Development of micro immunosensors to study genomic and proteomic biomarkers related to cancer and Alzheimer's disease

A report from the National Institutes of Health defines a disease biomarker as a “characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.” Early diagnosis is a crucial factor for incurable disease such as cancer and Alzheimer’s disease (AD). During the last decade researchers have discovered that biochemical changes caused by a disease can be detected considerably earlier as compared to physical manifestations/symptoms. In this dissertation electrochemical detection was utilized as the detection strategy as it offers high sensitivity/specificity, ease of operation, and capability of miniaturization and multiplexed detection. Electrochemical detection of biological analytes is an established field, and has matured at a rapid pace during the last 50 years and adapted itself to advances in micro/nanofabrication procedures. Carbon fiber microelectrodes were utilized as the platform sensor due to their high signal to noise ratio, ease and low-cost of fabrication, biocompatibility, and active carbon surface which allows conjugation with biorecognition moieties. This dissertation specifically focuses on the detection of 3 extensively validated biomarkers for cancer and AD. Firstly, vascular endothelial growth factor (VEGF) a cancer biomarker was detected using a one-step, reagentless immunosensing strategy. The immunosensing strategy allowed a rapid and sensitive means of VEGF detection with a detection limit of about 38 pg/mL with a linear dynamic range of 0–100 pg/mL. Direct detection of AD-related biomarker amyloid beta (Aβ) was achieved by exploiting its inherent electroactivity. The quantification of the ratio of Aβ1-40/42 (or Aβ ratio) has been established as a reliable test to diagnose AD through human clinical trials. Triple barrel carbon fiber microelectrodes were used to simultaneously detect Aβ1-40 and Aβ1-42 in cerebrospinal fluid from rats within a detection range of 100nM to 1.2μM and 400nM to 1μM respectively. In addition, the release of DNA damage/repair biomarker 8-hydroxydeoxyguanine (8-OHdG) under the influence of reactive oxidative stress from single lung endothelial cell was monitored using an activated carbon fiber microelectrode. The sensor was used to test the influence of nicotine, which is one of the most biologically active chemicals present in cigarette smoke and smokeless tobacco.

Effects of Endothelin 3 on the Tumor-Angiogenic Response in a Novel Melanoma Mouse Model

Skin cancer is the most common form of cancer in the United States. Melanoma is a particular type of skin cancer, which arises from the malignant transformation of melanocytes and generally exhibits a high propensity to metastasize. Melanoma progression is dependent on angiogenesis to deliver the oxygen and nutrients required to maintain the altered metabolism of rapidly proliferating tumorigenic cells. Recent studies have implicated the growth factor Endothelin 3 (Edn3) in melanoma progression and metastasis. The aim of this study was to examine the role that Edn3 plays in the angiogenesis of melanocytic lesions. For this purpose, Dct-Grm1 transgenic mice, which spontaneously acquire melanocytic lesions through the aberrant expression of the metabotropic glutamate receptor 1 (mGluR1), were crossed with K5-Edn3 transgenic mice that overexpress Edn3. Tumors in the Dct-Grm1/K5-Edn3 experimental population were examined and compared to the control Dct-Grm1 population using immuno-fluorescent staining targeted against the vascular endothelial cell marker CD31. Proteomic arrays were also used and identified changes in the expression of specific angiogenic factors. CD31 antibody staining results revealed an increased vascular density in Dct-Grm1/K5-Edn3 tumors compared with tumors from the Dct-Grm1 controls. Analysis of the relative expression of angiogenic proteins showed an upregulation of various vascular factors in tumors from the Dct-Grm1/K5-Edn3 population, including VEGF-B, MMP-8, MMP-9, and Angiogenin. These results suggest that endothelin signaling promotes angiogenesis in melanocytic lesions. Targeting the factors upregulated by Edn3 signaling may prove effective in hindering melanoma progression.

