bluuebunny/crossref_metadata_2025 huggingface dataset:
Down‐regulation of 3‐Hydroxy‐3‐Methyl Glutaryl Coenzyme‐A Reductase in HEPG2 Cells Using shRNA
Authors: Shah Kaitav Jayesh , Patel Khyati , Bhatt Vrushank , Boggeti Renuka , Ratna Warren N
Published: June 2021

3‐hydroxy‐3‐methyl glutaryl coenzyme‐A (HMG Co‐A) reductase is an enzyme that catalyzes the committing step in the pathway involved in the de novo biosynthesis of cholesterol in the liver. Pharmacotherapeutic management of hypercholestrolemic patients includes treatment with statins, mevalonate analogs, which are competitive inhibitors of HMG Co‐A reductase. To investigate the effectiveness of short hairpin RNA (shRNA)‐mediated targeted degradation of HMG Co‐A reductase mRNA as a means of inhibition of the expression of the enzyme, plasmid constructs containing sequences from three different regions of the HMG Co‐A mRNA were cloned into pGEM‐3Z vector. Each construct contained 21 nucleotide (nt) sense sequence and the corresponding antisense sequence separated by a 7‐nt spacer sequence. The shRNAs were synthesized by run‐off transcription using T7 RNA polymerase and transfected into human hepatocellular carcinoma (HEPG2) cells. RNA was extracted by NP‐40 lysis method and analyzed by RT‐PCR and Northern blotting. Western blot analysis was performed on whole cell lysates. All three shRNAs caused degradation of the HMG Co‐A mRNA, resulting in the inhibition of the expression of HMG Co‐A reductase, indicating the potential use of these shRNAs to down regulate HMG Co‐A reductase and consequently reduce cholesterol biosynthesis. This work was supported by AMS College of Pharmacy, Long Island University, NY.
Lactobacillus rhamnosus GG treatment potentiates intestinal hypoxia‐inducible factor, promotes intestinal integrity, prevents inflammation, and ameliorates alcohol‐induced liver injury
Authors: Wang Yuhua , Liu Yanlong , Kirpich Irina , McClain Craig , Feng Wenke
Published: June 2021

Gut‐derived endotoxin is a critical factor in the development and progression of alcoholic liver disease (ALD). Probiotics have been used to treat animal models of alcohol‐induced liver injury associated with gut leakiness and endotoxemia as well as human ALD. However, the mechanism(s) of their beneficial action are not well defined. We hypothesized that alcohol impaired the adaptive response induced hypoxia‐inducible factor (HIF), and that probiotic supplementation could attenuate this impairment and restore the barrier function in a mouse model of ALD by increasing HIF responsive proteins such as intestinal trefoil factor; thus reversing established ALD. C57BJ/6N mice were fed the Lieber deCarli diet containing 5% alcohol for 8 weeks. Lactobacillus rhamnosus GG (LGG) were supplemented in the last two weeks. LGG supplementation significantly reduced alcohol‐induced endotoxemia, hepatic steatosis, TNF‐α level and improved liver function. LGG restored alcohol‐induced reduction in the protein levels of HIF‐2α and intestinal trefoil factor and normalized mRNA expression of toll‐like receptors in the liver. In vitro studies using the Caco‐2 cell culture model showed that the addition of LGG supernatant prevented alcohol‐induced epithelial monolayer barrier dysfunction. Furthermore, gene silencing of HIF‐1α/2α abolished the LGG effects, indicating that the protective effect of LGG is HIF‐dependent. This study provides a mechanistic insight for utilization of probiotics for the treatment of ALD, and suggests a critical role for intestinal hypoxia and decreased trefoil factor in the development of ALD.
Long‐term effects of indocyanine green on lymphatic pump function
Authors: Weiler Michael , Dixon J. Brandon
Published: June 2021

Introduction

Near‐infrared (NIR) imaging with indocyanine green (ICG) has emerged as a potential technique for diagnosing lymphatic diseases, however, the long‐term effects of ICG on lymphatic function have not been studied in vivo. Therefore, we quantified the retention rate of ICG in lymphatic vessels and the effect of repeated ICG injections on lymphatic pump function.

Methods

Rats were given intradermal ICG injections and subsequently imaged using NIR to quantify lymphatic function as members of one of four treatment groups: (i) initial ICG injection with no follow‐up injection, (2) follow‐up ICG injection after 1 week, (3) 2 week follow‐up ICG injection, and (4) 1 month follow‐up ICG injection.

Results and Conclusions

ICG remained visible in rat collecting lymphatics for approximately two weeks, and NIR lymphatic function measurements after follow‐up ICG injections of 1 and 2 weeks showed significant reductions in fluid velocity, pulsatile flow frequency and clearance rate while no significant differences were observed after 1 month follow‐up ICG injections. These results indicate that ICG is retained in the local tissue space for an extended period and may suggest that lymphatic function is reduced during this time. ICG‐based NIR imaging diagnostics for lymphatic function, therefore, may necessitate a minimum of 1 month between subsequent ICG injections to avoid erroneous measurements of function.
RHO KINASE‐MEDIATED CALCIUM SENSITIZATION IN TONIC AND PHASIC CONTRACTIONS OF MESENTERIC COLLECTING LYMPHATIC VESSELS
Authors: Kurtz Kristine M , Souza-Smith Flavia M , Breslin Jerome W
Published: June 2021

