State of the Art and New Trends from the Second International StemNet Meeting.

The Second International StemNet (Federation of Stem Cell Research Associations) meeting took place on 18-20 October 2023 in Brescia (Italy), with the support of the University of Brescia and the Zooprophylactic Institute of Lombardy and Emilia Romagna. The program of the meeting was articulated in nine sections: (1) Biomedical Communication in Italy: Critical Aspects; (2) StemNet Next Generation Session; (3) Cell-Free Therapies; (4) Tips and Tricks of Research Valorisation; (5) Stem Cells and Cancer; (6) Stem Cells in Veterinary Applications; (7) Stem Cells in Clinical Applications; (8) Organoids and 3D Systems; (9) induced pluripotent stem cells (iPCS) and Gene Therapy. National and International speakers presented their scientific works, inspiring debates and discussions among the attendees. The participation in the meeting was high, especially because of the young researchers who animated all the sessions and the rich poster session.

Role of Epigenetics and Metabolomics in Predicting Endothelial Dysfunction in Type 2 Diabetes.

Type 2 diabetes (T2D) is a worldwide health problem and cardiovascular disease (CVD) is a leading cause of morbidity and mortality in T2D patients, making the prevention of CVD onset a major priority. It is therefore crucial to optimize diagnosis and treatment to reduce this burden. Endothelial dysfunction is one of the most important prognostic factors for CVD progression, thus novel approaches to identify the early phase of endothelial dysfunction may lead to specific preventive measures to reduce the occurrence of CVD. Nowadays, multiomics approaches have provided unprecedented opportunities to stratify T2D patients into endotypes, improve therapeutic treatment and outcome and amend the survival prediction. Among omics strategies, epigenetics and metabolomics are gaining increasing interest. Recently, a dynamic correlation between metabolic pathways and gene expression through chromatin remodeling, such as DNA methylation, has emerged, indicating new perspectives on the regulatory networks impacting cellular processes. Thus, a better understanding of epigenetic-metabolite relationships can provide insight into the physiological processes altered early in the endothelium that ultimately head to disease development. Here, recent studies on epigenetics and metabolomics related to CVD prevention potentially useful to identify disease biomarkers, as well as new therapies hopefully targeting the early phase of endothelial dysfunction are highlighted.

Essential Oils from Mediterranean Plants Inhibit In Vitro Monocyte Adhesion to Endothelial Cells from Umbilical Cords of Females with Gestational Diabetes Mellitus.

Essential oils (EOs) are mixtures of volatile compounds belonging to several chemical classes derived from aromatic plants using different distillation techniques. Recent studies suggest that the consumption of Mediterranean plants, such as anise and laurel, contributes to improving the lipid and glycemic profile of patients with diabetes mellitus (DM). Hence, the aim of the present study was to investigate the potential anti-inflammatory effect of anise and laurel EOs (AEO and LEO) on endothelial cells isolated from the umbilical cord vein of females with gestational diabetes mellitus (GDM-HUVEC), which is a suitable in vitro model to reproduce the pro-inflammatory phenotype of a diabetic endothelium. For this purpose, the Gas Chromatographic/Mass Spectrometric (GC-MS) chemical profiles of AEO and LEO were first analyzed. Thus, GDM-HUVEC and related controls (C-HUVEC) were pre-treated for 24 h with AEO and LEO at 0.025% v/v, a concentration chosen among others (cell viability by MTT assay), and then stimulated with TNF-α (1 ng/mL). From the GC-MS analysis, trans-anethole (88.5%) and 1,8-cineole (53.9%) resulted as the major components of AEO and LEO, respectively. The results in C- and GDM-HUVEC showed that the treatment with both EOs significantly reduced: (i) the adhesion of the U937 monocyte to HUVEC; (ii) vascular adhesion molecule-1 (VCAM-1) protein and gene expression; (iii) Nuclear Factor-kappa B (NF-κB) p65 nuclear translocation. Taken together, these data suggest the anti-inflammatory efficacy of AEO and LEO in our in vitro model and lay the groundwork for further preclinical and clinical studies to study their potential use as supplements to mitigate vascular endothelial dysfunction associated with DM.

Oleanolic acid rescues critical features of umbilical vein endothelial cells permanently affected by hyperglycemia.

