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Mural inflammation and neovascularization are characteristic pathologic features of abdominal aortic aneurysm (AAA) disease. Vascular endothelial growth factor receptor (VEGFR) expression may also mediate AAA growth and rupture. We examined VEGFR expression as a function of AAA disease progression in the Apolipoprotein E- deficient (Apo E−/−) murine AAA model.
Apo E−/− mice maintained on a high fat diet underwent continuous infusion with angiotensin II at 1000 ng/kg/min (Ang II) or vehicle (Control) via subcutaneous osmotic pump. Serial transabdominal ultrasound measurements of abdominal aortic diameter were recorded (n = 16 mice, 3 – 4 time points/ mouse) for up to 28 days. Near-infrared receptor fluorescent (NIRF) imaging was performed on Ang II mice (n = 9) and Controls (n=5) with scVEGF/Cy, a single-chain VEGF homo-dimer labeled with Cy5.5 fluorescent tracer (7–18 µg/mouse IV). NIRF with inactivated single chain VEGF/Cy tracer (scVEGF/In, 18 µg/mouse IV) was performed on two additional Ang II mice to control for non-receptor mediated tracer binding and uptake. Following image acquisition and sacrifice, aortae were harvested for analysis. An additional AAA mouse cohort received either an oral angiogenesis inhibitor or suitable negative or positive controls to clarify the significance of angiogenesis in experimental aneurysm progression.
Aneurysms developed in the suprarenal aortic segment of all Ang II mice. Significantly greater fluorescent signal was obtained from aneurysmal aorta as compared to remote, uninvolved aortic segments in Ang II scVEGF/Cy mice or AAA in scVEGF/In mice or suprarenal aortic segments in Control mice. Signal intensity increased in a diameter-dependent fashion in aneurysmal segments. Immunostaining confirmed mural VEGFR-2 expression in medial smooth muscle cells. Treatment with an angiogenesis inhibitor attenuated AAA formation while decreasing mural macrophage infiltration and CD-31+ cell density.
Mural VEGFR expression, as determined by scVEGF/Cy fluorescent imaging and VEGFR-2 immunostaining, increases in experimental AAAs in a diameter-dependent fashion. Angiogenesis inhibition limits AAA progression. Clinical VEGFR expression imaging strategies, if feasible, may improve real-time monitoring of AAA disease progression and response to suppressive strategies.
Abdominal aortic aneurysm (AAA) is a common and highly lethal, age-associated vascular degenerative disease1. Characteristic pathologic features present in both clinical and experimental aneurysms include marked elastin degradation and reduced medial vascular smooth muscle cell (SMC) cellularity in the presence of transmural inflammation and adventitial neovascularity. The exact sequence of and causal relationship between AAA-related pathologic events, as well as the time-dependent expression of mural pro-inflammatory mediators during clinical disease progression remain poorly understood 2.
Although much emphasis has been placed on the consequences of medial SMC loss and matrix degeneration during aneurysm pathogenesis, the contribution of adventitial neovascularization to human disease progression appears increasingly significant. At the time of surgical repair, aneurysm tissue demonstrates increased mural neovascularization compared to non-aneurysmal aorta3, and rupture foci demonstrate increased neovascularity compared to intact aneurysm segments4. In experimental aneurysms, pro-angiogenic mediator expression correlates with transmural inflammation and AAA progression5–8. In an effort to evaluate the utility of mural neovascularity monitoring as a surrogate marker for disease progression we tested the ability of in situ and ex vivo near infrared fluorescent imaging (NIRF) to identify vascular endothelial growth factor receptor (VEGFR) expression in experimental AAAs.
