Jessica E. Thorpe

Oscillating glucose is more deleterious to endothelial function and oxidative stress than mean glucose in normal and type 2 diabetic patients

OBJECTIVE—To explore the possibility that oscillating glucose may outweigh A1C levels in determining the risk for cardiovascular diabetes complications. RESEARCH DESIGN AND METHODS—A euinsulinemic hyperglycemic clamp at 5, 10, and 15 mmol/l glucose was given in increasing steps as a single “spike” or oscillating between basal and high levels over 24 h in normal subjects and type 2 diabetic patients. Flow-mediated dilatation, a marker of endothelial function, and plasma 3-nitrotyrosine and 24-h urinary excretion rates of free 8-iso PGF2α, two markers of oxidative stress, were measured over 48 h postclamp. RESULTS—Glucose at two different levels (10 and 15 mmol/l) resulted in a concentration-dependent fasting blood glucose–independent induction of both endothelial dysfunction and oxidative stress in both normal and type 2 diabetic patients. Oscillating glucose between 5 and 15 mmol/l every 6 h for 24 h resulted in further significant increases in endothelial dysfunction and oxidative stress compared with either continuous 10 or 15 mmol/l glucose. CONCLUSIONS—These data suggest that oscillating glucose can have more deleterious effects than constant high glucose on endothelial function and oxidative stress, two key players in favoring cardiovascular complications in diabetes. Concomitant vitamin C infusion can reverse this impairment.

Hypothesis: the ‘metabolic memory’, the new challenge of diabetes

Large randomized studies have established that early intensive glycaemic control reduces the risk of diabetic complications, both micro‐ and macrovascular. However, epidemiological and prospective data support a long‐term influence of early metabolic control on clinical outcomes. This phenomenon has recently been defined as ‘metabolic memory.’ Potential mechanisms for propagating this ‘memory’ are the non‐enzymatic glycation of cellular proteins and lipids, and an excess of cellular reactive oxygen and nitrogen species, in particular originated at the level of glycated‐mitochondrial proteins, perhaps acting in concert with one another to maintain stress signalling. Furthermore, the emergence of this ‘metabolic memory’ suggests the need for very early aggressive treatment aiming to ‘normalize’ glycaemic control and the addition of agents which reduce cellular reactive species and glycation in order to minimize long‐term diabetic complications.

Chronic Exposure to Arsenic in the Drinking Water Alters the Expression of Immune Response Genes in Mouse Lung

Background Chronic exposure to drinking water arsenic is a significant worldwide environmental health concern. Exposure to As is associated with an increased risk of lung disease, which may make it a unique toxicant, because lung toxicity is usually associated with inhalation rather than ingestion. Objectives The goal of this study was to examine mRNA and protein expression changes in the lungs of mice exposed chronically to environmentally relevant concentrations of As in the food or drinking water, specifically examining the hypothesis that As may preferentially affect gene and protein expression related to immune function as part of its mechanism of toxicant action. Methods C57BL/6J mice fed a casein-based AIN-76A defined diet were exposed to 10 or 100 ppb As in drinking water or food for 5–6 weeks. Results Whole genome transcriptome profiling of animal lungs revealed significant alterations in the expression of many genes with functions in cell adhesion and migration, channels, receptors, differentiation and proliferation, and, most strikingly, aspects of the innate immune response. Confirmation of mRNA and protein expression changes in key genes of this response revealed that genes for interleukin 1β, interleukin 1 receptor, a number of toll-like receptors, and several cytokines and cytokine receptors were significantly altered in the lungs of As-exposed mice. Conclusions These findings indicate that chronic low-dose As exposure at the current U.S. drinking-water standard can elicit effects on the regulation of innate immunity, which may contribute to altered disease risk, particularly in lung.

Long-Term Glycemic Control Influences the Long-Lasting Effect of Hyperglycemia on Endothelial Function in Type 1 Diabetes

