extfieldsofstudy: Biology Medicine

text:
Absence of calcium‐independent phospholipase A2β impairs platelet‐activating factor production and inflammatory cell recruitment in Trypanosoma cruzi‐infected endothelial cells

Abstract Both acute and chronic phases of Trypanosoma cruzi (T. cruzi) infection are characterized by tissue inflammation, mainly in the heart. A key step in the inflammatory process is the transmigration of inflammatory cells across the endothelium to underlying infected tissues. We observed increased arachidonic acid release and platelet‐activating factor (PAF) production in human coronary artery endothelial cells (HCAEC) at up to 96 h of T. cruzi infection. Arachidonic acid release is mediated by activation of the calcium‐independent phospholipase A2 (iPLA2) isoforms iPLA2β and iPLA2γ, whereas PAF production was dependent upon iPLA2β activation alone. Trypanosoma cruzi infection also resulted in increased cell surface expression of adhesion molecules. Increased adherence of inflammatory cells to T. cruzi‐infected endothelium was blocked by inhibition of endothelial cell iPLA2β or by blocking the PAF receptor on inflammatory cells. This suggests that PAF, in combination with adhesion molecules, might contribute to parasite clearing in the heart by recruiting inflammatory cells to the endothelium.

Introduction
Trypanosoma cruzi is the protozoan parasite responsible for Chagas' disease, which is associated with significant cardiac pathology. Over 10 million people worldwide are thought to be currently infected with T. cruzi, and about 300,000 infected individuals live in the United States (Bern and Montgomery 2009). The recent spread of the disease to several nonendemic countries is attributable to immigration from endemic areas, immunosuppression in the setting of organ transplantation, and blood transfusions from infected individuals. The acute phase of Chagas' disease may not cause symptoms, but in the chronic phase cardiac involvement occurs in 20-30% of infected individuals and may result in congestive heart failure, cardiac arrhythmias, and death (Rassi et al. 2000;Bern 2011). A long asymptomatic period separating acute and chronic phases is designated the indeterminate phase and may persist for decades.
Interactions between the host and pathogen during acute infection may determine the outcome of chronic Chagas' disease (Marinho et al. 1999). Parasite persistence reflected by the presence of T. cruzi antigens and DNA in the heart have been found to correlate with the intensity of chronic disease (Jones et al. 1993;Benvenuti et al. 2008), and it is therefore necessary to understand parasite-host interactions in the acute phase of Chagas' disease. A key pathological feature of T. cruzi infection is the intense cardiac inflammation in both acute and chronic stages. As a consequence of acute stage parasitemia, trypomastigotes migrate across endothelial barriers to infect underlying tissues, resulting in increased expression of vascular adhesion molecules and pro-inflammatory cytokines when T. cruzi infects endothelial cells (Huang et al. 1999;Michailowsky et al. 2004). Infection of the endothelium has a well-established role in the pathogenesis of Chagas' disease and contributes to increased platelet aggregation and thrombus formation (Rossi et al. 1984;Tanowitz et al. 1990).
Platelet-activating factor (PAF) is an important membrane phospholipid-derived inflammatory mediator expressed on the surface of endothelial cells, where it plays an important role in the recruitment, activation, and transmigration of leukocytes to sites of infection (Prescott et al. 2002). PAF is an acetylated alkyl ether glycerophosphocholine lipid species whose immediate precursor is produced by the action of phospholipase A 2 (PLA 2 ) enzyme(s), and PAF can elicit biological responses at concentrations as low as 10 À12 mol/L (Montrucchio et al. 2000). The PLA 2 family comprises enzymes that hydrolyze phospholipids at the sn-2 position to yield a free fatty acid and a 2-lysophospholipid. Lysophospholipid species of the structure 1-O-alkyl, 2-lyso-glycerophosphocholine (GPC) are designated lyso-PAF and when acetylated in the sn-2 position yield PAF (McHowat et al. 2001). When endothelial cell PAF interacts with the PAF receptor expressed on the surface of leukocytes (Shimizu et al. 1992), leukocytes become tethered to the endothelium and activated leukocytes can transmigrate into underlying tissues (Prescott et al. 2001).
We have shown that the major PLA 2 in cardiac endothelial cells is membrane associated, calcium-independent (iPLA 2 ), and specific for arachidonylated phospholipids (Creer and McHowat 1998). Two iPLA 2 isoforms (iPLA 2 b and iPLA 2 c) are expressed by cardiac endothelial cells (Sharma et al. 2011) and can be distinguished pharmacologically. The (R) enantiomer of the compound BEL (bromoenol lactone) preferentially inhibits iPLA 2 c, and (S)-BEL preferentially iPLA 2 b (Jenkins et al. 2002). In vitro studies using (R)-and (S)-BEL show that iPLA 2 b activation results in PAF production, which is required for neutrophil adherence to cardiac endothelium Sharma et al. 2011). Activated cardiac endothelial cells from wild-type and iPLA 2 c knockout mice produce PAF, but such cells from iPLA 2 b knockout mice fail to do so (Sharma et al. 2011). This suggests that iPLA 2 b may play an important role in recruiting inflammatory cells to the myocardium by enabling PAF production. Although downstream mediators generated from products of iPLA 2 action have been studied in Chagas' disease, there has been no examination of the contribution of individual iPLA 2 isoforms to these processes. We have therefore examined the contribution of endothelial cell iPLA 2 b to inflammatory cell recruitment following T. cruzi infection.

Materials and Methods
Human coronary artery endothelial cells Human coronary artery endothelial cells (HCAEC) were obtained from Lonza Walkersville, Inc. (Walkersville, MD). Cells were grown to confluence in EGM-2MV media obtained from Lonza (Walkersville, MD), with 5% fetal bovine serum (FBS). Cells were allowed to grow to confluence achieving a contact-inhibited monolayer of flattened, closely apposed endothelial cells in 4-5 days. After achieving confluence, cells were passaged in a 1:3 dilution and cells from passages 3-4 were used for experiments.

Mouse endothelial cell isolation
Animal protocols were in strict accordance with the National Institutes of Health guidelines for humane treatment of animals and were reviewed and approved by the Animal Care and Use Committee of Saint Louis University. Endothelial cells were isolated from mouse heart by collagenase digestion. The diced heart muscle was incubated in 2 mg/mL collagenase for 1 h at 37°C and the digested tissue was passed through a cell strainer. Cells were incubated with murine immunoglobulins to block Fc receptors and then incubated with anti-mouse platelet endothelial cell adhesion molecule-1 (PECAM-1) coupled to magnetic beads. Cells obtained were cultured until they reached confluence and sorted again using intercellular adhesion molecule-2 (ICAM-2) antibodies coupled with magnetic beads. The eluted cells were washed, resuspended in cell culture medium, and plated in culture. Nonadherent cells were removed the next day and cells were grown to confluence and passaged at a 1-3 dilution.

Parasitology
Tissue culture trypomastigotes (TCT) from the Brazil strain of T. cruzi were propagated in 3T3 mouse embryonic fibroblasts grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 2% neonatal calf serum (Eickhoff et al. 2010 ruptured following parasite multiplication, releasing an abundant number of parasites. The supernatant containing the parasites was collected, and parasite numbers determined using a Neubauer hemocytometer (Patterson Veterinary, Devens, MA). Cardiac endothelial cells, grown to confluence in the appropriate culture dish were counted and a multiplicity of infection (MOI) of 0.2 was used to infect cells. In selected experiments, parasites were killed by heating at 80°C for 10 min.

Phospholipase A 2 activity
Endothelial cells were suspended in 1 mL buffer containing (mmol/L): Sucrose 250, KCL 10, imidazole 10, ethylenediaminetetraacetic acid (EDTA) 5, dithiothreitol (DTT) 2 with 10% glycerol, pH 7.8 (PLA 2 activity buffer). The suspension was sonicated on ice six times for 10 sec (using microtip probe at 20% power output, 500 Sonic Dismembrator; Fisher Scientific, Pittsburgh, PA) and the sonicate centrifuged at 20,000g for 20 min to remove cellular debris and nuclei. The pellet was resuspended in activity buffer. PLA 2 activity was assessed by incubating enzyme (50 lg protein) with 100 lmol/L (16:0, [ 3 H]18:1) plasmenylcholine substrate in assay buffer containing (mmol/L): Tris 10, ethylene glycol tetraacetic acid (EGTA) 4, 10% glycerol, pH 7.0 at 37°C for 5 min in a total volume of 200 lL. The radiolabeled phospholipid substrate was introduced into the incubation mixture by injection in 5 lL ethanol to initiate the assay. Reactions were terminated by the addition of 100-lL butanol and released radiolabeled [ 3 H]oleic acid was isolated by the application of 25 lL of the butanol phase to channeled Silica Gel G plates, development in the petroleum ether/diethyl ether/acetic acid (70/30/1, v/v) and subsequent quantification by liquid scintillation spectrometry. Protein content of each sample was determined by the Lowry method utilizing freeze-dried bovine serum albumin as the protein standard.

