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First published online 12 September 2006
doi: 10.1242/jcs.03178

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Silencing profilin-1 inhibits endothelial cell proliferation, migration and cord morphogenesis

¹ Department of Bioengineering, University of Pittsburgh, 749 Benedum Hall, 3700 O'Hara Street, Pittsburgh, PA 15261, USA
² Department of Biochemistry, Ghent University, Albert Baertsoenkaai 3, 9000 Ghent, Belgium
³ Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA
⁴ Department of Pathology, University of Pittsburgh, School of Medicine, S-417 BST, 200 Lothrup Street, Pittsburgh, PA 15261, USA

View larger version (59K): [in a new window]   Fig. 1. Silencing Pfn1 expression affects the actin cytoskeleton in HUVECs: (A) Target specificity of Pfn1-siRNA was initially demonstrated by transfecting either control (C) or Pfn1-specific (P) siRNAs in MDA-MB-231 breast cancer cells that stably express either GFP or different point-mutants of GFP-Pfn1 (designated as mutant-1 and mutant-2 that carried a 2-base-pair alteration both within and outside of the targeting region of Pfn1-siRNA, respectively). Fluorescence micrographs of cells show that Pfn1-siRNA suppresses the expression of mutant-2, but not of mutant-1. Fluorescence of control GFP-expressing cells is not affected by Pfn1-siRNA treatment. Bar, 150 µm. (B) A bar graph shows time-dependent progressive loss of Pfn1 expression with nearly 97% suppression of Pfn1 expression 96 hours after Pfn1-siRNA transfection (data summarized based on immunoblot analyses of HUVEC extracts from three independent experiments). The inset shows the actual representative Pfn1-immunoblots of HUVEC extracts prepared at different time-points after siRNA transfection with GAPDH blot serving as the loading control. Additional immunoblot data shows no detectable Pfn2 expression under either experimental condition (purified Pfn2 serves the positive control for the immunoblot). (C) Rhodamine-phalloidin staining of HUVECs shows that silencing Pfn1 dramatically inhibits the formation of actin stress-fibers. Bar, 20 µm. (D) A bar graph displaying the relative (normalized with respect to the control cells) fluorescence intensity of phalloidin shows a 29% decrease in the average level of F-actin in Pfn1-deficient cells. These data were obtained from analyses of 640 control and 584 Pfn1-deficient cells from two independent experiments, the difference of which was found to be statistically significant (the asterisk indicates P<0.0001). (E) Immunoblots show comparable expression levels of actin and several ABPs such as VASP, mDia1, and N-WASP at 48, 72 and 96 hours after transfection.

View larger version (67K): [in a new window]   Fig. 2. Loss of Pfn1 expression alters cell-matrix and cell-cell adhesions in HUVECs: (A) Vinculin-immunostaining shows a dramatic reduction in FA formation when Pfn1 expression is silenced (C, control siRNA; P, Pfn1-siRNA). Bar, 20 µm. (B) A bar graph shows a significantly (P<0.001) higher FA density (number of FA/100 µm2 of cell area) in control cells (6.1±2.1) than in Pfn1-deficient cells (2.7±1.9). These data are based on analyses of 56 control and 69 Pfn1-deficient cells that were randomly selected from two independent experiments. (C,D) VE-cadherin and ZO-1 immunostaining show that VEGF stimulation causes loss of junctional staining of these proteins (arrow) and creation of paracellular holes (arrowhead) in control cells. VEGF-induced delocalization of VE-cadherin and ZO-1 from cell-cell junctions is significantly inhibited in Pfn1-deficient ECs. Bar, 20 µm. (E) Immunoblot data show no appreciable change in the expression of VE-cadherin and ZO-1 between control and Pfn1-siRNA treated cells (the GAPDH blot serves as the loading controls).

View larger version (10K): [in a new window]   Fig. 3. Silencing Pfn1 expression inhibits cell growth: (A) A line graph shows the ratio of the number of control (denoted by `C') to that of Pfn1-deficient cells (denoted by `P') in culture at 48, 72 and 96 hours after transfection. (These data were summarized from three independent experiments and P<0.0002.) (B) DAPI staining of cells shows normal nuclear morphology under either experimental condition. Bar, 20 µm.

