Embryonic stem cells overexpressing high molecular weight FGF2 isoform enhance recovery of pre-ganglionic spinal root lesion in combination with fibrin biopolymer mediated root repair.

BACKGROUND: Spinal ventral root avulsion results in massive motoneuron degeneration with poor prognosis and high costs. In this study, we compared different isoforms of basic fibroblast growth factor 2 (FGF2), overexpressed in stably transfected Human embryonic stem cells (hESCs), following motor root avulsion and repair with a heterologous fibrin biopolymer (HFB). METHODS: In the present work, hESCs bioengineered to overexpress 18, 23, and 31 kD isoforms of FGF2, were used in combination with reimplantation of the avulsed roots using HFB. Statistical analysis was conducted using GraphPad Prism software with one-way or two-way ANOVA, followed by Tukey's or Dunnett's multiple comparison tests. Significance was set at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. RESULTS: For the first set of experiments, rats underwent avulsion of the ventral roots with local administration of HFB and engraftment of hESCs expressing the above-mentioned FGF2 isoforms. Analysis of motoneuron survival, glial reaction, and synaptic coverage, two weeks after the lesion, indicated that therapy with hESCs overexpressing 31 kD FGF2 was the most effective. Consequently, the second set of experiments was performed with that isoform, so that ventral root avulsion was followed by direct spinal cord reimplantation. Motoneuron survival, glial reaction, synaptic coverage, and gene expression were analyzed 2 weeks post-lesion; while the functional recovery was evaluated by the walking track test and von Frey test for 12 weeks. We showed that engraftment of hESCs led to significant neuroprotection, coupled with immunomodulation, attenuation of astrogliosis, and preservation of inputs to the rescued motoneurons. Behaviorally, the 31 kD FGF2 - hESC therapy enhanced both motor and sensory recovery. CONCLUSION: Transgenic hESCs were an effective delivery platform for neurotrophic factors, rescuing axotomized motoneurons and modulating glial response after proximal spinal cord root injury, while the 31 kD isoform of FGF2 showed superior regenerative properties over other isoforms in addition to the significant functional recovery.

Impaired proliferation and migration of HUVEC and melanoma cells by human anti-FGF2 mAbs derived from a murine hybridoma by guided selection.

Disadvantages of using murine monoclonal antibodies (mAb) in human therapy, such as immunogenicity response, led to the development of technologies to transform murine antibodies into human antibodies. The murine anti-FGF2 3F12E7 mAb was proposed as a promising agent to treat metastatic melanoma tumors; once it blocks the FGF2, responsible for playing a role in tumor growth, angiogenesis, and metastasis. Considering the therapeutic potential of anti-FGF2 3F12E7 mAb and its limited use in humans due to its origin, we used this antibody as the template for a guided selection humanization technique to obtain human anti-FGF2 mAbs. Three Fab libraries (murine, hybrid, and human) were constructed for humanization. The libraries were phage-displayed, and the panning was performed against recombinant human FGF2 (rFGF2). The selected human variable light and heavy chains were cloned into AbVec vectors for full-length IgG expression into HEK293-F cells. Surface plasmon resonance analyses showed binding to rFGF2 of seven mAbs out of 20 expressed. Assays performed with these mAbs resulted in two that showed proliferation reduction and cell migration attenuation of HUVEC and SK-Mel-28 melanoma cells. In-silico analyses predicted that these two human anti-FGF2 mAbs interact with FGF2 at a similar patch of residues than the chimeric anti-FGF2 antibody, comprehending a region within the heparin-binding domains of FGF2, essential for its function. These results are comparable to those achieved by the murine anti-FGF2 3F12E7 mAb and showed success in the humanization process and selection of two human mAbs with the potential to inhibit undesirable FGF2 roles.

The guided selection humanization process enabled the production of 20 human mAbs anti-FGF2;Seven human anti-FGF2 mAbs showed binding to the rFGF2 antigen in the SPR binding assay;Two human anti-FGF2 mAbs inhibited the proliferation and migration of HUVEC and SK-Mel-28 cells and were predicted to contact the FGF2 at a similar patch of residues than the original mAb.

Impaired proliferation and migration of HUVEC and melanoma cells by human anti-FGF2 mAbs derived from a murine hybridoma by guided selection

Disadvantages of using murine monoclonal antibodies (mAb) in human therapy, such as immunogenicity response, led to the development of technologies to transform murine antibodies into human antibodies. The murine anti-FGF2 3F12E7 mAb was proposed as a promising agent to treat metastatic melanoma tumors; once it blocks the FGF2, responsible for playing a role in tumor growth, angiogenesis, and metastasis. Considering the therapeutic potential of anti-FGF2 3F12E7 mAb and its limited use in humans due to its origin, we used this antibody as the template for a guided selection humanization technique to obtain human anti-FGF2 mAbs. Three Fab libraries (murine, hybrid, and human) were constructed for humanization. The libraries were phage-displayed, and the panning was performed against recombinant human FGF2 (rFGF2). The selected human variable light and heavy chains were cloned into AbVec vectors for full-length IgG expression into HEK293-F cells. Surface plasmon resonance analyses showed binding to rFGF2 of seven mAbs out of 20 expressed. Assays performed with these mAbs resulted in two that showed proliferation reduction and cell migration attenuation of HUVEC and SK-Mel-28 melanoma cells. In-silico analyses predicted that these two human anti-FGF2 mAbs interact with FGF2 at a similar patch of residues than the chimeric anti-FGF2 antibody, comprehending a region within the heparin-binding domains of FGF2, essential for its function. These results are comparable to those achieved by the murine anti-FGF2 3F12E7 mAb and showed success in the humanization process and selection of two human mAbs with the potential to inhibit undesirable FGF2 roles.

