Anna Caretti

Carbamylated erythropoietin ameliorates the metabolic stress induced in vivo by severe chronic hypoxia

Ischemia and chronic hypoxia (CH) trigger a variety of adverse effects arising from metabolic stress that injures cells. In response to reduced O2, hypoxia-inducible factor 1α (HIF-1α) activates erythropoietin (Epo) as well as many other target genes that counteract the effects of O2 deficiency. Epo produced by the kidney stimulates erythrocyte production, leading to decreased HIF-1α production by improved tissue O2 delivery. However, Epo is produced by many other tissues, and it is currently unclear to what extent, if any, locally produced Epo modulates HIF-1α expression. Derivatives of Epo that possess tissue-protective activities but do not stimulate erythropoiesis [e.g., carbamylated Epo (CEpo)] are useful tools with which to determine whether exogenous Epo modulates HIF-1α in the absence of changes in hemoglobin concentration. We compared the effects of CH (6.5% O2 for 10 days) with or without CEpo administered by daily s.c. injection (10 μg/kg of body weight). CEpo administration did not alter the survival rate, weight loss, or increased hemoglobin concentration associated with CH. Therefore, CEpo does not directly suppress HIF-mediated erythropoiesis. CEpo does, however, prevent CH-induced neuronal increases of HIF-1α and Epo receptor-associated immunoreactivity (a measure of stress) while reducing the apoptotic index. In contrast, the myocardium did not exhibit increased HIF-1α expression during CH, although CEpo did reduce the apoptotic index. These observations therefore demonstrate that CEpo administration reduces the metabolic stress caused by severe CH, resulting in improved cellular survival independent of erythrocyte production.

Phosphodiesterase-5 Inhibition Abolishes Neuron Apoptosis Induced by Chronic Hypoxia Independently of Hypoxia-Inducible Factor-1α Signaling

Exposure to hypoxia triggers a variety of adverse effects in the brain that arise from metabolic stress and induce neuron apoptosis. Overexpression of the hypoxia-inducible factor-1α (HIF-1α) is believed to be a major candidate in orchestrating the cell’s defense against stress. To test the impact of HIF-1α on apoptosis during chronic hypoxia in vivo, we examined the protective effect of modulating the nitric oxide (NO)/cGMP pathway by sildenafil, a selective inhibitor of phosphodiesterase-5 (PDE-5). Male ICR/CD-1 mice were divided into 3 groups (n = 6/group): normoxic (21% O2), hypoxic (9.5% O2), and hypoxic with sildenafil (1.4-mg/kg intraperitoneal injections daily). At the end of the 8-day treatment period, the mice were euthanized and cerebral cortex biopsies were harvested for analyses. We found that sildenafil: (1) did not significantly alter the hypoxia-induced weight loss and hemoglobin increase, but did augment plasma nitrates+nitrites and the tissue content of cGMP and phosphorylated (P) NO synthase III; (2) reversed the hypoxia-induced neuron apoptosis (terminal deoxynucleotidyl transferase positivity and double-staining immunofluorescence, P = 0.009), presumably through increased bcl-2/Bax (P = 0.0005); and (3) did not affect HIF-1α, but rather blunted the hypoxia-induced increase in P-ERK1/2 (P = 0.0002) and P-p38 (P = 0.004). We conclude that upregulating the NO/cGMP pathway by PDE-5 inhibition during hypoxia reduces neuron apoptosis, regardless of HIF-1α, through an interaction involving ERK1/2 and p38.

Dihydroceramide delays cell cycle G1/S transition via activation of ER stress and induction of autophagy.

Dihydroceramides, the precursors of ceramides in the de novo sphingolipid synthesis, have been recently implicated in active signalling. We previously demonstrated that dihydroceramide accumulation, in response to treatment with the dihydroceramide desaturase inhibitor XM462, induced autophagy with no sign of cell death in the gastric carcinoma HCG27 cell line. Here we show that XM462 treatment induces a transient early increase in dihydroceramides that are successively metabolized into other sphingolipids. Dihydroceramides accumulation is associated with cyclin D1 expression modulation, delayed G1/S transition of cell cycle and increased autophagy. Moreover, XM462 treatment induces ER stress via the activation of the translation inhibitor eIF2α and the pro-survival transcriptional factor Xbp1. Exogenous addition of a short chain dihydroceramide analog reproduces the effects of endogenous accumulation of dihydroceramides, causing cell cycle delay of the G1/S transition, autophagy enhancement, eIF2α activation and Xbp1 splicing. Blocking autophagy with 3-methyladenine abrogates the effect of XM462 on cell cycle and reduces cell survival to XM462 treatment. Furthermore, the XM462-induced survival response is able to reduce etoposide toxicity in HCG27 and HCT116 cancer cells. Our data suggest a role of dihydroceramide in regulating cell proliferation and survival.

