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Inhibiting histone deacetylase 6 suppresses the proliferation of microvascular endothelial cells by epigenetically activating miR-375-3p, potentially contributing to bone loss during mechanical unloading

Journal of Translational Medicine volume 22, Article number: 811 (2024) Cite this article

Abstract

Background

Mechanical unloading-induced bone loss threatens prolonged spaceflight and human health. Recent studies have confirmed that osteoporosis is associated with a significant reduction in bone microvessels, but the relationship between them and the underlying mechanism under mechanical unloading are still unclear.

Methods

We established a 2D clinostat and hindlimb-unloaded (HLU) mouse model to simulate unloading in vitro and in vivo. Micro-CT scanning was performed to assess changes in the bone microstructure and mass of the tibia. The levels of CD31, Endomucin (EMCN) and histone deacetylase 6 (HDAC6) in tibial microvessels were detected by immunofluorescence (IF) staining. In addition, we established a coculture system of microvascular endothelial cells (MVECs) and osteoblasts, and qRT‒PCR or western blotting was used to detect RNA and protein expression; cell proliferation was detected by CCK‒8 and EdU assays. ChIP was used to detect whether HDAC6 binds to the miRNA promoter region.

Results

Bone mass and bone microvessels were simultaneously significantly reduced in HLU mice. Furthermore, MVECs effectively promoted the proliferation and differentiation of osteoblasts under coculture conditions in vitro. Mechanistically, we found that the HDAC6 content was significantly reduced in the bone microvessels of HLU mice and that HDAC6 inhibited the expression of miR-375-3p by reducing histone acetylation in the miR-375 promoter region in MVECs. miR-375-3p was upregulated under unloading and it could inhibit MVEC proliferation by directly targeting low-density lipoprotein-related receptor 5 (LRP5) expression. In addition, silencing HDAC6 promoted the miR-375-3p/LRP5 pathway to suppress MVEC proliferation under mechanical unloading, and regulation of HDAC6/miR-375-3p axis in MVECs could affect osteoblast proliferation under coculture conditions.

Conclusion

Our study revealed that disuse-induced bone loss may be closely related to a reduction in the number of bone microvessels and that the modulation of MVEC function could improve bone loss induced by unloading. Mechanistically, the HDAC6/miR-375-3p/LRP5 pathway in MVECs might be a promising strategy for the clinical treatment of unloading-induced bone loss.

Background

Mechanical unloading due to prolonged bed rest or exposure to long-duration spaceflight affects almost all physiological systems, especially bone loss, which has emerged as a critical factor limiting human spaceflight and public health concern worldwide. Angiogenesis plays crucial roles in bone growth, development, repair, and regeneration processes [1]. Several studies have demonstrated a positive correlation between the microvasculature within human bones and bone mineral density. Notably, osteoporosis was associated with a substantial reduction in bone microvessels in aged and ovariectomized mice [2,3,4]. However, the morphological alterations, functional changes, and underlying molecular mechanisms governing bone microvessels during unloading-induced bone loss remain incompletely understood. Investigating the reciprocal regulation between microvascular endothelial cells (MVECs) and osteoblasts holds promise for novel strategies aimed at mitigating and preventing osteoporosis under mechanical unloading conditions.

Given the intimate association between bone and microvessels, our study focused primarily on investigating the impact of simulated unloading on the functionality of MVECs and elucidating its underlying regulatory mechanisms. Our previous studies have shown that mechanical unloading can suppress MVEC proliferation and downregulate histone deacetylase 6 (HDAC6) expression in vitro [5]. HDAC6, a class IIB HDAC isoform, is involved in several diseases, including neurological disorders, various cancers, immunological conditions and other diseases [6]. Furthermore, downregulated HDAC6 induced by unloading could inhibit the proliferation of MVECs, as demonstrated in our previous studies. However, the underlying mechanism by which HDAC6 regulates proliferation remains unclear and requires further research. miRNAs are single-stranded noncoding RNAs 18–25 nucleotides in length that are associated with the posttranscriptional regulation of gene expression [7]. Various miRNAs, such as miR-155-5p, miR-27b-5p, and miR-503-5p are considered important regulators of endothelial cells through multiple pathways [5, 8,9,10]. miR-375-3p has been previously described as a tumor suppressor and is frequently downregulated in multiple types of cancer [11, 12]. However, miR-375 also acts as an oncogene in cancer by promoting tumor progression [13, 14]. Low-density lipoprotein-related receptor 5 (LRP5), a component of the Wnt ligand–receptor complex, is involved in activating the Wnt/β-catenin pathway [15]. LRP5 modulates vascular development in the retina and lung [16, 17]. Furthermore, LRP5 regulates angiogenesis and alveolar formation in the lung by modulating the expression of the angiopoietin receptor in ECs [18]. To the best of our knowledge, the roles of miR-375-3p and LRP5 in the regulation of MVEC function under mechanical unloading conditions have not been reported.

