RESUMEN
Fatty acid synthase (FASN) is responsible for the endogenous production of fatty acids from acetyl-CoA and malonyl-CoA. Its overexpression is associated with poor prognosis in human cancers including melanomas. Our group has previously shown that the inhibition of FASN with orlistat reduces spontaneous lymphatic metastasis in experimental B16-F10 melanomas, which is a consequence, at least in part, of the reduction of proliferation and induction of apoptosis. Here, we sought to investigate the effects of pharmacological FASN inhibition on lymphatic vessels by using cell culture and mouse models. The effects of FASN inhibitors cerulenin and orlistat on the proliferation, apoptosis, and migration of human lymphatic endothelial cells (HDLEC) were evaluated with in vitro models. The lymphatic outgrowth was evaluated by using a murine ex vivo assay. B16-F10 melanomas and surgical wounds were produced in the ears of C57Bl/6 and Balb-C mice, respectively, and their peripheral lymphatic vessels evaluated by fluorescent microlymphangiography. The secretion of vascular endothelial growth factor C and D (VEGF-C and -D) by melanoma cells was evaluated by ELISA and conditioned media used to study in vitro lymphangiogenesis. Here, we show that cerulenin and orlistat decrease the viability, proliferation, and migration of HDLEC cells. The volume of lymph node metastases from B16-F10 experimental melanomas was reduced by 39% in orlistat-treated animals as well as the expression of VEGF-C in these tissues. In addition, lymphatic vessels from orlistat-treated mice drained more efficiently the injected FITC-dextran. Orlistat and cerulenin reduced VEGF-C secretion and, increase production of VEGF-D by B16-F10 and SK-Mel-25 melanoma cells. Finally, reduced lymphatic cell extensions, were observed following the treatment with conditioned medium from cerulenin- and orlistat-treated B16-F10 cells. Altogether, our results show that FASN inhibitors have anti-metastatic effects by acting on lymphatic endothelium and melanoma cells regardless the increase of lymphatic permeability promoted by orlistat.
Asunto(s)
Cerulenina/farmacología , Ácido Graso Sintasas/antagonistas & inhibidores , Lactonas/farmacología , Vasos Linfáticos/efectos de los fármacos , Melanoma Experimental/prevención & control , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Inhibidores de la Síntesis de Ácidos Grasos/farmacología , Humanos , Linfangiogénesis/efectos de los fármacos , Metástasis Linfática , Vasos Linfáticos/metabolismo , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Orlistat , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor C de Crecimiento Endotelial Vascular/metabolismo , Factor D de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: Angiogenesis is an early stage of psoriatic lesion development, but less is known about lymphagiogenesis and its role in the development of psoriasis. OBJECTIVE: To examine the expression of specific lymphatic markers and lymphatic growth factors in untreated psoriatic skin, in the unaffected skin of patients and skin of healthy volunteers, as well as their alteration after treatment with an anti-TNF agent. METHODS: Immunohistochemistry for the lymphatic markers D2-40 and LYVE-1, in addition to the VEGF-C and VEGF-D growth factors, was performed in the skin biopsies of psoriatic lesions and adjacent non-psoriatic skin of 19 patients before and after treatment with etanercept, as well as in the skin biopsies of 10 healthy volunteers. RESULTS: The expressions of D2-40, VEGF-C and VEGF-D on lymphatic vessels underwent statistically significant increases in untreated psoriatic skin compared with non-lesional skin, in contrast to LYVE-1, which did not involve significant increase in expression in psoriatic skin. VEGF-C expression on lymphatic vessels diminished after treatment with etanercept. Moreover VEGF-C and VEGF-D staining on fibroblasts presented with higher expression in lesional skin than in non-lesional adjacent skin. CONCLUSION: Remodeling of lymphatic vessels possibly occurs during psoriatic lesion development, parallel to blood vessel formation. The exact role of this alteration is not yet clear and more studies are necessary to confirm these results. .
