Obesity-induced changes in cancer cells and their microenvironment: Mechanisms and therapeutic perspectives to manage dysregulated lipid metabolism.

Obesity has been closely related to cancer progression, recurrence, metastasis, and treatment resistance. We aim to review recent progress in the knowledge on the obese macroenvironment and the generated adipose tumor microenvironment (TME) inducing lipid metabolic dysregulation and their influence on carcinogenic processes. Visceral white adipose tissue expansion during obesity exerts systemic or macroenvironmental effects on tumor initiation, growth, and invasion by promoting inflammation, hyperinsulinemia, growth-factor release, and dyslipidemia. The dynamic relationship between cancer and stromal cells of the obese adipose TME is critical for cancer cell survival and proliferation as well. Experimental evidence shows that secreted paracrine signals from cancer cells can induce lipolysis in cancer-associated adipocytes, causing them to release free fatty acids and acquire a fibroblast-like phenotype. Such adipocyte delipidation and phenotypic change is accompanied by an increased secretion of cytokines by cancer-associated adipocytes and tumor-associated macrophages in the TME. Mechanistically, the availability of adipose TME free fatty acids and tumorigenic cytokines concomitant with the activation of angiogenic processes creates an environment that favors a shift in the cancer cells toward an aggressive phenotype associated with increased invasiveness. We conclude that restoring the aberrant metabolic alterations in the host macroenvironment and in adipose TME of obese subjects would be a therapeutic option to prevent cancer development. Several dietary, lipid-based, and oral antidiabetic pharmacological therapies could potentially prevent tumorigenic processes associated with the dysregulated lipid metabolism closely linked to obesity.

Soluble low-density lipoprotein receptor-related protein 1 as a surrogate marker of carotid plaque inflammation assessed by 18F-FDG PET in patients with a recent ischemic stroke.

BACKGROUND: 18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET) identifies carotid plaque inflammation and predicts stroke recurrence. AIM: Our aim was to evaluate the performance of soluble low-density lipoprotein receptor-related protein 1 (sLRP1) as an indicator of carotid plaque inflammation. METHODS: A prospective study was conducted among adult patients with recent (< 7 days) anterior circulation ischemic stroke and at least one atherosclerotic plaque in the ipsilateral internal carotid artery. Patients underwent an early (< 15 days from inclusion) 18F-FDG PET, and the maximum standardized uptake value (SUVmax) within the plaque was measured. sLRP1 levels were measured in plasma samples by ELISA. The association of sLRP1 with SUVmax was assessed using bivariate and multivariable linear regression analyses. Hazard ratios (HR) were estimated with Cox regression to evaluate the association between circulating sLRP1 and stroke recurrence. RESULTS: The study was conducted with 64 participants, of which 57.8% had ≥ 50% carotid stenosis. The multivariable linear and logistic regression analyses showed that sLRP1 was independently associated with (i) SUVmax within the plaque (ß = 0.159, 95% CI 0.062-0.257, p = 0.002) and (ii) a probability of presenting SUVmax ≥ 2.85 g/mL (OR = 1.31, 95% CI 1.00-1.01, p = 0.046), respectively. Participants with stroke recurrence showed higher sLRP1 levels at baseline [6447 ng/mL (4897-11163) vs. 3713 ng/mL (2793-4730); p = 0.018]. CONCLUSIONS: sLRP1 was independently associated with carotid plaque inflammation as measured by 18F-FDG PET in patients with recent ischemic stroke and carotid atherosclerosis.

Implantation of CPT1AM-expressing adipocytes reduces obesity and glucose intolerance in mice.

