Comparative study of serum proteomes in Legg-Calve-Perthes disease.

BACKGROUND: Legg-Calve-Perthes Disease (LCPD) is an idiopathic osteonecrosis of the developing femoral head complicated by pain and disability of the hip joint. To date, the pathological mechanisms of LCPD are not well-known. This study screened the changes in serum protein expression in patients with LCPD. METHODS: Age- and sex-matched serum samples from 10 control subjects and 10 patients with LCPD were compared using the isobaric tags for relative and absolute quantification (iTRAQ) technique. Gene ontology analyses, KEGG pathway and functional network analyses were performed. Proteins of interest with large differences in expression, S100-A8, alpha-1-acid glycoprotein 1, haptoglobin and apolipoprotein E, were compared by western blotting. RESULTS: The disease/control ratios showed 26 proteins were significantly differentially expressed (all p < 0.05). Including higher abundances of complement factor H (1.44), complement C4-B (1.45), isocitrate dehydrogenase [NAD] subunit alpha (2.7) alpha-1-acid glycoprotein 1 (1.87), heptoglobin (1.53) and Ig lambda-2 chain C regions (1.46), and lower levels of apolipoprotein E (0.50), apolipoprotein F (0.60), apolipoprotein C-III (0.69), S100-A8 (0.73), S100-A9 (0.75) and prothrombin (0.77) in LCPD than in controls. The alpha-1-acid glycoprotein 1 and haptoglobin increases, and apolipoprotein E and S100-A8 decreases were confirmed by western blot. KEGG pathway analysis revealed these proteins were related to the complement and coagulation cascades, Staphylococcus aureus infection, PPAR signaling, fat digestion and absorption, and vitamin digestion and absorption. Functional network analysis suggested that the proteins were involved in lipid regulation. CONCLUSIONS: The complement and coagulation cascades, and abnormal lipid metabolism may be involved in the pathogenesis of LCPD.

Increased circulating CD31+/CD42b-EMPs in Perthes disease and inhibit HUVECs angiogenesis via endothelial dysfunction.

AIMS: Endothelial microparticles (EMPs) are extracellular vesicles secreted by endothelial cells. The purpose of this research is to explore that the clinical significance and roles in angiogenesis and endothelial dysfunction of circulating microparticles in Perthes disease. MAIN METHODS: We collected platelet-poor plasma (PPP) from patients and controls, then microparticles (MPs) were extracted. Flow cytometry was performed to calculate the concentrations of CD31+/CD42b-, CD62E+ and CD31+/CD42b+ MPs. ELISA was performed to detect the expression level of biomarkers of endothelial dysfunction and inflammatory factors in plasma. In vitro experiments to evaluate the effect of circulating MPs and EMPs derived from IL-6-stimulated human umbilical vein endothelial cells (HUVECs) on angiogenesis and endothelial dysfunction. KEY FINDINGS: Our results revealed that the CD31+/CD42b- EMPs were significantly higher in Perthes disease group than in the control group. The Perthes-MPs being taken up by HUVECs promoted endothelial cell apoptosis, endothelial dysfunction and inhibited angiogenesis in vitro. Moreover, the level of IL-6 in plasma significantly increased in patients with Perthes, which was tightly correlated with the elevated level of circulating CD31+/CD42b- EMPs. IL-6 promoted HUVECs to secrete CD31+/CD42b- MPs, and EMPs derived from high concentration IL-6-stimulated (100 and 1000 pg/mL) HUVECs promoted endothelial cell apoptosis, endothelial dysfunction and inhibited angiogenesis. SIGNIFICANCE: In summary, our study suggests that circulating EMPs in the phenotypic spectrum revealed unique phenotypes of endothelial dysfunction, showing close correlation with the secretion of IL-6. These circulating EMPs may give rise to endothelial cell apoptosis, endothelial dysfunction and angiogenesis in Perthes disease.

The role and underlying mechanisms of microRNA­214 in Legg­Calvé­Perthes disease.

