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doi: 10.1242/10.1242/jcs.00600

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Thrombospondin signaling through the calreticulin/LDL receptor-related protein co-complex stimulates random and directed cell migration

¹ Department of Pathology, Division of Molecular and Cellular Pathology and The Cell Adhesion and Matrix Research Center, University of Alabama at Birmingham, Birmingham, AL 35294-0019, USA
² Research Service, Birmingham VA Medical Center, Birmingham, AL 35233-1996, USA
³ Department of Pathology, Division of Laboratory Medicine, University of Alabama at Birmingham, Birmingham, AL 35294-0019, USA

View larger version (28K): [in a new window]   Fig. 1. TSP1/hep I stimulate directed endothelial cell migration. (A) BAE cells were grown to near confluence, loaded with calcein AM, and plated onto a 96-well Neuro Probe chemotaxis chamber coated with vitronectin and fibronectin. Cells were stimulated to migrate towards increasing concentrations of soluble TSP1 for 5 hours. As a positive control, cells were also stimulated to migrate towards a bFGF gradient. Cells were scraped from the upper surface and cells migrating to the lower surface assayed by determining the remaining fluorescence in a plate reader. Migration was normalized to that seen with media alone and presented as fold stimulation of migration above baseline levels. n=4-6, *P<0.05, **P<0.01. (B) Cells were stimulated to migrate towards increasing concentrations of either the hep I peptide or a modified, inactive hep I peptide, and migration was assessed as described in (A). n=3, **P<0.01.

View larger version (27K): [in a new window]   Fig. 2. Hep I increases random migration in endothelial cells. (A) BAE cells were grown to near confluence, loaded with calcein AM, and plated onto a 96-well Neuro Probe chemotaxis chamber coated with vitronectin and fibronectin. Cells were stimulated to migrate in response to increasing concentrations of hep I, or the inactive, modified hep I peptide, in the absence of a gradient for 5 hours. Cells were scraped from the upper surface and cells migrating to the lower surface assayed by determining the remaining fluorescence in a plate reader. Migration was normalized to that seen with media alone and presented as fold stimulation of migration above baseline levels. n=3-4, *P<0.05, **P<0.01. (B) Cells were stimulated to migrate with increasing concentrations of TSP1 in the absence of a gradient and migration was assessed as described in (A). Treatment with 100 pM bFGF was included as a positive control in this assay. n=3, *P<0.05, **P<0.01.

View larger version (14K): [in a new window]   Fig. 3. TSP1/hep I-induced endothelial cell chemotaxis in the Dunn chamber. BAE cells were plated at low density under serum-free conditions onto glass coverslips coated with vitronectin and fibronectin. Cells were allowed to attach for 3 hours and loaded onto the Dunn Chamber in serum-free media. Serum-free media was removed from the outer well and media containing either serum-free media (A), hep I (100 nM) (B), TSP1 (7.8 nM) (C) or modified hep I (100 nM) (D) was added to the outer well. The chamber was sealed, and time-lapse video was taken of the cells over a 7-hour time span. Migration of individual cells was tracked using Metamorph software and individual tracks were analyzed for distance, displacement, directionality and orientation. Final positions of the cells are plotted with the initial point being the origin, and the top of the graph representing the outer well. Results are representative of at least three separate experiments. Average displacement () toward the outer well is given for each treatment. *P<0.05, **P<0.01.

View larger version (24K): [in a new window]   Fig. 4. Histogram for endothelial migration speeds following TSP1/hep I treatment. BAE cells were plated at low density under serum-free conditions onto glass coverslips coated with vitronectin and fibronectin. Cells were allowed to attach for 3 hours and loaded onto the Dunn Chamber in serum-free media (A), or media containing either hep I (100 nM) (B), TSP1 (7.8 nM) (C) or modified hep I (100 nM) (D). The chamber was sealed, and time-lapse video was taken of the cells over a 7-hour time span. Migration of individual cells was tracked using Metamorph software and migration speed was determined. Cells were separated into consecutive speed intervals and the percent of cells migrating at each interval was determined. At least 75 cells were analyzed per condition. n=3-4.

View larger version (23K): [in a new window]   Fig. 5. CRT-knockout fibroblasts do not migrate in response to TSP1/hep I. Wild-type and CRT-knockout (KO) MEFs were plated at low density under serum-free conditions onto glass coverslips coated with vitronectin and fibronectin. Cells were allowed to attach for 3 hours and loaded onto the Dunn Chamber in serum-free media (A,B), with or without 100 nM hep I (C,D), 7.8 nM TSP1 (E,F) or 0.1% FBS (G,H). The chamber was sealed, and time-lapse video was taken of the cells over a 7-hour time span. Migration of individual cells was tracked using Metamorph software and individual tracks were analyzed for distance, displacement, directionality and orientation. Final positions of the cells are plotted with the initial point being the origin. Results are representative of at least three separate experiments. Average displacement () is given for each treatment. *P<0.05, **P<0.01, ***P<0.001.

