For over half a century, biologists have been culturing cells in vitro, or in a dish, separate from the organisms they come from. Many of the natural features of a cell can be recapitulated in culture; however, our inability to specifically recreate their native environment is a significant hurdle in understanding countless cellular processes and associated diseases. Within an organism, cells take cues from surrounding tissues and cells, and from the kind of surface on which they exist. Such signals instruct the cells when they should divide, or differentiate into a new cell type, when they should move, and even when they should die. Creating a setting that is as close as possible to the natural environment of a cell is essential for truly understanding how cells work.
For Dr. Huan Sharon Wang, a recent PhD graduate from the department of Molecular, Cellular, and Developmental Biology (MCDB) at CU-Boulder, identifying and recapitulating the surface on which cells grow is a fundamental goal. She is interested in understanding cardiac valve diseases, specifically calcific aortic stenosis. In this disease, the valves of the aorta stiffen and calcify. This stiffness, known as fibrosis, can cause cardiac valves to become as stiff as bone, narrowing the amount of space for blood to flow through and impeding the heart’s ability to function normally.
Although the cause of calcific aortic stenosis is not entirely understood, it is thought that fibroblasts may contribute to disease progression. Fibroblasts are a type of cell residing in the valve. They are known as mechano-sensing cells, meaning that they can sense and respond to the stiffness of the surface on which they are growing.
In diseases such as calcific aortic stenosis, fibroblasts incorrectly differentiate into myofibroblasts. Myofibroblasts are not found in healthy hearts, and are normally present only after cardiac injury. They function in cardiac immune response and also produce stiffening proteins such as collagen. An increase in their number can contribute to stiffening the valve matrix.
Though it has been shown by others that matrix stiffness affects cardiac fibroblasts, Sharon wanted to understand how these cells molecularly sense their environment, and how that cue gets translated to a change toward myofibroblasts. As a graduate student being advised by both Dr. Leslie Leinwand in MCDB and Dr. Kristi Anseth in Chemical and Biochemical Engineering, Sharon had the expertise and perspectives from two diverse labs to guide her. This unique mentoring situation gave her the opportunity to explore the interdisciplinary area of tissue engineering.In response to this stiffened valve matrix, additional fibroblasts continue to differentiate into myofibroblasts, confounding the problem.
In order to get a better grasp on the cause of calcific aortic stenosis, Sharon studied fibroblast differentiation in response to both biochemical and biophysical factors, making use of special substrates called hydrogels to culture the cells. As opposed to a traditional cell culture dish made of hard plastic, hydrogels provide a surface of adjustable stiffness, and can offer a much softer area for cells to grow on. Therefore, using hydrogels allowed Sharon to provide an in vitro setting for her cells that was much closer to their natural environment than traditional cell culture techniques would allow.
Sharon recently published a paper in the Proceedings of the National Academy of Sciences (PNAS) entitled “Hydrogels preserve native phenotypes of valvular fibroblasts through an elasticity-related PI3K/Akt pathway.” For the publication, Sharon cultured a type of cardiac fibroblasts, called valvular interstitial cells (VICs), on hydrogels of various stiffnesses to determine their response. She noticed that when her cells were grown on either traditional cell culture plastic or a very stiff hydrogel, they lost their identity as fibroblasts and differentiated into myofibroblasts, similar to what happens in calcific aortic stenosis.
Sharon also discovered that there were some changes to the molecular behavior of the cells grown on hard surfaces. Notably, signaling through a pathway known as PI3K/AKT was strongly increased. This pathway has long been of interest to cancer researchers, because it is often abnormally up-regulated in cancerous cells. In cancer, its up-regulation decreases programmed cell death, or apoptosis, and contributes to the ability of malignant cells to grow uncontrollably. More relevant to Sharon’s project, however, was the fact that the PI3K/AKT pathway had previously been shown to be involved in cardiac hypertrophy, a condition in which the heart abnormally increases in size, reducing its ability to pump blood through the body.
A single signaling pathway can control many different biological processes in different contexts; although the PI3K/AKT pathway had previously been shown to function in cardiac tissue, no one had yet shown whether it could be contributing to diseases like calcific aortic stenosis. Using her hydrogel-based cell culture system, Sharon had an opportunity to explore whether the PI3K/AKT pathway may be involved in fibroblast differentiation.
If Sharon blocked activation of the PI3K/AKT pathway in her cells while they were being cultured on a stiff surface, they no longer became myofibroblasts. Conversely, if she simulated over-expression of the pathway in cells being cultured on a soft hydrogel, they started to differentiate into myofibroblasts, despite being on a soft surface. Therefore, it appears that cardiac fibroblasts sense the stiffness of their environment through the PI3K/AKT pathway. Sharon and her mentors also believe that the PI3K/AKT pathway could be an important mechanism that other mechano-sensing cells use to sense and respond to their environment.
Excitingly, the identification of the PI3K/AKT pathway as a mediator between the cell and its environment provides a possible therapeutic target for treating fibrotic diseases. The results of Sharon’s research indicate that repressing activation of the PI3K/AKT pathway in cardiac fibroblasts could prevent them from mistakenly differentiating into myofibroblasts.
Sharon is hopeful that this knowledge will lead to the development of treatments for calcific aortic stenosis, as there are currently no drug-based therapies for the disease. Now that this study has established a model system for studying cardiac fibroblast differentiation, researchers will be able to determine whether any drugs currently approved by the FDA are able to slow or stop inappropriate differentiation. In addition, this research opens the door for new drugs to be designed that specifically target the PI3K/AKT pathway.
On a more broad scale, Sharon’s research promotes our general understanding of the dynamic and critical interactions between cells and their matrix environment. This could potentially lead to better design for scaffold development for tissue regeneration. Organ transplantation, the existing technique for restoring organ function, has a high risk of failure, and requires the patient to continually take immuno-suppressants to avoid rejection. If scientists are able to more accurately recreate the surfaces on which organs develop, they may be able to regenerate tissue from the actual patient, an exciting possibility for the future of medicine.
By Jaimee Hoefert