Elena Piegari

Doxorubicin induces senescence and impairs function of human cardiac progenitor cells

The increasing population of cancer survivors faces considerable morbidity and mortality due to late effects of the antineoplastic therapy. Cardiotoxicity is a major limiting factor of therapy with doxorubicin (DOXO), the most effective anthracycline, and is characterized by a dilated cardiomyopathy that can develop even years after treatment. Studies in animals have proposed the cardiac progenitor cells (CPCs) as the cellular target responsible for DOXO-induced cardiomyopathy but the relevance of these observations to clinical settings is unknown. In this study, the analysis of the DOXO-induced cardiomyopathic human hearts showed that the majority of human CPCs (hCPCs) was senescent. In isolated hCPCs, DOXO triggered DNA damage response leading to apoptosis early after exposure, and telomere shortening and senescence at later time interval. Functional properties of hCPCs, such as migration and differentiation, were also negatively affected. Importantly, the differentiated progeny of DOXO-treated hCPCs prematurely expressed the senescence marker p16INK4a. In conclusion, DOXO exposure severely affects the population of hCPCs and permanently impairs their function. Premature senescence of hCPCs and their progeny can be responsible for the decline in the regenerative capacity of the heart and may represent the cellular basis of DOXO-induced cardiomyopathy in humans.

EGF-responsive rat neural stem cells: Molecular follow-up of neuron and astrocyte differentiation in vitro

Neural stem cells (NSCs) could be very useful for the “cell therapy” treatment of neurological disorders. For this reason basic studies aiming to well characterize the biology of NSCs are of great interest. We carried out a molecular and immunocytochemical analysis of EGF‐responsive NSCs obtained from rat pups. After the initial growth of NSCs as floating neurospheres in EGF‐containing medium, cells were plated on poly‐L‐ornithine‐coated dishes either in the presence or absence of EGF. We followed cell differentiation and apoptosis for 21 days in vitro and analyzed the expression levels of some genes having a major role in these processes, such as pRB, pRB2/p130, p27, and p53. We observed that EGF impairs neuronal differentiation. Furthermore, in the absence of mitogens, apoptosis, which appeared to proceed through the “p53 network,” was significantly lower than in the presence of EGF. The cyclin kinase inhibitor p27, while important for cell cycle exit, seemed dispensable for cell survival and differentiation.

Induction of apoptosis and differentiation in neuroblastoma and astrocytoma cells by the overexpression of Bin1, a novel Myc interacting protein.

Bin1 is a novel protein that specifically binds Myc and inhibits, at least in part, Myc transactivation. Bin1 seems to play a role in cell cycle control, acting as a tumor suppressor gene. Since MYC family genes play a regulatory role in the proliferation, differentiation, and apoptosis of the nervous system, we studied the effects of the overexpression of the Myc‐interacting protein, Bin1, in neuroblastoma and astrocytoma cell lines, which were chosen as neural cell system models. The major effects of BIN1 overexpression observed in undifferentiated neuroblastoma and astrocytoma cells were a significant reduction of cell growth, an increase in the G0/G1 cell population and the induction of apoptosis. The trigger of programmed cell death by Bin1 is described for the first time. Bin1 overexpression in undifferentiated cells did not induce any maturation process as neither neuronal nor astrocyte differentiation markers were upregulated in neuroblastoma and astrocytoma cells, respectively. On the other side, the effects of Bin1 overproduction in neuroblastoma and astrocytoma cells committed towards neuronal and astrocyte differentiation, respectively, were different from those observed in undifferentiated cells. Although we did not evidence any triggering of programmed cell death, we did notice a further induction towards more differentiated phenotypes. Our studies suggest that Bin1 overexpression in neuroblastoma and astrocytoma cells can result in one of the following pathways: (1) suppressed cell proliferation, (2) induced differentiation, or (3) apoptosis. Thus, it appears that Bin1 operates through different pathways that involve activation of different genes: the chosen pathway however will depend on the proliferating or differentiated state of the cell. J. Cell. Biochem. 74:313–322, 1999.