Skin wound healing is a physiological process that involves several cell types. Among them, endothelial cells are required for inflammation resolution and neo-angiogenesis, both necessary for tissue restoration after injury. Primary human umbilical vein endothelial cells (C-HUVECs) are derived from the umbilical cord. When women develop gestational diabetes, chronic exposure to hyperglycemia induces epigenetic modifications in these cells (GD-HUVECs), leading to a permanent pro-inflammatory phenotype and impaired angiogenesis in contrast to control cells. Oleanolic acid (OA) is a bioactive triterpenoid known for its epithelial cell migration promotion stimulation and higher tensile strength of wounds. However, the potentially anti-inflammatory and pro-angiogenic properties of OA are still under investigation. We tested OA on C- and GD-HUVECs under inflammatory conditions induced by low levels of the inflammatory cytokine TNF-α. Reduced expression of adhesion molecules VCAM1, ICAM1, and SELE was obtained in OA-pre-treated C- and GD-HUVECs. Additionally, protein VCAM1 levels were also decreased by OA. Coherently, monocyte adhesion assays showed that a lower number of monocytes adhered to GD-HUVEC endothelium under OA pre-treatment when compared to untreated ones. It is noteworthy that OA improved angiogenesis parameters in both phenotypes, being especially remarkable in the case of GD-HUVECs, since OA strongly rescued their poor tube formation behavior. Moreover, endothelial cell migration was improved in C- and GD-HUVECs in scratch assays, an effect that was further confirmed by focal adhesion (FA) remodeling, revealed by paxillin staining on immunocytochemistry assays. Altogether, these results suggest that OA could be an emergent wound healing agent due to its capacity to rescue endothelial malfunction caused by hyperglycemia.

Pharmacogenetics of Cardiovascular Prevention in Diabetes: From Precision Medicine to Identification of Novel Targets.

Pharmacogenetics-a branch of precision medicine-holds the promise of becoming a novel tool to reduce the social and healthcare burdens of cardiovascular disease (CVD) and coronary artery disease (CAD) in diabetes. The improvement in cardiovascular risk stratification resulting from adding genetic characteristics to clinical data has moved from the modest results obtained with genetic risk scores based on few genetic variants, to the progressively better performances of polygenic risk scores based on hundreds to millions of variants (CAD-PGRS). Similarly, over the past few years, the number of studies investigating the use of CAD-PGRS to identify different responses to cardio-preventive treatment has progressively increased, yielding striking results for lipid-lowering drugs such as proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors and statins. The use of CAD-PGRS to stratify patients based on their likely response to diabetes-specific interventions has been less successful, but promising results have been obtained with regard to specific genetic variants modulating the effects of interventions such as intensive glycemic control and fenofibrate. The finding of diabetes-specific CAD-loci, such as GLUL, has also led to the identification of promising new targets that might hopefully result in the development of specific therapies to reduce CVD burden in patients with diabetes. As reported in consensus statements from international diabetes societies, some of these pharmacogenetic approaches are expected to be introduced in clinical practice over the next decade. For this to happen, in addition to continuing to improve and validate these tools, it will be necessary to educate physicians and patients about the opportunities and limits of pharmacogenetics, as summarized in this review.

Estrogen Receptor 1 (ESR1) and the Wnt/ß-Catenin Pathway Mediate the Effect of the Coumarin Derivative Umbelliferon on Bone Mineralization.

Bone physiology is regulated by osteoblast and osteoclast activities, both involved in the bone remodeling process, through deposition and resorption mechanisms, respectively. The imbalance between these two phenomena contributes to the onset of bone diseases. Among these, osteoporosis is the most common metabolic bone disorder. The therapies currently used for its treatment include antiresorptive and anabolic agents associated with side effects. Therefore, alternative therapeutic approaches, including natural molecules such as coumarin and their derivatives, have recently shown positive results. Thus, our proposal was to investigate the effect of the coumarin derivative umbelliferon (UF) using an interesting model of human osteoblasts (hOBs) isolated from osteoporotic patients. UF significantly improved the activity of osteoporotic-patient-derived hOBs via estrogen receptor 1 (ESR1) and the downstream activation of ß-catenin pathway. Additionally, hOBs were co-cultured in microgravity with human osteoclasts (hOCs) using a 3D system bioreactor, able to reproduce the bone remodeling unit in bone loss conditions in vitro. Notably, UF exerted its anabolic role by reducing the multinucleated cells. Overall, our study confirms the potential efficacy of UF in bone health, and identified, for the first time, a prospective alternative natural compound useful to prevent/treat bone loss diseases such as osteoporosis.

