Regulatory effects of bio-intensity electric field on transformation of human skin fibroblasts / 中华烧伤杂志

Objective: To investigate the regulatory effects of bio-intensity electric field on the transformation of human skin fibroblasts (HSFs). Methods: The experimental research methods were used. HSFs were collected and divided into 200 mV/mm electric field group treated with 200 mV/mm electric field for 6 h and simulated electric field group placed in the electric field device without electricity for 6 h. Changes in morphology and arrangement of cells were observed in the living cell workstation; the number of cells at 0 and 6 h of treatment was recorded, and the rate of change in cell number was calculated; the direction of cell movement, movement velocity, and trajectory velocity within 3 h were observed and calculated (the number of samples was 34 in the simulated electric field group and 30 in 200 mV/mm electric field group in the aforementioned experiments); the protein expression of α-smooth muscle actin (α-SMA) in cells after 3 h of treatment was detected by immunofluorescence method (the number of sample was 3). HSFs were collected and divided into simulated electric field group placed in the electric field device without electricity for 3 h, and 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group which were treated with electric fields of corresponding intensities for 3 h. Besides, HSFs were divided into simulated electric field group placed in the electric field device without electricity for 6 h, and electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group treated with 200 mV/mm electric field for corresponding time. The protein expressions of α-SMA and proliferating cell nuclear antigen (PCNA) were detected by Western blotting (the number of sample was 3). Data were statistically analyzed with Mann-Whitney U test, one-way analysis of variance, independent sample t test, and least significant difference test. Results: After 6 h of treatment, compared with that in simulated electric field group, the cells in 200 mV/mm electric field group were elongated in shape and locally adhered; the cells in simulated electric field group were randomly arranged, while the cells in 200 mV/mm electric field group were arranged in a regular longitudinal direction; the change rates in the number of cells in the two groups were similar (P>0.05). Within 3 h of treatment, the cells in 200 mV/mm electric field group had an obvious tendency to move toward the positive electrode, and the cells in simulated electric field group moved around the origin; compared with those in simulated electric field group, the movement velocity and trajectory velocity of the cells in 200 mV/mm electric field group were increased significantly (with Z values of -5.33 and -5.41, respectively, P<0.01), and the directionality was significantly enhanced (Z=-4.39, P<0.01). After 3 h of treatment, the protein expression of α-SMA of cells in 200 mV/mm electric field group was significantly higher than that in simulated electric field group (t=-9.81, P<0.01). After 3 h of treatment, the protein expressions of α-SMA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were 1.195±0.057, 1.606±0.041, and 1.616±0.039, respectively, which were significantly more than 0.649±0.028 in simulated electric field group (P<0.01). Compared with that in 100 mV/mm electric field group, the protein expressions of α-SMA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly increased (P<0.01). The protein expressions of α-SMA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were 0.730±0.032, 1.561±0.031, and 1.553±0.045, respectively, significantly more than 0.464±0.020 in simulated electric field group (P<0.01). Compared with that in electric field treatment 1 h group, the protein expressions of α-SMA in electric field treatment 3 h group and electric field treatment 6 h group were significantly increased (P<0.01). After 3 h of treatment, compared with that in simulated electric field group, the protein expressions of PCNA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 100 mV/mm electric field group, the protein expressions of PCNA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 200 mV/mm electric field group, the protein expression of PCNA of cells in 400 mV/mm electric field group was significantly decreased (P<0.01). Compared with that in simulated electric field group, the protein expressions of PCNA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were significantly decreased (P<0.01); compared with that in electric field treatment 1 h group, the protein expressions of PCNA of cells in electric field treatment 3 h group and electric field treatment 6 h group were significantly decreased (P<0.05 or P<0.01); compared with that in electric field treatment 3 h group, the protein expression of PCNA of cells in electric field treatment 6 h group was significantly decreased (P<0.01). Conclusions: The bio-intensity electric field can induce the migration of HSFs and promote the transformation of fibroblasts to myofibroblasts, and the transformation displays certain dependence on the time and intensity of electric field.

