bluuebunny/crossref_metadata_2025 huggingface dataset:
Assembly of the prothrombinase complex on fibroblast surface, promoted by TSP1, results in cytokine release and CTGF upregulation
Authors: Rico Mario C , Rough James J , Del Carpio‐Cano Fabiola E , Safadi Fayez F , Kunapuli Satya P , DeLa Cadena Raul A
Published: June 2021

Activated factor X (FXa) and thrombin can upregulate the gene expression of connective tissue growth factor (CTGF) on fibroblasts. Since TF is expressed on fibroblasts, we hypothesized that fibroblasts may assemble the prothrombinase complex leading to CTGF upregulation. In addition, the effect of thrombospondin‐1 (TSP1) was evaluated. HS‐68 fibroblasts were incubated with purified FVII, FX, FV, FII and Ca++ in the presence and absence of TSP1 and generation of FXa and thrombin assessed with chromogenic substrates. SMAD pathway phosphorylation was detected via Western‐blot analysis. Pre‐incubation of FVII with fibroblasts lead to its activation by cell‐surface expressed TF, which in turn in the presence of FX, FV, FII and Ca++ lead to the generation of FXa (9.7 ± 0.8 nM) and thrombin (7.9 ± 0.04 U/mL x10‐3). Addition of TSP1 significantly enhanced thrombin generation (23.3 ± 0.7 U/mL x10‐3 p<0.05) but not FXa generation (8.5 ± 0.6 nM). FXa and thrombin generation lead to upregulation of CTGF. Interestingly TSP1 alone upregulated CTGF, TNFα and IL‐6. The upregulation of CTGF and IL‐6 was mediated via the TGFβ pathway as judged by phosphorylation of the SMAD pathway, which was blocked by a TGFβI receptor antagonist. FXa and thrombin‐ induced upregulation of CTGF was not blocked by TGFβI receptor antagonist. This mechanism may play an important role in human diseases associated with fibrosis and with atherosclerosis.
Development and Regeneration of the Inner Ear: Coordinating cell cycle and differentiation – Constraining Notch signaling.
Authors: Segil Neil
Published: June 2021

Generating the correct number and type of cells, in the correct ratio and spatial relationship is required for normal functional development of complex tissues. However, the mechanisms coordinating cell proliferation with growth and differentiation to achieve correct organ size and function remain a poorly understood aspect of embryonic development and postnatal regeneration. In this presentation, I will discuss our work on the molecular basis for the timing of cell cycle exit in the embryonic inner ear, and how this is coordinated with differentiation to produce the correct number of hair cell and supporting cell precursors to build a functional organ of Corti. In the second part of my talk I will discuss the mechanisms that govern cell fate decisions between sensory hair cells and the various supporting cell types in the developing organ of Corti. Specifically, I will focus on the role of Notch‐dependent and Notch‐independent mechanisms governing Hes and Hey gene expression, and their potential role in the maintenance of cell fate during subsequent maturation. Finally, I will discuss the potential role of these developmental processes in the failure of regeneration in the mammalian inner ear.
The vasodilatory peptide maxadilan increases vascular permeability due to activation of leukocytes in postcapillary venules
Authors: Svensjo Erik , Cruz Vanessa , Saraiva Elvira M , Bozza Marcelo T , Lerner Ethan A , Scharfstein Julio
Published: June 2021

The vasodilatory peptide maxadilan (MAX) isolated from the sand fly Lutzomyia longipalpis induces plasma leakage in postcapillary venules of the hamster cheek pouch (HCP) via PAC‐1‐receptor stimulation. Here we studied arteriolar dilation, plasma leakage (FITC‐dextran 150) and accumulation of rhodamine‐labeled leukocytes in the HCP induced by topical applications of MAX 134 nM alone or combined with 8 μM M‐65, a PAC1‐receptor antagonist. Arteriolar diameter, plasma leakage (FITC‐fluorescence) and leukocyte accumulation (rhodamine‐fluorescence) was measured in digital camera images with a computer software (AxioVision 4.4). Four groups of hamsters, saline control, MAX 134 nM, MAX + M‐65 and FITC‐dextran 40 + MAX were studied. Applications of MAX and M‐65 were made topically to the HCPs in 250 μl of saline and images were recorded for 60 minutes. There was no plasma leakage increase in the saline control group but a slight increase in leukocyte accumulation. In contrast there was a lasting increase in plasma leakage (180%, P < 0.05) and leukocyte accumulation (150%, P < 0.05) in the MAX‐group with peak responses at 40 min and 60 min, respectively, that was inhibited by M‐65 and FITC‐dextran 40. M‐65 also inhibited arteriolar dilation while there was no significant inhibition of dilation in the FITC‐dextran 40 group. Conclusion: PAC1‐receptor activation by maxadilan causes plasma leakage and leukocyte accumulation independent of its vasodilatory effect.
The roles of protein kinase C (PKC) epsilon and tetrahydrobiopterin (BH4)/dihydrobiopterin (BH2) related to endothelial nitric oxide synthase (eNOS) coupling/uncoupling in ischemia/reperfusion (I/R)
Authors: Perkins Kerry‐Anne , Zambrano Christopher , Pershad Sailesh , Chen Qian , McGraw Sloane , Adams Jovan , Young Lindon
Published: June 2021