Obesity and its associated metabolic comorbidities are a rising global health and social issue, with novel therapeutic approaches urgently needed. Adipose tissue plays a key role in the regulation of energy balance and adipose tissue-derived mesenchymal stem cells (AT-MSCs) have gained great interest in cell therapy. Carnitine palmitoyltransferase 1A (CPT1A) is the gatekeeper enzyme for mitochondrial fatty acid oxidation. Here, we aimed to generate adipocytes expressing a constitutively active CPT1A form (CPT1AM) that can improve the obese phenotype in mice after their implantation. AT-MSCs were differentiated into mature adipocytes, subjected to lentivirus-mediated expression of CPT1AM or the GFP control, and subcutaneously implanted into mice fed a high-fat diet (HFD). CPT1AM-implanted mice showed lower body weight, hepatic steatosis and serum insulin and cholesterol levels alongside improved glucose tolerance. HFD-induced increases in adipose tissue hypertrophy, fibrosis, inflammation, endoplasmic reticulum stress and apoptosis were reduced in CPT1AM-implanted mice. In addition, the expression of mitochondrial respiratory chain complexes was enhanced in the adipose tissue of CPT1AM-implanted mice. Our results demonstrate that implantation of CPT1AM-expressing AT-MSC-derived adipocytes into HFD-fed mice improves the obese metabolic phenotype, supporting the future clinical use of this ex vivo gene therapy approach.

LDL Receptor Regulates the Reverse Transport of Macrophage-Derived Unesterified Cholesterol via Concerted Action of the HDL-LDL Axis: Insight From Mouse Models.

RATIONALE: The HDL (high-density lipoprotein)-mediated stimulation of cellular cholesterol efflux initiates macrophage-specific reverse cholesterol transport (m-RCT), which ends in the fecal excretion of macrophage-derived unesterified cholesterol (UC). Early studies established that LDL (low-density lipoprotein) particles could act as efficient intermediate acceptors of cellular-derived UC, thereby preventing the saturation of HDL particles and facilitating their cholesterol efflux capacity. However, the capacity of LDL to act as a plasma cholesterol reservoir and its potential impact in supporting the m-RCT pathway in vivo both remain unknown. OBJECTIVE: We investigated LDL contributions to the m-RCT pathway in hypercholesterolemic mice. METHODS AND RESULTS: Macrophage cholesterol efflux induced in vitro by LDL added to the culture media either alone or together with HDL or ex vivo by plasma derived from subjects with familial hypercholesterolemia was assessed. In vivo, m-RCT was evaluated in mouse models of hypercholesterolemia that were naturally deficient in CETP (cholesteryl ester transfer protein) and fed a Western-type diet. LDL induced the efflux of radiolabeled UC from cultured macrophages, and, in the simultaneous presence of HDL, a rapid transfer of the radiolabeled UC from HDL to LDL occurred. However, LDL did not exert a synergistic effect on HDL cholesterol efflux capacity in the familial hypercholesterolemia plasma. The m-RCT rates of the LDLr (LDL receptor)-KO (knockout), LDLr-KO/APOB100, and PCSK9 (proprotein convertase subtilisin/kexin type 9)-overexpressing mice were all significantly reduced relative to the wild-type mice. In contrast, m-RCT remained unchanged in HAPOB100 Tg (human APOB100 transgenic) mice with fully functional LDLr, despite increased levels of plasma APO (apolipoprotein)-B-containing lipoproteins. CONCLUSIONS: Hepatic LDLr plays a critical role in the flow of macrophage-derived UC to feces, while the plasma increase of APOB-containing lipoproteins is unable to stimulate m-RCT. The results indicate that, besides the major HDL-dependent m-RCT pathway via SR-BI (scavenger receptor class B type 1) to the liver, a CETP-independent m-RCT path exists, in which LDL mediates the transfer of cholesterol from macrophages to feces. Graphical Abstract: A graphical abstract is available for this article.

Plasma circular RNA hsa_circ_0001445 and coronary artery disease: Performance as a biomarker.