In this study, we demonstrated that bone mass and bone microvessels were significantly reduced simultaneously in hindlimb-unloaded (HLU) mice. Similarly, MVECs effectively promoted the proliferation and differentiation of osteoblasts under coculture conditions in vitro. We firstly found that HDAC6 content was significantly reduced in the bone microvessels of HLU mice and that HDAC6 inhibited the expression of miR-375-3p through histone modification. Our study further revealed that miR-375-3p was upregulated and attenuated MVEC proliferation by directly targeting LRP5 expression under mechanical unloading conditions. Moreover, LRP5 was found to be essential for HDAC6/miR-375-3p axis-mediated MVEC proliferation during unloading and regulation of HDAC6/miR-375-3p axis in MVECs could affect osteoblast proliferation under coculture conditions, leading us to consider a preventive treatment for unloading-induced bone loss by regulating the HDAC6/miR-375-3p/LRP5 signaling pathway in MVECs.

Materials and methods

Cell culture and 2D clinorotation

The mouse brain microvascular endothelial (bEnd.3) cell line and mouse preosteoblast (MC3T3-E1) cell were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in high-glucose DMEM or α-MEM with 10% FBS (Gibco, MA, USA) and 1% penicillin/streptomycin (Gibco, MA, USA) at 37 Â°C in a 95% humidified incubator with 5% CO2. Cells at passages 4–8 were used in the experiments, and all the experiments were repeated 3 times.

2D clinorotation (developed by the Astronaut Research and Training Center of China, Beijing) was used to simulate the unloading environment of cells on the ground. bEnd.3 cells were seeded in a 25 cm2 rotating special culture flask at a density of 5 × 105 cells and cultured routinely. After the cells adhered to the wall, the culture flask was filled with medium and plugged, ensuring complete removal of bubbles. Finally, culture flasks were placed into the clinostat and rotated around their horizontal axis at 24 rpm. The control group was placed in the same environment without rotation.

Animals and HLU model

Male C57BL/6J mice were obtained from the Animal Center of Air Force Medical University (Xi’an, China) for use to build HLU model. At 6 weeks of age, these mice were randomly assigned to either a Control group or a HLU group and housed under standard conditions (22 Â°C, 12-hour light/dark cycle and 50–60% humidity). The HLU mice underwent tail suspension at an angle of 30° to the ground for 4 weeks, resulting in suspended hindlimbs while allowing free access to food and water. Following euthanasia, bilateral femurs and tibias were collected for further experimental analysis. All experiments adhered to regulations set by the Laboratory Animal Ethics Committee of Air Force Medical University.

Establishment of cell co-culture system

The osteoblasts were evenly distributed in 6-well plates at a cell density of 2 × 105 /mL. The MVECs were uniformly seeded on the bottom surface of a 0.4 Î¼m transwell chamber (Millipore, MA, USA), with a cell density adjusted to 2 × 105 /mL. Subsequently, the transwell chamber containing MVECs was inserted into the 6-well plate containing osteoblasts to establish a co-culture system with an osteoblast-to-endothelial cell ratio of 1:1. Osteoblasts cultured in isolation in the control group. The co-culture medium (a mixture of high glucose DMEM and α-MEM at a ratio of 1:1) supplemented with 10% FBS and 1% penicillin-streptomycin, and incubated at 37℃ incubator with CO2 concentration of 5%. The medium was refreshed every 2 days.

Quantitative real-time PCR (qRT-PCR) analysis

Total RNA of MVECs were extracted with RNAiso Plus (TaKaRa, Tokyo, Japan) and reverse transcribed to complementary DNA (cDNA). According to the standard protocol of the manufacturer, mRNA was reverse-transcribed using a Prime Script™ RT Master Mix Kit (TaKaRa, Tokyo, Japan) and miRNA was reverse-transcribed using a Mir-X miRNA First-Strand Synthesis Kit (TaKaRa, Tokyo, Japan). GAPDH or U6 small nuclear RNA was used as the reference gene. The mRNA expression levels were detected by qRT-PCR using SYBR ® Premix Ex Taq TM II (TaKaRa, Tokyo, Japan) and a CFX96 real-time PCR detection system (BIO-RAD, CA, USA). The primer sequences used in this study are shown in Supplementary Table1.

Western blotting analysis

Cells were lysed on ice, and total protein was sonicated using radio immunoprecipitation assay (RIPA) lysis buffer complemented with 2% proteinase and phosphatase inhibitor cocktail, and 1mM phenylmethylsulfonyl fluoride (Beyotime, Shanghai, China). The protein concentration was quantitated using the Pierceâ„¢ BCA Protein Assay Kit (Thermo Fisher Scientific, MA, USA). The proteins were subjected to NuPage Bis-Tris polyacrylamide gels (Invitrogen, MA, USA) and transferred to 0.45 Î¼m polyvinylidene fluoride (PVDF) membranes. After blocking the membranes in 5% skim milk for 2 h at room temperature, membranes were incubated at 4 Â°C for 8 h with primary antibodies: GAPDH (1:1000; Cell Signaling Technology, MA, USA), PCNA (1:1000; Cell Signaling Technology, MA, USA), RUNX2 (1:1000; Cell Signaling Technology, MA, USA), OCN (1:2000; Abcam, Cambridge, UK), LRP5 (1:1000; Cell Signaling Technology, MA, USA). After washing with TBST, the membranes were incubated in a peroxidase-conjugated secondary antibody (1:5000; ZS-GB-BIO, Beijing, China) for 2 h at room temperature, and ECL detection kit (Thermo Fisher Scientific, MA, USA) were used for protein detection. The intensities of blots were analyzed using ImageJ software.