Legg­Calvé­Perthes disease (LCPD) is a pediatric form of femoral head osteonecrosis with unknown etiology. MicroRNAs (miRs) have been revealed to serve an important role in LCPD. MiR­214 serves an important role in chondrogenesis. The aim of the present study was to investigate the potential role of miR­214 in LCPD and the underlying mechanisms. The expression levels of miR­214 and B­cell lymphoma 2 (Bcl­2)­associated X protein (Bax) in dexamethasone (DEX)­treated TC28 cells, and the femoral head cartilage tissues, serum and primary chondrocytes of patients with LCPD, and healthy individuals were determined via reverse transcription quantitative polymerase chain reaction and western blot analysis. A luciferase reporter assay was conducted to investigate the association between miR­214 and Bax, while cell viability was determined via an MTT assay, and flow cytometry was performed to investigate cell apoptosis. The results revealed that miR­214 was downregulated and Bax was upregulated in DEX­treated TC28 cells and tissues obtained from patients with LCPD. MiR­214 was demonstrated to directly target Bax and negatively regulate its expression. DEX administration significantly suppressed cell proliferation, promoted apoptosis and decreased the Bcl­2/Bax ratio in TC28 cells; overexpression of miR­214 induced opposing effects, which were reversed by Bax overexpression. In conclusion, the results indicated that miR­214 and Bax may be potential therapeutic targets for the future clinical treatment of LCPD.

The role of protein C deficiency in the etiology of Perthes disease.

BACKGROUND: Recent reports support the hypothesis that thrombophilia plays an important role in the pathogenesis of Perthes disease (PD) and the objective of this report is to show evidence of the role of protein C deficiency in the etiology of PD, based on a meta-analysis of current scientific literature. MATERIAL AND METHODS: Studies were selected in all languages over the last twenty years (1986 to 2006) in MEDLINE, LILACS and EMBASE data bases. The inclusion criteria involved controlled studies, those that presented protein C as a continuous variable, and studies conducted in children with Perthes disease. The fixed effect model for continuous data was used; differences between groups were assessed by the t-test, Z-test and Cochran Q test for independent data and the level of significance was p < 0.05. RESULTS: The selected studies involved 175 patients and 193 control subjects. The selected studies were shown to be heterogeneous, but there were no statistically significant differences in protein C levels between groups. CONCLUSION: The authors' findings were unable to support the hypothesis that protein C deficiency is associated with Perthes disease and that it may play an important role in the ethiopathogenesis of avascular necrosis of the femoral head in childhood.

Is VEGF under-expressed in Indian children with Perthes disease?

BACKGROUND: The role of vascular endothelial growth factor (VEGF) after ischaemic necrosis of the femoral head in Legg-Calve-Perthes disease (LCPD) has not been adequately studied in humans, especially in Indian population. Therefore, we aimed to evaluate the serum levels of VEGF-A in Indian children with various stages of LCPD and compare them with those of an age- and sex-matched control group of healthy children. METHODS: In this case-control study, we enrolled 42 children (below 14 years age) suffering from LCPD and 21 age- and sex-matched healthy controls. Patients were classified radiographically according to Waldenstrom's classification. Serum VEGF-A was estimated by sandwich enzyme-linked immunosorbent assay technique. The serum values were compared between the patient group and the control group, as well as between the Waldenstrom subgroups. Results were expressed as means with ranges or median with interquartile range. RESULTS: The mean age in the patient as well as the control group was 9 years (range 4-13 years). The median value (interquartile range) of serum VEGF-A was 162.5 pg/ml (673.75 pg/ml) in the patient group and 652 pg/ml (190.5 pg/ml) in the control group (p = 0.013). When compared between lower Waldenstrom stages (initial stage + stage of fragmentation) and higher Waldenstrom stages (re-ossification stage + stage of healing), the mean values of serum VEGF-A were 464.7 pg/ml (range 0-2211 pg/ml) and 301.1 pg/ml (range 0-1910 pg/ml), respectively (p = 0.305). CONCLUSIONS: VEGF is under-expressed in Indian children suffering from LCPD. As VEGF acts as a key regulator of endochondral ossification, our finding may open new therapeutic approaches to the disease. Also, serum VEGF may act as a valuable marker for the follow-up of the disease. Our study also provides baseline data about serum VEGF-A levels in Indian cohort of LCPD patients. Future multi-centre studies are warranted with a larger sample size to fully appreciate the patho-physiological changes in VEGF occurring in LCPD.

