Balance of growth factors in the human perinatal lung : Implications for physiological lung development and link to bronchopulmonary dysplasia

The aims of this Thesis was to evaluate the role of proangiogenic placental growth factor (PlGF), antiangiogenic endostatin and lymphangiogenic vascular endothelial growth factor (VEGF) -C as well as the receptors vascular endothelial growth factor receptor (VEGFR) -2 and VEGFR-3 during lung development and in development of lung injury in preterm infants. The studied growth factors were selected due to a close relationship with VEGF-A; a proangiogenic growth factor important in normal lung angiogenesis and lung injury in preterm infants. The thesis study consists of three analyses. I: Lung samples from fetuses, preterm and term infants without lung injury, as well as preterm infants with acute and chronic lung injury were stained by immunohistochemistry for PlGF, endostatin, VEGF-C, VEGFR-2 and VEGFR-3. II: Tracheal aspirate fluid (TAF) was collected in the early postnatal period from a patient population consisting of 59 preterm infants, half developing bronchopulmonary dysplasia (BPD) and half without BPD. PlGF, endostatin and VEGF-C concentrations were measured by commercial enzyme-linked immunosorbent assay (ELISA). III: Cord plasma was collected from very low birth weight (VLBW) (n=92) and term (n=48) infants in conjuncture with birth and endostatin concentrations were measured by ELISA. I: All growth factors and receptors studied were consistently stained in immunohistochemistry throughout development. For endostatin in early respiratory distress syndrome (RDS), no alveolar epithelial or macrophage staining was seen, whereas in late RDS and BPD groups, both alveolar epithelium and macrophages stained positively in approximately half of the samples. VEGFR-2 staining was fairly consistent, except for the fact that capillary endothelial staining in the BPD group was significantly decreased. II: During the first postnatal week in TAF mean PlGF concentrations were stable whereas mean endostatin and VEGF-C concentrations decreased. Higher concentrations of endostatin and VEGF-C correlated with lower birth weight (BW) and associated with administration of antenatal betamethasone. Parameters reflecting prenatal lung inflammation associated with lower PlGF, endostatin and VEGF-C concentrations. A higher mean supplemental fraction of inspired oxygen during the first 2 postnatal weeks (FiO2) correlated with higher endostatin concentrations. III: Endostatin concentrations in term infants were significantly higher than in VLBW infants. In VLBW infants higher endostatin concentrations associated with the development of BPD, this association remained significant after logistic regression analysis. We conclude that PlGF, endostatin and VEGF-C all have a physiological role in the developing lung. Also, the VEGFR-2 expression profile seems to reflect the ongoing differentiation of endothelia during development. Both endostatin and VEGFR-2 seem to be important in the development of BPD. During the latter part of the first postnatal week, preterm infants developing BPD have lower concentrations of VEGF-A in TAF. Our findings of disrupted VEGFR-2 staining in capillary and septal endothelium seen in the BPD group, as well as the increase in endostatin concentrations both in TAF and cord plasma associated with BPD, seem to strengthen the notion that there is a shift in the angiogenic balance towards a more antiangiogenic environment in BPD. These findings support the vascular hypothesis of BPD.

Role of GDNF and its Cross-Talk with Other Growth Factors in the Dopaminergic System

Parkinson´s Disease (PD) is a neurodegenerative movement disorder resulting from loss of dopaminergic (DA) neurons in substantia nigra (SN). Possible causative treatment strategies for PD include neurotrophic factors, which protect and in some cases restore the function of dopaminergic neurons. Glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors have been to date the most promising candidates for treatment of PD, demonstrating both neuroprotective and neurorestorative properties. We have investigated the role of GDNF in the rodent dopaminergic system and its possible crosstalk with other growth factors. We characterized the GDNF-induced gene expression changes by DNA microarray analysis in different neuronal systems, including in vitro cultured Neuro2A cells treated with GDNF, as well as midbrains from GDNF heterozygous (Hz) knockout mice. These microarray experiments, resulted in the identification of GDNF-induced genes, which were also confirmed by other methods. Further analysis of the dopaminergic system of GDNF Hz mice demonstrated about 40% reduction in GDNF levels, revealed increased intracellular dopamine concentrations and FosB/DeltaFosB expression in striatal areas. These animals did not show any significant changes in behavioural analysis of acute and repeated cocaine administration on locomotor activity, nor did they exhibit any changes in dopamine output following treatment with acute cocaine. We further analysed the significance of GDNF receptor RET signalling in dopaminergic system of MEN2B knock-in animals with constitutively active Ret. The MEN2B animals showed a robust increase in extracellular dopamine and its metabolite levels in striatum, increased tyrosine hydroxylase (TH) and dopamine transporter (DAT) protein levels by immunohistochemical staining and Western blotting, as well as increased Th mRNA levels in SN. MEN2B mice had increased number of DA neurons in SN by about 25% and they also exhibited increased sensitivity to the stimulatory effects of cocaine. We also developed a semi-throughput in vitro micro-island assay for the quantification of neuronal survival and TH levels by computer-assisted methodology from limited amounts of tissue. This assay can be applied for the initial screening for dopaminotrophic molecules, as well as chemical drug library screening. It is applicable to any neuronal system for the screening of neurotrophic molecules. Since our microarray experiments revealed possible GDNF-VEGF-C crosstalk we further concentrated on studying the neurotrophic effects of VEGF-C. We showed that VEGF-C acts as a neurotrophic molecule for the DA neurons both in vitro and in vivo, however without additive effect when used together with GDNF. The neuroprotective effect for VEGF-C in vivo in rat 6-OHDA model of PD was demonstrated. The possible signalling mechanisms of VEGF-C in the nervous system were investigated - infusion of VEGF-C to rat brain induced ERK activation, however no direct activation of RET signalling in vitro was found. VEGF-C treatment of rat striatum lead to up-regulation of VEGFR-1-3, indicating that VEGF-C can regulate the expression level of its own receptor. VEGF-C dopaminotrophic activity in vivo was further supported by increased vascular tissue in the neuroprotection experiments.

