Corcoran, Brendan

MacRae, Victoria

Phadwal, Kanchan

Tang, Qiyu

China Scholarship Council

CavalierHealth.org

Canine Health Foundation

American Kennel Club (AKC)

2024-07-09T10:20:59Z

2024-07-09T10:20:59Z

2024-07-09

Myxomatous mitral valve disease (MMVD) represents a predominant cardiac valvular pathology in both humans and canines, serving as a principal contributor to substantial morbidity and mortality across these species. Presently, therapeutic options to prevent, decelerate, or ameliorate the valvular degeneration attributed to MMVD are nonexistent. Consequently, an augmented comprehension of MMVD's pathogenetic mechanisms is imperative to facilitate the innovation of new therapeutic approaches for this condition in both humans and canines. The objective of this study was to elucidate the involvement of phosphoinositide 3-kinase (PI3K) signalling in the pathogenesis of canine MMVD by delineating the molecular dynamics governing the pathological phenotypic transition of valve interstitial cells (VICs) and related cellular processes within a 2D VIC culture system derived from clinical samples. High expression levels of transforming growth factor-β1 (TGF-β1) and PI3K were identified in myxomatous mitral valve tissues. Activation of the PI3K/AKT/mTOR pathway and an upsurge in TGF-β expressions were observed in cultured activated myofibroblasts (aVICs). TGF-β1 facilitated the conversion of quiescent VICs (qVICs) to aVICs through the enhancement of PI3K/AKT/mTOR signalling. Counteracting PI3K/AKT/mTOR signalling mitigated the myofibroblast transformation of aVICs by suppressing cellular senescence and fostering autophagy. Augmentation of mTOR/S6K signalling prompted the transition of senescent aVICs, characterized by a diminished propensity for apoptosis and autophagy. Targeted silencing of p70 S6K (RPS6KB1) mitigated cellular transition by reducing senescence, curtailing apoptosis, and augmenting autophagy. Hence, TGF-β-induced PI3K/AKT/mTOR signalling was implicated in MMVD development, significantly influencing myofibroblast differentiation, apoptosis, autophagy, and senescence within the context of MMVD. Next senescent aVICs were found to exhibit impaired autophagy as evidenced by compromised autophagy flux and immature autophagosomes. mTOR-dependent autophagy induced by rapamycin attenuated cell senescence and promoted the translocation of cyclin-dependent kinase inhibitors (CDKIs) p16INK4A (CDKN2A) and p21CIP1 (CDKN1A) from the nucleus to the cytoplasm. Furthermore, induction of autophagy by autophagy-related (ATG) gene overexpression restored autophagy flux and reduced p16INK4A and p21CIP1 expression and senescence-associated secretory phenotype (SASP). Conversely, autophagy deficiency induced p16INK4A and p21CIP1 accumulation and SASP, and ATG re-expression alleviated senescent phenotypic transformation, observed in both VICs and HEK293T cells. Notably, p16INK4A and p21CIP1 localized to autophagosomes and lysosomes after rapamycin treatment. p62/SQSTM1 was screened out as the autophagy receptor to selectively sequestrate p16INK4A and p21CIP1 cargoes for autophagic degradation. Finally canine induced pluripotent stem cells (ciPSCs) were developed in order to further investigate the important role of autophagy in stemness, senescence and cell differentiation of VICs. To conclude, aberrant activation of TGF-β induced PI3K signalling may drive the transformation of senescent aVICs from qVICs, SASP induction and autophagy impairment. Deficient autophagy in VICs accumulates CDKI p16INK4A and p21CIP1 and induces cell senescence. Activation of autohagy reverses senescent myofibroblast transformation and restores cellular function. These data will inform the development of therapeutic strategies for the treatment of MMVD in the dog and human, and for and for the management of other age-related degenerative disorders.

https://hdl.handle.net/1842/41966

http://dx.doi.org/10.7488/era/4689

en

The University of Edinburgh

Tang Q, Markby GR, MacNair AJ, Tang K, Tkacz M, Parys M, Phadwal K, MacRae VE, Corcoran BM. (2023). TGF-β-induced PI3K/AKT/mTOR pathway controls myofibroblast differentiation and secretory phenotype of valvular interstitial cells through the modulation of cellular senescence in a naturally occurring in vitro canine model of myxomatous mitral valve disease. Cell Prolif. 4:e13435

Phadwal K, Tang Q, Luijten I, Zhao JF, Corcoran B, Semple RK, et al. (2023). p53 Regulates Mitochondrial Dynamics in Vascular Smooth Muscle Cell Calcification. Int. J. Mol. Sci. 13;24(2):1643

McNair AJ, Markby GR, Tang Q, MacRae VE, Corcoran BM. (2023). TGF-β phospho antibody array identifies altered SMAD2, PI3K/AKT/SMAD, and RAC signalling contribute to the pathogenesis of myxomatous mitral valve disease. Front. Vet. Sci. Oct 16;10:1202001.

Tang Q, McNair AJ, Phadwal K, Macrae VE and Corcoran BM. (2022). The Role of Transforming Growth Factor-β signalling in Myxomatous Mitral Valve Degeneration. Front. Cardiovasc. Med. 9:872288

Phadwal K, Koo E, Jones RA, Forsythe RO, Tang K, Tang Q, Corcoran BM, Caporali A, MacRae VE. (2022). Metformin protects against vascular calcification through the selective degradation of Runx2 by the p62 autophagy receptor. J. Cell Physiol. 237(11):4303-4316

Myxomatous mitral valve disease

MMVD

PI3K signalling

valve interstitial cells

TGF-β signaling

canine MMVD

Role of PI3K signalling in canine myxomatous mitral valve disease (MMVD)

The role of PI3K signalling in canine myxomatous mitral valve disease (MMVD)

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy