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First published online 17 February 2004
doi: 10.1242/jcs.00969

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The bHLH TAL-1/SCL regulates endothelial cell migration and morphogenesis

¹ UMR 5535, Institut de Génétique Moléculaire, IFR122, 1919 Route de Mende
² INSERM U475, 99 rue Puech Villa, F 34000-Montpellier, France
³ UMR 5124, Institut de Génétique Moléculaire, IFR122, 1919 Route de Mende

View larger version (31K): [in a new window]   Fig. 1. TAL-1 protein expression in human endothelial cells. (A) pp48-50 is the only TAL-1 protein species detected in human proliferating endothelial cells (ECs). (Left) TAL-1 expression was assessed in whole cell lysates (30 µg) by immunoblotting with the 3BTL73 monoclonal antibody directed against the C terminus of the human TAL-1 protein (top). The blot was reprobed with an anti-GAPDH rabbit antiserum to evaluate protein loading. Expression was assessed in the following cells: K562, a human erythroleukemic cell line; ECV 304, an epithelial cell line established from a human bladder carcinoma; HUVEC, ECs derived from umbilical cord; HDMEC, ECs derived from dermal microvessels; HMEC-1, a cell line derived from HDMECs immortalized with large T antigen (Ades et al., 1992) and BMEC-1, a cell line established from bone marrow ECs immortalized with large T antigen (Candal et al., 1996). (Right) Expression of TAL-1 protein was assessed in circulating endothelial cells derived from umbilical cord blood (UCB-EC) at day 25 of culture (see Materials and Methods). The blot was reprobed for FLI-1 and VE-cadherin protein expression, two endothelial-specific markers, and for GAPDH to monitor loading. (B) Down-regulation of tal-1 expression in quiescent non-activated HUVECs. Proliferating HUVECs (Expo) were grown to confluence and were maintained either in complete medium for an additional 8 hours (Conf) or in medium containing only 5% FCS for 3 hours and 8 hours. (Left) Whole cell lysates were prepared at the indicated times after starvation and TAL-1 expression was monitored by western blot analysis using the 3BTL73 anti-TAL-1 monoclonal antibody. The same blot was reprobed with an anti-GAPDH rabbit antiserum for normalization of protein levels. TAL-1 and GAPDH proteins were both quantified by densitometry and TAL-1 protein content relative to GAPDH in one of two representative experiments is shown. The TAL-1 content from exponentially growing HUVECs (Expo) was arbitrarily set at 1. (Right) tal-1 mRNA expression was monitored by real-time PCR. The tal-1 mRNA signal was normalized to that of GAPDH. Data shown are the means ±s.d. of three independent RT-PCR experiments. tal-1 mRNA content from exponentially growing HUVECs was arbitrarily set at 1.

View larger version (67K): [in a new window]   Fig. 2. tal-1 expression is modulated during in vitro angiogenesis. Exponentially growing cells (HUVECs, HMEC-1 or UCB-ECs) were plated on Matrigel and cultured for 24 hours. (A) Photomicrographs of two-dimensional cultures of HUVECs on Matrigel demonstrating the progression of the tubular network. Initial plating on Matrigel corresponds to time 0 hour. (B-D) Cells were recovered from Matrigel at the indicated times to prepare whole cell extracts and TAL-1 expression was monitored by western blot analysis using the 3BTL73 anti-TAL-1 monoclonal antibody. The blots were reprobed with an anti-GAPDH rabbit antiserum for normalization of protein levels. TAL-1 and GAPDH proteins were both quantified by densitometry. TAL-1 content from exponentially growing ECs was arbitrarily set at 1. Representative autoradiographs of three independent experiments and the means ± s.d. of TAL-1 protein content relative to that of GAPDH are shown. (B) HUVECs; (C) UCB-ECs at day 30-33 of culture; (D) HMEC-1.

