HIF-1α Regulates Bone Homeostasis and Angiogenesis, Participating in the Occurrence of Bone Metabolic Diseases

In the physiological condition, the skeletal system’s bone resorption and formation are in dynamic balance, called bone homeostasis. However, bone homeostasis is destroyed under pathological conditions, leading to the occurrence of bone metabolism diseases. The expression of hypoxia-inducible factor-1α (HIF-1α) is regulated by oxygen concentration. It affects energy metabolism, which plays a vital role in preventing bone metabolic diseases. This review focuses on the HIF-1α pathway and describes in detail the possible mechanism of its involvement in the regulation of bone homeostasis and angiogenesis, as well as the current experimental studies on the use of HIF-1α in the prevention of bone metabolic diseases. HIF-1α/RANKL/Notch1 pathway bidirectionally regulates the differentiation of macrophages into osteoclasts under different conditions. In addition, HIF-1α is also regulated by many factors, including hypoxia, cofactor activity, non-coding RNA, trace elements, etc. As a pivotal pathway for coupling angiogenesis and osteogenesis, HIF-1α has been widely studied in bone metabolic diseases such as bone defect, osteoporosis, osteonecrosis of the femoral head, fracture, and nonunion. The wide application of biomaterials in bone metabolism also provides a reasonable basis for the experimental study of HIF-1α in preventing bone metabolic diseases.


Introduction
The skeletal system is an essential part of the human body. Under physiological conditions, osteoclasts and osteoblasts play a role in maintaining bone resorption and formation in dynamic balance [1]. Bone marrow mesenchymal stem cells (BMSCs) are important precursor cells of the osteoblast line [2]. Under appropriate conditions, BMSCs can differentiate into mesenchymal-derived cell types, such as chondrocytes, osteoblasts, and adipocytes, which play a crucial role in bone remodeling [3]. Osteoclasts derived from bone marrow-derived macrophages (BMMs) clear old or damaged bone under the trigger of osteocytes while releasing growth factors from the bone matrix, thus inducing BMSCs recruitment and migration to the repair area, resulting in BMSCs differentiation into osteoblasts and formation of new bone at specific sites [4,5]. However, under the pathological conditions of estrogen deficiency, abnormal mechanical stress, and drug side effects, the homeostasis of bone formation and bone resorption is disrupted, leading to osteoporosis, fracture, nonunion, or osteonecrosis of the femoral head [2]. Therefore, the regulation of bone formation and bone resorption is the basic strategy for preventing and treating bone metabolism-related diseases, in which osteoclasts and osteoblasts play a crucial role.
Hypoxia-inducible factor (HIF-1) is a pivotal transcriptional regulator of cell response to hypoxia, which is composed of HIF-1α and HIF-1β subunits, interacting with the hypoxia-responsive element (HRE) and further regulating the expression of target genes [6].
TWIST, as a transcriptional inhibitor of runt-related transcription factor 2 (RUNX2), is one of the downstream targets of HIF-1α, inhibiting BMSCs osteogenic differentiation by downregulating bone morphogenetic protein 2 (BMP2) and RUNX2 [57][58][59][60][61][62]. Mechano growth factor (MGF) is a splicing variant of insulin-like growth factor 1 (IGF-1), which can be used for autocrine tissue repair. Studies have shown that it promotes the growth and osteogenic differentiation of MSCs through the Phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway [63,64]. Furthermore, the MGF E peptide reversed the low expres-Cells 2022, 11, 3552 4 of 23 sion of HIF-1α under severe hypoxia via MEK-ERK1/2 and PI3K-Akt signal pathways, promoting BMSCs proliferation and osteogenic differentiation [65]. These results suggest that hypoxia degree and duration affect HIF-1α expression, regulating the biological behavior of BMSCs ( Figure 2). It is unclear whether TWIST, as a critical factor in connecting HIF-1α and osteogenic genes, results in the above contradictory results, but the mechanism of MGF E peptide reversing HIF-1α low expression induced by severe hypoxia has been elucidated.
Cells 2022, 11, x FOR PEER REVIEW 4 of 23 osteogenic differentiation of MSCs through the Phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway [63,64]. Furthermore, the MGF E peptide reversed the low expression of HIF-1α under severe hypoxia via MEK-ERK1/2 and PI3K-Akt signal pathways, promoting BMSCs proliferation and osteogenic differentiation [65]. These results suggest that hypoxia degree and duration affect HIF-1α expression, regulating the biological behavior of BMSCs ( Figure 2). It is unclear whether TWIST, as a critical factor in connecting HIF-1α and osteogenic genes, results in the above contradictory results, but the mechanism of MGF E peptide reversing HIF-1α low expression induced by severe hypoxia has been elucidated.

