| Name | Umberto |
| Surname | Galderisi |
| Institution | Università degli Studi della Campania Luigi Vanvitelli |
| Telephone | +39 081 566 75 85 |
| umberto.galderisi@unicampania.it | |
| Address | Dept. of Experimental Medicine, Via Luigi De Crecchio 7 – 80138 Napoli, Italy |
Following radiotherapy, bone sarcomas account for a significant percentage of recurring tumors. This risk is further increased in patients with hereditary retinoblastoma that undergo radiotherapy. We analyzed the effect of low and medium dose radiation on mesenchymal stromal cells (MSCs) with inactivated RB1 gene to gain insights on the molecular mechanisms that can induce second malignant neoplasm in cancer survivors. MSC cultures contain subpopulations of mesenchymal stem cells and committed progenitors that can differentiate into mesodermal derivatives: adipocytes, chondrocytes, and osteocytes. These stem cells and committed osteoblast precursors are the cell of origin in osteosarcoma, and RB1 gene mutations have a strong role in its pathogenesis. Following 40 and 2000 mGy X-ray exposure, MSCs with inactivated RB1 do not proliferate and accumulate high levels of unrepaired DNA as detected by persistence of gamma-H2AX foci. In samples with inactivated RB1 the radiation treatment did not increase apoptosis, necrosis or senescence versus untreated cells. Following radiation, CFU analysis showed a discrete number of cells with clonogenic capacity in cultures with silenced RB1. We extended our analysis to the other members of retinoblastoma gene family: RB2/P130 and P107. Also in the MSCs with silenced RB2/P130 and P107 we detected the presence of cells with unrepaired DNA following X-ray irradiation. Cells with unrepaired DNA may represent a reservoir of cells that may undergo neoplastic transformation. Our study suggests that, following radiotherapy, cancer patients with mutations of retinoblastoma genes may be under strict controls to evaluate onset of secondary neoplasms following radiotherapy.
Lysosomal storage disorders (LDS) comprise a group of rare multisystemic diseases resulting from inherited gene mutations that impair lysosomal homeostasis. The most common LSDs, Gaucher disease (GD), and Fabry disease (FD) are caused by deficiencies in the lysosomal glucocerebrosidase (GBA) and alpha-galactosidase A (GLA) enzymes, respectively. Given the systemic nature of enzyme deficiency, we hypothesized that the stem cell compartment of GD and FD patients might be also affected. Among stem cells, mesenchymal stem cells (MSCs) are a commonly investigated population given their role in hematopoiesis and the homeostatic maintenance of many organs and tissues. Since the impairment of MSC functions could pose profound consequences on body physiology, we evaluated whether GBA and GLA silencing could affect the biology of MSCs isolated from bone marrow and amniotic fluid. Those cell populations were chosen given the former's key role in organ physiology and the latter's intriguing potential as an alternative stem cell model for human genetic disease. Our results revealed that GBA and GLA deficiencies prompted cell cycle arrest along with the impairment of autophagic flux and an increase of apoptotic and senescent cell percentages. Moreover, an increase in ataxia-telangiectasia-mutated staining 1 hr after oxidative stress induction and a return to basal level at 48 hr, along with persistent gamma-H2AX staining, indicated that MSCs properly activated DNA repair signaling, though some damages remained unrepaired. Our data therefore suggest that MSCs with reduced GBA or GLA activity are prone to apoptosis and senescence due to impaired autophagy and DNA repair capacity.
Mesenchymal stromal cells (MSCs) are a heterogeneous population, which contain several cell phenotypes: mesenchymal stem cells, progenitor cells, fibroblasts and other type of cells. Previously, we identified unique stem cells that we named multilineage-differentiating stress enduring (Muse) cells as one to several percent of MSCs of the bone marrow, adipose tissue and dermis. Among different cell populations in MSCs, Muse cells, positive for pluripotent surface marker SSEA-3, may represent cells responsible for pluripotent-like property of MSCs, since they express pluripotency genes, able to differentiated into triploblastic cells from a single cells and are self-renewable. MSCs release biologically active factors that have profound effects on local cellular dynamics. A thorough examination of MSC secretome seems essential for understanding the physiological functions exerted by these cells in our organism and also for rational cellular therapy design. In this setting, studies on secretome of Muse cells may shed light on pathways that are associated with their specific features. Our findings evidenced that secretomes of MSCs and Muse cells contain factors that regulate extracellular matrix remodeling, ox-redox activities and immune system. Muse cells appear to secrete factors that may preserve their stem cell features, allow survival under stress conditions and may contribute to their immunomodulation capacity. In detail, the proteins belonging to protein kinase A signaling, FXR/RXR activation and LXR/RXR activation pathways may play a role in regulation of Muse stem cell features. These last 2 pathways together with proteins associated with antigen presentation pathway and coagulation system may play a role in immunomodulation.
