Cardiac Capillary Research Articles

2013 Late-Breaking Basic Science Abstracts

### 21998 #### Enhanced Myocardial Repair with CardioChimeras Pearl J Quijada, Jonathan D Cubillo, Claudio Staub, Mark A Sussman; San Diego State Univ, San Diego, CA Dual stem cell transplantation of c-kit positive cardiac progenitor cells (CPCs) and mesenchymal stem cells (MSCs) after infarction support modest improvements in cardiac function, however there are currently no reports substantiating a single stem cell type supporting both direct and indirect mechanisms of myocardial repair. Therefore we created a CardioChimera, a stem/progenitor cell formed by fusion between CPCs and MSCs, resulting in a unique progeny superior to either individual precursor. CardioChimeras were purified after cell fusion with Hemagglutinating virus of Japan and expanded clonally based on dual expression of fluorescent proteins mCherry and eGFP from CPCs and MSCs respectively. CardioChimeras are mono-nucleated, have comparable growth kinetics to parental CPCs and MSCs and display increased cellular size unrelated to cell cycle arrest and/or senescence. CardioChimeras have increased expression of cardiomyogenic lineage markers cardiac troponin T (2.5-fold), smooth muscle 22 (9.3-fold), and CD31 (10.7-fold) concomitantly associated with decreased c-kit protein expression (50%) relative to parent CPCs. Lineage commitment of CardioChimeras is bolstered by dexamethasone treatment measured by mRNA levels of cardiogenic genes and increases in active mitochondria (2.2-fold) after labeling with MitoTracker. Induction of apoptosis is blunted in cardiomyocytes co-cultured with CardioChimeras compared to co-culturing with CPCs and MSCs alone, or combination of non-fused parent cells. CardioChimeras enhance cardiomyocyte growth similar to parent MSCs owing to an increased propensity to secrete pro-growth factors. Collectively, CardioChimeras represent an adaptable cell therapy combining the beneficial properties of CPCs to undergo cardiac specific commitment as well as MSCs that foster an improved microenvironment with protective paracrine secretion. Clinically, CardioChimeras merge the application of distinct cell types into a single entity for increased engraftment, mitigation of inflammation and blunting the progression of heart failure by promoting myocardial regeneration. …

Importance of tumor suppressor gene p53-mediated endothelial cell apoptosis for cardiac angiogenesis and hypertrophy

Cardiac hypertrophy initially develops as adaptive response of the heart to increased workload, but may progress to organ dysfunction if continued over the long term. Previous studies suggested that the rarefication of cardiac endothelial cells may be causally involved in the development of heart failure. The aim of this study was to examine whether prevention of endothelial cell apoptosis, achieved by deletion of the tumor suppressor gene p53, may improve cardiac remodelling in response to increased afterload and prevent or postpone the transition to heart failure. Mice with endothelial cell-specific deletion of p53 (End.p53-KO) were generated by crossing p53flox/flox mice with mice expressing Cre recombinase under control of the tamoxifen-inducible, endothelial cell-specific promoter Tie2 (Tie2.ERT.Cre). Cardiac hypertrophy was induced using the transaortic constriction (TAC) model. In control mice, cardiac hypertrophy was accompanied by increased p53 protein expression (P<0.05 vs. sham) and elevated levels of apoptosis (P<0.01), as shown by TUNEL and activated caspase-3 staining. In contrast, reduced apoptotic cell numbers were detected in hearts of End.p53-KO mice after TAC (P<0.01 vs. controls). Moreover, End.p53-KO mice exhibited increased cardiac capillary density (P<0.001) and higher number of lectin-perfused blood vessels (P=0.0002). Endothelial p53 deletion also improved extracardiac angiogenesis in the unilateral hindlimb ischaemia model and was associated with enhanced reperfusion (P<0.001) and increased capillary density (P<0.001). In vitro analyses confirmed that inhibition or transient reduction of p53, achieved by preincubation of human microvascular endothelial cells with pifithrin-α or p53-specific small interfering RNAs, promoted endothelial sprouting (P<0.05). In addition to improved cardiac angiogenesis, hearts of End.p53-KO mice exhibited less interstitial fibrosis compared to those of controls (P<0.001) and expressed lower mRNA levels of p53-regulated target genes involved in extracellular matrix remodelling (including collagen, CTGF or PAI-1). Importantly, echocardiographical measurements revealed less severe fractional shortening reduction (P<0.05 vs. controls) and better survival in End.p53-KO mice 20 weeks after TAC. Taken together, our findings reveal that accumulation of p53 in endothelial cells contributes to the rarefication of blood vessels and the development of fibrosis during cardiac hypertrophy and that apoptotic endothelial cell death may be involved in the development of heart failure in response to chronic elevations of cardiac afterload.

