Published online before print December 28, 2006, doi: 10.1161/01.ATV.0000256458.82320.be
© 2007 American Heart Association, Inc.
Vascular Biology |
In Vivo Actions of Angiopoietins on Quiescent and Remodeling Blood and Lymphatic Vessels in Mouse Airways and Skin
From the National Research Laboratory of Vascular Biology and Department of Biological Sciences (K.E.K., C-H.C., H-Z.K., G.Y.K.), Korea Advanced Institute of Science and Technology, Daejeon, Korea; Cardiovascular Research Institute (P.B., D.M.M.), Comprehensive Cancer Center, and Department of Anatomy, University of California, San Francisco, Calif.
Correspondence to Gou Young Koh, Department of Biological Sciences, KAIST, 373-1, Guseong-dong, Daejeon, 305-701, Republic of Korea. E-mail gykoh{at}kaist.ac.kr
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Abstract |
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Objective— We investigated and compared the in vivo effects of all four angiopoietins (COMP-Ang1, Ang2, Ang3, and Ang4) on blood and lymphatic vascular remodeling in adult mice. We analyzed the microvasculature of trachea and ear skin, and compared quiescent skin microvasculature with that during wound healing.
Methods and Results— We were able to achieve similar levels of relatively long-term and sustained circulating expression of each angiopoietin using an adenoviral delivery system. Two weeks after treatment, we observed tracheal blood and lymphatic vascular enlargement, and lymphatic filopodia formation, with the following order of potency: COMP-Ang1>Ang3=Ang4>Ang2. Co-treatment with Ang2 attenuated Ang1-induced tracheal blood and lymphatic remodeling. In the normal ear skin, all angiopoietins induced blood vessel enlargement, whereas none induced lymphatic vascular remodeling. However, in the healing margin of ear skin wounds, all angiopoietins strongly induced lymphatic vascular enlargement and formation of lymphatic sprouts and filopodia, while they potentiated blood vascular enlargement. Co-treatment of Ang2 with Ang1 produced an additive effect on these changes.
Conclusion— This study, one of the first to our knowledge to characterize the in vivo actions of all 4 angiopoietins, may expand the current concepts for use of angiopoietins for therapeutic angiogenesis and lymphangiogenesis.
Angiopoietins 1, 2, 3, and 4 elicited different amounts of blood and lymphatic vascular remodeling in normal and wound healing tissues. COMP-Ang1 and Ang2 given together produced an amount of vascular remodeling in normal vessels intermediate between the individual angiopoietins but had additive effects on vascular remodeling in skin wounds.
Key Words: angiogenesis • angiopoietin • lymphangiogenesis • Tie2 • vascular remodeling
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Introduction |
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The discovery of angiopoietin (Ang) family proteins, Ang1, Ang2, Ang3, and Ang4, has provided insight into the molecular and cellular mechanisms of blood vessel formation and vascular protection.1–4 Ang3 and Ang4 were identified as interspecies orthologs between mouse and human.5 All angiopoietins are secreted proteins, and they bind to the endothelial cell tyrosine kinase receptor, Tie2, which is found primarily on blood endothelial cells and early hematopoietic stem cells.1,2 Recent reports indicate that in addition to the established receptor Tie2, angiopoietins may also signal through the related Tie1 receptor, integrins, or directly with the cell surface.6–10 However, the relative affinity, and signaling pathways in such interactions remain to be defined.
Knockout experiments have provided significant information toward understanding the role of Ang1, Ang2, and Tie2 in vivo, and has shown that this signaling system is indispensable for normal vessel development.11,12 Interestingly, mice lacking Ang2 are born alive and display a chylous leaking in the abdomen and thorax, suggesting a problem with lymphatic drainage.13 Postnatally, the hyaloid vasculature in the eye of mice lacking Ang2 fails to regress, suggesting that within a certain environment, Ang2 may in fact have an antagonistic effect on Tie2 signaling-induced blood vessel maturation.13 However, the role of Ang3 in vascular development has not yet been studied in this manner. Moreover, no disorder with a genetic deficiency of Ang4 has yet been reported in humans.
