bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1958: MicroRNA Mir-27b Rescues Impaired Angiogenic Function of Endothelial Progenitor Cells and Accelerates Wound Healing in Type 2 Diabetes
Authors: Wang Jie-Mei , Tao Jun , Chen Alex F
Published: July 2021

Endothelial progenitor cells (EPCs) play a key role in angiogenesis, which is dysfunctional in diabetes. MicroRNAs (miRNAs) are endogenous non-coding RNAs that regulate gene expression at the post-transcriptional level. However, whether miRNAs regulate EPC-mediated angiogenesis in diabetes is unknown. We tested the hypothesis that mir-27b rescues impaired EPC angiogenesis
in vitro
and
in vivo
via suppressing anti-angiogenic molecule thrombospondin-1 (TSP-1) in type 2 diabetes. Bone marrow-derived EPCs from adult male (C57BLKS/J, 9 weeks) type 2 diabetic db/db and their normal littermates db/+ mice (glucose 371.8±37.8 vs. 167.5±21.3 mg/dL, n=38, p<0.05) were used. miRNA processing enzyme Dicer in EPCs was decreased by >40% in db/db vs. db/+ mice (Western blot analysis, n=4 p<0.01), paralleled with >66% reduction of mir-27b expression (real-time PCR, n=4, p<0.05). Both TSP-1 mRNA and protein in EPCs were significantly higher in db/db vs. db/+ mice (real-time PCR, 130.1%, n=4, p<0.05, Western blot analysis, 127.4%, n=4 p<0.05), which were suppressed upon mir-27b mimic transfection (by 75%, real-time PCR and 69%, Western blot analysis, n=4 – 6, p<0.01). EPC-induced angiogenesis was decreased by >70% in db/db vs. db/+ mice (Matrigel tube formation assay, n=4, p<0.05), which was rescued upon mir-27b mimic transfection or silencing TSP-1 expression by its siRNA (both n=4, p<0.05). Furthermore, inhibition of mir-27b in normal EPCs increased their TSP-1 protein by 117.5% (n=6, p<0.05) and impaired their angiogenesis by 81.5% (n=4, p<0.01), both were reversed by silencing TSP-1 expression by its siRNA. Finally, excisional wound closure was markedly delayed in db/db vs. db/+ mice (4-mm punch biopsy, n=4, p<0.05), accompanied by impaired wound angiogenesis (perfusion index by Laser Doppler, n=4, p<0.05). Cell therapy of diabetic EPCs (3×10
5
cells) transfected with mir-27b mimic onto diabetic wounds significantly accelerated their closure rates (n=4, p<0.05 vs. diabetic EPCs alone), with a concomitant augmentation of
in vivo
wound angiogenesis (n=4, p<0.05). Mir-27b rescues impaired EPC angiogenesis and accelerates wound healing in type 2 diabetic mice, at least in part, via suppressing TSP-1 expression.

This research has received full or partial funding support from the American Heart Association, AHA Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).
Abstract 3427: Foxo1-Induced Stimulation of Autophagy Contributes to Cardiac Atrophy
Authors: Hariharan Nirmala , Kyoi Shiori , Usui Soichiro , Sadoshima Junichi
Published: July 2021

The Forkhead box-O (Foxo) family transcription factors mediate atrophy in both skeletal and cardiac muscles. Autophagy is a bulk degradation process, in which cytosolic proteins are sequestered by double membrane vesicles termed autophagosomes and degraded through lysosomes. Expression of Foxo1 is upregulated when the heart undergoes atrophy, such as during cardiac unloading. We hypothesized that Foxo1 increases autophagic flux, which in turn plays an essential role in mediating atrophy of cardiac myocytes. Adenovirus harboring Foxo1 (Ad-Foxo1) was transduced into neonatal rat ventricular myocytes (NRVMs). Foxo1 significantly reduced the cell size at baseline (−54%, p<0.01) and in response to phenylephrine (−61%, p<0.01) in NRVMs, consistent with the notion that Foxo1 stimulates atrophy in cardiac myocytes. Ad-Foxo1 increased LC3-II/LC3-I (3.3 fold vs Ad-LacZ, p<0.01) as well as GFP-LC3 dots (the number of dots/cell, 2.5 fold vs Ad-LacZ, p<0.05), established indicators of autophagy. Ad-Foxo1 significantly reduced protein expression of p62/SQSTM1 (−45% vs Ad-LacZ, p<0.05), a protein degraded through autophagy. Ad-Foxo1 also increased the autophagic flux, as determined by LC3-II/LC3-I (LacZ 0.69, Foxo1 6.92) and accumulation of p62/SQSTM1 (LacZ 0.57, Foxo1 1.18) in the presence of 50μM leupeptin and 500μM PMSF, lysosomal protease inhibitors. Both 3-methyladenine (3MA) and adenovirus harboring shRNA Beclin1 reduced both Foxo1-induced increases in LC3-II formation (LacZ 1.0, Foxo1 3.6, Foxo1+3MA 1.7, Foxo1+shBeclin1 1.3) and Foxo1-induced decreases in p62/SQSTM1, confirming that 3MA and shBeclin1 attenuate Foxo1-induced autophagy. Both 3MA and shRNA Beclin1 attenuated Foxo1 induced reduction in NRVM cell size (LacZ 100%; Foxo1 66%; Foxo1+3MA 81%, p<0.05; Foxo1+shBeclin1 89%, p<0.05) and the relative protein content (Foxo1+3MA vs Foxo1 p<0.05; Foxo1+shBeclin1 vs Foxo1 p<0.01), suggesting that that Foxo1-induced reduction in cell size is in part mediated by autophagy. In conclusion, these results suggest that Foxo1 stimulates autophagy in cardiac myocytes and that stimulation of the autophagy-lysosomal pathway plays an important role in mediating Foxo1-induced cardiac atrophy.
Abstract 3430: Left Ventricular Hypertrophy and Diastolic Dysfunction in Mice Lacking All Nitric Oxide Synthase Isoforms
Authors: Tsutsui Masato , Shibata Kiyoko , Shimokawa Hiroaki , Yatera Yasuko , Furuno Yumi , Nakata Sei , Sabanai Ken , Morishita Tsuyoshi , Tasaki Hiromi , Nakashima Yasuhide , Yanagihara Nobuyuki , Otsuji Yutaka
Published: July 2021

