Leucocyte and endothelial cell adhesion molecule expression in porcine histiocytic leiomyofibrosarcoma

A histiocytic sarcoma was present at birth in a pig. On the basis of ultra-structure and structural-protein composition (presence of alpha-smooth-muscle actin but not keratin), the sarcoma component was identified as a leiomyofibrosarcoma. Lipid-laden macrophages (histiocytes), which permeated the tumour in an apparently random fashion, were somewhat atypical in that they were negative for some macrophage markers; they gave a reaction, however, for CDw14. Despite its aggressive metastatic capacity, this tumour occurred almost exclusively in the subcutis, dermis and skeletal muscle. The tumour was extensively vascularized with many small capillaries which did not express E-selectin (CD69E), MHC class II or the L-selectin (CD69L) ligand, markers characteristic of inflamed (activated) endothelial cells in pig skin. Significant numbers of the histiocytes were positive for the integrins CD18 and VLA-4 (CD49d), indicating involvement of integrin pathways in the spread or growth, or both, of the leiomyofibrosarcoma. Most of the fibrous sarcoma cells also had extensive reactivity with an antibody to the standard variant form of CD44 (CD44s).     

Effects of aminoguanidine on adhesion molecule expression of human endothelial cells

The effect of aminoguanidine (AG) on the expression of adhesion molecules on nonactivated human umbilical vein endothelial cells (HUVEC) was investigated in vitro. Nonactivated HUVEC cultivated on long-term glycated fibronectin (FN) as compared to native FN showed a significant upregulation of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and CD31 which could be further promoted by long-term glycated bovine serum albumin. AG, at a concentration of 0.01 mol/l, caused an upregulation of ICAM-1 of 48 +/- 17.4% in HUVEC cultivated on gelatin. In contrast, VCAM-1 and E-selectin remained unaffected. At this concentration, formation of advanced glycation end products (AGE) was inhibited by 57%, as determined immunologically, and by 50%, as verified by AGE-specific fluorescence. A hypothesis concerning the upregulation of ICAM-1 by AG as compared to VCAM-1 is proposed relating to its relative redox insensitivity. Our results demonstrate that the beneficial effect of AG in reducing the risk of accelerated development of atherosclerosis in diabetic patients by inhibiting formation of AGE on matrix proteins such as FN might be hampered by its tendency to upregulate ICAM-1 on endothelial cells.     

Anti-inflammatory drugs and endothelial cell adhesion molecule expression in murine vascular beds

STATEMENT OF PROBLEM: There are discrepancies among researchers concerning the reliability and use of temporomandibular joint sounds. PURPOSE: This study examined the reliability of mandibular movements and sounds and determined the correlation between movements and sounds. MATERIAL AND METHODS: The mandibular movements of 35 subjects diagnosed with temporomandibular disorders were recorded with 2 CCD cameras, and sounds were recorded bilaterally with Panasonic electret condenser microphones in the ear canal. Subjects performed 3 movements, each repeated 5 times. RESULTS: Reliability of maximum movements across the 5 trials was good to excellent, with Intraclass Correlation Coefficients (ICC) between 0.76 and 0.91 for all movements except protrusion. Temporomandibular sound event counts were reliable for most movements, including vertical opening, protrusion, and right and left laterotrusion (ICCs between 0.41 and 0.81). Most subjects produced sound events either in 100% or in none of the trials. Reliability for sound events was better during protrusion (ICCs between 0.56 and 0.81) than vertical opening (ICCs 0.41 to 0.64). Subjects with sound events during vertical opening (followed by closing) were significantly more likely to have sound events during protrusion (followed immediately by vertical opening and closing) (P <.01). CONCLUSION: Temporomandibular sound events are generally reliable and warrant study regarding their use in classifying and diagnosing patients with temporomandibular disorders. Condylar translation, which occurs during both vertical opening and protrusion, appears to have a strong influence on the production of temporomandibular sound events.     

mRNA expression of glycolytic enzymes and glucose transporter proteins in ischemic myocardium with and without reperfusion

