© 1997 American Heart Association, Inc.
Articles |
Antifibrinolytic Properties of the Vascular Wall
Dependence on the History of Smooth Muscle Cell Doublings In Vitro and In Vivo
From the Departments of Cardiology (G.C., G.M., K.H.), Vascular Biology and Thrombosis Research (G.C., P.H., C.K., J.W., B.R.B.), and Cardiothoracic Surgery (G.L.), University of Vienna, Austria.
Correspondence to Günter Christ, MD, Department of Vascular Biology and Thrombosis Research, University of Vienna, Schwarzspanierstr17, A-1090 Vienna, Austria.
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Abstract |
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Abstract Increased expression of plasminogen activator inhibitor-1 (PAI-1) mRNA in atherosclerotic human arteries suggests a linkage between PAI-1 gene expression and cellular proliferation, the fundamental feature of atherosclerosis. To investigate whether smooth muscle cell (SMC) proliferation influences overall fibrinolytic properties of the vascular wall, we examined the effect of serial in vitro passaging of human SMCs on tissue plasminogen activator (TPA) and PAI-1 synthesis levels as well as the ability to modulate TPA and PAI-1 synthesis of human umbilical vein endothelial cells (HUVECs). As in vivo correlates for such late-passage cells in culture, SMCs derived from human atherosclerotic plaques were used, because they are thought to have already undergone numerous cell doublings. We observed an increase of PAI-1 secretion (from 591±106 to 2952±290 ng PAI-1·105 cells-1·24 h-1) with a concomitant fourfold to fivefold increase of PAI-1 mRNA levels, as well as a decrease of TPA secretion (from 118±34 to 8±1.3 ng TPA·105 cells-1·24 h-1) and a twofold to threefold decrease of TPA mRNA levels with increasing in vitro passage number (from passage 3 to 11) of normal pulmonary artery smooth muscle cells (PASMCs) (P<.05). SMCs derived from atherosclerotic plaques of coronary arteries (CASMCs) displayed higher levels of PAI-1 antigen synthesis (3093±507 ng PAI-1·105 cells-1·24 h-1) with an approximately twofold increase of PAI-1 mRNA levels, as well as decreased levels of TPA antigen synthesis (10±1.6 ng TPA·105 cells-1·24 h-1) with an
1.5- to 2-fold decrease of TPA mRNA levels in passage 1, compared with their counterparts derived from normal-appearing arterial tissue of the same vessel (1794±525 ng PAI-1·105 cells-1·24 h-1; 17±5 ng TPA·105 cells-1·24 h-1) (P<.001; P<.01). Incubation of HUVEC cultures with the 24-hour conditioned media (CM) of early-passage PASMCs decreased endothelial PAI-1 antigen synthesis by 42% (P<.001) and endothelial PAI-1 mRNA levels about twofold to threefold (P<.001), whereas by incubation with the 24-hour CM of late-passage PASMCs, endothelial PAI-1 antigen synthesis was upregulated by 68% (P=.001), with a concomitant twofold increase of endothelial PAI-1 mRNA levels (P<.001). The apparent MW of this heat- and acid-stable PAI-1 upregulating factor appears to be between 50 and 100 kD, as judged by ultrafiltration. Incubation of HUVEC cultures with the 24-hour CM of early-passage CASMCs derived from normal-appearing arterial tissue showed no significant influence on endothelial PAI-1 synthesis, whereas incubation with late-passage normal CASMCs, as well as early-passage atherosclerotic CASMCs from the same vessel, increased endothelial PAI-1 antigen secretion by 45% and 48% (P<.001), with a concomitant 1.5-fold to 2-fold increase of endothelial PAI-1 mRNA levels (P<.05). No significant change in endothelial TPA synthesis was observed by incubation with CM of either PASMCs (early or late passage) or CASMCs (atherosclerotic or normal). These data suggest that SMC proliferation is associated with (1) increased SMC PAI-1 synthesis as well as decreased TPA synthesis and (2) upregulation of endothelial PAI-1 synthesis by SMC CM. This phenomenon is observed with either late passages of normal PASMCs and CASMCs or early passages of atherosclerotic plaque CASMCs. This suggests that proliferating SMCs are a major regulator of the fibrinolytic potential within the vessel wall, thereby contributing to the thrombotic risk associated with the development of atherosclerosis.
