Cold Storage Preservation and Warm Ischaemic Injury to Isolated Arterial Segments: Endothelial Cell Injury

Injury to endothelial calls is thought to be important to the development of the vascular lesion of chronic rejection. It was the aim of this study to develop a semiquantitative method to assess endothelial injury in arterial grafts and to document the injury produced by cold storage preservation and additional warm ischaemia. Twelve- and 24-h cold preservation of rat aortic segments, together with an additional 1 h of warm ischaemia, were assessed. Electron micrographs of representative endothelial cells were scored for cytoplasmic, nuclear and mitochondrial injury. The overall injury score was obtained by addition of the individual scores. Storage for up to 24 h in University of Wisconsin (UW) and Terasaki did not produce any injury. Twenty-four hours of storage in Euro-Collins resulted in endothelial cell death. Injury occurred after 12 h of storage in Ross, Collins and normal saline, and the injury increased following 24 h of storage. One hour of warm ischaemia did not increase the injury. Injury to endothelial cells varies with the preservation solution used and the time of cold storage, so that both the type of solution and the storage time should be taken into account in clinical studies looking at the influence of cold ischaemia time and graft outcome.

Endothelial cell serine proteases expressed during vascular morphogenesis and angiogenesis

Many serine proteases play important regulatory roles in complex biological systems, but only a few have been linked directly with capillary morphogenesis and angiogenesis. Here we provide evidence that serine protease activities, independent of the plasminogen activation cascade, are required for microvascular endothelial cell reorganization and capillary morphogenesis in vitro. A homology cloning approach targeting conserved motifs present in all serine proteases, was used to identify candidate serine proteases involved in these processes, and revealed 5 genes (acrosin, testisin, neurosin, PSP and neurotrypsin), none of which had been associated previously with expression in endothelial cells. A subsequent gene-specific RT-PCR screen for 22 serine proteases confirmed expression of these 5 genes and identified 7 additional serine protease genes expressed by human endothelial cells, urokinase-type plasminogen activator, protein C,TMPRSS2, hepsin, matriptase/ MT-SPI, dipepticlylpepticlase IV, and seprase. Differences in serine protease gene expression between microvascular and human umbilical vein endothelial cells (HUVECs) were identified and several serine protease genes were found to be regulated by the nature of the substratum, ie. artificial basement membrane or fibrillar type I collagen. mRNA transcripts of several serine protease genes were associated with blood vessels in vivo by in situ hybridization of human tissue specimens. These data suggest a potential role for serine proteases, not previously associated with endothelium, in vascular function and angiogenesis.

High glucose-mediated effects on endothelial cell proliferation occur via p38 MAP kinase

The mitogen-activated protein ( MAP) kinases contribute to altered cell growth and function in a variety of disease states. However, their role in the endothelial complications of diabetes mellitus remains unclear. Human endothelial cells were exposed for 72 h to 5 mM ( control) or 25 mM ( high) glucose or 5 mM glucose plus 20 mM mannitol ( osmotic control). The roles of p38 and p42/44 MAP kinases in the high glucose-induced growth effects were determined by assessment of phosphorylated MAP kinases and their downstream activators by Western blot and by pharmacological inhibition of these MAP kinases. Results were expressed as a percentage ( means +/- SE) of control. High glucose increased the activity of total and phosphorylated p38 MAP kinase ( P < 0.001) and p42/44 MAP kinase ( P < 0.001). Coexposure of p38 MAP kinase blocker with high glucose reversed the antiproliferative but not the hypertrophic effects associated with high-glucose conditions. Transforming growth factor (TGF)-beta1 increased the levels of phosphorylated p38 MAP kinase, and p38 MAP kinase blockade reversed the antiproliferative effects of this cytokine. The high glucose-induced increase in phosphorylated p38 MAP kinase was reversed in the presence of TGF-beta1 neutralizing antibody. Although hyperosmolarity also induced antiproliferation (P < 0.0001) and cell hypertrophy (P < 0.05), there was no change in p38 activity, and therefore inhibition of p38 MAP kinase had no influence on these growth responses. Blockade of p42/44 MAP kinase had no effect on the changes in endothelial cell growth induced by either high glucose or hyperosmolarity. High glucose increased p42/44 and p38 MAP kinase activity in human endothelial cells, but only p38 MAP kinase mediated the antiproliferative growth response through the effects of autocrine TGF-beta1. High glucose-induced endothelial cell hypertrophy was independent of activation of the MAP kinases studied. In addition, these effects were independent of any increase in osmolarity associated with high-glucose exposure.