PAF assay
Endothelial cells grown in 12-well culture dishes were washed twice with Hanks' balanced salts solution containing NaCl 135 mmol/L, MgSO 4 0.8 mmol/L, HEPES (pH = 7.4) 10 mmol/L, CaCl 2 1.2 mmol/L, KCl 5.4 mmol/L, KH 2 PO 4 0.4 mmol/L, Na 2 HPO 4 0.3 mmol/L, and glucose 6.6 mmol/L and incubated with 50 lCi [ 3 H] acetic acid for 20 min. After the selected time interval for incubation with the appropriate agents, lipids were be extracted from the cells by the method of Bligh and Dyer (1959). The chloroform layer was concentrated by evaporation under N 2 , applied to a silica gel 60 thin layer chromatography (TLC) plate, and developed in chloroform/methanol/acetic acid/water (50/25/8/4 vol/vol). The region corresponding to PAF was scraped and radioactivity quantified using liquid scintillation spectrometry. Loss of PAF during extraction and chromatography was corrected for by adding a known amount of [ 14 C] PAF as an internal standard. [ 14 C] PAF was synthesized by acetylating the sn-2 position of lyso-PAF with [ 14 C] acetic anhydride using 0.33 mol/L dimethylaminopyridine as a catalyst. The synthesized [ 14 C] PAF was purified by high-performance liquid chromatography (HPLC).

Inflammatory cell adherence
RAW 264.7 cells were grown to confluence in DMEM with 10% FBS. Cell suspensions (10 9 10 6 /mL) were labeled with 4 lg/mL calcein-AM for 45 min at 37°C. Cells were washed three times with HEPES buffer, resuspended at a concentration of 4 9 10 6 /mL and 0.5 mL was added to confluent mouse cardiac myocyte cell layer. At the end of the incubation time, nonadherent cells were removed and adherent cells and cardiac myocytes were lysed in 1 mL of 0.2% Triton X-100. Calcein fluorescence in each sample was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. The percent cell adherence in each sample was calculated based upon the fluorescence measured in 0.5 mL of RAW 264.7 cell suspension.

Polymorphonuclear leukocyte (PMN) adherence
Blood (80 mL) was obtained from healthy adults and layered over an equal volume of Polymorphprep (AxisShield, Oslo, Norway) in 50-mL conical tubes. Tubes were spun at 500 g for 30 min at 20°C. The buffy coat at the sample-medium interface consisting of PMN was removed, washed, and resuspended in 5 mL of ice-cold Hank's balanced salt solution (HBSS), and cells were counted. HCAEC grown on a 12-mm plate were washed twice with HBSS. After appropriate pretreatment of endothelial cells or PMN, 0.5 mL of PMN suspension (4 9 10 6 cells/mL) in HBSS was added to each of the wells and incubated for 10 min at room temperature. Medium and unbound cells were removed and discarded. Plates were washed twice with prewarmed Dulbecco's phosphate-buffered saline (DPBS). Adherent PMN and endothelial cells were lysed in 1 mL of 0.2% Triton X-100. For maximal binding, a 0.5-mL aliquot of PMN suspension plus 0.5 mL of 0.2% Triton X-100 was used. To measure myeloperoxidase activity, we added 400 lL of cell lysate to 1 mL of phosphate-buffered saline (PBS), 1.2 mL of Hanks' buffer-bovine serum albumin (BSA), and 0.2 mL of 3,3-dimethoxybenzidine, and 0.2 mL of 0.05% H 2 O 2 was added. After 15 min, 0.2 mL of 1% NaN 3 was added to stop the reaction, and absorbance was measured using a 4050 ultraviolet/Visible spectrophotometer (Biochrom, Cambridge, UK) at 460 nm.

Surface expression of adhesion molecules
HCAEC were grown to confluence in 16-mm culture dishes and were incubated with T. cruzi (MOI 0.2) for up to 96 h. At the end of the incubation, cells were fixed with 1% paraformaldehyde and incubated overnight at 4°C. Cells were then washed three times with PBS and then blocked with Tris-buffered saline-Tween supplemented with 0.8% BSA (wt/vol) and 0.5% fish gelatin (wt/vol) for 1 h at 24°C. Appropriate primary antibody (1:50; Santa Cruz Biotechnology, Santa Cruz, CA) was used before treatment with horseradish peroxidase-conjugated rabbit anti-goat secondary antibody (1:5,000; Santa Cruz Biotechnology, Santa Cruz, CA). Subsequently, each well was incubated in the dark with the 3,3′,5,5′-tetramethylbenzidine liquid substrate system and color development measured at 450 nm.

Statistical analysis
Statistical comparison of values was performed by student's t-test or one-way analysis of variance with post hoc analysis performed using Dunnet's test. All results are expressed as mean AE SEM. Statistical significance was considered to be P < 0.05.

Trypanosoma cruzi infection of HCAEC
HCAEC were incubated with T. cruzi for up to 96 h and iPLA 2 activity, arachidonic acid release, PAF production, and expression of cell surface adhesion molecules were measured (Fig. 1). Trypanosoma cruzi (MOI 0.2) infection of HCAEC increased PLA 2 activity measured in the presence of 10 mmol/L EGTA (iPLA 2 activity) that was significant after 24 h of infection and remained increased over 96 h (Fig. 1A). Accompanying the increase in iPLA 2 activity, we measured a significant increase in arachidonic acid release (Fig. 1B) and PAF production (Fig. 1C). When HCAEC were incubated with heat-killed T. cruzi, no significant changes in iPLA 2 activity, arachidonic acid release, or PAF production were observed (Fig. 1A-C). Adhesion molecules expressed on the surface of endothelial cells could significantly contribute to inflammatory cell adherence and transmigration, and may work synergistically with PAF. After infection of HCAEC with T. cruzi, we observed increased cell surface expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin (Fig. 1C) prior to the observed increase in PAF production (Fig. 1C). As adhesion molecules are upregulated immediately after infection, they could contribute to the initial tethering of inflammatory cells to the endothelium prior to attachment and activation via the PAF-PAF receptor interaction. The involvement of multiple molecules highlights the importance of the recruitment of circulating leukocytes, and this redundancy may allow for compensation in the absence of any single adhesion molecule.
To determine which iPLA 2 isoform was involved in T. cruzi-mediated arachidonic acid and PAF production, we pretreated HCAEC with (R)-or (S)-BEL to inhibit iPLA 2 c or iPLA 2 b selectively (Jenkins et al. 2002) prior to infection with T. cruzi. Treatment with (R)-or (S)-BEL resulted in a significant reduction in basal iPLA 2 activity and inhibition of iPLA 2 activation in response to T. cruzi infection ( Fig. 2A). Inhibition of iPLA 2 b activity with (S)-BEL was significantly greater than corresponding concentrations of (R)-BEL used to inhibit iPLA 2 c ( Fig. 2A), suggesting that iPLA 2 b activity is predominant in HCAEC.
We measured arachidonic acid release and PAF production in the presence or absence of (R)-or (S)-BEL at 48 h post infection with T. cruzi. Endothelial cells showed a significant increase in arachidonic acid release (Fig. 2B) and PAF production (Fig. 2B) in response to T. cruzi infection. Although arachidonic acid release was inhibited by similar extent by (R)-or (S)-BEL (Fig. 2B), PAF production was unaffected by (R)-BEL pretreatment and completely inhibited by (S)-BEL pretreatment (Fig. 2C). Hence, our data suggest that T. cruzi-induced arachidonic acid release is mediated by activation of both iPLA 2 b and iPLA 2 c, whereas increased PAF production is entirely dependent upon iPLA 2 b activation.
To examine the effects of iPLA 2 b-mediated PAF production on inflammatory cell adherence to the endothelium,  (Fig. 2D). Pretreating HCAEC with (S)-BEL prior to infection resulted in a significant reduction in neutrophil adherence, and reduced adherence was also observed when PMN were treated with the PAF receptor antagonist CV3988 (Fig. 2D). Thus, the interaction of endothelial cell PAF with the PAF receptor on inflammatory cells is critical for inflammatory cell adherence to endothelium.
To further demonstrate involvement of iPLA 2 b activation in increased PAF production in T. cruzi-infected endothelium, cardiac endothelial cells were isolated from wild-type and iPLA 2 b knockout mice. Confluent monolayers of these cells were stained for coagulation factor VIII, which is an endothelial cell-specific marker expressed by more than 80% of the cultured cells (data not shown). After pretreatment with either (R)-or (S)-BEL, endothelial cells were infected with T. cruzi (MOI 0.2) for 48 h and PAF production was measured. Infection with T. cruzi resulted in a significant increase in PAF production by wild-type cardiac endothelial cells (Fig. 3A). Pretreatment of wild-type endothelial cells with (S)-BEL significantly inhibited the T. cruzi-induced production of PAF, whereas pretreatment with (R)-BEL had much less of an effect (Fig. 3A). No increase in PAF was observed when iPLA 2 b-KO cardiac endothelial cells were infected with T. cruzi. These data suggest that iPLA 2 b is required for mouse cardiac endothelial cell PAF production induced by infection with T. cruzi. Adherence of RAW 264.7 murine macrophage/monocyte cells to cardiac endothelial cells isolated from wild-type and iPLA 2 b-KO mice following infection with T. cruzi was determined (Fig. 3B). After 48 h, infected wild-type endothelial cells exhibited a significant increase in RAW 264.7 cell adherence compared to uninfected cells (Fig. 3B). Pretreatment with the iPLA 2 b inhibitor (S)-BEL resulted in a marked reduction in RAW 264.7 cell adherence to T. cruziinfected wild type cardiac endothelial cells (Fig. 3B). No increased adherence of RAW 264.7 cells to iPLA 2 b-KO cardiac endothelial cells was observed after infection with T. cruzi in the presence or absence of (S)-BEL (Fig. 3B). Pretreatment of RAW 264.7 cells with the PAF receptor antagonist CV3988 prior to addition to T. cruzi-infected endothelial cells resulted in complete inhibition of RAW 264.7 cell adherence to the cardiac endothelial cells under all experimental conditions (Fig. 3B). Thus, the increase in RAW 264.7 cell adherence to T. cruzi-infected endothelial cells requires iPLA 2 b-mediated PAF production.

