Endothelial cell-secreted bone targeting exosomes promoteangiogenesis coupling with osteogenesis via the PERK-ATF4-CRELD2 pathway

doi:10.21203/rs.3.rs-5289288/v1

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Endothelial cell-secreted bone targeting exosomes promoteangiogenesis coupling with osteogenesis via the PERK-ATF4-CRELD2 pathway

https://doi.org/10.21203/rs.3.rs-5289288/v1

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Objects: The role of endoplasmic reticulum (ER) stress in bone metabolism and the management of associated diseases has garnered significant interest. However, its role in regulating bone homeostasis and skeletal development remains largely unclear.

Results: This research revealed that human umbilical vein endothelial cells (HUVECs)-exosomes (Exos) enhanced the formation of osteoblast and angiogenic effects in vitro. Furthermore, in mice treated with HUVECs-Exos, osteoblast production, and CD31hiEmcnhi vessels were significantly increased. The mechanism by which HUVECs-Exos CRELD2 improved angiogenesis coupling with osteogenesis involved triggering the PERK-ATF4-CRELD2 pathway's ER stress.

Conclusion: HUVECs-Exos CRELD2 may be used as a potential bone metabolic disease nanodrug.

Endothelium

exosome

CRELD2

endoplasmic reticulum stress

angiogenesis

osteogenesis

An imbalance between the number and functionality of osteoclasts and osteoblasts leads to osteoporosis. H-type vessels are a unique subtype of blood vessels in the bone microenvironment and are closely associated with bone metabolism [1, 2]. It has been shown that osteoporosis can compromise the activity and number of H-type vessels, affecting the mechanisms involved in bone resorption and formation. Moreover, endothelial cells of the H-type vessels release RANKL, which facilitates different signaling pathways [3], such as the HIF-1α and Notch pathways [4, 5], via the RANKL-RANK signaling mechanism [6], therefore contributing to the control of angiogenesis and bone regeneration. This presents new targets for osteoporosis therapy, potentially enhancing bone quality and mitigating the disease by modulating the quantity or function of H-type vessels.

Extracellular vesicles, known as exosomes, have demonstrated significant promise in the management of osteoporosis. The therapeutic benefits of exosomes derived from mesenchymal stem cells (MSCs) can be enhanced [7]. They carry specific molecules, such as microRNA and proteins [8, 9], which can regulate the function of recipient cells, promote bone formation, and inhibit bone resorption. Exosomes that target CXCR4 can facilitate bone tissue-specific delivery of drugs, significantly contributing to the treatment of osteoporosis [10, 11]. Moreover, exosomes have also shown positive effects in promoting bone tissue regeneration, suppressing inflammation, and regulating immune reactions. However, studies regarding engineered exosomes' effect on H-type vessels are lacking.

Cysteine-rich with EGF-like domains 2 (CRELD2) has recently attracted greater scientific interest due to its involvement in various biological processes [12]. CRELD2 is a secretory factor and a resident protein of the endoplasmic reticulum (ER), whose expression and secretion are significantly induced by ER stress. The expression levels of CRELD2 fluctuate across several tissues, suggesting its significant and multifaceted impact in distinct tissue types. Research studies have shown that CRELD2 is related to the homeostasis of bone metabolism and is associated with pathological conditions such as chronic liver disease, cardiovascular disease, kidney disease, and cancer, all of which involve ER stress [13–15]. A recent study revealed that CRELD2 possesses a unique regulatory role regarding the differentiation of osteoclasts, which depends on the regulation of calcineurin-NFAT signaling and calcium release. The overexpression of CRELD2 impairs osteoclast differentiation, probably due to diminished calcium release, which reduces the activity of calcium-dependent phosphatases, leading to the inhibition of dephosphorylation, nuclear translocation, and activation of NFATc1 as an osteoclast transcription factor [16]. However, there is still inadequate knowledge of how CRELD2 activation affects osteoblast differentiation.

