Lipid mass spectrometry imaging and proteomic analysis of severe aortic stenosis.

Severe aortic stenosis (AS) is prevalent in adults ≥ 65 years, a significant cause of morbidity and mortality, with no medical therapy. Lipid and proteomic alterations of human AS tissue were determined using mass spectrometry imaging (MSI) and liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to understand histopathology, potential biomarkers of disease, and progression from non-calcified to calcified phenotype. A reproducible MSI method was developed using healthy murine aortic valves (n = 3) and subsequently applied to human AS (n = 2). Relative lipid levels were spatially mapped and associated with different microdomains. Proteomics for non-calcified and calcified microdomains were performed to ascertain differences in expression. Increased pro-osteogenic and inflammatory lysophosphatidylcholine (LPC) 16:0 and 18:0 were co-localized with calcified microdomains. Proteomics analysis identified differential patterns in calcified microdomains with high LPC and low cholesterol as compared to non-calcified microdomains with low LPC and high cholesterol. Calcified microdomains had higher levels of: apolipoproteins (Apo) B-100 (p < 0.001) and Apo A-IV (p < 0.001), complement C3 and C4-B (p < 0.001), C5 (p = 0.007), C8 beta chain (p = 0.013) and C9 (p = 0.010), antithrombotic proteins alpha-2-macroglobulin (p < 0.0001) and antithrombin III (p = 0.002), and higher anti-calcific fetuin-A (p = 0.02), while the osteoblast differentiating factor transgelin (p < 0.0001), extracellular matrix proteins versican, prolargin, and lumican ( p < 0.001) and regulator protein complement factor H (p < 0.001) were higher in non-calcified microdomains. A combined lipidomic and proteomic approach provided insight into factors potentially contributing to progression from non-calcified to calcific disease in severe AS. Additional studies of these candidates and protein networks could yield new targets for slowing progression of AS.

The brighter (and evolutionarily older) face of the metabolic syndrome: evidence from Trypanosoma cruzi infection in CD-1 mice.

BACKGROUND: Infection with Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, results in chronic infection that leads to cardiomyopathy with increased mortality and morbidity in endemic regions. In a companion study, our group found that a high-fat diet (HFD) protected mice from T. cruzi-induced myocardial damage and significantly reduced post-infection mortality during acute T. cruzi infection. METHODS: In the present study metabolic syndrome was induced prior to T. cruzi infection by feeding a high fat diet. Also, mice were treated with anti-diabetic drug metformin. RESULTS: In the present study, the lethality of T. cruzi (Brazil strain) infection in CD-1 mice was reduced from 55% to 20% by an 8-week pre-feeding of an HFD to induce obesity and metabolic syndrome. The addition of metformin reduced mortality to 3%. CONCLUSIONS: It is an interesting observation that both the high fat diet and the metformin, which are known to differentially attenuate host metabolism, effectively modified mortality in T. cruzi-infected mice. In humans, the metabolic syndrome, as presently construed, produces immune activation and metabolic alterations that promote complications of obesity and diseases of later life, such as myocardial infarction, stroke, diabetes, Alzheimer's disease and cancer. Using an evolutionary approach, we hypothesized that for millions of years, the channeling of host resources into immune defences starting early in life ameliorated the effects of infectious diseases, especially chronic infections, such as tuberculosis and Chagas disease. In economically developed countries in recent times, with control of the common devastating infections, epidemic obesity and lengthening of lifespan, the dwindling benefits of the immune activation in the first half of life have been overshadowed by the explosion of the syndrome's negative effects in later life.

High fat diet modulates Trypanosoma cruzi infection associated myocarditis.

