Etiological treatment of chronic Chagas disease: neglected 'evidence' by evidence-based medicine

No randomized clinical trials regarding the etiological treatment of chronic Chagas disease can be found in the medical literature. However, other 'evidence' sustaining the use of anti-Trypanosoma cruzi drugs for adult individuals with Chagas disease will be analyzed along with the limitations in evaluating the treatment efficacy. Today, the hypothesis of T. cruzi persistence in the target organs giving rise to the chronic inflammatory response is sustained. In addition, several experimental, pathological, nonrandomized clinical studies and studies based on the response or serological evolution (besides the clinical experience) demonstrate the role of T. cruzi in the pathogenesis of the chronic stage and the efficacy of etiological treatment to reduce the titers of antibodies and the progression of chronic Chagas heart disease. All of this supports the recommendation of treatment for every patient diagnosed with Chagas disease. The interpretation of this sum of evidence is not considered from the perspective of evidence-based medicine.     

Regulation of Trypanosoma cruzi infections in vitro and in vivo by transforming growth factor beta (TGF-beta)

The effects of transforming growth factor beta (TGF-beta) on interferon gamma-mediated killing of the intracellular protozoan parasite Trypanosoma cruzi and on the course of T. cruzi infection in mice were investigated. Spleen cells from mice with acute T. cruzi infections were found to produce elevated levels of biologically active TGF-beta in vitro, and the possibility that TGF-beta may mediate certain aspects of T. cruzi infection was then addressed. When mouse peritoneal macrophages were treated with TGF-beta in vitro, the ability of IFN-gamma to activate intracellular inhibition of the parasite was blocked. This occurred whether cells were treated with TGF-beta either before or after IFN-gamma treatment. TGF-beta treatment also blocked the T. cruzi-inhibiting effects of IGN-gamma on human macrophages. Additionally, treatment of human macrophages with TGF-beta alone led to increased parasite replication in these cells. The effects of TGF-beta on T. cruzi infection in vivo were then investigated. Susceptible C57BL/6 mice developed higher parasitemias and died earlier when treated with TGF-beta during the course of infection. Resistant C57BL/6 x DBA/2 F1 mice treated with TGF-beta also had increased parasitemias, and 50% mortality, compared with no mortality in infected, saline-treated controls. A single dose of TGF-beta, given at the time of infection, was sufficient to significantly decrease resistance to infection in F1 mice and to exacerbate infection in susceptible C57BL/6 mice. Furthermore, a single injection of TGF-beta was sufficient to counter the in vivo protective effects of IFN-gamma. We conclude that TGF-beta, produced during acute T. cruzi infection in mice, is a potent inhibitor of the effects of macrophage activating cytokines in vivo and in vitro and may play a role in regulating infection.     

Disruption of TGF-beta signaling in T cells accelerates atherosclerosis

Increasing evidence suggests that atherosclerosis is an inflammatory disease promoted by hypercholesterolemia. The role of adaptive immunity has been controversial, however. We hypothesized that proatherogenic T cells are controlled by immunoregulatory cytokines. Among them, TGF-beta has been implied in atherosclerosis, but its mechanism of action remains unclear. We crossed atherosclerosis-prone ApoE-knockout mice with transgenic mice carrying a dominant negative TGF-beta receptor II in T cells. The ApoE-knockout mice with disrupted TGF-beta signaling in T cells exhibited a sixfold increase in aortic lesion surface area, a threefold increase in aortic root lesion size, and a 125-fold increase in aortic IFN-gamma mRNA when compared with age-matched ApoE-knockout littermates. When comparing size-matched lesions, those of mice with T cell-specific blockade of TGF-beta signaling displayed increased T cells, activated macrophages, and reduced collagen, consistent with a more vulnerable phenotype. Ab's to oxidized LDL, circulating T cell cytokines, and spleen T cell activity were all increased in ApoE-knockout mice with dominant negative TGF-beta receptors in T cells. Taken together, these results show that abrogation of TGF-beta signaling in T cells increases atherosclerosis and suggest that TGF-beta reduces atherosclerosis by dampening T cell activation. Inhibition of T cell activation may therefore represent a strategy for antiatherosclerotic therapy.     

