Faculty Bibliography

Diaphanous 1 (DIAPH1), a member of the formin family, binds to the cytoplasmic domain of the receptor for advanced glycation end products (RAGE) and is required for RAGE signal transduction. Experiments employing genetic over-expression or deletion of Ager (the gene encoding RAGE) or its pharmacological antagonism, implicate RAGE in the pathogenesis of diabetes-associated nephropathy. We hypothesized that DIAPH1 contributes to pathological and functional derangements in the kidneys of diabetic mice. Here, we show that DIAPH1 is expressed in the human and murine diabetic kidney, at least in part in the tubulointerstitium and glomerular epithelial cells, or podocytes. To test the premise that DIAPH1 is linked to diabetes-associated derangements in the kidney, we rendered male mice globally devoid of Diaph1 or wild-type controls (C57BL/6 background), diabetic with streptozotocin. Control mice received equal volumes of citrate buffer. After six months of hyperglycemia, diabetic mice devoid of Diaph1 displayed significantly reduced mesangial sclerosis, podocyte effacement, glomerular basement thickening, and urine albumin/creatinine ratio, compared to diabetic mice expressing Diaph1. Analysis of whole kidney cortex revealed that deletion of Diaph1 in diabetic mice significantly reduced expression of genes linked to fibrosis and inflammation. In glomerular isolates, expression of two genes linked to podocyte stress, Gas1 and Cd36, was significantly attenuated in diabetic mice devoid of Diaph1 vs. controls, in parallel with significantly higher levels of Nes mRNA, a podocyte marker. Collectively, these data implicate DIAPH1 in the pathogenesis of diabetes-associated nephropathy and suggest that RAGE/DIAPH1 is a logical target for therapeutic intervention in this disorder.

PURPOSE/OBJECTIVE:The soluble receptor for advanced glycation end-products (sRAGE) and endogenous secretory RAGE (esRAGE) have been considered as biomarkers of several chronic diseases. However, the temporal reliability of their concentrations in the circulation is yet to be demonstrated. We evaluated whether a single measurement of serum sRAGE and esRAGE could serve as an estimate for usual serum levels in epidemiologic studies. METHODS:Serum sRAGE and esRAGE were measured using ELISAs in three yearly samples from 36 participants in the New York University Women's Health Study. The intraclass correlation coefficient (ICC) was used to evaluate temporal reliability. RESULTS:-transformed data. CONCLUSION/CONCLUSIONS:Our results indicate that a single measurement of serum sRAGE and esRAGE is a sufficiently reliable measure of their usual levels that can be used in epidemiologic studies.

Islet amyloid formation contributes to β-cell death and dysfunction in type-2 diabetes and to the failure of islet transplants. Amylin (Islet amyloid polypeptide, IAPP), a normally soluble 37 residue polypeptide hormone produced in the pancreatic β-cells, is responsible for amyloid formation in type-2 diabetes. The peptide is deficient in type-1 diabetes. Amylin normally plays an adaptive role in metabolism and the development of non-toxic, non-amyloidogenic, bioactive variants of human amylin are of interest for use as adjuncts to insulin therapy. Naturally occurring non-amyloidogenic variants are of interest for xenobiotic transplantation and because they can provide clues towards understanding the amyloidogenicity of human amylin. The sequence of amylin is well conserved among species, but sequence differences strongly correlate with in vitro amyloidogenicity and with islet amyloid formation in vivo. Bovine amylin differs from the human peptide at ten positions and is one of the most divergent among known amylin sequences. We show that bovine amylin oligomerizes, but is not toxic to cultured β-cells and is considerably less amyloidogenic than the human polypeptide and is only a low potency agonist at human amylin-responsive receptors. The bovine sequence contains several non-conservative substitutions relative to human amylin including His to Pro, Ser to Pro and Asn to Lys replacements. The effect of these substitutions is analyzed in the context of wild type human amylin; the results provide insight into their role in receptor activation, the mode of assembly of human amylin and into the design of soluble amylin analogs.

