Transcriptional regulation of the collagen alpha1(1) gene by hypoxia

This thesis investigates the molecular mechanisms of hypoxic regulation of the collagen α1(1) (COL1A1) gene. Collagen-1 is the most abundant extracellular protein in vertebrates. Transcription of this protein is normally under tight physiological control. However, under pathological conditions this fine control is lost leading to accumulation of collagen, scarring and fibrosis. Hypoxia, as a result of microvascular injury, has been implicated in the pathogenesis of fibrosis and studies in this laboratory had shown that hypoxia is a pro-fibrogenic stimulus increasing production and decreasing turnover of interstitial Collagen-1 in vitro. Hypoxia is a potent regulator of gene expression and hypoxia response elements (HRE) have been identified in a wide variety of genes. These HREs mostly, but not exclusively, contain consensus binding sites for the hypoxia-inducible factor-1 (HIF-1). Transfection experiments and deletion analysis of the COL1A1 promoter demonstrated that the HRE(s) for this gene lies between -220/+115. This evolutionary conserved region lacks any consensus HIF-1 binding sites suggesting HIF-1-independent transcriptional activation of COL1A1 in response to low O2. In addition TGF-β1, the preeminent fibrogenic stimulus, did not stimulate reporter gene activity in this region of COL1A1, indicating TGF-β1 independent hypoxic activation. Subsequent studies focussed on identification of c/s-acting DNA elements and transcription factors mediating the hypoxia-induced COL1A1 gene transcription: DNA elements; DNAse-l footprinting of the mouse COL1A1 -220/+115 with nuclear extracts from normoxic fibroblasts showed 2 protected regions. Hypoxic nuclear proteins demonstrated increased affinity to the promoter producing 11 protected areas representing higher transcriptional activity. Transcription factors: Nuclear extracts from normoxic and hypoxic fibroblasts were resolved on two dimensional gels and proteins identified by proteomics. Results of this study produced a broad spectrum of nuclear proteins therefore a more targeted approach was devised, where specific DNA binding proteins were identified by Southwestern blotting and characterised with proteomics. On Southwesterns, 7 normoxic nuclear proteins hybridised to the -220/+115 COL1A1 radiolabelled probe, while hypoxic nuclear extracts produced 12 strong bands, again illustrating higher transcriptional activity in hypoxia. Proteomic analysis of the bands identified several transcription factors with Sp1 displaying the highest increase in DNA binding in hypoxia. DNA-Protein interaction: Sequence scanning of COL1A1 -220/+115 revealed 3 putative Sp1 binding sites (-123/-114, -98/-82, -63/-53). Binding of Sp1 to the 2 more upstream sites has been previously reported. EMSAs and antibody supershifts showed Sp1 binding to the two upstream sites (-123/-114 and -98/-82) in normoxic and hypoxic nuclear extracts consistent with a role for this factor in basal and hypoxic transcriptional regulation of COL1A1. The binding of Sp1 to both sites increased in hypoxia with the most 5' site (-123/-114) showing the highest increase. The level of binding by normoxic and hypoxic nuclear proteins to the most proximal putative Sp1 binding site was weak. To assess the functional role of Sp1 the effect of inhibiting Sp1 binding to the COL1A1 promoter was examined. Mithramycin is a GC-specific DNA-binding drug, which selectively inhibits transcription of genes with GC-rich promoter. In transfection experiments mithramycin treatment completely inhibited the hypoxic induction of COL1A1 -220/+115 to a level blow the basal activity of the promoter. The increased binding of Sp1 to the promoter in hypoxia could be due to either upregulation of Sp1 with increased availability of Sp1 in the nucleus or to "activation" of Sp1 increasing the binding affinity of this factor for the promoter. By Western blotting there was no difference in nuclear Sp1 levels in hypoxia and normoxia suggesting that hypoxia increases Sp1 activation and DNA binding. In conclusion, these studies demonstrate Sp1 is an important mediator of hypoxia-induced COL1A1 gene transcription in fibroblasts acting via hypoxia response elements in the -220/+115 region of COL1A1

Analysis of the compatibility of dental implant systems in fibula free flap reconstruction

As a result of major ablative surgery, head and neck oncology patients can be left with significant defects in the orofacial region. The resultant defect raises the need for advanced reconstruction techniques. The reconstruction in this region is aimed at restoring function and facial contour. The use of vascularised free flaps has revolutionised the reconstruction in the head and neck. Advances in reconstruction techniques have resulted in continuous improvement of oral rehabilitation. For example, endosteal implants are being used to restore the masticatory function by the way of prosthetic replacement of the dentition. Implant rehabilitation usually leads to improved facial appearance, function, restoration of speech and mastication. Suitable dental implant placement’s site requires satisfactory width, height and quality of bone. Reconstruction of hard tissue defects therefore will need to be tailored to meet the needs for implant placement. The aim of this feasibility study was to assess the compatibility of five standard commercially available dental implant systems (Biomet 3i, Nobel Biocare, Astra tech, Straumann and Ankylos) for placement into vascularised fibula graft during the reconstruction of oromandibular region. Radiographs (2D) of the lower extremities from 142 patients in the archives of the Department of Radiology in University College London Hospitals (UCLH) were analysed in this study. These radiographs were from 61 females and 81 males. Additionally, 60 unsexed dry fibular bones, 30 right sided, acquired from the collection of the Department of Anatomy, University College London (UCL) were also measured to account for the 3D factor. In the right fibula (dry bone), 90% of the samples measured had a width of 13.1 mm. While in the left fibula (dry bone), 90% of the samples measured had a width of 13.3 mm. Fibulas measured on radiographs had a width of 14.3 mm in 90% of the samples. The length ranges of the dental implants used in this study were: 7-13 mm (Biomet 3i), 10-13 mm (Nobel biocare), 8-13 mm (Astra Tech), 8-12 mm (Straumann ) and 8-11 mm (Ankylos). This study reached a conclusion that the width of fibula is sufficient for placement of most frequently used dental implants for oral rehabilitation after mandibular reconstructive procedures

