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.

Diabetes and the abdominal aortic aneurysm.

OBJECTIVE: The aim of this review is to delineate the association between abdominal aortic aneurysms (AAAs) and diabetes mellitus. Mechanisms for the underlying association are then discussed. METHODS: A systematic review of the English-language literature using PubMed, EMBASE and Cochrane databases was undertaken up to September 2009. Studies reporting appropriate prevalence data were identified and a meta-analysis performed. RESULTS: Eleven studies were identified. The prevalence of diabetes mellitus in studied patients with AAA ranged from 6% to 14%. The prevalence of diabetes in control patients without AAA ranged from 17% to 36%. Pooled analysis suggested a reduced rate of diabetes amongst people with AAA compared to those without (OR 0.65, 0.60-0.70, p<0.001). CONCLUSIONS: Studies so far suggest a protective role for diabetes on the development of AAA. Further research is required to demarcate the underlying mechanisms for this possible association.

Hypoxic modulation of ca(2+) signaling in human venous and arterial endothelial cells.

Our understanding of vascular endothelial cell physiology is based on studies of endothelial cells cultured from various vascular beds of different species for varying periods of time. Systematic analysis of the properties of endothelial cells from different parts of the vasculature is lacking. Here, we compare Ca(2+) homeostasis in primary cultures of endothelial cells from human internal mammary artery and saphenous vein and how this is modified by hypoxia, an inevitable consequence of bypass grafting (2.5% O(2), 24 h). Basal [Ca(2+)]( i ) and store depletion-mediated Ca(2+) entry were significantly different between the two cell types, yet agonist (ATP)-mediated mobilization from endoplasmic reticulum stores was similar. Hypoxia potentiated agonist-evoked responses in arterial, but not venous, cells but augmented store depletion-mediated Ca(2+) entry only in venous cells. Clearly, Ca(2+) signaling and its remodeling by hypoxia are strikingly different in arterial vs. venous endothelial cells. Our data have important implications for the interpretation of data obtained from endothelial cells of varying sources.

Upregulated TRPC1 channel in vascular injury in vivo and its role in human neointimal hyperplasia.

Occlusive vascular disease is a widespread abnormality leading to lethal or debilitating outcomes such as myocardial infarction and stroke. It is part of atherosclerosis and is evoked by clinical procedures including angioplasty and grafting of saphenous vein in bypass surgery. A causative factor is the switch in smooth muscle cells to an invasive and proliferative mode, leading to neointimal hyperplasia. Here we reveal the importance to this process of TRPC1, a homolog of Drosophila transient receptor potential. Using 2 different in vivo models of vascular injury in rodents we show hyperplasic smooth muscle cells have upregulated TRPC1 associated with enhanced calcium entry and cell cycle activity. Neointimal smooth muscle cells after balloon angioplasty of pig coronary artery also express TRPC1. Furthermore, human vein samples obtained during coronary artery bypass graft surgery commonly exhibit an intimal structure containing smooth muscle cells that expressed more TRPC1 than the medial layer cells. Veins were organ cultured to allow growth of neointimal smooth muscle cells over a 2-week period. To explore the functional relevance of TRPC1, we used a specific E3-targeted antibody to TRPC1 and chemical blocker 2-aminoethoxydiphenyl borate. Both agents significantly reduced neointimal growth in human vein, as well as calcium entry and proliferation of smooth muscle cells in culture. The data suggest upregulated TRPC1 is a general feature of smooth muscle cells in occlusive vascular disease and that TRPC1 inhibitors have potential as protective agents against human vascular failure.

Characterisation of fractalkine/CX3CL1 and fractalkine receptor (CX3CR1) expression in abdominal aortic aneurysm disease.

