Nano-Hydroxyapatite Accelerates Vascular Calcification by Inhibiting Lysosomal Acidiﬁcation in Smooth Muscle Cells.

doi:10.21203/rs.3.rs-152330/v1

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Nano-Hydroxyapatite Accelerates Vascular Calcification by Inhibiting Lysosomal Acidiﬁcation in Smooth Muscle Cells.

https://doi.org/10.21203/rs.3.rs-152330/v1

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Background: Vascular calcification (VC) is a common characteristic of aging, diabetes, chronic renal failure, and atherosclerosis. The basic component of VC is hydroxyapatite (HAp). Nano-sized HAp (nHAp) has been identified as the initiator of pathological calcification of vasculature. However, whether nHAp can induce calcification in vivo and the mechanism of nHAp in the progression of VC remains unclear.

Results: We discovered that nHAp existed both in vascular smooth muscle cells (VSMCs) and their extracellular matrix (ECM) in the calcified arteries from patients. Synthetic nHAp had similar morphological and chemical properties as natural nHAp recovered from calcified artery. nHAp induced rapid progression of VC by stimulating osteogenic differentiation and accelerating mineralization of VSMCs in vitro. Synthetic nHAp could also directly induce VC in vivo. Mechanistically, nHAp was internalized into lysosome, which impaired lysosome vacuolar H⁺-ATPase for its acidification, therefore blocked autophagic flux in VSMCs. The accumulated autophagosomes and autolysosomes were converted into calcium-containing exosomes which were secreted into ECM and accelerated vascular calcium deposit. Inhibition of exosome release in VSMCs decreased calcium deposition.

Conclusions: Our results illustrated a novel mechanism of nHAp-induced vascular calcification. Understanding the role of nHAp in autophagy-lysosome-exosome pathway in SMCs could have great clinical significance in preventing the progression of VC.

Toxicology

nano-hydroxyapatite

vascular calcification

autophagy

lysosome

exosome

Vascular calcification (VC) is a detrimental pathology highly prevalent in aging, diabetes mellitus, atherosclerosis, and chronic renal failure. Development of VC involves aberrant calcium deposition in the vessel wall, apoptosis of vascular smooth muscle cells (VSMCs) and the phenotypic transformation of VSMCs into osteoblast-like cells.[1, 2]. During calcification, cytoplasmic Ca²⁺ and phosphate (Pi) incorporate with alkaline phosphatase (ALP) into exosomes that bud off the plasma membrane and associate with extracellular proteins, such as collagen. Crystals in the exosomes finally grow into nano-sized hydroxyapatite (nano-HAp or nHAp) [Ca₁₀(OH)₂(PO₄)₆][3]. Studies have shown that nHAp stimulated osteogenic transdifferentiation of SMCs in vitro and induced calcification on extracellular matrix (ECM) [4-6], but there was no experimental result in vivo. The above studies suggested that nHAp may lead to a pathological feedback loop causing more inflammation, cell death, phenotypic change, matrix degradation, and calcification. This may explain why calcification progresses rapidly once microcalcification exists. Whether nHAp can induce calcification in vivo and the underlying mechanism by which nHAp affects the VSMCs remain unclear.

Autophagy is a “housekeeping” subcellular process for lysosome-mediated turnover of damaged cytosolic materials that are engulfed by a double membraned phagophore to form an autophagosome. The autophagosomes fuse with single membraned acidic lysosomes to form autolysosomes, where the engulfed content is degraded and recycled [7]. Autophagy has been shown to regulate Ca²⁺ homeostasis in VSMCs. Studies proved that autophagy dysfunction contributes to VC[8, 9]. These studies demonstrated a protective role of autophagy in vascular disorders. Mice with VC had increased expression of the autophagy markers microtubule-associated light chain 3 II (LC3-II) and sequestosome 1 (SQSTM1/ p62), etc. in aortic VSMCs as compared to that in normal mice[10]. Evidence also demonstrated that increased cytosolic concentrations of Ca²⁺ induced autophagosome accumulation, which was caused by a decline in autophagic clearance via lysosomes[11, 12]. These studies indicated that impaired autophagy may contribute to VC, and lysosomal function may involve in VC. These data suggest that autophagy-lysosomal function plays an important regulatory role in vascular calcification.

Lysosomes are single membrane-enclosed organelles present in all eukaryotic cells that contain large numbers of hydrolytic enzymes. Lysosomes exhibit their maximal enzymatic activity at a low pH (pH ≤ 5) and digest extracellular material from the endocytic pathway and intracellular material from the autophagic pathway. Disorders of lysosome acidification disrupt the clearance of autophagy[13]. Nanoparticles could lead to impairment of both autophagy and lysosome[14]. However, whether and how nHAp affects autophagy and lysosome in VSMCs remain to be elucidated.

