FABP4 as a critical mediator in osteoporosis: inhibition strategies and therapeutic potential

doi:10.21203/rs.3.rs-5172644/v1

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FABP4 as a critical mediator in osteoporosis: inhibition strategies and therapeutic potential

https://doi.org/10.21203/rs.3.rs-5172644/v1

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Fatty acid-binding protein 4 (FABP4), a key lipid protein in metabolism and inflammation, has been suggested to be linked to osteoporosis (OP), though direct evidence is scarce. Here, we present the first clear evidence of FABP4's significant role in OP, supported by clinical data and comprehensive in vivo and in vitro experiments. Elevated serum FABP4 in OP patients inversely correlates with bone mineral density (BMD), with similar trends observed in OVX mice. While FABP4 does not influence osteoblast differentiation, it promotes osteoclast formation and bone resorption. The FABP4 inhibitor BMS309403, with an IC₅₀ of 0.89 µM, inhibits osteoclast differentiation by modulating calcium ions and suppressing the Ca²⁺-Calcineurin-NFATc pathway. Oral BMS309403 increased BMD in OVX mice, albeit less effectively than alendronate, whereas bone-targeted PLGA nanoparticles showed comparable efficacy to alendronate. This research identifies FABP4 as a promising therapeutic target for OP, with significant clinical implications.

Health sciences/Diseases/Endocrine system and metabolic diseases/Metabolic bone disease/Osteoporosis

Biological sciences/Drug discovery/Biomarkers/Predictive markers

Osteoporosis (OP) is the most common bone disease in the elderly, characterized by diminished bone mass and increased susceptibility to fragility fractures (1). Globally, approximately one-third of women and one-fifth of men over 50 are affected (2). With the aging global population, the incidence of OP is expected to rise significantly, posing a serious public health challenge (3). The fundamental mechanism of OP involves an imbalance in bone remodeling, where bone resorption surpasses bone formation, leading to bone loss (4). Current treatments address this imbalance: some drugs promote bone formation (e.g., teriparatide (5) ), while others inhibit bone resorption (e.g., alendronate (6, 7) and denosumab (8) ).

However, OP is a complex systemic metabolic disorder that can result from disruptions in bone remodeling triggered by factors such as inflammation (9), estrogen (10), lipid metabolism (11), glucose levels (12), and aging (13). Clinically, this manifests as a range of diseases that coexist with OP, including OP associated with rheumatoid arthritis (RA) (14), diabetes (15), glucocorticoid use (16), and other diseases (17). Therefore, exploring new mechanisms in OP is crucial for developing innovative treatments and improving patient outcomes by deepening our understanding of its complex pathophysiology and disease connections.

Fatty Acid-Binding Protein 4 (FABP4) is a key lipid-binding protein predominantly found in adipocytes and macrophages, essential for lipid metabolism through the transport and binding of fatty acids (18, 19). Previous research has identified FABP4 as a promising target for metabolic disorders such as obesity, insulin resistance, and diabetes (20). Recent studies have revealed its role in modulating inflammatory responses, with elevated levels of FABP4 correlating with inflammatory conditions such as arthritis (21, 22) and atherosclerosis (23). In 2022, Cai et al. (24) highlighted the exacerbating role of FABP4 from synovial M1 macrophages in RA. Interestingly, supplementary data indicated that feeding recombinant FABP4 protein to collagen-induced arthritis mice for eight weeks significantly increased osteoclasts (OCs) activity in the bone marrow. Subsequently, Cao et al. (25) demonstrated that the absence of Kindlin-2 in mouse adipocytes led to a notable increase in overall bone mass by inhibiting FABP4 expression. Despite not being the primary focus, these studies indirectly suggested a potential association between FABP4 and OP. If FABP4 emerges as a promising target for OP, it could become a key protein in both diabetes and RA-related OP. This would hold significant implications, enhancing our understanding of OP pathogenesis and offering the potential for developing FABP4 as a dual disease target for future treatments.

Currently, there is no direct research on the link between FABP4 and OP. Our study aims to investigate the impact of FABP4 on bone homeostasis and explore the potential of inhibiting FABP4 as a therapeutic approach for OP. We started with the negative correlation between serum levels of FABP4 and bone mineral density (BMD) in patients, and in vitro experiments confirmed that high FABP4 expression exacerbates OP by promoting bone resorption without affecting bone formation. Our findings indicated that FABP4 inhibitors can mitigate OCs activation through the Ca²⁺-Calcineurin-NFATc signaling pathway. Utilizing new bone targeting nanoparticles (NPs) for targeted delivery, we found that FABP4 inhibitor loaded NPs exhibit comparable efficacy to alendronate sodium (ALD) in treating OVX mice. This study is the first to confirm FABP4's role in OP, which is of significance.

