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PATHOLOGY Endothelial Cells Mount Genetic Response To Shifting Currents in the BloodFindings Provide Clues to New Heart-protecting Therapies More than a hundred years ago, researchers observed that atherosclerotic plaques had a habit of arising where the rush of blood is diverted from a linear path—for example, where the carotid artery forks and at other branch points and bends in the vasculature. At such points, blood flow can turn chaotic, creating eddies and other turbulent patterns. Many assumed that the heart-damaging plaques were wounds caused by the physical force of the miniature maelstroms as they beat against the thin layer of cells lining the artery walls.
Atherosclerotic plaques tend to form at branch points and curves in arteries (above, red patches). At such points, blood flow assumes a disturbed or turbulent pattern. Cells lining the artery are genetically responsive to these fluid mechanical disruptions, turning genes on and off. Endothelial cells translate fluid biomechanical stimuli into visible changes. When the cells are exposed to conditions of laminar flow (below, right) as opposed to steady flow (below, left), they undergo dramatic reorganization in their cytoskeleton as can be seen in the cells below, which were stained for the cytoskeletal protein actin. The actin gene is among those upregulated in cells experiencing laminar flow. Image adapted from original by Guillermo García-Cardeña. Photos courtesy of PNAS
That brute force scenario of plaque formation is being replaced by a more subtle and tantalizing picture, one in which endothelial cells play an unexpectedly active role. These cells, Michael Gimbrone and his colleagues have recently discovered, are exquisitely sensitive to disruptions in blood flow and steer a different genetic course depending on outside biomechanical conditions—captains of their fate rather than battered victims of external circumstances. Using a device that recreates the flow of blood outside the body, Guillermo García-Cardeña, Gimbrone, and colleagues monitored the genetic behavior of endothelial cells under two biomechanically different conditions—laminar linear flow and turbulent flow. Tracking more than 11,000 genes, they found that the cells exhibit remarkably different patterns of gene expression depending on the type of current, and hence the fluid mechanical forces, they experience. The Brigham and Women's Hospital researchers have even glimpsed these cells in the act of executing those genetic commands. For example, endothelial cells experiencing laminar flow were observed to ratchet up the expression of genes for three cytoskeletal proteins. Using a confocal fluorescent microscope, the scientists were able to observe the proteins being recruited to the internal scaffolding in those cells. Their findings appear in the April 10 Proceedings of the National Academy of Sciences. "It suggests that the cells are translating fluid biomechanical stimuli into real phenotypic changes," said García-Cardeña, HMS research fellow in pathology. They are also managing a torrent of other signals—for example, messages from blood-borne molecules such as hormones as well as from the extracellular matrix. "The sense we're getting is that every one of these endothelial cells is capable of integrating the various stimuli in its immediate environment, some of which are humoral, brought by the blood or secreted by another cell; some of which are physical, like the attachment to a surface; and some of which are these very dynamic fluid mechanical stimuli," said Gimbrone, the Elsie T. Friedman professor of pathology. "It integrates all of those and reads it out into a functional phenotype. What this study is opening for us is the ability to see that functional phenotype."
A team including (from left) Guillermo García-Cardeña, Keith Anderson, Brett Blackman, Jason Comander (seated), and Michael Gimbrone has been looking at how the physical forces of blood flow influence the activity of endothelial cells and development of atherosclerotic plaques. Photo by Steve Gilbert Understanding how endothelial cells respond to the fluid mechanical forces exerted by blood—which genes are upregulated and downregulated—could pave the way to new heart-protecting therapies. For example, if researchers knew which plaque-promoting genes are activated by conditions of turbulence, such as those occurring at the bifurcation in the carotid artery, they might find ways to turn those genes off and prevent the plaques from forming. "We're getting to a different era in pharmacogenetics, where we're going to reach in and turn on and off genes selectively, but that only works if you have a complete understanding of the normal mechanism of turning on and off genes," said Gimbrone. Flow GeneratorRescuing plaque-prone endothelial cells was a distant goal 20 years ago when Gimbrone set out to understand why plaques occur in the area of the carotid artery bifurcation. The more immediate challenge was to find a way to recreate in an experimental setting the fluid mechanical forces occurring at the bifurcation and elsewhere in the vasculature—a task solved by MIT engineer Forbes Dewey. During a sabbatical year in Gimbrone's lab, Dewey collaborated with Gimbrone to develop an apparatus—essentially a cone rotating over a petri dish layered with cultured endothelial cells. As the cone rotated through the culture medium, it recreated the fluid movments of the laminar and turbulent flows.Using the machine, which underwent further refinement, Gimbrone and his colleagues demonstrated that by varying the flow, they could change an endothelial cell's shape. Over the next few years, they looked for genetic changes that might be responsible for the shape alterations. They came up with a lengthy list, but it required a laborious process of monitoring genes one by one. "That's when Guillermo entered this evolving field and brought it to another level," Gimbrone said. García-Cardeña wanted to look at thousands of genes at once to compare gene expression under conditions of laminar versus turbulent shear stress. "Our question was, is the endothelial cell a fine enough machine to sense the difference?" García-Cardeña said. Acid TestUsing transcriptional profiling, which matches cDNA arrays to expressed mRNAs, the researchers monitored the activity of 11,397 genes after a 24-hour exposure to the two flow conditions. To compare genetic expression patterns, they utilized a Web-based program developed by Jason Comander, an MD–PhD student at HMS, and colleagues. The program, called ARGUS, revealed significant differences. More than 100 genes were expressed differently under the two conditions."We can tell exactly which genes are being up- or down-regulated," said García-Cardeña. Three genes that were upregulated under laminar stress were the genes for the cytoskeletal proteins myosin, actin, and plectin—proteins that became visible when the cells were exposed to laminar shear conditions (see figure). "It was very satisfying to see at a very specific site of the cell above the nucleus, after 24 hours of flow, this very nice formation of stress fibers," said García-Cardeña. Genes involved in cell cycle control, including Cyclin B1 and D1 were also expressed differently by endothelial cells under the two conditions. Sure enough, the cells displayed very different proliferative behavior—those under laminar shear stress replicated at a much lower rate than those experiencing turbulence. What is especially gratifying about the observation, say the researchers, is that more than 20 years ago, people found that endothelial cells in areas of carotid bifurcation had a very high proliferative index. "It suggests that the links we're trying to make are revealing mechanistic insights," García-Cardeña said. Now that the human genome has been sequenced, the researchers hope to add to their database of 11,397 genes and see how the remaining genes behave under different fluid mechanical conditions. To speed that effort, they have made their database and data analysis program available on a website (www.vessels.bwh.harvard.edu), which they hope will be added to by researchers in other labs. "It's a renewable resource to which more and more information will be added that will speak to what the genotype and phenotype of an endothelial cell are under a variety of conditions," Gimbrone said. "It will be limited only by our ability to imagine appropriate experimental designs." He hopes, ultimately, that the project fulfills the promise of the human genome project. "Once you get done understanding all the stars in the nighttime sky, you want to understand what the constellations are that are of biological relevance. That's what we're attempting," he said. —Misia Landau |