We have recently succeeded in developing mice in which all three nitric oxide synthase isoforms (nNOS, iNOS, and eNOS) are completely disrupted (
PNAS
2005). In this study, we examined cardiac morphology and function in those mice. Cardiac echocardiography and left ventricular (LV) hemodynamic measurement were performed in male wild-type (WT), singly nNOS
−/−
, iNOS
−/−
, eNOS
−/−
, and triply n/i/eNOS
−/−
mice at 2 and 5 months of age (n=5–8). At 2 months of age, no significant cardiac morphological or functional changes were detected in any strains studied. However, at 5 months of age, significant LV hypertrophy (wall thickness, mm) were noted in the triply n/i/eNOS
−/−
mice (1.3±0.1,
P
<0.01) and to a lesser extent in the singly eNOS
−/−
mice (1.1±0.1,
P
<0.05), but not in the singly nNOS
−/−
(0.8±0.1) or iNOS
−/−
mice (1.0±0.1), as compared with the WT mice (1.0±0.2). Furthermore, significant LV diastolic dysfunction (as evaluated by echocardiographic E/A ratio and by hemodynamic peak negative dP/dt), with preserved LV systolic function (as assessed by echocardiographic ejection fraction and by hemodynamic peak positive dP/dt), was noted only in the 5-month-old triply n/i/eNOS
−/−
mice (2.7±0.1 and 2505±60, both
P
<0.05), but not in any singly nNOS
−/−
(2.1±0.2 and 3833±402), iNOS
−/−
(2.0±0.1 and 3773±747), or eNOS
−/−
mice (2.0±0.3 and 2934±122), as compared with the WT mice (1.9±0.1 and 4038±344). In addition, significant cardiac fibrosis (fibrosis area, %, Masson-trichrome staining) was also detected only in the 5-month-old triply n/i/eNOS
−/−
mice (1.4±0.2,
P
<0.05) compared with the WT mice (0.3±0.1). Importantly, arterial blood pressure (mmHg, tail-cuff method) was significantly elevated in the triply n/i/eNOS
−/−
(143±3.1,
P
<0.05) than in the WT mice (104±7.3), but the hypertensive level was comparable to that in the singly eNOS
−/−
mice (140±8.5). Thus, mechanism(s) other than hypertension appears to be involved in the cardiac abnormalities of the triply n/i/eNOS
−/−
mice. These results provide the first evidence that genetic disruption of all NOSs results in LV hypertrophy and diastolic dysfunction in mice in vivo, suggesting a pivotal role of the NOS system in maintaining cardiac homeostasis.
Abstract 3438: Gene Expression Profiling of G Protein-Coupled Receptors in the Adult Mouse Heart
Authors: Moore-Morris Thomas , Varrault Annie , Mangoni Matteo E , Le Digarcher Anne , Negre Vincent , Journot Laurent , Couette Brigitte
Published: July 2021

The G protein-coupled receptors (GPCRs), one of the major targets of pharmaceutical drugs, are essential to many aspects of heart physiology including the regulation of heart rate, contractile force, cardiac remodelling and gene expression. The present study addresses the expression of endoGPCRs, all GPCRs excluding taste and odorant receptors, in each of the four cardiac chambers of adult mouse heart. High-throughput real-time RT-PCR was used to quantify 396 GPCRs and 7 associated proteins in right and left atria and ventricles. Statistical analysis of the most highly expressed transcripts showed that 47 were equally expressed throughout the heart and 89 were differentially expressed in at least one cardiac chamber. Among receptors uniformly expressed, we found well-characterized receptors such as the β2 adrenergic receptor, muscarinic M2 receptor, endothelin receptor type A (ETA) and purinoceptor P2Y2. Surprisingly enough, many of the most abundantly expressed GPCRs (e.g. belonging to the frizzled or chemokine receptor subfamilies) had not previously been identified in healthy heart. Repertoire analysis revealed genes specific to atria and ventricles. The atria exhibited the most specific signature with a lot of receptors not yet studied (receptors belonging to the EGF-like, Mucin-like, Decay-accelerating Factor and Mas-related subfamilies). No gene appeared to be significantly specific to the left or right side of the heart. RT-PCR data were confirmed at the protein level in all four cavities for several receptors and regulatory proteins including ETA, angiotensin receptor 1, protease-activated receptor 1, chemokine receptor CCR2, secreted frizzled related protein 1 and metabotropic glutamate receptor 1 (mGluR1). We also demonstrate that the mGluR1b splice variant is the sole highly expressed metabotropic glutamate receptor in adult mouse heart and is present and functional in isolated ventricular cardiomyocytes. Thus, large scale identification of the regional expression pattern of GPCRs in normal, and in the future in pathological conditions, might lead to the identification of new pharmacological targets for the treatment of human heart diseases.
Abstract 3464: Differential Effects of Progenitor Cell Populations on Myocardial Neovascularization and Left Ventricular Remodeling after Myocardial Infarction
Authors: Dubois Christophe L , Liu Xiashun , Marsboom Glenn , Depelteau Helène , Pokreisz Peter , Streb Witold , Chaothawee Lertlak , Maes Frederik , Gillijns Hilde , Pellens Marijke , Collen Desire , VandenDriessche Thierry , Chua Marinee , Van de Werf Frans , Bogaert Jan , Janssens Stefan
Published: July 2021

Background:
Cardiac progenitor cell transfer may improve myocardial repair after acute myocardial infarction (AMI), but responsible cell types and mechanisms remain uncertain.

Methods:
In a blinded, randomized, controlled study, we infused allogeneic porcine LentiGFP-infected mesenchymal stem cells (MSC, n=11, 10±2x10
6
CD29 – 44 –90 cells, capable of adipogenic and osteogenic differentiation), autologous late-outgrowth endothelial progenitor cells (EPC, n=10, 34±22x10
6
CD29 –31 cells, capable of tube formation) or vehicle (CON, n=10) in the CX artery 1 w after AMI induced by 90 min balloon occlusion. Hemodynamic and cine MRI measurements (dP/dt
max/min
and ejection fraction, EF) were performed at 1w and 7w follow-up. We measured LV remodeling from end-diastolic and end-systolic volumes (EDV, ESV) and quantified infarct size and transmurality on late enhanced images. We evaluated the secretome of cultured EPC and MSC and compared changes in relative upslope (RU) of first-pass perfusion images and vascular density on postmortem sections from infarct core and border zones.