Discussion
Previous studies have indicated that iPLA 2 b is responsible for the majority of cardiac endothelial cell iPLA 2 activity (Sharma et al. 2011). In previous studies, activation of endothelial cell iPLA 2 b by the proteases thrombin or tryptase results in PAF production (Sharma et al. 2011). PAF is considered an important mediator in regulating the transmigration of inflammatory cells across the endothelium and in facilitating their recruitment to areas of injury or infection (Prescott et al. 2002). Endothelial cell surface PAF interacts with the PAF receptor on the surface of inflammatory cells, and this results in activation, adhesion, and migration of leukocytes. Activated leukocytes generate reactive oxygen species, cytokines, and degradative enzymes at the site of injury in an attempt to restrict and eliminate the inflammatory stimulus, but these mediators can cause excessive inflammation that leads to tissue damage. Thus, these processes must be carefully regulated.
Our studies were designed to determine the role of endothelial cell iPLA 2 b during acute T. cruzi infection. Studies with HCAEC demonstrate that there is increased endothelial cell PAF production after infection with T. cruzi that requires iPLA 2 b ( Fig. 2A). A corresponding increase in PMN adherence to the endothelium is also observed in this context (Fig. 2D). Pharmacological inhibition of iPLA 2 b with (S)-BEL resulted in inhibition of PMN adherence to T. cruzi-infected HCAEC (Fig. 2D). Additionally, blockade of the PAF-R with the antagonist CV3988 on the PMN surface resulted in decreased adherence of neutrophils to the endothelium in both uninfected and T. cruzi-infected HCAEC (Fig. 2D). These results highlight the importance of the interaction between endothelial cell PAF and PAF-R on the inflammatory cell surface for adherence of inflammatory cells to the endothelium and hence their subsequent recruitment to the appropriate site.
Infecting HCAEC with T. cruzi resulted in an increase in cell surface expression of adhesion molecules as early as 4 h post infection, with a later increase in PAF production at 24 h. However, PMN adherence was not significantly increased until 48 h post infection. The time course of PMN adherence correlated with that for PAF accumulation rather than upregulation of adhesion molecules. There are multiple mediators involved in leukocyte adhesion and transmigration across the endothelium, and such biological redundancy underscores the importance of this process. Thus, even though our data indicate that PAF appears to be critical for this process in vitro, it is possible that its absence alone may not significantly affect cardiac inflammation in vivo. A number of reported observations indicate that adhesion molecules expressed on the endothelial cell surface are also important in leukocyte recruitment. Tanowitz et al. have demonstrated that infection of human umbilical vein endothelial cells (HUVECs) with T. cruzi results in NF-jB activation and that this leads to an increased expression of adhesion molecules (Huang et al. 1999). Michailowsky et al. (2004) have demonstrated that interferon c (IFNc) induces expression of adhesion molecules by vascular endothelium and that ICAM-1 plays a critical role in leukocyte migration in acute T. cruzi infection.
Chronic Chagas' disease is associated with cardiomyopathy involving impaired endothelial cell function that can contribute to microvascular abnormalities such as focal ischemia or microthrombus formation. Recently  Figure 3. PAF production (A) and RAW 264.7 cell adherence (B) in Trypanosoma cruzi infected (MOI 0.2, 48 h) wild-type or iPLA 2 b knockout endothelial cells following (R)-or (S)-BEL pretreatment (0.5 lmol/L, 10 min). *P < 0.05, **P < 0.01 when compared to uninfected cells. + P < 0.05, ++ P < 0.01 when comparing values in the presence or absence of BEL. Values shown are mean + SEM for six separate cell cultures. mice after 90 days of infection. Prolonged endothelial cell activation in chronic Chagas' disease may also be associated with increased PAF production as a result of iPLA 2 activation.
Studies using endothelial cells isolated from hearts of wild-type and iPLA 2 b knockout mice also indicated that iPLA 2 b is necessary for T. cruzi-induced PAF production (Fig. 3A). Wild-type cardiac endothelial cells produced greater amounts of PAF after infection with T. cruzi, and this was prevented by pretreatment with the iPLA 2 b inhibitor (S)-BEL. Endothelial cells isolated from iPLA 2 b-knockout mice failed to increase PAF production after T. cruzi infection. Similarly, mouse RAW 264.7 macrophage-like cells also exhibited increased adherence to mouse cardiac endothelial cells with the rise in iPLA 2 b-mediated PAF production that occurred after T. cruzi infection (Fig. 3B). Taken together, these studies suggest that iPLA 2 b deficiency results in reduced endothelial cell PAF production and that this might impair recruitment of inflammatory cells to cardiac tissue in the acute and chronic phases of Chagas' disease. Although our data indicate that the absence of iPLA 2 b results in impaired PMN adherence via reduced endothelial cell PAF production, iPLA 2 -mediated hydrolysis of membrane phospholipids results in the direct or indirect production of several inflammatory metabolites (Six and Dennis 2000;Burke and Dennis 2009;Dennis et al. 2011). Thus, we cannot rule out that other iPLA 2 b-catalyzed metabolites are not involved in PMN adherence. We have demonstrated previously that the absence of iPLA 2 b does not inhibit coronary artery endothelial cell eicosanoid generation completely (Sharma et al. 2011), but we have shown that lysoplasmenylcholine increases PMN adherence and cell surface expression of adhesion molecules ). Thus, it is likely that other downstream metabolites of iPLA 2 b-catalyzed membrane phospholipid hydrolysis may play a role in inflammatory cell adherence to the endothelium.
In conclusion, our data demonstrate that T. cruzi infection of endothelial cells results in iPLA 2 b activation and increased PAF production. Pretreatment of endothelial cells with an iPLA 2 b inhibitor or inflammatory cells with a PAF receptor antagonist demonstrate that blocking the PAF-PAF receptor interaction is sufficient to inhibit adherence of inflammatory cells to the endothelium in vitro; however, further studies are warranted to determine whether the absence of endothelial cell iPLA 2 b results in impaired inflammatory cell recruitment in vivo.


extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Medicine

text:
Role of Calcium Signaling in the Transcriptional Regulation of the Apicoplast Genome of Plasmodium falciparum

Calcium is a universal second messenger that plays an important role in regulatory processes in eukaryotic cells. To understand calcium-dependent signaling in malaria parasites, we analyzed transcriptional responses of Plasmodium falciparum to two calcium ionophores (A23187 and ionomycin) that cause redistribution of intracellular calcium within the cytoplasm. While ionomycin induced a specific transcriptional response defined by up- or downregulation of a narrow set of genes, A23187 caused a developmental arrest in the schizont stage. In addition, we observed a dramatic decrease of mRNA levels of the transcripts encoded by the apicoplast genome during the exposure of P. falciparum to both calcium ionophores. Neither of the ionophores caused any disruptions to the DNA replication or the overall apicoplast morphology. This suggests that the mRNA downregulation reflects direct inhibition of the apicoplast gene transcription. Next, we identify a nuclear encoded protein with a calcium binding domain (EF-hand) that is localized to the apicoplast. Overexpression of this protein (termed PfACBP1) in P. falciparum cells mediates an increased resistance to the ionophores which suggests its role in calcium-dependent signaling within the apicoplast. Our data indicate that the P. falciparum apicoplast requires calcium-dependent signaling that involves a novel protein PfACBP1.

