摘要:

We studied the influence of hypertensive (HT) serum on human umbilical vein endothelial cell (HUVEC) and on human umbilical artery smooth muscle cell (HUASMC) proliferation. We also tested the possible influence of the HT serum on vasoconstrictor substances released by HUVECs.Pools of HT and normotensive (NT) sera were collected and used to supplement cell culture medium. Both HUVECs and HUASMCs were conditioned with culture media supplemented with either HT or NT sera. After conditioning, both types of cells were tested for proliferative capacity. Under the same experimental conditions the HUVEC conditioned medium was assayed for angiotensin II (AII) and endothelin 1 (ET,). All and ET, were also assayed after acute exposure to HT serum of HUVEC preconditioned with NT serum.Our results showed that HUVEC and HUASMC conditioned with HT serum substantially enhanced cell proliferation.Preconfluent HUVECs conditioned with HT serum showed higher levels of AZZandlower levels of ET1compared with the controls in NT serum. These differences were reduced at confluence. HUVECs conditioned with NT serum showed a transient increase in ET1levels after acute exposure to HT serum.We suggest that serum from hypertensive patients is able to modulate vascular cell proliferation and possibly the production of vasoactive substances in culture.

ISSN:1062-3329    

DOI:10.3109/10623329609024693

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Apoptosis of endothelial cells (EC) is responsible for the removal of blood vessels during vascular remodelling. Cultured human umbilical vein EC (HUVEC) undergo apoptosis if deprived of either serum or adhesion. Apoptotic HUVEC rapidly loose adhesion and in this paper we describe the ultrastructure of detached apoptotic HWEC and human microvascular EC (HMEC). These cells displayed the formation of apoptotic bodies, nuclear condensation and nuclear fragmentation typical of apoptosis in other cell types. Ultrastructural changes occurred in parallel with the internucleosomal DNA cleavage characteristic of apoptosis. An important difference between apoptotic HWEC and other reported cells was the formation of vesicle-like canalicular structures confluent with the plasma membrane surface. These structures formed an interconnecting network throughout the apoptotic cell. Apoptotic HMEC also displayed this canalicular pattern. Continuity of the plasma membrane surface of apoptotic EC with these canaliculi was established using colloidal gold particles. Canalicularisation seemed to increase the mechanical fragility of apoptotic EC, facilitating the fragmentation of apoptotic cells to very small particles. EC are comparatively large, so that EC detached during trauma could act as potentially dangerous micro-thrombi. Since detached EC become apoptotic, it is suggested that the canalicular fragmentation represents an EC adaptation to reduce the micro-embolic potential of apoptotic EC.

ISSN:1062-3329    

DOI:10.3109/10623329609024694

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Substance P (SP) activates an outward K+current, membrane hyperpolarization and increases cytosolic Ca2+([Ca2+],) in porcine coronary artery endothelial cells (PCAECs). We tested the importance of Ca2+, CAMP, cGMP, arachidonic acid and G protein-linked pathways in modulating whole-cell K+currents activated by SP. Current or membrane potential and [Ca2+], were measured simultaneously in single cells in response to SP (10 nM), using the perforated patch technique and fura-2 microfluorimetry. Partial block of Kcachannels by D-tubocurarine resulted in a significant reduction in SP-induced membrane hyperpolarization and the plateau phase of the cell [Ca2+]iresponse. In PCAECs preloaded with BAPTA, SP failed to elevate [Ca2+]ior activate outward current. In Ca2+-free bath solution, SP induced transient elevations in [Ca2+]iand outward current. With SP still present, addition of 1 mM Ca2+to the bath after [Ca2+]ireturned to resting levels elevated [Ca2+]iand elicited outward current. Acute application of forskolin (20 pM), a CAMP activator, 8-bromo-cGMP (100 pM), a membrane-permeant analog of cGMP or arachidonic acid (200 pM) did not significantly affect the magnitude or time-course of SP-induced K+current. Open probability (P,) of single K+channels was measured in inside-out patches as a function of bath [Ca2+]. P0increased in a sigmoidal fashion with [Calf] but the relation was not significantly altered by GTP (200 pM), GDPPS (500 pM) or GTPyS (200 1M). These results suggest that SP-induced K+ current and subsequent hyperpolarization in PCAECs are important for regulation of Ca2+influx and can be triggered by Ca2+release from stores or by extracellular Ca2+entry. In addition, K+channel activity in PCAECs is primarily modulated by changes in [Ca2+], but not by CAMP, cGMP or arachidonic acid pathways and is not directly linked to a G protein.

ISSN:1062-3329    

DOI:10.3109/10623329609024695

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Increasing evidence suggests that nitric oxide (NO) plays a role in migraine pain. Thus, the NO donor glyceryl trinitrate provokes migraine attacks in sufferers and a greater dilatation of large arteries than in controls. Physiological NO-mediated dilatation of large arteries might therefore be abnormal in migraine. Recently an ultrasound method for the detection of shear stress stimulated endothelial NO release in large arteries in man has been described. Using this method we compared radial artery dilatation during reactive hyperaernia in 12 female sufferers of migraine without aura and 12 age- and sex- matched healthy subjects. No group differences were detected in these responses (peak dilatation 111±2% (mean±SEM) of baseline in migraineurs and 112±3% of baseline in the healthy control group, p = 0.79). These results indicate that shear stress stimulated endothelial NO release is normal in peripheral conduit arteries in patients suffering from migraine without aura. It remains to be settled whether the same is true in extra- and intracranial arteries.

