bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1118: Novel Lentiviral Vectors Bearing the Cardiac-Specific Promoter of the Sodium-Calcium Exchanger (NCX1) Report Cardiogenic Differentiation in Stem Cells
Authors: Barth Andreas S , Kizana Eddy , Terrovitis John , Dong Peihong , Smith Rachel R , Schneider Jay W , Leppo Michelle K , Zhang Yiqiang , Olson Eric N , Abraham Roselle , Marbán Eduardo
Published: July 2021

Background:
Cardiosphere-derived resident cardiac stem cells (CDCs) are readily isolated from adult hearts and confer functional benefit in animal models of heart failure. To study cardiogenic differentiation in CDCs, we developed a method to genetically label and selectively enrich for cells that have differentiated down the cardiomyocyte lineage.

Methods and Results:
Lentiviral vectors achieved significantly higher transduction efficiency (>90%) and longer-term transgene expression in human CDCs than any of the nine adeno-associated viral serotypes tested (AAV 1– 6, 2.5, 8, 9). To define the best reporter of cardiogenic differentiation, five cardiac-specific promoters (sodium-calcium exchanger [NCX1], L-type calcium channel α-subunit, α-myosin heavy chain, troponin I and myosin ventricular light chain) were subcloned upstream of GFP in lentiviral vectors. Cardiac specificity was assessed by transducing neonatal rat ventricular myocytes (NRVMs), HEK293, cardiac fibroblasts, skeletal myoblasts, endothelial and vascular smooth muscle cells with each vector and then measuring GFP fluorescence by flow cytometry. The cardiac NCX1-promoter conveyed the highest degree of cardiac specificity, with expression limited to NRVMs. At a multiplicity of infection of 35, NCX1-GFP vectors did not affect cell death or apoptosis of CDCs. Expression persisted for up to 6 months
in vitro
. NCX1-GFP positive CDCs subpopulations, were FACS-sorted and expanded
in vitro
, demonstrating enhanced expression of a variety of cardiac markers by real-time PCR. The utility of lentiviral vectors bearing the cardiac NCX1-promoter was further exemplified by treating CDCs with cardiogenic sulfonylhydrazones (SHZ). SHZ potently activated a NCX1-firefly luciferase reporter (211 ± 21% greater than control cells) and upregulated cardiac transcripts (including BNP, troponin I, α-tropomyosin, GATA-4).

Conclusion:
Lentiviral vectors carrying the NCX1 promoter represent a useful tool for genetically marking stem cells and their progeny that have differentiated down the cardiomyocyte lineage. The ability to selectively enrich for cardiomyocytes and their precursors has potential relevance for the development of cell-based therapies.
Abstract 1120: Akt Modified MSCs Secrete a Novel Stem Cell Paracrine Factor that Protects Injured Heart, Activates Cyclin Dependent Kinase and Phosphorylates/Activates Akt Signaling Pathway in Adult Rat Cardiomyocytes
Authors: Guo Jian , Mirotsou Maria , Huang Jing , Mu Hui , Zhang Lunan , Loiselle David , Zhang Zhiping , Pratt Richard E , Haystead Timothy , Dzau Victor J
Published: July 2021

Previously, we have shown that intracardiac injection of mesenchymal stem cell (MSC) overexpressing Akt (Akt-MSCs) could dramatically reduced infarct size and restored cardiac function in a paracrine manner. Microarray analysis of Akt-MSCs led to the identification of a novel transcript encoding a secreted protein. Cloning of the cDNA encoding this candidate gene into pET15b allowed the purification as a 6×His tagged recombinant protein. Interestingly, a 30 minute pre-incubation of the H9C2 myocytes with this protein (10nM) reduced H
2
O
2
induced apoptosis by 50% as measured by annexin V/PI staining on flow cytometry (n = 3, p<0.001). Moreover, this protein significantly prevent H
2
O
2
induced DNA fragmentation and inhibited Caspase 9 and Caspase 3/7 activities in adult rat cardiomyocytes by ~36% and ~42%, respectively (n=3, p<0.01). Furthermore, this protein dramatically inhibited the mitochondrial release of Cytochrome C and maintained mitochondrial Bcl-2 protein level. Intramyocardial injection of 1 μg of this novel protein dramatically reduced infarct size (−58%, n=10, p<0.001) and TUNEL positive nuclei (−69%, n=10, p<0.001) in a 30 min ischemia/24h reperfusion rat model. Fibrosis evidenced by collagen deposition was greatly reduced (−61%, n=8, p<0.001) 4 weeks after ischemia/reperfusion injury. Signaling studies demonstrated that this novel protein phosphorylated rat adult cardiomyocytes Akt
Thr308
but not Akt
Ser473
in a time-depended manner peaking at 30 min. Consequently, downstream Akt substrates, GSK3ß
Ser9
and Bad
Ser128
, are also phosphorylated at 30 min or at a later time point. In contrast to the traditional concept that PDK1 phosphorylates both Akt
Thr308
and Akt
Ser473
, our mass-spec and
in vitro
enzymatic analysis of rat cardiomyocytes stimulated with this factor suggests cyclin-dependent kinase 7 as being responsible for phosphorylation of Akt specifically at Thr 308 position. Further studies would be of great interest to interrogate whether this novel factor could activate other cyclin dependent kinase(s) and render the cardiomyocytes re-entering cell cycle. In conclusion, we have identified a novel paracrine factor from Akt-MSCs that mediates cardioprotective effects through activation of novel pathways involving Akt.
Abstract 1125: Mulberry Leaf Ameliorates The Production Of Adipocytokines By Inhibiting Oxidative Stress In White Adipose Tissue In db/db Mice
Authors: Sugimoto Masayuki , Arai Hidenori , Tamura Yukinori , Murayama Toshinori , Ono Koh , Ariyasu Hiroyuki , Akamizu Takashi , Kita Toru , Kamei Kaeko , Yokode Masayuki
Published: July 2021

