Long-Term Follow-Up of the Edmonton Protocol of Islet Transplantation in the United States.

We report the long-term follow-up of the efficacy and safety of islet transplantation in seven type 1 diabetic subjects from the United States enrolled in the multicenter international Edmonton Protocol who had persistent islet function after completion of the Edmonton Protocol. Subjects were followed up to 12 years with serial testing for sustained islet allograft function as measured by C-peptide. All seven subjects demonstrated continued islet function longer than a decade from the time of first islet transplantation. One subject remained insulin independent without the need for diabetic medications or supplemental transplants. One subject who was insulin-independent for over 8 years experienced graft failure 10.9 years after the first islet transplant. The remaining six subjects demonstrated continued islet function upon trial completion, although three had received a supplemental islet transplant each. At trial completion, five subjects were receiving insulin and two remained insulin independent, although one was treated with liraglutide. The median hemoglobin A1c was 6.3% (45 mmol/mol). All subjects experienced progressive decline in the C-peptide/glucose ratio. No patients experienced severe hypoglycemia, opportunistic infection, or lymphoma. Thus, although the rate and duration of insulin independence was low, the Edmonton Protocol was safe in the long term. Alternative approaches to islet transplantation are under investigation.

Symptoms of depression prospectively predict poorer self-care in patients with Type 2 diabetes.

AIMS: To examine prospectively the association of depression symptoms with subsequent self-care and medication adherence in patients with Type 2 diabetes mellitus. METHODS: Two hundred and eight primary care patients with Type 2 diabetes completed the Harvard Department of Psychiatry/National Depression Screening Day Scale (HANDS) and the Summary of Diabetes Self-Care Activities (SDSCA) at baseline and at follow-up, an average of 9 months later. They also self-reported medication adherence at baseline and at a follow-up. RESULTS: Baseline HANDS scores ranged from 0 to 27, with a mean score of 5.15 +/- 4.99. In separate linear regression models that adjusted for baseline self-care, patients with higher levels of depressive symptoms at baseline reported significantly lower adherence to general diet recommendations and specific recommendations for consumption of fruits and vegetables and spacing of carbohydrates; less exercise; and poorer foot care at follow-up (beta ranging from -0.12 to -0.23; P < 0.05). Similarly, each one-point increase in baseline HANDS score was associated with a 1.08-fold increase in the odds of non-adherence to prescribed medication at follow-up (95% confidence interval 1.001, 1.158, P = 0.047). Increases in depression scores over time also predicted poorer adherence to aspects of diet and exercise. CONCLUSIONS: Depressive symptoms predict subsequent non-adherence to important aspects of self-care in patients with Type 2 diabetes, even after controlling for baseline self-care. Although the relationship between symptoms of depression and poorer diabetes self-care is consistent, it is not large, and interventions may need to address depression and self-care skills simultaneously in order to maximize effects on diabetes outcomes.

Retinal haemodynamics in individuals with well-controlled type 1 diabetes.

AIMS/HYPOTHESIS: Abnormalities in retinal haemodynamics have been reported in patients with type 1 diabetes in advance of clinical retinopathy. These abnormalities could therefore be useful as early markers or surrogate endpoints for studying the microangiopathy. Since the DCCT, the increased focus on good glycaemic control is changing the natural history of diabetic retinopathy. Based on this, the aim of this study was to investigate whether patients with type 1 diabetes treated entirely or mostly in the post-DCCT era and tested in the absence of confounding factors show retinal haemodynamic abnormalities. METHODS: We measured retinal haemodynamics by laser Doppler flowmetry in 33 type 1 diabetic individuals with no or minimal retinopathy (age 30+/-7 years, duration of diabetes 8.8+/-4.6 years, 9% showing microaneurysms), and 31 age- and sex-matched non-diabetic controls. The study participants were not taking vasoactive medications, and blood glucose at the time of haemodynamic measurements was required to be between 3.8 and 11.1 mmol/l. RESULTS: HbA1c was 7.5+/-1.2% and blood glucose 7.7+/-2.8 mmol/l in these type 1 diabetic individuals, indicating relatively good glycaemic control. Retinal blood speed, arterial diameter and blood flow were not different between the diabetic individuals and the matched controls. CONCLUSIONS/INTERPRETATION: Type 1 diabetic patients with no or minimal retinopathy who maintain relatively good glycaemic control do not show abnormalities of the retinal circulation at steady state, even after several years of diabetes. In such patients it may be necessary to test the vascular response to challenges to uncover any subtle abnormalities of the retinal vessels.

