bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of the Dietary Supplement Protandim and 4‐ Hydroxytamoxifen on Pre‐malignant Human Breast Cancer Cells in 3D Culture
Authors: Dugan Allison , Carroll-Turpin Michelle , Crooks Samuel , Zhang Songlin , Mathis Michael , Kleiner-Hancock Heather E
Published: June 2021

Protandim is a dietary supplement with potent antioxidant properties. We hypothesized that Protandim would inhibit malignant progression of ductal carcinoma in situ (DCIS). A pre‐malignant human breast cancer cell line (MCF10DCIS.com) was grown in collagen type I for 1 week and then cultured for 2 more weeks in the presence or absence of a growth factor (GF) combination of TGF‐beta and CXCL12, 20ng/mL each. In the presence of GF, most cells exhibited long processes and formed interlacing networks and stellate aggregates. Numerous genes and cytokines involved in cancer progression, inflammation, and differentiation were up‐regulated. Bipotency was verified by IHC for pancytokeratin and alpha smooth muscle actin. Additional groups were dosed with 4‐hydroxytamoxifen (2.5 micromolar), Protandim (8 micrograms/mL), or vehicle (0.1% DMSO). In the Protandim and 4‐hydroxytamoxifen groups, more cells displayed a rounded shape with fewer processes. In the cytokine array, the following decreases by Protandim and 4‐hydroxytamoxifen (respectively) were observed: platelet‐derived growth factor (PGDF): 55% & 27%; interleukin 5 (IL‐5): 81% & 76%; monocyte chemotactic protein1 (MCP‐1): 84% & 76%; angiogenin: 68% & 77%; granulocyte‐macrophage colony‐stimulating factor (GM‐CSF): 63% & 44%; interleukin 6 (IL‐6): 77% & 79%. These results suggest Protandim may suppress DCIS progression. (NIH/NCRR)
The effects of nebivolol on the functional and structural changes in suramin‐induced preeclampsia‐like syndrome in rats
Authors: Soydan Guray , Guler Sebile , Cekic Edip Guvenc , Ayhan Arzu Saglam , Sargon Mustafa , Tuncer Meral
Published: June 2021

Suramin causes defective placentation in pregnant rats which induces placental deficiency. It has been aimed to investigate the effects of nebivolol, which is a β‐blocker drug, on suramin‐induced preeclampsia‐like syndrome in rats. Suramin was injected on 10th and 11th days of pregnancy. Blood pressures of rats were observed daily. Pregnancies were terminated on the 20th day. Fetuses, placentae, kidneys, urine and functional contractile responses of uterine arteries were investigated. Increased blood pressures and proteinuria were detected in suramin‐treated rats. Numbers and weights of fetuses and placentae were reduced. Acute tubuler necrosis was detected. Contractile responses to phenylephrine, angiotensin II, prostaglandin F2α and endothelin‐1 were increased, while KCl‐induced responses were not altered. Acethylcholine‐induced relaxations were inhibited. Nebivolol treatment reduced the increased blood pressures and proteinuria, partially improved acute tubular necrosis in kidneys, increased numbers and weights of fetuses and placentae. Nebivolol restored endothelial dysfunction in uterine artery. In preeclampsia‐like syndrome induced by suramin, nitric oxide (NO) appears to have a significant role in the pathogenesis of endothelial dysfunction in uterine arteries. These findings suggest that nebivolol restores suramin‐induced alterations in this model by its NO producing effect.
The Role of Eosinophils in Aspergillus fumigatus Lung Infection
Authors: Soriani Frederico , Russo Remo Castro , Nogueira Camila Rodrigues Chaves , de Sousa Lirlândia Pires , Rachid Milene , Teixeira Mauro Martins
Published: June 2021

Eosinophils are important in the pathophysiology of allergic diseases and in host immunity. Although microorganisms directly activate inflammatory cells, the mechanisms that trigger eosinophils activation and its functions in immune responses are unknown. Fungi are ubiquitous and may contribute to the development and exacerbation of pulmonary diseases. In this sense, the aim of this study was to unravel the role of eosinophils during Aspergillus fumigatus lung infection. Wild type (BALB/c) and ΔdblGATA mice were infected intranasally with 108 spores of an A. fumigatus wild type strain. Results show that ΔdblGATA mice had increased survival (40% versus 100% in WT mice). PMN influx in the lungs (1dpi) and airways (3 dpi) were greater in ΔdblGATA mice, but there was less protein leakage (3 dpi). Levels of eosinophils were very high in lungs and airways of WT mice after A. fumigatus infection but not detectable in ΔdblGATA mice. Airway levels of CXCL1, TNF‐α, CCL11, CCL2 and IL‐1β were reduced in ΔdblGATA mice. Interestingly, histopathological analysis did not show any differences between animals. Fungal burden was higher in ΔdblGATA mice than WT mice after 1 and 3 dpi. In conclusion, the absence of eosinophils leads to decreased production of key cytokines and increased fungal burden after infection with A. fumigatus. However, this is associated with enhanced neutrophil influx and survival, suggesting that eosinophils are crucial in setting the appropriate tone of the immune response to infection by A. fumigatus.

Financial Support: CNPq, Capes, FAPEMIG.
Class A Scavenger Receptors (SR‐A) mediate macrophage adhesion to glycated collagen and secretion of pro‐inflammatory mediators
Authors: Nikolic Dejan Miodrag , He Beixiang , Post Steven R
Published: June 2021