Recently, secretary autophagy as a new concept was presented in addition to the traditional degradative autophagy [15]. Autophagic proteins were found on matrix vesicles (MVs) that were secreted out of cells[15] . MV membranes are enriched in phosphatidylethanolamine[16, 17] which is also a main constituent of the mature autophagosome membrane[18]. These data suggest that autophagic vesicles can form MVs to be released into matrix outside of cells. In addition, one study proved that the calcium-containing MVs in osteoblasts were transported via lysosome and secreted by exocytosis, indicating that lysosome plays a central role in mineralization[19]. Studies have shown that MVs are enriched with a concoction of calcifying enzymes and crystals of calcium phosphate HAp mineral[16, 20]. Such MVs are released from osteogenic cells and facilitate HAp formation in ECM[21]. In context of atherosclerosis, VSMCs and macrophages are the primary source of these calcified MVs which are released into the collagen rich matrix in the intima to promote atherosclerotic calcification, directly leading to the formation of calcified plaques[22]. It is reasonable to speculate that MVs may be entwined with the network of autophagic vesicles either at the stage of their formation or release during the process of vascular calcification. Accumulating evidence indicates that matrix vesicles are secreted from multivesicular bodies and are enriched with the exosome markers CD63, CD9, CD81, and MHC I. Secretion of these exosome-like structures is regulated by sphingomyelin phosphodiesterase 3, and inhibition of this enzyme prevents vascular calcification[23]. Comparative proteomics showed that VSMC derived exosomes were compositionally similar to exosomes from other cell sources but also shared components with osteoblast-derived MVs including calcium-binding and extracellular matrix proteins[24]. Despite the clinical importance of exosomes in triggering vascular calcification, little is known of the link between exosomes biogenesis and VC induced by nHAp.

In this study, we demonstrated that nHAp induced VC through lysosomal impairment and blockage of autophagy flux in VSMCs and enhanced the release of calcifying-exosomes. We for the first time illustrated the effect of autophagy-lysosome impairment on VC, and highlighted the link between nHAp-induced deficiencies of lysosome activity and calcium deposition of VSMCs.

Characterization of Nanoparticles

Synthetic nHAp (purity 97%; particle size less than 100 nm) was obtained from Aladdin Biochemical Technology (Shanghai, China). Human-derived HAp crystals were prepared from samples of human carotid arteries removed by endarterectomy. The HAp crystals were dissected as described previously[27]. The hardened, obviously calcified regions were dissected from the atherosclerotic vessels and digested with collagenase (3 mg/mL) and elastase (1 mg/mL) overnight at 37°C. After digestion, the crystals were washed 3 times in Hanks’ Balanced Salt Solution. Samples were then placed on a 70-m sieve (Gibco) and washed further. The remaining crystals (＞70 um) were crushed using a pestle and mortar into Hanks’ Balanced Salt Solution. Washing of the crystals at this stage was achieved using ultracentrifugation at 100000 g to capture very small particles. The morphology and size of the crystals were characterized by scanning electron microscope (SU8010, HITACHI, Japan). The phase composition of the prepared nano-sized crystals was confirmed by X-ray diffraction (XRD, PANALYTICAL, The Netherlands) with Cu-Kα radiation and Fourier transform infrared (FT-IR) spectroscopy (FITR-8400S, SHIMADZU, Japan).

Cell Culture

Vascular SMCs were isolated from aortas of 8-week-old wild-type C57BL/6 mice (Slac Laboratory Animal). Briefly, the adventitia and endothelium were removed and the median layers were digested with 1 mg/ml collagenase type I (Worthington) and 0.714 U/ml elastase (Worthington) for about 1.5 h at 37°C. Cells were maintained in Dulbecco’s modified Eagle’s medium (Hyclone) containing 20% fetal bovine serum (Hyclone) and 100 U/mL penicillin, 100mg/mL streptomycin at 37°C in a humidified atmosphere with 5% CO₂. Cells were used between passage 3-5 in the experiments.

To determine the calcium deposition in vitro, VSMCs were treated with osteogenic medium (OM) containing 10 nmol/L dexamethasone (Sigma-Aldrich), 0.25 mmol/L L-ascobic acid (Sigma-Aldrich), 10 mmol/L β-glycerophosphate (Sigma-Aldrich), or/and 100 mg/ml HAp for 24 h, then washed with phosphate-buffered saline (PBS) to remove HAp, then continue to be cultured in OM or DMEM for 2 weeks.

Measurements of activity of Alkaline phosphatase (ALP).

The ATP activity of VSMCs was determined by using a luminescence ATP detection kit in accordance with the manufacturer's instructions (Beyotime Biotechnology,

Shanghai, China). After SMCs were cultured with HAp for 7 days, SMCs were washed with cold PBS, scraped into 100 μL of lysis buffer (0.2% NP-40 with l mM MgCl₂) and incubated for 30 min on ice. Then the lysis was centrifugated at 5000rpm for 5 min and the supernatant was collected. All samples were stored under -80°C until use for ALP assay which was performed following the manufacturer's instructions (Beyotime Biotechnology, Shanghai, China). By catalyzing p-nitrophenyl phosphate (pNPP) to pNP, ALP activity is referred as the change of absorbance at 405nm, and then normalized to the protein level. Unit Definition: One ALP DEA unit equals hydrolyzation of 1 mole of p-nitrophenyl phosphate per minute at pH 9.8 at 37℃.

