Heparin-Induced Overexpression of Basic Fibroblast Growth Factor, Basic Fibroblast Growth Factor Receptor, and Cell-Associated Proteoheparan Sulfate in Cultured Coronary Smooth Muscle Cells
Basic fibroblast growth factor (bFGF), a potent mitogen for arterial smooth muscle cells (SMCs), plays a pivotal role in the pathogenesis of arteriosclerosis and restenosis. Heparin in nanogram quantities may promote or even be required for binding of bFGF to its cognate receptor. Conversely, heparin in microgram doses is a strong inhibitor of arterial SMC replication in vitro and in vivo. Bovine coronary SMCs (cSMCs) express bFGF, bFGF receptor (FGF-R1), and cell membrane–integrated proteoheparan sulfate (HSPG). These three molecules are known to form a trimolecular complex that promotes signal transduction and mitogenesis. The bFGF synthesized by cSMCs is distributed to an intracellular and a pericellular compartment. Resting cultured cells retain about 80% of their bFGF intracellularly; 20% is found in the pericellular region. During proliferation, 70% to 80% of total bFGF is expressed in the pericellular compartment. Trypsinization generates soluble forms of the complex of bFGF with the ectodomains of the bFGF receptor and cell membrane–integrated HSPG in the pericellular compartment, thus allowing quantification of pericellular bFGF by a highly specific enzyme immunoassay. Standard heparin inhibits the proliferation of cSMCs by up to 80% in a concentration range between 10 and 100 μg/mL medium in a dose-dependent manner but increases the protein content of cSMCs compared with proliferating control cells. The heparin-induced increase in cellular protein content includes a 60% to 100% increase in the expression of pericellular bFGF, FGF-R1, and cell membrane–integrated HSPG. Thus, under heparin treatment, the heparan sulfate side chains of cell membrane–integrated HSPG incorporate more [35S]sulfate, and the proportion of [35S]heparan sulfate among total glycosaminoglycans increases from 36% to 52%. Fluorescence-activated cell sorting analysis and [3H]thymidine incorporation experiments provide evidence for multiple effects of heparin, including blocks at early and late checkpoints of the cell cycle in heparin-treated cells. These results indicate that heparin, despite its antiproliferative potency, stimulates the expression of all components of the bFGF system even in coronary SMCs in which growth is inhibited.
- Received August 21, 1995.
- Revision received February 29, 1996.
Luminal narrowing of coronary arteries seen after experimental vascular injury or angioplasty is considered to be due primarily to replication of the cSMCs.1 Recent data suggest that endogenous bFGF, known to be synthesized by aSMCs,2 plays a pivotal role in the stimulation of SMC replication in vivo3 4 5 and in vitro.2 6 7 Klagsbrun and Edelman,4 Lindner and Reidy,5 and Burgess and Maciag6 have shown (1) that bFGF released from aSMCs after injury is a potent mitogen, (2) that SMC replication appears to be correlated to arterial damage, and (3) that bFGF- or injury-induced SMC replication is significantly inhibited by anti-bFGF antibodies.
In previous studies,7 we have shown that bFGF expressed by cultured aSMCs in the G0 phase is retained primarily intracellularly but is delivered to the pericellular compartment during proliferation, reaching maximum values in the phase of exponential growth.
Heparin has a dual function in the replication of aSMCs. Recent data from several laboratories6 8 9 10 suggest that HSPG or heparin in nanogram quantities may promote or even be required for binding of bFGF to its cognate receptor and that the formation of a trimolecular complex of bFGF, FGF-R1, and HSPG is directly involved in bFGF signaling. Conversely, heparin in microgram doses is a strong inhibitor of SMC replication in vitro11 12 and in vivo.13 In 1977, Clowes and Karnovsky14 found that heparin suppresses SMC proliferation associated with damage of endothelial cells of the rat carotid artery. However, the mechanisms by which heparin inhibits the proliferation of SMCs are not well understood. Studies on cultured SMCs indicated that an excess of heparin competes with the native HS side chains of cell-associated HSPG for binding of bFGF but that exogenous heparin does not promote binding of bFGF to the FGF-R1 with resultant stimulation of mitogenic activity.15 In a rat carotid artery model of balloon catheter–induced injury, injection of heparin at the time of ballooning reduces SMC proliferation in part by removal of released bFGF from sites of injury.3 Experiments on the expression of c-fos and c-myc and studies on the metabolic block of the cell cycle under the influence of heparin have produced equivocal results.16 17 18 19 20
In the present study, we investigated the effect of growth-inhibitory doses of heparin on the compartmentalization of bFGF and on the expression of bFGF, FGF-R1, and HSPG in cSMCs in culture. All components were quantified by highly sensitive enzyme immunoassays. Our results show that heparin is a strong inducer of synthesis of bFGF, FGF-R1, and HSPG, even though the growth of cSMCs is simultaneously inhibited.