ISSN:1062-3329    

DOI:10.3109/10623329609024696

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

In order to study the signal transduction mechanism of endothelial perturbation, the effects of phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu), both protein kinase C (PKC) activators, on cultured human endothelial cell (EC) hydrogen peroxide (H2O2) generation, endocytotic activity, and cytoskeletal structure have been investigated. EC were incubated with 1-100 nM PMA, or PDBu, and cellular H2O2generation and endocytotic activity measured. PMA and PDBu exposure caused dose-dependent rises in EC H2O2production. Likewise, EC incubated with PMA and PDBu had dose-related endocytosis increases. Cytoskeletal inspection of 10 nM PMA-perturbed EC revealed structural remodeling with stress fiber formation. Similar cellular functional changes occur in EC exposed to high low-density lipoprotein (LDL) concentrations. Protein kinase C (PKC) inhibition with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) prevented cytoskeletal remodeling in PMA-stimulated EC. In difference to this inhibitory effect, H-7 and calphostin C, another PKC inhibitor, augmented EC H2O2generation and endocytotic activity. Similar cellular responses to H-7 and calphostin C occurred in high LDL-perturbed EC. These findings suggest that PKC activation contributes to the stress fiber formation mechanism, whereas H2O2generation and heightened endocytosis can occur by both PKC-dependent and PKC-independent mechanisms. Such cellular functional changes add to our understanding of endothelial perturbation, which has been hypothesized to be a major contributing factor in atherogenesis.

ISSN:1062-3329    

DOI:10.3109/10623329609024697

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Human umbilical vein endothelial cells (EC) metabolize arachidonic acid (AA) through three major pathways—cyclooxygenase, lipoxygenase and cytochrome P450 (P450) isozymes. Previously, we have shown that pathophysiological concentrations of native low density lipoprotein (n-LDL) increase EC P450-dependent epoxyeicosatrienoic acid (EET) production. The present study was designed to identify putative P450 isozymes involved in EC epoxidation of AA. Reverse transcription—polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) were employed to detect P450 2 family cDNA from EC mRNA. Degenerate primers complimenting 2 homologous regions from 6 different P450 2 families were designed to capture a 440 bp cDNA fragment corresponding to the heme binding region of P450 2 isozymes. RT-PCR of EC total RNA with these primers amplified a 440 bp fragment. After gel purification, the fragment was cloned, sequenced and found to share a high degree of identity with human liver P450 2C8 and 2C9. New primers were designed based on the nucleotide sequences of the 440 bp fragment and a third homologous upstream region from the 440 bp fragment. RT-PCR was used to capture 5′sequences upstream from the 440 bp fragment and lock-docking 3′-RACE was used to capture sequences downstream from the 440 bp fragment. These steps successfully expanded the number of nucleotides captured and sequenced to 1.4 kb. The partial clone was a homologous to human liver P450 2C8, sharing a>99.8% identity, and>86.1% with P450 2C9. When sequence analysis of independently cloned 440 bp fragments was performed, no other P450 2 isozymes were detected. To determine if P450 2C isozyme family members were capable of AA epoxidation, ECV-304 cells, which usually generate low levels of EETs were transfected with pcDNA3 plasmids containing DNA encoding functional P450 2C9. Transient P450 2C9 transfection of this eternal endothelial cell line increased EET production levels up to 4-fold, which was similar in magnitude to EET production by EC exposed to native low density lipoprotein (n-LDL). These data indicate that EC epoxidize AA by a P450 2C8 isozyme.

ISSN:1062-3329    

DOI:10.3109/10623329609024698

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

In vitro autoradiographic studies, using sections of human epicardial coronary arteries, have demonstrated dense [125I]-endothelin-l binding to the tunica media and regions of the adventitia. Micro-autoradiography has been employed to localise binding at the cellular level and immunohistochemistry has been used to identify specific cell types on adjacent tissue sections. Adventitial [125I]-ET-1 binding is predominantly to microvessels, including those supplying the perivascular nerves. This binding is markedly reduced in the presence of unlabelled ET-1 and the ETA/ETBreceptor antagonist bosentan. These results suggest that, apart from acting on vascular smooth muscle of the tunica media, the action of locally-released ET-1 may also be via adventitial microvessels, including those supplying the perivascular nerves.

ISSN:1062-3329    

DOI:10.3109/10623329609024699

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Vascular smooth muscle cells (VSMCs) activation and hyperplasia, the important etiologic factors in atherosclerosis development, are inhibitable by drugs which donate nitric oxide (NO). We tested the hypothesis that administration of ISDN (60 mg/day), a NO donor, would inhibit development of vascular hyperplasia in atherosclerotic minipigs. VSMC proliferation and NO release by endothelial cells (ECs) were investigated in freshly isolated cells from minipigs fed an atherogenic diet with oral ISDN treatment (n = 8) or without (n = 8), or a control diet (n = 8) for 4 months. In ECs, atherosclerosis depressed by 60% NO release and suppressed the L-arginine negative regulatory feedback of the NO-synthase. Concomitantly, a 4-fold increased proliferation (PCNA labelling) and a 2.3-fold activation (PDGF-BB labelling) were observed in atherosclerotic VSMCs. Long-term treatment of atherosclerotic animals with ISDN restored NO release and regulatory processes of NO synthase from ECs, and reduced by half the proliferation and the activation down to control levels in VSMCs. These data demonstrate that ISDN attenuates intimal hyperplasia and suggest this effect is NO dependent.

ISSN:1062-3329    

DOI:10.3109/10623329609024700

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor