Metformin alleviates the calcification of aortic valve interstitial cells through activating the PI3K/AKT pathway in an AMPK dependent way

Background Calcific aortic valve disease (CAVD) is the most prevalent valvular disease worldwide. However, no effective treatment could delay or prevent the progression of the disease due to the poor understanding of its pathological mechanism. Many studies showed that metformin exerted beneficial effects on multiple cardiovascular diseases by mediating multiple proteins such as AMPK, NF-κB, and AKT. This study aims to verify whether metformin can inhibit aortic calcification through the PI3K/AKT signaling pathway. Methods We first analyzed four microarray datasets to screen differentially expressed genes (DEGs) and signaling pathways related to CAVD. Then aortic valve samples were used to verify selected genes and pathways through immunohistochemistry (IHC) and western blot (WB) assays. Aortic valve interstitial cells (AVICs) were isolated from non-calcific aortic valves and then cultured with phosphate medium (PM) with or without metformin to verify whether metformin can inhibit the osteogenic differentiation and calcification of AVICs. Finally, we used inhibitors and siRNA targeting AMPK, NF-κB, and AKT to study the mechanism of metformin. Results We screened 227 DEGs; NF-κB and PI3K/AKT signaling pathways were implicated in the pathological mechanism of CAVD. IHC and WB experiments showed decreased AMPK and AKT and increased Bax in calcific aortic valves. PM treatment significantly reduced AMPK and PI3K/AKT signaling pathways, promoted Bax/Bcl2 ratio, and induced AVICs calcification. Metformin treatment ameliorated AVICs calcification and apoptosis by activating the PI3K/AKT signaling pathway. AMPK activation and NF-κB inhibition could inhibit AVICs calcification induced by PM treatment; however, AMPK and AKT inhibition reversed the protective effect of metformin. Conclusions This study, for the first time, demonstrates that metformin can inhibit AVICs in vitro calcification by activating the PI3K/AKT signaling pathway; this suggests that metformin may provide a potential target for the treatment of CAVD. And the PI3K/AKT signaling pathway emerges as an important regulatory axis in the pathological mechanism of CAVD.

Is Cell Regeneration and Infiltration a Double Edged Sword for Porcine Aortic Valve Deterioration: A Large Cohort of Histopathological Analysis

Background Bioprostheses are the commonest prostheses used for valve replacement in the western world. The major flaw of bioprostheses is the occurrence of structural valve deterioration (SVD). The objective of this study was to assess in a large cohort of patients the pathologic features of porcine aortic valve (PAV) SVD based on histomorphological and immunopathological features.Methods and materials 109 cases of resected PAV were observed grossly and histopathologically. The type and amount of infiltrated cells were evaluated in the different type of bioprosthetic SVD by immunohistochemical staining . Results The most common cause of SVD was calcification, leaflet dehiscence and tear (23.9%,19.3% and 18.3%, respectively). Immunohistochemical staining demonstrated that vimentin positive cells aggregated around the calcified area in calcified PAV. Macrophages infiltrated in the calcified, lacerated and dehiscence PAV. However, MMP-1 expression was mainly found in the lacerated PAV. The VIM(+)/SMA(-) and VIM(+)/CD31(-) cells were found in PAV. The endothelia rate of dehiscence leaflets were higher than that of calcified and lacerated leaflets. A large amount of CD31 positive cells aggregated in the spongy layer in the lacerated and dehiscence PAV. Conclusions Cell regeneration and infiltration is a double edged sword for the PAV deterioration. Valve interstitial cells (VIC) have essential role in PAV calcification. Macrophages infiltration maybe involve in the different type of SVD, but only MMP-1expression involves in leaflets laceration. VIM(+)/CD31(-) valve endothelial cells (VECs) protect the PAV against the formation of calcified and lacerated lesions. The existence of untransformed VECs maybe one of pathologic substrate of PAV tear and dehiscence, although they can prevent VICs activation and subsequent valve fibrosis and calcification.