Introduction
Malaria is the most deadly parasitic disease and yet it is still one of the most common infectious diseases in the tropical and subtropical region of the planet. Approximately 2 billion people (40% of the world's population) are at risk of malaria infection in over 90 countries. Each year 300-500 million cases are being reported out of which over one million cases result in death [1]. Artemisinin-based combination therapies (ACT) are presently recommended as the first line of malaria treatment and their effectiveness underline many major accomplishments of the world wide malaria control programs over the last decade. Unfortunately, there are alarming reports of reduced sensitivity to ACTs emerging in Southeast Asia that poses a major threat for the future [2][3][4]. Based on the experience with previously deployed antimalaria chemotherapeutics such as chloroquine (1950s) and antifolates in (1960s), a spread of artemisinin resistance around the world could erase all advances of the malaria control programs achieved in the recent years and bring the malaria epidemics to the pre-ACT era. Hence, discovery of new malaria intervention strategies is one of the highest research priorities for the future. For this purpose, understanding of unique biological processes that are essential for malaria parasites growth and development is crucial.
Calcium signaling in Plasmodium falciparum, the most dangerous species of the malaria parasites, may represent one such area for drug target explorations. Previous studies established that P. falciparum parasites utilize calcium signaling during their life cycle progression. This is demonstrated by the wide spectrum of genes encoding calciumdependent protein kinases and calmodulins present in the P. falciparum genome [5][6][7][8] but also by the tightly regulated cytoplasmic calcium concentration via intracellular calcium stores [9]. Calcium has been shown to be important for the parasite maturation [10,11] and for the vital parasitic processes such as invasion, gliding motility [11][12][13][14][15][16][17][18], and sexual stage development [19][20][21][22]. Nonetheless, many gaps remain in the comprehensive understanding of the role of calcium signaling in Plasmodium parasites. In particular, 2 BioMed Research International very little is known about the role of calcium signaling in transcriptional regulation of P. falciparum. Given that in other eukaryotic cell systems calcium-dependent transcription has a wide range of biological functions [23][24][25][26][27][28], it is reasonable to expect that in Plasmodium, calciumdependent intracellular signaling is also linked with transcription, regulating multiple mechanisms important for the parasite growth, development, and adaptation to its host environment.
One of the most widely used tools for studying calcium signaling in eukaryotic cells is calcium ionophores that are able to abolish electric potential and Ca 2+ gradients maintained at intracellular membranes, thereby mobilizing intracellular calcium stores. There are numerous examples where calcium ionophores have been used to explore calcium signaling events in eukaryotes including transcription. Genes encoding glucose-regulated proteins in hamster fibroblasts have been shown to be induced by A23187 mediated depletion of intracellular calcium stores [29]. It was also shown that an increase in the cytosolic calcium concentration induced by A23187 and ionomycin was able to trigger the commitment to differentiation and increased expression of erythroid genes in murine erythroleukemia cells [30]. Ionomycin was also shown to be able to induce calcium flux and thus expression of the T-cell CD7 gene [31]. The changes in the intracellular calcium concentration induced by calcium ionophores is also able to alter the induction of p33 gene expression by insulin [32]. There is strong experimental evidence that ionophore compounds can affect the internal calcium stores in Plasmodium parasites. First, calcium-imaging studies of P. berghei using fluorescent calcium indicators demonstrated that ionomycin increases cytoplasmic calcium concentrations from a nonacidic calcium-rich compartment and the alkalinized acidocalcisomes [33]. In P. falciparum, ionomycin has been shown to increase the cytoplasmic calcium concentration by releasing calcium from the intracellular calcium stores that include the parasitophorous vacuole (PV) [5,34]. Given the high activity of the calcium ionophores in the Plasmodium cells [35], these compounds provide a suitable tool for studies of calcium-dependent transcriptional processes during the malaria parasite development.
In this study we analyzed the transcriptional responses of P. falciparum parasites to two calcium ionophores, ionomycin and A23187, in order to evaluate the effect of changing calcium distribution within the cell. We show that both inhibitors induce overlapping but not identical changes of the P. falciparum transcriptome ranging from up-and downregulation of many genes of a narrow set of biochemical and cellular pathways (for ionomycin) to overall developmental arrests (for A23187). Both inhibitors, however, cause a strong inhibition of transcriptional activity of essentially all genes of the apicoplast genome. Focusing on this phenomenon, we identified a nuclear encoded apicoplast targeted protein (MAL13P1.156) that carries a calcium binding (EF hand) domain. Overexpression of MAL13P1.156 confers an increase in the resistance of P. falciparum parasites to ionomycin, which suggests that this protein might play a role in calciumdependent signaling pathway(s) in the apicoplast.

Transcriptional Responses of P. falciparum to Calcium
Ionophores. In the first step we wished to investigate genomewide gene expression responses of P. falciparum parasites to ionomycin and A23187, two calcium ionophore compounds whose effect (presumably) leads to a release of the internal calcium stores and thus altering Ca 2+ concentration in essentially all intracellular compartments. For this we carry out 6-hour treatments of highly synchronized P. falciparum culture at the schizont stage (∼30 hours after invasion, hpi) with 5 M of both ionophores (Dataset S1, available online at http://dx.doi.org/10.1155/2014/869401). Subsequent genomewide gene expression analysis (see materials and methods) revealed that both compounds induced significant transcriptional responses (Figure 1(a)). Intriguingly, in spite of the fact that both ionomycin and A23817 have an identical activity, transport of Ca 2+ across the membranes of eukaryotic cell compartments, the effect of these inhibitors on the P. falciparum transcriptome was not identical. The exposure of the P. falciparum parasites to A23817 led to differential expression of at least 2254 genes (1279 up-and 975 downregulated genes by >2-fold). As previously demonstrated, such broad transcriptional changes are typically not the reflections of specific transcriptional responses but rather a result of a developmental arrest [36,37]. To test this possibility, we utilized a recently developed algorithm that allows developmental stage evaluation ("IDC aging") by assessing the correlation of the entire expression profiles in each experimental time point to the high resolution P. falciparum IDC transcriptome [38]. Indeed, all five time points of the A23817 treatments have mapped to the 30 hpi which corresponds to the early schizont stage that was used as a starting point of all treatment experiments (Figure 1(b)). Hence the majority of the A23817induced differential expression corresponds to the mRNA differences between the starting/arrested parasite cultures compared to the untreated controls that progressed through the mid-to-late schizont stage normally (Figure 1(b)). In contrast to A23817, ionomycin did not arrest the Plasmodium schizont stage progression and the global transcriptional profiles of the five treatment time points mapped to the expected IDC timing (32-38 hpi). Instead, this compound induced a specific transcriptional response with 202 genes up-and 558 genes downregulated. Interestingly, the group of upregulated genes was statistically enriched for factors of host parasite interaction including a subgroup of the var gene family, the main antigenic determinants during P. falciparum infection. In contrast, the group of downregulated genes contained large number of factors of merozoite invasion including merozoite surface antigens, actomyosine motors, and resident proteins of invasion related organelles, rhoptry, microneme and dense granules (data not shown). In addition, 21 out of total 55 genes were predicted to play a role in the fatty acid synthesis in the apicoplast (as annotated by the Malaria Parasite Metabolic Pathway database [39]). This represents a strong statistical enrichment ( value ∼ 0) and indicates a specific effect of ionomycin on the function of the apicoplast.
Corroborating this observation, both A23817 and ionomycin caused a dramatic downregulation of the vast majority   of genes encoded by the plastid (apicoplast) genome. This is demonstrated by a unique narrow gene cluster with tightly correlated expression profiles (Pearson correlation > 0.93) that are characterized by a dramatic decrease of mRNA levels even in the early time points of the treatment (0.5 and 1 hour after treatment) (Figure 1(a), asterix). This is in sharp contrast with the majority of the ionophore-induced transcriptional differences that are gradual (Figure 1(a)). Visual inspections of these narrow gene clusters revealed a strong overrepresentation of plastid genome encoding genes. Total 19 out of the 29 plastid genes represented on the microarray were found downregulated as a result of both ionophore treatments ( Figure 1(c)) (10 remaining ORFs showed no expression). In addition, all plastid tRNAs (25) and one rRNA genes that were detected by the microarray analysis also show decreased levels in the ionophore treatments compared to the untreated cells ( Figure 1(c)). Moreover, two apicoplast encoded genes of the subunits of the putative apicoplast RNA polymerase (rpoB and rpoC) were downregulated by both ionophores. This is in contrast to the third subunit (rpoA) which is encoded by the nuclear genome (PF3D7 1307600) whose expression is unaffected by neither of the inhibitors (data not shown). This suggests that the Ca-dependent regulation of the apicoplast gene transcription is independent of the nuclear genome. Taken together, A23817 and ionomycin have a profound effect on the P. falciparum transcriptional cascade with the former causing a developmental arrest and the latter inducing specific transcriptional changes of the nuclear encoded genes. Besides this, both compounds have exhibited a strong inhibitory effect on the plastid transcription, downregulating essentially all genes encoded by the 35 kb DNA genome of this organelle.

Calcium Ionophores Neither Inhibit Apicoplast DNA Replication Nor
Interfere with the Normal Apicoplast Development. Given the consistency of the ionophore-mediated downregulation of the apicoplast genes, we hypothesize that transcriptional regulation in the P. falciparum plastid is sensitive to Ca 2+ concentration. To support this model, we wished to exclude the possibility that the decreased mRNA levels of the plastid genes are a simple reflection of discrepancies in the apicoplast development, particularly apicoplast DNA replication that is rapidly ongoing during the schizont stage. Hence we carried out comparative genomic hybridization (CGH) with total DNA isolated from schizonts treated by both calcium ionophores (Figure 2(a), Dataset S2). Even after 6 hours of treatment with both inhibitors, the apicoplast DNA content was not affected while the transcription was repeatedly downregulated as seen in the initial transcriptome analyses (Figure 2(a)). Interestingly, A23187 has much stronger effect on the mRNA level of the apicoplast genes reducing their content by median 3.24 ± 0.12 compared to ionomycin that caused reduction by 2.05 ± 0.15 after six hours of treatment.
According to previous studies on plant cells, calcium ionophores are able to inhibit protein import into the chloroplast, the orthologous endosymbiotic organelle of the apicoplast. Import of proteins with a cleavable signal peptide into the isolated intact chloroplasts can be inhibited by calcium ionophores as a consequence of emptying chloroplast calcium content [40]. In P. falciparum, 545 nuclear encoded proteinsare predicted to be imported into the apicoplast facilitating numerous metabolic and cellular processes associated with this compartment [41]. Inhibition of the import of these proteins will likely cause major disruptions to the apicoplast morphology as well as function. In order to investigate the effect of the ionophores on apicoplast proteins import, we utilize the acyl carrier protein (PfACP, PFB0385w) as a molecular marker of the apicoplast (Figure 2(b)). PfACP is one of the major apicoplast factors that are implicated in the type II fatty acid biosynthesis [42]. This protein carries an N-terminal signal sequence that targets its localization to the apicoplast [43]. For our study, we generate a transgenic P. falciparum cell line with PfACP episomal overexpression. Fluorescence microscopy analysis of the pattern of intracellular localizations of the PfACP in both ionomycin and A23187 treated cells is essentially identical to untreated cells.
Similarly neither of the ionophores affected the pattern of posttranslational processing of PfACP (Figure 2(b)). The fulllength apicoplast targeted proteins undergo processing by a stromal processing peptidase upon the import into the apicoplast as previously described [44]. In our results, the signal peptide cleavage is undisturbed even 6 hours after treatment (Figure 2(b)). Hence we conclude that in neither of the treatments we observe any major interference with the PfACP import into the apicoplast.
Based on previous studies of other eukaryotic systems, the ionophore-induced increases of cytosolic calcium concentrations are mediated by two possible mechanisms. These include an influx of calcium from the ambient media via native Ca 2+ channels and a phospholipase-C (PLC) mediated calcium release from the intracellular calcium stores [45]. In the next step, we wished to test whether the ionophore effect on the apicoplast transcription is mediated by a general influx of Ca 2+ from the extracellular medium into the parasite cytoplasm and subsequently to the apicoplast or whether it is associated with the redistribution of intracellular calcium concentration within the parasite cell. In a previous study Trypanosoma cruzi the causative agent of Chagas disease, it was shown that EGTA can be used to chelate the extracellular calcium, eliminating it of its influx into the ionophore treated cells and the majority of the phenotypic effect induced by the ionophores is due to redistribution of the intracellular of calcium [46]. Similar to these studies, we carried out additional transcriptome analyses where the parasites were treated with 5 M ionomycin and at the same time the extracellular calcium was depleted by chelation with 3 mM of EGTA. As expected, we observed a similar effect of the downregulation of apicoplast gene transcription that occurs to the same degree compared to cells grown in the calcium presence ( Figure 2(c), Dataset S3). These results suggest that the redistribution of the intracellular calcium such as release of the intracellular calcium stores and not the extracellular calcium influx is responsible for the observed ionophore effect on apicoplast transcription.
Taken together, these experiments show that neither of the ionophores caused dramatic disruptions of the apicoplast DNA replication or protein import. In addition, the microscopy studies did not detect any major abnormalities in the apicoplast morphology during its growth and division in the late schizont stages. Although the used techniques could not exclude subtle changes in the apicoplast morphology or protein content, these data suggest that the observed dramatic reduction of the apicoplast gene mRNA levels indeed represents a reduced transcriptional activity at the apicoplast genome. Moreover, the ionophore-induced transcriptional changes in the apicoplast are associated mainly (if not fully) with the intracellular stores of Ca 2+ . Hence, the apicoplast transcriptional regulation is sensitive to fluctuations of Ca 2+ concentration likely within the apicoplast itself.

Calcium Signaling in the Apicoplast.
Given the potential role of calcium on transcription of the apicoplast genes,   we wished to identify a protein factor(s) that may facilitate this phenomenon. For this we carried out bioinformatics analyses of all 545 plastid targeted nuclear encoded proteins [47,48] and identified MAL13P1.156, a calcium binding protein that contain a signal anchor with probability of 0.954 [49] and 5 of 5 positive tests for an apicoplast targeting peptide [47] at its C-terminus. A similar protein that lacks an apparent signal peptide but contains a signal anchor has been found to be targeted to the apicoplast via an independent bipartite signal targeting in Toxoplasma gondii [50,51] (Figure 3(e)). This suggests a putative calcium binding function of MAL13P1.156 and thus its role in calciumdependent signaling in the apicoplast such as sensing and/or buffering free Ca 2+ ions similar to its plant counterparts in the chloroplast [53].
TVAMDR--KT-LLSRERLERAFRMFDSDNS * * * * * * : : : : TPEKRV---TgCDPK1 --------- To investigate the biological function of MAL13P1.156, we constructed a fusion construct of this protein with a C-terminal GFP and subsequently generated a P. falciparum (3D7) transgenic cell line where this fusion protein is expressed episomally. Western blot analyses show a strong expression of this protein as a full-length fusion protein of 91 kDa (64 kDa full-length protein plus 27 kDa GFP) and a processed form ∼72 kDa (Figure 3(b)). This proteolytic cleavage is consistent with the signal peptide processing upon import to the apicoplast. Subsequently, immunofluorescence microscopy (IFA) of the transgenic cell line using the anti-GFP antibody shows the characteristic pattern of apicoplast localization with a single small compartment in the early schizonts, elongated branched formation in the late schizonts, and finally divided punctuate formations corresponding to new apicoplast precursors in the newly formed daughter merozoites (Figure 3(a)). Finally, IFA-based colocalization studies revealed a close proximity between the MAL13P1.156 labeled compartments and mitochondria labeled by mitotracker Red (Figure 3(c)). This is consistent with physical association of apicoplast and mitochondria in P. falciparum. Finally there is partial but significant colocalization between MAL13P1.156 and the apicoplast marker EF-Tu (Figure 3(d)). Based on these results we conclude that MAL13P1.156 localizes to the apicoplast and hence name this protein P. falciparum apicoplast calcium binding protein 1 (PfACBP1).
To investigate whether PfACBP1 plays a role in intracellular calcium homeostasis of the Plasmodium parasites we determined the sensitivity of the generated transgenic cell line to ionomycin. Here we hypothesize that MAL13P1.156 overexpressing parasites have a higher capacity to withstand ionomycin exposure and its effect as an intracellular calcium ion mobilizer that is presumably depleting the apicoplast of calcium. Overexpression of PfACBP1 would help to retain higher calcium concentration in the apicoplast via its calcium binding properties. To investigate this hypothesis, P. falciparum cells (mid schizont stage) were treated with 0, 0.25, 0.5 and 1 M of Ionomycin for 12-14 hrs until next invasion and the parasite survival was monitored by Giemsa smear microscopy. At 0.5 M of ionomycin (that roughly corresponds to the ionomycin IC50; see below), the survival of the PfACBP1 overexpressing cell line is ∼2-fold higher (P value 0.01) compared to the nontransfected parasites ( Figure 4). The survival rate of the transgenic cell line is comparable to the nontransfected parasites grown in the medium supplemented with 500 M of CaCl 2 . Here we assume that the extracellular calcium supplied in the growth medium reduces the severity of the calcium mobilization action of ionomycin ( Figure 4). This increase in the resistance of the PfACBP1 overexpressing parasites suggests its role in the calcium signaling in the plastid but also the fact that the calcium depletion from the apicoplast is a part to the toxic effect of ionophores in the P. falciparum parasites.

