DNA damage has been long recognized as causal factor for cancer development. When erroneous DNA repair leads to mutations or chromosomal aberrations affecting oncogenes and tumor suppressor genes, cells undergo malignant transformation resulting in cancerous growth. Genetic defects can predispose to cancer: mutations in distinct DNA repair systems elevate the susceptibility to various cancer types. However, DNA damage not only comprises a root cause for cancer development but also continues to provide an important avenue for chemo- and radiotherapy. Since the beginning of cancer therapy, genotoxic agents that trigger DNA damage checkpoints have been applied to halt the growth and trigger the apoptotic demise of cancer cells. We provide an overview about the involvement of DNA repair systems in cancer prevention and the classes of genotoxins that are commonly used for the treatment of cancer. A better understanding of the roles and interactions of the highly complex DNA repair machineries will lead to important improvements in cancer therapy. Erroneous repair of lesions can lead to mutations in the genome that can be inherited to daughter cells with deleterious consequences for individual’s health. As a consequence, eukaryotic cells have evolved a complex signaling network of repair processes known as the DNA damage response (DDR). The importance of DNA repair mechanisms is highlighted by the existence of many devastating human syndromes that are caused by defects in DDR genes. Notably, many of these mutations generally display increased sensitivity to DNA damaging agents and predispose to the development of specific cancer types . AlreadyXeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD) are rare autosomal recessive diseases caused by defects in the nucleotide excision repair (NER) pathway that protects the DNA molecule from the damage inflicted by UV irradiation . Indeed,The NER-associated diseases share an increased sun-sensitivity and freckling in the skin areas exposed to the sun but while XP is a skin cancer-prone (>1000-fold increase) disease  to Xeroderma pigmentosum, complementation group G (XPG); Nucleotide excision repair repairs the major lesions caused by UV light, the CPDs and 6-4PPs that distort the DNA double helix. A similar type of damage, as well resolved by NER, is caused by polycyclic aromatic hydrocarbons  and ε (in dividing cells) synthesize the new error-free sequence starting from the 3′-hydroxyl extremity generated by XPF-ERCC1. The remaining 3′-end incision is finally closed by Ligase I or III  generates a transiently open DNA structure by using the 3′-5′ and 5′-3′ nuclease activity of the two ATP-dependent helicases XPB and XPD . The funI or III .The TC-NER subpathway initiates when RNA polymerase stalls at a DNA lesion. XPC, which without XPE is incapable of binding to CPDs , is dispThe major regulators of the DDR are the two serine-threonine kinases ATM [ataxia telangiectasia (AT) mutated] and ATR (ATM and RAD3-related) which both belong, together with SMG-1 , DNA-PKcs  and mTOR , to the phosphonositide 3-kinase (PI3K)-related protein kinases (PIKKs) family. All of them share a conserved C-terminal kinase domain structure flanked by the FAT and FATC domains, two conserved regions, with high sequence similarity, regulating the kinase activity .The overlapping substrates of ATM and ATR comprise more than 700 different proteins mainly involved in DNA repair, cell cycle arrest, and transcription but also in developmental processes, immunity and intracellular protein traffic . Among tHumans carrying homozygous mutations (0.5–1%) in the ATM gene  suffer from the neurodegenerative disease AT, which is characterized by radiation sensitivity, chromosomal instability and predisposition to cancer. Up to 30% of AT patients develop lymphoid tumors since ATM play a critical role in the differentiation of T and B cells . CarrierHypomorphic mutations in ATR lead to Seckel syndrome. The main features of this disease are growth retardation, microcephaly and a characteristic “bird-headed” facial appearance . While gAlthough they share many substrates, ATM and ATR are activated in different ways. ATR is mainly induced upon DNA single strand breaks (SSBs) originated by replication fork stalling or as result of double strand breaks (DSBs) processing and NER activity. On the other hand, ATM primarily responds to DSBs caused by IR or ROS as well as breaks coming from physiological processes like meiosis, telomere maintenance, or immune system maturation (assembly of the T cell receptor and immunoglobulin genes via V(D)J recombination; In the ATR activation process: RPA, after coating the single strand DNA, recruits the ATR interacting protein (ATRIP). This complex helps to localize the site of damage  and to dAtaxia telangiectasia mutated is found in the nucleus of undamaged cells in the form of inactive dimers or higher order multimers, configuration that inhibit, by masking with the FAT domain, the kinase domain. Upon DNA damage, ATM undergoes autophosphorylation on residues Ser367, Ser1893, and Ser1981 with the last one located within the FAT domain. These posttranslational modifications result in dimer dissociation and release of active kinase monomers . Upon foAtaxia telangiectasia mutated exerts its survival function through the induction of cell cycle checkpoints. In the G1-S checkpoint, ATM phosphorylates the tumor suppressor p53 on S15 leading to the disruption of the inhibitory association with MDM2. Activated p53 induces p21, which binds to and inhibits the S-phase-promoting Cdk2-CyclinE complex .During the G2-M checkpoint, ATM phosphorylates monomers of CHK2 on Thr68 allowing the formation of CHK2 dimers that have as a main target the cell division cycle 25 homolog A (CDC25A). Phosphorylated CDC25A can finally be degraded by the proteasome and prevents cyclin-dependent kinase 2 (CDK2) and CDK1 dephosphorylation, which is required for progression through the cell cycle .DNA inter- and intrastrand crosslinks represent a dangerous form of damage blocking vital cellular processes like transcription and replication. The Fanconi anemia (FA) pathway is responsible to repair these aberrations arising in the DNA structure as a result of chemotherapeutics drugs treatment, like cisplatin or mitomycin C , or natuFanconi Anemia is a heterogeneous genetic disease, 16 different genes are involved in the establishment of the disorder and they can be divided in three major groups: the FA core complex, the I-D2 complex and downstream FA proteins. Eight proteins form the core complex, respectively named FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM while the I-D2 complex is constituted by FANCD2 and FANCI . In the Despite the fact that FANCD2 was shown to have intrinsic nuclease activity , other nThe FA pathway allows resolving the replication fork stalling by inducing the formation of a DSB and by coordinating the action of three critical repair mechanisms: translesion synthesis (TLS) bypasses the lesion and, after toxic adducts removal by NER, the gap is closed by homologous recombination (HR). The ID complex is finally able to leave the previously damaged area thanks to deubiquitination mediated by USP1 (ubiquitin specific peptidase 1) and UAF1 , DNA-dependent ATPase and 5′-3′ DNA helicase able to interact with BRCA1; FANCD1 (BRCA2), able to bind ssDNA and dsDNA and to stimulate RAD51 action; FANCN , required for FANCD1 stabilization and for the recruitment of BRCA2 and RAD51; and FANCO (RAD51C) involved in HJ resolution .C. elegans, DT40 chicken and mammalian cells by inhibiting NHEJ components  model. In the first one, also known as Double HJ model, the 5′ and 3′ ends of a DSB are resected by nucleases  and the 3′ ssDNA filament invades the intact sister chromatid, which is used as a template to repair the lesion. The displacement of the second strand results in the formation of a D-loop. The extension of the 3′ invading strand transforms the D-loop to a cross-shape structure known as HJ. The second 3′ overhang, not involved in the initial strand invasion, also produces a HJ with the homologous chromosome. This way of repair may result in the formation of chromosomal crossovers and principally takes place during meiosis . To avoiThe SDSA process shares all the steps of the classic HR repair model except for the absence of the D-loop structure formation . SDSA alBRCA1 and BRCA2 mutations are found in approximately 5–7% of all hereditary breast cancers  through its different interaction domains. Located at the N-terminus, the RING domain is the site for the interaction with BARD1 (BRCA-associated RING domain 1), a structurally-related protein responsible for BRCA1 stabilization and activity . The BRCBreast cancer susceptibility gene 1 is a component of three different multiprotein complexes involved in all cell cycle checkpoints: the BRCA1A complex , responsible to recruit BRCA1 to damaged sites; the BRCA1B complex (formed with BRIP1 and TOPBP1), mainly associated with replication-coupled DNA repair and the BRCA1C complex (formed together with CtIP and the MRN complex), which promotes HR despite NHEJ .BRCA1/BARD mutations cannot only fuel genome instability due to impaired HR activity, but also promote genome stability as recently shown in C. elegans mutants of the smc-5/6 complex that leads to replicative impediments and DSB formation at stalled replication forks  in tumorigenesis is highlighted by the fact that loss of expression of MMR proteins predispose to colorectal, gastric, endometrial and ovarian cancers and inherited defects in the MMR genes are associated with the most prevalent cancer syndrome in humans, the Lynch syndrome (LS), previously known as hereditary nonpolyposis colorectal cancer HNPCC; . MoreoveThe MMR pathway recognizes base–base mismatches and insertion-deletion loops IDLs;  originatδ and sealing of the nick by DNA ligase I , MutLβ (MLH1-MLH3), and MutLγ (MLH1-PMS1) can be recruited to form, with MutS, a ternary structure. The complex formed with MutLα is the most important in the MMR pathway, is able to translocate in both directions along the damaged area and to recruit proliferating cell nuclear antigen (PCNA), RFC, and EXO1 to perform the excision step . MutLβ fligase I .Being part of the replication fork, the MMR machinery operates mostly in dividing cells , nonetheMismatch repair dysfunction accounts for the mutator phenotype in which base substitution and frameshift mutations are highly increased due to microsatellite instability (MSI). Microsatellites are short tandem repeated DNA sequences of 1–4 base nucleotides spread all over the genome. Replication of these repeats has high error risk and when they are present in tumor suppressor genes, a defective repair may have detrimental consequences MSI; .S.S. John Harvey was unleashed in the Italian harbor of Bari during a German air raid. Physicians detected reduced white blood counts in autopsies following the incidence. It turned out that the vessel’s load of nitrogen mustard had attacked the white blood cells suggesting that leukemias could be targeted by nitrogen mustard therapy. Already a few years later the first alkylating agents were introduced to cancer therapy. Strikingly, it was found that effective chemotaxis such as nitrogen mustard and cisplatin evoke damage in nuclear DNA that then results in cell death. Therefore, DNA damage not only causes tumor development but could also battle cancers by impairing cancer growth and ultimately triggering the death of malignant cells  is one of the most widely used chemotherapeutic drugs. Its antitumor potential was discovered in the sixties by Escherichia coli cell division. Cisplatin soon drew interest in the scientific community and, after its efficacy was proven in mouse models , methionine and metallothioneins (MT) but its cytotoxic effect comes from the capacity to target DNA  is