Background
Avascular necrosis of the femoral head (ANFH) is a progressive disease characterized by necrosis and collapse of the bone. ANFH often ends in arthroplasty and has become a severe health problem worldwide. High-dose of GC administration is a common risk factor associated with ANFH due to a widespread use of GCs as adjuvant therapies in the treatment of many inflammatory and autoimmune diseases. Though the specific etiology of ANFH remains unclear, the pathophysiology of impaired circulation in the femoral head has been considered as one major cause of ANFH in patients [
1]. In GC-induced ANFH, insufficient neovascularization and elevated vascular permeability in the osteonecrotic lesions have been demonstrated [
2]. Based on this observation, many hypotheses have been proposed to address the pathogenesis of ANFH, such as apoptosis, oxidative stress, disorders of the vascular endothelium, blood coagulation disorders, fat embolism, and multiple-hit theory. However, none of these reached consensus, so far.
In 1997, the endothelial progenitor cell (EPC) was first reported by Asahara and colleagues [
3]. They described EPCs as a group of circulating cells which promoted angiogenesis and maintained vascular homeostasis. The role of EPCs in certain diseases has also been studied. The level of EPCs predicts the occurrence of cardiovascular events and may help to identify patients with a high risk of cardiovascular diseases [
4]. In ANFH, Sun et al. [
5] found transplantation of EPCs promoted vascularization and bone regeneration in the early stage of ANFH in a rabbit model. Though the anti-inflammatory effect of GCs helps to stabilize the endothelium, the effects of GCs on endothelial progenitor cells are still controversial. Grisar et al. [
6] found loss of EPC function and number in SLE patients and dexamethasone treatment significantly increased patients’ EPC population and CFU formation. Feng et al. [
7] found EPCs’ number was reduced in ANFH patients with different risk factors including the use of steroids. Paradoxically, Chen et al. [
8] reported an unchanged number of circulating EPCs in GC-induced ANFH patients. Moreover, as published literatures discussing GCs’ effect on EPCs were mostly based on in vivo studies, the direct effect of GCs on circulating EPCs in vitro has never been extensively studied. The change of EPCs in ANFH patients and the mechanisms of how GCs affect EPCs’ population and function still need further investigation.
In the current study, we investigated the population and function of circulating EPCs in ANFH patients by using a combination of EPC markers different from published literatures. In addition, we also studied and compared the effects of methylprednisolone and prednisolone on EPCs in vitro. We demonstrated for the first time that GCs showed significant inhibitory effect on EPC function of angiogenesis in vitro, while the growth of EPC was only inhibited under extremely high GC concentrations. We also demonstrated that steroids significantly downregulated CXC chemokine receptor 7 (CXCR7), which may be an important mechanism of suppressed EPCs’ angiogenesis after GC treatment in vitro.
Methods
Participant selection
Between March 2016 and September 2017, 10 adult male patients with glucocorticoid-induced ANFH (average age 37.97 years, ranging from 20 to 40 years) were recruited in the authors’ department. Ten healthy volunteers with similar age and body weight (average age 38.73 years, ranging from 20 to 40 years) were also recruited as a control group. Clinical backgrounds of all participants were summarized in Table
1. GC-induced ANFH was diagnosed by a long-term steroid use history, complaint of hip pain, and positive X-ray and magnetic resonance imaging [
1,
9]. Exclusion criteria included age under 18 years old, cancer, pregnancy, diabetes mellitus, current and previous bone infections, immunosuppressive drug therapy, history of inflammatory arthritis, cardiovascular diseases, impaired renal function, and mental health problems [
10,
11].
Table 1
Clinical characteristics of study subjects
Age (years) | 37.97 ± 8.99 | 38.73 ± 8.18 |
Body mass index (kg/m2) | 24.39 ± 4.00 | 23.67 ± 4.46 |
Systolic BP (mmHg) | 120.37 ± 13.12 | 109.57 ± 8.96 |
Diastolic BP (mmHg) | 79.40 ± 10.78 | 71.73 ± 7.07 |
Blood glucose (mmol/L) | 5.00 ± 0.48 | 4.99 ± 0.43 |
Isolation and culture of circulating EPCs
Peripheral blood sample (20 ml) was collected and centrifuged using the Ficoll-density gradient method. Buffy coat mononuclear cells (MNCs) were collected and washed with HANKs’ balanced salt solution twice. MNCs were then resuspended with EGM-2 medium (Lonza, San Diego, CA) and plated on human fibronectin-coated plates (BD Biosciences, Bedford, MA) at a density of 4 × 10
6 cells. Culture medium was changed every 3 days, and passage was performed when cell colonies were formed [
12,
13].
Characterization of EPCs
For flow cytometry, EPCs on day 7 were detached and resuspended in PBS. After Fc-blocking, 1.0 × 106 cells were incubated with conjugated anti-CD133-PE (BD Biosciences, San Jose, CA) and CD34-FITC (BD Biosciences, San Jose, CA) for 20 min at 4 °C. Cells were analyzed using Beckman Coulter FC500 flow cytometer. To observe DiI-ac-LDL uptake and FITC-UEA-1 binding, EPCs were incubated with 2.4 μg/mL Dil-Ac-LDL (Invitrogen-Molecular Probes, Eugene, OR) in EGM-2 medium at 37 °C for 4 h. Then, cells were fixed in 4% paraformaldehyde and further incubated with 20 μg/mL FITC-UEA-1 (Introvogen-Molecular Probes, Eugene, OR) for 1 h. For immunofluorescence staining, EPCs were fixed in 4% paraformaldehyde for 10 min, blocked in 5% normal goat serum, and then incubated with anti-CD133-PE (BD Biosciences, San Jose, CA) and anti-CD34-FITC (BD Biosciences, San Jose, CA) antibodies at 4 °C overnight. Fluorescence was observed under a confocal laser scanning microscope.
Migration assay
Migration assay was performed on the Zigmond chambers (Neuro Probe, Gaithersburg, MD). Twenty microliters of complete EGM-2 medium with EPCs from patients or healthy controls were seeded in the left Zigmond chamber and 20 μl complete EGM-2 medium containing only SDF-1α (200 ng/ml) was added to the right chamber. Cell migration was observed during the next 24-h culture and photographs were recorded every 5 min by a digital camera mounted on a light microscope. Migration distances of the cells were observed and quantified by using the Leica Q-Win software.
Cell cytotoxicity assay
The Cell Counting Kit-8 (CCK-8) assay was used to determine the effect of GCs on EPCs. EPCs were seeded at a density of 1 × 103 cells/well in a 96-well plate in triplicates and a series of concentrations (0.4, 4, 40, 400, and 4000 μg/ml) of GCs (prednisolone or methylprednisolone) were applied to the culture medium. After a 72-h culture, CCK-8 assay was performed according to the manufacturer’s instructions. Absorbance was observed at a wavelength of 450 nm by a microplate reader (Multiskan MK3; ThermoFisher Scientific, Waltham, MA).
Matrix Matrigel (BD Biosciences, San Jose, CA) was prepared according to the manufacturer’s instructions. After a 7-day culture, EPCs were collected and plated at a density of 5 × 104/well on top of Matrigel in a 96-well plate in triplicates. Cells were cultured with a series of concentrations (0, 0.4, 4, 40, and 400 μg/ml) of GCs (prednisolone or methylprednisolone) in the medium for 3 days. The diameters of closed network units formed by cells in six continuous visual fields were counted and analyzed by Leica Q-Win software.
