Donor-derived Cell-free DNA Evaluation in Pediatric Heart Transplant Recipients: A Single-center 12-mo Experience.

Background: Endomyocardial biopsy (EMB) is considered the gold-standard method to diagnose rejection after heart transplantation. However, the many disadvantages and potential complications of this test restrict its routine application, particularly in pediatric patients. Donor-derived cell-free DNA (dd-cfDNA), released by the transplanted heart as result of cellular injury, is emerging as a biomarker of tissue damage involved in ischemia/reperfusion injury and posttransplant rejection. In the present study, we systematically evaluated dd-cfDNA levels in pediatric heart transplant patients coming for follow-up visits to our clinic for 12 mo, with the aim of determining whether dd-cfDNA monitoring could be efficiently applied and integrated into the posttransplant management of rejection in pediatric recipients. Methods: Twenty-nine patients were enrolled, and cfDNA was obtained from 158 blood samples collected during posttransplant follow-up. dd-cfDNA% was determined with a droplet-digital polymerase chain reaction assay. EMB scores, donor-specific antibody measurements, and distress marker quantification were correlated with dd-cfDNA, together with echocardiogram information. Results: The percentage of dd-cfDNA increased when EMBs scored positive for rejection (P = 0.0002) and donor-specific antibodies were present (P = 0.0010). N-terminal pro-B-type natriuretic peptide and high-sensitive troponin I elevation were significantly associated with dd-cfDNA release (P = 0.02 and P < 0.0001, respectively), as were reduced isovolumetric relaxation time (P = 0.0031), signs of heart failure (P = 0.0018), and treatment for rejection (P = 0.0017). By determining a positive threshold for rejection at 0.55%, the test had a negative predictive value maximized at 100%. Conclusions: Collectively, results indicate that dd-cfDNA monitoring has a high negative prognostic value, suggesting that in heart transplanted children with dd-cfDNA levels of <0.55% threshold, protocol EMBs may be postponed.

Technical Advances in Circulating Cell-Free DNA Detection and Analysis for Personalized Medicine in Patients' Care.

Circulating cell-free DNA (cfDNA) refers to small fragments of DNA molecules released after programmed cell death and necrosis in several body fluids such as blood, saliva, urine, and cerebrospinal fluid. The discovery of cfDNA has revolutionized the field of non-invasive diagnostics in the oncologic field, in prenatal testing, and in organ transplantation. Despite the potential of cfDNA and the solid results published in the recent literature, several challenges remain, represented by a low abundance, a need for highly sensitive assays, and analytical issues. In this review, the main technical advances in cfDNA analysis are presented and discussed, with a comprehensive examination of the current available methodologies applied in each field. Considering the potential advantages of cfDNA, this biomarker is increasing its consensus among clinicians, as it allows us to monitor patients' conditions in an easy and non-invasive way, offering a more personalized care. Nevertheless, cfDNA analysis is still considered a diagnostic marker to be further validated, and very few centers are implementing its analysis in routine diagnostics. As technical improvements are enhancing the performances of cfDNA analysis, its application will transversally improve patients' quality of life.

Validation of a Simple, Rapid, and Cost-Effective Method for Acute Rejection Monitoring in Lung Transplant Recipients.

Despite advances in immunosuppression therapy, acute rejection remains the leading cause of graft dysfunction in lung transplant recipients. Donor-derived cell-free DNA is increasingly being considered as a valuable biomarker of acute rejection in several solid organ transplants. We present a technically improved molecular method based on digital PCR that targets the mismatch between the recipient and donor at the HLA-DRB1 locus. Blood samples collected sequentially post-transplantation from a cohort of lung recipients were used to obtain proof-of-principle for the validity of the assay, correlating results with transbronchial biopsies and lung capacity tests. The results revealed an increase in dd-cfDNA during the first 2 weeks after transplantation related to ischemia-reperfusion injury (6.36 ± 5.36%, p < 0.0001). In the absence of complications, donor DNA levels stabilized, while increasing again during acute rejection episodes (7.81 ± 12.7%, p < 0.0001). Respiratory tract infections were also involved in the release of dd-cfDNA (9.14 ± 15.59%, p = 0.0004), with a positive correlation with C-reactive protein levels. Overall, the dd-cfDNA percentages were inversely correlated with the lung function values measured by spirometry. These results confirm the value of dd-cfDNA determination during post-transplant follow-up to monitor acute rejection in lung recipients, achieved using a rapid and inexpensive approach based on the HLA mismatch between donor and recipient.