The role of circular RNAs (circRNAs) as biomarkers remains poorly characterized. Here, we investigated the performance of the circRNA hsa_circ_0001445 as a biomarker of coronary artery disease (CAD) in a real-world clinical practice setting. Plasma hsa_circ_0001445 was measured in a study population of 200 consecutive patients with suspected stable CAD who had undergone coronary computed tomographic angiography (CTA). Multivariable logistic models were constructed combining conventional risk factors with established biomarkers and hsa_circ_0001445. Model robustness was internally validated by the bootstrap technique. Biomarker accuracy was evaluated using the C-index. The integrated discrimination improvement (IDI) and net reclassification improvement (NRI) were also calculated. Risk groups were developed via classification tree models. The stability of plasma hsa_circ_0001445 was evaluated under different clinical conditions. hsa_circ_0001445 levels were associated with higher coronary atherosclerosis extent and severity with a 2-fold increase across tertiles (28.4%-50.0%). Levels of hsa_circ_0001445 were proportional to coronary atherosclerotic burden, even after comprehensive adjustment for cardiovascular risk factors, medications, and established biomarkers (fully adjusted OR = 0.432 for hsa_circ_0001445 as a continuous variable and fully adjusted OR = 0.277 for hsa_circ_0001445 as a binary variable). The classification of patients was improved with the incorporation of hsa_circ_0001445 into a base clinical model (CM) composed of conventional cardiovascular risk factors, showing an IDI of 0.047 and NRI of 0.482 for hsa_circ_0001445 as a continuous variable and an IDI of 0.056 and NRI of 0.373 for hsa_circ_0001445 as a binary variable. A trend toward higher discrimination capacity was also observed (C-indexCM  = 0.833, C-indexCM+continuous hsa_circ_0001445  = 0.856 and C-indexCM+binary hsa_circ_0001445  = 0.855). Detailed analysis of stability showed that hsa_circ_0001445 was present in plasma in a remarkably stable form. In vitro, hsa_circ_0001445 was downregulated in extracellular vesicles secreted by human coronary smooth muscle cells upon exposure to atherogenic conditions. In patients with suspected stable CAD referred for coronary CTA, plasma hsa_circ_0001445 improves the identification of coronary artery atherosclerosis.

Liver CPT1A gene therapy reduces diet-induced hepatic steatosis in mice and highlights potential lipid biomarkers for human NAFLD.

The prevalence of nonalcoholic fatty liver disease (NAFLD) has increased drastically due to the global obesity pandemic but at present there are no approved therapies. Here, we aimed to revert high-fat diet (HFD)-induced obesity and NAFLD in mice by enhancing liver fatty acid oxidation (FAO). Moreover, we searched for potential new lipid biomarkers for monitoring liver steatosis in humans. We used adeno-associated virus (AAV) to deliver a permanently active mutant form of human carnitine palmitoyltransferase 1A (hCPT1AM), the key enzyme in FAO, in the liver of a mouse model of HFD-induced obesity and NAFLD. Expression of hCPT1AM enhanced hepatic FAO and autophagy, reduced liver steatosis, and improved glucose homeostasis. Lipidomic analysis in mice and humans before and after therapeutic interventions, such as hepatic AAV9-hCPT1AM administration and RYGB surgery, respectively, led to the identification of specific triacylglyceride (TAG) specie (C50:1) as a potential biomarker to monitor NAFFLD disease. To sum up, here we show for the first time that liver hCPT1AM gene therapy in a mouse model of established obesity, diabetes, and NAFLD can reduce HFD-induced derangements. Moreover, our study highlights TAG (C50:1) as a potential noninvasive biomarker that might be useful to monitor NAFLD in mice and humans.

Annexin A6 modulates TBC1D15/Rab7/StARD3 axis to control endosomal cholesterol export in NPC1 cells.

Cholesterol accumulation in late endosomes is a prevailing phenotype of Niemann-Pick type C1 (NPC1) mutant cells. Likewise, annexin A6 (AnxA6) overexpression induces a phenotype reminiscent of NPC1 mutant cells. Here, we demonstrate that this cellular cholesterol imbalance is due to AnxA6 promoting Rab7 inactivation via TBC1D15, a Rab7-GAP. In NPC1 mutant cells, AnxA6 depletion and eventual Rab7 activation was associated with peripheral distribution and increased mobility of late endosomes. This was accompanied by an enhanced lipid accumulation in lipid droplets in an acyl-CoA:cholesterol acyltransferase (ACAT)-dependent manner. Moreover, in AnxA6-deficient NPC1 mutant cells, Rab7-mediated rescue of late endosome-cholesterol export required the StAR-related lipid transfer domain-3 (StARD3) protein. Electron microscopy revealed a significant increase of membrane contact sites (MCS) between late endosomes and ER in NPC1 mutant cells lacking AnxA6, suggesting late endosome-cholesterol transfer to the ER via Rab7 and StARD3-dependent MCS formation. This study identifies AnxA6 as a novel gatekeeper that controls cellular distribution of late endosome-cholesterol via regulation of a Rab7-GAP and MCS formation.

Circulating non-coding RNAs in biomarker-guided cardiovascular therapy: a novel tool for personalized medicine?

