Structural bases that underline Trypanosoma cruzi calreticulin proinfective, antiangiogenic and antitumor properties.

Microbes have developed mechanisms to resist the host immune defenses and some elicit antitumor immune responses. About 6 million people are infected with Trypanosoma cruzi, the protozoan agent of Chagas' disease, the sixth neglected tropical disease worldwide. Eighty years ago, G. Roskin and N. Klyuyeva proposed that T. cruzi infection mediates an anti-cancer activity. This observation has been reproduced by several other laboratories, but no molecular basis has been proposed. We have shown that the highly pleiotropic chaperone calreticulin (TcCalr, formerly known as TcCRT), translocates from the parasite ER to the exterior, where it mediates infection. Similar to its human counterpart HuCALR (formerly known as HuCRT), TcCalr inhibits C1 in its capacity to initiate the classical pathway of complement activation. We have also proposed that TcCalr inhibits angiogenesis and it is a likely mediator of antitumor effects. We have generated several in silico structural TcCalr models to delimit a peptide (VC-TcCalr) at the TcCalr N-domain. Chemically synthesized VC-TcCalr did bind to C1q and was anti-angiogenic in Gallus gallus chorioallantoic membrane assays. These properties were associated with structural features, as determined in silico. VC-TcCalr, a strong dipole, interacts with charged proteins such as collagen-like tails and scavenger receptors. Comparatively, HuCALR has less polarity and spatial stability, probably due to at least substitutions of Gln for Gly, Arg for Lys, Arg for Asp and Ser for Arg that hinder protein-protein interactions. These differences can explain, at least in part, how TcCalr inhibits the complement activation pathway and has higher efficiency as an antiangiogenic and antitumor agent than HuCALR.

Cerebrospinal fluid VEGF levels and angiogenic capacity as potential prognostic markers in patients with gliomas: a pilot study.

INTRODUCTION: Gliomas are tumors of the central nervous system. Despite new classifications, they are still divided in low and high-grade gliomas, being the latter of greater malignancy. The degree of malignancy is directly related with the angiogenic activity in tumoral tissues. We measured VEGF concentrations and angiogenic capacity in cerebrospinal fluid (CSF) from patients with high and low-grade gliomas. The purpose of this study was to find a biomarker that contributes in the differential diagnosis and prognosis of gliomas. METHODS: CSF was obtained from 19 individuals: 8 with low-grade gliomas, 6 with high-grade gliomas and 5 controls. VEGF concentration in CSF was measured by ELISA and the angiogenic capacity was measured by chick chorioallantoic membrane (CAM) test. RESULTS: The VEGF concentration was higher in patients with high-grade gliomas, compared to patients with low-grade gliomas and controls (2860 pg/mL ± 975 vs. 182.6 ± 37.1 and 47.4 ± 0.4, respectively). On the other hand, CSF from patients with high-grade gliomas generated a higher microvascular density (MVD) than patients with low-grade gliomas and controls (13.23 ± 0.6 vessels/9000µm2 vs. 9.3 ± 0.3 and 7.92 ± 0.2, respectively). Interestingly, there was not statistical differences in both VEGF levels and angiogenic capacity in patients with low-grade gliomas and controls. CONCLUSION: Together VEGF levels and angiogenic capacity in CSF can be used as a biological marker of gliomas malignancy.

Expresión de VEGF-A y VEGFR2 en Miocardio de Pollos Tratados con Ácido Acetilsalicílico (AAS) / VEGF-A and VEGFR2 Expression in Chicken Myocardium Treated with Acetylsalicylic Acid (AAS)

RESUMEN: Los niveles de VEGF y su unión a sus receptores son etapas claves en la regulación de la angiogénesis. El ácido acetilsalicílico (AAS), ampliamente utilizado en tratamiento post infarto al miocardio ha mostrado poseer un efecto antiangiogénico en modelos tumorales. Este efecto potencialmente contraproducente requiere ser estudiado en miocardio. El objetivo del presente trabajo es cuantificar el efecto de AAS y de ácido salicílico (AS) sobre la vascularización en membrana alantocoriónica (MAC) y sobre los niveles de VEGF-A y VEGFR2 en miocardio de embriones de pollo. Para ello, treinta fetos de pollo White Leghorn fueron instilados a los 10 días de gestación con 60 µL de DMSO 0,1 % (control) o conteniendo además 0,3 µmol de AAS o AS. A las 48 horas se realizó procesamiento histológico de MAC para recuento de vasos sanguíneos y de tejido cardíaco para cuantificar VEGF-A y VEGFR2 por inmunohistoquímica. La inmunorreactividad fue cuantificada mediante Image J. Tanto AAS como AS disminuyeron la densidad microvascular de MAC. En miocardio, AAS aunque no AS, disminuyó la concentración de VEGFR2. No hubo efecto sobre VEGF-A. En nuestro modelo experimental, fetos de pollo a los 10 días de gestación también se observó el efecto inhibidor de AAS sobre la angiogénesis en MAC. La disminución de VEGFR2 en cardiomiocitos sugiere que AAS también afecta la angiogénesis en miocardio sano, modificando la disponibilidad del receptor a VEGF. Estos hallazgos nos permiten postular que AAS podría interferir con la regeneración de tejido, en situaciones como post infarto al miocardio.

