bluuebunny/crossref_metadata_2025 huggingface dataset:
Focal actin tethering regulates E‐cadherin mobility in lung microvascular endothelial cells
Authors: Quadri Sadiqa K , Sun Li , Bhattacharya Jahar
Published: June 2021

Although cadherin mobility in the cell membrane determines barrier properties of the adherens junction, the manner in which actin regulates the mobility remains controversial. To test this hypothesis, we established confluent monolayers of rat lung microvascular endothelial cells (EC) over expressing full length GFP‐E‐cadherin. In single EC viewed by confocal microscopy, E‐cadherin fluorescence (Io) at the cell perimeter was two‐times higher at some (>3000 grey levels) versus other (<2000 grey levels) sites (mean±SE; n=20 cells). To determine E‐cadherin mobility, we photobleached GFP fluorescence to 50% of Io, then determined fluorescence recovery at 5 min (FRAP). At high and low fluorescence sites respectively, FRAP was 28±3% versus 45±3% of bleached fluorescence (P<0.05). However in monolayers treated with the actin depolymerizer, latrinculin B (10 nM) FRAP increased to 50±4% at high fluorescence sites (P<0.05), but remained unchanged elsewhere. Thus although baseline E‐cadherin mobility in the EC membrane varied inversely with E‐cadherin density, actin depolymerization increased mobility specifically at sites of high E‐cadherin density. Our findings show for the first time that sites of high E‐cadherin density are focal locations of actin‐cadherin tether formation. The extent of focal actin‐cadherin tethering might determine regulation of endothelial barrier properties (Support: HL36024).
Protective effects of adipose stem cells against cigarette‐smoke induced lung injury
Authors: Schweitzer Kelly , Johnstone Brian H. , Traktuev Dmitry , Garrison Jana , Rush Natalia I , Adamowicz Jeremy J , Cook Todd G , Feng Dongni , March Keith L. , Petrache Irina
Published: June 2021

Adipose‐derived stem cells (ASCs) express several critical determinants of lung rejuvenation, thus providing components necessary for tissues to recover from lung injury, such as that induced by cigarette smoke (CS) in emphysema. We evaluated the therapeutic effects of ASCs on CS‐induced lung injury. Mice were exposed to CS for 4 months; during the second half of CS exposure, mice were treated with 3.5 x 105 murine ASCs or saline (i.v. qow). Mice receiving ASCs were protected against CS‐induced alveolar space enlargement, measured by mean linear intercept (mean=30.72; sem=0.779; p=0.032). This effect was associated with decreased PMN lung inflammation, as well as AKT and p38 MAPK activation induced by CS exposure (p=0.028 and 0.0311). Alveolar (endothelial) cell barrier dysfunction may be an early injury marker induced by CS in the lung. Since ASCs are known to secrete vascular‐protective factors, we tested the ability of ASC supernatants to protect against CS‐induced endothelial cell permeability. CS caused a dose‐dependent decrease in primary mouse trans‐endothelial monolayer electrical resistance (64% at 5hr) that was significantly attenuated by ASC‐ media and by a specific p38 MAPK inhibitor (p=0.034 and 0.004). These results suggest a protective effect of ASCs from CS‐induced lung injury that may involve paracrine modulation of the AKT and p38 signaling pathways. Funding: IU Signature Center Grant.
Sedentary Aging is Associated with a Senescent Endothelial Cell Phenotype that is Ameliorated by Habitual Aerobic Exercise
Authors: Donato Anthony J , Chiang Rhea , Pierce Gary L , Lawson Brooke R , Seals Douglas R
Published: June 2021

Replicative senescence is used in culture as a model of vascular endothelial aging. However, the relevance to in vivo aging in humans and the influence of lifestyle factors such as habitual exercise are unknown. To gain insight, endothelial cells (EC) were extracted from the brachial arteries of young (YS: n=10; 24±1yr) and older (OS: n=18; 62±1yr) sedentary and older habitually exercising (OE: n=6; 62±1yr) healthy adults. Compared with YS, OS had larger EC size (10,072±317 vs. 9031±306 pxls, P<0.05), greater p53 protein expression (0.57±0.02 vs. 0.45±0.03 p53/HUVEC intensity, P<0.05, quantitative immunofluorescence), a transcription factor related to increased cell senescence, and lower expression of both nuclear hTERT (0.43±0.05 vs. 0.60±0.09 NhTERT/HUVEC intensity, P < 0.05), a measure of telomerase activity, and SIRT‐1 (0.41±0.03 vs. 0.52±0.05 SIRT‐1/ HUVEC intensity, P<0.05), a deacetylase linked to enhanced longevity. EC from OE more closely resembled those from YS, featuring smaller EC size (9075± 309, P<0.05), lower p53 (0.37±0.04, P<0.01) and greater NhTERT (0.60±0.08, P<0.05) vs. OS, although SIRT‐1 expression was not different (P=0.15). Our results indicate that sedentary, but not physically active, aging in humans is associated with development of a senescent vascular EC phenotype. Prevention of vascular endothelial senescence may be an important mechanism by which regular exercise protects against age‐related cardiovascular diseases.

NIH AG013038, AG022241, AG006537, AG029337, AG000279, RR00051
Cyclic GMP‐specific PDE5 in regulating pulmonary endothelial permeability
Authors: Zhu Bing , Xu Yan Chun , Rich Thomas C , Strada Samuel J
Published: June 2021