Thrombophilia and Legg-Calvé-Perthes disease: is it a causative factor and does it affect the severity of the disease?

Coagulation parameters were studied in a population of 118 children with Perthes disease in order to determine the possible role of thrombophilia as a causative factor for the disease and to determine if thrombophilia could affect its course. We found 27 children presenting one or more coagulation disorders. The statistical analysis concurs with previous findings of a relationship between Legg-Calvé-Perthes disease and an increased liability to thrombosis; however, no significant effect of thrombophilia on the severity of the disease could be demonstrated.

Alterations of serum osteocalcin levels in patients with Legg-Calvé-Perthes.

BACKGROUND: Legg-Calvé-Perthes disease (LCPD) is a paediatric form of osteonecrosis that ultimately heals but will cause femoral head and acetabular deformities. The purpose of this study was to investigate the early serum measurement of osteocalcin in children with femoral head necrosis compared with that of healthy children. METHODS: 20 patients with LCPD (4 girls and 16 boys) and 20 healthy volunteers (5 girls and 15 boys) were enrolled. All patients did not have additional treatment. Blood samples were obtained from all patients preoperatively in the morning. All cases had hip radiographs. The serum levels of osteocalcin comparisons between the LCPD patients and the healthy volunteers were performed using Wilcoxon signed-rank test. The Spearman rank correlation was used to assess correlation between LCPD grade and serum osteocalcin levels. Significance was set at p = 0.05. RESULTS: The 20 patients with LCPD (72.75 ± 24.92 ng/ml) had significantly higher serum osteocalcin levels compared with the healthy group (16.80 ± 4.04 ng/ml) (p<0.01). Serum osteocalcin levels of different LCPD grades were significantly different (Spearman's p = 0.540, p = 0.014). CONCLUSIONS: We observed a significant correlation between serum osteocalcin levels and LCPD. These results may be meaningful in clinical practice and to future studies.

Neutrophil to lymphocyte ratio may be a predictive marker of poor prognosis in Legg-Calvé-Perthes disease.

BACKGROUND: Legg-Calvé-Perthes disease (LCPD) is the idiopathic avascular necrosis of the femoral head in childhood. The pathologic changes seen in the femoral head are likely a result of vascular factors. Blood neutrophil to lymphocyte (N/L) ratio is a simple marker of subclinical inflammation. This study aims to to analyse the predictive ability of N/L ratio for the prognosis in LCPD patients. METHODS: Patients who had been diagnosed as LCPD from 2008 to 2014 were investigated retrospectively and 40 LCPD patients (33 male and 7 female) and 25 healthy age and sex-matched children (controls) were included in the study. LCPD patients were divided into 2 groups according to expected prognosis (good prognosis expected Herring A and B patients as Group I and poor prognosis expected Herring B-C and C patients as Group II) and healthy children (control) were included in Group III. All the patients' hematological markers were analysed. RESULTS: Mean age was 7.1 ± 2.0 years in group I (4.9-12 years), 8.3 ± 2.2 years (4-12.5 years) in group II and 7.8 ± 1.3 years (6-12 years) in group III. Mean values for Groups I, II and III for neutrophil to lymphocyte (N/L) ratio were 1.13 ± 0.65, 1.75 ± 0.95, 1.08 ± 0.37, respectively. The mean neutrophil to lymphocyte (N/L) ratio of Group II was higher than the other 2 groups (p = 0.003). CONCLUSIONS: N/L ratio may give us information about the natural course of LCPD and may be used as independent predictor of prognosis in patients with LCPD.

Legg-Calve-Perthes disease and thrombophilia.