Wound healing effect of a one-week Aloe Vera mouthwash: a pilot study / Efeito de reparação tecidual de uma semana de enxaguante bucal de Aloe Vera: um estudo piloto

Aloe Vera, a perennial Liliaceae plant, has medical, cosmetic, and wound-healing properties. Aloe vera has antioxidant, anti-cancer, anti-diabetic, and regenerative effects. Glucommannan increases collagen synthesis and aids healing after ginivectomy treatment. Natural mouthwashes may offer gingival wound healing efficacy with reduced side-effects when compared to Chlorhexidine. Objective: the objective of this clinical study was to compare the effects on wound healing of a one-week Aloe vera mouthwash with chlorhexidine mouthwash before gingivectomy surgical therapy. Material and Methods:a total of 45 individuals experiencing inflammatory gingival enlargement were included in the study. They underwent professional mechanical plaque removal and were then randomly divided into three groups. In group I, comprising 15 patients, participants were advised to utilize 100% Aloe vera juice as a mouthwash twice daily. Group II, also consisting of 15 patients, was instructed to use Chlorhexidine (0.2%) mouthwash twice daily. The Control group, which consisted of 15 patients, was recommended to use a placebo mouth rinse in addition to mechanical plaque removal. During the second visit, which occurred one week after the initial visit, the enlarged gingival tissue was surgically removed through scalpel gingivectomy. Immunohistochemical (IHC) analysis was performed on the excised tissue to measure the l evels of fibroblast growth factor-2. Results: when compared to the control group, Aloe vera showed significant differences regarding the expression of fibroblast growth factor-2(FGF-2), and highly significant differences in angiogenesis. At the same time, there were substantial differences in angiogenesis w ith no significant differences in the expression of FGF2 between Chlorhexidine and control groups. Conclusion: aloe vera has exhibited potential wound-healing effects as i t s ignificantly affected the IHC expression of FGF2 and angiogenesis when used as an adjunct to plaque control before gingivectomy surgical therapy (AU)

Aloe Vera, uma planta perene de Liliaceae, tem propriedades médicas, cosméticas e cicatrizantes. Aloe vera tem efeitos antioxidantes, anticancerígenos, antidiabéticos e regenerativos. O glucomanano aumenta a síntese de colágeno e auxilia na cicatrização a pós o tratamento de gengivectomia. Enxaguatórios bucais naturais podem oferecer efi cácia na reparação de feridas gengivais com efeitos colaterais reduzidos quando comparados à clorexidina. Objetivo:O objetivo deste estudo clínico foi comparar os efeitos na cicatrização de feridas de uma semana de enxaguatório bucal de Aloe vera com clorexidina antes da terapia cirúrgica de gengivectomia. Material e Métodos: um total de 45 indivíduos com aumento gengival inflamatório foram incluídos no estudo. Eles foram submetidos à remoção mecânica profissional da placa e foramdivididos aleatoriamente em três grupos. No grupo I, composto por 15 pacientes, os participantes foram orientados a utilizar 100% de suco de Aloe vera como enxaguante bucal duas vezes ao dia. O grupo II, também composto por 15 pacientes, foi instruído a usar enxaguante bucal com clorexidina (0,2%) duas vezes ao dia. O grupo controle, composto por 15 pacientes, foi recomendado o uso de enxaguatório bucal placebo além da remoção mecânica da placa. Durante a segunda visita, que ocorreu uma semana após a visita inicial, o tecido gengival aumentado foi removido cirurgicamente por meio de gengivectomia com bisturi. A análise imuno-histoquímica (IHC) foi realizada no tecido excisado para medir os níveis do fator de crescimento de fibroblastos-2 (FGF-2). Resultados: quando comparado ao grupo controle, o Aloe vera apresentou diferenças significativas em relação àexpressão do FGF-2, e diferenças altamente significativas na angiogênese. Ao mesmo tempo, houve diferenças substanciais na angiogênese, sem diferenças significativas na expressão de FGF-2 entre a clorexidina e os grupos controle. Conclusão: Aloe vera exibiu potenciais efeitos de cicatrização de feridas, pois afetou significativamente a expressão IHC de FGF-2 e a angiogênese quando usada como adjuvante no controle de placa antes da terapia cirúrgica de gengivectomia (AU)

The effect of scaffold hydroxyapatite derived from portunus pelagicus shell on the expression of fibroblast growth factor-2 (fgf-2) and bone morphogenetic proteins-2 (bmp -2) in the extraction sockets of cavia cobaya / El efecto del scaffold de hidroxiapatita derivada de portunus pelagicus sobre la expresión del factor de crecimiento de fibroblastos-2 (FGF-2) y las proteínas morfogenéticas óseas-2 (BMP-2) en los alvéolos dentarios de cavia cobaya

Objetive: To determine the expression of Fibroblast Growth Factor (FGF)-2 and Bone Morphogenetic Protein (BMP)-2 after application of scaffold hydroxyapatite from Rajungan crab shell (Portunus pelagicus) in the tooth extraction socket of Cavia cobaya. Material and Methods: This study used a post-test only control group design with 28 Cavia cobaya separated into two groups, control and treatment group. The left mandibular incisor was extracted, and socket preservation was conducted. A hydroxyapatite graft derived from crab shells was mixed with gelatin and eventually turned into a scaffold, which was afterward put into the extraction socket. After 7 days and 14 days, each group was terminated and examined using immunohistochemical staining to observe the expression of FGF-2 and BMP-2. One-Way Anova and Tukey HSD were used to examine the research data. Results: FGF-2 and BMP-2 expressions were observed higher in the group that received hydroxyapatite scaffold at the post-extraction socket than those in the group that did not receive hydroxyapatite scaffold. Conclusion: The application of a hydroxyapatite scaffold from Rajungan crab shell (Portunus pelagicus) to the tooth extraction socket can increase FGF-2 and BMP-2 expression.

