Faculty Bibliography

Background: RAGE and its ligands have been implicated in the pathogenesis of I/R injury through pathways of inflammation and apoptosis. We hypothesized that RAGE-directed quantitative imaging of myocardial uptake of 99mTc-anti-RAGE F(ab')2 in a mouse model of I/R can detect RAGE expression in the myocardium and show quantitative differences between early (18-20 h) and later times (48 h) after reperfusion. Methods: Twenty-four wild-type (WT) mice underwent left anterior descending coronary artery (LAD) occlusion for 30 min followed by reperfusion for 18-20 h (n = 8) or 48 h (n = 12) and 3 WT mice underwent sham operation without coronary intervention. Twenty-seven mice (including shams) were injected with 19.98 +/- 1.78 MBq 99mTc anti-RAGE F(ab')2 and 5 h later, mice were injected with 6.14 +/- 2.0 MBq 201Tl and immediately underwent dual isotope SPECT/CT imaging. Four WT mice were injected with control nonspecific F(ab')2 and similarly imaged at 18-20 h. At the completion of imaging, hearts were removed, radioactivity counted, and sectioned for histology. Results: The uptake of 99mTc-anti-RAGE F(ab')2 in the ischemic zone from the scans as mean percentage injected dose (%ID) was significantly greater at 18-20 h (5.7 +/- 2.1 x 10-3%) compared to 48 h (1.4 +/- 1.1 x 10-3%; p < 0.001) following reperfusion. Disease and antibody controls showed no focal uptake in the infarct. Gamma well counting of the myocardium from experiments performed at 18-20 h and 48 h post reperfusion supported the quantitative scan data. Immunohistochemical staining of the myocardium showed greater caspase-3 and RAGE staining at 18-20 h vs. 48 h (p = 0.04 and p = 0.01, respectively). On dual immunofluorescence, RAGE colocalized mainly with injured cardiomyocytes undergoing apoptosis. Conclusion: RAGE expression in reperfusion injury can be imaged in-vivo using a novel 99mTc-anti-RAGE F(ab')2. This imaging approach may be useful in evaluating contribution of RAGE to myocardial ischemic injury in diseases with increased expression such as diabetes

BACKGROUND: The receptor for advanced glycation end-products (RAGE) is implicated in pancreatic tumorigenesis. Activating Kras mutations and p16 inactivation are genetic abnormalities most commonly detected as pancreatic ductal epithelium progresses from intraepithelial neoplasia (PanIN) to adenocarcinoma (PDAC). OBJECTIVE: The aim of this study was to evaluate the effect of RAGE (or AGER) deletion on the development of PanIN and PDAC in conditional Kras ( G12D ) mice. MATERIALS AND METHODS: Pdx1-Cre; LSL-Kras ( G12D/+) mice were crossed with RAGE (-/-) mice to generate Pdx1-Cre; LSL-Kras ( G12D/+) ; RAGE (-/-) mice. Pdx1-Cre; LSL-Kras ( G12D/+); p16 ( Ink4a-/-) mice were crossed with RAGE (-/-) mice to generate Pdx1-Cre; LSL-Kras ( G12D/+); p16 ( Ink4a-/-); RAGE (-/-) mice. Pancreatic ducts were scored and compared to the relevant RAGE (+/+) controls. RESULTS: At 16 weeks of age, Pdx1-Cre; LSL-Kras ( G12D/+); RAGE (-/-) mice had significantly fewer high-grade PanIN lesions than Pdx1-Cre; LSL-Kras ( G12D/+); RAGE (+/+) controls. At 12 weeks of age, none of the Pdx1-Cre; LSL-Kras ( G12D/+); p16 ( Ink4a-/-); RAGE (-/-) mice had PDAC compared to a 45.5% incidence of PDAC in Pdx1-Cre; LSL-Kras ( G12D/+); p16 ( Ink4a-/-); RAGE (+/+) controls. Finally, Pdx1-Cre; LSL-Kras ( G12D/+); p16 ( Ink4a-/-); RAGE (-/-) mice also displayed markedly longer median survival. CONCLUSION: Loss of RAGE function inhibited the development of PanIN and progression to PDAC and significantly prolonged survival in these mouse models. Further work is needed to target the ligand-RAGE axis for possible early intervention and prophylaxis in patients at risk for developing pancreatic cancer