Patients’ BMD correlates negatively with their serum F4BP4 levels

Ovariectomy (OVX) in mice, leading to estrogen deficiency, is well-known for causing bone loss and increased marrow adiposity. However, the expression of FABP4 in the bone marrow of OVX mice is limited reported. Our study revealed significant trabecular bone loss 30 days post-ovariectomy, as shown by Micro-CT scans (Fig. 1A), and an increase in marrow adipose tissue, confirmed by paraffin sections and H&E staining (Fig. 1, B and D). Immunohistochemistry (IHC) demonstrated a marked upregulation of FABP4 in the bone marrow cavity (Fig. 1, C and E), suggesting changes in adipocyte function and possible effects on osteoblast activity.

Subsequent investigations included measuring serum levels of FABP4 and lipid profiles (total cholesterol (TC), triglycerides (TG), HDL, and LDL) in patients with OP (T-scores ≤ − 2.5) and non-OP (T-scores ≥ − 1) trauma fracture patients (Table S1). Our results showed that, among the lipid profile components, none of them were significantly abnormal in OP patients compared to non-OP individuals (Fig. S1A), and serum FABP4 levels were notably higher in OP patients (Fig. 1F). Correlation analysis revealed a significant negative relationship between BMD and serum FABP4 levels (Fig. 1G and S1B), indicating that higher FABP4 levels are associated with lower BMD. These findings provide compelling clinical evidence for FABP4's role in OP development.

FABP4 has no impact on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs)

Both bone marrow adipocytes and osteoblasts originate from BMSCs (26). An increase in bone marrow adiposity is associated with a decrease in bone mass (27). Initially, we hypothesized that FABP4 might inhibit osteogenic differentiation by promoting BMSCs to differentiate into adipocytes. To test this, we supplemented recombinant human FABP4 protein (25ng/mL or 50 ng/mL) during the 21-day induction of human BMSCs towards osteogenic differentiation, along with the concurrent addition of FABP4 protein and its inhibitor BMS309403 (5 µM or 10 µM) (Fig. 2A). Surprisingly, Alizarin Red staining revealed that neither the FABP4 protein nor its inhibitor affected the osteogenic differentiation capacity of BMSCs (Fig. 2B).

This led us to consider whether increased bone marrow adiposity elevates endogenous FABP4 levels within BMSCs, thereby reducing their osteogenic differentiation potential. To investigate this, we established stable BMSC cell lines overexpressing FABP4 (Fig. 2, C and D), achieving nearly a 4000-fold increase in FABP4 gene expression (Fig. 2E). However, when inducing osteogenic differentiation in these FABP4-overexpressing BMSCs, with or without BMS309403 (10 µM), the Alizarin Red staining results remained unchanged, indicating that FABP4 overexpression did not impact the osteogenic differentiation of BMSCs (Fig. 2, F and G).

These findings demonstrate that both endogenous and exogenously elevated levels of FABP4 do not hinder osteogenic differentiation. While this result may be disheartening, it directs us towards further research, suggesting that FABP4 might influence OCs differentiation.

FABP4 plays a crucial role in enhancing OCs differentiation

To assess the impact of exogenous FABP4 on the differentiation of bone marrow-derived macrophages (BMMs) into OCs, we first induced primary mouse bone marrow single-nucleus cells (BMNCs) into BMMs using M-CSF (28). During the subsequent induction of BMMs into OCs with RANKL, we introduced recombinant mouse FABP4 and its inhibitor BMS309403. Trap staining results revealed that the addition of just 25 ng/mL FABP4 significantly promoted the formation of OCs, and this effect could be reversed by BMS309403 (Fig. 3, A and B).

To further investigate the effect of increased endogenous FABP4 on OCs formation, we added a mixture of free fatty acids (FFA) to the culture medium (29). Western blot results indicated a significant increase in intracellular FABP4 expression in BMMs with 200X and 400X diluted FFA (Fig. 3, C and D). The subsequent addition of FFA and FABP4 inhibitors during OCs differentiation showed that FFA alone significantly increased the number of differentiated OCs, an effect that could be reversed by BMS309403 (Fig. 3, E and F).

