the molecular mechanism of inhibition remains undetermined at this time. (Supported by American Society of Pharmacognosy 1998 Research Starter Grant, NIH grant CA 67786 and FAPESP grants 01/03095‐0 and 05/60175‐2 to RGB and Midwestern University to AMM).

TOTAL
Asymmetric dimethylarginine (ADMA) elicits superoxide production in isolated arterioles via NAD(P)H oxidase
Authors: Veresh Zoltan , Racz Anita , Hamar Janos , Koller Akos
Published: June 2021

ADMA is thought to be an endogenous inhibitor of nitric oxide (NO) synthase, thus may decrease NO synthesis. However, our previous investigation showed that in isolated microvessels ADMA induced superoxide production, but the mechanisms remain unknown. We hypothesized that elevated level of exogenous ADMA, by activating vascular oxidases, elicits increased superoxide production, interfering with NO mediation of flow‐induced dilation. In the presence of indomethacin, isolated arterioles from rat gracilis muscle (∼ 150 m at 80 Hgmm) were incubated with ADMA (10−4 mol/L), which eliminated the dilations to increases in intraluminal flow (from max.: 28±1% to 7±2 %, p<0.05). However, in the presence of ADMA, the NAD(P)H oxidase inhibitor apocynin restored flow‐induced dilations, whereas the xanthine oxidase inhibitor oxypurinol was without effect. Superoxide dismutase plus catalase also restored dilations to flow, which were then abolished by inhibition of NOS. In addition, ADMA elicited significant constriction (from 162±4 μm to 143±4 μm at 80 Hgmm), which was prevented by prior incubation of arterioles with apocynin. Thus, we suggest that ADMA activates NAD(P)H oxidase, and the produced superoxide reduces the bioavailability of NO resulting in diminished flow‐induced dilation, a mechanism, which may contribute to the development of cardiovascular diseases associated with elevated levels of ADMA. (Grants: Hungarian Sci. Res. Funds/OTKA T48376, T67984; Health Sci. Council/ETT 364/2006 and Am. Heart Assoc. NE Aff. 0555897T, USA).
Regional characteristics in spontaneous rhythmic variations of cutaneous blood flow in human volunteers
Authors: Bari Ferenc , Bere Zsofia , Toth‐Szuki Valeria , Csorba Zsofia , Busija David W , Domoki Ferenc
Published: June 2021

Spontaneous, rhythmic variation of cutaneous blood flow (CuBF), – vasomotion, with dominant frequencies ranging from 1.5 to 20 cycles/min (cpm, 0.025–0.33 Hz) – was identified previously in the human forehead and forearm skin using laser‐Doppler flowmetry (LDF) (1). Our purpose was to extend these studies by investigating vasomotion in 15 different skin regions, and testing the reproducibility of the determination of vasomotion by LDF as compared with the determination of postocclusive hyperemia of the forearm skin. LDF data were recorded 3 times (0, 1, 12 months) in healthy young volunteers (20–22 years old, n=14) as described (1). Vasomotion was found in 11 skin regions. The dominant frequency of vasomotion significantly decreased from the midline to the more lateral skin areas, (mean±SEM, *p<0.05) from 7.9±0.3 (forehead) to 5.8±0.5* cpm (lateral thigh). Characteristics of vasomotion did not change over time (e.g.: on the templates 8.4±0.3, 8.3±0.4, and after 12 months 8.4±0.4, 8.3±0.4 cpm, respectively). In contrast, peak and area under the curve CuBF values of reactive hyperemia were highly variable during the repeated challenges. Our data indicate that the oscillations in the microcirculation exhibit a regional‐dependent pattern which seems largely time‐invariant whereas the parameters of reactive hyperaemia shows poor solidity. Ref: (1) Microvasc Res 70:121–8, 2005. Supported by OTKA K63401.
Potentiation of BK Channels by α5β1 Integrin Activation in Arteriolar Smooth Muscle
Authors: Davis Michael John , Yang Yan , Wu Xin , Gui Peichun , Sohma Yoshiro , Meininger Gerald A , Davis George E , Braun Andrew P
Published: June 2021

