bluuebunny/crossref_metadata_2025 huggingface dataset:
Physiological variations in plasma sphingomyelin (SM) inversely correlate with lecithin‐cholesterol acyltransferase (LCAT) activity
Authors: Subbaiah Papasani Venkata , Jiang Xian-cheng , Belikova Natalia , Aizezi Buzulagu , Huang Zhi Hua , Reardon Catherine A
Published: June 2021

We previously showed that sphingomyelin (SM), the second most abundant phospholipid in plasma, inhibits lecithin‐cholesterol acyltransferase (LCAT), the enzyme activity critical for HDL maturation, partly by competing with its substrate, phosphatidylcholine (PC). The physiological relevance of this inhibition, however, has not been investigated. In this study, using plasma from various genetic models of mice with naturally varying ratios of SM/PC, we show that this ratio is inversely correlated with the rate of cholesterol esterification by LCAT. Mice deficient in serine palmitoyltransferase, or SM synthase, both of which have reduced SM/PC ratios, showed significantly higher than normal LCAT activity when assayed with the endogenous substrates. On the other hand, LDL receptor deficient mice, and apo E knockout mice, both of which have high SM/PC ratios, had lower than normal LCAT activities. The enzyme levels in plasma, as assayed with an exogenous substrate, were not significantly different, except for a small decrease in apo E knockout mice. Plasma samples with high SM/PC ratios had lower percentage of 20:4, 22:5, and 22:6 cholesteryl esters (CE), and higher percentage of 18:1CE, showing that in vivo, the contribution of LCAT to plasma CE is diminished while that of liver ACAT is enhanced. These results show that plasma SM is a physiological modulator of cholesterol esterification and plasma CE composition.
Novel Mechanisms in Vascular Permeability
Authors: Fantin Alessandro
Published: June 2021

Neuropilin 1 (NRP1) is a transmembrane glycoprotein essential for blood vessel development in vertebrates. Best known for its ability to bind members of the vascular endothelial growth factor (VEGF) and class 3 semaphorin families through its extracellular domain, it also has a highly conserved cytoplasmic domain, which terminates in the SEA motif that binds the PDZ protein synectin/GIPC1/NIP. Previous studies in zebrafish embryos and tissue culture models raised the possibility that the SEA motif of NRP1 is essential for angiogenesis. Here, we describe the generation of mice that express NRP1 lacking the cytoplasmic domain and therefore the SEA motif (Nrp1cytoΔ/Δ mice). Our analysis of pre‐and postnatal vascular development and pathological neovascularisation revealed that vasculogenesis and angiogenesis are not affected by loss of the NRP1 cytoplasmic domain, which instead rely on the membrane‐anchored extracellular domain. In contrast, the NRP1 cytoplasmic domain was essential for VEGF‐induced vascular permeability and promoted VEGF‐dependent collateralisation after femoral artery ligation. Inhibiting signalling through the NRP1 cytoplasmic domain may be a suitable therapeutic approach to treat ischemic conditions where vessel leak exacerbates disease progression, but neoangiogenesis is required to alleviate tissue hypoxia.
Bile Acids Induce Diacyglycerol Kinase‐θ Expression in HepG2 liver Cells
Authors: Cai Kai , Sewer Marion B
Published: June 2021

Diacylglycerol kinases (DGKs) are intracellular lipid kinases that catalyze the conversion of diacylglycerol into phosphatidic acid (PA), thus regulating the intracellular concentrations of these two bioactive signaling lipids. We have found that DGK‐θ, the sole member of the type V family of DGK isoforms, is expressed in the nucleus of HepG2 cells. To determine the factors that regulate hepatic DGK‐θ expression, we examined the role of bile acid signaling in controlling DGK‐θ gene expression. We show that chenodeoxycholic acid and GW4064 induce DGK‐θ mRNA and protein expression in a dose‐dependent manner. Reporter gene studies using 1.5 kB of the DGK‐θ promoter fused to the luciferase gene revealed that the farnesoid X receptor (FXR) increases DGK‐ θ promoter activity, and that GW4064 potentiates FXR‐dependent reporter gene activity. The induction of nuclear DGK‐θ expression is concomitant with the increase in the nuclear production of PA. Given that the nuclear receptor liver receptor homolog‐1 (LRH‐1) binds phospholipids and regulates the bile acid metabolism, we postulate that increased nuclear DGK‐θ expression may modulate cholesterol homeostasis and bile acid metabolism by controlling nuclear PA production and LRH‐1 function.
Impact of Exposure to High Concentrations of Fructose on Cell Surface Chemistry and Carbohydrate Metabolism
Authors: Sacoman Juliana Lessa , Badish Lauren , Hollingsworth Rawle Irvine
Published: June 2021

We evaluated the impact high concentrations of fructose have on cell antigenicity and metabolism in vitro. Antigen composition and sugar pools in MEF and bEND‐3 cells were monitored when they were grown in either fructose or glucose. Cell antigenicity was monitored by the expression of ABO blood group antigens. MEF cells cultured in fructose exhibited an increase in A antigen expression compared to glucose. High concentrations of fructose lead to higher concentrations of fructose‐6‐phosphate, which accelerates hexosamine biosynthesis. Metabolic evaluation of carbohydrate pools by mass spectrometry also supports this. While the free sugar pool is similar in MEF cells treated with either sugar, it was noted that the glycoprotein/glycolipid pool is quite different. Cells cultured in fructose posses a 2:1 ratio of hexoses: hexosamines, while cells in glucose contain only hexoses in this pool, showing an increase in hexosamine concentrations in presence of fructose. bEND‐3 cells in fructose and glucose contained similar expression of all antigens monitored as well as carbohydrate pools although less dramatic shifts were also evident. From these results we can observe that fructose does impact hexosamine biosynthesis and therefore, cell antigenicity and carbohydrate metabolism but there is no universal response. While hexosamine production is greatly increased in MEF, which reflects in an increase of A antigens, the effects on bEND‐3 cells were minor. Changes in cell characteristics such as growth rate and tissue function may stem from exposure to different carbohydrates as major energy source.
The Inhibitory Effects of Curcumin and Coumarin Analogues on the NF‐kB Pathway in Heptocellular Carcinoma
Authors: Hanson Katelyn Austin , Hopkins Nancy
Published: June 2021