Understanding the mechanisms by which mesenchymal stromal cells (MSCs) interact with the physical properties (e.g. topography, charge, ζ-potential, and contact angle) of polymeric surfaces is essential to design new biomaterials capable of regulating stem cell behavior. The present study investigated the ability of two polymers (pHM1 and pHM3) with different positive surface charge densities to modulate the differentiation of MSCs into osteoblast-like phenotype via cell-cell ephrinB2/EphB4 signaling. Although pHM1 promoted the phosphorylation of EphB4, leading to cell differentiation, pHM3, characterized by a high positive surface charge density, had no significant effect on EphB4 activation or MSCs differentiation. When the MSCs were cultured on pHM1 in the presence of a forward signaling blocking peptide, the osteoblast differentiation was compromised. Our results demonstrated that the ephrinB2/EphB4 interaction was required for MSCs differentiation into an osteoblast-like phenotype and that the presence of a high positive surface charge density altered this interaction.
The term 'epigenetics' refers to heritable, reversible DNA or histone modifications that affect gene expression without modifying the DNA sequence. Epigenetic modulation of gene expression also includes the RNA interference mechanism. Epigenetic regulation of gene expression is fundamental during development and throughout life, also playing a central role in disease progression. The transforming growth factor β1 (TGF-β1) and its downstream effectors are key players in tissue repair and fibrosis, extracellular matrix remodelling, inflammation, cell proliferation and migration. TGF-β1 can also induce cell switch in epithelial-to-mesenchymal transition, leading to myofibroblast transdifferentiation. Cellular pathways triggered by TGF-β1 in thoracic ascending aorta dilatation have relevant roles to play in remodelling of the vascular wall by virtue of their association with monogenic syndromes that implicate an aortic aneurysm, including Loeys-Dietz and Marfan's syndromes. Several studies and reviews have focused on the progression of aneurysms in the abdominal aorta, but research efforts are now increasingly being focused on pathogenic mechanisms of thoracic ascending aorta dilatation. The present review summarizes the most recent findings concerning the epigenetic regulation of effectors of TGF-β1 pathways, triggered by sporadic dilative aortopathy of the thoracic ascending aorta in the presence of a tricuspid or bicuspid aortic valve, a congenital malformation occurring in 0.5-2% of the general population. A more in-depth comprehension of the epigenetic alterations associated with TGF-β1 canonical and non-canonical pathways in dilatation of the ascending aorta could be helpful to clarify its pathogenesis, identify early potential biomarkers of disease, and, possibly, develop preventive and therapeutic strategies.
Senescent cells secrete senescence-associated secretory phenotype (SASP) proteins to carry out several functions, such as sensitizing surrounding cells to senesce; immunomodulation; impairing or fostering cancer growth; and promoting tissue development. Identifying secreted factors that achieve such tasks is a challenging issue since the profile of secreted proteins depends on genotoxic stress and cell type. Currently, researchers are trying to identify common markers for SASP. The present investigation compared the secretome composition of five different senescent phenotypes in two different cell types: bone marrow and adipose mesenchymal stromal cells (MSC). We induced MSC senescence by oxidative stress, doxorubicin treatment, X-ray irradiation, and replicative exhaustion. We took advantage of LC-MS/MS proteome identification and subsequent gene ontology (GO) evaluation to perform an unbiased analysis (hypothesis free manner) of senescent secretomes. GO analysis allowed us to distribute SASP components into four classes: extracellular matrix/cytoskeleton/cell junctions; metabolic processes; ox-redox factors; and regulators of gene expression. We used Ingenuity Pathway Analysis (IPA) to determine common pathways among the different senescent phenotypes. This investigation, along with identification of eleven proteins that were exclusively expressed in all the analyzed senescent phenotypes, permitted the identification of three key signaling paths: MMP2 - TIMP2; IGFBP3 - PAI-1; and Peroxiredoxin 6 - ERP46 - PARK7 - Cathepsin D - Major vault protein. We suggest that these paths could be involved in the paracrine circuit that induces senescence in neighboring cells and may confer apoptosis resistance to senescent cells.
Restenosis is a complex pathophysiological disease whose causative mechanisms are not fully understood. Previous studies allowed us to demonstrate the efficacy of bone marrow mesenchymal stromal cells (MSCs) transplantation in limiting the pathophysiological remodeling in a model of arteriotomy-induced (re) stenosis. In the current research we studied the effectiveness of G-CSF treatment on male rate rats that were subjected carotid arteriotomy in order to evaluate a potentially effective non-invasive strategy that recapitulates the MSC-mediated recovery of injured vessels. WKY male rats were subjected carotid arteriotomy and given a nine day treatment (3 days pre- to 6 days post-arteriotomy) with G-CSF or saline. Carotids were harvested 7 and 30 days following arteriotomy (early- and late-phase, respectively). Although morphometrical analysis did not reveal differences in lumen narrowing between G-CSF- and PBS-carotids 30 days following arteriotomy, we detected a noticeable conservative effect of G-CSF treatment on vascular wall morphology. Histological and molecular analysis revealed an increase in cellularity within the tunica media with a concomitant increase of the VSMCs differentiation markers both at early- and late-phases of (re) stenotic response in G-CSF-treated carotids (Sm22-alpha, Myocd, and Smtn). These findings were accompanied by the downregulation of oxidative stress-related genes in G-CSF-injured rats. The effect exerted by G-CSF in our model of arteriotomy-induced (re) stenosis seemed support the recovery of the architecture of the tunica media of injured vessels by: (i) inducing VSMCs differentiation; and (ii) limiting the oxidative-stress response induced by arteriotomy.
