Horizontal gene transfer from human endothelial cells to rat
Cardiovascular Research (2008) 77, 534–543 doi:10.1093/cvr/cvm071 Horizontal gene transfer from human endothelial cells to rat cardiomyocytes after intracoronary transplantation ¨decke1, Alexander Assmann1, Andreas Wirrwar2, Sandra Burghoff1, Zhaoping Ding1, Stefanie Go 2 3 ¨ller2, Doris Buchholz , Olga Sergeeva , Cordula Leurs4, Helmut Hanenberg4, Hans-Wilhelm Mu 5 1 ¨rgen Schrader Wilhelm Bloch , and Ju 1 Institute for Heart and Circulatory Physiology, Heinrich Heine University Duesseldorf, Universitaetsstr. 1, 40225 Duesseldorf, Germany; 2Clinic for Nuclear Medicine, Heinrich Heine University Duesseldorf, Universitaetsstr. 1, 40225 Duesseldorf, Germany; 3 Department of Neurophysiology, Heinrich Heine University Duesseldorf, Universitaetsstr. 1, 40225 Duesseldorf, Germany; 4 Department of Pediatric Oncology Hematology and Immunology, Childrens Hospital, Heinrich Heine University Duesseldorf, Universitaetsstr. 1, 40225 Duesseldorf, Germany; and 5Department of Molecular and Cellular Sport Medicine, German Sport University, Cologne, Germany Received 18 May 2007; revised 18 September 2007; accepted 24 October 2007; online publish-ahead-of-print 13 November 2007 Time for primary review: 21 days KEYWORDS Aims Recent studies suggested that human umbilical vein endothelial cells (HUVECs) transdifferentiate into cardiomyocytes and smooth muscle cells in vitro. To test the functional relevance of this observation, we examined the transdifferentiation potential of HUVECs in vivo after intracoronary cell application in Wistar rats. Methods and results SPECT measurements (single photon emission computed tomography) revealed that 18% of 111In-labelled HUVECs infused by intracoronary delivery stably transplanted to the rat heart. For long-term tracking, HUVECs-expressing enhanced green fluorescent protein (EGFP) were infused. Two days following transplantation, HUVECs were positive for caspase-3. Within 3 days, EGFP was associated with individual cardiomyocytes. No labelling of endothelial and smooth muscle cells was observed. The total number of EGFP-labelled cardiomyocytes accounted for 58% of all initially trapped cells. These EGFP positive cells stained negatively for human mitochondrial proteins, but were positive for rat monocarboxylate transporter-1 protein (MCT-1). Furthermore, EGFP-mRNA was detected in these cells by single-cell RT–PCR (reverse transcription followed by polymerase chain reaction). After 21 days, EGFP positive cells were no longer observed. To investigate the underlying mechanism, we generated in vitro apoptotic bodies from EGFP-labelled HUVECs and found them to contain the genetic information for EGFP. Co-incubation of apoptotic bodies with neonatal rat cardiomyocytes caused cardiomyocytes to express EGFP. Conclusion When transplanted into the rat heart by efficient intracoronary delivery, EGFP-expressing HUVECs cause the exclusive but transient labelling of cardiomyocytes. Our in vivo findings suggest that it is not cell fusion and/or transdifferentiation that occurs under these conditions but rather a horizontal gene transfer of the EGFP marker via apoptotic bodies from endothelial cells to cardiomyocytes. Introduction Ventricular remodelling after myocardial infarction and subsequent development of heart failure cannot be prevented by the regenerative capacity of mature cardiomyocytes. Therefore, transplantation of beneficial cells is thought to be a promising therapeutic approach. At present, it is controversial whether a possible contribution of these cells to functional improvements of the injured * Corresponding author. Tel: +49 211 8112671; fax: +49 211 8112672. E-mail address: [email protected] heart is based on transplant-related angiogenesis, paracrine effects, or transdifferentiation into cardiomyocytes.1 Recent reports suggest that human umbilical vein endothelial cells (HUVECs) have the potential to transdifferentiate into heart-residing cells. For example, a significant fraction of HUVECs were shown to differentiate into smooth muscle like cells when the culture medium was deprived of fibroblast growth factor, a process which can be reversed by reapplicating the growth factor.2 Additionally, green fluorescent protein (GFP) expressing HUVECs were reported to transdifferentiate into cardiac muscle cells when cocultivated with freshly isolated neonatal cardiomyocytes.3 The colocalization of endothelial proteins Published on behalf of the European Society of Cardiology. All rights reserved. & The Author 2007. For permissions please email: [email protected]. Downloaded from by guest on July 6, 2015 Cell therapy; Transplantation; Stem cells; Catheter based stem cell