Neural stem cell self-renewal and differentiation are tightly regulated events during CNS development, leading to cell division into new neural stem cells or the formation of neurons and glial cells. This thesis focuses on the cellular responses induced by known and novel factors in neural stem and progenitor cells (NSPCs). Platelet-derived growth factor (PDGF) signaling has previously been implicated in NSPC regulation as well as in tumor formation. In order to evaluate the differentiation process and find new regulators of NSPCs a micro-array screen was performed, evaluating transcription during normal differentiation and the effect of PDGF-AA in this process. The transcriptional profile of PDGF-AA treated NSPCs was shown to be an intermediate between the profiles of neural stem cells and their progeny. The NSPC transcriptome was also found to have similarities with that of experimental glioma. A previously non-characterized transcript, the nuclear receptor binding protein 2 (NRBP2), was identified and found to be expressed in the developing and adult mouse brain and in medulloblastoma. NRBP2 down-regulation rendered neural progenitors sensitive to induced cell death. Different PDGF ligands interact with different combinations of PDGF receptors. Therefore NSPCs were stimulated with either PDGF-AA or -BB to further evaluate cellular responses with regard to the two specific isoforms. A divergent effect between the two isoforms in long-term proliferation and cell survival was found, with PDGF-BB being the most efficient stimulator. Stem cell factor (SCF) has previously been identified as a regulator in the hematopoietic system and we showed that SCF induces a migratory response in NSPCs. In addition, SCF positively affected cell survival but had no effect on NSPC differentiation. Insights into the regulatory mechanisms involved in neural stem cell signaling are needed to develop diagnostic tools and novel treatments.

The regulation of fluid (water) volume in the body is crucial for tissue homeostasis. The interstitial fluid, which comprises almost 20% of the body fluid, is stored in the loose connective tissue and its volume is actively regulated by components of this tissue. The loose connective tissue provides a path for fluid flow from capillaries to the tissue and lymphatics. This fluid is partially stored in the interstitium and the remainder is directed to the lymphatics. The fibroblasts in the loose connective tissue actively compact the fibrous extracellular matrix (ECM) through mechanotransduction via integrins. This in turn, maintains the interstitial fluid pressure and keeps the ground substance underhydrated. The interstitial fluid pressure is part of the forces that regulate the efflux of fluid from capillaries and keep the ground substance underhydrated. The underhydrated ground substance has a potential to take up fluid 3-fold the plasma volume. Therefore, the active contraction of the ECM via fibroblasts is crucial to prevent the risk of evacuation of fluid from capillaries. During pathologies, such as inflammation and carcinogenesis, the interstitial fluid pressure and hence the interstitial fluid volume is altered. The results presented in this thesis show that the signaling events downstream of αVβ3 integrin, collagen-binding β1 integrins, and platelet-derived growth factor receptor β, that induce cell-mediated matrix contraction, included paired function of PI3K and PLCγ, cofilin activation, actin turnover, and generation of actomyosin forces. Furthermore, the results highlight new potential roles for fibrin and αVβ3 integrins, for instance during clearance of edema. Notably, fibrin extravasation at inflammatory sites induced αVβ3 integrin-dependent matrix contraction, leading to normalization of the altered interstitial fluid volume. It also reprograms the expression of ECM-related genes and hence induces ECM turnover. Taken together, these results provide further insight into the regulatory mechanism through which the loose connective tissue actively regulates the interstitial fluid volume.

