FOXO1-mTOR pathway in vascular pericyte regulates the formation of type H vessels to control bone metabolism

Abstract

Background

As the population aging progresses, age-related osteoporosis has become one of the most common and severe chronic degenerative diseases. Due to insufficient understanding of its complex pathomechanisms, current clinical treatments often suffer from many negative effects. Type H vessels play critical role in bone remodeling owing to their specialized function in coupling angiogenesis and osteogenesis. Increasing evidences have shown a close association between the age-related decline of type H vessels and bone loss. However, the underlying mechanisms whereby the regression of type H vessels with aging remain largely unknown.

Methods

Col2-Cre^ERT/Foxo1^flox/flox mice and FOXO1 inhibitor (AS1842856) treated adult (6 months) and middle aged (10 months) mice were utilized for evaluating the variations in bone volume, bone microarchitecture and type H vessels through micro-CT scanning analysis, histological staining and immunofluorescence staining. In vitro tube-forming and scratch assays were applied to evaluate the angiogenic capacity of human umbilical vein endothelial cells (HUVECs) exposed to AS1842856 or conditioned culture milieu of Human Brain Vascular Pericytes (HBVPs). The expression of pericyte marker proteins, myofibroblast-related proteins and genes in inhibitors-stimulated HBVPs were detected via western blot analysis and Reverse transcription-quantitative PCR (RT-qPCR). Furthermore, perivascular myofibroblastic-like transformation was confirmed in AS1842856-treated animal models through immunofluorescence staining. We also constructed Adipoq-Cre/Foxo1^flox/flox conditional knockout mice and measured their bone mass and type H vessels by micro-CT and immunofluorescence staining. Mechanistic experiments in vitro were conducted via detection of mTOR signalling expression in HBVPs with pharmacological intervention (AS1842856 and rapamycin), genetic knockdown of Foxo1, or FOXO1-overexpression plasmid treatment, verified by RT-qPCR, western blot analysis and cellular immunofluorescence staining. In vivo validation was conducted on Adipoq-Cre/Foxo1^flox/flox mice using immunofluorescence staining. Finally, alterations in osteo-morphology and type H vessels were verified in AS1842856-treated and rapamycin-treated aged mouse models.

Results

This study identified FOXO1 in pericytes as key components for the formation of type H vessels. We found that FOXO1 expression in pericytes decreases with aging, and pharmacological blocking with AS1842856 promoted type H vessels degeneration and increased bone loss in adult and middle-aged mice, while rapamycin prevented the above pathology in middle-aged mice. We further showed that the loss of FOXO1 in Adipoq⁺ pericytes led to degeneration of type H vessels and bone loss in mice. Mechanistically, the inhibition of FOXO1 by AS1842856 or knockdown of Foxo1 by siRNAs activated mTOR signaling, thereby resulting in the myofibroblastic transformation of pericytes. Furthermore, blocking mTOR signaling by rapamycin rescued the above effects in vitro and in vivo.

Conclusion

Our findings uncover a hitherto unknown role of FOXO1 in maintaining the phenotype and function of pericytes, thereby promoting formation of type H vessels. This suggests that targeting the FOXO1-mTOR pathway in pericytes could be a potential therapeutic approach to overcome the regression of type H vessels and bone degeneration with aging.

The translational potential of this article

Our research uncovers a previously unidentified role of FOXO1 in preserving pericyte characteristics and promoting the development of type H vessels. Future translational research targeting the FOXO1-mTOR pathway in pericytes may provide new strategies for the prevention and treatment of age-related osteoporosis in the clinic.

Graphical abstract

1. Introduction

The aging population is leading to an increasing incidence of age-related osteoporosis, and consequently an increase in fragility fractures [1]. Epidemiological data reveal that 10.5 % of men and 9.7 % of women aged 40 and older in China [2] and approximately 20 % of men and 50 % of women aged 50 and older in the United States [3], suffered from osteoporotic fracture, a leading cause of disability in the elderly [4]. Most approved therapies for osteoporosis have focused on either suppressing bone resorption, such as bisphosphonates and calcitonin, or promoting bone formation by targeting anabolic pathways, such as recombinant parathyroid hormone and romosozumab-aqqg [5]. However, clinical data have shown that while these medications may significantly improve trabecular bone mass, they also have serious adverse effects, limiting their safety for long-term use [6,7]. Thus, age-related osteoporosis remains a major challenge, necessitating further research to identify new therapeutic targets to prevent or halt bone degeneration.

Bone remodeling requires a subtype of capillaries characterized by strong expression of CD31 and Endomucin (Emcn), which is termed type H vessels. These capillaries locate in the metaphysis close to the chondro-osseous junction and provide crucial niches for the maintenance of osteoprogenitors and hematopoietic stem cells [8,9]. Therefore, the number of type H vessels is critical to appropriate bone modeling, remodeling, and function [8]. Increasing evidences have shown that the decline in type H vessels with age correlates with age-related bone loss [8,10,11]. However, little is known about the mechanism underlying the reduction of type H vessels with aging. Recent studies have indicated that the loss of pericyte contributes to decreased capillary density and dysfunction in the aged brain [12,13]. As perivascular mural cells of capillaries, pericytes are essential for regulating capillary integrity, diameter, and blood flow [12,14]. Indeed, evidence has shown the critical role of pericytes in coupling angiogenesis with osteogenesis during bone remodeling and repair [15]. Despite the well-established endothelial–pericyte interaction in angiogenesis, further studies are necessary to elucidate the potential role and mechanism of pericytes in bone degeneration with aging.

Forkhead box O1 (FOXO1), a key transcription factor belonging to the subfamily member O of forkhead box family, is activated under stress conditions such as growth factor depletion and high levels of ROS, thereby regulating cell metabolism, differentiation, and transformation [16,17]. FOXO1, ubiquitously expressed in mammalian tissues, including bone, plays an important role in bone metabolism. Studies in mice have indicated a critical role of FOXO1 in osteoblastic differentiation and bone formation. Conditional knock-out of FOXO1, -3, and -4 in Osterix-positive progenitors increases the number of osteoblasts and bone mass [18]. Conversely, deletion of FOXO1 in Bglap-positive osteoblasts induces bone loss in young mice but improves bone mass in old mice [19]. Previous evidence has demonstrated that vessels are sensitive to changes in FOXO1 activity in endothelial cells. Loss of FOXO1 induces a profound vessel hyperplasia and enlargement, while overexpression of FOXO1 restricts vascular expansion and leads to hypobranching [17,20]. However, the role of FOXO1 in mediating age-associated bone loss remains unknown.

Here, we report a previously unknown fundamental function of FOXO1 in maintaining the phenotype of vascular pericytes by suppressing mTOR signaling. Pharmacological inhibition and genetic deletion of FOXO1 in pericytes may lead to the pericyte-to-myofibroblast transformation and the degeneration of type H vessels in the metaphyseal bone. Since FOXO1 in pericytes is essential for bone metabolism by maintaining the formation of type H vessels in bone, deficiency of perivascular FOXO1 contributes to age-related bone loss.

2. Materials and methods

2.1. Mice models and treatments

All animal experiments were performed according to the protocols approved by the Institutional Animal Care and Use Committee (IACUC) at Southern Medical University Nanfang Hospital, and all procedures were carried out following IACUC guidelines. Male C57BL/6 mice aged 6, 10, 12 months and 8 weeks were obtained from the Experimental Animal Center at Southern Medical University, and were housed under specific pathogen-free conditions (25 °C, 12-h light/dark cycle) with free access to food and water. All mice were acclimated to our animal facility for at least one week before use in the experiments.

To investigate the role of FOXO1 in bone metabolism during aging, we treated the adult (6 months old) and middle aged (10 months old) mice with FOXO1 inhibitor (AS1842856, HY-100596, MedChemExpress) at dose of 30 mg/kg per day, 5 days per week, or an equal volume of vehicle by oral gavage. After 2 months of treatment, mice at 8 and 12 months old were euthanized, and bilateral tibiae and femora were harvested for further analysis. To evaluate the role of mTOR signaling in vessel degeneration in bone, 12-month-old mice received 2 mg/kg rapamycin (HY-10219, MedChemExpress) by intraperitoneal injection on alternate days, while control mice were treated with the same volume of saline. After 4 weeks of treatment, mice were euthanized, tibiae and femurs were collected for further analysis.

To investigate the role of FOXO1 in bone metabolism, we crossed Col2-Cre^ERT mice (RRID: IMSR_JAX: 006774) with Foxo1^flox/+mice (Quote: TOS191231MG2-B, Stock No. 017986, Cyagen Bioscience Inc. China) to generate Col2-Cre^ERT/Foxo1^flox/flox mice. Ten-month-old Col2-Cre^ERT/Foxo1^flox/flox mice were injected intraperitoneally with tamoxifen (CAS# 10540-29-1, Sigma–Aldrich) at 75 mg/kg once a day for 5 consecutive days. After 8 weeks of treatment, mice were euthanized, and femurs were collected for further analysis.

