Long-Term and Sustained COMP-Ang1 Induces Long-Lasting Vascular Enlargement and Enhanced Blood Flow
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循环研究杂志 2005年第7期
the Biomedical Research Center and Department of Biological Sciences (C.-H.C., K.E.K., G.M.L., G.Y.K), Korea Advanced Institute of Science and Technology, Daejeon, Korea
the Department of Medicine (J.B., H.-S.J., D.-K.K.), Samsung Medical Center and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Korea
the Cardiovascular Research Institute, Comprehensive Cancer Center
Department of Anatomy (P.B., F.B., D.M.M.), University of California, San Francisco
the Department of Structural Analysis (N.M.), National Cardiovascular Center Research Institute, Suita, Osaka, Japan
the Department of Anatomy (J.K.), College of Medicine, The Catholic University of Korea Seoul
the Department of Physiology (B.H.J), College of Medicine, Chungnam National University Daejeon, Korea.
Abstract
Vascular enlargement is a characteristic feature of angiopoietin-1 (Ang1)-induced changes in adult blood vessels. However, it is unknown whether tissues having Ang1-mediated vascular enlargement have more blood flow or whether the enlargement is reversible. We have recently created a soluble, stable and potent Ang1 variant, COMP-Ang1. In the present study, we investigated the effects of varied dose and duration of COMP-Ang1 on vascular enlargement and blood flow in the tracheal microvasculature of adult mice and explored a possible mechanism of long-lasting vascular enlargement. We found that COMP-Ang1 administered by adenoviral vector induced long-lasting vascular enlargement and increased tracheal blood flow. In contrast, short-term administration of COMP-Ang1 recombinant protein induced transient vascular enlargement that spontaneously reversed within a month. In both cases, the vascular enlargement resulted from endothelial proliferation. The COMP-Ang1eCinduced vascular remodeling is mediated mainly through Tie2 activation. Sustained overexpression of Tie2 could participate in the maintenance of vascular changes. Together, our findings indicate that sustained treatment with COMP-Ang1 can produce long-lasting vascular enlargement and increased blood flow.
Key Words: angiopoietin-1 COMP-Ang1 vascular enlargement blood flow
Introduction
Angiopoietin-1 (Ang1) is known to be a ligand to Tie2 tyrosine kinase receptor expressed on endothelial cells.1 Ang1/Tie2 signaling is thought be involved in branching and remodeling of the primitive vascular network and in the recruitment of mural cells during development.2,3 Transgenic overexpression of Ang1 using the skin-specific keratin-14 promoter produces leakage-resistant and enlarged vessels with an increased number of endothelial cells in skin.4,5 Gene transfer of Ang1 into ischemic tissues produces notably enlarged blood vessels.6,7 Baffert et al recently identified that Ang1-induced vascular enlargement could be the result of endothelial proliferation in trachea mucosa.8 Thus, a cardinal feature of Ang1-induced vascular remodeling is vascular enlargement resulting from endothelial cell proliferation in adult animals.4eC8
Given that Ang1-induced therapeutic benefits correlated with vascular enlargement in the ischemic tissues,6,7,9 enhanced blood flow through blood vessels enlarged by Ang1 treatment could provide a great therapeutic benefit to ischemic peripheral tissues. However, it is not known whether the tissues having Ang1-mediated enlarged vessels have more blood flow. In addition, the effective dose and treatment period of Ang1 for inducing effective vascular enlargement is not known. Moreover, it is not known whether Ang1-mediated vascular enlargement regresses when Ang1 stimulation is withdrawn.
We have recently developed a soluble, stable, and potent Ang1 variant, COMP-Ang1.10 To create this protein, we replaced the amino-terminal portion of Ang1 with the short coiled-coil domain of cartilage oligomeric matrix protein (COMP). COMP-Ang1 is more potent than native Ang1 in phosphorylating the Tie2 receptor and signaling via Akt in primary cultured endothelial cells.10
In the present study, we investigated effects of period and dose of COMP-Ang1 on vascular enlargement and tissue blood flow in adult mice and investigated a possible mechanism for long-lasting vascular enlargement induced by long-term and sustained COMP-Ang1. To determine the underlying mechanism of COMP-Ang1eCstimulated vascular remodeling in adult mice, we focused on the microvasculature of the trachea, which is distinguished by its simplicity and monolayer structure. Our results indicate that long-term and sustained COMP-Ang1 produced by adenoviral delivery of COMP-Ang1 induces a long-lasting vascular enlargement and enhanced blood flow without enhanced pericyte recruitment in adult mice. Long-lasting Tie2 expression could be involved in the long-lasting vascular enlargement and enhanced blood flow.
Materials and Methods
Generation of COMP-Ang1 Recombinant Protein and Ade-COMP-Ang1
Recombinant Chinese hamster ovary cells expressing COMP-Ang1 (CA1eC2; production rate, 30 mg/L) were established as previously described.11 Recombinant adenovirus expressing COMP-Ang1 or LacZ was constructed using the pAdEasy vector system (Qbiogene). For additional Materials and Methods, see online data supplement at http://circres.ahajournals.org.
Animals, Treatment, and Measurement of Blood Pressure and Heart Rate
Specific pathogen-free FVB/N mice and Tie2-GFP transgenic mice (FVB/N)12 were purchased from Jackson Laboratory and bred in our pathogen-free animal facility. Male mice 8 to 10 weeks old were used for this study. Animal care and experimental procedures were performed under approval from the Animal Care Committees of the Korea Advanced Institute of Science and Technology. For protein treatment, 200 e of COMP-Ang1 recombinant protein or BSA dissolved in 50 e蘈 of sterile 0.9% NaCl was injected daily through the tail vein for 2 weeks. For adenoviral treatment, the indicated amount of Ade-COMP-Ang1, Ade-LacZ, or Ade-sTie2-Fc (generous gift from Drs Gavin Thurston and Ella Ioffe at Regeneron Pharmaceuticals, Terrytown, NY) diluted in 50 e蘈 of sterile 0.9% NaCl was injected intravenously through the tail vein. Systemic blood pressure and heart rate were measured under anesthesia.
Enzyme-Linked Immunosorbent Assay
Approximately 50 e蘈 of blood was obtained from the tail vein into a heparinized capillary tube at the indicated times. ELISA was adopted for precise detection of COMP-Ang1 in plasma.
Immunohistochemical Staining
Mice were anesthetized, perfused with 1% paraformaldehyde in PBS, and several organs including tracheas were removed. Tracheas and ear skins were immunostained as whole mounts, whereas other organs were immunostained as sections. Signals were visualized, and digital images were obtained with a Zeiss Apotome microscope and a Zeiss LSM 510 confocal microscope.