This research elucidated that exosomes originating from human umbilical vein endothelial cells (HUVECs) facilitated osteoblast formation and exhibited angiogenic effects in vitro. Moreover, in vivo results showed that osteoblast and CD31hiEmcnhi vessel formation were significantly enhanced following the administration of HUVECs-exosomes relative to their controls. The exosomal CRELD2 from HUVECs mechanistically promoted angiogenesis in conjunction with osteogenesis via inducing ER stress through the PERK-ATF4-CRELD2 pathway. Thus, the current study investigated the functions of exosomal CRELD2 in osteogenesis.

Cell culture

BMSCs were acquired from the femurs of 6-week-old mice (C57BL/6J) according to the established method [17]. The BMSCs were incubated at 37°C with CO₂ (5%) in α-MEM (Gibco, USA) with fetal bovine serum (10%; FBS) and penicillin-streptomycin (1%; Gibco, USA) added. The cells that did not attach after 72 hours were removed, while the cells that exhibited attachment to the culture flask were classified as BMSCs and subcultured as passage 1 (P1) on the seventh day. After the confluence reached 80%, these cells were subcultured. The BMSCs at the P3 stage were employed in the following assays.

The Dulbecco's Modified Eagle's Medium (DMEM high glucose, Sigma, USA), with antibiotics and FBS at concentrations mentioned before, were added to cultivate the HUVECs. The cells were incubated, as discussed above.

Exosome isolation

Exosome isolation was performed following the previously reported method [18]. In summary, for more than 24 h, HUVEC cells were grown in a complete medium with Exos-free FBS (SBI). Centrifugation was performed using the supernatant having HUVEC-Exos at 3,000 rpm (40 min) and then repeated at 20,000 rpm (60 min). After filtering out the supernatant, for 70 min at 120,000 × g, it was centrifuged again and then washed with PBS. Finally, suspended HUVEC-Exos in PBS were kept at -4°C for future use.

Exosome characterization

Using nanoparticle tracking analysis (NTA, Beckman Coulter, USA), the diameter distributions of exosomes were evaluated. Exosome morphology was examined via a transmission electron microscope (TEM, Hitachi, Japan). Through Western blot (WB) assay, the expression of certain labeled proteins—such as the surface markers specific to exosomes CD63 (1:1000, ab134045, Abcam), TSG101 (1:1000, ab125011, Abcam), and CD9 (1:1000, ab236630, Abcam)—was found on the exosome surface.

Exosome uptake assay

Red fluorescent dye was used to stain the HUVEC-Exos (PKH26, thermal, USA). The exosomes were extracted and rinsed with PBS after 20 min at 37°C. DAPI dye was used to stain the BMSC nuclei. BMSCs were co-cultured with PKH26-labeled exosomes for 24 h at 37°C, and the uptake of exosomes was monitored using a laser-scanning confocal microscope.

Mice and treatments

The Animal Research Committee of the General Hospital of the Southern Theatre Command of the PLA authorized the treatment of the animals and the experimental protocols. In this research, 12-month-old female C57BL/6 mice were utilized. At the animal center of the General Hospital of the Southern Theatre Command of the PLA, the mice were kept in barrier cages.

The osteoporosis (OP) model was generated by bilateral removal of mice ovaries after adaptive feeding for one week, according to previously reported [1]. Following this, OVX mice were administered with HUVEC-Exos or an equal volume of vehicle (without HUVEC-Exos) intravenously via the tail vein once weekly for 6 weeks.

Osteoblast differentiation and identification

BMSCs were grown in an osteogenic induction medium (Cyagen Biosciences Inc., China) to cause osteogenic differentiation and it was done in a twelve-well plate. The medium was changed every three days. On day 14, Alizarin red staining (ARS) or Alkaline phosphatase (ALP) staining was done to analyze mineralization activity.

For osteogenic identification, 20 min fixation was done using paraformaldehyde (4%) at day 14. Afterward, ALP and ARS staining was achieved via BCIP/NBT Alkaline Phosphatase Color Development Kit and Alizarin Red S Solution, according to the previously reported procedure [18].