Perinatal derivatives: How to best characterize their multimodal functions in vitro. Part C: Inflammation, angiogenesis, and wound healing.

Perinatal derivatives (PnD) are birth-associated tissues, such as placenta, umbilical cord, amniotic and chorionic membrane, and thereof-derived cells as well as secretomes. PnD play an increasing therapeutic role with beneficial effects on the treatment of various diseases. The aim of this review is to elucidate the modes of action of non-hematopoietic PnD on inflammation, angiogenesis and wound healing. We describe the source and type of PnD with a special focus on their effects on inflammation and immune response, on vascular function as well as on cutaneous and oral wound healing, which is a complex process that comprises hemostasis, inflammation, proliferation (including epithelialization, angiogenesis), and remodeling. We further evaluate the different in vitro assays currently used for assessing selected functional and therapeutic PnD properties. This review is a joint effort from the COST SPRINT Action (CA17116) with the intention to promote PnD into the clinics. It is part of a quadrinomial series on functional assays for validation of PnD, spanning biological functions, such as immunomodulation, anti-microbial/anti-cancer activities, anti-inflammation, wound healing, angiogenesis, and regeneration.

Effect of the Human Amniotic Membrane on the Umbilical Vein Endothelial Cells of Gestational Diabetic Mothers: New Insight on Inflammation and Angiogenesis.

One of the most relevant diabetes complications is impaired wound healing, mainly characterized by reduced peripheral blood flow and diminished neovascularization together with increased inflammation and oxidative stress. Unfortunately, effective therapies are currently lacking. Recently, the amniotic membrane (AM) has shown promising results in wound management. Here, the potential role of AM on endothelial cells isolated from the umbilical cord vein of gestational diabetes-affected women (GD-HUVECs), has been investigated. Indeed, GD-HUVECs in vivo exposed to chronic hyperglycemia during pregnancy compared to control cells (C-HUVECs) have shown molecular modifications of cellular homeostasis ultimately impacting oxidative and nitro-oxidative stress, inflammatory phenotype, nitric oxide (NO) synthesis, and bioavailability, thus representing a useful model for studying the mechanisms potentially supporting the role of AM in chronic non-healing wounds. In this study, the anti-inflammatory properties of AM have been assessed using a monocyte-endothelium interaction assay in cells pre-stimulated with tumor necrosis factor-α (TNF-α) and through vascular adhesion molecule expression and membrane exposure, together with the AM impact on the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-kB) pathway and NO bioavailability. Moreover, GD-HUVEC migration and tube formation ability were evaluated in the presence of AM. The results showed that AM significantly reduced TNF-α-stimulated monocyte-endothelium interaction and the membrane exposure of the endothelial vascular and intracellular adhesion molecules (VCAM-1 and ICAM-1, respectively) in both C- and GD-HUVECs. Strikingly, AM treatment significantly improved vessel formation in GD-HUVECs and cell migration in both C- and GD-HUVECs. These collective results suggest that AM positively affects various critical pathways in inflammation and angiogenesis, thus providing further validation for ongoing clinical trials in diabetic foot ulcers.

Circulating Extracellular Vesicles Are Increased in Newly Diagnosed Celiac Disease Patients.

Extracellular vesicles (EVs) are a class of circulating entities that are involved in intercellular crosstalk mechanisms, participating in homeostasis maintenance, and diseases. Celiac disease is a gluten-triggered immune-mediated disorder, characterized by the inflammatory insult of the enteric mucosa following local lymphocytic infiltration, resulting in villous atrophy. The goal of this research was the assessment and characterization of circulating EVs in celiac disease patients, as well as in patients already on an adequate gluten-free regimen (GFD). For this purpose, a novel and validated technique based on polychromatic flow cytometry that allowed the identification and enumeration of different EV sub-phenotypes was applied. The analysis evidenced that the total, annexin V+, leukocyte (CD45+), and platelet (CD41a+) EV counts were significantly higher in both newly diagnosed celiac disease patients and patients under GFD compared with the healthy controls. Endothelial-derived (CD31+) and epithelial-derived (EpCAM+) EV counts were significantly lower in subjects under gluten exclusion than in celiac disease patients, although EpCAM+ EVs maintained higher counts than healthy subjects. The numbers of EpCAM+ EVs were a statistically significant predictor of intraepithelial leukocytes (IEL). These data demonstrate that EVs could represent novel and potentially powerful disease-specific biomarkers in the context of celiac disease.

