bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for a Protective Role for Receptor Activity Modifying Protein‐1 (RAMP1) in Angiotensin II‐Induced Endothelial Dysfunction
Authors: Chrissobolis Sophocles , Zhang Zhongming , Kinzenbaw Dale A. , Russo Andrew F. , Faraci Frank M.
Published: June 2021

Angiotensin II (Ang II) plays a major role in hypertension and causes vascular oxidative stress and endothelial dysfunction. Calcitonin gene‐related peptide (CGRP), a potent vasodilator, reportedly has antioxidant properties in vascular cells in culture. CGRP receptors consist of a calcitonin‐like receptor and a receptor activity modifying protein‐1 (RAMP1). The aim of this study was to test if RAMP1 overexpression protects against Ang II‐induced endothelial dysfunction. Transgenic mice overexpressing human RAMP1 (hRAMP1) in most tissues (including blood vessels) were generated using a RAMP1 transgene under control of the CX1 promoter. Responses of carotid artery were examined in vitro following 22 hour incubation with vehicle or Ang II (10 nmol/L). Maximum relaxation to the endothelium‐dependent vasodilator acetylcholine (ACh) was impaired by ~40% in Ang II‐treated vs vehicle‐treated vessels from control mice (P<0.05). In contrast, in hRAMP1 mice, maximum relaxation to ACh was similar in vehicle and Ang II‐treated vessels (P>0.05), suggesting RAMP1 overexpression protects against Ang II‐induced endothelial dysfunction. Responses to nitroprusside were similar in both groups of mice and unaffected by Ang II treatment (P>0.05). RAMP1 may represent a therapeutic target to protect endothelial function in conditions where Ang II plays a major role, including hypertension.
Disruption of the sEH Gene Does Not Prevent Hypoxia‐Induced Right Ventricular Hypertrophy in Mice
Authors: Zhou Dan , Akiyama Taro , Sharma Neelam , Berger Joel , Alonso‐Galicia Maggie , Walker Matthew , Cook Lynn , Hargreaves Richard , Liu Haiying
Published: June 2021

It is reported that disruption of the sEH gene in mice decreases blood pressure and protects against ischemia/reperfusion injury in the brain and heart. Others found that sEH disruption reduced survival due to cardiac arrest in mice. We tested the hypothesis that disruption of sEH in mice protects against the development of hypoxia‐induced right ventricular hypertrophy (RVH). Cardiac magnetic resonance (CMR) imaging was conducted on wild‐type (WT) and sEH knockout (KO) mice housed under the hypoxia (10% O2) condition for 5 weeks. Serial short‐axis MR images of heart from apex to base covering both right (RV) and left ventricles (LV) were captured in real time. From these images, RV and LV end diastole volumes, ejection fraction and wall thickness were determined. Following 5 wks of hypoxia, WT mice developed hypoxia‐induced RVH characterized by enlarged RV volume, increased wall thickness and reduced ejection fraction. The KO mice also developed RVH under the same conditions, similar cardiac structural and functional changes as that of WT mice. Exposure to hypoxia for 5 wks had no impact on LV structure and function in WT mice. However, 7 out of 9 KO mice survived hypoxia while all WT mice survived. Among the 7, 2 KO mice showed enlarged LV end‐diastole volume accompanied by reduced ejection fraction. In summary, our data revealed that sEH deficiency is not cardio‐protective when mice are challenged with hypoxia.
Chronic cigarette smoke exposure causes hypertension, leukocyte‐specific reactive oxygen species (ROS) generation, endothelial dysfunction, and cardiac hypertrophy in mice
Authors: Talukder M.A. Hassan , Johnson Wesley , Varadharaj Saradhadevi , Lian Jiarui , Kearns Patrick , Druhan Lawrence , Liu Xiaoping , Zweier Jay
Published: June 2021

Cigarette smoking is a major risk factor for atherosclerotic cardiovascular diseases. While the association between smoking and atherosclerosis is well‐established, the underlying mechanisms are incompletely understood. Here, we report an in vivo mouse model of smoking‐induced cardiovascular disorders. Male C57BL/6 mice (8‐weeks) were exposed to whole body mainstream cigarette smoke (CS) by the SCIREQ "InExpose" smoking system (60 min/day, 5 days/week) for 8, 16, or 32 weeks. Mice were sacrificed 24‐hours after the last CS exposure. Blood pressure was measured and cardiac MRI was performed. In vitro aortic ring and isolated heart were used for vascular and cardiac function, respectively. Blood from smoker (SM) and nonsmoker (NSM) mice were investigated for nitric oxide (NO) decay rate and ROS generation. Compared to NSM mice at 32‐weeks of CS exposure (n = 4‐8): SM mice had significant weight loss and hypertension. Acetylcholine‐induced vasorelaxation was markedly shifted to the right and downwards; left ventricular (LV) mass and LV end diastolic volume were significantly larger with increased heart to body‐weight ratio. In the blood of SM mice, NO decay was increased by 15% and white blood cells demonstrated significantly higher ROS. Thus, the present in vivo mouse model provides evidence that CS exposure induces leukocyte activation, NO and endothelial dysfunction, hypertension, and cardiac hypertrophy.
Brief ACE Inhibition Produces Persistent Changes That Protect Heart but Not Kidney From L‐NAME Induced Damage
Authors: Hale Taben Mary , Robertson Susan , Burns Kevin D , deBlois Denis
Published: June 2021

ACE inhibitors reduce LV mass and cardiovascular‐renal fibrosis. The present study investigates whether brief ACE inhibition in SHR provides long‐term protection to LV and kidney from L‐NAME‐induced damage.

Adult SHR received the ACE inhibitor enalapril or tap water. One set of control (C2) and treated (E2) were sacrificed after 2 wks. The second set of control (C2+2) or treated (E2+2) SHR were sacrificed following a 2‐week washout and the final set of control (CL) and previously treated (EL) rats received L‐NAME (10 days) after washout period. At sacrifice, kidney and LV were weighed and formalin fixed.

