Orexin-A exerts equivocal role in atherosclerosis process depending on the duration of exposure : in vitro study

Orexin-A is a peptide hormone that plays a crucial role in feeding regulation and energy homeostasis. Diurnal intermittent fasting (DIF) has been found to increase orexin-A plasma levels during fasting hours, while Ramadan fasting which resembles DIF, has led to beneficial effects on endothelial function. Herein, we aimed to investigate the effects of orexin-A on the expression of molecules involved in the atherogenesis process: Monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) and tissue inhibitor of metalloproteinase-1 and 2 (TIMP-1 and TIMP-2), in human aortic endothelial cells (HAECs). HAECs were incubated with orexin-A at concentrations of 40 ng/mL, 200 ng/mL and 400 ng/mL for 6, 12 and 24 h. The mRNA levels of MCP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2 and orexin-1 receptor were measured by real-time qPCR. We also evaluated the MMP-2, p38, phospho-p38, NF-κΒ/p65 as well as TIMP-1 protein levels by Western blot and ELISA, respectively. MMP-2 activity was measured by gelatin zymography. Short-term 6-h incubation of HAECs with orexin-A at a high concentration (400 ng/mL) decreased MCP-1, MMP-2 expression, MMP-2/TIMP-1 ratio (p < 0.05), and MMP-2 activity, while incubation for 24 h increased MCP-1, MMP-2 expression (p < 0.05), MMP-2/TIMP-1 and MMP-2/TIMP-2 ratio (p < 0.01 and p < 0.05, respectively) as well as MMP-2 activity. The dual effects of orexin-A are mediated, at least in part, via regulation of p38 and NF-κΒ pathway. Orexin-A may have an equivocal role in atherosclerosis process with its effects depending on the duration of exposure

SGLT-2 Inhibitors in NAFLD: Expanding Their Role beyond Diabetes and Cardioprotection?

Non-alcoholic fatty liver disease (NAFLD) is an ‘umbrella’ term, comprising a spectrum ranging from benign, liver steatosis to non-alcoholic steatohepatitis, liver fibrosis and eventually cirrhosis and hepatocellular carcinoma. NAFLD has evolved as a major health problem in recent years. Discovering ways to prevent or delay the progression of NAFLD has become a global focus. Lifestyle modifications remain the cornerstone of NAFLD treatment, even though various pharmaceutical interventions are currently under clinical trial. Among them, sodium-glucose co-transporter type-2 inhibitors (SGLT-2i) are emerging as promising agents. Processes regulated by SGLT-2i, such as endoplasmic reticulum (ER) and oxidative stress, low-grade inflammation, autophagy and apoptosis are all implicated in NAFLD pathogenesis. In this review, we summarize the current understanding of the NAFLD pathophysiology, and specifically focus on the potential impact of SGLT-2i in NAFLD development and progression, providing current evidence from in vitro, animal and human studies. Given this evidence, further mechanistic studies would advance our understanding of the exact mechanisms underlying the pathogenesis of NAFLD and the potential beneficial actions of SGLT-2i in the context of NAFLD treatment

Estrogen Receptor Subtypes Elicit a Distinct Gene Expression Profile of Endothelial-Derived Factors Implicated in Atherosclerotic Plaque Vulnerability

In the presence of established atherosclerosis, estrogens are potentially harmful. MMP-2 and MMP-9, their inhibitors (TIMP-2 and TIMP-1), RANK, RANKL, OPG, MCP-1, lysyl oxidase (LOX), PDGF-β, and ADAMTS-4 play critical roles in plaque instability/rupture. We aimed to investigate (i) the effect of estradiol on the expression of the abovementioned molecules in endothelial cells, (ii) which type(s) of estrogen receptors mediate these effects, and (iii) the role of p21 in the estrogen-mediated regulation of the aforementioned factors. Human aortic endothelial cells (HAECs) were cultured with estradiol in the presence or absence of TNF-α. The expression of the aforementioned molecules was assessed by qRT-PCR and ELISA. Zymography was also performed. The experiments were repeated in either ERα- or ERβ-transfected HAECs and after silencing p21. HAECs expressed only the GPR-30 estrogen receptor. Estradiol, at low concentrations, decreased MMP-2 activity by 15-fold, increased LOX expression by 2-fold via GPR-30, and reduced MCP-1 expression by 3.5-fold via ERβ. The overexpression of ERα increased MCP-1 mRNA expression by 2.5-fold. In a low-grade inflammation state, lower concentrations of estradiol induced the mRNA expression of MCP-1 (3.4-fold) and MMP-9 (7.5-fold) and increased the activity of MMP-2 (1.7-fold) via GPR-30. Moreover, p21 silencing resulted in equivocal effects on the expression of the abovementioned molecules. Estradiol induced different effects regarding atherogenic plaque instability through different ERs. The balance of the expression of the various ER subtypes may play an important role in the paradoxical characterization of estrogens as both beneficial and harmful

