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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3139-3146.)
© 1997 American Heart Association, Inc.

Articles

Thrombomodulin Expression in Bovine Brain Capillaries

Anticoagulant Function of the Blood-Brain Barrier, Regional Differences, and Regulatory Mechanisms

Liang Wang; Nam D. Tran; Mamoru Kittaka; Mark J. Fisher; Steven S. Schreiber; ; Berislav V. Zlokovic

Correspondence to Berislav V. Zlokovic, MD, 2025 Zonal Ave, RMR 506, Los Angeles, Calif 90033. E-mail zlokovic{at}hsc.usc.edu

	Abstract

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Abstract Thrombomodulin (TM), a key cofactor of the TM-protein C pathway, is of major biologic significance for the antithrombotic properties of endothelial cells. Yet, there is uncertainty whether TM is expressed in brain and what mechanisms govern brain endothelial anticoagulant activity. In this study, bovine brain capillaries were used as an in vitro model of the blood-brain barrier to determine factors involved in the regulation of TM expression in cerebral vasculature. Quantitative competitive-polymerase chain reaction assay revealed significant regional differences in the amount of brain capillary TM mRNA, ie, cortical > cerebellar > pontine, consistent with the reverse transcription-polymerase chain reaction findings in which the abundance of TM mRNA was analyzed relative to ß-actin mRNA. Regional differences in TM mRNA brain capillary level correlated well with differences in protein C activation. The TM mRNA and activity were not detectable in brain parenchyma. Pathogenic mediators of ischemic stroke, interleukin 1ß (10 U/mL), and tumor necrosis factor (10 U/mL), produced a time-dependent decrease in brain capillary TM mRNA (t_1/2 of 2.1 and 3.9 hours, respectively) and reduced endothelial TM activity. Incubation of brain capillaries with retinoic acid (10 µmol/L) and dibutyryl cAMP (3 mmol/L) resulted in a 4-fold increase in TM mRNA at 4 and 8 hours, respectively, followed by an increase in protein C activation. We conclude that TM at the blood-brain barrier is likely to be an important physiologic anticoagulant in brain microcirculation. Its downregulation by cytokines may contribute to ischemic brain damage and potentially could be counteracted by retinoic acid and cAMP.

Key Words: thrombomodulin • blood-brain barrier • cytokines • retinoic acid • cAMP

	Introduction

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TM is an endothelial cell transmembrane glycoprotein and a key cofactor for the TM-protein C-protein S pathway, which is of major biologic significance for the antithrombotic properties of endothelial cells.^1–3 TM serves as a high-affinity receptor for thrombin on endothelial cell luminal surfaces. When bound to TM, thrombin changes its substrate specificity and the thrombin-TM complex is a potent activator of circulating protein C. APC functions as an antithrombotic enzyme by proteolitically degrading factors Va and VIIIa.^1–3 A fetal form of TM, fetomodulin, that is identical to TM plays an important role in brain development.⁴

TM offers major protection against a variety of thrombotic events. Anti-TM antibodies potentiate thrombin-induced thromboembolism in mice,⁵ while purified or recombinant TM protects against thromboembolism.^5,6 The APC provides protection against procoagulant and lethal effects in a sepsis model.⁷ Resistance to the effects of APC is closely linked to venous thrombosis in humans,⁸ while blockade of APC in a coronary occlusion model results in impaired cardiac outcome.⁹ Recently, lower levels of circulating APC have been suggested as an important risk factor in infection-associated stroke.¹⁰ Although thrombosis is of paramount importance in the pathophysiology of ischemic stroke, the mechanisms that govern anticoagulant activity in brain endothelium, ie, at the site of the BBB, are still poorly defined.^11–13

