Single-Cell RNA Sequencing Reveals Repair Features of Human Umbilical Cord Mesenchymal Stromal Cells.

RATIONALE: The chronic lung disease bronchopulmonary dysplasia (BPD) is the most severe complication of extreme prematurity. BPD results in impaired lung alveolar and vascular development and long-term respiratory morbidity, for which only supportive therapies exist. Umbilical cord-derived mesenchymal stromal cells (UC-MSCs) improve lung structure and function in experimental BPD. Results of clinical trials with MSCs for many disorders do not yet match the promising preclinical studies. A lack of specific criteria to define functionally distinct MSCs persists. OBJECTIVES: To determine and correlate single-cell UC-MSC transcriptomic profile with therapeutic potential. METHODS: UC-MSCs from five term donors and human neonatal dermal fibroblasts (HNDFs, control cells of mesenchymal origin) transcriptomes were investigated by single-cell RNA sequencing analysis (scRNA-seq). The lung-protective effect of UC-MSCs with a distinct transcriptome and control HNDFs was tested in vivo in hyperoxia-induced neonatal lung injury in rats. MEASUREMENTS AND MAIN RESULTS: UC-MSCs showed limited transcriptomic heterogeneity, but were different from HNDFs. Gene ontology enrichment analysis revealed distinct - progenitor-like and fibroblast-like - UC-MSC subpopulations. Only the treatment with progenitor-like UC-MSCs improved lung function and structure and attenuated pulmonary hypertension in hyperoxia-exposed rat pups. Moreover, scRNA-seq identified major histocompatibility complex class I as a molecular marker of non-therapeutic cells and associated with decreased lung retention. CONCLUSIONS: UC-MSCs with a progenitor-like transcriptome, but not with a fibroblast-like transcriptome, provide lung protection in experimental BPD. High expression of major histocompatibility complex class I is associated with reduced therapeutic benefit. scRNA-seq may be useful to identify subsets of MSCs with superior repair capacity for clinical application.

Prophylactic Administration of Mesenchymal Stromal Cells Does Not Prevent Arrested Lung Development in Extremely Premature-Born Non-Human Primates.

Premature birth is a leading cause of childhood morbidity and mortality and often followed by an arrest of postnatal lung development called bronchopulmonary dysplasia. Therapies using exogenous mesenchymal stromal cells (MSC) have proven highly efficacious in term-born rodent models of this disease, but effects of MSC in actual premature-born lungs are largely unknown. Here, we investigated thirteen non-human primates (baboons; Papio spp.) that were born at the limit of viability and given a single, intravenous dose of ten million human umbilical cord tissue-derived MSC per kilogram or placebo immediately after birth. Following two weeks of human-equivalent neonatal intensive care including mechanical ventilation, lung function testing and echocardiographic studies, lung tissues were analyzed using unbiased stereology. We noted that therapy with MSC was feasible, safe and without signs of engraftment when administered as controlled infusion over 15 minutes, but linked to adverse events when given faster. Administration of cells was associated with improved cardiovascular stability, but neither benefited lung structure, nor lung function after two weeks of extrauterine life. We concluded that a single, intravenous administration of MSC had no short- to mid-term lung-protective effects in extremely premature-born baboons, sharply contrasting data from term-born rodent models of arrested postnatal lung development and urging for investigations on the mechanisms of cell-based therapies for diseases of prematurity in actual premature organisms.

AMPA Receptor Antagonist CFM-2 Decreases Survivin Expression in Cancer Cells.

BACKGROUND: Glutamate receptors are widely expressed in different types of cancer cells. α-Amino-3- hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors are ionotropic glutamate receptors which are coupled to intracellular signaling pathways that influence cancer cell survival, proliferation, and migration. Blockade of AMPA receptors by pharmacologic compounds may potentially constitute an effective tool in anticancer treatment strategies. METHOD: Here we investigated the impact of the AMPA receptor antagonist CFM-2 on the expression of the protein survivin, which is known to promote cancer cell survival and proliferation. We show that CFM-2 inhibits survivin expression at mRNA and protein levels and decreases the viability of cancer cells. Using a stably transfected cell line which overexpresses survivin, we demonstrate that over-expression of survivin enhances cancer cell viability and attenuates CFM-2-mediated inhibition of cancer cell growth. RESULT: These findings point towards suppression of survivin expression as a new mechanism contributing to anticancer effects of AMPA antagonists.

Matrix metalloproteinase 9 regulates cell death following pilocarpine-induced seizures in the developing brain.

