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, 148 (2), 343-51

Homeobox B3 Promotes Capillary Morphogenesis and Angiogenesis

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Homeobox B3 Promotes Capillary Morphogenesis and Angiogenesis

C Myers et al. J Cell Biol.

Abstract

Endothelial cells (EC) express several members of the Homeobox (Hox) gene family, suggesting a role for these morphoregulatory mediators during angiogenesis. We have previously established that Hox D3 is required for expression of integrin alphavbeta3 and urokinase plasminogen activator (uPA), which contribute to EC adhesion, invasion, and migration during angiogenesis. We now report that the paralogous gene, Hox B3, influences angiogenic behavior in a manner that is distinct from Hox D3. Antisense against Hox B3 impaired capillary morphogenesis of dermal microvascular EC cultured on basement membrane extracellular matrices. Although levels of Hox D3-dependent genes were maintained in these cells, levels of the ephrin A1 ligand were markedly attenuated. Capillary morphogenesis could be restored, however, by addition of recombinant ephrin A1/Fc fusion proteins. To test the impact of Hox B3 on angiogenesis in vivo, we constitutively expressed Hox B3 in the chick chorioallantoic membrane using avian retroviruses that resulted in an increase in vascular density and angiogenesis. Thus, while Hox D3 promotes the invasive or migratory behavior of EC, Hox B3 is required for the subsequent capillary morphogenesis of these new vascular sprouts and, together, these results support the hypothesis that paralogous Hox genes perform complementary functions within a particular tissue type.

