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Neural deletion of Tgfbr2 impairs angiogenesis through an altered secretome.

Hellbach N, Weise SC, Vezzali R, Wahane SD, Heidrich S, Roidl D, Pruszak J, Esser JS, Vogel T - Hum. Mol. Genet. (2014)

Bottom Line: Blood vessels exhibited an atypical, clustered appearance were less in number and displayed reduced branching.HUVEC showed reduced migration towards CM of mutants compared with controls.These findings will be useful to further elucidate neurovascular interaction in general and to understand pathologies of the blood vessel system such as intracerebral haemorrhages, hereditary haemorrhagic telangiectasia, Alzheimeŕs disease, cerebral amyloid angiopathy or tumour biology.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University of Freiburg, 79104 Freiburg, Germany, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany.

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Impaired HUVEC migration through altered secretion of VEGFA, FGF2, IGF1, -2 and TGFβ. (A) Box-plot of end-point analyses of RTCA revealed reduced migration of HUVEC towards CM_cKO (red) compared with CM_Ctrl (green) of DT and VT (n = 20). The values for H_NB_suppl were set to 100% to normalize between different experiments and are therefore represented as an invariant grey line. (B and C) Growth curves of one representative experiment from DT and VT, respectively. Red line: CM_cKO, green line: CM_ctrl, grey line: H_NB_suppl control condition. (D and E) Box-plot representation of RTCA end-point analyses after supplementation with VEGFA alone, or in combination with FGF2. Addition of VEGFA alone rescued migration towards CM_cKO in VT-derived samples, but not in DT-derived samples (dark red). This is indicated by the loss of a significant difference between the treatment and control condition. Combined addition of VEGFA and FGF2 rescued migration in DT-derived samples, but not in VT-derived samples (light brown) (n = 3 for all experiments). (F and G) Box-plot of RTCA end-point analyses after supplementation with FGF2 alone or in combination with VEGFA. Addition of FGF2 rescued migration towards both VT- and DT-derived CM_cKO (dark red and light brown), also in the presence of VEGFA (n = 3 for all experiments). (H and I) Box-plot of RTCA end-point analyses after IGF1 addition rescued migration in DT-derived medium but had no significant effect in VT-derived medium (dark red). Combined addition of IGF1 with VEGFA and FGF2 did not improve migration further (light brown) (n = 3 DT, n = 4 VT). (J and K) Box-plot of RTCA end-point analyses after IGF2 supplementation. IGF2 addition alone did not rescue migration towards CM_cKO from both sources. Combined addition of IGF2 with VEGFA and FGF2 rescued in VT-derived CM_cKO (n = 4 DT, n = 5 VT). (L–M) Box-plot of end-point analyses after TGFβ1 supplementation, which impaired migration in the CM_Ctrl conditions, most strikingly in the presence of VEGFA and FGF2 (light and dark blue, n = 4). Data in A–M were normalized to H_NB_suppl (grey) of the respective experiment. All data were analysed by one-way ANOVA, followed by Sidak post-test.
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DDU338F7: Impaired HUVEC migration through altered secretion of VEGFA, FGF2, IGF1, -2 and TGFβ. (A) Box-plot of end-point analyses of RTCA revealed reduced migration of HUVEC towards CM_cKO (red) compared with CM_Ctrl (green) of DT and VT (n = 20). The values for H_NB_suppl were set to 100% to normalize between different experiments and are therefore represented as an invariant grey line. (B and C) Growth curves of one representative experiment from DT and VT, respectively. Red line: CM_cKO, green line: CM_ctrl, grey line: H_NB_suppl control condition. (D and E) Box-plot representation of RTCA end-point analyses after supplementation with VEGFA alone, or in combination with FGF2. Addition of VEGFA alone rescued migration towards CM_cKO in VT-derived samples, but not in DT-derived samples (dark red). This is indicated by the loss of a significant difference between the treatment and control condition. Combined addition of VEGFA and FGF2 rescued migration in DT-derived samples, but not in VT-derived samples (light brown) (n = 3 for all experiments). (F and G) Box-plot of RTCA end-point analyses after supplementation with FGF2 alone or in combination with VEGFA. Addition of FGF2 rescued migration towards both VT- and DT-derived CM_cKO (dark red and light brown), also in the presence of VEGFA (n = 3 for all experiments). (H and I) Box-plot of RTCA end-point analyses after IGF1 addition rescued migration in DT-derived medium but had no significant effect in VT-derived medium (dark red). Combined addition of IGF1 with VEGFA and FGF2 did not improve migration further (light brown) (n = 3 DT, n = 4 VT). (J and K) Box-plot of RTCA end-point analyses after IGF2 supplementation. IGF2 addition alone did not rescue migration towards CM_cKO from both sources. Combined addition of IGF2 with VEGFA and FGF2 rescued in VT-derived CM_cKO (n = 4 DT, n = 5 VT). (L–M) Box-plot of end-point analyses after TGFβ1 supplementation, which impaired migration in the CM_Ctrl conditions, most strikingly in the presence of VEGFA and FGF2 (light and dark blue, n = 4). Data in A–M were normalized to H_NB_suppl (grey) of the respective experiment. All data were analysed by one-way ANOVA, followed by Sidak post-test.