Discussion
Here we used transcriptional profiling to analyze the mode of action of ionophores (ionomycin and A23187) in order to gain more insights into the role of Ca 2+ signaling in P. falciparum. Although transcriptional profiling is known to be a powerful method to understand activities of small-molecule inhibitors in eukaryotic cells, in P. falciparum as well as other highly specialized pathogens, chemical or other types of external stimuli/perturbations do not always induce specific responses [37,54,55]. In P. falciparum, transcriptional responses to external perturbations range from low amplitude nonspecific changes in mRNA profiles to broad extensive transcriptional changes affecting the vast majority of the P. falciparum genes that are typically consistent with developmental arrests and/or induction of the sexual stages. Nonetheless, for several types of small molecule inhibitors, transcriptional responses are more specific, involving genes of direct or indirect targets [56,57]. In this study, we used two inhibitors that are known to have similar (or overlapping) effects on calcium distribution in the eukaryotic cell. Interestingly these two inhibitors exhibit dramatically different effects on the P. falciparum growth. While ionomycin affected expression of a narrow group of genes, with downregulation of plastid gene expression being most pronounced, A23187 caused a broad transcriptional shift that is consistent with a developmental arrest in the schizont stage. This is surprising given that both inhibitors have similar growth inhibitory effects on the P. falciparum cells with IC50 (inhibition concentration by 50%) 513 and 304 nM for ionomycin and A23187, respectively (data not shown). This could be either due to a stronger effect of A23187 on calcium redistribution in the cell and subsequently a more dramatic response manifested by the developmental arrest, or alternatively this discrepancy could be caused by A23187 interacting with additional molecular targets compared to ionomycin. Although more research is required to understand the molecular mechanism(s) that underlines the developmental arrests induced by various perturbations, these data further underline the overall diversity of P. falciparum transcriptional responses to external stimuli and their utility for systems biology approaches using the "guilty-by-association" principle [56,58,59]. The main activity of calcium ionophores is to carry Ca 2+ cations across the membranes down its concentration gradient [45]. In eukaryotic cells this causes cytoplasmic mobilizations of Ca 2+ that are released from the intracellular calcium stores, intracellular compartments in which calcium is sequestered under normal growth concentrations. Similar to other eukaryotic cells, the main intracellular calcium stores of apicomplexan parasites include the endoplasmic reticulum (ER), nuclei, and mitochondria as well as specialized acidic compartments, acidocalcisomes [60]. In addition to these canonical calcium stores, the parasitophorous vacuole (PV) (the lumen between the parasite plasma membrane (PPM) and the parasitophorous vacuolar membrane (PVM) diving the parasite cell from the host cell cytoplasm during their intraerythrocytic development) was shown to contain high concentrations of calcium [34]. It was proposed that the high concentration of calcium in the PV, estimated at ∼40 M, creates a calcium rich microenvironment that is essential for the parasite growth in the otherwise calcium poor erythrocyte cytoplasm, >100 nM. Expectedly, ionomycin causes a rapid efflux of calcium out of the PV into the parasite cytoplasm and to the ambient media and thus diminishing the concentration gradient at the parasite plasma membrane. In addition to the PV, ionomycin can also mobilize other intracellular calcium stores in the parasites (presumably ER, mitochondria, and acidocalcisomes) raising the calcium concentration in the parasite cytoplasm even further [34]. The rapid effect on the plastid genome transcription observed in our study suggests that the ionophores can affect the concentration of calcium diverting it from a steady state concentration that is likely essential for the proper function of this organelle. This model is supported by the fact that the ionomycin-mediated inhibition of the plastid transcription also occurs when the calcium is chelated from the ambient media (Figure 2(c)); hence, the ionophore-mediated calcium flow is directed away from the parasitized erythrocyte. Moreover, thapsigargin (THG) that increases cytoplasmic calcium by a specific release from the ER [34] has no effect on the plastid genome transcription (data not shown). Taken together these data suggest that in addition to calcisomes, ER, and mitochondria, the plastid can serves as another component of the internal calcium stores in the Plasmodium parasites.
There is mounting evidence that calcium signaling plays a major role in maintenance and genesis of the endosymbiotic organelles of eukaryotic cells. In the chloroplast of the plant cells, calcium is an essential signaling factor for at least three different functionalities: import of nuclear encoded proteins, vesicular transport system, and oxygenate photosynthesis (reviewed in [53]). Most of the calcium-dependent signal transduction in the chloroplast is believed to be facilitated by proteins that contain the "EF-hand" domain(s) [61]. Binding of calcium causes conformational changes to the EFhand proteins that subsequently results in increasing binding affinity to other interacting proteins or DNA sequences. Alternatively, calcium binding could cause cross-activation of enzymatic activities of additional domains present at the polypeptide such as protein kinases in calmodulins, calciumdependent protein kinases present in plants, and protists [62]. Here we identify a novel EF-hand protein in P. falciparum, PfACBP1, that is targeted to the plastid and its overexpression increases resistance of the parasite cells to ionomycin. In our bioinformatics analyses of the 545 plastid targeted proteins [47,48], PfACBP1 showed the highest homology to the EFhand consensus sequence which suggests its crucial role in calcium-mediated regulatory function(s) in this compartment. In future studies it will be intriguing to explore the role of PfACBP1, the highly conserved calcium binding protein, in regulation of plastid gene expression.
Until today, the plastid represents one of the most important targets for malaria chemotherapy [63]. A number of apicoplast functionalities can be targeted by specific wellestablished antimicrobial chemotherapeutics, some of which can be used for malaria treatment and prophylaxis. These chemotherapeutic strategies take advantage of the prokaryotic character of several basic apicoplast mechanisms including DNA replication, inhibited by fluoroquinolinone antibiotics [64]; RNA transcription inhibited by rifampicin [65]; and protein translation that can be blocked by clindamycin, azithromycin binding to 23S rRNA [66], and doxycycline and tetracycline binding to 16S rRNA [67]. All these compounds were shown to block apicoplast organellogenesis and division which leads to an absence of this compartment in the newly invaded parasite generations. Although these parasites could develop until the midstage of the (subsequent) IDC, the lack of apicoplast functionalities likely causes the ultimate cell death. Overall, this phenomenon, also known as the delayed death phenotype, is characteristic for most of these drugs with the exception of tetracycline that is believed to also affect the mitochondrion and kill the cells instantly [67]. In addition to rifampicin and other RNA synthesis blockers, doxycycline was also found to specifically and exclusively inhibit transcription of the apicoplast genome encoded genes [68]. This is somewhat surprising as the main mode of action doxycycline is blocking the apicoplast proteosynthesis. This suggests that transcriptional regulation of the apicoplast genes involves multiple components of intracellular signaling potentiality including apicoplast encoded proteins. Here we show that calcium-dependent signaling factors contribute to this regulation and that interference with these can also have toxic effect on the Plasmodium cells, albeit not via the delayed death mechanism.
Several biochemical pathways associated with the apicoplast are being explored as suitable drug targets for malaria chemotherapy. These include fatty acid type II (FASII) [69], nonmevalonate isoprenoid synthesis [70], apicoplast REDOX system [71], and heme synthesis [72]. Each of these pathways represents essential biological processes that take place within the endosymbiotic organelle and thus were retained through the evolution. With that, each pathway retained a certain portion of prokaryotic features that are being explored by malaria drug development efforts. The most remarkable example represents FASII that arrears to be the sole producer of fatty acids in Plasmodium cells as precursors of membrane synthesis and energy stores. Several inhibitors of FASII enzymes are being explored as suitable drug candidates including trichlosan (inhibitor of enoyl-ACP reductase, FabI) and thiolactomycine (inhibitor of betaketoacyl-ACP synthetase II and III, Fab II and III) (reviewed in [63]). Although the validity of this pathway as a drug target for blood stage parasites has been recently disputed by observations that apicoplast plays only a minor biochemical role during its asexual intraerythrocytic development [73]. FASII was found to be predominant and extremely important during the Plasmodium liver stage development [74]. Indeed, the Plasmodium liver stages appear to be highly sensitive to a FASII inhibitor hexachlorophene, as well as to rifampicin and tetracycline [74]. Given the importance of calciumdependent signaling in the apicoplast transcription it will be interesting to explore its potential as a new target for liver stage drug development which is one of the main objectives of the future programs for malaria control and elimination proposed for the next era of malaria-related research [75].

