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Endothelial-like cells expanded from CD34⁺ blood cells improve left ventricular function after experimental myocardial infarction

Ilka Ott^*,1, Ulrich Keller^,1, Martina Knoedler^*, Katharina S. Götze, Katharina Doss^*, Philipp Fischer, Katja Urlbauer, Gerlinde Debus, Nikolas von Bubnoff, Martina Rudelius, Albert Schömig^*, Christian Peschel and Robert A. J. Oostendorp^,2

^* I. and
III. Department of Medicine, Technical University Munich, Munich, Germany;
Department of Gynecology and Obstetrics, Krankenhaus Neuperlach, Munich, Germany; and
Department of Pathology, Technical University Munich, Germany

² Correspondence: III. Medizinische Klinik und Poliklinik, Technical University Munich, Ismaninger Strasse 22, Munchen 81675, Germany. E-mail: oostendorp{at}lrz.tum.de

SPECIFIC AIMS

Neovascularization in adults is the result of angiogenesis but involves bone marrow-derived endothelial progenitor cells (EPC) during vasculogenesis. EPCs isolated from peripheral blood, cord blood, and bone marrow contribute to neovascularization during vascular injury, ischemia, and tumor growth. We describe the expansion of endothelial-like cells from CD34+ cells derived from umbilical cord blood and apheresis from adult blood donors in vitro and investigate whether these culture-expanded cells increase vessel density and improve left ventricular function after acute myocardial infarction.

PRINCIPAL FINDINGS

1. Low frequency of endothelium-forming EPC, but high expansion potential
To study the endothelial fate of CD34⁺ cells from cord blood, we studied their ability to form endothelial colonies. During the first week of culture, only a few adherent CD34⁺ cells were detected. In most cultures, endothelial colony growth was observed after 2 wk. In a novel limiting dilution assay for CFU-EC, the frequency of CD34⁺ CB cells that formed endothelial colonies (CFU-EC) was estimated to be 1 in 8.7 x 10⁴ CD34⁺ cells. When EPC cultures were started with AC133⁺ cells, this frequency was much lower (1 in 3.0 x 0⁵ AC133⁺ cells), suggesting that, using our culture protocol, CD133⁺ cells less frequently form EC colonies than CD34⁺ cells. Despite the low CFU-EC frequency, 1 x 10⁶ CD34⁺ CB cells (4–11 EPC) generated up to 10²⁰ ECs in 60 days. Calculating from the number of CFU-EC, this would translate to 60 population doublings. In similar experiments using aphereisis products of G-CSF-mobilized patients we were unable to pinpoint the frequency of CFU-EC. We estimate that in these adult blood products, CFU-EC frequency is < 1 in 10⁶ CD34⁺ cells from adult apheresis-derived EC (PBEC). Calculating from a frequency of 1 in 10⁶, our results suggest that PBEC expand only 10 population doublings in 60 days. Thus, the expansion of CBEC appears to be much higher than that of PBEC; for this reason, the remainder of the experiments was carried out with CBEC.

2. Cord blood-derived endothelial-like cells express similar genes as HUVEC
Immunocytochemistry, real-time PCR, and macroarray analyses revealed a high degree of similarity of CB-derived endothelial-like cells (CBEC) and human umbilical vein endothelial cells (HuvEC). In a gene expression macroarray, we detected many EC-associated genes: vWF, CD31 (PECAM), CD102 (ICAM-2), CD105 (Endoglin), VEGF-R1 (Flt1), and Tie1. Lower signals were found for CD144 (VE-cadherin), CD146 (Muc18), EPAS-1, and PlGF. These results were confirmed using real time PCR. AC133 and VEGF-R2, markers of primary EPC, were not detected by macroarray or real-time PCR analyses. The cultured cells did not represent microvascular EC because they did not express CD36. Western blot analyses further revealed that CBEC and HuvEC express similar levels of the senescence-associated proteins p16^INK4a, p21^CIP1, p27^KIP1, or p53 and the telomere-associated TRF-2.

