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While the cellular mechanisms of atherosclerosis have been intensively studied, the mechanisms leading to preferential localization of atherosclerotic lesions are less well understood. To further define these mechanisms, endothelial cells from coronary arteries, i.e., vessels with frequent atheroscleritic lesions, were isolated and grown in vitro. In order to compare the reactions of both cell types, endothelial cells derived from microvessels of human hearts were isolated and cultured under identical conditions. Incubation of endothelial cells with oxidized LDL (75 µg/ml protein) induced a significant increase in PAI-1 activity (182%, p < 0.05) in coronary macrovascular endothelial cells. This stimulatory effect of ox-LDL was less significant in microvascular endothelial cells (144%, p < 0.05). n-LDL did not influence secreted PAI-1 activity. Stimulation with angiotensin II induced expression of E-selectin more effectively in coronary macrovascular than in microvascular endothelial cells. In addition, angiotensin II-induced E-selectin espression led to incresed E-selectin-dependent adhesion of HL60 cells to coronary macrovascular endothelial cells under flow conditions, while only little effect was observed with cardiac microvascular endothelial cells. In contrast, L-selectin-dependent adhesion, which has been shown to play an important role in inflammatory reactions, was preferentially observed in cardiac microvascular endothelial cells and could only be stimulated with TNFa, not by angiotensin II. Therefore, these cellular differences may in part explain specific properties of cardiac endothelial cells: Such that atherosclerotic lesions are localized in macrovascular vessel segments, whereas inflammatory responses are predominantly found in the microvasculature. Während die zellulären Mechanismen der Pathogenese der Arteriosklerose intensiv untersucht worden sind, ist über die Mechanismen, die zu einer bevorzugten Lokalisation arteriosklerotischer Läsionen in bestimmten Gefäßarealen führen, weniger bekannt. Zur Untersuchung dieser Mechanismen wurden Endothelzellen aus menschlichen Koronararterien, einem Gefäßbereich, in dem häufig arteriosklerotische Läsionen beobachtet werden, isoliert und kultiviert. Endothelzellen der Mikrozirkulation menschlicher Herzen wurden unter gleichen Bedingungen kultiviert und die Reaktion beider Zellarten verglichen. Inkubation der Zellen mit den in bezug auf die Bildung arteriosklerotischer Plaques besonders pathogenen oxidierten LDL induzierte in makrovaskulären koronaren Endothelzellen eine stärkere Zunahme der PAI-1-Aktivität (182%, p < 0,05) als in mikrovaskulären Zellen (144%, p < 0,05), während nichtoxidierte n-LDL die sezernierte PAI-1-Aktivität nicht beeinflußten. Nach Stimulation mit Angiotensin II (10-11–10-5M) zeigte sich auf makrovaskulären koronaren Endothelzellen eine stärkere Expression des Adhäsionsmoleküls E-Selectin als auf mikrovaskulären Endothelzellen. Die Angiotensin-II-induzierte E-Selectin-Expression führte in vitro auch funktionell zu einer erhöhten Adhäsion von HL60-Zellen unter Flußbedingungen. Dagegen wurde die für die Leukozytenrekrutierung im Rahmen inflammatorischer Reaktionen wichtige L-Selectin-abhängige Adhäsion von Angiotensin II nicht induziert. Stimulation mit TNFa führte überwiegend an mikrovaskulären Endothelzellen zum Auftreten einer L-Selectin-abhängigen Adhäsion. Die unterschiedliche Reaktion mikro- und makrovaskulärer kardialer Endothelzellen bietet eine Möglichkeit, zellspezifische Differenzierung und präferentielle Lokalisation arteriosklerotischer Läsionen in makrovaskulären Gefäßbereichen einerseits und das Auftreten entzündlicher Reaktionen im Bereich der Mikrozirkulation andererseits zu erklären.