Results:
While systolic function (dP/dt
max/min
and EF) at 7 w was similar between groups, LVEDV increased in MSC and CON pigs, but not in EPC pigs [+23.3 (25–75
th
percentile 1.7;27.9) and +26 ml/m
2
(17.9;31.9) vs +0.8 ml/m
2
(−10.1;18.8), resp,
P
<0.05]. A similar trend was noted for LVESV [+11.1 (10.4;15.4) and +10.7 ml/m
2
(5.9;14.5) in MSC and CON, resp], vs −1.8 ml/m
2
(−13.5;0.7) in EPC (
P
=0.06). After EPC transfer, infarct size decreased more in segments with >50% infarct transmurality (
P
=0.01 vs MSC and CON). Myocardial perfusion increased more in EPC than in MSC and CON [RU +72% (−2;119) vs −2% (−28;84) and +25% (−33;236), resp], and was associated with greater vascular density in infarct border zones (
P
=0.01) and with higher levels of pro-angiogenic growth factors, PLGF and FGF2 in EPC secretome (1730±1002 and 75±44 pg/10
6
cells resp vs 73±16 and 19±12 in MSC secretome,,n=5). No evidence of cell transdifferentiation was observed at 7w.

Conclusion:
Infusion of late-outgrowth EPCs after AMI limits negative LV remodeling but does not mediate cardiomyogenesis. EPC transfer may hold promise for heart failure prevention via paracrine matrix-modulating or pro-angiogenic effects.
Abstract 3465: Fkbp12.6 Overexpression in Mouse Cardiac Myocytes Offers Minor Protection Against Pressure Overload-Induced Cardiac Remodeling and Failure
Authors: Vinet Laurent , Pezet Mylène , Prévilon Miresta , Gellen Barnabas , Dachez Celine , Rouet-Benzineb Patricia , Mercadier Jean-Jacques
Published: July 2021

Alterations in RyR2 function is a hallmark of heart failure (HF). Decreased FKBP12.6 binding to RyR2 has been put forward to explain the diastolic SR Ca
2+
leakage observed in this condition. Previous work in the mouse has shown that cardiac FKBP12.6 overexpression protects against the development of myocardial infarction-induced heart failure. We tested the hypothesis that cardiac FKBP12.6 overexpression protects against transverse aortic constriction (TAC)-induced cardiac remodeling and failure. We used a mouse model of conditional cardiac-specific FKBP12.6 overexpression. Ten weeks after TAC, male transgenic mice (TG) and their wild-type controls (WT) underwent heart catheterization. Hemodynamic and gravimetric data are shown in the table

. Ventricular expression of the hypertrophic gene program and calcium handling proteins were assessed by real-time PCR and Western blot, respectively. Ten weeks after TAC, the mortality rate was 23% in WT and 13% in TG (14/60 vs 5/39, ns). The percentage of mice with HF, estimated on the presence of pulmonary oedema, was 42% in WT-TAC and 32% in TG-TAC (15/36 vs 7/22, ns). BNP mRNA level increased 2.8 fold in WT-TAC (p<0.01 vs WT-Shams) and 2.4 fold in TG-TAC (p<0.01 vs TG-Shams). α-skeletal actin mRNA level increased 4.3 fold in WT-TAC (p<0.001 vs WT-Shams) and 3.8 fold in TG-TAC (p<0.001 vs TG-Shams). β-MHC/α-MHC mRNA ratio increased 2.8 fold in WT-TAC (p<0.01 vs WT-Shams) and 4.3 fold in TG-TAC (p<0.05 vs TG-Shams). RyR2 protein level decreased by 58% in WT-TAC and 41% in TG-TAC (p<0.01 and p<0.05 vs sham-operated mice, respectively). SERCA2a protein level decreased by 29% in WT-TAC and 16% in TG-TAC (p<0.01 and p<0.05 vs sham-operated mice, respectively). No statistical difference was found between TG-TAC and WT-TAC for any of these parameters.

Conclusion: Cardiac FKBP 12.6 overexpression offers weak protection if any against TAC-induced cardiac remodeling and failure in the mouse.
Abstract 3646: Oxidative Stress-Mediated Thombospondin-2 Upregulation Contributes to Diabetic Endothelial Progenitor Cell Dysfunction via Matrix Metalloproteinase-9 Activation
Authors: Bae Ok-Nam , Chen Alex F
Published: July 2021

Endothelial progenitor cells (EPCs) play an essential role in angiogenesis but are dysfunctional in diabetes featuring excessive oxidative stress. Thrombospondin-2 (TSP-2) and matrix metalloproteinase-9 (MMP-9) possess potent anti-angiogenic properties, but their roles on EPC dysfunction in diabetes remain unknown. We tested the hypothesis that increased oxidative stress up-regulates TSP-2 and contributes to diabetic EPC dysfunction through MMP-9 activation in type 2 diabetes. Bone marrow-derived EPCs from adult male type 2 diabetic db/db mice (C57BLKS/J, 9 –13 weeks) and control db/+ mice on the same genetic background were used (blood glucose level, 451.0±8.2 mg/dL for db/db, n=38, vs. 141.3±3.7 mg/dL for db/+, n=28, p<0.01). TSP-2 mRNA and protein were both up-regulated in diabetic EPCs (3.43±0.41 folds of mRNA by qRT-PCR, n=6, p<0.01; 2.35±0.16 folds of protein by Western blot analysis, n= 5, p<0.01, vs. db/+). Silencing TSP-2 by its siRNA in diabetic EPCs improved their angiogenesis in vitro (Matrigel tube formation assay, n=4, p<0.05 vs. scrambled RNA). The increase of TSP-2 in diabetic EPCs was reversed by adenoviral vector-mediated overexpression of dominant-negative Rac1 (68±9%, n=5, p<0.05 vs. β-galactosidase reporter gene), which retards endogenous NADPH oxidase subunit Rac1 activity. Furthermore, both MMP-9 mRNA and enzymatic activity were significantly increased in diabetic EPCs (6.42±2.46 folds of mRNA by qRT-PCR, n=6, p<0.01; 1.37±0.08 folds activity by gelatin zymography, n=3, p<0.01, vs. db/+). The increased MMP-9 activity was significantly inhibited by silencing TSP-2 by its siRNA in diabetic EPCs (47±15%, n=5, p<0.05 vs. scrambled RNA). Our results suggest that up-regulation of TSP-2 mediated by increased oxidative stress contributes to dysfunction of EPC angiogenesis through MMP-9 activation in type 2 diabetes. These findings may provide a basis of targeting oxidative stress/TSP-2/MMP-9 pathway for restoring EPC angiogenic function and cell therapy of diabetic vascular complications.
Abstract 3656: Estradiol Improves Neural Functional Recovery After Nerve Injury by Reducing Inhibition of the Hedgehog-signaling Pathway
Authors: Sekiguchi Haruki , Ii Masaaki , Jujo Kentaro , Renault Marie-Ange , Thorne Tina , Clarke Trevor , Ito Aiko , Tanaka Toshikazu , Klyachko Ekaterina , Tabata Yasuhiko , Losordo Douglas W
Published: July 2021

