Huan Huang

Albert Einstein College of Medicine, New York, New York, United States

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Publications (39)137.15 Total impact

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    Yanfen Ma, Louis M Weiss, Huan Huang
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    ABSTRACT: Chagas disease caused by Trypanosoma cruzi is a major neglected tropical parasitic disease. The pathogenesis of this infection remains disputable. There is no suitable vaccine for the prevention. Attenuated live vaccines can provide strong protection against infection; however, there are the concerns about latent infection or reversion to virulence in such attenuated strains. A method to induce T. cruzi death would provide a critical tool for research into the pathophysiological mechanisms and provide a novel design of safe live attenuated vaccines. We established effective inducible systems for T. cruzi employing the degradation domain based on the Escherichia coli dihydrofolate reductase (ecDHFR). The DHFR degradation domain (DDD) can be stabilized by trimethoprim-lactate and can be used to express detrimental or toxic proteins. T. cruzi lines with Alpha-toxin, Cecropin A and GFP under the control of DDD with a hemagglutinin tag (HA) were developed. Interestingly, amastigotes bearing GFP-DDDHA, Alpha-toxin-DDDHA, Cecropin A-DDDHA and DDDHA all resulted in inducible cell death with these fusions, indicating that DDDHA protein is also detrimental to amastigotes. Furthermore, these strains were attenuated in mouse experiments producing no pathological changes and inoculation with these DDDHA strains in mice provided strong protection against lethal wild type infection. Copyright © 2015. Published by Elsevier Masson SAS.
    Microbes and Infection 04/2015; 51. DOI:10.1016/j.micinf.2015.04.003 · 2.73 Impact Factor
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    ABSTRACT: Trogocytosis was originally thought to be restricted to the interaction of cells of the immune system with cancer cells. Such membrane exchanges are probably a general process in cell biology, and membrane exchange has been demonstrated to occur between non-immune cells within an organism. Herein, we report that membrane and protein exchange, consistent with trogocytosis, between Trypanosoma cruzi (both the Brazil and Tulahuen strains) and the mammalian cells it infects. Transfer of labeled membrane patches was monitored by labeling of either parasites or host cells, i.e. human foreskin fibroblasts and rat myoblasts. Trypomastigotes and amastigotes transferred specific surface glycoproteins to the host cells along with membranes. Exchange of membranes between the parasite and host cells occurred during successful invasion. Extracellular amastigotes did not transfer membrane patches and were did not transfer either membranes or proteins to the host cells. Membrane exchange was also found to occur between interacting epimastigotes in cell-free culture and may be important in parasite-parasite interactions as well. Further studies should provide new insights into pathogenesis and provide targets for therapeutic intervention. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
    Microbes and Infection 11/2014; 17(1). DOI:10.1016/j.micinf.2014.10.009 · 2.73 Impact Factor
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    ABSTRACT: Trypanosoma cruzi infection in humans and experimental animals causes Chagas disease which is often accompanied by myocarditis, cardiomyopathy, and vasculopathy. T. cruzi-derived thromboxane A(2) (TXA(2)) modulates vasculopathy and other pathophysiological features of Chagasic cardiomyopathy. Here, we provide evidence that epimastigotes, trypomastigotes, and amastigotes of T. cruzi (Brazil and Tulahuen strains) express a biologically active prostanoid receptor (PR) that is responsive to TXA(2) mimetics, e.g. IBOP. This putative receptor, TcPR, is mainly localized in the flagellar membrane of the parasites and shows a similar glycosylation pattern to that of bona fide thromboxane prostanoid (TP) receptors obtained from human platelets. Furthermore, TXA(2)-PR signal transduction activates T. cruzi-specific MAPK pathways. While mammalian TP is a G-protein coupled receptor (GPCR); T. cruzi genome sequencing has not demonstrated any confirmed GPCRs in these parasites. Based on this genome sequencing it is likely that TcPR is unique in these protists with no counterpart in mammals. TXA(2) is a potent vasoconstrictor which contributes to the pathogenesis of Chagasic cardiovascular disease. It may, however, also control parasite differentiation and proliferation in the infected host allowing the infection to progress to a chronic state.
