Stimulation of Plasminogen Activator and Inhibitor in the Lymphatic Endothelium

Microvascular Research 60, 201–211 (2000) doi:10.1006/mvre.2000.2262, available online at http://www.idealibrary.com on

Stimulation of Plasminogen Activator and Inhibitor in the Lymphatic Endothelium Lee V. Leak,* Michael Saunders,† Agnes A. Day,‡ and Michael Jones§ *E. E. Just Laboratory of Cellular Biology, Department of Anatomy, College of Medicine, Howard University, Washington, DC 20059; †Department of Orthopedic Surgery, Boston University, Boston, Massachusetts 02118; ‡Department of Microbiology and Howard University Cancer Center, College of Medicine, Howard University, Washington, DC 20059; and §Laboratory of Animal Medicine and Surgery, National Heart, Lung and Blood Institute, Bethesda, Maryland 20892 Received January 6, 2000

Chromogenic assays, immunoblotting, and Northern blot hybridization methods were employed to assess the effects of various agonists on the production of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-1) by the lymphatic endothelium (LEC). Fibrin autography showed that plasminogen-dependent fibrinolytic activity occurred at M r of 110 kDa, which represents a complex of tPA with PAI-1, and 65- and 55-kDa bands corresponding to tPA and uPA, respectively. The fractionation of lymph collected from ovine lymphatic vessels also produced a prominent lytic band of ⬃110 kDa, suggesting the formation of PA/PAI complexes in lymph. The stimulation of various agonists produced large-scale increases in tPA mRNA, as shown by Northern blot hybridization analyses. The effects of ECGF, histamine, and LPS on the presence of tPA and on enhancing the levels of mRNA reached maximum activity at 4 h and declined to levels below that of controls by 8 h. However, phorbol-treated cells exhibited reduced levels of tPA mRNA at 4 h, but was significantly increased by 8 h. A large-scale increase in PAI-1 mRNA steady-state levels was also stimulated by the agonists used in these studies. Both the 3.4- and 2.4-kb species of PAI-1 mRNA were increased. These observations demonstrated that tPA and PAI-1 are produced and secreted by LEC monolayer cultures and are also present in lymph. © 2000 Academic Press

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Key Words: lymphatic endothelium; tissue plasminogen activator, tPA; plasminogen inhibitor, PAI-1; tPA mRNA; PIA-1 mRNA.

INTRODUCTION

Blood vessels form a circulatory system, which delivers nutrients, gases, and fluids to all components of the body. Lymphatic vessels, on the other hand, begin as blind-ended tubular vessels within the connective tissue spaces of the body and form a one-way drainage system for returning permeated plasma proteins and excess interstitial fluids and cells back to the bloodstream (Leak, 1971). Both systems are lined by a simple squamous endothelium. The blood vascular endothelium plays a vital role in maintaining the functional integrity of the vascular wall, through its involvement in the modulation of vascular tone and the maintenance of a nonthrombogenic vascular surface for the fluidity and flow of plasma and blood cells (Collen, 1980). In addition, the blood vascular endothelium has been shown to play a pivotal role in the regulation of endogenous fibrinolytic activity through the synthesis and secretion of plasminogen activators