associated with a reduced intracellular accumulation respect to wild type MEFs (The copper transporter 1 (CTR1) regulates cisplatin cellular uptake. Cisplatin treatment of ype MEFs  and CTR1ype MEFs . Copper ype MEFs . In addiype MEFs .Cisplatin resistance can also be established through the interaction with intracellular thiol-containing molecules such as GSH and MT. They can both sequester cisplatin within the cytoplasmic compartment and correlations between their expression level and cisplatin resistance were found in ovarian, cervical, lung, and bladder cancer cell lines .Cisplatin-induced DNA damage is primarily repaired by the FA pathway , as wellLike NER, also MMR deficiency compromises cisplatin-induced apoptotic signaling  and it wWhile cisplatin has a strong anti-cancer activity, it also exerts negative side effects like nephro- and neurotoxicity . The negcis-chloro groups, a bidentate dicarboxylate ligand, which slow down reactivity and unfavorable side effects. Carboplatin is actually used in the treatment of ovarian, head and neck, and lung tumors  and pyrimidine  nucleosides, the fundamental precursors of ATP, DNA, and RNA. This class of drugs is widely used in hematological malignancies and solid tumors and, as well, for the treatment of viral infections .Nucleoside analogs exert their cytotoxic activities after being incorporated into DNA and RNA molecules leading respectively to replication and transcription inhibition, or by directly interfering with critical enzymes such as polymerases, kinases, ribonucleotide reductases, methyltransferases, nucleoside phosphorylases, and thymidylate synthases . The celTargeting every proliferating cell, the lack of specificity of nucleoside analogs leads to negative side effects ranging from bone marrow suppression with immune system depletion to neurotoxicity. Concomitantly, targeted cells can also develop resistance to nucleoside analogs due to decreased activity of the dCK/dGK activating enzymes or by loss of expression of the NTs.Cytarabine or ara-c was the first nucleoside analog developed starting from modification of 2-deoxycytidine and approved by FDA in 1969 for acute myeloid leukemia (AML) treatment . Ara-c cD-configuration, Troxacitabine is a different kind of pyrimidine analog forming the opposite conformation, the β-l. Its uptake is not mediated by ENTs or CNTs and it is phosphorylated by a different type of kinase, the 3-phosphoglycerate kinase. Troxacitabine’s antiproliferative activity was demonstrated in clinical trials for both solid and hematological malignancies  or to strand breakage due to the formation of an apurinic/apyrimidinic (AP) site. On the other side, the two electrophilic sites of bifunctional agents can attack two different bases on the same or on opposite DNA filaments to form intra- or interstrand crosslinks, respectively, which potentially inhibit strand separation during replication or transcription.Alkylating agents are one of the oldest antineoplastic drugs. The first glimpse of a therapeutic potential of this class of compounds appeared during the first world war when it was noticed that people exposed to sulfur mustard, a chemical warfare, were developing bone marrow suppression and lymphoid aplasia . In 1949 adducts . Base al adducts . AlkylatDNA crosslinks can also be introduced as a result of the interaction between two adjacent bases previously modified by monofunctional agents . AlkylatNitrogen mustards represent the oldest group of bifunctional alkylating agents initially used to treat cancer patients. Due to the short half-life and high toxicity, the use of chloremethine, the progenitor of this class of compounds, is actually restricted to veterinary medicine but many of its derivatives were developed and are actually applied in the treatment of different neoplasias. Chlorambucil and Bendamustine are used for treating chronic lymphocytic leukemia (CLL). Melphalan, apart from being implied in breast and ovarian cancers, Hodgkin’s disease and neuroblastoma, is the standard treatment, in combination with prednisone, for multiple myeloma .Cyclophosphamide, the most used drug of this class of agents, possesses the broadest spectrum of anticancer activity. In addition to its beneficial role in hematological malignancies, it is also effective in the treatment of solid tumors like bladder, brain, breast, cervix, endometrium, lung, ovary, and testis . IfosfamBusulfan belongs to the class of alkyl sulfonates and is one of the most important bifunctional agent for the cure of chronic myelogenous leukemia CML; , lymphomThiotepa and altretamine are examples of another class of bifunctional alkylating agents, ethyleneimines. The first one is used for ovarian, breast, and bladder cancer , while tin vitro proliferation of different cancer cell lines  or direct reversal. BER is initiated by DNA glycosylases, which recognize and remove the DNA lesion with the concomitant formation of an abasic (AP) site. The AP site is then processed by specific endonucleases and the missing nucleotide is inserted by DNA polymerase-β. Sealing of the nick is performed by DNA ligase, which finally restores the DNA integrity  molecules as a substrate to form polymers of ADP ribose units (PAR) on target proteins. This post-translational modification, known as PARylation  and charged particles are the main forms of IR utilized in cancer therapy. X- and gamma rays represent widely used photon beams with low radiation charge generated respectively from electrons exciting devices and from the decay of radioactive substances like caesium, cobalt or radium. Once they enter the body, electromagnetic waves of photons do not stop on their targets but they keep going and affecting the surrounding healthy tissues by interacting with the electrons of other molecules. Moreover, the radiation dose decreases as the depth of penetration in the body increase .In photon therapy, most of the DNA damage is inflicted indirectly by the reaction with free radicals species formed upon ionization of water components. Of consequence, the availability of oxygen becomes one of the major limitations in treating solid tumors that are known to be hypoxic. To overcome this problem, chemical radiosensitizer that can react with free radicals in a similar way to oxygen have been developed. Nimorazole and Sanazole represent the best examples of oxygen mimicking drugs actually adopted in the clinic .Charged particles radiation therapy use cyclotron and synchrotron to accelerate electrons, protons or heavy ions like carbon causing direct DNA damage due to the higher linear energy transfer (LET) capacity. The large mass of protons and other charged particles, and their unique absorption profile (Bragg’s peak: maximum release of energy when the particles stop traveling through the body) minimize the lateral side scatter and inflict a more precise damage to the target . AlthougDNA damage occurs on a daily basis by endogenous and exogenous sources. Distinct DNA repair systems recognize and remove the lesions. When the damage remains unrepaired DNA damage checkpoints can halt the cell cycle or induce cellular senescence or apoptosis. Erroneous repair or replicative bypass of lesions can result in mutations and chromosomal aberrations. When mutations affect tumor suppressor genes or oncogenes, cell might transform into cancer cells. Therefore, DNA repair is essential for preventing tumor development. However, once a cancer has developed, DNA damage can be exploited to reduce cancerous growth and evoke apoptotic demise of cancer cells. Thus, chemo- and radiotherapies are still today, over 60 years after having been first introduced into tumor therapy, important strategies to fight cancer. Given the central role of genome instability in triggering and treating cancer, it is likely that genotoxic treatments will remain an important avenue of cancer therapy. Also the better understanding of DNA repair systems will allow therapies that specifically target selected repair pathways. It will be of particular importance to gain a deeper understanding how the various DNA repair systems interact with each other in the context of cellular homeostasis and DNA metabolism in order to optimize targeted approaches to cancer therapy.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In spite of major recent advances in diffuse intrinsic pontine glioma (DIPG) molecular characterization, this body of knowledge has not yet translated into better treatments. To date, more than 250 clinical trials evaluating radiotherapy along with conventional cytotoxic chemotherapy as well as newer biologic agents have failed to improve the dismal outcome when compared to palliative radiation alone. The biology of DIPG remained unknown until recently when the neurosurgical expertise along with the recognition by the scientific and clinical community of the importance of tissue sampling at diagnosis; ideally, in the context of a clinical trial and by trained neurosurgical teams to maximize patient safety. These pre-treatment tumor samples, and others coming from tissue obtained post-mortem, have yielded new insights into DIPG molecular pathogenesis. We now know that DIPG comprises a heterogeneous disease with variable molecular phenotypes, different from adult high-grade glioma, other non-pontine pediatric high-grade gliomas, and even between pontine gliomas. The discovery of histone H3.3 or H3.1 mutations has been an important step forward in understanding tumor formation, maintenance, and progression. Pharmacologic reversal of DIPG histone demethylation therefore offers an important potential intervention strategy for the treatment of DIPG. To date, clinical trials of newly diagnosed or progressive DIPG with epigenetic (histone) modifiers have been unsuccessful. Whether this failure represents limited activity of the agents used, their CNS penetration, redundant pathways within the tumor, or the possibility that histone mutations are necessary only to initiate DIPGs but not maintain their growth, suggest that a great deal still needs to be elucidated in both the underlying biology of these pathways and the drugs designed to target them. In this review, we will discuss the role of both epigenetic and genetic mutations within DIPG and the development of treatment strategies directed against the unique abnormalities present in this disease. Diffuse intrinsic pontine glioma (DIPG), the most frequent brainstem tumor in pediatrics, is one of the deadliest cancers among children. Until recently, diagnosis has been based on classic clinical and radiological presentation, with “no indication” for histologic confirmation in the vast majority of cases . This pr+/Vimentin+ neural pontine precursor-like cell as a candidate for DIPG cell of origin, linked both anatomically – restricted to the ventral pons – and temporally to the incidence of DIPG. Additionally, the same group proposed that dysregulation of the Hedgehog pathway is of relevance in tumor development  in the encoded histone H3.3 and H3.1. Interestingly, other pediatric midline high-grade gliomas share mutations in H3.3 but not the H3.1 mutations and cortical pediatric and adult malignant gliomas rarely share these mutations (ACVR1) that are not observed in other midline or cortical malignant gliomas  of mutually exclusive somatic mutations of utations . The ide0% of mut gliomas –14. Othed PDGFRA . Thus, id PDGFRA .H3F3A gene has gained considerable attention for its potential role in tumorigenesis, and therefore, as a good candidate for targeted therapy. As mentioned above, approximately 80% of DIPG tumors contain mutations in genes that encode histones (H3.3 or H3.1), proteins that package DNA into chromatin. These mutations, which change lysine 27 to methionine (K27M), are believed to sequester polycomb repressive complex 2 (PRC2), which normally represses gene expression through histone methylation. When