Human cardiac microvascular and macrovascular endothelial cells respond differently to oxidatively modified LDL.

Oxidation of low density lipoproteins (LDL) is considered a key event in the pathogenesis of atherosclerotic lesions. Disturbed generation of coagulatory and anticoagulatory factors by endothelial cells contributes to thrombosis and the progression of atherosclerosis in coronary arteries. In this study, the effects of native LDL (n-LDL) and oxidized LDL (ox-LDL) on human coronary endothelial cells were measured. The reaction of coronary endothelial cells to LDL were compared with those of cardiac microvascular endothelial cells grown under comparable conditions. LDL was isolated by ultracentrifugation and copper oxidized. The degree of oxidation was expressed as malondialdehyd (MDA) equivalents and was 0.78+/-0.14 nM MDA/mg LDL for native LDL and 13.63+/-1.18 nmol MDA/mg LDL for ox-LDL. Basal secretion of t-PA and PAI-1 activity were higher in macrovascular endothelial cells. Incubation of n-LDL in concentrations ranging from 3 to 100 microM/ml LDL-protein did not change t-PA-secretion, PAI-1 activity or procoagulant activity in both cell types. Ox-LDL (3 to 100 microM/ml LDL protein) decreased t-PA secretion in a concentration dependent manner from 30.9+/-1.7 to 13.7+/-30 ng/ml per 24 h per 10(6) cells (P < 0.01), increased PAI-1 antigen from 2772+/-587 to 4441+/-766 ng/ml per 24 h per 10(6) cells (P < 0.05) as well as PAI-1 activity from 34+/-6 to 55+/-9 AU/ml per 24 h per 10(6) cells (P < 0.05) in macrovascular endothelial cells but had only minor effects on microvascular endothelial cells. Procoagulant activity measured as coagulation time, similarly increased only in macrovascular endothelial cells from 197+/-6 to 76+/-6 s/24 h per 10(6) cells (P < 0.05). The effect on PAI-1 secretion showed a dependency to the degree of oxidation and could be completely blocked by the antioxidant probucol. The angiotensin converting enzyme (ACE), which represents an endothelial enzyme not related to coagulation, remained unchanged during incubation with ox-LDL. Basal ACE activity was higher in microvascular endothelial cells. The higher susceptibility of macrovascular endothelial cells to ox-LDL may partially determine the localization of thrombus formation and the development of atherosclerotic plaques in hyperlipidemic patients.

L-selectin-dependent leukocyte adhesion to microvascular but not to macrovascular endothelial cells of the human coronary system.

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-alpha (TNF-alpha)-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-alpha-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.

Isolation and characterization of macrovascular and microvascular endothelial cells from human hearts.

In vivo models to investigate mechanisms of local hemostasis in the macro- and microvascular coronary circulation are not available. Therefore, we established a culture system of human macro- and microvascular endothelial cells with high cellular yield and high endothelial cell purity. Microvascular endothelial cells from human hearts were isolated by enzymatic treatment of cardiac muscle preparations obtained during heart transplantation. The isolated microvessels were used to start cultures that were subsequently separated and purified from contaminating nonendothelial cells by paramagnetic beads linked to the lectin Ulex europaeus agglutinin I. Macrovascular endothelial cells were isolated from epicardial coronary arteries and purified by paramagnetic beads as well. With this method high purity (< 2% nonendothelial cells) was achieved as judged from fluorescence-activated cell sorting. Immunochemistry demonstrated the expression of several typical endothelial markers. The two endothelial cell types displayed functional heterogeneity in respect to bradykinin degradation and plasminogen activator inhibitor-1 activity. Thus the ability to selectively isolate and culture human macro- and microvascular cardiac endothelial cells provides a valuable tool to systematically investigate endothelial function in human hearts.

Cultivation and characterization of micro- and macrovascular endothelial cells from the human heart.

In order to investigate processes, such as atherosclerosis and inflammation in vitro, it is necessary to obtain viable and pure endothelial cell cultures from human hearts. To this end, endothelial cells were isolated and cultured from the micro- and macrovasculature of human hearts obtained during heart transplantation. Isolation of capillaries after enzymatic digestion of heart muscle provided a source of microvascular endothelial cells. Contaminating non-endothelial cells were removed by a new technique: paramagnetic beads linked to the lectin ulex europaeus I (UEA-I) were used to select endothelial cells. The resulting cultures contained less than 2% of non-endothelial cells, as judged from immunological staining and fluorescence-activated cell sorting. Both types of endothelial cell displayed typical endothelial properties. They were all positive for factor VIII-related antigen and expressed the endothelial-specific adhesion molecules, CD31 and E-selectin (ELAM-1), after stimulation with cytokines. In addition, they could be labelled with Dil-Ac-LDL, contained angiotensin converting enzyme activity and secreted tissue plasminogen activator, thus demonstrating that typical endothelial functions were preserved in culture.

Disambiguation of ditropic sentences: acoustic and phonetic cues.

In a previous study, we demonstrated that listeners were highly successful in identifying the intended meaning of spoken ditropic sentences (those which may carry either a literal or an idiomatic meaning) when speakers were instructed to convey the distinction. The present communication reports on acoustic and phonetic analyses carried out with the goal of identifying cues that distinguished the literal and idiomatic utterances. Certain prosodic differences were observed. Literal utterances were systematically longer than idioms. This was partly due to increased use of pauses, as well as to increased duration of major lexical items. Moreover, literal sentences were typically characterized by greater numbers of pitch contours (discernible rise-fall excursions of fundamental frequency) and open junctures than were idiomatic utterances. In addition to suprasegmental contrasts, articulatory distinctions--corresponding to lento-allegro phonological rules--were also observed. These distinctions directly reflect the structural differences intrinsic to the two types of utterances. A literal sentence is formulated by the organization of constituent words and phrases. Idioms, on the other hand, are holistic units, largely nontransparent to syntactic structure or the usual meaning of the lexical members.