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Lymphangiogenic gene therapy with minimal blood vascular side effects.

Saaristo A, Veikkola T, Tammela T, Enholm B, Karkkainen MJ, Pajusola K, Bueler H, Ylä-Herttuala S, Alitalo K - J. Exp. Med. (2002)

Bottom Line: Recent work from many laboratories has demonstrated that the vascular endothelial growth factor-C/VEGF-D/VEGFR-3 signaling pathway is crucial for lymphangiogenesis, and that mutations of the Vegfr3 gene are associated with hereditary lymphedema.However, as is the case with VEGF, high levels of VEGF-C cause blood vessel growth and leakiness, resulting in tissue edema.Importantly, adenoviral VEGF-C156S lacked the blood vascular side effects of VEGF and VEGF-C adenoviruses.

View Article: PubMed Central - PubMed

Affiliation: Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research, Biomedicum Helsinki, Helsinki University Central Hospital, University of Helsinki, Finland.

ABSTRACT
Recent work from many laboratories has demonstrated that the vascular endothelial growth factor-C/VEGF-D/VEGFR-3 signaling pathway is crucial for lymphangiogenesis, and that mutations of the Vegfr3 gene are associated with hereditary lymphedema. Furthermore, VEGF-C gene transfer to the skin of mice with lymphedema induced a regeneration of the cutaneous lymphatic vessel network. However, as is the case with VEGF, high levels of VEGF-C cause blood vessel growth and leakiness, resulting in tissue edema. To avoid these blood vascular side effects of VEGF-C, we constructed a viral vector for a VEGFR-3-specific mutant form of VEGF-C (VEGF-C156S) for lymphedema gene therapy. We demonstrate that VEGF-C156S potently induces lymphangiogenesis in transgenic mouse embryos, and when applied via viral gene transfer, in normal and lymphedema mice. Importantly, adenoviral VEGF-C156S lacked the blood vascular side effects of VEGF and VEGF-C adenoviruses. In particular, in the lymphedema mice functional cutaneous lymphatic vessels of normal caliber and morphology were detected after long-term expression of VEGF-C156S via an adeno associated virus. These results have important implications for the development of gene therapy for human lymphedema.

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Lymphangiogen-esis in AdVEGF-C156S and AdVEGF-C infected adult skin. Whole mount VEGFR-3 staining of the lymphatic vessels 1 wk (A–C) and 2 wk (D–F) after infection. Note enlarged lymphatic vessels (arrows) and sprouting or splitting lymphatic vessels (arrowheads) in AdVEGF-C156S (A) and AdVEGF-C (B) infected ears. 2 wk after infection, AdVEGF-C156S infected ear (D) contains large lymphatic vessels apparently still undergoing sprouting and splitting, whereas AdVEGF-C (E) infected ear is filled with small lymphatic sprouts. (G–I) Podoplanin stained tissue sections at 2 wk after adenoviral infection. Note the formation of a hyperplastic lymphatic network in response to adenoviral VEGF-C156S (G) and VEGF-C (H) compared with AdLacZ control (I). Sprouting and splitting of the vessels is especially clear in the AdVEGF-C infected ear (H). Scale bars: A–C, 60 μm; D–F, 220 μm; G–I, 70 μm.
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fig3: Lymphangiogen-esis in AdVEGF-C156S and AdVEGF-C infected adult skin. Whole mount VEGFR-3 staining of the lymphatic vessels 1 wk (A–C) and 2 wk (D–F) after infection. Note enlarged lymphatic vessels (arrows) and sprouting or splitting lymphatic vessels (arrowheads) in AdVEGF-C156S (A) and AdVEGF-C (B) infected ears. 2 wk after infection, AdVEGF-C156S infected ear (D) contains large lymphatic vessels apparently still undergoing sprouting and splitting, whereas AdVEGF-C (E) infected ear is filled with small lymphatic sprouts. (G–I) Podoplanin stained tissue sections at 2 wk after adenoviral infection. Note the formation of a hyperplastic lymphatic network in response to adenoviral VEGF-C156S (G) and VEGF-C (H) compared with AdLacZ control (I). Sprouting and splitting of the vessels is especially clear in the AdVEGF-C infected ear (H). Scale bars: A–C, 60 μm; D–F, 220 μm; G–I, 70 μm.

