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Am J Physiol Heart Circ Physiol 289: H251-H257, 2005; doi:10.1152/ajpheart.00206.2004
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Assembly, activation, and signaling by kinin-forming proteins on human vascular smooth muscle cells

Anthony N. Fernando,* Lawrence P. Fernando,* Yu Fukuda, and Allen P. Kaplan

Divisions of Pulmonary and Critical Care, Allergy and Clinical Immunology, Department of Medicine, Medical University of South Carolina, and Konishi-Medical University of South Carolina Institute for Inflammation Research, Charleston, South Carolina

Submitted 4 March 2004 ; accepted in final form 2 February 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cardiovascular disease is the number one cause of death in the United States. Vascular smooth muscle cells (VSMC) are an important constituent of the vessel wall that can bring about pathological changes leading to vascular disease. Depending on the environment, the function of VSMC can deviate profoundly from its normal contractile role. Despite advances in research, the underlying mechanisms that activate VSMC toward vascular disease are poorly understood. For the first time, we have observed that factor XII and high-molecular-weight kininogen, constituents of the blood plasma, can bind to VSMC in a Zn2+-dependent manner. In the presence of prekallikrein, this assembly of factor XII and high-molecular-weight kininogen on VSMC leads to the activation of prekallikrein to kallikrein with a rapid formation of bradykinin. The amount of bradykinin in the culture medium then decreases, presumably because of the presence of a kininase activity. p44/42 mitogen-activated protein kinase is rapidly phosphorylated in response to in situ-generated or in vitro-added bradykinin and is inhibited by bradykinin antagonist HOE-140. Binding of factor XII to VSMC also results in a concentration-dependent phosphorylation of p44/42 mitogen-activated protein kinase. This early mitogenic signal, which is also implicated in atherogenesis, may change the metabolic and proliferative activity of VSMC, which are key steps in the progression of atherosclerosis.

kallikrein; mitogen-activated protein kinases; vascular endothelial cells

PROTEINS OF THE PLASMA BRADYKININ (BK)-forming pathway have been shown to interact with microvascular endothelial cells, astrocytes (4, 5), neutrophils (31), platelets (8, 10), and human umbilical vein endothelial cells (HUVEC) by binding to proteins expressed along the cell surface (14). With the use of HUVEC as a model system, activation of the plasma kinin cascade along the cell surface has been demonstrated with conversion of prekallikrein (PK) to kallikrein and digestion of high-molecular-weight kininogen (HK) to release BK (32). Activation of kinin-forming proteins can occur in the presence and absence of factor XII. The latter circumstance requires cell-derived proteins as activators, two of which have been identified to be heat shock protein 90 (HSP90) (13) and prolylcarboxypeptidase (24).

During circumstances of endothelial cell injury, smooth muscle cells become exposed to plasma constituents, including those comprising the kinin-forming cascade (6a). Vascular smooth muscle cells (VSMC) are an important constituent of the arterial wall that participate in pathological changes leading to vascular disease. The normal function of VSMC is contractile in nature. However, given the right environment, the VSMC are capable of undergoing proliferation or apoptosis and synthesize cytokines, type I collagen, and matrix metalloproteinases as well as their tissue inhibitors, resulting in an expansion of matrix (3). Despite advances in research, the underlying mechanisms that activate VSMC toward vascular disease are poorly understood, and cardiovascular disease remains the number one cause of death in the United States. In this study we have examined the binding of the plasma BK-forming pathway proteins to human umbilical artery smooth muscle cells, as well as their activation to liberate BK. We have demonstrated that factor XII and HK binding to VSMC is saturable, reversible, and Zn2+ dependent with liberation of BK when PK is added. We also have observed activation of these cells as a result of this interaction, leading to phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) by both factor XII binding to the cells and interaction of liberated BK with B2 kinin receptors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
PK, factor XII, and HK were purchased from Enzyme Research Laboratories (South Bend, IN). SDS-PAGE analysis of factor XII and HK revealed a single band at 80 and 115 kDa, respectively, indicating that neither protein is cleaved (data not shown). Streptavidin conjugated to horseradish peroxidase (HRP), biotinylation, and tetramethyl benzidine (TMB), peroxidase substrate kits (Pierce, Rockford, IL); (4-amidophenyl)methanesulfonyl fluoride (APMSF; Calbiochem-Novabiochem, San Diego, CA); the kallikrein synthetic substrate H-D-propyl-L-phenylalanyl-L-arginine p-nitroaniline dihydrochloride (S2302; Kabi Pharmacia Hesper, Franklin, OH); ready-to-use 12% SDS-PAGE and polyvinylidene difluoride (PVDF) transfer membranes and 10x SDS-Tris glycine gel running buffer (Bio-Rad, Richmond, CA); and specific antibodies to total and phosphorylated forms of p44/42 (Cell Signaling Technology, Beverly, MA) were purchased as indicated. The antibody to BK was a gift from Dr. David Proud (Johns Hopkins University, Baltimore, MD). 125I-labeled [Tyr0]-BK was purchased from Peninsula Laboratories (San Carlos, CA).

