Plasma vascular endothelial growth factor in Japanese Type 2 diabetic patients with and without nephropathy.

AIM: To determine whether plasma vascular endothelial growth factor (VEGF) level is elevated in Type 2 diabetic patients with an early stage of diabetic nephropathy. METHODS: We studied 71 Japanese Type 2 diabetic patients with normal serum creatinine level (<100 micromol/l) (age 63.0 [60.3-65.6] years old, diabetes duration 15.6 [14.0-17.3] years, HbA1c 7.36% [7.06-7.66%], mean [95% confidence interval, CI]): normoalbuminuric patients (n=36); microalbuminuric patients (n=21); and proteinuric patients (n=14). Plasma VEGF concentration was measured by a quantitative sandwich enzyme immunoassay technique. RESULTS: Plasma VEGF concentration was not related to the degree of albuminuria: normoalbuminuric patients (25 [13-95] ng/l, median [25th-75th percentile]); microalbuminuric patients (33 [15-120] ng/l); and proteinuric patients (54 [17-107] ng/l). Plasma VEGF level in patients with retinopathy (25 [15-95] ng/l, n=30) was not elevated as compared to those without retinopathy (53 [14-126] ng/l, n=34). Plasma VEGF tended to correlated negatively with diabetes duration (R's=-.217, P=.0690) and HbA1c (R's=-.221, P=.0647), whereas there was no correlation between plasma VEGF level and age, serum creatinine or urinary albumin to creatinine ratio (ACR) of the patients, respectively. Plasma VEGF level in the group of patients with HbA1c equal to or below the median (<7.2%) was significantly higher than that in the group of patients with HbA1c above the median (>7.2%) (P<.05). CONCLUSIONS: The results suggested that Type 2 diabetic patients with microalbuminuria and those with retinopathy are not necessarily associated with an elevation of circulating plasma VEGF concentration. Plausible association between plasma VEGF level and glycemic control remains to be seen.

Analysis of cortisol secretion in hormonally inactive adrenocortical incidentalomas: study of in vitro steroid secretion and immunohistochemical localization of steroidogenic enzymes.

Adrenal incidentalomas have recently increased in incidence, and thus it has become important to establish clinical management of these patients. It is also important to evaluate whether these tumors are different from preclinical or overt Cushing's syndrome in their steroidogenesis. In this study, we therefore examined steroidogenesis of hormonally inactive adrenal incidentalomas via short-term culture of tumor specimens, in addition to an immunohistochemical study of steroidogenic enzymes. Five patients (two men and three women) diagnosed with adrenocortical incidentaloma without any clinical signs of adrenocortical hormonal excess except for hypertension and disturbed glucose tolerance, were recruited for this study. Hormonal findings, including circadian rhythms for cortisol and ACTH secretion, the response of ACTH to CRH infusion and results of dexamethasone suppression test were all within normal limits in these patients. Immunoreactivity for all steroidogenic enzymes involved in cortisol production was detected in tumor cells in all cases examined. Results of in vitro steroidogenesis analysis using short-term culture revealed that levels of cortisol secretion varied among the cases. There were no differences in the immunolocalization of steroidogenic enzymes and/or the levels of cortisol secretion between these hormonally inactive tumors and preclinical and/or overt Cushing's syndrome. Dehydroepiandrosterone-sulfotransferase (DHEA-ST) immunoreactivity in nonneoplastic regions was suppressed in one case in which the tumor secreted cortisol similar to preclinical and/or overt Cushing's syndrome. These results demonstrate that the levels of in vitro steroid production and/or the immunolocalization of steroidogenic enzymes in hormonally inactive adrenocortical tumors vary markedly and are not overtly different from those of preclinical and/or overt Cushing's syndrome.

The improvement of insulin resistance in patients with adrenal incidentaloma by surgical resection.