HLA-DRB1 mismatch-based identification of donor-derived cell free DNA (dd-cfDNA) as a marker of rejection in heart transplant recipients: A single-institution pilot study.

BACKGROUND: Donor-derived cell-free DNA (dd-cfDNA) is considered a reliable marker of organ damage with potential applications in the follow-up of transplant recipients. METHODS: In this work we present an assay based on the donor-recipient HLA-mismatch (human leukocyte antigen) at the HLA-DRB1 locus to monitor rejection by quantifying the percentage of dd-cfDNA using a droplet digital PCR (polymerase chain reaction) technique. A panel of probes targeting the HLA-DRB1 locus and covering >85% genetic variability was validated and used to assess dd-cfDNA levels in a prospective cohort of 19 adult heart transplant recipients (mean age 50.9±14.8 years). The assay was carried out on a total of 232 liquid biopsies collected at the same time as endomyocardial biopsy (EMB) during routine post-transplant follow-up. RESULTS: Results show a significant increase of dd-cfDNA related to ischemia-reperfusion injury (2.22±2.09%) and to acute cellular rejection (1.71±3.10%) compared to stable conditions (0.43±1.04%, p < 0.0001). On the contrary, no increase was observed during infections or vascular complications, underlining the potential role of this biomarker for rejection monitoring. With a cut-off of 0.11%, the test showed 70.8% specificity (95% CI, 58.17% - 81.40%) and 64.2% sensitivity (95% CI, 49.80% - 76.86%) in discriminating acute rejection from no rejection. CONCLUSIONS: These data demonstrate that this HLA mismatch-based droplet digital PCR method is effective for monitoring rejection in heart transplant recipients. Compared to next generation sequencing approaches, it is far more flexible, less expensive and provides faster results.

Differential Therapeutic Effect of Extracellular Vesicles Derived by Bone Marrow and Adipose Mesenchymal Stem Cells on Wound Healing of Diabetic Ulcers and Correlation to Their Cargoes.

Extracellular vesicles (EVs) derived from mesenchymal stem cells isolated from both bone marrow (BMSCs) and adipose tissue (ADSCs) show potential therapeutic effects. These vesicles often show a similar beneficial effect on tissue regeneration, but in some contexts, they exert different biological properties. To date, a comparison of their molecular cargo that could explain the different biological effect is not available. Here, we demonstrated that ADSC-EVs, and not BMSC-EVs, promote wound healing on a murine model of diabetic wounds. Besides a general similarity, the bioinformatic analysis of their protein and miRNA cargo highlighted important differences between these two types of EVs. Molecules present exclusively in ADSC-EVs were highly correlated to angiogenesis, whereas those expressed in BMSC-EVs were preferentially involved in cellular proliferation. Finally, in vitro analysis confirmed that both ADSC and BMSC-EVs exploited beneficial effect on cells involved in skin wound healing such as fibroblasts, keratinocytes and endothelial cells, but through different cellular processes. Consistent with the bioinformatic analyses, BMSC-EVs were shown to mainly promote proliferation, whereas ADSC-EVs demonstrated a major effect on angiogenesis. Taken together, these results provide deeper comparative information on the cargo of ADSC-EVs and BMSC-EVs and the impact on regenerative processes essential for diabetic wound healing.

Adipocyte-derived extracellular vesicles regulate survival and function of pancreatic ß cells.