Alizarin Red Staining

After cultured at specified conditions for 14 days, SMCs on dishes were fixed with 10% formalin for 30 min, then washed with double distilled water (ddH₂O) twice and incubated with Alizarin Red (Solarbio Life Sciences, China) for five minutes and washed with ddH₂O twice to remove the excessive dye. After examination and photography under a microscopy, the dye on the cells was extracted with 100ul Hexadecyl Pyridinium chloride (Sigma-Aldrich) and the optical density (OD) at 560 nm was measured using a microplate reader (Spectra MAX 190, Molecular Devices, USA).

von Kossa staining

A segment of the abdominal aorta was excised and fixed in 10% formalin for 24 h, then dehydrated, embedded in paraffin, and cut into 5-µm -thick sections. The slides were deparaffinized, dehydrated and washed in distilled water (ddH₂O),the samples were incubated with reagent A of von Kossa staining Kit (Solarbio Life Sciences, China) and placed under ultraviolet light for 10 min ,and then placed into reagent B for 2 min followed by staining with hematoxylin for the demonstration of cell nucleus.

Acridine orange (AO) staining

VSMCs were exposed to 100 μg/mL sHAP for 24 h. The cells were then stained with 5μg/mL acridine orange (Aladdin, China) for 20 min at 37°C and 5% CO₂. Then the cells were washed with PBS, followed by the addition of DMEM without serum to the cells. The fluorescence images of the cells were immediately acquired on a fluorescence microscope (Leica).

Analysis of EGFR degradation

After treated with nHAp for 24 h, VSMCs were incubated with serum-free DMEM containing 50 ng/ml of EGF for 0, 1, 2 and 3 h at 37 °C, and then the cells were either lysed and analyzed by western blot, or were fixed in 4% formaldehyde, stained with anti-EGFR antibody and imaged by fluorescence microscopy (Leica).

Calcium Quantification

To determine the calcium deposition in cells or exosomes, PBS-washed VSMCs on a dish or purified exosomes were dissolved in100 ul 0.6 N HCl for 48 h. Calcium content was measured by Calcium Assay Kit (Cayman Chemical, USA). The amount of calcium was normalized to the total amount of protein or the number of cells.

Exosome isolation

Exosomes were harvested by following a modified Exosome isolation protocol[52]. Confluent VSMCs were washed twice with PBS and cultured with DMEM medium with sHAp (100 μg/ml) for 3 days, and then the medium was collected and centrifuged at 10,000x g for 1 h to remove cells and apoptotic bodies. Exosomes were then harvested from the supernatant by centrifugation at 110,000x g for 70 min twice at 4℃ in an Ultracentrifuge (Optima L-90K; Beckman Coulter, USA). Exosomes were resuspended with PBS or 0.6N HCl for protein or calcium content measurement, respectively. Exos were observed by TEM (Tecnai G2 spirit, Thermo FEI, USA) which demonstrated typical cup shapes with diameters around 100 nm (Fig S11A). Dynamic light scattering analysis further confirmed that the average diameter of Exos was approximately 133nm (Fig S11B). Western blot confirmed the expression of the Exos’ marker proteins Alix, TSG101 and CD9 in Exos, but no GAPDH expression. While the VSMCs have no expression of CD9. And the above markers all don’t exist in the supernatants after ultracentrifugation (Fig S11C).

mRFP-GFP-LC3 Adenovirus Puncta Assay

For the analysis of autophagic flux, VSMCs were transfected with stubRFP-sensGFP-LC3 Adenovirus (JIKAI, China) following the manufacturer’s instructions. After 36 h，the transfected cells were starved or treated with 100ug/ml sHAp for 24 h. Then the cells were fixed with 4% paraformaldehyde in PBS and then stained with DAPI, and imaged using a fluorescence microscope (Leica, Germany).

Murine models of calcification

Two different murine models of calcification were created for this study. One is ectopic calcification on soft tissue inducing via injection of Matrigel mixed with GFP-labeled VSMCs and sHAp. The other is by smearing HAp on the abdominal aorta. Mice (C57BL/6, 3-month old) were anesthetized with isoflurane. The mice were shaved and the skin was disinfected with 75% ethanol prior to operation. For the first one, three longitudinal skin incisions about 1cm in length on the dorsal surface of each mouse were made, and subcutaneous pouches were created by blunt dissection. The implants were 400 μl in volume containing 5mg sHAp or 6 x 10⁶ SMCs or both, which was thoroughly mixed in advance, and were placed into pouch as following: left pouch, 200 μl sHAp + 200 μl Matrigel; right pouch, 100 μl sHAp + 100 μl GFP-labeled SMCs+200 μl Matrigel; lower pouch, 200 μl GFP-labeled SMCs+200 μl Matrigel. The incisions were closed with surgical sewing. For the second model, a longitudinal skin incision about 2cm in length on the abdominal surface of a mouse was made, and the abdominal aorta was isolated (between renal artery and the bifurcation of common iliac artery), the vascular adventitia was torn off. The artery was incubated with 0.9% NaCl, 0.5 M/l CaCl₂, or sHAp (25 mg/ml 0.9% NaCl) for 15min with the aid of Stereo Microscope (SZ61, Olympus, Japan). The incisions were closed with surgical sewing. Calcification in vivo was evaluated using micro-CT (U-CT-OI, Milab, USA).

Statistical analysis.

Data were expressed as means ± standard deviation (SD) and analyzed by Program Graph-Pad Prism version 6.0. Multiple comparisons were performed by one-way ANOVA followed by Bonferroni correction. A value of p < 0.05 was considered as statistically significant.

