Atherosclerotic cardiovascular disease (ASCVD) ranks among the most formidable threats to human health, standing as the foremost cause of global mortality. Mounting evidence reveals an essential role of inflammation in atherogenesis, aligning with Virchow’s groundbreaking inflammatory hypothesis of atherosclerosis proposed in 1856. A pivotal occurrence in the early stages of atherogenesis involves endothelial cell-mediated leukocyte infiltration. When triggered by atherogenic factors, endothelial cells express leukocyte adhesion molecules that interact with cognate ligands, facilitating the tethering, rolling, and arrest of circulating leukocytes. This cascade initiates vascular inflammation, culminating in the progression of atherosclerosis.

In recent years, long non-coding RNA (LncRNA) has garnered attention, characterized as RNA molecules surpassing 200 nucleotides, seemingly devoid of protein-encoding capabilities. Emerging evidence shows that LncRNAs, such as CARMN, NEXN-AS1, and MAARS, play critical roles in atherogenesis. Diverse modes of action have been identified for LncRNAs in the regulation of biological processes, such as functioning as a molecular sponge for microRNAs, a scaffold for transcriptional machine assembly, and a bridge or anchorage point for chromatin looping. Enhancer-associated LncRNA is a subset of LncRNA that is transcribed from the enhancer regions, which is able to regulate the transcription of its proximal genes, in addition to other epigenetic functions of LncRNAs. However, the specific roles of enhancer-associated LncRNA in the pathophysiology of atherosclerosis have not been charted so far.

In the latest issue of Circulation Research, Li et al. report new findings that PSMB8-AS1, an enhancer-associated LncRNA, promotes vascular inflammation and atherosclerosis through activating a transcriptional cascade of NONO (non-POU domain containing octamer binding)/PSMB9 (proteasome 20S subunit β9)/ZEB1 (zinc finger E-box-binding homeobox 1) [1]. Using an unbiased microarray analysis comparing human atherosclerotic plaques and normal carotid arteries, the authors identified 1353 differentially expressed enhancer-associated LncRNAs, among which 14 can also be detected in cultured human umbilical vein endothelial cells. PSMB8-AS1 is the top endothelium-enriched enhancer-associated LncRNA that is significantly elevated in human atherosclerotic plaques, notably, with the highest abundance found in advanced vulnerable plaques. Although the specific cell populations that contribute to PSMB8-AS1 abundance remain to be determined, cell culture studies show the expression of PSMB8-AS1 in endothelial cells and undetectable level in aortic smooth muscle cell or THP-1 monocytic cell line. Notably, a predominant nuclear localization in cultured endothelial cells suggests a chromatin-associated function of PSMB8-AS1. Using a PSMB8-AS1 knockin (KI) and Apoe (Apolipoprotein E) knockout (KO) mice (Apoe−/−PSMB8-AS1KI), the authors demonstrated that PSMB8-AS1 plays an active role in the development of Western diet-induced atherosclerosis, with significantly increased lesion burden at the aortic root, arch and brachiocephalic artery. These lesions have a reduced smooth muscle cell content but an increased macrophage infiltration, indicating the participation of PSMB8-AS1 in plaque vulnerability. Moreover, elevated levels of VCAM1 (vascular adhesion molecule 1) and ICAM1 (intracellular adhesion molecule 1) are detected in the plaques and sera of Apoe−/−PSMB8-AS1KI mice, indicating aggravated vascular inflammation in these mice. Consistently, in vitro gain- and loss-of-function studies revealed that PSMB8-AS1 induces adhesion of monocytes/macrophages to endothelial cells and elevates the levels of VCAM1 and ICAM1 in a PSMB9-dependent manner. In the human genome, there are four genes (TAP1, TAP2, PSMB8, PSMB9) proximal to the PSMB8-AS1 locus, among which only PSMB9 is upregulated by PSMB8_AS1. Crucially, PSMB9 knockdown attenuated the endothelial VCAM1 and ICAM1 abundance induced by PSMB8-AS1 overexpression in cultured endothelial cells. Impressively, the authors proved the athero-protective effects of PSMB9 in mice. In vivo depletion of Psmb9 reduced the size of atherosclerotic lesions, plaque vulnerability, and vascular inflammation in Apoe−/− mice. The major strength of this study resides in the rigorous and meticulous characterization of this pro-atherosclerotic transcriptional cascade initiated by a LncRNA. First, to understand how PSMB8-AS1 activates the transcription of PSMB9, the authors performed a chromatin isolation by RNA purification-mass spectrometry and identified 8 transcription factors which are bound to PSMB8-AS1. NONO, a transcription factor previously reported to induce plaque destabilization [2], was selected for experimental validation. Following fine mapping of binding sites/domains between PSMB8-AS1 and NONO and those between NONO and PSMB9 promoter, the authors provided convincing evidence for establishing a stimulatory role of PSMB8-AS1-NONO complex in PSMB9 transcription. Second, to dissect how PSMB9, which is recognized as an immunoproteasome component, elevates the expression of VCAM1 and ICAM1, the authors integrated bioinformatic inference of transcription factors for VCAM1 and ICAM1 with mass spectrometry detection of differentially expressed proteins in endothelial cells with PSMB9 knockdown; they identified ZEB1 as a transcription activator relaying the effects of PSMB9 on VCAM1 and ICAM1. Taken together, these new findings provide valuable insights into the previously undefined role and molecular mechanisms of PSMB8-AS1, an enhancer-associated LncRNA that regulates endothelial inflammation and atherosclerosis via eliciting an intricate transcriptional cascade, paving the way for developing treatment avenues for vascular diseases through the modulation of LncRNAs.