OBJECTIVE The objective of the study was to investigate the effect of different periods of hyperglycemia on the reversal of endothelial dysfunction by glucose normalization and antioxidant therapy. RESEARCH DESIGN AND METHODS Ten healthy subjects and three subgroups of 10 type 1 diabetic subjects were enrolled as follows: 1) patients within 1 month of diagnosis; 2) patients between 4.5 and 5.2 yr from diagnosis and with glycosylated hemoglobin levels 7% or greater since diagnosis; 3) patients between 4.8 and 5.4 yr from diagnosis and with glycosylated hemoglobin levels greater than 7% since diagnosis. Each patient participated in three experiments: 1) 24-h insulin treatment, achieving a near normalization of glycemia, together with the addition of the antioxidant vitamin C during the last 12 h; 2) 24-h vitamin C treatment with insulin treatment for the last 12 h; and 3) treatment with both vitamin C and insulin for 24 h. RESULTS Endothelial function, as measured by flow-mediated vasodilation of the brachial artery and levels of nitrotyrosine, an oxidative stress marker, were normalized by each treatment in subgroups 1 and 2. In the third subgroup, neither glucose normalization nor vitamin C treatment alone was able to normalize endothelial dysfunction or oxidative stress. Combining insulin and vitamin C, however, normalized endothelial dysfunction and nitrotyrosine. CONCLUSIONS This study suggests that long-lasting hyperglycemia in type 1 diabetic patients induces long-term alterations in endothelial cells, which may contribute to endothelial dysfunction and is interrupted only by both glucose and oxidative stress normalization.

Laboratory diet profoundly alters gene expression and confounds genomic analysis in mouse liver and lung.

Nutritional studies in laboratory animals have long shown that various dietary components can contribute to altered gene expression and metabolism, but diet alone has not been considered in whole animal genomic studies. In this study, global gene expression changes in mice fed either a non-purified chow or a purified diet were investigated and background metal levels in the two diets were measured by ICP-MS. C57BL/6J mice were raised for 5 weeks on either the cereal-based, non-purified LRD-5001 diet or the purified, casein-based AIN-76A diet, as part of a larger study examining the effects of low dose arsenic (As) in the diet or drinking water. Affymetrix Mouse Whole Genome 430 2.0 microarrays were used to assess gene expression changes in the liver and lung. Microarray analysis revealed that animals fed the LRD-5001 diet displayed a significantly higher hepatic expression of Phase I and II metabolism genes as well as other metabolic genes. The LRD-5001 diet masked the As-induced gene expression changes that were clearly seen in the animals fed the AIN-76A diet when each dietary group was exposed to 100 ppb As in drinking water. Trace metal analysis revealed that the LRD-5001 diet contained a mixture of inorganic and organic As at a total concentration of 390 ppb, while the AIN-76A diet contained approximately 20 ppb. These findings indicate that the use of non-purified diets may profoundly alter observable patterns of change induced by arsenic and, likely, by other experimental treatments, particularly, altering gene and protein expression.

Synthesis and Biological Activity of N4-phenylsubstituted-6-(2,4-dichloro phenylmethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines as Vascular Endothelial Growth Factor Receptor-2 Inhibitors and Antiangiogenic and Antitumor Agents

A series of eight N(4)-phenylsubstituted-6-(2,4-dichlorophenylmethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines 8-15 were synthesized as vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitors with varied substitutions in the phenyl ring of the 4-anilino moiety. In addition, five N(4)-phenylsubstituted-6-phenylmethylsubstituted-7H-pyrrolo[2,3-d]pyrimidin-4-amines 16-20 were synthesized to evaluate the importance of the 2-NH(2) moiety for multiple receptor tyrosine kinase (RTK) inhibition. Cyclocondensation of alpha-halomethylbenzylketones with 2,6-diamino-4-hydroxypyrimidine afforded 2-amino-6-(2,4-dichlorophenylmethyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one, 23 and reaction of alpha-bromomethylbenzylketones with ethylamidinoacetate followed by cyclocondensation with formamide afforded the 6-phenylmethylsubstituted-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-ones, 40-42, respectively. Chlorination of the 4-position and displacement with appropriate anilines afforded the target compounds 8-20. Compounds 8, 10 and 14 were potent VEGFR-2 inhibitors and were 100-fold, 40-fold and 8-fold more potent than the standard semaxanib, respectively. Previously synthesized multiple RTK inhibitor, 5 and the VEGFR-2 inhibitor 8 from this study, were chosen for further evaluation in a mouse orthotopic model of melanoma and showed significant inhibition of tumor growth, angiogenesis and metastasis.

