Background: Pulmonary arterial hypertension (PAH) is a lethal disease for which the fundamental molecular mechanisms are only partially understood. Existing therapies, which primarily focus on endothelial dysfunction, have limited effects on improving outcomes. Increases in pulmonary vascular resistance in PAH may be attributed to complex lung arterial remodeling which result in obliterative “plexiform” lesions, a pathological hallmark of this disease. Recent studies have shown that endothelial cell (ECs) apoptosis may be a central trigger for PAH, resulting in the emergence of growth-dysregulated and apoptosis-resistant ECs that contribute to the formation of complex neoplastic-like vascular lesions. However, the mechanism which links ECs apoptosis to dysregulated growth is not yet known. Previous studies in our lab have identified increased expression of translationally controlled tumor protein (TCTP) and its gene (TPT1), previously implicated in the transformation of neoplastic cells in cancer, and in blood outgrowth ECs from patients with PAH. Moreover, TCTP expression was found to be elevated in the lungs of patients with PAH, and tightly localized to complex arterial lesions. In addition, it was detected in obliterative intimal lesions of an experimental rat model of severe PAH.
Hypothesis: TCTP represents a central molecular mechanism linking ECs apoptosis to the emergence of growth-dysregulated lung vascular cells and occlusive, complex arterial remodelling in PAH.
Specific Hypotheses:
- Lentiviral overexpression of TCTP in human umbilical vein endothelial cells (HUVECs) and pulmonary artery smooth muscle cells (PASMCs) leads to a hyperproliferative and apoptosis-resistant phenotype.
- Overexpression of TCTP will increase its export into apoptotic extracellular vesicles, thereby augmenting cell-cell signalling between ECs and neighbouring SMCs.
Purpose: My objective was to examine the effects TCTP overexpression on ECs and SMCs survival in terms of proliferation and apoptosis, and TCTP release on the survival of nontransduced ECs and SMCs.
Methods and Results: The effect of TCTP overexpression on ECs growth and survival was studied using in vitro models. TCTP was overexpressed via a lentivirus vector in HUVECs and PASMCs. Compared to non-transfected or null transfected cells, TCTP overexpression led to increases in BrdU incorporation, consistent with hyper-proliferation, and decreases in caspase activity, consistent with apoptosis resistance. As well, TCTP was selectively exported into the conditioned media of apoptotic ECs, but not SMCs, despite similar levels of overexpression. In addition, the level of release was greater in serum starved conditioned media in comparison to the exosome fraction. Finally, our data demonstrates a selective effect of conditioned media (CM) from serum-starved ECs on PASMCs, but not ECs, in terms of an increase in proliferation and a decrease in apoptosis.
Conclusions: These support the idea that TCTP overexpression confers an increase in the survival of SMCs and HUVECs. Moreover, TCTP released from apoptotic ECs leads to a growth-dysregulated phenotype within SMCs (but not ECs) and may contribute to the formation of complex lung arterial lesions, leading to arteriolar obliteration in PAH. Finally, an increase in the level of TCTP expression via lentiviral transduction led to an increased TCTP export into the media, but this appeared to be mostly in the soluble portion, and less was associated with exosomes.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/38674 |
| Date | 10 January 2019 |
| Creators | Hassan, Dhiya |
| Contributors | Stewart, Duncan John |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
Page generated in 0.0021 seconds