Objetivo: Determinar la expresión del factor de crecimiento de fibroblastos (FGF)-2 y la proteína morfogenética ósea (BMP)-2 después de la aplicación de hidroxiapatita de andamio de caparazón de cangrejo Rajungan (Portunus pelagicus) en el alvéolo de extracción dental de Cavia cobaya. Material y Métodos: Este estudio utilizó un diseño de grupo de control solo posterior a la prueba con 28 Cavia cobaya separados en dos grupos, grupo de control y grupo de tratamiento. Se extrajo el incisivo mandibular izquierdo y se realizó la preservación del alvéolo. Un injerto de hidroxiapatita derivado de caparazones de cangrejo se mezcló con gelatina y se convirtió en un andamio, que luego se colocó en el alvéolo de extracción. Después de 7 días y 14 días, se terminó cada grupo y se examinó mediante tinción inmunohistoquímica para observar la expresión de FGF-2 y BMP-2. Se utilizaron One-Way Anova y Tukey HSD para examinar los datos de la investigación. Resultados: Las expresiones de FGF-2 y BMP-2 se observaron más altas en el grupo que recibió la estructura de hidroxiapatita en el alvéolo posterior a la extracción que en el grupo que no recibió la estructura de hidroxiapatita. Conclusión: La aplicación de un andamio de hidroxiapatita de caparazón de cangrejo Rajungan (Portunus pelagicus) al alvéolo de extracción dental puede aumentar la expresión de FGF-2 y BMP-2.

Efecto del factor de crecimiento fibroblástico dos (FGF-2), en la reducción de la senescencia en células madre mesenquimales aisladas de gelatina de Wharton / Fibroblastic growth factor two (FGF-2), in reducing the senescent effect on mesenchymal stem cell isolated from Wharton ́s jelly

Resumen Introducción. Las células madre mesenquimales han generado interés en la ingeniería de tejidos, debido a sus propiedades proliferativas y capacidad de reparación de tejidos, sin embargo, para un trasplante exitoso, es necesario aumentar el número de células mediante un cultivo in-vitro. Durante este proceso la capacidad proliferativa disminuye, provocando cambios en la morfología y funcionalidad celular y afectando la viabilidad del cultivo, este estado se conoce como senescencia celular y como posibles causales, se ha considerado el estrés oxidativo y la falta de factores de crecimiento. Objetivos: Evaluar el efecto de FGF-2 sobre la senescencia de un cultivo de células madre mesenquimales aisladas de gelatina de Wharton y su papel en la regulación del estrés oxidativo. Metodología. Se añadieron dosis de 3,5 y 7,5 ng de FGF-2 al cultivo. Durante los pasajes 5 y 7, se estimó tanto la senescencia celular como la presencia de ROS (especies reactivas de oxígeno). Resultados.Se obtuvo en el pasaje 5, una diferencia significativa del 99,5% entre el control (+) con respecto a los tratamientos con FGF-2, sin embargo, en el pasaje 7 se observó un aumento en la producción de la enzima ß-galactosidasa y cambios morfológicos, confirmando un estado senescente en el cultivo en todos los tratamientos evaluados. Conclusión. Las dosis utilizadas en este estudio contribuyeron positivamente a disminuir el proceso senescente en el cultivo celular, además se determinó, que el FGF-2 puede prolongar el tiempo de cultivo, retardando parcialmente la concentración de especies reactivas de oxígeno

AbstractIntroduction. Mesenchymal stem cells have been generated interest in tissue engineering, due to their proliferative properties and tissue repair capacity, however, for a successful transplant process, it is necessary to increase the number of cells in a culture expansion process. During this process the proliferative capacity is limited, causing changes in cell morphology and functionality affecting the viability of the culture, this state is known as cell senescence. Oxidative stress and deregulation of growth factors are considered as reasons. Aims. To evaluate the effect of FGF-2 on the senescence of a mesenchymal stem cells culture isolated from Wharton ́s jelly and its role in the regulation of oxidative stress. Methodology: 3,5 and 7,5 ng doses of FGF-2 were added to the culture medium from passage 2, then the senescence of the culture was evaluated and the presence of reactive oxygen species was determined during passages 5 and 7. Results. We observed that in passage 5, there is a significant difference 99.5% between the control (+) concerning the FGF-2 treatments, however, in passage 7, an increase in the production of the enzyme ß-galactosidase was observed and changes in morphology such as: increase in size and elongated shape of the cell, confirming a senescent state on the culture in all the treatments evaluated. Conclusion. The doses used in this study contributed positively to decrease this process in a cell culture, also, the FGF- 2 can prolong the cultivation time, partially decreasing the concentration of reactive oxygen species

Peri-implant health after supportive mucositis therapy is associated with increased levels of FGF-2