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)[25]. 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 (VSMC^sHAp) for 14 days, significantly more calcium deposition in VSMC^sHAp 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 CaCl₂were 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 VSMC^HAp (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 VSMC^sHAp, 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 VSMC^HAp 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 VSMC^sHAp (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[26]. 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 VSMC^sHAp (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 VSMC^sHAp (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 VSMC^sHAp, indicating weakened lysosomal proteolysis. This was further confirmed by western blot analysis showing that total cellular EGFR was retained in VSMC^sHAp 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 VSMC^sHAp as compared with that in control cells, indicating a reduced acidified- compartments in VSMC^sHAp.

In addition, more pre-matured cathepsin D (CTSD) at 43-kDa, a lysosomal protease, and less matured CTSD were detected in VSMC^sHAp 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 VSMC^HAp, 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 VSMC^sHAp (Fig 7E), suggesting a failure in the targeting of V-ATPase V1D subunit to lysosomes in VSMC^sHAp. 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 VSMC^sHAp 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 VSMC^sHAp than VSMCs (Fig 8B). Calcium content in Exos from VSMC^sHAp 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 VSMC^HAp. Exosome specific protein CD9, and autolysosome-associated LC3 and LAMP1 were correlatively reduced in Exos from VSMC^sHAp 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 VSMC^sHAp.

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 VSMC^sHAp. 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).

In this study, we demonstrated nHAp existed in calcified artery, and first observed internalization of nHAp into VSMC. Nano-HAp stimulated osteogenic differentiation and accelerated mineralization of VSMCs in vitro, and enhanced calcium deposits in artery in vivo. Such effect of nHAp on promoting VC was through altering lysosomal acidification and impairing the degradation function of thereafter formed autolysosomes. We first discovered that the blockage of autophagy flux by nHAp resulted in accumulation of autophagosomes and autolysosomes in VSMCs, which were turned into more calcium-containing exosomes released into ECM.

Nano-HAp could be both a result and cause of vascular calcification processes. HAp crystals had been isolated from human carotid atherosclerotic plaques[27] and suggested to contribute to early osteoblastic transdifferentiation of cardiovascular cells[28]. Here we detected nano-crystalline deposits in human calcified aorta, onset of Runx2 expression, and nHAp existence in the VSMC (Fig 1). And for the first time demonstrated that synthetic and human-derived HAp crystals were similar in morphology and characteristics. Both can accelerate calcium deposition of VSMCs via blockage of autophagy flux and accumulation of autophagic vacuoles. Studies have shown that nHAp promoted osteogenic differentiation of mesenchymal stem cells (MSCs) and osteoblasts with the increased expression of Runx2, OCN and ALP[29-33]. Such effect of nHAp on mineral deposition has been translated into clinical use of nHAp for bone repair[34]. The effect of HAp on VSMCs had also been studied, and upregulation of BMP-2 and OPN gene expression was found in VSMCs after treated with Pi-induced nanocrystals[35]. Calcifying nanoparticles (CNPs) extracted from human serum was found to induce accumulation of apoptotic bodies and accelerate vascular calcification[5]. CNPs were detectable in calcific but not healthy human arteries. They could propagate and induce mineral deposition in vitro with sufficient Pi[36]^,[4]. However, the above studies are lack in clinical evidence, thorough mechanism study and in vivo experiments. Our study confirmed that nano-sized HAp rested in calcified aorta, attached to the surrounding cells and internalized to the VSMC. Either sHAp or hHAp alone elevated the expression of Runx2 and OPN and the activity of ALP (Fig 2A and FigS3A-B), and expedited calcium deposition in VSMCs (Fig 2B). With additional Pi supplied in the osteogenic medium, HAp-induced calcification was significantly enhanced (Fig 2B), which is supported by the recently published report[6].

To confirm the effect of nHAp on calcification in vivo, sHAp was implanted underneath the skin or outside of aorta. Implantation of sHAp directly induced calcium deposition (Fig 3A-C). When nHAp was implanted along with VSMCs, more calcium deposition was observed (Fig 3C) and the implanted VSMCs were differentiated in osteogenic phenotypes with more Runx2 expression (Fig 3D), demonstrating that nHAp can induce VSMCs osteogenic differentiation in soft tissue. When nHAp was applied to the outside of abdominal aorta, significant calcification was observed in the vascular wall after 2 weeks (Fig 3E-G). According to previous studies, both CaCl₂ and CaPO₄ could induce calcification in the arterial segments[37]. Here, we employed CaCl₂as a positive control which indeed showed substantial calcification in artery (Fig 3A). This is the first time to demonstrate that nano-sized HAp induced direct vascular calcification in vivo.

Autophagy is a key regulator of cellular metabolism and homeostasis. Emerging evidence has demonstrated that autophagy plays an important role in vascular calcification. It has been documented that atorvastatin protected VSMCs from TGF-β1-stimulated calcification by inducing autophagy through suppression of the β-catenin pathway[9]. Another study demonstrated that estrogen inhibited osteoblastic differentiation of VSMCs in vitro and prevented arterial calcification in vivo by promoting autophagy through the ERα signaling pathway[38]. These studies demonstrated a protective role of autophagy in vascular calcification [10].Recent investigations point to the critical and defensive role of autophagy in the behavioral function of vascular cells against different insults[39], which was mainly focused on the death and phenotypic change of VSMCs without precise mechanism. Here, we found that nHAp was internalized by VSMCs (Fig 2B) and caused accumulation of autophagosomes (Fig 5A & S4A). It has great clinical significance to figure out whether blockage of autophagy flux contributes to nHAp-induced calcification.

