RESUMO
Vascular smooth muscle cells (VSMC) are highly specialized, and exhibit a contractile phenotype when mature and fully differentiated, being responsible for vessel homeostasis and blood pressure control. In response to pro-atherogenic stimuli VSMC alter their state of differentiation, increase proliferation and migration, resulting in SMC phenotypes ranging from contractile to synthetic. This variability is observed in cell morphology and expression level of marker genes for differentiation status. There is growing evidence that bone morphogenetic protein (BMP) signaling is involved in vascular diseases, including atherosclerosis. Here, we evaluated in vitro the role of specific agonists/antagonists belonging to the BMP pathway on dedifferentiation of VSMC harvested during early stages of atherosclerosis. RESULTS: Comparing primary VSMC isolated from aortas of susceptible ApoE-/- animals fed 8 weeks of western diet with their littermate controls fed usual diet, we observed that recombinant BMP4 was able to reduce SM22-alpha and alpha actin gene expression indicating dedifferentiation was under way. Unexpectedly, treatment with recombinant Gremlin-1, a known BMP antagonist, also reduced 4-6.5 folds gene expression of SM22-alpha, alpha-actin and, calponin, exclusively in VSMC from ApoE-/- animals, independently on the diet consumed. CONCLUSION: Our data show that BMP4 is capable of modulating of SM22-alpha and alpha actin gene expression, indicative of cell dedifferentiation in VSMC. Additionally, we report for first time that Gremlin-1 acts independently of the BMP pathway and selectively on VSMC from susceptible animals, reducing the expression of all genes evaluated.
Assuntos
Aterosclerose/patologia , Desdiferenciação Celular , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Animais , Aterosclerose/metabolismo , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Células Cultivadas , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fenótipo , Transdução de SinaisRESUMO
Mesenchymal stem cells (MSCs) though multipotent exhibit limited lifespan in vitro, with progressive reduction in capacity for self-renewal leading to irreversible arrest of cell division, which limits their use for therapeutic purposes. Human umbilical cord wall MSCs are easy to process and proliferate rapidly in culture, but variability of individual samples and impact upon in vitro expansion and aging processes is unknown. We compared isolation protocols to determine which one yields the highest number of viable cells with the best proliferation capacity. Three different protocols were tested: two were enzymatic procedures and one explant method. Isolated cells were evaluated in terms of proliferation, differentiation capacity, and phenotype. All samples were processed using one or more protocols. After passage 2 adherent cells displayed standard phenotypic and differentiation characteristics of MSCs, but our results show that isolating cells directly from Wharton's jelly is more advantageous. Cells obtained from explants presented similar characteristics to those from enzymatic protocols, but always reached proliferation arrest earlier, irrespective of initial population doubling times. From the same sample, cells obtained with enzymatic protocol ii reached later passages while exhibiting shorter doubling times in culture than cells from other protocols, that is, took longer to reach senescence. More important, each individual MSC sample exhibited different population doubling rates and reached senescence at different passages, irrespective of protocol. Thus, even when in strict conformity with procedures and quality control, each cord sample shows a unique behavior, a finding that should be taken into account when planning for therapeutic approaches.
Assuntos
Separação Celular/métodos , Células-Tronco Mesenquimais/citologia , Cordão Umbilical/citologia , Adipócitos/citologia , Adulto , Diferenciação Celular , Proliferação de Células , Forma Celular , Sobrevivência Celular , Células Cultivadas , Senescência Celular , Feminino , Humanos , Imunofenotipagem , Recém-Nascido , Masculino , Osteócitos/citologia , Gravidez , beta-Galactosidase/metabolismoRESUMO
OBJECTIVE: To calculate the positive likelihood ratio to determine whether telemedicine is able to optimize referral to the emergency department. METHODS: Unicenter study with 182 consecutive patients admitted to Hospital Israelita Albert Einstein due to respiratory symptoms. All patients were submitted to oxygen saturation measurement using the standard method Welch Allyn finger device vital sign monitor and a 2-minute evaluation (Binah.ai mobile application). The reproducibility of oxygen saturation measurements made with both methods was investigated using interclass correlation coefficients and analysis of dispersion. Bland-Altman plots were constructed and kappa concordance coefficients used to examine data normality. Accuracy was also estimated. RESULTS: Oxygen saturation measurement differences between methods were ≤2% in more than 85% of cases. The mean difference (bias) between methods was near zero (0.835; Bland-Altman analysis). Oxygen saturation measurements made using the Binah.ai mobile application had an average ability to detect patients with altered oxygen saturation levels compared to the conventional method (ROC analysis). The positive likelihood ratio of the mobile application was 6.23. CONCLUSION: Mobile applications for oxygen saturation measurement are accessible user-friendly tools with moderate impact on clinical telemedicine evaluation of patients with respiratory symptoms, and may optimize referral to the emergency department.
Assuntos
Aplicativos Móveis , Oxigênio , Humanos , Reprodutibilidade dos Testes , Oximetria/métodos , Curva ROCRESUMO
ABSTRACT Objective To calculate the positive likelihood ratio to determine whether telemedicine is able to optimize referral to the emergency department. Methods Unicenter study with 182 consecutive patients admitted to Hospital Israelita Albert Einstein due to respiratory symptoms. All patients were submitted to oxygen saturation measurement using the standard method Welch Allyn finger device vital sign monitor and a 2-minute evaluation (Binah.ai mobile application). The reproducibility of oxygen saturation measurements made with both methods was investigated using interclass correlation coefficients and analysis of dispersion. Bland-Altman plots were constructed and kappa concordance coefficients used to examine data normality. Accuracy was also estimated. Results Oxygen saturation measurement differences between methods were ≤2% in more than 85% of cases. The mean difference (bias) between methods was near zero (0.835; Bland-Altman analysis). Oxygen saturation measurements made using the Binah.ai mobile application had an average ability to detect patients with altered oxygen saturation levels compared to the conventional method (ROC analysis). The positive likelihood ratio of the mobile application was 6.23. Conclusion Mobile applications for oxygen saturation measurement are accessible user-friendly tools with moderate impact on clinical telemedicine evaluation of patients with respiratory symptoms, and may optimize referral to the emergency department.
RESUMO
Wharton's jelly mesenchymal stem cells (WJ-MSC) exhibit immunomodulatory effects on T cell response. WJ-MSC are easy to collect, process, and proliferate rapidly in culture, but information on the variability of individual cell samples impacting upon in vitro expansion, immunomodulatory potential, and aging processes is still lacking. We propose to evaluate the immunomodulatory cytokine profile and capacity to inhibit T cell proliferation of WJ-MSC progressing to replicative senescence in order to analyze if expected responses are affected. Our results show that the gene expression of immunomodulatory molecules varied among samples with no specific pattern present. In coculture, all WJ-MSC were capable of inhibiting mitogen-activated CD3+ T cell proliferation, although to different degrees, and each PBMC responded with a different level of inhibition. Thus, we suggest that each WJ-MSC displays unique behavior, differing in patterns of cytokine mRNA expression and immunomodulatory capacity. We believe that variability between samples may play a role in the effectiveness of WJ-MSC employed therapeutically.