Mulberry leaf (ML) is commonly used to feed silkworms. Previous study showed that ML ameliorates atherosclerosis. However, its mechanism is not completely understood. Because dysregulated production of adipocytokines is involved in the development of the metabolic syndrome and cardiovascular disease, we examined the effect of ML on the production of adipocytokines and metabolic disorders related to the metabolic syndrome, and compared its effect with that of a PPARγ agonist, pioglitazone (Pio). By treating obese diabetic db/db mice with ML, Pio, and their combination, we investigated the mechanism by which they improve metabolic disorders. In this study, db/+m (lean control) and db/db mice were fed a standard diet with or without 3% (w/w) ML and/or 0.01% (w/w) Pio for 12 weeks from 9 weeks of age. At the end of the experiment we found that ML decreased plasma glucose and triglyceride by 32% and 30%, respectively. Interestingly, administration of ML in addition to Pio showed additive effects; further 40% and 30% reduction in glucose and triglyceride compared with Pio treatment, respectively. Moreover, administration of ML in addition to Pio suppressed the body weight increase by Pio treatment and reduced visceral/subcutaneous fat ratio by 20% compared with control db/db mice. Importantly, ML treatment increased expression of adiponectin in white adipose tissue (WAT) by 40%, which was only found in db/db mice, not in control db/+m mice. Combination of ML and Pio increased plasma adiponectin concentrations by 25% and its expression in WAT by 17% compared with Pio alone. In contrast, ML decreased expression of TNF-α and MCP-1 by 25% and 20%, respectively, and the addition of Pio resulted in a further decrease of these cytokines by about 45%. To study the mechanism, we examined the role of oxidative stress. ML decreased the amount of lipid peroxides by 43% and the expression of NADPH oxidase subunits in WAT, which was consistent with the results of TNF-α and MCP-1. Thus our results indicate that ML ameliorates adipocytokine dysregulation by inhibiting oxidative stress in WAT of obese mice, and that ML may have a potential for the treatment of the metabolic syndrome as well as reducing adverse effects of Pio.
Abstract 1129: Obesity-related Elevations in Soluble P-selectin are Derived From Endothelium and Dependent on Expression of Leukocyte P-selectin Glycoprotein Ligand-1
Authors: Wickenheiser Kevin J , Bodary Peter F , Bahrou Kristina , Eitzman Daniel T
Published: July 2021

Background
: Obesity is associated with proinflammatory changes and an increased risk for vascular disease complications. The tissue source and mechanism by which soluble P-selectin (sPsel) is generated in obesity are unclear.

Methods and Results
: Soluble p-selectin (sPsel) levels were measured in the circulation from lean wild type and obese leptin receptor deficient mice (LepR−/−) at 4 and 10 weeks of age. In wild-type mice body weight increases from 13+/−2 to 20+/−3 grams over this time period while the body weight increases from 15+/−2 to 38+/−5 grams in LepR−/− mice. At 4 weeks of age sPsel levels were 103+/−8mg/mL in wild-type mice vs. 138+/−9 ng/mL in LepR−/− mice, p=0.048. By 10 wks of age sPsel increased to 112 +/− 2 in wild-type mice and 182 +/− 9 in LepR−/− mice, p=0.00005. In order to determine if the obesity-induced rise in sPsel is regulated by leukocyte Psgl-1, bone marrow transplantation was performed from Psgl+/+ or Psgl−/− donors into irradiated LepR−/−recipients. At 4 weeks post-transplant, sPsel levels were 166 +/−6 ng/mL in LepR−/− mice receiving Psgl+/+ marrow and 45 +/− 4 ng/mL in LepR−/− mice receiving Psgl−/− marrow, p=0.0000004. In order to determine if the sPsel in LepR−/− mice originated from the endothelium versus platelets, we transplanted Psel−/− bone marrow into irradiated LepR−/−mice. At 4 weeks post transplant, sPsel levels were 153 +/−3 ng/mL in LepR−/− mice receiving Psel−/− bone marrow and were not significantly different from LepR−/− mice receiving Psel+/+ bone marrow (166 +/−6 ng/mL, p=0.06). By 10 weeks post transplant, mice gained even more weight and levels were 377+/−51 ng/mL in LepR−/− mice receiving Psel+/+ bone marrow and 370+/−73 ng/mL in LepR−/− mice receiving Psel−/− bone marrow, p=0.87.

Conclusions
: These data suggest that the increase in sPsel observed in obesity is primarily derived from the endothelium and that this process is regulated by leukocyte Psgl-1.
Abstract 1138: Endogenous Adipocyte-Derived Factors Diminish Coronary Endothelial-Dependent Vasodilation Via Inhibition Of Nitric Oxide Synthase
Authors: Payne Gregory A , Borbouse Lena , Dick Gregory M , Tune Johnathan D
Published: July 2021

Adipocytokines may be the molecular link between obesity and vascular disease; however, effects of these factors on coronary vascular function have not been delineated. Accordingly, this study was designed to examine mechanisms by which endogenous adipocyte-derived factors impair coronary endothelial-dependent vasodilation
in vivo
. Experiments were conducted in open-chest anesthetized dogs (n = 16) before and during treatment with endogenous adipocyte-derived factors. Phosphate buffered saline was conditioned in a shaking water bath with parietal pericardial adipose tissue (3 g/ml) for 30 min at 37°C. The conditioned buffer was then filtered (0.2 μm) and infused directly into the coronary circulation (0.3 ml/min). Conditioned buffer did not significantly affect baseline coronary blood flow (0.50 ± 0.01 vs. 0.61 ± 0.05 ml/min/g, p = 0.68), mean arterial pressure (103 ± 6 vs. 96 ± 9 mmHg, p = 0.74), or heart rate (87 ± 13 vs. 110 ± 24 beats/min, p = 0.44). Conditioned buffer had no effect on responses to intracoronary angiotensin II (2.5 – 750 ng; 74 vs. 70% vasoconstriction). Under control conditions, bradykinin (0.03 – 3 μg/min) increased coronary blood flow (303 ± 65%) to 2.02 ± 0.31 ml/min/g in a dose-dependent manner. Conditioned buffer attenuated maximum bradykinin vasodilation to 1.64 ± 0.26 ml/min/g (167 ± 33% increase; p < 0.05). This decrease in endothelial-dependent dilation was not due to increases in superoxide production, as administration of the superoxide dismutase mimetic Tempol (10 mg/min, ic) did not improve bradykinin vasodilation (120 ± 27% increase; p < 0.05). Inhibition of nitric oxide synthase with L-NAME (150 μg/min, ic) reduced maximum bradykinin vasodilation to 0.93 ± 0.04 ml/min/g (p < 0.05) and endogenous adipocyte-derived factors had no further inhibitory effect (0.82 ± 0.09 ml/min/g, p = 0.24). These data indicate that endogenous adipocyte-derived factors diminish endothelial-dependent coronary vasodilation via inhibition of nitric oxide synthase rather than a reduction in nitric oxide bioavailability by superoxide. Our findings importantly link endogenous adipocyte-derived factors with pro-atherogenic coronary vascular dysfunction
in vivo
.
Abstract 1141: Adaptation of Coronary Arteriolar Dilations to The Simultaneous Presence of Obesity and Hypertension: Role of Increased Nitric Oxide Sensitivity
Authors: Bagi Zsolt , Fulop Tibor , Jebelovszki Eva , Erdei Nora , Szerafin Tamas , Forster Tamas , Koller Akos , Edes Istvan
Published: July 2021