SR‐A mediates macrophage adhesion to modified substrates via activation of pro‐inflammatory molecules including iPLA2. In this study we examined the extent to which SR‐A mediates macrophage adhesion to glycated collagen and whether this results in secretion of pro‐inflammatory arachidonic acid (AA) metabolites and cytokines. When compared to SR‐A‐/‐ macrophages, SR‐A+/+ macrophages displayed increased spreading on glycated collagen, but not on native collagen. Additionally, enhanced macrophage adhesion to glycated collagen was effectively abolished by a SR‐A “decoy” receptor. These results demonstrate that SR‐A mediates macrophage adhesion to glycated collagen. SR‐A‐mediated macrophage adhesion to glycated collagen resulted in a 3‐fold increase in PLA2‐mediated production of AA metabolites including PGE2, PGI2, and TXA2. Maximal PG production occurred during the first 60 min and returned to basal after 24 hrs. Inhibitor of iPLA2 prevented production of AA metabolites and abolished SR‐A‐mediated adhesion to glycated collagen. In addition, relative to SR‐A‐/‐ macrophages, SR‐A+/+ macrophages plated on glycated collagen for 24 hours secreted 2.5‐fold more TNFα. Thus, SR‐A may contribute to increased inflammation during diabetes by mediating macrophage adhesion to glycated collagen and secretion of pro‐inflammatory metabolites. Support: NIH‐HLR01‐076682 and Sturgis Charitable Trust.
Phytoestrogen genistein up‐regulates endothelial nitric oxide synthase expression via activation of cAMP‐responsive element‐binding protein in human aortic endothelial cells
Authors: Liu Dongmin , Si Hongwei , Jiang Hongling
Published: June 2021

We previously reported that genistein up‐regulates endothelial nitric oxide synthase (eNOS) and prevents hypertension in rats. However, how genistein regulates eNOS expression is unknown. Here, we show that genistein enhanced eNOS expression and nitric oxide (NO) synthesis in human aortic endothelial cells (HAECs). Inhibition of mitogen‐activated protein kinase, phosphatidylinositol 3‐OH kinase/Akt, or protein kinase C did not affect genistein‐enhanced NO synthesis. In addition, blockage of estrogen or prostanoid receptors failed to inhibit genistein‐stimulated eNOS expression. However, chemical or genetic inhibition of protein kinase A (PKA) completely abolished genistein‐stimulated eNOS expression and NO production in HAECs. Accordingly, genistein induced PKA activity and subsequent phosphorylation of cAMP‐responsive element‐binding protein (CREB). Suppression of CREB by siRNA transfection abolished genistein‐enhanced eNOS expression and NO production. Consistently, deletion of the cAMP responsive‐element (CRE) site within human eNOS promoter eliminated genistein‐stimulated eNOS promoter activity. These findings provide the first evidence that genistein directly targets cAMP/PKA/CREB/eNOS/NO signaling in ECs. This work was supported by grants from the AHA Mid‐Atlantic Affiliate (to DL) and the NCCAM of NIH (1R21AT004694 and 1R21AT002739 to DL).
Differential regulation of genes for intestinal cholesterol flux by epigallocatechin gallate (EGCG) and resveratrol (RES) in Caco‐2 cells: Potential role of histone deacetylases and sirtuins in intestinal cholesterol metabolism
Authors: Wegner Casey , Park Youngki , Koo Sung I , Lee Jiyoung
Published: June 2021

Despite well‐established cardio‐protective properties of phenolic compounds, their role in intestinal cholesterol metabolism is unclear. We investigated the effect of EGCG and RES on the transcriptional regulation of genes encoding intestinal cholesterol transporters and lipoprotein receptors in Caco‐2 cells. Cells treated with EGCG for 12 hr markedly increased ATP‐binding cassette transporter G5 (ABCG5) mRNA but decreased the expression of ABCA1, scavenger receptor class B type 1, Niemann‐Pick C1‐like 1 (NPC1L1), microsomal triglyceride transport protein and LDL receptor related protein 2 in a dose‐dependent manner (0–100 μM). In contrast, RES at 100 μM increased ABCA1 and NPClL1 mRNA. Of interest, EGCG significantly decreased the expression of all major histone deacetylase (HDAC), i.e., 1, 2, 3, 6, 7, 10, whereas RES had a minimal effect. Sirtuin (Sirt) 2–7 expression was also markedly repressed by EGCG but RES increased Sirt3, 4 and 5. The results indicate differential regulation of key transporters involved in intestinal cholesterol efflux by EGCG and RES. The over‐arching objective of this research will be to relate changes in the gene expression profile to the promotion of cholesterol flux from the basolateral to apical side of enterocytes. Importantly, this study suggests that expression of the aforementioned intestinal transporters may be linked the regulation of HDAC and Sirt in the intestine.

Grant Funding Source: Supported by USDA Hatch fund
Inhibitory Effects of Metabolites of Nobiletin and 5‐hydroxy Nobiletin on Human Lung Cancer Cells
Authors: Charoensinphon Noppawat , Zheng Jinkai , Dong Ping , Qiu Peiju , Tran Thony , Xiao Hang
Published: June 2021

Nobiletin (NBT) and 5‐hydroxy nobiletin (5HN) are unique polymethoxyflavones found in citrus fruits. We investigated the effects of NBT, 5HN, and their major metabolites, namely 4′‐hydroxy nobiletin (M1), 3′,4′‐dihydroxy nobiletin (M2), 3′,5‐dihydroxy nobiletin (M3), 4′,5‐dihydroxy nobiletin (M4), and 3′,4′,5‐trihydroxy nobiletin (M5) on human lung cancer cells. All metabolites showed much stronger inhibitory effects on lung cancer cells than NBT and 5HN. M3 produced the strongest inhibition among all metabolites. Cell cycle analyses revealed that M1 and M4 induced G0/G1 cell cycle arrest, while M3 and M5 caused G2/M cell cycle arrest after 24h treatments. Moreover, annexin V‐FITC/PI co‐staining assay showed that M2, M3 and M5 induced extensive apoptosis after 48h treatments. Consistent with these results, immunoblotting showed that M3 and M5 decreased expression levels of cyclinB1 and cdc‐2 (cdk‐1), while increased expression levels of p21(Cip1/Waf1). M1 and M4 decreased expression levels of cdk‐4. Furthermore, M2, M3, and M5 caused significant Increases in the expression levels of Bax, cleaved PARP, and cleaved caspase‐3. In contrast, NBT and 5HN did not cause any changes in these proteins at the same concentrations as the metabolites. In conclusion, our results demonstrated strong inhibitory effects of metabolites of NBT and 5HN on human lung cancer cells.