Endothelial cell dysfunction and nonalcoholic fatty liver disease (NAFLD) : a concise review

Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide. It is strongly associated with obesity, type 2 diabetes (T2DM), and other metabolic syndrome features. Reflecting the underlying pathogenesis and the cardiometabolic disorders associated with NAFLD, the term metabolic (dysfunction)-associated fatty liver disease (MAFLD) has recently been proposed. Indeed, over the past few years, growing evidence supports a strong correlation between NAFLD and increased cardiovascular disease (CVD) risk, independent of the presence of diabetes, hypertension, and obesity. This implies that NAFLD may also be directly involved in the pathogenesis of CVD. Notably, liver sinusoidal endothelial cell (LSEC) dysfunction appears to be implicated in the progression of NAFLD via numerous mechanisms, including the regulation of the inflammatory process, hepatic stellate activation, augmented vascular resistance, and the distortion of microcirculation, resulting in the progression of NAFLD. Vice versa, the liver secretes inflammatory molecules that are considered pro-atherogenic and may contribute to vascular endothelial dysfunction, resulting in atherosclerosis and CVD. In this review, we provide current evidence supporting the role of endothelial cell dysfunction in the pathogenesis of NAFLD and NAFLD-associated atherosclerosis. Endothelial cells could thus represent a “golden target” for the development of new treatment strategies for NAFLD and its comorbid CVD

Genetic and Diet-Induced Animal Models for Non-Alcoholic Fatty Liver Disease (NAFLD) Research

A rapidly increasing incidence of non-alcoholic fatty liver disease (NAFLD) is noted worldwide due to the adoption of western-type lifestyles and eating habits. This makes the understanding of the molecular mechanisms that drive the pathogenesis of this chronic disease and the development of newly approved treatments of utmost necessity. Animal models are indispensable tools for achieving these ends. Although the ideal mouse model for human NAFLD does not exist yet, several models have arisen with the combination of dietary interventions, genetic manipulations and/or administration of chemical substances. Herein, we present the most common mouse models used in the research of NAFLD, either for the whole disease spectrum or for a particular disease stage (e.g., non-alcoholic steatohepatitis). We also discuss the advantages and disadvantages of each model, along with the challenges facing the researchers who aim to develop and use animal models for translational research in NAFLD. Based on these characteristics and the specific study aims/needs, researchers should select the most appropriate model with caution when translating results from animal to human

Empagliflozin Attenuates Non-Alcoholic Fatty Liver Disease (NAFLD) in High Fat Diet Fed ApoE (-/-) Mice by Activating Autophagy and Reducing ER Stress and Apoptosis

