bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 297: Nkx2–5 Transcriptionally Activates C-kit-ligand And Regulates Cardiac Progenitor Cell Populations
Authors: Scotland Rebecca L , Shi Xiaozhong , Ferdous Anwarul , Kyba Michael , Garry Daniel J
Published: July 2021

C-kit-ligand, also known as stem cell factor, is expressed broadly and has a functional role during hematopoesis, gametogenesis, melanogenesis, mast cell growth and differentiation. Although the receptor for c-kit-ligand, c-kit, has been utilized as a marker to identify cardiac stem cell and progenitor cell populations, the transcriptional regulation and biological function of c-kit-ligand during cardiogenesis has not been defined. Here we demonstrate that c-kit-ligand is a novel downstream target of Nkx2–5. The homeodomain transcription factor, Nkx2–5, is one of the earliest markers of the cardiac lineage and mice lacking this transcription factor are nonviable. To identify potential Nkx2–5 downstream target genes, we utilized ES/EBs that were engineered to overexpress Nkx2–5 and undertook transcriptome analysis of embyroid bodies with and without Nkx2–5 induction. We observed a significant increase in c-kit-ligand expression following Nkx2–5 induction suggesting a role for Nkx2–5 in the activation of c-kit-ligand. Furthermore, analysis of the c-kit-ligand promoter revealed three evolutionarily conserved Nkx2–5 response elements, supporting the notion that Nkx2–5 is a transcriptional regulator of gene expression. We undertook transcriptional assays and transfected the c-kit-ligand promoter-luciferase reporter in the absence and presence of increasing amounts of Nkx2–5. We observed that Nkx2–5, in a dose dependent fashion, was a potent transcriptional activator of c-kit-ligand. These studies enhance our understanding of Nkx2–5 mediated transcriptional networks and further emphasize that Nkx2–5 is an important transcriptional regulator of cardiac progenitor cell populations.
Abstract 310: Thrombospondin 2 Protects Against Uncontrolled Cardiac Inflammation and Injury During Viral Myocarditis
Authors: Swinnen Melissa , Vanhoutte Davy , Pinto Yigal , Carmeliet Peter , Heymans Stephane
Published: July 2021

Background-The matricellular protein thrombospondin 2 (TSP2), a secreted extracellular matrix (ECM) glycoprotein, plays a crucial role in controlling cell-matrix interaction in tissue repair and cancer biology. Our previous data revealed that increased TSP2 reinforces the matrix and protects the against heart failure during hypertension. The present study investigated whether TSP2 may protects against exaggerated cardiac inflammation, injury and dysfunction during viral myocarditis.

Methods and Results-Therefore, mortality, cardiac inflammation and function were investigated in TSP2-null (KO) and wild type (WT) mice, and after adeno-associated virus (AAV)-9 mediated TSP-2 overexpression in WT, in a mouse model of coxsackievirus B3 (CVB3)-induced (106 CCID50 ip CBV3, 14 days) myocarditis. Absence of TSP2 resulted in an increased mortality in TSP2-null as compared with WT mice in both viral myocarditis (KO: 37,5% vs WT: 0%; n=8 per group). Histological analysis revealed increased cardiac inflammation (60 % increase of CD3-positive T-lymphocytes), necrosis and injury in TSP2-null compared to WT mice at 14 days of viral myocarditis. Ultrastructural analysis showed increased inflammation, matrix disruption and myocyte injury in absence of TSP2, together resulting in depressed systolic function (% FS; 40.1 ± 2.3 in WT vs. 24.6 ± 1.7 in KO mice, P<0.05) and increased cardiac dilatation (end-diastolic dimensions, mm; 2.6 ± 0.18 in WT vs. 3.6 ± 0.06 in KO mice, P<0.05) at 14 days at echocardiography. In concordance, AAV9-gene transfer of TSP2, with 50 % increase in cardiac TSP-2 expression, significantly reduced cardiac inflammation (60% reduction) and injury at 14 days as compared to control AAV9-GFP.

Conclusion-Together, these data clearly point towards a novel protective role for TSP2 against uncontrolled inflammation, myocardial injury and dysfunction in acute viral myocarditis.
Abstract 311: Poly(ADP-ribose) Polymerase-1 Inhibition Prevents High Fat Diet-induced Cardiac Hypertrophy by Modulating Matrix-destabilizing Proteases and Oxidative Stress
Authors: Hans Chetan P , Feng Yumei , Naura Amarjit , Zerfaoui Mourad , Troxclair Dana , Matrougui Khalid , Lazartigues Eric , Boulares Hamid
Published: July 2021

Background: We recently showed that PARP-1 is activated within atherosclerotic plaques and that inhibition of this enzyme, exerts protective effects against high fat diet-induced atherogenesis. Given the tight association between atherosclerosis and cardiac pathologies, we examined, whether PARP-1 is an active participant in high fat diet-induced cardiac hypertrophy and whether PARP-1 gene knockout prevents such pathological abnormalities.

Methods: Apolipoprotein (ApoE)−/− mice fed a high-fat diet were used as a model of atherosclerosis. C57BL/6 ApoE−/− mice that are deficient in PARP-1 (ApoE-PARP-1d−/−) were generated in our laboratory. Hemodynamic parameters were obtained by a small animal-adapted ultrasound imaging system.

Results: Hemodynamic parameters revealed that ApoE−/− mice on regular diet exhibited markers of cardiac hypertrophy. PARP-1 gene knockout markedly protected against dyslipidemia-induced cardiac hypertrophy. An increase in dyslipidemia burden further exacerbated the latter parameters in ApoE−/− mice. Inhibition of PARP-1 by gene deletion provided a significant protection against the parameters of cardiac hypertrophy. Histology showed enlarged hypertrophied cardiac myocytes in ApoE−/− mice with typical “boxcar-shaped” nuclei and high-fat diet caused separation of the myocardial fibers suggestive of dilated cardiomyopathy in these mice. PARP-1 gene deletion maintained the structural integrity of cardiac myocytes. MMP activity, measured by zymography, was dramatically increased in heart and aorta of ApoE−/− mice on high-fat diet compared to ApoE-PARP-1d−/− mice on similar diet regimen, which was accompanied by increased collagen degradation and mast cell degranulation. PARP-1 gene knockout was associated with increased TIMP-2 expression in various cells of vascular origin which seemed to neutralize the degenerative effects of MMP activity.

