The potassium channel blocker tetraethylammonium blocks the flow-induced increase in endothelial ICAM-1. We have investigated the subtype of potassium channel that modulates flow-induced increased expression of ICAM-1 on saphenous vein endothelium. Cultured human saphenous vein endothelial cells (HSVECs) or intact saphenous veins were perfused at fixed low and high flows in a laminar shear chamber or flow rig, respectively, in the presence or absence of potassium channel blockers. Expression of K(+) channels and endothelial ICAM-1 was measured by real-time polymerase chain reaction and/or immunoassays. In HSVECs, the application of 0.8 N/m(2) (8 dyn/cm(2)) shear stress resulted in a two- to fourfold increase in cellular ICAM-1 within 6 h (P < 0.001). In intact vein a similar shear stress, with pulsatile arterial pressure, resulted in a twofold increase in endothelial ICAM-1/CD31 staining area within 1.5 h (P < 0.001). Both increases in ICAM-1 were blocked by inclusion of 100 nM apamin in the vein perfusate, whereas other K(+) channel blockers were less effective. Two subtypes of small conductance Ca(2+)-activated K(+) channel (selectively blocked by apamin) were expressed in HSVECs and vein endothelium (SK3>SK2). Apamin blocked the upregulation of ICAM-1 on saphenous vein endothelium in response to increased flow to implicate small conductance Ca(2+)-activated K(+) channels in shear stress/flow-mediated signaling pathways.

Primary human endothelial cells have a finite life span in vitro. After 3–4 passages, they tend to de-differentiate and eventually reach senescence. This limits their use in studies of endothelial cell function. To overcome this, we have developed human saphenous vein endothelial cell lines (HSVEC lines). Two cell lines were produced by infection with pZipSVtsA58-U19 which encodes the simian virus 40 large T-antigen, and one cell line was obtained by transfection with pLXSN16E6E7, which encodes the human papillomavirus type 16 E6 and E7 genes. Two of the three HSVEC lines exhibited an extended life span in vitro and retained characteristic endothelial ‘cobblestone’ morphology. These cell lines expressed the known endothelial markers CD31 and vascular endothelial cadherin, and were able to bind Ulex europaeus lectin I, but they did not retain the expression of von Willebrand factor. Furthermore, one cell line was able to functionally up-regulate the expression of intercellular adhesion molecule-1 in response to stimulation with tumor necrosis factor α and was also able to incorporate acetylated low-density lipoprotein. Our results suggest that this latter HSVEC line will provide a useful resource to investigate selected responses of the vascular endothelium to physiological and pathological situations.

PURPOSEWe investigated the hypothesis that fibrinogen increased DNA synthesis (and cell proliferation) of smooth muscle cells (SMCs) cultured from human saphenous vein and that the increased DNA synthesis was attenuated when cells were cultured on polymeric collagen.METHODSSMCs were cultured from human saphenous vein on plastic, fibronectin, monomeric, and polymeric collagen. Fibrinogen products were prepared by proteolytic digestion. DNA synthesis was measured by bromodeoxyuridine incorporation into DNA, cell proliferation by cell counting, cyclic adenosine monophosphate by enzyme-linked immunosorbent assay, and fibrinopeptide B labeled with iodine 125 used for binding studies.RESULTSFibrin monomer (0.003-0.1 micromol/L) stimulated a concentration-dependent increase in DNA synthesis of up to 10-fold, which could be inhibited by the peptide Bbeta15-42. The stimulation of DNA synthesis was highest for cells cultured on plastic and lowest for cells cultured on type I collagen polymer. Much higher concentrations of fibrinogen (0.3-1 micromol/L) were required to effect similar increases in DNA synthesis. Fibrinogen had a particular effect to augment DNA synthesis, up to 14-fold, when cells were cultured on monomeric type I collagen. This augmented DNA synthesis was inhibited by a neutralizing antibody to urokinase-type plasminogen activator. Incubation of cells cultured on collagen monomer with fibrinogen resulted in production of fibrinopeptide B. Fibrinopeptide B (5 micromol/L) increased DNA synthesis by fourfold and had additive effects with fibrin monomer to increase DNA synthesis. Iodinated tyrosine fibrinopeptide B bound to SMCs (dissociation constant 0.6 micromol/L).CONCLUSIONCultured human saphenous vein SMCs appear to have high-affinity receptors for fibrin monomer and fibrinopeptide B, the engagement of which stimulates DNA synthesis. These mechanisms may be pertinent to the association between fibrinogen and vein graft stenosis in vivo.