Abstract This study aimed to analyze Fibroblast Growth Factor-2 (FGF-2) levels in the peri-implant crevicular fluid throughout supportive mucositis therapy. Twenty-six participants with Branemark protocol prosthesis were divided into two groups: the control group, characterized by healthy peri-implants, and the mucositis group, presenting a diagnosis of peri-implant mucositis. All participants underwent clinical examination, radiographic analysis, prosthesis removal, and non-invasive peri-implant therapy (mechanical debridement associated with chlorhexidine 0.12%) during a period of 36 days divided into three intervals. Peri-implant crevicular fluid samples were collected at each interval in order to analyze FGF-2 levels by immuno-enzymatic assay. The control and mucositis groups showed difference in keratinized mucosa. The smaller the range of keratinized mucosa the higher susceptibility of peri-implant mucositis. Throughout the treatment intervals, participants were diagnosed in different groups indicating whether or not the non-invasive therapy was able to treat peri-implant mucositis. There was a significant difference of FGF-2 levels between groups, with the higher FGF-2 levels in the control group (p=0.01). After supportive therapy, the mucositis group showed significantly increased FGF-2 levels (p<0.01) compared to initial levels. After 36 days of supportive therapy, there was a reduction of peri-implant mucositis from 70% to 23%. Clinical and laboratory outcomes showed a clear correlation since FGF-2 levels increased after 36 days. It was concluded that the therapy protocol was effective and promoted a regenerative reaction and FGF-2 can be considered a future target for peri-implant mucositis understanding.

Resumo Este estudo teve como objetivo analisar os níveis de FGF-2 no fluido crevicular peri-implantar durante a terapia de suporte da mucosite. Vinte e seis participantes com prótese protocolo Branemark foram divididos em dois grupos: o grupo controle, caracterizado por saúde peri-implanter, e o grupo mucosite, apresentando diagnóstico de mucosite peri-implantar. Todos os participantes foram submetidos a exame clínico, análise radiográfica, retirada da prótese e terapia não invasiva peri-implantar (debridamento mecânico associado à clorexidina 0,12%) durante um período de 36 dias, dividido em três intervalos. Amostras de fluido crevicular peri-implantar foram coletadas em cada intervalo para análise dos níveis de FGF-2, por ensaio imunoenzimático. Os grupos controle e mucosite não apresentaram diferença nos parâmetros clínicos, exceto para mucosa queratinizada. Ao longo dos intervalos de tratamento, os participantes foram diagnosticados em diferentes grupos, indicando se a terapia não invasiva era ou não capaz de tratar a mucosite peri-implantar. Houve diferença significativa dos níveis de FGF-2 entre os grupos, sendo os níveis de FGF-2 maiores no grupo controle (p = 0.01). Após a terapia de suporte, o grupo com mucosite apresentou níveis de FGF-2 significativamente aumentados (p <0.01) em comparação aos níveis iniciais. Após 36 dias de terapia de suporte, houve redução da mucosite peri-implantar de 70% para 23%. Os resultados clínicos e laboratoriais mostraram uma correlação clara, uma vez que os níveis de FGF-2 aumentaram após 36 dias. O protocolo de terapia foi eficaz e promoveu uma reação regenerativa. O FGF-2 pode ser considerado um alvo futuro para o tratamento da mucosite peri-implantar.

Could the photobiomodulation therapy induce angiogenic growth factors expression from dental pulp cells?

This study aimed to evaluate the effect of different photobiomodulation (PBM) radiant exposures on the viability, proliferation, and gene expression of pulp fibroblasts from human primary teeth (HPF) involved in the pulp tissue repair. HPF were irradiated with Laser InGaAlP (Twin Flex Evolution, MMOptics®) at 660-nm wavelength (red); single time, continuous mode, 0.04-cm2 laser tip area, and 0.225-cm laser tip diameter, keeping the distance of 1 mm between the laser beam and the cell culture. The doses used were between 1.2 and 6.2 J/cm2 and were evaluated at the 6 h, 12 h, and 24 h after PBM. MTT and crystal violet assays evaluated the cell viability and proliferation. RT-PCR verified VEGF and FGF-2 mRNA expression. A blinded examiner analyzed the data through two-way ANOVA followed by Tukey test (p < 0.05). The groups with higher powers (10 mW, 15 mW, 20 mW, and 25 mW), shortest application periods (10 s), and radiant exposures between 2.5 and 6.2 J/cm2 exhibited statistically higher viability than that of the groups with small power (5 mW), longer application period (50 s), and radiant exposure of 6.2 J/cm2 (p < 0.05). VEGF and FGF-2 mRNA expression were observed at the three evaluated periods (6 h, 12 h, and 24 h) and the highest expression was in the shortest period (p < 0.05). All radiant exposures maintained HPF viable. The period of 6 h after irradiation showed statistically greater gene expression for both growth factors than other periods. VEGF mRNA had no differences among the dosimetries studied. The best radiant exposures for FGF-2 gene expression were 2.5 J/cm2 and 3.7 J/cm2.

Photobiomodulation effect on angiogenic proteins produced and released by dental pulp cells.