Extracellular vesicles (EVs) are implicated in the crosstalk between adipocytes and other metabolic organs, and an altered biological cargo has been observed in EVs from human obese adipose tissue (AT). Yet, the role of adipocyte-derived EVs in pancreatic ß cells remains to be determined. Here, we explored the effects of EVs released from adipocytes isolated from both rodents and humans and human AT explants on survival and function of pancreatic ß cells and human pancreatic islets. EVs from healthy 3T3-L1 adipocytes increased survival and proliferation and promoted insulin secretion in INS-1E ß cells and human pancreatic islets, both those untreated or exposed to cytokines or glucolipotoxicity, whereas EVs from inflamed adipocytes caused ß cell death and dysfunction. Human lean adipocyte-derived EVs produced similar beneficial effects, whereas EVs from obese AT explants were harmful for human EndoC-ßH3 ß cells. We observed differential expression of miRNAs in EVs from healthy and inflamed adipocytes, as well as alteration in signaling pathways and expression of ß cell genes, adipokines, and cytokines in recipient ß cells. These in vitro results suggest that, depending on the physiopathological state of AT, adipocyte-derived EVs may influence ß cell fate and function.

Clinical exome sequencing is a powerful tool in the diagnostic flow of monogenic kidney diseases: an Italian experience.

BACKGROUND: A considerable minority of patients on waiting lists for kidney transplantation either have no diagnosis (and fall into the subset of undiagnosed cases) because kidney biopsy was not performed or histological findings were non-specific, or do not fall into any well-defined clinical category. Some of these patients might be affected by a previously unrecognised monogenic disease. METHODS: Through a multidisciplinary cooperative effort, we built an analytical pipeline to identify patients with chronic kidney disease (CKD) with a clinical suspicion of a monogenic condition or without a well-defined diagnosis. Following the stringent phenotypical and clinical characterization required by the flowchart, candidates meeting these criteria were further investigated by clinical exome sequencing followed by in silico analysis of 225 kidney-disease-related genes. RESULTS: By using an ad hoc web-based platform, we enrolled 160 patients from 13 different Nephrology and Genetics Units located across the Piedmont region over 15 months. A preliminary "remote" evaluation based on well-defined inclusion criteria allowed us to define eligibility for NGS analysis. Among the 138 recruited patients, 52 (37.7%) were children and 86 (62.3%) were adults. Up to 48% of them had a positive family history for kidney disease. Overall, applying this workflow led to the identification of genetic variants potentially explaining the phenotype in 78 (56.5%) cases. CONCLUSIONS: These results underline the importance of clinical exome sequencing as a versatile and highly useful, non-invasive tool for genetic diagnosis of kidney diseases. Identifying patients who can benefit from targeted therapies, and improving the management of organ transplantation are further expected applications.

Extracellular Vesicles From Adipose Stem Cells Prevent Muscle Damage and Inflammation in a Mouse Model of Hind Limb Ischemia: Role of Neuregulin-1.

OBJECTIVES: Critical hindlimb ischemia is a severe consequence of peripheral artery disease. Surgical treatment does not prevent skeletal muscle impairment or improve long-term patient outcomes. The present study investigates the protective/regenerative potential and the mechanism of action of adipose stem cell-derived extracellular vesicles (ASC-EVs) in a mouse model of hindlimb ischemia. Approach and Results: We demonstrated that ASC-EVs exert a protective effect on muscle damage by acting both on tissue microvessels and muscle cells. The genes involved in muscle regeneration were up-regulated in the ischemic muscles of ASC-EV-treated animals. MyoD expression has also been confirmed in satellite cells. This was followed by a reduction in muscle function impairment in vivo. ASC-EVs drive myoblast proliferation and differentiation in the in vitro ischemia/reoxygenation model. Moreover, ASC-EVs have shown an anti-apoptotic effect both in vitro and in vivo. Transcriptomic analyses have revealed that ASC-EVs carry a variety of pro-angiogenic mRNAs, while proteomic analyses have demonstrated an enrichment of NRG1 (neuregulin 1). A NRG1 blocking antibody used in vivo demonstrated that NRG1 is relevant to ASC-EV-induced muscle protection, vascular growth, and recruitment of inflammatory cells. Finally, bioinformatic analyses on 18 molecules that were commonly detected in ASC-EVs, including mRNAs and proteins, confirmed the enrichment of pathways involved in vascular growth and muscle regeneration/protection. CONCLUSIONS: This study demonstrates that ASC-EVs display pro-angiogenic and skeletal muscle protective properties that are associated with their NRG1/mRNA cargo. We, therefore, propose that ASC-EVs are a useful tool for therapeutic angiogenesis and muscle protection.

