Upregulation of non-ß cell-derived vascular endothelial growth factor A increases small clusters of insulin-producing cells in the pancreas.

Pancreatic ß cell-derived vascular endothelial growth factor A (VEGF-A) contributes to normal ß cell function. We therefore hypothesized that non-ß cell-derived VEGF-A may affect its properties in adult mice.We generated transgenic mice expressing human VEGF-A (hVEGF-A) in a visceral smooth muscle cell (SMC)-dominant manner under the control of the transgelin (Tagln/SM22α) promoter via a tamoxifen-induced Cre/loxP recombination system (SM-CreER(T2)/hVEGF mice).SM-CreER(T2)/hVEGF mice received tamoxifen orally followed by microscopic examination of their pancreas 4 weeks after the hVEGF-A induction. The number of clusters of insulin-producing cells (IPCs) in islets, pancreatic ducts, and individual IPCs were counted.The number of small IPC clusters (100-215 µm(2)) in the pancreas increased significantly in SM-CreER(T2)/hVEGF mice compared with SM-CreER(T2)(Ki) mice (473 out of 1 992 counts vs. 199 out of 976 counts, p<0.05), although total IPC area and the number of pancreatic duct IPCs, in proportion to exocrine area, were similar between the 2 groups. Although most small IPC clusters observed in SM-CreER(T2)/hVEGF mice were not accompanied by α and/or δ cells, some were attached to a single or a few α cells. An STZ-induced diabetic state in SM-CreER(T2)/hVEGF mice was slightly ameliorated, with only one point of significance 12 weeks after STZ administration, compared with SM-CreER(T2)(Ki) mice.Upregulation of non-ß cell-derived VEGF-A may alter the composition of pancreatic IPCs by increasing the number of small IPC clusters. These findings provide new information on the role of non-ß cell-derived VEGF-A to IPC regeneration and insulin production.

1,5-Anhydroglucitol attenuates cytokine release and protects mice with type 2 diabetes from inflammatory reactions.

1,5-anhydroglucitol (1,5-AG) decreases in diabetic patients and is used as a marker of glycemic control. Type 2 diabetic patients are susceptibile to lipopolysaccharides (LPS), which stimulate macrophages to release large quantities of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6. This study examines the effects of 1,5-AG on lung inflammation induced by LPS and consequent systemic inflammation to determine whether the decrease of 1,5-AG concentration induces susceptibility to LPS. Before the challenge with LPS (1 mg/kg in vivo and 500 ng/ml in vitro), we pretreated db/db mice and RAW264.7 cells with 1,5-AG at 38.5 mg/kg and 500 microg/ml, respectively. The levels of IL-6, TNF-alpha, macrophage chemoattractant protein (MCP)-1 and IL-1beta in the serum and in the cell supernatants were measured. We also measured macrophage recruitment and the expression of inducible nitric oxide synthase (iNOS) in pulmonary tissues. We found that 1,5-AG attenuated serum cytokine release and protected db/db mice from LPS-induced pulmonary inflammation. In addition, 1,5-AG suppressed cytokine release and iNOS expression by suppressing Akt/NF-kB activity in RAW264.7 cells. These results suggest that 1,5-AG may be a mediator in, as well as marker for diabetes, and 1,5-AG intake may confer tolerance to LPS in patients with type 2 diabetes.

Serum VEGF increases in diabetic polyneuropathy, particularly in the neurologically active symptomatic stage.

