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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 7, 2004 as doi:10.1096/fj.03-1179fje. |
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1ß1 and 2ß1 integrins
* Cutaneous Biology Research Center, Department of Dermatology, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts, USA;
Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA; and
ImClone Systems Incorporated, New York, New York, USA
2Correspondence: Cutaneous Biology Research Center, Department of Dermatology, Massachusetts General Hospital, Bldg. 149, 13th St., Charlestown, MA 02129, USA. E-mail: michael.detmar{at}cbrc2.mgh.harvard.edu
SPECIFIC AIMS
In surgical wounds, lymphatic vessel regeneration occurs about 1 wk after blood vascular regeneration; the reasons that repair-associated lymphangiogenesis occurs after angiogenesis are unknown. This study was performed to investigate the role of vascular endothelial growth factor-A (VEGF-A)—generally considered to be a blood vessel-specific angiogenesis factor—in lymphatic regeneration. The specific aims were to determine whether VEGF-A promotes repair-associated lymphangiogenesis and whether this involves up-regulation of distinct integrin receptors on lymphatic endothelium. Using a transgenic model for targeted overexpression of VEGF-A and systemic inhibition of VEGF signaling, our studies identify VEGF-A as a potent lymphangiogenesis factor and reveal that the lymphangiogenic activity of VEGF-A is mediated through the VEGFR-2. They identify a novel molecular mechanism by which VEGF-A, through up-regulation of the 
1 and the 2 integrins, promotes lymphatic endothelial cell migration in vitro and lymphangiogenesis in vivo. We propose that VEGF-A promotes lymphatic vasculature formation via activation of VEGFR-2 and that lineage-specific differences of integrin receptor expression contribute to the distinct dynamics of wound-associated angiogenesis and lymphangiogenesis.
PRINCIPAL FINDINGS
1. Increased angiogenesis and lymphangiogenesis in the granulation tissue of VEGF-A transgenic mice
For the first 3 wk after skin wounding, vascularization of the granulation tissue remained elevated in VEGF-A transgenic mice compared with their wild-type littermates (Fig. 1
A–F). Surprisingly, VEGF-A transgenic mice rapidly formed a lymphatic vasculature at the wound site. In the granulation tissue of wild-type mice, no lymphatic vessels were found at day 7 after wounding (Fig. 1A
) and only a few sprouting lymphatic vessels were observed by day 10. At day 14 and, more prominently, at day 21, lymphatic vessels were observed to bridge the wound area of wild-type mice, as visualized by staining for the lymphatic-specific hyaluronan receptor LYVE-1 (Fig. 1C, E
). Wounds of VEGF-A transgenic mice, in contrast, showed prominent lymphangiogenesis by day 7 (Fig. 1B
), and increased numbers of enlarged lymphatic vessels were observed throughout the first 3 wk after wound formation (Fig. 1D, F
). Morphometric analyses of CD31-labeled wound sections showed a significant increase of blood vessel density in VEGF-A transgenic mice at day 7 (P<0.01) and even at 3 wk postwounding (P<0.05; Fig. 1G
). The average vessel size and relative area of granulation tissue covered by blood vessels were increased in VEGF-A transgenic mice at all time points (Fig. 1H, I
). The granulation tissue of VEGF-A transgenic mice contained increased numbers of lymphatic vessels (Fig. 1)
, which were significantly enlarged (Fig. 1K, L
). No major differences in type I collagen mRNA expression were detected between the two genotypes by in situ hybridization, and quantitative real-time TaqMan RT-PCR revealed that expression levels of VEGF-C and VEGF-D were comparable in wounds of wild-type and of VEGF-A transgenic mice, although the expression level of VEGF-C was higher in the normal skin of VEGF-A transgenic mice than in wild-type mice. We found an 
2-fold increase in the number of macrophages in wounds obtained from VEGF-A transgenic mice at day 7 after wounding; at days 10 and 14 there was no major difference between wild-type and VEGF-A transgenic mice.
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2. Blockade of VEGFR-2 inhibits lymphangiogenesis
VEGFR-2 was strongly expressed by LYVE-1-positive lymphatic vessels within the granulation tissue whereas VEGFR-1 was not detected on LYVE-1-positive lymphatic vessels. At day 10 after wounding, mice treated with control IgG underwent initial lymphangiogenesis within the granulation tissue. In contrast, the ingrowth of lymphatic vessels was completely inhibited in mice treated with the anti-VEGFR-2 antibody DC101. Lymphangiogenesis within the granulation tissue was still absent at day 14 in antibody-treated mice, indicating that VEGFR-2 activation is necessary for efficient lymphangiogenesis during cutaneous wound healing.