LV mass was significantly reduced by enalapril and this difference between control and treated persisted throughout the study. In response to L‐NAME, the total number of proliferating cells, interstitial fibrosis, number of macrophages was decreased in the LV of SHR previously treated with enalapril. In contrast there was no difference in the glomerular size or degree of glomerulosclerosis.

The present study suggests that brief ACE inhibition may confer some protection against heart but not kidney disease, even in the absence of continued treatment.
Low dietary sodium intake is associated with enhanced vascular endothelial function in older adults with elevated baseline systolic blood pressure
Authors: Jablonski Kristen L , Gates Phillip E , Pierce Gary L , Seals Douglas R
Published: June 2021

Aging and elevated systolic blood pressure (SBP) are associated with impaired endothelium‐dependent dilation (EDD) and increased salt sensitivity. High dietary sodium impairs EDD in young rodents. We hypothesized that otherwise healthy middle‐aged and older adults (MA/O) with elevated systolic blood pressure (SBP; 130‐159 mmHg) consuming lower dietary sodium (DS) would demonstrate enhanced EDD compared with peers consuming higher DS. EDD was measured using flow‐mediated dilation (FMD) in MA/O (65±1 yr, mean±S.E.) who self‐reported lower (n=12; 73±6 mmol/d) or higher (n=13, 144±6 mmol/d) DS intake. Subjects were matched for potentially confounding characteristics including age, body mass, body mass index, SBP, diastolic BP, plasma lipids, fasting glucose, daily activity, peak oxygen uptake, and non‐DS related dietary profile. Brachial artery FMD was 34% higher (5.0±0.5 vs. 3.3±0.5 %, p<0.05) in subjects consuming low sodium, whereas endothelium‐independent dilation (brachial dilation to sublingual nitroglycerine) did not differ between groups (p=0.24). In the overall sample, FMD was inversely related to DS intake (r=‐0.53; p<0.01). These results suggest that DS intake may modulate EDD in M/O adults with elevated SBP. DS restriction may be an effective intervention to preserve vascular endothelial function with aging.

NIH AG013038, AG022241, AG006537, AG000279
Modelling volume‐loading hypertension with Guyton's model: role of whole body blood flow autoregulation
Authors: Montani Jean‐Pierre , Adair Thomas H. , Van Vliet Bruce N.
Published: June 2021

Computer simulations offer a unique way to test the role of whole body blood flow autoregulation (WBFAR) in the pathogenesis of volume‐loading hypertension since it is not possible to block WBFAR experimentally. Using Guyton's large circulatory model, we analyzed, in the presence or absence of WBFAR, the hemodynamic and fluid volume changes in three classical models of volume‐loading hypertension. In the first model, hypertension was induced by reducing renal mass to 30% of normal and increasing salt intake by 6‐fold. In the presence of WBFAR, the development of hypertension (+30 mmHg) was characterized by an initial increase in cardiac output (CO) by ~30% followed by a secondary increase in vascular resistance (+25%). In the absence of WBFAR, there was a similar long‐term increase in blood pressure (BP). However, a nearly 2‐fold increase in CO and an increase in extracellular fluid volume by more than 30% were observed whereas vascular resistance decreased by 30% owing to the mechanical distension of blood vessels by the increased BP. Qualitatively similar results were obtained in two other models of volume‐loading hypertension: aldosterone infusion and Goldblatt 1 kidney ‐ 1 clip hypertension. From the simulations one may conclude that autoregulation limits the amount of fluid retention required to increase BP in order to achieve salt balance, but does not affect the final level of BP. (Supported by HL‐51971)
Repeated taste exposure increases preferences for vegetables in elementary school aged children
Authors: Lakkakula Anantha , Zanovec Michael , Mann Nadine , Murphy Ellen , Pierce Sarah , Tuuri Georgianna
Published: June 2021

Children's food preferences play a major role in their food choices and consumption. This study aimed to increase children's preferences for fruit and vegetables by offering repeated opportunities to taste less liked vegetables. A total of 370 fourth and fifth grade students (44% boys; 61% fourth graders) attending low‐income, public elementary schools in southeastern Louisiana volunteered to participate. Once a week for 10 weeks children were given a taste of carrots, peas, tomatoes and bell peppers, and then asked to record their preference for each vegetable using a Likert‐type response scale. The percent of children that tasted all 10 times was 26% for carrots, 20.3% for peas, 19% for tomatoes, and 18.5% for bell peppers. Repeated measures ANOVAs indicated that repeated tasting improved preferences for all four vegetables: carrots (p<0.01); peas (p<0.01); tomatoes (p<0.01); bell peppers (p<0.05). A minimum of 6 tastings were needed to observe a change in preference score. At the end of 10 opportunities to taste, the percentage of children who had tasted at least 6 times was 74% for carrots, 70% for peas, 63% for tomatoes, and 60% for bell peppers. Repeated taste exposure is a promising strategy for promoting liking of less liked vegetables in children. This project was funded by the Baton Rouge Area Foundation, Blue Cross and Blue Shield of Louisiana Foundation, and the LSU AgCenter.