Considering that Adiponectin expressing cells (Adipoq⁺) are as abundant as both pericytes and stromal cells in the bone marrow [21], we crossed Adipoq-Cre mice (RRID: IMSR_JAX: 010803) with Foxo1^flox/flox mice, to investigate the role of FOXO1 in vasculature maintenance. The Adipoq-Cre/Foxo1^flox/flox mice and Foxo1^flox/flox mice in the same littermates were euthanized at 4 weeks of age, and femurs were harvest for comparative analysis. PCR analyses of genomic DNA were used to determine the genotypes of the mice using the following primers: Col2-Forward: 5’-cactgcgggctctacttcat-3’, Col2-Reverse: 5’-accagcagcacttttggaag-3’; Adipo-Forward: 5’-acggacagaagcattttcca-3’, Adipo-Reverse: 5’-ggatgtgccatgtgagtctg-3’; LoxP (Foxo1)-Forward: 5’-gctttcttctaatgccatctggtt-3’, LoxP (Foxo1)-Reverse: 5’-tattttagagctggcagaaagcac-3’.

2.2. Micro-computed tomography (micro-CT) analysis

For micro-CT analysis, femurs were obtained from mice, dissected free of soft tissue, fixed in 4 % paraformaldehyde for 24 h, and subsequently scanned by Bruker Micro-CT Skyscan 1276 system (Bruker, Kontich, Belgium). Scanning was performed with a voxel size of 5 μm, a voltage of 85 kV, a current of 200 mA, 1 mm Al filter and an integration time of 384 ms. Density measurements were calibrated using the manufacturer’s calcium hydroxyapatite (CaHA) phantom. Reconstruction was performed using NRecon software (version 1.7.4.2, SkyScan, Bruker). 3D images were obtained from contoured 2D images by methods based on distance transformation of the grayscale original images (CT vox; version 3.3.0, Bruker). Both 3D and 2D analyses were performed using software CT Analyser (version 1.18.8.0, Bruker). Analyses of the bone micro-architecture were carried out in a region of interest (ROI). The ROI of cancellous bone was defined as the area 0.45 mm from the epiphyseal growth plate with a length of 2 mm in the distal femora. A length of 1 mm ROI of the cortical bone was at the femoral mid-diaphysis 2.5 mm away from the distal growth plate. The parameters, including bone mineral density (BMD), trabecular bone volume fraction (BV/TV), trabecular number (Tb. N), trabecular thickness (Tb. Th), trabecular separation (Tb. Sp), trabecular bone pattern factor (Tb. Pf), cortical cross-sectional bone area (Cs. B. Area), and cortical cross-sectional thickness (Cs. Th) were calculated.

2.3. Histochemistry and immunohistochemistry analyses

Femurs were dissected free from attached muscle, and fixed overnight in 10 % formalin at 4 °C. After washing three times with ice-cold PBS, samples were decalcified at 4 °C using 10 % EDTA (pH 8.0) for 14 days, and finally processed for paraffin embedding. Coronal sections at 4-μm-thick were cut and processed for staining.

For hematoxylin and eosin (H&E) staining, bone sections were processed according to the standard protocols. Briefly, sections were stained by hematoxylin for 2 min and by eosin for 10 s.

For immunohistochemistry staining, after being deparaffinized, rehydrated and washed, sections were processed for heat-induced antigen retrieval (Tris–EDTA Buffer, pH 9.0). Sections were then incubated with 3 % hydrogen peroxide for 15 min to eliminate endogenous peroxidase activity, followed by incubation with 10 % goat serum (Cat. SL038, Solarbio, China) for 1 h to block unspecific antigens. Then, the sections were incubated with primary antibody against osteocalcin (Cat. ab93876, Abcam) overnight at 4 °C. Next day, after wash with PBST (PBS containing 0.1 % Triton X-100) for 3 times, sections were incubated with biotinylated secondary antibodies and Vectastain avidin and biotinylated horseradish peroxidase macromolecular complex (ABC) reagent (Cat. AK-5000, Vector Laboratories, Burlingame, USA) according to the manufacturer’s protocol. Immunoreactivity was assessed using a diaminobenzidine (DAB) substrate kit (Cat. SK4100, Vector Laboratories, Burlingame, USA). A microscope (BX63; Olympus, Tokyo, Japan) was used for observation and imaging to detect immunoreactivity. The number of positive-stained cells was calculated with Image J software (version 1.8.0, National Institutes of Health, Bethesda, MD, USA).

The activity of osteoclasts was detected by tartrate-resistant acid phosphatase (TRAP) staining kits (Cat. No. 294–67001, Wako, Japan) following the manufacturers’ instructions. In brief, rehydrated sections were incubated in TRAP staining solution in a moist chamber at room temperature for 10∼30 min. TRAP-positive multinucleated cells with at least three nuclei were counted under a microscope (BX63; Olympus, Tokyo, Japan).

2.4. Immunofluorescence analysis

For immunofluorescence staining, femoral samples were fixed with 4 % paraformaldehyde overnight, decalcified with daily changes of 10 % EDTA (pH 7.5) for 6 days, and incubated in 30 % sucrose overnight prior to embedding in optimal cutting temperature (OCT) compound (Cat. BL557A, Biosharp, China). Sections at 10-μm thickness of frozen-embedded samples were prepared for staining. Frozen sections were rewarmed and washed twice in PBS buffer containing 0.1 % Triton X-100 (Cat. T8200, Solarbio, China) for 10 min per time. After being blocked with 10 % goat serum for 1 h at room temperature, sections were incubated with primary antibodies overnight at 4 °C. Sections were then washed 5 times in PBS and then incubated with a secondary antibody for 1 h at room temperature in the dark. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI, Cat. E607303-0002, BBI Life Science, China). All sections were observed and imaged under a Zeiss LSM980 confocal microscope (Carl Zeiss, Germany), and the acquired images were analyzed using ZEN software (ZEN blue, Carl Zeiss).

For immunofluorescence in cell cultures, HBVPs were seeded at 1 × 10⁶ cells per dish in confocal 35-mm glass-bottomed petri dishes. When reaching 70 % confluency, cells were treated with 50 nM Foxo1-siRNA, 20 nM Rapamycin, or the same volume of vehicle. After 48 h of treatment, cells were washed three times with PBS buffer, fixed with pre-cooled 4 % paraformaldehyde solution for 20 min at room temperature, permeabilized with 0.5 % v/v Triton X-100 (PBST), blocked in 5 % goat serum, and incubated with primary antibodies. After washing, cells were incubated with secondary antibodies. Images were acquired using a confocal microscope (Zeiss LSM980, Carl Zeiss, Germany). Three fields per well were randomly chosen, and quantitative data were obtained from at least three independent experiments.

The antibodies used for immunofluorescence were Rabbit anti-CD31(1:200, #ab28364 Abcam), Rat anti-Endomucin (1:50, #sc-65495, Santa Cruz Biotechnology), Rabbit anti-FOXO1 (1:200, #18592-1-AP, Proteintech, China), Mouse anti-αSMA (1:400, #67735-1-Ig, Proteintech, China), Rabbit anti-αSMA (1:200, #14395-1-AP, Proteintech, China), Mouse anti-PDGFRβ (1:200, #ab69506, Abcam), Mouse anti-Phospho-mTOR (1:200, #67778-1-Ig, Proteintech, China), Rabbit anti-Phospho-mTOR (1:500, #80596-1-RR, Proteintech), Mouse anti-PDGFRα (1:200, #60045-1-Ig, Proteintech), Goat anti-Rabbit IgG Alexa Fluor™ 488 (1:200, #A-11008, Invitrogen), Goat anti-Rat IgG Alexa Fluor™ 594 (1:200, #A-11007, Invitrogen), Goat anti-Mouse IgG Alexa Fluor™ 647 (1:200, #A-21235, Invitrogen).

2.5. Serum P1NP and CTX-1 assays

Mice were anesthetized, and then a cardiac puncture was performed to collect whole blood. The blood was allowed to coagulate at room temperature for 2 h, followed by centrifugation for 15 min at 1000 × g. Serum was then separated immediately and stored in aliquots at −80 °C for further analysis. Biochemistry assays were performed with the Mouse Procollagen Ⅰ N-Terminal Peptide (P1NP) ELISA Kit (Cat. CSB-E12775m, CUSABIO, China) and Mouse cross linked C-telopeptide of type Ⅰ collagen (CTX-1) ELISA Kit (Cat. CSB-E12782m, CUSABIO, China). According to the manufacturer’s instructions, absorbance was measured at 450 nm, and concentrations were calculated based on a standard curve fitted by the software (CurveExpert, Version 1.40, Microsoft Corporation).