OBJECTIVE: Several recent studies have indicated that patients with adrenal incidentaloma often have disturbed glucose tolerance or/and hypertension. It is unclear whether these metabolic conditions could be caused by adrenal incidentaloma. We investigated the prevalence of disturbed glucose tolerance, hypertension and insulin resistance in the patients with non-functioning adrenal incidentaloma and evaluated the changes of the parameters such as glucose tolerance, blood pressure and insulin sensitivity after adrenalectomy. PATIENTS AND METHODS: Among 15 patients with incidentally discovered adrenal tumours in our department from 1996 to 1999, 4 patients were diagnosed as having pre-clinical Cushing's syndrome and the other 11 as having non-functioning tumours based on detailed endocrinological examinations including dexamethasone suppression testing. Four tumours with pre-clinical Cushing's syndrome and 8 tumours out of 11 patients with non-functioning tumours were diagnosed histopathologically as adrenocortical adenomas and the other 3 as of non-adrenal origin including a myelolipoma, an adrenal vascular cyst and an endothelioma. The prevalence of disturbed glucose tolerance was determined with an oral glucose tolerance test, and insulin sensitivity was evaluated by the method of steady state of plasma glucose (SSPG). RESULTS: All 12 patients with adrenocortical adenoma exhibited insulin resistance based on the SSPG (6.9-13.2 mmol/l). Before surgical removal of the tumours, the SSPG titre was relatively higher in the patients with pre-clinical Cushing's syndrome than in those with non-functioning with adrenocortical adenoma (mean value 11.65 vs. 8.99 mmol/l), whereas 2 of the 3 patients with non-adrenocortical tumours did not have insulin resistance. Among the 12 patients with adrenocortical adenoma, 7 (58%) and 9 (75%) patients exhibited hypertension and disturbed glucose tolerance, respectively. After removal of the tumours, SSPG of the patients with adrenocortical adenoma, but not that of the other 3 patients with non-cortical tumours, was significantly decreased compared to pre-adrenalectomy values. There are no significant differences in the changes of SSPG titres between in pre-clinical Cushing's syndrome and in non-functioning adrenocortical adenoma. Systolic blood pressure, but not diastolic blood pressure, was also significantly decreased in the patients with adrenocortical adenoma. CONCLUSION: High prevalences of disturbed glucose tolerance, insulin resistance and hypertension were found among the patients with non-functioning adrenocortical tumours. Adrenocortical adenoma may be one of the risk factors for insulin resistance that is believed to induce disturbed glucose tolerance and/or hypertension. Therefore, it is useful to evaluate insulin resistance for the patients with adrenal incidentalomas since results are likely to be helpful in deciding whether to remove the tumour by surgery.

SSPG titer is a diagnostic marker for adrenocortical adenoma in patients with non-functioning adrenal incidentaloma.

We analyzed insulin resistance-related clinical markers as well as imaging characteristics of adrenal masses in 11 patients with incidentally discovered adrenal tumors that were diagnosed as non-functioning according to the results of the 1 mg dexamethasone suppression test and then voluntarily resected. Histologic examination of the resected tumors revealed adrenocortical adenoma in eight patients, and non-cortical tumors: a myelolipoma. an endothelioma and an adrenal vascular cyst in three patients. All eight patients with adrenocortical adenoma exhibited insulin resistance as estimated by the steady-state plasma glucose (SSPG). The mean systolic blood pressure of the patients with adrenocortical adenoma was higher than that of the patients with non-cortical tumors, even though three of eight patients were normotensive. HOMA-R, EIRI on 75 g oGTT. diastolic blood pressure and serum triglyceride were not significantly different between the patients with adrenocortical adenomas and those with non-cortical tumors. Imaging characteristic such as tumor size, shape and heterogeneity in the mass did not distinguish these two groups either. These results suggest that the presence of insulin resistance estimated by SSPG may be a diagnostic marker for adrenocortical adenoma in patients with a non-functioning incidentaloma.

Improvement by the insulin-sensitizing agent, troglitazone, of abnormal fibrinolysis in type 2 diabetes mellitus.

