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European Journal of Heart Failure 2007 9(10):995-1002; doi:10.1016/j.ejheart.2007.07.008
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© 2007 European Society of Cardiology

Idiopathic dilated cardiomyopathy exhibits defective vascularization and vessel formation

Santiago Rouraa, Francesc Planasb, Cristina Prat-Vidala, Ruben Letab, Carolina Soler-Botijaa, Francesc Carrerasb, Anna Llacha, Leif Hove-Madsena, Guillem Pons Lladób, Jordi Farréa, Juan Cincaa,b,c and Antoni Bayes-Genisa,b,c,*

a Cardiology Service-Institut Català de Ciencies Cardiovasculars, Hospital Santa Creu i Sant Pau Barcelona, Spain
b Cardiology Service, Hospital Santa Creu i Sant Pau Barcelona, Spain
c Departament de Medicina, Universitat Autònoma de Barcelona Barcelona, Spain

* Corresponding author. Cardiology Service-ICCC, Hospital de la Santa Creu i Sant Pau, Departament de Medicina (Universitat Autónoma de Barcelona) C/ Sant Antoni Ma Claret 167, 08025 Barcelona, Spain. Tel.: +34 93 291 92 58; fax: +34 93 291 94 24. E-mail address: abayesgenis{at}santpau.es (A. Bayes-Genis).


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Background: Ultrastructural findings of idiopathic dilated cardiomyopathy (IDCM) include myocyte atrophy and myofilament loss, yet little is known about the vascular abnormalities present in IDCM.

Methods and results: Patients with IDCM and controls underwent multi-slice CT to examine length and diameter of epicardial vasculature. The levels of mobilizing cytokines and circulating EPCs were assessed by endothelial colony formation assay and flow cytometry. Immunohistochemistry and Western blot were used to examine microvessel density and expression of HIF-1{alpha} and β-catenin.

Main epicardial coronary arteries were shorter and smaller, and microvascular density was reduced in the epicardium in IDCM. Epicardial vessel paucity was associated with increased numbers of HIF-1{alpha}+ cells (46.8±13.1% vs. 19.4±9.4%, p=0.006) indicating local epicardial hypoxia and elevation of circulating VEGF-A (394pg/mL vs. 22pg/mL, p=0.001). The number of mobilized progenitors CD133+/VEGF-R2+ was 21-fold higher in IDCM compared with controls (6.5±3.3% vs. 0.3±0.2%; p<0.001). Moreover, this defective vascularization was associated with reduced myocardial expression of vascular β-catenin, an important angiogenic regulator.

Conclusions: This study shows defective vascularization and impaired vasculogenesis (the de novo vascular organization of mobilized endothelial progenitors) and angiogenesis (by which new blood vessels are formed from pre-existing mature endothelial cells) in human IDCM.

Key Words: Idiopathic dilated cardiomyopathy • Vasculogenesis • Angiogenesis • Multi-slice CT • EPC • β-catenin

Received April 25, 2007; Revised May 22, 2007; Accepted July 5, 2007


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Idiopathic dilated cardiomyopathy (IDCM) is a cardiac disease characterized by depressed contractility and increased ventricular chamber size in the absence of atherosclerotic coronary artery disease, valvular abnormalities or pericardial disease [1]. Characteristic pathologic findings, albeit not specific, include myocyte atrophy, interstitial fibrosis, myofilament loss, ultrastructural mitochondrial abnormalities, T-tubular dilation and presence of intracellular lipid droplets [2]. Common clinical manifestations are progressive heart failure and increased risk of sudden cardiac death.

Characterization of coronary artery tree and myocardial microvessels is important to understand the pathophysiology of IDCM. In patients with IDCM, analysis of digitized coronary arteriograms showed inappropriate coronary artery size with regard to left ventricular mass [3,4]. Moreover, a murine model of IDCM has revealed reduced myocardial capillary density [5]. Collectively, these data suggest impaired vascularization in IDCM.

