Doxorubicin-Induced Cardiac Senescence Is Alleviated Following Treatment with Combined Polyphenols and Micronutrients through Enhancement in Mitophagy.

Oxidative stress and impaired mitophagy are the hallmarks of cardiomyocyte senescence. Specifically, a decrease in mitophagic flux leads to the accumulation of damaged mitochondria and the development of senescence through increased ROS and other mediators. In this study, we describe the preventive role of A5+, a mix of polyphenols and other micronutrients, in doxorubicin (DOXO)-induced senescence of H9C2 cells. Specifically, H9C2 cells exposed to DOXO showed an increase in the protein expression proteins of senescence-associated genes, p21 and p16, and a decrease in the telomere binding factors TRF1 and TRF2, indicative of senescence induction. Nevertheless, A5+ pre-treatment attenuated the senescent-like cell phenotype, as evidenced by inhibition of all senescent markers and a decrease in SA-ß-gal staining in DOXO-treated H9C2 cells. Importantly, A5+ restored the LC3 II/LC3 I ratio, Parkin and BNIP3 expression, therefore rescuing mitophagy, and decreased ROS production. Further, A5+ pre-treatment determined a ripolarization of the mitochondrial membrane and improved basal respiration. A5+-mediated protective effects might be related to its ability to activate mitochondrial SIRT3 in synergy with other micronutrients, but in contrast with SIRT4 activation. Accordingly, SIRT4 knockdown in H9C2 cells further increased MnSOD activity, enhanced mitophagy, and reduced ROS generation following A5+ pre-treatment and DOXO exposure compared to WT cells. Indeed, we demonstrated that A5+ protects H9C2 cells from DOXO-induced senescence, establishing a new specific role for A5+ in controlling mitochondrial quality control by restoring SIRT3 activity and mitophagy, which provided a molecular basis for the development of therapeutic strategies against cardiomyocyte senescence.

The PTX3/TLR4 autocrine loop as a novel therapeutic target in triple negative breast cancer.

BACKGROUND: The pattern recognition receptor long pentraxin-3 (PTX3) plays conflicting roles in cancer by acting as an oncosuppressor or as a pro-tumor mediator depending on tumor context. Triple negative breast cancer (TNBC) represents the most aggressive histotype of breast cancer, characterized by the lack of efficacious therapeutic targets/approaches and poor prognosis. Thus, the characterization of new molecular pathways and/or alternative druggable targets is of great interest in TNBC. METHODS: The expression of PTX3 in BC tumor samples and in BC cell lines has been analyzed using the Gene Expression-Based Outcome for Breast Cancer Online (GOBO), qPCR, Western blot and ELISA assay. The contribution of tumor and stromal cells to PTX3 production in TNBC was assessed by analyzing single cell RNA sequencing data and RNAscope performed on TNBC tumor samples. In order to investigate the effects of PTX3 in TNBC, different cell lines were engineered to knock-down (MDA-MB-231 and BT549 cells) or overexpress (MDA-MB-468 and E0771 cells) PTX3. Finally, using these engineered cells, in vitro (including gene expression profiling and gene set enrichment analyses) and in vivo (orthotopic tumor models in immune-compromised and immune competent mice) analyses were performed to assess the role and the molecular mechanism(s) exerted by PTX3 in TNBC. RESULTS: In silico and experimental data indicate that PTX3 is mainly produced by tumor cells in TNBC and that its expression levels correlate with tumor stage. Accordingly, gene expression and in vitro results demonstrate that PTX3 overexpression confers a high aggressive/proliferative phenotype and fosters stem-like features in TNBC cells. Also, PTX3 expression induces a more tumorigenic potential when TNBC cells are grafted orthotopically in vivo. Conversely, PTX3 downregulation results in a less aggressive behavior of TNBC cells. Mechanistically, our data reveal that PTX3 drives the activation of the pro-tumorigenic Toll-like receptor 4 (TLR4) signaling pathway in TNBC, demonstrating for the first time that the PTX3/TLR4 autocrine stimulation loop contributes to TNBC aggressiveness and that TLR4 inhibition significantly impacts the growth of PTX3-producing TNBC cells. CONCLUSION: Altogether, these data shed light on the role of tumor-produced PTX3 in TNBC and uncover the importance of the PTX3/TLR4 axis for therapeutic and prognostic exploitation in TNBC.

The TFEB-TGIF1 axis regulates EMT in mouse epicardial cells.