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(tPA, uPA) 1 and plasminogen activator inhibitors (PAI-1, PAI-2), which are major physiologic inhibitors of tPA and uPA (Erickson et al., 1985; Kruithof et al., 1995). Therefore, the fibrinolytic system of the blood vascular endothelium consists of two opposing activities, which maintain a balance in blood fluidity by changing the level of activator or inhibitor or both (Mann and Krishnaswamy, 1992). Under physiological conditions fibrinolysis is a highly regulated process which occurs on the surface of a fibrin clot (Collen and Lijnen, 1991). Blood endothelial cells also express receptors that bind plasminogen activators and plasminogen, which appear to localize and enhance fibrinolytic activity (Ellis et al., 1991; Hajjar and Hamel, 1990). Although plasminogen is abundant in plasma, tPA and u-PA are found only in trace quantities, being produced and secreted only as needed by the vascular endothelium (Levin and Luskutoff, 1982). In addition, there is an increasing amount of data which suggest the participation of plasminogen activators (tPA and uPA) in many physiological and regulatory processes, such as tissue remodeling, cell migration, angiogenesis, ovulation, embryogenesis, brain function, and various pathological processes (Carmeliet et al., 1994; Collen and Lijnen, 1991; Dano et al., 1985). The constant propulsion of lymph through lymphatic vessels and a series of lymph nodes requires lymph to maintain its fluidity and suggests the existence of a coordinated synthesis and expression of proteases and inhibitors by the lymphatic endothelium (LEC). Although it has been known since the early 1900s that lymph coagulates (Howell, 1914), there is little information concerning the role of the lymphatic endothelium on the regulation of endogenous fibrinolytic activity to maintain the fluidity of lymph as it is propelled through lymphatic vessels and lymph nodes. The studies of Laschinger et al. (1990) demonstrated that bovine lymphatic endothelial cells in culture treated with TNF produced a four1

Abbreviations used: ECGF, endothelial cell growth factor; LEC, lymphatic endothelial cells; PA, plasminogen activator; tPA, tissuetype PA; uPA urokinase-like PA; PAI-1, PA inhibitor type 1; PAI-2, PA inhibitor type 2; PBS, phosphate-buffered saline; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; LPS, lipopolysaccharide; TPA, 12-O-tetradecanol phorbol-13-acetate.

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Leak et al.

fold increase in PAI-1 activity in the culture medium after 24 h. Likewise, TNF produced a fourfold increase in tPA-PAI-1 complexes in the conditioned media. Additionally, results from studies in our laboratory showed that cytokines and various agonists modulate PA and PAI activity in LEC in vitro (Leak et al., 1993). In the present report we have studied the effects of growth factors and various agonists on PA and PAI-1 proteins and mRNA levels in LEC.

MATERIALS AND METHODS Lymphatic endothelial cell isolation and culture. The procedures for the isolation and culture of LEC from ovine mesenteric lymphatic vessels were carried out as previously described (Leak and Jones, 1993, 1994). Cells were grown to confluence at 37°C in a humidified atmosphere of 5% CO 2 in air, subcultured on gelatin-coated six-well plates, and used at passages 3 to 6 for experiments to characterize the production and secretion of tPA and PAI-1. Stimulation of PA and PAI activity in LEC monolayer cultures. For experiments to characterize PA and PAI production by LEC, triplicate wells of LEC monolayer cultures grown in six-well plates (6 ⫻ 10 4 cells/cm 2) were washed twice in serum- and growth factor-free medium and exposed to fresh serum-free media with endothelial cell growth factor (ECGF), histamine, or lipopolysaccharide (LPS) (Sigma Chemical Co.) for the concentrations and time periods indicated. Cultures grown in serum- and growth factorfree media at various time periods were used as controls. To determine secreted PA activity, conditioned media from cells treated with and without growth factor and agonists for the various time periods were collected, centrifuged (10,000g for 5 min), and stored at ⫺80°C until used. Lymph collection. To determine the presence of PA/PAI in lymph, mesenteric lymphatic vessels were cannulated and lymph was collected prior to isolation of LEC from these vessels. However, since only limited quantities of lymph could be collected from these vessels, lymph was also collected from the efferent duct of the caudal mediastinal lymph node according

