In vitro study of vascular endothelial injury by activated platelets and its prevention

doi:10.1016/0021-9150(89)90092-0

Atherosclerosis

Volume 76, Issues 2–3, April 1989, Pages 95-101

https://doi.org/10.1016/0021-9150(89)90092-0 Get rights and content

Abstract

We performed an in vitro study to assess injury to vascular endothelial cells by platelets. Cultured endothelial cells isolated from fetal bovine aorta were used. Addition of human platelets, activated by collagen or lysed by sonication, to the culture dish resulted in dose- and time-dependent damage to the cells as estimated by [³H]adenine release. Analysis of [³H]adenine nucleotides by thin-layer chromatography on PEI-cellulose revealed decreased intracellular ATP content in the cells treated with platelet lysate. The medium contained AMP and adenosine, the latter increasing following the treatment of the cells. Of the substances released by the activated platelets, thromboxane A₂ (TxA₂) and serotonin caused cell damage. Platelet-derived growth factor (PDGF), however, did not damage the endothelial cells up to a concentration of 200 ng/ml. Pretreatment of the cells with methysergide (10⁻⁶ M) or ONO 3708 (10⁻⁵ M), a TxA₂ antagonist, only partially prevented the damage, while ZK 36374 (10⁻⁶ M), a prostacyclin analog, and 3-isobutyl-l-methylxanthine (IBMX; 10⁻³ M), a phosphodiesterase inhibitor, potently inhibited injury. We conclude that the substances released from activated platelets may injure endothelial cells in an additive or synergistic manner and that agents which produce effects that elevate cyclic AMP levels may protect the cells from damage induced by the platelets.

