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Hypoxia is a major stimulator of osteoclast formation and bone resorption
Abstract
Hypoxia is known to act as a general stimulator of cells derived from marrow precursors. We investigated the effect of oxygen tension on the formation and function of osteoclasts, the cells responsible for bore resorption, which are of promonocytic origin. Using 7- and 13-day cultures of mouse marrow cells on ivory discs, we found that reducing oxygen tension from the ambient atmospheric level of 20% by increasing the proportion of nitrogen caused progressive increases in the formation of multinucleated osteoclasts and resorption pits. Peak effects occurred in 2% oxygen, where stimulations of resorption up to 21-fold were measured. Significant stimulations of osteoclast formation and resorption were observed even in severely hypoxic cultures gassed with 0.2% oxygen. Short-term cultures of cells disaggregated from rat bones indicated that hypoxia did not alter the resorptive activity of mature osteoclasts, but reduced their survival or adherence. In 3-day organ cultures of mouse calvarial bones, exposure to 2% oxygen resulted in maximal, fivefold stimulation of osteoclast-mediated calcium release, an effect equivalent to that of prostaglandin E(2) (PGE(2)), a reference osteolytic agent. Hypoxia also caused a moderate acidosis in calvarial cultures, presumably as a result of increased anaerobic metabolism; this observation is significant because osteoclast activation is dependent on extracellular acidification. Our experiments reveal a previously-overlooked mechanism of considerable potential importance for the regulation of bone destruction. These findings may help explain the bone loss associated with a wide range of pathological states involving local or systemic hypoxia, and emphasize the importance of the vasculature in bone.
... Hypoxia was shown to be a major stimulator of osteoclast formation and bone resorption as a fourfold increase in osteoclast number and 21-fold increase in its activity was reported after exposure to 2% O 2 [123,[148][149][150]. A reduction in O 2 level to 2% increased the mean area of osteoclasts nearly 8-folds and exhibited a 13-fold increase in resorption lacunae in bone slices [151]. ...
... Osteoclasts require high amount of ATP for their bone-resorbing function, and therefore, excessive accumulation of mitochondrial ROS during low oxygen tension for longer periods may promote apoptosis and limit bone resorption [157]. A significant decrease in bone calcium content with increased osteoclast differentiation after exposure to low oxygen levels has been reported in rainbow trouts which is consistent with the report of a fivefold increase in osteoclast-mediated calcium release after exposure to 2% O 2 in a 3-day organ culture of mouse calvarial bones [148,158]. ...
... Several reports have similarly noted increased osteoclast differentiation following HIF-1α stimulation [6,164,165], while a study noted that HIF1-α inactivation through siRNA accelerated osteoclast cell fusion and HIF-1α induction moderately inhibited its differentiation [153]. In few reports, HIF-1α induction caused a small decrease in osteoclast numbers, which authors reasoned to the longer reoxygenation time compared to other studies [148][149][150][151]153]. Homozygous knockdown of PHD3 in animal models increased osteoclast formation, which was associated with increased expression of the differentiation marker NFATC1, which however, did not change the final number of osteoclasts formed but increased the final resorption activity [153,166]. ...
Deteriorated bone microarchitecture is a major health concern affecting millions worldwide, amounting to high mortality along with psychological, social, and economic burden. Hypoxia has been known to affect bone mineral metabolism in various in vitro and in vivo experiments in an inconclusive manner and only a few studies are available on natives or travelers of high altitude, pointing towards the deterioration of bone health. HIF proteins, fundamental to hypoxia signaling have also been shown to affect bone remodeling by mediating osteoblastogenesis and osteoclastogenesis but the underlying mechanism of this process is not clear. Most studies have been reported in men but only few in female, while it has been already established that estrogen plays a major role in protecting skeletal health and recent reports identify estrogen as a major player in determining bone quality in men as well. The tough terrain and lack of transport in these areas require optimal bone quality to be maintained for continuous locomotion and load-bearing capacity. The Wnt pathway is involved in load-induced bone formation and sclerostin; the inhibitor of this pathway has been reported to be regulated by both estrogen and HIF proteins. However, the hypobaric hypoxia-operated molecular mechanism regulating the bone quality and microarchitecture in both male and female is still not fully elucidated. Therefore, in this review, available literature on the bone health status under sustained hypoxic exposure focusing on the significance and crosstalk of HIF proteins, Wnt pathway, and estrogen are compiled and discussed to open new aspects of high-altitude bone health research.
... Hypoxia conditions, such as reduced oxygen partial pressure, poor oxygen diffusion, and perfusion environment, may trigger cellular hypoxic responses. Studies have demonstrated that both osteoblasts and osteoclasts are oxygen-sensing cells [14]. Pathological or environmental hypoxia can disrupt bone homeostasis, thereby affecting bone health [15,16]. ...
... However, the specific impact of hypoxia on osteoclast differentiation has been inconsistent across studies, with some reporting enhanced osteoclast differentiation and activity and others showing reduced osteoclastogenesis. Prior research has indicated that hypoxia could promote the differentiation of BMDMs in osteoclast, with the peak effect observed at 2% O 2 , resulting in a fourfold increase in the number of mature multinucleated cells and a 21-fold increase in resorptive activity [14,45]. Similar results were found for the effect of hypoxia on cat osteoclast activity [45]. ...
Osteoporosis, a prevalent metabolic bone disorder, is characterized by reduced bone density and increased fracture risk. The pathogenesis of osteoporosis is closely associated with an imbalance in bone remodeling, in which the resorption function of osteoclasts exceeds the formation function of osteoblasts. Hypoxia has been implicated in the promotion of osteoclast differentiation and the subsequent development of osteoporosis. The ubiquitin–proteasome system (UPS) and its regulatory enzymes, deubiquitinating enzymes (DUBs), play a significant role in bone homeostasis. In this study, we investigated the contribution and mechanism of Ubiquitin-specific protease 18 (USP18), a DUB, in osteoclast differentiation under hypoxic conditions. BMDMs and RAW264.7 cells were treated with RANKL to induce osteoclastogenesis and were subjected to overexpression or knockdown of USP18 under normoxic or hypoxia conditions. Osteoclast formation was assessed using TRAP staining, and the expression of osteoclast marker genes was determined using qRT-PCR. The activation of the NF-κB signaling pathway was evaluated using immunoblotting. We found that hypoxia significantly enhanced the differentiation of BMDMs and RAW264.7 cells into osteoclasts, accompanied by a notable downregulation of USP18 expression. The overexpression of USP18 inhibited RANKL-induced osteoclast differentiation, while the knockdown of USP18 promoted that process, unveiling the inhibitory effect of USP18 in osteoclastogenesis. Furthermore, the overexpression of USP18 rescued the hypoxia-induced increase in osteoclast differentiation. Mechanistic insights revealed that USP18 inhibits osteoclastogenesis by suppressing the NF-κB signaling pathway, with a potential target on TAK1 or its upstream molecules. This study indicates that hypoxia promotes osteoclast differentiation through the downregulation of USP18, which, in turn, relieves the suppression of the activation of the NF-κB signaling pathway. The USP18 emerges as a potential therapeutic target for osteoporosis treatment, highlighting the importance of the hypoxia–DUB axis in the pathogenesis of the disease.
... Majority of the adult hemoglobin is α₂ β₂ form 10 .Hemoglobin transports oxygen from the lungs to the cells of the body and also transports metabolically produced carbon dioxide and hydrogen ion from the body to the lungs 11 . Oxygen is one of the regulators of osteoclastic activity in bone resorption 12 . A change of oxygen availability is regulated by a transcription factor called hypoxia inducible factor (HIF-1). ...
... Erythropoietin stimulates the precursors of osteoclast cells and enhances bone loss 15 . Hypoxia also aggravates extracellular acidification as a result of anaerobic metabolism which activates the osteoclast cells and accelerates bone resorption 12 . ...
Background: Globally osteoporosis is a major public health concern. Various factors have influence on bone mass. Anemia is common in Bangladesh among the women of reproductive age group. This may be a contributing factor to low density of mineral of bone in young adult females. Aim: This research was performed to observe hemoglobin concentration’s association with density of bone mineral in adult females of Dhaka city. Materials and Method: This cross sectional study was performed in the Physiology Department of Dhaka Medical College, Dhaka between 2018 July and 2019 June. One hundred and twenty two female participants in the age range from 18 to 44 years were recruited for the study. The density of mineral of bone measurement was carried out for the eligible subjects after obtaining informed written consent. Data was collected and statistical analysis was done using One-way ANOVA, Bonferroni test and Pearson’s correlation coefficient (r) using SPSS for windows version 25.0. Results: The mean (± SD) hemoglobin concentration of group A, group B and group C were 9.87 ± 0.71, 11.44 ± 0.26, and 12.75 ± 0.75 g/dl respectively. The mean (± SD) bone mass density (BMD) T score of lumbar spine of group A, group B and group C were -0.70 ± 1.01, -0.13 ± 0.85, and -0.03 ± 0.92 respectively. The mean (± SD) BMD T score of femoral neck of group A, group B and group C were -0.75 ± 0.95, -0.19 ± 0.85, and -0.06 ± 0.84 respectively. We found statistically significant positive correlation (r= +0.284 and r= +0.366 respectively) between BMD T score of both lumbar spine and femoral neck with hemoglobin concentration (p=0.001 and p<0.001 respectively). Conclusion: A reduction in hemoglobin concentration may lead to reduced oxygen in blood with stimulation of proteosomal degradation ; osteoclastogenesis and inhibition of osteoblastic bone formation. These changes may cause decrease in bone mineral density. Therefore, early detection of anemia may help prevent reduction in density of bone mineral and maintain bone health. J Med Coll Women Hosp.2024;20(1):23-31
... Hypoxia conditions, such as reduced oxygen partial pressure, poor oxygen diffusion and perfusion environment, may trigger cellular hypoxic responses. Studies have demonstrated that both osteoblasts and osteoclasts are oxygen-sensing cells [14]. Pathological or environmental hypoxia can disrupt bone homeostasis, thereby affecting bone health [15,16]. ...
... Our study revealed that both sustained and transient hypoxia significantly promotes osteoclast differentiation and contributes to the pathogenesis of osteoporosis. Prior research has indicated that hypoxia could promote the differentiation BMDMs into osteoclast, with the peak effect observed at 2% O2, resulting in a fourfold increase in the number of mature multinucleated cells and a 21-fold increase in resorptive activity [14,46]. Similar results were found for the effect of hypoxia on cat osteoclast activity [46]. ...
Background: Osteoporosis, a prevalent metabolic bone disorder, is characterized by reduced bone density and increased fracture risk. The pathogenesis of osteoporosis is closely associated with an imbalance in bone remodeling, where the resorption function of osteoclasts exceeds the formation function of osteoblasts. Hypoxia has been implicated in the promotion of osteoclast differentiation and the subsequent development of osteoporosis. The ubiquitin proteasome system (UPS) and its regulatory enzymes, deubiquitinating enzymes (DUBs), play a significant role in bone homeostasis. In this study, we investigated the contribution and mechanism of USP18, a DUB, in osteoclast differentiation under hypoxic conditions. Methods: BMDMs and RAW264.7 cells were treated with RANKL to induce osteoclastogenesis and were subjected to overexpression or knockdown of USP18 under normoxic or hypoxia conditions. Osteoclast formation was assessed using TRAP staining, and the expression of osteoclast marker genes was determined by qRT-PCR. The activation of the NF-κB signaling pathway was evaluated by immunoblotting. Results: We found that hypoxia significantly enhanced the differentiation of BMDMs and RAW264.7 cells into osteoclasts, accompanied by a notable downregulation of USP18 expression. Overexpression of USP18 inhibited RANKL-induced osteoclast differentiation, while knockdown of USP18 promoted that process, unveiling the inhibitory effect of USP18 in osteoclastogenesis. Furthermore, overexpression USP18 rescued the hypoxia-induced increase in osteoclast differentiation. Mechanistic insights revealed that USP18 inhibits osteoclastogenesis by suppressing the NF-κB signaling pathway, with a potential target on TAK1 or its upstream molecules. Conclusion: Our study indicates that hypoxia promotes osteoclast differentiation through the downregulation of USP18, which in turn relieves the suppression on the activation of NF-κB signaling pathway. The USP18 emerges as a potential therapeutic target for osteoporosis treatment, highlighting the importance of the hypoxia-DUBs axis in the pathogenesis of the disease.
... A number of factors are known to affect bone quality and bone development, e.g. vitamin D levels, 25 presence of hypoxia, 26 physical activity, 21 and growth trajectory. 27 Previous reports have shown that vitamin D deficits are common among young patients with Fontan circulation. ...
... Furthermore, hypoxia has been suggested as a factor that could affect bone Cardiology in the Young development. 26 Most of the patients in our cohort were not hypoxic (<90%) at the time of the peripheral quantitative CT examination, and no associations were found between oxygen saturation and strength-strain index. However, age at total cavo-pulmonary connection surgery, which reflects time spent in hypoxia, was associated with a lower strength-strain index. ...
Objectives
Previous reports indicate bone deficits in patients with Fontan circulation. However, the consequences of these deficits on bone strength and when these changes occur are unclear.
Aim
To compare the tibial bone strength-strain index between young patients (6–19 years) with Fontan circulation and age- and sex-matched controls, and to determine strength-strain-index in subgroups of children (6–12 years) and adolescents (13–19 years) versus controls.
Method
The tibia was examined with peripheral quantitative CT. Based on the assessed data, bone strength-strain index was calculated in the lateral and anterior-posterior directions.
Results
Twenty patients with Fontan and twenty controls (mean age 13.0 ± 4.4 years; 50% females) were examined. Patients had a lower strength-strain index in the lateral direction compared to controls (808.4 ± 416.8mm ³ versus 1162.5 ± 552.1mm ³ , p = 0.043). Subgroup analyses showed no differences regarding strength-strain index in children (6–12 years) with Fontan circulation compared to controls. However, the adolescents (13–19 years) with Fontan circulation had lower strength-strain indexes in both the lateral and anterior-posterior directions compared to controls (1041.4 ± 299.8mm ³ versus 1596.4 ± 239.6mm ³ , p < 0.001, and 771.7 ± 192.4mm ³ versus 1084.9 ± 215.0mm ³ , p = 0.004). When adjusted for height, there were differences between patients (6–19 years) and controls in strength-strain indexes in both the lateral and anterior-posterior directions. In subgroup analyses, the results remained robust.
Conclusion
Young patients (6–19 years) with Fontan circulation have a lower strength-strain index in the tibia compared to controls. Subgroup analyses show that this deficit is mainly driven by the differences in adolescents (13–19 years), which might suggest that bone strength decreases with age.
... Additionally, iron affects bone through hypoxia, which is a condition of reduced oxygen supply to tissues. Hypoxia causes stimulate of bone resorption by increasing of osteoclastogenesis followed by osteogenesis [93,94]. In normoxia, prolyl hydroxylase enzyme activation is required by α-ketoglutarate, iron and molecular oxygen. ...
... A study in 2015 by Okito et al., reported that EPO stimulated bone resorption in mice [97]. In addition, stimulating osteoclast activity by acidosis leads to bone loss [93][94][95][96][97][98]. Hypoxia can lead to acidosis. ...
Iron and vitamin D deficiencies are some of the most common health problems in the world. Iron is essential in oxygen transport and participates in many enzymatic systems in the body, with important roles in vitamin D metabolism. Osteoporosis is one of the most prevalent chronic disease of the elderly in the world as well as in the Saudi population. The relationship between iron, vitamin D deficiency and bone health comes from clinical observations in iron overload patients who suffered bone loss. The opposite scenario, whether iron and vitamin D deficiencies affect bone metabolism, has not been fully addressed. This is of great interest, as this nutrient deficiency is a worldwide public health problem and at the same time osteoporosis and bone alterations are highly prevalent. The relationship between 25(OH)D and iron deficiencies with osteoporosis is unknown up to date. This review presents the current knowledge on nutritional iron and vitamin D deficiencies in bone remodeling, and discuss the link between iron and bone metabolism among postmenopausal women. Finally, it is hypothesized that chronic iron and vitamin D deficiencies induces bone resorption and risk of osteoporosis, thus complete recovery from anemia and its prevention should be promoted in order to improve quality of life including bone health. Several mechanisms are implicated; hence, further investigation on the possible impact of iron and vitamin D deficiencies on the development of osteoporosis is needed.
... During the early phase of bone defect healing, hypoxic signalling enhances osteogenic and angiogenic progenitor cell migration, osteogenesis and angiogenesis Wang et al., 2007). Other studies report on reduced osteoblast differentiation and activity (Utting et al., 2006) as well as increased osteoclastogenesis (Arnett et al., 2003) caused by oxygen deficiency. In the present study, hypoxic cultivation led to a reduced cell number of osteoblasts, osteocytes and osteoclasts in the triple cultures. ...
... These results matched with a downregulated expression of osteoclast markers CTSK and ACP5 in mouse macrophages (RAW264.7 cell line) as reported for cultivation in 2% O 2 (Ma et al., 2019). In contrast, it is known, that osteoclastogenesis of mouse bone marrow stem cells was stimulated by hypoxic cultivation (5% O 2 or 2% O 2 ) for 7 days (Arnett et al., 2003). However, the number of mature rat osteoclasts and their ability to adhere is reduced by cultivation with < 2% O 2 for 26 h without affecting the resorption activity (Arnett et al., 2003, p. 20). ...
An in vitro bone triple culture involving human primary osteoblasts, osteocytes and osteoclasts enables the investigation of bone healing factors, drugs or biomaterials in a model system for native bone tissue. The present study analyses the impact of Sr²⁺ as well as hypoxic cultivation (5% O2 content or chemically induced by Co²⁺) on bone cells. The three cell types were cultivated together in the presence of 100 µM Sr²⁺, hypoxic conditions or in the presence of 75 µM Co²⁺. After cultivation the cell types were separated and analysed on mRNA and protein level individually. In response to Sr²⁺ osteoblasts showed a downregulation of IBSP expression and a stimulation of ALP activity. Osteocyte gene marker expression of PDPN, MEPE, RANKL, OPG, osteocalcin and likewise the amount of secreted osteocalcin was reduced in the presence of Sr²⁺. Activity of osteoclast-specific enzymes TRAP and CAII was enhanced compared to the Sr²⁺ free control. Hypoxic conditions induced by both 5% O2 or a Co²⁺ treatment led to decreased DNA content of all bone cells and downregulated expression of osteoblast markers ALPL and IBSP as well as osteocyte markers PDPN, RANKL and OPG. In addition, Co²⁺ induced hypoxia decreased gene and protein expression of osteocalcin in osteocytes. In response to the Co²⁺ treatment, the TRAP gene expression and activity was increased. This study is the first to analyse the effects of Sr²⁺ or hypoxia on triple cultures with primary human bone cells. The investigated in vitro bone model might be suitable to reduce animal experiments in early stages of biomaterial and drug development.
... There is a significant association between OS and OP. Cardiovascular diseases, diabetes, and smoking are risk factors for OP that are linked to OS elevation [97,98]. ROS-mediated OS can seriously impair bone homeostasis and skeletal fragility [99,100]. ...
Biological aging is a substantial change that leads to different diseases, including osteoporosis (OP), a condition involved in loss of bone density, deterioration of bone structure, and increased fracture risk. In old people, there is a natural decline in bone mineral density (BMD), exacerbated by hormonal changes, particularly during menopause, and it continues in the early postmenopausal years. During this transition time, hormonal alterations are linked to elevated oxidative stress (OS) and decreased antioxidant defenses, leading to a significant increase in OP. Aging is significantly associated with an abnormal ratio of oxidant/antioxidant and modified nuclear factor erythroid-derived two related factor2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) pathway. OS adversely affects bone health by promoting osteoclastic (bone resorbing) activity and impairing osteoblastic (bone-forming cells). Nrf2 is critical in controlling OS and various cellular processes. The expression of Nrf2 is linked to multiple age-related diseases, including OP, and Nrf2 deficiency leads to unbalanced bone formation/resorption and a consequent decline in bone mass. Various drugs are available for treating OP; however, long-term uses of these medicines are implicated in diverse illnesses such as cancer, cardiovascular, and stroke. At the same time, multiple categories of natural products, in particular flavonoids, were proposed as safe alternatives with antioxidant activity and substantial anti-osteoporotic effects.
... Besides mechanical factors, vascularization and oxygen concentration within the PDL seem to also regulate cellular density and activation, contributing to PDL homeostasis [11,40,41]: hypoxia seems to activate osteoclastic activity, while increased perfusion and oxygen levels seem to activate osteoblastic activity. ...
Orthodontic tooth movement is the fundamental phenomenon underlying the treatment of dental malocclusions. For orthodontic treatment to be efficient and effective, the amount of force applied to the teeth for every kind of movement should be appropriately dosed, because it is associated with the risk of side effects and the treatment time. However, our knowledge of the complex cascade of events that transforms a mechanical stimulus into an ordinated bone remodeling is incomplete. Predictive theoretical numerical models could be of invaluable help in understanding the bone response to orthodontic loading and in studying the effects of complex orthodontic force systems. However, either short-term or evolutive predictive models showed a large heterogeneity of material properties and governing equations. The present review provides an outline of the physical and biochemical basis of orthodontic tooth movement with a focus around the periodontal ligament interface. The use of a standardized method for designing predictive models is advocated, and perspectives for future studies are presented.
... Moreover, oxygen is also a cosubstrate in this reaction, and under conditions of iron deficit, hypoxia is common [33]. Hypoxia-inducible factors (HIFs) have a myriad of actions and act in cells derived from bone marrow precursors, such as osteoclasts, finally resulting in bone resorption [34]. However, hypoxia is the main stimulus for erythropoietin (EPO) synthesis, and EPO regulates the formation of bone and stimulates hematopoiesis as well as osteopoiesis in the marrow [35], which could compensate for the inhibitory effect on collagen synthesis. ...
A cross-sectional study was performed in healthy adults (mean age 28 y, 67% women) whose habitual diet was an omnivore, lacto-ovo vegetarian, or vegan diet. The total sample (n = 297) was divided into two groups according to the parathormone (PTH) cut-off value of 65 pg/mL of either normal-PTH (n = 228) or high-PTH (n = 69). Vitamin D status (25-hydroxycholecalciferol, 25-OHD), PTH, and bone formation (bone alkaline phosphatase, BAP) and bone resorption (N-telopeptides of type I collagen, NTx) markers were determined. Hematocrit, erythrocytes, hemoglobin, platelets, serum iron, serum transferrin, transferrin saturation, and serum ferritin were also measured. In the total sample, 25-OHD and PTH were negatively correlated, and all subjects with high PTH presented vitamin D insufficiency (25-OHD < 75 nmol/L). High bone remodeling was observed in the high-PTH group, with significantly higher NTx and marginally higher BAP compared to the normal-PTH group. Hematocrit and ferritin were significantly lower in the high-PTH compared to the normal-PTH group. However, serum iron was higher in the high-PTH group, which was only observed for the lacto-ovo vegetarian and vegan subjects. It is concluded that both low vitamin D and low iron status are associated with elevated PTH and bone resorption, more in vegetarians than omnivores, which is in line with the hypothesis that chronic iron deficiency in adulthood mainly predisposes to osteoporosis in postmenopausal women and the elderly.
... Cigarette smoking is generally considered to down-regulate osteoblast function, resulting in suboptimal bone health and impaired formation of new bone (Pocock et al., 1989). Nicotine also increases carbon monoxide levels in arterial blood, contributing to tissue hypoxia, which induces osteoclast activity and bone resorption (Arnett et al., 2003;McDonough & Moffatt., 1999). The comorbidity of headaches with psychiatric disorders, mostly anxiety and depression, is a well-recognized phenomenon. ...
Previous studies have observed the protective effects of alcohol and the deleterious effects of smoking on chronic pain in the general population. Interestingly, alcohol and smoking were negatively associated with the severity and status of depression and anxiety. Meanwhile, depression and anxiety are associated with an increased perception of pain severity. However, little is known regarding the associations of alcohol and smoking with chronic pain in people with depression and anxiety. Depression was assessed with the Patient Health Questionnaire-9 (PHQ-9). Anxiety was measured with items based on the Generalized Anxiety Disorder-7 (GAD-7). We used logistic and liner regression models to examine associations between smoking, alcohol consumption, and 8 pain phenotypes, including (1) headache; (2) facial pain; (3) neck or shoulder pain; (4) back pain; (5) stomach or abdominal pain; (6) hip pain; (7) knee pain; and (8) multisite chronic pain. Additionally, we did subgroup analysis in the non-depression and anxiety groups and comorbid anxiety-depression group to test if there were differences. Our results found that alcohol consumption was negatively associated with headache in depression group (odds ratio (OR) = 0.71, P = 0.007), which was also found in anxiety group (OR = 0.63, P = 0.002), comorbid anxiety and depression group (OR = 0.68, P = 0.025), and non-depression and anxiety group (OR = 0.72, P = 0.002). We observed that smoking was positively associated with back pain in depression group (OR = 1.14, P = 0.016) and mild to moderate depression group (OR = 1.13, P = 0.049). Also, positive associations of smoking with hip pain were found in the anxiety group (OR = 1.37, P = 0.001) and comorbid anxiety and depression group (OR = 1.31, P = 0.022). Our research indicated that for participants with depression and anxiety, alcohol consumption was negatively associated with most of the pain phenotypes, while smoking was positively associated with most of the 8 pain phenotypes.
... We utilized inhibitors of cathepsin B (CA-074) and cathepsin L (SB 412515) in our panel, as well as inhibitors of cathepsin S (LY3000328) and cathepsin K (L-006,235). Despite the restricted tissue expression of cathepsins K and S 52-55 , hypoxia has been implicated in the regulation of cathepsin K and bone resorption [56][57][58][59] , while cathepsin S intriguingly retains activity across a broad pH range [60][61][62][63] , similar to the rabbit reticulocyte protease that cleaves HIF1αODD-GFP ( Supplementary Fig. S1). Cathepsin S inhibitor LY3000328 and Cathepsin K inhibitor L-006,235 showed negligible reduction in HIF1αODD-GFP cleavage at any concentrations tested (Fig. 4c). ...
The oxygen-labile transcription factor called hypoxia-inducible factor (HIF) is responsible for the cellular and organismal adaptive response to reduced oxygen availability. Deregulation of HIF is associated with the pathogenesis of major human diseases including cardiovascular disease and cancer. Under normoxia, the HIFα subunit is hydroxylated on conserved proline residues within the oxygen-dependent degradation domain (ODD) that labels HIFα for proteasome-mediated degradation. Despite similar oxygen-dependent degradation machinery acting on HIF1α and HIF2α, these two paralogs have been shown to exhibit unique kinetics under hypoxia, which suggests that other regulatory processes may be at play. Here, we characterize the protease activity found in rabbit reticulocytes that specifically cleaves the ODD of HIF1α but not HIF2α. Notably, the cleavage product is observed irrespective of the oxygen-dependent prolyl-hydroxylation potential of HIF1α, suggesting independence from oxygen. HIF1α M561T substitution, which mimics an evolutionary substitution that occurred during the duplication and divergence of HIF1α and HIF2α, diminished the cleavage of HIF1α. Protease inhibitor screening suggests that cysteine proteases cathepsins L and B preferentially cleave HIF1αODD, thereby revealing an additional layer of differential HIF regulation.
... Moreover, what does this mean for orthodontic treatment, which seems to require a hypoxic environment to a certain extent? [50,51] HIF-1α is discussed to enhance the bone-resorbing activity of mature osteoclasts [52] and as other studies found, this process is known to precede osteoclastogenesis [43,53]. For orthodontic tooth movement the mechanical deformation of ligament fibroblasts seems to be more important than hypoxia [43]. ...
Background
The aim of this study was to investigate the in vitro effect of the antirheumatic drug methotrexate (MTX) on biomechanically compressed human periodontal ligament fibroblasts (hPDLFs), focusing on the expression of interleukin 6 (IL-6), as its upregulation is relevant to orthodontic tooth movement.
Methods
Human PDLFs were subjected to pressure and simultaneously treated with MTX. Cell proliferation, viability and morphology were studied, as was the gene and protein expression of IL-6.
Results
Compared with that in untreated fibroblasts, IL-6 mRNA expression in mechanically compressed ligament fibroblasts was increased (two to sixfold; ****p < 0.0001). Under compression, hPDLFs exhibited a significantly more expanded shape with an increase of cell extensions. MTX with and without pressure did not affect IL-6 mRNA expression or the morphology of hPDLFs.
Conclusion
MTX has no effect on IL-6 expression in compressed ligament fibroblasts.
... Researchers have suggested that hypoxia, secondary inflammation, endothelial dysfunction, oxidative stress, sleep deprivation, and leptin resistance caused by OSA can interfere with normal bone metabolism and cause osteoporosis [9][10][11][12][13][14][15][16][17][18][19][20]. However, some researchers believe that intermittent hypoxia from OSA can stimulate the mobilization of mesenchymal stem cells and enhance the osteogenic effect in animal models [5] and that the mechanical load produced by the larger body weight of OSA patients may have a certain positive effect on bone formation [21]. ...
Background
Obstructive sleep apnea (OSA) and osteoporosis are both prevalent diseases with shared pathophysiological mechanisms and risk factors. However, the association between the two diseases is seldom studied. This study aimed to identify the link between OSA and bone metabolism.
Methods
Male participants aged 30–59-years who visited the sleep clinic were continuously recruited. Polysomnography was used to evaluate sleep and respiratory conditions. Blood samples were collected to detect metabolic, inflammatory and bone turnover indicators. High-resolution peripheral quantitative computer tomography was used to measure the non-dominant lateral radius and tibia.
Results
Ninety subjects were recruited. The cortical area (Ct.Ar) of tibia of the severe OSA group was significantly higher than that of the mild and moderate OSA groups ( P = 0.06 and P = 0.048). There were significant differences between the four groups in terms of total volumetric bone mineral density (vBMD) ( F = 2.990, P = 0.035), meta trabecular vBMD ( F = 3.696, P = 0.015), trabecular thickness (Tb.Th) ( F = 7.060, P = 0.000) and cortical thickness (Ct.Th) ( F = 4.959, P = 0.003). The mean values of the OSA groups were lower than control group. Hypopnea index and percentage of total sleep time with SpO 2 < 90% were both positively correlated with alkaline phosphatase ( R = 0.213, P = 0.044; R = 0.212, P = 0.045). Sleep efficiency was correlated with multiple indicators of the radius.
Conclusions
In non-elderly male populations, OSA patients tended to have lower vBMD, Tb.Th and Ct.Th than non-OSA patients. The negative effect of OSA may mainly affect the osteogenesis process, and is presumed to be related to sleep-related hypoxemia and sleep efficiency.
... These processes include aerobic cell metabolism, enzyme activity, collagen synthesis, and the regulation of several angiogenic genes [29]. Although widely studied in vitro [30], the in vivo effects of oxygen on the cells involved in bone healing have been underexplored. Recently, we developed a method for mapping intravascular oxygen saturation (SO 2 ) in vivo that combined multiwavelength IOS imaging with a vascular-centric estimation of optical path lengths [13]. ...
The healing of calvarial bone defects is a pressing clinical problem that involves the dynamic interplay between angiogenesis and osteogenesis within the osteogenic niche. Although structural and functional vascular remodeling (i.e., angiogenic evolution) in the osteogenic niche is a crucial modulator of oxygenation, inflammatory and bone precursor cells, most clinical and pre-clinical investigations have been limited to characterizing structural changes in the vasculature and bone. Therefore, we developed a new multimodality imaging approach that for the first time enabled the longitudinal (i.e., over four weeks) and dynamic characterization of multiple in vivo functional parameters in the remodeled vasculature and its effects on de novo osteogenesis, in a preclinical calvarial defect model. We employed multi-wavelength intrinsic optical signal (IOS) imaging to assess microvascular remodeling, intravascular oxygenation (SO2), and osteogenesis; laser speckle contrast (LSC) imaging to assess concomitant changes in blood flow and vascular maturity; and micro-computed tomography (μCT) to validate volumetric changes in calvarial bone. We found that angiogenic evolution was tightly coupled with calvarial bone regeneration and corresponded to distinct phases of bone healing, such as injury, hematoma formation, revascularization, and remodeling. The first three phases occurred during the initial two weeks of bone healing and were characterized by significant in vivo changes in vascular morphology, blood flow, oxygenation, and maturity. Overall, angiogenic evolution preceded osteogenesis, which only plateaued toward the end of bone healing (i.e., four weeks). Collectively, these data indicate the crucial role of angiogenic evolution in osteogenesis. We believe that such multimodality imaging approaches have the potential to inform the design of more efficacious tissue-engineering calvarial defect treatments.
... Numerous pathological conditions are characterized by a hypoxic microenvironment that correlates with disease progression and is frequently associated with bone loss, such as cancer and extra-skeletal conditions (e.g., obstructive pulmonary disease). The majority of these stimulating factors can create a hypoxic microenvironment, which then activates osteoclastogenesis (Arnett, 2010;Arnett et al., 2003;Dengler et al., 2014;Devraj et al., 2017;Muller et al., 2017;Vassilaki & Frakolaki, 2017) that plays a role in the formation of ECR. HIF-1 has a very short halflife due to being effectively degraded by ubiquitin in the proteasome when the oxygen condition is normal. ...
Background
The mechanism of action of root resorption in a permanent tooth can be classified as infection‐related (e.g., microbial infection) or non‐infection‐related (e.g., sterile damage). Infection induced root resorption occurs due to bacterial invasion. Non‐infection‐related root resorption stimulates the immune system through a different mechanism.
Objectives
The aim of this narrative review is to describe the pathophysiologic process of non‐infection‐related inflammatory processes involved in root resorption of permanent teeth.
Methods
A literature search on root resorption was conducted using Scopus (PubMed and Medline) and Google Scholar databases to highlight the pathophysiology of bone and root resorption in non‐infection‐related situations. The search included key words covering the relevant category. It included in vitro and in vivo studies, systematic reviews, case series, reviews, and textbooks in English. Conference proceedings, lectures and letters to the editor were excluded.
Results
Three types of root resorption are related to the non‐infection mechanism of action, which includes surface resorption due to either trauma or excessive orthodontic forces, external replacement resorption and external cervical resorption. The triggers are usually damage associated molecular patterns and hypoxia conditions. During this phase macrophages and clastic cells act to eliminate the damaged tissue and bone, eventually enabling root resorption and bone repair as part of wound healing.
Discussion
The resorption of the root occurs during the inflammatory phase of wound healing. In this phase, damaged tissues are recognized by macrophages and neutrophiles that secrete interlaukines such as TNF‐α, IL‐1, IL‐6, IL‐8. Together with the hypoxia condition that accelarates the secretion of growth factors, the repair of the damaged perioduntiom, including damaged bone, is initiated. If the precementum and cementoblast are injured, root resorption can occur.
Conclusions
Wound healing exhibits different patterns of action that involves immune stimulation in a bio‐physiological activity, that occurs in the proper sequence, with overlapping phases. Two pathologic conditions, DAMPs and hypoxia, can activate the immune cells including clastic cells, eliminating damaged tissue and bone. Under certain conditions, root resorption occurs as a side effect.
... Tissue hypoxia promotes osteoclastic activity. [17][18][19] The hyperactivation of the renin-angiotensin-aldosterone system, a key mechanism in the pathophysiology of HF, increases calcium and magnesium excretion and oxidative stress, which contribute to the elevation of PTH levels and the consequent potentiation of bone resorption. [1,2,20,21] Functional limitations and immobility caused by HF also generate bone loss. ...
Background:
This study aimed to evaluate the bone mineral density (BMD), trabecular bone score (TBS), and fracture history of middle-aged patients hospitalized for heart failure (HF), as well as analyze the association of these factors with cardiometabolic parameters and muscle strength.
Methods:
A cross-sectional study with patients aged 40 to 64 years hospitalized for HF was performed. Dual energy X-ray absorptiometry was performed to obtain BMD and TBS. Fracture history, handgrip strength (HGS), and clinical and laboratory cardiometabolic parameters of the patients were evaluated.
Results:
Altogether, 109 patients were evaluated (female 50.5%). Medians and interquartile ranges for age and length of hospital stay were 58.0 (53.0-61.0) years and 20.0 (11.0-32.0) days, respectively. Osteoporosis was observed in 15.6% of the patients, low TBS was observed in 22.8%, and 6 patients had a history of fragile fracture. No differences between the sexes regarding BMD (p=0.335) or TBS (p=0.736) classifications were observed. No association was observed between low BMD and HF classification (p>0.05) regarding the ejection fraction, ischemic etiology, or New York Heart Association Functional Classification. However, there was a significant association between high serum parathyroid hormone (PTH) and the presence of osteoporosis (62.5 [37.2-119.0] pg/mL vs. 34.2 [25.0-54.1] pg/mL; p=0.016). There was a negative correlation between serum PTH and TBS (r=-0.329, p=0.038) and a higher frequency of reduced HGS in patients with low TBS (92.3% vs. 50.0%; p=0.009).
Conclusions:
We found relevant frequencies of osteoporosis and bone microarchitecture degradation in middle-aged patients with HF, which were related to high serum PTH concentrations.
... Concerning the possible biological mechanisms between Hb and BMD, the bone-derived hormone fibroblast growth factor 23 (FGF-23), mainly produced by osteoblasts and osteocytes, can regulate erythropoiesis and may be an important link between bone metabolism and erythropoiesis (23). Besides, another study proposed that extracellular acidification and oxidative stress under hypoxemia caused by anemia could increase bone resorption and lead to lower BMD (24). Nevertheless, a prospective longitudinal study from United States failed to show the association between Hb level and lumbar spine or total hip BMD in the community-dwelling older individuals (25). ...
Introduction
In the elder population, both low hemoglobin (Hb)/anemia and osteoporosis (OP) are highly prevalent. However, the relationship between Hb and OP is still poorly understood. This study was to evaluate the correlation between Hb and OP in Chinese elderly population.
Methods
One thousand and sisty-eight individuals aged 55–85 years were enrolled into this cross-sectional study during June 2019–November 2019. Data on the demographics and clinical characteristics were recorded. Detections of complete blood count, liver/kidney function, glucose metabolism and lipid profile, and thoracolumbar X-ray were performed, and bone mineral density (BMD) at lumbar spine 1–4, femur neck, and total hip was measured by dual-energy X-ray absorptiometry (DXA). Univariate and multivariate linear regression analyses were employed to evaluate the correlation between Hb with BMD T-score. Logistic regression analysis was performed to access the correlation between different Hb levels and the odds ratio (OR) for OP.
Results
Compared with non-OP group, OP patients had lower level of Hb. Univariate linear regression analysis indicated Hb level was positively related to the BMD of lumbar spine 1–4, femur neck and total hip, and this relationship remained after adjusting confounding variables [gender, age, body mass index (BMI), diabetes mellitus (DM) and morphological vertebral fracture]. Logistic regression analysis showed the ORs for OP decreased with the increase of Hb. Compared with the subjects with the lowest quartile of Hb, the OR for OP in the highest quartile group was 0.60 (0.41–0.89) after adjusting for gender, age and BMI, and the OR for OP was 0.62 (0.41–0.92) after further adjustment for gender, age, BMI, DM, and lipid indexes.
Discussion
In conclusion, Lower Hb level is related to lower BMD in the elderly population. However, whether Hb level could be used to predict the risk of OP needs to be further determined in more longitudinal clinical studies.
... Furthermore, in humans, the resultant poor esthetics and function may negatively impact mental health and wellbeing [46]. Altered periodontal structures and microenvironment have been proposed as contributing factors of TR [47,48] yet the factors that activate osteoclasts/odontoclasts and draw them to root surfaces remain unknown. Our study found that fGMSCTR create (Fig. 4K), and NRF2 (Fig. 4L). ...
Gingiva-derived mesenchymal stromal cells (GMSCs) are multipotent cells characterized by multilineage differentiation potential, proliferative expansion, and unique immunomodulatory ability, making them attractive as a new treatment of periodontal regeneration. In this study, GMSCs obtained from the gingiva of healthy cats (HE) as well as from cats affected by tooth resorption (TR) were isolated and characterized. Feline GMSCs (fGMSCs) from HE patients exhibited fibroblast-like morphology, developed cellular body, specific growth pattern, high expansion, and proliferative potential as well as reduced senescence signature. fGMSCs demonstrated high s-100 and IL-10 positive cells, while simultaneously having low activity of IL-1. Moreover, high activity of ki-67 combined with reduced senescence markers were noted. In comparison, GMSCs from cats with TR exhibited enlarged nuclei and flat, irregular shape along with increased expression of CD44, s-100 and CD45 and downregulation of CD73. GMSCs from TR cats showed lower ability to form colonies, increased incidence of apoptosis, higher number of senescent cells, and reduced cell migration. Upregulation of pro-inflammatory cytokines was also noted in the TR group along with lower expression of mTOR and miR-17 and upregulation of miR-378. Mitochondrial dynamics, biogenesis and antioxidant properties are also negatively impacted in this group. Collectively, our findings suggest that GMSCs isolated from the gingiva of cats affected with TR have deteriorated functionality caused by impaired proliferation and growth and possibly mediated via mitochondrial dysfunction. fGMSCs or their EV’s should be further investigated for their role in the pathophysiology of TR in cats.
Graphical Abstract
... This number aligns with prior estimates from smaller studies in a different era, which have reported fracture rates as high as 50% following lung transplantation (25,46). Explanations for the high rate of bone disease include frequent and chronic glucocorticoid use for pulmonary disease, low BMI, relative immobility, kidney disease, and metabolic complications of advanced lung disease (e.g., hypoxia) (52). We speculate that glucocorticoids may add to risk of pathologic and traumatic fractures, which we found to be associated with low BMD and frailty. ...
Fractures negatively impact quality of life and survival. We hypothesized that recipient frailty score and genetic profile measured before transplant would predict risk of fracture after lung transplant. We conducted a retrospective cohort study of bone mineral density (BMD) and fracture among lung transplant recipients at a single center. The association between predictors and outcomes were assessed by multivariable time-dependent Cox models or regression analysis. Among the 284 participants, osteoporosis and fracture were highly prevalent. Approximately 59% of participants had posttransplant osteopenia, and 35% of participants developed at least 1 fracture. Low BMD was associated with a polygenic osteoporosis risk score, and the interaction between genetic score and BMD predicted fracture. Pretransplant frailty was associated with risk for spine and hip fracture, which were not associated with chronic lung allograft dysfunction or death. Chest fractures were the most frequent type of fracture and conferred a 2.2-fold increased risk of chronic lung allograft dysfunction or death (time-dependent P < .001). Pneumonia, pleural effusions, and acute rejection frequently occurred surrounding chest fracture. Pretransplant frailty and recipient genotype may aid clinical risk stratification for fracture after transplant. Fracture carries significant morbidity, underscoring the importance of surveillance and osteoporosis prevention.
... Along with modulating osteoblastogenesis, hypoxia is also a crucial inducer of osteoclastogenesis in both murine and human cell culture systems [58]. Previously reported studies showed that low oxygen tension (2%) enhances the differentiation of osteoclasts and bone resorption, whereas hyperoxia reduces osteoclastogenesis [59]. ...
