A STUDY OF HYPOXIA INDUCIBLE FACTOR AND RELATED GENES IN DISEASE IN MAN

In this study, the mRNA and protein levels of hypoxia inducible factor 1 (HIF-1α), and a number of genes regulated by hypoxia (VEGF, GLUT-1, p53), were determined in four breast carcinoma cell lines, peripheral blood mononuclear cells (PBMCs) of patients with breast cancer and Type 1 diabetes (TIDM), and in human breast and brain tumour tissue. Breast carcinoma cells and PMBCs from both patients and normal controls were exposed to hypoxia (≤1% 0 2) and/or high glucose. Both up-regulated and down-regulated HIF-1α, GLUT-1 and p53 mRNA expression was observed in the breast carcinoma cell lines exposed to hypoxia and/or high glucose, and in controls for osmolarity, confirming that hypoxic regulation of HIF-1α, p53 and possibly GLUT-1 occurs post-transcriptionally. Conversely, up-regulation of HIF- 1α and GLUT-1 mRNA was observed in patients with TIDM exposed to high glucose. The GLUT-1 mRNA up-regulation observed in patients without complications differed significantly from normal controls, where up to a 2 fold increase in expression was observed over that of patients with complications. This may indicate that the expression and function of glucose transporters differs in these patients, potentially leading to fewer complications. Investigation of breast and glial cell tumour tissue demonstrated that both HIF-1α and GLUT- 1 mRNA expression levels increase with disease progression, indicating that up-regulation of HIF-1α is partly at the transcriptional level (Søndergaard et al, 2002). Follow-up survival studies in all patients with glial cell tumours showed that HIF-1α protein expression is a significant prognostic factor in cumulative overall survival. An additional investigation of p53 or p73 polymorphisms in the development of carcinoma of the breast did not find that they were significant risk factors in the development of the disease in the British Caucasoid population. Further studies are required using larger sample populations investigating HIF-1α protein to determine the precise role of HIF-1 in the response to hypoxia and angiogenesis in disease in man

Genetic analysis of the microvascular complications of diabetes mellitus

There is increasing evidence to suggest that genetic factors are involved in the pathogenesis of microvascular complications in diabetes mellitus. Recent studies have suggested that genetic variations in the aldose reductase (ALR2) gene may contribute to the genetic susceptibility to microvascular complications. Aldose reductase is the first and rate-limiting enzyme of the polyol pathway and is implicated in the pathogenesis of diabetic microvascular disease (nephropathy, retinopathy and neuropathy). It has recently been shown that the three polymorphisms of the ALR2 gene are associated with susceptibility to microvascular complications in both TIDM and T2DM. The aim of this study was to investigate the CA dinucleotide repeat polymorphism (5'ALR2) that is located -2100bp and the C-106T substitution in the promoter region of the ALR2 gene, and also the A+ 11842C within intron 8 of the ALR2 gene itself. DNA from 285 Caucasoid patients with TIDM and well-defined microvascular disease and 120 normal healthy controls, as well as 60 Southern Indian patients with T2DM and 43 non diabetic controls were typed. The 5'ALR2 Z-2/X genotype was significantly increased in patients with nephropathy (n=92), retinopathy (n=160) and neuropathy (n=104) compared to those with no microvascular disease after 19 years duration of diabetes (uncomplicated, n=66) (46%, 41%, 42% vs. 24%, respectively). In contrast, the frequency of the Z+2/Y genotype (where Y is not Z-2) was significantly reduced in the patients with nephropathy, retinopathy and neuropathy compared to the uncomplicated (17%, 23%, 23% vs. 52%, respectively). Similar observations were made in the Southern Indian T2DM patients, however no significant differences were found. In the patients with TIDM the C-106 allele was associated with the Z-2 5'ALR2 allele. The C/Z-2 haplotype was present in 32% of the nephropaths, 32% of the retinopaths and 35% of the neuropaths compared to 11.5% of the uncomplicated. The A+ 11842 allele was also associated with the C-1 06 allele in TIDM patients with microvascular disease. The reported mitochondrial polymorphism (mt5178A/C) was not found in this. TIDM population, possibly due to differences in the background frequencies between ethnic groups. Family studies investigating the transmission of the 5'ALR2 and C-106T alleles from parents to offspring with diabetic nephropathy found preferential transmission of the Z-2 allele although this was not statistically significant. Functional studies of the activity of the ORE in TIDM patients with and without microvascular disease showed differences in the mean OREBP binding activity. OREB and OREC were found to have increased activity in response to hyperglycaemia in the complicated patients compared to the uncomplicated and normal controls. In conclusion, these results confirm the role of the aldose reductase gene in the genetic susceptibility to diabetic microvascular complications, and a possible role of the DI7S934 polymorphism in T2DM. These results also provide a novel insight into the role of the ORE of the ALR2 gene in the pathogenesis of diabetic microvascular complications. Further studies are now required to determine the molecular basis of these observations. Hopefully, in the future it will be possible to offer 'high risk' patients therapeutic intervention that will prevent the ravages of the long term complications of diabetes mellitus

