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Reconnected heart

Heart Institute team identifies possible genetic marker in the durability of bypass surgery

MARCOS GARUTIIn the 10th floor lab of InCor, the Heart Institute at the University of São Paulo, from where one gets a wonderful view of the São Paulo State capital, physician José Eduardo Krieger’s team is starting to unveil the source of a phenomenon that limits to about ten years the durability of some of the saphenous vein bypasses: the blockage, even if partial, of the grafts of segments of this vein from the leg, used to re-establish the flow of blood to the heart, a flow diminished by the accumulation of lipid plaques within the arteries that irrigate the organ. In a series of experiments with rats and human blood vessels, the InCor group has been discovering how physical factors change the programming of the cells of the grafted vein, when forced to work as an artery. This reprogramming may cause the vein to thicken too much and block the bypass a few years after the heart re-vascularization surgery.

This search has already resulted in the identification of several proteins involved in the thickening of the grafts, two of which are totally characteristic. “We believe that, with this type of research, we will get to one or more proteins that may be used as durability indicators for saphenous vein grafts, or as targets for increasing graft efficiency,” states Krieger. In a few years he hopes to produce a genetic test capable of forecasting whether the candidate for surgery has the tendency to develop saphenous vein occlusion and to develop treatments to minimize the problem. “We’re working towards discovering when and how to intervene,” he says.

Created in 1967 by Argentine physician René Favaloro, the saphenous vein bypass revolutionized heart surgery. In a long operation in which a 30-cm long cut was made in the patient’s chest and in which the ribs were pushed apart, Favaloro connected one end of a saphenous vein segment almost one hand-span long to the aorta artery and the other end to the part of the heart that was short of blood. Thus, he enabled the blood to go around the blockage and feed the body’s strongest and most resistant muscle once again. The heart contracts on average 100 thousand times a day, pumping nutrients and oxygen to all the body’s tissues. In the last 42 years, this procedure was improved and is repeated daily all over the world, prolonging the lives of millions – it is estimated that every year, 47 thousand heart re-vascularization surgeries are conducted in Brazil and another 450 thousand in the United States.

This solution, however, is not perfect and often one pays highly for transforming a vein, a blood vessel that specializes in carrying small volumes of blood under low pressure, to operate as an artery, with a flow that is about 10 times greater and pressure that is more than 20 times higher. The change in the vein’s operating conditions causes an excess thickening of its innermost cell layer. Consequently, the plaques of fat that generally take four to five decades to jeopardize the flow of blood along the heart’s arteries (the coronaries) form far faster and obstruct some 10 percent of the saphenous vein bypasses in just 10 years, requiring  new surgery to be undertaken. This proportion of blockages, which back in the early 1990’s was as high as 50 percent, but that has dropped thanks to changes in diet and cholesterol-lowering drugs, is still considered high.

“Though in most patients one can use arteries, such as the mammary arteries, to re-vascularize the heart muscle, the saphenous vein is an alternative often used because it involves a less invasive procedure, besides being a long blood vessel, which allows one to take several grafts,” explains Luís Alberto Dallan, an InCor surgeon that collaborates with Krieger’s team. “If we solve the saphenous vein bypass occlusion problem, we’ll solve the main issue of cardiovascular surgery.”

MARCOS GARUTIIt was almost by chance that the InCor group started looking into the thickening of saphenous vein bypasses five years ago. At the time, Ayumi Miyakawa, a medical researcher, worked with Krieger in the identification of genes activated within the cells of the innermost layer of the blood vessels – the endothelium – by the flow of blood. Just as river water licks the river banks as if it wanted to drag them along with the current, the blood flow tends to take along with it the cells on the inside of veins and arteries. This physical force, known as drag force, activates the system of the endothelium cells and modifies the production of a protein that controls the functioning of the blood vessel and blood pressure, the converting enzyme angiotensin. At the Genetics and Molecular Cardiology Laboratory, Ayumi had a device that simulated the drag, but she was dissatisfied with it. It was still necessary to somehow represent a second force to act upon blood vessel cells: the pressure of the blood against the walls of veins and arteries, which causes them to dilate every time the heart pulses. Ayumi asked the InCor bioengineering team to help her develop equipment capable of reproducing the two forces both independently and simultaneously. Once this device was ready, she realized that she could test saphenous vein grafts under similar conditions to those they would experience once grafted onto a heart.

