ABSTRACT Copper metabolism MURR1 domain protein 10 (COMMD10) regulates numerous biological processes that are essential for cellular homeostasis. However, the role of COMMD10 in angiogenesis and bone formation remains unexplored. We constructed a COMMD10 knockdown model in endothelial cells and determined the influence of COMMD10 on angiogenesis and bone formation. Our results indicate that COMMD10 knockdown enhances vascular formation by influencing the expression of genes and proteins related to angiogenesis in endothelial cells. In addition, endothelial cells expressing low levels of COMMD10 facilitate bone formation by secreting pro‐osteogenic factors. Further, the Rap1 signaling pathway is activated under low COMMD10 conditions. Double knockdown of RAP1B and COMMD10 attenuated the angiogenic ability of endothelial cells. In summary, our research demonstrates that low COMMD10 expression promotes angiogenesis and bone formation through the Rap1 signaling pathway. Keywords: angiogenesis, bone formation, COMMD10, endothelial cells, Rap1 __________________________________________________________________ We constructed a COMMD10 knockdown model in endothelial cells and determined the influence of COMMD10 on angiogenesis and osteogenesis. The Rap1 signaling pathway is activated under low COMMD10 conditions and further enhances vascular formation and secreting pro‐osteogenic factors. Double knockdown of RAP1B and COMMD10 attenuated the angiogenic ability of endothelial cells. In summary, our research demonstrates that low COMMD10 expression promotes angiogenesis and osteogenesis through the Rap1 signaling pathway. graphic file with name FBA2-7-e70016-g002.jpg 1. Introduction Bone formation is crucial for maintaining the homeostasis and function of the mammalian skeletal system. Dysfunctions in bone formation can lead to various bone diseases. Angiogenesis, the process of new blood vessels generating from pre‐existing vessels, is intimately linked to skeletal development and bone turnover. Blood vessels provide nutrients, oxygen, growth factors, and hormones necessary for osteogenesis to bone tissues, and have been found to provide essential signals that promote osteogenesis [[26]1, [27]2]. The copper metabolism MURR1 domain (COMMD) protein family includes 10 members (COMMD1–COMMD10) that are defined by the presence of a unique C‐terminal homology region termed the COMM domain [[28]3]. The COMMD family has been implicated as important for cellular homeostasis in lots of biological processes, such as the regulation of copper [[29]4] and sodium [[30]5] ions transport, nuclear factor‐κB signaling [[31]6, [32]7], cell cycle progression [[33]8], cell proliferation, and adaptation to hypoxia [[34]9]. Recently, the role of the COMMD family in mediating angiogenesis, especially in tumors, has been reported. In HCC patients, COMMD3 upregulation is related to vascular invasion. Knockdown of COMMD3 in hepatocellular carcinoma cells suppressed their migration and invasion, and the conditional medium of hepatocellular carcinoma cells decreases angiogenesis in endnotheilal cells [[35]10]. In renal cancer cells, COMMD5 decreases vascular endothelial growth factor (VEGF) production through downregulating HIF‐1α, ultimately inhibiting tumor angiogenesis [[36]11]. COMMD10 was found to bind and suppress hypoxia‐inducible factor 1α in hepatocellular carcinoma cells. These findings offer unprecedented insight into the potential role of COMMD10 in angiogenesis [[37]12]. In this study, COMMD10 was knocked down in endothelial cells using small interfering RNA (siRNA). We determined how COMMD10 knockdown in endothelial cells affected both angiogenesis and osteogenesis and explored the mechanisms underlying these effects. 