The mechanisms underlying the lymphatic contractile cycle, consisting of phasic contractions superimposed over tone, are poorly understood. We hypothesized that Rho kinase (ROCK) promotes lymphatic contractile activity. The spontaneous contractions of isolated, pressurized (2 cm H2O) rat mesenteric collecting lymphatics were studied. ROCK was inhibited with with H‐1152 (0.1–10μM). Vessel diameter and cytosolic free [Ca2+] were simultaneously measured in a subset of vessels loaded with Fura‐2 AM. Inhibition of ROCK increased end diastolic diameter and end systolic diameter in a concentration‐dependent manner, associated with decreased lymphatic tone at 1–10 μM H‐1152 (1 and 10 μM: 7.6 ± 2.6 and 5.8 ± 1.7 % vs. control: 22.5 ± 6.6 %, P<0.05 and P<0.01 respectively). H‐1152 also significantly decreased phasic contraction amplitude. Transient increases in cytosolic [Ca2+] preceded each phasic contraction. After H‐1152 was added, this pattern ceased for about a minute, followed by a return of the Ca2+ transients without the accompanying phasic contractions. H‐1152 caused no change in basal cytosolic [Ca2+] despite the decrease in tone. Our data suggest that ROCK is essential for both tonic constriction and phasic contractions in lymphatics. We propose that ROCK increases Ca2+ sensitivity of contractile molecules in lymphatics. Supported by NIH P20RR018766 and Louisiana Board of Regents D‐00004465‐2010.
Resolving inflammation in stroke through FPR2/ALX
Authors: smith helen k , gavins felicity
Published: June 2021

Ischaemia/reperfusion (I/R) injury in stroke can worsen patient outcome through excess inflammation. We investigated the pharmacological potential of an endogenous anti‐inflammatory mechanism, formyl peptide receptor 2/lipoxin receptor (FPR2/ALX; Fpr2/Fpr3 in mouse), in reducing I/R‐associated inflammation in a mouse model of stroke.

Mice underwent 5 min global cerebral ischaemia, followed by 40 min or 2 h reperfusion. FPR agonists—Ac2‐26 (Annexin A1 pharmacophore) and 15‐epi‐lipoxin A4—were administered at the start of reperfusion. Subsequent leukocyte‐endothelial (L‐E) interactions (characteristic of an inflammatory response) were quantified through a cranial window using intravital video microscopy.

15‐epi‐lipoxin A4 administration dramatically reduced L‐E interactions at 40 min reperfusion but less so at 2 h. Ac2‐26 treatment decreased leukocyte adhesion at 40 min and all L‐E interactions at 2 h. Combined treatment with Ac2‐26 plus FPR antagonists Boc2 or WRW4 (FPR2/ALX‐selective) abrogated the effects of Ac2‐26.

We show that activity through Fpr2/Fpr3 reduces L‐E interactions in a model of global cerebral I/R. These data provide evidence that FPR2/ALX could be a therapeutic target in stroke.

This was funded by the BBSRC.
Forty five minutes of ischemia and 24 h of reperfusion in the Wistar rat induce an acute pre‐renal injury which is not improved by 4 days of pre‐ischemia treatment with Spironolactone
Authors: Acuña Dulce Mariela Montes , López-Rodríguez Juan Francisco , Calvo-Turrubiartes Miriam Zaharí , Varela Norma Leticia Dávila , LLamazares-Azuara Lilia , Rodríguez-Martínez Manuel
Published: June 2021

To characterize what kind of renal injury is induced by 45 min of ischemia and 24 h of reperfusion (I/R) and assess the effect of 4 days of pre‐ischemia treatment with Spironolactone (Sp = 20 mg/kg/day/o.g.gavage) or Vehicle (V = 560μl/day/o.g. gavage) on this I/R model, Wistar male rats (~250g of bw) were random assigned to three groups: V+Sham‐I/R, V+Real‐I/R & Sp+Real‐I/R and the following parameters were measured: glomerular filtration rate (GFR = CCr = ml/min/100g), urinary protein excretion (UPrV = mg/24h/100g), fractional excretion of sodium (FENa = %), free water clearance (CH20 = ml/min/100g).

n
GFR
UPrV
FENa
CH2O

V+Sham‐I/R
8
0.51±0.04
7.2±0.6
2.1±0.8
−2.0±3.3

V+Real‐I/R
8
0.28±0.03#
16.2±3.0°
2.7±1.2
−3.4±3.0

Sp+Real‐I/R
8
0.12±0.03&
17.3±3.0°
15.7±5.5°*
−0.7±4.0

Values are mean ± SEM;

°p< 0.05,

p=0.01,

p=0.0001 vs. V+Sham‐I/R;

p<0.05,

p<0.005 vs. V+Real‐I/R

There were no differences among groups in 24h urinary nitrogen oxides excretion, renal malondialdehyde content or baseline renal superoxide production either.

Conclusions

45 min of ischemia and 24h of reperfusion in the Wistar rat induce an acute pre‐renal injury, which unexpectedly was not improved by 4 days of pre‐ischemia treatment with spironolactone. Supported by CO9‐FAI‐03‐20.20.
Neutrophil sling: A new cell‐autonomous adhesive structure enabling rolling at high shear
Authors: sundd prithu , Gutierrez Edgar , Pospieszalska Maria K , Koltsova Ekaterina , Kuwano Yoshihiro , Fukuda Satoru , Groisman Alex , Ley Klaus
Published: June 2021

Cell flattening and long tethers are known to facilitate neutrophil rolling at shear stresses of 6 dyn/cm2 or higher. When DiI‐stained mouse neutrophils were allowed to roll on P‐selectin in a microfluidic device at a shear stress of 6–10 dyn/cm2 and footprints recorded using quantitative dynamic footprinting (qDF), we found that long tethers did not retract, but instead appeared as ‘slings’ at the front of rolling neutrophils. Slings were confirmed in an acute model of inflammation in vivo using intravital microscopy of mouse cremaster. P‐selectin‐glycoprotein‐ligand (PSGL)‐1 was presented as discrete patches while integrin LFA‐1 was expressed over the entire length on slings. As neutrophils rolled forward, slings wrapped around the rolling neutrophils and underwent a step‐wise peeling from the P‐selectin substrate enabled by the tandem failure of PSGL‐1 patches. Failure of PSGL‐1 patches was concomitant with spurts in rolling velocity. This study reveals a previously undiscovered cell‐autonomous adhesive structure, “sling”, which enables neutrophils to roll at high shear stress during inflammation. This study was supported by the American Heart Association‐11SDG7340005 (P.S.) and NIH EB02185 (K.L.).
Novel Inhibition of Monocyte Attachment to Endothelial Cells Involves Hypersulfated Heparan Sulfate
Authors: Ali Mohamed , Phillips Shane
Published: June 2021