SUMMARY: The VEGF levels and its binding to its receptors are key stages in the regulation of angiogenesis. Acetylsalicylic acid (ASA), widely used in post-myocardial infarction treatment, has been shown to have an anti-angiogenic effect in tumor models. This potentially counterproductive effect requires to be studied in myocardium. The aim of this study is to quantify the effect of ASA and salicylic acid (SA) on the vascularization in chick allantochorionic membrane (CAM) and on the levels of VEGF-A and VEGFR2 in myocardium of chicken embryos. Thirty White Leghorn chicken fetuses were instilled at 10 days of gestation with 60 mL of 0.1 % DMSO (control) or also containing 0.3 mmol of ASA or SA. After 48 hours, CAM histological processing was performed to count blood vessels and heart tissue to quantify VEGFA and VEGFR2 by immunohistochemistry. Immunoreactivity was quantified by Image J. Both ASA and SA decreased CAM microvascular density. In myocardium, AAS, although not SA, decreased the concentration of VEGFR2. There was no effect on VEGF-A. In our experimental model, chicken fetuses at 10 days of gestation, the inhibitory effect of ASA on angiogenesis in CAM were also observed. The decrease in VEGFR2 in cardiomyocytes suggests that ASA also affects angiogenesis in healthy myocardium, modifying the availability of the receptor to VEGF. These findings allow us to postulate that ASA could interfere with tissue regeneration, when it is required, as post myocardial infarction.

Angiogenesis and tumor progression inhibition of cyclooxygenase-2 selective inhibitor celecoxib associated with poly (lactic-co-glycolic acid) in tumor cell line resistant to chemotherapy / Inhibición de angiogénesis y progresión tumoral por inhibidor selectivo de ciclooxigenasa-2 celecoxib asociado con ácido (poli láctico co-glicólico) en línea de células tumorales resistentes a quimioterapia

Although, antineoplastic therapies have now been developed reduction of tumor progression,itis necessarytofind new therapeutic alternatives to suppress angiogenesis.Thus celecoxib (Cx) has been used for its antiangiogenic action in combination with certain polymeric compounds such as poly (lactic co-glycolic acid) (PLGA) acid, which help to improve the bioavailability and avoid effects of long drug administrations. For this purpose we used a murine tumor modelinduced by mammary adenocarcinoma cells resistant to chemotherapy (TA3-MTXR). CX/PLGA inhibits the microvascular density, VEGF expression and cell proliferationinaddition to increased apoptosis (P <0.0001). Cx reduces tumor progression in a concentration of 1000 ppm associated with PLGA, reducing cell proliferation, the presence of VEGF and promoting apoptosis of multiresistant TA3 tumor cells.

Si bien actualmente se han desarrollado terapias antineoplásicas que permiten reducir de cierta manera el avance tumoral, es necesario buscar nuevas alternativas terapéuticas que permitan suprimir la angiogénesis. Es así como el Celecoxib (Cx) ha sido utilizado por su acción antiangiogénica en combinación con algunos compuestos poliméricos, tal como el ácido poli (láctico co-glicólico) (PLGA), el cual ayudaría a mejorar la biodisponibilidad y evitaría efectos derivados de largas administraciones del fármaco. Para tal efecto se ha utilizado un modelo tumoral murino, inducido por células tumorales de adenocarcinoma mamario resistente a la quimioterapia (TA3-MTXR). Los resultados indican que CX/PLGA inhibe la microvascularización, expresión de VEGF y la proliferación celular además del aumento de la apoptosis (P<0,0001). El efecto antitumoral del Cx está bien reportado en la literatura; este sumado a la microencapsulación con PLGA, aportarían un sistema de administración útil, ya que nos otorga una administración sostenida en el tiempo, los cual podría ayudar a mantener los niveles de droga durante un período más prolongado, lo cual sería beneficioso en la terapia tumoral.

