Impact of sirtuin-1 expression on H3K56 acetylation and oxidative stress: a double-blind randomized controlled trial with resveratrol supplementation

Abstract Aims Sirtuin-1 (SIRT-1) down-regulation in type 2 diabetes mellitus (T2DM) has been associated with epigenetic markers of oxidative stress. We herein aim to evaluate whether an increase in SIRT-1 expression affects histone 3 acetylation at the 56 lysine residue (H3K56ac) in T2DM patients randomly selected to receive either resveratrol (40 mg or 500 mg) or a placebo for 6 months. The primary outcome is changes in the H3K56ac level by variation in SIRT-1 expression and the secondary outcome is the evidence of association between SIRT-1 level, antioxidant markers (TAS), and metabolic variables. Methods and results At baseline, peripheral blood mononuclear cell H3K56ac values among the SIRT-1 tertiles did not differ. At trial end, SIRT-1 levels were significantly higher in patients receiving 500 mg resveratrol. At follow-up, patients were divided into tertiles of delta (trial end minus baseline) SIRT-1 value. Significant reductions in H3K56ac and body fat percentage were found in the highest tertile as were increased TAS levels. A multiple logistic regression model showed that the highest delta SIRT-1 tertile was inversely associated with variations in H3K56ac (OR = 0.66; 95% CI 0.44–0.99), TAS (OR = 1.01; 95% CI 1.00–1.02), and body fat percentage (OR = 0.75; 95% CI 0.58–0.96). Conclusions We provide new knowledge on H3K56ac and SIRT-1 association in T2DM. These data suggest that boosting SIRT-1 expression/activation may impact redox homeostasis in these patients. ClinicalTrials.gov Identifier NCT02244879. </jats:sec

PDGF-BB Carried by Endothelial Cell-Derived Extracellular Vesicles Reduces Vascular Smooth Muscle Cell Apoptosis in Diabetes

Endothelial cell-derived extracellular vesicles (CD31EVs) constitute a new entity for therapeutic/prognostic purposes. The roles of CD31EVs as mediators of vascular smooth muscle cell (VSMC) dysfunction in type 2 diabetes (T2D) are investigated herein. We demonstrated that, unlike serum-derived extracellular vesicles in individuals without diabetes, those in individuals with diabetes (D CD31EVs) boosted apoptosis resistance of VSMCs cultured in hyperglycemic condition. Biochemical analysis revealed that this effect relies on changes in the balance between antiapoptotic and proapoptotic signals: increase of bcl-2 and decrease of bak/bax. D CD31EV cargo analysis demonstrated that D CD31EVs are enriched in membrane-bound platelet-derived growth factor-BB (mbPDGF-BB). Thus, we postulated that mbPDGF-BB transfer by D CD31EVs could account for VSMC resistance to apoptosis. By depleting CD31EVs of platelet-derived growth factor-BB (PDGF-BB) or blocking the PDGF receptor β on VSMCs, we demonstrated that mbPDGF-BB contributes to D CD31EV-mediated bak/bax and bcl-2 levels. Moreover, we found that bak expression is under the control of PDGF-BB-mediated microRNA (miR)-296-5p expression. In fact, while PDGF-BB treatment recapitulated D CD31EV-mediated antiapoptotic program and VSMC resistance to apoptosis, PDGF-BB-depleted CD31EVs failed. D CD31EVs also increased VSMC migration and recruitment to neovessels by means of PDGF-BB. Finally, we found that VSMCs, from human atherosclerotic arteries of individuals with T2D, express low bak/bax and high bcl-2 and miR-296-5p levels. This study identifies the mbPDGF-BB in D CD31EVs as a relevant mediator of diabetes-associated VSMC resistance to apoptosis

Unacylated Ghrelin Promotes Skeletal Muscle Regeneration Following Hindlimb Ischemia via SOD-2-Mediated miR-221/222 Expression