Western blot assay
Normal EPCs were treated with 40 μg/ml methylprednisolone or the same volume of PBS for 24 h before harvest using a regular lysis buffer containing protease inhibitor. The total protein concentration was measured by Bradford assay (BioRad, CA, USA) following the instructions of the manufacturer. Thirty micrograms of protein of each sample was separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a PVDF membrane (EMD Millipore, Darmstadt, Germany). An immunoblotting analysis was performed. Briefly, the membranes were blocked with 5% Bovine Serum Albumin (Sigma-Aldrich, Missouri, USA) in Tris-buffered saline with 0.5% Tween 20 (TBST) for an hour at room temperature, then incubated with the appropriate primary antibody overnight. Primary antibodies were diluted as instructed by the manufacturers. Primary antibodies include anti-GAPDH (PTG, Rosemont, USA), anti-CXCR7 (Abcam, Cambridge, UK), anti-GSK3β (Abcam, Cambridge, UK), anti-phospho-GSK3β (Abcam, Cambridge, UK), anti-AKT (Cell Signaling Technology, Massachusetts, USA), anti-phospho-AKT (Abcam, Cambridge, UK), and anti-FYN (Abcam, Cambridge, UK). The membranes were washed with TBST buffer and then incubated with HRP-conjugated anti-rabbit or anti-mouse secondary antibody for an hour at room temperature. Then, the membranes were washed and exposed to the enhanced chemiluminescence.
Statistics
All data were expressed as mean values ± standard derivation with a 95% confidence interval. Statistical analysis was performed by paired Student’s t tests (two-tailed) or variance analysis using a SPSS 13.0 software and P < 0.05 was considered significant.
Discussion
The defect of microcirculatory balance is an important mechanism for the onset and progression of GC-induced ANFH since it results in chronic regional ischemia and endothelial cell impairment in the femoral head [
19‐
21]. Increasing evidences have shown that EPCs play a pivotal role in neovascularization and vascular repair, particularly under ischemic conditions [
22,
23]. Based on these findings, emerging studies have focused on the role of dysfunction of EPCs in the pathogenesis of ANFH. Though contradictive results have been shown for the number of circulating EPCs in ANFH patients, our study further proved that in GC-induced ANFH patients, both the number and function of circulating EPCs were significantly decreased. The changes of EPCs may result in a microcirculatory imbalance and contribute to the progression of the disease. Sun et al. [
5] performed EPC transplantation on rabbit glucocorticoid-induced ANFH model and found EPC transplantation combined with core decompression promoted the neovascularization and bone regeneration. Based on our and others’ studies, EPC transplantation may be a novel therapy to treat glucocorticoid-induced ANFH in the future.
Asahara et al. [
3] first described human circulating angioblasts as a population of cells which were able to differentiate into epithelium cells in vitro. These so-called epithelial progenitor cells significantly contributed to neovascularization after tissue ischemia in vivo. Though markers like CD34, VEGFR2, and CD133 are typically used to identify EPC, the definition of EPC is still controversial [
24]. Experimental results may be divergent when different combinations of EPC markers are used. In ANFH patients, Feng et al. [
7] found that the number of CD34
+VEGFR2
+ cells was decreased in peripheral blood of ANFH patients. In contrast, Chen and colleagues [
8] found no difference in the number of circulating CD133
+VEGFR2
+ cells between ANFH patients and healthy controls. Our study showed that in ANFH patients, the population of circulating CD133
+CD34
+cells was significantly reduced and EPCs’ functions were also impaired. By using a different combination of EPC markers, our results further proved that GC-induced ANFH patients had less circulating EPCs, which may be a major cause for the disease progression.
