Henrique Almeida

Superoxide dismutase expression in human cumulus oophorus cells

Success in assisted reproductive techniques (ART) is influenced by gamete and embryo quality but the assessment of these parameters has been thwarted by the lack of reliable biomarkers. Follicular fluid and cumulus oophorus cells may provide biomarkers due to their close relationship to the oocyte. These cells produce antioxidants and thus protect the oocyte from oxidative damage exerted by reactive oxygen species (ROS). ROS and antioxidants are known to intervene in reproductive physiology and pathology, but their roles are unclear. It is hypothesized that superoxide dismutase (SOD), a first line antioxidant enzyme, is associated with oocyte quality. Cells obtained in the course of ART for the treatment of infertility due to male factor or female pathology were processed for SOD intracellular isoforms (CuZnSOD and MnSOD) immunodetection, total SOD activity and isoforms content. Cells presented strong positive staining for CuZnSOD and MnSOD. SOD activity decreased with increasing female age but was increased in endometriosis and in ovulatory dysfunction. When male factor was the cause for infertility, successful ART was associated with higher SOD activity. Variations in SOD emphasize the relevance of oxidative stress in the oocyte maturation process. These variations also suggest that SOD is a potential biomarker for ART success.

Carbonylation of the cytoskeletal protein actin leads to aggregate formation

Protein carbonylation is a common feature in cells exposed to oxidants, leading to protein dysfunction and protein aggregates. Actin, which is involved in manifold cellular processes, is a sensitive target protein to this oxidative modification. T-cell proteins have been widely described to be sensitive targets to oxidative modifications. The aim of this work was to test whether the formation of protein aggregates contributes to the impaired proliferation of Jurkat cells after oxidative stress and to test whether actin as a major oxidation-prone cytoskeletal protein is an integral part of such protein aggregates. We used Jurkat cells, an established T-cell model, showing the formation of actin aggregates along with the decrease of proteasome activity. The presence of these protein aggregates inhibits Jurkat proliferation even under conditions not influencing viability. As a conclusion, we propose that an oxidative environment leads to actin aggregates contributing to T-cell cellular functional impairment.

Melanocortin 5 receptor activates ERK1/2 through a PI3K-regulated signaling mechanism

Melanocortin 5 receptor (MC5R) is a G protein coupled receptor (GPCR) with high affinity for alpha-melanocyte-stimulating hormone (alpha-MSH). To unravel some of the downstream cell-signaling pathways activated by this receptor, HEK293 cells were transiently and stably transfected with a vector encoding green fluorescent protein (GFP)-tagged MC5R. In these cells the receptor was correctly addressed to the cell surface and was functional, as shown by the MC5R-induced formation of intracellular cAMP. In fact, the MC5R agonist alpha-MSH induced a 10- or 16-fold increase (transient or stable cells, respectively) above the cAMP levels found in unstimulated cells. Moreover, in cells stably expressing MC5R-GFP, alpha-MSH promoted ERK1/2 phosphorylation in a dose-dependent manner (EC50=7.3 nM) with the maximal effect occurring after 5 min of agonist incubation. The signaling pathway conveyed through ERK1/2 is not linked to cAMP, since the phosphorylation of these kinases is unchanged by the inhibition of adenylyl cyclase. Also, ERK1/2 activation is not significantly affected by protein kinase A (PKA), protein kinase C (PKC) and protein kinase B or Akt (Akt/PKB) specific inhibitors. However, alpha-MSH-induced ERK1/2 activation is abolished by the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002. Altogether, these findings demonstrate that MC5R signals through a PI3K-regulated Akt-independent pathway leading to downstream activation of ERK1/2. The involvement of these MAPK suggests that MC5R could be implicated in cellular proliferation or differentiation mechanisms.

Aging and Orchidectomy Modulate Expression of VEGF Receptors (Flt‐1 and Flk‐1) on Corpus Cavernosum of the Rat

Abstract:  Aging and hypogonadic states are known risk factors for erectile dysfunction (ED), contributing together to vascular damage of penile tissue. In the present study, VEGF‐specific membrane receptor (VEGFR‐1/Flt‐1 and VEGFR‐2/Flk‐1) expression was studied by confocal immunofluorescence in the corpus cavernosum of control rats, rats aged 12 and 18 months, and orchidectomized Wistar rats (90 days of bilateral orchidectomy). Immunocytochemical results demonstrated VEGFR‐2 expression restricted to the endothelium in both control and orchidectomized rats. Aged animals (12 and 18 months) presented enlarged vessels with intense VEGFR‐2 endothelial staining. On the other hand, VEGFR‐1 was demonstrated in smooth muscle fibers, particularly in those that surround vessel endothelium, the endothelial expression being very low in control and orchidectomized rats. However, in the aged rats, a shift resulting in a VEGFR‐1 and VEGFR‐2 co‐localization in the endothelial cell was observed. The findings suggest an upregulation of VEGFR‐1 in the corpora cavernosa during aging in the rat, which is evident from an increased expression by endothelial cells.

