Endoglin, a co-receptor of the TGF-β superfamily, is predominantly expressed in endothelial cells and in some myeloid cells and implicated as a potential modulator of immune responses. We previously demonstrated that Endoglin heterozygous (Eng+/-) mice subjected to the dextran sulfate sodium colitis model developed persistent inflammation and epithelial ulceration, while Eng+/+ mice recovered following the acute phase of disease. Our aim was to assess potential alterations in distribution and number of immune cells, expression of inflammatory mediators and mechanisms of oxidative burst in Eng+/- mice. While the number of overall T, B and myeloid cells was unaltered between the genotypes, changes in neutrophil regulating cytokines and angiogenesis mediating factors were observed in Eng+/- mice. In addition, downregulation of phagocyte oxidative burst enzymes point to potential defects in microbial clearance in Eng+/- mice. These findings suggest a role for endoglin in regulating immune and vascular functions during inflammation.

Las plaquetas (PLTs) son protagonistas en la reparación de las heridas, ya que contienen factores de crecimiento (GFs), los cuales producen quimiotaxis, proliferación y diferenciación celular, neovascularización y deposición de matriz extracelular (ECM). Se ha propuesto la utilización de concentrados de plaquetas (PCs) autólogos para acelerar la reparación de las heridas y estimular la capacidad de regeneración de los tejidos lesionados. En los capitulos III y IV de esta tesis se presentan dos estudios clínicos sobre el efecto de un concentrado plaquetario (PC) autólogo en caballos con patología músculo-esquelética grave. Este PC fue preparado por medio de una nueva técnica de doble centrifugación en tubo. Una caracterización celular y molecular de este PC se presenta en el cápitulo V de ésta tesis. Se evaluó el efecto de la inyección intra-articular del PC en 7 caballos con enfermedad articular grave clasificada así según el grado de cojera (DL) y la efusión sinovial (JE). El PC produjo una mejoría estadísticamente significativa del DL y JE (p<0.05). La mejoría más notable fue observada a los dos meses de finalizado el tratamiento y permaneció hasta 8 meses después (ver cápitulo III). En el capitulo IV se evaluó el efecto del PC en 7 caballos con lesiones tendinosas (tendonitis del tendón flexor digital superficial (SDFT) y ligamentosas (desmitis del ligamento suspensorio (DSL)). Todos los caballos mejoraron significativamente su DL y la respuesta a la prueba de flexión (p<0.05). Los registros ultrasonográficos mejoraron en los caballos con tendinopatías, pero no cambiaron en los que tenían desmopatías. Dos caballos con tendinitis retornaron con exito a su nivel de competición sin recidivar. Un caballo con tendinosis crónica recidivó. El resto de pacientes con desmopatías volvieron a su nivel de competición pre-lesión. Se obtuvieron un promedio de 250 ± 71.8 x 106 plaquetas, 8.68 ± 3.78 leucocitos x 106 y 12515 ± 2443 pg de TGF- ?1/ml de PC. No se observaron signos clínicos adversos asociados con el tratamiento. En el cápitulo V se realizó un análisis celular y molecular del PC usado clínicamente (PC¬C), sangre entera y 4 fracciones de PCs adicionales (PC-A, PC-B y PC-D), los cuales son obtenidos durante la elaboración del PC-C. El objetivo fue evaluar el método de obtención de este PC, en el que son necesarios dos periodos de centrifugación. Todas las muestras fueron analizadas mediante citometría de flujo y determinación de los niveles de TGF- ?1. Las concentraciones de plaquetas para PC-A, PC-B, PC-C y PC-D fueron un 45%, 44%, 71% y 21%, respectivamente más altas en comparación con cada PC y la sangre entera. Las concentraciones de TGF- ?1 para PC-A, PC-B, PC-C y PC-D fueron un 38%, 44%, 44% and 37%, respectivamente más altas en comparación con cada PC y la sangre entera. Se concluyó que el método empleado para concentrar plaquetas es valido para producir PCs con niveles potencialmente terapéuticos de TGF-?1. Los resultados obtenidos en esta tesis abren un nuevo y prometedor campo de investigación para conocer los efectos clínicos y moleculares de los PCs en caballos con enfermedad crónica músculo-esquelética. Los resultados que se puedan obtener en caballos serán de gran valor para conocer el uso potencial de los PCs autólogos en seres humanos con similares patologías. / Platelets (PLTs) play a central role in wound healing, since they contain growth factors (GFs), which produce chemotaxis, cellular proliferation and differentiation, neovascularization, and extra¬cellular matrix (ECM) deposition. The use of autologous platelet concentrates (PCs) has been proposed to accelerate wound repair and to stimulate the regenerative capacity of injured tissues. Two clinical studies about the effect of an autologous platelet concentrate (PC) in horses with severe musculoskeletal pathology are presented in chapters III and IV of this thesis. The PC was prepared by a novel double centrifugation tube method. A cellular and molecular characterization of this PC is presented in chapter V of this thesis.The effect of the intraarticular injection of this PC in 7 horses with severe joint disease was evaluated on the basis on degree of lameness (DL) and joint effusion (JE). When PCs were injected into the joint a statistically significant improvement in both DL and JE (p<0.05) were observed. The most marked improvement was maximun 2 months after the last injection and persisted up to 8 months later (see chapter III). The clinical effect of PC injection in 7 horses with soft tissue musculoskeletal injuries namely: SDFT tendinopathy and desmopathy of the susensory ligament (DSL) was also evaluated (see the chapter IV). All the horses presented a clinical and a statistical (p<0.05) decrease of the DL and the response to flexion test. Ultrasonic appearance improved in the horses with SDFT lesions, but remained the same in the horses with DSL. Two horses with acute SDFT tendinopathy returned successfully to competition level without reinjury. One horse with chronic SDFT tendinopathy relapsed. The rest of the horses with DSL returned successfully to competition level without reinjury. A mean of 250 ± 71.8 x 106 platelets, 8.68 ± 3.78 leucocytes x 106, and 12515 ± 2443 pg TGF-?1 were obtained per ml of the PC. No adverse reactions resulted from this treatment. A cellular and a molecular study of the PC clinically used in this thesis (PC-C),of whole blood and of three additional PCs (PC-A, PC-B, and PC-D) obtained during the PC-C preparation were performed (see the chapter V) to compare the single and the double centrifugation tube methods for concentrating equine platelets. Whole blood and the 4 PCs were analyzed using flow cytometry for cellular quantification and determination of TGF- ?1 in all the samples. Platelet concentrations for PC-A, PC-B, PC-C and PC-D were 45%, 44%, 71% and 21% higher, respectively, compared to the same values for citrated whole blood samples. TGF- ?1 concentrations for PC-A, PC-B, PC-C and PC-D were 38%, 44%, 44% and 37% higher, respectively, compared to citrated whole blood sample values. In conclusion, the single and double centrifugation tube methods are reliable methods for concentrating equine platelets and obtaining potentially therapeutic TGF- ?1 levels. The results obtained in this thesis open a new encouraging research field on clinical and molecular effects of PCs in equine chronic musculoskeletal pathology. The future potential results obtained in horses can be of key value to determine the potenetial use of autologous PCs in human beings with chronic musculoskeletal pathology.