Curcumin, a principal compound found in the spice tumeric, has shown to be a natural inhibitor of the inflammatory nuclear factor‐kB pathway. NF‐kB is a transcription factor involved in the upregulation of pro‐inflammatory and anti‐apoptotic genes. In cancer cells, NF‐kB remains constitutively active, preventing the initition of apoptosis. By inhibiting NF‐kB activation and its translocation into the nucleus, it is possible to turn on this apoptotic pathway. Due to curcumin's low potency and poor absorption characteristics it's clinical potential remains limited, so newly developed curcumin analogues that can be administered effectively at lower doses and with relatively low toxcity have become the targets of much current research. Coumarin, which is found in pepper, has also been shown to cause apoptosis in liver cancer cell lines. This study focuses on the effects of novel curcumin and coumarin analogues on inhibiting the NF‐kB pathway and promoting apoptosis is human hepatocellular carcinoma cell lines (HepG2) and compare these to the inhibitory effects of natural curcumin, by conducting Western blot analysis to detect for individual proteins and caspase assays to differentiate between apoptosis and necrotic cell death
Validation of Sojasun®, a soy‐based fermented dessert, as an efficient source of calcium
Authors: Joubrel Gwénaële , Housez Béatrice , Cazaubiel Murielle , Coxam Véronique
Published: June 2021

Aging is associated with increased bone turnover and bone loss, mainly in menopausal women in relation with oestrogen deficiency. Women with low calcium (Ca) intake are at greater risk for bone loss. Soy products are naturally low Ca content, but often fortified with Ca at a level equivalent to many dairy foods. A double blind, randomized parallel‐arm clinical trial was carried out to compare the effects of calcium naturally present in yogurt vs. a calcium‐fortified soy‐based fermented dessert (Sojasun® containing Lipocal®, triphosphate Ca encapsulated with lecithin) on bone metabolism in early menopausal women (N = 44). Subjects consumed study products three times a day for 12 weeks. Both dairy and soy yoghurts contained 150 mg Ca per serving. Bone metabolism parameters (BALP for formation and CTX as a marker for resorption) were evaluated at baseline and end of treatment. A decline in bone turnover was noticed in the 2 groups, as observed by a decrease in bone parameters in both groups. No statistical differences were observed after 12 weeks of consumption, suggesting that both sources of calcium were equivalent. Supplementation with calcium seems to limit bone metabolism turnover, as shown by the reduction in bone metabolism parameters in both groups.
Association between total fat intake and depressive symptoms in Cuban Americans with and without type 2 diabetes
Authors: Exebio Joel C. , Zarini Gustavo G. , Duenas Carla , Huffman Fatma G.
Published: June 2021

The objective of this study was to examine the association between fat intake and depressive symptoms in Cuban Americans with and without type 2 diabetes (T2D). Subjects with (n=190) and without (n=175) T2D were recruited from randomly selected mailing list. Willett's Food Frequency Questionnaire was used to calculate fat intakes. Depressive symptoms were assessed using the Beck Depression Inventory. Analysis included descriptive statistics and linear regression. Depressive symptom was the dependent variable and separate models were run for total fat, saturated fat and trans fat intakes as independent variables. Calorie intake, diabetes status, age, gender, and BMI were included as covariates. Total fat, saturated fat, and trans fat intakes were all independent predictors of depressive symptoms (P=0.001, P=0.006, P=0.035, respectively). However, when adjusting for covariates, only total fat held its significance (R2=0.073, P=0.031). A one gram increase in total fat intake increased the depressive symptoms score by 0.061 units. High fat intake may be associated with higher depressive symptoms in Cuban‐Americans. Funding for this research was provided through an NIH/NIDDK sponsored grant.
Estimates of body composition during pregnancy using bioelectrical impedance analysis
Authors: Toro-Ramos Tatiana , Hoffman Daniel J. , Sichieri Rosely
Published: June 2021

Bioelectrical impedance analysis (BIA) is commonly used for body composition assessment, however prediction equations used by BIA equipment are developed in healthy populations from developed countries and the assumptions made regarding the body proportions may not apply to populations with altered body geometry, such as in pregnancy. BIA using body segments may estimate body composition better than whole body measurements in pregnant women. Study subjects included 209 pregnant women aged 15–40 years and between 18 to 22 weeks gestation. Percent fat mass (%FM) and fat‐free mass in kilograms (FFM) were estimated using whole body BIA and compared to the estimated parameters using two different sets of equations from body segment and whole body resistance (R) and impedance (Z). The estimates of %FM by the manufacturer were significantly lower (30% v. 34%) and significantly higher for FFM (47kg v. 43kg) compared to the equations by Baumgartner, (P<0.001) There was poor agreement between the body composition parameters estimated using prediction equations from segmental data and the parameters measured by whole body BIA. Thus, prediction equations based on segmental measurements and developed from non‐pregnant women are a poor estimate of body composition during pregnancy. Using whole body BIA may provide a more accurate estimate of maternal body composition.

