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The endomembrane homeostasis controls the phenotype switching of vascular smooth muscle cells

ABSTRACT: Background Vascular calcification contributes to cardiovascular disease. One important process is the phenotypic transition of vascular smooth muscle cells (SMCs) from a contractile to a mineralizing phenotype, releasing calcifying extracellular vesicles (EVs). The endolysosomal system is involved in EV biogenesis and regulates essential cell physiological functions. We hypothesize that alterations in endomembrane homeostasis affect SMC phenotypic identity and EV cargo, thereby regulating vascular calcification. Results In human calcified carotid plaques and calcifying SMCs, the abundance of FYVE-Type Zinc Finger Containing Phosphoinositide Kinase (PIKfyve), an essential lipid kinase, in the endomembrane maturation, is increased. Phosphatidylinositol 3-phosphate (PI3P) - the substrate of PIKfyve - was decreased in cellular membranes of calcifying SMCs (-40%) and recovered by Apilimod, a small molecule PIKfyve inhibitor. In vitro, Apilimod reduced the osteogenic features, like matrix mineralization (-77%), collagen secretion (-99%), and tissue non-specific alkaline phosphatase (TNAP) protein expression (-86%) in calcifying SMCs. Moreover, Apilimod-induced EVs exhibited fewer mineral-positive EVs and reduced aggregation potential (-80%), suggesting diminished EV calcification potential. A proteomic analysis of the EV cargo demonstrated that Apilimod reduces TNAP cargo, which was supported by western blot. In a murine atherosclerosis model, Apilimod reduced aortic calcification assessed by ex vivo osteosense imaging. Transcriptomics revealed phenotypic transitions of calcifying SMCs after PIKfyve inhibition. Apilimod promoted adipogenic markers (PPARG: +218%, FABP3: +2250%), fatty acid uptake (+86%), lipid droplets (OilRed, Raman spectroscopy), and increased the expression of macrophage-like SMC markers (CD68: +500%; LGALS3: +132%), suggesting an alternative SMC phenotype. Kinome and in silico analyses identified YAP1 deactivation as a potential mechanism. Re-activation of YAP1 partially antagonized Apilimod-dependent effects in calcifying SMCs. Conclusion PIKfyve inhibition inhibits the osteogenic transition of SMCs while promoting a phenotypic adaptation towards adipogenic/pro-inflammatory SMCs, which is partially mediated by YAP1. The phenotypic identity of calcifying SMCs is sensitive to alterations of the endomembrane homeostasis.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Christian Preisinger

LAB HEAD: Claudia Göttsch

PROVIDER: PXD060400 | Pride | 2026-01-17

REPOSITORIES: Pride

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			Action	DRS
	20250129_153951_ClGoe_exo_human_canonical_bovine_full_universalcontaminant.sne	Other
	ClGoe_exo_human_canonical_bovine_full_universalcontaminant_Report_BGS_Factory_Report.tsv	Tabular
	ClGoe_exo_human_canonical_bovine_full_universalcontaminant_Report_proteins.tsv	Tabular
	OR3_2023_08_26_ClGoe_exo_sDIA_r1_1.raw	Raw
	OR3_2023_08_26_ClGoe_exo_sDIA_r1_2.raw	Raw

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Project description:Vascular calcification is a complex process and has been associated with aging, diabetes, chronic kidney disease (CKD). Although there have been several studies studying the role of miRNAs (miRs) in bone osteogenesis, little is known about the role of miRs in vascular calcification and their role in the pathogenesis of vascular abnormalities. Matrix vesicles (MV) are known to play an important role in initiating vascular smooth muscle cell (VSMC) calcification. In the present study, we performed miRNA microarray analysis to identify the dysregulated miRs between MV and VSMC derived from CKD rats to understand the role of post-transcriptional regulatory networks governed by these miRNAs in vascular calcification and to uncover the differential miRNA content of MV. The percentage of miRNA to total RNA was increased in MV compared to VSMC. Comparison of expression profiles of miRNA by microarray demonstrated 33 miRs to be differentially expressed with the majority (~ 57%) of them down-regulated. Target genes controlled by differentially expressed miRNAs were identified utilizing two different complementary computational approaches Miranda and Targetscan to understand the functions and pathways that may be affected due to the production of MV from calcifying VSMC thereby contributing to the regulation of genes by miRs. We found several processes including vascular smooth muscle contraction, response to hypoxia and regulation of muscle cell differentiation to be enriched. Signaling pathways identified included MAP-kinase and wnt signaling that have previously been shown to be important in vascular calcification. In conclusion, our results demonstrate that miRs are concentrated in MV from calcifying VSMC, and that important functions and pathways are affected by the miRs dysregulation between calcifying VSMC and the MV they produce. This suggests that miRs may play a very important regulatory role in vascular calcification in CKD by controlling an extensive network of post-transcriptional targets. Compare miRNA from matrix vesicles to miRNA from vascular smooth muscle cells that gave rise to the matrix vesicles from 3 sets of MV and VSMC derived from 3 normal and 3 CKD rats

Project description:Tissue Factor Pathway Inhibitor-2 is Induced by Fluid Shear Stress in Vascular Smooth Muscle Cells and Affects Cell Proliferation and Survival Introduction: Vascular smooth muscle cells (SMCs) are exposed to fluid shear stress (FSS) after interventional procedures such as balloon-angioplasty. Whereas the effects of hemodynamic forces on endothelial cells are explored in detail, the influence of FSS on smooth muscle cell function is poorly characterized. Here, we investigated the effect of FSS on SMC gene expression and function. Methods: Laminar FSS of arterial level (14 dynes/cm2) was applied to SMC cultures for 24 h in a parallel-plate flow chamber. The effect of FSS on gene expression was first screened with microarray technology and the results further verified by real time (RT) PCR and immunoblotting. Protein expression was also studied in the rat carotid artery after balloon-injury and DNA synthesis and apoptosis was examined in SMCs in vitro. Results: Microarrays identified tissue-pathway inhibitor-2 (TFPI-2) as the most differentially expressed gene by FSS in cultured SMCs. The regulatory effect of FSS on the expression of TFPI-2 was confirmed by RT-PCR and immunobloting demonstrating a more than 400-fold increase in TFPI-2 expression in SMCs exposed to FSS compared to static controls and a consistent upregulation at the protein level. Functionally, SMC proliferation was decreased by FSS and recombinant TFPI-2 was found to inhibit SMC proliferation and induce SMC apoptosis as indicated by activation of caspase-3. In vivo, TFPI-2 expression was found to be up-regulated 5, 10 and 20 h after rat carotid balloon-injury and immunohistochemistry demonstrated TFPI-2 protein in luminal SMCs exposed to FSS in rat carotid intimal hyperplasia 10 days after balloon-injury. Conclusion: FSS strongly influence gene expression in cultured SMCs and induce TFPI-2 expression, which is also expressed after rat carotid balloon injury in luminal SMCs exposed to FSS. Functionally, TFPI-2 may play an important role in vessel wall repair by regulating SMC proliferation and survival. Further studies are needed to elucidate the mechanisms by which TFPI-2 control SMC function. 3 x 2 samples from a paired fluid shear stress experiment on smooth muscle cells.

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The endomembrane homeostasis controls the phenotype switching of vascular smooth muscle cells

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