2.6. Cell culture and conditional culture

The HBVPs and HUVEs cell lines were purchased from Zhongqiaoxinzhou Biological Technology Co., Ltd (Shanghai, China). For single culture, HBVPs were grown in pericyte medium (PM, Cat. #1201, ScienCell). HUVEs were cultured in DMEM medium (#11995, Gibco) containing 2 % FBS (fetal bovine serum, Gibco) and 100 U/mL penicillin-streptomycin (#15140-122, Thermo Fisher Scientific).

For conditional culture of HUVECs, the supernatant of HBVPs culture treated with various concentrations (0, 0.1 and 1 μM) of AS1842856 (HY-100596, MedChemExpress) for 48 h were collected. The supernatants were centrifugated at 1000 rpm for 5 min to remove large cell debris and subsequently filtered through a 0.22-μm filter. The filtered supernatants were then mixed with DMEM complete medium at ratio of 1:1 (v/v) and used as conditioned medium for HUVECs.

2.7. Tube formation assay

Chilled 24-well plates were coated with 150 μL Matrigel (Cat. # 356234; Corning) that was polymerized at 37 °C for 30∼60 min. HUVECs (6 × 10⁴ cells/well) were serum-starved overnight, trypsinized, and seeded in Matrigel-coated well with low serum (1 % fetal bovine serum). To evaluate whether blocking FOXO1 in HUVECs might influence its tube formation, we treated the HUVECs with various concentrations (0, 0.1 and 1 μM) of AS1842856. The formation of tubes was imaged on an inverted microscope (Olympus IX-73, Japan) after 12 h of treatment. To determine whether blocking FOXO1 in HBVPs might affect the tube formation of HUVECs, we treated HUVECs with the conditioned media collected from HBVPs treated with various concentrations (0, 0.1 and 1 μM) of AS1842856. Tube formation was captured on the inverted microscope at the indicated time points. Images were processed using the Angiogenesis Analyzer Program for ImageJ (version 1.8.0, NIH, USA) to obtain the total branching points and total tube length.

2.8. Scratch assay

HUVECs were cultured in 35-mm petri dishes at a density of 1 × 10⁶ cells per dish and incubated at 37 °C for 24 h. When cells reached a 90 % confluent monolayer, a 200-μL sterile tip was used to scratch a fixed-width gap in the cell monolayer. After washing away cell debris with PBS, cells were then incubated in conditioned media. Scratch closure was monitored under an inverted microscope (Olympus IX-73, Japan) at 0, 24, 48, and 72 h after scratching. The area the scratch was detected by ImageJ software (version 1.8.0, NIH, USA) and the healing rate was calculated to assess the migration ability of the HUVECs. The healing rate was calculated as follows: wound-healing rate (%) = (A₀ – A_n)/A₀ × 100 %, where A₀ and A_n represents the initial area and unhealed area of the wound, respectively.

2.9. RNA isolation and quantitative real-time PCR (qPCR) analysis

Total RNA of cells was extracted using AG RNAex Pro Reagent (#AG21101, Accurate biology, China) according to the manufacturer’s instructions. Reverse transcription into cDNA was performed using Hifair® Ⅲ 1st Strand cDNA Synthesis SuperMix for qPCR (#11141ES60, Yeasen, China). qPCR was performed using SYBR Green (#11202ES08, Yeasen, China) on a QuantStudio5 system (Applied Biosystems, USA) according to the manufacturer’s protocol. The GAPDH genes were used as an internal control. The relative amount of each gene was calculated using the 2-ΔΔCT method. PCR primers for samples from HBVPs were h-GAPDH Forward, 5’-cagcctcaagatcatcagca-3’, Reverse, 5’-tgtggtcatgagtccttcca-3’; h-VEGF Forward, 5’-gccttgccttgctgctctac-3’, Reverse, 5’-tgattctgccctcctccttctg-3’; h-ANG-1 Forward, 5’-agaaccttcaaggcttggttac-3’, Reverse, 5’-ggtggtagctctgtttaattgct-3’; h-PDGFRβ Forward, 5’-tgatgccgaggaactattcatct-3’, Reverse, 5’-tttcttctcgtgcagtgtcac-3’; h-ACTA2 Forward, 5’-aaaagacagctacgtgggtga-3’, Reverse, 5’-gccatgttctatcgggtacttc-3’; h-COL1A1 Forward, 5’-gagggccaagacgaagacatc-3’, Reverse, 5’-cagatcacgtcatcgcacaac-3’; h-TAGLN2 Forward, 5’-atcccaactggttccctaagaa-3’, Reverse, 5’-cccatctgtaacccgatcacg-3’; h-LOXL1 Forward, 5’-ggctgctatgacacctacaatg-3’, Reverse, 5’-gtagtgaatgttgcatctcacca-3’; h-FOXO1 Forward, 5’-ggatgtgcattctatggtgtacc-3’, Reverse, 5’-tttcgggattgcttatctcagac-3’; h-FOXO3a Forward, 5’-tcacgcaccaattctaacgc-3’, Reverse, 5’-cacggcttgcttactgaagg-3’; h-FOXO4 Forward, 5’-ggctgccgcgatcatagac-3’, Reverse, 5’-ggctggttagcgatctctgg-3’; h-FOXO6 Forward, 5’-gagttcgtggtggatgctga-3’, Reverse, 5’-gcttcttcttgctcgccttg-3’.

2.10. Western blot analysis

Total protein extracts from bone tissue or cells were prepared using radio immunoprecipitation assay lysis buffer (#PC101, Epizyme, Shanghai, China) containing protease inhibitor (#HY-K0010, MedChemExpress) and phosphatase inhibitor (#HY-K0023, MedChemExpress). Protein concentrations were quantified using a Pierce Bicinchoninic Acid protein assay kit (#23225, Thermo Fisher Scientific). Proteins were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (#IPVH00010, Immobilon P, Merck Millipore, Cork, Ireland). After being blocked in 5 % milk powder in TBST (Tris-buffered saline TBS buffer + 1 % Tween20) at room temperature for 1 h, the membranes were incubated with primary antibody at 4 °C overnight. The primary antibodies included Rabbit anti-β-Tubulin (1:5000, #10094-1-AP, Proteintech), Mouse anti-GAPDH (1:5000, #60004-1-Ig, Proteintech), Rabbit anti-FOXO1 (1:2000, #18592-1-AP, Proteintech), Mouse anti-PDGFRα (1:2000, #60045-1-Ig, Proteintech), Mouse anti-PDGFRβ (1:2000, #ab69506, Abcam), Mouse anti-αSMA (1:2000, #14395-1-AP, Proteintech), and Mouse anti-Phospho-mTOR (1:2000, #67778-1-Ig, Proteintech). The following day, membranes were washed three times in TBST, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h. The secondary antibodies were goat anti-mouse HRP (#HA1006, Huabio, China) and goat anti-rabbit HRP (HA1001, Huabio, China). Immunoblots were developed using Western Lightning Plus enhanced chemiluminescence (ECL) (#NEL105001EA, PerkinElmer, MA, USA), visualized and imaged using a chemiluminescence instrument (Guangzhou Ewell Bio-Technology, Guangzhou, China).

2.11. Plasmid transfection

The cDNA of human Foxo1 gene, based on NCBI Reference Sequence NM_002015.4, was cloned into the pcDNA3.1 vector to construct Foxo1/pcDNA3.1 for Foxo1 overexpression studies. The construction of Foxo1/pcDNA3.1 (#BW2266, BVA01) was prepared by Umine Biotechnology Co. LTD, Guangzhou. The primer sequences used for plasmid construction were: forward, 5’-atccgccaccatggccgaggcgcctcag-3’; reverse, 5’-ctcgaggcctgacacccagctatgtgtcg-3’. For plasmid transfection, HBVPs were seeded in 6-well plates at a density of 0.5∼1 × 10⁶/well. When the growth confluence reached 70∼80 %, cells were transfected with Foxo1/pcDNA3.1 or pCDNA3.1 (control) using Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific). After 48 h of transfection, cells were used for Western blot analysis.

2.12. siRNA transfection

Negative control siRNAs (siNC) and Foxo1-specific siRNAs were obtained from GenePharma (Shanghai, China). The sequences of Foxo1-targeting siRNAs were 5’-uuacuguuguuguccauggtt-3’ (si-Foxo1#1); 5’-auaguacuagcauuugagctt-3’ (si-Foxo1#2); 5’-uugucuugacacuguguggtt-3’ (si-Foxo1#3) and 5’-uuaucucagacagacugggtt-3’ (si-Foxo1#4). HBVPs at confluency of 50∼70 % were transfected with siRNAs using the Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific) following the manufacturer’s protocol. Six hours after transfection, the medium was replaced with fresh medium, with or without rapamycin (20 nM). Cells were collected for subsequent experiments 48 h after transfection.

