VCE-005.1, an hypoxia mimetic betulinic acid derivative, induces angiogenesis and shows efficacy in a murine model of traumatic brain injury.

One of the main global causes of mortality and morbidity is traumatic brain injury (TBI). Neuroinflammation and brain-blood barrier (BBB) disruption play a pivotal role in the pathogenesis of acute and chronic TBI onset. The activation of the hypoxia pathway is a promising approach for CNS neurodegenerative diseases, including TBI. Herein, we have studied the efficacy of VCE-005.1, a betulinic acid hydroxamate, against acute neuroinflammation in vitro and on a TBI mouse model. The effect of VCE-005.1 on the HIF pathway in endothelial vascular cells was assessed by western blot, gene expression, in vitro angiogenesis, confocal analysis and MTT assays. In vivo angiogenesis was evaluated through a Matrigel plug model and a mouse model of TBI induced by a controlled cortical impact (CCI) was used to assess VCE-005.1 efficacy. VCE-005.1 stabilized HIF-1α through a mechanism that involved AMPK and stimulated the expression of HIF-dependent genes. VCE-005.1 protected vascular endothelial cells under prooxidant and pro-inflammatory conditions by enhancing TJ protein expression and induced angiogenesis both in vitro and in vivo. Furthermore, in CCI model, VCE-005.1 greatly improved locomotor coordination, increased neovascularization and preserved BBB integrity that paralleled with a large reduction of peripheral immune cells infiltration, recovering AMPK expression and reducing apoptosis in neuronal cells. Taken together, our results demonstrate that VCE-005.1 is a multitarget compound that shows anti-inflammatory and neuroprotective effects mainly by preventing BBB disruption and has the potential to be further developed pharmacologically in TBI and maybe other neurological conditions that concur with neuroinflammation and BBB disruption.

Differences in Expression of IQSEC2 Transcript Isoforms in Male and Female Cases with Loss of Function Variants and Neurodevelopmental Disorder.

Pathogenic hemizygous or heterozygous mutations in the IQSEC2 gene cause X-linked intellectual developmental disorder-1 (XLID1), characterized by a variable phenotype including developmental delay, intellectual disability, epilepsy, hypotonia, autism, microcephaly and stereotypies. It affects both males and females typically through loss of function in males and haploinsufficiency in heterozygous females. Females are generally less affected than males. Two novel unrelated cases, one male and one female, with de novo IQSEC2 variants were detected by trio-based whole exome sequencing. The female case had a previously undescribed frameshift mutation (NM_001111125:c.3300dup; p.Met1101Tyrfs*5), and the male showed an intronic variant in intron 6, with a previously unknown effect (NM_001111125:c.2459+21C>T). IQSEC2 gene expression study revealed that this intronic variant created an alternative donor splicing site and an aberrant product, with the inclusion of 19bp, confirming the pathogenic effect of the intron variant. Moreover, a strong reduction in the expression of the long, but also the short IQSEC2 isoforms, was detected in the male correlating with a more severe phenotype, while the female case showed no decreased expression of the short isoform, and milder effects of the disease. This suggests that the abnormal expression levels of the different IQSEC2 transcripts could be implicated in the severity of disease manifestations.

Remission of Spontaneous Canine Tumors after Systemic Cellular Viroimmunotherapy.

Dogs with spontaneous tumors treated in veterinary hospitals offer an excellent opportunity for studying immunotherapies, including oncolytic viruses. Oncolytic viruses have advanced into the clinic as an intratumorally administered therapeutic; however, intravenous delivery has been hindered by neutralization in the blood. To circumvent this hurdle, mesenchymal stem cells have been used as a "Trojan horse." Here, we present the treatment of 27 canine patients with cancer with canine mesenchymal stem cells infected with ICOCAV17, a canine oncolytic adenovirus. No significant adverse effects were found. The response rate was 74%, with 14.8% showing complete responses, including total remissions of lung metastasis. We detected virus infection, stromal degeneration, and immune cell infiltration in tumor biopsies after 4 weeks of treatment. The increased presence of antiadenoviral antibodies in the peripheral blood of treated dogs did not appear to prevent the clinical benefit of this therapy. These data indicate that oncolytic viruses loaded in mesenchymal stem cells represent an effective cancer immunotherapy.Significance: The classical clinical limitations of antitumoral viroimmunotherapy can be overcome by use of mesenchymal stem cells.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/17/4891/F1.large.jpg Cancer Res; 78(17); 4891-901. ©2018 AACR.

Age-associated hydroxymethylation in human bone-marrow mesenchymal stem cells.

BACKGROUND: Age-associated changes in genomic DNA methylation have been primarily attributed to 5-methylcytosine (5mC). However, the recent discovery of 5-hydroxymethylcytosine (5hmC) suggests that this epigenetic mark might also play a role in the process. METHODS: Here, we analyzed the genome-wide profile of 5hmc in mesenchymal stem cells (MSCs) obtained from bone-marrow donors, aged 2-89 years. RESULTS: We identified 10,685 frequently hydroxymethylated CpG sites in MSCs that were, as in other cell types, significantly associated with low density CpG regions, introns, the histone posttranslational modification H3k4me1 and enhancers. Study of the age-associated changes to 5hmC identified 785 hyper- and 846 hypo-hydroxymethylated CpG sites in the MSCs obtained from older individuals. CONCLUSIONS: DNA hyper-hydroxymethylation in the advanced-age group was associated with loss of 5mC, which suggests that, at specific CpG sites, this epigenetic modification might play a role in DNA methylation changes during lifetime. Since bone-marrow MSCs have many clinical applications, and the fact that the epigenomic alterations in this cell type associated with aging identified in this study could have associated functional effects, the age of donors should be taken into account in clinical settings.

