The Extracellular RNA Communication Consortium: Establishing Foundational Knowledge and Technologies for Extracellular RNA Research.

The Extracellular RNA Communication Consortium (ERCC) was launched to accelerate progress in the new field of extracellular RNA (exRNA) biology and to establish whether exRNAs and their carriers, including extracellular vesicles (EVs), can mediate intercellular communication and be utilized for clinical applications. Phase 1 of the ERCC focused on exRNA/EV biogenesis and function, discovery of exRNA biomarkers, development of exRNA/EV-based therapeutics, and construction of a robust set of reference exRNA profiles for a variety of biofluids. Here, we present progress by ERCC investigators in these areas, and we discuss collaborative projects directed at development of robust methods for EV/exRNA isolation and analysis and tools for sharing and computational analysis of exRNA profiling data.

Small RNA Sequencing across Diverse Biofluids Identifies Optimal Methods for exRNA Isolation.

Poor reproducibility within and across studies arising from lack of knowledge regarding the performance of extracellular RNA (exRNA) isolation methods has hindered progress in the exRNA field. A systematic comparison of 10 exRNA isolation methods across 5 biofluids revealed marked differences in the complexity and reproducibility of the resulting small RNA-seq profiles. The relative efficiency with which each method accessed different exRNA carrier subclasses was determined by estimating the proportions of extracellular vesicle (EV)-, ribonucleoprotein (RNP)-, and high-density lipoprotein (HDL)-specific miRNA signatures in each profile. An interactive web-based application (miRDaR) was developed to help investigators select the optimal exRNA isolation method for their studies. miRDar provides comparative statistics for all expressed miRNAs or a selected subset of miRNAs in the desired biofluid for each exRNA isolation method and returns a ranked list of exRNA isolation methods prioritized by complexity, expression level, and reproducibility. These results will improve reproducibility and stimulate further progress in exRNA biomarker development.

MetfOrmin BenefIts Lower Extremities with Intermittent Claudication (MOBILE IC): randomized clinical trial protocol.

BACKGROUND: Peripheral artery disease (PAD) affects over 230 million people worldwide and is due to systemic atherosclerosis with etiology linked to chronic inflammation, hypertension, and smoking status. PAD is associated with walking impairment and mobility loss as well as a high prevalence of coronary and cerebrovascular disease. Intermittent claudication (IC) is the classic presenting symptom for PAD, although many patients are asymptomatic or have atypical presentations. Few effective medical therapies are available, while surgical and exercise therapies lack durability. Metformin, the most frequently prescribed oral medication for Type 2 diabetes, has salient anti-inflammatory and promitochondrial properties. We hypothesize that metformin will improve function, retard the progression of PAD, and improve systemic inflammation and mitochondrial function in non-diabetic patients with IC. METHODS: 200 non-diabetic Veterans with IC will be randomized 1:1 to 180-day treatment with metformin extended release (1000 mg/day) or placebo to evaluate the effect of metformin on functional status, PAD progression, cardiovascular disease events, and systemic inflammation. The primary outcome is 180-day maximum walking distance on the 6-min walk test (6MWT). Secondary outcomes include additional assessments of functional status (cardiopulmonary exercise testing, grip strength, Walking Impairment Questionnaires), health related quality of life (SF-36, VascuQoL), macro- and micro-vascular assessment of lower extremity blood flow (ankle brachial indices, pulse volume recording, EndoPAT), cardiovascular events (amputations, interventions, major adverse cardiac events, all-cause mortality), and measures of systemic inflammation. All outcomes will be assessed at baseline, 90 and 180 days of study drug exposure, and 180 days following cessation of study drug. We will evaluate the primary outcome with linear mixed-effects model analysis with covariate adjustment for baseline 6MWT, age, baseline ankle brachial indices, and smoking status following an intention to treat protocol. DISCUSSION: MOBILE IC is uniquely suited to evaluate the use of metformin to improve both systematic inflammatory responses, cellular energetics, and functional outcomes in patients with PAD and IC. TRIAL REGISTRATION: The prospective MOBILE IC trial was publicly registered (NCT05132439) November 24, 2021.

IL-4 polarized human macrophage exosomes control cardiometabolic inflammation and diabetes in obesity.

