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• W2763606722 abstract "In patients with chronic kidney and end-stage renal diseases, the major risk factor for progression of arterial calcification is the presence of existing (baseline) calcification. Here, we tested whether calcification of arteries is extended from calcified vascular smooth muscle cells (VSMCs) to adjacent normal cells by matrix vesicle–induced alteration of cell signaling. Matrix vesicles isolated from VSMC of rats with chronic kidney disease were co-cultured with VSMCs from normal littermates. Endocytosis of vesicles by recipient cells was confirmed by confocal microscopy. The addition of cellular matrix vesicles with characteristics of exosomes and low fetuin-A content enhanced the calcification of recipient VSMC. Further, only cellular-derived matrix vesicles induced an increase in intracellular calcium ion concentration, NOX1 (NADPH oxidase) and the anti-oxidant superoxide dismutase-2 in recipient normal VSMC. The increase in intracellular calcium ion concentration was due to release from endoplasmic reticulum and partially attributed to the activation of both NOX1 and mitogen-activated protein kinase (MEK1 and Erk1/2) signaling, since inhibiting both pathways blocked the increase in intracellular calcium ion in recipient VSMC. In contrast, matrix vesicles isolated from the media had no effect on the intracellular calcium ion concentration or MEK1 signaling, and did not induce calcification. However, media matrix vesicles did increase Erk1/2, although not to the level of cellular matrix vesicles, and NOX1 expression. Blockade of NOX activity further inhibited the cellular matrix vesicle–induced accelerated calcification of recipient VSMC, suggesting a potential therapeutic role of such inhibition. Thus, addition of cellular-derived matrix vesicles from calcifying VSMC can accelerate calcification by inducing cell signaling changes and phenotypic alteration of recipient VSMC. In patients with chronic kidney and end-stage renal diseases, the major risk factor for progression of arterial calcification is the presence of existing (baseline) calcification. Here, we tested whether calcification of arteries is extended from calcified vascular smooth muscle cells (VSMCs) to adjacent normal cells by matrix vesicle–induced alteration of cell signaling. Matrix vesicles isolated from VSMC of rats with chronic kidney disease were co-cultured with VSMCs from normal littermates. Endocytosis of vesicles by recipient cells was confirmed by confocal microscopy. The addition of cellular matrix vesicles with characteristics of exosomes and low fetuin-A content enhanced the calcification of recipient VSMC. Further, only cellular-derived matrix vesicles induced an increase in intracellular calcium ion concentration, NOX1 (NADPH oxidase) and the anti-oxidant superoxide dismutase-2 in recipient normal VSMC. The increase in intracellular calcium ion concentration was due to release from endoplasmic reticulum and partially attributed to the activation of both NOX1 and mitogen-activated protein kinase (MEK1 and Erk1/2) signaling, since inhibiting both pathways blocked the increase in intracellular calcium ion in recipient VSMC. In contrast, matrix vesicles isolated from the media had no effect on the intracellular calcium ion concentration or MEK1 signaling, and did not induce calcification. However, media matrix vesicles did increase Erk1/2, although not to the level of cellular matrix vesicles, and NOX1 expression. Blockade of NOX activity further inhibited the cellular matrix vesicle–induced accelerated calcification of recipient VSMC, suggesting a potential therapeutic role of such inhibition. Thus, addition of cellular-derived matrix vesicles from calcifying VSMC can accelerate calcification by inducing cell signaling changes and phenotypic alteration of recipient VSMC. Vascular calcification is highly prevalent in chronic kidney disease (CKD) and is a major cause of morbidity and mortality.1Chen N.X. Moe S.M. Vascular calcification: pathophysiology and risk factors.Curr Hypertens Rep. 2012; 14: 228-237Crossref PubMed Scopus (124) Google Scholar, 2Chen N.X. Moe S.M. Pathophysiology of vascular calcification.Curr Osteoporos Rep. 2015; 13: 