bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional characterization of β‐arrestin 1/ERK scaffolding complex downstream of PAR2
Authors: Min Jungah , DeFea Kathryn
Published: June 2021

β‐arrestin serves as a multifunctional scaffold protein that brings together MAPK cascade modules and spatially restricts them within the cell. Downstream of protease‐activated‐receptor‐2 (PAR‐2), ERK activity is prolonged by β‐arrestins at the plasma membrane and we hypothesize that this is dependent upon precise molecular interactions that are facilitated by interaction of the ERK module with β‐arrestins. We have shown that Raf binds directly to β‐arrestin 1 in vitro, but MEK1 and ERK2 bind indirectly to via Raf. Using bioluminescence resonance energy transfer (BRET), we have shown direct interaction of Raf with β‐arrestin‐1 in cells upon PAR‐2 activation. Furthermore, using recombinant proteins, we have determined that the C‐terminus of β‐arrestin‐1 directly binds Raf, while the N‐terminus of β‐arrestin binds Raf upon PAR‐2 activation, suggesting that the composition of the β‐arrestin/ERK scaffold is dependent upon the activating receptor. Expression of either N or C‐terminal β‐arrestin truncations partially rescued PAR‐2 stimulated ERK1/2 activation in β‐arrestin knockout cells; expression of both N and C‐terminal truncations together fully rescues PAR‐2 stimulated ERK1/2 activation. Taken together, β‐arrestin 1 recruits the ERK cascade modules via direct binding to Raf and subsequent recruitment of MEK1/2 and ERK1/2 through interactions with sites on both its N and C‐terminus.
RECEPTOR‐REGULATED INTERACTION OF ACTIVATOR OF G‐PROTEIN SIGNALING 4 AND GIALPHA
Authors: Oner Sukru Sadik , Maher Ellen , Breton Billy , Bouvier Michel , Blumer Joe B.
Published: June 2021

Activator of G Protein Signaling‐4 (AGS4), via its three G‐protein regulatory (GPR) motifs, is well positioned to modulate G‐protein signal processing by virtue of its ability to bind Gαi‐GDP subunits free of Gβγ. Apart from initial observations on the biochemical activity of the GPR motifs of AGS4 very little is known about the nature of the AGS4 – G‐protein interaction, how this interaction is regulated, the influence of the AGS4 – G‐protein interaction on signals emanating from G‐protein coupled receptors (GPCRs) or where the interaction takes place. As an initial approach to these questions, we evaluated the interaction of AGS4 with Gαi1 in living cells using bioluminescence resonance energy transfer (BRET). AGS4 and Gαi1 reciprocally tagged with either Renilla luciferase (RLuc) or YFP demonstrated saturable, specific BRET signals. BRET signals observed between AGS4‐RLuc and Gαi1‐YFP were reduced by GPCR activation and this agonist‐induced reduction in BRET was blocked by pertussis toxin. In addition, specific BRET signals were observed for AGS4‐RLuc and α2‐adrenergic receptor‐Venus which were Gαi‐dependent and reduced by agonist indicating that AGS4 – Gαi complexes are receptor‐proximal. These data suggest that AGS4 – Gαi complexes directly couple to a GPCR and may serve as substrates for receptor‐induced G‐protein activation.
Lipopolysaccharide (LPS) and induction of blood borne cell‐derived microvesicles
Authors: Xiong Jing , Miller Virginia M. , Jayachandran Muthuvel
Published: June 2021

Cell membrane‐derived microvesicles (MV) are shed into the circulation and reflect cell‐cell interaction, intravascular and intercellular signaling process. Infection increases risk for thrombosis and LPS signaling through toll‐like receptor 4 (TLR4) may activate cells to generate procoagulant (phosphatidylserine surface) MV. This study was designed to investigate MV production from leukocytes and platelets exposed to LPS in vitro. Blood from wild type (WT) and TLR4 gene deleted (dTLR4) mice was incubated with LPS (500ng/ml or 5ug/ml) for 6 hours. Flow cytometer was used to quantify MV using fluorophore conjugated cell specific antibodies plus annexin‐V. Leukocyte‐ and platelet‐derived MV showed a concentration dependent increase in the blood from WT mice; whereas in dTLR4 mice increased counts of leukocyte‐ and platelet‐derived MV were observed only at the higher concentration of LPS. At the higher concentration of LPS, numbers of leukocyte‐derived MV increased significantly and the mean number of platelet‐derived MV was two fold greater than under control conditions. These results indicate that low concentrations of LPS act through TLR4 but higher concentrations of LPS act through other receptors in addition to TLR4 to increase production of leukocyte‐ and platelet‐derived MV which could account in part for increased thrombotic risk with infection. Supported by AHA‐SDG 30503Z and Mayo Foundation.
Gender‐Specific Cardiovascular Advantages In Apolipoprotein E Knockout Mice and Effects Of Captopril Treatment On Late‐Stage Atherosclerotic Lesion Progression
Authors: Procknow Jesse D , Sprague Randy S , Stephenson Alan H
Published: June 2021

Apolipoprotein E‐deficient (apoE KO) mice develop aortic lesions when fed a high fat (Western) diet. Reports suggest that pre‐menopausal females are protected from adverse cardiovascular events brought on by an increase in circulating lipids. We hypothesized that there is gender‐afforded cardiovascular protection in apoE KO mice with established atherosclerotic lesions. Six‐week old apoE KO mice were fed a Western diet for 14 weeks and received 450 mg/ml collagen/100μM epinephrine (7.5 ml/kg, i.v.) at 20 weeks to assess cardiac risk. Some mice received captopril (25 mg/kg/day) for weeks 18–20. Western‐fed apoE KO mice had higher serum cholesterol levels (males 843.4 ± 40.7, females 991.6 ± 85.5 mg/dl) compared with chow‐fed wild‐type mice (WT, males 132.1 ± 4.5, females 95.6 ± 10.7 mg/dl, both p<0.01) and elevated aortic COX‐2 expression. Lesions were greater in Western‐fed apoE KO mice than in chow‐fed mice (males 20.50 ± 0.82 vs 3.38 ± 0.19%, females 20.57 ± 1.12 vs 3.29 ± 0.15%; both p<0.01), and were reduced in male (59.9%, p<0.01) and female (50.1%, p<0.01) captopril‐treated mice. Apo E KO females fed a Western diet had a higher survival percentage than their male counterparts. We conclude that cardiovascular protection is conferred on pre‐menopausal apoE KO female mice and that two weeks of captopril significantly reduces late‐stage atherosclerotic lesions in male and female apoE KO mice.
Involvement of NF‐κB/ERK and Sp1/p38 MAPK pathways in HNE‐induced 5‐lipoxygenase expression in murine macrophages
Authors: KIM Chi Dae , LEE Seung Jin , KIM Chae Eun , SEO Kyo Won , PARK Hye Mi , YUN Jung Wook , BAE Jin Ung
Published: June 2021

