Efecto del Medio Condicionado de la Línea Celular H9C2 Tratada con Dehidroepiandrosterona y Expuesta a Daño sobre la Motilidad de Células Troncales Mesenquimales / Effect of the Conditioned Medium from H9C2 Cell Line Treated with Dehydroepiandrosterone and Exposed to Damage on the Motility of Mesenchymal Stem Cells

RESUMEN: Las enfermedades cardiovasculares (ECV) son la principal causa de muerte a nivel mundial, donde la terapia con Células Troncales Mesenquimales (CTM) representa una alternativa para los pacientes que no logran recuperarse con los tratamientos actuales. El lograr que las CTM residentes se movilicen al órgano afectado representaría una ventaja para el manejo terapéutico de las ECV. La dehidroepiandrosterona (DHEA) es un precursor hormonal cuyos niveles disminuyen a lo largo de la vida, lo que se ha asociado al desarrollo de ECV. Diversos estudios han demostrado que el consumo de DHEA previene y mejora la condición cardiaca, aunque no se sabe si esto ocurre porque se ejerce un efecto en los cardiomiocitos y estos, a su vez, hacia las CTM. El objetivo del presente estudio fue determinar el efecto del medio condicionado procedente de la línea H9C2 pretratada con DHEA y sometida a daño, sobre la motilidad de CTM, llevando a cabo un ensayo de cierre de herida. El pretratamiento con DHEA y el daño en la línea H9C2, promueve la motilidad de CTM. El estímulo de la motilidad de CTM por un efecto indirecto de DHEA podría ser una estrategia terapéutica para el daño cardiaco.

ABSTRACT: Cardiovascular diseases (CVD) are the leading cause of death worldwide. Mesenchymal Stem Cell (MSC) therapy is an alternative for patients who cannot recover with current treatments. Ensure movilization of MSC to the affected organs would represent an advantage for therapeutic management of CVD. Dehydroepiandrosterone (DHEA) is a hormone precursor whose levels decrease throughout life, which has been associated with the onset of CVD. Several studies have shown that DHEA consumption, prevents and improves heart condition, although it is not known if this is because an effect on cardiomyocytes is exercised on these cells and this, in turn, to CTM. The aim of this study was to determine the effect of conditioned medium from H9C2 cell line pretreated with DHEA and subjected to damage, on the motility of CTM, performing a wound healing assay. Pretreatment with DHEA and damage to H9C2 cell line, promotes motility of CTM. Stimulation of CTM motility by an indirect effect of DHEA could be a therapeutic strategy for heart damage.

Efecto de la dehidroepiandroesterona y el acetónido de triamcinolona sobre la línea celular 3T3-L1 / Effect of dehydroepiandroesterone and triamcinolone acetonide on 3T3-L1 cell line

RESUMEN: La piel es el órgano más extenso en el ser humano, su integridad representa protección contra diferentes agentes químicos, biológicos y mecánicos. Las lesiones ocasionadas en este tejido se resuelven mediante la formación de una cicatriz, sin embargo, diferentes alteraciones moleculares pueden sobre estimular este proceso, lo que conlleva a la formación de cicatrices aberrantes (hipertrófica o queloide). El tratamiento más recomendado para este tipo de lesiones es la aplicación intralesional del acetónido de triamcinolona (AT) y por otro lado, la dehidroepiandrosterona (DHEA) es una pro-hormona que posee una gran variedad de efectos biológicos como: regulación de la síntesis de fibras de colágeno, protección celular, propiedades antitumorales, antiinflamatorias y antioxidante. En este trabajo, se estudió la combinación de AT-DHEA sobre la proliferación y muerte celular en la línea celular de fibroblastos 3T3-L1. Los resultados mostraron que la AT a 100 M y la DHEA a 1000 M inhiben la proliferación en un 50 y 40% respectivamente. La combinación de AT-DHEA (10000-10 M) inhibe la proliferación celular e inducen muerte celular programada, entonces esta combinación pudiera utilizarse en cicatrices hipertróficas o queloides para su eliminación.

ABSTRACT: The skin in the human is the largest organ, his integrity represents protection against various chemical, biological and mechanical agents. The injuries in this tissue are solved by forming a scar, however, different molecular alterations may overstimulate this process, leading to the formation of aberrant scars (hypertrophic or keloid). The most recommended treatment for such injuries is the intralesional application of triamcinolone acetonide (TA) and on the other hand, dehydroepiandrosterone (DHEA) is a pro-hormone that has a wide variety of biological effects such as regulation of the synthesis of collagen fibers, cell protection, anti-tumor properties, anti-inflammatory and antioxidant. In this paper, the combination of AT-DHEA on proliferation and cell death in fibroblast cell line 3T3-L1 was studied. The results showed that the AT 100 and 1000 M DHEA to inhibit proliferation by 50 and 40% respectively. The combination of AT-DHEA (10000-10 M) inhibits cell proliferation and induce programmed cell death, so this combination could be used in hypertrophic or keloid scars for disposal.

The altered expression of inflammation-related molecules and secretion of IL-6 and IL-8 by HUVEC from newborns with maternal inactive systemic lupus erythematosus is modified by estrogens.