372-380Crossref PubMed Scopus (72) Google Scholar, 3Chen N.X. O'Neill K.D. Chen X. Moe S.M. Annexin-mediated matrix vesicle calcification in vascular smooth muscle cells.J Bone Miner Res. 2008; 23: 1798-1805Crossref PubMed Scopus (119) Google Scholar The prevalence of calcification increases with worsening kidney disease4Mehrotra R. Adler S. Coronary artery calcification in nondialyzed patients with chronic kidney diseases.Am J Kidney Dis. 2005; 45: 963Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar; by the time patients reach the dialysis stage, 70%–80% have significant coronary artery calcification.5Kalpakian M.A. Mehrotra R. Vascular calcification and disordered mineral metabolism in dialysis patients.Semin Dial. 2007; 20: 139-143Crossref PubMed Scopus (47) Google Scholar On histology, medial calcification often begins to show itself as small areas within the medial layer. More-advanced lesions expand to become circumferential throughout the entire medial layer.6Moe S.M. O'Neill K.D. Duan D. et al.Medial artery calcification in ESRD patients is associated with deposition of bone matrix proteins.Kidney Int. 2002; 61: 638-647Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar Risk factors for the presence of calcification in patients on dialysis include older age, diabetes, and disordered mineral metabolism, including hyperphosphatemia and hypercalcemia.2Chen N.X. Moe S.M. Pathophysiology of vascular calcification.Curr Osteoporos Rep. 2015; 13: 372-380Crossref PubMed Scopus (72) Google Scholar However, patients who have calcification at the start of dialysis have greater progression compared with those who do not have calcification, despite similar clinical and biochemical risk factors.7Block G.A. Raggi P. Bellasi A. Kooienga L. Spiegel D.M. Mortality effect of coronary calcification and phosphate binder choice in incident hemodialysis patients.Kidney Int. 2007; 71: 438-441Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar This difference suggests that expansion of existing calcification may occur through different mechanisms than does initiation of vascular calcification. Studies from the past decade have led to increased understanding of the pathophysiology of vascular calcification. The VSMC must become synthetic with increased intracellular calcium8Berra-Romani R. Mazzocco-Spezzia A. Pulina M.V. Golovina V.A. Ca2+ handling is altered when arterial myocytes progress from a contractile to a proliferative phenotype in culture.Am J Physiol Cell Physiol. 2008; 295: C779-C790Crossref PubMed Scopus (176) Google Scholar, 9Rodenbeck S.D. Zarse C.A. McKenney-Drake M.L. et al.Intracellular calcium increases in vascular smooth muscle cells with progression of chronic kidney disease in a rat model.Nephrol Dial Transplant. 2016; 32: 450-458Google Scholar ([Ca2+]i) and downregulation of myocardin and alpha-smooth muscle actin,10Chen N.X. Kiattisunthorn K. O'Neill K.D. et al.Decreased MicroRNA Is Involved in the vascular remodeling abnormalities in chronic kidney disease (CKD).PLoS One. 2013; 8: e64558Crossref PubMed Scopus (89) Google Scholar followed by de-differentiation via upregulation of the “bone” (runt-related) transcription factor RUNX2.11Speer M.Y. Li X. Hiremath P.G. Giachelli C.M. Runx2/Cbfa1, but not loss of myocardin, is required for smooth muscle cell lineage reprogramming toward osteochondrogenesis.J Cell Biochem. 2010; 110: 935-947Crossref PubMed Scopus (110) Google Scholar, 12Moe S.M. Duan D. Doehle B.P. O'Neill K.D. Chen N.X. Uremia induces the osteoblast differentiation factor Cbfa1 in human blood vessels.Kidney Int. 2003; 63: 1003-1011Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar These transformed, or de-differentiated, synthetic VSMCs initiate calcification by synthesizing small (50–200 nm) vesicles that initiate calcification on the extracellular matrix. In bone, these vesicles are called matrix vesicles (MVs), as they were identified as being an integral part of the conversion of hypertrophic chondrocytes in epiphyses of bones as they develop into mineralized bone13Anderson H.C. Molecular biology of matrix vesicles.Clin Orthop Relat Res. 1995; : 266-280PubMed Google Scholar, 14Anderson H.C. Garimella R. Tague S.E. The role of matrix vesicles in growth plate development and biomineralization.Front Biosci. 