Renal Regenerative Potential of Extracellular Vesicles Derived from miRNA-Engineered Mesenchymal Stromal Cells.

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) possess pro-regenerative potential in different animal models with renal injury. EVs contain different molecules, including proteins, lipids and nucleic acids. Among the shuttled molecules, miRNAs have a relevant role in the pro-regenerative effects of EVs and are a promising target for therapeutic interventions. The aim of this study was to increase the content of specific miRNAs in EVs that are known to be involved in the pro-regenerative effect of EVs, and to assess the capacity of modified EVs to contribute to renal regeneration in in vivo models with acute kidney injuries. To this purpose, MSCs were transiently transfected with specific miRNA mimics by electroporation. Molecular analyses showed that, after transfection, MSCs and derived EVs were efficiently enriched in the selected miRNAs. In vitro and in vivo experiments indicated that EVs engineered with miRNAs maintained their pro-regenerative effects. Of relevance, engineered EVs were more effective than EVs derived from naïve MSCs when used at suboptimal doses. This suggests the potential use of a low amount of EVs (82.5 × 106) to obtain the renal regenerative effect.

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 ß 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.

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

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.

IL-3R-alpha blockade inhibits tumor endothelial cell-derived extracellular vesicle (EV)-mediated vessel formation by targeting the ß-catenin pathway.

The proangiogenic cytokine Interleukin-3 (IL-3) is released by inflammatory cells in breast and ovarian cancer tissue microenvironments and also acts as an autocrine factor for human breast and kidney tumor-derived endothelial cells (TECs). We have previously shown that IL-3-treated endothelial cells (ECs) release extracellular vesicles (EVs), which serve as a paracrine mechanism for neighboring ECs, by transferring active molecules. The impact of an anti-IL-3R-alpha blocking antibody on the proangiogenic effect of EVs released from TECs (anti-IL-3R-EVs) has therefore been investigated in this study. We have found that anti-IL-3R-EV treatment prevented neovessel formation and, more importantly, also induced the regression of in vivo TEC-derived neovessels. Two miRs that target the canonical wingless (Wnt)/ß-catenin pathway, at different levels, were found to be differentially regulated when comparing the miR-cargo of naive TEC-derived EVs (EVs) and anti-IL-3R-EVs. miR-214-3p, which directly targets ß-catenin, was found to be upregulated, whereas miR-24-3p, which targets adenomatous polyposis coli (APC) and glycogen synthase kinase-3ß (GSK3ß), was found to be downregulated. In fact, upon their transfer into the cell, low ß-catenin content and high levels of the two members of the "ß-catenin destruction complex" were detected. Moreover, c-myc downregulation was found in TECs treated with anti-IL-3R-EVs, pre-miR-214-3p-EVs and antago-miR-24-3p-EVs, which is consistent with network analyses of miR-214-3p and miR-24-3p gene targeting. Finally, in vivo studies have demonstrated the impaired growth of vessels in pre-miR-214-3p-EV- and antago-miR-24-3p-EV-treated animals. These effects became much more evident when combo treatment was applied. The results of the present study identify the canonical Wnt/ß-catenin pathway as a relevant mechanism of TEC-derived EV proangiogenic action. Furthermore, we herein provide evidence that IL-3R blockade may yield some significant advantages, than miR targeting, in inhibiting the proangiogenic effects of naive TEC-derived EVs by changing TEC-EV-miR cargo.

