Genetic risk converges on regulatory networks mediating early type 2 diabetes.

Type 2 diabetes mellitus (T2D), a major cause of worldwide morbidity and mortality, is characterized by dysfunction of insulin-producing pancreatic islet ß cells1,2. T2D genome-wide association studies (GWAS) have identified hundreds of signals in non-coding and ß cell regulatory genomic regions, but deciphering their biological mechanisms remains challenging3-5. Here, to identify early disease-driving events, we performed traditional and multiplexed pancreatic tissue imaging, sorted-islet cell transcriptomics and islet functional analysis of early-stage T2D and control donors. By integrating diverse modalities, we show that early-stage T2D is characterized by ß cell-intrinsic defects that can be proportioned into gene regulatory modules with enrichment in signals of genetic risk. After identifying the ß cell hub gene and transcription factor RFX6 within one such module, we demonstrated multiple layers of genetic risk that converge on an RFX6-mediated network to reduce insulin secretion by ß cells. RFX6 perturbation in primary human islet cells alters ß cell chromatin architecture at regions enriched for T2D GWAS signals, and population-scale genetic analyses causally link genetically predicted reduced RFX6 expression with increased T2D risk. Understanding the molecular mechanisms of complex, systemic diseases necessitates integration of signals from multiple molecules, cells, organs and individuals, and thus we anticipate that this approach will be a useful template to identify and validate key regulatory networks and master hub genes for other diseases or traits using GWAS data.

Genetic effects on liver chromatin accessibility identify disease regulatory variants.

Identifying the molecular mechanisms by which genome-wide association study (GWAS) loci influence traits remains challenging. Chromatin accessibility quantitative trait loci (caQTLs) help identify GWAS loci that may alter GWAS traits by modulating chromatin structure, but caQTLs have been identified in a limited set of human tissues. Here we mapped caQTLs in human liver tissue in 20 liver samples and identified 3,123 caQTLs. The caQTL variants are enriched in liver tissue promoter and enhancer states and frequently disrupt binding motifs of transcription factors expressed in liver. We predicted target genes for 861 caQTL peaks using proximity, chromatin interactions, correlation with promoter accessibility or gene expression, and colocalization with expression QTLs. Using GWAS signals for 19 liver function and/or cardiometabolic traits, we identified 110 colocalized caQTLs and GWAS signals, 56 of which contained a predicted caPeak target gene. At the LITAF LDL-cholesterol GWAS locus, we validated that a caQTL variant showed allelic differences in protein binding and transcriptional activity. These caQTLs contribute to the epigenomic characterization of human liver and help identify molecular mechanisms and genes at GWAS loci.

Human and rat skeletal muscle single-nuclei multi-omic integrative analyses nominate causal cell types, regulatory elements, and SNPs for complex traits.

Skeletal muscle accounts for the largest proportion of human body mass, on average, and is a key tissue in complex diseases and mobility. It is composed of several different cell and muscle fiber types. Here, we optimize single-nucleus ATAC-seq (snATAC-seq) to map skeletal muscle cell-specific chromatin accessibility landscapes in frozen human and rat samples, and single-nucleus RNA-seq (snRNA-seq) to map cell-specific transcriptomes in human. We additionally perform multi-omics profiling (gene expression and chromatin accessibility) on human and rat muscle samples. We capture type I and type II muscle fiber signatures, which are generally missed by existing single-cell RNA-seq methods. We perform cross-modality and cross-species integrative analyses on 33,862 nuclei and identify seven cell types ranging in abundance from 59.6% to 1.0% of all nuclei. We introduce a regression-based approach to infer cell types by comparing transcription start site-distal ATAC-seq peaks to reference enhancer maps and show consistency with RNA-based marker gene cell type assignments. We find heterogeneity in enrichment of genetic variants linked to complex phenotypes from the UK Biobank and diabetes genome-wide association studies in cell-specific ATAC-seq peaks, with the most striking enrichment patterns in muscle mesenchymal stem cells (∼3.5% of nuclei). Finally, we overlay these chromatin accessibility maps on GWAS data to nominate causal cell types, SNPs, transcription factor motifs, and target genes for type 2 diabetes signals. These chromatin accessibility profiles for human and rat skeletal muscle cell types are a useful resource for nominating causal GWAS SNPs and cell types.

RAGE and its ligands: from pathogenesis to therapeutics.