New blood vessel growth in the adult requires upregulation of several growth factors and cytokines [6]. Vascular endothelial growth factor (VEGF-A), through interaction with its specific receptors, promotes migration of bone marrow endothelial progenitor cells (EPCs) to a target organ and is of major importance for blood vessel formation [7,8]. Vasculogenesis from bone marrow-derived cells has been most extensively studied in animal models of myocardial infarction [9,10], limb ischaemia [11] and atherosclerotic plaque [12]. Despite the contribution of these cells to tissue revascularization in these experimental models, the importance of EPCs in restoring cardiac vascularization in patients with IDCM remains unknown.

The aims of this study were to examine in human IDCM: (1) the presence of defective cardiac vascularization and HIF-1{alpha} expression as a surrogate marker of hypoxia; (2) the circulating levels of VEGF-A and EPC mobilization required for vasculogenesis; and 3) the myocardial expression of β-catenin, an important angiogenic regulator.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Study population
2.1.1. Patients
Fifteen IDCM patients and eighteen healthy controls with no cardiovascular disorders were enrolled in the study. Eligible IDCM criteria included NYHA class II or more, LV end-diastolic diameter ≥ 60 mm and LV ejection fraction < 35%. IDCM was diagnosed at least one month prior to enrolment in the study (mean 39.9±28.3 months), patients did not have a previous history of myocarditis and were not receiving statin treatment. Age- and sex-matched individuals referred for a cardiovascular check-up, who had no evidence of cardiovascular disease by ECG and physical examination, constituted the control group. Informed consent was obtained and the study was approved by the local Ethics Committee.

2.1.2. Autopsy material
Myocardial samples for histomorphological analysis were obtained from our institution's tissue bank. IDCM myocardial samples were obtained from 15 hearts explanted during cardiac transplantation, and control myocardial samples were obtained at autopsy from 10 individuals who died of non-cardiac causes.

2.2. Cardiac vascularization
2.2.1. Multi-slice computed tomography (CT)
Multi-slice CT studies analyzed epicardial coronary vasculature, LV volumes, LV ejection fraction, and LV mass in the two study groups [13]. A Toshiba Aquilion 16 system (Toshiba Corporation, Medical Systems Company) completing a 360° rotation in 0.5 s, and able to acquire up to 32 0.5-mm thick axial slices in 1 s, was used. Patients' heart rates ranged from 47 to 105 bpm with a mean of 60 bpm. Acquisition of cardiac volume images was continuous (helical) during the entire cardiac cycle. Cardiac volume images were reconstructed at the end of acquisition using a dedicated software workstation (Vitrea2®, Vital Images, Plymouth, MN) provided with the following tools: axial images, oblique and curved multiplanar reconstructions (MPR), maximum intensity projections (MIP), and volume-rendered three-dimensional reconstructions.

The length of the main epicardial arteries and the number of side branches were measured on MPR. The diameter of epicardial arteries was measured using manual callipers as follows: left main was measured within 5 cm from the ostium; proximal left anterior descending (LAD) and left circumflex (LCX) within 1.5 cm after bifurcation; mid LAD and LCX within 1.5 cm after first diagonal and first obtuse marginal, respectively; distal LAD and LCX were measured within 1.5 cm after the largest diagonal or obtuse marginal branch merging from the distal third of LAD and LCX, respectively; proximal right coronary artery (RCA) was measured within 5 cm from the ostium, mid RCA after within 1.5 cm after the first marginal, and distal RCA was measured within 1.5 cm before the bifurcation to posterior descending artery and posterolateral branches [14]. Image resolution was of 0.8 mm. The number of side branches emerging from LAD (diagonal and septal arteries), LCX (obtuse marginal arteries), and RCA (right ventricular, posterolateral, and posterior descending arteries) was also assessed.

2.2.2. Microvascular density
The number of microvessels (≤ 200 µm) was counted blindly in high-power fields of 19.14 mm2 in both left ventricular epicardium (outer third of myocardial wall) and endocardium (inner third of myocardial wall) according to a previously reported method [15]. Three sections were analyzed from each individual, yielding 270 and 180 fields for IDCM and control samples, respectively. Counts were made at a sampling ratio of every 25 µm. Images were examined under light microscopy at x 400 magnification.