Biocompatibility assessment of biosorbable polymer coated nitinol alloys

Owing to an increased risk of aging population and a higher incidence of coronary artery disease (CAD), there is a need for more reliable and safer treatments. Numerous varieties of durable polymer-coated drug eluting stents (DES) are available in the market in order to mitigate in-stent restenosis. However, there are certain issues regarding their usage such as delayed arterial healing, thrombosis, inflammation, toxic corrosion by-products, mechanical stability and degradation. As a result, significant amount of research has to be devoted to the improvement of biodegradable polymer-coated implant materials in an effort to enhance their bioactive response. ^ In this investigation, magneto-electropolished (MEP) and a novel biodegradable polymer coated ternary Nitinol alloys, NiTiTa and NiTiCr were prepared to study their bio and hemocompatibility properties. The initial interaction of a biomaterial with its surroundings is dependent on its surface characteristics such as, composition, corrosion resistance, work of adhesion and morphology. In-vitro corrosion tests such as potentiodynamic polarization and electrochemical impedance spectroscopic (EIS) studies were conducted to determine the coating stability and longevity. In-vitro hemocompatibility studies and HUVEC cell growth was performed to determine their thrombogenic and biocompatibility properties. Critical delamination load of the polymer coated Nitinol alloys was determined using Nano-scratch analysis. Sulforhodamine B (SRB) assays were performed to elucidate the effect of metal ions leached from Nitinol alloys on the viability of HUVEC cells. Scanning Electron Microscopy (SEM), X-ray Photoelectron Spectroscopy (XPS), contact angle meter and X-ray diffraction (XRD) were used to characterize the surface of the alloys. ^ MEP treated and polymer coated (PC) Nitinol alloys displayed a corrosion resistant polymer coating as compared to uncoated alloys. MEP and PC has resulted in reduced Ni and Cr ion leaching from NiTi5Cr and subsequently low cytotoxicity. Thrombogenicity tests revealed significantly less platelet adhesion and confluent endothelial cell growth on polymer coated and uncoated ternary MEP Nitinol alloys. Finally, this research addresses the bio and hemocompatibility of MEP + PC ternary Nitinol alloys that could be used to manufacture blood contacting devices such as stents and vascular implants which can lead to lower U.S. healthcare spending.^

Theoretical Investigation of Intra- and Inter-cellular Spatiotemporal Calcium Patterns in Microcirculation

Microcirculatory vessels are lined by endothelial cells (ECs) which are surrounded by a single or multiple layer of smooth muscle cells (SMCs). Spontaneous and agonist induced spatiotemporal calcium (Ca2+) events are generated in ECs and SMCs, and regulated by complex bi-directional signaling between the two layers which ultimately determines the vessel tone. The contractile state of microcirculatory vessels is an important factor in the determination of vascular resistance, blood flow and blood pressure. This dissertation presents theoretical insights into some of the important and currently unresolved phenomena in microvascular tone regulation. Compartmental and continuum models of isolated EC and SMC, coupled EC-SMC and a multi-cellular vessel segment with deterministic and stochastic descriptions of the cellular components were developed, and the intra- and inter-cellular spatiotemporal Ca2+ mobilization was examined. Coupled EC-SMC model simulations captured the experimentally observed localized subcellular EC Ca2+ events arising from the opening of EC transient receptor vanilloid 4 (TRPV4) channels and inositol triphosphate receptors (IP3Rs). These localized EC Ca2+ events result in endothelium-derived hyperpolarization (EDH) and Nitric Oxide (NO) production which transmit to the adjacent SMCs to ultimately result in vasodilation. The model examined the effect of heterogeneous distribution of cellular components and channel gating kinetics in determination of the amplitude and spread of the Ca2+ events. The simulations suggested the necessity of co-localization of certain cellular components for modulation of EDH and NO responses. Isolated EC and SMC models captured intracellular Ca2+ wave like activity and predicted the necessity of non-uniform distribution of cellular components for the generation of Ca2+ waves. The simulations also suggested the role of membrane potential dynamics in regulating Ca2+ wave velocity. The multi-cellular vessel segment model examined the underlying mechanisms for the intercellular synchronization of spontaneous oscillatory Ca2+ waves in individual SMC. From local subcellular events to integrated macro-scale behavior at the vessel level, the developed multi-scale models captured basic features of vascular Ca2+ signaling and provide insights for their physiological relevance. The models provide a theoretical framework for assisting investigations on the regulation of vascular tone in health and disease.