Evaluation of a prototype Trypanosoma cruzi antibody assay with recombinant antigens on a fully automated chemiluminescence analyzer for blood donor screening

BACKGROUND: Chagas disease is caused by Trypanosoma cruzi, a protozoan parasite that can be transmitted by transfusion. The diagnosis of chronic T. cruzi infection is generally made by detecting specific antibodies that bind to parasite antigens. The aim of this study was to assess the sensitivity and specificity of a new serologic assay for antibodies to T. cruzi on a fully automated analyzer (PRISM, Abbott Laboratories). STUDY DESIGN AND METHODS: A prototype chemiluminescent immunoassay based on chimeric recombinant antigens and run on the automated PRISM system was developed for detecting antibodies to T. cruzi in human serum and plasma. Assay specificity was evaluated by testing samples from random blood donors and from a diverse group of specimens from persons with diseases or conditions often associated with false-positive reactions in T. cruzi assays. Sensitivity was determined by testing 377 geographically diverse T. cruzi antibody-positive specimens. RESULTS: Six of 7911 samples (0.08%) from random donors were repeatedly reactive in the prototype PRISM Chagas assay. One of these was reactive in three other tests, including the radioimmune precipitation assay and was presumed to be a true positive. Hence, the specificity was 99.94 percent (7905/7910) in the negative donor group studied. All 377 T. cruzi antibody-positive specimens were positive in the prototype assay and thus the sensitivity was 100 percent. CONCLUSION: The results obtained to date, in terms of sensitivity as well as specificity, strongly suggest that the PRISM Chagas assay should function well as a tool for screening blood for serologic evidence of T. cruzi infection.     

Disruption of transforming growth factor beta signaling by a novel ligand-dependent mechanism

Transforming growth factor (TGF)-beta is the prototype in a family of secreted proteins that act in autocrine and paracrine pathways to regulate cell development and function. Normal cells typically coexpress TGF-beta receptors and one or more isoforms of TGF-beta, thus the synthesis and secretion of TGF-beta as an inactive latent complex is considered an essential step in regula-ting the activity of this pathway. To determine whether intracellular activation of TGF-beta results in TGF-beta ligand-receptor interactions within the cell, we studied pristane-induced plasma cell tumors (PCTs). We now demonstrate that active TGF-beta1 in the PCT binds to intracellular TGF-beta type II receptor (TbetaRII). Disruption of the expression of TGF-beta1 by antisense TGF-beta1 mRNA restores localization of TbetaRII at the PCT cell surface, indicating a ligand-induced impediment in receptor trafficking. We also show that retroviral expression of a truncated, dominant-negative TbetaRII (dnTbetaRII) effectively competes for intracellular binding of active ligand in the PCT and restores cell surface expression of the endogenous TbetaRII. Analysis of TGF-beta receptor-activated Smad2 suggests the intracellular ligand-receptor complex is not capable of signaling. These data are the first to demonstrate the formation of an intracellular TGF-beta-receptor complex, and define a novel mechanism for modulating the TGF-beta signaling pathway.     

Transfusion-associated Chagas disease (American trypanosomiasis) in Mexico: implications for transfusion medicine in the United States

BACKGROUND: Trypanosoma cruzi, the protozoan cause of Chagas disease, causes life-long infection and is easily transmitted by blood transfusion. Our goals were to determine the prevalence of Chagas disease among donors in five Mexican blood banks, to look for evidence of transmission of T. cruzi by transfusion, and to evaluate two serologic assays for Chagas disease. STUDY DESIGN and METHODS: Blood samples from donors were tested initially with the Abbott Chagas EIA or the Meridian Chagas' IgG ELISA. Samples giving readings that were at least 50% of the cutoffs were run in a confirmatory radioimmune precipitation assay (RIPA), as were samples from recipients of blood products from RIPA-positive donors. RESULTS: The overall prevalence of Chagas disease was 1/133 (55/7,296; 0.75%). In addition, 4 of 9 surviving recipients of blood products from T. cruzi-infected donors were in turn infected. Using the manufacturers' recommended cutoffs, the sensitivity and specificity of the Abbott test were 92.0% (23/25) and 99.8% (2,865/2,872) respectively, and the corresponding values for the Meridian assay were 70.0% (21/30) and 100.0% (4,369/4,369). CONCLUSIONS: These findings indicate clearly that transfusion-associated transmission of T. cruzi is occurring in the study areas. Serologic testing of blood donors for Chagas disease should be performed there and in the rest of Mexico. The two screening assays evaluated may lack the accuracy necessary for blood donor testing when used as suggested by the manufacturers.     