HIF-1α Regulates Bone Homeostasis by Affecting Energy Metabolism
Hypoxia leads to the insufficient energy production of mitochondria and excessive reactive oxygen species (ROS), which damages mitochondria. The high expression of HIF-1α transforms energy metabolism from oxidative phosphorylation to glycolysis, reduces ROS production, and enhances mitochondria's tolerance to hypoxia injury, suggesting that hypoxia plays a regulatory role in energy metabolism by inducing adaptive high expression of HIF-1α [66,67]. HIF-1α is associated with various diseases by affecting the glycolysis pathways of tumors, inflammation, and immune cells and regulating biological processes such as cell proliferation, differentiation, migration, chemotaxis, phagocytosis, and apoptosis.

HIF-1α Regulates Bone Homeostasis by Affecting Energy Metabolism
Hypoxia leads to the insufficient energy production of mitochondria and excessive reactive oxygen species (ROS), which damages mitochondria. The high expression of HIF-1α transforms energy metabolism from oxidative phosphorylation to glycolysis, reduces ROS production, and enhances mitochondria's tolerance to hypoxia injury, suggesting that hypoxia plays a regulatory role in energy metabolism by inducing adaptive high expression of HIF-1α [66,67]. HIF-1α is associated with various diseases by affecting the glycolysis pathways of tumors, inflammation, and immune cells and regulating biological processes such as cell proliferation, differentiation, migration, chemotaxis, phagocytosis, and apoptosis.
Selective knockout of the VHL gene up-regulates HIF-1α in mouse osteoblasts. It promotes bone marrow angiogenesis, showing a significant increase in trabecular volume, which effectively reverses bone loss caused by estrogen deficiency and promotes long bone formation with high bone density and rich blood supply [127][128][129]. Zuo et al. [130] found that the cortical bone area of VHL-deficient mice increased significantly, which was attributed to the proliferation and osteogenic differentiation of BMSCs promoted by the VHL/HIF-1α/β-catenin pathway. In addition, VHL deficiency of osteocytes increases bone mass and bone marrow hematopoiesis by regulating HIF-1α/Wnt signal pathway [131].
Selective knockout of the VHL gene up-regulates HIF-1α in mouse osteoblasts. It promotes bone marrow angiogenesis, showing a significant increase in trabecular volume, which effectively reverses bone loss caused by estrogen deficiency and promotes long bone formation with high bone density and rich blood supply [127][128][129]. Zuo et al. [130] found that the cortical bone area of VHL-deficient mice increased significantly, which was attributed to the proliferation and osteogenic differentiation of BMSCs promoted by the VHL/HIF-1α/β-catenin pathway. In addition, VHL deficiency of osteocytes increases bone mass and bone marrow hematopoiesis by regulating HIF-1α/Wnt signal pathway [131]. These studies suggest that VHL/HIF-1α pathway is a pivotal mediator in regulating angiogenesis and osteogenic differentiation of BMSCs. In addition, VHL also plays a vital role in regulating bone morphogenesis. Although the absence of VHL does not change the differentiation direction of mesenchymal progenitor cells into chondrocytes during development, it damages the proliferation ability of chondrocytes, resulting in the structural collapse of the growth plate and affecting bone morphogenesis [132,133].
Type H ECs are a subtype of vascular endothelial cell with high expression of CD31 and Endomucin (EMCN) in the metaphyseal, distributing many bone progenitor cells around, which can differentiate into osteoblasts and osteocytes [149,150]. Two consecutive studies in Nature by the same team in 2014 found that Type H ECs mediate local vascular growth via HIF-1α/VEGF and provide clear signals for perivascular osteoblasts through the Notch signal pathway [150,151]. HIF-1α is highly expressed in Type H ECs of young rats, decreasing with aging, and is related to age-dependent bone loss, which can be reversed by DFO [152]. EC-specific inactivated VHL gene enhances Type H vessels angiogenesis and increases the number of bone progenitor cells by stabilizing HIF-1α in ECs [8]. In addition, hypoxia signals increase the number of Type H vessels and enhance endochondral angiogenesis and osteogenesis [150]. Osteostatin (OST), mainly expressed in bone marrow, induces BMSCs osteogenic differentiation and promotes the proliferation, migration, and angiogenesis of Type H ECs via the HIF-1α/VEGF pathway under hypoxia conditions [153,154]. These results suggest that HIF-1α/VEGF pathway regulates angiogenesis and osteogenesis by connecting Type H ECs and osteoblasts in the skeletal system ( Figure 4).