Cell transfection

Cells were seeded and cultured in 6-well plates overnight and transfected with pEX-HDAC6 (100 ng/µl), siRNA-LRP5 (80 nM), miR-375-3p mimic (40 nM), miR-375-3p inhibitor (80 nM) and their corresponding negative controls (GenePharma, Shanghai, China) using Lipofectamine 2000 (Invitrogen, MA, USA) following the manufacturer’s protocol. The sequences of miR-375-3p, HDAC6, and LRP5 are listed in Supplementary Table 2.

Cell proliferation assay

Cell proliferation was evaluated using a Cell Counting Kit-8 (CCK-8) assay (MISHU, Xi’an, China) based on the chromogenic reaction of WST-8 according to the standard protocol of the manufacturer. Cells were seeded in 96-well plates at a density of 2 × 104 cells/mL (100 µL/well) and incubated overnight at 37 Â°C. After 24 h of treatment, the cells were supplemented with 10 Âµl of CCK-8 reagent, incubated for 2 h at 37 Â°C, and detected at 450 nm using a microplate reader. Each group had six biological replicates, and all experiments were repeated at least three times.

5-Ethynyl-2′-deoxyuridine (EdU) labeling

EdU is a thymine analog that can replace thymine and be incorporated into newly duplicated DNA of S-phase cells and is commonly used for assessing DNA replication. Cells were seeded in 6-well plates at a density of 1 × 105 cells and incubated overnight. Then, each well of cells was supplemented with 50 µM EdU (Beyotime, Shanghai, China) and cultured for an additional 4 h at 37 Â°C. EdU measurements were conducted according to the manufacturer’s instructions. The ratio of EdU-positive cells to the total number of DAPI-positive cells (blue) indicates the EdU fusion rate. Each experiment was repeated three times, and three independent replicates were used.

Alkaline phosphatase (ALP) staining

Alkaline phosphatase staining was performed using the BCIP/NBT Alkaline Phosphatase chromogenic kit (Beyotime, Shanghai, China). Cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min. Fixed cells were again washed 3 times with PBS, and then BCIP/NBT staining working solution was added to each well, and the color reaction was terminated by incubation at room temperature in the dark for 1 h before washing with ddH2O. Cells were imaged with a digital camera.

Chromatin immunoprecipitation (ChIP) assay

ChIP assays were conducted according to the manufacturer’s protocol (SimpleChIP Enzymatic Chromatin IP Kit; Cell Signaling Technology, MA, USA). Cells were incubated with 1% formaldehyde to fix protein DNA complexes, washed with ice PBS and harvested Then the DNA was cut into approximately 100–900 bp DNA fragments by enzymatic hydrolysis. Cross-linked DNA was precipitated using HDAC6 antibodies (anti-HDAC6) (NOVUS, CO, USA), acetylated-histone H3K9 (Ac-H3K9) antibodies (Cell Signaling Technology, MA, USA). Anti-mouse IgG was used as a control for nonspecific binding. Immunoprecipitated DNA was analyzed by real-time PCR using promoter-specific primer of HDAC6 binding sites in the miR-375-3p promoter. Forward: 5′-tgttgttccagaggcgctcc-3′ and Reverse: 5′-tttgctcggggcaaatattgactcatg-3′.

Micro-CT

The tibiae of control and HLU mice were harvested, thoroughly cleaned, and the surrounding soft tissue was removed. Subsequently, they were fixed in 4% paraformaldehyde for 48 h before being scanned using Micro-CT (Perkin Elmer, USA). Bone mass and microstructure analysis were performed on a cubic region of interest (ROI) measuring approximately 2.5 × 2.5 × 3 mm³ located approximately 1.5 mm from the growth plate. Three-dimensional reconstruction of the ROI enabled measurements of bone mineral density (BMD), relative bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), bone surface area to bone volume ratio (BS/BV), and trabecular separation (Tb.Sp).

Immunofluorescence (IF) staining

Paraffin sections were deparaffinized and hydrated, followed by antigen retrieval and natural cooling. The slides were washed thrice with PBS for 5 min each time. After blocking with BSA for 30 min, the primary antibody was added and incubated in the refrigerator at 4℃ overnight. Following three washes with PBS, corresponding secondary antibodies were added and incubated for 1 h at room temperature in the dark. DAPI staining solution was then applied and incubated in the dark at room temperature for 10 min and sealed using an anti-fluorescence quench agent. Nuclei stained with DAPI appeared blue while EMCN and HDAC6 showed positive green signals, and CD31 exhibited red fluorescence. Primary antibodies used included CD31 (1:300, Servicebio, Wuhan, China), EMCN (1:200, Servicebio, Wuhan, China), HDAC6 (1:300, NOVUS, CO, USA). Secondary antibodies used included HRP-labeled goat anti-rabbit IgG (1:500, Servicebio, Wuhan, China) and Alexa Fluor 488 Labeled Goat Anti-Rabbit IgG (1:400, Servicebio, Wuhan, China).