Increased body weight and elevated blood pressure could lead to the development of hyperdynamic circulation requiring increased coronary blood flow. Thus, we aimed to characterize the effects of simultaneous presence of obesity and hypertension on the dilator function of human coronary arterioles. Agonist-induced dilations were assessed in pressurized coronary arterioles isolated from the right atrial appendages of patients (N=38), that underwent cardiac surgery. There were no significant differences in bradykinin (BK)-induced and the nitric oxide (NO)-donor, sodium-nitroprusside (SNP)-evoked dilations of coronary arterioles between normotensive and hypertensive lean patients. In contrast, in the obese, normotensive patients BK- and SNP-induced dilations were reduced (BK, 10
−7
M, lean:90±4%, obese:64±7%; SNP, 10
−6
M, lean:89±7%, obese:76±5%), whereas in the obese, hypertensive patients BK- and SNP-induced dilations were significantly enhanced, when compared to lean subjects (BK, 10
−7
M, lean:71±7%, obese:85±3%; SNP, 10
−6
M, lean:60±6%, obese:83±2%). Correspondingly, in hypertensive individuals, but not in those of normotensives, a positive correlation was found between body mass index (BMI) and bradykinin-induced (
P
=0.03, r=0.46), and also SNP-evoked (
P<
0.03, r=0.44) coronary dilations. To correlate coronary responses to those of peripheral vessels, in an additional 55 hypertensive patients flow-mediated (FMD) and nitroglycerin (NTG)-induced dilations were assessed by high-resolution ultrasound. In obese, hypertensive individuals both FMD and NTG-induced dilations were significantly greater (FMD:6.2±0.7%, NTG:17.2±0.9%), than in lean hypertensive patients (FMD:3.7±0.6%, NTG:13.6±1.1%). Correspondingly, both FMD and NTG-induced dilations were positively correlated with BMI, (
P
=0.02, r=0.31,
P
=0.03, r=0.29, respectively). These findings are the first to show that in humans obesity may lead to activation of an adaptive vascular mechanisms in hypertension, such as an increased sensitivity to NO, enhancing the dilator function of coronary and peripheral arterial vessels.
Abstract 1157: Coronary Endothelial Dysfunction In Mice With Conditional, Cardiomyocyte Specific Overexpression Of The Mineralocorticoid Receptor: Prevention By An Antioxidant Treatment
Authors: Favre Julie , Zhang Andi , Thuillez Christian , Jaisser Frèdèric , Richard Vincent
Published: July 2021

Deleterious effects of aldosterone excess have been demonstrated in cardiovascular diseases, and they might be linked in part to coronary vascular dysfunction. However, whether such vascular dysfunction is a cause or a consequence of the changes occurring in the cardiomyocytes is unclear. Moreover, the possible link between aldosterone-mediated effects on the cardiomyocyte on one hand and the coronary arteries on the other hand are unknown. Thus, we used a mouse model of conditional, cardiomyocyte-specific overexpression of human mineralocorticoid receptor (MR) (Ouvrard-Pascaud et al., Circulation 2005) and observed its effects on coronary endothelial function. Three months old transgenic (TG) mice were obtained after removing doxycycline treatment used to inhibit lethal embryonic MR overexpression. TG mice and their matched controls (Cont) were either untreated or treated with MR antagonist canrenoate (40 mg/kg/day) or with an antioxidant treatment (vitamin E 1% in chow and vitamin C 0.05% in water) for 1 month. Segments of left coronary arteries (diameter 180 –220 μm) were isolated and mounted in a wire myograph. After pre-contraction with serotonin, we assessed endothelium-mediated (either NO-dependent or independent) relaxations in response to increasing concentrations of acetylcholine (Ach) before and after incubation with a NOSynthase inhibitor (LNNA). Compared to control mice, cardiac MR overexpression induced decreased relaxing responses to Ach either without (maximal relaxation: Cont: 95±4% n=7; TG: 68±8% n=7 p<0.05) or with LNNA (Cont: 19±3% n=7; TG: 5±4% n=7 p<0.05). A one month treatment by canrenoate totally prevented this coronary dysfunction which was also prevented by a one month treatment with vitamin E/vitamin C. The endothelium-independent relaxing responses to the NO-donor sodium nitroprusside were similar in all groups.We thus demonstrate that an increase in MR expression, restricted to cardiomyocytes is sufficient to induce a coronary endothelial dysfunction mostly through an increase in reactive oxygen species production. This suggests for the first time that the phenotypic changes induced by aldosterone within the cardiomyocyte are sufficient to induce per se a secondary coronary vascular dysfunction.
Abstract 1158: Mitral Leaflet Remodeling: Response to Isolated Mitral Regurgitation
Authors: Stephens Elizabeth H , Nguyen Tom C , Itoh Akinobu , Miller David C , Grande-Allen K. Jane
Published: July 2021

Objectives:

Chronic mitral regurgitation (MR) adversely remodels the left ventricle. Although leaflet integrity is important for mitral coaptation, its response to isolated MR remains unknown. In a chronic ovine model, we hypothesize that isolated MR
also
adversely remodels the mitral leaflet.