A Model of Lung Tumor Angiogenesis in a Biomimetic Poly(ethylene glycol)-based Hydrogel System

Tumor angiogenesis is critical to tumor growth and metastasis, yet much is unknown about the role vascular cells play in the tumor microenvironment. A major outstanding challenge associated with studying tumor angiogenesis is that existing preclinical models are limited in their recapitulation of in vivo cellular organization in 3D. This disparity highlights the need for better approaches to study the dynamic interplay of relevant cells and signaling molecules as they are organized in the tumor microenvironment. In this thesis, we combined 3D culture of lung adenocarcinoma cells with adjacent 3D microvascular cell culture in 2-layer cell-adhesive, proteolytically-degradable poly(ethylene glycol) (PEG)-based hydrogels to study tumor angiogenesis and the impacts of neovascularization on tumor cell behavior.

In initial studies, 344SQ cells, a highly metastatic, murine lung adenocarcinoma cell line, were characterized alone in 3D in PEG hydrogels. 344SQ cells formed spheroids in 3D culture and secreted proangiogenic growth factors into the conditioned media that significantly increased with exposure to transforming growth factor beta 1 (TGF-β1), a potent tumor progression-promoting factor. Vascular cells alone in hydrogels formed tubule networks with localized activated TGF-β1. To study cancer cell-vascular cell interactions, the engineered 2-layer tumor angiogenesis model with 344SQ and vascular cell layers was employed. Large, invasive 344SQ clusters developed at the interface between the layers, and were not evident further from the interface or in control hydrogels without vascular cells. A modified model with spatially restricted 344SQ and vascular cell layers confirmed that observed 344SQ cluster morphological changes required close proximity to vascular cells. Additionally, TGF-β1 inhibition blocked endothelial cell-driven 344SQ migration.

Two other lung adenocarcinoma cell lines were also explored in the tumor angiogenesis model: primary tumor-derived metastasis-incompetent, murine 393P cells and primary tumor-derived metastasis-capable human A549 cells. These lung cancer cells also formed spheroids in 3D culture and secreted proangiogenic growth factors into the conditioned media. Epithelial morphogenesis varied for the primary tumor-derived cell lines compared to 344SQ cells, with far less epithelial organization present in A549 spheroids. Additionally, 344SQ cells secreted the highest concentration of two of the three angiogenic growth factors assessed. This finding correlated to 344SQ exhibiting the most pronounced morphological response in the tumor angiogenesis model compared to the 393P and A549 cell lines.

Overall, this dissertation demonstrates the development of a novel 3D tumor angiogenesis model that was used to study vascular cell-cancer cell interactions in lung adenocarcinoma cell lines with varying metastatic capacities. Findings in this thesis have helped to elucidate the role of vascular cells in tumor progression and have identified differences in cancer cell behavior in vitro that correlate to metastatic capacity, thus highlighting the usefulness of this model platform for future discovery of novel tumor angiogenesis and tumor progression-promoting targets.