transplantation; HUVECs; Horizontal gene transfer; Cardiac imaging; Multipinhole-SPECT Horizontal gene transfer from human endothelial cells to rat cardiomyocytes dishes containing DMEM (Gibco), 20% foetal bovine serum (Biochrom AG), penicillin G (100 U/mL), streptomycin (100 mg/mL) (Gibco). After 2 h at 378C non-adherent cells were replated. Apoptotic bodies were generated by incubation of HUVECs in Basal Medium (PromoCell) without supplements for 24 h.9 Where indicated, 0.5 mg/ml ethidium bromide or 0.5 mg/ml 4,6-diamidino-2-phenylindole (DAPI, Sigma) was added. Medium was then centrifuged for 10 min at 800 g, and the supernatant again for 20 min at 16 000 g. Apoptotic bodies were resuspended in complete medium. For cocultivation assay conditions used were: 25 000 3-day old neonatal rat cardiomyocytes were seeded onto sterile glas plates (Ø22 mm, Plano, Wetzlar, Germany) and apoptotic bodies from a 100 mm flask of EGFP-expressing HUVECs were added. Cells were incubated at 378C with fresh medium every 2 days. After 4 days, cells were harvested and fixed for immunofluorescence staining. Heart disintegration, single cell isolation, reverse transcription, and PCR analysis Hearts were perfused with 1 mg/ml Collagenase II (Biochrom AG) and 5 mM CaCl2, minced and incubated at 378C for 15 min. Cells were filtrated through a 100 mm Cell Strainer (BD Biosciences, Heidelberg, Germany), centrifuged and resuspended in PBS. Green fluorescent cells (excitation 485 nm, emission 530 nm) were sucked into a sterile glass electrode (tip diameter 2–3 mm) filled with 9 ml water. The content of the electrode was expelled into a reaction tube containing 7 ml of a reverse transcriptase mixture (Amersham Biosciences, Buckinghamshire, UK) and incubated at 378C for 1 h. DNA from HUVECs and apoptotic bodies was isolated using the DNeasy Tissue Kit (Qiagen GmbH, Hilden, Germany). PCR was performed using primers for EGFP (50 -acgtaaacggccacaa gttc-30 , 30 -cacatgaagcagcacgactt-50 ). Conditions for PCR amplification were: 30 s, 948C; 45 s, 578C; 75 s, 728C; 35 cycles. Methods The investigation conforms with the Declaration of Helsinki for use of human tissue or subjects. Cell culture and labelling HUVECs were harvested by collagenase (Biochrom AG, Berlin, Germany) and cultured to confluency in Basal Medium supplemented with endothelial single quots (PromoCell, Heidelberg, Germany) on gelatine-precoated culture flasks. Second passage HUVECs (1 106) were incubated for 5 min with 7 MBq 111In (37 MBq/mL; Tyco Healthcare, Neustadt/Donau, Germany) in serum free M199 medium (Gibco, Karlsruhe, Germany) at room temperature. Labelling efficiency was measured using a dose calibrator (MED Nuklear-Medizintechnik, Dresden, Germany). Vitality of cells 90 min post-labelling was 66–84% (n = 4) as tested by trypan blue exclusion. For stable expression of EGFP, second passage HUVECs were transduced with lentivirus derived from the HIV1-vector pGJ37 expressing EGFP under the control of the U3 promoter of spleen focus forming virus, a mutant of the Friend mink cell focus-forming virus. Viral particles were pseudotyped with the vesicular stomatitis virus glycoprotein G. HUVECs were washed (10 times) and passaged before usage. In vitro cocultivation assay Neonatal rat cardiomyocytes were isolated from ventricular muscle of newborn Wistar rats.8 Ventricles were minced in ice-cold PBS buffer and incubated in 1% trypsin-EDTA (Sigma, Taufkirchen, Germany) repeatedly at 378C for 15 min. Cell suspension was filtered through a 100 mm nylon mesh and seeded on 60 mm culture Cyclosporine A experiments HUVECs (1 104/cm2) were cultured on gelatine-precoated culture flasks in Basal Medium supplemented with endothelial single quots (PromoCell) and 6 mg/ml Cyclosporin (Sandimmunw, Novartis, Nu ¨rnberg, Germany). Medium was changed daily. After 7 days cells were fixed with 4% paraformaldehyde and 15% picric acid in PBS and applied to immunohistochemistry. Animal experiments The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The intracoronary transplantation of HUVECs was performed as previously described.6 Briefly, male Wistar rats (450–470 g) were intubated and anaesthetized by mechanical ventilation with isoflurane (1.5% v/v; Abbott, Wiesbaden, Germany). After induction of transient cardiac arrest [1 mM esmolol (Breviblocw, Gensia, San Diego, USA), 1 mM acetylcholin (Sigma) in 1 ml PBS] either 2.5 105 HUVEC (500 ml) or MEM (500 ml, sham) were gently injected into the coronaries. All animals received 30 mg/kg cyclosporine (Sandimmunw) the day prior to transplantation and 15 mg/kg/d cyclosporine daily thereafter. For planar scintigraphic measurements during and after cell transplantation, a double headed gamma camera (PRISM 2000XP, Philips/ Picker, Hamburg, Germany; 10 frames for 15 s followed by 10 frames for 5 min) equipped with a parallel hole collimator was used. At indicated time points, animals were anaesthetized and echocardiography (Hewlett Packard Sonos 5500 equipped with 15 MHz linear array probe) was performed. Downloaded from by guest on July 6, 2015 with cardiomyocytic proteins within one cell was interpreted as evidence for transdifferentiation. Final proof by using species-specific antibodies to discriminate between transdifferentiation and cell fusion was not provided.3 Welikson et al.4 re-investigated these latter results, but were not able to find transdifferentiated cardiomyocytes originated from HUVECs. Only 1.2% of all cells were positive for sarcomeric myosin and human nuclear antigen, but the majority of these cells was binucleated suggesting cell fusion between HUVECs and neonatal rat cardiomyocytes rather than transdifferentiation. The direct injection of HUVECs into the infarcted area of rat hearts led to an infiltration of macrophages and their subsequent phagocytosis.5 This was associated with improved left ventricular function without signs of HUVEC transdifferentiation and might be due to inflammatory responses.5 Given the divergent reports in the literature on endothelial cell plasticity, the present study explored the fate of HUVECs under in vivo conditions in the beating rat heart using a minimally invasive intracoronary delivery system for cardiac cell transplantation.6 We found a large fraction of the transplanted HUVECs having traversed the coronary vascular wall and reached the interstitial space. Thereafter, HUVECs became apoptotic and stained positive for active caspase-3, whereas the infiltration of macrophages was not observed. Finally, solely rat cardiomyocytes transiently expressed the HUVEC-introduced marker protein enhanced GFP (EGFP). In our study, we did not find evidence for cell fusion. Using species-specific antibodies, we ruled out the possibility of transdifferentiation, but suggest that horizontal gene transfer of EGFP through apoptotic bodies explains our finding. 535 536 S. Burghoff et al. Immunofluorescence and immunohistochemistry Statistics Data are expressed as mean + SEM. P , 0.05 was considered to be statistically significant. Total number of EGFP-expressing HUVECs (ntotal) was calculated using the following formula: ntotal ¼ nV A ðd þ tÞ where n is the number of counted cells per section, V the heart volume, A the area analysed, d the diameter of HUVECs, and t the thickness of the section.10,11 Results Distribution of human umbilical vein endothelial cells after intracoronary transplantation For the intracoronary delivery of HUVECs into rats, a minimally invasive catheter-based technique was applied.6 The position of the catheters during cell infusion is illustrated in Figure 1A, and a representative recording of aortic pressure during the intervention is shown in Figure 1B. In order to visualize the distribution pattern of the cells during and after intracoronary transplantation 111In-labelled HUVECs (2.5 105 cells in 0.5 ml, 2.8 MBq) were transplanted. During cell delivery (within 30 s) and the following 50 min, planar views of the distribution of radioactivity within the rat were obtained and permitted the calculation of the number of cells which accumulated within the heart. Fate of intracoronarely transplanted HUVEC One hour after cell delivery, EGFP-expressing HUVECs were found within the capillaries of the heart as evidenced by endothelial staining for caveolin-1 and detected by epifluorescence (Figure 3A and B). This result was confirmed when the staining was repeated using a rat specific anti-PECAM-1 antibody (data not shown). To determine the number of HUVECs within the heart, the number of green fluorescent cells in 6 consecutive slices (each 7 mm) was counted. The total number of all EGFP-expressing cells within the heart was calculated to be 17% of all initially transplanted HUVECs. To determine the origin of EGFP-expressing cells in the rat heart, species-specific antibodies were used. One hour after transplantation of EGFP-expressing HUVECs, green fluorescent cells stained positively with anti-human mitochondria antibody (Figure 3C). No crossreaction with rat endothelial cells and rat cardiomyocytes was observed. The anti-rat MCT-1 antibody did not stain EGFP positive HUVECs (Figure 3D). This antibody is well suited to detect rat myocardium, since no immunostaining was observed after omission of the first antibody (Figure 3E–G). One day after cell transplantation, EGFP-expressing cells have crossed the endothelial barrier, are still clearly positive for PECAM-1 and human mitochondrial proteins, and negative for rat MCT-1 (data not shown). Three and seven days after transplantation of EGFP-expressing HUVECs, all fluorescently labelled cells showed typical cardiomyocytic morphology and appeared to be well integrated in the myocardial tissue (Figure 4A). No green fluorescent cells were detected in the endothelium of rat coronary vessels. Careful examination of EGFP Downloaded from by guest on July 6, 2015 Hearts were fixed with 4% paraformaldehyde for 12 h, rinsed in 0.1 M PBS for 3 10 min, stored for 24 h in PBS solution with 18% sucrose and frozen at 2808C. Cryostat sections of 7 mm were cut from cryoprotected cardiac tissue. Cells were fixed by applying 4% paraformaldehyde for 20 min and then rinsed in PBS for 24 h. In case of immunohistochemical detection of EGFP samples were incubated in 3% H2O2 for 30 min and thereafter in 0.5 M NH4Cl, 0.25% Triton X-100 in 0.05 M TBS for 10 min. Blocking was performed in 5% BSA in 0.05 M TBS for 1 h at room temperature. Samples were marked with rabbit anti-human-mitochondria (pAB, 1:1000), chicken anti-rat-monocarboxylate transporter 1 (MCT-1) (pAB, 1:1000), mouse anti-human-mitochondria (for doubleimmunohistochemistry, mAb, 1:1000) (Chemicon, Temecula, USA), rat anti-mouse-CD31 (1:800, BD Pharmingen, San Jose, USA), anti-caveolin-1 (pAB, 1:500, Transduction, Lexington, USA), mouse anti-a-actinin (mAB, 1:400, Sigma), rabbit anti-GFP (pAB, 1:500, Santa Cruz, CA, USA), or rabbit active-caspase-3 antibody (for double-immunohistochemistry, BD Pharmingen, 1:500) in 0.8% BSA in TBS overnight at 48C. The secondary antibodies biotinylated goat anti-rabbit-IgG and goat anti-mouse-IgG (1:400, Dako, Hamburg, Germany), biotinylated sheep anti-rat-IgG (1:400, Amersham, LIFE SCIENCE, Little Chalfont, UK), biotinylated goat anti-rabbit-IgG (1:400, Dako), and biotinylated goat anti-chicken-IgG (1:400, Promega, Madison, USA) were used in 0.8% BSA for staining of samples. Extravidin Alexa 586 was used for visualization of fluorescence of antibody binding. Where indicated nuclei were counterstained with DAPI. Sections were analysed with confocal laser scanning microscopy (Zeiss, Oberkochen, Germany). For immunohistochemistry detection of goat antirabbit-IgG occured with extravidin–horseradish-peroxidase-complex (PRN1051, Amersham, 1:150, 60 min) and visualized by incubation with DAB/NiSO4-solution (15 min). For subsequent detection of goat anti-mouse-IgG streptavidin–alkaline-phosphate-complex (D0396, Dako, 1:400, 60 min) was used and visualized by Fuchsin substrate-chromagen system (Ko0624, Dako, 1 min). As depicted in Figure 1C, radioactivity over the heart reached a maximum of 36% of all cell associated radioactivity immediately after intracoronary cell infusion. Thereafter, radioactivity declined and reached a stable value (halftime corrected) of 18% after 40 min. Figure 1D illustrates that cell distribution was site specific, since 111In was found to be enriched over the heart. Similarly, threedimensional single photon emission computed tomography (SPECT) data showed a specific and uniform accumulation of labelled cells within the heart (data not shown). Haemodynamic analysis revealed no significant changes in ejection fraction and fractional shortening after HUVECs transplantation (Figure 2). In order to follow the cell fate for a longer period of time, HUVECs were transduced with the lentivirus pGJ3-CSCGW carrying the gene for EGFP.7 The endothelial character of EGFP-expressing HUVECs was fully maintained up to nine passages in culture as judged by the unchanged expression of PECAM-1, VE-Cadherin, E-Selectin, and VCAM-1 after induction with TNF-a (data not shown). Furthermore, EGFP expression did not stimulate PGE2 release into the medium, nor did it impair proliferation rate (data not shown). For the in vivo experiments, viral stocks were titrated to yield a transduction efficiency of maximal 30% EGFP positive HUVECs, corresponding statistically to one viral integration per transduced cell12 to ensure unchanged endothelial phenotype. When these cells were transplanted, EGFP positive cells were observed by epi-fluorescence from endocardium to epicardium 7 days after cell delivery (Figure 1E). Horizontal gene transfer from human endothelial cells to rat cardiomyocytes 537 Downloaded from by guest on July 6, 2015 Figure 1 Intracoronary transplantation and distribution of human umbilical vein endothelial cells in the rat heart. (A) Schematic representation showing the positions of extended arterial balloon (AB) in the aorta and venous balloon (VB) catheter in the right atrium of the rat heart. (B) Representative registration of aortic pressure during cell infusion. 