Published online Dec 12, 2015. doi: 10.5528/wjtm.v4.i3.113
Peer-review started: June 29, 2015
First decision: August 25, 2015
Revised: October 16, 2015
Accepted: November 10, 2015
Article in press: November 11, 2015
Published online: December 12, 2015
AIM: To verify in vivo relevance of the categorization of human vascular endothelial cells (VECs) into type-I (pro-proliferative) and type-II (anti-proliferative).
METHODS: Endothelial layers of murine femoral arteries were removed by wire injury (WI) operation, a common technique to induce arteriostenosis. Type-I and type-II VECs produced from human induced pluripotent stem cells (iPSCs), whose characters were previously determined by their effects on the proliferation of vascular smooth muscle cells in in vitro co-culture experiments, were mixed with Matrigel® Matrix. The mixtures were injected into subcutaneous spaces around WI-operated femoral arteries for the transplanted human iPSC-derived VECs (iPSdECs) to take a route to the luminal surface via vasa vasorum, a nutrient microvessel for larger arteries. Histologies of the femoral arteries were examined over time. The presence of human iPSdECs was checked by immunostaining studies using an antibody that specifically recognizes human VECs. Degrees of stenosis of the femoral arteries were calculated after three weeks. To determine the optimal experimental condition, xenotransplantation experiments were performed under various conditions using immunocompromised mice as well as immunocompetent mice with or without administration of immunosuppressants.
RESULTS: Because immunocompromised mice showed unexpected resistance to WI-induced arteriostenosis, we performed xenotransplantation experiments using immunocompetent mice along with immunosuppressant administrations. After one week, luminal surfaces of the WI-operated arteries were completely covered by human iPSdECs, showing the efficacy of our novel transplantation technique. After three weeks, type-I-iPSdECs-transplanted arteries underwent total stenosis, while type-II-iPSdECs-transplanted arteries remained intact. However, untransplanted arteries of immunosuppressant-treated mice also remained intact by unknown reasons. We found that transplanted human VECs had already been replaced by murine endothelial cells by this time, indicating that a transient existence of human type-II-iPSdECs on arterial luminal surfaces can sufficiently prevent the development of stenosis. Thus, we re-performed xenotransplantation experiments using immunocompetent mice without administrating immunosuppressants and found that arteriostenosis was accelerated or prevented by transplantation of type-I or type-II iPSdECs, respectively. Similar results were obtained from the experiments using human embryonic stem cell-derived VECs at early passages (i.e., type-II) and late passages (i.e., type-I).
CONCLUSION: Pro- and anti-stenosis capacities of type-I and type-II human iPSdECs were verified, respectively, promising a therapeutic application of allogenic iPSdECs.
Core tip: We previously reported that human vascular endothelial cells (VECs) were classified into two categories by their in vitro effects on the proliferation of vascular smooth muscle cells: Pro-proliferative VECs (type-I) and anti-proliferative VECs (type-II). Applying our new technique to transplant human VECs onto the luminal surface of endothelial layer-removed murine arteries, the in vivo relevance of the concept for VEC categorization was validated. Transplantation of pro-proliferative VECs (type-I) resulted in total stenosis while that of anti-proliferative VECs (type-II) completely blocked the development of arteriostenosis. Thus, pro-stenosis (type-I) and anti-stenotic (type-II) capacities were verified in vivo.