Mechanisms of right heart failure-A work in progress and a plea for failure prevention.

Mechanisms of right heart failure-A work in progress and a plea for failure prevention.

FTY720 Protects Cardiac Microvessels of Diabetes: A Critical Role of S1P1/3 in Diabetic Heart Disease

Background: Diabetes is associated with an increased risk of cardiac microvascular disease. The mechanisms by which this damage occurs are unknown. However, research suggests that signaling through the sphingosine-1-phosphates receptor 1 and 3 (S1P1/3) by FTY720, a sphiongolipid drug that is structually similar to SIP, may play a role in the treatment on cardiac microvascular dysfunction in diabetes. We hypothesized that FTY720 might exert the cardioprotective effects of S1P1 and S1P3 viaprotein kinase C-beta (PKCβ II) signaling pathway. Methodology/Principal Findings: Transthoracic echocardiography was performed to detect the change of cardiac function. Scanning and transmission electron microscope with lanthanum tracer were used to determine microvascular ultrastructure and permeability in vivo. Apoptosis was detected by TUNEL and CD31 dual labeling in paraffin-embedded sections. Laser capture miscrodissection was used to assess cardiac micovascular endothelial cells (CMECs) in vivo. RT-PCR and Western blot analysis were used to determine the mRNA levels and protein expression of S1P1, S1P3, and PKCβ II. In the diabetic rats vs. controls, cardiac capillaries showed significantly higher density; CD31 positive endothelial cells were significantly reduced; the apoptosis index of cardiac endothlial cells was significantly higher. And FTY720 could increase the expressional level of S1P1 and boost S1P3 trasnslocation from membrane to nuclear, then ameliorate cardiac microvascular barrier impairment and pathologic angiogenesis induced by diabetes. In addition, overexpression of PKCβ II significantly decreased the protective effect of FTY720. Conclusions: Our study represents that the deregulation of S1P1 and S1P3 is an important signalresponsible for cardiac microvascular dysfunction in diabetes. FTY720 might be competent to serve as a potential therapeutic approach for diabetic heart disease through ameliorating cardiac microvascular barrier impairment and pathologic angiogenesis, which might be partly dependent on PKCβII-mediated signaling pathway.

Swimming Training in Rats Increases Cardiac MicroRNA-126 Expression and Angiogenesis

MicroRNA (miRNA)-126 is angiogenic and has two validated targets: Sprouty-related protein 1 (Spred-1) and phosphoinositol-3 kinase regulatory subunit 2 (PI3KR2), negative regulators of angiogenesis by VEGF pathway inhibition. We investigated the role of swimming training on cardiac miRNA-126 expression related to angiogenesis. Female Wistar rats were assigned to three groups: sedentary (S), training 1 (T1, moderate volume), and training 2 (T2, high volume). T1 consisted of 60 min·d of swimming, five times per week for 10 wk with 5% body overload. T2 consisted of the same protocol of T1 until the eighth week; in the ninth week, rats trained for two times a day, and in the 10th week, rats trained for three times a day. MiRNA and PI3KR2 gene expression analysis was performed by real-time polymerase chain reaction in heart muscle. We assessed markers of training, the cardiac capillary-fiber ratio, cardiac protein expression of VEGF, Spred-1, Raf-1/ERK 1/2, and PI3K/Akt/eNOS. The cardiac capillary-fiber ratio increased in T1 (58%) and T2 (101%) compared with S. VEGF protein expression was increased 42% in T1 and 108% in T2. Cardiac miRNA-126 expression increased 26% (T1) and 42% (T2) compared with S, correlated with angiogenesis. The miRNA-126 target Spred-1 protein level decreased 41% (T1) and 39% (T2), which consequently favored an increase in angiogenic signaling pathway Raf-1/ERK 1/2. On the other hand, the gene expression of PI3KR2, the other miRNA-126 target, was reduced 39% (T1) and 78% (T2), and there was an increase in protein expression of components of the PI3K/Akt/eNOS signaling pathway in the trained groups. This study showed that aerobic training promotes an increase in the expression of miRNA-126 and that this may be related to exercise-induced cardiac angiogenesis, by indirect regulation of the VEGF pathway and direct regulation of its targets that converged in an increase in angiogenic pathways, such as MAPK and PI3K/Akt/eNOS.

