Expression Of Endothelial And Smooth Muscle Cells Markers During The Development Of Mouse Embryo

Background and objectiveVasculoproliferative disorders such as atherosclerosis, allograft vasculopathy, in-stent restenosis and restenosis after percutaneous balloon angioplasty, and so on, are common clinical pathogenesis. Investigation of origin of vascular smooth muscle cells (VSMCs) will be of help to the treatment of proliferative vessel disease. Previously, it was thought that embryonic VSMCs arise from at least two different lineages: one is the neuronal crest (Ectomesenchymal VSMCs), the other is the mesoderm (Mesenchymal VSMCs). Recently it is considered that VSMCs maybe arise from adult bone marrow-derived stem VSMCs and progenitor cells, circulating monocytes/macrophages, skeletal muscle adult stem cells, endothelial cell (ECs), etc. Yamashita J et al. confirmed that fetal liver kinase-1 (Flk-1, i.e. vascular endothelial growth factor receptor-2) positive cells derived from embryonic stem cells could differentiate into vascular ECs under the effect of vascular endothelial growth factor (VEGF), and into VSMCs under the regulation of platelet-derived growth factor-BB in vitro. So Flk-1-positive cells is considered as the common progenitor of vascular ECs and VSMCs, but this theory has no in vivo evidence. In this study, the relationship and expression of the endothelial markers, Flk-1, platelet endothelial cell adhesion molecule-1(PECAM-1/CD31), factorⅧ-related antigen, and VSMCs marker, Smooth Muscleα-actin(SMα-actin) during the early stage of angiogenesis were investigated by using immunohistochemical methods, in order to provide a new in vivo experimental evidence for origin ofVSMCs.Flk-1 is mainly located in vascular ECs, hematopoietic cells and its progenitor cells. It was reported that Flk-1 is first detected at mouse embryo 6.5-days post coition (dpc), it plays an important role in ECs and hematopoietic cells development, and it is abundantly detected in angiogenesis physiological processes or tumour pathological processes. But expression of Flk-1 in the mouse preimplantation embryos has never been reported. Therefore, expression and function of Flk-1 in the mouse preimplantation embryos development were investigated in this study. Methods1. Acquisition of 8.5 to 18.5-dpc mouse embryos: Females were housed overnight with males, and in the next morning females having pessary were pregnant, this day was called as 0.5-dpc. On 8.5 to 18.5-dpc, pregnant females were killed and mouse embryos were obtained.2. Acquisition of mouse preimplantation embryos: Females were injected with 10 U of pregnant mares'serum gonadotropin, followed by an injection of 5 U of human chorionic gonadotrop after 44~48 h, and then females were housed overnight with males. Embryos were obtained at the following stages: 1-cell stage (12-hours post coition, 12-hpc), 2-cell stage (36-hpc), 4-cell stage (48-hpc), 8-cell stage (60-hpc), blastula stage (84-hpc). 1 to 8-cell embryos were flushed from the oviducts of superovulated females, and morula and blastula were flushed from the uteri of females.3. 8.5 to 18.5-dpc embryos were fixed in 4% paraformaldehyde (PFA) for 3 h at room temperature, paraffin-embedded after routine dehydration methods, made into 5μm cross sections. Dorsal aorta sections in thoracic and abdominal cavity were used for section staining.4. Embryos were briefly washed in phosphatebuffered saline containing 3 mg/ml polyvinylpyrrolidone (PBS/PVP) and fixed in 2.5% PFA for 15 min at room temperature. Fixed embryos were stored at 4°C in PBS/PVP.5. Morphological changes of the dorsal aorta of 8.5 to 18.5-dpc mouse embryos were observed in sections stained with hematoxylin and eosin.6. Relationship between the expression of ECs markers Flk-1, CD31, factorⅧ-related antigen and VSMCs marker SMα-actin was investigated using immunohistochemical methods.7. 20 embryos from the preimplantation different stages were collected to Rnase-free eppendorf. Total mRNA was isolated by using mRNA Capture Kit. The captured mRNA could now be used directly for reverse transcription polymerase chain reaction (RT-PCR) to observe temporal pattern of Flk-1 mRNA expression.8. The protein expression and localization of Flk-1 in the preimplantation different stages embryos was detected through microdrops by using whole-mount immunocytochemistry on a leica laser-scanning confocal microscopy.9. To determine whether macromolecule antibody can breakthrough zona pellucida into embryos, we cultured 8-cell embryos in culture medium with 5 mg/L Flk-1 neutralization antibody for 12 h, and then added rhodamine conjugated affinipure goat anti-rat IgG into the medium.10. 