The roles of coupled or uncoupled eNOS mediating nitric oxide (NO) or hydrogen peroxide (H2O2) release respectively in I/R have not been evaluated in vivo. The effects of PKC epsilon activator or inhibitor peptides, which can increase or decrease eNOS activity respectively, combined with BH4 or BH2 were tested in isolated perfused rat hearts (ex vivo) and femoral arteries/veins (in vivo) that were subjected to I(20 min)/R(45 min). When given during reperfusion, the PKC epsilon activator combined with BH4 restored post‐reperfused cardiac function (p<0.05), increased NO release (p<0.05) and reduced H2O2 release in femoral veins (p<0.01) compared to controls, but when combined with BH2 resulted in compromised post‐reperfused cardiac function, decreased NO and increased H2O2 release in femoral veins compared to controls (p<0.05). The results suggest that the combination of PKC epsilon activator and BH4 promoted eNOS to function in its coupled state producing NO. However, the combination of PKC epsilon inhibitor with BH2 or BH4 significantly improved post‐reperfused cardiac function (p<0.05), reduced H2O2 release (p<0.05) and increased NO release (p<0.05) in femoral veins compared to controls. These results suggest that inhibition of eNOS uncoupling following I/R attenuates H2O2 release. This study was supported by NHLBI Grant 2R15HL‐76235‐02 and the Center for the Chronic Disorders of Aging at PCOM.
Protection of NADH‐linked Fe‐S clusters in cardiac mitochondria by ranolazine
Authors: Gadicherla Ashish K. , Antholine William E. , Heisner James S. , Camara Amadou K.S. , Aldakkak Mohammed , Yang Meiying , Boelens Age D. , Stowe David F.
Published: June 2021

Mitochondrial complex I (CI), composed of Fe‐S clusters numbered N1‐N5, is susceptible to damage by reactive oxygen species during cardiac ischemia reperfusion (IR). We used electron paramagnetic resonance (EPR) to assess how IR injury affects these Fe‐S clusters and if ranolazine (RAN), an antianginal drug, attenuates these effects. Guinea pig hearts were perfused with Krebs Ringer (KR) buffer and subjected to one of the following protocols: a) KR perfusion for 30 min, b) 30 min ischemia (I), c) 5 μM RAN for 1 min before ischemia (RAN+I), d) 30 min ischemia followed by 10 min reperfusion (IR) and e) 5 μM RAN for 1 min before 30 min ischemia and 10 min reperfusion (RAN+IR). Hearts were immediately ground in liquid N2 and the powder packed into EPR tubes. Changes in signal intensities at g=1.94, 1.86, and 1.89, assigned to N1b, N3, and N4, respectively, were recorded at 10°K (liquid He) at 9.6 GHz, 5 mW. Versus time control (100%), N1b signal decreased to 0% in the I hearts and was restored to 40%, 100%, and 70% in IR, RAN+I and RAN+IR hearts, respectively. N4 signal was 67% for I, 183% for RAN+I, 117% for IR, and 100% in RAN+IR hearts. N3 signal was 50% for both IR and RAN+IR hearts, and unaltered for I and RAN+I hearts (100%). These data suggest that RAN treatment before IR protects electron flux only through some Fe‐S centers. This finding may underlie a possible mechanism of RAN to attenuate electron flux in distinct Fe‐S clusters of CI.

This work was supported in part by grants from NIH HL089514 and HL073246; VA 8204‐05P, and AHA 0855940G.
Low dose nitrite enhances perfusion after fluid resuscitation from hemorrhagic shock
Authors: Cabrales Pedro
Published: June 2021

This study determines the systemic and microvascular hemodynamic consequences of administering a low dose sodium nitrite after fluid resuscitation from hemorrhagic shock. Hemodynamic responses to hemorrhagic shock and resuscitation were studied in the hamster window chamber model. Moderated hemorrhage was induced by arterial controlled bleeding of 50% of the blood volume (BV) and the hypovolemic state was maintained for one hour. Volume restitution was performed by infusion of 25% of BV using Hextend® (6% Hetastarch 670 kDa in lactated electrolyte solution) 10 min after fluid resuscitation 100μl of specific concentrations of sodium nitrite were infused. The experimental groups were named based on the nitrite concentration used, namely: 0 μM, 10 μM and 50 μM. Systemic parameters, microvascular hemodynamics and capillary perfusion (functional capillary density, FCD) were followed during entire protocol. Exogenous 10 μM nitrite maintained systemic and microhemodynamic conditions post fluid resuscitation from hemorrhagic shock, compared to 50 μM or no nitrite. A moderated increase in plasma nitrite during the early phase of resuscitation reversed arteriolar vasoconstriction and increased capillary perfusion and venous return, improving central cardiac function. Nitrite effects on resistance vessels, directly influenced intravascular pressure redistribution, sustained blood flow, and prevented tissue ischemia. In conclusion, increasing nitrite plasma bioavailability after fluid resuscitation from hemorrhagic shock can be a potential therapy to enhance microvascular perfusion and to improve overall outcome. This work was supported by Bioengineering Research Partnership grant R24‐HL64395 and grants R01‐HL62354, R01‐HL62318.
Reduction of Myocardial Reperfusion Injury by Mechanical Tissue Resuscitation (MTR) using a Bio‐absorbable Patch
Authors: Jordan James E , Mays Jennifer J. , Shelton Julie E , Thompson Edreca A , Bryant Allyson K , Morykwas Michael J , Argenta Louis C
Published: June 2021

Myocardial ischemia‐reperfusion injury (MI/R) follows a well defined sequence of early and late myocyte death. Most therapeutic strategies address only one facet of reperfusion injury. Mechanical resuscitation of injured tissue with sub‐atmospheric pressure reestablishes physiologic homeostasis and improves tissue survival. This study explores the use of absorbable material and a mechanical device to achieve resuscitation of myocardial tissue following MI/R.

Anesthetized swine were subjected to 80 min of left coronary occlusion and 3 hours of reperfusion. Animals were assigned to either:1) control or 2) treatment with 50mmHg of MTR during reperfusion using a bio‐absorbable patch. Both groups had equivalent (p=0.07) areas at risk (16±1.0% and 13.7±0.6%, respectively). MTR applied directly on the injured myocardium for 180 minutes significantly (p<0.002) reduced infarct size compared to controls: 14.2±3.1% vs 30.2±2.6%. This reduction in infarct size (53%) is comparable to that observed previously when commercially available, non‐absorbable patch materials (55–65% reduction in infarct size) were used.