Overexpression of angiopoietins using transgenic mice or adenoviral delivery also has provided information regarding the role of angiopoietins and their receptors in vivo.2,14,15 Transgenic overexpression of Ang1 induces vascular enlargement, increased recruitment of perivascular cells, and less vessel tortuosity without altering vascular permeability.14,15 We and others recently showed that adenoviral overexpression of Ang1 produced long-lasting and stable vascular enlargement and increased blood flow.16–19 Mice overexpressing Ang2 transgenically driven by Tie2 promoter display a separation defect between the endocardium and myocardium and impaired sprouting from developing vessels, supporting the notion that Ang2 acts as a naturally occurring antagonist of Ang1.2 However, overexpression of Ang3 or Ang4 using transgenic mice or adenoviral delivery to understand the role of these proteins in vivo has not been reported.
Therefore, in this study, we investigated and compared the in vivo roles of angiopoietins 1, 2, 3, and 4 in blood and lymphatic vascular remodeling in adult mice by overexpressing each angiopoietin using an adenoviral delivery system. To examine the vascular remodeling, we focused on the microvasculature of the trachea and ear skin, which are distinguished by their simple layered structure.16,18,19 In addition to the normal tissues, we further examined the effect of angiopoietins on vascular remodeling in ear skin during wound healing to see whether each angiopoietin exerts its action somewhat differently on blood and lymphatic remodeling at different local environment. Overexpression of native Ang1 often produces nonspecific effects in vivo because it is an insoluble, and sticky protein that easily forms aggregates.20 To avoid these nonspecific effects, we used COMP-Ang1 as an alternative to Ang1. COMP-Ang1 is a soluble, stable, and potent Ang1 recombinant chimera.20 Our results indicate that different angiopoietins elicited different vascular and lymphatic remodeling in normal and wound healing tissues. Interestingly, co-treatment of Ang2 produced an antagonistic or an agonistic effect on Ang1-induced blood and lymphatic vascular remodeling in different situations.
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Methods |
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Generation of Adenoviral Angiopoietins
Recombinant adenoviruses expressing COMP-Ang1, mouse Ang2, Ang3, Ang4, or ß-gal were constructed using the pAdEasy vector system (Qbiogene) as previously described.18
Animals and Treatment
Specific pathogen-free FVB/N mice and Tie2–green fluorescent protein transgenic mice (FVB/N) were purchased from Jackson Laboratory (Jackson Labs, Bar Harbor, Me) and bred in our pathogen-free animal facility. Male 7- to 8-week-old mice were used for this study. Animal care and experimental procedures were performed under approval from the Animal Care Committees of KAIST. For hole-punch assays, a 2.0-mm hole was made in the center of both ears of the mouse using a metal ear punch (Harvard Apparatus, Holliston, Mass). At 12 hours after wounding, 
1x109 pfu Ade-ß-gal, Ade-COMP-Ang1, Ade-Ang2, Ade-Ang3, Ade-Ang4, or Ade-COMP-Ang1 plus Ade-Ang2 diluted in 50 µL of sterile 0.9% NaCl was injected intravenously through the tail vein.
Enzyme-Linked Immunosorbent Assay
To detect circulating angiopoietins, we used an established enzyme-linked immunosorbent assay protocol.18
Histological and Morphometric Analysis
Immunofluorescence staining and morphometric analysis for blood and lymphatic vascular remodeling in whole mounted organ was performed as previously described.18,19
Statistics
Values presented are means±SD. Significant differences between means were determined by analysis of variance followed by the Student-Newman-Keuls test. Statistical significance was set at P<0.05 or P<0.01.