As soon as the surgeon finishes the suture and releases the flow of blood, the piece of vein that now starts bringing oxygen-rich blood from the aorta to the heart suffers a brutal impact. Consisting of three thin cell layers, the vein, which previously carried blood rich in carbon dioxide and withstood pressures of 5 to 20 mm of mercury (mmHg), now starts working under pressure 25 times greater (some 120 mmHg),  than if the person who got the graft did not have high blood pressure. It was already a known fact that, under the new circumstances, the vein becomes thicker after some time. What was not known was which physical factors and which genes triggered this transformation that, if excessive, can damage and hinder the functioning of the bypass.

For four days, Ayumi cultivated 2 centimeter-long segments of the saphenous veins of people who had been operated on by Dallan under two flow and pressure systems: the vein one, in which 5 ml of a nutrient-rich compound and oxygen pass through the blood vessel every minute, at an average pressure of 5 mmHg, and the artery one, with a 50 ml/second flow and pressure of 80 mmHg. On the very first day, transformations already materialized. The cells of the vein treated like an artery started showing signs of apoptosis (programmed death), whereas the veins maintained under low pressure and a low flow did not die.

It was an interesting result, which gave rise to even more curiosity. What would happen to the saphenous vein bypass after some more time? Were the changes observed under these artificial conditions similar to what takes place within live beings?

In the face of these doubts, Ayumi and Krieger decided to develop and experiment to reflect better on what happens to saphenous veins grafted onto human hearts. As it is obviously very complicated to get samples of these veins once the bypass is in place and the person has left the surgical center, the researchers devised an operation on rats in which one of the carotid arteries, which carry blood from the heart to the head, is linked to a jugular vein, which drains the brain. Then, they monitored for as long as three months the changes in the jugulars subjected to the high flow and pressure that is typical in the arteries.

Carotid artery in a rat treated with genetically modified protein to penetrate the cells more easily (shiny dots on the image on the side) and the carotid in a rodent that was given the normal version of the same protein (on the left)

Carotid artery in a rat treated with genetically modified protein to penetrate the cells more easily (shiny dots on the image on the side) and the carotid in a rodent that was given the normal version of the same protein (on the left)

Between the first and the third day, the cell death rate in the rodents’ jugular linked to the carotid rose sharply. After the first week, however, it changed: the dead cells started to be replaced by muscle cells typical of blood vessels’ external layers. Moreover, the ring of elastic fibers found only in arteries, separating the first and the second layer of cells, also started to form. “These are indications that the veins are trying to adapt to the conditions of their new environment,” explains Ayumi.

The problem is that often this adaptation gets out of control and, instead of making the vein stronger, ends up blocking it. Under the microscope, Ayumi and Thaiz Borin found that the innermost layer of the jugular, generally consisting of a line of cells, had become a hundred times thicker, whereas the two most external layers, comprised of contracting muscle cells, had only doubled.

During this cell transformation period, the activity level of some genes drew Krieger’s and Ayumi’s attention. One of them contains the recipe for p21, one of the proteins that inhibit cell reproduction. One week after the rodents’ carotid had been connected to the jugular, the level of p21 in the vein cells dropped to almost half of what was normal and remained low until the end of the experiment – indicating that the environment change had deactivated the gene.

Meanwhile, the gene responsible for making the protein CRP3, generally active only in arteries, was activated immediately after the rodents’ jugular started functioning under high pressure, carrying large volumes of blood – this gene also appeared to be active in human saphenous veins made to function as arteries. On the very first day, CRP3, the protein that is part of the structure that gives cells their shape, the cytoskeleton, started being produced in the jugular at levels similar to those found in arteries. Its production dropped slightly after the first month, but remained high throughout the experiment. Apparently, it was the increase in pressure  and not the drag force that triggered the cellular mechanism and stimulated the production of this protein, reports Luciene Gastalho Campos, in an article published in April of this year in Cardiovascular Research. “We believe that producing this protein is important for the initial remodeling of the veins, which must withstand the hemodynamic system in the arteries, because it strengthens the cellular structure,” says Ayumi.