Key Words: plasminogen activator inhibitor-1 • smooth muscle • endothelial cells • atherosclerosis
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Introduction |
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Plasminogen activator inhibitor type 1, the primary regulator of plasminogen activation in vivo,1 2 appears to have major clinical relevance. Elevated levels of plasma PAI-1 activity are associated with thrombotic disorders, such as deep venous thrombosis,3 disseminated intravascular coagulation,4 coronary artery disease,5 and myocardial infarction,6 whereas deficient plasma PAI-1 activity causes recurrent bleeding disorders.7 8 9 10
In addition to this apparently key role within thrombosis and hemostasis, PAI-1 seems to contribute to the development of atherosclerosis. Recent in situ hybridization studies demonstrate an increase of local PAI-1 mRNA expression predominantly in SMCs within atherosclerotic lesions of human arteries.11 12 13 Furthermore, there is evidence for a positive correlation of plasma PAI-1 activity with the early stage of atherosclerosis,14 as well as the risk of coronary restenosis after successful balloon angioplasty.15 These findings suggest a linkage of increased PAI-1 expression to the incidence of cellular proliferation within the developing atherosclerotic lesion as well as neointima formation after angioplasty.
Cell-cell interactions between ECs and SMCs have become a focus of interest during recent years. Various studies show the existence of complex regulatory systems concerning cell proliferation and replication and also PAI-1 expression via growth factor activation.16 17 18
We reported recently that early passages of SMCs have the ability to downregulate PAI-1 synthesis of ECs, thus regulating the fibrinolytic potential of the vascular wall.19 Because the regulation of PAI-1 expression seems to change with increasing number of cell doublings,20 21 we were interested in whether serial passaging of cultured SMCs in vitro influences the PAI-1 (and TPA) synthesis pattern of SMCs as well as their ability to modulate endothelial PAI-1 (and/or TPA) synthesis. Additionally, SMCs derived from human atherosclerotic plaques served as an in vivo correlate to such serially passaged cells in culture, because they are thought to have already undergone numerous cell doublings.22 In this report, we provide evidence that SMC PAI-1 synthesis increases and SMC TPA synthesis decreases with increasing passage number and that SMCs derived from atherosclerotic lesions express a priori higher PAI-1 and lower TPA levels than SMCs derived from normal tissue. Furthermore, we show that regulation of endothelial PAI-1 synthesis by SMCs changes with increasing number of cell doublings, resulting in an overall increase of the antifibrinolytic potential of the vessel wall.
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Methods |
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Cell Culture
HUVECs were isolated by mild collagenase treatment23 and grown under standard culture conditions as published previously.19 All HUVECs used in this study were between passages 2 and 4.
SMCs were isolated either from pulmonary arteries (n=4) by collagenase treatment and separation from ECs by fluorescence-activated cell sorter analysis as published previously19 or by the explant technique.24 SMCs from coronary arteries (obtained from heart explants of patients undergoing heart transplantation, n=3) were isolated by the explant technique. SMCs were characterized as published previously19 and grown to confluence under the same culture conditions as HUVECs. For serial passaging, PASMCs were split in a ratio ranging from 1:4 to 1:6.
Preparation of CM
CM were prepared as described previously.19 Briefly, confluent SMC cultures were washed twice with Hanks' balanced salt solution and given 1 mL of standard culture medium. After 24 hours, the CM were collected, centrifuged (1000g, 5 minutes) to remove cell debris, and immediately transferred to corresponding HUVEC cultures.
