New Mediators in the Crosstalk between Different Adipose Tissues.

Adipose tissue is a multifunctional organ that regulates many physiological processes such as energy homeostasis, nutrition, the regulation of insulin sensitivity, body temperature, and immune response. In this review, we highlight the relevance of the different mediators that control adipose tissue activity through a systematic review of the main players present in white and brown adipose tissues. Among them, inflammatory mediators secreted by the adipose tissue, such as classical adipokines and more recent ones, elements of the immune system infiltrated into the adipose tissue (certain cell types and interleukins), as well as the role of intestinal microbiota and derived metabolites, have been reviewed. Furthermore, anti-obesity mediators that promote the activation of beige adipose tissue, e.g., myokines, thyroid hormones, amino acids, and both long and micro RNAs, are exhaustively examined. Finally, we also analyze therapeutic strategies based on those mediators that have been described to date. In conclusion, novel regulators of obesity, such as microRNAs or microbiota, are being characterized and are promising tools to treat obesity in the future.

Increased let-7d-5p in non-alcoholic fatty liver promotes insulin resistance and is a potential blood biomarker for diagnosis.

BACKGROUND AND AIMS: The molecular mechanisms driving non-alcoholic fatty liver disease (NAFLD) are poorly understood; however, microRNAs might play a key role in these processes. We hypothesize that let-7d-5p could contribute to the pathophysiology of NAFLD and serve as a potential diagnostic biomarker. METHODS: We evaluated let-7d-5p levels and its targets in liver biopsies from a cross-sectional study including patients with NAFLD and healthy donors, and from a mouse model of NAFLD. Moreover, the induction of let-7d-5p expression by fatty acids was evaluated in vitro. Further, we overexpressed let-7d-5p in vitro to corroborate the results observed in vivo. Circulating let-7d-5p and its potential as a NAFLD biomarker was determined in isolated extracellular vesicles from human plasma by RT-qPCR. RESULTS: Our results demonstrate that hepatic let-7d-5p was significantly up-regulated in patients with steatosis, and this increase correlated with obesity and a decreased expression of AKT serine/threonine kinase (AKT), insulin-like growth factor 1 (IGF1), IGF-I receptor (IGF1R) and insulin receptor (INSR). These alterations were corroborated in a NAFLD mouse model. In vitro, fatty acids increased let-7d-5p expression, and its overexpression decreased AKT, IGF-IR and IR protein expression. Furthermore, let-7d-5p hindered AKT phosphorylation in vitro after insulin stimulation. Finally, circulating let-7d-5p significantly decreased in steatosis patients and receiver operating characteristic (ROC) analyses confirmed its utility as a diagnostic biomarker. CONCLUSIONS: Our results highlight the emerging role of let-7d-5p as a potential therapeutic target for NAFLD since its overexpression impairs hepatic insulin signalling, and also, as a novel non-invasive biomarker for NAFLD diagnosis.

Applicability of probabilistic graphical models for early detection of SARS-CoV-2 reactive antibodies after SARS-CoV-2 vaccination in hematological patients.

Prior studies of antibody response after full SARS-CoV-2 vaccination in hematological patients have confirmed lower antibody levels compared to the general population. Serological response in hematological patients varies widely according to the disease type and its status, and the treatment given and its timing with respect to vaccination. Through probabilistic machine learning graphical models, we estimated the conditional probabilities of having detectable anti-SARS-CoV-2 antibodies at 3-6 weeks after SARS-CoV-2 vaccination in a large cohort of patients with several hematological diseases (n= 1166). Most patients received mRNA-based vaccines (97%), mainly Moderna® mRNA-1273 (74%) followed by Pfizer-BioNTech® BNT162b2 (23%). The overall antibody detection rate at 3 to 6 weeks after full vaccination for the entire cohort was 79%. Variables such as type of disease, timing of anti-CD20 monoclonal antibody therapy, age, corticosteroids therapy, vaccine type, disease status, or prior infection with SARS-CoV-2 are among the most relevant conditions influencing SARS-CoV-2-IgG-reactive antibody detection. A lower probability of having detectable antibodies was observed in patients with B-cell non-Hodgkin's lymphoma treated with anti-CD20 monoclonal antibodies within 6 months before vaccination (29.32%), whereas the highest probability was observed in younger patients with chronic myeloproliferative neoplasms (99.53%). The Moderna® mRNA-1273 compound provided higher probabilities of antibody detection in all scenarios. This study depicts conditional probabilities of having detectable antibodies in the whole cohort and in specific scenarios such as B cell NHL, CLL, MM, and cMPN that may impact humoral responses. These results could be useful to focus on additional preventive and/or monitoring interventions in these highly immunosuppressed hematological patients.