Cell culture. Human umbilical artery smooth muscle cells (VSMC) were purchased from Clonetics (San Diego, CA) and cultured as instructed by the supplier. The cells were grown in smooth muscle growth medium (SmGM-2) supplemented with the necessary growth factors (insulin, human fibroblast growth factor, human epidermal growth factor; Bio-Whitaker, San Diego, CA) in a humidified incubator at 37°C with 5% CO2.

Inactivation of contaminating proteases from PK, HK, and factor XII. Any contaminating proteases were deactivated as described by Fernando et al. (4, 5). PK, factor XII, and HK were treated with APMSF (2 mM) for 90 min on ice to inactivate contaminating serine proteases and were then diluted in the binding buffer (10 mM HEPES, 137 mM NaCl, 4 mM KCl, 11 mM D-glucose, and 0.1% BSA, pH 7.4) and left on ice for an additional 1 h to inactivate the residual APMSF.

Biotinylation of HK and factor XII. HK and factor XII were biotinylated so that the binding of these proteins to the cell surface could be monitored via the color development system by using the peroxidase activity of HRP linked to streptavidin. Biotinylation of HK and factor XII was carried out as suggested by the manufacturer. The concentrations of the purified biotinylated proteins were determined by Coomassie Protein Assay reagent (Pierce Rockford, IL).

Binding of biotinylated HK or factor XII. The binding of the biotinylated proteins was carried out as described by Zhao et al. (32). The cells were washed with the binding buffer with or without Zn2+ (three times with 150 µl/well) and incubated with different amounts of biotinylated HK or factor XII for the indicated period of time in binding buffer with or without Zn2+ (50 µM). The unbound protein was washed off. The amount of bound biotinylated protein was determined by treatment with HRP-conjugated streptavidin (1 µl of a 1 mg/ml HRP-streptavidin solution diluted to 10 ml). The cells were washed three times with their respective binding buffers. The color was developed by the addition of 100 µl of a 1:1 mixture of H2O2 and TMB reagent. The peroxidase reaction was stopped with 100 µl of 2 M sulfuric acid, and the optical density at 450 nm (OD450) was read as a function of the concentration of bound protein in an automated ELISA plate reader.

Binding of fluorescent-labeled HK or factor XII. VSMC were grown in eight-well chamber slides. When the cells were ~75% confluent, the growth medium was changed to serum- and growth factor-free culture medium. The cells were washed with binding buffer and fixed with 5% paraformaldehyde. The cells were then incubated with FITC-labeled HK, rhodamine-labeled factor XII, or a mixture of both labeled proteins (5–10 µg/ml) for 1 h at room temperature and washed well with the binding buffer to remove nonspecifically bound proteins. The cells were observed using fluorescent microscopy.

Activation of PK on VSMC. PK activation was carried out on confluent cultures grown in 96-well tissue culture plates. The cells were incubated with S2302 (0.6 mM) and different combination of PK, factor XII, and HK (1 µg/ml) (see Fig. 5). The kinetics of formation of chromogenic ligand p-nitroaniline caused by the cleavage of S2302 by the in situ-formed kallikrein were monitored by reading the optical density at 405 nm in an automated ELISA plate reader.



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  		Fig. 5. Activation of kinin-forming proteins on VSMC. VSMC were grown in 96-well plates until they reached ~90% confluence. The cells were serum- and growth factor-starved for 18–24 h. The cells were incubated with the indicated combinations of prekallikrein (PK), factor XII, and HK (1 µg/ml) in the binding buffer containing Zn2+. All of the combinations received S2302 (0.6 mM), a chromogenic kallikrein substrate. The rate of conversion of PK to kallikrein was monitored as the cleavage of S2302 with increasing time by measuring the optical density at 405 nM in an automated ELISA plate reader. {bullet}, Factor XII and HK; {square}, factor XII and PK; {triangleup}, HK and PK; x, factor XII, HK, and PK. Results are representative of 3 values.

