Nano-sized hydroxyapatite exists in human calcified aorta
To study the relationship between nHAp and vascular calcification, human calcified aorta specimen was collected. Calcium deposition in both media and adventitia of the artery was detected (Fig 1A). Nano-sized HAp was observed on the surface of the vascular cells by scanning electron microscope (SEM) (Fig. 1B). Interestingly, we found that nHAp was adhered to the VSMC surface and there was nHAp in the lysosome of the cell (Fig 1C). In addition, abundant α-SMA positive cells expressing osteogenic marker Runx2 were observed on the calcified artery (Fig 1D). These findings indicate that nHAp internalization potentially contribute to the rapid progression of VC.
Characterization and cellular internalization of HAp crystals
To analyze the effect of HAp on calcification in SMCs, we used synthetic nHAp (sHAp) to mimic human naturally crystallized HAp (hHAp) that was derived from atherosclerotic plaques of human aorta. The crystalline structures of synthetic and hHAp crystals were similar in morphology under SEM (Fig 2A), both were homogeneous and rod-like crystals. The size is about 100 nm for sHAp and ~80 nm for hHAp crystals. The X-ray diffraction (XRD) spectrum of synthetic and hHAp crystals showed both had eight diffraction peaks of crystals (Fig 2B), consistent with the standard HAp (JCPDS09-0432). The samples were further investigated by Fourier transform infrared spectrum (FT-IR) (Fig 2C). Both crystals had the vibration peaks at 563, 603 and 1029 cm−1 which belonged to the asymmetric stretching vibration peaks of P-O in the PO43− groups. Vibration peak at 3421 cm−1 were attributed to the O-H stretching vibration in HAp. Both XRD and FT-IR spectra data confirmed that synthetic and hHAp are similar and well crystallized in pure-phase.
To study engraftment of the HAp crystals by VSMCs, HAp was dyed bright green by the fluorexon (Fig S1). As shown in Fig 1D, both hHAp and sHAp were internalized into VSMCs after the HAp crystals were mixed with VSMCs for 24 h. Under EM, irregularly aggregated HAp crystals in different size were visible inside cytoplasm of VSMCs after mixing cells with HAp for 24 h (Fig 2E). The images show that a number of HAp crystals were gathered in the lysosomes, autophagosomes and autolysosomes.
Nano-HAp induced osteogenic differentiation of VSMCs and calcification deposition
To evaluate the effect of HAp on osteogenic differentiation of VSMCs, osteogenic marker proteins were examined. Expression of ALP, Runx2 and OPN was all increased significantly after VSMCs were treated with sHAp (Fig 3A) or hHAp (Fig S2A) for 5 days as compared with those of the controls. The activity of ALP in VMSCs was also increased significantly after treated with HAp (Fig S2B).
After VSMCs were treated with osteogenic medium (OM) or/and sHAp (VSMCsHAp) for 14 days, significantly more calcium deposition in VSMCsHAp was observed (Fig 3B-D) as compared with VSMCs cultured in OM, whereas VSMCs under normal medium showed no calcium deposition. VSMCs treated with both OM and sHAp showed the maximal calcium deposition. Similar phenomena were also observed for VSMCs cultured with hHAp (Fig S2C-E). These data indicated that both sHAp and hHAp crystals could induce calcification of VSMCs, and HAp crystals accelerated the development of calcification compared with the normal OM. Such capacity was notably enhanced with OM. In view of the fact that human samples are hard to obtain, we may use sHAp instead of hHAp to conduct the following experiments on the basis of the above experiments.
Nano-HAp induced ectopic tissue calcification in vivo
To investigate whether nHAp can directly contribute to mineralization of ECM in vivo, Matrigel mixed with VSMCs or/and sHAp were subcutaneously implanted into the dorsum of C57BL/6 mice (Fig 4A). Greater calcification was detected using micro-CT at the region injected with both VSMCs and sHAp as compared to that with sHAp only (Fig 4B-C) after implantation for 1 month. No calcification was observed at the site injected with VMSCs only. Likewise, the calcium content in the recovered subcutaneous tissues with both VSMCs and sHAp was the highest among 3 groups (Fig 4B-C). Calcific deposits and cells were identified in the recovered subcutaneous tissues (Fig S3). Most of the cells in the recovered tissues with VSMCs and sHAp were GFP positive, indicating that they were the originally implanted VSMCs, and they were strong positive for osteogenic marker Runx2; whereas in the tissue with VSMCs only, significantly less Runx2 was detected in the GFP+ VSMCs (Fig 4D). As expected, neither GFP+ cells nor Runx2 were detected in the recovered tissues with sHAp only. These results confirmed that nHAp possessed the ability to induce osteogenic differentiation of VSMCs and ectopic matrix mineralization in vivo.
