Silvia Nistri

Oxidative Stress by Monoamine Oxidase Mediates Receptor-Independent Cardiomyocyte Apoptosis by Serotonin and Postischemic Myocardial Injury

Background— Serotonin (5-hydroxytryptamine [5-HT]), released by activated platelets during cardiac ischemia, is metabolized by the mitochondrial enzyme monoamine oxidase A (MAO-A). Because hydrogen peroxide is one of the byproducts of 5-HT degradation by MAO-A, we investigated the potential role of reactive oxygen species generated by MAOs in 5-HT-dependent cardiomyocyte death and post-ischemia-reperfusion cardiac damage. Methods and Results— Treatment of isolated adult rat cardiomyocytes with 5-HT induced intracellular oxidative stress and cell apoptosis. The apoptotic cascade triggered by 5-HT involves release of cytochrome c, upregulation of proapoptotic Bax protein, and downregulation of antiapoptotic Bcl-2 protein. These effects were prevented by inhibition of amine transporter or MAO, antioxidants, or iron chelation. In contrast, cardiomyocyte apoptosis was only slightly affected by the 5-HT2B receptor antagonist SB 206553. In vivo, inhibition of MAO-A largely reduced myocardial ultrastructural damage induced by 30 minutes of ischemia followed by 60 minutes of reperfusion in the rat heart. Cardioprotective effects of MAO inhibitors were associated with the prevention of postischemic oxidative stress, neutrophil accumulation, and mitochondrial-dependent cell death and were not reverted by SB 206553. Administration of MAO-A inhibitors during ischemia was still effective in preventing cardiac damage. Conclusions— Our results supply the first direct evidence that oxidative stress induced by MAO is responsible for receptor-independent apoptotic effects of 5-HT in cardiomyocytes and postischemic myocardial injury. These findings provide new insight into the mechanisms of 5-HT action in the heart and may constitute the basis for novel therapies.

Inducible Nitric Oxide Synthase Expression in Human Colorectal Cancer : Correlation with Tumor Angiogenesis

To investigate the potential involvement of the nitric oxide (NO) pathway in colorectal carcinogenesis, we correlated the expression and the activity of inducible nitric oxide synthase (iNOS) with the degree of tumor angiogenesis in human colorectal cancer. Tumor samples and adjacent normal mucosa were obtained from 46 surgical specimens. Immunohistochemical expression of iNOS, vascular endothelial growth factor (VEGF), and CD31 was analyzed on paraffin-embedded tissue sections. iNOS activity and cyclic GMP levels were assessed by specific biochemical assays. iNOS protein expression was determined by Western blot analysis. iNOS and VEGF mRNA levels were evaluated using Northern blot analysis. Both iNOS and VEGF expressions correlated significantly with intratumor microvessel density (r(s) = 0.31, P = 0.02 and r(s) = 0.67, P < 0.0001, respectively). A significant correlation was also found between iNOS and VEGF expression (P = 0.001). iNOS activity and cyclic GMP production were significantly higher in the cancer specimens than in the normal mucosa (P < 0.0001 and P < 0.0001, respectively), as well as in metastatic tumors than in nonmetastatic ones (P = 0.002 and P = 0.04, respectively). Western and Northern blot analyses confirmed the up-regulation of the iNOS protein and gene in the tumor specimens as compared with normal mucosa. NO seems to play a role in colorectal cancer growth by promoting tumor angiogenesis.

Relaxin up-regulates inducible nitric oxide synthase expression and nitric oxide generation in rat coronary endothelial cells

Relaxin (RLX) is a reproductive hormone with vasodilatatory properties on several organs, including the heart. RLX‐induced vasodilatation appears to depend on the stimulation of endogenous NO production. Here, we investigate whether RLX acts on rat coronary endothelial (RCE) cells in vitro by inducing changes of NO generation and, if so, to clarify the possible mechanism of action. RCE cells were treated for 24 h with vehicle (controls) or RLX, alone or in association with inhibitors of NO synthesis or dexamethasone, which inhibits transcription of NO synthase gene. In some experiments, inactivated RLX was given in the place of authentic RLX. Expression of NO synthase isozymes II and III was analyzed by immunocytochemistry, Western blot, and RT‐PCR. NO production was evaluated by the Griess reaction for nitrite and the NO‐sensitive fluorophore DAF‐2/DA. Agonist‐induced changes of intracellular Ca2+ transient were studied with the Ca2+‐sensitive fluorophore Fura 2‐AM. RLX was found to up regulate NOS II mRNA and protein and to stimulate intrinsic NO generation, likely through the activation of a dexamethasone‐sensitive transcription factor, and to decrease agonist‐induced intracellular Ca2+ transient. Conversely, RLX had negligible effects on NOS III expression. By these biological effects, RLX may afford significant protection against cardiovascular disease.