Discussion: Our results demonstrate that PARP-1 gene deletion protects against dyslipidemia-induced cardiac hypertrophy and dilatations. We also provide evidence that PARP-1 inhibition protects against these pathologies by increasing expression of TIMP-2 and subsequent inhibition of tissue-degrading proteases in a mouse model of atherosclerosis.
Abstract 313: Interleukin-6 Mediates Concentric Hypertrophy, Diastolic Dysfunction and Myocardial Fibrosis in Rats
Authors: Melendez Giselle C , Du Yan , Janicki Joseph S , Brower Gregory L
Published: July 2021

Induction of inflammatory cytokines has been implicated in the progression of myocardial remodeling and heart failure (HF). Increased levels of circulating TNF-α and interleukin-6 (IL-6) in patients with HF, suggest that these cytokines may be involved in the pathogenesis of heart disease. In previous studies we have shown that TNF-α is an important contributor to the adverse myocardial remodeling. TNF-α is known to mediate collagen degradation as well as in-series sarcomeric addition contributing to ventricular dilatation. However, the effects of IL-6 on cardiac remodeling in vivo have not been investigated. Accordingly, in this study we explore the hypothesis that up-regulation of IL-6 mediates adverse myocardial remodeling. To this end, a group of adult male Sprague Dawley rats was infused with IL-6 (2.5 μg/kg/hr, IP) for 7 days via osmotic minipump and compared to aged-matched shams. LV pressure, size, and function were measured using a blood-perfused isolated heart preparation. At the end of the experiment, hearts were weighed and analyzed for collagen volume fraction (CVF) and isolated cardiomyocyte size. The EDP-EDV (End Diastolic Pressure and Volume) relationship provided that IL-6 infusion produced LV stiffness and a clear tendency for a shift of the EDP-EDV curve to the left due to ventricular hypertrophy and diastolic dysfunction. LV weight differences demonstrate concentric hypertrophy (749 mg versus 660 mg in control hearts; p< 0.05) and a marked increase in interstitial collagen in the IL-6 infused hearts relative to that in control hearts (CVF of 6.2% vs. 1.7%, respectively; p< 0.001). The cardiomyocyte hypertrophy at the cellular level also reflected a concentric phenotype, with cells being significantly longer and thicker (18% and 32%, respectively; p< 0.01). These novel observations demonstrate a direct effect of IL-6 on cardiac remodeling in vivo, which in contrast to TNF-α, induces a dramatic myocardial fibrosis together with concentric cardiac hypertrophy. This suggests that IL-6 may contribute to the development of diastolic dysfunction, and as such could drive the transition to heart failure.
Abstract 324: RNAi Inhibition of Phosphodiesterase 1A Prevents Cold-Induced Hypertension and Cardiac Hypertrophy
Authors: Sun Zhongjie
Published: July 2021

The calcium/calmodulin-dependent phosphodiesterase 1A (PDE1A) hydrolyzes the intracellular second messengers cAMP and cGMP, which are known to play an important role in regulating vascular tone and smooth muscle cell (VSMC) proliferation. Intermittent exposure to cold (6 hr/day) results in hypertension and cardiac hypertrophy. Interestingly, PDE1A expression was up-regulated in resistance arteries, kidneys and heart of rats exposed to cold. We hypothesize that RNAi inhibition of PDE1A would attenuate cold-induced hypertension (CIH) and cardiac hypertrophy (CICH). We constructed PDE1A shRNA (PDE1A-shRNA) plasmid, Control-shRNA plasmid, and LacZ plasmid driven by a CMV promoter. AAV-2 was used for
in vivo
gene delivery. Five groups of rats were used to test the effect of in vivo inhibition of PDE1A expression on the development of CIH and CICH. Four groups received an intravenous injection of AAV.PDE1A-shRNA, AAV.Control-shRNA, AAV.LacZ, and PBS, respectively, before intermittent exposure to moderate cold temperature (5°C), while the last group received PBS and was kept at room temperature (25°C) as a control. Blood pressure (BP), monitored using a telemetry system, was increased significantly in all cold-exposed groups except the group treated with PDE1A-shRNA, indicating that PDE1A-shRNA prevented the development of CIH. PDE1A-shRNA enhanced hypothesive responses to acetylcholine in cold-exposed rats, indicating improved endothelial dysfunction. The MRI analysis indicated that the
in vivo
left ventricle wall thickness was also decreased significantly in the cold-exposed rats treated with PDE1A-shRNA. All animals were sacrificed at 10 weeks after gene delivery. PDE1A-shRNA significantly attenuated the cold-induced increases in heart weight, cardiomyocyte sizes, and vascular wall thickness. PDE1A-shRNA increased intracellular cGMP and cAMP levels and decreased VSMC cell proliferation and migration. PDE1A protein expression was significantly decreased, indicating effective silence of PDE1A gene. RNAi inhibition prevented CIH, CICH and cold-induced vascular dysfunction and hypertrophy. Thus, PDE1A plays a critical role in the pathogenesis of CIH and CICH.
Abstract 339: Long-term AAV6-βARKct Myocardial Gene Therapy Improves Cardiac Function Via Restoration Of β-Adrenergic Receptor Signaling And Neurohormonal Status Of The Failing Heart
Authors: Rengo Giuseppe , Lymperopoulos Anastasios , Zincarelli Carmela , Donniacuo Maria , Soltys Stephen , Rabinowitz Joseph E , Koch Walter J
Published: July 2021

BACKGROUND: The up-regulation of G protein-coupled receptor kinase-2 (GRK2) that is present in compromised myocardium contributes to dysfunctional β-adrenergic receptor (βAR) signaling and cardiac function in heart failure (HF). The peptide βARKct, which inhibits the activation of GRK2, has been shown in acute gene transfer experiments to rescue HF. This study was designed to evaluate chronic βARKct expression in post-myocardial infarction (MI) induced HF using stable myocardial gene delivery with adeno-associated virus serotype-6 (AAV6).