HIF-1α/VEGF Pathway Regulates Type H Vessels in Various Bone Metabolic Disease Models
Studies have shown that the number of Type H vessels in the long bone of aging mice decreased significantly, accompanied by bone progenitor cells decrease. The osteoporosis model of ovariectomized mice showed the same phenotype [152]. Local injection of tetramethylpyrazine into the bone marrow of aging mice directly induces the formation of Type H vessels. It improves bone homeostasis by regulating the AMPK/mTOR and HIF-1α/VEGF signal pathways, preventing and treating glucocorticoid-induced osteoporosis [155]. In addition, miR-210 significantly promotes HIF-1α and VEGF expression in BMSCs dose-and-time-dependently, upregulates alkaline phosphatase (ALP) and Osterix, inhibits peroxisome proliferators-activated receptors (PPARγ), and induces BMSCs differentiating into osteoblasts, improving osteoporosis caused by estrogen deficiency [156]. Similarly, miR-497~195 cluster improves senile osteoporosis by inducing Type H vessel angiogenesis coupling with osteogenesis via Notch and HIF-1α pathways [157]. In addition, matrix metalloproteinase-2 inhibitor 1 (MMP2-I1) could induce BMSCs osteogenesis differentiation and promote Type H vessel angiogenesis through the HIF-1α signal pathway, rescuing osteonecrosis, bone defect, as well as osteoporosis [158,159] (Figure 4). matrix metalloproteinase-2 inhibitor 1 (MMP2-I1) could induce BMSCs osteogenesis differentiation and promote Type H vessel angiogenesis through the HIF-1α signal pathway, rescuing osteonecrosis, bone defect, as well as osteoporosis [158,159] (Figure 4). Low-level laser therapy (LLLT) has a positive photobiological stimulation effect on cell proliferation, angiogenesis, osteogenic differentiation, bone regeneration, and fracture healing [160][161][162]. The relevant evidence shows that laser irradiation changes the mitochondrial membrane potential, promotes the oxidation of ferrous iron by inducing ROS production, and then inhibits HIF-1α degradation by inactivating the PHD, accompanied by VEGF and transforming growth factor-β (TGF-β) expression upregulation, promoting Type H vessel formation and BMSCs osteogenic differentiation, coupling angiogenesis and osteogenesis [163][164][165] (Figure 4).

