Epigenetic mechanisms in cardiovascular complications of diabetes: towards future therapies.

The pathophysiological mechanisms of cardiovascular disease and microvascular complications in diabetes have been extensively studied, but effective methods of prevention and treatment are still lacking. In recent years, DNA methylation, histone modifications, and non-coding RNAs have arisen as possible mechanisms involved in the development, maintenance, and progression of micro- and macro-vascular complications of diabetes. Epigenetic changes have the characteristic of being heritable or deletable. For this reason, they are now being studied as a therapeutic target for the treatment of diabetes and the prevention or for slowing down its complications, aiming to alleviate the personal and social burden of the disease.This review addresses current knowledge of the pathophysiological links between diabetes and cardiovascular complications, focusing on the role of epigenetic modifications, including DNA methylation and histone modifications. In addition, although the treatment of complications of diabetes with "epidrugs" is still far from being a reality and faces several challenges, we present the most promising molecules and approaches in this field.

Persistent epigenetic signals propel a senescence-associated secretory phenotype and trained innate immunity in CD34+ hematopoietic stem cells from diabetic patients.

BACKGROUND: Diabetes-induced trained immunity contributes to the development of atherosclerosis and its complications. This study aimed to investigate in humans whether epigenetic signals involved in immune cell activation and inflammation are initiated in hematopoietic stem/progenitor cells (HSPCs) and transferred to differentiated progeny. METHODS AND RESULTS: High glucose (HG)-exposure of cord blood (CB)-derived HSPCs induced a senescent-associated secretory phenotype (SASP) characterized by cell proliferation lowering, ROS production, telomere shortening, up-regulation of p21 and p27genes, upregulation of NFkB-p65 transcription factor and increased secretion of the inflammatory cytokines TNFα and IL6. Chromatin immunoprecipitation assay (ChIP) of p65 promoter revealed that H3K4me1 histone mark accumulation and methyltransferase SetD7 recruitment, along with the reduction of repressive H3K9me3 histone modification, were involved in NFkB-p65 upregulation of HG-HSPCs, as confirmed by increased RNA polymerase II engagement at gene level. The differentiation of HG-HSPCs into myeloid cells generated highly responsive monocytes, mainly composed of intermediate subsets (CD14hiCD16+), that like the cells from which they derive, were characterized by SASP features and similar epigenetic patterns at the p65 promoter. The clinical relevance of our findings was confirmed in sternal BM-derived HSPCs of T2DM patients. In line with our in vitro model, T2DM HSPCs were characterized by SASP profile and SETD7 upregulation. Additionally, they generated, after myeloid differentiation, senescent monocytes mainly composed of proinflammatory intermediates (CD14hiCD16+) characterized by H3K4me1 accumulation at NFkB-p65 promoter. CONCLUSIONS: Hyperglycemia induces marked chromatin modifications in HSPCs, which, once transmitted to the cell progeny, contributes to persistent and pathogenic changes in immune cell function and composition.

Reduction of Cardiac Fibrosis by Interference With YAP-Dependent Transactivation.

BACKGROUND: Conversion of cardiac stromal cells into myofibroblasts is typically associated with hypoxia conditions, metabolic insults, and/or inflammation, all of which are predisposing factors to cardiac fibrosis and heart failure. We hypothesized that this conversion could be also mediated by response of these cells to mechanical cues through activation of the Hippo transcriptional pathway. The objective of the present study was to assess the role of cellular/nuclear straining forces acting in myofibroblast differentiation of cardiac stromal cells under the control of YAP (yes-associated protein) transcription factor and to validate this finding using a pharmacological agent that interferes with the interactions of the YAP/TAZ (transcriptional coactivator with PDZ-binding motif) complex with their cognate transcription factors TEADs (TEA domain transcription factors), under high-strain and profibrotic stimulation. METHODS: We employed high content imaging, 2-dimensional/3-dimensional culture, atomic force microscopy mapping, and molecular methods to prove the role of cell/nuclear straining in YAP-dependent fibrotic programming in a mouse model of ischemia-dependent cardiac fibrosis and in human-derived primitive cardiac stromal cells. We also tested treatment of cells with Verteporfin, a drug known to prevent the association of the YAP/TAZ complex with their cognate transcription factors TEADs. RESULTS: Our experiments suggested that pharmacologically targeting the YAP-dependent pathway overrides the profibrotic activation of cardiac stromal cells by mechanical cues in vitro, and that this occurs even in the presence of profibrotic signaling mediated by TGF-ß1 (transforming growth factor beta-1). In vivo administration of Verteporfin in mice with permanent cardiac ischemia reduced significantly fibrosis and morphometric remodeling but did not improve cardiac performance. CONCLUSIONS: Our study indicates that preventing molecular translation of mechanical cues in cardiac stromal cells reduces the impact of cardiac maladaptive remodeling with a positive effect on fibrosis.

