Biomedical subjects

Subrata Chakrabarti

Publications and source records attributed to Subrata Chakrabarti.

53 records · Page 3Linked to original sources

PubMed2003

Ex vivo and extracorporeal perfusion with hDAF pig kidneys.

The present study was undertaken to determine whether human decay accelerating factor (hDAF) transgene would prevent hyperacute rejection (HAR) while perfused with human blood or extracorporeally in baboons. Four hDAF pig kidneys and three non-hDAF pig kidneys were perfused ex vivo with fresh human blood for 6 h. Additionally four hDAF pig kidneys and four non-hDAF pig kidneys were extracorporeally perfused in baboons and pigs, respectively, for 3 h. In ex vivo perfusion, the color of hDAF pig kidneys remained pink at the end of 6-h perfusion and they had normal histology, while non-hDAF kidneys developed HAR. HDAF pig kidneys had superior function over non-transgenic pig kidneys. Urine output was 17.31 +/- 3.70 ml/h for hDAF pig kidneys, and only 5.81 +/- 0.26 ml/h for non-hDAF kidneys (P < 0.05). Creatinine clearance was 1.16 +/- 1.24 ml/min for hDAF kidneys and 0.22 +/- 0.15 ml/min for non-hDAF kidneys (P < 0.05). Other functional data including potassium, urine specific density, and osmolality were normal in the hDAF kidneys, while in non-hDAF kidneys, serum potassium was elevated to over 9 mmol/l by the end of perfusion (P < 0.01). Non-hDAF kidneys also lost more sodium through urine than hDAF kidneys (173.67 +/- 14.05 mmol/l vs. 109 +/- 31 mmol/l, P < 0.05). In the extracorporeal perfusion, all the baboons tolerated the procedure well with normal hemodynamic and hemotologic profiles. These baboons were well until killed 42 to 56 days after perfusion, although their antiporcine antibodies were greatly elevated. We conclude that hDAF transgene protects against HAR, allowing the pig kidney to function normally while perfused with human blood, and that extracorporeal perfusion using hDAF pig kidneys is a safe procedure in baboons.

Animals↗

PubMed2003

Diabetes-induced activation of nuclear transcriptional factor in the retina, and its inhibition by antioxidants.

Oxidative stress is increased in the retina in diabetes, and long-term administration of antioxidants inhibits the development of retinopathy in diabetic rats. The purpose of this study is to determine how diabetes affects the activation of a redox-sensitive nuclear transcriptional factor in the retina, NF-kappaB, and its inhibition by antioxidants. Alloxan diabetic rats were assigned to receive standard diet or the diet supplemented with multiple antioxidants, including ascorbic acid, Trolox, dl alpha-tocopherol acetate, N-acetyl cysteine, beta-carotene, and selenium for up to 14 months. NF-kappaB activation, oxidative stress and nitric oxides were measured in the retina at 2, 8 and 14 months of diabetes. Retinal NF-kappaB was activated by about 60% at two months after induction of diabetes, remained activated for up to 14 months of diabetes, and the duration of diabetes had no effect on the intensity of NF-kappaB activation. Similarly, oxidative stress and nitric oxides were elevated by over 50% in the retina of rats diabetic for 14 months, and nitrotyrosine levels were elevated by over two folds. Administration of the antioxidants to the rats for the entire duration of diabetes inhibited activation of NF-kappaB and elevations in oxidative stress, nitric oxides and nitrotyrosine formation without ameliorating the severity of hyperglycemia. These in vivo results were confirmed by in vitro studies showing that high glucose activates NF-kappaB and elevates NO and lipid peroxides in both retinal endothelial cells and pericytes that can be inhibited by antioxidants. Thus, the results suggest that the activation of retinal NF-KB in diabetes is an early event in the development of retinopathy, and it remains active when the retinal capillary cell death is accelerating, and histopathology is developing. Beneficial effects of antioxidants on the development of diabetic retinopathy might involve inhibition of NF-kappaB activation and its downstream pathways in the retina.

Animals↗

PubMed2003

Increased endothelin-1 and endothelin receptor expression in myocytes of ischemic and reperfused rat hearts and ventricular myocytes exposed to ischemic conditions and its inhibition by nitric oxide generation.