Mentions: We then wanted to compare the lymphangiogenesis induced by viral delivery of VEGF-C156S and VEGF-C. For this purpose, adenoviruses encoding VEGF-C156S, VEGF-C, or LacZ were injected intradermally into the ears of nu/nu mice. The first sprouting lymphatic vessels were detected in the infected ear skin by VEGFR-3 whole mount staining 3 to 4 d after the adenoviral infection. The lymphatic sprouting was more abundant in the AdVEGF-C–infected skin than in the AdVEGF-C156S–infected skin at all time points studied. In the AdVEGF-C156S–infected skin the lymphatic vessels enlarged progressively, and 1 wk after the infection several lymphatic vessels appeared to be splitting to form new daughter vessels (Fig. 3 A). At this time point nearly all lymphatic vessels in the AdVEGF-C–infected skin exhibited sprouting and bridging; some enlarged vessels were also detected, whereas no signs of lymphangiogenesis were seen in the AdLacZ-infected control skin (Fig. 3, B and C). 2 wk after AdVEGF-C156S infection the lymphatic vessels network was vastly enlarged and most of the large vessels had formed new sprouts or bridges (Fig. 3 D). At the same time point the AdVEGF-C–infected skin was filled with very actively sprouting, relatively small lymphatic vessels, while only a few enlarged lymphatic vessels were detected (Fig. 3 E). A few new lymphatic sprouts were detected in only one AdLacZ-infected control mouse around the injection wound (n = 15; unpublished data), otherwise the lymphatic vasculature in the control skin remained unaltered (Fig. 3 F). The staining of histological sections for VEGFR-3, LYVE-1, and podoplanin confirmed that the sprouting of the lymphatic vessels was more efficient in the AdVEGF-C than in the AdVEGF-C156S infected samples (Fig. 3, G–I, and unpublished data). The newly formed lymphatic vessels regressed gradually with time as expression of the viral genes was downregulated, and by 8 t to 10 wk after the infection the lymphatic vessel network had returned to its normal architecture (unpublished data).


Lymphangiogenic gene therapy with minimal blood vascular side effects.

Saaristo A, Veikkola T, Tammela T, Enholm B, Karkkainen MJ, Pajusola K, Bueler H, Ylä-Herttuala S, Alitalo K - J. Exp. Med. (2002)

Lymphangiogen-esis in AdVEGF-C156S and AdVEGF-C infected adult skin. Whole mount VEGFR-3 staining of the lymphatic vessels 1 wk (A–C) and 2 wk (D–F) after infection. Note enlarged lymphatic vessels (arrows) and sprouting or splitting lymphatic vessels (arrowheads) in AdVEGF-C156S (A) and AdVEGF-C (B) infected ears. 2 wk after infection, AdVEGF-C156S infected ear (D) contains large lymphatic vessels apparently still undergoing sprouting and splitting, whereas AdVEGF-C (E) infected ear is filled with small lymphatic sprouts. (G–I) Podoplanin stained tissue sections at 2 wk after adenoviral infection. Note the formation of a hyperplastic lymphatic network in response to adenoviral VEGF-C156S (G) and VEGF-C (H) compared with AdLacZ control (I). Sprouting and splitting of the vessels is especially clear in the AdVEGF-C infected ear (H). Scale bars: A–C, 60 μm; D–F, 220 μm; G–I, 70 μm.
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Related In: Results  -  Collection