Neutral endopeptidase 24.11 (NEP) degrades vasoactive peptides, including natriuretic peptides, kinins, angiotensins, and endothelins. It contributes to the regulation of vascular tone and body fluid homeostasis. In the present study the expression of NEP was investigated in cultured human smooth muscle cells derived from umbilical veins (HSMC) and human coronary arteries (HCSMC). A constitutive NEP expression was found in growing and starved smooth muscle cells and was about 4 fold higher than in endothelial cells derived from umbilical veins. Treatment of smooth muscle cells with dexamethasone (0.01–0.1 μM Dex) and with the protein kinase C activator, phorbol myristate acetate (0.1 μM PMA), increased NEP mRNA by 3–4 fold and two fold, respectively. Dexamethasone (0.1 μM) and prednisolone (0.1 μM) increased protein concentrations of NEP and NEP‐activity after 3 days and continued to increase at 5 days, whereas PMA induced maximal increase of NEP concentrations after 48 hours. The effect of dexamethasone was concentration‐dependent and was comletely abolished by cycloheximide (10 μM), a protein synthesis inhibitor. The effect of PMA on NEP protein was completely blocked by protein kinase C inhibitors, calphostin C and H7 (both 10 μM). NEP 24.11 is constitutively expressed in human smooth muscle cells from unbilical veins and coronary arteries and is upregulated by glucocorticoids and by protein kinase C activation in these cells.

The breeding biology of Weddell seals (Leptonychotes weddellii) was studied at Drescher Inlet (Antarctica) during the austral late winter and spring 1986. The whelping season was estimated to last about 40 days, with a peak in pupping at late October. No post natal mortality was observed during the whole study. The attachment between parous females and their pups was strong during the first five weeks after parturition. From the third week on mothers spent more time in the water than the pups. Data on daily peak haul-out time of pups, post natal presence of pups on the ice and distribution of births during the whelping season, indicate that censuses should be carried out between 12.00 hours and 16.00 hours local time and during three weeks after the peak of the pupping season. Pups weaned between 6 and 7 weeks of age. Hormone concentrations (oestradiol-17ß and progesterone) indicated that females did not come into oestrus during lactation. Throughout the whole lactation period the mother dominates the relationship with her pup. Growth of the pups was linear during the first 5 weeks post partum, during which period birth weight (x=29.1kg) increased more than 3 1/2 times. Until the 6th week of age the pups gained on average 2.6 kg/day, the respective weight loss in cows was 5.8 kg/day. The latter represents an average loss of nearly 38% of the initial weight at parturition. During the course of lactation, suckling frequency decreased, whereas the length of each bout increased resulting in an approximately constant total suckling duration per week. Of the energy used by females during the first 5 weeks, 52% was consumed by pups. Pups only took milk and the cows did not feed at all during the whole period of observation. Therefore the realized growth in pups, 10 g/min of suckling, was totally derived from energy stored by the cows.

To characterize L-selectin–dependent cell adhesion to human vascular endothelium, human cardiac microvascular endothelial cells (HCMEC) and human coronary endothelial cells (HCEC) were isolated from explanted human hearts. The adhesion behavior of human (NALM-6) and mouse (300.19) pre-B cells transfected with cDNA encoding for human L-selectin was compared with that of the respective nontransfected cells in a flow chamber in vitro. More than 80% of the adhesion to tumor necrosis factor-α (TNF-α)–stimulated HCMEC at shear stresses <2 dyne/cm2 was L-selectin dependent and could be equally well blocked by an anti–L-selectin antibody or a L-selectin-IgG-chimera. No L-selectin dependent adhesion to HCEC could be shown. The L-selectin dependent adhesion to HCMEC was insensitive to neuraminidase, but greatly inhibited by addition of NaClO3 , which inhibits posttranslational sulfation and remained elevated for at least 24 hours of stimulation. E-selectin dependent adhesion of HL60 cells to HCMEC was blocked by neuraminidase, but not by NaClO3 and returned to control levels within 18 hours of HCMEC stimulation. It is concluded that microvascular, but not macrovascular endothelial cells express TNF-α–inducible sulfated ligand(s) for L-selectin, which differ from known L-selectin ligands, because sialylation is not required. The prolonged time course of L-selectin dependent adhesion suggests a role in sustained leukocyte recruitment into inflammatory sites in vivo.

The electrophysiological properties of human coronary endothelial cells (HCEC) of macro-and microvascular origin were studied using the whole-cell configuration of the patch-clamp technique. The membrane potential of confluent HCEC(-41.9±3.9 mV (mean±SEM, n=32) for macro-and -33.6±22.6 mV (n=64) for microvascular cells, respectively) was less negative than the K+ equilibrium potential. Inward currents of isolated cells at potentials below the K+ equilibrium potential were blocked by external Ba2+ (1 mM), inactivated due to time- and voltage-dependent block caused by external Na+, and their amplitudes were enhanced by increasing extracellular [K+]; these currents were identified as inwardly rectifying K+ currents. Some isolated cells displayed outwardly directed K+ currents which were abolished after replacement of Cs+ for K+ on both sides of the membrane. Voltage-dependent Ca2+ currents could not be observed in isolated HCEC. Hyperpolarizations induced by vasoactive agonists have been observed in some endothelial cells from different species. In contrast, extracellularly applied ATP (adenosine-5'-triphosphate) and ADP (adenosine-5'-diphosphate) at micromolar concentrations depolarized confluent HCEC, whereas adenosine had no effect on resting potentials (RP), indicating that the nucleotide-induced depolarizations were mediated via P2-purinoceptors. These depolarizations occurred even after replacement of N-methyl-D-glucamine for extracellular Na+, indicating that Ca2+-influx was involved. There were no marked differences in the electrophysiological properties between cells of macro-and microvascular origin.