Cigarette smoking alters the oral microbiome; however, the effect of alternative tobacco products remains unclear. Middle Eastern tobacco products like dokha and shisha, are becoming globally widespread. We tested for the first time in a Middle Eastern population the hypothesis that different tobacco products impact the oral microbiome. The oral microbiome of 330 subjects from the United Arab Emirates Healthy Future Study was assessed by amplifying the bacterial 16S rRNA gene from mouthwash samples. Tobacco consumption was assessed using a structured questionnaire and further validated by urine cotinine levels. Oral microbiome overall structure and specific taxon abundances were compared, using PERMANOVA and DESeq analyses respectively. Our results show that overall microbial composition differs between smokers and nonsmokers (p = 0.0001). Use of cigarettes (p = 0.001) and dokha (p = 0.042) were associated with overall microbiome structure, while shisha use was not (p = 0.62). The abundance of multiple genera were significantly altered (enriched/depleted) in cigarette smokers; however, only Actinobacillus, Porphyromonas, Lautropia and Bifidobacterium abundances were significantly changed in dokha users whereas no genera were significantly altered in shisha smokers. For the first time, we show that smoking dokha is associated to oral microbiome dysbiosis, suggesting that it could have similar effects as smoking cigarettes on oral health.

BACKGROUND:The receptor for advanced glycation end products (RAGE) is linked to cellular stress and inflammation during Alzheimer's disease (AD). RAGE signals through Diaphanous-1 (DIAPH1); however, the expression of DIAPH1 in the healthy and AD human brain has yet to be methodically addressed. OBJECTIVE:To delineate the cell- and disease-state specific expression of DIAPH1 in the human medial temporal cortex during healthy aging and AD. METHODS:We used semi-quantitative immunohistochemistry in the human medial temporal cortex paired with widefield and confocal microscopy and automated analyses to determine colocalization and relative expression of DIAPH1 with key cell markers and molecules in the brains of subjects with AD versus age-matched controls. RESULTS:We report robust colocalization of DIAPH1 with myeloid cells and increased expression during AD, which strongly correlated to increased neutral lipids and morphology of inflamed myeloid cells. DIAPH1 moderately colocalized with markers of endothelial cells, astrocytes, neurons, and oligodendrocytes. DISCUSSION/CONCLUSIONS:Our findings localize DIAPH1 particularly to myeloid cells in the CNS, especially in AD in the locations of lipid droplet accumulation, thereby implicating RAGE-DIAPH1 signaling in dysregulated lipid metabolism and morphological changes of inflamed myeloid cells in this disorder.

Our previous work has shown that the cytoplasmic tail (ct) of RAGE is essential for RAGE ligand-mediated signal transduction and consequent modulation of gene expression and cellular properties. RAGE signaling requires interaction of ctRAGE with the intracellular effector, mammalian diaphanous 1, or DIAPH1. Given the complex, multi-ligand nature of ligands binding to the extracellular domains of RAGE, we sought to discover small molecule inhibitors of the interaction of ctRAGE with DIAPH1. Prompted by our identification of 13 small molecules that block the interaction of ctRAGE-DIAPH1, we pursued structure-activity relationship experiments to identify analogues potentially able to delay or prevent the progression of RAGE-related chronic diseases, such as diabetes. In vitro binding studies Native tryptophan fluorescence experiments were conducted using 1mM of compound dissolved in phosphate buffer and DMSO. 10 nM ctRAGE solution was titrated from 0.1 nM-100 muM and the dissociation constants, Kd, were estimated from the changes in peak fluorescence intensities as a function of the free compound concentration. NMR experiments were performed operating at 1H frequencies of 500 MHz and 700 MHz. NMR samples contained 50 muM of [U-15N] ctRAGE and 10 muM of the compound. All spectra were collected at 298K, which yielded high quality NMR spectra of [U-15N] RAGE tail. Compound binding to ctRAGE was identified as at least a 0.01 ppm change in the ctRAGE chemical shift. Small molecule leads were identified and referred to as NYU 1-5 (Table 1). Ex vivo biological activity assays Inhibition of smooth muscle cell (SMC) migration induced by RAGE ligands after incubation with indicated NYU compounds. SMCs were grown to confluence and starved overnight. The next morning, the medium was removed, compounds were added and the monolayer was wounded using a p200 pipette tip and allowed to incubate for 1.5h. Following incubation, compounds were removed and fresh medium containing the RAGE ligand, CML-AGE (10 mug/ml) was added for 4h. Images were taken, measured and an area ingrowth of effective migrating cells was calculated (Table 1). In vivo studies (A) Delayed type hypersensitivity. Female CF-1 mice were sensitized over the left inguinal lymph node with a methylated bovine serum albumin emulsion. On day 19 and 20 after sensitization, mice received, by oral gavage, the test compounds (5 mg/kg/bw) or vehicle twice daily. Post-final compound injection, mBSA was injected into the left hind paw. Inflammation was assessed using a semiquantitative scoring system (Figure 1). (B) Myocardial infarction in diabetic mice. Male C57BL/6 mice were rendered diabetic with STZ and were diabetic for 2 months. Mice were subjected to left anterior descending coronary artery occlusion followed by reperfusion (LAD/reperfusion). After 48h, the hearts were excised. TTC and Evan's blue staining was used to measure infarct area. Mice received a total of 7 doses of either VEHICLE or an NYU compound (Figure 2). In summary, refined compounds that inhibit the interaction of ctRAGE with DIAPH1, which exhibit in vitro and in vivo inhibition of RAGE-dependent molecular processes, present attractive scaffolds for the development of therapeutics against RAGE-mediated diseases, such as those linked to diabetic complications and chronic inflammation. We conclude that these identified compounds hold significant potential as druggable scaffolds for further development and testing for the treatment of RAGE-related disorders