2. Methods 2.1. Cell Culture Mouse islet endothelial cells (MS‐1) purchased from Procell (China) were cultured in complete media containing high‐glucose Dulbecco's modified Eagle medium (DMEM, Gibco, USA) supplemented with 5% fetal bovine serum (FBS, Gibco) and 1% penicillin–streptomycin (Hyclone, USA). The immortalized mouse calvarial osteoblast cell line, MC3T3‐E1 subclone 14 cells (Procell, China), was grown in dedicated culture media supplied by Procell (MEMα+10% FBS + 1% penicillin–streptomycin). All cells were cultured at 37°C in a sterile, humidified incubator with 5% CO[2]. The cell medium was replaced every 2–3 days. A trypsin solution (Hyclone, USA) was used to pass cells when they reached approximately 80% confluency. 2.2. Transfection of siRNAs COMMD10 siRNA, RAP1B siRNA, and control siRNA were purchased from GenePharma (China). The COMMD10 siRNA sequences were as follows: sense 5′‐GGAGAUUCCCACGACUUCUTT‐3′ and antisense 5′‐AGAAGUCGUGGGAAUCUCCTT‐3′. The RAP1B siRNA sequences were as follows: sense 5′‐GUGAAUAUAAGCUCGUCGUTT‐3′ and antisense 5′‐ACGACGAGCUUAUAUUCACTT‐3′. Cells were plated in 6‐well plates and allowed to adhere for 24 h before transfection with siRNA using GP‐transfect‐Mate (GenePharma, China) in DMEM, in accordance with the manufacturer's protocol. After 4 h, the medium was replaced with fresh complete medium, and cells were harvested 24–72 h after transfection. To investigate the impact of Commd10 knockdown, endothelial cells were divided into two treatment groups: (1) cells transfected with COMMD10 siRNA (SiCOMMD10) and (2) cells transfected with negative control siRNA (NC). To confirm whether Rap1 signaling participated in the promotion of angiogenesis led by Commd10‐knockdown, endothelial cells were divided into three treatment groups: (1) cells transfected with NC siRNA at 8 and 32 h after seeding respectively, (2) cells transfected with NC and SiCOMMD10 at 8 and 32 h after seeding respectively, (3) cells transfected with RAP1B siRNA (SiRAP1B) and SiCOMMD10 at 8 and 32 h after seeding respectively. 2.3. Co‐Culture Assay To determine the influence of endothelial cell secretions on MC3T3‐E1 cells, Transwell co‐culture experiments were performed in 12‐well plates using inserts with a 0.4‐μm pore size (LABSELECT, China). MC3T3‐E1 cells in complete medium were added to the outer wells of the Transwell system, and endothelial cells transfected with COMMD10 siRNA or control siRNA in complete DMEM were seeded in the inner wells. After 72 h, the outer medium was replaced with an osteogenic induction medium, and cells were maintained in the osteogenic induction medium until harvested for further assays. MC3T3‐E1 cells were divided into two treatment groups: (1) cells co‐cultured with endothelial cells transfected with COMMD10 siRNA (SiCOMMD10) and (2) cells co‐cultured with endothelial cells transfected with a negative control siRNA (NC). 2.4. Transcriptome Sequencing and Analysis Endothelial cells were harvested using Trizol (Invitrogen). We constructed and sequenced transcriptome libraries as previously described. Significantly differentially expressed genes (DEGs) were defined as those genes with a fold‐change > 2 or < 0.5 and a p‐value < 0.05. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed for DEGs. 2.5. Quantitative Polymerase Chain Reaction (qPCR) Analysis Total RNA from endothelial cells was obtained using the FastPure Cell/Tissue Total RNA Isolation Kit (Vazyme, China). A NanoDrop 2000 spectrophotometer (Thermo Scientific, USA) was used to measure the concentration and purity of the RNA. Total RNA from each group was reverse transcribed (PrimeScript RT Reagent Kit, Accurate Biology, China). The relative expression levels of target genes were determined with the CFX Connect Real‐Time PCR Detection System (Bio‐Rad, USA) and the SYBR Green Pro Taq HS Premix Kit (Accurate Biology, China). Gapdh was utilized as the internal reference. The primers for the target genes are displayed in Table [38]1. TABLE 1. Quantitative PCR primers for target genes. Gene name Forward Reverse Gapdh AAGGCCGGGGCCCACTTGAA GGACTGTGGTCATGAGCCCTTCCA Commd10 GTGGTCTTCTATGGGTCAAGAA TCATTGTTCACTCCGAGTTGTA Fgfbp1 AATAAGCAGAGAAGCAGGACAT GCAGATATTTTTCTGTTTGCGC Adra2b CCTGGTCTCTCCTCTATCTTCT TTCTTCTGGTCCTGGACTGATC Vegfa ACCTCACCAAAGCCAGCACA GCTTTGTTCTATCTTTCTTTGGTCTGC Cfh GTATCAAAACGGATTGTGACGT TAACACATGTCACAGTGTCTGA Sema6a GGTTCAAGGAACAGAAATCACC CATGAGTGGATGCGTCTTAATG Igf1 