Endothelin-1 (ET-1) and nitric oxide (NO) exert opposite effects in the cardiovascular system, and there is evidence that the NO counters the potential deleterious effects of ET-1. We investigated whether NO affects the increased mRNA expression of ET-1 and endothelin receptors induced by (i) 30 min of ischemia with or without 30 min reperfusion in myocytes from isolated rat hearts or (ii) ischemic conditions (acidosis or hypoxia) in cultured rat neonatal ventricular myocytes. Ischemia with or without reperfusion produced more than a twofold increase in mRNA expression of ET-1 as well as the ET(A) and ET(B) receptor (P < 0.05), although these effects were completely blocked by the NO donor 3-morpholinosydnonimine (SIN-1; 1 microM). To assess the possible factors regulating ET expression, myocytes were exposed to acidosis (pH 6.8-6.2) or to hypoxic conditions in an anaerobic chamber for 24 h in the presence or absence of SIN-1. At all acidic pHs, ET-1 and ET(A) receptor mRNA expression was significantly (P < 0.05) elevated approximately threefold, although the magnitude of elevation was independent of the degree of acidosis. These effects were completely prevented by SIN-1. ET(B) receptor expression was unaffected by acidosis. Hypoxia increased ET-1 as well as ET(A) and ET(B) receptor expression threefold (P < 0.05), although this was unaffected by SIN-1. Our results demonstrate that myocardial ischemia and reperfusion upregulate the ET system, which is inhibited by NO. Although increased expression of the ET system can be mimicked by both acidosis and hypoxia, only the effects of the former are NO sensitive. NO may serve an endogenous inhibitory factor which regulates the expression of the ET system under pathological conditions.

Acidosis↗

PubMed2003

Endothelins in chronic diabetic complications.

Endothelins are widely distributed in the body and perform several vascular and nonvascular functions. Experimental data indicate abnormalities of the endothelin system in several organs affected in chronic diabetic complications. In support of this notion, it has been shown that endothelin-receptor antagonists prevent structural and functional abnormalities in target organs of diabetic complications in animal models. Alterations of plasma endothelin levels have also been demonstrated in human diabetes. This review discusses the role of endothelins in the pathogenesis of chronic diabetic complications. The current experimental evidence suggests that endothelin-receptor antagonism may potentially be an adjuvant therapeutic tool in the treatment of chronic diabetic complications.

Animals↗

PubMed2002

High glucose-induced, endothelin-dependent fibronectin synthesis is mediated via NF-kappa B and AP-1.

Human endothelial cells cultured under high glucose (HG) conditions were shown before to upregulate several basement membrane proteins, including fibronectin (FN), thus mimicking effects of diabetes. Using human macrovascular (HUVEC) and microvascular (HMEC) endothelial cell lines, we evaluated in the present study some of the key molecular signaling events involved in HG-induced FN overexpression. This expression was shown to be dependent on endogenous endothelin (ET) receptor-mediated signaling. We also examined the roles played by protein kinase C (PKC) and the transcription factors nuclear factor kappaB (NF-kappaB) and activating protein (AP)-1 with respect to such changes. HG, PKC activators, and ETs (ET-1 and ET-3) that increased FN expression also caused activation of NF-kappaB and AP-1. Inhibitors of both NF-kappaB and AP-1 prevented HG- and ET-induced FN production. ET receptor blockade also prevented these HG- and ET-mediated changes. The results of this study indicate that glucose-induced increased FN production in diabetes may be mediated via ET-dependent NF-kappaB and AP-1 activation.

Active Transport, Cell Nucleus↗

PubMed2002

A PCR-based amplification method retaining the quantitative difference between two complex genomes.

