Uric acid, an abundant metabolite in human plasma, is prone to oxidation in inflammatory conditions. Uric acid oxidation is associated with cardiovascular disease by inducing endothelial dysfunction, oxidative stress, and systemic and local inflammation. In an article published in the journal Antioxidants, scientists from the RIDC Redoxoma led by Flavia Meotti, a professor at the Instituto de Química at Universidade de São Paulo (USP), propose a mechanism to explain how uric acid causes vascular lesions that can lead to cardiovascular disease.

Our blood vessels are lined by the endothelium, which has complex functions for maintaining vascular homeostases, such as regulating blood flow, clotting, endothelial cell proliferation, and inflammatory and immune responses. The endothelium expresses an enzyme called peroxidasin, which is essential for endothelial cells survival and, mainly, for structuring the extracellular matrix, the set of molecules that unite and fix cells to form tissues.

The researchers showed that uric acid is oxidized by peroxidasin in endothelial cells, impairing the formation of the extracellular matrix and causing decreased cell adhesion and migration.

For Meotti, the results of the study elucidate both the role of uric acid in endothelial dysfunction and the functions of peroxidasin. “As peroxidasin is an enzyme that was recently described, all these findings are relevant to really understand what it is doing and how its reactivity can modify endothelial function.”

Peroxidasin

Peroxidases are enzymes that oxidize substrates in the presence of hydrogen peroxide (H₂O₂). Meotti’s group had already shown in previous studies that uric acid is oxidized by myeloperoxidase, generating the urate free radical and urate hydroperoxide, both capable of causing oxidative damage.

Peroxidasin is an enzyme from the myeloperoxidase family that is abundant in vascular tissue but was only described in the mid-1990s. The enzyme uses hydrogen peroxide to form hypobromous acid and promote collagen IV covalent crosslink, which structures the extracellular matrix. Because of its extracellular matrix-forming function, it is associated with the progression of some types of cancer. It is also linked to cardiovascular disease.

To investigate whether uric acid reacts with peroxidasin and compromises the enzyme’s function in human endothelial cells, the first step of the study was to find peroxidasin in the secretome (set of secreted proteins) of vascular endothelial cells (HUVEC), through proteomic analysis. “We used endothelial cells to show the physiological relevance of the process,” says Meotti.

Then, analyzing HEK293 overexpressing peroxidasin cells, the researchers confirmed that the enzyme oxidizes uric acid. And finally, they studied PFHR9 cells, which overproduce extracellular matrix. “We wanted to extract the matrix so we could see the effect of uric acid on it.” The results showed a decrease in collagen IV crosslinks in the extracellular matrix.

“After reacting with hydrogen peroxide, peroxidasin reacts with uric acid to form an intermediate, compound II, in which the enzyme gets trapped until it reacts with a second uric acid to return to its native state. The reaction of the first uric acid is faster but the reaction of the second one with compound II is much slower. This is a kind of inhibition because it slows down the enzyme, but it is not irreversible. The uric acid is also competing with the bromide, so the enzyme stops forming hypobromous acid and forms the urate free radical. At this point, the formation of extracellular matrix is reduced, because of this competition and the delay in the complete cycle of the enzyme”, explains the researcher.

Endothelial dysfunction

Endothelial dysfunction is a term that refers to the malfunction of the vascular endothelium, a condition that plays an important role in the development of cardiovascular disease. In collaboration with researchers from the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil), Meotti’s group had already published a clinical study showing that plasma levels of uric acid and allantoin, the most stable product of uric acid oxidation, are positively associated with carotid intima-media thickness, proving that uric acid oxidation is a relevant process in atherosclerosis.

Now, when investigating the cellular function of endothelial cells, the researchers observed that both uric acid and a peroxidasin inhibitor, phloroglucinol, impair cell adhesion and migration. The same is also seen in cells in which the insertion of an interfering RNA prevents the expression of the enzyme. With this, they proved that adhesion and migration are dependent on peroxidasin and that the effect of uric acid on these processes is also related to the inhibition of the enzyme.

Adhesion and migration are essential for tissue repair, angiogenesis, and maintenance of vascular integrity. According to Meotti, “these functional processes are important, for example, in the regeneration of a vessel. When you have atherosclerosis, the vessel is damaged by the inflammatory process, by the oxidizing species that are formed, and the adhesion and migration process collaborates with healing, helping the vessel to recover. So if you damage that too, it’s another mechanism that prevents the injury from resolving.”

The research was carried out during Bianca Dempsey’s Ph.D. and Litiele Cezar Cruz’s postdoctoral studies, both FAPESP grant recipients and the first authors of the article. Meotti points out that FAPESP’s financial support through the RIDC Redoxoma and the Young Investigator 2 grant was fundamental for the rigor and quality of the study.

The article Uric Acid Reacts with Peroxidasin, Decreases Collagen IV Crosslink, and Impairs Human Endothelial Cell Migration and Adhesion, by Bianca Dempsey, Litiele Cezar Cruz, Marcela Franco Mineiro, Railmara Pereira da Silva, and Flavia Carla Meotti, can be accessed here.