 
Western blot analysis of signaling proteins. The cells were grown in 12-well plates until the monolayers reached ~80–95% confluence. The growth medium was removed and supplemented with fresh culture medium containing BSA (0.1%) and Zn2+ (50 µM) but lacking the growth factors. The cells were further incubated for 20–24 h and treated (see Figs. 6 and 7). The cells were scraped into 100 µl of cell lysing buffer (0.6 M Tris, 10% glycerol, 5% 2-mercaptoethanol, and 5% SDS, pH 6.8). Equal amounts of the cell lysates were separated on SDS-PAGE, transferred to PVDF membranes, and probed with specific antibodies for total p44/42 MAPK protein and then for phospho-p44/42 MAPK protein. The signals were detected using autoradiography.



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  		Fig. 6. Activation of VSMC by in situ-generated bradykinin (BK). VSMC were grown as described in MATERIALS AND METHODS and made quiescent by replacing the growth medium with serum- and growth factor-free culture medium. The cells were treated at 37°C as shown in a cell culture incubator. A: control cells (group A) received no further treatment. Cells in the HOE-140 group (B) were treated with the B2 kinin receptor antagonist HOE-140 (10–5 M) for 40 min and then received no further treatment. Cells in group E received BK (10–9 M) for 7 min. HK, PK, and factor XII (each at 5 µg/ml) were added for 7 min (group D). Cells in groups C and F were preincubated with HOE-140 for 40 min, and then BK (group F) or HK, PK, and factor XII (group C) were added and cells were incubated for 7 min. Phosphorylation of p44/42 MAPK subunits were assessed using Western blot analysis by employing specific antibodies that detect total or phosphorylated (phospho) forms of p44/42 MAPK. B: band densities expressed as a fold increase relative to the control band. Results are means ± SD of 3 or more values.

 


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  		Fig. 7. Activation of VSMC by binding of factor XII. VSMC were grown in 6-well plates, and the growth medium was replaced with culture medium lacking serum and growth factors 20–24 h before stimulation. A: cells were incubated at 37°C with increasing concentrations of factor XII (A, 0; B, 1; C, 2.5; D, 5; E, 10; and F, 15 µg/ml) for 7 min. The cells were extracted, and phosphorylation of p44/42 MAPK subunits was investigated using Western blot analysis of the VSMC extracts by employing specific antibodies that detect total or phosphorylated (phospho) forms of p44/42 MAPK. B: band densities expressed as a fold increase relative to the control band. Results are means ± SD of 3 or more values.

 
Treatment of VSMC with kinin-forming proteins and storage of conditioned media. The cells (80–90% confluent) grown in serum- and growth factor-free culture medium were stimulated with a mixture of HK or a combination of HK, PK, and factor XII for 7 min, 1 h, or 24 h. The culture medium was collected, and 9 volumes of culture medium were mixed immediately with 1 volume of an inhibitor cocktail (20 mg/ml EDTA, 4 mg/ml polybrene, 10 mg/ml 1,10-phenantholene, 1.67 mg/ml aprotinin, and 5 mg/ml soy bean trypsin inhibitor) and centrifuged (900 g, 20 min, 4°C) to remove cells. The supernatants were stored at –80°C until measurements were made.

Radioimmunoassay for BK determination. Quantitation of BK was performed as described (19) with some modifications. Culture medium (50 µl) was precipitated at 4°C for 3 h by adding 100 µl of ethanol. The mixture was centrifuged for 20 min at 12,000 rpm, and the supernatants were collected and evaporated. The residue was dissolved in 500 µl of 100 mM sodium phosphate (pH 7.4), 3 mM 1,10-phenantholene, and 10 mM EDTA [radioimmunoassay (RIA) buffer] with 0.1% gelatin. After vigorous mixing with an equal volume of 1,1,2-trichlorotrifluoroethane, 100 µl of the aqueous fraction were incubated at 4°C overnight with anti-BK antibody and 125I-[Tyr0]-BK (10,000 cpm). The BK-antibody immunocomplexes were then precipitated with 12.5% polyethylene glycol 8,000 by using bovine IgG as a carrier protein. The resulting solution was centrifuged at 3,000 rpm for 30 min at 4°C. Radioactivity in the pellet was measured using a gamma counter (1282 Compugamma; LKB).