The attachment of monocytes to the endothelium involving Heparan Sulfate Proteoglycans (HSPG) is an early hallmark of cardiovascular disease. Previous studies indicate that increased cell surface expression and sulfation of HSPG increases monocyte attachment and contributes to disease. The purpose of this study was to test the hypothesis that protamine derived decapeptides with molecular binding tropism to hyper‐sulfated Heparan Sulfate inhibit monocyte attachment to endothelial cells. We implemented an in vitro endothelial cell flow system with live‐cell time‐lapse microscopy. Human Umbilical Vein Endothelial Cells (HUVECs) were activated with Tumor Necrosis Factor (TNF‐α 15 ng/ml for 24 hours). Monocytic leukemia cells THP‐1 were labeled with Calcein AM for fluorescence microscopy and flow cytometric determination of attachment to HUVEC under laminar flow (5 dynes/cm2). Treatment with marine‐protamines derived cationic peptides (PRTIB‐ONCMY:13‐22) and (PRT1A‐ONCMY:2‐11) at molar concentration of 50μM reduced THP‐1 attachment to HUVECs (60±9% reduction) and preferentially bound to activated HUVECs. Activated HUVECs exhibited increased uptake (35±12%) of the fluorescence labeled peptides compared to controls. We conclude that protamine derived peptides inhibit leukocytes attachment to activated endothelium and may be a novel therapeutic intervention for inflammatory conditions.
Insulin ameliorates myocardial inflammation and promotes recovery of T cell in murine experimental autoimmune myocarditis
Authors: Zhang Yuan , Zhuang Ran , Geng Chaoqun , Tian Na , Cai Xiaoqing , Lei Wen , Gao Feng
Published: June 2021

Aim

Glucose‐insulin‐potassium is a useful adjunct to myocarditis. Besides its essential action in energy metabolism, insulin also exerts an anti‐inflammatory effect. This study investigated the effect of insulin on myocardial inflammation in experimental autoimmune myocarditis (EAM) mice and its underlying mechanisms.

Methods

BALB/c mice were immunized subcutaneously with myocarditogenic peptide MyHC‐a and randomly divided into mock‐immunized, saline‐treated and insulin‐treated groups. T lymphoma cell line EL4 was used for in vitro study.

Results

Insulin reduced pro‐inflammatory cytokine IL‐1β with no changes of blood glucose and autoantibody production. Inflammatory infiltrates were less abundant in insulin‐treated hearts than those in saline‐treated hearts on day 21 after immunization. Meanwhile, insulin significantly reduced sera cTnI of EAM mice on day 14 and 21 (P<0.05). Interestingly, insulin promoted T cell recovery. CD3+ T cells in insulin‐treated mice proliferated more vigorously than those in saline‐treated mice without changing the memory/naïve T cell ratio. Moreover, ERK1/2 inhibitors blocked the insulin‐induced EL4 cells proliferation.

Conclusions

These data suggest that insulin exerts an anti‐inflammatory action in EAM and increases T cell proliferation by activating ERK1/2.

Supported by the State Key Program of National Natural Science Foundation of China (No. 81030005)
Differential Role of Complement in Microvascular Thrombosis in Two Models of Experimental Sepsis
Authors: Stark Ryan , Rumbaut Rolando
Published: June 2021

Bacterial infections can promote a pro‐inflammatory cascade via activation of the complement system; complement activation is associated with a pro‐thrombotic state. We showed previously that microvascular thrombosis is enhanced by two models of experimental sepsis, cecal ligation and perforation (CLP) and endotoxemia (induced by lipopolysaccharide, LPS). Thus, we tested the hypothesis that two key components of the complement system: factor C3 and the receptor for C5a (C5aR), mediate enhanced thrombosis in sepsis. We challenged C3‐ and C5aR‐deficient mice, or wild type control mice, with LPS or CLP. We then assessed the kinetics of photochemical injury‐induced thrombosis in mouse cremaster venules. While LPS promoted microvascular thrombosis in wild type mice as compared to saline controls, it had no effect on thrombosis in mice deficient in either C3 or C5aR (time to thrombotic occlusion was reduced by 37% in wildtype vs 9% in knockouts, p<0.05). In contrast, CLP enhanced thrombosis in both complement‐deficient strains as compared to sham‐operated controls. Our studies to date suggest differential roles of complement in microvascular thrombosis in endotoxemia and polymicrobial sepsis. The mechanisms accounting for this differential response remain to be clarified. Support: T32 HL007939‐07, and a Merit Review Grant from The Department of Veterans Affairs.
Simvastatin improves cardiovascular sympathetic modulation and endothelial function of resistance arteries from hypercholesterolemic mice
Authors: Moraes-Silva Ivana C , Souza LE , Rossoni LV , Irigoyen MC
Published: June 2021

Can simvastatin treatment (S) alter sympathetic cardiovascular control and mesenteric resistance arteries (MRA) relaxation of LDL receptor knock out (L) mice? Male L mice were treated with S (2 mg/kg, i.p., 7 days) or vehicle. C67Bl/6 mice were used as control (C). Total cholesterol (TC), blood pressure (BP) and autonomic modulation were analyzed. MRA rings were studied in an isometric myograph. Concentration‐response curves to Ach (0.01nM‐30μM) were obtained in rings incubated for 30 min with L‐NAME (100 μM), a NOS inhibitor, tetraethylammonium (TEA; 5mM), a K+ channel blocker, or with vehicle. TC was 3x higher and BP was increased (systolic:30%, diastolic:7%) in L vs. C whereas S had no effect. Cardiac autonomic balance, which was 3x higher, and sympathetic modulation to the vessels, 5x higher in L vs. C, were significantly reduced in L+S (17 and 57%, respectively). MRA % of relaxation to Ach was impaired in L (37±2%) vs. C (92±5%) while S restored it to 70±6%. In C MRA, L‐NAME and TEA inhibited Ach‐induced relaxation in 46±8% and 74±5%, respectively (p<0.05 L‐NAME vs. TEA). This inhibition was ~90±2% in L for both drugs (p<0.05 vs. C); while S shifted it to 70±7% of relaxation inhibition (p<0.05 vs. C and L). S partially restored MRA endothelial function in L mainly by modulating NO concomitantly with an improved autonomic cardiovascular control even without changes in systemic BP and lipid levels. Support: CNPq
Novel Laser Microsurgery and Imaging Techniques for the Longitudinal Study of Structural Adaptation of Microvascular Networks
Authors: Gruionu Gabriel , Munn Lance L.
Published: June 2021

