Biomedical subjects

Subrata Chakrabarti

Publications and source records attributed to Subrata Chakrabarti.

At least 37 records · Page 2Linked to original sources

PubMed2005

Peritransplant treatment with cobalt protoporphyrin attenuates chronic renal allograft rejection.

Allogen-independent injury contributes to chronic rejection in renal allografts and heme oxygenase-1 (HO-1) has been shown to be protective in a number of settings. This study evaluated the effect of renal allograft recipient HO-1 up-regulation on chronic rejection in a rat model. Rat (F344 to Lewis) renal transplantation recipients were grouped: (i) cyclosporine (CsA) alone (0.75 mg/kg s.c. x 10 day; n = 5); (ii) CsA + low dose cobalt protoporphyrin (CoPP) an HO-1 inducer (0.5 mg/kg i.p. on days -5,0,5; n = 13) and (iii) CsA + high dose CoPP (5.0 mg/kg i.p. on days -5,0,5; n = 8). Renal function was assessed by serum creatinine levels on day 140. Histopathologic changes in allografts were graded. Morphometric analyses performed to objectively quantify the vascular changes and glomerulosclerosis. HO-1 expression quantified by Western blot and both HO-1 and endothelin (ET-1) localized using immunohistochemistry. Recipients treated with CsA + high dose CoPP had significantly decreased cortical scarring, vascular hyalinization and intimal thickness. They also had a significant, dose-dependent, reduction in luminal obliteration and glomerulosclerosis by morphometric analyses. This freedom from chronic rejection in recipients treated with CoPP translated into quiescent grafts at postoperative day 140 with immunostaining and Western blot demonstrating decreased level of HO-1 versus controls (P = 0.012). In summary, the peritransplant up-regulation of HO-1 in renal allograft recipients significantly attenuates chronic rejection in rat renal allografts by inhibiting transplant vasculopathy.

Animals↗

PubMed2005

Chemokine receptor CXCR4-beta1 integrin axis mediates tumorigenesis of osteosarcoma HOS cells.

It is known that beta1 integrins mediate the migratory response of cells to chemokine stimulation. Also, both beta1 integrins and chemokines have roles in tumor development. In the present study, the beta1 integrin-chemokine axis is assessed using human osteosarcoma (HOS) transfectant cells expressing the CXCR4 receptor for chemokine SDF-1 (CXCL12). We first identified in vitro the specific beta1 integrins that mediated the migratory response to SDF-1 stimulation. Results showed that on collagen type I and laminin, the chemotactic response to SDF-1 was predominantly mediated by alpha2beta1 integrin. On fibronectin, SDF-1-stimulated chemotaxis involved both alpha4beta1 and alpha5beta1 integrins. A comparison of the transfectant clones expressing CXCR4 at low, intermediate, and high levels and the control transfectant revealed that the transfectant clones migratory response in vitro and their ability to form tumors in vivo was related to their levels of CXCR4 expression. In addition, treatment by injection with mAbs to CXCR4, integrin alpha2beta1, or integrin alpha5beta1 effectively inhibited the growth of HOS-CXCR4 transfectant cells in vivo. Therefore, our results show that the beta1 integrins that mediated the migratory response were also functionally linked to the enhanced tumor growth of CXCR4-expressing HOS transfectant cells.

Animals↗

PubMed2005

A point mutation in the iron-responsive element of the L-ferritin in a family with hereditary hyperferritinemia cataract syndrome.

BACKGROUND: Hereditary hyperferritinemia cataract syndrome is an autosomal dominant condition that is characterized by a high serum ferritin level and bilateral early-onset cataracts in the absence of iron overload. The genetic abnormality is identified as a mutation in the 5' regulatory region of the L-ferritin messenger RNA known as the iron-responsive element (IRE). The IRE controls ferritin synthesis in response to cytoplasmic iron pools by interacting with regulatory proteins called iron responsive proteins. Mutations in the IRE decrease its affinity for iron responsive proteins, leading to the constitutive synthesis of L-ferritin which results in hyperferritinemia and the intracellular accumulation of ferritin in the lens and eventual cataract formation. PATIENTS AND METHODS: A 22-year-old woman who was being investigated for hyperferritinemia was diagnosed with hereditary hyperferritinemia cataract syndrome after an extensive workup, including genetic testing for hemochromatosis and a liver biopsy to rule out iron overload. She developed anemia with phlebotomy treatments and subsequently developed symptomatic cataracts. The pedigree of her family affected with cataracts was consistent with an autosomal dominant transmission pattern. DNA was extracted from peripheral leukocytes of eight family members, four of whom were affected by cataracts. Polymerase chain reaction amplification of the 5' region of the L-ferritin gene was performed and a heterozygous point mutation (G32T) was identified in the bulge region of the IRE. CONCLUSION: The combination of early-onset cataracts and an elevated ferritin level should suggest this genetic syndrome.

