Radiotherapy is commonly used for treatment of thoracic and chest wall tumors. Although radiotherapy is effective against the cancer, it is also known to induce delayed damage in surrounded normal tissue, including cardiac damage [
Preclinical studies have demonstrated the involvement of radiation-induced microvascular damage in the development of cardiac injury. Radiation leads to endothelial cell loss, which results in a decrease in microvascular density. Radiation also activates thrombotic and inflammatory reactions in the remaining vessels and induces the development of fibrosis in the myocardium [
The development of new blood vessels, originating from precursor cells that differentiate into endothelial cells, is called vasculogenesis. Vasculogenesis is one of two processes, in addition to angiogenesis, by which new blood vessels are formed and which has been shown to be essential in tissue repair and remodeling during acute and chronic ischemic tissue damage [
Several studies have confirmed the benefit of BM-derived EPCs to restore tissue vascularization after ischemia in the myocardium and other organs [
Hereditary hemorrhagic telangiectasia (HHT) is a vascular disorder with a mutation in the transforming growth factor-beta (TGFbeta) signaling pathway. Patients suffer from dilated blood vessels, characterized by telangiectasis and epistaxis [
In our study we investigate whether radiation-induced microvascular damage can be diminished by revascularization of BM-derived EPCs and whether endoglin plays a role in this process.
Eng+/− C57BL/6 mice were originally obtained from H. Arthur (Institute of Human Genetics, International Centre for Life, Newcastle upon Tyne, UK) and subsequently bred in the Netherlands Cancer Institute. Male Eng+/+ mice aged 8–12 weeks were randomly allocated (after genotyping by PCR) to receive 16 Gy or 0 Gy to the heart. Mice were housed in a temperature-controlled room with 12-hour-light-dark cycle. Standard mouse chow and water were provided ad libitum. Irradiation was performed with 250 kV X-rays, operating at 12 mA and filtered with 0.6 mm Copper. The dose rate was 0.94 Gy/min with a field size of 10.6 × 15 mm (including the whole heart and up to 30% lung volume) and the rest of the mouse was shielded with lead. Mice were immobilized without anesthetics, in a prone position in acrylic perspex jigs. Four cohorts of animals were included for analyses at 40 weeks after treatment: age-matched controls (sham irradiated with 0 Gy and no transplantations), 16 Gy irradiation alone, and 16 Gy followed by transplantation with bone marrow-derived endothelial like progenitor cells (BM-derived EPCs) from either Eng+/+ or Eng+/− mice (Figure
Schedule overview. Schematic representation of Eng+/+ or Eng+/− BM-derived EPCs transplantation at both 16 weeks and 28 weeks after 16 Gy heart irradiation.
Each cohort typically comprised 10 to 15 mice (
Donor male Eng+/+ mice or Eng+/− littermates aged 8–12 weeks were killed with an overdose of CO2. Femurs, tibias, and ilia were surgically dissected, and the adhering tissues were completely removed. Both ends of the bones were excised, and bone marrow cells (BMCs) were harvested by flushing with Endothelial Cell Growth Medium2 (EGM-2), supplemented with fetal calf serum (0.02 mL/mL), VEGF (0.5 ng/mL), basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor-1, ascorbic acid, heparin, hydrocortisone, and antibiotics, using a 25-gauge needle (Promocell, Huissen, The Netherlands). The BMCs were gently resuspended with a 25-gauge needle in EGM-2 medium before culturing on 1% gelatine coated petri dishes (Sigma G9391, bovine skin) at 5% CO2 at 37°C. Adherent cells were gently washed with PBS at day 3 to remove unattached cells and fresh EGM-2 was added. This procedure was repeated every 2 days until day 14, at which time the BM-derived EPCs were identified by typical endothelial cell (EC) morphology. Petri dishes were washed once with PBS and 1 mL trypsin/EDTA (Promocell) was added. The released cells were counted in a CASY Model TT system (Roche, Almere, The Netherlands) and then re suspended at 106 cells in 100
Before injection, cells were washed with PBS and incubated with a fluorescent cell viability marker, CellTracker Orange CMTMR (5-(and-6)-(((4-Chloromethyl)Benzoyl)Amino) Tetramethylrhodamine) (Invitrogen, Breda, The Netherlands) for 30 minutes at 37°C. Incubation was at a concentration of 10
Typical endothelial cell morphology was identified by cobblestone-like appearance (CCD-B/W Microscope system with a motorized Zeiss AxioObserver Z1 camera, Zeiss, Sliedrecht, The Netherlands).
Phenol red-free Matrigel (Becton & Dickinson, Franklin lakes, USA) was added to a prechilled 24-well plate. The Matrigel was then solidified by incubation at 37°C for 1 hour. The BM-derived EPCs (200.000 cells/well) were suspended in 500
Cells were incubated overnight at 4°C with 2.5
No differences in expression of specific endothelial markers were observed between BM-derived EPCs that originated from Eng+/− and Eng+/+.
At termination of the experiment, the heart was perfused via the aortic arch (retrograde), under lethal sodium pentobarbital anesthesia (18 mg per mouse, i.p.), with PBS (frozen sections) or PBS followed by 1% paraformaldehyde (paraffin sections). The heart was then quickly excised before freezing on dry ice or immersion in 1% paraformaldehyde.