Invasive microsurgical techniques are currently used to induce collateral vessel formation by resecting or ligating a limited number of arteries feeding a vascular network. The objective of this study was to develop a non‐invasive technique for selectively blocking a specified number of small arterioles, venules and capillaries in a dense microvascular network such as the mouse dorsal skin fold chamber. We used a multiphoton laser to selectively ablate small vessel fragments through the glass window of the chamber in transgenic FVB/N mice, which express GFP in Tie‐2‐positive cells and Ds red in α‐SMA‐positive cells. Blood perfusion was visualized by i.v. injection of a fluorescence vascular tracer. We assessed diameter, length, and blood flow changes and tracked cellular dynamics during remodeling using time‐lapse fluorescence microscopy and Optical Frequency‐Domain Imaging (OFDI). The microsurgery did not interrupt blood flow to neighboring vessels or cause detectable tissue bleeding or damage. After an initial decrease, the diameter of upstream and downstream vessels returned to pre‐surgery levels within 24hours. In the following days, endothelial cells migrated to repair ablated regions of arterioles, eventually restoring flow. The new techniques can be further applied to study microvascular changes in normal and pathological (e.g. tumor) microvascular environments. Supported by NIH R01CA149285.
Impaired Blood Pressure Compensation after Hemorrhage in Obesity
Authors: Xiang Lusha , Clemmer John , Lu Silu , Lee Lynn , Hester Robert
Published: June 2021

Increased morbidity and mortality following severe hemorrhage have been shown in obese humans and animals, whereas the mechanisms are unknown. The compensatory mechanisms following hemorrhage involve an increase in total peripheral vascular resistance (TPR) along with capillary fluid shifts. We hypothesize that the cardiovascular response and capillary fluid shifts following hemorrhage are impaired in obese Zucker rats (OZ), an animal model of obesity. Blood pressure (BP), heart rate (HR), arteriolar and venous blood gas, and oxygen consumption (VO2) were measured in lean Zucker rats (LZ) and OZ (11–12 wk) before and 1 hour after 35% total blood volume loss. Cardiac output (CO) was estimated using the Fick principle. The basal BP, CO, HR, TPR, and HCT were not different between LZ and OZ. After the 1 hour recovery period, both LZ and OZ exhibited decreased BP, CO, VO2, and hematocrit (HCT) along with increased TPR. The BP recovery was impaired in OZ (64% ± 3% recovery from basal) as compared with LZ (79% ± 2% recovery from basal). The HR was restored in the LZ but was decreased in the OZ (Table). Additionally, OZ exhibited a lower TPR and a higher HCT than LZ (Table). These results suggest impaired blood pressure compensation in OZ following severe hemorrhage due to an attenuated vasoconstriction and capillary fluid shift. (Supported by NIH HL‐51971 and HL‐89581)
NOX4 upregulation increases superoxide and mitochondrial dysfunction in brain endothelial cells
Authors: Horne James N , Krause Diana N , Kemper Martin , Duckles Sue P , Weiss John H
Published: June 2021

Reactive oxygen species (ROS) are key contributors to cardiovascular pathophysiology. The primary generators of cell ROS are mitochondria and NADPH oxidases (NOX); however possible interactions between these two sources are not clear. We have shown in mouse brain endothelial cells (cultured bEND.3 cells) that mitochondrial superoxide increases with ischemic insults that cause cell death. We hypothesized that angiotensin II (ANGII), another promoter of vascular dysfunction, would increase mitochondrial ROS and do so via its known ability to activate NOX enzymes. We found ANGII (10uM) treatment 24 or 48 h of endothelial (bEND3) cells increased mitochondrial superoxide generation 3‐fold (using Mitosox dye) and decreased mitochondrial membrane potential (using TMRM). These effects were suppressed by diphenylene iodonium chloride (DPI), an inhibitor of all NOX isoforms, and VAS2870 (VAS), selective inhibitor of NOX4. ANGII increased protein levels of NOX4, but not NOX1 and NOX2. We found NOX4 localized to the mitochondria; NOX4 protein levels measured in isolated mitochondria were increased 1.5 fold by ANGII. Together, our data suggest a novel action of ANGII to increase mitochondrial superoxide in endothelial cells through increased expression and mitochondrial localization of NOX4, a constitutively active ROS generator. Chronically, this effect would lead to mitochondrial damage and endothelial dysfunction. Supported by US NIH RO1 HL50775
Lower body muscle tensing is an effective countermeasure to initial orthostatic hypotension induced cerebral hypo‐perfusion upon standing from a squatted position
Authors: Walsh Jeremy J , Bentley Robert F , Kellawan J Mikhail , Tschakovsky Michael E
Published: June 2021

PURPOSE

to examine the effects of 5 s of lower body muscle tensing (LBMT) after rising from squat on middle cerebral artery blood velocity (MCABV) following a squat‐to‐stand protocol.

METHODS

Healthy subject (n =8; 24.4 ±1.9 yrs) performed a squat‐to‐stand protocol. Upon standing participants performed either no manoeuvre (Control) or LBMT which was held for 5 s. MCABV (cm/s; surrogate for cerebral perfusion) was measured with transcranial Doppler ultrasound (TCD), mean arterial pressure (MAP) via finger photoplethysmography. Cerebral blood pressure (CBP) was calculated by measuring the hydrostatic column from the heart (MAP) to the TCD probe. Cerebral vascular conductance (CVC) was calculated from MCABV and CBP.

RESULTS

Data are mean ±SD. The drop in MAP upon rising from squat was −15.8 ±3.9 mmHg in Control vs. −6.02 ±10.5 mmHg in LBMT, p < 0.05. This represented a 62% attenuation of hypotension via LBMT. MCABV decreased by −11.8 ±3.07 cm/s in Control vs. −3.72 ±4.48 cm/s in LBMT, P <0.05, which represents a 68% attenuation of cerebral hypo‐perfusion.