Plasminogen Activators and Inhibitors Produced by LEC

to the methods of Staub et al. (1975), using NIH guidelines of the Institutional Animal Care and Use Committee. The lymph was centrifuged and the supernatant stored at ⫺80°C until used. Quantification of PA activity. A spectrophotometric assay utilizing colorimetric changes related to plasminogen conversion to plasmin was used to quantify plasminogen activator activity. The chromogenic assay based on the method of Gilboa et al. (1982) was used to quantitate plasminogen activator activity in conditioned medium of LEC and values were compared to those obtained with human tPA and uPA standards assayed in parallel. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and electrophoretic enzymography. Plasminogen activator activity in 30 ␮l/ml of conditioned media was analyzed by fractionation in SDS–PAGE for electrophoretic comigration with tPA and uPA as standards. In addition, aliquots (30 ␮l) of conditioned medium and lymph fractionated by SDS– PAGE were analyzed by fibrin autography for PA activity by fibrin–agar indicator gel plates (GranelliPiperno and Reich, 1978). In addition, zymographic analysis of plasminogen activators present in conditioned medium was undertaken with 10% polyacrylamide gel containing 0.1% SDS that was polymerized in the presence of plasminogen and casein (Niedbala and Stein-Pecarella, 1992). Immunoblotting. To determine the extent of tPA and PAI-1 immunoreactive proteins secreted by LEC after treatment with various agonists, aliquots (30 ␮l/ ml) of conditioned medium were mixed with SDS sample buffer and separated by 10% SDS–PAGE. Proteins were transferred electrophoretically onto nitrocellulose membranes (Amersham, Corp., Arlington Heights, IL) and blocked with 1% BSA/PBS/0.1% Tween 20 for 1 h or overnight at 4°C. The membranes were subsequently incubated with 1 ␮g/ml of primary antibody (goat anti-human melanoma tPA antibody or rabbit anti-PAI-1 antibody) in 1% BSA/PBS/ 0.1% Tween 20 for 1 h at room temperature or overnight at 4°C. Membranes were washed and incubated in anti-goat IgG- or anti-rabbit IgG-conjugated peroxidase for 30 min. After extensively washing the membranes, the immunoreactivity was detected on hyperfilm using ECL (ECL Kit, Amersham Corp.).

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Images produced on ECL-developed film were captured using the Kodak Digital Science ID Image Analysis software (Eastman Kodak Co., Rochester, NY) Lymph was also fractionated by SDS–PAGE and the proteins were transferred electrophoretically onto nitrocellulose and probed with anti-tPA and anti-PAI-1 antibodies as described above. Regulation of LEC PA and PAI-1 mRNA levels by growth factors and agonists. For experiments to determine PA and PAI-1 mRNA in the lymphatic endothelium, confluent cultures of LEC grown in 175-cm 2 flasks were washed twice in serum- and growth factor-free media and refed with fresh serum-free media containing ECGF (50 ng/ml), histamine (100 ␮M/ml), LPS (50 ng/ml), or the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA) (50 ng/ml) for 4 to 8 h. At the conclusion of each experiment, conditioned media were collected and stored at ⫺80°C until used for chromogenic assays or SDS zymograms. These experiments were repeated at least three times. For PA and PAI-1 mRNA analysis the cells were washed three times with PBS and total RNA was extracted from the cells as described below. RNA isolation, electrophoresis, and Northern blot hybridization. Following the treatment of LEC monolayers with the various agents, cultures were washed in PBS and total RNA was extracted using the Qiagen RNeasy Total RNA Kit System, according to the manufacturer’s protocol (Qiagen Inc., Chatsworth, CA). Approximately 4 to 5 ⫻ 10 7 cells per 175-cm 2 flask were used for each treatment. Following isolation, the RNA pellet was resuspended in diethylpyrocarbonate-treated water and the RNA concentration of each sample determined by measuring the A 260, assuming that 1 A 260 unit was 40 ␮g RNA. RNA (10 ␮g/lane) was denatured in 4-morpholinepropanesulfonic acid (MOPS)/formaldehyde at 65°C for 5 min and fractionated in a 1% agarose/formaldehyde (2.2 M) gel. Gels were washed in distilled water and stained with 1.0 ␮g/ml ethidium bromide to assess the integrity and equivalence of RNA concentrations among replicate samples using standard procedures (Sambrook et al., 1989). The fractionated RNA was transferred to nitrocellulose membranes by standard capillary blotting techniques (Sambrook et al., 1989) in transfer buffer. Following prehybridization, filters

