Circulating levels of AGEs and soluble RAGE isoforms are associated with all-cause mortality and development of cardiovascular complications in type 2 diabetes: a retrospective cohort study.

BACKGROUND: Advanced glycation end-products (AGEs) and their interaction with the receptor for advanced glycation end-products (RAGE) play a pivotal role in the development and progression of type 2 diabetes. In this retrospective cohort study, we explored the association of circulating levels of soluble RAGE (sRAGE) isoforms, i.e., endogenous secretory esRAGE and cleaved cRAGE, AGEs and their respective ratios with 15-year all-cause mortality in type 2 diabetes. METHODS: Baseline AGEs and sRAGE isoforms concentration were measured by ELISA in 362 patients with type 2 diabetes and in 125 age- and gender-matched healthy control subjects (CTR). Independent predictors of mortality were determined using Cox proportional-hazards models and used to build and validate a nomogram for all-cause mortality prediction in type 2 diabetes. RESULTS: AGEs, total sRAGE, cRAGE and the AGEs/sRAGE and AGEs/esRAGE ratios were significantly increased in patients with type 2 diabetes compared to CTR (p < 0.001). In CTR subjects, but not in type 2 diabetes patients, a significant negative correlation between cRAGE and age was confirmed (p = 0.003), whereas the AGEs/sRAGE (p = 0.032) and AGEs/cRAGE (p = 0.006) ratios were positively associated with age. At an average follow-up of 15 years (4,982 person-years), 130 deaths were observed. The increase in the AGEs/cRAGE ratio was accompanied by a higher risk of all-cause mortality in patients with type 2 diabetes (HR per each SD increment = 1.30, 95% CI 1.15-1.47; p < 0.001). Moreover, sRAGE was associated with the development of major adverse cardiovascular events (MACE) in type 2 diabetes patients without previous MACE (OR for each SD increase: 1.48, 95% CI 1.11-1.89). A nomogram based on age, sex, HbA1c, systolic blood pressure, and the AGEs/cRAGE ratio was built to predict 5-, 10- and 15-year survival in type 2 diabetes. Patients were categorized into quartiles of the monogram scores and Kaplan-Meier survival curves confirmed the prognostic accuracy of the model (log-rank p = 6.5 × 10- 13). CONCLUSIONS: The ratio between AGEs and the cRAGE isoform is predictive of 15-year survival in patients with type 2 diabetes. Our data support the assessment of circulating AGEs and soluble RAGE isoforms in patients with type 2 diabetes as predictors of MACE and all-cause mortality.

MicroRNA-34a: the bad guy in age-related vascular diseases.

The age-related vasculature alteration is the prominent risk factor for vascular diseases (VD), namely, atherosclerosis, abdominal aortic aneurysm, vascular calcification (VC) and pulmonary arterial hypertension (PAH). The chronic sterile low-grade inflammation state, alias inflammaging, characterizes elderly people and participates in VD development. MicroRNA34-a (miR-34a) is emerging as an important mediator of inflammaging and VD. miR-34a increases with aging in vessels and induces senescence and the acquisition of the senescence-associated secretory phenotype (SASP) in vascular smooth muscle (VSMCs) and endothelial (ECs) cells. Similarly, other VD risk factors, including dyslipidemia, hyperglycemia and hypertension, modify miR-34a expression to promote vascular senescence and inflammation. miR-34a upregulation causes endothelial dysfunction by affecting ECs nitric oxide bioavailability, adhesion molecules expression and inflammatory cells recruitment. miR-34a-induced senescence facilitates VSMCs osteoblastic switch and VC development in hyperphosphatemia conditions. Conversely, atherogenic and hypoxic stimuli downregulate miR-34a levels and promote VSMCs proliferation and migration during atherosclerosis and PAH. MiR34a genetic ablation or miR-34a inhibition by anti-miR-34a molecules in different experimental models of VD reduce vascular inflammation, senescence and apoptosis through sirtuin 1 Notch1, and B-cell lymphoma 2 modulation. Notably, pleiotropic drugs, like statins, liraglutide and metformin, affect miR-34a expression. Finally, human studies report that miR-34a levels associate to atherosclerosis and diabetes and correlate with inflammatory factors during aging. Herein, we comprehensively review the current knowledge about miR-34a-dependent molecular and cellular mechanisms activated by VD risk factors and highlight the diagnostic and therapeutic potential of modulating its expression in order to reduce inflammaging and VD burn and extend healthy lifespan.

Effects of RAGE Deletion on the Cardiac Transcriptome during Aging.

Cardiac aging is characterized by increased cardiomyocyte hypertrophy, myocardial stiffness, and fibrosis, which enhance cardiovascular risk. The receptor for advanced glycation end-products (RAGE) is involved in several age-related diseases. RAGE knockout (Rage-/-) mice show an acceleration of cardiac dimension changes and interstitial fibrosis with aging. This study identifies the age-associated cardiac gene expression signature induced by RAGE deletion. We analyzed the left ventricle transcriptome of 2.5-(Young), 12-(Middle age, MA), and 21-(Old) months-old female Rage-/- and C57BL/6N (WT) mice. By comparing Young, MA, and Old Rage-/- versus age-matched WT mice, we identified 122, 192, and 12 differently expressed genes, respectively. Functional inference analysis showed that RAGE deletion is associated with: (i) down-regulation of genes involved in antigen processing and presentation of exogenous antigen, adaptive immune response, and cellular responses to interferon beta and gamma in Young animals; (ii) up-regulation of genes related to fatty acid oxidation, cardiac structure remodeling and cellular response to hypoxia in MA mice; (iii) up-regulation of few genes belonging to complement activation and triglyceride biosynthetic process in Old animals. Our findings show that the age-dependent cardiac phenotype of Rage-/- mice is associated with alterations of genes related to adaptive immunity and cardiac stress pathways.