There is uncertainty in the literature whether TM is in fact expressed in brain. Some studies reported that TM protein is present in human brain exclusively in microvessels,^14,15 whereas others claimed its absence or very limited expression.^16–18 Recently it has been shown that TM is expressed in cultured bovine brain endothelial cells,¹³ and the presence of TM mRNA has been demonstrated in cerebral microvessels in adult bovine and rat brain.^19,20 The factors involved in TM regulation in cerebral vasculature, however, remain largely speculative. It is well documented that pathogenic mediators of stroke, IL-1ß, and TNF-^21,22 downregulate TM expression in peripheral vascular endothelial cells,^23–26 but it is not known whether these cytokines exert similar effects on brain endothelial TM, which in turn could contribute to ischemic brain damage. RA^27–29 and cAMP,^30–34 on the other hand, stimulate TM expression in systemic vascular endothelium and counteract the effects of IL-1ß and TNF-. Whether RA and increased intracellular cAMP levels may enhance the anticoagulant function of the BBB is unclear, but if yes, these strategies may have the potential to ameliorate focal ischemic brain injury. In this study, we used isolated bovine BCs as an in vitro model of the BBB^35,36 to address these questions.

	Methods

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Reagents
Recombinant human TNF- (2.7x10⁸ U/mg) and IL-1ß (6.7x10⁷ U/mg) were purchased from Genzyme Corporation. All-trans-RA, RA, and db-cAMP were obtained from Sigma Chemical Co. -Glutamyl transpeptidase activity was determined using a Sigma diagnostic assay. All other chemical reagents were purchased from Sigma Chemical Co, except where indicated.

Isolation of BCs
BCs were isolated from bovine cerebral cortical mantles, cerebellum, and pons by a modified mechanical homogenization technique.³⁷ Briefly, bovine brains were rinsed in ice-cold buffer B (mmol/L: 103 NaCl, 4.7 KCl, 2.5 CaCl₂, 1.2 KH₂PO₄, 1.2 MgSO₄, 15 HEPES; pH, 7.4). The cerebral cortical mantles, cerebellum, and pons were rapidly freed of meninges (ie, pial vessels) under sterile conditions, and the arteries of the circle of Willis, veins, and choroid plexi were discarded. Cerebral and cerebellar cortices and pons were used to isolate the capillaries. Homogenization of brain tissue in buffer A (buffer B + the following in mmol/L: 25 HCO₃, 10 glucose, 1 sodium pyruvate, and 0.156 dextran, M_r=64,000) with a hand-held Teflon homogenizer was followed by dextran density centrifugation at 5800g at 4°C. The pellet was resuspended in buffer A and passed over an 85-µm nylon mesh. Arterioles and venules remained on top of the mesh, while the capillaries, red cells, nuclei, and other debris were collected in the filtrate passing through the mesh. This filtrate was then passed over a 3x4-cm glass bead column (0.45-mm glass beads) with 44-µm nylon mesh at the bottom, and the column was washed with buffer B. The BCs adhere to the glass beads while the other contaminants pass unimpeded. Capillaries were recovered by repeated gentle agitation of the glass beads in buffer A, and the supernatant with capillaries was decanted and spun at 500g for 5 minutes to obtain the final pellet. The purity of the cerebral capillary preparation was checked by light and phase microscopy. The cerebral capillaries were free of adjoining brain tissue, and preparations consisted primarily of capillaries, but also contained minor amounts (5% to 10%) of small arterioles, as described.^36,37 The presence of no more than minimal detectable activity of a specific cerebrovascular marker, -glutamyl transpeptidase, was used to confirm the absence of contamination of capillary-depleted brain parenchyma with microvessels, as reported.³⁸

Incubation of BCs
Freshly isolated cortical capillaries were incubated in medium-199/Ham's F-12 media with 10% fetal calf serum, 1 mol/L Na pyruvate, 2 mg/L NaHCO₃, 2 mmol/L L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2.5 µg/mL Fungizone in six-well tissue culture plates in a humified 5% CO₂/95% air incubator at 37°C. Capillaries were incubated for 2, 4, 8, 12, 16, and 24 hours with 10 U/mL IL-1 ß, or 10 U/mL TNF-, or 10 µmol/L RA, and/or 3 mmol/L db-cAMP. Each experiment had its own control group incubated with the vehicle. Experiments were performed repeatedly (n=3) in triplicate.