    Parasitology Research 02/2013; 112(4). DOI:10.1007/s00436-012-3271-5 · 2.33 Impact Factor
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    ABSTRACT: Chagas disease, caused by Trypanosoma cruzi, is an important cause of morbidity and mortality primarily resulting from cardiac dysfunction, although T. cruzi infection results in inflammation and cell destruction in many organs. We found that T. cruzi (Brazil strain) infection of mice results in pancreatic inflammation and parasitism within pancreatic β-cells with apparent sparing of α cells and leads to the disruption of pancreatic islet architecture, β-cell dysfunction, and surprisingly, hypoglycemia. Blood glucose and insulin levels were reduced in infected mice during acute infection and insulin levels remained low into the chronic phase. In response to the hypoglycemia, glucagon levels 30 days postinfection were elevated, indicating normal α-cell function. Administration of L-arginine and a β-adrenergic receptor agonist (CL316, 243, respectively) resulted in a diminished insulin response during the acute and chronic phases. Insulin granules were docked, but the lack of insulin secretion suggested an inability of granules to fuse at the plasma membrane of pancreatic β-cells. In the liver, there was a concomitant reduced expression of glucose-6-phosphatase mRNA and glucose production from pyruvate (pyruvate tolerance test), demonstrating defective hepatic gluconeogenesis as a cause for the T. cruzi-induced hypoglycemia, despite reduced insulin, but elevated glucagon levels. The data establishes a complex, multi-tissue relationship between T. cruzi infection, Chagas disease, and host glucose homeostasis.
    American Journal Of Pathology 01/2013; DOI:10.1016/j.ajpath.2012.11.027 · 4.60 Impact Factor
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    ABSTRACT: Chagas' disease, caused by the parasite Trypanosoma cruzi, is an important cause of heart disease. Previous studies from this laboratory revealed that microvascular spasm and myocardial ischemia were observed in infected mice. Infection of endothelial cells with this parasite increased the synthesis of biologically active endothelin-1 (ET-1). Therefore, in the myocardium of T. cruzi-infected mice, we examined ET-1 expression and the p42/44-mito-gen activated protein kinase (MAPK)-AP-1 pathway that regulates the expression of ET-1. There was parasitism and myonecrosis in the myocardium of infected C57BL/6 mice. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed elevated mRNA expression of transcription factor AP-1 (c-jun and c-fos) and increased AP-1 DNA binding activity as determined by electrophoretic mobility shift assay (EMSA). Western blot analysis demonstrated an increase in the phosphorylated forms of extracellular signal-regulated kinase (ERK1/2). ET-1 mRNA was upregulated in the myocardium of infected mice. Immunohistochemical and immunoelectron microscopy using anti-ET-1 antibody detected increased expression in cardiac myocytes and endothelium of these mice.These data suggest that ET-1 contributes to chagasic cardiomyopathy and that the mechanism of the increased expression of ET-1 is a result of the activation of the MAPK pathway by T. cruzi infection. (C) 2000 Lippincott Williams & Wilkins, Inc.
    Journal of Cardiovascular Pharmacology 08/2012; 36(5). DOI:10.1097/00005344-200036001-00046 · 2.11 Impact Factor
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    Yan Fen Ma, Louis M Weiss, Huan Huang
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    ABSTRACT: Analysis of gene function in Trypanosoma cruzi is limited due to the absence of rapid, simple and reversible genetic tools to regulate gene and corresponding protein expression. We have designed a modified pTREX vector which uses an N-terminal fusion of a ligand-controlled destabilisation domain (ddFKBP) to a gene/protein of interest. This vector allows rapid and reversible protein expression and efficient functional analysis of proteins in different T. cruzi life cycle stages.