CONCLUSIONS

LBMT for the first 5 s upon rising from squat is an effective countermeasure for minimizing cerebral hypo‐perfusion. NSERC.
Enhanced external counterpulsation (EECP) increases GLUT‐4 protein expression, capillary density and glucose tolerance in patients with abnormal glucose tolerance
Authors: Martin Jeffrey S , Beck Darren T , Aranda Juan M , Braith Randy W
Published: June 2021

We sought to evaluate the effects of EECP therapy on outcomes of glucose tolerance and skeletal muscle morphology in patients with abnormal glucose tolerance (AGT).

Methods

Eighteen patients with AGT were randomly assigned (2:1 ratio) to receive either 7 weeks (35 1‐hour sessions) of EECP (n = 12) or 7‐weeks of standard care (control; n = 6). Biochemical assays, oral glucose tolerance, and skeletal muscle morphology were assessed before and after EECP or control.

Results

Following EECP, plasma vascular endothelial growth factor (VEGF) was increased (9.74 ± 1.59 to 17.04 ± 2.65 pg/mL, P<0.05). Vastus lateralis skeletal muscle biopsies demonstrated improvements in capillary density (454 ± 21 to 497 ± 19 mm−2, P<0.05) following EECP. There was no change in glucose uptake signaling pathways, but total GLUT‐4 protein expression was elevated (+47%, P<0.05) following EECP. Fasting (−16.9 ± 5.4 mg/dL) and 120‐min (−28.3 ± 7.3 mg/dL) plasma glucose from oral glucose tolerance testing were also improved following EECP (P<0.05). Further, the Matsuda index, a whole‐body composite insulin sensitivity index, increased (+21%, P<0.05).

Conclusions

EECP mediated increases in capillary density and GLUT‐4 in patients with AGT paralleled improvements in surrogates of insulin sensitivity and glucose tolerance.

This research was supported by the Department of Applied Physiology and Kinesiology at the University of Florida.
Pathogenesis of insulin resistance syndrome in Western‐style diets‐induced obese and non‐obese experimental animal models
Authors: Huang Jiung-Pang , Hung Li-Man
Published: June 2021

Western diet induces obesity and consequently leads to insulin resistance, diabetes, and cardiovascular disease (CVD) is threatening to become a global epidemic. Although, more than 70 % of insulin resistance patient is obese, insulin resistance also occurs in non‐obese individuals. Hence, the present study was aimed to characterize the insulin resistance syndrome in obese and non‐obese and further explore its pathogenesis. The animals were fed with different western‐styles diet for 12 week and divided into five groups: (1) chow diet control group, control; (2) high fat diet, HFat; (3) high fructose diet, HFru; (4) high cholesterol diet, HC; (5) high cholesterol diet plus with 10% fructose in drinking water, HCF. The insulin resistance syndrome was observed in both HFat‐obese and HCF‐nonobese rats. The phosphorylated Akt (thr308) protein level was increased in adipose tissues of four western‐styles diets compared to chow diet control. The phosphorylated Akt (Ser473) protein level was increased in the adipose tissues of HFat, whereas it was reduced in the adipose tissues of HC and HCF. The GLUT4 level was downregulated in the adipose tissue of HFat, HC and HCF. The crown‐like structures around adipocytes were observed especially in HC and HCF groups. Up‐regulated NFκB/IκB ratio showed the higher inflammation degree in HFat, HC, and HCF groups. The lower MnSOD and Cu/ZnSOD proteins exhibited a weaker antioxidative ability and resulted raising of in superoxide anion in HC and HCF adipose tissues. The cleaved caspase 3 protein increased implying that adipocytes underwent apoptosis process especially in HC groups. In conclusion, our results indicate the causal relationships between inflammation and insulin resistance in non‐obese insulin resistance.
IRE1‐alpha signaling can be related to palmitate‐induced insulin resistance in L6 myotubes
Authors: Silva Patricia Ebersbach , Poletto Ana Claudia , Anhe Gabriel Forato , Machado Ubiratan Fabres
Published: June 2021

Recent studies showed that elevated free fatty acids (FFAs) can induce endoplasmic reticulum (ER) stress, and this, in turn, plays a role in the development of insulin resistance (IR). This work aimed to verify the effects of palmitate on GLUT4 and ER stress proteins in L6 cells. GLUT4, IRE1‐alpha and TRAF2 proteins were analyzed in myotubes treated with 0,75mM of palmitate during 1 to 12 hours. The results showed that palmitate caused a significant reduction of the GLUT4 protein content in all treatment periods (control: 99.99±4.3, 2 hours: 66.37±7.2*, 4 hours: 65.03±8.0*, 6 hours: 67.16±8.8*, 12 hours: 58.88±5.5*), the analysis also showed an increase of IRE1‐alpha protein within 4 hours of incubation (control: 100.00±0.0, 4 hours: 184.16±49.45*) and an important increase of TRAF2 protein at the first hour of treatment (control: 100.00±0.0, 1 hour: 125.20±9.07*). Results were expressed in arbitrary units, n=4 to 6, *P<0.05. These data showed that palmitate impaired GLUT4 expression what is preceded by activation of IRE1alpha/TRAF2 pathway. This data reinforces the proposition that FFA‐induced ER stress is associated with IR probably by reducing GLUT4 content through mechanisms not fully identified.

Support: Fapesp 2010/09984‐5
Mammalian target of rapamycin regulate kidney oxygen consumption by controlling mitochondrial function via regulation of uncoupling protein 2
Authors: Sivertsson Ebba , Friederich Malou , Palm Fredrik
Published: June 2021

Excessive kidney oxygen consumption (QO2) without concomitantly increased delivery results in tissue hypoxia, which is a common pathway to the development of kidney disease, including diabetic nephropathy. Mammalian target of rapamycin (mTOR) regulates cell proliferation but also mitochondrial function. However, the role for kidney QO2 is presently unknown. We therefore investigated in vivo QO2 and also studied specific mechanisms using isolated mitochondria from control and diabetic rats.

