Faculty Bibliography

Macrophages play an essential role in regression of diabetic atherosclerosis. A key player in these processes is Receptor for Advanced Glycation End Products (RAGE), which binds specific ligands enriched in atherosclerotic plaques. Here, we sought to test the mechanisms by which deletion of Ager in diabetic mice accelerates regression of atherosclerosis in an aorta transplantation model in which donor aortic arches from diabetic Ldlr null Western diet-fed mice (CD45.2) are transplanted into diabetic wild-type (WT) or homozygous Ager null mice (CD45.1), thereby effecting lipid lowering and promoting regression, which was improved in Ager null vs. WT mice. We employed RNA sequencing to identify the transcriptional events by which RAGE mediates its effects in donor (CD45.2) and/or recipient (CD45.1) macrophages in diabetic regressing plaques. Our results suggest that critical gene expression profiles, including those genes involved in interferon signaling, particularly the gene encoding interferon regulating factor-7 (Irf7), monocyte/macrophage fate (recruitment, proliferation, apoptosis), and lipid metabolism, are beneficially modulated, at least in part, via Ager deletion particularly in the recipient CD45.1 macrophages in atherosclerosis regression. We tested the role of the IRF7 pathway and our findings, to date, include: 1) There is a decrease in IRF7+/ macrophage area in plaques retrieved from diabetic Ager null vs. diabetic WT mice; 2) Levels of IFN-gamma are lower in diabetic Ager null vs. WT diabetic recipient mice after transplantation; 3) Bone marrow derived macrophages (BMDMs) treated with 2% serum from Ldlr null mice fed Western diet resulted in significant upregulation of Irf7 in WT but not in Ager null BMDMs; and 4) Upon treatment with 2% serum from Ldlr mice fed Western diet, significant upregulation of genes involved in cholesterol transport (Abca1 and Abcg1) was only observed in Ager null but not WT BMDMs compared to treatment with serum from WT normolipidemic mice. These findings suggest that RAGE may affect lipid driven regulation of the interferon-signaling pathway to impair regression of diabetic atherosclerosis. These data may provide avenues for therapeutic strategies to accelerate regression of diabetic atherosclerosis

The Receptor for Advanced Glycation End Products (RAGE) is expressed by multiple cell types in the brain and spinal cord that are linked to the pathogenesis of neurovascular and neurodegenerative disorders, including neurons, glia (microglia and astrocytes) and vascular cells (endothelial cells, smooth muscle cells and pericytes). Mounting structural and functional evidence implicates the interaction of the RAGE cytoplasmic domain with the formin, Diaphanous1 (DIAPH1), as the key cytoplasmic hub for RAGE ligand-mediated activation of cellular signaling. In aging and diabetes, the ligands of the receptor abound, both in the central nervous system (CNS) and in the periphery. Such accumulation of RAGE ligands triggers multiple downstream events, including upregulation of RAGE itself. Once set in motion, cell intrinsic and cell-cell communication mechanisms, at least in part via RAGE, trigger dysfunction in the CNS. A key outcome of endothelial dysfunction is reduction in cerebral blood flow and increased permeability of the blood brain barrier, conditions that facilitate entry of activated leukocytes into the CNS, thereby amplifying primary nodes of CNS cellular stress. This contribution details a review of the ligands of RAGE, the mechanisms and consequences of RAGE signal transduction, and cites multiple examples of published work in which RAGE contributes to the pathogenesis of neurovascular perturbation. Insights into potential therapeutic modalities targeting the RAGE signal transduction axis for disorders of CNS vascular dysfunction and neurodegeneration are also discussed.

Macrophages accumulate prominently in the visceral adipose tissue (VAT) of obese humans and high fat diet (HFD) fed mice, and this is linked to insulin resistance and type II diabetes. While the mechanisms regulating macrophage recruitment in obesity have been delineated, the signals directing macrophage persistence in VAT are poorly understood. We previously showed that the neuroimmune guidance cue netrin-1 is expressed in the VAT of obese mice and humans, where it promotes macrophage accumulation. To better understand the source of netrin-1 and its effects on adipose tissue macrophage (ATM) fate and function in obesity, we generated mice with myeloid-specific deletion of netrin-1 (Ntn1^fl/flLysMCre^+/-; Ntn1^Δmac). Interestingly, Ntn1^Δmac mice showed a modest decrease in HFD-induced adiposity and adipocyte size, in the absence of changes in food intake or leptin, that was accompanied by an increase in markers of adipocyte beiging (Prdm16, UCP-1). Using single cell RNA-seq, combined with conventional histological and flow cytometry techniques, we show that myeloid-specific deletion of netrin-1 caused a 50% attrition of ATMs in HFD-fed mice, particularly of the resident macrophage subset, and altered the phenotype of residual ATMs to enhance lipid handling. Pseudotime analysis of single cell transcriptomes showed that in the absence of netrin-1, macrophages in the obese VAT underwent a phenotypic switch with the majority of ATMs activating a program of genes specialized in lipid handling, including fatty acid uptake and intracellular transport, lipid droplet formation and lipolysis, and regulation of lipid localization. Furthermore, Ntn1^Δmac macrophages had reduced expression of genes involved in arachidonic acid metabolism, and targeted LCMS/MS metabololipidomics analysis revealed decreases in proinflammatory eicosanoids (5-HETE, 6-trans LTB₄, TXB₂, PGD₂) in the obese VAT. Collectively, our data show that targeted deletion of netrin-1 in macrophages reprograms the ATM phenotype in obesity, leading to reduced adipose inflammation, and improved lipid handling and metabolic function.

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.

Guidelines to reduce cardiovascular risk in diabetes include aggressive LDL lowering, but benefits are attenuated compared to those in patients without diabetes. Consistent with this, we have reported in mice that hyperglycemia impaired atherosclerosis regression. Aldose reductase (AR) is thought to contribute to clinical complications of diabetes by directing glucose into pathways producing inflammatory metabolites. Mice have low levels of AR, thus, raising them to human levels would be a more clinically relevant model to study changes in diabetes under atherosclerosis regression conditions. Donor aortae from western diet-fed Ldlr

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.