Liver fibrosis, a major medical problem with significant morbidity and mortality, is considered as a wound-healing response to a variety of chronic stimuli. It is characterized by an excessive deposition of extracellular matrix (ECM) proteins, which disrupts the normal architecture of liver and ultimately leads to pathophysiological damage to liver. Hepatoma-derived growth factor (HDGF), a growth factor originally purified from hepatoma cells, is highly expressed in fetal hepatocytes and hepatoma. It is known to play multifunctional roles in mitogenesis, organogenesis, embryogenesis, and tumorigenesis. Its expression correlates with the proliferating state of hepatocellular carcinoma (HCC) and serves as a prognostic factor. Since liver fibrosis frequently occurs prior to HCC development, the specific aim of this study is to investigate the role of HDGF in the progression of liver fibrosis by using animal models of mice receiving either bile duct ligation surgery or carbon tetrachloride administration. Quantitative real-time PCR and Western blotting analysis showed a significant elevation of HDGF expression in both models. HDGF levels correlated with progression of liver fibrosis in a time-dependent manner as well as paralleled with the expression of other two fibrotic markers, transforming growth factor-b1 (TGF-b1) and pro-collagen type I, in fibrotic livers. Intriguingly, the over-expressed HDGF protein was localized mainly in perivenous hepatocytes of fibrotic livers. Besides, adenovirus-mediated HDGF gene delivery potentiated the production of TGF-b1 and pro-collagen type I, thereby enhancing the intrahepatic collagen matrix deposits as evidenced by Sirius red stain and morphometrical analysis. In cultured hepatocytes, TGF-b1 and HDGF mutually up-regulated their de novo synthesis only when grown on collagen-coated matrix, strongly suggesting that the TGF-b1- and/or HDGF-driven pro-fibrogenic signaling is collagen-dependent and a vicious circle may exist at the initial stage of hepatic fibrogenesis. Moreover, administration with recombinant HDGF stimulated BrdU uptake and synthesis of both a-smooth muscle actin and pro-collagen type I in cultured hepatic stellate cells, implicating that a mode of paracrinal action lies between these two cell types. In conclusion, HDGF plays a pro-fibrogenic role during liver fibrosis and blockade of HDGF pathway may potentially constitute the preventive or therapeutic strategies for chronic liver diseases.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on Feb. 2, 2010). Includes bibliographical references.