Renewing interest in the study of intermediate metabolism and cellular bioenergetics is brought on by the global increase in the prevalence of metabolic illnesses. Understanding of the mechanisms that integrate energy metabolism in the entire organism has significantly improved with the application of contemporary biochemical tools for quantifying the fuel substrate metabolism with cutting-edge mouse genetic procedures. Several unexpected findings in genetically altered mice have prompted research into the direction of intermediate metabolism of skeletal cells. These findings point to the possibility of novel endocrine connections through which bone cells can convey their energy status to other metabolic control centers. Understanding the expanded function of skeleton system has in turn inspired new lines of research aimed at characterizing the energy needs and bioenergetic characteristics of these bone cells. Bone-forming osteoblast and bone-resorbing osteoclast cells require a constant and large supply of energy substrates such as glucose, fatty acids, glutamine, etc., for their differentiation and functional activity. According to latest research, important developmental signaling pathways in bone cells are connected to bioenergetic programs, which may accommodate variations in energy requirements during their life cycle. The present review article provides a unique perspective of the past and present research in the metabolic characteristics of bone cells along with mechanisms governing energy substrate utilization and bioenergetics. In addition, we discussed the therapeutic inventions which are currently being utilized for the treatment and management of bone-related diseases such as osteoporosis, rheumatoid arthritis (RA), osteogenesis imperfecta (OIM), etc., by modulating the energetics of bone cells. We further emphasized on the role of GUT-associated metabolites (GAMs) such as short-chain fatty acids (SCFAs), medium-chain fatty acids (MCFAs), indole derivates, bile acids, etc., in regulating the energetics of bone cells and their plausible role in maintaining bone health. Emphasis is importantly placed on highlighting knowledge gaps in this novel field of skeletal biology, i.e., “Osteometabolism” (proposed by our group) that need to be further explored to characterize the physiological importance of skeletal cell bioenergetics in the context of human health and bone related metabolic diseases.
... In the present study, hypoxia was reported in all patients who suffered from the elevation of IL-6-induced cytokines storm. Of course, hypoxia is a major stimulator of bone resorption, inducing osteoclastogenesis, which is later followed by osteoblastogenesis [37,38]. The noticed acidosis resulting from hyponatremia resulting from hyperventilation may have an impact on feeling pain and osteoporosis where metabolic or local acidosis can induce osteoclast activation and bone loss [39]. ...
By 23 rd August 2020, 23,057,288 confirmed cases with SARS-CoV-2 that causes 800,906 deaths worldwide. The severe illness and high mortality are attributed to the dysregulation of hematopoiesis accompanied by cytokines storm. The severe illness and high mortality rate confirm the failure of the conventional strategy in the treatment of COVID19 patients. Also, lack of a specific vaccine or therapies target for SARS-CoV-2 infection, the drug repositioning in the treatment of COVID19 patients is the only opportunity to face this pandemic. Thus, we highlight the implication of a novel strategy of treatment based on the inhibition of IL-6, the major player in a cytokine storm, using Tocilizumab, in combination with micronutrients including Zinc, Selenium, vitamin C, and Glutathione. Application of Tocilizumab in infectious diseases is considered as a new treatment protocol since its usual uses are in autoimmune diseases. In the present study, we analyze the data for COVID19 patients who showed cytokine storm due to elevated level of IL-6 and suffered from severe illness, fever, ARDS, and transferred to ICU in Armed Forces Hospital. We found that Tocilizumab plus micronutrients improves the clinical outcomes in these critical cases of COVID19 patients. Statistical analyses of biochemical parameters including complete blood count to evaluate the efficacy of this combined therapy on counteracting the effect of SARS-CoV-2 on the differential of blood cell ratios, pro-inflammatory parameters (CRP, D-dimer, and ferritin), liver, cardiac, and kidney function were carried out. Finally, based on the success of Tocilizumab, an IL-6 inhibitor, in the treatment of COVID19 patients, we recommend using Cosentyx that is an inhibitor of IL-17, a partner of IL-6 in the inflammation process.
... Hypoxia-inducible factor (HIF) is a major transcription factor that induces cellular responses to hypoxic conditions (18). It is composed of a subunit regulated by the oxygen content [i.e., hypoxia-induced factor 1 α (HIF1α)], and a subunit of sustained expression [i.e., hypoxia-induced factor 1 beta (HIF1β)], which play pivotal roles in bone formation (19)(20)(21)(22). Other studies have shown that MAPKs and HIF1α influence each other (23)(24)(25); however, the effect of HIF1α on MAPKs in regulating osteoclast differentiation and activation is unknown. ...
Background:
Hypoxia (low-oxygen tension) and excessive osteoclast activation are common conditions in many bone loss diseases, such as osteoporosis, rheumatoid arthritis (RA), and pathologic fractures. Hypoxia-inducible factor 1 alpha (HIF1α) regulates cellular responses to hypoxic conditions. However, it is not yet known how HIF1α directly affects osteoclast differentiation and activation. This study sought to. explore the effects of HIF1α on osteoclast differentiation and it's molecular mechanisms.
Methods:
L-mimosine, a prolyl hydroxylase (PHDs) domain inhibitor, was used to stabilize HIF1α in normoxia. In the presence of receptor activator of nuclear factor-kB (NF-kB) ligand (RANKL), RAW264.7 cells were cultured and stimulated by treatment with L-mimosine at several doses to maintain various levels of intracellular HIF1α. The multi-nucleated cells were assessed by a tartrate-resistant acid phosphatase (TRAP) and F-actin ring staining assays. The osteoclast-specific genes, such as Cathepsin K, β3-Integrin, TRAP, c-Fos, nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), and matrix metallo-proteinase 9 (MMP9), were analyzed by real time-polymerase chain reaction (RT-PCR). The expression of relevant proteins was analyzed by Western blot.
Results:
L-mimosine increased the content of intracellular HIF1α in a dose-dependent manner, which in turn promoted RANKL-induced osteoclast formation and relevant protein expression by upregulating the mitogen-activated protein kinase (MAPK) pathways.
Conclusions:
Our findings suggest that HIF1α directly increases the osteoclast differentiation of RANKL-mediated RAW264.7 cells in vitro by upregulating the MAPK pathways.
... Regarding the possible mechanism of the association between OSAHS and decreased BMD: 1. OSAHS may lead to a state of vitamin D deficiency and induce secondary hyperparathyroidism, which may lead to bone demineralization and decreased BMD (34); 2. Hypoxia is closely related to changes in bone turnover, and recent in vitro studies have shown that lower nighttime oxygen levels are a feature of OSAHS, while hypoxia promotes osteoclast formation and activity while inhibiting osteoblast function, thus determining bone resorption (35,36). ...
Objective
This study is undertaken to explore the relationship between obstructive sleep apnea-hypopnea syndrome (OSAHS) and osteoporosis, including the relationship between OSAHS and osteoporosis incidence, lumbar spine bone mineral density (BMD), and lumbar spine T-score.
Method
Cochrane Library, PubMed, Embase, Web of Science, and other databases are searched from their establishment to April 2022. Literature published in 4 databases on the correlation between OSAHS and osteoporosis,lumbar spine BMD,lumbar spine T-score is collected. Review Manager 5.4 software is used for meta-analysis.
Results
A total of 15 articles are selected, including 113082 subjects. Compared with the control group, the OSAHS group has a higher incidence of osteoporosis (OR = 2.03, 95% CI: 1.26~3.27, Z = 2.90, P = 0.004), the lumbar spine BMD is significantly lower (MD = -0.05, 95% CI: -0.08~-0.02, Z = 3.07, P = 0.002), and the lumbar spine T-score is significantly decreased (MD = -0.47, 95% CI: -0.79~-0.14, Z = 2.83, P = 0. 005).
Conclusion
Compared with the control group, the OSAHS group has a higher incidence of osteoporosis and decreased lumbar spine BMD and T-score. In order to reduce the risk of osteoporosis, attention should be paid to the treatment and management of adult OSAHS, and active sleep intervention should be carried out.
... 14 HF is one of the several cardiovascular disorders that can cause a certain degree of tissue hypoxia. 14,15 Hypoxia causes a progressive increase in osteoclastogenesis and resorption in the bone tissue, which is one of the possible mechanisms that contribute to the development of osteoporosis in individuals with HF. 16 Hyperactivation of the renin-angiotensin-aldosterone system (RAAS) is the central feature in the pathophysiology of HF. RAAS acts as a compensatory mechanism in response to reduced cardiac function, which in the long run activates signalling pathways associated with myocyte hypertrophy and apoptosis, thus feeding a vicious cycle that culminates in myocardial function deterioration. ...
Heart failure (HF) is a prevalent clinical syndrome that causes significant physical limitations. Osteoporosis is also an important cause of loss of functionality, and it mainly affects women. There are several reports linking HF and osteoporosis, and both share risk factors. Most of the data available so far point to bone fragility as a consequence of HF, and several mechanisms have been identified to explain this relationship. Among the proposed pathophysiological mechanisms are the hyperactivation of the renin-angiotensin-aldosterone system and the increase in parathyroid hormone, functional limitation, production of inflammatory mediators and the use of drugs for HF. The role of osteoprotegerin has gained attention owing to its cardiovascular and skeletal effects, its observed deficiency during the postmenopausal period along with its compensatory increases in HF and severe osteoporosis. The objective of this review was to perform a literature search for the main evidence on skeletal impairment in HF, with emphasis on women. As for epidemiological studies, we selected data from 3 meta-analyses and 20 individual observational studies, which together showed the interrelationship between the two clinical conditions in terms of both decreased bone density and increased fracture risk. In conclusion, HF and osteoporosis are interrelated conditions mediated by complex pathophysiological mechanisms which may be more relevant for postmenopausal women, considered to be a vulnerable population for both cardiovascular diseases and bone fragility.
... It has been reported that hypoxia could reduce osteoblast matrix mineralization and bone formation due to the inhibition of the differentiation and activation of osteoblasts, although the results are contradictory [15,16]. In addition, hypoxia has also been associated with increased osteoclastogenesis and the resorptive capacity of osteoclasts [17,18]. The inhibition of osteoblast differentiation under conditions of hypoxia is associated with a reduced expression of Runx2 and Sp7, factors which are involved in osteoblast differentiation from multipotent mesenchymal cells [19][20][21]. ...
Hypoxia may be associated with alterations in bone remodeling, but the published results are contradictory. The aim of this study was to characterize the bone morphometry changes subject to hypoxia for a better understanding of the bone response to hypoxia and its possible clinical consequences on the bone metabolism. This study analyzed the bone morphometry parameters by micro-computed tomography (μCT) in rat and guinea pig normobaric hypoxia models. Adult male and female Wistar rats were exposed to chronic hypoxia for 7 and 15 days. Additionally, adult male guinea pigs were exposed to chronic hypoxia for 15 days. The results showed that rats exposed to chronic constant and intermittent hypoxic conditions had a worse trabecular and cortical bone health than control rats (under a normoxic condition). Rats under chronic constant hypoxia were associated with a more deteriorated cortical tibia thickness, trabecular femur and tibia bone volume over the total volume (BV/TV), tibia trabecular number (Tb.N), and trabecular femur and tibia bone mineral density (BMD). In the case of chronic intermittent hypoxia, rats subjected to intermittent hypoxia had a lower cortical femur tissue mineral density (TMD), lower trabecular tibia BV/TV, and lower trabecular thickness (Tb.Th) of the tibia and lower tibia Tb.N. The results also showed that obese rats under a hypoxic condition had worse values for the femur and tibia BV/TV, tibia trabecular separation (Tb.Sp), femur and tibia Tb.N, and BMD for the femur and tibia than normoweight rats under a hypoxic condition. In conclusion, hypoxia and obesity may modify bone remodeling, and thus bone microarchitecture, and they might lead to reductions in the bone strength and therefore increase the risk of fragility fracture.
... The findings of the present study could be relevant to clinical practice. In contrast to sustained hypoxia, which has been shown to have negative effects on bone metabolism (Arnett et al., 2003), moderate intermittent hypoxia combined with non-pharmacological treatments (such as exercise) has been considered a safe and non-invasive strategy (Bayer et al., 2017) and well tolerated by older adults (Liu et al., 2020). Moreover, it might achieve adaptations with less physical stress, which could be interesting to an aging population with pathologies or risk of injury (Pramsohler et al., 2017). ...
Purpose:
Aging leads to gradual irreversible decline in bone mass. As adherence to pharmacological treatment is poor, hypoxia combined with strength training has been suggested for therapeutic benefit for clinical populations. The present study investigated the effects of normobaric cyclic hypoxic exposure combined with resistance circuit training on bone of older adults.
Methods:
Healthy older adults (n = 50) were randomly assigned to a (1) control group (CON; n = 20), who were instructed to continue with their normal daily activities, (2) a group that performed resistance training in normoxia (RTN; n = 17) and (3) a group that performed resistance training in hypoxia (RTH; n = 13). During 24 weeks, RTH group performed resistance training with elastic bands under normobaric hypoxic conditions (16.1 % FiO2). A session of both exercise groups included nine exercises of several body areas with a structure of 3 sets × 12-15 repetitions per exercise, with a 1-minute rest between sets. Bone mineral density (g·cm-2) was measured using dual-energy X-ray absorptiometry. Bone turnover markers of formation (N-terminal propeptide of type I procollagen; PINP) and resorption (C-terminal telopeptide of type I collagen; bCTX) were analysed with enzyme-linked immunosorbent assay (ELISA) microplate reader.
Results:
Values of bCTX and bCTX/PINP significant decreased in RTN (bCTX: 47.79 %; p = 0.002; bCTX/PINP: 61.43 %; p = 0.007) and RTH (bCTX: 59.09 %; p = 0.001; bCTX/PINP: 62.61 %; p = 0.003) groups compared with CON group. Change in bone mineral density was not significantly different between groups. Based on clinically significant change, 23 % of the participants in the RTH group reached this value for femoral neck and trochanter bone mineral density (vs 0 % and 6 % of the RTN group, respectively).
Conclusions:
24-Weeks of normobaric cyclic hypoxic exposure combined with resistance circuit training has potential to generate positive effects on bone in older adults.
Trial registration number:
NCT04281264 (date of registration: February 24, 2020).
... The presence of MNGCs has also been suggested to cause chronic inflammation leading to failed osteogenesis and increased osteoclastic differentiation and activity [74]. Increased HIF1α is reported to regulate inflammation and polarize macrophages towards the pro-healing M2 phenotype via NF-κß [94][95][96][97][98]. Further, HIF1α is a positive regulator of bone formation, increasing osteoblastogenesis, enhancing bone defect healing in vivo [99][100][101], while selectively downregulating osteoclastogenesis [102][103][104], thus favouring bone deposition. Our findings reveal both nanozymes significantly upregulated HIF1α 24 h following exposure to IR. Das and colleagues [57] demonstrated that CeONPs activate HIF1α through modulation of intracellular O 2 levels; first extracting O 2 and then liberating it in the catalytic cycle: CeO 2-x + 0.5xO 2 ⇆ CeO 2 . ...