BACKGROUND: Surgical treatment of peripheral artery disease, even if successful, does not prevent reoccurrence. Under these conditions, increased oxidative stress is a crucial determinant of tissue damage. Given its reported antioxidant effects, we investigated the potential of unacylated‐ghrelin (UnAG) to reduce ischemia‐induced tissue damage in a mouse model of peripheral artery disease. METHODS AND RESULTS: We show that UnAG but not acylated ghrelin (AG) induces skeletal muscle regeneration in response to ischemia via canonical p38/mitogen‐actived protein kinase signaling UnAG protected against reactive oxygen species–induced cell injuries by inducing the expression of superoxide dismutase‐2 (SOD‐2) in satellite cells. This led to a reduced number of infiltrating CD68(+) cells and was followed by induction of the myogenic process and a reduction in functional impairment. Moreover, we found that miR‐221/222, previously linked to muscle regeneration processes, was up‐regulated and negatively correlated with p57(Kip2) expression in UnAG‐treated mice. UnAG, unlike AG, promoted cell‐cycle entry in satellite cells of mice lacking the genes for ghrelin and its receptor (GHSR1a). UnAG‐induced p38/mitogen‐actived protein kinase phosphorylation, leading to activation of the myogenic process, was prevented in SOD‐2–depleted SCs. By siRNA technology, we also demonstrated that SOD‐2 is the antioxidant enzyme involved in the control of miR‐221/222–driven posttranscriptional p57(Kip2) regulation. Loss‐of‐function experiments targeting miR‐221/222 and local pre–miR‐221/222 injection in vivo confirmed a role for miR‐221/222 in driving skeletal muscle regeneration after ischemia. CONCLUSIONS: These results indicate that UnAG‐induced skeletal muscle regeneration after ischemia depends on SOD‐2–induced miR‐221/222 expression and highlight its clinical potential for the treatment of reactive oxygen species–mediated skeletal muscle damage

Activated Stat5 trafficking Via Endothelial Cell-derived Extracellular Vesicles Controls IL-3 Pro-angiogenic Paracrine Action.

Soluble factors and cell-derived extracellular vesicles (EVs) control vascular cell fate during inflammation. The present study investigates the impact of Interleukin 3 (IL-3) on EV release by endothelial cells (ECs), the mechanisms involved in EV release and paracrine actions. We found that IL-3 increases EV release, which is prevented by IL-3Ralpha blockade. EVs released upon IL-3 stimulation were able to induce pro-angiogenic signals as shown by chromatin immunoprecipitation (ChIP) assay performed on the promoter region of cyclin D1 and tridimensional tube-like structure formation. We herein demonstrate that these effects rely on the transfer of miR-126-3p, pre-miR-126 and, more importantly, of activated signal transduction and activator of transcription 5 (pSTAT5) from IL-3-EV cargo into recipient ECs. We show, using the dominant negative form (ΔN)STAT5 and an activated STAT5 (1*6STAT5) constructs, that STAT5 drives IL-3-mediated EV release, miR-126-3p and pSTAT5 content. Finally, using EVs recovered from ΔNSTAT5 expressing ECs, we provide evidence that miR-126-3p and pSTAT5 trafficking is relevant for IL-3-mediated paracrine pro-angiogenic signals. These results indicate that IL-3 regulates EC-EV release, cargo and IL-3 angiogenic paracrine action via STAT5. Moreover, these results provide evidence that EC-derived IL-3-EVs can serve as pro-angiogenic clinical delivery wound healing devices

Unacylated ghrelin prevents mitochondrial dysfunction in a model of ischemia/reperfusion liver injury

Ischemia/reperfusion (I/R) injury is a common cause of liver dysfunction during hepatectomy, liver transplantation procedures and in generalized shock. Although effort has been dedicated to rescuing tissue damage in these clinical settings, there is still an urgent need for an effective treatment to protect the liver from the burden of I/R injury. In this study, we have investigated the potential clinical impact of unacylated-ghrelin (UnAG) in a liver I/R rat model. Particular attention has been paid to mitochondria. We demonstrate that UnAG was able to reduce the lag-phase time in response to ADP administration and increase oxygen consumption in ex vivo experiments using liver mitochondria recovered from rats subjected to I/R. Moreover, we found that UnAG rescued the expression of a key regulator of mitochondrial morphology and electron transport chain function; the optic atrophy 1 (Opa1) protein. Cytochrome c oxidase (COX), ATP synthase (complex V) activity and mitochondrial permeability transition pore (mPTP) opening were also affected by UnAG administration in vivo. An in vitro, hepatic I/R model was used to validate these data. We demonstrate that UnAG upregulates the expression of Cox subunit IV (CoxIV) and increases cellular ATP content. This results in Bcl-2 upregulation and protection against apoptosis. Opa1 silencing shows that Opa1 is crucial for a UnAG-induced increase in cellular ATP content, apoptosis resistance, Bcl-2 and CoxIV expression. Finally, we show that UnAG improves Opa1's interaction with MIC60 in the I/R setting, hinting at its role in cristae shape regulation. Our results demonstrate that UnAG administration rescues the intrinsic mitochondrial pathway triggered by I/R damage. Opa1's contribution in mediating this effect is also reported. This suggests that UnAG can interfere with mitochondrial dysfunction, via Opa1, in a preclinical liver I/R model. We therefore provide the rationale for exploiting UnAG as an alternative means to rescuing mitochondrial damage and organ dysfunction