METHODS AND RESULTS: In 12 week post-MI HF rats, we delivered βARKct or as a control, Green Fluorescent Protein, via direct intramyocardial injection. We also treated groups with concurrent administration of metoprolol. We found robust and long-lasting (up to 12 weeks post-delivery) transgene expression in the left ventricle (LV) and βARKct expression resulted in significantly improved global heart function as LV ejection fraction and ±dP/dt were increased, whereas LV end diastolic diameter and pressure were decreased. At the molecular level, cardiac βAR density and cAMP accumulation significantly improved over control groups. Fibrotic and hypertrophy markers, as well as heart-to-body weight ratio were markedly decreased by βARKct gene therapy indicating active reversal of adverse LV remodeling. For the first time, we found that chronic βARKct expression and normalization of cardiac βAR signaling led to a reduction of circulating levels of cardiotoxic neurohormones (catecholamines and aldosterone) demonstrating a potential additive mechanism of GRK2 inhibition. Concomitant metoprolol administration preserved the gain in inotropy achieved by βARKct, suggesting compatibility of these two therapeutic modalities, however, metoprolol alone only prevented the deterioration of cardiac function in HF.

CONCLUSIONS
: Chronic cardiac βARKct gene therapy for HF treatment via AAV6-mediated intracardiac gene delivery is feasible and results in improved cardiac function accompanied by restoration of βAR molecular abnormalities and amelioration of neurohormonal status of HF. These findings suggest βARKct gene therapy might be clinically applicable and of significant value for human HF treatment.

This research has received full or partial funding support from the American Heart Association, AHA Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).
Abstract 383: Depletion of Perivascular Macrophages of the Blood-Brain Barrier Reduces Sympathetic Drive in Rats after Myocardial Infarction
Authors: Yu Yang , Zhang Zhi-hua , Wei Shun-guang , Serrats Jordi , Weiss Robert M , Felder Robert B
Published: July 2021

Pro-inflammatory cytokines induce cyclooxygenase-2 (COX-2) activity and synthesis of prostaglandin E2 (PGE
2
) in perivascular macrophages of hypothalamic paraventricular nucleus (PVN). Cytokine-induced PGE
2
synthesis in the brain contributes to increased sympathetic nerve activity following myocardial infarction (MI). Perivascular macrophages can be selectively depleted by clodronate liposomes. Depletion of perivascular macrophages of the blood-brain barrier will reduce COX-2 expression and cytokine-induced sympathetic nerve activity in rats after MI. Clodronate liposomes (CLOD), control liposomes (LIPO) or vehicle (VEH, artificial CSF) were injected (1 μl/min over 40 minutes) into lateral ventricle in rats within 24 hours of MI, or normal rats. One week after injection, perivascular macrophages were completely destroyed and COX-2 immunoreactivity was eliminated in PVN of all CLOD-treated rats but not in LIPO or VEH-treated rats. Activated microglia were unaffected by CLOD. Compared with sham-operated rats (n=10), VEH-treated MI rats (n=11) had increased COX-2 (0.09±0.01* vs 0.05±0.01, normalized to GAPDH, *P<0.05), interleukin-1β (IL-1β, 0.48±0.06* vs 0.21±0.03) and tumor necrosis factor - α (TNF-α, 0.50±0.09* vs 0.22±0.08) mRNA in PVN, IL-1β (23±2* vs 15±1, pg/ml), TNF-α (5±1* vs 2±0, pg/ml), norepinephrine (1146±143* vs 450±66, pg/ml) levels in plasma, PGE
2
(1152±109* vs 480±84, pg/ml) in CSF and Fra-like-positive neurons (40±3* vs 21±2) in PVN. CLOD treatment of MI rats (n=11) normalized COX-2 mRNA in PVN and reduced CSF PGE
2
(by 30%#, #P<0.05 vs VEH), plasma norepinephrine (by 34%#) and Fra-like positive PVN neurons (by 29%#), but had no effects on IL-1β and TNF-αin PVN or plasma. In normal rats, CLOD treatment (n=9) reduced (P<0.05, vs VEH) sympatho-excitatory responses (renal sympathetic nerve activity, blood pressure and heart rate) induced by intracarotid artery injection of TNF-α (0.5 μg/kg). No effects were found in LIPO-treated MI or normal rats. Pro-inflammatory cytokines stimulate sympathetic excitation after MI by inducing COX-2 activity and PGE
2
production in perivascular macrophages of the blood-brain barrier.
Abstract 403: BMPER Is an Angiogenic Modulator that is Regulated by KLF-15 and FoxO3A and Controls Bone Morphogentic Protein Activity
Authors: Helbing Thomas , Heinke Jennifer , Volkmar Franziska , Wehofsits Leonie , Baar Kim-Miriam , Diehl Philipp , Bode Christoph , Patterson Cam
Published: July 2021

BMPER (bone morphogenetic protein [BMP] endothelial precursor cell derived regulator) is an extracellular protein, that interacts with BMPs and thereby modulates BMP dependent vasculogenesis and angiogenesis. Our previous observations suggest a complex regulation of BMPER expression. During embryogenesis BMPER is expressed at the time and at sites of vasculogenesis, whereas in the adult organism it is expressed in heart, lung and skin.