MTR of injured myocardium using an absorbable patch for a defined period of time can reduce tissue death following MI/R. This study reproduces the reduction of infarct size previously observed while utilizing novel, bio‐absorbable materials. The use of degradable material for the application of MTR may allow for longer treatment and better outcomes in the clinical setting while at the same time eliminate the need for a second operation to remove the device. This treatment strategy offers a simple, effective, clinically relevant, non‐pharmacologic approach to reducing injury following MI/R.
Disruption of the Intestinal Mucin Layer and Entry of Pancreatic Digestive Enzymes in Early Stages of Shock
Authors: Chang Marisol , Schmid‐Schönbein Geert W.
Published: June 2021

Our recent evidence shows that passage of digestive enzymes across the mucosal epithelial barrier in the intestine sets the stage for shock and multi‐organ failure. We hypothesize that the intestinal mucin layer provides a physical barrier that prevents the entry of serine proteases normally contained within the lumen of the intestine and during ischemic states the mucin layer becomes diluted allowing access of these enzymes into the intestinal wall. As a model of splanchnic ischemia we occluded the superior mesenteric and celiac arteries up to 30 minutes. We used Jejunal sections for: tissue zymography to quantify and visualize chymotrypsin entry and activity in the intestinal wall; immunohistochemistry to visualize goblet cell secreted mucin (mucin2) and membrane bound mucin (mucin13); and alcian blue staining to visualize the carbohydrate part of mucin. The results show low levels of chymotrypsin entry into the mucin layer and intestinal wall of normal non‐ischemic intestine. In contrast there is early swelling of the mucin layer and entry of chymotrypsin from the lumen of the intestine across the epithelium into the mucosa of ischemic intestine. These results indicate that dilution of the mucin layer allows the entry of powerful pancreatic digestive proteases across the intestinal barrier within minutes of ischemia. Supported by HL76180 and GM85072.
Resveratrol protects the renal microcirculation during sepsis
Authors: Holthoff Joseph H , Mayeux Philip R
Published: June 2021

Over 200,000 patients die annually from sepsis in the US. In severe cases, acute kidney injury (AKI) develops and increases mortality to near 70%. Sepsis induced by cecal‐ligation and puncture (CLP) in the mouse leads to renal microvascular dysfunction and the generation of reactive nitrogen species (RNS) in the capillary microenvironment of kidney. The polyphenol resveratrol (Rsv) has been shown to have potent anti‐oxidant properties. Thus, we propose that Rsv might protect the kidney against injury during sepsis by serving as an RNS scavenger. Rsv (30 mg/kg) was given to mice 6 h prior to CLP, at CLP, and again 6 h post‐CLP. At 18 h, CLP reduced renal capillary perfusion from 323 ± 14 μm/sec in sham to 222 ± 18 μm/sec in CLP (P<.05) and capillary volumetric flow from 11.8 ± 0.3 pl/sec in sham to 8.7 ± 0.5 pl/sec in CLP (P<.05) in the cortex as measured by intravital video microscopy. Rsv treatment prevented the decline in both renal perfusion and volumetric flow. Kidneys of septic mice also showed an increase in oxidation of dihydrorhodamine 123 compared to sham animals, indicating an increase in RNS generation. Rsv also blunted this increase. Collectively, these data indicate that Rsv can preserve the renal microvascular circulation and reduce RNS generation in the kidney during sepsis. Thus, Rsv or agents like it may have clinical application as a novel treatment to prevent sepsis‐induced renal injury. Supported by NIH DK075991.
Computational models of personalized medicine: leading indicators for angiogenesis therapies
Authors: Mac Gabhann Feilim , Popel Aleksander S.
Published: June 2021

Therapies targeting inhibition of vascular endothelial growth factors (VEGF) have been approved for use in combination with chemotherapy in multiple cancers; more inhibitors of the VEGF pathway are in trial. In comparison, despite promising pre‐clinical data, fourteen clinical trials targeting VEGF upregulation in coronary artery disease or peripheral artery disease have been unsuccessful. Using detailed computational models of VEGF and its receptors in healthy and diseased tissue, we analyze the reasons for the success and failure of pro‐angiogenic and anti‐angiogenic therapies, ranging from intravascular infusion of antibodies to local gene therapy. Intriguingly, using published data on interindividual variability, the model predicts that the outcome of each therapy depends on the individual to whom it is administered; patients can be divided into responders and non‐responders. For example, individuals with low levels of VEGFR1 are predicted to respond better to VEGF gene delivery than others. Further, the route of administration and frequency of delivery can also impact the result, and can be optimized. The computational tools developed here can be used to improve drug development and delivery at all lifecycle stages, from initial trials to post‐approval administration. Support provided by: NIH‐R33‐HL087351, NIH‐R01‐HL079653 and NIH‐R00‐HL093219.
Central TNF‐alpha enhances pressor hemodynamic response to acute stressor
Authors: Zera Tymoteusz , Ufnal Marcin , Szczepañska‐Sadowska Ewa
Published: June 2021

Growing evidence points to the role of brain TNF‐alpha in the cardiovascular control. The aim of the present study was to determine whether TNF‐alpha acting in the brain is involved in regulation of the cardiovascular responses to acute stressor (AS) (air puff applied on the head of a rat). The experiments were carried out on freely moving Sprague‐Dawley rats pretreated for 28 days with saline or TNF‐alpha blocker etanercept delivered from osmotic minipump through cannula implanted into the lateral cerebral ventricle (LCV). Measurements of mean arterial pressure (MAP) and heart rate (HR) were performed during 60 min of LCV infusion of saline (5μl/hr) or TNF‐alpha (200ng/5μl/hr) followed by AS. Three groups of rats were analyzed: saline pretreatment + saline infusion, saline pretreatment + TNF‐alpha infusion, etanercept pretreatment + saline infusion. Baseline MAP and HR did not differ among groups. Infusion of TNF‐alpha resulted in increase of baseline MAP without changes in HR and enhanced pressor response to AS. Etanercept pretreatment resulted in lower MAP increases to AS than the saline pretreatment group. In conclusion present results show pressor action of centrally administered TNF‐alpha and its involvement in regulation of pressor response to acute stressor.