Receptor for advanced glycation end products (RAGE) is an immunoglobulin-like receptor present on cell surface. RAGE binds to an array of structurally diverse ligands, acts as a pattern recognition receptor (PRR) and is expressed on cells of different origin performing different functions. RAGE ligation leads to the initiation of a cascade of signaling events and is implicated in diseases, such as inflammation, cancer, diabetes, vascular dysfunctions, retinopathy, and neurodegenerative diseases. Because of the significant involvement of RAGE in the progression of numerous diseases, RAGE signaling has been targeted through use of inhibitors and anti-RAGE antibodies as a treatment strategy and therapy. Here in this review, we have summarized the physical and physiological aspects of RAGE biology in mammalian system and the importance of targeting this molecule in the treatment of various RAGE mediated pathologies. Highlights Receptor for advanced glycation end products (RAGE) is a member of immunoglobulin superfamily of receptors and involved in many pathophysiological conditions. RAGE ligation with its ligands leads to initiation of distinct signaling cascades and activation of numerous transcription factors. Targeting RAGE signaling through inhibitors and anti-RAGE antibodies can be promising treatment strategy.

Influence of the COVID-19 Pandemic on Treatment Times for Acute Ischemic Stroke: The Society of Vascular and Interventional Neurology Multicenter Collaboration.

BACKGROUND AND PURPOSE: The pandemic caused by the novel coronavirus disease 2019 (COVID-19) has led to an unprecedented paradigm shift in medical care. We sought to evaluate whether the COVID-19 pandemic may have contributed to delays in acute stroke management at comprehensive stroke centers. METHODS: Pooled clinical data of consecutive adult stroke patients from 14 US comprehensive stroke centers (January 1, 2019, to July 31, 2020) were queried. The rate of thrombolysis for nontransferred patients within the Target: Stroke goal of 60 minutes was compared between patients admitted from March 1, 2019, and July 31, 2019 (pre-COVID-19), and March 1, 2020, to July 31, 2020 (COVID-19). The time from arrival to imaging and treatment with thrombolysis or thrombectomy, as continuous variables, were also assessed. RESULTS: Of the 2955 patients who met inclusion criteria, 1491 were admitted during the pre-COVID-19 period and 1464 were admitted during COVID-19, 15% of whom underwent intravenous thrombolysis. Patients treated during COVID-19 were at lower odds of receiving thrombolysis within 60 minutes of arrival (odds ratio, 0.61 [95% CI, 0.38-0.98]; P=0.04), with a median delay in door-to-needle time of 4 minutes (P=0.03). The lower odds of achieving treatment in the Target: Stroke goal persisted after adjustment for all variables associated with earlier treatment (adjusted odds ratio, 0.55 [95% CI, 0.35-0.85]; P<0.01). The delay in thrombolysis appeared driven by the longer delay from imaging to bolus (median, 29 [interquartile range, 18-41] versus 22 [interquartile range, 13-37] minutes; P=0.02). There was no significant delay in door-to-groin puncture for patients who underwent thrombectomy (median, 83 [interquartile range, 63-133] versus 90 [interquartile range, 73-129] minutes; P=0.30). Delays in thrombolysis were observed in the months of June and July. CONCLUSIONS: Evaluation for acute ischemic stroke during the COVID-19 period was associated with a small but significant delay in intravenous thrombolysis but no significant delay in thrombectomy time metrics. Taking steps to reduce delays from imaging to bolus time has the potential to attenuate this collateral effect of the pandemic.

Sequenceserver: A Modern Graphical User Interface for Custom BLAST Databases.

Comparing newly obtained and previously known nucleotide and amino-acid sequences underpins modern biological research. BLAST is a well-established tool for such comparisons but is challenging to use on new data sets. We combined a user-centric design philosophy with sustainable software development approaches to create Sequenceserver, a tool for running BLAST and visually inspecting BLAST results for biological interpretation. Sequenceserver uses simple algorithms to prevent potential analysis errors and provides flexible text-based and visual outputs to support researcher productivity. Our software can be rapidly installed for use by individuals or on shared servers.

Lysophosphatidic acid-RAGE axis promotes lung and mammary oncogenesis via protein kinase B and regulating tumor microenvironment.