Background:
Prior data indicate that estradiol (E2) favorably affects neovascularization in injured tissue, and that Sonic Hedgehog (Shh)-induced neovascularization is critical for nerve regeneration; however, little is known about the relationship between E2 and Shh signaling. We tested the hypothesis that E2 potentiates nerve regeneration via Shh-activated neoangiogen-esis.

Methods and Results:
Ovariectomized mice were subjected to unilateral sciatic nerve crush injury and treated with either E2 (via locally administered biodegradable polymer) or placebo (polymer alone). E2 treatment resulted in greater functional recovery of the nerve as evaluated by both exercise duration (P<0.05) and motor nerve conduction velocity (P<0.05), and intraneural vascularity was significantly higher in E2-treated animals than in the placebo-treatment group (P<0.05). Analyses of mRNA expression in the injured nerves of placebo-treated mice showed that the Shh pathway was activated immediately after injury, but Shh expression declined substantially within 7 days. Shh activation persisted in the E2-treatment group; Shh expression on day 7 was greater in E2-treated animals than in the placebo-treatment group (P<0.05). The expression of Hedgehog-interacting protein (HIP), an endogenous Shh inactivator, was significantly downregulated 24 hours after E2 treatment, and HIP downregulation was attenuated by the E2-receptor blocker ICI, suggesting that E2 augments Shh signaling both my enhancing Shh expression and by inhibiting HIP. Inhibition of HIP expression by E2 was also observed in vitro in Schwann cells and endothelial cells. The expression of Ptc1 (Shh receptor) and Gli1 (a downstream target of Shh) was evaluated in Ptc1-LacZ and Gli1-LacZ mice after nerve-crush injury. X-gal staining demonstrated that E2 treatment upregulated both Ptc1 and Gli1 expression within 3 days of surgery.

Conclusions:
E2 accelerates the functional recovery of injured nerves by enhancing angiogenesis. We propose that these effects occur in part through the inhibition of the Shh inactivator HIP, thereby preserving Shh activity after nerve injury. These findings may have identified a novel regulatory mechanism for nerve repair via E2-enhaced, Shh-mediated angiogenesis.
Abstract 3668: Endothelial Progenitor Cells Express Connexins which may be Important for the Functional Integration of Circulating Progenitor Cells into the Endothelium
Authors: Petoumenos Vasileios , Nickenig Georg , Werner Nikos
Published: July 2021

Bone marrow (BM)-derived endothelial progenitor cells (EPC) enhance endothelial cell (EC) repair after focal EC damage, improve endothelial dysfunction, and are an important cellular risk predictor for cardiovascular mortality. For an effective EC repair, functional integration of circulating EPC into the damaged endothelium is a pre-requisite. Here, we determine the importance of connexins (Cx) for the interaction with mature ECs. Expression of Cx was evaluated on magnetic-bead selected CD133+ cells using immunocytochemistry and flow cytometry. CD133+ cells expressed Cx 40 and Cx 43 but not Cx 37. Mononuclear cells were cultivated to obtain early outgrowth EPC or colony forming units-endothelial cells (CFU-EC). Cx 37, 40, and 43 were expressed on cultured EPC. Co-culture experiments with mature human coronary artery endothelial cells (HCAEC) and EPC demonstrated the development of Cx 40, and 43 positive gap-junctions between both cells types. In order to investigate whether the interaction between HCAEC and EPC results in a functional communication between cells, dye transfer experiments were performed. Di-Ac-LDL labelled EPC transformed into Di-Ac-LDL and Calcein AM double positive cells when co-cultured with Calcein AM positive HCAEC and vice versa. In order to study the role of connexins in reendothelialization, C57bl6 (wild-type, WT) mice were subjected to a focal endothelial cell denudation and repetitively treated with WT and Cx37−/− deficient mononuclear cells. In contrast to the treatment with WT mononuclear cells, treatment with Cx37−/− cells was not associated with enhanced reendothelialization. Furthermore, endothelial function of isolated aortic rings derived from Cx37−/− mice showed an impaired endothelial-dependent vasorelaxation compared to WT mice. We provide evidence that connexins are differentially expressed on immature naíve EPC and cultured early-outgrowth EPC. The expression of connexin 37, 40, and 43 may be required for the functional integration of endothelial progenitor cells into the damaged endothelium.
Abstract 3680: Transfusion-Induced Tissue Hypoxia is Ameliorated by
S
-nitrosohemoglobin Repletion of Stored Red Blood Cells
Authors: Diesen Diana L , Stamler Jonathan S
Published: July 2021

Transfusion of stored red blood cells (RBCs) is associated with a decrease in tissue oxygenation in animal models and with increased mortality and morbidity in patients. Recent studies have demonstrated that stored RBCs are deficient in vasodilatory ability and depleted of
S
-nitrosohemoglobin (SNO-Hb), and that renitrosylation
ex vivo
can increase SNO-Hb levels and restore vasoactivity. We have examined in a mouse model the extent to which transfusion impairs tissue oxygenation and whether SNO-Hb repletion can ameliorate that impairment. We report here that transfusion of (mouse) RBCs stored for 1 day or 1 week results in tissue hypoxia that is largely prevented by SNO-Hb repletion prior to transfusion (
1 day stored blood
: % decrease in oxygenation 58+/−10% untreated vs. 92+/−0.7% SNO-Hb repleted, p<0.05, n=3– 6;
1 week stored blood
: % decrease in oxygenation 66+/−10% untreated vs. 91+/−2.8% SNO-Hb repleted, p<0.05, n=3– 6). Storage of mouse blood beyond human expiration-equivalents (1 month) resulted in substantial lysis and the death of all mice transfused (native and SNO-Hb repleted blood, n=5). In conclusion, repletion of SNO-Hb ameliorates the decrease in tissue oxygenation that results from transfusion of untreated stored blood. Therefore, SNO-Hb repletion may provide a simple and efficacious method to reduce transfusion-related mortality and morbidity.
Abstract 3693: Early Growth Response Gene-1 Deficiency In Bone-marrow-derived Cells Reduces Macrophage Accumulation And Atherosclerotic Lesion Formation In A Hyperlipidemic Mouse Model
Authors: Preusch Michael R , Albrecht Claudia , Hofmann Gotz , Blessing Erwin , Katus Hugo A , Bea Florian
Published: July 2021