RGD peptides produce sustained vasodilation of rat skeletal muscle arterioles. We hypothesized that RGD ligands work through α5β1 integrin to modulate the activity of large conductance, Ca2+‐activated K+ (BK) channels in arteriolar smooth muscle. K+ currents were recorded in single arteriolar myocytes using whole‐cell and single‐channel patch clamp methods. Activation of α5β1 integrin by an insoluble antibody resulted in ∼30–50% increase in the amplitude of IBTX‐sensitive, whole‐cell K+ current. The endogenous α5β1 integrin ligand, fibronectin (FN; 10 μg/ml, 120 kD fragment), potentiated IBTX‐sensitive K+ current by ∼80%, an effect that was blocked by the c‐Src inhibitor, PP2 (0.1–1 μM). In cell‐attached or excised, inside‐out patches, the N•Po of a ∼240 pS K+ channel was significantly increased after FN application to the external surface of the patch through the recording pipette. FN also caused a leftward shift in the N•Po‐voltage relationship at constant [Ca2+], which was blocked by PP2. The results suggest that α5β1 integrin activation potentiates BK channel activity in vascular smooth muscle via a membrane‐delimited pathway through both Ca2+‐ and c‐Src‐dependent mechanisms.
Early‐phase cerebral arteriogenesis: Evidence of endothelial activation, vascular proliferation and local inflammation
Authors: Bondke Anja , Hillmeister Philipp , Duelsner André , Gruber Clemens , Buschmann Ivo R. , Lehmann Kerstin
Published: June 2021

Arteriogenesis holds potential as a novel treatment for ischemic vascular disease. Our study aimed at an identification of key processes of early‐phase cerebral arteriogenesis in vivo.

In a rat model of nonischemic cerebral hypoperfusion the posterior cerebral artery (PCA) constitutes a main collateral to the contralateral hemisphere. Following reversed flow, endothelial cell (EC) activation was assessed by electron microscopy. Gene expression during biomechanical EC activation was analyzed by cDNA microarray. Proarteriogenic vascular proliferation and local inflammation were evaluated.

PCA diameters increase significantly(+30%). Ultrastructural EC activation occurs at 3d postoperatively, followed by CD68+ cellular transmigration and sustained vessel wall proliferation. Of 164 genes deregulated, 48 contain shear stress response elements (SSREs) or NFkB binding sites in their promoter regions. Arteriogenesis leads to NFkB activation as shown by local NFkB p65 expression.

Cerebral arteriogenesis is initiated by EC activation through changed blood flow, facilitating monocyte influx and collateral proliferation. Gene expression is modified by biomechanical activation via SSREs and the pro‐inflammatory environment (NFkB activation, monocyte influx). Therapeutic arteriogenesis may aim at 1) biomechanical EC activation 2)vascular proliferation or 3)local monocyte function.
A role for angiotensin and oxidative stress in diabetes‐induced coronary artery remodeling
Authors: Katz Paige S , Souza Flavia M , Reed Ryan E , Lord Kevin C , Lucchesi Pamela A
Published: June 2021

Coronary artery (CA) remodeling may lead to increased arterial stiffness and decreased coronary flow reserve. The molecular mechanisms that dictate diabetes‐induced CA remodeling are undefined. This study established a link between oxidative stress, angiotensin II (Ang II), CA remodeling and stiffness in Type 2 diabetic mice. CA (<100 μ) were isolated from 16 wk heterozygous (Db/db) and diabetic (db/db) mice ± treatment with the NADPH‐oxidase inhibitor apocynin (5mM) or the AT1R blocker candesartan (1mg/kg/day). Mean arterial pressure, measured by telemetry, was similar between groups. CA were mounted on a pressure myograph for measurement of structural and passive mechanical properties. Significant inward remodeling was detected in db/db vs. Db/db, defined by increased wall thickness, media:lumen ratio, remodeling index and decreased compliance. This inward remodeling in db/db CA was associated with increased oxidative stress and collagen accumulation as measured by DHE and picrosirius red staining respectively. Immunoblot analysis indicted that basal JNK, ERK, and AKT phosphorylation were increased in db/db CA tissue lysates vs. Db/db. In vivo treatment with apocynin or candesartan reduced inward CA remodeling. These data suggest that increased oxidative stress and Ang II is critical for altered structural and functional remodeling of CA in Type 2 diabetes. Supported by HL056046 and P20RR18766 (PAL).
Inhibition of inducible nitric oxide synthase attenuates platelet activation and fibrinogen/fibrin deposition in pulmonary microvessels caused by lung ischemia‐reperfusion in rabbits
Authors: Roberts Andrew M. , Perez‐Abadia Gustavo , Conhaim Robert L. , Lominadze David
Published: June 2021