Cardiometabolic disease is an increasing cause of morbidity and death in society. While M1-like macrophages contribute to metabolic inflammation and insulin resistance, those polarized to an M2-like phenotype exert protective properties. Building on our observations reporting M2-like macrophage exosomes in atherosclerosis control, we tested whether they could serve to control inflammation in the liver and adipose tissue of obese mice. In thinking of clinical translation, we studied human THP-1 macrophages exposed to interleukin (IL)-4 as a source of exosomes (THP1-IL4-exo). Our findings show that THP1-IL4-exo polarized primary macrophages to an anti-inflammatory phenotype and reprogramed their energy metabolism by increasing levels of microRNA-21/99a/146b/378a (miR-21/99a/146b/378a) while reducing miR-33. This increased lipophagy, mitochondrial activity, and oxidative phosphorylation (OXPHOS). THP1-IL4-exo exerted a similar regulation of these miRs in cultured 3T3-L1 adipocytes. This enhanced insulin-dependent glucose uptake through increased peroxisome proliferator activated receptor gamma (PPARγ)-driven expression of GLUT4. It also increased levels of UCP1 and OXPHOS activity, which promoted lipophagy, mitochondrial activity, and beiging of 3T3-L1 adipocytes. Intraperitoneal infusions of THP1-IL4-exo into obese wild-type and Ldlr-/- mice fed a Western high-fat diet reduced hematopoiesis and myelopoiesis, and favorably reprogramed inflammatory signaling and metabolism in circulating Ly6Chi monocytes. This also reduced leukocyte numbers and inflammatory activity in the circulation, aorta, adipose tissue, and the liver. Such treatments reduced hepatic steatosis and increased the beiging of white adipose tissue as revealed by increased UCP1 expression and OXPHOS activity that normalized blood insulin levels and improved glucose tolerance. Our findings support THP1-IL4-exo as a therapeutic approach to control cardiometabolic disease and diabetes in obesity.

Female mice with apolipoprotein E4 domain interaction demonstrated impairments in spatial learning and memory performance and disruption of hippocampal cyto-architecture.

We have previously reported cognitive impairments in both young and old mice, particularly in female mice expressing mouse Arg-61 apoE, with a point mutation to mimic the domain interaction feature of human apoE4, as compared to the wildtype mouse (C57BL/6J) apoE. In this study, we further evaluated water maze performance in the female Arg-61 mice at an additional time point and then investigated related hippocampal cyto-architecture in these young female Arg-61 apoE mice vs. the wildtype mice. The results of behavioral performance consistently support our previous report that the young female Arg-61 apoE showed cognitive impairment versus C57BL/6J at the same age. The cyto-architectural results showed that volume of the granular cell layer (GCL) was significantly larger in both 5- and 10-month old Arg-61 apoE mice versus C57BL/6J mice. While the number of newborn calretinin-positive neurons was greater in the sub-granular zone (SGZ) in 5-month old Arg-61 mice, this number dropped significantly in 10-month old Arg-61 mice to a lower level than in age-matched C57BL/6J mice. In addition, the amyloid ß species was significantly higher in 5-month old Arg-61 mice versus age-matched C57BL/6J mice. In conclusion, impaired cognitive functions in female Arg-61 apoE mice appear correlated with larger GCL volume and higher calretinin-positive cell number and suggest a compensatory cellular response that may be related to amyloid beta perturbations early in life. Therefore this study suggests a novel cyto-architectural mechanism of apoE4-dependent pathologies and increased susceptibility of APOEε4 subjects to Alzheimer's disease.

An intracellular matrix metalloproteinase-2 isoform induces tubular regulated necrosis: implications for acute kidney injury.