OBJECTIVES: The aim of this study was to image expression of receptor for advanced glycation end products (RAGE) in a mouse model of myocardial reperfusion injury. BACKGROUND: RAGE and its ligands are implicated in the pathogenesis of ischemia/reperfusion injury and infarction. We hypothesized that RAGE-directed quantitative imaging of myocardial uptake of technetium-99m ((99m)Tc)-anti-RAGE F(ab')(2) in a mouse model of myocardial ischemic injury can detect RAGE expression and show quantitative differences between early (18 to 20 h) and later times (48 h) after reperfusion. METHODS: Twenty-five wild-type (WT) mice underwent left anterior descending coronary artery occlusion for 30 min. Mice were injected with 19.98 +/- 1.78 MBq of (99m)Tc anti-RAGE F(ab')(2) at 2 time points after reperfusion (at 18 to 20 h [n = 8] and at 48 h [n = 12]) and 5 h later with 6.14 +/- 2.0 MBq of thallium-201 ((201)Tl). Five WT mice were injected with nonspecific F(ab')(2) and (201)Tl 18 to 20 h after reperfusion. Six WT mice underwent sham operation without coronary intervention. After injection with (201)Tl, all mice immediately underwent dual isotope single-photon emission computed tomography/computed tomography. At completion of imaging, hearts were counted and sectioned. RESULTS: The uptake of (99m)Tc-anti-RAGE F(ab')(2) in the ischemic zone from the scans as mean percentage injected dose was significantly greater at 18 to 20 h (5.7 +/- 2.1 x 10(-3)%) as compared with at 48 h (1.4 +/- 1.1 x 10(-3)%; p < 0.001) after reperfusion. Disease and antibody controls showed no focal uptake in the infarct. Gamma well counting of the myocardium supported the quantitative scan data. By immunohistochemical staining there was greater caspase-3 and RAGE staining at 18 to 20 h versus at 48 h (p = 0.04 and p = 0.01, respectively). On dual immunofluorescence, RAGE colocalized mainly with injured cardiomyocytes undergoing apoptosis. CONCLUSIONS: RAGE expression in myocardial ischemic injury can be imaged in vivo using a novel (99m)Tc-anti-RAGE F(ab')(2). RAGE plays a role in several cardiovascular diseases and is a potential target for clinical imaging.

RAGE, the multiligand receptor of the immunoglobulin superfamily of cell surface molecules, is implicated in innate and adaptive immunity. Complement component C1q serves roles in complement activation and antibody-independent opsonization. Using soluble forms of RAGE (sRAGE) and RAGE-expressing cells, we determined that RAGE is a native C1q globular domain receptor. Direct C1q-sRAGE interaction was demonstrated with surface plasmon resonance (SPR), with minimum K(d) 5.6 muM, and stronger binding affinity seen in ELISA-like experiments involving multivalent binding. Pull-down experiments suggested formation of a receptor complex of RAGE and Mac-1 to further enhance affinity for C1q. C1q induced U937 cell adhesion and phagocytosis was inhibited by antibodies to RAGE or Mac-1. These data link C1q and RAGE to the recruitment of leukocytes and phagocytosis of C1q-coated material.

The Receptor for Advanced Glycation Endproducts (RAGE) is a scavenger ligand that binds glycated endproducts as well as molecules released during cell death such as S100b and HMGB1. RAGE is expressed on antigen presenting cells where it may participate in activation of innate immune responses but its role in adaptive human immune responses has not been described. We have found that RAGE is expressed intracellularly in human T cells following TCR activation but constitutively on T cells from patients with diabetes. The levels of RAGE on T cells from patients with diabetes are not related to the level of glucose control. It co-localizes to the endosomes. Its expression increases in activated T cells from healthy control subjects but bystander cells also express RAGE after stimulation of the antigen specific T cells. RAGE ligands enhance RAGE expression. In patients with T1D, the level of RAGE expression decreases with T cell activation. RAGE+ T cells express higher levels of IL-17A, CD107a, and IL-5 than RAGE- cells from the same individual with T1D. Our studies have identified the expression of RAGE on adaptive immune cells and a role for this receptor and its ligands in modulating human immune responses.

