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Molecular alarm

Team of researchers from Unifesp and Incor find an enzyme that behaves as a biological marker for hypertension: the protein only appears in someone who already has high blood pressure or shows a predisposition for having it one day

A silent effort for over 20 years may lead to an unprecedented advance in the history of the struggle against hypertension, or high blood pressure. Working almost as discretely as the sneaky way this illness takes hold of the organism, molecular biologist Dulce Casarini, from the Nephrology group of the Federal University of São Paulo (Unifesp), identified a new form of angiotensin I converting enzyme (ACE), which works as a biological marker for the disease in mice, and – here is the best part of the story – seems to perform the same role in human beings. We are speaking about the ACE with a molecular weight of 90 kDa (kiloDaltons, the unit of atomic mass), an alternative form of the enzyme discovered by Dulce in 1982.

Her studies show that hypertensive animals, or even healthy onea that are predisposed to develop high blood pressure, always carry this compound in their urine. Healthy animals, though, that have no tendency to develop high blood pressure, do not show this form of the enzyme. If the relation between the 90 kDa ACE and hypertension is also true for our species, Dulce will have discovered the first marker related to the appearance of hypertension. “Under the supervision of Frida Plavnik and Odair Marson, from the Nephrology group, we analyzed the urine of about 1500 persons with hypertension and 550 individuals with normal blood pressure.

All the scientific evidence leads us to believe that this form of the enzyme really is a marker for hypertension in humans” explains the researcher from Unifesp. The conclusion is always the same: anybody who has the marker and is still not hypertensive, may be hypertensive one day. Unless he or she changes the life style – quit smoking, starting to do exercises, reducing the consumption of alcohol, losing weight and reducing salt in the diet -, the increase in his blood pressure is just a question of time.

The evidence that 90 kDa ACE can be an efficient marker for hypertension is the fruit of cross-checking data from a series of studies of mice and human beings. Confronting the information supplied by the analysis of the urine of animals andof people with normal blood pressure andthose with hypertension, Dulce and José Eduardo Krieger, a doctor and director of the Laboratory of Genetics and Molecular Cardiology of the Heart Institute of the University of São Paulo (Incor/USP) and her main collaborator in the research, realized the key role played by this form of the enzyme.

Three situations appeared. Individuals known to be hypertensive would always show two kinds of ACE, one with a molecular weight of 90 kDa and the other of 65 kDa. The picture shown by individuals without high blood pressure was more complex: some would show only two forms of the enzyme, those of 65 and 190 kDa, and other would have three varieties of ACE (65, 90 and 190 kDa). By themselves, these results were not conclusive, but they did point to a good lead to be followed. The systematic monitoring of the two groups of persons without high blood pressure (those with two and those with three kinds of enzyme) – help to clear up the reasoning. Anybody who was healthy but whose urine had this variety of the enzyme would become hypertensive, after some time.

“In some cases, people started to show high blood pressure after three years”, says Dulce. Obviously, from the moment they become hypertensive, these individuals came to show in their urine just the two typical kinds of ACE found in this kind of patient, those of 65 and 90 kDa. To sum up all this work: the ECA of 90kDa behaves as hypertension marker for the general population. The test developed by Dulce is able to indicate the presence in human urine of each one of the three forms of ACE studied.

Blocked enzymes
In partnership with the teams of Krieger, Adriana Carmona and Eduardo Cilli, from Unifesp’s Department of Biophysics, Dulce developed a kit to measure the presence of the protein in human urine. In practice, if it is proven that 90kDa ACE is a biological marker for high blood pressure, the kit will acquire the status of a predictive test for hypertension. Without precedent in the world, the test may help doctors in the arduous task of identifying patients who are potentially hypertensive. Hypertension affects about 10% of the whole population on the planet, and it increases the risks of heart and kidney problems.

The researchers from São Paulo have now requested a patent over the use of the marker and the test in Brazil, which can detect the existence of the enzyme with the help of various laboratory techniques, like chromatography or mass spectrometry. The moment that the property register is granted them for Brazilian territory, which should happen soon, the team has its intellectual rights preserved for one year abroad. “We will take advantage of this moratorium period to lodge patent requests in the United States, Europe and Japan.” says Krieger.

With this temporary protection practically in their hands, the researchers, who had been keeping their discovery secret, decided to make public their work with the marker for hypertension. “We will be sending to various scientific magazines the six articles we are writing now”, says Dulce. For an illness with the peculiarities of hypertension, the creation of a test able to pick out people who are going to develop the disease is excellent news. With the justified fame of being a silent killer, high blood pressure gives no clue that it has taken hold of the organism. For a good while, the person feels nothing. When at last he notices something different and goes to see a doctor, the diagnosis is hypertension. The problem is that this verdict is always late.