Cytotoxic and Genotoxic Effects of Composite Resins on Cultured Human Gingival Fibroblasts.

The aim of the study was to evaluate the cytotoxic and genotoxic potential of five commercially available dental composite resins (CRs), investigating the effect of their quantifiable bisphenol-A-glycidyl-methacrylate (Bis-GMA) and/or triethylene glycol dimethacrylate (TEGDMA) release. Experiments were performed using the method of soaking extracts, which were derived from the immersion of the following CRs in the culture medium: Clearfil-Majesty-ES-2, GrandioSO, and Enamel-plus-HRi (Bis-GMA-based); Enamel-BioFunction and VenusDiamond (Bis-GMA-free). Human Gingival Fibroblasts (hGDFs) were employed as the cellular model to mimic in vitro the oral cavity milieu, where CRs simultaneously release various components. Cell metabolic activity, oxidative stress, and genotoxicity were used as cellular outcomes. Results showed that only VenusDiamond and Enamel-plus-HRi significantly affected the hGDF cell metabolic activity. In accordance with this, although no CR-derived extract induced a significantly detectable oxidative stress, only VenusDiamond and Enamel-plus-HRi induced significant genotoxicity. Our findings showed, for the CRs employed, a cytotoxic and genotoxic potential that did not seem to depend only on the actual Bis-GMA or TEGDMA content. Enamel-BioFunction appeared optimal in terms of cytotoxicity, and similar findings were observed for Clearfil-Majesty-ES-2 despite their different Bis-GMA/TEGDMA release patterns. This suggested that simply excluding one specific monomer from the CR formulation might not steadily turn out as a successful approach for improving their biocompatibility.

Pro-Osteogenic Properties of Violina pumpkin (Cucurbita moschata) Leaf Extracts: Data from In Vitro Human Primary Cell Cultures.

Traditional medicines rely mainly on use of plant extracts to mitigate or treat a wide range of disorders, including those that affect skeletal homeostasis. In this study, we investigated for the first time the potential pro-osteogenic effects of hexane, acetone and methanol extracts of the leaves of Cucurbita moschata, a very popular pumpkin cultivar in Western countries. We found that in Cucurbita moschata leaves, there are acetone-extractable substances-in particular, fatty acids such as 13-OH-9Z,11E,15E-octadecatrienoic acid (PU-13OH-FA), which is capable of both stimulating the function of human primary osteoblasts, which are responsible for bone formation, and inhibiting the differentiation of human osteoclasts, which are responsible for bone resorption. This dual effect was monitored by analyzing Runx2 expression, deposition of mineralized matrix, ALP activity, TRAP and actin ring staining respectively. This study suggests that bioactive chemicals from Cucurbita moschata leaves are potentially suitable as therapeutics for managing metabolic bone disorders such as osteoporosis and rheumatoid arthritis, and promoting tissue healing and functional recovery after bone fractures. The data we obtained increase knowledge on the biological activities of Cucurbita moschata, and in particular underline the potential benefits of consuming leaves which are a part of the plant currently little considered in the Western world.

Three-Dimensional Co-Culture System of Human Osteoblasts and Osteoclast Precursors from Osteoporotic Patients as an Innovative Model to Study the Role of Nutrients: Focus on Vitamin K2.