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FIG. 1. Phase-contrast micrograph illustrating the flattened appearance of a monolayer of cultured lymphatic endothelial cells. Original magnification, ⫻350.

were hybridized overnight at 60°C with 2.5 ⫻ 10 6 cpm/ml of each probe ( 32P-labeled heat-denatured cDNA probe). After hybridization, the filters were washed in 2⫻ NaCl/Na citrate, 0.1% SDS twice for 15 min at room temperature, followed by two washes in 0.1⫻ NaCl/Na citate, 0.1% SDS for 40 min at 60°C. Hybridization using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was done at 42°C as described (Sambrook et al., 1989). Following hybridization the filters were dried and exposed to autoradiographic film (Kodak XAR) at ⫺70°C with an intensifying screen for 1–5 days. All experiments were repeated at least three times. Preparation of probes. cDNA probes for tPA and PAI-1 were obtained from American Type Culture Collection (ATCC, Rockville, MD). The cDNA probes of tPA and PAI-1 were labeled with [ 32P]dCTP (3000 Ci/mM) by random hexamer primer extension (Finberg and Vogelstein, 1983). The tPA cDNA from human Bowers melanoma was cloned into the pBR322 vector (insert size, 1.77 kb from Genentec). Human PAI-1 was cloned in puC9 (insert size, 5.2 kb) (Bosman et al., 1988). A 1.2-kb PstI fragment of rat GAPDH cDNA (Fort et al., 1988) was used as an internal standard probe (Offringa et al., 1988). Quantitation was

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carried out with a ChemiImager using AlphaEase software (Alpha Innotech, San Leandro, CA). Results are expressed relative to control nontreated cultures at the same time point. Statistical analysis. All experiments and assays were carried out in triplicate. The results were analyzed with ANOVA or a paired Student’s test (Sigma Software). Differences were accepted as statistically significant at P ⬍ 0.05.

RESULTS Endothelial nature of LEC monolayers. Lymphatic endothelial cells isolated from ovine mesenteric lymphatic vessels were judged to be lymphatic endothelium by their ability to form monolayers with a cobblestone appearance, as depicted in phase-contrast images (Fig. 1). They also exhibited the expression of VEGFR-3 (FLT4) receptor kinase, which is unique for the lymphatic endothelium, as demonstrated by flow cytometric analysis (Leak et al., 1999). The close association of adjacent cells to form a continuous monolayer was confirmed in silver nitrate-treated monolay-

Plasminogen Activators and Inhibitors Produced by LEC

FIG. 2. Analysis by fibrin autography, after SDS– gel electrophoresis showed that multiple forms of PA were detected in the conditioned media from LEC treated with various agonists; lanes: 1, ECGF (50 ng/ml); 2, TPA (50 ng/ml); 3, LPs (50 ng/ml); 4, histamine 100 ␮M/ml); 5, lymph; 6, control. Plasminogen-dependent fibrinolytic activity occurred at 65 kDa, corresponding to tPA, 55 kDa, which represents uPA, and 110 kDa, representing a complex of tPA with PAI-1.

ers and transmission and scanning electron micrographs (Leak and Jones, 1993). LEC from confluent cultures also exhibit an intense reaction to Factor VIII-related antigens and LEC also require endothelial cell growth factor to maintain continued growth rates (Leak and Jones, 1993). Secretion of PA by LEC. The influences of various agonists on the production of t-PA and PAI-1 were examined in an effort to gain insight into the production and regulation of fibrinolytic components by the lymphatic endothelium. For these experiments, use was made of fibrin–agar indicator gel plates. With this method, fibrinolytic activity in fibrin–agar indicator gel represents PA activity as determined by tPA and uPA standards as well as other lytic bands that comigrate with them. No lytic bands occurred in replicate gels polymerized in the absence of plasminogen. Analysis of plasminogen-dependent fibrinolytic activity by fibrin autography after SDS– gel electrophoresis showed that multiple forms of PA were detected in the conditioned medium from LEC treated with various agonists (Fig. 2). PA activity in conditioned medium from LEC grown in serum heparin and growth factorfree medium (lane 6) showed plasminogen-dependent fibrinolytic activity with lytic bands at between 92 and 110 kDa representing a complex of tPA with PAI-1, a 65-kDa band corresponding to tPA, and a 55-kDa band which represents uPA (Fig. 2). The 65-kDa band (lane 6) exhibited in conditioned medium in the nonstimulated cells appeared as a very prominent band in the SDS–fibrin overlay gels (Fig. 2). Lane 1 depicts PA