Intracellular cGMP ([cGMP]i) regulates endothelial cell (EC) permeability. PDE5, an enzyme possessing cGMP hydrolytic activity, expresses discretely in pulmonary artery and microvascular ECs (PAECs/PMVECs). Indeed, without PDE5 expression, [cGMP]i in PMVECs is higher than it is in PAECs. Interestingly, PMVECs have tighter barrier junctions, with less permeability, than PAECs. However, the role of PDE5 in regulating endothelial barrier permeability is unknown. We questioned whether increases in [cGMP]i caused by decreasing PDE5 activity protects endothelial barrier junctions. Using labeled Dextran, we determined whether the permeability of ECs' monolayer is altered by PDE5 activity. Decrease of PDE5 activity in PAECs, either by siRNA or selective inhibitor, led to a decrease in barrier permeability. PDE5 inhibition also protected PAECs from a thrombin‐induced increases in permeability and barrier dysfunction. Overexpression of PDE5A1 in PMVECs reduced basal [cGMP]i. PDE5A1‐expressing cells showed increased basal permeability and thrombin‐triggered permeability which was not seen in wild‐type PMVECs. This cGMP‐regulated permeability requires PKG and its corresponding phosphorylation of VASP‐Ser239 and maintenance of cytoskeletal structure of F‐actin. Therefore, results suggest that PDE5 plays a key role in regulating endothelial barrier integrity in pulmonary artery (AHA 0665169).
FGF21 determined angiogenic phenotypes in pulmonary endothelial cells
Authors: Zhu Bing , Xu Yan Chun , Stevens Troy
Published: June 2021

Fibroblast growth factor 21 (FGF21) is a member of FGF superfamily with metabolic activities. The role of FGF21 in endothelium is unknown. We have reported a cGMP‐dependent FGF21 expression in pulmonary microvascular endothelial cells (PMVECs) under the regulation of cGMP‐PDE5 expression. Overexpression of PDE5A1 in PMVECs leads to a 20‐fold decrease of FGF21 mRNA due to a reduced intracellular [cGMP]i. PDE5A1‐expressing PMVECs have an attenuated angiogenic capability for network formation on regular Matrigel and exhibit a novel "branch‐like" phenotype. Herein, we tested FGF21 on endothelial cells phenotypes using a growth factor reducing (GFR)‐Matrigel. With a serum‐free DMEM, PDE5A1‐expressing PMVECs and controls were seeded on the GFR‐Matrigel. Results indicated that PDE5A1 cells and controls required more cell numbers on GFR Matrigel to form networks vs. regular Matrigel; two cell types were in a similar capability of network formation. FGF21 increased networks with cell proliferation and "branch‐like" phenotypes in PDE5A1‐expressing cells, which were not seen in PMVECs controls. FGF21 in promoting angigenic phenotypes for PDE5A1 cells was enhanced by KT5823 but inhibited by 8‐Br‐cGMP, suggest PKG activity was involved. Results suggest that a FGF21 related angiogenic phenotype in PMVECs is regulated by [cGMP]i for controlling its expression and signaling pathways (AHA 0665169/0865356).
Roles of focal adhesion kinase and paxillin in focal adhesion dynamics of living endothelial cells
Authors: Hu Ying‐Li , Li Yi‐Shuan , Chien Shu
Published: June 2021

Focal adhesion kinase (FAK) is a signaling protein that can activate the adaptor protein paxillin, which is also localized at focal adhesions (FAs) and associated with integrins, to influence the cytoskeleton and membrane protrusions, thereby regulating FA dynamics and cell motility. In living ECs transfected with GFP‐FAK and DsRed‐paxillin, concurrent monitoring of the intracellular dynamics of FAK and paxillin by time‐lapse video recording and dual‐color fluorescence imaging showed the co‐localization of FAK and paxillin at FAs. The ratio of the fluorescence intensities (FI) of FAK/paxillin was determined during assembly/disassembly of FAs. The FAK/paxillin FI ratio of FA sites was higher in the cytoplasm than in the perinuclear region. This ratio in the cytoplasm increased as the dynamics of FA sites became active, especially at the leading edge of the migrating EC. Our findings show the roles of FAK and paxillin in the assembly/disassembly of FAs and in cell adhesion. We conclude that both molecules play important roles in modulating the FA dynamics and cellular signaling during cell migration. This work was supported by NHLBI Research Grants HL‐080518 and HL‐085159 (S.C.).
Gender‐independent pattern of dyslipidemia and systemic oxidation in overweight children
Authors: Rosa Jaime , Oliver Stacy R , Pontello Andria M , Milne Ginger L , Johnson Sarah J , Sanchez Stephanie C , Flores Rebecca L , Bachmann Georgia V , Galassetti Pietro R
Published: June 2021

The incidence of pediatric obesity, a major risk factor for cardiovascular disease (CVD) and diabetes, has increased over the past decades. Gender differences in lipid distribution and oxidative status (a key biochemical link between obesity and CVD) are known to exist in the adult population. Little information is available, however, about gender effects on the interaction between oxidative stress and dyslipidemia in children.

We therefore measured systemic oxidative stress (F2‐isoprostanes, F2‐IsoP) and lipid variables in 98 male and female peripubertal children (43 normoweight, NW, 13.4±0.5 yrs, 25F, BMI%<85th; 55 overweight, OW, 12.7±2.5 yrs, 25F, BMI%=95th).

Overall, OW displayed elevated F2‐IsoP vs. NW (99±7 vs. 74±4, p<0.005) and this pattern was maintained across genders (see figure). OW also displayed alterations of all components of lipid metabolism compared to NW children (FFA 564±46 vs. 372±34, HDL‐C 1.3±0.04 vs. 1.5±0.03, LDL‐C 2.5±0.1 vs. 2.2±0.1, triglycerides 0.95±0.07 vs. 0.53±0.05, all p<0.01); again, as shown in the figure, the pattern of dyslipidemia was similar for both genders.

Our data indicate that alterations in lipid metabolism and the consequent increase in oxidative stress occur in obese children in a gender‐independent fashion, likely exposing both genders to a similar degree of increased future risk of diabetes and CVD.

