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. Author manuscript; available in PMC: 2021 Jun 17.
Published in final edited form as: Kidney Int. 2017 Oct 9;93(2):343–354. doi: 10.1016/j.kint.2017.07.019

Matrix vesicles induce calcification of recipient vascular smooth muscle cells through multiple signaling pathways

Neal X Chen 1, Kalisha D O’Neill 1, Sharon M Moe 1,2
PMCID: PMC8211355  NIHMSID: NIHMS1703789  PMID: 29032812

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.

Keywords: calcium, chronic kidney disease, mineral metabolism, vascular calcification

Vascular calcification is highly prevalent in chronic kidney disease (CKD) and is a major cause of morbidity and mortality.1–3 The prevalence of calcification increases with worsening kidney disease4; by the time patients reach the dialysis stage, 70%–80% have significant coronary artery calcification.5 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.6 Risk factors for the presence of calcification in patients on dialysis include older age, diabetes, and disordered mineral metabolism, including hyperphosphatemia and hypercalcemia.2 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.7 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 calcium8,9 ([Ca2+]i) and downregulation of myocardin and alpha-smooth muscle actin,10 followed by de-differentiation via upregulation of the “bone” (runt-related) transcription factor RUNX2.11,12 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 bone13,14

Over the past decade, appreciation of the role of vesicles in cell–cell communication in nonmineralized tissues.has also increased.15 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.16 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.3,17 Kapustin et al.18 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.18 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.18 We and other groups have found that the origin and content of these MVs appears to be a central determinant of their mineralization potential.3,19 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.20

Multiple studies have demonstrated that vesicles can be taken up by recipient cells (these have been reviewed21). 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).

RESULTS

Cellular-derived, but not media-derived, MVs enhanced calcification of recipient VSMC

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,3,22 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.,23 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.24 No nanotubes were identified by any imaging technique.25

Figure 1 |. Matrix vesicle (MV)—induced calcification of recipient VSMC.

Figure 1 |

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MVs were isolated from 4 sources of MVs: cellular-derived MVs (from chronic kidney disease [CKD] VSMC incubated with high phosphorus [5 mM β-glycerophosphate = [calcifying or Cal = black bars] or with normal phosphorus [no β-glycerophosphate] = control = Ct = white bars]) or media-derived MVs from calcifying or control CKD VSMC. Normal VSMC was incubated alone (hatched bar) or with cellular- or media-derived MV in the presence of β-glycerophosphate. Only the cellular origin of MV, regardless of whether they came from VSMC incubated in high or normal phosphorus, induced calcification of the recipient normal VSMC. Data are shown as mean ± SD (n = 3 MV sets from 3 CKD rats, with 3 cell cultures from each MV set, for a final n of 9). *P < 0.05, cellular MV versus VSMC alone or VSMC + media-derived MV.

Figure 2 |. Comparison of content of matrix vesicles (MVs) isolated from cells or media.

Figure 2 |

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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.

Matrix vesicles are endocytosed by recipient VSMC

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.

Figure 3 |. Both cellular and media MV are endocytosed by recipient VSMC.

Figure 3 |

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MV was isolated from CKD VSMC incubated with high-phosphorus media and then labeled with the membrane dye PKH26. The MVs were co-cultured with VSMC (a), Alexa 647–labeled dextran (b), and Alexa 488–labeled transferrin (c), and imaged by confocal microscopy (60X objective; bar = 50 μm). After 24 hours, endocytosis was observed with co-localization of the MV dye with dextran (d, light image; e, purple, merged image), indicating that MVs co-localize with ly- sosomes. We also examined cellular MV isolated from VSMC with normal phosphorus (Ct) and media-derived MV and found similar patterns of endocytosis. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

Cellular-derived MVs, but not media-derived MVs, increase intracellular calcium concentration in recipient VSMC

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).

Figure 4 |. Matrix vesicles (MVs) differ in their ability to increase intracellular calcium [Ca2+]i in recipient VSMC.