SIRT1 activation attenuates diastolic dysfunction by reducing cardiac fibrosis in a model of anthracycline cardiomyopathy

BACKGROUND Doxorubicin (DOXO) is an effective anti-neoplastic drug but its clinical benefits are hampered by cardiotoxicity. Oxidative stress, apoptosis and myocardial fibrosis mediate the anthracycline cardiomyopathy. ROS trigger TGF-β pathway that activates cardiac fibroblasts promoting fibrosis. Myocardial stiffness contributes to diastolic dysfunction, less studied aspect of anthracycline cardiomyopathy. Considering the role of SIRT1 in the inhibition of the TGF-β/SMAD3 pathway, resveratrol (RES), a SIRT1 activator, might improve cardiac function by interfering with the development of cardiac fibrosis in a model of DOXO-induced cardiomyopathy. METHODS F344 rats received a cumulative dose of 15 mg/kg of DOXO in 2 weeks or DOXO+RES (DOXO and RES, 2.5mg/kg/day, concomitantly for 2 weeks and then RES alone for 1 more week). The effects of RES on cardiac fibroblasts were also tested in vitro. RESULTS Along with systolic dysfunction, DOXO was also responsible of diastolic abnormalities. Myocardial stiffness correlated with fibroblast activation and collagen deposition. DOXO+RES co-treatment significantly improved ± dP/dt and, more interestingly, ameliorated end-diastolic pressure/volume relationship. Treatment with RES resulted in reduced fibrosis and fibroblast activation and, most importantly, the mortality rate was significantly reduced in DOXO+RES group. Fibroblasts isolated from DOXO+RES-treated rats, in which SIRT1 was upregulated, showed decreased levels of TGF-β and pSMAD3/SMAD3 when compared to cells isolated from DOXO-exposed hearts. CONCLUSIONS Our findings reveal a key role of SIRT1 in supporting animal survival and functional parameters of the heart. SIRT1 activation by interfering with fibrogenesis can improve relaxation properties of myocardium and attenuate myocardial remodeling related to chemotherapy.

Glutamate-induced ATP synthesis: relationship between plasma membrane Na+/Ca2+ exchanger and excitatory amino acid transporters in brain and heart cell models.

It is known that glutamate (Glu), the major excitatory amino acid in the central nervous system, can be an essential source for cell energy metabolism. Here we investigated the role of the plasma membrane Na+/Ca2+ exchanger (NCX) and the excitatory amino acid transporters (EAATs) in Glu uptake and recycling mechanisms leading to ATP synthesis. We used different cell lines, such as SH-SY5Y neuroblastoma, C6 glioma and H9c2 as neuronal, glial, and cardiac models, respectively. We first observed that Glu increased ATP production in SH-SY5Y and C6 cells. Pharmacological inhibition of either EAAT or NCX counteracted the Glu-induced ATP synthesis. Furthermore, Glu induced a plasma membrane depolarization and an intracellular Ca2+ increase, and both responses were again abolished by EAAT and NCX blockers. In line with the hypothesis of a mutual interplay between the activities of EAAT and NCX, coimmunoprecipitation studies showed a physical interaction between them. We expanded our studies on EAAT/NCX interplay in the H9c2 cells. H9c2 expresses EAATs but lacks endogenous NCX1 expression. Glu failed to elicit any significant response in terms of ATP synthesis, cell depolarization, and Ca2+ increase unless a functional NCX1 was introduced in H9c2 cells by stable transfection. Moreover, these responses were counteracted by EAAT and NCX blockers, as observed in SH-SY5Y and C6 cells. Collectively, these data suggest that plasma membrane EAAT and NCX are both involved in Glu-induced ATP synthesis, with NCX playing a pivotal role.

SIRT1 activation rescues doxorubicin-induced loss of functional competence of human cardiac progenitor cells

BACKGROUND The search for compounds able to counteract chemotherapy-induced heart failure is extremely important at the age of global cancer epidemic. The role of SIRT1 in the maintenance of progenitor cell homeostasis may contribute to its cardioprotective effects. SIRT1 activators, by preserving progenitor cells, could have a clinical relevance for the prevention of doxorubicin (DOXO)-cardiotoxicity. METHODS To determine whether SIRT1 activator, resveratrol (RES), interferes with adverse effects of DOXO on cardiac progenitor cells (CPCs): 1) human CPCs (hCPCs) were exposed in vitro to DOXO or DOXO+RES and their regenerative potential was tested in vivo in an animal model of DOXO-induced heart failure; 2) the in vivo effects of DOXO+RES co-treatment on CPCs were studied in a rat model. RESULTS In contrast to healthy cells, DOXO-exposed hCPCs were ineffective in a model of anthracycline cardiomyopathy. The in vitro activation of SIRT1 decreased p53 acetylation, overcame suppression of the IGF-1/Akt pro-survival and anti-apoptotic signaling, enhanced oxidative stress defense and prevented senescence and growth arrest of hCPCs. Priming with RES counterbalanced the onset of dysfunctional phenotype in DOXO-exposed hCPCs, partly restoring their ability to repair the damage with improvement in cardiac function and animal survival. The in vivo co-treatment DOXO+RES prevented the anthracycline-induced alterations in CPCs, partly preserving cardiac function. CONCLUSION SIRT1 activation protects DOXO-exposed CPCs and re-establishes their proper function. Pharmacological intervention at the level of tissue-specific progenitor cells may provide cardiac benefits for the growing population of long-term cancer survivors that are at risk of chemotherapy-induced cardiovascular toxicity.

