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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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Influence of Hox D3 on β3 integrin and uPA expression in endothelial cells. (A) Northern blot analysis of β3 integrin, β5 integrin, uPA, and Hox D3 mRNA levels from 20 μg total RNA from immortalized HUVEC stably transfected with  control (C) or Hox D3 (Hox) expression vectors. The lower panel  shows ethidium bromide staining of total RNA loaded for each  sample in the corresponding gel. (B) Northern blot analysis of β3  integrin, β5 integrin, and uPA mRNA expression levels from immortalized HUVECS transfected with control (C) or antisense  expression vectors for Hox D3 (AS). RNA (20 μg) from each  sample was loaded, and total RNA was visualized by ethidium  bromide staining of the corresponding gel. The lower box shows  RT-PCR of 1 μg total RNA from cells transfected with control  (C) or antisense expression vectors against Hox D3 (AS) using  primers for Hox D3 or GAPDH.
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Figure 2: Influence of Hox D3 on β3 integrin and uPA expression in endothelial cells. (A) Northern blot analysis of β3 integrin, β5 integrin, uPA, and Hox D3 mRNA levels from 20 μg total RNA from immortalized HUVEC stably transfected with control (C) or Hox D3 (Hox) expression vectors. The lower panel shows ethidium bromide staining of total RNA loaded for each sample in the corresponding gel. (B) Northern blot analysis of β3 integrin, β5 integrin, and uPA mRNA expression levels from immortalized HUVECS transfected with control (C) or antisense expression vectors for Hox D3 (AS). RNA (20 μg) from each sample was loaded, and total RNA was visualized by ethidium bromide staining of the corresponding gel. The lower box shows RT-PCR of 1 μg total RNA from cells transfected with control (C) or antisense expression vectors against Hox D3 (AS) using primers for Hox D3 or GAPDH.

Mentions: To determine whether the expression of Hox D3 influences expression of genes associated with the angiogenic phenotype of EC, a HUVEC cell line was stably transfected with a Hox D3 expression vector. Northern blot analysis shows that HUVECs overexpressing Hox D3 have significantly higher levels of steady-state β3 integrin and uPA mRNA levels as compared to cells transfected with control vectors. In contrast, mRNA encoding integrin β5, which is not upreglated during angiogenesis, did not differ in control or Hox D3-transfected cells (Fig. 2 A). To establish whether Hox D3 was required for EC expression of β3 integrin and uPA, HUVECs were stably transfected with Hox D3 antisense expression vectors. Compared to control-transfected cells, baseline levels of both β3 integrin and uPA mRNA were significantly reduced in cells expressing Hox D3 antisense. In contrast, levels of β5 integrin mRNA were not influenced by expression of Hox D3 antisense (Fig. 2 B).


Induction of the angiogenic phenotype by Hox D3.

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

Influence of Hox D3 on β3 integrin and uPA expression in endothelial cells. (A) Northern blot analysis of β3 integrin, β5 integrin, uPA, and Hox D3 mRNA levels from 20 μg total RNA from immortalized HUVEC stably transfected with  control (C) or Hox D3 (Hox) expression vectors. The lower panel  shows ethidium bromide staining of total RNA loaded for each  sample in the corresponding gel. (B) Northern blot analysis of β3  integrin, β5 integrin, and uPA mRNA expression levels from immortalized HUVECS transfected with control (C) or antisense  expression vectors for Hox D3 (AS). RNA (20 μg) from each  sample was loaded, and total RNA was visualized by ethidium  bromide staining of the corresponding gel. The lower box shows  RT-PCR of 1 μg total RNA from cells transfected with control  (C) or antisense expression vectors against Hox D3 (AS) using  primers for Hox D3 or GAPDH.
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Related In: Results  -  Collection

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Figure 2: Influence of Hox D3 on β3 integrin and uPA expression in endothelial cells. (A) Northern blot analysis of β3 integrin, β5 integrin, uPA, and Hox D3 mRNA levels from 20 μg total RNA from immortalized HUVEC stably transfected with control (C) or Hox D3 (Hox) expression vectors. The lower panel shows ethidium bromide staining of total RNA loaded for each sample in the corresponding gel. (B) Northern blot analysis of β3 integrin, β5 integrin, and uPA mRNA expression levels from immortalized HUVECS transfected with control (C) or antisense expression vectors for Hox D3 (AS). RNA (20 μg) from each sample was loaded, and total RNA was visualized by ethidium bromide staining of the corresponding gel. The lower box shows RT-PCR of 1 μg total RNA from cells transfected with control (C) or antisense expression vectors against Hox D3 (AS) using primers for Hox D3 or GAPDH.
Mentions: To determine whether the expression of Hox D3 influences expression of genes associated with the angiogenic phenotype of EC, a HUVEC cell line was stably transfected with a Hox D3 expression vector. Northern blot analysis shows that HUVECs overexpressing Hox D3 have significantly higher levels of steady-state β3 integrin and uPA mRNA levels as compared to cells transfected with control vectors. In contrast, mRNA encoding integrin β5, which is not upreglated during angiogenesis, did not differ in control or Hox D3-transfected cells (Fig. 2 A). To establish whether Hox D3 was required for EC expression of β3 integrin and uPA, HUVECs were stably transfected with Hox D3 antisense expression vectors. Compared to control-transfected cells, baseline levels of both β3 integrin and uPA mRNA were significantly reduced in cells expressing Hox D3 antisense. In contrast, levels of β5 integrin mRNA were not influenced by expression of Hox D3 antisense (Fig. 2 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