The study was supported by a grant from the Polish Ministry of Education and Science nr 0085/B/P01/2008/35
New applications of automated electrophysiology: heat activation of TRPV1 and action potentials of cardiomyocytes
Authors: Haedo Rodolfo , Haythornthwaite Ali , Stoelzle Sonja , Haarmann Claudia , Farre Cecilia , Guinot David , Kreir Mohamed , Steindl Juergen , Beckler Matthias , George Michael , Brueggemann Andrea , Fertig Niels
Published: June 2021

Automated patch clamp devices, with their increased throughput and high data quality, are finding their place in the market of automated electrophysiological measurements.

Unique features coupled with ease of use and higher throughput, make some of these planar patch clamp devices ideal tools for ion channel screening and safety testing. E.g., the ability of temperature control and rapid application of heated solution is an advantage in compound discovery, targeting TRPV ion channels. Discrimination of antagonising the ligand‐activated vs. the heat‐activated response of TRP channels could be important in the discovery of novel compounds with reduced side‐effects. Data will be shown of TRPV1 currents activated by heated solution and block of these responses by ruthenium red.

Another novel feature of the planar patch clamp device used is its ability to conduct parallel current‐clamp recordings. This in combination with the temperature control allows unique experiments of action potential recordings from stem‐cell derived cardiomyocytes, a system becoming increasingly important in drug safety testing. Current recordings from hERG, NaV, CaV and KV channels as well as action potential recordings and modulation of the latter by hERG active compounds, all done at 35¢a C will be shown.
Quantification of inflammatory and Toll‐like receptor gene expression in insulin resistant horses
Authors: Stokes Ashley Michelle , Carlisle Katherine , Madara Elise , McGeachy Matthew , Lavie Nicholas , Keowen Michael , Garza Frank , Andrews Frank
Published: June 2021

The objectives of this study were to quantify the expression of several inflammatory‐related genes from the Toll‐like receptor signaling pathway found in leukocyte samples taken from insulin resistant horses in comparison to normal horses. Genes of interest are Toll‐like receptors 2 and 4, (TLR2, 4), Cluster of Differentiation Factor 14 (CD‐14), Interleukin‐8 (IL‐8), Phosphoinositide 3‐kinase (PI3K), and nuclear factor NFKBIA inhibitor IkBa. Alterations should reflect chronic low‐grade inflammation resulting from insulin resistance as found in other species.

Objectives were to identify horses with insulin resistance and quantify expression of these genes in insulin resistant horses in comparison to normal horses.

Insulin resistance was determined in 21 horses using an IV glucose‐insulin tolerance test.

Total leukocyte RNA was extracted from a single baseline jugular venous sample from normal (n=13) and insulin resistant (n=8) adult horses. TaqMan RT‐PCR was used to examine the samples and quantify the genes of interest.

Significance was determined after calculating ΔΔCT values with a P‐value considered significant < 0.05.

There was significant down‐regulation of inflammatory inhibitors (IkBa, PI3K) and up‐regulation of indicators of TLR involvement and inflammation (TLR‐4, IL‐8) indicating the possibility of the existence of chronic inflammation in insulin resistant horses.
Actin tethering in endothelial junctions
Authors: Quadri Sadiqa K , Sun Lily , Bhattacharya Jahar
Published: June 2021

In rat lung microvascular endothelial cells (RLMEC), enhancement of cortical actin (F‐actin) increases E‐cadherin at intercellular junctions, thereby enhancing endothelial barrier properties (Quadri, 2003). The Y12F mutation on the actin‐binding protein, α‐actinin (MUT‐α‐actinin) abrogates F‐actin formation. To determine actin‐cadherin interactions, we established confluent RLMEC monolayers expressing GFP‐E‐cadherin. Confocal microscopy of EC junctions revealed regions of high (HD) and low (LD) E‐cadherin density. To determine E‐cadherin mobility, we photobleached GFP fluorescence to 60% of initial, then determined fluorescence recovery (FRAP) at 5 min. At HD versus LD sites, FRAP was respectively, 28±3% versus 50±3% of the pre‐bleached fluorescence (P<0.05), indicating that E‐cadherin mobility was considerably lower at HD than LD sites. In monolayer expressing MUT‐α‐actinin, E‐cadherin mobility increased to 50±4% at HD sites (P<0.05), while remaining unchanged at LD sites, indicating that E‐cadherin‐F‐actin linkages were formed at HD sites alone. Our findings indicate that in lung endothelial cells, sites of high E‐cadherin density are focal locations of actin‐cadherin tether formation. The tether function is mediated by α‐actinin, which is therefore, potentially a critical regulator of endothelial barrier properties (Support: HL36024).
Investigating the role of PECAM‐1 in regulating eNOS activity in response to flow
Authors: McCormick Margaret Elizabeth , Elms Shawn , Fulton David , Tzima Ellie
Published: June 2021