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activity in LEC-conditioned medium after treatment with ECGF. A prominent lytic band was produced at 65 kDa, while minor bands occurred at 92–110 and 55 kDa. The treatment of LEC with TPA (lane 2) led to a large increase in free uPA (55-kDa lytic band) and an increase in the PA/PAI complex. However, there was little to no free tPA (65-kDa lytic band) detected after the TPA treatment using this method. LPS (lane 3) and histamine (lane 4) treatment led to the production of reduced amounts of free uPA and PA/PAI complexes and little to no tPA activity was detected (Fig. 2). The fractionation of lymph (lane 5) collected from the efferent duct of ovine caudal mediastinal lymph nodes by the SDS–fibrin indicator gel method showed major lytic bands in the range of 92 to 110 kDa which suggest the formation of a PA/PAI complex. However, no free PA activity was detected in lymph using SDS–fibrin overlay gels (Fig. 2). Immunoblotting of PA and PAI-1 by LEC. Analysis of immunoblotting proteins for tPA using goat anti-human melanoma tPA showed that LEC treated with TPA, LPS, and histamine demonstrated secretion of 92-, 70-, 65-, and 33-kDa species. In addition, when tPA protein in lymph was immunoblotted using this anti-tPA antibody, a major band was produced at 65 kDa. Likewise, immunoreactive forms were also located at 110, 92, 70, and 33 kDa (Fig. 3A). To determine the secretion of PAI-1 protein by LEC we examined conditioned medium from similarly treated LEC monolayer cultures. Immunoblotting of PAI-1 protein using rabbit anti-PAI-1 antibody showed that LEC secreted high levels of 46- and 92-kDa forms. PAI-1 in lymph isolated by SDS–PAGE and immunoblotting using rabbit anti-PAI-1 antibodies produced a major band migrating at 46 kDa. Immunoreactive forms of 110, 75, and ⬃40 kDa were also demonstrated (Fig. 3B). Stimulation of PA activity with growth factor and agonists. To determine if LEC monolayers responded to various agonists by an enhanced production of PA in a manner similar to that of blood endothelium, we examined the response of various doses of histamine and LPS on tPA production. The levels of PA present in conditioned medium of LEC treated for 24 h with various concentrations of histamine (50 nM/ml to 100 ␮M/ml) were measured using the chro-

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associated with an increase in tPA mRNA levels. The apparent size of the tPA mRNA transcript was approximately 2.5 kb. Studies were carried out to determine if the treatment of LEC monolayer cultures with optimal doses of various agents were also accompanied by reciprocal changes in the level of mRNA. To

FIG. 3. (A) Immunoblotting analysis of immunoreactive proteins for tPA using anti-human melanoma tPA demonstrated species of 110, 92, 70, 65, and 35 kDa; lanes: 1, lymph; and CM from LEC treated with 2, histamine (100 ␮M/ml), 3, LPs (50 ng/ml), and 4, TPA (50 ng/ml). (B) Immunoblotting of PAI-1 protein using rabbit anti-PAI-1 antibody showed that LEC secreted high levels of 46- and 92-kDa forms. Similar results were obtained in the analysis of immunoreactive protein for PAI-1 in lymph; lanes: 1, lymph; and CM from LEC treated with 2, histamine, 3, LPS, and 4, TPA.