5‐LO is suggested as a modulator of atherosclerotic plaque instability, and co‐exist with 4‐hydroxynonenal (HNE) in macrophages in atherosclerotic lesions. To determine the potential role for HNE on the regulation of 5‐LO expression, the promoter activity of 5‐LO was evaluated in HNE‐stimulated macrophages. A genomic sequence of promoter 2.0 kb upstream of the transcription initiation site of mouse genomic DNA was amplified by PCR, and then the constructs containing a series of sequentially deleted fragments was fused to pGL3 vector. Luciferase assay showed that promoter region 50–213 bp upstream of transcription start site was responsible for HNE‐enhanced transcriptional activity of 5‐LO. The importance of NF‐κB and Sp1 in HNE‐induced 5‐LO expression was demonstrated by siRNA knockdown of NF‐κB and Sp1 as well as by a site‐directed mutagenesis. Among the MAPK pathways, HNE‐enhanced NF‐κB and Sp1 activities were attenuated by an ERK inhibitor, and a p38 MAPK inhibitor, respectively. Moreover, the HNE‐enhanced phosphorylation of ERK and p38 MAPK was significantly attenuated by an EGF receptor antagonist, but not by PDGF receptor antagonist. Collectively, these data suggest that 5‐lipoxygenase expression by HNE is regulated via EGF receptor‐mediated activation of NF‐κB/ERK and Sp1/p38 MAPK pathways.
Platelet function and relationship to coronary artery disease (CAD) risk in early menopausal women
Authors: Miller Virginia M. , Jayachandran Muthuvel , Litwiller Robert D. , Lahr Brian D. , Bailey Kent R , Heit John A. , Owen Whyte G.
Published: June 2021

Risk of CAD increases at menopause and platelets contribute to the etiology of CAD. We hypothesized that individual variation in platelet procoagulant activity at menopause correlates with the extent of CAD as reflected by coronary artery calcification (CAC). We developed several novel and global assays of platelet procoagulant activity: dense (ATP release) and alpha (P‐selectin expression) granule secretion and GPαIIbβ3 fibrinogen‐binding (PAC1 binding) to ADP stimulation, and prostaglandin E1 (PEG1) sensitivity as a surrogate for prostacyclin sensitivity. Platelets were prepared from venous blood collected from women screened for the Kronos Early Estrogen Prevention Study (n=125; mean±SD age=52.5±2.4 years) within 19.4±9.2 months of last menses. Baseline CAC score was determined. Of conventional CAD risk factors, only systolic blood pressure correlated with positive CAC score (n=23). ATP secretion tended to be less in women with positive CAC scores and was negatively correlated with blood glucose (P<0.001). P‐selectin expression and PAC1 binding increased with total cholesterol (P<0.05). PEG1 sensitivity increased with increasing age and waist circumference (P<0.05). These results indicate that platelet functions change with age and in association with conventional risk factors for the disease in women at menopause. Supported by the Kronos Longevity Research Institute and NIH HL90639.
Interleukin 17 promotes atherosclerosis and protects against aneurysmal rupture
Authors: Madhur Meena S , Funt Samuel A , Blinder Yelena , Harrison David G
Published: June 2021

Interleukin 17 (IL17) is a unique cytokine produced by Th17 cells, a novel subset of T lymphocytes distinct from Th1 and Th2 cells. Atherosclerosis and aneurysm formation are inflammatory diseases in which Th1 cytokines play an important role. The role of IL17 in atherosclerosis is poorly defined, however, and its role in aneurysm formation is unknown. We investigated the effect of IL17 on atherosclerosis and aneurysm formation by crossing IL17 and ApoE deficient mice. Mice were then subjected to either 3 months of high fat diet or 4 weeks of high fat diet plus Ang II infusion. Aortic atherosclerosis, as defined as percent lesion area, was reduced in IL17−/−/ApoE−/− mice compared to ApoE−/− controls after 3 months of high fat diet (15% vs 20%, n=11, p=0.03) as well as after 4 weeks of high fat diet plus Ang II infusion (13% vs 21%, n=4–5, p=0.02). High fat diet plus Ang II infusion induced a similar degree of abdominal aortic aneurysm (AAA) formation but a greater incidence of death due to AAA rupture in IL17−/−/ApoE−/− mice compared to ApoE−/− controls (31% vs 7%, n=14–16). Thus, IL17 promoted atherosclerosis but protected against aneurysmal rupture. IL17 could therefore represent a novel therapeutic target for treatment of atherosclerosis but its effect on AAA rupture must be taken into consideration when designing strategies that target IL17. Supported by NIH Grants P01HL58000, R01HL39006, P01HL58000, and ACTSI TL1 RR025010.
What can the zebrafish lateral line teach us about hearing?
Authors: Piotrowski Tatjana , Lush Mark , Duncan Robert
Published: June 2021