RT-PCR Analysis
Total RNA was extracted from bovine capillaries and capillary-depleted brains with TRIZOL reagent (GIBCO BRL). cDNA was synthesized by RT using 4 µg of total RNA in 20 µL of reaction buffer containing 1 mmol/L each dNTP, 25 mmol/L Tris-HCl (pH 8.3), 1 µg of oligo(dT), 25 mmol/L KCl, 5 mmol/L MgCl₂, 5 mmol/L dithiothreitol, 0.25 mmol/L spermidine, 10 U of RNase inhibitor (Boehringer Mannheim), and 10 U of AMV reverse transcriptase (Promega). The reaction was carried out at 42°C for 1 hour and terminated at 52°C for 40 minutes. The cDNA was stored at -20°C until use. PCR was performed with 2 µL of the RT reaction mixture, 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 8.3), 1 mmol/L MgCl₂, 0.5 mmol/L of each TM primer, 200 µmol/L each dNTP, and 1.0 U of Taq polymerase (Boehringer Mannheim) in a final volume of 50 µL. Amplification was carried out in a DNA thermal cycler (Perkin Elmer Corp). After an initial denaturation at 95°C for 5 minutes, samples were subjected to 30 cycles (95°C, 1 minute; 58°C, 1 minute; 72°C, 2 minutes). PCR products (10 µL) were analyzed by electrophoresis on a 1% agarose gel. The bovine thrombomodulin primers (National Biosciences Inc) correspond to nucleotides 228 to 256 (sense primer, 5'-CTCGGCAACTACACGTGCATCTGCGAG-3') and 907 to 935 (antisense primer, 5'-GCCACCACCAGAGACAGGCTTGCAATGG-3'), of the published sequence for bovine TM.³⁹ ß-Actin primers (forward primer, 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3'; reverse primer, 5'-CTAGAAGCATTTGCGGTGGACGATGGAGG3'; Stratagene) were used to amplify ß-actin mRNA as a housekeeping nonchanging control for gene expression.

The RT-PCR analysis was performed by comparing the relative change in TM mRNA expression to ß-actin. The number of cycles used³⁰ was within the linear range of the amplification response for both genes. The amplification reaction for each cDNA prepared from BCs and/or no RT blank was monitored by varying cycle numbers (cycle titration, 20, 25, 30, 35, 40, and 45), and 10 µL of the PCR product was analyzed by electrophoresis on 1% agarose gel. Gels were visualized with ethidium bromide staining and ultraviolet transillumination. Photographs were taken with positive/negative type 55 film, (Polaroid Corp), and the negatives were scanned with a Hoefer GS 300 scanning densitometer interfaced to an IBM PC computer with a DT 2805 analog and digital system (Data Translation).

QC-PCR
To validate whether the RT-PCR method as described above could be used for quantitative comparisons, the QC-PCR, as we described,¹³ was used in parallel in some experiments, and the results were compared with densitometric readings obtained with RT-PCR in which the change in TM mRNA level was expressed relative to ß-actin. QC-PCR tubes contained all the amplification reagents (described in the above section), a constant amount of target TM cDNA from each preparation, and serial dilutions of known concentrations of a competitor TM cDNA template. The reaction mixture was coamplified, as described above. The competitor cDNA template was prepared by site-directed mutagenesis.⁴⁰ A single base change of A to G at base pair 346 created a unique Sal I restriction site.

After coamplification, the PCR products were digested with Sal I: 10 µL of the PCR product, 1 U of Sal I restriction enzyme, and 2 µL of enzyme buffer incubated at 37°C for 2 hours. The digested competitor (591 bp) and target (707 bp) cDNAs were separated by electrophoresis on a 2% agarose gel, and visualized by ethidium bromide and ultraviolet transillumination. Negatives were prepared with a Polaroid (Polaroid Corp) and scanned by optical densitometry as above. Density readings of the target cDNA were multiplied by 591/707 to correct for differences in molecular weight. The ratio of amplified versus competitor cDNA optical densities was plotted as a function of competitor template concentration.¹³ The initial concentration of target cDNA was derived from the point at which the ratio of target and competitor cDNA optical density equaled 1.