Biocompatibility Assessment of Biosorbable Polymer Coated Nitinol Alloys

Owing to an increased risk of aging population and a higher incidence of coronary artery disease (CAD), there is a need for more reliable and safer treatments. Numerous varieties of durable polymer-coated drug eluting stents (DES) are available in the market in order to mitigate in-stent restenosis. However, there are certain issues regarding their usage such as delayed arterial healing, thrombosis, inflammation, toxic corrosion by-products, mechanical stability and degradation. As a result, significant amount of research has to be devoted to the improvement of biodegradable polymer-coated implant materials in an effort to enhance their bioactive response. In this investigation, magneto-electropolished (MEP) and a novel biodegradable polymer coated ternary Nitinol alloys, NiTiTa and NiTiCr were prepared to study their bio and hemocompatibility properties. The initial interaction of a biomaterial with its surroundings is dependent on its surface characteristics such as, composition, corrosion resistance, work of adhesion and morphology. In-vitro corrosion tests such as potentiodynamic polarization and electrochemical impedance spectroscopic (EIS) studies were conducted to determine the coating stability and longevity. In-vitro hemocompatibility studies and HUVEC cell growth was performed to determine their thrombogenic and biocompatibility properties. Critical delamination load of the polymer coated Nitinol alloys was determined using Nano-scratch analysis. Sulforhodamine B (SRB) assays were performed to elucidate the effect of metal ions leached from Nitinol alloys on the viability of HUVEC cells. Scanning Electron Microscopy (SEM), X-ray Photoelectron Spectroscopy (XPS), contact angle meter and X-ray diffraction (XRD) were used to characterize the surface of the alloys. MEP treated and polymer coated (PC) Nitinol alloys displayed a corrosion resistant polymer coating as compared to uncoated alloys. MEP and PC has resulted in reduced Ni and Cr ion leaching from NiTi5Cr and subsequently low cytotoxicity. Thrombogenicity tests revealed significantly less platelet adhesion and confluent endothelial cell growth on polymer coated and uncoated ternary MEP Nitinol alloys. Finally, this research addresses the bio and hemocompatibility of MEP + PC ternary Nitinol alloys that could be used to manufacture blood contacting devices such as stents and vascular implants which can lead to lower U.S. healthcare spending.

ROLE OF STAT1 AND CXCL10 IN THE TUMOUR IMMUNE MICROENVIRONMENT OF HIGH-GRADE SEROUS OVARIAN CANCER

High-grade serous ovarian cancer (HGSC) is the most prevalent epithelial ovarian cancer characterized by late detection, metastasis and resistance to chemotherapy. Previous studies on the tumour immune microenvironment in HGSC identified STAT1 and CXCL10 as the most differentially expressed genes between treatment naïve chemotherapy resistant and sensitive tumours. Interferon-induced STAT1 is a transcription factor, which induces many genes including tumour suppressor genes and those involved in recruitment of immune cells to the tumour immune microenvironment (TME), including CXCL10. CXCL10 is a chemokine that recruits tumour infiltrating lymphocytes (TILs) and exhibits angiostatic function. The current study was performed to determine the effects of differential STAT1 and CXCL10 expression on HGSC disease progression and TME. STAT1 expression and intratumoural CD8+ T cells were evaluated as prognostic and predictive biomarkers via immunohistochemistry on 734 HGSC tumours accrued from the Terry Fox Research Institute-Canadian Ovarian Experimental Unified Resource. The combined effect of STAT1 expression and CD8+ TIL density was confirmed as prognostic and predictive companion biomarkers in the second independent biomarker validation study. Significant positive correlation between STAT1 expression and intratumoral CD8+ TIL density was observed. The effects of enforced CXCL10 expression on HGSC tumour growth, vasculature and immune tumour microenvironment were studied in the ID8 mouse ovarian cancer cell engraftment in immunocompetent C57BL/6 mice. Significant decrease in tumour progression in mice injected with ID8 CXCL10 overexpressing cells compared to mice injected with ID8 vector control cells was observed. Multiplexed cytokine analysis of ascites showed differential expression of IL-6, VEGF and CXCL9 between the two groups. Endothelial cell marker staining showed differences in tumour vasculature between the two groups. Immune transcriptomic profiling identified distinct expression profiles in genes associated with cytokines, chemokines, interferons, T cell function and apoptosis between the two groups. These findings provide evidence that STAT1 is an independent biomarker and in combination with CD8+ TIL density could be applied as novel immune-based biomarkers in HGSC. These results provide the basis for future studies aimed at understanding mechanisms underlying differential tumour STAT1 and CXCL10 expression and its role in pre-existing tumour immunologic diversity, thus potentially contributing to biomarker guided immune modulatory therapies.