Mentions: Using real-time cell analysis (RTCA), we studied migration of HUVEC towards CM of both control and Tgfbr2-cKO and observed that migration was generally less compared with control conditions (H_NB_suppl) (Fig. 7A–C). HUVEC showed reduced chemotaxis towards Tgfbr2-cKO CM as compared with CM from controls. CM_cKO from VT interfered even more with migration in comparison with CM from DT (Fig. 7A–C). To study the contribution of several factors independently, we supplemented the CM with VEGFA, FGF2, IGF1, -2, TGFβ1, THBS2 and ADAMTS1, respectively, and assessed for potential restoration of HUVEC migration. End-point analyses of several independent RTCA experiments revealed that double supplementation with VEGFA and FGF2 significantly increased HUVEC migration towards CM_cKO and thus rescued the defect (Fig. 7D–G, Supplementary Material, Fig. S9Ca, b). Supplementation of CM from control cells with VEGFA and FGF2 together significantly improved migration in DT-derived cells as well, but had no effect on migration when applied to VT-derived cells. We also assessed the contribution of each factor individually. Addition of VEGFA improved migration towards DT—but not towards VT-derived CM from Tgfbr2-cKO. Supplementation of DT-derived CM_cKO resulted in near complete rescue onto the CM_Ctrl level (Fig. 7D and E, Supplementary Material, Fig. S9Cc, d). Addition of FGF2 alone improved migration to cKO_DT/VT, but not up to non-supplemented Ctrl_DT/VT conditions (Fig. 7F and G, Supplementary Material, Fig. S9Ce, f). Hence, the best condition to restore migration ability in Tgfbr2-cKO was a double supplementation with VEGFA and FGF2. Next, we supplemented the CM with IGF1 and -2, as we had observed increased expression of these factors in Tgfbr2-cKO in vivo. IGF1 increased migration of HUVEC only towards DT-derived medium, whereas addition to VT-derived medium had only a moderate effect. In DT-derived CM_cKO, but not in VT-derived CM_cKO, IGF1 supplementation rescued HUVEC migration even above CM_Ctrl condition (Fig. 7H and I, Supplementary Material, Fig. S9Cg, h). Addition of IGF2 had little effect under control conditions (CM_Ctrl_DT/VT). However, in accordance with existing literature (55), increasing amounts of IGF2 in CM_Ctrl_VT also displayed the pro-angiogenic effects of IGF2 using the RTCA assay (Supplementary Material, Fig. S9Aa, b). In CM from Tgfbr2_cKO, IGF2 improved migration towards VT-derived, but had no effect on DT-derived CM_cKO (Fig. 7J and K, Supplementary Material, Fig. S9Ci, j). In vivo, we discovered increased expression of IGF1 and -2 ligands. The potential to rescue the migration behaviour of HUVEC by addition of exogenous IGF in these experiments was therefore surprising. We hypothesized that bioavailability of the IGF ligands might be limited because of simultaneous increased expression levels of IGFBP2 and IGF2BP1, which might bind and sequester the IGF ligands. Addition of either IGFBP2 or IGF2BP1 did not have an observable impact on HUVEC migration towards mutant or control CM. Furthermore, the simultaneous addition of the ligands and binding proteins did not change migration behaviour (Supplementary Material, Fig. S9Da–d). These data suggested that the increased expression of IGFBP2 and IGF2BP1 did not contribute to impaired endothelial migration.Figure 7.


Neural deletion of Tgfbr2 impairs angiogenesis through an altered secretome.