OBJECTIVE: To verify the photobiomodulation effect on angiogenic proteins produced and released by dental human pulpal fibroblasts (HPFs). MATERIAL AND METHODS: HPFs were irradiated with 660-nm low-level laser at fluences of 2.5 J/cm2 and 3.7 J/cm2. The control group was not irradiated. MTT, crystal violet, and ELISA assays respectively verified viability, proliferation, and angiogenic protein (supernatant/lysate) at 6 h, 12 h, and 24 h after photobiomodulation. Capillary-like structure formation assay verified functional role. Two-way ANOVA/Tukey's test and ANOVA/Bonferroni's multiple comparisons test respectively verified cell viability/proliferation and intragroup and intergroup comparisons of protein synthesis (p < 0.05). RESULTS: Irradiated and non-irradiated HPFs showed statistically similar cell viability and proliferation pattern. Intragroup comparisons showed similar patterns of protein synthesis for all groups: VEGF-A, VEGF-C, and vascular endothelial growth factor receptor 1 (VEGFR1) increased significantly in the supernatant, while FGF-2 and VEGF-A increased significantly in the lysate. The lower fluence significantly increased BMP-9 (6 h) in the supernatant and VEGFR1 (6 h and 12 h) and VEGF-D (24 h) in the lysate, while the higher fluence significantly increased BMP-9 (6 h) in the supernatant and VEGFR1 (12 h) in the lysate. Regardless of the time, both fluences statistically downregulated placental growth factor (PLGF) and PDGF secretion. Both fluences statistically decreased VEGF-A secretion (24 h) and PLGF production (6 h). CONCLUSION: Photobiomodulation produced stimulatory effects on angiogenic protein secretion by pulp fibroblasts. In terms of photobiomodulation, over time, both fluences significantly increased the secretion of VEGF-A, VEGF-C, and VEGFR1 and significantly upregulated BMP-9 (6 h) in the supernatant; for capillary-like structure formation, the fluence of 2.5 J/cm2 was better than the fluence of 3.7 J/cm2. CLINICAL RELEVANCE: This study results addressed effective photobiomodulation parameters tailored for pulp angiogenesis.

Does photobiomodulation change the synthesis and secretion of angiogenic proteins by different pulp cell lineages?

This study aimed to compare the synthesis and secretion of VEGF-A, VEGF-C, VEGF-D, VEGFR1, VEGFR2, and FGF-2 between pulp fibroblasts from human primary teeth (HPF) and stem cell from human deciduous teeth (SHED) before and after photobiomodulation. HPF were obtained from explant technique and characterized by immunohistochemistry, while SHED were obtained from digestion technique and characterized by flow cytometry. HPF (control group) and SHED were plated, let to adhere, and put on serum starvation to synchronize the cell cycles prior to photobiomodulation. Then, both cell lineages were irradiated with 660-nm laser according to the following groups: 2.5 and 3.7 J/cm2. MTT and crystal violet assays respectively verified viability and proliferation. ELISA Multiplex Assay assessed the following proteins: VEGF-A, VEGF-C, VEGF-D, VEGFR1, VEGFR2, FGF-2, at 6, 12, and 24 h after photobiomodulation, in supernatant and lysate. Two-way ANOVA/Tukey test evaluated cell viability and proliferation, while angiogenic production and secretion values were analyzed by one-way ANOVA (P < .05). Statistically similar HPF and SHED viability and proliferation patterns occurred before and after photobiomodulation (P > .05). HPF exhibited statistically greater values of all angiogenic proteins than did SHED, at all study periods, except for FGF-2 (supernatant; 12 h); VEGFR1 (lysate; non-irradiated; 12 h); and VEGFR1 (lysate; non-irradiated; 24 h). Photobiomodulation changed the synthesis and secretion of angiogenic proteins by HPF. HPF produced and secreted greater values of all tested angiogenic proteins than did SHED before and after irradiation with both energy densities of 2.5 and 3.7 J/cm2.

MiR-424 overexpression protects alveolar epithelial cells from LPS-induced apoptosis and inflammation by targeting FGF2 via the NF-κB pathway.

Acute respiratory distress syndrome (ARDS) is a multifactorial, inflammatory lung injury disease with high morbidity and mortality. However, the underlying pathogenic mechanism remains unknown. In this study, lipopolysaccharide (LPS)-stimulated alveolar epithelial cells were used to mimic the inflammatory pathogenesis of ARDS in vitro. We here investigated the role of miR-424 in LPS-stimulated alveolar epithelial cells and found it to be substantially downregulated. Overexpression of miR-424 inhibited apoptosis and inflammation in LPS-stimulated alveolar epithelial cells, and the miR-424 inhibitor exhibited the opposite effect. A bioinformatic analysis revealed a potential binding site of miR-424 in the 3'-UTR of fibroblast growth factor 2 (FGF2). A luciferase reporter assay suggested that miR-424 targeted FGF2 in alveolar epithelial cells. The level of FGF2 protein was inhibited by miR-424 mimic, whereas was significantly upregulated after miR-424 suppression in LPS-stimulated alveolar epithelial cells. MiR-424 also exhibited the protective role in LPS-induced apoptosis and inflammation by directly targeting FGF2 via the NF-κB pathway. In conclusion, our results demonstrate that miR-424 had a protective role in LPS-induced apoptosis and inflammation of alveolar epithelial cells by targeting FGF2 via regulating NF-κB pathway. This might contribute novel evidence to help identify a therapeutic target for treating ARDS.

Effects of subcutaneous injection of ozone during wound healing in rats.

Fibroblast growth factor 2 (FGF2) regulates the wound repair process and it is secreted by inflammatory and endothelial cells, and by myofibroblasts. This study aimed to establish the expression patterns of FGF2 and myofibroblastic differentiation during wound healing in rats treated with subcutaneous ozone injection. We created full-thickness excisional wounds in rats, and the healing process was analyzed through morphometric analyses and digital quantification of immunoreactivity of smooth muscle actin and FGF2. Ozone therapy-treated wounds presented granulation tissue with a reduced number of inflammatory cells and greater dermal cellularity, and intense collagen deposition. FGF2 immunoreactivity, microvessel density, and amount of myofibroblasts were significantly higher in treated wounds compared to controls. In conclusion, it was demonstrated that subcutaneous injections of ozone accelerate and ameliorate wound repairing process. Moreover, injectable ozone therapy's action mechanism may be associated with FGF2 overexpression.