Figure 4 |

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(a) MV from cellular- or media-derived CKD VSMC in normal- (Ct) or high-phosphorus media (Cal) were added to VSMC labeled with the calcium Rhod-3 and [Ca2+]i assessed by fluorescence at various time points up to 4 hours, using a CLARIOstar high-performance microplate reader (BMG LABTECH, Cary, NC). The results demonstrate that cellular-derived MVs, regardless of phosphorus content, induce a progressive rise in [Ca2+]I, whereas media-derived MVs do not. (b) Cellular-derived MV from calcifying CKD VSMC were co-cultured with VSMC with and without the inositol1,4,5 triphosphate (IP3) inhibitor 2-aminoethoxydiphenyl borate (APB) (10 μM) or the L-type calcium channel inhibitor verapamil (10 μM) for 4 hours, and [Ca2+]i was assessed. The results reveal the attenuation of MV-mediated induction of [Ca2+]i with 2-APB but not verapamil, indicating that the rise in calcium was due to a release from intracellular stores. Data are shown as mean ± SD (n = 3 MV sets from 3 CKD rats, with 3 cell cultures from each MV set, for a final n of 9). Cal, calcified (MV isolated from CKD VSMC in high-phosphorus media); Ct, control (MV isolated from CKD VSMC in normal-phosphorus media). (a) *P < 0.05, cellular MV versus media MV or no MV, Ct MV or Cal MV; (b) *P < 0.05, MV versus no MV, treatment or no treatment; #P < 0.05, MV versus MV +2-APB.

Matrix vesicles activate MAPK signaling in recipient VSMC

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/Junamino-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.

Figure 5 |. Matrix vesicles (MVs) activate MAPK signaling in recipient VSMC.

Figure 5 |

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MV isolated from cellular-derived chronic kidney disease VSMC in high-phosphorus media were added to normal VSMC for 30 minutes, and total protein from the co-culture was isolated. The activation of MAPK was first assessed using a PathScan MAP Kinase Multi-Target Sandwich ELISA Kit. The results revealed an increase in phospho-MEK1 and phosphor-Erk1/2 compared to VSMC without MV (a). Western blot analysis confirmed cellular MV– but not media MV–induced phosphorylation of ERk1/2 (b) and MEK-1 (c) (normalized by total Erk1/2 and MEK) in recipient VSMC. Preincubation of the recipient VSMC with the MAPK (MEK/ERK) inhibitor U0126 (10 μM) for 12 hours partially attenuated an MV-induced rise in [Ca2+]i in the recipient VSMC (d). Data are shown as mean ± SD (n = 3 separate experiments). *P < 0.05, VSMC versus MV + VSMC, cellular or media MV; #P < 0.05, cellular MV versus media MV. (d) *P < 0.05, no MV versus MV; #P < 0.05, MV versus MV + U0126. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

Matrix vesicles modulate the expression of genes involved in VSMC differentiation and calcification in recipient VSMC

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.

Figure 6 |. Matrix vesicle (MV) induces late changes in genes involved in the differentiation and calcification in recipient VSMC.

Figure 6 |

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The addition of MV derived from chronic kidney disease (CKD) VSMC to recipient normal VSMC induced downregulation of the vascular smooth muscle marker sm22α (a), and upregulation of AT1R (a) and bone morphogenic protein-2 BMP-2 (b) at 7 days. No changes were observed at days 1 and 3 for these genes, and other genes were unaffected. Thus, MV induced late changes consistent with an osteoblast phenotype. Data are shown as mean ± SD (n = 3 MV sets from 3 CKD rats, with 3 cell cultures from each MV set, for a final n of 9). RUNX, (runt-related) transcription factor; * P < 0.05, MV versus no MV.

Matrix vesicle uptake induces NADPH oxidase signaling in recipient VSMC

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.26 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]).

Figure 7 |. Matrix vesicles (MVs) altered oxidative stress in recipient VSMC.

Figure 7 |

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The addition of cellular MV from calcifying chronic kidney disease (CKD) VSMC to healthy VSMC induced changes in NADPH oxidase isoform 1 (NOX1) expression, by 24 hours, that continued over time (top) and an increase in the antioxidant SOD-2, beginning at day 3 and continuing over time (bottom). Data are shown as mean ± SD (n = 3 MV sets from 3 CKD rats, with 3 cell cultures from each MV set, for a final n of 9). *P < 0.05, MV versus no MV.

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).

Figure 8 |. The role of NADPH oxidase (NOX) activity in cellular matrix vesicle (MV)–mediated cell signaling and calcification in recipient VSMC.