Support: NIH grants M01‐RR00827‐28, P01HD048721‐01A1.
siRNA mediated knockdown of ERbeta in vivo in magnocellular neurosecretory cells of the rat paraventricular nucleus
Authors: Doherty Faye C , Sladek Celia D
Published: June 2021

Estrogen receptor β (ERβ) is a transcription factor that is downregulated to undetectable levels in the nuclei of magnocellular neurosecretory cells (MNCs) in response to hyperosmolality or hypovolemia in rats. To determine if downregulation of ERβ allows upregulation of other genes in the MNCs during dehydration, we used siRNA to locally and acutely reduce ERβ expression in the paraventricular nucleus (PVN) of rats. This avoids the systemic and developmental effects of knockout models. We used an siRNA construct specific to ERβ and modified to enhance stability and nuclease resistance (siSTABLE, Dharmacon) as well as a corresponding non‐targeting construct. siRNA (6.48ng in 0.5ul PBS) was stereotaxically injected into PVN, using the ERβ‐specific construct on one side and the non‐targeting one on the other. On the side of the PVN injected with the ERβ‐specific construct, the number of ERβ‐positive nuclei was reduced to 50±13% (p=0.013; n=2) compared to the control side. This reduced expression is not due to dehydration since hematocrit in these rats (35±1% n=2) was lower than in dehydrated animals (41±1%, n=10; p=0.012), but not lower than in unmanipulated controls (37.8±0.7%, n=9; p=0.137). The effect of the siRNA is highly localized; misplaced injections in the caudal PVN did not effect ERβ expression in the MNCs. This supports the feasibility of using siRNA to reduce ERβ in discrete neuronal populations.
Estrogenic modulation of lysyl oxidase in adult cardiac fibroblasts
Authors: Voloshenyuk Tetyana G , Gardner Jason D
Published: June 2021

Lysyl oxidase (LOX) is necessary for the biosynthesis of functional collagen through its crosslinking of collagen and elastin. Increased LOX expression has been associated with hepatic, lung and kidney fibrosis, but the role of LOX in the heart is not known. Cardiac fibroblasts constitutively express pro‐LOX protein and bone morphogenic protein‐1, which cleaves pro‐LOX to generate active LOX and LOX‐propeptide. We hypothesized that LOX expression and activity in the heart is regulated by estrogen and may contribute to gender specific cardioprotection by altering collagen processing. Adult cardiac fibroblasts were isolated from ovariectomized (OX) and intact female rats. Cells were grown in media with 10% FBS, and serum starved prior to treatment with 17β‐estradiol. Fibroblasts from OX rats had the highest basal expression of pro‐LOX (200% vs. intact), which was suppressed by estrogen. In contrast, estrogen enhanced pro‐ and active LOX expression in fibroblasts from intact females. These findings indicate that changes in hormonal status alter cardiac fibroblast expression of pro‐form and active LOX. Future studies are warranted to evaluate the estrogenic modulation of LOX expression and activity in the heart, as well as the resulting effects on myocardial collagen and cardiac function.
Role of aldosterone in postmenopausal hypertension
Authors: Yanes Licy L
Published: June 2021

Postmenopausal women have higher blood pressure (BP) than premenopausal ones. However, the mechanisms that mediate the BP increase after menopause are unclear. The present study was performed to test the hypothesis that an increase in plasma aldosterone partially mediates hypertension in old female SHR (OF‐SHR), a rat model of postmenopausal hypertension. The mineralocorticoid receptor antagonist, eplerenone (100 mg/kg/12 h), was administered by gavage for 2 weeks to OF‐SHR (n=7), aged 16 mos, and to young female SHR (YF‐SHR), aged 4 mos. BP was measured by radiotelemetry. In another set of rats, adrenal glands and kidneys were removed for determination of aldosterone synthase and mineralocorticoid receptor mRNA expression by real‐time RT‐PCR . Blood samples were taken for determination of plasma aldosterone levels by ELISA. Eplerenone decreased BP (‐20 mm Hg) in OF‐SHR (p<0.05) but not in YF‐SHR. Plasma aldosterone was elevated in OF‐SHR compared with YF‐SHR (135.62±21.7 vs. 186.44±31.36 ng/dl, p<0.05), and mineralocorticoid receptor mRNA expression was decreased 50% in kidney of OF‐SHR. Aldosterone synthase mRNA expression was 2 fold increases in adrenal gland in OF‐SHR compared to YF‐SHR. This study shows that aldosterone plays an important role in mediating postmenopausal hypertension in OF‐SHR, likely due to adrenal aldosterone synthase‐mediated increases in plasma aldosterone. This study was supported by American Heart Association Scientist Development Award 0830239N.
Endothelin receptor blockade abolishes angiotensin II sensitivity in intrauterine growth restricted rats
Authors: Ojeda Norma B , Royals Thomas B , Alexander Barbara T
Published: June 2021

Our laboratory has developed a unique rat model of placental insufficiency that results in intrauterine growth restriction (IUGR), hypertension and an increased responsiveness to angiotensin II (ANG II). We previously demonstrated that activation of the endothelin system contributes to hypertension in IUGR. Therefore, we hypothesize that increased responsiveness to ANG II may be mediated by endothelin in IUGR. Rats were pretreated with RAS blockade (250 mg/L enalapril for 1 week to normalize blood pressure), then systemic and renal hemodynamics parameters were determined before and after acute infusion (30 min) of ANG II (100 ng/kg/min) and endothelin type A receptor blockade (ETRAB), (ABT‐627, 10ng/kg/min). ETARB abolished the increase in mean arterial pressure (MAP) and renal vascular resistance (RVR) and the decrease in renal blood flow (RBF) and glomerular filtration rate (GFR) induced by acute ANG II in IUGR. Thus, these data suggest endothelin may contribute to increased responsiveness to ANG II in IUGR and may serve as an underlying mechanism in IUGR hypertension.

* P < 0.05 vs. Control

† P < 0.05 vs. Counterpart

‡ P < 0.05 vs. Control + ABT‐627
Cell surface UT‐A1 is sequestered in lipid rafts and phosphorylated and ubiquitinated in non‐raft membrane domains
Authors: Feng Xiuyan , Yang Yuan , Fröhlich Otto , Klein Janet D , Sands Jeff M , Chen Guangping
Published: June 2021

