Expression of GLc3Man9GlcNac2-PP-Dol is a prerequisite for capillary endothelial cell proliferation

Protein N-glycosylation has been proposed to be intimately involved in the migration, proliferation and differentiation of endothelial cells. Using a synchronized, non-transformed capillary endothelial cell line from bovine adrenal medulla as a model, and the N-glycosylation inhibitor, tunicamycin, we have elucidated the molecular basis of the dolichol pathway in the angiogenic process. The synchronized culture required approximately 68 hrs. to complete one cell cycle, cells spending nearly 36 hrs. in G1 phase, 8 hrs. in S phase and 24 hrs. in G2 + M phase when maintained in 2% fetal bovine serum (heat-inactivated). The cell cycle however, was shortened due to a reduction of the G1 phase by 12-16 hrs. when the serum concentration was increased to 10%, or when beta FGF (1 or 10 nanogram) was added into the culture media containing 2% serum. Light microscopy and scanning electron microscopy both supported these proliferative responses. Serum concentration below 2% arrested cell proliferation and induced capillary lumen-like structure formation with 48 hrs. Expression of the blood clotting antigen factor VIII:C (a M-r 270, 000 dalton N-linked glycoprotein and a marker of our endothelial cells) preceded the endothelial cell proliferation and established a temporal relationship. Tunicamycin, an inhibitor of Glc(3)Man(9)GlcNAc(2)-PP-Dol biosynthesis, a prerequisite for N-linked protein glycosylation in the ER- inhibited the cell growth and proliferation in a time and dose-dependent manner with a concomitant accumulation of immunopositive, non-glycosylated factor Vm:C in the conditioned media. Tunicamycin also caused surface blebbing and induction of programmed cell death (PCD)(apoptosis) within 32 hrs. Absence of cellular growth and proliferation, surface blebbing and the induction of PCD in the presence of tunicamycin, provided conclusive evidence that normal expression of Glc(3)Man(9)GlcNAc(2)-PP-Dol is an essential event for capillary proliferation during angiogenesis.