Aims/hypothesis: SGLT-2 inhibitors (SGLT-2i) have been studied as potential treatments against NAFLD, showing varying beneficial effects. The molecular mechanisms mediating these effects have not been fully clarified. Herein, we investigated the impact of empagliflozin on NAFLD, focusing particularly on ER stress, autophagy and apoptosis. Methods: Five-week old ApoE(-/-) mice were switched from normal to a high-fat diet (HFD). After five weeks, mice were randomly allocated into a control group (HFD + vehicle) and Empa group (HFD + empagliflozin 10 mg/kg/day) for five weeks. At the end of treatment, histomorphometric analysis was performed in liver, mRNA levels of Fasn, Screbp-1, Scd-1, Ppar-γ, Pck-1, Mcp-1, Tnf-α, Il-6, F4/80, Atf4, Elf2α, Chop, Grp78, Grp94, Χbp1, Ire1α, Atf6, mTor, Lc3b, Beclin-1, P62, Bcl-2 and Bax were measured by qRT-PCR, and protein levels of p-EIF2α, EIF2a, CHOP, LC3II, P62, BECLIN-1 and cleaved CASPASE-8 were assessed by immunoblotting. Results: Empagliflozin-treated mice exhibited reduced fasting glucose, total cholesterol and triglyceride serum levels, as well as decreased NAFLD activity score, decreased expression of lipogenic enzymes (Fasn, Screbp-1c and Pck-1) and inflammatory molecules (Mcp-1 and F4/80), compared to the Control group. Empagliflozin significantly decreased the expression of ER stress molecules Grp78, Ire1α, Xbp1, Elf2α, Atf4, Atf6, Chop, P62(Sqstm1) and Grp94; whilst activating autophagy via increased AMPK phosphorylation, decreased mTOR and increased LC3B expression. Finally, empagliflozin increased the Bcl2/Bax ratio and inhibited CASPASE-8 cleavage, reducing liver cell apoptosis. Immunoblotting analysis confirmed the qPCR results. Conclusion: These novel findings indicate that empagliflozin treatment for five weeks attenuates NAFLD progression in ApoE(-/-) mice by promoting autophagy, reducing ER stress and inhibiting hepatic apoptosis

Επίδραση των οιστρογόνων σε παράγοντες που εμπλέκονται στην αποσταθεροποίηση της αθηρωματικής πλάκας: αποσαφήνιση μοριακών μηχανισμών