YAP activation promotes the transdifferentiation of cardiac fibroblasts to myofibroblasts in matrix remodeling of dilated cardiomyopathy

Yes-associated protein (YAP) is an important regulator of cellular proliferation and transdifferentiation. However, little is known about the mechanisms underlying myofibroblast transdifferentiation in dilated cardiomyopathy (DCM). We investigated the role of YAP in the pathological process of cardiac matrix remodeling. A classic model of DCM was established in BALB/c mice by immunization with porcine cardiac myosin. Cardiac fibroblasts were isolated from neonatal Sprague-Dawley rats by density gradient centrifugation. The expression levels of α-smooth muscle actin (α-SMA) and collagen volume fraction (CVF) were significantly increased in DCM mice. Angiotensin II (Ang II)-mediated YAP activation promoted the proliferation and transdifferentiation of neonatal rat cardiac fibroblasts, and this effect was significantly suppressed in the shRNA YAP + Ang II group compared with the shRNA Control + Ang II group in vitro (2.98±0.34 ×105 vs 5.52±0.82 ×105, P<0.01). Inhibition of endogenous Ang II-stimulated YAP improved the cardiac function by targeting myofibroblast transdifferentiation to attenuate matrix remodeling in vivo. In the valsartan group, left ventricular ejection fraction and fractional shortening were significantly increased compared with the DCM group (52.72±5.51% vs 44.46±3.01%, P<0.05; 34.84±3.85% vs 26.65±3.12%, P<0.01). Our study demonstrated that YAP was a regulator of cardiac myofibroblast differentiation, and regulation of YAP signaling pathway contributed to improve cardiac function of DCM mice, possibly in part by decreasing myofibroblast transdifferentiation to inhibit matrix remodeling.

Effects of renal dysfunction on healing of colonic anastomosis: experimental study in wistar rats / Efeitos da disfunção renal na cicatrização de anastomoses colônicas: estudo experimental em ratos wistar

ABSTRACT Background: Chronic kidney disease affects more than 500 million people worldwide. In this context, the uremic toxins present are related to worsening in tissue healing. Aim: Evaluate on healing of colonic anastomosis in uremic rats, serum and anatomopathological indicators, which may be related to the change tissue repair process. Methods: Twenty Wistar rats, were randomly separated into two groups. In the sham group they were submitted to 5/6 nephrectomy simulation in left kidney, simulation right nephrectomy, median laparotomy, colotomy and colorraphy. In the uremia group, they were submitted to 5/6 nephrectomy of the left kidney, total nephrectomy of the right kidney and median laparotomy, colotomy and colorraphy. Were collected for serum urea, creatinine and CRP dosages and the colonic segments were studied for evaluation of granulation tissue, collagen maturation, microvascular and myofibroblasts density, and cell viability. Through histochemical processing, microvascular density was evaluated by anti-CD34 monoclonal antibody marking, cell viability by cell proliferation nuclear antigen screening and myofibroblasts density with monoclonal anti-α-actin antibody. Computerized histometry was used for evaluations of collagens type I and III by the coloration of picrosirius. Results: The group submitted to nephrectomy 5/6, compared to the sham group, show urea increase (p<0.0000) and higher C reactive protein (p=0.0142). Decrease of granulation tissue formation (border reepithelialization p=0,0196, angiofibroblast proliferation p=0.0379), mean collagen I (p=0,0009) and collagen III (p=0,016), microvascular density (p=0,0074), cell proliferation nuclear antigen (p<0,0000) and myofibroblasts (p<0,0001). Conclusion: The uremia induced by nephrectomy 5/6 model establishes negative impact in the colonic wound healing.

RESUMO Racional: A doença renal crônica atinge mais de 500 milhões de pessoas em todo o mundo. Neste contexto, as toxinas urêmicas estão relacionadas ao comprometimento da cicatrização tecidual. Objetivo: Avaliar, na cicatrização de anastomoses colônicas de ratos urêmicos indicadores séricos e anatomopatológicos que possam estar relacionados com alteração do processo de reparação tissular. Métodos: Utilizaram-se 20 ratos Wistar divididos aleatoriamente em dois grupos. No grupo simulação eles foram submetidos à simulação da nefrectomia 5/6 do rim esquerdo, simulação de nefrectomia total do rim direito, laparotomia mediana, colotomia e colorrafia. No grupo uremia, eles foram submetidos à nefrectomia 5/6 do rim esquerdo, nefrectomia total do rim direito, laparotomia mediana, colotomia e colorrafia. Coletaram-se amostras de sangue para dosagens séricas da ureia, creatinina e proteína C reativa, e do cólon para processamentos histológicos e histoquímicos na avaliação do tecido de granulação, maturação de colágeno, densidade microvascular e de miofibroblastos, viabilidade celular cicatricial. Empregou-se a histometria computadorizada para as avaliações de colágenos tipos I e III, densidade microvascular pela marcação com anticorpo monoclonal anti-CD34, viabilidade celular pela pesquisa do antígeno nuclear de proliferação celular e a densidade de miofibroblastos com anticorpo monoclonal anti-α-actina. Resultados: O grupo submetido à nefrectomia 5/6, em comparação ao grupo simulação, demonstraram aumentos da ureia sérica (p<0,0000) e proteína C reativa (p=0,0142), redução da formação de tecido de granulação (reepitelização de bordas p=0,0196, proliferação angiofibroblástica p=0,0379), porcentagens de colágeno I (p=0,0009) e colágeno III (p=0,016), densidade microvascular (p=0,0074) e miofibroblastos (p<0,0001) e antígeno nuclear de proliferação celular (p<0,0000). Conclusão: A uremia induzida pelo modelo de nefrectomia 5/6 determina impacto negativo no processo de cicatrização colônico.

