8.4. De la terapia del juego a la terapia del videojuego
8.4.1. Características de la terapia del videojuego.
Mouse lung development is a complicated process consisting of many distinct stages. In my dissertation, I have extensively interrogated the question of whether three members of the class I HDAC family, HDAC1, 2, and 3 are functionally important for lung epithelial development and regeneration by carefully analyzing a series of mouse genetic models. These studies revealed distinct roles for different members of HDACs: Hdac1/2 are redundantly required for the early development of proximal lung epithelial progenitor cells (refer to Chapter 2), while Hdac3 is required for the proper formation of primitive alveolar saccules and remodeling of alveolar epithelial lineages right before birth (refer to Chapter 3). These results are consistent with the fact that Hdac1/2 are found in the same chromatin remodeling complexes whereas Hdac3 resides in different chromatin complexes. In this chapter, I will discuss several unresolved questions and the potential ways to address them.
What roles do class I HDACs play in lung mesodermal development?
The data presented in Chapter 2 and Chapter 3 have described how Hdac1/2 and Hdac3 directs lung epithelial development and regeneration. Since these HDAC proteins also appear to be highly expressed in the lung mesenchyme (as shown in Figure 2.1 and 3.1), it is possible that they are also crucial for lung mesodermal development.
To determine in which mesenchymal lineages these HDACs are expressed, we could double stain HDACs with various mesodermal markers, including
endothelial marker CD31, pericyte markers Pdgfrα and Pdgfrβ and smooth
muscle markers SM22. These data will inform us the contribution of class I HDACs to the development of mesodermal lineages.
To study whether class I HDACs are required for the development of lung
mesenchyme, we will utilize the Dermo1Cre line which has been shown to be
active in various lung mesenchymal lineages (115), and cross it with Hdac1flox,
Hdac2flox, Hdac3flox alleles. Histological sections on the lungs of Dermo1Cre : Hdac1/2f/f and Dermo1Cre : Hdac3f/f lungs from different embryonic stages will be examined for lung developmental defects. Based on the defects, we can further investigate the phenotype by immunostaining or qPCR for markers of cell
proliferation, apoptosis and various lung lineages. RNA-seq or microarray can be performed to further understand the molecular pathways that underlie these phenotypes. These studies are currently underway in our laboratory.
One caveat is that Dermo1Cre line is also widely active in the mesenchymal cells
of other organs, such as in the heart. Thus Dermo1Cre : Hdac1/2f/f and Dermo1Cre :
Hdac3f/f embryos might have a lethal heart defect that might preclude study of
lung development. In this case, individual tamoxifen-inducible lineage deleters such as Tbx4creERT2 and Tie2CreERT2 (116, 117), can be used to study the function of HDACs in lung mesodermal development.
Does Tgf-β signaling play an epithelial cell-specific role in promoting AT1
cell spreading and lung sacculation?
As shown in Chapter 3, pharmacological inhibition of Tgf-βsignaling reduced the
spreading area of AT1 cells in primary culture, suggesting that Tgf-βsignaling
may play a role in promoting AT1 cell spreading in a cell-autonomous manner. However, p-Smad2 staining was decreased in both lung epithelial cell and
mesenchymal cells in the Shhcre:Hdac3f/f mutants. Although our data showed that
the Shhcre:Hdac3f/f mutant AT1 cells have a cell-intrinsic defect in spreading, it is still unclear if a reduction of Tgf-βactivity in the mesenchymal cells has any contributions to the phenotype seen in the Shhcre:Hdac3f/f mutants. To address this question, we could utilize the Alk5flox (Tgf
βr1flox) allele (25) and cross it into
the lung epithelial and mesenchymal specific deleters including Shhcre and
Dermo1cre (or Tbx4CreERT2), respectively. We can then assess if the Shhcre:Alk5f/f
or Dermo1cre:Alk5f/f lungs exhibit defects in lung sacculation and AT1 cell spreading similar to Shhcre:Hdac3f/f mutants. These data will inform us more definitively whether Tgf-β signaling is important for lung sacculation and in which
cell lineages it is important.
Can AT1 cells exert a mechanical force to actively drive lung sacculation and alveologenesis?
