More and more pieces of evidence show that vascular system diseases are related to bone metabolism. Some scholars presented a concept of a bone-vascular axis to explore the correlation between them (1, 2). They supported that there exited cellular, endocrine, and metabolic signals that flow bidirectionally between the vasculature and bone, and vascular and skeletal disease may occur concurrently when dysmetabolic states perturbed the bone–vascular axis (2). Osteoporosis and atherosclerosis are the most common skeletal and vascular disease while also being the chronic systemic diseases and major public health problems worldwide (3). Epidemiology studies have linked osteoporosis and atherosclerosis, suggesting that postmenopausal women with osteoporosis are often accompanied by atherosclerosis (4). Decreased bone mineral density and osteoporotic fractures were significantly associated with the development of echogenic plaques in carotid artery (5). Regardless of no widely consensus on the exact cellular and molecular basis underlying the high comorbidity between osteoporosis and atherosclerosis, it has been testified that they shared risk factors, common pathogenesis and genetic factors, and a causal association (6).

Several risk factors involving ageing, postmenopausal status, smoking habit, physical inactivity, and alcohol intake were considered as the common factors shared by osteoporosis and atherosclerosis (7). Osteoblast activity and survival decline with aging, whereas osteoclast activity increases, contributing to the age-associated decline of bone mass (8). Simultaneously, atherosclerosis, a high prevalence and incidence disease in the elderly causing most heart attacks and strokes, is considered a hallmark of aging process (9). However, there is growing evidence that a potential link exists between osteoporosis and atherosclerosis beyond aging (10). Several pathophysiological mechanisms including inflammatory cytokines, lipid oxidation products, and vitamin D and K deficiency were identified for the interplay between the skeletal and vascular systems (7, 11). Inflammatory cytokines [including tumor necrosis factor–alpha (TNF-α) and interleukin (IL)-1, IL-6, and IL-17] and chemokines have been shown to be associated with atherosclerosis, increased cardiovascular morbidity and mortality, and increased bone loss (12, 13). Dyslipidemia, including elevated level of total serum cholesterol, triglycerides, and low-density lipoprotein (LDL) cholesterol, was considered to promote atherosclerosis progression and also influenced bone metabolism (14). In addition, there also exists similarity of the treatment for osteoporosis and atherosclerosis. Statins, cholesterol-lowering drugs in preventing and treating cardiovascular disease, have potential positive effects on bone mineral density and decreasing osteoporotic fracture (15). Bisphosphonate therapy for osteoporosis reduces progression of vascular calcification (16). These findings strongly suggested the interaction of these two pathological conditions, simultaneously exerting an influence on the development of osteoporosis and atherosclerosis. However, they were mainly from clinical perspectives, and few studies have investigated genomic relationship between osteoporosis and atherosclerosis.

Contemporary, the quick development of bioinformatics approaches allows us to get a better grasp of disease pathobiology more deeply from the genetic level (17). Recently, a system-level analysis of whole blood genome−wide expression data identified several enriched biological pathways and three genes (NOSIP, GXYLT2, and TRIM63) that were significantly associated with early traits of both osteoporosis and atherosclerosis, supporting the idea that they were comorbid based on transcriptomic evidence (18). However, the representativeness of their samples was limited, and studies integrating gene data from public databases with both osteoporosis and atherosclerosis are lacking. In view of this, the purpose of this study is to integrate and analyze gene data related to the pathogenesis of osteoporosis complicated with atherosclerosis from the public databases, which provide new insights into the biological mechanisms of these two diseases, and it will help to develop dual-purpose prevention methods. The research flowchart of this research was shown in Figure 1 .