Cross sections were cut at the level of the mid-horizontal plane of the heart from fixed paraffin-embedded tissues (4
An anti-CD31 antibody (1 : 50, Becton & Dickinson) was used to visualize cardiac vasculature. To determine functional changes in the microvasculature, a histochemical staining with Naphtol AS-MX/DMF and fast Blue BB salt was performed to detect endothelial cell alkaline phosphatase (ALP). Sections were also incubated with antibodies against von Willebrand Factor (vWF) (1 : 4000, Abcam, Cambridge, USA), as a marker of thrombotic changes. Within one time group all sections were processed identically, at the same time, with precisely the same incubation times for the primary and secondary antibody and DAB solution.
For quantification of microvascular changes, five random fields (40x objective) from transverse sections cut at the mid-horizontal plane of the heart were photographed with a CCD 2-Color Microscope system, including a Zeiss AxioCam color camera (AxiocamHRc, Zeiss, Göttingen, Germany). A computerized morphometry system (Leica Qwin V3) was used to quantify the microvascular density (MVD) of CD31 positive structures. Vessels beneath a size of 1.5 or above 200
Interstitial collagen was determined in the myocardium based on Sirius red staining. Photographs of the LV wall, excluding the septum, were taken using a 40x objective (Leica DFC320). Interstitial collagen was quantified in five randomly selected areas of the subendocardium and myocardium of the LV (40x objective) and results were expressed as percentage tissue positive for Sirius red relative to myocardial area. Morphometric parameters were analyzed using a computerized morphometry system (Leica Qwin V3, Leica, Rijswijk, The Netherlands).
Data are expressed as mean ± SEM and groups were compared using nonparametric Mann-Whitney exact
After 10–14 days in culture BM-derived EPCs from both Eng+/+ and Eng+/− mice exhibited cobblestone morphology (Figure
EPC characteristics by EPC culture assay and immunohistochemistry. (a) Morphological features of confluent EPCs; EPCs after 14 days in culture demonstrating distinct flat, spread out, cobblestone morphology. (b) EPCs plated on Matrigel; EPCs originated from Eng+/+ mice (c) and Eng+/− mice show initial capillary tube formation after 6 hours in Matrigel. (d) DiI-acLDL uptake in red and (e) binding of UEA-1 in EPCs (green) were analyzed by fluorescent microscope. Nearly all adherent cells bound UEA and internalized Ac-DiI-LDL. Original magnification 63. (f) Immunohistochemical staining with the endothelial marker CD31 (in red). Staining was analyzed by fluorescent microscopy. Original magnification ×40.
The BM-derived EPCs were then further examined for expression of endothelial cell markers using immunohistochemistry. Both Eng+/+ and Eng+/− BM-derived EPCs expressed endothelial markers CD31 and were bound to UEA-1 and took up Dil-ac-LDL (Figures
Analysis of frozen sections from mice transplanted with CellTracker Orange-labeled BM-EPCs revealed only a few BM-EPCs in the myocardium (data not shown). There were no differences between BM-EPCs transplantation of cells originated from either Eng+/− or Eng+/+ mice.
Microvascular density (MVD) decreased significantly after 16 Gy irradiation alone and the decline was not restored by treatment with BM-derived EPCs from either Eng+/+ mice or Eng+/− mice (Figure
Microvascular alterations after irradiation alone or treatment with BM-derived EPCs. (a) MVD per unit area expressed as number of microvessels per mm2. (b) ALP positive tissue area as % of total tissue. (c) vWF positive tissue area as % of total tissue
Cardiac fibrosis, established from the extent of collagen staining, was significantly increased after irradiation (Figure
Fibrotic changes after irradiation alone or treatment with BM-derived EPCs. Collagen positive tissue area as % of total tissue for animals treated with irradiation alone or with BM-derived EPCs.
In this study we investigated whether endothelial progenitor cells, derived from bone marrow of Eng+/+ or Eng+/− mice, can contribute to repair of radiation-induced microvascular injury. Our results indicate that microvascular damage was not improved by this approach but the development of radiation-induced fibrosis was inhibited by transplantation of BM-derived EPCs.
We demonstrated in a previous study that irradiation leads to microvascular damage, which progresses continuously over time, although cardiac function remained within normal ranges until sudden death of the mice. We hypothesized that compensatory mechanisms operate to maintain cardiac function until the extent of underlying damage overwhelmed these mechanisms [
Clinical studies have recently announced the safety of BM-EPCs transplantation, but their efficacy varies widely. This might be the result of uncertainties regarding the best method of administration, timing of administration, or cell type utilized. In our preclinical studies, we might not have chosen the ideal timing of administration of BM-derived EPCs to restore radiation-induced microvascular damage, or the number of cells may have been insufficient. In our study we cultured the mononuclear fraction in a manner that usually results in EPCs [
We conclude that radiation-induced endothelial cell damage and cell loss was not restored by transplantation of BM-derived EPCs. However, transplantation did reduce the amount of radiation-induced cardiac fibrosis. Endoglin deficiency in transplanted cells did not impair their ability to reduce fibrosis.
The authors declare that there is no conflict of interests.
The authors would like to thank Dr. Jack Cleutjens, Department of Pathology, University of Maastricht, for helping with the Leica Qwin morphometry system. Moreover, we would like to thank Professor Dr. Christine L. Mummery, Department of Anatomy and Embryology, University of Leiden, and Dr. Marion Scharpfenecker, Department of Biological Stress Response, the Netherlands Cancer Institute, for their helpful discussions.