Statistical analysis

All statistical analyses were conducted with SPSS 22.0 software and expressed as the mean ± standard deviation from at least three independent experiments. The statistical significance of differences between the control and treated groups was analyzed by Student’s t test and one-way ANOVA. P < 0.05 and P < 0.01 were considered statistically significant. All experiments were repeated three times.

Results

The content of microvessels in the tibia bone of mice was significantly reduced under unloading

Six-week-old mice were used to establish an HLU animal model. After four weeks of tail suspension, micro-CT scanning was performed to assess changes in bone microstructure and mass in the tibia. The results revealed that HLU mice exhibited a typical osteoporosis phenotype characterized by disrupted bone microstructure and significantly reduced bone mass (Fig. 1A). BMD, BV/TV, Tb.N and Tb.Th were significantly lower, whereas BS/BV and Tb.Sp were significantly higher in the HLU group than in the Con group (Fig. 1B). IF staining was performed to observe the expression and distribution of CD31 and EMCN in tibial microvessels after unloading. The results revealed that the microvessels in the Con group were organized in a columnar structure, whereas the microvessels in the HLU group were sparse and disorganized in a punctate distribution. Furthermore, there was a significant reduction in skeletal microvessel content within the tibia of HLU mice compared to Con mice (Fig. 1C).

Fig. 1
figure 1

Microvessel contents in the tibia bone of mice are significantly reduced under unloading. (A) Representative images of micro-CT and three-dimensional reconstructions of the proximal tibias of the mice in the Con and HLU groups (N = 5). (B) Three-dimensional measurements of the bone mineral density (BMD), relative bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), bone surface area to bone volume ratio (BS/BV), and trabecular separation (Tb.Sp) in the ROI regions of the proximal tibias of mice (N = 5). (C) Representative images of immunofluorescence (IF) staining of tibial sections from Con and HLU mice. CD31 (red) and EMCN (green) were used to detect type H vessels. Scale bar, 100 Î¼m. (D) Cell proliferation was evaluated by the CCK-8 assay (N = 3). The symbol *(Red) represents the Con vs. Coculture group. (E) Proliferating MC3T3-E1 cells were labeled with EdU (N = 3). Scale bar, 50 Î¼m. (F) PCNA, RUNX2, and OCN protein expression levels in MC3T3-E1 cells were analyzed by western blotting (N = 3). (H) Representative images of Alp staining in MC3T3-E1 cells (N = 3). *P < 0.05, **P < 0.01 vs. control

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To further explore the causes and underlying mechanisms of the decreased bone formation ability and bone microvascular content, we established a coculture system of MVECs and osteoblasts. After coculture with MVECs, a CCK-8 assay was performed on the osteoblasts. The growth curve revealed that the cell activity of the cocultured osteoblasts increased more significantly than that of the control cells did, and the EdU results were consistent with these findings (Fig. 1D and E). Western blotting further revealed a significant increase in PCNA protein expression in osteoblasts after coculture with MVECs, indicating a substantial increase in the proliferation ability of osteoblasts in the coculture group (Fig. 1F). Moreover, both the mRNA and protein expression levels of Runx2 and Ocn were significantly increased after coculture with MVECs (Fig. 1F and G). Additionally, ALP staining confirmed that the osteogenic differentiation ability of the coculture group was markedly enhanced (Fig. 1H). These results suggest that MVECs can effectively promote the proliferation and differentiation of osteoblasts under coculture conditions, which further deepened theenhances our understanding of the regulatory effect of MVECs on osteoblast function. Therefore, we focused on MVECs, aiming to ameliorate unloading-induced bone loss by modulating endothelial cell function.

HDAC6 expression is reduced in the bone microvessels of HLU mice and inhibits the expression of miR-375-3p

HDAC6, an epigenetic protein, is involved in regulating gene transcription. We found that HDAC6 mRNA expression was downregulated under clinorotation-unloading conditions, which was consistent with our previous studies [5] (Fig. 2B). To verify whether the changes in HDAC6 expression in the tibial microvessels of HLU mice were consistent with those at the cellular level, the expression of CD31 and HDAC6 in the tibial microvessels of mice in both groups was detected by IF, which revealed that HDAC6 expression was positive in both groups. However, the content of HDAC6 in the tibial microvessels of HLU mice was significantly lower than that in the tibial microvessels of control mice (Fig. 2A). A microarray assay was performed to detect differential miRNA expression after the transfection of siRNA-HDAC6 or the corresponding control into MVECs [5]. The expression of candidate miRNAs selected from the microarray data was further validated by qRT–PCR based on fold changes in expression after knocking down HDAC6 or unloading. The results indicated that miR-375-3p expression was significantly elevated in the siRNA-HDAC6 group, whereas overexpression of HDAC6 inhibited the expression of miR-375-3p (Fig. 2C). Furthermore, we detected the expression of miR-375-3p in MVECs under normal and clinorotation-unloading conditions respectively, and found that compared with MVECs in normal condition, the expression of miR-375-3p in MVECs continued to increase under clinorotation-unloading conditions (Fig. 2D). To further examine whether miR-375-3p expression is directly regulated by HDAC6 in MVECs, we performed a ChIP assay using an HDAC6 antibody. Compared with the control group, the group treated with anti-HDAC6 had markedly increased binding of HDAC6 to the miR-375-3p promoter region (Fig. 2E). We then knocked down HDAC6 in MVECs to perform a ChIP assay with an Ac-H3K9 antibody. HDAC6 knockdown increased histone acetylation in the miR-375-3p promoter region (Fig. 2F). These results illustrated that HDAC6 regulates the expression of miR-375-3p by reducing Ac-H3K9 in the promoter region of MVECs (Fig. 2G).

