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. 2004 Sep;72(9):5274–5282. doi: 10.1128/IAI.72.9.5274-5282.2004

Trypanosoma cruzi Infection Activates Extracellular Signal-Regulated Kinase in Cultured Endothelial and Smooth Muscle Cells

Shankar Mukherjee 1, Huan Huang 1, Stefka B Petkova 2, Chris Albanese 3, Richard G Pestell 3, Vicki L Braunstein 1, George J Christ 4,5, Murray Wittner 1, Michael P Lisanti 6, Joan W Berman 1,7, Louis M Weiss 1,8, Herbert B Tanowitz 1,8,*
PMCID: PMC517449  PMID: 15322023

Abstract

Trypanosoma cruzi infection causes cardiomyopathy and vasculopathy. We examined the consequence of this infection for the mitogen-activated protein kinase (MAPK) pathways, which regulate cell proliferation in cultured human umbilical vein endothelial and vascular smooth muscle cells. Infection of these cells resulted in activation of extracellular signal-regulated kinases 1and 2 (ERK1/2) but not c-Jun N-terminal kinase or p38 MAPK. Treatment of these cells with the MAPK kinase inhibitor PD98059 prior to infection blocked the increase in phosphorylated ERK1/2 seen with infection. Heat-killed parasites did not activate ERK1/2, indicating that activation of ERK1/2 was dependent on infection of these cells by live parasites. Furthermore, transfection with dominant-negative Raf(301) or Ras(N17) constructs reduced the infection-associated levels of phospho-ERK1/2, indicating that the activation of ERK1/2 involved the Ras-Raf-ERK pathway. Infection also resulted in an increase in activator protein 1 (AP-1) activity, which was inhibited by transfection with a dominant-negative Raf(301) construct. T. cruzi-infected endothelial cells secreted endothelin-1 and interleukin-1β, which activated ERK1/2 and induced cyclin D1 expression in uninfected smooth muscle cells. These data suggest a possible molecular paradigm for the pathogenesis of the vasculopathy and the cardiovascular remodeling associated with T. cruzi infection.

Chagas' disease is caused by infection with Trypanosoma cruzi. Although only 10 to 30% of T. cruzi-infected persons will develop chronic symptomatic Chagas' disease, the burden of mortality and disability in the countries where the disease is endemic is enormous (27, 53). Despite the reductions in transfusion-associated and vector-borne transmission of T. cruzi in many countries in recent years, this burden will be borne by the affected nations on a continuing basis, as millions of T. cruzi-infected persons gradually develop symptomatic Chagas' disease. The number of persons living in the United States with chronic T. cruzi infections has increased enormously in recent years. Data from the 2000 census indicate that more than 12 million Latin Americans from countries where Chagas' disease is endemic now reside in the United States. Over eight million of these immigrants are from Mexico and Central America, where the prevalence of T. cruzi infection is high (27).

There are several serious cardiovascular complications of this infection, including acute myocarditis and chronic cardiomyopathy (27). Pathological examination of the cardiovascular system in human and experimental acute chagasic myocarditis reveals inflammation, myonecrosis, vasculitis, and numerous parasite pseudocysts (53). In chronic chagasic heart disease, inflammation, fibrosis, myocytolysis, and vasculitis persist but there are few parasites in the infected tissues. T. cruzi infection induces vasculopathy as a result of damage to the endothelium. Important consequences of this endothelial damage include vasospasm and focal ischemia (16, 38, 42, 52).

T. cruzi infects many cell types in the cardiovascular system, including cardiac myocytes, cardiac fibroblasts, endothelial cells, and vascular smooth muscle cells. Vascular endothelial cells are one of the first types of cells to come in contact with T. cruzi. Endothelium that is damaged generally leads to vascular dysfunction. Infection plays a significant role in the alteration of a variety of important pathways, resulting in the development of cardiomyopathy and vasculopathy. For example, previous studies have reported increased expression of proinflammatory cytokines (51), chemokines (56), vascular adhesion molecules (24, 40), and nitric oxide synthase (6); activation of mitogen-activated protein kinases (MAPKs), transcription factors such as AP-1, NF-κB, and cell cycle regulatory molecules (21); and increased expression of endothelin-1 (ET-1) in T. cruzi infection both in vivo and in vitro (37, 38, 54, 64). All of these factors promote inflammation and vascular injury. Observations with experimental animals have demonstrated the presence of platelet thrombi, increased platelet aggregation, and elevated levels of thromboxane A2 in plasma (50), factors that promote vasospasm and platelet aggregation. Endothelial cells that are invaded may be destroyed (48), thereby exposing the underlying vascular smooth muscle cells to parasites and inflammatory mediators. The type of injury induced by T. cruzi may be viewed as similar to that which occurs following balloon angioplasty in which there is destruction of the endothelial cell layer and restenosis due to smooth muscle cell hyperplasia (18, 25, 26, 29, 59).