Association of diabetes-related emotional distress with diabetes treatment in primary care patients with Type 2 diabetes.

AIMS: To characterize the determinants of diabetes-related emotional distress by treatment modality (diet only, oral medication only, or insulin). METHODS: A total of 815 primary care patients with Type 2 diabetes completed the Problem Areas in Diabetes (PAID) Scale and other questions. We linked survey data to a diabetes clinical research database and used linear regression models to assess the associations of treatment with PAID score. RESULTS: PAID scores were significantly higher among insulin-treated (24.6) compared with oral-treated (17.8, P < 0.001) or diet-treated patients (14.7, P < 0.001), but not different between oral- vs. diet-treated patients (P = 0.2). Group scores remained similar, but the statistical significance of their differences was reduced and ultimately eliminated after sequential adjustment for diabetes severity, HbA(1c), body mass index, regimen adherence, and self-blood-glucose monitoring. Insulin-treated patients reported significantly higher distress than oral- or diet-treated patients on 16 of 20 PAID items. 'Worrying about the future' and 'guilt/anxiety when ... off track with diabetes' were the top two serious problems (PAID >or= 5) in all treatment groups. Not accepting diabetes diagnosis was a top concern for oral- and diet-treated patients, and unclear management goals distressed diet-treated patients. CONCLUSIONS: Primary care patients treated with insulin reported higher diabetes-related emotional distress compared with oral- or diet-treated patients. Greater distress was largely explained by greater disease severity and self-care burdens. To improve diabetes-specific quality of life, clinicians should address patients' sense of worry and guilt, uncertain acceptance of diabetes diagnosis, and unclear treatment goals.

The incremental costs of recommended therapy versus real world therapy in type 2 diabetes patients.

BACKGROUND: The goals of diabetes management have evolved over the past decade to become the attainment of near-normal glucose and cardiovascular risk factor levels. Improved metabolic control is achieved through optimized medication regimens, but costs specifically associated with such optimization have not been examined. OBJECTIVE: To estimate the incremental medication cost of providing optimal therapy to reach recommended goals versus actual therapy in patients with type 2 diabetes. METHODS: We randomly selected the charts of 601 type 2 diabetes patients receiving care from the outpatient clinics of Massachusetts General Hospital March 1, 1996-August 31, 1997 and abstracted clinical and medication data. We applied treatment algorithms based on 2004 clinical practice guidelines for hyperglycemia, hyperlipidemia, and hypertension to patients' current medication therapy to determine how current medication regimens could be improved to attain recommended treatment goals. Four clinicians and three pharmacists independently applied the algorithms and reached consensus on recommended therapies. Mean incremental medication costs, the cost differences between current and recommended therapies, per patient (expressed in 2004 dollars) were calculated with 95% bootstrap confidence intervals (CIs). RESULTS: Mean patient age was 65 years old, mean duration of diabetes was 7.7 years, 32% had ideal glucose control, 25% had ideal systolic blood pressure, and 24% had ideal low-density lipoprotein cholesterol. Care for these diabetes patients was similar to that observed in recent national studies. If treatment algorithm recommendations were applied, the average annual medication cost/patient would increase from 1525 to 2164 dollars. Annual incremental costs/patient increased by 168 dollars (95% CI 133-206 dollars) for antihyperglycemic medications, 75 dollars (57-93 dollars) for antihypertensive medications, 392 dollars (354-434 dollars) for antihyperlipidemic medications, and 3 dollars (3-4 dollars) for aspirin prophylaxis. Yearly incremental cost of recommended laboratory testing ranged from 77-189 dollars/patient. LIMITATIONS: Although baseline data come from the clinics of a single academic institution, collected in 1997, the care of these diabetes patients was remarkably similar to care recently observed nationally. In addition, the data are dependent on the medical record and may not accurately reflect patients' actual experiences. CONCLUSION: Average yearly incremental cost of optimizing drug regimens to achieve recommended treatment goals for type 2 diabetes was approximately 600 dollars/patient. These results provide valuable input for assessing the cost-effectiveness of improving comprehensive diabetes care.

