Unravelling the impact of aging on the human endothelial lncRNA transcriptome

Abstract

The incidence and prevalence of cardiovascular disease is highest among the elderly. There is a need to further understand the mechanisms behind endothelial cell aging in order to achieve vascular rejuvenation and minimize the onset of age-related vascular diseases. Long non-coding RNAs (lncRNAs) have been proposed to regulate numerous processes in the human genome, yet their function in vascular aging and their therapeutic potential remain largely unknown. This is primarily because the majority of studies investigating the impact of aging on lncRNA expression heavily rely on in vitro studies based on replicative senescence. Here, using a unique collection of young and aged endothelial cells isolated from native human arteries, we sought to characterize the age-related alterations in lncRNA expression profiles. We were able to detect a total of 4463 lncRNAs expressed in the human endothelium from which ∼17% (798) were altered in advanced age. One of the most affected lncRNAs in aging was the primate-specific, Prostate Cancer Associated Transcript (PCAT) 14. In our follow up analysis, using single molecule RNA FISH, we showed that PCAT14 is relatively abundant, localized almost exclusively in the nucleus of young endothelial cells, and silenced in the aged endothelium. Functionally, our studies proposed that downregulation of PCAT14 alters endothelial cell transcription profile and cell functions including endothelial cell migration, sprouting and inflammatory responses in vitro. Taken together, our data highlight that endothelial cell aging correlates with altered expression of lncRNAs, which could impair the endothelial regenerative capacity and enhance inflammatory phenotypes.

Keywords: PCAT14; aging; endothelium; lncRNA; regeneration.

FIGURE 1

Effect of aging on endothelial cell transcriptome. Bulk RNA-sequencing analysis was performed in human native young and aged arterial endothelial cells (n=6 per group, each sample is a pool of 5 different isolates). (A,C) Hierarchical clustering heatmap showing differentially expressed mRNAs (A) and lncRNAs (C). (B,D) Representative volcano plots showing differentially expressed mRNAs (B) and lncRNAs (D) (total mRNA variables = 15,379, total lncRNA variables = 4,463). (E) Chromosome distribution of differentially expressed (DE) lncRNAs detected as in (C) and (D). (F) Expression levels presented as ΔFPKM of lncRNA transcripts detected in panels (C) and (D).

FIGURE 2

PCAT14 expression, localization and impact on endothelial cell transcriptome. (A) UCSC Genome Browser showing the PCAT14 locus (hg38) and the poly (A)+ RNA-seq tracks from young and aged human native endothelial cells. (B) Maximum intensity projections of representative images of PCAT14 smRNA FISH on young and aged human native endothelial cells. Exon, gray; nucleus, blue, outlined with a dashed circle. Scale bar = 5 μm. RNA FISH was performed on 7 young and 4 aged samples. (C) Relative lncRNA levels of PCAT14 in young and aged native isolated arterial endothelial cells (n=3 per group, *p<0.05, Student’s t-test). (D) Relative PCAT14 expression normalized to CTL in Human umbilical vein endothelial cells (HUVEC, Passage 3) were transfected with GapmeR (50 nmol/L, 72 h) targeting PCAT14 (PCAT^KD) or a respective control (CTL). (E) Representative volcano plots showing differentially expressed transcripts (total variables = 22,059) in samples as in Figure 1D. (F) Hierarchical clustering heatmap showing the differentially expressed levels of PCAT14, endothelial and inflammatory markers in human native young and aged arterial endothelial cells (n=6 per group, each sample is a pool of 5 different isolations) and in human umbilical vein endothelial cells CTL or PCAT^KD as in (D). N.S: Not Significant; log₂FC: log₂ (Fold Change); p-val: p-value.

FIGURE 3

Effect of PCAT14 deletion on endothelial angiogenic responses and activation. Human umbilical vein endothelial cells (HUVEC, Passage 3) were transfected with a GapmeR (50 nmol/L, 72 h) targeting PCAT14 (PCAT^KD) or a respective control (CTL). (A) Percentage of endothelial cell confluence for up to 48 h. (B) Representative brightfield images and quantification following a wound scratch assay at 0 and 6 hours. (C) Representative brightfield images and quantification of endothelial cell sprouts on a 3D spheroid assay. (D) Representative brightfield images and quantification of THP-1 monocytes adhered to endothelial cells. As a positive control, endothelial cells treated with Interleukin 1β (Il-1β, 10 ng/mL, 4 h). n=3-6 biological replicates per group, experiments performed at least 2 times. Scale bar = 500 μm, *p<0.05, **p<0.01, Student’s t-test paired, N.S: Not Significant; log₂FC: log₂ (Fold Change); p-adj: p-value adjusted.