Grant Funding Source

Baton Rouge Area Foundation adn Blue Cross Blue Shield of Louisiana
Inhibition of telomerase activity contributes to HIV‐1‐induced activation of inflammatory responses and alteration of tight junction protein expression in human brain endothelial cells
Authors: Huang Wen , András Ibolya E. , Eum Sung Yong , Hennig Bernhard , Toborek Michal
Published: June 2021

Telomerase reverse transcriptase (TERT) is a catalytic rate‐limiting component of telomerase. Progressive telomere shortening has been observed to contribute to cell senescence. To determine whether TERT activity can contribute to HIV‐1 induced endothelial dysfunction, senescence of human brain endothelial cells (hCMEC/D3 cell line) was induced by inhibition of telomere activity via a siRNA directed against human TERT. These cells were then exposed to HIV‐infected Jurkat cells and evaluated for the induction of inflammatory mediators and the barrier function. Inhibition of hTERT resulted in upregulation of HIV‐1 induced mRNA and protein expressions of intercellular adhesion molecule (ICAM)‐1 and vascular cell adhesion molecule (VCAM‐1). In contrast, hTERT deficiency potentiated HIV‐induced downregulation of expression of tight junction proteins such as zonula occludens (ZO)‐1, ZO‐2, and claudin‐1. These alterations were associated with elevated transendothelial migration of HIV‐infected monocytes. The present findings indicate that cell senescence induced by inhibition of hTERT in brain endothelial cells may contribute to HIV‐1 induced endothelial dysfunction of brain blood barrier. Supported by MH63022, MH072567, and NS39254.
Novel model of brain metastasis formation: the influence of polychlorinated biphenyls on blood‐brain barrier integrity and brain metastasis
Authors: Seelbach Melissa Jessica , Chen Lei , Choi Yean Jung , Wei Yinan , Layne Joseph D. , Hennig Bernhard , Toborek Michal
Published: June 2021

Anatomically situated at the level of the cerebral capillary endothelium, the blood‐brain barrier (BBB) is a highly dynamic interface comprised of a network of tight junction (TJ) proteins that seal adjacent cells of apposing endothelium. Perturbations in BBB integrity have been associated with numerous CNS disease states, including brain metastasis. Polychlorinated biphenyls (PCBs) comprise a ubiquitous class of toxic substances associated with carcinogenic and tumor promoting effects. Previously, our laboratory has demonstrated that exposure to PCBs are effective in activating endothelial cells and inducing prometastatic properties, however less is known regarding the influence of PCBs in brain cancer metastasis in vivo. In the current study we demonstrate accumulation of PCB 153, PCB 126, and PCB 118 in brain tissue as early as 48 hours post‐systemic exposure (49 mg/kg). This accumulation corresponds with changes in BBB molecular properties. Additionally, we employ our external carotid artery injection (ECI) brain metastasis model, in which luciferase‐tagged tumor cells are introduced directly to the cerebravasculature, coupled with bioluminescence imagining techniques to investigate the role of PCBs in brain metastasis. These studies are critical in understanding the neurotoxic influence of PCBs in brain metastasis. Supported by NIEHS (P42ES07380)
Peripheral leptin administration to ob/ob mice decreases the gene expression of hypothalamic mu opioid receptors
Authors: Barnes Maria J , Primeaux Stefany D , Braymer Hugh D , Bray George A
Published: June 2021

Activation of mu opioid receptors make animals hyperphagic and increase their preference for a high fat diet. We have demonstrated that the gene and protein expression of this receptor population is significantly greater in the hypothalamus of obese animals. This warrants the consideration that the increased mu opioid receptors are potentiating the hyperphagia and the preference for a high fat diet that is associated with obesity. Utilizing ob/ob mice, we investigated the hypothesis that hypothalamic mu opioid receptors are increased in obesity due to an attenuation of leptin's anorexic effect. The gene expression of hypohtalamic mu opioid receptors was significantly greater in ob/ob mice compared to the control C57/B6 mice. Ob/ob mice given ad libitum access to a high fat diet for fourteen days gained more weight, were hyperphagic, had an increase in body fat, and a significantly greater gene expression of hypothalamic mu opioid receptors when compared to their low fat controls. When leptin was administered intraperitoneal (IP) to the ob/ob animals it resulted in a significant decrease in the gene expression of hypothalamic mu opioid receptors, body weight, food intake, and body fat percentage. Taken together, these data suggest that the development of leptin resistance in obese animals positively contributes to the increased gene expression of hypothalamic mu opioid receptors. DK R34‐32089
Changes in ECM proteins, decorin and biglycan, during adipogenesis in 3T3‐L1 cells and in adipose tissue of mice on a high fat diet
Authors: Ajuwon Kolapo M , Cruz Meliza , Buhman Kimberly
Published: June 2021

Adipogenic conversion requires an extensive remodeling of the extracellular matrix (ECM) to allow for adipocyte hypertrophy, hyperplasia and neovascularization of the tissue. Although ECM remodeling is known to be important in adipogenesis, details of the mechanisms and how this process determines the overall fate of the adipocyte and its functionality are poorly understood. However, it is known that the ECM plays significant role in obesity, inflammation and insulin resistance. We have examined the expression of 2 proteoglycans, decorin and biglycan, during adipogenic conversion of 3T3‐L1 cells and in obesity. Both decorin and biglycan are highly expressed in confluent adipocytes. However, after induction of adipogenesis, the mRNA expression of both proteoglycans falls precipitously to about 10% of the level at confluence. This reduced level of expression was noticeable 2 days after adipogenic induction and was sustained until day 4, after which the expression level begins to rise. Nevertheless, the expression at day 12 remains at approximately 50% of the level at confluence. Expression of both proteoglycans was also significantly induced in the epididymal adipose tissue of mice on a high fat diet compared to those on regular chow. Because both decorin and biglycan are differentially regulated during adipogenesis, they could play significant regulatory roles in adipogenesis and obesity.
Neuropeptide AF is associated with satiety in chicks
Authors: Newmyer Brandon Anthony , Cline Mark
Published: June 2021

Neuropeptide AF (NPAF), an 18 reside peptide, is a member of the RFamide family and has structural similarity to the satiety‐inducing neuropeptides FF and VF. However, to our knowledge the appetite associated effects of NPAF are unreported. Thus, chicks were intracerebroventricularly (ICV) injected with increasing doses of NPAF and food and water intake were measured. NPAF was associated with decreased food intake during the entire 180 minute observation period and decreased water intake from 60 to180 minutes post injection. Whole blood glucose concentration was also significantly reduced after ICV NPAF injection. In experiment two, chicks that received ICV NPAF displayed altered behavior patterns; sleep time and feeding pecks were increased while perch time and number of jumps were decreased. In conclusion, NPAF appears to be a novel regulator of appetite and modulator of other behaviors.
Verapamil reduces transepithelial sodium and potassium transport in H441 monolayers
Authors: Han Dongyun , Nie Hongguang , Gu Xiu , Ji Honglong
Published: June 2021