In our study, we found that both sHAp and hHAp crystals increased intracellular autophagosomes (Fig 5A, and Fig S4A) and elevated autophagic cargo proteins, including LC3-II and p62 in the VSMCs (Fig 5C,6A and Fig S4B), and the process is mTOR-independent. This was due to blockage of autophagic flux by nHAp-induced dysfunction of autolysosome degradation. The underlying mechanism was further elucidated that acidification of lysosome was inhibited by sHAp (Fig 7D). However, Wang, et al proved that nHAp modulated osteoblast differentiation by mediating

Autophagy via mTOR signaling pathway[40], which is inconsistent with our results. Our data suggest an involvement of an inhibitory effect of nHAp on the late stage of autophagy. This conclusion was supported from 3 lines of evidence. First, nHAp had an additive effect on rapamycin-induced production of LC3-II (Fig S5B); second, nHAp arrested the degradation of LC3 and p62, the selective autophagy substrates (Fig 6); third, nHAp prevented the maturation of lysosomes, leading to lysosomal dysfunction (Fig 7). Our results first demonstrate that nHAp-induced autophagy dysfunction by lysosomal impairment resulted in vascular calcification. Lysosomal impairment could be a general toxic mechanism of nanoparticles to cells [41, 42]. Previous studies testified lysosomal disfunction exacerbates vascular calcification[43, 44].This provides evidence to focus on lysosomal function in our study. Mechanism underlining nHAp-induced dysfunction of autolysosome degradation was further studied by analyzing lysosomal acidification. We found that nHAp impaired lysosome maturation by inhibiting lysosomal acidification without disturbing fusion between autophagosome and lysosome (Fig 6E). Degradation of internalized EGFR was impaired (Fig 7A & B). Maturation of lysosomal protease CTSD was inhibited (Fig 8C). Based on the dampened acidity of lysosome with a deficiency in CTSD maturation, we considered V-ATPase be a potential target of damage by nHAp. V-ATPases are membrane-embedded protein complexes that function as ATP hydrolysis-driven proton pumps. V-ATPases are the primary source of organellar acidification in all eukaryotes, making them essential for many fundamental cellular processes[45]. In our study, we detected a disruption in lysosomal anchoring of V-ATPase (Fig 7E). Although further studies are required, our data first suggest that nHAp-caused defect of lysosomal acidification may involve impairment in V-ATPase transport to lysosomes. This may provide us new idea that cyclic-3′,5′-adenosine monophosphate (cAMP) which restores lysosomal pH may control the progression of vascular calcification. As nHAp blocks autophagy flux by impairing autolysosome degradation (Fig 6), the accumulated autophagic vacuoles promoted calcium deposits. This was supported by our observation that either autophagy stimulator rapamycin or inhibitor CQ aggravated nHAp-induced calcification (Fig 7G), and both had an additive effect on the accumulation of LC3 when they were used with nHAp (Fig 6B and Fig S5B).

Previous study proved that blocking autophagy promotes more rampant calcification by promoting release of MVs[46], and VSMC vesicle release contributed to calcification[47]. It has been shown that MVs played a central role in skeletal mineralization as they served as a nidus for initiation of hydroxyapatite crystal formation[48]. MVs were also identified in human aortic calcification, demonstrating that vascular calcification and bone mineralization occur by similar mechanisms[49]. Proteomic studies showed similarities between exosomes from VSMCs and skeletal osteoblasts, supporting that vascular calcification recapitulates skeletal mineralization[50].We found that the exosomes released from VSMCs^nHAp contained more LC3 and LAMP1 than that from control VSMCs (Fig 8B). It has been reported that more “calcifying” exosomes were secreted from VSMCs after pro-calcifying stimulation, which induced VC procession[46]. Calcium binds with Pi to form hydroxyapatite nodes on the inner and outside of “calcifying” exosomes membranes, which further initializes mineral deposition[46]. Nollet et al. found that autophagosomes from calcified mouse osteoblasts are packed with calcified hydroxyapatite[51]. Our study also showed there were HAp crystals in autophagosomes in VSMCs^nHAp (Fig 1E). Autophagic proteins were detected in MVs released from valve interstitial cells [20]. These results suggest that exosomes may have a crosstalk with the network of autophagic vesicles either at the stage of their formation or release during the process of VC[7]. Our study showed the accumulation of autophagosomes and autolysosomes, the enhancement of ALP activity and the increased release of exosomes from VSMCs induced by HAp (Fig 8A). Based on these previous studies, we assume that the autophagic vesicles in VSMCs may be secreted out of the cells in the form of exosomes. We found that there were more exosomes released from VSMC^HAp as compared with the control (Fig8A, Fig S11A&B), which resulted in more calcium contents released into ECM (Fig 3B, &8C). GW4869 reduced not only the number of exosomes but also the protein content of LC3 and LAMP1, and calcium deposition as well (Fig 8D-G). All these confirmed our postulation that accumulated autophagosomes and autolysosomes could be converted into calcium-containing exosomes that were released into ECM (Fig 9). This agrees with the report showing that VSMC calcification is mediated by regulated exosome secretion[24]. However, more studies are needed to further explore the relationship between autophagosomes and exosomes in regards the VC process. Our study unveiled that once nano-crystals were formed, they would facilitate the more rapid progression of calcification, showing the positive feedback of nHAp in the development of VC. Therapies aim to either prevent initial nHAp formation or nHAp-induced autophagy blockage or exosome releasing may be useful to control VC.