Adult↗

PubMed2005

2-amino-phenoxazine-3-one attenuates glucose-induced augmentation of embryonic form of myosin heavy chain, endothelin-1 and plasminogen activator inhibitor-1 in human umbilical vein endothelial cells.

The aim of this study was to investigate the changes in mRNA level of embryonic form of myosin heavy chain (SMemb), endothelin-1 (ET-1) and plasminogen activator inhibitor-1 (PAI-1), which are considered to be involved in the angiogenesis and atherosclerosis in diabetic blood vessels, in human umbilical vein endothelial cells (HUVECs) caused by high ambient glucose, and the effects of 2-aminophenoxazine-3-one (Phx-3), which was produced by the reaction of bovine hemoglobin with o-aminophenol, on them. The mRNA level of SMemb, ET-1 and PAI-1 and the level of SMemb protein were extensively upregulated in HUVECs treated with high concentration of glucose (15 mM), compared with those in the cells with normal concentration of glucose (5 mM). The migration activity of HUVECs evaluated by the cell migration assay was accelerated by 15 mM glucose. When 10 microM Phx-3, at the concentration of which the proliferation of HUVECs was not affected, was administered to HUVECs with 15 mM glucose, the mRNA level of SMemb, ET-1 and PAI-1 and the level of SMemb protein were significantly downregulated to the normal levels in the cells. However, when 10 microM Phx-3 was administered to HUVECs with 5 mM of glucose, the mRNA level of SMemb, ET-1 and PAI-1 and the level of SMemb protein were not affected. The migration activity of HUVECs, which was accelerated by high glucose, was reversed by 10 microM Phx-3. The present results suggest that Phx-3 may be a drug to prevent the high glucose-associated endothelial damage, vascular angiogenesis in diabetic patients, by inhibiting the expression of angiogenic factors, such as SMemb, ET-1 and PAI-1, in the endothelial cells.

Cells, Cultured↗

PubMed2004

Compensatory mechanisms influence hemostasis in setting of eNOS deficiency.

The balance between thrombosis and hemorrhage is carefully regulated. Nitric oxide (NO) is an important mediator of these processes, as it prevents platelet adhesion to the endothelium and inhibits platelet recruitment. Although endothelial NO synthase (eNOS)-deficient mice have decreased vascular reactivity and mild hypertension, enhanced thrombosis in vivo has not been demonstrated. To determine the role of endogenous NO in hemostasis, a model of carotid arterial injury and thrombosis was performed using eNOS-deficient and wild-type mice. Paradoxically, the eNOS-deficient animals had a prolongation of time to occlusion compared with the wild-type mice (P < 0.001). Consistent with this finding, plasma markers suggesting enhanced fibrinolysis [tissue plasminogen activator (t-PA) activity and antigen and D-dimer levels] were significantly elevated in eNOS-deficient animals. Vascular tissue expression of t-PA and platelet activity levels were not altered. In endothelial cells, t-PA is stored in Weibel-Palade bodies, and exocytosis of these storage granules is inhibited by NO. Thus in the absence of NO, release of Weibel-Palade body contents (and t-PA) could be enhanced; this observation is also supported by increased von Willebrand factor levels observed in eNOS-deficient animals. In summary, although eNOS deficiency attenuates vascular reactivity and increases platelet recruitment, it is also associated with enhanced fibrinolysis due to lack of NO-dependent inhibition of Weibel-Palade body release. These processes highlight the complexity of NO-dependent regulation of vascular homeostasis. Such compensatory mechanisms may partially explain the lack of spontaneous thrombosis, minimally elevated baseline blood pressure, and normal life span that are seen in animals deficient in a pivotal regulator of vascular patency.

Animals↗

PubMed2004

Co-localization of stanniocalcin-1 ligand and receptor in human breast carcinomas.