Confocal microscopy images of F-actin staining in OCs revealed a more regular and distinct bone skeleton structure with abundant F-actin when stimulated by additional FABP4 or FFA compared to the group with only RANKL and M-CSF (Fig. S2A). Furthermore, we confirmed the influence of FABP4 on the bone-resorptive capabilities of mature OCs on bovine bone slices. The addition of FFA or FABP4 enhanced bone resorption, whereas the FABP4 inhibitor effectively blocked this process (Fig. 3G and Fig. S2B). All of these experiments robustly demonstrate that FABP4 significantly promotes the differentiation of OCs and enhances bone resorption.

FABP4 inhibitor BMS309403 shows comparable OCs differentiation inhibition to ALD

Given the significant expression of FABP4 in BMMs cells (Fig. 3C), we postulated that an FABP4 inhibitor could inhibit OCs differentiation even without external stimuli. The initial CCK8 assay revealed that BMS309403, at concentrations up to 50 µM, did not hinder the proliferation of BMMs cells (Fig. S3A). Subsequently, the successful validation of our hypothesis through trap staining after exposure to different doses of BMS309403 underscores the intricate role of FABP4 in OCs differentiation (Fig. 3H and Fig. S3B). Remarkably, the in vitro efficacy of BMS309403 in inhibiting OCs, comparable to that of the established clinical therapy ALD (Fig. S3, C and D), reinforces its therapeutic promise. With an IC₅₀ value of approximately 0.89 µM for BMS309403 and 0.44 µM for ALD (Fig. 3I), these results demonstrate the potency of BMS309403 in regulating OCs function.

FABP4 inhibitor suppresses OCs differentiation via Ca-Calcineurin-NFATc pathway

To further investigate the mechanism by which FABP4 inhibitors impede OCs differentiation, we performed transcriptome sequencing on three groups: BMMs cells, BMMs-induced OCs, and OCs treated with 1µM BMS309403 during induction (OCs + BMS). We initially analyzed differentially expressed genes using volcano plots for BMMs vs. OCs and OCs vs. OCs + BMS (Fig. 4A). A Venn diagram identified 107 genes upregulated 2-fold in BMMs-induced OCs and 284 genes downregulated 0.3-fold in OCs post-BMS309403 treatment, converging on 69 common genes for KEGG pathway analysis (Fig. 4B) and GO analysis (Fig. S4). The most affected KEGG pathways were OCs differentiation and calcium signaling (Fig. 4C). Cluster analysis of FPKM values (Fig. 4D) and subsequent qPCR validation (Fig. 4E) revealed significant downregulation of key genes (Oscar, Acp5, Fos, Nfatc1) following BMS309403 treatment. This was corroborated by Western Blotting results (Fig. 4F), which demonstrated suppression of TRAP, c-Fos, and NFATc1 protein levels (Fig. 4G), thus confirming that FABP4 inhibitors suppress OCs differentiation at both the transcriptional and translational levels. Regarding the critical role of the calcium signaling pathway highlighted by KEGG, we examined intracellular calcium levels in OCs, OCs + FABP4, OCs + FABP4 + BMS309403, and OCs + BMS309403 using the calcium fluorescent probe Fluo-4 AM via flow cytometry (Fig. 4H) and fluorescence microscopy (Fig. 4I). Results showed a significant increase in intracellular calcium levels in OCs with FABP4, which was reversed by BMS309403.

The calcium signaling pathway is crucial in OCs differentiation (30), whereby RANKL-RANK interaction activates downstream PLCγ, leading to elevated intracellular calcium concentrations and subsequent Calcineurin activation (31). This cascade dephosphorylates NFATc1, regulating transcription of key proteins such as TRAP, CTSK, and OSCAR in the nucleus (32). Our transcriptome analysis indicated no change in Plcγ gene expression post-BMS309403 treatment, supported by Western Blotting showing constant PLCγ levels, while the Calcineurin A and B subunits were significantly downregulated (Fig. 4, J and K). This suggests that FABP4 elevates calcium levels in OCs, thereby activating the Ca²⁺-Calcineurin-NFATc1 pathway to promote OCs differentiation, thus expanding our understanding of this cellular process.