Evaluation of the Abbott ARCHITECT Chagas prototype assay

Serologic tests are established tools for the diagnosis of Chagas disease applied to support a safe blood supply in endemic countries. However, sensitivity and specificity of most commercially available enzyme-linked immunosorbent assays (ELISAs) are not regarded as adequate enough to rely on a single assay to determine the Trypanosoma cruzi infection status of a blood donor or a patient. The overall assay performance is driven by the general choice of antigens and the actual antigen cocktail provided in the test. In this report we describe key performance data of the Abbott ARCHITECT Chagas prototype assay in comparison to the well-recognized bioMérieux ELISA cruzi assay. The ARCHITECT assay demonstrated superior specificity (99.99% versus 99.93%) and sensitivity (99.85% versus 98.38%), along with excellent precision, thus showing the potential to serve as single assay to determine the T. cruzi status of a given blood unit or diagnostic specimen on a fully automated instrument platform.     

Pharmacological characterization, structural studies, and in vivo activities of anti-chagas disease lead compounds derived from tipifarnib

Chagas disease, caused by the protozoan pathogen Trypanosoma cruzi, remains a challenging infection due to the unavailability of safe and efficacious drugs. Inhibitors of the trypanosome sterol 14α-demethylase enzyme (CYP51), including azole antifungal drugs, are promising candidates for development as anti-Chagas disease drugs. Posaconazole is under clinical investigation for Chagas disease, although the high cost of this drug may limit its widespread use. We have previously reported that the human protein farnesyltransferase (PFT) inhibitor tipifarnib has potent anti-T. cruzi activity by inhibiting the CYP51 enzyme. Furthermore, we have developed analogs that minimize the PFT-inhibitory activity and enhance the CYP51 inhibition. In this paper, we describe the efficacy of the lead tipifarnib analog compared to that of posaconazole in a murine model of T. cruzi infection. The plasma exposure profiles for each compound following a single oral dose in mice and estimated exposure parameters after repeated twice-daily dosing for 20 days are also presented. The lead tipifarnib analog had potent suppressive activity on parasitemia in mice but was unsuccessful at curing mice, whereas posaconazole as well as benznidazole cured 3 of 5 and 4 of 6 mice, respectively. The efficacy results are consistent with posaconazole having substantially higher predicted exposure than that of the tipifarnib analog after repeat twice-daily administration. Further changes to the tipifarnib analogs to reduce plasma clearance are therefore likely to be important. A crystal structure of a trypanosomal CYP51 bound to a tipifarnib analog is reported here and provides new insights to guide structure-based drug design for further optimized compounds.     

Detection of antibodies to Trypanosoma cruzi among blood donors in the southwestern and western United States. I. Evaluation of the sensitivity and specificity of an enzyme immunoassay for detecting antibodies to T. cruzi

BACKGROUND: Chagas' disease or American trypanosomiasis, caused by infection with Trypanosoma cruzi, is a significant health problem in Latin America. In the United States, transfusions of T. cruzi- contaminated blood from Latin American immigrants may represent the major source of Chagas' disease. STUDY DESIGN AND METHODS: A new enzyme immunoassay (EIA) for the detection of antibody to T. cruzi was evaluated in the sera of blood donors from the southwestern and western regions of the United States. Serum samples had been screened and were negative for all tests required. Specimens that were repeatedly reactive in the Chagas antibody EIA were analyzed for seroreactivity by a confirmatory EIA and by radioimmunoprecipitation assay. RESULTS: Fourteen of the 13,309 donor samples (0.105%) were confirmed as being positive for antibody to T. cruzi. The Chagas antibody EIA showed improved sensitivity over the Chagas IgG enzyme-linked immunosorbent assay and two indirect hemagglutination assays. The Chagas antibody EIA had a specificity of 99.98 percent with negative samples. The sensitivity of the Chagas antibody EIA was 100 percent (80/80) in xenodiagnosed specimens and 100 percent (50/50) in specimens positive by consensus (i.e., reactive in EIA, indirect hemagglutination assay, and immunofluorescence assays). CONCLUSION: This Chagas antibody EIA meets the need for accurate and rapid identification of seroreactive samples in low-prevalence or endemic populations.     

