Glowing Cholesterol A Game-Changer For Study Of Heart Disease

By Deborah Borfitz

September 13, 2019 | A new technique that lights up a protein shuttling LDL (aka “bad”) cholesterol around the circulatory system has made it financially feasible to do high-throughput screening for cardiovascular drugs as well as measure the size of LDL particles—an important but poorly-understood disease risk factor, according to Steven Farber, a researcher at the Carnegie Institution for Science.

The “glowing cholesterol” pulses through the bloodstream of tiny larval zebrafish, which have genes and phenotypes well matched to those in humans, says Farber, one of the developers of the LipoGlo system. LipoGlo used state-of-the-art genome engineering to tag the LDL-carrying Apolipoprotein-B (ApoB) with an engineered light-emitting protein called NanoLuc that’s over 100 times brighter than a similar enzyme in fireflies. This enabled the research team, which includes scientists at Johns Hopkins University, to use a robot to distribute thousands of larvae into 96-well plates to identify small molecules that lower LDL.

The system works because every LDL particle has only one ApoB protein, so scientists can instantly measure how much bad cholesterol is in the zebrafish by quantifying the light level coming from each larva using a camera placed over the assay plate, continues Farber. LipoGlo also enables them to monitor LDL movement, distribution, and particle dimensions.

LDL cholesterol correlates well with cardiovascular disease risk, so identifying ways to lower levels of it in the bloodstream would save lives, he notes. The Centers for Disease Control and Prevention reports that heart disease accounts for one-quarter of deaths nationally.

The ability to do high-throughput drug screening is cost-prohibitive in both people and mice, but not zebrafish, Farber says. The fish each run about $3 per year, while a mouse costs closer to $1 per day. Moreover, a single female zebrafish will produce 300 to 500 larvae every week.

After an article describing the methodology published in Nature Communications on July 31, it quickly joined the top 3% of most-read articles of a similar age, Farber reports. “People are very excited about our approach because it mixes the genetics of the [optically clear] zebrafish with human disease and is a new strategy. No one ever tagged ApoB before.” Less than five scientists anywhere are studying lipoprotein metabolism in the zebrafish, he adds.

A possible reason why ApoB was never tagged is that the protein is massive with complementary DNA that is almost 14,000 base pairs long. “If one is lucky enough to amplify the sequence using PCR [polymerase chain reaction], you cannot grow a plasmid containing the sequence in bacteria,” explains Farber. “The bacteria that do grow end up containing plasmids that are mutated.”

With the advent of gene editing technology, however, it is no longer necessary to take this type of approach, he continues. For the LipoGlo system, researchers use transcription activator-like effector nucleases (TALEN) to cut DNA precisely in a particular spot in the ApoB gene sequence and then a much smaller plasmid to insert the DNA sequence encoding the light-emitting protein.

Lead author on the published paper was James Thierer, a graduate student at Johns Hopkins who does research in Farber’s lab in Carnegie's Department of Embryology. Co-author Stephen Ekker, in the Department of Biochemistry and Molecular Biology at the Mayo Clinic, provided key technology for the TALEN assembly.

Farber says the idea for LipoGlo was “concocted” four years ago after he and Thierer attended a scientific meeting together and later learned to use the TALEN Technology from Ekker. Johns Hopkins and the Carnegie Institution currently hold the intellectual property on LipoGlo.

The search for new therapeutics using LipoGlo is being supported by a $3 million, five-year grant from the National Institutes of Health, Farber says. A portion of the funds goes to purchase the NanoLuc substrate from Promega for the proprietary reagent, which is many times brighter than the readily available luciferase substrate—an important consideration, given the exquisitely small quantities of material needing illuminating.

New Discoveries

LipoGlo has already led to the discovery of a mysterious gene called pla2g12b that has a huge impact on the size and number of ApoB-containing lipoproteins, Farber says. Only seven papers have ever been published on the gene. Further investigation on pla2g12b could help the team understand why heart disease runs in families or point to a new strategy for controlling lipoproteins in the bloodstream.

Existing life science literature is dominated by studies largely on a small subset of proteins (e.g., tumor protein p53) encoded by the genome, he says. Farber and his colleagues instead opted to do “forward genetics,” which involves randomly making mutations throughout the genome and then looking for animals with changes in lipid metabolism to figure out what gene was mutated. “This kind of approach is entirely unbiased by anything currently in the literature,” he says.

The researchers have two more papers in the works on new discoveries made using the LipoGlo system, Farber says, one of which describes a mutation in the microsomal triacylglycerol transfer protein (MTP) that is known to load different kinds of lipids into LDL particles. The characterization of this mutation in zebrafish lead them to make the same mutation in the human protein for studies in cell culture, the results of which point to a new strategy for treating cardiovascular disease.

New treatments are needed, especially for the subset of patients who don’t response to statin drugs, says Farber. The first major study of statins involving over 6,500 Scottish men with high plasma cholesterol found 31% fewer heart attacks in the statin versus non-statin group after five years. But many people still die from cardiovascular diseases because current risk scoring tools don’t factor in LDL particle size.

People with lower LDL readings may in fact be producing many very small LDL particles and have a higher risk of disease, says Farber. Conversely, some people with higher LDL cholesterol are making big particles that place them at lower risk. “We don’t understand the genes that affect particle size.”

In addition to identifying new therapeutics for treating lipid abnormalities, other potential applications for LipoGlo are to study areas beyond the blood where lipoproteins congregate, including the central nervous and musculoskeletal systems, Farber says. Humans with very high cholesterol can have tendon abnormalities, he notes. “No one has ever been able to study lipoproteins separate from the plasma until now.”