All protocols were reviewed and approved by the Administrative Panel on Laboratory Animal Care at Stanford University (http://labanimals.stanford.edu/). C57/Bl 6 Apo E −/− mice, 11–14 weeks of age, weighing 25–30 grams ere anesthetized with inhaled isoflurane for surgical and imaging procedures and recovered with free access to food and water. Chronic subcutaneous angiotensin II (Ang II) infusion was used to create suprarenal murine AAAs9. After sterile preparation, osmotic mini-pumps (Alzet Model 2004, Durect Corporation, Cupertino) were inserted underneath the dorsal skin, delivering 1000 ng/kg/min Ang II (Sigma Lot 056K51001) in normal saline. Control mice received mini-pumps loaded with vehicle alone. Following their procedures all mice were maintained on high fat diets (40% kcal lipid, D12109, Resource Diets, Inc. New Brunswick) for up to 28 days. Using these methods, postoperative mortality was less than 10%.
Transabdominal 40 MHz B-mode ultrasound (US) imaging was used to measure aortic diameter in vivo (supplemental methods section). Imaging was performed at intervals for up to 28 days following mini-pump implantation. Baseline suprarenal aortic diameter ranged from 0.8 – 0.85 mm; diameters > 1.15 mm following Ang II infusion (> 25% increase) were considered aneurysmal. Aortic diameters were measured in the longitudinal scan plane. Validation of US-determined aortic measurements using similar imaging protocols has been previously described10, 11.
At 21 to 28 days following mini-pump implantation, Ang II and Control mice underwent fluorescent imaging with scVEGF/Cy (Sibtech, Inc.), an engineered single chain (sc) VEGF homo-dimer, labeled with Cy5.5-maleimide (GE Healthcare, USA) as previously described12. Additional Ang II mice received inactivated scVEGF/Cy (scVEGF/In) to control for non-receptor mediated intracellular accumulation (supplemental methods section). Active or inactive scVEGF/Cy, 7–18 µg per mouse, was administered via penile vein injection approximately two hours prior to imaging sessions. Following anesthetic induction, in vivo fluorescent images were obtained and analyzed via the Maestro NIRF Imaging System (Cambridge Research Instruments Inc, Woburn, MA). Four mice (2 Ang II scVEGF/Cy, 1 Control scVEGF/Cy and 1 Ang II scVEGF/In) underwent additional fluorescent imaging with the eXplore Optix NIRF system (GE Healthcare). Postmortem in situ images were obtained following staged abdominal evisceration and subsequent bilateral nephrectomy. After the background signal was removed, scVEGF/Cy signal (signal) from regions of interest (ROI) within aneurysmal as well as grossly uninvolved distal aortic segments were calculated using respective system software. Fluorescent signal intensity reflects the counts per exposure time, normalized to area of ROI. The aortae were explanted and fixed immediately after in situ imaging. Following aortic explantation, fixation and sectioning, ex-vivo Cy5.5 fluorescence from representative aneurysmal and uninvolved aortic sections was confirmed using a 675/694 nm filtered Meiji MX6300 Fluorescence Microscope.
Following pump implantation, select mice were administered an angiogenesis inhibitor (Exel 0862, Exelixis Inc., S. San Francisco, CA, 10 mg/kg daily, n = 6), doxycycline (100 mg/kg, n = 4) or vehicle alone (n = 8), all by oral gavage, for 14 days. Mice were then euthanized for histologic analysis as described above. Doxycycline is known to limit experimental AAA progression and was used as a positive control2. The IC50’s (50% inhibitory concentration) of Exel 0862 were; FGFR1 = 3.7 nM; FGFR2 = 3.5 nM; Flt-1 = 13.6 nM; KDR = 2.4 nM; PDGFRa = 2.1 nM; and PDGFRb = 11.1 nM. Exel 0862 has a bioavailability in mice of 52% and a half life of 2.5 hours.