The effects of high glucose and atorvastatin on endothelial cell matrix production

Background Statins are known to enhance atherosclerotic plaque stability through influences on extracellular matrix homeostasis. Net matrix production reflects the relative balance of matrix production and degradation through enzymes such as matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitor of MMP (TIMPs). The effects of statins on endothelial cell production of these parameters following co-exposure with a proatherogenic stimulus such as high glucose are not known. Methods Human endothelial cells were exposed for 72 h to 5 mM> (control) or 25 mM (high) glucose +/- atorvastatin (1 mumol/l). Extracellular matrix homeostasis was assessed by measuring matrix metalloproteinase (MMP)-2 secretion, tissue inhibitor of MMP (TIMP)-1 and -2 secretion and net collagen IV production. Results were expressed as percentage +/- SEM of control values. Results Exposure to high glucose increased cellular collagen IV expression to 190.1 +/- 11.7% (P < 0.0001) of control levels. No change in MMP-2 secretion (111.6 +/- 5.2%; P > 0.05) was observed but both TIMP-1 and TIMP-2 expression were increased to 136.3 +/- 6.4% and 144.0 +/- 27.5%, respectively (both P < 0.05). The presence of atorvastatin in high glucose conditions reduced collagen IV expression to 136.1 +/- 20.6%. This was paralleled by increased secretion of MMP-2 to 145.8 +/- 7.8% (P < 0.01), increased TIMP-2 expression to 208.0 +/- 21.3% (P < 0.005 compared with high glucose) but no change in TIMP-1 expression (155.1 +/- 14.6%) compared with high glucose alone. The presence of atorvastatin in control conditions did not affect levels of collagen IV expression (114.5 +/- 13.2%). Conclusions Endothelial cell exposure to high glucose was associated with a MMP/TIMP profile that increased extracellular matrix production which was attenuated by concurrent exposure to atorvastatin. Consequently, a mechanism by which the atherosclerotic plaque regression that is observed in patients taking these drugs has been demonstrated.

Exercise and the endothelial cell

Regular exercise is known to be effective in the prevention and treatment of cardiovascular disease. Among the cardioprotectant mechanisms influenced by exercise, the endothelium is becoming recognised as a major target. Preservation of endothelial cell structure is vital for frictionless blood flow, prevention of macrophage and lipid infiltration and, ultimately, optimal vascular function. Exercise causes various kinds of mechanical, chemical and thermal stresses, and repeated exposure to these stresses may precondition the endothelial cell to future stresses through a number of different mechanisms. This review discusses stress-induced changes in endothelial cell morphology, biochemistry and components of platelet activation and cell adhesion that impact on endothelial cell structure. An enhanced understanding of the effects of exercise on the endothelial cell will assist in directing future research into the prevention of cardiovascular disease. (c) 2004 Elsevier Ireland Ltd. All rights reserved.

Bcl-2 in endothelial cells is increased by vitamin E and alpha-lipoic acid supplementation but not exercise training

Atherosclerotic plaque contains apoptotic endothelial cells with oxidative stress implicated in this process. Vitamin E and a-lipoic acid are a potent antioxidant combination with the potential to prevent endothelial apoptosis. Regular exercise is known to increase myocardial protection, however, little research has investigated the effects of exercise on the endothelium. The purpose of these studies was to investigate the effects of antioxidant supplementation and/or exercise training on proteins that regulate apoptosis in endothelial cells. Male rats received a control or antioxidant-supplemented diet (vitamin E and alpha-lipoic acid) and were assigned to sedentary or exercise-trained groups for 14 weeks. Left ventricular endothelial cells (LVECs) were isolated and levels of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax were measured. Antioxidant supplementation caused a fourfold increase in Bcl-2 (P < 0.05) with no change in Bax (P > 0.05). Bcl-2:Bax was increased sixfold with antioxidant supplementation compared to non-supplemented animals (P < 0.05). Exercise training had no significant effect on Bcl-2, Bax or Bcl-2:Bax either alone or combined with antioxidant supplementation (P > 0.05) compared to non-supplemented animals. However, Bax was significantly lower (P < 0.05) in the supplemented trained group compared to non-supplemented trained animals. Cultured bovine endothelial cells incubated for 24 h with vitamin E and/or a-lipoic acid showed the combination of the two antioxidants increased Bcl-2 to a greater extent than cells incubated with the vehicle alone. In summary, vitamin E and a-lipoic acid increase endothelial cell Bcl-2, which may provide increased protection against apoptosis. (c) 2005 Elsevier Ltd. All rights reserved