Correlates of health-related quality of life in type 2 diabetes.

AIMS/HYPOTHESIS: We assessed the impact of medical comorbidities, depression, and treatment intensity on quality of life in a large primary care cohort of patients with type 2 diabetes. METHODS: We used the Health Utilities Index-III, an instrument that measures health-related quality of life based on community preferences in units of health utility (scaled from 0=death to 1.0=perfect health), in 909 primary care patients with type 2 diabetes. Demographic and clinical correlates of health-related quality of life were assessed. RESULTS: The median health utility score for this population was 0.70 (interquartile range 0.39-0.88). In univariate analyses, older age, female sex, low socioeconomic status, cardiovascular disease, microvascular complications, congestive heart failure, peripheral vascular disease, chronic lung disease, depression, insulin use and number of medications correlated with decreased quality of life, while obesity, hypertension and hypercholesterolaemia did not. In multiple regression analyses, microvascular complications, heart failure and depression were most strongly related to decreased health-related quality of life, independently of duration of diabetes; in these models, diabetes patients with depression had a utility of 0.59, while patients without symptomatic comorbidities did not have a significantly reduced quality of life. Treatment intensity remained a significant negative correlate of quality of life in multivariable models. CONCLUSIONS/INTERPRETATION: Patients with type 2 diabetes have a substantially decreased quality of life in association with symptomatic complications. The data suggest that treatment of depression and prevention of complications have the greatest potential to improve health-related quality of life in type 2 diabetes.

Reversal of multidrug-resistance using Valspodar (PSC 833) and doxorubicin in osteosarcoma.

High-grade osteosarcoma is an extremely aggressive neoplasm, where over 80% of patients present with life-threatening micrometastases at diagnosis. Systemic control of the disease is therefore critical for the treatment of these patients and neoadjuvant chemotherapy using various drugs, including doxorubicin (DXR), which has been demonstrated to be the most effective regimen. Multidrug resistance (MDR) to some anticancer agents, including DXR, mediated by the MDR1 gene product P-glycoprotein (Pgp), has been shown to be a major cause of chemotherapy failure in osteosarcoma. We analyzed the effect of a cyclosporine A derivate Valspodar (PSC 833) on MDR human osteosarcoma cells. We also evaluated Pgp expression in sporadic appendicular canine osteosarcoma. Moreover, dogs were treated with combined administration of DXR and PSC 833. Several blood samples were collected for the determination of DXR and PSC 833 levels. PSC 833 induced a complete reversal of the resistant phenotype at concentrations compatible with the clinical use. Pgp was present in 12/18 (66.6%) of the cases. At the time of DXR administration, adequate blood concentrations of PSC 833, to provide a complete MDR reversal, were obtained without clinical or laboratory findings of toxicity. Combination therapy with DXR and PSC 833 allowed a 30% decrease in DXR dose infusion with equivalent therapeutic exposure. The high incidence of Pgp expression in osteosarcoma confers to the study a rationale for an effective regimen based on down-modulation of MDR.

Clinical inertia in the management of Type 2 diabetes metabolic risk factors.