The Role of Mesothelial Cells in Liver Development, Injury, and Regeneration

Mesothelial cells (MCs) cover the surface of visceral organs and the parietal walls of cavities, and they synthesize lubricating fluids to create a slippery surface that facilitates movement between organs without friction. Recent studies have indicated that MCs play active roles in liver development, fibrosis, and regeneration. During liver development, the mesoderm produces MCs that form a single epithelial layer of the mesothelium. MCs exhibit an intermediate phenotype between epithelial cells and mesenchymal cells. Lineage tracing studies have indicated that during liver development, MCs act as mesenchymal progenitor cells that produce hepatic stellate cells, fibroblasts around blood vessels, and smooth muscle cells. Upon liver injury, MCs migrate inward from the liver surface and produce hepatic stellate cells or myofibroblast depending on the etiology, suggesting that MCs are the source of myofibroblasts in capsular fibrosis. Similar to the activation of hepatic stellate cells, transforming growth factor β induces the conversion of MCs into myofibroblasts. Further elucidation of the biological and molecular changes involved in MC activation and fibrogenesis will contribute to the development of novel approaches for the prevention and therapy of liver fibrosis.

Progress in myofibroblast and its application in forensic medicine / 法医学杂志

The myofibroblasts have dual characteristics of smooth muscle cells and fibroblasts. In repairing tissular wound, myofibroblasts are involved in fibrogenesis and remodeling the extracellular matrix of the fibrotic cascades reaction. The review describes the morphological characteristics and biological behaviors of myofibroblasts and the application of skin wound age determination, which may provide reference for research in forensic medicine.

Estudo imuno-histoquímico da presença de miofibroblastos e da expressão do fator transformador de crescimento-beta1, interferon gama, mataloproteinase de matriz 13 e indutor de metaloproteinases de matriz em lesões odontogênicas epiteliais / Immunohistochemical study of the presence of myofibroblasts and the expression of transforming growth factor-beta1, interferon gamma, mataloproteinase matrix 13 and matrix metalloproteinase inducer in odontogenic epithelial lesions