Celecoxib/PLGA suprime angiogénesis y metástasis pulmonar de un cáncer mamario murino experimental / Celecoxib/PLGA suppresses angiogenesis and lung metastasis of murine experimental breast cancer

La angiogénesis y metástasis son eventos esenciales en el proceso de invasión tumoral. Su relación íntima los hace buenos blancos en la terapia antitumoral. El objetivo fue analizar el patrón de metástasis pulmonar y angiogénesis, luego de la aplicación del antiangiogénico Celecoxib microencapsulado en ácido poli(láctico-co-glicólico) en ratones. Se utilizó un modelo de tumor experimental, inducido por células TA3-MTX-R, en 18 ratones, separados en 3 grupos de 6 animales, los cuales fueron tratados con dos presentaciones de Celecoxib en administración intramuscular (Grupo Control; Grupo Cx 1000 ppm y Grupo Cx 1000 ppm+PLGA). Los ratones fueron sacrificados y procesados histológicamente para ser teñidos con H&E y Tricrómico de Arteta. El estudio reveló que el pulmón muestra una marcada heterogeneidad, y un patrón de metástasis perivascular; además, Celecoxib asociado a ácido poli(láctico-co-glicólico) redujo la invasión tumoral y angiogénesis en el pulmón. Los resultados son similares a descripciones parciales realizadas previamente y son comparables a otras líneas tumorales, siendo celecoxib/ácido poli(láctico-co-glicólico) un candidato potencial en la terapia antitumoral.

Angiogenesis and metastasis are critical events on the tumor invasion process. Their close association is related as a good target in antitumor therapy. The aim was to analyze lung metastasis pattern and angiogenesis following application of microencapsulated Celecoxib with poli(lactic-co-glycolic) acid in mice. An experimental tumor model was assessed, induced by TA3-MTX-R cells, in 18 mice, separated in 3 groups of 6 animals and treated with 2 intramuscular Celecoxib presentations (Group Control; Group Cx 1000 ppm and Group Cx 1000 ppm+PLGA). Mice were sacrificed and histologically processed to stain slides with H&E and Arteta Trichromic. The study revealed that the lung showed a significant heterogeneity, and a perivascular metastasis pattern; moreover, Celecoxib associated to poli(lactic-co-glycolic) acid reduces tumor invasion and pulmonary angiogenesis. The results are similar to partial previous descriptions and are comparable to other tumor lines, concluding that Celecoxib/poli(lactic-co-glycolic) acid is a potential candidate in antitumor therapy.

Ácido acetilsalicílico (AAS) inhibe angiogénesis en membrana alantocorionica de pollo (MAC) / Acetyl-salicylic acid inhibits angiogenesis in alanto chorionic chicken membrane

Introducción: El ácido acetilsalicílico (AAS) es ampliamente utilizado en el manejo de patología cardiovascular. En modelos "in vitro" el AAS restringe la angiogénesis, atribuyéndose este efecto al bloqueo de ciclooxigenasa-1, manteniendo íntegra la zona adhe-rente endotelial, citotoxicidad directa y otras vías de señalización. Hipótesis: El AAS en concentración terapéutica antiplaquetaria utilizada en humanos ejerce un efecto antiangiogénico en modelo de membrana alantocorió-nica de pollo (MAC). Objetivo: Comparar la capacidad antiangiogénica del AAS en distintas concentraciones en MAC utilizando como punto de comparación la angiogénesis fisiológica de la MAC. Método: Se incubaron 46 huevos fecundados de gallinas White Leghorn, en cámara temperada a 37°C, provenientes del Instituto de Salud Pública de Chile. Mediante procedimiento descrito por Ribatti (2006), se instiló sobre filtro de metilcelulosa 10uL de Dimetilsulfóxido al 0.1% + m199, sin fármaco al control, asociado a AAS y ácido salicílico (AS) a los grupos de estudio en concentraciones 2mM y 5 mM. Posteriormente se fijó y analizó la muestra en forma ciega. Resultados: El promedio de vasos del control fue 21.8. Para el grupo AAS 2mM y 5mM fue 11.3 y 10, siendo para el grupo AS 2mM y 5mM 15.6 y 12.4. El análisis estadístico mediante ANOVA y t-Student muestra que todos los grupos que recibieron fármacos tuvieron una disminución significativa en el numero de vasos sanguíneos en relación al grupo control. No hubo diferencias significativas entre ambo grupos de AAS. El AS demostró tener mayor potencia antiangiogénica dosis dependiente. Discusión: En este estudio se demuestra que el AAS ejerce un efecto antiangiogénico en concentración terapéutica en condiciones fisiológicas de un modelo "in vivo".