Activation of HIF-1 by metallothionein contributes to cardiac protection in the diabetic heart

Metallothionein (MT) protects against heavy metal-induced cellular damage and may participate in other fundamental physiological and pathological processes, such as antioxidation, proliferation, and cell survival. Previously, we have shown that elevation of MT by transgene or by induction with zinc protects the heart against diabetic cardiomyopathy by mechanisms such as antidiabetes-induced oxidative stress and inactivation of glycogen synthase kinase-3, which mediates glucose metabolism. We also reported that MT overexpression rescued the diabetic-induced reduction of hypoxia-inducible factor (HIF)-1α, which plays an important role in glucose utilization and angiogenesis. Here, we showed that overexpression of MT increased hexokinase (HK)-II expression under control conditions and attenuated diabetes-decreased HK-II expression. Glycolytic flux assay demonstrated that MT increased glycolysis output in high glucose-containing media-cultured H9c2 cells. The diabetes-induced reduction in cardiac capillaries was also attenuated by MT overexpression. Furthermore, MT induction significantly increased HIF-1 expression under both control and diabetic conditions. Moreover, in the present study, we demonstrated that MT-enhanced HIF-1α activity is likely through a mechanism of protein nuclear translocation. These results suggest that MT induces HIF-1α expression, leading to increased HK-II in the diabetic heart.

Ramipril and Telmisartan Exhibit Differential Effects in Cardiac Pressure Overload-Induced Hypertrophy Without an Additional Benefit of the Combination of Both Drugs

We aimed to characterize different cellular effects of angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin 1 (AT1) receptor blockers (ARBs) as mono- or combination therapy in cardiac pressure overload. Methods and C57B1/6 mice received either the ACEI ramipril (2.5 mg/kg body weight), the ARB telmisartan (20 mg/kg body weight), or the combination. In all groups, pressure overload was induced by transverse aortic constriction (TAC). Cardiac hypertrophy (heart weight/tibia length) induced by TAC was reduced in all 3 treatment groups, with the most pronounced effect in the telmisartan group. The cardiomyocyte short-axis diameter and cardiac fibrosis were increased by TAC and similarly reduced by ACEI, ARB, and the combination therapy. The TAC-induced increase in the number of proliferating Ki67(pos) cardiomyocytes and noncardiomyocytes was reduced more potently by ACEI than by ARB. Four days of drug treatment induced a significant increase in Scal(pos)/VEGFR1(pos) endothelial progenitor cells (EPCs) in all animals in the treated SHAM groups. After 1 day of aortic constriction, only ramipril increased EPC numbers; after 5 weeks, telmisartan monotherapy did not change the EPC levels compared to vehicle or the combination therapy but raised it compared to ramipril. Neither TAC nor one of the therapies changed the number of cardiac capillaries per cardiomyocytes. ACE inhibition and AT1 receptor blockade have beneficial effects in remodeling processes during cardiac pressure overload. There are small differences between the 2 therapeutical approaches, but the combination therapy has no additional benefit.

Moderate exercise training promotes adaptations in coronary blood flow and adenosine production in normotensive rats

OBJECTIVES:Aerobic exercise training prevents cardiovascular risks. Regular exercise promotes functional and structural adaptations that are associated with several cardiovascular benefits. The aim of this study is to investigate the effects of swimming training on coronary blood flow, adenosine production and cardiac capillaries in normotensive rats.METHODS:Wistar rats were randomly divided into two groups: control (C) and trained (T). An exercise protocol was performed for 10 weeks and 60 min/day with a tail overload of 5% bodyweight. Coronary blood flow was quantified with a color microsphere technique, and cardiac capillaries were quantified using light microscopy. Adenine nucleotide hydrolysis was evaluated by enzymatic activity, and protein expression was evaluated by western blot. The results are presented as the means ± SEMs (p<0.05).RESULTS:Exercise training increased the coronary blood flow and the myocardial capillary-to-fiber ratio. Moreover, the circulating and cardiac extracellular adenine nucleotide hydrolysis was higher in the trained rats than in the sedentary rats due to the increased activity and protein expression of enzymes, such as E-NTPDase and 5′-nucleotidase.CONCLUSIONS:Swimming training increases coronary blood flow, number of cardiac capillaries, and adenine nucleotide hydrolysis. Increased adenosine production may be an important contributor to the enhanced coronary blood flow and angiogenesis that were observed in the exercise-trained rats; collectively, these results suggest improved myocardial perfusion.