2.3. Immunohistochemistry
To analyze the expression of HIF-1{alpha} and β-catenin by immunohistochemistry, dewaxed sections were processed using a previously described method with minor modifications [16]. Antibodies used were: mouse anti-human HIF-1{alpha} (1:5000) (Abcam Ltd, Cambridge, UK), and mouse anti-human β-catenin (1:40) (BD Transduction Laboratories). Signals were analyzed using a Nikon ES-400 microscope at x 400 magnification. HIF-1{alpha}+ cells were counted in the epicardium and endocardium at a sampling ratio of every 25 µm. Results are expressed as a percentage of HIF-1{alpha}-positive cells/total number of cells.

2.4. Assays of mobilizing cytokines
Plasma VEGF-A and VEGF-soluble receptor-1 (sVEGF-R1) were assayed by ELISA according to the manufacturers' instructions (BLK diagnostics for VEGF-A, and Bender MedSys. for sVEGF-R1). Triplicate assays were calibrated against VEGF-A (16-1000 pg/mL) and sVEGF-R1 (0.16-10 ng/mL) standards. Assay sensitivity was 11 pg/mL and 0.1 ng/mL, respectively.

2.5. Circulating EPCs
2.5.1. Isolation of peripheral blood mononuclear cells
A 30-mL sample of venous blood was processed within 4 h after collection for EPC isolation according to a modification of a previous report [17]. Total blood sample was diluted 1:1 with calcium- and magnesium-free phosphate-buffered saline (PBS) (Gibco), containing 2% bovine serum albumin, 0.6% sodium citrate (Sigma Chemical, St. Louis, MO) and 1% penicillin-streptomycin (Gibco). Diluted cell suspension was layered over 15 mL of 1.077 g/mL Lymphoprep– solution (Nycomed) and centrifuged at 800 g for 20 min. Mononuclear cells collected at the interphase were recovered and washed to remove platelets and contamination by plasma proteins with 3 volumes of balanced salt solution, corresponding to a 1:9 mix of Solution A (0.145 M Tris-HCl pH 7.6, 0.1% Anhydrous D-glucose, 5x10– 5 M CaCl2, 9.8x10– 4 M MgCl2, 5.4x10– 3 M KCl) and Solution B (0.14 M NaCl). Cells were resuspended in growth medium consisting of {alpha}-MEM supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin-streptomycin. These cells were then studied for endothelial colony formation and expression of endothelial-specific markers.

2.5.2. CFU-E assay
An initial pre-plating of 5 million mononuclear cells in a fibronectin-coated six-well plate was performed at 37 °C in 5% CO2 in air to eliminate mature circulating endothelial cells. At 48 h, non-adherent cells were collected, counted and 7x105 cells were re-plated onto new fibronectin-coated plates for final assessment of colony number seven days after re-plating. A colony of EPCs consisted of multiple thin, flat cells emanating from a central cluster of rounded cells. Samples were independently analyzed by two observers to assess reproducibility. Inter-observer correlation was 0.91 and intraclass correlation, i.e. a single observer who analyzed two blood samples from the same subject at least one week apart, was 0.95.

Specific monoclonal antibodies (mAbs) for VEGF-R2 (R&D Systems), CD34, and vWF (BD Biosciences) were used to assess endothelial lineage marker expression by immunofluorescence.

2.5.3. Flow cytometry
A suspension of 106 mononuclear cells was incubated in PBS containing 0.5% FBS and 1% antibiotics with 10 µL of both mouse anti-CD133 and anti-VEGF-R2 mAbs (R&D Systems) conjugated with FITC and PE, respectively, for 15 min at room temperature. The number of positive cells was compared with that of IgG isotype controls and quantified using a FACSVantage cytometer (BD Biosciences).

2.6. Protein extraction and Western blot
Each myocardial sample was agitated in 2 mL of ice-cold lysis buffer (20 mM Hepes pH 7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 1 mM PMSF, 1 µg/mL aprotinin and 1 µg/mL leupeptin) with a Polytron homogenizer for 30 min on ice. Supernatant, separated by centrifugation at 14,000 rpm for 30 min at 4 °C, was designated as the cytoplasmic fraction and stored frozen at – 80 °C until further use. Cell remnants were solubilized in 20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 2.5 mM EGTA and 1% SDS (membrane/cytoskeletal fraction). Proteins were resolved by SDS-PAGE using a specific mAb against human β-catenin (1/500) (BD Transduction Laboratories). Protein bands were visualized using ECL (Amersham Biosciences) and quantified using an Imaging Densitometer GS670 and the Kodak Digital Science 1D software (Kodak). Results are expressed as arbitrary units per µg of protein.