Several natural compounds, such as vitamin K2, have been highlighted for their positive effects on bone metabolism. It has been proposed that skeletal disorders, such as osteoporosis, may benefit from vitamin K2-based therapies or its regular intake. However, further studies are needed to better clarify the effects of vitamin K2 in bone disorders. To this aim, we developed in vitro a three-dimensional (3D) cell culture system one step closer to the bone microenvironment based on co-culturing osteoblasts and osteoclasts precursors obtained from bone specimens and peripheral blood of the same osteoporotic patient, respectively. Such a 3-D co-culture system was more informative than the traditional 2-D cell cultures when responsiveness to vitamin K2 was analyzed, paving the way for data interpretation on single patients. Following this approach, the anabolic effects of vitamin K2 on the osteoblast counterpart were found to be correlated with bone turnover markers measured in osteoporotic patients' sera. Overall, our data suggest that co-cultured osteoblasts and osteoclast precursors from the same osteoporotic patient may be suitable to generate an in vitro 3-D experimental model that potentially reflects the individual's bone metabolism and may be useful to predict personal responsiveness to nutraceutical or drug molecules designed to positively affect bone health.

Comprehensive Search for Novel Circulating miRNAs and Axon Guidance Pathway Proteins Associated with Risk of ESKD in Diabetes.

BACKGROUND: Mechanisms underlying the pro gression of diabetic kidney disease to ESKD are not fully understood. METHODS: We performed global microRNA (miRNA) analysis on plasma from two cohorts consisting of 375 individuals with type 1 and type 2 diabetes with late diabetic kidney disease, and targeted proteomics analysis on plasma from four cohorts consisting of 746 individuals with late and early diabetic kidney disease. We examined structural lesions in kidney biopsy specimens from the 105 individuals with early diabetic kidney disease. Human umbilical vein endothelial cells were used to assess the effects of miRNA mimics or inhibitors on regulation of candidate proteins. RESULTS: In the late diabetic kidney disease cohorts, we identified 17 circulating miRNAs, represented by four exemplars (miR-1287-5p, miR-197-5p, miR-339-5p, and miR-328-3p), that were strongly associated with 10-year risk of ESKD. These miRNAs targeted proteins in the axon guidance pathway. Circulating levels of six of these proteins-most notably, EFNA4 and EPHA2-were strongly associated with 10-year risk of ESKD in all cohorts. Furthermore, circulating levels of these proteins correlated with severity of structural lesions in kidney biopsy specimens. In contrast, expression levels of genes encoding these proteins had no apparent effects on the lesions. In in vitro experiments, mimics of miR-1287-5p and miR-197-5p and inhibitors of miR-339-5p and miR-328-3p upregulated concentrations of EPHA2 in either cell lysate, supernatant, or both. CONCLUSIONS: This study reveals novel mechanisms involved in progression to ESKD and points to the importance of systemic factors in the development of diabetic kidney disease. Some circulating miRNAs and axon guidance pathway proteins represent potential targets for new therapies to prevent and treat this condition.

Effect of TNFα stimulation on expression of kidney risk inflammatory proteins in human umbilical vein endothelial cells cultured in hyperglycemia.

We recently identified a kidney risk inflammatory signature (KRIS), comprising 6 TNF receptors (including TNFR1 and TNFR2) and 11 inflammatory proteins. Elevated levels of these proteins in circulation were strongly associated with risk of the development of end-stage kidney disease (ESKD) during 10-year follow-up. It has been hypothesized that elevated levels of these proteins in circulation might reflect (be markers of) systemic exposure to TNFα. In this in vitro study, we examined intracellular and extracellular levels of these proteins in human umbilical vein endothelial cells (HUVECs) exposed to TNFα in the presence of hyperglycemia. KRIS proteins as well as 1300 other proteins were measured using the SOMAscan proteomics platform. Four KRIS proteins (including TNFR1) were down-regulated and only 1 protein (IL18R1) was up-regulated in the extracellular fraction of TNFα-stimulated HUVECs. In the intracellular fraction, one KRIS protein was down-regulated (CCL14) and 1 protein was up-regulated (IL18R1). The levels of other KRIS proteins were not affected by exposure to TNFα. HUVECs exposed to a hyperglycemic and inflammatory environment also showed significant up-regulation of a distinct set of 53 proteins (mainly in extracellular fraction). In our previous study, circulating levels of these proteins were not associated with progression to ESKD in diabetes.

The Dual Role of Vitamin K2 in "Bone-Vascular Crosstalk": Opposite Effects on Bone Loss and Vascular Calcification.