Grant Funding Source : NIH: CA139174, AICR: 10A044
Serotonin 5‐HT2A receptor activation potently inhibits TNF‐α mediated inflammation in vivo, and blocks the development of asthma
Authors: Nau Felix Joseph , Yu Banging , Miller Justin , Ahlert Terry , Cormier Stephania , Nichols Charles D.
Published: June 2021

Here, we show that activation of serotonin 5‐HT2A receptors has potent anti‐inflammatory effects in whole animal models of inflammation. Systemic administration of the 5‐HT2A agonist, R‐DOI, potently inhibits the inflammatory effects of TNF‐α mediated inflammation and decreases expression of key proinflammatory markers like ICAM1, CCL5, MCP1, as well as circulating levels IL6. These effects are potent in vascular tissues like the aortic arch, and superpotent in the gut, where R‐DOI doses as low as 0.01 mg/kg completely blocked the proinflammatory effects of TNF‐α. Importantly, pre‐administration of the 5‐HT2A selective antagonist, M100907, blocked the anti‐inflammatory effects of DOI. Next, we examined the effects of 5‐HT2A receptor activation on a common human inflammatory disease, allergic asthma. We found that in a mouse model of allergic asthma, 5‐HT2A receptor activation completely and potently blocked the development of airways hyper‐responsiveness, eosinophilia, inflammation, and mucus overproduction. Taken together, our results have defined a novel role for serotonin and 5‐HT2A receptors in inflammatory processes, and suggest that activation of 5‐HT2A receptors represent a novel therapeutic strategy for treating chronic human inflammatory diseases. Supported by: NIH R21HL095961.
Investigation of Hypersensitivity to PEGylated Particles For Use in Drug Delivery
Authors: Simmons Ken , Nejati Elham , Johnson Rebecca , Pearce Robert , Albrecht Ralph , Mecozzi Sandro
Published: June 2021

Nanoparticles have shown promise in the field of drug delivery. Many have the potential to improve the solubility and safety‐profile of our current drugs. Polyethylene glycol is commonly used as the hydrophilic segment in many polymer‐based nanoparticle systems. There have been reports of hypersensitivities to PEGylated particles. In order for these novel drug delivery systems to reach clinical use, it is essential to investigate the mechanism behind their allergic‐like responses. This study examines hypersensitivities to PEGylated particles in a canine model. Dogs are a very sensitive model for this type of allergy, which is thought to be caused through either direct complement activation or an antibody‐based mechanism. We have tested a number of PEG‐fluorocarbon polymers using this model and have performed blood analysis pre and post injection. Current results indicate an antibody‐mediated pathway is likely responsible for this type of hypersensitivity.

This project was supported by the UW Institute for Clinical and Translational Research funded through a National Institutes of Health Clinical and Translational Science Award, 1UL1 RR025011, and the STEP program administered by the Wisconsin Alumni Research Foundation.
Oral administration of interferon‐τ reduces adiposity in Zucker diabetic fatty rats
Authors: Tekwe Carmen D. , Lei Jian , Yao Kang , Li Xilong , Rezaei Reza , Dahanayaka Sudath , Meininger Cynthia J. , Carroll Raymond J. , Bazer Fuller W. , Wu Guoyao
Published: June 2021

The Zucker diabetic fatty (ZDF) rat spontaneously develops type‐2 diabetes at 7 to 8 weeks of age. This study tested the hypothesis that oral administration of Interferon‐ô (IFNT) reduces obesity and delay the onset of diabetes in ZDF rats. Beginning at 4 weeks of age, 18 male ZDF rats were fed a Purina 5008 diet and received drinking water containing 0, 330 and 660 ng IFNT/100 mL (n=6/group), providing 0, 4 and 8 μg IFNT/kg BW per day. At 12 weeks of age, rats were euthanized to obtain tissues. Water or food intake did not differ (P > 0.05) among the three groups of rats. Compared to the control group, oral administration of 8 μg IFNT/kg BW per day reduced (P < 0.05) the body weight of the rats by 6.2%, the onset of diabetes by 8.5 days, concentrations of branched‐chain amino acids by 28%, and white adipose tissue by 40%, while increasing (P < 0.05) brown adipose tissue (BAT) in the interscapular region by 46%. Oral administration of 8 μg IFNT/kg BW per day enhanced (P < 0.05): 1) glucose oxidation in the liver, BAT, and white adipose tissue; 2) oleate oxidation in the liver, gastrocnemius muscle and BAT; and 3) leucine transamination in skeletal muscle. None of the measured variables differed (P > 0.05) between the rats receiving oral administration of 0 and 4 μg IFNT/kg BW per day. Our results indicate that IFNT at 8 μg IFNT/kg BW per day reduced obesity and improved the metabolic profile in ZDF rats. (Supported by AHA and NIH grants)
Biodistribution of mesenchymal stem cells in the treatment of experimental caustic esophageal injury
Authors: Caliskan Bahadir , Kantarcioglu Murat , Guven Ahmet , Karacali Ozgur , Ural Ali Ugur , Surer Ilhami
Published: June 2021

Aim

In our study, we investigate experimentally biodistribution of mesenchymally stem cells (MSCs) after injection in early period of caustic esophageal injury especially due to alkaline agents or toher various chemical substances ingestions for accidentally drink.

Method and Material

In the study, 23 Spraque Dawley rats in total of 3 groups were used. Our study groups designed as Positron Emission Tomograhy (PET) group (n=12), control group (n=7) and MSCs/bone marrow production group (n=4). Caustic esophageal injury created by injection of %20 NaOH solution. This method is described by Gehanno and Guedon. After 24 hours caustic esophageal injury, Fluorodeoxyglucose (FDG) marked MSCs dose at 1 million cells/rat injected into rats tail vein. One hour and 4 hours after injection PET imaging was performed.

Results

At first hour after FDG injection, FDG uptake was observed in our rats brain, muscle and bladder tissue and FDG marked MSCs settled in the lung. On the other hand at 4 hours after injection FDG marked MSCs settled at lung and also migrated to injured esophageal tissue. When screening with barium esophageal gavage, damaged esophageal tissue in the migration of MSCs marked with FDG was confirmed.