Migration and homing are critical for circulating EPCs to function in distal organs and ischemic tissues. Stromal cell-derived factor-1 (SDF-1) is a chemokine considered to play an important role in the recruitment of EPCs for ischemic neovascularization [
25]. In our study, we for the first time used the Zigmond chamber with SDF-1 as the chemo-attractant to evaluate the migration of EPCs. Instead of measuring the number of cells moved through the pores towards the chemoattractant in transwells [
7,
8], the Zigmond chamber allows accurate quantification of the cells’ migration distance and orientation by utilizing high-resolution light microscopy. By creating a SDF-1 concentration gradient, Zigmond chamber mimics the physiological environment in which circulating EPCs migrated towards distal organs in the peripheral blood. Transwell studies have shown that less EPCs migrated to VEGF in ANFH patients. By directly visualizing the trace of migration and quantification of moving distance, our study further demonstrated that EPCs’ migration ability was significantly impaired in ANFH patients when SDF1 was used as the attractant.
Though the use of steroids may affect the number and function of circulating EPCs in vivo, the direct effect of GCs on EPCs in vitro has not been extensively studied. Grisar et al. [
26] showed that dexamethasone administration led to significantly increased CFU formation of EPCs in vivo. Aschbacher et al. [
27] found cortisol impaired circulating angiogenic cell (CAC) migratory function and VEGF secretion in vitro. In our study, the effects of two common synthetic glucocorticoids, prednisolone and methylprednisolone, on EPCs in vitro were investigated. We demonstrated that only extremely high concentrations (0.4 and 4 mg/ml) of GCs significantly inhibited cell growth while low to medium concentrations (0.4 to 40 μg/ml) showed no effect. Since the peak serum concentrations of GCs after a high dose of oral prednisolone or pulse intravenous methylprednisolone generally range from 7 to 34 μg/ml [
28], our results suggest that regular dosage of clinical steroid administration may never reach a serum concentration high enough to directly suppress the growth of EPCs. The lower number of EPCs in ANFH patients may be attributed to an indirect effect of prolonged exposure to GCs or other mechanisms. However, GCs significantly suppressed tube formation by EPCs in vitro even at low concentrations. With increased concentrations, the inhibitory effects of both synthetic GCs became more prominent. These results suggest that patients with long-term steroids treatment may have significantly suppressed neovascularization because of dysfunction of EPCs. The impaired angiogenic function of EPCs may also be an important mechanism in the progression of GC-induced ANFH.
Though GCs directly inhibit angiogenesis by EPCs in vitro, the underlying mechanism is not clear. Emerging evidences have indicated that CXCR7 is critical in the regulation of EPCs’ survival, angiogenic, and migratory functions in human and mouse [
16‐
18,
29]. CXCR7 antagonist significantly blocked human EPCs’ tube formation in vitro [
16]. In the current study, we also found that methylprednisolone downregulated the expression of CXCR7 in EPCs in vitro. Attenuated Akt/GSK-3
β phosphorylation and increased Fyn expression were also found after methylprednisolone treatment. Akt and GSK-3β/Fyn are important molecules downstream of CXCR7 and were responsible for the angiogenic function of EPCs [
18,
30,
31]. Upregulation of Fyn may increase the degradation of Nrf2, which causes oxidative stress damage and subsequent impairment of angiogenesis [
18]. These results further suggested that downregulation of CXCR7 and downstream Akt/GSK-3
β pathway is a novel mechanism of how GCs impair EPCs’ angiogenic function. In addition, as a receptor of SDF-1, downregulation of CXCR7 in EPCs by GCs may also explain why EPCs from GC-induced ANFH patients had decreased migratory ability when SDF-1 was used as chemo-attractant.
Conclusion
In conclusion, our study showed that glucocorticoid-induced ANFH patients have reduced the number and impaired functions of circulating EPCs. GCs did not show a significant effect on the growth of EPCs in vitro except extremely high concentrations of GCs. However, GCs significantly impaired EPCs angiogenic function in vitro, even at low concentrations. These results indicated that EPC dysfunction may be an important reason for the development and progression of GC-induced ANFH. In addition, our study also suggested that downregulation of CXCR7 and its downstream Akt/GSK-3β/Fyn pathway in EPCs might be a novel mechanism of how GCs suppress EPCs’ angiogenesis.
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