D28-25 RNAseq Analyses of Co-cultured Human Smooth Muscle and Endothelial Cells Expressing Sphingosine Kinase and a Dominant Negative Mutant

Background

Pulmonary vascular remodeling is characterized in part by enhanced proliferation of pulmonary artery smooth muscle cells (PASMCs) and their resistance to apoptosis. Sphingolipid signaling has been implicated this remodeling of the vasculature. A dominant negative (DN) mutation in human SPHK1 has previously been shown to enhance inflammation and attenuates the anti-apoptotic effects of sphingosine -1 phosphate (S1P). We hypothesized that paracrine effectors from SPHK1 and the DN mutant differentially act on HPAEC to influence cellular processes leading to altered vascular maintenance and cell apoptosis/autophagy.

Methods

Primary human pulmomary artery smooth muscle (PASMC) and pulmonary artery endothelial (HPAEC) cells were grown under routine growth conditions using co-culture inserts (Corning). Wild type SPHK1 cDNA or a dominant negative mutant (G96D) were cloned into lentivirus (VectorBuilder) and transduced into confluent PASMC monolayers with HPAEC monolayers on the opposite side of the insert. After 72hr, co-cultured cells were washed, harvested, and used for RNAseq analyses.

Results

SPHK1 overexpression in smooth muscle cells caused the dysregulation of several genes in HPAEC involved in micro- and macro-autophagy, pyroptosis, ferroptosis, and interferon signaling pathways. Network analyses showed both an increase in metabolic enzymes and transcription factors involved in maintenance of the vascular architecture, blood vessel morphogenesis, and vasodilation of blood vessels. Dysregulation of lncRNAs were observed which have been implicated in transcription and gene expression regulation. Expression of the DN mutant resulted in changes in cellular functions and maintenance, and cell death and survival. SPHK1 overexpression caused a significant downregulation of Mage Family Member D4 (MAGED4) transcript (-2.2 fold change; p < 0.05) that was attenuated in co-culture experiments using the DN mutant (2.9 fold change; p < 0.01).

Conclusions

SPHK1 dysregulation in co-cultured smooth muscle cells revealed a potential convergence of metabolic signaling pathways with a transcription program regulating the maintenance of the pulmonary vasculature and cardiac morphogenesis. MAGED4 expression suggests modulation of the autophagy/apoptosis in endothelial cells.

This abstract is funded by: NIH