Measurement of Tracheal Tissue Blood Flow
After the mice were anesthetized, a type N flowprobe (Transonic Systems Inc, Ithaca, NY) was placed on tracheal wall along second, third, and forth cartilage rings without applying pressure, as this would occlude the vessels and reduce perfusion in the area of interest. The flowprobe was kept in place on the position of the highest sensitivity by a micromanipulator and connected to a laser-Doppler flowmeter (model BLF21; Transonic Systems Inc), which can measure microcirculation in 1 mm3 of tissue for real-time assessment of perfusion (mL/min per 100 g of tissue).
Morphometric Measurements and Statistics
Morphometric measurements of the vessel diameters and area densities in mouse trachea were made as previously described.13 For each trachea, the numbers of PH3-immunopositive endothelial cells, platelet/endothelial cell adhesion molecule (PECAM)-1eCimmunopositive blood vessels, and desmin/NG2-immunopositive pericytes were measured in 5 regions, each 0.21 mm2 in area. Values were expressed per millimeter squared. Values presented are mean±SD. Significance of differences between mean was tested by analysis of variance followed by the StudenteCNewmaneCKeuls test. Statistical significance was set at P<0.05.
Results
Systemic Adenoviral COMP-Ang1 Produces Differential Enlargements of Blood Vessels in Mouse Tracheal Mucosa
For in vivo treatments with COMP-Ang1, we developed a stable Chinese hamster ovary cell line (CA1eC2) which produces COMP-Ang1 at 30 mg/L. The potency, solubility, oligomerization status, and stability of the COMP-Ang1 produced from CA1eC2 are similar to those of COMP-Ang1 produced from COS-7 cells transiently transfected with plasmid vector containing the COMP-Ang1 gene10 (data not shown). Adult mice were treated with a daily intravenous injection of 200 e of COMP-Ang1 recombinant protein or BSA through the tail vein for 2 weeks, then blood vessels in the tracheal mucosa were visualized with PECAM-1 immunostaining (Figure 1). Six segments of the microvasculature were distinguished by their position in the vascular hierarchy and differences in endothelial cell morphology.14 Enlargement of tracheal blood vessels was found in mice that received COMP-Ang1 in the following descending order of effect: postcapillary venules>capillaries>collecting venules>venules>terminal arterioles (Figure 1B). No significant change was noted in segmental arterioles. These phenomena were observed in all individuals of several mouse strains studied (FVB/N, C57BL/6, BALB/c, BALB/c-nu, C3H/HeJ). No changes in the sizes or shape of tracheal blood vessels were found in mice that received BSA.
Short-Term and Intermittent Circulating COMP-Ang1 Induces Reversible Enlargement of Postcapillary Venules and Arterioles in Tracheal Vessels
When 200 e of COMP-Ang1 recombinant protein was injected intravenously into adult male mice, circulating COMP-Ang1 level peaked immediately after injection (3.75 minutes), then declined, and returned almost to the control level 3 to 4 hours after treatment (Figure 2A, left). The half-life (t1/2) of circulating COMP-Ang1 was 11.8 minutes. Daily intravenous injection of 200 e of COMP-Ang1 for 1 week in mice produced an 2.0-fold enlargement of postcapillary venules and a 1.4-fold enlargement of terminal arterioles in the trachea (Figure 2). The COMP-Ang1eCinduced enlargement of postcapillary venules, collecting venules, venous end of capillaries, venules, and terminal arterioles were further increased up to 2 weeks on continuation of daily injection of COMP-Ang1 for up to 2 weeks. However, COMP-Ang1eCinduced enlarged blood vessels returned gradually to normal after discontinuation of the COMP-Ang1 treatment (Figure 2). One month after discontinuation of the COMP-Ang1 treatment, a second round of treatment with a daily intravenous injection of 200 e of COMP-Ang1 for 2 weeks induced similar enlargements of tracheal vessels, again in a reversible manner (data not shown). In comparison, the diameters of tracheal vessels were indistinguishable between the control and experimental periods in tracheal vessels of mice treated with BSA (data not shown). These results indicate that short-term spikes of circulating COMP-Ang1 induce reversible enlargement of some tracheal vessels.
Long-Term and Sustained Circulating COMP-Ang1 Induces Long-Lasting Enlargement of Postcapillary Venules and Terminal Arterioles in Tracheal Vessels
As an alternative method for systemic treatment with COMP-Ang1, an adenoviral vector encoding the COMP-Ang1 gene (Ade-COMP-Ang1) was developed. As a control, an adenoviral vector encoding the LacZ gene (Ade-LacZ) was developed. The potency, solubility, oligomerization status, and stability of the COMP-Ang1 produced from HEK293 cells transduced with Ade-COMP-Ang1 are similar to that of COMP-Ang1 produced from COS-7 cells transiently transfected with plasmid vector containing the COMP-Ang1 gene10 (data not shown). Adult mice were treated with 1x109 pfu Ade-COMP-Ang1 or Ade-LacZ. At multiple times more than a period of 16 weeks, circulating plasma COMP-Ang1 levels were measured, and blood vessels in tracheal mucosa were visualized with PECAM-1 immunostaining (Figure 3). Circulating COMP-Ang1 increased as early as 12 hours after treatment, peaked at 1 to 2 weeks, declined gradually thereafter, and returned to control levels at 6 weeks after treatment (Figure 3A). The peak concentrations of circulating COMP-Ang1 were 3.5 to 4.5 e. Significant enlargement of postcapillary venules, capillaries (distinctively, only the venous end of capillaries was enlarged), collecting venules, and terminal arterioles, but not segmental arterioles, was noticeable at 1 week after the Ade-COMP-Ang1 treatment (Figure 3B). The vascular enlargements induced by Ade-COMP-Ang1 increased further for up to 6 weeks and then reached a plateau (Figure 3A and 3B). For example, the diameter of postcapillary venules increased 4.3-fold at 2 weeks, 6.0-fold at 4 weeks, and 6.8-fold at 6 weeks (Figure 3A). The enlargement of terminal arterioles was also significant beginning at 1 week after the treatment and increased in a time-dependent manner. However, the increase in diameter in terminal arterioles was less than that in postcapillary venules (Figure 3A and 3B). Importantly, the size of Ade-COMP-Ang1eCinduced enlarged blood vessels did not significantly decrease for as long as 16 weeks after the treatment, although circulating COMP-Ang1 returned to the control level at 6 weeks after treatment (Figure 3A). In comparison, diameters of tracheal vessels in mice treated with Ade-LacZ were indistinguishable between the control and experimental periods (data not shown). Using a laser-Doppler flowmeter, tracheal tissue blood flows were measured at 2 weeks (the peak level of circulating COMP-Ang1) and 16 weeks (undetectable level of circulating COMP-Ang1) after Ade-LacZ or Ade-COMP-Ang1 treatment. At 2 weeks, tracheal tissue blood flow was increased 25% in the mice treated with Ade-COMP-Ang1 compared with the mice treated with Ade-LacZ (Figure 3C and 3D). At 16 weeks, importantly, increased tracheal tissue blood flow by Ade-COMP-Ang1 was not significantly changed (Figure 3C and 3D). These results indicate that long term and sustained circulating COMP-Ang1 treatment induces long-lasting enlargement of tracheal blood vessels with long-lasting enhancement of tissue blood flow in the adult mice.