Stanniocalcin-1 (STC1) is a new polypeptide hormone that has metabolic effects on target cell mitochondria. Recent studies have shown that the STC1 gene is upregulated in primary breast tumors and co-expressed with the estrogen receptor. In this report we have demonstrated the histological co-localization of STC1 and its receptor in invasive and non-invasive human mammary gland ductal carcinomas. Analysis of 58 malignant breast biopsies revealed that STC1 and its receptor co-localized to cancer cells in 91% of cases. The study therefore reveals that in breast carcinomas STC1 signals in an autocrine feedback loop and opens up the possibility that it may be sequestered by neoplastic cells in much the same manner as it is by non-malignant cells. The data further supports the notion that STC1 plays a role in breast cancer and that it may prove to be a novel diagnostic and prognostic marker, and potential therapeutic target.

Adult↗

PubMed2004

Vascular endothelial growth factor in diabetes induced early retinal abnormalities.

Increased vascular permeability and blood flow alterations are characteristic features of diabetic retinal microangiopathy. The present study investigated vascular endothelial growth factor (VEGF) and its interactions with endothelin (ET) 1 and 3, endothelial, and inducible nitric oxide synthase (eNOS, iNOS) in mediating diabetes induced retinal vascular dysfunction. Male Sprague Dawley rats with streptozotocin (STZ) induced diabetes, with or without VEGF receptor signal inhibitor SU5416 treatment (high or low dose) were investigated after 4 weeks of follow-up. Colour Doppler ultrasound of the ophthalmic/central retinal artery, retinal tissue analysis with competitive RT-PCR and microvascular permeability were studied. Diabetes caused increased microvascular permeability along with increased VEGF mRNA expression. Increased vascular permeability was prevented by SU5416 treatment. Diabetic animals showed higher resistivity index (RI), indicative of vasoconstriction with increased ET-1 and ET-3 mRNA expression, whereas eNOS and iNOS mRNA expressions were un-affected. SU5416 treatment corrected increased RI via increased iNOS in spite of increased ET-1, ET-3 and VEGF mRNA expression. Cell culture (HUVEC) studies indicate that in part, an SU5416 induced iNOS upregulation may be mediated though a MAP kinase signalling pathway. The present data suggest VEGF is important in mediating both vasoconstriction and permeability in the retina in early diabetes.

Animals↗

PubMed2004

Glycoprotein IIb/IIIa inhibition enhances platelet nitric oxide release.

INTRODUCTION: Platelet aggregates form by fibrinogen binding to the membrane receptor glycoprotein IIb/IIIa (GPIIb/IIIa). While GPIIb/IIIa inhibitors block fibrinogen-platelet binding, stimulation of other functionally important platelet receptors may still occur. Blocking the GPIIb/IIIa receptor prevents platelet aggregation but not activation and the subsequent effect on other platelet pathways is largely unknown. MATERIALS AND METHODS: As activated platelets release reactive oxygen species that may influence thrombosis or vascular function, the effect of GPIIb/IIIa inhibitors on the platelet release of nitric oxide (NO) and superoxide was determined using an electrochemical detector and luminescence, respectively. Location of relevant platelet proteins and the interaction between platelets and leukocytes in the presence or absence of GPIIb/IIIa inhibition was determined. RESULTS: Although incubation with GPIIb/IIIa inhibitors completely abolished platelet aggregation, stimulation dependent NO release was significantly enhanced. Superoxide is known to alter the bioavailability of NO, and its contribution to the GPIIb/IIIa dependent increase in NO release was determined. In the presence of GPIIb/IIIa inhibitors, platelet superoxide release was significantly decreased. Preincubation with GPIIb/IIIa inhibitors also modified aggregation induced membrane translocation of the platelet proteins, endothelial NO synthase (eNOS) and NADPH oxidase (p67phox and p47phox), known to contribute to the generation of NO and superoxide, respectively. In the presence of leukocytes, abciximab incubation led to enhanced NO release and attenuated superoxide generation. CONCLUSION: These observations suggest that the pharmacological effects of GPIIb/IIIa antagonists on platelet function, apart from inhibition of aggregation, may contribute to their efficacy.

Abciximab↗

PubMed2004

Extracellular signal-regulated kinase (ERK) in glucose-induced and endothelin-mediated fibronectin synthesis.