Representative aneurysmal and non-aneurysmal aortic segments were selected for pressure perfusion fixation at 100 mmHg with 4% PFA and embedded in paraffin. Aortic tissues were processed and embedded in paraffin. Blocks were sectioned at 4 µm for H&E and elastic Masson (EM) and immunohistochemical (IHC) staining. Sections were incubated for one hour at room temperature in primary antibody solutions including: a) rabbit anti-mouse smooth muscle α-actin (SM-α) polyclonal antibody to stain for smooth muscle cells (1 µg/mL, Lab Vision., Fremont, CA); b) rabbit anti-mouse CD31 polyclonal antibody to stain for endothelial cells (1 µg/mL, Lab Vision); c) rat anti-mouse MAC-2 monoclonal antibody to stain for macrophages (0.5 µg/mL, Cedarlane, Burlington, NC); and d) rabbit anti-mouse VEGFR-1 and VEGFR-2 polyclonal antibodies (1 µg/mL, Lab Vision.) to stain for VEGF receptors. After staining with the primary antibodies, samples were incubated with the appropriate biotinylated secondary antibodies (Dako, Inc. & Biocare Medical, Concord, CA) for 30 minutes at room temperature. Color development was performed using the DAB color development system (Dako Inc.). For CD31/VEGFR-2, CD31/VEGFR-1, and SM-α/VEGFR- double staining, the DAB color development system (Dako, Inc.) was used for BrdU (brown color) and the Fast-red color development system (Biocare Medical) was used for GFAP (red color).
Statistical analyses were performed with StatView (SAS, Cary, NC) software. Data were expressed as mean +/− standard error unless otherwise noted. Individual signal data were compared via 1-way ANOVA using the Fisher PLSD correction for multiple comparisons. The Pearson coefficient was used to calculate the correlation between aortic diameter and signal intensity. Differences were considered significant at p<0.05.
Sixteen mice underwent NIRF fluorescence imaging with either scVEGF/Cy (9 Ang II, 5 Control) or scVEGF/In (2 Ang II). Due to intrinsic visceral and renal autofluorescence, AAA-specific signal was not discernable during transabdominal in vivo imaging. Therefore, subsequent signal acquisition and dosimetry calculations were obtained following evisceration and total nephrectomy (Figure 1). Using the Maestro system, signal intensity in aneurysmal aortic segments was significantly higher (0.342 ± 0.045, mean ± SE) than that obtained from remote, non-aneurysmal segments in Ang II mice (0.160 ± 0.048) and aortae from Control (0.091 ± 0.043) and Ang II scVEGF/In mice (0.08 ± 0.02, all p < 0.05, Figure 2). Similar results were obtained using the eXplore Optix system on both whole vessel (Supplemental Figure I) as well as tomographic aortic reconstructions (Supplemental Figure II) from suprarenal aneurysms as well as non-involved segments of the more distal aorta. Suprarenal aortic signal intensity correlated closely with US-determined aortic diameter obtained immediately prior to laparotomy (Figure 3).
Evidence for Cy5.5 co-localization (representing scVEGF/Cy uptake) within areas of VEGFR-2 expression identified by IHC was confirmed via fluorescence microscopy (Figures 4A, B). More Cy5.5 fluorescence and VEGFR-2 expression were present in aneurysmal as compared to non-aneurysmal aortic segments within the same animal (Figures 4C, D). Unexpectedly, maximal Cy5.5 fluorescence was evident in the best preserved, least involved regions of the aortic circumference within aneurysmal sections. No co-localization with VEGFR-2 expression was noted in scVEGF/In mice (Figures 4E, F).
Ang II aneurysmal aortic segments demonstrated characteristic regions of medial lamellar disruption (Figures 5A) and transmural mononuclear inflammation (Figure 5B) in isolated quadrants as previously described13. VEGFR-2 expression was evident circumferentially, and was most apparent in the areas of intact media within aneurysmal sections (Figure 5C). Co-staining with smooth muscle alpha-actin confirmed maximal VEGFR-2 expression within medial smooth muscle cells (Figure 5D). Non-aneurysmal aortae from control and experimental mice demonstrated intact elastic lamellae, minimal inflammation and reduced VEGFR-2 expression (Figure 5E, F). Increased transmural VEGFR-1 expression was also present in aneurysmal aortic segments but was limited to the intima in non-aneurysmal aortae (Supplemental Figure III A, B).