Asunto(s)
Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Anticuerpos Monoclonales de Origen Murino/análisis , Vasos Linfáticos/patología , Psoriasis/tratamiento farmacológico , Factores de Necrosis Tumoral/antagonistas & inhibidores , Factores de Crecimiento Endotelial Vascular/análisis , Proteínas de Transporte Vesicular/análisis , Anticuerpos Monoclonales de Origen Murino/efectos de los fármacos , Biopsia , Biomarcadores/análisis , Inmunohistoquímica , Inmunoglobulina G/uso terapéutico , Factores Inmunológicos/uso terapéutico , Linfangiogénesis/efectos de los fármacos , Vasos Linfáticos/efectos de los fármacos , Psoriasis/metabolismo , Psoriasis/patología , Valores de Referencia , Receptores del Factor de Necrosis Tumoral/uso terapéutico , Estadísticas no Paramétricas , Piel/efectos de los fármacos , Piel/patología , Factores de Crecimiento Endotelial Vascular/efectos de los fármacos , Proteínas de Transporte Vesicular/efectos de los fármacosRESUMEN
BACKGROUND: Angiogenesis is an early stage of psoriatic lesion development, but less is known about lymphagiogenesis and its role in the development of psoriasis. OBJECTIVE: To examine the expression of specific lymphatic markers and lymphatic growth factors in untreated psoriatic skin, in the unaffected skin of patients and skin of healthy volunteers, as well as their alteration after treatment with an anti-TNF agent. METHODS: Immunohistochemistry for the lymphatic markers D2-40 and LYVE-1, in addition to the VEGF-C and VEGF-D growth factors, was performed in the skin biopsies of psoriatic lesions and adjacent non-psoriatic skin of 19 patients before and after treatment with etanercept, as well as in the skin biopsies of 10 healthy volunteers. RESULTS: The expressions of D2-40, VEGF-C and VEGF-D on lymphatic vessels underwent statistically significant increases in untreated psoriatic skin compared with non-lesional skin, in contrast to LYVE-1, which did not involve significant increase in expression in psoriatic skin. VEGF-C expression on lymphatic vessels diminished after treatment with etanercept. Moreover VEGF-C and VEGF-D staining on fibroblasts presented with higher expression in lesional skin than in non-lesional adjacent skin. CONCLUSION: Remodeling of lymphatic vessels possibly occurs during psoriatic lesion development, parallel to blood vessel formation. The exact role of this alteration is not yet clear and more studies are necessary to confirm these results.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/análisis , Vasos Linfáticos/patología , Psoriasis/tratamiento farmacológico , Inhibidores del Factor de Necrosis Tumoral , Factores de Crecimiento Endotelial Vascular/análisis , Proteínas de Transporte Vesicular/análisis , Adulto , Anticuerpos Monoclonales de Origen Murino/efectos de los fármacos , Biomarcadores/análisis , Biopsia , Etanercept , Femenino , Humanos , Inmunoglobulina G/uso terapéutico , Inmunohistoquímica , Factores Inmunológicos/uso terapéutico , Linfangiogénesis/efectos de los fármacos , Vasos Linfáticos/efectos de los fármacos , Masculino , Persona de Mediana Edad , Psoriasis/metabolismo , Psoriasis/patología , Receptores del Factor de Necrosis Tumoral/uso terapéutico , Valores de Referencia , Piel/efectos de los fármacos , Piel/patología , Estadísticas no Paramétricas , Factores de Crecimiento Endotelial Vascular/efectos de los fármacos , Proteínas de Transporte Vesicular/efectos de los fármacosRESUMEN
BACKGROUND: Angiogenesis and lymphangiogenesis are the processes of neovascularization that evolve from preexisting blood and lymphatic vessels. There are few studies on angiogenesis and none on lymphangiogenesis in leprosy. Thus, the role of neovascularization in the pathophysiological mechanisms of the disease was studied across the spectrum of leprosy, its reactional states and its residual lesions. METHODOLOGY/PRINCIPAL FINDINGS: Seventy-six biopsies of leprosy skin lesions and seven healthy controls were selected. Fifty-five serum samples were used for the detection of CD105 by ELISA. Histological sections were stained with antibodies against CD31 (blood and lymphatic vessels), D2-40/podoplanin (lymphatic vessels), and CD105/endoglin (neovessels). Microvessels were counted in 100 high-power fields (400x) and the number of vessels was evaluated in relation to the extension of the inflammatory infiltrate (0-3), to the bacillary index (0-6) and to the clinical forms. Angiogenesis, as marked by CD31 and CD105, was observed across the leprosy spectrum, compared with the controls. Additionally, there was a positive correlation between these markers with extension of the infiltrate (p <0.0001). For D2/40, lymphangiogenesis was observed in the tuberculoid form (p <0.0001). There was no statistical significance for values of CD105 detected in plasma by ELISA. CONCLUSIONS/SIGNIFICANCE: Angiogenesis is present across the spectrum of leprosy and in its reactional forms. The increase in the number of vessels, as detected by CD31 and CD105 staining, is related to the extension of the inflammatory infiltrate. Samples from reactional lesions have a higher number of CD31+ and CD105+ stained vessels, which indicates their involvement in the pathophysiological mechanisms of the reactional states. The regression of lesions is accompanied by the regression of neovascularization. Drugs inhibiting angiogenesis may be relevant in the treatment of leprosy, in addition to multidrugtherapy, and in the prevention of the development of reactions.
