Fig 1: Transcriptional microarray of ACTN4 and HLA-DPA1 knockdown revealed gene expression and pathway changes. A, ACTN4 and HLA-DPA1 transcript expression under siRNA-driven ACTN4 and HLA-DPA1 knockdown in PAECs. B, ACTN4 and HLA-DPA1 transcript expression under lentivirus forced overexpression of ACTN4 with and without pretreatment with IL-1ß in PAECs. C and D, Top 15 upregulated and downregulated genes after ACTN4 and HLA-DPA1 knockdown in PAECs. E, Pathways enriched under ACTN4 and HLA-DPA1 knockdown using Ingenuity Pathway Analysis software. n=3. F, CLDN1 transcript expression under siRNA mediated ACTN4 and HLA-DPA1 knockdown. G, CLDN1 transcript expression under IL-1ß treatment. H and I, Representative images of Group 1 PAH and control human lungs and monocrotaline treated and control rats stained with immunofluorescent probes for CLDN1 (red), CD31 (green), a-SMA (gray), nuclear counterstained with DAPI (blue), and imaged by confocal microscopy. Scale bar: 50 µm. N=6 to 8 per group. Data represented as mean±SEM. Significance determined by permuted t test or permuted ANOVA. a-SMA indicates alpha smooth muscle actin; ACTN4, alpha actinin 4; CLDN-1, claudin-1; ERK, extracellular signal-regulated kinase; HLA, human leukocyte antigen; IL-1ß, interleukin-1ß; IL-6, interleukin-6; ILK, integrin-linked kinase; JAK/STAT, Janus kinase/signal transducer and activator of transcription; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; NF-?B, nuclear factor kappa B; PAEC, pulmonary arterial endothelial cell; PAH, pulmonary artery hypertension; RFP, red fluorescent protein; RhoA, ras homolog family member A; and TGF-ß, transforming growth factor beta.
Fig 2: ACTN4-HLA-DPA1-CLDN1 axis affects angiogenesis, an endothelial phenotype associated with PAH. A, Tube formation assay after siACTN4, siHLA-DPA1, and siNC knockdown in PAECs. B, Tube formation assay after LV-ACTN4 and LV-NC overexpression in PAECs under vehicle and IL-1ß treatment. C, Tube formation assay after LV-CLDN1 overexpression in PAECs. D, Tube formation assay after siCLDN1 under vehicle and IL-1ß treatment. Scale bar: 500 µm. n=5 to 8 per group. Data are represented as mean±SEM. Significance determined by permuted t test or permuted ANOVA. ACTN4 indicates alpha actinin 4; CLDN-1, claudin-1; HLA, human leukocyte antigen; IL-1ß, interleukin-1ß; LV-ACTN4, lentivirus-mediated ACTN4; LV-CLDN1, lentivirus-mediated CLDN1; LV-NC, lentivirus negative control; PAEC, pulmonary arterial endothelial cell; PAH, pulmonary arterial hypertension; siACTN4, siRNA-mediated ACTN4; siCLDN1, CLDN1 knockdown; siHLA-DPA1, siRNA-mediated HLA-DPA1; and siNC, siRNA-mediated negative control.
Fig 3: Model of the rs9277336-ACTN4-HLA-DPA1 genomic axis in pulmonary arterial hypertension.In this study, we defined the pathogenic activity of functional SNP rs9277336, entailing the allele-specific binding of ACTN4, which controls expression of the neighboring HLA-DPA1 gene. Through inflammatory or genetic means, downregulation of this ACTN4-HLA-DPA1 regulatory axis promotes endothelial pathophenotypes, in part via CLDN1 expression, thus providing a mechanistic explanation for the association between this SNP and PAH outcomes. ACTN4 indicates alpha actinin 4; CLDN-1, claudin-1; HLA, human leukocyte antigen; IL-1ß, interleukin-1ß; PAH, pulmonary arterial hypertension; and SNP, single nucleotide polymorphism.
from Charles River for Lenti-CMV-CLDN1, lentiviral particles
Titer: >10^8 IU/mL