Implication of miR-155-5p and miR-143-3p in the Vascular Insulin Resistance and Instability of Human and Experimental Atherosclerotic Plaque.

(1) Background: Cardiovascular diseases (CVDs) are the main cause of death in developed countries, being atherosclerosis, a recurring process underlying their apparition. MicroRNAs (miRNAs) modulate the expression of their targets and have emerged as key players in CVDs; (2) Methods: 18 miRNAs were selected (Pubmed and GEO database) for their possible role in promoting atherosclerosis and were analysed by RT-qPCR in the aorta from apolipoprotein E-deficient (ApoE-/-) mice. Afterwards, the altered miRNAs in the aorta from 18 weeks-ApoE-/- mice were studied in human aortic and carotid samples; (3) Results: miR-155-5p was overexpressed and miR-143-3p was downregulated in mouse and human atherosclerotic lesions. In addition, a significant decrease in protein kinase B (AKT), target of miR-155-5p, and an increase in insulin-like growth factor type II receptor (IGF-IIR), target of miR-143-3p, were noted in aortic roots from ApoE-/- mice and in carotid plaques from patients with advanced carotid atherosclerosis (ACA). Finally, the overexpression of miR-155-5p reduced AKT levels and its phosphorylation in vascular smooth muscle cells, while miR-143-3p overexpression decreased IGF-IIR reducing apoptosis in vascular cells; (4) Conclusions: Our results suggest that miR-155-5p and miR-143-3p may be implicated in insulin resistance and plaque instability by the modulation of their targets AKT and IGF-IIR, contributing to the progression of atherosclerosis.

Potential role of insulin receptor isoforms and IGF receptors in plaque instability of human and experimental atherosclerosis.

BACKGROUND: Clinical complications associated with atherosclerotic plaques arise from luminal obstruction due to plaque growth or destabilization leading to rupture. We previously demonstrated that overexpression of insulin receptor isoform A (IRA) and insulin-like growth factor-I receptor (IGF-IR) confers a proliferative and migratory advantage to vascular smooth muscle cells (VSMCs) promoting plaque growth in early stages of atherosclerosis. However, the role of insulin receptor (IR) isoforms, IGF-IR or insulin-like growth factor-II receptor (IGF-IIR) in VSMCs apoptosis during advanced atherosclerosis remains unclear. METHODS: We evaluated IR isoforms expression in human carotid atherosclerotic plaques by consecutive immunoprecipitations of insulin receptor isoform B (IRB) and IRA. Western blot analysis was performed to measure IGF-IR, IGF-IIR, and α-smooth muscle actin (α-SMA) expression in human plaques. The expression of those proteins, as well as the presence of apoptotic cells, was analyzed by immunohistochemistry in experimental atherosclerosis using BATIRKO; ApoE-/- mice, a model showing more aggravated vascular damage than ApoE-/- mice. Finally, apoptosis of VSMCs bearing IR (IRLoxP+/+ VSMCs), or not (IR-/- VSMCs), expressing IRA (IRA VSMCs) or expressing IRB (IRB VSMCs), was assessed by Western blot against cleaved caspase 3. RESULTS: We observed a significant decrease of IRA/IRB ratio in human complicated plaques as compared to non-complicated regions. Moreover, complicated plaques showed a reduced IGF-IR expression, an increased IGF-IIR expression, and lower levels of α-SMA indicating a loss of VSMCs. In experimental atherosclerosis, we found a significant decrease of IRA with an increased IRB expression in aorta from 24-week-old BATIRKO; ApoE-/- mice. Furthermore, atherosclerotic plaques from BATIRKO; ApoE-/- mice had less VSMCs content and higher number of apoptotic cells. In vitro experiments showed that IGF-IR inhibition by picropodophyllin induced apoptosis in VSMCs. Apoptosis induced by thapsigargin was lower in IR-/- VSMCs expressing higher IGF-IR levels as compared to IRLoxP+/+ VSMCs. Finally, IRB VSMCs are more prone to thapsigargin-induced apoptosis than IRA or IRLoxP+/+ VSMCs. CONCLUSIONS: In advanced human atherosclerosis, a reduction of IRA/IRB ratio, decreased IGF-IR expression, or increased IGF-IIR may contribute to VSMCs apoptosis, promoting plaque instability and increasing the risk of plaque rupture and its clinical consequences.

