Research identifies drug capable of preventing complications from hemolysis

AGÊNCIA FAPESP/DICYT Patients with sickle cell anemia frequently suffer from hemolysis, the destruction of red blood cells leading to the release of hemoglobin into the blood stream.

Recent studies have shown that hemolysis is a key factor in the aggravation of sickle cell disease. Recurrent hemolysis can produce chronic inflammation and vaso-occlusive crises, which cause intense pain and may lead to infarction in any part of the body, as well as organ damage.

In an article published in the August 6 issue of the journal Blood and highlighted in an editorial, researchers from the University of Campinas (UNICAMP) in São Paulo, Brazil, show that some of the complications resulting from hemolysis can be prevented with hydroxyurea. The drug is already used in chronic treatment of sickle cell disease, but in this case its purpose would be different.

“We believed hydroxyurea was underused and designed a clinical trial to evaluate the drug’s efficacy against acute inflammation due to hemolysis and in the treatment of vaso-occlusive crises,” said Nicola Conran, a researcher at UNICAMP’s Blood Center and a co-author of the article.

The research was conducted with support from FAPESP during Camila Bononi de Almeida’s postdoctoral work, which was supervised by Conran, under the aegis of a Thematic Project coordinated by Fernando Ferreira Costa, a professor at UNICAMP.

As Conran explained, sickle cell anemia is a hereditary disease characterized by a genetic change in hemoglobin, the protein that gives blood its red color and is responsible for carrying oxygen in the circulatory system.

The mutation makes hemoglobin polymerize after it releases oxygen, so the red blood cells take on a sickle-like shape. The deformed cells become rigid and tend to adhere to the vascular endothelium – the inner surface of blood vessels – hindering blood flow to tissue.

Hydroxyurea has been used in chronic treatment of sickle cell disease with the aim of increasing the production of fetal hemoglobin. The main oxygen transport protein in the fetus and newborn, fetal hemoglobin, mitigates the polymerization of genetically altered hemoglobin and the risk of vaso-occlusion.

Previous research in mice with sickle cell anemia by the group at UNICAMP’s Blood Center has shown that a higher dose of hydroxyurea is effective in acute treatment of vaso-occlusive crises (read more at http://agencia.fapesp.br/16495), activating a nitric oxide-dependent cell signaling channel that facilitates vasodilation and prevents interaction between white and red blood cells so that the latter do not adhere to the endothelium.

In their new study, the group performed experiments with healthy mice showing that this “nitric oxide-donating effect” of hydroxyurea can also help combat the inflammation caused by hemolysis.

“When hemoglobin is released into the bloodstream, it consumes nitric oxide inside blood vessels and this causes vasoconstriction, hindering circulation. Our experiments showed that this process, called hyperhemolysis, leads almost immediately to systemic inflammation, probably owing to nitric oxide consumption,” Conran said.

The experiment

To induce hemolysis in healthy mice, the researchers injected water intravenously. The rupturing of red blood cells led in approximately 15 minutes to a systemic inflammatory state comparable to that seen in the group that did not receive water as a hemolytic stimulus but instead received an injection of TNFα, recognized in the scientific literature as a pro-inflammatory cytokine.

The degree of inflammation was measured using an in vivo imaging system (IVIS) thanks to the collaboration of researchers at the Center for Cell-Based Therapy (CTC), one of the Research, Innovation and Dissemination Centers (RIDCs) funded by FAPESP. The CTC belongs to the University of São Paulo’s Ribeirão Preto Medical School (FMRP-USP).

IVIS injects into the animals a probe molecule that recognizes and binds to substances produced by activated defense cells. When binding occurs, the probe emits quantifiable light. The more leukocytes and neutrophils are activated, the more light is emitted.

“To be sure that hemolysis was occurring, we dosed the level of hemoglobin in the rodents’ blood plasma and found that it increased after water injection, reaching the same level as that seen in animals with sickle cell anemia,” Almeida said.

Vascular inflammation was measured in terms of white cell adherence to the endothelium using intravital microscopy. “We observed blood circulating in the translucent skin of the mouse’s scrotal sac, where we were able to see cells migrating to the inflamed site and adhering to the walls of blood vessels,” Almeida said.

A different group of mice received hydroxyurea at the same time that water was injected into the bloodstream. When the tests were repeated, the results showed that, although red blood cells were destroyed and hemoglobin was released into the bloodstream, both the inflammatory process and the resulting adherence of cells to the inner surface of blood vessels were significantly inhibited.

Range of possibilities

According to Conran, destruction of red blood cells may be caused by other factors in addition to genetic defects in hemoglobin such as those present in sickle cell anemia, including drugs, physical trauma, transfusion of incompatible blood types, and malaria, thalassemia, sepsis, and other diseases.

“The use of hydroxyurea may help combat the complications deriving from hemolysis in several of these conditions, but that’s something we need to explore further. This study opens up a wide range of possibilities for new investigations,” Conran said.