1, inflation of VB; 2, inflation of AB; 3, cardiac arrest by esmolol and ACh; 4, injection of human umbilical vein endothelial cells; 5, deflation of AB and VB, injection of epinephrin and cardiopulmonary resuscitation; 6, normalization of aortic pressure. (C) Time course of radioactivity in the heart during the delivery of 111In-labelled human umbilical vein endothelial cells. (D) Whole body distribution of human umbilical vein endothelial cells 45 min after transplantation. Bar = 1 cm. (E) Representative overview over the left ventricle from endocardium to epicardium showing an even distribution of enhanced green fluorescent protein-expressing cells 7 days post-transplantation. Bar = 200 mm. positive cells using laser scanning microscopy and epifluorescence detection revealed mononucleated cells in investigated heart slices (7 mm), and mono- and binucleated EGFP-expressing cells in dispersed cell preparation from transplanted hearts (Figure 4A–C). Green fluorescent cells stained positive for muscle protein a-actinin, thereby confirming a cardiomyocytic phenotype (Figure 4D). Using the same calculation procedure as described above, the total 538 S. Burghoff et al. To exclude the possibility that cyclosporine might have altered the endothelial phenotype, HUVECs were cultured for 7 days (n = 3) using culture medium that contained the highest measured cyclosporine concentration in vivo [6 mg/ ml; 4 + 2 mg/ml (n = 3)]. After this period of time, HUVECs were still positive for PECAM-1 and negative for a-actinin and sm-actin (data not shown). Apoptosis of human umbilical vein endothelial cells and enhanced green fluorescent protein gene transfer to rat cardiomyocytes number of EGFP-expressing cardiomyocytes within the heart after 7 days was estimated to be 58% of all HUVECs trapped within the heart at 50 min after transplantation. To define the species affiliation of EGFP-expressing cells, 3 and 7 days after transplantation immunofluorescence revealed that green fluorescing cells were also positive for rat MCT-1 protein (Figure 4E), but did not stain for human mitochondrial proteins (Figure 4F), whereas the antibody is well suited to detect human cardiac cells (Figure 4H). In order to demonstrate EGFP transcripts in EGFP-expressing rat cardiomyocytes, single cell PCR on such cells was performed. To this end, cells were separated from a heart that had received EGFP-expressing HUVECs 6 days ago, isolated single EGFP positive cardiomyocytes, and performed reverse transcription followed by PCR. As shown in the representative Figure 4G, EGFP-expressing rat cardiomyocytes display a positive signal demonstrating the existance of EGFP-mRNA within rat cardiomyocytes, whereas control cardiomyocytes did not. Interestingly, we found dot like staining for human mitochondrial proteins ubiquitiously distributed and aligned to the cellular membrane of cardiomyocytes (Figure 4I), a phenomenon not observed in sham operated rats (inset Figure 4I). Expression of EGFP in rat cardiomyocytes was transient. Three weeks after transplantation of labelled HUVECs, no EGFP-expressing cells were detected within the heart using epi-fluorescence. Also, there was no longer a positive dot like staining of the anti-human mitochondria antibody after this period of time (data not shown). Discussion This study investigated the in vivo transdifferentiation potential of HUVECs. To this end, we transplanted 111In or EGFP-labelled HUVECs via the coronary arteries into rat hearts and found that 18% of the infused HUVECs were retained evenly distributed within the heart immediately Downloaded from by guest on July 6, 2015 Figure 2 Ejection fraction and fractional shortening of rat hearts after transplantation of enhanced green fluorescent protein-expressing human umbilical vein endothelial cells over 3 weeks. Rats were measured under isofluoran anaesthesia before operation (n = 8), after operation (n = 8) and at time points of sacrifice (n = 2 for days 1, 3, 7 and n = 1 for day 21). During operation either enhanced green fluorescent protein-expressing human umbilical vein endothelial cells (filled square) or medium (filled circle) were intracoronarely infused resulting in no significant functional changes. In order to study the mechanism of disappearance of HUVECs in vivo, hearts were excised 2 days after cell transplantation and analysed for apoptosis by immunohistochemistry. As shown in Figure 5A and B, only hearts that had received HUVECs contained caspase-3 activated cells. Figure 5C and D shows two representative apoptotic cells stained for active-caspase-3 (black) and human mitochondrial proteins (fuchsin-red). As can be seen cells are double positive and show an apoptotic morphology. Since apoptotic bodies have been reported to be capable of transferring genetic information to target cells13,14 