OBJECTIVES: Fractalkine (CX3CL1) promotes adhesion and extravasation of leucocytes through interactions with fractalkine receptor (CX3CR1) expressed on CD56+/CD16+ NK cells and CD8+ T cells. The current study aims to test the hypothesis the CX3CL1-CX3CR1 interaction contributes to the inflammatory infiltrate in AAA tissue. DESIGN AND METHODS: Immunohistochemistry (IHC) was used to define expression of CX3CR1 in AAA tissue. Multi-parametric flow cytometry (FC) was used to determine CX3CR1 expression on T-cells (CD3+) and NK cells (CD56+) from AAA tissue and peripheral blood of AAA patients and healthy controls. Regulation of CX3CL1 expression by vascular endothelial (vEC) and smooth muscle cells (vSMC) was examined in vitro using primary cell cultures. RESULTS: CX3CR1+ cells were detected in 19/28 AAA tissue samples and predominately localised in the adventitia. PBMCs from patients with AAA demonstrated higher percentages of CX3CR1+ NK cells (60.0-88.6%) and T cells (7.5-39.4%) compared with healthy controls. Furthermore, the frequency of CX3CR1+ NK cells (91%) and T cells (94%) in inflammatory AAA tissue were higher than in atherosclerotic AAA tissue. The pro-inflammatory cytokine TNFalpha increased expression of fractalkine by vSMC and vEC. CONCLUSION: CX3CL1+ and CX3CR1+ cells are present in AAA disease and their interaction may contribute to the recruitment of inflammatory cells seen in AAA tissue.

Trauma related guilt cognitions partially mediate the relationship between PTSD symptom severity and functioning among returning combat veterans.

Trauma related guilt, a distressing emotion associated with negative cognitions regarding one's actions or inaction during a traumatic event, is common among individuals with posttraumatic stress disorder (PTSD). We hypothesized that trauma related guilt cognitions would partially explain the relationship between PTSD symptom severity and functioning. The sample consisted of 254 combat veterans or active duty military personnel who served in Operation Enduring Freedom, Operation Iraqi Freedom or Operation New Dawn (OEF/OIF/OND) who consented to participate in a larger PTSD treatment study. Results revealed a significant relationship between PTSD severity and guilt cognitions (standardized ß = 0.40), as well as PTSD and overall functioning (ß = 0.49). Guilt cognitions (ß's = 0.13 to 0.32) were significantly associated with nearly all domains of functioning, including overall functioning (ß = 0.27), and partially explained the relationship between PTSD and functioning. This study lends support to the addition of guilt as a symptom of PTSD in the DSM-5 as it contributes significantly to functional impairment even when accounting for other symptoms of PTSD, although co-occurring mental health problems may also contribute to functional impairments associated with PTSD. Future studies are needed to investigate whether reductions in traumatic guilt are related to improved functional outcomes in PTSD treatments.

Hypoxic regulation of Ca2+ signalling in astrocytes and endothelial cells.

Acute hypoxia is well known to modulate plasmalemmal ion channels in specific tissue types, thereby modulating [Ca2+]i. Alternative mechanisms by which acute hypoxia could modulate [Ca2+]i are less well explored, particularly in non-excitable cells. Here, we describe experiments employing microfluorimetric recordings from Fura-2-loaded rat cortical astrocytes and human saphenous vein endothelial cells designed to explore any effects of hypoxia (pO2 20-30 mmHg) on [Ca2+]i. In both cell types, hypoxia evoked small rises of [Ca2+]i in the majority of cells during perfusion with a Ca(2+)-free solution, indicating hypoxia can release Ca2+ from an intracellular pool. Capacitative Ca2+ entry was observed when Ca2+ was subsequently restored to the extracellular solution. These effects were abolished by pre-treatment of cells with thapsigargin or prior application of inositol 1,4,5-trisphosphate (IP3)-generating agonists. Antioxidants fully prevented this effect of hypoxia in both cell types. Mitochondrial uncoupling significantly enhanced the effects of hypoxia in astrocytes, yet markedly suppressed the effects of hypoxia in endothelial cells. Our findings indicate that hypoxia can modulate [Ca2+]i in non-excitable cells; most importantly, it can evoke Ca2+ release from intracellular stores via a mechanism which involves reactive oxygen species. The involvement of mitochondria in this effect appears to be tissue specific.

The development of an in vitro flow model of human saphenous vein graft intimal hyperplasia.