81st Annual Meeting of the American Thyroid Association SHORT CALL ABSTRACTS

81st Annual Meeting of the American Thyroid Association SHORT CALL ABSTRACTS

In This Issue

HomeCirculation ResearchVol. 109, No. 3In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Originally published22 Jul 2011https://doi.org/10.1161/RES.0b013e31822b3313Circulation Research. 2011;109:245AKAP150 in LQT8 (p 255)Cheng et al propose a way to combat arrhythmias in Timothy syndrome.Download figureDownload PowerPointA mutation in the cytoplasmic loop of calcium channel protein, Cav1.2a, is known to cause Timothy syndrome, which is characterized by structural heart defects, arrhythmia, and autism spectrum disorders. The disease is also known as long QT syndrome 8, because it is associated with a prolonged depolarization-to-repolarization (QT) interval. It is not clear how the mutation causes channel dysfunction, but Cheng et al wondered if interaction with a cytoplasmic anchor protein called AKAP150 may be involved. They made transgenic mice that expressed the mutant channel and lacked AKAP150, and found these mice were protected from cardiac hypertophy and arrhythmia. In vitro analysis revealed that mutant channels functioned normally in the absence of AKAP150. The anchor appears to stabilize the mutant channels in the open configuration and might also promote clusters of mutant channels to open together – both of which would increase calcium influx and thus delay repolarization. In short, the removal of the perfectly normal AKAP150 protein fixed arrhythmias caused by the mutant Cav1.2a. When it comes to possible therapies, say the authors, disrupting the interaction between these two proteins might be sufficient.PGF Regulates Cardiac Adaptation (p 272)Placental growth factor helps stressed hearts to adapt, reportAccornero et al.Download figureDownload PowerPointDespite its name, placental growth factor (PGF) is found in many tissues, including the heart. PGF is elevated in human heart tissue and in cultured cardiomyocytes under low oxygen conditions. It is also elevated in the blood of patients after heart attack or during ischemic cardiomyopathy. Accornero et al also wanted to know the role of PGF in such stress conditions. The team made mice that overexpressed PGF in cardiomyocytes, and compared the effect of heart stress in these mice with wild type mice and with mice that lacked PGF. When the hearts of these mice were subjected to pressure overload, the animals overexpressing PGF exhibited greater cardiac capillary growth, fibroblast proliferation and cardiomyocyte hypertrophy, and were protected from heart failure. On the other hand, mice that lacked PGF died of heart failure within a week. Even though the extra PGF was produced by cardiomyocytes, it was the capillary cells and fibroblasts that were directly affected, while cardiomyocyte hypertrophy was a secondary response. The protective effect of PGF may make it a useful therapeutic agent but the authors caution that trials with PGF's close cousin VEGF for ischemic heart disease have yet to show promise.Antiangiogenic VEGF165b in Systemic Sclerosis (p e14)Manetti et al suggest a way to boost blood vessel renewal in patients suffering from scleroderma.Download figureDownload PowerPointScleroderma is a rare and devastating autoimmune disease that is characterized by widespread fibrosis and vascular damage. The chronic vessel damage causes tissue ischemia, which would normally lead to the production of proangiogenic factors. Paradoxically, scleroderma patients have high levels of a known proangiogenic factor, VEGF, but angiogenesis is still impaired. Manetti et al have recently worked out why. They found that scleroderma patients produce a variant of VEGF (VEGF165b) that is antiangiogenic. This variant, which is produced by alternative splicing, was discovered only recently, and previous studies of patient tissues had not differentiated between this variant and its proangiogenic form. A splicing factor called SRp55, known to drive production of VEGF165b, was also upregulated in these patients. And both SRp55 and VEGF165b were upregulated by TGF-β1. TGF-β1 is known to promote fibrosis in scleroderma patients. Thus, the new data suggests TGF-β1 drives the vascular damage as well. Lastly, the team showed that blocking VEGF165b could stimulate angiogenesis in patient-derived vascular cells, so the hope is that such an approach might also work in patients.Written by Ruth Williams Previous Back to top Next FiguresReferencesRelatedDetails July 22, 2011Vol 109, Issue 3 Advertisement Article InformationMetrics © 2011 American Heart Association, Inc.https://doi.org/10.1161/RES.0b013e31822b3313 Originally publishedJuly 22, 2011 PDF download Advertisement

A Potential Role of Distinctively Delayed Blood Clearance of Recombinant Adeno-associated Virus Serotype 9 in Robust Cardiac Transduction