AIM: To identify the relationship between vascular endothelial growth factor (VEGF) and diabetic polyneuropathy (DPN). METHODS: Two hundred and twenty diabetic patients participated, 113 with DPN and 107 without DPN. All patients were also classified according to the four stages of DPN (no neuropathy: stage 0; asymptomatic neuropathy: stage 1; symptomatic neuropathy: stage 2; disabling neuropathy: stage 3). Serum VEGF concentration was measured using an enzyme-linked immunosorbent assay (ELISA) and levels between the patients with and without DPN and also between the different stages of DPN, were compared. RESULTS: The mean serum VEGF level in all patients was 264.6 +/- 218.8 pg/ml. The mean serum VEGF level was higher in patients with DPN (310.1 +/- 224.3 pg/ml) than in the patients without DPN (216.5 +/- 204.0 pg/ml, P = 0.0014). Serum VEGF was higher in the 'symptomatic' stage (stage 2, 364.8 +/- 225.9 pg/ml) in comparison with the 'asymptomatic' (stage 1, 256.7 +/- 224.4 pg/ml, P = 0.015) and 'disabling' (stage 3, 180.3 +/- 109.4 pg/ml, P = 0.042) stages. The mean serum VEGF level in patients with diabetic retinopathy (261.1 +/- 210.6 pg/ml) and in patients with diabetic nephropathy (241.5 +/- 185.7 pg/ml) was not increased. CONCLUSIONS: The serum VEGF level is increased in patients with DPN, particularly in patients in the neurologically active 'symptomatic' stage.

Measles virus receptors.

Measles virus (MV) has two envelope glycoproteins, the hemagglutinin (H) and fusion protein, which are responsible for attachment and membrane fusion, respectively. Signaling lymphocyte activation molecule (SLAM, also called CD150), a membrane glycoprotein expressed on immune cells, acts as the principal cellular receptor for MV, accounting for its lymphotropism and immunosuppressive nature. MV also infects polarized epithelial cells via an as yet unknown receptor molecule, thereby presumably facilitating transmission via aerosol droplets. Vaccine and laboratory-adapted strains of MV use ubiquitously expressed CD46 as an alternate receptor through amino acid substitutions in the H protein. The crystal structure of the H protein indicates that the putative binding sites for SLAM, CD46, and the epithelial cell receptor are strategically located in different positions of the H protein. Other molecules have also been implicated in MV infection, although their relevance remains to be determined. The identification of MV receptors has advanced our understanding of MV tropism and pathogenesis.

The role of water channel aquaporin 3 in the mechanism of TNF-alpha-mediated proinflammatory events: Implication in periodontal inflammation.

Aquaporin 3 (AQP3) is the predominant water channel protein in human keratinocytes and acts as an inflammatory mediator in some lesions. A chronic, inflammatory process of periodontitis is related with a dramatic change of surrounding fluid homeostasis to plasma extravasation. The exact pattern of aquaporin (AQP) water channel expression and its mechanism in periodontal disease is still unknown. We describe herein an up-regulated AQP3 expression in the epithelial lesion with chronic periodontitis and its functional role. The levels of AQP3 expression in inflamed gingival epithelial tissues were significantly higher than those of healthy subjects. Consistent with these results, AQP3 expression (i.e., levels of mRNA and protein) in cultured rat primary gingival epithelial cells and the human gingival epithelial cell line Ca9-22 were strongly increased in response to TNF-alpha treatment through the 55 kDa TNF-alpha receptor (TNFR I). In this context, small interfering RNA- (siRNA)-mediated "aqp-3 gene silencing," which could reduce AQP3 expression by more than 65%, significantly attenuated selected proinflammatory events of ICAM-1 expression induced by TNF-alpha in Ca9-22. A sixfold increase in leukocyte adherence to TNF-alpha-stimulated epithelial cells was demonstrated by an adherence assay (P < 0.001) and pretreatment with AQP3 siRNA and anti-ICAM-1 antibody reduced leukocyte retention by 85% (P < 0.001). Our study indicates for the first time a novel important mode in the regulation of the inflammatory response through TNF-alpha/TNFR I ligation at the site of epithelial lesions by specialized membrane channel AQP3 and ICAM-1 protein, which is closely implicated in the development of periodontitis mechanisms.

Tumor necrosis factor-alpha stimulates gingival epithelial cells to release high mobility-group box 1.