2.7. Statistical analysis
Descriptive statistics were used to summarize baseline characteristics. Differences between IDCM and controls were assessed by Student's t-test or Mann-Whitney U rank sum test when variables were non-Gaussian. Pearson's correlation coefficient was used to assess the association between CFU-E and plasma VEGF-A levels. p values < 0.05 between the groups were considered significant. All computations were performed with SigmaStat software (version 3.1).


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. Study subjects
We studied 15 patients with IDCM and 18 controls matched for age (44±11 years vs. 49±15 years), sex (85% vs. 80% males), height, weight, and body surface area (1.9±0.14 m2 vs. 1.85±0.17 m2). Multi-slice CT showed that patients with IDCM had larger left ventricular (LV) end-diastolic (252±115 mL vs. 131±41 mL, p<0.001) and end-systolic (200±116 mL vs. 59±23 mL, p<0.001) volumes, lower LV ejection fraction (23±13% vs. 55±9%, p<0.001) and greater LV mass (160±46 g vs. 96±30 g, p<0.001) than control subjects.

3.2. Defective cardiac vascularization and tissue hypoxia
The length and diameter of epicardial coronary arteries in absolute values and values adjusted for LV mass in the IDCM and control groups are given in Tables 1 and 2, respectively. As compared with controls, patients with IDCM had shorter vessels, including the left main, LAD, LCX, and RCA after adjustment for LV mass. Among side branches, only the second right ventricular branch was significantly shorter after adjustment for LV mass in IDCM (Table 1). Table 2 shows that the diameter of the left main and proximal, mid, and distal segments of LAD, LCX, and RCA after adjustment for LV mass was significantly smaller in patients with IDCM.


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  		Table 1 Length of coronary arteries

 


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  		Table 2 Diameter of coronary arteries

 
Patients with IDCM had a marked side-branch reduction of LAD and LCX compared with controls (LAD: 4.1±1.3 vs. 5.6±2.1; p=0.04; and LCX: 2.0±1.5 vs. 3.1±1.3; p=0.03, respectively). RCA side-branching was comparable among both groups (2.1±1.2 vs. 2.5±1.8, p=0.6). Fig. 1A and B shows volume-rendered 3-D reconstructions representative of a patient with IDCM, and a control subject, respectively. In patients with IDCM, the epicardial coronary arteries are not adequately sized to the larger LV mass, and side-branch paucity is apparent.


Figure 01
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  		Fig. 1 Cardiac vascularization and HIF-1{alpha} expression. Multi-slice CT 3-D reconstructions of a control subject (A) and a patient with IDCM (B). Marked epicardial coronary artery mismatch for left ventricular (LV) mass and side-branch reduction is observed in IDCM. (C) Histograms showing capillaries/field in the epicardium and endocardium from control and IDCM samples. A significant reduction in microvessel density was found in the epicardium of patients with IDCM. (D) Representative immunohistochemical HIF-1{alpha} expression in control and IDCM. (E) Histograms showing HIF-1{alpha} expression (as the percentage of HIF-1{alpha}+ cells/total number of cells) in the epicardium and endocardium of control and IDCM samples.

 
In addition to large epicardial coronary artery analysis, we studied microvascular density (vessels less than 200 µm in size, which accomplish and regulate myocardial oxygen delivery) in myocardial samples obtained from explanted hearts with IDCM and from decedents of non-cardiac causes (controls). Myocardium with IDCM showed significantly lower epicardial microvascular density than controls, yet endocardial microvascular density was comparable in the two groups (Fig. 1C). These data in humans are in agreement with the reduced myocardial capillary density found in a murine model of IDCM caused by mitochondrial dysfunction [5].