For characterization of SMC-derived PAI-1 upregulating activity, serum-free CM were subjected to heat treatment for 5 minutes at 95°C, acidification (pH 2.0 for 1 hour at 4°C) with 10 mol/L HCl (readjustment with 10 mol/L NaHCO3), and incubation with antibodies (100 µg/mL) against TGF-ß (polyclonal neutralizing rabbit anti-human TGF-ß antibody) and bFGF (polyclonal neutralizing goat anti-human bFGF antibody; both, R&D Systems). For size determination of the PAI-1 stimulating factor(s), serum-free late-passage SMC CM were filtered through membranes of various MW cutoff (10 kD, 50 kD, 100 kD; Vivaspin 500 concentrator; Vivascience Ltd) before incubation with HUVEC monolayers.
Assay for PAI-1 and TPA Antigen
Antigen in the CM was determined by specific ELISA using monoclonal antibodies recognizing active, latent, and complexed PAI-1 or free as well as complexed TPA (Technoclone).
Preparation of RNA and Northern Blots
Total RNA was prepared by acid guanidinium thiocyanate–phenol–chloroform extraction.25 Samples of RNA (20 µg) were electrophoretically separated on 1.2% agarose gels containing 6% formaldehyde. Capillary transfer to Duralon-UV membranes (Stratagene) was performed overnight with 10xSSC followed by UV cross-linking. A simplified hybridization technique using 5% SDS according to Virca et al26 was used. Briefly, blots were prehybridized with 50 mmol/L PIPES (pH 6.5), 100 mmol/L NaCl, 50 mmol/L sodium phosphate (pH 7.0), 1 mmol/L EDTA (pH 7.0), and 5.0% SDS for 15 minutes at 56°C. Hybridization with fresh buffer (as above) containing 106 cpm/mL (Cerenkov radiation) of random-primed [
-32P]dCTP-labeled (Boehringer Mannheim) probes for either human PAI-1 (a 1.4-kb EcoR1-BglII fragment of PCR-amplified cDNA), human TPA (a 1.5-kb Sma I–HindIII cDNA fragment), or rat GAPDH (a 1.3-kb Pst I cDNA fragment) was carried out overnight at 56°C. Blots were rinsed for 10 minutes in 50 mL of 5% SDS/1x SSC at room temperature, washed twice for 20 minutes in 400 mL at 56°C, air-dried, and exposed to Kodak XAR x-ray films (Eastman Kodak) with intensifying screens. Quantitative analysis was performed by densitometry (Hirschmann Elscript 400).
Statistical Analysis
The results are reported as mean±SD. Student's unpaired t test was used for determination of significance levels, except for the serial passaging experiments, for which ANOVA followed by Duncan's multiple range test was used.
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Results |
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Influence of Serial In Vitro Propagation on PAI-1 and TPA Synthesis of PASMCs
Increasing in vitro PASMC passage number was accompanied by an increase of PAI-1 antigen secretion. SMC passage 3 secreted 591±106 ng PAI-1·105 cells-1·24 h-1, SMC passage 5 secreted 878±117 ng PAI-1·105 cells-1·24 h-1, SMC passage 7 secreted 1505±349 ng PAI-1·105 cells-1·24 h-1, SMC passage 9 secreted 2439±347 ng PAI-1·105 cells-1·24 h-1, and SMC passage 11 secreted 2952±290 ng PAI-1·105 cells-1·24 h-1 into the CM (Fig 1A
). On the steady-state levels of PAI-1 mRNA, an increase of up to fivefold of both the 3.2- and 2.2-kb forms occurred (Fig 1B). ANOVA (F=70; P<.00001) followed by Duncan's test with a significance level of .05 showed significant differences between all passages.
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In contrast, TPA secretion decreased with increasing passage number. SMC passage 3 secreted 118±34 ng TPA·105 cells-1·24 h-1, SMC passage 5 secreted 105±31 ng TPA·105 cells-1·24 h-1, SMC passage 7 secreted 44±20 ng TPA·105 cells-1·24 h-1, SMC passage 9 secreted 12±3 ng TPA·105 cells-1·24 h-1, and SMC passage 11 secreted 8±1.3 ng TPA·105 cells-1·24 h-1 into the CM (Fig 1C
). On the steady-state levels of TPA mRNA, a twofold to threefold decrease occurred (Fig 1D). ANOVA followed by Duncan's test with a significance level of .05 showed significant differences after the seventh passage (antigen) and ninth passage (mRNA).
