Biomedical subjects

M A Gimbrone

Publications and source records attributed to M A Gimbrone.

At least 145 records · Page 8Linked to original sources

PubMed1986

Phorbol diester modulates alpha-adrenergic receptor-coupled calcium efflux and alpha-adrenergic receptor number in cultured vascular smooth muscle cells.

The phorbol ester 12-O-tetradecanoyl phorbol-13-acetate was used to probe the role of protein kinase-C in the modulation of alpha-adrenergic receptor-coupled calcium efflux in cultured vascular smooth muscle cells derived from rabbit aorta. Exposure of cells to 12-O-tetradecanoyl phorbol-13-acetate for 6 minutes caused a concentration-related (maximum effect at greater than or equal to 10 nM) increase in calcium-45 efflux from preloaded cells. Maximum calcium-45 efflux stimulated by 12-O-tetradecanoyl phorbol-13-acetate was equivalent to maximum norepinephrine-stimulated calcium-45 efflux, and maximally effective concentrations of 12-O-tetradecanoyl phorbol-13-acetate and norepinephrine together were no more potent than either drug alone. Exposure of cells to 12-O-tetradecanoyl phorbol-13-acetate for periods of 1-24 hours resulted in complete loss of norepinephrine-stimulated calcium-45 efflux and a much slower, concentration-related reduction in alpha-adrenergic receptor number, with a maximum reduction of 50-60% at 12-O-tetradecanoyl phorbol-13-acetate concentrations greater than or equal to 10 nM. Twenty-four hours of exposure to 12-O-tetradecanoyl phorbol-13-acetate (10 nM) and norepinephrine (10 microM) together caused no greater reduction in [3H]prazosin binding than did norepinephrine alone. 12-O-tetradecanoyl phorbol-13-acetate had no effect on [3H]prazosin-binding affinity. These data suggest an important role for protein kinase-C in both the acute excitation-contraction coupling of vascular smooth muscle alpha-adrenergic receptors, and in the long-term modulation of vascular alpha-adrenergic receptor responsiveness.

Animals↗

PubMed1986

Regulation of the fibrinolytic system of cultured human vascular endothelium by interleukin 1.

We examined the effects of human interleukin 1 (IL-1) on the production of fibrinolytic components by cultured human vascular endothelium. Conditioned media collected from IL-1-treated (5 U/ml, 24 h) monolayers exhibited decreased tissue-type plasminogen activator (tPA) activity and increased plasminogen activator inhibitor (PAI) activity, as assessed by fibrin and reverse fibrin-autography. Quantitative immunological assays revealed a 35% decrease in tPA antigen and a 360% increase in active PAI antigen, after incubation for 24 h with 0.6 U/ml IL-1. Maximal effects (approximately 50% decrease in tPA antigen; 400-800% increase in active PAI antigen) were observed with 2.5-5 U/ml IL-1. Changes in tPA and PAI reached a maximum at approximately 24 h and persisted for greater than 48 h. IL-1 induction of endothelial procoagulant activity was more rapid and transient, peaking by 6 h and subsiding by 24 h. Natural monocyte-derived IL-1 and two species of recombinant IL-1 had comparable effects. Heat and polymyxin-B treatments differentiated IL-1 actions from those of endotoxin, which promoted similar endothelial alterations. IL-1 effects on endothelial procoagulant and fibrinolytic activities may contribute to the generation and maintenance of fibrin in pathophysiological settings in vivo.

Blood Coagulation Tests↗

PubMed1986

Incorporation of platelet and leukocyte lipoxygenase metabolites by cultured vascular cells.

