Mechanism(s) by which pigment epithelium-derived factor regulate angiogenesis

Pigment Epithelium-derived Factor (PEDF), a natural protein possessing both neuroprotective and anti-angiogenic properties, is a very unique and attractive candidate as a therapeutic agent in the management of pathological neovascular diseases, such as tumours, age-related macular degeneration (AMD) and diabetic retinopathy. While it is well-known that PEDF can exert powerful effects on various tissues and cells, the underlying mechanism of PEDF's action is not well understood. This study investigated the relationship between vascular endothelial growth factor (VEGF)/PEDF and VEGFR-1 /VEGFR-2 by exploring Presenilin-l (PS-l) dependent regulated intramembrane proteolysis (RIP). Work on this non-classical pathway was initiated by Cai et al., (2006) using in vitro models of bovine retinal microvascular endothelial cells (BRMECs). Current study used BRMECs and human retinal pigment epithelial (HRPE) cells. In this study, BRMECs and HRPE cells were isolated and cultured. BRMECs were used as an angiogenic cell type while HRPE cells were used as an angiogenic regulator cell type. The characteristics of endothelial and epithelial cells and the localisation of VEGFR-1, VEGFR-2 and PS in BRMECs and HRPE cells were determined using immunocytochemistry techniques. The effects of VEGF and PEDF on VEGFR-1, VEGFR-2 and PS were assessed using immunocytochemistry and Western blotting, y-secretase activity in BRMECs and HRPE cells treated with various growth factors were analysed using a y-secretase activity kit. The role of VEGF on the production of PEDF and the expression of VEGFR-1, VEGFR-2 and PS in HRPE cells was investigated at both the transcriptional and translational levels. The techniques, VEGF-small interfering ribonucleic acid (VEGF-siRNA), reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and Enzyme-linked immunosorbent assay (ELISA) were used for the investigation. Results obtained from the project showed that PEDF had a regulatory role in the counterbalance of VEGFR-1 and VEGFR-2 expression in cultured BRMECs. PEDF upregulated y-secretase activity and PS-1 expression in BRMECs while VEGF acted as an antagonist of the effect of PEDF. In contrast, in HRPE cells, VEGF upregulated y-secretase activity and PEDF acted as an antagonist of the effect of VEGF. VEGF-siRNA induced a reduction of PEDF at both transcriptional and protein levels and a reduction of VEGFR-1 at the protein level. The effects of VEGF and PEDF on VEGFR-1 and VEGFR-2 may be cell type dependent. This study strengthens the view that PEDF can exert different regulatory effects on the same molecule (s) in different cell types. PEDF acts either antagonistically to VEGF or synergistically dependent upon the target molecule. Deciphering the cellular and molecular mechanisms underlying these interactions will not only contribute to our understanding of PEDF's action but also provide the foundation to maximise the therapeutic potential of this protein.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

BMP9 is a potent inducer of chondrogenesis, volumetric expansion and collagen type II accumulation in bovine auricular cartilage chondroprogenitors

Reconstruction of the outer ear currently requires harvesting of cartilage from the posterior of the auricle or ribs leading to pain and donor site morbidity. An alternative source for auricular reconstruction is in vitro tissue engineered cartilage using stem/progenitor cells. Several candidate cell-types have been studied with tissue-specific auricular cartilage progenitor cells (AuCPC) of particular interest. Whilst chondrogenic differentiation of competent stem cells using growth factor TGFβ1 produces cartilage this tissue is frequently fibrocartilaginous and lacks the morphological features of hyaline cartilage. Recent work has shown that growth factor BMP9 is a potent chondrogenic and morphogenetic factor for articular cartilage progenitor cells, and we hypothesised that this property extends to cartilage-derived progenitors from other tissues. In this study we show monoclonal populations of AuCPCs from immature and mature bovine cartilage cultured with BMP9 produced cartilage pellets have 3-5-fold greater surface area in sections than those grown with TGFβ1. Increased volumetric growth using BMP9 was due to greater sGAG deposition in immature pellets and significantly greater collagen accumulation in both immature and mature progenitor pellets. Polarised light microscopy and immunohistochemical analyses revealed that the organisation of collagen fibrils within pellets is an important factor in the growth of pellets. Additionally, chondrocytes in BMP9 stimulated cell pellets had larger lacunae and were more evenly dispersed throughout the extracellular matrix. Interestingly, BMP9 tended to normalise the response of immature AuCPC monoclonal cell lines to differentiation cues whereas cells exhibited more variation under TGFβ1. In conclusion, BMP9 appears to be a potent inducer of chondrogenesis and volumetric growth for AuCPCs a property that can be exploited for tissue engineering strategies for reconstructive surgery though with the caveat of negligible elastin production following 21-day treatment with either growth factor