Effects of FABP4 inhibitor on bone resorption in OVX mouse OP model

To further validate the efficacy of FABP4 inhibitors in combating OP in vivo, we established a bilateral ovariectomy-induced OP mouse model to simulate postmenopausal OP. Seven days post-surgery, mice were divided into four groups: Sham, Model, ALD (positive control), and BMS309403. The Sham group underwent minor fat tissue removal around the ovaries. After 28 days of daily oral gavage, mice were euthanized, and serum, uterus, tibia, and femur samples were collected for further analysis (Fig. 5A). Uterine atrophy confirmed the success of the surgical model (Fig. S5A). Dynamic weight changes showed a significant increase in body weight post-ovariectomy, with slight decreases observed in the ALD and BMS groups compared to the Model group, maintaining overall stability (Fig. S5B). ELISA analysis revealed a significant increase in serum FABP4 levels in the untreated Model group, while FABP4 levels notably decreased in the ALD and BMS groups (Fig. 5B). These results reaffirmed the close association between FABP4 levels and OP progression. Subsequently, we assessed serum levels of the bone formation marker PINP (Fig. 5C) and the bone resorption marker CTX1 (Fig. 5D). Similar to ALD, FABP4 inhibitors had no effect on bone formation but exhibited inhibitory effects on bone resorption. Micro-CT assessment of bone mass at the distal femur demonstrated significant inhibition of OP progression in ovariectomized mice treated with FABP4 inhibitors (Fig. 5E). 3D analysis indicated that oral administration of FABP4 inhibitors significantly increased BMD (Fig. 5F) and trabecular thickness (Tb.Th, Fig. 5G). Results from TRAP staining further confirmed the inhibitory effect of BMS309403 on bone resorption in vivo (Fig. 5, I and J). Additionally, HE staining (Fig. S5, C and D)and IHC analysis of FABP4 protein expression (Fig. S5, E and F) in bone marrow fat tissue revealed no significant difference between the BMS and ALD treatments, suggesting that the lack of impact on osteogenic and adipogenic differentiation of BMSCs may account for this.

Differential efficacy of FABP4 inhibitor and ALD in OVX mouse OP model

FABP4 protein is extensively distributed across various tissues in the body, including adipose tissue, liver, intestines, heart, and muscles (33). Consequently, developing FABP4 inhibitors presents targeting challenges, as these inhibitors may affect FABP4 in multiple tissues, leading to unpredictable systemic effects. Furthermore, the pharmacokinetic properties of these inhibitors could be influenced by the ubiquitous expression of FABP4, impacting their efficacy and safety.

In our in vivo study, the oral dose of ALD (1.3 mg/kg) followed clinical standards (7), whereas the dose of BMS309403 (10 mg/kg) was based on prior preclinical doses for diabetes treatment (34). Although BMS309403 demonstrated comparable activity to ALD in vitro, in vivo results showed that even at 7.7 times the dose of ALD, BMS309403 did not achieve the same efficacy. Specifically, compared to ALD, FABP4 inhibitors had no significant effect on trabecular number (Tb.N, Fig. 5H) and bone volume/tissue volume ratio (BV/TV, Fig. S5G) in OVX mice. Additionally, Western blot analysis of various OVX mice tissues revealed substantial FABP4 presence in muscles, white adipose tissue (WAT), brown adipose tissue (BAT), heart, and kidneys, while bone marrow showed minimal presence (Fig. S6). This highlights the critical need to enhance the bone-targeting capabilities of FABP4 inhibitors for their potential clinical application in treating OP.

Developing novel bone-targeted PLGA nanoparticles with click chemistry

The structural similarity of bisphosphonates to inorganic phosphates in bone allows them to establish strong bonds with hydroxyapatite (HA) crystals on bone surfaces. Studies have shown that bisphosphonate-modified PLGA-PEG nanoparticles (NPs) enable controlled drug release, proving effective in addressing diverse bone conditions (35). In this study, bisphosphonate-conjugated PLGA-PEG (PLGA-PEG-Ald) was synthesized via click chemistry using PLGA-PEG-N3 and DBCO-ALD (Fig. 6A). The structure was confirmed by FT-IR (Fig. S7) and ¹H NMR (Fig. S8). Previous research has demonstrated that NPs assembled from polymers with 20% PLGA-PEG-Ald exhibit superior stability and bone-targeting ability (35). Building on these findings, incorporating the FABP4 inhibitor BMS309403 into Ald-BMS-NPs, composed of 20% PLGA-PEG-Ald and 80% PLGA-PEG, shows promise, with NPs made entirely of PLGA-PEG referred to as PLGA-BMS-NPs (Fig. 6B).

