The role of smooth muscle cells in calcification of atherosclerotic plaques

Sammanfattning: Calcification (CALC) is a predominant feature of late-stage cardiovascular disease (CVD) but responsible mechanisms and its contribution to the risk of clinical events remain unclear. Formation of highly mineralized extracellular matrix (ECM) leads to progressive aortic valve stenosis (AVS) and has been identified as a surrogate marker for atherosclerotic disease burden. However, we previously found enrichment of genes associated with CALC in atherosclerotic carotid lesions from asymptomatic patients and in patients on statin therapy. This thesis aimed to investigate how CALC correlates with gene expression profiles in human specimens of carotid plaques as well as AVS and functionally characterize the underlying mechanisms associated to osteogenic phenotypic transformation of structural cells. Study I explored gene expression profiles and biological pathways related to macro-CALC in human carotid lesions, estimated by computed tomography (CT). Microarray profiling, bioinformatic analysis and histological validation based on high- vs low-CALC plaques revealed upregulation of smooth muscle cell (SMC) markers in high-CALC plaques, whereas macrophage markers were downregulated. The most enriched processes in high-CALC plaques were related to SMC differentiation and ECM organization, while inflammation, lipid transport and chemokine signaling were repressed. Proteoglycan 4 (PRG4) was identified as the most upregulated gene in association with plaque CALC and found in the ECM overlapping with SMA, CD68 and tartrate-resistant acid phosphatase (TRAP) positive cells. Study II characterized PRG4 in the context of AVS and aortic valve CALC. Transcriptomic, histological and immunohistochemical (IHC) analysis of human aortic valves from patients undergoing aortic valve replacement showed significant upregulation of PRG4 in thickened and CALC regions of aortic valves compared with healthy regions. In addition, PRG4 positively associated with mRNA expression of proteins involved in cardiovascular CALC. Treatment of human valve interstitial cells (VICs) with recombinant human PRG4 (rhPRG4) enhanced phosphate (Pi) induced CALC and increased bone morphogenetic protein 2 (BMP2) expression. Study III analyzed the role of PRG4 in vascular remodeling and intimal CALC. PRG4, detected by IHC, localized to SMCs in early human intimal thickening, while in advanced lesions it was found in the ECM, surrounding macro-CALC. In vivo mouse and rat models showed increased Prg4 expression in SMCs during intimal hyperplasia, correlating with osteogenic markers and transforming growth factor b (Tgfb). Moreover, PRG4 was enriched around intimal plaque CALC in apolipoprotein E deficient mice (ApoE-/-) on warfarin. In vitro, PRG4 was induced in primary human vascular SMCs by TGFb and calcifying conditions, while SMC markers were repressed. Silencing experiments showed that PRG4 expression was driven by transcription factors mothers against decapentaplegic homolog 3 (SMAD3) and SRY-box transcription factor 9 (SOX9). The addition of rhPRG4 increased ectopic SMC calcification, while arresting cell migration, proliferation and osteogenic transformation. Study IV assessed the influence of biomechanical forces, related to atherosclerotic carotid macro-CALC, on SMC phenotype and plaque stability. Finite element modeling (FEM), based on preoperative CT images, identified that biomechanical stretch was significantly reduced in close proximity to macro-CALC, while pathologically increased levels were observed within distant soft tissues. In vitro modeling of these conditions revealed upregulation of markers for SMC differentiation and contractility under low stretch but also impeded SMC alignment and increased osteogenic transdifferentiation. In contrast, high-stretch in combination with calcifying conditions induced rapid SMC apoptosis, suggesting a contribution to atherosclerotic plaque stabilization by the load-bearing capacities of macro-CALC. Overall, this thesis demonstrates that vascular disease pathology related to CALC can be comprehensively described by linking clinical diagnostics and underlying biological processes via in silico analysis, thereby contributing to the basic understanding of disease progression and patient specific phenotypic variability.