It is known that ischemia commonly increases exogenous glucose utilization by accelerating glucose uptake and flux rates through the Embden-Meyerhof pathway. Constitutive enzymes regulate the rate of glycolysis and in turn are regulated by product inhibition and allosteric controls. The purpose of this report was to test whether mRNA abundance for select glycolytic enzymes, and glucose transport proteins, is also modified. Six intact working pig hearts with coronary flow controlled by extracorporeal perfusion were compared at the following conditions: (1) aerobic control perfusion; (2) ischemia affected by a 60% decrease in left anterior descending (LAD) coronary perfusion: (3) ischemia again affected by a 60% decrease in LAD flow followed by a 40-min interval of aerobic reflow; (4) an intermittent ischemia and reflow protocol including four cycles of similar LAD flow reductions (5 min per cycle) interspersed with 15-20 min of aerobic reperfusion; (5) a 4-day model designed to produce myocardial chronic hibernation: and (6) mild ischemia induced by a 40% decrease in LAD flow for 85 min to produce certain adaptations compatible with short-term hibernation. In each heart, mRNA abundance was measured from LAD and circumflex (LCF) perfused myocardium for hexokinase, phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase and the two glucose transporter isomers, GLUT 4 and GLUT 1. mRNA data from LAD myocardium in intervention hearts were normalized to those from LAD tissue in the control heart (LADc) and with LCF values in the same intervention hearts. Signal variance around unity in the LAD tissue, with respect to that of the LCF myocardium, in the control heart compared closely (44 and 41% in two separate runs, respectively). GLUT 1/GLUT 4 ratios in the LAD and LCF beds of this heart also agreed closely. LAD/LADc ratios were increased for hexokinase (1.69), phosphofructokinase (3.69), and glyceraldehyde-3-phosphate dehydrogenase (2.29) in the ischemia heart and for phosphofructokinase (3.90), glyceraldehyde-3-phosphate dehydrogenase (2.20), GLUT 4 (1.55) and GLUT 1 (2.20) in the ischemia/reflow heart. There was no evidence of excess signal in the intermittent ischemia/reflow, chronic hibernation, or mild ischemia hearts. Altered signal from LCF myocardium was also suggested. These data indicate that mRNA abundance for select glycolytic enzymes and transporter proteins is increased in ischemic myocardium with or without reperfusion and offers a possible mechanism for increased protein activity in settings of diminished regional coronary flow.     

Differential regulation of endothelial cell adhesion molecule expression by nitric oxide donors and antioxidants

Although nitric oxide (NO) and antioxidants inhibit adhesion molecule expression, their inhibitory effects on nuclear factor kappaB (NF-kappaB) activation may differ. The NO donors, but not 8-bromo-cGMP, decreased tumor necrosis factor alpha (TNF-alpha)-induced VCAM-1, ICAM-1, and E-selectin expression by 11-70%. In contrast, NAC completely abolished VCAM-1 and E-selectin expression and decreased ICAM-1 expression by 56%. Gel shift assays demonstrate that NF-kappaB activation was inhibited by both NO and antioxidants. The activation of NF-kappaB involves the phosphorylation and degradation of its cytoplasmic inhibitor IkappaB-alpha by 26S proteasomes. The 26S proteasome inhibitor MG132 prevented the degradation of phosphorylated IkappaB-alpha. NAC inhibited IkappaB kinase (IKK) activity and prevented IkappaB-alpha phosphorylation and degradation. In contrast, NO did not inhibit IKK activity, IkappaB-alpha phosphorylation, or IkappaB-alpha degradation. However, NO, but not antioxidants, induced IkappaB-alpha promoter activity. The inhibitory effects of NO on adhesion molecule expression, therefore, differs from that of antioxidants in terms of the mechanism by which NF-kappaB is inactivated.     

Lymphocytes mediate TNF-alpha-induced endothelial cell adhesion molecule expression: studies on SCID and RAG-1 mutant mice

