Direct conversion of somatic cells towards oligodendroglial lineage cells: A novel strategy for enhancement of myelin repair.

Oligodendrocyte precursor cells (OPCs) are considered as the main cell source for myelination in the central nervous system. Following demyelination, proliferation, migration, and differentiation capability of endogenous OPCs remarkably increase leading to remyelination in damaged areas. Despite the beneficial impacts of resident OPCs for myelin repair, the capacity of endogenous repair is low and insufficient. Therefore, several strategies have been developed to improve endogenous myelin repair. Although stem cell therapy has been introduced as a promising strategy for neurodegenerative disorders, but several limitations such as cell rejection, teratoma formation, and ethical concerns have hampered the extensive application of stem cells in clinic. In recent years, direct conversion of fully differentiated somatic cells into desired cells in the lesion area has opened a new era in regenerative medicine. In addition to direct reprogramming of somatic cells to neurons, recent evidence have also demonstrated that somatic cells, including fibroblasts and astrocytes, can be directly reprogrammed to OPC-like cells by overexpression of some specific transcription factors, microRNAs, or application of small molecules. Interestingly, induced OPCs differentiated to myelinating oligodendrocytes that could effectively ensheath the host axons. In the present review article, the current advancements in direct conversion of somatic cells towards oligodendroglial cells have been discussed both in vitro and in vivo.

Regulation of von Willebrand Factor Gene in Endothelial Cells That Are Programmed to Pluripotency and Differentiated Back to Endothelial Cells.

Endothelial cells play a central role in physiological function and pathophysiology of blood vessels in health and disease. However, the molecular mechanism that establishes the endothelial phenotype, and contributes to its signature cell type-specific gene expression, is not yet understood. We studied the regulation of a highly endothelial-specific gene, von Willebrand factor (VWF), in induced pluripotent stem cells generated from primary endothelial cells (human umbilical vein endothelial cells [HUVEC] into a pluripotent state [HiPS]) and subsequently differentiated back into endothelial cells. This allowed us to explore how VWF expression is regulated when the endothelial phenotype is revoked (endothelial cells to HiPS), and re-established (HiPS back to endothelial cells [EC-Diff]). HiPS were generated from HUVECs, their pluripotency established, and then differentiated back to endothelial cells. We established phenotypic characteristics and robust angiogenic function of EC-Diff. Gene array analyses, VWF chromatin modifications, and transacting factors binding assays were performed on the three cell types (HUVEC, HiPS, and EC-Diff). The results demonstrated that generally cohorts of transacting factors that function as transcriptional activators, and those that contribute to histone acetylation and DNA demethylation, were significantly decreased in HiPS compared with HUVECs and EC-Diff. In contrast, there were significant increases in the gene expression levels of epigenetic modifiers that function as methyl transferases in HiPS compared with endothelial cells. The results demonstrated that alterations in chromatin modifications of the VWF gene, in addition to expression and binding of transacting factors that specifically function as activators, are responsible for establishing endothelial specific regulation of the VWF gene. Stem Cells 2019;37:542-554.

Facio-genital dysplasia-5 regulates matrix adhesion and survival of human endothelial cells.

OBJECTIVE: The function of the endothelial cell (EC)-enriched Rho family guanine nucleotide exchange factor, facio-genital dysplasia-5 (FGD5), is poorly understood. We sought to determine whether FGD5 regulates endothelial cytoskeletal reorganization and angiogenesis. METHODS AND RESULTS: We observed that FGD5 is expressed in primary human EC isolated from sites across the vasculature. Inhibition of FGD5 expression using RNA interference decreased the protein by ≈70%. In 3-dimensional vascular endothelial growth factor-stimulated angiogenesis in vitro, FGD5-deficient endothelial sprout protrusion was markedly blunted versus nonsilenced controls. FGD5 knockdown impaired adhesion to fibronectin and collagen IV and remodeling of matrix adhesion complexes. Similarly, monolayer electric impedance was decreased, and impedance increased at a slower rate after seeding FGD5-deficient cells versus controls, reflecting decreased EC spreading. Further, FGD5 plays a role in cell survival, because expression of cleaved caspase-3 was increased in FGD5-deficient EC after loss of cell-matrix contacts, and proapoptotic tumor necrosis factor-α stimulation elicited EC with subdiploid DNA content among FGD5-deficient EC. Mechanistically, the phosphatidylinositol 3-kinase/Akt pathway that regulates both adhesive and survival signal transduction pathways requires FGD5. Vascular endothelial growth factor-stimulated Akt phosphorylation and downstream forkhead box protein-O1 inactivation is inhibited by FGD5 loss. CONCLUSIONS: FGD5 regulates endothelial adhesion, survival, and angiogenesis by modulating phosphatidylinositol 3-kinase signaling.

