Inhibition of Phosphate-Induced Vascular Smooth Muscle Cell Osteo-/Chondrogenic Signaling and Calcification by Bafilomycin A1 and Methylamine

Abstract

Background/Aims: Excessive phosphate concentrations trigger vascular calcification, an active process promoted by osteoinduction of vascular smooth muscle cells (VSMCs) with increased expression and activity of transcription factor RUNX2 (Core-binding factor α1, CBFA1), alkaline phosphatase (ALPL), TGFß1, transcription factor NFAT5, and NFAT5-sensitive transcription factor SOX9. The osteoinductive signaling and vascular calcification of hyperphosphatemic klotho-hypomorphic mice could be reversed by treatment with NH₄Cl, effects involving decrease of TGFß1 and inhibition of NFAT5-dependent osteoinductive signaling. Known effects of NH₄Cl include alkalinization of acidic cellular compartments. The present study explored whether osteo-/chondrogenic signaling could be influenced by alkalinization of acidic cellular compartments following inhibition of the vacuolar H⁺ ATPase with bafilomycin A1 or following dissipation of the pH gradient across the membranes of acidic cellular compartments with methylamine. Methods: Primary human aortic smooth muscle cells (HAoSMCs) were treated with high phosphate to trigger osteo-/chondrogenic signaling and calcification in the absence or presence of bafilomycin A1 or methylamine. Calcium content was determined using a QuantiChrom Calcium assay, ALP activity by a colorimetric assay and transcript levels by quantitative RT-PCR. Results: High phosphate increased significantly the calcium deposition, CBFA1 and ALPL mRNA expression as well as alkaline phosphatase activity in HAoSMCs, all effects ameliorated by both, bafilomycin A1 and methylamine. High phosphate further significantly up-regulated the mRNA levels of TGFB1, NFAT5 and SOX9, effects significantly blunted by additional treatment with bafilomycin A1 or methylamine. Treatment of HAoSMCs with human TGFß1 protein or high phosphate up-regulated NFAT5, SOX9, CBFA1 and ALPL mRNA expression to similarly high levels which could not be further increased by combined treatment with high phosphate and TGFß1. Bafilomycin A1 failed to reverse the osteo-/chondrogenic signaling triggered by high phosphate together with TGFß1. Conclusions: Inhibition of the vacuolar H⁺ ATPase or dissipation of the pH gradient across the membranes of acidic cellular compartments both disrupt osteo-/chondrogenic signaling and calcium deposition in VSMCs, observations supporting the hypothesis that vascular calcification requires acidic cellular compartments.

SGK1 induces vascular smooth muscle cell calcification through NF-κB signaling

“…Accordingly, we demonstrated that SGK1 mRNA and protein expression in VSMCs was upregulated by dexamethasone and aldosterone as well as phosphate exposure. Phosphate exposure triggers upregulation of TGF-β and BMP-2, which augment the osteo-/chondrogenic transdifferentiation of VSMCs (32,33). Treatment with either TGF-β or BMP-2 increases SGK1 expression.…”

Defective recruitment of motor proteins to autophagic compartments contributes to autophagic failure in aging

“…We opted for this combination as we have extensively shown that at these concentrations and for the duration of our studies (always <6 hr) vesicular fusion was not affected (no additive effect of vinblastine) and that the entire effect can be attributed to reduced degradation at the lysosomal compartment (Klionsky, Elazar, Seglen & Rubinsztein, 2008). In addition, this combination does not have an impact on calcium stores nor does it induce increased autophagosome biogenesis as described for other agents and longer treatments (Alesutan et al., 2015; Rubinsztein et al., 2009). Morphometric analysis of autophagic compartments by electron microscopy was carried out as described previously (Bejarano et al., 2014).…”

Roles of High Mobility Group Box 1 in Cardiovascular Calcification

“…HMGB1 facilitates assembly of nuclear proteins and participates in DNA replication, recombination, transcription and repair [26, 27]. However, upon cell activation, injury or death, HMGB1 is translocated outside of the cell [9, 28]. Recent evidence reveals that the initial translocation of HMGB1 from the nuclear to the cytoplasm is regulated by JAK/STAT1-mediated acetylation [29], with subsequent extracellular release being partly controlled by dsRNA-activated protein kinase R (PKR)/inflammasome-mediated pyroptosis [30].…”

“…Inflammatory cytokines induce generation of reactive oxygen species (ROS), which have also been implicated in cardiovascular calcification [8]. Moreover, the pH of acidic cellular compartments also plays a role in osteo-/chondrogenic transformation and calcification of VSMCs [9]. …”