2.13. Statistical analysis

Statistical analyses were performed using SPSS 19.0 software (IBM, USA). Values in graphs were expressed as means ± SEM. Levene’s test examined variance homogeneity of data to confirm the normality and homogeneity. For two-group comparisons, unpaired Student’s t-test (equal variances) or Mann–Whitney U test (unequal variances) was used to evaluate the statistical difference. For multiple group comparisons, one-way analysis of variance (ANOVA) was used, and the differences between groups were evaluated using post hoc multiple comparisons with Tukey test. However, Dunnett T3’s test was applied when the populations variances were not equal. A value of P < 0.05 was used as the criterion for statistical significance.

3. Results

3.1. FOXO1 inhibition aggravates bone loss in mice

To investigate the role of FOXO1 in bone metabolism, we first examined the protein levels of FOXO1 in tibiae of mice at 3, 10, and 24 months of age. Western blot results showed that the FOXO1 levels in 24-month-old mice were dramatically decreased relative to 3- and 10-month-old mice (Fig. 1a and b). We then treated adult and middle-aged mice with AS1842856, an inhibitor of FOXO1 for 2 months. Micro-CT analysis and three-dimensional reconstruction of trabecular bone in the distal femur revealed a decrease in BV/TV in 12-month-old mice compared with 8-month-old ones (Fig. 1c and e). The reduction in bone mass was principally due to decreased trabecular number (Tb. N) and trabecular thickness (Tb. Th) (Fig. 1f and g), alongside increased trabecular separation (Tb. Sp) and trabecular pattern factor (Tb. Pf) (Fig. 1h and i). Importantly, AS1842856 treatment exacerbated reductions in both BMD and BV/TV in 8- and 12-month-old mice (Fig. 1d and e). The decreased BV/TV by AS1842856 treatment was mainly attributed to reduced Tb. N rather than Tb. Th (Fig. 1f and g). AS1842856 treatment increased Tb. Sp only in 8-month-old mice but increased Tb. Pf in the bone in both 8- and 12-month-old mice (Fig. 1h and i). In contrast, micro-CT analysis of the femur diaphysis showed no differences in cortical thickness or cortical bone cross-sectional area (Supplementary Fig. S1a-c). To further explore the cellular mechanism that might mediate the bone loss induced by AS1842856, we performed immunohistochemical staining for Ocn, an osteoblastic marker, and histochemistry staining for TRAP, an osteoclastic marker. Results showed that AS1842856 treatment reduced the number of Ocn positive-stained cells on the surface of trabecular bone, while increased the number of TRAP positive-stained cells in the bone in both 8- and 12-month-old mice (Fig. 1j–l). In consistent, 12-month-old mice had lower level of P1NP, a bone formation marker, compared with 8-month-old mic, and AS1842856 treatment further decreased serum P1NP levels in both age groups (Fig. 1m). Additionally, AS1842856 treatment significantly increased the level of bone resorption marker CTX-1 in 12-month-old mice, with no effect observed in 8-month-old mice (Fig. 1n). These results indicate that decreased expression levels of FOXO1 may contribute to the age-related bone loss and that pharmacological blocking FOXO1 with AS1842856 may aggregate bone loss by suppressing anabolism while promoting catabolism of trabecular bone.

Figure 1.

FOXO1 expression decreases with age and AS1842856 treatment may aggravate bone loss in mice.a, b. Western blotting (a) and quantitative analysis (b) of FOXO1 expression in tibias of 3-, 10- and 24-month-old mice. n = 6/group. ∗P < 0.05; ∗∗P < 0.01. c. Representative microcomputed tomography (micro-CT) images of the distal femur and trabeculae after 2 months of treatment with FOXO1 inhibitor (AS1842856, 30 mg/kg, 5 days of continuous gavage with 2 days interval) in 8- and 12-month-old mice. Scale bars represent 200 μm. d-i. Quantitative analysis of three-dimensional structural parameters, including bone mineral density (BMD) (d), trabecular bone volume fraction (BV/TV) (e), trabecular number (Tb. N) (f), trabecular thickness (Tb. Th) (g), trabecular separation (Tb. Sp) (h), and trabecular pattern factor (Tb. Pf) (i). n = 6/group. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. j. Representative images of immunohistochemical staining (upper panel) for osteocalcin (Ocn) and tartrate-resistant acid phosphatase (TRAP) staining (lower panel) in the femoral metaphysis of 8- and 12-month-old mice after 2 months of AS1842856 treatment. Scale bars: 20 μm (upper panel), 50 μm (lower panel). k. Quantitative analysis of osteocalcin-positive (Ocn⁺) cells on the surface of bone trabeculae (N. Ocn⁺/mm). n = 5/group. ∗∗∗P < 0.001. l. Quantitative analysis of TRAP⁺ cells per bone surface (N. TRAP⁺/mm). n = 5/group. ∗P < 0.05, ∗∗∗P < 0.001. m, n. Serum bone formation marker P1NP (m) and resorption marker CTX-1 (n). n = 6/group. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Data are represented as mean ± SEM. Each data point represents one animal. Statistical comparisons were made using one-way ANOVA with Tukey’s post-hoc test if variances were equal (b, d, e, f, g, k, l, m and n); otherwise, Dunnett’s T3 test was utilized (h and i).

Given that type II collagen (Col2)-positive cells, defined as skeletal stem cells, chondrogenic and osteoblastic linage cells, contribute to both bone development and metabolism [22], we constructed Col2-Cre^ERT/Foxo1^f/f conditional knockout (CKO) mice to investigate the function of FOXO1 in bone metabolism. Foxo1 was knocked out in these mice at 10 months of age following tamoxifen induction. Unexpectedly, we found that knock out of Foxo1 in Col2⁺ cells had no effect on bone mass or the microstructure of trabecular and cortical bone in the femurs of CKO mice (Supplementary Fig. S2a-i). These data suggest that FOXO1 in Col2⁺ cells may not be essential for trabecular or cortical bone metabolism in older mice.

3.2. FOXO1 inhibition exacerbates the degeneration of type H vessels

Type H vessels (CD31^hiEmcn^hi vessels) are thought to be intimately associated with the bone metabolic microenvironment [23]. Thus, we applied immunofluorescence double staining to label type H vessels in femoral tissues from 8- and 12-month-old mice after AS1842586 treatment. The results exhibited that inhibition of FOXO1 decreased the number of H-type vessels (yellow) in both 8- and 12-month-old mice, with a 22.83 % reduction in type H vessels area in 8-month-old mice bone and a more pronounced 70.29 % reduction in 12-month-old mice (Fig. 2a and b). To investigate whether the reduction in type H vessels might be associated with the down-regulated expression of FOXO1 in endothelial cells, we performed double-fluorescence staining to show the localization of FOXO1 and Emcn. We found a distinct localization of Emcn⁺ and FOXO1⁺ cells, with some FOXO1⁺ cells located closely to Emcn⁺ cells. Notably, there was a significant decline in Emcn⁺ cells in the femoral metaphysis of 12-month-old mice relative to 8-week-old mice (Fig. 2c and d). Furthermore, a notable loss of FOXO1⁺ cells around Emcn⁺ vessels and in cell nuclei was evident in the femoral metaphysis of 12-month-old mice (Fig. 2c, e, and f). Interestingly, we found that FOXO1 was colocalized with PDGFRβ, a marker for pericyte of vessels, and both the total PDGFRβ expression and the FOXO1 expression in PDGFRβ⁺ cells declined dramatically with aging (Fig. 2g–i). These data indicate that the reduction in FOXO1 levels in pericytes of vessels contributes to the degeneration of type H vessels.

Figure 2.

FOXO1 inhibition byAS1842856exacerbates degeneration of type H vessels in the long bone of mice. a. Representative images of immunofluorescent staining of type H vessels in the femoral metaphysis of 8-month-old (8M) and 12-month-old (12M) mice treated with AS1842856 or vehicle. Scale bars represent 50 μm. b. Quantitative statistical plots of the fluorescent signal of type H vessels (area mm²). n = 6/group. ∗P < 0.05, ∗∗∗P < 0.001. c. Representative images of the fluorescence distribution of FOXO1 around the Emcn⁺ vessels in the metaphyseal area below the growth plate in distal femurs of mice at 8W and 12M of age. Scale bars represent 50 μm. d-f. Quantitative analysis of Emcn⁺ area in metaphyseal region below the growth plate (d), relative fluorescence intensity (FI) of FOXO1 surrounding Emcn⁺ vessels (e), and relative FI of nuclear-localized FOXO1 (f). n = 6/group. ∗∗∗P < 0.001. g-i. Representative images (g) and quantitative analysis (h and i) of the fluorescence intensity of co-localized PDGFRβ and FOXO1 in the femoral metaphysis of mice at 8W and 12M of age. Scale bars: 50 μm. n = 6/group. ∗∗∗P < 0.001. Data are represented as mean ± SEM. Each data point represents one animal. Student’s t-test was used for the statistical analysis of two groups (d, e, f, h and i). One-way ANOVA with Tukey’s post-hoc test was used for the statistical analysis in four groups (b).