Effects of the extracorporeal shock wave therapy on the skin: an experimental study.

Extracorporeal shock wave therapy (ESWT) has been extensively studied for its multiple biological properties, and although it is widely applied in esthetical procedures, little is known about its effects on the epidermis and dermis. In this study, a histological and immunohistochemical study of the effects of ESWT was performed on rat skin. Forty-five female rats were treated with one or two sessions of ESWT and sacrificed on days 1, 7, 14, and 21 after treatment. The samples were histologically processed and then morphometric analyses were performed to assess the epidermis, dermis, and subcutaneous fat tissue thickness. Immunohistochemical reactions were also performed against the antibodies: basic fibroblastic growth factor (FGF2), its receptor (FGFR1), and α-smooth muscle actin. Slides were scanned and digitally assessed, to determine the microvessel density (MVD) and digital scoring of the immunohistochemical staining. The results showed that ESWT produced a significantly higher collagen content, MVD, and epidermis and dermis thickness than the control, non-treated group. Both in epidermis and dermis, FGF2 was overexpressed in the ESWT-treated groups, whereas FGFR1 was increased only in the group treated with two ESWT sessions at 21-days post-treatment. The ESWT-treated groups have also shown diminished thickness of subcutaneous fat tissue. In conclusion, ESWT induces neocollagenesis and neoangiogenesis, and upregulates the FGF2 expression, particularly in the groups treated with two sessions. Furthermore, it was demonstrated that overexpression of FGF2 on skins treated with ESWT seems to be a key role on its mechanism of action.

Expansion and phenotypic changes of mouse bone marrow mesenchymal cells cultured with FGF-2 and facial nerve-conditioned medium / Expansión y cambios fenotípicos de células mesenquimales de médula ósea de ratón cultivadas con FGF-2 y medio condicionado por el nervio facial

Mesenchymal cells (MCs) exhibit great regenerative potential due to their intrinsic properties and ability to restore tissue function, either directly through transdifferentiation or indirectly through paracrine effects. This study aimed to evaluate morphometric and phenotypic changes in MCs grown with facial nerve-conditioned medium in the presence or absence of fibroblast growth factor 2 (FGF-2). For quantitative phenotypic analysis, the expression of GFAP, OX-42, MAP-2, β-tubulin III, NeuN, and NF-200 was analyzed by immunocytochemistry. Cells cultured with facial nerve-conditioned medium in the presence of FGF-2 expressed GFAP, OX-42, MAP-2, β-tubulin III, NeuN, and NF-200. On average, the area and perimeter of GFAP-positive cells were higher in the group cultured with facial nerve-conditioned medium compared to the group cultured with conditioned medium and FGF-2 (p=0.0001). This study demonstrated the plasticity of MCs for neuronal and glial lineages and opens up new research perspectives in cell therapy and trans.differentiation.

Las células mesenquimales (CM) exhiben un gran potencial regenerativo debido a sus propiedades intrínsecas y la capacidad de restaurar la función del tejido, ya sea directamente, a través de la transdiferenciación, o indirectamente, a través de efectos parácrinos. Este estudio tuvo como objetivo evaluar los cambios morfométricos y fenotípicos en CM cultivadas con medio condicionado por nervio facial en presencia o ausencia de factor de crecimiento de fibroblastos 2 (FGF-2). Para el análisis fenotípico cuantitativo, se analizó la expresión de GFAP, OX-42, MAP-2, β-tubulina III, NeuN y NF-200 mediante inmunocitoquímica. Las células cultivadas con medio condicionado por el nervio facial en presencia de FGF-2 expresaban GFAP, OX-42, MAP-2, β-tubulina III, NeuN y NF-200. En promedio, el área y el perímetro de las células positivas para GFAP fueron mayores en el grupo cultivado con medio condicionado por el nervio facial en comparación con el grupo cultivado con medio acondicionado y FGF-2 (p = 0,0001). Este estudio demostró la plasticidad de CM para linajes neuronales y gliales y abre nuevas perspectivas de investigación en terapia celular y transdiferenciación.

Deficiency of fibroblast growth factor 2 (FGF-2) leads to abnormal spermatogenesis and altered sperm physiology.

In previous studies, we described the presence of fibroblast growth factor 2 (FGF-2) and its receptors (FGFRs) in human testis and sperm, which are involved in spermatogenesis and in motility regulation. The aim of the present study was to analyze the role of FGF-2 in the maintenance of sperm physiology using FGF-2 knockout (KO) mice. Our results showed that in wild-type (WT) animals, FGF-2 is expressed in germ cells of the seminiferous epithelium, in epithelial cells of the epididymis, and in the flagellum and acrosomal region of epididymal sperm. In the FGF-2 KO mice, we found alterations in spermatogenesis kinetics, higher numbers of spermatids per testis, and enhanced daily sperm production compared with the WT males. No difference in the percentage of sperm motility was detected, but a significant increase in sperm concentration and in sperm head abnormalities was observed in FGF-2 KO animals. Sperm from KO mice depicted reduced phosphorylation on tyrosine residues (a phenomenon that was associated with sperm capacitation) and increased acrosomal loss after incubation under capacitating conditions. However, the FGF-2 KO males displayed no apparent fertility defects, since their mating with WT females showed no differences in the time to delivery, litter size, and pup weight in comparison with WT males. Overall, our findings suggest that FGF-2 exerts a role in mammalian spermatogenesis and that the lack of FGF-2 leads to dysregulated sperm production and altered sperm morphology and function. FGF-2-deficient mice constitute a model for the study of the complex mechanisms underlying mammalian spermatogenesis.