Current clinical guidelines emphasize the unmet need for technological innovations to guide physician decision-making and to transit from conventional care to personalized cardiovascular medicine. Biomarker-guided cardiovascular therapy represents an interesting approach to inform tailored treatment selection and monitor ongoing efficacy. However, results from previous publications cast some doubts about the clinical applicability of biomarkers to direct individualized treatment. In recent years, the non-coding human transcriptome has emerged as a new opportunity for the development of novel therapeutic strategies and biomarker discovery. Non-coding RNA (ncRNA) signatures may provide an accurate molecular fingerprint of patient phenotypes and capture levels of information that could complement traditional markers and established clinical variables. Importantly, ncRNAs have been identified in body fluids and their concentrations change with physiology and pathology, thus representing promising non-invasive biomarkers. Previous publications highlight the translational applicability of circulating ncRNAs for diagnosis and prognostic stratification within cardiology. Numerous independent studies have also evaluated the potential of the circulating non-coding transcriptome to predict and monitor response to cardiovascular treatment. However, this field has not been reviewed in detail. Here, we discuss the state-of-the-art research into circulating ncRNAs, specifically microRNAs and long non-coding RNAs, to support clinical decision-making in cardiovascular therapy. Furthermore, we summarize current methodological and conceptual limitations and propose future steps for their incorporation into personalized cardiology. Despite the lack of robust population-based studies and technical barriers, circulating ncRNAs emerge as a promising tool for biomarker-guided therapy.

Circulating microRNAs in suspected stable coronary artery disease: A coronary computed tomography angiography study.

OBJECTIVES: To explore the diagnostic performance of circulating microRNAs (miRNAs) as biomarkers in patients with suspected stable coronary artery disease (CAD). METHODS: Plasma samples were collected from 237 consecutive patients referred for coronary computed tomography angiography (CCTA). Presence, extension and severity of coronary stenosis were evaluated using the indexes: presence of diameter stenosis ≥ 50%, segment involvement score (SIS), segment stenosis score (SSS) and 3-vessel plaque score. A panel of 10 miRNAs previously associated with CAD was analysed using RT-qPCR. Multivariate analyses were used to analyse the associations between biomarkers and indexes. Discrimination was evaluated using the area under the ROC curve (AUC). Decision trees were generated using chi-squared Automatic Interaction Detector (CHAID) prediction models. RESULTS: After comprehensive adjustment including cardiovascular risk factors, medication use, confounding factors and protein-based biomarkers (hs-TnT and hs-CRP), several circulating miRNAs were inversely associated with coronary atherosclerosis extension (SIS and 3-vessel plaque score) and severity (SSS). In the whole population, circulating miRNAs showed a poor discrimination value for all indexes (AUC = 0.539-0.644) and did not increase the discrimination capacity of a clinical model of coronary stenosis presence, extension and severity based on conventional cardiovascular risk factors. Conversely, the inclusion of circulating miRNAs in decision trees produces models that improve the classification of cases and controls in specific patient subgroups. CONCLUSIONS: This study identifies a group of circulating miRNAs that failed to improve the discrimination capacity of cardiovascular risk factors but that has the potential to define specific subpopulations of patients with suspected stable CAD.

Human ApoA-I Overexpression Enhances Macrophage-Specific Reverse Cholesterol Transport but Fails to Prevent Inherited Diabesity in Mice.

Human apolipoprotein A-I (hApoA-I) overexpression improves high-density lipoprotein (HDL) function and the metabolic complications of obesity. We used a mouse model of diabesity, the db/db mouse, to examine the effects of hApoA-I on the two main functional properties of HDL, i.e., macrophage-specific reverse cholesterol transport (m-RCT) in vivo and the antioxidant potential, as well as the phenotypic features of obesity. HApoA-I transgenic (hA-I) mice were bred with nonobese control (db/+) mice to generate hApoA-I-overexpressing db/+ offspring, which were subsequently bred to obtain hA-I-db/db mice. Overexpression of hApoA-I significantly increased weight gain and the incidence of fatty liver in db/db mice. Weight gain was mainly explained by the increased caloric intake of hA-I-db/db mice (>1.2-fold). Overexpression of hApoA-I also produced a mixed type of dyslipidemia in db/db mice. Despite these deleterious effects, the overexpression of hApoA-I partially restored m-RCT in db/db mice to levels similar to nonobese control mice. Moreover, HDL from hA-I-db/db mice also enhanced the protection against low-density lipoprotein (LDL) oxidation compared with HDL from db/db mice. In conclusion, overexpression of hApoA-I in db/db mice enhanced two main anti-atherogenic HDL properties while exacerbating weight gain and the fatty liver phenotype. These adverse metabolic side-effects were also observed in obese mice subjected to long-term HDL-based therapies in independent studies and might raise concerns regarding the use of hApoA-I-mediated therapy in obese humans.