Autoimmunity in Chagas' disease. Identification of cardiac myosin-B13 Trypanosoma cruzi protein crossreactive T cell clones in heart lesions of a chronic Chagas' cardiomyopathy patient.

Heart tissue destruction in chronic Chagas' disease cardiomyopathy (CCC) may be caused by autoimmune recognition of heart tissue by a mononuclear cell infiltrate decades after Trypanosoma cruzi infection. Indirect evidence suggests there is molecular mimicry between T. cruzi and heart tissue. In murine models of CCC, antibodies and CD4+ T cells recognize myosin, the major heart protein. We recently identified a heart-specific epitope of cardiac myosin heavy chain (residues 1442-1447, AAALDK) that is crossreactive with a homologous sequence (AAAGDK) of the immunodominant T. cruzi antigen B13. Furthermore, cardiac myosin-B13 crossreactive antibodies are present in 100% CCC patients vs 14% asymptomatic T. cruzi-seropositive individuals (P = 2.3 x 10(-6)), suggesting a role for molecular mimicry between cardiac myosin and B13 in CCC pathogenesis. In this paper, we obtained heart-infiltrating T cell clones from CCC patients to assess whether molecular mimicry between cardiac myosin and B13 is directly involved in the genesis of heart lesions. We identified T cell clones derived from CCC heart lesions simultaneously responsive to cardiac myosin heavy chain (but not skeletal myosin heavy chain) and B13 T. cruzi protein, but could not find T cell clones primarily reactive to any T. cruzi antigen. Together with the association of myosin-B13 crossreactive antibodies with CCC, the present data strongly suggest the relevance of molecular mimicry between cardiac myosin and the T. cruzi protein B13 in the pathogenesis of heart lesions in chronic Chagas' disease cardiomyopathy.     

Interleukin 10 and interferon gamma regulation of experimental Trypanosoma cruzi infection

Studies were undertaken to determine whether interleukin 10, (IL-10) a cytokine shown to inhibit interferon gamma (IFN-gamma) production, was involved in Trypanosoma cruzi infections in mice. Exogenous IFN-gamma protects mice from fatal infection with T. cruzi. Furthermore, resistant B6D2 mice developed fatal T. cruzi infections when treated with neutralizing anti-IFN-gamma monoclonal antibody (mAb). Thus, endogenous as well as exogenous IFN-gamma is important in mediating resistance to this parasite. Because both T. cruzi-susceptible (B6) and -resistant (B6D2) mouse strains produced IFN-gamma during acute infection, we looked for the concomitant production of mediators that could interfere with IFN-gamma-mediated resistance to T. cruzi. We found that IL-10-specific mRNA was produced in the spleens of mice with acute T. cruzi infections. In addition, spleen cell culture supernatants from infected B6 mice, and to a lesser extent B6D2 mice, elaborated an inhibitor(s) of IFN-gamma production. This inhibitor(s) was neutralized by anti-IL-10 mAb. These experiments demonstrated the production of biologically active IL-10 during T. cruzi infection. In further studies in vitro, it was shown that IL-10 blocked the ability of IFN-gamma to inhibit the intracellular replication of T. cruzi in mouse peritoneal macrophages. Thus, in addition to its known ability to inhibit the production of IFN-gamma, IL-10 (cytokine synthesis inhibitory factor), may also inhibit the effects of IFN-gamma. These experiments demonstrate that IL-10 is produced during infection with a protozoan parasite and suggest a regulatory role for this cytokine in the mediation of susceptibility to acute disease.     

Specific antibodies to Trypanosoma cruzi among blood donors in Los Angeles, California

BACKGROUND: Trypanosoma cruzi, the cause of Chagas' disease, is often transmitted by transfusion in Latin America. Previous studies showed that at least 1 in 1000 eligible blood donors at the Los Angeles County+University of Southern California (LAC+USC) Medical Center Blood Bank had specific antibodies to T. cruzi. In June 1993, serologic screening of prospective allogeneic donors at epidemiologic risk for T. cruzi infection was begun voluntarily. STUDY DESIGN AND METHODS: The risk of T. cruzi infection in all eligible donors was assessed by questionnaire. At-risk donors were screened serologically for antibodies to T. cruzi with an enzyme immunoassay, and confirmatory testing was done with a radioimmunoprecipitation assay. RESULTS: During the 29-month study period 1311 (39.5%) of 3320 donors were judged to be at risk for T. cruzi infection. Seven donors (1/475) were reactive by an enzyme immunoassay, and six of these seven (1/ 553) were positive in a radioimmunoprecipitation assay. All radioimmunoprecipitation assay- positive donors had been born in countries in which Chagas' disease is endemic. One person in this group had received a transfusion in his homeland. CONCLUSION: These results demonstrate that a substantive proportion of eligible blood donors at our institution have antibodies specific for T. cruzi and that a commercially available assay can be used to detect these antibodies. Our data suggest that the risk of transmission of T. cruzi by transfusion could be eliminated by serologic testing limited to persons born in or transfused in countries in which Chagas' disease is endemic.     