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.

Liraglutide preserves CD34+ stem cells from dysfunction Induced by high glucose exposure.

BACKGROUND: Glucagon like peptide-1 receptor agonists (GLP-1RAs) have shown to reduce mortality and cardiovascular events in patients with type 2 diabetes mellitus (T2DM). Since the impairment in number and function of vasculotrophic circulating CD34+ hematopoietic stem progenitor cells (HSPCs) in T2D has been reported to increase cardiovascular (CV) risk, we hypothesized that one of the mechanisms whereby GLP-1 RAs exert CV protective effects may be related to the ability to improve CD34+ HSPC function. METHODS: In cord blood (CB)-derived CD34+ HSPC, the expression of GLP-1 receptor (GLP-1R) mRNA, receptor protein and intracellular signaling was evaluated by RT-qPCR and Western Blot respectively. CD34+ HSPCs were exposed to high glucose (HG) condition and GLP-1RA liraglutide (LIRA) was added before as well as after functional impairment. Proliferation, CXCR4/SDF-1α axis activity and intracellular ROS production of CD34+ HSPC were evaluated. RESULTS: CD34+ HSPCs express GLP-1R at transcriptional and protein level. LIRA treatment prevented and rescued HSPC proliferation, CXCR4/SDF-1α axis activity and metabolic imbalance from HG-induced impairment. LIRA stimulation promoted intracellular cAMP accumulation as well as ERK1/2 and AKT signaling activation. The selective GLP-1R antagonist exendin (9-39) abrogated LIRA-dependent ERK1/2 and AKT phosphorylation along with the related protective effects. CONCLUSION: We provided the first evidence that CD34+ HSPC express GLP-1R and that LIRA can favorably impact on cell dysfunction due to HG exposure. These findings open new perspectives on the favorable CV effects of GLP-1 RAs in T2DM patients.

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.

Prothrombin is a binding partner of the human receptor of advanced glycation end products.

The receptor for advanced glycation end products (RAGE) plays a key role in mammal physiology and in the etiology and progression of inflammatory and oxidative stress-based diseases. In adults, RAGE expression is normally high only in the lung where the protein concentrates in the basal membrane of alveolar Type I epithelial cells. In diseases, RAGE levels increase in the affected tissues and sustain chronic inflammation. RAGE exists as a membrane glycoprotein with an ectodomain, a transmembrane helix, and a short carboxyl-terminal tail, or as a soluble ectodomain that acts as a decoy receptor (sRAGE). VC1 domain is responsible for binding to the majority of RAGE ligands including advanced glycation end products (AGEs), S100 proteins, and HMGB1. To ascertain whether other ligands exist, we analyzed by MS the material pulled down by VC1 from human plasma. Twenty of 295 identified proteins were selected and associated to coagulation and complement processes and to extracellular matrix. Four of them contained a γ-carboxyl glutamic acid (Gla) domain, a calcium-binding module, and prothrombin (PT) was the most abundant. Using MicroScale thermophoresis, we quantified the interaction of PT with VC1 and sRAGE in the absence or presence of calcium that acted as a competitor. PT devoid of the Gla domain (PT des-Gla) did not bind to sRAGE, providing further evidence that the Gla domain is critical for the interaction. Finally, the presence of VC1 delayed plasma clotting in a dose-dependent manner. We propose that RAGE is involved in modulating blood coagulation presumably in conditions of lung injury.

Diabetes Induces a Transcriptional Signature in Bone Marrow-Derived CD34+ Hematopoietic Stem Cells Predictive of Their Progeny Dysfunction.

