Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

The University of Sydney

We examined the ability of pyridoxamine (PM), an inhibitor of formation of advanced glycation end products (AGEs) and lipoxidation end products (ALEs), to protect against diabetes-induced retinal vascular lesions. The effects of PM were compared with the antioxidants vitamin E (VE) and R-alpha-lipoic acid (LA) in streptozotocin-induced diabetic rats. Animals were given either PM (1 g/l drinking water), VE (2,000 IU/kg diet), or LA (0.05%/kg diet). After 29 weeks of diabetes, retinas were examined for pathogenic changes, alterations in extracellular matrix (ECM) gene expression, and accumulation of the immunoreactive AGE/ALE N( epsilon )-(carboxymethyl)lysine (CML). Acellular capillaries were increased more than threefold, accompanied by significant upregulation of laminin immunoreactivity in the retinal microvasculature. Diabetes also increased mRNA expression for fibronectin (2-fold), collagen IV (1.6-fold), and laminin beta chain (2.6-fold) in untreated diabetic rats compared with nondiabetic rats. PM treatment protected against capillary drop-out and limited laminin protein upregulation and ECM mRNA expression and the increase in CML in the retinal vasculature. VE and LA failed to protect against retinal capillary closure and had inconsistent effects on diabetes-related upregulation of ECM mRNAs. These results indicate that the AGE/ALE inhibitor PM protected against a range of pathological changes in the diabetic retina and may be useful for treating diabetic retinopathy.

The AGE inhibitor pyridoxamine inhibits development of retinopathy in experimental diabetes.

Chemical modification of proteins by advanced glycation and lipoxidation end products is implicated in the pathogenesis of macrovascular disease in aging and diabetes. To identify biomarkers of the lipoxidative modification of protein, we studied the oxidation of phospholipids in the presence of the model protein RNase A and compared protein-bound products formed in these reactions with those formed during oxidation of plasma proteins. Metal-catalyzed oxidation of 1-palmitoyl-2-arachidonoyl-phosphatidylcholine or 1-palmitoyl-2-linoleoyl-phosphatidylcholine in the presence of RNase led to the loss of amino groups in RNase and the incorporation of phosphate, hexanoate, pentanedioate, nonanedioate, and palmitate into protein. Protein-bound palmitate and phosphate correlated strongly with one another, and protein-bound pentanedioate and nonanedioate, derived from arachidonate and linoleate, respectively, accounted for approximately 20% of the cross-linking of lipid phosphorus to protein. Similar results were obtained on oxidation of total plasma or isolated LDL. We conclude that alkanedioic acids are quantitatively important linkers of oxidized phospholipids to proteins and that measurement of protein-bound phosphate and long-chain fatty acids may be useful for assessing long-term lipid peroxidative damage to proteins in vivo. Analyses of plasma proteins from control and diabetic patients indicated significant increases in lipoxidative modification of protein in diabetic compared with control subjects.

Chemical modification of proteins during peroxidation of phospholipids.

Plasma from two diabetic rat models and human diabetic patients was analyzed to investigate the hypothesis that enhanced oxidative stress in diabetes promotes lipid-derived protein modification. We evaluated the nonenzymatic modification of plasma protein by oxidized phospholipids, including measurement of protein-bound pentanedioate, nonanedioate, and hexanoate, all derived from oxidation of phospholipid polyunsaturated fatty acids. Generally pentanedioate was higher in diabetic compared with nondiabetic control groups, and nonanedioate was also higher in the diabetic rat models. We conclude that diabetes is associated with higher levels of phospholipid-derived protein modification in both animal models and human diabetes. Their role in the development of diabetes vascular complications warrants further research.

Lipid-derived modifications of plasma proteins in experimental and human diabetes.

Circulating low molecular weight (<10 kDa) fluorophores (LMW-F) measured by non-specific fluorescence spectroscopy may detect small advanced glycation end-products (AGEs) not recognized by other assays. This longitudinal study assessed correlates of LMW-F and predictive power of LMW-F levels for vascular health in Type 1 diabetes (T1DM) patients.

Longitudinal analysis of low-molecular weight fluorophores in type 1 diabetes mellitus.

To determine if levels of coated-platelets, which are potentially pro-thrombotic, are increased in end-stage renal disease patients on haemodialysis, a condition associated with high cardiovascular disease risk.

Increased coated-platelet levels in chronic haemodialysis patients.

Amyloid fibrils and amyloid-like structures are implicated in atherosclerosis via macrophage activation and inflammation. A common property of amyloid-like structures is their ability to induce thioflavin T (ThT) fluorescence. We measured ThT fluorescence in serum and related these levels to traditional cardiovascular risk factors and non-invasive measures of vascular dysfunction (elasticity). In addition, chemically modified serum components that contribute to serum ThT fluorescence were explored and identified.