Fractalkine enhances oligodendrocyte regeneration and remyelination in a demyelination mouse model.

Demyelinating disorders of the central nervous system (CNS) occur when myelin and oligodendrocytes are damaged or lost. Remyelination and regeneration of oligodendrocytes can be achieved from endogenous oligodendrocyte precursor cells (OPCs) that reside in the adult CNS tissue. Using a cuprizone mouse model of demyelination, we show that infusion of fractalkine (CX3CL1) into the demyelinated murine brain increases de novo oligodendrocyte formation and enhances remyelination in the corpus callosum and cortical gray matter. This is achieved by increased OPC proliferation in the cortical gray matter as well as OPC differentiation and attenuation of microglia/macrophage activation both in corpus callosum and cortical gray matter. Finally, we show that activated OPCs and microglia/macrophages express fractalkine receptor CX3CR1 in vivo, and that in OPC-microglia co-cultures fractalkine increases in vitro oligodendrocyte differentiation by modulating both OPC and microglia biology. Our results demonstrate a novel pro-regenerative role of fractalkine in a demyelinating mouse model.

Native PLGA nanoparticles regulate APP metabolism and protect neurons against ß-amyloid toxicity: Potential significance in Alzheimer's disease pathology.

Biodegradable poly(lactic-co-glycolic acid)(PLGA) nanoparticles have been used extensively in delivering drugs to target tissues due to their excellent biocompatibility. Evidence suggests that PLGA-conjugated drugs/agents can attenuate pathology in cellular/animal models of Alzheimer's disease (AD), which is initiated by increased level/aggregation of amyloid ß (Aß) peptide generated from amyloid precursor protein (APP). The beneficial effects were attributed to conjugated-drugs rather than to PLGA nanoparticles. Interestingly, we recently reported that PLGA without any drug/agent (native PLGA) can suppress Aß aggregation/toxicity. However, very little is known about the internalization, subcellular localization or effects of PLGA in neurons. In this study, using primary mouse cortical neurons, we first showed that native PLGA is internalized by an energy-mediated clathrin-dependent/-independent pathway and is localized in endosomal-lysosomal-autophagic vesicles. By attenuating internalization, PLGA can protect neurons against Aß-mediated toxicity. Additionally, PLGA treatment altered expression profiles of certain AD-associated genes and decreased the levels of APP, its cleaved products α-/ß-CTFs and Aß peptides in mouse as well as iPSC-derived neurons from control and AD patients. Collectively, these results suggest that native PLGA not only protects neurons against Aß-induced toxicity but also influences the expression of AD-related genes/proteins - highlighting PLGA's implication in normal and AD-related pathology.

Significance of native PLGA nanoparticles in the treatment of Alzheimer's disease pathology.

Alzheimer's disease (AD) is believed to be triggered by increased levels/aggregation of ß-amyloid (Aß) peptides. At present, there is no effective disease-modifying treatment for AD. Here, we evaluated the therapeutic potential of FDA-approved native poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles on Aß aggregation and in cellular/animal models of AD. Our results showed that native PLGA can not only suppress the spontaneous aggregation but can also trigger disassembly of preformed Aß aggregates. Spectroscopic studies, molecular dynamics simulations and biochemical analyses revealed that PLGA, by interacting with the hydrophobic domain of Aß1-42, prevents a conformational shift towards the ß-sheet structure, thus precluding the formation and/or triggering disassembly of Aß aggregates. PLGA-treated Aß samples can enhance neuronal viability by reducing phosphorylation of tau protein and its associated signaling mechanisms. Administration of PLGA can interact with Aß aggregates and attenuate memory deficits as well as Aß levels/deposits in the 5xFAD mouse model of AD. PLGA can also protect iPSC-derived neurons from AD patients against Aß toxicity by decreasing tau phosphorylation. These findings provide unambiguous evidence that native PLGA, by targeting different facets of the Aß axis, can have beneficial effects in mouse neurons/animal models as well as on iPSC-derived AD neurons - thus signifying its unique therapeutic potential in the treatment of AD pathology.