BACKGROUND: Receptor for advanced glycation end products (RAGE) is a multi-ligand transmembrane receptor of the immunoglobulin superfamily. Lysophosphatidic acid (LPA) is a ligand for RAGE and is involved in physiological and pathophysiological conditions including cancer. However, RAGE-LPA axis is unexplored in lung and mammary cancer. METHODS: RAGE was silenced in A549, MDA MB-231 and MCF7 using RAGE shRNA. For in vitro tumorigenesis, we performed wound healing, colony formation, cell proliferation and invasion assays. Evaluation of expression of oncogenes, EMT markers and downstream signaling molecules was done by using western blot and immunohistochemistry. For subcellular expression of RAGE, immunofluorescence was done. In vivo tumorigenesis was assessed by intraperitoneal injection of cancer cells in nude mice. RESULTS: Here we show RAGE mediated profound increase in proliferation, migration and invasion of lung and mammary cancer cells via LPA in Protein kinase B (PKB) dependent manner. LPA mediated EMT transition is regulated by RAGE. In vivo xenograft results show significance of RAGE in LPA mediated lung and mammary tumor progression, angiogenesis and immune cell infiltration to tumor microenvironment. CONCLUSION: Our results establish the significance and involvement of RAGE in LPA mediated lung and mammary tumor progression and EMT transition via RAGE. RAGE-LPA axis may be a therapeutic target in lung and mammary cancer treatment strategies. Video Abstract.

Lysophosphatidic acid converts monocytes into macrophages in both mice and humans.

Monocytes and macrophages represent critical arms of the innate immune system and are considered regulators and effectors of inflammation and the innate immune response. Monocytes can mobilize from bone marrow, traffic to their required destination, and differentiate into effector cells, depending on the local tissue environment, to perform multiple roles during infection or inflammation, making them important components of body's immune defense. Macrophages have diverse roles in tissue homeostasis, development, and tissue repair following injury. Adult bone marrow monocytes can give rise to tissue-resident macrophages during infection or inflammatory reactions, besides self-replication of tissue resident macrophages. Lysophosphatidic acid (LPA), a lipid by-product of autotaxin activity, is involved in cancer, vascular defects, and neural tissue, but is largely unexplored in immune system. Here, we reveal an unexpected function of LPA that transfigures CD11b+ murine monocytes into F4/80+ macrophages. LPA-stimulated Akt/mTOR signaling is critical for LPA-mediated macrophage development in mice. Additionally, transcriptome analysis reveals that PPARγ is the key transcriptional regulator in the development of LPA-induced macrophages. In humans, LPA mediates macrophage formation following similar pathways. These findings identify a critical role for LPA in regulating innate immune system.

Risk factors for recoarctation of aorta after Norwood procedure in patients with hypoplastic left heart syndrome.

BACKGROUND: Recoarctation (reCoA) of the aorta is a common complication afer the Norwood procedure. Untreated, it can lead to failure of the systemic ventricle and death. The main goal of the study is to define risk factors of reCoA after the Norwood procedure in hypoplastic left heart syndrome (HLHS). METHODS: We retrospectively analyzed the pre-, intra- and postoperative data of 96 successive patients who underwent the Norwood procedure between 2007 and 2011. In case of reCoA balloon angioplasty was performed. We analyzed and compared the data of the patients with reCoA and without reCoA using the StatSo STATISTICA TM 10 software. RESULTS: ReCoA was noted in 23 patients (33.3%). This complication was diagnosed 95.1 days (49-156 days) on the average a er the Norwood procedure. Balloon angioplasty successfully allowed for decreasing the mean gradient across the site of the narrowing from the average 27.5 mmHg to the average 9.7 mmHg (p = 0.008) and enlarged the neo-isthmus by the average of 2 mm (p <0.05). The risks factors seemed to be the diameter of the ascending aorta OR = 7.82 (p = 0.001), atresia of the mitral valve OR = 7.00 (p = 0.003) and atresia of the aortic valve - OR = 6.22 (p = 0.002). CONCLUSION: Balloon angioplasty seems to be an effective intervention in case of reCoA. A low diameter of the native ascending aorta (<=3mm) and the presence of atresia of the mitral and/or aortic valve should intensify the vigilance of a cardiologist in the search for signs of reCoA of the aorta.

Initial experience with intracorporeal continuous ow LVAD in pediatric patients in Poland.