Increased extracellular matrix protein deposition and basement membrane thickening are important features of diabetic angiopathy. One key matrix protein that has been shown to be instrumental in basement membrane thickening is fibronectin (FN). We have previously demonstrated that glucose-induced increased expression of endothelin-1 (ET-1), may in part, be responsible for increased FN expression via nuclear factor-kappaB (NF-kappaB) and activating protein (AP-1) activation. The present study was aimed at elucidating the mechanism of ET-1 with respect to mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway activation and glucose-induced FN upregulation. Human endothelial cells were exposed to either low (5 mM) or high (25 mM) glucose levels. Cells in low glucose were also treated with ET-1 peptide (5 nM). In addition, we treated cells exposed to high glucose levels with specific MAPK/ERK inhibitor PD098059 (50 microM), dual ET-receptor antagonist, bosentan (10 microM), and PKC blocker, chelerythrine (1 microM). Following incubation period, RNA and total proteins were extracted for RT-PCR for FN and immunoblot analysis of MAPK/ERK activation. Confocal microscopy was performed for analysis of FN protein and nuclear localization of activated Elk. Electrophoretic mobility shift assay was carried out to detect NF-kappaB and AP-1 activation. Our data demonstrates that high glucose-induced upregulation of FN messenger RNA and protein levels occur via activation of MAPK/ERK pathway, which was prevented by treatment of cells with bosentan, PD098059 and PKC blocker chelerythrine. Confocal microscopy demonstrated nuclear localization of phospho-Elk protein. Glucose-induced FN expression was also associated with protein kinase C, NF-kappaB, and AP-1 activation. These results suggested that glucose-induced, ET- and PKC-dependent, upregulation of FN is, in part, mediated via MAPK/ERK activation.

Alkaloids↗

PubMed2004

Pro-oxidant role of heme oxygenase in mediating glucose-induced endothelial cell damage.

Oxidative damage to the vascular endothelial cells may play a crucial role in mediating glucose-induced cellular dysfunction in chronic diabetic complications. The present study was aimed at elucidating the role of glucose-induced alteration of highly inducible heme oxygenase (HO) in mediating oxidative stress in the vascular endothelial cells. We have also investigated the interaction between HO and the nitric oxide (NO) system, and its possible role in alteration of other vasoactive factors. Human umbilical vein endothelial cells (HUVECs) were exposed to low (5mmol/l) and high (25mmol/l) glucose levels. In order to determine the role of HO in endothelial dysfunction and to elucidate a possible interaction between the HO and NO systems, cells were exposed to HO inducer (hemin, 10 micromol/l), HO antagonist (SnPPIX, 10 micromol/l), and NO synthase blocker (L-NAME, 200 micromol/l) with or without NO donor (arginine, 1 mmol/l). mRNA expression of HO and NO isoforms was measured by real time RT-PCR. HO activity was measured by bilirubin production and cellular oxidative stress was assessed by 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nitrotyrosine staining. We also determined the expression of vasoactive factors, endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF). In the endothelial cells, glucose caused upregulation of HO-1 expression and increased HO activity. A co-stimulatory relationship between HO and NO was observed. Increased HO activity also associated with oxidative DNA and protein damage in the endothelial cells. Furthermore, increased HO activity augmented mRNA expression of vasoactive factors, ET-1 and VEGF. These data suggest that HO by itself and via elaboration of other vasoactive factors may cause endothelial injury and functional alteration. These findings are of importance in the context of chronic diabetic complications.

Endothelin-1↗

PubMed2004

Heme-oxygenase-mediated iron accumulation in the liver.

Heme oxygenase (HO) isozymes, HO-1 and HO-2, catalyze the conversion of heme to iron, carbon monoxide, and biliverdin. The present study was aimed at elucidating the role of the HO system in iron accumulation and oxidative stress in the liver. We have also studied the regulation of an iron exporter, ferroportin-1 (FPN-1), as an adaptive response mechanism to increased iron levels. Sprague-Dawley rats were treated with HO inducer hemin or HO inhibitor tin-protoporphyrin IX (SnPPIX) for 1 month. A portion of liver tissues was subjected to RT-PCR for HO-1, HO-2, and FPN-1 gene expression as well as an HO activity assay. Paraffin-embedded tissues were stained for iron with Prussian blue. Hepatic iron concentration was measured by High Resolution-Inductively Coupled Plasma-Mass Spectrometry. 8-hydroxy-2'-deoxyguanosine (8-OHdG) stain, a sensitive and specific marker of oxidative DNA damage, was performed to assess oxidative stress. Hemin treatment led to augmented HO expression and activity in association with increased iron accumulation and oxidative stress. FPN-1 expression was also found to be upregulated. SnPPIX treatment reduced HO activity, intracellular iron levels, and oxidative stress as compared to controls. Our data provides evidence of increased HO activity as an important pro-oxidant mechanism leading to iron accumulation in the liver.