IMMUNE-ANGIOGENESIS MECHANISMS ASSOCIATED WITH PORCINE PREGNANCY SUCCESS AND FAILURE

Spontaneous fetal loss (25-40%) leading to decrease in litter size is a significant concern to the pork industry. A deficit in the placental vasculature has emerged as one of the important factors associated with fetal loss. During early pig pregnancy, the endometrium becomes enriched with immune cells recruited by conceptus-derived signals including specific chemokine stimuli. These immune cells assist in various aspects of placental development and angiogenesis. Recent evidence suggests that microRNAs (miRNAs: small non-coding RNAs that regulate gene expression) regulate immune cell development and their functions. In addition, intercellular communication including exchange of biomolecules (e.g. miRNAs) between the conceptus and endometrium regulate key developmental processes during pregnancy. To understand the biological significance of immune cell enrichment, regulation of their functions by miRNAs and transfer of miRNAs across the maternal fetal-interface, we screened specific sets of chemokines and pro- and anti-angiogenic miRNAs in endometrial lymphocytes (ENDO LY), endometrium, and chorioallantoic membrane (CAM) isolated from conceptus attachment sites (CAS) during early, gestation day (gd)20 and mid-pregnancy (gd50). We report increased expression of selected chemokines including CXCR3 and CCR5 in ENDO LY and CXCL10, CXCR3, CCL5, CCR5 in endometrium associated with arresting CAS at gd20. Some of these differences were also noted at the protein level (CXCL10, CXCR3, CCL5, and CCR5) in endometrium and CAM. We report for the first time significant differences for miRNAs involved in immune cell-derived angiogenesis (miR-296-5P, miR-150, miR-17P-5P, miR-18a, and miR-19a) between ENDO LY associated with healthy and arresting CAS. Significant differences were also found in endometrium and CAM for some miRNAs (miR-17-5P, miR-18a, miR-15b-5P, and miR-222). Finally, we confirm that placenta specific-exosomes contain proteins and 14 select miRNAs including miR-126-5P, miR-296-5P, miR-16, and miR-17-5P that are of relevance to early implantation events. We further demonstrated the bidirectional exosome shuttling between porcine trophectoderm cells (PTr2) and porcine aortic endothelial cells (PAOEC). PTr2-derived exosomes were able to modulate the endothelial cell proliferation that is crucial for the establishment of pregnancy. Our data unravels the selected chemokines and miRNAs associated with immune cell-regulated angiogenesis and reconfirm that exosome mediated cell-cell communication opens-up new avenues to understand porcine pregnancy.

Masson’s tumor: a soft tissue tumor simulating a tendon cyst. Case report

Introduction. Intravascular papillary endothelial hyperplasia (Masson's hemangioma or Masson’s tumor) is a benign vascular disease with an exuberant endothelial proliferation in normal blood vessels. Although relatively uncommon, its correct diagnosis is important because it can clinically be like both benign lesions and malignant neoplasms. We present a case of intravascular proliferative endothelial hyperplasia simulating a tendon cyst both clinically and on ultrasound. Case report. A 74-year old Caucasian female presented with a 4-month history of soreness and swelling in the fourth finger of the right hand. Ultrasound showed an oval mass with fluid content, referred to a tendon cyst. A wide surgical excision was subsequently performed. The final histological diagnosis was Masson’s tumor. Discussion. The pathogenesis of intravascular papillary endothelial hyperplasia is still unclear but the exuberant endothelial cell proliferation might be stimulated by an autocrine loop of endothelial basic fibroblast growth factor (bFGF) secretion. There are three types of papillary endothelial hyperplasia: primary, or intravascular; secondary, or mixed; and extravascular. The main differential diagnosis is against pyogenic granuloma, Kaposi sarcoma, hemangioma, and angiosarcoma. Conclusions. Masson's tumor can be like both benign lesions and malignant neoplasms clinically and on ultrasound. For this reason, the right diagnosis can be made only by histology, which reveals a papillary growth composed of hyperplastic endothelial cells supported by delicate fibrous stalks entirely confined within the vascular lumen.