Thrombomodulin Cofactor Assay
Endothelial cell TM activity was assayed from capillary cellular homogenates by measuring the increase in protein C activation as reported.^26,41,42 After incubation with cytokines, RA, and/or db-cAMP, capillaries were washed with phosphate-buffered saline three times (centrifugation at 10 000 rpm for 5 minutes), the microvascular pellet was sonicated, resuspended in phosphate-buffered saline and spun three times at 10 000 rpm for 5 minutes. Microvascular homogenate samples were assayed for protein content by the method of Lowry et al.⁴³ A standard amount of microvascular proteins from each sample (35 µg) was incubated in 100 µL of assay buffer containing 50 mmol/L Tris-HCl (pH 8.0), 2 mmol/L CaCl₂, 0.1 mol/L NaCl, 0.1% bovine serum albumin, 0.6% Triton X-100, and 2.2 nmol/L of bovine thrombin (Enzyme Research Laboratory), and 65 nmol/L bovine protein C (Enzyme Research Laboratory) for 60 minutes at 37°C. The reaction was terminated by adding antithrombin III (5 µg) and heparin (5 U), and the reaction mixture was centrifuged at 10 000 rpm for 5 minutes. Equal volumes of the reaction mixture and a chromogenic substrate (S-2366, Kabi Pharmacia) were added to a 96-well microtiter plate. Optical densities were measured with a spectrophotometer (EL-311SX, BioTek Instruments) at 405 nm.

Statistical Analysis
Results were compared by analysis of variance. P<.05 was taken to be statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Brain Compartmental and Regional Distribution of TM mRNA
The presence of TM mRNA transcript was initially demonstrated in bovine cortical BCs (Fig 1). RT-PCR performed on BCs and capillary-depleted cortical brain parenchyma indicated the presence of TM mRNA transcript only in BCs (Fig 1A). Neither TM mRNA nor TM activity (data not shown) was detectable in capillary-depleted brain parenchyma from cortex or other regions. As indicated by -glutamyl transpeptidase activity, the contamination of capillary-depleted cortical brain parenchyma by brain microvessels was minimal and <0.5%, as we reported.³⁸ The relative amounts of mRNA for ß-actin gene were comparable in the vasculature and capillary-depleted cortical brain (Fig 1B).

View larger version (30K): [in this window] [in a new window]   Figure 1. The presence of TM mRNA transcript in freshly isolated cortical bovine BCs, and its absence from capillary-depleted brain cortex (CD-B). A, Agarose gel (1%) stained with ethidium bromide showing RT-PCR results obtained with TM-specific primers that correspond to nucleotides 228 to 256 (sense) and 907 to 935 (antisense) of the published sequence of bovine TM in BC (lane 1) and CD-B (lane 2). As an internal control for nonchanging gene expression, the amplification using ß-actin primers in BCs (lane 3) and CD-B (lane 4) is shown. B, Relative abundance of TM and ß-actin mRNA from BCs (solid bars) and CD-B (hatched bars) determined by scanning densitometry. Values are mean±SD, n=5. Values in BCs are arbitrarily set as 1.

We then performed QC -PCR (Fig 2) to determine TM mRNA expression from brain regional capillary preparations. Cortical capillaries expressed the highest concentration of TM mRNA (mean±SD), 24.3±1.2 pg; compared with cerebellar, 19.8±5.7 pg, or pontine, 5.5±0.5 pg. The QC-PCR findings were consistent with the RT-PCR analysis performed on the same BC samples demonstrating reduced TM mRNA expression in the BCs from cerebellum and pons (Fig 3). When values in cortical BCs were arbitrarily set as 1, the RT-PCR and QC-PCR analysis of the regional abundance of BC TM mRNA revealed, in fact, similar results (Figs 2 and 3). For example, in the cerebellum the level of TM mRNA in BCs was 0.85 (RT-PCR) and 0.82 (QC-PCR) of the cortical level, and in the pons the level of TM mRNA in BCs was 0.24 (RT-PCR) and 0.23 (QC-PCR) of the cortical level. As there were no significant differences between the levels of TM mRNA determined by RT-PCR and QC-PCR, in experiments described below the RT-PCR analysis was used as a simpler, less time-consuming. and less-expensive method.