Methods and Results: We have cloned the mouse BMPER promoter and appropriate deletion constructs into pGL3 to regulate luciferase expression. As predicted in silicio, we found that Sp1 and Sp1-like transcription factors such as the krueppel-like factors (KLFs) regulate BMPER transcription. KLF-15 resulted in a 4.5 fold upregulation. Accordingly, BMPER expression was inhibited by the Sp-1/SP-1 like inhibitor mitramycin A. Site specific mutation of a proximal KLF-15 binding site reduced the effect of KLF-15 on BMPER expression. Along the same lines, knock down of KLF-15 in HUVEC by siRNA reduced BMPER expression. The transactivating effect of KLF-15 could be competed away by coexpression of Sp-1 suggesting that both factors may compete for the same binding site in the BMPER promoter. In EMSA, an oligo representing a well characterized KLF-15 binding site in the AceCs2 promoter but not an oligo encoding for a NFkappa-B site competed with the oligo coding for the KLF-15 site in the BMPER promoter. In contrast FoxO3A, a member of the FoxO family of transcription factors, serves as an inhibitor of BMPER expression, as shown by gain and lack of FoxO3A experiments. Additionally, we found that BMPER stimulates angiogenesis in a BMP-4 dependent manner in several in vitro and in vivo assays. Vice versa, BMPER is necessary for BMP-4 to exert is angiogenic activity on endothelial cells.

Conclusion: BMPER is upregulated by KLF-15 and inhibited by FoxO3a. BMPER has angiogenic activity and is a key modulator of the BMP pathway.
Abstract 405: Deficiency of p38α MAPK in Macrophages Promotes Apoptosis and Plaque Necrosis in Advanced Murine Atherosclerotic Lesions
Authors: Seimon Tracie A , Wang Yibin , Han Seongah , Senokuchi Takafumi , Kuriakose George , Tall Alan , Tabas Ira
Published: July 2021

Endoplasmic reticulum (ER) stress occurs in macrophage-rich areas of advanced atheroscle-rotic lesions and contributes to macrophage apoptosis and subsequent plaque necrosis. Therefore, signaling pathways that alter ER stress-induced apoptosis may affect the development of advanced atherosclerosis. Here we show that Western diet-fed
Apoe
−/−
mice deficient in macrophage p38α MAPK have a marked increase in apoptosis and plaque necrosis. We found a significant increase in the mean necrotic area in the p38α-deficient lesions (46,700 μm
2
versus 18,965 μm
2
in the control mice, p < 0.008). Quantification of the data revealed an approximate 51% increase in the percent of apoptotic cells in the p38α-deficient lesions. The macrophage-p38α-deficient lesions also exhibited a 30% decrease in collagen content and a 40% decrease in fibrous cap thickness. Consistent with our
in vivo
data, we found that ER stress-induced apoptosis in cultured macrophages was markedly accelerated under conditions of p38 inhibition, and this effect was further enhanced in the setting of insulin resistance. Mechanistic experiments revealed that p38 inhibition causes suppression of the pro-survival Akt pathway
in vitro
and
in vivo
. We found that inhibition of Akt with LY294002 enhances apoptosis under conditions of ER stress. Furthermore, expression of a constitutively active Akt, myristoylated-Akt, blocked the enhancement of apoptosis that occurred with p38 inhibition, but did not inhibit apoptosis induced by ER stress itself. In view of clinical trials exploring the use of p38 inhibitors in inflammatory diseases, our results raise the possibility that these drugs may promote plaque progression in subjects with advanced atherosclerotic lesions.

This research has received full or partial funding support from the American Heart Association, AHA Founders Affiliate (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont).
Abstract 410: Mitochondrial Uncoupling with CCCP Reverses Acute Hyperglycemia Induced Endothelial Dysfunction in Human Arterioles
Authors: Wang Jingli , Gutterman David D , Widlansky Michael E
Published: July 2021

Background
: Acute hyperglycemia (HG) impairs endothelial function, which may be related to hyperpolarization of the inner mitochondrial membrane and excessive production of mitochondrial superoxide (ROS). Mitochondrial uncoupling agents could potentially reverse endothelial dysfunction due to HG in intact human arterioles.

Methods
: Human adipose arterioles were incubated in either euglycemic (NG, 5mM) or hyperglycemic (HG, 33mM) buffer for 4 hours. Vessels were then exposed to increasing doses of acetylcholine (ACh) in the presence and absence of

NADPH oxidase inhibitor gp91ds-tat or

CCCP, (100 nM), a mitochondrial membrane uncoupling agent.

Vascular superoxide levels were also measured after incubation with NG and HG using a chemiluminescent probe (L-012) in the absence and presence of 1 and 2 above.

Results
: ACh-induced vasodilation was impaired by HG vs. NG control (P<0.001). This impairment was modestly reversed by gp91 ds-tat (P< 0.001), but endothelial function was completely restored to the level observed under euglycemic conditions by CCCP (P=0.008 vs. HG, P>0.90 vs. NG). Endothelium-dependent vasodilation in HG vessels was greater after CCCP compared to gp91ds-tat (P<0.001). L-NAME inhibited all dilation to ACh indicating endothelium-dependence (data not shown). HG induced an increase in superoxide relative to NG (P<0.001), which was similarly suppressed by gp91ds-tat (P = 0.001) and CCCP (P=0.002).

Conclusion
: HG-induced endothelial dysfunction is reversed with partial uncoupling of mitochondrial oxidative phosphorylation. Mitochondrial uncoupling more than NADPH oxidase blockade improves endothelial dysfunction due to HG.
Abstract 414: Endothelial dysfunction of Epicardial Resistance Arteries in Pigs after Coronary Artery Implant of Paclitaxel-Eluting Stents
Authors: Li Jinsheng , Pendyala Lakshmana , Shinke Toshiro , Chen Jack P , Chronos Nicolas , Robinson Keith , Hou Dongming
Published: July 2021

Background:
Abnormal endothelium-dependent vasorelaxation of coronary arteries adjacent to drug-eluting stent (DES) implants has been reported and may be associated with late stent thrombosis. However, epicardial resistance vessel vasomotor function in the distal perfusion bed of DES has not been studied. We tested small vessel function 1 mo after coronary artery paclitaxel-eluting stent (PES) implant.

Methods:
Pigs (n=9) received overlapping bare metal stent (BMS) and PES as well as naïve vessels. One month later, myocardium distal to the stented region, as well as similar location in non-stented vessel (nSV) was harvested. Eighteen epicardial resistance arteries (lumen diameter ~ 250μm) were isolated under a dissecting microscope and mounted on a myograph apparatus. Vasoconstriction (KCl, PGF2α & ET-1), endothelial-dependent relaxation (EDdR) response to substance P (SP), and endothelial-independent relaxation (EDiR) to sodium nitroprosside (SNP) were measured.