BACKGROUND: Trypanosoma cruzi, the causative agent of Chagas disease, has high affinity for lipoproteins and adipose tissue. Infection results in myocarditis, fat loss and alterations in lipid homeostasis. This study was aimed at analyzing the effect of high fat diet (HFD) on regulating acute T. cruzi infection-induced myocarditis and to evaluate the effect of HFD on lipid metabolism in adipose tissue and heart during acute T. cruzi infection. METHODOLOGY/PRINCIPAL FINDINGS: CD1 mice were infected with T. cruzi (Brazil strain) and fed either a regular control diet (RD) or HFD for 35 days following infection. Serum lipid profile, tissue cholesterol levels, blood parasitemia, and tissue parasite load were analyzed to evaluate the effect of diet on infection. MicroPET and MRI analysis were performed to examine the morphological and functional status of the heart during acute infection. qPCR and immunoblot analysis were carried out to analyze the effect of diet on the genes involved in the host lipid metabolism during infection. Oil red O staining of the adipose tissue demonstrated reduced lipolysis in HFD compared to RD fed mice. HFD reduced mortality, parasitemia and cardiac parasite load, but increased parasite load in adipocytes. HFD decreased lipolysis during acute infection. Both qPCR and protein analysis demonstrated alterations in lipid metabolic pathways in adipose tissue and heart in RD fed mice, which were further modulated by HFD. Both microPET and MRI analyses demonstrated changes in infected RD murine hearts which were ameliorated by HFD. CONCLUSION/SIGNIFICANCE: These studies indicate that Chagasic cardiomyopathy is associated with a cardiac lipidpathy and that both cardiac lipotoxicity and adipose tissue play a role in the pathogenesis of Chagas disease. HFD protected mice from T. cruzi infection-induced myocardial damage most likely due to the effects of HFD on both adipogenesis and T. cruzi infection-induced cardiac lipidopathy.

Adipocytes in both brown and white adipose tissue of adult mice are functionally connected via gap junctions: implications for Chagas disease.

Adipose tissue serves as a host reservoir for the protozoan Trypanosoma cruzi, the causative organism in Chagas disease. Gap junctions interconnect cells of most tissues, serving to synchronize cell activities including secretion in glandular tissue, and we have previously demonstrated that gap junctions are altered in various tissues and cells infected with T. cruzi. Herein, we examined the gap junction protein connexin 43 (Cx43) expression in infected adipose tissues. Adipose tissue is the largest endocrine organ of the body and is also involved in other physiological functions. In mammals, it is primarily composed of white adipocytes. Although gap junctions are a prominent feature of brown adipocytes, they have not been explored extensively in white adipocytes, especially in the setting of infection. Thus, we examined functional coupling in both white and brown adipocytes in mice. Injection of electrical current or the dye Lucifer Yellow into adipocytes within fat tissue spread to adjacent cells, which was reduced by treatment with agents known to block gap junctions. Moreover, Cx43 was detected in both brown and white fat tissue. At thirty and ninety days post-infection, Cx43 was downregulated in brown adipocytes and upregulated in white adipocytes. Gap junction-mediated intercellular communication likely contributes to hormone secretion and other functions in white adipose tissue and to nonshivering thermogenesis in brown fat, and modulation of the coupling by T. cruzi infection is expected to impact these functions.

Markers of oxidative stress in adipose tissue during Trypanosoma cruzi infection.

The protozoan parasite Trypanosoma cruzi causes Chagas disease. Cardiac and adipose tissues are among the early targets of infection and are sites of persistent infection. In the heart and adipose tissue, T. cruzi infection results in an upregulation of pro-inflammatory mediators. In the heart, infection is associated with an increase in the markers of oxidative stress. To date, markers of oxidative stress have not been evaluated in adipose tissue in this infection. Brown and white adipose tissues were obtained from CD-1 mice infected with the Brazil strain of T. cruzi for 15, 30, and 130 days post infection. Protein carbonylation and lipid peroxidation assays were performed on these samples. There was an upregulation of these markers of oxidative stress at all time-points in both white and brown adipose tissue. Determinants of anti-oxidative stress were downregulated at similar time-points. This increase in oxidative stress during T. cruzi infection most likely has a deleterious effect on host metabolism and on the heart.

Trypanosoma cruzi infection results in an increase in intracellular cholesterol.

Chagasic cardiomyopathy caused by Trypanosoma cruzi is a major health concern in Latin America and among immigrant populations in non-endemic areas. T. cruzi has a high affinity for host lipoproteins and uses the low density lipoprotein receptor (LDLr) for invasion. Herein, we report that T. cruzi infection is associated with an accumulation of LDL and cholesterol in tissues in both acute and chronic murine Chagas disease. Similar findings were observed in tissue samples from a human case of Chagasic cardiomyopathy. T. cruzi infection of cultured cells displayed increased invasion with increasing cholesterol levels in the medium. Studies of infected host cells demonstrated alterations in their cholesterol regulation. T. cruzi invasion/infection via LDLr appears to be involved in changes in intracellular cholesterol homeostasis. The observed changes in intracellular lipids and associated oxidative stress due to these elevated lipids may contribute to the development of Chagasic cardiomyopathy.

Alterations in glucose homeostasis in a murine model of Chagas disease.

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 ß-cells with apparent sparing of α cells and leads to the disruption of pancreatic islet architecture, ß-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 ß-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 ß-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.

Current understanding of immunity to Trypanosoma cruzi infection and pathogenesis of Chagas disease.