Transmural neovessel formation was evident within aneurysmal segments as demonstrated by CD-31 immunostaining (Figure 6A). By comparison, few neovessels were present in non-aneurysmal aortic segments from either Ang II or Control mice (Figure 6B). Co–staining studies of VEGFR-2 and CD-31 confirmed increased VEGFR-2 expression in areas of neovessel formation (Figure 6C). Within aneurysmal segments there was an abundance of adventitial neovessel formation in intact quadrants (Figure 6D) compared to limited constitutive cells and neovascularity present within disrupted regions (Figure 6E). CD-31 and VEGFR-1 co-staining (Supplemental Figure III C) also demonstrated the presence of VEGFR-1 expression in areas of adventitial neovascularization (Supplemental Figure III D).
Treatment with either Exel 0862 or doxycycline significantly decreased AAA diameter at 14 days compared to vehicle alone (Exel 0862 0.8±0.2 mm; Doxycycline 0.94±0.2 mm, Vehicle 1.3±0.2, p < 0.05 Vehicle vs. Exel 0862, Doxycycline). Aortae from both Exel 0862 and Doxycycline mice demonstrated decreased mural inflammation (Exel 0862 2.4±0.5 MAC-2 cells/high power field; Doxycycline 5.6 ±1.6; and Vehicle 36.8±8.5;, p < 0.05 Vehicle vs. Exel 0862, Doxycycline). CD-31+ cells (as a surrogate for neovascularity) were less prevalent in the Exel 0862 treated group vs. all others. (Exel 0862 0.7±0.2 CD-31+ cells/high power field; Doxycycline 10.4 ± 3.5; and Vehicle 11.0±3.5 ;, p < 0.05 AI vs. Vehicle, Doxycycline.)
In situ NIRF imaging with scVEGF/Cy consistently localized VEGFR expression to regions of concomitant aneurysmal degeneration in experimental AAAs. NIRF signal intensity correlated well with both aneurysm location and diameter. The fidelity of this co-localization was confirmed with ex-vivo fluorescence microscopy. Both VEGFR-1 and 2 expression were increased within aneurysmal aortic segments. VEGFR-2 was most intensely expressed in medial smooth muscle cells in intact aortic medial segments adjacent to and across from sites of maximal inflammation and medial lamellar disruption. Similarly, CD31, as a surrogate for neovessel formation was most abundantly expressed in intact adventitial segments with increased VEGFR-1 and 2 expression in proximity to areas of aneurysmal disruption. The mechanistic significance of neovessel formation in AAA progression was confirmed by profound reductions in mural inflammation and neovascularity following treatment with a broad-spectrum oral angiogenesis agent.
Mural neovascularization is recognized as a salient pathologic feature of human AAA disease and may catalyze clinical progression.3. Conversely, neovascularization may also play a role in protective and/or compensatory mural remodeling at other stages of human disease3, 4, 7, 8, 14, 15. In our experimental construct, functional activity of VEGFR-1 as measured by scVEGF/Cy tracer uptake correlated well with aortic diameter. In addition, treatment with an angiogenesis inhibitor with competitive activity against VEGFR-1 and 2 as well as other vascular growth factor receptors significantly reduced mural inflammation and disease progression. Our data suggests that VEGFR expression and activity is mechanistically relevant to experimental AAA disease, and that VEGFR imaging may provide a useful window on disease progression16–23. If translatable to the human condition, these techniques or suitable alternatives may provide more accurate and timely guidance for suppressive medical therapies than absolute AAA diameter or growth rate alone.
Increased aortic VEGFR expression within aneurysmal segments was confirmed using two complementary fluorescence imaging systems as well as fluorescence microscopy and IHC. While NIRF signal intensity correlated well with increasing aortic diameter, alternative explanations include both increased aortic tissue mass/wall thickness as well as increased neovessel density per unit tissue. To control for intensity per unit area, tomographic in situ NIRF reconstructions were obtained in both axial and coronal planes. This technique confirmed increased signal intensity within aneurysmal planar sections, supporting our initial interpretation (e.g. signal production reflected VEGF receptor expression per unit area).
