Patrycja Kłos

Perinatal exposure to lead induces morphological, ultrastructural and molecular alterations in the hippocampus.

The aim of this paper is to examine if pre- and neonatal exposure to lead (Pb) may intensify or inhibit apoptosis or necroptosis in the developing rat brain. Pregnant experimental females received 0.1% lead acetate (PbAc) in drinking water from the first day of gestation until weaning of the offspring; the control group received distilled water. During the feeding of pups, mothers from the experimental group were still receiving PbAc. Pups were weaned at postnatal day 21 and the young rats of both groups then received only distilled water until postnatal day 28. This treatment protocol resulted in a concentration of Pb in rat offspring whole blood (Pb-B) below the threshold of 10 μg/dL, considered safe for humans.We studied Casp-3 activity and expression, AIF nuclear translocation, DNA fragmentation, as well as Bax, Bcl-2 mRNA and protein expression as well as BDNF concentration in selected structures of the rat brain: forebrain cortex (FC), cerebellum (C) and hippocampus (H). The microscopic examinations showed alterations in hippocampal neurons.Our data shows that pre- and neonatal exposure of rats to Pb, leading to Pb-B below 10 μg/dL, can decrease the number of hippocampus neurons, occurring concomitantly with ultrastructural alterations in this region. We observed no morphological or molecular features of severe apoptosis or necrosis (no active Casp-3 and AIF translocation to nucleus) in young brains, despite the reduced levels of BDNF. The potential protective factor against apoptosis was probably the decreased Bax/Bcl-2 ratio, which requires further investigation. Our findings contribute to further understanding of the mechanisms underlying Pb neurotoxicity and cognition impairment in a Pb-exposed developing brain.

Increased circulating endothelial progenitor cells in patients with haemorrhagic and ischaemic stroke: The role of Endothelin-1

Ischaemic stroke induces endothelial progenitor cell (EPC) mobilisation from bone marrow into peripheral blood. Circulating EPCs play an important role in post-injury regeneration of vasculature, whereas endothelial cells (ECs) have been shown to reflect endothelial damage and may be responsible for increased Endothelin-1 (ET-1) expression. We investigated herein the association between numbers of circulating ECs and EPCs, the levels of soluble factors regulating their migration and function, and the clinical outcome in patients with haemorrhagic (HS) or ischaemic stroke (IS). Sixteen patients with HS and eighteen with IS were assessed during the first 24h, day 3, and day 7 after stroke and compared them with twenty-three control subjects. We found elevated EPC and EC concentrations using flow cytometry and increase in VEGF, SDF-1, HGF, and ET-1 plasma levels by ELISA in the HS patients, while ET-1 mRNA expression in peripheral blood cells was elevated in the IS patients. Significant correlations were observed between EPCs or ECs and Big ET-1 protein or mRNA levels in HS but not in the IS patients. We suggest that ET-1 may play a role in pathophysiology of stroke and subsequent EPC mobilisation; however, further studies aimed at the precise elucidation of this issue are required.

Different populations of circulating endothelial cells in patients with age-related macular degeneration: a novel insight into pathogenesis.

PURPOSE Circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) may serve as novel markers of endothelial dysfunction. The presence and clinical implications of CECs and the expression of endothelin (ET)-1, one of the most potent vasoconstrictors, have not been evaluated in patients with the neovascular form of age-related macular degeneration (AMD). This study was conducted to determine the different populations of endothelial cells (ECs) in the peripheral blood of AMD patients and to correlate these findings with the expression of ET-1 and the cytokines and growth factors responsible for EC migration and function. METHODS Peripheral blood samples were collected from 29 patients with diagnosed neovascular AMD and from 38 healthy control subjects. CD133(-)CD144(+) CECs and CD34(+)CD133(+)CD144(+) EPCs were counted and analyzed by flow cytometry. The intracellular expression of ET-1 in peripheral blood nuclear cells (PBNCs) was studied by using qRT-PCR, Western blot, and immunocytofluorescence assays, and ET-1, IGF-1, VEGF, SDF-1, and HGF plasma concentrations were measured in enzyme-linked immunosorbent assays. RESULTS Increased CECs and EPCs were found in the AMD patients compared with the counts in healthy individuals. The expression of intracellular ET-1 was significantly elevated in PBNCs from the AMD patients compared with the control subjects. In addition a significantly higher plasma concentration of IGF-1 was observed, but a lower SDF-1 level in the group of AMD patients. CONCLUSIONS These findings suggest that circulating endothelial cells, together with high ET-1 content, may contribute to the development of AMD. Further prospective investigations on the mechanism involved may be relevant to the potential treatment of this disease.

Stem Cells are mobilized from the bone marrow into the peripheral circulation in response to retinal pigment epithelium damage--a pathophysiological attempt to induce endogenous regeneration.