Chagas disease caused by Trypanosoma cruzi remains an important neglected tropical disease and a cause of significant morbidity and mortality. No longer confined to endemic areas of Latin America, it is now found in non-endemic areas due to immigration. The parasite may persist in any tissue, but in recent years, there has been increased recognition of adipose tissue both as an early target of infection and a reservoir of chronic infection. The major complications of this disease are cardiomyopathy and megasyndromes involving the gastrointestinal tract. The pathogenesis of Chagas disease is complex and multifactorial involving many interactive pathways. The significance of innate immunity, including the contributions of cytokines, chemokines, reactive oxygen species, and oxidative stress, has been emphasized. The role of the components of the eicosanoid pathway such as thromboxane A(2) and the lipoxins has been demonstrated to have profound effects as both pro- and anti-inflammatory factors. Additionally, we discuss the vasoconstrictive actions of thromboxane A(2) and endothelin-1 in Chagas disease. Human immunity to T. cruzi infection and its role in pathogen control and disease progression have not been fully investigated. However, recently, it was demonstrated that a reduction in the anti-inflammatory cytokine IL-10 was associated with clinically significant chronic chagasic cardiomyopathy.

Recent developments in the interactions between caveolin and pathogens.

The role of caveolin and caveolae in the pathogenesis of infection has only recently been appreciated. In this chapter, we have highlighted some important new data on the role of caveolin in infections due to bacteria, viruses and fungi but with particular emphasis on the protozoan parasites Leishmania spp., Trypanosoma cruzi and Toxoplasma gondii. This is a continuing area of research and the final chapter has not been written on this topic.

Mechanisms of Trypanosoma cruzi persistence in Chagas disease.

Trypanosoma cruzi infection leads to development of chronic Chagas disease. In this article, we provide an update on the current knowledge of the mechanisms employed by the parasite to gain entry into the host cells and establish persistent infection despite activation of a potent immune response by the host. Recent studies point to a number of T. cruzi molecules that interact with host cell receptors to promote parasite invasion of the diverse host cells. T. cruzi expresses an antioxidant system and thromboxane A(2) to evade phagosomal oxidative assault and suppress the host's ability to clear parasites. Additional studies suggest that besides cardiac and smooth muscle cells that are the major target of T. cruzi infection, adipocytes and adipose tissue serve as reservoirs from where T. cruzi can recrudesce and cause disease decades later. Further, T. cruzi employs at least four strategies to maintain a symbiotic-like relationship with the host, and ensure consistent supply of nutrients for its own survival and long-term persistence. Ongoing and future research will continue to help refining the models of T. cruzi invasion and persistence in diverse tissues and organs in the host.

Response of adipose tissue to early infection with Trypanosoma cruzi (Brazil strain).

Brown adipose tissue (BAT) and white adipose tissue (WAT) and adipocytes are targets of Trypanosoma cruzi infection. Adipose tissue obtained from CD-1 mice 15 days after infection, an early stage of infection revealed a high parasite load. There was a significant increase in macrophages in infected adipose tissue and a reduction in lipid accumulation, adipocyte size, and fat mass and increased expression of lipolytic enzymes. Infection increased levels of Toll-like receptor (TLR) 4 and TLR9 and in the expression of components of the mitogen-activated protein kinase pathway. Protein and messenger RNA (mRNA) levels of peroxisome proliferator-activated receptor γ were increased in WAT, whereas protein and mRNA levels of adiponectin were significantly reduced in BAT and WAT. The mRNA levels of cytokines, chemokines, and their receptors were increased. Nuclear Factor Kappa B levels were increased in BAT, whereas Iκκ-γ levels increased in WAT. Adipose tissue is an early target of T. cruzi infection.

Chagas heart disease: report on recent developments.

Chagas disease, caused by the parasite Trypanosoma cruzi, is an important cause of cardiac disease in endemic areas of Latin America. It is now being diagnosed in nonendemic areas because of immigration. Typical cardiac manifestations of Chagas disease include dilated cardiomyopathy, congestive heart failure, arrhythmias, cardioembolism, and stroke. Clinical and laboratory-based research to define the pathology resulting from T. cruzi infection has shed light on many of the cellular and molecular mechanisms leading to these manifestations. Antiparasitic treatment may not be appropriate for patients with advanced cardiac disease. Clinical management of Chagas heart disease is similar to that used for cardiomyopathies caused by other processes. Cardiac transplantation has been successfully performed in a small number of patients with Chagas heart disease.

Curcumin treatment provides protection against Trypanosoma cruzi infection.