2005; 10: 822-837Crossref PubMed Scopus (241) Google Scholar Over the past decade, appreciation of the role of vesicles in cell–cell communication in nonmineralized tissues.has also increased.15Camussi G. Deregibus M.C. Tetta C. Paracrine/endocrine mechanism of stem cells on kidney repair: role of microvesicle-mediated transfer of genetic information.Curr Opin Nephrol Hypertens. 2010; 19: 7-12Crossref PubMed Scopus (135) Google Scholar Vesicles are heterogeneous and originate from the endosome or plasma membrane of cells. Although nomenclature and isolation techniques vary, vesicles can be released through outward budding of the plasma membrane (known as “shedding microvesicles”) or inward budding of the endosomal membrane, resulting in the formation of multivesicular bodies.16Abels E.R. Breakefield X.O. Introduction to extracellular vesicles: biogenesis, RNA cargo selection, content, release, and uptake.Cell Mol Neurobiol. 2016; 36: 301-312Crossref PubMed Scopus (819) Google Scholar We have previously characterized differences between vesicles isolated from the media and the cells of calcifying bovine VSMCs: those from the media contain fetuin-A and do not readily mineralize, whereas those from cells do not contain fetuin-A and do mineralize.3Chen N.X. O'Neill K.D. Chen X. Moe S.M. Annexin-mediated matrix vesicle calcification in vascular smooth muscle cells.J Bone Miner Res. 2008; 23: 1798-1805Crossref PubMed Scopus (119) Google Scholar, 17Chen N.X. Kircelli F. O'Neill K.D. Chen X. Moe S.M. Verapamil inhibits calcification and matrix vesicle activity of bovine vascular smooth muscle cells.Kidney Int. 2010; 77: 436-442Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar Kapustin et al.18Kapustin A.N. Chatrou M.L. Drozdov I. et al.Vascular smooth muscle cell calcification is mediated by regulated exosome secretion.Circ Res. 2015; 116: 1312-1323Crossref PubMed Scopus (330) Google Scholar also compared vesicles from the media of calcifying human VSMC and found a similar proteomic profile to that of both cellular and media vesicles from osteoblasts.18Kapustin A.N. Chatrou M.L. Drozdov I. et al.Vascular smooth muscle cell calcification is mediated by regulated exosome secretion.Circ Res. 2015; 116: 1312-1323Crossref PubMed Scopus (330) Google Scholar Exosome production was increased by factors of clinical significance in CKD: increased extracellular calcium, tumor necrosis factor-α, and platelet-derived growth factor BB. They further identified these vesicles to be enriched with tetraspanins (CD9, CD63, and CD81), indicating origination from multivesicular bodies, and found such multivesicular bodies in calcified human arteries.18Kapustin A.N. Chatrou M.L. Drozdov I. et al.Vascular smooth muscle cell calcification is mediated by regulated exosome secretion.Circ Res. 2015; 116: 1312-1323Crossref PubMed Scopus (330) Google Scholar We and other groups have found that the origin and content of these MVs appears to be a central determinant of their mineralization potential.3Chen N.X. O'Neill K.D. Chen X. Moe S.M. Annexin-mediated matrix vesicle calcification in vascular smooth muscle cells.J Bone Miner Res. 2008; 23: 1798-1805Crossref PubMed Scopus (119) Google Scholar, 19Kapustin A.N. Davies J.D. Reynolds J.L. et al.Calcium regulates key components of vascular smooth muscle cell-derived matrix vesicles to enhance mineralization.Circ Res. 2011; 109: e1-e12Crossref PubMed Scopus (277) Google Scholar This unique function, depending on content, is further supported by findings that vesicles isolated from atherosclerotic plaque (macrophage derived) and medial arterial calcification also differ in content.20Reid D.G. Shanahan C.M. Duer M.J. et al.Lipids in biocalcification: contrasts and similarities between intimal and medial vascular calcification, and boneby NMR.J Lipid Res. 2012; 53: 1569-1575Crossref PubMed Scopus (29) Google Scholar Multiple studies have demonstrated that vesicles can be taken up by recipient cells (these have been reviewed21Mulcahy L.A. Pink R.C. Carter D.R. Routes and mechanisms of extracellular vesicle uptake.J Extracell Vesicles. 