Osteoporosis (OP) and vascular calcification (VC) represent relevant health problems that frequently coexist in the elderly population. Traditionally, they have been considered independent processes, and mainly age-related. However, an increasing number of studies have reported their possible direct correlation, commonly defined as "bone-vascular crosstalk". Vitamin K2 (VitK2), a family of several natural isoforms also known as menaquinones (MK), has recently received particular attention for its role in maintaining calcium homeostasis. In particular, VitK2 deficiency seems to be responsible of the so-called "calcium paradox" phenomenon, characterized by low calcium deposition in the bone and its accumulation in the vessel wall. Since these events may have important clinical consequences, and the role of VitK2 in bone-vascular crosstalk has only partially been explained, this review focuses on its effects on the bone and vascular system by providing a more recent literature update. Overall, the findings reported here propose the VitK2 family as natural bioactive molecules that could be able to play an important role in the prevention of bone loss and vascular calcification, thus encouraging further in-depth studies to achieve its use as a dietary food supplement.

Predialysis and Dialysis Therapies Differently Affect Nitric Oxide Synthetic Pathway in Red Blood Cells from Uremic Patients: Focus on Peritoneal Dialysis.

Red blood cells (RBCs) have been found to synthesize and release both nitric oxide (NO) and cyclic guanosine monophosphate (cGMP), contributing to systemic NO bioavailability. These RBC functions resulted impaired in chronic kidney disease (CKD). This study aimed to evaluate whether predialysis (conservative therapy, CT) and dialysis (peritoneal dialysis, PD; hemodialysis, HD) therapies used during CKD progression may differently affect NO-synthetic pathway in RBCs. Our data demonstrated that compared to PD, although endothelial-NO-synthase activation was similarly increased, HD and CT were associated to cGMP RBCs accumulation, caused by reduced activity of cGMP membrane transporter (MRP4). In parallel, plasma cGMP levels were increased by both CT and HD and they significantly decreased after hemodialysis, suggesting that this might be caused by reduced cGMP renal clearance. As conceivable, compared to healthy subjects, plasma nitrite levels were significantly reduced by HD and CT but not in patients on PD. Additionally, the increased carotid intima-media thickness (IMT) values did not reach the significance exclusively in patients on PD. Therefore, our results show that PD might better preserve the synthetic NO-pathway in CKD-erythrocytes. Whether this translates into a reduced development of uremic vascular complications requires further investigation.

Plasma from obese children increases monocyte-endothelial adhesion and affects intracellular insulin signaling in cultured endothelial cells: Potential role of mTORC1-S6K1.

Childhood obesity is characterized by the loss of vascular insulin sensitivity along with altered oxidant-antioxidant state and chronic inflammation, which play a key role in the onset of endothelial dysfunction. We previously demonstrated a reduced insulin-stimulated Nitric Oxide (NO) bioavailability in Human Umbilical Vein Endothelial cells (HUVECs) cultured with plasma from obese pre-pubertal children (OB) compared to those cultured with plasma of normal-weight children (CTRL). However, mechanisms underlying endothelial dysfunction in childhood obesity remains poorly understood. Hence, the present study aimed to better investigate these mechanisms, also considering a potential involvement of mammalian Target Of Rapamycin Complex1 (mTORC1)-ribosomal protein S6 Kinase beta1 (S6K1) pathway. OB-children (N = 32, age: 9.2 ± 1.7; BMI z-score: 2.72 ± 0.31) had higher fasting insulin levels and increased HOMA-IR than CTRL-children (N = 32, age: 8.8 ± 1.2; BMI z-score: 0.33 ± 0.75). In vitro, HUVECs exposed to OB-plasma exhibited significant increase in Reactive Oxygen Species (ROS) levels, higher vascular and intercellular adhesion molecules exposure, together with increased monocytes-endothelial interaction. This was associated with unbalanced pro- and anti-atherogenic endothelial insulin stimulated signaling pathways, as measured by increased Mitogen Activated Protein Kinase (MAPK) and decreased Insulin Receptor Substrate-1 (IRS-1)/protein kinase B (Akt)/ endothelial NO Synthase (eNOS) phosphorylation levels, together with augmented S6K1 activation. Interestingly, inhibition of mTORC1-S6K1 pathway using rapamycin significantly restored the IRS-1/Akt/eNOS activation, suggesting a feedback regulation of IRS-1/Akt signal through S6K1. Overall, our in vitro data shed light on new mechanisms underlying the onset of endothelial dysfunction in childhood obesity.