Doxorubicin targets multiple players: A new view of an old problem☆

Graphical abstract Figure. No Caption available. ABSTRACT Anthracycline cardiotoxicity remains a serious problem in paediatric and adult cancer survivors, and the advancement of cardio‐oncology is a necessary step for an effective care of the patients that experience adverse cardiovascular effects. In this review, we discuss the multiple instruments used by clinicians that constitute the current strategies for primary and secondary prevention aiming at contrasting the onset of early and late doxorubicin‐induced cardiotoxic events. The importance of early detection of cardiotoxicity and the following pharmacological therapy has been acknowledged with the emphasis put on impaired diastolic function, an increasingly recognized precocious sign of doxorubicin cardiotoxicity with an emerging scientific and clinical interest. We highlight the involvement of progenitor cells of cardiac and extra‐cardiac origin as well as multiple cardiac cell types (fibroblasts and vasculature cells), focusing on molecular signals involved in cellular injury and response. Oxidative stress, DNA damage, senescence and cell death are established mechanisms driving anthracycline toxicity, but the comprehension of their relative weight on affecting specific cell type behaviour remains to be consolidated. The contribution of these crucial stressors and the emerging tools for preserving cell function are discussed.

Cardiac shock wave therapy: assessment of safety and new insights into mechanisms of tissue regeneration

Although low‐energy extracorporeal cardiac shock wave (ECSW) therapy represents an attractive non‐invasive treatment option for ischaemic heart disease, the precise mechanisms of its action and influence on the cardiac tissue remain obscure. The goal of this study was to evaluate the effects of SW application on cardiac function and structure. Four‐month‐old Fisher 344 rats were subjected to ECSW therapy. Echocardiographic measurements of cardiac function were performed at baseline and at 1 and 3 months after treatment. Signs of inflammation, apoptosis and fibrosis were evaluated by immunohistochemistry in the control and treated hearts. ECSW application did not provoke arrhythmia or increase the troponin‐I level. At all time points, the left ventricular ejection fraction and fractional shortening remained stable. Histological analysis revealed neither differences in the extracellular matrix collagen content nor the presence of fibrosis; similarly, there were no signs of inflammation. Moreover, a population of cardiac cells that responded eagerly to ECSW application in the adult heart was identified; c‐kit–positive, Ki67‐positive, orthochromatic cells, corresponding to cardiac primitive cells, were 2.65‐fold more numerous in the treated myocardium. In conclusion, non‐invasive ECSW therapy is a safe and effective way of activating cardiac stem cells and myocardial regeneration. Because many factors influence cellular turnover in the ischaemic myocardium during the course of ischaemic heart disease, cardiac remodelling, and heart failure progression, studies to identify the optimal treatment time are warranted.

pRb2/p130 Gene Overexpression Induces Astrocyte Differentiation

There are many data on the activity of the RB gene in neural differentiation and apoptosis, but the role of pRb2/p130 in neuronal and glial maturation has been far less investigated. To elucidate the role of pRb2/p130 in astrocyte development we overexpressed this protein in astrocytoma and normal astrocyte cultures by adenoviral-mediated gene transfer. In astrocytoma cells, p130/RB2 overexpression resulted in a significant reduction of cell growth and in an increased G(0)/G(1) cell population. We did not observe any induction of programmed cell death as determined by TUNEL reaction. Interestingly, pRb2/p130 overexpression induced astrocyte differentiation. Astrocyte cell cycle arrest and differentiation seemed to proceed through a way distinct from the p53 pathway.

Doxorubicin cardiotoxicity and target cells: a broader perspective

The cardiotoxicity of doxorubicin is becoming an interdisciplinary point of interest given a growing population of cancer survivors. The complex and not completely understood pathogenesis of this complication makes difficult to design successful preventive or curative measures. Although cardiomyocyte has been considered a classical cellular target, other cells including various types of undifferentiated cells are involved in myocardial homeostasis. Such perspective may shed light on previously unrecognized aspects of cardiotoxicity and promote new experimental and clinical cardioprotective strategies. In this review, different cellular targets of doxorubicin are discussed with the focus on cardiac progenitor cells, oxidative stress, DNA damage, senescence and apoptosis all of which contribute to their compromised functional properties.