Prevalent role of the insulin receptor isoform A in the regulation of hepatic glycogen metabolism in hepatocytes and in mice.

AIMS/HYPOTHESIS: In the postprandial state, the liver regulates glucose homeostasis by glucose uptake and conversion to glycogen and lipids. Glucose and insulin signalling finely regulate glycogen synthesis through several mechanisms. Glucose uptake in hepatocytes is favoured by the insulin receptor isoform A (IRA), rather than isoform B (IRB). Thus, we hypothesised that, in hepatocytes, IRA would increase glycogen synthesis by promoting glucose uptake and glycogen storage. METHODS: We addressed the role of insulin receptor isoforms on glycogen metabolism in vitro in immortalised neonatal hepatocytes. In vivo, IRA or IRB were specifically expressed in the liver using adeno-associated virus vectors in inducible liver insulin receptor knockout (iLIRKO) mice, a model of type 2 diabetes. The role of IR isoforms in glycogen synthesis and storage in iLIRKO was subsequently investigated. RESULTS: In immortalised hepatocytes, IRA, but not IRB expression induced an increase in insulin signalling that was associated with elevated glycogen synthesis, glycogen synthase activity and glycogen storage. Similarly, elevated IRA, but not IRB expression in the livers of iLIRKO mice induced an increase in glycogen content. CONCLUSIONS/INTERPRETATION: We provide new insight into the role of IRA in the regulation of glycogen metabolism in cultured hepatocytes and in the livers of a mouse model of type 2 diabetes. Our data strongly suggest that IRA is more efficient than IRB at promoting glycogen synthesis and storage. Therefore, we suggest that IRA expression in the liver could provide an interesting therapeutic approach for the regulation of hepatic glucose content and glycogen storage.

Protective role of oleic acid against cardiovascular insulin resistance and in the early and late cellular atherosclerotic process.

BACKGROUND: Several translational studies have identified the differential role between saturated and unsaturated fatty acids at cardiovascular level. However, the molecular mechanisms that support the protective role of oleate in cardiovascular cells are poorly known. For these reasons, we studied the protective role of oleate in the insulin resistance and in the atherosclerotic process at cellular level such as in cardiomyocytes (CMs), vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). METHODS: The effect of oleate in the cardiovascular insulin resistance, vascular dysfunction, inflammation, proliferation and apoptosis of VSMCs were analyzed by Western blot, qRT-PCR, BrdU incorporation and cell cycle analysis. RESULTS: Palmitate induced insulin resistance. However, oleate not only did not induce cardiovascular insulin resistance but also had a protective effect against insulin resistance induced by palmitate or TNFα. One mechanism involved might be the prevention by oleate of JNK-1/2 or NF-κB activation in response to TNF-α or palmitate. Oleate reduced MCP-1 and ICAM-1 and increased eNOS expression induced by proinflammatory cytokines in ECs. Furthermore, oleate impaired the proliferation induced by TNF-α, angiotensin II or palmitate and the apoptosis induced by TNF-α or thapsigargin in VSMCs. CONCLUSIONS: Our data suggest a differential role between oleate and palmitate and support the concept of the cardioprotector role of oleate as the main lipid component of virgin olive oil. Thus, oleate protects against cardiovascular insulin resistance, improves endothelial dysfunction in response to proinflammatory signals and finally, reduces proliferation and apoptosis in VSMCs that may contribute to an ameliorated atherosclerotic process and plaque stability.