BACKGROUND AND OBJECTIVE: High-mobility-group box 1 functions as a late-phase inflammatory mediator. It can be released extracellularly by macrophages and necrotic cells through lipopolysaccharide and tumor necrosis factor-alpha. The objective of this study was to clarify the source of high-mobility-group box 1 in chronic periodontitis tissues and tumor necrosis factor-alpha-stimulated gingival epithelial cells, and subsequently elucidate its inducible inflammatory pathway. MATERIAL AND METHODS: Chronic periodontitis and healthy gingival sections were stained for high-mobility-group box 1 by immunohistochemistry and immunofluorescence. The amounts of high-mobility-group box 1 released into the gingival crevicular fluid and supernatants from gingival epithelial cells stimulated by tumor necrosis factor-alpha were examined by western blot. The phosphorylation of mitogen-activated protein kinases (MAPKs) in gingival epithelial cells was also examined. RESULTS: High-mobility-group box 1 was detected in the cytoplasm and nucleus of gingival epithelial cells with periodontitis. Western blotting revealed a significant increase in high-mobility-group box 1 expression in the gingival crevicular fluid from periodontitis patients. High-mobility-group box 1 production in gingival epithelial cells was increased following stimulation with tumor necrosis factor-alpha. The molecular dialogue between tumor necrosis factor-alpha and gingival epithelial cells involved modulation of the activities of p38MAPK, Jun N-terminal kinase and p44/42. Interestingly, only phosphorylation of p38MAPK contributed to more than half of the signaling initiated by tumor necrosis factor-alpha-elicited high-mobility-group box 1 release. CONCLUSION: High-mobility-group box 1 is continuously released from the gingival epithelial cells modulated by tumor necrosis factor-alpha. These findings imply that high-mobility-group box 1 expression and possibly p38MAPK constitute important features in periodontitis.

Highly accumulated platelet vascular endothelial growth factor in coagulant thrombotic region.

BACKGROUND: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific potent mitogen that induces angiogenesis and microvascular hyperpermeability. Recently, it has been reported that megakaryocytes and platelets contain VEGF in their cytoplasm. OBJECTIVES: To elucidate and confirm the bioactivity and role of VEGF in platelets (platelet VEGF), which may be closely related to vascular thrombosis and atherosclerosis. METHODS: The VEGF localization in megakaryocytes on bone marrow smears was analyzed by immunofluorescence and confocal laser scanning microscopic analysis. The intracellular VEGF expressed in platelets was determined by flow cytometric analysis. Platelet-rich plasma and washed platelets were used to analyze the secretion of VEGF during platelet aggregation by thrombin or gelatinase A (matrix metalloproteinase-2) stimulation. Immunohistochemical studies for VEGF in the thrombotic region were performed. RESULTS AND CONCLUSIONS: Megakaryocytes and platelets are a very rich source of circulating VEGF. Gelatinase A, which is closely associated with vascular remodeling, enhances the VEGF levels released from platelets. VEGF was clearly detected in the fibrin nets of a thrombus. Taken together, platelet VEGF is bioactive as a direct angiogenic growth factor, and may play a very important role in wound healing and atherosclerosis in conjunction with other platelet cytokines such as platelet-derived growth factor, platelet-derived endothelial cell growth factor, transforming growth factor (TGF)-alpha, and TGF-beta.

The agonist of the protease-activated receptor-1 (PAR) but not PAR3 mimics thrombin-induced vascular endothelial growth factor release in human smooth muscle cells.

Thrombin, a serine protease generated by the activation of the blood coagulation cascade following vessel injury, induces vascular endothelial growth factor-(VEGF) release. However, the molecular mechanism of thrombin-induced VEGF release is largely unknown. Anagonist of protease-activated receptor-i (PARI), SFLL-RNPNDKYEPF, mimicked thrombin-induced VEGF release in human vascular smooth muscle (HVSM) cells, as determined by enzyme-linked immunosorbent assay, reverse transcriptase-polymerase chain reaction, and Northern blotting. In contrast, the agonist of PAR3, TFR- GAP, did not affect VEGF release or expression. SFLL-RNPNDKYEPF, but not TFRGAP, up-regulated [Ca2-]i.Moreover, the calcium ionophone A23187 was found to trigger VEGF release in HVSM cells. Thrombin-inducedVEGF release was blocked by anti-thrombin, heparin, a synthetic thrombin receptor inhibitor E5510, the calcium chelator BAPTA, the protein kinase C inhibitor calphostin C, and the MEK1/2 inhibitor U0126. Thus, our data show that thrombin caused VEGF release via PARI activation in a manner dependent on [Ca2+]i and p44/42 downstream from the receptor activation.