Conclusion

We demonstrated that after 4 hours MSCs injection via peripheral vein for treatment of esophageal injury due to caustic chemical substances, MSCs settled at lung and small amount of MSCs migrated to injured area (homing). Our findings seems to support of homing feature of MSCs in acute injured tissue when infused via peripheral vein.
Roles of MOP and NOP receptors in regulating buprenorphine‐induced physiological responses in monkeys
Authors: Cremeans Colette M , Gruley Erin , Kyle Donald J , Ko Mei-Chuan
Published: June 2021

Buprenorphine is known as a mu opioid receptor (MOP) partial agonist, but its analgesia was attenuated by activation of nociceptin/orphanin FQ peptide (NOP) receptors in rodents. The aim of this study was to investigate the roles of MOP and NOP receptors in regulating buprenorphine‐induced physiological responses in monkeys. Systemic buprenorphine (0.01–0.1 mg/kg) dose‐dependently produced antinociception, respiratory depression, and itch/scratching responses. A MOP antagonist, naltrexone 0.03 mg/kg, produced similar degrees of rightward shifts of buprenorphine's dose‐response curves for all three endpoints and confirmed that they were MOP‐mediated effects. In contrast, a NOP antagonist, J‐113397 0.1 mg/kg, did not change buprenorphine‐induced effects, indicating that there were no functional NOP receptors in buprenorphine‐induced actions. More importantly, a NOP agonist Ro 64–6198 enhanced buprenorphine‐induced antinociception, and the dose‐addition analysis revealed that systemic combination of buprenorphine and Ro 64–6198 produced superadditive antinociceptive effects. These findings provided functional evidence that co‐activation of MOP and NOP receptors produced synergistic antinociception in primates. This study strongly supports the therapeutic potential of mixed MOP/NOP agonists as innovative analgesics (Supported by USPHS grant AR‐059193).
Regulation of 3‐methylindole metabolism by nuclear receptors
Authors: Gray Matthew A , Squires E. James
Published: June 2021

Regulation of 3‐methylindole metabolism by nuclear receptors 3‐methylindole (3MI) is a known pneumotoxin that is found in naturally in fecal matter and is a major component of tobacco smoke; it is also a major component of boar taint, the accumulation of malodorous compounds in pigs that negatively impacts meat quality. We studied the effects transactivation of the constitutive androstane receptor (CAR), pregnane X receptor (PXR), and farnesoid X receptor (FXR) on gene expression and the metabolism of 3MI in porcine hepatocytes. Real‐time PCR was used to determine the expression of key genes in agonist treated porcine hepatocytes, while the production of major 3MI metabolites over time was quantified by HPLC. FXR transactivation significantly (p<0.05) increased the expression of CYP2E1, which is crucial to 3MI metabolism, 1.29 (1.19,1.40) fold over the untreated controls. PXR transactivation increased CYP2A19 expression 1.24 (1.14, 1.36) and CYP2C49 expression 4.51 (3.64, 5.57) fold; both of these enzymes have been shown to play a role in 3MI metabolism in pigs. FXR transactivation stimulated the formation of 6‐hydroxy‐3‐methylindole, a metabolite crucial in the 3MI excretion pathway, by 1.86 fold over the untreated controls. CAR and PXR transactivation did not affect overall 3MI metabolism or the formation of major metabolites. FXR may thus play an important role in regulating the excretion of 3MI, thus helping prevent toxicity, as well as improving meat quality in boars.
Acute and Repeated Dose Toxicity Studies of Methanol Extract of Bauhinia variegata Leaves
Authors: Kulkarni Yogesh Anant , Warrier Sona
Published: June 2021

Bauhinia variegata Linn. (Caesalpiniaceae), an imperative medicinal plant in traditional system of medicine of India is known to have several therapeutic activities. The present study was carried out to determine the safety of methanol extract of Bauhinia variegata leaves (MEBV) after acute and repeated dose administration in rodents, as per OECD guidelines. For acute toxicity study, MEBV was administered orally to female Swiss albino mice at doses of 300 and 2000 mg/kg. For repeated dose toxicity study, Wistar rats of either sex were orally administered with MEBV at the dose of 1000 mg/kg/day for a period of 28 days. The effects of MEBV on body weight, food and water consumption, hematological parameters, biochemical parameters and histopahology of important organs were studied. In acute toxicity study, administration of the MEBV at selected doses did not produce any mortality or significant changes in the general behaviour. The no‐observed adverse effect level (NOAEL) of methanol extract was found to be 2000 mg/kg. In repeated dose toxicity study, no mortality was observed at selected dose during study period of 28 days. There were no significant differences in the general condition, body weight, food and water intake in treated animals when compared with control animals. 28 days repeated administration of MEBV did not show any significant changes in haematological and biochemical parameters in experimental animals. Histopathology of important organs showed normal structure. Thus, the methanol extract of Bauhinia variegata leaves was found safe in acute and repeated dose toxicity studies when tested in mice and rats.
Inquisitive doctor, reluctant patient: the story of Alexis St. Martin's gastric fistula and America's first physiologist, Dr. William Beaumont, who discovered gastric juice and the physiology of digestion (1822–1833; video shown by permission of the Mackinac Island State Park Commission)
Authors: Dean Jay B.
Published: June 2021

On June 6, 1822, inside the American Fur Company's store on Mackinac Island, epicenter for the fur trading enterprise in the Great Lakes region, a French Canadian voyageur named Alexis St. Martin (1794–1880) was accidently shot in the stomach. Dr. William Beaumont (1785–1853), Post‐Surgeon at Fort Mackinac (1820–1825), attended to St. Martin's injuries over the next 3 years, but his stomach wound never closed resulting in a permanent gastric fistula. Recognizing a serendipitous opportunity to study digestion, the doctor began tying small bits of food to string and lowering them through the fistula into St. Martin's stomach, recording how long it took to digest the food. Beaumont conducted 238 experiments intermittently over 8 years at Fort Mackinac, MI (1825); Fort Niagara, NY (1825); Fort Crawford, WI (1829–31), and Washington D.C. (1832–33). In 1833 he published his discoveries in “Experiments and observations on the gastric juice and the physiology of digestion”. Beaumont's pioneering studies established the field of digestive physiology, identifying HCl as the important element in gastric juice along with 50 other conclusions from his experiments (5.5 min video will be shown) (USF).
Changes in the circulating 1,25(OH)D‐to‐25(OH)D ratio correlate with IFN‐gamma alterations
Authors: Barker Tyler , Trawick Roy H
Published: June 2021