THE AIM: The aim of the study is to present the initial experience with continuous flow left ventricle assist device (CF-LVAD) in pediatric patients with BSA below 1.5 m2. MATERIAL AND METHODS: Between 2016 and 2017, CF-LVAD (the Heartware System) have been implanted in three pediatric patients in the Department of Pediatric Cardiac Surgery, Jagiellonian University, Krakow, Poland. The indications for initiating CF-LVAD were end-stage congestive heart failure due to dilated cardiomyopathy in all children. RESULTS: Implanted patients have had BSA of 1.09, 1.42, 1.2 m2, and 37, 34, 34 kg of body weight and the age 12, 11, 12 years, respectively. The time of support was 550 days in two patients and 127 in another one, and is ongoing. The main complication has been driveline infection. CONCLUSION: The outcomes from our single-center experience using the HeartWare CF-LVAD have been excellent with a low incidence of complication and no necessity to reoperation in our patients. Children could be successfully and safely discharged home.

Treatment effect with anti-RAGE F(ab')2 antibody improves hind limb angiogenesis and blood flow in Type 1 diabetic mice with left femoral artery ligation.

We investigated treatment with a receptor for advanced glycation endproduct (RAGE) blocking antibody on angiogenic response to hind limb ischemia in diabetic mice. Streptozotocin treated C57BL/6 mice received either murine monoclonal anti-RAGE F(ab')2 intraperitoneally (n=10) or saline (n=9) for 9 weeks. Diabetic plus 10 non-diabetic C57BL/6 mice underwent left femoral artery ligation and 5 days later angiogenesis imaging with (99m)Tc-Arg-Gly-Asp (RGD) nanoSPECT/CT. Twenty-four days later, hind limb blood flow was measured with ultrasound, the mice were euthanized, and tissue was taken for immunohistochemistry. The angiogenic imaging signal in ischemic limbs was higher in RAGE-ab treated versus saline treated mice at day 5 (3.1±1.4 vs 1.68±0.35, p=0.02) and blood flow was higher at day 24 (1.49±0.5 vs 0.61±0.39, p=0.04). Immunohistochemistry of ischemic muscles showed greater capillary density in the RAGE-ab treated group versus the vehicle-treated group (p<0.001) (NS from non-diabetic mice). In conclusion, treatment with anti-RAGE F(ab')2 in diabetic mice improves neovascularization in the ischemic leg.

An in vitro Comparison of Endodontic Medicaments Propolis and Calcium Hydroxide alone and in Combination with Ciprofloxacin and Moxifloxacin against Enterococcus Faecalis.

AIM: To evaluate and compare the antimicrobial properties of propolis and calcium hydroxide alone and in combination with ciprofloxacin and moxifloxacin against Enterococcus faecalis (E. Faecalis). MATERIALS AND METHODS: The laboratory study was carried out to test the effectiveness of propolis and calcium hydroxidealone as well as in combination with the established endodontic medicaments (moxifloxacin and ciprofloxacin). The various combinations were-group 1: propolis, group 2: calcium hydroxide, group 3: moxifloxacin, group 4: ciprofoxacin, group 5: propolis + moxifloxacin, group 6: propolis + Ciprofloxacin, group 7: calcium hydroxide + ciprofloxacin, group 8: calcium hydroxide + moxifloxacin. The efficacy of these medicaments was tested by checking for the zone of inhibition for the specific strain (ATCC 29212) of E. faecalis at different time intervals, i.e. 24, 48 and 72 hours. RESULTS: Mean zone of inhibition was maximum in group V (21.94 ± 4.26) followed by group VI (18.80 ± 1.93), group I (18.71 ± 4.26), group VIII (15.88 ± 2.59), group III (14.91 ± 1.00), group VII (14.57 ± 2.17), group IV (13.91 ± 1.00) and minimum in group II (12.89 ± 2.14). Mean zone of inhibition was found to be maximum at 72 hours and minimum at 24 hours. At all time intervals, the combination of Propalis with Moxifocacin showed the maximum antimicrobial efficacy. CONCLUSION: On the basis of the results of the present study, it can be concluded that propolis and calcium hydroxide show synergistic effect with moxifloxacin and ciprofloxacin against E. Faecalis. Propolis in combination with antibiotics and alone is more effective than calcium hydroxide. CLINICAL SIGNIFICANCE: Since propolis alone and in combination with antibiotics was observed to be more effective than calcium hydroxide, propolis can be considered as an intracanal medicament when compared to traditional calcium hydroxide.

The receptor for advanced glycation end products (RAGE) specifically recognizes methylglyoxal-derived AGEs.

