Felipe Massó

High-sucrose diet increases ROS generation, FFA accumulation, UCP2 level, and proton leak in liver mitochondria

Obesity, a risk factor for insulin resistance, contributes to the development of type 2 diabetes and cardiovascular diseases. The relationship between increased levels of free fatty acids in the liver mitochondria, mitochondrial function, and ROS generation in rat model of obesity induced by a high-sucrose diet was not sufficiently established. We determined how the bioenergetic functions and ROS generation of the mitochondria respond to a hyperlipidemic environment. Mitochondria from sucrose-fed rats generated H(2)O(2) at a higher rate than the control mitochondria. Adding fatty acid-free bovine serum albumin to mitochondria from sucrose-fed rats significantly reduced the rate of H(2)O(2) generation. In contrast, adding exogenous oleic or linoleic acid exacerbated the rate of H(2)O(2) generation in both sucrose-fed and control mitochondria, and the mitochondria from sucrose-fed rats were more sensitive than the control mitochondria. The increased rate of H(2)O(2) generation in sucrose-fed mitochondria corresponded to decreased levels of reduced GSH and vitamin E and increased levels of Cu/Zn-SOD in the intermembrane space. There was no difference between the levels of lipid peroxidation and protein carbonylation in the two types of mitochondria. In addition to the normal activity of Mn-SOD, GPX and catalase detected an increased activity of complex II, and upregulation of UCP2 was observed in mitochondria from sucrose-fed rats, all of which may accelerate respiration rates and reduce generation of ROS. In turn, these effects may protect the mitochondria of sucrose-fed rats from oxidative stress and preserve their function and integrity. However, in whole liver these adaptive mechanisms of the mitochondria were inefficient at counteracting redox imbalances and inhibiting oxidative stress outside of the mitochondria.

17β-estradiol inhibits the adhesion of leukocytes in TNF-α stimulated human endothelial cells by blocking IL-8 and MCP-1 secretion, but not its transcription

Inflammation, and especially mononuclear cell adhesion to endothelium, is an important physiopathological component of atherosclerosis. Since coronary heart disease in women of reproductive age and/or with estrogen replacement therapy is reduced, our aim was to determine if 17β-estradiol had a regulatory effect on the adhesion of lymphocytes to the endothelium. We performed U-937 cells adhesion assays in TNF-α-stimulated HUVECs, and we also quantitated IL-8 and MCP-1 in culture supernatants, in the presence or not of 17β-estradiol. The presence of α- and β-estrogen receptors was determined by Western blot and RT-PCR, respectively, whereas the transcription of both chemokines was evaluated by RT-PCR. The results showed a 35% decrease in the adhesion of U-937 monocyte cells to TNF-α-stimulated HUVECs, and a 54% and 65% inhibition of TNF-α-induced IL-8 and MCP-1 secretion by physiological and physiologically high doses of 17β-estradiol. The hormone did not affect the transcription of both chemokine genes. Tamoxifen reverted the inhibitory effect induced by 17βestradiol. In conclusion, 17β-estradiol modifies the adhesion of leukocytes to endothelial cells by inhibiting the secretion, but not the gene transcription, of proinflammatory chemokines.

Statin-induced inhibition of MCF-7 breast cancer cell proliferation is related to cell cycle arrest and apoptotic and necrotic cell death mediated by an enhanced oxidative stress.

Statins have antiproliferative and anti-tumoral effects in MCF-7 cells. We determined the effect of statins upon MCF-7 cell cycle, toxicity, cell death, reactive oxygen species (ROS) production and mitochondrial membrane potential. Fluvastatin, simvastatin and atorvastatin inhibited cell proliferation. Antiproliferation was associated with a decrease in the DNA synthesis and a cell cycle arrest in the G1 and G2/M phases. A loss in the mitochondrial membrane potential was observed with fluvastatin. Statins induced increase in ROS production that was associated with cell death, which was abrogated by the antioxidant NAC. Our results suggest that the cytotoxic effect observed is mediated by an oxidative stress.

Dehydroepiandrosterone inhibits the proliferation of human umbilical vein endothelial cells by enhancing the expression of p53 and p21, restricting the phosphorylation of retinoblastoma protein, and is androgen- and estrogen-receptor independent.

Dehydroepiandrosterone (DHEA), a steroid hormone, modified the proliferation of human umbilical vein endothelial cells in a dose‐dependent manner. Its inactive sulfate ester (DHEA‐S) and two of its metabolites – estradiol and testosterone – had no inhibitory effect at physiological concentrations. Antiproliferation was associated with arrest in the G1 phase of the cell cycle, but not with cell death, as evaluated by cleavage of poly(ADP‐ribose) polymerase and exposure of phosphatidylserine. The effect was not blocked by inhibitors of androgen or estrogen receptors. DHEA diminished the levels of phosphorylated retinoblastoma protein and increased the expression of p53 and p21 mRNAs. These results show that DHEA inhibits endothelial cell proliferation by regulating cell cycle relevant proteins through a cytoplasmic steroid hormone‐independent pathway.

Reduced NO synthesis and eNOS mRNA expression in endothelial cells from newborns with a strong family history of type 2 diabetes

A deficient synthesis of nitric oxide (NO) may play a role in the early endothelial dysfunction of healthy humans with a strong family history of type 2 diabetes (DM2). In this study, we evaluate the intracellular synthesis of NO and the expression of eNOS transcripts in human umbilical vein endothelial cells (HUVECs), exposed to high glucose concentrations, of healthy newborns with (experimental) and without (control) a strong family history of DM2.