Another gene that seemed more active than normal, both in the rodents’ jugular linked to the carotid and in the blood vessels used in saphenous vein bypasses, is interleukin-1 beta, a protein in the body’s defense system that is produced during inflammation. Soon after surgery, the rats’ jugular cells started making about 20 times more interleukin-1 beta than when this vein faced low blood pressure and flow. “This environment fosters the development of atherosclerosis [an accumulation of plaques of fat on the blood vessels’ walls], which may be aggravated further when the rates of blood glucose and cholesterol are high,” says Ayumi. During the three months of the study, this protein’s levels remained five times higher than normal. Ayumi also measured the rate of interleukin-1 beta in the saphenous veins of people who had died from other causes during the first week of heart re-vascularization surgery or one to five years later. She found higher levels of this inflammation protein, especially in the first week after surgery.
Analyzing the genetic material of these individuals, she and Krieger discovered that the highest levels were produced by people who had some sort of alteration – the replacement of a single nitrogen base, the building blocks of the DNA molecular ? in the position -511 of the two copies of the interleukin gene. Those who had one normal gene and one altered gene made intermediate levels and people with two healthy copies of the gene made low levels of interleukin. “Because they modulate the amount of interleukin in human saphenous veins used as arteries, these variants may work as a genetic marker of how the saphenous vein bypass will evolve,” states Krieger.

Despite such promising results, years of work will still be needed until one gets a genetic test that can indicate the durability of a saphenous vein bypass. While the experiments on the viability of such a test advance, the InCor team is thinking of ways to control the functioning of the genes and to extend the benefits of the surgery that uses the saphenous vein to replace the function of diseased coronaries, a problem that kills 7.2 million people a year in the world.

They have already shown that at least one of them works. Using bacteria, Krieger’s team managed to make a recombined version of p27, a protein from the p21 family that can also bring cell reproduction to a halt. In an experiment conducted jointly with the teams of Ana Maria de Oliveira and Leandra Ramalho, from USP in Ribeirão Preto, the InCor group grafted a silicon ring around the carotids of rats that gradually released the altered protein to make it penetrate the cells more easily. Two weeks after surgery, cell multiplication was lower in the artery of the animals treated with recombined p27 than in those that were given an innocuous protein, according to the data presented in an article in Therapeutic Advances in Cardiovascular Disease. “This is an intervention strategy that proved to be capable of controlling cell reproduction only in the area in which it needs to be blocked,” Krieger tells us. The InCor team is also planning to use proteins in association with stents, a type of spring implanted within a blood vessel to keep it open, or with the balloons used in catheterization treatment to stimulate or inhibit cell reproduction, as necessary, and increase the durability of saphenous vein bypasses.

The projects
1.
Identification of genes with differentiated expression in saphenous vein submitted to the arterial regime (00/09485-7); Type Post-doctoral grant (Ayumi Miyakawa); Coordinator José Eduardo Krieger – InCor; Investment R$ 195,776.52 (FAPESP).
2. Differently expressed genes in model of arterialization of vein graft in rats (03/01828-0); Type Doctoral grant (Thaiz Ferraz Borin); Coordinator José Eduardo Krieger – InCor; Investment R$ 98,222.52 (FAPESP).
3. Study of arterial hypertension: molecular and functional characterization of the cardiovascular system (01/00009-0); Type Thematic Project; Coordinator Eduardo Moacyr Krieger – InCor; Investment R$ 6,111,202.31 (FAPESP).
4. Identification and characterization of genes linked to the arterialization of human vein grafts; Type Regular Research Awards; Coordinator José Eduardo Krieger – InCor; Investment R$ 50,000.00 (CNPq)

Scientific articles
CAMPOS, L.C. et al. Induction of CRP3/MLP expression during vein arterialization is dependent on stretch rather than shear stressCardiovascular Research. April 2009.
NEUKAMM, B. et al. Local TAT-p27Kip1 fusion protein inhibits cell proliferation in rat carotid arteriesTherapeutic Advances in Cardiovascular Disease. v. 2, p. 129-136. June 2008.
BORIN, T. F. et al. Apoptosis, cell proliferation and modulation of cyclin-dependent kinase inhibitor p21cip1 in vascular remodeling during vein arterialization in the rat. International Journal of Experimental Pathology. v. 90, p. 328-337. June 2009.

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