Collaboration Uncovers New Clues About What Triggers A Stroke

By Deborah Borfitz 

September 7, 2022 | Researchers at Tulane University and Ochsner Health have characterized carotid plaque taken from patients within days of a stroke, confirming that ruptures are associated with the thinning of the fibrous cap stabilizing the fatty deposits as well as suggesting that B cells play a bigger role than has previously been appreciated. Tissues from those patients were also found to contain messenger RNA that could be behind loss of the protective layer of connective tissue preventing plaques from breaking open, according to Cooper Woods, Ph.D., associate professor of physiology and medicine at Tulane University School of Medicine. 

Previous studies have relied on carotid artery samples obtained from patients long after they had a stroke or heart attack, or after their death. The level of B cells in these histological samples was typically scarce, says Woods. 

But, as covered in a study that published recently in Scientific Reports (DOI: 10.1038/s41598-022-17546-9), the role of B lymphocytes and interferons in atherosclerotic plaque rupture now appear worth further examination. The hope is that the finding will spur exploration of how manipulating B cell activity in plaque might help prevent strokes and heart attacks, Woods says. 

Woods and his chief collaborator, Ochsner Health’s vascular and endovascular surgeon Hernan Bazan, M.D., will be focused on building the best algorithm for predicting ruptures and identifying the drug targets likely to have the most benefit. “In the case of therapeutics, it’s tricky because some of the things that might prevent fibrous cap thinning and hopefully stabilize a plaque can also accelerate plaque development elsewhere in the vasculature.” says Woods.  

Atherosclerosis takes many years to develop, he explains. “We get this inflammatory response to the cholesterol being deposited in the arteries, and over time plaques form around that cholesterol and grow.” The plaques can be clinically silent, or stable, and cause no problems until the fourth or fifth decade of life (depending partly on genetics and lifestyle) when they become unstable and thus vulnerable to rupturing. 

When the fibrous cap at long last breaks, the buildup of cholesterol and fat and dead cell material from longtime battles with the immune system gets released into the bloodstream, continues Woods. “When that happens, a lot of little clots form downstream.” 

When the destination is the brain, the clots can cause an ischemic stroke. In the coronary circulation, they can instead block oxygen from getting to the cardiac tissue and produce a heart attack. 

Matter Of Timing 

In the new study, the researchers compared the transcriptomes of asymptomatic and recently (within five days) ruptured carotid plaques to determine the molecular mechanisms active at the time of rupture. The plaques were obtained from 11 patients undergoing carotid endarterectomy (CEA), a necessarily small study population due to the expense of RNA sequencing, Woods explains.  

“The goal with these types of studies is to identify in a high throughput manner what genes might be of interest and then follow up on those with studies in larger groups,” he continues. Preliminary findings in this case were validated using traditional droplet digital PCR methods in a larger and separate group of 16 patients with recently ruptured carotid plaques. 

Recruitment for the study was tricky since the plaque samples from symptomatic individuals had to be obtained so quickly after they presented in the emergency room with an ischemic cerebrovascular event, such as a stroke, transient ischemic attack, or impairment of vision, says Woods. That meant the consulting vascular surgeon had to proceed with CEA surgery within a few days to remove the plaque to prevent another such event from occurring.  

Bazan’s group at Ochsner Health likes to do CEA procedures in an urgent setting, typically no more than two to three days after a stroke, Woods says. “Traditionally, it has been done weeks or months later, after the patient has stabilized, gone home, and recovered from the stroke the best they’re going to.” 

Over the last decade, a growing number of surgeons have come to believe that for preventive purposes, the sooner they get the plaque out of the carotid artery the better the outcomes for patients, he adds. “Scientifically to me, [Ochsner’s approach] was an attractive model... because it was giving a picture of what was going on at the time of rupture.” 

Mapping Relationships 

Woods and Bazan previously used the same sequencing strategy to identify lipids elevated in patients who recently had a stroke (DOI: 10.1016/j.plefa.2017.08.007). They also found that the ratio of a circular RNA (284) to a microRNA (221) in serum could potentially be a diagnostic biomarker of carotid plaque rupture and stroke (Circulation: Cardiovascular Genetics, DOI: 10.1161/CIRCGENETICS.117.001720), as might the expression of certain genes detectable in the bloodstream.  

The latest study is part of a larger effort to map the relationships between the noncoding microRNA in these plaques and the resulting changes in the messenger RNA, says Woods. “We have a couple of other databases from the same types of patients and so we are looking at how a comorbidity like diabetes might alter the signals that we saw in the current paper.” Having diabetes puts people at two to four times greater risk of heart attack and stroke, he notes. 

Another set of investigations underway is looking at the causal role of noncoding RNA in the changes seen in coding RNA in the present study. “We think that it may be more fruitful to have a larger set of targets for therapeutics and diagnostics,” Woods says. Yet another research project is seeking to understand if changes in blood flow in the carotids might be responsible for the observed changes in RNA and thus the thinning of the fibrous cap.