Induced osteogenic differentiation of human smooth muscle cells as a model of vascular calcification

Abstract

Vascular calcification is a severe pathological event in the manifestation of atherosclerosis. Pathogenic triggers mediating osteogenic differentiation of arterial smooth muscle cells (SMC) in humans remain insufficiently understood and are to a large extent investigated in animal models or cells derived thereof. Here, we describe an in vitro model based on SMC derived from healthy and diseased humans that allows to comprehensively investigate vascular calcification mechanisms. Comparing the impact of the commonly used SMC culture media VascuLife, DMEM, and M199, cells were characterised by immunofluorescence, flow cytometry, qPCR, and regarding their contractility and proliferative capacity. Irrespective of the arterial origin, the clinical background and the expansion medium used, all cells expressed typical molecular SMC marker while contractility varied between donors. Interestingly, the ability to induce an osteogenic differentiation strongly depended on the culture medium, with only SMC cultured in DMEM depositing calcified matrix upon osteogenic stimulation, which correlated with increased alkaline phosphatase activity, increased inorganic phosphate level and upregulation of osteogenic gene markers. Our optimized model is suitable for donor-oriented as well as broader screening of potential pathogenic mediators triggering vascular calcification. Translational studies aiming to identify and to evaluate therapeutic targets in a personalized fashion would be feasible. Vascular calcification due to osteogenic differentiation of arterial smooth muscle cells (SMC) is a severe pathological event in atherosclerosis. Massive, bone-like or brittle, amorphous calcifications can occlude the lumen, cause atherosclerotic plaque rupture, thrombus formation and infarction or result in failure of the arterial wall 1-6. Deposition of calcified matrix within the vessel wall was described as an active, SMC-driven process 5,7. This process is initially marked by the switch of SMC from their contractile phenotype towards a synthetic state, in which proliferation and migration is enhanced 8-12. The ability of SMC for phenotypic switching 1,9 is important during vascular repair, but makes them also susceptible to pathological changes 8,9,12-14 , including their differentiation into osteoblast-and chondrocyte-like cells or acquisition of macrophage and foam cell markers 1,11,13-22. Several studies investigated the processes and key factors involved in the osteogenic differentiation of mammalian SMC, subsequent deposition of mineralized matrix and vascular calcification 20,21,23-25. These studies provide evidence that an active inhibition of calcification and the prevention of pro-osteogenic gene expression is crucial for retention of the SMC phenotype 7,23,26-28. Conversely, it was found that SMC-mediated calcification is enhanced by an inflammatory environment, oxidative stress, hypercalcaemia and hyperphosphatemia, as well as apoptosis, and senescence 1,25,29-31. Despite the fact that these pathogenic triggers link atheroscleros...

FHL5 controls vascular disease-associated gene programs in smooth muscle cells

“…Based on the perturbation of vascular remodeling and ossification pathways supported by RNA-seq, we next hypothesized that FHL5 may regulate vascular calcification to mediate CAD/MI risk. To this end, we treated SMCs with an osteogenic cocktail as done previously 46,47 . We observed increased mineral deposition quantified by alizarin red staining upon FHL5 and FHL5-NLS overexpression ( Figure 3G ).…”

Osteomodulin attenuates smooth muscle cell osteogenic transition in vascular calcification

“…Similarly, exogenous administration of OMD protein solely was sufficient to activate SMCs into a synthetic phenotype, inducing endogenous OMD expression and ECM reorganisation, possibly via SMAD3 transcription factor, while generally preventing other changes in expression of SMC markers and markers of osteoblastic or inflammatory transition. To this end, OMD was also upregulated after exposure to high Pi and osteogenic media, strong drivers of osteoblastic transdifferentiation of SMCs, 63 suggesting that OMD could serve as a mechanosensitive marker in reaction to vascular ECM changes. In addition, silencing of OMD gene expression modulated SMC phenotype by inducing the osteogenesis‐related genes and therefore, enhancing the ECM calcification.…”

The Vessels-Bone Axis: Iliac Artery Calcifications, Vertebral Fractures and Vitamin K from VIKI Study

“…However, that study was conducted in a subsample of the general population that was relatively young (aged 56 ± 11 years) and free from clinical cardiovascular disease, thus possibly less prone to the development of the medial calcifications common in iliac arteries [ 33 ]. It is documented that the wall of muscular or predominantly muscular arteries, such as the iliac and femoral arteries, is more susceptible to develop calcification [ 20 , 21 , 22 ].This could be related to the higher density of smooth muscle cells present in the wall of these vessels, which are those that have eventually undergone bone- and cartilage-like phenotypic changes under several in vitro experimental conditions [ 33 , 34 ]. Our results indirectly corroborate this hypothesis.…”