The design and discovery of water soluble 4-substituted-2,6-dimethylfuro[2, 3-d]pyrimidines as multitargeted receptor tyrosine kinase inhibitors and microtubule targeting antitumor agents

The design, synthesis and biological evaluations of fourteen 4-substituted 2,6-dimethylfuro[2,3-d]pyrimidines are reported. Four compounds (11-13, 15) inhibit vascular endothelial growth factor receptor-2 (VEGFR-2), platelet-derived growth factor receptor β (PDGFR-β), and target tubulin leading to cytotoxicity. Compound 11 has nanomolar potency, comparable to sunitinib and semaxinib, against tumor cell lines overexpressing VEGFR-2 and PDGFR-β. Further, 11 binds at the colchicine site on tubulin, depolymerizes cellular microtubules and inhibits purified tubulin assembly and overcomes both βIII-tubulin and P-glycoprotein-mediated drug resistance, and initiates mitotic arrest leading to apoptosis. In vivo, its HCl salt, 21, reduced tumor size and vascularity in xenograft and allograft murine models and was superior to docetaxel and sunitinib, without overt toxicity. Thus 21 affords potential combination chemotherapy in a single agent.

Design, synthesis and biological evaluation of substituted pyrrolo[2,3-d]pyrimidines as multiple receptor tyrosine kinase inhibitors and antiangiogenic agents.

Direct and indirect involvement of receptor tyrosine kinases (RTKs) in tumor growth and metastasis makes them ideal targets for anticancer therapy. A paradigm shift from inhibition of single RTK to inhibition of multiple RTKs has been recently demonstrated. We designed and synthesized eight N(4)-phenylsubstituted-6-(2-phenylethylsubstituted)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines as homologated series of our previously published RTK inhibitors. We reasoned that increased flexibility of the side chain, which determines potency and selectivity, would improve the spectrum of RTK inhibition. These compounds were synthesized using a bis-electrophilic cyclization to afford substituted pyrrolo[2,3-d]pyrimidines followed by chlorination and substitution at the 4-position with various anilines. Five additional compounds of this series were previously reported by Gangjee et al.(1) with activities against IGFR only. Their synthesis, characterization and biological activities against a variety of other RTKs are reported in this study for the first time. The biological evaluation, in whole cell assays, showed several analogs had remarkable inhibitory activity against epithelial growth factor receptor (EGFR), vascular endothelial growth factor receptor-1 (VEGFR-1), platelet-derived growth factor receptor-beta (PDGFR-beta), the growth of A431 cells in culture, and in the chicken embryo chorioallantoic membrane (CAM) angiogenesis assay. The inhibitory data against the RTKs in this study demonstrate that variation of the 6-ethylaryl substituents as well as the N(4)-phenyl substituents of these analogs does indeed control both the potency and specificity of inhibitory activity against RTKs. In addition, homologation of the chain length of the 6-substituent from a methylene to an ethyl increases the spectrum of RTK inhibition. New multi-RTK inhibitors (8, 12) and potent inhibitors of angiogenesis (15, 19) were identified with the best compound, N(4)-(3-trifluromethylphenyl)-6-(2-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (15), with an IC(50) value of 30nM in the CAM angiogenesis inhibition assay.

Effect of acute hyperglycaemia, long‐term glycaemic control and insulin on endothelial dysfunction and inflammation in Type 1 diabetic patients with different characteristics

Diabet. Med. 27, 911–917 (2010)

Hypoxia triggers a HIF‐mediated differentiation of peripheral blood mononuclear cells into osteoclasts

BACKGROUND The source and mechanisms leading to osteoclast (OC) generation during tooth movement are not clearly understood. We hypothesized that during tooth movement, OC differentiate from peripheral blood mononuclear cells (PBMNC) downstream of the global hypoxia-inducible transcription factor hypoxia-inducible factor (HIF)-1α. OBJECTIVE The objective of this study was to demonstrate up-regulation of OC growth factors from osteoblasts (OB) and subsequent conversion of PBMNC into functional OC under hypoxic stress. MATERIAL AND METHODS Human primary PBMNC were cocultured with/without OB and subjected to either hypoxia (2.5% O2) or normoxia (21% O2) over 14 days. Levels of HIF, vascular endothelial growth factor (VEGF) and receptor activator for nuclear factor kappa-β ligand (RANKL) were measured. Conversion of PBMNC into OC was measured using resorption and TRAP assays. RESULTS Functional OC were only observed in response to hypoxia during coculture of PBMNC and OB and only after up-regulation of HIF, VEGF and RANKL in the hypoxic conditions. YC-1, a HIF inhibitor, reduced OC formation in response to hypoxia. CONCLUSION Hypoxia triggers the differentiation of PBMNC into functional OC in the presence of OB in a HIF-dependent manner as would occur during orthodontic loading of the periodontal ligament space.