Exploring molecular profiles of calcification in aortic vascular smooth muscle cells and aortic valvular interstitial cells

Cardiovascular calcification can occur in vascular and valvular structures and is commonly associated with calcium deposition and tissue mineralization leading to stiffness and dysfunction. Based on shared risk factors and end stage pathologies, calcific aortic valve disease (CAVD) and coronary artery calcification (CAC) are often considered as one disease, and similarly treated. However, the clinical conditions associated with each phenotype can be different, suggesting multifaceted pathologies. To better understand diversity in molecular and cellular processes that underlie calcification in vascular and valvular structures, we exposed aortic vascular smooth muscle cells (AVSMCs) and aortic valve interstitial cells (AVICs) to calcific stimuli including high (2.5mM) phosphate and osteogenic media (OM) treatments in vitro. Consistent with clinical observations made by others, we show that AVSMCs are more susceptible to calcification than AVICs, and this process is mediated by cell-specific and treatment-specific molecular responses. RNA-seq analysis demonstrates that in response to calcific-stimuli, both AVSMCs and AVICs activate a robust ossification-program, although the signaling pathways, cellular processes and osteogenic-associated markers involved are diverse. In addition, VIC-mediated calcification appears to involve biological processes related to osteo-chondro differentiation and down regulation of actin cytoskeleton genes, that are not observed in VSMCs. Furthermore, are findings suggest that signaling pathways involved in cardiovascular cell calcification are dependent on the calcific-stimuli, including a requirement of PI3K signaling for OM-induced calcification, and not 2.5mM Phosphate. Together, this study provides a wealth of information suggesting that the pathogenesis of cardiovascular calcifications may be significantly more diverse than previously appreciated. Porcine aortic valve interstitial cells (pAVICs) and aortic vascular smooth muscle cells (pAVSMCs) were cultured as previously described and harvested either 3 (d3) or 5 (d5) days after treatment in conditioned media (CM), osteogenic media (OM), or 2.5mM phosphate (Pi). 200ng total RNA was extracted using the RNeasy Mini Kit from Qiagen (#74104), quantified using NANOdrop, and sent to Psomagen, Rockville, MD for analyses. Amplified cDNA libraries suitable for sequencing were prepared from 200 (ng) of DNA-free total RNA using the Universal Plus mRNA-Seq Library Prep Kit (NuGEN Technologies, Inc. #0508-96).  The RNA Integrity Number (RIN) was determined using Ribogreen on a TapeStation to measure concentration and quality, with a value of >7.0 meeting threshold. Libraries were prepared using the TruSeq Stranded mRNA LT Sample Prep kit and sequenced on an Illumina platform. Raw FASTQs were split into files containing ~40,000,000 total reads and checked for quality using the FASTX-Toolkit. The reads were filtered (removing sequences that did not pass Illumina’s quality filter) and trimmed based on the quality results (3 nucleotides at the left end of the R1 reads and 1nt at the left end of the R2 reads). Trimmed reads were mapped to reference genome (Sus Scrofa, GCF_000003025.6_Sscrofa11.1) with HISAT2. After the read mapping, Stringtie was used for transcript assembly. Expression profiles were calculated for each sample and transcript/gene as read count and FPKM (Fragment per Kilobase of transcript per Million mapped reads).