Carola Meier

Transplantation of human umbilical cord blood cells mediated beneficial effects on apoptosis, angiogenesis and neuronal survival after hypoxic-ischemic brain injury in rats

Transplantation of human umbilical cord blood (hucb) cells in a model of hypoxic-ischemic brain injury led to the amelioration of lesion-impaired neurological and motor functions. However, the mechanisms by which transplanted cells mediate functional recovery after brain injury are largely unknown. In this study, the effects of hucb cell transplantation were investigated in this experimental paradigm at the cellular and molecular level. As the pathological cascade in hypoxic-ischemic brain injury includes inflammation, reduced blood flow, and neuronal cell death, we analyzed the effects of peripherally administered hucb cells on these detrimental processes, investigating the expression of characteristic marker proteins. Application of hucb cells after perinatal hypoxic-ischemic brain injury correlated with an increased expression of the proteins Tie-2 and occludin, which are associated with angiogenesis. Lesion-induced apoptosis, determined by expression of cleaved caspase-3, decreased, whereas the number of vital neurons, identified by counting of NeuN-positive cells, increased. In addition, we observed an increase in the expression of neurotrophic and pro-angiogenic growth factors, namely BDNF and VEGF, in the lesioned brain upon hucb cell transplantation. The release of neurotrophic factors mediated by transplanted hucb cells might cause a lower number of neurons to undergo apoptosis and result in a higher number of living neurons. In parallel, the increase of VEGF might cause growth of blood vessels. Thus, hucb transplantation might contribute to functional recovery after brain injury mediated by systemic or local effects.

Neuroglial activation and Cx43 expression are reduced upon transplantation of human umbilical cord blood cells after perinatal hypoxic-ischemic injury

Glial cells play a crucial role in the pathomechanism of perinatal hypoxic-ischemic brain injury (HI) and are involved in the maintenance of a chronic state of inflammation that causes delayed neuronal damage. Activation of astrocytes is one factor prolonging brain damage and contributing to the formation of a glial scar that limits neuronal plasticity. In this context, the major astrocytic gap junction protein Connexin 43 (Cx43) has been ascribed various functions including regulation of astrocytic migration and proliferation. Here, we investigate glial responses like microglia/macrophages and astrocytic activation in a rat model of neonatal HI and characterize changes of these parameters upon transplantation of human umbilical cord blood cells (hUCB). As an alleviation of motor function in lesioned rats has previously been described in transplanted animals, we analyze the putative correlation between motor function and glial activation over time. The lesion-induced impairment of motor function, assessed by forelimb use bias, muscle strength and distal spasticity, was alleviated upon transplantation of hUCB short and long term. HI induced an acute inflammatory reaction with activation of microglia/macrophages and reactive astrogliosis associated with perilesional upregulation of Cx43 that slowly declined during the chronic post-ischemic phase. hUCB transplantation accelerated the regression of inflammatory events, narrowed the perilesional astrocytic wall and led to a downregulation of the investigated astrocytic proteins. Thus, in the immature brain, hUCB may indirectly reduce secondary cell death upon hypoxia-ischemia and facilitate post-ischemic plasticity through the attenuation of reactive gliosis. This article is part of a Special Issue entitled Electrical Synapses.

Umbilical cord blood cell transplantation after brain ischemia—From recovery of function to cellular mechanisms

Cell transplantation has been proposed as a potential approach to the treatment of neurological disorders. One cell population of interest consists of human umbilical cord blood (hUCB) cells, which have previously been shown to be useful for reparative medicine in haematological diseases. However, hUCB cells are also capable of differentiating into various non-haematopoietic cells, including those of the neural lineage. Moreover, hUCB cells can secrete numerous neurotrophic factors and modulate immune function and inflammatory reaction. Several studies on animal models of ischemic brain injury have demonstrated the potential of hUCB cells to minimize damage and promote recovery after ischemic brain injury.This review focuses on the treatment of both stroke and perinatal hypoxic-ischemic brain injury using hUCB cells. We discuss the therapeutic effects demonstrated after hUCB cell transplantation and emphasize possible mechanisms counteracting pathophysiological events of ischemia, thus leading to the generation of a regenerative environment that allows neural plasticity and functional recovery. The therapeutic functional effects of hUCB cells observed in animal models make the transplantation of hUCB cells a promising experimental approach in the treatment of ischemic brain injury. Together with its availability, low risk of transplantation, immaturity of cells, and simple route of application, hUCB transplantation may stand a good chance of being translated into a clinical setting for the therapy of ischemic brain injury.