Background: Acetylsalicylic acid (ASA) is widely used in the treatment of various cardiovascular disorders. In vitro, AAS decreases angiogenesis, through cyclo-oxigenase-1 blockade while keeping active the adherent endothelial zone, direct toxicity and other signaling pathways. Hypothesis: AAS at therapeutic anti plaquetary doses exerts an anti-angiogenic effect in the alanto choronic chicken membrane (ACM) Method: 46 fertilized eggs form White Leghorn hens were incubated at 37oC. 10 uL of 0.1% Dimethyl sulfoxide +Ml 19 with no drug were used as control, while experimental groups received ASA and Salicylic acid (SA), 2mM. After fixation, samples were analyzed in a blind fashion Results: The mean number of vessels was 21.8 for controls, 11.3 and 10 for ASA 2mM and ASA 5mM, respectively. Corresponding values for SA 2 and SA 5mM were 15.6 and 12.4, respectively. Thus, a statistically significant (ANOVA and Student's t) decrease in the number of vessels was observed in both ASA groups. SA showed had a greater potential for anti-angiogenesis in a dose dependent way. Conclusion: This study shows that ASA in therapeutic concentrations has an anti-angiogenic effect in a physiologic model in vivo.

Efecto antimetastásico de celecoxib/PLGA en un modelo murino de adenocarcinoma mamario TA3-MTX-R / Antimetastatic effect of celecoxib/PLGA in a TA3-MTX-R murine breast adenocarcinoma model

La metástasis es el proceso de propagación de un foco cancerígeno a un órgano distinto de aquel en que se inició; ocurriendo generalmente por vía sanguínea o linfática. La localización más frecuentes de las metástasis son los órganos más irrigados como el cerebro, pulmones, hígado, huesos y glándulas suprarrenales. El objetivo fue analizar el patrón de metástasis hepática de tumor TA3-MTX-R, luego de la aplicación del antiangiogénico Celecoxib microencapsulado en PLGA en ratones, así como la disminución de áreas metastásicas a nivel lobulillar. Se utilizó un modelo de tumor experimental, inducido por células TA3-MTX-R, en 18 ratones, separados en 3 grupos de 6 animales, los cuales fueron tratados con dos presentaciones de Celecoxib en administración intramuscular (Grupo 1, Control: TA3-MTX-R; Grupo 2: TA3-MTX-R+Cx y Grupo 3: TA3-MTX-R+Cx/PLGA). Los ratones fueron sacrificados y procesados histológicamente para ser teñidos con H&E y Tricrómico de Arteta. El estudio reveló que el higado muestra una marcada heterogeneidad, y un patrón de metástasis perivascular y neovascularización central y periférica. Además, Celecoxib redujo significativamente la invasión tumoral en el hígado (p<0,0001). Los resultados son similares a descripciones parciales realizadas previamente y son comparables a otras líneas tumorales. Creemos que la vía de administración del fármaco es crítica para la interpretación de los resultados. Los hallazgos son importantes para la discusión de otras investigaciones en donde Celecoxib es usado como un fármaco antiangiogénico.

Metastasis is the propagation process of a cancerous focus to an organ other than that in which it started; usually occurring through blood or lymphatic route. The most common sites of metastases are the organs most irrigated such as the brain, lungs, liver, bones and adrenal glands. The objective was to analyze the pattern of liver tumor metastasis TA3-MTX-R, after application of antiangiogenic Celecoxib microencapsulated in PLGA in mice and decreased metastatic to lobular level areas. An experimental model of tumor induced TA3-MTX-R cells was used, 18 mice divided into 3 groups of 6 animals, which were treated with two presentations Celecoxib intramuscular (Group 1, control was used -R; Group 1: TA3-MTX-R+Cx and Group 3: TA3-MTX-R+Cx/PLGA). The mice were sacrificed and processed histologically to be stained with H&E and Arteta trichrome. The study revealed that the liver shows a marked heterogeneity, and a pattern of perivascular metastasis and central and peripheral neovascularization. Furthermore, Celecoxib significantly reduced tumor invasion in the liver (p <0.0001). The results are similar to partial descriptions made previously and are comparable to other tumor lines. It is believed that the route of administration of the drug is critical for the interpretation of the results. These are important for the discussion of other investigations in which Celecoxib is used as an antiangiogenic drug.