GAGGGGCTTTTACTTCAACAAG TACATCTCCAGTCTCCTCAGAT Ceacam1 ACATTATCTATGGTCCGGACAC GTGACAGAGTTATTGACGAAGC Rflnb CTATAGTCCCGAACCCAGCATC GTCAAACTCAACTCCTTCGC Ccl5 GTATTTCTACACCAGCAGCAAG TCTTGAACCCACTTCTTCTCTG Egfr TGAGTTCTCTGAGTGCAACTAG GAATGCGTCATCTATGTTGTCC Fgf18 AAGTACTCTGGTTGGTATGTGG CTTGGTGACTGTGGTGTATTTG Itgb2 AAAGTGACACTTTACTTGCGAC GAGGTAGTACAGATCAATGGGG Fgfr1 GGAGGCTACAAGGTTCGCTATGC GCTGGTAGGTGTGGTTGATGCTC Pard6b CATCTTCATATCTCGGCTCGTC CTCTTCCCGGACACTTCTATAC Rap1b TGCAGGAACGGAGCAATTCACAG TGATGGAGTAAACCAGAGCGAAGC [39]Open in a new tab 2.6. Western Blot Total proteins were extracted from endothelial cells using the Total Protein Extraction Kit (SAB, USA) and quantified with Coomassie Brilliant Blue staining (Bradford Protein Assay Kit, TIANGEN, China). For each group, 40 μg of protein was isolated by 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Proteins were transferred to 0.45‐μm polyvinylidene difluoride membranes, and the membranes were gently shaken in 5% skim milk (Bio‐Rad) for 60 min at room temperature. The membranes were then immersed in a solution of primary antibodies at 4°C for 24 h, followed by a solution of secondary antibodies for 1 h at room temperature. The primary antibodies included COMMD10 (Abcam, UK, 1:1000), RAP1A + B (Abcam, UK, 1:5000), FGFBP1 (Abclonal, China, 1:1000), IGF1 (Abclonal, China, 1:1000), FGF18 (Abcam, China, 1:1000), Tubulin (Abclonal, China, 1:5000), and GAPDH (SAB, USA, 1:5000). 2.7. Tube Formation Assay Matrigel (Corning, USA) was seeded into a 48‐well plate on ice, followed by incubation at 37°C for 30 min to facilitate Matrigel polymerization. Endothelial cells transfected with either COMMD10 siRNA or control siRNA were seeded onto the Matrigel. Images were acquired after incubating for 4, 8 or 12 h. Semi‐quantitative analyses of the junction, node, tube number, and total tube length were conducted using ImageJ software. 2.8. 3D Spheroid Sprout Assay of Angiogenesis The MS‐1 cells transfected with either COMMD10 siRNA or control siRNA were suspended in Methocol (Sigma, USA) solution with a density of 20,000/mL. Endothelial cell‐methocel solution was then pipetted onto a square Petri dish for hanging drop formation to form a cell spheroid. After collection, the spheroids were embedded in methocel containing 20% FBS and mixed with collagen. Observations were made after 24 h of culturing, and the number and length of sprouts from cell spheroids were measured. Semi‐quantitative analyses were conducted using ImageJ software. 2.9. Alkaline Phosphatase (ALP) Staining After induction for 7 days, ME3T3‐E1 cells were fixed in 4% paraformaldehyde (Biosharp, China). ALP stain was applied using the BCIP/NBT ALP color development kit (Beyotime, China). 2.10. Statistics Microsoft Excel was used to compile the data, and GraphPad Prism 8 software was used to perform statistical analyses. All data passed the Shapiro–wilk test to confirm their normality and lognormality. A one‐way analysis of variance was employed for comparisons among more than two groups, whereas a two‐tailed t‐test was utilized for comparisons between two groups. Data are displayed as the mean ± the standard deviation. Significant results are defined as p < 0.05. 3. Results 3.1. Verification of siCOMMD10 Real‐time PCR and Western blot analyses were conducted to determine the effects of siCOMMD10. We found that both mRNA and protein levels of COMMD10 were significantly decreased 72 h after transfection in the SiCOMMD10 group compared with the NC group (Figure [40]1). FIGURE 1. FIGURE 1 [41]Open in a new tab (A) Gene expression of Commd10. (B) Protein expression of COMMD10 was qualitatively and semi‐quantitatively analyzed. *p < 0.01, **p < 0.01. 3.2. COMMD10 Knockdown Promotes Angiogenesis in Endothelial Cells The effects of COMMD10 knockdown in endothelial cells were explored using transcriptome sequencing and analysis. A volcano plot shows the DEGs identified when comparing SiCOMMD10 and NC endothelial cells (Figure [42]2A). GO enrichment analysis revealed DEGs associated with the term “positive regulation of blood vessel endothelial cell proliferation involved in sprouting angiogenesis” (Figure [43]2B), and KEGG pathway analysis suggested that the Rap1 signaling pathway might mediate the angiogenic process (Figure [44]2C). FIGURE 2. FIGURE 2 [45]Open in a new tab (A) Volcano map of differentially expressed genes (DEGs). (B) Gene ontology (GO) analysis. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. To further explore the effects of COMMD10 knockdown on angiogenesis, a heat map was generated showing the expression profiles of angiogenesis‐related genes [[46]13] (Figure [47]3A). Proangiogenic genes (Hmox1, Chrd, Igf1, Ephb2, Fgf18, Fgfbp1, Adra2b, Itgb2) were upregulated in SiCOMMD10 endothelial cells compared with NC endothelial cells, whereas genes inhibiting angiogenesis (Cfh, Sema6a, Ptn) were downregulated. To validate the transcriptome sequencing analysis results, gene and protein expression were evaluated using qPCR and Western blot analyses. COMMD10 knockdown was associated with the upregulated expression of proangiogenic genes (Fgfbp1, Adra2b; Figure [48]3B) and proteins (FGFBP1; Figure [49]3C) and the downregulated expression of genes inhibiting angiogenesis (Cfh, Sema6a; Figure [50]3B). FIGURE 3. FIGURE 3 [51]Open in a new tab COMMD10 regulates the expression of angiogenesis‐related genes and proteins in endothelial cells; COMMD10 knockdown in endothelial cells increased tube formation. (A) Heat map showing expression levels of genes associated with angiogenesis. (B) Expression of proangiogenic genes (Fgfbp1, Adra2b, Vegfa) and anti‐angiogenic genes (Cfh, Sema6a) (n = 3 per group). (C) Protein expression level of FGFBP1 was increased upon COMMD10 knockdown. (D) Representative 3D spheroid angiogenesis assay images. (E) Sprouts genesis of cell spheroids was quantified by measuring the number of sprouts per spheroid and the sprout length of all sprouts per spheroid (n = 6 per group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The 3D spheroid sprout assay of angiogenesis was conducted to determine how COMMD10 knockdown affects the angiogenic functions of endothelial cells. During 24 h observation, the spheroids in the SiCOMMD10 group grew more and longer sprouts than in the NC group(Figure [52]3D,E), indicating that endothelial cells in the SiCOMMD10 group displayed higher measures of angiogenic ability than those in the NC group. These results indicate that low COMMD10 expression levels facilitate angiogenesis in endothelial cells. 3.3. COMMD10 Knockdown Promotes the Pro‐Osteogenic Ability of Endothelial Cells Because angiogenesis and osteogenesis are usually coupled [[53]14, [54]15], we determined whether COMMD10 knockdown also affects the pro‐osteogenic functions of endothelial cells. A heat map showing the expression profiles of secreted factors related to osteogenesis was generated (Figure [55]4A). Factors that promote osteogenesis were upregulated in endothelial cells in the SiCOMMD10 group compared with those in the NC group, whereas factors that inhibit osteogenesis were downregulated. In addition, the expression levels of related genes and proteins were evaluated using qPCR and Western blot analyses. Compared with endothelial cells in the NC group, endothelial cells in the SiCOMMD10 group displayed upregulation of pro‐osteogenic factors at the mRNA (Igf1 and Ceacam1 [[56]16, [57]17]; Figure [58]4B) and protein levels (insulin‐like growth factor 1 [IGF1]; Figure [59]4C), whereas factors that inhibit osteogenesis were downregulated. FIGURE 4. FIGURE 4 [60]Open in a new tab COMMD10 knockdown increased the pro‐osteogenic capabilities of endothelial cells. (A) Heat map showing the relative mRNA expression levels of factors associated with osteogenesis. (B) Expression of pro‐osteogenic factors (Igf1, Ceacam1) and anti‐osteogenic factors(Rflnb, Ccl5, Egfr) (n = 3 per group). (C) Insulin‐like growth factor 1 (IGF1) protein expression level increased upon COMMD10 knockdown. (D) Alkaline phosphatase (ALP) staining of MC3T3‐E1 cells co‐cultured with endothelial cells. *p < 0.05, **p < 0.01, ***p < 0.001. To further confirm the osteogenic effects of COMMD10 knockdown in endothelial cells, co‐culture experiments were conducted, and ALP staining was performed. The results of ALP staining indicated the endothelial cells in the SiCOMMD10 group displayed enhanced pro‐osteogenic capabilities compared with those in the NC group (Figure [61]4D). 