Aspectos estruturais, farmacológicos e biológicos de fucanas da alga marrom sargassum vulgare

The present study examines the chemical composition and their effects on free radicals, inflammation, angiogenesis, coagulation, VEGF effects and cellular proliferation of a polysaccharides from alga Sargassum vulgare. The sulfated polysaccharide was extracted from brown seaweed by proteolysis with enzymes maxataze. The presence of proteins and sugars were observed in crude polysaccharides. Fractionation of this crude extract was made with growing concentration of acetone (0.3-1.5 v) and produced four groups of polysaccharides. Anionic polysaccharides from brown seaweed Sargassum vulgare, SV1and PSV1 were fractionated (SV1) and purified (PSV1), and displayed with high total sugars and sulfate content and very low level of protein. This fucan SV1 contains low levels of protein and high carbohydrate and sulfate content. This polysaccharides prolonged activated partial thromboplastin time (aPTT) at 50 μg (>240 s). SV1 was found to have no effect on prothrombin time (PT), corresponding to the extrinsic pathway of coagulation. SV1 exhibits high antithrombotic action in vivo, with a concentration ten times higher than heparin. Polysaccharides from S. vulgare promoted direct inhibition enzymatic activity of thrombin and stimulated enzymatic activity of FXa. SV1 showed optimal inhibitory activity of thrombin (50.2±0.28%) at a concentration of 25 μg/mL. Its antioxidant action on scavenging radicals by DPPH was (22%), indicating the polymer has no cytotoxic action (hemolytic) on ABO and Rh blood types in different erythrocyte groups and displays strong anti-inflammatory action on all concentrations tested in the carrageenan-induced paw edema model, demonstrated by reduced edema and cellular infiltration. Angiogenesis is a dynamic process of proliferation and differentiation. It requires endothelial proliferation, migration, and tube formation. In this context, endothelial cells are a preferred target for several studies and therapies. The antiangiogenic efficacy of polysaccharides was examined in vivo in the chick chorioallantoic membrane (CAM) model by using fertilized eggs. Decreases in the density of the capillaries were assessed and scored. The results showed that SV1 and PSV1 have an inhibitory effect on angiogenesis. These results were also confirmed by inhibition tubulogenesis in rabbit aorta endothelial cell (RAEC) in matrigel. These compounds were assessed in Apoptosis assay (Annexin V - FITC / PI) and cell viability by MTT assay of RAEC. These polysaccharides do not affect the viability and do not have apoptotic or necrotic action. RAEC cell when incubated with SV1 and PSV1showed inhibition of VEGF secretion, observed when compounds were incubated at 25, 50 and 100 μg/μL. The VEGF secretion with the RAEC cell line for 24 h, was more effective for PSV1 at 50 μg/μL(71.4%) than SV1 100 μg/μL (75.9%). SV1 and PSV1 had an antiproliferative action (47%) against tumor cell line HeLa. Our results indicate that these sulfated polysaccharides have antiangiogenic and antitumoral actions

The Effect of Architecture and Shear Stress on Endothelialization of 3D Printed Vascular Networks

Despite significant progress in the field of tissue engineering within the last decade, a number of unsolved problems still remain. One of the most relevant issues is the lack of proper vascularization that limits the size of engineered tissues to smaller than clinically relevant dimensions. In particular, the growth of engineered tissue in vitro within bioreactors is plagued with this challenge. Specifically, the tubular perfusion system bioreactor has been used for large scale bone constructs; however these engineered constructs lack inherent vasculature and quickly develop a hypoxic core, where no nutrient exchange can occur, thus leading to cell death. Through the use of 3D printed vascular templates in conjunction with a tubular perfusion system bioreactor, we attempt to create an endothelial cell monolayer on 3D scaffolds that could potentially serve as the foundation of inherent vasculature within these engineered bone grafts.

O papel da lisofosfatidilcolina na ativação do inflamassoma NLRP3 e sua relação com a formação de células gordurosas

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Molecular, 2016.

Antiviral activity and mechanism of action of arbidol against Hantaan virus infection

Purpose: To investigate the activity and mechanism of action of arbidol against Hantaan virus (HTNV) activity by modulating inflammation via TLR-4 pathway. Methods: HUVEC cells infected with HTNV 76-118 were treated with serially diluted arbidol solutions at -2h (2 h before viral infection, pre-treatment mode), 0 h (at the same time as viral infection, simultaneous treatment mode) or 2 h (2 h after viral infection, post-treatment mode). The transcript levels of TLR4 were detected by semi-quantitative reverse transcription-PCR (RT-PCR) at 6, 12, 18, and 24 h later. The levels of iNOS and TNF-α were examined using enzyme-linked immunosorbent assay (ELISA). Results: Pre-treatment with arbidol, rather than simultaneous treatment or post-treatment, effectively inhibited up-regulation of cellular TLR4 expression (up to 40 ± 6.1 % inhibition) and activity of supernatant iNOS induced by HTNV infection(up to 44.1 ± 9.4 % inhibition), as well as in a LPSstimulated inflammatory endothelial cell. Arbidol decreased the elevated TNF-α levels induced by LPS stimulation. Conclusion: These results are the first evidence that arbidol modulates viral PRRs signaling and its consequential inflammatory cytokine/chemokine response during hantavirus infection.