The biosynthesis of the selectin-ligand sialyl Lewis x in colorectal cancer tissues is regulated by fucosyltransferase VI and can be inhibited by an RNA interference-based approach

Sialyl Lewis x (sLex) is a selectin ligand whose overexpression in epithelial cancers mediates metastasis formation. The molecular basis of sLex biosynthesis in colon cancer tissues is still unclear. The prerequisite for therapeutic approaches aimed at sLex down-regulation in cancer, is the identification of rate-limiting steps in its biosynthesis. We have studied the role of α1,3-fucosyltransferases (Fuc-Ts) potentially involved in sLex biosynthesis in specimens of normal and cancer colon as well as in experimental systems. We found that: (i) in colon cancer, but not in normal mucosa where the antigen was poorly expressed, sLex correlated with a Fuc-T which, like Fuc-TVI, was active on 3sialyllactosamine at a low concentration (Fuc-T(SLN)); (ii) competitive RT-PCR analysis revealed that the level of Fuc-T mRNA expression in both normal and cancer colon was Fuc-TVI>Fuc-TIII>Fuc-TIV; Fuc-TV and Fuc-TVII expression was negligible; (iii) sLex was expressed only by the gastrointestinal cell lines displaying both Fuc-TVI mRNA and Fuc-T(SLN) activity, but not by those expressing only Fuc-TIII mRNA; (iv) transfection with Fuc-TVI cDNA, but not with Fuc-TIII cDNA, induced sLex expression in gastrointestinal cell lines; (v) Fuc-TVI knock-down with specific siRNA induced down-regulation of Fuc-TVI mRNA and Fuc-T(SLN) activity and a dramatic inhibition of sLex expression. These data indicate that in colon cancer tissues Fuc-TVI is a key regulator of sLex biosynthesis which can be the target of RNA-interference-based gene knock-down approaches.

Protein modulation in mouse heart under acute and chronic hypoxia

Exploring cellular mechanisms underlying beneficial and detrimental responses to hypoxia represents the object of the present study. Signaling molecules controlling adaptation to hypoxia (HIF‐1α), energy balance (AMPK), mitochondrial biogenesis (PGC‐1α), autophagic/apoptotic processes regulation and proteomic dysregulation were assessed. Responses to acute hypoxia (AH) and chronic hypoxia (CH) in mouse heart proteome were detected by 2‐D DIGE, mass spectrometry and antigen–antibody reactions. Both in AH and CH, the results indicated a deregulation of proteins related to sarcomere stabilization and muscle contraction. Neither in AH nor in CH the HIF‐1α stabilization was observed. In AH, the metabolic adaptation to lack of oxygen was controlled by AMPK activation and sustained by an up‐regulation of adenosylhomocysteinase and acetyl‐CoA synthetase. AH was characterized by the mitophagic protein Bnip 3 increment. PGC‐1α, a master regulator of mitochondrial biogenesis, was down‐regulated. CH was characterized by the up‐regulation of enzymes involved in antioxidant defense, in aldehyde bio‐product detoxification and in misfolded protein degradation. In addition, a general down‐regulation of enzymes controlling anaerobic metabolism was observed. After 10 days of hypoxia, cardioprotective molecules were substantially decreased whereas pro‐apoptotic molecules increased accompained by down‐regulation of specific target proteins.

Role of Sphingolipids in the Pathobiology of Lung Inflammation

Sphingolipid bioactivities in the respiratory airways and the roles of the proteins that handle them have been extensively investigated. Gas or inhaled particles or microorganisms come into contact with mucus components, epithelial cells, blood barrier, and immune surveillance within the airways. Lung structure and functionality rely on a complex interplay of polar and hydrophobic structures forming the surfactant layer and governing external-internal exchanges, such as glycerol-phospholipids sphingolipids and proteins. Sphingolipids act as important signaling mediators involved in the control of cell survival and stress response, as well as secreted molecules endowed with inflammation-regulatory activities. Most successful respiratory infection and injuries evolve in the alveolar compartment, the critical lung functional unit involved in gas exchange. Sphingolipid altered metabolism in this compartment is closely related to inflammatory reaction and ceramide increase, in particular, favors the switch to pathological hyperinflammation. This short review explores a few mechanisms underlying sphingolipid involvement in the healthy lung (surfactant production and endothelial barrier maintenance) and in a selection of lung pathologies in which the impact of sphingolipid synthesis and metabolism is most apparent, such as acute lung injury, or chronic pathologies such as cystic fibrosis and chronic obstructive pulmonary disease.