BACKGROUND: Thrombophilia has previously been identified as a potential etiologic factor in Legg-Calve-Perthes disease. We prospectively studied the association between Legg-Calve-Perthes disease and coagulation abnormalities by comparing seventy-two children who had the disease with 197 healthy controls. METHODS: A nonselected, consecutive series of seventy-two patients with Legg-Calve-Perthes disease (mean age [and standard deviation], 6.6 +/- 2.6 years) was studied in their order of referral and compared with 197 healthy controls (mean age, 7.6 +/- 5.1 years). Assays were done for factor-V Leiden, prothrombin G20210A, methylenetetrahydrofolate reductase C677T, and plasminogen activator inhibitor-1 4G/5G gene mutations. Levels of anticardiolipin antibodies immunoglobulin G and M (IgG and IgM), homocysteine, protein C, protein S, antithrombin III, and plasminogen activator inhibitor-1 were also measured. RESULTS: The factor-V Leiden mutation was more common in the patients (eight of seventy-two) than in the controls (seven of 197) (chi-square = 5.7, p = 0.017). After we controlled for the false-discovery rate, the case-control difference remained significant (p = 0.017). The odds ratio for the development of Legg-Calve-Perthes disease in the presence of the factor-V Leiden mutation was 3.39 with a 95% confidence interval of 1.18 to 9.73. A high level of anticardiolipin antibodies (IgG and/or IgM) was found in nineteen of the seventy-two patients compared with twenty-two of the 197 controls (chi-square = 9.5, p = 0.002). After we controlled for the false-discovery rate, the case-control difference remained significant (p = 0.002). The odds ratio of patients with Legg-Calve-Perthes disease having one or more abnormalities in factor V, anticardiolipin antibody IgG, or anticardiolipin antibody IgM as opposed to normal values for all three variables was 3.29 (95% confidence interval, 1.73 to 6.24; p = 0.0003). CONCLUSIONS: Two thrombophilic risk factors, the factor-V Leiden mutation and anticardiolipin antibodies, are associated with Legg-Calve-Perthes disease, an association that may reflect causality. LEVEL OF EVIDENCE: Prognostic study, Level II-1 (retrospective study). See Instructions to Authors for a complete description of levels of evidence.

Prospective reevaluation of the association between thrombotic diathesis and legg-perthes disease.

BACKGROUND: Legg-Perthes disease is associated with ischemia of the capital femoral epiphysis in children. Thrombophilia has been implicated as a potential cause of the condition, and screening of patients with Legg-Perthes disease for thrombophilia has been recommended. We analyzed the value of screening for inherited thrombophilia in patients with Legg-Perthes disease by examining the association between Legg-Perthes disease and abnormalities in the thrombotic pathway. METHODS: A random series of consecutive patients with Legg-Perthes disease were prospectively enrolled in this study. Assays for the detection of factor-V Leiden mutation and the plasma concentrations of protein C, protein S, antithrombin III, and lipoprotein (a) were performed on plasma samples from children with Legg-Perthes disease, and the results were compared with those for pooled plasma from normal controls. Plasma concentrations below the 95% midrange of the control values were classified as protein deficiencies. The estimated population frequency of each coagulation abnormality was compared with the proportion of the study group with the corresponding abnormality. RESULTS: The proportion of abnormalities observed in the study group did not differ from the estimated population frequency for protein C, protein S, antithrombin III, or factor-V Leiden mutation. A lipoprotein (a) level of >30 mg/dL (>1.07 micro mol/L) was found in 16% of the study group. CONCLUSIONS: Our data do not suggest that thrombotic diatheses due to deficiency of protein C, protein S, or antithrombin III or due to factor-V Leiden mutation are major causes of Legg-Perthes disease. The elevated levels of lipoprotein (a) in children with Legg-Perthes disease suggest that they may be at risk for atherosclerosis as adults.

Thrombotic and fibrinolytic alterations in the aseptic necrosis of femoral head.

Recent reports seem to support the role of the thrombophilia and decreased fibrinolysis in the aetiopathogenesis of aseptic necrosis of bone. In the present study, haemostatic disturbances were analysed in adults (n = 49) and patients in childhood (Perthes disease) (n = 47) with aseptic necrosis of the femoral head. Fibrinolytic parameters (in vitro clot lysis, plasminogen, plasmatic plasminogen activator inhibitor-1 activity, D-dimer) along with lipoprotein (a) [Lp(a)] and fibrinogen were measured. von Willebrand factor, platelet activation and some thrombophilic factors (activated protein C resistance and factor V Leiden mutation, protein C, protein S activity) were also determined. Impaired fibrinolysis, an increased Lp(a) level along with slow clot lysis and increased platelet activation were found in adult cases. We detected five cases of factor V Leiden mutations (one heterozygotic and four homozygotic) among patients with Perthes disease. The clinical course of the heterozygous case was similar to the usual form of Perthes disease. The most severe form of Perthes disease has been observed in homozygous factor V Leiden mutation cases. The mutation of factor V Leiden per se probably does not induce the development of aseptic necrosis of bone tissue in childhood, but it does play a role in its acceleration. Homozygous factor V Leiden mutation definitely runs a more severe course. On the other hand, in adult cases, the disturbances of haemostasis, impaired fibrinolysis, elevated Lp(a) level, increased platelet activation and slight elevation of fibrinogen might have clinical relevance. Further studies should focus on proving the role of the haemostatic alterations in the pathogenesis of severe forms of aseptic bone necrosis. The use of antithrombotic drugs in order to slow the process of aseptic necrosis also has to be addressed in future surveys.