MAPKs are structurally related serine threonine kinases that include the extracellular signal-regulated kinases 1 and 2 (ERK1/2), the c-Jun N-terminal kinase (JNK), and p38 MAPK and that integrate numerous extracellular signals, resulting in regulation of cell proliferation, differentiation, and cell survival (4, 11, 30, 44, 57). A variety of mitogenic factors such as epidermal growth factor, insulin-like growth factor, and platelet-derived growth factor activate ERK1/2. This activation involves the participation of Ras, Raf, and MAPK kinase (MEK), which phosphorylate ERK1/2. Downstream of ERK are the transcription factors Elk-1, AP-1, and activating transcription factor 2 (ATF2). JNK and p38 MAPK are activated by environmental stresses such as UV light, heat shock, and the presence of tumor necrosis factor alpha and Fas ligand (30, 44, 57, 63). JNK activates transcription factors including Elk-1, AP-1, and ATF2. Injury to the myocardium caused by hypoxia, ischemia, and ischemia-reperfusion causes increased expression of cytokines and activation of MAPKs and the transcription factors NF-κB and AP-1 (2, 35, 46). Activation of ERK1/2, with the subsequent increase in cyclin D1 expression, is associated with cellular proliferation and hypertrophy in the cardiovascular system, resulting in cardiomyopathy and vasculopathy (25, 47).

In the present study, we report that T. cruzi infection of endothelial cells and vascular smooth muscle cells preferentially activates the Ras-Raf-ERK-cyclin D1 pathway. Our data also underscore the role of ET-1 and cytokines in the pathogenesis of T. cruzi-induced vasculopathy. The delineation of these pathways may lead to an understanding of the nature of the parasite-host relationship and may provide useful targets for adjunctive therapy for the treatment of Chagas' disease.

MATERIALS AND METHODS

Reagents.

The MEK-1 inhibitor PD98059 (2′-amino-3′-methoxyflavone) was purchased from Alexis Biochemicals (San Diego, Calif.). Phorbol myristate acetate (PMA), ET-1, and anisomycin were obtained from Sigma (St. Louis, Mo.). Endothelin receptor A (ETA) antagonist (BQ123) was obtained from the American Peptide Company (Sunnyvale, Calif.), and interleukin-1 (IL-1) and IL-1 receptor antagonist (IL-1ra) were purchased from R & D Systems (Minneapolis, Minn.). Antibodies to phosphorylated ERK1/2, JNK, p38, and total ERK1/2 were obtained from Cell Signaling Technology (Beverley, Mass.). Anti-cyclin D1 antibody (DCS-6) was obtained from Neo Markers (Fremont, Calif.). The expression vectors containing dominant-negative Raf(301), Ras(N17), and JNK(K-R), the empty vector (pSRα), and the luciferase reporter plasmid containing four TPA (12-O-tetradecanoyl phorbol-13-acetate)-responsive elements (TRE) in tandem (4X TRE-luciferase plasmid) were kindly provided by Shu Chien and Michael Karin of the University of California, San Diego, La Jolla (33). pEGFP-C1, expressing a green fluorescent protein, was obtained from Clontech (Palo Alto, Calif.). All other chemicals were of the highest purity available from commercial suppliers.

Cultivation of parasites.

The Tulahuen strain of T. cruzi was maintained by syringe passage in A/J mice (Jackson Laboratories, Bar Harbor, Maine). Trypomastigotes were maintained and harvested from L6E9 myoblasts as previously described (43). In the experiments involving heat-killed trypomastigotes, the parasites were killed by exposure to 75°C for 20 min.

Isolation and cultivation of human vascular smooth muscle cells.

Homogeneous explant smooth muscle cell cultures were obtained from human corporal vascular smooth muscle cells (5, 8, 67). Briefly, sections were placed in low-glucose Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Grand Island, N.Y.) containing 1% penicillin-streptomycin solution (Invitrogen). Tissue was washed and cut into 1- to 2-μm-thick pieces and placed in tissue culture dishes with sufficient nutrient medium to prevent drying. After the explants had attached to the substrate, usually within 1 to 2 days, more culture medium was added. When the cells had migrated from the explant and undergone division, they were detached using 0.05% trypsin and 0.02% EDTA at 37°C for 5 min. Cells were subsequently grown in DMEM containing 10% fetal bovine serum (FBS), 2 mM l-glutamine, and 1% PenStrep in a humidified atmosphere of 5% CO2-95% air at 37°C. Cellular homogeneity was verified by immunofluorescent staining using monoclonal antibodies to human smooth muscle myosin. Only passages 2 through 4 were used in all the experiments.