Novel drug development opportunity for relaxin in acute myocardial infarction: evidences from a swine model

The hormone relaxin has been shown to cause coronary vasodilation and to prevent ischemia/reperfusion‐induced cardiac injury in rodents. This study provides evidence that relaxin, used as an adjunctive drug to coronary reperfusion, reduces the functional, biochemical, and histopathological signs of myocardial injury in an in vivo swine model of heart ischemia/reperfusion, currently used to test cardiotropic drugs for myocardial infarction. Human recombinant relaxin, given at reperfusion at doses of 1.25, 2.5, and 5 µg/kg b.wt. after a 30‐min ischemia, caused a dose‐related reduction of key markers of myocardial damage (serum myoglobin, CK‐MB, troponin T) and cardiomyocyte apoptosis (caspase 3, TUNEL assay), as well as of cardiomyocyte contractile dysfunction (myofibril hypercontraction). Compared with the controls, relaxin also increased the uptake of the viability tracer 201Thallium and improved ventricular performance (cardiac index). Relaxin likely acts by reducing oxygen free radical‐induced myocardial injury (malondialdehyde, tissue calcium overload) and inflammatory leukocyte recruitment (myeloperoxidase). The present findings show that human relaxin, given as a drug to counteract reperfusion‐induced cardiac injury, affords a clear‐cut protection to the heart of swine with induced myocardial infarction. The findings also provide background to future clinical trials with relaxin as adjunctive therapy to catheter‐based coronary angioplasty in patients with acute myocardial infarction.

Paracrine effects of transplanted myoblasts and relaxin on post-infarction heart remodelling.

In the post‐infarcted heart, grafting of precursor cells may partially restore heart function but the improvement is modest and the mechanisms involved remain to be elucidated. Here, we explored this issue by transplanting C2C12 myoblasts, genetically engineered to express enhanced green fluorescent protein (eGFP) or eGFP and the cardiotropic hormone relaxin (RLX) through coronary venous route to swine with experimental chronic myocardial infarction. The rationale was to deliver constant, biologically effective levels of RLX at the site of cell engraftment. One month after engraftment, histological analysis showed that C2C12 myoblasts selectively settled in the ischaemic scar and were located around blood vessels showing an activated endothelium (ICAM‐1‐,VCAM‐positive). C2C12 myoblasts did not trans‐differentiate towards a cardiac phenotype, but did induce extracellular matrix remodelling by the secretion of matrix metalloproteases (MMP) and increase microvessel density through the expression of vascular endothelial growth factor (VEGF). Relaxin‐producing C2C12 myoblasts displayed greater efficacy to engraft the post‐ischaemic scar and to induce extracellular matrix re‐modelling and angiogenesis as compared with the control cells. By echocardio‐graphy, C2C12‐engrafted swine showed improved heart contractility compared with the ungrafted controls, especially those producing RLX. We suggest that the beneficial effects of myoblast grafting on cardiac function are primarily dependent on the paracrine effects of transplanted cells on extracellular matrix remodelling and vascularization. The combined treatment with myoblast transplantation and local RLX production may be helpful in preventing deleterious cardiac remodelling and may hold therapeutic possibility for post‐infarcted patients.

Relaxin prevents cardiac fibroblast-myofibroblast transition via notch-1-mediated inhibition of TGF-β/Smad3 signaling.

