Published online before print May 31, 2007, doi: 10.1161/CIRCRESAHA.106.143594
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© 2007 American Heart Association, Inc.
Integrative Physiology |
Angiopoietin-2 Impairs Revascularization After Limb Ischemia
From the Institute of Neurology (Y.R., J.D., M.H.H.S., K.H.P.), Frankfurt University Medical School, Germany; Max-Planck Institute for Heart & Lung Research (M.H., S.T., W.S.), Bad Nauheim, Germany; Institute of Biochemistry II (M.H.H.S.), Frankfurt University Medical School, Germany; and Sunnybrook & Women’s Research Institute (D.J.D.), Toronto, Canada.
Correspondence to Prof Karl H. Plate, Institute of Neurology, Deutschordenstrasse 46, 60528 Frankfurt, Germany. E-mail karl-heinz.plate{at}kgu.de
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Abstract |
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Angiopoietins play important roles in the formation of neovessels and complex vascular networks. Angiopoietin (Ang)-1 and Ang-2 belong to a family of growth factors that display opposing effects on the activation of Tie2 (tyrosine kinase with immunoglobulin and epidermal growth factor homology domain 2). Endothelial Ang-2 expression is associated with vessel destabilization and regulates a balance between vascular regression and growth. To elucidate, in particular, the role of Ang-2 after arterial artery occlusion in the mouse limb, we applied a transgenic animal model with targeted Ang-2 expression in endothelial cells. We show here that restoration of blood flow in Ang-2:Tie1 transgenic mice is dramatically impaired when Ang-2 expression is induced in the vasculature. The defective restoration of perfusion in Ang-2 transgenic mice is evidenced by reduced collateral artery growth, which typically occurs to compensate for flow deficits after occlusion of the large conductance artery. Furthermore, reduced movement capacities and higher incidents of necrosis are consequently observed in the transgenic limbs as compared with controls. Mechanistically, the observed effects are attributed to defective smooth muscle cell recruitment in Ang-2 transgenic mice. Moreover, distinct Ang-2 levels in the genetically modified animals clearly correlated with the magnitude of reduced perfusion. In conclusion, our studies define Ang-2 as an important molecule for the progression of collateral artery growth and angiogenesis during ischemia and suggest precise Ang-2 dosage activities to accomplish blood vessel growth.
Key Words: angiopoietins • Tie2 • collateral artery growth • hindlimb ischemia • angiopoietin transgenic mice
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Introduction |
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Molecular events underlying the formation of new blood vessels during angiogenesis and arteriogenesis are complex and involve the coordinated interaction of numerous growth factors and their corresponding receptors.1,2 The angiopoietin (Ang)/Tie system has been reported to be critically involved in disease progression through the activation of signaling pathways that control angiogenic remodeling.3–7 Although Ang-1 and Ang-2 share similar binding affinities for the Tie2 receptor (the tyrosine kinase with immunoglobulin and epidermal growth factor [EGF] homology domain 2 receptor), they have opposing effects on receptor activation. Ang-1 induces receptor phosphorylation and contributes to blood vessel stabilization by the recruitment of periendothelial cells.5,6 Ang-2 antagonizes the actions of Ang-1 and, depending on the presence of vascular endothelial cell growth factor (VEGF)-A, is associated with blood vessel growth or regression.7,8 Ang-1 is constitutively expressed in normal adult tissues, whereas Ang-2 is upregulated only at sites of vascular remodeling to allow the vessel to revert to a more plastic state.8 As such, the development of a functional vasculature requires the spatiotemporal expression of growth factors that regulate endothelial cell proliferation, migration, and differentiation.
Collateral artery growth and angiogenesis are both targets for gene therapies. Combined treatment with VEGF-A and Ang-19,10 has shown improved reperfusion after occlusion of main limb conductance arteries. During cerebral ischemia, Ang-2 has been shown to promote the formation of neovessels.11 To investigate the role of Ang-2 during adult neovascularization in more detail, we generated endothelial cell–specific Ang-2 double transgenic mice that allowed the inducible regulation of Angiopoietin expression. We aimed to identify actions of Ang-2 in ischemic tissues and to test the hypothesis that imbalances in the ratio of Ang-1:Ang-2 might interfere with Ang-2–mediated effector functions in endothelial cells in vivo.