Grant Funding Source : The American Egg Board
Anthropometric antecedents of metabolic syndrome and its components among university students
Authors: Nichols Selby , Edwards Anna-Marie
Published: June 2021

Metabolic syndrome is major risk factor for coronary heart disease (CHD) ‐ a major cause of illness and death in the Caribbean. In this study, we investigated the anthropometric antecedent of metabolic syndrome and its components in a health population of university students. Participants had anthropometry measured using standard procedures. In addition, they had fasting blood sugar (FBS), blood pressure (BP), HDL cholesterol and triglyceride levels measured using portable hand held devices. Our results suggest that 5 percent of participants met the criteria for MS while 12, 22 and 25 percent respectively met the criteria for elevated FBS, BP and triglycerides. 22% had low levels of HDL cholesterol. Several measures of adiposity were significantly inversely associated with HDL cholesterol. Weight, BMI and waist circumference were significantly positively associated with BP. Our findings suggest that although the prevalence of MS was low in this population, the levels of single MS components were relatively higher and may be influenced by anthropometry.
Fructooligosaccharides (FOS) enhance beneficial effects of a flavonoid, quercetin‐3‐glucoside (Q3G), on insulin sensitivity in rats fed high sucrose diet
Authors: Phuwamongkolwiwat Panchita , Suzuki Takuya , Hira Tohru , Hara Hiroshi
Published: June 2021

An enhancement FOS on improvement of glucose tolerance and insulin sensitivity by a flavonoid glycoside, Q3G, and their mechanisms were studied. Rats were fed a sucrose‐based diet with or without 0.3% Q3G, 5% FOS, or 0.3% Q3G+5% FOS, while dextrin‐based diet was used for a normal reference group. OGTTs were conducted on day 14, 28, 45. Flavonoid levels in the aortic blood were measured by LC/MS. Body weight gain was lowered in Q3G+FOS group without changes in food intake. Abdominal fat weights and cecal pH were reduced in both the FOS‐fed groups without any effects of Q3G. Combination of Q3G and FOS only reduced blood glucose levels at 60 min in all OGTTs. HOMA‐IR showed an improvement in the Q3G+FOS group on day 14 and 28. On day 45, the value was improved in the individual FOS or Q3G group, and was further improved in the FOS+Q3G group. A much higher blood level of quercetin was observed in the FOS+Q3G group compared with the Q3G group, which may contribute the combination effects of Q3G and FOS. Secretion of GLP‐1 observed in the small intestinal loops of anesthetized rats and in vivo experiment were increased by addition of quercetin to luminal fluid containing FOS. This may involve in the improvement of glucose tolerance. In conclusion, FOS increases bioavailability of Q3G, and enhanced beneficial effects of Q3G on glucose tolerance and insulin sensitivity, which have a potential for prevention of type 2 diabetes.

Grant Funding Source : Graduate School of Agriculture, Hokkaido University
PH‐427 reduces the survival of fat‐treated pancreatic cancer cells
Authors: Cantoria Mary Jo Castro , Meuillet Emmanuelle
Published: June 2021

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and lethal cancer. A large number of epidemiological studies have linked type‐2 diabetes, obesity and metabolic syndrome, characterized by insulin resistance with an increased risk for developing PDAC. The IGF‐IR/PI3K/AKT survival pathway has been suggested to be the major signaling pathway involved in these processes. Indeed, AKT is a key signaling pathway in PDAC as it mediates cancer cell survival and proliferation. We have developed the AKT Pleckstrin Homology (PH) domain inhibitor, PH‐427, which prevents AKT activation and possess anti‐tumor activities in vivo models of pancreatic cancer. Hence, the objectives of this study were 1) to develop a cellular model for obesity‐driven pancreatic cancer and 2) to determine the effects of PH‐427 on cell viability of pancreatic cancer cells exposed to a fatty environment. BxPC3 cells were incubated in RPMI media supplemented with cholesteryl hemisuccinate (CHS) for two weeks. We show that CHS induced an insulin resistance state whereby AKT phosphorylation is reduced upon IGF‐I stimulation as compared to control cells. Moreover, MTT assays indicated that CHS‐treated cells were more sensitive to PH‐427 treatment as compared to control cells (IC50= 10.9±3.7 versus 15.2±1.5□M). These preliminary data suggest the promising potential use of this novel drug for the treatment of obesity‐induced PDAC.

Grant Funding Source : None
Resveratrol decreases inflammation and oxidative stress in the mdx mouse model of duchenne muscular dystrophy
Authors: Gordon Bradley Scott , Weed Patti , Learner Emily , Schoenling Drew , Kostek Matthew C
Published: June 2021

Duchenne Muscular Dystrophy (DMD) is a genetic disease characterized by muscle damage, oxidative stress, chronic inflammation, and fibrosis. Resveratrol (RES) is an antioxidant and anti‐inflammatory. We have shown that RES improves muscle function in the mdx mouse model of DMD, and others have shown resveratrol decreases fibrosis and oxidative stress in older mdx mice. However, its effect on pathology in young mdx mice is unknown. The purpose of this study was to investigate the effect of resveratrol on muscle pathology in young mdx mice. RES (100 mg/kg) or vehicle was administered to 4–5 week old mdx mice everyday for 10 days or every other day for 8 weeks. Muscle fiber integrity, inflammation, and oxidative stress were assessed by H&E staining and 4‐HNE content. Total inflammation was reduced 21 ± 6% (p < 0.05) after 10 days of treatment with no change in oxidative stress. After 8 weeks of RES treatment, centrally located nuclei were reduced 12 ± 4% (p < 0.05), oxidative stress measured through 4‐HNE content decreased 2 ± 0.13 fold (p < 0.05), and total inflammation and fibrosis did not change. We conclude that RES enhances muscle membrane integrity by reducing inflammation during the peak pathological period and long term oxidative stress. Therefore, resveratrol could be a treatment for boys with DMD. This project was funded by The Center for Alternative Medicine at The University of South Carolina School of Medicine.

