Endothelial cell injury by high glucose and heparanase is prevented by insulin, heparin and basic fibroblast growth factor

BACKGROUND: Uncontrolled hyperglycemia is the main risk factor in the development of diabetic vascular complications. The endothelial cells are the first cells targeted by hyperglycemia. The mechanism of endothelial injury by high glucose is still poorly understood. Heparanase production, induced by hyperglycemia, and subsequent degradation of heparan sulfate may contribute to endothelial injury. Little is known about endothelial injury by heparanase and possible means of preventing this injury. OBJECTIVES: To determine if high glucose as well as heparanase cause endothelial cell injury and if insulin, heparin and bFGF protect cells from this injury. METHODS: Cultured porcine aortic endothelial cells were treated with high glucose (30 mM) and/or insulin (1 U/ml) and/or heparin (0.5 μg/ml) and /or basic fibroblast growth factor (bFGF) (1 ng/ml) for seven days. Cells were also treated with heparinase I (0.3 U/ml, the in vitro surrogate heparanase), plus insulin, heparin and bFGF for two days in serum free medium. Endothelial cell injury was evaluated by determining the number of live cells per culture and lactate dehydrogenase (LDH) release into medium expressed as percentage of control. RESULTS: A significant decrease in live cell number and increase in LDH release was found in endothelial cells treated with high glucose or heparinase I. Insulin and/or heparin and/or bFGF prevented these changes and thus protected cells from injury by high glucose or heparinase I. The protective ability of heparin and bFGF alone or in combination was more evident in cells damaged with heparinase I than high glucose. CONCLUSION: Endothelial cells injured by high glucose or heparinase I are protected by a combination of insulin, heparin and bFGF, although protection by heparin and/or bFGF was variable

Exchange bias in Co-Cr2O3 nanocomposites

The possibility of using exchange bias in ferromagnetic-antiferromagnetic system to over come the effect of superparamagnetism in small cobalt nanoparticles is explored. We have prepared Co-Cr2O3 nanocomposite powders using a chemical method. It is shown that in this system the effect of superparamagnetism in cobalt nanoparticles could be overcome. The superparamagnetic blocking temperature of 3 nm cobalt particles has been increased to above room temperature. The choice of Cr2O3 is vital as its TN is higher compared to other antiferromagnetic materials used for this purpose such as CoO. The field cooled and zero field cooled hysteresis measurements of the samples confirm the existence of exchange bias interaction in this system.Comment: 17 pages including 4 figures and 1 tabl

Heparin inhibits endothelin-1 production in cultured rat mesangial cells

Heparin inhibits endothelin-1 production in cultured rat mesangial cells. The present study was designed to examine whether heparin inhibits basal or stimulated endothelin-1 production by arginine vasopressin (AVP) and platelet-derived growth factor (PDGF) in cultured rat mesangial cells. In addition, the reversibility of the heparin effect on mesangial cell endothelin-1 production was examined. AVP and PDGF stimulated endothelin-1 secretion in a concentration-dependent manner in these cells. Heparin (10 to 100 U/ml) exhibited concentration-related inhibition of AVP- and PDGF-stimulated endothelin-1 secretion. Heparin also had weak but significant inhibitory effects on basal endothelin-1 secretion in these cells. The protein kinase (PKC)-activating phorbor ester, phorbor myristate acetate (PMA), stimulated endothelin-1 secretion and heparin inhibited PMA-stimulated endothelin-1 secretion. In addition, the inhibitory effect of heparin was completely abolished in PKC-depleted mesangial cells. Mesangial cells which were exposed to a high concentration (100 U/ml) of heparin for 24 hours were capable of producing endothelin-1 after a short lag period of removal of heparin from the culture medium. These mesangial cells also showed recovery of responses to AVP and PDGF by secreting a significantly greater amount of endothelin-1 than the non-stimulated level. These results indicate that heparin potently inhibits mesangial cell endothelin-1 production, especially when stimulated by AVP or PDGF. This inhibitory effect of heparin is probably PKC dependent, and reversible

Identification of R368H as a predominant CYP1B1 allele causing primary congenital glaucoma in Indian patients