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fig3: Lymphangiogen-esis in AdVEGF-C156S and AdVEGF-C infected adult skin. Whole mount VEGFR-3 staining of the lymphatic vessels 1 wk (A–C) and 2 wk (D–F) after infection. Note enlarged lymphatic vessels (arrows) and sprouting or splitting lymphatic vessels (arrowheads) in AdVEGF-C156S (A) and AdVEGF-C (B) infected ears. 2 wk after infection, AdVEGF-C156S infected ear (D) contains large lymphatic vessels apparently still undergoing sprouting and splitting, whereas AdVEGF-C (E) infected ear is filled with small lymphatic sprouts. (G–I) Podoplanin stained tissue sections at 2 wk after adenoviral infection. Note the formation of a hyperplastic lymphatic network in response to adenoviral VEGF-C156S (G) and VEGF-C (H) compared with AdLacZ control (I). Sprouting and splitting of the vessels is especially clear in the AdVEGF-C infected ear (H). Scale bars: A–C, 60 μm; D–F, 220 μm; G–I, 70 μm.
Mentions: We then wanted to compare the lymphangiogenesis induced by viral delivery of VEGF-C156S and VEGF-C. For this purpose, adenoviruses encoding VEGF-C156S, VEGF-C, or LacZ were injected intradermally into the ears of nu/nu mice. The first sprouting lymphatic vessels were detected in the infected ear skin by VEGFR-3 whole mount staining 3 to 4 d after the adenoviral infection. The lymphatic sprouting was more abundant in the AdVEGF-C–infected skin than in the AdVEGF-C156S–infected skin at all time points studied. In the AdVEGF-C156S–infected skin the lymphatic vessels enlarged progressively, and 1 wk after the infection several lymphatic vessels appeared to be splitting to form new daughter vessels (Fig. 3 A). At this time point nearly all lymphatic vessels in the AdVEGF-C–infected skin exhibited sprouting and bridging; some enlarged vessels were also detected, whereas no signs of lymphangiogenesis were seen in the AdLacZ-infected control skin (Fig. 3, B and C). 2 wk after AdVEGF-C156S infection the lymphatic vessels network was vastly enlarged and most of the large vessels had formed new sprouts or bridges (Fig. 3 D). At the same time point the AdVEGF-C–infected skin was filled with very actively sprouting, relatively small lymphatic vessels, while only a few enlarged lymphatic vessels were detected (Fig. 3 E). A few new lymphatic sprouts were detected in only one AdLacZ-infected control mouse around the injection wound (n = 15; unpublished data), otherwise the lymphatic vasculature in the control skin remained unaltered (Fig. 3 F). The staining of histological sections for VEGFR-3, LYVE-1, and podoplanin confirmed that the sprouting of the lymphatic vessels was more efficient in the AdVEGF-C than in the AdVEGF-C156S infected samples (Fig. 3, G–I, and unpublished data). The newly formed lymphatic vessels regressed gradually with time as expression of the viral genes was downregulated, and by 8 t to 10 wk after the infection the lymphatic vessel network had returned to its normal architecture (unpublished data).

Bottom Line: Recent work from many laboratories has demonstrated that the vascular endothelial growth factor-C/VEGF-D/VEGFR-3 signaling pathway is crucial for lymphangiogenesis, and that mutations of the Vegfr3 gene are associated with hereditary lymphedema.However, as is the case with VEGF, high levels of VEGF-C cause blood vessel growth and leakiness, resulting in tissue edema.Importantly, adenoviral VEGF-C156S lacked the blood vascular side effects of VEGF and VEGF-C adenoviruses.

View Article: PubMed Central - PubMed

Affiliation: Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research, Biomedicum Helsinki, Helsinki University Central Hospital, University of Helsinki, Finland.

ABSTRACT
Recent work from many laboratories has demonstrated that the vascular endothelial growth factor-C/VEGF-D/VEGFR-3 signaling pathway is crucial for lymphangiogenesis, and that mutations of the Vegfr3 gene are associated with hereditary lymphedema. Furthermore, VEGF-C gene transfer to the skin of mice with lymphedema induced a regeneration of the cutaneous lymphatic vessel network. However, as is the case with VEGF, high levels of VEGF-C cause blood vessel growth and leakiness, resulting in tissue edema. To avoid these blood vascular side effects of VEGF-C, we constructed a viral vector for a VEGFR-3-specific mutant form of VEGF-C (VEGF-C156S) for lymphedema gene therapy. We demonstrate that VEGF-C156S potently induces lymphangiogenesis in transgenic mouse embryos, and when applied via viral gene transfer, in normal and lymphedema mice. Importantly, adenoviral VEGF-C156S lacked the blood vascular side effects of VEGF and VEGF-C adenoviruses. In particular, in the lymphedema mice functional cutaneous lymphatic vessels of normal caliber and morphology were detected after long-term expression of VEGF-C156S via an adeno associated virus. These results have important implications for the development of gene therapy for human lymphedema.

Show MeSH
Related in: MedlinePlus