Control and streptozotocin‐diabetic rats were administered rapamycin (0.15 mg/day) for 14 days. Kidney function was measured under Inactin anesthesia.

Inhibition of mTOR increased kidney QO2 (+121% and +31%) and decreased tubular transport efficiency (−50% and −36%) in both controls and diabetics without affecting glomerular filtration rate. mTOR inhibition increased QO2 of isolated mitochondria by inducing uncoupling via uncoupling protein‐2.

In conclusion, mTOR inhibition increases kidney QO2 by inducing mitochondrial uncoupling, which may aggravate tissue hypoxia. Thus, mTOR inhibition in patients with pre‐existing nephropathy may accelerate the progression of disease.
The Reduction of Renal Injury in Diabetic Dahl Salt‐sensitive Rats with Insulin is Associated with Decreased MMP Activity
Authors: Slaughter Tiffani Nicole , Spires Denisha , Rucker Carlos , Wells Adrienne , Kyle Patrick , Roman Richard , Williams Jan Michael
Published: June 2021

The current study examined whether treatment with a therapeutic dose of insulin would reduce the progression of renal injury in Dahl salt‐sensitive (SS) rats treated with streptozotocin (STZ). Nine week‐old SS rats were treated with (1) vehicle, (2) STZ (50 mg/kg, i.p.) to induce diabetes and given an insulin implant (2 U/day, s.c) to maintain plasma glucose between 300–400 mg/dL, or (3) STZ and two insulin implants (4 U/day, s.c.) to normalize plasma glucose levels. Rats were fed a low sodium diet containing 0.3% NaCl to minimize the development of hypertension. At 18 weeks of age, proteinuria increased to 302±36 mg/day in STZ SS rats versus 103±13 mg/day in vehicle SS rats. Treatment with a therapeutic dose insulin prevented with severe development of proteinuria in STZ SS rats (101±14 mg/day). The kidneys of STZ SS rats exhibited glomeruloslcerosis and renal fibrosis and the development of renal injury was associated with elevations in matrix metalloproteinase‐2 and ‐9 (MMP‐2 and ‐9) activities in the renal cortex when compared to vehicle‐SS rats. However, administrations of the therapeutic dose of insulin markedly improved the renal histopathology and normalized renal cortical MMP‐2 and ‐9 activities. These results suggest that controlling plasma glucose levels slow the progression of renal disease in this model. The study was supported by a PHRMA Starter grant and NIH grants HL36279 and HL29587.
Protection of Hippocampal Neuronal Cells from Buthionine Sulfoximine‐induced Oxidative Stress by Food‐derived Phase 2 Detoxifying Enzyme Inducers
Authors: Kim Jong-Sang , Lim Ji Sun , Seo Jiyeon , Jung Chaelim , Han Jung-Hwa , Park In-Sil , Kang Hye-ryung , Kim Seong Soon
Published: June 2021

The brain is sensitive to oxidative stress because it consumes a large amount of oxygen and has much polyunsaturated fatty acids. The dysfunction of the antioxidant/detoxification system may lead to neurodegenerative diseases such as Alzheimerˈs disease, Parkinsonˈs disease, and brain cerebral ischemia. Buthionine sulfoximine (BSO) is known to inhibit gamma‐GCS which is a precursor of glutathione. The imbalance of the oxidized/reduced forms of glutathione generates excessive reactive oxygen species and leads to oxidative stress in neuronal cells. Phytochemicals, present in a variety of plants, have numerous bioactive effects such as anticancer, anti‐inflammatory, and antioxidant activities. We hypothesized that phase 2 enzyme inducers protect neuronal cells from BSO‐induced oxidative damage. In order to test the hypothesis, HT22 neuronal cells pretreated with the known phase 2 enzyme inducers including dehydroglyasperin C (DGC) and acerogenin A was exposed to BSO. We also examined whether the expression of the antioxidant/phase 2 detoxifying enzymes is mediated by the nuclear translocation of Nrf2. DGC and acerogenin A were found to protect the neuronal cells from BSO‐induced toxicity, probably due to the induction of antioxidant enzymes such as heme oxygenase1 (HO‐1), glutathione reductase (GR), and NAD(P)H:quinone oxidoreductase 1 (NQO1) which, in turn, was mediated by Nrf2 signaling pathway.
Drug transporter expression and function in primary cultures of human renal epithelial cells
Authors: Jenkinson Sarah Elizabeth , Brown Colin
Published: June 2021

Primary epithelial cell cultures are an important model for studying drug transporters in vitro. They can be used as a replacement for animal models and possibly are more suitable due to variation in transporter expression between animal and human tissue. The aim of this study was to validate the use of primary epithelial cell cultures as a model for studying drug transporter expression and function in human kidney.

Primary epithelial cells were isolated from human kidney tissue samples and cultured for 7 to 10 days. qPCR was used to determine mRNA expression of key transporters in the primary cells. Cells were grown on permeable filter supports to create polarised monolayers and transport of a range of radio‐labelled compounds was tested across the cell monolayers. Dye retention assays were used to determine the presence of functional efflux transporters.

Expression of mRNA for a range of prototypic transporters including OCT‐2, MDR1 and MRP was confirmed. Basolateral to apical transport of various compounds including metformin and indomethacin was shown suggesting possible elimination via the kidney. Hoechst 33342 retention assay showed expression of functional MDR1. CMFDA dye retention assay showed presence of MK571 sensitive transporters, likely to be MRPs. These findings show that primary cell cultures are a good model for studying drug transporter expression and function in human kidney.
Probing the Structure of the hSGLT1 Sugar Binding Site
Authors: Gorraitz Edurne , Loo Donald D.F. , Hirayama Bruce A. , Jiang Xuan , Wright Ernest M.
Published: June 2021