Data analysis. All data points represent the mean of three or more values and their standard deviations. The mean and standard deviations were calculated using the computer program StatView 4.5. The significance levels were also calculated via ANOVA (Scheffé's test) using StatView 4.5. P values <0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Zn2+ on kinetics and concentration-dependant binding of HK and factor XII to human VSMC. It was shown previously that Zn2+ is necessary for the binding of HK (22, 29) and factor XII (17) to HUVEC microvascular endothelial cells and astrocytes (4, 5). Therefore, we investigated the requirement of Zn2+ for HK and factor XII to bind to VSMC as described in MATERIALS AND METHODS. VSMC were incubated with biotinylated HK (10.0 nM; Fig. 1A) or factor XII (12.5 nM; Fig. 2A) in the binding buffer (50 µl/well) in the presence or absence of Zn2+ (50 µM) from 0 to 160 min in 20-min increments. In a second experiment, VSMC were incubated with increasing concentrations of biotinylated HK (Fig. 1B) or factor XII (Fig. 2B) for 90 min in the binding buffer (50 µl/well) in the absence or presence of Zn2+ (50 µM). The absorbance at 450 nm, which is proportional to the bound biotinylated HK or factor XII, increased in the presence of Zn2+ significantly compared with the corresponding values of absorption in the absence of Zn2+ (P < 0.05). In the absence of Zn2+, the binding of HK and factor XII was minimal and near background levels, because there was no change in OD450. The maximum concentrations of HK and factor XII that bound to VSMC were 12.5 and 15 nM, respectively. The optimum binding of both proteins was observed after 80–100 min of incubation.



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  		Fig. 1. Binding of high-molecular-weight kininogen (HK) to vascular smooth muscle cells (VSMC). VSMC were grown in 96-well plates until they reached >95% confluence. The cells were growth factor- and serum-starved for 18–24 h. The binding of HK was carried out by incubating the cells in binding buffer with or without Zn2+ for different periods of time (A) or with increasing concentrations of biotinylated HK for 90 min (B). The amount of bound biotinylated HK was determined by measuring the optical density at 450 nm by employing a peroxidase-dependent color reaction with streptavidin-linked horseradish peroxidase (HRP). Results presented are means ± SD of 3 values for each data point. Dashed lines represent the binding curve in the presence of Zn2+; solid lines represent the binding curve in absence of Zn2+.

 


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  		Fig. 2. Binding of factor XII to VSMC. VSMC were grown in 96-well plates until they reached ~95% confluence. The cells were growth factor- and serum-starved for 18–24 h. Biotinylated factor XII was added for different time periods (A) or at increasing concentrations (B). The amount of bound biotinylated factor XII was determined by measuring the optical density at 450 nm by employing a peroxidase-dependent color reaction with streptavidin-linked HRP. Results presented are means ± SD of 3 values for each data point. Dashed lines represent the binding curve in the presence of Zn2+; solid lines represent the binding curve in the absence of Zn2+.

 
Reversibility of binding of proteins with VSMC. Incubation of VSMC for 90 min with biotinylated protein leads to the binding of the protein (A, HK; C, factor XII; Fig. 3). When 200-fold molar excess of the nonbiotinylated protein was added further for 1 h, the amount of bound biotinylated HK (B) and factor XII (D) were significantly decreased (P < 0.05), suggesting reversibility of the binding of HK and factor XII with VSMC.



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  		Fig. 3. VSMC grown in 96-well plates were incubated with either biotinylated HK (12.5 nM) or biotinylated factor XII (15 nM) for 90 min, and then a 200-fold molar excess of the nonbiotinylated protein was added and further incubated for 1 h. The binding of each protein was determined as described in text. OD450, optical density measured at 450 nm. Results presented are means ± SD of 3 values for each data point. A and C, biotinylated HK and factor XII binding, respectively; B and D, biotinylated HK and factor XII binding, respectively, after treatment of nonbiotinylated protein; 200-fold molar excess for 1 h.