Survivin expression promotes VEGF-induced tumor angiogenesis via PI3K/Akt enhanced ß-catenin/Tcf-Lef dependent transcription.

Early in cancer development, tumour cells express vascular endothelial growth factor (VEGF), a secreted molecule that is important in all stages of angiogenesis, an essential process that provides nutrients and oxygen to the nascent tumor and thereby enhances tumor-cell survival and facilitates growth. Survivin, another protein involved in angiogenesis, is strongly expressed in most human cancers, where it promotes tumor survival by reducing apoptosis as well as favoring endothelial cell proliferation and migration. The mechanisms by which cancer cells induce VEGF expression and angiogenesis upon survivin up-regulation remain to be fully established. Since the PI3K/Akt signalling and ß-catenin-Tcf/Lef dependent transcription have been implicated in the expression of many cancer-related genes, including survivin and VEGF, we evaluated whether survivin may favor VEGF expression, release from tumor cells and induction of angiogenesis in a PI3K/Akt-ß-catenin-Tcf/Lef-dependent manner. Here, we provide evidence linking survivin expression in tumor cells to increased ß-catenin protein levels, ß-catenin-Tcf/Lef transcriptional activity and expression of several target genes of this pathway, including survivin and VEGF, which accumulates in the culture medium. Alternatively, survivin downregulation reduced ß-catenin protein levels and ß-catenin-Tcf/Lef transcriptional activity. Also, using inhibitors of PI3K and the expression of dominant negative Akt, we show that survivin acts upstream in an amplification loop to promote VEGF expression. Moreover, survivin knock-down in B16F10 murine melanoma cells diminished the number of blood vessels and reduced VEGF expression in tumors formed in C57BL/6 mice. Finally, in the chick chorioallantoid membrane assay, survivin expression in tumor cells enhanced VEGF liberation and blood vessel formation. Importantly, the presence of neutralizing anti-VEGF antibodies precluded survivin-enhanced angiogenesis in this assay. These findings provide evidence for the existance of a posititve feedback loop connecting survivin expression in tumor cells to PI3K/Akt enhanced ß-catenin-Tcf/Lef-dependent transcription followed by secretion of VEGF and angiogenesis.

Celecoxib decreases growth and angiogenesis and promotes apoptosis in a tumor cell line resistant to chemotherapy.

BACKGROUND: During the last few years it has been shown in several laboratories that Celecoxib (Cx), a non-steroidal anti-inflammatory agent (NSAID) normally used for pain and arthritis, mediates antitumor and antiangiogenic effects. However, the effects of this drug on a tumor cell line resistant to chemotherapeutical drugs used in cancer have not been described. RESULTS: Cx reduces angiogenesis in the chick embryonic chorioallantoic membrane assay (CAM), inhibits the growth and microvascular density of the murine TA3-MTXR tumor, reduces microvascular density of tumor metastases, promotes apoptosis and reduces vascular endothelial growth factor (VEGF) production and cell proliferation in the tumor. CONCLUSION: The antiangiogenic and antitumor Cx effects correlate with its activity on other tumor cell lines, suggesting that Prostaglandins (PGs) and VEGF production are involved. These results open the possibility of using Celecoxib combined with other experimental therapies, ideally aiming to get synergic effects.

Human survivin and Trypanosoma cruzi calreticulin act in synergy against a murine melanoma in vivo.

Immune-based anti-tumor or anti-angiogenic therapies hold considerable promise for the treatment of cancer. The first approach seeks to activate tumor antigen-specific T lymphocytes while, the second, delays tumor growth by interfering with blood supply. Tumor Associated Antigens are often employed to target tumors with therapeutic drugs, but some are also essential for tumor viability. Survivin (Surv) is a member of the inhibitor of apoptosis protein family that is considered a Tumor Associated Antigen important for cancer cell viability and proliferation. On the other hand, Trypanosoma cruzi (the agent of Chagas' disease) calreticulin (TcCRT) displays remarkable anti-angiogenic properties. Because these molecules are associated with different tumor targets, we reasoned that immunization with a Surv-encoding plasmid (pSurv) and concomitant TcCRT administration should generate a stronger anti-tumor response than application of either treatment separately. To evaluate this possibility, C57BL/6 mice were immunized with pSurv and challenged with an isogenic melanoma cell line that had been pre-incubated with recombinant TcCRT (rTcCRT). Following tumor cell inoculation, mice were injected with additional doses of rTcCRT. For the combined regimen we observed in mice that: i). Tumor growth was impaired, ii). Humoral anti-rTcCRT immunity was induced and, iii). In vitro rTcCRT bound to melanocytes, thereby promoting the incorporation of human C1q and subsequent macrophage phagocytosis of tumor cells. These observations are interpreted to reflect the consequence of the following sequence of events: rTcCRT anti-angiogenic activity leads to stress in tumor cells. Murine CRT is then translocated to the external membrane where, together with rTcCRT, complement C1 is captured, thus promoting tumor phagocytosis. Presentation of the Tumor Associated Antigen Surv induces the adaptive anti-tumor immunity and, independently, mediates anti-endothelial cell immunity leading to an important delay in tumor growth.

