Periodic Reporting for period 3 - Restoring Roots (Pulmonary hypertension: “aberrant” mimicry of lung vascular morphogenesis?)
Periodo di rendicontazione: 2023-10-01 al 2025-03-31
The lung's primary function is to oxygenate the blood, which is accomplished by coordinating multiple processes within the lungs. The airways transport air to the alveoli, while the pulmonary artery transports deoxygenated blood from the heart to the alveoli. The oxygenated blood reaches the heart through the pulmonary veins, and the heart then circulates the blood into the systemic circulation. The pulmonary artery and vein, collectively referred to as the pulmonary vessels, perform an essential function in oxygenating blood. Problems with the pulmonary vessel have a devastating effect on human health and occasionally even result in mortality. Pulmonary arterial hypertension (PAH) is a disease that affects the function of the pulmonary vessels. During the disease, smooth muscles in the pulmonary vessel begin to proliferate, causing the vessel to close, eventually obstructing the pulmonary vessel and causing an accumulation of pressure in the heart, at which point the heart ceases to function. One of the primary causes of PAH is the proliferation of smooth muscle cells. PAH is a progressive disease that typically affects the elderly. Paediatric pulmonary arterial hypertension (PPAH) could also develop in neonates and children, according to research conducted over the past ten years, although it is unclear how PPAH is developing. PAH is a devastating disease; despite significant advances in the treatment of pulmonary arterial hypertension (PAH), there are still obstacles to reaching optimal outcomes. Ambrisentan, tadalafil, and treprostinil, which target the endothelin, nitric oxide, and prostacyclin pathways, respectively, are the only approved remedies for PAH patients that provide symptomatic relief. In the field of pulmonary hypertension, effective and curative therapeutic interventions still need investigation. Even though PAH and PPAH share a similar system, the causes of these diseases appear distinct. The greatest challenge in our laboratory was determining 1) the distinction between PAH and PPAH and 2) the causes of PPAH development. This project's overarching objective was comprehensively comprehending the PAH and PPAH therapy envelope. In addition, by researching PAH and PPAH, we gain an in-depth understanding of how pulmonary vessels form after birth, a topic that has been neglected for decades. This knowledge will aid in developing a treatment for PAH and several other pulmonary diseases.
We used isolated adventitial fibroblast from the pulmonary arteries of patients with idiopathic PAH. Adventitial fibroblast (FBs) is one of the primary mesenchymal cell types involved in the vascular remodelling during the PAH. Hyper proliferation and less apoptosis are maintained in PAH-FB compared to the donor-FB in ex-vivo cell culture, and it allows us to perform different molecular biology technics such as qPCR, ATAC and RNAseq. Our analysis indicates that SOX9, TBX4 and TBX5 overexpressed in the remodelled pulmonary vessel in PAH patients. These proteins are usually expressed during embryonic development. ERC grant's primary Aim 1 was to delineate the role of these proteins in PAH. With the help of RNA interference, we downregulated the TBX4, TBX5 or SOX9 in PAH-FBs and PAH-SMC, which are the main cells involved in vascular remodelling. Our results indicate that the single base pair deletion of TBX4 and SOX9 significantly impacted suppressing the hyperproliferative phenotype of PAH-FBs. Moreover, the simultaneous knockdown of all three transcription factors (TBX4, TBX5, and SOX9) significantly (P 0.001) suppressed the hyperproliferative phenotype of PAH-FBs. We also observed that individual down-regulation of TBX4, TBX5, or SOX9 caused a decrease in the transcription of ACTA2 and PDE5A, as well as modulating the expression of TF genes involved in the mesenchymal transcriptional network, such as ETS1, paired-like homeodomain 1 (PITX1), and FGF10. Suggesting that overexpression of TBX4, TBX5 and SOX9 in pulmonary artery mesenchymal cells may trigger the vascular remodelling during the development of PAH (Chelladurai et al. Sci Transl Med. 2022 Jun 8;14(648):eabe5407). The next major aim of the ERC project is to identify the role of TBX4/TBX5/SOX9 in developing PH in neonates/newborns. People widely accepted that vascular remodelling is the major cause of PH development. However, in the case of pediatric PH, it is not clear how it develops. Lack of a model system was one of the key issues we ran into when investigating PH of the newborn (PPHN). Generating TBX4 knockout and mutant mice, we identified the key role of Tbx4 in postnatal pulmonary vascularization and PPHN.
Altogether
1) We identified key mesenchymal transcriptional networks that coordinate cellular hyperplasia in PAH
2) We generated Tbx4 mutant mice responsible for developing PPHN/childhood associated PH in humans.
3) Our data suggest that the fundamental reason for developing PH in Tbx4 mutant patients is the lack of vessel formation during lung development.
4) Developed a novel imaging method that helps to quantify the pulmonary vessel in neonatal mice, we provided novel insight into the pulmonary vessel development.
Altogether
1) We identified key mesenchymal transcriptional networks that coordinate cellular hyperplasia in PAH
2) We generated Tbx4 mutant mice responsible for developing PPHN/childhood associated PH in humans.
3) Our data suggest that the fundamental reason for developing PH in Tbx4 mutant patients is the lack of vessel formation during lung development.
4) Developed a novel imaging method that helps to quantify the pulmonary vessel in neonatal mice, we provided novel insight into the pulmonary vessel development.
At the end of the project, we will define the contribution of developmental transcription factors (TBX4/TBX5/SOX9) to the vascular remodeling processes underlying neonatal and adult PH.
Furthermore, uncovering the TBX4/TBX5/SOX9-epigenetic circuitry by profiling epigenetic landscape in both “pathological remodeling” and the loss of “physiological reverse remodeling” in PPHN, I aim to harness novel intervention strategies (locus specific epigenetic modifications) to reverse aberrant remodeling and foster regeneration of lung vasculature.
Furthermore, uncovering the TBX4/TBX5/SOX9-epigenetic circuitry by profiling epigenetic landscape in both “pathological remodeling” and the loss of “physiological reverse remodeling” in PPHN, I aim to harness novel intervention strategies (locus specific epigenetic modifications) to reverse aberrant remodeling and foster regeneration of lung vasculature.