With the increasing emergence of genome-wide analysis technologies (including comparative genomic hybridization (CGH), expression profiling on microarrays, differential display (DD), subtractive hybridization, and representational difference analysis (RDA)), there is frequently a need to amplify entire genomes or cDNAs by PCR to obtain enough material for comparisons among target and control samples. A major problem with PCR is that amplification occurs in a nonlinear manner and reproducibility is influenced by stray impurities. As a result, when two complex DNA populations are amplified separately, the quantitative relationship between two genes after amplification is generally not the same as their relation before amplification. Here we describe balanced PCR, a procedure that faithfully retains the difference among corresponding amplified genes by using a simple principle. Two distinct genomic DNA samples are tagged with oligonucleotides containing both a common and a unique DNA sequence. The genomic DNA samples are pooled and amplified in a single PCR tube using the common DNA tag. By mixing the two genomes, PCR loses the ability to discriminate among the different alleles and the influence of impurities is eliminated. The PCR-amplified pooled samples can be separated using the DNA tag unique to each individual genomic DNA sample. The principle of this method has been validated with synthetic DNA, genomic DNA, and cDNA applied on microarrays. By removing the bias of PCR, this method allows a balanced amplification of allelic fragments from two complex DNAs even after three sequential rounds of PCR. This balanced PCR approach should allow genetic analysis in minute laser-microdissected tissues, paraffin-embedded archived material, or single cells.

DNA, Complementary↗

PubMed2002

Noninvasive prediction of cirrhosis in C282Y-linked hemochromatosis.

The aim of the present study was to examine the predictive accuracy of noninvasive clinical and biochemical variables associated with cirrhosis among patients with C282Y homozygous hemochromatosis. Sixteen clinical and laboratory variables were recorded at the time of diagnosis in 193 Canadian C282Y homozygous patients. All patients underwent percutaneous liver biopsy and 27 (14%) had biopsy specimen-proven cirrhosis. Prediction of cirrhosis was assessed first by univariate regression analysis. Variables significantly related to cirrhosis were then evaluated by stepwise linear multivariate regression. Receiver operating characteristic curve analysis of the most informative variables from multivariate analysis was then used to devise a clinically applicable index for the noninvasive prediction of cirrhosis. This index was then validated in 162 C282Y homozygous patients in France. Ferritin, blood platelets, and aspartate transaminase (AST) level were selected for the clinical index. The combination of ferritin levels of 1,000 microg/L or greater, platelet levels of 200 x 10(9)/L or less, and AST levels above the upper limit of normal led to a correct diagnosis of cirrhosis in 77% of Canadian patients. In the French patients, this led to a correct diagnosis of cirrhosis in 90%. In conclusion, in C282Y homozygous patients, a combination of easily measured laboratory variables (ferritin, platelets, AST) can be used to make the diagnosis of cirrhosis in approximately 81% of cases, reducing the need for liver biopsy.

Adult↗

PubMed2002

A nomogram to predict C282Y hemochromatosis.

Genetic testing of hemochromatosis has not been widely used as a diagnostic test because of unawareness of its existence and concerns about genetic discrimination. We developed a nomogram for the prediction of C282Y homozygotes for hemochromatosis from transferrin saturation and ferritin using Bayes theorem. The results of transferrin saturation and C282Y genotyping were available for 8,572 participants (5,042 men, and 3,530 women). The study group included patients in population-screening projects, referred cases, and family members. Likelihood ratios were calculated for transferrin saturation in predicting C282Y homozygotes. Pretest probabilities were estimated on the basis of serum ferritin concentration, and a predictive nomogram for men and women was created with the use of Bayes' theorem. In the highest-risk region of the nomogram in men, the probability of C282Y hemochromatosis was 89.7% (95% confidence interval = 85.1-94.3); in the lowest-risk zone it was 1.1% (0.4-1.9). The corresponding regions in women were 88.9% in the high zone (95% confidence interval = 77.0-100.0) and 6.5% in the lowest (95% confidence interval = 4.9-8.1). This approach allows the clinician to predict the probability of a patient's being a C282Y homozygote over a wide range of ferritin and transferrin saturation values instead of above a particular threshold.

Amino Acid Substitution↗

PubMed2002

Natural history of C282Y homozygotes for hemochromatosis.

PURPOSE: To study the clinical outcomes of subjects who are homozygous for the C282Y mutation of the hemochromatosis gene. SUBJECTS AND METHODS: All patients referred to a tertiary referral centre for hemochromatosis were included. The study also included 16 C282Y homozygotes detected in a population screening study. RESULTS: The study comprised 277 C282Y homozygotes, including 16 nonexpressing C282Y homozygotes. The mean follow-up period was 7.3 years (range zero to 44 years). The actuarial survival rates of C282Y homozygotes at five, 10 and 20 years were 95%, 93% and 66%, respectively. Life-threatening diseases (cirrhosis, hepatocellular carcinoma, diabetes, heart disease) were present in 36% of male C282Y homozygotes and 19% of female C282Y homozygotes. Cirrhosis of the liver and diabetes were the major clinical symptoms affecting long term survival. Only one nonexpressing homozygote required venesection therapy during the follow-up period. CONCLUSIONS: Long term survival is excellent in C282Y homozygotes diagnosed and treated before the development of cirrhosis and diabetes.

