Therapeutic benefit of bone marrow-derived endothelial progenitor cell transplantation after experimental aneurysm embolization with coil in rats

Abstract

Aneurysm embolization with coil is now widely used clinically. However, the recurrence of aneurysms after embolization has always plagued neurosurgeons because the endothelial layer of the aneurysm neck loses its integrity after being embolized by coil. Bone marrow-derived endothelial progenitor cells (BM-EPCs) could be incorporated into injured endothelium and differentiate into mature endothelial cells during vascular repairing processes. The aim of our study is to explore the effects of BM-EPCs on aneurysm repairing and remodeling in a rat embolization model of abdominal aortic aneurysm. BM-EPC proliferation, migration and tube formation were not affected by super-paramagnetic iron oxide nanoparticle (SPIO) labeling compared to the controls (p>0.05). The number of SPIO-labeled cells greatly increased in EPC transplanted rats compared to that of phosphate buffered saline treated rats. SPIO-labeled EPC (SPIO-EPC) are mainly located in the aneurysm neck and surrounded by fibrous tissue. A histology study showed that the aneurysm orifice was closed with neointima and the aneurysm was filled with newly formed fibrous tissue. The SPIO-EPC accumulated in the aneurysm neck, which accelerated focal fibrous tissue remodeling, suggesting that BM-EPCs play a crucial role in repairing and remodeling the aneurysm neck orifice.

Figure 1. Embolization aneurysm specimen handling process.

(a) specimens obtained after cell transplantation for about 6 weeks (b) tissues were sectioned using an isomet low speed saw at 1000 µm intervals in a coronal orientation after being dehydrated and embedded (c), the tissue slices contained a large amount of coils under the microscope (d), the coils were extracted carefully by micro-tweezers under the microscope (e), after coil extraction and re-embedding, the tissue sections were processed into 5 mm slices by a microtome.

Figure 2. BM-EPC culture and identification.

A. Photomicrographs showing that after 5 days of culturing, BM-EPC isolated from rat bone marrow, displayed spindle-like attached cells and formed colonies (a), Bar = 140 µm. Higher magnification showed that these cells performed endothelial cell morphology (b), Bar = 140 µm. B. Putative BM-EPCs were stem cell marker CD34⁺ (red color) and were able to take in Ulex europeus agglutinin-5 (green color. c). They also were DiI-acLDL⁺(red color) and fluorescein isothiocyanate-Ulex europeus agglutinin-1⁺(green color), which were (d) co-localized in >95% cells, Bar = 100 µm.

Figure 3. BM-EPC SPIO labeling.

Photomicrographs showing BM-EPCs labeled with super-paramagnetic iron oxide nanoparticles (SPIO) in vitro pre-incubation (a), post-incubation (b). Bar = 140 µm. Prussian blue staining showing that the cell cytoplasm contained a large number of SPIO (blue color) and the nucleus displayed pink after nuclear fast red staining. Bar graph showing quantification of cell viability following BM-EPC incubation with various SPIO concentrations of 5 µg/ml, 10 µg/ml, 15 µg/ml, 20 µg/ml, 40 µg/ml.

Figure 4. Animal AAA model.

Rat abdominal aortic aneurysm embolization model and MRA: The arrow in the figure (a) shows the aneurysm without embolization and the embolism aneurysm is demonstrated by the arrow in the figures (b) and (d).

Figure 5. BM-EPCs involved in aneuryam repairing.

HE staining showing that the tissue repairation of aneurysm neck in EPC transplantation group (b) is better than in HUVEC transplantation group (c) and PBS control group (a). Prussian blue staining showing SPIO-labeled cells located in the aneurysm neck of all rats in the EPC transplantation group (e), in contrast to the HUVEC transplantation group (f) and control group (d). Bar = 100 µm.