Protein atlas of the human vascular extracellular matrix

Abstract

Extracellular matrix (ECM) remodelling is a hallmark of many cardiovascular diseases. Importantly, haemodynamic environments and disease propensity differ across vessels. A comprehensive ECM characterisation across non-diseased vascular territories, however, has not been performed. Here, I used human aortas, internal mammary arteries, radial arteries and saphenous veins (n=10 each) collected during surgery to characterise the vascular ECM by untargeted proteomics followed by targeted proteomics and glycoproteomics to quantify and investigate vascular ECM and glycosylation changes. 454 ECM and ECM-associated proteins were identified in the human vasculature, of which 184 were core ECM proteins. Arteries were rich in network-forming collagens IV and VIII which confer flexibility. In contrast, fibrillar collagens, conferring stiffness were reduced in aortas compared to the other vessels. Hyalectan proteoglycans (aggrecan and versican) providing viscoelasticity related to blood pressure, were abundant in aortas. IMAs, renowned for their resistance to atherosclerosis, showed reduced levels of proatherogenic small leucine rich proteoglycans (SLRPs) and increased levels of collagen XVIII, including its antiangiogenic and anti-atherosclerotic proteolytic fragments, endostatin and neostatin. Veins contained high levels of basement membrane (BM) glycoproteins (i.e. laminins and nidogen-2) and collagen VI, critical for BM stability, compared to arteries. For quantification, I selected proteotypic peptides to create a targeted panel of 125 ECM proteins, and applied it to aortas with (n=7) and without (n=6) type 2 diabetes (T2DM). These changes were validated in carotid endarterectomy samples from patients with (n=34) and without (n=70) T2DM. Targeted proteomics revealed higher levels of laminins β2, α4 and α5, and collagen α3(VI), in aortas of patients with T2DM compared to controls. This increase in BM proteins was also evident in carotid endarterectomy specimen from T2DM patients. Additionally, an increase in the SLRPs, biglycan, lumican, mimecan and prolargin was found, further corroborating the finding that the reduction in SLRPs in IMAs may relate to their decreased susceptibility for plaque formation. Differences in glycan composition on ECM proteins were detected in T2DM, which was supported by an increase in glycosylation enzymes. I provide the first comprehensive characterisation of the vascular ECM, revealing distinct ECM components of different vascular territories, which may influence disease propensity. Our targeted proteomics method allowed detection of changes in ECM proteins even before manifestation of overt atherosclerotic lesions in patients with T2DM, supporting the role of BM proteins, not only in diabetic micro-, but also macrovascular pathology. Additionally, I performed the first glycoproteomic characterisation of the ECM in the human diabetic aortas, demonstrating that T2DM alters the glycosylation status of vascular ECM proteins.

Date of Award	1 Jul 2021
Original language	English
Awarding Institution	King's College London
Supervisor	Manuel Mayr (Supervisor) & Cathy Shanahan (Supervisor)