The disability, mortality and costs due to ionizing radiation (IR)-induced osteoporotic bone fractures are substantial and no effective therapy exists. Ionizing radiation increases cellular oxidative damage, causing an imbalance in bone turnover that is primarily driven via heightened activity of the bone-resorbing osteoclast. We demonstrate that rats exposed to sublethal levels of IR develop fragile, osteoporotic bone. At reactive surface sites, cerium ions have the ability to easily undergo redox cycling: drastically adjusting their electronic configurations and versatile catalytic activities. These properties make cerium oxide nanomaterials fascinating. We show that an engineered artificial nanozyme composed of cerium oxide, and designed to possess a higher fraction of trivalent (Ce³⁺) surface sites, mitigates the IR-induced loss in bone area, bone architecture, and strength. These investigations also demonstrate that our nanozyme furnishes several mechanistic avenues of protection and selectively targets highly damaging reactive oxygen species, protecting the rats against IR-induced DNA damage, cellular senescence, and elevated osteoclastic activity in vitro and in vivo. Further, we reveal that our nanozyme is a previously unreported key regulator of osteoclast formation derived from macrophages while also directly targeting bone progenitor cells, favoring new bone formation despite its exposure to harmful levels of IR in vitro. These findings open a new approach for the specific prevention of IR-induced bone loss using synthesis-mediated designer multifunctional nanomaterials.
... Although no clear mechanism of the relationship between hemoglobin levels and osteoporosis has been established, there are several possible explanations. First, on the one hand, relative hypoxia caused by decreased hemoglobin may increase the differentiation and activity of osteoclasts by increasing the expression of hypoxia-inducible factors and osteoclast-specific factors and the formation of extracellular acidic environment, resulting in increased bone resorption 29,30,[34][35][36][37] On the other hand, the effects of hypoxia on osteoblasts, including inhibition of proliferation and differentiation and alteration of bone mineralization, suppress bone formation. 38 Second, we learned that the interaction between bone cells and hematopoietic cells has been observed in animal models. ...
Purpose:
To explore the relationship between hemoglobin levels and osteoporosis and the risk factors for osteoporosis in patients with type 2 diabetes mellitus (T2DM).
Patients and methods:
A cross-sectional study was conducted in 495 T2DM adults. Medical data were collected from electronic medical records.
Results:
T2DM patients with osteoporosis had significant lower hemoglobin levels (P < 0.001). Spearman correlation analysis and logistic regression analysis showed that age, female, body mass index (BMI), smoking, drinking and hemoglobin levels were significantly associated with osteoporosis in T2DM patients (all P < 0.05). After adjustment for BMI, diabetic duration, estimated glomerular filtration rate (eGFR), smoking and drinking, a significant association between hemoglobin levels and osteoporosis was observed in T2DM males aged 50 years and older (odds ratio [OR] = 0.978, 95% confidence interval [CI]: 0.958-0.998, P = 0.030). Compared to Q3 groups with normal hemoglobin levels, Q1 groups with anemia had an odd of osteoporosis increased 2.9-fold in T2DM men after adjustment for age, BMI, diabetic duration, eGFR, smoking and drinking (P = 0.032).
Conclusion:
Hemoglobin levels were associated with the presence of osteoporosis in T2DM men, especially in those aged 50 years and older.
... The presence of MNGCs has also been suggested to cause chronic inflammation leading to failed osteogenesis and increased osteoclastic differentiation and activity [74]. Increased HIF1α is reported to regulate inflammation and polarize macrophages towards the pro-healing M2 phenotype via NF-κß [94][95][96][97][98]. Further, HIF1α is a positive regulator of bone formation, increasing osteoblastogenesis, enhancing bone defect healing in vivo [99][100][101], while selectively downregulating osteoclastogenesis [102][103][104], thus favouring bone deposition. Our findings reveal both nanozymes significantly upregulated HIF1α 24 h following exposure to IR. Das and colleagues [57] demonstrated that CeONPs activate HIF1α through modulation of intracellular O 2 levels; first extracting O 2 and then liberating it in the catalytic cycle: CeO 2-x + 0.5xO 2 ⇆ CeO 2 . ...
The disability, mortality and costs due to ionizing radiation (IR)-induced osteoporotic
bone fractures are substantial and no effective therapy exists. Ionizing radiation
increases cellular oxidative damage, causing an imbalance in bone turnover that is
primarily driven via heightened activity of the bone-resorbing osteoclast. We
demonstrate that rats exposed to sublethal levels of IR develop fragile, osteoporotic
bone. At reactive surface sites, cerium ions have the ability to easily undergo redox
cycling: drastically adjusting their electronic configurations and versatile catalytic
activities. These properties make cerium oxide nanomaterials fascinating. We show
that an engineered artificial nanozyme composed of cerium oxide, and designed to
possess a higher fraction of trivalent (Ce 3+ ) surface sites, mitigates the IR-induced
loss in bone area, bone architecture, and strength. These investigations also
demonstrate that our nanozyme furnishes several mechanistic avenues of protection
and selectively targets highly damaging reactive oxygen species, protecting the rats
against IR-induced DNA damage, cellular senescence, and elevated osteoclastic
activity in vitro and in vivo . Further, we reveal that our nanozyme is a previously unreported key regulator of osteoclast formation derived from macrophages while also
directly targeting bone progenitor cells, favoring new bone formation despite its
exposure to harmful levels of IR in vitro . These findings open a new approach for the
specific prevention of IR-induced bone loss using synthesis-mediated designer
multifunctional nanomaterials.
... Furthermore, it is a crucial transcription factor for osteoclast differentiation [17,18]. Moreover, recent evidence suggests that a hypoxic environment and HIF-1α are involved in periodontal inflammatory responses [19][20][21], angiogenesis in mesenchymal stem cells [22], osteoclast formation [23], and the activation of several microRNAs (miRNAs) implicated in essential cell functions and epigenetic modifications [24]. In addition, we have shown that NFAT5 participates in the hypoxia response, but it is independent of HIF-1α [25] and switches the upregulation of inducible-form nitric oxidases synthase by hypoxia [26]. ...
Hypoxia associated with inflammation are common hallmarks observed in several diseases, and it plays a major role in the expression of non-coding RNAs, including microRNAs (miRNAs). In addition, the miRNA target genes for hypoxia-inducible factor-1α (HIF-1α) and nuclear factor of activated T cells-5 (NFAT5) modulate the adaptation to hypoxia. The objective of the present study was to explore hypoxia-related miRNA target genes for HIF-1α and NFAT5, as well as miRNA-20a, miRNA-30e, and miRNA-93 expression in periodontitis versus healthy gingival tissues and gingival mesenchymal stem cells (GMSCs) cultured under hypoxic conditions. Thus, a case-control study was conducted, including healthy and periodontitis subjects. Clinical data and gingival tissue biopsies were collected to analyze the expression of miRNA-20a, miRNA-30e, miRNA-93, HIF-1α, and NFAT5 by qRT-PCR. Subsequently, GMSCs were isolated and cultured under hypoxic conditions (1% O2) to explore the expression of the HIF-1α, NFAT5, and miRNAs. The results showed a significant upregulation of miRNA-20a (p = 0.028), miRNA-30e (p = 0.035), and miRNA-93 (p = 0.026) in periodontitis tissues compared to healthy gingival biopsies. NFAT5 mRNA was downregulated in periodontitis tissues (p = 0.037), but HIF-1α was not affected (p = 0.60). Interestingly, hypoxic GMSCs upregulated the expression of miRNA-20a and HIF-1α, but they downregulated miRNA-93e. In addition, NFAT5 mRNA expression was not affected in hypoxic GMSCs. In conclusion, in periodontitis patients, the expression of miRNA-20a, miRNA-30e, and miRNA-93 increased, but a decreased expression of NFAT5 mRNA was detected. In addition, GMSCs under hypoxic conditions upregulate the HIF-1α and increase miRNA-20a (p = 0.049) expression. This study explores the role of inflammatory and hypoxia-related miRNAs and their target genes in periodontitis and GMSCs. It is crucial to determine the potential therapeutic target of these miRNAs and hypoxia during the periodontal immune–inflammatory response, which should be analyzed in greater depth in future studies.
... Notably, hypoxia affected the function and formation of osteoclasts, the cells responsible for bone resorption, due to an increase extracellular anaerobic metabolism, causing osteoclast activation and stimulating osteoclast formation and resorption, leading to bone loss. 37,38 However, our study did not find an independent association between cyanosis and low BMD, similar to two recent studies in adults with complex CHD. 6 ...
Background and Aims
Recent studies have highlighted the increased risk of low bone mineral density (BMD) in adults with cardiovascular disease. However, little is known about BMD in adults with congenital heart disease (CHD), particularly in developing countries. We hypothesized that factors related to BMD would lead to a high prevalence of low BMD in adults with CHD. This study aimed to determine the prevalence of low BMD and its related factors in Vietnamese adults with CHD.
Methods
We conducted a cross‐sectional study of 73 adults diagnosed with CHD in Vietnam. Low BMD was classified based on their site‐specific Z‐scores and T‐scores at the posteroanterior lumbar spine and left proximal femur. Logistic regression analyses were performed to evaluate factors related to low BMD.
Results
Low BMD was confirmed in one‐third of the adults with CHD. There were trends of more bone loss in certain parts of the body than in others, with the prevalence of low BMD at the sites of the lumbar vertebrae (L1‒L4) and left proximal femur (femoral neck, trochanteric femur, and intertrochanteric area) of 43.9%, 31.8%, 28.8%, 33.3%, 8.8%, 1.5%, and 6.1%, respectively. The prevalence of low BMD in the lumbar spine was significantly higher than that in the left proximal femur (34.3% vs. 2.9%, p < 0.001). Moreover, the prevalence of low BMD was significantly higher in adults with CHD than in those without polycythemia and vitamin D deficiency (55.6% vs. 20.9%, p = 0.001 and 46.2% vs. 19.4%, p = 0.002, respectively). A stratified multivariate logistic regression analysis revealed that low BMD was associated with polycythemia (odds ratio: 4.72; 95% confidence interval: 1.64–13.58, p = 0.004).
Conclusions
Low BMD is common among adults with CHD in Vietnam and related to polycythemia.
... Another mechanism that can be involved is hypoxia, which is very frequent in anemic subjects in which oxygen supply to tissues is generally markedly reduced. It has been described that hypoxia is able to induce bone resorption, because it may increase osteoclasts activity which subsequently induces an increase in osteoblasts activation and function (52). Thus, it has been hypothesized that chronic iron deficiency may induce increased bone resorption and increase the risk of osteoporosis (47). ...
In these recent years many people are adopting a vegetarian type diet due to the numerous positive health effects of this regimen such as the reduction of the incidence of many chronic disorders like diabetes, hypertension, obesity and cancer. However this diet is quite restrictive and so it could be possible to have a deficiency in some specific nutrients, increasing the risk of osteoporosis and fractures. Although there are conflicting results on the effects of the vegetarian diet on bone health and fracture incidence, it is always recommendable in vegetarian people to have an adequate intake of calcium and vitamin D, through an increased intake of supplements, natural and fortified foods, an adequate intake of protein, fruit, vegetables, as well as vitamin B12. The aim of this literature review is to revise the actual knowledge of the effect of some nutrients and vegetarian diets on bone health.
... BMC Musculoskeletal Disorders (2022) 23:682 intermittent hypoxia may play an important role in determining the consequences of OSA. Hypoxia is also known to downregulate osteogenic differentiation and directly stimulate osteoclast formation and bone resorption [4,5]. A recent meta-analysis indicated that OSA is strongly associated with an increased risk of osteoporosis in cohort studies [6]. ...
Background
Hypoxia in obstructive sleep apnea (OSA) patients during sleep may have an effect on bone metabolism. Few data regarding evaluation of bone metabolism in young individuals diagnosed with OSA. In this study, we aim to identify the association between bone mineral density and OSA in young men (≤ 40 years old of age).
Methods
Consecutive male subjects who underwent polysomnography were enrolled. Serum calcium, 25-hydroxyvitamin-D3, β-isomerized form C-terminal telopeptide of type I collagen, osteocalcin and procollagen type 1 N-propeptide were measured in all participants, and bone mineral density (BMD) at lumbar spine (L1-L4), femoral neck and hip total were determined by dual energy X-ray absorption (DXA).
Results
The population consisted of 85 subjects (mean age 35.53 years). The BMD at lumbar spine (L1-L4) in moderate OSA patients was higher than control and severe OSA group significantly ( p = 0.036). After adjustment for confounding factors, stepwise multiple linear regression analyses showed LaSO 2 (β = 0.340, p = 0.008) as an independent explanatory variable for Lumbar L1–L4 BMD, LaSO 2 (β = 0.304, p = 0.037), BMI (β = 0.393, p = 0.008) for femur neck BMD and BMI (β = 0.720, p = 0.002) for hip total BMD. Conclusions.
Our finding indicated that there was a relationship between OSA and bone metabolism in younger men, and moderate OSA-related hypoxia positively related with BMD. This study also showed that different degrees of recurrent hypoxia had different effects on bone metabolism, a finding that required further investigation.
The vasculature of the skeletal system is crucial for bone formation, homoeostasis and fracture repair, yet the diversity and specialization of bone-associated vessels remain poorly understood. Here we identify a specialized type of post-arterial capillary, termed type R, involved in bone remodelling. Type R capillaries emerge during adolescence around trabecular bone, possess a distinct morphology and molecular profile, and are associated with osteoprogenitors and bone-resorbing osteoclasts. Endothelial cell-specific overexpression of the transcription factor DACH1 in postnatal mice induces a strong increase in arteries and type R capillaries, leading to local metabolic changes and enabling trabecular bone formation in normally highly hypoxic areas of the diaphysis. Indicating potential clinical relevance of type R capillaries, these vessels respond to anti-osteoporosis treatments and emerge during ageing inside porous structures that are known to weaken compact bone. Our work outlines fundamental principles of vessel specialization in the developing, adult and ageing skeletal system.