Epithelial-mesenchymal transition (EMT) is a complex and pivotal process involved in organogenesis and is related to several pathological processes, including cancer and fibrosis. During heart development, EMT mediates the conversion of epicardial cells into vascular smooth muscle cells and cardiac interstitial fibroblasts. Here, we show that the oncogenic transcription factor EB (TFEB) is a key regulator of EMT in epicardial cells and that its genetic overexpression in mouse epicardium is lethal due to heart defects linked to impaired EMT. TFEB specifically orchestrates the EMT-promoting function of transforming growth factor (TGF) ß, and this effect results from activated transcription of thymine-guanine-interacting factor (TGIF)1, a TGFß/Smad pathway repressor. The Tgif1 promoter is activated by TFEB, and in vitro and in vivo findings demonstrate its increased expression when Tfeb is overexpressed. Furthermore, Tfeb overexpression in vitro prevents TGFß-induced EMT, and this effect is abolished by Tgif1 silencing. Tfeb loss of function, similar to that of Tgif1, sensitizes cells to TGFß, inducing an EMT response to low doses of TGFß. Together, our findings reveal an unexpected function of TFEB in regulating EMT, which might provide insights into injured heart repair and control of cancer progression.

HMGB1-Mediated Activation of the Inflammatory-Reparative Response Following Myocardial Infarction.

Different cell types belonging to the innate and adaptive immune system play mutually non-exclusive roles during the different phases of the inflammatory-reparative response that occurs following myocardial infarction. A timely and finely regulation of their action is fundamental for the process to properly proceed. The high-mobility group box 1 (HMGB1), a highly conserved nuclear protein that in the extracellular space can act as a damage-associated molecular pattern (DAMP) involved in a large variety of different processes, such as inflammation, migration, invasion, proliferation, differentiation, and tissue regeneration, has recently emerged as a possible regulator of the activity of different immune cell types in the distinct phases of the inflammatory reparative process. Moreover, by activating endogenous stem cells, inducing endothelial cells, and by modulating cardiac fibroblast activity, HMGB1 could represent a master regulator of the inflammatory and reparative responses following MI. In this review, we will provide an overview of cellular effectors involved in these processes and how HMGB1 intervenes in regulating each of them. Moreover, we will summarize HMGB1 roles in regulating other cell types that are involved in the different phases of the inflammatory-reparative response, discussing how its redox status could affect its activity.

miR-200c-3p Regulates Epitelial-to-Mesenchymal Transition in Epicardial Mesothelial Cells by Targeting Epicardial Follistatin-Related Protein 1.

Recent findings suggest that epithelial to mesenchymal transition (EMT), a key step during heart development, is involved in cardiac tissue repair following myocardial infarction (MI). MicroRNAs (miRNAs) act as key regulators in EMT processes; however, the mechanisms by which miRNAs target epicardial EMT remain largely unknown. Here, by using an in vitro model of epicardial EMT, we investigated the role of miRNAs as regulators of this process and their potential targets. EMT was induced in murine epicardial-mesothelial cells (EMCs) through TGF ß1 treatment for 48, 72, and 96 h as indicated by the expression of EMT-related genes by qRT-PCR, WB, and immunofluorescence. Further, enhanced expression of stemness genes was also detected. Among several EMT-related miRNAs, miR-200c-3p expression resulted as the most strongly suppressed. Interestingly, we also found a significant upregulation of Follistatin-related protein 1 (FSTL1), a miR-200c predicted target already identified as a potent cardiogenic factor produced by epicardial cells that promotes regeneration following MI. Dual-luciferase reporter assay demonstrated that miR-200c-3p directly targeted the 3'-untranslated region of FSTL1 in EMCs. Consistently, WB analysis showed that knockdown of miR-200c-3p significantly increased FSTL1 expression, whereas overexpression of miR-200c-3p counteracted TGF ß1-mediated FSTL1 upregulation. Importantly, FSTL1 silencing maintained epithelial features in EMCs, despite EMT induction by TGF ß1, and attenuated EMT-associated traits, including migration and stemness. In conclusion, epicardial FSTL1, an important cardiogenic factor in its secreted form, induces EMT, stemness, and migration of EMCs in a miR-200c-3p dependent pathway.

MicroRNAs in Cancer Treatment-Induced Cardiotoxicity.