2014; 3Crossref PubMed Scopus (1483) Google Scholar). Given the pathologic appearance of vesicles in areas of vascular calcification in vivo and the role in calcification in vitro, we hypothesized that the transmission of vesicles from CKD cells to normal cells would facilitate calcification of the recipient cells and serve as a model of the extension or propagation of calcification observed in patients who have CKD. Given the parallel pathophysiology of both physiologic and pathologic calcification, we use the term matrix vesicles (MVs). We compared 4 sources of MVs: cellular-derived MVs (from CKD VSMC incubated with either high-level phosphorus [calcifying] or normal-level phosphorus [control]) or media-derived MVs from calcifying or control CKD VSMC. These MVs were added to recipient normal rat VSMC as a co-culture and incubated with calcification media (high phosphorus) for 7 days. The results (Figure 1) revealed that both cellular sources of MVs from CKD VSMC (whether they were derived from donor VSMC incubated in normal-level or high-level phosphorus) induced calcification of the recipient VSMC incubated in high-phosphorus media. In contrast, MVs isolated from the media of cultured VSMC had no effect on calcification of recipient VSMC. These results suggest that the source of MVs, rather than the phosphorus conditions of the VSMC from which the MVs are derived, is what affects calcification. Figure 2a demonstrates that the MVs isolated from both sources of cellular VSMC contained annexin II, V, and VI, with higher expression in VSMCs that were incubated with additional phosphorus (calcifying). In contrast, the MVs isolated from the media had a lower level of annexins, and no differences were found whether they were from cells incubated in high-phosphorus media or not (Figure 2a and b). As we and others have found previously,3Chen N.X. O'Neill K.D. Chen X. Moe S.M. Annexin-mediated matrix vesicle calcification in vascular smooth muscle cells.J Bone Miner Res. 2008; 23: 1798-1805Crossref PubMed Scopus (119) Google Scholar, 22Reynolds J.L. Joannides A.J. Skepper J.N. et al.Human vascular smooth muscle cells undergo vesicle-mediated calcification in response to changes in extracellular calcium and phosphate concentrations: a potential mechanism for accelerated vascular calcification in ESRD.J Am Soc Nephrol. 2004; 15: 2857-2867Crossref PubMed Scopus (758) Google Scholar vesicles isolated from the cell media contained markedly increased fetuin-A. But again, whether the originating CKD VSMC cells were incubated with or without phosphorus made little difference (Figure 2a and b). Both cellular and media MVs from calcifying (high-phosphorous) conditions contain the exosomal tetraspanins CD63, CD81, and CD9, but cellular MVs are enriched with CD63, whereas media MVs are enriched with CD81 and CD9 (Figure 2a and b). These findings are consistent with those of Lotvall et al.,23Lotvall J. Hill A.F. Hochberg F. et al.Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles.J Extracell Vesicles. 2014; 3: 26913Crossref PubMed Scopus (1701) Google Scholar who reported that although various types of extracellular vesicles contain many common exosome-enriched markers, such as tetraspanins, the relative proportions of these markers seem to vary in the different types. Despite these differences, examination by electron microscopy (Figure 2c) showed that both cellular and media MVs are membrane-bound vesicles of approximately 100 nm in diameter, consistent with the size of exosomes as described in the literature.24Thery C. Regnault A. Garin J. et al.Molecular characterization of dendritic cell-derived exosomes. Selective accumulation of the heat shock protein hsc73.J Cell Biol. 1999; 147: 599-610Crossref PubMed Scopus (856) Google Scholar No nanotubes were identified by any imaging technique.25Vallabhaneni K.C. Haller H. Dumler I. Vascular smooth muscle cells initiate proliferation of mesenchymal stem cells by mitochondrial transfer via tunneling nanotubes.Stem Cells Dev. 2012; 21: 3104-3113Crossref PubMed Scopus (134) Google ScholarFigure 2Comparison of content of matrix vesicles (MVs) isolated from cells or media. MVs isolated from the same 4 sources as in Figure 1 were analyzed for content of annexin II(36 kDa), annexin V (36 kDa), annexin VI (47-51 kDa), fetuin-A (59 kDa), CD63 (core protein, MV 26 kDa), CD81 (22–26 