Blood flow plays an integral role in both the regulation of vascular homeostasis as well as the development of diseases such as atherosclerosis. We have recently shown that platelet endothelial cell adhesion molecule‐1 (PECAM‐1) plays a central role in the endothelial response to flow. Although it is unclear how PECAM‐1 transduces the shear‐stress signal, in vitro experiments indicate that upon the initiation of flow, PECAM‐1 becomes rapidly tyrosine phosphorylated and stimulates downstream signals. Numerous pathways have been suggested including the activation of endothelial nitric oxide synthase (eNOS). Importantly, flow is the major activator of eNOS phosphorylation on Ser1179. The goal of the present study is to investigate the relationship between PECAM‐1 and eNOS. Our data suggest that PECAM‐1 is required for the flow induced activation of eNOS as well as regulation of eNOS phosphorylation and localization basally. In the absence of PECAM‐1, ECs are unable to phosphorylate eNOS in response to shear stress. However, PECAM‐1 knockout ECs have increased basal eNOS phosphorylation as well as an approximately 4‐fold increase in basal NO production. In addition to alterations in eNOS activity, PECAM‐1 also appears to regulate the localization of eNOS within ECs. Therefore PECAM‐1 is required for both the basal regulation as well as flow‐induced activation of eNOS. Support from NIH grants HL069768 and HL088632.
3‐Azidopropylamine is an excellent substrate of lysyl oxidase
Authors: Pestov Nikolay B , Okkelman Irina A , Kutiakov Sergey V , Prokhorenko Igor A , Korshun Vladimir A
Published: June 2021

Lysyl oxidase (LOX) is a copper‐containing amino oxidase which physiological function is oxidative deamination of lysine side chains in collagen, elastin etc for subsequent formation of protein‐protein cross‐links. Substrate specificity of LOX was characterized in numerous studies and it is well known that LOX can utilize various primary mono‐ and diamines such hexylamine or 1,5‐diaminopentane. Here we report that introduction of an azide group can greatly affect substrate properties. 3‐Azidopropylamine has been synthesized from bromopropylamine and purified by distillation. Both LOX prepared from sheep aorta and recombinant mouse LOX overexpressed in E. coli showed highly efficient oxidation of 3‐azidopropylamine measured as release of hydrogen peroxide. Kinetics is complex with substrate inhibition at high concentrations: apparent Ki 2.5 mM and apparent Ks 13 μM, the latter being much better than that for any other known low molecular weight substrate. Hypothetically, azidoalkylamines have these remarkable properties due to interaction of the azide group with LOX‐bound copper. Interestingly, 3‐azidopropylamine has no overt cell toxicity below millimolar concentrations and, therefore, may be used for in vivo studies. Supported by MCB Program of the Presidium of Russian Academy of Sciences.
Compensatory response of cardiac lysyl oxidase to chronic volume overload
Authors: Gardner Jason D , Voloshenyuk Tetyana G , Bradley Jessica M
Published: June 2021

Volume overload causes extensive remodeling of the ventricle, with structural and functional alterations. These alterations include disruption of extracellular collagen, ventricular hypertrophy and dilatation, and eventual heart failure. The collagen crosslinking enzyme, lysyl oxidase (LOX), is necessary for collagen maturation, but little is known about its role in the remodeling heart. We hypothesized that chronic volume overload induced remodeling and dysfunction are accompanied by elevations of cardiac LOX protein expression and activity. Surgically induced volume overload and sham‐operated rats were studied at 4, 14 and 21 wks post‐surgery. Ultrasound was used to evaluate cardiac function. Left ventricular (LV) LOX expression and activity were determined by Western blot and Amplex red assay, respectively. Collagen content was determined by picrosirius red assay. Volume overload caused progressive ventricular dilatation and dysfunction. LV LOX protein expression was elevated after 4, 14 and 21 wks of volume overload (30%, 65% and 92% increases vs. sham, p<0.05). This elevation correlated with increased LOX activity at 4 and 14 wks, but not at 21 wks. Despite increased LOX expression and activity, a progressive loss of collagen was observed. These data indicate that compensatory increases in LOX occur in response to volume overload, but are insufficient to prevent collagen loss and dilatation.
Macrophage PPAR‐γ in Post‐Myocardial Infarct remodeling
Authors: Katwa Laxmansa C , Nichols Marshall W , Virag Jitka AI , Carr Chris J , Gould Amanda N , Lust Robert M
Published: June 2021

Macrophages play key role in initial tissue remodeling by engulfing debris and releasing cytokines at the site of injury. Currently, the contribution of macrophage PPARγ in response to myocardial infarction (MI) and post‐MI remodeling is unknown and the effect of early cytokine signals on the post‐MI expression of collagens, MMPs (Matrix metalloproteinases) and TIMPs (tissue inhibitors of MMP) is unclear. The study objective was to examine the role of macrophage PPARγ in post‐MI remodeling, particularly collagen I & III, MMP, and TIMP expression using macrophage specific PPARγ knockout (KO) and wild type (WT) mice. Protein and RNA were extracted from 28‐day post‐MI heart tissues from WT and PPARγ‐KO mice for western blot and real‐time PCR analysis. qPCR results showed a significant increase (p<0.05) in expression of collagens I and III, MMPs 9 and 13, and TIMPs 1 and 2 in the 28‐day post‐MI PPARγ‐KO model compared to the WTMI group. The level of MMP 2, although not significantly elevated, showed an increasing trend in the PPARγ‐KOMI compared to WTMI group. Western blot showed similar increasing trends in TIMP 1 and 2 expressions in the PPARγ‐KOMI group. Our results indicate that PPARγ could be influencing the initial signals that alter post‐MI remodeling by regulating the expression of collagens (I, III), MMP 9, TIMPs 1 and 2.
Functional heterogeneity in vascular remodeling (MMP‐9−/− and PAR‐1−/+) in hyperhomocysteinemic (CBS‐/+) and diabetic (Akita, Ins2−/+) mice.
Authors: Givvimani Srikanth , Jala Ramanjamma , Mishra Paras Kumar , Sen Utpal , Tyagi Neetu , Qipshidze Natia , Munjal Charu , Kandel Madhavi , Tyagi Suresh.C
Published: June 2021