Intranasal Delivery of Plasma and Platelet Growth Factors Using PRGF-Endoret System Enhances Neurogenesis in a Mouse Model of Alzheimer’s Disease

[EN] Neurodegeneration together with a reduction in neurogenesis are cardinal features of Alzheimer’s disease (AD) induced by a combination of toxic amyloid-β peptide (Aβ) and a loss of trophic factor support. Amelioration of these was assessed with diverse neurotrophins in experimental therapeutic approaches. The aim of this study was to investigate whether intranasal delivery of plasma rich in growth factors (PRGF-Endoret), an autologous pool of morphogens and proteins, could enhance hippocampal neurogenesis and reduce neurodegeneration in an amyloid precursor protein/presenilin-1 (APP/PS1) mouse model. Neurotrophic and neuroprotective actions were firstly evident in primary neuronal cultures, where cell proliferation and survival were augmented by Endoret treatment. Translation of these effects in vivo was assessed in wild type and APP/PS1 mice, where neurogenesis was evaluated using 5-bromodeoxyuridine (BdrU), doublecortin (DCX), and NeuN immunostaining 5 weeks after Endoret administration. The number of BrdU, DCX, and NeuN positive cell was increased after chronic treatment. The number of degenerating neurons, detected with fluoro Jade-B staining was reduced in Endoret-treated APP/PS1 mice at 5 week after intranasal administration. In conclusion, Endoret was able to activate neuronal progenitor cells, enhancing hippocampal neurogenesis, and to reduce Aβ-induced neurodegeneration in a mouse model of AD.

The effect of vitamins C and E on some of growth factors of cultured Acipenser ruthenus

The effect of vitamins C and E on some of growth factors of cultured Acipenser ruthenus was studied in this thesis. For this purpose diets supplemented with a combination of 100 and 400 mg/kg vitamin C, L-ascorbyl-2-polyphosphate and 100 and 400 mg/kg vitamin E, D-alpha-tocopherol,were each fed to sterlet in 2 replications for 15 weeks. Fifteen fish with average weight of 350.92±14.28 gr were distributed to each of 18 tanks after adaptation with experimental diet. After 5 weeks, there were significant differences in RBC, ESR, HCT and differential counting of white blood cells among the treatments (P<0.05), but there was no significant difference in the amount of WBC among the treatments (P>0.05). After 10 weeks, there were significant differences in the amounts of Monocytes, Lymphocytes and Eosinophils (P<0.05), but there was no significant differences in the amount of other hematologic factors (P>0.05). At the end of the experiment (15th week) only WBC, RBC, Monocyte, lymphocyte and neutrophil showed significant differences between the treatments (P<0.05) and other hematologic factors did not show any significant differences between the treatments (P>0.05). The results of biochemical indices analysis showed significant differences (P<0.05) among treatments for all of the parameters for 5th weeks, total protein and glucose for 10th week and only cholesterol for 15th week. The carcass analysis at the end of experiment showed that only the amount of carbohydrate, protein and ash were significantly difference between the treatments (P<0.05). The results of growth parameters at the end of 3th, 9th, 12th and 15th week showed significant differences between the treatments (P<0.05), but at the end of 6th week only GR was significantly different between the treatments (P<0.05). After concerning acute stress test including reduce water volume, cutting the aeration for 30 minutes at the end of experiment, cortisol and glucose significantly increased (P<0.05) compared with prestress period, but the lowest response to the stressor was observed in fish fed by E400 C 400 mg/kg. On the other hand the survival was 100% during the experiment and no mortality was occurred during this period. Results of this study indicate that Vitamins C and E can have remarkable effects on hematological, biochemical and growth indices in different growth periods. So regarding the effects of vitamin C and E on some growth indices, it seems that the diet containing E400 C 100 mg/kg can be considered as the optimum diet in the rearing condition for this weigth range of fish.

