Masayasu Kitano

Clinical significance of vascular endothelial growth factor and hepatocyte growth factor in multiple myeloma

Summary. Angiogenesis is a crucial process in the progression of multiple myeloma (MM). Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) are multifunctional cytokines that potently stimulate angiogenesis including tumour neovascularization. Serum levels of VEGF and HGF were measured in 52 patients with MM by enzyme‐linked immunosorbent assay (ELISA). Serum levels of VEGF and HGF were elevated in MM patients compared with healthy controls (VEGF: mean 0·31 ng/ml and 0·08 ng/ml respectively, P < 0·01; HGF: mean 2·17 ng/ml and 0·45 ng/ml, respectively, P < 0·001). In serial samples taken after chemotherapy, serum VEGF and HGF levels were correlated with M‐protein levels. Serum levels of VEGF were higher in patients with extramedullary plasmacytomas than in patients without them (P < 0·05). They were also significantly higher in a group of patients who showed poor response to chemotherapy (P < 0·01). Serum levels of HGF were higher in patients with complications such as anaemia, hypercalcaemia and amyloidosis than in patients without these complications (P < 0·01, P < 0·05, P < 0·05 respectively). Both serum VEGF and HGF levels were significant predictors of mortality (P = 0·01, P = 0·02, respectively, log‐rank test). The present study demonstrated that serum levels of VEGF and HGF are significantly elevated and dependent on the severity of MM, suggesting that measurement of VEGF and HGF may be useful for assessing disease progression and for predicting the response to chemotherapy in MM patients.

Enhanced production of osteopontin in multiple myeloma: clinical and pathogenic implications

Summary. In this study, we examined osteopontin (OPN) production in myeloma cells and plasma OPN levels in multiple myeloma (MM) patients. We assessed OPN production in bone marrow cells (BMCs) by immunocytochemistry and enzyme‐linked immunosorbent assay (ELISA). We also assessed OPN production in various B‐cell malignant cell lines, including three myeloma cell lines by reverse transcription polymerase chain reaction (RT‐PCR) and Western blotting. In addition, we measured plasma OPN concentrations by ELISA in 30 MM patients, 21 monoclonal gammopathy of undetermined significance (MGUS) patients and 30 healthy volunteers. As a result, in an immunocytochemical study, abundant OPN was detected in BMCs from overt MM patients, whereas no OPN was detected in BMCs from patients with other haematological diseases, including MGUS. Cultured BMCs from overt MM patients produced more OPN than those from patients with either smouldering MM or MGUS. Myeloma cell lines spontaneously produced OPN. Plasma OPN levels of MM patients were significantly higher than those of MGUS patients and healthy volunteers (P < 0·05). Moreover, they correlated with both progression and bone destruction of the disease (P < 0·05). These suggest that myeloma cells actively produce OPN, which possibly contributes to osteoclastic bone resorption in MM. Plasma OPN levels may be a useful biomarker for assessing bone destruction in MM and distinguishing MM from MGUS or smouldering MM.

Role of Sphingosine 1-Phosphate in the Pathogenesis of Sjögren’s Syndrome

Primary Sjögren’s syndrome (SS) is an autoimmune disease characterized by inflammatory mononuclear cell infiltration and destruction of epithelial cells of lacrimal and salivary glands. Sphingosine 1-phosphate (S1P) and signaling through its receptor S1P1 have been implicated in many critical cellular events including inflammation, cancer, and angiogenesis. This study was undertaken to examine the role of S1P1 signaling in the pathogenesis of primary SS. S1P1 and sphingosine kinase 1, which converts sphingosine to S1P, were detected in the cytoplasm of inflammatory mononuclear cells, vascular endothelial cells, and epithelial cells in all labial salivary glands by immunohistochemistry. The expression of S1P1 in inflammatory mononuclear cells was enhanced in advanced stages of primary SS. S1P enhanced proliferation and IFN-γ production by CD4+ T cells. The enhancing effect of S1P on IFN-γ production by CD4+ T cells was stronger in patients with primary SS than in healthy controls. S1P also enhanced Fas expression and Fas-mediated caspase-3 induction in salivary gland epithelial cells. IL-6 expression was detected in the cytoplasm of inflammatory mononuclear cells and ductal epithelial cells and was enhanced in advanced stages of primary SS. Furthermore, both IFN-γ and S1P augmented IL-6 secretion by salivary gland epithelial cells. These effects of S1P were inhibited by pretreatment of pertussis toxin. Our data reveal that S1P1 signaling may modulate the autoimmune phenotype of primary SS by the action of immune as well as epithelial cells.