kDA), and CD9 (24 kDa) by Western blot (a) with quantification of band intensity normalized by Ponceau S (b). MVs isolated from cells had increased expression of annexins and CD63, but neglible fetuin-A compared to that from MVs isolated from media. Isolation from cells in high-phosphorus (calcifying) media in general increased expression. In contrast, in the MVs isolated from the media, there was high fetuin-A content, high levels of CD81 and CD9, and no differences when isolated from VSMC with and without calcifying (high-phosphorus) media. Transmission electron microscopy revealed that both cellular (c, left) and media MV (c, right) show uniform size of 100 mm–diameter, membrane-bound vesicles. Data are shown as mean ± SD (n = 3 separate experiments). Cal, calcified (MV isolated from CKD VSMC in high-phosphorus media); Ct, control (MV isolated from CKD VSMC in normal-phosphorus media); *P < 0.05, Ct MV versus Cal MV same source (cellular MV or media MV); #P < 0.05, cellular MV versus media MV, same condition (control or calcifying/high-phosphorus). Black bar = 200 nm (c). To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine if VSMC can uptake MVs, we labeled MVs with the membrane fluorescent dye PKH26 and examined uptake by confocal microscopy. The results revealed that MVs added to recipient VSMC were endocytosed by the VSMC (Figure 3a; red) and co-localized with Alexa 647–labeled dextran (Figure 3b; blue) but not transferrin (Figure 3c, green) after 24 hours. This finding indicates that once they are endocytosed, MVs become located in lysosome (Figure 3d and 3e, purple). Additional studies revealed that MVs derived from media are similarly endocytosed, and MVs from VSMC incubated in normal- versus high-phosphorus media do not differ (data not shown). Thus, the endocytosis of the MVs by recipient cells is similar and does not depend on MV content. To determine if endocytosed MVs induced cell-signaling changes in the recipient VSMC, we examined MV-mediated alterations of intracellular calcium concentration ([Ca2+]i) in recipient VSMC. The addition of cellular MVs (regardless of whether they were sourced from calcifying [high-phosphorus] or control [normal-phosphorus] CKD VSMC) to recipient VSMC increased the [Ca2+]i by 60 minutes, with a continued increase over the 4 hours tested (Figure 4a). In contrast, the addition of media-derived MVs had no effects on [Ca2+]i (Figure 4b). We therefore continued the study using only cellular-derived MVs from calcifying CKD VSMC. To confirm the results, VSMCs were labeled with calcium fluorescence dye Fluo-4 and MV-induced calcium transients in VSMCs examined using spinning-disk microscopy. The results revealed that cellular MVs increased calcium fluorescence intensity (Supplemental Figure S1), confirming our time-course experiments. The MV-induced increase in [Ca2+]i in recipient VSMC was partially mediated by inositol 1,4,5 triphosphate (IP3)–induced [Ca2+]i release, as treatment with 2-aminoethoxydiphenyl borate (2-APB) reduced the MV-induced increase in [Ca2+]i (indicating release of calcium from sarcoplasmic reticulum), but using verapamil to block the external entry of calcium into the L-type calcium channel had no effect (Figure 4b). To determine the role of MVs on MAPK signaling in VSMC, cellular or media MVs were isolated from calcifying CKD VSMC. First, cellular MVs were incubated with normal recipient VSMC at various time points, and the activation of MAPK was assessed using a PathScan MAP Kinase Multi-Target Sandwich ELISA kit (Cell Signaling Technology, Danvers, MA). The activity of phospho-Erk1/2 and phosphoMEK1 was found to be increased at 30 minutes and remained similarly increased at 2 hours and 4 hours in VSMC (30-minute time point is shown in Figure 5a). However, MVs had no significant effect on activation of phosphor-p38 MAPK and phosphor-stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK). To compare the role of cellular versus media MVs on the activation of MAPK in recipient VSMC, a Western blot analysis was used, and results confirmed cellular MV–induced phosphorylation of Erk1/2 (Figure 5b) and MEK1 (Figure 5c). In contrast, media MVs had no effect on phosphorylation of MEK1, and they slightly increased phosphorylation