We previously reported that UT‐A1 mainly targets to lipid raft pools in both a heterologous expression system (UT‐A1‐MDCK cells) and in native rat IMCD suspensions. In this study, we investigated the possible role of lipid rafts in UT‐A1 regulation. Disruption of cell membrane lipid rafts by methyl‐β ‐cyclodextrin in Xenopus oocytes significantly increased UT‐A1 urea transport activity. When stimulated with forskolin, the distribution of UT‐A1 in the cell membrane fraction was shifted from lipid raft to non‐lipid fractions in UT‐A1‐MDCK cells. We then examined whether the UT‐A1 that was moved away from the lipid fraction could represent an activated form. We metabolically labeled UT‐A1‐MDCK cells with 32P‐orthophosphorylate; then treated the cells with 10 μM forskolin for 10 min. High levels of UT‐A1 phosphorylation were found in non‐lipid rafts. Dowregulation of UT‐A1 is accompanied via ubiquitination by E3 ligase MDM2. We further investigated cell surface UT‐A1 degradation. UT‐A1 293 cells were transfected with pCW7‐His‐ubiquitin. Ubiquitinated UT‐A1 was found in non‐lipid rafts. Interestingly, UT‐A1 ubiquitin ligase MDM2 is only identified in the non‐lipid raft fraction, which is consistent with the predominance of UT‐A1 ubiquitination and degradation outside of rafts. These findings suggest a new mechanism for regulation of UT‐A1 at the cell surface. UT‐A1 urea transport activity regulation involves movement between lipid and non‐lipid populations. Functional UT‐A1 is translocated in non‐lipid factions. UT‐A1 in rafts has low clearance. Lipid rafts sequester UT‐A1 away from the degradation machinery located in non‐raft membrane domains (MDM2).
LC‐MS/MS analysis of differential centrifugation fractions from native inner medullary collecting duct (IMCD) of rat
Authors: Sachs Aaron N , Pisitkun T , Hoffert J D , Yu M‐J , Knepper M A
Published: June 2021

We have performed LC‐MS/MS analysis of differential centrifugation fractions from rat IMCD to evaluate the utility of differential centrifugation in assessing trafficking of the water channel aquaporin‐2 (AQP2). Freshly prepared IMCD suspensions were homogenized and subjected to sequential centrifugations at 1K g, 4K g, 17K g, and 200K g. Preliminary immunoblotting confirmed that the ratio of AQP2 in the 17K fraction to the 200K fraction underwent an increase in response to the vasopressin analog dDAVP, largely due to a reduction in the 200K fraction. Immunoblotting revealed that the major phosphorylated forms of AQP2 were present in both the 17K and 200K fractions. LC‐MS/MS analysis showed that markers of 'intracellular vesicles' were present in both the 17K and 200K fractions. In contrast, plasma membrane proteins were predominantly present in the 4K and 17K fractions. Proteins associated with several multiprotein complexes (e.g. proteasomes and actin‐related protein 2/3 complex) were virtually exclusively present in the 200K fraction. Overall, we identified 656 proteins, including 189 not previously present in the IMCD Proteome Database. The data show that both the 17K and 200K fractions are highly heterogeneous and cannot be equated with 'plasma‐membrane' and 'intracellular vesicle' fractions, respectively. Alternative methods for assessment of AQP2 trafficking are recommended.
GCN2 activation by urea protects cells from osmotic stress
Authors: Cai Qi , Brooks Heddwen L.
Published: June 2021

General control non‐derepressible‐2 (GCN2) is one kinase known to be involved in the integrated stress response (ISR), and GCN2 activation (via phosphorylation) occurs in response to a variety of environmental stresses. GCN2 then phosphorylates eIF2α, a key step in ISR. Renal inner medullary cells are constantly exposed to high and variable concentrations of urea as a result of the urinary concentrating mechanism. To determine whether high concentrations of urea cause ISR in medullary cells, mIMCD3 cells were subjected to 250, 300 or 400 mM urea. GCN2 was rapidly activated by urea in a time‐dependent and dose‐dependent manner. At 250‐300mM urea GNC2 activation was reversible, however, when exposed to 400mM urea GCN2 activation persisted (~16 hours). eIF2α phosphorylation was also dose‐dependent. To investigate the effect of GCN2 activity on osmotic tolerance, GCN2 ‐/‐ and +/+ MEF cells were used. GCN2 ‐/‐ MEF cells were more susceptible to osmotic stress; 77% ± 5.0 of GCN2 +/+ MEF cells survived 300mM urea; in contrast, only 55% ± 9.6 of GCN2 ‐/‐ survived (P<0.05). We conclude that GCN2 is activated by urea and contributes to renal medullary cells' ability to survive osmotic stress.
Properties of the zinc transporter ZIP14 suggest a role in cellular uptake of nontransferrin‐bound iron (NTBI) characteristic of iron‐overload conditions
Authors: Pinilla Tenas Jorge J , Sparkman Brian K , Illing Anthony C , Liuzzi Juan P , Cousins Robert J , Knutson Mitchell D , Mackenzie Bryan
Published: June 2021

Iron‐overload conditions (e.g., thalassemia, hereditary hemochromatosis) are characterized by the appearance in plasma of nontransferrin‐bound iron (NTBI), and result in cardiomyopathy, diabetes, hepatic cancer and cirrhosis. Since the zinc transporter ZIP14 (SLC39A14) is strongly expressed in liver, heart, and pancreas‐major sites of organ damage in iron overload‐we tested the hypothesis that ZIP14 transports free iron. We expressed murine ZIP14 in Xenopus oocytes and examined its activity using radiotracer assays and the voltage clamp. ZIP14 stimulated the uptake of 55Fe2+, 54Mn2+, 65Zn2+ and 109Cd2+ by 50‐ to 1,000‐fold over that in control oocytes. ZIP14‐mediated 55Fe2+ uptake was saturable (K0.5 for Fe2+ of 2.6 ± 0.6 μM), temperature‐dependent (activation energy, Ea = 14 ± 2 kcal.mol−1), and inhibited by Co2+, Mn2+, and Zn2+. Ca2+ and HCO−3 stimulated 55Fe2+ transport. These data agree well with those obtained from NTBI uptake measurements in perfused rat liver [Wright et al (1986) J Biol Chem 261, 10909], and suggest that ZIP14‐mediated iron transport may be of pathophysiological relevance in iron overload. We observed no currents associated with metal‐ion transport in oocytes expressing ZIP14 and conclude that ZIP14 is not rheogenic (i.e., net charge movement is zero). Our data reveal that ZIP14 is a complex, broad‐scope metal‐ion transporter capable of mediating cellular NTBI uptake.
Role of prostaglandins in the loss of lymphatic contractile function in the experimental guinea pig model of TNBS‐induced ileitis
Authors: Rehal Sonia , Roizes Simon , von der Weid Pierre‐Yves
Published: June 2021

The lymphatic vessel pumping is critical in maintaining tissue fluid homeostasis, particularly during edematous stress, such as inflammation. Loss of this function (i.e. dilated hyporesponsive lymphatic vessels) previously observed during experimental intestinal inflammation could be mediated by prostaglandins, which are heavily involved in the intestinal inflammatory response and potently alter lymphatic contractility. We thus evaluated the ability of lymphatics to synthesize and respond to prostaglandins in physiological and inflammatory conditions.