Real-time PCR

With the aid of the TRIzol (Thermo, USA) reagent, total RNA was extracted from BMSCs treated with Negative Control (N.C.) and HUVEC-Exos. To reverse transcribe the cDNA, the ReverTra Ace qPCR RT Kit (TOYOBO, Japan) was utilized. A MasterCycler was used to carry out the reaction. To evaluate the expression levels of mRNA of different genes, quantitative real-time PCR (qRT-PCR) was performed in a LightCycler480 System (Roche, CH) using the LightCycler480 software 1.5.1.62 SP3. The comparative CT approach was used to examine the relative mRNA levels after they were adjusted to those of GAPDH mRNA.

The mRNA primers were procured from Sangon Biotech Co., Ltd, Shanghai. The following primer sequences were employed in the qRT-PCR: GAPDH: forward, 5′- AAT GGA TTT GGA CGC ATT GGT-3′ and reverse, 5′- TTT GCA CTG GTA CGT GTT GAT-3′; Alp: forward, 5′-CAG CGG GTA GGA AGC AGT TTC-3′ and reverse, 5′-CCC TGC ACC TCA TCC CTG A-3′; BGLAP: forward, 5′-CTG ACC TCA CAG ATG CCA AGC-3′ and reverse, 5′-TGG TCT GAT AGC TCG TCA CAA G-3′; Creld2: forward, 5′-GCC AGG AAG AAT TTC GGT GG-3′ and reverse, 5′-CAT GAT CTC CAG AAG CCG GAT-3′.

Tube formation assay

The 24-well plates covered with Matrigel (Corning, USA) were seeded with BMECs (1 × 10⁴ cells per well), which were then cultivated in MCDB131 media under various treatment conditions. The cellular observations were made with an inverted microscope (Leica) six hours later. ImageJ software was used to measure each parameter, including total branching points, total loop length, and total tube length.

Scratch wound assay

After being seeded onto a 24-well plate, the BMECs (1 × 10⁴ cells/well) were maintained until confluence. Following that, under an inverted microscope (Leica), the monolayer of cells was scratched via a sterile pipette tip. Afterward, the cells were cultured in MCDB131 media with several therapeutic conditions. The images of the wounds were taken immediately, 0 h, 3 h, and 12 h later. ImageJ software was utilized to measure the migration rate in the area.

Transwell migration assays

Under varying treatment conditions, BMECs (1 × 10⁴ cells per well) were transferred to a 24-well transwell plate (Corning, NY, USA) with an 8 µm pore size moved to its upper chamber [19]. The bottom compartment was then filled with the complete media. The unmigrated cells were removed from the membranes using cotton swabs after 12 hours. The migrated cells were treated using crystal violet (0.5%) dye for 3 min and counted via an optical microscope. ImageJ software was used to calculate the rate of migration.

Micro-CT analysis

OVX mice femur dissection samples were scanned and examined using micro-CT (Quantum GX, PE). The scanner was configured with 20 µm per pixel resolution, a voltage of 100 kV, and a current of 600 µA. Utilizing software for data analysis (CTAn v2.0), image reconstruction (NRecon v1.8), and three-dimensional model visualization (µCTVol v2.2), the characteristics of the distal femoral metaphyseal trabecular bone were examined.

Immunofluorescence staining

To investigate the angiogenesis of H-type vessels in paraformaldehyde solution (4%), the femur tissues were dissected and left overnight. Afterward, the decalcification was performed for 21 days using EDTA (10%; pH = 7.4) solution. Slices of 30 µm size were made from the frozen bone tissues. After 30 min of incubation in Triton X-100 (0.3%), the cells and sections were blocked with BSA solution. Following that, CD31 and endomucin antibodies (Santa Cruz, 1:50) were used to stain these samples. Following this, the fluorescence-conjugated secondary antibodies (Santa Cruz, 1:500) were utilized for detection, and DAPI was used to stain the nuclei.