Identification of new biophysical markers for pathological ventricular remodelling in tachycardia-induced dilated cardiomyopathy.

Our aim was to identify biophysical biomarkers of ventricular remodelling in tachycardia-induced dilated cardiomyopathy (DCM). Our study includes healthy controls (N = 7) and DCM pigs (N = 10). Molecular analysis showed global myocardial metabolic abnormalities, some of them related to myocardial hibernation in failing hearts, supporting the translationality of our model to study cardiac remodelling in dilated cardiomyopathy. Histological analysis showed unorganized and agglomerated collagen accumulation in the dilated ventricles and a higher percentage of fibrosis in the right (RV) than in the left (LV) ventricle (P = .016). The Fourier Transform Infrared Spectroscopy (FTIR) 1st and 2nd indicators, which are markers of the myofiber/collagen ratio, were reduced in dilated hearts, with the 1st indicator reduced by 45% and 53% in the RV and LV, respectively, and the 2nd indicator reduced by 25% in the RV. The 3rd FTIR indicator, a marker of the carbohydrate/lipid ratio, was up-regulated in the right and left dilated ventricles but to a greater extent in the RV (2.60-fold vs 1.61-fold, P = .049). Differential scanning calorimetry (DSC) showed a depression of the freezable water melting point in DCM ventricles - indicating structural changes in the tissue architecture - and lower protein stability. Our results suggest that the 1st, 2nd and 3rd FTIR indicators are useful markers of cardiac remodelling. Moreover, the 2nd and 3rd FITR indicators, which are altered to a greater extent in the right ventricle, are associated with greater fibrosis.

Circular RNAs in Blood.

Recent advances in RNA sequencing and bioinformatic analysis have allowed the development of a new research field: circular RNAs (circRNAs). These members of the non-coding transcriptome are generated by backsplicing, which results in a covalently closed, single-stranded RNA molecule. To date, thousands of circRNAs have been identified in different human cell types. CircRNAs are evolutionarily conserved, highly stable, cell-/developmental stage-specific and have longer half-lives compared with linear RNAs. Interestingly, different studies have demonstrated that circRNAs are abundantly expressed in the bloodstream. In this chapter, we review the current knowledge of circRNA biology in blood cells and the cell-free compartment, including extracellular vesicles. The potential clinical application of blood circRNAs in the biomarker and therapy fields is also discussed. Finally, perspectives for future studies are proposed.

Translating the microRNA signature of microvesicles derived from human coronary artery smooth muscle cells in patients with familial hypercholesterolemia and coronary artery disease.

AIMS: To analyze the impact of atherogenic lipoproteins on the miRNA signature of microvesicles derived from human coronary artery smooth muscle cells (CASMC) and to translate these results to familial hypercholesterolemia (FH) and coronary artery disease (CAD) patients. METHODS: Conditioned media was collected after exposure of CASMC to atherogenic lipoproteins. Plasma samples were collected from two independent populations of diagnosed FH patients and matched normocholesterolemic controls (Study population 1, N=50; Study population 2, N=24) and a population of patients with suspected CAD (Study population 3, N=50). Extracellular vesicles were isolated and characterized using standard techniques. A panel of 30 miRNAs related to vascular smooth muscle cell (VSMC) (patho-)physiology was analyzed using RT-qPCR. RESULTS: Atherogenic lipoproteins significantly reduced levels of miR-15b-5p, -24-3p, -29b-3p, -130a-3p, -143-3p, -146a-3p, -222-3p, -663a levels (P<0.050) in microvesicles (0.1µm-1µm in diameter) released by CASMC. Two of these miRNAs, miR-24-3p and miR-130a-3p, were reduced in circulating microvesicles from FH patients compared with normocholesterolemic controls in a pilot study (Study population 1) and in different validation studies (Study populations 1 and 2) (P<0.050). Supporting these results, plasma levels of miR-24-3p and miR-130a-3p were also downregulated in FH patients compared to controls (P<0.050). In addition, plasma levels of miR-130a-3p were inversely associated with coronary atherosclerosis in a cohort of suspected CAD patients (Study population 3) (P<0.050). CONCLUSIONS: Exposure to atherogenic lipoproteins modifies the miRNA profile of CASMC-derived microvesicles and these alterations are reflected in patients with FH. Circulating miR-130a-3p emerges as a potential biomarker for coronary atherosclerosis.