Tie2 Activation Is Involved in COMP-Ang1eCInduced Vascular Remodeling
To determine the involvement of Tie2 activation in COMP-Ang1eCinduced vascular remodeling, the mice were pretreated with 1x108 pfu or 5x108 pfu Ade-sTie2-Fc at 24 hours before 1x108 pfu Ade-COMP-Ang1 treatment. Two weeks later, the diameters of postcapillary venules and terminal arterioles were measured. Pretreatment with 1x108 pfu or 5x108 pfu Ade-sTie2-Fc suppressed COMP-Ang1eCinduced vascular remodeling to the following extent: 46.5±7.7% or 93.5±6.4% in postcapillary venules and 59.7±6.6% or 95.1±5.7% in terminal arterioles, respectively (Figure 3E and 3F). These data indicate that COMP-Ang1eCinduced vascular remodeling is mainly mediated through Tie2 activation in adult tracheal vessels.
Long-Term and Sustained Circulating COMP-Ang1 Induces Various Vascular Remodeling in Different Organ
Both mice treated with Ade-LacZ (1x109 pfu) and those treated with Ade-COMP-Ang1 (1x109 pfu) appeared generally healthy, as they gained weight normally. However, the skin of mice treated with Ade-COMP-Ang1 appeared strikingly redder than the skin of mice treated with Ade-LacZ, beginning 10 to 14 days after the treatment. The Ade-COMP-Ang1eCinduced skin redness persisted for as long as 16 weeks after the treatment (Figure 4). Sixteen weeks after the treatment, skin color in hair-sparse portions such as the face, hands, soles, penis, and tail of mice treated with Ade-COMP-Ang1 were distinctly redder than those of mice treated with Ade-LacZ. Blood vessels of the ear and capillaries of the heart, adrenal cortex, and liver of the mice treated with Ade-COMP-Ang1 were enlarged (Figures 4 and 5 ). More PECAM-1eCpositive endothelial cells were present in the lung, heart, liver, and renal medulla of mice treated with Ade-COMP-Ang1 compared with the mice treated with Ade-LacZ (Figures 4 and 5 and online Figure I in the data supplement). However, blood vessels of the renal cortex, including glomeruli, and intestinal villi of the mice treated with Ade-COMP-Ang1 and the mice treated with Ade-LacZ were indistinguishable. In addition, the body weights, systemic blood pressures, and heart rates of the 2 groups of mice were indistinguishable. These results indicate that long-term and sustained circulating COMP-Ang1 treatment induces long-lasting tissue-specific vascular remodeling in different blood vessels without notable changes in systemic blood pressure and heart rate (online Table I).
Induction of Tie2 Could Be Involved in Permanent Changes of COMP-Ang1eCInduced Vascular Remodeling
Based on these observations, we asked whether Tie2 expression was more abundant in postcapillary venules than terminal arterioles in mouse trachea. Therefore, we examined the extent of Tie2 expression using transgenic mice with Tie2 promotereCdriven green fluorescent protein (GFP).12 In the tracheal mucosa of adult mice, Tie2 expression was not detectable in most endothelial cells of postcapillary venules, whereas it was moderately expressed in endothelial cells of terminal and precapillary arterioles of tracheal vessels (Figure 6). Thus, differential enlargement of tracheal vessels on COMP-Ang1 stimulation is not dependent on the extent of Tie2 expression. However, Tie2 expression was markedly increased in endothelial cells of collecting venules, venules, postcapillary venules, and capillaries at 2 weeks after the Ade-COMP-Ang1 treatment (Figure 6), which is somewhat consistent with a recent report with Ade-Ang1.8 Tie2 expression was further increased in endothelial cells of the same vessels at 16 weeks after the Ade-COMP-Ang1 treatment (Figure 6). In contrast, Tie2 expression was not changed in any endothelial cells of enlarged tracheal vessels at 2 weeks after the recombinant COMP-Ang1 protein treatment (Figure 6). Area densities of Tie2 expression in a given microscopic field area (0.22 mm2) for arterioles, capillaries, and venules in tracheal mucosa were 8.2±1.7, 2.8±0.4, and 3.3±0.6% (mean±SD from 4 mice), respectively, after Ade-LacZ treatment (at 2 weeks); 7.6±1.9, 3.1±0.5, and 3.7±0.6% after COMP-Ang1 protein treatment (at 2 weeks); 11.3±2.2, 10.3±1.7, and 28.1±5.4% after Ade-COMP treatment (at 2 weeks); and 13.3±2.7, 18.2±3.5, and 47.7±7.2 after Ade-COMP treatment (at 16 weeks). In addition, Tie2 expression was notably higher in the enlarged veins of abdominal skin and the sinusoidal capillaries in liver of the mice treated with Ade-COMP-Ang1 than the mice treated with Ade-LacZ at 16 weeks after the treatment (online Figure II). Thus, Tie2 expression in venular and capillary endothelial cells could be induced with long-term and sustained Tie2 stimulation induced by Ade-COMP-Ang1 but not with short-term spiked Tie2 stimulation induced by recombinant COMP-Ang1 protein.
COMP-Ang1eCInduced Vascular Enlargement Could Result From Circumferential Endothelial Cell Proliferation
COMP-Ang1eCinduced enlargement of blood venules appears to result from endothelial cell proliferation rather than vasodilation or endothelial cell hypertrophy because the endothelial cells were normal in size (Figure 7A and 7B). To test this possibility, we examined by immunostaining the number of endothelial cells positive for phosphohistone H3 (nuclear protein of diving cell). Numerous phosphohistone H3eCpositive immunostained endothelial cells were detected in various portions including postcapillary venules, capillaries, collecting venules, venules, and terminal arterioles of tracheal vessels at 4 days and 2 weeks after the Ade-COMP-Ang1 treatment (Figure 7D, 7F, and 7I) or after recombinant COMP-Ang1 protein treatment (data not shown). However, almost no phosphohistone H3eCpositive endothelial cells were detected in any portion of tracheal vessels at 4 days or 2 and 16 weeks after the Ade-LacZ treatment and at 16 weeks after the Ade-COMP-Ang1 treatment (Figure 7C, 7E, 7G, and 7I). These findings indicate that vascular enlargement induced by COMP-Ang1 is more likely to result from endothelial cell proliferation depending on concentration of circulating COMP-Ang1 than from vasodilation or endothelial cell hypertrophy.