TNF-alpha is known to elicit a rapid increase in the expression of specific endothelial cell adhesion molecules (ECAMs) within different vascular beds. The aim of this study was to determine whether lymphocytes contribute to the increased ECAM expression elicited by TNF-alpha. A dual radiolabeled mAb technique was used to quantify constitutive and TNF-alpha-induced expression of ICAM-1, VCAM-1, E-selectin, and P-selectin in different vascular beds (lung, heart, stomach, mesentery, small intestine, large intestine, and muscle) in wild-type and SCID mice. In reconstitution experiments, either whole splenocytes, T cell-enriched splenocytes, or B cell-enriched splenocytes were injected into SCID mice 48 h before TNF-alpha administration. Although the constitutive expression of ECAMs differed only slightly between wild-type and SCID mice, TNF-alpha-induced ECAM expression was markedly blunted in SCID mice compared with wild-type mice. This blunted response to TNF-alpha was also demonstrated for VCAM-1 in recombination activating gene (RAG)-1 mutant mice. Reconstitution studies revealed that administration of 50 x 10(6) splenocytes in SCID mice at 48 h before cytokine treatment restored the TNF-alpha-induced expression of VCAM-1 to levels normally observed in wild-type mice. Reconstitution with T cell- but not B cell-enriched splenocytes, also restored the TNF-alpha-induced expression of VCAM-1 in SCID mice to wild-type levels. These results implicate circulating T lymphocytes as modulators of the increased ECAM expression elicited by TNF-alpha.     

Role of ICE-like proteases in endothelial cell hypoxic and reperfusion injury

Because of its location between blood and tissue, the endothelium is particularly vulnerable to hypoxic/reperfusion injury, but the mechanisms responsible for this injury are not known. A number of recent findings suggest that hypoxia and reperfusion injures neuronal cells via apoptosis. Apoptosis has recently been shown to depend on the activation of a class of proteases with homology to Interleukin-1 beta converting enzyme (ICE) protease. Therefore, we examined the effect of specific inhibitors of ICE-like proteases on hypoxic and reperfusion injury in cultured EAhy926 endothelial cells. Pretreatment of cells with ICE inhibitor II (Ac-YVAD-CMK), ICE inhibitor III (Z-Asp-2,6-dichlorobenzoyloxy-methylketone-Z-Asp-CH2-DCB+ ++), or ICE inhibitor IV (Ac-YVKD-CHO) (all at 10-100 microM) did not protect cells from hypoxic injury. However, pretreatment of cells with ICE inhibitor III and to a lesser extent with ICE inhibitor II, but not with ICE inhibitor IV, protected cells from reperfusion injury. The protective effect of ICE inhibitor III was not dependent upon pH, but was associated with decreased release of arachidonic acid from cells. These findings suggest that reperfusion injury to EAhy926 endothelial cells involves ICE-like proteases. The identity of the protease(s) is not known but it does not appear to be a YAMA-type protease based upon ICE inhibitor specificity. Our data also indicate that a potential target of this protease is phospholipase A2 (PLA2).     

Redox transformations of spin probes during interaction with isolated myocardium in ischemic and reperfusion conditions

It has been shown, that a certain part of spin labels TEMPO, TEMPAMINE or TEMPOL undergoes redox-transformations during the perfusion of rat isolated working hearts. The kinetic parameters of the redox-transformations depend on the duration of the ischemia period.     

Neutralization of TNF by the antibody cA2 reveals differential regulation of adhesion molecule expression on TNF-activated endothelial cells

Upregulation of adhesion proteins plays an important role in mediating inflammation. The induction of adhesive molecules has been well studied, but the reversibility of their expression has not been well characterized. A neutralizing anti-TNF monoclonal antibody (cA2) was used to study the down regulation of TNF-induced E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) on cultured human umbilical vein endothelial cells (HUVECs). Addition of cA2 following TNF stimulation of HUVECs enhanced the rate of E-selectin and VCAM-1 down-regulation from the cell surface and also reduced steady state E-selectin and VCAM-1 mRNA levels. The cA2-mediated disappearance of E-selectin, but not VCAM-1 protein was microtubule and not microfilament dependent. Neutralization of TNF only slightly reduced ICAM-1 cell surface levels following initial TNF stimulation, suggesting a slower turnover of ICAM-1 compared to E-selectin and VCAM-1. Microtubule inhibition during TNF stimulation partially inhibited E-selectin, VCAM-1 and ICAM-1 mRNA upregulation. VCAM-1 and ICAM-1 cell surface expression were similarly partially inhibited, however, E-selectin levels were unaffected, presumably due to the dual, opposing effect of inhibiting protein expression and inhibiting internalization. Microfilament inhibition during protein induction specifically inhibited the maximal expression of VCAM-1 protein and mRNA, without affecting E-selectin or ICAM-1. These data support the notion that E-selectin, VCAM-1, and ICAM-1 expression are differentially regulated on HUVECs and suggest that TNF neutralizing therapies may be effective because of their ability to reduce the levels of pre-existing adhesion proteins.     