Methods:

29 sheep were randomized to either control (CTRL, n=11) or experimental groups (HOLE, n=18). In HOLE, a 2.8 – 4.8mm diameter hole was punched in the middle scallop of the posterior mitral leaflet. MR grade was followed by echocardiography. At 12 weeks, immunohistochemistry (IHC) was performed on anterior and posterior mitral leaflet cross-sections to demonstrate active matrix remodeling, including markers for active collagen synthesis synthesis and cross-linking prolyl 4-hydroxylase (P4H), heat shock protein 47 (HSP47), and lysyl oxidase (LOX) and matrix metalloproteinases (MMPs 1, 2, 9, 13) largely responsible for matrix degradation implicated in valvular pathologies. The proteoglycans biglycan (BGN), decorin (DC), and versican (VC), the glycosaminoglycan hyaluronan (HA), as well as fibrillin (F), and collagen III (col 3), all of which regulate matrix organization, were also visualized using IHC. A semi-quantitative intensity and delineation grading scale (from 0 to 3) was used to quantify differences between the HOLE and CTRL leaflets.

Results:

At 12 weeks, MR grade was greater in HOLE v CTRL (0.4±0.4 v 3.0±0.8, p < 0.001). In HOLE, valve matrix remodeling reduced hole diameter (69.5%32±.8%, p<0.001). The overall intensity of P4H, MMP13, and HSP47 decreased (1.02 v 1.30, p<0.02) while BGN, DC, and HA staining delineation increased 34 fold around the hole compared to CTRL (1.22 v 0.36, p<0.01). In the anterior leaflet of HOLE, P4H, LOX, MMPs, F, col 3, and HA were increased (1.43 v 1.24), while VC, DC, and BGN were decreased (1.66 v 1.84) compared to CTRL (both p≤0.05).

Conclusions:

In HOLE, significant remodeling was evident in the posterior leaflet near the site of injury; moreover expression of matrix turnover markers increased in the anterior leaflet due to the chronic low pressure LV volume overload. This adverse remodeling in the anterior leaflet in response to MR may explain how “MR begets MR,” providing insight for reparative surgical techniques.
Abstract 1165: Impact of Gender on Serial Atherosclerotic Changes in Response to Established Medical Therapies
Authors: Kalidindi Srinivasa R , Hsu Amy , Moon Keon-Woong , Tuzcu E. Murat , Nissen Steven E , Nicholls Stephen J
Published: July 2021

Background:
While the importance of coronary artery disease in females has become increasingly recognized, little is known regarding the impact of gender with regard to changes in arterial wall dimensions with progression and regression of atherosclerosis. This study investigated the remodeling response of the artery wall accompanying changes in atheroma burden in response to use of medical therapies, stratified according to gender.

Methods:
1533 patients (27.5% female) underwent serial intravascular ultrasound evaluation of a single coronary artery in the context of clinical trials that assess the impact of medical therapies on plaque progression. The relationship between gender and remodeling of the arterial wall at baseline and its serial change in association with plaque progression and regression were studied.

Results:
Females were older (59 v 57 years, p<0.01), had a higher body mass index (31.5 v 29.5 kg/m
2
, p<0.01), were more likely to have hypertension (86 v 71.5%, p<0.01) and metabolic syndrome (57 v 49%, p<0.01) and less likely to have a history of smoking (57.5 v 73.5%, p=0.01) and myocardial infarction (27.5 v 35.5%, p<0.01). After adjusting for body surface area, females demonstrated a trend towards smaller external elastic membrane (EEM) (226.3 v 234.3 mm
3
, p=0.09) and larger lumen (143.7 v 137.7 mm
3
, p=0.01) volumes. The remodeling index at the most diseased site did not differ between genders (0.95 v 0.95, p=0.95). No differences were observed between genders with regard to changes in EEM (−5.6 v −6.2 mm
3
, p=0.29) and lumen (−4.9 v −4.5 mm
3
, p=0.82) volumes and remodeling index (−0.02 v −0.03, p=0.43) in response to use of medical therapies. Similarly, there were no differences between genders with regard to the percentage of patients undergoing expansion (34.7 v 35.5%, p=0.86) or contraction (20.4 v 21.8%, p=0.69) of lumen volume in association with regression of atherosclerotic plaque.

Conclusion:
A similar pattern of remodeling of the arterial wall was observed between genders in association with serial changes in atheroscle-rotic plaque. This further highlights our understanding of the pathological interactions between atherosclerosis and the arterial wall in females.
Abstract 1172: Neointima Formation is Dependent on p38α Mitogen-Activated Protein Kinase
Authors: Proctor Brandon M , Muslin Anthony J
Published: July 2021

Neointima formation frequently occurs after arterial injury and is responsible for substantial human morbidity. We previously demonstrated that the intracellular linker protein Grb2 is required for neointima formation, and that Grb2 regulates p38α mitogen-activated protein kinase (MAPK) activation in vascular smooth muscle cells (SMCs). In this work, the role of p38α MAPK in neointima formation was examined. In vitro experiments showed that pharmacological inhibition of p38 MAPK activity in cultured SMCs blocked platelet-derived growth factor (PDGF)-stimulated DNA replication and cell proliferation. Specifically, in control SMCs, overnight stimulation with PDGF induced an 11.8-fold increase in thymidine incorporation and a 1.9 fold increase in cell number. However, inhibition of p38 MAPK activity reduced PDGF-induced thymidine incorporation to 2.8-fold (P = 0.0006) and completely blocked PDGF-stimulated cell proliferation (P = 0.0001). Also, p38 MAPK activity was required for PDGF-induced inactivation of the retinoblastoma tumor suppressor protein, Rb, and induction of mini-chromosome maintenance protein-6 (MCM6), a fundamental regulator of DNA replication. Next, compound transgenic mice were generated with doxycycline (Dox)-inducible, SMC-specific expression of a dominant-negative form of p38α MAPK (SMC-DN-p38α ). Dox induced robust expression of DN-p38α mRNA and protein in the aorta and carotid arteries of compound transgenic mice, and inactivation of native, arterial p38 MAPK. SMC-DN-p38α and single transgenic, control mice were subjected to carotid injury by use of an epoxy resin-beaded probe. After 21 days, control mice developed robust neointima formation that frequently resulted in an occlusive lesion with a mean neointima/media ratio of 2.62 (N = 8). In contrast, SMC-DN-p38α mice were resistant to the development of neointima. Specifically, neointima/media ratio was reduced to 0.63 for SMC-DN-p38α mice (N = 12; P = 0.045). In addition, compared to control mice, injured carotid arteries of SMC-DN-p38α mice showed defective p38 MAPK activation in SMCs of the tunica media. Our results demonstrate that vascular SMC p38α MAPK is required for neointima formation after arterial injury.
Abstract 1173: Gq Signaling Mediates High Blood Pressure and Hypertrophy in Two Divergent Models of Hypertension
Authors: Harris David M , Cohn Heather I , Pesant Stephanie , Zhou Rui-Hai , Eckhart Andrea D
Published: July 2021