Heparin sodium salt (grade II) from porcine intestinal mucosa (170 USP units/mg) and cycloheximide were purchased from Sigma Chemical Co. [35S]Sodium sulfate (carrier free, 0.8 to 1.5 TBq/mg sulfur) and [6-3H]thymidine (1.10 TBq/mmol) were obtained from Amersham Buchler. Chondroitin ABC lyase was a product of Seikagaku Corp. Cell culture media were purchased from Seromed; FCS and soybean trypsin inhibitor were supplied by Boehringer-Mannheim. The bFGF ELISA system was a product of Amersham International. Standard chromatography media were from Pharmacia LKB Technology. All other chemicals were of analytical grade or the best grade available and were purchased from Boehringer-Mannheim, Merck, or Serva. The monoclonal antibody (MAB125) against the FGF-R1 was obtained from Chemicon International Inc. This monoclonal antibody also cross-reacts with the bek gene product to a lesser degree. All other antibodies were purchased from Sigma.
Primary cultures of cSMCs were isolated from explants of bovine coronary blood vessels and cultured in medium with 10% FCS at 37°C in a humidified, 5% CO2/95% air atmosphere as described previously.21 Cultures of the third to sixth passages were used in the experiments. Cell growth was measured by cell counting. In labeling experiments, the low-sulfate culture medium BME (basal medium Eagle) contained 925 kBq/mL of [35S]sulfate. Cells were labeled for 48 hours. The time interval between processing of cells for ELISA (see below) and measurement was <1 hour. [3H]Thymidine (74 kBq) was added 8 hours before the end of the experiment.
To assay for inhibitory activity of heparin, 25 000 cSMCs were seeded into Petri dishes (diameter, 35 mm) in medium supplemented with 10% FCS. After 6 hours, the medium was replaced by fresh medium containing 10% FCS and different heparin concentrations (25 to 100 μg/mL). The control cultures were exposed to medium supplemented with 10% FCS or 0.1% FCS. Ninety-six hours later, the cell numbers were measured in duplicate samples with a Coulter counter.
Determination of DNA Content by Flow Cytometry
About 5×105 cSMCs were trypsinized for 5 minutes at 37°C. For analysis of nuclear DNA, the released cells were stained with propidium iodide with the Cycle Test Plus DNA Reagent Kit from Becton Dickinson (catalogue No. 340242). The DNA content was analyzed by flow cytometry on a fluorescence-activated cell sorter. To determine the proportion of cells in the G0/G1, S, and G2/M phases of the cell cycle, the resulting fluorescence histograms of the DNA content were analyzed by the gaussian fitting method.22
DNA was prepared from nuclei isolated from 3×106 cells according to Tata.23
For determination of bFGF compartmentalization, cultured cells were processed as follows.
1. For pericellular bFGF, the medium of cultured SMCs in 35-mm-diameter Petri dishes at the specified growth status was removed and the cell layer was washed exhaustively with PBS and trypsinized (0.3 mL 0.05% trypsin/0.02% EDTA in PBS). After addition of 0.15 mL of 0.042 mol/L soybean trypsin inhibitor, the cells and the trypsin digest were separated by centrifugation. The supernatant containing the pericellular bFGF was stored at 4°C for analysis (enzyme immunoassay) and further experiments.
2. For intracellular bFGF, the trypsinized cells were washed with PBS and lysed in 200 μL PBS containing protease inhibitors according to Oegema et al24 and 1 mmol/L PMSF. The lysate was subjected to repeated (three times) freezing and thawing, ultrasonication for 5 seconds (50 W), and centrifugation. The supernatant containing the intracellular bFGF was used for analysis and further experiments.