Glomerular endothelial PI3 kinase-α couples to VEGFR2, but is not required for eNOS activation.

Vascular endothelial growth factor (VEGF)-dependent signals are central to many endothelial cell (EC) functions, including survival and regulation of vascular tone. Akt and endothelial nitric oxide synthase (eNOS) activity are implicated to mediate these effects. Dysregulated signaling is characteristic of endothelial dysfunction that sensitizes the glomerular microvasculature to injury. Signaling intermediates that couple VEGF stimulation to eNOS activity remain unclear; hence, we examined the PI3 kinase isoforms implicated to regulate these enzymes. Using a combination of small molecule inhibitors and RNAi to study responses to VEGF in glomerular EC, we observed that the PI3 kinase p110α catalytic isoform is coupled to VEGFR2 and regulates the bulk of Akt activity. Coimmunoprecipitation experiments support a physical association of p110α with VEGFR2. Downstream, Akt-mediated FOXO1 phosphorylation in EC is regulated by p110α. The p110δ isoform contributes a minor amount of VEGF-stimulated Akt activation. However, we observe no effect of p110α or p110δ to regulate VEGF-stimulated eNOS activation via Akt-mediated phosphorylation on eNOS Ser1177, or NO-mediated vasodilation of the afferent arteriole ex vivo. VEGFR2-stimulated eNOS activation and NO production are inhibited by Compound C, an inhibitor of AMP-stimulated kinase, independent of PI3 kinase signaling. PI3 kinase-α/δ-mediated signaling downstream of VEGFR2 activation regulates Akt-dependent survival signals, but our data suggest it is not required to activate eNOS or to elicit NO production in glomerular EC.

Endothelial IQGAP1 regulates efficient lymphocyte transendothelial migration.

Leukocyte movement from the blood to the tissues is a fundamental process in acute and chronic inflammatory diseases. While the role of endothelial cells (EC) to recruit leukocytes to sites of inflammation is well known, the mechanisms that control remodeling of EC shape and adhesive contacts during leukocyte transendothelial migration (TEM) are not completely understood. We studied the role of IQGAP1, an adaptor protein that binds to filamentous-actin and microtubules (MT) at interendothelial junctions, during lymphocyte TEM. EC IQGAP1 knockdown decreases MT tethered to the adherens junction, and decreases lymphocyte TEM to approximately 70% (p<0.05) versus control. Similarly, loss of adherens junction-associated MT induced by brief nocodazole (ND) treatment decreases lymphocyte TEM to approximately 65% of control (p<0.01). Confocal microscopy imaging indicates that EC IQGAP1 knockdown and MT depolymerization both result in lymphocyte accumulation above the vascular endothelial cadherin (VE-cadherin) junctions and reduces the fraction of adherent lymphocytes that complete diapedesis across interendothelial cell junctions. However, we observe no change in VE-cadherin gap formation underlying adherent lymphocytes among control, IQGAP1 knockdown, or MT depolymerised EC monolayers. These data indicate that IQGAP1 contributes to MT stability at endothelial junctions. Further, IQGAP1 is involved in junction remodeling required for efficient lymphocyte diapedesis, independent of VE-cadherin gap formation.

In Silico Analysis to Explore Lineage-Independent and -Dependent Transcriptional Programs Associated with the Process of Endothelial and Neural Differentiation of Human Induced Pluripotent Stem Cells.