Old and New Biomarkers Associated with Endothelial Dysfunction in Chronic Hyperglycemia.

Chronic hyperglycemia and vascular damage are strictly related. Biomarkers of vascular damage have been intensively studied in the recent years in the quest of reliable cardiovascular risk assessment tools able to facilitate risk stratification and early detection of vascular impairment. The present study is a narrative review with the aim of revising the available evidence on current and novel markers of hyperglycemia-induced vascular damage. After a discussion of classic tools used to investigate endothelial dysfunction, we provide an in-depth description of novel circulating biomarkers (chemokines, extracellular vesicles, and epigenetic and metabolomic biomarkers). Appropriate use of a single as well as a cluster of the discussed biomarkers might enable in a near future (a) the prompt identification of targeted and customized treatment strategies and (b) the follow-up of cardiovascular treatment efficacy over time in clinical research and/or in clinical practice.

Role of Polyphenols and Carotenoids in Endothelial Dysfunction: An Overview from Classic to Innovative Biomarkers.

Nowadays, the dramatically increased prevalence of metabolic diseases, such as obesity and diabetes mellitus and their related complications, including endothelial dysfunction and cardiovascular disease, represents one of the leading causes of death worldwide. Dietary nutrients together with healthy lifestyles have a crucial role in the endothelium health-promoting effects. From a growing body of evidence, active natural compounds from food, including polyphenols and carotenoids, have attracted particular attention as a complementary therapy on atherosclerosis and cardiovascular disease, as well as preventive approaches through the attenuation of inflammation and oxidative stress. They mainly act as radical scavengers by promoting a variety of biological mechanisms, such as improvements in endothelial function, blood pressure, platelet activity, and insulin sensitivity, and by modulating various known biomarkers. The present review highlights the role of polyphenols and carotenoids in early endothelial dysfunction with attention to their beneficial effect in modulating both classical and recent technologically generated emerging biomarkers. These, alone or in combination, can play an important role in the prediction, diagnosis, and evolution of cardiovascular disease. However, a main challenge is to speed up early and prompt new interventions in order to prevent or slow down disease progression, even with an adequate intake of bioactive compounds. Hence, there is an urgent need of new more validated, appropriate, and reliable diagnostic and therapeutic biomarkers useful to diagnose endothelial dysfunction at an earlier stage.

Association of the 1q25 Diabetes-Specific Coronary Heart Disease Locus With Alterations of the γ-Glutamyl Cycle and Increased Methylglyoxal Levels in Endothelial Cells.

A chromosome 1q25 variant (rs10911021) has been associated with coronary heart disease (CHD) in type 2 diabetes. In human umbilical vein endothelial cells (HUVECs), the risk allele "C" is associated with lower expression of the adjacent gene GLUL encoding glutamine synthase, converting glutamic acid to glutamine. To further investigate the mechanisms through which this locus affects CHD risk, we measured 35 intracellular metabolites involved in glutamic acid metabolism and the γ-glutamyl cycle in 62 HUVEC strains carrying different rs10911021 genotypes. Eight metabolites were positively associated with the risk allele (17-58% increase/allele copy, P = 0.046-0.002), including five γ-glutamyl amino acids, ß-citryl-glutamate, N-acetyl-aspartyl-glutamate, and ophthalmate-a marker of γ-glutamyl cycle malfunction. Consistent with these findings, the risk allele was also associated with decreased glutathione-to-glutamate ratio (-9%, P = 0.012), decreased S-lactoylglutathione (-41%, P = 0.019), and reduced detoxification of the atherogenic compound methylglyoxal (+54%, P = 0.008). GLUL downregulation by shRNA caused a 40% increase in the methylglyoxal level, which was completely prevented by glutamine supplementation. In summary, we have identified intracellular metabolic traits associated with the 1q25 risk allele in HUVECs, including impairments of the γ-glutamyl cycle and methylglyoxal detoxification. Glutamine supplementation abolishes the latter abnormality, suggesting that such treatment may prevent CHD in 1q25 risk allele carriers.