The study of Indian shrimp larvae feeding by enriched rotifer with vitamin C and unsaturated fatty acid (epa, dha) and its effects on growth factors, survival rate and environmental stresses

In this experiment, the feeding of Indian white shrimp larvae by unenriched rotifers (treatment 1) and enriched with highly unsaturated fatty acid (treatment 2) and highly unsaturated fatty acid along with vitamin C (treatment 3) on the growth factors, survival and resistance against salinity and formalin stress tests were studied and their differences with control treatment including newly hatched Artemia nauplii is compared. In this the study four treatments in a completely randomized design with 3 replicates per treatment were used. Farming of shrimp larvae of Zoea II to postlarvae 5 was done in 20 liter plastic bucket. Present results indicated that growth factors and survival rate of stage Zoea II to postlarvae 1 in treatments 1, 2 and 3 improve rather than control in which this case was due to optimal size rotifer rather than Artemia nauplii. Also, treatments 2 and 3 feeding with oil liver cod emulsion enriched rotifer have the highest concentration of DHA (mg/g DW) and the ratio DHA/EPA in which due to have shown the highest growth factors and a significant difference (P<0.05) with treatments 1 and control. The highest survival at stage PL1 were observed in treatment 3 that was enriched with ascorbyl palmitate in which have to the synergistic properties of vitamin C rather than treatments 2, 1 and control and showed a significant difference (P<0.05). But in stage PL5 the highest amount of growth and survival rates were related to control treatment which showed a significant difference (P<0.05) with other treatments that control has higher size rather than treatments 1, 2 and 3. Also, among experiment treatments that the two treatments 2 and 3 due to enrichment had higher growth and survival rates compared with treatment 1 in which their differences have also been significant (P<0.05). In the case of stress tests, results indicated that the highest survival rate has been reported when specimens were offered a diet containing high levels of highly unsaturated fatty acids with vitamin C. So that in stage PL1 in the salinity stress tests 10 and 20 ppt the highest survival rate was observed in treatment 3. As for the second, treatment 2 showed a significant difference (P<0.05) with treatment 3. It is worth mentioning that treatment 3 showed a higher survival rate compared to treatment 2 due to the synergistic properties of vitamin C. The difference between these two treatments with treatment 1 and control was also significant. No significant difference was observed in formalin stress test 100 ppm in this stage between treatments 3 and 2 which shows the highest survival rate. But their difference with treatments 1 and control was significant (P<0.05). Also, in stage PL5 in the salinity stress tests 10 and 20 ppt the highest survival rate was observed in treatment 3 which showed no significant difference (P<0.05) with control treatment. While their difference in the amount of survival rate with treatment 1 and 2 was significant (P<0.05). In this stage, the highest observed survival rate in formalin stress test 100 ppm included treatments control, 3 and 2 among which there were no significant differences (P<0.05). While the difference between these three treatments with treatment 1 was significant.

Destabilization of basic fibroblast growth factor by oligodeoxynucleotides

The effects of oligodeoxynucleotide (ODN) on the conformation of basic fibroblast growth factor (bFGF) were studied by spectral method. The results showed that ODN destabilized the protein.

Study on growth factors of intermetallic layer within hot-dipped 25% Al-Zn alloy coating on steel

25%Al-Zn alloy coating performs better than hot dip galvanized coating and 55%Al-Zn-Si coating with regard to general seawater corrosion protection. This study deals with the interfacial intermetallic layer's growth, which affects considerably the corrosion resistance and mechanical properties of 25%Al-Zn alloy coatings, by means of three-factor quadratic regressive orthogonal experiments, The regression equation shows that the intermetallic layer thickness decreases rapidly with increasing content of Si added to the Zn-Al alloy bath, increases with rise in bath temperature and prolonging dip time. The most effective factor that determined the thickness of intermetallic layer was the amount of Si added to Zn-Al alloy bath, while the effect of bath temperature and dip time on the thickness of intermetallic layer were not very obvious.

Chondrogenesis of adult stem cells from adipose tissue and bone marrow: induction by growth factors and cartilage-derived matrix.

OBJECTIVES: Adipose-derived stem cells (ASCs) and bone marrow-derived mesenchymal stem cells (MSCs) are multipotent adult stem cells with potential for use in cartilage tissue engineering. We hypothesized that these cells show distinct responses to different chondrogenic culture conditions and extracellular matrices, illustrating important differences between cell types. METHODS: Human ASCs and MSCs were chondrogenically differentiated in alginate beads or a novel scaffold of reconstituted native cartilage-derived matrix with a range of growth factors, including dexamethasone, transforming growth factor beta3, and bone morphogenetic protein 6. Constructs were analyzed for gene expression and matrix synthesis. RESULTS: Chondrogenic growth factors induced a chondrocytic phenotype in both ASCs and MSCs in alginate beads or cartilage-derived matrix. MSCs demonstrated enhanced type II collagen gene expression and matrix synthesis as well as a greater propensity for the hypertrophic chondrocyte phenotype. ASCs had higher upregulation of aggrecan gene expression in response to bone morphogenetic protein 6 (857-fold), while MSCs responded more favorably to transforming growth factor beta3 (573-fold increase). CONCLUSIONS: ASCs and MSCs are distinct cell types as illustrated by their unique responses to growth factor-based chondrogenic induction. This chondrogenic induction is affected by the composition of the scaffold and the presence of serum.