 
Binding of HK and factor XII to human VSMC. Next, the binding of HK and factor XII to VSMC was analyzed using fluorescence microscopy. The cells were grown in eight-well chamber slides. The cells in the chamber that were reacted with a mixture of FITC-labeled HK and rhodamine-labeled factor XII (Fig. 4A) showed a yellowish color, suggesting the colocalization of both proteins. The cells that were reacted with either rhodamine-labeled factor XII (Fig. 4B) or FITC-labeled HK (Fig. 4C) in the presence of Zn2+ (50 µM) bound to each of the proteins distinctively. In the absence of Zn2+, the binding of both proteins was minimal and the fluorescence signal was not detectable (Fig. 4D, mixture of factor XII and HK; Fig. 4E, factor XII alone; Fig. 4F, HK alone).



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  		Fig. 4. Binding of fluorescent-labeled HK and factor XII to the cell surface. A: cells incubated with a mixture of FITC-labeled HK and rhodamine-labeled factor XII (x400). B: cells incubated with rhodamine-labeled factor XII in the presence of 50 µM Zn2+ (x400). C: cells incubated with FITC-labeled HK in the presence of 50 µM Zn2+ (x400). D–F: cells treated with fluorescent-tagged proteins in the same order as in A–C in the absence of Zn2+. Results are representative of 3 or more observations.

 
Assembly of kinin-forming system components and activation of PK on VSMC. The Zn2+-dependent assembly of kinin-forming proteins on HUVEC leads to the activation of the kinin-forming cascade (12). Therefore, we next checked whether the Zn2+-dependent assembly of kinin-forming proteins on VSMC could lead to its activation. The rate of activation of PK to kallikrein was measured by incubating the cells grown in 96-well plates with different combinations of PK, factor XII, and HK. The highest rate of activation was seen when PK, factor XII, and HK were incubated together, followed by PK and factor XII in the absence of HK (Fig. 5). The results demonstrate that PK, factor XII, and HK can assemble along the surface of the smooth muscle cells, leading to the conversion of PK to kallikrein. PK also can be activated in the absence of HK, but the activation takes place at a much slower rate. The role of HK is known to orient PK for activation (9, 25), and it does so by forming a 1:1 molar complex with HK (18).

Assembly of kinin-forming proteins on VSMC leads to rapid formation of BK. The cells were first serum- and growth factor-starved when they were ~80% confluent and then treated at 37°C with HK or with HK, PK, and factor XII at 2.5 µg/ml for 7 min, 1 h, or 24 h as indicated Table 1. The culture medium was collected and analyzed using RIA, and the results expressed as the means of triplicate values are shown in Table 1. There was a rapid formation of BK in the presence of the three proteins HK, PK, and factor XII. The amount of BK decreased within an hour of stimulation, suggesting that a BK-degrading activity is present in the cells.


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  		Table 1. Analysis of BK in conditioned medium

 
Phosphorylation of VSMC p44/42 MAPK by BK and in situ-generated BK. Next, we tested whether activated PK can cleave HK and generate BK to evoke a signal through B2 kinin receptors. It has been shown that HK, the precursor for BK, and B2 kinin receptors are present on VSMC (3, 20). The B2 kinin receptor is a member of the G protein-coupled receptor family and, when stimulated, is known to activate MAPK (16). The cells were extracted and analyzed with specific antibodies that react with total p44/42 MAPK and then with the phosphorylated form of p44/42 MAPK by using Western blot analysis as described in MATERIALS AND METHODS. The signal with the antibody that detects specifically the total amount of p44/42 MAPK was equal in intensity with all the treatment regiments. As shown in Fig. 6, A and B, the signals for the phosphorylated forms of p44/42 MAPK in cells treated with BK (E) or with HK, PK, and factor XII (D) were higher than in control cells (A) or cells treated with HOE-140 alone (B). However, prior treatment with HOE-140 reduced the effect of both BK (F) and the combination of HK, PK and factor XII (C) significantly (P < 0.05). Thus HOE-140, a standard B2 kinin receptor antagonist, inhibits BK signaling through the B2 kinin receptor. Therefore, the phosphorylation of p44/42 MAPK seen with a mixture of HK, PK, and factor XII is caused by the formation of BK, and it can be concluded that in vitro addition of BK as well as in situ generation of BK by the assembly of kinin-forming proteins on VSMC can phosphorylate p44/42 MAPK. Intact HK or PK did not phosphorylate p44/42 MAPK significantly (data not shown).

