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This study aimed to evaluate the effects of a new photodynamic protocol (ALAD-PDT) on primary human osteoblasts (hOBs). The ALAD-PDT protocol consists of a heat-sensitive gel with 5% 5-delta aminolevulinic acid commercialized as Aladent (ALAD), combined with 630 nm LED. For this purpose, the hOBs, explanted from human mandible bone fragments, were used and treated with different ALAD concentrations (10%, 50%, 100% v/v) incubated for 45 min and immediately afterwards irradiated with a 630 nm LED device for 7 min. The untreated and unirradiated cells were considered control (CTRL). The cellular accumulation of the photosensitizer protoporphyrin IX (PpIX), the proliferation, the alkaline phosphatase (ALP) activity, and the calcium deposition were assessed. All concentrations (10, 50, 100%) determined a significant increment of PpIX immediately after 45 min of incubation (0 h) with the highest peak by ALAD (100%). The consequent 7 min of light irradiation caused a slight decrease in PpIX. At 48 h and 72 h, any increment of PpIX was observed. The concentration 100% associated with LED significantly increased hOB proliferation at 48 h (+ 46.83%) and 72 h (+ 127.75%). The 50% and 100% concentrations in combination to the red light also stimulated the ALP activity, + 12.910% and + 14.014% respectively. The concentration 100% with and without LED was selected for the assessment of calcium deposition. After LED irradiation, a significant increase in calcium deposition was observed and quantified (+ 72.33%). In conclusion, the ALAD-PDT enhanced proliferation, the ALP activity, and mineralized deposition of human oral osteoblasts, highlighting a promising potential for bone tissue regeneration.

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.

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.

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.

Extracellular vesicles (EVs) are a heterogenous population of plasma membrane-surrounded particles that are released in the extracellular milieu by almost all types of living cells. EVs are key players in intercellular crosstalk, both locally and systemically, given that they deliver their cargoes (consisting of proteins, lipids, mRNAs, miRNAs, and DNA fragments) to target cells, crossing biological barriers. Those mechanisms further trigger a wide range of biological responses. Interestingly, EV phenotypes and cargoes and, therefore, their functions, stem from their specific parental cells. For these reasons, EVs have been proposed as promising candidates for EV-based, cell-free therapies. One of the new frontiers of cell-based immunotherapy for the fight against refractory neoplastic diseases is represented by genetically engineered chimeric antigen receptor T (CAR-T) lymphocytes, which in recent years have demonstrated their effectiveness by reaching commercialization and clinical application for some neoplastic diseases. CAR-T-derived EVs represent a recent promising development of CAR-T immunotherapy approaches. This crosscutting innovative strategy is designed to exploit the advantages of genetically engineered cell-based immunotherapy together with those of cell-free EVs, which in principle might be safer and more efficient in crossing biological and tumor-associated barriers. In this review, we underlined the potential of CAR-T-derived EVs as therapeutic agents in tumors.

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.