Cobalt
Co 2+ stabilizes HIF-1α by inactivating PHD in a normoxic environment, commonly used as a hypoxia inducer [80]. It has been confirmed that BMSCs treated with Co 2+ promote osteogenesis and angiogenesis in bone defect areas [85]. Co-doped HA significantly upregulates HIF-1α and VEGF in MG-63 cells, then promotes cell cycle progression and proliferation and induces osteocyte differentiation [190]. HA as a prosthesis coating triggers survival osteogenic gene signals, rescues the inhibition of osteoblasts and osteoclasts activity caused by Co 2+ and chromium (Cr) ions concentration elevation around the prosthesis through regulating the HIF-1α signal pathway and endocytic/cytoskeletal gene [191]. Co-doped BG scaffolds with different mesoporous or chemical modifications of the upregulation of VEGF, HIF-1α, and osteogenic genes in BMSCs, promote angiogenesis and osteogenic differentiation, as well as inhibit chondrogenic differentiation [86,192,193]. The hydrogel fiber scaffold composed of collagen and alginate achieves a more substantial bone repair by carrying Co 2+ and BMP2 [194]. In addition, combined organic and inorganic biomaterials doped with Co 2+ , such as collagen glycosaminoglycan (CG)-BG and calcium alginate (CA)-Nano-HA, also have an excellent ability to promote bone tissue regeneration [195,196].

Other Trace Elements
Zn commonly presents in all tissues, body fluids, and organs of the human body-more than 95% of which are present in cells and involved in the transcription of DNA [197]. Under 1% O 2 conditions, Zn 2+ inhibits HIF-1α expression, promoting migration and proliferation and delaying the senescence of BMSCs [198]. Ti scaffolds coated on Mg significantly stimulate the proliferation, adhesion, extracellular matrix mineralization, and ALP activity of MC3T3-E1 cells via the JAK1/STAT1/HIF-1α pathway, as well as improve the proliferation, adhesion, tubular formation, scratch healing and Transwell ability of human umbilical vein endothelial cells (HUVECs) through HIF-1α/VEGF pathway [199,200]. Zn/Mg co-implanted Ti scaffold (Zn/Mg-PIII) upregulates magnesium transporter 1 (MAGT1) in HUVECs, promotes Mg 2+ influx, and activates the HIF-1α/VEGF pathway, inducing angiogenesis. In addition, Zn/Mg-PIII upregulates integrin α1 and integrin β1 to promote rat BMSCs adhesion and spread, promoting Runx2, ALP, and OCN expression in BMSCs by inducing Zn 2+ and Mg 2+ to recruit into cells. Compared with traditional Ti scaffolds, the Zn/Mg-PIII enhanced bone formation, angiogenesis, and antibacterial activity [201]. The ions released from Si-based biomaterials upregulate HIF-1α. The porous TiO 2 coating doped with a small amount of Si significantly enhances HUVECs angiogenesis, while excessive Si doping will impair the vascular response [202]. Monte et al. [203] confirmed that 0.5mM, as an appropriate concentration of Si 4+ , enhanced HUVECs viability by alleviating oxidative stress damage and promoted HUVECs proliferation, migration, and angiogenesis through upregulating HIF-1α. Furthermore, mesoporous silica nanoparticles (MSN) loaded with platelet-derived growth factor BB (PDGF-BB) significantly stimulate the "HIF-1α-VEGF-Ang-1 axis" and up-regulate osteogenesis-related genes in BMSCs, inducing osteogenesis and angiogenesis [204].
These results suggest that suitable biomaterials induce BMSCs migration and attachment to the bone repair zone by stably releasing trace elements such as Cu 2+ and Co 2+ and upregulate HIF-1α/VEGF and osteogenic genes, coupling angiogenesis and osteogenesis, achieving bone regeneration ( Figure 5). nanoparticles (MSN) loaded with platelet-derived growth factor BB (PDGF-BB) significantly stimulate the "HIF-1α-VEGF-Ang-1 axis" and up-regulate osteogenesis-related genes in BMSCs, inducing osteogenesis and angiogenesis [204].
These results suggest that suitable biomaterials induce BMSCs migration and attachment to the bone repair zone by stably releasing trace elements such as Cu 2+ and Co 2+ and upregulate HIF-1α/VEGF and osteogenic genes, coupling angiogenesis and osteogenesis, achieving bone regeneration ( Figure 5). Figure 5. Application of HIF-1α in biomaterials. Organic/inorganic biomaterials can stabilize HIF-1α by doping trace elements such as copper (Cu) and Co or carrying chemical drugs such as dimethyloxalylglycine (DMOG) and DFO. The signal pathways involved include Wnt/β-catenin, ERK1/2, PI3K/AKT, and JAK1/STAT1, which upregulate downstream osteogenic and angiogenic genes.