Isolation and culture of human umbilical vein endothelial cells.

Human umbilical vein endothelial cells were obtained from umbilical cords (19, 51) and cultured in medium consisting of complete Medium 199 (Invitrogen) with 20% newborn calf serum (Invitrogen), 5% human serum, 25 μg of heparin (Sigma)/ml, and 1.5 mM glutamine and supplemented with either 7.5 μg of endothelial cell growth supplement (Sigma)/ml or 12 μg of bovine brain extract (Clonetics, Walkersville, Md.)/ml. The cultures were maintained at 37°C in 5% CO2. Cell type was confirmed by the cobblestone morphology and by positive immunofluorescence with factor VIII antibodies (>95% positive). Confluent monolayers were prepared in 100-mm-diameter tissue culture plates coated with 0.2% gelatin. Only passages 2 through 4 were used for this study.

Infection of cells.

Trypomastigotes were harvested from the supernatants of infected myoblasts as previously described (43). When grown to near confluence, human umbilical vein endothelial cells or smooth muscle cells were infected with a multiplicity of infection of 1.5 to 2.0:1. After 48 h of exposure, the parasites were washed off. The approximate percent parasitisms at 24, 48, and 72 h were 20, 50, and 80%, respectively, as determined by Giemsa staining.

Treatment of smooth muscle cells with PD98059.

Smooth muscle cells were grown to 70 to 80% confluence in low-glucose DMEM with 10% FBS. The cells were pretreated with 50 μM PD98059 for 2 h in DMEM with 1% FBS, followed by infection with T. cruzi. The cells were harvested 24, 48, and 72 h postinfection and processed for immunoblotting.

Treatment of smooth muscle cells with conditioned medium obtained from T. cruzi-infected endothelial cells.

Conditioned media obtained at 48 h from infected and uninfected endothelial cells were filtered through 0.22-μm-pore-size filter units to remove any parasites and diluted 1:1 with fresh medium. They were then layered onto growing smooth muscle cells and incubated for a further 48 h. In some experiments, the smooth muscle cells were preincubated with either ET-1 receptor blocker (ETA) BQ123 (10 μM) or IL-1ra (10 ng/ml) for 4 h at 37°C followed by the addition of the conditioned medium.

Transfection of dominant-negative expression vectors and luciferase assay.

Smooth muscle cells and endothelial cells were transfected with the expression vectors containing dominant-negative Raf(301), Ras(N17), and JNK(K-R) genes in the presence of antibiotics and serum by using Gene PORTER 2 (Gene Therapy Systems, Inc., San Diego, Calif.) (51). Briefly, cells were plated 24 h before transfection onto 60-mm-diameter plates. At 70 to 80% confluence, they were transfected with 2.5 μg of dominant-negative expression vectors. For the luciferase assay, the cells were cotransfected with 2.5 μg of dominant-negative Raf(307) expression vector and 4X TRE-luciferase plasmid. The cells were also transfected with 200 ng of pEGFP-C1 to monitor successful gene transfer and transfection efficiency. Transfection was carried out for 2 h at 37°C and 5% CO2. The cells were allowed to recover for an additional 24 h with fresh medium, after which they were infected with 1.5 × 106 trypomastigotes. In the control experiments, the cells were transfected with dominant-negative Raf(301) or the empty vector (pSRα) and then either treated with 100 nM PMA or left unstimulated for 10 min at 37°C.

The luciferase assay was performed with the luciferase assay system (Promega, Madison, Wis.) by using the manufacturer's protocol. Light units from the luciferase activity were measured at room temperature with an AutoLumat LB 953 luminometer (EG&G, Berthold, Tenn.). Luciferase activity was measured for the initial 10 s of the reaction, and the values were expressed in arbitrary light units.

Immunoblot analysis.

To prepare protein lysates from endothelial and smooth muscle cells, cells were incubated on ice with lysis buffer (21) containing protease inhibitors (Complete Mini EDTA free; Roche, Indianapolis, Ind.) for 15 min and lysed by sonication for 20 s on ice. After centrifugation at 17,000 × g for 15 min, the supernatant was collected and the protein concentration was measured with protein assay reagent (Bio-Rad, Hercules, Calif.). For detection of phospho-ERK1/2, cells were lysed in boiling lysis buffer to neutralize phosphatases. Samples (30 μg of protein) were separated by sodium dodecyl sulfate (SDS)-10% polyacrylamide gel electrophoresis and transferred onto nitrocelullose membrane by the wet-gel transfer method. Membranes were then probed with either rabbit anti-phospho-specific ERK1/2, JNK, p38, or anti-ERK1/2 antibodies at a dilution of 1:1,000 and anti-rabbit horseradish peroxidase-conjugated secondary antibodies (dilution of 1:5,000). Cyclin D1 expression was determined with rabbit anti-cyclin D1 antibody at a dilution of 1:1,000 and anti-rabbit horseradish peroxidase-conjugated secondary antibodies (dilution of 1:5,000). The bound antibodies were detected using enhanced chemiluminescence by the ECL detection method (Amersham Biosciences, Buckinghamshire, United Kingdom). For detection of equal loading (as a control), either the immunoblots were washed in stripping buffer (100 mM β-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl [pH 6.8]) for 30 min at 50°C or equal loading gels were used in parallel and probed with specific control antibodies.