Os miofibroblastos são células que apresentam um fenótipo híbrido exibindo características morfológicas de fibroblastos e de células musculares lisas, sendo a aquisição de tal fenótipo denominada diferenciação, passando então a expressar a a-SMA, a qual é importante na identificação dessas células. Estudos têm sugerido que os miofibrobíastos apresentam relação com a agressividade de diversas lesões e que o seu processo de diferenciação estaria relacionado à expressão do TGF-pl e do IFN-y atuando, respectivamente, no estímulo e na inibição dessa diferenciação. O objetivo deste trabalho foi investigar o papel dos miofibroblastos em lesões odontogênicas epiteliais, relacionando-os à agressividade das lesões e analisar por meio da imuno-histoquímica. a expressão do TGF-pl e IFN-y no processo de diferenciação, além da análise da MMP-13 que é ativada por miofibroblastos e do indutor de metaloproteinases de matriz (EMMPRIN) como precursor desta MMP. A amostra foi constituída por 20 ameloblastomas sólidos, 10 ameloblastomas unicfsticos, 20 ceratocistos odontogênicos e 20 tumores odontogênícos adenomatóides. Para a avaliação dos miofibroblastos, foram quantificadas as células imunorreativas ao anticorpo a-SMA presentes no tecido conjuntivo, próximo ao tecido epitelial. As expressões de TGF-pl, IFN-y, MMP-13 e EMMPRIN, foram avaliadas no componente epitelial e no conjuntivo, estabelecendo-se o percentual de imunorreatividade e atribuindo-se escores de 0 a 4. A análise dos miofibroblastos evidenciou maior concentração nos ameloblastomas sólidos (média de 30,55), seguido pelos ceratocistos odontogênicos (22,50), ameloblastomas unicísticos (20,80) e tumores odontogênicos adenomatóides (19,15) com valor de p= 0,001. Não foi encontrada correlação significativa entre TGF-pl e IFN-y no processo de diferenciação dos miofibroblastos, bem como na relação entre a quantidade de miofibroblastos e a expressão da MMP-13. Constatou-se, correlação estatística entre MMP-13 e TGF-pi (r= 0,087; p= 0,011) além de significante correlação entre MMP-13 e IFN-y (r=0,348; p=0,003). Entre EMMPRÍN e MMP-13 verificou-se significância (r= 0,474; p<0,001) assim como entre EMMPRIN e IFN-y (r=0,393; p=0,001). A maior quantidade de miofibroblastos evidenciada nos ameloblastomas sólidos, ceratocistos odontogênicos e ameloblastomas unicísticos sugere que estas células podem ser um dos fatores responsáveis para um comportamento biológico mais agressivo destas lesões, embora a população de miofibroblastos não tenha apresentado correlação com TGF- -pi, IFN-y ,MMP-13 e EMMPRIN. Quanto a correlação evidenciada entre MMP-13 e TGF-pl, isto pode sugerir um papel indutor do TGF-pl para a expressão da MMP-13, assim como os resultados deste estudo reforçam a relação bem estabelecida do EMMPRIN como indutor da MMP-13. Constatou-se também relação entre EMMPRIN e IFN-y assim como entre MMP-13 e IFN-y sugerindo, dessa forma, um sinergismo na ação anti-fibrótica desses marcadores.

Myofibroblasts are cells that exhibit a hybrid phenotype, sharing the morphoíogical characteristics of fibroblasts and smooth muscle cells, which is acquired during a process called differentiation. These cells then start to express a-SMA, a marker that can be used for their identification. Studies suggest that myofibroblasts are related to the aggressiveness of different tumors and that TGF-pl and IFN-y play a role in myofibroblast differentiation, stimulating or inhibiting this differentiation, respectively. The objective of this study was to investigate the role of myofibroblasts in epithelial odontogenic tumors, correlating the presence of these cells with the aggressiveness of the tumor. Immunohistochemistry was used to evaluate the expression of TGF-pl and IFN-y in myofibroblast differentiation, as well as the expression of MMP-13, which is activated by myofibroblasts, and of EMMPRIN (extracellular matrix metalloproteinase inducer) as a precursor of this MMP. The sample consisted of 20 solid ameloblastomas, 10 unicystic ameloblastomas, 20 odontogenic keratocysts, and 20 adenomatoid odontogenic tumors. For evaluation of myofibroblasts, anti-a-SMA-immunoreactive cells were quantified in connective tissue close to the epithelium. Immunoexpression of TGF-pl, IFN-y, MMP-13 and EMMPRIN was evaluaíed in the epithelial and connective tissue components, attributing scores of 0 to 4. The results showed a higher concentration of myofibroblasts in solid ameloblastomas (mean of 30.55), followed by odontogenic keratocysts (22.50), unicystic ameloblastomas (20.80), and adenomatoid odontogenic tumors (19.15) (p=0.00). No significant correlation between TGF-pl and IFN-y was observed during the process of myofibroblast differentiation. There was also no correlation between the quantity of myofibroblasts and MMP-13 expression. Significant correlations were found between MMP-13 and TGF-pi (r=0.087; p=0.01 1), between MMP-13 and ÍFN-y (r=0.348; p=0.003), as well as between EMMPRIN and MMP-13 (r=0.474; /xO.001) and between EMMPRIN and IFN-y (r=0.393; p=0.00). The higher quantity of myofibroblasts observed in solid ameloblastomas, odontogenic keratocysts and unicystic ameloblastomas suggests that these cells are one of the factors responsible for the more aggressive biological behavior of these tumors, although the myofibroblast population was not correlated with TGF-01, IFN-y, MMP-13 or EMMPRIN. The correlation between MMP-13 and TGF-pl suggests that the latter induces the expression of this metalloproteinase. The present results also support the well-established role of EMMPRIN as an inducer of MMP-13. Furthermore, the relationship between EMMPRIN and IFN-y and between MMP-13 and IFN-y suggests synergism in the antifibrotic effect of these markers.