Figure 8 |

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To determine the role of NOX activity in cellular MV–induced cell signaling and calcification, cellular MVs were added to recipient VSMC in the presence or absence of NOX1 (isoform 1) and NOX4 (isoform 4) activity blocker GKT137831 (4 μM) and intracellular calcium concentration ([Ca]I), and calcification was determined. The results demonstrated that the blockade of NOX1 and NOX4 activity attenuated the cellular MV–induced rise in [Ca]i (a) and calcification of recipient VSMC (b). Data are shown as mean ± SD (n = 3 MV sets from 3 rats with chronic kidney disease, with 3 cell cultures from each MV set, for a final n of 9). *P < 0.05, cellular MV versus no MV. #P < 0.05, MV versus MV + GKT137831.

DISCUSSION

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.27

Previous studies have found that the MAPK–ERK signaling pathway is important in VSMC proliferation/differentiation28 and in de-differentiation of smooth muscle cells to osteochondrogenic (RUNX2 expressing) cells in arteries,29 whereas phosphorus-induced calcification acts primarily30,31 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 VSMCs32 Such mitochondrial stress leads to excess H2O2 and O2- that can modify sulfhydryl groups of cysteine residues of signaling pathways and calcium transport proteins,33 including the sarcoplasmic reticulum calcium ATPase; SERCA),34,35 phospholipase C (PLC)- IP336, and the ryanodine receptor (RyR).37 Our studies confirmed that the rise in intracellular calcium was from an endoplasmic source, because 2-APB, an inhibitor of IP3, blocked the increase in [Ca2+]i in response to uptake of cellular MVs. We have previously found that freshly isolated VSMC from the CKD animals used in this study have a progressive rise in [Ca+2]I, with increasing severity of CKD.9 Such changes may be induced by many uremic toxins, including parathyroid hormone,38 FGF23,39,40 and angiotensin II41, all of which are both elevated in patients who have CKD and known to alter VSMC phenotype and/or calcification.

In contrast to the late changes in cell-differentiation marker RNA expression in recipient VSMC, changes in NOX1 expression were observed by day 1 in the cellular MV–VSMC co-cultures, and changes in [Ca2+]i can upregulate NOX1 and vice versa.42 Given the absence of changes in protein expression of NOX1, we inhibited NOX activity with GKT137831 and reduced calcification. Thus, NOX appears to play an important role in cellular MV–induced calcification; this finding is further supported by our data revealing that GKT137831 also blocked the increase in [Ca2+]i (but not MAPK) in the cellular MV–VSMC co-cultures. The family of NADPH oxidases generates superoxide and other reactive oxygen species (ROS) at both the plasma and ER membranes, with variable expression of isoforms, depending on the cultured condition and species.27 The generation of ROS, in turn, alters [Ca2+]i and mitochondrial function,42,43 but we did not see a change in downstream mitrochondrial components with the uptake of cellular MV by VSMC. This lack of change may be due to countereffects of antioxidants, as we observed that the mitrochondrial SOD-2 was upregulated by 3 days in cellular VSMC–MV co-cultures. We saw a similar upregulation of NOX1 RNA expression in media MV–VSMC co-cultures, although media MV did not induce a rise in [Ca2+]i, MEK, SOD-2 expression, or calcification. Thus, despite similar effects on NOX1 RNA expression by both the cellular and media MV, the downstream signaling and calcification differ. These results suggest distinct intracellular trafficking of the media MV compared with the cellular MV, before both end up in lysosomes, and that these differences are important in calcification. Additionally, the difference in results may be due to variations in NOX1 activity, as changes in expression do not always equate with activity. Dai et al.44 found that high-phosphorus media induced autophagy of VSMCs and that this was protective of calcification. However, upregulation of SOD2, with overexpression of SOD2 or the administration of an SOD2 mimic, reduced autophagy. More work is needed to fully illuminate the downstream pathways.