Systemic lupus erythematosus (SLE) predominantly affects women, especially those in reproductive age. Genetic contributions to disease susceptibility as well as immune dysregulation, particularly persistent inflammatory responses, are considered essential features. Our aim was to determine whether human umbilical vein endothelial cells (HUVEC) isolated from healthy newborns to women with inactive SLE show inflammation-related abnormalities that might lead to an early development of SLE in the offsprings. HUVEC isolated from six women with inactive SLE were stimulated with 2.5 ng/mL of TNF-alpha and/or physiological and pharmacological doses of 17-I(2) estradiol (E2). Then the expression of VCAM-1, ICAM-1, E-selectin, toll-like receptor-9 (TLR-9), heat shock protein 70 (HSP70) and HSP90 were measured. The concentrations of IL-6, IL-8, and IL-10 were also determined in maternal serum and in TNF-alpha stimulated and non-stimulated HUVEC culture supernatant. HUVEC from children with no family history of autoimmune disease served as controls. Our results showed that in HUVEC from SLE+ mothers, a constitutively low expression of adhesion molecules was enhanced by TNF-alpha treatment. The E2 (1 ng/mL) increased the expression of adhesion molecules but had no effect upon TNF-alpha-treated cells. IL-6 was constitutively higher in SLE+ HUVEC, whereas IL-8 was lower; E2 treatment diminished the latter. The E2 had no effect upon IL-6 and IL-8 secretions in TNF-alpha-treated cells. SLE+ HUVEC showed a disordered cytoskeleton and overexpressed HSP70, HSP90, and TLR-9. Our results indicate that endothelial cells of newborns to SLE+ mothers are in a proinflammatory condition which can be upregulated by estrogens.

[Role of tumor necrosis factor alpha in atherosclerosis]. / Participación del factor de necrosis tumoral-alpha en la aterosclerosis.

We describe tumour necrosis factor alpha and its role in the development of the atherosclerotic lesion, and detail the effects of this cytokine upon vascular endothelial cells under normal and high risk conditions. We propose that TNF-alpha performs a central role in the progression of the lesion since, once the endothelial cell feedback regulatory mechanisms are altered, there is an increase in the microenvironment TNF-alpha concentration, which together with some of the already well known risk factors, generates an environment that favours and perpetuates the development of the atheromatous lesion.

Ceramide mimics tumour necrosis factor-alpha in the induction of cell cycle arrest in endothelial cells. Induction of the tumour suppressor p53 with decrease in retinoblastoma/protein levels.

Tumour necrosis factor (TNF)-alpha induces a transient increase in N-octanoylsphingosine (C8-ceramide) which has been postulated as an intracellular mediator in TNF-alpha signalling. We tested the ability of C8-ceramide to reproduce the TNF-alpha-mediated interference with endothelial cell proliferation and DNA synthesis. TNF-alpha (10 ng.mL-1) and C8-ceramide (20 microM) inhibited the incorporation of [3H]thymidine into DNA and led to an accumulation of cells in the G1 phase of the cell cycle. When the responses of the tumour suppressors p53 and RB were analysed, it was found that TNF-alpha and C8-ceramide induced increased expression of p53. Treatment with TNF-alpha or C8-ceramide lead to a significant decrease in total retinoblastoma protein (RB) content that correlated with high levels of p53. These results suggest that p53 and RB may complement each other in their contribution to cell cycle arrest. TNF-alpha prevented RB phosphorylation whereas C8-ceramide did not interfere with this process, suggesting that it follows a ceramide-independent pathway.

[Hematopoietic growth factors induce proliferation of non-hematopoietic tumor cells]. / Los factores de crecimiento hematopoyéticos estimulan la proliferación de células tumorales no hematopoyéticas.

OBJECTIVE: To evaluate the effect of hematopoietic growth factors (HGF) on the proliferation of non-hematopoietic cells such as fibroblasts and epithelial cells of normal and tumoral origin. METHODS: The lymphoid factor IL-2 and the myeloid HGF assayed were IL-3, G-CSF, GM-CSF and M-CSF. The cellular proliferation was determined by measuring the amount of crystal violet dye incorporation by cells through spectrophotometry. RESULTS: All myeloid HGF tested stimulated the proliferation of cell lines 5637, CaLo and L-929. These results suggest that the stromal cells can be induced to proliferate by myeloid growth factors hinting to a bilateral interaction between these two types of cells as it is known that stromal cells in turn secrete HGF. We also observed that for the mouse fibroblastic and epithelial cells, IL-2 was unable to induce proliferation in normal cells but had a strong effect on transformed cells. Finally we discuss our observation that tumour cells responded to IL-2 as a possible mechanism for an immune escape by these cells through IL-2 depletion.

Interference with c-myc expression and RB phosphorylation during TNF-mediated growth arrest in human endothelial cells.

Incorporation of [3H]-thymidine into DNA in non-synchronized cultures of human endothelial cells was blocked by a 24 h exposure to TNF in a dose dependent manner that resulted in accumulation of cells in G1, as assayed by flow cytometry analysis of DNA content. Proliferation restarted when cells were replated in the absence of TNF. Northern analysis of c-myc mRNA in synchronized untreated cultures showed a transient increase previous to DNA synthesis that was decreased with TNF treatment. Western analysis of the retinoblastoma gene product RB in untreated synchronized cultures showed reduced electrophoretic mobility during the transition from G1 to S, congruent with RB inactivation by phosphorylation. TNF treatment prevented RB retardation and reduced total levels of RB protein. Taken together our results show that the TNF-mediated block of endothelial proliferation correlates with deficient activation of the G1 events necessary for entry into S, despite the presence of serum and endothelial mitogens.