Cellular mechanisms by which proinsulin C-peptide prevents insulin-induced neointima formation in human saphenous vein.

AIMS/HYPOTHESIS: Endothelial cells (ECs) and smooth muscle cells (SMCs) play key roles in the development of intimal hyperplasia in saphenous vein (SV) bypass grafts. In diabetic patients, insulin administration controls hyperglycaemia but cardiovascular complications remain. Insulin is synthesised as a pro-peptide, from which C-peptide is cleaved and released into the circulation with insulin; exogenous insulin lacks C-peptide. Here we investigate modulation of human SV neointima formation and SV-EC and SV-SMC function by insulin and C-peptide. METHODS: Effects of insulin and C-peptide on neointima formation (organ cultures), EC and SMC proliferation (cell counting), EC migration (scratch wound), SMC migration (Boyden chamber) and signalling (immunoblotting) were examined. A real-time RT-PCR array identified insulin-responsive genes, and results were confirmed by real-time RT-PCR. Targeted gene silencing (siRNA) was used to assess functional relevance. RESULTS: Insulin (100 nmol/l) augmented SV neointimal thickening (70% increase, 14 days), SMC proliferation (55% increase, 7 days) and migration (150% increase, 6 h); effects were abrogated by 10 nmol/l C-peptide. C-peptide did not affect insulin-induced Akt or extracellular signal-regulated kinase signalling (15 min), but array data and gene silencing implicated sterol regulatory element binding transcription factor 1 (SREBF1). Insulin (1-100 nmol/l) did not modify EC proliferation or migration, whereas 10 nmol/l C-peptide stimulated EC proliferation by 40% (5 days). CONCLUSIONS/INTERPRETATION: Our data support a causative role for insulin in human SV neointima formation with a novel counter-regulatory effect of proinsulin C-peptide. Thus, C-peptide can limit the detrimental effects of insulin on SMC function. Co-supplementing insulin therapy with C-peptide could improve therapy in insulin-treated patients.

Hypoxic remodelling of Ca2+ stores does not alter human cardiac myofibroblast invasion.

Cardiac fibroblasts are the most abundant cell type in the heart, and play a key role in the maintenance and repair of the myocardium following damage such as myocardial infarction by transforming into a cardiac myofibroblast (CMF) phenotype. Repair occurs through controlled proliferation and migration, which are Ca(2+) dependent processes, and often requires the cells to operate within a hypoxic environment. Angiotensin converting enzyme (ACE) inhibitors reduce infarct size through the promotion of bradykinin (BK) stability. Although CMF express BK receptors, their activity under the reduced O(2) conditions that occur following infarct are entirely unexplored. Using Fura-2 microfluorimetry on primary human CMF, we found that hypoxia significantly increased the mobilisation of Ca(2+) from intracellular stores in response to BK whilst capacitative Ca(2+) entry (CCE) remained unchanged. The enhanced store mobilisation was due to a striking increase in CMF intracellular Ca(2+)-store content under hypoxic conditions. However, BK-induced CMF migration or proliferation was not affected following hypoxic exposure, suggesting that Ca(2+) influx rather than mobilisation is of primary importance in CMF migration and proliferation.

Diabetic ketoacidosis--a possible complicating factor in deaths associated with drug overdose: two case reports.

Two cases are described where diabetic ketoacidosis was found in conjunction with significant levels of prescription drugs. Case 1: A 45-year-old woman with a history of insulin-dependent diabetes mellitus was found to have Armanni-Ebstein nephropathy characteristic of hyperglycaemia with a vitreous humour glucose level of 63.1 mmol/L and a beta-hydroxybutyrate level of 14.25 mmol/L. Ancillary toxicological evaluation revealed a lethal level of sertraline (2.5 mg/L), with an elevated level of methadone (0.23mg/L). Death was due to diabetic ketoacidosis complicating mixed drug toxicity. Case 2: A 27-year-old man with a history of insulin-dependent diabetes mellitus was found to have Armanni-Ebstein nephropathy with a vitreous humour glucose level of 51.7 mmol/L and a beta-hydroxybutyrate level of 18.6mmol/L. Ancillary toxicological evaluation revealed a potentially lethal level of methadone of 0.39mg/L. Death was attributed to diabetic ketoacidosis complicating methadone toxicity. These cases demonstrate a situation where drug toxicity led to diabetic ketoacidosis resulting in death most likely from a combination of factors. Measuring vitreous humour glucose and beta-hydroxybutyrate levels is important in individuals with histories, or scene evidence, of insulin-dependent diabetes mellitus, in addition to toxicological screening when there is evidence of possible drug taking. It appears that drug intoxication in both cases had impaired the ability to administer insulin, resulting in the development over time of diabetic ketoacidotic states. Lethal mechanisms were, therefore, more complex than simple drug toxicity or diabetic ketoacidosis in isolation.

A rapid fatal erythroleukemia caused by J2E cells can be treated ex vivo with erythropoietin.

The J2E cell line is an immature erythroid line which terminally differentiates in response to erythropoietin (epo), producing mature, hemoglobin-synthesizing red blood cells. We have shown that when these cells were injected into mice a rapid and fatal erythroleukemia developed with symptoms of severe anemia and hepatosplenomegaly. Southern blotting demonstrated that the leukemic cells were the introduced J2E cells. In addition to spleen and liver, the bone marrow was a major site of leukemic cell infiltration, and when grown in vitro leukemic cells from bone marrow remained responsive to erythropoietin. We reasoned, therefore, that treatment of mice with this hormone should alleviate the erythroleukemia, but regular injections of epo in vivo failed to arrest the progress of the disease. However, when bone marrow from leukemic mice was exposed continuously to the hormone ex vivo, before reinfusion into naive recipients, a marked extension in life span was observed. It was concluded that ex vivo epo treatment could be used therapeutically for J2E cell erythroleukemias.