[3H]Thymidine incorporation assay.

Smooth muscle cells were seeded into 24-well plates and incubated for 6 h with conditioned medium obtained at 48 h from infected or uninfected endothelial cells. The cells were supplemented with [3H]thymidine (Amersham) at 2 μCi/ml and kept for an additional 12 h. The reaction was terminated by three washes with phosphate-buffered saline and addition of 10% ice-cold trichloroacetic acid for 20 min. Finally, the cells were washed once in cold phosphate-buffered saline and solubilized with 0.1 N NaOH-0.1% SDS for 1 h at 37°C. Cell lysates (200 μl) were used to determine the incorporation of [3H]thymidine into DNA with a liquid scintillation counter (Packard Tri-Carb 2100 TR; GMI, Inc., Albertville, Minn.).

Statistical analysis.

All experiments were performed at least three times. The Student t test was performed on all quantitative data.

RESULTS

Activation of ERK1/2 in infected endothelial and smooth muscle cells.

In the endothelial cells, activation of ERK1/2 (demonstrated by increased levels of phospho-ERK) was observed at 24 h and the levels were increased at 48 and 72 h postinfection, indicating that there was persistent activation of this pathway (Fig. 1A). A similar pattern of ERK1/2 activation was observed in infected smooth muscle cells (Fig. 1B). Levels of total ERK1/2 remained unchanged among all the samples, indicating that infection resulted in activation of ERK1/2 without an increase in de novo synthesis of ERK protein. Interestingly, activation of p38 MAPK and stress-activated protein (SAP)/JNK was not observed even after 72 h of infection (Fig. 1A). These MAPKs were also not activated at 2, 6, and 12 h postinfection in either smooth muscle or endothelial cells (data not shown). In order to investigate whether the primary cultured endothelial cells and smooth muscle cells had a defect in activating p38 MAPK and SAP/JNK, we treated these cell types with anisomycin for 60 and 120 min. Anisomycin treatment activated both p38 MAPK and SAP/JNK (Fig. 1C), indicating that these cells are capable of activating these MAPK pathways (3).

FIG. 1.

FIG. 1.

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Representative immunoblot analysis of MAPKs in T. cruzi-infected endothelial and smooth muscle cells. Human umbilical vein endothelial cells (HuVEC) (A) and smooth muscle cells (SMC) (B) were probed with antibodies directed against phospho-p38, JNK, and ERK1/2. Total ERK was used as a control. Note that only phospho-ERK1/2 expression was increased over the course of infection, and not that of p38 and SAP/JNK kinases. Uninf, uninfected; Inf, infected. (C) Activation of p38 and JNK by anisomycin. All experiments were performed at least three times.

Heat-killed trypomastigotes incubated with smooth muscle cells did not cause significant activation of ERK1/2 (Fig. 2), indicating that live parasites are necessary for the activation of ERK1/2. In addition, the anti-phospho-ERK1/2 and anti-total ERK1/2 antibodies used in this study did not react with parasite lysates (data not shown).

FIG. 2.

FIG. 2.

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Live trypomastigotes (Trps) activated ERK1/2 in endothelial cells, but heat-killed trypomastigotes and uninfected (Uninf) cells did not. This experiment was repeated three times with similar results. SMC, smooth muscle cells.

Infection-associated ERK1/2 activation in endothelial and smooth muscle cells is via the activation of MEK-1.

The mechanism of ERK1/2 activation was examined using PD98059, a selective cell-permeable MEK-1 inhibitor (1, 15). Pretreatment of cells with 50 μM PD98059 prior to infection significantly inhibited the phosphorylation of ERK1/2 in infected cells (Fig. 3), indicating that activation of ERK1/2 is indeed the result of MEK-1 activation. MEK-1 is an upstream component in the Ras-Raf-ERK pathway. PD98059 had no effect on the growth of the parasite in culture and did not affect the infectivity of the parasite (data not shown).

FIG. 3.

FIG. 3.

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Effect of PD98059 on T. cruzi-infected smooth muscle and endothelial cells. Smooth muscle cells (SMC) and human umbilical vein endothelial cells (HuVEC) were pretreated with 50 μM PD98059 for 2 h (see Materials and Methods) and then infected with trypomastigotes. Lysates obtained from infected cells at 24, 48, and 72 h postinfection were processed for immunoblotting with antibodies to phospho-ERK1/2. PD98059 treatment significantly reduced phosphorylation of ERK1/2 in infected cells compared to that in the untreated infected cells. The units used are arbitrary values obtained after densitometric analysis of an immunoblot experiment. The data presented are from a representative experiment; three separate experiments were performed, all of which yielded similar results.

Role of Ras and Raf in activation of phospho-ERK1/2 in T. cruzi infection.

To analyze the mechanism of activation of ERK, we transfected endothelial and smooth muscle cells with dominant-negative Raf(301), Ras(N17), and JNK(K-R) expression vectors. Transfection efficiency was estimated to be 20% in endothelial cells and 35% in smooth muscle cells as judged by control pEGFP-C1 transfection (data not shown), which also indicated that these cells were viable. In both cell types, the dominant-negative Raf construct was able to significantly reduce the abundance of phospho-ERK1/2, indicating proper transcription and translation of the transferred genes (Fig. 4A, lane 4). Furthermore, when the transfected cells were infected for 48 h, there was a reduction of the infection-associated increase in the activation of ERK1/2 by 2- and 1.8-fold (P < 0.05; n = 3) (Fig. 4A, lane 5) in smooth muscle and endothelial cells, respectively, compared to that in the control (Fig. 4A, lane 2). To further investigate the upstream regulators of this pathway, we transfected smooth muscle cells with dominant-negative Ras(N17) and JNK(K-R). Although there was no significant change in levels of phospho-ERK1/2 in cells transfected with JNK, there was a decrease in the level of phospho-ERK1/2 in cells transfected with dominant-negative Ras(N17) (Fig. 4B), indicating that the activation of ERK1/2 in infected cells followed the Ras-Raf-ERK pathway.

FIG. 4.

FIG. 4.

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Transfection of endothelial and smooth muscle cells with dominant-negative Ras, Raf, and JNK vectors. (A) Both smooth muscle cells (SMC) and human umbilical vein endothelial cells (HuVEC) were transfected with dominant-negative (DN) Raf(301) and then infected with trypomastigotes for 48 h. In both cases, infected cells transfected with dominant-negative Raf demonstrated decreased ERK1/2 activity compared to that in the control infected cells (lane 5 versus lane 2). Lane 1 represents uninfected (Uninf) cells. Inf, infected; Et PI, empty plasmid. (B) Transfection of smooth muscle cells with dominant-negative JNK(K-R) vector showed no significant effect on phospho-ERK1/2 expression in uninfected (DN Jnk) versus infected (DN Jnk+Inf) cells compared to transfection with dominant-negative Ras(N17). Empty plasmid transfection had no effect on phospho-ERK1/2 expression. (C) In control experiments, both smooth muscle cells and endothelial cells were transfected with dominant-negative Raf(301) and treated with PMA, a known inducer of ERK1/2. In transfected cells, there was a decreased level of phospho-ERK1/2 compared to that in cells transfected and induced with the empty plasmid, indicating proper transcription and translation of the dominant-negative expression vector. All experiments were performed at least three times. +, present; −, absent.

As a positive control, we transfected both endothelial cells and smooth muscle cells with Raf(301) and treated them with PMA (a known activator of ERK). In both cell types, dominant-negative Raf(301) was able to effectively reduce the levels of phospho-ERK1/2 but not to the same extent. This may have been due to differences in transfection efficiencies or in biological responses between the cell types (Fig. 4C). A positive control utilizing PMA demonstrated the efficiency of the transferred genes in blocking the Raf-ERK pathway in both endothelial cells and smooth muscle cells.

Increase in infection-associated AP-1 activity is mediated through Raf.