We have previously characterized MVs isolated from cell lysate and cell culture media of bovine VSMC,3 demonstrating that MVs isolated from the cell culture media contain high concentrations of fetuin-A, whereas MVs isolated from cells do not. These findings were confirmed in the present study in rat VSMC, and the presence of fetuin-A may be another explanation for why media vesicles do not induce calcification. We also found that the fetuin-A–containing rat vesicles lacked the tetraspanin exosome marker CD63, an important marker used to isolate exosomes from human fluids.45 Thus, the media vesicles in cell culture may not be the equivalent of circulating vesicles isolated using this technique from human fluids. By contrast, our cellular-derived MVs were characteristic of exosomes derived from multivesicular bodies. Recently, Kapustin et al.18 identified such bodies in areas of calcification of arterial rings incubated with high calcium or from that of dialysis patients. Fetuin-A is a known inhibitor of VSMC and osteoblast mineralization in vitro,46–48 and when present in vesicles, it renders them incapable of calcium uptake.47 Fetuin-A prevents the initial calcium apatite formation by preferentially trafficking the calcium and phosphorus into fetuin-A–containing calciprotein particles in the circulation.49 The differences in [Ca+2]i in response to MVs that contain versus do not contain fetuin-A in the present study may be one mechanism by which differences in calcification potential of these MVs occur. We have previously demonstrated that the calcification activity of MVs can be inhibited by decreasing [Ca2+]i with the annexin calcium channel inhibitor K201, either in the VSMCs from which the MVs are isolated, or in the MVs themselves.3,17

In summary, we have demonstrated that cellular–derived MVs isolated from CKD rat VSMC can facilitate the calcification of recipient VSMC from normal rats. Both cellular and media MVs can be endocytosed by recipient normal VSMC. However, only cellular MVs induced an increase in [Ca2+]i from endoplasmic reticulum in the recipient VSMC (Figure 9). The increase in [Ca2+]i is partially attributed to the activation of both NOX and MAPK signaling, as inhibiting either pathway blocked the increase in [Ca2+]i in recipient VSMC. Blockade of NOX activity further inhibited the cellular MV–induced accelerated calcification of recipient VSMC, but it did not completely abrogate calcification, suggesting that other pathways are important. In addition, the MV–VSMC co-cultures were in high-phosphorus media, and thus the phosphorus may activate cellular de-differentiation via upregulation of RUNX250 and/or autophagy.44 The latter may be inhibited by the upregulation of SOD-2.44 Both of these pathways would further increase calcification. In contrast, media MV did not induce an increase in [Ca2+]i in recipient VSMC, perhaps because the increase in NOX was not offset by an increase in SOD-2. Understanding the differences in the cell signaling induced by different forms of MVs that lead to the presence or absence of calcification in neighboring cells is important in stopping the progression of calcification in vivo.

Figure 9 |. Overall hypothesis for mechanism of VSMC calcification.

Figure 9 |

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In the present study, MVs (matrix vesicles) isolated from chronic kidney disease (CKD) VSMC (red circles) were added to normal recipient VSMC. Three major signaling pathways were noted: (1) MAPK signaling (yellow box)—specifically, increased MEK1 and ERK signaling; (2) increased intracellular calcium from sarcoplasmic reticulum stress (purple box); and (3) increased NOX mRNA activity, and increased SOD2 mRNA. GKT1387831 inhibited both NOX1 (NADPH oxidase isoform 1) and NOX4 (NADPH oxidase isoform 4) activity and the increase in intracellular calcium, resulting in decreased VSMC calcification. We do not know if the increased ERK activity also contributed, as cells could not be cultured with inhibitors long enough to reveal a direct role. In addition to the present study, other investigators have found that elevated phosphorus50 plays a critical role in inducing calcification through the MAPK p38 pathway.51 Increased phosphorus also increases autophagy, and this appears to protect against calcification. However, increased SOD2 can counteract this protective effect.44 Together, these effects facilitate the propagation of calcification in CKD. Black lines = experiments done in present study; blue lines = experiments from the literature; dashed lines = inhibition.

METHODS

Animal models and cell culture

Primary rat VSMCs were isolated from a model of CKD–mineral bone disorder, and the Cy/+ rat and its normal littermates. This model spontaneously develops all 3 manifestations of the disease/disorder: biochemical abnormalities, extraskeletal calcification, and abnormal bone52,53 The VSMCs were isolated from the descending thoracic aorta of the rats with CKD and healthy rats, by the explant method, as described previously.54 To induce calcification, the VSMCs were treated with calcification media (5 mM β-glycerophosphate [which is converted to phosphorus], 1 U/ml fetal alkaline phosphatase, and 15% FBS)54; control cultures did not have the β-glycerophosphate but had normal media phosphorus levels. Co-culture experiments (as described below in section of Matrix vesicle–VSMC co-culture) were always in the presence of β-glycerophosphate.