3. Culture-expanded CD34⁺-derived EC generate vascular structures in ischemic myocardium
To find out whether the expanded EPC-derived progeny could be useful for therapeutic vasculogenesis, we evaluated whether these cells engraft in vivo and generate new vessels in ischemic myocardium. CBEC were transplanted into the ischemic myocardium of nude rats. After 7 and 14 days, animals were killed and the hearts studied for the presence of human cells. Figure 1 A, B show control stains of human-specific CD31 antibody that stains only human cells (Fig. 1B ), not rat endothelial cells (Fig. 1A ). Using this antibody, numerous vascular structures resembling developing capillaries and arterioles derived from human cells were observed adjacent to infarcted myocardium. Some of these vascular structures contained blood cells in their lumen, suggesting that anastomosis had occurred with the spared coronary vessels (Fig. 1C ). After 7 days of transplantation, some of the human endothelial-like cells within vessel walls expressed the proliferation marker Ki67 (Fig. 1D ), indicating that the proliferative capacity of the transplanted human cells is not impaired in the rat environment.

View larger version (142K): [in this window] [in a new window]   Figure 1. Detection of human endothelial-like cells after transplantation of EPC-derived human EC into ischemic myocardium of athymic nude rats. Cryosections of control tissues of nontransplanted rats were stained with the anti-human CD31 monoclonal antibody. This antibody did not show any reactivity in the infarcted area of mock (PBS)-transplanted (A) nude rats. In contrast, pelleted human EPC-derived progeny stained strongly with this same antibody (B). C) Cryosections from nude rats transplanted with human endothelial-like cells. To induce myocardial infarction, the left anterior descending coronary artery was ligated for 30 min, followed by reperfusion. Immediately thereafter, EPC-derived cells in 100µl PBS were injected into 5 sites of the ischemic myocardium. The presence of nucleated human EC-like cells was demonstrated 7 days after infarction using a human specific anti-human CD31 antibody (C). Arrows indicate nucleated EC. Some transplanted human CD31+ cells (brown) show positivity for the proliferation marker Ki67 (red) as pointed out with the arrows (D).

4. Culture-expanded endothelial-like cells improve left ventricular function after myocardial infarction
We next investigated whether culture expanded CFU-EC might have therapeutic significance. We determined whether the transplanted EC-like cells increased vessel density or improved ventricular heart function after myocardial infarction. Morphometric analyses showed that the vessel density within the infarct area was significantly increased in nude rats transplanted with CBEC (Fig. 2 A). Echocardiographic analysis revealed that 2 wk after myocardial infarction left ventricular ejection fraction of control animals decreased from 56 + 0.7% to 43 ± 1% (n=8), whereas in animals transplanted with EPC-derived cells ejection fraction decreased only from 55 ± 0.7% to 50 ± 1.6% (n=8, P<0.01 (unpaired t test) compared with ejection fraction in mock-transplanted animals) (Fig. 2B ).

View larger version (14K): [in this window] [in a new window]   Figure 2. Transplanted EC-like cells increase vessel density and improve left ventricular function after acute myocardial infarction (AMI). Culture expanded EC-like cells were transplanted into the ischemic myocardium as described in Fig. 1 ; 2 wk later rats were investigated using echocardiography. Subsequently, rats were killed and the hearts stained with anti-CD31 (cross-reactive with both human and rat CD31). A) Transplanted human cells increase the number of CD31+ vessels per microscopic field. B) Analysis of the echocardiographic results. End diastolic (EDA) and end systolic (ESA) ejection areas were determined and the left ventricular ejection fraction was calculated (EF=(EDA-ESA)/EDA) from these values.

Thus, local transplantation of EPC-derived progeny in acute myocardial infarction improved ischemia-induced myocardial damage most likely is due to enhanced vessel regeneration. These findings suggest that expanded EPC-derived progeny may be suitable for therapeutic vasculogenesis in ischemic disorders.

