Endothelial cell migration on Vn and uPA/PAI-1 co-localization are affected by a uPAR^-/- deficiency: (A) Absence of uPAR expression decreased EC migration on Vn but not on collagen. Quiescent WT and uPAR^-/- ECs were plated to confluency on Vn- and collagen-coated 6-well dishes and a scratch induced. Migration was induced by the presence of VEGF (10 ng/ml) and images of the scratch area were acquired immediately after the scratch and after 24 hr of incubation at 37°; C/6.5% CO₂. The number of cells that had migrated in the scratch area was counted and graphed as percent of migrated WT cells. The graph represents the mean ± SEM of three independent assays each performed in triplicate. Significance levels (*) indicates p value of < 0.05 between WT and uPAR^-/- ECs. (B) uPA/PAI-1 co-localization is cytoplasmic in migratory uPAR^-/- ECs. Fixed cells were stained with antibodies against uPA (green, Alexa Fluor 488) and PAI-1 (red, Alexa Fluor 647). Co-localization of uPA/PAI-1 in WT cells was observed along the focal adhesions or cell membrane (arrows). (C) In the uPAR^-/- ECs uPA/PAI-1 co-localization was observed as extensive blobs within the cytoplasm (arrows).

Actin cytoskeleton organization is circular in uPAR^-/- ECs: Lack of uPAR expression induces changes in cell morphology and actin cytoskeleton. Quiescent WT (A, C) and uPAR^-/- (B, D) ECs were plated on Vn and allowed to adhere for 4 hr and then stained for actin with phalloidin-FITC. Cells stained for actin (green) and the nucleus (blue) were imaged using a confocal microscope. Images obtained using a 30× objective (A, B) revealed morphological differences between WT and uPAR^-/- ECs. Images at higher magnification (100× objective) (C, D) demonstrated that while the WT cells showed polarized actin formation, the uPAR^-/- ECs maintained an atypical concentric actin organization.

In vitro angiogenesis is impaired in uPAR^-/- ECs: (A) WT cells on cytodex beads and embedded in a fibrin matrix were stained for actin using phalloidin-TRITC (red) and DNA (blue) and imaged using a 100× objective. After 24 hr incubation the bead was surrounded by adherent cells which were elongated. (B) Enhanced elongation of sprouts was observed from uPAR^-/- ECs adherent on the beads and these sprouts appeared to be thinner. (C) Sprout elongation by WT cells in the presence of 10 ng/ml of VEGF. Boxed area shows the development of robust lamellipodia formation of WT cells. (D) Elongation process after 24 hr for uPAR^-/- ECs in the presence of 10 ng/ml of VEGF. Boxed area shows that the sprouts derived from the uPAR^-/- ECs are thinner and show truncated lamellipodia formation.

Tubulogenesis is impaired in uPAR^-/- ECs: Bead-based fibrin gel angiogenesis was observed at 96 hr and the cells were stained for actin using phalloidin-TRITC (red) and DNA (blue) and imaged using a 20× objective. Formation of capillary-like structures at 96 hr in WT cells in control (A) assay and in the presence of 10 ng/ml VEGF was observed (C). In beads plated with uPAR^-/- ECs, highly branched sprouts were observed but were not organized to form capillary-like structures in the absence or presence of VEGF (B, D).

A simplified schematic depiction of the role of uPAR in angiogenesis: The uPAR/Pro-uPA interaction leads to the generation of active uPA on the cell surface. This complex binds to vitronectin in the extracellular matrix, allowing interaction with its transmembrane partners, the α/β integrins. This leads to a cascade of activation events resulting in tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin molecules. Through recruitment of other adaptor molecules, such as Src and p130Cas-CRK complex (not shown), Rac is activated. Activated Rac then induces actin filament assembly leading to membrane protrusion and motility. Formation of focal adhesion complexes enables cellular adhesion and migration. The uPA/uPAR complex also generates the serine protease, plasmin that degrades the extracellular matrix (ECM) thereby stimulating conditions for migration and proliferation. The transmembrane partnership between uPA/uPAR and α/β integrins also activates mitogen activated kinase signaling molecules, MEK and ERK1/2, as well as the phosphoinositide 3-kinase (PI3K)/Akt signaling axis. Thus uPAR-dependent multiple signaling events regulates cellular adhesion, proliferation, and migration, events associated with angiogenesis.

An uPAR deficiency alters cell adhesion to Vn and collagen: (A) uPAR deficiency promotes EC adhesion to Vn as observed by enhanced adhesion of uPAR^-/- ECs compared to WT cells. WT and uPAR^-/- cells on BSA serve as a control for this assay. (B) Enhanced adhesion of uPAR^-/- ECs on collagen at 4 hr. (C) Adhesion of uPAR^-/- ECs is similar to WT cells when plated on fibronectin. BSA served as a negative control matrix. The cell counts represent the mean ± SEM of three independent assays each performed in triplicate (40× field). Significance levels (*) indicates p value of < 0.05 between WT and uPAR^-/- ECs.

uPAR^-/- ECs exhibited altered proliferation on Vn: (A) WT and uPAR^-/- ECs were seeded on Vn-coated plates at a density of 1 × 10⁵ cells/ml as described. Graph represents total cell counts measured at 24 and 48 hr (mean ± SEM) of three independent assays. At both time points proliferation of uPAR^-/- ECs was significantly diminished compared to WT cells. p values between WT and uPAR^-/- ECs at both time points were <0.05. (B) Proliferation of WT and uPAR^-/- ECs is similar on collagen-coated plates at 24 and 48 hr.

uPAR^-/- ECs demonstrated changes in focal adhesion proteins FAK(P-Tyr925), Pax(P-Tyr118), integrins, and signal transduction: (A) WT cells adherent on Vn for 4 hr were stained for FAK(P-Tyr925) (green) and the nucleus (blue) and were imaged using an 100× objective. FAK(P-Tyr925) was found to be localized on focal adhesions (arrow) in WT cells. (B) uPAR^-/- ECs stained for FAK(P-Tyr925) demonstrated that FAK(P-Tyr925) was present not only along the cellular membrane (arrow) but also centrally in the cytoplasm (arrow). (C) Similar to (A), WT cells adherent on Vn were stained for Pax(P-Tyr118) (green) and DNA (blue). Images (100× objective) demonstrated that Pax(P-Tyr118) is present mainly along the focal adhesions and lamellipodia (arrow) in WT cells. Robust lamellipodia formation was observed. (D) Pax(P-Tyr118) (green) localization in uPAR^-/- ECs adherent on Vn occurred along the focal adhesions (arrow) of the circularly shaped cell as well as centrally within the cytoplasm (arrow). Lack of lamelllipodia formation was observed in uPAR^-/- ECs (B, D). (E) Immunoblot analyses of focal adhesion proteins and STAT1. Quiescent WT and uPAR^-/- ECs were seeded on Vn-coated 6-well plates and allowed to adhere for 4 hr. The medium was aspirated and the cell lysates were utilized for analyses. Levels of integrins β1, β3, and FAK(P-Tyr925) were determined by IP of 100 μg of cell lysates. Levels of integrin β1 and FAK(P-Tyr925) were enhanced in uPAR^-/- ECs compared to WT cells, while levels of integrin β3, total FAK, and paxillin were similar between WT and uPAR^-/- ECs. uPAR^-/- ECs showed decreased levels of STAT1 compared to WT cells. Tubulin served as a loading control. (F) The graph shows the levels of FAK(P-Tyr925/FAK), STAT1/tubulin, and Integrin β1/tubulin between WT and uPAR^-/- ECs. The values obtained represent the mean ± SEM of three independent assays. Significance levels (*) indicates p value of < 0.05 between WT and uPAR^-/- cells.

Quantification of in vitro tube formation assay: (A) Quantification of the number of cells adherent on the beads. Increased number of uPAR^-/- ECs adhered to the beads compared to WT cells, both in the absence or presence of VEGF. (B) Increased number of sprouts/bead was observed in uPAR^-/- cells in the absence or presence of VEGF compared to WT cells. (C) uPAR^-/- cells demonstrated increased sprout length compared to WT ECs in the absence and presence of VEGF. Ten beads/treatment/experiment/genotype were analyzed and the graph represents the mean ± SEM of three independent assays. (*) Denotes significance between control WT and uPAR^-/- ECs and (**) identifies significance between WT and uPAR^-/- ECs in the presence of VEGF. p value were < 0.05 between WT and uPAR^-/- cells.

Effect of a uPAR deficiency on angiogenesis in an in vivo Matrigel assay: Matrigel containing 10 ng/ml VEGF was implanted s.c. and recovered on day 14 after implantation, fixed, and stained for smooth muscle α-actin to observe capillary formation. (A) Representative image of Matrigel implant excised from WT mouse shows the presence of robust and well-developed capillaries (arrows). (B) Representative image of Matrigel implant excised from uPAR^-/- mouse shows the presence of several truncated structures that have not been organized to form a functional capillary (arrows).