Hematopoietic stem/progenitor cells (HSPCs) participate in cardiovascular (CV) homeostasis and generate different types of blood cells including lymphoid and myeloid cells. Diabetes mellitus (DM) is characterized by chronic increase of pro-inflammatory mediators, which play an important role in the development of CV disease, and increased susceptibility to infections. Here, we aimed to evaluate the impact of DM on the transcriptional profile of HSPCs derived from bone marrow (BM). Total RNA of BM-derived CD34+ stem cells purified from sternal biopsies of patients undergoing coronary bypass surgery with or without DM (CAD and CAD-DM patients) was sequenced. The results evidenced 10566 expressed genes whose 79% were protein-coding genes, and 21% non-coding RNA. We identified 139 differentially expressed genes (p-value < 0.05 and |log2 FC| > 0.5) between the two comparing groups of CAD and CAD-DM patients. Gene Set Enrichment Analysis (GSEA), based on Gene Ontology biological processes (GO-BP) terms, led to the identification of fourteen overrepresented biological categories in CAD-DM samples. Most of the biological processes were related to lymphocyte activation, chemotaxis, peptidase activity, and innate immune response. Specifically, HSPCs from CAD-DM patients displayed reduced expression of genes coding for proteins regulating antibacterial and antivirus host defense as well as macrophage differentiation and lymphocyte emigration, proliferation, and differentiation. However, within the same biological processes, a consistent number of inflammatory genes coding for chemokines and cytokines were up-regulated. Our findings suggest that DM induces transcriptional alterations in HSPCs, which are potentially responsible of progeny dysfunction.

The Janus face of HMGB1 in heart disease: a necessary update.

High mobility group box 1 (HMGB1) is a ubiquitous nuclear protein involved in transcription regulation, DNA replication and repair and nucleosome assembly. HMGB1 is passively released by necrotic tissues or actively secreted by stressed cells. Extracellular HMGB1 acts as a damage-associated molecular pattern (DAMPs) molecule and gives rise to several redox forms that by binding to different receptors and interactors promote a variety of cellular responses, including tissue inflammation or regeneration. Inhibition of extracellular HMGB1 in experimental models of myocardial ischemia/reperfusion injury, myocarditis, cardiomyopathies induced by mechanical stress, diabetes, bacterial infection or chemotherapeutic drugs reduces inflammation and is protective. In contrast, administration of HMGB1 after myocardial infarction induced by permanent coronary artery ligation ameliorates cardiac performance by promoting tissue regeneration. HMGB1 decreases contractility and induces hypertrophy and apoptosis in cardiomyocytes, stimulates cardiac fibroblast activities, and promotes cardiac stem cell proliferation and differentiation. Interestingly, maintenance of appropriate nuclear HMGB1 levels protects cardiomyocytes from apoptosis by preventing DNA oxidative stress, and mice with HMGB1cardiomyocyte-specific overexpression are partially protected from cardiac damage. Finally, higher levels of circulating HMGB1 are associated to human heart diseases. Hence, during cardiac injury, HMGB1 elicits both harmful and beneficial responses that may in part depend on the generation and stability of the diverse redox forms, whose specific functions in this context remain mostly unexplored. This review summarizes recent findings on HMGB1 biology and heart dysfunctions and discusses the therapeutic potential of modulating its expression, localization, and oxidative-dependent activities.

miR-34a: A Promising Target for Inflammaging and Age-Related Diseases.

The term "inflammaging" describes the chronic, low-grade systemic inflammation that occurs during physiological aging in the absence of an overt infection [...].

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.

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.

Non-oxidizable HMGB1 induces cardiac fibroblasts migration via CXCR4 in a CXCL12-independent manner and worsens tissue remodeling after myocardial infarction.

Myocardial infarction (MI) is a major health burden worldwide. Extracellular High mobility group box 1 (HMGB1) regulates tissue healing after injuries. The reduced form of HMGB1 (fr-HMGB1) exerts chemotactic activity by binding CXCL12 through CXCR4, while the disulfide form, (ds-HMGB1), induces cytokines expression by TLR4. Here, we assessed the role of HMGB1 redox forms and the non-oxidizable mutant (3S) on human cardiac fibroblast (hcFbs) functions and cardiac remodeling after infarction. Among HMGB1 receptors, hcFbs express CXCR4. Fr-HMGB1 and 3S, but not ds-HMGB1, promote hcFbs migration through Src activation, while none of HMGB1 redox forms induces proliferation or inflammatory mediators. 3S is more effective than fr-HMGB1 in stimulating hcFbs migration and Src phosphorylation being active at lower concentrations and in oxidizing conditions. Notably, chemotaxis toward both proteins is CXCR4-dependent but, in contrast to fr-HMGB1, 3S does not require CXCL12 since hcFbs migration persists in the presence of the CXCL12/CXCR4 inhibitor AMD3100 or an anti-CXCL12 antibody. Interestingly, 3S interacts with CXCR4 and induces a different receptor conformation than CXCL12. Mice undergoing MI and receiving 3S exhibit adverse LV remodeling owing to an excessive collagen deposition promoted by a higher number of myofibroblasts. On the contrary, fr-HMGB1 ameliorates cardiac performance enhancing neoangiogenesis and reducing the infarcted area and fibrosis. Altogether, our results demonstrate that non-oxidizable HMGB1 induce a sustained cardiac fibroblasts migration despite the redox state of the environment and by altering CXCL12/CXCR4 axis. This affects proper cardiac remodeling after an infarction.

