Annalisa Merlino

Postconditioning induces an anti-apoptotic effect and preserves mitochondrial integrity in isolated rat hearts.

Postconditioning (PostC) may limit mitochondrial damage and apoptotic signaling. We studied markers of apoptosis and mitochondrial protection in isolated rat hearts, which underwent a) perfusion without ischemia (Sham), b) 30-min ischemia (I) plus 2-hour reperfusion (R), or c) PostC protocol (5 intermittent cycles of 10-s reperfusion and 10-s ischemia immediately after the 30-min ischemia). Markers were studied in cytosolic (CF) and/or mitochondrial (MF) fractions. In CF, while pro-apoptotic factors (cytochrome c and caspase-3) were reduced, the anti-apoptotic markers (Bcl-2 and Pim-1) were increased by PostC, compared to the I/R group. Accordingly, phospho-GSK-3beta and Bcl-2 levels increased in mitochondria of PostC group. Moreover, I/R reduced the level of mitochondrial structural protein (HSP-60) in MF and increased in CF, thus suggesting mitochondrial damage and HSP-60 release in cytosol, which were prevented by PostC. Electron microscopy confirmed that I/R markedly damaged cristae and mitochondrial membranes; damage was markedly reduced by PostC. Finally, total connexin-43 (Cx43) levels were reduced in the CF of the I/R group, whereas phospho-Cx43 level resulted in higher levels in the MF of the I/R group than the Sham group. PostC limited the I/R-induced increase of mitochondrial phospho-Cx43. Data suggest that PostC i) increases the levels of anti-apoptotic markers, including the cardioprotective kinase Pim-1, ii) decreases the pro-apoptotic markers, e.g. cytochrome c, iii) preserves the mitochondrial structure, and iv) limits the migration of phospho-Cx43 to mitochondria.

Arachidonic Acid–Induced Ca2+ Entry Is Involved in Early Steps of Tumor Angiogenesis

Growth factor–induced intracellular calcium signals in endothelial cells regulate cytosolic and nuclear events involved in the angiogenic process. Among the intracellular messengers released after proangiogenic stimulation, arachidonic acid (AA) plays a key role and its effects are strictly related to calcium homeostasis and cell proliferation. Here, we studied AA-induced intracellular calcium signals in endothelial cells derived from human breast carcinomas (B-TEC). AA promotes B-TEC proliferation and organization of vessel-like structures in vitro. The effect is directly mediated by the fatty acid without a significant contribution of its metabolites. AA induces Ca2+i signals in the entire capillary-like structure during the early phases of tubulogenesis in vitro. No such responses are detectable in B-TECs organized in more structured tubules. In B-TECs growing in monolayer, AA induces two different signals: a Ca2+i increase due to Ca2+ entry and an inhibition of store-dependent Ca2+ entry induced by thapsigargin or ATP. An inhibitor of Ca2+ entry and angiogenesis, carboxyamidotriazole, significantly and specifically decreases AA-induced B-TEC tubulogenesis, as well as AA-induced Ca2+ signals in B-TECs. We conclude that (a) AA-activated Ca2+ entry is associated with the progression through the early phases of angiogenesis, mainly involving proliferation and tubulogenesis, and it is down-regulated during the reorganization of tumor-derived endothelial cells in capillary-like structures; and (b) inhibition of AA-induced Ca2+ entry may contribute to the antiangiogenic action of carboxyamidotriazole. (Mol Cancer Res 2008;6(4):535–45)

Hydrogen sulfide regulates intracellular Ca2+ concentration in endothelial cells from excised rat aorta.

Hydrogen sulphide (H2S) is a recently discovered gasotransmitter that may regulate a growing number of endothelial functions, including nitric oxide (NO) release, proliferation, adhesion and migration, which are the key steps of angiogenesis. The mechanism whereby H2S impacts on endothelial physiology is still unclear: however, the aforementioned processes are driven by an increase in intracellular Ca2+ concentration ([Ca2+]i). In the present study, we exploited the excised rat aorta to gain insights into the regulation of [Ca2+]i by H2S within in situ endothelial cells (ECs). Sodium hydrosulphide (NaHS), a H2S donor, caused an elevation in [Ca2+]i, which disappeared in absence of extracellular Ca2+. NaHSinduced Ca2+ inflow was sensitive to high doses of Gd3+, but not BTP-2. Inhibition of the reverse-mode of the Na+-Ca2+ exchanger (NCX), with KB-R7943 or upon removal of extracellular Na+, abrogated the Ca2+ response to NaHS. Moreover, NaHS-elicited Ca2+ entry was significantly reduced by TEA and glybenclamide, which hinted at the involvement of ATP-dependent K+ (KATP) channels. Conversely, NaHS-evoked Ca2+ signal was not affected by the reducing agent, dithiothreitol. Acute addition of NaHS hindered both Ca2+ release and Ca2+ entry induced by ATP, a physiological agonist of ECs. Consistently, inhibition of endogenous H2S synthesis with DL-propargylglycine impaired ATP-induced Ca2+ inflow, whereas it did not affect Ca2+ mobilization. These data provide the first evidence that H2S may stimulate Ca2+ influx into ECs by recruiting the reverse-mode of NCX and KATP channels. In addition, they show that such gasotransmitter may modulate the Ca2+ signals elicited by physiological stimuli in intact endothelium.