mogenic assay. Histamine induced an increase in tPA activity by threefold at a 100 nM concentration and treatment with 500 nM and 100 ␮M concentrations induced tPA activity of greater than twofold in LEC cultures (Fig. 4A). Treatment of LEC cultures with LPS (1 to 50 ng/ml) produced a concentration-dependent increase in tPA activity by LEC (Fig. 4B). Effects of growth factors and agonists on tPA mRNA expression in LEC monolayer cultures. The enhancement of tPA production and release in blood endothelial cell cultures by various agonists have been

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FIG. 4. Monolayer cultures of LEC were treated with various concentrations of histamine or grown in serum-free medium, as indicated in these bar graphs. After 24 h the conditioned medium was removed and assayed for plasminogen activator activity (tPA) using a chromogenic assay. The treatment of LEC cultures with histamine (50 nM/ml) induced increases in tPA activity, as shown in A. The treatment of LEC with LPS (1 to 50 ng/ml) also produced an increased induction of tPA in the conditioned medium, as shown in B. Values represent the means ⫾ SE of results from triplicate experiments.

Plasminogen Activators and Inhibitors Produced by LEC

FIG. 5. LEC monolayer cultures were incubated with various agonists and the cell layers were used for RNA isolation, as described under Materials and Methods. Northern hybridization were performed using tPA, PAI-1, and GAPDH cDNA probes. Treatment of LEC with ECGF, lane 1, TPA, lane 2, LPS, lane 3, and histamine, lane 4, showed enhanced levels of tPA mRNA after 4 h. After 8 h the mRNA levels for TPA-treated cells, lane 5, were greatly enhanced, while LPS, lane 6, histamine, lane 7, and ECGF, lane 8, showed a marked reduction in tPA mRNA levels.

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FIG. 7. Northern hybridization analysis of PAI-1 mRNA of LEC treated with various agonists for 4 h (lanes 1– 8). A large-scale increase in PAI-1 mRNA steady-state levels was stimulated by these agonists; lanes: 1, negative control; 2, ECGF; 3, histamine; 4, LPS; 5, TPA. Both 3.4- and 2.4-kb species of PAI-1 mRNA were increased.

determine the effects of growth factors and various agonists on the expression of tPA and PAI-1 genes, total cellular RNA was extracted from confluent LEC monolayer cultures that were treated with ECGF, histamine, TPA, and LPS and hybridized with specific single-strand cDNA probes. Changes in tPA mRNA levels were examined by Northern blot and slot blot hybridization at 4 and 8 h. Quantitation of changes in mRNA was assessed by densitometric analysis of the developed autoradiograms. When LEC monolayer cultures were placed in serum- and growth factor-free medium, tPA mRNA lev-

els were shown to peak at 4 h. After 8 h the control level decreased by half. ECGF produced a 1.5-fold increase in tPA mRNA levels at 4 h. Histamine produced a 1.7-fold increase and LPS stimulated a 1.3-fold increase after 4 h (Figs. 5 and 6). The treatment of LEC with TPA for 4 h showed no increase over that of the controls (Figs. 5 and 6). However, after 8 h of treatment with ECGF, histamine and LPS there was a marked decrease in tPA mRNA levels when compared to those of four h. On the other hand the treatment of LEC with TPA produced a 2.3-fold increase in tPA mRNA levels between 4 and 8 h (Figs. 5 and 6).

FIG. 6. Effects of various agonists on tPA mRNA in LEC. This bar graph of representative data obtained from the tPA Northern blots. Scanning densitometry was performed on autoradiographs and the intensity of the tPA mRNA signal compared with that of GAPDH. Results are means ⫾ range and were plotted relative to level of densitometry measurement from triplicate experiments.

FIG. 8. Effects of various agonists on PAI-1 mRNA in LEC. This bar graph represents data obtained from the PAI-1 Northern blots. Scanning densitometry was performed on autoradiographs and the intensity of the tPA mRNA signal compared with that of GAPDH. Results are means ⫾ range and were plotted relative to level of densitometry measurement from triplicate experiments.