Animals↗

PubMed2004

Oncofetal fibronectin in diabetic retinopathy.

PURPOSE: Imbalance between extracellular matrix protein synthesis and degradation is a key feature of diabetic retinopathy. Fibronectin, a predominant constituent of the extracellular matrix, has been shown to undergo alternative splicing to produce embryonic isoforms in various pathologic conditions, such as fibrotic diseases and tumorigenesis. Two such isoforms, oncofetal fibronectin variants that are characterized by the inclusion of the oncofetal domains A and B, were the focus of the present study. METHODS: The expression of oncofetal fibronectin variants was determined in human vitreous samples obtained from patients undergoing vitrectomy for proliferative diabetic retinopathy and nondiabetes-associated ocular conditions such as macular hole. In addition, an animal model of chronic diabetes and cultured endothelial cells was used to elucidate the mechanistic basis for this aberrant expression of oncofetal fibronectin. RESULTS: Expression of fibronectin containing the oncofetal domain B was upregulated in the vitreous of patients with diabetic retinopathy. CONCLUSIONS: Use of a well-established animal model of chronic diabetic complications and cultured endothelial cells showed that diabetes-induced upregulation of oncofetal fibronectin is, in part, dependent on hyperglycemia-induced transforming growth factor-beta1 and endothelin-1. Furthermore, the data suggest that oncofetal fibronectin is involved in endothelial cell proliferation.

Aged↗

PubMed2004

Potential contributory role of H-Ras, a small G-protein, in the development of retinopathy in diabetic rats.

Hyperglycemia is thought to be the underlying factor in the development of diabetic retinopathy, but the mechanisms involved remain partially understood. Diabetes increases oxidative stress, and reactive oxygen species affect the interactions between a small-molecular- weight G-protein, H-Ras, and several of its effector proteins. The purpose of this study was to examine the regulatory role of H-Ras in glucose-induced apoptosis of retinal endothelial cells. The expressions of H-Ras and its effector protein (Raf-1) were compared in the retina obtained from diabetic rats (2-8 months' duration) and age-matched normal rats and in retinal endothelial cells exposed to high-glucose medium. The effect of inhibition of H-Ras function on glucose-induced capillary cell death and the potential involvement of oxidative stress in diabetes-induced activation of H-Ras were also determined. The expressions of H-Ras and Raf-1 were increased in the retina in diabetes, and antioxidant therapy prevented diabetes-induced increased protein and mRNA expressions. The inhibitors of Ras farnesylation inhibited glucose-induced nitric oxides and apoptosis in isolated retinal endothelial cells. Thus, the results suggest that functional activation of H-Ras might be one of the signaling steps involved in glucose-induced capillary cell apoptosis and supports the role of H-Ras in the development of retinopathy in diabetes.

Animals↗

PubMed2003

Alteration in CD45RBhi/CD45RBlo T-cell ratio following CD45RB monoclonal-antibody therapy occurs by selective deletion of CD45RBhi effector cells.

Tolerance induction by CD45RB monoclonal antibody (mAb) in murine allograft models is associated with an alteration in the CD45RBlo/CD45RBhi T-cell ratio in favor of CD45RBlo T cells, which can function as regulatory cells and promote tolerance. It has been proposed that inversion of the CD45RBhi/CD45RBlo normal T-cell ratio by mAb can occur by down-regulation of CD45RB surface molecules expressed by T cells. Because CD45RB mAb infusion can lead to a reduction in peripheral T cells, we tested whether other mechanisms might participate in the inversion of the CD45RBhi/CD45RBlo ratio, including apoptosis of CD45RBhi cells. We report that CD45RB mAb led to rapid elimination of both CD4+ and CD8+ T cells in vitro. Importantly, CD45RB mAb selectively eliminated CD45RBhi T cells without affecting the viability of CD45RBlo T cells. Furthermore, the death of T cells occurred with a reduction in mitochondrial transmembrane potential and DNA fragmentation but with little evidence of nuclear condensation and cell shrinkage typically found with cells undergoing apoptosis. We propose that CD45RB mAb therapy may promote a dominant regulatory T-cell population that has the capacity to inhibit rejection by the selective elimination of CD45RBhi effector T cells. This occurs by a process that does not involve the classic morphologic features of apoptosis. Strategies that facilitate an inversion of the CD45RBhi/CD45RBlo T-cell subset ratio may improve the efficacy of CD45RB mAb, and therapeutic measures that prevent deletion of CD45RBhi T cells may need to be avoided to achieve tolerance clinically.