3. For total bFGF, cultured cells were processed as in 2, but trypsinization was omitted.
4. For heparin-releasable bFGF, in independent experiments, cells at the specified growth status were washed with PBS and incubated with 500 μL of a 0.01% heparin solution in PBS for 20 minutes. The heparin solution that contained a part of the pericellular bFGF (see above) was stored at 4°C for enzyme immunoassay.
bFGF Enzyme Immunoassay
The enzyme immunoassay uses the quantitative “sandwich” enzyme immunoassay technique. The assay system (Amersham Life Science, RPN 2158) was described previously.7
HSPG from freshly obtained bovine aorta was prepared according to Schmidt and Buddecke21 : 1 mg of the purified HSPG was dissolved in 0.5 mL PBS. Antibodies were raised by primary subcutaneous injection of the solution into male rabbits. After 2 weeks, boost injections were administered subcutaneously every 2 weeks until a satisfactory response was obtained.
HSPG Enzyme Immunoassay
The cell-associated HSPG concentration was determined with an enzyme immunoassay as described by Boehringer Mannheim Biochemica (catalogue No. 1471678), with the exception that a polyclonal antibody against bovine arterial tissue HSPG21 was used. The samples and the standard (2 to 60 ng HSPG/mL) were suspended in 0.1 mol/L Tris/HCl, pH 7.4, and 0.3 mol/L MgCl2.
FGF-R1 Enzyme Immunoassay
For enzyme immunoassay of FGF-R1 protein, the cells were processed as described for the HSPG enzyme immunoassay (see above). The samples were incubated with a mouse anti–FGF-R1 monoclonal antibody (see “Methods”). A horseradish peroxidase–conjugated monoclonal anti-mouse IgM (μ-chain specific) was used as second antibody. After complete staining, the FGF-R1 content was determined in an ELISA reader at 405 nm within 20 minutes.
Isolation of Cell-Associated HS
Cells were labeled with [35S]sulfate for 48 hours, washed several times with PBS, and digested with papain (12 U crystallized papain No. P4762, Sigma) after addition of cysteine (0.005 mol/L) and EDTA (0.01 mol/L). The digest containing the cell-associated [35S]glycosaminoglycans was processed as described previously.7
Chondroitin sulfate and dermatan sulfate were assayed with chondroitin lyase ABC according to Saito et al.25 Cell counting and protein determination were performed by standard methods.
Results are expressed as mean±SD of the specified number of experiments carried out on different cultures of cSMCs in duplicate or triplicate. Statistical significance was assessed with Student's paired t test.
Expression and Compartmentalization of bFGF in cSMCs
Cultured bovine cSMCs express 3 to 12 ng bFGF/mg cell protein (75 to 280 pg/105 cells), depending on their growth status. The bFGF is distributed between an intracellular and a pericellular compartment. The latter can be released from the cell surface by trypsinization. In immunoblot analysis, both intracellular and pericellular bFGF were identified as dominant 18-kD double bands (data not shown). Since trypsinization also removes the ectodomains of the FGF-R1 and the cell membrane–integrated HSPG, which together form a trypsin-resistant complex with bFGF, pericellular bFGF can also be quantified by an enzyme immunoassay without loss due to proteolysis.
Fig 1⇓ shows that the amounts of both pericellular and intracellular bFGF change during transition of cSMCs from exponential growth to confluence. Trypsinized cells lack pericellular bFGF (day 0 in Fig 1⇓). However, there is a rapid regeneration of the pericellular bFGF during proliferation, which reaches 70% to 80% of total bFGF at day 3 to 4 (P<.01) and declines to 10% to 20% of total bFGF with increasing cell density at day 10 (P<.01 in relation to day 3). During this period, the concentration of intracellular bFGF shows a sharp decrease, with a minimum at day 3 to 4 (P<.01), but increases again and reaches maximal values at confluent growth status. The fall of total bFGF within the first days of culturing (Fig 1B⇓) might result from intracellular proteolytic degradation. Release from the cells into the culture medium during this period could be excluded, since no bFGF was detected in the culture medium. Native FCS does not contain detectable amounts of bFGF. Proteolytic degradation of bFGF within the medium could be excluded (see below).
About 20% of the bFGF appearing at the cell surface during exponential growth (days 1 to 4) may be removed by incubation of the washed cells with PBS containing 100 μg/mL heparin for 20 minutes (Fig 1⇑). However, after incubation of the cells under culture conditions for 4 days in the presence of heparin, no bFGF could be detected in the culture medium.