Results:
Angiographic late lumen loss at 1-mo was less for PES than BMS (p=0.002). Epicardial resistance artery relaxation was attenuated in the perfusion distribution of PES, as maximal EDdR in response to SP was 56.0±7.0% compared to vessels isolated from the perfusion distribution of BMS (85.0±7.0%) and non-stented coronary segments (107±2.0%, P=0.034 vs. PES). Maximal EDiR in response to SNP, however, was similar among the groups (90.0±6.0%, PES; 98.0±2.0%, BMS; 123±5.0%, nSV; P=NS respectively). Similarly contraction to endothelin-1 was not affected by stent type (3.95±0.56%, PES; 3.07±0.65%, BMS; 3.01±0.36%, nSV; ratio of contraction ET-1/KCl; P=NS respectively).

Conclusions:
Epicardial resistance artery endothelial-dependent vasomotor dysfunction is present one month after overlapping coronary PES implant in swine. Derangement of microcirculatory vasomotor function may also be present with DES implant in humans and be associated with a spectrum of adverse clinical sequelae.
Abstract 424: Vascular Smooth Muscle Cell Expression of S100a12 in Vivo Induces Enhanced Oxidative Stress & Vascular Remodeling
Authors: Bowman Marion Hofmann , Wilk Jeannine , Kim Gene , Zhang Yanmin , Rehman Jalees , McNally Elizabeth
Published: July 2021

S100A12 is a small calcium binding protein that is a signal transduction ligand of the receptor for advance glycation endproducts (RAGE). S100A12, like RAGE, is expressed in the vessel wall of atherosclerotic vasculature, particularly in smooth muscle cells (SMC). While RAGE has been extensively implicated in inflammatory states such as atherosclerosis, the role of S100A12 is less clear. We tested the hypothesis that expression of human S100A12 directly exacerbates vascular inflammation. Several lines of Bl6/J transgenic mice (tg) expressing human S100A12 in SMC under control of the SM22a promoter were generated. Primary aortic SMC from tg and wild type (wt) littermates were isolated and analyzed for (i) proliferation using MTS/Formazan Assay and BrdU incorporation, (ii) oxidative stress using using flow cytometry with MitoSOX antibody, oxidative DNA damage using immunofluorescence microscopy with anti-8-oxo-dG antibody, and NF-kB activation measured by EMSA and (iii) cytokine expression measured by IL-6 ELISA. Furthermore, the aortas from tg and wt mice were examined.

Results: Tg but not wt SMC expressed S100A12 protein. Tg SMC had a significant 1.9 to 2.7 fold increase in conversion of MTS into Formazan at 24–96 hours likely reflective of increased metabolic activity since BrdU incorporation into DNA was less in tg compared to wt SMC (4% vs 21% positive BrdU nuclei, p <0.05). Tg SMC showed significantly higher levels of mitochondrial generated ROS, nuclear staining for oxidative DNA damage which was not detected in the nuclei of wt SMC’s, and a 2.5 fold increase in NFkB activity. IL-6 production at baseline was higher in tg SMC’s (615 vs 213 pg/ml, p< 0.05) and increased dramatically after LPS treatment (10 ng/ml) in tg SMC’s (2130 vs 415 pg/ml). Histologic examination of the thoracic aorta at 10 weeks of age revealed increased collagen deposition in the aortic media with fragmentation and disarray of elastic fibers. In vivo ultrasound revealed a progressive dilation of the aortic arch from age 10 weeks to 16 weeks of age (1.27 to 1.60 mm, p<0.05) in tg but not in wt littermate mice (1.30 to 1.33 mm, p=0.1). These data reveal the novel finding that targeted expression of human S100A12 in SMC modulates oxidative stress, inflammation and vascular remodeling.
Abstract 428: Regression of Abdominal Aortic Aneurysms by Targeting Toll-Like Receptor 2
Authors: Yan Huimin , Ma Yonggang , Mi Su , Cui Bing , Zhang Xiaowei , Cai Wenfeng , Lv Xiaoxi , Wang Qingqing , Yan Jun , Xin Bingmu , Wang Xiaoxing , Wang Ziyan , Li Yongchao , Yang Hongzhen , Hu Zhuowei
Published: July 2021

Abdominal aortic aneurysm (AAA), a chronic inflammatory disorder, results in progressive expansion and rupture of the aorta with high mortality among elderly people. There is increasing evidence that both innate and adaptive immunity tightly regulate the progress of AAA. We recently find that TLR2 plays a critical role in the regulation of wound-repairing process after tissue injury caused by the molecules of pathogen-associated molecular pattern or damage-associated molecular pattern. We wondered if TLR2 had a role in mediating calcium-induced inflammatory responses to participate in the development of AAA in a mice model of CaCl
2
-induced AAA. We found that selectively blocking TLR2 activity using a TLR2-neutralizing antibody not only prevented the development of AAA but also caused regression of well established AAA elicited by incubation of calcium. Furthermore, TLR2 deficient mice failed to develop AAA and had reduced aortic expansion and internal elastic lamina degradation, validating the roles of TLR2 in the development of AAA. The prophylactic and therapeutic effects of blocking TLR2 activity were accompanied by a significant inhibition of vascular fibrosis and inflammation because targeting TLR2 decreased the expressions of α-SMA, matrix metalloproteinase-2/9, and several Th1 and Th2 cytokines such as IFNγ, IL-10, IL-6, TGFβ1 as well as transcription factor Stat3/p-Stat3 in the vascular tissue of AAA. Targeting TLR2 also specifically decreased the expression and extracellular distribution of high-mobility group box 1 and heat shock protein 70, the endogenous TLR2 ligands that sustain chronic inflammation and vascular injury, in the aortic tissue of AAA. In conclusion, our studies demonstrate that TLR2 plays a critical role in the pathogenesis of calcium-induced AAA and highlight unifying mechanisms that blocking TLR2 activity can inhibit specific immune responses induced by calcium and subsequently by HMGB1/HSP70 to modify injury-induced abnormal vascular remodeling. TLR2 is a promising therapeutic target for the prevention and treatment of AAA.
Abstract 429: Rosiglitazone Reduces Development and Rupture of Experimental Aortic Aneurysms
Authors: Deb Rajdeep , Torsney Evelyn , Thompson Matthew M , Jones Alun , Howe Franklyn , Cockerill Gillian W
Published: July 2021