Thioflavin T fluorescence in human serum: correlations with vascular health and cardiovascular risk factors.

Kallistatin, a serpin widely produced throughout the body, has vasodilatory, anti-angiogenic, anti-oxidant, and anti-inflammatory effects. Effects of diabetes and its vascular complications on serum kallistatin levels are unknown.

Increased serum kallistatin levels in type 1 diabetes patients with vascular complications.

An assay of plasma 1,5-anhydroglucitol was evaluated. Assay CVs, effects of four plasma freeze-thaw cycles, glucose up to 80 mmol/L and triglycerides up to 20 mmol/L were acceptable. 1,5-anhydroglucitol levels were significantly lower in diabetic vs. non-diabetic subjects and correlated inversely with renal function, but not with HbA1c.

Plasma 1,5 anhydroglucitol levels, a measure of short-term glycaemia: assay assessment and lower levels in diabetic vs. non-diabetic subjects.

Post-translational modification of proteins in diabetes, including formation of advanced glycation end products (AGEs) are believed to contribute to vascular dysfunction and disease. Impaired function of the endothelium is an early indicator of vascular dysfunction in diabetes and as many endothelial cell processes are dependent upon intracellular [Ca(2+)] and Ca(2+) signaling, the aim of this study was to examine the acute effects of AGEs on Ca(2+) signaling in bovine aortic endothelial cells (BAEC). Ca(2+) signaling was studied using the fluorescent indicator dye Fura-2-AM. AGEs were generated by incubating bovine serum albumin with 0-250 mM glucose or glucose-6-phosphate for 0-120 days at 37°C. Under all conditions, the main AGE species generated was carboxymethyl lysine (CML) as assayed using both gas-liquid chromatograph-mass spectroscopy and high-performance liquid chromatography. In Ca(2+)-replete solution, exposure of BAEC to AGEs for 5 min caused an elevation in basal [Ca(2+)] and attenuated the increase in intracellular [Ca(2+)] caused by ATP (100 μM). In the absence of extracellular Ca(2+), exposure of BAEC to AGEs for 5 min caused an elevation in basal [Ca(2+)] and attenuated subsequent intracellular Ca(2+) release caused by ATP, thapsigargin (0.1 μM), and ionomycin (3 μM), but AGEs did not affect extracellular Ca(2+) entry induced by the re-addition of Ca(2+) to the bathing solution in the presence of any of these agents. The anti-oxidant α-lipoic acid (2 μM) and NAD(P)H oxidase inhibitors apocynin (500 μM) and diphenyleneiodonium (1 μM) abolished these effects of AGEs on BAECs, as did the IP3 receptor antagonist xestospongin C (1 μM). In summary, AGEs caused an acute depletion of Ca(2+) from the intracellular store in BAECs, such that the Ca(2+) signal stimulated by the subsequent application other agents acting upon this store is reduced. The mechanism may involve generation of reactive oxygen species from NAD(P)H oxidase and possible activation of the IP3 receptor.

Advanced glycation end products acutely impair ca(2+) signaling in bovine aortic endothelial cells.

Associations of semicarbazide-sensitive amine oxidase (SSAO) activity with renal and vascular function, oxidative stress, glycaemia and diabetes complications were determined.

Plasma semicarbazide-sensitive amine oxidase activity in type 1 diabetes is related to vascular and renal function but not to glycaemia.

Circulating fibroblast growth factor 21 (FGF21) levels are often elevated in obesity, dyslipidaemia, insulin resistance and type 2 diabetes. This study investigated the relationship of plasma FGF21 levels with cardiovascular events in patients with type 2 diabetes.

The relationship of fibroblast growth factor 21 with cardiovascular outcome events in the Fenofibrate Intervention and Event Lowering in Diabetes study.

This cohort study evaluates whether the association between low-grade inflammation and incident depressive symptoms is independent of abdominal obesity and metabolic disturbances.

The longitudinal association between inflammation and incident depressive symptoms in men: the effects of hs-CRP are independent of abdominal obesity and metabolic disturbances.

Andrzej Januszewski

Biomarkers Laboratory,
NHMRC Clinical Trials Centre,
Faculty of Medicine,
Biomarkers Laboratory, NHMRC Clinical Trials Centre, Faculty of Medicine

The University of Sydney

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Andrzej Januszewski

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The University of Sydney

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Biomarkers Laboratory,
NHMRC Clinical Trials Centre,
Faculty of Medicine,
Biomarkers Laboratory, NHMRC Clinical Trials Centre, Faculty of Medicine