Regenerative Healing in Mice
In the process of carrying out an autoimmunity experiment, the Heber-Katz research team noted that in the MRL strain of mice, punched ear holes used for long term identification rapidly closed without any sign of scarring. Besides lack of scarring when the ear hole closed, a blastema formed and new hair follicles and cartilage grew back, processes not generally seen in adult mammals though thought to be part of a regenerative process seen in amphibians.
The laboratory has been actively pursuing the identification of genes involved in this trait along with the mechanisms that allow this healing to take place. They found that the matrix metalloproteinases are upregulated early after wounding and just prior to blastema formation along with enhanced remodeling responses and breakdown of the basement membrane.
They showed that components of the inflammatory response are increased, that dedifferentiation markers like Nanog are expressed, and that an embryonic-like metabolic state is used. And this laboratory has shown that a single gene, p21CIP/WAF1, when deleted from the animal, can produce a regenerative mouse. Most recently, they have been working on the profound effects of dietary fat on regeneration, identifying genes like the molecule Pref-1 (or preadipocyte factor) which is upregulated late after wounding and just as the blastema is beginning to redifferentiate into mature cells.
These studies are currently being applied to a model of breast cancer .
Recent Scientific Advances
1. The role of inflammation on regeneration
The role of inflammatory responses in the MRL mouse regenerative response was recognized early due to the fact that the MRL mouse displays autoimmune sequelae for multiple diseases. The laboratory found that inflammatory cells enhanced the regenerative response, were involved with potent tissue remodeling responses and enhanced MMP responses. Current studies involve the examination of TLRs as well as HIF1a in the induction of regeneration and is being investigated in collaboration with Dr. P. Messersmith (Northwestern University). These studies have been recently funded by a Eureka grant and a separate RO1 from the NIDCR, NIH.
2. Metabolism and regeneration
Analysis of the metabolic state of the adult MRL mouse has shown that these mice use aerobic glycolysis (an embryonic metabolism) as a primary source of energy and as the resting metabolic state in contrast to oxidative phosphorylation, traditionally seen in adult mammals. Aerobic glycolysis is also the typical metabolic state used by tumor cells, stem cells, and importantly, of regenerating amphibians, supporting stem cell renewal and the de-differentiated state. These studies are continuing to determine the exact cause of this metabolic state and if conversion to this state will lead to a regenerative healing response. Studies have been carried out in collaboration with Dr. R. Naviaux (UCSD).
3. Genetics of Regeneration
A genetic analysis of this healing response has been carried out since 1996, using both microsatellite and then SNP mapping to identify the genetic loci involved. Most recently, fine mapping studies of the healing trait to narrow genetic intervals to less than 1cM has been advanced using recombinant inbred strains and F34 advanced intercross lines, as well as through gene expression data. These studies are continuing in collaboration with Drs. E. Blankenhorn (Drexel Univ) and J. Cheverud (Wash Univ).
4. Cell cycle Regulation and Regeneration
Cell cycle bias appears to be key to the regenerative response. The Heber-Katz Laboratory has shown that p21CIP/WAF1 protein is down in MRL mouse cells, DNA damage is up, and a G2M pause is seen in rapidly dividing MRL cells in culture. This work has shown that a p21 KO non-regenerative mouse replicates the healing seen in the MRL and represents a key mechanistic piece of the regeneration puzzle in mammals. Further studies have indicated that p53, an activator of p21, is not involved but that the TGFb pathway which also can activate p53, is. These studies were done in collaboration with the P. Lieberman laboratory at the Wistar Institute.
5. Heart Regeneration
Besides ear hole closure, he Heber-Katz research group examined the possibility of regeneration in heart tissue of the MRL mouse. A cryoinjury made to to the right ventricle of the MRL heart led to nearly complete replacement of injured tissue with new dividing cardiomyocytes and functional recovery as determined by echocardiography, and the appearance of stem cell markers, This work is continuing by examination of various mutant mice.
6. Spinal Cord Regeneration
Studies were also carried out to determine if the MRL could regenerate CNS tissue including the spinal cord and optic nerve. . The Heber-Katz laboratory found that the MRL mouse showed unusual healing and axonal growth through injury sites. They also identified scar tissue as a key blocking element in axonal re-growth. Thus, spinal cord transection where fibroblastic infiltrates were kept to a minimum resulted in recovery of function or coordinated walking within 3 weeks. They identified one molecule, apolipoprotein E, along with its receptors, which appear to be upregulated during a regenerative response.