3.4. COMMD10 Knockdown May Promote Angiogenesis via Rap1 Signaling KEGG pathway enrichment analysis revealed that Rap1 signaling may mediate the COMMD10‐regulated functions of endothelial cells. We examined the expression levels of genes and proteins associated with the Rap1 signaling pathway. Moreover, both mRNA (Fgf18, Itgb2, Fgfr1 and Pard6b [[62]18, [63]19]; Figure [64]5A) and protein expression levels (FGF18, Figure [65]5B) of Rap1‐associated factors were upregulated in the SiCOMMD10 group compared with the NC group, indicating that COMMD10 may regulate angiogenesis via Rap1 signaling in endothelial cells. FIGURE 5. FIGURE 5 [66]Open in a new tab COMMD10 knockdown may promote angiogenesis via Rap1 signaling. (A) Expression of genes in the Rap1 signaling pathway. COMMD10 knockdown increased the expression of Fgf18, Itgb2, Fgfr1, and Pard6b (n = 3 per group). (B) Expression of protein in the Rap1 signaling pathway. COMMD10 knockdown increased the expression levels of fibroblast growth factor 18 (FGF18). (C) Commd10 and Rap1b mRNA expression levels. Commd10 was significantly knocked down in the SiCOMMD10 group and DK group. Rap1b gene was only significantly knocked down in the DK group (n = 3 per group). (D) The angiogenesis‐related genes Fgfbp1 and Vegfa were expressed significantly higher in the SiCOMMD10 group, while no significant differences were found between the NC group and DK group. (E) The expression level of the secretory protein gene Igf1 was lower in the DK group than in the SiCommd10 group. The expression level of the secretory protein gene inhibiting osteogenesis, Ccl5, was significantly upregulated after Rap1b knockdown (n = 3 per group). “ns”= no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Furthermore, we inhibited the expression of Rap1 signaling through transfection of Rap1b siRNA. In this section, three treatment groups were set: (1) NC group: cells transfected with NC siRNA at 8 and 32‐h after seeding respectively, (2) SiCommd10 group: cells transfected with NC and SiCOMMD10 at 8 and 32‐h after seeding respectively, and (3) double knockdown (both rap1b and commd10 knockdown, DK group): cells transfected with RAP1B siRNA (SiRAP1B) and SiCOMMD10 at 8 and 32‐h after seeding respectively. Under the conditions of transfection with either NC or siRAP1B, Commd10 could still be knocked down, and the knockdown efficiency was not significantly affected. However, the expression level of the Rap1b gene in endothelial cells transfected with siRAP1B was significantly lower compared to the other two groups (Figure [67]5C). When comparing the relative expression levels of angiogenesis‐related genes, the endothelial cells with simultaneous knockdown of Rap1b and Commd10 showed significantly lower expression of Fgfbp1 and Vegfa genes compared to the NC group and SiCommd10 group (Figure [68]5D). The expression level of the secretory protein gene promoting osteogenesis, like Igf1, was lower in the DK group than in the SiCommd10 group. Conversely, the expression level of the secretory protein gene inhibiting osteogenesis, like Ccl5, was significantly upregulated after Rap1b knockdown (Figure [69]5E). In terms of the angiogenic phenotype, we observed that the angiogenic capacity of endothelial cells in the DK group was lower than that in the SiCommd10 group and showed no significant difference compared to the control group, either by tube formation or 3D spheroid assay (Figure [70]6). These results suggest that the Rap1 signaling pathway may play a critical role in promoting angiogenesis and osteogenic functions during the process of Commd10 knockdown in endothelial cells. FIGURE 6. FIGURE 6 [71]Open in a new tab Rap1b knockdown attenuated the angiogenic ability improvement of Commd10‐knockdown endothelial cells. (A) Representative tube formation images. (B) Tube networks were quantified by measuring the node, junction, tube number, and total tube length (n = 3 per group). All parameters in the DK group had no significant differences compared to the NC group. (C) Representative 3D spheroid angiogenesis assay images. (D) Sprouts genesis of cell spheroids was quantified by measuring the number of sprouts per spheroid and the sprout length of all sprouts per spheroid (n = 3 per group). All parameters in the DK group had no significant differences compared to the NC group. “ns”= no significance, *p < 0.05, **p < 0.01. 