Use of a nested polymerase chain reaction (N-PCR) to detect Trypanosoma cruzi in blood samples from chronic chagasic patients and patients with doubtful serologies

Chagas disease, caused by Trypanosoma cruzi, is an important endemic illness in Latin America. Serologic tests for T. cruzi detection in blood are sensitive, but their specificity is unsatisfactory. Direct detection of parasites in blood, either by xenodiagnosis or hemoculture, is highly specific but of low sensitivity. Molecular assays such as the Polymerase chain reaction (PCR), which amplifies certain repetitive sequences of nuclear DNA has been used as a good alternative tool for T. cruzi detection in human blood. The present study aimed to test PCR diagnosis in chagasic chronic patients and doubtful serologic patients attended in GEDOCH (Chagas Disease Study Group/UNICAMP, Brazil). A 149 bp fragment originated from nuclear DNA was specifically detected in chronic chagasic patients. The results of these tests were compared with serologic diagnosis performed using standard techniques and xenodiagnosis. We found that 43 out of 50 patients previously serodiagnosed as chagasic were positive using the N-PCR method. Thirteen of 30 patients with doubtful serologic results were confirmed as positive by N-PCR. Our results suggest that the N-PCR may be a complementary tool to serology in the diagnosis of Chagas disease, and that it is usefull for parasite detection in patients with chronic disease and patients with doubtful serologic results.     

TGF-beta switches from tumor suppressor to prometastatic factor in a model of breast cancer progression

The TGF-beta signaling network plays a complex role in carcinogenesis because it has the potential to act as either a tumor suppressor or a pro-oncogenic pathway. Currently, it is not known whether TGF-beta can switch from tumor suppressor to pro-oncogenic factor during the course of carcinogenic progression in a single cell lineage with a defined initiating oncogenic event or whether the specific nature of the response is determined by cell type and molecular etiology. To address this question, we have introduced a dominant negative type II TGF-beta receptor into a series of genetically related human breast-derived cell lines representing different stages in the progression process. We show that decreased TGF-beta responsiveness alone cannot initiate tumorigenesis but that it can cooperate with an initiating oncogenic lesion to make a premalignant breast cell tumorigenic and a low-grade tumorigenic cell line histologically and proliferatively more aggressive. In a high-grade tumorigenic cell line, however, reduced TGF-beta responsiveness has no effect on primary tumorigenesis but significantly decreases metastasis. Our results demonstrate a causal role for loss of TGF-beta responsiveness in promoting breast cancer progression up to the stage of advanced, histologically aggressive, but nonmetastatic disease and suggest that at that point TGF-beta switches from tumor suppressor to prometastatic factor.     

Blocking Smad7 restores TGF-beta1 signaling in chronic inflammatory bowel disease

TGF-beta1 functions as a negative regulator of T cell immune responses, signaling to target cells using the Smad family of proteins. We show here that Smad7, an inhibitor of TGF-beta1 signaling, is overexpressed in inflammatory bowel disease (IBD) mucosa and purified mucosal T cells. Both whole tissue and isolated cells exhibit defective signaling through this pathway, as measured by phospho-Smad3 immunoreactivity. Specific antisense oligonucleotides for Smad7 reduce Smad7 protein expression in cells isolated from patients with IBD, permitting the cells to respond to exogenous TGF-beta1. TGF-beta1 cannot inhibit proinflammatory cytokine production in isolated lamina propria mononuclear cells from patients with Crohn disease (CD), but inhibition of Smad7 restores TGF-beta1 signaling and enables TGF-beta1 to inhibit cytokine production. In inflamed mucosal tissue explants from patients with CD, inhibition of Smad7 also restores p-Smad3 and decreases proinflammatory cytokine production, an effect that is partially blocked by anti-TGF-beta1. These results show that Smad7 blockade of TGF-beta1 signaling helps maintain the chronic production of proinflammatory cytokines that drives the inflammatory process in IBD and that inhibition of Smad7 enables endogenous TGF-beta to downregulate this response.     

