Periodic Reporting for period 2 - Grolaries (Cartilage progenitor cells for growth plate regeneration)
Okres sprawozdawczy: 2021-01-01 do 2021-12-31
Growth plate (GP) injuries result in growth arrest, formation of a “bony bar” and angular limb deformities in children. Novel therapeutic approaches directed towards prevention of bone formation and growth arrest have to integrate cellular grafts, biomaterials and growth factors with the ultimate goal of recapitulating the complex zonal organization of the growth plate. One endogenous source of cartilage progenitor cells is thought to be the resting zone of the growth plate. Until now, the lack of specific marker(s) for the resting zone restricted the examination of this population. Currently, the evaluation of potential strategies for growth and cartilage disorders can mainly be achieved in vivo, therefore we proposed to use genetic modified mice to characterize the GP population and to characterize its development. Mice are the most appropriate model to use for several reasons: 1) GP is a tissue hardly approachable in vitro 2) we aim to characterize the GP population, and GP dynamics during the process of growing for which there is no optimal in vitro assays 3) we chose PHEX hemizygous mice for the study of GP dynamics because it is a well stablished model of X-linked hypophosphatemia (XLH), which has being used for decades to study growth plate ossification. An other thing to keep in mind is that the standard treatments for XLH patients do not completely rescue the rickets and bone deformities. Serious side effects such as nephrocalcinosis and hyperparathyroidism have also been observed. Antagonizing FGF23 activity with antibodyes (Burosumab treatment) is a recent and very promising therapy. However, this treatment requires at least a monthly infusion, is very costly and alleviates symptoms in many patients but not all. Hence, it is of the utmost necessity to identify more affordable strategies for therapy. Dr. Santos’ Laboratory demonstrated that inhibiting the MAPK pathway (FGF23 downstream pathway) in PHEX mice partially rescues growth impairment by normalizing the GP structure, specifically in the hypertropy zone (Fuente et al., 2019). Nevertheless, it is not completely understood how FGF23 inhibition affects GP dynamics or how this is translated into a growth rescue and whether this treatment would be suitable for paediatric patients. Consequently, we will utilize the PHEX mice and FGF23 to 1) gain a better understanding of GP development and 2) look for alternative therapies to antagonize FGF23 activity.
(Aim 1-2)A manuscript has been published in Nature Communication under the title of ¨A FoxA2+ long-term stem cell population is necessary for growth plate cartilage regeneration after injury¨. We have demonstrated that FoxA2+ cells exhibit high clonogenicity and longevity. FoxA2+ cell number expand in response to trauma (Figure 1A and 1B) and the data suggest that these cells participate in the production of hyaline cartilage, allowing for successful cartilage regeneration . Furthermore, long-term ablation of FoxA2+ cells lead to premature GP hypertrophy and growth arrest. Unlike other cartilage regions such as articular cartilage, GP has the ability to regenerate. However, a better understanding of GP dynamics and the role of premature chondrocyte hypertrophy still need to be elucidated (Aim 2 and 3). Firstly, I am proposing to use the PHEX mice (an animal model for XLH already present in Dr. Carsten Wagner’s laboratory) to study the GP development and secondly, explore FGF23 inhibition strategies as an alternative therapy for growth cartilage disorders (Aim 2 and 3).
(Aim 2-3) The following activities regarding with the study of GP dynamics and developing of new drugs,were performed at the University of Zurich in colaboration with the University of Oviedo:
Mice experiments on XLH animal model with potential drugs for XLH treatment
Cell culture experiments using potential drugs for XLH treatment
Collection and analysis of data
Writing a manuscript to Bone Research: "Blocking FGF23 function with modified C-terminal FGF23 peptides as a novel therapy for X-linked hypophosphatemia rickets"
Preliminary data shows that C-terminal and N-terminal FGF23 peptides are able to improve tibia length and growth plate structure in PHEX mice compared with control PHEX mice and higher value than the already published one cFGF23, rescuing in part the GP aberrations acting predominantly on the hypertrophic zone (HZ) (see table bellow) (Figure 1C and 1D).Our data indicate that treatment with the Nmod and Cmod peptides partially reversed part of the pathological symptoms of XLH. The corrections of the profound GP morphological abnormalities are independent of the degree of hipophophatemia and indicate a direct relationship between FGF23 and cartilage diferenciation. Thereby these drugs emerged as a promising new therapy to reduce the risk of bone deformities in XLH patients and regulate cartialage diferenciation, critical factor in other more prevalent diseases such as osteoarthitis. We also hypothesis that in order to improve bone quality normalization of FGF23 is not enaought and a normalization in the Pi levels should be approached.