Antagonistic effect of TNF-alpha and insulin on uncoupling protein 2 (UCP-2) expression and vascular damage.

BACKGROUND: It has been reported that increased expression of UCP-2 in the vasculature may prevent the development of atherosclerosis in patients with increased production of reactive oxygen species, as in the diabetes, obesity or hypertension. Thus, a greater understanding in the modulation of UCP-2 could improve the atherosclerotic process. However, the effect of TNF-α or insulin modulating UCP-2 in the vascular wall is completely unknown. In this context, we propose to study new molecular mechanisms that help to explain whether the moderate hyperinsulinemia or lowering TNF-α levels might have a protective role against vascular damage mediated by UCP-2 expression levels. METHODS: We analyzed the effect of insulin or oleic acid in presence or not of TNF-α on UCP-2 expression in murine endothelial and vascular smooth muscle cells. At this step, we wondered if some mechanisms studied in vitro could be of any relevance in vivo. We used the following experimental models: ApoE-/- mice under Western type diet for 2, 6, 12 or 18 weeks, BATIRKO mice under high-fat diet for 16 weeks and 52-week-old BATIRKO mice with o without anti-TNF-α antibody pre-treatment. RESULTS: Firstly, we found that TNF-α pre-treatment reduced UCP-2 expression induced by insulin in vascular cells. Secondly, we observed a progressive reduction of UCP-2 levels together with an increase of lipid depots and lesion area in aorta from ApoE-/- mice. In vivo, we also observed that moderate hyperinsulinemic obese BATIRKO mice have lower TNF-α and ROS levels and increased UCP-2 expression levels within the aorta, lower lipid accumulation, vascular dysfunction and macrovascular damage. We also observed that the anti-TNF-α antibody pre-treatment impaired the loss of UCP-2 expression within the aorta and relieved vascular damage observed in 52-week-old BATIRKO mice. Finally, we observed that the pretreatment with iNOS inhibitor prevented UCP-2 reduction induced by TNF-α in vascular cells. Moreover, iNOS levels are augmented in aorta from mice with lower UCP-2 levels and higher TNF-α levels. CONCLUSIONS: Our data suggest that moderate hyperinsulinemia in response to insulin resistance or lowering of TNF-α levels within the aorta attenuates vascular damage, this protective effect being mediated by UCP-2 expression levels through iNOS.

Dysregulation of micro-RNA 143-3p as a Biomarker of Carotid Atherosclerosis and the Associated Immune Reactions During Disease Progression.

Atherosclerosis commonly remains undiagnosed until disease manifestations occur. The disease is associated with dysregulated micro(mi)RNAs, but how this is linked to atherosclerosis-related immune reactions is largely unknown. A mouse model of carotid atherosclerosis, human APOB100-transgenic Ldlr-/- (HuBL), was used to study the spatiotemporal dysregulation of a set of miRNAs. Middle-aged HuBL mice with established atherosclerosis had decreased levels of miR-143-3p in their carotid arteries. In young HuBL mice, early atherosclerosis was observed in the carotid bifurcation, which had lower levels of miR-15a-5p, miR-143-3p, and miR-199a-3p, and higher levels of miR-155-5p. The dysregulation of these miRNAs was reflected by specific immune responses during atheroprogression. Finally, levels of miR-143-3p were 70.6% lower in extracellular vesicles isolated from the plasma of patients with carotid stenosis compared to healthy controls. Since miR-143-3p levels progressively decrease when transitioning between early and late experimental carotid atherosclerosis, we propose it as a biomarker for atherosclerosis.

Potential protective role of let-7d-5p in atherosclerosis progression reducing the inflammatory pathway regulated by NF-κB and vascular smooth muscle cells proliferation.