The encapsulation efficiency of BMS309403 in Ald-BMS-NPs and PLGA-BMS-NPs is 43.94% and 30.43%, respectively, with drug loading rates of 4.21% and 2.95%, significantly higher than reported in existing literature (35). The particle surface Zeta potential of Ald-BMS-NPs is approximately − 21.6 mV, while that of PLGA-BMS-NPs is around − 19.0 mV, indicating successful enrichment of bisphosphonate ions on Ald-BMS-NPs' surface (Fig. 6C). The particle size of Ald-BMS-NPs is uniform, with an average size of about 183.3 ± 2.8nm (Fig. 6D), slightly larger than that of PLGA-BMS-NPs at 183.3 ± 2.8nm (Fig. 6E).

Then in vitro CCK-8 experiments showed that different concentrations of Ald-BMS-NPs, PLGA-BMS-NPs, and empty Ald-NPs did not exhibit cytotoxicity towards BMMs cells (Fig. S9A). The BMS309403 encapsulated in Ald-BMS-NPs demonstrated slow release in physiological saline at pH 7.0 for at least 72 hours, with a slightly faster release rate compared to PLGA-BMS-NPs (Fig. 6F). By introducing a small amount of fluorescent molecule Cy5 into both NPs, Ald-BMS-Cy5-NPs and PLGA-BMS-Cy5-NPs were obtained. Under confocal microscopy, both types of nanoparticles were observed to be readily taken up by BMSCs after 30 minutes (Fig. 6G) of co-culturing and had increased accumulation within the cells after 6 hours (Fig. S9B).

Subsequently, the bone-targeting efficacy of Ald-BMS-NPs was confirmed through both in vitro and in vivo assessments. Initially, Ald-BMS-NPs and PLGA-BMS-NPs were incubated with nano-HA for 6 hours to assess their binding capability, as visualized with TEM imaging (Fig. 6H). This revealed strong adhesion of Ald-BMS-NPs to HA. Furthermore, Cy5-labeled NPs were co-cultured with bovine bone slices, illustrating increased binding of Ald-BMS-NPs (Fig. S10A). In vivo validation using IVIS revealed Ald-BMS-NPs exhibited significantly enhanced bone targeting compared to PLGA-BMS-NPs (Fig. S10B and C). IVIM imaging at 24- and 48-hours post-injection highlighted preferential accumulation of Ald-BMS-Cy5-NPs in the bone marrow, underscoring their potent bone-targeting capabilities (Fig. S10D).

In conclusion, our click-chemistry-derived PLGA nanoparticles demonstrate precise bone and bone marrow targeting, uniform size, efficient drug loading, controlled release, and are tailored for targeted FABP4 inhibitor delivery in vivo.

Bone-targeted NPs delivery of FABP4 inhibitor shows superior anti-OP effect in OVX mouse model

To further validate the in vivo therapeutic efficacy of bone-targeted Ald-BMS-NPs loaded with FABP4 inhibitors, we re-established the OVX-induced osteoporotic mouse model. Seven days post-bilateral ovariectomy, C57BL/6J mice were divided into groups: Sham (saline), Model (saline), ALD (10 mg/kg), Free BMS309403 (10 mg/kg), Ald-BMS-NPs (10 mg/kg of BMS), and empty Ald-NPs (equivalent PLGA content to Ald-BMS-NPs). All groups received bi-weekly intraperitoneal injections over four weeks (Fig. 7A). Body weight was monitored bi-daily throughout the treatment period (Fig. S11). At the conclusion of the experiment, tibia were subjected to Micro-CT analysis. BMD data revealed no improvement in the OVX group treated with empty Ald-NPs. In contrast, intraperitoneal BMS309403 significantly increased BMD. Notably, the Ald-BMS-NPs group exhibited significant BMD enhancement, with BMD values approximating those of the Sham group (Fig. 7B). Micro-CT reconstruction figures indicated reduced trabecular bone loss and increased bone mass in the Ald-BMS-NPs group compared to the ALD and Free BMS309403 groups (Fig. 7C). The 3D analysis data further demonstrated significant improvements in Tb.Sp (Fig. 7D), BV/TV (Fig. 7E), and Tb.N (Fig. 7F) in the Ald-BMS-NPs group. While statistically similar to the ALD group, the Ald-BMS-NPs group showed modestly superior values. Additionally, Ald-BMS-NPs significantly improved Tb.Sp (Fig. 7G), an effect not observed in the ALD group.