Relationship among LRP1 expression, Pyk2 phosphorylation and MMP-9 activation in left ventricular remodelling after myocardial infarction.

Left ventricular (LV) remodelling after myocardial infarction (MI) is a crucial determinant of the clinical course of heart failure. Matrix metalloproteinase (MMP) activation is strongly associated with LV remodelling after MI. Elucidation of plasma membrane receptors related to the activation of specific MMPs is fundamental for treating adverse cardiac remodelling after MI. The aim of current investigation was to explore the potential association between the low-density lipoprotein receptor-related protein 1 (LRP1) and MMP-9 and MMP-2 spatiotemporal expression after MI. Real-time PCR and Western blot analyses showed that LRP1 mRNA and protein expression levels, respectively, were significantly increased in peri-infarct and infarct zones at 10 and 21 days after MI. Confocal microscopy demonstrated high colocalization between LRP1 and the fibroblast marker vimentin, indicating that LRP1 is mostly expressed by cardiac fibroblasts in peri-infarct and infarct areas. LRP1 also colocalized with proline-rich tyrosine kinase 2 (pPyk2) and MMP-9 in cardiac fibroblasts in ischaemic areas at 10 and 21 days after MI. Cell culture experiments revealed that hypoxia increases LRP1, pPyk2 protein levels and MMP-9 activity in fibroblasts, without significant changes in MMP-2 activity. MMP-9 activation by hypoxia requires LRP1 and Pyk2 phosphorylation in fibroblasts. Collectively, our in vivo and in vitro data support a major role of cardiac fibroblast LRP1 levels on MMP-9 up-regulation associated with ventricular remodelling after MI.

Extracellular vesicles do not contribute to higher circulating levels of soluble LRP1 in idiopathic dilated cardiomyopathy.

Idiopathic dilated cardiomyopathy (IDCM) is a frequent cause of heart transplantation. Potentially valuable blood markers are being sought, and low-density lipoprotein receptor-related protein 1 (LRP1) has been linked to the underlying molecular basis of the disease. This study compared circulating levels of soluble LRP1 (sLRP1) in IDCM patients and healthy controls and elucidated whether sLRP1 is exported out of the myocardium through extracellular vesicles (EVs) to gain a better understanding of the pathogenesis of the disease. LRP1 α chain expression was analysed in samples collected from the left ventricles of explanted hearts using immunohistochemistry. sLRP1 concentrations were determined in platelet-free plasma by enzyme-linked immunosorbent assay. Plasma-derived EVs were extracted by size-exclusion chromatography (SEC) and characterized by nanoparticle tracking analysis and cryo-transmission electron microscopy. The distributions of vesicular (CD9, CD81) and myocardial (caveolin-3) proteins and LRP1 α chain were assessed in SEC fractions by flow cytometry. LRP1 α chain was preferably localized to blood vessels in IDCM compared to control myocardium. Circulating sLRP1 was increased in IDCM patients. CD9- and CD81-positive fractions enriched with membrane vesicles with the expected size and morphology were isolated from both groups. The LRP1 α chain was not present in these SEC fractions, which were also positive for caveolin-3. The increase in circulating sLRP1 in IDCM patients may be clinically valuable. Although EVs do not contribute to higher sLRP1 levels in IDCM, a comprehensive analysis of EV content would provide further insights into the search for novel blood markers.

K Domain CR9 of Low Density Lipoprotein (LDL) Receptor-related Protein 1 (LRP1) Is Critical for Aggregated LDL-induced Foam Cell Formation from Human Vascular Smooth Muscle Cells.