COMP-Ang1eCInduced Postcapillary Venule Enlargement Is Not Accompanied by Pericyte Recruitment
Ang1 is known to be a strong growth factor for pericyte recruitment to nascent endothelial cells during vasculogenesis in physiological and pathological conditions.3eC5 Therefore, we examined the interaction between endothelial cells and pericytes in the enlarged blood vessels of the trachea by double-immunostaining for endothelial cells and pericytes at 4 weeks after Ade-LacZ or Ade-COMP-Ang1 treatment. The interaction of endothelial cells and pericytes in most of tracheal blood vessels (except postcapillary venules) in mice that received Ade-COMP-Ang1 was similar to that in mice that received Ade-LacZ (Figure 8). Although less interaction of endothelial cells with pericytes was found on the enlarged postcapillary venules than elsewhere, the number of pericytes of the enlarged postcapillary venules was similar to the control postcapillary venules (Figure 8). Thus, COMP-Ang1 did not promote pericyte recruitment to the COMP-Ang1eCinduced enlarged venules in the trachea.
Discussion
The most important and novel finding in this study is that enlargement of tracheal blood vessels and enhancement of tracheal tissue blood flow induced by long-term and sustained exposure to COMP-Ang1 had not regressed for up to 16 weeks, despite the fact that exposure to COMP-Ang1 had already been discontinued at 6 to 7 weeks in adult mice. In comparison, enlargement of tracheal blood vessels induced by short-term intermittent exposure to COMP-Ang1 regressed on discontinuation of recombinant COMP-Ang1 treatment. Therefore, long-lasting vascular enlargement and enhancement of blood flow can be achieved by long-term and sustained exposure to COMP-Ang1.
Like other therapeutic proteins, circulating COMP-Ang1 rapidly disappeared in the plasma, probably because of its trapping by the Tie2 receptor of lung endothelial cells.15 However, we were able to achieve long-term (>4 weeks) and sustained (>1000 ng/mL) circulating COMP-Ang1 in mice by a single intravenous injection of 1x109 pfu Ade-COMP-Ang1. Throughout these experiments, we learned that long-term (6 weeks) and sustained exposure to COMP-Ang1 produced long-lasting enlargement of postcapillary venules and terminal arterioles in the tracheal mucosa, whereas short-term (2 weeks) and intermittent exposure to COMP-Ang1 produced reversible enlargements of these vessels. Similar to our results, another study found that long-term (4 weeks) sustained exposure to vascular endothelial growth factor (VEGF) produced long-lasting acquired vascular remodeling in liver, whereas short-term (2 weeks) sustained exposure to VEGF produced reversible vascular remodeling.16 What are the major mechanisms and factors that produce long-lasting and reversible vascular remodeling Is there a threshold stimulation of Tie2 by COMP-Ang1 that can produce permanent enlargement Our results suggest that auto-amplification of Tie2 expression by treatment with COMP-Ang1 above a certain dose and exposure period could be one of the mechanisms. Once Tie2 expression is activated by a long-term and excess exposure to COMP-Ang1, after discontinuation of COMP-Ang1, the subsequent activation of Tie2 might be achieved by endogenous circulating Ang1 or increased shear stress caused by increased blood flow.17 However, auto-amplification of Tie2 expression cannot be achieved below a certain dose and exposure period of COMP-Ang1, as evidenced by the experiments with intravenous administration of COMP-Ang1 recombinant protein. Therefore, the dose and the exposure period of COMP-Ang1 or VEGF should be considered in any therapeutic approaches where permanent vascular enlargements are needed to alleviate dysfunctions of ischemic tissues.
Tie1, an endothelial-specific receptor tyrosine kinase, shares a high degree of homology with Tie2. Although Tie1 was isolated more than a decade ago,18 no ligand had been found to activate it. Recently, Saharinen et al demonstrated that COMP-Ang1 stimulated Tie1 phosphorylation in cultured endothelial cells.19 Moreover, they showed that COMP-Ang1eCinduced Tie1 activation was amplified via Tie2 and was more efficient than native Ang1- and Ang4-induced Tie1 activation. Thus, COMP-Ang1 and Ang1 are now known to be activating ligands for both Tie1 and Tie2. However, our data indicate that COMP-Ang1eCinduced vascular remodeling in adult tracheal vessels is mainly mediated through activation of Tie2, not by Tie1. (See expanded Discussion section in the online data supplement.)
Although Ang1 induces vascular enlargement and has therapeutic benefits to ischemic tissues in several experimental animal models,6,7,9 little is known about whether the vascular enlargement is accompanied by enhanced blood flow. Our results showed that COMP-Ang1eCinduced vascular enlargement was accompanied by enhanced tissue blood flow in the trachea. Therefore, enhanced blood flow through arteriolar and venular enlargements induced by COMP-Ang1 could provide a great therapeutic benefit to ischemic peripheral tissues. In fact, Ang1-induced vessel enlargement is a unique characteristic among many growth factors. Our immunohistological examination of phosphohistone H3 revealed that COMP-Ang1eCinduced vascular enlargements were evidently the result of endothelial proliferation, which is consistent with a recent report.14 Thus, arteriolar and venular enlargements are achieved mainly by circumferential endothelial proliferation, which is a unique phenomenon and is different from multidirectional endothelial cell proliferation during vasculogenesis and angiogenesis. Moreover, our results revealed that different organs showed different sensitivities to long-term and sustained COMP-Ang1. In fact, blood vessels in the skin, heart, adrenal cortex, and liver, among other organs, are relatively sensitive to the COMP-Ang1eCinduced vascular enlargement. Therefore, COMP-Ang1 could provide a great therapeutic benefit to patients with delayed skin wound healing and ischemic heart diseases through its ability to promote vascular remodeling. Nevertheless, the mice treated with long-lasting and sustained COMP-Ang1 did not show any significant changes in body weight, systemic blood pressure, or heart rate. More detailed analysis will be necessary to clarify how it is possible that the mice with enlarged blood vessels caused by long-term and sustained COMP-Ang1 have normal blood pressure and heart rate.
Ang1 is known to be a strong growth factor for pericyte recruitment to nascent endothelial cells during development.3eC5 This Ang1-induced pericyte recruitment is related to the Ang1-induced antileakage effect on VEGF and proinflammatory stimuli.5 However, our results show a lower number and poorer covering of pericytes in COMP-Ang1eCinduced enlarged postcapillary venules. In fact, in a mouse model that completely blocks pericyte recruitment to developing vessels by injection of antagonistic monoclonal antibody against platelet-derived growth factor receptor-, Ang1 is able to restore a hierarchical architecture of growing blood vessels and rescues retinal edema and hemorrhage even in the absence of pericyte recruitment.20 Thus, COMP-Ang1 may be able to assemble endothelial cells in a frame of hierarchical architecture without pericyte recruitment in the COMP-Ang1eCinduced enlarged blood vessels.
In conclusion, long-lasting vascular enlargement and enhancement of blood flow can be achieved by long-term and sustained exposure to COMP-Ang1.
Acknowledgments
Supported, in part, by the Bio-Challenge Program and the National Research Laboratory Program (2004-02376 to G.Y.K.) of the Korean Ministry of Science and Technology, the Korea Health R&D Project (0405-DB01-0104-0006 to G.Y.K.), the Ministry of Health & Welfare, and the Korea Science and Engineering Foundation (R01-2004-000-10045-0 to B.H.J.). Also supported by National Institutes of Health grants HL-24136 and HL-59157 from the National Heart, Lung and Blood Institute (to D.M.D.).