The role of coagulation abnormalities in the development of Perthes' disease.

Recent reports have suggested an association between Perthes' disease and an underlying thrombophilic or hypofibrinolytic tendency. In Northern Ireland there is a high incidence of Perthes' disease (11.7 per 100,000 or 1 in 607 children) in a stable paediatric population. We reviewed 139 children with Perthes' disease and compared them with a control group of 220 aged- and gender-matched healthy primary schoolchildren with similar racial and ethnic backgrounds. There were no significant deficiencies of antithrombotic factors protein C, protein S, antithrombin III or resistance to activated protein C. A total of 53 (38.1%) of the children with Perthes' disease had a prolonged activated partial thromboplastin time (>38) compared with 13 (5.9%) of the control group (p < 0.001). Our findings have shown that using standard assays, thrombophilia secondary to antithrombotic factor deficiency or resistance to activated protein does not appear to be an aetiological factor for Perthes' disease. The cause of the prolonged activated partial thromboplastin time, usually associated with a clotting factor deficiency, is under further investigation.

Does thrombophilia play an aetiological role in Legg-Calvé-Perthes disease?

Heritable thrombophilic disorders have been proposed as one of the causes for Legg-Calvé-Perthes disease. A total of 62 patients diagnosed with this disease between 1988 and 1997 and 50 controls were screened for thrombophilia. The incidence and relationship of thrombophilia to the severity of the disease were evaluated. One patient and none of the controls had protein S deficiency. One of the control group and one of the patients had protein C deficiency with the latter child also having a combined deficiency with a mutant factor V gene. The number of children with a mutant factor V gene, protein C deficiency, who were homozygous for the C 677T polymorphism of methylenetetrahydrofolate reductase or were heterozygous for mutant G20210A prothrombin did not differ statistically in the study and the control groups. No patient had antithrombin deficiency or positive lupus anticoagulant. We found no correlation between thrombophilia and the extent of the disease. The most common risk factors for arteriovenous thromboembolism showed no statistical significance in our patients compared with the control group or with the general population. These data do not confirm an aetiological role for thrombophilia in Perthes' disease.

Perthes' disease and the relevance of thrombophilia.

Recent work has suggested that thrombophilia may be an aetiological factor in up to 50% of children with Perthes' disease, and that up to 75% may have a coagulopathy. Our aim was to test these findings in the local population of children with Perthes' disease and attempt to correlate them with the severity of the condition. In 64 children there were only eight (12%) with low levels of clotting proteins, as defined by normal paediatric ranges. Of these eight, only five could be said to show any thrombophilic tendency.

Serum immunoglobulin in Perthes' disease.

Serum immunoglobulin concentrations in 41 children with Perthes' disease and 82 age and sex matched controls were measured by radial immunodiffusion. Significant increases in IgG and IgM were seen in children with Perthes' disease.

Absence of congenital prethrombotic disorders in children with Legg-Perthes disease.

Resistance to activated protein C (RPCA) and other congenital prethrombotic disorders have been recently reported to be strongly associated with Legg-Perthes disease. RPCA and deficiencies of protein C, protein S, and antithrombin III were sought in 22 children with Legg-Perthes disease. Detection of the factor V Leiden mutation was found in children with RPCA. Twenty-two healthy children paired by age and sex served as controls. The prevalence of congenital prethrombotic disorders was not found to differ significantly among patients with Legg-Perthes disease and among control subjects. Only one patient had RPCA; this patient was heterozygous for the factor V Leiden mutation. Twenty patients and all the control subjects had entirely normal coagulation results. The authors conclude that unless more data become available, RPCA and deficiencies of protein C, protein S, and antithrombin III should not be considered associated with Legg-Perthes disease.