Despite a major interest in understanding how the endothelial cell phenotype is established, the underlying molecular basis of this process is not yet fully understood. We have previously reported the generation of induced pluripotent stem cells (iPS) from human umbilical vein endothelial cells and differentiation of the resulting HiPS back to endothelial cells (Ec-Diff), as well as neural (Nn-Diff) cell lineage that contained both neurons and astrocytes. Furthermore, the identities of these cell lineages were established by gene array analysis. Here, we explored the same arrays to gain insight into the gene alteration processes that accompany the establishment of endothelial vs. non-endothelial neural cell phenotypes. We compared the expression of genes that code for transcription factors and epigenetic regulators when HiPS is differentiated into these endothelial and non-endothelial lineages. Our in silico analyses have identified cohorts of genes that are similarly up- or downregulated in both lineages, as well as those that exhibit lineage-specific alterations. Based on these results, we propose that genes that are similarly altered in both lineages participate in priming the stem cell for differentiation in a lineage-independent manner, whereas those that are differentially altered in endothelial compared to neural cells participate in a lineage-specific differentiation process. Specific GATA family members and their cofactors and epigenetic regulators (DNMT3B, PRDM14, HELLS) with a major role in regulating DNA methylation were among participants in priming HiPS for lineage-independent differentiation. In addition, we identified distinct cohorts of transcription factors and epigenetic regulators whose alterations correlated specifically with the establishment of endothelial vs. non-endothelial neural lineages.

Cross-sectional analysis of peripheral blood mononuclear cells in lymphopenic and non-lymphopenic relapsing-remitting multiple sclerosis patients treated with dimethyl fumarate.

BACKGROUND: Relapsing-remitting multiple sclerosis (RRMS) is an autoimmune disorder of the central nervous system. Dimethyl Fumarate is a disease-modifying medication used to treat RRMS patients that can induce lymphopenia. We aimed to immunophenotype peripheral blood mononuclear cells (PBMC) in RRMS patients cross-sectionally and examine the characteristics and modifications of lymphopenia over time. METHODS: Characterization of PBMC was done by multiparametric flow cytometry. Patients had been on treatment for up to 4 years and were grouped into lymphopenic (DMF-L) and non-lymphopenic (DMF-N) patients. RESULTS: Lymphopenia affected the cell population changes over time, with other patient characteristics (gender, age, and previous treatment status) also having significant effects. In both lymphopenic and non-lymphopenic patients, PBMC percentages were reduced over time. While overall T and B cells frequencies were not affected, males, older patients and untreated patients had significant changes in B cell subpopulations over time. CD4+ to CD8+T cell ratio increased significantly in lymphopenic patients over time. CD4-CD8-T cell population was similarly reduced in both lymphopenic and non-lymphopenic patients, over time. While the monocyte and NK overall populations were not changed, non-classical monocyte subpopulation decreased over time in lymphopenic patients. We also found CD56-CD16+ and CD56-CD16- NK cells frequencies changed over time in lymphopenic patients. Immune populations showed correlations with clinical outcomes measured by EDSS and relapse rate. Analysis of the overall immunophenotype showed that, while groups divided by other patient characteristics showed differences, the lymphopenia status overrode these differences, resulting in similar immunophenotype within DMF-L. CONCLUSIONS: Our data provide evidence that under the same therapy, lymphopenia affects how the immunophenotype changes over time and can override the differences associated with other patient characteristics and possibly mask other significant changes in the immune profile of patients.

Multimodal peripheral fluid biomarker analysis in clinically isolated syndrome and early multiple sclerosis.

BACKGROUND: Increasing evidence suggests that various inflammatory, immunological and metabolic pathways are altered in the clinically isolated syndrome (CIS) of multiple sclerosis (MS). Moreover, recent diagnostic criteria have made possible the very early diagnosis of MS. We evaluated multiple fluid biomarkers in people with early MS and CIS. METHODS: We measured blood levels of cytokines, matrix metalloproteinases (MMPs), serum metabolomics and immune cell immunophenotyping in participants in the Trial of Minocycline in a Clinically Isolated Syndrome of Multiple Sclerosis. RESULTS: When compared with healthy controls, people with early MS/CIS had higher levels of eotaxin, MCP-3, IL-1 receptor antagonist, IL-1ß, IL-9 and IP-10, as well as MMPs 1, 8 and 9. In metabolomics analysis, the alanine, aspartate and glutamate metabolism and the synthesis and degradation of ketone bodies pathways were altered compared to healthy controls. There were no differences in lymphocyte subpopulation numbers. Out of all these biomarkers, only MMP-1 was able to differentiate between early MS and CIS, and was found to correlate with lesion volume and gadolinium enhancing lesions on MRI. CONCLUSION: The immunological and metabolic profile of CIS and early MS is remarkably similar, supporting that these are a continuum of a common underlying pathophysiological process.