Internal Ribosomal Entry Site (IRES) Activity Generates Endogenous Carboxyl-terminal Domains of Cx43 and Is Responsive to Hypoxic Conditions

Background: Protein fragments of the gap junction Cx43 regulate cellular functions, including resistance to hypoxic stress. Results: Hypoxia-sensitive IRES activity within the coding region of Cx43 is responsible for generating carboxyl-terminal domains. Conclusion: Endogenous fragments of Cx43 seem to convey important non-junctional functions. Significance: Learning how fragments of gap junction proteins are generated is crucial for understanding their functions. Connexin43 (Cx43) is the most abundant gap junction protein in higher vertebrate organisms and has been shown to be involved in junctional and non-junctional functions. In addition to the expression of full-length Cx43, endogenously produced carboxyl-terminal segments of Cx43 have been described and have been suggested to be involved in manifold biological functions, such as hypoxic preconditioning and neuronal migration. Molecular aspects, however, behind the separate generation of carboxyl-terminal segments of Cx43 have remained elusive. Here we report on a mechanism that may play a key role in the separate production of these domains. First, stringent evidence derived from siRNA treatment and specific knockouts revealed significant loss of the low molecular weight fragments of Cx43. By applying a dicistronic vector strategy on transfected cell lines, we were able to identify putative IRES activity (nucleotides 442–637) in the coding region of Cx43, which resides upstream from the nucleotide sequence encoding the carboxyl terminus (nucleotides 637–1149). Functional responsiveness of the endogenous expression of Cx43 fragments to hypoxic/ischemic treatment was evaluated in in vitro and in vivo models, which led to a significant increase of the fastest migrating form (20 kDa) under conditions of metabolic deprivation. By nano-MS spectrometry, we achieved stringent evidence of the identity of the 20-kDa segment as part of the carboxyl-terminal domain of full-length Cx43. Our data prove the existence of endogenously expressed carboxyl-terminal domains, which may serve as valuable tools for further translational application in ischemic disorders.

Changes in Interleukin-1 alpha serum levels after transplantation of umbilical cord blood cells in a model of perinatal hypoxic-ischemic brain damage.

Transplantation of human umbilical cord blood (hUCB) cells is a potential approach for the treatment of perinatal hypoxic-ischemic brain injury. Neurological and motor deficits resulting from the brain lesion are ameliorated upon transplantation. The molecular mechanisms underlying these improvements are currently being unravelled. One parameter identified as part of the beneficial effects of hUCB cells is the reduction of brain inflammation. It is, however, unclear whether the modulation of brain inflammation is due to local or systemic effects of hUCB cells. In this study, the effects of hUCB cell transplantation in a model of perinatal hypoxic-ischemic brain injury were investigated at the systemic level by measurement of serum levels of pro-inflammatory cytokines by multiplex bead arrays. Two days after induction of the brain damage, levels of the pro-inflammatory cytokines Interleukin-1α (IL-1α), Interleukin-1β (IL-1β), and Tumor necrosis factor α (TNFα) were increased in the serum of rats. Application of hUCB cells, in turn, correlated with a reduced elevation of serum levels of these pro-inflammatory cytokines. This decrease was accompanied by a reduced expression of CD68, a marker protein of activated microglia/macrophages in the brain. Therefore, systemic modulation of the immune response by hUCB cells could represent one possible mechanism of how these cells might mediate their beneficial effects. Creation of a regenerative environment with reduced inflammation might account for the functional regeneration observed upon hUCB cell treatment in lesioned animals.

Bronchopulmonary dysplasia in a double-hit mouse model induced by intrauterine hypoxia and postnatal hyperoxia: Closer to clinical features?

Despite increased survival of very preterm newborns, bronchopulmonary dysplasia (BPD) remains a major threat, as it affects long-term pulmonary function and neurodevelopmental outcome. Recent research focused on mechanisms of lung repair. Animal models of BPD in term rodents use postnatal hyperoxia in order to mimic features observed in very preterm human neonates: reduced alveolarization and impaired septal architecture without profound inflammatory changes. In contrast, BPD in very preterm human neonates involves prenatal hits e.g. infections and growth restriction plus postnatal ventilation. BPD induced in rodents by postnatal hyperoxia also exhibits reduced alveolarization however without septal pathology but with marked inflammation. We therefore aimed to establish an animal model combining prenatal growth restriction (FiO₂ 0.1 for 4 days) with postnatal hyperoxia (FiO₂ 0.7 for 2 weeks). In double-hit mice the development was retarded: body weight and length, lung and brain weight were significantly reduced by day P14 compared with normoxic controls. Histomorphometric analysis revealed reduced alveolarization and increased septal thickness without pronounced inflammatory lesions. A down-regulation of SftpB and SftpC genes was observed in double-hit animals compared with controls. Thus, we established a new model of BPD using pre- and postnatal hits.