Berlin, Freie Universität, Thesis (doctoral), 2002.

Pulmonary fibrosis is a potentially deadly pathology characterized by excessive deposition of extracellular matrix (ECM), increased tissue stiffness, and loss of tissue structure and function. Recent evidence has suggested epithelial to mesenchymal transition (EMT), the transdifferentiation of an epithelial cell into a mesenchymal fibroblast, is one mechanism that results in the accumulation of myofibroblasts and excessive deposition of ECM. EMT is a highly orchestrated process involving the integration of biochemical signals from specific integrin mediated interactions with ECM proteins and soluble growth factors including TGFβ. TGFβ, a potent inducer of EMT, can be activated by cell contraction mediated mechanical release of the growth factor from a macromolecular latent complex. Therefore, TGFβ activity and subsequent EMT may be influenced by both the biochemical composition and biophysical state of the surrounding ECM. Based on these knowns it was first investigated how changes in the biochemical composition of the matrix and changes in tissue rigidity together modulate EMT due to changes in epithelial cell contraction and TGFβ activation. Here we show that integrin specific interactions with fibronectin (Fn) variants displaying both the RGD and PHSRN binding sites facilitate cell binding through α3β1 and α5β1 integrins, and that these interactions maintain an epithelial phenotype despite engagement of increased tissue rigidities. Conversely, Fn fragments that facilitate cell binding through αv integrins drive TGFβ activation and subsequent EMT even while engaging soft underlying substrates. Adding to the complexity of studying mechanisms that contribute to pulmonary fibrosis, is exposure of the lung to injuries from environmental particulates. Therefore, we investigated how EMT is altered in response to particulate matter (PM). Here we show that PM exposure further drives TGFβ activation, EMT, and increases intracellular levels of reactive oxygen species (ROS). Additionally, cells binding the ECM through α5β1 and α3β1 integrins only partially recover an epithelial phenotype, suggesting ROS may be a secondary driver of TGFβ and EMT. Taken together these results suggest dynamic changes to the ECM microenvironment are major contributors to the control of EMT responses and provide insights into the design of biomaterial-based microenvironments for control of epithelial cell phenotype.

Immunotherapy has been proposed as an alternative to conventional cancer therapies due to its reduced toxicity and ability to induce long-lasting anti-tumor immune responses. Dendritic cell (DC) vaccination is one immune-based anti-cancer strategy that has received attention due to the ability of DC to process and present antigen to T lymphocytes to initiate immune responses. However, the clinical efficacy of DC-based immunotherapy against established cancers in humans has been extremely low and despite recent advances, objective response rates in DC vaccine trials are rarely above 10%. This lack of efficacy is due in part to immunosuppressive factors, such as transforming growth factor &beta (TGF-&beta), present in the tumor microenvironment that promote tumor immune escape. Therefore, TGF-&beta represents a major barrier to effective cancer immunotherapy and strategies to neutralize this cytokine may lead to more efficacious DC vaccines.In this study, we employed two small molecule transforming growth factor &beta receptor type I (T&betaRI/ALK5) kinase inhibitors (HTS466284 and SM16) in combination with DC vaccines to treat established TGF-&beta-secreting 4T1 mammary tumors. The results demonstrate that while both inhibitors blocked the effects of TGF-&beta in vitro, HTS466284 by itself or in combination with DC vaccination was unable to consistently control the growth and metastasis of established 4T1 tumors. In contrast, SM16 inhibited the growth of established tumors when delivered orally and suppressed the formation of pulmonary metastases when delivered orally or via daily intraperitoneal (i.p.) injection. The efficacy of SM16 was dependent on cellular immunity as this drug had no effect in immunodeficient SCID mice. Furthermore, orally delivered SM16 in combination with DC vaccination led to complete tumor regression in several mice that correlated with increased T cell infiltration of the primary tumor and enhanced in vitro IFN-gamma production and tumor-specific cytolytic activity by splenocytes. Finally, a suboptimal dose of SM16 that failed to control primary tumor growth on its own synergized with DC vaccination to inhibit the growth of established 4T1 tumors. These findings suggest that blockade of TGF-&beta signaling using a small molecule T&betaRI/ALK5 kinase antagonist may be an effective strategy to bolster the efficacy of DC-based cancer vaccines.