AT1 cells are extremely thin and spread over an extensive surface area to cover 95% of the alveolar respiratory surface of a mature lung. They are an integral part of the thin interface that mediates the efficient diffusion of oxygen into the pulmonary capillaries. My studies presented in Chapter 3 suggest that Hdac3 plays a critical role in promoting the spreading and remodeling of AT1 cells
during saccular formation. One interesting phenomenon we observed during lung sacculation is the simultaneous occurrence of AT1 cell spreading and expansion of distal airspace, which raises the question whether the spreading of AT1 cells can exert a mechanical force to contribute to the expansion of distal airways.
To address this question, we will utilize traction force microscopy (TFM) to measure the force exerted by the primary Pdpn+ EpCAM+ AT1 cells to the substrate in culture. Previous studies have suggested that interstitial fibroblasts play a crucial role in driving the formation of primary and secondary alveoli (118).
We could measure the force between the Pdgfrα+ lung fibroblasts and substrate
in parallel and compare to that exerted by the AT1 cells. Given that the ShhCre :
Hdac3f/f AT1 cells have defects in spreading in culture, we will perform TFM on
these mutant cell and test if they display a lack of the capacity to exert force on the substrate. In addition, if the Shhcre:Alk5f/f mutant lungs also have defects in AT1 cell spreading in vivo, we could also perform TFM on the Shhcre:Alk5f/f
mutant AT1 cells in culture to test if epithelial specific loss of Tgf-βsignaling can lead to a defect in force-generating ability.
Does Hdac3 play a role in the regeneration of new alveoli after lung injury?
In Chapter 3, I have demonstrated that Hdac3 mediated repression of miR-17-92
and subsequent Tgf-β activation is required for the proper spreading of AT1 cells
and distal airspace during late lung development. Previous studies have
established a well-known role for Tgf-βsignaling in promoting lung fibrosis (105).
Thus, it is possible that Hdac3 might also play an important role in promoting lung fibrosis given its link to Tgf-βsignaling during lung development.
Our preliminary data using the SftpcCreER;Hdac3f/f mutant mice showed that these
mice had no gross lung phenotype during homeostasis. However, these mutant lungs exhibited a significantly reduced fibrotic response to bleomycin injury,
suggesting that the Hdac3-miR-17-92-Tgf-βmolecular cascade may also play a
role during adult injury repair. Interestingly, staining for the AT1 cell marker Aqp5
showed that the SftpcCreER;Hdac3f/f mutant AT1 cells displayed a more cuboidal
and less flattened morphology, consistent with what we have observed with ShhCre : Hdac3f/f lungs during the developmental stage.
For future studies, we will try to confirm the down-regulation of Tgf-βsignaling in
confirm whether the newly regenerated SftpcCreER;Hdac3f/f mutant AT1 cells are defective in spreading after bleomycin injury by testing more animals, or perform TFM on these mutant AT1 cell culture to see if they have an impaired ability to generate a mechanical force.
An interesting phenotype exhibited by the SftpcCreER;Hdac3f/f mutant mice was that they showed both a resistance to fibrotic response and a defect in AT1 cell
spreading. If Tgf-βsignaling is confirmed to be reduced in these mutant lungs,
then there would arise an intriguing question of how exactly this pathway
functions during lung injury repair. It is possible that activation of Tgf-βsignaling is important for the proper spreading of AT1 cells and regeneration of new alveoli
after injuries. On the other hand, excessive Tgf-βsignaling promotes the
activation of fibroblasts and collagen deposition, leading to the deleterious
fibrosis that would disrupts alveoli function. In this case, it might suggest that Tgf-
βsignaling has both a positive and a negative impact on the regenerative
process, which should be taken into consideration when we use Tgf-βinhibitors
to treat pulmonary fibrosis.
Concluding Remarks
In this dissertation, I have investigated the functional roles of three members of class I HDAC family during lung development and regeneration. In Chapter 2, I showed that Hdac1/2 are redundantly required for the development and
regeneration of Sox2+ proximal lung endoderm progenitors via direct repression of Bmp4 and cell cycle inhibitors including Rb1/p21/p16. In Chapter 3, I
demonstrated that Hdac3 plays a critical role in promoting alveolar epithelial remodeling and distal airway expansion during lung sacculation and
alveologenesis. These studies provide some of the first evidences that lung development and regeneration is regulated by chromatin remodeling factors, and that distinct epigenetic factors can play very different physiological roles. These studies also provide evidence suggesting that deregulated HDAC activity can contribute to the pathogenesis of adult lung diseases such as COPD, and it will be exciting to see if these studies and future findings translate into potential therapies for human diseases.
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