AIMS: Delays in the initiation and intensification of medical therapy may be one reason patients with diabetes do not reach evidence-based goals for metabolic control. We assessed intensification of medical therapy over time, comparing the management of hyperglycaemia, hypertension, and hyperlipidaemia. METHODS: Prospective cohort study of 598 adults with Type 2 diabetes receiving primary care in an academic medical centre from May 1997 to April 1999. We assessed whether patients failing to achieve standard treatment goals for haemoglobin A1c (HbA1c), systolic blood pressure (SBP), or low density lipoprotein (LDL) cholesterol when last measured during 12 months (Year 1, 5/97-4/98) had increases in their corresponding medical regimen during the following 12 months (Year 2, 5/98-4/99). RESULTS: Among untreated patients in Year 1, seven of 12 (58%) of those above goal for HbA1c were initiated on medical therapy in Year 2, compared with 16 of 48 (34%) above SBP goal (P=0.02) and 26 of 115 (23%) above LDL cholesterol goal (P=0.02). Among patients on therapy and above goal, 124 of 244 (51%) patients with elevated HbA1c had their regimen increased in Year 2, compared with 85 of 282 (30%) with elevated SBP (P<0.001) and 22 of 79 (30%) with elevated LDL cholesterol (P<0.001). From Year 1 to Year 2 there was a decline in the overall proportion of patients above goal for LDL cholesterol (from 58% to 45%, P=0.002) but not for HbA1c or blood pressure. CONCLUSIONS: Greater initiation and intensification of pharmaceutical therapy, particularly for elevated blood pressure or cholesterol, may represent a specific opportunity to improve metabolic control in Type 2 diabetes.

Neurovisual abnormalities preceding the retinopathy in patients with long-term type 1 diabetes mellitus.

BACKGROUND: Changes in the retina caused by diabetes may lead to visual impairment in dim light, even with good visual acuity and visual fields. To evaluate the visual abnormalities preceding the retinopathy in patients with type 1 diabetes mellitus (DM), we applied electrophysiological methods. METHODS: The visual evoked responses were recorded with sinusoidally modulated vertical gratings at 10 spatial frequencies presented sequentially on a high-resolution monitor in patients with type 1 DM and in normal volunteers. Similarly, the contrast visual evoked responses of 10 contrast levels were recorded at five spatial frequencies. Both amplitudes at the second harmonic were calculated by discrete Fourier transform. The visual acuity and contrast thresholds were determined objectively. RESULTS: There was dissociation between the Snellen and the estimated visual evoked response acuity measurements in patients with diabetes (r2=0.077, P=0.44). The saturation phenomena were observed at lower levels of contrast stimuli than in normal individuals at. 1.0, 2.0, 4.0 and 8.0 cycles per degree (P=0.0001). The contrast sensitivity function was deeply abnormal in all tested patients despite the metabolic control. The values of the area under the curve of the visual evoked response amplitude-contrast level function at five spatial frequencies were smaller in patients with DM than in the control group (P<0.05) at all spatial frequencies tested. CONCLUSIONS: Patients with type 1 DM without retinopathy showed significant lower amplitude of the visual evoked responses at all spatial frequencies tested, with the saturation phenomena observed at lower level of contrast stimuli. In addition, there was a dissociation between the sweep visual evoked responses and the Snellen acuity measurements. A significant and nonselective neuronal visual loss involving the visual pathway precedes the ophthalmoscopically detectable retinopathy in patients with type 1 DM.

Clozapine, diabetes mellitus, weight gain, and lipid abnormalities: A five-year naturalistic study.