Phosphorylation of VSMC p44/42 MAPK by binding of factor XII. Both factor XII and HK have been shown to interact with membrane proteins such as gC1qR, cytokeratin 1, and urokinase plasminogen activator receptor (uPAR) in endothelial cells (17), and these proteins may initiate signaling from cell surfaces when they interact with their ligands. Therefore, we next investigated whether addition of factor XII could evoke a signal leading to phosphorylation of p44/42 MAPK in VSMC. Figure 7, A and B, shows that the addition of increasing amounts of factor XII (A–F) led to a significantly increased phosphorylation of p44/42 MAPK at concentrations greater than 1 µg/ml (P < 0.05) and was dependent on the concentration of factor XII. Therefore, factor XII binding can result in signaling independent of BK formation. HK, when tested similarly, did not phosphorylate p44/42 MAPK significantly (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Human arterial smooth muscle cells can contribute to inflammation as well as to the development of vascular disease. If the endothelium is injured, VSMC can then become exposed to plasma constituents; thus we investigated whether the presence of kinin-forming system proteins can assemble and activate on these cells. Our data indicate that factor XII and HK bind with the VSMC surface in the presence of Zn2+ in a time-dependent, reversible, and saturable manner. Therefore, the binding is specific and the data are similar to data reported in the literature for HUVEC, microvascular endothelial cells, and astrocytes (4, 5, 22, 29). The requirement of Zn2+ for the binding of these proteins to HUVEC has been further investigated in great detail by replacing BSA in the binding buffer with an agent that does not bind to Zn2+, such as gelatin(32). A much lower concentration of free Zn2+ (0.3 µM) was sufficient for the optimal binding of HK, which was well below physiological Zn2+ levels (10–25 µM). However, in BSA-containing buffers, much higher Zn2+ levels were required for the optimal binding of HK (32). VSMC are not directly exposed to blood plasma, which has a lower physiological concentration of free Zn2+. During vascular injury, collagen, leukocytes, and VSMC could come into contact at the site of the atherosclerotic plaque and become an extra source of Zn2+ to support the assembly of the kinin-forming proteins. Interestingly, it has been observed (17) that platelets in the presence of collagen can become activated to release extra amounts of Zn2+ resembling that is seen during vascular remodeling in atherosclerosis. Studies with HUVEC, microvascular endothelial cells, and astrocytes have clearly identified the protein nature of these binding sites, which have been attributed to three proteins, namely, gC1qR, cytokeratin 1, and uPAR (17). These proteins have been identified by specific antibody inhibition of factor XII and HK binding, sequencing of binding proteins isolated from solubilized membranes by ligand-affinity chromatography, and binding studies with purified and cloned proteins using ligand blotting (14). However, in this report we have not tried to characterize these proteins in VSMC.

The Zn2+-dependent assembly of the kinin-forming proteins activates PK to form kallikrein. Zhao et al. (32) previously observed that PK gets activated to kallikrein on cell-bound HK and can be inhibited by antipain. Unlike HUVEC, VSMC are not directly exposed to blood plasma, and it is reasonable to believe that if exposed to kinin-forming proteins, the concentration would be very low. Therefore, we used a concentration of 1 µg/ ml (equivalent to 10 nM) of the three proteins. In the absence of factor XII, we saw a slow activation of PK to kallikrein when HK was present, which may be due to cell-derived HSP90 (13) or prolylcarboxypeptidase (24) interacting with PK that is attached to HK (14). In vitro autoactivation of PK also has been suggested (27). In the presence of factor XII, PK was converted to kallikrein at a much faster rate. It is not certain whether this augmentation of activation is due to autoactivation of factor XII (24a) or activation of factor XII through formation of kallikrein via HSP90 or prolylcarboxypeptidase, or both. The highest rate of activation was observed in the presence of all three proteins, suggesting that assembly of all three components would bring about the most favorable conformation for the activation of the kinin-forming system.

In the next series of experiments, we incubated the VSMC with 2.5 µg/ml factor XII, PK, and HK for 10 min, 1 h, or 24 h and measured the concentration of liberated BK. We observed a peak concentration of BK within 10 min of incubation. The BK concentration diminished rapidly, likely because of the presence of a kinin-degrading enzyme. Our results are in agreement with the observations of Zhao et al. (32), who observed that the amount of BK liberated by the assembly of PK and HK was ~50% higher if the incubation was done in the presence of lisinopril, an angiotensin-converting enzyme inhibitor.