Osteoporosis and atherosclerosis are both widely prevalent disorders and often seen concurrently, exerting a severe impact on public health (29). The common pathophysiological mechanisms between osteoporosis and atherosclerosis have been attracting intense research interest around the world. Previous studies identified some molecular lipid species associated with early markers of both osteoporosis and atherosclerosis based on the Young Finns Study cohort, supporting the osteoporosis and atherosclerosis comorbidity hypothesis (14, 30). Latest research found that platelet-derived growth factor–BB (PDGF-BB), secreted from preosteoclasts, worked as an important mediator of vascular stiffening in response to aging and metabolic stress (31). Extracellular vesicles derived from aged bone matrix during bone resorption promote bone marrow mesenchymal stem cells adipogenesis rather than osteogenesis and augment calcification of vascular smooth muscle cells (32). These results have deepened our knowledge of the concept of a bone-vascular axis and may help to reveal the molecular mechanism between osteoporosis and atherosclerosis. However, it seems that few studies have explore the common pathogenesis of osteoporosis and atherosclerosis on a genetic level. To our knowledge, this is the first time to investigate osteoporosis and atherosclerosis comorbidity hypothesis by integrating data from a variety of public databases to identify the common mechanisms of osteoporosis and atherosclerosis.

Global gene expression studies can help us better understand the specific pathobiology between osteoporosis and atherosclerosis. The results of WGCNA and DEG analysis showed that immune and inflammatory response–related functions may play an important role in both osteoporosis and atherosclerosis. At the same time, we found that T-cell differentiation and inflammatory signaling pathways were also enriched in the enrichment analysis results of common genes from public disease databases. Therefore, an inflammatory environment caused by the immune and inflammatory response may be a common feature in the pathophysiology of osteoporosis and atherosclerosis.

Under physiological conditions, the bone homeostasis is regulated by osteoblast-mediated bone formation and osteoclast-mediated bone resorption through receptor activator of nuclear factor–κB (RANK), ligand for a RANK receptor (RANKL), and osteoprotegerin (OPG) interactions (33). Any imbalance of this control leads to an increase in the activity of osteoclast, resulting in osteoporosis. Although initially, it was thought that hormonal imbalance was the leading cause of osteoporosis, the role of the immune system in osteoporosis slowly came into view with advanced studies (34, 35). In pathological states such as rheumatoid arthritis, multiple sclerosis, osteoarthritis, and bone tumors, a host of inflammatory cytokines and activated immune cells disrupts this balance in favor of osteoclast-mediated bone resorption (36). Accumulated pieces of evidence suggested that immune and inflammatory response induced by both innate and adaptive immune cells can affect bone metabolism through several pathways.

Immune cell activation often goes along with inflammatory mediators’ production, such as reactive oxygen species, and pro-inflammatory cytokines and chemokines, which directly or indirectly influence bone metabolism and promote the development of osteoporosis (37). In innate immune system, pro-inflammatory cytokines such as TNF-a and IL-6 can stimulate the polarization of macrophages into M1 macrophages (both an inflammatory phenotype and a precursor of osteoclast), associating with bone catabolic activity (38). The monocytes show high levels of C-C chemokine receptor 2 (CCR2) in inflammatory microenvironment and can serve as osteoclast precursor, participating in bone remodeling by producing cytokines (39). In addition, dendritic cells and neutrophils can activate T cells, and the activated T cells produce cytokines and soluble factors that participate in bone resorption process (40). In adaptive immune system, T cells are major players of it and the results of our enrichment analysis were also involved in T_H1, T_H2, and T_H17 cell differentiation and inflammatory factor–related pathways. Previous studies have found that T_H1 and T_H2 cells inhibit osteoclastogenesis via secreting IFN-γ and IL-4 cytokines and thus act as an osteoprotective role (41). Lower serum levels of IFN-γ and IL-4 cytokines in postmenopausal osteoporotic patients further suggest its osteoprotective role (42). However, a study suggested that IFN-γ can also promote osteoblasts generation by inducing expression of RANKL on activated T cells and thus plays a dual role in bone remodeling (43). T_H17 cells are osteoclastogenic subsets of T cells and can produce proinflammatory cytokine, including RANKL, TNF-α, IL-17, and IL-6, all of which augment osteoclastogenesis (44). Moreover, enhancement in the number of T_H17 cells and enhanced expression of proinflammatory cytokines (IL-6, TNF-α, RANKL, and IL-17) were observed in osteoporotic mice (45). Recent research suggested that “pyroptosis” of osteoblast (a programmed cell death mechanism) correlates with inflammation and contributes to excessive differentiation of osteoclasts via producing NLRP3 and also IL-1β and IL-18 (46). In summary, the concept of osteoimmunology was consistent with the enrichment results above in our study. Zhang et al. (47) performed a GO analysis of genes corresponding to differentially expressed proteins in osteoporosis and also found that immune inflammation and related functions were significantly enriched, consistent with our analysis.