The prevalence of cardiovascular diseases (CVDs) is increasing in the last decades, even is the main cause of death in first world countries being atherosclerosis one of the principal triggers. Therefore, there is an urgent need to decipher the underlying mechanisms involved in atherosclerosis progression. In this respect, microRNAs dysregulation is frequently involved in the progression of multiple diseases including CVDs. Our aim was to demonstrate that let-7d-5p unbalance could contribute to the pathophysiology of atherosclerosis and serve as a potential diagnostic biomarker. We evaluated let-7d-5p levels in vascular biopsies and exosome-enriched extracellular vesicles (EVs) from patients with carotid atherosclerosis and healthy donors. Moreover, we overexpressed let-7d-5p in vitro in vascular smooth muscle cells (VSMCs) to decipher the targets and the underlying mechanisms regulated by let-7d-5p in atherosclerosis. Our results demonstrate that let-7d-5p was significantly upregulated in carotid plaques from overweight patients with carotid atherosclerosis. Moreover, in EVs isolated from plasma, we found that let-7d-5p levels were increased in carotid atherosclerosis patients compared to control subjects specially in overweight patients. Receiver Operating Characteristic (ROC) analyses confirmed its utility as a diagnostic biomarker for atherosclerosis. In VSMCs, we demonstrated that increased let-7d-5p levels impairs cell proliferation and could serve as a protective mechanism against inflammation by impairing NF-κB pathway without affecting insulin resistance. In summary, our results highlight the role of let-7d-5p as a potential therapeutic target for atherosclerosis since its overexpression induce a decrease in inflammation and VSMCs proliferation, and also, as a novel non-invasive diagnostic biomarker for atherosclerosis in overweight patients.

Concerted expression of the thermogenic and bioenergetic mitochondrial protein machinery in brown adipose tissue.

Brown adipose tissue (BAT) is specialized in non-shivering thermogenesis through the expression of the mitochondrial uncoupling protein-1 (UCP1). In this paper, we describe the relationship between UCP1 and proteins involved in ATP synthesis. By the use of BATIRKO mice, which have enhanced UCP1 expression in BAT, an increase in ATP synthase as well as in ubiquinol cytochrome c reductase levels was observed. Alterations in mitochondrial mass or variations in ATP levels were not observed in BAT of these mice. In addition, using a protocol of brown adipocyte differentiation, the concerted expression of UCP1 with ATP synthase was found. These two scenarios revealed that increases in the uncoupling machinery of brown adypocites must be concomitantly followed by an enhancement of proteins involved in ATP synthesis. These concerted changes reflect the need to maintain ATP production in an essentially uncoupling cell type.

Oxidative Stress Modulation by ncRNAs and Their Emerging Role as Therapeutic Targets in Atherosclerosis and Non-Alcoholic Fatty Liver Disease.

Atherosclerosis and non-alcoholic fatty liver disease (NAFLD) are pathologies related to ectopic fat accumulation, both of which are continuously increasing in prevalence. These threats are prompting researchers to develop effective therapies for their clinical management. One of the common pathophysiological alterations that underlies both diseases is oxidative stress (OxS), which appears as a result of lipid deposition in affected tissues. However, the molecular mechanisms that lead to OxS generation are different in each disease. Non-coding RNAs (ncRNAs) are RNA transcripts that do not encode proteins and function by regulating gene expression. In recent years, the involvement of ncRNAs in OxS modulation has become more recognized. This review summarizes the most recent advances regarding ncRNA-mediated regulation of OxS in atherosclerosis and NAFLD. In both diseases, ncRNAs can exert pro-oxidant or antioxidant functions by regulating gene targets and even other ncRNAs, positioning them as potential therapeutic targets. Interestingly, both diseases have common altered ncRNAs, suggesting that the same molecule can be targeted simultaneously when both diseases coexist. Finally, since some ncRNAs have already been used as therapeutic agents, their roles as potential drugs for the clinical management of atherosclerosis and NAFLD are analyzed.

Syndecan-4 proteoliposomes enhance revascularization in a rabbit hind limb ischemia model of peripheral ischemia.

Regenerative therapeutics for treating peripheral arterial disease are an appealing strategy for creating more durable solutions for limb ischemia. In this work, we performed preclinical testing of an injectable formulation of syndecan-4 proteoliposomes combined with growth factors as treatment for peripheral ischemia delivered in an alginate hydrogel. We tested this therapy in an advanced model of hindlimb ischemia in rabbits with diabetes and hyperlipidemia. Our studies demonstrate enhancement in vascularity and new blood vessel growth with treatment with syndecan-4 proteoliposomes in combination with FGF-2 or FGF-2/PDGF-BB. The effects of the treatments were particularly effective in enhancing vascularity in the lower limb with a 2-4 increase in blood vessels in the treatment group in comparison to the control group. In addition, we demonstrate that the syndecan-4 proteoliposomes have stability for at least 28 days when stored at 4°C to allow transport and use in the hospital environment. In addition, we performed toxicity studies in the mice and found no toxic effects even when injected at high concentration. Overall, our studies support that syndecan-4 proteoliposomes markedly enhance the therapeutic potential of growth factors in the context of disease and may be promising therapeutics for inducing vascular regeneration in peripheral ischemia. STATEMENT OF SIGNIFICANCE: Peripheral ischemia is a common condition in which there is a lack of blood flow to the lower limbs. This condition can lead to pain while walking and, in severe cases, critical limb ischemia and limb loss. In this study, we demonstrate the safety and efficacy of a novel injectable therapy for enhancing revascularization in peripheral ischemia using an advanced large animal model of peripheral vascular disease using rabbits with hyperlipidemia and diabetes.

Role of miR-15a-5p and miR-199a-3p in the inflammatory pathway regulated by NF-κB in experimental and human atherosclerosis.

BACKGROUND: Cardiovascular diseases (CVDs) prevalence has significantly increased in the last decade and atherosclerosis development is the main trigger. MicroRNAs (miRNAs) are non-coding RNAs that negatively regulate gene expression of their target and their levels are frequently altered in CVDs. METHODS: By RT-qPCR, we analysed miR-9-5p, miR-15a-5p, miR-16-5p and miR-199a-3p levels in aorta from apolipoprotein knockout (ApoE-/- ) mice, an experimental model of hyperlipidemia-induced atherosclerosis, and in human aortic and carotid atherosclerotic samples. By in silico studies, Western blot analysis and immunofluorescence studies, we detected the targets of the altered miRNAs. RESULTS: Our results show that miR-15a-5p and miR-199a-3p are significantly decreased in carotid and aortic samples from patients and mice with atherosclerosis. In addition, we found an increased expression in targets of both miRNAs that participate in the inflammatory pathway of nuclear factor kappa B (NF-κB), such as IKKα, IKKß and p65. In human vein endothelial cells (HUVECs) and vascular smooth muscle cells (VSMCs), the overexpression of miR-15a-5p or miR-199a-3p decreased IKKα, IKKß and p65 protein levels as well as NF-κB activation. On the other hand, miR-15a-5p and miR-199a-3p overexpression reduced ox-LDL uptake and the inflammation regulated by NF-κB in VSMCs. Moreover, although miR-15a-5p and miR-199a-3p were significantly increased in exosomes from patients with advanced carotid atherosclerosis, only in the ROC analyses for miR-15a-5p, the area under the curve was 0.8951 with a p value of .0028. CONCLUSIONS: Our results suggest that the decrease of miR-199a-3p and miR-15a-5p in vascular samples from human and experimental atherosclerosis could be involved in the NF-κB activation pathway, as well as in ox-LDL uptake by VSMCs, contributing to inflammation and progression atherosclerosis. Finally, miR-15a-5p could be used as a novel diagnostic biomarker for advanced atherosclerosis.

Safety of COVID-19 vaccination in patients with previous cerebral venous sinus thrombosis.