Vitamin D status decreases during inflammation, and interferon (IFN)‐γ is an inflammatory cytokine that regulates vitamin D metabolism in isolated immune cells. However, it is unknown if perturbations in circulating vitamin D metabolites correlate with IFN‐γ changes after anterior cruciate ligament (ACL) reconstruction, a surgery followed by local and systemic cytokine increases. Therefore, the purpose of this study was to correlate circulating 25‐hydroxyvitamin D (25(OH)D), 1,25‐dihydroxyvitamin D (1,25(OH)D) and 1,25(OH)D‐to‐25(OH)D ratio changes to IFN‐γ alterations in patients after ACL surgery. Plasma 25(OH)D, 1,25(OH)D and IFN‐γ concentrations were measured in fasting blood draw samples obtained from ACL injured patients (n = 12) pre‐surgery and 90‐m, 3‐d and 7‐d after surgery. Despite no change in 1,25(OH)D or the 1,25(OH)D‐to‐25(OH)D ratio, 25(OH)D concentrations decreased significantly (p < 0.05) at 90‐m and 3‐d. Strikingly, 1,25(OH)D (r = 0.37, p < 0.05) and the 1,25(OH)D‐to‐25(OH)D ratio (r = 0.52, p < 0.05) changes correlated with increasing IFN‐γ, suggesting that the conversion of 25(OH)D to 1,25(OH)D increases with IFN‐γ in the circulation. Thus, extending and translating from previous isolated immune cell results, we conclude that IFN‐γ contributes to the regulatory conversion of vitamin D to its active hormonal form in the circulation.
Activation of leptin receptors in the NTS contributes to the elevated blood pressure in obesity via inhibiting NTS melanocortin signaling
Authors: Sekizawa Shin-ichi , Chen Chao
Published: June 2021

Recent evidence suggests that leptin selective resistance (i.e., resistance to the weight‐reducing but not the pressor actions of leptin) contributes to obesity‐hypertension. Actions of leptin on hypothalamic POMC neuron‐induced increase in BP are well recognized. The role of endogenous leptin in obesity‐hypertension is not as clear. We hypothesized that in obesity, elevated leptin increases BP by decreasing excitability of NTS 2nd‐order baroreceptive neurons via inhibiting NTS melanocortin signaling. SD rats were randomly assigned to a control or high fat diet group (45% fat). High fat diet increased body weight by 27%, BP by 18%, and plasma leptin level by 163% compared to age matched control rats (p<0.05). In anesthetized rats, microinjections of a leptin antagonist into the NTS decreased (−14±7 mmHg, p<0.05) BP in high fat rats (n=4) but not in the control rats (n=5). Pre‐injection of a MC4R antagonist blocked the leptin antagonist‐induced decreased BP in obese rats (n=3, p<0.05). Using patch‐clamp techniques in NTS slices, leptin induced membrane hyperpolarization in control group but not in high fat rats (p<0.05), an effect that was blocked by a MC4R antagonist. Data suggest that 1) NTS is an important site contributing to the leptin selective resistance seen in obesity‐hypertension; and 2) leptin‐induced inhibition of POMC neurons in the NTS is an important pathway contributing to obesity‐hypertension.
Dynamics of focal adhesion kinase and paxillin in lamellipodial protrusion of migrating endothelial cells
Authors: Hu Ying-Li , Lu Shaoying , Wang Yingxiao , Chien Shu
Published: June 2021

The dynamics of focal adhesions (FAs) can regulate cell migration, intracellular signaling, and remodeling of actin filaments. FA turnover is dependent on focal adhesion kinase (FAK) and paxillin, which are involved in FA disassembly and formation. We used time‐lapse double‐color imaging to monitor concurrently the spatial‐temporal dynamics of FAK and paxillin in live endothelial cells (ECs) co‐transfected with GFP‐FAK and DsRed‐paxillin. In comparison to fluorescence intensity (FI) of paxillin‐FAs, FI of FAK‐FAs was markedly higher at cell front (lamellipodial protrusion), approximately equal at cell center, and lower at cell rear. Consistently, the mean FAK/Paxillin FI ratio from multiple cells is highest at cell front (mean±SE: 4.73±0.48), almost equal to 1 at cell center (0.95±0.11), and lowest at cell rear (0.64±0.05). In ECs co‐transfected with RFP‐actin and GFP‐FAK, FAK is found to be associated with actin filaments at cell leading edge, but paxillin shows minimal presence. Our results suggest that FAK‐FAs are assembled in lamellipodial protrusion earlier than paxillin. While both FAK and paxillin play important roles in modulating FA dynamics during cell migration, the FAK associated with actin filaments promotes FA formation ahead of paxillin at the cell leading edge. This work was supported by NHLBI Research Grants HL‐104402 and HL‐106579 (S.C.).
Endogenous H2S opposes vasoconstriction in conjunction with NO in rat aorta
Authors: Paredes Daniel A , Jackson-Weaver Olan , DeLeon Xavier , Kanagy Nancy
Published: June 2021