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Results |
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Systemic Overexpression of Angiopoietins
For systemic administration of angiopoietins in vivo, adult mice were treated with 1x109 pfu Ade-COMP-Ang1, Ade-Ang2, Ade-Ang3, Ade-Ang4, or Ade-ß-gal, which are hereafter referred as COMP-Ang1, Ang2, Ang3, Ang4, and control, unless otherwise specified. The circulating plasma level of each angiopoietin was measured at several time points over 6 weeks. All circulating angiopoietins increased similarly as early as 1 day after treatment, peaked at 5 to 7 days, declined gradually thereafter, and returned to control levels at 6 weeks after treatment (supplemental Figure I, available online at http://atvb.ahajournals.org). The peak concentrations of all 4 circulating angiopoietins were
2.6 to 3.2 µg/mL. All mice treated with any of the adenoviral constructs appeared generally healthy and gained weight normally. The systemic blood pressures and heart rates among the groups of mice at 2 and 6 weeks after the treatment were indistinguishable.
Angiopoietins Remodel Quiescent Tracheal Blood and Lymphatic Vessels
To examine the effect of angiopoietins on blood and lymphatic vascular remodeling, we first focused on the microvasculature of the trachea, which is distinguished by its simplicity and monolayer structure.16,18 To visualize blood and lymphatic vascular remodeling, immunofluorescence staining for the blood vessel endothelial cell marker, platelet–endothelial cell adhesion molecule-1 (CD31), and the lymphatic vessel endothelial cell marker, lymph vessel endothelial hyaluronan receptor-1 was applied to whole mounted tissues of trachea. Two weeks after the treatment, differential enlargement of postcapillary venules, venous end of capillaries, collecting venules, terminal arterioles, and segmental arterioles were observed (Figure 1A). The diameters of terminal arterioles and postcapillary venules were increased 1.7- and 3.7-fold by COMP-Ang1; 1.1- and 1.3-fold by Ang2; 1.4- and 1.7-fold by Ang3; and 1.5- and 1.6-fold by Ang4, compared with those treated by control (Figure 1). Ang4 was almost equipotent as mouse Ang3 in its action on mouse receptors in vivo. However, co-treatment with Ang2 attenuated the COMP-Ang1-induced enlargement of segmental arterioles and postcapillary venules by 42% and 68% (Figure 1). In addition, noticeable but differential increases in the numbers of filopodia (length exceeding 10 µm) on lymphatic vessels were observed (Figure 1A). The number of filopodia on lymphatic vessels was increased 20.3-fold by COMP-Ang1; 4.6-fold by Ang2; 7.6-fold by Ang3; and 8.5-fold by Ang4 compared with those treated by control (Figure 1). However, co-treatment with Ang2 attenuated the COMP-Ang1-induced increase in the number of filopodia on lymphatic vessels by 31% (Figure 1). We next examined whether the remodeled blood and lymphatic vessels in the trachea were normalized when all circulating angiopoietins were returned to control. Six weeks after the treatment, compared with the blood vessels at 2 weeks after the treatment, COMP-Ang1 and Ang2 maintained similar levels of enlargement of the blood vessels, while the levels of blood vessel enlargement induced by Ang3 and Ang4 had decreased (supplemental Figure IIA). In addition, the numbers of filopodia on lymphatic vessels had decreased (supplemental Figure IIA). Co-treatment with Ang2 attenuated COMP-Ang1–induced enlargement of segmental arterioles and postcapillary venules by 28% and 30%, and COMP-Ang1–induced increased number of filopodia on lymphatic vessels by 54% (supplemental Figure II). Thus, each angiopoietin elicited a differential agonistic effect on blood and lymphatic vascular remodeling in tracheal vessels at different time points. When COMP-Ang1 and Ang2 were given together, the resulting enlargement was half-way between that of either angiopoietin given alone.