Cell Culture, Drug Treatment, and DNA Microarray.
All treatment experiments were carried out with the P. falciparum 3D7 strain. Calcium ionophore treatments were carried out as follows: highly synchronized P. falciparum cultures were treated with 5 M of calcium ionophores, ionomycin and A23187 (Sigma) at the schizont stage for 30 minutes, 1 hour, 2 hours, 4 hours, and 6 hours. Total RNA from each of the time points was isolated and aminoallyl-cDNA was synthesized using reverse transcriptase system (Fermentas). Subsequently cDNA made from the treated and untreated parasites were labeled with Cy5 (GE-Amersham). A reference pool was made by mixing equal amount of RNA from the parasites collected at 6 hours interval throughout the 48 hours life cycle and was labeled with Cy3 (GE-Amersham). The samples were then hybridized on a spotted cDNA chip platform comprising 10166 MOEs representing 5363 coding sequences [36]. The data was normalized and filtered with the condition, signal intensity > background intensity + 2 SD of background intensity using NOMAD (http://derisilab.ucsf.edu). Hierarchical clustering of the log-transformed ratios was then done using Cluster (Eisen lab) [76] and visualized using Treeview (Eisen lab) [76]. Pathway analysis was done based on the hyper geometric and binomial probability distribution and pathways which had a P value of <0.01 were considered significant. For the extracellular calcium chelation experiment, the medium was treated with 3 mM of EGTA and the ionomycin treatment was carried out later on at 5 M concentration. Hybridization and data analysis were carried out as above.

Comparative Genomic Hybridization (CGH).
Comparative genomic hybridization was carried out with total DNA isolated from the untreated parasites and the parasites treated with the calcium ionophores (ionomycin and A23187 at 5 M concentration) at 1 hour, 4 hours, and 6 hours after treatment.
3 g of the total DNA from each of the samples was subjected to klenow (NEB) reaction as described before [77]. Treated DNA labeled with Cy3 was then hybridized against untreated DNA labeled with Cy5. Hybridization and Data analysis were done as described above for the cDNA hybridization.

Transfection.
Transfection of 3D7 parasites was performed as described before [78]. Two lines of transgenic parasites episomally expressing GFP fused to the C-terminus of acyl carrier protein (ACP) and MAL13P1.156, respectively, were developed. ACP and MAL13P1.156 were amplified from the 3D7 genomic DNA using the following primers: pARL vector [79] was modified for generating the required constructs. Stevor gene was replaced with ACP and MAL13P1.156 for generating pARL-ACP-GFP and pARL-MAL13P1.156, respectively, under the control of PfCRT promoter and hDHFR as the selectable marker.

Fluorescence Microscopy.
Parasites expressing ACP-GFP episomally was used for the import inhibition experiment. Smears were made from parasites exposed to ionomycin and A23187 1 hour, 2 hours, 4 hours, and 6 hours after invasion. The smears were fixed in 4% paraformaldehyde and, after staining with DAPI, were viewed under a fluorescence microscope. For the localization and colocalization experiments, immunofluorescence assay was done using anti-GFP (mouse anti-GFP, Santacruz Biotech) and anti-tuFA (rabbit anti-tuFA, kindly given by Dr. Saman Habib, CDRI, India).

Western Blots.
Proteins from the crude parasite lysates were separated on a 10% SDS polyacrylamide gel. The resolved proteins were then transferred to a nitrocellulose membrane. The blots were then probed with rabbit anti-GFP antibody which was in turn detected with anti-rabbit IgG conjugated with HRP. 4.6. Drug Assay. Nontransfectants, nontransfectants with 500 M CaCl 2 in the medium, and the transfectants overexpressing MAL13P1.156 were exposed to ionomycin at 0, 0.25, 0.5, and 1 M concentrations in the schizont stage. The newly invaded rings were counted on giemsa stained smears under a light microscope. The percentage of rings was plotted against the inhibitory concentrations.


Preclinical humanized mouse model with ectopic ovarian tissues

The aim of the present study was to establish human ovarian stroma within the mouse subcutaneously, in order for the resulting stroma to serve as a useful preclinical tool to study the progression of human ovarian cancer in a humanized ovarian microenvironment. Normal human ovarian tissues were subcutaneously implanted into severe combined immunodeficient (SCID) mice and then the implants were identified by immunohistochemistry. The implants became vascularized and retained their original morphology for about 4 weeks following implantation. Immunohistochemical staining for cytokeratin-7 confirmed the ovarian origin of the epithelial cells. CD34 staining demonstrated human-derived vessels. Positive estrogen receptor and partially-positive progesterone receptor staining indicated the estrogen and progesterone dependence of the implants. Only vascular pericytes expressed α-smooth muscle actin, indicating the normal ovarian origin of the xenografts. Human ovarian tissue successfully survived in SCID mice and retained its original properties. This humanized mouse model may be used as preclinical tool to investigate ovarian cancer.

Introduction
Despite rapid advances in the understanding of ovarian carcinogenesis, ovarian cancer remains the most common cause of mortality from gynecological malignancies and the fifth most common cause of cancer mortalities in females in the United States (1,2). Since mouse models of ovarian cancer can mimic the clinical processes of human ovarian cancer, several models have been developed during the past decades to promote in vivo research on human ovarian cancer (3)(4)(5)(6). However, a major limitation of these models is the lack of a human microenvironment and an inaccurate replication of the interactions between ovarian cancer cells and the human ovarian microenvironment (7,8), which is now known to play a crucial role in the regulation of tumor growth and metastasis (9,10).
To precisely mimic the clinical processes of human cancer, an increasing number of humanized factors have been gradually added to these mouse models. The development of 'humanized' mice is now an important research tool for the in vivo study of human biological processes (11). Since the first study reported that severe combined immunodeficient (SCID) mice were able to be successfully engrafted with human tissues, normal and neoplastic human tissues have been successfully engrafted into SCID mice (12). The implantation of human tissue xenografts in immunodeficient mice has provided insight into the biology of human cancer, autoimmunity and infectious diseases (11). Proia and Kuperwasser (13) reproducibly established functionally normal breast tissue in mice by implanting reduction mammoplasty tissue samples in an orthotopic xenograft model. The endogenous mouse epithelium was cleared, and comixed human epithelial and stromal cells were implanted to construct a humanized mouse model. However, whether this model may be used to understand normal human breast development or tumorigenesis remains unknown. Bankert et al (14) described a humanized mouse model of ovarian cancer that recapitulated the solid tumor progression, ascites formation and metastasis observed in patients. In this model, the tumor and tumor stroma were successfully engrafted into the peritoneal cavity of SCID mice. This model may be used to determine how fibroblasts and lymphocytes within the tumor microenvironment contribute to tumor growth and metastasis. However, this humanized mouse model focused solely upon the engraftment of ovarian cancer tissues, not cancer cells grown in a human microenvironment.
In previous studies, orthotopic implantation of human breast tissues in mice was demonstrated to result in a novel mouse model with a human mammary microenvironment (15), while the implantation of human gastric tissues in mice resulted in a novel mouse model with a human gastric microenvironment (16). To the best of our knowledge, there have been no studies investigating the implantation of normal human ovarian tissue in a SCID mouse model. Therefore, the aim of the present study was to develop a novel protocol for the establishment of human ovarian stroma within a mouse model subcutaneously. It was hypothesized that the resulting stroma may serve as a useful preclinical tool that may allow the progression of human ovarian cancer to be investigated in a humanized ovarian microenvironment.

Materials and methods
Animals and materials. A total of 28 female SCID mice (age, 5-6 weeks; C.B-17IcrCrl-scid-bgBR) were purchased from the Model Animal Research Center of Nanjing University (MARC, Nanjing, China). The mice were housed under specific pathogen-free, temperature-controlled conditions. Their cages, bedding and drinking water were autoclaved and changed regularly. Food was sterilized by irradiation. The mice were maintained in a daily cycle of alternating 12-h periods of light and darkness. All experimental procedures were conducted according to the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of Nanjing Medical University (Nanjing, China).
Normal, human, noncancerous ovarian tissues were obtained from patients that had undergone a total hysterectomy with prophylactic oophorectomy at the First Affiliated Hospital of Nanjing Medical University. The use of human samples in the study was approved by the Committee for Ethical Review of Research at Nanjing Medical University, according to the ethical guidelines of the Declaration of Helsinki and each patient provided informed consent to participate in the study.