Hydrogen sulfide (H2S) is an endogenous vasodilator produced in the vasculature by cystathionine γ‐lyase (CSE) from L‐cysteine (Cys). Studies suggest interaction between NO and H2S to potentiate each other's effects. We hypothesized that H2S‐induced relaxation in rat aorta requires activation of nitric oxide synthase (NOS). In aortic ring segments, U46619‐contraction was reversed by Cys (1 μM). Inhibiting CSE with β‐cyano‐alanine, (BCA, 100 μM) diminished Cys‐induced dilation by 50%. Inhibiting NOS (L‐NNA, 100 μM) or disrupting the endothelium lowered Cys ‐ induced dilation by 80% and 85% respectively, suggesting Cys ‐ induced dilation is mediated in part through NOS. CSE inhibition diminished acetylcholine (ACH)‐induced dilation by 40% whereas either L‐NNA or endothelium disruption completely abolished dilation. U‐46619 induced greater contraction in BCA treated arteries vs. controls suggesting endogenous H2S opposes contraction. Endothelium disruption or inhibition of both NOS and CSE also increased contraction by U‐46619. H2S production measured in aortic segments was diminished by BCA and augmented by NOS inhibition. In aorta, CSE was primarily expressed in the endothelium. Our results indicate that ACH stimulates H2S production in the endothelium by CSE and dilation to H2S requires NOS. Furthermore, basal production of H2S appears to be inhibited by NOS.
Vascular eNOS is reduced in conjunction with increased calpain activity in dystrophin‐deficient human endothelial cells
Authors: Li Jia-Ling , Shen Guo-Liang , Thomas Gail D
Published: June 2021

Endothelial nitric oxide synthase (eNOS) is reduced in arteries of mdx mice, a model of Duchenne and Becker muscular dystrophy (DMD/BMD) caused by mutation of the dystrophin gene. Whether this finding translates to human endothelial cells is an important unanswered question, as reduced vascular NO could contribute to vasospasm or ischemic injury in vulnerable dystrophin‐deficient skeletal and cardiac muscle. Loss of dystrophin in skeletal muscle leads to activation of the cysteine protease calpain. We therefore hypothesized that calpain would be activated and eNOS, a known calpain substrate, would be reduced in dystrophin‐deficient endothelial cells. In cultured human aortic endothelial cells (HAECs), we found that siRNA‐mediated knockdown of dystrophin was accompanied by an increase in eNOS mRNA (+58 ± 10%) but a decrease in eNOS protein (−53 ± 20%). Acute dystrophin knockdown had no effect on the level of calpain‐1 protein, but increased calpain activity by 67 ± 20%. In endothelial cells obtained by intravascular venous biopsy of patients with BMD, preliminary findings show that eNOS protein also is reduced in the setting of chronic dystrophin deficiency. Taken together, these data indicate that loss of dystrophin in endothelial cells secondarily decreases eNOS, which may be due to calpain‐mediated proteolysis. Supported by CSMC.
CaMKII regulates intracellular calcium stores of native endothelial cells from mesenteric arteries
Authors: Toussaint Fanny , Charbel Chimène , Blanchette Alexandre , Villeneuve Louis R. , Ledoux Jonathan
Published: June 2021

Ca2+ homeostasis is crucial for endothelial function and the regulation of vascular tone. The Ca2+‐dependent kinase CaMKII is activated by Ca2+ pulsars and might play an important role in endothelial Ca2+ homeostasis. Thus, the involvement of endothelial CaMKII in the regulation of intracellular Ca2+ dynamics has been investigated in native endothelial cells of mesenteric arteries. Acute exposure to KN‐93 (10 μM), a CaMKII inhibitor, has little if any effect on Ca2+ pulsars frequency or amplitude. However, CaMKII inhibition induces pulsar sites recruitment as more pulsars sites are active in the presence of KN‐93 compared to control (25%). Interestingly, in absence of extracellular Ca2+, KN‐93 appears to empty intracellular Ca2+ stores. Indeed, exposure to ionomycin, to empty endoplasmic reticulum in absence of extrallular Ca2+, provoked a large and transient intracellular Ca2+ increase. However, preincubation with KN‐93 abolished the ionomycin‐induced Ca2+ transient. These results suggest that CaMKII might inhibit IP3R receptors and/or phosphorylates phospholamban to regulate ER Ca2+ content. Confocal microscopy revealed that CaMKIIα & β are colocalized with IP3R1 in native endothelium. Our data suggest that CaMKII is an important regulator of endothelial Ca2+ homeostasis and may represent a novel target for the treatment of pathologies like hypertension.

Supported by FRSQ, CFI, CIHR and HSFC.
Evidence for a role of endothelial‐cell derived PLGF in paracrine communication
Authors: Xiang Lingjin , Lloyd Pamela G.
Published: June 2021

Placenta growth factor (PLGF) is a vascular endothelial growth factor (VEGF)‐family protein that mediates arteriogenesis (adaptive collateral artery remodeling) via a monocyte‐dependent mechanism. In a previous study of 10 different endothelial cell (EC) and vascular smooth muscle cell (SMC) lines, we found that EC express much higher levels of PLGF mRNA and protein than do SMC. The high level of PLGF expression by EC was surprising, as the related factor VEGF is expressed mainly in tissue surrounding EC, rather than in EC. PLGF has been shown not to be mitogenic for EC. Therefore, we hypothesized that the high level of PLGF expression in EC might be important for supporting other EC functions, such as survival, migration, and tube formation. To test this hypothesis, we knocked down PLGF expression in human coronary artery endothelial cells (HCAEC) using siRNA and performed functional assays. Knockdown of PLGF did not affect EC viability. Migration of EC towards VEGF and tube formation in GelTrex were likewise unaffected by PLGF knockdown. Survival of human coronary artery smooth muscle cells (HCASMC) was also unaffected by PLGF siRNA treatment. These results suggest that the high expression of PLGF by EC likely mediates paracrine communication between EC and non‐vascular cells, such as circulating monocytes. Further studies are needed to characterize the secretion of PLGF by EC. Funding: NIH R01 HL084494 (PL).
Increasing pressure in newborn carotid arteries promotes endothelial cell maturation
Authors: Heberlein Katherine R , Flavahan Sheila , Mozayan Mansoor , Lindgren Isa , Flavahan Nicholas A
Published: June 2021