View larger version (30K): [in this window] [in a new window]   Figure 2. QC-PCR analysis of TM mRNA transcript in BCs prepared from different brain regions. A constant amount of unknown target cDNA from cortical, cerebellar, and pontine capillaries was added to PCR tubes containing 10-fold serial dilutions of a competitor cDNA template. After amplification, the PCR products were resolved on a 2% agarose gel. A, QC-PCR analysis of TM mRNA transcript in cortical BCs. Lanes 1 to 6, 103, 102, 101, 100, 10-1, and 10-2 pg of initial competitor template, respectively. With decreasing amounts of competitor cDNA added, there is a decrease of competitor PCR products (591 bp) and a concomitant increase in unknown target PCR products (707 bp). Target and competitor denote target and competitor PCR products with 707 and 591 bp, respectively. B, QC-PCR analysis of TM mRNA transcript in cortical BCs. The optical densities of unknown target and competitor PCR products from gel illustrated in A are plotted against the concentration of competitor template (picograms). The initial concentration of the unknown target cDNA can be derived from the point at which the optical density of the unknown target equals that of the competitor. The same DNA was analyzed in triplicate. C, Amount of TM mRNA (picograms) in BCs from cortex, cerebellum, and pons determined by QC-PCR as explained in "Methods" and illustrated in A and B. Values are mean±SD, n=3.

View larger version (45K): [in this window] [in a new window]   Figure 3. Regional levels of TM mRNA in BCs from cortex, cerebellum, and pons determined by RT-PCR analysis relative to ß-actin mRNA. The BC samples analyzed by RT-PCR in Fig 3 were also assayed by QC-PCR (Fig 2). A, Agarose gel (1%) stained with ethidium bromide showing results of PCR amplifications with ß-actin primers in BC from cortex (lane 1), cerebellum (lane 2), and pons (lane 3) is shown on the left. The amplification using TM-specific primers in these BC samples from cortex (lane 4), cerebellum (lane 5), and pons (lane 6) is shown on the right. The ß-actin and TM PCR products were 313 and 707 bp, respectively. B, There was no difference in the intensity of ß-actin signal from these samples (ACTIN, left). The abundance of TM mRNA was determined relative to ß-actin mRNA by scanning densitometry (TM, right). Values are mean±SD, n=3. Values in cortical BCs are arbitrarily set as 1.

The regional differences in BC TM mRNA level correlated well with differences in TM BC activity determined in BC cellular homogenates by measuring the increase in protein C activation. For example, in BCs from the cortex and pons, the protein C activities expressed as optical density determined at 405 nm were 2.54±0.26 and 0.72±0.07 A₄₀₅/min (mean±SD, n=3), respectively.

Effects of Immunomodulators on BC TM mRNA and Activity
Addition of IL-1ß to freshly isolated cortical BCs produced significant downregulation of TM mRNA with a t_1/2 of 2.1 hours compared with control values obtained when BCs were incubated for the same periods of time with vehicle only. Incubation with vehicle did not result in significant changes in either TM or ß-actin mRNA levels within 24 hours of incubation (data not shown). The TM mRNA signal was undetectable from 12 to 24 hours of incubation with IL-1ß (Fig 4A). The levels of ß-actin mRNA were not altered during the 24-hour period of incubation with IL-1ß (data not shown). A decrease in TM mRNA was accompanied by reduced BC TM activity (Fig 4B), but the TM activity was still measurable and about 20% of control values after 12 hours of incubation with IL-1ß.