Purpose: To investigate the predominant mutation in the CYP1B1 gene in patients in India with primary congenital glaucoma (PCG), using PCR-restriction fragment length polymorphism (RFLP) methods and to characterize the molecular defect in two generations of an affected family. Methods: DNA samples from 146 patients with PCG from 138 pedigrees were analyzed for several distinct mutations in CYP1B1 by PCR-RFLP. Results: PCR-RFLP screening revealed that 30.8% of patients were positive for any one of the six mutations (376insA, 528G→A, 923C→T, 959G→A, 1449G→A, and 1514C→A), and 17.8% of the patients were found to have the rarely reported mutation R368H (1449G→A). All mutations were confirmed by DNA sequencing. Conclusions: The results suggest extensive allelic heterogeneity in the Indian patients with PCG, with the predominant allele being R368H among the 146 Indian patients tested. It appears possible to use this approach for carrier detection in pedigrees with a positive family history and in population screening. The approach also offers a method for rapid screening of potential carriers and affected individuals

Globally, CYP1B1 mutations in primary congenital glaucoma are strongly structured by geographic and haplotype backgrounds

Purpose: To obtain a global perspective on the distribution and evolution of CYP1B1 mutations in primary congenital glaucoma (PCG) worldwide. Methods. Five intragenic single-nucleotide polymorphisms in CYP1B1-R48G, A119S, V432L, D449D, and N453S-were used to generate haplotype data from 138 Indian patients with PCG and 132 ethnically matched normal controls, which were then analyzed in conjunction with data from other populations. Maximum-likelihood estimates of haplotype frequencies were estimated from the genotype data. Subsets of patients and normal control subjects were also genotyped with respect to eight short tandem repeat (STR) markers around the CYP1B1 locus (D2S305, D2S165, D2S367, D2S2259, D2S391, D2S3337, D2S23678, and D2S286), to gain evolutionary insights. Results: Common mutations in CYP1B1 that are causal of PCG occurred on a uniform haplotype background among Indian patients, which is completely distinct from the modal haplotype background found among unaffected control subjects. Comparison of these data with data from other global regions reveals strong clustering of CYP1B1 mutations by geographic and haplotype backgrounds. The two distinct modal haplotypes found among Indian patients with PCG and control subjects are both ancient with ages of similar magnitudes, as indicated by large variances in the number of repeats at eight STR loci. Together with data from chimpanzee and normal control subjects from India and other global regions, it was possible to make a parsimonious reconstruction of the evolution of these haplotypes. Conclusions: The strong association of specific haplotypes with some predominant CYP1B1 mutations underlying PCG and the observed geographical clustering, probably due to founder effects, may be useful for predictive testing

Novel mutation in FOXC1 wing region causing Axenfeld-Rieger anomaly

Purpose: To determine the possible molecular genetic defect underlying Axenfeld-Rieger anomaly (ARA) and to identify the pathogenic mutation causing this anterior segment dysgenesis in an Indian pedigree. Methods: The FOXC1 gene was amplified from genomic DNA of members of an ARA-affected family and control subjects using four novel sets of primers. The amplicons were directly sequenced, and the sequences were analyzed to identify the disease-causing mutation. Results: A heterozygous novel missense mutation was identified in the coding region of the FOXC1 gene in all three patients in this family. Consistent with the autosomal dominant inheritance pattern, the mutation segregated with the disease phenotype and was fully penetrant. The mutation was found in the wing region of the highly conserved forkhead domain of the FOXC1 gene and resulted in a very severe phenotype leading to blindness. Conclusions: This is the first study to demonstrate that a mutation in the FOXC1 wing region can cause an anterior segment dysgenesis of the eye. This mutation resulted in blindness in the ARA-affected family, and the findings suggest that the FOXC1 wing region has a functional role in the normal development of the eye. Moreover, this is the first study from India to report the genetic etiology of Axenfeld-Rieger anomaly. Genotype-phenotype correlations of FOXC1 may help in establishing the disease prognosis and also in understanding the clinical and genetic heterogeneity associated with various anterior segment dysgenesis caused by this gene