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Angiopoietins Remodel Blood Vessels but not Lymphatic Vessels of Mouse Ear Skin
We also examined the effect of angiopoietins on blood and lymphatic vascular remodeling in the outer marginal portion of ear skin, which is also distinguished by its simple layered structure. Two weeks after the treatment, differential enlargement and increased densities of blood vessels in the ear skin were observed (Figure 2A). The densities of blood vessels increased 2.0-fold by COMP-Ang1; 1.4-fold by Ang2; 1.7-fold by Ang3; and 1.5-fold by Ang4 compared with those treated by control (Figure 2). Co-treatment with Ang2 attenuated the COMP-Ang1–induced increased density of blood vessels by 31% (Figure 2). In addition to the skin, differential enlargements and increased densities of blood vessels were observed in several organs, including liver and brain at 2 weeks after the treatment (supplemental Figure III). Six weeks after the treatment, the enlargement and increased densities of the blood vessels induced by COMP-Ang1 or Ang2 had not changed significantly, whereas the enlargements and increased densities of blood vessels induced by Ang3 and Ang4 had decreased (Figure 2). Co-treatment with Ang2 did not attenuate the COMP-Ang1–induced increased density of blood vessels (Figure 2). In comparison, densities of lymphatic vessels in the outer marginal area of ear skin were unchanged by 2 weeks or 6 weeks of treatment with any of the angiopoietins (Figure 2A and 2C). Moreover, none of the angiopoietin resulted in lymphatic sprouting or filopodia (Figure 2A). Thus, at 2 and 6 weeks, each angiopoietin induced differential amounts remodeling on quiescent blood vessels, but not on lymphatic vessels, in ear skin. Interestingly, co-treatment with Ang1 and Ang2 produced an amount of blood vessel enlargement in ear skin roughly midway between the values for the individual angiopoietins.
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Angiopoietins Remodel Blood and Lymphatic Vessels at Margins of Healing Wounds
To investigate the effect of angiopoietins on blood and lymphatic remodeling during wound healing in vivo, we made hole-punch injuries to the ears of adult mice and treated them with each angiopoietin as described. Two weeks after the treatment, mice treated with any of the angiopoietins had more but differentially enlarged and denser blood and lymphatic vessels than the mice treated with control (Figure 3A). This effect was seen at the margin of the wound healing area. Moreover, the mice treated with any of the angiopoietins had more blood and lymphatic vessels having sprouting and filopodia than the mice treated with control in the margin of the wound healing area (Figure 3A). Overall, blood and lymphatic vessel densities in the healing margin were increased 1.6- and 1.5-fold by COMP-Ang1; 1.3- and 1.5-fold by Ang2; 1.4- and 1.5-fold by Ang3; and 1.4- and 1.4-fold by Ang4 (Figure 3). Intriguingly, in contrast to the action of Ang2 on vessels of trachea and normal ear skin, COMP-Ang1 and Ang2 had additive effects on vessels in skin wounds, increasing the densities of blood and lymphatic vessels by 28% and 50% above the value for COMP-Ang1 alone (Figure 3). Six weeks after the treatment, in the margin of wound healing area, the mice treated with COMP-Ang1 had more enlarged and denser blood vessels than the mice treated with control adenovirus, whereas the mice treated with Ang2, Ang3, or Ang4 had slightly enlarged and more densely packed blood vessels than mice treated with control (Figure 4). The mice treated with COMP-Ang1 plus Ang2 had less densely packed blood vessels than the mice treated with COMP-Ang1 but more than after Ang2 alone (Figure 4). No differences were found in lymphatic densities after the various treatments (Figure 4). Moreover, sprouting and filopodia were not observed in the marginal lymphatic vessels of the mice treated with any of the angiopoietins. Thus, each angiopoietin initially caused relatively strong enlargement of blood and lymphatic vessels at the margin of healing wounds in ear skin, but the enlargement subsequently disappeared. Unlike what was observed in the trachea, Ang1 and Ang2 had additive effects on blood and lymphatic vessels during the initial period of wound healing in ear skin.