Early growth response gene-1 (Egr-1), a prototype of a family of zinc-finger transcription factors, is a master regulator of many genes which play important roles in cardiovascular diseases. Within atherosclerotic lesions Egr-1 is expressed in several cell types, such as smooth muscle cells, endothelial cells and monocytes/macrophages. Since macrophages play a pivotal role in atherosclerotic lesion development, this study investigated the effects of Egr-1-deficiency within bone-marrow derived cells on the development of atherosclerosis in a hyperlipidemic mouse model. Therefore we transplanted bone-marrow from Egr-1 deficient mice and wild type controls into lethally irradiated low-density lipoprotein receptor deficient mice. After 20 weeks on a western diet atherosclerotic lesions within the aortic sinus and gene expression of inflammatory genes in the aortas of the recipients were evaluated. Mice receiving Egr-1 deficient bone-marrow had less atherosclerotic lesion development compared with mice receiving wild type bone-marrow (318 736 ± 98 910μm
2
vs. 404 539 ± 92 408μm
2
, p<0.05). The size of the necrotic core within the lesions was also reduced. Immunohistochemistry revealed that mice receiving Egr-1 deficient bone-marrow had less macrophages in comparison to controls. Gene expression analysis in the aortas of the mice demonstrated reduced expression of Vascular Cell Adhesion Molecule (VCAM-1), an important adhesion molecule during the development of atherosclerosis. These results were validated with in vitro studies where Egr-1-deficient peritoneal macrophages revealed less VCAM-1 mRNA expression after stimulation with lipopolysaccharide in comparison to wildtype macrophages. This study demonstrates that bone-marrow derived Egr-1 promotes macrophage accumulation and atherosclerotic lesion development possible over an increased expression of VCAM-1.
Abstract 3753: Critical Role of Scavenger Receptor-BI Expressing Bone Marrow-Derived Endothelial Progenitor Cells in the Attenuation of Allograft Vasculopathy by Human Apo A-I Transfer
Authors: Feng Yingmei , van Eck Miranda , Van Craeyveld Eline , Jacobs Frank , Van Linthout Sophie , Erdel Martin , Tjwa Marc , De Geest Bart
Published: July 2021

Background: Accelerated endothelial regeneration mediated by enhanced endothelial progenitor cell (EPC) incorporation may attenuate the development of allograft vasculopathy.

Hypothesis: We investigated the hypothesis that modulation of EPC biology and attenuation of allograft vasculopathy by increased HDL cholesterol following human apo A-I (AdA-I) transfer requires scavenger receptor (SR)-BI expression in bone marrow-derived EPCs.

Methods: Bone marrow transplantations with SR-BI+/+ or SR-BI−/− bone marrow were performed 4 weeks before gene transfer or saline injection. E1E3E4-deleted vectors containing a hepatocyte-specific human apo A-I expression cassette or containing no expression cassette were injected via the tail vein. Two weeks later, a common carotid artery of a female Balb/c donor mouse was transplanted paratopically into male recipient C57BL/6 mice. To analyse EPC incorporation, sex mismatch bone marrow transplantations were performed in female C57BL/6 mice and incorporated EPCs were quantified by in situ hybridization for the murine Y-chromosome.

Results: Following AdA-I transfer, the number of circulating EPCs increased 2.0-fold (p<0.0001) at different time-points in C57BL/6 mice transplanted with SR-BI+/+ bone marrow but was unaltered in mice with SR-BI−/− bone marrow. The effect of HDL on EPC migration in vitro requires signaling via SR-BI and extracellular signal-regulated kinases (ERK) and is dependent on increased NO production in EPCs. Human apo A-I transfer 2 weeks before paratopic artery transplantation reduced intimal area at day 21 3.7-fold (p<0.001) in mice with SR-BI+/+ bone marrow but had no effect in mice with SR-BI−/− bone marrow. The number of CD31 positive endothelial cells lining the lumen and the number of incorporated EPCs was increased 3.0-fold (p<0.001) and 9.7-fold (p<0.001), respectively, in AdA-I treated chimeric SR-BI+/+ mice compared to control mice with SR-BI+/+ bone marrow. Endothelial regeneration and EPC incorporation was not increased after AdA-I transfer in chimeric SR-BI−/−mice.

Conclusion: Human apo A-I transfer-mediated endothelial regeneration to prevent allograft vasculopathy is strictly dependent on SR-BI expressing bone marrow-derived EPCs.
Abstract 3768: The Deficiency of Ryanodine Receptor Type 2 Accelerates Transition from Adaptive Cardiac Hypertrophy to Dysfunction during Pressure Overload
Authors: Liang Yanyan , Zou Yunzeng , Niu Yuhong , Ma Zhen , Li Jiming , Sun Aijun , Toko Haruhiro , Akazawa Hiroshi , Ge Junbo
Published: July 2021