    International journal for parasitology 11/2011; 42(1):33-7. DOI:10.1016/j.ijpara.2011.11.002 · 3.40 Impact Factor
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    ABSTRACT: Mitogen-activated protein kinase (MAPK) pathways are major signal transduction systems by which eukaryotic cells convert environmental cues to intracellular events, such as cell proliferation and differentiation. Toxoplasma gondii is an obligate intracellular protozoan that is both a human and animal pathogen. This Apicomplexan causes significant morbidity and mortality in immune-competent and immune-compromised hosts. In humans, the most common manifestations of T. gondii infections are chorioretinitis in congenital infection and encephalitis in immune-compromised patients, such as patients with advanced AIDS. We have identified a T. gondii homolog of the MAPK family that we have called TgMAPK2. Sequence analyses demonstrated that TgMAPK2 has homology with lower eukaryotic ERK2 but has significant differences from mammalian ERK2. TgMAPK2 has an open reading frame of 2,037 bp, 678 amino acids, and its molecular weight is 73.1 kDa. It contains the typical 12 subdomains of a MAPK and has a TDY motif in the dual phosphorylation and activation subdomains. This suggests that TgMAPK2 may play an important role in stress response. recombinant TgMAPK2 was catalytically active and was not inhibited by a human ERK2 inhibitor, FR180204. A partial TgMAPK2 lacking the ATP-binding motifs GxGxxGxV was successfully regulated by a ligand-controlled destabilization domain (ddFKBP) expression vector system in T. gondii. Since TgMAPK2 is significantly different from its mammalian counterpart, it may be useful as a drug target. This work establishes a foundation for further study for this unique kinase.
    Cell cycle (Georgetown, Tex.) 10/2011; 10(20):3519-26. DOI:10.4161/cc.10.20.17791 · 5.01 Impact Factor
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    ABSTRACT: Gap junction channels provide intercellular communication between cells. In the heart, these channels coordinate impulse propagation along the conduction system and through the contractile musculature, thereby providing synchronous and optimal cardiac output. As in other arrhythmogenic cardiac diseases, chagasic cardiomyopathy is associated with decreased expression of the gap junction protein connexin43 (Cx43) and its gene. Our studies of cardiac myocytes infected with Trypanosoma cruzi have revealed that synchronous contraction is greatly impaired and gap junction immunoreactivity is lost in infected cells. Such changes are not seen for molecules forming tight junctions, another component of the intercalated disc in cardiac myocytes. Transcriptomic studies of hearts from mouse models of Chagas disease and from acutely infected cardiac myocytes in vitro indicate profound remodelling of gene expression patterns involving heart rhythm determinant genes, suggesting underlying mechanisms of the functional pathology. One curious feature of the altered expression of Cx43 and its gene expression is that it is limited in both extent and location, suggesting that the more global deterioration in cardiac function may result in part from spread of damage signals from more seriously compromised cells to healthier ones.
    Advances in Parasitology 01/2011; 76:63-81. DOI:10.1016/B978-0-12-385895-5.00003-7 · 4.36 Impact Factor
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    ABSTRACT: Mice carrying a defective leptin receptor gene (db/db mice) are metabolically challenged and upon infection with Trypanosoma cruzi (Brazil strain) suffer high mortality. In genetically modified db/db mice, (NSE-Rb db/db mice), central leptin signaling is reconstituted only in the brain, which is sufficient to correct the metabolic defects. NSE-Rb db/db mice were infected with T. cruzi to determine the impact of the lack of leptin signaling on infection in the absence of metabolic dysregulation. Parasitemia levels, mortality rates, and tissue parasitism were statistically significantly increased in infected db/db mice compared with those in infected NSE-Rb db/db and FVB wild-type mice. There was a reduction in fat mass and blood glucose level in infected db/db mice. Plasma levels of several cytokines and chemokines were statistically significantly increased in infected db/db mice compared with those in infected FVB and NSE-Rb db/db mice. These findings suggest that leptin resistance in individuals with obesity and diabetes mellitus may have adverse consequences in T. cruzi infection.