In summary, we report nHAp could directly induce VC in vivo and put new light on the mechanism by which nHAp promotes VC. Nano-HAp internalizes into VSMCs, damages lysosomal V-ATPase and then impairs lysosomal acidification, thus causing blockage of autophagy flux and release of Ca²⁺-containing exosomes, eventually resulted in calcium deposition on ECM. Our results suggest that targeting this novel autophagy-lysosome-exosome pathway may help to control vascular calcification development.

Nano-HAp, nHAp: Nano-sized hydroxyapatite; VC: Vascular calcification. VSMCs: vascular smooth muscle cells. ALP: alkaline phosphatase; Runx2: Runt-related transcription factor 2; OPN: Osteopontin; MVs: Matrix vesicles; SEM: Scanning electron microscope; TEM: Transmission electron microscopy; XRD: X-ray diffraction; FTIR: Fourier transform infrared; SQSTM1, p62: sequestosome 1; EEA1: Early endosomal antigen 1; RAB7: Ras-related protein 7; LAMP1: Lysosomal-associated membrane protein 1; EGFR: Epidermal growth factor receptor; CQ: Chloroquine; RAPA: Rapamycin; 3-MA: 3-methyladenine; CTSD: Cathepsin-D; AO: Acridine orange; Exos: Exosomes.

Acknowledgements

The authors would like to acknowledge Prof Wei Liu (Department of Biochemistry and Molecular Biology; Program in Molecular cell Biology; Zhejiang University school of Medicine) for kindly providing some advices on the project and reviewing the paper.

Funding

This work was supported by the National Natural Science Foundation of China Grant (No. 81570251 to HY).

Authors’ contributions

QL and YL performed experiments, analyzed data, and prepared the manuscript; QL, YL, YZ and PPX analyzed data and prepared the figures; YZ, JYZ, PPX, WWJ, WJJ and MYC performed statistical analysis; RKT designed the experimental study, and revised the manuscript; HY designed the experimental study, prepared and revised the manuscript. All authors read and approved the final manuscript.

Availability of data and materials

The datasets used and/or analyzed during this study are available from the

corresponding authors on reasonable request.

Ethics approval and consent to participate

Animals were treated humanely and all experimental protocols were approved by the Committee on Animal Use and Care of Zhejiang University. All the methods in the present study were performed according to the approved guidelines.

Consent for publication

Both authors of this paper have read and agreed to its content and are accountable for its accuracy and integrity. This research is original and is not currently under consideration by another journal.

Declaration of competing interest

The authors declare no competing financial interest.

Author details

¹Department of Cardiology, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China, 310009; ²The Affiliated Cardiovascular Hospital of Qingdao University, Qingdao, Shandong Province, China, 266071; ³Department of Chemistry, Zhejiang University, Hangzhou, Zhejiang Province, China, 310027.

