A USF Health preclinical study indicates nanoparticles containing a micro-RNA switch offers promising biotechnology to advance the fight against atherosclerotic cardiovascular disease

Illustration of angioplasty with a stent

Tampa, FL (Feb. 9, 2021) – Percutaneous coronary intervention (PCI), commonly known as angioplasty with a stent, opens clogged arteries and saves lives. Despite its benefit in treating atherosclerosis that causes coronary artery disease, this common minimally invasive procedure still poses severe complications for some patients.

Angioplasty involves inflating a balloon at the tip of a catheter to compress fatty deposits (plaques) against the artery wall, thereby restoring blood flow to the narrowed or blocked vessels. The image-guided procedure is often combined with the placement of either uncoated stents — tiny expandable mesh devices– or stents coated with slowly-released antiproliferative drugs. The drug-eluting stents help avert the growth of scar tissue (smooth muscle cell proliferation) in the artery so that the vessel does not eventually close again, known as restenosis.

However, current antiproliferative drugs indiscriminately inhibit the growth of all nearby cells, including the layer of endothelial cells lining the blood vessels. These endothelial cells prevent blood clots (thrombosis) within the stent and the formation of more plaques (neoatherosclerosis), which can trigger a heart attack or sudden cardiac death.

Focused on tackling this treatment complication, University of South Florida Health (USF Health) Morsani College of Medicine researchers recently developed a next-generation nanotherapy. Their preclinical findings are detailed in a study published Feb. 2 in Molecular Therapy.

Hana Totary-Jain, PhD, USF Health associate professor of molecular pharmacology and physiology, was principal investigator for the nanotherapy study.

The nanotherapy comprised of a nontoxic peptide known as p5RHH and a synthetic messenger RNA (mRNA) that carries the genetic instructions, or code, needed by cells to make proteins. By simply mixing up the p5RHH with the mRNA, they spontaneously self assemble into compacted nanoparticles that specifically target the injured regions of the arteries in mouse models mimicking angioplasty. The nanoparticles contain an microRNA switch added to the mRNA.

“One of the main challenges of cardiovascular disease remains the delivery of targeted therapies specifically to the plaque regions and the cells that form plaques, including the smooth muscle cells and inflammatory cells — without affecting the endothelial cells or the healthy regions,” said the study’s principal investigator Hana Totary-Jain, PhD, an associate professor of molecular pharmacology physiology at USF Health Morsani College of Medicine.

To do this, the researchers used mRNA that encodes for p27 protein, which blocks cell growth, and added to the mRNA an endothelial cell-specific microRNA to generate a microRNA switch. The design of this microRNA switch allowed the researchers to turn on the mRNA in smooth muscle cells to inhibit their growth and the formation of restenosis. It also enabled them to turn off the mRNA in endothelial cells so these cells could grow uninhibited and quickly heal the damaged blood vessel.

John Lockhart, PhD, was the paper’s lead author.

“If we can come up with an antiproliferative therapy that specifically targets the cardiovascular smooth muscles cells and the infiltrating inflammatory cells but spares the endothelial cells – which we’ve done with the design of our microRNA switches – then we should be able to achieve the therapeutic effects of drug-eluting stents without the downside of thrombosis and neoatherosclerosis,” said the paper’s lead author John Lockhart, PhD, who worked on the study as a doctoral student at USF Health Molecular Pharmacology and Physiology. Dr. Lockhart is continuing his postdoctoral training at Moffitt Cancer Center.

The latest study builds upon previous research by Dr. Totary-Jain, indicating that a microRNA-based therapy worked better than drug-eluting stents in a rat model of angioplasty. That work used an adenovirus vector to carry the cell-selective therapy to injured arteries. In this study the viral vector was replaced with a nanoparticle alternative – a change needed to avoid safety concerns and advance the therapy toward use in patients.

The investigational nanoparticles were injected into mice with arteries mimicking post-angioplasty vessel injury every three days for two weeks (5 doses total). Mice treated with the nanoparticles containing the miRNA switch had significantly reduced restenosis and completely restored endothelial cell growth in the injured artery, compared to animals treated with nanoparticles containing mRNA without the miRNA switch, the researchers report.

Above: Injured control artery treated with near infrared florescent protein, depicts restenosis in center. Below: Injured artery treated with the microRNA switch nanotherapy shows open artery (no restenosis) and clear endothelial cell layer marked in green. | Images courtesy of Hana Totary-Jain, USF Health

In addition, the nanoparticles efficiently delivered its mRNA cargo, without degradation, solely to regions of the artery where endothelial cells were damaged. The particles did not toxically accumulate either in the cells of healthy organs (the liver, spleen. lungs or kidneys), or in uninjured arteries adjacent to those requiring treatment. The researchers observed no adverse reactions or outcomes in mice treated with the nanoparticles.

Overall, the findings suggest that the miRNA-switch nanoparticles could be applied clinically to selectively prevent restenosis after PCI by specifically targeting areas of endothelial cell damage to allow quicker cell regrowth and repair of injured arteries.

The USF Health researchers next plan to investigate the potential of the microRNA-switch nanoparticles to directly treat atherosclerotic plaques, thereby eliminating the need for PCI.

“Cardiovascular disease is still the number one cause of death,” said Dr. Totary-Jain, a member of the USF Health Heart Institute. “This research offers promise for the development of novel biomolecular therapies to advance the fight against coronary artery disease and peripheral artery disease,”

One person dies of cardiovascular disease every 36 seconds in the U.S., according to the Centers for Disease Control and Prevention.