The endothelium of newborn arteries has an unusual phenotype, which includes immature cell:cell junctions, prominent stress fibers and reduced NO‐mediated endothelial dilation to acetylcholine (Ach). To investigate mechanisms regulating postnatal endothelial maturation, carotid arteries were isolated from newborn (postnatal day 1, P1), P7 and P21 mice, and analyzed in a myograph at a transmural pressure (P™) of 20 mmHg (mean blood pressure BP at P1). Ach caused dilation in maturing (P7, P21), but not newborn arteries. This was associated with increased Akt activity in maturing arteries. Indeed, inhibition of PI3K/Akt (wortmannin, LY294002) markedly reduced dilation to Ach at P7. A brief, transient exposure of P1 arteries to increased P™ (50 mmHg, equivalent to systolic BP at P7, 60 min) uncovered powerful dilation to Ach. The effect of increased P™ to rescue Ach responses in P1 arteries was prevented by inhibition of PI3K/Akt or by a VE‐cadherin function‐blocking antibody, which disrupts endothelial adherens junctions. The antibody also inhibited Ach responses in P7 arteries. Our results suggest that maturation of newborn arterial endothelium, including increased NO‐mediated endothelial dilation, may be induced by increases in BP in the early postnatal period. Furthermore, the effect of increasing pressure may be mediated by increased VE‐Cadherin signaling at adherens junctions. Funded by NIH HL102715
RATE OF DESENSITIZATION OF CORONARY VASOCONSTRICTION INDUCED BY POLYMERS OF ANGIOTENSIN II IS DETERMINED BY THE POLYMER MOLECULAR WEIGHT
Authors: Torres-Tirado David , Rubio Rafael
Published: June 2021

We have shown that Angiotensin II (AngII) does not diffuse across the vessel wall, remains intravascularily and acts solely in coronary endothelial luminal membrane AngII type 1 receptors (CELM‐AT1R) as do large size, non‐diffusible, polymer of AngII (AngII‐Pol, 15,000 kDa). A sustained intracoronary infusion (SCI) of AngII causes a transient coronary vasoconstriction (V) effect that reaches a maximum, decays gradually and by 2 min is back to control (desensitization; DES). These effects are due to CELM‐AT1R density decrease. In contrast, SCI of AngII‐Pol causes a sustained V and CELM‐AT1R density remains constant explaining why AngII‐Pol did not cause DES. These findings lead us to hypothesize: the rate of DES (RDES) is inversely proportional to molecular weight of AngII‐Pol (MWAngII‐Pol). To test this hypothesis we synthesized AngII‐Pols of various MW (kDa): 1, 9, 37, 438, 3140, 12566. AngII moieties were bound to Pol by its amino group. In Langendorff constant flow perfused isolated guinea pig hearts, AngII‐Pols were separately SCI for 16 min. We found RDES vary inversely with MWAngII‐Pol. Also a primary SCI of AngII causes that a secondary AngII administration gives a depressed response (tachyphylaxis, T). However, in the case of a primary SCI of AII‐Pols the T effect also varied inversely with MWAngII‐Pol. These results confirm our hypothesis and indicate that MWAngII‐Pol is the determining factor for RDES and T and that the chemical alteration of AngII into AngII‐Pol is not. Possibly RDES and T dependence on MWAngII‐Pol results from an inversely proportional CELM‐AT1R rate of internalization.
Role of superoxide anion in coronary endothelial dysfunction in type 2 diabetic mice
Authors: Cho Young-Eun , Pangare Meenal , Makino Ayako
Published: June 2021

Coronary endothelial dysfunction leads to the increase in coronary blood flow and decrease in capillary density, and subsequently increases the incident of cardiac ischemia. Excess superoxide production (O2‐) has maladaptive effects on cellular functions in many tissues and organs. The objective of this study is to identify the role of O2‐ in coronary endothelial dysfunction in type 2 diabetic mice. Acetylcholine (ACh)‐induced relaxation (indicative of endothelium‐dependent relaxation) was significantly attenuated in diabetic coronary arteries (CAs) compared with control CAs. The pharmacological approach reveals that NO‐dependent relaxation, but not endothelial‐derived hyperpolarization‐dependent relaxation, is decreased in diabetic CAs. In the presence of Tempol (a O2‐scavenger), ACh‐induced relaxation in diabetic CAs is increased to the level seen in control CAs. In addition, mouse coronary endothelial cells (MCECs) isolated from diabetic mice exhibit significant increase in mitochondrial O2‐production and decrease in Mn‐SOD protein expression in comparison to those in control MCECs. These results suggest that the excess O2‐production in mitochondria attenuates the bioavailability of NO and subsequently decreases ACh‐induced relaxation in CAs in diabetic mice. This research was supported by grant DK083506.
HYDROGEN SULFIDE (H2S) IMPROVES THE RENAL FUNCTION IN A COMBINED STATE OF HYPERTENSION AND DIABETES
Authors: Rathore Hassaan Anwer , Din Fiaz ud , Abdullah Mohammed H , Zubaid Munavvar , Abdullah Nor Azizan , Johns Edward J
Published: June 2021

This study investigated the action of H2S on mean arterial pressure (MAP) and renal function in a combined state of hypertension and diabetes.

Groups (n=6) of spontaneously hypertensive rats (SHR) or Wistar Kyoto (WKY) rats received vehicle, streptozotocin (STZ)(40mg/kg) or STZ plus Sodium Hydrogen Sulphide (NaHS), at 56μmol/Kg i.p daily for 35 days; therafter, MAP and cortical blood perfusion (CBP) were measured in ketamine + xylazine anaesthetized rats. Data, mean±SEM, were subjected to ANOVA and significance set at p<0.05.

MAP was similar in the WKY (104±1 mmHg), WKY‐STZ and WKY‐STZ‐NaHS groups as was CBP (163±2 BPU). MAP of SHR‐STZ was higher than SHR (145±2 vs 142±2 mmHg) but was lower in SHR‐STZ‐NaHS at 132±3mmHg (P<0.05). CBP in the SHR‐STZ was lower than the SHR (138±3 vs 150±3BPU) but was 12% higher than SHR‐STZ (P<0.05) in SHR‐STZ‐NaHS. GFR in the WKY‐STZ‐NaHS was higher (P<0.05) than the WKY‐STZ (1.8±0.07 vs 0.7±0.02 ml/min/100g). In the SHR‐STZ‐NaHS GFR was higher (P<0.05) than in SHR‐STZ, 2±0.1 vs 1.2±0.1 ml/min/100g.