Adolescent↗

PubMedDate not supplied

Contributions of endothelin-1 and sodium hydrogen exchanger-1 in the diabetic myocardium.

BACKGROUND: Endothelin-1 (ET-1) and sodium hydrogen exchanger-1 (NHE-1) are important mediators of several disease processes affecting the heart, especially relating to myocardial ischemia. There is evidence that their actions may be interrelated. Their contributions to diabetic heart disease have not been extensively documented. Accordingly, the aim of this study was to investigate the interactive roles of ET-1 and NHE-1 in the pathogenesis of diabetic cardiomyopathy, a significant cause of morbidity in diabetic patients. METHODS: Streptozotocin-induced diabetic Sprague Dawley rats were treated with NHE-1 blocker cariporide or dual ET(A)/ET(B) blocker bosentan and were subsequently studied one, three and six months after induction of diabetes. These animals were compared with nondiabetic rats as well as with diabetic rats on poor blood glucose control. RESULTS: Diabetes leads to hyperglycemia, reduced body weight gain and increased glycated hemoglobin levels. These animals exhibited focal myocardial fibrosis and increased ejection fraction, in association with a tendency to increased left ventricular wall thickness and heart weight, after six months of follow-up, both bosentan and cariporide prevented these responses. Diabetes also caused significant increases in ET-1 mRNA and protein expression in the heart at all time points, which was further augmented by cariporide treatment for three months. Diabetes did not affect either mRNA or protein expression of NHE-1, although these did decrease in hearts of diabetic animals treated with bosentan for six months. CONCLUSIONS: These results indicate an important contribution of both ET-1 and NHE-1 in the pathogenesis of diabetic cardiomyopathy. These data suggest that NHE-1 may act as a downstream mediator in the production of ET-induced functional and structural changes in the myocardium in diabetes.

Animals↗

PubMedDate not supplied

The role of the sodium hydrogen exchanger-1 in mediating diabetes-induced changes in the retina.

BACKGROUND: The sodium hydrogen exchanger (NHE) is a transmembrane protein responsible for alkalinization and control of intracellular acidosis by the removal of hydrogen and the subsequent influx of sodium. Our investigation attempts to determine the role of NHE-1 in the pathogenesis of early retinal microangiopathy due to diabetes. METHODS: Diabetes was induced in male Sprague-Dawley rats with a single intravenous streptozotocin injection (65 mg/kg). To examine the duration-dependent changes in NHE-1 expression, retinas from 1-, 6- and 12-week diabetic animals were analyzed. To examine the functional consequences of NHE-1 inhibition in comparison with good blood glucose control, diabetic rats were randomly assigned to poorly controlled diabetic, well-controlled diabetic, poorly controlled diabetic with cariporide groups and were compared with nondiabetic controls after six weeks. Cariporide is an orally active inhibitor of NHE-1 (6000 ppm in rat chow). At the end of the treatment period, color Doppler ultrasound was used to determine the resistivity index (RI) of the central retinal artery. The mRNA expression of endothelin (ET) isoforms 1 and 3, inducible and endothelial nitric oxide synthase (iNOS and eNOS respectively) and NHE-1 were examined. NHE-1 distribution was localized with immunohistochemistry. RESULTS: All diabetic animals showed hyperglycemia, increased glycated hemoglobin and lower body weight gain compared to nondiabetic controls. Diabetes caused an increased RI, indicative of retinal vasoconstriction, which was corrected by both cariporide treatment and good glucose control. NHE-1 was localized in the endothelium of the retinal microvasculature and the neuronal and glial components. NHE-1 mRNA expression was unchanged after 1 week and increased after 6 and 12 weeks of diabetes. Furthermore, a diabetes-induced upregulation of ET-1 and ET-3 mRNA expression after six weeks was corrected with cariporide treatment. NHE-1 inhibition of diabetic animals upregulated iNOS mRNA levels, although expression of eNOS and iNOS mRNA were not altered in poorly controlled diabetes. Improved blood glucose control with higher doses of insulin also corrected diabetes-induced increased RI by upregulating eNOS and iNOS mRNA expression. CONCLUSIONS: The results of the study suggest that NHE-1 may be involved in the regulation of several vasoactive modulators that contribute to functional alterations in diabetic retinal microangiopathy.