Imatinib mesylate inhibited rat adjuvant arthritis and PDGF-dependent growth of synovial fibroblast via interference with the Akt signaling pathway

Overgrowth of the synovium plays an important role in the pathogenesis of rheumatoid arthritis (RA). Platelet-derived growth factor (PDGF) is one of the most potent mitogenic factors of synovial cells, and imatinib mesylate (imatinib) is a specific inhibitor of the PDGF receptor tyrosine kinase. The aim of this study was to elucidate the anti-rheumatic activity of imatinib. The in vivo effects of imatinib were assessed by evaluating the sequential manifestation of adjuvant-induced arthritis in rats using paw volume and clinical scores. Imatinib was found to inhibit rat adjuvant-induced arthritis, but the inhibitory effects were incomplete. To confirm the mechanism of anti-rheumatic-activity of imatinib, we assessed the in vitro effects of imatinib on the proliferation of RA synovial fibroblast-like cells (RASFs) using a MTT assay. Intracellular signaling of PDGF was evaluated by Western blot analysis. Platelet-derived growth factor was found to induce a significant proliferation of RASFs, while imatinib inhibited PDGF-induced proliferation of RASF. Imatinib also inhibited PDGF-induced phosphorylation of the PDGF receptor and Akt, whereas constitutive activated extracellular signal-regulated kinase was not inhibited by imatinib. In contrast, imatinib did not inhibit transforming growth factor β- and basic fibroblast growth factor-induced proliferation of RASF. Oral administration of imatinib ameliorated adjuvant-induced arthritis in rats, and it inhibited PDGF-induced RASF proliferation through disruption of the PDGF-R to Akt kinase signaling pathway. Because imatinib cannot inhibit the non-PDGF-dependent proliferation of RASFs, the anti-rheumatic effect of imatinib may be incomplete. The development of inhibitors of RASF proliferation may lead to the successful treatment of RA.

Sphingosine 1-phosphate (S1P)/S1P receptor 1 signaling regulates receptor activator of NF-κB ligand (RANKL) expression in rheumatoid arthritis.

Sphingosine 1-phosphate (S1P)/S1P receptor 1 (S1P1) signaling plays an important role in synovial cell proliferation and inflammatory gene expression by rheumatoid arthritis (RA) synoviocytes. The purpose of this study is to clarify the role of S1P/S1P1 signaling in the expression of receptor activator of NF-κB ligand (RANKL) in RA synoviocytes and CD4(+) T cells. We demonstrated MH7A cells, a human RA synovial cell line, and CD4(+) T cells expressed S1P1 and RANKL. Surprisingly, S1P increased RANKL expression in MH7A cells and CD4(+) T cells in a dose-dependent manner. Moreover, S1P enhanced RANKL expression induced by stimulation with TNF-α in MH7A cells and CD4(+) T cells. These effects of S1P in MH7A cells were inhibited by pretreatment with PTX, a specific Gi/Go inhibitor. These findings suggest that S1P/S1P1 signaling may play an important role in RANKL expression by MH7A cells and CD4(+) T cells. S1P/S1P1 signaling of RA synoviocytes is closely connected with synovial hyperplasia, inflammation, and RANKL-induced osteoclastogenesis in RA. Thus, regulation of S1P/S1P1 signaling may become a novel therapeutic target for RA.

Biphasic anti-osteoclastic action of intravenous alendronate therapy in multiple myeloma bone disease

Multiple myeloma is a malignancy of plasma cells with osteolytic bone destruction. Bisphosphonates inhibit osteoclast activity and are widely used for the treatment of myeloma bone disease. We analyzed the changes in urinary cross-linked N-telopeptides of collagen (u-NTx) and urinary calcium (u-Ca) after bisphosphonate alendronate therapy in ten patients with myeloma bone disease. In all patients, the levels of u-Ca and u-NTx decreased within a week. After the maximum decrease of u-NTx, u-NTx started increasing in half of the patients. However, this further increase in u-NTx decreased again without any additional therapy. Disease severity and pretreatment u-NTx concentrations did not differ between patients with and without the rebound. Patients who did not have rebound had decreased bone marrow monocytes and decreased serum concentrations of interleukin 18, which is produced by monocytes. Our results suggest that impaired activity of monocytes, which are possible osteoclast precursors, is related to reduced bone destruction in multiple myeloma.

Correlation between salivary epidermal growth factor levels and refractory intraoral manifestations in patients with Sjögren's syndrome

Abstract Objective. To assess changes in salivary epidermal growth factor (EGF) levels and the correlation between these levels and the severity of intraoral manifestations in Sjögrens syndrome (SS). Methods. Forty SS patients and 23 controls were enrolled. Salivary EGF concentration was measured using an enzyme-linked immunosorbent assay, and intraoral manifestations were evaluated using a short version of the Oral Health Impact Profile (OHIP-14). The associations among salivary flow rate, EGF levels and the severity of intraoral manifestations were analyzed. Results. The total salivary EGF output was significantly decreased in the SS patients compared with the controls (9237.6 ± 8447.0 vs. 13296.9 ± 7907.1 pg/10 min, respectively, p = 0.033). In the SS patients, total EGF output and salivary flow rate showed a strong positive correlation (rs = 0.824, p = 0.0005), while total EGF output and disease duration showed a negative correlation (rs = −0.484, p = 0.008). Further, total EGF output was significantly correlated with the OHIP-14 score (rs = −0.721, p = 0.012). Conclusions. The salivary flow rate and EGF levels are decreased in SS, and this deterioration in saliva quality causes refractory intraoral manifestations. Our findings have provided new therapeutic targets for SS.