Using real time PCR, we analyzed mesenteric lymphatics, arteries, veins, mesothelium and ileum obtained from control animals, TNBS‐treated guinea pigs and their sham counterpart for the following mRNA transcripts: COX‐1, COX‐2, prostacyclin synthase, prostaglandin E (PGE) synthase 1 and 2, prostacyclin receptor, and PGE2 receptors EP1, EP2, EP3 and EP4. We also measured PGE2 content in lymphatics by ELISA.

When compared to sham and control animals, we observed an upregulation of COX‐2 mRNA and a downregulation of PGE synthase 2 and EP4 receptor mRNAs in lymphatics and ileum of TNBS‐treated animals. Preliminary data also showed an increase in PGE2 content in lymphatics from TNBS‐treated animals.

Our results further suggest alteration of PGE2 function in lymphatics of TNBS‐treated guinea pigs

Supported by the Crohn's and Colitis Foundation of Canada
C‐terminal domains are required for ZO‐1 stabilization and tight junction assembly
Authors: Shen Le , Yu Dan , Turner Jerrold R.
Published: June 2021

Zonula occludens (ZO) proteins direct assembly of epithelial tight junctions (TJs). While critical functions have been defined for N‐terminal PDZ domains, a central SH3‐GUK module, and the C‐terminal actin‐binding region (ABR), the remaining ZO‐1 C‐terminus has not been characterized. The aim of this study was to determine if the ZO‐1 C‐terminus participates in TJ assembly. Epithelial cells were stably transfected with full‐length ZO‐1 tagged with GFP at the N‐terminus (GFP‐ZO‐1) as well as a GFP‐tagged deletion mutant lacking the ABR (ZO‐1δABR) and a GFP‐tagged truncation mutant lacking the ABR and remaining C‐terminus (ZO‐11‐1152). ZO‐11‐1152 markedly delayed epithelial barrier development as well as peak transepithelial resistance (TER) achieved. In contrast, ZO‐1δABR did not delay assembly and only slightly diminished peak TER. Morphologic analysis showed that GFP‐ZO‐11‐1152, but not GFP‐ZO‐1 or GFP‐ZO‐1δABR, localized to the TJ inefficiently and interfered with junctional recruitment of endogenous ZO‐1 and β‐catenin. We have previously used fluorescence recovery after photobleaching to show that GFP‐ZO‐1 stability at the TJ increases as monolayers mature. GFP‐ZO‐11‐1152 stabilization did not occur with maturation, but GFP‐ZO‐1δABR stabilized normally. These data suggest that undefined domains within the ZO‐1 C‐terminus are essential for TJ assembly and maturation.
Blockade of STAT3 does not alter acute injury after hepatic ischemia/reperfusion in mice
Authors: Clarke Callisia Nathelee , Tevar Amit , Edwards Michael , Lentsch Alex B
Published: June 2021

Background

STAT3 is a critical transcription factor regulating the protective effects of liver ischemic preconditioning. However, its role in hepatic ischemia/reperfusion (I/R) injury is unclear. Here we investigated whether blockade of STAT3 phosphorylation altered the injury response to hepatic I/R injury.

Methods

Mice were subjected to 90 minutes of hepatic ischemia followed by reperfusion with or without ip injection of 5mg/kg AG490 (selective JAK2 inhibitor) or 10mg/kg of Stattic (direct inhibitor of STAT3 phosphorylation). Mice were sacrificed at 24 hr post reperfusion and liver injury (serum ALT), histology and tissue neutrophil accumulation (liver MPO) measured.

Results

Neither Stattic nor AG490 had any effect on liver injury induced by I/R. However, Stattic significantly reduced neutrophil accumulation.

Conclusion

The data suggest that STAT3 is not relevant to acute liver injury after I/R. However, it may be involved in pathways leading to recruitment of neutrophils.
Acute resistance exercise effects on IGF‐1 signaling cascade downstream pathway
Authors: song taejeong , Farrar Roger P
Published: June 2021

Illuminating muscle hypertrophy mechanisms were main issues for over a decade. Now it is recognized that IGF‐1 downstream pathways are key molecules to increase muscle mass by activating gene transcription, protein synthesis and satellite cell proliferation and differentiation. But the activation timing and patterns of these pathways after resistance exercise is still controversial.

The purpose of this study is to investigate the time course and trend of IGF‐1 signaling downstream after acute ladder climbing exercise. Male Sprague‐Dawley rats, 384g, were divided into control (CON), one bout exercised (1B) and three bouts exercised (3B) group and trained once per every three days then sacrificed at 3, 6 and 12 hours after last bout of exercise. Rats were fasted 3 hours before last exercise session and received standardized nutriment supplement via gavage (5m/kg) immediately after the last session.

