Andrea Roberts

Though development of the coronary vasculature is a critical event during embryogenesis, the molecular mechanisms that regulate its formation are not well characterized. Two unique approaches were used to investigate interactions between cardiac myocytes and proepicardial (PE) cells, which are the coronary anlagen. One of these experimental approaches used a 3-D collagen scaffold system on which specific cell-cell and cell-matrix interactions were studied. The other approach used a whole heart culture system that allowed for the analysis of epicardial to mesenchymal transformation (EMT). The VEGF signaling system has been implicated previously as an important regulator of coronary development. Our results demonstrated that a specific isoform of VEGF-A, VEGF(164), increased PE-derived endothelial cell proliferation and also increased EMT. However, VEGF-stimulated endothelial cells did not robustly coalesce into endothelial tubes as they did when cocultured with cardiac myocytes. Interestingly, blocking VEGF signaling via flk-1 inhibition reduced endothelial tube formation despite the presence of cardiac myocytes. These results indicate that VEGF signaling is complex during coronary development and that combinatorial signaling by other VEGF-A isoforms or other flk-1-binding VEGFs are likely to regulate endothelial tube formation.

Abstract Mass spectrometry imaging (MSI) is a rapidly advancing technology that provides mapping of the spatial molecular landscape of tissues for a variety of analytes. Matrix-assisted laser desorption/ionization (MALDI)-MSI is commonly employed, however, confident in situ identification and accurate quantification of analytes remain challenging. We present a novel imaging methodology combining trapped ion mobility spectrometry (TIMS)-based parallel accumulation-serial fragmentation (PASEF) with MALDI ionization for targeted imaging parallel reaction monitoring (iprm-PASEF). We investigated the spatial distribution of lipids and metabolites in liver tissues from male wild-type and CD38 knockout mice (CD38 -/- ). CD38, an enzyme involved in nicotinamide adenine dinucleotide (NAD⁺) metabolism, significantly influences liver metabolic function and contributes to age-related NAD⁺ decline. Although CD38 deletion previously was linked to improved metabolic phenotypes, the underlying spatial metabolic mechanisms are poorly understood. The spatial iprm-PASEF workflow enabled confident identification and differentiation of lipid isomers at the MS2 fragment ion level and confirmed increased NAD + and decreased adenosine diphosphate ribose (ADPR), a by-product of NAD + hydrolysis, in CD38 -/- livers. This approach provided confident, specific, and robust MS2-based identification and quantification of fragment ions in spatial MSI experiments. Additionally, the innovative iprm-PASEF opens unprecedented opportunities for spatial metabolomics and lipidomics, offering spatially resolved insights into molecular mechanisms.