PRC2 is functionally inactivated, genes that should be silent are expressed, which is thought to drive cell transformation   .in vivo experiment with the use of athymic  mice harboring brainstem K27M glioma xenografts. The drug showed significant reduction in the growth of K27M tumors engrafted in mouse brainstem and significantly extended animal survival with evidence of decreased proliferation and augmented apoptosis , since the driving target identified in the tumor at diagnosis – and directing therapy – may be gone or altered at progression.Novel clinical trial design and combination of agents with different mechanism of action may offer a better chance for tumor control in this deadly entity.Many questions remain to be answered in the understanding of this disease, including the cancer stem cell and appropriate context for DIPG initiation, as well as which mutations and epigenetic alterations contribute to tumor maintenance and progression. The high incidence and exclusivity of histone H3 mutations to DIPG highlights the importance of identifying the true cell of origin that is susceptible to these mutations, as well as how the signaling pathways cooperate to drive gliomagenesis in a specific developmental context. It is now well recognized that epigenetic changes influence many of the hallmarks of cancer, such as malignant self-renewal, differentiation blockade, evasion of cell death, and tissue invasiveness , 30. ThuThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Kiss1 at puberty onset, and early experience-dependent programming of Avp, a major stress gene. At the genome-scale, genomic imprinting can be epigenetically switched on or off at select genes in a tightly controlled temporospatial manner and provides a versatile mechanism for dosage regulation of genes with important roles in stem cell quiescence or differentiation. Moreover, retrotransposition in neural progenitors provides an intriguing example of an epigenetic-like switch, which is stimulated by bivalently marked neurodevelopmental genes and possibly results in increased genomic flexibility regarding unprecedented challenge. Overall, we propose that molecular epigenetic switches illuminate the catalyzing function of epigenetic mechanisms in guiding dynamic changes in gene expression underpinning robust transitions in cellular and organismal phenotypes as well as in the mediation between dynamically changing environments and the static genetic blueprint.Epigenetic mechanisms encode information above and beyond DNA sequence and play a critical role in brain development and the long-lived effects of environmental cues on the pre- and postnatal brain. Switch-like, rather than graded changes, illustrate par excellence how epigenetic events perpetuate altered activity states in the absence of the initial cue. They occur from early neural development to maturation and can give rise to distinct diseases upon deregulation. Many neurodevelopmental genes harbor bivalently marked chromatin domains, states of balanced inhibition, which guide dynamic “ON or OFF” decisions once the balance is tilted in response to developmental or environmental cues. Examples discussed in this review include neuronal differentiation of embryonic stem cells (ESC) into progenitors and beyond, activation of  The field of molecular epigenetics has evolved rapidly over the past 25 years Allis,  and has Even so the identification and characterization of these mechanisms has illuminated our insight into the molecular basis of certain epigenetic phenomena, a formal definition of epigenetics remains still a matter of debate. More recently, Adrian Bird suggested that epigenetic events pertain to “the structural adaptation of chromosomal regions so as to register, signal or perpetuate altered activity states” Bird, . This unThis broad definition has been criticized by Mark Ptashne Ptashne, , among oA central issue behind this ongoing discussion on the quality of epigenetic mechanisms is causality: are DNA and histone modifications the driving force behind differences in chromatin states and transcription, or are differences in these modifications mostly downstream to dynamic processes such as transcription and nucleosome remodeling , which preferentially recognizes hemimethylated DNA  promoters of genes on the active copy of the X chromosome vs. the inactive one and imprinting control regions —a pathway suggested to operate in mouse brain  and Polycomb group (PcG) proteins play important roles in early development due to their chromatin-modifying activities  and DNA tiling arrays in mouse ESCs and resulted in the enrichment of various groups of developmental genes including the Genome-wide analysis by combining ChIP with next-generation sequencing (ChIP-seq) showed that almost all promoter CGIs of high CpG density were marked by H3K4me3, whereby ~22% contained at the same time H3K27me3, and were weakly expressed  caused the resolution of ~675 bivalent domains concomitant with the formation of ~550 new ones  and type III iodothyronine deiodinase (Dio3) are both expressed from the paternal inherited allele and map on chromosome 14q32 and distal chromosome 12 in human and mice, respectively  genes map 80 kb apart and flank an intergenic DMR consisting of tandem repeats that represent the only region at the domain marked by germ line-derived paternal methylation  show hypermethylation on the paternal and hypomethylation on the maternal allele, respectively , particularly in NSCs and astrocytes of the germinal niche, but not in their differentiated derivatives such as neuroblasts and parenchymal astrocytes. Astrocytes residing in the SVZ predominantly produce the secreted form of Dlk1, whereas NSCs predominantly contain the membrane bound form of Dlk1. Co-culture with naïve, but not with Dlk1-deficient astrocytes enhances NSC proliferation indicating that both forms are important for neurogenesis  migrate via a “cut-and-copy” mechanism that leads to the replication and insertion into new places in the genome  although they disrupt genomic nucleotide sequence Bird,  a limitaThe existence of mobile elements is not new although they were thought to be active only in germ cells, pluripotent cells, and cancer tissues. Therefore, the recent discovery that they increase their activity during the differentiation of neural progenitor cells (NPCs) into neurons came as a surprise  in a pulsatile mode during sleep, which triggers re-awakening of the hypothalamic-pituitary-gonadal (HPG) axis. Contrary to widely held believe, this reproductive axis is first active during prenatal and early postnatal life, a period known as neonatal “mini-puberty”, to be turned off and stay dormant throughout childhood via inhibitory inputs to the hypothalamus , which is transported by the bloodstream to the gonads and stimulates steroidogenesis  or interacting partners . All of these genes, except Rnf2, were subject to increased methylation at puberty indicating that the PcG complex undergoes functionally relevant epigenetic changes with the onset of puberty and that this repressive gene complex is a key factor in mediating central inhibition. At the same time, methylation at the Kiss1 promoter stayed unaltered  concomitantly with the active marks H3K4me3 and H3K9, 14ac thus matching the criteria of a bivalent promoter  signaling has been suggested to play a critical role to translate photoperiodic information into the reproductive neuroendocrine system even so thyroid secretion of the prohormone T4 is unaltered by seasonal change  via deiodinase type 2 (DIO2) or the receptor-inactive enantiomer via deiodinase type 3 (DIO3) are tightly regulated by changes in photoperiod  secretion, that in turn, controls the reproductive neuroendocrine system Reiter, . Moleculdio3 expression couple to epigenetic mechanisms —via the retinohypothalamic tract and direct circadian rhythms in a number of peripheral tissues. Various nutrient sensors further calibrate peripheral clocks by coupling metabolic states to histone modifications regulating clock activity  and cryptochome (CRY) family members representing the negative regulatory output of the circadian clock system. Chromatin remodelers and histone modifications act in concert with transcriptional cycles in establishing the oscillation pattern of molecular clocks  to genome-wide promoter tiling arrays evidenced significant changes between mice entrained to 22-h vs. 24-h days. These changes included a total of 1,294 differentially methylated (hypo- or hyper-methylated) promoter regions of genes relevant to synaptogenesis, axonal guidance, and neurohormone signaling, which correlated with changes in clock genes and global gene transcription  can also enduringly change the genetically determined period of circadian behavior in adult mice —play an important role in epigenetic programming of the hypothalamic-pituitary-adrenal (HPA) axis, the major stress system in mammals.A body of studies over the last years has provided compelling evidence that various environmental conditions Bird, , includiEarly-life adversity denotes different situations such as parental maladjustment , interpersonal loss , and maltreatment  among others  1–10) causes lasting hyperactivity of the HPA axis marked by heightened corticosterone secretion under resting conditions and hyperresponsiveness to acute stressors applied in later life. These neuroendocrine signs were accompanied by distinct behavioral changes including memory deficits in an inhibitory avoidance task and increased immobility in the forced swim test . There are, however, many sensitive periods depending upon the question   and both together determine future gene expression and phenotype.The identification of bivalent domains provides persuasive evidence for molecular epigenetic switches in gene expression in neurodevelopment and beyond. Once this dynamic equilibrium between activation and repression is tilted, robust “ON or OFF” decisions take place. Examples discussed in this review include neuronal differentiation of ESCs, activation of Still, the clear cut effects of molecular epigenetic switches on cellular or animal phenotypes seem to translate into more graded responses in human populations. A plausible explanation for this discrepancy is the high degree of genetic homogeneity in conjunction with well-defined experimental conditions in most cell culture and animals studies. In this respect, genetic heterogeneity and different life-experiences are likely sources for phenotypic variation in response to molecular epigenetic switches in human (Heim and Binder, In contrast to ELS, light entrainment represents a less complex but meaningful stimulus for resetting genetically encoded circadian cycles. Plastic changes in DNA methylation showed a remarkable flexibility of this mark and associate with reversible switches in circadian behavior. Future studies are still necessary to elucidate the precise order of epigenetic events in the timing of molecular clocks.Furthermore, epigenetic “ON or OFF” switches at imprinted genes provide a versatile mechanism for dosage regulation of genes with an important role in balancing stem cell quiescence vs. proliferation. These changes occur in a temporospatial manner through so far incompletely understood mechanism across the genome at several loci (Hoffmann et al., Lastly, retrotransposition in NPCs provides an intriguing example of an epigenetic-like switch occurring stochastically and/or in response to genomic stress. Activation of retrotransposition results from the expression of developmental TF, which are under the control of bivalent domains. While strengthening the importance of bivalent domains for epigenetic switches, these findings also indicate that their functions may extend to increased flexibility regarding “unanticipated challenges that are not precisely programmed” (McClintock, Overall, we suggest that the concept