Several case-control studies have shown a negative correlation between diabetes and aortic aneurysm. Using an animal model of aneurysm, and cell cultures, we investigated the effects of Rosiglitazone (RGZ), a drug used in the treatment of type II diabetes. Twenty-eight 12 month old ApoE-deficient mice were randomised to three groups. All animals had an osmotic pump inserted subcutaneously on the flank, the positive control group and treatment group released Angiotensin-II (Ang-II) (1μg/min/kg), the sham operated group released saline. The treatment group received RGZ (10mg/kg/day) in their drinking water one week before inserting the pumps and continuously during the following 28 day release of Ang-II. Plasma was collected to measure the lipid profile and determine concentration of IL-6 and active MMP-2 and MMP-9. Gross pathology was evaluated using video micrometry. Expression of proteins and RNAs were analysed using immunohistochemistry, ELISA and quantitative real time RT-PCR. The effect of RGZ on Ang-II induced DNA-binding of transcription factor Egr-1 was determined using electrophoretic mobility shift assays of human aortic smooth muscle cell cultures. In comparison to the positive control group of animals, treatment with Rosiglitazone inhibited the occurrence of fatal rupture (5/10 vs 0/10) and reduced maximal dilatation of the aorta (5.6 ± 0.26 vs 2.6 ± 0.4 mm
2
). RGZ reduced the concentration of total cholesterol but no significant difference in the atherosclerotic burden was found between groups. RGZ inhibited the angiotensin II-induced tissue expression of AT1a receptor, but had no affect on increased mRNA expression of AT2 receptor, MMP-2 or MMP-9. Plasma protein concentrations of IL6, and MMP-9 were increased by Ang-II and decreased by RGZ. The amount of collagen deposited in the supra-renal region of the abdominal aortae was increased in animals treated with RGZ. RGZ inhibited Ang-II induced DNA binding of the transcription factor, early growth response-1 (Egr-1) in human smooth muscle cells. These data suggest that RGZ reduces the incidence of development and rupture in this murine model of aneurysm through AT1a mediated effects on transcriptional regulation of genes which determine phenotypic status of the vessel wall.
Abstract 430: Inhibition of Aneurysm Development by Intra-peritoneal Application of Ribbon-type Decoy Oligodeoxynucleotide against Nuclear Factor-κB and Ets in a Rat Model
Authors: Miyake Takashi , Aoki Motokuni , Fujiwara Yoshikazu , Makino Hirofumi , Morishita Ryuichi
Published: July 2021

As elective surgical or endovascular repair is not effective to improve survival of small abdominal aortic aneurysm (AAA), the development of a novel therapeutic approach for treating small AAA is awaited. We have previously reported the regressive effect of chimeric decoy oligodeoxynucleotides (ODN) against NFκB and ets in an experimental AAA model. However, local application of conventional phosphorothioated chimeric decoy ODN (PC-ODN) was necessary due to easily degradation. Therefore, we developed a novel chimeric decoy ODN with a ribbon-shaped circular structure (RC-ODN) to stabilize decoy ODN against nucleases. Transfection of RC-ODN inhibited MMP-2 expression in VSMC, but not MMP-9 expression. In contrast, in cultured THP-1 cells, MMP-9 expression was suppressed by RC-ODN, whereas MMP-2 expression was not altered. Interestingly, these inhibitory effects of RC-ODN were more potent than that of PC-ODN (n=8, P<0.05). Next, intra-peritoneal application of RC-ODN was performed in an elastase-induced rat AAA model. One week after injection, FITC-labeled RC-ODN were detected in migrating macrophages into aneurysm wall. In addition, both NFκB and ets activity were suppressed by RC-ODN. Four weeks after treatment, RC-ODN treatment inhibited the aortic dilatation as compared to control or scrambled decoy ODN transfer (n=6, P<0.05), while PC-ODN treatment failed to prevent aneurysm formation. Interestingly, a significant preservation of elastic fibers was observed with RC-ODN treatment, accompanied by a reduction of expression of MMP-9, MMP-12, cathepsin B and cathepsin K (P<0.05), while the recruitment of macrophages was not inhibited. Finally, to consider the clinical application, we investigated the effect of intravenous injection of RC-ODN. Four weeks after infusion of angiotensin II in apolipoprotein E-deficient mice, intravenous injection of RC-ODN (1 mg/Kg) was performed 3 times per week. RC-ODN treatment showed a tendency to decrease the size of AAA, but not significant. In conclusion, the present study provided a novel strategy to treat AAA using intra-peritoneal application of RC-ODN in a rat model. Further development of this strategy would be useful to treat human AAA, as a minimally invasive molecular therapy.
Abstract 472: Cardiac Fibroblasts Regulate Myocardial Proliferation and Ventricular Formation through β1 Integrin Signaling
Authors: Ieda Masaki , Tsuchihashi Takatoshi , Ivey Kathryn , Srivastava Deepak
Published: July 2021