The hormone relaxin (RLX) is produced by the heart and has beneficial actions on the cardiovascular system. We previously demonstrated that RLX stimulates mouse neonatal cardiomyocyte growth, suggesting its involvement in endogenous mechanisms of myocardial histogenesis and regeneration. In the present study, we extended the experimentation by evaluating the effects of RLX on primary cultures of neonatal cardiac stromal cells. RLX inhibited TGF-β1-induced fibroblast-myofibroblast transition, as judged by its ability to down-regulate α-smooth muscle actin and type I collagen expression. We also found that the hormone up-regulated metalloprotease (MMP)-2 and MMP-9 expression and downregulated the tissue inhibitor of metalloproteinases (TIMP)-2 in TGF-β1-stimulated cells. Interestingly, the effects of RLX on cardiac fibroblasts involved the activation of Notch-1 pathway. Indeed, Notch-1 expression was significantly decreased in TGF-β1-stimulatedfibroblasts as compared to the unstimulated controls; this reduction was prevented by the addition of RLX to TGF-β1-stimulated cells. Moreover, pharmacological inhibition of endogenous Notch-1 signaling by N-3,5-difluorophenyl acetyl-L-alanyl-2-phenylglycine-1,1-dimethylethyl ester (DAPT), a γ-secretase specific inhibitor, as well as the silencing of Notch-1 ligand, Jagged-1, potentiated TGF-β1-induced myofibroblast differentiation and abrogated the inhibitory effects of RLX. Interestingly, RLX and Notch-1 exerted their inhibitory effects by interfering with TGF-β1 signaling, since the addition of RLX to TGF-β1-stimulated cells caused a significant decrease in Smad3 phosphorylation, a typical downstream event of TGF-β1 receptor activation, while the treatment with a prevented this effect. These data suggest that Notch signaling can down-regulate TGF-β1/Smad3-induced fibroblast-myofibroblast transition and that RLX could exert its well known anti-fibrotic action through the up-regulation of this pathway. In conclusion, the results of the present study beside supporting the role of RLX in the field of cardiac fibrosis, provide novel experimental evidence on the molecular mechanisms underlying its effects.

Mesenchymal stromal cells affect cardiomyocyte growth through juxtacrine Notch-1/Jagged-1 signaling and paracrine mechanisms: clues for cardiac regeneration.

The possibility to induce myocardial regeneration by the activation of resident cardiac stem cells (CSCs) has raised great interest. However, to propose endogenous CSCs as therapeutic options, a better understanding of the complex mechanisms controlling heart morphogenesis is needed, including the cellular and molecular interactions that cardiomyocyte precursors establish with cells of the stromal compartment. In the present study, we co-cultured immature cardiomyocytes from neonatal mouse hearts with mouse bone marrow-derived mesenchymal stromal cells (MSCs) to investigate whether these cells could influence cardiomyocyte growth in vitro. We found that cardiomyocyte proliferation was enhanced by direct co-culture with MSCs compared with the single cultures. We also showed that the proliferative response of the neonatal cardiomyocytes involved the activation of Notch-1 receptor by its ligand Jagged-1 expressed by the adjacent MSCs. In fact, the cardiomyocytes in contact with MSCs revealed a stronger immunoreactivity for the activated Notch-intracellular domain (Notch-ICD) as compared with those cultured alone and this response was significantly attenuated when MSCs were silenced for Jagged-1. The presence of various cardiotropic cytokines and growth factors in the conditioned medium of MSCs underscored the contribution of paracrine mechanisms to Notch-1 up-regulation by the cardiomyocytes. In conclusions these findings unveil a previously unrecognized function of MSCs in regulating cardiomyocyte proliferation through Notch-1/Jagged-1 pathway and suggest that stromal-myocardial cell juxtacrine and paracrine interactions may contribute to the development of new and more efficient cell-based myocardial repair strategies.

Bone Marrow Mesenchymal Stromal Cells Stimulate Skeletal Myoblast Proliferation through the Paracrine Release of VEGF