Extracellular calcium increases fibroblast growth factor 2 gene expression via extracellular signal-regulated kinase 1/2 and protein kinase A signaling in mouse dental papilla cells

Abstract We previously reported that elevated extracellular calcium (Ca2+) levels increase bone morphogenetic protein 2 expression in human dental pulp (hDP) cells. However, it is unknown whether extracellular Ca2+ affects the expression of other growth factors such as fibroblast growth factor 2 (FGF2). Objective: The present study aimed to examine the effect of extracellular Ca2+ on FGF2 gene expression in hDP and immortalized mouse dental papilla (mDP) cells. Materials and Methods: Cells were stimulated with 10 mM CaCl2 in the presence or absence of cell signaling inhibitors. FGF2 gene expression was assessed using real-time polymerase chain reaction. The phosphorylation status of signaling molecules was examined by Western blotting. Results: Extracellular Ca2+ increased FGF2 gene expression in mDP and hDP cells. Gene expression of the calcium-sensing receptor and G protein-coupled receptor family C group 6 member A, both of which are extracellular Ca2+ sensors, was not detected. Ca2+-mediated Fgf2 expression was reduced by pretreatment with the protein kinase A (PKA) inhibitor H-89 or extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 but not by pretreatment with the protein kinase C inhibitor GF-109203X or p38 inhibitor SB203580. Extracellular Ca2+ increased PKA activity and ERK1/2 phosphorylation. Ca2+-induced PKA activity decreased by pretreatment with PD98059. Conclusions: These findings indicate that elevated extracellular Ca2+ levels led to increased Fgf2 expression through ERK1/2 and PKA in mDP cells and that this mechanism may be useful for designing regenerative therapies for dentin.

Fibroblast growth factor 2 (FGF2) is present in human spermatozoa and is related with sperm motility. The use of recombinant FGF2 to improve motile sperm recovery.

Fibroblast growth factors (FGFs) and their receptors (FGFRs) regulate several functions of somatic cells. In a previous work, we reported FGFR expression in human spermatozoa and their involvement in motility. This study aimed to evaluate the presence and localization of fibroblast growth factor 2 (FGF2) in human spermatozoa, to determine the relationship of FGF2 levels with conventional semen parameters and to assess the effect of recombinant FGF2 (rFGF2) on sperm recovery in a selection procedure. Western immunoblotting analysis using an antibody against FGF2 revealed an 18-kDa band in sperm protein extracts. The protein was immunolocalized in the sperm flagellum and acrosomal region, as well as in all germ cells. Sperm FGF2 levels, assessed by flow cytometry, showed a positive (p < 0.05) correlation with sperm concentration, motility, total sperm number and total motile cells per ejaculate. Moreover, samples with abnormal motility depicted diminished (p < 0.01) FGF2 levels compared to those with normal motility. Spermatozoa exposed to rFGF2 bound the protein, exhibited higher (p < 0.05) total and motile sperm recoveries, and increased (p < 0.01) kinematic parameters after the swim-up. Findings herein presented lead to consider sperm FGF2 level as a potential marker of sperm quality, and rFGF2 as a supplement for improving sperm recovery in selection techniques.

Transgenic human embryonic stem cells overexpressing FGF2 stimulate neuroprotection following spinal cord ventral root avulsion.

Ventral root avulsion (VRA) triggers a strong glial reaction which contributes to neuronal loss, as well as to synaptic detachment. To overcome the degenerative effects of VRA, treatments with neurotrophic factors and stem cells have been proposed. Thus, we investigated neuroprotection elicited by human embryonic stem cells (hESC), modified to overexpress a human fibroblast growth factor 2 (FGF-2), on motoneurons subjected to VRA. Lewis rats were submitted to VRA (L4-L6) and hESC/FGF-2 were applied to the injury site using a fibrin scaffold. The spinal cords were processed to evaluate neuronal survival, synaptic stability, and glial reactivity two weeks post lesion. Then, qRT-PCR was used to assess gene expression of ß2-microglobulin (ß2m), TNFα, IL1ß, IL6 and IL10 in the spinal cord in vivo and FGF2 mRNA levels in hESC in vitro. The results indicate that hESC overexpressing FGF2 significantly rescued avulsed motoneurons, preserving synaptic covering and reducing astroglial reactivity. The cells were also shown to express BDNF and GDNF at the site of injury. Additionally, engraftment of hESC led to a significant reduction in mRNA levels of TNFα at the spinal cord ventral horn, indicating their immunomodulatory properties. Overall, the present data suggest that hESC overexpressing FGF2 are neuroprotective and can shift gene expression towards an anti-inflammatory environment.

