RAMP2-AS1 Regulates Endothelial Homeostasis and Aging

Figures

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  FIGURE 1. PCA analysis and aging trajectory of ECs subject to PS and OS. The first 3 principal components of time-series RNA-sequencing data plotted against time (hours) for protein coding genes (A) and non-coding genes (B) of two replicates (OS 1, 2; PS 1, 2). (A) The first principal component (PC1) is correlated with time. The second component (PC2) includes intermittent spiking signals associated with the expression of mitochondrial genes, and the third component (PC3) shows divergent trajectories between OS and PS conditions. (B) Same as A except for non-coding genes. Divergent trajectories are shown in the second component (PC2). The third component is not clearly interpretable.

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  FIGURE 2. Identification of RAMP2 and RAMP2-AS1 as top candidates. (A,B) PS/OS, ATV/control, and TNFα/control log2 fold changes in expression of top 50 protein-coding genes (PCGs), ranked by absolute values of PC3; non-coding genes (NCGs), ranked by absolute values of PC2. RAMP2 and RAMP2-AS1 are labeled in red showing induction by PS and ATV and suppression by TNFα. (C) Top 15 Biological Pathway terms from pathway enrichment analysis of top 200 PCGs, plotted with -log10 P-value. (D) Genomic structures of RAMP2-AS1 and RAMP2, wherein blocks depict exons, horizontal lines resemble introns, and arrowheads indicate the opposite transcriptional directions of RAMP2-AS1 and RAMP2; aligned with RNA-seq measurements along RAMP2 and RAMP2-AS1 in HUVECs. (E) RNA levels of RAMP2, RAMP2-AS1, and EZH1, plotted with PS/OS log2 fold changes across 10 time points, from 1 to 24 h under PS or OS. (F) Spearman correlation between RAMP2-AS2 and RAMP2 RNA levels in 44 human mesenteric arterial intimae quantified by qPCR, plotted with log2-transformed relative gene expression.

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  FIGURE 3. RAMP2-AS1 inhibition suppresses RAMP2 expression. (A) Design of LNAs and qPCR primers for RAMP2-AS1 transcripts. (B,C) HUVECs were transfected with scramble (Scr) or locked nucleic acid (LNA) GapmeRs against RAMP2-AS1 (LNA1, hereafter labeled “LNA”) at 25 nM, before treatment with ATV (5 μM) or DMSO. RAMP2-AS1 and RAMP2 mRNA levels were analyzed by qPCR. (D,E) Baseline HUVEC transfected with Scr and LNA were subjected to immunoblotting with specific antibodies as indicated. Representative blots (D) and densitometry analysis based on three experiments (E). Data are representative of mean ± SEM from 5 (B,C) and 3 (D,E) independent experiments with each experiment performed in duplicates. ∗P < 0.05 based on ANOVA, followed by Tukey’s post hoc test (B,C) and two-tailed t-test (E).

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  FIGURE 4. RAMP2-AS1 inhibition promotes cell senescence. HUVECs were transfected with scramble or RAMP2-AS1 LNA GapmeRs and then used for SA-β-gal staining and apoptosis assay. (A) Representative images of SA-β-gal staining (green indicates positive staining) of ECs transfected with scramble or RAMP2-AS1 LNA GapmeRs. Scale bars: 200 μm (upper images) and 100 μm (lower images). (B) Three randomly selected views were captured from each of the duplicates per condition per experiment and percentage of ECs with SA-β-gal positive staining were calculated. ∗P < 0.05 based on Student’s t-test. Data represent mean ± SEM from five independent experiments. (C,D) Apoptotic cells were quantified by Annexin V staining using flow cytometry. Representative scatter plots show in (C) and quantification of Annexin V+ cells expressed in percentage (%) in (D). Data represent mean ± SEM based on three independent experiments.

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  FIGURE 5. RAMP2-AS1 inhibition impairs EC angiogenic capacity. HUVECs were transfected with scramble or RAMP2-AS1 LNA GapmeRs for 48 h and then used in 3D spheroid sprouting assay. (A) Representative images showing the sprouting from the EC aggregates. (B–D) Quantitative analysis of EC sprouts after 72 h in hydrogel using three parameters as indicated. Ten aggregates were analyzed per condition in each experiment. *p < 0.05 based on Student’s t-test. Data are represented as mean ± SEM from three independent experiments.

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  FIGURE 6. RNA-seq profiling of transcriptome change caused by RAMP2-AS1 knockdown. HUVECs were transfected with RAMP2-AS1 LNA or scramble control in biological replicates. (A) Number of genes showing DE in RNA-seq with indicated P-value cutoffs. Genes showing DE with P < 0.1 are in cyan and those with P < 0.05 are in purple. (B) Top 15 enriched pathways ranked by -log10 (p-value). Pathways belonging to the same family are grouped into similar colors; angiogenesis in cyan, inflammatory response in blue, unfolded protein response pathways in red, apoptosis in yellow, and ECM remodeling in purple. (C) Log2 FC (RAMP2-AS1 LNA vs. scramble control) of select genes involved in angiogenesis, inflammation, or unfolded protein response in RAMP2-AS1 knockdown vs. scramble control.