Acute kidney injury (AKI) causes severe morbidity, mortality, and chronic kidney disease (CKD). Mortality is particularly marked in the elderly and with preexisting CKD. Oxidative stress is a common theme in models of AKI induced by ischemia-reperfusion (I-R) injury. We recently characterized an intracellular isoform of matrix metalloproteinase-2 (MMP-2) induced by oxidative stress-mediated activation of an alternate promoter in the first intron of the MMP-2 gene. This generates an NH2-terminal truncated MMP-2 (NTT-MMP-2) isoform that is intracellular and associated with mitochondria. The NTT-MMP-2 isoform is expressed in kidneys of 14-mo-old mice and in a mouse model of coronary atherosclerosis and heart failure with CKD. We recently determined that NTT-MMP-2 is induced in human renal transplants with delayed graft function and correlated with tubular cell necrosis. To determine mechanism(s) of action, we generated proximal tubule cell-specific NTT-MMP-2 transgenic mice. Although morphologically normal at the light microscopic level at 4 mo, ultrastructural studies revealed foci of tubular epithelial cell necrosis, the mitochondrial permeability transition, and mitophagy. To determine whether NTT-MMP-2 expression enhances sensitivity to I-R injury, we performed unilateral I-R to induce mild tubular injury in wild-type mice. In contrast, expression of the NTT-MMP-2 isoform resulted in a dramatic increase in tubular cell necrosis, inflammation, and fibrosis. NTT-MMP-2 mice had enhanced expression of innate immunity genes and release of danger-associated molecular pattern molecules. We conclude that NTT-MMP-2 "primes" the kidney to enhanced susceptibility to I-R injury via induction of mitochondrial dysfunction. NTT-MMP-2 may be a novel AKI treatment target.

Apolipoprotein E enhances microRNA-146a in monocytes and macrophages to suppress nuclear factor-κB-driven inflammation and atherosclerosis.

RATIONALE: Apolipoprotein E (apoE) exerts anti-inflammatory properties that protect against atherosclerosis and other inflammatory diseases. However, mechanisms by which apoE suppresses the cellular activation of leukocytes commonly associated with atherosclerosis remain incompletely understood. OBJECTIVE: To test the hypothesis that apoE suppresses inflammation and atherosclerosis by regulating cellular microRNA levels in these leukocytes. METHODS AND RESULTS: An assessment of apoE expression among such leukocyte subsets in wild-type mice revealed that only macrophages and monocytes express apoE abundantly. An absence of apoE expression in macrophages and monocytes resulted in enhanced nuclear factor-κB signaling and an exaggerated inflammatory response on stimulation with lipopolysaccharide. This correlated with reduced levels of microRNA-146a, a critical negative regulator of nuclear factor-κB signaling. Ectopic apoE expression in Apoe(-/-) macrophages and monocytes raised miR-146a levels, whereas its silencing in wild-type cells had an opposite effect. Mechanistically, apoE increased the expression of transcription factor purine-rich PU-box-binding protein 1, which raised levels of pri-miR-146 transcripts, demonstrating that apoE exerts transcriptional control over miR-146a. In vivo, even a small amount of apoE expression in macrophages and monocytes of hypomorphic apoE mice led to increased miR-146a levels, and inhibited macrophage proinflammatory responses, Ly-6C(high) monocytosis, and atherosclerosis in the settings of hyperlipidemia. Accordingly, cellular enrichment of miR-146a through the systemic delivery of miR-146a mimetics in Apoe(-/-)Ldlr(-/-) and Ldlr(-/-) mice attenuated monocyte/macrophage activation and atherosclerosis in the absence of plasma lipid reduction. CONCLUSIONS: Our data demonstrate that cellular apoE expression suppresses nuclear factor-κB-mediated inflammation and atherosclerosis by enhancing miR-146a levels in monocytes and macrophages.

A screen in mice uncovers repression of lipoprotein lipase by microRNA-29a as a mechanism for lipid distribution away from the liver.

UNLABELLED: Identification of microRNAs (miRNAs) that regulate lipid metabolism is important to advance the understanding and treatment of some of the most common human diseases. In the liver, a few key miRNAs have been reported that regulate lipid metabolism, but since many genes contribute to hepatic lipid metabolism, we hypothesized that other such miRNAs exist. To identify genes repressed by miRNAs in mature hepatocytes in vivo, we injected adult mice carrying floxed Dicer1 alleles with an adenoassociated viral vector expressing Cre recombinase specifically in hepatocytes. By inactivating Dicer in adult quiescent hepatocytes we avoided the hepatocyte injury and regeneration observed in previous mouse models of global miRNA deficiency in hepatocytes. Next, we combined gene and miRNA expression profiling to identify candidate gene/miRNA interactions involved in hepatic lipid metabolism and validated their function in vivo using antisense oligonucleotides. A candidate gene that emerged from our screen was lipoprotein lipase (Lpl), which encodes an enzyme that facilitates cellular uptake of lipids from the circulation. Unlike in energy-dependent cells like myocytes, LPL is normally repressed in adult hepatocytes. We identified miR-29a as the miRNA responsible for repressing LPL in hepatocytes, and found that decreasing hepatic miR-29a levels causes lipids to accumulate in mouse livers. CONCLUSION: Our screen suggests several new miRNAs are regulators of hepatic lipid metabolism. We show that one of these, miR-29a, contributes to physiological lipid distribution away from the liver and protects hepatocytes from steatosis. Our results, together with miR-29a's known antifibrotic effect, suggest miR-29a is a therapeutic target in fatty liver disease.