Epicardial vessel paucity was associated with increased numbers of HIF-1{alpha}+ cells in this region (46.8±13.1% vs. 19.4±9.4%, p=0.006) (Fig. 1D) indicating local epicardial hypoxia. The number of HIF-1{alpha}+ cells in the endocardium was comparable in the two groups (30.3±9.5% vs. 22.3±7.5%, p=0.18) (Fig. 1E). HIF-1{alpha} is a surrogate marker of hypoxia, which in turn modulates VEGF-A synthesis and secretion [18-20].

3.3. Circulating levels of VEGF-A and EPC mobilization: vasculogenesis response
Up to an 18-fold increase in VEGF-A was documented in patients with IDCM (394 pg/mL vs. 22 pg/mL, p=0.001). The level of circulating sVEGF-R1, a high affinity VEGF antagonist, was not significantly different between the study groups (1.2 ng/mL vs. 0.9 ng/mL; p=0.8).

In patients with IDCM, the number of endothelial progenitors assessed as CFU-E was markedly higher compared with controls (median of 30, range: 2-53 and 12, range: 9-21, respectively; p<0.001). Moreover, a positive association was found between plasma VEGF-A levels and the number of CFU-E (r=0.44; p=0.035). CFU-E showed endothelial cell lineage markers such as CD34, VEGF-R2, and vWF (Fig. 2A).


Figure 02
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  		Fig. 2 Mobilization of endothelial progenitor cells. (A) CFU-E expressing the endothelial lineage markers CD34, VEGF-R2, and vWF. (B) Analysis of mobilized EPCs by flow cytometry. Representative dot blots showing circulating CD133+/VEGF-R2+ cells in a patient with IDCM and a control.

 
The percentage of circulating CD133+/VEGF-R2+ cells, an EPC fraction that can proliferate and differentiate into mature adherent endothelial cells in vitro [6], was assessed by flow cytometry. Patients with IDCM had a 21-fold increase in circulating CD133+/VEGF-R2+ cells compared with controls (6.5±3.3% vs. 0.3±0.2%; p<0.001) (Fig. 2B). Similar results were observed by Gill et al. after vascular injury [21], vascular trauma induced by burn injury or surgical manipulation resulted in rapid mobilization of CD133+/VEGF-R2+ EPCs into the peripheral circulation.

3.4. Myocardial expression of β-catenin: angiogenic response
In the presence of impaired vasculogenesis, we asked whether angiogenesis was also altered in IDCM. Myocardial β-catenin, an important angiogenic regulator, was examined. Immunohistochemistry showed abundant myocardial β-catenin at the intercalated discs of both IDCM patients and control subjects. In contrast, vascular β-catenin expression was almost absent in IDCM (Fig. 3A). Western blotting confirmed that β-catenin was significantly reduced in both Triton-soluble (cytoplasmic) and Triton-insoluble (membrane/cytoskeletal) fractions from IDCM tissue (p=0.008 and p=0.005, respectively) (Fig. 3B).


Figure 03
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  		Fig. 3 Myocardial expression of β-catenin. (A) β-catenin and CD31 immunostaining. β-catenin expression in intercalated discs is similar in controls and in IDCM, but is reduced in vascular endothelial cells from IDCM. Scale bars=10 µm. (B) Representative Western blots (n=3 in both IDCM and control groups) showing β-catenin in myocardial Triton-insoluble (I) and Triton-soluble (S) fractions. Densitometry of β-catenin bands (n=5 and n=7 in IDCM and controls, respectively).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
This study shows that even though patients with IDCM had abundant mobilization of circulating EPCs, their hearts show impaired vascularization characterized by shorter length and smaller diameter of the major coronary segments adjusted to LV mass and lower epicardial microvascular density. We also found that this defective vascularization was associated with reduced expression of β-catenin in myocardial vessels. Collectively, these data indicate that both vasculogenesis (the de novo vascular organization of mobilized endothelial progenitors) and angiogenesis (by which new blood vessels are formed from pre-existing mature endothelial cells) are altered in human IDCM.