Grant Funding Source : Center of Alternative Medicine University of South Carolina School of Medicine
Intestinal damage in weanling pigs following a nutritional insult is transient
Authors: Levesque Crystal L , de Ridder Karl , Skinner Larry D , de Lange Cornelius FM
Published: June 2021

Forty pigs were used to evaluate the long‐term effect of nutritional insult on measures of intestinal development in weanling pigs. Pigs were weaned at 21±2 days of age and fed diets ad libitum containing either high (H) or low (L) quality protein sources for 6 wks. Growth performance was monitored weekly. Pigs were killed at wk 2 and 4. Sections of proximal jejunum and distal ileum were taken for histology and ileal digesta was sampled. Pigs on L had lower (P = 0.002) daily gain in wks 1–3 but not different in wks 4–6 compared to H pigs. Similarly, feed efficiency in pigs on L was lower (P< 0.05) during wks 2–3 but not different in wks 4–6. In the proximal jejunum, villus height was lower (P = 0.03) at wk 2 in pigs on L (356 vs 427 μm) but not different at wk 4 (527 vs 492 μm). There was no difference in crypt depth but villus height:crypt depth ratio increased (P = 0.04) from wk 2 to wk 4 (1.7 vs 2.2) in pigs on L and did not change in pigs on H (2.3 vs 2.5). Pigs on H tended to have a greater (P < 0.17) proportion of proliferating cells in the jejunum at wk 2 and 4. In wk 2, digesta amine concentration was higher (P = 0.06) in pigs on L. Diet quality did not impact gut morphology or cell proliferation at the distal ileum. Low quality protein sources reduced overall growth performance in weanling pigs but the negative effect of protein quality on gut morphology was transient. Recovery in feed utilization in pigs on L was likely due to rapid recovery of intestinal function.
Effect of Dabigatran and Rivaroxaban on thrombomodulin mediated activation of protein C and thrombin activated fibrinolysis inhibitor (TAFI)
Authors: Fareed Jawed , Hoppensteadt Debra , Cunanan Josephine , Lewis Bruce
Published: June 2021

Introduction

The new oral anticoagulant agents Dabigatran and Rivaroxaban target thrombin and factor Xa to mediate their anticoagulant effects perspectively. This study was designed to investigate the effects of active form Dabigatran and Rivaroxaban on the activation process of protein C and TAFI by thrombin‐thrombomodulin complex.

Materials and Methods

The active form of Dabigatran was synthesized. While Rivaroxaban was extracted from commercially available tablets. Both agents were dissolved in appropriate solution matrices at a stock concentration of 100ug/ml. Thrombin‐thrombomodulin mediated activation of protein C and TAFI were measured using specific chromogenic substrate based methods at a concentration of 0–10ug/ml in different matrices.

Results

Dabigatran produced a strong inhibition of the generation of both the activated protein C and TAFI (IC50<1.0ug/ml) whereas Rivaroxaban did not produce any inhibition of the activation of either of these proteases. Dabigatran also inhibited the amidolytic actions of thrombin‐thrombomodulin complex whereas Rivaroxaban did not produce any effect. Neither Dabigatran nor Rivaroxaban produced a direct inhibition of activated protein C or TAFI at concentrations of up to 10ug/ml.

Conclusions

The persistent inhibition of thrombin and its regulatory effects by Dabigatran may differentiate its pharmacologic profile from Rivaroxaban.
High Affinity Binding of Dynorphin A‐(2–13) at the Bradykinin‐2 Receptor
Authors: Hall Sara Michelle , Rankin David R. , Cai Alice , Hruby Victor J. , Lee Yeon Sun , Lai Josephine
Published: June 2021

Experimental evidence has shown that dynorphin A can interact with bradykinin receptors to activate calcium influx through a non‐opioid mechanism. The apparent affinity and potency of the non‐opioid fragment, dynorphin A‐(2–13), at the bradykinin‐2 receptor (B2R) were moderate (Ki ~ 1 μM and EC50 ~ 500 nM). This suggests that the interaction of dynorphin A and its proteolytic fragments with the B2R may be physiologically relevant only during up regulation of endogenous dynorphin A. Here we report that dynorphin A's interaction with the B2R is surprisingly sensitive to pH within a narrow physiological range.

Competitive binding against [3H]kallidin showed the highest affinity of dynorphin A‐(2–13) at pH 6.8 (5 fold, and 100 fold higher than that at pH 7.4 and pH > 7.4, respectively). The pH sensitivity likely contributed to an initial underestimation of dynorphin's affinity at the receptor. Also the distinct structure‐activity relationship of dynorphin A analogs suggest that dynorphin A can interact with bradykinin receptors specifically and with high affinity. Its higher affinity for B2R at lower pH may have relevance in chronic inflammatory pain.

This study is supported by NIH grant P01 DA 006248.
Arginase 1 as a Therapeutic Target in Diabetic Retinopathy
Authors: Elms Shawn Christopher , Flores-Toque Haroldo , Caldwell R William , Caldwell Ruth
Published: June 2021

Objective

Ischemic injury is a prominent feature of diabetic retinopathy (DR), but the underlying molecular mechanisms are not understood. In healthy individuals, nitric oxide (NO) produced by NO synthase (NOS) maintains normal blood flow. The urea cycle enzyme arginase (Arg) can limit NO formation by competing for L‐arginine, a substrate for both enzymes. Our objective is to investigate the role of Arg in diabetes‐induced retinal vascular dysfunction.

Method

Using a novel mouse funduscope capable of imaging the retinal microcirculation, we examined the role of Arg expression/activity in STZ diabetes‐ induced retinal vascular dysfunction.

Results

Our studies showed that endothelial dependent vasorelaxation is markedly impaired in arterioles of STZ diabetic mice (60% of control) as Arg activity and expression are increased. The vascular dysfunction is largely prevented in Arg I+/− mice (85% of control) or mice treated with the specific Arg inhibitor ABH, thus indicating Arg as a mediator of diabetes‐induced decreases in endothelial dependent vasorelaxation.