Recombinant adeno-associated virus serotype 9 (rAAV9) vectors show robust in vivo transduction by a systemic approach. It has been proposed that rAAV9 has enhanced ability to cross the vascular endothelial barriers. However, the scientific basis of systemic administration of rAAV9 and its transduction mechanisms have not been fully established. Here, we show indirect evidence suggesting that capillary walls still remain as a significant barrier to rAAV9 in cardiac transduction but not so in hepatic transduction in mice, and the distinctively delayed blood clearance of rAAV9 plays an important role in overcoming this barrier, contributing to robust cardiac transduction. We find that transvascular transport of rAAV9 in the heart is a capacity-limited slow process and occurs in the absence of caveolin-1, the major component of caveolae that mediate endothelial transcytosis. In addition, a reverse genetic study identifies the outer region of the icosahedral threefold capsid protrusions as a potential culprit for rAAV9's delayed blood clearance. These results support a model in which the delayed blood clearance of rAAV9 sustains the capacity-limited slow transvascular vector transport and plays a role in mediating robust cardiac transduction, and provide important implications in AAV capsid engineering to create new rAAV variants with more desirable properties.

Effects of des-aspartate-angiotensin I on myocardial ischemia-reperfusion injury in rats

The present study investigated the actions of des-aspartate-angiotensin I (DAA-I) on infarct size and three early inflammatory events in acute myocardial ischemia-reperfusion injury in rats. The rationale was based on earlier findings showing that chronic daily administration of DAA-I attenuated infarct size of ischemic-reperfused rat heart, and cardiac hypertrophy in pressure overload rats. Anesthetized rats were subjected to 45 min of ischemia and 5 h of reperfusion. Infarcted area, serum creatine kinase, and tissue myeloperoxidase activity were determined. The expression of intercellular adhesion molecule-1 (ICAM-1) was also investigated by immunohistochemistry and Western blotting. Intravenous administration of DAA-I at 5 min post reperfusion reduced myocardial infarct size by 45.5%, lowered serum creatine kinase activity, decreased myeloperoxidase activity in cardiac tissue, and inhibited the expression of ICAM-1 in cardiac capillary endothelium. The maximum effective dose was 1013 pmol/kg, and the cardioprotective actions of DAA-I were blocked by indomethacin. The data showed that the cardioprotection accorded by DAA-I was the result of its anti-inflammatory actions on early inflammatory processes in myocardial ischemia-reperfusion injury. The anti-inflammatory processes were indomethacin sensitive and probably mediated by prostaglandins.

Renin inhibition mitigates anti-angiogenesis in spontaneously hypertensive rats

Spontaneously hypertensive rats (SHRs) are characterized by capillary rarefaction, which may contribute to blood pressure elevation. We hypothesized that capillary rarefaction involves a suppressed angiogenesis; renin inhibition influences anti-angiogenesis homeostasis by acting on angiopoietins; transient renin blockade reduces anti-angiogenesis thereby ameliorating long-lasting blood pressure and cardiac hypertrophy in SHRs. First, serum angiopoietin-1 and angiopoietin-2 were measured in 2-month old normotensive Wistar-Kyoto rats (WKYs) and SHRs after renin inhibition (aliskiren: 1 and 10 mg/kg per day) or placebo. Second, 4-week old SHRs were prehypertensively treated with aliskiren (1 and 10 mg/kg per day) or placebo for 4 weeks. After 4 weeks of 'drug holiday' 12-week old SHRs were given L-nitro-arginine methyl ester (L-NAME) (25 mg/kg per day) for a 4-week interval to promote capillary rarefaction. Thereafter, mean arterial pressure (MAP), cardiac remodeling, capillary density, pAkt/Akt as marker for cellular survival, pro-angiogenic genes and systemic angiopoietins were investigated. Baseline angiopoietin levels were similar between WKYs and SHRs. Renin inhibition increased angiopoietin-1 in SHR and reduced angiopoietin-2 in both WKY and SHR blood pressure independently. Prehypertensive renin inhibition reduced MAP and cardiac hypertrophy in adult SHRs. This was associated with higher cardiac capillary density, pAkt/Akt, pro-angiogenic expression pattern and serum angiopoietin-1, whereas angiopoietin-2 was lower as compared to vehicle-pretreated SHRs. These results were independent of prehypertensive blood pressure lowering by aliskiren. We conclude that renin inhibition modulates anti-angiogenesis signaling independently of blood pressure by increasing angiopoietin-1/angiopoietin-2 ratio. This promotes in SHR stabilization of endothelial cells, favors pro-angiogenic action and consequently results in higher capillary density.