At this point, high blood pressure has already caused damage to the cardiovascular system and often to the kidneys as well. According to the World Health Organization, there are about 600 million people in the world with high blood pressure. In Brazil, 20% of adults and half of those aged 50 or over have hypertension. The black population is also more affected than the others. One third of the deaths on the planet is due to heart problems, aggravated by hypertension.

It has been known for decades that the angiotensin I converting enzyme performs a crucial role in setting off hypertension. The ACE acts on the so-called renin-angiotensin system, which is important for the equilibrium of the cardiovascular function. Produced by the kidneys, renin is a proteolytic enzyme that is capable of breaking up large proteins into smaller compounds. Its function is to act on angiotensinogen, a substratum, and a larger protein, which originates an inactive hormone, angiotensin I.

How does the ACE hormone come into the story? When blood passes through the lungs, the ACE transforms harmless angiotensin I into the dangerous angiotensin II, perhaps hypertension’s biggest villain. This second form of angiotensin makes the arteries constrict more and stimulates the release of hormones that raise blood pressure. The greater part of anti-hypertensive drugs, like captopril and enalapril, inhibits the ACE. They are medicines that prevent the action of this enzyme, which, in short, prevents angiotensin I from being converted into angiotensin II.

Intriguing results
Because of its decisive participation in the mechanism that raises blood pressure, ACE is the subject of many studies. Various forms of ACE, with different molecular weights, have already been found in human urine. The most common of them all is the one with 190 kDa. Dulce discovered two new kinds, the candidate for a marker of hypertension, 90 kDa, and one with 65 kDa. “I do not know how no one has noticed this before”, Dulce admits. “It is so simple to measure.”

The first time she detected the presence of 90 kDa ACE in human urine, almost 20 years ago, Dulce thought that the measurement was the fruit of a technical artifact, the jargon used by laboratory personnel to describe the observation of an illusory phenomenon, caused by some inaccuracy in the method employed, or, sometimes, in the apparatus used in the experiment. In plain English, she thought that the detection of this form of the enzyme was the result of some procedural error. Time went by, she started other research in molecular biology, but she never forgot this intriguing experience.

In the 90’s, during one of her two post-graduate studies she carried out in France, Dulce had the opportunity of discussing the matter with Pierre Corvol and François Alhenc-Gelas, two world authorities on hypertension and the discoverers of the two actives centers of ACE. They carried out new analyses with human urine, and the result was the same: the Brazilian really had found a new form of ACE, not described in scientific literature. It was still not known what the discovery would be useful for, but Corvol encouraged her to carry on with her research with the enzyme. And, in a kind of gentlemen’s agreement, he assured her that no one from his team would begin any study of 90 kDa ACE. The promise was kept.

Back in Brazil, in 1994, Dulce continued her research and entered into a partnership with Kriger, from Incor, who was already studying the genetic bases of hypertension. As the hypothesis that the 90 kDa ACE was a marker for hypertension took shape, both started to adopt a very critical attitude towards the possible discovery. “We started to think of experiments that could show that we were wrong” says Krieger. This “devil’s advocate” approach had the aim of looking for answers to some of the questions that most troubled the researchers. If it seemed so simple to identify this form of the enzyme in human urine, why was it that no one had found it before? Could it be just a coincidence that the enzyme in its 90 kDa form “persecuted” those with, or with a tendency for, high blood pressure?

To see if they could find some fault in the hypertension marker theory, Dulce and Krieger carried out several studies, above all with rodents. They saw, for example, that healthy mice that had a tendency towards high blood pressure – which therefore had 90 kDa ACE -, would continue to show this form of the enzyme, even when they were receiving treatment to prevent high pressure from developing. Regardless of showing the disease or not, the animals would maintain the biological marker, as it was to be expected.

In another work, the researchers literally squeezed with clips the arteries of animals with normal blood pressure that showed no predisposition towards it (and which lacked the enzyme that was a candidate for a biological marker). The intention, which was successful, was to create hypertension in mice that would not suffer from high blood pressure in a natural way. The rats really did become hypertensive, but even so did not start showing the enzyme. In short, it all went according to the expectations, to the joy of the researchers. “Happily, we were unable to prove ourselves wrong, and it all seemed too good to be true”, was Krieger’s comment.

Tests in Vitória
What was still missing to prove that 90 kDa ACE is a marker for human hypertension? In general terms, the researchers need to produce longer term studies to show the prevalence of this form of the enzyme in various generations of the same family, made up of individuals both hypertensive and without high blood pressure. With this intent, further research will be carried out in Unifesp’s Kidney and Hypertension Hospital and at Incor. Straight away, Dulce and Krieger have at their disposal 1600 urine samples from the population of Vitória (in the state of Espírito Santo), to analyze the efficiency of the marker. The material is part of a study coordinated by José Geraldo Mill, from the Federal University of Espírito Santo (Ufes), which, jointly with the Vitória’s Department for Health, mapped out the principal cardiovascular risk factors among the inhabitants of the capital city of the state of Espírito Santo.