Alternations of ryanodine receptor type 2 (RyR-2) associate with cardiac hypertrophy and dysfunction, but the mechanism remains elusive. We here examined this by using the mice with heterozygous reduction of
RyR-2
gene (
RyR-2
+/−
) and their littermate wild type ones (
RyR-2
+
/
+
). The mutation induced an impairing of Ca
2+
release from sarcoplasmic reticulum (SR) but did not affect growth and morphology of the cardiomyocytes at basal condition. When pressure overload was imposed, comparing to
RyR-2
+
/
+
mice, the
RyR-2
+/−
mice displayed an attenuated cardiac hypertrophy and contractibility, the increased death of cardiomyocytes, further down-regulated expression of
RyR-2
, the different reprogramming of SR Ca
2+
-ATPase 2, L-type Ca
2+
channel and Na
1+
/Ca
2+
exchanger expressions, and a unchanged vasculature reduction in the heart at 3 weeks. Additionally, the decrease of binding of FKBP12.6 to RyR-2 and the increase in phosphorylation of RyR-2 by protein kinase A were aggravated in the loaded
RyR-2
+/−
heart. Furthermore, activation of protein kinase B/Akt and calcineurin by pressure overload was declined whereas that of Ca
2+
/calmudulin-dependent protein kinase II unchanged in the
RyR-2
+/−
heart comparing the
RyR-2
+
/
+
one. These results suggest that reduction of RyR-2 attenuated cardiac hypertrophy but accelerated the development of cardiac dysfunctions through disturbed Ca
2+
homeostasis, impaired activation of Akt and calcineurin and increased cardiomyocyte death during pressure overload.
Abstract 3775: Cardiomyocyte-Specific Deletion of Inhibitor of Kappa-B Kinase-β (IKKβ) In Vivo Reveals Essential Role in Calcium Handling
Authors: Ashida Noboru , Coggins Matthew , Shi Jianru , Wang Bo , del Monte Federica , Liao Ronglih , Rosenzweig Anthony
Published: July 2021

The serine-threonine kinase, IKKβ, is required in most settings for activation of NF-κB transcription factors, which regulate a wide range of essential processes from cell survival to inflammation. In the heart, while IKKβ and NF-κB are activated in many diseases conditions, whether this activation is adaptive or deleterious remains unclear. To determine the role of IKKβ in the heart, we generated cardiac-specific knockout mice utilizing cardiomyocyte (CM)-specific expression of either Cre or an activatable Cre-fusion protein, producing constitutive (c) or tamoxifen-inducible (i) cardiac-specific IKKβ knockouts (CSIKO), respectively. cCSIKO were born at the expected Mendelian frequency and developed normally. IKKβ expression was dramatically reduced in both cCSIKO and (after tamoxifen) iCSIKO hearts. As expected, CM NF-κB activation after in vivo LPS treatment was markedly impaired in both cCSIKO and iCSIKO mice. Interestingly, both lines had cardiac dysfunction. No evidence of increased apoptosis was observed by TUNEL staining in cCSIKO. Studies in isolated CM from cCSIKO demonstrated reduced decreased peak shortening (2.3% vs. 3.7%) and calcium transient amplitude (0.30 vs 0.46, p<0.03 for both) without a change in normalized kinetics. Protein expression of the calcium handling proteins (L-type calcium channel [LTCC], calsequestrin, sodium-calcium exchanger, and phospho- to dephosphorylated ryanodine receptors) was unchanged in whole heart lysates. However confocal immunohistochemistry demonstrated a markedly abnormal distribution of the LTCC α2 subunit in CSIKO hearts despite intact T-tubules and dyads by electron microscopy. In conclusion, conditional deletion of IKKβ reveals an essential and unanticipated role in regulating CM calcium handling that appears mediated by alterations in LTCC subunit trafficking. Understanding the role of IKKβ and NF-κB in regulating cardiac function has important practical implications for therapeutic strategies targeting this pathway.
Abstract 3785: An Involvement of a Novel G-protein Activator on Hypoxia-Induced Apoptosis of Cardiomyocytes and its Interaction with Connexin 43
Authors: Sato Motohiko , Honda Takashi , Jiao Qibin , Kurotani Reiko , Toyota Eiji , Lanier Stephen M , Ishikawa Yoshihiro
Published: July 2021

Recently, we identified a novel receptor-independent G-protein activator, Activator of G-protein signaling 8 (AGS8), from a cDNA library of rat hearts subjected to repetitive transient ischemia. AGS8 mRNA was induced in the ischemic myocardium and it regulated Gβγ signaling. In the face of myocardial ischemia and/or ischemia/reperfusion, AGS8 may be involved in signaling events associated with adaptation of the cardiomyocyte to ischemic events. As an initial step to address this issue, we examined the influence of AGS8 on survival of cultured cardiomyocyte following hypoxia. Cultured rat neonatal cardiomyocytes (NCM) were exposed to hypoxia for 6h, 24 h or hypoxia (6h)/reoxygenation (18h) following transfection of AGS8 siRNA. Apoptosis was determined by DNA end-labeling or detection of changes of mitochondrial membrane potential. Interestingly, silencing of AGS8 inhibited apoptosis of cardiomyocytes induced by hypoxia alone (57.8±3.4%, p<0.05 vs. control-siRNA, at 24 h, mean±SEM) as well as hypoxia/reoxygenation (36.4±4.0%, p<0.05 vs. control-siRNA, mean±SEM). Transfection of pcDNA::AGS8 by virus envelope increased apoptosis of NCM (151%±9.6%, p<0.05 vs. vector group, mean±SEM) following hypoxia/reoxygenation, but not NCM cultured in normoxia. Affinity purified AGS8 antibody recognized immunoreactive signals enriched at the cell-cell interface of ventricular cardiomyocytes and NCM, regions also enriched in the gap junction protein connexin 43 (CX43). Indeed, AGS8 and CX43 co-immunoprecipitated from left ventricle lysates. The level of CX43 protein was decreased by overexpression of AGS8 in NCM, whereas suppression of AGS8 expression increased the level of CX43. Subsequent studies indicated that AGS8 stimulated phosphorylation of CX43 in a Gβγ dependent manner following transient expression in COS7 cells. As the level and/or phosphorylation of CX43 may influence apoptosis of cardiomyocytes, these studies suggest a potential mechanism by which AGS8 induction in the face of ischemia may provide a component of the signaling events involved in adaptation and suggest that targeted disruption of the AGS8 cascade may protect the myocardium from ischemic injury.
Abstract 3860: Arrhythmia Vulnerability of Aged Haploinsufficient Cx43 Mice is Determined by Heterogeneous Downregulation of Cx43 Combined with Increased Fibrosis
Authors: Jansen John A , van Veen Toon A , Bosch Astrid A , van der Nagel Roel , Vos Marc A , de Bakker Jacques M , van Rijen Harold V
Published: July 2021

Background: Reduced gap junction expression and increased collagen deposition are commonly found in ventricles of electrically remodeled diseased hearts. Their interactive contribution to slow conduction and increased arrhythmogeneity is, however, unclear. In this study, we investigated the effect of increased fibrosis with normal or reduced levels of the ventricular gap junction protein Cx43 on impulse propagation and arrhythmogeneity.

