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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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Colocalization of  Hox D3 and αvβ3 integrin in  endotheliomas in vivo. (A)  H&E-stained cross-section  of tissue infected by Hox D3-expressing retrovirus shows  an abnormally large capillary-like structure filled with  erythrocytes. (B) Immunofluorescent staining with  LM609 against αvβ3 in the  corresponding serial section  shows strong positive staining in both the endothelial  component (ec) of this vascular structure and in hematopoietic cells (h) contained  within. (C) In situ hybridization of retrovirally expressed  Hox D3 in corresponding serial section showing widespread expression in epithelium (ep), endothelial cells  (ec), connective tissue (c),  and hematopoietic cells (h).  (D) Control in situ hybridization in a serial section performed with a sense riboprobe for Hox D3. Bar, 20 μm.
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Figure 5: Colocalization of Hox D3 and αvβ3 integrin in endotheliomas in vivo. (A) H&E-stained cross-section of tissue infected by Hox D3-expressing retrovirus shows an abnormally large capillary-like structure filled with erythrocytes. (B) Immunofluorescent staining with LM609 against αvβ3 in the corresponding serial section shows strong positive staining in both the endothelial component (ec) of this vascular structure and in hematopoietic cells (h) contained within. (C) In situ hybridization of retrovirally expressed Hox D3 in corresponding serial section showing widespread expression in epithelium (ep), endothelial cells (ec), connective tissue (c), and hematopoietic cells (h). (D) Control in situ hybridization in a serial section performed with a sense riboprobe for Hox D3. Bar, 20 μm.

Mentions: During the late stages of angiogenesis, integrin αvβ3 and uPA are down regulated as EC reestablish an intact BM and form a lumen, suggesting that Hox D3 may be required only during the initial stages of angiogenesis. Maintaining expression of Hox D3 therefore might be expected to prolong expression of an invasive phenotype and interfere with normal vascular maturation and/or remodelling. To test this possibility, we constructed replication-defective avian retroviral vectors designed to constitutively express human Hox D3 in vivo. Transfection of a transformed viral packaging cell line Q4dh with Hox D3 proviral vectors yielded a retrovirus capable of infecting proliferating embryonic chick cells and driving expression of human Hox D3 in these cells. When grafted onto 10-d chick embryo CAM's, transformed virus-producing Q4dh cells generate solid tumors in these animals. The continued production of infectious virus leads to infection of adjacent, proliferating cells including tumor-associated EC. As shown in Fig. 5 A, maintenance of Hox D3 expression in these tissues caused blood vessel malformations, reminiscent of endotheliomas, which appeared as abnormal capillary-like structures several times greater in diameter than large capillaries and were filled with hematopoietic cells. A majority of the EC and certain hematopoietic cells within these endotheliomas remained positive for αvβ3, providing evidence that Hox D3 was active in these tissues (Fig. 5 B). To confirm the presence of Hox D3 in these vascular structures, we performed in situ hybridization using probes that detect retrovirally produced human Hox D3 but do not detect endogenous chick Hox genes. Retroviral-mediated Hox D3 expression was observed in a wide variety of tissues including epithelium, connective tissue, hematopoietic cells, and endothelial cells (Fig. 5 C). In contrast to the widespread viral infection and expression of Hox D3, αvβ3 integrin was only detected in EC and some hematopoietic cells, suggesting this expression was highly specific.


Induction of the angiogenic phenotype by Hox D3.

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

Colocalization of  Hox D3 and αvβ3 integrin in  endotheliomas in vivo. (A)  H&E-stained cross-section  of tissue infected by Hox D3-expressing retrovirus shows  an abnormally large capillary-like structure filled with  erythrocytes. (B) Immunofluorescent staining with  LM609 against αvβ3 in the  corresponding serial section  shows strong positive staining in both the endothelial  component (ec) of this vascular structure and in hematopoietic cells (h) contained  within. (C) In situ hybridization of retrovirally expressed  Hox D3 in corresponding serial section showing widespread expression in epithelium (ep), endothelial cells  (ec), connective tissue (c),  and hematopoietic cells (h).  (D) Control in situ hybridization in a serial section performed with a sense riboprobe for Hox D3. Bar, 20 μm.
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Figure 5: Colocalization of Hox D3 and αvβ3 integrin in endotheliomas in vivo. (A) H&E-stained cross-section of tissue infected by Hox D3-expressing retrovirus shows an abnormally large capillary-like structure filled with erythrocytes. (B) Immunofluorescent staining with LM609 against αvβ3 in the corresponding serial section shows strong positive staining in both the endothelial component (ec) of this vascular structure and in hematopoietic cells (h) contained within. (C) In situ hybridization of retrovirally expressed Hox D3 in corresponding serial section showing widespread expression in epithelium (ep), endothelial cells (ec), connective tissue (c), and hematopoietic cells (h). (D) Control in situ hybridization in a serial section performed with a sense riboprobe for Hox D3. Bar, 20 μm.
Mentions: During the late stages of angiogenesis, integrin αvβ3 and uPA are down regulated as EC reestablish an intact BM and form a lumen, suggesting that Hox D3 may be required only during the initial stages of angiogenesis. Maintaining expression of Hox D3 therefore might be expected to prolong expression of an invasive phenotype and interfere with normal vascular maturation and/or remodelling. To test this possibility, we constructed replication-defective avian retroviral vectors designed to constitutively express human Hox D3 in vivo. Transfection of a transformed viral packaging cell line Q4dh with Hox D3 proviral vectors yielded a retrovirus capable of infecting proliferating embryonic chick cells and driving expression of human Hox D3 in these cells. When grafted onto 10-d chick embryo CAM's, transformed virus-producing Q4dh cells generate solid tumors in these animals. The continued production of infectious virus leads to infection of adjacent, proliferating cells including tumor-associated EC. As shown in Fig. 5 A, maintenance of Hox D3 expression in these tissues caused blood vessel malformations, reminiscent of endotheliomas, which appeared as abnormal capillary-like structures several times greater in diameter than large capillaries and were filled with hematopoietic cells. A majority of the EC and certain hematopoietic cells within these endotheliomas remained positive for αvβ3, providing evidence that Hox D3 was active in these tissues (Fig. 5 B). To confirm the presence of Hox D3 in these vascular structures, we performed in situ hybridization using probes that detect retrovirally produced human Hox D3 but do not detect endogenous chick Hox genes. Retroviral-mediated Hox D3 expression was observed in a wide variety of tissues including epithelium, connective tissue, hematopoietic cells, and endothelial cells (Fig. 5 C). In contrast to the widespread viral infection and expression of Hox D3, αvβ3 integrin was only detected in EC and some hematopoietic cells, suggesting this expression was highly specific.

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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Related in: MedlinePlus