Androgen depletion in humans leads to cavernous tissue reorganization and upregulation of Sirt1-eNOS axis.

Aging and physiological androgen decay leads to structural changes in corpus cavernosum (CC) that associate with erectile function impairment. There is evidence that such changes relate to nitric oxide (NO) bioavailability, an endothelial compound produced by the action of endothelial NO synthase (eNOS), and is regulated by sirtuin-1 (Sirt1), a NAD+-dependent protein deacetylase. Taking into account the reduced NO synthesis observed in aging and erectile dysfunction, we aimed to characterize human CC of androgen-deprived, young, and aged individuals postulating that androgen deprivation induces modifications similar to those observed in aging. Human penile fragments were collected from young individuals submitted to male-to-female sex reassignment procedure, who undergone an androgen deprivation chemical regimen, from young organ donors and from aged patients submitted to penile deviation surgery. They were processed for histomorphometric analysis of smooth muscle (SM) and connective tissues (CT), and dual-immunofluorescence of alpha-actin/vWf or Sirt1, and endothelin-1/eNOS. Estrogen receptors were analyzed by immunohistochemistry and semiquantification of Sirt1, eNOS, and phospho-Akt was assayed by Western blotting. Androgen withdrawal, similarly to aging, leads to a noteworthy reduction of SM-to-CT ratio in CC. However, in contrast to young and aged, a significant increase in penile Sirt1 expression accompanied by an increase in total eNOS expression was observed in androgen-depleted individuals. No changes were evidenced in phospho-Akt system and estrogen receptors were undetectable. These findings indicate that Sirt1 regulates the expression of eNOS in human CC employing mechanisms influenced by androgen depletion.

Copper ability to induce premature senescence in human fibroblasts.

Human diploid fibroblasts (HDFs) exposed to subcytotoxic concentrations of oxidative or stressful agents, such as hydrogen peroxide, tert-butylhydroperoxide, or ethanol, undergo stress-induced premature senescence (SIPS). This condition is characterized by the appearance of replicative senescence biomarkers such as irreversible growth arrest, increase in senescence-associated β-galactosidase (SA β-gal) activity, altered cell morphology, and overexpression of several senescence-associated genes. Copper is an essential trace element known to accumulate with ageing and to be involved in the pathogenesis of some age-related disorders. Past studies using either yeast or human cellular models of ageing provided evidence in favor of the role of intracellular copper as a longevity modulator. In the present study, copper ability to cause the appearance of senescent features in HDFs was assessed. WI-38 fibroblasts exposed to a subcytotoxic concentration of copper sulfate presented inhibition of cell proliferation, cell enlargement, increased SA β-gal activity, and mRNA overexpression of several senescence-associated genes such as p21, apolipoprotein J (ApoJ), fibronectin, transforming growth factor β-1 (TGF β1), insulin growth factor binding protein 3, and heme oxygenase 1. Western blotting results confirmed enhanced intracellular p21, ApoJ, and TGF β1 in copper-treated cells. Thus, similar to other SIPS-inducing agents, HDF exposure to subcytotoxic concentration of copper results in premature senescence. Further studies will unravel molecular mechanisms and the biological meaning of copper-associated senescence and lead to a better understanding of copper-related disorder establishment and progression.

Age-related changes in the inner zone of the adrenal cortex of the rat—a morphologic and biochemical study

