Multimodality Imaging of Aortic Valve Calcification and Function in a Murine Model of Calcific Aortic Valve Disease and Bicuspid Aortic Valve.

Calcific aortic valve disease (CAVD) is a prevailing disease with increasing occurrence and no known medical therapy. Dcbld2-/- mice have a high prevalence of bicuspid aortic valve (BAV), spontaneous aortic valve calcification, and aortic stenosis (AS). 18F-NaF PET/CT can detect the aortic valve calcification process in humans. However, its feasibility in preclinical models of CAVD remains to be determined. Here, we sought to validate 18F-NaF PET/CT for tracking murine aortic valve calcification and leveraged it to examine the development of calcification with aging and its interdependence with BAV and AS in Dcbld2-/- mice. Methods: Dcbld2-/- mice at 3-4 mo, 10-16 mo, and 18-24 mo underwent echocardiography, 18F-NaF PET/CT (n = 34, or autoradiography (n = 45)), and tissue analysis. A subset of mice underwent both PET/CT and autoradiography (n = 12). The aortic valve signal was quantified as SUVmax on PET/CT and as percentage injected dose per square centimeter on autoradiography. The valve tissue sections were analyzed by microscopy to identify tricuspid and bicuspid aortic valves. Results: The aortic valve 18F-NaF signal on PET/CT was significantly higher at 18-24 mo (P < 0.0001) and 10-16 mo (P < 0.05) than at 3-4 mo. Additionally, at 18-24 mo BAV had a higher 18F-NaF signal than tricuspid aortic valves (P < 0.05). These findings were confirmed by autoradiography, with BAV having significantly higher 18F-NaF uptake in each age group. A significant correlation between PET and autoradiography data (Pearson r = 0.79, P < 0.01) established the accuracy of PET quantification. The rate of calcification with aging was significantly faster for BAV (P < 0.05). Transaortic valve flow velocity was significantly higher in animals with BAV at all ages. Finally, there was a significant correlation between transaortic valve flow velocity and aortic valve calcification by both PET/CT (r = 0.55, P < 0.001) and autoradiography (r = 0.45, P < 0.01). Conclusion: 18F-NaF PET/CT links valvular calcification to BAV and aging in Dcbld2-/- mice and suggests that AS may promote calcification. In addition to addressing the pathobiology of valvular calcification, 18F-NaF PET/CT may be a valuable tool for evaluation of emerging therapeutic interventions in CAVD.

Positron Emission Tomography Imaging of Vessel Wall Matrix Metalloproteinase Activity in Abdominal Aortic Aneurysm.

BACKGROUND: Matrix metalloproteinases (MMPs) play a key role in the pathogenesis of abdominal aortic aneurysm (AAA). Imaging aortic MMP activity, especially using positron emission tomography to access high sensitivity, quantitative data, could potentially improve AAA risk stratification. Here, we describe the design, synthesis, characterization, and evaluation in murine AAA and human aortic tissue of a first-in-class MMP-targeted positron emission tomography radioligand, 64Cu-RYM2. METHODS: The broad spectrum MMP inhibitor, RYM2 was synthetized, and its potency as an MMP inhibitor was evaluated by a competitive inhibition assay. Toxicology studies were performed. Tracer biodistribution was evaluated in a murine model of AAA induced by angiotensin II infusion in Apolipoprotein E-deficient mice. 64Cu-RYM2 binding to normal and aneurysmal human aortic tissues was assessed by autoradiography. RESULTS: RYM2 functioned as an MMP inhibitor with nanomolar affinities. Toxicology studies showed no adverse reaction in mice. Upon radiolabeling with Cu-64, the resulting tracer was stable in murine and human blood in vitro. Biodistribution and metabolite analysis in mice showed rapid renal clearance and acceptable in vivo stability. In vivo positron emission tomography/computed tomography in a murine model of AAA showed a specific aortic signal, which correlated with ex vivo measured MMP activity and Cd68 gene expression. 64Cu-RYM2 specifically bound to normal and aneurysmal human aortic tissues in correlation with MMP activity. CONCLUSIONS: 64Cu-RYM2 is a first-in-class MMP-targeted positron emission tomography tracer with favorable stability, biodistribution, performance in preclinical AAA, and importantly, specific binding to human tissues. These data set the stage for 64Cu-RYM2-based translational imaging studies of vessel wall MMP activity, and indirectly, inflammation, in AAA.

Homeostatic, Non-Canonical Role of Macrophage Elastase in Vascular Integrity.