Sympathetic blockade prevents the decrease in cardiac VEGF expression and capillary supply in experimental renal failure

Uremic cardiomyopathy of men and rodents is characterized by lower myocardial capillary supply that in rats could be prevented by central and peripheral blockade of the sympathetic nervous system. The underlying pathomechanisms remain largely unknown. We investigated whether alterations of cardiac vascular endothelial growth factor (VEGF) gene and protein expression were involved. In our long-term experiment, we analyzed whether VEGF gene and protein expression was altered in the heart of male Sprague-Dawley rats with either sham operation (sham, n=10) or subtotal nephrectomy (SNX, n=10). In our short-term experiment (17 sham, 24 SNX), the effect of a putative downregulation of sympathetic nervous activity by surgical renal denervation (interruption of renal afferent pathways) on cardiac gene expression of VEGF, flt-1, and flk-1 and on myocardial capillary supply was analyzed. In the long-term study, cardiac capillary supply and vascular endothelial growth factor gene and protein expression were significantly lower in SNX than in sham. In the short-term experiment, cardiac VEGF mRNA expression was significantly lower in untreated SNX (4,258±2,078 units) than in both sham groups (11,709±4,169 and 8,998±4,823 units); this decrease was significantly prevented by renal denervation (8,190±3,889, P<0.05). We conclude that cardiac VEGF gene and protein expression is reduced in experimental renal failure, and this may be considered as one potential reason for impaired myocardial adaptation under the situation of cardiac hypertrophy. The beneficial effect of sympathetic downregulation on cardiac structure and function in renal failure may be at least in part explained by increased cardiac VEGF gene expression.

Endothelial dysfunction induced by antibodies against angiotensin AT1 receptor in immunized rats

To investigate the association between autoantibodies against angiotensin AT1 receptor (AT1-AAs) and endothelial dysfunction in vivo. Rat models with AT1 receptor antibodies (AT1-Abs) were established by active immunization for nine months. Lactate dehydrogenase (LDH) activity was regarded as an indicator of cell necrotic death. Endothelin-1 (ET-1) in the sera of rats was determined and endothelium-dependent vasodilatation was detected in isolated thoracic aorta. Endothelial intercellular adhesion molecule-1 (ICAM-1) expression in aorta endothelium was assessed using confocal microscopy. Coronary artery endothelial ultrastructure was observed. IgGs in the immunized group significantly increased the LDH activity (0.84±0.17 vs 0.39±0.12, P<0.01 vs vehicle group IgGs)in incubated human umbilical vein endothelial cells through AT1 receptor. Higher content of ET-1 occurred in the immunized rats than that of the vehicle group, and reached two peaks at month 3 (27±4 ng/L, P<0.01) and month 7 (35±5 ng/L, P<0.01), respectively. In addition, aortic endothelium-dependent vasodilatation was attenuated; endothelial ICAM-1 level was markedly increased and cardiac capillary endothelium was damaged following immunization. Our study demonstrated that AT1-Abs contributed to endothelial dysfunction in vivo, which was a potential mechanism through which the antibodies play vital roles in related diseases.

Abstract 4572: Reduced Cardiac Progenitors in the Right Ventricle of Cyanotic Heart Lesions: Insights into RV Adaptation

The RV in hypoplastic left heart syndrome (HLHS) and Tetralogy of Fallot (TOF) is exposed to hypoxia and abnormal load which may lead to RV dysfunction despite surgical repair. The ability of hypoxia to upregulate angiogenic signaling in the RV is not known. We measured vascular endothelial growth factor (VEGF) mediated regulation of cardiac progenitors in the RV of patients with HLHS and TOF. Methods: RV myocardial samples were obtained from 6 pts with HLHS (age 0.96±1.5 yrs), 6 pts with TOF (age 3.5±6.3 yrs) and 9 age-matched controls with structurally normal hearts (age 0.16±0.3 yrs) at surgery/transplant/autopsy. The following were measured: VEGF, thymosin β-4 (recruits progenitors), Nkx2.5 (myocyte precursor), Flk-1 (smooth muscle progenitor), CD34 (endothelial progenitor), and von willebrand factor (vWf) (endothelial marker). Results: (i) VEGF and thymosin β-4 expression was lower in HLHS vs controls. This was associated with reduced cardiac progenitors and lower myocardial capillary density vs controls (0.14±0.01 vs 0.55±0.16, p&lt;0.05). (ii) VEGF expression was preserved but thymosin β-4 was reduced in RV in TOF. This was associated with preserved myocyte progenitors but reduction in endothelial and smooth muscle lineages and reduced capillary density vs controls (0.13±0.03 vs 0.55±0.16, p&lt;0.05) (Figure 1 ). Conclusion: Hypoxia fails to induce an angiogenic response in the RV in cyanotic heart lesions. This may be due to reduced VEGF (HLHS) or impaired coupling of VEGF to thymosin β-4 (TOF). This maladaptation may contribute to post-operative RV dysfunction and lower RV regenerative capacity in later life. Figure 1: Reduced capillary density (red) in HLHS and TOF