OBJECTIVE: The goal of this 5-year naturalistic study of patients treated with clozapine was to examine the incidence of treatment-emergent diabetes mellitus in relation to other factors, including weight gain, lipid abnormalities, age, clozapine dose, and treatment with valproate. METHOD: Data on age, gender, race, diagnosis, family history of diabetes, and age at clozapine initiation were collected from medical records of 82 outpatients with schizophrenia or schizoaffective disorder. Clozapine dose, data on use of valproate, and laboratory test results were recorded at 6-month intervals. RESULTS: The mean age at the time of clozapine initiation of the 82 patients was 36.4 years; 26.8% of the patients were women, and 91.5% were Caucasian. The mean baseline weight was 175.5 lb, and the mean body mass index was 26.9 kg/m(2). Thirty patients (36.6%) were diagnosed with diabetes during the 5-year follow-up. Weight gain, use of valproate, and total daily dose of clozapine were not significant risk factors for developing diabetes mellitus. Patients experienced significant weight gain that continued until approximately month 46 from initiation of clozapine. There was a nonsignificant increase in total serum cholesterol and a significant increase in serum triglycerides level. CONCLUSIONS: The results support the hypotheses that patients treated with clozapine experience significant weight gain and lipid abnormalities and appear to be at increased risk for developing diabetes.

The expression of Met/hepatocyte growth factor receptor gene in giant cell tumors of bone and other benign musculoskeletal tumors.

Overexpression of the hepatocyte growth factor receptor (Met/HGF receptor), a transmembrane tyrosine kinase encoded by the MET proto-oncogene, is involved in transformation and invasive behavior of human carcinomas and sarcomas. We have previously found that bone sarcomas express high levels of Met/HGF receptor while in some cases the ligand HGF is co-expressed with the receptor, activating an autocrine loop. In this study, we analyzed 40 biopsy samples of a collection of giant cell tumors and other rare benign tumors of bone for expression of the MET proto-oncogene. These included nonossifying fibromas, osteoblastomas, desmoplastic fibromas of bone, chondroblastomas, and giant cell tumors of bone. Snap frozen samples were tested for the MET and HGF gene expression by immuno-histochemistry and Western blotting with anti-MET antibodies and RT-PCR. Over 50% of all cases scored positive for MET expression being constantly positive in recurrent or locally aggressive lesions. Sporadic co-expression of the Met/HGF receptor and ligand is also demonstrated. Met/HGF receptor expression in benign bone neoplasms suggests its early involvement in sarcomagenesis.

Megestrol-induced clinical adrenal insufficiency.

OBJECTIVE: To describe a case of megestrol-induced adrenal insufficiency. METHODS: A patient who received megestrol and then developed pituitary-adrenal axis abnormalities is described. The literature is reviewed. RESULTS: The patient developed megestrol-induced Cushingoid features. She developed adrenal insufficiency when megestrol was stopped. CONCLUSION: Megestrol can induce alterations of the pituitary-adrenal axis in some patients. It is important to consider a diagnosis of adrenal insufficiency in patients with symptoms of fatigue, hypotension, and asthenia who have been treated with megestrol.

MET oncogene aberrant expression in canine osteosarcoma.

The objective of this study was to investigate the role of the MET oncogene in canine osteosarcoma. Seven large-breed dogs affected by spontaneous skeletal osteosarcoma underwent en bloc tumor excision. Total RNA was extracted from frozen tumor samples and assessed for expression of the MET oncogene by Northern blot analysis. Five of seven biopsy samples expressed high levels of the MET oncogene; its expression in the primary tumors was comparable with that previously identified in primary osteosarcomas in humans. A lung metastasis from one of the dogs expressed MET at a higher level than did its primary tumor. Spontaneously arising osteosarcoma in dogs clinically and pathologically mimics the corresponding disease in humans. We previously demonstrated that the MET oncogene was aberrantly expressed in a high percentage of human osteosarcomas. The results of the current study also provide a molecular parallel between the tumors in dogs and humans. This in vivo model may be helpful in evaluating new strategies for therapy against osteosarcoma.

Immediate feedback of HbA1c levels improves glycemic control in type 1 and insulin-treated type 2 diabetic patients.