Zebrafish and the majority of other aquatic vertebrates possess hair cells not only in the vestibular sensory epithelia of the ear but also in their skin. These hair cells are part of a sensory system called the lateral line, so‐named because the sensory organs are arranged in lines on the trunk and around the eyes. The function of the sensory lateral line is to detect water motion and initiate the appropriate behavioral response for capturing prey, avoiding predators and schooling. The sensory organs are composed of support cells surrounding hair cells that are very similar to the hair cells of the inner ear of vertebrates. Importantly, despite the unusual location of the hair cells on the trunk, lateral line and ear hair cells develop and differentiate by similar stereotyped developmental mechanisms. For example, mutations in genes causing deafness in humans also disrupt hair cell function in the zebrafish lateral line and vestibular system (reviewed in Nicolson T., 2005). However, in contrast to human inner ear hair cells, the lateral line hair cells of zebrafish regenerate after damage and are accessible to experimental manipulation and visualization. We are therefore employing zebrafish as a valuable new model system to study signaling pathways that underly the development, as well as regeneration of hair cells.
Cellular junction remodeling underlies the role for the planar cell polarity pathway in directional cochlear extension
Authors: Chen Ping , Ren Dongdong , Chacon‐Heszele Maria , Kelly Micahel C , Qian Dong
Published: June 2021

The mammalian hearing organ, the organ of Corti, is suspended along the spiraled cochlear duct and consists of stereotypical arrays of uniformly oriented sensory hair cells interdigitated with supporting cells. During development, postmitotic precursor cells of the organ of Corti undergo cellular rearrangement, resulting in its extension as well as precise patterning of sensory and non‐sensory cells and uniform orientation of hair cells. It is thought that the vertebrate planar cell polarity (PCP) pathway regulates both the cellular rearrangement for the extension of the organ of Corti and the uniform orientation of sensory hair cells.

The objectives of this study are to characterize cellular behavior and morphology during terminal differentiation of the organ of Corti, and to investigate the mechanism underlying cellular rearrangements for the extension of the organ of Corti.

We examined cellular geometry, cellular contacts and junctions in the wild‐type developing organ of Corti. We also examined the development of the organ of Corti in mouse mutants defective in an adheren junction component, p120, or in an essential PCP component, Vangl2. We are carrying out live imaging to record cellular behavior and geometric change during terminal differentiation of the organ of Corti in culture.

Our results show that the developing organ of Corti undergoes unique cellular intercalation for its directional extension. Proper remodeling of cell‐cell junctions is required for cochlear extension, and the role for the PCP pathway in cochlear extension is likely mediated by cell adhesion molecules and independent of the role for PCP pathway in hair cell orientation.
Pentoxifylline Reduces Endothelial Activation Induced by Cytokine and HIV Derived Peptides: Implications for HIV‐Associated Cardiovascular Disease
Authors: Kim Chul , Gupta Samir , Rajashekhar Gangaraju , Taylor Brian , Gollahalli Nagesh , Renwick Abby , Clauss Matthias
Published: June 2021

HIV infection is associated with increased serum levels of inflammatory markers, endothelial dysfunction, and cardiovascular events. Here we investigated the hypothesis that HIV infection enhances the activity of proinflammatory cytokines on endothelial cell activation, and assessed anti‐inflammatory treatment with pentoxifylline (PTX). Flow‐mediated dilation (FMD) was evaluated for 8 weeks in 9 HIV‐infected patients treating with PTX 400mg. Recombinant IFN‐γ and TNF‐α, either alone or together with HIV‐TAT protein was added to primary human endothelial cells in vitro. Endothelial cells were also treated with the anti‐inflammatory PTX to assess modulation of IP‐10 and VCAM‐1for gene expression through qRT‐PCR, and protein production by ELISA or using immunofluorescence. We demonstrate that HIV‐1 positive patients have decreased endothelial function as assessed by FMD compared with HIV‐1 negative patients, and that FMD can be improved by treating with PTX. In vitro, the expression levels of IP‐10 and VCAM‐1 genes in endothelial cells were significantly enhanced by HIV‐TAT when combined with IFN‐γ or TNF‐α, respectively and were significantly reduced by co‐treatment with PTX. Similarly, protein production of IP‐10 and VCAM‐1 was also increased by HIV‐TAT in combination with IFN‐γ or TNF‐α treatment, respectively, compared with single IFN‐γ or TNF‐α treatment, and significantly reduced by PTX. These results extend our understanding of the roles of HIV and systemic inflammatory mediators on endothelial inflammation and may explain the beneficial effects of PTX on endothelial function in HIV patients.
Leukocyte adhesion and its contribution on the local oxidative and nitrosative stresses in microcirculation
Authors: Kar Saptarshi , Kavdia Mahendra
Published: June 2021

In diabetes, a number of adhesion molecules are over expressed leading to leukocyte adhesion and subsequent transmigration in the vessel wall. This is one of the early stages for vascular inflammation. These adherent or transmigrating leukocytes release high levels of nitric oxide (NO) and superoxide thereby increasing local oxidative and nitrosative stresses. In the current study, we developed a computational model to understand the effect of leukocyte adhesion and transmigration on the profiles of oxidative and nitrosative stress inducing free radicals NO, superoxide and peroxynitrite in a blood vessel. The results are presented for three cases: 1) A normal case with relatively low amount of leukocyte adhesion, 2) An oxidative stress case with leukocyte adhesion and transmigration and 3) A severe oxidative stress case with reduced SOD levels. The results indicate a significant increase in peroxynitrite concentration and decrease in NO concentration for the severe oxidative stress case with respect to the normal case. Decreasing NO formation could impair vasodilation whereas increasing peroxynitrite formation may initiate oxidation of sulfydryl groups in proteins and lipids and nitration of amino acids resulting in damage to the endothelial and smooth muscle cells of the blood vessel, which can contribute to the occurrence of cardiovascular related diseases. Supported by NIH R01 HL084337.
Stressed microvilli and long tethers in rolling, tight adhesion zones and aft trunks in arresting neutrophils revealed using Total Internal Reflection Fluorescence Microscopy (TIRFM)
Authors: Sundd Prithu , Gutierrez Edgar , Pospieszalska Maria , Groisman Alexander , Ley Klaus
Published: June 2021