Background Hyperglycemia-induced oxidative stress and inflammation are potent drivers of atherosclerotic cardiovascular disease (ASCVD). Gestational diabetes (GDM) is characterized by chronic hyperglycemia during pregnancy and may represent a clinical model to study the mechanisms of oxidative stress and inflammation induced by hyperglycemia. GDM is associated with a range of adverse perinatal and long-term outcomes for both mother and offspring. In this perspective, it is emerging a putative association between maternal GDM and offspring's epigenetic trait. Purpose To investigate the link between histone modifications, oxidative stress and inflammatory phenotype as well as the transmission of epigenetic signatures to the offspring. Methods We analyzed peripheral blood mononuclear cells (PBMC) from GDM and control mothers as well as human umbilical vein endothelial cells (HUVEC) and cord blood mononuclear cells (CBMC) isolated from newborn umbilical cords obtained at delivery from both groups. Histone methyltransferase MLL1-dependent trimethylation of histone 3 at lysine 4 amino residue (H3K4me3) on NF-kB p65 subunit promoter region was assessed by chromatin immunoprecipitation (ChIP) and real-time qPCR in HUVEC, PBMC and CBMC, respectively. MLL1 and downstream inflammatory and redox genes were determined by real-time qPCR and immunocytochemistry in the presence and in the absence of MLL1 inhibitor MM-102. Measurement of reactive oxygen species (ROS) was performed by electron spin resonance spectroscopy. Results For the first time, we demonstrated a significant increase of MLL1 expression with subsequent MLL1-induced upregulation of NF-kB p65 gene via H3K4me3 in GDM as compared to control cells. MLL1-driven epigenetic remodeling of NF-kB p65 promoter is upstream to the activation of inflammatory pathway. Indeed, treatment with MM-102 decreased H3K4me3 and blunted expression of NF-kB p65 as well as VCAM-1, MCP-1 and IL-6 genes. We also found that expression of ROS scavenger aldehyde dehydrogenase 2 is reduced, whereas pro-oxidant nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit NOX4 is upregulated. Interestingly, the increased ROS generation observed in GDM is involved in the upregulation of MLL1 as shown by the restoring effect of antioxidant vitamin C on MLL1 expression levels. Conclusions Our results suggest that a complex interplay between oxidative stress and histone modifications are responsible for the GDM maternal inflammatory and oxidative phenotypes and its transmission to the offspring. The deciphering of epigenetic-induced chromatin remodelling opens the perspective for pharmacological reprogramming of adverse chromatin changes to reduce the burden of early development of metabolic phenotypes and ASCVD. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): University G. d'Annunzio MIUR fundings Schematic figure

Current research on dental implants has mainly focused on the influence of surface roughness on the rate of osseointegration, while studies on the development of surfaces to also improve the interaction of peri-implant soft tissues are lacking. To this end, the first purpose of this study was to evaluate the response of human gingival fibroblasts (hGDFs) to titanium implant discs (Implacil De Bortoli, Brazil) having different micro and nano-topography: machined (Ti-M) versus sandblasted/double-etched (Ti-S). The secondary aim was to investigate the effect of the macrogeometry of the discs on cells: linear-like (Ti-L) versus wave-like (Ti-W) surfaces. The atomic force microscopy (AFM) and scanning electron microscopy (SEM) analysis showed that the Ti-S surfaces were characterized by a significantly higher micro and nano roughness and showed the 3D macrotopography of Ti-L and Ti-W surfaces. For in vitro analyses, the hGDFs were seeded into titanium discs and analyzed at 1, 3, and 5 days for adhesion and morphology (SEM) viability and proliferation (Cck-8 and MTT assays). The results showed that all tested surfaces were not cytotoxic for the hGDFs, rather the nano-micro and macro topography favored their proliferation in a time-dependent manner. Especially, at 3 and 5 days, the number of cells on Ti-L was higher than on other surfaces, including Ti-W surfaces. In conclusion, although further studies are needed, our in vitro data proved that the use of implant discs with Ti-S surfaces promotes the adhesion and proliferation of gingival fibroblasts, suggesting their use for in vivo applications.

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.

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.

Myo-inositol (Myo) improves insulin resistance, glucose metabolism, and helps gestational diabetes (GDM) management. GDM is associated with a pro-inflammatory state and increased oxidative stress, which are both involved in vascular damage in diabetes. Our aim was to study Myo anti-inflammatory/antioxidant potential effects on an in vitro model of human umbilical vein endothelial cells (HUVECs). To this end, monocyte cell adhesion to HUVECs, adhesion molecule membrane exposure, and oxidative stress levels were determined in cells from control (C-) and GDM women treated during pregnancy either with diet only (GD-) or with diet plus Myo (GD+Myo). To deeply study the vascular effects of Myo, the same evaluations were performed in C- and GD-HUVECs following 48 h in vitro stimulation with Myo. Notably, we first observed that GD-HUVECs obtained from women assuming Myo supplementation exhibited a significantly decreased number of monocytes that adhered to endothelial cells, less adhesion molecule exposure, and lower intracellular reactive oxygen species (ROS) levels in the basal state as compared to GD-HUVECs obtained from women treated by diet only. This Myo anti-inflammatory/antioxidant effect was confirmed by 48 h in vitro stimulation of GD-HUVECs as compared to controls. Altogether, these results strongly suggest that Myo may exert protective actions against chronic inflammation induced by endothelial dysfunction in diabetes.