Although proteinase activated receptor‐1 (PAR‐1) and matrix metalloproteinase‐9 (MMP‐9) play significant role in vasculogenesis and remodeling in hyperhomocysteinemia (HHcy) and diabetes, the mechanism remains nebulous. To test the hypothesis that differential vasculogenesis and remodeling in different vascular beds in hyperhomocysteinemia and diabetes was an adaptive phenomenon during pathogenesis, we measured vascular density in heart, lung, liver, kidney, intestinal mesentery and brain of mice lacking PAR‐1, MMP‐9, CBS and Insulin‐2 gene(Ins2), and compared with wild type C57Bl/6J mice. The vascular density was detected by X‐ray angiography using KODAK 4000 MM image station by infusing barium sulfate solution through carotid artery and jugular vein at constant flow rate of 200 micro.lit/min for 5 min using syringe pump. Coronary vascular density in the hearts of CBS−/+ (HHcy) and Ins2−/+ (Akita, type‐1 diabetic mice) was decreased while lung vasculature was increased in CBS−/+ and MMP‐9−/−. There was decrease in vascular density in liver and renal vasculature of CBS−/+ and Akita mice. Vascular density in brain and intestinal mesentery was decreased in CBS−/+ mice. Collectively these findings suggest that metabolic derangement in diabetes and hyperhomocysteinemia caused chronic decline/rarefaction in vasculature.
Regulates Early Fibroblast Responses to Myocardial Infarction
Authors: McCurdy Sarah M. , Dai Qiuxia , Bradshaw Amy D. , Lindsey Merry L.
Published: June 2021

Secreted protein, acidic, and rich in cysteine (SPARC) is a matricellular protein that helps collagen fibrils assemble. Collagen deposition to form a scar is a key event following a myocardial infarction (MI). Accordingly, we examined wild type (WT; n=22) and SPARC deficient (SPARC‐null; n=25) mice at day 3 post‐MI. Day 0 unoperated WT (n=28) and SPARC‐null (n=20) mice served as controls. Infarct size was similar between the two groups (52±2% for WT and 47±2% for SPARC‐null; p=n.s.). WT mice increased end diastolic volumes, but SPARC‐null mice showed increased end diastolic volumes to a lesser extent, indicating reduced remodeling. Interestingly, collagen levels in the SPARC‐null mice were 66% of the wild type values, indicating reduced collagen deposition in the absence of SPARC. Fibroblasts isolated from the infarct region of the null mice showed differential expression of several ECM genes, including MMP‐3, TIMP‐2, CTGF, and TGFβ. Our data suggests that SPARC deletion is beneficial in the early time post MI by regulating fibroblast activation, but whether this benefit persists through longer times needs to be examined. In conclusion, SPARC plays a major role in cardiac wound healing by regulating fibroblast function.
Folic Acid Mitigated Cardiac Dysfunction by Normalizing the Levels of Tissue Inhibitor of Metalloproteinase and homocysteine‐metabolizing enzymes Post myocardial Infarction in Mice.
Authors: Qipshidze Natia , Tyagi Neetu , Sen Utpal , Givvimani Srikanth , Metreveli Naira , Abe Oluwasegun A , Lominadze David , Tyagi Suresh C
Published: June 2021

Myocardial infarction (MI) results in significant metabolic derangement in myocardium. Homocysteine (Hcy), a non‐protein amino acid, was generated during nucleic acid methylation and methionine de‐methylation. However, the role of Hcy metabolism in derangement of in myocardial injury after MI is not known. Folic acid (FA) decreased Hcy levels by remethylating the Hcy to methionine, by 5‐methylene tetrahydrofolate reductase (5‐MTHFR). The derangement in Hcy metabolism/clearance led to hyperhomocysteinemia (HHcy) and cardiovascular dysfunction; and MI resulted in significant metabolic derangement. The fraction shortening (FS) was 58% (sham) and 27% (MI). FA supplementation resulted in significantly preserving FS to 41%. The ejection fraction was closely paralleled the FS. The calcium transients and contractile function were found to be lower after MI but markedly improved in MI hearts with FA supplementation. The levels of TIMP were decreased significantly in MI hearts. Interesting, the decrease in TIMP expression in MI was mitigated after FA treatment. The decreased levels of cystathionine beta synthase (CBS), cystathionine gamma lyase (CSE), and 5‐MTHFR in MI hearts were ameliorated by FA‐treatment. The present study suggests that FA supplementation results in significant preservation of myocardial function in mouse heart after MI, in part, by homocysteine metabolic pathway.
Inhibition of Phosphodiesterases Prevents Mitochondrial Permeability Transition Pore Opening by Inactivating GSK‐3β via PKA and PKG in Cardiac Cells
Authors: Chanoit Guillaume , Lee SungRyul , McIntosh Rachel , Zvara David A. , Xu Zhelong
Published: June 2021

Both protein kinase A (PKA) and protein kinase G (PKG) can inactivate glycogen synthase kinase 3β (GSK‐3β), and inactivation of GSK‐3â may lead to cardioprotection through suppression of the mitochondrial permeability transition pore (mPTP) opening. The aim of this study was to test if inhibition of phosphodiesterases (PDEs) can moderate the pore opening via GSK‐3β. IBMX (1‐methyl‐3‐isobutylxanthine), a non‐selective inhibitor of PDEs, prevented H2O2‐induced mPTP opening in cardiac cells. IBMX was not able to inhibit the pore opening in cells transfected with the constitutively active GSK‐3β mutant, suggesting that IBMX prevents the pore opening by inactivating GSK‐3β. IBMX reduced reperfusion injury in a GSK‐3β –dependent manner. In support, IBMX significantly increased GSK‐3β phosphorylation at Ser9, an effect that was reversed by both the PKA inhibitor H89 and the PKG inhibitor KT5823, implying that IBMX inactivates GSK‐3β via PKA and PKG. IBMX also activated both PKA and PKG. IBMX failed to prevent the pore opening in cells treated with H89 or PKA siRNA, suggesting a role of PKA in the action of IBMX. Similarly, KT5823 and PKG siRNA also reversed the protective effect of IBMX. In conclusion, inhibition of PDEs prevents the mPTP opening by inactivating GSK‐3β through both PKA and PKG. Inhibition of PDEs may serve a useful approach to attenuate reperfusion injury.