Shroff R, Long DA, Shanahan C. Mechanistic insights into vascular calcification in CKD. J Am Soc Nephrol. 2013;24(2):179–89.
Villa-Bellosta R, Millan A, Sorribas V. Role of calcium-phosphate deposition in vascular smooth muscle cell calcification. Am J Physiol Cell Physiol. 2011;300(1):C210-20.
Demer LL, Tintut Y. Vascular calcification: pathobiology of a multifaceted disease. Circulation. 2008;117(22):2938–48.
Liu Y, Zhang L, Ni Z, Qian J, Fang W. Calcium Phosphate Crystals from Uremic Serum Promote Osteogenic Differentiation in Human Aortic Smooth Muscle Cells. Calcif Tissue Int. 2016;99(5):543–55.
Hunter LW, Charlesworth JE, Yu S, Lieske JC, Miller VM. Calcifying nanoparticles promote mineralization in vascular smooth muscle cells: implications for atherosclerosis. Int J Nanomedicine. 2014;9:2689–98.
Liu H, Huang LH, Sun XY, Ouyang JM. High-phosphorus environment promotes calcification of A7R5 cells induced by hydroxyapatite nanoparticles. Mater Sci Eng C Mater Biol Appl. 2020;107:110228.
Phadwal K, Feng D, Zhu D, MacRae VE. Autophagy as a novel therapeutic target in vascular calcification, Pharmacol Ther (2019) 107430.
Chen R, Xu Y, Zhong W, Li B, Yang P, Wang ZQ, Shao C, Wang CP, Yan JC, Activation of CD137 Signaling Enhances Vascular Calcification through c-Jun N-Terminal Kinase-Dependent Disruption of Autophagic Flux, Mediators Inflamm 2018 (2018) 8407137.
Liu D, Cui W, Liu B, Hu H, Liu J, Xie R, Yang X, Gu G, Zhang J, Zheng H. Atorvastatin protects vascular smooth muscle cells from TGF-beta1-stimulated calcification by inducing autophagy via suppression of the beta-catenin pathway, Cell Physiol Biochem 33(1) (2014) 129 – 41.
Frauscher B, Kirsch AH, Schabhüttl C, Schweighofer K, Kétszeri M, Pollheimer M, Dragun D, Schröder K, Rosenkranz AR, Eller K. P. Eller, Autophagy Protects From Uremic Vascular Media Calcification. Front Immunol. 2018;9:1866.
Williams A, Sarkar S, Cuddon P, Ttofi EK, Saiki S, Siddiqi FH, Jahreiss L, Fleming A, Pask D, Goldsmith P, O'Kane CJ, Floto RA, Rubinsztein DC. Novel targets for Huntington's disease in an mTOR-independent autophagy pathway. Nat Chem Biol. 2008;4(5):295–305.
Ganley IG, Wong PM, Gammoh N, Jiang X. Distinct autophagosomal-lysosomal fusion mechanism revealed by thapsigargin-induced autophagy arrest. Mol Cell. 2011;42(6):731–43.
Mindell JA. Lysosomal acidification mechanisms. Annu Rev Physiol. 2012;74:69–86.
Stern ST, Adiseshaiah PP, Crist RM. Autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity. Part Fibre Toxicol. 2012;9:20.
Papandreou ME, Tavernarakis N. Autophagy and the endo/exosomal pathways in health and disease, Biotechnol J 12(1) (2017).
Golub EE. Role of matrix vesicles in biomineralization. Biochim Biophys Acta. 2009;1790(12):1592–8.
Golub EE. Biomineralization and matrix vesicles in biology and pathology. Semin Immunopathol. 2011;33(5):409–17.
Yin Z, Pascual C, Klionsky DJ. Autophagy: machinery and regulation. Microb Cell. 2016;3(12):588–96.
Iwayama T, Okada T, Ueda T, Tomita K, Matsumoto S, Takedachi M, Wakisaka S, Noda T, Ogura T, Okano T, Fratzl P, Ogura T, Murakami S. Osteoblastic lysosome plays a central role in mineralization. Sci Adv. 2019;5(7):eaax0672.
Cui L, Houston DA, Farquharson C, MacRae VE. Characterisation of matrix vesicles in skeletal and soft tissue mineralisation. Bone. 2016;87:147–58.
Buchet R, Pikula S, Magne D, Mebarek S. Isolation and characteristics of matrix vesicles. Methods Mol Biol. 2013;1053:115–24.
Chistiakov DA, Myasoedova VA, Melnichenko AA, Grechko AV, Orekhov AN, Calcifying Matrix Vesicles and Atherosclerosis, Biomed Res Int 2017 (2017) 7463590.
Leopold JA. Vascular calcification: Mechanisms of vascular smooth muscle cell calcification. Trends Cardiovasc Med. 2015;25(4):267–74.
Kapustin AN, Chatrou ML, Drozdov I, Zheng Y, Davidson SM, Soong D, Furmanik M, Sanchis P, De Rosales RT, Alvarez-Hernandez D, Shroff R, Yin X, Muller K, Skepper JN, Mayr M, Reutelingsperger CP, Chester A, Bertazzo S, Schurgers LJ, Shanahan CM. Vascular smooth muscle cell calcification is mediated by regulated exosome secretion. Circ Res. 2015;116(8):1312–23.
Ha SW, Jang HL, Nam KT, Beck GR Jr. Nano-hydroxyapatite modulates osteoblast lineage commitment by stimulation of DNA methylation and regulation of gene expression. Biomaterials. 2015;65:32–42.
Yu L, Chen Y, Tooze SA. Autophagy pathway: Cellular and molecular mechanisms. Autophagy. 2018;14(2):207–15.
Ewence AE, Bootman M, Roderick HL, Skepper JN, McCarthy G, Epple M, Neumann M, Shanahan CM, Proudfoot D. Calcium phosphate crystals induce cell death in human vascular smooth muscle cells: a potential mechanism in atherosclerotic plaque destabilization. Circ Res. 2008;103(5):e28–34.
Bertazzo S, Gentleman E, Cloyd KL, Chester AH, Yacoub MH, Stevens MM. Nano-analytical electron microscopy reveals fundamental insights into human cardiovascular tissue calcification. Nat Mater. 2013;12(6):576–83.