A STUDY OF THE MOLECULAR GENETICS OF RENAL CELL CARCINOMA IN MAN

The aim was to measure the expression levels of hypoxia inducible factor 1-α (HJF-lα), glucose transporter one (GLUT-1), and vascular endothelial growth factor (VEGF) isoforms 165 and 189 mRNA in patients with renal cell carcinoma (RCC). Patients with RCC underwent radical nephrectomy at Derriford Hospital, Plymouth. Tumour as well as matched normal tissue from the kidney was harvested, snap frozen and stored in liquid nitrogen, and used to quantitate VEGF 165, VEGF 189, GLUT-1 and HJF-1α mRNA expression using ribonuclease protection assays, and quantified using a Phosphor-imager system. The VEGF 165 isoform was increased in the tumour tissue in comparison with the adjacent normal tissue (3.05 vs. 1.56 P=0.00002) as was the VEGF 189 isoform (2.41 vs. 1.43 P=0.0002). Forty four patients were analysed for the expression of HIF-lα and GLUT-1 with statically significant differences seen between the tumour tissues with respect to the normal tissue for both HIF-lα (1.34 vs. 1.10 P=0.01) and GLUT-1 (1.99 vs. 1.63 P=0.003). Hypoxia inducible factor I (HIF-1) is a key regulator of genes involved in the hypoxic response. HIF consists of alpha and beta subunits, with the alpha subunit being degraded under normoxic conditions and stabilised under hypoxia. Polymorph isms in exon 12 of the HIF gene have been recently been identified consisting of nucleotide changes (C+ 1772T and G+ 1790A) resulting in an amino acid substitution from Proline 582 to Serine, and Alanine 588 to Threonine respectively. These polymorphisms are found within the oxygen-dependent degradation domain (ODD) of the HIF-lα protein when transcribed which is important in the oxygen regulation of the protein via hydroxylation of the proline residue at position 564 (P564) by HIF-α- prolylhydroxylase (HIF-PH). The regulation of HIF-1α by this method is a novel way of regulating the levels of the protein, and polymorphisms in the ODD of HIF-lα may affect the ability of VHL to direct the alpha subunit for destruction. We have genotyped 146 patients and 288 controls for the G+ 1790A, and 160 patients and 162 controls for the C+ 1772T polymorphisms respectively. We found an increase in both the GA and CC (P<0.00001 and P= 0.00002) genotypes in our patients with renal cell carcinoma, and a decrease in GG and CT (P<0.00001 and P=0.00002) genotypes respectively. Haplotype analysis revealed there to be an increase in the T-A and C-A haplotypes (P=0.00008, and P=0.02) and a decrease in the T-G haplotype P=0.01. No statistical difference was found for the other haplotypes. We have shown that these HIF-lα polymorphisms are present in RCC with increased frequency and may play an important role in the disease process, leading to increased angiogenesis in the tumour. Vascular endothelial growth factor (VEGF) is a highly specific mitogen that is able to stimulate the proliferation of endothelial cells. There have been a number of findings linking the expression of VEGF with RCC, with it also being used to assess the prognosis of the disease. Polymorphisms in the VEGF gene have been recently identified. A possible link between promoter polymorphisms and expression of mRNA isoforms has been found in a variety of cytokines. Certain polymorphisms in renal cell carcinoma patients can lead to an up regulation of the expression of the mRNA, and may be a factor in the highly vascularized nature of the tumours studied. The aim was to investigate the frequency of polymorphisms within the VEGF gene (C-2578A) in 173 patients with RCC and 142 normal controls. No differences were seen between the patients and control populations, and the polymorphism did not correlate with Robson stage, Fuhrman grade or age and gender. Although a trend was seen between the C- 2578A polymorphism and expression of VEGF mRNA species in RCC patients