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With increasing social competition, the effect of psychological stress on related diseases cannot be neglected, such as cardiovascular, cerebrovascular, endocrine, and mental diseases. When humans are under chronic stress, permanent changes in their physiological responses can lead to disease through immune responses (Rief et al., 2001) and platelet activation (Kishi and Numano, 1989). Stimulation of adrenalin excretion by psychological stress can increase circulating formaldehyde levels, which may be involved in the initiation of endothelial injury, and, subsequently, angiopathy (Yu et al., 1997).
Laminaria japonica is a popular seafood and medicinal plant in China. Laminaria japonica is used in traditional Chinese medicine to treat and prevent hypertension and edema.
The vascular protective activity and mechanism of sulfated polysaccharides were studied in adrenalin-induced vascular endothelial damage in rats after psychological stress (PS). Vehicle (sham and PS groups), sulfated polysaccharide from Laminaria japonica (LP; 1 mg/kg and 5 mg/kg) and enoxaparin sodium (1 IU/kg, reference drug) were all administered for 10 days. Behavioral changes were recorded. Plasma levels of adrenalin, cortisol, monoamine oxidase (MAO), semicarbazide-sensitive amine oxidase (SSAO), formaldehyde, H₂O₂, nitric oxide (NO), endothelin-1 (ET-1), 6-keto-prostaglandin F_1a (6-keto-PGF_1a), and thromboxane B₂ (TXB₂) were measured. Endothelium-dependent relaxation of the thoracic aorta was measured and transmission electron microscopy of aortic vessels was performed.
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Honokiol is a bioactive compound extracted from the Chinese medicinal herb Magnolia officinalis. We recently demonstrated that honokiol inhibited arterial thrombosis through stimulation of prostacyclin (PGI₂) generation and endothelial cell protection. The current study is designed to investigate its mechanism of stimulation of PGI₂ generation and cell protection. 6-keto-PGF1α, the stable metabolite of PGI₂, in the media of rat aortic endothelial cells was measured with radioimmunoassay kits. Indomethacin, an inhibitor of cyclooxygenase (COX) and tranylcypromine, a prostacyclin synthease inhibitor were used to ascertain the target enzyme affected by honokiol. Prostacyclin synthease protein levels in endothelial cells were determined by Western blot analysis using an anti-PGI₂ synthease rabbit polyclonal antibody. Flow cytometry was used to quantify the apoptotic cells and spectrophotometry was used to test the caspase-3 activity. Honokiol (0.376–37.6 μM) increased the level of 6-keto-PGF1α in the media of normal endothelial cells. It counteracted the inhibitory effect of tranylcypromine on the PGI₂ generation, but did not influence the effect of indomethacin; evidently, honokiol up-regulated the expression of prostacyclin synthease in the endothelial cells. These effects showed perfect concentration-dependent behavior. In addition, at lower concentration (0.376–3.76 μM), honokiol significantly decreased the percentage of apoptotic endothelial cells induced by oxidized low-density lipoprotein (ox-LDL) and significantly lowered the activity of caspase-3 stimulated by ox-LDL. A high dose of honokiol (37.6 μM), however, failed to influence either of them. In conclusion, honokiol augments PGI₂ generation in normal endothelial cells; its effect on PGI₂ generation attributes to up-regulation of prostacyclin synthease expression; its cell protection may be correlated with its inhibition on apoptosis of endothelial cells. These findings have partly revealed the molecular mechanism of honokiol on inhibiting arterial thrombosis.
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The neuropeptide substance P induces many events associated with neurogenic inflammation; it alters vascular permeability and calibre, and releases a relaxant factor from vascular endothelium; it also stimulates the release of interleukins, tumour necrosis factor and arachidonic acid (Lotz et al., 1988). There is some evidence that 5-HT itself may have a direct effect on the endothelium when released by activated platelets (Kishi and Numano, 1989). In addition to these direct effects, oxidative deamination of 5-HT by SSAO generates hydrogen peroxide (Callingham and Barrand, 1987), which has many potential functions within cells (Parchment, 1993; Stangel et al., 1996).
The semicarbazide-sensitive amine oxidase (EC 1.4.3.6; SSAO) from crude homogenates of human dental pulp was shown to catalyse the oxidative deamination of 5-hydroxytryptamine (serotonin; 5-HT) with a K_m of 318±52 μM. In this respect the human enzyme resembles that in pig dental pulp, but differs from SSAO in all other tissues studied, which are inactive towards 5-HT. A method is described for obtaining intact dental pulp in which the anatomical details are preserved. Extracted teeth are frozen in dry ice and later defrosted rapidly before being fractured in a mechanical vice, facilitating pulp removal. Immunohistochemistry showed SSAO in the odontoblast layer, nerve fibres and blood vessels. The presence of SSAO in nerves in dental pulp appears to be unique. Tryptophan hydroxylase, a key enzyme in 5-HT synthesis, was also demonstrated in nerves and the odontoblast layer of human dental pulp.
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Transforming growth factor-β₁ released from platelet α-granules may preserve endothelial functions in injured vessels. However, direct evidence is lacking regarding how this cytokine modifies the antithrombotic function of injured endothelial cells. We performed an in vitro study to investigate the effects of transforming growth factor-β₁ on platelet functions in the presence of cultured endothelial cells exposed to anoxia-reoxygenation injury. Cultured bovine aorta endothelial cells were placed in an anoxic chamber (0.5% O₂, 5% CO₂) for 60 minutes followed by a 90-minute reoxygenation. Collagen (2 μg/mL)-induced platelet aggregation (10⁸ platelets/mL), as determined by impedance aggregometry, was potently inhibited in the presence of control endothelial cells (17.4±3.3 Ω) at a concentration of 5×10⁴ cells/mL, as compared to their absence (68.2±2.2 Ω). Inhibition of platelet aggregation was attenuated in endothelial cells exposed to anoxia-reoxygenation (54.6±2.5 Ω). However, preincubation of endothelial cells with transforming growth factor-β₁ (1.0 ng/mL) for 16 hours partially recovered the inhibitory capability of platelet aggregation by injured endothelial cells (40.6±3.8 Ω). Cell viability, confirmed by a trypan blue dye exclusion test, was similar (93–96%), including control, 1.0 ng/mL transforming growth factor-β₁- and/or anoxia-reoxygenation-pretreated cells. The capability of platelet inhibition was restored when the endothelial cells were preincubated for 4 hours or more. Restoration of antiplatelet capacity in endothelial cells by transforming growth factor-β₁ was suggested to be due to several mechanisms, including an increase in nitric oxide synthase activity, up-regulation of prostacyclin release, and restoration of adenosine triphosphate diphosphohydrolase activity, which was attenuated by anoxia-reoxygenation pretreatment. In summary, transforming growth factor-β₁ released from activated platelets may play a compensatory role in the preservation of endothelial functions to inhibit platelet activation.

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Research paperIn vitro study of vascular endothelial injury by activated platelets and its prevention

Abstract

Atherosclerosis

Atherosclerosis

Atherosclerosis

J. Biol. Chem.

J. Biol. Chem.

Lancet

Am. J. Cardiol.

Eur. J. Pharmacol.

The pathogenesis of atherosclerosis — an update

N. Engl. J. Med.

Interaction of platelets and leukocytes with vascular endothelium: In vitro studies

Ann. N.Y. Acad. Sci.

Arterial wall shear and distribution of early atheroma in man

Nature

Chemotherapy of atherosclerosis

Jpn. Cir. J.

Research paper
In vitro study of vascular endothelial injury by activated platelets and its prevention