Fig. 2
figure 2

HDAC6 expression is reduced in the bone microvessels of HLU mice and inhibits the expression of miR-375-3p. (A) Representative images of IF staining of tibia sections from Con and HLU mice. DAPI staining is represented in blue, HDAC6 is represented by positive green signal, and CD31 is represented by positive red signal. Scale bar, 100 Î¼m. (B) qRT‒PCR analysis of HDAC6 mRNA expression under unloading conditions (N = 3). (C) miR-375-3p expression was analyzed by qRT‒PCR after transfecting siRNA-HDAC6, pEX-HDAC6 or the corresponding controls in MVECs (N = 3). (D) miR-375-3p mRNA levels were analyzed by qRT‒PCR in MVECs cultured under clinorotation conditions for 24–72 h (N = 3). (E) ChIP‒qRT‒PCR assay to assess the interaction of HDAC6 with the miR-375-3p promoter (N = 3). (F) ChIP‒qRT‒PCR assay to assess the enrichment of histone deacetylation by HDAC6 in the miR-375-3p promoter region (N = 3). (G) Schematic diagram of HDAC6 acting on the miR-375 promoter region *P < 0.05, **P < 0.01 vs. control

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Mir-375-3p silencing promotes MVEC proliferation and partially mitigates their proliferation under mechanical unloading conditions

To further study the role of miR-375-3p in MVECs, we transfected a miR-375-3p mimic or inhibitor into MVECs. The results showed that miR-375-3p suppressed both PCNA protein expression and cell viability, as measured by the CCK-8 assay (Fig. 3A and B). Furthermore, the number of EdU-positive cells was reduced in the miR-375-3p mimic group, whereas the miR-375-3p inhibitor had the opposite effect (Fig. 3C). These data showed that miR-375-3p negatively regulates MVEC proliferation.

Fig. 3
figure 3

miR-375-3p silencing promotes MVEC proliferation and partially mitigates their proliferation under mechanical unloading conditions. (A) PCNA protein expression in MVECs was analyzed by western blotting (N = 3). (B) Cell proliferation was evaluated by the CCK-8 assay (N = 3). The symbol *(Red) represents the mimic-NC vs. mimic-375 group; the symbol *(Green) represents the inhibitor-NC vs. inhibitor-375 group. (C) Proliferating MVECs were labeled with EdU (N = 3). Scale bar, 50 Î¼m. (D) The results of the EdU incorporation assay were analyzed by confocal microscopy (N = 3). Scale bar, 50 Î¼m. (E) PCNA protein levels in MVECs were analyzed by western blotting (N = 3). (F) Cell proliferation was evaluated by a CCK-8 assay at 24–72 h (N = 3). The symbol * (black) represents the Con group vs. the Clino group; the symbol * (blue) represents the Clino plus inhibitor-NC group vs. the Clino plus inhibitor-375 group. *P < 0.05, **P < 0.01 vs. control

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To explore whether mechanical stimuli regulate MVEC proliferation through miR-375-3p, we transfected a miR-375-3p inhibitor into MVECs and then cultured the cells under clinorotation–unloading conditions. Inhibition of miR-375-3p partially reversed the reduction in PCNA protein expression under clinorotation conditions (Fig. 3E). CCK-8 and EdU labeling assays showed that miR-375-3p inhibition promoted the growth of MVECs (Fig. 3D and F). These results revealed that miR-375-3p might be involved in regulating MVEC proliferation under mechanical unloading conditions.

Mir-375-3p is negatively correlated with LRP5 under mechanical unloading conditions

To further identify the molecular mechanisms of miR-375-3p-mediated cell proliferation, bioinformatics analysis using TargetScan and literature review analysis suggested that LRP5, which is related to cellular proliferation, is a potential target of miR-375-3p. After transfection with the miR-375-3p mimic or inhibitor, qRT‒PCR and western blotting were performed to detect the expression of LRP5. Compared with the NC treatment, treatment with the miR-375-3p inhibitor significantly increased the expression of LRP5 in MVECs, whereas the miR-375-3p mimic inhibited LRP5 expression (Fig. 4A and B). We further validated the binding between miR-375-3p and LRP5 using dual-luciferase reporter assays. Compared with the LRP5 3’-UTR MUT vector, miR-375-3p overexpression markedly attenuated the luciferase activity of the LRP5 3’-UTR WT vector, confirming that miR-375-3p directly targets LRP5 (Fig. 4C and D). These results suggest that LRP5 is an important target for miR-375-3p and regulates the proliferation of MVECs.

Fig. 4
figure 4

miR-375-3p is negatively correlated with LRP5 under mechanical unloading conditions. (A) qRT‒PCR analysis of LRP5 mRNA expression in MVECs after transfection with mimic-375, inhibitor-375 or the negative control. (B) Western blotting analysis of the protein expression of LRP5 in MVECs after transfection. (C) After 293T cells were treated with mimic-375-3p and the corresponding controls for 48 h, the luciferase activities of the LRP5 WT and MUT reporters were evaluated. (N = 3). (D) Schematic representation of the luciferase reporters containing LRP5 3′-UTR WT or MUT sequences. (E) Western blotting analysis of the LRP5 protein levels under mechanical unloading conditions. (F) Western blotting analysis of PCNA protein levels in MVECs after transfection with si-LRP5 (N = 3). (G) Cell proliferation was evaluated by a CCK-8 assay (N = 3). (H) The results of the EdU incorporation assay were analyzed by confocal microscopy (N = 3). *P < 0.05, **P < 0.01 vs. control

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We then explored whether LRP5 was involved in clinorotation-induced cell proliferation and found that the protein expression of LRP5 decreased significantly under mechanical unloading (Fig. 4E). We then transfected LRP5 siRNA into MVECs and found that knockdown of LRP5 decreased the protein expression level of PCNA (Fig. 4F). CCK-8 and EdU labeling assays consistently showed that inhibition of LRP5 expression suppressed MVEC proliferation (Fig. 4G and H).

LRP5 is associated with the mir-375-3p-mediated inhibition of MVEC proliferation

To confirm whether the suppression of proliferation induced by miR-375-3p depends on LRP5 in MVECs, a miR-375-3p inhibitor and siRNA-LRP5 or its negative control were cotransfected into MVECs. We found that inhibitor-375-3p significantly increased PCNA protein levels, whereas cotransfection with siRNA-LRP5 significantly decreased this effect (Fig. 5A). The results of the CCK-8 and EdU labeling assays were consistent with these findings, as siRNA-LRP5 disrupted the growth of MVEC proliferation induced by inhibitor-375-3p (Fig. 5B and C). Taken together, LRP5 might partially counteract the miR-375-3p-mediated inhibition of MVEC proliferation during unloading, providing a scientific basis for the development of more effective measures to reduce the number of bone microvessels caused by unloading.

Fig. 5
figure 5

LRP5 is associated with the miR-375-3p-mediated inhibition of MVEC proliferation. (A) PCNA protein levels were measured by western blotting after cotransfecting inhibitor-375-3p and siRNA-LRP5 into MVECs (N = 3). (B) Cell proliferation was evaluated by a CCK-8 assay (N = 3). (C) The results of the EdU incorporation assay were analyzed by confocal microscopy (N = 3). Scale bar, 50 Î¼m. *P < 0.05, **P < 0.01 vs. control

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LRP5 is essential for HDAC6/miR-375-3p axis-mediated MVEC proliferation during unloading

Our data demonstrated that HDAC6 suppressed miR-375-3p and that LRP5 was the target of miR-375-3p. We further explored whether HDAC6 could positively regulate the expression of LRP5, and the results showed that HDAC6 promoted the protein expression of LRP5 under normal conditions (Fig. 6A). Moreover, to further verify whether HDAC6 could regulate LRP5 via inhibiting miR-375-3p under clinorotation unloading, we cotransfected pEX-HDAC6, mimic-375-3p, and their negative controls into MVECs. Western blotting analyses demonstrated that HDAC6 partially reversed the reduction in LRP5 expression induced by clinorotation unloading in MVECs through miR-375-3p (Fig. 6B). In addition, siRNA-LRP5 reversed the proliferation induced by pEX-HDAC6 during unloading, as indicated by the results of the PCNA protein expression, CCK-8 and EdU labeling assays (Fig. 6C-E). In conclusion, LRP5 was responsible for HDAC6/miR-375-3p axis-mediated MVEC proliferation during unloading.

Fig. 6
figure 6

LRP5 is essential for HDAC6/miR-375-3p axis-mediated MVEC proliferation during unloading. (A) LRP5 protein levels in MVECs were analyzed by western blotting after transfection with HDAC6 siRNA or pEX-HDAC6 (N = 3). (B) LRP5 protein expression was analyzed by western blotting after MVECs were transfected with pEX-HDAC6 alone or pEX-HDAC6 plus mimic-375 and subjected to clinorotation unloading (N = 3). (C) Cell proliferation was evaluated by a CCK-8 assay at 24–72 h (N = 3). (D) The results of the EdU incorporation assay were analyzed by confocal microscopy (N = 3). Scale bar, 50 Î¼m. (E) PCNA protein expression was analyzed by western blotting after the cells were transfected with pEX-HDAC6 alone or pEX-HDAC6 plus siRNA-LRP5 and subjected to clinorotation unloading (N = 3). *P < 0.05, **P < 0.01 vs. control