Animals↗

PubMed2003

Endothelin-1 promotes migration and induces elevation of [Ca2+]i and phosphorylation of MAP kinase of a human extravillous trophoblast cell line.

A highly proliferative, migratory and invasive subpopulation of human placental trophoblasts, known as extravillous trophoblasts (EVT), invades the uterus and its vasculature, to establish an adequate exchange of key molecules between the maternal and fetal circulation. Our earlier studies provided evidence for a positive regulation of migration/invasion of EVT by an autocrine factor IGFII and a paracrine, decidua-derived factor IGFBP1. The present study examined the role played by endothelin (ET)-1, also produced at the fetal-maternal interface, and its receptor subtypes ET(A) and ET(B) in the regulation of human EVT cell functions. We utilized an in vitro propagated EVT cell line (HTR-8/SVneo) which exhibits the phenotypic and functional characteristics of EVT in situ. Reverse transcription-PCR with primers specific for prepro-ET-1, ET(A) and ET(B) cDNAs demonstrated the expression of all these genes in HTR-8/SVneo cells. While proliferation was not influenced, migration of these cells through porous Transwell membranes was stimulated by exogenous ET-1. ET-1 also induced biphasic elevation of cytosolic free Ca(2+) concentrations ([Ca(2+)](i)) consisting of an initial transient followed by a sustained plateau, as measured by spectrofluorimetry. The dependence of the Ca(2+) response on phospholipase C (PLC) was demonstrated by its abrogation in the presence of PLC inhibitor U73122. Furthermore, ET-1 treatment of EVT cells rapidly stimulated phosphorylation of MAP kinase (ERK1/2). By using ET receptor antagonists and agonists, it was shown that both ET(A) and ET(B) receptors were responsible for the effects of ET-1 on migration, [Ca(2+)](i) and MAPK phosphorylation. Thus, ET-1 may represent an autocrine/paracrine mediator of invasive trophoblast function.

Adenylyl Cyclases↗

PubMed2003

Differential activation of NF-kappa B and AP-1 in increased fibronectin synthesis in target organs of diabetic complications.

Increased extracellular matrix protein production leading to structural abnormalities is a characteristic feature of chronic diabetic complications. We previously showed that high glucose in endothelial cell culture leads to the upregulation of basement membrane protein fibronectin (FN) via an endothelin (ET)-dependent pathway involving activation of NF-kappaB and activating protein-1 (AP-1). To delineate the mechanisms of basement membrane thickening, we used an animal model of chronic diabetes and evaluated ET-dependent activation of NF-kappaB and AP-1 and subsequent upregulation of FN in three target organs of chronic diabetic complications. After 3 mo of diabetes, retina, renal cortex, and myocardium demonstrated increased FN mRNA and increased ET-1 mRNA expression. Increased FN expression was shown to be dependent on ET receptor-mediated signaling, as the increase was prevented by the dual ET receptor antagonist bosentan. NF-kappaB activation was most pronounced in the retina, followed by kidney and heart. AP-1 activation was also most pronounced in the retina but was similar in both kidney and heart. Bosentan treatment prevented NF-kappaB activation in the retina and heart and AP-1 activation in the retina and kidney. These data indicate that, although ETs are important in increased FN production due to diabetes, the mechanisms with respect to transcription factor activation may vary depending on the microenvironment of the organ.

Actins↗

PubMed2003

Improvement in human decay accelerating factor transgenic porcine kidney xenograft rejection with intravenous administration of gas914, a polymeric form of alphaGAL.