Figures

Figure 1
Figure 1
Expression of Hox B3 in HMEC cultured under different conditions. A, Semiquantitative RT-PCR of Hox B3 (left) and GAPDH (right) in HMEC grown in the presence (+) or absence (−) of BM (Matrigel) shows the relative levels of the 1.3-kb transcript encoding Hox B3 in EC under each of these conditions. The 0.6-kb transcript for GAPDH was used to normalize levels of RNA used in this analysis. B, Semiquantitative RT-PCR for Hox B3 in HMEC cultured in the presence or absence bFGF (50 ng/ml) for 24 h.
Figure 2
Figure 2
Antisense against Hox B3 impairs BM-induced capillary morphogenesis in EC. A, Western blot for Hox B3 in 50 μg total protein lysates from immortalized HMEC-1 stably transfected with empty vector (control) or plasmid expressing antisense against human Hox B3 (Hox B3 AS). B, Capillary morphogenesis of HMEC-1 after culturing on thick BM (Matrigel) after 18 h. The panel on the left shows normal capillary morphogenesis occurring in HMEC-1 stably transfected with empty vector (Control). The panel on the right shows impaired capillary morphogenesis in HMEC-1 transfected with antisense against Hox B3 (Hox B3 AS). The arrow shows representative groups of cells that remain in clusters and fail to elongate and form contacts with adjacent cells.
Figure 3
Figure 3
Expression of ephrin A1 is selectively reduced in EC lacking Hox B3. A, The upper panel shows similar amounts of a 120-kD band corresponding to EphA2 recovered by immunoprecipitation from control transfected (Control) HMEC-1 and HMEC-1 transfected with antisense against Hox B3 (AS). The middle panel shows subsequent blotting with an antiphosphotyrosine antibody and reveals a reduced level of phosphorylation of the EphA2 receptor in cells transfected with antisense against Hox B3. The lower panel shows Western blotting with a polyclonal antibody against ephrin A1 (1:500 dilution) performed on 20 μg total EC lysates from HMEC-1 transfected with control plasmid (Control) or antisense against Hox B3 (AS) and reveals a marked reduction in expression of the ∼28-kD ephrin A1 protein. B, Northern blot analysis using 10 μg total RNA isolated from control transfected HMEC-1 (Control) or HMEC-1 transfected with antisense against Hox B3 (AS). The upper panel shows reduced levels of ephrin A1 mRNA in AS as compared with control cells. The middle and lower panels show the same blot reprobed with cDNAs corresponding to either ephrin B1 or integrin β3, respectively. The bottom panel shows the corresponding rRNA loading controls.
Figure 5
Figure 5
Overexpression of Hox B3, but not Hox D3, enhances expression of ephrin A1 in HMEC-1. A, Comparison of HMEC-1 transfected with empty vector (Control) or CMV-driven Hox B3 expression plasmids (Hox B3). Western blots for expression of Hox B3 (top) and ephrin A1 (bottom) from 40 μg total protein lysates from control transfected HMEC-1 or those overexpressing Hox B3. B, Immunoprecipitation of 300 μg of lysates from control of Hox B3 transfected cells using an ephA2 antibody yields similar levels of the ephA2 receptor (top), whereas subsequent blotting with an antiphosphotyrosine antibody shows an increased degree of phosphorylation of the receptor in cells overexpressing Hox B3 (bottom). C, Northern blot analysis of ephrin A1 mRNA levels (top) from 10 μg total RNA isolated from HMEC-1 transfected with either Hox B3 (Hox B3), or with empty vector (Control). Lower panel shows corresponding ribosomal RNA (rRNA) loading control for each cell type. D, Northern blot analysis for β3 integrin and ephrin A1 mRNA levels in 10 μg total RNA isolated from in HMEC-1 transfected with CMV-driven Hox D3 expression plasmids (Hox D3) or empty vector (Control). Lower panel shows corresponding levels of ribosomal RNA (rRNA) visualized with ethidium bromide. E, The influence of Hox gene expression on EC migration. Migration of HMEC-1 transfected with control plasmid (□), Hox B3 (▪), or Hox D3 . ) was assessed after 5 h in modified Boyden chambers coated with 20 μg/ml fibrinogen. Data are expressed as the mean ± SD (n = 3). **P < 0.05.
Figure 5
Figure 5
Overexpression of Hox B3, but not Hox D3, enhances expression of ephrin A1 in HMEC-1. A, Comparison of HMEC-1 transfected with empty vector (Control) or CMV-driven Hox B3 expression plasmids (Hox B3). Western blots for expression of Hox B3 (top) and ephrin A1 (bottom) from 40 μg total protein lysates from control transfected HMEC-1 or those overexpressing Hox B3. B, Immunoprecipitation of 300 μg of lysates from control of Hox B3 transfected cells using an ephA2 antibody yields similar levels of the ephA2 receptor (top), whereas subsequent blotting with an antiphosphotyrosine antibody shows an increased degree of phosphorylation of the receptor in cells overexpressing Hox B3 (bottom). C, Northern blot analysis of ephrin A1 mRNA levels (top) from 10 μg total RNA isolated from HMEC-1 transfected with either Hox B3 (Hox B3), or with empty vector (Control). Lower panel shows corresponding ribosomal RNA (rRNA) loading control for each cell type. D, Northern blot analysis for β3 integrin and ephrin A1 mRNA levels in 10 μg total RNA isolated from in HMEC-1 transfected with CMV-driven Hox D3 expression plasmids (Hox D3) or empty vector (Control). Lower panel shows corresponding levels of ribosomal RNA (rRNA) visualized with ethidium bromide. E, The influence of Hox gene expression on EC migration. Migration of HMEC-1 transfected with control plasmid (□), Hox B3 (▪), or Hox D3 . ) was assessed after 5 h in modified Boyden chambers coated with 20 μg/ml fibrinogen. Data are expressed as the mean ± SD (n = 3). **P < 0.05.