The Future Challenge of Reactive Oxygen Species (ROS) in Hypertension: From Bench to Bed Side.

Reactive oxygen species (ROS) act as signaling molecules that control physiological processes, including cell adaptation to stress. Redox signaling via ROS has quite recently become the focus of much attention in numerous pathological contexts, including neurodegenerative diseases, kidney and cardiovascular disease. Imbalance in ROS formation and degradation has also been implicated in essential hypertension. Essential hypertension is characterized by multiple genetic and environmental factors which do not completely explain its associated risk factors. Thereby, even if advances in therapy have led to a significant reduction in hypertension-associated complications, to interfere with the unbalance of redox signals might represent an additional therapeutic challenge. The decrease of nitric oxide (NO) levels, the antioxidant activity commonly found in preclinical models of hypertension and the ability of antioxidant approaches to reduce ROS levels have spurred clinicians to investigate the contribution of ROS in humans. Indeed, particular effort has recently been devoted to understanding how redox signaling may contribute to vascular pathobiology in human hypertension. However, although biomarkers of oxidative stress have been found to positively correlate with blood pressure in preclinical model of hypertension, human data are less convincing. We herein provide an overview of the most relevant mechanisms via which oxidative stress might contribute to the pathophysiology of essential hypertension. Moreover, alternative approaches, which are directed towards improving antioxidant machinery and/or interfering with ROS production, are also discussed.

Empagliflozin Limits Myocardial Infarction in Vivo and Cell Death in Vitro: Role of STAT3, Mitochondria, and Redox Aspects.

Empagliflozin (EMPA), a drug approved for type 2 diabetes management, reduced cardiovascular death but is unknown if it reduces myocardial infarction. We sought to investigate: (i) the effect of EMPA on myocardial function and infarct size after ischemia/reperfusion in mice fed with western diet (WD), (ii) the underlying signaling pathways, (iii) its effects on cell survival in rat embryonic-heart-derived cardiomyoblasts (H9C2) and endothelial cells (ECs). To facilitate the aforementioned aims, mice were initially randomized in Control and EMPA groups and were subjected to 30 min ischemia and 2 h reperfusion. EMPA reduced body weight, blood glucose levels, and mean arterial pressure. Cholesterol, triglyceride, and AGEs remained unchanged. Left ventricular fractional shortening was improved (43.97 ± 0.92 vs. 40.75 ± 0.61%) and infarct size reduced (33.2 ± 0.01 vs. 17.6 ± 0.02%). In a second series of experiments, mice were subjected to the above interventions up to the 10th min of reperfusion and myocardial biopsies were obtained for assessment of the signaling cascade. STAT3 was increased in parallel with reduced levels of malondialdehyde (MDA) and reduced expression of myocardial iNOS and interleukin-6. Cell viability and ATP content were increased in H9C2 and in ECs. While, STAT3 phosphorylation is known to bestow infarct sparing properties through interaction with mitochondria, we observed that EMPA did not directly alter the mitochondrial calcium retention capacity (CRC); therefore, its effect in reducing myocardial infarction is STAT3 dependent. In conclusion, EMPA improves myocardial function and reduces infarct size as well as improves redox regulation by decreasing iNOS expression and subsequently lipid peroxidation as shown by its surrogate marker MDA. The mechanisms of action implicate the activation of STAT3 anti-oxidant and anti-inflammatory properties.

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.

Stem Cell-Derived, microRNA-Carrying Extracellular Vesicles: A Novel Approach to Interfering with Mesangial Cell Collagen Production in a Hyperglycaemic Setting.