This study evaluated abnormal fibrinolysis in diabetic patients in terms of the pathophysiological significance and reversibility by oral hypoglycemic agents. Forty-seven patients with type 2 diabetes mellitus were randomly treated for 4 weeks with glibenclamide (n = 23) or troglitazone (n = 24). Before and after treatment, glycemic control, steady-state plasma glucose and insulin (SSPG and SSPI, respectively), and markers of fibrinolysis (tissue plasminogen activator [tPA] and plasminogen activator inhibitor-1 [PAI-1]) were analyzed in each patient. Pretreatment plasma PAI-1 in diabetic patients, but not tPA, was well correlated with the severity of retinopathy assessed by the fluorescence technique. Four weeks of treatment with troglitazone significantly decreased hemoglobin A1c (HbA1c), SSPG, and PAI-1 without an alteration of tPA. The troglitazone-induced decrease in plasma PAI-1 (50.3 v28.8 micromol/L; P < .05) was correlated with HbA1c (8.80% v7.21%, r = .539, P < .01) and SSPG (16.2 v 8.97 mmol/L, r = .562, P < .01) but not with SSPI. In contrast, treatment with glibenclamide for 4 weeks also reduced the HbA1c titer to almost the same extent as troglitazone (1.38% v 1.59%), but did not change the plasma PAI-1 or SSPG titer. These results suggest that an abnormal fibrinolytic state, especially overproduction of PAI-1, may be a pathogenic factor in the development of diabetic complications such as retinopathy, which may be improved by correction of the insulin resistance with troglitazone.

Enhancement by homocysteine of plasminogen activator inhibitor-1 gene expression and secretion from vascular endothelial and smooth muscle cells.

In order to elucidate the relationship between homocysteine and the fibrinolytic system, we examined the effect of homocysteine on plasminogen activator inhibitor-1 (PAI-1) and tissue-type plasminogen activator (tPA) gene expression and protein secretion in cultured human vascular endothelial and smooth muscle cells in vitro. PAI-1 mRNA and secreted protein levels were both enhanced by homocysteine in a dose dependent manner, with significant stimulation of PAI-1 secretion observed at concentrations greater than 0.5 mM homocysteine. In contrast, secretion and mRNA expression of tPA were not significantly altered by homocysteine stimulation. Secretion of TGFbeta (transforming growth factor beta) and TNFalpha (tumor necrosis factor alpha), possible regulators of PAI-1 expression and secretion, were not stimulated by treatment with 1.0 mM homocysteine. These results suggests that hyperhomocysteinemia-induced atherosclerosis and/or thrombosis may be caused by homocysteine-induced stimulation of PAI-1 gene expression and secretion in the vasculatures by a mechanism independent from paracrine-autocrine activity of TGFbeta and TNFalpha.

A patient with preclinical Cushing's syndrome and excessive DHEA-S secretion having unilateral adrenal carcinoma and contralateral adenoma.

We report a case of preclinical Cushing's syndrome in a 54-year-old male associated with bilateral adrenocortical tumours. Physical findings and general laboratory data were unremarkable except for mild hypertension (158/90 mmHg) and impaired glucose tolerance. Endocrinological evaluation revealed the presence of autonomous cortisol secretion including unsuppressible serum cortisol by 8 mg dexamethasone test (11 microg/dl), high serum DHEA-S (3580 ng/ml, normal: 400-3500) and increased urinary 17-KS excretion (31.0-35.8 mg/day, normal: 5.8-21.3). CT scan demonstrated the presence of tumours in both adrenals and bilateral adrenalectomy was subsequently performed. Histological examination of the resected specimens revealed an adrenocortical carcinoma on the right side and an adenoma on the left side with noticeable cortical atrophy in non-neoplastic adrenals. Immunohistochemical study of steroidogenic enzymes demonstrated that all the steroidogenic enzymes involved in cortisol biosynthesis were expressed in both right and left adrenal tumours. Enzymatic activities of 21, 17alpha, 18, 11beta-hydroxylases were detected in both right and left adrenals except for the absence of 11beta-hydroxylase activity in the left adrenal adenoma. Results of in vitro tissue steroidogenesis examined in short-term tissue culture of the specimens revealed no significant differences between carcinoma and adenoma in cortisol production, but the production of adrenal androgens in carcinoma was significantly higher than that in adenoma, which may indicate the importance of evaluating adrenal androgen levels in patients with adrenocortical neoplasms.

Thiazolidinediones down-regulate plasminogen activator inhibitor type 1 expression in human vascular endothelial cells: A possible role for PPARgamma in endothelial function.