H3K4me1 marks DNA regions hypomethylated during aging in human stem and differentiated cells.

In differentiated cells, aging is associated with hypermethylation of DNA regions enriched in repressive histone post-translational modifications. However, the chromatin marks associated with changes in DNA methylation in adult stem cells during lifetime are still largely unknown. Here, DNA methylation profiling of mesenchymal stem cells (MSCs) obtained from individuals aged 2 to 92 yr identified 18,735 hypermethylated and 45,407 hypomethylated CpG sites associated with aging. As in differentiated cells, hypermethylated sequences were enriched in chromatin repressive marks. Most importantly, hypomethylated CpG sites were strongly enriched in the active chromatin mark H3K4me1 in stem and differentiated cells, suggesting this is a cell type-independent chromatin signature of DNA hypomethylation during aging. Analysis of scedasticity showed that interindividual variability of DNA methylation increased during aging in MSCs and differentiated cells, providing a new avenue for the identification of DNA methylation changes over time. DNA methylation profiling of genetically identical individuals showed that both the tendency of DNA methylation changes and scedasticity depended on nongenetic as well as genetic factors. Our results indicate that the dynamics of DNA methylation during aging depend on a complex mixture of factors that include the DNA sequence, cell type, and chromatin context involved and that, depending on the locus, the changes can be modulated by genetic and/or external factors.

Dopamine mobilizes mesenchymal progenitor cells through D2-class receptors and their PI3K/AKT pathway.

As the nervous system exerts direct and indirect effects on stem cells mobilization and catecholamines mobilize hematopoietic stem cells, we hypothesized that dopamine might induce mesenchymal progenitor cells (MPCs) mobilization. We show that dopamine induced in vitro MPCs migration through D2-class receptors, and their alternative phosphoinositide 3-kinase/Akt pathways. Also, administration of catecholamines induced in vivo mobilization of colony-forming unit-fibroblast in mice. In contrast, in vitro and in vivo MPCs migration was suppressed by D2-class receptors antagonists and blocking antibodies, consistent with dopamine signaling pathway implication. In humans, patients treated with L-dopa or catecholaminergic agonists showed a significant increase of a MPC-like population (CD45-CD31-CD34-CD105+) in their peripheral blood. These findings reveal a new link between catecholamines and MPCs mobilization and suggest the potential use of D2-class receptors agonists for mobilization of MPCs in clinical settings.

In vivo ectopic implantation model to assess human mesenchymal progenitor cell potential.

Clinical interest on human mesenchymal progenitor cells (hMPC) relies on their potential applicability in cell-based therapies. An in vitro characterization is usually performed in order to define MPC potency. However, in vitro predictions not always correlate with in vivo results and thus there is no consensus in how to really assess cell potency. Our goal was to provide an in vivo testing method to define cell behavior before therapeutic usage, especially for bone tissue engineering applications. In this context, we wondered whether bone marrow stromal cells (hBMSC) would proceed in an osteogenic microenvironment. Based on previous approaches, we developed a fibrin/ceramic/BMP-2/hBMSCs compound. We implanted the compound during only 2 weeks in NOD-SCID mice, either orthotopically to assess its osteoinductive property or subcutaneously to analyze its adequacy as a cell potency testing method. Using fluorescent cell labeling and immunohistochemistry techniques, we could ascertain cell differentiation to bone, bone marrow, cartilage, adipocyte and fibrous tissue. We observed differences in cell potential among different batches of hBMSCs, which did not strictly correlate with in vitro analyses. Our data indicate that the method we have developed is reliable, rapid and reproducible to define cell potency, and may be useful for testing cells destined to bone tissue engineering purposes. Additionally, results obtained with hMPCs from other sources indicate that our method is suitable for testing any potentially implantable mesenchymal cell. Finally, we propose that this model could successfully be employed for bone marrow niche and bone tumor studies.

Enrichment of neural-related genes in human mesenchymal stem cells from neuroblastoma patients.

Neuroblastoma (NB) is one of the most common pediatric solid tumors and, like most human cancers, is characterized by a broad variety of genomic alterations. Although mesenchymal stem cells (MSCs) are known to interact with cancer cells, the relationship between MSCs and metastatic NB cancer cells in bone marrow (BM) is unknown. To obtain genetic evidence about this interaction, we isolated ΒΜ-derived MSCs from children with NB and compared their global expression patterns with MSCs obtained from normal pediatric donors, using the Agilent 44K microarrays. Significance analysis of microarray results with a false discovery rate (FDR) <5% identified 496 differentially expressed genes showing either a 2-fold upregulation or downregulation between both groups of samples. Comparison of gene ontology categories of differentially expressed genes revealed the upregulation of genes categorized as 'neurological system process', 'cell adhesion', 'apoptosis', 'cell surface receptor linked signal transduction', 'intrinsic to membrane' and 'extracellular region'. Among the downregulated genes, several immunology-related terms were the most abundant. These findings provide preliminary genetic evidence of the interaction between MSCs and NB cancer cells in ΒΜ as well as identify relevant biological processes potentially altered in MSCs in response to NB.