Abstract 4128189: CD44 Knockdown Attenuates the Abnormal Cellular Phenotype of BMPR2-Deficient Primary Human Pulmonary Artery Endothelial Cells

Background: BMPR2 gene silencing in primary human pulmonary artery endothelial cells (PAECs) produces a proliferative, hypermigratory PAH-like cellular phenotype. Genome-wide expression profiling in BMPR2 -silenced cells uncovered CD44 , a cancer stem cell marker associated with tumor progression and metastasis, among the top upregulated transcripts. Therapeutic strategies that block CD44 or reduce its expression are currently in various stages of development for cancer. Thus, mechanistic studies investigating the contribution of CD44 to vascular remodeling in PAH may reveal new therapeutic targets. Hypothesis: Upregulation of CD44 contributes to the abnormal phenotype of BMPR2-deficient PAECs and thus may contribute to vascular remodeling in PAH. Aims: Determine the effect of CD44 -silencing on gene expression and cell proliferation in BMPR2-deficient PAECs. Methods: Commercially available primary, human PAECs were transfected with scrambled control (siCTRL) or gene specific siRNA(s). PAH patient-derived (N=24) and failed donor-derived (N=11) PAECs were obtained from the PHBI. mRNA and protein expression were determined by quantitative RT-PCR and Western blotting, respectively. Cell proliferation was assessed by BrdU incorporation 96h after siRNA transfection. Data was analyzed using t-tests or ANOVA with post hoc pairwise comparisons. Results: Consistent with our previous findings, CD44 protein expression was increased 4-fold in BMPR2 -silenced PAECs 48h following siRNA transfection (P<0.0001 vs siCTRL; N=10 unique donors). Genes involved in endothelial-mesenchymal transition and cell proliferation were also increased in BMPR2 -silenced PAECs including HMGA1 , ID1 , SNAI1, and SNAI2 (P<0.01 for all vs siCTRL; N=5 independent experiments) and co-silencing CD44 attenuated their upregulation (P<0.05 for all vs siBMPR2 alone). Importantly, CD44 knockdown reduced PAEC proliferation in the absence (P=0.01 vs siCTRL) and presence of BMPR2 deficiency (P=0.002 vs siBMPR2 alone). Like BMPR2 -silenced PAECs, CD44 mRNA levels were higher in PAH patient-derived versus failed donor control PAECs (P=0.07). Conclusions: CD44 knockdown corrected aberrant gene expression associated with endothelial-mesenchymal transition and reduced cellular proliferation in BMPR2-deficient PAECs. Thus, endothelial CD44 upregulation may contribute to pathologic vascular remodeling in PAH and represents an unexplored therapeutic target.