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Each Angiopoietin Increases Tie2 Expression in Blood Vessels, but not Lymphatic Vessels, at the Wound Margin of Ear Skin
Based on these observations, we asked whether changes in Tie2 expression were involved in angiopoietin-induced remodeling of blood and lymphatic vessels in ear skin. Transgenic mice with Tie2 promoter-driven green fluorescent protein were used to address this issue. In the outer margin of normal ear skin of adult mice, faint Tie2 expression was detected in endothelial cells of arterioles and capillaries, but not in endothelial cells of venules or lymphatics (Figure 5 and supplemental Figure IV). Tie2 expression was increased by COMP-Ang1 (supplemental Figure IV), consistent with a previous report.18 At the margin of the wound healing portion of ear skin, Tie2 expression was strong in endothelial cells of arterioles and capillaries after control treatment (Figure 5). Furthermore, angiopoietins differentially increased Tie2 expression during wound healing in the endothelial cells of blood vessels (Figure 5). In addition, Tie2 expression was notably higher in the outer portion than in the inner portion of the marginal healing region. However, even in this situation, Tie2- green fluorescent protein was not detected in endothelial cells of lymphatic vessels (Figure 5 and supplemental Figure IV).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The central aim of this study was to investigate and compare the in vivo roles of COMP-Ang1, Ang2, Ang3, and Ang4 in blood and lymphatic vascular remodeling in adult mice. To this end, we used an adenoviral delivery system for systemic administration of each angiopoietin and analyzed the microvasculature of the trachea and ear skin. Each angiopoietin evoked different amounts of vascular and lymphatic remodeling at different time periods in normal and wound healing tissues. Intriguingly, co-treatment with COMP-Ang1 and Ang2 produced an amount of vascular remodeling in normal tracheal and skin vessels intermediate between the individual angiopoietins but had additive actions on vascular remodeling in skin wounds. These results provide evidence that each angiopoietin exerts its action somewhat differently on blood and lymphatic remodeling in vivo based on local environment.
A single intravenous injection of 1x109 pfu adenoviral vector produced approximately similar levels of long-term and sustained circulating angiopoietins in mice. Consistent with our recent report,18 COMP-Ang1 produced long-lasting (>6 weeks) enlargement of tracheal and skin blood vessels, although circulating COMP-Ang1 was no longer detected at 6 weeks. Ang3 and Ang4 produced comparable enlargements of blood vessels at 2 weeks, consistent with results obtained previously in a corneal micropocket assay.21 Thus, human Ang4 was active on mouse receptors in vivo. However, in contrast to COMP-Ang1, the Ang3- and Ang4-induced enlargements of blood vessels gradually decreased over time, and the reduction paralleled the circulating levels of Ang3 and Ang4. Ang2-induced enlargement of blood vessels was mild at 2 weeks after the treatment; however, in tracheal blood vessels, but not skin blood vessels, were the enlargement was maintained at 6 weeks. Thus, their changes did not parallel the circulating level of Ang2. These differences among effects of angiopoietins might be attributed to different binding, affinity, or activation of Tie2, Tie1, integrins, or signaling via the cell surface directly.6–10 A more detailed analysis will be necessary to determine how different angiopoietins produce different vascular remodeling in vivo.
Ang1-induced blood vessel enlargement is a distinctive characteristic that has not been reported for angiogenic growth factors.14–18 Recent reports16–18 indicate that Ang1-induced vascular enlargements achieved mainly by circumferential endothelial proliferation, which is different from multi-directional endothelial cell proliferation during vasculogenesis and angiogenesis. Given that Ang2, Ang3, and Ang4 also induce blood vessel enlargement with similar morphology, the blood vessel enlargement induced by Ang2, Ang3, and Ang4 might also be achieved mainly by circumferential endothelial proliferation. Ang1-induced vascular enlargement was accompanied with increased tissue blood flow in trachea and skin.18,19 Increased blood flow induced by Ang2, Ang3, and Ang4 correlated with the extent of arteriolar and venular enlargement in the trachea and ear skin (unpublished observations). It will be important to determine whether each of the four angiopoietins can reverse tissue ischemia by increasing blood flow through enlarged blood vessels.