Matrix vesicle isolation

Cellular-derived MVs were isolated from CKD rat VSMCs by collagenase digestion with sequential centrifugation, as previously described.3 In brief, cells were incubated with crude collagenase (500 U/ml, type IA, MilliporeSigma, St Louis, MO) in a solution of 0.25 M sucrose, 0.12 M NaCl, 0.01 M KCl, and 0.02 M Tris buffer, with a pH of 7.45, at 37°C for 3 hours. The digests were centrifuged at 800 g and 30,000 g, to remove cell debris, apoptotic bodies, and microsomes, respectively. The supernatant was centrifuged at 250,000 g to pellet the MVs, followed by resuspension in TBS (pH 7.6) with 0.25 M sucrose. In some experiments, MVs were also isolated from the cultured cell media (media MVs), as we have described previously.3 The media were decanted and spun at 30,000 g for 10 minutes, followed by centrifugation at 250,000 g for 30 minutes at 4°C, and MV was isolated from the supernatant. The MVs were quantified by protein concentration (Bio-Rad Laboratories, Hercules, CA).

Western blotting

Western blotting analysis was performed as previously described.3 The blots were incubated with antibody against annexin II, V, and VI, and CD63, CD81, and CD9 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) or fetuin-A (1:2000; a gift from Dr. Willi Jahnen-Dechent, University Hospital, Aachen, Germany) overnight at 4°C, followed by incubation, with peroxidase-conjugated secondary antibody (1:5000 dilution), and immunodetection, with an enhanced chemiluminescence kit (GE Healthcare Bio-Sciences, Pittsburgh, PA). The band intensity was analyzed by the ChemiDoc MP Imaging System (Imaging Lab 4.0, Bio-Rad Laboratories, Hercules, CA) and normalized to total protein expression using Ponceau S.

Transmission electron microscope for MVs

Analysis using a transmission electron microscope was performed by the Electron Miscroscopy Center at Indiana University School of Medicine (Indianapolis, IN). Briefly, 20 ug of cellular or media MVs were fixed with 10% glutaraldehyde and then 300 mesh nickel for-mvar/carbon–coated grids (Electron Microscopy Sciences, Hatfield, PA) were placed under the MV solutions and allowed to absorb, over the course of a weekend, at 4°C. The grids were then taken out of the solution and allowed to dry for approximately 1 minute; then, they were negative stained for 10 seconds, using Nanovan (Nanoprobes, Yaphank, NY). The grids were viewed on a Tecnai Spirit BioTwin TEM (Thermo Fisher Scientific, Hillsboro, OR), and images were taken with a CCD (charge-coupled device) camera (Advanced Microscopy Techniques, Woburn, MA).

Matrix vesicle–VSMC co-culture

To determine if MVs from CKD animal–derived VSMCs enhanced calcification of recipient healthy animal–derived VSMC, co-culture experiments were done. The normal VSMCs were incubated with and without 10 μg of MVs, always in the presence of β-glycer-ophosphate (to provide phosphorus, which is needed for calcification) for up to 7 days. The media were removed, and cells were incubated in 0.6N hydrochloric acid for 24 hours; calcification was determined colorimetrically by the o-cresolphthalein complex one method (calcium Reagent Set, Pointe Scientific, Canton, MI), as previously described.3 In some experiments, MV–-VSMC co-cultures were treated with or without an inhibitor of the IP3 receptor (2-APB; Calbiochem, Darmstadt, Germany), MAPK inhibitor U0126 (Cell Signaling Technology, Danvers, MA), or NOX1 and NOX4 inhibitor GKT137831 (BioVision, Milpitas, CA).

Matrix vesicle endocytosis in recipient VSMCs

Labeling of MVs was performed using membrane fluorescent dye PKH26 (PKH26 Red Fluorescent Cell Linker Kit, MilliporeSigma, St Louis, MO) and added to cultured recipient VSMC; the uptake was examined using confocal microscopy at various time points with an MRC-1024 laser-scanning confocal microscope (Bio-Rad Laboratories, Hercules, CA), as previously described.55,56 To determine the colocalization of MVs with endosomes or lysosomes, Alexa 488–labeled transferrin (which labels recycling endosomes) and Alexa 647–labeled dextran (which labels lysosomes) were also added to the co-cultures (Molecular Probes, Carlsbad, CA).