4. Discussion Bone remodeling is a tightly regulated process that coordinates the resorption and formation of skeletal tissue and is essential for ensuring the integrity of the skeleton. Angiogenesis and bone formation are coupled during bone development and repairing [[72]14, [73]15]. COMMD10 plays roles in the control of several biological processes, including Staphylococcus clearance [[74]20], inflammation inhibition [[75]21], Na^+ transport [[76]22], and other essential biological processes [[77]3, [78]12, [79]23, [80]24]. However, the roles of COMMD10 during angiogenesis and osteogenesis have not yet been identified. Here, we examined the effects of COMMD10 knockdown in endothelial cells. We found that COMMD10 knockdown increased the expression of proangiogenic genes and proteins in endothelial cells and promoted tube formation in vitro. In addition, we found that endothelial cells in the SiCOMMD10 group secreted more pro‐osteogenic factors than endothelial cells in the NC group, which further enhanced osteogenesis in MC3T3‐E1 cells. Compared with the NC group, the SiCOMMD10 group displayed upregulated expression of Rap1 signaling‐related genes and proteins, suggesting that Rap1 signaling may mediate the effects of COMMD10. Our findings indicate that COMMD10 knockdown in endothelial cells promotes angiogenesis and the coupling of angiogenesis with osteogenesis. We sequenced the transcriptome and analyzed the complete gene expression profile of endothelial cells with low COMMD10 expression levels. Our findings suggest that COMMD10 regulates the expression of angiogenesis‐related genes. The qPCR and Western blot results validated the transcriptomic sequencing results. Specifically, Fgfbp1, Adra2b, and Vegfa were upregulated, whereas Cfh and Sema6a were downregulated. FGFBP1, a well‐established proangiogenic factor [[81]25], is a secreted carrier protein that serves as an extracellular chaperone that binds and activates FGFs (FGF1, 2, 7, 10, and 22) in a reversible and non‐covalent manner. Upon binding with FGFBP1, FGF2 is released from the extracellular matrix and binds with FGF receptors to activate angiogenesis and tissue repair [[82]26]. Alpha‐2B adrenergic receptor (ADRA2B) promotes angiogenesis by suppressing VEGF receptor type 1 (VEGFR1, Flt1). Flt1 and its soluble splice variant (sFlt1) are antiangiogenic molecules that bind VEGF with high affinity, preventing the VEGF‐mediated activation of other receptor subtypes [[83]27]. Complement factor H (CFH) inhibits angiogenesis by reducing endothelial cell migration [[84]28]. Similarly, semaphorin 6A (SEMA6A) blocks VEGF‐mediated endothelial cell migration [[85]29, [86]30]. In the current study, we found increased levels of Ffgbp1, Adra2b, and Vegfa and decreased Cfh and Sema6a levels in endothelial cells in the SiCOMMD10 group compared with those in the NC group. These results were further confirmed through in vitro angiogenic assays. Our results suggest that low expression levels of COMMD10 promote angiogenesis. To confirm the effects of endothelial cell secretions on bone regeneration, we examined the expression of bone formation factors in endothelial cells. IGF1 plays multiple roles in enhancing bone formation through its actions on osteoblast lineage cells, including promoting cell migration [[87]16, [88]31] and stimulating osteoblast differentiation [[89]32, [90]33]. Carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1) promotes bone regulation by inhibiting osteoclastogenesis [[91]17]. Refilin B (RFLNB) may be involved in the negative regulation of bone mineralization during bone maturation, based on inference from the biological aspect of ancestor analysis. Chemokine C—C motif ligand 5 (CCL5) can induce chemotaxis in pre‐osteoclasts and promote their differentiation into osteoclasts [[92]34, [93]35]. Epidermal growth factor receptor (EGFR) enhances the proliferation of mesenchymal progenitor cells, pre‐chondrocytes, and pre‐osteoblasts, resulting in a larger pool of precursor cells, but EGFR inhibits terminal differentiation toward mature osteoblasts [[94]36]. In the present study, we found increased expression of Igf1 and Ceacam1 and decreased expression of Rflnb, Ccl5, and Egfr in endothelial cells in the SiCOMMD10 group compared with those in the NC group. These results indicate that endothelial cells with low COMMD10 levels may have a pro‐osteogenic function. Furthermore, we conducted ALP staining of MC3T3‐E1 cells co‐cultured with endothelial cells; the results were consistent with the qPCR and Western blot assays. Finally, we examined the potential mechanisms underlying the observed effects of COMMD10 knockdown. KEGG pathway analysis indicated that the Rap1 signaling pathway may be a contributing factor. Therefore, we examined the expression levels of genes and proteins in the Rap1 signaling pathway. Fgf18 and Igf1 encode growth factors that activate the Rap1 signaling pathway [[95]37]. FGFR1 is a receptor tyrosine kinase. Par‐6 family cell polarity regulator beta (PARD6B) and ITGB2 are downstream components of the Rap1 signaling pathway [[96]19]. Upregulation of these genes and proteins indicates the activation of the Rap1 signaling pathway in endothelial cells with COMMD10 knockdown, suggesting that Rap1 might be involved in the regulation of angiogenesis and bone formation by COMMD10. On the other hand, when both COMMD10 and RAP1B were knocked down, the angiogenic capacity of endothelial cells was significantly weaker than when only COMMD10 was knocked down. The expression of pro‐angiogenic genes and proteins also showed the same trend, while the expression levels of osteogenesis‐related genes and proteins were significantly lower than when only COMMD10 was knocked down. These results indicate that when the RAP1 signaling pathway is inhibited, the ability of COMMD10 knockdown to promote angiogenesis and osteogenesis in endothelial cells is suppressed, further supporting that the RAP1 signaling pathway may play a key role in the effects of COMMD10 knockdown. This work demonstrates that low COMMD10 levels in endothelial cells facilitate angiogenesis. In addition, endothelial cells with low COMMD10 levels secrete factors that promote bone formation. Furthermore, we demonstrated that the angiogenic functions observed for endothelial cells with low COMMD10 expression may be regulated by the Rap1 signaling pathway, which could be further explored in future studies. 5. Conclusion We examined the roles played by COMMD10 in angiogenesis and bone formation. We found that endothelial cells with low COMMD10 expression promoted angiogenesis and facilitated bone formation. Finally, we found that the Rap1 signaling pathway might mediate these effects of low COMMD10 expression. Author Contributions Formal analysis, methodology, validation, software and visualization, investigation, resources, data curation, conceptualization: P.L. and Y.L. Writing – original draft preparation: Y.L. Writing – review and editing, funding acquisition: P.L. All authors have read and agreed to the published version of the manuscript. Conflicts of Interest The authors declare no conflicts of interest. Funding: This research was funded by Beijing Stomatological Hospital, Capital Medical University Young Scientist Program (No. YSP‐2022‐09‐15). Those who funded the research had no role in the study design, data collection, analysis, publication decision, or manuscript preparation. Data Availability Statement The data that support the findings of this study are available in the Materials and Methods and Results of this article. References