The receptor for advanced glycation endproducts (RAGE) was first described as a signal transduction receptor for advanced glycation endproducts (AGEs), the products of nonenzymatic glycation and oxidation of proteins and lipids that accumulate in diabetes and in inflammatory foci. The discovery that RAGE was a receptor for inflammatory S100/calgranulins and high mobility group box 1 (HMGB1) set the stage for linking RAGE to both the consequences and causes of types 1 and 2 diabetes. Recent discoveries regarding the structure of RAGE as well as novel intracellular binding partner interactions advance our understanding of the mechanisms by which RAGE evokes pathological consequences and underscore strategies by which antagonism of RAGE in the clinic may be realized. Finally, recent data tracking RAGE in the clinic suggest that levels of soluble RAGEs and polymorphisms in the gene encoding RAGE may hold promise for the identification of patients who are vulnerable to the complications of diabetes and/or are receptive to therapeutic interventions designed to prevent and reverse the damage inflicted by chronic hyperglycemia, irrespective of its etiology

Diabetes mellitus types 1 and 2 (DM1 and DM2) and/or hypertension (HTN) can contribute to cognitive decline, cerebral atrophy and white matter abnormalities in humans. Adult rat models of streptozotocin-induced DM1 and genetic strains of DM2 and HTN were used to investigate relative contributions of DM and HTN for alterations in cerebral structure and function as well as insulin receptor biology using cognitive testing, magnetic resonance imaging (MRI), and histological and molecular methods. The effects of DM1 or DM2 were generally similar. DM was associated with earlier onset of cognitive impairment than with HTN alone. DM was independently correlated with brain atrophy, whereas HTN had minimal effects on brain volume. The combination of DM and HTN led to identifiable mild hippocampal neuronal loss while either DM or HTN led to synaptic loss. Only DM led to downregulation of the insulin receptor pathways' activation. In contrast, only HTN was associated with vascular luminal reduction and restricted cerebral perfusion on MRI. The impacts of DM and HTN in the brain differ, while their separate contributions can lead to some additive adverse effects within rodent brain grey matter.

Aging men and women display both increased incidence of cardiovascular disease and complications of myocardial infarction and heart failure. We hypothesized that altered glucose metabolism, in particular, flux of glucose via the polyol pathway (PP) may be responsible, in part, for the enhanced vulnerability of aging myocardium to ischemic injury, even in the absence of superimposed disease processes linked to PP flux, such as diabetes. To test our hypothesis, we determined the expression and products of PP enzymes aldose reductase (AR) and sorbitol dehydrogenase (SDH) in hearts from Fischer 344 aged (26 months) and young (4 months) rats subjected to global ischemia followed by reperfusion in the presence or absence of blockers of PP and the measures of ischemic injury and functional recovery were determined. Expression and activities of AR and SDH were significantly higher in aged vs. young hearts, and induction of ischemia further increased AR and SDH activity in the aged hearts. Myocardial ischemic injury was significantly greater in aged vs. young hearts, and blockade of AR reduced ischemic injury and improved cardiac functional recovery on reperfusion in aged hearts. These data indicate that innate increases in activity of the PP enzymes augment myocardial vulnerability to I/R injury in aging, and that blockers of PP protect the vulnerable aging hearts

OBJECTIVE: There are several pathways that mediate the aberrant metabolism of glucose and that might induce greater vascular damage in the setting of diabetes. The polyol pathway mediated by aldose reductase (AR) has been postulated to be one such pathway. However, it has been reported that AR reduces toxic lipid aldehydes and, under some circumstances, might be antiatherogenic. METHODS AND RESULTS: Atherosclerosis development was quantified in 2 lines of transgenic mice expressing human AR (hAR) crossed on the apolipoprotein E knockout background. The transgenes were used to increase the normally low levels of this enzyme in wild-type mice. Both generalized hAR overexpression and hAR expression via the Tie 2 promoter increased lesion size in streptozotocin diabetic mice. In addition, pharmacological inhibition of AR reduced lesion size. CONCLUSIONS: Although in some settings AR expression might reduce levels of toxic aldehydes, transgenic expression of this enzyme within the artery wall leads to greater atherosclerosis