An Investigation for Genetic Susceptibility to Type 1 Diabetes Mellitus and its Microvascular Complications

Long-term exposure to diabetes mellitus is associated with metabolic abnormalities such as chronic hyperglycemia and redox imbalance. Uncontrolled hyperglycaemia and genetic factors are implicated in the pathogenesis of diabetic microvascular diseases. Genetic mutation through the genes coding for enzymes involved in glucose metabolism and immuno-regulatory mechanisms may contribute to the susceptibility to type I diabetes mellitus (TIDM) and its chronic microvascular diseases. Previous studies have shown that the transcription factor, nuclear factor kappa 8 (NFκB) and heat shock proteins (HSPs) are two redox-sensitive cellular responses of most immune and inflammatory diseases including diabetes and its late vascular complications. NFκB promotes the transcription of a wide array of proinflammatory mediators and adhesion molecules. HSPs are proposed to have a cytoprotective effect; in contrast they have the capacity to promote pathogenic processes. In this study, polymerase chain reaction (PCR)-based microsatellite analysis and restriction fragment length polymorphism (RFLP) analysis were used to genotype the genes coding for NFκB, HSP70-A2, sorbitol dehydrogenase (SORD) and protein kinase C- β(PKC-β). The A10 allele of the NFκB gene and H3 and H7 alleles of the HSP70-A2 gene were identified as risk markers of TIDM (P< 0.01). These alleles were not associated with microvascular complications. No evidence of associations was obtained between either PKC-β or SORD genes with TIDM and its late complications. Uncontrolled hyperglycaemia may alter the transcription mechanism of many genes, which control vascular homeostasis. The electrophoresis mobility shift assay (EMSA) was used to assess the transcription factor, heat shock factor-1 (HSF-1) and NFκB-DNA binding activity in response to a concentration of 31 mM D- glucose in peripheral blood mononuclear cells (PBMCs) from patients with TIDM with and without microvascular complications. Hyperglycaemia induced significant increases in both NFκB and HSF-1-DNA binding activities in PBMCs from patients (p= 0.003 and 0.017 respectively). The protein activity was more pronounced in PBMCs from patients with microvascular complications. Hyperglycaemia-induced NFκB-DNA binding activity was correlated to that of HSF-1 (p< 0.0 I). Patients with TIDM with microvascular complications demonstrated a significant increase in NFκB-DNA binding activity compared to patients with a short duration of diabetes (SD) or diabetic controls (DC) (p= 0.003 and p = 0.047 respectively). A significant positive correlation was found between the duration of diabetes and hyperglycaemia-induced NFκB-DNA binding activity (p=0.035). These results suggest that hyperactive flux through the polyol pathway is relevant to hyperglycaemia-induced protein activity since the aldose reductase inhibitors (ARIs) zopolrestat and sorbinil reduced HSF-1 and NFκB-DNA binding activity in PBMCs. In conclusion, these results suggest that NFκB and HSP70-A2 genes may contribute to the genetic susceptibility to TIDM. Uncontrolled hyperglycaemia in diabetes may alter the transcription mechanism and magnify the proinflammatory responses, which accelerate the development of diabetic microvascular complications

Hypoxia-Inducible Factor-1 as a Therapeutic Target in Endometrial Cancer Management

In the Western world, endometrial cancer (EC) is the most common malignant tumor of the female genital tract. Solid tumors like EC outgrow their vasculature resulting in hypoxia. Tumor hypoxia is important because it renders an aggressive phenotype and leads to radio- and chemo-therapy resistance. Hypoxia-inducible factor-1α (HIF-1α) plays an essential role in the adaptive cellular response to hypoxia and is associated with poor clinical outcome in EC. Therefore, HIF-1 could be an attractive therapeutic target. Selective HIF-1 inhibitors have not been identified. A number of nonselective inhibitors which target signaling pathways upstream or downstream HIF-1 are known to decrease HIF-1α protein levels. In clinical trials for the treatment of advanced and/or recurrent EC are the topoisomerase I inhibitor Topotecan, mTOR-inhibitor Rapamycin, and angiogenesis inhibitor Bevacizumab. Preliminary data shows encouraging results for these agents. Further work is needed to identify selective HIF-1 inhibitors and to translate these into clinical trials

