Dr. Lisa Fortier
Arthritis is costly to the equine industry in loss of athletic performance and expenses incurred for treatment. Although there are many causes of arthritis, aging is the greatest risk factor associated with its occurrence. Cartilage in horses is considered fully mature and therefore aging at about the age of 2 years, so aging studies apply to all athletic horse populations. Articular cartilage has a limited capacity for self-regeneration in part due to its sparse cellularity. This is magnified during aging because as tissue ages, cells reach senescence at which point they can no longer divide to replace injured/dead cells and maintain normal tissue homeostasis. During aging, not only are chondrocytes rendered unable to divide, but they lose their ability to respond to growth factors, and the type of proteins that are synthesized and excreted into the matrix are altered. Combined, these changes render aged cartilage susceptible to the development of arthritis.
The inability of aged cells to divide is termed replicative senescence which is initiated by shortening of telomeres. The enzyme telomerase is responsible for maintaining long telomeres within cells thereby permitting cellular replication. Although telomerase activity has been found to be low or absent in somatic (non-reproductive system) cells, ectopic expression of telomerase has been shown to extend the lifespan of normal human cells, even those that had reached 80% of their lifespan. Two members of the Rho-subfamily of GTPase proteins (Cdc42 and Rac) have been shown to increase telomerase activity in somatic cells, and our laboratory previously determined that Cdc42 and Rac are important in maintaining cartilage homeostasis during aging. Understanding senescence and the role of Rho-sbufamily GTPase proteins in chondrocytes during aging might provide insight into novel mechanisms to enhance the regenerative capacity of cartilage.
Our hypothesis for this study is that telomerase activity decreases with increasing age in articular chondrocytes, and that telomerase levels can be restored by increasing expression of Rho-subfamily GTPases. In the first Specific Aim, we will determine how telomerase activity alters with age in equine chondrocytes. The second Specific Aim is designed to determine the effect of activation of the Rho-subfamily GTPases (Cdc42, Rac, and Rho) on telomerase activity. We will use a commercially available telomerase kit to quantitatively measure telomerase activity in chondrocytes from horses aged 1 day to 28 years. We will then introduce GTPases (Cdc42, Rac, and Rho) into chondrocyte populations that have lost telomerase activity in order to determine if verexpression of the Rho-subfamily GTPases can restore telomerase activity in equine articular chondrocytes. We we also determine the functional significance of increased telomerase on the ability of chondrocytes to divide, respond to growth factors, and synthesize cartilage matrix proteins in a similar pattern to young chondrocytes.
We anticipate that increasing telomerase in chondrocytes will increase their ability to replicate thereby increasing the number of cells contributing to regeneration of damaged cartilage matrix. In addition, increased telomerase should minimize biochemical changes in synthesis of the cartilage matrix that are hallmark signs of aging and render aged articular cartilage susceptible to the development of arthritis.
Successful completion of this project should elucidate novel information regarding senescence in aging articular cartilage. This knowledge will be important as we move forward in generating techniques to restore aged, damaged articular cartilage, and for the treatment of arthritis.