This study was supported by NIHR01HL08336.
Ranolazine delays Ca2+‐induced mitochondrial permeability transition pore opening and membrane potential depolarization in guinea pig heart mitochondria
Authors: Aldakkak Mohammed , Camara Amadou K.S. , Heisner James S. , Gadicherla Ashish K. , Boelens Age D. , Yang Meiying , Dash Ranjan K. , Stowe David F.
Published: June 2021

Ranolazine (RAN) is an antianginal drug that may block a late Na+ current in cardiac cells during ischemia. We showed previously that RAN improved cardiac function and reduced ROS emission during ischemia and reperfusion (IR). Here we tested if these effects of RAN are associated with delay in opening of the mitochondrial (m) permeability transition pore (PTP) and membrane potential (ΔΨm) depolarization induced by incrementally adding buffer CaCl2 to mitochondrial suspension. Guinea pig hearts were perfused with RAN (5 μM) for 1 min followed by 30 min ischemia and 120 min reperfusion to assess m[Ca2] and infarct size (IS) in intact hearts, or reperfused for only 10 min to isolate mitochondria. ΔΨm and matrix Ca2+ uptake were measured using fluorescence spectrophotometry with rhodamine 123 and indo‐1. We found that m[Ca2+] increased less during early ischemia in RAN (296±25 nM) vs control hearts (436±34 nM), and m[Ca2+] was less in RAN treated (280±39 nM) vs control hearts (429±45 nM) during early reperfusion. RAN reduced IS (24±3%) vs control (37±3%). More buffer CaCl2 (20–25%) was required to open PTP and to induce full ΔΨm depolarization in RAN vs control hearts. Reduced ROS emission during ischemia may delay cytosolic Na+, Ca2+ loading, and consequently mCa2+ loading. The delay in PTP opening by Ca2+ in RAN treated hearts could result from improved mitochondrial bioenergetics with increased capacity to extrude Ca2+.

This work was supported in part by the American Heart Association [0355608Z, 0855940G to D.F. Stowe]; the National Institutes of Health [K01 HL73246 to A.K.S. Camara, R01 HL089514 to D.F. Stowe]
Role of Superoxide in endothelium‐dependent dilation of soleus feed arteries in young and old rats
Authors: Trott Daniel Wayne , Seawright John W , Woodman Christopher R
Published: June 2021

We tested the hypothesis that superoxide (O2−) contributes to age‐induced endothelial dysfunction in rat soleus feed arteries (SFA). SFA from young (4 mo) and old (24 mo) Fischer 344 rats were isolated and cannulated for examination of vasodilator responses to flow and acetylcholine (ACh) in the absence or presence of an NAD(P)H oxidase inhibitor (DPI; 10μM) or a O2− scavenger (Tiron; 10mM). Flow‐ and ACh‐induced dilations were attenuated in old SFA. Addition of DPI and Tiron improved flow‐ but not Ach‐induced dilation in old SFA. In young SFA, DPI and Tiron had no effect on flow‐induced dilation and attenuated ACh‐induced dilation. To determine if O2− contributes to endothelium derived hyperpolarizing factor‐ (EDHF) mediated, ACh‐induced dilation, dilator responses were assessed in the presence of L‐NNA (300μM), indomethacin (5μM), and/or Tiron. In young SFA, incubation with L‐NNA+Indo+Tiron attenuated ACh‐induced dilation to a greater extent than L‐NNA+Indo or Tiron alone. Immunobloting revealed that protein content of NAD(P)H subunits gp91phox and p67phox were greater in old SFA compared to young. These results suggest that O2− contributes to impaired flow‐induced dilation in old SFA. In contrast O2− appears to play a role EDHF mediated dilation in response to ACh. Supported by AHA 0765043Y, and a Huffines Institute Research Grant.
Modifications of coronary vascular reactivity due to a non‐enzymatic reaction between vitamin C and imidazole
Authors: Tiwari Kanchan , Kipp Brian , Sylvester Francis
Published: June 2021

The objective of this study was to evaluate the role of superoxide (O2•−) resulting from the reaction between imidazole and vitamin C in altering the vascular reactivity of coronary arteries. It was hypothesized that a potential non‐enzymatic redox reaction between vitamin C and imidazole results in the production of O2•− and alters vascular reactivity. Left anterior descending coronary arteries (LAD) from porcine hearts were dissected, mounted in organ baths and passively loaded with 1.5 g of tension. Following a 5‐hour equilibration with control (Krebs‐Henseleit solution), vitamin C (2mM), imidazole (200mM), or combination of vitamin C and imidazole, LADs were treated with increasing concentrations of potassium chloride (KCl) and sodium nitroprusside (SNP) to assess changes in vascular reactivity. Incubation with the combination of vitamin C and imidazole as well as imidazole alone attenuated vascular responses to KCl and SNP. To detect the presence of O2•− in coronary arteries, a fluorescent dye‐based dihydorethidine (DHE) assay was utilized. An increase in the O2•− levels was observed in the arteries incubated with vitamin C and imidazole. The results from these studies may provide basic information on non‐enzymatic sources of superoxide in the vasculature and their effect on coronary artery function. This research was supported by a Grand Valley State University Presidential Grant.
Local regulation of L‐type calcium channels by reactive oxygen species
Authors: Amberg Gregory C , Earley Scott , Glapa Stephanie A
Published: June 2021