Matrix metalloproteinases (MMPs) are involved in tissue repair, cell death and morphogenesis. We investigated the role of the gelatinases MMP-2 and MMP-9 in the pathogenesis of neuronal death induced by prolonged seizures in the developing brain. Seven-day-old rats, MMP-9 knockout mice and transgenic rats overexpressing MMP-9 received intraperitoneal injections of pilocarpine, 250 mg/kg, to induce seizures. After 6-72 h pups were sacrificed, tissue from different brain regions was isolated and expression of MMP-9 mRNA and protein was analyzed by real-time PCR or Western blot. Additionally, brains were fixed and processed for TUNEL-staining, immunohistochemistry and in situ zymography. We found increased numbers of TUNEL-positive cells 24 h after pilocarpine-induced seizures, most pronounced in cortical areas and the dentate gyrus, and less pronounced in thalamus. At 6-24 h, MMP-9 mRNA levels showed significant elevation compared to sham-treated controls; this effect resolved by 48 h, whereas MMP-2 mRNA levels remained stable. Cortical gelatinolytic activity, monitored by in situ zymography, was enhanced following pilocarpine-induced seizures. The MMP inhibitor GM 6001 ameliorated cell death following pilocarpine-induced seizures in infant rats. MMP-9 knockout mice were less susceptible to seizure-induced brain injury. Transgenic rats overexpressing MMP-9 were equally susceptible to seizure-induced brain injury as wild type rats. Our results suggest a significant contribution of MMP-9 to cell death after pilocarpine-induced seizures in the developing brain. As indicated by Western blot analysis, MMP-9 activation may be linked to activation of the Erk/CREB-pathway. The findings implicate involvement of MMP-9 in the pathophysiology of brain injury following seizures in the developing brain.

Silencing of selected glutamate receptor subunits modulates cancer growth.

BACKGROUND: Emerging evidence supports a role for glutamate in the biology of cancer. We studied the impact of glutamate receptor subunit silencing on cancer phenotype. MATERIALS AND METHODS: Different fragments of the coding region for ionotropic glutamate receptor AMPA 4 (GLUR4), ionotropic glutamate receptor N-methyl D-aspartate 1 (NR1), ionotropic glutamate receptor kainate 5 (KA2) and ionotropic glutamate receptor N-methyl D-aspartate 2D (NR2D) were stably transfected into human TE671, RPMI8226 and A549 cell lines. Resulting changes in cell proliferation, migration and mRNA expression of genes that determine cancer phenotype were assayed. RESULTS: Decreased expression of GLUR4 markedly increased cancer cell proliferation, whereas decreased expression of NR1 markedly reduced the propensity of cancer cells to proliferate. Knockdown of KA2 and NR2D did not influence cancer phenotype. Gene silencing of GLUR4 modulated the mRNA expression of various genes in these cancer cell lines, as determined with the Human Cancer PathwayFinder™ PCR Array. Knockdown of GLUR4 influenced the expression and function of genes involved in invasion and metastasis, tumour suppressor genes, oncogenes and adhesion genes. CONCLUSION: The findings suggest that glutamate receptor subunits on cancer cells are linked to biochemical pathways that regulate malignant phenotype.

Glutamate receptors in laryngeal cancer cells.

AIM: Despite recent improvements in treatment strategies, the results of chemotherapy in patients with advanced squamous cell carcinoma of the larynx are not satisfactory. Thus, the development of new approaches which influence specific metabolic pathways are needed. In the last decade, evidence has emerged implicating a role for glutamate as a signal mediator in tumors. MATERIALS AND METHODS: The presence of glutamate receptor subunits in two laryngeal cancer cell lines (RK33 and RK45) was evaluated by means of end-point PCR, real-time PCR, and immunocytochemistry. RESULTS: Glutamate receptor subunits are differentially expressed in laryngeal cancer cell lines. In addition, we show that selected ionotropic glutamate receptor antagonists and metabotropic glutamate receptor 5 antagonist inhibit proliferation of laryngeal cancer cells. Glutamate antagonists also affected activity of extracellular signal-regulated kinases 1/2 in tumor cells. CONCLUSION: Signaling through glutamate receptors may influence growth of laryngeal cancer cells and may constitute an adjunctive therapeutic target.

Matrix metalloproteinases 2 and 9 fail to influence drug-induced neuroapoptosis in developing rat brain.

Matrix metalloproteinases (MMPs) play an essential role in tissue repair, cell death, and morphogenesis. The aim of the present study was to investigate potential involvement of selected MMPs in the pathogenesis of neuronal apoptosis induced by the NMDA antagonist MK-801 (dizocilpine) or the GABA(A) agonist phenobarbital in infant rats, transgenic rats overexpressing MMP-9 and MMP-9 knockout mice. Seven-day-old rats or knockout mice received intraperitoneal injections of MK-801, 1 mg/kg, or phenobarbital, 50 mg/kg. At different survival intervals following administration of the compounds (1-72 h), pups were sacrificed, tissue from different brain regions was isolated, and the expression and activity of MMP-2 and MMP-9 were analyzed by real-time PCR, western blot, and zymography. In addition, brains were fixed and processed for TUNEL staining. In all the brain regions analyzed, we found an increased number of TUNEL-positive cells 24 h after administration of MK-801. After treatment, we detected no significant increase in MMP-2 or MMP-9 mRNA expression in cortical areas. No changes in the MMP-9 protein expression or gelatinolytic activity of MMP-2 were observed in conjunction with MK-801 or phenobarbital-induced neuroapoptosis in any brain region analyzed. The extent of neurodegeneration induced by MK-801 or phenobarbital was not altered in MMP-9 transgenic rats and was increased in MMP-9 knockout mice compared to wild-type rats and mice. Treatment with the panmetalloproteinase inhibitor GM6001 did not confer protection against MK-801-induced apoptotic cell death in the developing rat brain. Our results suggest that activation of MMP-9 and MMP-2 does not contribute to pathogenesis of neuronal apoptosis caused by NMDA antagonists or GABA(A) agonists in the developing rat and mouse brain.