The ratio of FHL muscle mass (mg) to body weight (g) in 3B (1.69±0.1, P < 0.01) is increased significantly comparing to CON (1.49±0.05) and 1B (1.53±0.09). pAkt is peaked at 3B3 (+114%) and both pmTOR (+106%) and pp70S6K (+119%) are the most activated at 1B3. pERK shows peak at 3B12 (+80%). There are no changes of total Akt, mTOR, p70S6K and ERK. These data support that mTOR and p70S6K can be activated independent to AKT and are respond early during training.
Progesterone or dihydrotestosterone induced relaxation of female guinea pig gallbladder strips is mediated by different mechanisms
Authors: Kline Loren , Karpinski Edward
Published: June 2021

Progesterone (P) and dihydrotestosterone (DHT) each relax cholecystokinin octapeptide (CCK) induced tension. The relaxation was shown to be concentration dependent for both P and DHT. An in vitro technique was used to determine which second messenger system(s) mediate the observed relaxation. Statistical analysis used either paired t‐tests or analysis of variance. To determine if the PKA/cAMP second messenger system mediated the P‐ or DHT‐induced relaxation, PKA inhibitor 14‐22 amide myristolated (PKA‐IM; 180 nM) was used. PKA‐IM caused a significant (p<0.01) decrease in P‐induced relaxation (58.0+3.5 vs 53.3+3.4%), but had no significant effect on DHT‐induced relaxation. When the PKC inhibitors bisindolymaleimide IV (0.5 µM) and chelerythrine Cl (5 µM) were used together, a significant (p<0.01) reduction in P‐induced relaxation (57.6+3.3 vs 39.3+4.1%) was observed. These agents had no significant effect on the DHT‐induced relaxation. The PKG inhibitor, KT5823 (585 nM), had no significant effect on either P‐ or DHT‐induced relaxation. 2‐APB (126 µM), an inhibitor of IP3 induced intracellular Ca2+ release, significantly (p<0.001) decreased the amount of CCK‐induced tension, but had no significant effect on either the P‐ or DHT‐induced relaxation. Genistein (10 µM), a protein tyrosine kinase inhibitor, significantly (P<0.001) decreased the amount of CCK‐induced tension with no significant decrease in either P‐ or DHT‐induced relaxation. The results suggest that both the PKA/cAMP and PKC second messenger systems mediated the P‐induced relaxation of CCK‐induced tension in female guinea pig gallbladder strips. The results also show that the DHT‐induced relaxation is not mediated by the same second messenger pathways as P and is the subject of current investigations.
Oral mucosal and periodontal ligament mechanoreceptors (PDLMs) affect antral motility differently
Authors: Lorber Mortimer
Published: June 2021

Do oral mucosal mechanoreceptors affect antral motility? PDLMs are activated by tooth movements in chewing. These also occur during simulated mastication, activating antral motility (Can J Physiol Pharmacol 78:29, 2000) and gastric emptying (J Dent Res 81:302, 2002). Because a mouthful of food contacts all oral tissues, the antral motility effects of stimulating 8 oral mucosal sites under pentobarbital were sought. 9 groups of 8 adult female rats fasted 18h. Visual observations avoided disturbing the stomach for cannula or transducer attachment. All studies had 3 five minute periods: baseline, procedure, and post‐procedure. The mucosal procedure was 5 min of gentle movement of a teflon rod against: the mandibular gingiva, anterior & posterior tongue & its undersurface, the oral floor, buccal mucosa, & hard & soft palates. PDLMs were activated by opening & closing the jaw so opposing molars contacted each other extremely gently. This simulated mastication was done 30 times in 12 sec with a 3 sec rest. This sequence was repeated 20 times in 5 min. Statistical analysis: ANOVA & Bonferroni. No mucosal site affected antral motility; their ANOVA P values were 0.173 to 0.969. By contrast, PDLM activation had ANOVA P = 0.010. Its 3 period values (mean + s.d.) were 2.88 + 2.47, 5.50 + 4.11, 8.00 + 2.00. Between the first and last values P = 0.003. PDLMs are the sole basis of periodontogastric reflexes. Personal research support.
No effect of diabetes on intramyocellular diglyceride accumulation in human skeletal muscle
Authors: Anastasiou Costas A , Kavouras Stavros A , Lentzas Yannis , Gova Afrodite , Sidossis Labros S , Melidonis Andreas
Published: June 2021

Intramyocellular lipids may be linked to insulin resistance. We aimed to investigate the effect of adiposity and diabetes on intramyocellular diglycerides (DG) and triglycerides (TG). The study participants were stratified according to the presence of diabetes and obesity into three groups: non‐diabetic, obese (nDiab‐Ob, n=19), diabetic, non‐obese (Diab‐nOb, n=4) and diabetic, obese subjects (Diab‐Ob, n=11). Intramyocellular lipids were determined in biopsies from the vastus lateralis under fasting. Glycemic control and insulin resistance were assessed by an oral glucose tolerance test and the homeostatic model. Diab‐Ob had significantly higher intramyocellular TG, compared to nDiab‐Ob (66.7±23.8 vs. 18.4±4.4 nmol/mg dry tissue, p<0.05), while no difference was observed between nDiab‐Ob and Diab‐nOb. DG levels were not significantly different among the study groups (1.7±0.3, 1.1±0.2 and 1.9±0.7 nmol/mg dry tissue for nDiab‐Ob, Diab‐nOb and Diab‐Ob respectively). Monounsaturated fatty acids were the major constituent of TG in all groups, while DG contained mainly saturated fatty acids. No correlation was found between intramyocellular TG or DG levels and insulin resistance. A modest correlation was found between monounsaturated fatty acids in the TG and the area under the glucose curve (R=0.383, p<0.05). Our data do not provide evidence for a major role of intramyocellular DG in insulin resistance.
Enhanced cardiac fibroblast proliferation and reduced myofibroblast content in the early diabetic myocardium
Authors: Shamhart Patricia Ellen , Luther Daniel , Hodson Ben , Koshy John , Ohanyan Vahagn , Hodnichak Cheryl , Meszaros J. Gary
Published: June 2021

Diabetic patients are prone to developing myocardial fibrosis and suffer from decreased wound healing capabilities. The purpose of this study was to determine whether diabetes alters cardiac fibroblast (CF) activity, myofibroblast content, and overall function of the myocardium in a 6 week streptozotocin‐induced (STZ) type 1 diabetic model. The initial stages of dilated cardiomyopathy were evident: echocardiography revealed a 58.6% increase in corrected LV chamber volume despite no obvious hypertrophy in the STZ rats. We observed increased proliferation of CFs isolated from the diabetic heart vs. those from control hearts. Microarray analysis of mRNA from whole left ventricles revealed down‐regulation of known inhibitors of proliferation, p53 and p21, in the STZ group (p<0.05), consistent with our proliferation data. We explored the potential signaling underlying the down‐regulation of these genes, utilizing whole heart protein lysates and Western analysis. Activated AKT, a signal that inhibits p53, was elevated in the stz group. Surprisingly, the STZ group contained lower levels of the myofibroblast marker α‐SMA (34.6% control, p<0.05) and higher levels of desmin (192% control, P<0.05). These data suggest that early stage diabetic hearts have fewer myofibroblasts which may compromise wound healing, but contain more proliferative CFs which predisposes the diabetic myocardium to fibrosis.
Vasopeptidase Inhibitor Improves Coronary Dysfunction in Zucker Obese but not Zucker Diabetic Fatty (ZDF) Rats
Authors: Oltman Christine L. , Kleinschmidt Travis L. , Coppey Lawrence J. , Davidson Eric P. , Yorek Mark A.
Published: June 2021

