Researchers at the University of Wisconsin–Madison have uncovered new evidence in a decades-old genetic mystery, discovering how multiple genetic variations in a long-mysterious region of the human genome can leave people at higher risk of cardiovascular disease.
Thanks to Genome Wide Association Studies (GWAS), scientists have long known that genetic variations in the 9p21.3 locus, a portion of the human chromosome 9, contribute to the increased risk of developing coronary artery disease (CAD), also known as atherosclerosis of the coronary arteries. This locus presents in the human population as “risk” (increasing risk for CAD) or “non-risk” (protective). However, it was not well understood why, or how, almost a hundred genetic variations in this region lead to an increased risk for CAD. Shrouded in mystery for nearly 20 years, researchers can now confirm that variations in 9p21.3 affect vascular smooth muscle cells biology. Smooth muscle cells are one of the main components of the artery wall. The risk version of the 9p21.3 locus induces these important cells to acquire features of bone or cartilage cells, leading to the hardening, or calcification of vascular tissue within the heart.
Understanding how 9p21.3 functions to increase risk for CAD is the first step in potentially preventing the damaging effects of these variations and, perhaps one day, offering those with this genetic predisposition therapeutic strategies to block this pathway.
This long awaited breakthrough, published in Arteriosclerosis, Thrombosis, and Vascular Biology, was discovered through the use of Induced pluripotent stem cells (iPSCs), which are adult living donor cells that are reprogrammed into embryonic-like pluripotent cells. These pluripotent stem cells have the remarkable ability to become nearly any cell in the body. In this study, researchers programmed iPSCs into vascular cells, a methodology that finally allowed scientists to unlock the mystery of the 9p21.3 CAD locus.
“For multiple reasons, this region has been very hard to study,” says Valentina Lo Sardo, the lead researcher on this project, an assistant professor of Cellular and Regenerative Biology, and a Stem Cell and Regenerative Medicine Center member. “This region is only present in the genome of humans and a few non-human primates, so you cannot make an animal model that faithfully recapitulates the genetics of the human species. What we used instead is something that is very much human specific, which is iPSC. The fact that we can make iPSC from any living donors and from donors that have a specific genotype at this particular region is so important for us. I think it’s an extremely powerful tool.”
Lo Sardo’s lab differentiated the iPSCs into vascular smooth muscle cells and studied how the cells of donors carrying the risk variants at the 9p21.3 locus behaved vs those of donors who carry the protective variants. They discovered that smooth muscle cells carrying the risk version of 9p21.3 behaved differently, shifting in a cell state to resemble the features of cells seen in cartilage and bone.
“This was quite remarkable,” says Lo Sardo. “What we found is that the cells change their transcriptional makeup, and they are in between smooth muscle and these osteochondrogenic [or bone-like] cells. We discovered that these cells are more prone to calcification. And this is very important, because CAD is basically atherosclerosis [a hardening of the arteries] and calcification of the arteries is one of the hallmarks of this type of pathology.” Lo Sardo’s group used genome editing to remove the risk version of 9p21.3 and were able to see that the cells revert to a “healthy” status, showing that “disarming” the risk effect of this genomic region may be valuable to reenabling smooth muscle cells to exert their function.
Lo Sardo notes that CAD is a complex disease influenced by multiple factors including environmental factors, lifestyle choices, and genetics, but in the case of 9p21.3, she discovered that variations in this genomic region can promote calcification without the influence of any outside factors.
“By using iSPCs, we have cells in vitro that are not in a tissue and don’t have any other surrounding cell types. They are not subjected to blood flow or other inputs because they are just in a dish. So, just on their own, they have this capacity to turn into calcified cells. This is truly remarkable, because other studies have observed that people that carry the risk version of 9p21.3 are more prone to have calcified atherosclerotic plaques,” says Lo Sardo. “For me it’s amazing that our model of genetic risk in a dish is so close in resembling what happens in the native tissue”.