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the Department of Medicine (J.B., H.-S.J., D.-K.K.), Samsung Medical Center and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Seoul, Korea
the Cardiovascular Research Institute, Comprehensive Cancer Center
Department of Anatomy (P.B., F.B., D.M.M.), University of California, San Francisco
the Department of Structural Analysis (N.M.), National Cardiovascular Center Research Institute, Suita, Osaka, Japan
the Department of Anatomy (J.K.), College of Medicine, The Catholic University of Korea Seoul
the Department of Physiology (B.H.J), College of Medicine, Chungnam National University Daejeon, Korea.
Abstract
Vascular enlargement is a characteristic feature of angiopoietin-1 (Ang1)-induced changes in adult blood vessels. However, it is unknown whether tissues having Ang1-mediated vascular enlargement have more blood flow or whether the enlargement is reversible. We have recently created a soluble, stable and potent Ang1 variant, COMP-Ang1. In the present study, we investigated the effects of varied dose and duration of COMP-Ang1 on vascular enlargement and blood flow in the tracheal microvasculature of adult mice and explored a possible mechanism of long-lasting vascular enlargement. We found that COMP-Ang1 administered by adenoviral vector induced long-lasting vascular enlargement and increased tracheal blood flow. In contrast, short-term administration of COMP-Ang1 recombinant protein induced transient vascular enlargement that spontaneously reversed within a month. In both cases, the vascular enlargement resulted from endothelial proliferation. The COMP-Ang1eCinduced vascular remodeling is mediated mainly through Tie2 activation. Sustained overexpression of Tie2 could participate in the maintenance of vascular changes. Together, our findings indicate that sustained treatment with COMP-Ang1 can produce long-lasting vascular enlargement and increased blood flow.
Key Words: angiopoietin-1 COMP-Ang1 vascular enlargement blood flow
Introduction
Angiopoietin-1 (Ang1) is known to be a ligand to Tie2 tyrosine kinase receptor expressed on endothelial cells.1 Ang1/Tie2 signaling is thought be involved in branching and remodeling of the primitive vascular network and in the recruitment of mural cells during development.2,3 Transgenic overexpression of Ang1 using the skin-specific keratin-14 promoter produces leakage-resistant and enlarged vessels with an increased number of endothelial cells in skin.4,5 Gene transfer of Ang1 into ischemic tissues produces notably enlarged blood vessels.6,7 Baffert et al recently identified that Ang1-induced vascular enlargement could be the result of endothelial proliferation in trachea mucosa.8 Thus, a cardinal feature of Ang1-induced vascular remodeling is vascular enlargement resulting from endothelial cell proliferation in adult animals.4eC8
Given that Ang1-induced therapeutic benefits correlated with vascular enlargement in the ischemic tissues,6,7,9 enhanced blood flow through blood vessels enlarged by Ang1 treatment could provide a great therapeutic benefit to ischemic peripheral tissues. However, it is not known whether the tissues having Ang1-mediated enlarged vessels have more blood flow. In addition, the effective dose and treatment period of Ang1 for inducing effective vascular enlargement is not known. Moreover, it is not known whether Ang1-mediated vascular enlargement regresses when Ang1 stimulation is withdrawn.
We have recently developed a soluble, stable, and potent Ang1 variant, COMP-Ang1.10 To create this protein, we replaced the amino-terminal portion of Ang1 with the short coiled-coil domain of cartilage oligomeric matrix protein (COMP). COMP-Ang1 is more potent than native Ang1 in phosphorylating the Tie2 receptor and signaling via Akt in primary cultured endothelial cells.10
In the present study, we investigated effects of period and dose of COMP-Ang1 on vascular enlargement and tissue blood flow in adult mice and investigated a possible mechanism for long-lasting vascular enlargement induced by long-term and sustained COMP-Ang1. To determine the underlying mechanism of COMP-Ang1eCstimulated vascular remodeling in adult mice, we focused on the microvasculature of the trachea, which is distinguished by its simplicity and monolayer structure. Our results indicate that long-term and sustained COMP-Ang1 produced by adenoviral delivery of COMP-Ang1 induces a long-lasting vascular enlargement and enhanced blood flow without enhanced pericyte recruitment in adult mice. Long-lasting Tie2 expression could be involved in the long-lasting vascular enlargement and enhanced blood flow.
Materials and Methods
Generation of COMP-Ang1 Recombinant Protein and Ade-COMP-Ang1
Recombinant Chinese hamster ovary cells expressing COMP-Ang1 (CA1eC2; production rate, 30 mg/L) were established as previously described.11 Recombinant adenovirus expressing COMP-Ang1 or LacZ was constructed using the pAdEasy vector system (Qbiogene). For additional Materials and Methods, see online data supplement at http://circres.ahajournals.org.
Animals, Treatment, and Measurement of Blood Pressure and Heart Rate
Specific pathogen-free FVB/N mice and Tie2-GFP transgenic mice (FVB/N)12 were purchased from Jackson Laboratory and bred in our pathogen-free animal facility. Male mice 8 to 10 weeks old were used for this study. Animal care and experimental procedures were performed under approval from the Animal Care Committees of the Korea Advanced Institute of Science and Technology. For protein treatment, 200 e of COMP-Ang1 recombinant protein or BSA dissolved in 50 e蘈 of sterile 0.9% NaCl was injected daily through the tail vein for 2 weeks. For adenoviral treatment, the indicated amount of Ade-COMP-Ang1, Ade-LacZ, or Ade-sTie2-Fc (generous gift from Drs Gavin Thurston and Ella Ioffe at Regeneron Pharmaceuticals, Terrytown, NY) diluted in 50 e蘈 of sterile 0.9% NaCl was injected intravenously through the tail vein. Systemic blood pressure and heart rate were measured under anesthesia.
Enzyme-Linked Immunosorbent Assay
Approximately 50 e蘈 of blood was obtained from the tail vein into a heparinized capillary tube at the indicated times. ELISA was adopted for precise detection of COMP-Ang1 in plasma.
Immunohistochemical Staining
Mice were anesthetized, perfused with 1% paraformaldehyde in PBS, and several organs including tracheas were removed. Tracheas and ear skins were immunostained as whole mounts, whereas other organs were immunostained as sections. Signals were visualized, and digital images were obtained with a Zeiss Apotome microscope and a Zeiss LSM 510 confocal microscope.
Measurement of Tracheal Tissue Blood Flow
After the mice were anesthetized, a type N flowprobe (Transonic Systems Inc, Ithaca, NY) was placed on tracheal wall along second, third, and forth cartilage rings without applying pressure, as this would occlude the vessels and reduce perfusion in the area of interest. The flowprobe was kept in place on the position of the highest sensitivity by a micromanipulator and connected to a laser-Doppler flowmeter (model BLF21; Transonic Systems Inc), which can measure microcirculation in 1 mm3 of tissue for real-time assessment of perfusion (mL/min per 100 g of tissue).