View larger version (14K): [in this window] [in a new window]   Figure 4. Time-dependent downregulation of TM mRNA and TM activity in cortical BCs by IL-1ß (10 U/mL). A, Agarose gel (1%) stained with ethidium bromide showing RT-PCR results with TM-specific primers, and TM mRNA abundance relative to ß-actin plotted against the time of incubation. BCs were incubated with IL-1ß for 2, 4, 8, 12, 16, and 24 hours as indicated by their respective lane number marked on the top of the gel. The levels of ß-actin mRNA were not altered by these incubations compared with control values at time zero (not shown). Values in the presence of IL-1ß are normalized to the respective control values obtained with incubation of BCs with vehicle only between 2 and 24 hours. B, TM activity was determined from BC cellular homogenates by measuring the increase in protein C activation as described in "Methods." Protein C activity expressed as optical density determined at 405 nm/min is plotted against the time of incubation. Values are mean±SD, n=3.

Addition of TNF- produced a similar time-dependent decrease of TM mRNA, but with a somewhat longer t_1/2 of 3.9 hours compared with control values (Fig 5A). As with IL-1ß, the levels of ß-actin mRNA were not altered during a 24-hour incubation with TNF- (data not shown). There was progressive reduction in TM activity, and both TM mRNA and TM activities were undetectable after 16 hours of incubation (Fig 5B). Changes in the levels of TM activity followed decreases in TM mRNA, but some minor discrepancies were also noted; for example, between 4 and 8 hours there was a significant drop in TM mRNA level, but no significant difference was found in TM activity (Fig 5B).

View larger version (14K): [in this window] [in a new window]   Figure 5. Time-dependent downregulation of TM mRNA and TM activity in cortical BCs by TNF- (10 U/mL). A, Agarose gel (1%) stained with ethidium bromide showing RT-PCR results with TM-specific primers, and TM mRNA abundance relative to ß-actin plotted against the time of incubation. BCs were incubated with TNF- for 2, 4, 8, 12, 16, and 24 hours as indicated by their respective lane number marked on the top of the gel. The levels of ß-actin mRNA were not altered by these incubations compared with control values at time zero (not shown). Values in the presence of TNF- are normalized to the respective control values obtained with incubation of BCs with vehicle only between 2 and 24 hours. B, TM activity was determined from BC cellular homogenates by measuring the increase in protein C activation as described in "Methods." Protein C activity expressed as optical density determined at 405 nm/min is plotted against the time of incubation. Values are mean±SD, n=3.

Effects of RA and db-cAMP on BC TM mRNA and Activity
A time course evaluation indicated that RA amplifies TM mRNA by 4.2-fold after 4 hours (Fig 6A), followed by a decline in TM mRNA; after 24 hours a 2-fold increase was still observed in comparison with a control incubated with vehicle only (Fig 6B). The TM activity followed a trend of TM mRNA increase, and a 2.6-fold maximal increase was found at 4 hours (Fig 6B). This effect was sustained through 12 hours, but declined at 24 hours toward the values at the beginning of incubation. As with IL-1ß and TNF-, RA did not produce significant changes in the levels of ß-actin mRNA within 24 hours of incubation (data not shown).

View larger version (16K): [in this window] [in a new window]   Figure 6. Time course of upregulation of TM mRNA and TM activity in cortical BCs by RA (10 µmol/L). A, Agarose gel (1%) stained with ethidium bromide showing RT-PCR results with TM-specific primers, and TM mRNA abundance relative to ß-actin plotted against the time of incubation. BCs were incubated with RA for 2, 4, 8, 12, 16, and 24 hours as indicated by their respective lane number marked on the top of the gel. The levels of ß-actin mRNA were not altered by these incubations compared with control values at time zero (not shown). Values in the presence of RA are normalized to the respective control values obtained with incubation of BCs with vehicle only between 2 and 24 hours. B, TM activity was determined from BC cellular homogenates by measuring the increase in protein C activation as described in "Methods." Protein C activity expressed as optical density determined at 405 nm/min is plotted against the time of incubation. Values are mean±SD, n=3.