Photoinactivation of Trypanosoma cruzi in red cell suspensions with thiopyrylium.

Chagas disease, endemic in rural areas of Mexico, Central and South America, is caused by the protozoan parasite, Trypanosma cruzi, which is spread by the Reduviid bug and also by transfusion or organ transplant. Transmission of the organism from asymptomatic donors to immunocompromised recipients, leads to clinically apparent disease. With recent immigration patterns, T. cruzi is now becoming an increasing problem in non-endemic areas of North America and Europe. Blood screening tests for T. cruzi are being developed, and one test is currently licensed by the United States Food and Drug Administration and has been implemented in some US blood centers. This study alternatively investigates the potential for a novel DNA-intercalating photosensitizer, thiopyrylium (TP), to inactivate T. cruzi in red cell suspensions. With complete inactivation using 6.3 microM of TP and 1.1J/cm(2) red light treatment, results suggest that the organism is highly sensitive to photoinactivation under conditions much less stringent than those that have been previously demonstrated to maintain red cell (RBC) properties during 42 day storage.     

TGF-beta signaling is required for the function of insulin-reactive T regulatory cells

We have previously isolated insulin-reactive Tregs from diabetic NOD mice designated 2H6, from which TCR transgenic mice were generated. The T cells from these 2H6 transgenic mice recognize insulin but have suppressive properties in vitro. They protect NOD mice in vivo from spontaneous development of diabetes and adoptive transfer of disease caused by polyclonal diabetogenic spleen cells as well as the highly diabetogenic monoclonal BDC2.5 TCR transgenic T cells that recognize an islet granule antigen. Using cells from both NOD and BDC2.5 mice that express a dominant-negative TGF-beta receptor type II (TGF-betaDNRII), we show that 2H6 T cells protected from disease by producing TGF-beta and that the ability of the target diabetogenic T cells to respond to TGF-beta was crucial. We further demonstrate that TGF-beta signaling in 2H6 cells was important for their protective properties, as 2H6 cells were unable to protect from adoptive transfer-induced diabetes if they were unable to respond to TGF-beta. Thus, our data demonstrate that insulin-specific regulatory cells protect from diabetes by virtue of their production of TGF-beta1 that acts in an autocrine manner to maintain their regulatory function and acts in a paracrine manner on the target cells.     

Pioglitazone induces apoptosis in human vascular smooth muscle cells from diabetic patients involving the transforming growth factor-beta/activin receptor-like kinase-4/5/7/Smad2 signaling pathway

Alterations in vascular wall remodeling are a typical complication in type 2 diabetes mellitus due to an imbalance between cell proliferation and apoptosis. In this context, we have previously shown that vascular smooth muscle cells (VSMC) from diabetic patients were resistant to induced apoptosis. Thiazolidinediones, such as pioglitazone, seem to exert direct antiatherosclerotic effects on type 2 diabetes. Here, we aimed to study whether pioglitazone was able to induce apoptosis in VSMC from diabetic patients (DP) and, if so, whether the transforming growth factor (TGF)-beta1/Smad-2 pathway was involved. We isolated human internal mammary artery VSMC from patients who had undergone coronary-artery bypass graft. Pioglitazone (100 microM) induced apoptosis in human VSMC from diabetic and nondiabetic patients (NDP), analyzed by DNA fragmentation and by degradation of Bcl-2, in high-glucose-containing medium (15 and 25 mM). This apoptotic effect was inhibited by the activin receptor-like kinase-4/5/7/Smad2 inhibitor 4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide (SB-431542), denoting that the TGF-beta1/Smad-2 pathway was involved. Pioglitazone rapidly increased the extracellular TGF-beta1 levels and concomitantly induced phosphorylation of Smad2 in VSMC from DP and NDP. Thus, we demonstrated that pioglitazone induced apoptosis in human VSMC from DP, which are strongly resistant to the induced apoptosis. This effect of pioglitazone might contribute in the treatment of alterations of vascular remodeling in type 2 diabetes mellitus.