Atherosclerosis is a chronic disease with an autoimmune component due to its accompaniment with a chronic, low-grade inflammatory response that attracts cells of the innate and adaptive immune systems into the atherosclerotic plaque (48). The important role of immunity and inflammation in atherosclerosis has been demonstrated by overwhelming experimental and clinical evidence. In our study, the analysis of positively correlated modules in atherosclerosis also supported this view. A genome-wide association study for coronary artery disease identified some locus linked to inflammation (49). The most robust genetic association has been identified for single-nucleotide polymorphisms in the 9p21 locus, which has been implicated in regulation of IFN-γ signaling (50). Oxidized LDL (oxLDL), the well-established precursor particles of atherosclerosis, can trigger inflammation of the arterial wall by binding to Toll-like receptors (TLRs) (51), and it can also reduce osteoblast viability (52).

In the vessel wall, oxLDL and inflammatory statement elicit an influx of monocytes that differentiate into macrophages and then into foam cells, accumulate intracellular cholesterol, and produce inflammatory mediators (53). CD4⁺ T cells are also recruited to the forming lesion, and single-cell data from human atherosclerotic plaques showed that the majority of CD4⁺ T cells in the plaque are T_H1 and T_H2 cells (54). In our study, we similarly found a significant enrichment for T_H1 and T_H2 cell differentiation pathway. Proinflammatory mediators such as IFN-γ, TNF-α, IL-2, and IL-3, produced by T_H1 and T_H2 cells, can activate macrophages and other plaque cells and thereby accelerate the inflammatory response (54). Knocking IFN-γ and its receptor protects mice from atherosclerosis (55). In addition, T_H17 cells also can be induced by oxLDL and constitute a minor population in plaques, promoting the vascular inflammation (56). Ongoing inflammatory and hemodynamic assaults on the atherosclerotic lesion might eventually cause local dysfunction or breakdown of endothelial integrity. One study identified the DEGs in atherosclerosis, compared with healthy controls. The bioinformatics analysis showed that immune response, inflammatory response, and vascular smooth muscle contraction were the unique gene signatures in atherosclerosis, which were consistent with our analysis (57).

As mentioned above, we found that immune and inflammatory response were enrich in both atherosclerosis and osteoporosis, and most of common genes obtained from WGCNA, DEGs, and three public databases were associated with immune response. The immune-associated genes in both atherosclerosis and osteoporosis from WGCNA mainly included TREM1, CYBB, CCR1, CD83, CD52, IL7R, and THBS1. The immune-associated genes from DEGs mainly included TNF, ITGB2, CD74, CCR5, and MAF. The immune-associated genes from three public databases mainly included IFNG, IL2, IL4, CCL2, TGFB1, and TLR2. Therefore, the occurrence and development of atherosclerosis and osteoporosis are complicated and these genes may provide clues for the common underlying mechanisms between them. Suppression of inflammation may have beneficial effects on bone and on vasculature.

Then, six hub genes (COL1A1, IBSP, CTSD, RAC2, MAF, and THBS1) were obtained through the integration of multisource databases and they were all significantly upregulated in both the osteoporosis and atherosclerosis group compared with the control group. According to CTD, COL1A1 was closely associated with osteoporosis and atherosclerosis and it had the highest inference score and reference count. COL1A1 is mainly involved in bone matrix formation, coding for collagen type 1 which is the most abundant extracellular protein in bone and serves as an indicator of bone formation (58). Mutations in COL1A1 gene have been demonstrated to be responsible for the autosomal dominant form of osteogenesis imperfecta, with severe osteoporosis (59). Studies have confirmed that COL1A1 is overexpressed in artery at animals with atherogenic diet and the increase was contributed to the inflammatory process and the activate the cytokine TGF-β, which supports the fibrotic process through expressing COL1A1 (60). In addition, previous studies have shown that COL1A1 may influence the prognosis in tumors by affecting infiltrating immune cells (61). Therefore, COL1A1 may be involved in the pathogenesis of osteoporosis and atherosclerosis by influencing bone formation, vascular fibrosis, immune response, but these related molecular mechanisms warrant additional investigation. Thrombospondin-1 (THBS1), an immune-associated gene, can affect endothelial cell proliferation, migration, and apoptosis by antagonizing the activity of VEGF, participating in the regulation of vascular formation (62). The latest research suggests that CD68⁺ macrophages activate TGF-β1 by expression and secretion of THBS1 and consequently induce bone information (63). Importantly, THBS1 was closely related to inflammatory response and is found to be elevated in inflammatory processes (64). However, few studies directly analyze the role of THBS1 in osteoporosis and atherosclerosis, which emphasizes its importance in future research. Other hub genes were also involved in the pathogenesis of osteoporosis and atherosclerosis by regulating different BPs and believe that the specific mechanism is worthy of further exploration.

Finally, by constructing miRNAs–genes network, we found that let-7g had the most target genes and it also involved in immune and inflammatory response. It was reported that the let-7g was significantly downregulated in patients with recent osteoporotic fractures and it could enhance osteoblast formation in vitro and in vivo by targeting HMGA2 (65). In addition, it also can regulate osteoblast formation by targeting COL1A2 (66). More importantly, high-fat diet can suppress let-7g expression (67) and activate NF-κB signaling pathway, which serves as a pivotal mediator of inflammatory responses (68). let-7g can reduce macrophage transformation and alleviates foam cell apoptosis by suppressing NF-κB pathways to prevent atherosclerosis (69). Because the expression of let-7g is regulated by a number of cellular elements which are associated with inflammatory states and chemokine release (70), it is possible that inflammatory conditions contribute to the relative paucity of let-7g resulting in reducing osteogenic formation and increasing endothelial and foam cell apoptosis, in favor of both osteoporosis and atherosclerosis. In view of the critical role of let-7g in inflammatory response, let-7g might be an important potential target for the treatment of osteoporosis and atherosclerosis.

In conclusion, our work revealed that the immune and inflammatory response might be a common susceptible factor for both osteoporosis and atherosclerosis and identified novel gene candidates who could be used as biomarkers or as potential therapeutic targets. It may provide some clues for detailed molecular mechanisms underlying bone–vascular axis. However, the results of our study need to be further verified in cell or animal experiments, which will be critical direction for future research.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

LM, CM, and YL contributed to the conception of the study; LM, CM, ZW, and JL performed the analysis; CZ, WN, ZC, and WH contributed significantly to analysis and manuscript preparation; LM and CM performed the data analyses and wrote the manuscript; LM, WH, ZC, CZ and YL helped perform the analysis and constructive discussions. YL, CZ, and ZC gained the founding; All authors contributed to the article and approved the submitted version.

This research was supported by Guangzhou Science and Technology Bureau (Grant No. 202102020930), National Natural Science Foundation of China (Grant No. 82104883, No. 81573996), Natural Science Foundation of Guangdong Province (Grant No. 2021A1515011484), Traditional Chinese Medicine Bureau of Guangdong Province (Grant No. 20221136) and Double First-rate Discipline and High-level University Construction Projects of Guangzhou University of Chinese Medicine (Grant No. 2021xk46).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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