of Erk1/2. Further, inhibition of MAPK activity, via preincubation of the normal VSMC with MEK1 and Erk1/2 inhibitor U0126, decreased cellular MV–induced elevation of [Ca2+]i (Figure 5d). The effect of inhibition of MAPK on calcification could not be assessed, due to toxicity to cells undergoing prolonged incubation. The co-culture of cellular CKD MVs with VSMCs decreased the recipient VSMC gene expression of SMA22) and increased expression of angiotensin receptor 1 (AT1R) at day 7 (Figure 6a), but not day 1 or 3. The addition of MVs had no effect on the recipient VSMC expression of myocardin at any time point (Figure 6a). The MVs also increased the expression of bone morphogenic protein-2 (BMP-2) in recipient VSMC at day 7, but not day 1 and 3, and had no effect on the expression of RUNX2 or osteocalcin (Figure 6b). These results reveal that the addition of MVs alter some, but not all, of the genes known to be important in calcification after 7 days, but not at earlier time points. Altered intracellular calcium signaling can induce changes in mitochondrial function and oxidative stress, and vice versa. We first examined the expression of NADPH oxidase (NOX) isoforms in cultured VSMC and found that the expression of NOX1 and NOX4 was increased during calcification of CKD VSMC (Supplemental Figure S2). We thus examined the effect of cellular MV on the expression of NOX1 and NOX4 in recipient VSMC after 1, 3, and 7 days. The expression of NOX1 in recipient normal VSMC was increased at all 3 time points (Figure 7a, top), but NOX4 had no effect at any time point (not shown); NOX4 is known to be constitutively active in VSMC.26Clempus R.E. Sorescu D. Dikalova A.E. et al.Nox4 is required for maintenance of the differentiated vascular smooth muscle cell phenotype.Arterioscler Thromb Vasc Biol. 2007; 27: 42-48Crossref PubMed Scopus (272) Google Scholar However, MV did not increase NOX1 protein levels at any time point (data not shown). We then examined the expression of the antioxidant superoxide dismutase, and found that the addition of cellular MV to VSMC increased the expression of SOD-2 at days 3 and 7, but not day 1 (Figure 7a, bottom). However, no increase was found in expression of SOD-1 in VSMC at any of the 3 time points (data not shown). Assessment of mitochondrial function in cellular MV–VSMC co-culture by western blot analysis, using total oxidative phosphorylation cocktail antibodies revealed no changes in any of the mitochondrial subunits (Supplemental Figure S3). We then examined the expression of NOX1 in VSMC co-cultured with media-derived MV and found increased NOX1, but no change in SOD-2, after 3 days (for NOX1 expression, no MV = 1.35 ± 0.10; media MV = 2.23 ± 0.39 [P < 0.01]); for SOD-2 expression, no MV = 0.85 ± 0.07; media MV = 1.07 ± 0.22 (not significant]). To determine the role of NOX activity in cellular MV–mediated signaling and calcification in recipient VSMCs, cellular MVs were added to normal VSMC in the presence or absence of the specific NOX1 or NOX4 activity inhibitor, GKT137831, and [Ca2+]i and MAPK signaling was determined. Inhibition of NOX activity reduced the cellular MV–induced increase in [Ca2+]i. (Figure 8a ) but had no effect on MAPK signaling (for phosphor-MEK1: MV = 1.37 ± 0.02 AU; MV + GKT137821 = 1.40 ± 0.03 AU). Confirming the importance of NOX activity in calcification, the addition of GKT137831 to co-cultures partially reduced cellular MV–induced calcification of recipient normal VSMC (Figure 8b). Matrix vesicles have a critical role in the initiation of mineral deposition in skeletal tissues. In the current study, we demonstrated endocytosis of cellular-derived MV isolated from CKD VSMC by recipient normal VSMCs, with a rise in [Ca2+]i, an increase in MEK1 and ERK1/2 MAPK signaling, and accelerated calcification. In contrast, MV isolated from the media had no effect on [Ca2+]i or MEK1 signaling, and did not induce calcification; however, media MV did increase ERK1/2, although not to the level that cellular MV did.We further found that inhibition of MEK1/ERK1/2 signaling with the specific inhibitor U0126 reduced cellular MV–induced alteration of [Ca2+]i in recipient VSMC. In contrast, we did not see a change in p38 or JNK signaling. Taken together, these results suggest that MEK1 is the predominant MAPK signaling pathway for both the increased [Ca2+]i and calcification in the cellular MV–VSMC co-cultures.27Briones A.M. Tabet F. Callera G.E. et al.Differential regulation of Nox1, Nox2 and Nox4 in vascular smooth muscle cells from WKY and SHR.J Am Soc Hypertens. 