miRNA Expression Profiling Uncovers a Role of miR-139-5p in Regulating the Calcification of Human Aortic Valve Interstitial Cells

Calcific aortic valve disease (CAVD) is the most common structural heart disease, and the morbidity is increased with elderly population. Several microRNAs (miRNAs) have been identified to play crucial roles in CAVD, and numerous miRNAs are still waiting to be explored. In this study, the miRNA expression signature in CAVD was analyzed unbiasedly by miRNA-sequencing, and we found that, compared with the normal control valves, 152 miRNAs were upregulated and 186 miRNAs were downregulated in calcified aortic valves. The functions of these differentially expressed miRNAs were associated with cell differentiation, apoptosis, adhesion and immune response processes. Among downregulated miRNAs, the expression level of miR-139-5p was negatively correlated with the osteogenic gene RUNX2, and miR-139-5p was also downregulated during the osteogenic differentiation of primary human aortic valve interstitial cells (VICs). Subsequent functional studies revealed that miR-139-5p overexpression inhibited the osteogenic differentiation of VICs by negatively modulating the expression of pro-osteogenic gene FZD4 and CTNNB1. In conclusion, these results suggest that miR-139-5p plays an important role in osteogenic differentiation of VICs via the Wnt/β-Catenin pathway, which may further provide a new therapeutic target for CAVD.

Carbon Nanotubes Substrates Alleviate Pro-Calcific Evolution in Porcine Valve Interstitial Cells

The quest for surfaces able to interface cells and modulate their functionality has raised, in recent years, the development of biomaterials endowed with nanocues capable of mimicking the natural extracellular matrix (ECM), especially for tissue regeneration purposes. In this context, carbon nanotubes (CNTs) are optimal candidates, showing dimensions and a morphology comparable to fibril ECM constituents. Moreover, when immobilized onto surfaces, they demonstrated outstanding cytocompatibility and ease of chemical modification with ad hoc functionalities. In this study, we interface porcine aortic valve interstitial cells (pVICs) to multi-walled carbon nanotube (MWNT) carpets, investigating the impact of surface nano-morphology on cell properties. The results obtained indicate that CNTs significantly affect cell behavior in terms of cell morphology, cytoskeleton organization, and mechanical properties. We discovered that CNT carpets appear to maintain interfaced pVICs in a sort of “quiescent state”, hampering cell activation into a myofibroblasts-like phenotype morphology, a cellular evolution prodromal to Calcific Aortic Valve Disease (CAVD) and characterized by valve interstitial tissue stiffening. We found that this phenomenon is linked to CNTs’ ability to alter cell tensional homeostasis, interacting with cell plasma membranes, stabilizing focal adhesions and enabling a better strain distribution within cells. Our discovery contributes to shedding new light on the ECM contribution in modulating cell behavior and will open the door to new criteria for designing nanostructured scaffolds to drive cell functionality for tissue engineering applications.

IL-8 promotes the calcification of human aortic valve interstitial cells, which is prevented through antagonizing CXCR1 and CXCR2 receptors

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

Hypoxic Culture Maintains Cell Growth of the Primary Human Valve Interstitial Cells with Stemness

The characterization of aortic valve interstitial cells (VICs) cultured under optimal conditions is essential for understanding the molecular mechanisms underlying aortic valve stenosis. Here, we propose 2% hypoxia as an optimum VIC culture condition. Leaflets harvested from patients with aortic valve regurgitation were digested using collagenase and VICs were cultured under the 2% hypoxic condition. A significant increase in VIC growth was observed in 2% hypoxia (hypo-VICs), compared to normoxia (normo-VICs). RNA-sequencing revealed that downregulation of oxidative stress-marker genes (such as superoxide dismutase) and upregulation of cell cycle accelerators (such as cyclins) occurred in hypo-VICs. Accumulation of reactive oxygen species was observed in normo-VICs, indicating that low oxygen tension can avoid oxidative stress with cell-cycle arrest. Further mRNA quantifications revealed significant upregulation of several mesenchymal and hematopoietic progenitor markers, including CD34, in hypo-VICs. The stemness of hypo-VICs was confirmed using osteoblast differentiation assays, indicating that hypoxic culture is beneficial for maintaining growth and stemness, as well as for avoiding senescence via oxidative stress. The availability of hypoxic culture was also demonstrated in the molecular screening using proteomics. Therefore, hypoxic culture can be helpful for the identification of therapeutic targets and the evaluation of VIC molecular functions in vitro.