    The Journal of Infectious Diseases 10/2010; 202(7):1104-13. DOI:10.1086/656189 · 5.78 Impact Factor
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    ABSTRACT: Mitogen-activated protein kinase (MAPK) pathways are major signal transduction systems by which eukaryotic cells convert environmental cues to intracellular events such as proliferation and differentiation. We have identified a Trypanosoma cruzi homologue of the MAPK family that we have called TcMAPK2. Sequence analyses demonstrates TcMAPK2 has high homology with lower eukaryotic ERK2 but has significant differences from mammalian ERK2. Enzymatic assays of both recombinant TcMAPK2 and native protein obtained by immunoprecipitation using anti-TcMAPK2 demonstrated that both preparations of TcMAPK2 were catalytically active. Immunofluorescence analysis of the subcellular localization of TcMAPK2 determined it is mainly cytoplasmic in epimastigotes, along the flagella in trypomastigotes and on the plasma membrane of intracellular amastigotes. Phosphorylated TcMAPK2 was highest in trypomastigotes and lowest in amastigotes. Recombinant TcMAPK2 was able to phosphorylate the recombinant protein of a cAMP specific phosphodiesterase. Overexpression of TcMAPK2 in epimastigotes inhibited growth and development leading to death. TcMAPK2 has an important role in the stress response of the parasite and may be important in regulating proliferation and differentiation.
    Cell cycle (Georgetown, Tex.) 07/2010; 9(14):2888-96. DOI:10.4161/cc.9.14.12372 · 5.01 Impact Factor
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    ABSTRACT: Protein kinase A (PKA) has been suggested as a regulator of stage differentiation in Trypanosoma cruzi. Using a yeast two-hybrid system we have begun to characterize the downstream substrates of T. cruzi PKA. We identified several members of the trans-sialidase super family by this approach. Immunoprecitation demonstrated that a TcPKAc monoclonal antibody was able to pull-down proteins recognized by trans-sialidase antibodies as well as a SA85-1.1 antibody and vice versa. An in vitro phosphorylation assay demonstrated that PKA phosphorylated the recombinant protein of an active trans-sialidase. In addition, a phospho-(Ser/Thr) PKA substrate antibody detected bands on immunoblot analysis of trans-sialidase antibody precipitated proteins from parasite lysate and the media of L(6)E(9) myoblasts infected with trypomastigotes as well as from a SA85-1.1 antibody precipitated proteins from parasite lysate. Immunofluorescence analysis suggested that some TcPKAc localizes to the plasma membrane surface of trypomastigotes. The identified trans-sialidases have PKA consensus phosphorylation sites located near the endoplasmic reticulum retention motif in the N-terminal. These data support that PKA phosphorylates trans-sialidase super family members in vivo.
    Microbes and Infection 05/2010; 12(10):716-26. DOI:10.1016/j.micinf.2010.04.014 · 2.73 Impact Factor
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    ABSTRACT: Cyclic AMP-protein kinase A (PKA) signaling is important for the growth and differentiation of Trypanosoma cruzi. Immunofluorescence suggests that PKA can associate with the plasma membrane of trypomastigotes. We found that the PKA regulatory subunit interacts with several P-type ATPases. These P-type ATPases may play a role in anchoring PKA to the plasma membrane in T. cruzi.
    The American journal of tropical medicine and hygiene 07/2009; 80(6):941-3. · 2.74 Impact Factor
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    ABSTRACT: Chagas disease is caused by the parasite Trypanosoma cruzi. The critical initial event is the interaction of the trypomastigote form of the parasite with host receptors. This review highlights recent observations concerning these interactions. Some of the key receptors considered are those for thromboxane, bradykinin, and for the nerve growth factor TrKA. Other important receptors such as galectin-3, thrombospondin, and laminin are also discussed. Investigation into the molecular biology and cell biology of host receptors for T. cruzi may provide novel therapeutic targets.
    Parasitology Research 04/2009; 104(6):1251-60. DOI:10.1007/s00436-009-1383-3 · 2.33 Impact Factor
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    ABSTRACT: Infection with Trypanosoma cruzi causes megasyndromes of the gastrointestinal (GI) tract. We used magnetic resonance imaging (MRI) to monitor alterations in the GI tract of T. cruzi-infected mice, and to assess the role of nitric oxide (NO) in the development of intestinal dilation. Brazil strain-infected C57BL/6 wild-type (WT) mice exhibited dilatation of the intestines by 30 days post-infection. Average intestine lumen diameter increased by 72%. Levels of intestinal NO synthase (NOS) isoforms, NOS2 and NOS3, were elevated in infected WT mice. Inflammation and ganglionitis were observed in all infected mice. Intestinal dilation was observed in infected WT, NOS1, NOS2, and NOS3 null mice. This study demonstrates that MRI is a useful tool to monitor intestinal dilation in living mice and that these alterations may begin during acute infection. Furthermore, our data strongly suggests that NO may not be the sole contributor to intestinal dysfunction resulting from this infection.