Conclusion

Our data indicate that increases in Arg protein/activity are involved in diabetes‐induced retinal vascular dysfunction. Using this novel imaging technology to measure vascular function in vivo in conjunction with arginase inhibitors offers a novel methodology for pharmacological research on retinal microvascular disease.
Cathelicidin encoded Lactococcus lactis protects against Helicobacter pylori infection and inflammation in mice
Authors: Zhang Lin , Cho Chi Hin
Published: June 2021

Cathelicidin has been shown to modulate microbial growth, wound healing, and cancer development. However, its role in Helicobacter pylori (H. pylori) infection and inflammation in stomachs remains unexplored. This study sought to elucidate the actions of mouse cathelicidin (mCRAMP) in protection against H. pylori infection and inflammation in mice. Cathelicidin wild‐type (Cnlp+/+) and knockout (Cnlp−/−) mice were given orally once every two days with mCRAMP‐encoded Lactococcu lactis (L. lactis) for two weeks before H. pylori challenge in stomachs. This treatment continued for a further two months. Stomachs were then excised for assessment of H. pylori infection and inflammation. Results showed that L. lactis could harbor and secrete mCRAMP in the gastric mucosa. In the absence of cathelicidin, Cnlp−/− mice exhibited stronger H. pylori colonization together with higher inflammation score and marked tumor necrosis factor (TNF‐α) and interleukin‐6 (IL‐6) expressions in the gastric mucosa. H. pylori infection however by itself stimulated gastric epithelium‐ and neutrophil‐derived mCRAMP production in Cnlp+/+ mice. To further confirm the importance of cathelicidin on these pathological conditions, pre‐treatment with mCRAMP‐encoded L. lactis significantly increased mucosal mCRAMP level in both types of animals and reduced H. pylori infection and also TNF‐α and IL‐6 mRNA expressions. These findings indicate that the food‐grade probiotic encoded with cathelicidin protects against H. pylori infection and gastritis in vivo. (Grant support: Research Fund for Control of Infectious Diseases, Food and Health Bureau, Hong Kong)
Mechanistic and pharmacologic role of placental growth factor in experimental ulcerative colitis in rats
Authors: Deng Xiaoming , Xiong Ximing , Khomenko Tetyana , Chen Longchuan , Szabo Sandor , Sandor Zsuzsanna
Published: June 2021

We demonstrated that vascular endothelial growth factor accelerated the healing of experimental duodenal ulcer but aggravated ulcerative colitis (UC), probably because of the differences in ulcer healing. Since placental growth factor (PlGF) enhances abnormal (“pathologic”) angiogenesis, we measured its expression and role in experimental UC. Groups of rats were given iodoacetamide (IA) to cause UC and they were euthanized at 0.5, 2, 6, 12, 24, 48, and 84 hr after IA. Additional groups of rats were given neutralizing anti‐PlGF antibody, IgG (50ug/rat, i.m.) or saline on 2nd and 5th days and euthanized 7th day after IA when the extent of UC was evaluated macroscopically and by microscopy. Mucosal scrapings of distal colon were obtained at autopsy. Expression of PlGF was measured by Western blotting.

Results

Expression of PlGF protein was enhanced at 2 hr, with further increases (+50–80%) until 7 days. Inhibition of PlGF significantly reduced the levels of colonic PlGF, i.e., 2‐ to 3‐folds compared to control. Colonic lesions were also decreased from 323.2±44.1 (controls) to 209.8±31.2 mm2 (anti‐PlGF) (p < 0.05).

Conclusions

1) The sustained increase in expression of PlGF in UC may indicate an abnormal (pathologic) angiogenesis occurring in the development of UC. 2) Inhibition of PlGF attenuated experimental UC development. 3) Thus, PlGF may play a pathogenic & therapeutic role in the development & healing of UC.

(This work was supported by the Veterans Health Administration Merit Review Grants to Zs. Sandor and S. Szabo)
CGRP released from perivascular sensory nerves down‐regulates NO signaling through activation of pannexin‐based hemichannels in mesenteric resistance arteries
Authors: Gaete Pablo Simón , Lillo Mauricio , Pérez Francisco , Figueroa Xavier
Published: June 2021

It is thought that sensory nerve fibers contribute to the control of vasomotor tone by the release of calcitonin gene‐related peptide (CGRP). We stimulated the isolated rat arterial mesenteric bed with 1μM capsaicin or 100nM CGRP for 20 min and assessed the changes in nitric oxide (NO) signaling and hemichannel activity. Hemichannel activity was evaluated by ethidium or Lucifer Yellow (LY) uptake, expression and phosphorylation of eNOS by Western blot and NO‐dependent vasodilation with acetylcholine (ACh). Mesenteric arteries did not show detectable hemichannel activity in control conditions, but stimulation with either capsaicin or CGRP induced a robust ethidium or LY uptake that was maintained for >1 hr and was inhibited by probenecid, a pannexin hemichannel blocker. Capsaicin treatment induced a decrease in eNOS expression and eNOS phosphorylation at serine 1177 that reached significance 1–3 hrs after stimulation. Accordingly, the NO‐dependent ACh‐induced vasodilation was abolished 1 hr after capsaicin application, which was prevented by treatment with probenecid. These results suggest that CGRP release from capsaicin‐sensitive sensory nerves induces a prolonged activation of pannexin‐based hemichannels in endothelial cells, which leads to a reduction in NO signaling in mesenteric resistance arteries.

FONDECYT 1100850, FONDECYT 1090757, FONDECYT 1111033, Anillos ACT‐71
Agmatine‐mediated Arterial Relaxation is impaired in Salt‐Sensitive Hypertension
Authors: Gadkari Tushar , Cortes Natalie , Tsoukias Nikolaos , Joshi Mahesh
Published: June 2021

Background

We have shown that agmatine may initiate signal transduction by NO synthesis via α‐2 AR binding. Since NO activity is compromised in hypertension, we investigate whether agmatine mediated relaxation is perturbed in hypertension.

Methods

Six week old Dahl rats separated in two diet groups (0.49% NaCl and 4% NaCl) developed hypertension after 11 weeks on high salt. Isolated, cannulated, pressurized 2nd order mesenteric arterioles were preconstricted to norepinephrine in a vessel chamber at 37°C. Vascular reactivity to agmatine was tracked in real time on inverted microscope. mRNA was isolated and RT‐PCR expression of α‐2A‐AR, α‐2B‐AR and eNOS was carried out.

