Caporali, Andrea

Hadoke, Patrick

Martello, Andrea

2019-07-15T11:04:43Z

2019-07-15T11:04:43Z

2019-07-06

Autophagy is an essential quality control function of the cell. It selectively degrades harmful protein aggregates and/or damaged organelles, enabling maintenance of cellular homeostasis. Basal autophagy also mediates proper cardiovascular function. A variety of cardiovascular risk factors cause defective autophagy, consequently, pharmacotherapy with compounds to re-establish physiological levels of autophagy and stimulate pro-survival effects in the vasculature is an emerging strategy for cardiovascular disease. Therefore, it is crucial for the design of new pharmacological therapy the identification of novel key genes involved in the regulation of autophagy in vascular cells. In two recent studies Caporali et al showed how miR-503 might be considered a suppressor of postishemic neovascularization in type I diabetes and suggested some critical genes targeted by miR-503 whom downregulation may be crucial for the detrimental effect on endothelial cells and pericyte. Among the putative targets of miR-503 not prioritized in the previous studies but of potential interest for endothelial cell functions there was TCHP. Trichoplein (TCHP) was first identified as a keratin binding protein in 2005 and was described having a role in cancer progression, cellular apoptosis, cell cycle, primary cilium formation, mitochondria fragmentation and mitochondria-endoplasmic reticulum tethering. Using biochemical and bioinformatics approaches, I have demonstrated that TCHP is a novel target of miR-503. The data presented in this thesis will address the hypothesis that TCHP regulates endothelial function through the control of autophagy. TCHP expression in ECs was reduced when cultivated in high glucose in combination with low growth factors condition. Notably, ECs sorted from mice with Type-1 diabetes showed low levels of Tchp compared to non-diabetic mice. Knockdown of TCHP in ECs in vitro was characterized by marked changes in the tubulin cytoskeleton organization, resulting in an impaired microtubule network, reduced EC migration, reduced barrier function and impaired EC sprouting. Loss of TCHP function in ECs in vitro led to a defective autophagy, resulting in accumulation of SQSTM1/p62 and unfolded protein aggregates. Therefore, blocking autophagic flux in ECs resulted in premature cellular senescence as demonstrated by the appearance of β- galactosidase staining, the senescence-associated secretory phenotype (SASP) and increased expression of p16INK4A. This phenotype was associated with activation of the mTOR pathway and could be rescued using mTOR inhibitors. Thus, Torin-1 improved the migratory capacity of TCHP knockdown cells and rescued their senescent phenotype. In addition, ultrastructure analysis, immunofluorescence and epidermal growth factor pulse chase experiments revealed substantial abnormalities in the endosome and lysosome compartments in TCHP knock down cells. Remarkably, Tchp knockout mice exhibited a decreased cardiac vascularization and a significant accumulation of SQSTM1/p62 in cardiac vessels and cardiomyocytes, as a demonstration of defective autophagic flux. Finally, I observed loss of TCHP and an increase of SQSTM1/p62 and aggregates in the vessel wall derived-ECs, from patients with vascular dysfunction and premature coronary artery disease. Remarkably ECs from patients exhibited impaired migration and increased expression of SASP, resembling the phenotype of TCHP knockdown cells. In line with this, function in EC from patients was rescued by Torin-1 treatment. Taken together, these results reveal for the first time the pivotal role played by TCHP in the vascular endothelium and identify a new mechanism by which pathological conditions, through the silencing of TCHP, lead to the endothelial dysfunction. Importantly, this study highlights a fundamental link between EC function and cellular proteostasis, through the control of autophagy, and suggests a new therapeutic direction in cardiovascular disease.

http://hdl.handle.net/1842/35782

en

The University of Edinburgh

2020-07-06

TCHP

Trichoplein

autophagy

endothelial dysfunction

PCDA

Premature Coronary Artery Disease

SASP

Senescence-associated Secretory Phenotype

p62/SQSTM1

HUVECs

proteostasis

inflammation

Role of Trichoplein in endothelial cell function and autophagy

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy

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