INTRODUCTION: Cerebral venous sinus thrombosis (CVST) after coronavirus (COVID-19) vaccination has been reported. There are no data about thrombosis risk in prior CVST patients. The objective of the study was to describe short-term serious adverse events to COVID-19 vaccines in patients with history of CVST. MATERIAL AND METHODS: We present an observational prospective study of patients with known CVST who received COVID-19 vaccination. Serious event rates within 30 days after second dose vaccination (except one dose for Janssen) were evaluated, including recurrences, hospital admission and death. RESULTS: The 62 vaccinated patients received: BNT162b2 (Pfizer-BioNTech) in 43 patients (69.4%), mRNA-1273 (Moderna) in 7 patients (11.3%), AZD1222 (ChAdOx1) in 7 patients (11.3%) and Ad26.COV2.S (Janssen) in 5 patients (8.1%). There were no thrombotic recurrences within 30 days of vaccination (95% confidence interval, 0.0-5.8). There was one death (1.6%), not attributable to the vaccine. CONCLUSIONS: COVID-19 vaccines are safe for previous CVST patients.

Considerations on diagnosis and surveillance measures of PTEN hamartoma tumor syndrome: clinical and genetic study in a series of Spanish patients.

BACKGROUND: The limited knowledge about the PTEN hamartoma tumor syndrome (PHTS) makes its diagnosis a challenging task. We aimed to define the clinical and genetic characteristics of this syndrome in the Spanish population and to identify new genes potentially associated with the disease. RESULTS: We reviewed the clinical data collected through a specific questionnaire in a series of 145 Spanish patients with a phenotypic features compatible with PHTS and performed molecular characterization through several approaches including next generation sequencing and whole exome sequencing (WES). Macrocephaly, mucocutaneous lesions, gastrointestinal polyposis and obesity are prevalent phenotypic features in PHTS and help predict the presence of a PTEN germline variant in our population. We also find that PHTS patients are at risk to develop cancer in childhood or adolescence. Furthermore, we observe a high frequency of variants in exon 1 of PTEN, which are associated with renal cancer and overexpression of KLLN and PTEN. Moreover, WES revealed variants in genes like NEDD4 that merit further research. CONCLUSIONS: This study expands previously reported findings in other PHTS population studies and makes new contributions regarding clinical and molecular aspects of PHTS, which are useful for translation to the clinic and for new research lines.

Rhamnan sulfate reduces atherosclerotic plaque formation and vascular inflammation.

OBJECTIVE: While lipid-lowering drugs have become a mainstay of clinical therapy these treatments only slow the progression of the disease and can have side effects. Thus, new treatment options are needed to supplement the effects of lipid lowering therapy for treating atherosclerosis. We examined the use of an inexpensive and widely available marine polysaccharide rhamnan sulfate as an oral therapeutic for limiting vascular inflammation and atherosclerosis. METHODS AND RESULTS: We found rhamnan sulfate enhanced the barrier function of endothelial cells, preventing the deposition of LDL and maintaining barrier function even in the presence of glycocalyx-degrading enzymes. Rhamnan sulfate was also found to bind directly to FGF-2, PDGF-BB and NF-κB subunits with high affinity. In addition, rhamnan sulfate was a potent inhibitor of NF-κB pathway activation in endothelial cells by TNF-α. We treated ApoE-/- mice with a high fat diet for 4 weeks and then an addition 9 weeks of high fat diet with or without rhamnan sulfate. Rhamnan sulfate reduced vascular inflammation and atherosclerosis in both sexes of ApoE-/- mice but had a stronger therapeutic effect in female mice. Oral consumption of rhamnan sulfate induced a significant decrease in cholesterol plasma levels in female mice but not in male mice. In addition, there was a marked reduction in inflammation for female mice in the liver and aortic root in comparison to male mice. CONCLUSIONS: Rhamnan sulfate has beneficial effects in reducing inflammation, binding growth factors and NF-κB, enhancing endothelial barrier function and reducing atherosclerotic plaque formation in ApoE-/- mice.

Concerted regulation of non-alcoholic fatty liver disease progression by microRNAs in apolipoprotein E-deficient mice.