Hellbach N, Weise SC, Vezzali R, Wahane SD, Heidrich S, Roidl D, Pruszak J, Esser JS, Vogel T - Hum. Mol. Genet. (2014)

Impaired HUVEC migration through altered secretion of VEGFA, FGF2, IGF1, -2 and TGFβ. (A) Box-plot of end-point analyses of RTCA revealed reduced migration of HUVEC towards CM_cKO (red) compared with CM_Ctrl (green) of DT and VT (n = 20). The values for H_NB_suppl were set to 100% to normalize between different experiments and are therefore represented as an invariant grey line. (B and C) Growth curves of one representative experiment from DT and VT, respectively. Red line: CM_cKO, green line: CM_ctrl, grey line: H_NB_suppl control condition. (D and E) Box-plot representation of RTCA end-point analyses after supplementation with VEGFA alone, or in combination with FGF2. Addition of VEGFA alone rescued migration towards CM_cKO in VT-derived samples, but not in DT-derived samples (dark red). This is indicated by the loss of a significant difference between the treatment and control condition. Combined addition of VEGFA and FGF2 rescued migration in DT-derived samples, but not in VT-derived samples (light brown) (n = 3 for all experiments). (F and G) Box-plot of RTCA end-point analyses after supplementation with FGF2 alone or in combination with VEGFA. Addition of FGF2 rescued migration towards both VT- and DT-derived CM_cKO (dark red and light brown), also in the presence of VEGFA (n = 3 for all experiments). (H and I) Box-plot of RTCA end-point analyses after IGF1 addition rescued migration in DT-derived medium but had no significant effect in VT-derived medium (dark red). Combined addition of IGF1 with VEGFA and FGF2 did not improve migration further (light brown) (n = 3 DT, n = 4 VT). (J and K) Box-plot of RTCA end-point analyses after IGF2 supplementation. IGF2 addition alone did not rescue migration towards CM_cKO from both sources. Combined addition of IGF2 with VEGFA and FGF2 rescued in VT-derived CM_cKO (n = 4 DT, n = 5 VT). (L–M) Box-plot of end-point analyses after TGFβ1 supplementation, which impaired migration in the CM_Ctrl conditions, most strikingly in the presence of VEGFA and FGF2 (light and dark blue, n = 4). Data in A–M were normalized to H_NB_suppl (grey) of the respective experiment. All data were analysed by one-way ANOVA, followed by Sidak post-test.
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DDU338F7: Impaired HUVEC migration through altered secretion of VEGFA, FGF2, IGF1, -2 and TGFβ. (A) Box-plot of end-point analyses of RTCA revealed reduced migration of HUVEC towards CM_cKO (red) compared with CM_Ctrl (green) of DT and VT (n = 20). The values for H_NB_suppl were set to 100% to normalize between different experiments and are therefore represented as an invariant grey line. (B and C) Growth curves of one representative experiment from DT and VT, respectively. Red line: CM_cKO, green line: CM_ctrl, grey line: H_NB_suppl control condition. (D and E) Box-plot representation of RTCA end-point analyses after supplementation with VEGFA alone, or in combination with FGF2. Addition of VEGFA alone rescued migration towards CM_cKO in VT-derived samples, but not in DT-derived samples (dark red). This is indicated by the loss of a significant difference between the treatment and control condition. Combined addition of VEGFA and FGF2 rescued migration in DT-derived samples, but not in VT-derived samples (light brown) (n = 3 for all experiments). (F and G) Box-plot of RTCA end-point analyses after supplementation with FGF2 alone or in combination with VEGFA. Addition of FGF2 rescued migration towards both VT- and DT-derived CM_cKO (dark red and light brown), also in the presence of VEGFA (n = 3 for all experiments). (H and I) Box-plot of RTCA end-point analyses after IGF1 addition rescued migration in DT-derived medium but had no significant effect in VT-derived medium (dark red). Combined addition of IGF1 with VEGFA and FGF2 did not improve migration further (light brown) (n = 3 DT, n = 4 VT). (J and K) Box-plot of RTCA end-point analyses after IGF2 supplementation. IGF2 addition alone did not rescue migration towards CM_cKO from both sources. Combined addition of IGF2 with VEGFA and FGF2 rescued in VT-derived CM_cKO (n = 4 DT, n = 5 VT). (L–M) Box-plot of end-point analyses after TGFβ1 supplementation, which impaired migration in the CM_Ctrl conditions, most strikingly in the presence of VEGFA and FGF2 (light and dark blue, n = 4). Data in A–M were normalized to H_NB_suppl (grey) of the respective experiment. All data were analysed by one-way ANOVA, followed by Sidak post-test.