Anti-inflammatory action of lipid nanocarrier-delivered myriocin: therapeutic potential in cystic fibrosis

BACKGROUND Sphingolipids take part in immune response and can initiate and/or sustain inflammation. Various inflammatory diseases have been associated with increased ceramide content, and pharmacological reduction of ceramide diminishes inflammation damage in vivo. Inflammation and susceptibility to microbial infection are two elements in a vicious circle. Recently, sphingolipid metabolism inhibitors were used to reduce infection. Cystic fibrosis (CF) is characterized by a hyper-inflammation and an excessive innate immune response, which fails to evolve into adaptive immunity and to eradicate infection. Chronic infections result in lung damage and patient morbidity. Notably, ceramide content in mucosa airways is higher in CF mouse models and in patients than in control mice or healthy subjects. METHODS The therapeutic potential of myriocin, an inhibitor of the sphingolipid de novo synthesis rate limiting enzyme (Serine Palmitoyl Transferase, SPT),was investigated in CF cells and mice models. RESULTS We treated CF human respiratory epithelial cells with myriocin, This treatment resulted in reduced basal, as well as TNFα-stimulated, inflammation. In turn, TNFα induced an increase in SPT in these cells, linking de novo synthesis of ceramide to inflammation. Furthermore, myriocin-loaded nanocarrier, injected intratrachea prior to P. aeruginosa challenge, enabled a significant reduction of lung infection and reduced inflammation. CONCLUSIONS The presented data suggest that de novo ceramide synthesis is constitutively enhanced in CF mucosa and that it can be envisaged as pharmacological target for modulating inflammation and restoring effective innate immunity against acute infection. GENERAL SIGNIFICANCE Myriocin stands as a powerful immunomodulatory agent for inflammatory and infectious diseases.

Chronic systemic hypoxia promotes LNCaP prostate cancer growth in vivo

Solid tumors contain underperfused regions where hypoxia‐inducible factor‐1α (HIF‐1α) over‐expression induces hypoxia adaptation and cell proliferation. We test the hypothesis that systemic hypoxia promotes prostate cancer growth in vivo and examine HIF‐1α centrality in this effect.

Heart HIF-1α and MAP Kinases During Hypoxia: Are They Associated In Vivo?:

To study the in vivo dynamics of hypoxia-inducible factor 1α (HIF-1α), master regulator of O2-dependent gene expression, and mitogen-activated protein kinases (MAPKs) in the hypoxic myocardium, Sprague-Dawley rats (n = 4 to 6 per group) were exposed to 1-hr hypoxia (10% O2), 23-hr hypoxia, and 23-hr hypoxia, followed by reoxygenation. HIF-1α increased 15-fold after 1-hr hypoxia, remained constant for 23 hrs, and returned to baseline on reoxygenation. Extracellular signal–regulated kinases (ERK1/2) were unchanged throughout. Phosphorylated p38 increased 4-fold after 1-hr hypoxia and returned to baseline within 23-hr hypoxia. The activity of stress-activated protein kinases/c-Jun NH2-terminal kinases (JNKs), measured as phosphorylated c-Jun, increased 3-fold after 1-hr hypoxia and remained sustained afterward. Furthermore, HIF-1α was halved in rats that were administered with the p38 inhibitor SB202190 and made hypoxic for 1 hr. In conclusion, although very sensitive to the reoxygenation, HIF-1α is overexpressed in vivo in the hypoxic myocardium, and its acute induction by hypoxia is correlated with that of p38.

Resveratrol: A potential challenger against gastric cancer

Gastric cancer (GC) is the fourth most common cancer and the second leading cause of cancer-related mortality in the world. Late diagnosis and classical therapeutic approaches such as surgery, chemotherapy and radiotherapy make this disease a still threatening tumor. Genetic asset, environmental stress, dietary habit and infections caused by Helicobacter pylori (H. pylori) are the major causes concurring to GC initiation. A common mechanism is induction of radicals resulting in gastric mucosal injury. A regular food intake of antioxidant and radical scavenging agents has been proposed to exert protection against tumorigenesis. Resveratrol belongs to the polyphenol flavonoids class of antioxidants produced by a restricted number of plants. Resveratrol exerts bactericidal activity against H. pylori and is a powerful antioxidant, thus acting as a tumor preventive agent. Resveratrol intracellular signaling results in growth arrest and apoptosis, so that it can be directed against tumor progression. Resveratrol therapeutic potential against GC initiation and progression are reviewed here.