Methods: 11 Cx43fl/fl (Control) and 13 Cx43CreER(T)/fl mice (expressing only 50% of Cx43 protein compared to control; Cx43HZ) were aged to 20 months. Epicardial activation mapping (208 electrode terminals) of right (RV) and left (LV) ventricle was performed on Langendorff perfused hearts. Effective refractory period (ERP) was determined by premature stimulation and arrhythmia inducibility was tested by 1–3 premature stimuli and burst pacing. Epicardial conduction velocity longitudinal (CVL) and transverse (CVT) to fiber orientation and inhomogeneity of conduction was determined during S1S1 pacing (150 ms). Cx43, N-cadherin expression, and tissue collagen content was determined by (immuno)histology and Western blotting.

Results: Sustained ventricular arrhythmias were induced in 0/11 control and 8/13 Cx43HZ mice (p<0.01). CVL and CVT were unchanged, except for CVT in RV, which was decreased from 48.2±2 to 39.3±1.8 cm/s (Control vs Cx43HZ, p<0.05). In RV, ERP was decreased from 78.2±6.0 to 53.1±3.3 ms (Control vs Cx43HZ, p<0.05). In both RV and LV, inhomogeneity of conduction was significantly higher in Cx43HZ mice with arrhythmias (VT+), compared to both Control and Cx43HZ mice without arrhythmias (VT−). Cx43 expression levels were comparable between VT+ and VT- Cx43HZ mice, though strongly reduced compared to Control. However, Cx43 distribution was very heterogeneous in VT+ mice with large areas devoid of Cx43 while N-cadherin was unaffected, indicating the presence of intact intercalated disks. Compared to Control interstitial fibrosis was increased in Cx43HZ mice, but more pronounced in VT+ Cx43HZ mice when compared to Cx43HZ VT- mice.

Conclusions: Combined heterogeneous reduction of Cx43 and increased fibrosis strongly enhance arrhythmogenic vulnerability in aged haploinsufficient Cx43 mice.
Abstract 3932: Central Role Of Toll-like Receptor 4 In Oxidized Low Density Lipoprotein-and Lipopolysaccharide-Induced Bone Morphogenetic Protein-2 Expression In Human Coronary Artery Endothelial Cells
Authors: Su Xin , Johnson Thomas R , Ao Lihua , Paschall Bruce L , Yang Xiaoping , Cleveland Joseph C , Fullerton David A , Meng Xianzhong
Published: July 2021

Previous studies demonstrated that osteogenic mediators, in particular bone morphogenetic protein-2 (BMP-2), play an important role in the progression of atherosclerosis and vascular calcification. Although it is known that the atherogenic factor oxidized low density lipoprotein (oxLDL) up-regulates BMP-2 expression in endothelial cells, the signaling mechanisms involved are not well understood. In macrophages, oxLDL up-regulates Toll-like receptor 4 (TLR4) expression, and this innate immunoreceptor appears to be involved in oxLDL-induced macrophage actin polymerization. We earlier found that stimulation of TLR4 with lipopolysac-charide (LPS) increases cellular BMP-2 protein levels in human aortic valve interstitial cells. We

hypothesized

that the TLR4 pathway plays a central role in mediating BMP-2 expression in human coronary artery endothelial cells (CAECs). The

purposes

of this study were to examine whether TLR4 mediates oxLDL- and/or LPS-induced BMP-2 expression in human CAECs, and to determine whether the p38 and/or p44/42 MAPKs are involved.

Methods and results

: Stimulating human CAECs with LPS (E. coli 0111:B4, 200 ng/ml) for 24 h up-regulated BMP-2 protein expression. Similarly, stimulation with oxLDL (from human plasma, CuSO
4
-oxidized, LPS-free, 40–160 μg/ml) for 24 h induced BMP-2 expression in a dose-dependent manner. Pretreatment with either TLR4-neutralizing antibody or TLR4 siRNA significantly attenuated oxLDL-induced BMP-2 expression and abrogated the effect of LPS on BMP-2 expression. Over-expression of TLR4 enhanced the cellular BMP-2 response to oxLDL and LPS. Further, immunofluorescent staining co-localized oxLDL with TLR4. Although LPS induced ICAM-1 expression in human CAECs, oxLDL had no effect, indicating that oxLDL and LPS have different pro-inflammatory effects. BMP-2 expression was associated with activation of p38 and p44/42 MAPKs. Inhibiting p44/42, but not p38, reduced BMP-2 expression.

Conclusions:

In human CAECs, TLR4 plays a central role in regulating BMP-2 expression induced by either oxLDL or LPS, and the signaling mechanism involves the p44/42 MAPK pathway. These novel findings underscore an important role of TLR4 in atherosclerosis and vascular calcification.
Abstract 3943: Adrenomedullin Induces Lymphangiogenesis and Ameriolates Secondary Lymphedema
Authors: Jin DongHao , Yamahara Kenich , Harada Kazuhiko , Ohnishi Shunsuke , Kangawa Kenji , Nagaya Noritoshi
Published: July 2021

Background:

Adrenomedullin (AM) stimulates angiogenesis by activation of extracellular signal-regulated kinase (ERK) and Akt in vascular endothelial cells (EC). Vascular endothelial growth factor C and D were major lymphangiogenesis factors, which enhance lymphatic EC activity via ERK and Akt pathways. Therefore, we investigated whether AM promotes lymphangiogenesis through activation of lymphatic EC and improves secondary lymphedema.

Methods and Results:

RT-PCR analysis revealed that human lymphatic microvascular EC (HLMVEC) expressed mRNAs of all AM receptor complexes including calcitonin-receptor-like receptor (CRLR) and receptor-activity-modifying proteins (RAMP) 1–3. AM (10
−9
-10
−7
M) significantly enhanced proliferation of HLMVEC (MTS assay; 194 ± 4% in 10
−7
M, P < 0.05) with accumulation of intracellular cAMP in a dose dependent manner. Transwell migration assay revealed that AM (10
−9
-10
−7
M) enhanced the chemotactic activity of HLMVEC (204 ± 7% in 10
−7
M, P < 0.05). Proliferation of HLMVEC induced by AM (10
−7
M) or a cAMP agonist, 8-Br-cAMP (10
−4
M) was significantly inhibited by mitogen-activated protein kinase kinase (MEK) inhibitors, PD98059 (5 × 10
−6
M) and U0126 (10
−6
M). Western blot analysis revealed that AM (10
−7
M) and 8-Br-cAMP (10
−4
M) induced the phosphorylation of ERK in HLMVEC. A mouse model of tail lymphedema was developed in 8 –10 weeks old male BALB/c mice, and human AM (0.05 μg/kg/min) was delivered for 14 days using an osmotic minipump, which was subcutaneously implanted. AM significantly decreased the tail thicknesses at day 14 to 16 postoperation (Tail diameter at day 14; control vs. AM treatment, 4.19 ± 0.13 vs. 3.79 ± 0.08 mm, P = 0.023). Immunohistological analysis of injury sites using anti-LYVE-1 and anti-vWF antibodies revealed that AM treatment increased the number of lymphatic vessels (104 ± 15 vs. 188 ± 15 / mm
2
, P = 0.007) and vascular vessels (230 ± 11 vs. 315 ± 15 / mm
2
, P = 0.003).