Hypertension (HBP) is associated with increased levels of AngII and NE, which bind to Gq-coupled receptors. Many animal models and humans with HBP have increased levels of G protein receptor kinase 2 (GRK2) in vascular smooth muscle(VSM), which causes HBP. We tested whether inhibiting VSM Gq signaling by expressing of an intracellular peptide (GqI) decreased HBP and cardiac hypertrophy, in two divergent murine models of HBP: the 2 kidney, 1 clip (2K1C)model and a genetic mouse model that overexpresses G protein receptor kinase 2 (GRK2) in VSM.

Methods:
GqI mice and their non-transgenic littermate controls (C) were subjected to 2K1C. Conscious BP was measured via radiotelemetry and echo(m-mode) was performed before and 4 wks post 2K1C. Increasing doses of AngII were added to aortic rings. To test the efficacy of GqI in the 2K1C model, we also treated mice with Losartan, an AngII-receptor blocker. We crossbred GRK2 mice (have HBP) with GqI. We measured BP, echo, and did immunohistology on these hearts.

Results:
GqI mice had lower BP vs. C after 2K1C (111 ± 3 vs. 125 ± 4 mmHg, n = 8,8). Although, there was a decrease in BP in GqI mice following 2K1C, cardiac hypertrophy still occurred. There were similar increases in ventricular wall thickness (VWT) between C and GqI 4 weeks post 2K1C (C: 1.00 ±.05 vs. .79 ±.03 mm, n = 10,15; GqI: .93 ± .03 vs. .76 ± .02 mm, n = 20,19). There were also similar decreases in cardiac function after 2K1C measured by fractional shortening (FS) in C (35.7 ± 1.3 vs. 26.0 ± 1.9%, n = 10,15) and GqI (34.2 ± 0.8 vs. 25.9 ± 1.4, n = 20,19). The dose response curve to AngII in aortic rings was right-shifted 2.4 fold in GqI mice vs. C, suggesting decreased AngII sensitivity in GqI mice. Losartan normalized VWT in both C and GqI after 2K1C (0.77 ± .03 and 0.77 ± .02 mm) and FS (32.9 ± 0.7 and 32.0 ± 0.9%, n = 10,8). Hybrid GRK2/GqI mice had decreased BP vs. GRK2 (87.5 ± 6.3 vs. 111.6 ± 3.1 mmHg, n = 5,7). Hybrid GRK2/GqI mice also had less VSM hypertrophy than GRK2 (23.8 ± 1.9 vs. 32.9 ± 2.5 um, n = 6,3).

Conclusions:
We show that Gq signaling is important in 2 models of HBP. It appears that Gq signaling contributes to high BP through both AngII signaling and VSM hypertrophy. These data suggests that inhibition of VSM Gq signaling may provide future therapeutic strategies to treat HBP and HF.
Abstract 1185: Role Of Sphingosine-1-Phosphate (S1P) In Insulin Signaling.
Authors: Nicholl Suzanne M , Roztocil Elisa , Davies Mark G
Published: July 2021

A failure to increase glucose disposal into peripheral tissues in response to insulin leads to impaired insulin signaling and an inability to uptake glucose leading to the onset of insulin resistance, a major contributing factor to diabetes. We examined the role of sphingosine-1-phosphate (S1P) in insulin signaling and its ability to regulate glucose uptake in skeletal muscle cells. S1P, a sphingolipid found in abundance in the circulation, has been implicated in not only mediating crosstalk with other signaling pathways but has also been implicated in insulin resistance. We hypothesize that S1P interacts with post-receptor insulin signaling to increase glucose disposal in an in vitro model of insulin resistance using differentiated mouse skeletal C2C12 myotubes. Our data demonstrates that S1P (10μM) increases basal glucose levels similar to that observed in response to insulin (100nM) under conditions of low glucose (** p < 0.005: n = 3). Conversely, high glucose conditions completely inhibit both insulin and S1P stimulated glucose uptake (*p < 0.01:n = 3). Pre-incubation with S1P does not augment insulin-induced glucose uptake (***p < 0.001:n = 3), suggesting that S1P does not act via a separate signaling pathway. This is confirmed by our data demonstrating that S1P-induced glucose uptake is abrogated by Cytochalasin B (*p < 0.001:n = 3). In addition, the PI3-K inhibitors, LY294002 and Wortmannin, the Akt inhibitor, AKT2 and the p38MAPK inhibitor, SB203580 significantly inhibited glucose uptake in response to S1P, demonstrating their importance in S1P-induced glucose uptake (*p < 0.05:n = 3). S1P2 and S1P3 receptor expression were upregulated in response to insulin (~2-fold over basal) under low glucose conditions suggesting that insulin may regulate S1P signaling via one or both of these receptors. S1P increased serine phosphorylation of IRS1, both at serine 307 and serines 636/639 maximally after 15 minutes of stimulation. This data has important clinical implications in patients with metabolic syndrome who have impaired skeletal muscle glucose disposal due to insulin resistance and will help guide present and future therapy for patients who have this rapidly growing disease.
Abstract 1186: Overexpression Of Endothelial Lipase In The Liver Promotes The Clearance Of Plasma Lipids But Accelerates Liver Steatosis In A Mouse Model Of Metabolic Syndrome
Authors: Yasuda Tomoyuki , Ishida Tatsuro , Kojima Yoko , Tanaka Hanayo , Okada Takeaki , Fukuda Akira , Hara Tetsuya , Inoue Michihiko , Hirata Kenichi
Published: July 2021

