T. cruzi-induced Changes in Cardiac Endothelial Cells

Publication Date

July 2019

Location

LSU Health Medical Education Building

Document Type

Abstract

Start Date

26-7-2019 9:00 AM

End Date

26-7-2019 12:00 PM

Description

Chagas’ Disease is a parasitic infection caused by the kinetoplastid parasite, Trypanosoma cruzi and is endemic throughout South & Central America, and Mexico. It is estimated that as many as 8 million individuals are infected worldwide, including more than 300,000 currently residing in the USA with associated costs of over $9M. T. cruzi is most commonly transmitted by triatomine (aka “kissing”) bugs, but can also be transmitted congenitally, via blood transfusion, and through ingestion of food & drink. Acute Chagas’ is an inflammatory condition resulting in mild to moderate signs and symptoms lasting several weeks or months, but often goes undiagnosed. Untreated disease may advance to include serious cardiovascular and/or digestive pathology. Up to 30% of those infected with T. cruzi will develop Chronic Chagas’ Disease (CCD) involving life-threatening cardiomyopathy. Treatments options are currently limited to only 2 drugs, both associated with limited efficacy and side-effects, and no vaccine is currently available. Symptoms of CCD include cardiac hypertrophy and dysfunction caused by excessive inflammation and fibrosis. While several cell types have been shown to contribute to this pathology, any potential involvement by cardiac endothelial cells is largely unknown. In this study, we aim to identify changes in cardiac endothelial phenotypes that may result in increased pro-fibrotic functions. As endothelial cells are known to undergo endothelial-to-mesenchymal transition (EndMT), we hypothesize that T. cruzi infection induces EndMT in cardiac endothelium, leading to increased cardiac fibrosis. We have chosen to develop a multicellular, 3D model of chronic T. cruzi infection. In order to establish this model, we must first identify a baseline of gene expression profiles in both control and infected cell types, as well as validate our reagents- in 2D culture. For this report we have utilized Quantitative Reverse Transcriptase Polymerase Chain Reaction (QRTPCR) to measure changes in gene expression, and immunocytochemistry (ICC) to characterize changes in protein expression in both cardiac microvascular endothelial cells (cMVEC) and cardiac fibroblasts (cFb) in 2D mono- and co-cultures. We find that cMVEC infection with T. cruzi tissue-culture-derived trypomastigotes (TcTs) results in both early and late changes in gene expression. The pro-inflammatory genes TGFβ and IL-1β are rapidly increased at early timepoints, as are the EndMT-associated transcription factors SNAIL and SLUG. By 24 hrs post-infection, expression of key endothelial markers had dropped, although no upregulation of fibrosis-associated genes was observed. We also stained fixed cell cultures for characterization of both endothelial and fibroblast cellular markers. We found that our current reagents (Abs, etc) successfully identified key markers on both cell types as evidenced by immunofluorescent microscopy. These findings in a 2D model set baseline parameters for the identification of potential EndMT-associated changes in cellular phenotypes as we move forward into our 3D model.

Comments

Mentor: Douglas Johnston, PhD, Microbiology, Immunology, and Parasitology

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Jul 26th, 9:00 AM Jul 26th, 12:00 PM

T. cruzi-induced Changes in Cardiac Endothelial Cells

LSU Health Medical Education Building

Chagas’ Disease is a parasitic infection caused by the kinetoplastid parasite, Trypanosoma cruzi and is endemic throughout South & Central America, and Mexico. It is estimated that as many as 8 million individuals are infected worldwide, including more than 300,000 currently residing in the USA with associated costs of over $9M. T. cruzi is most commonly transmitted by triatomine (aka “kissing”) bugs, but can also be transmitted congenitally, via blood transfusion, and through ingestion of food & drink. Acute Chagas’ is an inflammatory condition resulting in mild to moderate signs and symptoms lasting several weeks or months, but often goes undiagnosed. Untreated disease may advance to include serious cardiovascular and/or digestive pathology. Up to 30% of those infected with T. cruzi will develop Chronic Chagas’ Disease (CCD) involving life-threatening cardiomyopathy. Treatments options are currently limited to only 2 drugs, both associated with limited efficacy and side-effects, and no vaccine is currently available. Symptoms of CCD include cardiac hypertrophy and dysfunction caused by excessive inflammation and fibrosis. While several cell types have been shown to contribute to this pathology, any potential involvement by cardiac endothelial cells is largely unknown. In this study, we aim to identify changes in cardiac endothelial phenotypes that may result in increased pro-fibrotic functions. As endothelial cells are known to undergo endothelial-to-mesenchymal transition (EndMT), we hypothesize that T. cruzi infection induces EndMT in cardiac endothelium, leading to increased cardiac fibrosis. We have chosen to develop a multicellular, 3D model of chronic T. cruzi infection. In order to establish this model, we must first identify a baseline of gene expression profiles in both control and infected cell types, as well as validate our reagents- in 2D culture. For this report we have utilized Quantitative Reverse Transcriptase Polymerase Chain Reaction (QRTPCR) to measure changes in gene expression, and immunocytochemistry (ICC) to characterize changes in protein expression in both cardiac microvascular endothelial cells (cMVEC) and cardiac fibroblasts (cFb) in 2D mono- and co-cultures. We find that cMVEC infection with T. cruzi tissue-culture-derived trypomastigotes (TcTs) results in both early and late changes in gene expression. The pro-inflammatory genes TGFβ and IL-1β are rapidly increased at early timepoints, as are the EndMT-associated transcription factors SNAIL and SLUG. By 24 hrs post-infection, expression of key endothelial markers had dropped, although no upregulation of fibrosis-associated genes was observed. We also stained fixed cell cultures for characterization of both endothelial and fibroblast cellular markers. We found that our current reagents (Abs, etc) successfully identified key markers on both cell types as evidenced by immunofluorescent microscopy. These findings in a 2D model set baseline parameters for the identification of potential EndMT-associated changes in cellular phenotypes as we move forward into our 3D model.