Abstract 5570: β-2 Adrenergic Receptors Overexpression Promotes Angiogenesis in the Failing Myocardium through Activation of Vascular Endothelial Growth Factor/Akt Transduction Pathway

β-2 adrenergic receptor (β-2AR) plays a pivotal role in endothelial cell proliferation and promotes angiogenesis in response to hindlimb ischemia. Desensitization/uncoupling of cardiac β-2AR represents a well established phenomenon in heart failure (HF). In this study we aimed to investigate the role of β-2AR in the impairment of angiogenic processes occurring in the failing myocardium. To this purpose, we explored the angiogenic effects of β-2AR in a rat model of post-myocardial infarction (MI) HF. Cardiac adenoviral-mediated β-2AR overexpres-sion has been obtained via direct intramyocardial injection (4 x 10 11 PFU) 4 weeks after MI. We also extended our observation to β-2AR knockout (β2KO) mice developing HF after large anterior MI (infarct size 45 ± 6%). Cardiac β-2AR overexpression in HF rats markedly increased capillary and arteriolar length density, and induced larger left ventricular (LV) hypertrophic responses than that observed in untreated HF animals. Such LV adaptive remodelling was associated to reduced LV diameters and improved cardiac LV fractional shortening (FS) and +dP/dt. At molecular level, cardiac β-2AR gene transfer induced sustained activation of p-Akt and S6 kinase (S6K) phosphorylation and increased p-eNOS and VEGF levels that were instead reduced in untreated HF rats. In β2KO mice, we found a ~25% reduction of cardiac capillary and arteriolar density compared to WT mice. In β2KO mice, MI was associated with a dramatically higher 21-day mortality compared to WT (70% vs 30%, respectively). Survivors β2KO mice showed increased LV end diastolic and systolic diameters, and worse global cardiac contractility compared to controls. Cardiac Akt activation, S6K phosphorylation, p-eNOS, and VEGF-A production were significantly blunted in HF β2KO mice. Conclusions. β-2ARs play an important role in regulating angiogenic responses in post-MI HF hearts. The activation of Akt signal transduction pathway seems to be strongly involved in these mechanisms.

Synchrotron Microangiography of the Rat Heart Using the Langendorff Model

Background and Objectives: The ability to study microvessels of a beating heart in real time at the level of the capillary is essential for research. However, there are no proven methods currently available to achieve this. The conventional absorption-contrast agents have limitations for studying capillaries. Microangiography with using synchrotron phase-contrast X-ray technology and no contrast agent has recently been reported on. We tried to verify this previous report, and we wanted to visualize the microvessels of a rat heart using air as a contrast agent. Materials and Methods: We made the Langendorff apparatus in a hutch of the Pohang Accelerator Laboratory. The images were obtained with a white beam and a monochromatic beam. The visual images were magnified using 3× and 20× optical microscope lenses, and the images were captured with a charge-coupled device camera. Results: We could not duplicate the previously reported findings in which microvessels were visualized without the use of contrast agent. But with using air as a contrast agent, the microvasculature of rat hearts was clearly identified at a spatial resolution of 1.2 μm. Air being absorbed inside a capillary was also observed. Vessels under 10 μm diameter were unable to be visualized with using iodine as a contrast agent. Conclusion: Phase contrast imaging already allows spatial resolution of 1 μm, which is enough to inspect capillaries. We were able to obtain images of cardiac capillaries with using air as a contrast agent. Yet air has the fatal limitations in that it causes embolism and ischemia. A more suitable contrast agent or imaging method needs to be developed in order to study the microvessels of a beating heart. (Korean Circ J 2008;38:462-467)