Effect of CM From Different PASMC Passages on PAI-1 and TPA Synthesis of HUVECs
Incubation of confluent monolayers of HUVECs with 100% 24-hour CM of PASMC passages 3 through 5 for 24 hours resulted in mean decreases of 42% (P<.001) and 28% (P=.004), respectively, whereas on incubation with the 24-hour CM of SMC passages 7 through 9, mean increases of 54% (P=.002) and 68% (P=.001), respectively, occurred (Fig 2A). This effect appears to be specific for PAI-1, because synthesis of TPA (data not shown) and total protein synthesis as shown previously19 remain unchanged. Furthermore, incubation of HUVECs with SMC CM of any passage number had no visible influence on the endothelial morphology (phase contrast microscopy), nor could any change in EC viability (trypan blue exclusion) be observed. On the steady-state levels of PAI-1 mRNA, a twofold to threefold (P<.001) downregulation of endothelial PAI-1 mRNA by incubation with early-passage SMC CM, and an approximately twofold upregulation (P<.001) of the steady-state levels of both the 3.2- and 2.2-kb forms of endothelial PAI-1 mRNA by incubation with late-passage SMC CM were found (Fig 2B).
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Partial characterization of the SMC-derived PAI-1 stimulating factor(s) included acidification, heat treatment, ultrafiltration with various MW cutoff membranes (10 kD, 50 kD, 100 kD), and blocking experiments with antibodies against TGF-ß and bFGF. Neither heat treatment, acidification, nor incubation with antibodies against TGF-ß and bFGF influenced SMC CM-stimulating activity significantly. The apparent MW of the PAI-1 stimulating factor(s) by ultrafiltration appears to be between 50 and 100 kD, because ultrafiltration of late-passage SMC CM with 50-kD-cutoff membranes completely abolished the observed effect, whereas 100-kD-cutoff membranes did not influence PAI-1 stimulation significantly (Table).
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Increase of PAI-1 and Decrease of TPA Synthesis of CASMCs Derived From Atherosclerotic Plaque
Cultures of CASMCs derived from atherosclerotic lesions showed, in passage 1, higher levels of PAI-1 antigen secretion into the CM (3093±507 ng PAI-1·105 cells-1·24 h-1) compared with cultures of passage 1 CASMCs derived from normal areas of the same vessel (1794±525 ng PAI-1·105 cells-1·24 h-1) (P<.001) (Fig 3A). On the mRNA level, a corresponding increase of approximately two times the steady-state levels of both the 3.2- and 2.2-kb forms of CASMC PAI-1 mRNA (P<.001) was observed (Fig 3B).
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In contrast, TPA antigen secretion by passage 1 CASMCs derived from atherosclerotic lesions was reduced (10±1.6 ng TPA·105 cells-1·24 h-1) compared with cultures of passage 1 CASMCs derived from normal areas of the same vessel (17±5 ng TPA·105 cells-1·24 h-1) (P<.01) (Fig 3C). On the mRNA level, a corresponding decrease about 1.5 to 2 times the steady-state levels of CASMC TPA mRNA (P<.01) was observed (Fig 3D).
Effect of CM From CASMCs of Different Origins (Atherosclerotic Lesion Versus Normal Area) on PAI-1 and TPA Synthesis of HUVECs
Incubation of confluent monolayers of HUVECs with 100% 24-hour CM of passage 1 CASMCs derived from atherosclerotic plaque for 24 hours resulted in a mean increase of PAI-1 antigen secretion of 48% (P<.001), whereas by incubation with 100% 24-hour CM of passage 1 CASMCs derived from normal-appearing areas of the same vessel, no significant alteration of the endothelial PAI-1 secretion occurred. After serial passaging of CASMCs derived from normal-appearing areas, incubation with 24-hour CM of passage 7 CASMCs resulted in a mean increase of PAI-1 antigen secretion of 45% (P<.001) (Fig 4A). Endothelial TPA synthesis remained unchanged in all cases (data not shown). The changes of PAI-1 antigen secretion were also reflected on the steady-state levels of PAI-1 mRNA. By incubation with 100% 24-hour CM of CASMCs derived from atherosclerotic plaques for 24 hours, an approximately twofold upregulation of the steady-state levels of both the 3.2- and 2.2-kb forms of endothelial PAI-1 mRNA could be demonstrated (P<.05; Fig 4B). By incubation with the 24-hour CM of early passages (passages 1 through 3) of CASMCs derived from normal areas, no significant change of endothelial PAI-1 mRNA occurred, whereas late passages (passage 7) increased endothelial PAI-1 mRNA by about the same extent (1.5-fold to 2-fold) as early passages of CASMCs derived from atherosclerotic lesions of the same vessel (Fig 4C).
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Discussion |
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Inappropriately high expression of PAI-1 correlates with the progression of atherosclerosis. This hypothesis is supported by several studies demonstrating the presence of high PAI-1 mRNA levels within atherosclerotic lesions.11 13 Furthermore, in patients with coronary artery disease as well as early atherosclerosis, high PAI-1 plasma levels have been found.5 6 14 In fact, an increase in plasma PAI-1 as well as enhanced expression of PAI-1 mRNA has been implicated in the process of restenosis after balloon dilatation.15 27 Therefore, it was assumed that high PAI-1 levels would attenuate fibrinolysis and in turn create a prothrombotic state that might result in pathological fibrin deposition, thus promoting accumulation of extracellular matrix and atheroma formation.
The origin of plasma PAI-1 under normal and pathological conditions is still unknown.12 SMCs as well as ECs are sources of PAI-1 synthesis in normal and atherosclerotic arteries in vivo.11 13 On stimulation with various stimuli (eg, lipopolysaccharide, interleukin-1, TGF-ß, bFGF, platelet-derived growth factor, angiotensin II, thrombin), PAI-1 biosynthesis of ECs and SMCs is further upregulated.28 29 30 31 32 33 34 Therefore, it is conceivable that at the site of vessel wall injury, eg, atherosclerotic lesions, high PAI-1 expression is found. In addition, at the same sites, SMC proliferation is seen, indicative of the ongoing atherosclerotic process. In fact, the same mediators that are able to upregulate SMC PAI-1 biosynthesis are also either potent mitogens (eg, platelet-derived growth factor)22 or suspected "hypertrophy" hormones and/or growth factors (eg, angiotensin II)35 36 37 for SMCs.
We have shown previously19 that low-passage SMCs are capable of downregulating endothelial PAI-1 synthesis, indicating a possible regulatory link between SMCs and ECs. Because of the above-mentioned strong correlation between SMC proliferation and increased PAI-1 expression, we were interested in whether the increased level of PAI-1 mRNA found within the thickened intima of atherosclerotic lesions in vivo correlates with a passage-dependent modulation of the SMC-EC interaction.
We could, in fact, show that SMC proliferation, mimicked by serial propagation of SMCs in vitro, is associated with an increase of SMC PAI-1 mRNA expression and PAI-1 antigen synthesis (as well as a decrease in TPA synthesis). Consistent with these findings, SMCs derived from atherosclerotic lesions, which are thought to have undergone higher numbers of cell doublings in vivo,22 express a priori higher levels of PAI-1 synthesis (as well as lower levels of TPA synthesis) compared with SMCs derived from normal-appearing arterial tissue of the same vessel. Furthermore, we could demonstrate that regulation of endothelial PAI-1 synthesis (but not endothelial TPA synthesis) by CM of SMCs is affected by the number of SMC doublings: low-passage SMCs downregulate PAI-1 synthesis in ECs twofold to threefold, whereas high-passage SMCs upregulate the steady-state levels of endothelial PAI-1 mRNA about fivefold, a phenomenon that cannot be explained by a simple nonspecific toxic effect. Consistent with the regulation of PAI-1 by in vitro passaged SMCs, CM of early-passage SMCs derived from atherosclerotic arterial tissue increase steady-state levels of endothelial PAI-1 mRNA about twofold, whereas CM of SMCs derived from normal-appearing arterial tissue of the same vessel exhibit this effect only after serial passaging.
A variety of PAI-1–upregulating factors (including growth factors like bFGF and TGF-ß29 ) have been identified in SMCs.38 Although heat and acid stability in our cell culture system would point toward TGF-ß as the stimulating agent,39 blocking experiments with antibodies against TGF-ß and bFGF, as well as MW obtained by ultrafiltration, rather precluded involvement of these factors. However, we would like to point out that ultrafiltration gives only a rough estimate of the MW and cutoffs are given instead for concentrating molecules with MW greater than the cutoff. Because of the cutoff sizes available, we could not use a different, more precise technology. In addition, we cannot completely exclude the possibility that TGF-ß might be at least partially involved, because treatment with anti–TGF-ß antibodies caused a slight, statistically not significant reduction in PAI-1 upregulating activity.
The passage-dependent increase of SMC PAI-1 expression as well as the altered regulation of endothelial PAI-1 expression by SMCs might be caused by a natural senescence process,40 41 which is associated with altered expression of components of the fibrinolytic system by human cells.20 21 42 43 44 At the sites of atherosclerotic lesions, SMC proliferation is thought to be accelerated, which in turn would cause such a senescence process. This phenomenon does not seem to reflect a general tissue culture artifact, because senescent fibroblasts in vivo,21 as well as primary cultures of SMCs derived from atherosclerotic lesions (thought to have already undergone numerous cell doublings in vivo), exhibit a priori elevated levels of PAI-1 expression. Furthermore, induction of endothelial PAI-1 expression by serially passaged cells does not seem to reflect a tissue culture phenomenon, because serially passaged fibroblasts are not capable of upregulating endothelial PAI-1 synthesis.45
The lack of downregulatory activity in the CM of SMCs isolated from histologically "normal" tissue in the vicinity of inflammatory or thrombotic processes might be due to localized activation of the endothelium,11 leading to functional changes (morphologically not visible), because activation of the endothelium is known to precede the appearance of histologically defined atherosclerotic lesions.46 Therefore, the lack of downregulatory activity of SMCs derived from normal-appearing arterial tissue may reflect already incipient atherogenesis within the vessel wall. Results obtained in another study,47 showing a stimulation of endothelial PAI-1 expression by vascular SMCs of different origin, might also be explained by such a passage dependency.
Increased PAI-1 expression at the site of vessel wall injury might therefore be triggered by the originally activating stimulus such as thrombin, growth factors, macrophages,48 or mechanical activation, which lead to overexpression of PAI-1 in ECs and SMCs as well as SMC proliferation causing "senescent" SMCs. Such "senescent" SMCs would express high PAI-1 as well as low TPA levels by themselves and would also upregulate PAI-1 in ECs, thereby leading to a feed-forward mechanism rendering the vessel wall antifibrinolytic, facilitating local thrombus formation and, in turn, apparent atherosclerosis (Fig 5). Conversely, high PAI-1 expression at the site of vessel wall injury might also reflect a negative feedback mechanism causing decreased local SMC migration. Such a mechanism is suggested by the low neointima formation found in transgenic mice overexpressing PAI-1 and the significantly accelerated neointima formation and neointimal accumulation of SMCs found in PAI-1 knockout mice.49 Furthermore, recent evidence suggests that PAI-1 inhibits SMC growth by blocking integrin vß3 binding to vitronectin.50
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Regardless of the possible pathogenetic role of PAI-1 in vascular disease, our data provide evidence for an endogenous mechanism correlating the amount of locally expressed PAI-1 with the number of SMC doublings. Furthermore, these findings not only support the hypothesis that increased PAI-1 gene expression is linked to the cellular proliferative response characteristic of developing atherosclerotic lesions but also point toward the proliferating SMCs as a major regulator of PAI-1 biosynthesis within the vessel wall and therefore as major factors for the development of thrombotic, often fatal complications associated with atherosclerosis.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by grants from the Herzfelder'sche Familienstiftung, the Österreichischer Herzfond, and the Austrian Fund for the Promotion of Scientific Research Project F509.
Received March 26, 1996; accepted December 16, 1996.
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References |
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