Matrix vesicle–mediated alteration of intracellular calcium

To determine if MVs alter the [Ca]i in recipient VSMC, normal VSMCs were seeded in 96-well culture plates and labeled with the Rhod-3 Calcium Imaging Kit (Molecular Probes, Carlsbad, CA) for 30 minutes. The MVs were then added to the VSMC, and the acute change in [Ca2+]i in VSMC was assessed by fluorescence at various time points up to 4 hours, using the CLARIOstar high-performance microplate reader (BMG LABTECH, Cary, NC). Results were confirmed using spinning-disk microscopy.57

Effect of MVs on MAPK signaling in VSMC

To determine if MVs affect MAPK signaling, recipient VSMCs were incubated with and without 10 μg MVs for 30 minutes, 2 hours, and 4 hours, at 37°C; total protein from the co-culture was isolated using lysis buffer as previously described.54 The activation of MAPK was assessed using a PathScan MAP Kinase Multi-Target Sandwich ELISA Kit. To confirm the MAPK signaling by cellular and media MVs, Western blot analyses were performed. Briefly, 20 μg of protein was loaded on 10% SDS-PAGE, and the blots were incubated with antibody against Phospho-MEK1 or Phospho-Erk1/2 (1:500; Cell Signaling Technology, Danvers, MA) overnight, at 4°C, followed by incubation, with peroxidase-conjugated secondary antibody (1:5000 dilution), and immunodetection, with the Enhanced Chemiluminescence Prime Western Blotting Detection Reagent (Amersham Biosciences, Piscataway, NJ). For loading control, western blotting analysis was also performed using antibodies against total MEK1 or total Erk1/2 (1:100; Cell Signaling Technology, Danvers, MA). The band intensity was analyzed using a ChemiDoc MP Imaging System (Imaging Lab 4.0), and MAPK activation was quantified by normalizing phosphorylated MAPK to total MAPK.

RNA isolation, quantification, and real-time PCR

Total RNA from MV–VSMC co-culture was isolated using miRNeasy Mini Kit (Qiagen, Hilden, Germany). Target-specific PCR primers were obtained from Applied Biosystems (Foster City, CA). The gene expression of bone morphogenic protein 2 (BMP-2), RUNX-2, osteocalcin, Sm22α, myocardin, NADPH oxidase isoform 1 and 4 (NOX1 and NOX4), angiotensin II type I receptor (AT1R) and SOD 1 and 2 was analyzed using real-time PCR with a Taqman gene expression assay system (TaqMan MGP probes, FAM dye-labeled, Applied Biosystems, Foster City, CA) using ViiA 7 systems.10 The cycle number at which the amplification plot crosses the threshold was calculated (CT); the ΔΔCT method was used to analyze the relative changes in mRNA expression; and normalization was performed by beta-actin, as previously described.10

Statistical Analyses

Statistical analysis was conducted by ANOVA and within-group comparisons using Fisher’s post hoc analysis (StatView, SAS Institute, Cary, NC). The results are expressed as means ± SD, with P < 0.05 considered to be significant.

Supplementary Material

Supplemental Fig 1

Figure S1. MV increases [Ca]i in recipient VSMC. To determine if MV induces alteration of [Ca]i in recipient VSMC, VSMC were loaded with calcium fluorescence dye Fluo-4 and incubated with or without MV. The calcium transients in VSMC were examined using spinning disc microscopy quantified using Metamorph software. The tracing shows fold-change versus time zero after bleach correction and normalized with VSMC without MV. The results confirm our results by a calcium Rhod-3 Calcium Imaging kit assessed with a plate reader and demonstrate MV-induced [Ca]i in recipient VSMC over time. Fluo4, green-fluorescent calcium indicator.

Supplemental Fig 2

Figure S2. The expression of NOX1 and 4 in control or calcified VSMC. VSMC isolated from CKD rats was incubated with control (normal phosphorus) or calcification (high phosphorus) media for 10 days, and total RNA was isolated for real time PCR. The results demonstrated that the expression of NOX1 and 4 are significantly increased in calcified VSMC compared to noncalcified VSMC. Data are shown as mean ± SD (n = 3 separate experiments, with 3 cell cultures from each set for final n of 9). *P < 0.05, control versus calcified. NOX1, NADPH oxidase isoform 1; NOX4, NADPH oxidase isoform 4.