Morphometric Measurements and Statistics
Morphometric measurements of the vessel diameters and area densities in mouse trachea were made as previously described.13 For each trachea, the numbers of PH3-immunopositive endothelial cells, platelet/endothelial cell adhesion molecule (PECAM)-1eCimmunopositive blood vessels, and desmin/NG2-immunopositive pericytes were measured in 5 regions, each 0.21 mm2 in area. Values were expressed per millimeter squared. Values presented are mean±SD. Significance of differences between mean was tested by analysis of variance followed by the StudenteCNewmaneCKeuls test. Statistical significance was set at P<0.05.
Results
Systemic Adenoviral COMP-Ang1 Produces Differential Enlargements of Blood Vessels in Mouse Tracheal Mucosa
For in vivo treatments with COMP-Ang1, we developed a stable Chinese hamster ovary cell line (CA1eC2) which produces COMP-Ang1 at 30 mg/L. The potency, solubility, oligomerization status, and stability of the COMP-Ang1 produced from CA1eC2 are similar to those of COMP-Ang1 produced from COS-7 cells transiently transfected with plasmid vector containing the COMP-Ang1 gene10 (data not shown). Adult mice were treated with a daily intravenous injection of 200 e of COMP-Ang1 recombinant protein or BSA through the tail vein for 2 weeks, then blood vessels in the tracheal mucosa were visualized with PECAM-1 immunostaining (Figure 1). Six segments of the microvasculature were distinguished by their position in the vascular hierarchy and differences in endothelial cell morphology.14 Enlargement of tracheal blood vessels was found in mice that received COMP-Ang1 in the following descending order of effect: postcapillary venules>capillaries>collecting venules>venules>terminal arterioles (Figure 1B). No significant change was noted in segmental arterioles. These phenomena were observed in all individuals of several mouse strains studied (FVB/N, C57BL/6, BALB/c, BALB/c-nu, C3H/HeJ). No changes in the sizes or shape of tracheal blood vessels were found in mice that received BSA.
Short-Term and Intermittent Circulating COMP-Ang1 Induces Reversible Enlargement of Postcapillary Venules and Arterioles in Tracheal Vessels
When 200 e of COMP-Ang1 recombinant protein was injected intravenously into adult male mice, circulating COMP-Ang1 level peaked immediately after injection (3.75 minutes), then declined, and returned almost to the control level 3 to 4 hours after treatment (Figure 2A, left). The half-life (t1/2) of circulating COMP-Ang1 was 11.8 minutes. Daily intravenous injection of 200 e of COMP-Ang1 for 1 week in mice produced an 2.0-fold enlargement of postcapillary venules and a 1.4-fold enlargement of terminal arterioles in the trachea (Figure 2). The COMP-Ang1eCinduced enlargement of postcapillary venules, collecting venules, venous end of capillaries, venules, and terminal arterioles were further increased up to 2 weeks on continuation of daily injection of COMP-Ang1 for up to 2 weeks. However, COMP-Ang1eCinduced enlarged blood vessels returned gradually to normal after discontinuation of the COMP-Ang1 treatment (Figure 2). One month after discontinuation of the COMP-Ang1 treatment, a second round of treatment with a daily intravenous injection of 200 e of COMP-Ang1 for 2 weeks induced similar enlargements of tracheal vessels, again in a reversible manner (data not shown). In comparison, the diameters of tracheal vessels were indistinguishable between the control and experimental periods in tracheal vessels of mice treated with BSA (data not shown). These results indicate that short-term spikes of circulating COMP-Ang1 induce reversible enlargement of some tracheal vessels.
Long-Term and Sustained Circulating COMP-Ang1 Induces Long-Lasting Enlargement of Postcapillary Venules and Terminal Arterioles in Tracheal Vessels
As an alternative method for systemic treatment with COMP-Ang1, an adenoviral vector encoding the COMP-Ang1 gene (Ade-COMP-Ang1) was developed. As a control, an adenoviral vector encoding the LacZ gene (Ade-LacZ) was developed. The potency, solubility, oligomerization status, and stability of the COMP-Ang1 produced from HEK293 cells transduced with Ade-COMP-Ang1 are similar to that of COMP-Ang1 produced from COS-7 cells transiently transfected with plasmid vector containing the COMP-Ang1 gene10 (data not shown). Adult mice were treated with 1x109 pfu Ade-COMP-Ang1 or Ade-LacZ. At multiple times more than a period of 16 weeks, circulating plasma COMP-Ang1 levels were measured, and blood vessels in tracheal mucosa were visualized with PECAM-1 immunostaining (Figure 3). Circulating COMP-Ang1 increased as early as 12 hours after treatment, peaked at 1 to 2 weeks, declined gradually thereafter, and returned to control levels at 6 weeks after treatment (Figure 3A). The peak concentrations of circulating COMP-Ang1 were 3.5 to 4.5 e. Significant enlargement of postcapillary venules, capillaries (distinctively, only the venous end of capillaries was enlarged), collecting venules, and terminal arterioles, but not segmental arterioles, was noticeable at 1 week after the Ade-COMP-Ang1 treatment (Figure 3B). The vascular enlargements induced by Ade-COMP-Ang1 increased further for up to 6 weeks and then reached a plateau (Figure 3A and 3B). For example, the diameter of postcapillary venules increased 4.3-fold at 2 weeks, 6.0-fold at 4 weeks, and 6.8-fold at 6 weeks (Figure 3A). The enlargement of terminal arterioles was also significant beginning at 1 week after the treatment and increased in a time-dependent manner. However, the increase in diameter in terminal arterioles was less than that in postcapillary venules (Figure 3A and 3B). Importantly, the size of Ade-COMP-Ang1eCinduced enlarged blood vessels did not significantly decrease for as long as 16 weeks after the treatment, although circulating COMP-Ang1 returned to the control level at 6 weeks after treatment (Figure 3A). In comparison, diameters of tracheal vessels in mice treated with Ade-LacZ were indistinguishable between the control and experimental periods (data not shown). Using a laser-Doppler flowmeter, tracheal tissue blood flows were measured at 2 weeks (the peak level of circulating COMP-Ang1) and 16 weeks (undetectable level of circulating COMP-Ang1) after Ade-LacZ or Ade-COMP-Ang1 treatment. At 2 weeks, tracheal tissue blood flow was increased 25% in the mice treated with Ade-COMP-Ang1 compared with the mice treated with Ade-LacZ (Figure 3C and 3D). At 16 weeks, importantly, increased tracheal tissue blood flow by Ade-COMP-Ang1 was not significantly changed (Figure 3C and 3D). These results indicate that long term and sustained circulating COMP-Ang1 treatment induces long-lasting enlargement of tracheal blood vessels with long-lasting enhancement of tissue blood flow in the adult mice.