In the course of our experiments, we studied the restoration of perfusion in experimentally induced limb ischemia in Ang-2:Tie1 transgenic mice. We provide evidence that endothelium-specific Ang-2 transgenic mice display a reduced capacity to compensate for flow deficits after arterial artery occlusion in the mouse hindlimb. Impaired blood flow recovery in these mice is evidenced by defective collateral artery growth and SMC recruitment. In summary, we define Ang-2 dosage as a critical parameter necessary to balance blood vessel growth and regression.
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Materials and Methods |
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Transgenic Animals
Tie1-tTA driver and Tetos- human Ang-2 responder transgenes were generated as previously described.12 Genotypes of Tie1-tTA lines crossed with Tetos-Ang-2 lines were determined by PCR (expanded Materials and Methods section in the online data supplement, available at http://circres.ahajournals.org).
The present study was performed in accordance with the German Legislation on the Protections of Animals and the Guide for the Care and Use of Laboratory Animals with permission of the Regierungspräsidium Darmstadt. Transgenic and control animals (CD1) were used at 6 to 12 weeks of age. Wild-type (WT) littermates that inherited 1 or no transgenes served as experimental controls.
Hindlimb Surgery
The surgical procedure was performed as previously described.13 Briefly, the right femoral artery was exposed and ligated distally to the origin of the arteria profunda femoris. Ang-2 expression was induced in adult animals at the day of hindlimb surgery. One representative experiment with 8 animals per group is shown (n=5).
Laser Doppler Imaging
Relative blood flow to the foot was measured under standardized conditions by laser Doppler imaging (LDI) as described.13 Measurements were performed pre– and post–artery ligation, additionally on postoperative days 3, 7, 14, and 21. The right-to-left ratio (occluded-to-nonoccluded leg) was calculated for each animal.
Oxygen Saturation of Hemoglobin
Oxygen saturation of hemoglobin in foot pads was determined by the hemoglobin absorption spectrum as previously described.13 Measurements were performed directly after LDI. The right-to-left ratio was calculated for each individual mouse.
Foot Movement Score
To assess the functional recovery of limbs, we used a scoring system based on active foot movement of individual mice: use of the leg, score 1; active foot use, score 2; use of complete foot or spreading of toes, score 3; unrestricted movement, score 4. Additionally, we scored the severity of necroses in WT and transgenic animals to assess mice that had to be euthanized during the course of the experiment.
Tissue Sampling
Tissue sampling and morphometric analysis of collateral artery growth were performed as described.13 Briefly, vasculature was perfused with adenosine-containing buffer to induce vasodilatation followed by 2% paraformaldehyde. Adductor muscles were cryopreserved, sectioned, and processed for immunohistochemistry staining with isolectin B4 and 
smooth muscle actin (online data supplement). Image analyses were performed using a Nikon confocal microscope (LSM C1S1).
In Situ Hybridization
In situ hybridization was performed as previously described14 using mouse VEGF-A164, mouse Ang-2 (recognizing mouse and human), and mouse Tie2 cDNA templates.
Immunoprecipitation and Immunoblotting
Lungs and adductor muscles from transgenic mice were frozen in liquid nitrogen and processed to immunoprecipitation essentially as described15 using Tie2 antibody (4G8, Chemicon; online data supplement).
Enzyme-Linked Immunosorbent Assay
Human Ang-2 ELISA (R&D Systems) was performed according to the instructions of the manufacturer. Blood was obtained by cardiac puncture.
Statistical Analysis
Data were analyzed by Student’s t test. P<0.05 was considered statistically significant. Data are means±SEM.
