Abstract
Background/Introduction
Inflammation is a key feature of calcific aortic valve stenosis (CAVS) against which there is currently no pharmacological treatment.
Purpose
To verify the hypothesis that interleukin-8 (IL-8), a pro-inflammatory factor involved in arterial calcifications, also promotes the calcification of human aortic valve interstitial cells (hVICs).
Methods
Primary hVICs were isolated from healthy pieces of aortic valves harvested from patients undergoing surgical valve replacement. They were cultured in a pro-calcifying condition (Pi-3.8mM) with or without IL-8 (5 to 50 pg/ml) for up to 21 days. Calcification was analysed by alizarin red staining and calcium content was measured with the o-cresolphthalein complexone method. The viability of hVICs was verified by the MTT assay. The expression of osteogenic (BMP2, OPN, osterix and ALP) and myofibrotic (alpha-SMA, collagen-1, collagen-3 and elastin) markers as well as that of metalloproteases (MMP-2, -9 and -12) was analysed by RT-qPCR. The expression of IL-8 receptors, CXCR-1 and CXCR-2 was evaluated by Western blot and flow cytometry, and the effects of IL-8 were tested in the presence or absence of SCH527123, an antagonist of CXCR-1 and CXCR-2. Finally, the expression of CXCR-1 and -2 and elastin was analysed by immunohistochemistry in the calcified and non-calcified areas of human aortic valve samples. All of these experiments were carried out from valves of at least 5 different donors and a P<0.05 was considered statistically significant.
Results
IL-8 (15 pg/mL) caused a significant ∼2-fold increase in the calcification of hVICs in the Pi condition, compared to the Pi-only condition, without modulation of cell viability. In the presence of Pi, IL-8 exposure significantly stimulated the expression of the transcripts of elastin and MMP-12, an elastase, and reduced that of OPN, a well-known inhibitor of calcification. The effects of IL-8 on hVICs calcification and on the expression of MMP-12, elastin and OPN transcripts were significantly prevented by the addition of SCH527123. In addition, the expression of CXCR-1 and -2 was confirmed in histological samples of human aortic valves. This expression was more pronounced in calcified areas compared to non-calcified areas and co-localized with degraded elastin.
Conclusion
IL-8 promoted the calcification of hVICs in culture. This effect was significantly prevented by antagonizing CXCR-1 and CXCR-2 IL-8 receptors, which we showed for the first time to be expressed by human VICs and aortic valves of patients with CAVS. Further studies are underway to clarify the cellular mechanisms involved.
FUNDunding Acknowledgement
Type of funding sources: Public Institution(s). Main funding source(s): Fédération Hospitalo-Universitaire REMOD-VHF
Matrix Gla protein negatively regulates calcification of human aortic valve interstitial cells isolated from calcified aortic valves
