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Induction of the angiogenic phenotype by Hox D3.

Boudreau N, Andrews C, Srebrow A, Ravanpay A, Cheresh DA - J. Cell Biol. (1997)

Bottom Line: Expression of Hox D3, in the absence of bFGF, resulted in enhanced expression of integrin alphavbeta3 and uPA.In fact, sustained expression of Hox D3 in vivo on the chick chorioallantoic membrane retained EC in this invasive state and prevented vessel maturation leading to vascular malformations and endotheliomas.Therefore, Hox D3 regulates EC gene expression associated with the invasive stage of angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, USA. nboudrea@nsc.vcu.edu

ABSTRACT
Angiogenesis is characterized by distinct phenotypic changes in vascular endothelial cells (EC). Evidence is provided that the Hox D3 homeobox gene mediates conversion of endothelium from the resting to the angiogenic/invasive state. Stimulation of EC with basic fibroblast growth factor (bFGF) resulted in increased expression of Hox D3, integrin alphavbeta3, and the urokinase plasminogen activator (uPA). Hox D3 antisense blocked the ability of bFGF to induce uPA and integrin alphavbeta3 expression, yet had no effect on EC cell proliferation or bFGF-mediated cyclin D1 expression. Expression of Hox D3, in the absence of bFGF, resulted in enhanced expression of integrin alphavbeta3 and uPA. In fact, sustained expression of Hox D3 in vivo on the chick chorioallantoic membrane retained EC in this invasive state and prevented vessel maturation leading to vascular malformations and endotheliomas. Therefore, Hox D3 regulates EC gene expression associated with the invasive stage of angiogenesis.

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Hox D3 mediates bFGF-induced expression of β3 integrin and uPA. (A) RT-PCR of 1 μg total RNA from HUVEC 24 h after  treatment with 20 ng/ml bFGF (+bFGF) using primers for Hox D3 or GAPDH. (B) Northern blot analysis of β3 integrin, β5 integrin,  and uPA mRNA levels in HUVEC transfected with control plasmid (C) or antisense against Hox D3 (AS) after 24 h with (+) or without (−) addition of 20 ng/ml bFGF. (C) Western blot of cyclin D1 in immortalized HUVEC transfected with control or antisense against  Hox D3. After 24 h in serum-free M199, samples were collected at 2, 4, and 8 h after addition of 20 ng/ml bFGF. 10 μg total cell lysate  from each time point was separated on 12% SDS-PAGE, transferred to nylon membranes, and probed with a polyclonal antibody  against human cyclin D1.
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Figure 4: Hox D3 mediates bFGF-induced expression of β3 integrin and uPA. (A) RT-PCR of 1 μg total RNA from HUVEC 24 h after treatment with 20 ng/ml bFGF (+bFGF) using primers for Hox D3 or GAPDH. (B) Northern blot analysis of β3 integrin, β5 integrin, and uPA mRNA levels in HUVEC transfected with control plasmid (C) or antisense against Hox D3 (AS) after 24 h with (+) or without (−) addition of 20 ng/ml bFGF. (C) Western blot of cyclin D1 in immortalized HUVEC transfected with control or antisense against Hox D3. After 24 h in serum-free M199, samples were collected at 2, 4, and 8 h after addition of 20 ng/ml bFGF. 10 μg total cell lysate from each time point was separated on 12% SDS-PAGE, transferred to nylon membranes, and probed with a polyclonal antibody against human cyclin D1.

Mentions: The angiogenic cytokine bFGF leads to expression of both uPA and β3 integrin in EC, which subsequently contribute to the invasive properties of EC during the early stages of angiogenesis (Moscatelli et al., 1985; Enenstein et al., 1992; Brooks et al., 1994; Sepp et al., 1994). To determine whether Hox D3 could influence the bFGF-mediated induction of integrin αvβ3 and uPA, EC were made quiescent by culturing on BM and treating with or without bFGF and analyzed for expression of mRNA encoding these proteins. As shown in Fig. 4, exposure of HUVECs to bFGF led to an increase in Hox D3 expression after 8 h, which was accompanied by increased β3 integrin and uPA steady-state mRNA levels by 24 h (Fig. 4, A and B). In contrast, in EC expressing antisense against Hox D3, the ability of bFGF to induce high levels of expression of β3 integrin and uPA mRNA expression was attenuated. Importantly, integrin β5 mRNA was not altered in either the control or antisense-transfected cells in the presence or absence of bFGF (Fig. 4, A and B).


Induction of the angiogenic phenotype by Hox D3.