To assess the potential impact of NPs on liver and kidney functions, we analyzed serum markers of liver enzymes (ALT, AST, ALP, γ-GT) and kidney function indicators (UREA, CREA, UA). Results indicated no abnormalities in liver function markers across all groups, except for one mouse in the Ald-BMS-NPs group with elevated AST levels. Kidney function markers were also within normal ranges, except for increased UA levels in the ALD group (Fig. 7H). Histological examination of the heart, liver, spleen, lungs, and kidneys in all groups, using H&E staining, revealed no organic changes (Fig. S12).

These findings demonstrate that bone-targeted NPs loaded with FABP4 inhibitors not only provide excellent anti-OP effects but also exhibit high safety, offering strong evidence for the therapeutic potential of FABP4 inhibitors in OP treatment.

Bone and adipose tissues share numerous metabolic pathways and significantly influence each other (27, 37). Imbalances in lipid metabolism, involving products like fatty acids and cholesterol, can impact bone density (38), with fatty acids providing energy (39) and cholesterol acting as a precursor for vitamin D, which is essential for calcium absorption and bone health (40). Adipose tissue secretes hormones and cytokines, such as leptin (41) and adiponectin (42), that affect bone cell function and metabolism. Therefore, targeting lipid metabolism in anti-osteoporosis drug research could address both metabolic syndrome and osteoporosis. While peroxisome proliferator-activated receptor-γ (PPAR-γ) agonists, such as rosiglitazone (43) and pioglitazone (44), which are used to treat type 2 diabetes, can decrease bone density by influencing adipocyte and osteocyte differentiation. In this study, we has revealed that FABP4, a lipid protein involved in fatty acid transport and metabolism, can promote bone resorption to exacerbate OP. This may clarify why patients with metabolic syndrome, especially those with high FABP4 levels, such as diabetics, are frequently more susceptible to OP (15). Therefore, investigating FABP4’s role in bone metabolism and developing effective FABP4 inhibitors is crucial for OP prevention and treatment.

An important finding in this study was that FABP4 inhibitors suppress OCs differentiation by inhibiting the Ca²⁺-Calcineurin-NFATc signaling pathway. When intracellular calcium levels rise, calmodulin binds to Ca²⁺, forming a complex that activates calcineurin (45). This leads to the dephosphorylation and nuclear translocation of the NFATc transcription factor, which then regulates genes associated with OCs differentiation (46). The study shows that overexpression of FABP4 can increase Ca²⁺ concentration in OCs precursors. This may be due to FABP4's role in binding and transporting fatty acids, altering the intracellular lipid environment and calcium ion flux, thus promoting NFATc activation. This discovery is the first to confirm that FABP4 influences OCs differentiation via the Ca²⁺-Calcineurin-NFATc pathway, providing significant insights into the mechanisms of OCs differentiation.

By inducing OCs differentiation with the FABP4 inhibitor BMS309403, we determined its inhibitory potency (IC₅₀ = 0.89 µM), comparable to the clinical drug ALD. This suggests that BMS309403 has significant clinical potential. However, despite promising preclinical results, the clinical trials of FABP4 inhibitors are still in their early stages due to the widespread distribution of FABP4 in adipose tissue (33). Nonetheless, as FABP4 is a key biomarker for metabolic syndrome, the development of highly selective FABP4 inhibitors remains a major focus of research (47, 48). Our study demonstrated that oral administration of FABP4 inhibitors effectively improves bone density in mice, indicating the potential for these inhibitors to simultaneously address inflammation, insulin resistance, and OP. Additionally, advancements in drug delivery systems suggest that bone-targeting NPs carrying FABP4 inhibitors can achieve therapeutic effects comparable to existing clinical OP drugs. This significantly broadens the potential clinical applications for FABP4 inhibitors.

In summary, our study demonstrates that FABP4 exacerbates OP by promoting bone resorption, as proven by clinical and experimental data. The FABP4 inhibitor BMS309403 shows activity comparable to ALD in vitro, inhibiting OC differentiation by reducing calcium ion concentration and suppressing the Ca²⁺-Calcineurin-NFATc pathway. Oral administration of FABP4 inhibitors significantly increased bone mass in OVX mice, although it was less effective than ALD. Bone-targeting nanoparticles delivering the FABP4 inhibitor demonstrated efficacy comparable to ALD. This research establishes a basis for using FABP4 inhibitors in treating OP-related diseases, with significant practical implications.