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.

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 β-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.

Cardiovascular and metabolic bone diseases are demanding health problems with high morbidity and mortality [...]

Human umbilical cord endothelial cells (HUVECs) obtained from women affected by gestational diabetes (GD-HUVECs) display durable pro-atherogenic modifications and might be considered a valid in vitro model for studying chronic hyperglycemia effects on early endothelial senescence. Here, we demonstrated that GD- compared to C-HUVECs (controls) exhibited oxidative stress, altered both mitochondrial membrane potential and antioxidant response, significant increase of senescent cells characterized by a reduced NAD-dependent deacetylase sirtuin-1 (SIRT1) activity together with an increase in cyclin-dependent kinase inhibitor-2A (P16), cyclin-dependent kinase inhibitor-1 (P21), and tumor protein p53 (P53) acetylation. This was associated with the p300 activation, and its silencing significantly reduced the GD-HUVECs increased protein levels of P300 and Ac-P53 thus indicating a persistent endothelial senescence via SIRT1/P300/P53/P21 pathway. Overall, our data suggest that GD-HUVECs can represent an "endothelial hyperglycemic memory" model to investigate in vitro the early endothelium senescence in cells chronically exposed to hyperglycemia in vivo.

Background: Glucosamine (GlcN) functions as a building block of the cartilage matrix and its multifaceted roles in promoting joint health has been extensively investigated. On the contrary, the role of GlcN in osteogenesis and bone tissue is poorly understood, mainly due to the lack of adequate experimental models. Consequently, the benefit of GlcN in bone disorders remains controversial. In order to broaden the pharmacological relevance and potential therapeutic/nutraceutic efficacy of GlcN, we investigated the effect of GlcN on human primary osteoclasts (hOCs) and osteoblasts (hOBs) that were grown with 2D traditional methods or co-cultured in a more complex culture system one step closer to the in vivo bone microenvironment, consisting in a three-dimensional (3D) dynamic system.Methods: Osteoclasts derived from peripheral blood monocytes (hMCs) of either healthy or osteoarthritis (OA) donors, were used and kept in culture with osteoclastogenic inducers. hMCs were also combined with hOBs in a 3D dynamic co‐culture system by using RCCS-4TM bioreactor in osteogenic medium without osteoclastogenic inducers. In this condition osteoclastogenesis was supported by hOBs and sizeable self assembling aggregates were obtained. The differentiated osteoclasts were evaluated by the tartrate-resistant acid phosphatase assay, osteogenic differentiation was monitored by analyzing mineral matrix deposition through Alizarin Red staining, and expression of osteogenic markers through RT-qPCR. The cells were treated with DONA® Crystalline Glucosamine Sulfate (i.e. the original GlcN sulfate product).Results: DONA® was effective in decreasing the hOCs differentiation and function. Osteoclasts from OA donors were more sensitive than those from healthy donors. At the same time, DONA® showed anabolic effects on osteoblasts both in 2D conventional cell culture and in osteoclasts/osteoblasts 3D dynamic co-culture system.Conclusions: Here we demonstrated the effectiveness of a 3D dynamic co-culture system to provide useful information on the spectrum of action of GlcN on bone microenvironment. This can pave the way to better define the potential applications of a compound such as GlcN which is positioned between pharmaceuticals and nutraceuticals. Therefore, based on our observations, we hypothesize that GlcN could have potential benefits either in the treatment of osteopenic diseases such as osteoporosis, or in the bone health maintenance.

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.