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FIG. 9. The mRNA level for the constitutively expressed gene (GAPDH) is shown in this figure. Lanes 1– 8 are the same as those in Fig. 7.

Effect of agonist on PAI-1 mRNA expression in LEC. Northern blot analysis of PAI-1 mRNA obtained from LEC monolayers treated with various agents is shown in Fig. 7. While the basal level of the 2.4-kb species of PAI-1 mRNA was higher than that of the 3.4-kb species in the control, the treatment of LEC with various agonists for 4 h showed that both the 3.4- and 2.4-kb species of PAI-1 mRNA were increased. The 3.4-kb species was increased 2.6-fold with ECGF, 4.6-fold with histamine, 3.4-fold with LPS, and 4.0-fold with TPA. The results showed that mRNA levels for 2.4-kb species increased 1.3-fold when treated with ECGF, 2.1-fold for histamine, 3.2-fold for LPS, and 3.2-fold for TPA. The relative amounts of mRNA for each kilobase species after each treatment are shown in Fig. 8. Figure 9 shows Northern blot analysis of GAPDH mRNA from experiments similar to those indicated in Fig. 7.

DISCUSSION The constant propulsion of lymph through lymphatic vessels, and through a series of lymph nodes, before being returned to the blood vessels requires lymph to remain in a fluid state. Lymph coagulation in various disease states, such as lymph thrombi produced by chronic filarial nematode infection and severe lymphedema (Fader and Ewert, 1986; Witte and Witte, 1995), provide elegant testimony to the importance of lymph fluidity in the maintenance of fluid homeostasis throughout the body for stable cardiovascular function. Various agents, including hormones, growth factors (Medcalf et al., 1988; Montesano et al., 1986), and cytokines (Niedbala and Stein-Pecarella, 1992; van Hinsbergh et al., 1990), have been shown to

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induce PA and PAI activities in the blood endothelium as well as in a number of other cell types (Fukumoto et al., 1994). In the present study we describe the secretion of tPA and PAI-1 by LEC monolayer cultures and the presence of activator and inhibitor in lymph. In addition, we also assess the effects of various agonists on tPA and PAI-1 secretion and mRNA expression by the lymphatic endothelium. In this study we assessed the production of tPA and PAI-1 which accumulated in conditioned media of LEC cultures grown in serumand growth factor-free medium and in cultures stimulated with various agonists. LEC was shown to produce a steady level of PA and PAI activities that were also partitioned into a tPA/PAI-1 complex. It is of interest to note that conditioned media examined from each treatment (ECGF, TPA, LPS, and histamine) exhibited a fribinolytic band in SDS–zymograms which corresponded to the tPA/PAI-1 complex. This was also exhibited in lymph collected from ovine caudal mediastinal and mesenteric lymphatic vessels. These observations are in agreement with other studies which demonstrated the formation of PA/PAI complexes (Santell and Levin, 1988; Santell et al., 1992). The complexing of continuously secreted PA and PAI provides a mechanism for maintaining a balance between activator and inhibitor in lymph that is constantly being transported from the various organ systems of the body for return to the blood circulation. The treatment of LEC with ECGF, histamine, LPS, and TPA caused an enhanced secretion of tPA into the conditioned medium of LEC monolayer cultures. The stimulatory effects of these agents on tPA production in LEC was shown to be the result of large-scale increases in tPA mRNA as demonstrated by Northern blot hybridization studies. After 4 h of treatment with histamine a pronounced elevation in tPA mRNA was produced. There was also an increase in tPA mRNA levels with ECGF and LPS after 4 h, but to a lesser degree than that of histamine. It is significant that TPA did not stimulate the secretion of tPA mRNA levels above that of the negative control at the 4-h treatment period. However, by 8 h, the production of tPA mRNA by ECGF, histamine, and LPS had declined below that of the control levels. In contrast TPAtreated cells showed enhanced levels of tPA mRNA by 8 h. The delayed production in tPA mRNA levels after