The action of interleukin-1 alpha (IL-1α) and tumor necrosis factor alpha (TNFα) on the production of biologically active granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) was studied and the secretion into the supernatant of human endothelial cell cultures was quantified by enzyme-linked immunosorbent assay (ELISA). In unstimulated endothelial cells low concentrations of G-CSF and GM-CSF were measured by ELISA. Stimulation for 24 h with either IL-1α or TNFα caused a concentration-dependent increase of both colony stimulating factors in the supernatant. Range of IL-1α studied: 0.1-100 U/ml; maximum CSF induced: G-CSF 142 + 16 ng/6 × 105 cells, GM-CSF 18.5 ± 3.3 ng/6 × 105 cells. Range of TNFα studied: 10-2000 U/ml. 4 maximum CSF induced: G-CSF: 36 ± 6 ng/6 × 105 cells, GM-CSF 16.7 ± 1.5 ng/6 × 10 cells. The dosages of both factors were varied to study the combined action of IL-1α and TNFα on CSF production. IL-1α (0.1-10 U/ml) combined with TNFa (10-1000 U/ml) enhanced the effect onCSFproductionina superadditivemannercomparedwith theeffect of TNFaorIL-1α alone. Higher concentrations of IL-1α (10 U/ml) with TNFa (10-1000 U/ml) inhibited G-CSF production but not GM-CSF production. IL-1α was more potent as an inducer of G-CSF than TNFa at doses approximately equipotent in causing GM-CSF production in human endothelial cells. Quantitative determination of G-CSF and GM-CSF revealed that the production of these CSFs was differently regulated by IL-1α and TNFα and demonstrated a superadditive effect by the two cytokines on G-CSF and GM-CSF production.

Activation of vascularß-adrenergic receptors prevents an increase in vascular permeability caused by free radicals or inflammatory peptides. Methylxanthines seem to have similar protective effects on vascular endothelium. In the present study we investigated the effect of theophylline on theß-adrenergic receptor expression and cAMP concentrations in cultured endothelial and smooth muscle cells from bovine aorta. Comparable values forß-receptor density and binding affinity were detected in both cell types. Isoproterenol induced significant downregulation ofß-receptors in endothelial (BAEC: -60.5%) and smooth muscle cells (BASMC: -52.5%; P < 0.01). Incubation of endothelial cells with theophylline (4 µg/ml and 40 µg/ml) for 24 hours did not affectß-receptor expression, whereas in smooth muscle cells theß-receptor density was reduced for -31.5% and -28.7, respectively. In endothelial cells a transient effect on cAMP concentrations was observed after stimulation with isoproterenol (1 µM), but no effect was found in theophylline treated endothelial cells. Stimulation of intact smooth muscle cells with isoproterenol and theophylline (4 µg/ml and 40 µg/ml) resulted in a significant increase of cAMP concentrations after 60 and 240 minutes. The present data suggest a novel, celltype specific effect of theophylline on theß-adrenergic receptor expression in vascular smooth muscle cells in vitro.

Endothelial cells of the arterial vascular system and the heart contain straight actin filament bundles, of which there are few, if any, in the venous endothelium1–4. Since stress fibre-containing endothelial cells within the vascular system tend to be located at sites exposed to particularly high shear stress of blood flow, we have investigated, in an experimental rheological system (Fig. 1), the response of the endothelial actin filament skeleton to controlled levels of fluid shear stress. Here we report that endothelial stress fibres can be induced by a 3-h exposure of confluent monolayer cultures of human vascular endothelium to a fluid shear stress of 2 dynes cm−2, approximately the stress occurring in human arteries in vivo. Fourfold lower levels of shear stress that normally occur only in veins, had no significant effect on the endothelial actin filament system. The formation of endothelial stress fibres in response to critical levels of fluid shear stress is probably a functionally important mechanism that protects the endothelium from hydrodynamic injury and detachment.