Mentions: Using real-time cell analysis (RTCA), we studied migration of HUVEC towards CM of both control and Tgfbr2-cKO and observed that migration was generally less compared with control conditions (H_NB_suppl) (Fig. 7A–C). HUVEC showed reduced chemotaxis towards Tgfbr2-cKO CM as compared with CM from controls. CM_cKO from VT interfered even more with migration in comparison with CM from DT (Fig. 7A–C). To study the contribution of several factors independently, we supplemented the CM with VEGFA, FGF2, IGF1, -2, TGFβ1, THBS2 and ADAMTS1, respectively, and assessed for potential restoration of HUVEC migration. End-point analyses of several independent RTCA experiments revealed that double supplementation with VEGFA and FGF2 significantly increased HUVEC migration towards CM_cKO and thus rescued the defect (Fig. 7D–G, Supplementary Material, Fig. S9Ca, b). Supplementation of CM from control cells with VEGFA and FGF2 together significantly improved migration in DT-derived cells as well, but had no effect on migration when applied to VT-derived cells. We also assessed the contribution of each factor individually. Addition of VEGFA improved migration towards DT—but not towards VT-derived CM from Tgfbr2-cKO. Supplementation of DT-derived CM_cKO resulted in near complete rescue onto the CM_Ctrl level (Fig. 7D and E, Supplementary Material, Fig. S9Cc, d). Addition of FGF2 alone improved migration to cKO_DT/VT, but not up to non-supplemented Ctrl_DT/VT conditions (Fig. 7F and G, Supplementary Material, Fig. S9Ce, f). Hence, the best condition to restore migration ability in Tgfbr2-cKO was a double supplementation with VEGFA and FGF2. Next, we supplemented the CM with IGF1 and -2, as we had observed increased expression of these factors in Tgfbr2-cKO in vivo. IGF1 increased migration of HUVEC only towards DT-derived medium, whereas addition to VT-derived medium had only a moderate effect. In DT-derived CM_cKO, but not in VT-derived CM_cKO, IGF1 supplementation rescued HUVEC migration even above CM_Ctrl condition (Fig. 7H and I, Supplementary Material, Fig. S9Cg, h). Addition of IGF2 had little effect under control conditions (CM_Ctrl_DT/VT). However, in accordance with existing literature (55), increasing amounts of IGF2 in CM_Ctrl_VT also displayed the pro-angiogenic effects of IGF2 using the RTCA assay (Supplementary Material, Fig. S9Aa, b). In CM from Tgfbr2_cKO, IGF2 improved migration towards VT-derived, but had no effect on DT-derived CM_cKO (Fig. 7J and K, Supplementary Material, Fig. S9Ci, j). In vivo, we discovered increased expression of IGF1 and -2 ligands. The potential to rescue the migration behaviour of HUVEC by addition of exogenous IGF in these experiments was therefore surprising. We hypothesized that bioavailability of the IGF ligands might be limited because of simultaneous increased expression levels of IGFBP2 and IGF2BP1, which might bind and sequester the IGF ligands. Addition of either IGFBP2 or IGF2BP1 did not have an observable impact on HUVEC migration towards mutant or control CM. Furthermore, the simultaneous addition of the ligands and binding proteins did not change migration behaviour (Supplementary Material, Fig. S9Da–d). These data suggested that the increased expression of IGFBP2 and IGF2BP1 did not contribute to impaired endothelial migration.Figure 7.

Bottom Line: Blood vessels exhibited an atypical, clustered appearance were less in number and displayed reduced branching.HUVEC showed reduced migration towards CM of mutants compared with controls.These findings will be useful to further elucidate neurovascular interaction in general and to understand pathologies of the blood vessel system such as intracerebral haemorrhages, hereditary haemorrhagic telangiectasia, Alzheimeŕs disease, cerebral amyloid angiopathy or tumour biology.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University of Freiburg, 79104 Freiburg, Germany, Faculty of Biology, University of Freiburg, 79104 Freiburg, Germany.

Show MeSH
Related in: MedlinePlus