Down-regulation of platelet endothelial cell adhesion molecule-1 results in thrombospondin-1 expression and concerted regulation of endothelial cell phenotype

Abstract

bEND.3 cells are polyoma middle T-transformed mouse brain endothelial cells that express very little or no thrombospondin-1, a natural inhibitor of angiogenesis, but express high levels of platelet endothelial cell adhesion molecule-1 (PECAM-1) that localizes to sites of cell-cell contact. Here, we have examined the role of PECAM-1 in regulation of bEND.3 cell proliferation, migration, morphogenesis, and hemangioma formation. We show that down-regulating PECAM-1 expression by antisense transfection of bEND. 3 cells has a dramatic effect on their morphology, proliferation, and morphogenesis on Matrigel. There is an optimal level for PECAM-1 expression such that high levels of PECAM-1 inhibit, whereas moderate levels of PECAM-1 stimulate, endothelial cell morphogenesis. The down-regulation of PECAM-1 in bEND.3 cells resulted in reexpression of endogenous thrombospondin-1 and its antiangiogenic receptor CD36. The expression of the vascular endothelial growth factor receptors flk-1 and flt-1, as well as integrins and metalloproteinases (which are involved in angiogenesis), were also affected. These observations are consistent with the changes observed in proliferation, migration, and adhesion characteristics of the antisense-transfected bEND.3 cells as well as with their lack of ability to form hemangiomas in mice. Thus, a reciprocal relationship exists between thrombospondin-1 and PECAM-1 expression, such that these two molecules appear to be constituents of a "switch" that regulates in concert many components of the angiogenic and differentiated phenotypes of endothelial cells.

Figure 1

Analysis of steady-state PECAM-1, TS1, and CD36 mRNA levels in antisense-transfected cells. Poly(A⁺) RNA was prepared from logarithmically growing cells, size fractionated on 1.2% agarose-formaldehyde gel, and transferred to ζ-probe membrane. The mRNAs for PECAM-1, TS1, and CD36 were detected by using specific cDNA probes for PECAM-1, TS1, and CD36. This blot was also probed with the cDNA for GAPDH to control for loading. The bEND are parental cells; the bEND pREP8 and bEND pMEP4 are vector-transfected controls; the bEND pREP8/BAMASPOP and bEND pMEP4/BAMASPOP are the population of transfected cells; the bEND pREP8/BAMASPOP2× are the population of transfected cells enriched twice for cells that lacked PECAM-1; and the bEND pREP8/BAMAS47, pMEP4/BAMAS20, -21, and -36 are clones of antisense-transfected cells.

Figure 2

Phase micrograph of bEND.3 parental cells transfected with (A) pREP8, (B) pREP8/BAMAS5, (C) pMEP4, and (D) pMEP4/BAMAS21.

Figure 3

Analysis of the steady-state flk-1 and flt-1 mRNA levels in antisense-transfected cells. The Northern blot described in Figure 2 was striped and probed with specific cDNAs for murine flk-1, flt-1, and GAPDH to control for loading.

Figure 4

Differentiation of antisense-transfected bEND.3 cells that express different levels of PECAM-1 in three-dimensional Matrigel cultures after 24 h. The left panels illustrate the phase micrograph and the right panels display the FACS analysis of the same cells: A, pMEP4 (control); B, pMEP4/BAMAS20; C, pMEP4/BAMAS21; and D, pMEP4/BAMAS36. These results are representative of more than a dozen similar experiments performed with vector or antisense PECAM-1-transfected clones of bEND.3 cells.

Figure 5

Analysis of the steady-state α_v, β₁, β₃, and β₅ integrins mRNA levels in antisense-transfected cells. Poly(A⁺) RNA was prepared from antisense-transfected bEND.3 cells as described in Figure 2 and analyzed by Northern blot. The blot was probed with specific cDNA probes for α_v, β₁, β₃, β₅, and GAPDH to control for loading.

Figure 6

Cell adhesion of antisense-transfected bEND.3 cells to vitronectin. Cells were prepared as described in MATERIALS AND METHODS and examined for their ability to adhere to different concentrations of vitronectin. These experiments were repeated at least twice with several clones of antisense-transfected cells with identical results.

Figure 7

Analysis of the steady-state c-jun, collagenase, and stromelysin-1 mRNA levels in antisense-transfected cells. The poly(A⁺) RNA prepared from antisense-transfected cells was analyzed by Northern blot. Blots were probed with specific cDNAs for c-jun, collagenase, stromelysin-1, and GAPDH to control for loading.