View larger version (88K): [in a new window]   Fig. 3. Effect of wild-type and bas TAL-1 overexpression on in vitro angiogenesis. (A) Western blot analysis showing ectopic overexpression of wild-type (wt) and bas TAL-1 protein in adenovirus-infected HUVECs. ECs were co-infected with Ad-Tet-Off and one of the following: Ad-TRE-wtTAL-1, Ad-TRE-bas or the control Ad-TRE-LacZ. Whole cell lysates were prepared 20 hours after infection. Note the intensities of wt and bas TAL-1 detected in 1 µg of whole cell lysate derived from infected cells as compared to that of endogenous TAL-1 detected in 30 µg of whole cell lysate derived from Ad-LacZ-infected cells (LacZ) or non-treated cells (NT). (B) Electrophoretic mobility shift analysis (EMSA) of TAL-1 DNA binding activity. A double stranded 32P-labeled oligonucleotide probe containing the TAL-1 E-box consensus sequence was incubated with nuclear extracts from HUVECs. (Left) EMSA showing the binding of TAL-1/E endogenous heterodimers to the TAL-1 E-box in nuclear extract from HUVEC (10 µg). The specificity of the interaction was controlled by adding a 100-fold excess of unlabeled TAL-1 E-box oligonucleotides (comp). Additionally, the presence of TAL-1/E heterodimers was verified by supershifting the complexes with an anti-TAL-1 antibody (TAL) or disrupting them with an anti-E47 antibody (E47). An irrelevant monoclonal antibody (12CA5) was added as a negative control. (Right) Ectopic TAL-1 DNA binding on E-box sequences was monitored in the presence or absence of a supershifting anti-TAL-1 mAb. Experimental conditions precluded the detection of endogenous TAL-1/E heterodimers in control LacZ-infected HUVECs (only 3 µg of nuclear extract was used). Note the absence of TAL-1/E DNA complex in nuclear extract from ECs infected with Ad-TRE-bas. (C) The effect of wt or bas TAL-1 overexpression on in vitro angiogenesis. Angiogenesis was monitored using adenovirus-infected ECs plated on Matrigel. Representative photographs of HUVECs after 90 minutes and 20 hours of plating are shown.

View larger version (22K): [in a new window]   Fig. 4. Effects of wild-type and bas TAL-1 overexpression on cell proliferation and migration. (A) 7x104 HUVECs were seeded onto collagen-coated 12-well plates and infected with Ad-Tet-Off and with either Ad-LacZ or Ad-wtTAL-1 or Ad-bas and cultured in complete medium. Cell numbers were monitored at 72 hours post-infection by direct counting. The number of LacZ-infected ECs was arbitrarily set at 100. The values are the mean±s.d. of independent infection experiments (n=10); statistical significance relative to Ad-LacZ infected cells is indicated: *P<0.05 (Student's t-test). (B) HUVECs or UCB-ECs were co-infected with Ad-Tet-Off and with Ad-TRE-wtTAL-1, Ad-TRE-bas or Ad-TRE-LacZ. Twenty hours post-infection, 4x104 cells were placed in the upper wells of 8 µm cell culture inserts in serum-free medium. The lower wells contained either control medium or chemoattractant medium. After 90 minutes, cells were fixed and labeled with propidium iodide. Migration was quantified by counting the cells that had migrated through the inserts in 6 randomly selected fields (original magnification, x10). The number of cells that had migrated through the inserts towards a serum-free medium was considered as background and was arbitrarily set at 1. The values are the mean±s.d. of three independent infection experiments; statistical significance relative to Ad-LacZ infected cells is indicated: *P<0.05; **P<0.01; ***P<0.001 (Student's t-test).

View larger version (58K): [in a new window]   Fig. 5. Effects of wild-type and bas TAL-1 ectopic expression on in vivo angiogenesis. Male SCID mice were injected subcutaneously at three sites with 0.5 ml Matrigel mixed with bFGF, heparin and Ad-Tet-Off together with each Ad-TRE. Injection sites were chosen such that each animal received the three Ad-TRE-LacZ, wtTAL-1 and bas. Three experiments were carried out with five or six animals. Matrigel plugs were removed after 6 days, photographed and processed for histology. Macroscopic observations. Representative photographs of three Matrigel implants formulated with the indicated adenovirus harvested from two individual mice belonging to two different experimental groups. Note the increase in angiogenesis (as demonstrated by brown/red color) in wt-TAL-1-transduced implants and its inhibition in -bas-transduced implants (almost white) as compared to control LacZ implants. Histological analyses. (Top) Representative images of Matrigel plug sections after Hematoxylin and Eosin staining; capillary structures with lumen (indicated by arrows) were more numerous in wtTAL-1 and very rare in bas as compared to control implants (LacZ). Higher magnification (x20) shows the presence of red blood cells within these structures only in wtTAL-1- and LacZ- but not in bas-formulated implants. (Bottom) Low magnification image (x5) illustrating the very large vascular structures with large vascular cavities engorged with red blood cells observed in wt-TAL-transduced implants. (C) Immunohistochemical staining with anti-CD31 antibody. Representative photographs illustrating the infiltration of CD31-positive ECs (appearing in brown) and their different organization within the three types of Matrigel implants.

View larger version (74K): [in a new window]   Fig. 6. Effects of TAL-1 knockdown on in vitro angiogenesis. HUVECs were transfected with tal-1 or control siRNA and analyzed 24 hours after the second transfection for TAL-1 protein content or in vitro angiogenesis. tal-1 silencing in endothelial cells by RNA interference. TAL-1 expression was assessed in whole cell lysates (30 µg) by immunoblotting with the anti-TAL-1 mAb 3BTL73. The blot was reprobed with an anti-GAPDH rabbit antiserum to control protein loading. Effect of TAL-1 knockdown on in vitro angiogenesis. Representative photographs of transfected HUVECs after 4 hours of plating on Matrigel.

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