3.3. FOXO1 inhibition in pericytes hinders angiogenesis

To evaluate the role of FOXO1 in angiogenesis, we first stimulated HUVECs with different concentrations of AS1842856. Scratch wound healing assays showed that the migration ability of HUVECs was enhanced with increasing inhibitor concentration (Supplementary Fig. S3a and b). However, Matrigel tube-forming results showed that AS1842856 had little effect on the number of tubule branch points or the length of tubule branches formed by HUVECs (Supplementary Fig. S3c-e). Considering that pericytes are important for maintaining vascular integrity and promoting angiogenesis through their close physical and paracrine contacts with endothelial cells [24,25], we further evaluated the role of pericytic FOXO1 in angiogenesis. Conditional medium (CM) from cultures of HBVPs treated with various concentrations of AS1842856 was collected and subjected to HUVECs culture for Matrigel tube-forming assay. We found that AS1842856-treated CM dose- and time-dependently suppressed tube formation, as revealed by reduced tube length and branch points (Fig. 3a–c). Consistently, scratch assay revealed that migration of HUVECs was suppressed notably by AS1842856-treated CM (Fig. 3d and e). In keeping with the above findings, mRNA expression of VEGF and ANG-1, essential factors for angiogenesis [26], were down-regulated notably by AS1842856 treatment in HBVPs (Fig. 3f and g). The above data indicate that FOXO1 in pericytes plays a critical role in angiogenesis by regulating angiogenesis-associated factors.

Figure 3.

FOXO1 inhibition in pericytes prevents angiogenesis.a-c. Representative images (a) and quantitative analyses (b and c) of tube-forming assays of HUVECs. HUVECs were cultured in conditional medium (CM) from HBVPs treated with various with concentrations of AS1842856 (0, 0.1, 1 μM), and collected after 8, 12 and 20 h. Scale bars represent 200 μm. n = 4/group. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. d, e. Representative images (d) and wound healing rates (healed area/scratch area%) (e) in the scratch assay showing HUVECs migration under different conditional cultures. Scale bars represent 500 μm. n = 4/group. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. f, g. qPCR analysis of VEGF (f) and ANG-1 (g) mRNA expression in HBVPs treated with various concentrations of AS1842856 for 48 h. n = 4/group. ∗P < 0.05. Data are represented as mean ± SEM and individual points represent independent wells of cultured cells. Statistical comparisons were made using one-way ANOVA with Tukey’s post-hoc test (b, c and f) or Dunnett’s T3 test (e and g).

3.4. Reduction of FOXO1 activates the pericyte-to-myofibroblast transformation

Given that PDGFRβ, a pericyte marker, is important in regulating the expression of VEGF and ANG-1 [[27], [28], [29]], we detected the effect of AS1842856 on the expression of PDGFRβ in HBVPs. Results showed that AS1842856 treatment significantly down-regulated mRNA and protein expression of PDGFRβ (Fig. 4a–c). Interestingly, with increasing concentrations of AS1842856, HBVPs gradually turned into elongated, shuttle-shaped cells in a “fiber-like” interwoven arrangement (Fig. 4d). Indeed, AS1842856 treatment significantly up-regulated the mRNA expression of fibroblastic marker genes including ACTA2, COL1A1, TAGLN2 and LOXL1 (Fig. 4e–h). Additionally, the protein levels of αSMA and PDGFRα, which are myofibroblastic marker genes, were also notably up-regulated by AS1842856 treatment (Fig. 4i–k). To further determine whether FOXO1 inhibition might promote the pericyte-myofibroblast transformation in vivo, we detected the location and expression of αSMA in the bones of mice treated with either vehicle or AS1842856. In 12-month-old mice, αSMA expression around Emcn-labeled vessel-like structures was up-regulated, while Emcn⁺ endothelial cells lost their strip-like morphology in the bone compared with the 8-month-old mice (Fig. 4l–n). AS1842856 treatment further enhanced αSMA expression around these vessel-like structures in bone (Fig. 4l–n). These data therefore support the notion that FOXO1 is essential for maintaining pericyte phenotype and function, thus, loss of FOXO1 may promote the pericyte-to-myofibroblast transformation.

Figure 4.

Blocking FOXO1 using AS1842856 in pericytes promote their myofibroblastic transformation.a. qPCR analysis of PDGFRβ mRNA expression in HBVPs treated with various concentrations of AS1842856 for 48 h. n = 4/group. ∗P < 0.05, ∗∗∗P < 0.001. b, c. Western blot analysis (b) and relative levels (c) of PDGFRβ in HBVPs treated with AS1842856 (1 μM) for 24, 48 and 72 h. n = 4/group. ∗P < 0.05, ∗∗∗P < 0.001. d. Representative images of light field showing fibroblastic morphological changes of HBVPs in response to increasing concentrations of AS1842856 for 48 h. Scale bars represent 100 μm. e-h. qPCR analysis of myofibroblast-related gene mRNA levels (ACTA2, COL1A1, TAGLN2 and LOXL1) in HBVPs treated with various concentrations of AS1842856. n = 3/group. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. i-k. Western blotting (i) and quantitative analysis (j and k) of myofibroblastic markers (αSMA and PDGFRα) in HBVPs. n = 4/group. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. l. Representative immunofluorescence images of αSMA (green) and Emcn (red) in the femoral metaphysis of 8- and 12-month-old mice after AS1842856 or vehicle treatment for 2 months. Scale bars represent 50 μm. m, n. Quantitative analysis of αSMA fluorescence signal area (m) and intensity (n) around Emcn⁺ vessels. n = 6/group. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Data are represented as mean ± SEM and each data point represents an individual cell or animal sample. Statistical comparisons were made using one-way ANOVA with Tukey’s post-hoc test (equal variances) (a, e, h and k) or Dunnett’s T3 test (unequal variances) (c, f, g, j, m and n).

3.5. FOXO1 knockout in pericytes impedes osteogenesis and angiogenesis in mice

Recent study has identified that adiponectin (Adipoq)-positive cells, a unique subpopulation of cells in the bone marrow mesenchymal lineage, exist abundantly as pericytes and stromal cells that form a ubiquitous 3D network inside the marrow cavity [21]. Therefore, Adipoq-Cre mice were crossed with Foxo1^f/f mice to delete Foxo1 in Adipoq⁺ cells. We found that Adipo-Cre/Foxo1^f/f mice were distinguishable from Foxo1^f/f control mice by their shorter body and limbs (Supplementary Fig. S4a). We next assessed the effect of Foxo1 deficiency on mice bone microstructure. Micro-CT analysis revealed a notable loss in trabecular bone and a reduction in cortical bone thickness in Adipoq-Cre/Foxo1^f/f mice (Fig. 5a, Supplementary Fig. S4b). Quantification confirmed significant decreases in BMD and BV/TV in Adipoq-Cre/Foxo1^f/f mice (Fig. 5b and c). The reduced bone mass was attributed to decrease in both trabecular number and thickness, coupled with a notable increase in trabecular separation (Fig. 5d–g). Additionally, both cortical bone thickness and cross-sectional area of the femoral diaphysis were significantly reduced in Adipoq-Cre/Foxo1^f/f mice (Fig. 5a, h and i). The reduction in bone mass was mainly due to impaired bone formation, as indicated by lower serum levels of P1NP in Adipoq-Cre/Foxo1^f/f mice compared with Foxo1^f/f mice (Fig. 5j), while CTX-1 levels remained similar between genotypes (Fig. 5k). Thus, the data support that FOXO1 in Adipoq⁺ cells are functionally important for supporting bone formation.

Figure 5.

Knock-out ofFoxo1inAdipoq⁺ cells leads to bone loss and degeneration of type H vessels.a. Representative micro-CT images of femoral trabeculae and cortical bone in 4-week-old Adipo-Cre/Foxo1^f/f and Foxo1^f/f mice. Scale bars: 200 μm (upper panel), 500 μm (lower panel). b-i. Quantitative analysis of three-dimensional structural parameters: bone mineral density (BMD) (b), trabecular bone volume fraction (BV/TV) (c), trabecular number (Tb. N) (d), trabecular thickness (Tb. Th) (e), trabecular separation (Tb. Sp) (f), trabecular pattern factor (Tb. Pf) (g), crossectional thickness (Cs. Th) (h), and crossectional bone area (Cs. B. Area) (i). n = 7/group. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. j, k. Statistical analysis of serologic bone formation markers P1NP (j) and bone resorption marker CTX-1 (k). n = 7/group. ns, no significance (P ≥ 0.05), ∗∗P < 0.01. l, m. Representative images (l) and quantitative analysis (m) of immunofluorescence staining for Emcn (green) and CD31 (red) in the femurs of 4-week-old Adipo-Cre/Foxo1^f/f and Foxo1^f/f mice. Scale bars represent 50 μm. n = 6/group, ∗∗∗P < 0.001. n. Representative immunofluorescence images of perivascular PDGFRβ (purple) and αSMA (red) in the femurs of 4-week-old Adipo-Cre/Foxo1^f/f and Foxo1^f/f mice. Scale bars represent 50 μm. o, p. Relative fluorescence intensity (a. u.) of PDGFRβ and αSMA per Emcn⁺ area in the field of view. n = 6/group. ∗P < 0.05, ∗∗∗P < 0.001. Data are represented as mean ± SEM. Each data point represents one animal. Statistical comparisons were made using Student’s t-test.