    The American journal of tropical medicine and hygiene 12/2008; 79(5):760-7. · 2.74 Impact Factor
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    ABSTRACT: Protein kinase A (PKA) is an important mediator of many signal transduction pathways that occur in eukaryotic cells, and it has been implicated as a regulator of stage differentiation in Trypanosoma cruzi. To evaluate the importance of the PKA catalytic subunit of T. cruzi (TcPKAc), a gene encoding a PKA inhibitor (PKI) containing a specific PKA pseudosubstrate, R-R-N-A, was subcloned into a pTREX vector and introduced into epimastigotes by electroporation. Expression of PKI has a lethal effect in this parasite. Similarly, a pharmacological inhibitor, H89, killed epimastigotes at a concentration of 10 muM. To understand the biology of PKA, identification of the particular substrates of this enzyme is essential. Using a yeast two-hybrid system, 38 candidates interacting with TcPKAc were identified. Eighteen of these were hypothetical proteins with unknown functions, while the others had putative or known functions. The entire open reading frames of eight genes presumably important in regulating T. cruzi growth, adaptation, and differentiation, including a type III PI3 kinase (Vps34), a putative PI3 kinase, a putative mitogen-activated extracellular signal-regulated kinase, a cyclic AMP (cAMP)-specific phosphodiesterase (PDEC2), a hexokinase, a putative ATPase, a DNA excision repair protein, and an aquaporin were confirmed to interact with TcPKAc in the yeast Saccharomyces cerevisiae under the highest stringency selection conditions, and PKA phosphorylated the recombinant proteins of these genes. Taken together, these findings demonstrate the importance of cAMP-PKA signaling in this organism.
    Infection and immunity 09/2008; 76(10):4757-63. DOI:10.1128/IAI.00527-08 · 4.16 Impact Factor
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    ABSTRACT: Gap junction proteins (connexins) are required for myocardial function, since they allow intercellular transmission of current carrying ions and signaling molecules. Previous studies demonstrated that rat cardiac myocytes infected with Trypanosoma cruzi lost gap junctional communication and decreased automaticity. We infected mouse cardiac myocytes with trypomastigotes of the Y strain of T. cruzi and observed alterations in connexin43 (Cx43) distribution. One hour post infection Cx43 levels were significantly increased. However, at longer time points post infection there was a significant loss of Cx43 staining in membranes of infected cardiac myocytes. Interestingly, there was also a significant reduction in myocardial Cx43 protein levels during acute infection. These data indicate that T. cruzi infection alters Cx43 expression both in vitro and in vivo. Disruptions in Cx43 may contribute to the pathogenesis of cardiac electrical alterations observed in T. cruzi infection.
    Microbes and Infection 02/2008; 10(1):21-8. DOI:10.1016/j.micinf.2007.09.017 · 2.73 Impact Factor
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    ABSTRACT: Chagas' disease is caused by infection with the parasite Trypanosoma cruzi. We report that infected, but not uninfected, human endothelial cells (ECs) released thromboxane A(2) (TXA(2)). Physical chromatography and liquid chromatography-tandem mass spectrometry revealed that TXA(2) is the predominant eicosanoid present in all life stages of T. cruzi. Parasite-derived TXA(2) accounts for up to 90% of the circulating levels of TXA(2) in infected wild-type mice, and perturbs host physiology. Mice in which the gene for the TXA(2) receptor (TP) has been deleted, exhibited higher mortality and more severe cardiac pathology and parasitism (fourfold) than WT mice after infection. Conversely, deletion of the TXA(2) synthase gene had no effect on survival or disease severity. TP expression on somatic cells, but not cells involved in either acquired or innate immunity, was the primary determinant of disease progression. The higher intracellular parasitism observed in TP-null ECs was ablated upon restoration of TP expression. We conclude that the host response to parasite-derived TXA(2) in T. cruzi infection is possibly an important determinant of mortality and parasitism. A deeper understanding of the role of TXA(2) may result in novel therapeutic targets for a disease with limited treatment options.