The metabolic syndrome includes high triglyceride (TG) and low HDL-cholesterol (HDL-C) levels in the plasma, and often accompanies steatosis in the liver. Endothelial lipase (EL) is a phospholipase that regulates HDL metabolism. EL is expressed by hepatocytes, while the function of EL in the liver has not been identified. Here, we examined the role of EL in the liver using a mouse model of metabolic syndrome. The EL expression in the liver was analyzed by real-time PCR. It revealed that liver EL expression was significantly increased in obese and diabetic db/db mice compared to that of control mice. To examine the significance of the EL upregulation in the liver, we injected the recombinant adenovirus encoding human EL into mice. The EL overexpression in the liver resulted in a significant decrease in plasma HDL-C, TG, and free fatty acid levels. Interestingly, the EL overexpression in the liver increased liver weight and liver TG content both in wild-type and db/db mice. In db/db mice, particularly, EL overexression accelerated the formation of steatosis by increasing the mRNA level of fatty acid synthase. These findings indicate that EL expression is increased in the liver in the metabolic syndrome. The upregulation of EL promotes the uptake of plasma lipids by hepatocytes, and accelerates the progression of steatosis in db/db mice. Thus EL may play a role in the genesis of steatosis as well as dyslipidemia in the metabolic syndrome.
Abstract 1201: Lack of Dehydroepiandrosterone-sulfate (DHEAS) contributes to CD4 T Cell-Induced Endothelial Apoptosis in Perimenopausal Women
Authors: Suzuki Yuki , Sato Kayoko , Gomita Keiko , Nuki Toshiaki , Hagiwara Nobuhisa , Kasanuki Hiroshi
Published: July 2021

Background:
Postmenopausal women develop atherosclerosis faster and have higher incidence of acute coronary syndromes. Endogenous dehydroepiandrosterone-sulfate (DHEAS) is a major source of estrogen for women after menopause. Low levels of DHEAS is also a known risk factor for coronary atherosclerotic disease in men. However, the causal relationship between DHEAS levels in women and acute coronary syndromes (ACS) is not fully understood. In this study, we examined whether CD4 T cells from perimenopausal women (PMW) with low levels of DHEAS induce endothelial apoptosis that is prone to develop plaque erosion.

Methods and Results:
Fresh CD4 T cells were isolated from 45 PMW within 5 years after menopause (mean age 53.9 ± 6.0 years) and from 40 women with regular menstruation cycles (NC, mean age 34.0 ± 7.4 years). DHEAS levels in plasma by ELISA correlated negatively with age (R = 0.500, P < 0.02). DHEAS was lower in PMW than in NC (P < 0.0001). Estrogen receptor α (ESR1) and estrogen receptor β (ESR2) mRNA of CD4 T cells in PMW were lower compared to NC by real time PCR (P < 0.05). DHEAS levels correlated significantly with ESR2 transcripts (R = 0.789, P < 0.0001), but not with ESR1. Measurement of fragmentation of DAPI-binding nuclear proteins showed that CD4 T cells from PMW induce significant human umbilical vein endothelial cells (HUVEC) apoptosis compared to those from NC (P<0.0001). DHEAS levels correlated negatively with CD4 T cell-induced HUVEC apoptosis (R = 0.499, P < 0.02). Treatment with 17β-estradiol showed reduction of CD4 T cell-induced apoptosis in PMW. Pure estrogen receptor antagonist ICI 172,780 abolished estrogen protection.

Conclusions:
Our results showed that the aging marker of DHEAS was lower in perimenopausal women, and that the low levels DHEAS, a endogenous major source of estrogen after menopause, induced CD4 T cell-induced endothelial apoptosis may be associated with ESR2. We concluded that low levels of DHEAS in perimenopausal women can be one factor that contributes to the acceleration of plaque erosion in atherosclerotic plaque and acute coronary syndromes.
Abstract 1209: Interleukin-6 Reduces Myocardial Glucose Metabolism by Altering AMPK, Insulin Signaling, and PKC-𝛉 in Mice
Authors: Zhang Zhiyou , Ko Hwi Jin , Jung Dae Young , Ma Zhexi , Kim Jason K
Published: July 2021

Increasing evidence implicates the role of inflammation in the pathogenesis of diabetes and complications. Inflammatory cytokines (IL-6, TNF-α) are elevated in obese diabetic subjects, and are shown to modulate glucose metabolism in peripheral organs. In this report, we examined the effects of IL-6 on cardiac metabolism and insulin action in vivo. Male C57BL/6 mice were intravenously treated with IL-6 (16 ng/hr) or saline (control) for 2 hrs, and [
14
C]2-deoxyglucose was intravenously injected in awake mice to measure myocardial glucose metabolism (n=9~10). Hyperinsulinemic-euglycemic clamps (2.5 mU/kg/min insulin infusion) were also performed in IL-6 or saline-treated mice (n=4~5) to measure cardiac insulin action. Acute treatment with IL-6 caused a 25% increase in myocardial STAT3 activity and significantly reduced basal myocardial glucose metabolism (Fig. 1

; *
P<
0.05). IL-6 treatment also reduced insulin-stimulated glucose uptake in heart, and these effects were associated with marked decreases in AMPK activity (Thr-phosphorylation of AMPK; Fig. 2

) and IRS-1 tyrosine phosphorylation (Fig. 3

). Acute IL-6 treatment increased myocardial expression of PKC-𝛉, which has been shown to mediate insulin resistance in peripheral organs (Fig. 4

). These results indicate that IL-6 is a potent negative regulator of myocardial glucose metabolism and insulin action, and the underlying mechanism may involve IL-6 mediated activation of PKC-𝛉 and defects in AMPK and insulin signaling activity. Thus, our findings suggest a potential role of IL-6 in the pathogenesis of diabetic heart failure.
Abstract 1210: Interferon-gamma-induced CD40/CD40 Ligand Pathway Activation Mediates Atherosclerotic Plaque Progression and Destabilization in ApoE-Knockout Mice
Authors: IKEDA AYAMI , Kai Hisashi , Koga Mitsuhisa , Imaizumi Tsutomu
Published: July 2021

Background:
A triggering role of interferon-gamma (IFN-g) is suggested in atherosclerotic plaque formation by activating inflammatory process. Soluble mutant of IFN-g receptor (sIFNgR) blocks IFN-g-induced activation of the target cells in vivo. The immune mediator CD40 ligand (CD40L) and its receptor CD40 are crucial for initiation of plaque formation and for progression and destabilization of advanced plaque by producing monocyte chemoattracting protein-1 (MCP-1) and matrix metalloproteinase-9 (MMP-9). We assessed the hypothesis that postnatal inhibition of intrinsic IFN-g would reduce and stabilize advanced plaques via inhibition of the CD40L/CD40 pathway.

Methods and Results:
From 8-week old (w), apoE-knockout (apoEKO) mice were fed daily high-fat diet. At 12 w when aortic plaques had been established, mice were randomized to 2 groups: sIFNgR-treated mice received gene transfer of naked DNA plasmid encoding sIFNγR into the thigh muscle twice at 12 and 14 w. Mock-treated mice received the empty plasmid. At 16 w, mock-treated apoEKO mice showed progression of aortic plaque. They had massive oil red O-stained lipid core and bulky MOMA2-labeled macrophage accumulation accompanied by thin Azan-stained fibrous cap with little smooth muscle cells (SMCs). These findings demonstrated markedly low plaque stability. sIFNgR treatment reduced plaque area by more than 50% versus mock-treated mice. Moreover, sIFNgR treatment stabilized the plaque by decreasing lipid and macrophage areas and increasing the areas of SMCs and collagen. In mock-treated mice, CD40 and CD40L as well as MCP-1 and MMP-9 were markedly upregulated in aortic plaque at 16 w, which were inhibited by sIFNgR treatment. sIFNgR treatment not only reduced the mobilization of peritoneal macrophages but also attenuated the CD40L/CD40 expressions of macrophages. Circulating monocyte count and serum cholesterol levels were not affected by sIFNgR treatment.

Conclusions:
It was suggested that intrinsic IFN-g function blocking reduced and stabilized advanced plaque in apoEKO mice in part by inhibiting CD40L/CD40 pathway. Targeting IFN-g may be a new strategy for regression and stabilization of atherosclerosis.
Abstract 228: Short-term Cyclosporine Therapy Increases Ischemia-induced Endothelial Progenitor Cell Mobilization Through Manipulation Of The CD26 System
Authors: Wang Chao Hung , Cherng Wen-Jin , Hsieh I-Chang , Yang Ning-I , Yeh Chi-Hsiao
Published: July 2021

Cyclosporine A (CsA), an immunosuppressant, has remarkably improved the short-term success rate of transplantation. The CD26/dipeptidylpeptidase IV (DPP IV) system plays a critical role in mobilizing endothelial progenitor cells (EPCs) from bone marrow. This study investigated whether CsA manipulates CD26/DPP IV activity and increases EPC mobilization. C57 BL/6 mice were divided into control and CsA-treated groups. Circulating EPCs were enumerated before and after hindlimb ischemia was induced. Levels of stroma-derived factor-1α (SDF-1α), stem cell factor (SCF), vascular endothelial grow factor (VEGF), and granulocyte-colony stimulating factor (G-CSF) were measured in the blood. Compared to the controls, CsA treatment significantly increased the serum levels of SDF-1α and SCF (p < 0.001), but not of VEGF or G-CSF after ischemic stress. The CsA group displayed a significant increase in the number of circulating EPCs (with a 7-fold increase in sca-1
+
KDR
+
cells and a 1.5-fold increase in c-kit
+
CD31
+
cells versus the controls at 18 h after ischemia was induced, both p < 0.05).
In vivo
, CsA also caused 2- and 5-fold increases in the numbers of bone marrow-derived EPCs incorporated into the Matrigel and ischemic limbs, respectively (p < 0.05). In the peripheral blood, CsA significantly decreased CD26
+
cell numbers (p < 0.001) and attenuated the plasma CD26/DPP IV activity (p < 0.001). CsA did not alter the acitivty of MMP-2 or MMP-9
in vitro
or
in vivo
. Compared to the controls, short-term CsA treatment significantly improved the perfusion of ischemic limbs and decreased the spontaneous digital amputation rate. In conclusions, this study demonstrated that CsA manipulates the mobilization of bone marrow-derived EPCs into the circulation via the CD26/DPP IV system. Short-term CsA treatment has beneficial effects on angiogensis of ischemic tissues.
Abstract 1268: Small Molecule Targeting Of gβγ Reduces β-adrenergic Receptor Desensitization And Normalizes Cardiac Dysfunction In An Acute Heart Failure Model
Authors: Casey Liam , Nwakanma Frances , Vorobiof Gabriel , Dunaevsky Olga , Smrcka Alan V , Blaxall Burns C
Published: July 2021