Erratum: Author Correction: Repression of phagocytosis by human CD33 is not conserved with mouse CD33.

[This corrects the article DOI: 10.1038/s42003-019-0698-6.].

Endothelial cells of different organs exhibit heterogeneity in von Willebrand factor expression in response to hypoxia.

BACKGROUND AND AIMS: We have previously demonstrated that in response to hypoxia, von Willebrand factor (VWF) expression is upregulated in lung and heart endothelial cells both in vitro and in vivo, but not in kidney endothelial cells. The aim of our current study was to determine whether endothelial cells of different organs employ distinct molecular mechanisms to mediate VWF response to hypoxia. METHODS: We used cultured human primary lung, heart and kidney endothelial cells to determine the activation of endogenous VWF as well as exogenously expressed VWF promoter in response to hypoxia. Chromatin immunoprecipitation and siRNA knockdown analyses were used to determine the roles of VWF promoter associated transacting factors in mediating its hypoxia response. Platelet aggregates formations in vascular beds of mice were used as a marker for potential functional consequences of hypoxia-induced VWF upregulation in vivo. RESULTS: Our analyses demonstrated that while Yin Yang 1 (YY1) and specificity protein 1 (Sp1) participate in the hypoxia-induced upregulation of VWF specifically in lung endothelial cells, GATA6 mediates this process specifically in heart endothelial cells. In both cell types, the response to hypoxia involves the decreased association of the NFIB repressor with the VWF promoter, and the increased acetylation of the promoter-associated histone H4. In mice exposed to hypoxia, the upregulation of VWF expression was concomitant with the presence of thrombi in heart and lung, but not kidney vascular beds. CONCLUSIONS: Heart and lung endothelial cells demonstrated VWF upregulation in response to hypoxia, using distinct mechanisms, while this response was lacking in kidney endothelial cells.

Repression of phagocytosis by human CD33 is not conserved with mouse CD33.

CD33 is an immunomodulatory receptor linked to Alzheimer's disease (AD) susceptibility via regulation of phagocytosis in microglia. Divergent features between human CD33 (hCD33) and murine CD33 (mCD33) include a unique transmembrane lysine in mCD33 and cytoplasmic tyrosine in hCD33. The functional consequences of these differences in restraining phagocytosis remains poorly understood. Using a new αmCD33 monoclonal antibody, we show that mCD33 is expressed at high levels on neutrophils and low levels on microglia. Notably, cell surface expression of mCD33 is entirely dependent on Dap12 due to an interaction with the transmembrane lysine in mCD33. In RAW264.7 cultured macrophages, BV-2 cultured microglia, primary neonatal and adult microglia, uptake of cargo - including aggregated Aß1-42 - is not altered upon genetic ablation of mCD33. Alternatively, deletion of hCD33 in monocytic cell lines increased cargo uptake. Moreover, transgenic mice expressing hCD33 in the microglial cell lineage showed repressed cargo uptake in primary microglia. Therefore, mCD33 and hCD33 have divergent roles in regulating phagocytosis, highlighting the importance of studying hCD33 in AD susceptibility.

Characterization of lymphopenia in patients with MS treated with dimethyl fumarate and fingolimod.

OBJECTIVE: Lymphopenia is a common occurrence of disease-modifying therapies (DMTs) for relapsing-remitting MS (RRMS). The aim of this study was to dissect the prevalence of various lymphocyte subsets in patients with RRMS treated with 2 DMTs commonly associated with lymphopenia, dimethyl fumarate (DMF), and fingolimod (FTY). METHODS: Multicolor flow cytometry and multiplex assays were used to identify up to 50 lymphocyte subpopulations and to examine the expression of multiple cytokines in selected patients. We compared patients untreated (NT) or treated with FTY or DMF who did (DMF-L) or did not (DMF-N) develop lymphopenia. RESULTS: All FTY patients developed lymphopenia in both T-cell and B-cell compartments. CD41 T cells were more affected by this treatment than CD81 cells. In the B-cell compartment, the CD271IgD2 subpopulation was reduced. T cells but not B cells were significantly reduced in DMF-L. However, within the B cells, CD271 cells were significantly lower. Both CD41 and CD81 subpopulations were reduced in DMF-L. Within the remaining CD41 and CD81 compartments, there was an expansion of the naive subpopulation and a reduction of the effector memory subpopulation. Unactivated lymphocyte from DMF-L patients had significantly higher levels of interferon-γ, interleukin (IL)-12, IL-2, IL-4, IL-6, and IL-1ß compared with DMF-N. In plasma, TNFß was significantly higher in DMF-N and DMF-L compared with NT, whereas CCL17 was significantly higher in DMF-L compared with NT and DMF-N. CONCLUSIONS: This study shows that different treatments can target different lymphocyte compartments and suggests that lymphopenia can induce compensatory mechanisms to maintain immune homeostasis.