Diabetes-induced hyperglycemia increases the extracellular concentration of methylglyoxal. Methylglyoxal-derived hydroimidazolones (MG-H) form advanced glycation end products (AGEs) that accumulate in the serum of diabetic patients. The binding of hydroimidozolones to the receptor for AGEs (RAGE) results in long-term complications of diabetes typified by vascular and neuronal injury. Here we show that binding of methylglyoxal-modified albumin to RAGE results in signal transduction. Chemically synthesized peptides containing hydroimidozolones bind specifically to the V domain of RAGE with nanomolar affinity. The solution structure of an MG-H1-V domain complex revealed that the hydroimidazolone moiety forms multiple contacts with a positively charged surface on the V domain. The high affinity and specificity of hydroimidozolones binding to the V domain of RAGE suggest that they are the primary AGE structures that give rise to AGEs-RAGE pathologies.

Formin mDia1 mediates vascular remodeling via integration of oxidative and signal transduction pathways.

RATIONALE: The mammalian diaphanous-related formin (mDia1), governs microtubule and microfilament dynamics while functioning as an effector for Rho small GTP-binding proteins during key cellular processes such as adhesion, cytokinesis, cell polarity, and morphogenesis. The cytoplasmic domain of the receptor for advanced glycation endproducts binds to the formin homology 1 domain of mDia1; mDia1 is required for receptor for advanced glycation endproducts ligand-induced cellular migration in transformed cells. OBJECTIVE: Because a key mechanism in vascular remodeling is the induction of smooth muscle cell migration, we tested the role of mDia1 in this process. METHODS AND RESULTS: We report that endothelial denudation injury to the murine femoral artery significantly upregulates mDia1 mRNA transcripts and protein in the injured vessel, particularly in vascular smooth muscle cells within the expanding neointima. Loss of mDia1 expression significantly reduces pathological neointimal expansion consequent to injury. In primary murine aortic smooth muscle cells, mDia1 is required for receptor for advanced glycation endproducts ligand-induced membrane translocation of c-Src, which leads to Rac1 activation, redox phosphorylation of AKT/glycogen synthase kinase 3ß, and consequent smooth muscle cell migration. CONCLUSIONS: We conclude that mDia1 integrates oxidative and signal transduction pathways triggered, at least in part, by receptor for advanced glycation endproducts ligands, thereby regulating pathological neointimal expansion.

Signal transduction in receptor for advanced glycation end products (RAGE): solution structure of C-terminal rage (ctRAGE) and its binding to mDia1.

The receptor for advanced glycation end products (RAGE) is a multiligand cell surface macromolecule that plays a central role in the etiology of diabetes complications, inflammation, and neurodegeneration. The cytoplasmic domain of RAGE (C-terminal RAGE; ctRAGE) is critical for RAGE-dependent signal transduction. As the most membrane-proximal event, mDia1 binds to ctRAGE, and it is essential for RAGE ligand-stimulated phosphorylation of AKT and cell proliferation/migration. We show that ctRAGE contains an unusual α-turn that mediates the mDia1-ctRAGE interaction and is required for RAGE-dependent signaling. The results establish a novel mechanism through which an extracellular signal initiated by RAGE ligands regulates RAGE signaling in a manner requiring mDia1.

Pharmacoeconomic Analysis of Fixed-Dose Combinations of Proton Pump Inhibitors Available in India.

Introduction The global proton pump inhibitors (PPIs) market was valued at US$ 2.9 billion in 2020 and is expected to exhibit a compound aggregated growth rate of 4.30% during the forecast period (2020-2027), as they are regularly prescribed for many gastrointestinal disorders, and the treatment usually lasts for a longer period. PPIs are usually combined with antiemetics and prokinetic drugs. The price of PPIs for the same combination varies a lot, which can lead to a lot of financial burden on the patients. Objective To evaluate the cost ratio and percentage cost variation of commonly used PPIs in various combinations. Methodology The cost of different brands of commonly used PPIs in combination with other drugs was analyzed in our study. A total of 21 different combinations (10 capsules/tablets for oral use) were tabulated by referring to the "Monthly Index of Medical Specialities" October-December 2021, and 1mg online pharmacy. The cost ratio and percentage cost variation for various brands of a particular strength and dosage form were calculated and compared. Cost ratio > 2 and cost variation > 100% were considered significant. Results The results show a huge variation (1788.88%) in costs of different brands with the highest being rabeprazole 20 mg and domperidone 10 mg (cost ratio: 18.88, percentage cost variation: 1788.88%) in oral formulation, followed by pantoprazole 40 mg and itopride 150 mg. The minimum cost ratio (1.35) and percentage cost variation (1.35%) is for pantoprazole 40 mg and levosulpiride 75 mg. Logistic regression analysis between the number of brands and percentage cost variation gives an R2 value of 0.0923. Conclusion There is a wide variation in the prices of PPIs available in the market, which can inadvertently increase the financial burden of therapy on patients. Physicians need to be made aware of these price differences so that they can choose the best available alternative for patients, which can help in increasing compliance with the prescribed drugs.