BACKGROUND: Matrix metalloproteinase (MMP)-12 is highly expressed in abdominal aortic aneurysms and its elastolytic function has been implicated in the pathogenesis. This concept is challenged, however, by conflicting data. Here, we sought to revisit the role of MMP-12 in abdominal aortic aneurysm. METHODS: Apoe-/- and Mmp12-/-/Apoe-/- mice were infused with Ang II (angiotensin). Expression of neutrophil extracellular traps (NETs) markers and complement component 3 (C3) levels were evaluated by immunostaining in aortas of surviving animals. Plasma complement components were analyzed by immunoassay. The effects of a complement inhibitor, IgG-FH1-5 (factor H-immunoglobulin G), and macrophage-specific MMP-12 deficiency on adverse aortic remodeling and death from rupture in Ang II-infused mice were determined. RESULTS: Unexpectedly, death from aortic rupture was significantly higher in Mmp12-/-/Apoe-/- mice. This associated with more neutrophils, citrullinated histone H3 and neutrophil elastase, markers of NETs, and C3 levels in Mmp12-/- aortas. These findings were recapitulated in additional models of abdominal aortic aneurysm. MMP-12 deficiency also led to more pronounced elastic laminae degradation and reduced collagen integrity. Higher plasma C5a in Mmp12-/- mice pointed to complement overactivation. Treatment with IgG-FH1-5 decreased aortic wall NETosis and reduced adverse aortic remodeling and death from rupture in Ang II-infused Mmp12-/- mice. Finally, macrophage-specific MMP-12 deficiency recapitulated the effects of global MMP-12 deficiency on complement deposition and NETosis, as well as adverse aortic remodeling and death from rupture in Ang II-infused mice. CONCLUSIONS: An MMP-12 deficiency/complement activation/NETosis pathway compromises aortic integrity, which predisposes to adverse vascular remodeling and abdominal aortic aneurysm rupture. Considering these new findings, the role of macrophage MMP-12 in vascular homeostasis demands re-evaluation of MMP-12 function in diverse settings.

Differential BMP Signaling Mediates the Interplay Between Genetics and Leaflet Numbers in Aortic Valve Calcification.

Expression of a neuropilin-like protein, DCBLD2, is reduced in human calcific aortic valve disease (CAVD). DCBLD2-deficient mice develop bicuspid aortic valve (BAV) and CAVD, which is more severe in BAV mice compared with tricuspid littermates. In vivo and in vitro studies link this observation to up-regulated bone morphogenic protein (BMP)2 expression in the presence of DCBLD2 down-regulation, and enhanced BMP2 signaling in BAV, indicating that a combination of genetics and BAV promotes aortic valve calcification and stenosis. This pathway may be a therapeutic target to prevent CAVD progression in BAV.

Computed tomography imaging of macrophage phagocytic activity in abdominal aortic aneurysm.

Inflammation plays a major role in the pathogenesis of several vascular pathologies, including abdominal aortic aneurysm (AAA). Evaluating the role of inflammation in AAA pathobiology and potentially outcome in vivo requires non-invasive tools for high-resolution imaging. We investigated the feasibility of X-ray computed tomography (CT) imaging of phagocytic activity using nanoparticle contrast agents to predict AAA outcome. Methods: Uptake of several nanoparticle CT contrast agents was evaluated in a macrophage cell line. The most promising agent, Exitron nano 12000, was further characterized in vitro and used for subsequent in vivo testing. AAA was induced in Apoe-/- mice through angiotensin II (Ang II) infusion for up to 4 weeks. Nanoparticle biodistribution and uptake in AAA were evaluated by CT imaging in Ang II-infused Apoe-/- mice. After imaging, the aortic tissue was harvested and used from morphometry, transmission electron microscopy and gene expression analysis. A group of Ang II-infused Apoe-/- mice underwent nanoparticle-enhanced CT imaging within the first week of Ang II infusion, and their survival and aortic external diameter were evaluated at 4 weeks to address the value of vessel wall CT enhancement in predicting AAA outcome. Results: Exitron nano 12000 showed specific uptake in macrophages in vitro. Nanoparticle accumulation was observed by CT imaging in tissues rich in mononuclear phagocytes. Aortic wall enhancement was detectable on delayed CT images following nanoparticle administration and correlated with vessel wall CD68 expression. Transmission electron microscopy ascertained the presence of nanoparticles in AAA adventitial macrophages. Nanoparticle-induced CT enhancement on images obtained within one week of AAA induction was predictive of AAA outcome at 4 weeks. Conclusions: By establishing the feasibility of CT-based molecular imaging of phagocytic activity in AAA, this study links the inflammatory signal on early time point images to AAA evolution. This readily available technology overcomes an important barrier to cross-sectional, longitudinal and outcome studies, not only in AAA, but also in other cardiovascular pathologies and facilitates the evaluation of modulatory interventions, and ultimately upon clinical translation, patient management.

Hydroxamate-Based Selective Macrophage Elastase (MMP-12) Inhibitors and Radiotracers for Molecular Imaging.

Macrophage elastase [matrix metalloproteinase (MMP)-12] is the most upregulated MMP in abdominal aortic aneurysm (AAA) and, hence, MMP-12-targeted imaging may predict AAA progression and rupture risk. Here, we report the design, synthesis, and evaluation of three novel hydroxamate-based selective MMP-12 inhibitors (CGA, CGA-1, and AGA) and the methodology to obtain MMP-12 selectivity from hydroxamate-based panMMP inhibitors. Also, we report two 99mTc-radiotracers, 99mTc-AGA-1 and 99mTc-AGA-2, derived from AGA. 99mTc-AGA-2 displayed faster blood clearance in mice and better radiochemical stability compared to 99mTc-AGA-1. Based on this, 99mTc-AGA-2 was chosen as the lead tracer and tested in murine AAA. 99mTc-AGA-2 uptake detected by autoradiography was significantly higher in AAA compared to normal aortic regions. Specific binding of the tracer to MMP-12 was demonstrated through ex vivo competition. Accordingly, this study introduces a novel family of selective MMP-12 inhibitors and tracers, paving the way for further development of these agents as therapeutic and imaging agents.