2011; 5: 137-153Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar Previous studies have found that the MAPK–ERK signaling pathway is important in VSMC proliferation/differentiation28Ding H.T. Wang C.G. Zhang T.L. Wang K. Fibronectin enhances in vitro vascular calcification by promoting osteoblastic differentiation of vascular smooth muscle cells via ERK pathway.J Cell Biochem. 2006; 99: 1343-1352Crossref PubMed Scopus (52) Google Scholar and in de-differentiation of smooth muscle cells to osteochondrogenic (RUNX2 expressing) cells in arteries,29Speer M.Y. Yang H.Y. Brabb T. et al.Smooth muscle cells give rise to osteochondrogenic precursors and chondrocytes in calcifying arteries.Circ Res. 2009; 104: 733-741Crossref PubMed Scopus (432) Google Scholar whereas phosphorus-induced calcification acts primarily30Chavkin N.W. Chia J.J. Crouthamel M.H. Giachelli C.M. Phosphate uptake-independent signaling functions of the type III sodium-dependent phosphate transporter, PiT-1, in vascular smooth muscle cells.Exp Cell Res. 2015; 333: 39-48Crossref PubMed Scopus (68) Google Scholar, 31Huang J. Huang H. Wu M. et al.Connective tissue growth factor induces osteogenic differentiation of vascular smooth muscle cells through ERK signaling.Int J Mol Med. 2013; 32: 423-429Crossref PubMed Scopus (22) Google Scholar through p38. In the present study, we found some changes in gene expression consistent with a switch from a vascular to an osteoblast-like phenotype after the addition of MV to recipient normal VSMC (downregulation of vascular smooth muscle marker sm22α, and upregulation of BMP-2 and AT1R). However, these changes were observed only at 7 days and thus are unlikely to be due to the immediate MEK1/ERK signaling observed within 30 minutes of the addition of MV; for the same reason, they also are unlikely to be the major mechanism by which calcification is enhanced by cellular MV. However, the late differentiation of the VSMC is still likely to be critically important in calcification, and perhaps the addition of MV augments these changes through other mechanisms. Alteration of [Ca2+]i-induced ER stress may also be important in the pathogenesis of vascular calcification. The ER stress markers Grp78 (78-kDa glucose-regulated protein), Grp94 (94-kDa glucose-regulated protein), and CHOP (C/-EBP homologous protein) were found in calcified artery from rats treated with vitamin D, which is known to increase [Ca2+]i and calcification in VSMCs32Duan X. Zhou Y. Teng X. Tang C. Qi Y. Endoplasmic reticulum stress-mediated apoptosis is activated in vascular calcification.Biochem Biophys Res Commun. 2009; 387: 694-699Crossref PubMed Scopus (67) Google Scholar Such mitochondrial stress leads to excess H2O2 and O2- that can modify sulfhydryl groups of cysteine residues of signaling pathways and calcium transport proteins,33Trebak M. Ginnan R. Singer H.A. Jourd'heuil D. Interplay between calcium and reactive oxygen/nitrogen species: an essential paradigm for vascular smooth muscle signaling.Antioxid Redox Signal. 2010; 12: 657-674Crossref PubMed Scopus (106) Google Scholar including the sarcoplasmic reticulum calcium ATPase; SERCA),34Tong X. Hou X. Jourd'heuil D. Weisbrod R.M. Cohen R.A. Upregulation of Nox4 by TGF{beta}1 oxidizes SERCA and inhibits NO in arterial smooth muscle of the prediabetic Zucker rat.Circ Res. 2010; 107: 975-983Crossref PubMed Scopus (91) Google Scholar, 35Wu K.D. Bungard D. Lytton J. 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• W2763606722 created "2017-10-20" @default.
• W2763606722 creator A5018885816 @default.
• W2763606722 creator A5036155533 @default.
• W2763606722 creator A5074659892 @default.
• W2763606722 date "2018-02-01" @default.
• W2763606722 modified "2023-10-14" @default.
• W2763606722 title "Matrix vesicles induce calcification of recipient vascular smooth muscle cells through multiple signaling pathways" @default.
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