We previously showed that lung ischemia‐reperfusion (IR) causes platelet adhesion and pulmonary microvascular constriction. Experiments were done to examine the role of platelet activation and fibrinogen/fibrin (Fg/Fb) deposition in vascular dysfunction during pulmonary IR. In anesthetized rabbits with open chest, we ventilated the lungs and examined subpleural microvessels in the right lung by video microscopy during brief periods when the lungs were statically inflated (10 cm H2O). IR was caused by placing a removable clip on the right pulmonary artery for 2 h. During reperfusion, we injected albumin labeled with fluorescein (green) and a fluorescently labeled (red) polyclonal antibody to Fg/Fb, via the right atrium. Enhanced platelet adhesion and Fg/Fb deposition were found during IR and were associated with sites of albumin leakage. These effects were attenuated by infusion of an iNOS activity inhibitor (1400W) before IR. Confocal microscopy of tissue samples confirmed associations between platelets, Fg/Fb, and albumin leakage. Our data suggest that platelet activation and Fb/Fg deposition are associated with elevated iNOS activity and may be a cause of microvascular dysfunction during lung IR.

Supported in part by The American Lung Association‐Kentucky to AMR and by NIH grant# HL080394 to DL.
Influence of acute graded hypoxia on the bronchial circulation in healthy humans
Authors: Ceridon Maile L , Snyder Eric M , Olson Thomas P , Hulsebus Minelle L , Johnson Bruce D
Published: June 2021

Reduced partial pressures of inspired O2 may influence the bronchial circulation through a number of mechanisms (e.g. altered pulmonary and systemic vascular pressures, lung fluid regulation, airway cooling & drying from increased ventilation). The present study examined the impact of acute, graded hypoxia on bronchial blood flow (Qaw) in eight healthy adults (28 ± 9 years). Subjects were exposed to inspired O2 levels resulting in arterial O2 saturation (SaO2) values of 90% and 80% for 15 min each. To control for the mild hyperventilation that accompanies hypoxia, subjects then mimicked the 80% SaO2 ventilation (VE) rate with 21% FIO2 and again with CO2 added to the inspirate to maintain normocapnia. Qaw and cardiac output (CO) were measured using soluble gas techniques. Also measured were alveolar‐capillary conductance (Dm, index of lung fluid balance), pulmonary capillary blood volume (Vc), and systemic mean (MAP) and pulmonary arterial pressures (PAP). Hypoxia resulted in minimal change in MAP, a small rise in PAP, minimal change in CO and Vc, and a rise in Dm. There was also a non significant decrease in Qaw with hypoxia. Mild hyperventilation resulted in a significant fall in Qaw, but when normocapnia was maintained, Qaw returned to baseline values. These data suggest that acute, graded hypoxic exposure does not significantly impact Qaw, however, the hypocapnia associated with mild hyperventilation reduces Qaw.
Glutathione Improves Impaired Responses of Cerebral Arterioles in Type 1 Diabetes
Authors: Arrick Denise M , Mayhan William G , Sun Hong
Published: June 2021

Our goal was to examine whether glutathione improves impaired nitric oxide synthase‐dependent responses of cerebral arterioles during Type 1 diabetes. We measured diameter of cerebral arterioles in nondiabetic and diabetic (streptozotocin; 50 mg/kg) rats to nitric oxide synthase‐dependent, acetylcholine and adenosine 5’‐diphosphate (ADP) and a nitric oxide synthase‐independent agonists, nitroglycerin. We measured responses before and during topical application of glutathione (5 mM). In addition, we measured superoxide production by brain tissue in nondiabetic and diabetic rats. In nondiabetic rats, acetylcholine and ADP produced dilatation of cerebral arterioles. However, there was significant impairment in reactivity of arterioles to acetylcholine and ADP in diabetic rats. Vasodilatation to nitroglycerin was similar in nondiabetic and diabetic rats. Glutathione did not alter baseline diameter of cerebral arterioles, but significantly improved impaired nitric oxide synthase‐dependent vasodilatation in diabetic rats. Superoxide production was increased under basal states in diabetic rats and glutathione restored superoxide levels to that observed in nondiabetic rats. Our findings suggest that glutathione selectively improves endothelial dysfunction of cerebral arterioles during Type 1 diabetes.
Cerebral microvascular dysfunction and inflammation following venous sinus thrombosis in mice.
Authors: Nagai Mutsumi , Terao Satoshi , Yilmaz Gokhan , Granger D. Neil
Published: June 2021