Immunosuppression With FTY720 Reverses Cardiac Dysfunction in Hypomorphic ApoE Mice Deficient in SR-BI Expression That Survive Myocardial Infarction Caused by Coronary Atherosclerosis.

AIMS: We recently reported that immunosuppression with FTY720 improves cardiac function and extends longevity in Hypomorphic ApoE mice deficient in scavenger receptor Type-BI expression, also known as the HypoE/SR-BI(­/­) mouse model of diet-induced coronary atherosclerosis and myocardial infarction (MI). In this study, we tested the impact of FTY720 on cardiac dysfunction in HypoE/SR-BI(­/­) mice that survive MI and subsequently develop chronic heart failure. METHODS/RESULTS: HypoE/SR-BI(­/­) mice were bred to Mx1-Cre transgenic mice, and offspring were fed a high-fat diet (HFD) for 3.5 weeks to provoke hyperlipidemia, coronary atherosclerosis, and recurrent MIs. In contrast to our previous study, hyperlipidemia was rapidly reversed by inducible Cre-mediated gene repair of the HypoE allele and switching mice to a normal chow diet. Mice that survived the period of HFD were subsequently given oral FTY720 in drinking water or not, and left ventricular (LV) function was monitored using serial echocardiography for up to 15 weeks. In untreated mice, LV performance progressively deteriorated. Although FTY720 treatment did not initially prevent a decline of heart function among mice 6 weeks after Cre-mediated gene repair, it almost completely restored normal LV function in these mice by 15 weeks. Reversal of heart failure did not result from reduced atherosclerosis as the burden of aortic and coronary atherosclerosis actually increased to similar levels in both groups of mice. Rather, FTY720 caused systemic immunosuppression as assessed by reduced numbers of circulating T and B lymphocytes. In contrast, FTY720 did not enhance the loss of T cells or macrophages that accumulated in the heart during the HFD feeding period, but it did enhance the loss of B cells soon after plasma lipid lowering. Moreover, FTY720 potently reduced the expression of matrix metalloproteinase-2 and genes involved in innate immunity-associated inflammation in the heart. CONCLUSIONS: Our data demonstrate that immunosuppression with FTY720 prevents postinfarction myocardial remodeling and chronic heart failure.

Inhibition of p38 Mitogen-Activated Protein Kinase Enhances the Apoptosis Induced by Oxidized Low-Density Lipoprotein in Endothelial Progenitor Cells.

Oxidized low-density lipoprotein (oxLDL) is an important risk factor in the development of atherosclerosis. oxLDL has been shown to decrease endothelial progenitor cell (EPC) number by inducing apoptosis. p38 mitogen-activated protein kinase (MAPK) was shown to be activated by oxLDL and participated in the regulation of EPC number and function. However, the role of p38 remains unknown. Here, we show that oxLDL-induced p38 phosphorylation in EPCs is time and dose dependent. Treatment with antioxidant N-acetyl cysteine restored oxLDL-induced p38 phosphorylation to basal levels. LOX-1-blocking antibody also significantly decreased oxLDL-induced p38 phosphorylation. Interestingly, TUNEL staining showed that pretreatment with the p38 inhibitor SB203580 further increased oxLDL-induced apoptosis in EPCs. In accordance with these findings, pretreatment with SB203580 further attenuated Akt phosphorylation in EPCs challenged with oxLDL, indicating an interaction between Akt and p38 MAPK pathways. In agreement, inhibition of p38 MAPK further attenuated Akt phosphorylation and increased apoptosis in EPCs isolated from hypercholesterolemic ApoE-/- mice. In conclusion, p38 MAPK serves as an anti-apoptotic pathway by supporting Akt activity when EPCs are challenged with oxLDL.

Hyperglycemia impairs atherosclerosis regression in mice.

