Author + information
- Vivek Nanda, PhD∗ (, )
- Sophia Xiao,
- Jianqin Ye, PhD and
- Nicholas J. Leeper, MD
- ↵∗Address for correspondence:
Dr. Vivek Nanda, Department of Surgery, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, California 94305.
It is now known that the internalization and transcytosis of low density lipoprotein (LDL) in the vessel wall occurs through molecular pathways independent of the LDL receptor. In a study recently published in Nature Communications, investigators cross-referenced results from a genome-wide ribonucleic acid interference screen with targets identified in publicly-available genome-wide association studies datasets to identify activin-like kinase 1 as a novel driver of this process. This approach has relevance to the field of atherosclerosis, and could be used as a model for the prioritization of future “hits” in large-scale genomic screens.
- activin-like kinase 1
- endothelial cells
- genome-wide association studies
- genome-wide RNAi screen
One of the earliest changes observed during atherogenesis is the accumulation of plasma-derived apolipoprotein B–containing lipoproteins, such as low-density lipoprotein (LDL) and chylomicron remnants in the subendothelial region of the vessel wall (1). Because LDL particles are predominantly internalized via the low-density lipoprotein receptor (LDLR) pathways (2), investigators have been relentless in designing therapies aimed at removing circulating LDL from the blood via this receptor.
However, signs of accelerated atherosclerosis in patients carrying loss-of-function mutations specific to the LDLR (3) have revealed the existence of alternate mechanisms regulating the internalization and retention of LDL in the vessel wall. In a study recently published in Nature Communications, Kraehling et al. (4) have addressed this gap in knowledge by identifying a novel molecular pathway responsible for LDL transport into and across the endothelium (Central Illustration).
The authors began by performing an unbiased genome-wide ribonucleic acid (RNA) interference screen designed to target over 18,000 genes in cultured endothelial cells, after which gene hits were cross-referenced to publicly available genome-wide association studies (GWAS). Using such an approach, activin-like kinase 1 (ALK1), a transforming growth factor-B-type 1 receptor highly expressed in endothelial cells, was identified as a candidate gene regulating LDL uptake independent of LDLR activity. These findings were validated at the cellular level through classical ALK1-specific knockdown and overexpression studies. Together, these data provide the first evidence that ALK1 is pivotal in regulating LDL uptake into endothelial cells in an LDLR-independent manner. Further, the authors reported that LDL taken up via this mechanism does not lead to lysosomal degradation, implying that ALK1 does not affect sterol sensing.
Next, the authors performed a series of binding assays showing a direct interaction between LDL and the ectodomain of ALK1. Interestingly, this binding was observed not to compete with that of previously demonstrated LDLR-LDL binding, therefore reinforcing the concept that ALK1 mediates LDL uptake in a manner that is independent of the LDLR. This was followed by a series of complementary assays, which included total internal reflectance microscopy, to demonstrate that ALK-1 mediates LDL movement from the apical side to the basolateral membrane of the endothelium. Last, in vivo data generated in endothelial-specific ALK1-deficient mice on an LDLR background showed reduced LDL uptake in the aortic wall compared with control mice. These studies provide clear evidence that ALK1 mediates LDL internalization and transcytosis, and suggest relevance to cardiovascular disease.
Although there continues to be a surge in the development of lipid-lowering therapies, such as the PCSK9 inhibitors (5), it is clear that many of these agents promote benefit via a molecular mechanism that relies upon the LDLR, potentially explaining the diminishing returns being seen with newer cholesterol-lowering medicines. Given the high residual risk patients experience even on maximum dose statin therapy, there is a clear need to understand and then translationally target novel pathways that mediate LDL transportation into and across the endothelium independently of the LDLR. The finding that ALK1 fulfills the criteria of being LDLR- and sterol sensing–independent suggests that these pathways could be exciting new targets for the development of novel antiatherosclerotic therapies.
Perhaps more importantly, this new study provides a road map that can help demystify the data generated with high-throughput “omics” approaches, such as genome-wide RNA interference screens and association studies. Recently, such studies have identified vast numbers of candidate proteins, microRNAs, and long noncoding RNAs that are of unclear significance or are poorly studied. Here, the authors cross-referenced screening hits with leads confirmed in publicly available genomic datasets as a method to prioritize candidates for additional study. This approach helped eliminate potentially irrelevant leads and allowed for the rapid focus on a pathway that explained the phenotype in the cell of interest. Such a winnowing may prove useful as next-generation “omics” studies continue to identify causal pathways in regions of the genome that may be devoid of obvious leads and help investigators avoid the pursuit of factors that may not have human relevance.
Dr. Nanda’s work was supported by the American Heart Association (15POST21310005). Dr. Leeper’s work was supported by the National Institutes of Health (R01HL12337002). All other authors have reported that they have no relationships relevant to the contents of this paper to disclose.
All authors attest they are in compliance with human studies committees and animal welfare regulations of the authors’ institutions and Food and Drug Administration guidelines, including patient consent where appropriate. For more information, visit the JACC: Basic to Translational Science author instructions page.
- Received March 15, 2017.
- Accepted March 15, 2017.
- 2017 The Authors
- Tabas I.,
- Williams K.J.,
- Boren J.
- Vasile E.,
- Simionescu M.,
- Simionescu N.
- Kraehling J.R.,
- Chidlow J.H.,
- Rajagopal C.,
- et al.