This important study also brings several unaddressed questions. First, what are the non-endothelial cell types targeted by PSMB8-AS1? It is very intriguing to note that the increase in immunoactivities of VCAM1 and ICAM1 in PSMB8-AS1 KI mice is not confined in the endothelium but rather diffused through the whole lesion, implicating the effects of PSMB8-AS1 on lesional SMCs and infiltrated monocytes. Moreover, serum lipids are also impacted in PSMB8-AS1 KI mice. Indeed, Li et al. managed to address the contribution of PSMB8-AS1 in the endothelial compartment by showing that endothelial-restricted expression of PSMB8-AS1 via an adeno-associated virus carrying an Icam2 promoter-based expression cassette of PSMB8-AS1 phenocopied the pro-inflammatory functions in Apoe−/− mice. Nevertheless, it remains conceivable that PSMB8-AS1 executes its pro-atherogenic effects by modulating the functions of immune cells, SMCs, or even hepatocytes. Second, is it possible to employ PSMB8-AS1 as a druggable target to treat atherosclerosis? The present study is mainly built on the data from the PSMB8-AS1 KI model. As PSBM8-AS1 has been recognized as a human-specific transcript so far, the authors took considerable efforts to study Gm20496, an antisense transcript of mouse Psmb8, however, showing no effects on mouse atherogenesis. Therefore, one can choose to take a close examination of the downstream target of PSMB8-AS1, such as NONO, PSMB9, or ZEB1. As the β1i subunit of the immunoproteasome, PSMB9 can be targeted using a highly specific small-chemical inhibitor UK-101, which may circumvent the plaque-destabilizing effects of non-specific proteasome inhibition. Of note, both NONO, in SMCs, and ZEB1, in myeloid cells, have been previously reported for their functional roles in vascular injury and atherosclerosis [3]. In fact, the current study is the first report for their involvement in endothelial biology in the context of atherosclerosis. Further studies should be warranted to understand if an endothelial-specific approach is required to inhibit these two transcription factors in atherogenesis. Third, since PSMB8-AS1 KI or viral delivery of PSMB8-AS1 is initiated before the onset of diet-induced atherosclerosis, the impact of PSMB8-AS1 on plaque stability or surface integrity of established atherosclerosis remains to be determined.

The long non-coding RNA PSMB8-AS1 plays a pivotal role in exacerbating vascular inflammation and expediting the progression of atherosclerosis through the NONO/PSMB9/ZEB1 pathway. During the early phases of atherogenesis, endothelial cells initiate an increase in leukocyte infiltration. Within these cells, PSMB8-AS1 elevates PSMB9 expression by recruiting the NONO (non-POU domain-containing octamer-binding protein) and binding to the PSMB9 promoter region. This upregulation of PSMB9 subsequently enhances the expression of the ZEB1 transcription factor. ZEB1, in turn, promotes the transcription of VCAM1 and ICAM1, crucial surface adhesive molecules in mediating monocyte and macrophage adhesion to endothelial cells. This adhesion is a key event in triggering and sustaining vascular inflammation, thereby accelerating the onset and development of atherosclerosis.