Tie2 Activation Is Involved in COMP-Ang1eCInduced Vascular Remodeling
To determine the involvement of Tie2 activation in COMP-Ang1eCinduced vascular remodeling, the mice were pretreated with 1x108 pfu or 5x108 pfu Ade-sTie2-Fc at 24 hours before 1x108 pfu Ade-COMP-Ang1 treatment. Two weeks later, the diameters of postcapillary venules and terminal arterioles were measured. Pretreatment with 1x108 pfu or 5x108 pfu Ade-sTie2-Fc suppressed COMP-Ang1eCinduced vascular remodeling to the following extent: 46.5±7.7% or 93.5±6.4% in postcapillary venules and 59.7±6.6% or 95.1±5.7% in terminal arterioles, respectively (Figure 3E and 3F). These data indicate that COMP-Ang1eCinduced vascular remodeling is mainly mediated through Tie2 activation in adult tracheal vessels.
Long-Term and Sustained Circulating COMP-Ang1 Induces Various Vascular Remodeling in Different Organ
Both mice treated with Ade-LacZ (1x109 pfu) and those treated with Ade-COMP-Ang1 (1x109 pfu) appeared generally healthy, as they gained weight normally. However, the skin of mice treated with Ade-COMP-Ang1 appeared strikingly redder than the skin of mice treated with Ade-LacZ, beginning 10 to 14 days after the treatment. The Ade-COMP-Ang1eCinduced skin redness persisted for as long as 16 weeks after the treatment (Figure 4). Sixteen weeks after the treatment, skin color in hair-sparse portions such as the face, hands, soles, penis, and tail of mice treated with Ade-COMP-Ang1 were distinctly redder than those of mice treated with Ade-LacZ. Blood vessels of the ear and capillaries of the heart, adrenal cortex, and liver of the mice treated with Ade-COMP-Ang1 were enlarged (Figures 4 and 5 ). More PECAM-1eCpositive endothelial cells were present in the lung, heart, liver, and renal medulla of mice treated with Ade-COMP-Ang1 compared with the mice treated with Ade-LacZ (Figures 4 and 5 and online Figure I in the data supplement). However, blood vessels of the renal cortex, including glomeruli, and intestinal villi of the mice treated with Ade-COMP-Ang1 and the mice treated with Ade-LacZ were indistinguishable. In addition, the body weights, systemic blood pressures, and heart rates of the 2 groups of mice were indistinguishable. These results indicate that long-term and sustained circulating COMP-Ang1 treatment induces long-lasting tissue-specific vascular remodeling in different blood vessels without notable changes in systemic blood pressure and heart rate (online Table I).
Induction of Tie2 Could Be Involved in Permanent Changes of COMP-Ang1eCInduced Vascular Remodeling
Based on these observations, we asked whether Tie2 expression was more abundant in postcapillary venules than terminal arterioles in mouse trachea. Therefore, we examined the extent of Tie2 expression using transgenic mice with Tie2 promotereCdriven green fluorescent protein (GFP).12 In the tracheal mucosa of adult mice, Tie2 expression was not detectable in most endothelial cells of postcapillary venules, whereas it was moderately expressed in endothelial cells of terminal and precapillary arterioles of tracheal vessels (Figure 6). Thus, differential enlargement of tracheal vessels on COMP-Ang1 stimulation is not dependent on the extent of Tie2 expression. However, Tie2 expression was markedly increased in endothelial cells of collecting venules, venules, postcapillary venules, and capillaries at 2 weeks after the Ade-COMP-Ang1 treatment (Figure 6), which is somewhat consistent with a recent report with Ade-Ang1.8 Tie2 expression was further increased in endothelial cells of the same vessels at 16 weeks after the Ade-COMP-Ang1 treatment (Figure 6). In contrast, Tie2 expression was not changed in any endothelial cells of enlarged tracheal vessels at 2 weeks after the recombinant COMP-Ang1 protein treatment (Figure 6). Area densities of Tie2 expression in a given microscopic field area (0.22 mm2) for arterioles, capillaries, and venules in tracheal mucosa were 8.2±1.7, 2.8±0.4, and 3.3±0.6% (mean±SD from 4 mice), respectively, after Ade-LacZ treatment (at 2 weeks); 7.6±1.9, 3.1±0.5, and 3.7±0.6% after COMP-Ang1 protein treatment (at 2 weeks); 11.3±2.2, 10.3±1.7, and 28.1±5.4% after Ade-COMP treatment (at 2 weeks); and 13.3±2.7, 18.2±3.5, and 47.7±7.2 after Ade-COMP treatment (at 16 weeks). In addition, Tie2 expression was notably higher in the enlarged veins of abdominal skin and the sinusoidal capillaries in liver of the mice treated with Ade-COMP-Ang1 than the mice treated with Ade-LacZ at 16 weeks after the treatment (online Figure II). Thus, Tie2 expression in venular and capillary endothelial cells could be induced with long-term and sustained Tie2 stimulation induced by Ade-COMP-Ang1 but not with short-term spiked Tie2 stimulation induced by recombinant COMP-Ang1 protein.
COMP-Ang1eCInduced Vascular Enlargement Could Result From Circumferential Endothelial Cell Proliferation
COMP-Ang1eCinduced enlargement of blood venules appears to result from endothelial cell proliferation rather than vasodilation or endothelial cell hypertrophy because the endothelial cells were normal in size (Figure 7A and 7B). To test this possibility, we examined by immunostaining the number of endothelial cells positive for phosphohistone H3 (nuclear protein of diving cell). Numerous phosphohistone H3eCpositive immunostained endothelial cells were detected in various portions including postcapillary venules, capillaries, collecting venules, venules, and terminal arterioles of tracheal vessels at 4 days and 2 weeks after the Ade-COMP-Ang1 treatment (Figure 7D, 7F, and 7I) or after recombinant COMP-Ang1 protein treatment (data not shown). However, almost no phosphohistone H3eCpositive endothelial cells were detected in any portion of tracheal vessels at 4 days or 2 and 16 weeks after the Ade-LacZ treatment and at 16 weeks after the Ade-COMP-Ang1 treatment (Figure 7C, 7E, 7G, and 7I). These findings indicate that vascular enlargement induced by COMP-Ang1 is more likely to result from endothelial cell proliferation depending on concentration of circulating COMP-Ang1 than from vasodilation or endothelial cell hypertrophy.
COMP-Ang1eCInduced Postcapillary Venule Enlargement Is Not Accompanied by Pericyte Recruitment
Ang1 is known to be a strong growth factor for pericyte recruitment to nascent endothelial cells during vasculogenesis in physiological and pathological conditions.3eC5 Therefore, we examined the interaction between endothelial cells and pericytes in the enlarged blood vessels of the trachea by double-immunostaining for endothelial cells and pericytes at 4 weeks after Ade-LacZ or Ade-COMP-Ang1 treatment. The interaction of endothelial cells and pericytes in most of tracheal blood vessels (except postcapillary venules) in mice that received Ade-COMP-Ang1 was similar to that in mice that received Ade-LacZ (Figure 8). Although less interaction of endothelial cells with pericytes was found on the enlarged postcapillary venules than elsewhere, the number of pericytes of the enlarged postcapillary venules was similar to the control postcapillary venules (Figure 8). Thus, COMP-Ang1 did not promote pericyte recruitment to the COMP-Ang1eCinduced enlarged venules in the trachea.