To investigate the activation of ER stress in vivo, the dissected femur tissues were placed in paraformaldehyde solution (4%) overnight and allowed to decalcify for 21 days in an EDTA (10%; pH = 7.4) solution. The xylene and graduated ethanol were used to dewax and rehydrate formalin-fixed and paraffin-embedded (FFPE) tissues. To retrieve antigens, slides were pressure-cooked in either citrate buffer (0.01M citric acid; pH = 6.0) or Tris-EDTA buffer (10 mM Tris-Cl and 1 mM EDTA; pH = 9.0). Next, these samples were stained with ATF4 (Abcam; 1:100) or Pperk (Abcam; 1:100) antibodies. Afterward, the secondary antibodies (Santa Cruz, 1:500) coupled with fluorophore were used for detection. Nuclear staining was done with DAPI.

H&E staining

Following their isolation from mice, the bone samples were soaked in paraformaldehyde solution (4%), in EDTA (10%; pH = 7.4, Sigma, USA) for 21 days the sample was decalcified, and later paraffin was embedded to make staining sections. Moreover, paraformaldehyde solution (4%) was applied to the kidney, liver, heart, lung, spleen, and colon of the mice before they were embedded in paraffin. For the following assay, after overnight incubation at 37°C, the paraffin slides were first dewaxed and rehydrated using a gradient of ethanol in water. Finally, an H&E (Solarbio, G1120) staining kit was used to evaluate the parameters of osteoporosis in mice.

Western blot (WB) analysis

The methods for WB were performed as previously mentioned [20]. For 15 minutes, at 4 ℃ the whole protein lysate was centrifuged (13,500 rpm). To measure protein quantities, a bicinchoninic acid (BCA) test kit (Beyotime, China) was utilized. On 10–15% SDS-PAGE gels, equal volumes (20–30 µg) of protein samples were loaded, resolved, and then transferred to nitrocellulose membranes (Millipore, USA). After one hour of blocking in nonfat milk (5%) in TBST, the membranes were incubated at 4°C with primary antibodies overnight, including GAPDH (1: 1000, ab8245, Abcam), ATF4 (1:1000, ab216839, Abcam), Pperk (1:1000, ab192591, Abcam), CRELD2 (1:1000, 27017-1-AP, Proteintech), CD63 (1:1000, 25682-1-AP, Proteintech), CD9 (1:1000, 20597-1-AP, Proteintech), and HSP70 (1:1000, 10995-1-AP, Proteintech). After washing in TBST solution thrice, the membranes were incubated for one hour at ambient temperature with anti-mouse or anti-rabbit secondary antibodies.

Statistical analysis

As mentioned in the figure legends, all results were displayed as mean ± standard deviation (SD). For statistical analysis, IBM SPSS Statistics 20.0 was utilized. For a single comparison, an unpaired or paired Student's t-test was performed; for multiple comparisons across groups, a two-way analysis of variance (ANOVA) with Tukey's post hoc test was employed. The threshold for statistical significance was set at p < 0.05.

BMSC osteogenesis was stimulated by HUVEC-Exos.

Transmission electron microscopy was utilized to examine the spherical shape and approximately 100–200 nm diameter of exosomes produced from HUVECs (Fig. 1A). The exosomes' size and size distribution were found to be around 100–200 nm, according to the nanoparticle tracking study (Fig. 1B). In contrast to not expressing GAPDH, the WB revealed that the isolated exosomes displayed three exosome markers: HSP70, CD63, and CD9. The isolated exosomes matched the requirements for exosome identification, according to the results above.