Animals↗

PubMedDate not supplied

Endothelin-mediated remodeling in aortas of diabetic rats.

BACKGROUND: Smooth muscle cells proliferation and extracellular matrix (ECM) protein deposition are key features of diabetic macroangiopathy. In the present study, we have studied the role of endothelin(A) (ET(A)) receptor, the predominant receptor on smooth muscle cells, in diabetes-induced vascular hypertrophy and remodeling. METHODS: Streptozotocin-induced diabetic rats were administrated a selective ET(A) receptor antagonist, TBC3214, for 26 weeks. Following treatment, aortas were harvested and subjected to gene expression and morphometric analyses. We quantified fibronectin (FN) and plasminogen activator inhibitor-1 (PAI-1) expression as indicators of increased ECM protein synthesis. ET-1, ET-3, transforming growth factor-beta1 (TGF-beta1) and angiotensinogen mRNA levels were measured to elucidate genes involved in FN expression. We have investigated an embryonic splice variant of FN, oncofetal FN, and nonmuscle myosin heavy chain (SMemb) as vascular remodeling indicators. RESULTS: Our results show that diabetes leads to upregulation of FN, PAI-1, ET-1, ET-3, TGF-beta1 and angiotensinogen mRNA levels in association with increased medial thickness. Immunohistochemical analyses revealed concurrent protein level changes. Diabetes also upregulated oncofetal FN and SMemb mRNA levels. Treatment with TBC3214 attenuated the mRNA levels of several genes and prevented increased medial thickness. CONCLUSIONS: These results indicate that diabetes-induced vascular hypertrophy and remodeling is associated with reexpression of embryonic forms of FN and myosin heavy chain. Such changes are ET-dependent and may be mediated via TGF-beta1 and angiotensin.

Animals↗

PubMedDate not supplied

Expression of ferroportin in hemochromatosis liver.

Iron-regulated transporter protein 1 (IREG1 or ferroportin 1) is a transmembrane iron transporter that has been described in macrophages and hepatocytes. Ferroportin mutations have been described to result in hepatic iron overload in human pedigrees. The role of hepatic ferroportin in the pathogenesis of C282Y-linked hemochromatosis has not been clearly established. The objective was to study the expression of ferroportin mRNA and protein in C282Y-linked hemochromatosis liver and in controls. Human liver biopsies were stained with an anti-ferroportin antibody and quantitation of ferroportin at 62 kDa was done by Western blotting. mRNA was studied by real time RT-PCR. Ferroportin protein expression was increased in C282Y homozygotes (n = 23) compared to wild-type patients (n = 37) (P < 0.003). There was no significant correlation between ferroportin protein or mRNA expression (n = 25) and liver iron concentration or serum ferritin. Immunohistochemical staining demonstrated ferroportin in hepatocytes and macrophages. In conclusion, ferroportin protein is increased in iron-loaded hemochromatosis liver. The increase in ferroportin protein without an increase in mRNA is consistent with iron-mediated translational regulation through the 5'IRE in the mRNA.

Adult↗

PubMedDate not supplied

C-peptide and retinal microangiopathy in diabetes.