The effect of peroxisome proliferator-activated receptor (PPAR) gamma activators, thiazolidinediones, on plasminogen activator type 1 (PAI-1) was examined in cultured human umbilical vein endothelial cells (HUVEC). Tumor necrosis factor alpha (TNF-alpha) enhanced PAI-1 secretion and mRNA expression by approximately 2-fold. The thiazolidinediones, troglitazone and pioglitazone, decreased basal and TNF-alpha-stimulated PAI-1 secretion and mRNA expression in HUVEC in a dose-dependent fashion. PPARgamma mRNA in HUVEC could be detected by reverse transcriptase-polymerase chain reaction using specific primers. These results suggest that PPARgamma may regulate PAI-1 expression in HUVEC and that thiazolidinediones have a therapeutic potential for improving endothelial dysfunction observed in insulin resistance.

Endoscopy in the diagnosis of small intestinal tumors.

The importance of endoscopy in the diagnosis of small intestinal tumors was evaluated in 15 patients with small intestinal tumors treated in our hospital. Two tumors were benign, and 13 were malignant (carcinoma in 5 patients, malignant lymphoma in 5 and leiomyosarcoma in 3). The presence of lesions could be determined by X-rays before surgery, but definitive diagnoses were difficult. When preoperative endoscopy of the small intestine was possible accurate preoperative diagnoses could be made based on the endoscopic findings and biopsies taken under direct vision. Endoscopy is therefore very important for the diagnosis of small intestinal tumors. It is necessary to develop small intestinal endoscopes that are easier to insert.

Association of Helicobacter pylori and diffuse type gastric cancer.

The major purpose of this study was to evaluate the association of Helicobacter pylori and diffuse type gastric cancer (DGC) clinicopathologically (study 1). The second aim was to investigate genetic differences of H. pylori in patients with DGC and intestinal type cancer (IGC) (study 2). The prevalence of H. pylori and the types of histopathological changes were evaluated in resected early gastric cancer (DGC; 25 patients, IGC; 25 patients). Genetic differences of H. pylori in DGC patients (n = 19) and IGC patients (n = 22) were analyzed by polymerase chain reaction (PCR) methods in terms of restriction fragment length polymorphism patterns of the ureB gene and cagA gene positive rates. All patients had evidence of H. pylori infection in the resected stomach, but the positive rate for H. pylori in the area surrounding cancer was 52% (in DGC; 56%, IGC; 48%). But in 40.0% of DGC cases (10/25), mucosal atrophy and intestinal metaplasia were rarely seen in the area surrounding cancer and the positive rate of H. pylori was 80.0% (8/10), in contrast, in 60.0% of IGC cases (15/25), atrophy and metaplasia were progressed and positive rate of H. pylori was 26.7% (4/15) in the area. UreB gene products from 89.5% of DGC cases (17/19) were unable to be digested by Spe I. 31.8% of products from IGC cases (7/22) were also unable to be digested by Spe I, but the positive rate of cagA gene in this group was higher than other groups. The high prevalence of H. pylori infection in DGC patients suggests that H. pylori plays a role in the pathogenesis of DGC, but in the stomach with DGC, it is considered atrophy and intestinal metaplasia are not so implicated in H. pylori, compared with IGC. A genetic specificity of H. pylori in DGC and IGC was indicated by the results, suggesting that H. pylori may play different roles in the pathogenesis of DGC and IGC.

Bacteria closely resembling Helicobacter pylori detected immunohistologically and genetically in resected gallbladder mucosa.

A microorganism with close immunohistological and genetic resemblance to Helicobacter pylori was found in the resected gallbladder mucosa of a 41-year-old woman. The woman was admitted to hospital complaining of fever and right hypochondrial pain. Cholecystectomy was carried out under the diagnosis of gallstones and cholecystitis. A microorganism resembling H. pylori (stained with H&E, Giemsa, and Wartin-Starry) was detected incidentally on pathological examination. The microorganism was also positive for immunohistochemical staining. An amplification reaction was seen on genetic examination by the polymerase chain reaction (PCR) method (urease beta-genes). Our findings suggest that H. pylori may be present in tissues other than gastric mucosa.