When arachidonic acid metabolism is studied during platelet-endothelial interactions in vitro, the predominant cyclooxygenase end products of each cell type (thromboxane B2 and 6-keto-prostaglandin-F1 alpha, respectively) are essentially completely recovered in the cell-free supernatants of these reactions. In contrast, 50% of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), the major lipoxygenase metabolite from platelets, is released into the cell-free supernatant. In investigating the basis of this observation, we have found that platelet lipoxygenase metabolites were generated to the same extent during these coincubations but became rapidly incorporated into the endothelial cells. The endothelial cell-associated 12-HETE was present not only as free fatty acid, but was also incorporated into cellular phospholipids and triglycerides. When purified 3H-12-HETE, 3H-5-HETE (the major hydroxy acid lipoxygenase product of leukocytes), and 3H-arachidonic acid (the common precursor of these metabolites) were individually incubated with suspensions of cultured bovine aortic endothelial cells or smooth muscle cells, different patterns of intracellular lipid distribution were found. In endothelial cells, 12-HETE was incorporated equally into phospholipids and triglycerides, whereas 5-HETE was incorporated preferentially into triglycerides, and arachidonic acid was incorporated into phospholipids. In smooth muscle cells, both 12-HETE and 5-HETE showed more extensive incorporation into triglycerides. The rapid and characteristic incorporation and esterification of platelet and leukocyte monohydroxy fatty acid lipoxygenase products by endothelial and smooth muscle cells suggests a possible physiologic role for these processes in regulating vascular function.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed1985

Phorbol ester and 1-oleoyl-2-acetylglycerol inhibit angiotensin activation of phospholipase C in cultured vascular smooth muscle cells.

Angiotensin II acts on cultured rat aortic vascular smooth muscle cells (VSMC) to induce the rapid, phospholipase C-mediated generation of inositol trisphosphate from phosphatidylinositol 4,5-bisphosphate and mobilization of intracellular Ca2+. sn-1,2-Diacylglycerol, the other major product of inositol phospholipid breakdown, is known to activate protein kinase C, but its role in angiotensin II action on VSMC has not been defined. We report herein that, in cultured VSMC prelabeled with [3H]myoinositol, brief incubations (2-5 min) with 4 beta-phorbol 12-myristate 13-acetate (PMA) (1-100 nM) or 1-oleoyl-2-acetylglycerol (10-100 microM), two potent activators of protein kinase C, inhibit subsequent angiotensin II (100 nM)-induced increases in phosphatidylinositol 4,5-bisphosphate breakdown and inositol trisphosphate formation. In addition, pretreatment of VSMC with either PMA (IC50 approximately 1 nM) or 1-oleoyl-2-acetylglycerol (IC50 approximately 7.5 microM) also markedly inhibits angiotensin II (1 nM)-stimulated increases in cytosolic free Ca2+, as measured with the calcium-sensitive fluorescent indicator quin 2, or 45Ca2+ efflux. Neither PMA nor 1-oleoyl-2-acetylglycerol initiated phosphatidylinositol 4,5-bisphosphate breakdown or Ca2+ flux by itself. PMA treatment (10 nM, 5 min) did not influence the number or affinity of 125I-angiotensin II-binding sites in intact cells. These data suggest that one function of angiotensin II-generated sn-1,2-diacylglycerol in vascular smooth muscle may be to modulate, by protein kinase C-mediated mechanisms, angiotensin II receptor coupling to phospholipase C.

Aminoquinolines↗

PubMed1985

Renin expression by vascular endothelial cells in culture.

Cultured bovine aortic endothelial cells were examined for renin activity by biochemical, immunological, and immunohistochemical techniques. When cell sonicates were incubated with renin substrate, linear generation of angiotensin I was observed (1.12 +/- 0.2 ng angiotensin I/10(6) cells per hr). The effect of pH on this activity was similar to that of bovine renal renin, and renin antibodies inhibited a large portion of the enzymatic activity. Furthermore, immunofluorescence microscopy with antirenin antisera confirmed the presence of renin within these cells. Biosynthetic radiolabeling, followed by immunoprecipitation, demonstrated de novo synthesis of a renin precursor in the endothelial cells, which was processed to a more mature protein. Thus, bovine aortic endothelial cells in culture contain and biosynthesize renin, a key component of the renin-angiotensin system. The expression of renin activity by endothelium may contribute to the local regulation of vascular tone.

Angiotensin I↗

PubMed1985

Epidermal growth factor, a vascular smooth muscle mitogen, induces rat aortic contraction.

