Università degli Studi di Milano, 2015-12-09

The hypoxic condition determines several functional consequences that ultimately lead to cellular death and irreversible damage to cardiac myocytes. Under hypoxic condition, cells activate several protective pathways; among them, HIF-1? plays a key role in controlling cellular response to hypoxia at molecular level. However, HIF-1? regulatory mechanisms are extremely complex. On these premises, the present work was based on the hypothesis that NEU3 sialidase, a glycolytic enzyme ubiquitously expressed over the plasmatic membrane, can have a regulatory activity on HIF-1? expression in hypoxic/ischemic cardiac myocytes. The experiments performed in this in-vitro model allowed us to draw the following conclusions: 1) Endogenous NEU3 sialidase expression and activity are up-regulated in murine skeletal muscle cells (C2C12) upon oxygen starvation, leading to a signaling cascade resulting in the activation of HIF-1?. 2) Moreover, induced overexpression of NEU3 significantly increases HIF-1? expression and cell resistance to hypoxic stress, whereas NEU3 silencing causes the opposite effects and renders myoblasts more susceptible to apoptosis. 3) The hypoxia-driven activation of NEU3 sialidase can activate the EGFR prosurvival signaling pathway by controlling the content of ganglioside GM3. Furthermore, we demonstrated that NEU3 overexpression causes a reduction of ganglioside GM3, which is known to block EGFR autophosphorylation. Then resulted were extended from skeletal muscle to cardiac myocytes, particularly aiming to ascertain the role of NEU3 in activating the human cardiomyocyte response to hypoxia. Particularly, we evaluated if NEU3 activation occurred in human cardiomyocytes using two different models: 1) A model of acute cardiac ischemia achieved during aortic cross-clamp time and extracorporeal circulation in adult patient submitted to cardiac surgical procedures. 2) A model of chronic hypoxia in neonates and young patients operated for cyanogen congenital cardiac defects. In the acute model of cardiac ischemia, we harvested a sample of right atrial appendage just before and after aortic cross-clamping, during routine adult cardiac surgery procedure. However, no significant activation of NEU3 and HIF-1? was evident in cardiac sample harvested before and after aortic cross-clamping. In our opinion there are several possible explanations for the lack of NEU3 and HIF-1? increased expression in the cardiac surgery model. First, it is possible that in the in-vivo setting the mean aortic cross-clamp time was too short (mean time = 79 minutes) to elucidate the same response that we observed in the in vitro model, where the cells were incubated under hypoxic conditions for at least 12 hours. Secondly, and most important in our opinion, the technique of myocardial protection, especially cardioplegic arrest and hypothermia, by protecting the myocardium from the ischemic injury could have limited NEU3 and HIF-1? expression in our samples. To overcome these limitation, in the final part of my PhD program we evaluated HIF-1? and NEU3 expression in a human in-vivo model of chronic cardiac hypoxia, studying patients affected by cyanotic cardiac defects submitted to surgical correction. In this model of chronic hypoxia, we observed a significant increase in NEU3 expression and activity in cyanotic patients. Furthermore, a significant increase of EGFR was observed, supporting the hypothesis that this signaling pathway is upregulated by the sialidase NEU3. Indeed we observed an increase in expression of genes downstream of EGFR, both related to cellular proliferation (ERK and p38) and to apoptosis resistance (AKT and p70S6K). Finally we observed a significant activation of HIF-1? and of its downstream genes. Another important aspect of cellular adaptation to hypoxia is the metabolic switch between oxidative and glycolytic metabolism, the so-called “Pasteur effect”. In the present study we found that the glycolytic enzymes Glucose transporter Glut1, the Aldolase and the GAPDH were significantly enhanced in the cyanotic group which in turn demonstrates that the myocardium of patients affected by cyanogen cardiac defects is metabolic adapted to chronic hypoxia. In conclusion, the results of this PhD project support the hypothesis of a physiological role of NEU3 in mediating cellular response to hypoxic stress. It is interesting to underline that NEU3 activation is mediated by ganglioside GM3 on cellular membrane. Indeed, an increase in NEU3 level determines a reduction of GM3, which is a well know inhibitor of EGF receptor. On these premises, to mimic the effects of NEU3 activation, it could be possible to inhibit GM3 synthesis, in example by the selective inhibition of the sialyltransferase involved in the last passage of its synthesis. In this direction, our laboratory is performing some experiments with small chemical molecules, designed for blocking selectively the GM3 synthesis with the aim of activating the endogenous response to hypoxic stress.

diritti: info:eu-repo/semantics/openAccess
tutor: L. Anastasia; coordinatore: F. Bonomi; direttore: S. Sonnino
Settore BIO/12 - - Biochimica Clinica e Biologia Molecolare Clinica

Tesi di dottorato. | Lingua: Inglese. | Paese: | BID: TD16002198