OBJECTIVE: Although the role of blood flow has been investigated in animal models of intimal hyperplasia, there have been no detailed studies in intact human vein owing to the difficulties in designing a suitable laboratory model. The aim of this study was to develop a flow model of human vein graft intimal hyperplasia. METHODS: Organ cultures of human saphenous vein were exposed to laminar flow by culturing in a closed circulatory system under predetermined conditions of venous and arterial shear stress for 14 days. Following fixation and processing, paraffin sections were immunostained and neointimal thicknesses measured. RESULTS: It was found that arterial flow completely inhibited neointima formation, but venous flow only partly suppressed the response when compared with vein cultured under static conditions. These results are in agreement with previous in vivo studies in a primate graft model, where increased shear stress inhibited intimal proliferation. CONCLUSION: The endothelial cell is believed to be the key mediator of haemodynamic effects which influence smooth muscle cell proliferation, and the flow rig developed in this study offers the potential to study inter-cellular interactions within the intact vessel. Furthermore, this method provides the facility to study the effects of different flow conditions on segments of vein from the same patient. This model has scope for further development and sophistication which may ultimately lead to increasing our understanding of the aetiology of vein graft stenoses, and hence formulation of preventative strategies.

The role of big endothelin-1 in colorectal cancer.

INTRODUCTION: Changes in liver blood flow caused by an unknown splanchnic vasoconstrictor have been noted in colorectal cancer patients with liver metastases. This prospective study was performed to assess whether plasma levels of big endothelin-1 (big ET-1) were raised in patients with colorectal cancer. METHODS: Plasma samples from peripheral vein of patients who underwent surgery for primary colorectal cancer (n=60) and those with known colorectal liver metastases (n=45) for a period of 15 months were taken prior to treatment and compared to age- and sex-matched controls (n=20). Plasma samples were analysed by using a single-step sandwich enzyme immunoassay. Immunohistochemistry and in situ hybridisation were also performed on tumour sections to investigate the expression of ET-1 by cancer cells. RESULTS: The median (range) plasma concentration of big ET-1 in controls was 2.1 pg/mL (1.2-13.4 pg/mL). The median (range) plasma concentration of big ET-1 in colorectal cancer patients with no overt hepatic metastases was 3.8 pg/mL (1.2-15.8 pg/mL), p=0.002, and the median (range) plasma concentration of big ET-1 in colorectal cancer patients with hepatic metastases was 5.2 pg/mL (1.7-30 pg/mL), p=0.0001; both were significantly elevated compared to the control group. A significant difference in immunostaining for big ET-1 was noted between paired normal colonic mucosa (median score-1) and tumour sections (median score-3), p=0.01. CONCLUSION: This study has demonstrated elevated concentrations of big ET-1 in colorectal cancer patients, especially in those with hepatic metastases. Upregulation of ET activity in colorectal cancer could be inferred by the increased immunostaining of big ET-1 in cancer cells. Therefore, plasma big ET-1 levels should be evaluated as a potential tumour marker for the identification of hepatic metastases at an earlier stage.

The renal toxicity of (R)-3-chlorolactate in the rat.

The renal toxicity of (R,S)-3-chlorolactate has been shown to be due to the (R)-isomer which, when administered to rats, induces diuresis and glucosuria. The metabolic activity of isolated tubule cells, prepared from rat kidney, was inhibited by (R)-3-chlorolactate and the action of the compound was localised as affecting mitochondrial metabolism. Studies with kidney mitochondria pin-pointed the site of action as being involved with the oxidative metabolism of malate but not the inhibition of mitochondrial malate dehydrogenase. The effects of oxalate, a metabolite of (R)-3-chlorolactate, and of (R,S)-3-chlorolactaldehyde on renal tubule cells was investigated. While some degrees of inhibition of metabolic activity were evident, these compounds were not responsible for the toxic effects produced by (R)-3-chlorolactate.

The effect of the isomers of alpha-cholorohydrin and racemic beta-chlorolactate on the rat kidney.

The (R)- and (S)-isomers of the male antifertility agent alpha-chlorohydrin have been synthesized. When administered to rats, the (R)-isomer induced a period of diuresis and glucosuria, whereas the (S)-isomer, which possesses the antifertility activity, had no detrimental action on the kidney. Neither of the isomers of alpha-chlorohydrin nor those of an active analogue, 3-amino-1-chloropropan-2-ol, had any inhibitory activity on the oxidative metabolism of glucose or lactate in isolated kidney tubules. However, beta-chlorolactate, a metabolite common to both compounds, inhibited the oxidation of glucose, lactate, pyruvate and glutamate to CO2. It is proposed that the antifertility action of the (S)-isomers of alpha-chlorohydrin and 3-amino-1-chloropropan-2-ol is unrelated to the renal toxicity of the (R)-isomers, a toxic action involving the inhibition of oxidative metabolism by (S)-beta-chlorolactate or a further product of this metabolite.