Cancer treatment has made significant progress in the cure of different types of tumors. Nevertheless, its clinical use is limited by unwanted cardiotoxicity. Aside from the conventional chemotherapy approaches, even the most newly developed, i.e., molecularly targeted therapy and immunotherapy, exhibit a similar frequency and severity of toxicities that range from subclinical ventricular dysfunction to severe cardiomyopathy and, ultimately, congestive heart failure. Specific mechanisms leading to cardiotoxicity still remain to be elucidated. For instance, oxidative stress and DNA damage are considered key players in mediating cardiotoxicity in different treatments. microRNAs (miRNAs) act as key regulators in cell proliferation, cell death, apoptosis, and cell differentiation. Their dysregulation has been associated with adverse cardiac remodeling and toxicity. This review provides an overview of the cardiotoxicity induced by different oncologic treatments and potential miRNAs involved in this effect that could be used as possible therapeutic targets.

FGF Trapping Inhibits Multiple Myeloma Growth through c-Myc Degradation-Induced Mitochondrial Oxidative Stress.

Multiple myeloma, the second most common hematologic malignancy, frequently relapses because of chemotherapeutic resistance. Fibroblast growth factors (FGF) act as proangiogenic and mitogenic cytokines in multiple myeloma. Here, we demonstrate that the autocrine FGF/FGFR axis is essential for multiple myeloma cell survival and progression by protecting multiple myeloma cells from oxidative stress-induced apoptosis. In keeping with the hypothesis that the intracellular redox status can be a target for cancer therapy, FGF/FGFR blockade by FGF trapping or tyrosine kinase inhibitor impaired the growth and dissemination of multiple myeloma cells by inducing mitochondrial oxidative stress, DNA damage, and apoptotic cell death that were prevented by the antioxidant vitamin E or mitochondrial catalase overexpression. In addition, mitochondrial oxidative stress occurred as a consequence of proteasomal degradation of the c-Myc oncoprotein that led to glutathione depletion. Accordingly, expression of a proteasome-nondegradable c-Myc protein mutant was sufficient to avoid glutathione depletion and rescue the proapoptotic effects due to FGF blockade. These findings were confirmed on bortezomib-resistant multiple myeloma cells as well as on bone marrow-derived primary multiple myeloma cells from newly diagnosed and relapsed/refractory patients, including plasma cells bearing the t(4;14) translocation obtained from patients with high-risk multiple myeloma. Altogether, these findings dissect the mechanism by which the FGF/FGFR system plays a nonredundant role in multiple myeloma cell survival and disease progression, and indicate that FGF targeting may represent a therapeutic approach for patients with multiple myeloma with poor prognosis and advanced disease stage. SIGNIFICANCE: This study provides new insights into the mechanisms by which FGF antagonists promote multiple myeloma cell death. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/11/2340/F1.large.jpg.

Cardiac Repair: The Intricate Crosstalk between the Epicardium and the Myocardium.

BACKGROUND: Substantial evidences support the hypothesis that the epicardium has a role in cardiac repair and regeneration in part providing, by epithelial to mesenchymal transition (EMT), progenitor cells that differentiate into cardiac cell types and in part releasing paracrine factors that contribute to cardiac repair. Besides cell contribution, a significant paracrine communication occurs between the epicardium and the myocardium that improves the whole regenerative response. Signaling pathways underlying this communication are multiple as well as soluble factors involved in cardiac repair and secreted both by myocardial and epicardial cells. Most recently, extracellular vesicles, i.e. exosomes, that accumulate in the pericardial fluid (PF) and are able to transport bioactive molecules (cytosolic proteins, mRNAs, miRNAs and other non-coding RNAs), have been also identified as potential mediators of epicardial-mediated repair following myocardial injury. CONCLUSION: This mini-review provides an overview of the epicardial-myocardial signaling in regulating cardiac repair in ischemic heart diseases. Indeed, a detailed understanding of the crosstalk between myocardial and epicardial cells and how paracrine mechanisms are involved in the context of ischemic heart diseases would be of tremendous help in developing novel therapeutic approaches to promote cardiomyocytes survival and heart regeneration following myocardial infarction (MI).

HMGB1-mediated apoptosis and autophagy in ischemic heart diseases.

