Proteomics study of endothelial cells exposed to cyclic stretch and estrogen
thesisposted on 2022-03-28, 10:56 authored by Nurul Farhana Jufri
Hypertension and estrogen deficiency are known risk factors that contribute to the incidence of cardiovascular and cerebrovascular disease. It is thought that hypertensive risk due to excessive stretching of vascular walls and the loss of a protective effect from estrogen causes injury and weakness of the vascular structure that may lead to atherosclerosis, coronary heart disease, aneurysm and/or stroke. Previously, estrogen has been demonstrated to exert a protective effect on the vascular system as it has anti-inflammatory and anti-atherogenic properties, reducing reactive oxygen species production and increasing mitochondria efficiency. Excessive mechanical tensile stretch, as occurs during hypertension, is associated with cellular injury and endothelial dysfunction. However, the results of the combination of these positive and negative effects from both stimuli are still unclear and warrant further investigation. Thus, this thesis aims to investigate the proteomic profile of human cerebral microvascular endothelial cells (HCMEC) subjected to either cyclic mechanical tensile stretch regimes and /or 17-estradiol using proteomics. Two types of relative quantitative mass spectrometry-based proteomic methods have been applied, namely label-free shotgun proteomics and Isobaric Tag for Relative and Absolute Quantification (iTRAQ). Pathological cyclic stretch at 20% intensity resulted in significantly greater proteome changes compared to physiological cyclic stretch at 5% intensity independent of the time period. Pathological cyclic stretch for two hrs resulted in changes to proteins associated with (i) contractile dysfunction and (ii) inflammation. In contrast, pathological cyclic stretch for 18 hrs altered proteins potentially associated with aneurysm pathophysiology (e.g., titin and apolipoprotein B100). These two proteins have been previously observed to be dysregulated in aneurysm samples. To further investigate the effects of 17β-estradiol present during cyclic stretch on HCMECs, estrogen receptors were initially identified using immunocytochemistry. Estrogen receptor beta and G protein-coupled estrogen receptor were both identified as expressed on HCMEC. The interaction of these receptors with 17β-estradiol was confirmed using a proximity ligation assay. Finally, proteomic analysis was conducted on HCMECs exposed to 17β-estradiol and pathological cyclic stretch using iTRAQ. An unique protein profile was observed with HCMEC exposed to 17β-estradiol only and HCMECs exposed to both 17β-estradiol and pathological cyclic stretch. This included proteins that might be involved in antioxidant properties, cell-to-cell adhesion and structural activity that may indicate 17β-estradiol's ability to preserve cellular integrity during exposure to high stretch intensity physical challenges. In conclusion, this study provides compelling proteomic evidence for underlying protein changes induced by cyclic stretch and 17β-estradiol. 17β-estradiol may have a stronger effect on HCMEC protein's modulation in comparison to mechanical cyclic stretch and it may exert a protective effect on the cells from consequences of pathological cyclic stretch. This model may reflect protein regulation that may occur during hypertension and has potential in developing estrogen-like compounds in a future therapeutic approach.