Diabetic patients are known to be more susceptible to atherosclerosis and its associated cardiovascular complications. However, the effects of hyperglycemia on atherosclerosis regression remain unclear. We hypothesized that hyperglycemia impairs atherosclerosis regression by modulating the biological function of lesional macrophages. HypoE (Apoe(h/h)Mx1-Cre) mice express low levels of apolipoprotein E (apoE) and develop atherosclerosis when fed a high-fat diet. Atherosclerosis regression occurs in these mice upon plasma lipid lowering induced by a change in diet and the restoration of apoE expression. We examined the morphological characteristics of regressed lesions and assessed the biological function of lesional macrophages isolated with laser-capture microdissection in euglycemic and hyperglycemic HypoE mice. Hyperglycemia induced by streptozotocin treatment impaired lesion size reduction (36% versus 14%) and lipid loss (38% versus 26%) after the reversal of hyperlipidemia. However, decreases in lesional macrophage content and remodeling in both groups of mice were similar. Gene expression analysis revealed that hyperglycemia impaired cholesterol transport by modulating ATP-binding cassette A1, ATP-binding cassette G1, scavenger receptor class B family member (CD36), scavenger receptor class B1, and wound healing pathways in lesional macrophages during atherosclerosis regression. Hyperglycemia impairs both reduction in size and loss of lipids from atherosclerotic lesions upon plasma lipid lowering without significantly affecting the remodeling of the vascular wall.

Inducible ApoE gene repair in hypomorphic ApoE mice deficient in the low-density lipoprotein receptor promotes atheroma stabilization with a human-like lipoprotein profile.

OBJECTIVE: To study atherosclerosis regression in mice after plasma lipid reduction to moderately elevated apolipoprotein B (apoB)-lipoprotein levels. APPROACH AND RESULTS: Chow-fed hypomorphic Apoe mice deficient in low-density lipoprotein receptor expression (Apoe(h/h)Ldlr(-/-)Mx1-cre mice) develop hyperlipidemia and atherosclerosis. These mice were studied before and after inducible cre-mediated Apoe gene repair. By 1 week, induced mice displayed a 2-fold reduction in plasma cholesterol and triglyceride levels and a decrease in the non-high-density lipoprotein:high-density lipoprotein-cholesterol ratio from 87%:13% to 60%:40%. This halted atherosclerotic lesion growth and promoted macrophage loss and accumulation of thick collagen fibers for up to 8 weeks. Concomitantly, blood Ly-6C(high) monocytes were decreased by 2-fold but lesional macrophage apoptosis was unchanged. The expression of several genes involved in extracellular matrix remodeling and cell migration was changed in lesional macrophages 1 week after Apoe gene repair. However, mRNA levels of numerous genes involved in cholesterol efflux and inflammation were not significantly changed at this time point. CONCLUSIONS: Restoring apoE expression in Apoe(h/h)Ldlr(-/-)Mx1-cre mice resulted in lesion stabilization in the context of a human-like ratio of non-high-density lipoprotein:high-density lipoprotein-cholesterol. Our data suggest that macrophage loss derived in part from reduced blood Ly-6C(high) monocytes levels and genetic reprogramming of lesional macrophages.

The immunosuppressant FTY720 prolongs survival in a mouse model of diet-induced coronary atherosclerosis and myocardial infarction.

FTY720, an analogue of sphingosine-1-phosphate, is cardioprotective during acute injury. Whether long-term FTY720 affords cardioprotection is unknown. Here, we report the effects of oral FTY720 on ischemia/reperfusion injury and in hypomorphic apoE mice deficient in SR-BI receptor expression (ApoeR61(h/h)/SRB1(-/- mice), a model of diet-induced coronary atherosclerosis and heart failure. We added FTY720 (0.3 mg·kg(-1)·d(-1)) to the drinking water of C57BL/6J mice. After ex vivo cardiac ischemia/reperfusion injury, these mice had significantly improved left ventricular (LV) developed pressure and reduced infarct size compared with controls. Subsequently, ApoeR61(h/h)/SRB1(-/-) mice fed a high-fat diet for 4 weeks were treated or not with oral FTY720 (0.05 mg·kg(-1)·d(-1)). This sharply reduced mortality (P < 0.02) and resulted in better LV function and less LV remodeling compared with controls without reducing hypercholesterolemia and atherosclerosis. Oral FTY720 reduced the number of blood lymphocytes and increased the percentage of CD4+Foxp3+ regulatory T cells (Tregs) in the circulation, spleen, and lymph nodes. FTY720-treated mice exhibited increased TGF-ß and reduced IFN-γ expression in the heart. Also, CD4 expression was increased and strongly correlated with molecules involved in natural Treg activity, such as TGF-ß and GITR. Our data suggest that long-term FTY720 treatment enhances LV function and increases longevity in mice with heart failure. These benefits resulted not from atheroprotection but from systemic immunosuppression and a moderate reduction of inflammation in the heart.