Material and method

Study design

The primary aim of this study is to validate FABP4 as a promising therapeutic target for OP. Serum levels of FABP4 in patients were measured using ELISA to determine its role in exacerbating OP, either by inhibiting bone formation or promoting bone resorption. This investigation included Alizarin Red S staining for BMSC osteogenic differentiation and TRAP staining for the differentiation of bone marrow mononuclear cells into OCs. Additionally, we developed an ovariectomized mouse model to assess the efficacy of a FABP4 inhibitor in treating OP, mainly utilizing Micro-CT and histological TRAP staining. Results indicated inconsistent efficacy of the FABP4 inhibitor across in vivo and in vitro settings. To address this discrepancy, we developed bone-targeting drug-loaded nanoparticles and evaluated their distribution using IVIS and IVIM imaging, aiming to achieve targeted delivery of the FABP4 inhibitor to bone tissue.

Animal study

Ten- to 12-week-old, female C57/BL/6J mice were purchased from BesTest Bio-Tech (Zhuhai, China). All mice were kept in a pathogen-free environment at Shenzhen university. Mice were accommodated in group housing under conditions of 20–24°C temperature and 40–60% humidity, following a 12-hour light/12-hour dark cycle. All animal handling and use was approved by Institutional Animal Care and Use Committee (IACUC) at Shenzhen University Medical School on May 30, 2023. The registration number is IACUC-202300048.

Initially, all mice were anesthetized with isoflurane and their fur was shaved. A 1 cm incision was carefully made in the skin and peritoneum, 1 cm away from the spine. For model mice, both ovaries were surgically removed and ligated, whereas in the control group, a minor fat deposit was excised. Subsequently, the incision was meticulously sutured, and iodine was applied for disinfection.

Human study

Human studies were reviewed and conducted in accordance with the approval of the ethics committee at Shenzhen University General Hospital (NCT number: KYLLMS-17). Written informed consent was obtained from all participants. Individual characteristics are detailed in Tables S1.

To assess bone health, we measured adult BMD at the hip, including the total hip, femoral neck, and femoral metaphysis, in accordance with ISCD guidelines. BMD values were expressed in milligrams per square centimeter, and T-scores and Z-scores were calculated relative to local population norms. For the human serum samples analysis, a total of 15 patients who were diagnosed of Non-OP and OP at Shenzhen University General Hospital in Shenzhen, China. Patients with a history of systemic autoimmune diseases or severe infectious diseases were excluded from the study. After 2 hours fast, blood samples were drawn and centrifuged at 3000 rpm for 5 minutes. The plasma samples were then stored at − 80°C until further analysis.

Primary cell culture

BMNCs were isolated from 6- to 8-week-old C57BL/6J mice by dissecting intact tibia and femur post-euthanasia. The bone marrow cavity was washed with DMEM complete medium, and cells were filtered and separated using an animal bone marrow mononuclear cell isolation kit. After overnight culture in αMEM complete medium (Gibco, cat#10091148), non-adherent cells were supplemented with 50 ng/mL of M-CSF (Peprotech, cat#315-02) for adhesion and differentiation into BMMs.

OCs formation

The BMNCs were differentiated into BMMs using 50 ng/mL of M-CSF for two days. This was followed by the addition of 50 ng/mL of M-CSF and 100 ng/mL of sRANKL (Peprotech, cat#315-02) for an additional 4 days to promote their differentiation into OCs. The culture medium was refreshed every two days throughout the process. Subsequently, cells were stained following the protocol of the TRAP Staining Kit (Servicebio, cat#G1050), with cell nuclei stained using hematoxylin.

Bone resorption activity on mature OCs

Cow bone slices (TuyuanBiotech, China)were arranged in a 96-well plate, and BMNCs were added to each well at a density of 5×104 cells. The cells were then cultured according to the OCs culture protocol for 11 days, with the medium refreshed every other day. Drawing from existing literature, the bone slices underwent a series of treatments: rinsing with distilled water, immersion in 1% ammonia solution under ultrasound for 30 minutes, dehydration, and drying using a gradient of alcohols prior to field emission scanning electron microscope (ZEISS, SUPRA55).

RNA-seq studies

BMMs, OCs, and OCs + BMS309403 (MCE, cat#HY-101903) groups were collected. The cells were lysed using TRIZOL reagent and sent to BGI Genomics for RNA extraction and bulk mRNA sequencing. The raw sequencing data analysis was performed as previously described. The BMMs group consisted of cells induced with M-CSF for two days. The OCs group consisted of BMMs induced with both M-CSF and sRANKL. The OCs + BMS309403 group consisted of BMMs induced with M-CSF, sRANKL, and 1 µM BMS309403.

