Michael A. Ihnat

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.

Novel STAT3 Phosphorylation Inhibitors Exhibit Potent Growth-Suppressive Activity in Pancreatic and Breast Cancer Cells

The constitutive activation of signal transducer and activator of transcription 3 (STAT3) is frequently detected in most types of human cancer where it plays important roles in survival, drug resistance, angiogenesis, and other functions. Targeting constitutive STAT3 signaling is thus an attractive therapeutic approach for these cancers. We have recently developed novel small-molecule STAT3 inhibitors, known as FLLL31 and FLLL32, which are derived from curcumin (the primary bioactive compound of turmeric). These compounds are designed to bind selectively to Janus kinase 2 and the STAT3 Src homology-2 domain, which serve crucial roles in STAT3 dimerization and signal transduction. Here we show that FLLL31 and FLLL32 are effective inhibitors of STAT3 phosphorylation, DNA-binding activity, and transactivation in vitro, leading to the impediment of multiple oncogenic processes and the induction of apoptosis in pancreatic and breast cancer cell lines. FLLL31 and FLLL32 also inhibit colony formation in soft agar and cell invasion and exhibit synergy with the anticancer drug doxorubicin against breast cancer cells. In addition, we show that FLLL32 can inhibit the induction of STAT3 phosphorylation by IFNalpha and interleukin-6 in breast cancer cells. We also show that administration of FLLL32 can inhibit tumor growth and vascularity in chicken embryo xenografts as well as substantially reduce tumor volumes in mouse xenografts. Our findings highlight the potential of these new compounds and their efficacy in targeting pancreatic and breast cancers that exhibit constitutive STAT3 signaling.

'Glycaemic variability': a new therapeutic challenge in diabetes and the critical care setting.

Diabet. Med. 27, 862–867 (2010)

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.

Intraretinal leakage and oxidation of LDL in diabetic retinopathy.

PURPOSE The pathogenesis of diabetic retinopathy (DR) is not fully understood. Clinical studies suggest that dyslipidemia is associated with the initiation and progression of DR. However, no direct evidence supports this theory. METHODS Immunostaining of apolipoprotein B100 (ApoB100, a marker of low-density lipoprotein [LDL]), macrophages, and oxidized LDL was performed in retinal sections from four different groups of subjects: nondiabetic, type 2 diabetic without clinical retinopathy, diabetic with moderate nonproliferative diabetic retinopathy (NPDR), and diabetic with proliferative diabetic retinopathy (PDR). Apoptosis was characterized using the TUNEL assay. In addition, in cell culture studies using in vitro-modified LDL, the induction of apoptosis by heavily oxidized-glycated LDL (HOG-LDL) in human retinal capillary pericytes (HRCPs) was assessed. RESULTS Intraretinal immunofluorescence of ApoB100 increased with the severity of DR. Macrophages were prominent only in sections from diabetic patients with PDR. Merged images revealed that ApoB100 partially colocalized with macrophages. Intraretinal oxidized LDL was absent in nondiabetic subjects but present in all three diabetic groups, increasing with the severity of DR. TUNEL-positive cells were present in retinas from diabetic subjects but absent in those from nondiabetic subjects. In cell culture, HOG-LDL induced the activation of caspase, mitochondrial dysfunction, and apoptosis in HRCPs. CONCLUSIONS These findings suggest a potentially important role for extravasated, modified LDL in promoting DR by promoting apoptotic pericyte loss.

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.

Single Agents with Designed Combination Chemotherapy Potential: Synthesis and Evaluation of Substituted Pyrimido[4,5-b]indoles as Receptor Tyrosine Kinase and Thymidylate Synthase Inhibitors and as Antitumor Agents

Combinations of antiangiogenic agents (AAs) with cytotoxic agents have shown significant promise in cancer treatment, and several such clinical trials are currently underway. We have designed, synthesized, and evaluated two compounds that each inhibit vascular endothelial growth factor receptor-2 (VEGFR-2) and platelet-derived growth factor receptor-beta (PDGFR-beta) for antiangiogenic effects and also inhibit human thymidylate synthase (hTS) for cytotoxic effects in single agents. The synthesis of these compounds involved the nucleophilic displacement of the common intermediate 5-chloro-9H-pyrimido[4,5-b]indole-2,4-diamine with appropriate benzenethiols. The inhibitory potency of both these single agents against VEGFR-2, PDGFR-beta, and hTS is better than or close to standards. In a COLO-205 xenograft mouse model, one of the analogs significantly decreased tumor growth (tumor growth inhibition (TGI) = 76% at 35 mg/kg), liver metastases, and tumor blood vessels compared with a standard drug and with control and thus demonstrated potent tumor growth inhibition, inhibition of metastasis, and antiangiogenic effects in vivo. These compounds afford combination chemotherapeutic potential in single agents.

Enhanced angiogenesis of modified porcine small intestinal submucosa with hyaluronic acid-poly(lactide-co-glycolide) nanoparticles: from fabrication to preclinical validation.

Hyaluronic acid-poly(de-co-glycolide) nanoparticles (HA-PLGA NPs) were synthesized to stabilize the porous structure of porcine small intestinal submucosa (SIS), to improve surface biocompatibility and to enhance performance in tissue regeneration. HA-PLGA NPs were characterized for size, zeta potential, surface morphology, and HA loading. Human microvascular endothelial cells responded to HA-PLGA NPs and HA-PLGA modified SIS (HA-PLGA-SIS) with elevated cell proliferation. HA-PLGA-SIS significantly enhanced neo-vascularization in an in ovo chorioallantoic membrane angiogenesis model. The angiogenic capability of the newly fabricated HA-PLGA-SIS was tested in a canine bladder augmentation model. Urinary bladder augmentation was performed in beagle dogs following hemi-cystectomy using HA-PLGA-SIS. The regenerated bladder was harvested at 10 weeks post augmentation and vascularization was evaluated using CD31 immunohistochemical staining. Bladder regenerated with HA-PLGA-SIS had significantly higher vascular ingrowth compared to unmodified SIS. This study shows that HA-PLGA NPs may represent a new approach for modifying naturally derived SIS biomaterials in regenerative medicine.