We next examined whether deficiency of Foxo1 in Adipoq⁺ cells might impair the type H vessel formation in bone. Double-fluorescence staining for Emcn and CD31 showed that the area of Emcn⁺CD31⁺ type H vessels was significantly reduced in the bone of Adipoq-Cre/Foxo1^f/f mice (Fig. 5l and m). Consistent with the effects of pharmacological inhibition of FOXO1 by AS1842856, knocking out Foxo1 in Adipoq⁺ cells resulted in notably down-regulated expression of PDGFRβ along Emcn⁺ endothelial cells and a significant increase in αSMA⁺ cells in vessel-like structures in bone (Fig. 5n–p). Thus, FOXO1 in Adipoq⁺ pericytic cells is essential for the formation of type H vessels in bone, and loss of FOXO1 may stimulate the transformation of pericytes to myofibroblastic cells, thereby impairing both type H vessel and bone formation.

3.6. Blocking FOXO1 in HBVPs may stimulate the vascular pericytes-to-myofibroblasts transformation by activating mTOR signaling

Accumulating evidence shows that mTOR signaling is involved in myofibroblastic transformation [30]. A recent study has demonstrated the critical role of mTOR signaling in mediating pericyte-myofibroblast transition in choroidal neovascularization [31]. To investigate the possible interplay between FOXO1 and mTOR in regulating vascular pericyte-myofibroblast transition, we examined the expression of FOXO1 and mTOR in HBVPs treated with AS1842856 or rapamycin. Western blot assays revealed that blocking mTOR signaling by rapamycin did not change FOXO1 expression at any of the three time points studied. However, inhibition of FOXO1 using AS1842856 notably stimulated the phosphorylation of mTOR (p-mTOR) at 24 h and 48 h of treatments (Fig. 6a–c). To confirm the regulatory role of FOXO1 in mTOR activation, we used Foxo1/pcDNA3.1 for the overexpressed FOXO1 in HBVPs (Supplementary Fig. S5a). Results showed that overexpression of FOXO1 suppressed p-mTOR levels in HBVPs (Fig. 6d and e). These data indicate that down-regulation of FOXO1 may lead to activation of mTOR signaling in pericytes.

Figure 6.

FOXO1 maintains the phenotype of pericytes by suppressing mTOR signaling.a-c. Western blotting (a) and quantitative analysis of p-mTOR (b) and FOXO1 (c) in HBVPs after 24, 48, and 72 h of treatment with AS1842856 (1 μM) or Rapamycin (10 nM). n = 4/group. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. d, e. Western blotting (d) and quantitative analysis (e) of p-mTOR in HBVPs after transfection with Foxo1/pcDNA3.1 or pcDNA3.1. n = 4/group. ∗∗P < 0.01. f-h. Representative cell immunofluorescence staining and quantification of the mean fluorescence intensity for αSMA (green) and nuclear-localized p-mTOR (red) in HBVPs after indicated treatments. Nuclei were stained with DAPI. Scale bars represent 20 μm. n = 4/group. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. i-k. Western blotting (i) and quantitative analysis (j and k) of protein levels of αSMA and PDGFRα in HBVPs after indicated treatment for 48 h. n = 4/group. ∗∗P < 0.01, ∗∗∗P < 0.001. l-n. Western blotting (l) and quantitative analysis of changes on αSMA (m) and PDGFRα (n) in HBVPs after 48 h of transfection with plasmids. n = 4/group. ∗∗P < 0.01, ∗∗∗P < 0.001. Data are displayed as mean ± SEM. Each data point represents an independent cell sample. Statistical comparisons were made using one-way ANOVA with Tukey’s post-hoc test (equal variances) (b, c, h, j, k, m and n) or Dunnett’s T3 test (unequal variances) (e and g).

To further determine whether blocking FOXO1 may stimulate pericyte-myofibroblast transition by activating mTOR signaling, we treated HBVPs with AS1842856, rapamycin, or a combination of both. Based on the morphological characteristics of HBVPs, while rapamycin treatment alone had no effect on HBVPs morphology, it blocked the effect of AS1842856-induced spindle-like morphology in HBVPs (Supplementary Fig. S5b). Western blot analysis confirmed that rapamycin treatment blocked αSMA expression induced by AS1842856 and suppressed PDGFRα expression (Supplementary Fig. 5c and 5dSupplementary Fig. S5c and d). Given that FOXO proteins are a family of transcription factors with four members in mammals, including FOXO1, FOXO3a, FOXO4, and FOXO6 [32,33], we evaluated whether AS1842856 might have non-specific effect on the other family members. Results showed that AS1842856 had no effect on the mRNA expression of other FOXO members (Supplementary Fig. S5e-g). Thus, these data indicate that AS1842856 activates mTOR signaling specifically by blocking FOXO1, thereby stimulates pericyte-myofibroblast transition.

To further specify the role of FOXO1 in pericyte-myofibroblast transition, we designed siRNAs against Foxo1 and selected the siRNA#2 fragment with the highest knockdown efficiency for subsequent experiments (Supplementary Fig. S6a and b). This siRNA fragment had no effects on the mRNA expression of Foxo3a, Foxo4, and Foxo6 in HBVPs (Supplementary Fig. S6c). Immunofluorescence staining data showed that Foxo1 knockdown promoted αSMA expression and the nucleus location of p-mTOR in HBVPs, while rapamycin blocked the above effects (Fig. 6f–h). Consistently, western blot analysis confirmed that rapamycin treatment blocked the up-regulation of αSMA expression induced by Foxo1-siRNA and concurrently suppressed PDGFRα expression (Fig. 6i–k). Furthermore, overexpression of FOXO1 significantly suppressed the expression of αSMA and PDGFRα, similar to the effects observed with rapamycin (Fig. 6l–n). Thus, these suggest that the upregulation of mTOR signaling contributes to the pericytes-to-myofibroblasts transformation in the context of FOXO1 deficiency.

3.7. FOXO1 suppresses the vascular pericyte-to-myofibroblast transformation by inhibiting mTOR signaling in murine bone

To confirm the regulatory role of FOXO1 in mTOR signaling in vascular pericytes in vivo, we detected p-mTOR levels in vascular pericytes of Adipoq-Cre/Foxo1^f/f mice by immunofluorescence staining. Results showed a notable up-regulation of PDGFRα expression in metaphyseal bone area (Fig. 7a and b), and a significant increase in p-mTOR expression in PDGFRα⁺ cells of Adipoq-Cre/Foxo1^f/f mice compared with Foxo1^f/f mice (Fig. 7a and c). In addition, 12-month-old mice displayed higher levels of PDGFRα and p-mTOR in the metaphyseal bone area compared with 8-month-old mice (Fig. 7d–f). Moreover, AS1842856 treatment resulted in a significant increase in both PDGFRα and p-mTOR levels, with a pronounced increase in p-mTOR level in PDGFRα⁺ cells (Fig. 7d–g). These data suggest that FOXO1 deficiency activates mTOR signaling in PDGFRα⁺ cells.

Figure 7.

FOXO1 in PDGFRα⁺pericytes maintains type H vessels by inhibiting mTOR signaling in mice bone.a. Representative immunofluorescence images of PDGFRα (green) and p-mTOR (red) in the femoral metaphysis of 4-week-old Adipo-Cre/Foxo1^f/f and Foxo1^f/f mice. Nuclei were stained DAPI. Scale bars represent 50 μm. b, c. Quantitative analysis of PDGFRα⁺ fluorescence intensity (b) and PDGFRα⁺ p-mTOR⁺ colocalization fluorescence intensity (c). n = 6/group. ∗∗∗P < 0.001. d. Representative immunofluorescence images of PDGFRα (red) and p-mTOR (green) in 8- and 12-month-old mice treated with AS1842856. Nuclei were stained DAPI. Scale bars represent 50 μm. e-g. Quantitative analysis of PDGFRα⁺ (e), phosphorylated mTOR⁺ (f), and their colocalization (g) fluorescence signal intensities. n = 6/group. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. Data are represented as mean ± SEM. Each data point represents one animal. Statistical comparisons were made using Student’s t-test (equal variances) (b and c) or one-way ANOVA with Tukey’s post-hoc test (equal variances) (e, f and g).