View larger version (47K): [in a new window]   Fig. 4. Loss of Pfn1 expression affects HUVEC spreading: (A) Phase contrast images of HUVECs seeded 1 hour after plating on a substrate that is pre-coated with 100 µg/ml matrigel show higher proportion of spreading cells (appear phase dense) in the control group (denoted by `C'). Impaired cell spreading was evident from round morphology of majority of Pfn1-deficient cells (denoted by `P'). Bar, 100 µm. (B) A bar graph plotting the percentage of spreading cells at two different time-points (30 minutes and 1 hour) and for two different coating concentrations of matrigel clearly shows increased spreading efficiency of the control cells (these data were pooled from the analyses of approximately 800-1000 cells for each experimental condition from two independent experiments). The asterisk indicates P<0.001. (C) Pfn1-deficient cells were still found to be much less flat and spread-out compared with the control cells at 22 hours after cell-seeding on matrigel-coated substrates. Bar, 50 µm.

View larger version (38K): [in a new window]   Fig. 5. Loss of Pfn1 inhibits HUVEC migration in wound healing assay. (A) Representative images of the wound margins immediately and 12 hours after wounding show significant impairment in wound closure by HUVECs due to loss of Pfn1 expression (C, control siRNA; P, Pfn1-siRNA). Bar, 100 µm. (B) A higher magnification of the wound margin indicates closer association of Pfn1-deficient cells. Bar, 100 µm. (C) A bar graph plotting the relative efficiency of wound closure shows Pfn1-siRNA treatment inhibited wound-closure by 25%, 36% and 47% when evaluated at 48, 72 and 96 hours after transfection, respectively. (These data are summarized from three independent experiments and the asterisk indicates P<0.002.)

View larger version (42K): [in a new window]   Fig. 6. Effect of silencing Pfn1 expression on single cell migration: (A) A typical time-lapse imaging experiment shows directed migration of control cells (denoted by `C') involving directed protrusion and significant net cell translocation. By contrast, Pfn1-deficient cells (denoted by `P') produce small, randomly directed protrusion with much less net cell translocation (the direction of protrusion is indicated by the arrow). Bar, 30 µm. (B) Trajectories of individual cells from the frame-by-frame analyses of the centroid of cell nuclei show a significantly (P<0.001) larger net distance traveled by the control cells (43.6±26.0 µm) compared with the Pfn1-deficient cells (11.3±9.6 µm) during the 90-minute observation period (migration data of 27 control and 21 Pfn1-deficient cells from a total of three independent experiments were pooled for the analysis). (C) Representative plots of change in protrusion direction between successive image frames () from a typical experiment are shown side-by-side to display much larger oscillation of in Pfn1-deficient cells (these plots were generated based on motility data of five control and six Pfn1-deficient cells in one experiment). (D) The bar graph compares the standard deviation values of for the entire 90-minute observation period (these data are based on the average values calculated for 14 control and 19 Pfn1-deficient cells - those cells that either barely protruded or came in contact with a neighboring cell at any point during the course of the experiment were excluded from the analysis). The asterisk indicates P<0.0001.

View larger version (68K): [in a new window]   Fig. 7. Silencing Pfn1 alters the localization of VASP in migrating HUVECs. In control cells (denoted by `C'), VASP is predominantly localized at the focal adhesions and actin stress-fibers, and has a punctate distribution at the leading edge. Pfn1-deficient cells (denoted by `P') have much stronger localization of VASP at the leading edge. The inset in each panel shows a magnified view of the boxed region depicting differences in VASP localization at the leading edge between the two experimental conditions. Bar, 10 µm.

View larger version (45K): [in a new window]   Fig. 8. Effect of silencing Pfn1 on early cord morphogenesis of HUVECs. (A) Control HUVECs (denoted by `C') form prominent cord-like structures on polymerized matrigel by 8 hours after plating. Cord formation is significantly inhibited in the case of Pfn1-deficient cells (denoted by `P'). Bar, 100 µm. (B) A bar graph shows significantly higher number of nodes involving at least three branches in the control cells compared with the same in Pfn1-deficient cells (the graph summarizes data from a total of four independent experiments; the asterisk indicates P<0.001).

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