Studies on BK-mediated signaling in HUVEC or in VSMC have been limited to in vitro studies in which synthesized BK is incubated with cells. The B2 kinin receptor is a member of the G protein-coupled receptor family and, when stimulated, is known to activate MAPK (16). Studies by Zhao et al. (32) employing HUVEC demonstrated that BK liberated from in situ assembly of HK and PK could lead to the formation of nitric oxide. In our studies, we asked the question whether the in situ generation of BK by the assembly of the kinin-forming system proteins could interact with the B2 kinin receptor, leading to a phosphorylation of p44/42 MAPK. We observed a rapid phosphorylation of the p44/42 MAPK that was inhibited by HOE-140, a B2 kinin receptor antagonist. The results show a close resemblance to the results of the studies done on rat VSMC with in vitro-added BK (30).

The cell surface proteins gC1qR, cytokeratin 1, and uPAR, which interact with factor XII and HK in HUVEC (14), can initiate signaling when their respective ligands interact with these proteins. Therefore, we checked whether any of the components of the kinin-forming proteins could contribute to the phosphorylation of p44/42 MAPK and found that factor XII was able to phosphorylate p44/42 MAPK in a concentration-dependent manner. We consistently observed a higher degree of inhibition of the phosphorylation of p44/42 MAPK by HOE-140 in the in vitro BK-treated VSMC than in the cells that were stimulated with in situ-generated BK by the assembly of the three proteins. This residual phosphorylation may be attributed to the contribution from factor XII toward the phosphorylation of p44/42 MAPK. Interestingly, we noted that the inhibition of phosphorylation of p44/42 MAPK by HOE-140 was not complete when we assembled all three proteins on VSMC, whereas BK-stimulated phosphorylation was more completely inhibited by HOE-140. Supporting our observations, it was also reported previously that factor XII can act as a peptide growth factor and promote the proliferation of a smooth muscle cell line (A10 ATCC) (7) and hepatocytes (HepG2 cell line) (23) via phosphorylation of p44/42 MAPK. Factor XII has domains that show structural similarity to other proteins. Of particular interest are the two distinct domains of factor XII that bear strong homology to epidermal growth factor (EGF) (7, 23). The mitogenic activity of factor XII may be caused by the interaction of these two domains with the EGF receptor (EGF-R). Although it has been postulated that factor XII might be acting via surface proteins other than EGF-R, the data presented are not conclusive (7). In HUVEC, factor XII is known to bind with the cell surface proteins gC1qR, cytokeratin 1, and uPAR, and binding to uPAR was particularly predominant (Mohanseenivasan V, Joseph K, and Kaplan AP, unpublished observations). The signaling by factor XII could be through uPAR, because uPAR can function as a receptor to initiate signaling when urokinase plasminogen activator interacts with it (15). Interestingly, signaling through both EGF-R and uPAR involves phosphorylation of MAPK p44/42 proteins (15). The functional significance of the phosphorylation of MAPK p44/42 toward the development of atherosclerosis is noteworthy. It has been shown that signaling through MAPK phosphorylation in VSMC can induce TGF-{beta}1 expression at the transcriptional level. TGF-{beta}1 can then induce the transcription of type I collagen as well as matrix-degrading enzymes and their inhibitors, which can lead to fibrosis in blood vessels (3, 6). Also, phosphorylation of MAPK p44/42 proteins can signal either cell proliferation or apoptosis (2). Therefore, phosphorylation of MAPK p44/42 proteins may serve as a mitogenic signal or, alternatively, an apoptotic signal for VSMC. Both proliferating (21, 26) and apoptotic (1) VSMC have been observed in atherosclerotic lesions. Future studies are need to examine whether this p44/42 MAPK phosphorylation leads to the formation of products such as type I collagen, cytokines, and matrix metalloproteinases and their tissue inhibitors, which are key players in vascular remodeling.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. P. Kaplan, Medical Univ. of South Carolina, Dept. of Medicine/Pulmonary, PO Box 250623, 96 Jonathan Lucas St., Charleston, SC 29425 (E-mail: kaplana{at}musc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* A. N. Fernando and L. P. Fernando contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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