In the last year, the promising features of mesenchymal stem cells (MSCs), including their regenerative properties and ability to differentiate into diverse cell lineages, have generated great interest among researchers whose work has offered intriguing perspectives on cell-based therapies for various diseases. Currently the most commonly used adult stem cells in regenerative medicine, MSCs, can be isolated from several tissues, exhibit a strong capacity for replication in vitro, and can differentiate into osteoblasts, chondrocytes, and adipocytes. However, heterogeneous procedures for isolating and cultivating MSCs among laboratories have prompted the International Society for Cellular Therapy (ISCT) to issue criteria for identifying unique populations of these cells. Consequently, the isolation of MSCs according to ISCT criteria has produced heterogeneous, nonclonal cultures of stromal cells containing stem cells with different multipotent properties, committed progenitors, and differentiated cells. Though the nature and functions of MSCs remain unclear, nonclonal stromal cultures obtained from bone marrow and other tissues currently serve as sources of putative MSCs for therapeutic purposes, and several findings underscore their effectiveness in treating different diseases. To date, 493 MSC-based clinical trials, either complete or ongoing, appear in the database of the US National Institutes of Health. In the present article, we provide a comprehensive review of MSC-based clinical trials conducted worldwide that scrutinizes biological properties of MSCs, elucidates recent clinical findings and clinical trial phases of investigation, highlights therapeutic effects of MSCs, and identifies principal criticisms of the use of these cells. In particular, we analyze clinical trials using MSCs for representative diseases, including hematological disease, graft-versus-host disease, organ transplantation, diabetes, inflammatory diseases, and diseases in the liver, kidney, and lung, as well as cardiovascular, bone and cartilage, neurological, and autoimmune diseases.
Senescent cells secrete several molecules that help to prevent the progression of cancer. However, cancer cells can also misuse these secreted elements to survive and grow. Since the molecular and functional bases of these different elements remain poorly understood, we analyzed the effect of senescent mesenchymal stromal cell (MSC) secretome on the biology of ARH-77 myeloma cells. In addition to differentiating in mesodermal derivatives, MSCs have sustained interest among researchers by supporting hematopoiesis, contributing to tissue homeostasis, and modulating inflammatory response, all activities accomplished primarily by the secretion of cytokines and growth factors. Moreover, senescence profoundly affects the composition of MSC secretome. In this study, we induced MSC senescence by oxidative stress, DNA damage, and replicative exhaustion. While the first two are considered to induce acute senescence, extensive proliferation triggers replicative (i.e., chronic) senescence. We cultivated cancer cells in the presence of acute and chronic senescent MSC-conditioned media and evaluated their proliferation, DNA damage, apoptosis, and senescence. Our findings revealed that senescent secretomes induced apoptosis or senescence, if not both, to different extents. This anti-tumor activity became heavily impaired when secretomes were collected from senescent cells previously in contact (i.e., primed) with cancer cells. Our analysis of senescent MSC secretomes with LC-MS/MS followed by Gene Ontology classification further indicated that priming with cancer profoundly affected secretome composition by abrogating the production of pro-senescent and apoptotic factors. We thus showed for the first time that compared with cancer-primed MSCs, naïve senescent MSCs can exert different effects on tumor progression.
A sharp definition of what a senescent cell is still lacking since we do not have in depth understanding of mechanisms that induce cellular senescence. In addition, senescent cells are heterogeneous, in that not all of them express the same genes and present the same phenotype. To further clarify the classification of senescent cells, hints may be derived by the study of cellular metabolism, autophagy and proteasome activity. In this scenario, we decided to study these biological features in senescence of Mesenchymal Stromal Cells (MSC). These cells contain a subpopulation of stem cells that are able to differentiate in mesodermal derivatives (adipocytes, chondrocytes, osteocytes). In addition, they can also contribute to the homeostatic maintenance of many organs, hence, their senescence could be very deleterious for human body functions. We induced MSC senescence by oxidative stress, doxorubicin treatment, X-ray irradiation and replicative exhaustion. The first three are considered inducers of acute senescence while extensive proliferation triggers replicative senescence also named as chronic senescence. In all conditions, but replicative and high IR dose senescence, we detected a reduction of the autophagic flux, while proteasome activity was impaired in peroxide-treated and irradiated cells. Differences were observed also in metabolic status. In general, all senescent cells evidenced metabolic inflexibility and prefer to use glucose as energy fuel. Irradiated cells with low dose of X-ray and replicative senescent cells show a residual capacity to use fatty acids and glutamine as alternative fuels, respectively. Our study may be useful to discriminate among different senescent phenotypes.