The quantitative recovery of the individual bFGF fractions was assessed in independent experiments. Authentic (recombinant) bFGF (15 pg) was added to each 200 μL of the intracellular and pericellular bFGF-containing fraction. The subsequent quantitative bFGF enzyme immunoassay revealed a correspondingly higher bFGF concentration and a recovery of the added exogenous bFGF of 95% to 98%.
Exogenous bFGF added to cell-free culture medium could be quantitatively recovered after 24 hours at 37°C.
The bFGF contents of cSMCs and aortic SMCs are similar,7 but slight quantitative differences were found in the RNA content (23.6±4.1 in cSMCs versus 16.7±2.5 pg/cell in aortic SMCs) and in the susceptibility to the inhibitory action of heparin. Dose-response curves revealed maximal inhibition at 100 μg/mL heparin. At this concentration, aortic SMCs displayed 50% to 60% and cSMCs 75% to 80% inhibition of proliferation.
Heparin Inhibits Cell Proliferation but Increases the Expression of bFGF, FGF-R1, and HSPG
Autocrine/paracrine growth stimulation of cSMCs by bFGF requires the expression of FGF-R1 and the cell membrane–integrated HSPG,10 which has been shown to possess bFGF-binding capacity. Table 1⇓ shows the presence of all components required for biological activity of bFGF in proliferating cSMCs. On a molar basis, the HSPG is expressed in a 50-fold excess over bFGF. The figures for FGF-R1 refer to arbitrary units and do not reflect absolute values.
Heparin treatment of cSMCs appeared to significantly increase the level of endogenous bFGF (P<.01). The data in Table 2⇓ show an increase of total bFGF from 90 pg/105 cells (control) to 240 pg/105 cells after exposure to 50 μg/mL heparin for 4 days. The pericellular bFGF increased to a greater extent than intracellular bFGF, with a low statistical significance (P<.05). A comparison of the bFGF content per cell of control and heparin-treated cultures reveals that the bFGF content of controls decreases from time 0 to day 4, whereas the bFGF content of heparin-treated cells remains fairly constant. However, the content of protein (Table 2⇓), DNA (Table 3⇓), and [35S]glycosaminoglycans is doubled over the same time period in heparin-treated cells, indicating de novo synthesis processes under the influence of heparin (see “Discussion”).
As is known from numerous previous studies,11 12 13 heparin inhibits the proliferation of arterial SMCs in a dose-dependent fashion. As Fig 2⇓ shows, in our bioassay system, heparin causes inhibition of cSMC proliferation by about 70%. A statistically significant reduced cell number (P<.01) is detectable in a range of 25 to 100 μg/mL heparin. Under these conditions, heparin not only increases the expression of bFGF (Table 2⇑) but also causes overexpression of FGF-R1 and of the cell-associated HSPG, both of which are required for the biological activity of bFGF. The data in Fig 2⇓ indicate a continuous elevation of bFGF, FGF-R1, and HSPG with increasing heparin concentration. Statistically significant differences (P<.01) for all components were calculated at 50 μg/mL heparin and higher concentrations. The increase of these components is part of a general increase of cell protein, which was found to be 38 μg/105 cells at 50 μg/mL heparin in comparison with 25 μg/105 cells in controls (P<.02).
The heparin-induced increase of bFGF, FGF-R1, and HSPG levels cannot be elicited by other sulfated glycosaminoglycans. Castellot et al11 showed for the first time that other artery-specific glycosaminoglycans such as chondroitin 4- and 6-sulfate, dermatan sulfate, or hyaluronan in doses up to 100 μg/mL have absolutely no inhibitory effect on SMC growth and division. We have confirmed this in previous studies.26 27
The HSPG values in Fig 2⇑ are based on quantitative enzyme immunoassay determination of the protein core. To explore the biosynthesis of the HS side chains, the influence of heparin on the incorporation of [35S]sulfate into the total and individual glycosaminoglycans was investigated. Table 4⇓ indicates the stimulation of [35S]sulfate incorporation into the total glycosaminoglycans and the shift in the distribution pattern of HS and chondroitin sulfate/dermatan sulfate, the latter comprising the side chains of decorin, biglycan, and versican. It is evident from statistical values (P<.02) that the proportion of HS to chondroitin sulfate/dermatan sulfate increases (Table 4⇓).