Increased VEGFR-1 and 2 expression was present within aneurysmal aortic segments. VEGFR-1 was expressed in both the intima and adventitia; VEGFR-2 in the media and adventitia. VEGFR-2 co-localized to medial alpha-actin positive cells. Although alpha-actin may not be expressed exclusively in differentiated vascular smooth muscle cells24, the marked regional localization of VEGFR-2 within the media strongly suggested SMC expression. Until recently VEGF and VEGFR were believed to be expressed exclusively by endothelial cells (ECs). Circulating endothelial progenitor cell populations are increased in AAA patients25 and VEGF is known to stimulate endothelial progenitor cell mobilization26. However, recent in vitro 27, 28, animal modeling29 and human tissue 30 studies demonstrate SMC VEGFR expression and migration in response to VEGF stimulation. Human and mouse embryonic stem cells differentiate into vascular smooth muscle cells that express VEGFR-231. While vascular VEGFR-2 expression is minimal under quiescent conditions32, increased expression has been demonstrated in human and animal models of other disease states33, 34. The presence of VEGFR-2 within human aortic smooth muscle cells in vitro has also been demonstrated 35. The finding in the present study of maximal VEGFR-2 expression in aortic medial cells remote from areas of advanced inflammation and lamellar disruption was also unexpected, providing stimulus for further research into mechanisms for aneurysm initiation and localization in this model.
The relative paucity of both VEGFR-2 and CD-31 + cells in disrupted mural quadrants may be a consequence of aortic dissection and contained rupture, both characteristic features of this AAA model. Imaging at earlier intervals may have revealed alternative expression patterns prior to medial disruption. Indeed, the cellular status of relatively intact quadrants adjacent to areas of medial disruption within aneurysmal segments are probably most representative of the relative neovascularity present in these areas just prior to medial disruption, and as such are more relevant to mechanisms involved in early disease progression.
Other limitations related to these modeling methods are well known and detailed elsewhere36. Confirming the timing, localization and quantification of VEGFR expression in complementary AAA models may provide additional insights regarding the validity of these observations to human disease. The additional surgical exposure required for either the porcine pancreatic elastase infusion or abluminal calcium chloride application models will likely increase the total area of VEGFR expression present throughout the wound, however, reducing the specificity and relevance of receptor localization and quantification for disease monitoring. The limited sensitivity of currently available transabdominal fluorescence imaging systems (due in part to intrinsic autofluorescence) made aortic exposure and evisceration necessary for adequate visualization. Proof of concept has clearly been established by this study, however, and further technical refinements may substantially improve efficacy and provide practical applications to human disease monitoring. Although tomographic imaging demonstrated increased tissue signal production per unit area, in practical terms the specificity of signal intensity as a function of tissue mass vs. increased receptor expression is a secondary consideration due to the fact that enlarging aneurysms increase aortic tissue mass as a function of disease progression.
In conclusion, we demonstrate the feasibility of VEGFR expression imaging in experimental AAAs as a surrogate for disease progression. These findings lend further support to prior observations regarding the potential significance of VEGF, VEGF receptor activity and medial and adventitial neovascularization in AAA pathogenesis. Similar or complementary molecular imaging strategies, if proven relevant to human disease, may expand diagnostic and therapeutic alternatives for AAA disease.
a) This work was supported by the National Institutes of Health 2 R01 HL064338-07) (2 RO1 EB000898-04), (2R44CA113080), (2R44CA113080), an American Heart Association Western States Affiliate Postdoctoral Fellowship Award (0625171Y), Exelixis Inc. and GE Healthcare. b) Both Mary Gerritsen PhD and Geoffrey Schultz MD made substantial contributions to the design and analysis of these experiments. c) Conflict of Interest: Marina Backer is an employee at Sibtech, Inc, and Joseph Backer is the CEO of Sibtech, Inc. Mary Gerritsen is an executive officer at Exelixis Inc.