we next prepared apoptotic bodies from EGFP-expressing HUVECs with DAPI and ethidium bromide-labelled DNA and evaluated in a first step their composition (Figure 6A–D). Isolated apoptotic bodies contained nucleic acids, but did not contain visible amounts of EGFP. PCR analysis confirmed that apoptotic bodies from EGFP-expressing HUVECs, but not from non-transduced HUVECs, carry the gene for EGFP (Figure 6E). To investigate whether apoptotic bodies can be taken up by cardiomyocytes studies with rat neonatal cardiomyocytes were performed. Coincubation of rat cardiomyocytes with apoptotic bodies derived from EGFP-expressing HUVECs for 4 days clearly revealed EGFP-expressing cells as shown using immunohistochemistry (Figure 6F and G). These cells are positive for rat MCT-1 (Figure 6H–K, epi-fluorescence) and myosin-heavy-chain but negative for human mitochondrial proteins (data not shown). Even after incubation of apoptotic bodies with DNAse, in order to exclude the possibility of EGFP plasmid transfer, rat MCT-1 positive cells were detected (data not shown). Gene transfer was a rather rare event: we found 8–10 EGFP positive cells per culture plate. It should be noted that despite optimized culture conditions only cardiomyocytes that incorporated the label lost their typical morphology and viability, whereas EGFP negative cardiomyocytes in the same culture plate appeared structurally unaltered. To exclude viral shuttle to cardiomyocytes, supernatant from HUVECs was added to HEK293T and HT1080 cells, which are easily transduced by viruses, and incubated for 5 days. FACS analysis did not reveal any green fluorescing cells (data not shown) indicating the absence of active virus in the supernatant of our HUVEC cultures. Horizontal gene transfer from human endothelial cells to rat cardiomyocytes 539 Downloaded from by guest on July 6, 2015 Figure 3 Localization of enhanced green fluorescent protein-expressing human umbilical vein endothelial cells immediately after intracoronary application as detected by epi-fluorescence. (A) Enhanced green fluorescent protein-expressing human umbilical vein endothelial cells (green) are shown in rat myocardium. (B) Visualization of capillary endothelium by staining against caveolin-1 (red, visualized with Alexa 586) demonstrates intravascular localization of the enhanced green fluorescent protein positive cells. (C ) Immunostaining with anti-human mitochondria antibody (red, visualized with Alexa 586) reveals the human origin of enhanced green fluorescent protein positive cells by overlay of enhanced green fluorescent protein and Alexa 586 fluorescence (yellow). (D) Rat specific anti-MCT-1 antibody (red, visualized with Alexa 586) detected only rat cardiomyocytes, whereas enhanced green fluorescent protein positive cells were negative. (E–G) The secondary antibody alone did not stain rat myocardium. Shown is an enhanced green fluorescent protein-expressing human umbilical vein endothelial cell in rat heart tissue where enhanced green fluorescent protein is green (E), secondary antibody is red (F) and (G) shows both images merged. Bars = 5 mm. after transplantation. After 2 days, HUVECs underwent apoptosis and after 3 days EGFP was exclusively associated with rat cardiomyocytes. Using species-specific antibodies and confocal microscopy, we excluded HUVECs transdifferentiation and cell fusion with cardiomyocytes as the underlying cause. We rather provide evidence that apoptosis and apoptotic bodies generated from transplanted HUVECs were responsible for directly transmitting nucleic acids coding for EGFP to rat cardiomyocytes. Numerous reports suggested that endothelial cells are able to transdifferentiate into different cell types such as smooth muscle cells and myocytes. Although transdifferentiation of human endothelial progenitor cells into cardiomyocytes appears to be an extremely rare event,15 these cells give rise to skeletal muscle cells when transplanted into mice.16 In contrast, in coculture experiments with neonatal rat cardiomyocytes, no convincing evidence of transdifferentiation into cardiomyocytes was obtained.17 Mature bovine aortic endothelial cells are also able to transdifferentiate into smooth muscle cells in vitro.18 Similarly, HUVECs can form smooth muscle-like cells after cultivation without fibroblast growth factor.2 In the present study, we found no evidence for transdifferentiation of HUVECs under in vivo conditions, despite an efficient transfer of EGFP from HUVECs to cardiomyocytes. Species-specific antibodies did not detect human protein within the target cells, 540 S. Burghoff et al. Downloaded from by guest on July 6, 2015 Figure 4 Fate of enhanced green fluorescent protein-expressing human umbilical vein