Introduction: Pre-menopausal women have a significantly lower risk for cardiovascular disease while postmenopausal women and women with impaired ovarian function have a greater chance of developing cardiovascular disease than premenopausal women of the same age. The time since menopause and the age at which estrogen administration has been initiated seems to be important factor to declare the protective/harmful effect of hormone replacement therapy (E2) on atherosclerosis. Among the key vessel wall components in the later stages of atherogenic process are endothelial cells (ECs). During the stages of plaque instability/rupture, metalloproteinases (MMP2 and MMP9), their inhibitors (TIMP2 and TIMP1), RANK, RANKL, OPG, MCP1, lysyl oxidase (LOX), PDGF, and ADAMTS4 play critical role. Moreover, recent studies suggest the implication of the oncosuppression gene 53 and its transcriptional target p21 in the atherogenesis process as well as in the plaque calcification through OPG regulation. Therefore in this study We aimed to investigate i) the effect of estrogens alone and in the present of low grade inflammation (2ng TNF-α) on the expression of the above molecules in ECs and ii) which type of estrogen receptor mediates these effects iii) If p21 is involved in atherosclerosis plaque formation and stability in the presence of estrogens.Methods: Human aortic endothelial cells (HAECs) were cultured, in the absence or in the presence of estradiol (E2) at various concentrations and for various incubation times without or with pre-incubation with TNFα (resembling a low grade inflammation state). The mRNA levels of all the aforementioned genes and the protein level of MCP-1, TIMP-2 and OPG were assessed by real-time PCR and Elisa respectively. The MMP-2 and MMP-9 gelatinase activity was measured by gelatin zymography assay .The experiments were repeated in either ER-α or ER-β transfected or p21 silenced HAECs.Results: HAECs did not express ER-α and ER-β while they express G-protein coupled estrogen receptor 30 (GPR30). Incubation with E2 at low concentrations induced an increase in LOX and MCP1 mRNA expression. Zymography revealed that E2 induced a down regulation of active MMP2, dose-dependently. Incubation with E2 following pretreatment with TNF-α induced a marginal increase in MMP9 and TIMP1 expression and a ten times increase in MCP1. The MMP-2 activity was also increased significantly after pre-incubation of cells with TNF-α followed by co-incubation with E2. In ERα-transfected HAECs, incubation with E2 led to up-regulation of TIMP1 and TIMP2 and a marginal increase of LOX and MCP1 mRNA levels. Moreover, E2 increased the MCP-1 and TIMP-1 protein levels and reduced the MMP-2 gelatinase activity in ER-α transfected cells. In ER-β-transfected HAECs, E2 increased the expression of LOX while decreased the expression of PDGF. The MCP-1 protein level was significantly reduced in this experimental condition. In addition, incubation of cells silenced for p21 with E2 results in reduction of MCP-1, LOX and ADAMTs-4 mRNA levels. On the contrary the mRNA level of PDGF was elevated significantly after incubation of p21 silenced cells with various concentration of E2.Conclusion: E2 induced different effects regarding atherogenic plaque instability through different ERs. The balance of expression of the various ER subtypes may play an important role in the paradoxical characterization of estrogens as both beneficial and harmful. The P21 expression it may play a crucial role in Plaque formation and stability/instability through regulation of MCP-1 and LOX, ADAMTS-4 and PDGF expression.Εισαγωγή: Οι προ-εμμηνοπαυσιακές γυναίκες έχουν σημαντικά χαμηλότερο κίνδυνο εμφάνισης καρδιαγγειακής νόσου συγκριτικά με τις μετεμμηνοπαυσιακές γυναίκες ή τις γυναίκες με επηρεασμένη ωοθηκική λειτουργία της ίδιας ηλικίας. Η ηλικία εμμηνόπαυσης όπως και η ηλικία έναρξης της θεραπείας υποκατάστασης με οιστραδιόλη φαίνεται να παίζουν σημαντικό ρόλο στον κίνδυνο αθηροσκλήρωσης. Σε επίπεδο ιστού τα ενδοθηλιακά κύτταρα συμμετέχουν ενεργά στη δημιουργία αθηροσκλήρωσης . Στην δημιουργία της πλάκας και στην αστάθειά της σημαντικό ρόλο παίζουν οι μεταλλοπρωτεινάσες (MMP2 και MMP9), οι αναστολείς τους (TIMP2 και TIMP1), και τα μόρια RANK, RANKL, OPG, MCP1, lysyl oxidase (LOX), PDGF, και ADAMTS4. Πρόσφατες μελέτες έχουν δείξει οτι το ογκοκατασταλτικό γονίδιο p53 και ο μεταγραφικός του στόχος p21 εμπλέκονται στην ασβεστοποίηση της πλάκας μέσω της ρύθμισης του OPG. Σκοπός της παρούσας εργασίας είναι : α) η μελέτη της επίδρασης των οιστρογόνων είτε μόνων τους είτε παρουσία παραγόντων φλεγμονής (2ng TNF-α) στην έκφραση των παραπάνω μορίων στα ενδοθηλιακά κύτταρα ii) η μελέτη των υποδοχέων μέσω των οποίων τα οιστρογόνα ασκούν αυτές τις δράσεις iii) αν το p21 εμπλέκεται στον σχηματισμό και την σταθεροποίηση της αθηρωματικής πλάκας παρουσία των οιστρογόνων. Mέθοδοι Ανθρώπινα ενδοθηλιακά κύτταρα της αορτής καλλιεργήθηκαν παρουσία ή απουσία οιστραδιόλης σε διάφορες συγκεντρώσεις και για διαφορετικό χρόνο επώασης παρουσία ή όχι του TNFα. Μελετήθηκαν τα επίπεδα mRNA των παραπάνω γονιδίων όπως και πρωτεΐνης των MCP-1, TIMP-2 και OPG με real-time PCR and Elisa αντίστοιχα. Η ενζυμική δραστικότητα των MMP-2 and MMP-9 μελετήθηκε με την δοκιμασία της gelatin zymography. Tα πειράματα επαναλήφθηκαν σε ER-α ή ER-β διαμολυσμένα ενδοθηλιακά κύτταρα και σε κύτταρα διαμολυσμένα με p21 siRNA. Αποτελέσματα: Τα HAECs δεν εκφράζουν τους ER-α και ER-β ενώ εκφράζουν G-protein coupled estrogen receptor 30 (GPR30). Η καλλιέργεια των κυττάρων με χαμηλές συγκεντρώσεις E2 είχε σαν αποτέλεσμα την αύξηση της έκφρασης του mRNA των γονιδίων LOX and MCP1. Η ζυμογραφία έδειξε οτι η Ε2 προκάλεσε καταστολή της MMP2 με δοσοεξαρτώμενο τρόπο. Η επώαση με Ε2 παρουσία του TNF-α είχε σαν αποτέλεσμα την οριακή αύξηση της έκφρασης των MMP9 και TIMP1 και τον δεκαπλασιασμό της έκφρασης του MCP1 με δοσοεξαρτώμενο τρόπο. Επιπρόσθετα, η ενεργότητα της MMP2 αυξήθηκε σημαντικά μετά την επώαση με Ε2 παρουσία του TNF-α. Σε ΕR-a διαμόλυσμένα κυττάρα, η έκθεση στην Ε2 οδήγησε σε επαγωγή της έκφρασης του mRNA των γονιδίων TIMP1 and TIMP2 και οριακή άυξηση των LOX and MCP1. Επίσης η έκθεση στην Ε2 αυτών των διαμολυσμένων κυττάρων οδήγησε σε αύξηση των πρωτεϊνικών επιπέδων των MCP-1 και TIMP-1 και σε μείωση της ενζυμικής ενεργότητας της MMP2. Η Ε2 επίσης οδήγησε σε αύξηση των επιπέδων mRNA του γονιδίου LOX και σε μείωση της έκφρασης του PDGF στα ER-β-διαμολυσμένα HAECs. Τα πρωτεϊνικά επίπεδα της MCP-1 ήταν σημαντικά μειωμένα στα διαμολυσμένα ER-β κύτταρα παρουσία Ε2. Επίσης η επώαση των κυττάρων με αποσιώπηση του p21 γονιδίου, με Ε2 οδήγησε σε μείωση των επιπέδων mRNA των MCP -1, LOX και ADAMTs-4 γονιδίων. Σε αντίθεση τα mRNA επίπεδα του PDGF ήταν σημαντικά αυξημένα μετά απο επώαση αυτών των κυττάρων σε διαφορετικές συγκεντρώσεις Ε2. Συμπεράσματα: Η Ε2 είχε διαφορετικές επιδράσεις στην αστάθεια της αθηρωματικής πλάκας μέσω των υποδοχέων της. Η ισορροπία της έκφρασης των διάφορων τύπων των υποδοχέων ER μπορεί να επεξηγήσει τον παράδοξο χαρακτηρισμό των οιστρογόνων σαν ωφέλιμα και βλαβερά ταυτόχρονα. Η έκφραση του P21 μπορεί να παίξει σημαντικό ρόλο στον σχηματισμό της αθηρωματικής πλάκας μέσω της ρύθμισης της έκφρασης των MCP-1, LOX, ADAMTS-4 και PDGF γονιδίων