Boudreau N, Andrews C, Srebrow A, Ravanpay A, Cheresh DA - J. Cell Biol. (1997)

Hox D3 mediates bFGF-induced expression of β3 integrin and uPA. (A) RT-PCR of 1 μg total RNA from HUVEC 24 h after  treatment with 20 ng/ml bFGF (+bFGF) using primers for Hox D3 or GAPDH. (B) Northern blot analysis of β3 integrin, β5 integrin,  and uPA mRNA levels in HUVEC transfected with control plasmid (C) or antisense against Hox D3 (AS) after 24 h with (+) or without (−) addition of 20 ng/ml bFGF. (C) Western blot of cyclin D1 in immortalized HUVEC transfected with control or antisense against  Hox D3. After 24 h in serum-free M199, samples were collected at 2, 4, and 8 h after addition of 20 ng/ml bFGF. 10 μg total cell lysate  from each time point was separated on 12% SDS-PAGE, transferred to nylon membranes, and probed with a polyclonal antibody  against human cyclin D1.
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Related In: Results  -  Collection

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Figure 4: Hox D3 mediates bFGF-induced expression of β3 integrin and uPA. (A) RT-PCR of 1 μg total RNA from HUVEC 24 h after treatment with 20 ng/ml bFGF (+bFGF) using primers for Hox D3 or GAPDH. (B) Northern blot analysis of β3 integrin, β5 integrin, and uPA mRNA levels in HUVEC transfected with control plasmid (C) or antisense against Hox D3 (AS) after 24 h with (+) or without (−) addition of 20 ng/ml bFGF. (C) Western blot of cyclin D1 in immortalized HUVEC transfected with control or antisense against Hox D3. After 24 h in serum-free M199, samples were collected at 2, 4, and 8 h after addition of 20 ng/ml bFGF. 10 μg total cell lysate from each time point was separated on 12% SDS-PAGE, transferred to nylon membranes, and probed with a polyclonal antibody against human cyclin D1.
Mentions: The angiogenic cytokine bFGF leads to expression of both uPA and β3 integrin in EC, which subsequently contribute to the invasive properties of EC during the early stages of angiogenesis (Moscatelli et al., 1985; Enenstein et al., 1992; Brooks et al., 1994; Sepp et al., 1994). To determine whether Hox D3 could influence the bFGF-mediated induction of integrin αvβ3 and uPA, EC were made quiescent by culturing on BM and treating with or without bFGF and analyzed for expression of mRNA encoding these proteins. As shown in Fig. 4, exposure of HUVECs to bFGF led to an increase in Hox D3 expression after 8 h, which was accompanied by increased β3 integrin and uPA steady-state mRNA levels by 24 h (Fig. 4, A and B). In contrast, in EC expressing antisense against Hox D3, the ability of bFGF to induce high levels of expression of β3 integrin and uPA mRNA expression was attenuated. Importantly, integrin β5 mRNA was not altered in either the control or antisense-transfected cells in the presence or absence of bFGF (Fig. 4, A and B).

Bottom Line: Expression of Hox D3, in the absence of bFGF, resulted in enhanced expression of integrin alphavbeta3 and uPA.In fact, sustained expression of Hox D3 in vivo on the chick chorioallantoic membrane retained EC in this invasive state and prevented vessel maturation leading to vascular malformations and endotheliomas.Therefore, Hox D3 regulates EC gene expression associated with the invasive stage of angiogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, USA. nboudrea@nsc.vcu.edu

ABSTRACT
Angiogenesis is characterized by distinct phenotypic changes in vascular endothelial cells (EC). Evidence is provided that the Hox D3 homeobox gene mediates conversion of endothelium from the resting to the angiogenic/invasive state. Stimulation of EC with basic fibroblast growth factor (bFGF) resulted in increased expression of Hox D3, integrin alphavbeta3, and the urokinase plasminogen activator (uPA). Hox D3 antisense blocked the ability of bFGF to induce uPA and integrin alphavbeta3 expression, yet had no effect on EC cell proliferation or bFGF-mediated cyclin D1 expression. Expression of Hox D3, in the absence of bFGF, resulted in enhanced expression of integrin alphavbeta3 and uPA. In fact, sustained expression of Hox D3 in vivo on the chick chorioallantoic membrane retained EC in this invasive state and prevented vessel maturation leading to vascular malformations and endotheliomas. Therefore, Hox D3 regulates EC gene expression associated with the invasive stage of angiogenesis.

Show MeSH
Related in: MedlinePlus