Arterial smooth muscle voltage‐dependent L‐type calcium channels can operate in a protein kinase C alpha (PKC alpha)‐dependent, high open probability mode resulting in sites of sustained calcium influx called “persistent calcium sparklets.” However, the molecular mechanisms underlying sustained PKC alpha‐dependent calcium channel activity are poorly understood. Here we tested the hypothesis that reactive oxygen species enhance arterial smooth muscle calcium influx by inducing persistent calcium channel (i.e. sparklet) activity. Consistent with this, oxidant exposure increased arterial wall calcium and tone in pressurized cerebral arteries. Using TIRF microscopy and patch clamp electrophysiology, we found that reactive oxygen species promoted localized persistent L‐type calcium channel activity in isolated arterial myocytes via activation of PKC alpha. Furthermore, we observed that punctate subcellular generation of endogenous reactive oxygen species preceded and co‐localized with persistent L‐type calcium channel activity. Taken together, these data suggest that local oxidative activation of PKC alpha‐dependent L‐type channel calcium influx represents a functionally relevant convergence of oxidant and calcium signaling pathways that contribute to calcium entry and arterial tone. This work is supported by the American Heart Association and the National Institutes of Health.
Cyclin D1 is a bona fide target gene of NFATc1 and is sufficient in the mediation of injury‐induced vascular wall remodeling
Authors: Wang Dong , Karpurapu Manjula , Van Quyen Dong , Kundumani‐Sridharan Venkatesh , Pulusani Srinidhi , Rao Gadiparthi N
Published: June 2021

Platelet‐derived growth factor BB (PDGF‐BB) induced cyclin D1 expression in a time‐ and NFAT‐dependent manner in human aortic smooth muscle cells (HASMCs) and blockade of NFATs prevented HASMC DNA synthesis and their cell cycle progression from G1 to S phase. Selective inhibition of NFATc1 by its siRNA also blocked HASMC proliferation and migration. Characterization of cyclin D1 promoter revealed the presence of several NFAT binding sites and the site at −1333 was found to be sufficient in mediating PDGF‐BB‐induced cyclin D1 promoter‐reporter gene activity. In addition to its role in cell cycle progression, cyclin D1 mediated HASMC migration in NFATc1‐dependent manner. Balloon injury‐induced cyclin D1‐CDK4 activity requires NFAT activation and adenovirus‐mediated transduction of cyclin D1 was found to be sufficient to overcome the blockade effect of NFATs by VIVIT on balloon injury‐induced vascular wall remodeling events, including SMC migration from medial to luminal region, their proliferation in the intimal region and neointima formation. Together, these results provide another mechanistic evidence for the role of NFATs, particularly NFATc1 in the regulation of HASMC proliferation and migration as well as vascular wall remodeling. NFATc1 could be a potential therapeutic target against the re‐narrowing of artery after angioplasty.

This work was supported by a grant from NIH (HL069908).
BAY 41‐2272 reduces vascular smooth muscle cell growth via PK‐G & PK‐A signals
Authors: Brown Trisha A , Mendelev Natalia N. , Fox Jonathan C. , Joshi Chintamani N. , Tulis David A.
Published: June 2021

In the cardiovascular system, abnormal growth of vascular smooth muscle (VSM) is intimately involved in both primary disease and during complications following therapeutic intervention. In turn, approaches aimed at modifying the cell signaling pathways responsible for VSM growth are scientifically significant. The objective of the current study is to investigate novel routes for cyclic nucleotide‐based control of VSM growth. We hypothesize that BAY 41‐2272 (BAY), a recently characterized soluble guanylate cyclase (sGC) stimulator and inducer of cyclic guanosine monophosphate (cGMP) synthesis, has capacity to reduce VSM cell proliferation and migration and that these occur through protein kinase‐G (PK‐G) and protein kinase‐A (PK‐A) pathways. In rat thoracic aorta primary VSM cells (passage ≤ 6), BAY reduces proliferation and inhibits migration using a modified Boyden chemotaxis assay and an in vitro scrape wounding injury model. Cyclic nucleotide assays and Western blot analyses reveal that BAY induces both the cGMP and cAMP signaling pathways, and inhibitor studies suggest that BAY operates through both PK‐G and PK‐A. These novel findings show that BAY is capable of reducing VSM growth through stimulation of parallel cGMP and cAMP systems and suggest that BAY may prove therapeutic for the treatment of cardiovascular diseases.

Research Support: NHLBI NIH Award Numbers R01HL081720 and R01HL081720‐03S1.
An ethyl acetate extract from a subaleurone layer of Japanese rice inhibits angiotensin ll–induced signal transduction and hypertrophy in vascular smooth muscle cells
Authors: Takaguri Akira , Utsnomiya Hirotoshi , Kono Ryohei , Akazawa Shin‐ichi , Eguchi Satoru
Published: June 2021

Objective

Rice has been consumed well in many countries. However, whether rice contains an ingredient useful to prevent cardiovascular diseases remains unknown. We have shown that angiotensin II (AngII) induces hypertrophy and migration of vascular smooth muscle cells (VSMCs) through activation of epidermal growth factor receptor (EGFR) and Rho/Rho‐kinase. A subaleurone layer of rice is rich in oligosaccharide and dietary fibers. In this study, we examined the effects of ethyl acetate extract from a subaleurone layer on AngII‐induced signal transduction and hypertrophy in VSMCs.

Methods and Results

VSMCs were prepared from the aorta of 12‐week‐old male Sprague Dawley rats. Phosphorylation and expression of the signaling proteins were evaluated by immuoblotting. Pretreatment of the ethyl acetate extract (100 μg/ml) inhibited AngII (100 nM)‐induced EGFR transactivation and phosphorylation of a ROCK substrate, myosin phosphatase target subunit‐1. Also, AngII‐induced c‐fos expression was inhibited by the ethyl acetate extract. Thus, AngII‐induced hypertrophy was significantly inhibited by the ethyl acetate extract.

Conclusion

These data suggest that an ethyl acetate extract from a subaleurone layer might have a protective effect toward cardiovascular diseases by interfering with signal transduction induced by enhanced rennin‐angiotensin system