INTRODUCTION/BACKGROUND:Self-reported tobacco use in the United Arab Emirates is among the highest in the region. Use of tobacco products other than cigarettes is widespread, but little is known about specific behavior use patterns. There have been no studies that have biochemically verified smoking status. METHODS:The UAE Healthy Future Study (UAEHFS) seeks to understand the causes of non-communicable diseases through a 20,000-person cohort study. During the study pilot, 517 Emirati nationals were recruited to complete a questionnaire, provide clinical measurements and biological samples. Complete smoking data were available for 428 participants. Validation of smoking status via cotinine testing was conducted based on complete questionnaire data and matching urine samples for 399 participants, using a cut-off of 200ng/ml to indicate active smoking status. RESULTS:Self-reported tobacco use was 36% among men and 3% among women in the sample. However, biochemical verification of smoking status revealed that 42% men and 9% of women were positive for cotinine indicating possible recent tobacco use. Dual and poly-use of tobacco products was fairly common with 32% and 6% of the sample reporting respectively. CONCLUSIONS:This is the first study in the region to biochemically verify tobacco use self-report data. Tobacco use in this study population was found to be higher than previously thought, especially among women. Misclassification of smoking status was more common than expected. Poly-tobacco use was also very common. Additional studies are needed to understand tobacco use behaviors and the extent to which people may be exposed to passive tobacco smoke. IMPLICATIONS/CONCLUSIONS:This study is the first in the region to biochemically verify self-reported smoking status.

The role of high-density lipoprotein (HDL) function and advanced glycation end products (AGEs) in HIV-related atherosclerotic cardiovascular disease (CVD) is unclear. Both glycation and oxidation (HDLox) are major modifications of HDL that can alter its composition and function. Therefore, we explored the longitudinal association of HDLox with progression of glycation, as evaluated by measurement of circulating forms of receptor for AGE that predict morbidity (soluble Receptors for Advanced Glycation Endproducts [sRAGE], endogenous secretory Receptors for Advanced Glycation Endproducts [esRAGE]), in people with HIV-1 (PWH; HIV-1) and uninfected (HIV-1) individuals.We retrospectively assessed if levels of plasma sRAGE and esRAGE and HDL function (reduced antioxidant function is associated with increased HDL lipid hydroperoxide content; HDLox) in a subset of participants (n = 80) from a prospective 3-year study (AIDS Clinical Trials Group A5078). Primary outcomes were baseline and yearly rates of change over 96 of 144 weeks (Δ) in HDLox in HIV-1 versus uninfected HIV-1 controls (noted as HIV-1).Higher baseline levels of sRAGE in PWH on effective anti-retroviral therapy and with low CVD risk, but not in HIV-1 persons, were independently associated with higher HDLox. EsRAGE, but not sRAGE, had consistent inverse relationships with ΔHDLox in both HIV-1 and HIV-1 persons at baseline. In HIV-1 but not in HIV-1 persons, ΔHDLox had positive and inverse relationships with ΔRAGE and ΔesRAGE, respectively.Glycation and oxidation of HDL may contribute to impaired HDL function present in PWH.