Celecoxib decreases growth and angiogenesis and promotes apoptosis in a tumor cell line resistant to chemotherapy

BACKGROUND: During the last few years it has been shown in several laboratories that Celecoxib (Cx), a non-steroidal anti-inflammatory agent (NSAID) normally used for pain and arthritis, mediates antitumor and antiangiogenic effects. However, the effects of this drug on a tumor cell line resistant to chemotherapeutical drugs used in cancer have not been described. Herein we evaluate the angiogenic and antitumor effects of Cx in the development of a drug-resistant mammary adenocarcinoma tumor (TA3-MTXR). RESULTS: Cx reduces angiogenesis in the chick embryonic chorioallantoic membrane assay (CAM), inhibits the growth and microvascular density of the murine TA3-MTXR tumor, reduces microvascular density of tumor metastases, promotes apoptosis and reduces vascular endothelial growth factor (VEGF) production and cell proliferation in the tumor. CONCLUSION: The antiangiogenic and antitumor Cx effects correlate with its activity on other tumor cell lines, suggesting that Prostaglandins (PGs) and VEGF production are involved. These results open the possibility of using Celecoxib combined with other experimental therapies, ideally aiming to get synergic effects.

Angiogenic potential of the cerebrospinal fluid (CSF) of patients with high-grade gliomas measured with the chick embryo chorioallantoic membrane assay (CAM).

High-grade gliomas are highly vascularized tumors. Neo-angiogenesis plays a key role in tumor growth and resistance to therapy. A cerebrospinal fluid (CSF) sample could be a useful way to obtain pro-angiogenic predictive or prognostic markers at different stages of the disease. As a first step we looked for pro-angiogenic activity in the CSF of patients with high-grade gliomas. We performed the chicken embryo chorio-allantoic membrane (CAM) assay to study the angiogenic potential of the cerebrospinal fluid (CSF), obtained either by lumbar puncture (LP) or craniotomy from six patients with high-grade brain tumors (three glioblastoma (WHO grade IV), one anaplastic oligodendroglioma (WHO grade III), two anaplastic ganglioglioma (WHO grade III)), and four healthy controls. Significantly increased neo-angiogenesis was observed on the surface of the growing CAM in the 6 patients with high-grade gliomas compared to controls (3.69 ± 1.23 versus 2.16 ± 0.97 capillaries per area (mean ± SD), p<0.005). There was no statistical difference related to the hystological grade of the tumor (WHO grade III or IV), previous treatment (radio-chemotherapy plus temozolomide, temozolomide alone or no treatment), or the site of CSF sample (surgery or lumbar puncture). Our results suggest a pro-angiogenic potential in the CSF of patients with high-grade gliomas.

Angiogenic potential of the cerebrospinal fluid (CSF) of patients with high-grade gliomas measured with the chick embryo chorioallantoic membrane assay (CAM)

High-grade gliomas are highly vascularized tumors. Neo-angiogenesis plays a key role in tumor growth and resistance to therapy. A cerebrospinal fluid (CSF) sample could be a useful way to obtain pro-angiogenic predictive or prognostic markers at different stages of the disease. As a first step we looked for pro-angiogenic activity in the CSF of patients with high-grade gliomas. We performed the chicken embryo chorio-allantoic membrane (CAM) assay to study the angiogenic potential of the cerebrospinal fluid (CSF), obtained either by lumbar puncture (LP) or craniotomy from six patients with high-grade brain tumors (three glioblastoma (WHO grade IV), one anaplastic oligodendroglioma (WHO grade III), two anaplastic ganglioglioma (WHO grade III)), and four healthy controls. Significantly increased neo-angiogenesis was observed on the surface of the growing CAM in the 6 patients with high-grade gliomas compared to controls (3.69 ± 1.23 versus 2.16 ± 0.97 capillaries per area (mean ± SD), p<0.005). There was no statistical difference related to the hystological grade of the tumor (WHO grade III or IV), previous treatment (radio-chemotherapy plus temozolomide, temozolomide alone or no treatment), or the site of CSF sample (surgery or lumbar puncture). Our results suggest a pro-angiogenic potential in the CSF of patients with high-grade gliomas.