Morphological and biochemical studies of human beta-mannosidosis: identification of a novel beta-mannosidase gene mutation.

BACKGROUND: There are seven well-known lysosomal storage diseases that produce angiokeratoma corporis diffusum clinically. beta-Mannosidosis (MANB1; OMIM248510), first reported in humans in 1986, is a rare hereditary lysosomal storage disease caused by a deficiency of the enzyme beta-mannosidase. Since then, 13 cases of beta-mannosidase deficiency in ten families have been described. A human beta-mannosidase mutation has been reported only by Alkhayat et al. in 1998. OBJECTIVES: To clarify its pathogenesis we did electron microscopic, biochemical and molecular biological investigations of a Japanese patient with beta-mannosidosis. METHODS: Ultrastructural analyses, enzyme assays, cell culture and mRNA and genomic DNA were sequenced to find mutations in the beta-mannosidase gene. RESULTS: Electron microscopy of skin biopsy specimens from the patient showed cytoplasmic vacuolation of lysosomes in blood and lymph vessels, endothelial cells, fibroblasts, secretory portions of eccrine sweat glands, neural cells and basal keratinocytes in the epidermis. This vacuolation was also observed in cultured keratinocytes and fibroblasts. Assays of seven enzyme activities in plasma and cultured skin fibroblasts showed a marked decrease of beta-mannosidase activity. Sequencing the beta-mannosidase cDNA revealed a four-base (ATAA) insertion between exons 7 and 8, resulting in a frameshift at codon 321 and termination at codon 325. Analysis of the patient's genomic DNA revealed a novel homozygous A(+1)-->G splice site mutation in intron 7. CONCLUSIONS: To our knowledge, this is the first case of beta-mannosidosis reported in Japan and the second report in which a gene mutation is identified. The biological importance of beta-mannose moieties in glycoproteins in basal keratinocytes is suggested.

Expression of vascular endothelial growth factor in tuberculous meningitis.

The pathogenesis of tuberculous meningitis is still unclear. Recently, vascular endothelial growth factor (VEGF) was found to be associated with inflammatory diseases and we found the increased serum level of VEGF in pulmonary tuberculosis. We hypothesized that VEGF might be associated with the pathogenesis of tuberculous meningitis and measured serum and cerebrospinal fluid (CSF) levels of VEGF in 28 patients with tuberculous meningitis and 31 non-tuberculous infectious meningitis patients (13 bacterial meningitis patients, eight fungal meningitis patients and 10 patients with viral meningitis) before therapy. We examined the CSF VEGF levels 3 months after in 12 tuberculous meningitis patients. The serum and CSF levels of VEGF were significantly higher in tuberculous meningitis than in other meningitis. The decrease in titer of CSF VEGF paralleled the clinical improvement of tuberculous meningitis. Immunohistochemical staining of autopsied brains demonstrated the presence of VEGF in the inflammatory mononuclear cells of the dense fibroconnective tissue both in the subarachnoid space and surrounding the vasculitis lesion. We found the expression of VEGF in tuberculous meningitis and think that VEGF reflects its activity.

Circulating levels of MMP-1, -2, -3, -9, and TIMP-1 are increased in POEMS syndrome.