To evaluate the impact of mTOR on age-associated degeneration of type H vessels, we administered rapamycin to middle-aged mice. The results showed that rapamycin significantly increased the number of type H vessels in the metaphyseal bone of middle-aged mice (Fig. 8a and b). Furthermore, rapamycin treatment led to a significant increase in PDGFRβ expression, a pericytes marker, and a dramatic decrease in αSMA expression, a myofibroblast marker (Fig. 8c–e). H&E staining and quantification showed that rapamycin significantly elevated the number of osteoblasts in the metaphyseal bone of middle-aged mice (Supplementary Fig. S7a and b). In consistent, micro-CT analysis revealed improvements in both trabecular and cortical bone mass in these mice (Fig. 8f). Quantitative analysis demonstrated that rapamycin treatment significantly increased BMD and BV/TV (Fig. 8g and h), primarily due to increased Tb.Th and decreased Tb.Sp, with no significant change in Tb. N (Fig. 8i–k). Although cortical thickness remained unchanged, the cortical bone area was improved by rapamycin treatment (Fig. 8l and m). Serum biochemistry assays showed that rapamycin significantly increased circulating P1NP levels while decreased CTX-1 levels in middle-aged mice (Fig. 8n and o). Together, these results indicate that FOXO1 is an essential factor for maintaining the phenotype of vascular pericyte by suppressing mTOR signaling.

Figure 8.

Rapamycin diminishes pericyte-myofibroblast transformation and ameliorates type H vessels degeneration and bone loss in mice.a, b. Representative images (a) and quantitative analysis (b) of the immunofluorescence staining of type H vessels (CD31 and Emcn) in the femoral epiphysis of 12-month-old mice treated with rapamycin (2 mg/kg) for 4 weeks. Nuclei were stained with DAPI. Scale bars represent 50 μm. n = 6/group. ∗P < 0.05. c-e. Representative images (c) and quantitative analysis (d and e) of the immunofluorescence staining of PDGFRβ⁺ (purple) and αSMA⁺ (green) in the femoral epiphysis of mice. Nuclei were stained with DAPI. Scale bars represent 50 μm. n = 6/group. ∗∗∗P < 0.001. f. Representative micro-CT reconstructions of trabecular and cortical bone in the femoral epiphysis of mice. Scale bars represent 200 μm. g-m. Quantitative analysis of micro-CT structural parameters: bone mineral density (BMD) (g), trabecular bone volume fraction (BV/TV) (h), trabecular number (Tb. N) (i), trabecular thickness (Tb. Th) (j), trabecular separation (Tb. Sp) (k), crossectional thickness (Cs. Th) (l), and cross-sectional bone area (Cs. B. Area) (m). n = 6/group. ns, no significance (P ≥ 0.05), ∗P < 0.05, ∗∗P < 0.01. n, o. Quantitative ELISA analysis of serum P1NP (n) and CTX-1 (o). n = 6/group. ∗P < 0.05, ∗∗∗P < 0.001. Data are represented as mean ± SEM. Each data point represents one animal. Statistical comparisons were made using Student’s t test (b, d, e, h, i, j, k, l, m, n and o) or Mann–Whitney U test (unequal variances) (g).

4. Discussion

FOXO1, an important “protective gene” [34], plays a critical role in preventing cellular and organismal aging [35]. However, its function in bone degeneration is still unclear. In this study, we demonstrated that the transformation of pericytes into myofibroblast, induced by FOXO1 deficiency in pericytes, is a core mechanism underlying age-associated reduction in type H vessels and trabecular bone loss. Furthermore, we uncovered a new mechanism by which FOXO1 might preserve the phenotype of pericytes by suppressing TORC1 signaling. Our findings of the roles of FOXO1 in regulating bone metabolism have significantly extended upon previous studies that primarily focused on osteogenic activity and proliferation of osteoprogenitors [18,19,36].

Our study highlights the fact that the function of FOXO1 in bone metabolism is highly dependent on cellular context. Previous evidence showed that specific deletion of FOXO1 in mature osteoblasts using Col1a1-Cre or broad deletion of Foxo1, -3, and -4 in early progenitors using Mx1-Cre caused loss of bone mass [37,38]. Conversely, deletion of Foxo1, -3, and -4 in committed osteoblasts using Osterix-Cre led to increased bone mass [18]. Recent evidence showed that knockout of Foxo1 using Bglap-Cre led to bone loss in young mice but alleviated age-associated bone loss in older mice [19]. However, it is somehow unexpected that deletion of Foxo1 using Col2-Cre (ERT) had no effect on bone mass in 10-month-old mice. It is important to note that Col2-targeted cells contribute not only to chondrocytes and perichondrial cells in growth cartilage but also a majority of osteoblast and mesenchymal progenitors in metaphyseal bone [21,22]. Considering the distinct functions of FOXO1 in osteoblastic lineage cells at different stages [19,37,38], these discrepant findings might be attributed to multiple cell types within the Col2-linage, including mesenchymal progenitors, osteoblasts, osteocytes, and adipocytes in adult and old mice [21,22,39]. Furthermore, our observation that pharmacological inhibition of FOXO1 leads to significant bone loss in both adult and middle-aged mice suggests that FOXO1 in non-Col2⁺ bone marrow cells may play a critical role in modulating bone metabolism.

Our study has revealed a critical role of FOXO1 in maintaining type H vessels in aging bone. Although it is well recognized that type H vessels in bone are fundamentally important for bone modeling and remodeling [23], and age-related bone loss is associated with the decline of type H vessels and the concomitant reduction in osteoprogenitors [8,10,40], the molecular mechanisms driving the age-associated degeneration of type H vessels are poorly understood. Earlier studies have indicated that FOXO1 is an essential regulator of vascular growth. For instance, either full or epithelial-specific knockout of Foxo1 induces embryonic lethality due to incomplete vascular development [41,42]. Consistent with this notion, here we show that pharmacological inhibition of FOXO1 decreases the number of type H vessels in adult mice and exacerbates this reduction in middle-aged mice, correlating with severe bone loss. Our study suggests that FOXO1 may be critical to the stability of type H vessels, thereby maintaining the bone mass. In consistent with a recent study indicating decreased FOXO1 mRNA expression in the bone with aging [19], we observed a decreased protein level of FOXO1 in the bone of aged mice. Interestingly, while pharmacological inhibition of FOXO1 using AS1842856 did not directly affect tubule formation in HUVECs, conditional medium from AS1842856-treated pericytes impaired tubule formation in HUVECs. Additionally, our study found decreased FOXO1 expression in PDGFRβ⁺ pericytes surrounding type H vessels. Furthermore, conditional knockout of Foxo1 in Adipoq⁺ cells led to reduced type H vessels and a considerable reduction in bone mass, suggesting that FOXO1 reduction in pericytes may mediate the degeneration of type H vessels in aged mice.

It is important to note that mice harboring Adipoq-Cre can also target the floxed Foxo1 in white and brown adipose tissue, in addition to bone marrow [43]. While specific silencing of Foxo1 in Adipoq⁺ cells promote browning of adipose tissue and activates metabolic pathways [44,45], it has been shown that fat transplantation does not rescue distorted bone marrow vasculature and bone loss after ablating Adipoq⁺ cells [21]. Thus, Foxo1 deficiency in peripheral fat tissues may not be involved in the changes in bone vessels and bone mass. Additionally, Adipoq⁺ cells are as abundant as both pericytes and stromal cells in the bone marrow [21]. Besides modulating osteogenesis and osteoclastogenesis [[46], [47], [48], [49], [50]], bone marrow Adipoq⁺ cells have recently been revealed to play functional role in regulating type H vessel formation by secreting osteopontin [47]. Therefore, the potential role of FOXO1 in bone marrow Adipoq⁺ stromal cells regarding the formation of type H vessels in vivo cannot be disregarded. Despite this, our present data demonstrate that the reduction of FOXO1 in pericytes is at least one of the important mechanisms underlying the degeneration of type H vessels with aging.

Our findings have provided a new understanding of the mechanism by which FOXO1 maintains the pericytes phenotype in type H vessels by suppressing mTOR signaling. As essential components of the microvascular vessel wall, pericytes are one of the main sources of VEGFA and ANG-1, which are critical factors for vessel formation and stabilization [[51], [52], [53]]. Our findings have identified FOXO1 as a regulatory factor for the expression of VEGF and ANG-1 in pericytes, with a critical role of in endothelial tubule formation and angeiogenesis. Recent studies have demonstrated an age-dependent decline in vascular density and a loss of pericytes, accompanied by a transformation from pericytes to fibroblasts, contributing to the loss of pericytes in tissues [54]. Likewise, our work shows a reduction in PDGFRβ⁺ pericytes and an increase in fibroblasts along the of vessel walls in the metaphyseal bone of aged-mice or mice treated with FOXO1 inhibitors. Additionally, our finding further identifies FOXO1 as a key upstream inhibitor of mTORC1, both in vivo and in vitro, thereby supporting the phenotype and function of pericytes. Therefore, down-regulation of FOXO1 in pericytes is at least one of the underlying mechanisms whereby type H vessels degenerate upon transformation of pericytes-to-fibroblasts.

mTORC is a critical signaling molecule implicated in tissue degeneration and metabolism-related pathology [30]. A collection of rapamycin derivatives, the mTORC inhibitors, has been approved for anticancer treatment in clinic [55,56]. In addition, growing clinical and laboratory evidence has highlighted rapamycin derivatives as promising treatments for age-associated neurodegenerative diseases [57,58]. However, the exact role of mTORC in bone metabolism varies under different conditions. Inhibition of mTOR signaling using rapamycin or genetic inactivation of mTORC1 by deleting Raptor in pre-osteoblasts has been revealed to induce bone loss in mice [59,60]. Conversely, an increased level of mTOR can suppress both bone formation and angiogenesis in osteoporotic animal models, and osteocyte-specific down-regulation or pharmacological inhibition of mTORC signaling has been shown to increase bone mass [61,62]. The conflicting effects of blocking mTORC1 on bone metabolism may be due to critical roles of mTORC1 signaling in various cell types in bone tissue. Here, our study provides strong evidence that long-term rapamycin treatment at a low dose ameliorate age-associated degeneration in bone. A recent study has implicated mTOR signaling in the pericyte-myofibroblast transition in subretinal fibrosis in age-related macular degeneration [31]. Our studies extend this finding that mTORC1 signaling mediates the pericyte-myofibroblast transition in type H vessels in aged bone. It will be interesting to further investigate how FOXO1 suppresses mTOR signaling in pericytes.