The USF Health research was supported by grants from the National Institutes of Health. Samuel Wickline, MD, director of the USF Health Heart Institute, and Hua Pan, PhD, assistant professor at the Heart Institute, collaborated on the study.

The vape flavorings so popular with kids and young adults are cardiotoxic and disrupt the heart’s normal electrical activity, a University of South Florida Health preclinical study finds

TAMPA, Fla (Dec. 11, 2020) — The appealing array of fruit and candy flavors that entice millions of young people take up vaping can harm their hearts, a preclinical study by University of South Florida Health (USF Health) researchers found.

Mounting studies indicate that the nicotine and other chemicals delivered by vaping, while generally less toxic than conventional cigarettes, can damage the lungs and heart. “But so far there has been no clear understanding about what happens when the vaporized flavoring molecules in flavored vaping products, after being inhaled, enter the bloodstream and reach the heart,” said the study’s principal investigator Sami Noujaim, PhD, an associate professor of molecular pharmacology and physiology at the USF Health Morsani College of Medicine.

In their study published Nov. 20 in the American Journal of Physiology- Heart and Circulatory Physiology, Dr. Noujaim and colleagues report on a series of experiments assessing the toxicity of vape flavorings in cardiac cells and in young mice. “The flavored electronic nicotine delivery systems widely popular among teens and young adults are not harm-free,” Dr. Noujaim said. “Altogether, our findings in the cells and mice indicate that vaping does interfere with the normal functioning of the heart and can potentially lead to cardiac rhythm disturbances.”

Dr. Noujaim’s laboratory is among the first beginning to investigate the potential cardiotoxic effects of the many flavoring chemicals added to the e-liquids in electronic nicotine delivery systems, or ENDS. He recently received a five-year, $2.2-million grant from the NIH’s National Institute of Environmental Health Sciences to carry out this laboratory research. Commonly called e-cigarettes, ENDS include different products such as vape pens, mods, and pods.

Sami Noujaim, PhD, of  USF Health Molecular Pharmacology and Physiology, is investigating the potential cardiotoxicity of flavoring chemicals added to vaping e-liquids using preclinical models, including “cardiac cells in a dish.”  | Photo by Allison Long, USF Health Communications

Vaping involves inhaling an aerosol created by heating an e-liquid containing nicotine, solvents such as propylene glycol and vegetable glycerin, and flavorings. The vaping device’s battery-powered heat converts this e-liquid into a smoke-like aerosolized mixture (e-vapor). Manufacturers tout e-cigarettes as a tool to help quit smoking, but evidence of their effectiveness for smoking cessation is limited, and they are not FDA approved for this use. E-cigarettes contain the same highly addictive nicotine found in tobacco products, yet many teens and young adults assume they are safe.

Among the USF Health study key findings:

• In mouse cardiac muscle cells (HL-1 cells), the researchers tested the toxicity of three different, popular flavors of e-liquid: fruit flavor, cinnamon, and vanilla custard. All three were toxic to HL-1 cells exposed to e-vapor bubbled into the laboratory dish where the cells were cultured.

• Cardiac cells derived from human pluripotent stem cells were exposed to three distinct e-vapors. The first e-vapor containing only solvent interfered with the electrical activity and beating rate of cardiac cells in the dish. A second e-vapor with nicotine added to the solvent increased the toxic effects on these cells. The third e-vapor comprised of nicotine, solvent, and vanilla custard flavoring (the flavor previously identified as most toxic) augmented damage to the spontaneously beating cells even more. “This experiment told us that the flavoring chemicals added to vaping devices can increase harm beyond what the nicotine alone can do,” Dr. Noujaim said.

• Healthy young mice implanted with tiny electrocardiogram devices were exposed to 60 puffs of vanilla-flavored e-vapor five days a week, for 10 weeks. Heart rate variability (HRV) – that is, fluctuations in the time interval between successive heartbeats – decreased in these test mice compared to the control mice that inhaled only puffs of air under the same regimen. A sophisticated analysis by the USF Health researchers showed that vaping interfered with normal HRV in the mice by disrupting the autonomic nervous system’s control of heart rate (the acceleration and slowing down of heartbeats), Dr. Noujaim said.

• Finally, mice exposed to vaping were more prone to an abnormal and dangerous heart rhythm disturbance known as ventricular tachycardia compared to control mice.

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Whether the mouse findings will translate to people is unknown. Dr. Noujaim emphasizes that more preclinical and human studies are needed to further determine the safety profile of flavored ENDS and their long-term health effects.

A partial government ban on flavored e-cigarettes aimed at stopping young people from vaping focused on enforcement against flavored e-cigarettes with pre-filled cartridges, like those produced by industry leader JUUL. However, teens quickly switched to newer disposable e-cigarettes still sold in a staggering assortment of youth-appealing fruity and dessert-like flavors.

“Our research matters because regulation of the vaping industry is a work in progress,” Dr. Noujaim said.  “The FDA needs input from the scientific community about all the possible risks of vaping in order to effectively regulate electronic nicotine delivery systems and protect the public’s health. At USF Health we will continue to examine how vaping may adversely affect cardiac health.”

In 2020, 3.6 million U.S. youths still used e-cigarettes, and among current users, more than eight in 10 reported using flavored varieties, according to the Centers for Disease Control and Prevention.

The USF Health study reported in the AJP-Heart and Circulatory Physiology was supported in part by the NIH’s National Heart Lung and Blood Institute.

Vanilla flavored e-liquid for an electronic cigarette