Growth factors and cox2 in wound healing: an experimental study with ehrlich tumors / Avaliação dos fatores de crescimento e cox-2 na cicatrização de camundongos com neoplasia de ehrlich

ABSTRACT Background: Healing is an innate biological phenomenon, and carcinogenesis acquired, but with common humoral and cellular elements. Carcinogenesis interferes negatively in healing. Aim: To evaluate the histological changes in laparotomy scars of healthy Balb/c mice and with an Ehrlich tumor in its various forms of presentation. Methods: Fifty-four mice were divided into three groups of 18 animals. First group was the control; the second had Ehrlich tumor with ascites; and the third had the subcutaneous form of this tumor. Seven days after tumor inoculation, all 54 mice were submitted to laparotomy. All of the animals in the experiment were operated on again on 7th day after surgery, with resection of the scar and subsequent euthanasia of the animal. The scars were sent for histological assessment using immunohistochemical techniques to evaluate Cox-2 (cyclooxygenase 2), VEGF (vascular endothelial growth factor) and FGF (fibroblast growth factor). Semi-quantitatively analysis was done in the laparotomy scars and in the abdominal walls far away from the site of the operation. Results: Assessing the weight of the animals, the correct inoculation of the tumor and weight gain in the group with tumoral ascites was observed. The histological studies showed that groups with the tumor showed a statistically significant higher presence of Cox-2 compared to the control. In the Cox-2 analysis of the abdominal wall, the ascites group showed the most significant difference. VEGF did not present any significant differences between the three groups, regardless of the site. The FGF showed a significant increase in animals with the tumor. Conclusion: Histological findings in both laparotomy scar and the abdominal wall showed that with Ehrlich's neoplasia there was an exacerbated inflammatory response, translated by more intense expression of Cox-2 and greater fibroblast proliferation, translated by more intense expression of FGF, that is, it stimulated both the immediate inflammatory reactions, observed with Cox-2 reactions, and late scarring by fibroblasts and FGF.

RESUMO Racional: A cicatrização é fenômeno biológico inato, e a carcinogênese adquirido, mas com elementos humorais e celulares comuns. A carcinogênese interfere de forma negativa na cicatrização. Objetivo: Avaliar as modificações histológicas nas cicatrizes laparotômicas de camundongos Balb/c sadios como controles, e com a neoplasia de Ehrlich, em suas diferentes formas de apresentação. Métodos: Foram utilizados 54 camundongos, divididos em três grupos de 18 animais cada um. O primeiro era controle; o segundo com a neoplasia de Ehrlich em sua forma ascítica; e o terceiro na forma subcutânea. Sete dias após a inoculação do tumor, todos os 54 camundongos foram submetidos à laparotomia e reoperados no sétimo dia de pós-operatório, com ressecção da cicatriz e posterior eutanásia. As cicatrizes foram encaminhadas para estudo histológico com técnicas imunoistoquímicas para avaliar Cox-2 (ciclo-oxigenase 2), VEGF (fator de crescimento do endotélio vascular) e FGF (fator de crescimento dos fibroblastos) e analisadas de forma semiquantitativana tanto na cicatriz laparotômica como na parede abdominal mais distante do local operado. Resultados: Avaliando o peso, observou-se a correta inoculação do tumor e o aumento de peso no grupo com a neoplasia na modalidade ascítica. Os estudos histológicos mostraram que os grupos com a neoplasia apresentaram maior presença da Cox-2 em relação ao controle, estatisticamente significante. No estudo da Cox-2 da parede abdominal foi o local em que o grupo ascítico apresentou a diferença mais expressiva. O VEGF não apresentou diferenças significantes entre os três grupos, independentemente do local estudado. O FGF teve aumento significante nos animais com neoplasia. Conclusão: Os achados histológicos encontrados tanto na cicatriz das laparotomias quanto na parede abdominal mostraram que com a neoplasia de Ehrlich houve resposta inflamatória exacerbada, traduzida por expressão mais intensa da Cox-2 e maior proliferação fibroblástica, traduzida por expressão mais intensa do FGF, ou seja, estimulou tanto as reações inflamatórias imediatas, observadas nas reações da Cox-2, como nas cicatriciais tardias com os fibroblastos e o FGF.

Combined effects of melatonin and FGF-2 on mouse preosteoblast behavior within interconnected porous hydroxyapatite ceramics - in vitro analysis

ABSTRACT Objective Biocompatible materials such as interconnected porous hydroxyapatite ceramics (IP-CHA) loaded with osteogenic cells and bioactive agents are part of an evolving concept for overcoming craniofacial defects by use of artificial bone tissue regeneration. Amongst the bioactive agents, melatonin (MEL) and basic fibroblast growth factor (FGF-2) have been independently reported to induce osteoblastic activity. The present in vitro study was undertaken to examine the relationship between these two bioactive agents and their combinatory effects on osteoblastic activity and mineralization in vitro. Material and Methods Mouse preosteoblast cells (MC3T3-E1) were seeded and cultured within cylindrical type of IP-CHA block (ø 4x7 mm) by vacuum-assisted method. The IP-CHA/MC3T3 composites were subjected to FGF-2 and/or MEL. The proliferation assay, alkaline phosphatase enzyme activity (ALP), mRNA expressions of late bone markers, namely Osteocalcin (OCN) and Osteopontin (OPN), and Alizarin Red staining were examined over a period of 7 days. Results FGF-2 mainly enhanced the proliferation of MC3T3-E1 cells within the IP-CHA constructs. MEL mainly induced the mRNA expression of late bone markers (OCN and OPN) and showed increased ALP activity of MC3T3 cells cultured within IP-CHA construct. Moreover, the combination of FGF-2 and MEL showed increased osteogenic activity within the IP-CHA construct in terms of cell proliferation, upregulated expressions of OCN and OPN, increased ALP activity and mineralization with Alizarin Red. The synergy of the proliferative potential of FGF-2 and the differentiation potential of MEL showed increased osteogenic activity in MC3T3-E1 cells cultured within IP-CHA constructs. Conclusion These findings indicate that the combination of FGF-2 and MEL may be utilized with biocompatible materials to attain augmented osteogenic activity and mineralization.