8-cell embryos were cultured in normal medium or medium containing 5 mg/L Flk-1 neutralization antibody or 5 mg/L Flk-1 unneutralization antibody, respectively. After embryos were incubated for 36 h, blastula formation rate was counted and tested by X2 analysis, and deemed significantly diversity when P﹤0.05.11. The protein expression and localization of VEGF in the 8-cell embryos was detected through microdrops by using whole-mount immunocytochemistry on a leica laser-scanning confocal microscopy. Results1. Left and right dorsal aorta could be found in thoracic and abdominal cavity of 8.5-dpc mouse embryos; there was a single dorsal aorta in abdominal cavity, left and right dorsal aorta in thoracic cavity of 9.5-dpc mouse embryos. Flk-1 and CD31 were expressed in monolayer cells of the dorsal aorta of 9.5-dpc mouse embryos, but SMα-actin andⅧ-related antigen were not expressed.2. Left and right dorsal aorta could be found at atrium level in thoracic cavity of 10.5-dpc mouse embryos; there was a single dorsal aorta at ventricle level in thoracic cavity and in abdominal cavity. Flk-1, CD31, SMα-actin andⅧ-related antigen were expressed in monolayer cells of the dorsal aorta of 10.5-dpc mouse embryos.3. The dorsal aorta wall consisted of multilayer cells at 11.5 dpc. Endothecium cells expressed Flk-1 instead of SMα-actin; meanwhile,exothecium cells expressed SMα-actin instead of Flk-1. There was no clear boundary between the dorsal aorta wall and the surrounding mesenchymal cells. Some scattered SMα-actin-positive cells could be seen around the dorsal aorta.4. After 11.5 dpc, VSMCs of the dorsal aorta grew in number and transformed from irregular cells into spindle-shaped cells. Flk-1 remained positive in ECs, and SMα-actin in VSMCs. There was a clear boundary between the dorsal aorta wall and the surrounding mesenchymal tissue. No SMα-actin-positive cells could be seen surrounding the dorsal aorta.5. Flk-1 transcript was most abundant in the 8-cell embryos. After 28 cycles of RT-PCR amplification, Flk-1 transcript was only observed in the 8-cell embryos. With further amplification (32 cycles), Flk-1 expression could be detected in the 4-cell embryos. Flk-1 transcript was not detected in 1-cell embryos, 2-cell embryos, morula and blastula.6. There was green fluorescence on the cell membrane of 4-cell embryos, but no fluorescence in the cytoplasm and nucleus. Flk-1 protein expression was restricted to the cell-cell borders and especially embryos'outer margin. There was stronger green fluorescence on the cell membrane of 8-cell embryos than 4-cell embryos. Flk-1 protein expression was restricted to the same position as 4-cell embryos. 1-cell embryos, 2-cell embryos, morula and blastula did not express Flk-1.7. After incubated in the medium containing 5 mg/L Flk-1 neutralization antibody for 12 h, 8-cell embryos were took out, incubated in the second antibody. As a result, green fluorescence was found inside the zona pellucida showing that macromolecule antibody could breakthrough zona pellucida into embryos.8. After 8-cell embryos were incubated in the medium containing 5 mg/L Flk-1 neutralization antibody for 36 h, 36 h blastula formation rate of three groups: control group 80.95%(17/21), antibody control group 81.81%(18/22), experiment group 40.00%(10/25). There was significant diversity between experiment group and blank control group, and no significant diversity between blank control group and antibody control group. At the same time, we found that 90% of embryos could develop into blastula after embryos were incubated in medium containing 5 mg/L Flk-1 neutralization antibody for 48 h.9. VEGF was expressed in 8-cell embryos. Red fluorescence was restricted to the cytoplasm, not in the cell membrane and nucleus.Conclusion1. The earliest derivation of the dorsal aorta VSMCs of the mouse embryo can be from Flk-1, CD31-positive cells; thereafter, VSMCs can derive from recruitment and differentiation of the surrounding mesenchymal cells.2. The expression of Flk-1 varies in different stages of the preimplantation embryos, the function of which may be involved in the development of preimplantation embryos.