Inflammation, angiogenesis, and lymphangiogenesis are crucial to the wound healing process.22,23 Accordingly, ligands and their receptors that participate in these processes, including vascular endothelial growth factor (VEGF)/VEGF receptor and angiopoietin/Tie2, are dynamically upregulated.23,24 Each angiopoietin including Ang2 led to robust angiogenesis and lymphangiogenesis in healing wounds in ear skin. Thus, angiopoietins can increase both angiogenesis and lymphangiogenesis, possibly mediated through increased activation of angiogenic signaling during wound healing. Ang2 is involved in vessel regression, abnormal vascular leakage, and inflammatory reactions through mainly or partly by antagonistic action to Ang1 on Tie2.2,24,25 Because Ang2 regresses and destabilizes blood vessels in the absence of VEGF, whereas Ang2 leads to robust blood vessel formation in the presence of VEGF, such as tumor and inflammatory angiogenesis,24 Ang2 could lead to robust blood vessel formation during wound healing with locally high levels of VEGF. Moreover, COMP-Ang1 and Ang2 had additive effects on angiogenesis and lymphangiogenesis in wounds, but Ang2 had antagonistic effects on COMP-Ang1-induced vascular remodeling in quiescent tracheal and skin vessels. Thus, our study showed that Ang2 could act as antagonist and agonist to COMP-Ang1 depends on different situations.
What are the major mechanisms and factors that produce angiopoietin-induced lymphatic vascular remodeling? Recently, 2 reports26,27 clearly indicate that Ang1 can drive lymphangiogenesis through Tie2 receptor signaling, possibly by activation of VEGF receptor 3.26,27 However, in our study, angiopoietin-induced lymphatic remodeling occurred at the margin of healing wounds. Given that Ang1-induced lymphangiogenesis has been observed after certain manipulations, it is possible that a minor inflammation could be a prerequisite for angiopoietin-induced lymphangiogenesis. In fact, lymphangiogenesis in adulthood is closely involved in inflammation progression.28 Although immunohistochemical studies revealed that Tie2 may be expressed in some lymphatic endothelial cells of normal intestine or in pathologic skin,26,27 our study using Tie2–green fluorescent protein mouse revealed that Tie2 is not expressed at detectable levels in lymphatic endothelial cells of normal trachea and ear skin, or even in the margin of the wound healing region of ear skin. Therefore, we suggest that angiopoietin-induced lymphangiogenesis could be resulted indirectly from concomitant increased requirement for drainage of interstitial fluid caused by the increased density of capillaries induced by angiopoietins, activation of VEGF receptor 3 or other mechanisms.
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Acknowledgments |
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Sources of Funding
This work was supported by the Korea Science and Engineering Foundation (KOSEF) through the National Research Laboratory Program (2004-02376, G.Y.K.) and the World Leading Scientists Program (R02-2004-000-10120-0, GYK) funded by the Ministry of Science and Technology and by US National Institutes of Health grants HL-24136 and HL-59157 from the National Heart, Lung, and Blood Institute (D.M.M.).
Disclosures
None.
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Footnotes |
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Original received August 4, 2006; final version accepted November 19, 2006.
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References |
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 C. Jang, Y. J. Koh, N. K. Lim, H. J. Kang, D. H. Kim, S. K. Park, G. M. Lee, C. J. Jeon, and G. Y. Koh Angiopoietin-2 Exocytosis Is Stimulated by Sphingosine-1-Phosphate in Human Blood and Lymphatic Endothelial Cells Arterioscler Thromb Vasc Biol, March 1, 2009; 29(3): 401 - 407. [Abstract] [Full Text] [PDF]
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