Stereo-selective inhibition of transient receptor potential TRPC5 cation channels by neuroactive steroids.

BACKGROUND AND PURPOSE: Transient receptor potential canonical 5 (TRPC5) channels are widely expressed, including in the CNS, where they potentiate fear responses. They also contribute to other non-selective cation channels that are stimulated by G-protein-coupled receptor agonists and lipid and redox factors. Steroids are known to modulate fear and anxiety states, and we therefore investigated whether TRPC5 exhibited sensitivity to steroids. EXPERIMENTAL APPROACH: Human TRPC5 channels were conditionally expressed in HEK293 cells and studied using intracellular Ca2+ measurement, whole-cell voltage-clamp and excised patch techniques. For comparison, control experiments were performed with cells lacking TRPC5 channels or expressing another TRP channel, TRPM2. Native TRPC channel activity was recorded from vascular smooth muscle cells. KEY RESULTS: Extracellular application of pregnenolone sulphate, pregnanolone sulphate, pregnanolone, progesterone or dihydrotestosterone inhibited TRPC5 activity within 1-2min. Dehydroepiandrosterone sulphate or 17ß-oestradiol had weak inhibitory effects. Pregnenolone, and allopregnanolone, a progesterone metabolite and stereo-isomer of pregnanolone, all had no effects. Progesterone was the most potent of the steroids, especially against TRPC5 channel activity evoked by sphingosine-1-phosphate. In outside-out patch recordings, bath-applied progesterone and dihydrotestosterone had strong and reversible effects, suggesting relatively direct mechanisms of action. Progesterone inhibited native TRPC5-containing channel activity, evoked by oxidized phospholipid. CONCLUSIONS AND IMPLICATIONS: Our data suggest that TRPC5 channels are susceptible to relatively direct and rapid stereo-selective steroid modulation, leading to channel inhibition. The study adds to growing appreciation of TRP channels as non-genomic steroid sensors.

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 effect of six different statins on the proliferation, migration, and invasion of human smooth muscle cells.

Intimal hyperplasia (IH) can occur after any vascular injury and results from smooth muscle cells (SMC) proliferation, migration, and invasion into the subintimal space. The purpose of this study was to investigate the effect of six different statins on the proliferation, migration, and invasion of human venous SMC. The statins were all used at their Cmax concentrations. SMCs were used to construct growth curves in the presence of 10% fetal calf serum or 10% fetal calf serum supplemented with the six statins. Migration and invasion experiments were performed using modified Boyden chambers. The invasion experiments were performed using Matrigel coated plates. We found that all of the statins significantly inhibited SMC proliferation compared to the platelet-derived growth factor control (ranging from fluvastatin 33% of control to pravastatin 72% of control, P = 0.03). SMC migration through uncoated polycarbonate membranes in presence of the six statins was significantly reduced (ranging from lovastatin 43% to pravastatin 57% of control, P = 0.006). All six statins also significantly reduced SMC invasion (ranging from fluvastatin 65% to simvastatin 87% of control, P = 0.002). We conclude that the inhibitory effect of statins on SMC proliferation, migration, and invasion is a class, rather than drug specific effect.

A comparison of six statins on the development of intimal hyperplasia in a human vein culture model.

OBJECTIVE: Intimal hyperplasia (IH) threatens the patency of up to 35% of saphenous vein (SV) bypass grafts. In addition to reducing cholesterol levels, statins may modulate smooth muscle cell proliferation and migration. Statins inhibit matrix metalloproteinase (MMP) activity. We therefore investigated the effect of six statins on neointimal formation and MMP activity in human SV organ culture. STUDY DESIGN: Human SV specimens were cultured for 14 days in the presence of six different statins and subsequently processed for measurement of neointimal thickness and MMP activity. The drug concentrations chosen corresponded to the manufacturers' Cmax. RESULTS: The six statins all significantly reduced IH development (P = 0.004) in association with reduced expression of proMMP-2 and 9 (P = 0.03) and reduced activity of activated MMP-2 and 9 (P = 0.03). CONCLUSION: This study suggests that the potential benefit of statins in reducing IH is a class effect and not confined to specific statins. The reduction of IH produced by statins may in part be due to their inhibition of MMPs.