Trypanosoma cruzi, the etiologic agent of Chagas disease, causes an acute myocarditis and chronic cardiomyopathy. The current therapeutic agents for this disease are not always effective and often have severe side effects. Curcumin, a plant polyphenol, has demonstrated a wide range of potential therapeutic effects. In this study, we examined the effect of curcumin on T. cruzi infection in vitro and in vivo. Curcumin pretreatment of fibroblasts inhibited parasite invasion. Treatment reduced the expression of the low density lipoprotein receptor, which is involved in T. cruzi host cell invasion. Curcumin treatment of T. cruzi-infected CD1 mice reduced parasitemia and decreased the parasitism of infected heart tissue. This was associated with a significant reduction in macrophage infiltration and inflammation in both the heart and liver; moreover, curcumin-treated infected mice displayed a 100% survival rate in contrast to the 60% survival rate commonly observed in untreated infected mice. These data are consistent with curcumin modulating infection-induced changes in signaling pathways involved in inflammation, oxidative stress, and apoptosis. These data suggest that curcumin and its derivatives could be a suitable drug for the amelioration of chagasic heart disease.

Energy transfer in "parasitic" cancer metabolism: mitochondria are the powerhouse and Achilles' heel of tumor cells.

It is now widely recognized that the tumor microenvironment promotes cancer cell growth and metastasis via changes in cytokine secretion and extracellular matrix remodeling. However, the role of tumor stromal cells in providing energy for epithelial cancer cell growth is a newly emerging paradigm. For example, we and others have recently proposed that tumor growth and metastasis is related to an energy imbalance. Host cells produce energy-rich nutrients via catabolism (through autophagy, mitophagy, and aerobic glycolysis), which are then transferred to cancer cells to fuel anabolic tumor growth. Stromal cell-derived L-lactate is taken up by cancer cells and is used for mitochondrial oxidative phosphorylation (OXPHOS) to produce ATP efficiently. However, "parasitic" energy transfer may be a more generalized mechanism in cancer biology than previously appreciated. Two recent papers in Science and Nature Medicine now show that lipolysis in host tissues also fuels tumor growth. These studies demonstrate that free fatty acids produced by host cell lipolysis are re-used via beta-oxidation (beta-OX) in cancer cell mitochondria. Thus, stromal catabolites (such as lactate, ketones, glutamine and free fatty acids) promote tumor growth by acting as high-energy onco-metabolites. As such, host catabolism, via autophagy, mitophagy and lipolysis, may explain the pathogenesis of cancer-associated cachexia and provides exciting new druggable targets for novel therapeutic interventions. Taken together, these findings also suggest that tumor cells promote their own growth and survival by behaving as a "parasitic organism." Hence, we propose the term "Parasitic Cancer Metabolism" to describe this type of metabolic coupling in tumors. Targeting tumor cell mitochondria (OXPHOS and beta-OX) would effectively uncouple tumor cells from their hosts, leading to their acute starvation. In this context, we discuss new evidence that high-energy onco-metabolites (produced by the stroma) can confer drug resistance. Importantly, this metabolic chemo-resistance is reversed by blocking OXPHOS in cancer cell mitochondria with drugs like Metformin, a mitochondrial "poison." In summary, parasitic cancer metabolism is achieved architecturally by dividing tumor tissue into at least two well-defined opposing "metabolic compartments:" catabolic and anabolic.

Adipose tissue, diabetes and Chagas disease.

Adipose tissue is the largest endocrine organ in the body and is composed primarily of adipocytes (fat cells) but also contains fibroblasts, endothelial cells, smooth muscle cells, macrophages and lymphocytes. Adipose tissue and the adipocyte are important in the regulation of energy metabolism and of the immune response. Adipocytes also synthesize adipokines such as adiponectin which is important in the regulation of insulin sensitivity and inflammation. Infection of mice with Trypanosoma cruzi results in an upregulation of inflammation in adipose tissue that begins during the acute phase of infection and persists into the chronic phase. The adipocyte is both a target of infection and a reservoir for the parasite during the chronic phase from which recrudescence of the infection may occur during periods of immunosuppression.

Evidence for Trypanosoma cruzi in adipose tissue in human chronic Chagas disease.

Trypanosoma cruzi the cause of Chagas disease persists in tissues of infected experimental animals and humans. Here we demonstrate the persistence of the parasite in adipose tissue from of three of 10 elderly seropositive patients with chronic chagasic heart disease. Nine control patients had no parasites in the fat. We also demonstrate that T. cruzi parasitizes primary adipocytes in vitro. Thus, in humans as in mice the parasite may persist in adipose tissue for decades and become a reservoir of infection.