of molecular epigenetic switches illuminates the catalyzing function of epigenetic mechanisms in the mediation between dynamically changing environments and the static genetic blueprint. In the context of Waddington’s epigenetic landscapes of valleys and mountains, molecular epigenetic switches are guiding dynamic changes in gene expression underlying robust changes in cellular and organismal phenotypes. Ultimately, molecular epigenetic switches may also serve to reconfigure Waddington’s epigenetic landscape for better or for worse by lowering the threshold for transitions between distinct developmental trajectories and increasing flexibility regarding unanticipated challenges. Although of potential benefit, such changes are also likely to increase the risk for human disease.All authors contributed to conception and writing of this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The complement system is a component of the immune system involved in both recognition and response to pathogens, and it is implicated in an increasing number of homeostatic and disease processes. It is well documented that reperfusion of ischemic tissue results in complement activation and an inflammatory response that causes post-reperfusion injury. This occurs following cerebral ischemia and reperfusion and triggers secondary damage that extends beyond the initial infarcted area, an outcome that has rationalized the use of complement inhibitors as candidate therapeutics after stroke. In the central nervous system, however, recent studies have revealed that complement also has essential roles in synaptic pruning, neurogenesis, and neuronal migration. In the context of recovery after stroke, these apparent divergent functions of complement may account for findings that the protective effect of complement inhibition in the acute phase after stroke is not always maintained in the subacute and chronic phases. The development of effective stroke therapies based on modulation of the complement system will require a detailed understanding of complement-dependent processes in both early neurodegenerative events and delayed neuro-reparatory processes. Here, we review the role of complement in normal brain physiology, the events initiating complement activation after cerebral ischemia-reperfusion injury, and the contribution of complement to both injury and recovery. We also discuss how the design of future experiments may better characterize the dual role of complement in recovery after ischemic stroke. Following cerebral ischemia, most patients reperfuse at least part of the ischemic area , and theIn the CNS, as in other organs and tissues, complement plays a key role in the inflammatory reaction following ischemia and reperfusion. Disruption of the blood–brain barrier following ischemic stroke allows access of complement proteins to the brain, although proteins of the complement system can be locally produced by the cells of the CNS –8. ComplIn addition to its well-documented role in host defense, complement plays important roles in various other physiologic and homeostatic functions, such as immune complex catabolism, the clearance of dead and dying cells, and the modulation of adaptive immune responses. Complement is also involved in various developmental and regenerative processes, and is integrated with multiple other biological systems and pathways [reviewed in Ref. ]. Comple−/− mice  in the presence of astrocytes, and did not contribute to neuroprotection against Kainaite-induced excitotoxicity  and investigating how complement inhibition affects outcome in models of stroke. There follows a summary of studies that have been performed in vitro model for cerebral ischemia, a procedure that results in both apoptotic and necrotic cell death. Upon hypoxic insult, neuronal cultures have been shown to overexpress several complement proteins. Both mRNA and protein levels of C1q were elevated in rat neuronal cells exposed to hypoxia, and newly produced C1q preferentially deposited on hypoxic neurons, serving as both a primary opsonin and an activator of the complement cascade , the only approved treatment for acute stroke. As a plasma protease, t-PA is capable of proteolytically activating components of the complement system via the recently recognized extrinsic pathway. In support of this, an early study reported that after cerebral embolization rabbits treated with t-PA had higher levels of C3 and C5 compared to vehicle . InteresFollowing stroke, damage to the blood–brain barrier will allow access of hematogenous complement proteins to the CNS, but there is evidence that increased local expression of complement proteins contribute to secondary injury after ischemia. For example, expression of C1q, C3, and CD11b (a component of complement receptor 3 expressed on phagocytes) is upregulated in response to ischemia in models of transient MCAO , 60, 81.In an influential paper on the application of complement inhibition in stroke, Huang et al. described the use of soluble complement receptor 1 (sCR1) and its sialyl Lewis × glycosylated form (sCR1-sLex) to study the effect of systemic complement inhibition and selectin-targeted complement inhibition on outcomes after murine stroke. Soluble CR1 inhibits all complement pathways at the C3 activation step, and the sLex carbohydrate moiety binds to both P and E selectin, adhesion molecules that are upregulated on activated endothelium. Following MCAO and reperfusion, sCR1 modestly reduced ischemic injury while sCR1-sLex markedly diminished infarct volume, improved neurological outcome, and inhibited leukocyte migration . Thus, dAnother strategy used to target complement inhibition to an anatomical site is to link a complement inhibitor to a recombinant portion of complement receptor type 2 (CR2). Ligands for CR2 are C3 activation products that become covalently attached at sites of complement activation . ComplemThe sCR1 and Crry inhibitors described above are pan complement inhibitors. Interventional studies in experimental models of stroke have also been performed with C1-inhibitor (C1-inh) that inhibits only the classical and lectin pathways. In various ischemic stroke models, C1-inh has been shown to decrease infarct volume , 70, 80,Further investigation has indeed demonstrated an important role for the lectin pathway in murine models of stroke. MBL deficiency resulted in significantly reduced infarct volumes, neurological impairment, and C3 deposition, whereas reconstitution of MBL-deficient mice with MBL resulted in larger infarct volumes . Two addin vivo expression of injury  showed significant improvements in infarct volume and neurological outcomes, with less granulocyte infiltration. Furthermore, the effect was reversed when C3-deficient mice were reconstituted with C3 . A simil−/−) mice. The intestinal target of this mAb was identified as non-muscle myosin  must be met. In brief, there should be consistent pre-clinical data in different animal species and in animal cohorts that represent the human stroke predisposed population. Such populations include both sexes, adult and elderly age groups, and patients with comorbidities such as hypertension and diabetes . Thus, sStudies on gene polymorphisms at complement gene loci, associated polymorphisms at the C5 and factor H gene loci with increased incidence of cerebrovascular accidents , 107. AmThe predominant focus of studies on complement and stroke has been on the role of complement in perpetuating the inflammatory cascade and contributing to neuronal death. By comparison, very few studies have investigated the putative protective role that complement may play in ischemic rescue and ischemia-induced neurogenesis. The great majority of reports investigate acute-phase outcomes where complement blockade prevents activation of pro-inflammatory mechanisms. However, as indicated by a few studies, the same favorable outcomes may not occur in the subacute and chronic phases after stroke, when complement opsonins may be important for mediating anti-inflammatory events such as removal of debris, and when anaphylatoxins may contribute to homeostatic pro-survival and neuro-regenerative effects. It will be important to investigate how manipulation of the complement system impacts subacute and chronic outcomes, as well as to evaluate areas away from the ischemic core. Investigation of chronic outcomes may reveal a role for complement in maintaining neuronal survival after acute damage has been resolved, and possible mechanisms may include stimulation of neuronal growth factor production by astrocytes and microglia , 36, or Going forward, the design of experimental models to assess longer endpoints and multiple outcome measures after stroke may reveal a more diverse role for complement in ischemic injury and rescue. Future studies should also investigate the role of different complement components in early and delayed pathophysiological events. One area of investigation that has received little attention is the role of complement opsonins in early neuronal and synaptic loss after stroke, as well as the role of these opsonins in the resolution of inflammation and synaptic re-wiring that can occur later. Future studies should also address the interplay between complement opsonins and immune cells bearing receptors for these opsonins, especially infiltrating and resident macrophages/microglia; these opsonins may also play a role in driving macrophage polarization toward an anti-inflammatory and pro-regenerative phenotype. We also have an incomplete understanding of the role of the anaphylatoxins in stroke. They are implicated in recruiting and activating immune cells acutely, as well as stimulating neuronal genesis and migration in delayed phases after stroke. It is also not clear whether C3a and C5a have redundant, overlapping, or possibly divergent effects. Tools to address these questions are available in the form of genetically deficient mice and inhibitors that block the generation of specific complement activation products or that antagonize complement receptors, and it will be important for future studies to better evaluate long-term effects and outcomes after stroke.As mentioned in Section “The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


No randomised controlled trial has been successfully conducted during an influenza pandemic. The ASAP trial (Adjuvant Steroids in Adults with Pandemic Influenza) aims to use the novel strategy of set-up in advance of an influenza pandemic, and ‘hibernation’ in readiness for activation in a pandemic. Upon activation, the trial needs to recruit the first participant within 4 weeks, and to complete recruitment of 2200 participants within the first pandemic wave of approximately 6 weeks.Approvals are in place for 40 sites being maintained in ‘hibernation’. Three key programmes of work during hibernation have been identified: 1) Regular review to ensure compliance with current regulations, viability of Investigational Medicinal Product (IMP) and appropriate trial materials, 2) Planning for the ‘pre-activation’ phase , 3) Clinical Trials Unit (CTU) planning for activation.Regular review includes detailed annual checks of trial site readiness and interest, review of trial materials and contracts, replacement of expiring IMP, maintenance of trial oversight committees and reporting to stakeholders. Planning for pre-activation includes packaging of IMP, developing of training materials, and logistical aspects of trial delivery including changes in key trial staff. The CTU is also developing an operational plan to support activation and reporting.Long term maintenance of a clinical trial in readiness for a public health emergency is extremely challenging. Careful planning and detailed procedures should enable rapid activation and delivery of this trial. Anticipating future uncertainties is a key facet of trial maintenance during hibernation.