Patients with heart failure (HF) have elevated levels of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), leading to macrophage activation, impaired microvascular function, changes in muscle contraction and fibrosis development. In addition, these proinflammatory cytokines promote bone resorption, thereby increasing the incidence of bone fractures. The aim of the study is to evaluate the effect of tumor necrosis factor-alpha receptor types 1 and 2 (TNF-α-SR1 and TNF-α-SR2) on fracture probability in individuals with osteoporosis who have concurrent chronic heart failure (HF) and type 2 diabetes mellitus (T2DM). The study was conducted on 178 women aged 50 to 65 years. Among these women, 48 were in group 1 and had both heart failure and type 2 diabetes. Group 2 included 93 patients with osteoporosis and heart failure and group 3 included 37 women with osteoporosis, heart failure and type 2 diabetes. Thirty-five postmenopausal women without any clinical and instrumental signs of cardiovascular pathology or osteoporosis were selected as the control group. The levels of TNF-α-SR1 and TNF-α-SR2 receptors in patient groups 1-3 were significantly higher (p<0.01) than in the control group, as were their concentrations. In addition, it was found that the levels of both receptors were significantly higher in group 3 than in patients in groups 1 and 2. To evaluate the levels of TNF-α-SR1 and TNF-α-SR2, the data were divided into four quartiles (Q1-Q4) based on the increase in the concentrations of these markers. For TNF-α-SR2, an increasing risk gradient was observed, with the odds ratio (OR) for osteoporosis events increasing from 1.4 to 12.83, but the risk was statistically significant only for Q3-Q4. For Q4 TNF-α-SR1 levels, a significant increase in the risk of an adverse osteoporosis outcome at 36 months was also found (OR=5.25, p=0.038). In addition, the use of loop diuretics was found to be a predictor of high risk of osteoporotic fractures in patients with chronic HF (JR=6.29, 95% CI=1.45-17.26, p=0.015). Postmenopausal women with chronic HF and elevated levels of soluble TNF-α-SR1 and TNF-α-SR2 receptors are at higher risk for osteoporosis and adverse disease progression. In addition, the use of loop diuretics in these individuals is associated with an increased risk of osteoporotic fractures.
As a common clinical disease, fracture is often accompanied by pain, swelling, bleeding as well as other symptoms and has a high disability rate, even threatening life, seriously endangering patients’ physical and psychological health and quality of life. Medical practitioners take many strategies for the treatment of fracture healing, including Traditional Chinese Medicine (TCM). In the early stage of fracture healing, the local fracture is often in a state of hypoxia, accompanied by the expression of hypoxia inducible factor-1α (HIF-1α), which is beneficial to wound healing. Through literature mining, we thought that hypoxia, HIF-1α and downstream factors affected the mechanism of fracture healing, as well as dominated this process. Therefore, we reviewed the local characteristics and related signaling pathways involved in the fracture healing process and summarized the intervention of TCM on these mechanisms, in order to inspirit the new strategy for fracture healing, as well as elaborate on the possible principles of TCM in treating fractures based on the HIF molecular mechanism.
Objective
This research aimed to elucidate the molecular mechanisms underlying the periodontitis‐associated bone loss, with particular emphasis on the contributory role of hypoxic microenvironment in this process.
Background
Periodontitis generally causes alveolar bone loss and is often associated with a hypoxic microenvironment, which affects bone homeostasis. However, the regulating mechanism between hypoxia and jaw metabolism remains unclear. Hypoxia triggers autophagy, which is closely related to osteogenic differentiation, but how hypoxia‐induced autophagy regulates bone metabolism is unknown. HDAC6 and FOXO1 are closely related to bone metabolism and autophagy, respectively, but whether they are related to hypoxia‐induced bone loss and their internal mechanisms is still unclear.
Methods
Established rat nasal obstruction model and hypoxia cell model. Immunohistochemistry was performed to detect the expression and localization of HDAC6 and FOXO1 proteins, analysis of autophagic flux and transmission electron microscopy was used to examine the autophagy level and observe the autophagosomes, co‐immunoprecipitation and chromatin immunoprecipitation were preformed to investigate the interaction of HDAC6 and FOXO1.
Results
Hypoxia causes increased autophagy and reduced osteogenic differentiation in rat mandibles and BMSCs, and blocking autophagy can attenuate hypoxia‐induced osteogenic differentiation decrease. Moreover, hypoxia dissociated the FOXO1‐HDAC6 complex and accumulated them in the nucleus. Knocking down of FOXO1 or HDAC6 alleviated hypoxia‐induced autophag y elevation or osteogenic differentiation reduction by binding to related genes, respectively.
Conclusion
Hypoxia causes mandibular bone loss and autophagy elevation. Mechanically, hypoxia dissociates the FOXO1‐HDAC6 complex and aggregates them in the nucleus, whereas HDAC6 decreases osteogenic differentiation and FOXO1 enhances autophagy to inhibit osteogenic differentiation.
Study objectives:
Obstructive sleep apnea (OSA) and low bone mineral density (BMD) are two prevalent conditions with a significant negative impact on patients' well-being and quality of life. Recent research consistently showed low BMD at different bone sites in male patients with OSA. Although the efficacy of continuous positive airway pressure (CPAP) treatment for OSA has been widely demonstrated, the evidence for understanding its impact on BMD and other bone-related outcomes is insufficient. The aim of this observational study was to investigate the effect of twelve months of CPAP treatment on lumbar and femur BMD and bone-related serum biomarkers in male patients with severe OSA.
Methods:
Sixty patients (mean age: 55.1±9.9 years) were consecutively included and underwent BMD measurement with dual-energy x-ray absorptiometry at baseline and after 12 months of CPAP treatment. Vitamin D, parathyroid hormone (PTH), and calcium serum levels were examined at the same time points.
Results:
Significant increase in BMD in the L1 (p<0.001, d=0.27) and L2 (p<0.001, d=0.26) vertebrae was observed after CPAP treatment, along with an increase in vitamin D (p<0.001, d=0.71) and calcium (p<0.001, d=0.73) levels and a decrease in PTH levels (p<0.001, d=0.60). The increase in BMD in L1 was significantly correlated with the decrease in PTH serum levels (r=-0.50, p<0.001).
Conclusions:
Overall, these findings showed that beneficial OSA treatment might restore bone health, and support CPAP treatment as a feasible strategy to improve BMD in male patients with severe OSA. Accordingly, diagnosing and targeting OSA may be warranted in the treatment of male patients with undetermined osteopenia and osteoporosis.
The osteochondral unit comprises the articular cartilage (90%), subchondral bone (5%) and calcified cartilage (5%). All cells present at the osteochondral unit that is ultimately responsible for matrix production and osteochondral homeostasis, such as chondrocytes, osteoblasts, osteoclasts and osteocytes, can release adenine and/or uracil nucleotides to the local microenvironment. Nucleotides are released by these cells either constitutively or upon plasma membrane damage, mechanical stress or hypoxia conditions. Once in the extracellular space, endogenously released nucleotides can activate membrane-bound purinoceptors. Activation of these receptors is fine-tuning regulated by nucleotides' breakdown by enzymes of the ecto-nucleotidase cascade. Depending on the pathophysiological conditions, both the avascular cartilage and the subchondral bone subsist to significant changes in oxygen tension, which has a tremendous impact on tissue homeostasis. Cell stress due to hypoxic conditions directly influences the expression and activity of several purinergic signalling players, namely nucleotide release channels (e.g. Cx43), NTPDase enzymes and purinoceptors. This review gathers experimental evidence concerning the interplay between hypoxia and the purinergic signalling cascade contributing to osteochondral unit homeostasis. Reporting deviations to this relationship resulting from pathological alterations of articular joints may ultimately unravel novel therapeutic targets for osteochondral rehabilitation. At this point, one can only hypothesize how hypoxia mimetic conditions can be beneficial to the ex vivo expansion and differentiation of osteo- and chondro-progenitors for auto-transplantation and tissue regenerative purposes.
Osteoporosis is an age-related disease of bone metabolism marked by reduced bone mineral density and impaired bone strength. The disease causes the bones to weaken and break more easily. Osteoclasts participate in bone resorption more than osteoblasts participate in bone formation, disrupting bone homeostasis and leading to osteoporosis. Currently, drug therapy for osteoporosis includes calcium supplements, vitamin D, parathyroid hormone, estrogen, calcitonin, bisphosphates, and other medications. These medications are effective in treating osteoporosis but have side effects. Copper is a necessary trace element in the human body, and studies have shown that it links to the development of osteoporosis. Cuproptosis is a recently proposed new type of cell death. Copper-induced cell death regulates by lipoylated components mediated via mitochondrial ferredoxin 1; that is, copper binds directly to the lipoylated components of the tricarboxylic acid cycle, resulting in lipoylated protein accumulation and subsequent loss of iron-sulfur cluster proteins, leading to proteotoxic stress and eventually cell death. Therapeutic options for tumor disorders include targeting the intracellular toxicity of copper and cuproptosis. The hypoxic environment in bone and the metabolic pathway of glycolysis to provide energy in cells can inhibit cuproptosis, which may promote the survival and proliferation of various cells, including osteoblasts, osteoclasts, effector T cells, and macrophages, thereby mediating the osteoporosis process. As a result, our group tried to explain the relationship between the role of cuproptosis and its essential regulatory genes, as well as the pathological mechanism of osteoporosis and its effects on various cells. This study intends to investigate a new treatment approach for the clinical treatment of osteoporosis that is beneficial to the treatment of osteoporosis.
The hypoxia pathway not only regulates the organism to adapt to the special environment, such as short-term hypoxia in the plateau under normal physiological conditions, but also plays an important role in the occurrence and development of various diseases such as cancer, cardiovascular diseases, osteoporosis. Bone, as a special organ of the body, is in a relatively low oxygen environment, in which the expression of hypoxia-inducible factor (HIF)-related molecules maintains the necessary conditions for bone development. Osteoporosis disease with iron overload endangers individuals, families and society, and bone homeostasis disorder is linked to some extent with hypoxia pathway abnormality, so it is urgent to clarify the hypoxia pathway in osteoporosis to guide clinical medication efficiently. Based on this background, using the keywords “hypoxia/HIF, osteoporosis, osteoblasts, osteoclasts, osteocytes, iron/iron metabolism”, a matching search was carried out through the Pubmed and Web Of Science databases, then the papers related to this review were screened, summarized and sorted. This review summarizes the relationship and regulation between the hypoxia pathway and osteoporosis (also including osteoblasts, osteoclasts, osteocytes) by arranging the references on the latest research progress, introduces briefly the application of hyperbaric oxygen therapy in osteoporosis symptoms (mechanical stimulation induces skeletal response to hypoxic signal activation), hypoxic-related drugs used in iron accumulation/osteoporosis model study, and also puts forward the prospects of future research.
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.
Macrophages are ubiquitous in the stromal compartment of tissues under normal physiological conditions and the number of these cells increases markedly with the onset and progression of many pathological states. The mechanisms underlying this response are well described in such conditions as wound healing and malignant tumors, where tissue-specific signals enhance the extravasation of blood monocytes and their subsequent differentiation into macrophages. Recent evidence suggests that macrophages may also be stimulated by microenvironmental factors present in diseased tissues to perform distinct, tissue-specific activities. One such factor, hypoxia (low oxygen tension), results from insufficient vascular perfusion of a given tissue. Various studies have shown that experimental hypoxia alters the morphology, expression of cell surface markers, viability, phagocytosis, metabolic activity, and release of cytokines by macrophages. Here we review the evidence for these macrophage responses to hypoxia, the involvement of co-stimuli, and their implications for the role of macrophages in various disease processes. Because the intracellular mechanisms mediating the effects of hypoxia on gene expression in other cell types have been characterized recently, we discuss their possible involvement in the effects of hypoxia on gene expression in macrophages.
Recombinant (r) and natural human (h) macrophage colony-stimulating factor (M-CSF, CSF-1) have been considered poor stimulators of macrophage progenitor cells present in human marrow, although they are potent stimulators of these cells in mouse marrow. We compared the growth characteristics of rhM-CSF-responsive human macrophage progenitor cells placed in semisolid agarose or agar culture medium and incubated for 14 days at ambient (approximately 20%) or lowered (5%) O2 tension. By itself, rhM-CSF was found to be a good stimulator of macrophage colony formation by human bone marrow cells cultured in agarose but not in agar; this growth was enhanced by incubation at 5% O2. Maximal numbers (up to 115/10(5) nonadherent low density cells plated) of macrophage colonies (50 to greater than 500 cells per colony) were stimulated by 500 to 1,000 units rhM-CSF/mL, with 1/2 maximal numbers stimulated by 250 to 500 units/mL. With agarose as the support medium, rhM-CSF was two- to fourfold more active on mouse than on human macrophage colony formation, in contrast to previous reports of 10- to 100-fold greater activity when agar was used as the support medium. Using nonadherent low density T lymphocyte-depleted human bone marrow cells growing in agarose at 5% O2, greater than additive effects on colony formation were observed when 31 to 500 units rhM-CSF were used in combination with either 10 ng rh interleukin-1 alpha (IL-1 alpha), 20, or 200 units rh granulocyte-macrophage (GM)-CSF or rhG-CSF. The agarose assay system should be useful for evaluating factors regulating the proliferation of human macrophage progenitor cells in vitro and during clinical trials with rhM-CSF.
Disaggregated chick osteoclasts sedimented onto bovine cortical bone slices excavate deep and sharply defined resorption lacunae that stain intensely with toluidine blue. We have used this observation to develop a simple light microscopic method for quantifying the bone resorptive activity of chick osteoclasts in vitro. Using this technique, we have found that disaggregated chick osteoclasts are strikingly less sensitive than rat osteoclasts to salmon calcitonin (sCT). Bone resorption by rat osteoclasts was completely abolished by synthetic sCT at a concentration of 10 pg/ml. In contrast, sCT at concentrations up to 100 micrograms/ml did not significantly inhibit bone resorption by chick osteoclasts. Bone resorption by chick osteoclasts could, however, be inhibited by prostaglandin E2 at concentrations of 10(-6) M or more. In time-lapse video experiments, the motility of rat osteoclasts was rapidly inhibited by sCT (5-50 pg/ml), prostaglandins I2 and E2 (less than or equal to 10(-4) M), and Bu2cAMP (2.5 X 10(-4) M), whereas chick osteoclasts failed to show such a response. These findings suggest that CT does not play an important role in the regulation of osteoclastic activity in the chick and may explain why injection of the hormone has generally not been found to evoke an acute hypocalcemic response in birds. This fundamental difference in response to CT may limit the utility of chick osteoclast systems as models of mammalian bone resorption.
Acidosis has been implicated in the pathogenesis of various metabolic bone diseases. Experimental acid ingestion is known to stimulate the release of bone mineral in humans and increase osteoclastic resorption surfaces in animals, but the mechanism has remained unclear. We have investigated the effect of medium pH on osteoclastic activity in vitro using a new method that allows direct measurement of bone resorption. Osteoclasts were disaggregated from neonatal rat long bones and allowed to settle onto thin slices of bovine cortical bone. After 24-h incubation in medium 199, adjusted to the required pH, bone slices were fixed, and the number and surface area of resorption pits per slice were determined. Reduction in medium pH from 7.4 to 6.8 resulted in a 14-fold increase in the mean area resorbed per bone slice (P less than 0.01). Five- and 9-fold increases were seen at pH 7.2 and 7.0, respectively. Incubation of bone cells with human PTH-(1-34) (100 ng/ml) did not significantly enhance resorption at either pH 7.4 or 6.4, whereas human calcitonin (50 pg/ml) produced near-total inhibition. Mean osteoclast and mononuclear cell numbers were unaltered by all treatments. This is the first direct demonstration that low pH stimulates cell-mediated bone resorption, an observation that may have considerable physiological and pathophysiological significance.