OBJECTIVE: Accurate and reliable HbA1c results can be obtained at the time of the office visit by using benchtop analyzers. We tested the hypothesis that immediately available HbA1c results could improve glycemic control by changing physician or patient behavior or both. RESEARCH DESIGN AND METHODS: A randomized controlled trial was conducted in 201 type 1 and insulin-treated type 2 diabetic patients attending an academic diabetes center. HbA1c levels, changes in insulin therapy, and use of health care resources were assessed during a 12-month follow-up period. RESULTS: HbA1c levels decreased significantly at 6 and 12 months in the immediate assay group (-0.57 +/- 1.44 and -0.40 +/- 1.65%, respectively; P < 0.01) but did not change in the control group (-0.11 +/- 0.79 and -0.19 +/- 1.16%, respectively; NS). The changes were similar for both type 1 and type 2 diabetic patients. There were no differences in the rates of hypoglycemic events or use of health care resources. CONCLUSIONS: In the setting of a controlled randomized trial, the immediate feedback of HbA1c results at the time of patient encounters resulted in a significant improvement of glycemic control at 6-month follow-up and persisted for the 12-month study. The introduction of this assay was positively received by both patients and physicians.

Cytoskeletal changes induced by excess extracellular matrix impair endothelial cell replication.

Thickening of basement membranes is an early and characteristic feature of diabetic vessels, but its consequences on the properties of vascular cells remain undefined. We investigated whether and how excess extracellular matrix (ECM) alters the replication of vascular endothelial cells in vitro. To test the effects of endogenous excess matrix, human umbilical vein endothelial cells (HUVEC) were plated on ECM produced under culture conditions (high ambient glucose) that increase ECM synthesis. Four of six HUVEC isolates plated on such ECM yielded a lower cell number (68 +/- 18%) than cells plated on control ECM. Growth inhibition was observed in HUVEC cultured on elevated concentrations (10 and 50 microg/ml) of exogenous fibronectin, when compared with HUVEC plated on tissue culture plastic or 0.25, 1.0, and 5.0 microg/ml fibronectin; the decreased replication was attributable to delayed transit through the G1 phase of the cell cycle. HUVEC grown on both 1 and 10 microg/ml fibronectin exhibited a modest upregulation of the fibronectin-specific integrin receptor alpha5beta1, and increased attachment to fibronectin substratum. However, unique to the HUVEC plated on growth-inhibitory concentrations of fibronectin was a redistribution in situ of integrins and vinculin to form more numerous focal adhesions, and an increased polymerization of cytoskeletal actin to form stress fibers. Concentrations (0.01 microg/ml) of cytochalasin D intended to prevent excess actin polymerization prevented the growth inhibition. Thus, excess ECM hampers endothelial cell replication in vitro through increased cell-ECM adhesion and attendant cytoskeletal rearrangements. These phenotypic changes provide probes to test whether cell-ECM interactions are altered in diabetic vessels in a direction that may compromise orderly endothelial cell renewal and its antithrombogenic function.

Altered levels of scavenging enzymes in embryos subjected to a diabetic environment.

Maternal diabetes during pregnancy is associated with an increased rate of congenital malformations in the offspring. The exact molecular etiology of the disturbed embryogenesis is unknown, but an involvement of radical oxygen species in the teratological process has been suggested. Oxidative damage presupposes an imbalance between the activity of the free oxygen radicals and the antioxidant defence mechanisms on the cellular level. The aim of the present study was to investigate if maternal diabetes in vivo, or high glucose in vitro alters the expression of the free oxygen radical scavenging enzymes superoxide dismutase (CuZnSOD and MnSOD), catalase and glutathione peroxidase in rat embryos during late organogenesis. We studied offspring of normal and diabetic rats on gestational days 11 and 12, and also evaluated day-11 embryos after a 48 hour culture period in 10 mM or 50 mM glucose concentration. Both maternal diabetes and high glucose culture caused growth retardation and increased rate of congenital malformations in the embryos. The CuZnSOD and MnSOD enzymes were expressed on gestational day 11 and both CuZnSOD, MnSOD and catalase were expressed on day 12 with increased concentrations of MnSOD transcripts when challenged by a diabetic milieu. There was a good correlation between mRNA, protein, and activity levels, suggesting that the regulation of these enzymes occurs primarily at the pretranslational level. Maternal diabetes in vivo and high glucose concentration in vitro induced increased MnSOD expression, concomitant with increased total SOD activity, and a tentative decrease in catalase expression and activity in the embryos. These findings support the notion of enhanced oxidative stress in the embryo as an etiologic agent in diabetic teratogenesis.

