Obtaining and characterizing extracellular vesicles from human prostate stromal cells derived from patients with benign prostatic hyperplasia
Benign Prostatic Hyperplasia (BPH) is the result of excessive cellullar proliferation in the prostatic transition zone, that compresses the urethra, causing the symptoms of the disease. Evidence supports that atherogenic environment could contribute to the development and progression of BPH, increas...
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| Formato: | Artículo revista |
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Universidad Nacional Córdoba. Facultad de Ciencias Médicas. Secretaria de Ciencia y Tecnología
2022
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| Acceso en línea: | https://revistas.unc.edu.ar/index.php/med/article/view/38980 |
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I10-R327-article-38980 |
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ojs |
| institution |
Universidad Nacional de Córdoba |
| institution_str |
I-10 |
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R-327 |
| container_title_str |
Revista de la Facultad de Ciencias Médicas de Córdoba |
| format |
Artículo revista |
| topic |
extracellular vesicles prostatic stromal cells differential ultracentrifugation vesículas extracelulares Células estromales prostáticas Ultracentrifugación diferencial . |
| spellingShingle |
extracellular vesicles prostatic stromal cells differential ultracentrifugation vesículas extracelulares Células estromales prostáticas Ultracentrifugación diferencial . Roldán Gallardo, FF Solla , ED Lopez Seoane, M Maldonado, CA Quintar , AA Obtaining and characterizing extracellular vesicles from human prostate stromal cells derived from patients with benign prostatic hyperplasia |
| topic_facet |
extracellular vesicles prostatic stromal cells differential ultracentrifugation vesículas extracelulares Células estromales prostáticas Ultracentrifugación diferencial . |
| author |
Roldán Gallardo, FF Solla , ED Lopez Seoane, M Maldonado, CA Quintar , AA |
| author_facet |
Roldán Gallardo, FF Solla , ED Lopez Seoane, M Maldonado, CA Quintar , AA |
| author_sort |
Roldán Gallardo, FF |
| title |
Obtaining and characterizing extracellular vesicles from human prostate stromal cells derived from patients with benign prostatic hyperplasia |
| title_short |
Obtaining and characterizing extracellular vesicles from human prostate stromal cells derived from patients with benign prostatic hyperplasia |
| title_full |
Obtaining and characterizing extracellular vesicles from human prostate stromal cells derived from patients with benign prostatic hyperplasia |
| title_fullStr |
Obtaining and characterizing extracellular vesicles from human prostate stromal cells derived from patients with benign prostatic hyperplasia |
| title_full_unstemmed |
Obtaining and characterizing extracellular vesicles from human prostate stromal cells derived from patients with benign prostatic hyperplasia |
| title_sort |
obtaining and characterizing extracellular vesicles from human prostate stromal cells derived from patients with benign prostatic hyperplasia |
| description |
Benign Prostatic Hyperplasia (BPH) is the result of excessive cellullar proliferation in the prostatic transition zone, that compresses the urethra, causing the symptoms of the disease. Evidence supports that atherogenic environment could contribute to the development and progression of BPH, increasing its incidence and aggressiveness. The BPH hyperproliferative state requires coordinated communication between the different components that maintain a permissive microenvironment. Thus, extracellular vesicles (EVs) have become relevant as intercellular communication regulators in multiple processes. It has been described that oxidized-LDL molecule (OxLDL) would be involved in numerous signaling pathways; including signaling by EVs promoting cellullar proliferation. EVs, may contain diverse biomolecules that confer them functions in cellular communication. In addition, it has been reported that EVs derived from different cell types are capable to mediate proliferative and inflammatory effects.
In this context, we proposed as objectives: 1) to evaluate the effect of OxLDL, simulating an atherogenic state, on cell proliferation in primary cultures of human prostate stromal cells (HPSC) from patient samples (n=8) from the Sanatorio Allende of Córdoba; 2) to isolate EVs from primary cultures by differential ultracentrifugation and analyze their production and release into the medium in treatments with OxLDL vs. vehicle; 3) to morphologically characterize EVs by transmission electron microscopy (TEM) and confirm the identity and presence of exosomes, through CD63 immuno-staining using colloidal gold.
It was observed that OxLDL (20μM) produced a significant increase in cell proliferation rate compared to vehicle. EVs were obtained by differential ultracentrifugations (2k, 10k, 150k pellets) and visualized by TEM using negative staining. HPSC from patients with BPH showed a very low frequency of EVs released, with OxLDL inducing a 10-fold increase, especially in the 15-20nm fraction (p<0.001). Ultrastructurally, these EVs exhibited a spherical and concave appearance, compatible with exosomes. Therefore, they were positively verified by CD63 immunostaining.
