Oxygen-mediated basic fibroblast growth factor (FGF2) effects on adult human dermal fibroblasts

This thesis investigates the effects of low oxygen culture conditions and fibroblast growth factor-2 (FGF2) on adult human dermal fibroblasts. It was previously shown that low oxygen and FGF2 culture conditions lead to an extension of proliferative lifespan, low-level activation of stem cell genes, and global transcriptional changes in adult human dermal fibroblasts. Additionally, an increased in vivo tissue regenerative response can be observed when human muscle-derived fibroblasts grown with FGF2 and low oxygen are implanted into mouse muscle injury, leading to a decrease in collagen deposition and scar formation and increase of functional skeletal muscle regeneration, including formation of Pax7+ muscle stem cells. These findings led to an analysis of key cellular oxygen sensors, hypoxia inducible factors (HIFs) and their role in this regenerative response. Directly linking these factors with the regenerative response, I have shown, with knockdown experiments, that HIF-2&alpha; is required for the increased proliferative capability and decreased senescence of human dermal fibroblasts (hDFs) induced by hypoxia. I have also determined that low oxygen causes an early and transient increase of HIF-1&alpha; and late and sustained increase of HIF-2&alpha; protein accompanied by increased nuclear translocation. Using overexpression and knockdown approaches via lent-virus, I determined that HIF-2&alpha; appears to modulate FGF2 signaling through the FGF receptors. First, under low oxygen conditions, exogenous FGF2 led to downregulation of endogenous FGF2, which can be mimicked by overexpression of HIF-2&alpha;. In ambient oxygen we didn't see this effect. Second, HIF-2&alpha; overexpression appears to lead to increases in FGFR1 phosphorylation and consequently increased ERK1/2 phosphorylation, and increases in the expression of heparan sulfate modifying enzymes (NDST1, NDST2, and EXTL2). Lastly, sustained supplementation with FGF2 in low oxygen inhibits receptor-mediated FGF2 signaling. To understand these effects at the transcriptional level, using microarray technology, we identified oxygen-mediated FGF2 effects on genes involved in cell survival and proliferation. Through bioinformatics analyses, I determined that genes involved in wound healing (extracellular matrix genes, adhesion molecules, cytokines) are upregulated in FGF2 treated fibroblasts grown under low oxygen. By utilizing a gain-of-function approach, we were able to assess the effects of altered HIF-2&alpha; activity on the expression of Oct4, Sox2, Nanog, Rex1, and Lin28 in adult hDFs. The results indicate that overexpression of the HIF-2&alpha; transcription factor increases Oct4 mRNA, but not Oct4 protein, levels, and had no effect on Nanog and Lin28 proteins. HIF-2&alpha; overexpression also mediated FGF2 induction of Sox2 and Rex1 proteins of higher molecular weight. This thesis expands our knowledge about effects of low oxygen and FGF2 on adult human dermal fibroblasts and explains in part, how FGF2 under low oxygen conditions may lead to increased proliferation, extended life span, regenerative competency and increased developmental plasticity of adult hDFs.</p

Aberrant Synthesis of Indole-3-Acetic Acid in Saccharomyces cerevisiae Triggers Morphogenic Transition, a Virulence Trait of Pathogenic Fungi

Many plant-associated microbes synthesize the auxin indole-3-acetic acid (IAA), and several IAA biosynthetic pathways have been identified in microbes and plants. Saccharomyces cerevisiae has previously been shown to respond to IAA by inducing pseudohyphal growth. We observed that IAA also induced hyphal growth in the human pathogen Candida albicans and thus may function as a secondary metabolite signal that regulates virulence traits such as hyphal transition in pathogenic fungi. Aldehyde dehydrogenase (Ald) is required for IAA synthesis from a tryptophan (Trp) precursor in Ustilago maydis. Mutant S. cerevisiae with deletions in two ALD genes are unable to convert radiolabeled Trp to IAA, yet produce IAA in the absence of exogenous Trp and at levels higher than wild type. These data suggest that yeast may have multiple pathways for IAA synthesis, one of which is not dependent on Trp

FGF2-induced effects on transcriptome associated with regeneration competence in adult human fibroblasts

BACKGROUND: Adult human fibroblasts grown in low oxygen and with FGF2 supplementation have the capacity to tip the healing outcome of skeletal muscle injury – by favoring regeneration response in vivo over scar formation. Here, we compare the transcriptomes of control adult human dermal fibroblasts and induced regeneration-competent (iRC) fibroblasts to identify transcriptional changes that may be related to their regeneration competence. RESULTS: We identified a unique gene-expression profile that characterizes FGF2-induced iRC fibroblast phenotype. Significantly differentially expressed genes due to FGF2 treatment were identified and analyzed to determine overrepresented Gene Ontology terms. Genes belonging to extracellular matrix components, adhesion molecules, matrix remodelling, cytoskeleton, and cytokines were determined to be affected by FGF2 treatment. CONCLUSIONS: Transcriptome analysis comparing control adult human fibroblasts with FGF2-treated fibroblasts identified functional groups of genes that reflect transcriptional changes potentially contributing to their regeneration competence. This comparative transcriptome analysis should contribute new insights into genes that characterize cells with greater regenerative potential

Phytochemical Profiles and In Vitro Immunomodulatory Activity of Ethanolic Extracts from Galium aparine L.