The authors quantitatively measured levels of matrix metalloproteinases (MMP), tissue inhibitor of metalloproteinases (TIMP), and vascular endothelial growth factor (VEGF) in blood samples of POEMS syndrome. Circulating levels of MMP-1, -2, -3, -9, and TIMP-1 were more increased in patients with POEMS syndrome than in patients with other neurologic disorders or in healthy controls. Serum levels of VEGF and TIMP-1 were strongly correlated with each other. Increased circulating levels of MMP-1, -2, -3, -9, and TIMP-1 may lead to a better understanding the pathogenesis of POEMS syndrome.

[Vascular endothelial growth factor and Crow-Fukase syndrome].

Serum vascular endothelial growth factor (VEGF) is highly elevated in patients with Crow-Fukase syndrome (CFS) and is well correlated with the clinical manifestations of CFS. In circulating blood, VEGF is specifically stored in platelets and released during platelet aggregation. To clarify the role of VEGF in the pathomechanism of CFS, we transplanted VEGF secretion tumors in nude mice and studied the pathological findings in these mice. Prominent edema with elevated serum VEGF were found. Organomegaly was also found in liver, spleen and kidney. Pathological findings in these organs were similar to those found in autopsies of CFS patients. In peripheral nerve, mild intraneural edema was seen, however, neuropathy was not prominent. These findings suggest that elevated VEGF may be closely correlated with generalized edema (anasarca). However, it is also important to consider factors such as cytokines and other T cell functions that, in association with VEGF, may be the cause of neuropathy in CFS.

Expression of vascular endothelial growth factor in pulmonary aspergilloma.

OBJECTIVE: Vascular endothelial growth factor (VEGF), a cytokine associated with malignant neoplasms, is increased in a number of inflammatory and infectious disorders. But little is known about VEGF in pulmonary aspergilloma (PA). In order to clarify the predictive value of VEGF in PA, we examined serum VEGF levels in PA patients as well as expression of VEGF in the PA lesion. Patients Twenty-one patients with PA (6 with hemoptysis and the rest without hemoptysis) were investigated. METHODS: In all patients, serum VEGF levels were measured by enzyme-linked immunosorbent assay. We examined the statistical correlations of serum VEGF levels with the ratio of affected area to total lung volume and VEGF with PaO2 levels. In patients with hemoptysis, we measured the serum VEGF levels three months after the initiation of therapy. We examined the expression of VEGF in the resected lung tissue of 4 patients with PA using immunohistochemistry. RESULTS: Serum VEGF levels showed a significant positive correlation with the ratio of affected to total lung area and a significant negative correlation with PaO2 levels. Serum VEGF levels in patients with hemoptysis were significantly higher than in those without hemoptysis. The serum levels of VEGF in patients with hemoptysis decreased significantly in parallel with the alleviation of hemoptysis. Immunohistochemical staining demonstrated the expression of VEGF in alveolar macrophages in the lesion of PA patients. CONCLUSION: We suggest that VEGF can be used as a serum marker to determine the extent of the lesion, hypoxia and hemoptysis in PA.

Serum levels of vascular endothelial growth factor dependent on the stage progression of lung cancer.

STUDY OBJECTIVE: In lung cancer, vascular endothelial growth factor (VEGF) is an important cytokine and is correlated with tumor vessel density, malignant pleural effusions, and coagulation-fibrinolysis factors in vitro. We investigated the correlation between serum VEGF level and stage progression in lung cancer to study the predicted value of VEGF level. We also studied whether coagulation-fibrinolysis factors and PaO(2) levels, which are also important factors for the prediction of the clinical course, are correlated with VEGF. METHODS: Forty-nine patients with lung cancer were investigated prospectively. VEGF levels of sera and malignant effusions, and plasma concentrations of coagulation-fibrinolysis factors were measured by enzyme-linked immunosorbent assay. We measured PaO(2) levels in all patients at rest. RESULTS: Serum levels of VEGF were increased significantly according to stage progression. Additionally, plasma concentrations of D dimer, thrombin-antithrombin complex (TAT), and tissue plasminogen activator/plasminogen activator inhibitor type I complex were elevated significantly according to stage progression. The serum VEGF level had a significant positive correlation with the TAT and D dimer levels. Serum VEGF levels had a significant negative correlation with PaO(2) levels. The incidence of cerebral vascular disorder was significantly higher in the patients with systemic hypoxemia than in those without (p<0.05). Mean VEGF levels in malignant effusions in eight patients (five with pleural effusions, two with pericardial effusions, and one with both) were extremely high, especially in pericardial effusions ([mean +/- SD] pleural effusions, 531.9+/-285.4 pg/mL; pericardial effusion, 3,071.6+/-81.3 pg/mL). CONCLUSION: We predict that in lung cancer, VEGF production and the abnormality of the coagulation-fibrinolysis system differ depending on the stage of progression of disease. Serum VEGF levels would be affected by PaO(2) levels in lung cancer.