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Regulation of HDAC6 and mir-375-3p expression in MVECs can affect osteoblast proliferation under coculture conditions

To further examine whether the function of MVECs affect osteoblast function in vitro, we cotransfected si-HDAC6, miR-375-3p inhibitor or its negative control in MVECs to change the expression of HDAC6 and miR-375-3p, and then cocultured with osteoblast. For functional assays, we found that the expression of PCNA were reduced in coculture + si-HDAC6 group compared with co-culture + NC-si group, whereas the reduction of PCNA caused by si-HDAC6 was restored after transfected with inhibitor-375 (Fig. 7A). Furthermore, CCK-8 and EdU labeling assays were performed to evaluate the rate of osteoblast growth and proliferation. Compared to coculture + NC-si group, HDAC6 knockdown in MVECs caused a markedly decrease in the growth and proliferation of osteoblasts, while miR-375-3p inhibitor was partially reversed the suppression of osteoblast proliferation induced by si-HDAC6 (Fig. 7B and C). These results suggested that regulation of HDAC6 and miR-375-3p expression in MVECs could affect osteoblast proliferation under co-culture conditions, deepening the understanding of the association between MVECs regulation of osteoblast function and bone formation.

Fig. 7
figure 7

Regulation of HDAC6 and miR-375-3p expression in MVECs can affect osteoblast proliferation under coculture conditions. (A) PCNA protein expression was analyzed by western blotting after transfection with si-HDAC6 alone or pEX-HDAC6 plus inhibitor-375 and coculture with osteoblasts (N = 3). (B) Cell proliferation was evaluated by a CCK-8 assay at 24–72 h (N = 3). (C) The results of the EdU incorporation assay were analyzed by confocal microscopy (N = 3). Scale bar, 50 Î¼m. *P < 0.05, **P < 0.01 vs. control

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Discussion

Mechanical unloading-induced bone loss is one of the main limitations of long-term space flight and has attracted widespread attention from researchers. Angiogenesis and bone formation are closely linked in the growth, development, repair, and reconstruction of bones. Endothelial cells in bone can release cytokines that regulate the function of osteoblasts and promote the formation of new bone [19, 20]. Conversely, osteoblasts also secrete various cytokines to influence the activity of endothelial cells and angiogenesis [21]. In this study, we observed that unloading led to bone loss along with a significant decrease in the number of bone microvessels. Furthermore, we demonstrated that coculturing osteoblast cells with MVECs significantly increased their proliferation compared with monoculture conditions alone, providing a basis for preventing and treating osteoporosis as well as constructing vascularized tissue-engineered bones.

Our previous studies demonstrated that mechanical unloading inhibited MVEC proliferation and downregulated the expression of HDAC6, which is involved in multiple biological processes, including apoptosis, proliferation, autophagy and migration, through interactions with several histone or nonhistone proteins [22, 23]. In addition, our study found that the HDAC6 content was significantly reduced in the bone microvessels of HLU mice, which is consistent with the cellular results. It has been reported that HDAC6 serves as a crucial regulator of chromatin maintenance and function by regulating histone acetylation. In addition to directly regulating mRNA gene expression, the inhibition of HDAC6 activity has been shown to regulate miRNA expression in a variety of human diseases [24, 25]. Our previous microarray assay showed that the level of miR-375-3p was significantly elevated after the transfection of siRNA-HDAC6 in MVECs. Knocking down HDAC6 for ChIP assays illustrated that HDAC6 suppression increased histone acetylation in the miR-375-3p promoter region, indicating that HDAC6 inhibited the expression of miR-375-3p through regulating histone acetylation in MVECs.

miR-375 was first known as a pancreatic islet-specific miRNA that regulated insulin secretion [26]. The expression of miR-375 is reduced in multiple types of tumors, and it functions as a tumor suppressor through the dysregulation of transcription factors, aberrant promoter methylation, and aberrant histone deacetylation [27]. Recent studies have reported that hypermethylation of promoter DNA frequently leads to downregulation of miR-375 in different human cancers, such as hepatocellular carcinoma and esophageal squamous cell carcinoma [28, 29]. However, the role of miR-375-3p in MVEC proliferation, especially under unloading conditions, has not been reported. In this study, we found that the level of miR-375-3p continued to increase, peaked at 72 h under clinorotation–unloading conditions, and inhibited MVEC proliferation. For therapeutic consideration, we transfected a miR-375-3p inhibitor into cells and then mechanically unloaded them. The results of the PCNA protein expression, CCK-8 and EdU labeling assays indicated that miR-375-5p knockdown could reverse the suppression of MVEC proliferation induced by mechanical unloading. Furthermore, we demonstrated that miR-375-3p could inhibit MVEC proliferation by negatively regulating LRP5.