Glutamate receptors in pediatric tumors of the central nervous system.

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS). Experimental evidence indicates that glutamate receptor antagonists may limit tumor growth. This study explores expression of glutamate receptor subunits in pediatric CNS tumors. Samples from eight ependymomas, four glioblastomas, six medulloblastomas and eight low grade astrocytomas were analysed. RNA was used for semiquantitative and quantitative RT-PCR. We examined expression of NMDA receptor subunits NR1-NR3B, AMPA receptor subunits GluR1-GluR4, kainate receptor subunits GluR5-GluR7, KA1, KA2 and metabotropic receptor subunits mGluR1-8. Paraffin embedded samples were immunohistochemically stained for selected subunits. All glutamate receptor subunits were differentially expressed in the tumors examined. Expression of NR2D, NR3A, KA1, GluR4, mGluR1, mGluR4, mGluR5 and mGluR6 was higher in the high grade tumors compared to human brain (HB). In low grade astrocytomas expression of glutamate receptor subunits was comparable or lower than in HB. Immunohistochemistry revealed expression of several glutamate receptor subunit proteins in tumor specimen. This study demonstrates expression of glutamate receptor subunits in pediatric CNS tumors. Together with experimental evidence indicating that interference with glutamate signalling may suppress tumor growth, our findings suggest that adjunctive treatment with glutamate receptor modulators may be a feasible therapeutic option for pediatric patients with CNS tumors.

Expression of glutamate receptor subunits in human cancers.

Emerging evidence suggests a role for glutamate and its receptors in the biology of cancer. This study was designed to systematically analyze the expression of ionotropic and metabotropic glutamate receptor subunits in various human cancer cell lines, compare expression levels to those in human brain tissue and, using electrophysiological techniques, explore whether cancer cells respond to glutamate receptor agonists and antagonists. Expression analysis of glutamate receptor subunits NR1-NR3B, GluR1-GluR7, KA1, KA2 and mGluR1-mGluR8 was performed by means of RT-PCR in human rhabdomyosarcoma/medulloblastoma (TE671), neuroblastoma (SK-NA-S), thyroid carcinoma (FTC 238), lung carcinoma (SK-LU-1), astrocytoma (MOGGCCM), multiple myeloma (RPMI 8226), glioma (U87-MG and U343), lung carcinoma (A549), colon adenocarcinoma (HT 29), T cell leukemia cells (Jurkat E6.1), breast carcinoma (T47D) and colon adenocarcinoma (LS180). Analysis revealed that all glutamate receptor subunits were differentially expressed in the tumor cell lines. For the majority of tumors, expression levels of NR2B, GluR4, GluR6 and KA2 were lower compared to human brain tissue. Confocal imaging revealed that selected glutamate receptor subunit proteins were expressed in tumor cells. By means of patch-clamp analysis, it was shown that A549 and TE671 cells depolarized in response to application of glutamate agonists and that this effect was reversed by glutamate receptor antagonists. This study reveals that glutamate receptor subunits are differentially expressed in human tumor cell lines at the mRNA and the protein level, and that their expression is associated with the formation of functional channels. The potential role of glutamate receptor antagonists in cancer therapy is a feasible goal to be explored in clinical trials.

The role of matrix metalloproteinases in infant traumatic brain injury.

Matrix metalloproteinases (MMPs) play an essential role in tissue repair, cell death and morphogenesis and may constitute therapeutic targets for acute brain injuries. In this study, we investigated the expression of 72 kDa and 92 kDa collagenases MMP-2 and MMP-9 at transcriptional, functional and protein expression level following traumatic brain injury in infant rats. Seven-day-old Wistar rats were subjected to head trauma using a weight drop device. Pups were sacrificed at defined time points (2-72 h) after trauma and brains were processed for molecular studies (semiquantitative and real-time PCR, Western blot, gelatin zymography) and histology. Trauma triggered widespread cell death in the cortex, basal ganglia and white matter. mRNA levels for MMP-2 and -9 were increased in the brain at 12-72 h after trauma. Protein expression of the analyzed MMPs and activity of MMP-2 were increased at 12 h and peaked at 24 h after trauma. Intraperitoneal injection of GM6001 (Ilomastat), an MMP inhibitor, 2 h after trauma, substantially attenuated traumatic brain injury in a dose-dependent manner. These findings causally link the MMPs to trauma-induced neuronal cell death in the immature rodent brain. MMPs might serve as useful targets for therapeutic approaches aimed at preserving neuronal function in the immature brain in the context of mechanical injury.