Label-free single-vesicle based surface enhanced Raman spectroscopy: A robust approach for investigating the biomolecular composition of small extracellular vesicles.

Small extracellular vesicles (sEVs) are cell-released vesicles ranging from 30-150nm in size. They have garnered increasing attention because of their potential for both the diagnosis and treatment of disease. The diversity of sEVs derives from their biological composition and cargo content. Currently, the isolation of sEV subpopulations is primarily based on bio-physical and affinity-based approaches. Since a standardized definition for sEV subpopulations is yet to be fully established, it is important to further investigate the correlation between the biomolecular composition of sEVs and their physical properties. In this study, we employed a platform combining single-vesicle surface-enhanced Raman spectroscopy (SERS) and machine learning to examine individual sEVs isolated by size-exclusion chromatography (SEC). The biomolecular composition of each vesicle examined was reflected by its corresponding SERS spectral features (biomolecular "fingerprints"), with their roots in the composition of their collective Raman-active bonds. Origins of the SERS spectral features were validated through a comparative analysis between SERS and mass spectrometry (MS). SERS fingerprinting of individual vesicles was effective in overcoming the challenges posed by EV population averaging, allowing for the possibility of analyzing the variations in biomolecular composition between the vesicles of similar and/or different sizes. Using this approach, we uncovered that each of the size-based fractions of sEVs contained particles with predominantly similar SERS spectral features. Indeed, more than 84% of the vesicles residing within a particular group were clearly distinguishable from that of the other EV sub-populations, despite some spectral variations within each sub-population. Our results suggest the possibility that size-based EV fractionation methods produce samples where similarly eluted sEVs are correlated with their respective biochemical contents, as reflected by their SERS spectra. Our findings therefore highlight the possibility that the biogenesis and respective biological functionalities of the various sEV fractions may be inherently different.

The EV antibody database: An interactive database of curated antibodies for extracellular vesicle and nanoparticle research.

Antibodies are critical tools for research into extracellular vesicles (EVs) and other extracellular nanoparticles (ENPs), where they can be used for their identification, characterization, and isolation. However, the lack of a centralized antibody platform where researchers can share validation results thus minimizing wasted personnel time and reagents, has been a significant obstacle. Moreover, because the performance of antibodies varies among assay types and conditions, detailed information on assay variables and protocols is also of value. To facilitate sharing of results on antibodies that are relevant to EV/ENP research, the EV Antibody Database has been developed by the investigators of the Extracellular RNA Communication Consortium (ERCC). Hosted by the ExRNA Portal (https://exrna.org/resources/evabdb/), this interactive database aggregates and shares results from antibodies that have been tested by research groups in the EV/ENP field. Currently, the EV Antibody Database includes modules for antibodies tested for western Blot, EV Flow Cytometry, and EV Sandwich Assays, and holds 110 records contributed by 6 laboratories from the ERCC. Detailed information on antibody sources, assay conditions, and results is provided, including negative results. We encourage ongoing expert input and community feedback to enhance the database's utility, making it a valuable resource for comprehensive validation data on antibodies and protocols in EV biology.

Apolipoprotein E4 domain interaction accelerates diet-induced atherosclerosis in hypomorphic Arg-61 apoe mice.