Hypoxia mimetics restore bone biomineralisation in hyperglycaemic environments

Diabetic patients have an increased risk of fracture and an increased occurrence of impaired fracture healing. Diabetic and hyperglycaemic conditions have been shown to impair the cellular response to hypoxia, via an inhibited hypoxia inducible factor (HIF)-1α pathway. We investigated, using an in vitro hyperglycaemia bone tissue engineering model (and a multidisciplinary bone characterisation approach), the differing effects of glucose levels, hypoxia and chemicals known to stabilise HIF-1α (CoCl2 and DMOG) on bone formation. Hypoxia (1% O2) inhibited bone nodule formation and resulted in discrete biomineralisation as opposed to the mineralised extracellular collagen fibres found in normoxia (20% O2). Unlike hypoxia, the use of hypoxia mimetics did not prevent nodule formation in normal glucose level. Hyperglycaemic conditions (25 mM and 50 mM glucose) inhibited biomineralisation. Interestingly, both hypoxia mimetics (CoCl2 and DMOG) partly restored hyperglycaemia inhibited bone nodule formation. These results highlight the difference in osteoblast responses between hypoxia mimetics and actual hypoxia and suggests a role of HIF-1α stabilisation in bone biomineralisation that extends that of promoting neovascularisation, or other system effects associated with hypoxia and bone regeneration in vivo. This study demonstrates that targeting the HIF pathway may represent a promising strategy for bone regeneration in diabetic patients

Enhanced photodynamic therapy and fluorescence imaging using gold nanorods for porphyrin delivery in a novel in vitro squamous cell carcinoma 3D model

Nanocomposites of gold nanorods (Au NRs) with the cationic porphyrin TMPyP (5,10,15,20-tetrakis(1- methyl 4-pyridinio)porphyrin tetra(p-toluenesulfonate)) were investigated as a nanocarrier system for photodynamic therapy (PDT) and fluorescence imaging. To confer biocompatibility and facilitate the cellular uptake, the NRs were encapsulated with polyacrylic acid (PAA) and efficiently loaded with the cationic porphyrin by electrostatic interaction. The nanocomposites were tested with and without light exposure following incubation in 2D monolayer cultures and a 3D compressed collagen construct of head and neck squamous cell carcinoma (HNSCC). The results showed that Au NRs enhance the absorption and emission intensity of TMPyP and improve its photodynamic efficiency and fluorescence imaging capability in both 2D cultures and 3D cancer constructs. Au NRs are promising theranostic agents for delivery of photosensitisers for HNSCC treatment and imaging

Molecular Modeling of 5HT(2A) Receptor - Arylpiperazine Ligands Interactions

In this paper, we report the molecular modeling of the 5HT(2A) receptor and the molecular docking of arylpiperazine-like ligands. The focus of the research was on explaining the effects the ligand structure has on the binding properties of the 5HT(2A) receptor and on the key interactions between the ligands and the receptor-binding site. To see what the receptor-ligand interactions were, various substituents were introduced in one part of the ligand, keeping the rest unchanged. In this way, using a docking analysis on the proposed 5HT(2A) receptor model, we identified key receptor-ligand interactions and determined their properties. Those properties were correlated with experimentally determined binding affinities in order to determine the structure to activity relationship of the examined compounds

Interactions of N-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-2-aryl-2-yl-acetamides and 1-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-3-aryl-2-yl-ureas with dopamine D-2 and 5-hydroxytryptamine 5HT(1A) receptors

It is suggested that the ratio of dopamine D-2 to 5-hydroxytryptamine 5-HT1A activity is an important parameter that determines the efficiency of antipsychotic drugs. Here we present the synthesis of N-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-2-aryl-2-yl-acetamides and 1-{[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-3-aryl-2-yl-ureas and their structure-activity relationship studies on dopamine D-2 and 5-hydrohytryptamine 5-HT1A receptors. It was shown that ligand selectivity and affinity strongly depends on their topology and the presence of a pyridyl group in the head of molecules. Molecular modeling studies using homology modeling and docking simulation revealed a rational explanation for the ligand behavior. The observed binding modes and receptor-ligand interactions provided us with a clue for optimizing the optimal selectivity towards 5-HT1A receptors