Hypoxic inhibition of human cardiac fibroblast invasion and MMP-2 activation may impair adaptive myocardial remodelling.

Cardiac fibroblasts account for up to two-thirds of the total number of cells in the normal heart and are responsible for extracellular matrix homoeostasis. In vitro, type I collagen, the predominant myocardial collagen, stimulates proteolytic activation of constitutively secreted proMMP-2 (pro-matrix metalloproteinase-2). This occurs at the cell membrane and requires formation of a ternary complex with MT1-MMP (membrane-type-1 MMP) and TIMP-2 (tissue inhibitor of metalloproteinases-2). Following MI (myocardial infarction), normally quiescent fibroblasts initiate a wound healing response by transforming into a proliferative and invasive myofibroblast phenotype. Deprivation of oxygen to the myocardium is an inevitable consequence of MI; therefore this reparative event occurs under chronically hypoxic conditions. However, species and preparation variations can strongly influence fibroblast behaviour, which is an important consideration when selecting experimental models for provision of clinically useful information.

The major erythroid DNA-binding protein GATA-1 is stimulated by erythropoietin but not by chemical inducers of erythroid differentiation.

The expression of the major erythroid DNA-binding protein GATA-1 was studied during erythropoietin and chemically induced erythroid differentiation of J2E and murine erythroleukemia (MEL) cells. An increase in GATA-1 mRNA levels was observed shortly after hormonal stimulation of J2E cells, due to increased transcription and not stabilization of the short-lived transcripts. Concomitantly, an increase in protein capable of binding to the GATA motif was detected. Premature removal of erythropoietin from culture resulted in submaximal GATA-1, globin, and hemoglobin production. In contrast, differentiation of J2E cells initiated by sodium butyrate resulted in a sudden depletion of GATA-1 transcripts. Similarly, dimethyl sulphoxide induction of MEL cells produced a transient decrease in GATA-1 mRNA. Surprisingly, these decreases in mRNA were not reflected in alterations to GATA-1 protein content, or the ability of the transcription factor to bind DNA. We concluded from this study that the sequence of events initiated by erythropoietin was not followed during chemical stimulation of erythroid differentiation.

Fatal childhood vascular injuries associated with seat belt use.

The deaths of two children who were passengers in motor vehicles involved in accidents were directly attributable to vascular injuries derived from seat belts. In the first case, a 10-year-old boy died as a result of abdominal aortic transection by a lap seat belt, and in the second case a 15-year-old boy died as a result of transection of his common carotid artery by a lap-shoulder seat belt. Although these cases demonstrate rare fatalities associated with seat belt use, there is no doubt that seat belts have significantly reduced mortality and morbidity from traffic accidents. Although it is possible that a fatal outcome might have occurred in each of these cases from other injuries that might have been sustained had seat belts not been worn, appropriate positioning and size of seat belt harnesses might have avoided the lethal injuries.

Retrovirally-produced erythropoietin effectively induces differentiation and proliferation of J2E erythroid cells.

The J2E erythroid cell line terminally differentiates in response to recombinant human erythropoietin (rHu-epo). Here we demonstrate that retrovirally produced monkey erythropoietin (Zen-epo) is also capable of inducing the final maturation steps of J2E cells in a dose-dependant manner. The hormone committed J2E cells to a pathway of accelerated proliferation, haemoglobin synthesis and morphological changes including enucleation. However, only 6 h exposure to Zen-epo was required to initiate maximum haemoglobin synthesis 72 h later. Significantly, the synthesis of haemoglobin was achieved by concomitantly activating globin production, haem synthesis and iron uptake. Methylcellulose assays revealed that the vast majority of clonogenic cells were able to respond to Zen-epo. These data show that retrovirally manufactured erythropoietin is an effective stimulus for the erythropoietic development of J2E cells in vitro.

Erythropoietin exerts transcriptional and translational control over globin synthesis in J2E cells.

The J2E erythroid cell line, generated by transforming fetal liver cells, terminally differentiates in response to erythropoietin (epo). The cells expressed both adult and embryonic globin genes, although considerably more adult globin was produced, and transcripts for both species rose following exposure to epo. A 6-fold increase in transcription of the adult alpha and beta maj globin genes was observed after hormonal stimulation, which resulted in a substantial accumulation of mRNA. In addition, a modest but transient rise in translation enabled a 6-fold elevation in globin protein to occur. Concurrently, the total heme content rose markedly, enhancing hemoglobin synthesis 10-fold. The prosthetic group complexed entirely with globin proteins, and the hemoglobin produced was present as fully functional oxyhemoglobin, capable of gaseous exchange. We concluded, therefore, that hemoglobin synthesis in epo-induced J2E cells normally results from the coordinate stimulation of heme and globin synthesis. However, some mutant clones emerged where concomitant increases in globin and heme were not observed. Despite similar profiles for the appearance of hemoglobin and equivalent amounts of the oxygen carrier, several noticeable differences in globin synthesis were detected between epo-induced J2E cells and DMSO-stimulated murine erythroleukemia cells, i.e., the types of globin genes expressed, patterns of mRNA and protein production, and translation rates. These results demonstrate that the J2E cells provide a useful model system for investigating the molecular mechanisms of epo-initiated hemoglobin synthesis.