Figure 4.

Effects of LOXL4 on angiogenesis. (a) LOXL4 expression and microvessel density in DC, GG and KCOT stroma. Immunohistochemical analysis of LOXL4 (a1, a2, a4, a5). Few LOXL4-positive fibroblasts were observed in GG (a1, a2) or DC (a4, a5). (a7, a8) Most of the KCOT stromal fibroblasts were strongly LOXL4-positive. (a3, a6) Few CD105-positive microvessels were observed adjacent to the epithelium of normal GG (a3) and dentigerous cysts (a6). A high density of strongly CD105-positive microvessels was found in the stroma immediately beneath the epithelium lining KCOT (a9). The intensity of LOXL4 protein expression was consistent with the density of CD105-positive microvessels in GG (a3), DC (a6) and KCOT (a9). Scale bar=100 µm. (b) Effects of LOXL4 on the proliferation and migration of HUVECs. (b1, b2) There was a significant increase in the expression of LOXL4 both in intracellular and extracellular of HUVECs transfected with LOXL4 than transfected with empty plasmid. (b3) Transient LOXL4 transfection increased cell growth, cell proliferation was measured with Cell Counting Kit-8 at 6, 12, 24, 72 and 96 h. (b4) HUVECs migration ability was assessed by scratching assay with or without LOXL4 transfection. Representative images are shown (magnification: ×100). (b5) HUVECs migration ability was also tested by Boyden Chamber assay with or without LOXL4 transfection. Representative images are shown (magnification: ×200). Each treatment was triplicate and six fields were randomly chosen from each well for counting mean number of migrated cells. Data are presented as mean±s.e.m., *P<0.05, **P<0.01, Student's t-test. DC, dentigerous cysts; ELISA, enzyme-linked immunosorbent assay; GG, gingival primary stromal fibroblast cells; HUVEC, human umbilical vein endothelial cell; KCOT, keratocystic odontogenic tumors; LOXL4, lysyl oxidase-like 4; OD, optical density; Ctl, control.