Low density lipoprotein receptor-related protein (LRP1) mediates the internalization of aggregated LDL (AgLDL), which in turn increases the expression of LRP1 in human vascular smooth muscle cells (hVSMCs). This positive feedback mechanism is thus highly efficient to promote the formation of hVSMC foam cells, a crucial vascular component determining the susceptibility of atherosclerotic plaque to rupture. Here we have determined the LRP1 domains involved in AgLDL recognition with the aim of specifically blocking AgLDL internalization in hVSMCs. The capacity of fluorescently labeled AgLDL to bind to functional LRP1 clusters was tested in a receptor-ligand fluorometric assay made by immobilizing soluble LRP1 "minireceptors" (sLRP1-II, sLRP1-III, and sLRP1-IV) recombinantly expressed in CHO cells. This assay showed that AgLDL binds to cluster II. We predicted three well exposed and potentially immunogenic peptides in the CR7-CR9 domains of this cluster (termed P1 (Cys(1051)-Glu(1066)), P2 (Asp(1090)-Cys(1104)), and P3 (Gly(1127)-Cys(1140))). AgLDL, but not native LDL, bound specifically and tightly to P3-coated wells. Rabbit polyclonal antibodies raised against P3 prevented AgLDL uptake by hVSMCs and were almost twice as effective as anti-P1 and anti-P2 Abs in reducing intracellular cholesteryl ester accumulation. Moreover, anti-P3 Abs efficiently prevented AgLDL-induced LRP1 up-regulation and counteracted the down-regulatory effect of AgLDL on hVSMC migration. In conclusion, domain CR9 appears to be critical for LRP1-mediated AgLDL binding and internalization in hVSMCs. Our results open new avenues for an innovative anti-VSMC foam cell-based strategy for the treatment of vascular lipid deposition in atherosclerosis.

Hypoxia-driven sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) downregulation depends on low-density lipoprotein receptor-related protein 1 (LRP1)-signalling in cardiomyocytes.

The maintenance of sarcoplasmic reticulum Ca(2+) ATPase (SERCA2) activity is crucial for cardiac function and SERCA2 is dramatically reduced in the heart exposed to hypoxic/ischemic conditions. Previous work from our group showed that hypoxia upregulates the phosphorylated form of the Ca(2+)-dependent nonreceptor protein tyrosine kinase (PTK) proline-rich tyrosine kinase 2 (pPyk2) protein levels in a low-density lipoprotein receptor-related protein (LRP1)-dependent manner. Pyk2 in turn may modulate SERCA2 in cardiomyocytes although this remains controversial. We therefore aimed to investigate the role of LRP1 on hypoxia-induced SERCA2 depletion in cardiomyocytes and to establish LRP1 signalling mechanisms involved. Western blot analysis showed that hypoxia reduced SERCA2 concomitantly with a sustained increase in LRP1 and pPyk2 protein levels in HL-1 cardiomyocytes. By impairing hypoxia-induced Pyk2 phosphorylation and HIF-1α accumulation, LRP1 deficiency prevented SERCA2 depletion and reduction of the sarcoplasmic reticulum calcium content in cardiomyocytes. Moreover, the inhibition of Pyk2 phosphorylation (with the Src-family inhibitor PP2) or the specific silencing of Pyk2 (with siRNA-anti Pyk2) preserved low HIF-1α and high SERCA2 levels in HL-1 cardiomyocytes exposed to hypoxia. We determined that the LRP1/Pyk2 axis represses SERCA2 mRNA expression via HIF-1α since HIF-1α overexpression abolished the protective effect of LRP1 deficiency on SERCA2 depletion. Our findings show a crucial role of LRP1/Pyk2/HIF-1α in hypoxia-induced cardiomyocyte SERCA2 downregulation, a pathophysiological process closely associated with heart failure.

Intratumor cholesteryl ester accumulation is associated with human breast cancer proliferation and aggressive potential: a molecular and clinicopathological study.