Previous studies have shown endothelial dysfunction in coronary and mesenteric near resistance arteries in Zucker obese and ZDF rats. Our hypothesis is that vascular dysfunction is due in part to upreglulation of neutral endopeptidase and treatment with a vasopeptidase inhibitor will improve vascular function. 20‐week‐old Zucker obese and 12‐week‐old ZDF rats were treated with the vasopeptidase inhibitor AVE7688 (500mg/kg) for 12 weeks. Afterwards, coronary and mesenteric function was evaluated using an isolated vessel preparation. Vessels (75 ‐ 175µm intraluminal diameter and ~1 mm in length) were studied under hydrostatic pressure (40mm Hg) and no flow, and internal diameter was measured. Dilator responses were evaluated for acetylcholine (Ach, 10−10‐10−4 M), and calcitonin gene‐related peptide (CGRP, 10−11‐10−9 M) after constriction with U46619.

Results

ACh (10μM) responses were attenuated in arteries from untreated rats compared to responses from lean rats (coronary: Zucker obese = 49.4%, ZDF = 50.7% decrease and mesenteric: Zucker obese = 27.4%, ZDF = 33.5% decrease). ACh response was augmented in coronary vessels from AVE7688 treated Zucker obese but not ZDF rats when compared to vessels from untreated rats. Our data suggest that coronary artery endothelial dysfunction is improved when Zucker obese but not ZDF rats were treated with AVE7688. Supported by VA Merits to Dr. Oltman and Dr. Yorek.
Maintenance of contractile properties despite impaired skeletal muscle oxidative metabolism in diet‐induced obesity.
Authors: Shortreed Karin Elizabeth , Krause Matthew P , Huang Julianna H , Dhanani Dili , Ceddia Rolando , Hawke Thomas J
Published: June 2021

Diet‐induced obesity (DIO) is increasing in prevalence among youth; a time where robust muscle growth occurs. We hypothesized that consumption of a high fat diet (HFD) in early adulthood would negatively impact mouse muscle metabolism and contractile function. HFD (~60% kcal fat for 8 weeks) induced an obese and insulin resistant phenotype with reduced relative muscle mass (72 ± 1.9%) compared to control.

Using isolated single muscle fibers for analysis, HFD induced a significant impairment in palmitate and glucose oxidation (72.8 ± 6.6% and 61.8 ± 9.1% of control respectively). This novel technique was validated in intact muscle. Impaired insulin responses were noted for glucose metabolism and glycogen synthesis in HFD muscle vs. control, while glucose and palmitate uptake was not altered between groups. Preliminary RT‐PCR results demonstrate no difference in CPT‐1, PGC‐1α, PPARα or UCP‐2 expression. Further gene and protein analysis into the mechanisms underlying changes in fat uptake and oxidation are currently underway. Despite metabolic impairments, HFD muscle exhibited no loss of maximal contractile force or alteration in fatigue rates.

Our data suggest that HFD skeletal muscle is highly adaptive in the face of impaired substrate oxidation, as demonstrated by the preservation of contractile properties. These findings contribute to our understanding of the impact of DIO on developing skeletal muscle.
Impact of lipopolysaccharide (LPS) on insulin action in the conscious mouse (C57BL6/j)
Authors: Mulligan Kimberly Xaviera , Otero Yolanda , Malabanan Carlo , Bracy Deanna , McGuinness Owen P.
Published: June 2021

The impact of lipopolysaccharide (LPS) on tissue glucose uptake has been well characterized in the rat. LPS increased basal glucose uptake in both insulin‐responsive (e.g. muscle, fat) and non‐insulin responsive tissues (e.g. macrophages), while insulin‐stimulated tissue glucose uptake was decreased. In the present study, we assessed the effects of LPS in 12‐week old chronically catheterized conscious male mice (C57BL6/j). Mice received an intravenous bolus of either LPS (10ug/g body weight) or saline 1‐h after a fast began. Four hours later either a 2‐hr euglycemic (120mg/dl) hyperinsulinemic clamp was performed or saline was infused. After 100mins, a bolus of 2‐deoxy [14C] glucose was given to determine tissue‐specific glucose uptake. As LPS impairs insulin clearance, insulin levels were matched by infusing 4mU·kg−1·min−1 in mice that received saline, and 2.5mU·kg−1·min−1 in mice that received LPS. LPS decreased the basal glucose uptake by the heart (37%), soleus (21%), gastrocnemius (32%) and vastus lateralis (62%), while the adipose tissue was unaffected. In the insulin‐stimulated state the glucose infusion rate (GIR) was decreased by ~30% in animals that received LPS (54±3 vs. 38±6 mg/kg/min). Insulin‐stimulated tissue‐specific glucose uptake was decreased in the heart (32%), gastrocnemius (53%), vastus lateralis (57%), soleus (31%), and adipose tissue (44%). In summary, LPS decreased basal glucose uptake and impaired insulin‐stimulated glucose uptake in a tissue‐specific manner in the C57BL6/j mouse. While LPS impairs insulin‐stimulated glucose uptake in both the rat and mouse, the impact of LPS on basal metabolism differs. (Support DK43748 and DK59637)
Effect of shRNA inhibition of ζ‐crystallin expression on renal expression of phosphoenolpyruvate carboxykinase and glutaminase
Authors: Spond Julie Ann , Curthoys Norm P
Published: June 2021