Verapamil‐induced noncardiogenic pulmonary edema is a common clinical disorder, the pathogenetic mechanisms remain unknown. In this study, we investigated the effects of verapamil on transepithelial short‐circuit currents (Isc) in confluent H441 monolayers and alveolar fluid clearance in vivo. Intratracheal administration of verapamil (100 μM) reduced alveolar fluid clearance by 41 ± 6% (P<0.05, n=4). The Ussing chamber studies showed that apical application of verapamil reduced amiloride‐sensitive Isc in a dose‐dependent manner (Ki=26 μM). This inhibitory effect was also observed when 100 μM verapamil was added to the basolateral side of intact monolayers (from 20 ± 4 to 12 ± 2 μA/cm2, P<0.05, n=4). Further studies showed the direct inhibitory effects on transapical (from 41 ± 1 to 28 ± 3 μA/cm2, P<0.05, n=4) and transbasolateral Isc (61 ± 1 to 52 ± 2 μA/cm2, P<0.05, n=4) in permeabilized monolayers. The inhibitors of Kv (clofilium), KCa (clotrimazole), and KATP (glibenclamide) channels decreased amiloride‐sensitive Isc levels by 54 ± 5%, 32 ± 5%, and 21 ± 1%, respectively (P<0.05, n=4). In the presence of these K+ channel inhibitors, verapamil‐inhibitable Isc fraction was significantly decreased. Importantly, the openers for Kv (pyrithione), KCa (1‐EBIO), and KATP channels (minoxidil) recovered verapamil‐induced decrease in Isc. Taken together, our results for the first time demonstrate that inhibition of K+ channels may be a critical mechanism for the pathogenesis of verapamil‐induced lung edema.
Red blood cell‐induced proinflammatory lung endothelial signaling in hypoxia
Authors: Huertas Alice , Das Shonit , Lindert Jens , Yiming Maimaiti , Bhattacharya Sunita , Bhattacharya Jahar
Published: June 2021

To determine endothelial (EC) mechanisms underlying hypoxia‐induced lung inflammation, we considered that red blood cell (RBC)‐derived H2O2 diffuses to lung EC (Kiefmann. Blood. 2008). Hence, H2O2 might induce proinflammatory signaling causing EC expression of the leukocyte adhesion receptor, E‐selectin. To test this possibility, we exposed isolated mouse lungs, perfused with RBC‐containing buffer to 4 hours of normoxia (21% O2) or hypoxia (8% O2). Then using our reported methods (Yiming. AJRCMB. 2008), we infused the pulmonary artery with chilled collagenase and trypsin to obtain 104 cells/lung in the left atrial effluent. We dual labeled the cells with fluorophore‐conjugated mAbs (10μg/ml) against E‐selectin as well as the EC marker, von Willebrand factor. Finally, using flow cytometry, we determined cell fluorescence. Exposing lungs to hypoxia increased EC E‐selectin three times above the normoxic control (p<0.05; n=3). Importantly, the H2O2 hydrolyzing agent, catalase (50 U/ml) blocked the hypoxia‐induced E‐selectin enhancement (p<0.05, n=3). Moreover, in lungs perfused with RBC‐free buffer, hypoxia failed to increase EC E‐selectin above control (p<0.05, n=3). We conclude that in hypoxia, RBC‐derived H2O2 activates E‐selectin expression in lung EC. These responses might underlie initiation of lung vascular inflammation (Support: HL36024).
Effect of G‐CSF on pulmonary emphysema
Authors: Ito Hiromichi , Matsushita Shonosuke , Ishikawa Shigemi , Sakakibara Yuzuru
Published: June 2021

Purpose

G‐CSF is a cytokine to stimulate the bone marrow to produce granulocytes and stem cells. Recently it has demonstrated that G‐CSF mobilize the endothelial progenitor cell (EPC) to peripheral circulation and pluripotency of EPC for tissue repair, we investigated the effect of G‐CSF on pulmonary emphysema (PE).

Methods

Male Wistar Rat (6 wks, n=18) were divided into two groups; Control (PE; n=9) group and G‐CSF group (PE treated with G‐CSF; n=9). PE was induced by the injection of porcine elastase (200 U/kg) through trachea. G‐CSF was injected subcutaneously (100 microgram/kg/day, 12 days). Number of EPCs in peripheral blood was calculated at 13 days after initiation of G‐CSF injection by flow cytometry. Seven weeks after from injection of elastase, rats were sacrificed and evaluated by histopathology (HE and Elastica Van Gieson stain). PE was evaluated by the number of crossing alveolar wall in lattice with 100 micrometer.

Results

EPC was significantly increased by the use of G‐CSF (G‐CSF 4822 vs. Control 2814 per micro liter, p<0.05). The number of crossing alveolar wall is significantly preserved in G‐CSF treated rats compared with non‐treated rats (G‐CSF 2.660.29 vs. Control 1.660.33, p<0.0005).

Conclusion

G‐CSF preserved the alveolar structure in pulmonary emphysema significantly at least in part due to increase of endothelial progenitor cells in peripheral circulation.
Hypoxia‐induced proliferation via epidermal growth factor receptor signaling in pulmonary endothelial cells
Authors: Toby Inimary T , Cui Hongmei , Chen Bernadette , Nelin Leif D
Published: June 2021