Conclusions:

AM enhanced proliferation and migration of lymphatic EC through activation of cAMP/MEK/ERK pathway. Infusion of AM induced lymphangiogenesis and improved secondary lymphedema in the mouse model of tail lymphedema. These results indicate that AM might represent a novel therapeutic strategy for treatment of lymphedema.
Abstract 5355: An Alpha-1A-Adrenergic Receptor Subtype Agonist Prevents Cardiomyopathy Without Increasing Blood Pressure
Authors: Chan Trevor , Dash Rajesh , Simpson Paul C
Published: July 2021

Background:
Alpha-1-adrenergic receptor (AR) agonists classically increase blood pressure (BP). Among the 3 alpha-1-AR subtypes, A, B, and D, the alpha-1A is required for cardiac protection in a knockout (KO) mouse, and is sufficient for protection of cultured cardiac myocytes, via ERK activation. However, it is unknown if activation of the alpha-1A-subtype by a drug can protect the heart in vivo.

Hypothesis:
At a dose that does not increase BP, an agonist selective for the alpha-1A-AR subtype can prevent cardiomyopathy.

Methods:
We gave the alpha-1A agonist A61603 (A6) to 11 week-old wild type (WT) male C57Bl6J mice by osmotic minipump. We induced cardiomyopathy with a single dose of doxorubicin (DOX) (25 mg/kg IP), a cardiotoxic cancer drug. We measured BP by tail cuff, activated (phosphorylated, P)-ERK by immunoblot, heart mRNAs by RT-qPCR, fractional shortening (FS) by echocardiog-raphy (ECHO), myocyte necrosis by serum creatine kinase (CK), apoptosis by TUNEL stain, and fibrosis by sirius red stain.

Results
: In dose-finding experiments (0.01–100 ug/kg/d), A6 at 10 ng/kg/d over 7 days had no effect on daily tail cuff BP (average mmHg Vehicle 115 ± 4; A6 119 ± 4), but increased heart P-ERK (1.7-fold) and the mRNAs for beta-MyHC and ANF (5-fold). A6 at higher doses increased BP. Next, A6 at the non-hypertensive dose (10 ng/kg/d) or vehicle was infused over 7 days after a single DOX injection. The
TABLE
shows that DOX caused cardiomyopathy, with reduced survival and FS, and increased necrosis, apoptosis, and fibrosis (data are mean ± SE). A6 prevented all of these abnormalities. In alpha-1A-subtype KO mice, DOX caused increased apoptosis and mortality compared with WT mice, and A6 had no beneficial effect (not shown), indicating specificity of A6.

Conclusions:
A very low dose of an agonist selective for the alpha-1A-AR subtype can activate cardiac survival signaling (P-ERK), induce cardiac fetal genes, and prevent DOX-induced cardiomyopathy, all without increasing BP.
Abstract 5368: Igf +hgf Enhance Sca-1
+
/cd31
−
Cell Therapy in Hearts with Postinfarction Lv Remodeling
Authors: Wang Xiaohong , Braunlin Elizabeth , Hu Qingsong , Lee Joseph , Li Qinglu , Xiong Qiang , Jameel Mohammad N , Zhang Jianyi
Published: July 2021

Insulin-like growth factor 1 (IGF-1) and hepatocyte growth factor (HGF) are two potent cell survival factors in response to hypoxia and oxidative stress. We have previously shown that transplantation of Sca-1
+
/CD31
−
cells can improve LV function in hearts with acute myocardial infarction (AMI) although the engraftment rate was low. We hypothesized that supplementation of IGF-1 and HGF at delivery of cellular therapy can enhance the beneficial effects of Sca-1
+
/CD31
−
cells in the injured heart by increasing engraftment rate and endogenous cardiac cell survival. Adenovirus nuclear LacZ-labeled Sca-1
+
/CD31
−
cells (1x10
6
) were injected into the ischemic area after LAD ligation in BalbC mice. Recombinant mouse IGF-1+HGF (100ng) was added to the cell suspension prior to the injection. The LV function (ECHO) and in vivo myocardial bioenergetics (
31
P-NMR spectroscopy) were assessed 4 weeks after AMI and cell transplantation. Normal mice (Normal, n=6), and 2 control groups of LAD ligation (MI, n=6), and MI plus Sca-1
+
/CD31
−
cell transplantation (Cell, n=6), were compared to AMI mice with Sca-1
+
/CD31
−
cells transplantation plus IGF-1+HGF (Cell+IGF-1+HGF, n=6). Sca-1
+
/CD31
−
cells formed viable grafts and improved LV contractile function (EF: Control, 53.3+/−3.2; MI, 18.9+/−3.9; Cell, 29.1+/−5.1, p<0.05) and myocardial bioenergetics (PCr/ATP: Control, 2.13+/−0.09; MI, 1.21+/−0.09; Cell, 1.72+/−0.12; p<0.01). IGF+HGF significantly further enhanced the benefits of the cell transplantation as evidenced by significantly increased EF to 38.3+/−3.1% (p<0.05), which was accompanied by a higher cell engraftment rate (p<0.05). Using the
in vitro
co culture of Sca-1
+
/CD31
−
cells with HL-1 cells labeled by Vybrant CFDA SE cell tracer kit, we observed both IGF+HGF and Sca-1
+
/CD31
−
cells can inhibit TNFα and hypoxia induced HL-1 cell apoptosis (Control, 7+/−1%; TNFα, 24+/−1%; IGF+HGF+TNFα, 10+/−1%; n=6, P<0.001) and caspase 3 expression. These data demonstrate that IGF-1+HGF could serve as an adjuvant to cell transplantation for myocardial restoration.