Statins for the prevention of vein graft stenosis: a role for inhibition of matrix metalloproteinase-9.

Saphenous vein (SV) grafts are commonly used to bypass coronary arteries that are diseased due to atherosclerosis. However, the development of intimal hyperplasia in such grafts can lead to patency-threatening stenosis and re-occlusion of the vessel. The proliferation and migration of smooth muscle cells (SMC) play key roles in the development of intimal hyperplasia, and an agent that inhibits both of these processes therefore has therapeutic potential. A prerequisite for SMC proliferation and migration in vivo is degradation of the basement membrane, achieved by secretion of the matrix-degrading gelatinases matrix metalloproteinase-2 (MMP-2) and MMP-9. Statins are cholesterol-lowering drugs that also have direct effects on SMC function. Here we report that neointima formation in organ-cultured human SV segments is inhibited by simvastatin, an effect that is associated with reduced MMP-9 activity. Additionally, our work shows that simvastatin not only inhibits proliferation, but importantly also inhibits invasion (migration through a matrix barrier), of cultured human SV SMC. Thus simvastatin treatment appears to inhibit neointima formation as a result of combined inhibition of SMC proliferation and invasion. The potential intracellular mechanisms by which statins affect SMC proliferation and migration, and thus attenuate intimal hyperplasia, are discussed, with particular emphasis on the role of MMP-9.

Is the nephrotoxicity of (R)-3-chlorolactate in the rat caused by 3-chloropyruvate?

1. When (R, S)-[3-36 Cl]chlorolactate was administered to male rats, two radioactive constituents were excreted in the urine. These were identified as 36Cl- and [3-36 Cl]chlorolactate which was subsequently shown to be essentially the (S)-isomer. 2. Analysis of the urinary oxalate content from rats receiving either (R)- or (S)-3-chlorolactate revealed that elevated levels were produced by the (R)-isomer whereas normal levels followed the administration of the (S)-isomer. 3. Treatment of (R,S)-3-chlorolactate with a modified Fenton's oxidizing system produced oxalate and an intermediate which was identified as 3-chloropyruvate. 4. 3-Chloropyruvate is a potent nephrotoxin in the rat producing a brief phase of diuresis when administered, increasing the urinary excretion of oxalate and inhibiting the oxidative metabolic capability of rat kidney tubules and rat kidney mitochondria in vitro. 5. Both (R)-3-chlorolactate and 3-chloropyruvate were shown to be inhibitors of the commercially-available pyruvate dehydrogenase complex. 6. 3-Chloropyruvate inhibits kidney mitochondrial metabolism possibly at the pyruvate dehydrogenase complex level and appears to be a metabolite of (R)- but not (S)-3-chlorolactate.

Endothelin-1 is a mediator of intimal hyperplasia in organ culture of human saphenous vein.

BACKGROUND: Endothelin-1 (ET-1) is a powerful vasoconstrictor and a potent mitogen for vascular smooth muscle cells. Excessive smooth muscle cell proliferation is a feature of intimal hyperplasia, the pathological lesion of vein graft stenosis. This study investigates the role of ET-1 in isolated human saphenous vein smooth muscle cells and also in an organ culture of the human saphenous vein. METHODS: Growth-arrested human saphenous vein smooth muscle cells were stimulated with ET-1 and proliferation quantified by [3H]thymidine uptake in these cells compared with unstimulated control cells. Organ cultures of the human saphenous vein were established with endothelium intact, with endothelium denuded, and with endothelium denuded and the culture medium supplemented with ET-1. RESULTS: ET-1 stimulated DNA synthesis in isolated smooth muscle cells in a dose-dependent manner with half-maximal stimulation at 1 nmol/l. Addition of ET-1 to denuded vein caused a significant increase in median (range) neointimal thickness, from 4 (0-19) to 20 (4-30) microns (P < 0.05). In veins cultured with ET-1 a parallel increase in median (range) neointimal proliferation index, from 21 (0-31) to 33 (23-46) per cent (P < 0.05), was also observed. CONCLUSION: These results demonstrate that ET-1 is a mediator of intimal hyperplasia in human saphenous vein in vitro. Endothelin receptor antagonists may therefore be of therapeutic value in the modulation of vein graft intimal hyperplasia.