Arginase activity is involved in cellular proliferation via ornithine production and its subsequent metabolism to proline and/or polyamines. Ornithine decarboxylase (ODC) is the rate‐controlling enzyme in polyamine biosynthesis. We have previously shown that epidermal growth factor receptor (EGFR) is important in hypoxia‐induced cellular proliferation in endothelial cells. We hypothesized that hypoxia would activate EGFR which would in turn up‐regulate arginase expression and thereby promote cellular proliferation. We studied human pulmonary microvascular endothelial cells (hPMVEC) kept in either normoxia (20% O2 , 5% CO2) or hypoxia (1% O2, 5% CO2) for 24 hours. Arginase II protein expression was ~3 fold greater in hypoxia than in normoxia, and this hypoxia‐induced increase was attenuated by inhibition of EGFR using AG1478 (30nM). Urea production was also greater in hypoxia than in normoxia, and this hypoxia‐induced increase was completely prevented by EGFR inhibition. The mRNA levels of ODC were ~2.5 fold greater in hypoxia than in normoxia, and treatment with AG1478 prevented the hypoxia‐induced increase in ODC mRNA. Pharmacological inhibition of either arginase or ODC prevented hypoxia‐induced cellular proliferation. Our data demonstrate that EGFR tyrosine kinase activity is critical for hypoxia‐induced hPMVEC proliferation via up‐regulation of arginase and ODC.
Hypoxia upregulates arginase 2 in human endothelial cells
Authors: Krotova Karina , Zharikov Sergey , Patel Jawaharlal M , Block Edward R
Published: June 2021

We studied the effects of hypoxia on arginase expression and activity in human endothelial cells (EC). We found that in human lung microvascular EC and human pulmonary artery EC only one isoform‐ arginase 2 (Arg2) ‐is expressed and hypoxia upregulates the activity as well as mRNA and protein levels of Arg 2.

In EC arginase shares and competes for the substrate L‐arginine with nitric oxide synthase (NOS). Through regulation of substrate availability for NOS arginase is able to modulate nitric oxide (NO) production. To evaluate the role of Arg 2 in regulation of NO production under hypoxia we compared NO output (RFL‐6 reporter assay) in cells with normal and silenced Arg 2. Cells treated with the arginase inhibitor BEC or with an Arg 2 siRNA produced more available NO both in normoxia and especially in hypoxia.

We sought to determine the signaling pathways involved in the upregulation of Arg 2. Since HIF is a family of transcriptional factors activated by hypoxia, we tested the possibility of involvement of HIF‐1 and HIF‐2 in regulation of Arg 2 under hypoxia. The silencing of HIF‐2 but not HIF‐1 prevented the activation of Arg 2 by hypoxia.

This work was supported by Florida DOH (KK), VA and NIH (JMP).
Inflammasome: A Pivotal Role in hyperoxia‐induced acute lung injury?
Authors: Kolliputi Narasaiah , Shaik Rahamthulla , Waxman Aaron B
Published: June 2021

The inflammasome is a novel protein complex that stimulates caspase‐1 activation to promote the processing and secretion of pro‐inflammatory cytokines. We hypothesized that hyperoxia induced K+ efflux activates inflammasome through P2X7 receptors (P2X7R) leading to inflammation in hyperoxic acute lung injury (HALI). To test this hypothesis in vitro, we isolated alveolar macrophages from murine lung tissue, exposed cells to hyperoxia (95% oxygen and 5% CO2) and characterized the expression and activation of inflammasome components. We also analyzed the expression of the same molecules in alveolar macrophages obtained from the mice exposed to hyperoxia (100 % oxygen) for 72 hours. Our results showed that hyperoxia induced a significant increase in P2X7 activation and K+ efflux that was associated with inflammasome activation and release of pro‐inflammatory cytokines. P2X7 agonist, ATP enhances hyperoxia induced inflammasome activation and P2X7 antagonist, oxidized ATP inhibits hyperoxia induced inflammasome activation. However, when ATP was scavenged with apyrase, hyperoxia induced inflammasome activation was significantly decreased indicating that the possible involvement of P2X7R. Interestingly inflammasome silence abrogates hyperoxia‐induced secretion of pro‐inflammatory cytokines in‐vitro. Our studies suggest that hyperoxia induces K+ efflux through P2X7 receptor and results in inflammasome activation and secretion of pro‐inflammatory cytokines.
Adenosine A2b receptors unexpectedly activate cardioprotective kinases through Gi rather than Gs
Authors: Kuno Atsushi , Walker Sheree , Dost Turhan , Cohen Michael V. , Downey James M.
Published: June 2021

We reported that A2b receptors in the heart couple to the survival kinases Akt and ERK and that in preconditioned heart the A2b receptor′s sensitivity to adenosine is increased by PKC so endogenous adenosine becomes protective. We explored the behavior of these receptors in a simple model of human embryonic kidney cells stably transfected with human A2b receptors. Incubation of these cells with the A2b‐potent agonist 5′‐(N‐ethylcarboxamido) adenosine (NECA) caused a dose‐dependent activation of Akt and ERK as determined by western blotting with phospho‐specific antibodies. NECA also increased cAMP consistent with the previously reported Gs‐coupling of A2b receptors. The A2b‐selective antagonist MRS1754 blocked NECA′s ability to activate the kinases confirming an A2b mechanism. Activation of PKC with phorbol 12‐myristate 13‐acetate increased the sensitivity to NECA 10‐fold as compared to cells in which PKC was blocked with chelerythrine thus confirming PKC′s effect on receptor signaling. However, when Gs was inhibited with cholera toxin or the Gs‐antagonist NF449, the ability of NECA to activate the kinases was actually enhanced. Pertussis toxin abolished the ability of either NECA or the highly selective A2b agonist BAY60‐6583 to activate the kinases indicating that the A2b receptor activates the kinases by a Gi‐rather than a Gs‐coupled mechanism. Supported by HL20648
Adherens junction protein, p120 catenin, represses transcriptional activity of endothelial cells
Authors: Lum Hazel , Cheng Feng , O'Donnell James J. , Im Hee‐jeong , Holian Oksana
Published: June 2021