Blood brain barrier (BBB) dysfunction and brain edema are major consequences of cerebral venous sinus thrombosis (CVST) that underlie the poor clinical outcome in patients with this condition. However, relatively little is known about the mechanisms that link CVST to this vasogenic edema. We have developed a murine model of CVST wherein BBB permeability, brain water content, and leukocyte‐endothelial cell adhesion (LECA) can be monitored at different times (3–48 hrs) following thrombus formation. A cranial window was created over the superior sagittal sinus (SSS) of C57Bl/6 mice. CVST was induced by placing a ferric chloride (20%) soaked 6‐0 silk suture onto the SSS for 5 minutes. The adhesion of rhodamine‐6G labeled leukocytes, BBB permeability to Evans blue, and water content were measured. Brain edema was observed at 48 hr, but not 3 hr, after CVST. Both BBB permeability and LECA were increased at both 3 and 48 hrs following CVST. At least one refluxing cortical vein was noted in 3 of 5 mice in the 48‐hr group. More LECA was noted in these venules with retrograde (reflux) perfusion. Our findings indicate that CVST can be reproducibly induced in mouse brain, with an increased BBB permeability and brain edema that characterizes the human condition. The CVST‐induced increases in BBB permeability and edema are accompanied by LECA, suggesting a possible role for inflammation in these responses. (P01 DK43785)
Increased Cholesterol is Vital to Impairment of TRPV1 Channel‐Mediated Relaxation of Coronary Artery In Ossabaw Miniature Swine with Metabolic Syndrome
Authors: Bratz Ian N. , Neeb Z. N. , Alloosh M. , Sturek M.
Published: June 2021

TRPV1 channels are implicated in vascular regulation. Ossabaw swine fed excess high fat/cholesterol diet develop metabolic syndrome (MetS), however studies find high‐fructose diets elicit MetS without dyslipidemia. We tested the hypothesis that dyslipidemia is an important component for eliciting dysfunction of endothelial cell (EC) TRPV1 channels signaling. Coronary arteries were studied from male Ossabaw pigs fed standard chow (lean, n = 8) or excess kcal diets for 28 wk: fructose to induce MetS (n = 6) or high fat/cholesterol/fructose to induce MetS with added dyslipidemia (DMetS, n = 8), defined by increased LDL or total/HDL cholesterol. The TRPV1 channel agonist capsaicin, dose‐dependently relaxed coronary rings from lean pigs (EC50 = 0.12 ± 0.04 μM), which was attenuated in MetS pigs (EC50 = 10 ± 21 μM) and severely attenuated in DMetS pigs (EC50: ~ 1mM). Fura‐2 imaging revealed capsaicin generated calcium influx in healthy EC, which was attenuated in MetS and to a greater extent in DMetS. Coronary TRPV1 protein levels were decreased only in DMetS (lean~MetS>DMetS). Conclusions: Capsaicin‐induced relaxation of coronary conduit artery occurs via EC and TRPV1 channel signaling, which is disrupted in MetS and to a much greater extent in DMetS, suggesting lipids are important regulators of EC TRPV1‐mediated functions. Support: NIH RR013223, HL062552, T32 HL079995.
Effects of ischemic heart disease and exercise training on mechanisms of nitric oxide production
Authors: XIE WEI , Widmer A. Jimmy , Widmer R. Jay , Parker Janet L. , Heaps Cristine L.
Published: June 2021

Control and collateral‐dependent coronary arteries of exercise trained swine display enhanced nitric oxide‐dependent bradykinin‐mediated relaxation. For this study, we hypothesized that exercise training elicits cellular adaptations to enhance nitric oxide production in coronary arteries of ischemic hearts. Ameroid constrictors were placed around the proximal left circumflex coronary artery (LCX) of adult female Yucatan miniswine. Collateral‐dependent (LCX) and control (nonoccluded left anterior descending) arteries (∼1–2 mm diam) were isolated from sedentary and exercise trained (treadmill; 14 wks) pigs. Endothelial cells were dissociated and intracellular Ca2+ (fura‐2) responses to bradykinin were studied. Caveolin‐1 and eNOS protein levels were evaluated by immunoblot. Exercise training significantly enhanced bradykinin‐stimulated Ca2+ levels in cells of both control and collateral‐dependent arteries. Caveolin‐1 protein was significantly elevated in collateral‐dependent arteries of sedentary pigs; an increase that was reversed with exercise training. eNOS protein was not altered by occlusion or exercise training. Our studies suggest that increased bradykinin‐stimulated endothelial Ca2+ levels and reduced caveolin‐1 protein contribute to improved bradykinin‐mediated relaxation in both control and collateral‐dependent arteries of exercise trained swine. NIH HL64931
Effect of high salt diet and antioxidants on vascular function of mesenteric arteries
Authors: Raffai Gábor , Lombard Julian H
Published: June 2021