Fibronectin overexpression in retinal microvessels of patients with diabetes.

PURPOSE: To examine whether fibronectin is synthesized in the adult human retina and whether diabetes alters its amount, level of expression, and pattern of splicing. METHODS: Retinas were obtained after death from 24 patients (age, 64 +/- 6 years) with 8 +/- 5 years of diabetes and 24 age-matched control subjects. Retinal localization of fibronectin and its amount in retinal vessels were examined with the immunoperoxidase technique. Fibronectin mRNA in the total retina was studied with reverse transcriptase-polymerase chain reaction using primers encompassing the alternatively spliced exon ED-A. The levels of fibronectin mRNA were measured relative to an internal standard (beta-actin mRNA). To detect and measure fibronectin expression in microvessels, retinal trypsin digests were studied by in situ hybridization. RESULTS: In the adult human retina, fibronectin is present exclusively in the walls of the vessels, including capillaries, and in the internal limiting membrane. In diabetic vessels, immunoreactive fibronectin was increased 1.4-fold over levels in control vessels (P = 0.05). Fibronectin is synthesized locally, and the predominant mRNA species excludes the ED-A region. The splicing pattern was not altered by diabetes. However, in the retinas of patients with diabetes, the levels of fibronectin mRNA were increased 1.3-fold over levels in control retinas (P = 0.028), whereas actin mRNA levels were similar in the two groups. Levels of fibronectin mRNA showed a substantial increase in the microvessels of patients with diabetes (0.49 +/- 0.18 grains/cell versus 0.15 +/- 0.12 grains/cell in control microvessels; P = 0.009. CONCLUSIONS: Fibronectin is synthesized in the adult human retina. Diabetes increases the amount of fibronectin in retinal vessels and upregulates its expression without changing the splicing pattern of the ED-A segment. Increased synthesis and deposition of fibronectin by microvascular cells may modify the interactions of cells and matrix with functional consequences relevant to the lesions of retinopathy.

The effects of high glucose on human endothelial cell growth and gene expression are not mediated by transforming growth factor-beta.

BACKGROUND: Because accumulation of extracellular matrix is a prominent characteristic of the microangiopathy that complicates long-term diabetes, a pathogenetic role for transforming growth factor beta (TGF-beta) is being considered. Having observed that glucose levels mimicking diabetic hyperglycemia induce in vitro endothelial cell overexpression of extracellular matrix molecules, decreased replication, and increased levels of TGF-beta mRNA, we have examined whether the effects of high glucose are mediated by autocrine TGF-beta. EXPERIMENTAL DESIGN: TGF-beta levels were measured by bioassay in the media conditioned by human umbilical vein endothelial cells cultured in the presence of high (30 mM) or normal (5 mM) glucose concentrations. The effect of high glucose was tested on the proliferation of two epithelial cell lines, one (Mv1Lu) exquisitely sensitive to TGF-beta and the other (DR mutants) insensitive to the cytokine. To examine whether high glucose and TGF-beta affect cellular programs in a similar manner, the effects of high glucose and exogenous TGF-beta were compared on proliferation and gene expression of endothelial cells. RESULTS: Media conditioned by endothelial cells cultured in high or normal glucose contained similar amounts of TGF-beta (4.9 +/- 3.5 and 3.7 +/- 2.5 ng/10(6) cells, respectively (mean +/- SD)), all in the latent form. The replication of parental Mv1Lu cells and their DR mutants was decreased by high glucose to the same extent. Whereas the inhibitory effect of high glucose on endothelial cell replication was reversible, that of TGF-beta was not. Both perturbations induced up-regulation of fibronectin expression, but the effects were additive. Only TGF-beta induced overexpression of Type IV collagenase. CONCLUSIONS: These combined observations indicate that (a) endothelial cells exposed to high glucose do not secrete TGF-beta in excess of control cells, (b) there are growth-inhibitory effects of high glucose that are independent of TGF-beta, and (c) high glucose and TGF-beta exert their effects through distinct pathways and at different loci.