Probenecid reduces infection and inflammation in acute Pseudomonas aeruginosa pneumonia

The activation of inflammasome signaling mediates pathology of acute Pseudomonas aeruginosa pneumonia. This suggests that the inflammasome might represent a target to limit the pathological consequences of acute P. aeruginosa lung infection. Pannexin-1 (Px1) channels mediate the activation of caspase-1 and release of IL-1β induced by P2X7 receptor activation. The approved drug probenecid is an inhibitor of Px1 and ATP release. In this study, we demonstrate that probenecid reduces infection and inflammation in acute P. aeruginosa pneumonia. Treatment of mice prior to infection with P. aeruginosa resulted in an enhanced clearance of P. aeruginosa and reduced levels of inflammatory mediators, such as IL-1β. In addition, probenecid inhibited the release of inflammatory mediators in murine alveolar macrophages and human U937 cell-derived macrophages upon bacterial infection but not in human bronchial epithelial cells. Thus, Px1 blockade via probenecid treatment may be a therapeutic option in P. aeruginosa pneumonia by improving bacterial clearance and reducing negative consequences of inflammation.

17β-Estradiol Protects 7-Day Old Rats From Acute Brain Injury and Reduces the Number of Apoptotic Cells

Objective: To test a possible neuroprotective activity of 17β-estradiol in the neonatal rat brain exposed to hypoxic-ischemia (controlled hypoxia after unilateral carotid artery ligation). Methods: Seven-day-old Wistar rats underwent ligation of the left common carotid artery followed by 80 minutes hypoxia in 8% oxygen inducing an ipsilateral brain damage. Seven days later (d14), brains were analyzed quantitatively using a macroscopic and microscopic score for structural damage, hemisphere volumes were calculated, and immunohistochemistry for cleaved-caspase-3 (marker for apoptotic cells) was performed. Animals from the study group (n = 19) received 17β-estradiol (0.05 µg/g body weight intraperitoneally) before (−64, −40, and –16 hours) and after (+3 hours) the hypoxia (hour 0: start of the hypoxia) and the control group (n = 21) received mock treatment. Results: Of the 21 pups, 13 in the NaCl group had macroscopically a severe brain damage and 7 of 19 animals in the study group encountered only discrete to mild lesions. Microscopic brain damage in the study group was significantly lower (score 1.5 ± 0.7 vs 2.8 ± 0.8, P < .05). The determined volumes of the affected hemisphere were significantly lower in the NaCl group than in the treatment group. The numbers of apoptotic cells in both hemispheres was equal in the estradiol group, but in the control group, there were significantly more apoptotic cells in the affected hemisphere (control group: ipsilateral: 1435 ± 653 vs contralateral: 143 ± 57 cells, P < .05). Discussion: 17β-Estradiol protects newborn rat brains from hypoxic–ischemic injury, in terms of both microscopic cell injury and apoptosis.

The novel surfactant protein SP-H enhances the phagocytosis efficiency of macrophage-like cell lines U937 and MH-S

BackgroundSurfactant proteins (SP) secreted by alveolar type 2 cells, play an essential role in maintaining the air-liquid barrier of the lung and are also involved in the opsonisation and clearance of bacteria by phagocytes. We have recently described a novel surfactant protein, SP-H (SFTA3). Expression of SP-H was earlier demonstrated to be upregulated by LPS and negatively regulated by IL-1β and IL-23 in vitro. The influence of SP-H on phagocytosis was measured using a murine and a human phagocytic cell line and fluorescent latex beads.FindingsSP-H markedly increases phagocytosis in vitro in the murine-derived alveolar macrophage cell lines MH-S and in human-derived differentiated U937 cells.ConclusionIt can be assumed that SP-H is involved in regulating phagocytic activity of macrophages. SP-H is a new player in pulmonary host defence.

Pannexin-1 channels show distinct morphology and no gap junction characteristics in mammalian cells

Pannexins (Panx) are proteins with a similar membrane topology to connexins, the integral membrane protein of gap junctions. Panx1 channels are generally of major importance in a large number of system and cellular processes and their function has been thoroughly characterized. In contrast, little is known about channel structure and subcellular distribution. We therefore determine the subcellular localization of Panx1 channels in cultured cells and aim at the identification of channel morphology in vitro. Using freeze-fracture replica immunolabeling on EYFP-Panx1-overexpressing HEK 293 cells, large particles were identified in plasma membranes, which were immunogold-labeled using either GFP or Panx1 antibodies. There was no labeling or particles in the nuclear membranes of these cells, pointing to plasma membrane localization of Panx1-EYFP channels. The assembly of particles was irregular, this being in contrast to the regular pattern of gap junctions. The fact that no counterparts were identified on apposing cells, which would have been indicative of intercellular signaling, supported the idea of Panx1 channels within one membrane. Control cells (transfected with EYFP only, non-transfected) were devoid of both particles and immunogold labeling. Altogether, this study provides the first demonstration of Panx1 channel morphology and assembly in intact cells. The identification of Panx1 channels as large particles within the plasma membrane provides the knowledge required to enable recognition of Panx1 channels in tissues in future studies. Thus, these results open up new avenues for the detailed analysis of the subcellular localization of Panx1 and of its nearest neighbors such as purinergic receptors in vivo.