Supplemental Fig 3

Figure S3. The expression of OXPHOS complexes in MV-VSMC co-cultures. MVs isolated from CKD VSMC were co-cultured with normal VSMC and total protein isolated at 1 day and 3 days for the examination of levels of OXPHOS complexes in recipient VSMC by Western blot using total OXPHOS antibodies cocktail (Abcam). The results demonstrated MV had no effect on the levels of OXPHOS complexes in recipient VMSC. Data are shown as mean ± SD (n = 3 MV sets from 3 CKD rats, with 3 cell cultures from each MV set for final n of 9). ATPSA, ATP synthase; C, complex; MTCO, mitochondrial cytochrome c-oxidase; MV, matrix vesicle; NDFUB8, NADH ubiquinone dehydrogenase 1β subcomplex 8; SDHB, succinate dehydrogenase complex iron sulfur subunit B; UQCRC2, ubiquinol-cytochrome c reductase core protein II.

supp. figure legends

ACKNOWLEGEMENTS

This work was funded by a Veterans Administration Merit Award from the United States Department of Veterans Affairs, Biomedical Laboratory Research and Development Service (SMM) and the National Institutes of Health O’Brien Center P30-DK079312. The authors are grateful to Gosia Kamocka and Seth Winfree for assistance with imaging.

Footnotes

DISCLOSURE

All the authors declared no competing interests.

SUPPLEMENTARY MATERIAL

Supplementary material is linked to the online version of the paper at www.kidney-international.org.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Fig 1

Figure S1. MV increases [Ca]i in recipient VSMC. To determine if MV induces alteration of [Ca]i in recipient VSMC, VSMC were loaded with calcium fluorescence dye Fluo-4 and incubated with or without MV. The calcium transients in VSMC were examined using spinning disc microscopy quantified using Metamorph software. The tracing shows fold-change versus time zero after bleach correction and normalized with VSMC without MV. The results confirm our results by a calcium Rhod-3 Calcium Imaging kit assessed with a plate reader and demonstrate MV-induced [Ca]i in recipient VSMC over time. Fluo4, green-fluorescent calcium indicator.

NIHMS1703789-supplement-Supplemental_Fig_1.tif (12.8MB, tif)
Supplemental Fig 2

Figure S2. The expression of NOX1 and 4 in control or calcified VSMC. VSMC isolated from CKD rats was incubated with control (normal phosphorus) or calcification (high phosphorus) media for 10 days, and total RNA was isolated for real time PCR. The results demonstrated that the expression of NOX1 and 4 are significantly increased in calcified VSMC compared to noncalcified VSMC. Data are shown as mean ± SD (n = 3 separate experiments, with 3 cell cultures from each set for final n of 9). *P < 0.05, control versus calcified. NOX1, NADPH oxidase isoform 1; NOX4, NADPH oxidase isoform 4.

NIHMS1703789-supplement-Supplemental_Fig_2.tif (2.9MB, tif)
Supplemental Fig 3

Figure S3. The expression of OXPHOS complexes in MV-VSMC co-cultures. MVs isolated from CKD VSMC were co-cultured with normal VSMC and total protein isolated at 1 day and 3 days for the examination of levels of OXPHOS complexes in recipient VSMC by Western blot using total OXPHOS antibodies cocktail (Abcam). The results demonstrated MV had no effect on the levels of OXPHOS complexes in recipient VMSC. Data are shown as mean ± SD (n = 3 MV sets from 3 CKD rats, with 3 cell cultures from each MV set for final n of 9). ATPSA, ATP synthase; C, complex; MTCO, mitochondrial cytochrome c-oxidase; MV, matrix vesicle; NDFUB8, NADH ubiquinone dehydrogenase 1β subcomplex 8; SDHB, succinate dehydrogenase complex iron sulfur subunit B; UQCRC2, ubiquinol-cytochrome c reductase core protein II.

NIHMS1703789-supplement-Supplemental_Fig_3.tif (3.7MB, tif)
supp. figure legends
NIHMS1703789-supplement-supp__figure_legends.docx (12.6KB, docx)

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