Endothelin-1 and functional tissue factor: a possible relationship with severity in primary pulmonary hypertension.

Abstract. Although dysfunctional endothelium, vasoconstriction, and in situ thrombosis are characteristics of primary pulmonary hypertension, the role that plasma vasomotor and coagulation factors play in this phenomenon are not completely understood. The aim of this work was to ascertain the diagnostic value of endothelin-1, thrombomodulin, tissue factor, and tissue factor pathway inhibitor, as well as their correlation with endothelial dysfunction in primary pulmonary hypertension patients. We analyzed the plasmatic concentration and chromogenic of the above-mentioned molecules using immunoenzymatic techniques. Patients were divided into responders and nonresponders on the basis of their hemodynamic response to a vasodilator trial. We found a continuous increase in endothelin-1 levels and a continuous decrease in functional tissue factor in the control group, responders, and nonresponders, respectively. Moreover, the patients showed a moderate decrease in thrombomodulin levels compared with the control group, without statistical significance. These results support a previous description of a decrease in thrombomodulin levels in primary pulmonary hypertension patients and suggests that an alteration of endothelin-1 and functional tissue factor could be related to a worsening of endothelial function and, indirectly, with the clinical severity of primary pulmonary hypertension.

HUVECs from newborns with a strong family history of diabetes show diminished ROS synthesis in the presence of high glucose concentrations.

A family history of type 2 diabetes mellitus (DM) increases the probability to develop DM and endothelial dysfunction. The probable mechanism involves augmented reactive oxygen species (ROS) synthesis. The aim of this study was to evaluate the synthesis of ROS in human umbilical vein endothelial cells (HUVECs) obtained from healthy newborns with (experimental) and without (control) a strong family history of type 2 DM, exposed to different glucose concentrations.

Protein C activation peptide inhibits the expression of ICAM-1, VCAM-1, and interleukin-8 induced by TNF-a in human dermal microvascular endothelial cells

Activated protein C (APC) is generated from the cleavage of protein C by thrombin coupled to throm- bomodulin and, subsequently, is released as protein C activation peptide (papC). The aim of this study was to evaluate the effect of papC on human dermal microvascular endothelial cells (HMEC-1), activated with 5 ng/ /mL TNF-a. Flow cytometry showed that papC inhibited the expression of VCAM-1 and ICAM-1, after activa- tion with TNF-a. Similarly, RT-PCR analysis revealed that 2 and 4 pM papC inhibited the expression of VCAM-1 and IL-8 mRNA in TNF-a-treated HMEC-1. In addition, the expression of endothelial nitric oxide synthase (eNOS) increased in HMEC-1 treated with papC, compared to those without treatment. Furthermore, Jurkat cell adhesion to HMEC-1 induced by TNF-a was significantly inhibited after the addition of papC, compared to HMEC-1 without papC (p = 0.03). Finally, a control peptide analog to papC showed no effect on the expression of ICAM and VCAM on the surface of HMEC-1. In conclusion, our results suggest that papC exerts anti- inflammatory effects on endothelial cells. (Folia Histochemica et Cytobiologica 2012, Vol. 50, No. 3, 407-413)

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-α and/or physiological and pharmacological doses of 17-β 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-α 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-α treatment. The E2 (1 ng/mL) increased the expression of adhesion molecules but had no effect upon TNF-α-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-α-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.

Effects of mebendazole on protein biosynthesis and secretion in human-derived fibroblast cultures☆

Previous results of our group revealed that mebendazole, a broad spectrum anthelmintic drug with antimicrotubular properties, used for the treatment of liver cirrhosis, decreased total collagen content and biosynthesis in liver upon treatment. In the present study, we have evaluated the effects of mebendazole (5-50 micrograms/mL) on protein synthesis, secretion, and deposition in human-derived fibroblast cultures. The results showed a decrease in cell viability (18.5 +/- 0.9%) at 50 micrograms/mL. [3H]Thymidine incorporation diminished gradually with increasing mebendazole concentrations, reaching a plateau (53.67%) between 30 and 50 micrograms/mL. In late logarithmic phase cultures, the drug caused a decrease of [3H]proline incorporation (43.10%) and collagen biosynthesis (58.61%) in the extracellular matrix. This correlated with an increase in radioactivity in total proteins (51.28%) of the intracellular fraction. Similar results were obtained when mebendazole was assayed in post-confluent fibroblast cultures. The electrophoretic patterns of the extracellular matrix showed a decrease of radioactive collagenous components (alpha chains and beta dimers). By contrast, in the intracellular fraction an increase of radioactive collagen precursors (pro alpha chains) was observed. Immunofluorescence studies and immunotransfer analysis, using polyclonal anti-type I collagen antibodies, revealed an accumulation of intracellular collagen which included: collagen pro alpha chains, alpha chains, and low molecular weight peptides. The results obtained suggest that mebendazole interferes with the transcellular mobilization of proteins, resulting in a decrease of secretion and deposition of extracellular matrix proteins, and an accumulation of intracellular collagenous components. The intracellular accumulation of newly synthesized proteins could cause a feedback regulation in fibroblast cultures.