Acute myocardial infarction (MI) and its consequences are the most common and lethal heart syndromes worldwide and represent a significant health problem. Following MI, apoptosis has been generally seen as the major contributor of the cardiomyocyte fate and of the resultant myocardial remodeling. However, in recent years, it has been discovered that, following MI, cardiomyocytes could activate autophagy in an attempt to protect themselves against ischemic stress and to preserve cardiac function. Although initially seen as two completely separate responses, recent works have highlighted the intertwined crosstalk between apoptosis and autophagy. Numerous researches have tried to unveil the mechanisms and the molecular players involved in this phenomenon and have identified in high-mobility group box 1 (HMGB1), a highly conserved non-histone nuclear protein with important roles in the heart, one of the major regulator. Thus, the aim of this mini review is to discuss how HMGB1 regulates these two responses in ischemic heart diseases. Indeed, a detailed understanding of the crosstalk between apoptosis and autophagy in these pathologies and how HMGB1 regulates them would be of tremendous help in developing novel therapeutic approaches aimed to promote cardiomyocyte survival and to diminish tissue injury following MI.

HMGB1 and repair: focus on the heart.

High-mobility group box 1 (HMGB1) is one of the most abundant proteins in eukaryotes and the best characterized damage-associated molecular pattern (DAMP). The biological activities of HMGB1 depend on its subcellular location, context and post-translational modifications. Inside the nucleus, HMGB1 is engaged in many DNA events such as DNA repair, transcription regulation and genome stability; in the cytoplasm, its main function is to regulate the autophagic flux while in the extracellular environment, it possesses more complicated functions and it is involved in a large variety of different processes such as inflammation, migration, invasion, proliferation, differentiation and tissue regeneration. Due to this pleiotropy, the role of HMGB1 has been vastly investigated in various pathological diseases and a large number of studies have explored its function in cardiovascular pathologies. However, in this contest, the precise mechanism of action of HMGB1 and its therapeutic potential are still very controversial since is debated whether HMGB1 is involved in tissue damage or plays a role in tissue repair and regeneration. The main focus of this review is to provide an overview of the effects of HMGB1 in different ischemic heart diseases and to discuss its functions in these pathological conditions.

Long Pentraxin 3-Mediated Fibroblast Growth Factor Trapping Impairs Fibrosarcoma Growth.

Fibrosarcomas are soft tissue mesenchymal tumors originating from transformed fibroblasts. Fibroblast growth factor-2 (FGF2) and its tyrosine-kinase receptors (FGFRs) play pivotal roles in fibrosarcoma onset and progression, FGF2 being actively produced by fibroblasts in all stages along their malignant transformation to the fibrosarcoma stage. The soluble pattern recognition receptor long pentraxin-3 (PTX3) is an extrinsic oncosuppressor whose expression is reduced in different tumor types, including soft tissue sarcomas, via hypermethylation of its gene promoter. PTX3 interacts with FGF2 and other FGF family members, thus acting as a multi-FGF antagonist able to inhibit FGF-dependent neovascularization and tumor growth. Here, PTX3 overexpression significantly reduced the proliferative and tumorigenic potential of fibrosarcoma cells in vitro and in vivo. In addition, systemic delivery of human PTX3 driven by the Tie2 promoter inhibited the growth of fibrosarcoma grafts in transgenic mice. In a translational perspective, the PTX3-derived small molecule FGF trap NSC12 prevented activation of the FGF/FGFR system in fibrosarcoma cells and reduced their tumorigenic activity in vivo. In conclusion, impairment of the FGF/FGFR system by FGF trap molecules may represent a novel therapeutic approach for the treatment of fibrosarcoma.

Long Pentraxin-3 Modulates the Angiogenic Activity of Fibroblast Growth Factor-2.

Angiogenesis, the process of new blood vessel formation from pre-existing ones, plays a key role in various physiological and pathological conditions. Alteration of the angiogenic balance, consequent to the deranged production of angiogenic growth factors and/or natural angiogenic inhibitors, is responsible for angiogenesis-dependent diseases, including cancer. Fibroblast growth factor-2 (FGF2) represents the prototypic member of the FGF family, able to induce a complex "angiogenic phenotype" in endothelial cells in vitro and a potent neovascular response in vivo as the consequence of a tight cross talk between pro-inflammatory and angiogenic signals. The soluble pattern recognition receptor long pentraxin-3 (PTX3) is a member of the pentraxin family produced locally in response to inflammatory stimuli. Besides binding features related to its role in innate immunity, PTX3 interacts with FGF2 and other members of the FGF family via its N-terminal extension, thus inhibiting FGF-mediated angiogenic responses in vitro and in vivo. Accordingly, PTX3 inhibits the growth and vascularization of FGF-dependent tumors and FGF2-mediated smooth muscle cell proliferation and artery restenosis. Recently, the characterization of the molecular bases of FGF2/PTX3 interaction has allowed the identification of NSC12, the first low molecular weight pan-FGF trap able to inhibit FGF-dependent tumor growth and neovascularization. The aim of this review is to provide an overview of the impact of PTX3 and PTX3-derived molecules on the angiogenic, inflammatory, and tumorigenic activity of FGF2 and their potential implications for the development of more efficacious anti-FGF therapeutic agents to be used in those clinical settings in which FGFs play a pathogenic role.