Levels of vascular endothelial growth factor and hepatocyte growth factor in sera of patients with rheumatic diseases

Abstract Angiogenesis plays an important role in the progression of rheumatic disease. We measured the levels of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in sera from patients with rheumatic diseases and investigated whether these angiogenic factors would be useful in the evaluation of rheumatic diseases. Serum VEGF and HGF levels were determined using ELISA in 128 patients with rheumatic diseases and in 11 healthy controls. Serum VEGF and HGF levels were significantly higher in patients with rheumatic diseases compared to healthy controls [VEGF, 312 ± 20 pg/ml versus 61 ± 8 pg/ml (mean ± SE), P < 0.001; HGF, 935 ± 36 pg/ml versus 413 ± 49 pg/ml, P < 0.01]. Serum VEGF and HGF levels were significantly elevated in patients with adult Stills disease (VEGF, 1021 ± 258 pg/ml; HGF, 1500 ± 295 pg/ml) and were relatively increased in patients with active rheumatoid arthritis (RA) (VEGF, 359 ± 94 pg/ml) and systemic sclerosis (SSc) (VEGF, 356 ± 43 pg/ml; HGF, 1294 ± 224 pg/ml). HGF levels correlated with the clinical course and disease severity in rheumatic disease patients. VEGF levels correlated with the presence of Raynauds phenomenon (P < 0.05), interstitial lung disease (ILD) (P < 0.05), and serum KL-6 levels (P < 0.01), whereas HGF levels correlated with cryoglobulinemia (P < 0.05), ILD (P < 0.05), serum C-reactive protein (CRP) (P < 0.05), thrombomodulin (P < 0.05), and KL-6 levels (P < 0.05) in rheumatic disease patients. VEGF levels correlated with the skin scores and KL-6 levels in SSc patients and also correlated with the disease activity of RA patients. These data suggest that serum VEGF and HGF levels are related to rheumatic disease activity and the presence of complications. Analysis of VEGF and HGF may be useful in the clinical evaluation of rheumatic disease patients.

A comparative proteomics study of a synovial cell line stimulated with TNF-α.

To elucidate the pathogenesis of rheumatoid arthritis (RA), we used proteomic analysis to determine the protein profile in a synovial cell line, MH7A, established from patients with RA. Proteins were extracted from MH7A cells that were or were not stimulated with tumor necrosis factor‐α (TNF‐α), and then analyzed on a liquid chromatography/mass spectrometry system equipped with a unique long monolithic silica capillary. On the basis of the results of this proteomic analysis, we identified 2650 proteins from untreated MH7A cells and 2688 proteins from MH7A cells stimulated with TNF‐α. Next, we selected 269 differentially produced proteins that were detected only under TNF‐α stimulation, and classified these proteins by performing gene ontology analysis by using DAVID as a functional annotation tool. In TNF‐α‐stimulated MH7A cells, we observed substantial production of plasminogen‐activator inhibitor 2 and apoptosis‐regulating proteins such as BH3‐interacting domain death agonist, autophagy protein 5, apolipoprotein E, and caspase‐3. These results indicate that the upregulation of plasminogen‐activator inhibitor 2 and apoptosis‐regulating proteins in synovial cells in response to TNF‐α stimulation might represent a predominant factor that contributes to the pathogenesis of RA.

Rapid decrease in salivary epidermal growth factor levels in patients with Sjögren's syndrome: A 3-year follow-up study

Objectives. To assess changes in salivary epidermal growth factor (EGF) levels within three years and investigate the correlation between these changes and the severity of intraoral manifestations in patients with Sjögrens syndrome (SS). Methods. Twenty-three SS patients (14 primary SS and 9 secondary SS) and 14 controls were followed up for three years. Salivary EGF concentration was measured using an enzyme-linked immunosorbent assay, and intraoral manifestations were evaluated using a short version of the Oral Health Impact Profile (OHIP-14). Changes in salivary flow rate, EGF level, and severity of intraoral manifestations were analyzed, along with associations among them. Results. The OHIP-14 score significantly increased and the total salivary EGF output significantly decreased after three years in the SS group (10.2 ± 8.8 vs. 12.6 ± 9.2, p = 0.040; 10158.4 ± 9820.9 vs. 8352.8 ± 7813.3 pg/10 min, p = 0.032), though the salivary flow rate did not change. The decrease in total EGF output was especially high in patients with long disease duration and poor oral health-related quality of life (OHRQoL). In patients with poor OHRQoL, the change in total EGF output significantly correlated with the OHIP-14 score (r = − 0.847, p = 0.008). However, there was no correlation between the change in salivary flow rate and the OHIP-14 score. Conclusions. The rapid decrease in salivary EGF level contributes to the progression of intraoral manifestations of SS.