The microRNA-34a-Induced Senescence-Associated Secretory Phenotype (SASP) Favors Vascular Smooth Muscle Cells Calcification.

The senescence of vascular smooth muscle cells (VSMCs), characterized by the acquisition of senescence-associated secretory phenotype (SASP), is relevant for VSMCs osteoblastic differentiation and vascular calcification (VC). MicroRNA-34a (miR-34a) is a driver of such phenomena and could play a role in vascular inflammaging. Herein, we analyzed the relationship between miR-34a and the prototypical SASP component IL6 in in vitro and in vivo models. miR-34a and IL6 levels increased and positively correlated in aortas of 21 months-old male C57BL/6J mice and in human aortic smooth muscle cells (HASMCs) isolated from donors of different age and undergone senescence. Lentiviral overexpression of miR-34a in HASMCs enhanced IL6 secretion. HASMCs senescence and calcification accelerated after exposure to conditioned medium of miR-34a-overexpressing cells. Analysis of miR-34a-induced secretome revealed enhancement of several pro-inflammatory cytokines and chemokines, including IL6, pro-senescent growth factors and matrix-degrading molecules. Moreover, induction of aortas medial calcification and concomitant IL6 expression, with an overdose of vitamin D, was reduced in male C57BL/6J Mir34a-/- mice. Finally, a positive correlation was observed between circulating miR-34a and IL6 in healthy subjects of 20-90 years. Hence, the vascular age-associated miR-34a promotes VSMCs SASP activation and contributes to arterial inflammation and dysfunctions such as VC.

High Phosphate-Induced JAK-STAT Signalling Sustains Vascular Smooth Muscle Cell Inflammation and Limits Calcification.

Vascular calcification (VC) is an age-related complication characterised by calcium-phosphate deposition in the arterial wall driven by the osteogenic transformation of vascular smooth muscle cells (VSMCs). The JAK-STAT pathway is an emerging target in inflammation. Considering the relationship between VC and inflammation, we investigated the role of JAK-STAT signalling during VSMC calcification. Human aortic smooth muscle cells (HASMCs) were cultured in high-inorganic phosphate (Pi) medium for up to 7 days; calcium deposition was determined via Alizarin staining and colorimetric assay. Inflammatory factor secretion was evaluated via ELISA and JAK-STAT members' activation using Western blot or immunohistochemistry on HASMCs or calcified aortas of Vitamin D-treated C57BL6/J mice, respectively. The JAK-STAT pathway was blocked by JAK Inhibitor I and Von Kossa staining was used for calcium deposits in murine aortic rings. During Pi-induced calcification, HASMCs released IL-6, IL-8, and MCP-1 and activated JAK1-JAK3 proteins and STAT1. Phospho-STAT1 was detected in murine calcified aortas. Blocking of the JAK-STAT cascade reduced HASMC proliferation and pro-inflammatory factor expression and release while increasing calcium deposition and osteogenic transcription factor RUNX2 expression. Consistently, JAK-STAT pathway inhibition exacerbates mouse aortic ring calcification ex vivo. Intriguingly, our results suggest an alternative link between VSMC inflammation and VC.

Soluble Receptor for Advanced Glycation End-products regulates age-associated Cardiac Fibrosis.

Myocardial aging increases the cardiovascular risk in the elderly. The Receptor for Advanced Glycation End-products (RAGE) is involved in age-related disorders. The soluble isoform (sRAGE) acts as a scavenger blocking the membrane-bound receptor activation. This study aims at investigating RAGE contribution to age-related cardiac remodeling. We analyzed the cardiac function of three different age groups of female Rage-/- and C57BL/6N (WT) mice: 2.5- (Young), 12- (Middle-age, MA) and 21-months (Old) old. While aging, Rage-/- mice displayed an increase in left ventricle (LV) dimensions compared to age-matched WT animals, with the main differences observed in the MA groups. Rage-/- mice showed higher fibrosis and a larger number of α-Smooth Muscle Actin (SMA)+ cells with age, along with increased expression of pro-fibrotic Transforming Growth Factor (TGF)-ß1 pathway components. RAGE isoforms were undetectable in LV of WT mice, nevertheless, circulating sRAGE declined with aging and inversely associated with LV diastolic dimensions. Human cardiac fibroblasts stimulated with sRAGE exhibited a reduction in proliferation, pro-fibrotic proteins and TGF-beta Receptor 1 (TGFbR1) expression and Smad2-3 activation. Finally, sRAGE administration to MA WT animals reduced cardiac fibrosis. Hence, our work shows that RAGE associates with age-dependent myocardial changes and indicates sRAGE as an inhibitor of cardiac fibroblasts differentiation and age-dependent cardiac fibrosis.