    Journal of Experimental Medicine 05/2007; 204(4):929-40. DOI:10.1084/jem.20062432 · 13.91 Impact Factor
  • Molecular and Biochemical Parasitology 11/2006; 149(2):242-5. DOI:10.1016/j.molbiopara.2006.05.008 · 2.24 Impact Factor
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    ABSTRACT: Endothelin has been implicated in the pathogenesis of experimental and human Chagas' disease (American trypanosomiasis). In the present study, we tested the effect of bosentan, an antagonist of both ET(A) and ET(B) endothelin receptors, on parasitemia, histopathology (heart and diaphragm), heart levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-10, interferon (IFN)-gamma, CCL2, CCL3 and CCL5, and the serum levels of nitrate/nitrite (NOx). Bosentan treatment was accompanied by a significant increase in parasitemia and tissue parasitism or inflammation. In vehicle-treated rats, Trypanosoma cruzi infection increased the cardiac levels of TNF-alpha, IFN-gamma and IL-10, at day 9 post inoculation, and the TNF-alpha remained elevated until day 13. The infection also caused a significant increase in the cardiac levels of the chemokines CCL2 (9, 13 and 18 days) and CCL3 (13 and 18 days). Bosentan-treatment had no significant effect on the infection-associated increase in IFN-gamma and chemokine concentrations. There was a lower increase in IL-10 at day 9 and this was mirrored by a greater increase of TNF-alpha at day 13, in comparison with vehicle-treated rats. These latter findings correlated well with the enhanced inflammatory process in hearts of bosentan-treated infected rats. Bosentan treatment reduced the infection-associated increase in NOx serum concentration. Altogether, our data suggest that ET action on ET(A) and ET(B) receptors may play a role in the initial control of T. cruzi infection in rats probably by interfering in NO production.
    Microbes and Infection 08/2006; 8(8):2113-9. DOI:10.1016/j.micinf.2006.03.017 · 2.73 Impact Factor
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    ABSTRACT: Trypanosoma cruzi infection causes cardiomyopathy and vasculopathy. Previous studies have demonstrated that infection of human umbilical vein endothelial and smooth muscle cells resulted in activation of extracellular signal-regulated kinase (ERK). In the present study, smooth muscle cells were infected with trypomastigotes, and immunoblot analysis revealed an increase in the expression of cyclin D1 and proliferating cell nuclear antigen (PCNA), important mediators of smooth muscle cell proliferation. Interestingly, after infection, the expression of caveolin-1 was reduced in both human umbilical vein endothelial cells and smooth muscle cells. Immunoblot and immunohistochemical analyses of lysates of carotid arteries obtained from infected mice revealed increased expression of PCNA, cyclin D1, its substrate, phospho-Rb (Ser780), and phospho-ERK1/2. The expression of the cyclin-dependent kinase inhibitor p21(Cip1/Waf1), caveolin-1, and caveolin-3 was reduced in carotid arteries obtained from infected mice. There was an increase in the abundance of pre-pro-endothelin-1 mRNA in the carotid artery and aorta from infected mice. The ET(A) receptor was also elevated in infected arteries. ERK activates endothelin-1, which in turn exerts positive feedback activating ERK, and cyclin D1 is a downstream target of both endothelin-1 and ERK. There was significant incorporation of bromodeoxyuridine into smooth muscle cell DNA when treatment was with conditioned medium obtained from infected endothelial cells. Taken together, these data suggest that T. cruzi infection stimulates smooth muscle cell proliferation and is likely a result of the upregulation of the ERK-cyclin D1-endothelin-1 pathway.
    Infection and Immunity 02/2006; 74(1):152-9. DOI:10.1128/IAI.74.1.152-159.2006 · 4.16 Impact Factor