BACKGROUND: The present study was undertaken to determine whether intravenous administration of GAS914, a polymeric form of alphaGal, would minimize porcine kidney xenograft rejection in baboons. Human decay accelerating factor renal xenografts were transplanted into 16 baboon recipients. METHODS: Baseline immunosuppression for all groups included cyclosporine A, cyclophosphamide, SDZ-RAD, and methylprednisolone. Group 1 received only baseline immunosuppression; group 2 animals received low-dose GAS914 with baseline immunosuppression; group 3 animals received high dose GAS914 with high-dose baseline immunosuppression; and animals from group 4 received high-dose GAS914 and low-dose baseline immunosuppression. RESULTS: None of the animals in this study developed hyperacute rejection. Intravenous administration of GAS914 significantly reduced xenoreactive antibodies as measured by antiporcine hemolytic assays and anti-Gal (immunoglobulin [Ig] G and IgM) antibody assays. Rejection was less severe in the GAS914-treated group. Only 25% (3 of 12) of GAS914-treated animals were killed as a result of rejection, whereas 75% (three of four) of non-GAS914-treated animals were killed because of terminal rejection (P<0.01). Protocol biopsies demonstrated that the degree of acute humoral xenograft rejection (AHXR) was reduced in the GAS914-treated animals compared with non-GAS914-treated animals. CONCLUSION: The intravenous administration of GAS914 reduces xenoreactive antibody levels and reduces the degree of porcine kidney xenograft rejection, but does not improve survival. AHXR and drug toxicity remain major barriers to the long-term success of xenotransplantation.

Adrenal Cortex Hormones↗

PubMed2003

Heme oxygenase in diabetes-induced oxidative stress in the heart.

Diabetic cardiomyopathy is responsible for substantial morbidity and mortality in the diabetic population. Increased oxidative stress has been associated with the pathogenesis of chronic diabetic complications including cardiomyopathy. Multiple biochemical mechanisms have been proposed to increase oxidative stress in diabetes. The present study was aimed at elucidating the role of a potent oxidative and cellular stress-responsive system, the heme oxygenase (HO) system, in the heart in diabetes. Streptozotocin-induced diabetic rats were treated with a potent inhibitor of HO system, tin protoporphyrin IX (SnPPIX, 50 micromol/kg/d), and were compared with untreated diabetic and non-diabetic animals. All treatments began at the onset of diabetes, 48 h after injection of streptozotocin along with the confirmation of hyperglycemia. Animals were euthanized after 1 week and 1 month of treatment, and heart tissues were harvested. Frozen tissues were subjected to HO-1 and HO-2 mRNA expression by real-time RT-PCR and HO activity determination. Paraffin-embedded tissue sections were used for immunohistochemical analysis of HO-1 and HO-2. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) stain, a sensitive and specific marker of DNA damage, was preformed to assess damage induced by oxidative stress. In addition, tissue sections were subjected to histochemical analysis for iron. We further examined non-diabetic animals treated with a direct HO agonist, hemin (50 mg/kg/d). A possible relationship between the HO and the nitric oxide (NO) pathways was also considered by studying the mRNA levels of endothelial nitric oxide synthase (NOS) and inducible NOS, and by measuring the amount of NOS products. Our results demonstrate no significant alterations of the HO system following 1 week of diabetes. However, 1 month of diabetes caused increased oxidative stress as demonstrated by higher levels of 8-OHdG-positive cardiomyocytes (80% positive as compared to 11.25% in controls), in association with increased HO isozyme mRNA (2.7-fold increase as compared to controls) and protein expression, and augmented HO activity (759.3 as compared to 312.3 pmol BR/h/mg protein in controls). Diabetic rats further demonstrated increased number of cardiomyocytes with stainable iron. SnPPIX treatment resulted in reduced number of 8-OHdG-positive cardiomyocytes (19.5% as compared to 80% in diabetics) in parallel with reduced HO activity (569.7 as compared to 759.3 pmol BR/h/mg protein in diabetics). Non-diabetic rats treated with HO-agonist hemin exhibited abnormalities similar to diabetic rats. Our results provide the first direct demonstration that diabetes-induced oxidative stress in the heart is, in part, due to upregulated HO expression and activity. These results provide evidence of pro-oxidant activity of HO in the heart in diabetes, which could be mediated by increased redox-active iron.

8-Hydroxy-2'-Deoxyguanosine↗