Netrin-1 Protein, Small Derivatives Show Promise to Lessen PH in Mice

Patricia Inácio, PhD avatar

by Patricia Inácio, PhD |

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An under-the-skin infusion of a protein called netrin-1 — originally found to regulate nervous system development — shows promise for lessening pulmonary hypertension (PH), a mouse study suggests.

Additionally, the scientists found that three small peptides derived from netrin-1 also are a potential therapy for easing PH.

The protein and the peptides — short chains of amino acids, the building blocks of proteins — acted in a two-way manner, boosting the levels of nitric oxide and lessening oxidative stress, also a hallmark of the disease. Nitric oxide, called NO, is a potent vasodilator, meaning it’s a compound that widens blood vessels.

“Our data for the first time demonstrate intriguing findings that netrin- 1 and netrin-1 derived small peptides can be used as novel and robust therapeutics for the treatment of PH,” the scientists wrote.

The study, “Novel and robust attenuating effects on pulmonary hypertension of Netrin-1 and netrin-1-derived small peptides,” was published in the journal Redox Biology.

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PH is characterized by a narrowing of the blood vessels, called the pulmonary arteries, that carry blood from the heart to the lungs. This causes an increase in blood pressure within these arteries, and leads to problems with the heart’s right ventricle, which pumps blood to the lungs.

Oxidative stress, meanwhile, occurs when damaging reactive oxygen species (ROS), also called free radicals, outweigh the body’s antioxidant defenses.

A deficiency of NO and oxidative stress are both factors known to contribute to PH.

Pulmonary vasodilators like sildenafil — sold as Revatio or in a generic alternative in the U.S. — that increase NO have been shown to ease PH symptoms. However, their lack of impact on oxidative stress could contribute to their limited efficacy, according to researchers at UCLA, in California.

In a previous study, the scientists showed that netrin-1, known to regulate the behavior of a number of nerve cells, was able to boost NO production and attenuate or reduce the effects of oxidative stress.

Now, the team investigated the therapeutic effects of netrin-1 and its derived small peptides in a mouse model of PH triggered by exposure to low oxygen levels (hypoxia).

The animals received netrin-1 or three netrin-1-derived small peptides — V1, V2, or V3 — for three weeks as a subcutaneous (under-the-skin) infusion. Each compound was delivered using an implanted minipump at a continuous rate of 15 nanograms per day (ng/day).

Results showed that both the mean pulmonary artery pressure (mPAP) and right ventricular systolic pressure (RVSP) — a measure that estimates the pressure in the artery that supplies blood to the lungs — were attenuated after treatment with netrin-1 and its derived small peptides.

Animals in the hypoxia group alone served as controls. While the mean mPAP of these mice was 42.96 mmHg, treatment with netrin-1 lowered it to 28.98 mmHg. Similar results were seen with the peptides. Specifically, the mean mPAP was 23.83 mmHg with V1, 25.74 mmHg with V2 peptide, and 29.35 mmHg with V3.

RVSP decreased from a mean value of 43.77 mmHg in the control group to 32.19 mmHg after netrin-1 — a value similar to that seen in control animals under a normal amount of oxygen. The effect of the peptides was even more pronounced: 31.50 mmHg with V1, 33.10 mmHg with V2, and 31.23 mmHg with V3.

Treatment with netrin-1 or the small peptides also attenuated right ventricular hypertrophy (enlargement) and lessened blood vessel remodeling in the lungs, a hallmark of PH.

The researchers analyzed the animals’ lung tissue for markers of cell proliferation formation of smooth muscle, namely the proliferative cell nuclear antigen (PCNA) and the smooth muscle alpha-actin (SMA).

First, they confirmed the expression of both markers was increased in animals with PH-like disease compared with controls.

Treatment with netrin-1 or the peptides led to a marked decrease in the levels of both PCNA and SMA. The same was seen for deposits of collagen, a major molecule involved in scarring. Collagen detection was significantly attenuated by treatment with netrin-1 or its peptides.

Finally, the team evaluated whether the availability of NO within the lungs had changed after treatment. Using a NO-specific fluorescent probe, the researchers observed that while NO availability was decreased in PH animals, treatment with netrin-1 or the peptides rescued NO levels substantially or completely.

These changes were accompanied by reductions in the levels of ROS, namely superoxide.

Overall, “these dual mechanisms of increasing NO bioavailability and decreasing oxidative stress at the same time, underlie robust protective effects on PH of netrin- 1 and its derived small peptides, which are different from existing medications that primarily target NO signaling alone,” the researchers wrote.

“These observations would no doubt facilitate rapid translation of benchtop discoveries to clinical practice for the treatment of PH, for which effective therapies are urgently in need,” the team concluded.