Neutrophils are covered with microvilli. P‐selectin‐PSGL‐1 bonds are predicted to form when the microvillus tip approaches the P‐selectin substrate to within 70 nm. A microfluidic device with a substrate coated with P‐selectin/ICAM‐1 was perfused with blood from mice expressing GFP in neutrophils. Rolling interactions were studied at realistic wall shear stresses (τw = 6–8 dynes/cm2) using TIRFM. Following bond formation, microvilli undergo compression approaching the substrate to within 25 nm near the cell center. At the trailing edge, the P‐selectin‐PSGL‐1 bonds stretch to 125–150 nm before they dissociate. Rolling is stabilized by the formation of 3–4 long (up to 16 μm) tethers (life time~1–10s). Adding the chemokine CXCL1 to the substrate induces neutrophil arrest and formation of a trunk‐like structure that stretches up to 10 μm behind the cell. The closest contact (area~1–3 μm2) between the arrested neutrophil and the substrate is always found near the cell center. Its distance from the substrate (44 nm) corresponds to the length of the ICAM‐1‐LFA‐1 bond. These results identify the molecular and cellular dimensions of neutrophils during rolling and transition to arrest, thus providing a framework for the biomechanical analysis of this fundamental process. This work was supported by 09POST2230093 from American Heart Association (P.S) and NIH EB 02185 (K.L).
Exposure to ambient ultrafine particulate matter exacerbates cardiac ischemia/reperfusion injury in a cyclosporin‐sensitive manner.
Authors: Sloan Ruben C , Frasier Chad R , Cathey Brook L , Cascio Wayne E , Lust Robert M , Devlin Robert B , Brown David A , Wingard Christopher J
Published: June 2021

The purpose of this study was to investigate the effects of exposure to ultrafine particulate matter (UFPM;<0.1 μm) on ischemia‐reperfusion injury in isolated rat hearts. Male Sprague‐Dawley rats were randomly assigned into two groups and exposed to either saline or UFPM by intra‐tracheal instillation. Twenty‐four hours after exposure hearts underwent global ischemia (20 minutes) and reperfusion (2 hours). At the end of the protocol, infarct size was measured and reported as percent of zone at risk (ZAR). In rats exposed to UFPM, infarct size was larger than saline‐exposed counterparts (67±4 vs. 49±3% of the ZAR, respectively; P<0.01). The increased infarct size in UFPM‐treated animals was significantly attenuated when cyclosporin‐A was administered in the perfusate during the protocol. Infarct sizes in cyclosporin‐treated hearts were 52±6 and 45±3% ZAR for UFPM and saline hearts, respectively. There were no differences observed between groups in hemodynamic variables or propensity for arrhythmia. Our results show that exposure to UFPM increases the susceptibility of the isolated ischemic heart to reperfusion injury, and that the expansion of the infarct in UFPM‐exposed animal hearts may involve the activity of the mitochondrial permeability transition pore.
Mitochondrial Depolarization Is Not responsible for Cardioprotection During Ischemic Preconditioning
Authors: Lee Daniel , Quarrie Ricardo , Cramer Brandon , Steinbaugh Gregory , Pfeiffer Douglas R , Zweier Jay L , Crestanello Juan A
Published: June 2021

Mitochondrial (mito) respiratory electron transfer chain (RTC) is a major source of reactive oxygen species (ROS) during ischemia reperfusion (IR). Mito membrane potential (Δψ) may affect ROS production by changing the redox status of the RTC. Mild uncoupling can lead to oxidation of the RTC and decrease ROS production. It is unclear how ischemic preconditioning (IPC) decreases mito ROS generation. We examined the changes in mito Δψ and its correlation to ROS production in IR and IPC mito.

Isolated rat hearts (n=6/group) were subjected to 1) CONTROL: 30 minutes (min) of equilibration (EQ), 30 min of ischemia (I), and 30 min of reperfusion (RP), or 2) IPC: 10 min of EQ, two 5 min episodes of I and RP, 30 min of I, and 30 min of RP. Mitos were isolated at end I and end RP. ROS production was assessed by EPR spectroscopy using DMPO as spin trap. Δψ was measured using a TPP electrode. Data is expressed as mean±sem.

ROS production by Complex I & III (arbitrary units/mg protein)
Δψ (‐mV)

End I
End RP
End I
End RP

CONTROL
2220±22
3524±340
121±8
150±2

IPC
1878±175
2479±296*
147±7*
160±3*

p<0.05 vs. CONTROL

Ischemia led to lower Δψ in IPC and CONTROL. Δψ recovered during reperfusion. However, Δψ remained higher in IPC at end I and end RP. IPC decreases ROS production during RP. Membrane depolarization was not associated with lower ROS production. We conclude that membrane depolarization is not responsible for the cardioprotective effect of IPC.
Interstitial nitric oxide concentrations are elevated in rat gastrocnemius muscle following recovery from femoral occlusion
Authors: Fabris Sergio , Smith Ashley A , Parlett Brittney , MacLean David A
Published: June 2021

The present study examined the effects of ischemia‐reperfusion on the interstitial levels of nitric oxide (NO) and adenosine (Ado). Eight anesthetized rats had two microdialysis fibers inserted into the gastrocnemius muscle of each leg and perfused with Ringers (control) or 500 μM AOPCP (5'ectonucleotidase inhibitor). Following baseline collections both femoral arteries were clamped for 60 min (samples at 20, 40 and 60 min) and then released (samples at 20 min). With Ringers perfusion there were no increases in interstitial NO (baseline = 4.2±0.4 μM) during ischemia but NO was elevated (P<0.05) following reperfusion (5.4±0.5 μM). Prior to occlusion AOPCP treatment resulted in an increase (P<0.05) in NO from 4.3±0.6 to 5.5±0.5 μM as compared to Ringers. No further increases in NO were observed with AOPCP during ischemia, but NO was further elevated (P<0.05) following reperfusion (6.5±0.6 μM). Interstitial NO concentrations were higher (P<0.05) during AOPCP treatment compared to Ringers at all time points. There were no changes in interstitial Ado during the study. These data show that NO and Ado are not altered during femoral occlusion, but NO is elevated during reperfusion and that NO is even higher during ischemia‐reperfusion when Ado production is inhibited. These findings suggest that NO plays an important role in interstitially mediating vasodialtion during skeletal muscle reperfusion.

