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exposed PS the fundamental trigger of angiogenesis



Schroit, Fidler, MD Anderson.



Apoptotic Cells Initiate Endothelial Cell Sprouting via Electrostatic Signaling


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http://cancerres.aacrjournals.org/cgi/content/full/65/24/11529



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Apoptotic Cells Initiate Endothelial Cell Sprouting via Electrostatic Signaling

Zhang Weihua, Rachel Tsan, Alan J. Schroit and Isaiah J. Fidler

Department of Cancer Biology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas





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.


Results

Nonproliferating endothelial cells sprout toward apoptotic cells. Culturing mildly trypsinized confluent rat glomerular, endothelial cell monolayers resulted in the generation of sprouts that extended from a small number of cells. These sprouts extended from endothelial cells at the margin of the monolayer to rounded and partially detached residual cells that remained in the denuded areas (Fig. 1A and B). To determine whether these "attracting" cells were indeed apoptotic, the cells were assessed for exposure of phosphatidylserine and DNA fragmentation by their ability to bind Annexin V (Fig. 1C-F) and TUNEL staining (Fig. 1G and H), respectively. Fluorescence microscopy showed that the attracting cells were apoptotic. Further examination of the cultures by scanning electron microscopy revealed that in contrast to the rough surface of the nonsprouting endothelial cells, attracting cells had a relatively smooth surface (Fig. 1I). To test whether the correlation between sprouts and apoptotic cells is significant, we counted the number of sprouting cells and the number of sprouting cells coupled with apoptotic cells in five randomly selected areas. The average number of sprouting cells was 37, and all had sprouts pointing to apoptotic cells. We did not find any endothelial cells with sprouts that did not point toward apoptotic cells. The distance between the attracting and sprouting cells was typically <200 µm.

To unequivocally determine whether apoptotic cells were indeed the initiating stimulus for sprout formation, single cells along the monolayer margin were triggered into apoptosis by microinjection of cytochrome c into the cytosol. Figure 2A and B shows that within 20 minutes after injection of cytochrome c (red fluorescence), a directional sprout pointing towards the cytochrome c–positive cell began to form from an opposing endothelial cell. Surprisingly, apoptotic cells were also required for the maintenance of existing sprouts. This can be seen from results showing that removal of the sprout-initiating apoptotic cell caused the sprout to retract back into the main cell body of the sprouting endothelial cell

Apoptosis was induced in the melanoma side of the plate by serum starvation for 48 hours. No endothelial cell sprouting was observed as long as the two compartments were separate. Removal of the barrier resulted in the formation of endothelial cell sprouts within 12 hours. Importantly, although some intermixing between cell populations was unavoidable, only apoptotic (caspase positive) melanoma cells attracted sprouts (Fig. 2E and F). We also cultured epithelial origin cells, MCF7 (breast cancer cells), and Du145 (prostate cancer cells) in the presence of apoptotic cells. We did not observe formation of sprouts.

Initiation of sprouting by electrostatic signaling. Several distinct mechanisms could be responsible for apoptosis-dependent endothelial cell sprouting. These include a cell expansion proliferation–dependent mechanism, activation of VEGF and/or EGFRs known to participate in angiogenesis, or specific chemotactic factors released by dying cells (13). To determine whether sprouting was dependent on endothelial cell proliferation, mixed viable/apoptotic endothelial cell cultures were stained with PCNA antibody. Figure 3A shows that sprout-producing endothelial cells were nonproliferating (PCNA negative) and closest to but not adjacent to the attracting cells.

To determine whether VEGF or EGF regulate sprouting, trypsinized endothelial cell monolayer was incubated in the presence of AEE788, a potent competitive inhibitor of both EGFR and VEGFR phosphorylation (14). Figure 3 shows that although both EGFR and VEGFR phosphorylation were effectively inhibited (Fig. 3B), sprout formation was not inhibited (Fig. 3C). Surprisingly, AEE788 decreased the fraction of PCNA-positive proliferating endothelial cells (Fig. 3C) and increased the density of sprouts (Fig. 3D) and the fraction of TUNEL-positive apoptotic cells (Fig. 3E and F).

To test whether concentration gradients of diffusible compounds might be responsible for directional sprouting, the formation concentration gradient was prevented by incubating trypsinized endothelial cells on a horizontal shaker. As shown in Fig. 4A, culturing cells with constant shaking did not prevent the formation of directional sprouting. To rule out that there was no invisible preexisting physical cell-cell contact between the apoptotic cell and sprouting endothelial cells, scratches were made on the surface of the culture dish between attracting apoptotic and sprouting endothelial cells. Scratches did not disturb the extension of the sprout (Fig. 4B and C), and similar to the results shown in Fig. 2, the sprouts retracted only in response to removal of the attracting apoptotic cell (Fig. 4D).

Because apoptosis is associated with an increase in net negative cell surface charge (15, 16), the possibility exists that the initiation of endothelial cell sprouts could be dependent on specific electrostatic charge interactions between the attracting apoptotic cell and the sprouting endothelial cells. To dissipate the polarity differential between the apoptotic and sprouting endothelial cells, trypsinized endothelial cell cultures were treated with cationized or anionized ferritin, respectively. Binding of ferritin to the cells was determined by staining with ferritin antibodies. Figure 4 shows that anionized ferritin bound exclusively to sprouting endothelial cells (Fig. 4E and F). Cationized ferritin, on the other hand, bound only to attracting apoptotic cells (Fig. 4G and H). Interestingly, the addition of cationized (Fig. 4I) but not anionized (Fig. 4J) ferritin to the cultures prevented sprout initiation. In contrast, addition of anionized but not cationized ferritin to endothelial cells bearing established sprouts caused the extending sprout to retract back into the main cell body within 90 to 100 minutes. Ferritin-induced sprout reversal, however, was ineffective once a sprout reached the attracting apoptotic cell (Fig. 4K and L).