G-protein coupled receptors (GPCRs) constitute the largest family of membrane receptors, with high potential for drug discovery. These receptors can be activated by a panel of different ligands including ions, hormones, small molecules, and vasoactive peptides. Among those, angiotensins [angiotensin II (AngII) and angiotensin 1–7] are the major biologically active products of the classical and alternative renin-angiotensin system (RAS). These peptides bind and activate three different subtypes of GPCRs, namely AT1, AT2, and Mas receptors, to regulate cardiovascular functions. Over the past decade, the contribution of several RAS components in tumorigenesis has emerged as a novel important concept, AngII being considered as harmful and Ang1–7 as protective against cancer. Development of selective ligands targeting each RAS receptor may provide novel and efficient targeted therapeutic strategies against cancer. In this review, we focus on breast cancer to summarize current knowledge on angiotensin receptors , and discuss the potential use of angiotensin receptor agonists and antagonists in clinics. Figure 1). All Angiotensin peptides bind to GPCRs, namely AT1, and AT2 receptors for AngII and Mas receptor for Ang(1–7), but activate distinct signaling pathways leading to different and often opposite cellular effects . It is generally accepted that the classical ACE/AngII/AT1 axis promotes most of the RAS actions on cardiovascular, renal, and cerebral functions, and that these effects are counteracted by activation of the AT2 receptor  in breast cancer, and discuss the potential use of angiotensin receptor agonists and antagonists in malignancy.G-protein coupled receptors (GPCRs) of the renin-angiotensin system (RAS) system are activated by angiotensin peptides which are produced by angiotensin converting enzyme (ACE). ACE inhibitors (ACEi) have been assessed in cancer to characterize angiotensin functions in tumor growth, angiogenesis, and metastasis, and were shown to inhibit cell proliferation and tumor progression in several cancer models . Functional studies indicated that over expression of AT1 in breast cancer cells promotes cell invasion in vitro and tumor growth in vivo in absence of any stimulation by the AT1 agonist AngII  in 10–20% of invasive breast tumors , all of Table 1) raising the question of the functional effect of AT2 over expression in breast cancer. To specifically examine this question, we generated a human metastatic breast cancer cell line stably expressing high amounts of human AT2 receptors at the plasma membrane and no detectable AT1 levels . These results need to be validated at the protein level but one can speculate that Mas/Ang1–7 axis is present and targetable in breast tumors. Of interest, in orthotopic breast cancer xenograft models, administration of Ang1–7 efficiently reduced tumor volume and weight  are currently approved for the treatment of hypertension  are under development but preclinical studies are still needed to elucidate AT2 functions in cancer and to adequately target this receptor, in particular in breast cancer.Compound C21 is the only non-peptide orally active AT2 receptor agonist tested in several preclinical models with broad potential indication . C21 hasFigure 1; Targeting Mas receptor is a promising therapeutic option. Ang1–7 was evaluated in phase I clinical study involving eighteen patients with advanced solid tumors from colon, lung, pancreas, prostate, and soft tissues . Only miAngiotensin GPCRs appear as good therapeutic targets expressed at the cell surface and thus easily targetable with pharmacological drugs or humanized blocking antibodies. ARBs are already widely used to treat hypertension and may be safely used in other therapeutic applications, such as breast cancer, in combination with standard treatments. Targeting angiotensin receptors may be promising in several aspects, since beneficial effects of ARBs and Ang1–7 were observed on both tumor cells and tumor microenvironment . ARBs anFuture studies should be designed to further evaluate angiotensin receptor levels in breast tumors, in an attempt to select a population of patients that express high levels of AT1 or Mas receptors and may thus benefit from GPCR-targeted therapy.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


As the first drug to modulate the sphingolipid signaling pathway, it was marketed in 2010 for the treatment of multiple sclerosis (MS). At that time, immunomodulation was widely accepted as the key mechanism of fingolimod’s efficacy in MS. But given the excellent passage of this lipophilic compound into the brain and its massive brain accumulation as well as the abundant expression of S1P receptors on brain cells, it is conceivable that fingolimod also affects brain cells directly. Indeed, a seminal study showed that the protective effect of fingolimod in experimental autoimmune encephalitis (EAE), a murine MS model, is lost in mice lacking the S1P1 receptor on astrocytes, arguing for a specific role of astrocytic S1P signaling in MS. In this review, we discuss the role of sphingolipid mediators and their metabolizing enzymes in neurologic diseases and putative therapeutic strategies arising thereof.Sphingolipids are a fascinating class of signaling molecules derived from the membrane lipid sphingomyelin. They show abundant expression in the brain. Complex sphingolipids such as glycosphingolipids (gangliosides and cerebrosides) regulate vesicular transport and lysosomal degradation and their dysregulation can lead to storage diseases with a neurological phenotype. More recently, simple sphingolipids such ceramide, sphingosine and sphingosine 1-phosphate (S1P) were discovered to signal in response to many extracellular stimuli. Forming an intricate signaling network, the balance of these readily interchangeable mediators is decisive for cell fate under stressful conditions. The immunomodulator fingolimod is the prodrug of an S1P receptor agonist. Following receptor activation, the drug leads to downregulation of the S1P The more complex cerebrosides) are required for cell functions such as cell recognition, vesicular transport and lysosomal degradation and their dysregulation can lead to storage diseases with a neurological phenotype . From the point-of-view of drug development, the differential receptor expression patterns in different organs, a modulation of receptor expression in disease states, as well as the differential coupling to G proteins, may afford a sufficient degree of specificity to make S1P receptors drugable targets , and maintenance of endothelial barriers  has made a very long and interesting journey from traditional asian medicine to a drug tested in large scale international multicenter randomized-controlled placebo-or standard of care-controlled trials proving its efficacy in the treatment of multiple sclerosis (MS)  did not provide sufficient immunosuppression to allow reduction of co-immunosuppressants and was not superior to standard care. There was an increased incidence of macular edema and transient decreases in heart rate  activity in mast cells and therefore prostaglandin and thromboxane secretion , they meet their antigen and incite inflammatory demyelination leading to subacute neurological symptoms that can remit and, given the chronicity of the disease, relapse. In the early inflammatory phase of the disease, immunomodulation with beta-interferons or glatiramer acetate have been shown to slow disease progression. In later stages of the disease, neurodegenerative processes also play a role.Fingolimod was approved by the regulatory authorities worldwide as the first oral agent to treat relapsing-remitting multiple sclerosis (RRMS) after the successful completion of two phase III studies in 2010 showed its efficacy. FREEDOMS , neurons (synapsin-cre) and astrocytes (GFAP-cre). They found that S1P1-deficiency on astrocytes led to an attenuation of EAE and that the protective effect of fingolimod in EAE was lost in mice with astrocytic but not with neuronal S1P1 deletion , transient ischemia, inhalational anesthetics such as isoflurane or inflammatory agents such as bacterial lipopolysaccharide (LPS). HP is based on a hypoxia-induced stabilization of hypoxia-inducible factor (HIF) that is continuously degraded by an oxygen-sensing degradative pathway under normoxic conditions. Stabilized by hypoxia, HIF, which exists in three isoforms, binds to hypoxia-responsive elements (HRE) of hypoxia-regulated genes to promote their transcription. Besides hypoxia and ischemia, also non-hypoxic stimuli such as isoflurane  is often denoted as “the most untreatable cause of stroke” because there is no causal treatment and it is charged with a high mortality. Noteworthy, Asian populations display higher rates of ICH, with ICH representing over 30% of all first ever strokes in China in a meta-analysis of population-based studies  orally for 3 consecutive days  is the most common age-related neurodegenerative disorder and shows a rising prevalence along with global trends towards increasing life spans. AD leads to a cognitive decline that is attributable to neuronal loss predominantly affecting higher cortical functions  whereas cognitive working tempo and drive seem unaltered. Patients are often able to maintain an unremarkable “façade” even in later stages of the disease when brain atrophy, predominantly of the hippocampus and the temporal lobe can be seen in clinical brain imaging. Currently, there is no causal treatment of this devastating disease. While the disease-triggering mechanisms are yet unknown, the neuropathological hallmarks of AD consist of the deposition of amyloid-β (Aβ) plaques and the formation of neurofibrillary tangles consisting of hyperphosphorylated tau protein  ameliorated spatial learning and memory in the Morris water maze test at the end of the 2 week course and reduced neuronal damage and caspase-3 activation in the hippocampus  on the production of Aβ1-40 (decreased) and Aβ1-42 (increased), thus showing that fingolimod affects Aβ levels also in vivo  could be inhibited by S1P3 receptor antagonism. SphK1 is highly expressed in mossy fibers of the hippocampus and LTP was significantly impaired in SphK1 knockout mice, showing that SphK1 is required for hippocampal S1P generation. Congruently, mice with genetic deletion of SphK1 showed an impairment of spatial learning and memomory in the Morris water maze. Interestingly, somewhat opposing findings have recently been described by Hait et al.  and protease-activated receptors (PAR) in hippocampal learning processes  is an inherited neurodegenerative brain disease with autosomal-dominant inheritance. It is characterized by mutations of the huntingtin gene with extended CAG trinucleotide repeats encoding a dysfunctional