Management of hematologic malignancies in the era of COVID-19 pandemic: Pathogenetic mechanisms, impact of obesity, perspectives, and challenges

SIMPLE SUMMARY: Obesity is epidemiologically and likely, causally related to various hematological cancers, while both conditions may predispose to severe SARS-CoV-2 infection. The COVID-19 pandemic brought about a variety of obstacles with respect to numerous aspects in the management of hematological malignancies. In the present overview, the evidence linking obesity with the development of hematological cancers and their role as risk factors for severe COVID-19 is critically appraised. Furthermore, the various challenges which emerged during the the pandemic are reviewed, regarding not only the treatment of the underlying hematological malignancies themselves, but also the prevention and therapeutic management of SARS-CoV-2 infection in this patient group. Lastly, we discuss further unresolved issues which need to be addressed in order to optimize the management of patients with hematologic cancers in the course of ongoing COVID-19 pandemic. ABSTRACT: The COVID-19 pandemic brought about an unprecedented societal and healthcare system crisis, considerably affecting healthcare workers and patients, particularly those with chronic diseases. Patients with hematologic malignancies faced a variety of challenges, pertinent to the nature of an underlying hematologic disorder itself as well as its therapy as a risk factor for severe SARS-CoV-2 infection, suboptimal vaccine efficacy and the need for uninterrupted medical observation and continued therapy. Obesity constitutes another factor which was acknowledged since the early days of the pandemic that predisposed people to severe COVID-19, and shares a likely causal link with the pathogenesis of a broad spectrum of hematologic cancers. We review here the epidemiologic and pathogenetic features that obesity and hematologic malignancies share, as well as potential mutual pathophysiological links predisposing people to a more severe SARS-CoV-2 course. Additionally, we attempt to present the existing evidence on the multi-faceted crucial challenges that had to be overcome in this diverse patient group and discuss further unresolved questions and future challenges for the management of hematologic malignancies in the era of COVID-19