Taken together, our findings shed light on the role of FOXO1 in regulating both the phenotype and function of pericytes, thereby promoting the formation of type H vessels. Moreover, we provide novel insights into the mechanisms by which FOXO1 preserves phenotype of pericytes by suppressing mTOR signaling and retains its function in angiogenesis by regulating the expression of VEGF and ANG-1.

Ethics statement

All animal experiments were performed according to the protocols approved by the Institutional Animal Care and Use Committee (IACUC) (NFYY-2020-0933) at Southern Medical University Nanfang Hospital, and all procedures were carried out following IACUC guidelines.

Authorship

All persons who meet authorship criteria are listed as authors, and all authors certify that they have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript. Each author certifies that this material or part thereof has not been published in another journal, that it is not currently submitted elsewhere, and that it will not be submitted elsewhere until a final decision regarding publication of the manuscript in Journal of Orthopaedic Translation has been made.

Indicate the specific contributions made by each author (list the authors’ initials followed by their surnames, e.g., Y.L. Cheung). The name of each author must appear at least once in each of the three categories below.

Section I

The authors whose names are listed immediately below certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.

Authors: C.Y. Cheng, M.Y. Deng, C.B. Cheng, H.T. Wu, Y.T. Wang, M.C. Lu, Z.L. Yao, K.Q. Li, X.R. Zhang, B. Yu.

Section II

The authors whose names are listed immediately below report the following details of affiliation or involvement in an organization or entity with a financial or non-financial interest in the subject matter or materials discussed in this manuscript. Please specify the nature of the conflict on a separate sheet of paper if the space below is inadequate.

None

CRediT authorship contribution statement

Conceptualization, X.R. Zhang, B. Yu and C.Y. Cheng; Methodology, mouse experiments, C.Y. Cheng, M.Y. Deng and H.T. Wu; In vitro experiments, C.Y. Cheng, M.Y. Deng and C.B. Cheng; Collection of samples from individuals and analysis, Y.T. Wang, M.C. Lu and C.B. Cheng; Formal analysis, K.Q. Li and Z.L. Yao; Writing–Original Draf, C.Y. Cheng and M.Y. Deng; Writing–Review & Editing, X.R. Zhang and C.Y. Cheng; Funding Acquisition, B. Yu, X.R. Zhang and K.Q. Li; Project Administration and Supervision, X.R. Zhang and B. Yu.

Declaration of competing interest

The authors declare no conflict of interest that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Fig. S1.

AS1842856 treatment has no effect on femoral cortical bone.a. Representative micro-CT images of cross-section of mid-shaft femurs from 8- and 12-month-old mice after 2 months of treatment with AS1842856. b, c. Quantitative analysis of cortical bone thickness (b) and cross-sectional area (c). n = 6/group. ns, no significance (P ≥ 0.05). Data are represented as mean ± SEM. Each data point represents one animal. One-way ANOVA with Tukey’s post-hoc test was used to compare the groups.

Fig. S2.

Conditional knockout of Foxo1 has no effect on bone mass in aged Col2-Cre^ERT/Foxo1^f/f mice. a. Representative coronal and cross-sectional micro-CT images of the femurs from 12-month-old Col2-Cre^ERT/Foxo1^f/f mice after tamoxifen induction (75 mg/kg/day for 5 days with intraperitoneal injection) for 8 weeks. b-h. Quantitative analysis of microstructural parameters including bone mineral density (BMD) (b), trabecular bone volume fraction (BV/TV) (c), trabecular number (Tb. N) (d), trabecular thickness (Tb. Th) (e), trabecular separation (Tb. Sp) (f), crossectional thickness (Cs. Th) (g), and crossectional bone area (Cs. B. Area) (h). n = 7/group. ns, no significance (P ≥ 0.05). i. Representative images of H&E staining of the femurs from 12-month-old Col2-Cre^ERT/Foxo1^f/f mice after tamoxifen induction. Scale bars from top to bottom represent 200 μm, 100 μm, and 100 μm, respectively. Data are represented as mean ± SEM. Each data point represents one animal. The Student’s t test (b, e, f, g and h) was used for equal variances and the Mann-Whitney U test (c and d) was used for unequal variances.

Fig. S3.

Effects of AS1842856 treatment on vascular endothelial cell migration and tube-forming ability.a, b. Representative images (a) and wound healing rate (healed area/scratch area %) (b) in HUVECs scratch assay under various concentrations of AS1842856 (0, 0.1, 1 μM). Scale bars represent 500 μm. n = 4/group. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. c. Representative images of tube-forming experiments with various concentrations of AS1842856 (0, 0.1, 1 μM) stimulating HUVECs for 12 h. Scale bars represent 200 μm. d, e. Quantification of tubular branching points (d) and length (e) formed by HUVECs. n = 4/group. ns, no significance (P ≥ 0.05). Data are represented as mean ± SEM and individual points represent independent wells of cultured cells. For multiple comparisons between groups of three, one-way ANOVA with Tukey’s post-hoc test was used if variances were equal (d); otherwise, Dunnett’s T3 test was utilized (b and e).

Fig. S4.

Deletion of Foxo1 in Adipoq⁺ cells lead to a shorter body length in mice.a. Representative photographs of 4-week-old Adipo-Cre/Foxo1^f/fand Foxo1^f/f mice from the same litter. b. Representative micro-CT images of the coronal and transverse sections of the femurs.

Fig. S5.

Rapamycin treatment blocks AS1842856-induced myofibroblast transition in HBVPs. a. Relative abundance of FOXO1 mRNA at 24 h following transfection of Foxo1/pcDNA3.1 overexpression plasmids in HBVPs. n = 4/group. ∗P < 0.05. b. Representative phase-contrast micrographs of HBVPs after indicated treatments. Scale bars represent 100 μm. c, d. Western blotting (c) and quantitative analysis (d) of the protein levels of αSMA and PDGFRα in HBVPs after indicated treatment for 48 h. n = 4/group. ∗P < 0.05, ∗∗P < 0.01. e-g. qPCR analysis of the mRNA levels of FOXO3a, FOXO4 and FOXO6 in HBVPs treated with various concentrations of AS1842856. n = 4/group. ns, no significance (P ≥ 0.05). Data are expressed as mean ± SEM, with each data point representing an individual cell sample. Statistical comparisons were made using one-way ANOVA with Tukey’s post-hoc test (equal variances) (d, e, f and g) or Dunnett’s T3 test (unequal variances) (a).

Fig. S6.

The effectiveness of siRNA fragments targeting Foxo1. a. Relative abundance of FOXO1 mRNA in HBVPs at 24 h following transfection of siRNAs. n = 4/group. ∗P < 0.05. b. Western blotting analysis of FOXO1 protein levels in HBVPs at 48 h after transfection of siRNAs. c. Relative mRNA expression levels of FOXO3a, FOXO4, and FOXO6 in HBVPs at 24 h after Foxo1-siRNA (#2) transfection. n = 4/group. ns, no significance (P ≥ 0.05). Data are expressed as mean ± SEM, with each data point representing an individual cell sample. The Dunnett’s T3 post-hoc test was used to compare the five groups of unequal variances (a). The Student’s t-test was used for statistical analysis of the two groups with equal variances (c).

Fig. S7.

Histomorphologic changes in the femoral epiphysis of rapamycin-treated mice. a. Representative images of H&E staining in the femoral metaphysis of 12-month-old mice treated with Rapamycin (2 mg/kg) for 4 weeks. Black arrows show osteoblasts on the surface of trabecular bone. Scale bars in the upper and lower panels represent 200 μm and 100 μm, respectively. b. Quantitative analysis of osteoblast number per trabecular bone surface (N. Ob/mm). n = 6/group, ∗∗∗ P < 0.001.