In this work, a correlative morphologic and biochemical study on the effects of ageing on the rat adrenal Inner Zone (IZ) was made. Male Wistar rats were studied at 2, 6, 12, 18 and 24 months. Structural data of Zona Fasciculata (ZF) showed age-related increase in cell volume (P < 0.05), decrease in mitochondria (P < 0.01) and smooth endoplasmic reticulum (SER) volumes, and increase in lipid droplets (P < 0.01) and lipofuscin granules (P < 0.01) volumes. In Zona Reticularis, the main change observed was the increase in lipofuscin granules (P < 0.001). Serum corticosterone from unstimulated rats increased until 12 months but decreased thereafter (P < 0.01), to levels below those from 2-month-old rats. Similarly, plasma adrenocorticotrophic hormone (ACTH) presented a maximum at 12 months, followed by a decrease to levels higher than at 2 months (P < 0.05). In rats injected either with only ACTH or dexamethasone, before ACTH stimulation, corticosterone level had a maximum at 12 months. In aged rats, serum high density lipoprotein (HDL) and adrenal cholesterol ester increased significantly (P < 0.05 and P < 0.01, respectively), whereas adrenal corticosterone decreased. Products of lipid peroxidation, assayed with the thiobarbituric acid reaction and fluorimetry showed an age-related increase (P < 0.05). The age-related decrease in mitochondria and SER volumes is consistent with the decrease of serum corticosterone. The increase in lipid droplet and HDL and the reduction of adrenal corticosterone level correlate with the increase of adrenal cholesterol ester content. These suggest a continued uptake of steroid precursor but a reduced steroid synthesis. On the whole, the data provide evidence for an age-related reduced functional ability of IZ and particularly of ZF.

ER Stress Response in Human Cellular Models of Senescence

The aging process is characterized by progressive accumulation of damaged biomolecules in the endoplasmic reticulum, as result of increased oxidative stress accompanying cellular senescence. In agreement, we hypothesized that WI-38 human cellular models of replicative senescence and stress-induced premature senescence (SIPS) induced by hydrogen peroxide (H2O2-SIPS) or copper sulfate (CuSO4-SIPS) would present endoplasmic reticulum chaperoning mechanisms impairment and unfolded protein response activation. Results show that in replicative senescence and CuSO4-SIPS, immunoglobulin binding protein, calnexin, protein disulfide isomerase, and ER oxireductin-1 levels adjust to restore proteostasis and inositol-requiring enzyme-1 (IRE1)-, activating transcription factor 6 (ATF6)-, and pancreatic ER kinase (PERK)-mediated unfolded protein response are activated. However, H2O2-SIPS does not exhibit IRE1 and ATF6 pathways activation but a PERK-mediated upregulation of CCAAT/enhancer-binding protein homologous protein, showing that CuSO4-SIPS mimics better the endoplasmic reticulum molecular events of replicative senescence than H2O2-SIPS. Moreover, unfolded protein response activation is required for both SIPS models induction, because PERK and IRE1 inhibitors decreased senescence-associated beta-galactosidase appearance. In CuSO4-SIPS, the decrease in senescence levels is associated with PERK-driven, but IRE1 independent, cell cycle arrest while in H2O2-SIPS cell proliferation is PERK independent. These results add a step further on the molecular mechanisms that regulate senescence induction; moreover, they validate CuSO4-SIPS model as a useful tool to study cellular stress responses during aging, hoping to postpone age-related health decline.

Intracellular signaling mechanisms of the melanocortin receptors: current state of the art

The melanocortin system is composed by the agonists adrenocorticotropic hormone and α, β and γ-melanocyte-stimulating hormone, and two naturally occurring antagonists, agouti and agouti-related protein. These ligands act by interaction with a family of five melanocortin receptors (MCRs), assisted by MCRs accessory proteins (MRAPs). MCRs stimulation activates different signaling pathways that mediate a diverse array of physiological processes, including pigmentation, energy metabolism, inflammation and exocrine secretion. This review focuses on the regulatory mechanisms of MCRs signaling, highlighting the differences among the five receptors. MCRs signal through G-dependent and independent mechanisms and their functional coupling to agonists at the cell surface is regulated by interacting proteins, namely MRAPs and β-arrestins. The knowledge of the distinct modulation pattern of MCRs signaling and function may be helpful for the future design of novel drugs able to combine specificity, safety and effectiveness in the course of their therapeutic use.

Actin carbonylation: from cell dysfunction to organism disorder.

Protein carbonylation is an important event in the context of proteostasis because of its frequency, non-enzymatic nature and irreversible effects. The carbonylation of proteins disturbs their function and leads to protein aggregates, which may precede cellular senescence and cell death. Actin, an evolutionarily conserved cytoskeletal protein that is involved in important cellular processes, is one of the proteins most susceptible to carbonylation. Conditions resulting in oxidative stress are likely to lead to its carbonylation, loss of function and aggregate formation. In this review, we summarise actin susceptibility to carbonylation, as verified in cell free extracts, cell lines and animal models, and review its fate through the activation of cell mechanisms aimed at removing damaged proteins. Their insufficient activity may underlie age-related diseases and the ageing process. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.