Ventricular cardiomyocyte proliferation is essential to support increasing hemodynamic demands during late cardiogenesis. The increase in thickness of the ventricle is achieved by greater proliferation in the compact myocardium than in the trabecular myocardium. Disruption of this process leads to a disease known as ventricular non-compaction. Although epicardial-derived signals may contribute to the proliferative process, the factors and cell types responsible for development of the compact layer are unclear. In particular, the function of embryonic cardiac fibroblasts, derivatives from epicardium, and their secreted factors are largely unknown. By analyzing cell markers, we found that cardiac fibroblasts appeared in the compact myocardium at embryonic day 12.5 and increased in number coincident with growth of the compact myocardium. Using a novel co-culture system, we found that embryonic cardiac fibroblasts were much more effective in inducing cardiomyocyte proliferation than adult cardiac fibroblasts. A genome-wide screen and siRNA knockdown experiments revealed that fibronectin and collagen were embryonic cardiac fibroblast-specific signals that promoted cardiomyocyte proliferation through β1 integrin signaling in primary culture. In vivo, cardiomyocyte expression of β1 integrin was upregulated as cardiac fibroblasts appeared in the heart and secreted extracellular matrix proteins. In agreement with a critical role for β1 integrin, cardiomyocyte-specific deletion of β1 integrin in mice resulted in reduced myocardial proliferation and ventricular compaction, with downregulation of cyclinD1 and cyclinE1 and upregulation of cyclinG2 and p21, leading to prenatal death. These findings demonstrate that cardiac fibroblasts regulate cardiomyocyte proliferation and ventricular compaction through extracellular matrix/β1 integrin signaling.
Abstract 483: Targeting HDL Quality Rather than Quantity: Providing the Mechanistic Rationale for the Pharmacogenomic Interaction between the Haptoglobin Genotype and Vitamin E on Cardiovascular Disease in Individuals with Diabetes Mellitus
Authors: Asleh Rabea O , Blum Shany , Kalet-Litman Shiri , Aviram Michael , Levy Andrew P
Published: July 2021

Objective.
Pharmacogenomics is a key component of personalized medicine. ICARE, a prospective placebo controlled study, recently demonstrated vitamin E could dramatically reduce CVD in individuals with Diabetes Mellitus (DM) and the Haptoglobin (Hp) 2–2 genotype (40% of DM individuals). However, due to the large number of clinical trials which failed to demonstrate benefit from vitamin E coupled with the lack of a mechanistic explanation for why vitamin E should be beneficial only in DM individuals with the Hp 2–2 genotype, enthusiasm for this pharmacogenomic paradigm has been limited. In this study we sought to provide such a mechanistic explanation based on the hypothesis that the Hp 2–2 genotype and DM interact to promote HDL oxidative modification and dysfunction.

Research Design and Methods.
Clearance of
125
I-Hp 1 or
125
I-Hp 2-hemoglobin (Hb) complexes were assessed in non-DM and DM mice after injection of the complexes in the tail vein. Hb binding to HDL was assessed in HDL isolated by immunoprecipitation. Oxidative modification of HDL was assessed by measuring HDL associated lipid peroxides and redox active iron. HDL function was assessed based on its ability to promote cholesterol efflux from macrophages. A crossover placebo controlled study in Hp 2–2 DM humans and in Hp 1–1 and Hp 2–2 DM mice assessed the ability of vitamin E to reduce oxidative modification of HDL and improve HDL function.

Results.
In DM mice, the half-life of the Hp 2-Hb complex was dramatically increased. Immunoprecipitation studies demonstrated specific binding of Hp-Hb complex to HDL with over 25% of the injected Hp 2-Hb complex associating with HDL. Hb was found to be associated with HDL in all Hp 2–2 DM individuals and mice by western blot. Redox active iron and lipid peroxides associated with HDL were significantly increased and HDL function was remarkably impaired in Hp 2–2 DM individuals and mice. Vitamin E significantly decreased oxidative modification of HDL and improved HDL function in Hp 2–2 DM but had no effect in Hp 1–1 DM.

Conclusions.
In Hp 2–2 DM individuals, there is an increased amount of Hb bound to HDL which results in an oxidative modification of HDL and HDL dysfunction. Vitamin E significantly improves the quality of HDL and may explain the exclusive CVD benefit in this cohort.
Abstract 484: Large Tumor Suppressor 2, A Downstream Target of Mammalian Sterile 20 Like Kinase 1, Is an Endogenous Regulator of Myocyte Size and Apoptosis in the Heart
Authors: Matsui Yutaka , Nakano Noritsugu , Takagi Hiromitsu , Shao Dan , Gao Shumin , Hong Chull , Zhai Peiyong , Yabuta Norikazu , Nojima Hiroshi
Published: July 2021

Mammalian sterile 20-like kinase 1 (Mst1) plays an important role in mediating apoptosis and inhibiting hypertrophy in the heart. Mst1 forms a complex with hWW45 and Large tumor suppressor 2 (Lats2), thereby activating Lats2. Lats2 dose dependently increased apoptosis in cultured cardiac myocytes and transgenic mice with cardiac specific expression of Lats2 exhibited a cardiac dysfunction, suggesting that stimulation of Lats2 leads to increased cell death and cardiac dysfunction. In order to elucidate the function of endogenous Lats2 in the heart, we generated adenovirus harboring dominant-negative Lats2 (Ad-DN-Lats2) and transgenic mice with cardiac specific expression of dominant-negative Lats2 (Tg-DN-Lats2). Ad-DN-Lats2 prevented induction of apoptosis and inhibition of hypertrophy by Mst1 in cultured cardiac myocytes, suggesting that Lats2 mediates the function of Mst1. In Tg-DN-Lats2, both LV weight/tibia length (LVW/TL; 4.34 vs 3.68, N=9, N=7, p<0.01) and RV weight/tibia length (1.15 vs 0.88, N=9, N=7, p<0.01) were greater than in NTg. Echocardiographically determined LV function was normal in Tg-DN-Lats2 at baseline except that LV wall thickness was greater in Tg-DN-Lats2 than in NTg (LV posterior wall thickness; 0.97 vs 0.79 mm, N=10, N=7, p<0.05). Since expression of Lats2 is increased in response to pressure overload (8 fold), we examined whether upregulation of endogenous Lats2 during pressure overload works as a negative feedback mechanism for cardiac hypertrophy. Tg-DN-Lats2 mice exhibited greater LVW/TL than NTg after transverse aortic constriction (TAC) (5.9 vs 4.7 mm, N=9, N=7, p<0.05). Increases in LVW/TL (+37% vs 27%, N=9, N=7, p<0.05) and LV cross sectional area (+25% vs +16%, N=5, N=5, p<0.05) after TAC were greater in Tg-DN-Lats2 than those in NTg. Moreover, Tg-DN-Lats2 exhibited significantly lower cardiac myocyte apoptosis than NTg after TAC. These results suggest that Lats2 is a negative regulator of cardiac hypertrophy at baseline and in response to pressure overload, and a positive mediator of cardiac myocyte apoptosis under pressure overload. In conclusion, Lats2 is an evolutionarily conserved negative regulator of organ size: namely inhibiting hypertrophy while stimulating apoptosis in the heart.