Aβ oligomer-induced neurotoxicity has become an important area of therapeutic development in treating Alzheimer’s disease. Platelet-derived growth factor (PDGF) has been shown to be able to protect neurons against several neuronal insults such as ischemia and HIV1 toxin induced cytotoxicity. These neuroprotective effects correlate well with our previous results that demonstrate the neuroprotective effects of PDGF-BB, one of the PDGF receptor ligand subtypes, against NR2B containing NMDA receptor induced excitotoxicity, a possible underlying cause of Aβ oligomer induced synaptic dysfunction and neuronal death. This project examines the neuroprotective effect of PDGF-BB against Aβ1-42 oligomer induced cytotoxicity in both SH-SY5Y cells and primary hippocampal neurons. Cell viability was monitored by MTT assay and the affected signaling pathways were examined using pharmacological methods and Western blotting. The results demonstrated that Aβ1-42 oligomer elicited a dose-dependent toxicity with a sign of saturation at higher dosages, PDGF-BB failed to protect neurons against Aβ1-42 oligomer induced cytotoxicity. In contrast, Aβ1-42 oligomers strongly inhibit PDGF-BB induced mitogenesis in both SH-SY5Y cells and primary neurons. Further investigation using Western blotting to measure PDGF receptor expression and phosphorylation in SH-SY5Y cells showed that Aβ1-42 oligomer can inhibit PDGF-BB induced phosphorylation of PDGF β-receptor on Tyr1021, a site that is crucial for PLCγ mediated mitogenesis. These findings not only explained the poor neuroprotective effect elicited by PDGF-BB against Aβ1-42 oligomers, but also led to a novel hypothesis that Aβ1-42 oligomer may interfere with neurotrophic factor induced neuronal survival, either selectively or perhaps globally. Further exploration on this hypothesis will be able to shed light on this potentially novel mechanism of pathogenesis in Alzheimer’s disease.

Cardiovascular disease, which involves the cardiac, cerebrovascular and peripheral vascular system, is the major cause of morbidity and mortality in the western world. Changes in the vascular microenvironment trigger cascades of molecular events involving altered signaling, transcription and translation of a gene. The aim of this thesis was to increase our understanding on the molecular regulation of activated vascular smooth muscle cells. The first study looking at PDGF-D expression provides new insights into the regulatory mechanisms controlling the phosphorylation of Sp1. Studies performed identified three amino acids in Sp1 (Thr668, Ser670 and Thr681) that is phosphorylated by PKC-zeta activated by AngII. In the second study, the translational regulatory role of a novel gene YrdC induced by injury was investigated. Current knowledge of translational regulators controlling altered gene expression is little and studies in this thesis shows a splice variant of YrdC playing an important role in controlling mRNA translation and thus protein synthesis in the context of injury. The final study investigated in this study was the increased expression of the apoptotic FasL by the activation of GATA6. Although FasL has been extensively studied over the years, this is the first study linking a GATA factor with FasL in any cell type and provides key insights into the transcriptional events underpinning FasL-dependent SMC apoptosis following exposure to AngII.

Bioactive agents are proteins that regulate cellular activities including cell migration, proliferation, differentiation and matrix synthesis. Over the last decades there has been a focused effort to understand how these agents influence repair or regeneration of bony defects. Platelet derived growth factor (PDGF) has potent chemotactic and angiogenic properties. Bone morphogenetic protein (BMP) is a known factor for osteoblasts. This study evaluated the impact of recombinant human PDGF and BMP-2 on resolution of critical bony defects (2 mm) using mouse calvarial bone cultures. Calvaria from 5-7 day neonatal CD-1 mice were dissected and cultured in Dulbecco’s Modified Eagle’s Medium under sterile conditions. In the first experiment, two different delivery systems to deliver PDGF - freeze-dried bone allograft and beta- tricalcium phosphate were compared. The second experiment analyzed bone formation in response to BMP-2 in the presence or absence of freeze-dried bone allograft. The media was changed every 2 days and the spent media were analysed for calcium release. At the end of three weeks the calvaria were processed for histological observation, biochemical analyses and neutral red staining. The results show higher bone formation in response to BMP-2 than PDGF. The presence of allograft inhibits this response. We found B-TCP to be a better delivery agent for PDGF compared to freeze-dried bone allograft. The histologic assessment showed development of new bone through intramembranous pathway that replicates native bone development in presence of BMP-2. In conclusion our study proves that incorporation of two bioactive agents- PDGF and BMP-2 in an osteoconductive scaffold can induce repair and new bone formation in mouse calvarial bone cultures. / 2020-07-30T00:00:00Z