We investigated the effect of small shifts in extracellular pH on the resorptive activity of rat osteoclasts in vitro. Osteoclast-containing mixed bone cell populations disaggregated from neonatal rat long bones were cultured for 26 h at low density on bovine bone disks in HCO-3/CO2-buffered medium modified by the addition of small amounts of protons as HCl (2.5-15.0 mEq/L) or hydroxyl ions as NaOH (2.5-10.0 mEq/L). Little or no bone resorption occurred when ambient pH was above about 7.30; at pH 7.00, osteoclasts exhibited near-maximal activity. The greatest changes were associated with a pH difference of as little as 0.10 unit, between pH 7.25 and pH 7.15. Over this narrow range, the average number of resorption pits formed on each bone wafer increased sixfold. The steep sigmoidal pH response curve indicates that very slight alterations in ambient hydrogen ion concentration can effectively "switch on" or "switch off" rat osteoclasts in vitro.
There is increasing evidence that extracellular ATP acts directly on bone cells via P2 receptors. In normal rat osteoclasts, ATP activates both non‐selective cation channels and Ca ²⁺ ‐dependent K ⁺ channels. In this study we investigated the action of ATP on the formation of osteoclasts and on the ultimate function of these cells, namely resorption pit formation.
We found that ATP stimulated resorption pit formation up to 5.6‐fold when osteoclast‐containing bone cell populations from neonatal rats were cultured for 26 h on ivory discs, with a maximum effect occurring at relatively low concentrations (0.2‐2 μM). The stimulatory effect of ATP was amplified greatly when osteoclasts were activated by culture in acidified media (pH 6.9‐7.0). Pit formation by acid‐activated osteoclasts in the absence of ATP was inhibited by apyrase, an ecto‐ATPase and by suramin, an antagonist of P2 receptors.
Over the same concentration range at which rat osteoclast activation occurred (0.2‐2 μM), ATP also enhanced osteoclast formation in 10 day mouse marrow cultures, by up to 3.3‐fold, with corresponding increases in resorption pit formation. Higher concentrations of ATP (20–200 μM) reduced or blocked osteoclast formation. Adenosine, a P1 receptor agonist, was without effect on either osteoclast activation or formation.
These results suggest that low levels of extracellular ATP may play a fundamental role in modulating both the resorptive function and formation of mammalian osteoclasts.
Angiogenesis is essential for the increased delivery of oxygen and nutrients required for the reparative processes of bone healing. Vascular endothelial growth factor (VEGF), a potent angiogenic growth factor, has been implicated in this process. We have previously shown that hypoxia specifically and potently regulates the expression of VEGF by osteoblasts. However, the molecular mechanisms governing this interaction remain unknown. In this study, we hypothesized that the hypoxic regulation of VEGF expression by osteoblasts occurs via an oxygen-sensing mechanism similar to the regulation of the erythropoietin gene (EPO). To test this hypothesis, we examined the kinetics of oxygen concentration on osteoblast VEGF expression. In addition, we analyzed the effects of nickel and cobalt on the expression of VEGF in osteoblastic cells because these metallic ions mimic hypoxia by binding to the heme portion of oxygen-sensing molecules. Our results indicated that hypoxia potently stimulates VEGF mRNA expression. In addition, we found that nickel and cobalt both stimulate VEGF gene expression in a similar time- and dose-dependent manner, suggesting the presence of a hemelike oxygen-sensing mechanism similar to that of the EPO gene. Moreover, actinomycin D, cycloheximide, dexamethasone, and mRNA stabilization studies collectively established that this regulation is predominantly transcriptional, does not require de novo protein synthesis, and is not likely mediated by the transcriptional activator AP-1. These studies demonstrate that hypoxia, nickel, and cobalt regulate VEGF expression in osteoblasts via a similar mechanism, implicating the involvement of a heme-containing oxygen-sensing molecule. This may represent an important mechanism of VEGF regulation leading to increased angiogenesis in the hypoxic microenvironment of healing bone.
Extracellular nucleotides acting through P2 receptors elicit a wide range of responses in many cell types. There is increasing evidence that adenosine triphosphate (ATP) may function as an important local messenger in bone and cartilage. In this study, we used immunocytochemistry, employing novel polyclonal antibodies against P2X(1-7) receptors, and in situ hybridization, using oligonucleotide probes corresponding to P2X(2,4) and P2Y(2,4) messenger RNAs (mRNAs), to localize P2 receptors on undecalcified bone sections and on cultured osteoblasts and osteoclasts. We provide the first direct evidence that the P2X(2) receptor subtype is expressed on osteoclasts, osteoblasts, and chondrocytes. We also obtained evidence for the expression of P2X(5) and P2Y(2) receptors on osteoblasts and chondrocytes, and for P2X(4) and P2X(7) receptors on osteoclasts. Our results confirm earlier reports of P2Y(2) and P2X(4) expression in human osteoclastoma and rabbit osteoclasts, respectively, and are consistent with ATP responses observed on bone cells using electrophysiological techniques. Our novel finding that P2X(2) is expressed by osteoclasts is of particular interest. P2X(2) is the only P2 receptor subtype that requires extracellular acidification to show its full sensitivity to ATP, and our recent functional studies have shown that the stimulatory action of ATP on resorption pit formation by mature osteoclasts is amplified greatly at low pH. These findings point to fundamental new mechanisms for the local modulation of bone resorption.
We examined the effects of HCO(3)(-) and CO(2) acidosis on osteoclast-mediated Ca(2+) release from 3-day cultures of neonatal mouse calvaria. Ca(2+) release was minimal above pH 7.2 in control cultures but was stimulated strongly by the addition of small amounts of H(+) to culture medium (HCO(3)(-) acidosis). For example, addition of 4 meq/l H(+) reduced pH from 7.12 to 7.03 and increased Ca(2+) release 3.8-fold. The largest stimulatory effects (8- to 11-fold), observed with 15-16 meq/l added H(+), were comparable to the maximal Ca(2+) release elicited by 1,25-dihydroxyvitamin D(3) [1, 25(OH)(2)D(3); 10 nM], parathyroid hormone (10 nM), or prostaglandin E(2) (1 microM); the action of these osteolytic agents was attenuated strongly when ambient pH was increased from approximately 7.1 to approximately 7.3. CO(2) acidosis was a less effective stimulator of Ca(2+) release than HCO(3)(-) acidosis over a similar pH range. Ca(2+) release stimulated by HCO(3)(-) acidosis was almost completely blocked by salmon calcitonin (20 ng/ml), implying osteoclast involvement. In whole mount preparations of control half-calvaria, approximately 400 inactive osteoclast-like multinucleate cells were present; in calvaria exposed to HCO(3)(-) acidosis and to the other osteolytic agents studied, extensive osteoclastic resorption, with perforation of bones, was visible. HCO(3)(-) acidosis, however, reduced numbers of osteoclast-like cells by approximately 50%, whereas 1,25(OH)(2)D(3) treatment caused increases of approximately 75%. The results suggest that HCO(3)(-) acidosis stimulates resorption by activating mature osteoclasts already present in calvarial bones, rather than by inducing formation of new osteoclasts, and provide further support for the critical role of acid-base balance in controlling osteoclast function.
VEGF is produced by osteoblasts and has been postulated to function as an angiogenic stimulus during normal skeletal development and in fracture repair. In this study, we characterized the molecular mechanisms by which experimental hypoxia increases VEGF mRNA in human MG63 osteoblast-like cells. Exposure of MG63 cells to 1% O(2) for 24 h resulted in a four-fold increase in VEGF mRNA. Immunoblotting of nuclear extracts demonstrated a time-dependent increase in the level of the Hif-2alpha protein, which preceded the rise in VEGF mRNA. To determine the effect of hypoxia on VEGF gene transcription, MG63 cells were transiently transfected with a segment of the VEGF promoter construct fused to luciferase and then exposed to 1% O(2). Hypoxia induced VEGF promoter activity five-fold by 24 h. Forced expression of Hif-2alpha, but not Hif-1alpha, increased both basal and hypoxia induced VEGF promoter activity. By contrast, the ability of the VEGF reporter to respond to hypoxia or recombinant Hif-2alpha was abolished in cells transfected with a VEGF promoter construct containing a mutation in the hypoxia response element. In summary, exposure of osteoblast-like cells to hypoxia induces VEGF expression via induction of Hif-2alpha and transcriptional activation of the VEGF promoter.
Hypoxia-inducible factor (HIF) is a transcriptional complex that plays a central role in the regulation of gene expression
by oxygen. In oxygenated and iron replete cells, HIF-α subunits are rapidly destroyed by a mechanism that involves ubiquitylation
by the von Hippel–Lindau tumor suppressor (pVHL) E3 ligase complex. This process is suppressed by hypoxia and iron chelation,
allowing transcriptional activation. Here we show that the interaction between human pVHL and a specific domain of the HIF-1α
subunit is regulated through hydroxylation of a proline residue (HIF-1α P564) by an enzyme we have termed HIF-α prolyl-hydroxylase
(HIF-PH). An absolute requirement for dioxygen as a cosubstrate and iron as cofactor suggests that HIF-PH functions directly
as a cellular oxygen sensor.
There is increasing evidence that extracellular nucleotides act on bone cells via P2 receptors. This study investigated the action of ADP and 2-methylthioADP, a potent ADP analog with selectivity for the P2Y(1) receptor, on osteoclasts, the bone-resorbing multinuclear cells. Using three different assays, we show that ADP and 2-methylthioADP at nanomolar to submicromolar levels caused up to fourfold to sixfold increases in osteoclastic bone resorption. On mature rat osteoclasts, cultured for 1 day on polished dentine disks, peak effects on resorption pit formation were observed between 20 nM and 2 microM of ADP. The same concentrations of ADP also stimulated osteoclast and resorption pit formation in 10-day mouse marrow cultures on dentine disks. In 3-day explant cultures of mouse calvarial bones, the stimulatory effect of ADP on osteoclast-mediated Ca(2+) release was greatest at 5-50 microM and equivalent to the maximal effects of prostaglandin E(2). The ADP effects were blocked in a nontoxic manner by MRS 2179, a P2Y(1) receptor antagonist. Using in situ hybridization and immunocytochemistry, we found evidence for P2Y(1) receptor expression on both osteoclasts and osteoblasts; thus, ADP could exert its actions both directly on osteoclasts and indirectly via P2Y(1) receptors on osteoblasts. As a major ATP degradation product, ADP is a novel stimulator of bone resorption that could help mediate inflammatory bone loss in vivo.
Loss of mechanical loading, or disuse, rapidly precipitates locally mediated bone resorption. However, the pathway by which this process is initiated and mediated is poorly understood. In this study, we used a complementary in vivo and in vitro approach to determine whether disuse-induced osteocyte hypoxia resulted in upregulation of the hypoxia-dependent transcription factor HIF-1alpha. We found that acute disuse (1-5 days) resulted in a significant increase in the percentage of osteocytes staining positive for HIF-1alpha vs. normal bone (30.9 +/- 6.1 vs. 14.1 +/- 3.8%) and that this response was uniform around the cortex. In addition, we found that acute oxygen deprivation (4-12 h of 2% O2) resulted in a 2.1- to 3.7-fold upregulation of HIF-1alpha protein expression in MLO-Y4 osteocyte-like cells compared with cells cultured in parallel under normal oxygen conditions. Given known HIF-1alpha targets genes, we suggest that osteocyte hypoxia and subsequent upregulation of hypoxia-dependent pathways may serve to initiate and mediate disuse-induced bone resorption.
Mammalian cells respond to changes in oxygen availability through a conserved pathway that is regulated by the hypoxia-inducible factor (HIF). The alpha subunit of HIF is targeted for degradation under normoxic conditions by a ubiquitin-ligase complex that recognizes a hydroxylated proline residue in HIF. We identified a conserved family of HIF prolyl hydoxylase (HPH) enzymes that appear to be responsible for this posttranslational modification. In cultured mammalian cells, inappropriate accumulation of HIF caused by forced expression of the HIF-1alpha subunit under normoxic conditions was attenuated by coexpression of HPH. Suppression of HPH in cultured Drosophila melanogaster cells by RNA interference resulted in elevated expression of a hypoxia-inducible gene (LDH, encoding lactate dehydrogenase) under normoxic conditions. These findings indicate that HPH is an essential component of the pathway through which cells sense oxygen.
Recombinant (r) and natural human (h) macrophage colony-stimulating factor (M-CSF, CSF-1) have been considered poor stimulators of macrophage progenitor cells present in human marrow, although they are potent stimulators of these cells in mouse marrow. We compared the growth characteristics of rhM-CSF-responsive human macrophage progenitor cells placed in semisolid agarose or agar culture medium and incubated for 14 days at ambient (approximately 20%) or lowered (5%) O2 tension. By itself, rhM-CSF was found to be a good stimulator of macrophage colony formation by human bone marrow cells cultured in agarose but not in agar; this growth was enhanced by incubation at 5% O2. Maximal numbers (up to 115/10(5) nonadherent low density cells plated) of macrophage colonies (50 to greater than 500 cells per colony) were stimulated by 500 to 1,000 units rhM-CSF/mL, with 1/2 maximal numbers stimulated by 250 to 500 units/mL. With agarose as the support medium, rhM-CSF was two- to fourfold more active on mouse than on human macrophage colony formation, in contrast to previous reports of 10- to 100-fold greater activity when agar was used as the support medium. Using nonadherent low density T lymphocyte-depleted human bone marrow cells growing in agarose at 5% O2, greater than additive effects on colony formation were observed when 31 to 500 units rhM-CSF were used in combination with either 10 ng rh interleukin-1 alpha (IL-1 alpha), 20, or 200 units rh granulocyte-macrophage (GM)-CSF or rhG-CSF. The agarose assay system should be useful for evaluating factors regulating the proliferation of human macrophage progenitor cells in vitro and during clinical trials with rhM-CSF.