To test whether negative surface charge is the primary stimulus by which endothelial cells are triggered to sprout, we attempted to initiate charge-dependent sprouting in an apoptotic cell–free system. For this purpose, Sepharose beads coated with anionized (negative charged) or cationized (positive charged) ferritin and negatively charged phosphatidylserine or neutral phosphatidylcholine vesicles were added to trypsinized endothelial cell cultures. Figure 5A shows that negatively charged but not positively charged (Fig. 5B) Sepharose beads initiated sprouting. Similarly, negatively charged (Fig. 5C) but not neutral (Fig. 5D) vesicles initiated sprouting. Collectively, these results suggest that the negatively charged cell surface expressed in apoptotic cells provides the initiating stimulus that induces the formation of endothelial cell sprouts.

Endothelial cell membrane hyperpolarization and cytoskeleton reorganization triggered by phosphatidylserine phospholipid vesicles. Because endothelial cell membrane is rich in ion channels (20) and is polarized in resting cells (18), we tested whether distant static negative charge can alter the membrane potential of endothelial cells. Dual wavelength imaging analysis (19) of endothelial cells labeled with membrane potential sensitive dye (di-8-ANEPPS) revealed the hyperpolarization of endothelial cell exposed to phosphatidylserine vesicles. This was detected within 15 minutes after exposure to the vesicles (Fig. 6A-D) and preceded the appearance of sprouts (Fig. 6E). No transmembrane potential changes were detected in the control cells.

Because alteration in membrane potential can trigger cytoskeleton reorganization (20), we tested whether endothelial cell sprouting was associated with reorganization of the cytoskeleton by staining with FITC-conjugated phalloidin. Reorganization of the cytoskeleton occurred during different stages of sprouting (Fig. 7). Nonsprouting endothelial cells exhibited a nondirectional distribution (Fig. 7A), whereas in sprouting endothelial cells, the cytoskeleton polarized (Fig. 7B), elongated in a parallel manner (Fig. 7C), and finally concentrated at the tip of the sprout (Fig. 7D). Because Ca2+ flux changes have been shown to regulate cytoskeleton reorganization (21), we tested whether calcium channel blockers can inhibit sprout formation. Figure 7E and F shows that pretreatment of endothelial cells with protopine completely inhibited phosphatidylserine vesicle–induced sprout formation.


Discussion

Angiogenesis in development, wound healing, and neoplasia is dependent on the initiation of a defined sequence of events that include endothelial cell migration toward the region of (re)vascularization, endothelial cell proliferation, and finally, reorganization into blood carrying tubules. Although the critical initiating event for the generation of new blood vessels has been attributed to the production of diffusible growth factors that stimulate endothelial cell migration and proliferation, recent data suggests that endogenous electric fields may also participate in this process (22). Indeed, alterations in electric fields are associated with wounding where they persist until repair is complete (23).

Cells undergoing apoptosis undergo dramatic intracellular and membrane alterations. In particular, the normally asymmetrical transmembrane distribution of membrane phospholipids reorganizes in such a manner that phosphatidylserine, normally localized exclusively in the cell's inner membrane leaflet, redistributes to the outer membrane leaflet. The expression of anionic phospholipid results in increasingly negative surface charge (24) commonly identified by the ability of the cells to bind Annexin V (25). The data presented here show that endothelial cells produce sprouts in direct response to cell surface electrostatic charge on apoptotic cells. Studies carried out in the presence of the tyrosine kinase inhibitor, AEE788, a dual inhibitor of EGFR and VEGFR phosphorylation, revealed that sprout formation was independent of activation of the VEGFR and EGFR. In addition, continuous agitation to prevent formation of solute concentration gradients failed to affect sprout formation. Taken together, these data indicate that sprout formation towards attracting apoptotic cells is growth factor/growth factor receptor independent.

All the attracting cells were Annexin V positive and bound cationized ferritin that, when added during the early stages of sprout formation, was inhibitory. This suggests that a net negative surface charge is required for the initiation of sprout formation. Additional evidence in support of the concept that cell sprouting can be initiated by electrostatic charge comes from experiments showing that negatively charged beads and phosphatidylserine-containing vesicles (Fig. 5) also initiated sprouting. In contrast to negatively charged apoptotic cells that bound cationized ferritin, sprouting endothelial cells bound anionized ferritin, suggesting that the sprouting cell has a strong positively charged surface. Although the source and nature of the positive surface charge on sprouting endothelial cells remains unclear, this finding is consistent with reports that endothelial cells derived from angiogenic macrovascular tissues elongate and migrate toward the cathode in a direct current electric field (26, 27).

Using dual-wavelength imaging, we found that endothelial cells respond to distant negative charges by altering membrane potential and becoming hyperpolarized, a phenomenon similar to what occurs in neuronal cells following exposure to electrical fields (19). The nature of endothelial cell membrane hyperpolarization seems unrelated to potassium and sodium channels since glyburide, charybdotoxin (potassium channel blocker), and amiloride (sodium channel blocker) were without effect (data not shown). However, preincubation endothelial cells with calcium channel blocker but no other channel blockers (data not shown) did inhibit sprout formation, indicating that calcium signaling is critical to sprout formation. Ion channel blockers did not reverse preformed sprouts (data not shown).

In conclusion, the data presented here provide evidence that apoptosis is not only important for marking the cell for elimination by phagocytes but also triggers a sequence of events important for angiogenesis and vascular remodeling.



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