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.