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Results |
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Targeted Expression of Ang-2 in Endothelial Cells In Vivo
To address functions of Ang-2 during pathological angiogenesis, we specifically expressed Ang-2 in the mouse vasculature using a tetracycline-based transgenic system (Figure 1A).16 The driver line included the Tie1 promoter3 to drive expression of the tetracycline-responsive transactivator (tTA) in endothelial cells.16 This line was crossed to the responder line harboring human Ang-2 driven by the tetracycline-responsive promoter. Ang-2:Tie1 double transgenic (DT) animals were identified by PCR from tail biopsies (Figure 1B) and obtained in a mendelian ratio. Because of lethality of transgenic animals during gestation, doxycycline was administrated to repress Ang-2 and omitted at the day of hindlimb surgery. In our setting, before transgene expression during experimental ischemia, the vasculature of Ang-2 DT mice was normal (not shown). However, as reported previously, continuous Ang-2 expression in the liver resulted in vascular and lymphatic abnormalities.12 Animals were examined for transgene expression by in situ hybridization and ELISA (Figure 1C and 1D). Specifically, Ang-2 message was localized in the vasculature of Ang-2 DT mice (Figure 1C). Ang-2 transgene was expressed in nonischemic tissues when doxycycline was omitted and repressed when animals were fed doxycycline in the drinking water. In situ hybridization analyses revealed strong Ang-2 message in Ang-2 DT animals (Figure 1C; probe specific for human and mouse Ang-2). Intrinsic Ang-2 is readily induced in activated endothelial cells in mice and thus can be detected at low levels in ischemic muscles of WT animals (Figure 1C). Ang-2 was not expressed in tissues and nonischemic muscles of WT mice (data not shown; Figure 1C). High amounts of human Ang-2 protein accumulated in the serum of Ang-2 DT animals, as evidenced by ELISA (Figure 1D). In summary, using a transgenic system, we have generated a tool that allowed induction of imbalances in the ratio of Ang-1 and Ang-2 and specific investigation of the relative contribution of Ang-2 during limb ischemia in vivo.
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Reduced Flow Recovery in Ang-2:Tie1 Mice After Artery Occlusion
We next aimed to address the involvement of the Ang-2/Tie2 pathway in mouse ischemic limbs. After femoral artery occlusion in Ang-2 DT mice, blood flow was quantified at different time points using LDI. In detail, LDI analyses at day 3, 7, 14, and 21 after artery ligation revealed significantly reduced flow characteristics as compared with the nonligated limb in both Ang-2 DT and control animals (Figure 2A and 2B). Typically, continuous recovery of flow was indicated from day 3 (Figure 2A and 2B) after ischemia. However, blood flow recovery was severely attenuated in Ang-2 DT mice compared with littermate control animals over the observation period (Figure 2A and 2B). Next, we determined the hemoglobin oxygen saturation of the ligated versus the unaffected limb (Figure 2C). In Ang-2 DT transgenic mice, oxygen saturation was significantly reduced (compared with WT) starting from day 3. Recovery of oxygen saturation was attenuated over the 21 day observation period and did not reach preoperative numbers (Figure 2C). In contrast, control animals compensated oxygen saturation deficits from day 3 post operation (Figure 2C). Thus, our findings implicate a negative regulatory role for Ang-2 during arteriogenesis when continuously expressed in endothelial cells.
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Reduced Movement Capacity in Transgenic Mice Overexpressing Ang-2
As a functional parameter to assess the progress of collateral artery growth, we identified a score to assess active foot movement of the occluded limb. As shown in Figure 3A, as early as day 3 after collateral artery ligation, the movement score was significantly reduced in Ang-2 DT mice as compared with WT control animals. In addition, Ang-2 DT exhibited massive signs of necrosis, which were less frequent in the control group (Figure 3B). These results additionally support the observed negative impact of Ang-2 on blood flow recovery after femoral artery occlusion in the Ang-2 DT mice.
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Tie2 Receptor Activation in Ang-2 Transgenic Mice
In the quiescent vasculature, Tie2 is constitutively activated through binding of Ang-1.17 Ang-2 has been well recognized to antagonize the actions of Ang-1.7 To provide evidence that Ang-2 negatively interfered with tyrosine kinase receptor activation in our transgenic system, we investigated Tie2 phosphorylation. As shown in Figure 4, Tie2 message and protein are present in ischemic limbs (Figure 4A) and lungs (Figure 4B) of Ang-2 DT and WT animals as well as in lungs of Ang-1:Tie1 transgenic mice, which served as additional (positive) controls. During ischemia, Tie2 becomes upregulated in the adductor muscle (Figure 4A; compare to nonischemic control). Tie2 receptor density is very low in adductor muscles, and activation is not easily determined in the ischemic tissue itself. Therefore, we specifically chose lung tissue with high receptor densities17 to determine Tie2 phosphorylation (Figure 4C). Tie2 was phosphorylated in WT mice but not in Ang-2 DT mice, as evidenced by immunoprecipitation and Western blot analyses (Figure 4C). Furthermore, in Ang-1:Tie1 transgenic mice (which were designed using the same strategies as described for Ang-2:Tie1 mice), Tie2 phosphorylation was increased, as compared with WT controls (Figure 4C).
