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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

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).
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Figure 2: 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).

Mentions: Since cell migration and proliferation are coupled to adhesive properties of cells on the ECM, migration and proliferation assays on Vn and collagen were performed. A scratch assay was employed to assess the cellular motility of ECs from both genotypes in the presence of Vascular Endothelial Cell Growth Factor (VEGF), which is a major facilitator of both physiological and pathological angiogenesis. Cell migration of uPAR-/- ECs was diminished 60% compared to WT cells when plated on Vn (Figure 2A). However, migratory function of the uPAR-/- ECs was not affected on collagen. ECM proteolysis is controlled by the inhibitor, plasminogen activator inhibitor-1 (PAI-1), which forms a 1:1 complex with uPA bound to uPAR, and catalyzes the vesicular internalization of the uPA/PAI-1 complex. This results in eventual degradation of both uPA and PAI-1 [31]. Primary ECs express both uPA and PAI-1 [24], and since migration of uPAR-/- cells is impaired on Vn, co-localization of uPA and PAI-1 by immunofluorescence on cells migrating in the presence of VEGF was performed. In migrating WT cells, uPA and PAI-1 co-localization was observed along the focal adhesions at the advancing front of the cell (indicated by arrows) (Figure 2B). Co-localization of uPA/PAI-1 in the uPAR-/- ECs was observed abundantly within the cytoplasm (indicated by arrows), with very little staining at focal adhesion points (Figure 2C). ERK1/2 activation levels were not affected in uPAR-/- ECs compared to WT cells (data not shown) indicating that changes in angiogenic function in these cells are regulated by other signaling pathways.


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

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

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: 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).
Mentions: Since cell migration and proliferation are coupled to adhesive properties of cells on the ECM, migration and proliferation assays on Vn and collagen were performed. A scratch assay was employed to assess the cellular motility of ECs from both genotypes in the presence of Vascular Endothelial Cell Growth Factor (VEGF), which is a major facilitator of both physiological and pathological angiogenesis. Cell migration of uPAR-/- ECs was diminished 60% compared to WT cells when plated on Vn (Figure 2A). However, migratory function of the uPAR-/- ECs was not affected on collagen. ECM proteolysis is controlled by the inhibitor, plasminogen activator inhibitor-1 (PAI-1), which forms a 1:1 complex with uPA bound to uPAR, and catalyzes the vesicular internalization of the uPA/PAI-1 complex. This results in eventual degradation of both uPA and PAI-1 [31]. Primary ECs express both uPA and PAI-1 [24], and since migration of uPAR-/- cells is impaired on Vn, co-localization of uPA and PAI-1 by immunofluorescence on cells migrating in the presence of VEGF was performed. In migrating WT cells, uPA and PAI-1 co-localization was observed along the focal adhesions at the advancing front of the cell (indicated by arrows) (Figure 2B). Co-localization of uPA/PAI-1 in the uPAR-/- ECs was observed abundantly within the cytoplasm (indicated by arrows), with very little staining at focal adhesion points (Figure 2C). ERK1/2 activation levels were not affected in uPAR-/- ECs compared to WT cells (data not shown) indicating that changes in angiogenic function in these cells are regulated by other signaling pathways.

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