Population-scale skeletal muscle single-nucleus multi-omic profiling reveals extensive context specific genetic regulation.

Skeletal muscle, the largest human organ by weight, is relevant to several polygenic metabolic traits and diseases including type 2 diabetes (T2D). Identifying genetic mechanisms underlying these traits requires pinpointing the relevant cell types, regulatory elements, target genes, and causal variants. Here, we used genetic multiplexing to generate population-scale single nucleus (sn) chromatin accessibility (snATAC-seq) and transcriptome (snRNA-seq) maps across 287 frozen human skeletal muscle biopsies representing 456,880 nuclei. We identified 13 cell types that collectively represented 983,155 ATAC summits. We integrated genetic variation to discover 6,866 expression quantitative trait loci (eQTL) and 100,928 chromatin accessibility QTL (caQTL) (5% FDR) across the five most abundant cell types, cataloging caQTL peaks that atlas-level snATAC maps often miss. We identified 1,973 eGenes colocalized with caQTL and used mediation analyses to construct causal directional maps for chromatin accessibility and gene expression. 3,378 genome-wide association study (GWAS) signals across 43 relevant traits colocalized with sn-e/caQTL, 52% in a cell-specific manner. 77% of GWAS signals colocalized with caQTL and not eQTL, highlighting the critical importance of population-scale chromatin profiling for GWAS functional studies. GWAS-caQTL colocalization showed distinct cell-specific regulatory paradigms. For example, a C2CD4A/B T2D GWAS signal colocalized with caQTL in muscle fibers and multiple chromatin loop models nominated VPS13C, a glucose uptake gene. Sequence of the caQTL peak overlapping caSNP rs7163757 showed allelic regulatory activity differences in a human myocyte cell line massively parallel reporter assay. These results illuminate the genetic regulatory architecture of human skeletal muscle at high-resolution epigenomic, transcriptomic, and cell state scales and serve as a template for population-scale multi-omic mapping in complex tissues and traits.

RAGE binds C1q and enhances C1q-mediated phagocytosis.

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 µM, 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.

Activation of the ROCK1 branch of the transforming growth factor-beta pathway contributes to RAGE-dependent acceleration of atherosclerosis in diabetic ApoE-null mice.

RATIONALE: The multiligand RAGE (receptor for advanced glycation end products) contributes to atherosclerosis in apolipoprotein (Apo)E-null mice. OBJECTIVE: To delineate the specific mechanisms by which RAGE accelerated atherosclerosis, we performed Affymetrix gene expression arrays on aortas of nondiabetic and diabetic ApoE-null mice expressing RAGE or devoid of RAGE at nine weeks of age, as this reflected a time point at which frank atherosclerotic lesions were not yet present, but that we would be able to identify the genes likely involved in diabetes- and RAGE-dependent atherogenesis. METHODS AND RESULTS: We report that there is very little overlap of the genes that are differentially expressed both in the onset of diabetes in ApoE-null mice, and in the effect of RAGE deletion in diabetic ApoE-null mice. Pathway-Express analysis revealed that the transforming growth factor-beta pathway and focal adhesion pathways might be expected to play a significant role in both the mechanism by which diabetes facilitates the formation of atherosclerotic plaques in ApoE-null mice, and the mechanism by which deletion of RAGE ameliorates this effect. Quantitative polymerase chain reaction studies, Western blotting, and confocal microscopy in aortic tissue and in primary cultures of murine aortic smooth muscle cells supported these findings. CONCLUSIONS: Taken together, our work suggests that RAGE-dependent acceleration of atherosclerosis in ApoE-null mice is dependent, at least in part, on the action of the ROCK1 (rho-associated protein kinase 1) branch of the transforming growth factor-beta pathway.