Cryptorchidism is a male congenital disorder with an unspecified, multifactorial etiology. This review evaluated the strength of select factors in the development of cryptorchidism to better understand its etiology. The strength of relationship between factors and their respective functions during testicular descent was evaluated. Factors evaluated in the causal pathway include the signaling mechanisms Desert Hedgehog (DHH), Insulin-like Hormone 3 (INSL3) and Platelet-Derived Growth Factor (PDGF), as well as sex hormone regulation (androgen: estrogen ratio, aromatase expression). Articles supporting a factor in testicular descent were evaluated and scored. These scores were summed to create the “Step Score” for each step in the causal pathway. An arrow system was developed which ranked the strength of each pathway step as either “weak”, “moderate” or “strong”. Thus, step scores and the strength of factors in the pathological pathway were determined: DHH (15-moderate), PDGF (10-weak), INSL3 (24-strong) and Androgen: Estrogen ratio, Aromatase (23-strong). The pathological pathway produced by this review represents a literature based perspective of the research regarding cryptorchidism etiology. Literature indicates that prenatal exposure to endocrine disrupting chemicals in animals and humans may lead to abnormal genital development. Recently, prenatal maternal cigarette smoke was demonstrated to be a risk factor for cryptorchidism. This controversial finding was explored in the context of endocrine disrupting chemicals. However, literature has provided very little evidence in support of this hypothesis and more research is needed to better evaluate prenatal maternal smoking as a risk factor for undescended testis.

Platelet-derived growth factor-C (PDGF-C) and its tyrosine kinase receptor PDGFRɑ have been shown to contribute to several key processes during central nervous system (CNS) development, including normal vascularization and formation of cerebral ventricles and basal membrane of the meninges. Due to redundancy between PDGF-C and PDGF-A, PDGF-C specific roles are sometimes masked and difficult to determine. Using the double mutant    Pdgfc-/-;PdgfraGFP/+ mouse (Mus musculus) strain we were able to detect and examine a new, undescribed phenotype of PDGF-C impaired mice, namely cerebellar malformations. These mutant mice displayed an upwards rotation of the cerebellar vermis with a severe posterior vermis hypoplasia and an enlarged fourth ventricle, suggesting PDGF-C/PDGFRɑ signaling as a novel candidate to induce Dandy-Walker malformation (DWM). Due to suspected cerebellar vascular malformation a quantification of diameter, density and number of vessels were performed. A significant increase (P < 0.05) of the number and density of vascular bed in the cerebellar nuclei was detected, however the vessel diameter was not significantly different (P > 0.05) in Pdgfc-/-;PdgfraGFP/+ mice in comparison with the control. Through immunofluorescence staining we detected discontinuation of the ependyma in the acute angle of the ventricular zone adjacent to the rhombic lip, interfacing the fourth ventricle and cerebellar anlagen. We further noted ectopic expression of rhombic lip derived cells in the ventricular zone, suggesting a misguided migration due to ablation of PDGF-C. We conclude that PDGF-C is an essential player in normal cerebellar development.

No description available.