mutant huntingtin protein (mHtt). mHtt forms aggregates that are cytotoxic and interfere with many physiological cell functions, such as the expression of neurotrophins, e.g., BDNF  lead to increased levels of BDNF in affected brain structures and even to an amelioration of motor impairment and survival. This was the first description of a fingolimod-elicited production of the neurotrophin BDNF which could be attributed primarily to neurons by immunohistochemistry and cell culture experiments while a relevant contribution of astrocytes could be excluded  or structural  origin. While epilepsies are well controllable by anticonvulsant medication in the majority of cases, series of recurrent seizures or non-terminating status epilepticus represent a serious threat for patients with epilepsy. The processes happening in the propagation from single seizures to prolonged epileptic activity have been characterized in experimental models and emcompass cell death, axonal and dendritic plasticity, neurogenesis and neuroinflammation  are the most malignant forms with aggressive growth and invasion of the surrounding brain tissue. Due to their infiltrating nature, they cannot be completely excised and the majority will recur locally  drug to treat gliomas, whose progression is yet barely controlled by the current standard therapies.A putative therapeutic effect of fingolimod in the context of glioma research was evaluated as early as 2001 (Sonoda et al., According to experimental data, the sphingolipid signaling pathway seems to be of central relevance in the pathophysiology of many diverse neurological diseases. The versatile sphingosine analog fingolimod, prodrug of the S1P-modulating fingolimod phosphate, provides scientifically proven reduction of relapse rate and amelioration of disease progression in MS. But data from experimental animal models suggest that fingolimod has additional therapeutic applications in store. Many of the diseases reviewed here follow a fatal course and treatment options yet are scarce and their efficacy often short-lived. Given the growing body of experience with fingolimod accumulated by clinical neurologists and the favorable safety profile in the indication of MS, there is hope for a translation of some of these experimental findings into the clinics.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


During recent years different types of adult stem/progenitor cells have been successfully applied for the treatment of many pathologies, including cardiovascular diseases. The regenerative potential of these cells is considered to be due to their high proliferation and differentiation capacities, paracrine activity, and immunologic privilege. However, therapeutic efficacy of the autologous stem/progenitor cells for most clinical applications remains modest, possibly because of the attenuation of their regenerative potential in aged patients with chronic diseases such as cardiovascular diseases and metabolic disorders. In this review we will discuss the risk factors affecting the therapeutic potential of adult stem/progenitor cells as well as the main approaches to mitigating them using the methods of regenerative medicine. The elasticity of blood vessels declines with age along with an increase in their stiffness, which predetermines the progression of arterial hypertension. As people age, their adipose tissue mass increases, while their muscle volume decreases, leading to the development of insulin resistance, the most important pathogenic factor of T2DM. Aging is also associated with comorbidities, the simultaneous presence of two or more different diseases, often with chronic long-lasting progression. The most frequent age-associated comorbidities confounding each other are CAD and T2DM and obesity, arterial hypertension, and T2DM.6The target affected by the most CVD risk factors is the blood vessel wall. Endothelial dysfunction is considered to be the key pathogenic mechanism of angiopathies associated with CAD and T2DM. It should be noted that endothelial dysfunction develops as a result of the interaction of different risk factors, such as insulin resistance, hyperglycemia, and dyslipidemia. The long-term presence of these factors affects endothelial cells and promotes their apoptosis, which leads to the nitric oxide (NO) production failure. As a consequence, the vasodilatation and anti-aggregation functions of the endothelium are dysregulated along with its ability to inhibit smooth muscle cell proliferation. These factors potentiate atherosclerosis progression, forming the morphological basis of CAD.8Hyperglycemia is the main pathogenic factor of T2DM, but it also underlies CVD. First, it accelerates the progression of atherosclerosis and macroangiopathies; second, it affects the microvasculature network. This effect is manifested particularly in the disturbance of neovascularization processes in different tissues. In the retina, excessive angiogenesis leads to leaky vessels and diabetic retinopathy, while in the skeletal muscles and myocardium, adaptive angiogenesis is insufficient, which promotes the development of severe ischemia, persistent trophic ulcers, and amputations.9–14 Several types of cells are involved in the neovascularization processes of ischemic tissues. Endothelial cells; their circulating progenitors , which mobilize from the bone marrow as a response to the ischemia and vessel damage; and progenitor cells located within the vessel wall, including multipotent mesenchymal stem/stromal cells (MSCs), interact with each other and contribute to vasculature repair and regeneration.15–17Despite the obvious success in treating CAD and T2DM, all available therapeutic approaches are intended to restrict disease progression. A novel promising approach for the treatment of ischemic diseases is cell-based therapy using autologous stem/progenitor cells.18MSCs, derived from the bone marrow, adipose tissue (ADSCs), or other tissues, are considered a promising tool for cell-based therapy due to their high proliferative and differential potency, ability to stimulate the growth of new blood vessels and nerves, and especially their production of multiple cytokines and growth factors. These cells also secrete plasminogen activators and matrix metalloproteases (MMPs), which actively participate in the remodeling of extracellular matrix (ECM) and the proteolytic release and activation of some growth factors sequestered in the ECM. MSCs serve as a component of the vessel wall in all tissues and play an essential role in the vasculature network development and support in both normal and pathological conditions.19–22 and their safety and feasibility have been demonstrated, but the clinical effectiveness of these protocols was relatively modest and could not corroborate the promising results of preclinical studies .42Influence of aging on stem/progenitor cells is well studied for MSCs. Bone marrow-derived MSCs from aged donors were shown to have worse proliferation and differentiation capacity43 In poorly differentiated cells, telomere length is maintained due to the high activity of telomerase. Telomerase also has some telomere-independent functions. Telomerase activity was shown to be the highest in stem and tumor cells and was detected to a certain degree in many kinds of progenitor cells.44 Its activity is repressed as stem cells start to differentiate.37 Age-associated telomere shortening in MSCs has been shown.30 According to our data, relative telomere length decreases with age in both murine and human ADSCs.46 Interestingly, telomeres in “aged” stem/progenitor cells are still longer than telomeres in somatic cells from the same tissues,27 which could be explained by the lower proliferation activity of stem/progenitor cells or by special mechanisms of telomere-shortening prevention.Using the appropriate age markers is important for the evaluation of aging impact to the properties of stem/progenitor cells. One marker is the relative telomere length, which indicates the number of cell divisions. Telomere shortening is considered to be the main causal mechanism for replicative cell senescence and age-associated telomere damage, and the diminution of the telomere “capping” function and associated p53 activation have emerged as prime instigators of tissue stem/progenitor cells functional decline.47 In accord with telomere shortening in ADSCs from aged patients and old mice, we observed a decrease in proliferation activity of ADSCs with age as well as fewer actively proliferating cells.46Age-dependent decrease of stem/progenitor cell proliferation activity is considered to be related to higher expression of cell cycle inhibitors like p16INK4a, p21, p53, and p19ARF or inhibition of their degradation.24Because stem/progenitor cells persist in tissues throughout life, albeit mostly in a quiescent state, they experience age-related long-term exposure to genotoxic insults from both endogenous and exogenous sources. Accordingly, accumulation of DNA damage in aged stem/progenitor cells has been noted in several studies. The DNA damage response pathways necessary for stabilizing the genomic integrity may have reduced activity in stem cells with age. Accumulation of DNA damage in aged stem cells could cause stem cell senescence or apoptosis and alterations in stem cell self-renewal and differentiation.28 Different factors like hypoxia inducible factor-1 alpha (HIF-1α), ataxia telangiectasia mutated (ATM) protein, Bmi-1, and FoxO family factors may change ROS level in the cells.27 In bone marrow–derived MSCs obtained from aged patients, the activity of superoxide scavenger (superoxide dismutase) is decreased and the levels of ROS, NO, and oxidized and glycosylated proteins are increased.30 Supporting the hypothesis that ROS generation may promote stem cell aging, studies of aged human stem/progenitor cells, including MSCs, neural stem cells, and others, have found that excessive cellular ROS concentrations lead to abnormal proliferation, malignancy, and compromised stem cell self-renewal and differentiation capacity.24 The role of ROS in aging-related changes of ADSC properties was also demonstrated.48Oxidative stress and its regulators play an important role in aging of stem/progenitor cells. Increasing production of reactive oxygen species (ROS) and weakening of the antioxidant protective system in cells promote oxidative damage and lead to the loss of redox control, which affects ROS-regulated biological processes such as growth, proliferation, migration, apoptosis, differentiation, and so forth.49 and an age-related deficit of mitochondrial function leading to respiratory chain dysfunction in stem/progenitor cells is actively being investigated. These effects are considered to result largely from an accumulation of mutations in