View larger version (48K): [in a new window]   Fig. 6. Differences in fibroblast migration to TSP1 and hep I. (A) Wild-type and CRT-knockout (KO) MEF migration in response to serum-free media, with or without hep I (100 nM), TSP1 (7.8 nM) or FBS (0.1%), was assessed as previously described. Average cell migration speed under each condition was determined by dividing total migration distance by the total time of the assay. At least 75 cells were analyzed for each condition. n=3-4, *P<0.05, **P<0.01. (B) Percent of cells migrating under each condition was determined by dividing the number of cells migrating at least 0.06 microns/minute by the total number of cells. At least 75 cells were analyzed for each condition. n=3-4, *P<0.05, **P<0.01. (C) Total cellular displacement was determined as the difference between final cell position and initial cell position. At least 75 cells were analyzed for each condition. n=3-4, *P<0.05, **P<0.01, ***P<0.001. (D) Directionality of cell migration was determined by dividing the total cellular displacement by the total distance of migration. Under this method, a purely directional response will show a ratio of 1, whereas increasingly random migration responses will show ratios approaching zero. At least 75 cells were analyzed for each condition. n=3-4, *P<0.05, **P<0.01, ***P<0.001.

View larger version (46K): [in a new window]   Fig. 7. LRP-knockout fibroblasts are migration deficient. (A) Wild-type and LRP-knockout (KO) MEF migration in response to serum-free media, with or without hep I (100 nM), TSP1 (7.8 nM) or FBS (0.1%), was assessed as previously described. Average cell migration speed under each condition was determined by dividing total migration distance by the total time of the assay. At least 75 cells were analyzed for each condition. n=3-4, *P<0.05, **P<0.01. (B) Percent of cells migrating under each condition was determined by dividing the number of cells migrating at least 0.06 microns/minute by the total number of cells. At least 75 cells were analyzed for each condition. n=3-4, *P<0.05, **P<0.01. (C) Total cellular displacement was determined as the difference between final cell position and initial cell position. At least 75 cells were analyzed for each condition. n=3-4, *P<0.05, **P<0.01, ***P<0.001. (D) Directionality of cell migration was determined by dividing the total cellular displacement by the total distance of migration. Under this method, a purely directional response will show a ratio of 1, whereas increasingly random migration responses will show ratios approaching zero. At least 75 cells were analyzed for each condition. n=3-4, *P<0.05, **P<0.01, ***P<0.001.

View larger version (23K): [in a new window]   Fig. 8. RAP pretreatment blocks TSP1/hep I-induced endothelial cell migration. BAE cells were plated at low density under serum-free conditions onto glass coverslips coated with vitronectin and fibronectin. Cells were allowed to attach for 3 hours. Some coverslips were pretreated for 30 minutes with the LRP inhibitor receptor-associated protein (RAP) at 50 nM. Coverslips were loaded onto the Dunn Chamber in serum-free media (A,C,E,G) or serum-free media containing RAP (B,D,F,H), as well as 100 nM hep I (C,D), 7.8 nM TSP1 (E,F) or 0.1% FBS (G,H). The chamber was sealed, and time-lapse video was taken of the cells over a 7-hour time span. Migration of individual cells was tracked using Metamorph software and individual tracks were analyzed for distance, displacement, directionality and orientation. Final positions of the cells are plotted with the initial point being the origin. Results are representative of at least three separate experiments. Average displacement () is given for each treatment. *P<0.05, **P<0.01.

View larger version (33K): [in a new window]   Fig. 9. Hep I modulates aFGF/bFGF-induced endothelial cell chemotaxis. BAE cells were plated at low density under serum-free conditions onto glass coverslips coated with vitronectin and fibronectin. Cells were allowed to attach for 3 hours. Some coverslips were pretreated for 30 minutes with 100 nM hep I or 7.8 nM TSP1. Coverslips were loaded onto the Dunn Chamber in serum-free media (A,D,G) or serum-free media containing either 100 nM hep I (B,E,H) or 7.8 nM TSP1 (C,F,I). Media was removed from the outer well and corresponding media containing aFGF (67 pM) (D,E,F; Movies 4 and 6, available at jcs.biologists.org/supplemental) or bFGF (61 pM) (G,H,I; Movies 5 and 7, available at jcs.biologists.org/supplemental) was then loaded into the outer well establishing a chemical gradient. The chamber was sealed, and time-lapse video was taken of the cells over a 7-hour time span. Migration of individual cells was tracked using Metamorph software and individual tracks were analyzed for distance, displacement, directionality and orientation. Final positions of the cells are plotted with the initial point being the origin and the top of the graph representing the outer well. Results are representative of at least six separate experiments. Average displacement towards the outer well (X) is given for each treatment. *P<0.05, **P<0.01.