3. VEGF-A promotes lymphangiogenesis via the 1 and 2 integrins
Lymphatic vessels lie in close contact with interstitial collagen fibers in vivo, and cultured lymphatic endothelial cells (LEC) express higher levels of the 1 and 2 integrin subunits, but not of the 5 or V subunit, than blood vessel endothelial cells. Therefore, we investigated whether VEGF-A might promote lymphangiogenesis via up-regulation of the 1 and 2 integrins, the principal endothelial receptors for interstitial collagens. Treatment of cultured human LEC with VEGF-A potently up-regulated mRNA expression of the 1 and 2 integrins (8-fold and 2.5-fold, respectively). VEGF treatment strongly promoted LEC cord formation after overlay with type I collagen in vitro, whereas blockade of the 1 or 2 integrin by incubation of LEC with blocking antibodies significantly inhibited VEGF-A-induced LEC cord formation. Combined blockade of the 1 and 2 integrin resulted in enhanced inhibition of LEC cord formation to levels observed in LEC that had not been treated with VEGF-A. Treatment with VEGF-A stimulated haptotactic LEC migration toward type I collagen, and blockade of integrin subunits 1 and 2, but not of integrin vß3, significantly inhibited LEC migration toward type I collagen. To investigate whether 1 and 2 integrins might play a major role in VEGF-A-mediated lymphangiogenesis in vivo, immunodeficient mice were injected s.c. with Matrigel that contained SK-MEL-2 cells stably transfected with a control vector or a vector that expressed murine VEGF-A164 cDNA. Lymphatic vessel formation was greatly increased surrounding the VEGF-A-expressing implants and VEGF-A-induced lymphangiogenesis was completely inhibited by treatment with integrin 1- and 2-blocking antibodies. In a wound healing study, systemic treatment with anti-1 and anti-2 antibodies completely inhibited the sprouting of lymphatic vessels into the wound granulation tissue; wound-associated lymphangiogenesis was not affected in IgG-treated controls.
CONCLUSIONS AND SIGNIFICANCE
Whereas it has been generally thought that VEGF-A selectively acts as a (blood) vascular growth factor, its effect on the formation of lymphatic vessels has remained unclear. Surprisingly, we found that chronic transgenic delivery of VEGF-A potently promoted the growth of wound-associated lymphatic vessels. In accordance with our findings that VEGFR-2 was expressed by cultured lymphatic endothelial cells, we show here that VEGFR-2, but not VEGFR-1, is expressed by wound-associated lymphatic vessels, indicating that the lymphangiogenic effect of VEGF-A was mediated by VEGFR-2. This hypothesis was confirmed by studies with the VEGFR-2-blocking antibody DC101, revealing that systemic blockade of VEGFR-2 completely blocked lymphatic sprouting into the granulation tissue. The only other lymphangiogenesis factors identified are VEGF-C and VEGF-D, which signal via receptors VEGFR-2 and VEGFR-3. Although there was an increase of VEGF-C mRNA expression in normal unwounded skin of VEGF-A transgenic mice, we did not detect higher levels of either factor during wound healing, as determined by quantitative real-time RT-PCR and in situ hybridization. VEGF-A is therefore a novel lymphangiogenesis factor that promotes wound-associated lymph vessel formation through VEGFR-2 signaling. We found that the growth of newly formed lymphatic vessels into the wound granulation tissue occurred 1 wk later than the sprouting of blood vessels. Excisional wounds are characterized by the formation of a fibrin- and fibronectin-rich granulation tissue during the first week after wounding, which is gradually replaced by collagenous scar tissue. The vascular endothelial cells in this tissue express the Vß3 integrin, a cellular receptor for fibrin and fibronectin that regulates their migration and vessel-forming activities during the early stages of wound healing. This led us to investigate whether cell lineage-specific expression of integrin receptors might contribute to the distinct dynamics of wound-associated (blood) vascularization and lymphangiogenesis. Transcriptional profiling of cultured human blood vascular and lymphatic endothelial cells revealed a predominant expression of the Vß3 integrin by blood vascular endothelium; the 1 integrin, which pairs with the ß1 subunit to form a receptor for collagens, was more strongly expressed by the lymphatic endothelium.
VEGF-A potently up-regulated the expression of the 1 and 2 integrins in cultured human LEC, promoting their capacity to form cords and haptotactic migration. Blockade of both these integrins in vitro inhibited VEGF-A-induced cord formation and LEC migration toward collagen but not toward fibronectin. Systemic blockade of both the 1ß1 and 2ß1 integrins potently inhibited wound-associated lymphangiogenesis in vivo and blocked the lymphangiogenesis surrounding VEGF-A-producing Matrigel implants. Together, these findings provide a mechanism for the delayed development of lymphatic vasculature during wound healing compared with blood vascularization. Angiogenic blood vessels up-regulate expression of the fibrin/fibronectin receptor integrin Vß3, promoting their migration and invasion into the early fibrin- and fibrinogen-rich wound granulation tissue. In contrast, LEC predominantly express the collagen integrin receptors and therefore depend on the formation of the collagen-enriched matrix found in later-stage wounds. They are, however, unable to invade the fibrin/fibronectin-rich matrix that is prevalent in early wounds. The results of the present study identify VEGF-A as a new lymphangiogenesis factor and identify a novel molecular mechanism by which VEGF-A—through up-regulation of the 1 and 2 integrin subunits—promotes lymphatic vessel formation. We propose that VEGF-A promotes lymphatic vasculature formation via activation of VEGFR-2 on lymphatic endothelium and that lineage-specific differences of integrin receptor expression contribute to the distinct dynamics of wound-associated angiogenesis and lymphangiogenesis (Fig. 2
). It is important to create additional experimental models that allow the conditional inactivation of genes involved in lymphatic vessel formation to improve our understanding of neo-lymphangiogenesis and its function in tissue regeneration after wound formation.
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FOOTNOTES
1 Y.-K.H. and B.L.-A. contributed equally to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1179fje; doi: 10.1096/fj.03-1179fje
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