Lo Sardo shared that they also discovered that as the cells calcify more, they lose their ability to do vascular remodeling. For example, when you have a vascular injury, healthy vascular smooth muscle cells will migrate and just cover the wound, but with the risk version of 9p21.3 the cells are not able to actually migrate and therefore heal.
“Smooth muscle cells also migrate during the formation of atherosclerotic plaque, and believe it or not, this is actually good,” says Lo Sardo. “Through migration these cells can make almost a blanket above the plaque and keep it from rupturing and causing damage. We think that by reducing their migration capacity, smooth muscle cells with the risk 9p21.3 could result in more unstable, deleterious plaques. So, taken together, you have cells that are not able to do vascular remodeling, not able to maintain a healthy vasculature, are prone to calcify and not promoting stable atherosclerotic plaques. I guess this is overall not an ideal mix.”
This discovery is a significant step towards understanding the role of this genomic region as well as confirmation that iPSCs offer an extremely accurate way to study complex disease and complex human genetics. This research also has an impact on the cardiovascular genomics field and has paved the way for dissecting other regions of the genome that are still “function-orphan”. The human genome carries thousands of variants that increase the risk of developing CAD, but they are extremely difficult to study, because most of the time, like the 9p21.3, these regions are “gene deserts”, meaning they do not contain genes.
“This is challenging, because we don’t know how they affect our body and the cells of the blood vessel in particular,” says Lo Sardo. “Many of these regions in the human genome are also not conserved in other animals, so the “cardiovascular risk problem”, is very much something hard to model in mice, or other model organisms. We think this is the key to unravel and discover new biology that is specific to humans, new mechanism of CAD that can only be explained by using model that recapitulate the complex genetics of humans”.
Lo Sardo says she sees this project expanding in many ways, as it provides the key to map cardiovascular genetic risk and help advance precision medicine.
“People often ask, ‘how similar are the iPSC cells to what you can find in the human body?’’ says Lo Sardo. “So, we did this analysis, and we matched our smooth muscle cells derived in vitro with smooth muscle cells from human heart arteries, and they are remarkably similar. This was an incredible result, because it makes us confident that whatever we find, it’s actually relevant, that we can faithfully recapitulate the biology of smooth muscle cells and their remarkable capacity to adapt to different stimuli just in a dish!”
Clint Miller, PhD, an associate professor in the Department of Genome Sciences, and Department of Biochemistry and Molecular Genetics at the University of Virginia, a leading expert in cardiovascular genomics, assisted with the human genomics and native tissue portion of the research, helping to confirm the accuracy of the iPSC derived smooth muscle cells. Lo Sardo is also grateful to colleagues across campus who assisted with this research, including the support she received from members of the Stem Cell and Regenerative Medicine Center. Of significant note, is the work of Elsa Salido, a genetics PhD candidate in Lo Sardo’s lab who is the first author on this paper.
“This research is the work of my first graduate student,” says Lo Sardo. “It was the first project in the lab, and my first graduate student was actually able to accomplish most of this in her first three years. She has been extremely productive, and it is extremely rewarding seeing a young scientist accomplishing so much in such a short time. Her passion for science and genetics is something special. I’m very proud and grateful to have her in my lab.”
Salido completed genetics research as an undergraduate with a focus on plants, but this was her first exposure to human genetic research. She says that the ability to take someone’s blood and make stem cells from it just blew her away, both in terms of the fact that it’s possible, and in the array of potential research and therapeutic applications that could come as a result of this methodology.
“I think the strategy we’re taking with 9p21.3, stepping away from observational studies to put together this model where we can do gene editing and really tightly control the variables, is laborious, but invaluable to untangling these constellations of disease-causing elements,” says Salido. “It allows us to move beyond correlation and draw a conclusive cause-effect relationship between a genetic variant and specific, disease-relevant functional impacts, showing not just that this genetic variants increases risk of disease, but how. It’s so exciting to have linked 9p21.3 to this phenomenon that we see during coronary artery disease. Finding a real, concrete, cause-effect relationship between 9p21.3 and a phenotype we know is disease-relevant feels like discovering a major clue in a decade-old mystery!”
A shortened version of this story is also available on wisc.edu.