(Aim 2-3) The following activities regarding with the study of GP dynamics and developing of new drugs,were performed at the University of Zurich in colaboration with the University of Oviedo:
Mice experiments on XLH animal model with potential drugs for XLH treatment
Cell culture experiments using potential drugs for XLH treatment
Collection and analysis of data
Writing a manuscript to Bone Research: "Blocking FGF23 function with modified C-terminal FGF23 peptides as a novel therapy for X-linked hypophosphatemia rickets"
Preliminary data shows that C-terminal and N-terminal FGF23 peptides are able to improve tibia length and growth plate structure in PHEX mice compared with control PHEX mice and higher value than the already published one cFGF23, rescuing in part the GP aberrations acting predominantly on the hypertrophic zone (HZ) (see table bellow) (Figure 1C and 1D).Our data indicate that treatment with the Nmod and Cmod peptides partially reversed part of the pathological symptoms of XLH. The corrections of the profound GP morphological abnormalities are independent of the degree of hipophophatemia and indicate a direct relationship between FGF23 and cartilage diferenciation. Thereby these drugs emerged as a promising new therapy to reduce the risk of bone deformities in XLH patients and regulate cartialage diferenciation, critical factor in other more prevalent diseases such as osteoarthitis. We also hypothesis that in order to improve bone quality normalization of FGF23 is not enaought and a normalization in the Pi levels should be approached.
The proposed study was based on the concept that FoxA2 transcription factor marks a population of cartilage stem cells located in the resting zone of the growth plate (GP). GP is a cartilaginous tissue located at the end of all long bones and responsible for growth in length. Since the beginning of this project I have developed a new murine model of physeal injury, which recapitulates the Salter-Harris fracture type I, and displays efficient regeneration of the injured growth plate. Using this model we have trace FoxA2+ cells seeing that this population increase during GP regeneration. This cells has Steam cell qualities and might be a new source for cartilage regeneration therapies.The second half of the project, where gaining a better understanding of the GP dynamics was proposed to be examined PHEX murine model of and was performed at the University of Zurich. At the completion of this project, we hope to be able to translate the newly acquired basic science knowledge into clinical application, which would potentially reform the current treatments of injured growth plate cartilage and other bone growth disorders.
We chose XLH as a model disease because despite being considered a rare disorder, is the most common hereditary rickets in the clinic. Even though the most relevant phenotypic characteristic is growth plate deformities that lead to growth impairment, to date the underlying mechanisms are not widely studied and most studies are carried out in adults and focus on bone metabolism. In the fallowing year we aim to better understanding the mechanism that operate in the GP and how FGF23 related therapies direct affect its development. The techniques developed in this project will allow to analyze the mechanisms and dynamics of the growth cartilage development and repair. The results of this study will therefore be easily transferable to the clinic and can be applied to other pathologies with a high socio-sanitary impact, such as joint osteoarthritis, where chondrocyte development and differentiation also play a relevant role. Likewise, the project will facilitate the research training of the group's pre-doctoral members and is laying the foundations for possible future projects.
We chose XLH as a model disease because despite being considered a rare disorder, is the most common hereditary rickets in the clinic. Even though the most relevant phenotypic characteristic is growth plate deformities that lead to growth impairment, to date the underlying mechanisms are not widely studied and most studies are carried out in adults and focus on bone metabolism. In the fallowing year we aim to better understanding the mechanism that operate in the GP and how FGF23 related therapies direct affect its development. The techniques developed in this project will allow to analyze the mechanisms and dynamics of the growth cartilage development and repair. The results of this study will therefore be easily transferable to the clinic and can be applied to other pathologies with a high socio-sanitary impact, such as joint osteoarthritis, where chondrocyte development and differentiation also play a relevant role. Likewise, the project will facilitate the research training of the group's pre-doctoral members and is laying the foundations for possible future projects.