SOX18 directly interacts with MEF2C in endothelial cells

Recently, we demonstrated that mutations in the Sry-related HMG box gene Sox18 underlie vascular and hair follicle defects in the mouse allelic mutants ragged (Ra) and RaJ. Ra mice display numerous anomalies in the homozygote including, oedema, peritoneal secretions, and are almost completely naked. Sox18 and the MADS box transcription factor, Mef2C, are expressed in developing endothelial cells. Null mutants in Sox18 and Mef2c display overlapping phenotypic abnormalities, hence, we investigated the relationship between these two DNA binding proteins. We report here the direct interaction between MEF2C and SOX18 proteins, and establish that these proteins are coexpressed in vivo in endothelial cell nuclei. MEF2C expression potentiates SOX18-mediated transcription in vivo and regulates the function of the SOX18 activation domain. Interestingly, MEF2C fails to interact or co-activate transcription with the Ra or RaJ mutant SOX18 proteins. These results suggest that MEF2C and SOX18 may be important partners directing the transcriptional regulation of vascular development. (C) 2001 Academic Press.

Evidence for a non-antioxidant, dose-dependent role of alpha-lipoic acid in caspase-3 and ERK2 activation in endothelial cells

Endothelial cell apoptosis contributes to atherosclerosis and may be exacerbated by oxidative stress. Results from clinical trials using antioxidant supplementation are equivocal and could be enhanced by antioxidants with additional non-antioxidant properties such as a-lipoic acid and alpha-tocopherol. The aim of this study was to investigate the effects of these antioxidants on cytoprotective pathways and endothelial apoptosis. Endothelial cells were incubated with alpha-lipoic acid and alpha-tocopherol, alone or in combination, prior to incubation with H2O2 or staurosporine. alpha-lipoic acid pre-treatment alone increased caspase-3 activity in a dose-dependent manner. Both H2O2 and staurosporine increased DNA fragmentation and caspase-3 activity and pre-treatment of cells with a-lipoic acid and/or a-tocopherol failed to prevent stress-induced apoptosis. Neither antioxidant treatments nor apoptotic inducers alone altered expressions of BcI-2, Bax, HSP70 or pERK1/2 or pJNK. alpha-lipoic decreased pERK2 in staurosporine-treated cells in a dose-dependent manner. These findings indicate that pre-incubation with alpha-lipoic acid and alpha-tocopherol, alone or in combination, does not protect against oxidative- or non-oxidative-induced apoptosis in endothelial cells. Moreover, we have demonstrated a non-antioxidant, dose-dependent role of alpha-lipoic acid in caspase-3 and ERK2 activation. These data provide an insight and indicate caution in the use of high doses of alpha-lipoic acid as an antioxidant.

Accumulation and toxicity of monophenyl arsenicals in rat endothelial cells

Clark 1 (diphenylarsine chloride) and Clark 2 ( diphenylarsine cyanide) were used as chemical weapon agents (CWA), and the soil contamination by these CWA and their degraded products, diphenyl and phenyl arsenicals, has been one of the most serious environmental issues. In a series of comparisons in toxicity between trivalent and pentavalent arsenicals we investigated differences in the accumulation and toxicity of phenylarsine oxide (PAO(3+)) and phenylarsonic acid (PAA(5+)) in rat heart microvascular endothelial cells. Both the cellular association and toxicity of PAO(3+) were much higher than those of PAA(5+), and LC50 values of PAO(3+) and PAA(5+) were calculated to be 0.295 muM and 1.93 mM, respectively. Buthionine sulfoximine, a glutathione depleter, enhanced the cytotoxicity of both PAO(3+) and PAA(5+). N-Acetyl-L-cysteine (NAC) reduced the cytotoxicity and induction of heme oxygenase-1 (HO-1) mRNA in PAO(3+)-exposed cells, while NAC affected neither the cytotoxicity nor the HO-1 mRNA level in PAA(5+)-exposed cells. The effect of NAC may be due to a strong affinity of PAO(3+) to thiol groups because both NAC and GSH inhibited the cellular accumulation of PAO(3+), but PAA(3+) increased tyrosine phosphorylation levels of cellular proteins. These results indicate that the inhibition of protein phosphatases as well as the high affinity to cellular components may confer PAO(3+) the high toxicity.

