Tufts researchers find first genomic biomarkers in extracellular vesicles

The research provides a greater understanding of myxomatous mitral valve disease and heart failure in dogs.

Important biomarkers have been found in extracellular vesicles in dogs with myxomatous mitral valve disease (MMVD) and congestive heart failure, according to researchers at Cummings School of Veterinary Medicine at Tufts University in Grafton, Mass.

This is the first biomarker discovery based on extracellular vesicles in a veterinary disease; genomic material (microRNA, or miRNA) was isolated in small extracellular vesicles, or exosomes, circulating in blood, according to Cummings. Researchers hope the findings will provide understanding into the molecular basis, diagnosis, and therapy for MMVD in dogs, as well as mitral valve prolapse (MVP), a similar human disease.

In analyzing circulating exosome miRNA (Ex-miRNA), researchers discovered that in dogs with MMVD, expression levels change as heart failure develops and progresses, and as dogs age.

Researchers also found that Ex-miRNA expression level changes seem more specific to disease states than the measure of miRNA from plasma without attention to the isolation of Ex-miRNA, suggesting that Ex-miRNA may offer an approach that improves current methods of monitoring patients with heart disease, yet relies on readily available samples such as blood and urine, according to the study.

MMVD in dogs closely resembles MVP in humans. For dogs, MMVD is the most common acquired cardiac disease and cause of congestive heart failure (CHF), making up two-thirds of all cardiac cases. The disease’s prevalence in older small breed dogs reaches 100 percent. Previous studies have shown that once in congestive heart failure, dogs have a median survival time between one and nine months.

“There are currently no medical treatments available to delay the progression of these valvular diseases,” said Vicky Yang, DVM, Ph.D., research assistant professor in cardiology and regenerative medicine at Cummings School and the first author on the study. “While these new molecular signatures in exosomes require further study, the findings could open doors to novel molecular targets to slow or halt the progression of mitral valve disease to heart failure.”

Cummings stated that the first goal of its study was to isolate and identify changes in circulating Ex-miRNA expression levels in dogs with MMVD or MMVD with congestive heart failure (MMVD-CHF) compared to normal, healthy dogs with no cardiac disease. Researchers also examined the effect of age in control dogs. The second goal was to understand the range of miRNA isolated from Ex-miRNA compared to plasma miRNA, to see if Ex-miRNA are more disease relevant.

The study enrolled 47 dogs; each dog received a physical exam and an echocardiographic evaluation, and dogs with observed or history of respiratory signs also received chest radiographs. The dogs were then divided into three groups based on radiographs and echocardiogram: normal healthy dogs, dogs with asymptomatic MMVD, and dogs with MMVD and history of CHF. Dogs with other systemic diseases were excluded. Whole blood samples were collected from each dog for two types of analysis.

Plasma miRNA was isolated from blood samples of 27 enrolled dogs divided into three groups: normal dogs with no age limit, dogs with asymptomatic MMVD, and dogs with MMVD-CHF.

Plasma Ex-miRNA was isolated from blood samples from 27 enrolled dogs divided into four groups: young, normal dogs less than 7 years old; old, normal dogs 7 years old or older; dogs with asymptomatic MMVD; and dogs with MMVD-CHF.

Results showed that exosomal miR-181c and miR-495 significantly increased in dogs with MMVD-CHF compared to the other three groups, according to the researchers. Exosomal miR-9 increased in dogs with MMVD and MMVD-CHF compared to older normal dogs. Exosomal miR-599 decreased in dogs with MMVD compared to older normal dogs. In total plasma, 58 miRNAs were deemed significantly different between normal dogs, dogs with MMVD and dogs with MMVD-CHF. In contrast to Ex-miRNA, none of the miRNA in total plasma remained statistically significant if the false discovery rate was below 15 percent.

“These results suggest that Ex-miRNA expression changes may be more specific to disease states than total plasma miRNA,” said Andy Hoffman, DVM, Ph.D., professor and director of the Regenerative Medicine Laboratory at Cummings School and corresponding author on the study. “These plasma Ex-miRNAs show great promise as biomarkers for MMVD disease monitoring and may also help us understand the pathophysiology of the disease, and subsequently devise more specific molecular therapies that can halt disease progression.”

The research was supported by the Shipley Foundation and Tufts Companion Animal Health Fund.

Read the full results in the Journal of Extracellular Vesicles here.

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