The effect of synthetic malaria pigment (beta-haematin) on adhesion molecule expression and interleukin-6 production by human endothelial cells

The formalin test was used to measure the analgesia induced by restraint in male and female rats. Animals were restrained for 30 min or left undisturbed in their cage and then (1) killed immediately to collect blood for hormonal determinations; or (2) subcutaneously injected with formalin in the hind paw (or sham-injected), introduced to an open field for recording of behaviour, and killed at the end of this procedure. In both experiments, corticosterone was found to be higher in females. In Experiment 1, the ability of restraint to be stressful was confirmed by the increase in corticosterone in both sexes and by the decrease of testosterone in males. In Experiment 2, restraint-treatment induced a reduction in licking and flexing that was limited to the second phase. The reduction occurred in different periods and to a different degree in the two sexes; it was greater in females. Spontaneous behaviours showed sex differences in restraint-treated but not in formalin-treated animals. The results show that the hormonal effects observed after restraint are not present after the formalin test and that the marked analgesia observed with phasic painful stimuli does not occur with a longer-lasting one such as that induced by formalin, after which only partial and short-lasting effects were observed.     

Kinetics of adhesion molecule expression and spatial organization using targeted sampling fluorometry

Cellular interactions with the vascular wall under flow conditions are controlled, in part, by the density of adhesion molecules on endothelial cells. The spatial arrangement and absolute levels of these molecules over the endothelium are therefore important determinants of cellular localization. Many biochemical and functional studies have characterized the interactions between leukocytes and endothelial monolayers, but no reliable method has been reported for quantifying the spatial expression of adhesion molecules on intact endothelial cell monolayers. We report the development of targeted sampling fluorometry (TSF), which uses standard immunostaining, fluorescence microscopy and digital image analysis techniques to analyze cell surface molecule expression on a cell-by-cell basis. This technique is performed on an intact monolayer and results in cellular intensity distributions that reflect spatial heterogeneity in adhesion molecule expression. We demonstrate the use of targeted sampling fluorometry in a study of the kinetics of tumor necrosis factor alpha-induced activation of human umbilical vein endothelial cell monolayers and show that the spatial patterns of adhesion molecule expression correlate with the locations of bound lymphocytes.     

Effect of Rodiola rosacea on the resistance of isolated heart from stressed-out rats to ischemic and reperfusion injury

This study was performed to investigate the capability of iontophoretic delivery of prostaglandin E1 (PGE1) and the effect of this treatment on the viability of skin flaps on the rat dorsum model as described by Hammond and Ronald in 1993. The PGE1 level in the tissue sample under the electrode was assessed with radioimmunoassay. The iontophoretic treatment (for 20 minutes at 4 mA) with PGE1 solution (20 micrograms PGE1 in 2 ml saline) showed a significant increase of PGE1 in the flap tissue under the negative electrode (p < 0.05). Very little increase was observed after the iontophoresis of saline alone. This was not statistically significant. Iontophoretic treatment was given for 5 consecutive days following flap elevation. The survival area on the seventh postoperative day was significantly greater in the PGE1-treated flaps than that of either the control or saline-treated flaps. These results confirm the beneficial effect of iontophoretic treatment with PGE1 on the augmentation of skin flap viability.     

Time course of coronary endothelial healing after injury due to ischemia and reperfusion