Exposure to the cAMP analog db-cAMP resulted in a maximal 4-fold increase in TM mRNA by 8 hours (Fig 7A), followed by a decline in TM mRNA level, and about a 2-fold increase relative to control values between 16 and 24 hours (Fig 7B). The peak 2.3-fold increase in TM activity occurred at 4 hours. The effect was sustained through 4 hours, but began to decline toward control values between 12 and 16 hours (Fig 7B). At 24 hours with db-cAMP, the TM activity dropped by about 50% below control in spite of the 2-fold increase in TM mRNA. No changes in the levels of ß-actin mRNA were observed within 24 hours of incubation with db-cAMP (data not shown).

View larger version (15K): [in this window] [in a new window]   Figure 7. Time course of upregulation of TM mRNA and TM activity in cortical BC by db-cAMP (3 mmol/L). A, Agarose gel (1%) stained with ethidium bromide showing RT-PCR results with TM-specific primers and TM mRNA abundance relative to ß-actin plotted against the time of incubation. BCs were incubated with RA for 2, 4, 8, 12, 16, and 24 hours as indicated by their respective lane number marked on the top of the gel. The levels of ß-actin mRNA were not altered by these incubations compared with control values at time zero (not shown). Values in the presence of RA are normalized to the respective values obtained with incubation of BCs with vehicle only between 2 and 24 hours. B, TM activity was determined from BC cellular homogenates by measuring the increase in protein C activation as described in "Methods." Protein C activity expressed as optical density determined at 405 nm/min is plotted against the time of incubation. Values are mean±SD, n=3.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Present data and recent studies^13,19,20 have provided compelling evidence that functionally active TM is expressed and produced by the central nervous system microvascular endothelium and is likely to be an important physiologic anticoagulant in brain microcirculation. Neither TM mRNA nor TM cofactor activity was found in capillary-depleted brain parenchyma (Fig 1). Although conflicting results have been reported on TM expression in brain vasculature, all studies agree that TM is not expressed on astrocytes and neurons in vivo.^14–18 The expression of TM on neonatal astrocytes in vitro⁴ could be linked to its role during brain development to protect neurons from the excess of thrombin's serine protease activity before the tight junctions of the BBB are completely formed.

BCs may be contaminated by astrocytes,^35,36 but it is unlikely that the TM expression observed in this study is on astrocytes, as the TM signal or activity was not found in astrocyte-rich brain parenchyma. Regional amounts of BC TM mRNA have been demonstrated for the first time (Fig 3), and both the RT-PCR and quantitative QC-PCR analysis have revealed similar differences in TM mRNA regional brain BC levels (Figs 2 and 3). This correlated well with regional BC differences in the level of protein C activation determined in cellular homogenates of these capillaries. A previous immunocytochemical study in human brain has suggested that the abundance of TM antigen was reduced in several subcortical regions where brain infraction is common.¹⁴ It is possible that decreased level of BC TM mRNA and reduced endothelial TM activity in certain brain regions may reflect reduced anticoagulant capacity in those local brain microcirculations that could favor thrombosis as in other organs.^8,9 Low circulating levels of APC may predispose to larger strokes and arterial or venous cerebral thrombosis,^9,44 while downregulation of BC TM by stroke risk factors in rats was shown to be associated with enhanced ischemic brain injury.²⁰

Proinflammatory cytokines, IL-1ß and TNF-, are produced by stimulated astrocytes^45,46 and during an ischemic brain insult,^47,48 when they act as key pathogenic mediators of ischemic brain damage.^21,22,49 Both cytokines suppress endothelial cell surface anticoagulant TM activity in the systemic circulation,^23–27 which leads to the formation of fibrin strands on the endothelial cell surface.^23,50 It has also been shown that microvascular fibrin deposition and obstruction during early focal cerebral ischemia and reperfusion contribute to brain parenchymal damage.⁵⁰ This study demonstrates for the first time that IL-1ß and TNF- downregulate TM mRNA and reduce the rate of protein C activation in BCs (Figs 4 and 5). These effects of cytokines are not unexpected since qualitatively similar results were obtained in vascular systemic endothelial cells, ie, in HUVECs.^27,51 Whether brain endothelial cells are more sensitive to cytokine-mediated downregulation than HUVECs remains to be determined. It is likely that these two cytokines may elicit comparable effects on the BBB in vivo under pathologic conditions associated with activation of astrocytes and during cerebral ischemia. Thus, suppression of BBB anticoagulant activity along with tissue factor-initiated coagulation,⁵⁰ may facilitate fibrin formation in brain during ischemia and contribute to ischemic brain damage.