The crystal structure of vSGLT shows that sugar is bound in the center of the protein separated from the extra‐ and intracellular compartments by gates. We have investigated the sugar binding vestibule of the human Na+/glucose cotransporter hSGLT1 by labeling residues in the sugar binding site with tetramethylrhodamine methanethiosulfonate (TMR‐MTS). H83, T287, and Y290 were individually mutated to cysteine, expressed in Xenopus oocytes, and their access to external TMR‐MTS was monitored by simultaneous fluorescence and electrophysiological recordings. Treatment with TMR‐MTS completely inhibited sugar transport but only in the presence of external Na+. Voltage‐jumps elicited changes in rhodamine fluorescence which followed the presteady state charge movements for T287C and Y290C. Labeling blocked the H83C capacitive currents. There was no effect of external sugar on the fluorescence but, in contrast, rhodamine fluorescence changes were dependent on the external Na+. In summary, external TMR‐MTS gains access to the sugar binding site and blocks sugar binding but not sodium‐dependent partial reactions. We conclude that external Na+ triggers a conformational change in hSGLT1 to open the external gate and permit sugar binding. This large conformational change can accommodate molecules with a volume up to 1,700 Å3, i.e. TMR‐MTS or >50 water molecules. Support by NIH grant DK 19567 to E.M.W.
Nadph oxidase regulates alveolar epithelial sodium channel (ENaC) activity and lung fluid balance in vivo via O2‐ signaling
Authors: Helms My N , Goodson Preston , Jain Lucky , Koval Michael
Published: June 2021

To define roles for reactive oxygen species (ROS) and ENaC in maintaining lung fluid balance in vivo, we used multispectral and X‐ray imaging of C57BL/6J mouse lungs. We found that inhibition of NADPH oxidase with a Rac‐1 inhibitor, NSC23766, resulted in alveolar flooding which correlated with a decrease in lung ROS production in vivo. Consistent with a role for Nox2 in alveolar fluid balance, Nox2−/− mice showed increased retention of airspace fluid as compared to wild type controls. Interestingly, X‐ray analysis of C57BL/6J lungs IT instilled with LPS showed an acute stimulation of lung fluid clearance and ROS production in vivo which was abrogated by the ROS scavenger tetramethylpiperidine‐N‐oxyl (TEMPO). Acute application of LPS increased the activity of 20pS non‐selective ENaC channels by individual rat type 1 cells; the average number of channel and single channel open probability (NPo) increased from 0.14 ±0.04 to 0.62 ±0.23. Application of TEMPO to the same cell attached recording caused an immediate significant decrease in ENaC NPo to 0.04 ±0.03. These data demonstrate that, in vivo, ROS has the capacity to stimulate lung fluid clearance by increasing ENaC activity. NIH R00‐HL‐09222601awarded to MH.
Endoplasmic Reticulum Stress in Cardiovascular and Metabolic Control during DOCA‐Salt Treatment
Authors: Grobe Justin L , Hilzendeger Aline M , Siel Rick B , Davis Deborah R , Davisson Robin L , Mark Allyn L , Sigmund Curt D
Published: June 2021

Chronic exogenous deoxycorticosterone acetate and high dietary sodium (DOCA‐salt model) results in hypertension and elevated resting metabolic rate (RMR), through activation of the brain renin‐angiotensin system. We hypothesized that endoplasmic reticulum (ER) stress within the brain mediates the cardiovascular and metabolic consequences of DOCA‐salt treatment. C57Bl/6J mice were instrumented with radiotelemetric blood pressure probes and infusion pumps to deliver the chemical chaperone Tauroursodeoxycholic Acid (TUDCA, 5.28 ug/day) or aCSF vehicle into the lateral cerebral ventricle (ICV) and treated concurrently for three weeks with DOCA‐salt. Mean arterial pressure (24 hour MAP: aCSF 114.0 ± 3.0, n=13 vs TUDCA 116.5 ± 7.4, n=15 mmHg) and heart rate (528 ± 10 vs 529 ± 6 BPM) did not differ before treatments. Following DOCA‐salt, MAP increased comparably (+14.4 ± 5.3 vs +12.7 ± 8.6 mmHg), but ICV TUDCA suppressed the bradycardic effect of DOCA‐salt (−81 ± 15 vs −44 ± 9 BPM, P<0.05), supporting a possible role for ER stress in baroreflex regulation. RMR at thermoneutrality was lower with ICV TUDCA than with aCSF after 3 weeks of DOCA‐salt (DOCA+aCSF 3.38 ± 0.07, n=9 vs DOCA+TUDCA 3.16 ± 0.06, n=11 mL O2/100g/min, P=0.03), but body mass did not diverge (+0.90 ± 0.42 vs +0.91 ± 0.23 g/3 wk). These data support potential roles for brain ER stress in both the cardiovascular and metabolic effects of DOCA‐salt treatment.
Electromyographic chewing pattern following a MCA photothrombotic stroke
Authors: Condrey Jillian , Adams Sherry , Davenport Paul W.
Published: June 2021

Stroke is t commonly associated with dysphagia, altered chew patterns and breathing difficulties which can result in aspiration pneumonia. A middle cerebral artery (MCA) stroke causes cerebral ischemia which affects the insular and somatosensory cortexes. We hypothesized that MCA photochemical thrombotic stroke will disrupt chewing motor patterns in conscious rats. Animals were implanted with EMG electrodes into the right and left masseters, right and left cranial digastricus muscles and the right and left caudal digastricus muscles. The masseter contracts when the mouth is closed and the digastricus muscles contract when the mouth is open. Recordings are taken pre stroke and the right and left masseter muscles EMG patterns were synchronous bilaterally. The diagastricus muscles were bilaterally synchronous and contracted after masseter contraction. Right MCA stroke was induced by injection of a photosensitive dye, Rose Bengal, which is followed with focal laser irradiation of the artery to form a photothrombosis stroke. Within 48 hours post stroke, right and left masseter EMG activity was out of phase with the left masseter contracting synchronously with the digastricus muscles. These results demonstrate the right MCA stroke alters the coordination of chewing muscle motor activity.
Plasma Aldosterone Levels Increase After Cerebral Ischemia and Mineralocorticoid Receptor Antagonism at the Time of Reperfusion Reduces the Size of the Infarct
Authors: Dams Carla Maria , Sinclair Elaine Bliley , Dorrance Anne McLaren
Published: June 2021