Background and Purpose —Research for infectious agents in the etiology of atherosclerosis has identified Chlamydia pneumoniae as a possible candidate. While there is evidence of an association between presence of this microorganism and atherosclerosis, it is unclear whether infection has a genuinely etiologic role in this disease, whether its presence influences clinical outcomes, and, if so, at which stages of disease this occurs. We have approached this issue in patients with advanced carotid artery atherosclerosis using molecular biological detection methods and clinically relevant indicators of pathology in carotid artery atheroma to determine whether the presence of C pneumoniae correlates with plaque instability. Methods — C pneumoniae was detected with the use of a sensitive nested polymerase chain reaction. Preoperative embolization and preoperative infarcts were recorded with the use of transcranial Doppler insonation of the middle cerebral artery and cerebral CT, respectively. Results — C pneumoniae DNA was detected in 25.5% of a cohort of 98 symptomatic patients. There was no significant difference in plaque stability as measured by embolization rates between the chlamydial-positive and -negative specimens. There was also no correlation between the number of ipsilateral hemispheric infarcts in the territory of the middle cerebral artery and chlamydial status. Conclusions —This study confirms that C pneumoniae is a common finding in atherosclerotic plaques of the carotid artery but suggests that the presence of the infectious organism has little detectable impact on plaque instability when measured by clinically significant markers. This raises important questions for the rationale of antibiotic therapy in atherosclerosis.

BACKGROUND:There is an increasing body of evidence linking the human pathogen Chlamydia pneumoniae with atherosclerosis. METHODS:A Medline-based review of the literature was carried out. RESULTS AND CONCLUSION:Seroepidemiological studies have revealed the possibility that evidence of infection with C. pneumoniae and atherosclerotic disease are related. Studies on human tissue have demonstrated that evidence of the organism can be found in human atherosclerotic tissue by both direct and indirect methods significantly more often than in control vascular tissue. Using animal models it is possible to show that C. pneumoniae can be disseminated haematogenously following pulmonary infection and that it shows a tropism for atherosclerotic tissue. In vitro work has demonstrated that the organism is capable of infecting, surviving and multiplying in cells of the human vascular wall, and that it can provoke a cell-mediated cytokine response which has implications both locally and systemically. Two clinical trials of macrolide antibiotics have demonstrated that they confer increased cardiovascular protection in patients following myocardial infarction. Adequately powered trials are needed to establish the therapeutic role of antibiotics in peripheral arterial disease.

Myotonic dystrophy (DM) is associated with an underlying CTG trinucleotide repeat expansion at a locus on chromosome 19q13.3. We have determined the repeat length in 23 DM patients with varying clinical severity of symptoms and various sizes of repeat amplification. We confirm that as in previous studies there is no strong correlation between repeat length and clinical symptoms but find that the repeat length in peripheral blood cells of patients increases over a time span of five years indicating continuing mitotic instability of the repeat throughout life. Repeat length progression does not appear to be indicative of clinical progression but age probably is. The degree of expansion correlates with the initial repeat size and 50% of the patients with continuing expansions showed clinical progression of their disease symptoms over the five year study period.

This review aims to summarize the current understanding of the biology of maedi-visna virus (MVV), the prototype virus of the family lentivirinae. The paper provides a short overview of the historical background to the discovery of MVV. Detailed descriptions of the structure and organization of the MW genome and of the virion encoded polypeptides are given and the MVV life cycle in vitro and in vivo are compared and contrasted and the tropism of the virus discussed. The clinical consequences of infection are considered and the mode of transmission, immune response to the virus and possible mechanisms of pathogenesis are discussed.

The envelope glycoprotein gp135 of the ovine lentivirus maedi visna virus (MVV) is the main target for neutralising antibody in vivo, however little is known about the specific regions of gp135 which elicit this neutralising response. We have used the polymerase chain reaction (PCR) to generate overlapping fragments of the gp135 gene which have been expressed as fusion proteins in the yeast Ty-VLP system. These fusion proteins have been used to analyse the antibody response to gp135 in MVV infected sheep and we are able to identify at least three distinct regions of gp135 to which antibodies are directed. The approach described in this paper provides a rapid and simple method of generating overlapping fusion proteins with which to carry out epitope mapping studies.