This study compared vascular function in mesenteric arteries of male Sprague‐Dawley rats fed high salt (HS, 4% NaCl) or low salt (LS, 0.4% NaCl) diet. External diameter of perfused and superfused vessels was recorded at 75 mmHg. Norepinephrine (NE) sensitivity and acetylcholine dilation (of NE precontracted vessels) were determined. The effect of acute antioxidant application (bath) and chronic (drinking water) tempol treatment on vascular relaxation was tested. EC50 of LS (4.1±0.95μM) and HS (3.2×0.85μM) vessels to NE was not different. Vasorelaxation was suppressed in HS (6.2±2.8%) compared to LS (16.9±2.6%). Extraluminal (EL) tempol did not improve endothelial function in HS (5.7±2.5%) and reduced vasorelaxation in LS (2.7±4%). In contrast, intraluminal (IL) tempol [similarly to other EL antioxidants (PEG‐SOD, PEG‐SOD+tempol, PEG‐catalase+tempol)] ameliorated impaired relaxation in HS (10.5±2.3%) without affecting LS responses (13.5±2.8%). Chronic tempol treatment did not influence vasorelaxation responses in LS (10.9±4.8%), but prevented the deleterious effect of HS on endothelial function (13.5±3%). These results indicate that HS diet impairs vascular relaxation by increasing endothelial oxidative stress. EL tempol (unlike IL tempol and other EL antioxidants) cannot reduce such an oxidative stress, but might restrict responsiveness to vasodilator stimuli. (NIH #HL‐65289 and #HL‐72920).
Artery‐specific gene expression changes following chronic L‐NAME administration in Yucatan miniswine
Authors: Prior Barry , McAllister Richard M. , Laughlin M. Harold
Published: June 2021

Nitric oxide (NO) is an important vasodilator and signaling molecule in the arterial tree. The bioavailability of NO is dependent in part upon its production and interaction with other reactive oxygen species (ROS). We sought to examine select gene expression changes following systemic administration (30 d) of the nitric oxide synthase (NOS) inhibitor L‐NAME (8.0 mg/kg/d) in Yucatan miniswine (~30 kg). After sacrifice ~1 cm segments of the right coronary and femoral arteries were dissected, cleaned and stripped of endothelial cells. Total RNA from smooth muscle and endothelial cells was isolated and 1 ug was used for cDNA synthesis using random primers. Real time PCR (SYBR green) was performed using primers specific for endothelial NOS (eNOS), gp22phox and catalase with 18S rRNA used for normalization. Results suggested that within endothelial cells gene expression was higher in the femoral than in the coronary artery. In coronary endothelial cells, L‐NAME administration tended to increase catalase gene expression while eNOS and gp22phox appeared relatively unchanged. In contrast, L‐NAME tended to decrease all gene expression in femoral artery endothelial cells. The data suggest that regional differences along the arterial tree exist in relation to NO and ROS production between control and NOS‐inhibited states. These apparent differences may be in response to differences in shear stress along the arterial tree.
Transgene expression in neurons of rat nucleus tractus solitarii and pterygopalatine ganglion using adeno‐associated viral vectors
Authors: Lin Li‐Hsien , Taktakishvili Otar , Dragon Deidre Nitschke , Talman William T.
Published: June 2021

Adeno‐associated viral vector (AAV) has distinct advantages over other viral vectors in delivering genes of interest. AAV transfects mainly neurons, produces no toxicity or inflammatory responses and yields long‐term transgene expression. Previously we showed that adenovirus (Ad) mainly transfects glial cells and feline immunodeficiency virus (FIV) mainly transfects neurons in the nucleus tractus solitarii (NTS). However, gene transduction in the pterygopalatine ganglion (PPG) using either Ad or FIV has been unsatisfactory. In this study, we tested the hypothesis that AAV serotype 2 (AAV2) selectively transfects NTS and PPG neurons. Using double‐label fluorescent immunostaining combined with confocal microscopy we examined expression of the reporter gene enhanced green fluorescent protein (eGFP) after microinjecting AAV2eGFP into rat NTS or PPG. Robust expression of eGFP was observed in many cells of the NTS and PPG after introduction of AAV2eGFP into each. The majority of eGFP expressing NTS cells contained neuronal marker protein gene product 9.5 (PGP9.5)‐immunoreactivity (IR) but not glial marker glial fibrillary acidic protein (GFAP)‐IR. Similarly, the majority of eGFP expressing neurons in the PPG contained PGP9.5‐IR. We conclude that its neuronal selectivity and affinity make AAV2 a superior viral vector for transduction of NTS and PPG neurons.