The lipophilic dye Dio was used to label the HUVEC-Exos to study how osteogenesis was modulated in vitro and whether HUVEC-Exos were able to transfer to BMSCs. Exosome uptake by BMSCs occurred, according to immunofluorescence labeling (Fig. 1D). Moreover, treated BMSCs in the same volume with PBS or HUVEC-Exos were investigated. It was identified that as HUVEC-Exos concentration increased, osteoblast differentiation was up-regulated (Fig. 1E, 1F). Furthermore, on day 14, it was shown that HUVEC-Exos improved the mRNA expression of ALP and BGLAP in comparison to their negative controls (Fig. 1G). Overall, it was possible to infer that HUVEC-Exos stimulated osteoblast differentiation.

BMEC angiogenesis was enhanced by HUVEC-Exos.

To ascertain the function of HUVEC-Exos in endothelial cells in vitro, mouse BMECs were subjected to either HUVEC-Exos or PBS in an equivalent amount. BMECs treated with HUVEC-Exos were seen to exhibit an increased number of vascular-like structures in the tube formation experiment when compared to the negative controls (Fig. 2A). Furthermore, quantitative analyses showed that HUVEC-Exos improved the total loop length, branching points, and tube length (Fig. 2B). Moreover, as demonstrated by the scratch wound assay and Transwell experiment, HUVEC-Exos significantly increased cell migration in comparison to the negative controls (Fig. 2C-2F). These results show that HUVECs-Exos amplified the angiogenic impact on BMECs.

HUVEC-Exos promoted CD31 ^hi Emcn ^hi vessels and bone development in OVX mice.

To examine the in vivo role of HUVECs-Exos, the tissue distribution of HUVEC-Exos in mice was assessed after intravenous administration. Ex vivo fluorescent imaging showed that the DIR-labeled HUVECs-Exos could be detected mainly in mouse liver and spleen after intravenous injection for 3 h. The liver and lungs continued to accumulate significant fluorescence signals nine hours later. HUVECs-Exos can be delivered to the bone since the fluorescence signal of the limbs was much increased (Fig. 3A); however, it was still weaker than that of the liver and lungs.

To examine the impact of HUVEC-Exos in vivo, an osteoporosis mouse model was developed via OVX, resulting in a significant decrease in H-type blood vessels. Following that, for eight weeks, OVX mice received PBS or HUVEC-Exos intravenously in the exact volume through the tail vein thrice a week. OVX mice injected with HUVEC-Exos exhibited increased bone mass in the trabecular bone, as evidenced by decreased bone volume fraction (BV/TV), trabecular thickness (Tb. Th), trabecular number (Tb. N), and trabecular separation (Tb. Sp) between the bone surface and BV (Fig. 3B, 3C). Moreover, OVX mice injected with HUVEC-Exos showed a considerable increase in CD31 and EMCN double-positive endothelium in the bone when compared to their negative controls (Fig. 4D, 4E). OVX mice's bone marrow regularly showed a large number of fat vacuoles when stained with H&E; however, the administration of HUVEC-Exos inhibited the formation of fat cells (Fig. 4F, 4G). Thus, these results indicate that CD31^hiEmcn^hi vessels and osteoblast development were promoted in OVX mice injected with HUVEC-Exos.

HUVEC-Exosomal CRELD2 promoted osseous formation coupling with angiogenesis via up-regulating ER stress from the PERK⁃ATF4-CRELD2 pathway.

Genes that were differently expressed were selected for statistical examination and RNA sequencing to explore the molecular mechanism by which HUVEC-Exos alleviates bone loss. It was found that CRELD2 was highly significant in the HUVEC-Exos group than in the PBS group (Fig. 4A). Furthermore, CRELD2 expression was higher in BMSCs treated with HUVEC-Exos relative to the negative controls on 14th day (Fig. 4B). Consistently, it was observed that HUVEC-Exos increased endogenous levels of HDAC7 protein expression (Fig. 4C).