Activation of transcriptional factor ZBTB16 expression during osteogenic differentiation of mesenchymal stem cells

Aim. Calcified aortic valve stenosis is the third leading cause of cardiovascular disease. The mechanisms underlying this process remain unclear, however, it is known that they are largely similar to the formation of bone tissue during embryonic development, as well as in the postnatal period during regeneration. There is evidence for the involvement of Zinc Finger and BTB Domain Containing 16 (ZBTB16) in skeletal development. At the same time, a number of studies carried out on different types of cell cultures indicate a contradictory and ambiguous effect of ZBTB16 on RUNX2 expression. Thus, the aim of this study was to investigate the dynamic variability of ZBTB16 expression, as well as its role in aortic valve calcification.Methods. The study used different types of mesenchymal cells cultures - aortic valve interstitial cells, umbilical cord mesenchymal stem cells, ligament stem cells and dental pulp stem cells. Changes in ZBTB16 and RUNX2 expression levels under the influence of osteogenic stimuli, as well as during exogenous activation of ZBTB16, were analyzed using real-time PCR. Expression levels of some osteogenic markers - BMP2,4, COL1A1, IBSP, DLX2, PDK4 - were analyzed in the interstitial cells of the aortic valve.Results. The results of the study indicate that a significant increase in the expression of ZBTB16 is observed during the induction of osteogenic differentiation of various cell cultures - interstitial cells of the aortic valve, mesenchymal stem cells of the umbilical cord, stem cells of the ligaments and dental pulp. Apparently, the processes of osteogenic differentiation of aortic valve interstitial cells, in the presence of dexamethasone in cultivation medium, are provided through RUNX2-dependent signaling for the further activation of osteogenic markers.Conclusion. The study of modulation of cellular signals by ZBTB16, when activating or suppressing the work of a transcriptional factor, in the future may bring us closer to the ability to enhance the regenerative abilities of bone tissue cells or, conversely, prevent calcification of the aortic valve tissues.

Decreased Glucagon-Like Peptide-1 Is Associated With Calcific Aortic Valve Disease: GLP-1 Suppresses the Calcification of Aortic Valve Interstitial Cells

Objectives: This study explores the concentration and role of glucagon-like peptide-1 (GLP-1) in calcific aortic valve disease (CAVD).Background: Calcific aortic valve disease is a chronic disease presenting with aortic valve degeneration and mineralization. We hypothesized that the level of GLP-1 is associated with CAVD and that it participates in the calcification of aortic valve interstitial cells (AVICs).Methods: We compared the concentration of GLP-1 between 11 calcific and 12 normal aortic valve tissues by immunohistochemical (IHC) analysis. ELISA was used to measure GLP-1 in serum of the Control (n = 197) and CAVD groups (n = 200). The effect of GLP-1 on the calcification of AVICs and the regulation of calcific gene expression were also characterized.Results: The GLP-1 concentration in the calcific aortic valves was 39% less than that in the control non-calcified aortic valves. Its concentration in serum was 19.3% lower in CAVD patients. Multivariable regression analysis demonstrated that GLP-1 level was independently associated with CAVD risk. In vitro, GLP-1 antagonized AVIC calcification in a dose- and time-dependent manner and it down-regulated RUNX2, MSX2, BMP2, and BMP4 expression but up-regulated SOX9 expression.Conclusions: A reduction in GLP-1 was associated with CAVD, and GLP-1 participated in the mineralization of AVICs by regulating specific calcific genes. GLP-1 warrants consideration as a novel treatment target for CAVD.