Galium aparine L., family Rubiaceae, is a widely spread species in the Galium genus. The herb of G. aparine is part of folk remedies and dietary supplements. In this study, we analyzed the chemical composition and immunomodulatory activities of G. aparine herb ethanolic extracts obtained from the plant material by maceration with 20%, 60% or 96% ethanol. The contents of hydroxycinnamic acid derivatives, flavonoids and polyphenols were determined spectrophotometrically, with extractives and polysaccharides quantified gravimetrically. The qualitative composition was studied using UHPLC-DAD-MS/MS analysis; isolation not previously described in G. aparine quercetin rhamnoglucoside was carried out through column chromatography, and the immunomodulatory activity of extracts was determined in the reaction of lymphocyte blast transformation. Major constitutes of extracts were iridoids, i.e., monotropein, 10-desacetylasperulosidic acid and asperulosidic acid; p-hydroxybenzoic acid; hydroxycinnamic acid derivatives, i.e., 3-O-caffeoylquinic, 5-O-caffeoylquinic, 3,4-O-dicaffeoylquinic, 3,5-O-dicaffeoylquinic, 4,5-O-dicaffeoylquinic acids and caffeic acid derivatives; flavonoids, i.e., rutin, quercetin 3-O-rhamnoglucoside-7-O-glucoside, and isorhamnetin 3-O-glucorhamnoside. Significantly, quercetin 3-O-rhamnoglucoside-7-O-glucoside was first isolated and identified in Galium species so far investigated. All G. aparine herb ethanolic extracts stimulate the transformational activity of immunocompetent blood cells, with 96% ethanolic extract being the most active. The data obtained necessitate further research into the mechanisms of immunomodulatory activity of extracts from G. aparine herb

Immunomodulatory Activity and Phytochemical Profile of Infusions from Cleavers Herb

Extracts from aerial parts of G. aparine (cleavers) constitute a herbal remedy with monography in British Herbal Pharmacopeia. On the European market, there are several drugs and food supplements consisting of Galium extracts. In folk medicine, cleavers was used topically in Europe, Asia, and the Americas to treat skin diseases. In several remedies, cleavers is also listed as an immunomodulatory active herb influencing the defense response of the human body. The aim of this study was to investigate the immunostimulatory activity and antioxidant potential in vitro of a raw infusion of cleavers and bioactive fractions. The functional activity of lymphocytes in the reaction of the lymphocyte blast transformation (RLBT) method was used for immunomodulatory activity assays and direct scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO), and hydrogen peroxide (H2O2) was chosen for the examination of antioxidant activity. It was shown that both the raw extract and fractions show significant immunostimulatory and scavenging activities. The obtained data partially justify the traditional use of cleavers as topical remedy for skin infections and for wounds

RT-PCR for expression of embryonic stem cell specific genes.

<p>OCT4, NANOG and CRIPTO1 in human ESCs during culture (A), during 21 days after induction of hESC differentiation (B), and in adult human dermal fibroblasts cultured in 2% oxygen with FGF2 supplementation (C). Restriction digest of 646 bp OCT4 amplicon from human embryonic stem cells (D), control fibroblasts (E), and iPSCs (F). ApaI restriction digest of OCT4 amplicon in fibroblasts grown in 2% oxygen and FGF2 supplementation (G), and various transformed, multipotent and differentiated cells (H). NCCIT – teratocarcinoma, NTERA2– teratocarcinoma, SHSY – neuroblastoma, SMCs – smooth muscle cells, HUVECs – human umbilical vein endothelial cells, hMSCs – human mesenchymal stem cells. Only the embryonic OCT4A contains ApaI restriction site.</p

Expression and Differentiation between OCT4A and Its Pseudogenes in Human ESCs and Differentiated Adult Somatic Cells

<div><p>The POU5F1 gene codes for the OCT4 transcription factor, which is one of the key regulators of pluripotency. Its transcription, alternative splicing, and alternative translation leading to the synthesis of the active, nuclear localized OCT4A has been described in detail. Much less, however, is known about actively transcribed OCT4 pseudogenes, several of which display high homology to OCT4A and can be expressed and translated into proteins. Using RT-PCR followed by pseudogene-specific restriction digestion, cloning, and sequencing we discriminate between OCT4A and transcripts for pseudogenes 1, 3 and 4. We show that expression of OCT4 and its pseudogenes follows a developmentally-regulated pattern in differentiating hESCs, indicating a tight regulatory relationship between them. We further demonstrate that differentiated human cells from a variety of tissues express exclusively pseudogenes. Expression of OCT4A can, however be triggered in adult differentiated cells by oxygen and FGF2-dependent mechanisms.</p></div