Atherosclerotic arteries have enhanced reactivity to vasoconstrictors, which suggests that features of the atherosclerotic process itself may result in this abnormal responsiveness. Since vascular smooth muscle proliferation is a prominent feature of atherosclerosis, we postulated that vasoactive agonists and smooth muscle mitogens may share certain common cellular mechanisms of action which potentially contribute to this hyperreactivity. To test this hypothesis, we studied the effects of epidermal growth factor (EGF), a well-characterized mitogen, on rat aortic vascular smooth muscle, both in intact aortic strips and in culture. EGF caused contraction (EC50 = 19 nM) of rat aortic strips which maximally was equivalent to 40% of that induced by angiotensin II, a potent vasoconstrictor. EGF increased 45Ca efflux (EC50 = 3 nM) from cultured rat aortic smooth muscle cells, which was an effect shared by angiotensin II and thought to reflect increased cytosolic-free calcium concentration. EGF (7.5 nM) also stimulated growth of these cultured cells to the same extent as 10% calf serum. These results demonstrate that EGF is both a vasoconstrictor and mitogen for rat aortic smooth muscle cells. The similarities in the effects of EGF and angiotensin II suggest that certain common intracellular mechanisms of action may exist for vasoactive agonists and growth factors which may contribute to the altered vasoreactivity of atherosclerotic vessels.

Animals↗

PubMed1985

Interleukin 1 acts on cultured human vascular endothelium to increase the adhesion of polymorphonuclear leukocytes, monocytes, and related leukocyte cell lines.

Increased leukocyte adhesion to the endothelial lining of blood vessels is an essential event in inflammation and the pathogenesis of certain vascular diseases. We have studied the effect of interleukin 1 (IL-1), an inflammatory/immune mediator, on endothelial-leukocyte adhesion using quantitative in vitro assays. Selective pretreatment of cultured human umbilical vein endothelial monolayers with IL-1 (5 U/ml, 4 h) resulted in an 18.3 +/- 2.6-fold increase in human peripheral blood polymorphonuclear leukocyte (PMN) adhesion (mean +/- SEM, n = 16) and a 2.6 +/- 0.3-fold increase in monocyte adhesion (n = 7) over basal levels. IL-1-treated endothelial monolayers also supported increased adhesion of the promyelocytic cell line HL-60 and the monocytelike cell line U937 (33.0 +/- 6.0-fold, n = 6 and 4.9 +/- 0.5-fold, n = 15, respectively). In contrast, selective IL-1 pretreatment of leukocytes, or the addition of IL-1 during the adhesion assay, did not alter endothelial-leukocyte adhesion. Conditioned medium from IL-1-treated endothelial cultures also did not promote leukocyte adhesion to untreated monolayers. IL-1 induction of endothelial adhesivity was concentration dependent (maximum, 10 U/ml), time dependent (peak, 4-6 h), and reversible, was blocked by cycloheximide (10 micrograms/ml) or actinomycin D (5 micrograms/ml) but not by acetylsalicylic acid (100 microM), and occurred without detectable endothelial cell damage. IL-1 treatment of SV40-transformed human endothelial cells and dermal fibroblasts did not increase their adhesivity for leukocytes. These data suggest that IL-1 can act selectively on human vascular endothelium to increase its adhesivity for circulating blood leukocytes, and thus to localize leukocyte-vessel wall interactions at sites of inflammation in vivo.

Cell Adhesion↗

PubMed1985

Cultured vascular smooth muscle cells: an in vitro system for study of alpha-adrenergic receptor coupling and regulation.

Cultured vascular smooth muscle cells derived by enzymatic dissociation of rabbit aortic media were used for study of alpha 1-adrenergic receptors (AAR) and receptor-coupled calcium flux. AAR were characterized by binding of the alpha 1-selective radioligand [3H]prazosin, and norepinephrine-stimulated 45calcium efflux was measured as an index of AAR-mediated calcium flux. The binding of [3H]prazosin to a crude cellular homogenate was to a single, saturable site of high affinity (Kd = 0.15 nM, Bmax = 75-125 fmol/mg protein), which was stereo-specific and exhibited the appropriate potency order for competition by agonists. Prazosin (Kd = 0.07 nM) was approximately 3000-fold more potent than the alpha 2-selective antagonist yohimbine (Kd = 222 nM), both of which exhibited Hill coefficients of one, indicative of binding to a single receptor type. In intact cells, norepinephrine caused a concentration-related (EC50 = 100 nM) increase in 45calcium efflux which was blocked by low concentrations of prazosin (IC50 approximately equal to 0.1 nM), but not yohimbine (IC50 greater than 100 nM). An alpha 1-selective concentration of prazosin (100 nM) fully blocked norepinephrine-stimulated 45calcium efflux, and therefore, it appears that this effect does not involve alpha 2-adrenergic receptors. Treatment of cultured cells with 1-norepinephrine for 48 h caused a concentration-related decrease in both AAR density and maximum norepinephrine-stimulated 45calcium efflux. This cultured vascular smooth muscle cell system provides several advantages for the study of alpha-adrenergic receptor coupling and regulation.