To further confirm the effect of nHAp on vessel calcification, we applied sHAp to the out surface of mouse abdominal aorta whose adventitia was torn off. Calcification dots on the aorta being applied with either sHAp or CaCl2 were detected by enhanced micro-CT (Fig 4E) or directly viewed under stereomicroscope (Fig 4F) 14 days after the surgery, while application of control NaCl to aorta resulted in no calcium deposits, which was further confirmed by the von Kossa staining of the vessels (Fig 4G). Significantly more Runx2 was co-localized with SMCs marker a-SMA on the aorta treated with sHAp or CaCl2 as compared to that treated with NaCl (Fig 4H). These results indicate that nHAp could induce osteogenic differentiation of SMCs and vascular calcification in vivo.
Nano-HAp induced accumulation of autophagosomes in VSMCs
Under TEM, we observed increased number of double-membrane autophagosomes in VSMCHAp (Fig 5A and Fig S4A) as compared to the control cells, indicating that internalization of HAp could cause the accumulation of autophagic organelles. Indeed, almost 4-fold more LC3, a classical marker for autophagy, was detected on the calcified artery than that on the control (Fig 5B). More puncta formation of endogenous LC3 was observed in VSMCsHAp, while less and evenly dispersed LC3 throughout the cytoplasm was seen in the control cells (Fig 5C). Furthermore, significantly higher LC3-II was detected in VSMCHAp than the untreated controls (Fig 5D, and Fig S4B), indicating that HAp stimulated the conversion of cytoplasmic LC3-I to membrane-conjugated LC3-II. Taken together, these data demonstrate that both types of HAp crystals induced accumulation of autophagosomes in VSMCs.
The classical pathway of autophagy induction involves inhibition of mTOR, usually induced by energy reduction. We found that sHAp did not alter the phosphorylation of either mTOR or AMPK (Fig S5A), suggesting that sHAp stimulates formation of autophagosomes through a pathway different from starvation-induced mTOR inhibition. Rapamycin, an inhibitor of mTOR, which induces autophagy, additively increased LC3-II level in VSMCsHAp (Fig S5B), further confirming that sHAp-stimulated production of autophagosomes involves a pathway independent of mTOR inhibition.
Nano-HAp inhibited autophagic degradation without effecting fusion of autophagosome with lysosome.
Accumulation of autophagosomes could be a result of autophagy induction or blockage of autophagosome clearance which involves fusion of autophagosomes with lysosomes and thereafter degradation of the resulted autolysosomes. S-HAp did not increase the mRNA of LC3 (Fig S6A), indicating that HAp-induced increase in LC3 was not due to enhanced LC3 expression for more autophagosome formation.
Protein sequestosome 1 (p62) is a selective autophagic receptor that is incorporated into autophagosomes and preferentially degraded along with other substrates by lysosomal hydrolysis. Level of p62 protein was increased after VSMCs were treated with sHAp or hHAp (Fig 6A, and Fig S4B). Immunostaining showed more cytoplasmic p62 proteins were assembled into aggregates in VSMCsHAp (Fig S7). Such effect of HAp on accumulation of p62 was not due to more p62 expression as its mRNA level was not different from that in the untreated cells (Fig S6B). When chloroquine (CQ), an inhibitor of autophagosome-lysosome fusion, was added into the culture, both LC3II and p62 were additively elevated (Fig 6B). These data indicate that nHAp could block the autophagy flux.
When 3-methyladenine (3-MA), an inhibitor of autophagosome synthesis, was added to the starved cells, LC3-II was significantly reduced; however, such 3-MA mediated reduction of LC3-II did not occur in VSMCsHAp (Fig 6C), also suggesting that nHAp blocks autophagysome degradation.
Autophagic flux was further examined by transferring mRFP-GFP-LC3 fused genes using adenoviral vector. Under starvation, fusion of autophagosomes with lysosome lowered the pH that quenches the GFP signal (Fig 6D). When the cells were cultured with sHAp, starvation-induced quenching of GFP fluorescence was significantly reduced, confirming that sHAp blocks autophagic ﬂux and autophagic degradation.
To investigate the formation of autolysosomes, co-localization of autophagosomal LC3 with lysosome-anchored Ras-related protein 7 (Rab7), a GTPase, was examined to implicate the fusion of autophagosome with lysosome. LC3 was poorly colocalized with Rab7 in control VSMCs, while starvation enhanced the colocalization, demonstrating the formation of autolysosome (Fig 6E). Nano-HAp treatment of VSMCs resulted in a similar colocalization of LC3 and Rab7 as that of starved cells, indicating that the formation of autolysosomes was not affected by nHAp. This was further confirmed by colocalization of LC3 with the lysosomal associated membrane protein 1 (LAMP1) (Fig S8).
Nano-HAp impaired lysosomal degradation through inhibiting lysosomal acidiﬁcation.
Autophagy is a degradation process in cooperation with lysosomes. Since nHAp does not affect the fusion of autophagosome with lysosome, we then examined the function of lysosomes. The expression of early endosomal antigen 1 (EEA1), and LAMP1 both increased in VSMCs after sHAp treatment (Fig S10), indicating that sHAp may enter VSMCs through endosome/lysosome pathway. Entry of sHAp into lysosome was observed after mixing sHAp with VSMCs for 24 h (Fig 7A), which could impair the function of lysosomes. We then evaluated the ability of lysosomal degradation using a degradation assay of epidermal growth factor receptor (EGFR). EGFR was localized primarily on cell surface and was internalized and clustered into puncta after binding with its ligand EGF. Clustered EGFR was observed at 1 h post EGF treatment, and then was degraded later in the control VSMCs (Fig 7A). However, such EGFR puncta were retained in VSMCsHAp, indicating weakened lysosomal proteolysis. This was further confirmed by western blot analysis showing that total cellular EGFR was retained in VSMCsHAp while a timely reduction of EGFR was observed in the control cells (Fig 7B). These data indicate that nHAp impairs lysosomal degradation capacity.
Next, we examined the acidification ability of lysosomes since acidification is essential for the maturation and activation of most lysosomal enzymes. Acridine orange (AO) staining was used to assess the acidification of lysosomes. In cytosol, AO exists in nonprotonated monomeric form and emits green fluorescence. When it enters acidic lysosomes, the protonated form of AO aggregates and fluoresces bright red. As shown in Fig 7C, red fluorescence of AO was dramatically reduced in VSMCsHAp as compared with that in control cells, indicating a reduced acidified- compartments in VSMCsHAp.
In addition, more pre-matured cathepsin D (CTSD) at 43-kDa, a lysosomal protease, and less matured CTSD were detected in VSMCsHAp in comparison with that in the control cells (Fig 7D), indicating a suppression of the conversion from pre-matured to matured form.
To understand the alteration of acidification and subsequent lysosome immaturation in VSMCHAp, the subcellular localization of V-ATPase, an ATP-driven proton pump that imports protons into lysosomal lumen for the acidification of the compartment was examined. V1D subunit of V-ATPase was colocalized strongly with lysosome membrane protein LAMP1 in the control cells, while this colocalization was disrupted in VSMCsHAp (Fig 7E), suggesting a failure in the targeting of V-ATPase V1D subunit to lysosomes in VSMCsHAp. These results demonstrated that nHAp impairs lysosomal acidiﬁcation.
To investigate the relationship between autophagy and calcification, we utilized autophagy stimulator rapamycin (RAPA) and autophagy inhibitor chloroquine (CQ) （Fig S9）. RAPA and CQ themselves did not affect calcification in VSMCs (Fig 7G). When sHAp was added along with either RAPA or CQ, stronger Alizarin red staining with more calcium deposition in VSMCs was detected (Fig 7G), suggesting that both RAPA and CQ enhanced the calcification induced by sHAp.
Nano-HAp promoted release of exosomes containing LC3 and LAMP1.
Since exosome budding off plasma membrane has been proposed as a mechanism of calcification, and the interplay of autophagy and exosomal secretion was reported recently, we postulated that the accumulated autophagosomes and autolysosomes can be converted into exosomes and then secreted out of cells to lead calcification. To confirm that, exosomes (Exos) were purified from the conditioned medium of VSMCs and characterized (Fig S11). More Exos (18.8 ± 0.6 mg/107 cells) were harvested from VSMCsHAp than that from VSMCs (7.5 ± 0.5 mg/107 cells) (Fig 8A). There were more exosome specific marker CD9 as well as autolysosome-associated LC3 and LAMP1 proteins in Exos from VSMCsHAp than VSMCs (Fig 8B). Calcium content in Exos from VSMCsHAp was significantly higher (139.7 ± 1.6 ng/107 cells) than that from VSMCs (64.4 ± 1.6 ng/107cells) (Fig 8C). When GW4869, an inhibitor of neutral sphingomyelinase (nSMase), was added to the culture to block Exos release, significantly less Exos were harvested from VSMCHAp. Exosome specific protein CD9, and autolysosome-associated LC3 and LAMP1 were correlatively reduced in Exos from VSMCsHAp treated with GW4869 (Fig 8D and E), as well as the calcium content (Fig 8F). Accordingly, when VSMC were treated with sHAp in the presence of GW4869, calcium deposits were significantly reduced as compared with VSMCsHAp.
To confirm that sHAp could be carried out of the cells via Exos after they were internalized into VSMCs, fluorexon-labeled sHAp were cultured with VSMCs. Fluorescence signal was detected in the Exos purified from the conditional medium of VSMCsHAp. These data suggest that nHAp promoted release of Exos from VSMCs, and these Exos were at least partly originated from the accumulated autophagic vesicles which were resulted from the blockage of autophagy flux by nHAp (Fig 8H).