Molecular mechanisms of cardioprotective effects mediated by transplanted cardiac ckit+ cells through the activation of an inflammatory hypoxia-dependent reparative response.

The regenerative effects of cardiac ckit+ stem cells (ckit+CSCs) in acute myocardial infarction (MI) have been studied extensively, but how these cells exert a protective effect on cardiomyocytes is not well known. Growing evidences suggest that in adult stem cells injury triggers inflammatory signaling pathways which control tissue repair and regeneration. Aim of the present study was to determine the mechanisms underlying the cardioprotective effects of ckit+CSCs following transplantation in a murine model of MI. Following isolation and in vitro expansion, cardiac ckit+CSCs were subjected to normoxic and hypoxic conditions and assessed at different time points. These cells adapted to hypoxia as showed by the activation of HIF-1α and the expression of a number of genes, such as VEGF, GLUT1, EPO, HKII and, importantly, of alarmin receptors, such as RAGE, P2X7R, TLR2 and TLR4. Activation of these receptors determined an NFkB-dependent inflammatory and reparative gene response (IRR). Importantly, hypoxic ckit+CSCs increased the secretion of the survival growth factors IGF-1 and HGF. To verify whether activation of the IRR in a hypoxic microenvironment could exert a beneficial effect in vivo, autologous ckit+CSCs were transplanted into mouse heart following MI. Interestingly, transplantation of ckit+CSCs lowered apoptotic rates and induced autophagy in the peri-infarct area; further, it reduced hypertrophy and fibrosis and, most importantly, improved cardiac function. ckit+CSCs are able to adapt to a hypoxic environment and activate an inflammatory and reparative response that could account, at least in part, for a protective effect on stressed cardiomyocytes following transplantation in the infarcted heart.

SIRT1-SIRT3 Axis Regulates Cellular Response to Oxidative Stress and Etoposide.

Sirtuins are conserved NAD+ -dependent deacylases. SIRT1 is a nuclear and cytoplasmic sirtuin involved in the control of histones a transcription factors function. SIRT3 is a mitochondrial protein, which regulates mitochondrial function. Although, both SIRT1 and SIRT3 have been implicated in resistance to cellular stress, the link between these two sirtuins has not been studied so far. Here we aimed to unravel: i) the role of SIRT1-SIRT3 axis for cellular response to oxidative stress and DNA damage; ii) how mammalian cells modulate such SIRT1-SIRT3 axis and which mechanisms are involved. Therefore, we analyzed the response to different stress stimuli in WT or SIRT1-silenced cell lines. Our results demonstrate that SIRT1-silenced cells are more resistant to H2 O2 and etoposide treatment showing decreased ROS accumulation, γ-H2AX phosphorylation, caspase-3 activation and PARP cleavage. Interestingly, we observed that SIRT1-silenced cells show an increased SIRT3 expression. To explore such a connection, we carried out luciferase assays on SIRT3 promoter demonstrating that SIRT1-silencing increases SIRT3 promoter activity and that such an effect depends on the presence of SP1 and ZF5 recognition sequences on SIRT3 promoter. Afterwards, we performed co-immunoprecipitation assays demonstrating that SIRT1 binds and deacetylates the transcription inhibitor ZF5 and that there is a decreased interaction between SP1 and ZF5 in SIRT1-silenced cells. Therefore, we speculate that acetylated ZF5 cannot bind and sequester SP1 that is free, then, to increase SIRT3 transcription. In conclusion, we demonstrate that cells with low SIRT1 levels can maintain their resistance and survival by increasing SIRT3 expression. J. Cell. Physiol. 232: 1835-1844, 2017. © 2016 Wiley Periodicals, Inc.