Renal expression of phosphoenolpyruvate carboxykinase (PEPCK) and glutaminase (GA) significantly increases during the adaptive response to metabolic acidosis. This process results from selective stabilization of their mRNAs and is mediated by AU‐sequences that function as pH‐response elements (pHRE). ζ‐crystallin is one protein known to bind in vitro to the pHRE. A siRNA specific for porcine ζ‐crystallin was used to determine if this interaction is necessary for the turnover or stabilization of PEPCK and GA mRNAs. If so, effective knockdown of ζ‐crystallin expression would alter the expression of GA and PEPCK. To achieve significant knockdown, a shRNA construct was designed, cloned into a pSilencer vector (Ambion), and stably expressed in LLC‐PK1‐F+ pig kidney cells. Resulting data established that constitutive expression of the shRNA in the selected cells decreased ζ‐crystallin expression to less than 1% of normal. However, the knockout of ζ‐crystallin had no effect on PEPCK and GA levels. Further studies established that when ζ‐crystallin was knocked down, the levels of HuR and AUF1, two other proteins that bind to the pHRE, also remained unchanged. In addition, ζ‐crystallin knockdown had no effect on expression of C/EBPβ, a transcription factor required for basal expression of PEPCK. Therefore, the binding of ζ‐crystallin is not necessary for the in vivo turnover or stabilization of the GA or PEPCK mRNAs.
Nucleomics: Targeting and Genetic Analysis of Nutrient‐Sensing Cells
Authors: LaPresto Lindsay , Barthelson Roger , Cates Julia , Brooks Heddwen L , Galbraith David W , Lynch Ronald M
Published: June 2021

Pancreatic β‐cells are the most abundant nutrient sensing cell (NSC) type with fewer seen in the hypothalamus and intestine. It is difficult to study NSC's because they are embedded within other cell types in these organs. We showed that glucokinase (GK), a key enzyme in glucose metabolism in β‐cells, is also expressed in the other NSC's. To study NSC's, we developed a gene construct coding for a nuclear targeted fluorescent protein (FP) with a GK promoter to drive expression. Fluorescent nuclei can be rapidly extracted and purified by cell sorting, and the RNA isolated from these nuclei used to evaluate NSC specific gene expression. MIN6 β‐cells were transfected with this plasmid, and targeting of the FP to nuclei of only GK expressing cells was verified by microscopy. Clonal cell lines expressing the construct are now being expanded. Nuclei were isolated after cell disruption, and differential density centrifugation. All nuclei were labeled with a DNA probe DAPI (blue) and all stained nuclei were sorted by FACS, with NSC specific nuclei purified in the FP window. Results show that NSC nuclei and RNA can be isolated away from other cells, thereby providing a method for NSC specific gene analysis. This study will guide development of a mouse line in which changes in β‐cell (and nutrient sensing cells in general) gene expression can be studied during the onset of obesity and diabetes.
Modeling Regulation of Mitochondrial Free Ca2+ by Metabolite Dependent Ca2+ Buffering
Authors: Dash Ranjan K , Thompson Matthew D. , Vinnakota Kalyan C , Haumann Johan , Aldakkak Mohammed , Camara Amadou KS , Stowe David F , Beard Daniel A
Published: June 2021

Mitochondrial free Ca2+ ([Ca2+]m) is regulated by cation fluxes via Ca2+ uniporter (CU), Na+/Ca2+ exchanger (NCE), Na+/H+ exchanger (NHE), and Ca2+/H+ exchanger (CHE) as well as via Ca2+ buffering by mitochondrial proteins. However, the regulation of [Ca2+]m via metabolite dependent dynamic Ca2+ buffering in the matrix during transient state‐3 respiration is not well known. To gain a quantitative understanding of this Ca2+ buffering, we developed a computational model by integrating our recent biophysical models of CU, NCE, NHE and CHE into our existing model of mitochondrial bioenergetics. The model also accounts for binding and buffering of cations with metabolites, including ATP, ADP and Pi. Experiments were performed to spectrofluorometrically measure [Ca2+]m, pHm, ΔΨm and NADH redox state in guinea pig heart mitochondria suspended in Na+ and Ca2+ free buffer (ensured by ~50 μM EGTA) with 0.5 mM pyruvic acid (HPyr). Dynamics were inferred with various addition of CaCl2 (0, 10, 25 μM CaCl2; 16, 88, 130 nM [Ca2+]m) followed by 250 μM ADP with or without ANT and F1F0‐ATPase blockers. Model analysis of the data on (i) initial decrease of [Ca2+]m with addition of HPyr, and (ii) transient increases of [Ca2+]m with addition of ADP suggests metabolite dependent dynamic [Ca2+] buffering in the matrix. This model will be helpful to understand mechanisms by which [Ca2+]m regulates mitochondrial energy metabolism.
Phosphorylation of the Tight Junction Protein Occludin on Ser490 Regulates Barrier Function and Contributes to Growth Control
Authors: Runkle E. Aaron , Sundstrom Jeffrey M. , Gonsar Kristin B. , Antonetti David A.
Published: June 2021

Occludin was the first transmembrane tight junction protein identified but its function remains unclear. Occludin content correlates with barrier properties but gene deletion studies demonstrate it is not essential for formation of tight junctions. Recent data suggest occludin contributes to growth control. Our laboratory has shown that growth factor treatment of vascular endothelial cells alters barrier properties and leads to phosphorylation and redistribution of occludin and occludin Ser490 has been identified as a specific growth factor‐induced phosphorylation site. Here, our goal was to test the hypothesis that occludin Ser490 phosphorylation regulates barrier function in epithelial cells and contributes to control of cell growth. MDCK cell lines that stably overexpress wild type, Ser490 mutated to Ala (S490A) to block or Asp (S490D) to mimic phosphorylation were characterized. Occludin S490D promoted while S490A reduced growth rates as determined by cell count and DNA synthesis. Occludin S490A reduced permeability to 467Da TAMRA and Arg and increased electrical resistance. Finally, in a Ca2+ switch assay, S490A enhanced junction recovery rates while S490D recovered similar to empty vector. These results were further supported using confocal microscopy. Thus, phosphorylation of occludin on Ser490 regulates both barrier function and contributes to growth control in MDCK cells.

Supported by grants from NIH 012021 and American Heart Association (AHA) 0815468D.