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Study reveals how blood flow force prevents clogged arteries

August 26, 2008

Smoking and high cholesterol don't effect all blood vessels equally

Machines on cell surfaces, mechanical and lifeless as bed springs, protect blood vessels by responding to blood flow force, according to research published today in the Journal of Cell Biology. By sensing and reacting to force, such machines interfere with inflammatory pathways central to atherosclerosis, the leading cause of heart attack and stroke, the authors said. The next set of studies, already underway, seeks to "tweak" the process with the goal of designing a new class of therapies.




In recent years, researchers determined that mechanical force alone can kick off biochemical reactions which contribute to both health and disease. When applied to human bone during weightlifting, for instance, force can trigger biochemical reactions that thicken bone. In the current example, blood flow creates frictional force, called "shear stress," as it moves along the cells that line blood vessel walls. Secondly, each time the heart pumps, related pressure changes create a second, simultaneous force that stretches vessel walls.

The fast, steady blood flow (high frictional force) and the relatively straightforward stretch patterns seen in the straight portions of blood vessels have been shown to somehow protect those areas against atherosclerosis. At the many points where one vessel branches into two, however, blood flow is disturbed and slowed, like a river hitting a sharp bend. Shear stress is reduced with unsteady flow, as is stretching force. Going into the current study, the presumption was that the two forces, shear stress and stretch, created by steady blood flow trigger protective biochemical signals, and that those signals are interrupted where vessels branch. Which proteins were involved, and which biochemical signals sent in response to mechanical force, remained unclear.

"In a longstanding mystery, smoking, high cholesterol and lack of exercise should drive disease to develop evenly throughout all blood vessels, but in reality atherosclerotic lesions tend to cluster where vessels branch," said Keigi Fujiwara, Ph.D., professor of Medicine within the Aab Cardiovascular Research Institute (CVRI) at the University of Rochester Medical Center. "Our study provides new insight into why, and suggests how the protection afforded by the force of steady flow and simple stretch might be strengthened with drugs."

Life is Complicated

The study of shear stress, and the effect of mechanical force in general, in cells has been hampered in the past because it can only be studied in live tissues. Where else will blood create frictional force as flows over live cells? In this scenario, however, it is difficult to separate out signals transmitted in response to force from the flurry of biochemical signals happening all around them, researchers said. Thus, the current study examined the effects of stretch instead. The rationale: stretch-related mechanisms are simpler and, if purely activated by force, should occur in a lifeless system (zero biochemical signals). In addition, the same proteins respond to many kinds of force in cells lining blood vessels, so studying stretch should provide insight into shear stress.

To study stretch, the team built an elastic silicon membrane and coated it with collagen, the protein framework that cells stick to as they form tissues. Researcher next grew a layer of endothelial cells that line blood vessels on the collagen until they formed a uniform layer with each cell touching its neighbor. At the last minute, the team used detergent to remove the plasma membrane that would usually surround the endothelial cells, leaving behind protein skeletons that could no longer grow nor receive biochemical signals. In short, the remaining "cells" were no longer technically alive. Researchers then applied force to the model, stretching the silicon membrane, which stretched the collagen layer, which stretched the cells grown on it and the links between them by as much as 25 percent.

One of the candidates for a hypothetical molecule that should convert physical force into biochemical messages was a protein called platelet endothelial cell adhesion on molecule-1 (PECAM-1). When researchers stretched the model, PECAM-1 was indeed phosphorylated at two key points in its amino acid chain. Phosphorylation, the attachment of a phosphate group to a protein chain, is used by cells to switch on biochemical signals. In the study, PECAM-1 phoshorylation occurred in a lifeless model purely as a result of mechanical stretching force, which presumably pushed PECAM-1 and a nearby, built-in enzyme close enough to each other for phosphorylation to occur.

In addition, the team, via a series of clever eliminations, also identified from a library of 244 candidates the single enzyme, called Fyn, which phosphorylates PECAM-1 in response to force. Only when production of Fyn was greatly reduced, removing it from the stretch model, did PECAM-1 phosphorylation no longer respond to stretching force. When Fyn expression was silenced in live endothelial cells in a related study, PECAM-1 phosphorylation no longer responded to shear stress either.

The medical relevance of the study comes from a hypothesis that the phoshorylation of PECAM-1 enables it to activate, via signaling partners, the extracellular signal-regulated kinase (ERK) pathway. Past Aab CVRI studies suggest that ERK activation inhibits the expression of pro-inflammatory genes in endothelial cells. Experts once believed that atherosclerosis developed when too much cholesterol clogged arteries with fatty deposits in vessel walls called plaques. Today most agree that the reaction of the body's immune system to fatty build-up, as much as the build-up itself, creates risk for heart attack (complete blockage of an artery). Immune cells traveling with the blood mistake fatty deposits for intruders, akin to bacteria, and attack. Resultant inflammation makes plaques more likely to cut off flow.

The work was supported in part by grants from the National Institutes of Health and the American Heart Association.

"Obviously, we should all be exercising to get our hearts pumping fast, which increases blood flow force through our vessels," said Fujiwara. "Beyond that, genetic engineers may in the future design gene therapy that delivers genes only into cells at blood vessel branch points to increase PECAM-1 signaling. Alternatively, drug designers might create super-active Fyn, amplifying the signaling effects of PECAM-1 phosphorylation to protect vulnerable areas of blood vessels from inflammation."

University of Rochester Medical Center





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