Les macrophages (MΦ), des cellules clefs de la réponse immunitaire peuvent répondre à des contaminants environnementaux comme les particules diesel (DEP), des polluants atmosphériques récemment classés cancérigènes pour l'Homme. Les MΦ sont des cellules hétérogènes et plastiques qui s'activent en fonction de leur microenvironnement soit en MΦ M1 (dits classiquement activés ou pro-inflammatoires) sous l'effet de l'INFγ et du LPS soit en MΦ M2 (dits alternativement activés ou réparateurs) sous l'effet de l'IL-4 et/ou de l'IL-13. Les effets des DEP sur la polarisation M1/M2 des MΦ restent peu documentés. Nous avons dans un premier temps caractérisé l'expression des marqueurs des MΦ différenciés in vitro en présence de M-CSF à partir de monocytes humains et polarisés en sous-type M1 ou M2. Nos principaux résultats montrent que les MΦ différenciés au M-CSF considérés comme des MΦ anti-inflammatoires, sont en réalité capables de s'activer vers un phénotype M1 après une stimulation au LPS/IFNγ. De plus, les marqueurs mis en évidence au cours de ce travail ont permis d'évaluer l'impact d'extraits organiques de DEP (DEPe) sur la polarisation des MΦ et plus généralement sur leur physiologie. Les DEPe altèrent l'expression de certains marqueurs M1 et M2 des MΦ, sans toutefois provoquer d'inhibition globale des processus de polarisation M1 et M2 ou de transition d'un phénotype vers un autre. Cette altération du phénotype est associée à une diminution de la réponse inflammatoire LPS-dépendante dans les MΦ M1 et des capacités chimiotactiques des MΦ M2. Les DEPe diminuent la sécrétion de certaines cytokines et chimiokines comme l'IL-6, l'IL-12p40 et le CCL18 via l'activation d'AhR et/ou de Nrf2. Parallèlement, nous montrons que les MΦ M1 et M2 exposés aux DEPe sécrètent le platelet deried growth factor B (PDGF-B), un facteur de croissance profibrosant, via l'activation d'AhR en quantité suffisante pour stimuler la prolifération de fibroblastes pulmonaires. Au total, ces travaux démontrent que les DEP possèdent des propriétés immunotoxiques vis-à-vis de la physiologie des macrophages humains polarisés in vitro. Cette immunotoxicité pourrait participer aux effets délétères de ces contaminants environnementaux urbains sur la santé humaine. / Macrophages (MΦ), well-known to play a key role in immune response, also respond to environmental toxic chemicals such as diesel exhaust particles (DEP), an air pollutant recently classified as carcinogenic to humans. MΦ are heterogeneous and plastic cells which activate according to their microenvironment into either an M1 subtype (so called classically activated or pro-inflammatory) under IFNγ and LPS stimulation or an M2 subtype (so called alternatively activated or anti-inflammatory) under IL-4 and/or IL-13 stimulation. However, potential effects of DEPs on M1/M2 MΦ polarization remain poorly documented. First, we characterized the expression marker of in vitro M-CSF-differentiated MΦ from human monocytes and activated into the M1 or M2 subtypes. Our main results show that M-CSF-generated MΦ considered as anti-inflammatory are actually able to switch to an M1 phenotype after IFNγ/LPS stimulation. Furthermore, the markers identified in this study were used to assess the impact of organic extracts of DEP (DEPe) on MΦ polarization and more generally on their physiology. DEPe alter some M1 and M2 markers expressed by polarized MΦ, without causing the overall inhibition of the M1 and M2 polarization process or the switch to a different phenotype. This phenotype alteration is associated with a decrease in the LPS-dependent inflammatory response in M1 MΦ and the chemotactic capacities in M2 MΦ. DEPe decrease the secretion of some cytokines and chemokines such as IL-6, IL-12p40 and CCL18 via AhR and/or Nrf2 activation. At the same time, we show that M1 and M2 MΦ in response to DEPe are able to secrete a sufficient level of a pro-fibrotic growth factor, the platelet derived growth factor B (PDGF-B) via AhR activation, leading to stimulation of lung fibroblast proliferation. Finally, these works show that DEPe have immunotoxic properties with regards to the physiology of human in vitro polarized MΦ. This immunotoxicity may then contribute to the deleterious effects of these urban environmental contaminants on human health.

Stem cells are endowed with unique qualities: they can both self-renew and give rise to new mature cell types. Central nervous system (CNS) stem cells can give rise to neurons and glia. What factors regulate stem cell fate decisions? Identifying signals that are involved in the regulation of CNS stem cell proliferation, survival, differentiation and migration is fundamental to the understanding of CNS development. In addition, this knowledge hopefully will contribute to more efficient therapies of CNS damages and diseases. The focus of this thesis was to investigate mechanisms of CNS stem cell proliferation and differentiation. We have studied the role for platelet-derived growth factor (PDGF) in these cellular events both in vitro and in vivo. Previous reports have shown that PDGF are implicated in brain tumorigenesis and also supports neuronal differentiation of CNS stem cells. We have found that PDGF promotes survival and proliferation of immature neurons, thereby supporting neuronal differentiation. The intracellular Ras/ERK signaling pathway probably mediates the mitogenic activity of PDGF. In contrast, neuronal differentiation is not dependent on the Ras/ERK pathway. A genetic expression profile of stem cells during their differentiation was obtained. This microarray analysis suggests that PDGF-treated stem cells are at an intermediate stage between proliferation and differentiation. Furthermore, we generated transgenic mice that overexpress Pdgf-b in neural stem cells. Preliminary data indicate no signs of enhanced proliferation of immature neurons. Instead, increased apoptosis was detected in the developing striatum. The results presented in this thesis show how CNS stem cells are regulated by PDGF. PDGFs are widely expressed in the developing CNS and also in some brain tumors, which are thought to arise from CNS stem cells. Thus, this knowledge may contribute to an increased understanding of brain tumorigenesis in addition to normal CNS development.