Reprogramming of HUVECs into induced pluripotent stem cells (HiPSCs), generation and characterization of HiPSC-derived neurons and astrocytes.

Neurodegenerative diseases are characterized by chronic and progressive structural or functional loss of neurons. Limitations related to the animal models of these human diseases have impeded the development of effective drugs. This emphasizes the need to establish disease models using human-derived cells. The discovery of induced pluripotent stem cell (iPSC) technology has provided novel opportunities in disease modeling, drug development, screening, and the potential for "patient-matched" cellular therapies in neurodegenerative diseases. In this study, with the objective of establishing reliable tools to study neurodegenerative diseases, we reprogrammed human umbilical vein endothelial cells (HUVECs) into iPSCs (HiPSCs). Using a novel and direct approach, HiPSCs were differentiated into cells of central nervous system (CNS) lineage, including neuronal, astrocyte and glial cells, with high efficiency. HiPSCs expressed embryonic genes such as nanog, sox2 and Oct-3/4, and formed embryoid bodies that expressed markers of the 3 germ layers. Expression of endothelial-specific genes was not detected in HiPSCs at RNA or protein levels. HiPSC-derived neurons possess similar morphology but significantly longer neurites compared to primary human fetal neurons. These stem cell-derived neurons are susceptible to inflammatory cell-mediated neuronal injury. HiPSC-derived neurons express various amino acids that are important for normal function in the CNS. They have functional receptors for a variety of neurotransmitters such as glutamate and acetylcholine. HiPSC-derived astrocytes respond to ATP and acetylcholine by elevating cytosolic Ca2+ concentrations. In summary, this study presents a novel technique to generate differentiated and functional HiPSC-derived neurons and astrocytes. These cells are appropriate tools for studying the development of the nervous system, the pathophysiology of various neurodegenerative diseases and the development of potential drugs for their treatments.

Endothelial PI 3-kinase activity regulates lymphocyte diapedesis.

Lymphocyte recruitment to sites of inflammation involves a bidirectional series of cues between the endothelial cell (EC) and the leukocyte that culminate in lymphocyte migration into the tissue. Remodeling of the EC F-actin cytoskeleton has been observed after leukocyte adhesion, but the signals to the EC remain poorly defined. We studied the dependence of peripheral blood lymphocyte transendothelial migration (TEM) through an EC monolayer in vitro on EC phosphatidylinositol 3-kinase (PI 3-kinase) activity. Lymphocytes were perfused over cytokine-activated EC using a parallel-plate laminar flow chamber. Inhibition of EC PI 3-kinase activity using LY-294002 or wortmannin decreased lymphocyte TEM (48 +/- 6 or 34 +/- 7%, respectively, vs. control; mean +/- SE; P < 0.05). Similarly, EC knockdown of the p85alpha regulatory subunit of PI 3-kinase decreased lymphocyte transmigration. Treatment of EC with jasplakinolide to inhibit EC F-actin remodeling also decreased lymphocyte TEM to 24 +/- 10% vs. control (P < 0.05). EC PI 3-kinase inhibition did not change the strength of lymphocyte adhesion to the EC or formation of the EC "docking structure" after intercellular adhesion molecule-1 ligation, whereas this was inhibited by jasplakinolide treatment. A similar fraction of lymphocytes migrated on control or LY-294002-treated EC and localized to interendothelial junctions. However, lymphocytes failed to extend processes below the level of vascular endothelial (VE)-cadherin on LY-294002-treated EC. Together these observations indicate that EC PI 3-kinase activity and F-actin remodeling are required during lymphocyte diapedesis and identify a PI 3-kinase-dependent step following initial separation of the VE-cadherin barrier.