The dentate gyrus is a region that hosts most of the hippocampal cells in mammals. Nevertheless, its role in spatial memory remains poorly understood, especially in light of the recently-studied phenomenon of adult hippocampal neurogenesis and its possible role in aging and chronic brain disease. We found that chronic over-expression of transforming growth factor beta1 (TGFbeta1), a cytokine involved in neurodegenerative disease, results in several modifications of brain structure, including volumetric changes and persistent astrogliosis. Furthermore, TGFbeta1 over-expression affects the generation of new neurons, leading to an increased number of neurons in the dentate gyrus and deficits in spatial memory acquisition and storage in aged mice. Nonetheless, reducing neurogenesis via pharmacological treatment impairs spatial memory in juvenile mice but not in adult or aged mice. This suggests that the addition of new cells to hippocampal circuitry, and not the increased plasticity of these cells, is the most relevant role of neurogenesis in spatial memory. We tested this idea by modifying proliferation in the dentate gyrus at several ages using multiple techniques and evaluating the incorporation of newborn neurons into hippocampal circuitry. We found that all granule neurons, recently generated or not, have the same probability of being incorporated. Therefore, the number of new neurons participating in memory circuits is proportional to their availability. Our conclusion is that adult-generated cells have the same functional relevance as those generated during development. Together, our data show that the dentate gyrus is important for memory processing and that adult neurogenesis may be relevant to its functionality by optimizing the number of neurons for memory processing. The equilibrium between neurogenesis and optimal dentate gyrus size is disrupted when TGFbeta1 is chronically increased, which occurs in neurodegenerative pathologies, leading to cognitive impairment in aged animals.

The dentate gyrus is a region that hosts most of the hippocampal cells in mammals. Nevertheless, its role in spatial memory remains poorly understood, especially in light of the recently-studied phenomenon of adult hippocampal neurogenesis and its possible role in aging and chronic brain disease. We found that chronic over-expression of transforming growth factor beta1 (TGFbeta1), a cytokine involved in neurodegenerative disease, results in several modifications of brain structure, including volumetric changes and persistent astrogliosis. Furthermore, TGFbeta1 over-expression affects the generation of new neurons, leading to an increased number of neurons in the dentate gyrus and deficits in spatial memory acquisition and storage in aged mice. Nonetheless, reducing neurogenesis via pharmacological treatment impairs spatial memory in juvenile mice but not in adult or aged mice. This suggests that the addition of new cells to hippocampal circuitry, and not the increased plasticity of these cells, is the most relevant role of neurogenesis in spatial memory. We tested this idea by modifying proliferation in the dentate gyrus at several ages using multiple techniques and evaluating the incorporation of newborn neurons into hippocampal circuitry. We found that all granule neurons, recently generated or not, have the same probability of being incorporated. Therefore, the number of new neurons participating in memory circuits is proportional to their availability. Our conclusion is that adult-generated cells have the same functional relevance as those generated during development. Together, our data show that the dentate gyrus is important for memory processing and that adult neurogenesis may be relevant to its functionality by optimizing the number of neurons for memory processing. The equilibrium between neurogenesis and optimal dentate gyrus size is disrupted when TGFbeta1 is chronically increased, which occurs in neurodegenerative pathologies, leading to cognitive impairment in aged animals.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.