Prostaglandin E2 (PGE2) and the cytokines interleukin (IL) 1α and tumor necrosis factor (TNF)α increase bone resorption in vivo, but the effect of these agents on osteoclastic bone resorption has never been studied in an in vitro human system. Our recently described human bone marrow culture system, in which osteoclasts are generated (vitronectin and calcitonin receptor-positive cells which resorb bone), was used to study the effects of these agents. Addition of indomethacin to macrophage colony-stimulating factor (M-CSF)-treated cultures nearly abolished osteoclast parameters, indicating that prostaglandins are virtually essential for human osteoclast formation. Additionally, PGE2 dose responsively increased osteoclast numbers and bone resorption. The effects of M-CSF and PGE2 are independent, as demonstrated by unaltered PGE2 concentrations in culture supernatants in spite of the dose-responsive increase in osteoclast parameters in response to M-CSF. The generation of osteoclasts in the presence of PGE2 occurred in favor of CD 14-positive macrophage formation.
IL 1α and TNFα increased osteoclast parameters in a dose-responsive manner. Maximum stimulation yielded culture supernatant levels of PGE2 approximately the same as those concentrations of exogenous PGE2 that dramatically induced osteoclast formation. This osteoclast-inducing effect was inhibited both by indomethacin and by the specific inhibitor of inducible prostaglandin G/H synthase, NS-398, and this was reversed by addition of exogenous PGE2. These results demonstrate unequivocally that IL 1α and TNFα enhance human osteoclast formation and suggest that they mediate their effects through PGE2.
VEGF is produced by osteoblasts and has been postulated to function as an angiogenic stimulus during normal skeletal development and in fracture repair. In this study, we characterized the molecular mechanisms by which experimental hypoxia increases VEGF mRNA in human MG63 osteoblast-like cells. Exposure of MG63 cells to 1% O2 for 24 h resulted in a four-fold increase in VEGF mRNA. Immunoblotting of nuclear extracts demonstrated a time-dependent increase in the level of the Hif-2α protein, which preceded the rise in VEGF mRNA. To determine the effect of hypoxia on VEGF gene transcription, MG63 cells were transiently transfected with a segment of the VEGF promoter construct fused to luciferase and then exposed to 1% O2. Hypoxia induced VEGF promoter activity five-fold by 24 h. Forced expression of Hif-2α, but not Hif-1α, increased both basal and hypoxia induced VEGF promoter activity. By contrast, the ability of the VEGF reporter to respond to hypoxia or recombinant Hif-2α was abolished in cells transfected with a VEGF promoter construct containing a mutation in the hypoxia response element. In summary, exposure of osteoblast-like cells to hypoxia induces VEGF expression via induction of Hif-2α and transcriptional activation of the VEGF promoter.
IGF-I is known to stimulate the expression of oxygen- and nutrient-sensitive genes in several cell types. In this study we investigated the signaling pathways and transcriptional mechanisms that mediate IGF-I induction of vascular endo- thelial growth factor (VEGF) expression in human osteoblast- like cells. IGF-I (50 ng/ml) induced a rapid increase (3-fold) in VEGF mRNA in osteoblasts that was accompanied by an in- crease in the level of hypoxia-inducible factor-2 (HIF-2) protein without changes in HIF-2 mRNA expression. These effects were mimicked by chemical inhibition of proteosomal degradation of HIF-2. Transcriptional activation of a prox- imal VEGF promoter-luciferase construct was greatly en- hanced by cotransfection with an HIF-2, but not an HIF-1, construct. IGF-I acutely stimulated Akt phosphorylation, which was abolished by pretreatment of cells with the PI3K inhibitor LY294002. Pretreatment of the cells with LY294002 also greatly attenuated IGF-I induction of HIF-2 and blunted IGF-I-induced VEGF promoter activity. Finally, forced ex- pression of a constitutively active PI3K expression construct induced VEGF promoter to levels similar to those observed with IGF-I alone. These data indicate that IGF-I, by activation of the PI3K pathway, induces VEGF expression in osteoblasts through a transcriptional control mechanism common to those that activate VEGF and other hypoxia response genes. (Endocrinology 143: 420 - 425, 2002)
Numerous studies have demonstrated the critical role of angiogenesis for successful osteogenesis during endochondral ossification and fracture repair. Vascular endothelial growth factor (VEGF), a potent endothelial cell-specific cytokine, has been shown to be mitogenic and chemotactic for endothelial cells in vitro and angiogenic in many in vivo models. Based on previous work that (1) VEGF is up-regulated during membranous fracture healing, (2) the fracture site contains a hypoxic gradient, (3) VEGF is up-regulated in a variety of cells in response to hypoxia, and (4) VEGF is expressed by isolated osteoblasts in vitro stimulated by other fracture cytokines, the hypothesis that hypoxia may regulate the expression of VEGF by osteoblasts was formulated. This hypothesis was tested in a series of in vitro studies in which VEGF mRNA and protein expression was assessed after exposure of osteoblast-like cells to hypoxic stimuli. In addition, the effects of a hypoxic microenvironment on osteoblast proliferation and differentiation in vitro was analyzed. These results demonstrate that hypoxia does, indeed, regulate expression of VEGF in osteoblast-like cells in a dose-dependent fashion. In addition, it is demonstrated that hypoxia results in decreased cellular proliferation, decreased expression of proliferating cell nuclear antigen, and increased alkaline phosphatase (a marker of osteoblast differentiation). Taken together, these data suggest that osteoblasts, through the expression of VEGF, may be in part responsible for angiogenesis and the resultant increased blood flow to fractured bone segments. In addition, these data provide evidence that osteoblasts have oxygen-sensing mechanisms and that decreased oxygen tension can regulate gene expression, cellular proliferation, and cellular differentiation.
Breakdown or absence of vascular oxygen delivery is a hallmark of many common human diseases, including cancer, myocardial infarction, and stroke. The chief mediator of hypoxic response in mammalian tissues is the transcription factor hypoxia-inducible factor 1 (HIF-1), and its oxygen-sensitive component HIF-1α. A key question surrounding HIF-1α and the hypoxic response is the role of this transcription factor in cells removed from a functional vascular bed; in this regard there is evidence indicating that it can act as either a survival factor or induce growth arrest and apoptosis. To study more closely how HIF-1α functions in hypoxia in vivo, we used tissue-specific targeting to delete HIF-1α in an avascular tissue: the cartilaginous growth plate of developing bone. We show here the first evidence that the developmental growth plate in mammals is hypoxic, and that this hypoxia occurs in its interior rather than at its periphery. As a result of this developmental hypoxia, cells that lack HIF-1α in the interior of the growth plate die. This is coupled to decreased expression of the CDK inhibitor p57, and increased levels of BrdU incorporation in HIF-1α null growth plates, indicating defects in HIF-1α-regulated growth arrest occurs in these animals. Furthermore, we find that VEGF expression in the growth plate is regulated through both HIF-1α-dependent and -independent mechanisms. In particular, we provide evidence that VEGF expression is up-regulated in a HIF-1α-independent manner in chondrocytes surrounding areas of cell death, and this in turn induces ectopic angiogenesis. Altogether, our findings have important implications for the role of hypoxic response and HIF-1α in development, and in cell survival in tissues challenged by interruption of vascular flow; they also illustrate the complexities of HIF-1α response in vivo, and they provide new insights into mechanisms of growth plate development.
The effects of providing low oxygen tension in the gas phase of two different types of cell culture systems were investigated. The clonal growth of granulocyte-macrophage progenitor cells in an agar culture system was improved markedly by incubation within a low oxygen tension gas phase (48 mmHg--6.8%) instead of the conventional air (135 mmHg--19%), the effects being measured by increases in numbers of colony forming cells detected and in the colony sizes. The increased efficiency of colony formation was observed both with mouse and human marrow cells. A similar effect was observed in a liquid adherence culture system with primary cultures of foetal mouse fibroblasts both at clonal and higher cell densities.
Growth of hematopoietic stem and progenitor cells found in the mononuclear cell (MNC) fraction of human cord blood and bone marrow was evaluated under atmospheres containing reduced (5%) and normal (20%) oxygen tension. Cord blood MNC were grown in suspension and on preestablished irradiated bone marrow stromal layers, whereas bone marrow MNC were used to initiate one-step long-term bone marrow cultures (LTBMC). Reduced oxygen tension resulted in a substantial increase in both the number and frequency of colony-forming cells observed in all three types of long-term hematopoietic cultures (LTHC) studied. At various time points under low oxygen, progenitor cell numbers were as much as 12-fold, 3-fold, and 4-fold higher for granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), and granulocyte erythrocyte macrophage megakaryocyte colony-forming units (CFU-GEMM), respectively. In addition to these numerical increases, progenitor cells were maintained for 1-2 weeks longer under low oxygen conditions. Reduced oxygen tension also increased total cell numbers by as much as fivefold in cord blood suspension cultures, but this effect on total cell numbers was less pronounced in cultures containing a stromal layer. The rate of irradiated stromal layer degeneration, as judged by cell counts and microscopic examination, was reduced under low oxygen. Finally, the beneficial effect of reduced oxygen was comparable to the effect of an irradiated stromal layer for maintaining cord blood progenitor cells in LTHC. These results indicate that low oxygen, which better approximates the in vivo environment, enhances the growth and maintenance of both stromal and progenitor cells for a longer period of time in vitro.
Osteoclastic differentiation is induced from hematopoietic cells in the presence of 1,25-(OH)2D3 by stromal cells that are present in bone but not in hematopoietic spleen. Recent evidence suggests that prostaglandins (PGs) are essential for this process. In this communication we describe experiments in which we have examined further the role of PGE2 in osteoclast formation. We found a marked reduction in basal, 1,25-(OH)2D3, and IL-3-induced production of calcitonin receptor (CTR)-positive cells and bone resorption by cyclooxygenase inhibitors, which was restored by PGE2 addition. Although some stromal cell types (ST2 cells) that support osteoclast formation from spleen cells produced PGs in response to 1,25-(OH)2D3, others (ts8 and calvarial cells) did not, either alone or in combination with spleen cells. On the other hand, both bone marrow and spleen cells produced amounts of PGE2 in response to 1,25-(OH)2D3 that were sufficient to account for osteoclast formation. Osteoclast-inductive ts8 cells were able to support osteoclast formation from spleen cells in the presence of 1,25-(OH)2D3 or PGE2 even if devitalized. Incubation of ts8 cells in these agents before devitalization did not avoid the requirement for the presence of PGE2 or 1,25-(OH)2D3 during subsequent incubation with spleen cells. Thus, hematopoietic cells produce sufficient PGE2 for osteoclast formation, and the PGE2 thus produced acts on hematopoietic precursors, which can be induced in the presence of PGE2 to express CTR and resorb bone on contact with osteoclast-inductive stromal cells. The ability of osteoclast-inductive cells to support osteoclast formation appears not to rest on their ability to produce, induce, or respond to PGE2.
We tested acid and basic fibroblast growth factor (aFGF and bFGF), members of the heparin binding FGF family, for their ability to stimulate bone resorption as measured by the release of previously incorporated 45Ca from cultured fetal rat long bones in the presence and absence of heparin. Purified low-molecular-weight heparin (LMW heparin) at 5-125 micrograms/ml had no direct stimulatory effect. There was little effect from aFGF (10(-11)-10(-8) M) alone, but increased resorption was observed in the presence of LMW heparin. With bFGF, increased bone resorption was observed at 10(-9) M but not at 10(-8) M. The stimulatory effects of aFGF and bFGF in the presence of LMW heparin were not blocked by the addition of indomethacin (10(-6) M), which blocks prostaglandin production, or hydroxyurea (10(-3) M), which blocks DNA synthesis. However, pretreatment with aphidicolin (3 x 10(-5) M), a potent inhibitor of DNA synthesis, blocked the effect of acid FGF and diminished the effect of bFGF. These results indicate that both aFGF and bFGF can stimulate bone resorption by a prostaglandin-independent mechanism, particularly in the presence of heparin. The activation of FGF-mediated bone resorption by heparin could play a role in producing the osteoporosis that has been described with heparin therapy and mastocytosis.
The influence of low oxygen tension on the clonal growth of hemopoietic stem cells in vitro was examined. The numbers of colonies of neutrophil, macrophage, and eosinophil progenitors (CFU-C), derived from human bone marrow, increased at a rate 1.7 times higher in low oxygen tension (7% O2) than in a gas phase that contained air (19% O2). The erythroid (BFU-E) and multipotential (CFU-mix) progenitors increased about 2.4 times in 7% O2, and the increase in the composed cell type of mixed colonies showed no rate difference in either gas phase. Under atmospheric conditions, a mouse mast cell progenitor (CFU-mast) formed colonies, with the addition of 2-mercaptoethanol (2-ME). Under low oxygen tension, the CFU-mast formed colonies without 2-ME, but a further enhancement was observed with the addition of 2-ME. Blood gas analysis of human bone marrow showed a pO2 of 51.8 +/- 14.5 mmHg, which was closed to O2 tension in a gas phase culture media containing 7% O2. This data shows that the physiological O2 tension enhances hemopoietic stem cell proliferation in vitro, and that a part of the enhancing effect by 2-ME is due to a prevention of O2 toxicity at 19% O2.
In an effort to develop more nearly optimal conditions for the clonal growth of hemopoietic stem cells (CFC, BFU-E, CFU-mix and Mast-CFC) iv vitro, I examined the influence of low oxygen tension (7% O2) on plating efficiency. The numbers of colonies derived from human bone marrow CFC increased by 1.7 fold in 7% O2 than under a gas phase containing air (19% O2). Bursts obtained from bone marrow BFU-E and mixed colonies from CFU-mix increased by about 2.5 fold in 7% O2. Total cell count of mixed colonies showed that the average cell per colony gassed with 7% O2 were 900 compared with 511 in 19% O2. However the subpopulation of CFC and composed cell type of mixed colonies were not different in the two gas phase. With mouse spleen cell in 7% O2 a dramatic increase in Mast-CFC numbers without 2-ME and decreased enhancement by 2-ME were seen. Blood gas analysis of human bone marrow showed a Po2 of 51.8 +/- 14.5 mmHg, which was close to O2 tension in culture media of gas phase contained 7% O2. These data showed that physiological O2 tension enhances hemopoietic stem cell proliferation in vitro, and that part of the enhancing effect by 2-ME is due to a prevention of O2 toxicity at 19% O2.
The effect of various oxygen tensions on the synthesis and degradation of bone collagen has been studied biochemically in tissue culture. The incorporation of H3-proline from the tissue culture medium into collagen H3-hydroxyproline has been used as an index of collagen synthesis, and the release of hydroxyproline into the tissue culture medium as an index of collagen degradation. The results show that at low oxygen tensions the rate of collagen synthesis exceeds that of degradation. At higher oxygen tensions, both the rates of synthesis and degradation increase. At an oxygen tension of 50%, the rates of synthesis and degradation are approximately equal. Under the latter conditions evidence is also presented which suggests that the bone collagen present in the calvaria at the time of explantation is more susceptible to resorption than the collagen synthesized during culture.