Activity and expression of the Na+/H+ exchanger in human endothelial cells cultured in high glucose.

Establishing whether high ambient glucose affects the plasma membrane Na+/H+ exchanger is relevant to understanding the adverse effects of high glucose on cell replication and the mechanisms of the increased exchanger activity encountered in diabetic patients with nephropathy. In 8 primary and 15 first-passage isolates of human endothelial cells cultured in 30 mmol/l glucose for 8.7 +/- 2.3 and 15.8 +/- 2.3 days, respectively, we determined Na+/H+ exchanger activity and mRNA levels. Activity was determined by measuring 22Na+ influx in the presence or absence of dimethylamiloride (DMA) after intracellular acidification. We also measured fibronectin mRNA because fibronectin provides signals for cell replication through the Na+/H+ antiporter. Control cells grown in 5 mmol/l glucose showed at morphologic confluency a total Na+ influx (in nmol.mg protein-1.min-1) of 10.1 +/- 3.2 in primary and 11.7 +/- 2.2 in first subculture, which was reduced to 5.3 +/- 0.3 in the presence of DMA. Paired cultures exposed to 30 mmol/l glucose and exhibiting pHi and cell densities identical to controls showed in both primary and first subculture a reduction in total Na+ influx (delta = -0.98 +/- 0.93 nmol.mg protein-1.min-1 p < 0.005) whereas DMA-resistant Na+ influx was identical to that of control. Neither chronic hypertonicity nor acute exposure to high glucose mimicked the effects of chronic high glucose. The level of the Na+/H+ exchanger isoform 1 (NHE-1) mRNA was unchanged by high glucose whereas fibronectin mRNA levels were increased 1.5-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Vascular wall von Willebrand factor in human diabetic retinopathy.

PURPOSE: To reconstruct the role played by vascular endothelium in the elevation of circulating von Willebrand factor (vWf) in diabetic patients with microangiopathy and, specifically, to determine whether storage and synthesis of vWf is altered in diabetic retinal vessels. METHODS: Trypsin digests were prepared from retinas obtained post mortem from 11 patients (age 62 +/- 9 years, mean +/- SD) with 9 +/- 5 years of diabetes and 12 nondiabetic control subjects matched for age and sex. Trypsin digests were inspected for the presence of lesions of diabetic retinopathy; vWf protein was localized by indirect immunofluorescence; and vWf mRNA levels were studied by in situ hybridization. RESULTS: vWf immunofluorescence was present in vessels of all sizes. The granular fluorescence was localized to the endothelial cell cytoplasm. Pattern and intensity of staining in diabetic microvessels and large vessels were similar to those observed in the vessels of nondiabetic subjects. The amount of vWf mRNA detected by in situ hybridization in retinal endothelial cells was similar in diabetic (0.92 +/- 0.32 grains/cell) and control (0.91 +/- 0.42 grains/cell) microvessels. Likewise, no differences were observed in vWf mRNA levels in the large vessels of diabetic (0.073 +/- 0.034% grain area) and control (0.069 +/- 0.018 grain area) subjects. CONCLUSIONS: These observations are compatible with the occurrence in diabetes of the slow release of endothelial vWf through the pathway of vWf secretion not linked to synthesis, ie, the regulated pathway.