Discussion
The most important and novel finding in this study is that enlargement of tracheal blood vessels and enhancement of tracheal tissue blood flow induced by long-term and sustained exposure to COMP-Ang1 had not regressed for up to 16 weeks, despite the fact that exposure to COMP-Ang1 had already been discontinued at 6 to 7 weeks in adult mice. In comparison, enlargement of tracheal blood vessels induced by short-term intermittent exposure to COMP-Ang1 regressed on discontinuation of recombinant COMP-Ang1 treatment. Therefore, long-lasting vascular enlargement and enhancement of blood flow can be achieved by long-term and sustained exposure to COMP-Ang1.
Like other therapeutic proteins, circulating COMP-Ang1 rapidly disappeared in the plasma, probably because of its trapping by the Tie2 receptor of lung endothelial cells.15 However, we were able to achieve long-term (>4 weeks) and sustained (>1000 ng/mL) circulating COMP-Ang1 in mice by a single intravenous injection of 1x109 pfu Ade-COMP-Ang1. Throughout these experiments, we learned that long-term (6 weeks) and sustained exposure to COMP-Ang1 produced long-lasting enlargement of postcapillary venules and terminal arterioles in the tracheal mucosa, whereas short-term (2 weeks) and intermittent exposure to COMP-Ang1 produced reversible enlargements of these vessels. Similar to our results, another study found that long-term (4 weeks) sustained exposure to vascular endothelial growth factor (VEGF) produced long-lasting acquired vascular remodeling in liver, whereas short-term (2 weeks) sustained exposure to VEGF produced reversible vascular remodeling.16 What are the major mechanisms and factors that produce long-lasting and reversible vascular remodeling Is there a threshold stimulation of Tie2 by COMP-Ang1 that can produce permanent enlargement Our results suggest that auto-amplification of Tie2 expression by treatment with COMP-Ang1 above a certain dose and exposure period could be one of the mechanisms. Once Tie2 expression is activated by a long-term and excess exposure to COMP-Ang1, after discontinuation of COMP-Ang1, the subsequent activation of Tie2 might be achieved by endogenous circulating Ang1 or increased shear stress caused by increased blood flow.17 However, auto-amplification of Tie2 expression cannot be achieved below a certain dose and exposure period of COMP-Ang1, as evidenced by the experiments with intravenous administration of COMP-Ang1 recombinant protein. Therefore, the dose and the exposure period of COMP-Ang1 or VEGF should be considered in any therapeutic approaches where permanent vascular enlargements are needed to alleviate dysfunctions of ischemic tissues.
Tie1, an endothelial-specific receptor tyrosine kinase, shares a high degree of homology with Tie2. Although Tie1 was isolated more than a decade ago,18 no ligand had been found to activate it. Recently, Saharinen et al demonstrated that COMP-Ang1 stimulated Tie1 phosphorylation in cultured endothelial cells.19 Moreover, they showed that COMP-Ang1eCinduced Tie1 activation was amplified via Tie2 and was more efficient than native Ang1- and Ang4-induced Tie1 activation. Thus, COMP-Ang1 and Ang1 are now known to be activating ligands for both Tie1 and Tie2. However, our data indicate that COMP-Ang1eCinduced vascular remodeling in adult tracheal vessels is mainly mediated through activation of Tie2, not by Tie1. (See expanded Discussion section in the online data supplement.)
Although Ang1 induces vascular enlargement and has therapeutic benefits to ischemic tissues in several experimental animal models,6,7,9 little is known about whether the vascular enlargement is accompanied by enhanced blood flow. Our results showed that COMP-Ang1eCinduced vascular enlargement was accompanied by enhanced tissue blood flow in the trachea. Therefore, enhanced blood flow through arteriolar and venular enlargements induced by COMP-Ang1 could provide a great therapeutic benefit to ischemic peripheral tissues. In fact, Ang1-induced vessel enlargement is a unique characteristic among many growth factors. Our immunohistological examination of phosphohistone H3 revealed that COMP-Ang1eCinduced vascular enlargements were evidently the result of endothelial proliferation, which is consistent with a recent report.14 Thus, arteriolar and venular enlargements are achieved mainly by circumferential endothelial proliferation, which is a unique phenomenon and is different from multidirectional endothelial cell proliferation during vasculogenesis and angiogenesis. Moreover, our results revealed that different organs showed different sensitivities to long-term and sustained COMP-Ang1. In fact, blood vessels in the skin, heart, adrenal cortex, and liver, among other organs, are relatively sensitive to the COMP-Ang1eCinduced vascular enlargement. Therefore, COMP-Ang1 could provide a great therapeutic benefit to patients with delayed skin wound healing and ischemic heart diseases through its ability to promote vascular remodeling. Nevertheless, the mice treated with long-lasting and sustained COMP-Ang1 did not show any significant changes in body weight, systemic blood pressure, or heart rate. More detailed analysis will be necessary to clarify how it is possible that the mice with enlarged blood vessels caused by long-term and sustained COMP-Ang1 have normal blood pressure and heart rate.
Ang1 is known to be a strong growth factor for pericyte recruitment to nascent endothelial cells during development.3eC5 This Ang1-induced pericyte recruitment is related to the Ang1-induced antileakage effect on VEGF and proinflammatory stimuli.5 However, our results show a lower number and poorer covering of pericytes in COMP-Ang1eCinduced enlarged postcapillary venules. In fact, in a mouse model that completely blocks pericyte recruitment to developing vessels by injection of antagonistic monoclonal antibody against platelet-derived growth factor receptor-, Ang1 is able to restore a hierarchical architecture of growing blood vessels and rescues retinal edema and hemorrhage even in the absence of pericyte recruitment.20 Thus, COMP-Ang1 may be able to assemble endothelial cells in a frame of hierarchical architecture without pericyte recruitment in the COMP-Ang1eCinduced enlarged blood vessels.
In conclusion, long-lasting vascular enlargement and enhancement of blood flow can be achieved by long-term and sustained exposure to COMP-Ang1.
Acknowledgments
Supported, in part, by the Bio-Challenge Program and the National Research Laboratory Program (2004-02376 to G.Y.K.) of the Korean Ministry of Science and Technology, the Korea Health R&D Project (0405-DB01-0104-0006 to G.Y.K.), the Ministry of Health & Welfare, and the Korea Science and Engineering Foundation (R01-2004-000-10045-0 to B.H.J.). Also supported by National Institutes of Health grants HL-24136 and HL-59157 from the National Heart, Lung and Blood Institute (to D.M.D.).
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