An unhealthy diet is considered to be one of the main causes for increases in obesity and chronic diseases. Food choices are frequently influenced by food systems and environments along with the availability and affordability of healthy and sustainable food. In this context, a major contemporary challenge lies in improving these aspects in order to support healthy dietary choices. Hence, to address this issue, here, we propose a small-scale approach called SANI (Italian for “healthy”) which involves experts in science and marketing. Two typical agri-foods of the Abruzzo area (center of Italy), tomato sauce and extra virgin olive oil, are characterized as high-quality products in terms of their nutrient content, absence of chemical contaminants (chromatographic, spectrophotometric, and magnetic resonance techniques), and ecological footprint (lifecycle assessment and carbon footprint). Hence, their consumption is promoted, with strict attention being paid to several aspects of the food system, such as production, processing, distribution, labeling, and promotion, as well as marketing strategies and dissemination activities. Overall, these SANI actions, especially labeling and dissemination, have proven to be a valuable learning tool for consumers moving toward more conscious consumption, which can be extended and applied to additional food products. Future applications of similar research strategies in a wider context could positively affect human and environmental health.

A chromosome 1q25 variant (rs10911021) has been associated with coronary heart disease (CHD) in type 2 diabetes (T2D). 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 g-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 g-glutamyl amino acids, b-citryl-glutamate, N-acetyl-aspartyl-glutamate, and ophthalmate - a marker of g-glutamyl cycle malfunction. Consistent with these findings, the risk allele was also associated with decreased glutathione/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 down-regulation by shRNA caused a 40% increase in 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 g-glutamyl cycle and methylglyoxal detoxification. Glutamine supplementation abolishes the latter abnormality, suggesting that such treatment may prevent CHD in 1q25 risk allele carriers.

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, β-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.

The cardiovascular benefits of fibrates have been shown to be heterogeneous and to depend on the presence of atherogenic dyslipidemia. We investigated whether genetic variability in the PPARA gene, coding for the pharmacological target of fibrates (PPAR-alpha), could be used to improve the selection of patients with type 2 diabetes who may derive cardiovascular benefit from addition of this treatment to statins. We identified a common variant at the PPARA locus (rs6008845, C/T) displaying a study-wide-significant influence on the effect of fenofibrate on major cardiovascular events (MACE) among 3,065 self-reported White subjects treated with simvastatin and randomized to fenofibrate or placebo in the Action-to-Control-Cardiovascular-Risk-in-Diabetes (ACCORD) Lipid Trial. T/T homozygotes (36% of participants) experienced a 51% MACE reduction in response to fenofibrate (HR=0.49; 95%C.I. 0.34-0.72) whereas no benefit was observed for other genotypes (p for interaction=3.7x10-4). The “rs6008845-by-fenofibrate” interaction on MACE was replicated in African-Americans from ACCORD (N=585, p=0.02) and in external cohorts (ACCORD-Blood-Pressure, ORIGIN, and TRIUMPH, total N=3059, p=0.005). Remarkably, rs6008845 T/T homozygotes experienced a cardiovascular benefit from fibrate even in the absence of atherogenic dyslipidemia. Among these individuals, but not among carriers of other genotypes, fenofibrate treatment was associated with lower circulating levels of CCL11 - a pro-inflammatory and atherogenic chemokine also known as eotaxin (p for rs6008845-by-fenofibrate interaction=0.003). The Genotype-Tissue Expression (GTEx) dataset revealed regulatory functions of rs6008845 on PPARA expression in many tissues. In summary, we have found a common PPARA regulatory variant that influences the cardiovascular effects of fenofibrate and that could be used to identify T2D patients who would derive benefit from fenofibrate treatment, in addition to those with atherogenic dyslipidemia.