DFO
As an iron chelating agent, DFO stabilizes HIF-1α by inhibiting PHD activity. Poly (lactic-co-glycolic acid) (PLGA) loaded with DFO significantly stimulates angiogenesis and BMSCs osteogenic differentiation, promoting osteoporotic bone defect repair [205]. DFO also effectively induces bone regeneration in true bone ceramic (TBC) scaffolds, promoting angiogenesis and segmental bone defect repair [206,207]. Titania nanotube (TNT) diameter dependently enhances BMSCs proliferation and mineralization ability. As a drug carrier loaded with DFO, TNT promotes angiogenesis and BMSCs osteogenic gene expression by activating the HIF-1α signal pathway by continuously and stably releasing DFO, then stimulates BMSCs adhesion and proliferation and affecting HUVECs growth behavior [61,208]. DFO also significantly improves the surface roughness and hydrophilicity of the polydopamine (PDOPA) membrane, which is more conducive to the attachment, proliferation, and spread of MC3T3-E1 cells and HUVECs, thus achieving bone repair [209] (Figure 5).

DMOG
DMOG, as an inhibitor of PHD, reduces HIF-1α degradation. β-TCP loaded with DMOG significantly enhances the angiogenic activity of BMSCs, thus repairing skull defects [210]. Implantation of ASCs on porous β-TCP-alginate-gelatin scaffolds containing DMOG induces angiogenesis and bone repair in rat skulls [211]. MSN slowly releases Si 4+ to activate ALP, OCN, RUNX2, and OPN expression in human BMSCs and to stimulate BMSCs osteogenic differentiation, while MSN loaded with DMOG upregulates angiogenic genes in BMSCs by stabilizing HIF-1α, coupling osteogenesis and angiogenesis, which play a role in repairing bone defects [212]. Similarly, mesoporous bioactive glass  As an iron chelating agent, DFO stabilizes HIF-1α by inhibiting PHD activity. Poly (lactic-co-glycolic acid) (PLGA) loaded with DFO significantly stimulates angiogenesis and BMSCs osteogenic differentiation, promoting osteoporotic bone defect repair [205]. DFO also effectively induces bone regeneration in true bone ceramic (TBC) scaffolds, promoting angiogenesis and segmental bone defect repair [206,207]. Titania nanotube (TNT) diameter dependently enhances BMSCs proliferation and mineralization ability. As a drug carrier loaded with DFO, TNT promotes angiogenesis and BMSCs osteogenic gene expression by activating the HIF-1α signal pathway by continuously and stably releasing DFO, then stimulates BMSCs adhesion and proliferation and affecting HUVECs growth behavior [61,208]. DFO also significantly improves the surface roughness and hydrophilicity of the polydopamine (PDOPA) membrane, which is more conducive to the attachment, proliferation, and spread of MC3T3-E1 cells and HUVECs, thus achieving bone repair [209] (Figure 5).

Application of Tissue Engineering Combined with Gene Mutation Technique to HIF-1α in Bone Defects Repair
Angiogenic and osteogenic gene upregulation in normoxic conditions can be achieved by transplanting HIF-1α-modified BMSCs into bone defect areas [217]. Studies have shown that gene mutation technology effectively improves HIF-1α activity, promoting the applica-tion in bone metabolic diseases. Compared with wild-type HIF-1α, the EVs derived from BMSCs modified by mutant HIF-1α achieve a better osteogenic and angiogenic ability, thus rescuing steroid-induced osteonecrosis of the femoral head [218]. Furthermore, β-TCP scaffold loaded the rat BMSCs-derived EVs carrying mutant HIF-1α promotes angiogenesis and BMSCs proliferation and osteogenic differentiation [219]. Ca-Mg phosphate cement (CMPC) scaffold carrying the structural active form (CA5) of HIF-1αachieves higher osteogenic activity in repairing bone defects [220,221]. The gelatin sponge (GS) loaded the BMSCs transfecting CA5 upregulates angiogenic genes in BMSCs, promoting angiogenesis in the bone defect area [222]. These results suggest that tissue engineering combined with gene mutation effectively promotes bone regeneration and may be used as an effective treatment for bone metabolism diseases.