Given that CRELD2 participates in the ER stress response [21], the investigation was conducted to determine whether exosomes induce the unfolded protein response (UPR), a biological reaction to ER stress. In OVX mice administered with HUVEC-Exos, elevated amounts and enhanced nuclear localization of the ATF4 (transcription factor), which is modulated by PERK signaling at the translational level [22, 23], were observed compared to the negative controls (Fig. 4D, 4E). OVX mice injected with HUVEC-Exos consistently showed increased phosphorylation of PERK Thr 980 (related to activation) in comparison to their negative controls. Furthermore, ATF4 and phosphorylation of PERK were higher in HUVEC-Exos-treated BMSCs in contrast to the negative controls on day 14 (Fig. 4F). Moreover, it was shown that HUVEC-Exos increased endogenous levels of ATF4 and phosphorylation of PERK protein expression (Fig. 4G). Thus, these data suggest that signaling through PERK was enhanced by HUVEC-Exos.

CRELD2 promoted osteoblast differentiation and angiogenic effects in vitro.

BMSCs were treated with either CRELD2 recombinant protein or PBS in the same volume to ascertain the function of CRELD2 in vitro. The CRELD2 recombinant protein significantly raised the levels of CRELD2 expression (Fig. 5A). Furthermore, it was found that CRELD2 recombinant protein enhanced ALP staining and mineral deposition in vitro (Fig. 5B, 5C). According to these in vitro results, CRELD2 stimulated the differentiation in osteoblast.

Mouse BMECs were treated with HUVEC-Exos or PBS in the same volume to ascertain the function of CRELD2 in endothelial cells in vitro. In the tube formation experiment, it was identified that BMECs treated with CRELD2 recombinant protein displayed more vascular-like structures than their negative controls (Fig. 5D). Furthermore, quantitative studies demonstrated that CRELD2 improved total loop length, branching points, and tube length (Fig. 5E). Moreover, compared to the negative controls, the scratch wound and Transwell assay demonstrated a more significant increase in cellular migration for CRELD2 (Fig. 5D-5H). These results suggest that CRELD2 boosted BMECs' angiogenic effects.

CRELD2 enhanced bone development in vivo.

OVX mice were given PBS or CRELD2 recombinant protein intravenously in the exact quantities thrice a week for eight weeks via the tail vein to study the function of CRELD2 in vivo. For trabecular bone, OVX mice injected with CRELD2 recombinant protein showed higher trabecular thickness, bone volume fraction, and trabecular number, as well as lower bone surface/BV trabecular separation (Fig. 6A, 6B). Moreover, OVX mice injected with CRELD2 showed a significant increase in CD31 and EMCN double-positive endothelium in the bone in comparison to the negative controls (Fig. 6C, 6D). OVX mice's bone marrow consistently showed a large number of fat vacuoles when stained with H&E; however, the administration of CRELD2 inhibited the accumulation of fat cells (Fig. 6E, 6F). Thus, these results indicate that CRELD2 enhanced bone formation in vivo.

Administration of exosomal CRELD2 promotes angiogenesis and osteogenesis in vivo.

By using bioengineering technology, exosomes may be engineered to express certain markers or deliver therapeutic drugs, making them novel vehicles to be utilized for bone metabolic disorders therapy [24]. Therefore, the delivery of CRELD2 recombinant protein into exosomes was enhanced by ultrasound and constructed exosomal CRELD2. Afterward, HUVEC-Exos or HUVEC-Exos^CRELD2 were intravenously administrated in OVX mice via their tail veins. A higher bone volume fraction trabecular number, and trabecular thickness, as well as lower bone surface/BV trabecular separation, was observed in OVX mice injected with HUVEC-Exos^CRELD2 in comparison to the negative controls (Fig. 7A, 7B). Furthermore, OVX mice injected with HUVEC-Exos^CRELD2 showed a significant rise in CD31 and EMCN double-positive endothelium in the bone in relation to the negative controls (Fig. 7C, 7D). In the bone marrow of OVX mice, H&E staining revealed a large number of fat vacuoles, but HUVEC-Exos^CRELD2 therapy prevented the fat cells from accumulating (Fig. 7E, 7F). Thus, these results indicate that exosomal CRELD2 promotes osteogenesis and angiogenesis in vivo.