mitochondrial DNA damaged by elevated ROS or other mechanisms.49 Aside from primary mitochondrial lesions , secondary alterations in mitochondrial function driven by age-related cellular and metabolic changes may also contribute to the aging process.49A direct relationship between mitochondrial dysfunction and aging has been suggested by many studies,32 According to our data, the angiogenic potential of ADSCs is significantly impaired during aging. We have shown that ADSCs isolated from old mice (18 months), along with expressing age markers , have an impaired ability to stimulate blood vessel growth on in vitro and in vivo models of angiogenesis compared to ADSCs from young animals (1–2 months).45 Similar results were obtained with human ADSCs, including in patients with cardiovascular pathologies.46 We demonstrated that the mechanisms of age-associated decline of ADSC angiogenic activity include decreased production of key pro-angiogenic factors such as VEGF, placental growth factor (PlGF), hepatocyte growth factor (HGF), angiopoetin-1, and angiogenin.46 It should be noted that age-associated differences in the expression of pro-angiogenic factor genes were not found,46 indicating that posttranscriptional mechanisms, such as regulation by microRNA,50 age-associated protein misfolding, and so forth, could underlie the decreased secretion of angiogenic factors by ADSCs from aged patients.Paracrine activity of stem/progenitor cells, including MSCs, changes with age. Interleukin (IL)-6 secretion by bone marrow–derived MSCs co-cultured with T cells decreased in aged patients.51 These results are consistent with those we previously obtained from ADSCs from young and old mice.45 The findings could reflect the adaptive reaction of stromal cells to the age-associated ECM changes within the blood vessel wall52 and increasing levels of pro-inflammatory factors and ROS. Also considering the regulatory role of the uPA system in growth factor–induced endothelial cell migration and invasion and stimulation of angiogenesis in ischemic tissues,53 we can speculate that its activation in ADSCs from aged patients might be a compensatory response to the reduction of pro-angiogenic factor secretion.Apart from the angiogenic growth factor effects, ECM remodeling is crucial for successful angiogenesis. ECM remodeling and direction of cell migration for vessel wall formation are regulated by multiple factors such as urokinase (uPA) and its receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1), MMPs, and so forth. Analyzing the expression of factors involved in ECM remodeling and vascular cell migration and invasion, we found that mRNA levels of uPA, uPAR, and PAI-1 as well as uPAR surface expression along with the activation of MMP-2 and MMP-9 were higher in ADSCs from aged patients with CAD.Given the total evidence, aging could essentially affect the properties of stem/progenitors cells, thereby diminishing the effectiveness of autologous cell therapy. Testing of cell material before use may be required along with developing effective approaches for pretreatment or modification of stem/progenitor cells from aged patients to enhance their therapeutic potential. Some of these approaches will be discussed below.11 Aging is also associated with EPC dysfunction that further negatively affects the neovascularization and angiogenesis in tissues.54Since MSC are considered to be the components of vessel wall and take part in its repair after injury, cellular modifications due to aging can be an important pathogenic factor of age-related diseases such as atherosclerosis, diabetes, and arterial hypertension.We can conclude that organismal aging is a complex process in which resident stem/progenitor cells are involved both as a cell reservoir for the repair and regeneration of tissues and as targets exposed to the multiple local and systemic stimuli of the aged organism. These changes should be considered during the development of cell-based therapy using different types of autologous stem/progenitor cells.55–57 Among the pathologies affecting the functionality of stem and progenitor cells, autoimmune diseases, such as systemic sclerosis and systemic lupus, may be included.58During recent years a substantial amount of data have shown that chronic pathologies, including CAD and T2DM, affect the properties of stem/progenitor cells. It should be noted that widespread risk factors associated with CAD  may have an impact on the decrease in number and/or functional activity of stem and progenitor cells.59–61 and metabolic disorders including obesity and T2DM.62–65 Van Ark et al.66 demonstrated that in patients with T2DM both EPCs and circulating angiogenic cells levels were reduced and the ratio between EPCs and smooth muscle progenitor cells was disturbed, which may translate into reduced vascular repair capacity, thereby promoting macrovascular disease in T2DM.Numerous studies showed that the number, proliferation activity, ability for adhesion, migration, and angiogenic properties of EPCs are significantly impaired in patients with CAD35 showed that the number of ADSCs obtained from 50 patients with different vascular pathologies was relatively consistent independent of age and comorbidities like obesity, T2DM, and so forth. Similar results were obtained by Madonna et al.57 in 42 patients with different grades of cardiovascular risk. However, ADSCs isolated from adipose tissue of patients with T2DM were shown to have lower proliferation activity and were less responsive to pro-angiogenic stimuli such as hypoxia.67 In patients with obesity, impaired differentiation potential of ADSCs and a decline in their ability to stimulate blood vessel growth were observed.68 In a study performed by Vecellio et al.,69 MSCs obtained from the heart tissue of patients with T2DM were characterized by a reduced proliferation rate, diminished phosphorylation at histone H3 serine 10 (H3S10P), decreased differentiation potential, and premature cellular senescence compared to the control group.According to the results of several studies the number of MSCs is unlikely to be decreased in patients with cardiovascular diseases, but their regenerative potential may be attenuated. Thus, Harris et al.70 We showed that ADSCs from the patients with CAD (n=32) and CAD+T2DM (n=28) had similar morphology and immunophenotype, preserved adipogenic and osteogenic differentiation potency, and higher proliferation activity, but shorter telomeres compared to ADSCs from control patients without established chronic pathologies (n=19). These findings might reflect the depletion of progenitor cell compartment in the presence of chronic pathologies such as CAD and T2DM.We analyzed how ADSC properties are changed in patients with CAD and T2DM.HIF-1a gene expression. The activity of HIF-1a gene expression in its turn directly mediates VEGF gene expression. Moreover, by reducing eNOS the high glucose level inhibits the production of NO in endothelial and vascular smooth muscle cells, which is known as a downstream mediator of several angiogenic factors including angiopoietin-1.72Analyzing ADSCs as a tool for therapeutic angiogenesis we found that the angiogenic potential of ADSCs obtained from patients with CAD and with CAD+T2DM was decreased compared to patients without established cardiovascular pathologies independent of such factors as age and sex. We also did not see any statistically significant evidence that T2DM had an additional impact on the decline of ADSC angiogenic activity when combined with CAD. Since a paracrine mechanism is considered to be the main regulator of the beneficial effects of ADSCs, we examined the ability of ADSC to secrete some key angiogenic and anti-apoptotic growth factors. Interestingly, we revealed significantly higher production of some pro-angiogenic factors by ADSCs: VEGF and HGF for patients with CAD and HGF and PlGF for patients with CAD+T2DM, whereas the angiogenic activity of all products secreted by ADSCs from patients with both CAD and CAD+T2DM was significantly decreased compared to the control group. Absence of an elevation in the VEGF level in ADSC-conditioned medium from patients with CAD+T2DM may be associated with hyperglycemia. A high glucose level reduces endothelial nitric oxide synthase (eNOS) and stimulates inducible NOS (iNOS) expression that can inhibit THBS-1 gene expression is significantly increased in ADSCs from patients with CAD and CAD+T2DM, but we could not confirm this finding on the protein level. However, statistical analysis revealed negative correlation between mRNA THBS-1 expression and angiogenic activity of summary products secreted by ADSCs from patients with CAD and CAD+T2DM. It allows us to assume that some indirect mechanisms of THSP-1 participation in the angiogenic effects of ADSCs could be discussed.The process of angiogenesis depends on the intricate balance between angiogenic and angiostatic factors. We speculated that the main cause of impaired angiogenic potential of ADSCs obtained from patients with CAD and CAD+T2DM was the increased level of the angiogenesis inhibitors. Among others we analyzed production of endostatin and thrombospondin-1 (THBS1) by ADSCs and showed that 73–75 Acosta et al.76 showed that ADSCs obtained from patients with T2DM have less fibrinolytic ability because they secrete more PAI-1 and less tissue activator of plasminogen and D-dimer. This situation led to the development of microthrombotic complications when these cells were used to treat critical low limb ischemia. PAI-1 actively interacted with uPA, an important factor of extracellular proteolysis that not only specifically cleaves plasminogen and converts it into plasmin, causing activation of different MMPs, but also initiates intracellular signaling upon binding to its receptor on the cell surface. It therefore plays multiple roles in vascular remodeling and angiogenesis. We propose that PAI-1 produced by ADSCs exerts anti-angiogenic effects also through the inhibition of uPA.As for the factors involved in ECM remodeling, we found that the mRNA level of PAI-1 as well as its secretion by ADSCs was significantly increased in groups of patients with CAD and CAD+T2DM. PAI-1 is one of the primary regulators of the fibrinolytic system, and it has a crucial effect on cell migration and adhesion. A high level of PAI-1 was linked with a high risk of CAD, diabetes, and obesity. PAI-1 can both promote and inhibit vascular remodeling, but its role in angiogenesis and tissue regeneration is still controversial. The balance between these two mechanisms may depend on a disease state. Plasma PAI-1 is closely correlated with such factors as hypoxia, glucose-related signaling molecules, inflammatory cytokines, triacylglycerol, and insulin.77 who have shown that PAI-1 inhibition