BACKGROUND: The metabolic effect of intratumor cholesteryl ester (CE) in breast cancer remains poorly understood. The objective was to analyze the relationship between intratumor CE content and clinicopathological variables in human breast carcinomas. METHODS: We classified 30 breast carcinoma samples into three subgroups: 10 luminal-A tumors (ER+/PR+/Her2-), 10 Her-2 tumors (ER-/PR-/Her2+), and 10 triple negative (TN) tumors (ER-/PR-/Her2-). We analyzed intratumor neutral CE, free cholesterol (FC) and triglyceride (TG) content by thin layer chromatography after lipid extraction. RNA and protein levels of lipid metabolism and invasion mediators were analyzed by real time PCR and Western blot analysis. RESULTS: Group-wise comparisons, linear regression and logistic regression models showed a close association between CE-rich tumors and higher histologic grade, Ki-67 and tumor necrosis. CE-rich tumors displayed higher mRNA and protein levels of low-density lipoprotein receptor (LDLR) and scavenger receptor class B member 1 (SCARB1). An increased expression of acetyl-Coenzyme A acetyltransferase 1 (ACAT1) in CE-rich tumors was also reported. CONCLUSIONS: Intratumor CE accumulation is intimately linked to proliferation and aggressive potential of breast cancer tumors. Our data support the link between intratumor CE content and poor clinical outcome and open the door to new antitumor interventions.

Amyloid-ß increases metallo- and cysteine protease activities in human macrophages.

BACKGROUND/AIMS: Amyloid-ß (Aß) plays a crucial role in the onset and progression of atherosclerosis. Macrophages are a source of matrix metalloproteinases (MMPs), cysteine proteases and transforming growth factor (TGF)-ß1 in the vascular wall. The aims of this study were to analyze the capacity of Aß peptide (1-40) (Aß40), Aß peptide (1-42) (Aß42) and fibrillar Aß42 (fAß42) to modulate the expression and activity of MMP-9, MMP-2 and tissue inhibitor of MMP-1 (TIMP-1) in human monocyte-derived macrophages (HMDM). Additionally, we analyzed whether Aß internalization alters the secretion of cathepsin S (CatS) and TGF-ß1 by macrophages. METHODS: HMDM were exposed to native and fibrillar Aß. MMPs and TIMP-1 expression was analyzed by real-time PCR, and MMP abundance by zymography. Protein levels of precursor and active forms of CatS were analyzed by Western blot and TGF-ß1 levels by ELISA. RESULTS: Aß40, Aß42 and especially fAß42 strongly induced MMP-9/MMP-2 levels. Moreover, we showed enhanced active CatS and reduced TGF-ß1 protein levels in the secretome of Aß42 and fAß42-exposed macrophages. CONCLUSIONS: Aß can regulate the proinflammatory state of human macrophages by inducing metallo- and cysteine protease levels and by reducing TGF-ß1 secretion. These effects may be crucial in atherosclerosis progression.

Signature of subclinical femoral artery atherosclerosis in peripheral blood mononuclear cells.

BACKGROUND: Peripheral arterial disease is a relevant public health problem associated with increased risk of morbimortality. Most of the patients with this condition are asymptomatic. Therefore, the development of accessible biochemical markers seems to be necessary to anticipate diagnosis. Our hypothesis is that asymptomatic subjects with objectively confirmed femoral artery atherosclerosis could be distinguished from control subjects by gene expression analysis in peripheral blood mononuclear cells (PBMC). MATERIALS AND METHODS: A total of 37 asymptomatic males over 50 years old were recruited at the University Clinic of Navarra (Spain). Nineteen participants were free from atherosclerotic vascular disease and 18 participants presented subclinical femoral artery atherosclerosis defined by means of Doppler ultrasound. PBMC were isolated from blood and the RNA extracted. A panel of atherosclerotic-related genes were evaluated by Taqman low-density array. RESULTS: In univariate logistic regression models, we found a direct relationship between IL4, ITGAM and TLR2 expression levels in PBMC and femoral atherosclerosis, even when the models were adjusted for age and hypertension prevalence. Multivariate logistic regression models showed that elevated IL4 expression levels were intimately associated with subclinical femoral atherosclerosis after adjusting for the same potential confounders. CONCLUSIONS: Current data suggest that gene expression in PBMC, in particular IL4 expression, could be a useful tool in the diagnosis of femoral artery atherosclerosis in asymptomatic patients. Furthermore, in patients with no differences in cardiovascular risk factors except for hypertension, the results point to the immune and inflammatory deregulation as a feature of subclinical peripheral atherosclerosis.