Apoptotic cells initiate endothelial cell sprouting via electrostatic signaling

Abstract

Angiogenesis, the development of new blood vessels from preexisting vessels, is crucial to tissue growth, repair, and maintenance. This process begins with the formation of endothelial cell sprouts followed by the proliferation and migration of neighboring endothelial cells along the preformed extensions. The initiating event and mechanism of sprouting is not known. We show that the phenotypic expression of negatively charged membrane surface in apoptotic cells initiates the formation of directional endothelial cell sprouts that extend toward the dying cells by a mechanism that involves endothelial cell membrane hyperpolarization and cytoskeleton reorganization but is independent of diffusible molecules.

Fig. 1

Apoptotic cells initiate EC sprouting. (A, B) EC were cultured to 100% confluency and then partially trypsinized to produce denuded areas. The inner periphery contained partially detached cells, where EC sprouted (arrows) toward cells with typical apoptotic morphology (arrowheads). (C-F) The sprouts (arrow) extending toward the attracting cell (arrowhead) that was positive for annexin V binding (white arrows crossing images, green color). (G, H) TUNEL staining revealed that some attracting cells (arrowhead) are TUNEL-positive (green color). (I) Scanning electronic microscopy shows that the surfaces of the attracting cells (arrowheads) are smoother than the relatively rough surfaces of the neighbor cells (double-head arrow). Bar = 50 μM.

Fig. 2

Induction and removal of apoptotic attracting cells. (A) Cytc microinjection into viable EC (arrowhead) together with red fluorescent dye (arrowhead in the inset). (B) Twenty min after microinjection of cytc, an EC formed a sprout toward the injected cell (arrow). (C, D) Removal of the attracting apoptotic cell (arrowhead) resulted in retraction of the extending sprout within 15 min (arrows). (E) Confluent EC were co-cultured with apoptotic GFP-labeled K-1735 melanoma cells. An EC sprout (insert, enlarged view of the sprout) directed toward the apoptotic K-1735 melanoma cell (arrowhead, dashed line). (F) The apoptotic status of the K-1735 cell (yellow color) was confirmed by caspase 3 staining with Texas Red conjugated secondary antibody and merged with the green GPF image of K-1735 cells. Bar = 50 μM.

Fig. 3

Non-proliferating EC sprout independent VEGF and EGF pathways. (A) PCNA staining (arrowheads, brown color) of control endothelial cells with sprouts showing that the cells that sprout are PCNA-negative (arrow, blue color stained with Hematoxylin) and nearest to, but not adjacent to, the attracting cells. (B) Western blot analysis showing inhibition of VEGFR (pVEGFR) and EGFR (pEGFR) phosphorylation with AEE788. Total VEGFR (tVEGFR) and EGFR (tVEGFR) served as controls. (C) PCNA staining of endothelial cells treated with AEE788 showing more sprouts (arrowheads) and less proliferating cells as compared with control (A). (D) Statistical analysis of the number of sprouts in control and AEE788-treated EC (P<0.001). (E) Statistical analysis of the number of proliferating cells and apoptotic cells (identified with TUNEL assay) in control and AEE788-treated EC (P<0.001). (F) Co-localization of sprouts (arrows) with TUNEL-positive cells (arrowheads, green color) in AEE788-treated EC. Bar = 50 μM.

Fig. 4

Absence of mediation of the signaling between an attracting and sprouting cell by diffusible molecules and electrostatic characterization of apoptotic and sprouting cells. (A) Continuous horizontal shaking did not prevent sprout formation (arrow) toward its attracting cell (arrowhead). (B, C) Scratching the surface of the culture dish between an attracting cell (arrowheads) and sprouting cell (arrows) did not disrupt the pre-existing sprout (C, 30 min after B). (D) Removal of the attracting cell led to shrinkage of the sprout (15 min after C). The sprouting EC bound anionized ferritin (E, F, arrow), whereas the apoptotic cell bound cationized ferritin (G, H, arrow). (I) At the early stage of the process, cationized ferritin completely blocked the formation of sprouts, and (J) anionized ferritin failed to block the formation of sprouts (arrow). (K, L) The addition of anionized ferritin regressed the sprout before it reached the apoptotic cell (arrows; 100 min between K and I) but did not regress an established sprout (arrows with diamond head). Bar = 50 μM.

Fig. 5

Induction of EC sprouting by negatively-charged Sepharose beads and negatively-charged phospholipid vesicles. (A) A single negatively-charged Sepharose bead (asterisk) located in the vicinity of a denuded area of partially trypsinized EC induced sprout formation (arrows). (B) Positively-charged beads failed to initiate sprouting. (C) Negatively-charged PS vesicles (asterisk) induced formation of a sprout from the nearby EC (arrow), and (D) neutral vesicles did not. Bar = 50 μM.

Fig. 6

PS vesicles trigger EC membrane hyperpolarization prior to sprout formation. EC exposed to PS (asterisk) (A, C, and D) had hyperpolarized membranes as indicated by the increased red emission intensity (arrows) within 15 min after addition of the vesicles (B, D, arrows, note that the fluorescence shifted from green to yellow). Sprout formation was detected 30 to 40 min after exposure to the vesicles (A, C and E, arrows). Control EC did not show significant morphological changes during a 15-min monitoring (F, H), and their corresponding membrane potentials remained unchanged (G, I). Bar=20μm.

Fig. 7

Cytoskeleton re-organization and involvement of calcium signaling during sprouting. The cytoskeleton of non-sprouting ECs is non-directional (A), whereas the cytoskeleton of sprouting cells is polarized (B), extended in a parallel manner (C) at the early stage, and concentrated at the tip of sprout at the late stage (D). Control ECs formed sprouts (arrows) in response to PS vesicles (asterisk) (E), while the pre-incubation of EC with the calcium channel blocker, protopine, inhibited sprout formation (F). Bar=20μm.