Purpose: Stem cell regeneration of damaged tissue has recently been reported in many different organs. Here, we investigated the mobilization of different stem/progenitor cell (SPC) populations into the peripheral blood (PB), their subsequent homing to the injured retina (IR) and contribution to its regeneration in a retinal pigment epithelium (RPE) damage model induced by sodium iodate (NaIO3). Methods: Mobilization of SPCs was evaluated by flow cytometry. SPCs distribution in IR was assessed using bone marrow (BM)-derived GFP+Lin− cells transplanted intravenously into NaIO3-treated C57Bl/6 mice. The quantity of the chemokine SDF-1 in PB and IR was measured by ELISA and qRT-PCR, respectively. Apoptosis (TUNEL assay), cell proliferation (PCNA analysis) as well as functional retinal activity (electroretinogram) were examined at several time points after NaIO3 administration. Results: Mobilization of SPCs along with the highest cell proliferation and massive apoptosis within IR were observed on the third day after NaIO3 administration. Similarly, donor GFP+Lin− cells were detected in the retina as soon as day 4 after NaIO3 injection. Plasma levels of SDF-1 did not differ significantly in mice exposed to NaIO3 compared to healthy controls, however mRNA for SDF-1 was overexpressed locally in IR. Functional retinal recovery was not achieved. Conclusion: Our study provides evidence that BM SPCs egress into PB and home to the injured retina, but are not capable of restoring its function. These results indicate that if the range of retinal destruction is profound, endogenous regeneration is ineffective and may ultimately require adjuvant therapeutic transplantation of specific SPCs subpopulations.

Growth factors and their receptors derived from human amniotic cells in vitro.

In vitro studies have shown that amnion-produced growth factors participated in angiogenesis, re-epithelialization, and immunomodulation. The aim of our study was to investigate the growth factors and receptors produced by human amnion tissue and amniotic cells. Human amnions (hAM) were isolated, and amnion circles were dissected for in vitro analysis. Some amnion fragments were digested by the use of different methods to obtain two cell fractions, which were analysed for mesenchymal and epithelial cell markers. Amniotic circles and human amniotic cell fractions were cultured in a protein-free medium. Proteins secreted into the culture medium were analysed with a human growth factor antibody array. Conditioned culture media were added to human umbilical vein epithelial cells (HUVECs) to test for stimulation of migration (scratch test) and proliferation (Ki67 expression). Fraction 1 cells expressed both cytokeratin and mesenchymal cell markers which indicated that it was composed of a mixture of human amnion epithelial cells (hAECs) and mesenchymal stromal cells (hAMSCs). Fraction 2 cells mainly expressed cytokeratin and, therefore, were designed as hAECs. Secretion of proteins by the cultured cells increased with time. The hAM cultures secreted EGF-R, IGF, and IGFBP-2,-3 and -6; Cell Fraction 1 secreted NT-4, whereas Cell Fraction 2 secreted G-CSF, M-CSF, and PDGF. Conditioned media of hAM cultures stimulated HUVECs migration. We have showed for the first time that human amnions and amniotic cells secreted IGFBP-6, MCSF-R, PDGF-AB, FGF-6, IGFBP-4, NT-4, and VEGF-R3. We found that Cell Fraction 1, Cell Fraction 2, and the whole amnion secreted different proteins, possibly due to different proportions of amnion-derived cells and different cell-cell interactions. The hAM cell factors remained functional in vitro and induced intensified migration of HUVECs. The growth factors and receptors found in amnion or amniotic cell media might be used for regenerative medicine.

Neural stem/progenitor cells circulating in peripheral blood of patients with neovascular form of AMD: a novel view on pathophysiology

BackgroundThe neovascular form of age-related macular degeneration (AMD) manifested with choroidal neovascularization (CNV) is one of the leading causes of rapid and irreversible visual loss. Recent reports suggest that bone marrow-derived stem/progenitor cells (SPCs) play a crucial role in the development and progression of the disease. The purpose of this study was to investigate whether or not undifferentiated non-haematopoietic stem cells, including those capable of differentiating into neural phenotypes, play a role in the pathological state of CNV formation.MethodsPeripheral blood samples were collected from 46 patients diagnosed with CNV and from 46 controls. The CXCR4+Lin-CD45- stem cells were counted and analysed by flow cytometry. Using qRT-PCR and immunocytofluorescence, the expression of early neural and glial cell markers (β-III-tubulin, nestin, and glial fibrillary acidic protein) in the sorted cells was analysed, and correlated with plasma concentrations of stromal cell-derived factor 1 (SDF-1) (enzyme-linked immunosorbent assay), which is a pivotal chemokine that regulates the trafficking of SPCs.ResultsWe found that the number of circulating CXCR4+Lin-CD45- cells did not differ in patients with active CNV as compared to the controls. However, we noticed significant intracellular overexpression of β-III-tubulin in the cells derived from AMD patients. Moreover, we observed significantly lower SDF-1 plasma levels in neovascular AMD patients compared to healthy individuals.ConclusionsOur findings suggest that neural progenitor cells, together with low SDF-1 concentrations, may play a considerable role in the process of AMD progression. Further investigations aimed at the precise elucidation of these issues may help with the future development of effective prevention against, and the treatment of, this disease.