endothelial cells 7 days after intracoronary application. Detection of enhanced green fluorescent protein positive (green) and of Alexa 586 fluorescing cells (red) in rat myocardium 7d after human umbilical vein endothelial cell transplantation (A–F, I ) and in human myocardium (G). (A–C ) Merged image of enhanced green fluorescent protein positive cells either on heart sections (A) or dispersed cells (B, C) and 4,6-diamidino-2-phenylindole staining of the nuclei (blue) show the typical shape of cardiomyocytes and their nuclei (arrows). (D) a-Actinin staining revealed a cross-striation like pattern in enhanced green fluorescent protein positive cells. (E, F ) enhanced green fluorescent protein positive cells are stained by rat specific anti-MCT-1 antibody (E), whereas there was no immunostaining against human mitochondrial proteins in enhanced green fluorescent protein positive cells (F ). (G) Single cell PCR using primer against enhanced green fluorescent protein on enhanced green fluorescent protein-expressing cardiomyocyte, enhanced green fluorescent protein negative cardiomyocyte, enhanced green fluorescent protein-expressing human umbilical vein endothelial cell and water as control. The amplified fragment contains 187 bp. (H ) Clear mitochondrial staining of a specimen from human heart, using anti-human mitochondria protein antibody. (I ) Dot like immunostaining (arrows) in the interstitium of the rat heart 7 days after human umbilical vein endothelial cell transplantation when stained with anti-human mitochondria antibody (I ). In the heart of sham operated rats, no specific staining with anti-human mitochondria antibody appears (inset Figure 1). Bar (A) = 50 mm, (B)–(I ) = 20 mm. Horizontal gene transfer from human endothelial cells to rat cardiomyocytes 541 rather all EGFP positive cardiomyocytes were of rat phenotype. It should be noted that HUVECs are much smaller than cardiomyocytes and transdifferentiation would have required considerable cell growth within the short period of 3 days in addition to being well integrated within the texture of the heart. Another possible explanation for EGFP-expressing rat cardiomyocytes is fusion of HUVECs with cardiomyocytes. In accordance with data in the literature,19,20 we found rat ventricular cardiomyocytes often to be binucleated and to some extent mononucleated. Similarly, we found mono- and binucleated cells to be green fluorescent both in heart slices and dispersed cardiomyocytes. In all cases however, we did not detect human mitochondrial proteins within green fluorescent cardiomyocytes, definitely ruling out the possibility of cell fusion. We always noted that EGFP-expression in cardiomyocytes exceeded that of HUVECs despite the fact that the single volume of cardiomyocytes is by far higher than that of HUVECs suggesting amplification of the EGFP message within the target cell. Indeed, we were able to detect EGFPspecific mRNA within green fluorescent rat cardiomyocytes. This clearly shows the transfer of nucleic acids coding for EGFP from HUVECs to rat cardiomyocytes in vivo. The transfer of proteins from one cell type to another might also be achieved by nanotubular connections between cells.21–23 In our study, we did not find microscopic evidence for nanotubes between HUVECs and cardiomyocytes neither in vitro nor in vivo. If nanotubes were at all involved in the transfer of label in our experiments, this would have required the transport of nucleic acids since we detected mRNA and observed signal amplification in the target cell. Yet, nanotubular transport of nucleic acids has not been reported so far. Blomer et al.24 observed a viral shuttle from lentivirally transduced cardiomyocytes to recipient fibroblasts. Viral shuttle took place when cocultivation was started immediately after viral transduction and infectiosity was lost when the virus was incubated at 378C for several hours before transduction and after passaging the cells. In contrast, our cells were passaged once and kept in the incubator for several days in addition to extensive washing steps and still showed a transduction rate which is unlikely to result from direct viral shuttle. In addition, we did not find evidence for infectiosity when supernatants from EGFP-expressing HUVECs were cultured with HEK293T or HT1080 cells. This makes it unlikely that viral particles Downloaded from by guest on July 6, 2015 Figure 5 Immunostaining of activated-caspase-3 and human mitochondrial proteins in rat hearts 2 days after intracoronary application of medium or human umbilical vein endothelial cells. (A) Control hearts perfused with cell free medium did not stain against activated-caspase-3. (B) Hearts which received human umbilical vein endothelial