Increased extracellular matrix (ECM) protein deposition and capillary basement membrane (BM) thickening are characteristic features of diabetic retinal microangiopathy. Recent observations in the authors' laboratories suggest that high glucose in endothelial cells as well as diabetes causes up-regulation of total fibronectin (FN), as well as extradomain-B (EDB) containing the spliced variant of FN, oncofetal FN, in the retina. This splice variant is normally absent in mature adult tissues and is believed to be involved in angiogenesis. In this study, the authors have investigated the role of C-peptide in the production of ECM proteins and capillary BM thickening in the retina of diabetic rats. They investigated retinas from poorly controlled diabetic BB/Wor rats with or without C-peptide treatment as well as those from age-matched nondiabetic control rats after 8 months of diabetes. In addition, the authors investigated retinas from BBDRZ/Wor rats, a model of type 2 diabetes. Following a treatment period of 8 months, retinal tissues were harvested for gene expression and histological analyses. In the retinas of diabetic BB/Wor rats, a significant increase of oncofetal FN was demonstrated compared to control rats. C-peptide treatment of BB/Wor rats completely prevented such increase. Furthermore, retinas from BBDRZ/Wor rats, did not exhibit any such alteration in oncofetal FN expression. The authors further examined retinal capillary BM thickening using ultrastructural morphometry. C-peptide treatment was ineffective in preventing the diabetes-induced increase in capillary BM thickness. The authors' previous studies of cultured endothelial cells demonstrated that oncofetal FN synthesis is, at least in part, mediated via transforming growth factor-beta (TGF-beta) and endothelin-1 (ET-1). Hence, they examined these two transcripts in the retina of these animals. Diabetes caused significant increase in mRNA expression of ET-1 and TGF-beta, which was not prevented by C-peptide treatment. Hence it appears that C-peptide is effective in preventing diabetes-induced oncofetal FN expression and that these effects are not mediated via ET-1 or TGF-beta. In conclusion, these data suggest that C-peptide is involved in regulating ECM protein composition. Furthermore, normalization of diabetes-induced oncofetal FN up-regulation may suggest importance of C-peptide in advanced alterations in diabetic retinopathy such as angiogenesis.

Animals↗

PubMedDate not supplied

Endothelin-1-mediated alteration of metallothionein and trace metals in the liver and kidneys of chronically diabetic rats.

In the present study, the role of endothelin-1 (ET-1) on alterations of hepatic and renal metallothionein (MT) and trace metals (Zn, Cu, and Fe) were investigated in streptozotocin (STZ)-induced diabetic rats. Diabetic rats, age- and sex-matched controls, as well as control and diabetic animals on a dual ETA/ETB receptor blocker, bosentan, were investigated after 6 months of follow-up. MT was measured by cadmium-heme assay. Metals were measured by atomic absorption spectrometer. ET-1 mRNA was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) technique. Hepatic and renal ET-1 mRNA was increased in diabetic rats as compared to control rats, along with an increase in both hepatic and renal MT proteins. The increased hepatic MT protein level was associated with decreases in hepatic Cu and Fe, whereas increased renal MT was associated with increases in renal Cu and Fe accumulation. Zn levels were unaltered in both organs in diabetic rats. Bosentan treatment partially prevented the increase in MT levels in both liver and kidney, along with reduced serum creatinine and increased urinary creatinine levels. Further bosentan treatment corrected the increased Cu and Fe levels in the kidney in diabetic rats, but reduced hepatic Cu and Fe levels. No significant effects of bosentan treatment on nondiabetic rats were observed. The data suggest that the possible effects of ET antagonism in diabetes may be mediated via changes in MT and trace metals.

Animals↗

PubMedDate not supplied

Alteration of endothelins: a common pathogenetic mechanism in chronic diabetic complications.

Endothelin (ET) peptides perform several physiological, vascular, and nonvascular functions and are widely distributed in a number of tissues. They are altered in several disease processes including diabetes. Alteration of ETs have been demonstrated in organs of chronic diabetic complications in both experimental and clinical studies. The majority of the effects of ET alteration in diabetes are due to altered vascular function. Furthermore, ET antagonists have been shown to prevent structural and functional changes induced by diabetes in animal models. This review discusses the contribution of ETs in the pathogenesis and the potential role of ET antagonism in the treatment of chronic diabetic complications.

Diabetes Complications↗

PubMedDate not supplied

Growth factors in proliferative diabetic retinopathy.

Many growth factors are implicated in the pathogenesis of proliferative diabetic retinopathy. Alteration of growth factors and their receptors in diabetes has been shown in both experimental and clinical studies. Sustained hyperglycemia resulting from long-standing diabetes leads to several biochemical abnormalities that consequently result in retinal hypoxia. Retinal oxygenation state regulates various growth factors that promote angiogenesis in order to meet the oxygen demands of the tissue. However, unregulated expression of these growth factors and induction of complex cascades leading to augmentation of other proangiogenic factors, which may not be regulated by tissue oxygenation, leads to uncontrolled retinal neovascularization and blindness in diabetic patients.

Animals↗