The Role of Other Factors in Bone Homeostasis Regulation by HIF-1α
Toll-like receptor 2 (TLR2) is essential in regulating the immune response. Previous studies have shown that TLR2 activation promotes tissue angiogenesis and wound healing [223]. Furthermore, TLR2 significantly enhances HIF-1α and BMP-2 expression in BMSCs, upregulating the downstream osteogenic and angiogenic genes [224]. Epidermal growth factor (EGF) participates in proliferation, differentiation, adhesion, and survival processes. As a ligand of EGF, betacellulin (BTC) stabilizes HIF-1α, stimulating osteogenic progenitor cell proliferation while inhibiting differentiation [225,226]. As a demethylase, fat mass and obesity-associated protein (FTO) regulates the balance between adipogenic and osteogenic differentiation of BMSCs [227]. However, FTO induces BMSCs osteogenic differentiation by upregulating HIF-1α under mechanical stress conditions [228].
Icariin (ICA) is the main active component of Epimedium, which induces BMSCs migration by activating the SDF-1α/HIF-1α/CXCR4 pathway [229]. Safflower yellow (SY), as the main component of the traditional Chinese medicine safflower, promotes blood circulation and removes blood stasis [230]. In addition, SY also plays a pivotal role in osteogenesis and angiogenesis via the VHL/HIF-1α/VEGF signal pathway [231]. Curcumin, which exists in turmeric, significantly rescues hypoxia and reoxygenation (H/R) damage of BMSC through inhibiting mitochondrial ROS accumulation, which may be related to HIF-1α instability, the exchange protein activated by cAMP-1 (Epac1) and Akt activation and ERK1/2 and p38 inactivation [232]. Salidroside (SAL), as the main bioactive component of Rhodiola, significantly promotes the proliferation, migration, and angiogenesis of HUVECs through the HIF-1α/VEGF pathway and induces EC germination from the metatarsal [233]. Statins are traditional lipid-lowering drugs that promote fracture healing and bone defect repair [234][235][236]. Furthermore, simvastatin may induce BMSCs and endothelial progenitor cell migration via the HIF-1α/BMP-2 pathway, promoting bone defect healing [237].

Conclusions
Currently, the clinical treatment of bone metabolic diseases includes surgery and drug therapy. Bone transplantation in treating bone defects, joint prosthesis replacement in treating osteonecrosis of the femoral head, and plates and screws in treating fractures have achieved good results. However, it is difficult for drugs to reverse osteoporosis and prevent complications. Therefore, it is urgent to study the mechanism of bone metabolic disorder and implement effective intervention measures. With the effects of HIF-1α having been widely studied, its role in angiogenesis and bone homeostasis regulation has aroused the interest of researchers. HIF-1α regulates angiogenesis and osteoclasts and osteoblasts differentiation in various mechanisms, in which the epigenetic regulation of ncRNA also plays an important role. Based on the beneficial effects of HIF-1α coupling angiogenesis and osteogenesis and the application of biomaterials in bone metabolic diseases, new scaffold materials loaded with trace elements or drugs involved in the HIF-1α pathway may reach a better bone regeneration effect. Although HIF-1α has been widely studied in bone metabolism, there are still some problems to be explored. The bidirectional regulation of hypoxia on HIF-1α expression and the effect of HIF-1α on osteoclast differentiation have not been fully elucidated. It is necessary to study the mechanism of these opposite effects further and explore the effectiveness of HIF-1α in preventing bone metabolic diseases, laying a foundation for further clinical application.
Author Contributions: Conceptualization, L.Q. and J.T.; data curation, G.L.; writing-original draft preparation, W.C.; writing-review and editing, W.C., P.W. and L.Q.; supervision, F.Y. All authors have read and agreed to the published version of the manuscript.