Campi G, Cristofaro F, Pani G, Fratini M, Pascucci B, Corsetto PA, Weinhausen B, Cedola A, Rizzo AM, Visai L, Rea G. Heterogeneous and self-organizing mineralization of bone matrix promoted by hydroxyapatite nanoparticles. Nanoscale. 2017;9(44):17274–83.
Liu C, Zhai H, Zhang Z, Li Y, Xu X, Tang R. Cells Recognize and Prefer Bone-like Hydroxyapatite: Biochemical Understanding of Ultrathin Mineral Platelets in Bone. ACS Appl Mater Interfaces. 2016;8(44):29997–30004.
Ha S-W, Park J, Habib MM, Beck GR. Nano-Hydroxyapatite Stimulation of Gene Expression Requires Fgf Receptor, Phosphate Transporter, and Erk1/2 Signaling. ACS Appl Mater Interfaces. 2017;9(45):39185–96.
Bezerra F, Ferreira MR, Fontes GN, da Costa Fernandes CJ, Andia DC, Cruz NC, da Silva RA, Zambuzzi WF. Nano hydroxyapatite-blasted titanium surface affects pre-osteoblast morphology by modulating critical intracellular pathways. Biotechnol Bioeng. 2017;114(8):1888–98.
Cao S, Li H, Li K, Lu J, Zhang L. In vitro mineralization of MC3T3-E1 osteoblast-like cells on collagen/nano-hydroxyapatite scaffolds coated carbon/carbon composites. J Biomed Mater Res A. 2016;104(2):533–43.
Zhou K, Yu P, Shi X, Ling T, Zeng W, Chen A, Yang W, Zhou Z. Hierarchically Porous Hydroxyapatite Hybrid Scaffold Incorporated with Reduced Graphene Oxide for Rapid Bone Ingrowth and Repair. ACS Nano. 2019;13(8):9595–606.
Sage AP, Lu J, Tintut Y, Demer LL. Hyperphosphatemia-induced nanocrystals upregulate the expression of bone morphogenetic protein-2 and osteopontin genes in mouse smooth muscle cells in vitro. Kidney Int. 2011;79(4):414–22.
Miller VM, Rodgers G, Charlesworth JA, Kirkland B, Severson SR, Rasmussen TE, Yagubyan M, Rodgers JC, Cockerill FR 3rd, Folk RL, Rzewuska-Lech E, Kumar V, Farell-Baril G, Lieske JC. Evidence of nanobacterial-like structures in calcified human arteries and cardiac valves. Am J Physiol Heart Circ Physiol. 2004;287(3):H1115-24.
Yamanouchi D, Morgan S, Stair C, Seedial S, Lengfeld J, Kent KC, Liu B. Accelerated aneurysmal dilation associated with apoptosis and inflammation in a newly developed calcium phosphate rodent abdominal aortic aneurysm model. J Vasc Surg. 2012;56(2):455–61.
Peng YQ, Xiong D, Lin X, Cui RR, Xu F, Zhong JY, Zhu T, Wu F, Mao MZ, Liao XB, Yuan LQ. Oestrogen Inhibits Arterial Calcification by Promoting Autophagy Sci Rep. 2017;7(1):3549.
Hughes WE, Beyer AM. Vascular autophagy in physiology and pathology. Am J Physiol Heart Circ Physiol. 2019;316(1):H183-h185.
Wang R, Hu H, Guo J, Wang Q, Cao J, Wang H, Li G, Mao J, Zou X, Chen D, Tian W. Nano-Hydroxyapatite Modulates Osteoblast Differentiation Through Autophagy Induction via mTOR Signaling Pathway. J Biomed Nanotechnol. 2019;15(2):405–15.
Wang J, Yu Y, Lu K, Yang M, Li Y, Zhou X, Sun Z. Silica nanoparticles induce autophagy dysfunction via lysosomal impairment and inhibition of autophagosome degradation in hepatocytes. Int J Nanomedicine. 2017;12:809–25.
Zhao X, Wei S, Li Z, Lin C, Zhu Z, Sun D, Bai R, Qian J, Gao X, Chen G, Xu Z. Autophagic flux blockage in alveolar epithelial cells is essential in silica nanoparticle-induced pulmonary fibrosis. Cell Death Dis. 2019;10(2):127.
Cai Y, Wang XL, Flores AM, Lin T, Guzman RJ. Inhibition of endo-lysosomal function exacerbates vascular calcification. Sci Rep. 2018;8(1):3377.
Sudo R, Sato F, Azechi T, Wachi H. 7-Ketocholesterol-induced lysosomal dysfunction exacerbates vascular smooth muscle cell calcification via oxidative stress. Genes Cells. 2015;20(12):982–91.
Vasanthakumar T, Rubinstein JL. Structure and Roles of V-type ATPases. Trends Biochem Sci. 2020;45(4):295–307.
Zhang C, Zhang K, Huang F, Feng W, Chen J, Zhang H, Wang J, Luo P, Huang H. Exosomes, the message transporters in vascular calcification. J Cell Mol Med. 2018;22(9):4024–33.
Reynolds JL, Joannides AJ, Skepper JN, McNair R, Schurgers LJ, Proudfoot D, Jahnen-Dechent W, Weissberg PL, Shanahan CM. 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(11):2857–67.
Anderson HC. Vesicles associated with calcification in the matrix of epiphyseal cartilage. J Cell Biol. 1969;41(1):59–72.
Anderson HC. Mineralization by matrix vesicles, Scan Electron Microsc (Pt 2) (1984) 953–64.
Tintut Y, Demer LL. Exosomes: nanosized cellular messages. Circ Res. 2015;116(8):1281–3.
Nollet M, Santucci-Darmanin S, Breuil V, Al-Sahlanee R, Cros C, Topi M, Momier D, Samson M, Pagnotta S, Cailleteau L, Battaglia S, Farlay D, Dacquin R, Barois N, Jurdic P, Boivin G, Heymann D, Lafont F, Lu SS, Dempster DW, Carle GF. V. Pierrefite-Carle, Autophagy in osteoblasts is involved in mineralization and bone homeostasis. Autophagy. 2014;10(11):1965–77.
Théry C, Amigorena S, Raposo G, Clayton A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids, Curr Protoc Cell Biol Chap. 3 (2006) Unit 3.22.

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Nano-Hydroxyapatite Accelerates Vascular Calcification by Inhibiting Lysosomal Acidiﬁcation in Smooth Muscle Cells.

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