Results and Conclusion

In normotensive Dahl rats, the vessel in response to agmatine attaining a maximal relaxation of 90.41±1.69 % with an EC50 of 107.69±13.54 μM (n=3). However, in hypertensive Dahl rats, these relaxations were significantly compromised with maximal relaxation of 19.76±2.44 % (n=3) and EC50 was undeterminable. In RT‐PCR, the mRNA levels of eNOS, α‐2A‐AR, and α‐2B‐AR were significantly lower in high‐salt samples. Agmatine‐initiated relaxation is compromised in hypertension. This work was supported in part by NIH grant SC1HL095101

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Shedding of the Endothelial Glycocalyx and the Resistance to Flow in Capillaries
Authors: Lipowsky Herbert H , Gao Lujia , Lescanic Anne
Published: June 2021

The endothelial glycocalyx serves as a barrier to transvascular exchange of fluid, macromolecules, and leukocyte‐endothelium (EC) adhesion during the inflammatory process. Shedding of glycosaminoglycans and proteoglycans of the glycocalyx occur in response to several agonists. To elucidate the hemodynamic effects of glycan shedding on capillary resistance to flow during inflammation, intestinal mesentery (rat) was superfused with 10−7 M fMLP. Shedding was quantified by reductions of fluorescently labeled lectin (BS‐1) bound to the EC and reductions in thickness of the barrier to infiltration of 70 kDa dextran (Dx70) on the EC surface. Red cell velocities (two‐slit technique), pressure drops (dual servo‐null method), and capillary hematocrit (direct cell counting) were measured in parallel experiments. Glycocalyx thickness decreased by 80 nm. Capillary resistance rose 18% due to diminished deformability of activated WBCs and fell significantly 26% over a 30 min period, as glycans were shed from the EC surface to increase effective capillary diameter while capillary hematocrit and anatomical diameter remained invariant. This decrease in capillary resistance mitigated the increase in resistance due to diminished WBC deformability and hence these concurrent rheological events may be of equal importance in affecting capillary flow during the inflammatory process.

Supported by R01‐HL39286‐20.
The effect of systemic anaphylaxis on mesenteric lymph flow in anesthetized rats
Authors: Shibamoto Toshishige , Zhang Wei , Kuda Yuhichi , Kurata Yasutaka
Published: June 2021

The anaphylaxis‐induced fluid extravasation in splanchnic vascular bed was determined by measuring the mesenteric lymph flow (Qlym) during anaphylactic shock in anesthetized rats. Blood pressure (BP), portal venous pressure (PVP), and Qlym were measured. An injection of the ovalbumin antigen into the sensitized rats (n=10) caused a decrease in BP from 125 to 37 mmHg at 10 min, while an increase in PVP by 21 cm H2O at 2 min. Qlym increased 3.3‐fold baseline at 2 min, followed by a return to the baseline within 12 min. The lymph protein concentrations increased after antigen, which indicated increased vascular permeability. In the non‐sensitized rats (n=10), PVP was mechanically increased in a manner similar to that of the antigen‐challenged rats. Unexpectedly, this PVP elevation caused a similar increase in Qlym, but the lymph protein concentration decreased. In separate sensitized rats (n=5), the mesenteric arterial flow was much decreased after antigen, as compared with the PVP elevated rats (n=5), suggesting reduced splanchnic vascular surface area during anaphylaxis. In conclusion, systemic anaphylaxis in rats causes an increase in Qlym by increased vascular hydrostatic pressure due to portal hypertension, and by increased vascular permeability. However, this increased extravasation is relatively small probably due to decreased vascular surface area, and is observed transiently only soon after antigen.
ER stress induction increases NADPH oxidase and reduces eNOS activity in endothelial cells
Authors: Galán Maria , Kassan Modar , Alkhafaf Qassim , Trebak Mohamed , Matrougui Khalid
Published: June 2021

Cardiovascular diseases are associated with endoplasmic reticulum (ER) stress. We have demonstrated that induction of ER stress in C57BL/6J, by the injection of tunicamycin, impairs vascular endothelium‐dependent relaxation (unpublished data).

In this study, we aimed to determine the mechanism by which ER stress reduces nitric oxide synthase activity.

We incubated mice primary coronary arterioles endothelial cells with tunicamycin (1 ug/mL) for 6 h in the presence or absence of two ER stress inhibitors (Tudca, 500 ug/mL and PBA, 10 mM). Tunycamicin increased the expression of phosphorylated PERK and eIF2alpha, CHOP and ATF6, which were reduced with ER stress inhibitors. The induction of ER stress enhanced NADPH oxidase activity and Nox1/Nox4 mRNA levels associated with an increase of reactive oxygen species, which were reduced by ER stress inhibitors. Phosphorylated eNOS expression was reduced with tunicamycin and rescued by ER stress inhibition. The transfection of endothelial cells with a reporter plasmid containing the eNOS promoter revealed that ER stress induction reduced eNOS promoter activity by 50%, which was rescued by ER stress inhibition.

Conclusion

ER stress enhances oxidative stress and reduces eNOS promoter activity and phosphorylated eNOS, which are responsible for endothelial dysfunction.
Endoplasmic reticulum stress induces sarco/endoplasmic reticulum calcium ATPase and alters calcium homeostasis in the vasculature
Authors: Spitler Kathryn M. , Giachini Fernanda R. , Webb R. Clinton
Published: June 2021

