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A deficiency of uPAR alters endothelial angiogenic function and cell morphology.

Balsara RD, Merryman R, Virjee F, Northway C, Castellino FJ, Ploplis VA - (2011)

Bottom Line: This study focuses on the effect of uPAR deficiency (uPAR-/-) on angiogenic function and associated cytoskeletal organization.VEGF-enriched Matrigel implants from uPAR-/- mice demonstrated a lack of mature vessel formation compared to WT mice.Collectively, these results indicate that a uPAR deficiency leads to decreased angiogenic functions of endothelial cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: W, M, Keck Center for Transgene Research, University of Notre Dame, 230 Raclin-Carmichael Hall, Notre Dame, Indiana 46556, USA. vploplis@nd.edu.

ABSTRACT
The angiogenic potential of a cell requires dynamic reorganization of the cytoskeletal architecture that involves the interaction of urokinase-type plasminogen activator receptor (uPAR) with the extracellular matrix. This study focuses on the effect of uPAR deficiency (uPAR-/-) on angiogenic function and associated cytoskeletal organization. Utilizing murine endothelial cells, it was observed that adhesion, migration, proliferation, and capillary tube formation were altered in uPAR-/- cells compared to wild-type (WT) cells. On a vitronectin (Vn) matrix, uPAR-/- cells acquired a "fried egg" morphology characterized by circular actin organization and lack of lamellipodia formation. The up-regulation of β1 integrin, FAK(P-Tyr925), and paxillin (P-Tyr118), and decreased Rac1 activation, suggested increased focal adhesions, but delayed focal adhesion turnover in uPAR-/- cells. This accounted for the enhanced adhesion, but attenuated migration, on Vn. VEGF-enriched Matrigel implants from uPAR-/- mice demonstrated a lack of mature vessel formation compared to WT mice. Collectively, these results indicate that a uPAR deficiency leads to decreased angiogenic functions of endothelial cells.

No MeSH data available.


Related in: MedlinePlus

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.
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Figure 5: 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.

Mentions: Since adhesion of ECs to the ECM requires formation of focal adhesions that form cell-matrix junctions that aid in anchoring cytoskeletal proteins to the matrix [35], focal adhesion kinase (FAK) and paxillin, which are key components of focal adhesions that become phosphorylated when cells adhere to the ECM [36], were examined. The cellular distributions of FAK(P-Tyr925) and Pax(P-Tyr118) were evaluated in WT and uPAR-/- ECs. After 4 hr adhesion on Vn, FAK(P-Tyr925) was observed as small punctate signals, mainly in the cytoplasmic region of the WT cells, with very little staining observed on the membrane edge (Figure 5A). However, in uPAR-/- ECs FAK(P-Tyr925) was prominently localized in the central adhesion regions with some localization on the membrane edge (Figure 5B). Additionally, elevated levels of FAK(P-Tyr925) were observed in uPAR-/- ECs by immunoblotting (Figure 5E) and densitometric analyses (Figure 5F). Phosphorylation of FAK at Tyr925 as well as at other sites (Tyr407, 576, 577, and 861) is Src-dependent [37-40] and is essential for regulating F-actin assembly and maintaining cell adhesion dynamics [41]. Phosphorylation of endogenous FAK at Tyr925 is known to exclude FAK from focal adhesion points [42], which is observed in WT cells. Endogenous FAK(P-Tyr925) has been implicated in focal adhesion disassembly resulting in normal focal adhesion turnover and hence a migratory phenotype for the cell. On the other hand hyperphosphorylation of FAK(P-Tyr925) in uPAR-/- EC resulted in increased cytoplasmic localization of FAK, but not total exclusion of FAK from focal adhesions. This suggests that the levels of FAK(P-Tyr925) in focal adhesions are still sufficient to promote adhesion of the cells to Vn.


A deficiency of uPAR alters endothelial angiogenic function and cell morphology.

Balsara RD, Merryman R, Virjee F, Northway C, Castellino FJ, Ploplis VA - (2011)

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.
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Figure 5: 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.
Mentions: Since adhesion of ECs to the ECM requires formation of focal adhesions that form cell-matrix junctions that aid in anchoring cytoskeletal proteins to the matrix [35], focal adhesion kinase (FAK) and paxillin, which are key components of focal adhesions that become phosphorylated when cells adhere to the ECM [36], were examined. The cellular distributions of FAK(P-Tyr925) and Pax(P-Tyr118) were evaluated in WT and uPAR-/- ECs. After 4 hr adhesion on Vn, FAK(P-Tyr925) was observed as small punctate signals, mainly in the cytoplasmic region of the WT cells, with very little staining observed on the membrane edge (Figure 5A). However, in uPAR-/- ECs FAK(P-Tyr925) was prominently localized in the central adhesion regions with some localization on the membrane edge (Figure 5B). Additionally, elevated levels of FAK(P-Tyr925) were observed in uPAR-/- ECs by immunoblotting (Figure 5E) and densitometric analyses (Figure 5F). Phosphorylation of FAK at Tyr925 as well as at other sites (Tyr407, 576, 577, and 861) is Src-dependent [37-40] and is essential for regulating F-actin assembly and maintaining cell adhesion dynamics [41]. Phosphorylation of endogenous FAK at Tyr925 is known to exclude FAK from focal adhesion points [42], which is observed in WT cells. Endogenous FAK(P-Tyr925) has been implicated in focal adhesion disassembly resulting in normal focal adhesion turnover and hence a migratory phenotype for the cell. On the other hand hyperphosphorylation of FAK(P-Tyr925) in uPAR-/- EC resulted in increased cytoplasmic localization of FAK, but not total exclusion of FAK from focal adhesions. This suggests that the levels of FAK(P-Tyr925) in focal adhesions are still sufficient to promote adhesion of the cells to Vn.

Bottom Line: This study focuses on the effect of uPAR deficiency (uPAR-/-) on angiogenic function and associated cytoskeletal organization.VEGF-enriched Matrigel implants from uPAR-/- mice demonstrated a lack of mature vessel formation compared to WT mice.Collectively, these results indicate that a uPAR deficiency leads to decreased angiogenic functions of endothelial cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: W, M, Keck Center for Transgene Research, University of Notre Dame, 230 Raclin-Carmichael Hall, Notre Dame, Indiana 46556, USA. vploplis@nd.edu.

ABSTRACT
The angiogenic potential of a cell requires dynamic reorganization of the cytoskeletal architecture that involves the interaction of urokinase-type plasminogen activator receptor (uPAR) with the extracellular matrix. This study focuses on the effect of uPAR deficiency (uPAR-/-) on angiogenic function and associated cytoskeletal organization. Utilizing murine endothelial cells, it was observed that adhesion, migration, proliferation, and capillary tube formation were altered in uPAR-/- cells compared to wild-type (WT) cells. On a vitronectin (Vn) matrix, uPAR-/- cells acquired a "fried egg" morphology characterized by circular actin organization and lack of lamellipodia formation. The up-regulation of β1 integrin, FAK(P-Tyr925), and paxillin (P-Tyr118), and decreased Rac1 activation, suggested increased focal adhesions, but delayed focal adhesion turnover in uPAR-/- cells. This accounted for the enhanced adhesion, but attenuated migration, on Vn. VEGF-enriched Matrigel implants from uPAR-/- mice demonstrated a lack of mature vessel formation compared to WT mice. Collectively, these results indicate that a uPAR deficiency leads to decreased angiogenic functions of endothelial cells.

No MeSH data available.


Related in: MedlinePlus