Increased serum level of vascular endothelial growth factor in pulmonary tuberculosis.

Pulmonary tuberculosis, one of the granulomatous diseases, has few serological markers for its activity. Recently, an increased serum level of vascular endothelial growth factor (VEGF) was detected in patients with Crohn's disease, also a granulomatous disease. We hypothesized that VEGF might be associated with the pathogenesis of pulmonary tuberculosis. We investigated the serum level of VEGF in 43 patients with active pulmonary tuberculosis, 29 patients with old tuberculosis, and 25 patients with acute bronchitis. We were able to examine the serum VEGF levels every 3 mo for a period of 6 mo in seven patients with active pulmonary tuberculosis. We examined the presence of VEGF in the resected lungs of three patients with active pulmonary tuberculosis by immunohistochemistry. The serum levels of VEGF were significantly higher in patients with active pulmonary tuberculosis than in patients with old tuberculosis and acute bronchitis. The decrease in titer of serum VEGF paralleled the clinical improvement of patients with pulmonary tuberculosis. Immunohistochemical staining of the resected lungs demonstrated the presence of VEGF in alveolar macrophages surrounding the lesion. Therefore, VEGF may be associated with the pathogenesis of pulmonary tuberculosis.

Highly concentrated vascular endothelial growth factor in platelets in Crow-Fukase syndrome.

We report a marked difference in concentration of vascular endothelial growth factor (VEGF) between serum and plasma in patients with Crow-Fukase syndrome (CFS). The serum/plasma VEGF levels in 4 CFS patients were 8,634/152, 5,203/176, 3,724/127, and 868/13 pg/ml, respectively. We also showed that platelets were a major source of this VEGF and that VEGF was released during platelet aggregation by physiological stimulation. It is suggested that in CFS, local VEGF concentration is markedly elevated by aggregation of platelets containing excessive VEGF and their adhesion to vascular walls, resulting in excessive physiological activities of VEGF. Our findings provide important information for developing more effective therapeutic trials.

VEGF is causative for pulmonary hypertension in a patient with Crow-Fukase (POEMS) syndrome.

We report a case of Crow-Fukase (POEMS) syndrome associated with pulmonary hypertension (PH). In this case, the concentration of vascular endothelial growth factor (VEGF) was extremely high in the serum, and the levels of IL-1beta, IL-6, TNF-alpha, and thiamine, which were thought in past reports to be mediators of PH in Crow-Fukase syndrome, were normal. After prednisolone therapy, PH disappeared with a dramatic decrease in serum VEGF. Our results suggest that VEGF is closely correlated with PH in Crow-Fukase syndrome.

Three cases of polyarteritis nodosa cutanea and a review of the literature.

We describe three cases of polyarteritis nodosa cutanea (PNC) showing necrotizing arteritis and only cutaneous lesions without systemic symptoms or visceral involvement for eleven, six, and three years after the onset of the disease. Since it was first described, there has been continuous controversy as to whether PNC progresses to systemic PN. Some cases have been described which had begun with a cutaneous lesion and progressed to the systemic form 19 and 18 years after the onset of the disease, so we believe that long term follow-up of this disease is essential.