Among the nuclear targets of ERK1/2 in the cardiovascular system is the transcription factor AP-1 that mediates the inflammatory process, thereby contributing to cardiac remodeling after myocardial injury. In order to investigate whether AP-1 is the downstream target for activated ERK1/2, we tested the activation and subsequent binding of AP-1 to four TRE sites by using a luciferase reporter assay for smooth muscle cells. The four tandem TRE sequences in the 4X TRE-luciferase plasmid bind the transcription factor AP-1. The AP-1 transcriptional complex is made of either homo- or heterodimers of the members of the Fos (c-Fos, Fra-1, Fra-2, and Fos B) and Jun (c-Jun, Jun B, and Jun D) families. A marked increase in luciferase activity was observed in infected cells cotransfected with empty plasmid and the 4X TRE-luciferase construct (Fig. 5, lane 2). Cotransfection with dominant-negative Raf(301) and 4X TRE-luciferase significantly reduced luciferase activity (Fig. 5, lane 3). When cells cotransfected with Raf(301) were infected with T. cruzi, there was a significant reduction of the luciferase activity but total abolishment was not observed. This finding may be explained by the fact that the transfection efficiencies for endothelial cells and smooth muscle cells were lower (20 and 35%, respectively) than the infection efficiency (which was 50%). The relative decrease in luciferase activity (Fig. 5, lane 4) compared to that in the control (Fig. 5, lane 2) was probably a consequence of the activation of ERK1/2 in those cells that were infected but not transfected. Similar results were obtained using endothelial cells (data not shown).

FIG. 5.

FIG. 5.

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Activation of AP-1 in T. cruzi-infected (Inf) smooth muscle cells. Smooth muscle cells were cotransfected with dominant-negative (DN) Raf(301) and the 4X TRE-luciferase reporter plasmid. The transfected cells were infected with T. cruzi for 48 h and harvested for the luciferase assay. Cells transfected with empty plasmid (Et PI) when infected showed increased luciferase activity (lane 2). In Raf(301)-transfected infected cells, significant decrease in luciferase activity was observed (lane 4) (P < 0.05). Luciferase activity was measured in cell lysates and expressed as arbitrary units per microgram of protein. The results shown are the means ± standard deviations of results from four experiments and are presented as fold increases in luciferase activity over the baseline level seen in lane 1. RLU, relative light units; AP-1 luc, AP-1 luciferase; +, present; −, absent.

Interaction of T. cruzi-infected endothelial cells with smooth muscle cells.

Previous studies from our laboratory have shown that the synthesis of both ET-1 (64) and IL-1β (51), known to cause smooth muscle cell proliferation (10), is increased in T. cruzi-infected endothelial cells. Smooth muscle cells were incubated with filtered conditioned medium obtained from infected or uninfected endothelial cells at a dilution of 1:1 for 48 h. Immunoblot analysis revealed activation of ERK1/2 and increased levels of cyclin D1 after 48 h in those smooth muscle cells that were incubated with medium from infected endothelial cells but not in those cells incubated with medium obtained from uninfected cells (Fig. 6A). In addition, infection of smooth muscle cells caused activation of ERK1/2.

FIG. 6.

FIG. 6.

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Representative immunoblot analysis of MAPKs and cyclin D1 in T. cruzi-infected endothelial and smooth muscle cells. (A) Representative immunoblot analysis of phospho-ERK levels in smooth muscle cells incubated with media obtained from T. cruzi-infected (Inf Med) and uninfected (Uninf Med) endothelial cells. Medium obtained from infected endothelial cells (48 h) was filtered and then applied to smooth muscle cells (SMC) at a dilution of 1:1. After an incubation period of 48 h, there was activation of ERK1/2. The activation of ERK1/2 was reduced by the pretreatment of smooth muscle cell cultures with either BQ123 (ETA receptor blocker) or IL-1ra. A similar increase in cyclin D1 levels was also observed with infected conditioned medium. Smooth muscle cells infected with trypomastigotes (SMC + Trps) activated ERK1/2 as expected. Total ERK1/2 was unchanged in all experiments. (B) Immunoblot analysis demonstrating activation of ERK1/2 in smooth muscle cells treated with either ET-1, IL-1β, or ET-1 and IL-1β or infected with trypomastigotes (Trps). All experiments were performed in triplicate.

When smooth muscle cells were pretreated with either ETA receptor blocker BQ123 or IL-1 receptor blocker (IL-1ra), there was a significant reduction in the levels of phospho-ERK1/2 and in cyclin D1 expression (Fig. 6A). Treatment with the combination of BQ123 and IL-1ra caused a significant reduction in the levels of phospho-ERK1/2 and in cyclin D1 expression that was similar to that caused by either agent alone (data not shown). These data suggest that the presence of ET-1 and IL-1 in the infected medium activates ERK1/2 in smooth muscle cells. Interestingly, the activation of ERK1/2 results in the increased expression of cyclin D1, an essential component for smooth muscle cell proliferation. These data are consistent with our previous observation that while uninfected endothelial cells synthesize ET-1 constitutively, infection causes a dramatic increase in its synthesis. Incubation of ET-1 and IL-1β with uninfected smooth muscle cell cultures resulted in the activation of ERK1/2 (Fig. 6B), as did infection of smooth muscle cells with trypomastigotes (Fig. 6A and B).

