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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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Expression of Hox D3, β3 integrin, and uPA in quiescent and proliferative EC. (A) Morphology of HUVEC cultured  in the presence (+) or absence (−) of reconstituted BM after 24 h  in M199 containing 5% FCS. (B) RT-PCR of 1 μg mRNA from  HUVEC cultured for 48 h with (+) or without (−) reconstituted  BM using primers for Hox D3 or GAPDH. (C) Western (top)  and Northern (bottom) blot analysis of integrin β3 expression in  HUVEC cultured in the presence (+) or absence (−) of BM for  48 h. Northern blots were reprobed for uPA mRNA expression.  The bottom panel shows ethidium bromide staining of total RNA  loaded in the corresponding gel.
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Figure 1: Expression of Hox D3, β3 integrin, and uPA in quiescent and proliferative EC. (A) Morphology of HUVEC cultured in the presence (+) or absence (−) of reconstituted BM after 24 h in M199 containing 5% FCS. (B) RT-PCR of 1 μg mRNA from HUVEC cultured for 48 h with (+) or without (−) reconstituted BM using primers for Hox D3 or GAPDH. (C) Western (top) and Northern (bottom) blot analysis of integrin β3 expression in HUVEC cultured in the presence (+) or absence (−) of BM for 48 h. Northern blots were reprobed for uPA mRNA expression. The bottom panel shows ethidium bromide staining of total RNA loaded in the corresponding gel.

Mentions: EC cultured on basement membrane adopt a distinct morphology reminiscent of capillaries in vivo (Fig. 1 A, + BM), withdraw from the cell cycle, and cease DNA synthesis within 24 h (Kubota et al., 1988). This “differentiated phenotype” contrasts the characteristic cobblestone morphology associated with proliferating EC cultured in the absence of BM (Fig. 1 A, −BM). RT-PCR of mRNA from cultured EC detected the presence of a number of Hox gene mRNA's including Hox A4, B3, B4, B5, and D3 (data not shown). Previous studies have linked Hox D3 with expression of β3 integrin mRNA in erythroleukemia cells (Tanaguchi et al., 1995). This β3 subunit is common to integrin αvβ3, which is highly expressed by angiogenic EC (Brooks et al., 1994), and prompted us to examine whether Hox D3 expression was related to the expression of αvβ3 on EC. We therefore performed quantitative RT-PCR analysis of EC cultured in the presence (+) or absence (−) of BM for 36 h. As shown in Fig. 1 B, Hox D3 expression was detected in proliferating EC but not in quiescent EC cultured on BM. Northern and Western blot analysis also shows that high levels of both β3 integrin mRNA and protein were preferentially expressed by proliferating EC. Similarly, uPA mRNA was also highly expressed in proliferating but not quiescent EC (Fig. 1 C). Thus, while quiescent EC do not express detectable levels of Hox D3, β3 integrin, or uPA mRNA, EC in the absence of BM express Hox D3 and genes associated with the angiogenic phenotype.


Induction of the angiogenic phenotype by Hox D3.

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

Expression of Hox D3, β3 integrin, and uPA in quiescent and proliferative EC. (A) Morphology of HUVEC cultured  in the presence (+) or absence (−) of reconstituted BM after 24 h  in M199 containing 5% FCS. (B) RT-PCR of 1 μg mRNA from  HUVEC cultured for 48 h with (+) or without (−) reconstituted  BM using primers for Hox D3 or GAPDH. (C) Western (top)  and Northern (bottom) blot analysis of integrin β3 expression in  HUVEC cultured in the presence (+) or absence (−) of BM for  48 h. Northern blots were reprobed for uPA mRNA expression.  The bottom panel shows ethidium bromide staining of total RNA  loaded in the corresponding gel.
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Related In: Results  -  Collection

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Figure 1: Expression of Hox D3, β3 integrin, and uPA in quiescent and proliferative EC. (A) Morphology of HUVEC cultured in the presence (+) or absence (−) of reconstituted BM after 24 h in M199 containing 5% FCS. (B) RT-PCR of 1 μg mRNA from HUVEC cultured for 48 h with (+) or without (−) reconstituted BM using primers for Hox D3 or GAPDH. (C) Western (top) and Northern (bottom) blot analysis of integrin β3 expression in HUVEC cultured in the presence (+) or absence (−) of BM for 48 h. Northern blots were reprobed for uPA mRNA expression. The bottom panel shows ethidium bromide staining of total RNA loaded in the corresponding gel.
Mentions: EC cultured on basement membrane adopt a distinct morphology reminiscent of capillaries in vivo (Fig. 1 A, + BM), withdraw from the cell cycle, and cease DNA synthesis within 24 h (Kubota et al., 1988). This “differentiated phenotype” contrasts the characteristic cobblestone morphology associated with proliferating EC cultured in the absence of BM (Fig. 1 A, −BM). RT-PCR of mRNA from cultured EC detected the presence of a number of Hox gene mRNA's including Hox A4, B3, B4, B5, and D3 (data not shown). Previous studies have linked Hox D3 with expression of β3 integrin mRNA in erythroleukemia cells (Tanaguchi et al., 1995). This β3 subunit is common to integrin αvβ3, which is highly expressed by angiogenic EC (Brooks et al., 1994), and prompted us to examine whether Hox D3 expression was related to the expression of αvβ3 on EC. We therefore performed quantitative RT-PCR analysis of EC cultured in the presence (+) or absence (−) of BM for 36 h. As shown in Fig. 1 B, Hox D3 expression was detected in proliferating EC but not in quiescent EC cultured on BM. Northern and Western blot analysis also shows that high levels of both β3 integrin mRNA and protein were preferentially expressed by proliferating EC. Similarly, uPA mRNA was also highly expressed in proliferating but not quiescent EC (Fig. 1 C). Thus, while quiescent EC do not express detectable levels of Hox D3, β3 integrin, or uPA mRNA, EC in the absence of BM express Hox D3 and genes associated with the angiogenic phenotype.

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