Abstract 865: Gata-4 Is An Angiogenic Survival Factor Of The Infarcted Heart

Recent data suggest that the cardiac-restricted transcription factor GATA-4 is an anti-apoptotic factor required for adaptive responses as well as a key regulator of hypertrophy and hypertrophy-associated genes in the heart. As a leading cause of chronic heart failure, reversal of post-infarction left ventricular remodeling represents an important target for therapeutic interventions. Here we studied the role of GATA-4 as a mediator of post-infarction remodeling. Rats were subjected to experimental myocardial infarction (MI) by ligating the left anterior descending coronary artery (LAD). Ligation of the LAD decreased the DNA binding activity of GATA-4 by 69 % at day 1 after MI (P&lt;0.001, n=7– 8) as assessed by gel mobility shift assays. At 2 weeks the GATA-4 DNA binding was significantly upregulated (2.4-fold, P&lt;0.05, n=7), and returned to baseline at 4 weeks. To determine the functional role of GATA-4, rats underwent LAD ligation followed by peri-infarct intramyocardial delivery of adenoviral vector expressing GATA-4. Hearts treated with the GATA-4 gene transfer exhibited significantly increased ejection fraction (58±5% vs. 38±3% in LacZ-treated control animals with MI, P&lt;0.001, n=8 –9) and fractional shortening (28±3% vs. 16±1%, P&lt;0.001, n=8 –9) 2 weeks after MI. Accordingly, the infarct size was significantly reduced (26±4% vs. 45±4%, P&lt;0.01, n=8 –9). To determine the cardioprotective mechanisms of GATA-4, the number of cardiac stem cells, apoptotic cardiomyocytes and capillaries were assessed. The number of capillaries (59±4/field vs. 48±3/field, P&lt;0.051, n=7– 8) and c-kit positive stem cells (13±5 cells vs. 4±2 cells, P&lt;0.05, n=7– 8) were increased in GATA-4 treated hearts, and a tendency to decreased apoptosis was observed in TUNEL-stained histological sections. These results indicate that the reversal of reduced GATA-4 activity prevents adverse post-infarction remodeling through increased angiogenesis, recruitment of cardiac stem cells and anti-apoptosis. GATA-4-based gene transfer may represent a novel, efficient therapeutic approach for heart failure.

Abstract 1115: Impaired Angiogenic Potential of Cardiac Progenitor Cells in Mice with a Cardiomyocyte-Restricted Knock Out of STAT3

Mice with a cardiomyocyte-restricted knock out of STAT3 (KO: alpha-MHC-Cre tg/+; STAT3 flox/flox ) show a continuous decrease of the cardiac capillary density and develop heart failure beyond the age of 9 months. We sought to determine the paracrine influence of cardiomyocyte STAT3 on the endothelial differentiation potential of cardiac progenitor cells (CPC) of the adult mouse heart. Sca-1 + CPC were isolated from male mice hearts by MACS separation. STAT3 was entirely deleted in cardiomyocytes of KO mice, while CPC from KO showed normal expression of STAT3 (confirmed by PCR and Western blot). No difference in the total number of CPC per heart was observed between wildtype (WT: STAT3 flox/flox ) and KO mice. FACS analysis revealed a reduced number of endothelial progenitor cells (as defined by coexpression of Sca-1, CD31 and CD38, −25%, P&lt;0.05) among CPC from KO compared to CPC from WT. The differentiation potential of CPC from WT and KO was analyzed during in vitro culture on fibronectin-coated plates. After 4 weeks of culture RT-PCR for CD31 and immunohistochemistry (IHC) for endothelial cell (EC) marker tie2 and isolectin B4 was performed. CPC from WT showed markedly more efficient EC differentiation and tube formation compared to CPC from KO (p&lt;0.01). In contrast, adipocyte differentiation was enhanced in CPC from KO (p&lt;0.05, oil red staining and RT-PCR). Proliferation capacity of CPC from KO was reduced by 33% (p&lt;0.01) as compared to CPC from WT. Microarray results of freshly isolated CPC were consistent with the differences in EC and adipocyte differentiation (i.e. prostaglandin E receptor 3 up 2.3-fold in CPC from WT, Lipocalin-2 up 2.7-fold in CPC from KO). We did not observe cardiomyocyte differentiation (IHC for alpha-sarcomeric actinin; RT-PCR for Nkx 2.5, alpha-MHC, or alpha-skeletal actin) of CPC from both genotypes, neither in vitro by addition of oxytocin, 5-AZA, DMSO, nor following intramyocardial injection of CPC in vivo. Conclusion: STAT3-dependent paracrine mediators released from cardiomyocytes are determinants of differentiation and vasculogenic properties of new EC derived from cardiac progenitor cells. The identification of these factors may offer new approaches to enforce the endogenous vasculogenic repair potential of the adult heart.