One point that is still obscure is the origin of the 90 kDa ACE. For the time being, the researchers do not know the biological mechanism that leads to its formation. They do not know if the enzyme is derived from a larger protein, perhaps from ACEs with higher molecular weights that could be broken down by some enzyme, or if it is the result of some genetic alteration. In this second case, a mutation in the gene responsible for the codification of the enzyme could lead to the synthesis of the 90 kDa ACE.

Practically discarded is the hypothesis that this variant of the enzyme is the fruit of some “renal event”. That is, originating from the process of filtering carried out by the kidneys. The fact that Dulce had already identified in mice the 90 kDa ACE in various kinds of tissues – in the lungs, kidneys, pancreas, heart and aorta – help exclude this hypothesis. After all, if it is present invarious parts of the body, this form ofenzyme cannot be the fruit of filtering by the kidneys. In the course of the work to elucidate the origins of this mysterious compound, Krieger hopes not only to prove that the enzyme is a good marker of hypertension in humans, but also, who knows, to show that it may be directly related to the genesis of high blood pressure. “At any rate, we will be more than satisfied if we manage to show that the enzyme is in fact a marker”, says Krieger.

The identification of a possible marker for hypertension reinforces the tradition of Brazilian science for producing good news in this sector. In the 60’s, researcher Sergio Henrique Ferreira, from USP’s School of Medicine in Ribeirão Preto discovered the BPF (bradykinin potentiation factor) molecule, which originated captopril, one of the medicines most used in the world to control high blood pressure. Unfortunately, in those days, there was no concern over requesting patents, and the profits that arose from the achievement ended up going into the purses of the foreign laboratory that produced the medicine.

At the beginning of this year, yet another contribution: the Butantan Applied Toxicology Center, one of the Centers for Research, Innovation and Dissemination (Cepids) financed by FAPESP, patented the active principle of another molecule, evasin, which may be used to produce an anti-hypertensive drug. Now, in a clear sign of the new times, in which priority is given to defending the interests of Brazilian research, it was the turn of the Unifesp and Incor group to make its contribution to the fight against hypertension. “We didn’t patent the marker and the kit with the thought of making money from it”, says Dulce. “What we want to avoid is someone profiting from our work and, in the future, from our having to pay royalties to use products derived from our own efforts”.

In September, when she was in Paris and told her friend Pierre Corvol (and to whom she took the dough for the quintessentially Brazilian cheese bun) of the latest results of her research into the marker, the researcher from Unifesp heard the following comment: “It is a pity, for us (for not discovering it before)”. The good humored reaction of the current president of the scientific board of Inserm (the National Institute of Health and Medical Research) sizes up well the dimensions of the impact of the work by the Brazilian researchers on the struggle against hypertension.

Twelve by eight for the average person
Almost everybody has heard the doctor say that 120 by 80 is a good pressure. What does this mean? Before understanding the meaning of the two figures, the very concept of blood pressure has to be understood. When it beats, the heart pumps blood through the arteries, so taking nutrients to the tissues. The force that the flow of blood exercises on the walls of the arteries is called blood pressure.Everyone, obviously, has blood pressure, and the ideal is that it should be in the region of 120 by 80 for most of the time.

But what about the two figures? The popular apparatus that measures pressure, the sphygmomanometer, registers the maximum and minimum forces that the blood pressures the arteries with. The first figure, with a higher number, also called the systolic pressure, is the peak of the pressure of the blood attained in the inside of the vessels when the heart contracts. The diastolic pressure – or minimum – is the level of the final blood pressure attained when the heart relaxes. The unit used universally to measure blood pressure is millimeters of mercury. In colloquial language in Brazil, the units are usually dropped off, and people just say “twelve by eight”.

When the arteries narrow, the heart has to pump more strongly for the blood to run through the vessels and reach the tissues. This makes the blood pressure become permanently higher, overloading the heart muscle. If it frequently reaches the levels of 14 (during systole), 9 (diastole) or both, the pressure of classified as high. High blood pressure has no cure, but it can be controlled with medicine and a healthier life style: quit smoking, doing exercises, losing weight, reducing the consumption of salt and alcohol.

With the exception of a few kinds of high pressure that have their origin in specific problems, like kidney or endocrinous gland disorders, nine out of ten cases of the disease have no known cause. All that is known is that there is a hereditary component. The children of hypertensive parents show a higher risk of developing the disease. Blacks are also preferential victims of hypertension. In medicine, the view that prevails is that the disease is a problem with complex origins. High blood pressure is not the result of the action of a single gene or mechanism, but of a set of factors that are still obscure.

The project
Molecular Characterization of the Angiotensin I Converting Enzyme (99/01531-0); Modality: Regular line of assistance for research; Coordinator: Dulce Casarini – Unifesp; Investment: R$ 193,214.88