Ischemic stroke causes blood brain barrier (BBB) breakdown by an unknown mechanism. Upregulation of tight junction proteins Claudin‐5 (CLN‐5) and Occludin (OCC) is linked to increased BBB permeability with hypoxia. Chronic MR antagonism reduces stroke damage but the acute effects of antagonism are unknown. We hypothesized that aldo increases post‐stroke, and acute MR antagonism reduces damage by regulating CLN‐5 and OCC. Wistar Kyoto (WKY) rats had 2 hours (h) of middle cerebral artery occlusion (MCAO) and reperfusion (R) (n=8), control rats had sham surgery (CON; n=4). Plasma was collected before and, 6 and 48 h post‐surgery. Aldo, measured by ELISA was increased 6 h after MCAO (848±60 vs 449±87 pg, MCAO vs CON, p<0.05) and remained elevated at 48 h (1104±48 vs 625±100, MCAO vs CON, p<0.05). To study acute MR inhibition, WKY rats had 3 h of MCAO and 45 h of R. Spironolactone (SPIR; 25mg/kg I.P.) was given at R (n=11), CON rats received vehicle (n=11). SPIR reduced the infarct size. CLN‐5 mRNA expression, assessed by qRT‐PCR was reduced by SPIR; OCC mRNA unchanged. Interestingly, OCC and CLN‐5 protein expression were decreased by SPIR. These data suggest that aldo increases after stroke and MR inhibition may prevent the damage caused by ischemia by maintaining BBB integrity

Protein
mRNA

%HI
CLN‐5
OCC
CLN‐5
OCC

SPIR
25.7±3.5*
0.2±0.1*
2.2±0.3*
0.2±0.04*
0.2±0.1

CON
35.1± 5.0
0.6±0.1
5.8±1.3
0.4±0.1
0.4±0.04

Mean±SE,

p<0.05
Effects of nordihydroguaiaretic acid and dimethylsulfoxide on experimental cerebral ischemia
Authors: Villanueva Cleva , Aguilar Jenner , Guevara-Balcazar Gustavo , Alva Arturo , Medina-Santillan Roberto , Kross Robert David
Published: June 2021

Cerebral ischemia was produced in male Wistar rats through the permanent ligation of the right carotid and the right middle cerebral arteries. Three groups of rats (n=8) were treated, one hour prior to cerebral ischemia, with: 1) Nothing (control group), 2) An NDGA (10–30 mg/Kg, sc) aqueous solution of dimenthylsulfoxide (DMSO:water 1:3), and 3) DMSO. Eight hours thereafter, the animals were neurophysiologically evaluated and sacrificed by anesthesia overdose. The brain was sliced and incubated in 1% tetrazolium red to evaluate edema and infarct volume. Compared to the control, both the NDGA‐DMSO solution and the DMSO itself significantly decreased edema and infarct volume. Since there was no difference between these treatments, it was concluded that DMSO but not NDGA provided the protection. An additional group was treated with DMSO one hour after cerebral ischemia. The protective effect of DMSO was similar before and after ischemia. This protective effect after ischemia suggests that DMSO could be a useful drug not only for the prevention but also for the treatment of cerebral ischemia. This study was supported by grants SIP20110949, SIP20110954 and CONACYT083090
Melatonin affects the expression of mitochondrial fission‐fusion proteins in cultured neurons
Authors: Wappler Edina A , Katz Paige S , Katakam Prasad V.G , Busija David W
Published: June 2021

Melatonin (MT) is cytoprotective in different rodent brain ischemia models. MT also increases mitochondrial complex protein I and IV activities, displays antioxidant effects, and stabilizes mitochondrial (mito) membrane potential. However, the effect of MT on mito dynamics has not been examined. Primary rat cortical neurons were isolated from E18 Sprague Dawley fetuses, and were exposed to OGD followed by up 3, 6, or 24 hr reoxygenation. Mito dynamics was investigated by measuring the expression of mito fission protein dynamin‐related peptide 1(Drp1) and fusion proteins mitofusin‐1 and ‐2 (Mfn1, Mfn2) following MT treatment ± OGD. Protective effect of MT treatment was assessed by cell viability assay. We also investigated the effects of MT receptor (MTR) 1 and 2 blocker, Luzindol and MTR3 blocker, prazosin together with MT treatment. MT protected neurons following OGD and this was unaffected by MTR blockers. MT increased mfn‐1 expression in controls, whereas it did not change mfn‐2 expression. MT also increased Drp‐1 expression after OGD that was further increased by both MTRs. In conclusion, MT affords cytoprotection possibly by modulating mitochondrial dynamics in neurons both under normoxic and anoxic conditions. In addition, MTR blockers also influence mitochondrial fission‐fusion proteins; however, they do not alter neuroprotective effect of MT.
Physiological basis of poorer respiratory outcomes for males following preterm birth
Authors: Harding Richard , Ishak Noreen , Hanita Takushi , Sozo Foula , De Matteo Robert
Published: June 2021

Our aim was to identify the physiological basis for the “male disadvantage” in respiratory function in preterm infants, using an ovine model of preterm birth in which survival of males is less than in males. At ~125 days of gestation (DGA; term ~147DGA), fetal sheep underwent preparatory surgery. Ewes received lowdose betamethasone (5.7mg i.m.) at 131DGA. At 133DGA, lambs were delivered via caesarean section and were studied for 8 hours with no ventilatory support. Arterial and intrapleural pressures were recorded continuously and arterial blood was sampled every 15min. Lambs were then euthanized for static lung compliance measurement. At 6–8h after preterm birth, males had significantly lower arterial pH and higher PaCO2 than females (p<0.05). No differences were observed in lactate, glucose and mean arterial pressure. Static lung compliance was 30% lower in males than in females (p=0.012). There was a positive correlation between static lung compliance and mean pH (from 6–8h) and a negative correlation with mean PaCO2 (both p<0.05). Male preterm lambs have stiffer lungs than females at the same gestational age, leading to CO2 retention and acidemia, and is the likely cause of the “male disadvantage”. The lower lung compliance of males is likely a result of differences in surfactant composition.

Funding was provided by the National Health and Medical Research Council of Australia.