Implantation of human ovarian tissues in mice.
Under sterile conditions, normal human ovarian tissues were sliced into small sections (~4x4x4 mm in size). Three small sections of normal human ovarian tissue were randomly selected for subsequent histological examination; the other pieces were stored in phosphate-buffered saline at 4˚C until implantation into the mice.
Prior to implantation, the mice were anesthetized with an intraperitoneal injection of 1% pentobarbital sodium (10 µl/g body weight; Sigma-Aldrich, Steinheim, Germany). The surgical procedure was performed as previously described, with certain modifications (9,10). In brief, 5-6 mm incisions were made using a scalpel on the skin of the left mid-dorsal flank, through which four or five small sections of human ovarian tissue were subcutaneously implanted. The procedure was finished within 6 h following the prophylactic oophorectomy. All the mice received gentamicin in the drinking water (800,000 U/l) for up to one week following implantation. A total of 14 SCID mice were implanted with xenografts from one individual patient, while an additional 14 SCID mice were implanted with xenografts from two patients in this phase of the study where the xenografts come from three human patients.
Gross observation and specimen collection. Following implantation, the ovarian xenografts were subjected to weekly gross examinations. Mice were sacrificed at one, two or four weeks following surgery, and the human xenografts, including the mouse tissue surrounding the implanted human ovarian tissues, were harvested for histological assessment and immu-nohistochemical analysis. The harvested specimens were fixed in 10% formalin for examination.
Histological and immunohistochemical examination. Specimens were dehydrated and embedded in paraffin. Sections (4-µm thick) were stained with hematoxylin and eosin and examined under a microscope. For immunohistochemical staining, the specimens were deparaffinized and rehydrated using xylene and graded alcohol. Sections were mounted on slides, subjected to antigen retrieval and then incubated with rabbit monoclonal antibodies against human estrogen receptor (ER), progesterone receptor (PR) and CD34 (Maxim Biotech, San Francisco, CA, USA), as well as human cytokeratin (CK)-7, CK-20 and α-smooth muscle actin (CK-7, CK-20 ,α-SMA; LabVision, Kalamazoo, MI, USA). Immunocomplexes were visualized using the diaminobenzidine method and sections were counterstained with hematoxylin. Negative controls were serial sections processed without the primary antibody. These reagents exhibited no cross-reactivity between the relevant species (mouse and human). All histological examinations were performed by one experienced pathologist.

Results
Acceptance of xenografts in SCID mice. From one week following the implantation, the ovarian xenografts were subjected to weekly gross examinations. The embedded human ovarian tissues survived in the SCID mouse hosts for up to six weeks. No infection or tissue rejection reactions were observed at the time of tissue harvest, and all the implants became vascularized and survived well on the sheath of mouse muscles in the subcutaneous tissue ( Fig. 1A and B).
Histological examination. Histological evaluation revealed that the morphology of the human ovarian tissues embedded in the SCID mice was similar to the morphology of the original tissues prior to implantation. The viability of the cells within the xenografts was confirmed by the intact nature of the cell membranes, normal granulation of the cytoplasm and normal size and staining characteristics of the nucleus ( Fig. 2A and B). In a number of the engrafted ovarian tissues, which were derived from a perimenopausal female, the follicular architecture of the original tissues was retained ( Fig. 2C and D). However, ovarian tissues embedded in the nude mice in the preliminary experiments were unable to survive, and histological evaluation indicated that the specific morphology of the ovarian tissues was not retained in the implanted tissues (Fig. 2E).
Immunohistochemical analysis. Immunohistochemical analysis demonstrated that the harvested specimens were positive for ER and partially positive for PR, indicating that the inoculated ovarian tissues had remained estrogen-and progesterone-dependent, similar to the original tissues ( Fig. 3A and B). Positive staining was also observed for anti-human CK-7, indicating that the majority of the ovarian epithelium in the original and transplanted tissues was derived from the human ovarian tissues (Fig. 3C and D). Positive staining for anti-human CD34 indicated that the implanted ovarian tissues were well vascularized and remained viable (Fig. 3E). In addi-tion, α-SMA, which is often expressed in cancer-associated fibroblasts, was only detected in the vascular pericytes and not in the ovarian epithelium or ovarian stroma, indicating that the transplanted tissues were normal ovarian tissues similar to the original tissue (Fig. 3F).

Discussion
Complex biological processes often require in vivo analysis, and a number of important research advances have been made using mice as a model for the study of various biological systems. However, mice are not the same as humans, and in vivo studies of human biology are severely limited by ethical and technical constraints. There is a growing need for animal models, which can be used to construct in vivo studies of human cells, tissues and organs without putting individuals at risk. Humanized mice, or mouse-human chimeras, have been developed to overcome these constraints and are now an important research tool for the in vivo study of human cells and tissues (11). Humanized mice are defined as immunodeficient mice engrafted with hematopoietic cells or tissues, or mice that transgenically express human genes (11). The development of mice that are 'humanized' by the engraftment of human tissues provides an opportunity for the in vivo study of human biological processes, which may otherwise not be possible. In the present study, a novel protocol was developed for the establishment of normal human ovarian stroma within the subcutaneous tissues of mice. It was hypothesized that the implanted stroma would accurately mimic the human ovarian microenvironment, and result in a humanized mouse model that may potentially serve as a useful preclinical tool for investigating the progression of human ovarian cancer.
A key factor in the success of this engraftment and the long-term survival of human xenografts is the use of SCID mice. Failure to develop mature T and B lymphocytes and the lack of an immune system makes SCID mice an ideal model for cell and tissue-transfer experiments. As a classical model, the subcutaneous mouse model has been widely used for a number of years. In the present study, normal, human ovarian tissues were subcutaneously implanted into SCID mice with the aim of developing a novel human ovarian tissue-derived mouse model.
Numerous studies have used mice with xenografts implanted under the renal capsule (17). However, in the present study, human ovarian tissue was embedded subcutaneously in the SCID mice. The advantages of subcutaneous implantation include a plentiful blood supply at the implantation site and minimal surgical damage to the tissues. Between day 7 and 14 following implantation, neovascularization was observed in the implanted tissue. This model has the potential to improve the understanding of the crosstalk between tissue stroma and the epithelium, as well as the factors involved in tumor initiation and progression. The humanized ovarian microenvironment established in this mouse model offers a basis to study the proliferation of human ovarian cancer cells in a human ovarian milieu.
A normal, human tissue microenvironment contains three-dimensional intercellular interactions between the stroma cells, epithelial cells and cytokines. These are essential for maintaining epithelial polarity and modulating the growth inhibition of normal cells (18,19). However, when cancer cells emerge in normal tissue, they produce a range of stroma-modulating factors to activate the stromal cells in their microenvironment. The activated stromal cells then secrete matrix metalloproteinases and cytokines to promote tumor growth, stimulate angiogenesis, increase inflammatory responses and induce cell differentiation (20)(21)(22). Therefore, the normal tissue microenvironment is disrupted and transforms into the tumorous tissue microenvironment.
In the present study, the epithelial cells in the xenografts were shown to be derived from normal ovarian tissues, as these cells were positive for CK-7, which is diffusely distributed throughout the cytoplasm of ovarian cells. Furthermore, human-derived vessels within the implants remained viable for a period of four weeks following implantation. Positive staining for the ER and partially positive staining for the PR indicated that the inoculated ovarian tissues continued to be estrogen-and progesterone-dependent. α-SMA was found to be expressed only in the vascular pericytes and not in the epithelium or stromal fibroblasts, indicating that the subcutaneous xenografts of human ovarian tissues in the SCID mice consisted of cells derived from normal tissues. These observations indicated that the implanted human ovarian tissues survived well in the SCID mice.
The results of the present study indicate that the humanized SCID mouse ovarian transplant model has the potential to serve as a useful preclinical tool, which may be used to build a humanized mouse model of ovarian cancer to investigate the efficacy of chemotherapeutic strategies. The absence of an immune system is one of the major limitations inherent in this type of model. However, SCID mice are likely to continue to be critical in establishing xenograft models that recapitulate the tumor growth and spread observed in patients.
In conclusion, the present study demonstrated that human ovarian tissue successfully survives in a SCID mouse host and retains the properties of the original normal ovarian tissues. In addition, the experimental protocol used in the current study may be used to establish a humanized mouse model of ovarian cancer for preclinical research.