We then tested the ability of conditioned medium obtained from T. cruzi-infected or uninfected endothelial cells to cause smooth muscle cell proliferation. Smooth muscle cells were incubated with conditioned medium for 6 h, followed by further incubation with [3H]thymidine for 12 h. There was a dramatic increase in [3H]thymidine uptake with infected medium compared to that with the uninfected medium (Fig. 7). Moreover, this increase in [3H]thymidine uptake was significantly reduced (P < 0.05) when the smooth muscle cells were preincubated with ETA receptor blocker BQ123.

FIG. 7.

FIG. 7.

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[3H]Thymidine incorporation into smooth muscle cells (SMC) incubated with conditioned media obtained from infected (Inf Med) and uninfected (Uninf Med) endothelial cells. Smooth muscle cells were treated with conditioned media for 6 h, followed by addition of [3H]thymidine, and kept for 12 h. The incorporation of [3H]thymidine into DNA was measured by liquid scintillation counting. A significant increase in [3H]thymidine uptake (lane 2) was noted with infected medium compared to that with the uninfected control (lane 1) (P < 0.05). When the smooth muscle cells were pretreated with BQ123, the uptake of [3H]thymidine was blocked (lanes 3 and 4). Incubation of smooth muscle cells with PMA increased [3H]thymidine uptake (lane 5). Counts per minute were normalized with the protein content of the cell lysates. All data are expressed as means ± standard deviations.

DISCUSSION

Infection with T. cruzi causes vasculitis, cardiovascular remodeling, and vascular dysfunction. Since the vasculature is composed of endothelial and smooth muscle cells, both of these cell types are of interest in any investigation of vascular dysfunction. Previous studies from our laboratory demonstrated that infection of endothelial cells results in increased synthesis of the powerful vasoconstrictor ET-1 (54, 64) and cytokines such as IL-1β (51), activation of NF-κB, and increased expression of vascular adhesion molecules (22). In the present study, we continued our examination of the consequences of infection of endothelial and smooth muscle cells with trypomastigotes and of the functional relationship between these two cell types. These studies allow the assessment of the specific responses of endothelial cells and vascular smooth muscle cells without the confounding variables present in in vivo models (Fig. 7).

T. cruzi infection of endothelial and smooth muscle cells resulted in sustained activation ERK1/2 but not of p38 MAPK and JNK. When cells were pretreated with the MEK-1 inhibitor PD98059 prior to infection, there was a decrease in the activation of ERK1/2. In addition, infection of cells that had been transfected with an Elk-1 reporter demonstrated activation of Elk-1 (unpublished observation). In order to investigate upstream mechanisms of T. cruzi-induced ERK and AP-1 activation, cells were cotransfected with dominant-negative Raf(301) expression vector along with the 4X TRE-luciferase reporter gene. There was a significant reduction in the activation of ERK1/2 and AP-1 in those infected cells that were transfected with dominant-negative Raf(301). A similar result was obtained with the Ras(N17) vector. These data indicate that activation of ERK1/2 in infected endothelial and smooth muscle cells probably follows the Ras-Raf-ERK pathway. Furthermore, studies using conditioned medium obtained from infected endothelial cells demonstrated that ET-1 and IL-1β contribute to the activation of ERK1/2 in smooth muscle cells.

Cardiovascular dysfunction is an important characteristic of chagasic cardiomyopathy. Previous studies demonstrated activation of myocardial ERK1/2 and AP-1 and increased expression of ET-1 and cyclin D1 during murine T. cruzi infection (24). Those studies implicated nonmyocyte cell populations such as the cells of the vasculature as contributors to myocardial dysfunction. The notion that Chagas' disease is a vasculopathy is not new (41, 42). It is believed that alterations in vascular function ultimately lead to ischemia in those areas of the myocardium or other organs that are subserved by branches of the affected vessels.

An important model of vascular injury that has been extensively studied is balloon angioplasty. Injury to the vasculature from balloon angioplasty causes activation of ERK1/2, AP-1, cyclins, and cyclin-dependent kinases (18, 25, 29, 59), as well as ET-1 (14, 62) and inflammatory pathways (especially those of NF-κB and IL-1β) (31, 61). This injury is associated with smooth muscle cell proliferation, resulting in restenosis, and is reduced by treatment with inhibitors of the MAPK, ET-1, and cyclin pathways (7, 25, 28, 47, 62). Likewise, the observations reported herein also suggest an important role for ET-1 and IL-1β in the vasculopathy caused by T. cruzi infection. This is similar to the reported roles for ET-1 and IL-1β in the pathogenesis of cardiac myocyte hypertrophy and myocardial dysfunction (23, 36). IL-1β may induce the synthesis of ET-1 (12, 32), which has also been demonstrated to act as an inducer of proinflammatory cytokines, and both ET-1 and IL-1β are capable of activating ERK1/2 (Fig. 7) (60, 65, 66). In addition, cyclin D1, an important mediator of cell proliferation, is a downstream target of ERK1/2 and ET-1 (49, 66).