LRP5 is generally considered a Wnt coreceptor that has been implicated in activating the canonical Wnt/β-catenin pathway. After binding to Wnt ligands, LRP5 synergizes with Frizzled to activate the Wnt/β-catenin signaling pathway and prevent cytoplasmic β-catenin ubiquitination and degradation, resulting in β-catenin nuclear translocation and activation of Wnt target genes [30]. Canonical Wnt signaling is related to tumorigenesis, angiogenesis, blood‒brain barrier formation, and neuronal development. As a positive regulator of cell proliferation, LRP5 is a widely studied molecule that plays an important role in inducing angiogenesis. A recent study reported that knocking down LRP5 significantly inhibited the proliferation of HepG2 cells by disrupting the stability of nucleoporin 37 and may lead to subsequent destruction of the integrity of the nuclear pore complex [31]. Furthermore, mice deficient in the Wnt coreceptor LRP5 exhibited a lack of deep retinal blood vessels [32]. LRP5 stimulates pulmonary vascular development in neonatal mice and compensatory lung growth after PNX in adult mice and controls multiple angiogenic pathways, such as angiopoietin–Tie2, vascular endothelial growth factor (VEGF)–VEGFR2, and neuropilin [33]. Overexpression of LRP5, which restores the expression of angiogenic factors, could reverse the age-induced degeneration of vascular and alveolar morphogenesis. Our study showed that LRP5 was downregulated under mechanical unloading and associated with the miR-375-3p-mediated inhibition of MVEC proliferation. In addition, we further verified that HDAC6 promoted the protein expression of LRP5 and that LRP5 was responsible for HDAC6/miR-375-3p axis-mediated MVEC proliferation during unloading.

Conclusion

Disuse bone loss may be closely related to a reduction in the number of bone microvessels. Mechanistically, HDAC6 inhibited miR-375-3p expression through decreasing histone acetylation in the miR-375-3p promoter region in MVECs. Furthermore, silencing miR-375-3p promoted MVEC proliferation by directly targeting LRP5 expression and effectively attenuated the negative effects induced by clinorotation (Fig. 8). Our study revealed that the HDAC6/miR-375-3p/LRP5 signaling pathway in MVECs provides new ideas for the treatment and prevention of bone mass loss induced by unloading.

Fig. 8
figure 8

A schematic diagram of the HDAC6/miR-375-3p/LRP5 pathway regulating clinorotation unloading-induced inhibition of MVEC growth. Suppression of HDAC6 expression promoted miR-375-3p expression, thereby inhibiting the expression of LRP5

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Data availability

The datasets are available from the corresponding author on request.

Abbreviations

ALP:

Alkaline phosphatase

BMD:

Bone mineral density

BS/BV:

Bone surface area to bone volume ratio

ChIP:

Chromatin immunoprecipitation

CCK-8:

Cell Counting Kit-8

EMCN:

Endomucin

HDAC6:

Histone deacetylase 6

IF:

Immunofluorescence

LRP5:

Low-density lipoprotein-related receptors 5

MVECs:

Microvascular Endothelial cells

EdU:

5-Ethynyl-2′-deoxyuridine

BV/TV:

Relative bone volume

Tb.Th:

Trabecular thickness

Tb.N:

Trabecular number

Tb.Sp.:

Trabecular separation

VEGF:

Vascular endothelial growth factor

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Acknowledgements

Not applicable.

Funding

The present study was supported by grants from the National Natural Science Foundation of China (grant nos. 31971171 and 82302112) and Key R&D plan of Shaanxi Province (grant nos. 2023-YBSF-272).

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Author notes

  1. Liqun Xu, Lijun Zhang and Gaozhi Li contributed equally to this work.

Authors and Affiliations

  1. The Key Laboratory of Aerospace Medicine, Ministry of Education, Air Force Medical University, Xi’an, Shaanxi, 710032, China

    Liqun Xu, Lijun Zhang, Gaozhi Li, Xiaoyan Zhang, Quan Sun, Zebing Hu, Xinsheng Cao, Yixuan Wang, Fei Shi & Shu Zhang

  2. Department of Otolaryngology Head and Neck Surgery, Bethune International Peace Hospital, Shijiazhuang, Hebei, 050081, China

    Lijun Zhang

  3. The 94498th Unit of Chinese PLA, Nanyang, Henan, 473000, China

    Gaozhi Li

  4. Department of Otolaryngology Head and Neck Surgery, Western Theater Air Force Hospital of PLA, Chengdu, Sichuan, 610065, China

    Xiaoyan Zhang

  5. Department of Gastroenterology, The 940th Hospital of Joint Logistics Support Force of Chinese PLA, Lanzhou, 730050, China

    Yixuan Wang

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Contributions

Material preparation, data collection and analysis were performed by Xu L, Zhang L and Li G. Study conception and design was performed by Zhang S and Shi F. Figure design and table making were performed by Zhang X, Sun Q and Hu Z. Supervision and Funding acquisition were performed by Shi F, Wang Y and Cao X. The first draft of the manuscript was written by Xu L and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Xu, L., Zhang, L., Li, G. et al. Inhibiting histone deacetylase 6 suppresses the proliferation of microvascular endothelial cells by epigenetically activating miR-375-3p, potentially contributing to bone loss during mechanical unloading. J Transl Med 22, 811 (2024). https://doi.org/10.1186/s12967-024-05608-7

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Journal of Translational Medicine

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