An early report from the late 1940s found a direct correlation between the diameter of the coronary arteries and LV mass [22]. More recently, studies in patients with hypertrophic cardiomyopathy have reported that the size of the coronary arteries was inappropriate with regard to the degree of left ventricular hypertrophy [4,23], suggesting that a mismatch between artery size and LV mass contributed to myocardial hypoperfusion and ischaemia [23]. In the present study in patients with IDCM, we also found a mismatch between coronary artery size and LV mass. After adjustment for LV mass, the length and diameter of the major coronary arteries were significantly shorter and smaller in patients with IDCM. Despite methodological differences, measurements of lumen diameter by multi-slice CT agree with those obtained angiographically [14,24,25].

Endothelial dysfunction is a prominent feature in cardiovascular diseases. Thus, the crucial role played by EPCs in the maintenance and recovery of diseased myocardium is being increasingly investigated [26-28]. As we have shown, the vasculogenesis switch is on in IDCM, but EPCs appear unable to restore myocardial revascularization, likely due to multifactorial reasons. First, the higher amounts of deposited collagen, sparsely distributed within the myocardium and forming areas of patchy fibrosis characteristic of IDCM [29], may act as a physical barrier for functional EPC incorporation. Second, circulating EPCs may not be mature enough to incorporate into damaged tissue [6]. Lastly, impaired EPC incorporation may be due to deficiencies in key myocardial signalling pathways required for the selective attraction of these progenitor cells [30,31]. Unfortunately, it is impossible to investigate these issues in a mechanistic way in patients. The future development of animal models with the characteristics of human IDCM may help decipher each of these mechanisms as well as the kinetics of this strong cellular mobilization. Valgimigli et al. reported a biphasic increase in progenitor cells during heart failure with elevation and depression in the early and advanced phases, respectively [32].

Moreover, defective vascularization in IDCM was associated with reduced expression of vascular β-catenin which directly enhances the survival, proliferation and migration of mature endothelial cells [33,34]. β-catenin is commonly distributed in two separate pools in the cell. An insoluble pool at the plasma membrane binds to VE-cadherin and {alpha}-catenin, and a soluble cytosolic pool that is involved in angiogenic pathways. In this study, the soluble β-catenin pool was markedly reduced indicating limited activation, proliferation, and migration of endothelial cells from pre-existing vessels required for angiogenesis response [35]. In addition, the downregulation observed for insoluble β-catenin in IDCM may also affect endothelial cell survival and the stability of endothelial cell-cell contacts promoting decreased microvascular density.

It remains to be established whether the reduced capillary density and subsequent underperfusion of the already diseased heart contributes to, or is a marker of, the progression of LV dysfunction in patients with IDCM. Myocardial perfusion studies using positron emission tomography have demonstrated that patients with DCM have significantly impaired myocardial perfusion at rest and an impaired perfusion reserve [36]. These results, together with the finding that impaired vasodilator capacity is associated with increased risk of heart failure progression and death [37], suggest that restoration of adequate vascularization in DCM may be a therapeutic target in these patients.

We must point out that the autopsy material used in this study came from end-stage IDCM patients and, therefore, the reported histological findings should be interpreted with caution in IDCM patients not requiring cardiac transplants.

Based on our results, cardiac revascularization in IDCM may not be a matter of additional exogenous delivery of progenitor cells, but rather to promote a conducive microenvironment for homing and functional incorporation of the mobilized EPCs. More studies are required to evaluate the differentiation of mobilized EPCs into functional mature vessels in vivo, a process that may be mediated by cardiac-specific growth factors, proteases, and matrix proteins.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
Red de Investigación Cooperativa en Trasplante Cardiaco del Ministerio de Sanidad (C03/03), Ministerio de Educación y Ciencia (SAF2004-08044-C03-01), Universitat Autònoma de Barcelona (EME2004-06) and Societat Catalana de Cardiologia. We also appreciate support from Novartis, Fundació Daniel Bravo Andreu and Fundació Roviralta.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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K. V Arom, P. Ruengsakulrach, M. Belkin, and M. Tiensuwan
Intramyocardial Angiogenic Cell Precursors in Nonischemic Dilated Cardiomyopathy
Asian Cardiovasc Thorac Ann, August 1, 2009; 17(4): 382 - 388.
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