Multimodal Analytical Tools to Enhance Mechanistic Understanding of Aortic Valve Calcification

Abstract

This review focuses on technologies at the core of calcific aortic valve disease (CAVD) and drug target research advancement, including transcriptomics, proteomics, and molecular imaging. We examine how bulk RNA sequencing and single-cell RNA sequencing have engendered organismal genomes and transcriptomes, promoting the analysis of tissue gene expression profiles and cell subpopulations, respectively. We bring into focus how the field is also largely influenced by increasingly accessible proteome profiling techniques. In unison, global transcriptional and protein expression analyses allow for increased understanding of cellular behavior and pathogenic pathways under pathologic stimuli including stress, inflammation, low-density lipoprotein accumulation, increased calcium and phosphate levels, and vascular injury. We also look at how direct investigation of protein signatures paves the way for identification of targetable pathways for pharmacologic intervention. Here, we note that imaging techniques, once a clinical diagnostic tool for late-stage CAVD, have since been refined to address a clinical need to identify microcalcifications using positron emission tomography/computed tomography and even detect in vivo cellular events indicative of early stage CAVD and map the expression of identified proteins in animal models. Together, these techniques generate a holistic approach to CAVD investigation, with the potential to identify additional novel regulatory pathways.

Figure 1

Roadmap of advances in technology and understanding of disease pathophysiology. A PubMed query of “calcific aortic valve disease” yielded 5461 results from 1944 to 2023, showing the increasing trend of published research that paralleled the increased understanding of earlier stages of calcific aortic valve disease (CAVD). Within this 80-year period, the ability to analyze CAVD improved alongside development of novel techniques and groundbreaking technological advancements—tomography, RNA sequencing, near-infrared fluorescence, and proteomics to name a few—leading to the increase in discoveries made and benchmark findings published. This increase in novel technologies corresponds to a focal shift in the study of disease pathophysiology, with current technology and methodologies facilitating early stage CAVD investigation.^,,, , , , , , , BAV, bicuspid aortic valve; TAV, tricuspid aortic valve. Images obtained from BioRender.com (Toronto, ON, Canada).

Figure 2

Visualization of calcification ex vivo and in vivo using multiple imaging modalities. A: Co-injection of spectrally distinct fluorescent probes in apolipoprotein E (ApoE)–deficient mice visualizes a correlation between macrophages and osteogenesis in the root, arch, and abdominal aorta (ex vivo fluorescence reflectance imaging). B: Intravital near-infrared fluorescent molecular imaging was performed in ApoE-deficient mice using a calcified carotid artery to visualize the macrophages (macrophages, green) and calcium deposition (osteogenesis, red). The individual imaging signals were superimposed on one another (merged), showing the colocalization between macrophages and calcification in diseased tissue (in vivo near-infrared fluorescence molecular imaging). Scale bars: 3 mm (A); 250 μm (B). Adapted from Aikawa et al.

Figure 3

Immunofluorescent verification of layer-specific proteins. Microlayer atlas of calcific aortic valve disease (CAVD) tissue. Immunofluorescence corroborates layer proteomic findings and visualizes the localization of apolipoprotein B (ApoB), proline-arginine rich end leucine rich repeat protein (PRELP), sulfatase 1 (SULF1), and procollagen C-endopeptidase enhancer 2 (PCOLCE2) to the fibrosa layer; glial fibrillary acidic protein (GFAP) to the spongiosa; and cysteine and glycine rich protein 1 (CSRP1), calponin-1 (CNN1), and alpha-2-glycoprotein 1, zinc-binding (AZGP1) to the ventricularis valvular interstitial cells. Scale bars = 100 μm. Adapted from Schlotter et al.

Figure 4

The multi-omics landscape for calcific aortic valve disease (CAVD). Molecular profiling enabled by the three -omics fields that encompass the central dogma of biology, DNA (epigenetics) to multiple gene–expressed RNA (mRNA) (transcriptomics) to protein (proteomics), and the integration of animal models, imaging technologies, and cell culture models to enable the understanding of CAVD. Molecules of interest, epigenetics, and the further development of animal models represent part of the frontiers for CAVD research. Most studies to date have relied on human aortic valves retrieved from valve-replacement surgeries. ApoC-III, apolipoprotein C-III; HDL, high-density lipoprotein; MAOA, monoamine oxidase A; PALMD, palmdelphin; RNA-seq, RNA sequencing; scRNA-seq, single-cell RNA sequencing; vWF, von Willebrand factor.