Antiangiogenic and antitumor effects of Trypanosoma cruzi Calreticulin.

BACKGROUND: In Latin America, 18 million people are infected with Trypanosoma cruzi, the agent of Chagas' disease, with the greatest economic burden. Vertebrate calreticulins (CRT) are multifunctional, intra- and extracellular proteins. In the endoplasmic reticulum (ER) they bind calcium and act as chaperones. Since human CRT (HuCRT) is antiangiogenic and suppresses tumor growth, the presence of these functions in the parasite orthologue may have consequences in the host/parasite interaction. Previously, we have cloned and expressed T. cruzi calreticulin (TcCRT) and shown that TcCRT, translocated from the ER to the area of trypomastigote flagellum emergence, promotes infectivity, inactivates the complement system and inhibits angiogenesis in the chorioallantoid chicken egg membrane. Most likely, derived from these properties, TcCRT displays in vivo inhibitory effects against an experimental mammary tumor. METHODOLOGY AND PRINCIPAL FINDINGS: TcCRT (or its N-terminal vasostatin-like domain, N-TcCRT) a) Abrogates capillary growth in the ex vivo rat aortic ring assay, b) Inhibits capillary morphogenesis in a human umbilical vein endothelial cell (HUVEC) assay, c) Inhibits migration and proliferation of HUVECs and the human endothelial cell line Eahy926. In these assays TcCRT was more effective, in molar terms, than HuCRT: d) In confocal microscopy, live HUVECs and EAhy926 cells, are recognized by FITC-TcCRT, followed by its internalization and accumulation around the host cell nuclei, a phenomenon that is abrogated by Fucoidin, a specific scavenger receptor ligand and, e) Inhibits in vivo the growth of the murine mammary TA3 MTXR tumor cell line. CONCLUSIONS/SIGNIFICANCE: We describe herein antiangiogenic and antitumor properties of a parasite chaperone molecule, specifically TcCRT. Perhaps, by virtue of its capacity to inhibit angiogenesis (and the complement system), TcCRT is anti-inflammatory, thus impairing the antiparasite immune response. The TcCRT antiangiogenic effect could also explain, at least partially, the in vivo antitumor effects reported herein and the reports proposing antitumor properties for T. cruzi infection.

Comparative in vivo antiangiogenic effects of calreticulin from Trypanosoma cruzi and Homo sapiens sapiens.

Angiogenesis is a complex multi-step process of neovascularization arising from preexisting blood vessels whose generation is regulated by pro- and anti-angiogenic factors. Both Trypanosoma cruzi calreticulin (TcCRT) and its human counterpart (HuCRT) are antiangiogenic. This is the first report where the TcCRT and HuCRT anti-angiogenic properties are compared in vivo. In the chick embryonic chorioallantoid membrane assay (CAM) and at equimolar concentrations, TcCRT displayed significantly higher antiangiogenic activities than its human counterpart. LPS had marginal effects at the concentrations present in the recombinant protein preparations and the TcCRT antiangiogenic effects were largely inhibited by specific polyclonal antibodies, thus, reinforcing the fact that the observed TcCRT effects can be attributed to the parasite-derived molecule and not to the endotoxin. The antiangiogenic TcCRT effects correlate with its anti-tumor in vivo effects, as recently shown in our laboratory.

Betamethasone inhibits tumor development, microvessel density and prolongs survival in mice with a multiresistant adenocarcinoma TA3.

Tumor resistance to traditional cancer treatments poses an important challenge to modern science. Thus, angiogenesis inhibition is an important emerging cancer treatment. Many drugs are tested and corticosteroids have shown interesting results. Herein we investigate the effect on microvessel density, survival time and tumoral volume of mice with TA3-MTX-R tumors. Twenty six mice were inoculated with l x l06 tumor cells; 4-5 days after injection, six mice were injected with PBS (group A) and twenty mice were treated with ß-met (group B). All animals from Group A died on day 22. Group B was divided into Bl (treated discontinued) and B2 (treated daily) and observed until day 88. All mice were processed for histo-immunohistochemical analysis and the blood vessels were counted. A decrease in microvessel density and tumoral volume and longer survival times were observed in the treated group. We propose that the antiangiogenic ß-met effect explains, at least partially, its tumor inhibitory properties. As an important perspective, we will experimentally combine these strategies with those recently described by us with regard to the important antiangiogenic-antitumor effects of Trypanosoma cruzi calreticulin. Since the molecular targets of these strategies are most likely different, additive or synergic effects are envisaged.