in vivo under ischemic conditions increases the activity of pro-angiogenic factors such as VEGF-A and fibroblast growth factor (FGF)-2 and leads to the stimulation of angiogenesis and improved restoration of tissue perfusion. However, MMP-9 deficiency and VEGF-A blockade reversed the PAI-1 inhibitor-mediated neovascularization within the ischemic niche. Some experts have suggested that a PAI-1 blockade and an increase in MMP-9 gene expression under ischemic conditions could be promising molecular approach in the therapeutic angiogenesis.78 It should be noted that we did not find significant increase in the MMP-9 mRNA level in ADSCs from patients with cardiovascular pathologies in our study. Therefore, we assume that high expression of PAI-1 and insufficient activity of MMP-9 and, for diabetic patients, VEGF may be one of the mechanisms of impaired angiogenic potential of ADSCs from patients with CAD and CAD+T2DM.Importantly, we demonstrated that by neutralizing only one factor in ADSC-conditioned medium—PAI-1—we could partially restore the angiogenic activity of ADSCs obtained from patients with chronic diseases. Our data are corroborated by the results obtained by Tashiro et al.79 We have previously found that endothelial cells cultured in hyperglycemia modeling conditions (25 mM of glucose) have an impaired ability for VEGF- and serum-induced migration as well as forming capillary-like structures on Matrigel in vitro. To reveal the possible mechanisms of these effects we analyzed the expression of VEGF receptors (VEGFR) on endothelial cells because this growth factor specifically induced adaptive angiogenesis in ischemic tissues. We observed fivefold decrease in VEGFR1 expression and more than twofold decrease in VEGFR2 expression in the endothelial cells cultured in hyperglycemic conditions compared to the standard conditions.80One of the main adverse factors for cells from patients with T2DM is hyperglycemia. Several studies have shown that long-term exposure of vascular cells to a high level of glucose causes dysfunction and promotes apoptosis.in vitro was significantly decreased. We evaluated the transcriptome of ADSCs cultured in the standard conditions or in the presence of high glucose (25 mM) and found the most significant changes in gene expression of ephrin receptors, vitronectin, and plexin domain 1 contained protein (decreased in hyperglycemic conditions) and leptin, tumor necrosis factor α (TNFα), plasminogen, and angiopoetin-like factor 3 (increased in hyperglycemic conditions). However, we failed to find any significant changes in gene expression of key angiogenesis-related factors and their receptors associated with high-glucose culturing. To explain the decreased angiogenic activity of ADSCs cultured in hyperglycemic conditions, we focused on the elevated level of plasminogen in these cells. It is known that high expression of plasminogen in tumors is associated with activation of MMPs that perform cleave plasminogen to generate angiostatin, an angiogenesis inhibitor. This mechanism might also be realized in ADSCs, impairing their angiogenic potential. A combination of increased expression of TNFα and angiopoetin-like factor 3 also could affect ADSC angiogenic activity because these factors stimulate apoptosis of endothelial cells. Taken together, long-term incubation of vascular cells in hyperglycemic conditions affect both endothelial cells and ADSC functional properties, which can be one of the reasons for insufficient adaptive angiogenesis in patients with diabetes.81We also analyzed the influence of hyperglycemia on progenitor cells localized in the perivascular niche, such as MSCs. Culturing of ADSCs in high glucose (25 mM) medium didn't affect their proliferation activity, viability, or migratory properties, but the ability of the total secreted products of these cells to stimulate capillary-like tube formation 83 EPCs play a very important role in the repair of damaged endothelium and the revascularization of ischemic tissues. T2DM has been shown to affect EPC mobilization from the bone marrow, resulting in a lower amount of EPCs in circulation and a distortion of angiogenesis and normal repair processes in injured tissues.84 This promotes the progression of ischemic diseases and adversely affects the prognosis of patients with T2DM.It should be emphasized that EPC number and functional properties are also significantly changed in patients with metabolic disorders.+/CD34+, CD133+/CD34+/VEGF-R2+, CD133+/VEGF-R2+, CD34+/VEGF-R2+.85–87 However, some investigators have demonstrated that the subpopulation identified as CD45−VEGFR2+(KDR)CD34+ cells consists of mostly leukocyte progenitors, particularly monocytes.89 Contradictory data about the immunophenotype of EPCs is explained by the substantial overlap of surface markers between the endothelial and hematopoetic progenitors and differences in the protocols and flow cytometry analysis.No consensus exists about the EPC phenotype for the identification of these cells. In clinical practice, the most frequently used markers are CD133+ cells, including hematopoetic stem/progenitor cells as well as EPCs in the peripheral blood of patients with CAD and with CAD+T2DM. We found that EPC numbers were significantly higher in the patients with CAD only compared to the healthy control age- and sex-matched subjects. This increase could be caused by the stimulated mobilization of these cells from bone marrow in response to tissue ischemia. But in patients with CAD+T2DM, such an elevation was not observed, suggesting the possible distortion of EPC mobilization in T2DM.Given the association of both CAD and T2DM with endothelial dysfunction and tissue ischemia, EPCs might serve as biomarkers of severity and prognosis in patients with such pathologies. We analyzed total populations of CD3458 Similar data were obtained by Churdchomjan et al.84Analyzing EPC numbers based on the severity of T2DM, we found that in patients with compensated and subcompensated diabetes, the level of EPCs was similar to the normal level, but patients with decompensated diabetes and comparable severity of CAD had significantly lower numbers of these cells, which could indicate impaired mobilization of EPCs from bone marrow. We showed that EPC numbers were negatively correlated with glucose levels.Altogether ample evidence exists that chronic diseases such as CAD and T2DM severely affect not only the fully terminated endothelial cells, but also stem/progenitor cells involved in vascular remodeling in normal and pathological conditions. Further investigation of the influence of the mechanisms of chronic diseases on the quantitative and functional characteristics of stem/progenitor cells is important for the development of effective methods for diagnosis and correction of micro- and macroangiopathies.91Taking into account the results of numerous studies discussed, it is important to know the changes of patient's stem/progenitor cells to develop the methods of individual pretreatment of autologous cell material to improve its therapeutic potential.92–97 Thus, culture of bone marrow–derived MSC in hypoxic conditions before transplantation improved the survival of these cells in damaged tissues as well as their ability to stimulate blood vessel growth.98Hypoxic preconditioning is one of the most accessible and widespread approaches to treat stem/progenitor cells before transplantation to enhance their resistance to the ischemic stimuli and stimulate production of angiogenic factors in these cells. Many studies have shown that hypoxia stimulates angiogenic properties of bone marrow–derived MSCs and ADSC.99 Moreover, we found that after hypoxic preconditioning the balance between pro- and anti-angiogenic factors was shifted to the more pro-angiogenic profile in ADSCs obtained from both young and aged donors. The preconditioning enhances the ability of ADSCs to stimulate angiogenesis and stabilize the newly forming vessels.94 Despite the many advantages of using hypoxic preconditioning as a pretreatment procedure for autologous stem/progenitor cells, length of hypoxia, oxygen level, and number of hypoxia/reperfusion cycles vary from study to study, so the optimal pattern of hypoxic preconditioning remains open for discussion.In several studies, including our work, even short-term (2–3 days) exposure of ADSCs to low oxygen levels (1%–5%) stimulated their proliferation and angiogenic activity.ex vivo using the specific induction medium100 or sorted by the specific markers like CD34.101 Several growth factors and chemokines could be added to the culture medium of stem/progenitor cells, such as basic FGF (bFGF), epidermal growth factor, TNFα, insulin-growth factor-1, bone morphogenetic protein-2, and so forth, to enhance the viability, proliferation, migratory properties, and therapeutic potential of these cells.102For some purposes stem/progenitor cells could be initially differentiated 103 or with protein kinase p38 inhibitor,104 or by culturing the cells in the presence of melatonin to increase their viability and proliferation activity and stimulate angiogenic factor production and angiogenic potential after transplantation to the ischemic kidney parenchyma.105 Lutolf et al.106 described a novel approach to restore the regenerative potential of aged stem/progenitor muscle cells: p38 inhibition combined with bioengineered modeling of the cell microenvironment.106Another approach includes modulation of intracellular signal cascades, for example, by pretreating stem/progenitor cells with statins that activate the Akt/eNOS pathway within the cells31 have demonstrated that MSCs from old animals could have their capability for renewal and osteogenic potential restored by being cultured on ECM produced by the cells of young donors. Similarly, EPCs isolated from old rat peripheral blood recover their functions in vitro and in vivo after being cultured in the presence of young animal's serum.36Sun et al.108 Modification of stem/progenitor cells with other factors improved their ability for homing , increased viability , and stimulated paracrine function .91 Several factors could be combined for modification; for example, using MMP-3 tissue inhibitor109 or telomerase110 with VEGF to modify MSCs obtained from aged donors allowed significant improvement of cell regenerative potential.One of the most promising tools for stem/progenitor cell pretreatment is genetic modification of these cells. Through the use of different vectors, various genetic constructions could be efficiently inserted to ADSCs. ADSCs modified by VEGF and HGF produced significantly higher amount of these factors and had enhanced angiogenic activity.Aging and chronic diseases including CVD and diabetes substantially affect stem/progenitor cells of adult organism . Such co