Role of Calcium Channels in the Protective Effect of Hydrogen Sulfide in Rat Cardiomyoblasts

Background: Hydrogen sulfide contributes to the reduction of oxidative stress-related injury in cardiomyocytes but the underlying mechanism is still unclear. Aims: Here we investigated the role of voltage-operated calcium channels (VOCCs) as mediators of the beneficial effect of H₂S against oxidative stress in cultured rat cardiomyoblasts (H9c2). Methods: Intracellular calcium signals were measured by fluorimetric live cell imaging and cell viability by colorimetric assay. Results: Treatment with H₂S donor (NaHS 10 µM) or Nifedipine (10 µM) decreased resting intracellular calcium concentration [Ca]_i, suggesting that L-type VOCCs are negatively modulated by H₂S. In the presence of Nifedipine H₂S was still able to lower [Ca]_i, while co-incubation with Nifedipine and Ni²⁺ 100 µM completely prevented H₂S-dependent [Ca]_i decrease, suggesting that both L-type and T-type VOCCs are inhibited by H₂S. In addition, in the same experimental conditions, H₂S triggered a slow increase of [Ca]_i whose molecular nature remains to be clarified. Pretreatment of H9c2 with NaHS (10 µM) significantly prevented cell death induced by H₂O₂. This effect was mimicked by pretreatment with L-Type calcium channel inhibitor Nifedipine (10 µM). Conclusions: The data provide the first evidence that H₂S protects rat cardiomyoblasts against oxidative challenge through the inhibition of L-type calcium channels.

HYPOXIA AND HYDROGEN SULFIDE DIFFERENTIALLY AFFECT NORMAL AND TUMOR-DERIVED VASCULAR ENDOTHELIUM

Background endothelial cells play a key role in vessels formation both under physiological and pathological conditions. Their behavior is influenced by blood components including gasotransmitters (H2S, NO and CO). Tumor cells are subjected to a cyclic shift between pro-oxidative and hypoxic state and, in this scenario, H2S can be both cytoprotective and detrimental depending on its concentration. H2S effects on tumors onset and development is scarcely studied, particularly concerning tumor angiogenesis. We previously demonstrated that H2S is proangiogenic for tumoral but not for normal endothelium and this may represent a target for antiangiogenic therapeutical strategies. Methods in this work, we investigate cell viability, migration and tubulogenesis on human EC derived from two different tumors, breast and renal carcinoma (BTEC and RTEC), compared to normal microvascular endothelium (HMEC) under oxidative stress, hypoxia and treatment with exogenous H2S. Results all EC types are similarly sensitive to oxidative stress induced by hydrogen peroxide; chemical hypoxia differentially affects endothelial viability, that results unaltered by real hypoxia. H2S neither affects cell viability nor prevents hypoxia and H2O2-induced damage. Endothelial migration is enhanced by hypoxia, while tubulogenesis is inhibited for all EC types. H2S acts differentially on EC migration and tubulogenesis. Conclusions these data provide evidence for a great variability of normal and altered endothelium in response to the environmental conditions.

Corrigendum to “Postconditioning induces an anti-apoptotic effect and preserves mitochondrial integrity in isolated rat hearts” [Biochim. Biophys. Acta. 1787 (2009) 794–801]

Corrigendum to “Postconditioning induces an anti-apoptotic effect and preserves mitochondrial integrity in isolated rat hearts” [Biochim. Biophys. Acta. 1787 (2009) 794–801] Claudia Penna⁎, Maria-Giulia Perrelli, Stefania Raimondo, Francesca Tullio, Annalisa Merlino, Francesca Moro, Stefano Geuna, Daniele Mancardi, Pasquale Pagliaro a Department of Clinical and Biological Sciences, University of Turin, Italy b Istituto Nazionale Ricerche Cardiovascolari, Bologna, Italy