Supported by NSERC
Substrate availability for both endothelial nitric oxide synthase (eNOS) and glucose‐6‐phosphate dehydrogenase (G6PD) result in enzyme dysfunction as a result of ischemia/reperfusion injury: the role of NADPH in endothelial dysfunction.
Authors: Reyes Levy A , Dumitrescu Cristian , Biondi Roberto , Druhan Lawrence , Zweier Jay Louis
Published: June 2021

Ischemia/Reperfusion (IR) injury causes dysfunction in the coronary vascular endothelium. Endothelial nitric oxide synthase is a critical enzyme involved in endothelial function. Previously, we showed endothelial dysfunction occurs without direct damage to eNOS, but rather via depletion of the essential eNOS cofactor, tetrahydrobiopterin (BH4). Repletion of BH4 lost during IR partially restored endothelial function. Recently, we have found that 30 min of IR injury also results in the depletion of the eNOS substrate NADPH, to ~47% of pre‐injury levels. In isolated hearts repletion of NADPH restored endothelial function (as measured by coronary flow) to near pre‐ischemic levels, highlighting the significance the IR‐induced decrease in NADPH. Our current work shows levels of NADPH are altered throughout IR injury, rising ~20% above control values at 10 min ischemia (increasing from 49.2±2.2 to 61.2±3.5 nmoles/gram tissue), and then decreasing to below baseline (31.8±7.8 nmoles/gram tissue) 20 min post‐ischemia. Glucose‐6‐phosphate dehydrogenase (G6PD) is the rate limiting enzyme in the production of NADPH. We observed that while total G6PD activity is not changed during IR injury, it is limited by substrate availability. In conclusion, the modulation of NADPH throughout IR injury, partially due to a change in substrate availability of G6PD, gives new insights to the intracellular response to IR injury.
Classical transient receptor potential channel 6 (TRPC6) is essential for ischemia‐reperfusion injury of the lung
Authors: Dietrich Alexander , Sydykov Akylbek , Kalwa Hermann , Storch Ursula , Fuchs Beate , Ghofrani Hossein A. , Gudermann Thomas W. , Weissmann Norbert
Published: June 2021

Lung endothelial damage is a characteristic feature of ischemia‐reperfusion (I/R) injury. However the molecular steps involved in the loss of endothelial integrity and subsequent invasion of immune cells are still poorly understood. Most notably, in isolated lungs from TRPC6‐deficient mice we observed a resistance to I/R injury, while lungs from wild‐type mice displayed pulmonary edema formation and organ dysfunction. To differentiate between the role of TRPC6 in circulating and/or resident leukocytes versus endothelial cells, TRPC6‐deficient bone marrow cells were transplanted into lethally irradiated wild‐type mice and vice versa. Based on these experiments in chimeric mice it is evident that endothelial but not leukocytic TRPC6 deficency protects from I/R injury of the lung. Therefore, pulmonary endothelial cells from wild‐type and TRPC6‐deficient mice were isolated and further analyzed. TRPC1, TRPC4 and TRPC6 were the predominantly expressed TRPC isoforms in these cells. Primary isolated endothelial cells from TRPC6‐deficient lungs show reduced ischemia‐induced Ca2+ influx and subsequent actin stress‐fiber formation as well as a decreased cellular shape change when compared to wild‐type cells. Thus, TRPC6 represents a promising novel pharmacological target to control critical early stages of I/R injury in the lung. Supported by the von‐Behring‐Röntgen Stiftung.
Compartmentalized nitric oxide signaling at the myoendothelial junction can regulate gap junction communication
Authors: Straub Adam C. , Johnstone Scott R. , Best Angela K. , Palmer Lisa , Isakson Brant E.
Published: June 2021

Myoendothelial junctions (MEJ) couple endothelial cells (EC) and vascular smooth muscle cells (SMC) in resistance vessels via gap junctions (GJ). Throughout the MEJ, caveolin‐1, which regulates endothelial nitric oxide synthase (eNOS) derived nitric oxide (NO), is extensively localized. Therefore, we tested the hypothesis that eNOS is compartmentalized at the MEJ and that NO can regulate heterocellular communication by S‐nitrosylation of connexin (Cx) proteins. To test this, we immunolabeled for eNOS and identified its expression at the MEJ in vivo (by TEM) and in our vascular cell co‐culture (VCCC) model (by confocal). We found increased eNOS phosphorylation at serine 1177 at the MEJ compared to the EC monolayer. This eNOS phosphorylation was selectively increased at the MEJ upon stimulation of SMCs with phenylephrine (PE) but not at the EC monolayer. Using the biotin switch assay, we determined the degree of S‐nitrosylated Cx43 at the MEJ. One minute after PE stimulation of SMCs, de‐nitrosylation of Cx43 was observed, followed by progressive re‐nitrosylation up to 20 min post‐PE stimulation. Functionally, this correlated with an inhibition of UV‐uncaged IP3 movement from SMC to EC 1 min after PE stimulation, which was restored 20 mins post PE stimulation. These results suggest that S‐nitrosylation of Cx proteins likely maintain open GJ channels and can contribute to heterocellular communication at the MEJ.
Nuclear factor‐ kappa B expression and matrix metalloproteinase activity in hypertension
Authors: Wu Kwan‐I , Schmid‐Schönbein Geert W.
Published: June 2021

Our recent evidences suggest that inflammation of the spontaneously hypertensive rat (SHR) is associated with an uncontrolled matrix metalloproteinase (MMP) activity. We hypothesize that the transcription factor NF‐κB is overexpressed in the SHR and involved in the overexpression of MMPs.

