Chronic High Glucose Concentration Induces Inflammatory and Remodeling Changes in Valvular Endothelial Cells and Valvular Interstitial Cells in a Gelatin Methacrylate 3D Model of the Human Aortic Valve

Abstract

Calcific aortic valve disease (CAVD), a degenerative disease characterized by inflammation, fibrosis and calcification, is accelerated in diabetes. Hyperglycemia contributes to this process by mechanisms that still need to be uncovered. We have recently developed a 3D model of the human aortic valve based on gelatin methacrylate and revealed that high glucose (HG) induced osteogenic molecules and increased calcium deposits in a pro-osteogenic environment. To further understand the events leading to calcification in diabetic conditions in CAVD, we analyzed here the inflammatory and remodeling mechanisms induced by HG in our 3D model. We exposed valvular endothelial cells (VEC) and interstitial cells (VIC) to normal glucose (NG) or HG for 7 and 14 days, then we isolated and separated the cells by anti-CD31 immunomagnetic beads. The changes induced by HG in the 3D model were investigated by real-time polymerase chain reaction (RT-PCR), Western blot, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence. Our results showed that HG induced expression of different cytokines, cell adhesion molecules and matrix metalloproteinases in VEC and VIC. In addition, protein kinase C was increased in VEC and VIC, indicating molecular mechanisms associated with HG induced inflammation and remodeling in both valvular cells. These findings may indicate new biomarkers and targets for therapy in diabetes associated with CAVD.

Crosstalk of Diabetic Conditions with Static Versus Dynamic Flow Environment—Impact on Aortic Valve Remodeling

“…Under dynamic flow cultivation, ACTA2 expression remained unchanged. However, unchanged ACTA2 expression by HG is in line with our previous observations in VIC in a 2D culture model [16] and it is further in line with reports from other groups on VIC in a gelatin methacrylate 3D model [24] and also on cardiac fibroblasts [25]. Unaltered ACTA2 expression in our bioreactor system might therefore be indicative for a balanced stress level.…”

“…However, VIC in the latter model have been co-cultured with valvular endothelial cells [31]. Discrepancies between this model and our models thus might not only be due to mechanical differences but also might also be ascribed to the lack of VEC in our models, since VEC are known to communicate with VIC [32] possibly through protein kinase C under HG [24]. Direct comparisons of static vs. dynamic environment under basal conditions have shown a slight trend towards lower gene expression levels of SPP1, contrary to our previous observations in a similar setting [28].…”

“…Since 2D cultivation of VIC resulted in a significant increase in COL1A1 expression under HG + HI treatment [16], this remodeling marker remains preserved in more complex 3D models. Nevertheless, other collagen types, such as collagen type 3 as well as MMP1/13 might be altered, since others have reported the induction of these markers by HG [24]. Independent from diabetic treatment, basal expression of MMP2 was significantly higher under dynamic flow cultivation compared to static cultivation; this is indicative of an enhanced level of catabolic matrix degradation in AV tissue [33].…”

Reproducible In Vitro Tissue Culture Model to Study Basic Mechanisms of Calcific Aortic Valve Disease: Comparative Analysis to Valvular Interstitials Cells

“…The co-culturing techniques also have some disadvantages, because maintenance of the quality and stability of the population of co-cultured species is a tedious task [ 28 ]. However, recently described novel 3D models with human VICs, which are suitable to investigate VIC phenotype changes as a result of both communication with valvular endothelial cells and exposure to pathological stimuli, provide promising tools to better understand the valve cell biology and pathological mechanisms underlying CAVD [ 29 , 30 ].…”

Property-Tuneable Microgels Fabricated by Using Flow-Focusing Microfluidic Geometry for Bioactive Agent Delivery

“…In addition, the surface of GM supports cell adherence and growth [6]. This, along with the possibility of GM to undergo polymerisation in mild conditions, enables GM to be widely adopted as scaffolds for cell encapsulation [7][8][9]. In fact, the properties of GM as mentioned above are favourable not only for tissue engineering but also for bioactive agent delivery.…”