Psychosocial development and premorbid skeletal growth in Legg-Calvé-Perthes disease: a study of nineteen patients.

We studied psychosocial development and skeletal growth in 19 newly diagnosed patients with Legg-Calvé-Perthes disease (LCPD). Eleven patients had problems in visuospatial skills and five of 12 school-aged children had learning difficulties. The growth velocity of the patients was evaluated from 4 years before until 2 years after the diagnosis was made. Eight patients had a catch-up growth with +1.2 (0.9-1.7) delta SDS score (SDS: mean and ranges) before the diagnosis. Four patients with short stature and retarded bone age slightly diminished their growth velocity. Overnight serum growth hormone (GH) concentration and insulin-like growth factor I (IGF-I) levels were examined in the first nine consecutive patients. One patient had a high and another had a low mean GH concentration level, whereas all patients had IGF-I levels within normal limits. These results suggest that different kinds of growth disturbances may be associated with LCPD.

[Diagnostic significance of certain cellular immune system disorders in children with Perthes disease]. / Diagnosticheskoe znechenie nekotorykh narushenii kletochnogo zvena immunnoi sistemu u detei s bolezn'iu Pertesa.

Data are submitted to the effect that parameters associated with the cell link of the immune system should allow some judgement about character and degree of time-related immunological abnormalities in children with Perthes disease. In the earliest stages of the illness immunological abnormalities are in large measure related to the presence of foci of chronic bacterial infection as evidenced by a high NBT-test. The above immunological abnormalities aggravate the course of aseptic necrosis of the hip joint or they serve as a trigger mechanism of its development.

[Mechanism of early metabolism disorders and immunological reactivity in children with Perthes disease]. / O mekhanizme rannikh narushenii obmena i immunologicheskoi reaktivnosti u detei s bolezn'iu Pertesa.

Submitted in the paper are data to the effect that parameters of certain biochemical indices for blood plasma and cell link of the immune system could allow some judgement about the character of the pathological process in children with Perthes disease. Autoimmune inflammation caused by foci of chronic infection in the organism aggravates the course of aseptic necrosis in the proximal portion of the thighbone or else is a trigger mechanism of its development.

Effects of circulating endothelial progenitor cells, serum vascular endothelial growth factor and hypogammaglobulinemia in Perthes disease.

OBJECTIVE: The aim of this study was to investigate Legg-Calvé-Perthes disease (PD) pathogenesis by comparing absolute circulating endothelial progenitor cell (EPC) counts, serum levels of vascular endothelial growth factor-A (VEGF-A) and immunoglobulins between PD patients and controls. METHODS: The study included 28 PD cases (mean age: 8 ± 3.8) and 25 healthy age-matched control subjects. EPC, serum VEGF-A and immunoglobulin levels were measured in peripheral blood samples. Comparisons and correlation analysis were performed. RESULTS: In the PD group, 17 subjects were in the fragmentation stage and 11 in the healing stage. Four patients had bilateral disease and 14 had hypogammaglobulinemia. Median EPC count of the PD group was 80 and was significantly higher than those of the control group (p=0.011). No significant difference was determined in serum VEGF-A levels (p=0.354). EPC count were inversely correlated with serum IgG levels of the PD group (r=0.403, p=0.03). Absolute EPC count was also significantly higher in the fragmentation stage than in the healing stage and were also greater in bilaterally affected than in unilaterally affected patients. Circulating EPC count was correlated to the serum VEGF-A levels in patients with fragmentation stage of PD (r=0.605, p=0.01) and in those with hypogammaglobulinemia (r=0.599, p=0.001). CONCLUSION: High EPC count at the fragmentation stage of PD and relatively higher counts in bilateral disease suggest that EPC may be a valuable marker in the diagnosis and follow-up of PD. Additional studies are needed to explain the strong correlation between EPC and serum VEGF-A level in the fragmentation stage and in the presence of hypogammaglobulinemia.