The growth status of SMCs also influences the bFGF expression. Under culture conditions with low serum (0.1% FCS), the concentration of total bFGF is higher after a 4-day culture period (169 pg/105 cells) than in cells cultured in the presence of 10% FCS (90 pg/105 cells). However, the bFGF values are significantly higher in heparin-inhibited cells (240 pg/105 cells, see Table 2⇑), even in the presence of 10% FCS.
Heparin Has Multiple Inhibitory Effects on the Cell Cycle
Experiments were performed to identify the phase at which heparin acts in the cell cycle to inhibit cSMC proliferation. cSMCs cultured for 96 hours in the presence of 50 μg/mL heparin or control cells were submitted to flow cytometry after staining with propidium iodide and DNA determination. Table 3⇑ shows that most of the control cells are in the G0/G1 phase, fewer heparin-treated cells were in the G0/G1 phase, and there was an increase in the proportion of the cells in the G2/M phase from 9.0% (control cells) to 27.2% (heparin-treated cells). The shift in the ratio of G0+G1/G2+M phase (Table 3⇑) indicates that at least one target of heparin is late in the cell cycle. The increase of the DNA content of cSMCs under the influence of heparin from 7.7 to 15.8 pg/cell clearly illustrates at least a partial transition of the cells through the S phase in the presence of heparin.
The results obtained by use of the fluorescence-activated cell sorter (Table 3⇑) have also been examined by [3H]thymidine experiments. The cells were cultured in the presence or absence of 50 μg/mL heparin, and [3H]thymidine incorporation was determined at times specified in Fig 3⇓. [3H]Thymidine addition was 8 hours before the end of the experiments. The data in Fig 3⇓ reveal a partial block of entry of the cells into the S phase at all time points, indicating that heparin, in addition to its G2/M target, is also capable of blocking the cell cycle at an early phase.
The growth status of arterial SMCs can be modulated by heparin in two different ways. On the one hand, heparin has high-affinity binding capacity to bFGF and may promote its mitogenic activity.28 On the other, heparin is a potent antiproliferative agent for vSMCs in vitro11 12 and in vivo after vascular injury.13 29 30 31 However, the precise mechanism by which heparin inhibits cell division is poorly understood.
We examined the action of heparin on cSMCs and found that, despite its antiproliferative capacity, heparin produces an overexpression of bFGF, FGF-R1, and the cell-associated HSPG. Cell cycle analysis by fluorescence-activated cell sorter scanning, [3H]thymidine experiments, and DNA determination showed that the metabolic block in response to heparin occurs at multiple targets, including blocks at early and late phases of the cell cycle. Despite the inhibited mitosis, the SMCs produce an excess of cellular protein, including all components of the bFGF system. However, the overexpression of the bFGF system does not result in cell division.
The bFGF content per cell is significantly higher in heparin-treated than in control cells (Fig 2⇑), but this is due primarily to the proliferation-induced decrease of bFGF concentration of control cells (Fig 1⇑), whereas the bFGF content of heparin-treated cells remains fairly constant (200 pg/105 cells at day 0, Fig 1B⇑; 240 pg/105 cells at day 4, Fig 2⇑ and Table 2⇑). The question of whether the bFGF content of heparin-treated cells, like the control cells, shows an intermediate decrease was addressed by an independent experiment. When cells were incubated with a combination of heparin and 2.5 μmol/L cycloheximide, the bFGF synthesis was suppressed (180 pg/105 cells), so that at day 4 the bFGF content (A.S.-R. et al, 1995, unpublished results) is essentially lower, as in heparin-treated cells in the absence of cycloheximide (270 pg/105 cells). This indicates that even in the presence of heparin, the bFGF content of trypsinized cells goes down during the first days of culturing. Further evidence for a de novo synthesis of bFGF under the influence of heparin could be provided by bFGF labeling and pulse-chase experiments.
No conclusions can be drawn as to whether the synthesis of the components of the bFGF system occurs in the G1 or G2 phase. However, a metabolic block of the cell cycle in the G1 phase provoked by chlorate (Ch. Schriever et al, 1995, unpublished results) also leads to overexpression of all components of the bFGF system.