Animals↗

PubMed1985

Nonlinear relationship between alpha 1-adrenergic receptor occupancy and norepinephrine-stimulated calcium flux in cultured vascular smooth muscle cells.

To determine the relationship between vascular alpha 1-adrenergic receptor occupancy and receptor-coupled calcium flux, we have studied [3H]prazosin binding and l-norepinephrine-induced 45Ca efflux in cultured vascular smooth muscle cells isolated from the rabbit aorta. In a crude cellular homogenate, [3H]prazosin bound to a single high affinity site (Kd = 0.096 nM; Bmax = 105 +/- 15 fmol/mg of protein), whereas l-norepinephrine (NE) binding was best described by a two-site model with 43 +/- 8% of sites of high affinity (KH = 92 +/- 3 nM) and 57 +/- 7% of sites of low affinity (KL = 7460 +/- 4330 nM). NE-stimulated 45Ca efflux was concentration-dependent (EC50 = 108 nM) and potently inhibited by prazosin (IC50 = 0.15 nM), but not yohimbine (no inhibition at 10 microM). For the total receptor pool identified by [3H]prazosin binding, the relationship between receptor occupancy by NE and NE-stimulated 45Ca efflux was markedly nonlinear, such that 50% of maximum NE-stimulated efflux occurred with occupancy of only approximately 7% of receptors. Likewise, following irreversible inactivation of 69 +/- 5% of receptors by phenoxybenzamine, maximal NE-stimulated 45Ca efflux was decreased by only 8 +/- 2%. These two experimental approaches provide direct evidence for the presence in cultured rabbit aortic smooth muscle cells of a sizable pool of alpha 1-adrenergic receptors in excess of those needed for maximum NE-stimulated 45Ca efflux. This evidence of "spare" receptors, together with the finding of two affinity states of agonist binding, raises the possibility of functional heterogeneity of alpha 1-adrenergic receptors in this system.

Animals↗

PubMed1985

Interleukin-1 activation of vascular endothelium. Effects on procoagulant activity and leukocyte adhesion.

Interleukin-1 (IL-1), an inflammatory/immune mediator, acts directly and selectively on cultured human vascular endothelial cells to alter two important functional properties. First, IL-1 induces endothelial cell biosynthesis and surface expression of a tissue factor-like procoagulant activity. Second, IL-1 dramatically increases the adhesiveness of the endothelial cell surface for human peripheral blood polymorphonuclear leukocytes (6-42-fold increase) and monocytes (2-5-fold increase), as well as the related leukocyte cell lines HL-60 and U937. These IL-1 effects are concentration-dependent (maximum, 5-10 U/ml), time-dependent (peak 4-6 hours), and reversible. Cycloheximide and actinomycin D block these IL-1 actions on endothelium, which suggests the requirement for de novo protein synthesis. Human-monocyte-derived IL-1, cell-line--derived IL-1, and recombinant IL-1 exhibited comparable biologic activities in our assays, whereas two other mediators, IL-2 and immune interferon, were without effect. IL-1 stimulated procoagulant activity and leukocyte adhesion in human endothelial cells cultured from both umbilical veins and adult saphenous veins but not in other cultured cell types, including SV-40-transformed human endothelial cells and human dermal fibroblasts. Similar actions of IL-1 on vascular endothelium in vivo may contribute to the development of intravascular coagulation and enhanced leukocyte--vessel wall adhesion at sites of inflammation.