Heart failure (HF) is a debilitating disease with poor prognosis, despite substantial therapeutic advances in the past two decades. Excess signaling through cardiac G-protein Gβγ subunits is an important component of HF pathophysiology. They recruit elevated levels of cytosolic G-protein coupled receptor kinase 2 (GRK2, a.k.a. βARK1) to agonist-stimulated β-ARs in HF, leading to chronic β-AR desensitization and down-regulation; these events are all hallmarks of HF. Previous data has suggested that inhibiting Gβγ signaling and its interaction with GRK2 could be of therapeutic value in HF. We recently developed a novel small molecule targeting strategy to selectively inhibit Gβγ binding interactions, and identified several Gβγ inhibitory small molecules (Bonacci et al, Science, 2006). In particular, we identified compound M119, which blocked the interaction of purified Gβγ and GRK2 in vitro. To validate this activity in a cellular setting, we isolated adult mouse cardiomyocytes, where M119 significantly reduced β-AR-mediated membrane recruitment of GRK2. Furthermore, M119 significantly enhanced both adenylyl cyclase activity and cardiomyocyte contractility at baseline and in response to β-AR agonist stimulation. Upon finding that the compound was biologically available following intraperitoneal injection in mice, we investigated whether the observed in vitro effects in isolated adult cardiomyocytes would translate to in vivo effects on cardiac function. Initial investigations were pursued in an acute pharmacologic HF model (30 mg/kg/day isoproterenol for seven days). Importantly, concurrent once daily injections of M119 normalized cardiac function, morphology and GRK2 expression in this acute HF model. Collectively, our study has identified a small molecule Gβγ inhibitor capable of reducing β-AR desensitization, thereby enhancing β-AR-mediated isolated cardiomyocyte contractility and, importantly, normalizing cardiac function and morphology in an acute HF model in vivo. Our data suggest a promising therapeutic role for small molecule inhibition of Gβγ in the treatment of HF.
Abstract 1271: IKKβ/NF-κB Pathway Protects Hearts from Hemodynamic Stress Mediated through Regulating MnSOD Expression
Authors: Hikoso Shungo , Otsu Kinya , Yamaguchi Osamu , Takeda Toshihiro , Taniike Masayuki , Nakai Atsuko , Ohmiya Shigemiki , Mizote Isamu , Matsumura Yasushi , Nishida Kazuhiko , Hori Masatsugu
Published: July 2021

Objectives:
We have previously reported that NF-κB contributes to GPCR agonist-induced hypertrophy in cultured cardiomyocytes. However, the
in vivo
role of this pathway in the pathogenesis of cardiac remodeling remains to be elucidated. Although IκB kinase β (IKKβ)/NF-κB pathway is a major negative regulator of cell death, it can sensitize cells to death-inducing stimuli in some instances, thus it can be either anti- or pro-apoptotic. In this study, we aimed to clarify the role of IKKβ/NF-κB signaling in cardiac remodeling using cardiac-specific IKKβ deficient mice.

Methods and Results:
We crossed mice bearing an
IKK
β

flox

allele with mice expressing the Cre recombinase under the control of the myosin light chain 2v promoter (
MLC2v-Cre
+/−
) to generate
IKK
β

flox/flox

;
MLC2v-Cre
+/−
mice (conditional knockout:CKO). Then, CKO mice (n=14) and control littermates bearing
IKK
β

flox/flox

(CTRL, n=14) were subjected to pressure overload by means of transverse aortic constriction (TAC). EMSA analysis revealed NF-κB DNA binding activity after TAC had attenuated in CKO hearts. One week after TAC, echocardiography showed significantly lower left ventricular fractional shortening (26.9±2.7% vs. 41.4±0.9%, p<0.01), and higher left ventricular end-diastolic dimension (4.02±0.14 mm vs. 3.47±0.08 mm, p<0.01) and lung weight/body weight ratio (11.1±1.4 vs. 5.5±0.1, p<0.01) in CKO mice compared with CTRL mice, indicating the development of heart failure in CKO mice. Number of apoptotic cells had increased in CKO hearts after TAC, suggesting that the enhanced apoptosis is a cause for heart failure. The expression levels of MnSOD mRNA and protein after TAC, which is one of NF-κB target genes, were significantly lower in CKO than those in CTRL mice. As a consequence, oxidative stress and JNK activation in CKO hearts after TAC had significantly increased compared with those in CTRL heart, suggesting that increased oxidative stress and enhanced JNK activity resulted in cardiomyocyte apoptosis in CKO hearts.

Conclusion:
These results show that IKKβ/NF-κB pathway in cardiomyocyte plays a protective role mediated through attenuation of oxidative stress and JNK activation in response to pressure overload.
Abstract 1281: Human Adipose Tissue-derived Stem Cells Potently Preserve Cardiac Function Resulting From Permanently Induced Myocardial Ischemia
Authors: Cai Liying , Johnstone Brian H , Cook Todd G , March Keith L
Published: July 2021

Background
The use of stem cells has gained much interest as a potential therapeutic approach for repair of damage caused by a variety of cardiac insults. We and others have demonstrated the ability of pluripotent adipose-derived stem cells (ASCs), to promote repair in ischemic skeletal tissues. Here we demonstrate that ASCs also stimulate a robust functional improvement following acute myocardial infarction (MI) in rats.

Methods and Results
ASCs were harvested from human subcutaneous adipose tissue. ASCs were characterized in vitro before in vivo testing. Growth and metabolic activity of human microvascular endothelial cells (HMVEC) cultured in growth-factor deficient minimal medium (MM) increased 1.7-fold when supplemented with a 1:1 mixture of ASC CM (
p
<0.01). Sprout formation by HMVECs and migration of endothelial progenitor cell (EPC) was enhanced by 2.1 and 2.0-fold, repectively, when ASC CM was added to MM (
p
<0.01). Following demonstration of potency in vitro, the ASCs were evaluated for the ability to protect and rescue ischemic myocardium in an athymic nude rat following permanent ligation of the proximal LAD region. Immediately after ligation 10
6
ASCs in 100 μl saline or carrier alone was injected into 2 sites of the peri-infarct region, then at 4 and 28 d heart function was evaluated echocardiography using a Visualsonics Vivo770. ASC-treated rats consistently exhibited better cardiac function at 1 month compared to the saline control group. LV ejection fraction of the ASC group was 56 ± 7% (mean ± SEM) vs 37 ± 3% for the control (p<0.04). Fractional shortening was 32 ± 5% (ASC) vs 19 ± 2% (p<0.04). LV volumes both at end-diastolic and end-systolic stages were lower in ASC group (311 ± 17 μl and 139 ± 21 μl, respectively) than saline group (391 ± 30 μl and 249 ± 27 μl) (p<0.03). Anterior wall thinning was attenuated in ASC group (1.6 ± .08 mm vs 1.2 ± .2, at end-diastole, p<0.03). Post-mortem histological analysis demonstrated that ASC treated hearts had lower fibrosis (26 ± 6% vs 34 ± 6%; p<0.05).

Conclusion
We have demonstrated that ASCs have great potential as a cell therapy to preserve heart function following ischemic insult. Given the abundant source of ASCs, therapies with these cells have a higher potential for widespread adoption compared to more rare cell types.