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.

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.

Modulation of soluble receptor for advanced glycation end-products (RAGE) isoforms and their ligands in healthy aging.

The receptor for advanced glycation end-products (RAGE) recognizes several ligands involved in inflammatory diseases. Two circulating soluble isoforms exist: esRAGE derived from alternative splicing and cRAGE generated by the membrane-bound RAGE (FL-RAGE) proteolysis. Together, esRAGE and cRAGE constitute sRAGE and function as decoy receptors preventing FL-RAGE/ligands binding.We determined serum concentration of both, esRAGE and cRAGE, and their ligands AGEs, HMGB1 and S100A8/A9 in a healthy population of 169 subjects aged 20-90 years. cRAGE showed a negative (r=-0.375, P<0.0001) while AGEs (r=0.160, P=0.0384) and S100A8/A9 (r=0.207, P=0.0091) a positive correlation with age. esRAGE did not change during aging and inversely correlated with Hemoglobin, ALT, insulin, HOMA index, Waist-Hip ratio (W/H), Waist Circumference (WC) and positively with AGEs. cRAGE exhibited also an inverse correlation with WC, W/H, PAI-1, HMGB1, AGEs and S100A8/A9. Age, W/H, HMGB1, S100A8/A9 and AGEs are independent predictors of cRAGE, whereas W/H and AGEs associate with esRAGE. Treatment of cells with glycated albumin reduced cRAGE production and upregulated FL-RAGE.These results indicate that in a healthy population cRAGE is a biomarker of aging while esRAGE represents a more reliable marker of obesity and insulin resistance. Hence, sRAGE isoforms levels could be differentially associated with age-related diseases risk factors.

miR-34a Promotes Vascular Smooth Muscle Cell Calcification by Downregulating SIRT1 (Sirtuin 1) and Axl (AXL Receptor Tyrosine Kinase).

Objective- Vascular calcification (VC) is age dependent and a risk factor for cardiovascular and all-cause mortality. VC involves the senescence-induced transdifferentiation of vascular smooth muscle cells (SMCs) toward an osteochondrogenic lineage resulting in arterial wall mineralization. miR-34a increases with age in aortas and induces vascular SMC senescence through the modulation of its target SIRT1 (sirtuin 1). In this study, we aimed to investigate whether miR-34a regulates VC. Approach and Results- We found that miR-34a and Runx2 (Runt-related transcription factor 2) expression correlates in young and old mice. Mir34a+/+ and Mir34a-/- mice were treated with vitamin D, and calcium quantification revealed that Mir34a deficiency reduces soft tissue and aorta medial calcification and the upregulation of the VC Sox9 (SRY [sex-determining region Y]-box 9) and Runx2 and the senescence p16 and p21 markers. In this model, miR-34a upregulation was transient and preceded aorta mineralization. Mir34a-/- SMCs were less prone to undergo senescence and under osteogenic conditions deposited less calcium compared with Mir34a+/+ cells. Furthermore, unlike in Mir34a+/+ SMC, the known VC inhibitors SIRT1 and Axl (AXL receptor tyrosine kinase) were only partially downregulated in calcifying Mir34a-/- SMC. Strikingly, constitutive miR-34a overexpression to senescence-like levels in human aortic SMCs increased calcium deposition and enhanced Axl and SIRT1 decrease during calcification. Notably, we also showed that miR-34a directly decreased Axl expression in human aortic SMC, and restoration of its levels partially rescued miR-34a-dependent growth arrest. Conclusions- miR-34a promotes VC via vascular SMC mineralization by inhibiting cell proliferation and inducing senescence through direct Axl and SIRT1 downregulation, respectively. This miRNA could be a good therapeutic target for the treatment of VC.

MicroRNA-34a Induces Vascular Smooth Muscle Cells Senescence by SIRT1 Downregulation and Promotes the Expression of Age-Associated Pro-inflammatory Secretory Factors.

Arterial aging is a major risk factor for the occurrence of cardiovascular diseases. The aged artery is characterized by endothelial dysfunction and vascular smooth muscle cells altered physiology together with low-grade chronic inflammation. MicroRNA-34a (miR-34a) has been recently implicated in cardiac, endothelial, and endothelial progenitor cell senescence; however, its contribution to aging-associated vascular smooth muscle cells phenotype has not been explored so far. We found that miR-34a was highly expressed in aortas isolated from old mice. Moreover, its well-known target, the longevity-associated protein SIRT1, was significantly downregulated during aging in both endothelial cells and vascular smooth muscle cells. Increased miR-34a as well as decreased SIRT1 expression was also observed in replicative-senescent human aortic smooth muscle cells. miR-34a overexpression in proliferative human aortic smooth muscle cells caused cell cycle arrest along with enhanced p21 protein levels and evidence of cell senescence. Furthermore, miR-34a ectopic expression induced pro-inflammatory senescence-associated secretory phenotype molecules. Finally, SIRT1 protein significantly decreased upon miR-34a overexpression and restoration of its levels rescued miR-34a-dependent human aortic smooth muscle cells senescence, but not senescence-associated secretory phenotype factors upregulation. Taken together, our findings suggest that aging-associated increase of miR-34a expression levels, by promoting vascular smooth muscle cells senescence and inflammation through SIRT1 downregulation and senescence-associated secretory phenotype factors induction, respectively, may lead to arterial dysfunctions.

The receptor for advanced glycation end-products (RAGE) is only present in mammals, and belongs to a family of cell adhesion molecules (CAMs).

The human receptor for advanced glycation endproducts (RAGE) is a multiligand cell surface protein belonging to the immunoglobulin superfamily, and is involved in inflammatory and immune responses. Most importantly, RAGE is considered a receptor for HMGB1 and several S100 proteins, which are Damage-Associated Molecular Pattern molecules (DAMPs) released during tissue damage. In this study we show that the Ager gene coding for RAGE first appeared in mammals, and is closely related to other genes coding for cell adhesion molecules (CAMs) such as ALCAM, BCAM and MCAM that appeared earlier during metazoan evolution. RAGE is expressed at very low levels in most cells, but when expressed at high levels, it mediates cell adhesion to extracellular matrix components and to other cells through homophilic interactions. Our results suggest that RAGE evolved from a family of CAMs, and might still act as an adhesion molecule, in particular in the lung where it is highly expressed or under pathological conditions characterized by an increase of its protein levels.