NF‐κB expression level and translocation were compared between Wistar Kyoto rat and SHR kidney, heart, and brain. In addition, the animals were treated with a NF‐κB inhibitor, pyrrolidine dithiocarbamate (PDTC), for ten weeks. To determine the correlation between NF‐κB and MMP, MMP‐2 and −9 activities were examined after treatment.

Immunohistochemistry showed that NF‐κB expression is significantly increased in SHR in renal glomerular and tubulointerstitial areas and brain hypothalamus compared to that in WKY (p<0.05), but not in myocardium and cerebral cortex. Significant NF‐κB translocation into the nucleus was found in renal tubular cells, cardiac muscle cells, and arterioles of kidney and brain in SHR.

After PDTC treatment, the systolic blood pressure in SHR was suppressed by 36 % as compared to no significant difference in WKY rats. NF‐κB expression level in treated‐SHR was also decreased in renal glomerular and tubulointerstitial areas and hypothalamus compared to non‐treated animals (p <0.05). Furthermore, MMP‐2 activity was significantly reduced by PDTC treatment in both WKY and SHR. The enhanced MMP‐9 activity in SHR was also significantly decreased by PDTC (p <0.01). These results suggest NF‐κB is an important transcription factor in the SHR and may be responsible for its enhanced MMP activity.

Supported by HL 10881
Impaired Arteriolar Dilation in a Mouse Model of Familial Hypercholesterolemia: Impact of Chronic Exercise and Anti‐Cholesterol Therapy
Authors: Stapleton Phoebe A. , Goodwill Adam G. , Morrisette Michael R. , James Milinda E. , Frisbee Jefferson C.
Published: June 2021

Hypercholesterolemia is a strong risk factor for negative cardiovascular outcomes and, while mechanistic benefits of ameliorative therapies have not been fully elucidated, it has been clearly demonstrated that evolution of familial hypercholesterolemia (FH) can culminate in profound impairments to microvascular function. To interrogate thus, we hypothesized that FH‐induced increases in oxidant stress and inflammation will impair skeletal muscle resistance arteriolar dilation due to a decreased NO bioavailability. Vascular responses to NO‐dependent stimuli were attenuated with FH, and responses were improved by antioxidant treatment, suggesting oxidant scavenging of NO. Chronic ingestion of ezetimibe/simvastatin did not restore vascular reactivity with FH, but improved inflammation through reductions to TNF‐α, IL‐6, and CRP. Chronic swimming exercise caused minor improvements to vascular dilation, but improved antioxidant protection through increased SOD‐1 and catalase expression. Combination therapy improved dilator reactivity, and lowered inflammation and oxidant stress. Taken together, these observations suggest that genetic dyslipidemia creates a condition wherein endothelial function is compromised, negatively impacting dilator responses. Interventions which can reduce both oxidant stress and chronic inflammation appear to be effective in improving endothelial function.
Erythrocytes (RBCs) of humans with Type 2 diabetes (DM2) release ATP in response to activation of the prostacyclin receptor (IPR) but not in response to low PO2
Authors: Knebel Stephanie , Ellsworth Mary , Bowles Elizabeth , Sprague Randy , Stephenson Alan
Published: June 2021

The ability of RBCs to release ATP when exposed to low O2 permits these cells to participate in the matching of O2 delivery with demand in skeletal muscle. We previously reported that ATP release in response to low PO2 requires activation of the G protein, Gi and that both low O2‐induced ATP release and Gai2 expression are impaired in DM2 RBCs. Since human RBCs also express the Gs‐coupled prostacyclin receptor (IPR), whose activation similarly results in ATP release, we hypothesized that, in contrast to low O2 tension, IPR‐mediated ATP release is preserved in RBCs of humans with DM2. Healthy human (HH) and DM2 RBCs were isolated and incubated with iloprost (ILO, 1 μM), an IPR agonist. We determined that ILO increased ATP release by 328 ± 92% in HH RBCs (n=13, P<0.05) and by 557±149% (n=10, P<0.01), a value which was significantly greater than that observed for HH RBCs. Interestingly, in DM2 patients with good glycemic control (HbA1c = 7, n=3), ILO‐induced ATP release did not differ from that of HH. However, in individuals with poor glycemic control (HbA1c = 9.4±0.4, n=7), ILO‐induced ATP release was significantly greater (771±150%). These results demonstrate that IPR‐mediated ATP release is conserved in DM2 with the magnitude of ATP release in response to activation of the IPR receptor pathway increasing as glycemic control worsens. Funded by NIH grants HL‐64180 and HL‐89094.
Age‐related changes in stress‐induced cardiovascular and lumbar sympathetic nerve activity responses
Authors: Erdos Benedek , Basgut Bilgen , Whidden Melissa , Kirichenko Nataliya , Tumer Nihal
Published: June 2021

Central angiotensin II (AngII) signaling plays an important role in stress‐induced cardiovascular and sympathetic responses. However, the effects of age and high salt (HS) diet on central AngII‐mediated mechanisms are not known. We compared restraint stress‐induced BP, HR and lumbar sympathetic nerve activity (LSNA) responses in young and old rats under control conditions, after central infusion of losartan (15 μg/μL/hour), an AngII type‐1 receptor inhibitor, and after feeding rats with 8% HS diet for 10 days. BP, HR and LSNA were measured in young (5 mo) and old (27 mo) Fischer 344 x Brown Norway rats using radiotelemetry. Restraint stress was induced by placing animals in acrylic cylinder restrainers. Old rats had significantly higher resting BP and lower HR compared with young. Restraint stress induced rapid BP, HR and LSNA increases in both young and old; however, changes were significantly reduced in the old rats. Losartan significantly inhibited stress‐induced BP increases in young rats, while it had no effect on HR responses. In contrast, in the old rats, both BP and HR responses remained unaffected by losartan. HS diet feeding increased baseline BP both in young and old rats, with a more pronounced effect in old animals; however, it did not significantly alter restraint‐stress‐induced responses. In conclusion, resting BP increases with age, but hypertensive responses to stress diminish with age due to a decline in AngII‐mediated central mechanisms.
Peripheral vascular responses to an orthostatic challenge and plasma volume in Parkinson's disease patients with orthostatic hypotension
Authors: Hopman Maria TE , Thijssen Dick HJ , Esselink Rianne AJ , van Aalst Michiel , Seeger Joost PH , Bloem Bas R , Groothuis Jan T
Published: June 2021