Mesenchymal stromal cells (MSCs) are the leading cell candidates in the field of regenerative medicine. These cells have also been successfully used to improve skeletal muscle repair/regeneration; however, the mechanisms responsible for their beneficial effects remain to be clarified. On this basis, in the present study, we evaluated in a co-culture system, the ability of bone-marrow MSCs to influence C2C12 myoblast behavior and analyzed the cross-talk between the two cell types at the cellular and molecular level. We found that myoblast proliferation was greatly enhanced in the co-culture as judged by time lapse videomicroscopy, cyclin A expression and EdU incorporation. Moreover, myoblasts immunomagnetically separated from MSCs after co-culture expressed higher mRNA and protein levels of Notch-1, a key determinant of myoblast activation and proliferation, as compared with the single culture. Notch-1 intracellular domain and nuclear localization of Hes-1, a Notch-1 target gene, were also increased in the co-culture. Interestingly, the myoblastic response was mainly dependent on the paracrine release of vascular endothelial growth factor (VEGF) by MSCs. Indeed, the addition of MSC-derived conditioned medium (CM) to C2C12 cells yielded similar results as those observed in the co-culture and increased the phosphorylation and expression levels of VEGFR. The treatment with the selective pharmacological VEGFR inhibitor, KRN633, resulted in a marked attenuation of the receptor activation and concomitantly inhibited the effects of MSC-CM on C2C12 cell growth and Notch-1 signaling. In conclusion, this study provides novel evidence for a role of MSCs in stimulating myoblast cell proliferation and suggests that the functional interaction between the two cell types may be exploited for the development of new and more efficient cell-based skeletal muscle repair strategies.

Ceramide: A Key Signaling Molecule in a Guinea Pig Model of Allergic Asthmatic Response and Airway Inflammation

Although mechanisms involved in the pathogenesis of asthma remain unclear, roles for oxidative/nitrosative stress, epithelial cell apoptosis, and airway inflammation have been documented. Ceramide is a sphingolipid with potent proinflammatory and proapoptotic properties. This study aimed at determining whether increased formation of ceramide contributes to the development of airway inflammation and hyper-responsiveness, using a well characterized in vivo model of allergic asthmatic response and airway inflammation in ovalbumin-sensitized guinea pigs. Aerosol administration of ovalbumin increased ceramide levels and ceramide synthase activity in the airway epithelium associated with respiratory abnormalities, such as cough, dyspnea, and severe bronchoconstriction. These abnormalities correlated with nitrotyrosine formation in the airway epithelium and oxidative/nitrosative stress, epithelial cell apoptosis, and airway inflammation evident by the infiltration of neutrophils and eosinophils in lung tissues, mast cell degranulation, and release of prostaglandin D2 and proinflammatory cytokines. Inhibition of de novo ceramide synthesis with the competitive and reversible inhibitor of ceramide synthase fumonisin B1 (0.25, 0.5 and 1 mg/kg b.wt.), given i.p. daily for 4 days before allergen challenge, attenuated nitrotyrosine formation and oxidative/nitrosative stress, epithelial cell apoptosis, and airway inflammation while improving the respiratory and histopathological abnormalities. These results implicate ceramide in the development of allergic asthmatic response and airway inflammation. Strategies aimed at reducing the levels of ceramide and downstream events should yield promising novel anti-asthmatic agents.

Relaxin inhibits lipopolysaccharide-induced adhesion of neutrophils to coronary endothelial cells by a nitric oxide-mediated mechanism.

Neutrophil margination within blood vessels is an early finding during myocardial ischemia and can result in myocardial tissue injury. This phenomenon depends on the endothelial expression of adhesion molecules, which allow leukocyte extravasation. The hormone relaxin (RLX) was found to protect against experimental myocardial injury and to reduce neutrophil extravasation into the inflamed tissues. This study addresses the role of RLX in down‐regulating endothelial adhesiveness to neutrophils and the possible involvement of NO, an anti‐adhesive molecule, in the mechanism of action of RLX. Lipopolysaccharide (LPS)‐primed rat coronary endothelial (RCE) cells and neutrophils were co‐cultured and their adhesion was quantified in the absence and presence of RLX, alone or together with the NO‐synthase inhibitor l‐NMMA. Inactivated RLX was used as control for specificity of the RLX effect. A 24‐h incubation of LPS‐primed RCE cells with RLX (60 and 600 ng/ml) caused a significant reduction of adherent neutrophils and of endothelial expression of the adhesion molecules P‐selectin and VCAM‐1 protein and mRNA, evaluated by immunohistochemistry, Western blot, and RT‐PCR. These effects of RLX were blunted by l‐NMMA and were not reproduced by inactivated RLX. These findings suggest that RLX has anti‐inflammatory properties that could be of benefit in ischemic heart disease.