Milk is a complex fluid required for development, nutrition and immunological protection to the newborn offspring. Interestingly, latest finding proved the presence of novel stem cell population in human milk with multilineage differentiation potential. Given that little is known about cellular milk content in other mammalian species such as bovine, the purpose of our study was to isolate and characterize a potential stem cell-like population in bovine milk. In detail, we first analyzed the phenotype of the isolated cells able to grow in plastic adherence and then their capability to differentiate into osteogenic, chondrogenic, and adipogenic lineages. Bovine milk stem cells (bMSCs) resulted plastic adherent and showed a heterogeneous population with epithelial and spindle-shaped cells. Successively, their immunophenotype indicated that bovine milk cells were positive for the typical epithelial markers E-cadherin, cytokeratin-14, cytokeratin-18, and smooth muscle actin. Notably, a subset (30%-40%), constantly observed in purified milk cells, showed the typical mesenchymal surface antigens CD90, CD73, and CD105. Furthermore, the same percentage of bMSCs expressing CD90, CD73, and CD105 presented the stemness markers SOX2 and OCT4 translocated in their nuclei. Finally, our data showed that bMSCs were able to differentiate into osteoblasts, chondroblasts, and adipocytes. In addition, the flow cytometry analysis revealed the presence of a subpopulation of events characterized by typical extracellular vesicles (EVs, size 0.1-1 μm), which did not contain nuclei and were positive for the same markers identified on the surface of bMSCs (CD73, CD90, and CD105), and thus might be considered milk cell-derived EVs. In conclusion, our data suggest that bovine milk is an easily available source of multipotent stem cells able to differentiate into multiple cell lineages. These features can open new possibilities for development biology and regenerative medicine in veterinary area to improving animal health.

Endothelial cell (EC) dysfunction has been reported in cystic fibrosis (CF) patients. Thus, the availability of CF EC is paramount to uncover mechanisms of endothelial dysfunction in CF. Using collagenase digestion, we isolated cells from small fragments of pulmonary artery dissected from non-CF lobes or explanted CF lungs. These cells were a heterogeneous mixture, containing variable percentages of EC. To obtain virtually pure pulmonary artery endothelial cells (PAEC), we developed an easy, inexpensive, and reliable method, based on the differential adhesion time of pulmonary artery cells collected after collagenase digestion. With this method, we obtained up to 95% pure non-CF and CF-PAEC. Moreover, we also succeed at immortalizing both PAEC and CF-PAEC, which remained viable and with unchanged phenotype and proliferation rate over the 30th passage. These cells recapitulated cystic fibrosis transmembrane conductance regulator expression and functions of the parental cells. Thus, we isolated for the first time endothelial cells from CF patients, providing a valuable tool to define the emerging role of EC in CF lung and vascular disease.

Menaquinones, also known as Vitamin K2 family, regulate calcium homeostasis in a 'bone-vascular cross-talk' and recently received particular attention for their positive effect on bone formation. Given that the correlation between menaquinones and bone metabolism to date is still unclear, the objective of our study was to investigate the possible role of menaquinone-4 (MK-4), an isoform of the menaquinones family, in the modulation of osteogenesis. For this reason, we used a model of human amniotic fluid mesenchymal stem cells (hAFMSCs) cultured both in two-dimensional (2D) and three-dimensional (3D; RCCS™bioreactor) in vitro culture systems. Furthermore, to mimic the 'bone remodelling unit' in vitro, hAFMSCs were co-cultured in the 3D system with human monocyte cells (hMCs) as osteoclast precursors. The results showed that in a conventional 2D culture system, hAFMSCs were responsive to the MK-4, which significantly improved the osteogenic process through γ-glutamyl carboxylase-dependent pathway. The same results were obtained in the 3D dynamic system where MK-4 treatment supported the osteoblast-like formation promoting the extracellular bone matrix deposition and the expression of the osteogenic-related proteins (alkaline phosphatase, osteopontin, collagen type-1 and osteocalcin). Notably, when the hAFMSCs were co-cultured in a 3D dynamic system with the hMCs, the presence of MK-4 supported the cellular aggregate formation as well as the osteogenic function of hAFMSCs, but negatively affected the osteoclastogenic process. Taken together, our results demonstrate that MK-4 supported the aggregate formation of hAFMSCs and increased the osteogenic functions. Specifically, our data could help to optimize bone regenerative medicine combining cell-based approaches with MK-4 treatment.