Orthostatic hypotension (OH) is present in 50% of Parkinson's disease (PD) patients. A reduced peripheral vasoconstriction and an inadequate plasma volume may contribute to OH in these patients. Indeed, plasma volume expansion is considered as an effective therapy. The objective was to compare baseline plasma volume and peripheral vascular responses to an orthostatic challenge in PD patients with OH (PD+OH, n=15) with PD patients without OH (PD‐OH, n=17) and controls (C, n=15). We examined superficial femoral artery blood flow using duplex ultrasound during a 60° head‐up tilt (HUT). Leg vascular resistance (LVR) was calculated. In a subset (9 PD+OH, 9 PD‐OH and 8 C), we determined plasma volume by indicator dilution method with radio‐labeled albumin. We found a lower baseline LVR in PD+OH (0.6±0.3 mmHg/ml/min) compared with PD‐OH (1.2±0.6 mmHg/ml/min) and C (1.3±0.5 mmHg/ml/min). LVR increased during 60° HUT to a similar magnitude in all groups (PD+OH +0.3 to 0.9±0.1; PD‐OH +0.5 to 1.7±0.3; C +0.7 to 2.1±0.3 mmHg/ml/min). Plasma volume was larger in PD+OH (62±3 ml/kg) compared with PD‐OH (54±6 ml/kg) and C (54±8 ml/kg). In conclusion, PD+OH have a lower LVR in combination with a higher plasma volume compared with PD‐OH and C. Although PD+OH increase LVR during 60° HUT to a similar extend as the other groups, they are unable to maintain their blood pressure in an upright position, possibly due to the attenuated basal LVR.
Role of iNOS in Intrinsic Myocyte Dysfunction during Progression of Left Ventricular Hypertrophy
Authors: Kim Song‐Jung , Chavez Jaime , Crystal George J.
Published: June 2021

Our objective was to assess the role of inducible nitric oxide synthase (iNOS) in the intrinsic myocyte dysfunction in the compensated and uncompensated stages of left ventricular hypertrophy (LVH). 19 dogs were divided into three groups, Control (CON; n =7), LVH (n=7), and LVH/HF (n= 5), and instrumented for measurements of hemodynamics and LV function. LVH was induced by chronic aortic banding. Progression into LVH/HF was achieved by rapid ventricular pacing. Cardiac myocytes were isolated so that intrinsic contractile function (percent contraction; %C) could be assessed by video imaging. LV systolic pressure and wall stress, and LV wall thickness were increased 2‐fold in LVH compared to CON. LV end‐diastolic pressure was increased 4‐fold and LV dP/dt max was decreased by 30% in LVH compared to LVH/HF. %C was reduced by 30 % in LVH compared CONT (9.4±0.3%). However, in similarity to CON, 1 mM L‐arginine (the substrate for iNOS) caused no change in %C in LVH. Furthermore, expression of iNOS (Western blot) was not increased in LVH relatve to CON. %C was further reduced in LVH/HF. In contrast to LVH, L‐arginine caused marked decrease in %C in LVH/HF, which was blunted by specific iNOS inhibitor AMT. iNOS expression was increased more than 3‐fold in LVH/HF. Taken together, our results suggest that iNOS does not play a role in the intrinsic myocyte dysfunction during compensated LVH, but does so during the uncompensated stage.
Short term STZ‐induced diabetes reduced left ventricular dysfunction and promoted morphometric, immune and antioxidant adaptations after myocardial infarction in rats
Authors: Candido Georgia Orsi , Mendes Roberta Hack , Rodrigues Bruno , Rosa Kaleizu Teodoro , de Souza Leandro Ezequiel , Di Petta Antonio , Sammoniya Paulina , Belló‐Klein Adriane , De Angelis Kátia , Irigoyen Maria Claudia
Published: June 2021

We verified if left ventricular (LV) dysfunction induced by myocardial infarction (MI) in STZ‐diabetic rats was related to collagen deposition, inflammatory response and antioxidant enzymes profile. Male Wistar rats were divided: Control (C), Diabetes (D), MI (I) and diabetes +MI (DI). MI area was similar between I (36±6%) and DI (30±4%) groups at the end of protocol (5 days after MI surgery). However, ejection fraction (I: 38±4 vs. DI: 48±5%), fractional shortening (I: 25±2 vs. DI: 37±2%), and E/A ratio were improved in DI in comparison with I rats. Collagen (by picrosirius red stain) (25%) and myocardium inflammatory cells quantifications (by Hematoxin‐Eosin) (71%) was reduced in DI as compared with I animals; while heart antioxidants superoxide dismutase (25%) and catalase (10%) were increased in DI when compared to I rats. In addition, a positive correlation was observed between collagen and inflammatory cells quantifications (r=0.7; p<0.0001) in all groups. This study showed that the attenuation of LV dysfunction observed in DI rats is probably due to a compensatory mechanism associated with morphometric and immune adaptations, as well as an increase of antioxidant enzymes activity. These data suggest an important role of heart glucose availability in myocardial protection after an ischemic event.

FAPESP
Imidazolines as single agent therapy for diabetics with hypertension
Authors: Edwards Lincoln Paul , Crane Louis , Baziard‐Mouysset Genevieve , Brown‐Bryan TerryAnn
Published: June 2021

Pharmacologic treatment of many diabetics also involves treating hypertension. This requires taking multiple drugs, which increases the risk of drug interaction, and decrease compliance and glycemic control. Imidazoline compounds have the potential to act as single agent therapy to treat diabetics with hypertension, thereby positively impacting compliance. We hypothesize that imidazoline compounds act centrally to lower blood pressure and decrease hyperglycemia by stimulating glucose uptake.