Comparative in vivo antiangiogenic effects of calreticulin from Trypanosoma cruzi and Homo sapiens sapiens

Angiogenesis is a complex multi-step process of neovascularization arising from preexisting blood vessels whose generation is regulated by pro- and anti-angiogenic factors. Both Trypanosoma cruzi calreticulin (TcCRT) and its human counterpart (HuCRT) are antiangiogenic. This is the first report where the TcCRT and HuCRT anti-angiogenic properties are compared in vivo. In the chick embryonic chorioallantoid membrane assay (CAM) and at equimolar concentrations, TcCRT displayed significantly higher antiangiogenic activities than its human counterpart. LPS had marginal effects at the concentrations present in the recombinant protein preparations and the TcCRT antiangiogenic effects were largely inhibited by specific polyclonal antibodies, thus, reinforcing the fact that the observed TcCRT effects can be attributed to the parasite-derived molecule and not to the endotoxin. The antiangiogenic TcCRT effects correlate with its anti-tumor in vivo effects, as recently shown in our laboratory.

Betamethasone inhibits tumor development, microvessel density and prolongs survival in mice with a multiresistant adenocarcinoma TA3

Tumor resistance to traditional cancer treatments poses an important challenge to modern science. Thus, angiogenesis inhibition is an important emerging cancer treatment. Many drugs are tested and corticosteroids have shown interesting results. Herein we investigate the effect on microvessel density, survival time and tumoral volume of mice with TA3-MTX-R tumors. Twenty six mice were inoculated with lxlO6 tumor cells, 4-5 days after injection, six mice were injected with PBS (group A) and twenty mice were treated with p-met (group B). All animals from Group A died on day 22. Group B was divided into Bl (treated discontinued) and B2 (treated daily) and observed until day 88. All mice were processed for histo-immunohistochemical analysis and the blood vessels were counted. A decrease in microvessel density and tumoral volume and longer survival times were observed in the treated group. We propose that the antiangiogenic p-met effect explains, at least partially, its tumor inhibitory properties. As an important perspective, we will experimentally combine these strategies with those recently described by us with regard to the important antiangiogenic-antitumor effects of Trypanosoma cruzi calreticulin. Since the molecular targets of these strategies are most likely different, additive or synergic effects are envisaged.

An in vivo role for Trypanosoma cruzi calreticulin in antiangiogenesis.

Angiogenesis leads to neovascularization from existing blood vessels. It is associated with tumor growth and metastasis and is regulated by pro- and antiangiogenic molecules, some of them currently under clinical trials for cancer treatment. During the last few years we have cloned, sequenced and expressed a Trypanosoma cruzi calreticulin gene (TcCRT). Its product, TcCRT, a 45 kDa protein, is more than 50% identical to human CRT (HuCRT). TcCRT, present on the surface of trypomastigotes, binds both C1q and mannan binding lectin and inhibits the classical activation pathway of human complement. Since TcCRT is highly homologous to a functional antiangiogenic fragment from HuCRT (aa 120-180), recombinant (r) and native (n) TcCRT were tested in their antiangiogenic effects, in the chick embryonic chorioallantoid membrane (CAM) assay. Both proteins mediated highly significant antiangiogenic effects in the in vivo CAM assay. This effect was further substantiated in experiments showing that the plasmid construct pSecTag/TcCRT also displayed significant antiangiogenic properties, as compared to the empty vector. Most likely, the fact that antiangiogenic substances act preferentially on growing neoplasic tissues, but not on already established tumors, is due to their effects on emerging blood vessels. The results shown here indicate that TcCRT, like its human counterpart, has antiangiogenic properties. These properties may explain, at least partly, the reported antineoplasic effect of experimental T. cruzi infection.

Does Trypanosoma cruzi calreticulin modulate the complement system and angiogenesis?

Calreticulin, a calcium-binding protein that is highly conserved in its multiple functions, is present in a wide spectrum of subcellular compartments in virtually every cell of higher organisms. In this article, we propose a dual role for parasite calreticulin, with emphasis on the Trypanosoma cruzi model. By modulating the vertebrate complement system, calreticulin might provide the parasite with an effective immune-escape mechanism. Alternatively, by inhibiting angiogenesis, the parasite molecule might protect the host from ongoing neoplasic aggressions. Many questions are still unanswered, particularly those regarding the consequences that these interactions could have in vivo for both the parasite and the host.