Figure 4
Figure 4
Addition of recombinant ephrin A1/Fc fusion protein helps restore BM-induced capillary morphology in EC lacking Hox B3. A, Immunoprecipitation of EphA2 from 300 μg of protein lysates from HMEC-1 transfected with empty vector (Control) or antisense against Hox B3 (AS) and treated with 250 ng/ml of preclustered ephrin A1 for 20 min. Subsequent blotting with a 1:500 dilution of the polyclonal antibody against EphA2 reveals similar amounts of the 120-kD ephA2 receptor recovered after immunoprecipitation and separation by SDS-PAGE on 7.5% acrylamide gels. B, Duplicate samples from control or Hox B3 antisense transfected cells (AS) with or without the addition of 250 ng/ml of preclustered recombinant ephrin A1 (+ephrinA1) were immunoprecipitated using the anti-EphA2 antibody and separated by SDS-PAGE and blotted with a 1:1,000 dilution of an mAb against phosphotyrosine (anti-PO4 tyr). Both control and Hox B3 AS transfected cells showed a significant increase in levels of phosphotyrosine in the 120-kD band corresponding to immunoprecipitated EphA2. C, Shows morphology of control transfected HMEC-1 (Control), HMEC-1 transfected with antisense against Hox B3 (AS), or antisense cells treated with 250 ng/ml of clustered ephrin A1 (AS +ephrinA1) 18 h after plating of 0.5 × 106 cells in 60-mm culture dishes coated with 400 μl of BM (Matrigel).
Figure 4
Figure 4
Addition of recombinant ephrin A1/Fc fusion protein helps restore BM-induced capillary morphology in EC lacking Hox B3. A, Immunoprecipitation of EphA2 from 300 μg of protein lysates from HMEC-1 transfected with empty vector (Control) or antisense against Hox B3 (AS) and treated with 250 ng/ml of preclustered ephrin A1 for 20 min. Subsequent blotting with a 1:500 dilution of the polyclonal antibody against EphA2 reveals similar amounts of the 120-kD ephA2 receptor recovered after immunoprecipitation and separation by SDS-PAGE on 7.5% acrylamide gels. B, Duplicate samples from control or Hox B3 antisense transfected cells (AS) with or without the addition of 250 ng/ml of preclustered recombinant ephrin A1 (+ephrinA1) were immunoprecipitated using the anti-EphA2 antibody and separated by SDS-PAGE and blotted with a 1:1,000 dilution of an mAb against phosphotyrosine (anti-PO4 tyr). Both control and Hox B3 AS transfected cells showed a significant increase in levels of phosphotyrosine in the 120-kD band corresponding to immunoprecipitated EphA2. C, Shows morphology of control transfected HMEC-1 (Control), HMEC-1 transfected with antisense against Hox B3 (AS), or antisense cells treated with 250 ng/ml of clustered ephrin A1 (AS +ephrinA1) 18 h after plating of 0.5 × 106 cells in 60-mm culture dishes coated with 400 μl of BM (Matrigel).
Figure 6
Figure 6
Retrovirally expressed Hox B3 promotes tumor-induced angiogenesis in vivo. 5 × 106 quail fibrosarcoma-derived viral packaging cells transfected with empty vector (CK) or viral vectors expressing Hox B3 were grafted on 10-d-old CAMs. A and B, The resulting small tumors and associated vasculature in the CAMs in response to grafting 5 × 106 quail fibrosarcoma cells producing empty replication-defective retroviruses (CK). C and D, Increased vascular density associated with tumors produced on the CAM in response to addition of 5 × 106 quail fibrosarcoma cells producing replication-defective retroviruses expressing Hox B3 (CKHoxB3). The arrows show areas of angiogenic sprouting from larger vessels observed primarily in CAMs infected with retrovirus expressing Hox B3.
Figure 7
Figure 7
Increased capillary density correlates retrovirally expressed Hox B3. Immunofluorescence staining of serial 7-μM sections of CAMs harvested 72 h after application of fibrosarcoma cells producing empty retrovirus (CK) or retrovirus expressing Hox B3 with a His-6 epitope tag fused to the COOH terminus (+Hox B3). Staining with an antibody against von Willebrand factor (A and D) shows a relative increase in endothelial cell density in membranes exposed to Hox B3. Staining of serial sections with an antibody against the 6×His epitope tag fused to Hox B3 (B and D) reveals positive staining (arrows) in areas associated with increased vascular density. Areas showing strong autofluorescence within the tumor cores are indicated by the letter t. Corresponding DAPI nuclear staining is shown for both tissues (C and E).

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