NPs self-assembly, drug loading and characterization

Weigh 4mg of PLGA(50:50, 20K)-PEG2000-Ald, 16mg of PLGA(50:50, 20K)-PEG2000, and 1mg of BMS309403 in 1mL of dichloromethane. Add 1mL of 2% PVA solution and sonicate the mixture for 15 minutes using a probe sonicator (50%, 10s on/10s off intervals) (SCIENTZ, China). Subsequently, introduce the emulsion into 13mL of 0.2% PVA solution and sonicate for an additional 15 minutes at 50% power with the same intervals. The solution is left to stir at room temperature at 500rpm overnight. After centrifugation at 800g for 10 minutes, discard the sediment, and then discard the supernatant after subjecting it to centrifugation at 20000g for 30 minutes. Dissolve the sediment in 1mL of ultrapure water, centrifuge again at 800g for 10 minutes, and collect the supernatant as Ald-BMS-NPs. NPs formed without PLGA(50:50, 20K)-PEG2000-Ald are known as PLGA-BMS-NPs. Adding 0.015mg of Cy5 under the same conditions yields Ald-BMS-Cy5-NPs and PLGA-BMS-Cy5-NPs. NPs formed without BMS309403 are denoted as Empty Ald-NPs following the same procedure.

Transfer 100uL of NPs loaded with BMS309403, disrupt the emulsion with 900uL of acetonitrile, centrifuge the mixture, and collect the supernatant for HPLC (SHIMADZU, LC-20ADxr) analysis to assess drug loading and encapsulation efficiency. Utilize a Malvern particle size analyzer (ZETASIZER NANO ZS) to determine the size and zeta potential of the NPs. Capture the morphology of the NPs using transmission electron microscopy (TEM, G2 Spirit Biotwin).

Statistical analyses

Data are presented as mean ± SD, with sample sizes specified in the figure legends. Group differences were assessed using unpaired Student’s t-tests or one-way/two-way analysis of variance (ANOVA) followed by Tukey’s tests, as indicated. In vivo experiments utilized 5 to 10 mice per condition. Statistical significance was defined as a P-value ≤ 0.05. All statistical analyses were carried out using GraphPad Prism 8 software.

Funding: This work was supported by National Natural Science Foundation of China (82174033 to Y.W., 81970761 to H.R.T.).

Authorship contributions: Conceptualization, Q.X., H.R.T. and Y.W., Methodology, Q.X., X.F.D., J.H.L., Y.N.S., Y.F.L., and T.L., Investigation, T.L.W., Validation and Data Curation, Q.X., X.F.D, Z.J.H., and Z.K.K., Resources, B.S., Writing – Original Draft, Q.X., X.F.D and Y.W.; Writing – Review & Editing, Q.X.; Supervision, H.R.T.; Funding Acquisition, H.R.T. and Y.W.

Competing interests: The authors declare that they have no competing interests.

Data and materials availability: All data associated with this study are present in the paper or the supplementary materials. The RNA-seq raw data were uploaded publicly in the GEO datasets via GSE273951.

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FABP4OPsupplementarymaterial.docx

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FABP4 as a critical mediator in osteoporosis: inhibition strategies and therapeutic potential

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Abstract

Figures

Introduction

Results

Patients’ BMD correlates negatively with their serum F4BP4 levels

FABP4 has no impact on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs)

FABP4 plays a crucial role in enhancing OCs differentiation

FABP4 inhibitor BMS309403 shows comparable OCs differentiation inhibition to ALD

FABP4 inhibitor suppresses OCs differentiation via Ca-Calcineurin-NFATc pathway

Effects of FABP4 inhibitor on bone resorption in OVX mouse OP model

Differential efficacy of FABP4 inhibitor and ALD in OVX mouse OP model

Developing novel bone-targeted PLGA nanoparticles with click chemistry

Bone-targeted NPs delivery of FABP4 inhibitor shows superior anti-OP effect in OVX mouse model

Discussion

Material and method

Study design

Animal study

Human study

Primary cell culture

OCs formation

Bone resorption activity on mature OCs

RNA-seq studies

NPs self-assembly, drug loading and characterization

Statistical analyses

Declarations

References

Additional Declarations

Supplementary Files

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