The adherens junction‐associated protein, p120 catenin (p120ctn) belongs to the family of armadillo repeat containing proteins which harbor dual functions of cell‐cell adhesion and signaling to the nucleus. In endothelium, while p120ctn is most characterized for cell‐cell adhesion, virtually nothing is known of this latter function. We investigated whether p120ctn in endothelial cells has signaling function to the nucleus with use of minimal promoter reporter systems. Results show that siRNA‐mediated silencing of endogenous p120ctn increased luciferase activity 1.5‐2.0‐fold in cells transfected with reporter plasmids containing binding elements for transcription factors NFκB (pNFκB‐Luc,) or AP‐1 (pAP‐1‐Luc). The 120ctn loss also significantly increased promoter reporter activity of pro‐inflammatory genes, MMP‐1 [p(‐562)MMP‐1‐Luc]and ICAM‐1 [p(‐1393)ICAM‐1‐Luc]. The increased promoter reporter activity was accompanied by activation of ERK1/2 as detected by anti‐phospho‐ERK1/2(Thr202/Tyr204) Ab. Further, inhibition of ERK1/2 with MEK1 inhibitor (PD 98059) prevented nuclear translocation of endogenous p65 subunit of NFκB as well as the increased luciferase activity of p(‐562)MMP‐1‐Luc. In conclusion, loss of p120ctn was a potent stimulus of transcriptional activity, suggesting a novel transcription repression function by endothelial cells. The study is supported by NIH HL093715.
Production of Osteopontin and Osteocalcin by MG63 Cells In Response to Bone Morophogenetic Proteins and IGF‐1
Authors: Benghuzzi Ham A , Tucci Michelle A
Published: June 2021

Bone morphogenetic proteins (BMPs) are a group of structurally related proteins in the transforming growth factor‐beta (TGF‐beta) family which have been shown to stimulate bone formation in vivo. The hypothesis of this study was that osteoblast‐like cells (OBs) treated with BMP would have similar levels of secretion of osteopontin (OPN) and osteocalcin (OCN) to that of OBs treated with IGF‐1. Specifically, the aims of this study were to evaluate osteoblast‐like cells (MG‐63 cell line) for secretion of both OPN and OCN after treatment with DBM, OP‐1, or insulin‐like growth factor‐1 (IGF‐1) compared to control, at 24, 48, and 72 hours. After each incubation period, ELISA kits were used to determine the levels of osteopontin and osteocalcin production. The results clearly demonstrated a rise of OCN at 48 hours and a fall at 72 hours for all samples, including control groups. However, there was no significant difference between groups (p > 0.05). With the OPN assay, results showed no significant difference between groups until 72 hours (p = 0.022), where the IGF‐1 group was significantly higher than the Control and (p = 0.021, respectively). This information is important for understanding the signaling pathways that may be innervated in the osteoblast following stimulation with growth factors.
Hyperhomocysteinemia induces matrix disruption and oxidative stress in inner ear
Authors: Kundu Soumi , Tyagi Neetu , Kumar Munish , Tyagi Suresh C
Published: June 2021

Increasing number of clinical data suggests lower folic acid (FA) and high homocysteine (Hcy) causing hearing impairment. We analyzed hyperhomocysteinemia (HHcy) related matrix remodeling, oxidative stress and blood flow which may contribute to impaired hearing. We used wild type (WT) and heterozygous Cystathione beta‐synthase (CBS) mice. CBS () has higher level of plasma Hcy and exhibits some pathological features of HHcy. Expression of matrix metalloprotease (MMPs), their inhibitors, the TIMPs and nitrotyrosine (NT), p22phox and NOX‐3 were analyzed by western blotting and RT‐PCR. Masson Trichrome staining was done for collagen. Western blot analyses revealed increased level of active MMP‐2, MMP‐14 protein in CBS () mice. MMP‐9, MMP‐12 and MMP‐13 mRNA were higher in CBS () than the WT. Nitrotyrosine and p22phox was higher in CBS (), whereas NOX‐3 level showed moderate increase in CBS () as compared to WT. Staining for collagen was higher in WT as compared to CBS (). Hair cells were disorganized in CBS () along with hypertrophy as compared to WT. Our data suggested increased disruption of intercellular matrix resulting in damage and disorganized cellular structure in cochlea. Moreover an increased level of oxidative stress may also contribute to impaired hearing threshold.
The role of the Rho kinase isoforms in fibroblast contractility
Authors: Cayemberg Amy E , Wakatsuki Tetsuro
Published: June 2021

The Rho kinases, ROCK I and ROCK II, are homologous effectors of the small GTPase RhoA. Rho kinases are known to mediate contraction in non‐muscle cells such as fibroblasts. However, the specific contribution of each isoform has yet to be determined as current chemical inhibitors do not distinguish between the isoforms. To investigate whether ROCK I and ROCK II have distinct roles in fibroblast contraction, we transfected human foreskin fibroblasts (HFF) with lentiviral vectors containing shRNA targeting each isoform. We were able to reduce ROCK I protein expression in HFF cells by 65 percent, and ROCK II expression by 50 percent, as confirmed by Western blot. These transfected HFFs were then embedded in a type I collagen matrix to produce engineered tissues for quantifying cellular contractility. Tissues with reduced ROCK I expression showed a 75 percent decrease in contractile force compared to control tissues following stimulation with 10µM lysophosphatidic acid (LPA). Contractility of HFF tissues expressing decreased levels of ROCK II was comparable to control tissues. These results suggest that ROCK I may be preferentially activated by LPA in fibroblasts to cause contraction, while ROCK II may not be essential to fibroblast contractility.
Serum vitamin C and the prevalence of vitamin C deficiency in the United States: 2003‐2004 National Health and Nutrition Examination Survey (NHANES)
Authors: Schleicher Rosemary Lawless , Carroll Margaret D , Ford Earl S , Lacher David A
Published: June 2021