Finally, we conclude that OxLDL would favor cell proliferation, increase and release of EVs, which would participate in cell communication and maintenance of the permissive environment, propitious to progression and increased aggressiveness of BPH. |
| publisher |
Universidad Nacional Córdoba. Facultad de Ciencias Médicas. Secretaria de Ciencia y Tecnología |
| publishDate |
2022 |
| url |
https://revistas.unc.edu.ar/index.php/med/article/view/38980 |
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I10-R327-article-389802024-04-15T16:14:45Z Obtaining and characterizing extracellular vesicles from human prostate stromal cells derived from patients with benign prostatic hyperplasia Obtención y caracterización de vesículas extracelulares a partir de células estromales prostáticas humanas derivadas de pacientes con hiperplasia prostática benigna . Roldán Gallardo, FF Solla , ED Lopez Seoane, M Maldonado, CA Quintar , AA extracellular vesicles prostatic stromal cells differential ultracentrifugation vesículas extracelulares Células estromales prostáticas Ultracentrifugación diferencial . Benign Prostatic Hyperplasia (BPH) is the result of excessive cellullar proliferation in the prostatic transition zone, that compresses the urethra, causing the symptoms of the disease. Evidence supports that atherogenic environment could contribute to the development and progression of BPH, increasing its incidence and aggressiveness. The BPH hyperproliferative state requires coordinated communication between the different components that maintain a permissive microenvironment. Thus, extracellular vesicles (EVs) have become relevant as intercellular communication regulators in multiple processes. It has been described that oxidized-LDL molecule (OxLDL) would be involved in numerous signaling pathways; including signaling by EVs promoting cellullar proliferation. EVs, may contain diverse biomolecules that confer them functions in cellular communication. In addition, it has been reported that EVs derived from different cell types are capable to mediate proliferative and inflammatory effects. In this context, we proposed as objectives: 1) to evaluate the effect of OxLDL, simulating an atherogenic state, on cell proliferation in primary cultures of human prostate stromal cells (HPSC) from patient samples (n=8) from the Sanatorio Allende of Córdoba; 2) to isolate EVs from primary cultures by differential ultracentrifugation and analyze their production and release into the medium in treatments with OxLDL vs. vehicle; 3) to morphologically characterize EVs by transmission electron microscopy (TEM) and confirm the identity and presence of exosomes, through CD63 immuno-staining using colloidal gold. It was observed that OxLDL (20μM) produced a significant increase in cell proliferation rate compared to vehicle. EVs were obtained by differential ultracentrifugations (2k, 10k, 150k pellets) and visualized by TEM using negative staining. HPSC from patients with BPH showed a very low frequency of EVs released, with OxLDL inducing a 10-fold increase, especially in the 15-20nm fraction (p<0.001). Ultrastructurally, these EVs exhibited a spherical and concave appearance, compatible with exosomes. Therefore, they were positively verified by CD63 immunostaining. Finally, we conclude that OxLDL would favor cell proliferation, increase and release of EVs, which would participate in cell communication and maintenance of the permissive environment, propitious to progression and increased aggressiveness of BPH. La Hiperplasia Prostática Benigna (HPB) surge a consecuencia de la proliferación celular excesiva de la zona de transición prostática que comprime la uretra, provocando la sintomatología propia de la enfermedad. Evidencias sostienen que el entorno aterogénico podrían contribuir al desarrollo y progresión de la HPB, aumentando su incidencia y agresividad. El estado hiperproliferativo de la HPB requiere una coordinada comunicación entre los diferentes componentes que mantienen un microambiente permisivo. Así, las vesículas extracelulares (EVs) han tomado relevancia como reguladores de comunicación intercelular en múltiples procesos. Se ha descrito que la molécula de LDL-oxidada (OxLDL) estaría involucrada en diversas vías de señalización; incluida la señalización por EVs promoviendo la proliferación celular. Las EVs pueden contener diversas biomoléculas que les confieren funciones en comunicación intercelular y, además, se reportaron efectos proliferativos e inflamatorios mediados por EVs derivadas de distintos tipos celulares. Es este contexto, propusimos como objetivos: 1) evaluar el efecto de OxLDL, simulando un estado aterogénico, sobre la proliferación celular en cultivos primarios de células estromales prostáticas humanas (HPSC) de muestras de pacientes (n=8) del Sanatorio Allende de Córdoba; 2) aislar EVs de cultivos primarios mediante ultracentrifugaciones diferenciales y analizar su producción y liberación al medio en tratamientos con OxLDL vs. vehículo; 3) caracterizar morfológicamente EVs mediante microscopía electrónica de transmisión (TEM) y confirmar la identidad y presencia de exosomas, a través de inmuno-marcación de CD63 utilizando oro coloidal. Se observó que OxLDL (20μM) produjo un aumento significativo en la tasa de proliferación celular respecto al vehículo. EVs fueron obtenidas mediante utracentrifugaciones diferenciales (pellets 2k, 10k, 150k) y visualizadas por TEM utilizando tinción negativa. Las HPSC de pacientes con HPB mostraron una frecuencia muy baja de EVs liberadas, con OxLDL induciendo un aumento de 10 veces, especialmente en la fracción 15-20nm (p<0,001). Ultraestructuralmente, estas EVs exhibieron una apariencia esférica y cóncava, compatible con exosomas; luego fueron verificados positivamente por inmuno-marcación de CD63. Finalmente, podemos concluir que OxLDL favorecería la proliferación celular, incremento y liberación de EVs, las cuales participarían en comunicación celular y mantención del ambiente permisivo propicio para la progresión y aumento de agresividad de la HPB. . Universidad Nacional Córdoba. Facultad de Ciencias Médicas. Secretaria de Ciencia y Tecnología 2022-10-26 info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion texto texto . https://revistas.unc.edu.ar/index.php/med/article/view/38980 Revista de la Facultad de Ciencias Médicas de Córdoba.; Vol. 79 No. Suplemento JIC XXIII (2022): Suplemento JIC XXIII Revista de la Facultad de Ciencias Médicas de Córdoba; Vol. 79 Núm. Suplemento JIC XXIII (2022): Suplemento JIC XXIII Revista da Faculdade de Ciências Médicas de Córdoba; v. 79 n. Suplemento JIC XXIII (2022): Suplemento JIC XXIII 1853-0605 0014-6722 http://creativecommons.org/licenses/by-nc/4.0 |