Calcium homeostasis is tightly regulated in the vasculature. The sarco/endoplasmic reticulum (ER) Ca2+‐ATPase (SERCA) is responsible for maintaining a relatively low cytosolic Ca2+ concentration. The ER is a multifunctional organelle and is easily perturbed resulting in ER stress. When ER stress occurs, the ER initiates a cellular signaling network, the unfolded protein response (UPR) to alleviate this stress. We tested the hypothesis that during ER stress increased SERCA function would lead to increased Ca2+‐induced contraction and elevated ER Ca2+ stores in the aorta of male mice. Male CD‐1 mice were treated with tunicamycin (TM, 100mg/kg, 8hrs, i.p.), an inducer of ER stress or vehicle (saline, 125ul, 8hrs, i.p.). TM treatment significantly upregulated aortic SERCA2b expression. Using a wire myograph, aortic rings of TM treated mice had an increased Ca2+‐induced contraction when compared to control (99.7%±2.6% vs. 69.4%±2.7%) in the presence of thapsigargin (TG, 1μM), an inhibitor of SERCA. Caffeine (10mM) induced depletion of intracellular Ca2+ stores measured as a transient contraction, was greater in aortic rings from TM treated mice (62.1%±5.1% vs. 44.1%±3.9%) compared to controls which was completely abolished in the presence of TG (6.8%±1.7% vs 7.4%±1.1%). Therefore augmented SERCA activity following ER stress may be a mechanisms altering Ca2+ homeostasis in the vasculature.
Higher aortic wave reflection is mediated in part by greater autonomic support in older women
Authors: Kluck Breann Nicole , Barnes Jill N , Harvey Ronee E , Curry Timothy B , Hart Emma C , Charkoudian Nisha , Joyner Michael J , Casey Darren P
Published: June 2021

Aortic wave reflection increases with age, and is influenced by numerous factors, including sympathetic nerve activity. The purpose of this study was to examine the effect of autonomic blockade using trimethaphan camsylate on indices of aortic wave reflection in young (23±4 yr) and older postmenopausal (57±3 yr) women and determine if aging alters these interactions. Arterial tonometry was used to non‐invasively measure arterial waveforms from the radial artery before and during trimethaphan infusion. Aortic augmentation index (AIx), augmented pressure, and left ventricular wasted energy (Ew) were higher in older women compared to young women at baseline (p<0.05). Mean arterial pressure was similar between young (93±5 mmHg) and older women (91±2 mmHg) at baseline. AIx (Δ −12±5%), augmented pressure (Δ −11±4 mmHg), and Ew (Δ −2364±1020 dyne·cm2·sec) decreased after autonomic blockade in older women (p<0.05 for all). In addition, the age‐related differences in AIx and aortic wave reflection persisted during autonomic blockade (p<0.05). In conclusion, autonomic blockade reduces augmentation index and aortic wave reflection in older women. Our results suggest that sympathetic activity contributes to the higher aortic wave reflection observed in older women.
Involvement of the Endothelial Nitric Oxide Pathway and Leukocyte Infiltration in Secondhand Smoke Exposure‐Induced Vascular Endothelial Dysfunction and Hypertension
Authors: El-Mahdy Mohamed A , Abdelghany Tamer M , Hemann Craig , Varadharaj Saradhadevi , Esmat Ahmed , El-Sherbiny Gamal A , Zweier Jay L
Published: June 2021

Loss of nitric oxide (NO) production due to dysfunction of endothelial NO synthase (eNOS) occurs in a wide range of cardiovascular disease and has been linked to secondhand smoke (SHS)‐induced vascular endothelial dysfunction (VED). Our aim was to investigate the mechanisms of SHS‐induced VED. C57BL/6 mice were exposed for up to 48 weeks to SHS generated from 3R4F reference research cigarettes using the Teague smoking machine. SHS‐exposed (SHSE) mice showed persistent hypertension and impairment of acetylcholine‐induced endothelium‐dependent relaxation of thoracic aorta. Expression of NAD(P)H oxidase subunits p22phox and gp91phox increased in aortas of SHSE mice with concomitant superoxide production. H&E stained aortic sections showed evidence of leukocyte infiltration in SHSE mice. Plasma of SHSE mice showed significant depletion of tetrahydrobiopterin (BH4). Western blotting and immunohistochemistry showed a decrease in eNOS expression in SHSE mice aortas. Also, phosphorylation of eNOS and Aktwas decreased in SHSE mice. In conclusion, SHS induces leukocyte infiltration that triggers NAD(P)H oxidase over‐expression and ROS generation which leads to depletion of BH4 and eNOS uncoupling, resulting in VED and hypertension. Overall, our study provides important insights toward understanding contributions of SHS exposure to the genesis of cardiovascular disease. (NIH # HL38324 to JLZ)
ENaC Activity is Increased By Collecting Duct‐Specific Endothelin B Receptor Knockout
Authors: Bugaj Vladislav , Mironova Elena , Kohan Donald , Stockand James
Published: June 2021

Collecting duct (CD)‐derived endothelin‐1 (ET‐1) acting via endothelin B (ETB) receptors favors Na+ excretion. Compromise of ET‐1 signaling or ETB receptors in the CD cause Na+ retention and increase blood pressure. Activity of ENaC is limiting for Na+ reabsorption in the CD. To test for ETB receptor regulation of ENaC, we combined patch clamp method with CD‐specific knockout of endothelin receptors. We tested how ET‐1 signaling via specific ET receptors influences ENaC activity under differing dietary Na+ regimens. ET‐1 significantly decreased ENaC activity in CD isolated from wt and CD ETA KO mice but not CD ETB KO and CD ETA/B KO mice. ENaC activity was inversely related to dietary Na+ intake. ENaC activity in CD ETB KO and CD ETA/B KO mice tended to be elevated under all dietary Na+ regimens compared to wt and CD ETA KO mice, reaching significance with high (2%) Na+‐feeding. In CD ETB KO and ETA/B KO mice ENaC activity is less responsive to changes in dietary Na+ than wt being inappropriately elevated in the presence of high Na+. These results show that the bulk of ET‐1 inhibition of ENaC activity is mediated by the ETB receptor. In addition, they could explain the Na+ retention and elevated blood pressure observed in CD ET‐1 KO, CD ETB KO and CD ETA/B KO mice consistent with ENaC regulation by ET‐1 via ETB receptors contributing to the antihypertensive and natriuretic effects of the local endothelin system in the mammalian CD.
Transcriptional regulation of renal NCX1 by IFNγ in colitis
Authors: Radhakrishnan Vijayababu M , Thurston Robert D , Larmonier Claire B , Fritz Anne , Ramalingam Rajalakshmy , Kiela Pawel R , Ghishan Fayez K
Published: June 2021