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We next asked whether different Ang-2 concentrations would result in differences with regard to Tie2 activation. As demonstrated in Figure IIIA of the online data supplement, Ang-2 DT animals with the highest Ang-2 serum levels showed lower phosphorylation signals in lungs as compared with Ang-2 medium–expressing or WT animals. Thus, we provide evidence that continuous Ang-1 signaling is important to maintain Tie2 activation and that our transgenic mouse model is valid to investigate the antagonizing functions of Ang-2 in adult mice in vivo.
The repertoire of proangiogenic growth factors during ischemia involves the Ang/Tie system as well as other growth factors, such as VEGF-A and fibroblast growth factor-2. As previously reported,2,18 VEGF-A expression was upregulated during experimental ischemia. In our transgenic system, VEGF-A was similarly induced in ischemic muscles of Ang-2 DT and WT animals as compared with nonischemic controls (Figure 4C) and thus might be able to synergize with Ang-2 as evidenced from the literature2,19
Ang-2 Negatively Interfered With Collateral Artery Growth
A trigger for arteriogenesis is altered shear stress, which enables a complex cascade of molecular and cellular events, leading to increased vessel diameter and thickness. To identify mechanisms by which Ang-2 interfered with reperfusion in ischemic limbs in more detail, we determined collateral artery sizes. Representative images taken from occluded limbs in Ang-2 DT as compared with WT control animals are shown in Figure 5A. Morphometric evaluations of Ang-2 DT collateral arteries revealed a significant reduction in collateral artery size when compared with control animals (Figure 5B). Thus, Ang-2 seems to negatively influence collateral vessel growth and diameter, which is typically increased after limb occlusion.
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Defective SMC Coverage in Ang-2 DT Animals
The results presented above indicate that Ang-2 seems to influence endothelial/smooth muscle cell (SMC) interaction in collateral arteries. To explain how Ang-2 might act to negatively interfere with collateral growth and perfusion, we hypothesized that Ang-2 may inhibit SMC migration in the hindlimb model. Therefore, we investigated SMC coverage of collateral arteries in occluded muscles of Ang-2 transgenic and WT mice. As evidenced in Figure 6A, we observed significant differences in the collateral artery wall thickness in Ang-2 DT mice. Additionally, the number of SMCs per vessel area was significantly reduced in Ang-2 DT (Figure 6B). It is well established that increased levels of Ang-2 can lead to vessel destabilization and loss of perivascular cells during pathological angiogenesis.20 As indicated in Figure 6, our analyses revealed that Ang-2 interfered with SMC investment in ischemic limbs. Collectively we showed that continuous expression of Ang-2 results in reduced SMC coverage of large conductance arteries in the mouse hindlimb and consequently leads to reduced perfusion after ischemia.
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Ang-2 Dosage Critically Influenced the Magnitude of Reperfusion in Ang-2 Transgenic Mice
The balance of Ang-1 and Ang-2 during pathological angiogenesis must be precisely regulated to obtain functional new blood vessels. To explain the negative impact of Ang-2 on flow properties in Ang-2 transgenic mice, we reasoned that Ang-2 level might play an important role. Therefore, we investigated Ang-2 serum concentrations in DT animals (Figure 7A). We observed different Ang-2 level in transgenic animals as evidenced by ELISA (Figure 7A). We next wanted to determine whether different Ang-2 levels would result in diverse capacities to compensate for flow deficits among WT, Ang-2 medium–, and Ang-2 high–expressing animals. Mice displaying the highest amounts of Ang-2 showed the strongest defect in restoration of perfusion (Figure 7B). Furthermore, we observed concentration-dependent differences in reperfusion, with Ang-2 medium–expressing animals showing intermediate and Ang-2 WT animals displaying the best recovery effects (Figure 7B and supplemental Figure I). Additionally, among the different groups investigated, Ang-2 high–expressing animals showed high incidents of necrosis and signs of inflammation (Figure 3B and supplemental Figure II), indicative for deleterious effects of high Ang-2 levels.