Canine mesenchymal stem cells are neurotrophic and angiogenic:an in vitro assessment of their paracrine activity

Mesenchymal stem cells (MSCs) have been used in cell replacement therapies for connective tissue damage, but also can stimulate wound healing through paracrine activity. In order to further understand the potential use of MSCs to treat dogs with neurological disorders, this study examined the paracrine action of adipose-derived canine MSCs on neuronal and endothelial cell models. The culture-expanded MSCs exhibited a MSC phenotype according to plastic adherence, cell morphology, CD profiling and differentiation potential along mesenchymal lineages. Treating the SH-SY5Y neuronal cell line with serum-free MSC culture-conditioned medium (MSC CM) significantly increased SH-SY5Y cell proliferation (P < 0.01), neurite outgrowth (P = 0.0055) and immunopositivity for the neuronal marker βIII-tubulin (P = 0.0002). Treatment of the EA.hy926 endothelial cell line with MSC CM significantly increased the rate of wound closure in endothelial cell scratch wound assays (P = 0.0409), which was associated with significantly increased endothelial cell proliferation (P < 0.05) and migration (P = 0.0001). Furthermore, canine MSC CM induced endothelial tubule formation in EA.hy926 cells in a soluble basement membrane matrix. Hence, this study has demonstrated that adipose-derived canine MSC CM stimulated neuronal and endothelial cells probably through the paracrine activity of MSC-secreted factors. This supports the use of canine MSC transplants or their secreted products in the clinical treatment of dogs with neurological disorders and provides some insight into possible mechanisms of action.

Eficacia de terapias biológicas de tipo anti-angiogénico en el cáncer colorrectal metastásico revisión sistemática de la literatura

Introducción: El cáncer colorrectal (CCR) se encuentra entre los 5 tipos de cáncer con mayor incidencia a nivel mundial. Alrededor del 20% de los casos son diagnosticados en estadios metastásico, donde el tratamiento inicialmente era quimioterapia con una supervivencia global a 5 años de 12 a 14 meses. Es así que se investiga el papel de la angiogénesis tumoral, orientado al desarrollo de terapias, implementando su uso en estadios avanzados. Metodología: Se realizó una búsqueda sistemática en las bases de datos Embase, PubMed, SciELO y LILIACS con términos estandarizados a través de la herramienta MeSH y DECS bajo los lineamientos establecidos en las guías de revisiones sistemáticas y meta-análisis (Manual Cochrane). Se tomaron estudios clínicos aleatorizados controlados con pacientes con CCR metastásico, que hayan recibido quimioterapia sola o combinada con terapias antiangiogénicas, publicados en inglés y español entre el 2003 y 2013. Resultados: 6 artículos cumplieron con criterios de inclusión. Estos reportaron 15.8 meses en promedio de supervivencia global en el tratamiento de quimioterapia asociada a terapias biológicas frente a 14.4 meses con solo quimioterapia. Los eventos adversos de tipo vascular aumentaron más en el grupo de antiangiogénicos, reportando muertes debidas a perforaciones intestinales. Conclusiones: Los regímenes de quimioterapia asociadas a terapias antiangiogénicas brindan una mayor supervivencia global y libre de progresión, al igual que mayor número de tasas de respuesta. Son terapias con eventos adversos importantes pero que deberá seleccionarse bien al paciente para disminuir su riesgo de eventos. Palabras claves: Cáncer colorrectal metastásico, terapia anti-angiogénica, quimioterapia en segunda línea, receptor del factor de crecimiento de endotelio vascular, supervivencia global.