Adhesiveness↗

PubMed1984

Thrombospondin interactions with fibronectin and fibrinogen. Mutual inhibition in binding.

Thrombospondin synthesized and secreted by human endothelial cells in culture binds specifically to fibronectin immobilized on Sepharose beads. It can also bind to immobilized platelet-derived thrombospondin but not to immobilized gelatin or albumin. These interactions are not dependent on the presence of divalent cations or of other secreted materials. Purified platelet thrombospondin binds to fibronectin and fibrinogen immobilized on plastic surfaces with dissociation constants of 1.12 +/- 0.37 X 10(-7) M and 1.27 +/- 0.41 X 10(-7) M respectively, and to thrombospondin immobilized on plastic with dissociation constant of 4.82 +/- 1.01 X 10(-7) M. The affinities of interaction are not significantly affected by removal of divalent cations. Soluble fibrinogen inhibits binding of thrombospondin to fibronectin regardless of which of the latter two is surface-bound. Thrombospondin-fibronectin interaction is also inhibited by soluble thrombospondin. The binding of soluble thrombospondin to surface-bound fibrinogen is inhibited both by soluble fibronectin and soluble fibrinogen. These results suggest that thrombospondin plays a role both in platelet-platelet aggregation and in platelet-substratum adhesion, and that it may also take part in the construction of the extracellular matrix.

Binding, Competitive↗

PubMed1984

Interleukin 1 (IL-1) induces biosynthesis and cell surface expression of procoagulant activity in human vascular endothelial cells.

Human monocyte-derived interleukin 1 (IL-1) was found to be a potent inducer of procoagulant activity in cultured human vascular endothelium. IL-1-induced human umbilical vein endothelial cell procoagulant activity (HEC-PCA) was transiently expressed, manifest in intact cell monolayers, and required protein synthesis. Data obtained with coagulation factor-deficient plasma and a goat anti-human apoprotein III antiserum suggested that most, if not all, of IL-1-induced endothelial cell procoagulant activity is tissue factor-like. IL-1 induction of HEC-PCA may be important in the pathogenesis of intravascular coagulation in a variety of immunological and inflammatory conditions.

Blood Coagulation↗

PubMed1984

Interactions of T lymphocytes with human vascular endothelial cells: role of endothelial cells surface antigens.

We have studied the interactions of peripheral blood T lymphocytes with cultured human vascular endothelial cells, focusing upon endothelial cell surface antigens important for T cell recognition. Under standard culture conditions endothelial cells express class I but not class II major histocompatibility complex (MHC) antigens. However, class II antigens may be induced by activated T cells or T cell products, including the lymphokine immune interferon. Immune interferon concomitantly increases class I antigen expression and causes a change in cell shape. In addition to vascular endothelial cells, we have found that vascular smooth muscle cells and human dermal fibroblasts may also be induced by immune interferon to express class II antigens. All known human class II antigens are induced (i.e. HLA-DR, DC and SB) as is the associated invariant chain. Induced antigen expression in these cells is stable over several days, although mRNA levels decline rapidly upon withdrawal of interferon. Vascular and stromal cell class II antigens are functional, in that they can be recognized by cytolytic and helper T cell clones. Several non-MHC antigens are also involved in the recognition of endothelial and stromal cells by T cells. We propose a model for the role of inducible class II molecules on endothelium and stromal cells in vivo: The induction of class II MHC antigens on endothelial cells, locally mediated by activated T cells, enables endothelium to present an immunogenic cell surface structure, comprised of antigen plus self class II polymorphic determinants, which in turn, serves to recruit additional antigen-specific T cells from the circulation into the site of a developing cell mediated immune response. Class II molecules on stromal cells, also induced locally at the site of a developing response, confers immune accessory function on these cells and may serve to augment and sustain a T cell response.

Antigen-Presenting Cells↗

PubMed1984

Immune interferon activates multiple class II major histocompatibility complex genes and the associated invariant chain gene in human endothelial cells and dermal fibroblasts.

Immune interferon (IFN-gamma) increases the surface expression of HLA-A,B antigens and induces the surface expression of HLA-DR antigens on vascular endothelial cells and dermal fibroblasts. Here we report that IFN-gamma induces parallel expression of two other class II major histocompatibility complex (MHC) antigens, SB and DC. Maximal surface expression of all three antigens is reached in 4-6 days, and HLA-DR and -SB are induced to a higher level of expression than HLA-DC. For all three class II antigens, induction is marked by the de novo appearance of detectable transcripts of class II heavy and light chains and of the non-MHC-encoded invariant chain, suggestive of the transcription of multiple previously silent genes. Class I message levels and antigen expression are also increased by IFN-gamma at similar rates but from initial levels that are 50% of maximal. After removal of IFN-gamma, class II antigen expression persists for at least 4 days, while mRNA levels decrease rapidly. The parallel induction and persistence of the several class II MHC antigens may be important in conferring immune accessory function on vascular and stromal cells.

Endothelium↗

PubMed1984

Myosin in cultured vascular smooth muscle cells: immunofluorescence and immunochemical studies of alterations in antigenic expression.

Vascular smooth muscle cells (VSMC) in the rat mesenteric artery show specific immunofluorescent staining with antisera against purified human uterine myosin (ASMM) but not human platelet myosin (APM). However, in primary cultures produced by enzymatic dissociation of this vessel, VSMC stain specifically with both ASMM and APM within 5 h after plating and throughout growth to confluence (4-10 d). In confluent cultures, APM staining remains bright while ASMM staining is reduced in intensity in most cells. In contrast, cellular myosin content, determined by quantitative SDS PAGE, is comparable in confluent and growing cultures. Immunoprecipitation of high salt extracts of cultured VSMC with ASMM and APM yields myosins with the same mobilities on SDS PAGE. When serial, exhaustive precipitations are performed with one antiserum, followed by reprecipitation with the other, myosin in subconfluent and confluent VSMC cultures is exhaustively precipitated by either antiserum, thus indicating complete immunological cross-reactivity. These results might be explained by synthesis of a new myosin isoform reactive with both ASMM and APM. However, the development of APM staining in cultured VSMC did not require protein synthesis. Therefore, it is more likely that the changes in immunofluorescent staining observed in vitro reflect conformational alterations, perhaps related to cytoskeletal rearrangements. These changes in myosin antigenic expression may be relevant to the problem of VSMC phenotypic modulation both in vitro and in vivo.

Animals↗

PubMed1984

Regulation of myocardial and vascular alpha-adrenergic receptor affinity. Effects of guanine nucleotides, cations, estrogen, and catecholamine depletion.

The threshold sensitivity of cardiovascular tissues to alpha-adrenergic stimulation is determined largely by the affinity of alpha-adrenergic receptors for agonists. To determine whether changes in alpha-adrenergic receptor affinity could contribute to the regulation of cardiac and vascular responsiveness, we used the alpha-adrenergic-selective radioligands, [3H]prazosin, [3H]-WB-4101, and [3H]rauwalscine, to study and contrast the determinants of alpha-adrenergic receptor affinity in myocardium and vascular smooth muscle from the rat. In both tissues, l-epinephrine binding to the alpha 1-adrenergic receptor describes a shallow curve suggesting more than one affinity state. Computer analysis of binding to myocardial alpha 1-receptors indicates that 15% are of high affinity (Kd = 11 nM) and 85% are of low affinity (Kd = 400 nM). Expression of high affinity sites is magnesium dependent (maximum effect, 5-10 mM), and suppressed by the guanosine 5'-triphosphate analogue Gpp(NH)p (maximum effect, 1 mM) and sodium (maximum effect, 100-200 mM). In vascular smooth muscle, agonist-binding curves are also shallow and exhibit a similar response to that of Gpp(NH)p. Basal receptor affinity in myocardium is significantly higher (5.4-fold) than in vascular smooth muscle. Unlike vascular smooth muscle, in which alpha 1-adrenergic receptor affinity is increased by estrogen or reserpine treatment of the animal, the receptor in myocardium is unaffected by these treatments. In vascular smooth muscle, following reserpine-induced increase in alpha-adrenergic receptor affinity, the Gpp(NH)p effect is still present. Thus, alpha 1-adrenergic receptors in both myocardium and vascular smooth muscle exist in two affinity states and are subject to regulation by several factors, including guanine nucleotides, mono- and divalent cations, tissue of origin, sex hormones, and the level of sympathetic stimulation. Potentially, alterations in alpha 1-adrenergic receptor affinity, independent of a change in receptor number, may play an important role in the regulation of cardiovascular tissue responsiveness to catecholamines.

Animals↗

PubMed1984

Unidirectional transfer of prostaglandin endoperoxides between platelets and endothelial cells.

An important determinant of platelet-vessel wall interactions is the local balance of production of endothelial prostacyclin (PGI2) and platelet thromboxane (TX) A2, labile eicosanoids with opposing effects on hemostasis. Disputed evidence suggests that platelet-derived prostaglandin endoperoxide intermediates may be utilized as substrates for vascular PGI2 synthesis. Using several different approaches, we have found that platelets can transfer endoperoxides to cultured endothelial cells for efficient conversion to PGI2, but a reciprocal transfer of endothelial endoperoxides for utilization by platelet thromboxane synthetase does not occur under the same experimental conditions. However, platelets can utilize arachidonic acid released by endothelial cells for lipoxygenase metabolism. We have directly demonstrated the production of [3H]6-keto-PGF1 alpha (the breakdown product of [3H]PGI2) by aspirin-treated endothelial cells in the presence of platelets stimulated with [3H]arachidonic acid. In coincubation experiments using either arachidonate or ionophore A23187 as a stimulus, radioimmunoassay of the net production of arachidonic acid metabolites showed that 6-keto-PGF1 alpha generation by aspirin-treated endothelial cells in the presence of platelets may actually exceed its generation by uninhibited endothelial cells alone. In functional assays, platelet aggregation was inhibited in the presence of aspirin-treated endothelial cells after stimulation with either arachidonate or ionophore A23187. In contrast, the inverse experiments, using aspirin-treated platelets and uninhibited endothelial cells, failed to demonstrate platelet utilization of endothelial endoperoxides for TXA2 production by any of the above methods. These studies thus provide evidence that efficient unidirectional transfer and utilization of platelet-derived endoperoxides for endothelial PGI2 production can occur. This process may serve to amplify PGI2 generation adjacent to areas of vascular injury and permit tight localization of platelet plug formation at these sites.

Animals↗

PubMed1984

Influence of hemodynamic forces on vascular endothelial function. In vitro studies of shear stress and pinocytosis in bovine aortic cells.

The relationships between fluid shear stress, a physiologically relevant mechanical force in the circulatory system, and pinocytosis (fluid-phase endocytosis) were investigated in cultured bovine aortic endothelial cells using a specially designed apparatus. Continuous exposure to steady shear stresses (1-15 dyn/cm2) in laminar flow stimulated time- and amplitude-dependent increases in pinocytotic rate which returned to control levels after several hours. After 48 h continuous exposure to steady shear stress, removal to static conditions also resulted in a transient increase in pinocytotic rate, suggesting that temporal fluctuations in shear stress may influence endothelial cell function. Endothelial pinocytotic rates remained constant during exposure to rapidly oscillating shear stress at near physiological frequency (1 Hz) in laminar flow. In contrast, however, a sustained elevation of pinocytotic rate occurred when cells were subjected to fluctuations in shear stress amplitude (3-13 dyn/cm2) of longer cycle time (15 min), suggesting that changes in blood flow of slower periodicity may influence pinocytotic vesicle formation. As determined by [3H]thymidine autoradiography, neither steady nor oscillating shear stress stimulated the proliferation of confluent endothelial cells. These observations indicate that: (a) alterations in fluid shear stress can significantly influence the rate of formation of pinocytotic vesicles in vascular endothelial cells, (b) this process is force- and time-dependent and shows accommodation, (c) certain patterns of fluctuation in shear stress result in sustained elevation of pinocytotic rate, and (d) shear stresses can modulate endothelial pinocytosis independent of growth stimulation. These findings are relevant to (i) transendothelial transport and the metabolism of macromolecules in normal endothelium and (ii) the role of hemodynamic factors in the localization of atherosclerotic lesions in vivo.

Animals↗