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treatment with phorbol esters observed in LEC monolayer cultures is similar to the observations of Levin et al. (1989). Thus the changes in tPA mRNA also reflect variations in the production and secretion of tPA proteins. The present studies also demonstrated that the stimulation of PAI-1 production by the various agents was due to large-scale increases in PAI-1 mRNA. Both PAI-1 transcripts, 3.4 and 2.4 kb in length, were rapidly elevated, with increases being detected at 4 h of treatment with the above agents. However, when compared to basal levels the transcripts for the 3.4-kb species showed the greatest increase for each treatment, with histamine and TPA being more potent than the other agents. While the overall increase in the 2.4-kb species was lower than that in the 3.4-kb species the treatment of cells with LPS and TPA induced larger increases for the 2.4-kb species. The results demonstrated in this study are consistent with the observation of Sawdey et al. (1989), who showed that various cytokines markedly enhanced the steady-state levels of both RNA species of PAI-1. The elevation of steady-state PAI-1 mRNA levels in response to various growth factors and cytokines has also been reported in a variety of cultured cell types (Kooistra et al., 1994; Kruithof et al., 1995). The basal levels of the 2.4- and 3.4-kb transcripts were maintained at a ratio of 2:1. However, during treatment with ECGF and histamine the ratio approached 1:1, indicating that ECGF may exert a greater stimulatory effect on the 3.4 transcript when compared to the control. For LPS- and TPA-treated cells the ratios for the 2.4- to 3.4-kb species were similar to that of control (2:1 and 1.8:1, respectively). The present observations demonstrate that tPA and PAI-1 are produced and secreted by LEC monolayer cultures. In addition, lymph contained free tPA and PAI-1 as well as PA/PAI complexes when analyzed by immunoblotting methods. While it has been shown that tPA and uPA occur only in trace quantities in plasma and are produced and secreted only as needed within the blood vascular lumen (Levin and Luskutoff, 1982), the presence of free tPA and PAI-1 in lymph suggests an active and constant secretion of proteases and protease inhibitor by the lymphatic endothelium. This physiological function

differentiates the lymphatics from blood vessels. Previous studies on the fibrinolytic activity of lymph during acute venous hypertension on canine hind limb lymph showed increased lymph flow and its concentration of fibrinogen (Leach and Browse, 1985). Studies with chronic venous hypertension produced similar effects. In spite of this increase transexudation of fibrinogen across the interstitial space during venous hypertension, the fibrinolytic activity of lymph did not change under either acute or chronic conditions (Leach and Browse, 1985). In light of the current studies, this stable fibrinolytic activity of lymph could be accounted for by the ability of the lymphatic endothelium to regulate the fibrinolytic activity of lymph through the active secretion of proteases and protease inhibitors directly into lymph. Although these investigators (Leach and Browse, 1985) suggested that the contribution of lymph reflected those of the interstitial fluid and that the contribution of the lymphatic wall to the composition of lymph was minimal, data provided in the present study provide support for the concept that the lymphatic endothelium plays a pivotal role in the regulation of endogenous fibrinolytic activity within the lymphatic vascular lumen for the maintenance of lymph fluidity. Likewise, the continuous delivery of lymph to the systemic circulation with large amounts of PA and PAI suggests that the lymphatic endothelium also serves as an additional source for proteases as well as their inhibitors. This additional supply of proteases and inhibitors may also play a major role in the regulation of the fluidity of venous blood that is continuously returned to the heart. These findings provide additional evidence that the lymphatic endothelium not only lines lymphatic vessels but also contributes to the production of lymph components, including the secretion of plasminogen activator and inhibitor, two proteins which also play significant roles in cardiovascular function.

ACKNOWLEDGMENTS The authors express their thanks to Mr. Fan Hongtao and Mr. Haile F. Yancy for technical assistance. This work was supported in

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210 part by Grant S06GM08016, funded by NIGMS, NIH, and The American Heart Association, Mid-Atlantic Affiliate.

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