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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

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.
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Figure 7: 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.

Mentions: We show that uPAR-/- cells are altered in their angiogenic properties, e.g., proliferation, migration, and adhesion compared to WT cells when plated on Vn. To determine whether these altered angiogenic functions translate into an inability of the uPAR-/- ECs to form capillary sprouts and lumen, a microcarrier-based fibrin gel angiogenesis assay was performed in vitro. WT and uPAR-/- ECs adherent to collagen-coated cytodex beads were embedded in a fibrin matrix [56,57], which is known to induce EC tube formation in the presence or absence of VEGF. Capillary morphogenesis was observed after 24 and 96 hr incubation and stained for actin and nuclei. After 24 hr, sprouting or elongation of WT and uPAR-/- ECs was observed in the absence (Figure 6A,B) or presence of VEGF (Figure 6C,D). However, the number of uPAR-/- cells adhering to the microcarrier bead was higher (Figure 7A) and in consonance with our observation that these cells adhere more on collagen compared to WT cells (Figure 1B). Assessment of sprout number and length of sprouts per bead revealed that, in the absence of VEGF, uPAR-/- ECs developed more sprouts and the sprout length was longer compared to WT cells (Figure 7B,C). At higher magnification it was observed that WT ECs developed robust filopodia or finger-like projections at the end of the sprout after 24 hr, but the uPAR-/- ECs developed sprouts that were thinner with poorly developed filopodia structures (Figure 6, boxed areas). The presence of VEGF (10 ng/ml) did not appear to significantly enhance the number of cells adherent to the beads or the number and length of sprouts of WT cells (Figure 7A,B,C). However, sprout length of uPAR-/- ECs was increased in the presence of VEGF (Figure 7C).


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

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

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

License
Show All Figures
getmorefigures.php?uid=PMC3105951&req=5

Figure 7: 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.
Mentions: We show that uPAR-/- cells are altered in their angiogenic properties, e.g., proliferation, migration, and adhesion compared to WT cells when plated on Vn. To determine whether these altered angiogenic functions translate into an inability of the uPAR-/- ECs to form capillary sprouts and lumen, a microcarrier-based fibrin gel angiogenesis assay was performed in vitro. WT and uPAR-/- ECs adherent to collagen-coated cytodex beads were embedded in a fibrin matrix [56,57], which is known to induce EC tube formation in the presence or absence of VEGF. Capillary morphogenesis was observed after 24 and 96 hr incubation and stained for actin and nuclei. After 24 hr, sprouting or elongation of WT and uPAR-/- ECs was observed in the absence (Figure 6A,B) or presence of VEGF (Figure 6C,D). However, the number of uPAR-/- cells adhering to the microcarrier bead was higher (Figure 7A) and in consonance with our observation that these cells adhere more on collagen compared to WT cells (Figure 1B). Assessment of sprout number and length of sprouts per bead revealed that, in the absence of VEGF, uPAR-/- ECs developed more sprouts and the sprout length was longer compared to WT cells (Figure 7B,C). At higher magnification it was observed that WT ECs developed robust filopodia or finger-like projections at the end of the sprout after 24 hr, but the uPAR-/- ECs developed sprouts that were thinner with poorly developed filopodia structures (Figure 6, boxed areas). The presence of VEGF (10 ng/ml) did not appear to significantly enhance the number of cells adherent to the beads or the number and length of sprouts of WT cells (Figure 7A,B,C). However, sprout length of uPAR-/- ECs was increased in the presence of VEGF (Figure 7C).

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