CONCLUSIONS AND SIGNIFICANCE

We show that endothelial-like cells derived from cord blood CD34⁺ cells can be expanded beyond 50 population doublings. These endothelial-like cells are not transformed, as they do not express detectable levels of hTERT. Culture-expanded endothelial-like cells show normal regulation of molecules involved in cellular senescence, as well as telomere-associated molecules, compared with primary umbilical vein-derived endothelial cells. Our results suggest that CD34⁺ cells contain more EPC than CD133⁺ cells, a surface marker reportedly found on EPC and not mature EC. Thus, one might argue that the EC-forming CD34⁺ cells represent mature cells. However, in vitro-derived mature EC from early outgrowth EC colonies reportedly undergo only a 6-fold expansion, whereas EC derived from late outgrowth EC colonies can be expanded to 10²⁰ cells and originate from the bone marrow. Therefore, late outgrowth ECs are thought to derive from EPC or angioblast-like cells. We used a similar outgrowth assay and found that starting with 10⁶ CD34⁺ cells, EPC-derived cells from CB can be expanded up to 10¹⁵ cells within 60 days. This strongly suggests that the EC we expanded from CB and PB are derived from nontransformed angioblast-like cells or EPCs within the isolated CD34⁺ population.

Experimental studies showed that in acute myocardial infarction, autologous transplantation of EPC from circulating mononuclear cells improves left ventricular function. First clinical studies demonstrated the feasibility of percutaneous intracoronary application of bone marrow mononuclear cells or EPC into the target vessel in patients with acute myocardial infarction. The functional significance of EPC transplantation remains to be shown in larger studies. We show that expanded EPC-derived progeny improve left ventricular heart function after reperfused myocardial infarction. Together with our finding that these cells can easily be genetically modified using lentiviral transduction methods, this establishes the usefulness of expanded EPC-derived progeny from cord blood for therapeutic vasculogenesis.

Our present study is in accordance with observations of other investigators that transplant-related increase in vessel density improves left ventricular function. However, the nature of this improvement is not completely understood. Clinical studies have shown that in addition to infarct size, patency of the infarct-related artery or collateral flow are major determinants of ventricular remodeling. Within the infarcted myocardium, the capillary microcirculation may be plugged from aggregated platelets, monocytes, and macrophages. Thus, salvage of viable cardiomyocytes may be impaired. Previous studies have shown that marrow-derived EPC might contribute to local angiogenic and inflammatory reactions by the release of cytokines and chemokines, whereas vasculogenesis might prevail over time. CD34⁺ EPC from G-CSF mobilized peripheral blood increased vasculogenesis and reduced myocyte apoptosis. EPC may not be responsible only for the formation of new vessels, but may recruit local cells. Thus, increased vessel density stimulated by EPC and EPC-derived progeny may alter myocardial remodeling and stimulate local vasculogenesis, with decreased reduction of left ventricular function after myocardial infarction.

Our results indicate that EC-like cells expanded from cord blood CFU-EC form vessel-like structures and improve vessel density in ischemic myocardium and left ventricular function after myocardial infarction. It may be a disadvantage having to first expand cells for > 3 wk before transplantation can be attempted. However, it has been reported that myocardial bone marrow transplantation showed a therapeutic effect even 4 wk after transplantation. If this holds true in humans, expansion of sufficient cell numbers seems feasible. Expanded cells derived from cord blood or aphereses of adult blood donors might also be therapeutic for patients with chronic ischemic disease such as in severe coronary artery disease or peripheral occlusive disease. Thus, CFU-EC-derived progeny may be suitable for therapeutic vasculogenesis in ischemic disorders.

View larger version (26K): [in this window] [in a new window]   Figure 3. EC-like cells can be expanded in culture from CD34+-derived CFU-EC. These expanded cells will improve vessel density as well as improve left ventricular function after myocardial infarction.

FOOTNOTES

¹ These authors contributed equally to this work.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3219fje; doi: 10.1096/fj.04-3219fje

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This Article Full Text (PDF) Supplemental Data All Versions of this Article: 19/8/992 04-3219fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ott, I. Articles by Oostendorp, R. A. J. Search for Related Content PubMed PubMed Citation Articles by Ott, I. Articles by Oostendorp, R. A. J.