Circulating hematopoietic stem cell count is a valuable predictor of prematurity complications in preterm newborns

BackgroundThe frequency of preterm labour has risen over the last few years. Hence, there is growing interest in the identification of markers that may facilitate prediction and prevention of premature birth complications. Here, we studied the association of the number of circulating stem cell populations with the incidence of complications typical of prematurity.MethodsThe study groups consisted of 90 preterm (23–36 weeks of gestational age) and 52 full-term (37–41 weeks) infants. Non-hematopoietic stem cells (non-HSCs; CD45-lin-CD184+), enriched in very small embryonic-like stem cells (VSELs), expressing pluripotent (Oct-4, Nanog), early neural (β-III-tubulin), and oligodendrocyte lineage (Olig-1) genes as well as hematopoietic stem cells (HSCs; CD45+lin-CD184+), and circulating stem/progenitor cells (CSPCs; CD133+CD34+; CD133-CD34+) in association with characteristics of prematurity and preterm morbidity were analyzed in cord blood (CB) and peripheral blood (PB) until the sixth week after delivery. Phenotype analysis was performed using flow cytometry methods. Clonogenic assays suitable for detection of human hematopoietic progenitor cells were also applied. The quantitative parameters were compared between groups by the Mann–Whitney test and between time points by the Friedman test. Fisher’s exact test was used for qualitative variables.ResultsWe found that the number of CB non-HSCs/VSELs is inversely associated with the birth weight of preterm infants. More notably, a high number of CB HSCs is strongly associated with a lower risk of prematurity complications including intraventricular hemorrhage, respiratory distress syndrome, infections, and anemia. The number of HSCs remains stable for the first six weeks of postnatal life. Besides, the number of CSPCs in CB is significantly higher in preterm infants than in full-term neonates (p < 0.0001) and extensively decreases in preterm babies during next six weeks after birth. Finally, the growth of burst-forming unit of erythrocytes (BFU-E) and colony-forming units of granulocyte-macrophage (CFU-GM) obtained from CB of premature neonates is higher than those obtained from CB of full-term infants and strongly correlates with the number of CB-derived CSPCs.ConclusionWe conclude that CB HSCs are markedly associated with the development of premature birth complications. Thus, HSCs ought to be considered as the potential target for further research as they may be relevant for predicting and controlling the morbidity of premature infants. Moreover, the observed levels of non-HSCs/VSELs circulating in CB are inversely associated with the birth weight of preterm infants, suggesting non-HSCs/VSELs might be involved in the maturation of fetal organism.

Alteration of Selected Neurotrophic Factors and their Receptor Expression in Mouse Brain Response to Whole-Brain Irradiation

Ionizing radiation can significantly affect brain function in children and young adults, particularly in the hippocampus where neurogenic niches are located. Injury to normal tissue is a major concern when whole-brain irradiation (WBI) is used to treat central nervous system (CNS) tumors, and the pathogenesis of this injury remains poorly understood. We assessed the expression of selected neurotrophins (NTs) and NT receptors (NTRs) in brains of young mice after a single 10 Gy gamma-ray exposure using morphological and molecular analyses [qRT-PCR, Western blot, immunohistochemistry (IHC)] to evaluate WBI-induced injury in its acute phase. Activity of the NT–NTR axes was examined by analysis of ERK and Akt phosphorylation. Using Nissl staining of hippocampus slices to visualize morphological changes, and TUNEL assay and active caspase-3 detection to assess apoptotic cell death, we found evidence of apoptosis and degenerative changes in hippocampal tissue after WBI. Shortly after WBI, we also observed significant overexpression of several NTs (BDNF, NT-3, NGF and GDNF) and NTRs (TrkA, TrkB, TrkC, GFRα-1, and p75NTR) compared to control animals. The upregulated NT and NTR proteins, in part, originated from two analyzed neurogenic areas: the subgranular zone of the hippocampal dentate gyrus and the subventricular zone, as confirmed by IHC. Finally, components of intracellular signaling pathways, including Akt and MAPK, were activated in acute phase after WBI. Given the role of NTs in diverse biological mechanisms, including maintenance and growth of neurons in the adult brain, our findings of altered expression of neurotrophins and their receptors in brain tissue shortly after irradiation suggest that these molecules play a vital role in the pathophysiology of the acute phase of WBI-induced injury.