Trypanosoma cruzi utilizes the host low density lipoprotein receptor in invasion.

BACKGROUND: Trypanosoma cruzi, an intracellular protozoan parasite that infects humans and other mammalian hosts, is the etiologic agent in Chagas disease. This parasite can invade a wide variety of mammalian cells. The mechanism(s) by which T. cruzi invades its host cell is not completely understood. The activation of many signaling receptors during invasion has been reported; however, the exact mechanism by which parasites cross the host cell membrane barrier and trigger fusion of the parasitophorous vacuole with lysosomes is not understood. METHODOLOGY/PRINCIPAL FINDINGS: In order to explore the role of the Low Density Lipoprotein receptor (LDLr) in T. cruzi invasion, we evaluated LDLr parasite interactions using immunoblot and immunofluorescence (IFA) techniques. These experiments demonstrated that T. cruzi infection increases LDLr levels in infected host cells, inhibition or disruption of LDLr reduces parasite load in infected cells, T. cruzi directly binds recombinant LDLr, and LDLr-dependent T. cruzi invasion requires PIP2/3. qPCR analysis demonstrated a massive increase in LDLr mRNA (8000 fold) in the heart of T. cruzi infected mice, which is observed as early as 15 days after infection. IFA shows a co-localization of both LDL and LDLr with parasites in infected heart. CONCLUSIONS/SIGNIFICANCE: These data highlight, for the first time, that LDLr is involved in host cell invasion by this parasite and the subsequent fusion of the parasitophorous vacuole with the host cell lysosomal compartment. The model suggested by this study unifies previous models of host cell invasion for this pathogenic protozoon. Overall, these data indicate that T. cruzi targets LDLr and its family members during invasion. Binding to LDL likely facilitates parasite entry into host cells. The observations in this report suggest that therapeutic strategies based on the interaction of T. cruzi and the LDLr pathway should be pursued as possible targets to modify the pathogenesis of disease following infection.

Crucial role of the central leptin receptor in murine Trypanosoma cruzi (Brazil strain) infection.

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.

Interactions of Encephalitozoon cuniculi polar tube proteins.

Microsporidia are eukaryotic, obligate intracellular organisms defined by small spores that contain a single invasion organelle, the polar tube, which coils around the interior of the spore. When these parasites infect host cells, the polar tube is discharged from the anterior pole of the spore, pierces the cell, and transfers sporoplasm into the cytoplasm of the host. Three polar tube proteins (PTP1, PTP2, and PTP3) have been identified in this structure. The interactions of these proteins in the assembly and function of the polar tube are not known. This study was undertaken to examine the protein interactions of the Encephalitozoon cuniculi polar tube proteins (EcPTPs). Immunofluorescence and immunoelectron microscopy confirmed the colocalization of EcPTP1, EcPTP2, and EcPTP3 to the polar tube. Experiments using cross-linkers indicated that EcPTP1, EcPTP2, and EcPTP3 form a complex in the polar tube, which was confirmed by immunoprecipitation using EcPTP1 antiserum. Yeast two-hybrid analysis revealed that full-length EcPTP1, EcPTP2, and EcPTP3 interact with each other in vivo. Both the N and C termini of EcPTP1 were involved in these interactions, but the central region of this protein, which contains a repetitive motif, was not. Further studies of polar tube proteins and their structural interactions may help elucidate the formation of the polar tube during the invasion process.

Transcriptomic alterations in Trypanosoma cruzi-infected cardiac myocytes.

Trypanosoma cruzi infection is a major cause of cardiomyopathy. Previous gene profiling studies of infected mouse hearts have revealed prominent changes in gene expression within many functional pathways. This variety of transcriptomic changes in infected mice raises the question of whether gene expression alterations in whole hearts are due to changes in infected cardiac myocytes or other cells or even to systemic effects of the infection on the heart. We employed microarrays to examine infected cardiac myocyte cultures 48 h post-infection. Statistical comparison of gene expression levels of 7624 well annotated unigenes in four independent cultures of infected and uninfected myocytes detected substantial (>or=1.5 absolute fold changes) in 420 (5.5%) of the sampled genes. Major categories of affected genes included those involved in immune response, extracellular matrix and cell adhesion. These findings on infected cardiac myocytes in culture reveal that alterations in cardiac gene expression described in Chagas disease are the consequence of both direct infection of the myocytes themselves as well as resulting from the presence of other cell types in the myocardium and systemic effects of infection.