The shedding-derived soluble receptor for advanced glycation endproducts sustains inflammation during acute Pseudomonas aeruginosa lung infection.

BACKGROUND: The membrane-bound isoform of the receptor for advanced glycation end products (FL-RAGE) is primarily expressed by alveolar epithelial cells and undergoes shedding by the protease ADAM10, giving rise to soluble cleaved RAGE (cRAGE). RAGE has been associated with the pathogenesis of several acute and chronic lung disorders. Whether the proteolysis of FL-RAGE is altered by a given inflammatory stimulus is unknown. Pseudomonas aeruginosa causes nosocomial infections in hospitalized patients and is the major pathogen associated with chronic lung diseases. METHODS: P. aeruginosa was injected in Rage-/- and wild-type mice and the impact on RAGE expression and shedding, levels of inflammation and bacterial growth was determined. RESULTS: Acute P. aeruginosa lung infection in mice induces a reduction of the active form of ADAM10, which determines an increase of FL-RAGE expression on alveolar cells and a concomitant decrease of pulmonary cRAGE levels. This was associated with massive recruitment of leukocytes and release of pro-inflammatory factors, tissue damage and relocation of cRAGE in the alveolar and bronchial cavities. The administration of sRAGE worsened bacterial burden and neutrophils infiltration. RAGE genetic deficiency reduced the susceptibility to P. aeruginosa infection, mitigating leukocyte recruitment, inflammatory molecules production, and bacterial growth. CONCLUSIONS: These data are the first to suggest that inhibition of FL-RAGE shedding, by affecting the FL-RAGE/cRAGE levels, is a novel mechanism for controlling inflammation to acute P. aeruginosa pneumonia. sRAGE in the alveolar space sustains inflammation in this setting. GENERAL SIGNIFICANCE: RAGE shedding may determine the progression of inflammatory lung diseases.

The mitochondrial lncRNA ASncmtRNA-2 is induced in aging and replicative senescence in Endothelial Cells.

Age-associated cardiovascular diseases are at least partially ascribable to vascular cell senescence. Replicative senescence (RS) and stress-induced premature senescence (SIPS) are provoked respectively by endogenous (telomere erosion) and exogenous (H2O2, UV) stimuli resulting in cell cycle arrest in G1 and G2 phases. In both scenarios, mitochondria-derived ROS are important players in senescence initiation. We aimed to define whether a mtDNA-transcribed long-non-coding-RNA (lncRNA), ASncmtRNA-2, has a role in vascular aging and senescence. Aortas of old mice, characterized by increased senescence, showed an increment in ASncmtRNA-2 expression. In vitro analysis of Endothelial Cells (EC) and Vascular Smooth Muscle Cells (VSMC) established that ASncmtRNA-2 is induced in EC, but not in VSMC, during RS. Surprisingly, ASncmtRNA-2 is not upregulated in two different EC SIPS scenarios, treated with H2O2 and UV. The p16 gene displayed similar ASncmtRNA-2 expression patterns, suggesting a possible co-regulation of the two genes. Interestingly, the expression of two miRNAs, hsa-miR-4485 and hsa-miR-1973, with perfect homology to the double strand region of ASncmtRNA-2 and originating at least in part from a mitochondrial transcript, was induced in RS, opening to the possibility that this lncRNA functions as a non-canonical precursor of these miRNAs. Cell cycle analysis of EC transiently over-expressing ASncmtRNA-2 revealed an accumulation of EC in the G2/M phase, but not in the G1 phase. We propose that ASncmtRNA-2 in EC might be involved in the RS establishment by participating in the cell cycle arrest in G2/M phase, possibly through the production of hsa-miR-4485 and hsa-miR-1973. This article is part of a Special Issue entitled: Mitochondria.

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.

BPIFB4 and its longevity-associated haplotype protect from cardiac ischemia in humans and mice.