HMGB1 Inhibits Apoptosis Following MI and Induces Autophagy via mTORC1 Inhibition.

Exogenous High Mobility Group Box-1 protein (HMGB1) has been reported to protect the infarcted heart but the underlying mechanism is quite complex. In particular, its effect on ischemic cardiomyocytes has been poorly investigated. Aim of the present study was to verify whether and how autophagy and apoptosis were involved in HMGB1-induced heart repair following myocardial infarction (MI). HMGB1 (200 ng) or denatured HMGB1 were injected in the peri-infarcted region of mouse hearts following acute MI. Three days after treatment, an upregulation of autophagy was detected in infarcted HMGB1 treated hearts compared to controls. Specifically, HMGB1 induced autophagy by significantly upregulating the protein expression of LC3, Beclin-1, and Atg7 in the border zone. To gain further insights into the molecular mechanism of HMGB1-mediated autophagy, WB analysis were performed in cardiomyocytes isolated from 3 days infarcted hearts in the presence and in the absence of HMGB1 treatment. Results showed that upregulation of autophagy by HMGB1 treatment was potentially related to activation of AMP-activated protein kinase (AMPK) and inhibition of the mammalian target of rapamycin complex 1 (mTORC1). Accordingly, in these hearts, phospho-Akt signaling pathway was inhibited. The induction of autophagy was accompanied by reduced cardiomyocyte apoptotic rate and decreased expression levels of Bax/Bcl-2 and active caspase-3 in the border zone of 3 days infarcted mice following HMGB1 treatment. We report the first in vivo evidence that HMGB1 treatment in a murine model of acute MI might induce cardiomyocyte survival through attenuation of apoptosis and AMP-activated protein kinase-dependent autophagy. J. Cell. Physiol. 232: 1135-1143, 2017. © 2016 Wiley Periodicals, Inc.

Exosomal clusterin, identified in the pericardial fluid, improves myocardial performance following MI through epicardial activation, enhanced arteriogenesis and reduced apoptosis.

BACKGROUND: We recently demonstrated that epicardial progenitor cells participate in the regenerative response to myocardial infarction (MI) and factors released in the pericardial fluid (PF) may play a key role in this process. Exosomes are secreted nanovesicles of endocytic origin, identified in most body fluids, which may contain molecules able to modulate a variety of cell functions. Here, we investigated whether exosomes are present in the PF and their potential role in cardiac repair. METHODS AND RESULTS: Early gene expression studies in 3day-infarcted mouse hearts showed that PF induces epithelial-to-mesenchymal transition (EMT) in epicardial cells. Exosomes were identified in PFs from non-infarcted patients (PFC) and patients with acute MI (PFMI). A shotgun proteomics analysis identified clusterin in exosomes isolated from PFMI but not from PFC. Notably, clusterin has a protective effect on cardiomyocytes after acute MI in vivo and is an important mediator of TGFß-induced. Clusterin addition to the pericardial sac determined an increase in epicardial cells expressing the EMT marker α-SMA and, interestingly, an increase in the number of epicardial cells ckit(+)/α-SMA(+), 7days following MI. Importantly, clusterin treatment enhanced arteriolar length density and lowered apoptotic rates in the peri-infarct area. Hemodynamic studies demonstrated an improvement in cardiac function in clusterin-treated compared to untreated infarcted hearts. CONCLUSIONS: Exosomes are present and detectable in the PFs. Clusterin was identified in PFMI-exosomes and might account for an improvement in myocardial performance following MI through a framework including EMT-mediated epicardial activation, arteriogenesis and reduced cardiomyocyte apoptosis.

Generation of cardiac progenitor cells through epicardial to mesenchymal transition.

The epithelial to mesenchymal transition (EMT) is a biological process that drives the formation of cells involved both in tissue repair and in pathological conditions, including tissue fibrosis and tumor metastasis by providing cancer cells with stem cell properties. Recent findings suggest that EMT is reactivated in the heart following ischemic injury. Specifically, epicardial EMT might be involved in the formation of cardiac progenitor cells (CPCs) that can differentiate into endothelial cells, smooth muscle cells, and, possibly, cardiomyocytes. The identification of mechanisms and signaling pathways governing EMT-derived CPC generation and differentiation may contribute to the development of a more efficient regenerative approach for adult heart repair. Here, we summarize key literature in the field.