Mechanism(s) by which pigment epithelium-derived factor regulate angiogenesis

Pigment Epithelium-derived Factor (PEDF), a natural protein possessing both neuroprotective and anti-angiogenic properties, is a very unique and attractive candidate as a therapeutic agent in the management of pathological neovascular diseases, such as tumours, age-related macular degeneration (AMD) and diabetic retinopathy. While it is well-known that PEDF can exert powerful effects on various tissues and cells, the underlying mechanism of PEDF's action is not well understood. This study investigated the relationship between vascular endothelial growth factor (VEGF)/PEDF and VEGFR-1 /VEGFR-2 by exploring Presenilin-l (PS-l) dependent regulated intramembrane proteolysis (RIP). Work on this non-classical pathway was initiated by Cai et al., (2006) using in vitro models of bovine retinal microvascular endothelial cells (BRMECs). Current study used BRMECs and human retinal pigment epithelial (HRPE) cells. In this study, BRMECs and HRPE cells were isolated and cultured. BRMECs were used as an angiogenic cell type while HRPE cells were used as an angiogenic regulator cell type. The characteristics of endothelial and epithelial cells and the localisation of VEGFR-1, VEGFR-2 and PS in BRMECs and HRPE cells were determined using immunocytochemistry techniques. The effects of VEGF and PEDF on VEGFR-1, VEGFR-2 and PS were assessed using immunocytochemistry and Western blotting, y-secretase activity in BRMECs and HRPE cells treated with various growth factors were analysed using a y-secretase activity kit. The role of VEGF on the production of PEDF and the expression of VEGFR-1, VEGFR-2 and PS in HRPE cells was investigated at both the transcriptional and translational levels. The techniques, VEGF-small interfering ribonucleic acid (VEGF-siRNA), reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and Enzyme-linked immunosorbent assay (ELISA) were used for the investigation. Results obtained from the project showed that PEDF had a regulatory role in the counterbalance of VEGFR-1 and VEGFR-2 expression in cultured BRMECs. PEDF upregulated y-secretase activity and PS-1 expression in BRMECs while VEGF acted as an antagonist of the effect of PEDF. In contrast, in HRPE cells, VEGF upregulated y-secretase activity and PEDF acted as an antagonist of the effect of VEGF. VEGF-siRNA induced a reduction of PEDF at both transcriptional and protein levels and a reduction of VEGFR-1 at the protein level. The effects of VEGF and PEDF on VEGFR-1 and VEGFR-2 may be cell type dependent. This study strengthens the view that PEDF can exert different regulatory effects on the same molecule (s) in different cell types. PEDF acts either antagonistically to VEGF or synergistically dependent upon the target molecule. Deciphering the cellular and molecular mechanisms underlying these interactions will not only contribute to our understanding of PEDF's action but also provide the foundation to maximise the therapeutic potential of this protein

Discovering Domain Disentanglement for Generalized Multi-source Domain Adaptation

A typical multi-source domain adaptation (MSDA) approach aims to transfer knowledge learned from a set of labeled source domains, to an unlabeled target domain. Nevertheless, prior works strictly assume that each source domain shares the identical group of classes with the target domain, which could hardly be guaranteed as the target label space is not observable. In this paper, we consider a more versatile setting of MSDA, namely Generalized Multi-source Domain Adaptation, wherein the source domains are partially overlapped, and the target domain is allowed to contain novel categories that are not presented in any source domains. This new setting is more elusive than any existing domain adaptation protocols due to the coexistence of the domain and category shifts across the source and target domains. To address this issue, we propose a variational domain disentanglement (VDD) framework, which decomposes the domain representations and semantic features for each instance by encouraging dimension-wise independence. To identify the target samples of unknown classes, we leverage online pseudo labeling, which assigns the pseudo-labels to unlabeled target data based on the confidence scores. Quantitative and qualitative experiments conducted on two benchmark datasets demonstrate the validity of the proposed framework

Influence of six digestion methods on the determination of polystyrene microplastics in organisms using the fluorescence intensity

Microplastic pollution has become a global environmental problem and is a cause of great concern. To evaluate the biological effects of microplastics, microplastics in organisms need to be accurately quantified. The quantification of microplastics in organisms using the fluorescence intensity is common; the digestion of biological samples is an important pretreatment method. However, the microplastics may be destroyed by digestion, which affects the fluorescence intensity of the microplastics and results in large deviations between measured and true values. In this study, six commonly used digestive agents were studied: KOH, NaOH, H2O2, HNO3, HNO3: hcl, and HNO3: HClO4. The effect of different digestion methods on the fluorescence intensity and surface morphology of microplastics was studied and the most suitable protocol was selected. The results show that, among the six different digestion methods, KOH digestion(100 g·L-1, 60℃)has the least influence on the fluorescence intensity of the microplastics and does not affect their surface morphology. The other five digestion methods lead to different degrees of reduction of the fluorescence intensity of microplastics and damage the microplastics' surface(aggregation, bubbles, scratches, and depressions). In addition, the KOH digestion method was used to extract microplastics from biological samples. The recovery rate was≥96.3%±0.5%, indicating that the KOH digestion method is suitable for fluorescent microplastics in biological samples