Moxonidine (5nmol) and S43126 (5nmol), were injected cumulatively into the brain of spontaneously hypertensive rats, followed by measurement of blood pressure. HEK 293 kidney cells were treated with moxonidine (1μM) and glucose uptake measured. The imidazoline receptor antisera selective (IRAS) protein was overexpressed in HEK 293 cells, followed by treatment with moxonidine. Western blot was used to determine the phosphorylation of protein kinase B.

Our results showed that both S43126 and moxonidine lowered arterial pressure by 20mmHg and 45mmHg respectively. Moxonidine caused a greater than 6 fold increase in glucose uptake at 60 min of treatment in HEK 293 cells. Cells overexpressing the IRAS protein showed a greater phosphorylation of PKB after treatment with moxonidine.

These data suggest that imidazoline compounds may be developed as alternative therapy to treat diabetics with hypertension.
Chronic and acute postprandial hyperglycemia enhances the gene expressions of inflammatory cytokines in the peripheral leukocytes in rats
Authors: Goda Toshinao , Fujimoto Saki , Tanaka Yutaro , Fukaya Nanae , Shimada Masaya , Mochizuki Kazuki
Published: June 2021

Postprandial hyperglycemia is thought to cause inflammation in many tissues. In this study, we examined whether the gene expression of inflammatory cytokines in peripheral leukocytes are altered by chromic and/or acute postprandial hyperglycemia. Seven‐week‐old male Sprague‐Dawley rats received a high‐fat (64 energy%) diet for 77 days to induce an insulin resistance. Blood was collected from tail vein, and the total RNA extracted from peripheral leukocytes was subjected to microarrays and real‐time RT‐PCR. In another experiment, rats treated with streptozotocin (35 mg/kg BW) received an oral dose of sucrose (2 g/kg BW) in the presence or absence of α‐glucosidase inhibitor, miglitol, and the gene expressions of peripheral leukocytes were analyzed thereafter. Microarray analysis of the genes in peripheral leukocytes showed that rats with chronic postprandial hyperglycemia had greater expressions of the genes coding inflammatory cytokines (IL‐1β, S100 a8/a9) and those related to host defense. Oral sucrose loading in rats with glucose intolerance led to a 2‐fold increase in IL‐1β and TNF‐α mRNA levels in peripheral leukocytes within 3h, which was abolished by addition of miglitol to the sucrose solution. These results suggest that both chromic and acute postprandial hyperglycemia enhances the gene expression of inflammatory cytokines in peripheral leukocytes.
Fresh Frozen Plasma Induces Nitric Oxide Production Through AMPK‐Akt‐mediated eNOS Activation
Authors: Deng Xiyun , Doursout Marie‐Francoise , Cao Yanna , Duan Chaojun , He Guangchun E. , Kozar Rosemary A. , Holcomb John B. , Ko Tien C.
Published: June 2021

Hemorrhagic shock (HS) is the leading cause of potentially preventable death in trauma. Fresh frozen plasma (FFP) has been used to treat HS in trauma centers with survival benefits. The beneficial effects of FFP have been attributed to its coagulation properties; however, we propose that FFP has direct beneficial effects on vascular function. Activation of endothelial nitric oxide synthase (eNOS) induces NO, a key mediator of normal endothelial function. We hypothesize that part of the beneficial effects of FFP is due to its ability to induce NO production. In a standardized rat HS‐resuscitation model, we found that resuscitation with FFP resulted in an earlier and greater induction of NO in the sera compared to standard crystalloid lactated Ringer. In vitro, FFP induced NO in the conditioned media of human pulmonary microvascular endothelial cells (HPMECs) and dermal lymphatic endothelial cells (HDLECs). Western blotting revealed that FFP induced activation of AMPKα1, Akt1 and eNOS in HPMECs and HDLECs. eNOS activation was confirmed in the lung tissues of FFP‐resuscitated HS rats. Pretreatment with Compound C (AMPK inhibitor), LY294002 (PI3K/Akt inhibitor) or L‐NAME (NOS inhibitor) inhibited FFP‐induced eNOS activation and NO production in HPMECs. Together, this study reveals a novel mechanism by which FFP exerts its vasoprotective effect during HS.
Pre‐ and postconditioning with the mitochondrial ATP‐sensitive potassium (mitoKATP) channel opener BMS‐191095 protects cerebral endothelial cells against oxygen glucose deprivation (OGD)
Authors: Institoris Adam , Gaspar Tamas , Katakam Prasad V. , Domoki Ferenc , Bari Ferenc , Busija David W.
Published: June 2021

We have shown that the selective mitoKATP channel opener BMS‐191095 protects cultured rat cortical neurons against lethal OGD via acute and delayed preconditioning. We have tested if BMS‐191095 also protects rat brain endothelial cells (RBEC) against lethal OGD, in vitro. Primary RBECs were pretreated with BMS‐191095 (10, 20, 30μM) for 1 day or 1 hour (h) before and 1h immediately after 2h of OGD. OGD reduced viability from 100% to 53±1% but 1 day pretreatment and 1h post‐treatment dose dependently increased viability to 76±2% and 74±1%, respectively (n=16/group; p<0.05). 1h pretreatment was ineffective. Protection could be abolished using the phosphoinositide 3‐kinase (PI3K) inhibitor wortmannin (47±1% and 54±1%; n=16‐16), but not with the selective mitoKATP channel blocker 5‐hydroxydecanoate (66±1% and 71±1%; n=16‐16). Western blot analysis showed a rapid phosphorylation of Akt and ERK 1/2 which peaked at 15 minutes after BMS‐191095 application. Treatment for 24h did not change manganese superoxide dismutase and endothelial nitric oxide synthase levels. In summary, BMS‐191095 prevents OGD‐induced death of RBECs likely by mechanisms involving the PI3K‐Akt and ERK 1/2 pathways.