The proresolution lipid mediator lipoxin (LX)A4 bestows protective bioactions on endothelial cells. We examined the impact of LXA4 on transcellular endothelial signaling via microRNA (miR)-containing microvesicles. We report LXA4 inhibition of MV release by TNF-α-treated HUVECs, associated with the down-regulation of 18 miR in endothelial microvesicles (EMVs) and the up-regulation of miR-126-5p, both in HUVECs and in EMVs. LXA4 up-regulated miR-126-5p by ∼5-fold in HUVECs and promoted a release of microvesicles (LXA4-EMVs) that enhanced miR-126-5p by ∼7-fold in recipient HUVECs. In these cells, LXA4-EMVs abrogated the up-regulation of VCAM-1, induced in recipient HUVECs by EMVs released by untreated or TNF-α-treated HUVECs. LXA4-EMVs also reduced by ∼40% the expression of SPRED1, which we validated as an miR-126-5p target, whereas they stimulated monolayer repair in an in vitro wound assay. This effect was lost when the EMVs were depleted of miR-126-5p. These results provide evidence that changes in miR expression and microvesicle packaging and transfer represent a mechanism of action of LXA4, which may be relevant in vascular biology and inflammation.-Codagnone, M., Recchiuti, A., Lanuti, P., Pierdomenico, A. M., Cianci, E., Patruno, S., Mari, V. C., Simiele, F., Di Tomo, P., Pandolfi, A., Romano, M. Lipoxin A4 stimulates endothelial miR-126-5p expression and its transfer via microvesicles.

A variant on chromosome 1q25 (rs10911021) has been previously associated with coronary artery disease (CAD) in individuals with T2D. In human umbilical vein endothelial cells (HUVECs), the 1q25 risk allele (‘C’) was found to be associated with decreased expression of the adjacent gene GLUL encoding the enzyme glutamine synthase converting glutamate to glutamine. To further investigate the mechanisms through which the 1q25 locus modulates CAD risk in T2D, we performed a global metabolomics study (LC-MS, Metabolon) in 62 HUVECs (TT=11, CT=21, CC=30). Among 597 metabolites detected in HUVEC lysates, 23 attained p In summary, we have identified several metabolic pathways potentially mediating the 1q25 locus effect on CAD risk in T2D. These include impairment of the γ-glutamyl cycle, possibly increasing susceptibility to oxidative stress, and downregulation of PUFA derivatives and sphingomyelins. Further studies of these pathways may lead to the identification of novel therapeutic targets to reduce CAD risk in T2D. Disclosure C. Pipino: None. H. Shah: None. K. Park: None. S. Monti: None. C. Mendonca: None. G.L. King: Research Support; Self; Sanofi. A. Pandolfi: None. A. Doria: Research Support; Self; Sanofi-Aventis. Funding Sanofi-Aventis

Diabetes is associated with vascular inflammation, endothelial dysfunction, and oxidative stress, promoting the development of cardiovascular diseases (CVD). Several studies showed that a carotenoid-rich diet is associated to a reduced cardiovascular risk in healthy and diabetic subjects, although the mechanisms of action are still unknown. Here, the potential role of β-carotene (BC) and lycopene (Lyc) in human endothelial cells isolated from human umbilical cord vein (HUVECs) of women with gestational diabetes (GD) and respective controls (C) has been investigated. Results showed that BC and Lyc reduced the tumor necrosis factor alpha- (TNF-α-) stimulated monocyte-endothelium interaction (adhesion assay), membrane exposure (flow cytometry), and total expression levels (Western blot) of VCAM-1 and ICAM-1 in both cell types. Moreover, the treatment with BC and Lyc reduced the TNF-α-induced nuclear translocation of NF-κB (image flow cytometry) by preserving bioavailability of nitric oxide (NO, flow cytometry, and cGMP EIA kit assay), a key vasoactive molecule. Notably, BC and Lyc pretreatment significantly reduced peroxynitrite levels (flow cytometry), contributing to the redox balance protection. These results suggest a new mechanism of action of carotenoids which exert vascular protective action in diabetic condition, thus reinforcing the importance of a carotenoid-rich diet in the prevention of diabetes cardiovascular complications.

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.