The improved renal function with NaHS was likely due to the vasodilator effect of H2S reducing renovascular resistance and improving the renal blood flow in hypertension concomitant with diabetes in rats.
Impact of High‐Fructose Consumption on the Canine Cardiovascular System
Authors: Kertowidjojo Elizabeth , Qanud Khaled , Kandhi Sharath , Wolin Michael , Hintze Thomas H
Published: June 2021

Many studies have shown the correlation between fructose consumption (FC) and detrimental effects on the cardiovascular system. We hypothesized that the effects of FC are mediated by an overactivation of NADPH oxidase (Nox), which increases superoxide (O2−) production, effectively reducing NO bioavailability. We explored this hypothesis using male mongrel dogs (N=6) fed 60% fructose diet for 49 days. Hemodynamic measurements and echocardiography showed increases in blood pressure (122±1/81±3 to 157±3/117±7 mmHg) with no significant changes in coronary blood flow, shortening fraction, left ventricular pressure, or dP/dt. Furthermore, though no significant changes were seen in stroke volume or cardiac output, total peripheral resistance was significantly increased (36 ± 4 to 60 ± 7 mmHg). Preliminary data also showed an increase in glucose, lactate, and O2 uptake by the heart and a temporal increase in plasma insulin and Ang II. The ability of bradykinin to reduce myocardial O2 consumption ex vivo was impaired by FC, an effect reversed by apocynin or tiron, a Nox inhibitor and O2− scavenger, respectively. Finally, quantification of O2− by lucigenin chemiluminescence showed a significant increase of O2− in FC heart. In conclusion, FC lowers NO bioavailability through the generation of O2−, which alters the control of arterial pressures and disrupts the metabolism of the normal myocardium. Supported by PO43023.
Hydrogen Sulphide Influences the Excitability of Neurons in the Paraventricular Nucleus of the Hypothalamus
Authors: Khademullah Charline Sahara , Ferguson Alastair
Published: June 2021

Hydrogen sulphide (H2S) has been observed as having a protective role against gastro‐intestinal inflammation. H2S‐catalyzing enzymes are expressed in the paraventricular nucleus of the hypothalamus (PVN). Previous findings suggest that, if stimulated, the PVN can produce inflammatory responses in the GI tract. To understand the effects of H2S in the PVN, we examined the effects of the H2S donor, sodium hydrogen sulphide (NaHS) on the excitability of PVN neurons. Whole‐cell current clamp recordings from rat PVN neurons in slice preparations were performed. Bath application of 50 μM NaHS influenced 90% (9/10) of cells tested within approximately 45s. of administration. 100% of the responsive cells exhibited a hyperpolarizing response (n=9, −15.42 ± 2.55mV). Of these cells, 89% returned to baseline after approximately 15 mins. Four these neurons were identified as parvocellular neurons and 5 were identified as magnocellular neurons, both displayed similar responses to NaHS. These findings shed light on the role that NaHS plays in the PVN to modify the membrane potential of neurons and in turn, may affect the possible pathways associated with the PVN that are involved in inflammatory effects.

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Trypsin and MMP‐9 Levels and Activity Increase in Plasma, Peritoneal Space, and Vital Organs during Hemorrhagic Shock
Authors: Altshuler Angelina E , Penn Alexander H , Yang Jessica , Kim Ga-Ram , Schmid-Schonbein Geert W
Published: June 2021

Hemorrhagic shock (HS) is a major cause of death after trauma. During HS, perfusion to the intestine and pancreas are reduced, resulting in increased intestinal mucosal permeability, pancreatitis, and potential leakage of serine proteases (i.e. trypsin‐a potent matrix metalloproteinase [MMP] activator) into the circulation. We hypothesize that after HS, both trypsin and MMP‐9 enter the circulation, peritoneal space, and appear in peripheral organs. Male Wistar rats were grouped into No‐HS or HS (N=5). Mean arterial blood pressure of HS animals was reduced to 35 mmHg (2 hr) followed by resuscitation of shed blood (2 hr). Plasma and peritoneal lavage fluid were obtained before and after HS. The heart, lung, liver, and brain were collected. Protease activities in plasma and peritoneal fluid were measured by plate zymography (trypsin‐like and MMP‐9 specific substrates) or gelatin gel zymography. Pancreatic trypsin and MMP‐9 levels were identified by immunoblot. Trypsin‐like activities were elevated in plasma and peritoneal space fluid after HS (p<0.05). Immunoblot for pancreatic trypsin detected elevated levels in the plasma, peritoneal fluid, and lungs. MMP‐9 activity and protein levels were elevated in all compartments after HS (p<0.05). In HS, trypsin may be transported from the permeable intestine and/or pancreas into the periphery and may contribute to MMP‐9 activation.
Bufodienolides in pig plasma and urine
Authors: Darlington Daniel N. , Gann Donald S
Published: June 2021

Water and electrolyte balance is regulated in large part by Na/K ATPase. Inhibition of this enzyme leads to cellular depolarization, a rise in extracellular K and intracellular Na, cellular swelling, and has been associated with shock, hypertension, bipolar disorder, schizophrenia, depression and diabetes. Recently, marinobufogenin, a bufodienolide, has been identified in human and rat plasma and is a strong inhibitor of Na/K ATPase. To determine what bufodienolides are in pig, 1000ml of pooled pig plasma and 500ml of urine was extracted using 1× volume ethyl acetate/ 4× volume acetonitrile, dried, de‐conjugated with glucoronidase/sulfatase, and extracted by affinity chromatography (Digibind®, GlaxoSmithKline). Extract was run on Reverse Phase High Pressure Liquid Chromatography (Beckman Coulter). Chromatographic peaks were identified as marinobufogenin, bufalin, resibufogenin and cinobufagen by 1) co‐elution of standards, 2) UV spectrum and 3) mass spectroscopy of collected fractions. These data show that bufodienolides, potent inhibitors of Na/K ATPase, exist in plasma and urine of pig. Supported by the ARMY MRMC.