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Discussion |
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Ang/Tie2 signaling has been intensively studied during embryonic and postnatal vessel remodeling. Ang-1 is widely expressed in normal adult tissues, and constitutive Ang-1/Tie2 signaling is critical for endothelial cell survival and vessels quiescence. Ang-2 has been identified as an antagonizing molecule opposing the functions of Ang-1, thereby mediating vessel destabilization by disengagement of endothelial and perivascular cells. The tightly regulated interplay of both Tie2 ligands seems to be critical, as evidenced in a number of pathological settings of postnatal blood vessel growth in vivo. In this study, we provide evidence that Ang-2 is a critical regulator of postnatal vessel remodeling in the mouse hindlimb.
The importance of Ang-2 for the regulation of vessel remodeling has been shown in models of pathological angiogenesis.2 Depending on the presence of Ang-1 and Ang-2, the effects on the newly formed vasculature and their state of maturity are very heterogeneous.20 Therefore, we reasoned that dosage of Angiopoietins within the tissue might be very critical. To obtain more detailed knowledge about the actions of Ang-2 during adult blood vessel growth, we generated Ang-2 transgenic mice. Using the Tie1 promoter, a transgenic system was designed to induce Ang-2 expression in the vasculature. With the aid of this system, we were able to identify important functions of Ang-2, such as interference with collateral artery and SMC growth, in mouse ischemic limbs.
The relative contribution of angiopoietins during ischemia has not been studied intensively and remains elusive to date. Ang-1 gene transfer has been shown to augment neovascularization during myocardial infarction in the rat.21 Notably, in the presence of VEGF-A, effects of Ang-1 on vascularization and perfusion are even more profound.9,10,22 Results from these reports are in line with the well-established vessel stabilizing functions of Ang-1.2,20 Redirection of blood flow after occlusion of the major conductance collateral artery necessitates blood vessel stabilization to compensate increased shear forces on the vascular wall.1 Consequently, collateral artery ligation leads to tissue hypoxia (at distant sites) and upregulation of VEGF-A. In turn, both factors are known to upregulate Ang-2 in vitro.23,24 In addition, we and others have shown that Ang-2 is upregulated during cerebral11 and myocardial ischemia25,26 within the first 24 hours after occlusion and remained elevated for weeks after initial infarction.25,26 Therefore, we reasoned that Ang-2 might act in collaboration with other growth factors to promote arteriogenesis. The findings reported here disclose a mechanism by which continuous (and elevated) expression of Ang-2 in the vasculature negatively interfered with collateral artery growth after artery occlusion in the mouse limb. This might be attributable, in part, to the competition of Ang-2 with Ang-1 to interfere with receptor signaling, as Tie2 is no longer phosphorylated in the presence of Ang-2 in DT mice. Elevated Ang-2 expression is necessary to revert the vasculature to a more plastic state and provides destabilization signals to promote sprouting and remodeling.19 However, increased Ang-2 dosage can lead to vessel regression as evidenced by disrupted vasculature in mice overexpressing Ang-2 in endothelial cells.7 Consequently, continuous switch of Ang-2 in our transgenic mice resulted in reduced receptor phosphorylation, indicative for the antagonizing role of Ang-2. Moreover, we observed decreased Tie2 phosphorylation in the presence of different Ang-2 concentrations. Precise dosage of Ang-1 and Ang-2 therefore appears mandatory for vessel growth, as previously demonstrated in experimental tumors and as shown here after limb ischemia.
One study27 reported differential sensitivity of newly formed versus existing retinal vessels to regressive effects of Ang-2 and hypothesized that mature vessels acquire different types of survival signals, with only some of them might be interrupted by Ang-2. However, in the hindlimb model we used, de novo synthesis of collaterals is not indicated. More likely, preexisting arterioles are recruited, remodeled, and invested by SMCs to adapt to altered flow characteristics.1 In our experiments, we observed increased incidents of necrosis in the Ang-2 transgenic mice, indicative of destructive functions if Ang-2 dosage is elevated. Ang-1 expression in tissues is constitutive and necessary to maintain the vasculature at the quiescent state.17 During pathological angiogenesis (and arteriogenesis), Ang-2 is highly upregulated and interferes with Ang-1-mediated Tie2 phosphorylation and survival. Interestingly, Ang-2 has been shown to be stored in Weibel–Palade bodies in endothelial cells and is thus instantly available to interfere with Tie2 signaling.28 As such, a shift in the Ang-1:Ang-2 ratio in favor of Ang-2 critically interfered with destabilization of the vasculature during arteriogenesis as demonstrated in this study.