The present results suggest that the effects of both cytokines are mediated at the transcriptional level, which is followed by decreases in TM cofactor activity. Previous reports in HUVECs suggested that TNF- reduces TM transcription, but does not appear to alter TM mRNA stability or protein degradation.^24,52 Other factors may contribute to TM antigen brain endothelial depletion in vivo as shown for neutrophils.²⁶ This study does not suggest a rapid release of TM antigen that would precede TM mRNA downregulation (Figs 4 and 5). It is also unlikely that some minor discrepancies between TM mRNA and TM activity reductions (Fig 5) are due to post-transcriptional mechanisms, such as changes in internalization and endothelial degradation of TM antigen.^53,54 It is possible that these cytokines (eg, TNF-) decrease TM brain endothelial expression by reducing intracellular cAMP content,²⁵ via a cAMP-responsive element in the TM gene,⁵⁵ as in other types of endothelial cells.

RA and cAMP enhance TM biosynthesis in peripheral vascular endothelial cells via elevation of TM mRNA.^27–34 Similar effects were observed in this study in BCs (Figs 6 and 7). RA upregulates TM mRNA transcription via an RA-responsive element located in the 5'-flanking region of the human TM gene.⁵⁶ A cAMP-responsive element in the TM gene has been identified in the 3'-untranslated region.⁵⁵ Thus, the accessibility of the RA-responsive and cAMP-responsive elements to the binding proteins induced in endothelial cells and in different types of blood cells may control TM expression.²⁸ RA at a plasma concentration of 10 nmol/L⁵⁷ may antagonize TM downregulation in HUVECs exposed to TNF-.²⁷ Our preliminary results confirm similar activity of RA in BCs (data not shown), raising the possibility that RA may normally stimulate cerebrovascular TM expression. At pharmacologic concentrations (Fig 6) RA exhibited a more rapid effect on BCs than on HUVECs.²⁹ As circulating RA is taken up by the retinol-binding protein at the BBB,⁵⁸ BCs in vivo are likely to respond to RA treatment by elevating TM activity that may counteract downregulation by cytokines.

Drugs that increase intracellular endothelial cAMP content, such as isoproterenol and forskolin (ie, stimulators of adenylate cyclase activity) or rolipram (ie, inhibitor of cAMP phosphodiesterase activity)^59,60 have a potential to enhance anticoagulant mechanisms in brain. This could be important under pathologic conditions when cytokines and stroke risk factors,^61,62 transform the BBB from an actively anticoagulant into a procoagulant membrane.¹¹ Strategies to increase cAMP levels at the BBB may not only improve the TM anticoagulant function, but also could suppress the procoagulant tissue factor and plasminogen-activator inhibitor-1, while stimulating the production of tissue plasminogen activator, a key fibrinolytic enzyme.^63,64 All these events in turn could restore the antithrombotic properties of the BBB and ameliorate ischemic brain damage.

	Selected Abbreviations and Acronyms

 

APC = activated protein C BBB = blood-brain barrier BC = brain capillary db-cAMP = dibutyryl cAMP HUVEC = human umbilical vein endothelial cells IL-1ß = interleukin 1ß PCR = polymerase chain reaction QC = quantitative-competitive RA = retinoic acid RT = reverse transcription TM = thrombomodulin TNF- = tumor necrosis factor

	Acknowledgments

 
This work was supported by National Institutes of Health Research Grant NS-31945 and the Hoover Foundation.

	Footnotes

 
Departments of Neurosurgery (L.W., M.K., B.V.Z.) and Neurology (N.D.T., M.J.F., S.S.), and Division of Neurosurgery, Childrens Hospital (B.V.Z.), University of Southern California School of Medicine, Los Angeles Calif 90033.

Received June 2, 1997; accepted August 26, 1997.

	References

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