Extracellular vesicles (EVs) that are derived from stem cells are proving to be promising therapeutic options. We herein investigate the therapeutic potential of EVs that have been derived from different stem cell sources, bone-marrow (MSC) and human liver (HLSC), on mesangial cells (MCs) exposed to hyperglycaemia. By expressing a dominant negative STAT5 construct (ΔNSTAT5) in HG-cultured MCs, we have demonstrated that miR-21 expression is under the control of STAT5, which translates into Transforming Growth Factor beta (TGFß) expression and collagen production. A number of approaches have been used to show that both MSC- and HLSC-derived EVs protect MCs from HG-induced damage via the transfer of miR-222. This resulted in STAT5 down-regulation and a decrease in miR-21 content, TGFß expression and matrix protein synthesis within MCs. Moreover, we demonstrate that changes in the balance between miR-21 and miR-100 in the recipient cell, which are caused by the transfer of EV cargo, further contribute to providing beneficial effects. Interestingly, these effects were only detected in HG-cultured cells. Finally, it was found that HG reduced the expression of the nuclear encoded mitochondrial electron transport chain (ETC) components, CoxIV. It is worth noting that EV administration can rescue CoxIV expression in HG-cultured MCs. These results thus demonstrate that both MSC- and HLSC-derived EVs transfer the machinery needed to preserve MCs from HG-mediated damage. This occurs via the horizontal transfer of functional miR-222 which directly interferes with damaging cues. Moreover, our data indicate that the release of EV cargo into recipient cells provides additional therapeutic advantages against harmful mitochondrial signals.

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.

Skewed Epigenetics: An Alternative Therapeutic Option for Diabetes Complications.

Vascular complications are major causes of morbidity and mortality in type 2 diabetes patients. Mitochondrial reactive oxygen species (ROS) generation and a lack of efficient antioxidant machinery, a result of hyperglycaemia, mainly contribute to this problem. Although advances in therapy have significantly reduced both morbidity and mortality in diabetic individuals, diabetes-associated vascular complications are still one of the most challenging health problems worldwide. New healing options are urgently needed as current therapeutics are failing to improve long-term outcomes. Particular effort has recently been devoted to understanding the functional relationship between chromatin structure regulation and the persistent change in gene expression which is driven by hyperglycaemia and which accounts for long-lasting diabetic complications. A detailed investigation into epigenetic chromatin modifications in type 2 diabetes is underway. This will be particularly useful in the design of mechanism-based therapeutics which interfere with long-lasting activating epigenetics and improve patient outcomes. We herein provide an overview of the most relevant mechanisms that account for hyperglycaemia-induced changes in chromatin structure; the most relevant mechanism is called "metabolic memory."

Unacylated ghrelin induces oxidative stress resistance in a glucose intolerance and peripheral artery disease mouse model by restoring endothelial cell miR-126 expression.

Reactive oxygen species (ROS) are crucial in long-term diabetes complications, including peripheral artery disease (PAD). In this study, we have investigated the potential clinical impact of unacylated ghrelin (UnAG) in a glucose intolerance and PAD mouse model. We demonstrate that UnAG is able to protect skeletal muscle and endothelial cells (ECs) from ROS imbalance in hind limb ischemia-subjected ob/ob mice. This effect translates into reductions in hind limb functional impairment. We show that UnAG rescues sirtuin 1 (SIRT1) activity and superoxide dismutase-2 (SOD-2) expression in ECs. This leads to SIRT1-mediated p53 and histone 3 lysate 56 deacetylation and results in reduced EC senescence in vivo. We demonstrate, using small interfering RNA technology, that SIRT1 is also crucial for SOD-2 expression. UnAG also renews micro-RNA (miR)-126 expression, resulting in the posttranscriptional regulation of vascular cell adhesion molecule 1 expression and a reduced number of infiltrating inflammatory cells in vivo. Loss-of-function experiments that target miR-126 demonstrate that miR-126 also controls SIRT1 and SOD-2 expression, thus confirming its role in driving UnAG-mediated EC protection against ROS imbalance. These results indicate that UnAG protects vessels from ROS imbalance in ob/ob mice by rescuing miR-126 expression, thus emphasizing its potential clinical impact in avoiding limb loss in PAD.