Vascular endothelial cells respond to activation signals by angiogenesis and/or inflammation, indicating common signaling pathways. In a recent study, we have linked the Ang/Tie2 system to the regulation of inflammatory processes.29 Using a number of inflammatory models, we showed that Ang-2–deficient mice30 are no longer able to respond to inflammatory stimuli. In contrast, Ang-1 acts the opposite preventing leakiness and inflammation.31 In the ischemic hindlimb model, inflammatory cells are important regulators.13 We hypothesized that Ang-2 might be responsible for the recruitment of hematopoietic cells into ischemic limbs. However, the number of CD45 and CD11b+ cells in adductors of Ang-2 transgenic animals was not increased.
Another possible mechanistic explanation for the reduced reperfusion in Ang-2 transgenic animals might involve the recruitment/proliferation of SMCs, ie, important regulators of collateral artery growth.1 Maturation of vessels occurs as newly formed tubes recruit and become coated by mural cells such as SMCs and pericytes. This association results in vessels stabilization and quiescence. During ischemia, SMC growth is characteristic to adapt altered flow characteristics of collateral arteries.1 Mechanistically, the involvement of Ang-1 in the recruitment/proliferation of SMCs to endothelial cells is still not solved. Because Tie2 is largely specific to endothelial cells, it has been speculated that Ang-1 activates Tie2 on endothelial cells, which then produce factors such as platelet-derived growth factor to recruit mesenchymal cells to the newly formed vessel.32 However, in some reports Tie2 has been shown to be present on SMCs or their precursors.33–35 When SMCs are subjected to migration in vitro, Ang-1 has been shown to promote recruitment across transwell filter membranes, possibly directly via Tie2 which is upregulated by VEGF-A.33,34 In our hands, induction of Ang-2 resulted in reduced SMC coverage of collateral arteries after ischemia. Defective or reduced coverage with perivascular has been described in models of pathological angiogenesis such as in tumors.20 As demonstrated here, this concept also applies to other models of adult vessel growth, such as arteriogenesis.
Despite the progress in basic research and preclinical animal studies of therapeutic angiogenesis, problems in translating experimental findings into beneficial clinical approaches still exist.18 For example, microenvironmental doses of VEGF have recently shown to be critical for beneficial outcome after ischemia.36 Activities of angiopoietins appear to be context dependent, and doses have to be carefully balanced to accomplish pro- and antiangiogenic activities. Ang-1 has been shown to promote functional neovascularization and improvement of reperfusion after myocardial ischemia in the swine37 and, together with VEGF, decrease the myocardial infarct size in rats.38 Combinational therapies with VEGF and Ang-1 therefore seem to be protective and enhance arteriogenesis in a number of animal and preclinical models.9,10,21,22 However, VEGF also interfered with leakiness and induced inflammation, with negative outcome on reperfusion after ischemia.39 Similarly, Ang-2 has been shown to contribute to increased permeability and inflammation.29 As such, and as indicated by the results of our study, the balance and dosage of growth factors must be carefully evaluated for potential clinical use.
In conclusion, the findings reported here highlight the importance of precise Ang dosage during pathological blood vessel growth after ischemia. Ongoing experiments using vascular-specific, inducible Ang-2 transgenic mice are being conducted to understand Ang/Tie signaling in other models of pathological angiogenesis, such as subcutaneous or intracranial tumors, to mechanistically understand the actions of Ang-1 and Ang-2 on their tyrosine kinase receptor. Findings from studies of the Ang/Tie2 system, in conjunction with other growth factor systems, may provide useful clinical implications for vascular diseases.
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Acknowledgments |
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We thank Dr S. Martin for help with the hindlimb surgeries. A. Scholz is gratefully acknowledged for continuous experimental support.
Sources of Funding
This work was supported by German Research Foundation (Deutsche Forschungsgemeinschaft) grants SFB/TR23 C1 (to K.H.P. and Y.R.) and A4 (M.H.H.S.).
Disclosures
None.
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Footnotes |
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Original received October 26, 2006; revision received May 1, 2007; accepted May 23, 2007.
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