Long-living individuals (LLIs) escape age-related cardiovascular complications until the very last stage of life. Previous studies have shown that a Longevity-Associated Variant (LAV) of the BPI Fold Containing Family B Member 4 (BPIFB4) gene correlates with an extraordinarily prolonged life span. Moreover, delivery of the LAV-BPIFB4 gene exerted therapeutic action in murine models of atherosclerosis, limb ischemia, diabetic cardiomyopathy, and aging. We hypothesize that downregulation of BPIFB4 expression marks the severity of coronary artery disease (CAD) in human subjects, and supplementation of the LAV-BPIFB4 protects the heart from ischemia. In an elderly cohort with acute myocardial infarction (MI), patients with three-vessel CAD were characterized by lower levels of the natural logarithm (Ln) of peripheral blood BPIFB4 (p = 0.0077). The inverse association between Ln BPIFB4 and three-vessel CAD was confirmed by logistic regression adjusting for confounders (Odds Ratio = 0.81, p = 0.0054). Moreover, in infarcted mice, a single administration of LAV-BPIFB4 rescued cardiac function and vascularization. In vitro studies showed that LAV-BPIFB4 protein supplementation exerted chronotropic and inotropic actions on induced pluripotent stem cell (iPSC)-derived cardiomyocytes. In addition, LAV-BPIFB4 inhibited the pro-fibrotic phenotype in human cardiac fibroblasts. These findings provide a strong rationale and proof of concept evidence for treating CAD with the longevity BPIFB4 gene/protein.

Ischemic wound revascularization by the stromal vascular fraction relies on host-donor hybrid vessels.

Nonhealing wounds place a significant burden on both quality of life of affected patients and health systems. Skin substitutes are applied to promote the closure of nonhealing wounds, although their efficacy is limited by inadequate vascularization. The stromal vascular fraction (SVF) from the adipose tissue is a promising therapy to overcome this limitation. Despite a few successful clinical trials, its incorporation in the clinical routine has been hampered by their inconsistent results. All these studies concluded by warranting pre-clinical work aimed at both characterizing the cell types composing the SVF and shedding light on their mechanism of action. Here, we established a model of nonhealing wound, in which we applied the SVF in combination with a clinical-grade skin substitute. We purified the SVF cells from transgenic animals to trace their fate after transplantation and observed that it gave rise to a mature vascular network composed of arteries, capillaries, veins, as well as lymphatics, structurally and functionally connected with the host circulation. Then we moved to a human-in-mouse model and confirmed that SVF-derived endothelial cells formed hybrid human-mouse vessels, that were stabilized by perivascular cells. Mechanistically, SVF-derived endothelial cells engrafted and expanded, directly contributing to the formation of new vessels, while a population of fibro-adipogenic progenitors stimulated the expansion of the host vasculature in a paracrine manner. These data have important clinical implications, as they provide a steppingstone toward the reproducible and effective adoption of the SVF as a standard care for nonhealing wounds.

Inhibitor of NF-kappa B kinases alpha and beta are both essential for high mobility group box 1-mediated chemotaxis [corrected].

Inhibitor of NF-kappaB kinases beta (IKKbeta) and alpha (IKKalpha) activate distinct NF-kappaB signaling modules. The IKKbeta/canonical NF-kappaB pathway rapidly responds to stress-like conditions, whereas the IKKalpha/noncanonical pathway controls adaptive immunity. Moreover, IKKalpha can attenuate IKKbeta-initiated inflammatory responses. High mobility group box 1 (HMGB1), a chromatin protein, is an extracellular signal of tissue damage-attracting cells in inflammation, tissue regeneration, and scar formation. We show that IKKalpha and IKKbeta are each critically important for HMGB1-elicited chemotaxis of fibroblasts, macrophages, and neutrophils in vitro and neutrophils in vivo. By time-lapse microscopy we dissected different parameters of the HMGB1 migration response and found that IKKalpha and IKKbeta are each essential to polarize cells toward HMGB1 and that each kinase also differentially affects cellular velocity in a time-dependent manner. In addition, HMGB1 modestly induces noncanonical IKKalpha-dependent p52 nuclear translocation and p52/RelB target gene expression. Akin to IKKalpha and IKKbeta, p52 and RelB are also required for HMGB1 chemotaxis, and p52 is essential for cellular orientation toward an HMGB1 gradient. RAGE, a ubiquitously expressed HMGB1 receptor, is required for HMGB1 chemotaxis. Moreover, IKKbeta, but not IKKalpha, is required for HMGB1 to induce RAGE mRNA, suggesting that RAGE is at least one IKKbeta target involved in HMGB1 migration responses, and in accord with these results enforced RAGE expression rescues the HMGB1 migration defect of IKKbeta, but not IKKalpha, null cells. Thus, proinflammatory HMGB1 chemotactic responses mechanistically require the differential collaboration of both IKK-dependent NF-kappaB signaling pathways.