Effect of disrupted SOX18 transcription factor function on tumor growth, vascularization, and endothelial development

Background. The growth of solid tumors depends on establishing blood supply; thus, inhibiting tumor angiogenesis has been a long-term goal in cancer therapy. The SOX18 transcription factor is a key regulator of murine and human blood vessel formation. Methods: We established allograft melanoma tumors in wild-type mice, Sox18-null mice, and mice expressing a dominant-negative form of Sox18 (Sox18RaOp) (n = 4 per group) and measured tumor growth and microvessel density by immunohistochemical analysis with antibodies to the endothelial marker CD31 and the pericyte marker NG2. We also assessed the affects of disrupted SOX18 function on MCF-7 human breast cancer and human umbilical vein endothelial cell (HUVEC) proliferation by measuring BrdU incorporation and by MTS assay, cell migration using Boyden chamber assay, and capillary tube formation in vitro. All statistical tests were two-sided. Results: Allograft tumors in Sox18-null and Sox18RaOp mice grew more slowly than those in wild-type mice (tumor volume at day 14, Sox18 null, mean = 486 mm(3), 95% confidence interval [CI] = 345 mm(3) to 627 mm(3), p = .004; Sox18RaOp, mean = 233 mm(3), 95% CI = 73 mm(3) to 119 mm(3), p < .001; versus wild-type, mean = 817 mm(3), 95% CI = 643 mm(3) to 1001 mm(3)) and had fewer CD31- and NG2-expressing vessels. Expression of dominant-negative Sox18 reduced the proliferation of MCF-7 cells (BrdU incorporation: MCF-7(Ra) = 20%, 95% CI = 15% to 25% versus MCF-7 = 41%, 95% CI = 35% to 45%; P = .013) and HUVECs (optical density at 490 nm, empty vector, mean = 0.46 versus SOX18 mean = 0.29; difference = 0.17, 95% CI = 0.14 to 0.19; P = .001) compared with control subjects. Overexpression of wild-type SOX18 promoted capillary tube formation of HUVECs in vitro, whereas expression of dominant-negative SOX18 impaired tube formation of HUVECs and the migration of MCF-7 cells via the disruption of the actin cytoskeleton. Conclusions: SOX18 is a potential target for antiangiogenic therapy of human cancers.

Simultaneous detection of multiple antigens with triple immunolabeling

Immunolabeling is commonly used to localize antigens within frozen or paraffin tissue sections. We modified existing immunolabeling techniques to allow the detection of three antigens simultaneously within the one tissue section. The approach relies on the use of three monoclonal antibodies in sequential immunoperoxidase staining steps, each with colored substrates, resulting in the deposition of black, brown, and rose stains. The method is rapid and does not require novel techniques or materials. In this report, we demonstrate the colocalization of mast cell tryptase, neurofilament protein, and CD31 (platelet-endothelial cell adhesion molecule) or laminin in normal human skin and normal buccal mucosa, as an illustration of the power and simplicity of the multiple antigen localization technique.

Experimental methods for studying drug uptake in the head and brain

A number of techniques have been developed to study the disposition of drugs in the head and, in particular, the role of the blood-brain barrier (BBB) in drug uptake. The techniques can be divided into three groups: in-vitro, in-vivo and in-situ. The most suitable method depends on the purpose(s) and requirements of the particular study being conducted. In-vitro techniques involve the isolation of cerebral endothelial cells so that direct investigations of these cells can be carried out. The most recent preparations are able to maintain structural and functional characteristics of the BBB by simultaneously culturing endothelial cells with astrocytic cells,The main advantages of the in-vitro methods are the elimination of anaesthetics and surgery. In-vivo methods consist of a diverse range of techniques and include the traditional Brain Uptake Index and indicator diffusion methods, as well as microdialysis and positron emission tomography. In-vivo methods maintain the cells and vasculature of an organ in their normal physiological states and anatomical position within the animal. However, the shortcomings include renal acid hepatic elimination of solutes as well as the inability to control blood flow. In-situ techniques, including the perfused head, are more technically demanding. However, these models have the ability to vary the composition and flow rate of the artificial perfusate. This review is intended as a guide for selecting the most appropriate method for studying drug uptake in the brain.

SOX18 and the transcriptional regulation of blood vessel development

SOX18 is a transcription factor that is transiently expressed in nascent endothelial cells during embryonic development and adult neovascularization. This protein belongs to the SOX family of transcription factors, ih,which are proving to be some of the key regulators of cell-type specification in the vertebrate embryo. Natural mutations in the Sox18 gene have been shown to result to cardiovascular dysfunction, in some cases leading to death. Available evidence thus implicates Sox18 as an important regulator of vascular development, most likely playing a key role in endothelial cell specification. However; the genetic knockout of Sox18 in mice has produced a confounding result that complicates our understanding of the molecular mode of action of the SOX18 protein. We speculate that Sox18 inky act in a redundant fashion with closely related genes such as Sox7 and/or Sox17. (C) 2001, Elsevier Science Inc.