Although ET-1 was previously regarded solely as a spasmogen, it is now also recognized as a proinflammatory cytokine (45, 55) since it primes neutrophils, stimulates neutrophils to release elastase, and activates mast cells (45, 55). In addition, ET-1 stimulates monocytes to produce IL-1β, IL-8, IL-6, tumor necrosis factor alpha, transforming growth factor β, and granulocyte-macrophage colony-stimulating factor and upregulates the expression of vascular adhesion molecules (55). High levels of ET-1 are found in alveolar macrophages and polymorphonuclear leukocytes (45).

The incubation of uninfected smooth muscle cells with supernatants obtained from infected endothelial cells activated ERK1/2 and increased the expression of cyclin D1. Pretreatment of uninfected smooth muscle cells with either the ETA or IL-1 receptor blocker significantly reduced the levels of phospho-ERK1/2 and cyclin D1, important mediators of smooth muscle cell proliferation. Collectively, these observations indicate that ET-1 and IL-1, released from the infected endothelial cells, are important factors in the activation of the Ras-Raf-ERK pathway in uninfected smooth muscle cells.

The MAPK pathways play an important role in cellular regulation, leading to immune responses, cellular proliferation, and microbial invasion of mammalian hosts. Several studies have examined the activation of MAPK pathways resulting from parasite-host interactions. These studies have underscored the notion that alterations in these pathways are dependent on the experimental design and the nature of the parasite employed. For example, Leishmania major has developed strategies to evade host defense mechanisms by interfering with MAPK-mediated signal transduction in the host cell through the inhibition of ERK activation (20). Macrophages infected with L. donovani fail to synthesize proinflammatory cytokines and also have impaired MAPK signal transduction pathways (39). However, Theileria parva causes cell proliferation as a result of the activation of MAPKs and of the transcription factors AP-1 and ATF2 (13). Signaling through cellular and parasite MAPK pathways is also important for Toxoplasma gondii invasion (58).

In the present study, we demonstrated that T. cruzi infection of endothelial cells and vascular smooth muscle cells activated the Ras-Raf-ERK pathway. It has been reported that the trans-sialidase of T. cruzi can activate ERK1/2 (9); however, our data demonstrated that heat-killed parasites failed to activate the ERK1/2 pathway. These observations taken together indicate that parasite invasion and replication and parasite-derived substances are probably necessary for activation of ERK1/2.

The activation of ERK by T. cruzi may have multiple effects in cells, resulting in downstream activation of transcription factors such as Elk-1 and AP-1 and expression of downstream target genes such as the ET-1 and cyclin D1 genes (Fig. 8). Synthesis of cytokines and ET-1 promotes inflammation and cell proliferation. There are AP-1 consensus sites for interactions with the ET-1 gene (34) (Fig. 8). Thromboxane A2, a powerful vasoconstrictor, is also elevated in the plasma of T. cruzi-infected mice (50), and thromboxane A2 has been reported to induce phosphorylation of ERK1/2 in smooth muscle cells (17). Thus, there are several possible mechanisms for activation of ERK1/2 and subsequent cell proliferation in the setting of Chagas' disease (Fig. 8).

FIG. 8.

FIG. 8.

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Proposed pathway by which T. cruzi infection of vasculature causes vasculopathy and cardiomyopathy. This diagram demonstrates the complicated interrelationship between ERK, ET-1, and cyclin D1 and their effect on smooth muscle cell proliferation, eventual vasculopathy, and cardiomyopathy. The specific blockers used in this study and their points of action are also shown. The arrows in black boxes indicate specific proteins upregulated as a result of infection. DN, dominant negative.

We recognize that it is not possible to completely recapitulate the complicated relationships between endothelial cells and smooth muscle cells by using conditioned media in a laboratory setting. However, the observations in this paper demonstrate that T. cruzi induced an increase in the synthesis of ET-1 and IL-1β by stimulation of ERK1/2 and cyclin D1, which leads to cell proliferation, vasospasm, reduced blood flow, and ischemia. These pathways may provide important targets for modulating the progression of the disease.

Acknowledgments

This work was supported by grants to H.B.T. (NIH AI-12770, AI-52739, and HL-073732). M.P.L. was supported by grants from the National Institutes of Health, the Susan G. Komen Breast Cancer Foundation, and the American Heart Association and by the Hirschl/Weil-Caulier Career Scientist Award. J.W.B. was supported in part by National Institutes of Health grant PO AI0551519.

Editor: W. A. Petri, Jr.

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