Previous studies on the mechanism of the growth-inhibitory action of heparin gave controversial results. Castellot et al31 and Pukac et al17 concluded from in vitro studies that the proliferation of rat aorta SMCs was inhibited during the G0/G1 phase of the cell cycle. Wright et al16 suggested that in murine 3T3 fibroblasts, heparin suppresses the induction of c-fos and c-myc mRNA at a site distal to activation of the protein kinase C. Also, Bennet et al18 showed for vSMCs that heparin-induced growth arrest was accompanied by downregulation of c-myc protein mRNA. In contrast, Hamon et al19 found that in heparin-treated rabbits, the level of expression of c-myc, c-fos, and c-jun was not associated with an overall decrease after balloon denudation of the aorta. The level of these proto-oncogenes was similar in heparin and control groups. Therefore, it was concluded that the antiproliferative effect of heparin must be due to an effect on events of the cell cycle that do not involve the G0/G1 phase. Furthermore, Grainger et al20 found that heparin at a concentration between 5 and 100 μg/mL inhibited the proliferation of vSMCs but did not affect the entry of the cells into the S phase. In accordance with our data, heparin-treated cell populations contained significantly more cells in the G2/M phase of the cell cycle than control populations. The possible explanation for these wide variations in the effect of heparin on vSMCs could be the much higher concentration of heparin required to inhibit the entry of cells into the S phase. Castellot et al31 also found that 100 μg/mL heparin allowed 71% of the cells a (delayed) entry into the S phase (compared with 83% of control cells), but at high doses (400 μg/mL heparin) only 12% were able to enter the S phase. Collectively, all studies, including the data of Table 4⇑ and Fig 3⇑, suggest a concentration-dependent function of heparin that may have multiple targets for its antiproliferative mechanism of action, including blocks at early and late checkpoints of the cell cycle.
About 20% of the pericellular bFGF can be released from the cell surface by a short incubation of cSMCs with 100 μg/mL heparin (see “Methods”), and the released bFGF can be detected in the medium. However, after 4 days of exposure of the cells to heparin, no bFGF is present in the culture medium. This may be explained by the finding that vSMCs possess numerous high-affinity binding sites for heparin.32 In the presence of exogenous heparin, half of the bound heparin is internalized within 2 hours, with the remainder being internalized in 1 to 2 days.32 33 The heparin-bound bFGF of the culture medium might be cleared via this receptor-mediated pathway. Internalization of bFGF may also occur via the bFGF-binding capacity of cell surface HSPG.34 The rapid turnover of HSPG suggests that HS-mediated internalization may merely reflect the fact that HS-bound bFGF is simply carried along as the cell surface HSPG is turned over. This provides the mechanism for clearing bFGF that accumulates in the pericellular environment through interaction with HS.
The accumulation of extracellular proteoglycans is an important component of the development of intimal thickening in response to artery injury. Our study indicates that heparin can stimulate proteoglycan synthesis of cSMCs irrespective of their state of proliferation. Thus, in heparin-treated cSMCs, the [35S]sulfate incorporation into the cell-associated proteoglycans increased by 100%; in addition, the distribution pattern of sulfated glycosaminoglycans shifted toward a higher percentage of HS. An increased [35S] radioactivity in cell-associated HS and chondroitin sulfate of bovine aortic SMCs in the presence of 100 μg/mL heparin was also observed by Vijayagopal et al,35 but in the rat carotid artery subjected to balloon catheter injury, heparin treatment (0.3 mg·kg−1·h−1 IV) did not significantly alter the steady state mRNA levels for the HS-containing proteoglycans perlecan and syndecan in intimal-medial preparations.36
Our results have important clinical implications. Restenosis after coronary angioplasty remains a significant problem. Experimental work suggests that appropriate use of heparin may be beneficial through inhibition of cSMC proliferation, but limited clinical studies to date have failed to show therapeutic efficacy.37 A possible explanation for this lack could be our finding that heparin therapy causes an accumulation of bFGF, FGF-R1, and HSPG, which might also occur at the site of angioplasty-induced injury. Since the heparin effect is reversible, a rebound effect triggered by the elevated bFGF level after the end of heparin therapy cannot be excluded.
Selected Abbreviations and Acronyms
|bFGF||=||basic fibroblast growth factor|
|ELISA||=||enzyme-linked immunosorbent assay|
|FCS||=||fetal calf serum|
|SMC||=||smooth muscle cell|
The work was supported by the Deutsche Forschungsgemeinschaft (SFB 310/B6). The authors thank Barbara Glaß and Marc Levejohann for skillful technical assistance. We are grateful to Dr Paul Cullen for helpful discussion of the manuscript.
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