In NHANES III (1988‐1994), 11.6±0.8% (SE) of the US population was vitamin C deficient based on serum concentrations <11.4 ìmol/L. The distribution of serum vitamin C concentrations in the United States and the prevalence of deficiency in selected subgroups for 2003‐2004 were estimated. Total serum ascorbic acid (vitamin C) was measured in 7,277 non‐institutionalized civilians aged 6 years and older from the cross‐sectional, nationally representative NHANES 2003‐2004. The overall age‐adjusted mean from the square‐root transformed (SM) concentration was 51.4 ìmol/L (95% confidence interval: 48.4‐54.6). The highest concentrations were found in children and older persons. Within each race/ethnic group, females had higher concentrations than males (p<0.05). Smokers′ mean concentrations were one‐third lower than those of nonsmokers′(SM: 37.7 vs. 56.2 ìmol/L; p<0.001). Obese adults had serum ascorbic acid concentrations that were 14‐21% lower than those of overweight or healthy weight adults (p<0.01). The overall prevalence of age‐adjusted ascorbic acid deficiency was 7.10.9% (SE). Smokers were at risk for deficiency more than three times as often as nonsmokers. In 2003‐2004, the population weighted distribution of serum ascorbic acid concentrations in the United States showed an improvement in vitamin C status, and the prevalence of vitamin C deficiency was significantly lower than during NHANES III.
Antioxidant activity of folic acid: From mechanism of action to clinical application
Authors: Atteia Bashir Mahmoud Rezk , El‐Kak Abd El‐Aziz Atteia , Lucchesi Pamela A. , Delafontane Patrice
Published: June 2021

The presumed protective effect of folic acid (FA) on the pathogenesis of cardiovascular, hematological and neurological diseases and cancer has been associated with the antioxidant activity of FA. The actual antioxidant activity of FA depends on its consumption and reduction into its reduced forms. The more the FA is supplemented and/or reduced, the higher its antioxidant activity becomes. In rats subjected to acute cardiac volume overload, there was progressive left ventricular (LV) hypertrophy, and reduced capillary density which was prevented by FA supplementation (30 mg/kg). This effect was associated with prevention of apoptosis of endothelial cells as demonstrated by downregulation of active caspase‐3 expression. Moreover, FA prevented collagen loss and decreased both mast cell degranulation and MT1‐MMP expression. The antioxidant effect of FA was reflected in elevation of GSH/GSSG ratio and a reduction of protein nitration. Furthermore, in dyslipidemic patients with stable angina pectoris, FA supplementation (10 mg/day) for three months improved endothelial dysfunction and decreased carotid intima‐media thickness. Our results indicate that the beneficial effects of FA may in part be due to its potent antioxidant activity.
Human serum as an alternative for amniotic fluid stem cells culture media for clinical use
Authors: Duarte Sergio , Debes Adriana de Aguiar , Cavaglieri Rita de Cássia , Janz Felipe de Lara , Lopes Marco Antonio Borges , Bydlowski Sérgio Paulo , Zugaib Marcelo
Published: June 2021

Objective

to examine growth, phenotypic characteristics, gene expression and differentiation of amniotic fluid stem cells (AFSC) using human serum in culture media.

Introduction

AFSC are promising candidates for cell‐based therapies. They are obtained by prenatal screening, without raising the ethical concerns associated with human embryonic research. One major obstacle for their clinical use is the biosafety of fetal calf serum (FCS).

Methods

Amniotic fluid cells obtained from 2nd trimester amniocentesis were grown in α‐MEM+human serum20%. Attached cells were cultured and counted under microscope, investigated about the expression of Oct‐4, SOX2 and Nanog, by RT‐PCR and about phenotype by FACS‐analysis. Induced osteogenic and chondrogenic differentiation were confirmed with Alizarine Red and H&E stains respectively.

Results

AFSC showed high proliferation ratios; expressed Oct‐4, SOX2 and Nanog; stained positive for CD29, CD44, CD90, CD105 and negative for CD14, CD31, CD34, CD45, CD106 and CD133; showed osteogenic and chondrogenic potential.

Conclusions

Growth of AFSC in a FCS‐free medium is feasible. Can be isolated easily, proliferate quickly, show a mesenchymal phenotype and have osteogenic and chondrogenic potential. Moreover, Oct‐4, SOX2 and Nanog, expressed by AFSC, are associated with the pluripotency and for the maintenance of stem cells.
Acute alcohol intoxication inhibits the hematopoietic precursor cell response to Escherichia coli bacteremia
Authors: Siggins Robert Wade , Welsh David A , Bagby Gregory J , Raasch Caroline E , Happel Kyle I , Nelson Steve , Zhang Ping
Published: June 2021

Bacterial infection causes bone marrow hematopoietic activity to shift toward granulopoiesis which is a critical event for enhancing host defense. This study investigated the hematopoietic precursor cell (HPC) response to bacteremia and how alcohol affects this response. Acute alcohol intoxication was induced in Balb/c mice 30 min prior to initiation of E. coli bacteremia. Bacteremia significantly increased the number of lineage (lin)‐c‐kit+Sca‐1+ (LKS+) cells, which was suppressed by alcohol. LKS+ cells isolated from bacteremic mice had increased CFU‐GM activity compared to controls. Both enhanced proliferation of LKS+ cells as reflected by BrdU incorporation, and phenotypic inversion of lin‐c‐kit+Sca‐1‐ (LKS‐) cells accounted for the increase in LKS+ cells following bacteremia. Bacteremia increased plasma TNF‐α, which was inhibited by alcohol. Culture of LKS‐ cells with TNF‐α caused a dose‐dependent increase in conversion of these cells to LKS+ cells, which was attenuated by alcohol. Sca‐1 mRNA expression in cultured LKS‐ cells was up‐regulated following TNF‐α stimulation. E. coli clearance was impaired by alcohol, causing increased mortality in these animals. These data show that alcohol inhibits the HPC response to bacteremia which may serve as one mechanism underlying the impaired host defense in alcohol abusers with severe bacterial infections. Supported by NIH grants AA09803 and HL76100.
An estrogen metabolite, 2‐methoxyestradiol, promotes coronary artery dilation and inhibits breast cancer proliferation
Authors: Hill Brent J.F. , Schlicker Bonnie , Jordan Renee , Runge Steven W.
Published: June 2021