NCX1 is a Na+/Ca2+ exchanger which is believed to provide a key route for basolateral Ca2+ efflux in the renal epithelia, thus contributing to renal Ca2+ reabsorption. Altered mineral homeostasis, including intestinal and renal Ca2+ transport may represent a significant component of the pathophysiology of bone mineral density loss associated with Inflammatory Bowel Diseases (IBD). The objective of our research was to investigate the effects of acute (TNBS) and chronic colitis (adoptive transfer of CD4+IL10−/− T cells) and related inflammatory mediators on renal NCX1 expression. We tested the effects of selected cytokines (TNF, IFNγ and IL‐1β) on NCX1 expression and gene promoter activity in vitro using mIMCD3 cells. Colitis was associated with increased fractional Ca2+ excretion and with decreased renal NCX1 expression, as examined by real‐time RT‐PCR, Western blotting, and immunofluorescence. In mIMCD3 cells, IFNγ significantly reduced NCX1 mRNA and protein expression. Similar effects were observed in cells transiently transfected with a reporter construct bearing ‐350 bp fragment of kidney‐specific NCX1 gene promoter. These results strongly suggest an impaired renal Ca2+ handling in experimental colitis. Negative regulation of NCX1‐ mediated renal Ca2+ absorption by IFNγ may significantly contribute to the altered Ca2+ homeostasis in IBD patients and to IBD‐associated loss of bone mineral density.
Physiological aspects of renal K+:Cl− cotransporters (KCCs) regulation
Authors: MELO ZESERGIO CARLO , Vazquez Norma , Gamba Gerardo , Mercado Adriana
Published: June 2021

Little is known about the role of KCCs in renal physiology. KCCs are inactive in isotonic conditions and become active in hypotonicity, associated with dephosphorylation of the cotransporters in threonines residues 991 and 1048 of KCC3. Using Xenopus laevis oocytes we evaluated the functional properties of wild type KCC3 or KCC4 (WT) and double mutants KCC3 or KCC4 T991A/T1048A (TA2). In contrast to WT, the mutants TA2 exhibits constitutive activity under isotonic conditions. However, similar to WT, TA2s were further activated by hypotonicity and this activation was prevented by WNK3. In addition, the catalytically inactive WNK3 that activates WT in isotonicity also further increases the activity of TA2. These data suggest that besides T991/T1048 additional dephosphorylation sites are required for full activation of KCC3 or KCC4 by hypotonicity. On the other hand, we assessed the expression of KCC3 and KCC4 at the protein level in Wistar rat models of metabolic acidosis or streptozotocin‐induced diabetes mellitus. KCC4 expression is increased by metabolic acidosis in renal medulla, while KCC3 expression is increased during diabetes mellitus in renal cortex. These observations support a role for KCC4 in acid secretion by intercalated cells in which its expression has been confirmed, and for KCC3 in the response of proximal tubule cells to an increase in glucose concentration in the ultrafiltrate.
WNK4 is required for the Angiotensin II activation of NCC in vivo
Authors: Castaneda-Bueno Maria , Cervantes-Perez Luz Graciela , Vazquez Norma Hilda , Bobadilla Norma , Alessi Dario , Gamba Gerardo
Published: June 2021

In Xenopus laevis oocytes and mpkDCT cells, WNK4 is required for modulation of NCC activity by angiotensin II (AngII). Additionally, in rats AngII promotes NCC phosphorylation independently of aldosterone. To define the requirement of WNK4 in AngII regulation of NCC in vivo, we used a complete WNK4‐knockout (WNK4−/−) mouse strain generated by targeted disruption in Pfizer Inc. Control littermates and WNK4−/− mice of 14 weeks were studied. WNK4 mRNA and protein expression were absent in WNK4−/− mice, which exhibited a mild Gitelman's like syndrome, with normal blood pressure, together with increased plasma renin activity, and reduction of NCC expression and phosphorylation at T‐58. Immunohistochemistry revealed normal morphology of distal convoluted tubule. Low salt diet or AngII infusion for four days induced SPAK and NCC phosphorylation at S‐383 and T‐58, respectively, in control littermates, but not in WNK4−/−. Low K+ diet aggravated hypokalemia, but induced SPAK phosphorylation in WNK4−/− mice. Thus, absence of WNK4 in vivo precludes the NCC and SPAK phosphorylation promoted by low salt diet or AngII infusion, but not by hypokalemia, suggesting that AngII action on NCC occurs by signaling through a WNK4‐SPAK dependent mechanism.
Role of SPAK in Mediating Regulation of NCC in the Kidney in Response to Low K Diet
Authors: Liu Jie , Coleman Richard A. , Grimm P. Richard , Delpire Eric , Welling Paul A. , Wade James B.
Published: June 2021

The NaCl cotransporter (NCC) is stimulated by low K diet by an unknown mechanism. Since recent work has shown that the STE20/SPS‐1‐related proline‐alanine‐rich protein kinase (SPAK) can function to stimulate NCC by phosphorylation of specific N‐terminal sites, we investigated if the NCC response to low K diet is mediated by SPAK. Using phospho‐specific antibodies in Western blot and immunolocalization studies of WT and SPAK−/− mice fed a low K or control diet for 4 days, we found that Low K diet more than doubled total NCC expression and phosphorylation of NCC at the T58 site. This was associated with an increase in total SPAK expression in cortical homogenates and an increase in phosphorylation of SPAK at the T383 activation site. Changes in NCC, pNCC and pSPAK in response to low K diet were absent in SPAK knock‐out mice. These findings show that SPAK is a critical mediator of increased NCC expression and activation by phosphorylation in the DCT in response to low K diet. Supported by grants from the National Institutes of Health (1RC1DK086817 to PAW, DK32839 to JBW, GM074771 to ED and 5T32HL072751 to PRG).