This study demonstrated that HUVEC-Exos facilitated pro-angiogenic effects on BMECs and promoted osteoblast formation. OVX mice were developed, and it was shown that those administered with HUVEC-Exos in vivo had enhanced bone development. Moreover, it was established that the HUVEC-Exosomal CRELD2 pathway enhanced ER stress via the PERK-ATF4-CRELD2 mechanism, facilitating bone formation in conjunction with angiogenesis. Furthermore, exosomal CRELD2 treatment ameliorated bone loss and osteoporosis.

This study demonstrated that HUVEC-Exos facilitates angiogenesis along with osteoblast differentiation. A collection of cell-derived exosomes has been reported to influence osteoblast differentiation [25]. For instance, in a rat model of nonunion, BMSCs-Exos stimulated angiogenesis and osteogenesis to improve fracture repair [26], and LNCaP cell exosomes use miR-375 to encourage osteoblast differentiation [27]. To ascertain whether these exosomes have a therapeutic role in osteoporosis-targeted therapy, additional investigation is necessary.

In disorders like chondrodysplasias, the ER-resident chaperone CRELD2 is strongly active [28]. Furthermore, during osteoclast differentiation, CRELD2 is a novel controller of calcineurin-NFAT signaling and calcium release [16]. It was determined in this study that CRELD2 encouraged the connection of osseous development with angiogenesis. The roles of ER stress and UPR in osteoclastogenesis and bone resorption have been shown in several earlier investigations [29]. SIRT1 downregulation promotes osteoclastogenesis and osteolysis through the regulation of ER stress [30], and RANKL-induced osteoclast formation in BMMs is linked to the PERK-eIF2α signaling pathway [31]. The aforementioned results imply that the genes implicated in osteoclastogenesis may potentially govern the process of bone development. This suggestion might offer novel perspectives on the molecular processes underlying bone formation.

Exosome engineering involves converting natural exosomes into effective drug-delivery platforms. Various strategies have been used to enhance exosome production, targeting, and therapeutic efficacy. In this study, the efficiency of exosome-loading drugs was improved via ultrasound. However, a series of processes can be applied to exosomal drug loading strategies. The most often used exosomal surface protein with a targeting motif is LAMP-2B, whose N-terminus is visible on the exosomal surface, and it facilitates the insertion of targeting sequences [32, 33]. In this study, due to the limitation of time and funds, only one exosomal drug delivery method was selected, which may lead to some bias in the results.

HUVECs-exosomal CRELD2 enhanced angiogenesis coupling with osteogenesis via down-regulating ER stress from PERK⁃ATF4-CRELD2 pathway. Thus, HUVECs-exosomal CRELD2 can serve as a promising nanodrug for bone metabolic disease therapy.

Competing interests

The authors declare that they have no conflicts of interest.

Consent for publication

Not applicable.

Ethics approval and consent to participate

This study was approved by the Medical Research Ethics Board of the General Hospital of Southern Theater Command of PLA (No. 20231101). All methods were carried out in accordance with relevant guidelines and regulations.

Funding declaration

The work is supported by the Guangzhou science and technology project (2024A03J0690, 2023A03J0693). the Natural Science Foundation of Guangdong Province (2022A1515010307, 2024A1515012050)

Availability of Data and Material

The datasets generated and/or analysed during the current study are not publicly available due to patient privacy but are available from the corresponding author upon reasonable request.

Acknowledgements

None.

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No competing interests reported.

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Endothelial cell-secreted bone targeting exosomes promoteangiogenesis coupling with osteogenesis via the PERK-ATF4-CRELD2 pathway

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Abstract

Figures

Introduction

Methods and materials

Cell culture

Exosome isolation

Exosome characterization

Exosome uptake assay

Mice and treatments

Osteoblast differentiation and identification

Real-time PCR

Tube formation assay

Scratch wound assay

Transwell migration assays

Micro-CT analysis

Immunofluorescence staining

H&E staining

Western blot (WB) analysis

Statistical analysis

Results

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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