Asunto(s)
Lepra/tratamiento farmacológico , Lepra/fisiopatología , Neovascularización Patológica/metabolismo , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD/metabolismo , Biopsia , Estudios de Casos y Controles , Niño , Endoglina , Femenino , Humanos , Inflamación/metabolismo , Inflamación/fisiopatología , Linfangiogénesis/efectos de los fármacos , Masculino , Glicoproteínas de Membrana/metabolismo , Microcirculación , Persona de Mediana Edad , Neovascularización Patológica/tratamiento farmacológico , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Receptores de Superficie Celular/metabolismo , Estudios Retrospectivos , Piel/patología , Adulto JovenRESUMEN
BACKGROUND: Angiogenesis and lymphangiogenesis are the processes of neovascularization that evolve from preexisting blood and lymphatic vessels. There are few studies on angiogenesis and none on lymphangiogenesis in leprosy. Thus, the role of neovascularization in the pathophysiological mechanisms of the disease was studied across the spectrum of leprosy, its reactional states and its residual lesions. METHODOLOGY/PRINCIPAL FINDINGS: Seventy-six biopsies of leprosy skin lesions and seven healthy controls were selected. Fifty-five serum samples were used for the detection of CD105 by ELISA. Histological sections were stained with antibodies against CD31 (blood and lymphatic vessels), D2-40/podoplanin (lymphatic vessels), and CD105/endoglin (neovessels). Microvessels were counted in 100 high-power fields (400x) and the number of vessels was evaluated in relation to the extension of the inflammatory infiltrate (0-3), to the bacillary index (0-6) and to the clinical forms. Angiogenesis, as marked by CD31 and CD105, was observed across the leprosy spectrum, compared with the controls. Additionally, there was a positive correlation between these markers with extension of the infiltrate (p <0.0001). For D2/40, lymphangiogenesis was observed in the tuberculoid form (p <0.0001). There was no statistical significance for values of CD105 detected in plasma by ELISA. CONCLUSIONS/SIGNIFICANCE: Angiogenesis is present across the spectrum of leprosy and in its reactional forms. The increase in the number of vessels, as detected by CD31 and CD105 staining, is related to the extension of the inflammatory infiltrate. Samples from reactional lesions have a higher number of CD31+ and CD105+ stained vessels, which indicates their involvement in the pathophysiological mechanisms of the reactional states. The regression of lesions is accompanied by the regression of neovascularization. Drugs inhibiting angiogenesis may be relevant in the treatment of leprosy, in addition to multidrugtherapy, and in the prevention of the development of reactions.
Asunto(s)
Humanos , Masculino , Femenino , Niño , Adolescente , Adulto , Persona de Mediana Edad , Anciano , Anciano de 80 o más Años , Adulto Joven , Piel/patología , Biopsia , Glicoproteínas de Membrana/metabolismo , Antígenos CD/metabolismo , Estudios de Casos y Controles , Estudios Retrospectivos , Receptores de Superficie Celular/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Linfangiogénesis/efectos de los fármacos , Endoglina , Inflamación/fisiopatología , Inflamación/metabolismo , Lepra/fisiopatología , Lepra/tratamiento farmacológico , Microcirculación , Neovascularización Patológica/metabolismo , Neovascularización Patológica/tratamiento farmacológicoRESUMEN
Although the antiinflammatory and antiangiogenic properties of dexamethasone and acetylsalicylic acid have been studied extensively, their effects on lymphangiogenesis in regenerating tissues remain mostly unknown. We studied in rats the pharmacological modulation and the effect of a remote inflammatory stimulus on the lymphatic regeneration upon damage after a surgical procedure. A micronized purified flavonoid fraction bearing antiinflammatory and lymphagogue properties, was also used. An incisional wound and interruption of the afferent lymphatic vessels to the popliteal and axillary lymph nodes of adult rats were made in dorsal thigh and hypochondrium, respectively. The progress of lymphatic regeneration was evaluated 3, 7, 14 and 21 days after surgery. (99m)Tc-dextran lymphoscintigraphy and Evans blue dye uptake were used to evaluate the lymphatic flow and the kinetics of lymphatic regeneration. In control conditions, lymphatic regeneration took 14 days to be accomplished. In the presence of a remote inflammatory response, which conceivably yielded inflammatory mediators to the incised lymphatic vessels, that time was shortened to 7 days. In both conditions, lymphatic regeneration was inhibited by dexamethasone and acetylsalicylic acid and accelerated by the micronized purified flavonoid fraction. These findings indicate that lymphatic regeneration in an incisional wound may be significantly modulated by dexamethasone, aspirin and a micronized purified flavonoid fraction, and these results call our attention for the possibility to pharmacologically stimulate the recovery of a lymphatic failure due to a traumatic event, or to inhibit its function in order to limit the lymphatic spread of cytokines or neoplastic cells.