BACKGROUND: Although it has been demonstrated in short-term preparations that ischemia with early reperfusion results in coronary vascular injury manifested by abnormal endothelium-dependent relaxation and increased permeability to plasma proteins, it has not been clear whether these abnormalities are permanent or reversible. METHODS AND RESULTS: In a canine model, regional coronary ischemia was accomplished by 1 hour of left anterior descending coronary artery ligation, and follow-up studies were performed after reperfusion for 1 hour, 48 hours, 2 weeks, or 9 weeks. Vasorelaxation was measured in vitro with preconstricted epicardial coronary artery rings subjected to increasing concentrations of the endothelium-dependent vasodilator ADP and the endothelium-independent vasodilator nitroprusside. At 1 and 48 hours of reperfusion, relaxation of rings from the ischemic reperfused artery to ADP was blunted, but relaxation to nitroprusside was normal. At 2 weeks there was a nonsignificant trend toward a blunted response to ADP in the ischemic/reperfused rings, and at 9 weeks a completely normal response to ADP was observed. Coronary microvascular permeability was assessed by measurement of protein leak index (PLI), by using a double-isotope technique with autologous radiolabeled transferrin and erythrocytes. At 1 and 48 hours of reperfusion there were substantial increases in PLI in the previously ischemic regions, indicative of increased extravascular transferrin. There was a small increase in PLI at 2 weeks but a completely normal measurement at 9 weeks. Electron microscopy of ischemic/reperfused vessels demonstrated endothelial cell swelling and other abnormalities in epicardial arteries and the microcirculation at 48 hours of reperfusion but normal endothelium at 2 weeks of reperfusion. CONCLUSIONS: After 1 hour of regional coronary ischemia, coronary endothelial injury occurs early in reperfusion with abnormalities in epicardial coronary artery endothelium-dependent relaxation, coronary microvascular permeability, and both epicardial coronary artery and microvascular histology. This pattern of injury persists for at least 48 hours, but there is partial functional and complete histological recovery within 2 weeks and complete functional recovery within 9 weeks.     

Impaired neutrophil function in chronic renal failure--dysregulation of surface adhesion molecule expression and phagocytosis

To elucidate the impaired neutrophil function in patients with chronic renal failure, we analyzed the expression of the adhesion molecules, LAM-1, LFA-1, Mac-1, gp150/95 and phagocytosis activity of neutrophils in predialysis and hemodialysis patients by flow cytometry. Further, the response to granulocyte colony-stimulating factor (G-CSF), N-formyl-methionyl-leucyl-phenylalanine (fMLP) and tumor necrosis factor alpha (TNF alpha) were investigated. In hemodialysis patients, the expression of LAM-1 was decreased and that of MAC-1 was increased, indicating the activation of neutrophils. Also in predialysis patients, the same condition of "low LAM-1, high MAC-1" was observed, but to a lesser degree. Phagocytosis activity was significantly decreased in hemodialysis patients, whereas the neutrophils of predialysis patients showed almost the same phagocytosis activity compared to the controls. The responses to G-CSF, fMLP, TNF alpha were significantly reduced both in hemodialysis and predialysis patients. The inadequate activation of neutrophils and impaired response to stimulation may play an important role in uremic patients with regard to increased susceptibility to infections.     

Effects of intravenous methylprednisolone therapy on leukocyte and soluble adhesion molecule expression in MS

Intravenous methylprednisolone (IVMP) may inhibit inflammatory cell recruitment to active MS lesions by effects on leukocyte or endothelial cell adhesion molecule expression. We investigated 15 MS patients in relapse receiving a 5-day course of IVMP (500 mg/day) and 15 normal subjects. Patients' blood samples were obtained pretreatment, at 6 and 24 hours after the first dose, and 48 hours after completion of therapy. Levels of L-selectin, leukocyte functional antigen 1 (LFA-1), Mac-1, and very late activation antigen 4 (VLA-4) expression were determined on alphabeta and gammadelta T cells and monocytes by dual-color immunofluorescent flow cytometry. Serum levels of soluble (s) L-selectin, sE-selectin, soluble intercellular adhesion molecule 1 (sICAM-1) and soluble vascular cell adhesion molecule 1 (sVCAM-1) were measured by ELISA. There was a marked decrease in the T-cell and monocyte counts at 6 hours after therapy, with recovery to baseline at 24 to 48 hours. Adhesion molecule expression was normal on circulating T cells and monocytes in active MS. IVMP resulted in significant changes in the percent adhesion molecule expression on monocytes: increased L-selectin expression at 24 hours, decreased Mac-1 expression at 6 hours, and decreased VLA-4 expression at 6 hours and 24 hours following treatment. T-cell adhesion molecule expression was unaffected by the therapy. Serum sE-selectin was reduced at 6 hours and 24 hours following treatment. IVMP alters the distribution and kinetics of monocyte adhesion molecule expression and endothelial cell release of E-selectin, which may limit monocyte recruitment to areas of tissue destruction in MS.