An Exploration of the Widely Observed Mechanisms Permitting Freeze Tolerance & the Potential of Cope’s Gray Treefrog, Dryophytes chrysoscelis

Over 80% of the Earth’s surface is exposed to seasonal cold temperatures less than 5.0oC. Ectotherms implement a variety of strategies to survive seasonal, or permanent, cold exposure. Some of the most common overwintering strategies are migration, hibernation, and freeze avoiding behavior. However, freeze tolerance is a minority choice among ectotherms. This strategy permits organisms to survive between 50.0 to 70.0% of their total body water volumes frozen primarily in extracellular spaces for up to several months at a time. Freeze tolerant organisms undergo minimal supercooling of their body fluids to ensure ice formation is slow and produce a wide variety of specific proteins to control the size of ice crystals forming in the body. Freezing gives rise to severe physiological stressors which must be mitigated in order to survive freezing and thawing. While freeze tolerance is not fully understood, a growing body of evidence highlights several core tenants of this complex physiological process. When a non-freeze tolerant organism freezes, osmotic stress caused by the removal of pure water to form ice crystals in the extracellular fluid causes cells to shrink. As ice crystals thaw, water is rapidly reintroduced into the extracellular fluid causing local hypotonicity. Consequently, cells experience a rapid influx of water molecules, inducing acute cell swelling which progresses and ultimately causes cell lysis which leads to irreparable damage to an organism’s tissues and organs. Many freeze-tolerant animals combat dehydration stress by the seasonal accumulation or rapid mobilization at ice-nucleation of colligative cryoprotectants that diffuse across cell membranes through specific integral transmembrane proteins in order to limit cellular volume changes. Cope’s gray treefrog, Dryophytes chrysocelis, is a treefrog capable of freezing 65% of its total body water content for extended periods of time during harsh winter months. This treefrog is unique because it is the only known freeze-tolerant anuran which mobilizes glycerol as a cryoprotectant. Glycerol diffuses moves across cell membranes through integral transmembrane protein channels called aquaglyceroporins. This thesis presents a comprehensive literature review which focuses primarily on proposed cellular mechanisms that mitigate dehydration stress caused by the formation of pure ice crystals, as well as anoxic and oxidative stresses caused by freezing-induced ischemia and subsequent blood reperfusion during thawing. This thesis also proposes further research to elucidate vital information about the mechanisms permitting D. chrysoscelis’ freeze tolerance. Finally, the biomedical application of human organ and tissue cryopreservation is discussed, and an argument is presented that glycerol may be a superior cryoprotectant to use in future cryopreservation studies

Applications of Ketogenic Diets in Patients with Headache: Clinical Recommendations

Headaches are among the most prevalent and disabling neurologic disorders and there are several unmet needs as current pharmacological options are inadequate in treating patients with chronic headache, and a growing interest focuses on nutritional approaches as non-pharmacological treatments. Among these, the largest body of evidence supports the use of the ketogenic diet (KD). Exactly 100 years ago, KD was first used to treat drug-resistant epilepsy, but subsequent applications of this diet also involved other neurological disorders. Evidence of KD effectiveness in migraine emerged in 1928, but in the last several year's different groups of researchers and clinicians began utilizing this therapeutic option to treat patients with drug-resistant migraine, cluster headache, and/or headache comorbid with metabolic syndrome. Here we describe the existing evidence supporting the potential benefits of KDs in the management of headaches, explore the potential mechanisms of action involved in the efficacy in-depth, and synthesize results of working meetings of an Italian panel of experts on this topic. The aim of the working group was to create a clinical recommendation on indications and optimal clinical practice to treat patients with headaches using KDs. The results we present here are designed to advance the knowledge and application of KDs in the treatment of headaches