cells show activated-caspase-3 positive cells, indicating apoptosis. (arrows) (C, D) Two representative double-immunochemical images of hearts after human umbilical vein endothelial cell infusion with antibodies against human mitochondrial proteins (violet-red) and active-caspase-3 (black) providing evidence that transplanted human umbilical vein endothelial cells undergo apoptosis in vivo. The cell in (C ) shows a progressed state of apoptosis. Bars (A) and (B) = 100 mm, (C ) and (D) = 50 mm. 542 S. Burghoff et al. Downloaded from by guest on July 6, 2015 Figure 6 Genetic information for enhanced green fluorescent protein-expression is transferred to rat cardiomyocytes via apoptotic bodies. DNA from enhanced green fluorescent protein-expressing human umbilical vein endothelial cells and non-transduced human umbilical vein endothelial cells was labelled with ethidium bromide and apoptotic bodies were isolated. (A–D) show apoptotic bodies stained with 4,6-diamidino-2-phenylindole (A) and ethidium bromide (B) in brightfield (C ) and all images merged (D). Bar = 5 mm. (E) Gel electrophoresis of PCR products from DNA isolated from enhanced green fluorescent protein-expressing human umbilical vein endothelial cells, non-transduced human umbilical vein endothelial cells, and their respective apoptotic bodies (apt.B.) to detect enhanced green fluorescent protein. (F–G) Chromogenic detection of an enhanced green fluorescent protein-expressing neonatal rat cardiomyocyte after addition of apoptotic bodies derived from enhanced green fluorescent protein-expressing human umbilical vein endothelial cells 4 days ago (F ) and control of non-expressing cells (G). Bar = 50 mm. (H–K ) The same experiment as in (F ) showing neonatal rat cardiomyocytes-expressing enhanced green fluorescent protein (green, H ), staining positive for rat MCT-1 (red, I ) and both images merged (K ). Bar = 10 mm. adhered to HUVEC membranes and became incorporated into apoptotic bodies. Secondly, we did not achieve stable EGFP-expression in recipient cells, which argues against virus shuttle as well. The partial transformation of apoptotic cells into phosphatidylserine-containing apoptotic bodies has been reported earlier.25 In cell culture, apoptotic bodies were shown to increase the number and differentiation state of endothelial progenitor cells.9 In a study by Holmgren et al.,14 the cellular uptake of DNA from apoptotic bodies resulted in the transfer of the foreign genomic DNA to the nucleus of the phagocytosing cell and the expression of the marker gene at the protein and mRNA level. Even whole chromosomes or fragments thereof have been reported to be transferred by this pathway.13 Our data strongly suggest that the in vivo transfer of the EGFP label to cardiomyocytes was preceded by apoptosis of transplanted HUVECs. Not only contained HUVECs activatedcaspase-3 as an apoptotic signal 2 days after transplantation which were absent 1 day later, we also found that all EGFP-expressing cells were rat cardiomyocytes at that time point. Thus, our in vivo and in vitro data are in support of the view that apoptotic bodies derived from EGFP-expressing HUVECs transferred the genetic information for EGFP to cardiomyocytes. Since the expression of EGFP does not persist, we assume that the transferred DNA does not integrate into the chromosomes of the recipient cell and is subsequently removed. Should additionally mRNA be taken up, degradation of the transferred material can be expected. Also note that not all genes are equally transferred, since we did not find human mitochondrial proteins in rat cardiomyocytes. Whether this phenomenon is restricted to virus-inserted genes is presently unclear. EGFP has been widely used in the literature for stable cell-labelling in order to follow cell migration and/or phenotypic changes. Certainly, without additional control experiments, the sole association of the EGFP label with a cell of different phenotype provides no evidence for the Horizontal gene transfer from human endothelial cells to rat cardiomyocytes mechanism involved. Although in the past, transdifferentation and cell fusion were considered to be the main alternatives in studies with endothelial cells, the present study shows that horizontal gene transfer via apoptotic bodies is an important third alternative to be considered. Conflict of interest: none declared. Funding Forschungskommission of the Heinrich Heine University Duesseldorf (9772228 to S.B.), DFG (SFB612 to J.S.). References 11. Weibel ER. Stereological Methods Vol. 2: Theoretical Foundations. London: Academic Press; 2007. 12. Kustikova OS, Wahlers A, Kuhlcke K, Stahle B, Zander AR, Baum C et al. 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