The prevalence of non-alcoholic fatty liver disease (NAFLD) is constantly increasing, and altered expression of microRNAs (miRNAs) fosters the development and progression of many pathologies, including NAFLD. Therefore, we explored the role of new miRNAs involved in the molecular mechanisms that trigger NAFLD progression and evaluated them as biomarkers for diagnosis. As a NAFLD model, we used apolipoprotein E-deficient mice administered a high-fat diet for 8 or 18 weeks. We demonstrated that insulin resistance and decreased lipogenesis and autophagy observed after 18 weeks on the diet are related to a concerted regulation carried out by miR-26b-5p, miR-34a-5p, miR-149-5p and miR-375-3p. We also propose circulating let-7d-5p and miR-146b-5p as potential biomarkers of early stages of NAFLD. Finally, we confirmed that circulating miR-34a-5p and miR-375-3p are elevated in the late stages of NAFLD and that miR-27b-3p and miR-122-5p are increased with disease progression. Our results reveal a synergistic regulation of key processes in NAFLD development and progression by miRNAs. Further investigation is needed to unravel the roles of these miRNAs for developing new strategies for NAFLD treatment. This article has an associated First Person interview with the joint first authors of the paper.

Severe Hepatic Insulin Resistance Induces Vascular Dysfunction: Improvement by Liver-Specific Insulin Receptor Isoform A Gene Therapy in a Murine Diabetic Model.

BACKGROUND: Cardiovascular dysfunction is linked to insulin-resistant states. In this paper, we analyzed whether the severe hepatic insulin resistance of an inducible liver-specific insulin receptor knockout (iLIRKO) might generate vascular insulin resistance and dysfunction, and whether insulin receptor (IR) isoforms gene therapy might revert it. METHODS: We studied in vivo insulin signaling in aorta artery and heart from iLIRKO. Vascular reactivity and the mRNA levels of genes involved in vascular dysfunction were analyzed in thoracic aorta rings by qRT-PCR. Finally, iLIRKO mice were treated with hepatic-specific gene therapy to analyze vascular dysfunction improvement. RESULTS: Our results suggest that severe hepatic insulin resistance was expanded to cardiovascular tissues. This vascular insulin resistance observed in aorta artery from iLIRKO mice correlated with a reduction in both PI3K/AKT/eNOS and p42/44 MAPK pathways, and it might be implicated in their vascular alterations characterized by endothelial dysfunction, hypercontractility and eNOS/iNOS levels' imbalance. Finally, regarding long-term hepatic expression of IR isoforms, IRA was more efficient than IRB in the improvement of vascular dysfunction observed in iLIRKO mice. CONCLUSION: Severe hepatic insulin resistance is sufficient to produce cardiovascular insulin resistance and dysfunction. Long-term hepatic expression of IRA restored the vascular damage observed in iLIRKO mice.

miRNA Dysregulation in the Development of Non-Alcoholic Fatty Liver Disease and the Related Disorders Type 2 Diabetes Mellitus and Cardiovascular Disease.

According to the World Health Organization, the continuing surge in obesity pandemic creates a substantial increase in incidences of metabolic disorders, such as non-alcoholic fatty liver disease (NAFLD), type 2 diabetes mellitus, and cardiovascular disease. MicroRNAs (miRNAs) belong to an evolutionarily conserved class of short (20-22 nucleotides in length) and single-stranded non-coding RNAs. In mammals, miRNAs function as critical post-transcriptional negative regulators involved not only in many biological processes but also in the development of many diseases such as NAFLD and comorbidities. More recently, it has been described that cells can secrete miRNAs in extracellular vesicles, transported by body fluids, and uptaken by other tissues regulating gene expression. Therefore, this could be a mechanism of signaling involved not only in physiological pathways but also in the development of diseases. The association of some miRNA expression profiles with certain disorders has made them very interesting molecules for diagnosis, prognosis, and disease management. The finding of specific miRNA signatures to diagnose NAFLD and related diseases could anticipate the risk of development of related complications and, actually, it is the driving force of present health strategies worldwide. In this review, we have included latest advances in knowledge about the miRNAs involved in the development of NAFLD and related diseases and examined how this knowledge could be used to identify new non-invasive biomarkers and new pharmacological interventions.