Transcriptional profiling of HMGB1-induced myocardial repair identifies a key role for Notch signaling.

Exogenous high-mobility group box 1 protein (HMGB1) administration to the mouse heart, during acute myocardial infarction (MI), results in cardiac regeneration via resident c-kit(+) cell (CPC) activation. Aim of the present study was to identify the molecular pathways involved in HMGB1-induced heart repair. Gene expression profiling was performed to identify differentially expressed genes in the infarcted and bordering regions of untreated and HMGB1-treated mouse hearts, 3 days after MI. Functional categorization of the transcripts, accomplished using Ingenuity Pathway Analysis software (IPA), revealed that genes involved in tissue regeneration, that is, cardiogenesis, vasculogenesis and angiogenesis, were present both in the infarcted area and in the peri-infarct zone; HMGB1 treatment further increased the expression of these genes. IPA revealed the involvement of Notch signaling pathways in HMGB1-treated hearts. Importantly, HMGB1 determined a 35 and 58% increase in cardiomyocytes and CPCs expressing Notch intracellular cytoplasmic domain, respectively. Further, Notch inhibition by systemic treatment with the γ-secretase inhibitor DAPT, which blocked the proteolytic activation of Notch receptors, reduced the number of CPCs, their proliferative fraction, and cardiomyogenic differentiation in HMGB1-treated infarcted hearts. The present study gives insight into the molecular processes involved in HMGB1-mediated cardiac regeneration and indicates Notch signaling as a key player.

Vascular endothelial cells and dysfunctions: role of melatonin.

Several pathological conditions, including hypertension, atherosclerosis, diabetes, ischemia/reperfusion injury and nicotine-induced vasculopathy, are associated with vascular endothelial dysfunction characterized by altered secretory output of endothelial cells. Therefore there is a search for molecules and interventions that could restore endothelial function, in particular augmenting NO production, reducing the generation of free radicals and vasoconstrictors and preventing undesired inflammation. The pineal hormone melatonin exhibits several endothelium protective properties: it scavenges free radicals, activates antioxidant defence enzymes, normalizes lipid and blood pressure profile and increases NO bioavailability. Melatonin improved vascular function in experimental hypertension, reducing intimal infiltration and restoring NO production. Melatonin improved the NO pathway also in animal models for the study of diabetes and prevented NO down-regulation and adhesive molecules up-regulation in nicotine-induced vasculopathy. The protection against endothelial damage, vasoconstriction, platelet aggregation and leukocyte infiltration might contribute to the beneficial effects against ischemia-reperfusion injury by melatonin. Therefore, melatonin administration has endothelium-protective potential in several pathological conditions. Nevertheless, it still needs to be established, whether melatonin is able to revert already established endothelial dysfunction in these conditions.

Aging and vascular dysfunction: beneficial melatonin effects.

Aging is characterized by a progressive deterioration of physiological functions and metabolic processes. In aging and in diseases associated with the elderly, the loss of cells in vital structures or organs may be related to several factors. Sirtuin1 (SIRT1) is a member of the sirtuin family of protein deacetylases involved in life span extension; however, its involvement in the aging is not yet completely defined. Recently, melatonin, a pleiotropic molecule, shown to activate SIRT1 in primary neurons of young animals, as well as in aged neurons of a murine model of senescence. Melatonin is known to modulate oxidative stress-induced senescence and pro-survival pathways. We treated 6- and 15-week-old apolipoprotein E (APOE)-deficient mice (APOE 6w and 15w) with two melatonin formulations (FAST and RETARD) to evaluate their anti-aging effect. Morphological changes in vessels (aortic arch) of APOE mice were evaluated SIRT1, p53, endothelial nitric oxide synthase (eNOS), and endothelin-1 (ET-1) markers. We demonstrate that SIRT1 and eNOS decresed in APOE mice between 6 and 15 weeks and that aging induced an elevated expression of p53 and ET-1 in APOE animals. Melatonin improved the impairment of endothelial damage and reduced loss of SIRT1 and eNOS decreasing p53 and ET-1 expression. The RETARD melatonin preparation caused a greater improvement of vessel cytoarchitecture. In summary, we indicate that SIRT1-p53-eNOS axis as one of the important marker of advanced vascular dysfunctions linked to aging. Finally, we suggest that extended-release melatonin (RETARD) provides a more appropriate option for contrasting these dysfunctions compared with rapid release melatonin (FAST) administration.