Aortic valve calcification is an inflammatory disease
Elena Aikawa M.D., Ph.D.
Center for Molecular Imaging Research,
Massachusetts General Hospital,
Harvard Medical School
E-mail: eaikawa@mgh.harvard.edu
Calcific aortic valve stenosis, the most common valvular heart disease, has no therapeutic options other than surgical valve replacement performed approximately in 85,000 patients in the United States and 275,000 worldwide annually.1 Conventional imaging modalities can identify advanced late-stage calcification, yet, no current imaging methods can spatially resolve and quantify the dynamic pro-osteogenic molecular mechanisms and tissue mineralization at the earlier stages of disease.2 Our recent studies addressed this unmet scientific need by applying molecular imaging to the calcifying valvulo/vasculopathy of apoE-deficient (apoE-/-) mice and linked cardiovascular inflammation and calcification.3,4 Emerging molecular imaging approach monitors biological processes in vivo whereas conventional techniques offer only anatomical and structural information. We employed molecular imaging to investigate the mechanisms of calcific aortic valve disease using a comprehensive set of recently developed near-infrared fluorescent (NIRF) imaging agents. Detection of early molecular changes in aortic valves in vivo will aid in identification of targets for effective diagnostic and therapeutic strategies to prevent aortic stenosis.
The flexion area of the aortic leaflets near the attachment of the aortic root encounters highest mechanical forces.5 In addition, various proinflammatory molecules induce valve endothelial cell activation resulting in the macrophage infiltration. Using phage display–derived peptide sequences and multimodal nanoparticles, we previously developed a novel VCAM-1–targeted imaging agent.6 Our recent study using MRI ex vivo clearly demonstrated that distribution of VCAM-1 targeted agent mostly occurs in valve commissures (Figure 1). These results demonstrate that endothelial cell activation/damage initiates at the regions of increased mechanical stresses, and suggest that inflammatory cells enter the leaflets via circulation in response to endothelial cell activation or injury.
Figure 1. Endothelial cell activation occurs in the commissures of diseased aortic valves visualized by ex vivo MRI. Left: Long axis view shows the aortic arch and root. Dotted line demonstrates slice position of short-axis view. Middle: Short axis view shows signal reduction caused by uptake of VCAM-1-targeted nanoparticles. Right: Color-coded signal intensities (red) show focused uptake of VCAM-1 in commissures (arrows). Aikawa E et al, Circulation 2007;115:377-386.
Clinical evidence suggests that coronary atherosclerosis and aortic valve stenosis share similar epidemiologic risk factors such as age, sex, hypercholesterolemia and hypertension. Our study found that apoE-/- mice have early aortic valve lesions similar to atherosclerotic plaques located on the aortic site in the base (commissure) or in the middle portion (line of coaptation) of the leaflet, the areas subjected to the highest mechanical stresses. Magnetofluorescent nanoparticles and activatable imaging agents demonstrated that predominant cellular components of early lesions were macrophages containing activated matrix-degrading enzymes such as gelatinases (MMP-2 and MMP-9) and cathepsins (B and K).3 We previously showed that macrophage-derived proteolytic enzymes and cysteine endoproteases play critical roles in the pathogenesis of various cardiovascular diseases including atherosclerosis7,8 and myxomatous valve degeneration,9 and therefore represent useful targets for cardiovascular molecular imaging. Excessive levels of MMPs and cathepsins elaborated by macrophages may contribute to collagen, crucial determinant of valve durability, and elastin degradation leading to structural morphological abnormalities. Indeed, these features of inflammation-induced remodeling detected by molecular imaging associated with valvular dysfunction determined by MRI in vivo.3 Therefore, this advanced imaging approach for detection of proteolytic enzymes derived from activated macrophages in the early aortic valve lesions provides biological readout of inflammation in diseased valves may predict future risk of aortic valve stenosis.
We previously reported that myofibroblast-like cells, due to their plasticity, respond to various stimuli by activation and sequential phenotypic differentiation.1,9-14 Accumulating evidence also suggests that vascular and valvular myofibroblast-like cells may acquire an osteoblastic phenotype and that pro-atherogenic stimuli may promote expression of bone-regulating proteins (e.g., alkaline phosphatase, osteopontin, osteocalcin, osteonectin, collagen I and II, bone morphogenic proteins), transcription factors (e.g., Runx2/Cbfa1, Osterix), and eventually valvular and vascular calcification.3,15-17 Fluorescence microscopy on cryosections colocalized bisphosphonate-derivatized NIRF osteogenic signals with alkaline phosphatase activity, bone-regulating protein expression (e.g., osteopontin, osteocalcin, osteonectin), transcription factors (e.g., Runx2/Cbfa1, Osterix), and hydroxyapatite nanocrystals, while routine histological methods showed no evidence of calcification.3 Furthermore, NIRF imaging agent visualized osteogenic activity that was otherwise undetectable by X-ray computed tomography.4 Simultaneous imaging of osteogenic activity and macrophages colocalized calcification in inflamed lesions in aortic valves of apoE-/- mice (Figure 2).
Figure 2. Ex vivo imaging using multichannel laser scanning fluorescence microscopy performed on the excised aortic root as shown on the schematic diagram. Multicolor fluorescence imaging visualized simultaneously two different biological processes: osteogenesis (750 nm, red) and inflammation (680nm, green). Aikawa E et al, Circulation 2007;115:377-386.
Using the atherosclerotic artery as a model of in vivo calcification, we quantified the kinetics of inflammation and calcification.4 Three-dimensionally reconstructed in vivo images, obtained sequentially in the same mouse, visualized simultaneously spatial association between inflammation (green) and calcification (red, Figure 3). Intravital dual-channel fluorescence microscopy further monitored osteogenic changes in inflamed carotid arteries at 20 and 30 weeks of age and revealed that both macrophage burden and osteogenesis concomitantly increased during plaque progression (p<0.01) and decreased after anti-inflammatory statin treatment (p<0.05) (Figure 3, graph). These results support our hypothesis on inflammation-triggered calcification and, suggest that treatment of calcifying valvulo/vasculoparty earlier in its process prevents the progression of calcification.
Figure 3. Macrophages and calcification were imaged in vivo simultaneously and sequentially using multichannel, intravital fluorescence microscopy in carotid artery of apoE-/- mice at 20 and 30 weeks of age (Ap20; Ap30). Detection of osteogenic activity employed OS750 agent (red). A macrophage-targeted, spectrally distinct NIRF nanoparticle (green) visualized inflammation. Ap30 St denotes statin-treated mice. Aikawa E et al, Circulation 2007;116:2841-2850.
Collectively, results of our studies indicate that precalcified lesions in aortic valves develop in the setting of endothelial injury, inflammation and proteolytic activity, features also typical of atherosclerotic plaques. Therefore, modification of atherosclerotic risk factors such as dyslipidemia may slow the progression of aortic valve calcification when introduced early. To accomplish this tack in clinical settings, molecular imaging of osteogenic activity and microcalcifications should play important roles via identification of risk valves while disease is silent, and monitoring valvular osteogenesis during therapeutic interventions.
References
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