The cardioprotective benefit of estrogen (E2) has been well documented; however, its use in hormone replacement therapy has accentuated the development of breast cancer in some women. The purpose of this study was to compare the E2 metabolite, 2‐methoxyestradiol (2‐MeOH), with E2 on coronary arterial tone. Coronary arteries were dissected out of hearts obtained from female pigs, sectioned into 4 mm rings, and suspended in organ baths. A 60‐minute pre‐incubation using 10 [micro]M E2 and 2‐MeOH similarly inhibited a KCl and endothelin‐1‐induced contraction by 50% and 36%, respectively; thus, demonstrating inhibition of Ca2+ influx. Also, the K+ channel blocker, TEA (1 mM), enhanced the KCl‐induced contraction by 20 % after the pre‐incubation with 1 [micro]M 2‐MeOH, but not 10 [micro]M 2‐MeOH, this suggests 2‐MeOH stimulates Ca2+‐activated K+ channels. Breast cancer (MCF‐7) cells were incubated over a 6 day period with 1 nM E2 and 2‐MeOH in a normoxic and hypoxic environment. In contrast to E2, cell counts indicated that 2‐MeOH decreased the cell number by 39% and 32% in normoxic and hypoxic conditions, respectively. Overall, our data suggest that 2‐MeOH may be a therapeutic alternative in hormone replacement therapy because of its apoptotic effect on breast cancer and its similarity to the E2 cardioprotective effect on coronary tone. Support: NCRR of the NIH, Grant #P20 RR‐16460
Physiological Significance of a Coronary Stenosis Assessed from Pulsatile Resistance Index at Baseline Flow
Authors: Siebes Maria , Kolyva Christina , Verhoeff Bart‐Jan , Nolte Froukje , Spaan Jos A.E. , Piek Jan J.
Published: June 2021

Coronary pressure and flow waveforms change in the presence of a stenosis. We exploited the pulsatile nature of these hemodynamic signals to assess functional coronary stenosis severity.

In 29 patients, aortic pressure (Pa), and distal coronary pressure (Pd) and flow velocity (v) were recorded at rest and during maximal hyperemia in a diseased vessel. An index of the dynamic signal energy per cycle was defined as the root mean square of the recorded time‐varying signal after removing the mean. The Pulsatile Velocity Index (PVI) and respective Pulsatile Pressure Indices (PPIa and PPId) were calculated and a stenosis Pulsatile Resistance Index was derived as PRI = (PPId ‐ PPIa)/PVI. Average stenosis resistance SR = (Pa‐Pd)/v and fractional flow reserve (FFR) = Pd/Pa during maximal hyperemia served as gold standards.

Hyperemic SR was a major determinant of PPId (p<0.0001), while PVI remained relatively unaffected (r = 0.3). PRI at rest was strongly correlated to both hyperemic SR (p<0.0005) and FFR (p<0.002), with separate relationships for left and right coronary arteries, reflecting different compression of intramural vessels.

We conclude that the pulsatile energy of v, Pa and Pd can be utilized to characterize the functional significance of a coronary stenosis without inducing vasodilation. A practical advantage is that PRI is not affected by pressure drift.

(Support: NHF grants 2000.090 and 2006B145)
The effects of successive air and trimix dives on human cardiovascular function
Authors: Dujic Zeljko , Marinovic Jasna , Ljubkovic Marko , Obad Ante , Bakovic Darija , Breskovic Toni
Published: June 2021

The use of trimix has significantly increased among diver population. However, the data indicating how the field trimix dives at most common depths affect the cardiovascular function are sparse. The purpose of this study was to investigate the cardiovascular effects of trimix dives and compare them with the air dives, as well as determine whether the repetition of dives in successive days affects their extent. Eight professional divers performed four dives in consecutive days where the dive depth was progressively increased to the maximum of 55 m, and the divers used air in the 1st dive, nitrox 25 for the 2nd, and trimix 20/30 for the 3rd and 4th dive. After each dive, a significantly decreased left ventricular ejection fraction and fractional shortening, as well as increased end‐systolic volume without a change in end‐diastolic volume were found, indicating a depressed systolic function of the left heart. Assessment of the ratio between pulmonary artery acceleration time and right ventricular ejection time revealed an increase in PAP after all the dives. The current study shows that the cardiovascular effects of trimix dives do not differ from those of the dives with compressed air. However, it suggests that even a very safe and conservative trimix diving profile exerts significant cardiovascular effects.

This study was supported by the Croatian Ministry of Science, Education and Sports.
Chronic intermittent hypoxia enhances habituation of the arousal response to acute hypoxia in developing rodents
Authors: Darnall Robert A , McWilliams Samuel , Schneider Robert , Tobia Christine
Published: June 2021

Arousal from sleep is a critical apnea recovery mechanism. Rat pups progressively increase their time to arousal (latency) during repeated exposures to hypoxia (short term habituation‐STH). We hypothesized that postnatal exposure to chronic intermittent hypoxia (CIH) would increase the mean time (over several acute hypoxia exposures) to arousal (long term habituation‐LTH) and would accentuate STH, especially at P15, an age equivalent to the time of the highest incidence of Sudden Infant Death Syndrome (SIDS). Pups and dam were exposed to CIH for a total of 25 days. Each pup was removed at P5, P15, and P25, and exposed to four 3‐min trials of 5% oxygen alternating with 6‐min periods of normoxia. During the hypoxia trials, arousal latency progressively increased in both the Control and CIH groups (STH‐P=0.024). There was a mean increase in latency only in the CIH group (LTH) (P=0.027), most prominent at P15 (<0.001) and P25 (P=0.007). At P15, but not at P5 or P25, the STH was also accentuated in the CIH group (P=0.039). There were corresponding effects on arousal thresholds (inspired O2 concentration and oxyhemoglobin saturation at the time of arousal) and complex effects on heart rate. These results support the hypothesis that exposure to chronic intermittent hypoxia accentuates habituation of the arousal response in developing rat pups, especially in early infancy, and is an important mechanism in SIDS. P01 HD36379.