This research has received full or partial funding support from the American Heart Association, AHA Founders Affiliate (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont).
Abstract 486: Allogeneic Mesenchymal Stem Cells in Cellular Cardiomyoplasty: Mechanisms of Cardiac Repair
Authors: Hatzistergos Konstantinos E , Quevedo Henry , Oskouei Behzad N , Hu Qinghua , Mazhari Ramesh , Zambrano Juan P , Valdes David , Feigenbaum Gary S , Rodriguez Jose R , Margitich Irene S , Zimmet Jeffrey M , Revilla Concepción , Heldman Alan W , McNiece Ian , Hare Joshua M
Published: July 2021

Background-

Although there is significant enthusiasm for cell-based therapies, whether administered cells differentiate into cardiomyocytes (CM) or exert their effects solely by paracrine signaling remains controversial. We hypothesized that allogeneic bone marrow-derived mesenchymal stem cells (aMSCs) regenerate the infarcted heart by cell autonomous mechanisms involving differentiation into myocytes, endothelial cells, vascular smooth muscle cells and the stimulation of endogenous precursor cell proliferation.

Methods and Results
-

Seventeen female swine underwent myocardial infarction. Three days later, they received intramyocardial injections of male GFP
pos
aMSCs (n=7), placebo (n=7) or no injection (n=3). Animals were euthanized at 24h (n=1 aMSCs, n=1 placebo), 72h (n=3 aMSCs, n=3 placebo) and 2 weeks (n=3 aMSCs, n=3 placebo, n=3 controls) after injections. Within 24h, engrafted aMSCs exhibited lineage commitment into CM (GATA4, α-sarcomeric actinin) but not vascular precursors. By 72h, CM differentiation rates increased substantially (GFP
pos
/GATA
pos
: 23.4±3.2% of GFP
pos
cells at 72h
vs
13.4±5.4% at 24h) as well as vascular lineage committment (GFP
pos
/KDR/Factor VIII
pos
: 1.3±0.4%). Importantly, aMSCs migrated throughout ischemic but not viable myocardium. Two weeks later, MSCs had differentiated into mature vessels and CM. Cardiac regeneration was accompanied by recruitment of endogenous c-kit
pos
cells at infarct (IZ) and border (BZ) zones of the treated animals, compared both to placebo and control (p≤0.001). In contrast to the untreated animals, c-kit cells were localized in clusters in the IZ and BZ of the treated hearts and formed connexin-43 and N-cadherin connections with each other and the GFP
pos
aMSCs, resembling stem cell niches. The number of the c-kit
pos
/GATA-4
pos
cells within the niches was significantly higher at the BZ of the treated hearts (3.9±1.6%) compared to placebo and control (p≤0.05), indicative of an endogenous repair mechanism.

Conclusions-

Together these findings offer important mechanistic insight into the basis of aMSCs ability to achieve post-MI cardiac regeneration, and illustrate that the cells exert both cell dependent and host dependent effects.
Abstract 511: Mrp8 And Mrp14,Endogenous Activators of Toll-lLke Receptor4, are Associated with Rupture-Prone Human Atherosclerotic Plaques
Authors: Ionita Mihaela G , Pasterkamp Gerard , deKleijn Dominique
Published: July 2021

Objectives
: Atherosclerosis is a chronic, complex inflammatory process and is the underlying cause of stroke and myocardial infarction due to rupture of the atherosclerotic plaque leading to acute occlusion of the artery in the brain or heart. Macrophages, infiltrating atherosclerotic lesions, abundantly express Mrp8 and Mrp14. Recently Mrp8, Mrp14 and the complex Mrp8/14 have been identified as endogenous ligands of Tlr-4.The role of Tlr-4 in the development and progression of the atherosclerotic plaque is well recognized and it is associated with a rupture-prone plaque phenotype. Expression of Mrps in human plaques and its relation to plaque phenotype is unknown. For this, we investigated the levels of Mrp8, Mrp14 and Mrp8/14 complex in a large number of human atherosclerotic plaques.

Methods and results
: Mrp8, Mrp14 and Mrp8/14 were quantified by ELISAs in human carotid endarterectomy specimens (186 patients) and plaque phenotype was determined by immunohistochemistry. Mrp levels were higher in the unstable (58 fibro-atheromatous, 64 atheromatous) compared to the stable (64 fibrous) plaques: Mrp8
p
=
0.001
; Mrp14
p
=
0.001
; Mrp8/14
p
=
0.01
. Concomitantly, Mrp8, Mrp14 and Mrp8/14 were associated with characteristics of unstable plaques: more macrophages (
p
=
0.024; p
=
0.002; p
=
0.076
), less smooth muscle cells (
p
=
0.041; p
=
0.001; p
=
0.074
), larger lipid core (
p
=
0.001; p
=
0.001; p=0.004
), less collagen (
p
=
0.440; p
=
0.011; p
=
0.372
). Furthermore, Mrp plaque levels were positively correlated with the pro-inflammatory cytokines (IL-6 and IL-8) and matrix metalloproteinsases (MMP2, MMP8 and MMP9) plaque levels. EDA, marker of stable plaques, was negatively associated with Mrps plaque levels. Histological analysis revealed that Mrps are expressed by a subgroup of plaque macrophages localized in the plaque cap and shoulder, the most rupture-prone sites of an atherosclerotic plaque.

Conclusions:
We show that Mrp8, Mrp14 and Mrp8/14 are strongly associated with the histological characteristics and inflammatory status of human rupture-prone plaques and identify Mrps as a potential marker for rupture-prone plaques.