OBJECTIVE: Apolipoprotein (apo) E4 is an established risk factor for atherosclerosis, but the structural components underlying this association remain unclear. ApoE4 is characterized by 2 biophysical properties: domain interaction and molten globule state. Substituting Arg-61 for Thr-61 in mouse apoE introduces domain interaction without molten globule state, allowing us to delineate potential proatherogenic effects of domain interaction in vivo. METHODS AND RESULTS: We studied atherosclerosis susceptibility of hypomorphic Apoe mice expressing either Thr-61 or Arg-61 apoE (ApoeT(h/h) or ApoeR(h/h)mice). On a chow diet, both mouse models were normolipidemic with similar levels of plasma apoE and lipoproteins. However, on a high-cholesterol diet, ApoeR(h/h) mice displayed increased levels of total plasma cholesterol and very-low-density lipoprotein as well as larger atherosclerotic plaques in the aortic root, arch, and descending aorta compared with ApoeT(h/h) mice. In addition, evidence of cellular dysfunction was identified in peritoneal ApoeR(h/h) macrophages which released lower amounts of apoE in culture medium and displayed increased expression of major histocompatibility complex class II molecules. CONCLUSIONS: These data indicate that domain interaction mediates proatherogenic effects of apoE4 in part by modulating lipoprotein metabolism and macrophage biology. Pharmaceutical targeting of domain interaction could lead to new treatments for atherosclerosis in apoE4 individuals.

ApoE suppresses atherosclerosis by reducing lipid accumulation in circulating monocytes and the expression of inflammatory molecules on monocytes and vascular endothelium.

OBJECTIVE: We investigated atheroprotective properties of apolipoprotein (apo) E beyond its ability to lower plasma cholesterol. We hypothesized that apoE reduces atherosclerosis by decreasing lipid accumulation in circulating monocytes and the inflammatory state of monocytes and the vascular endothelium. METHODS AND RESULTS: We developed mice with spontaneous hyperlipidemia with and without plasma apoE. Hypomorphic apoE mice deficient in low-density lipoprotein receptor (Apoe(h/h)Ldlr(-/-)) were compared to Apoe(-/-)Ldlr(-/-) mice. Despite 4-fold more plasma apoE than WT mice, Apoe(h/h)Ldlr(-/-) mice displayed similar plasma cholesterol as Apoe(-/-) Ldlr(-/-) mice but developed 4-fold less atherosclerotic lesions by 5 months of age. The aortic arch of Apoe(h/h)Ldlr(-/-) mice showed decreased endothelial expression of ICAM-1, PECAM-1, and JAM-A. In addition, Apoe(h/h)Ldlr(-/-) mice had less circulating leukocytes and proinflammatory Ly6C(high) monocytes. These monocytes had decreased neutral lipid content and reduced surface expression of ICAM-1, VLA-4, and L-Selectin. Apoe(h/h)Ldlr(-/-) mice displayed increased levels of apoA1-rich HDL that were potent in promoting cellular cholesterol efflux. CONCLUSIONS: Our findings suggest that apoE reduces atherosclerosis in the setting of hyperlipidemia by increasing plasma apoA1-HDL that likely contribute to reduce intracellular lipid accumulation and thereby the activation of circulating leukocytes and the vascular endothelium.

Apolipoprotein E regulation of myeloid cell plasticity in atherosclerosis.

PURPOSE OF REVIEW: Apolipoprotein (apo) E is a multifunctional protein that has long been recognized for its ability to safeguard against atherosclerosis. Among its pleiotropic roles known to suppress atherosclerosis, mechanisms by which apoE regulates cells of the immune system have remained elusive. Because atherosclerosis is a chronic inflammatory disease that remains on the rise, understanding in more detail how apoE controls immune cell activation and function is of much interest. RECENT FINDINGS: Literature reported in the past year introduces apoE as a regulator of monocyte and macrophage plasticity. Through signals delivered by its interaction with cell surface receptors, apoE has been shown to influence the polarity and inflammatory phenotypes of the macrophage. By promoting cellular cholesterol efflux in a cell autonomous manner and through its ability to enhance HDL function in hyperlipidemic plasma, apoE is now known to suppress atherosclerosis by controlling myeloid cell proliferation, monocyte activation and their capacity to infiltrate the vascular wall. Lastly, the structural basis for apoE isoform-specific effects in macrophage dysfunction and atherosclerosis susceptibility is beginning to emerge. SUMMARY: Collectively, these findings introduce a new dimension to our understanding of how apoE links lipoprotein biology to monocyte and macrophage function in atherosclerosis susceptibility.

ApoE expression in macrophages communicates immunometabolic signaling that controls hyperlipidemia-driven hematopoiesis & inflammation via extracellular vesicles.

While apolipoprotein E (apoE) expression by myeloid cells is recognized to control inflammation, whether such benefits can be communicated via extracellular vesicles is not known. Through the study of extracellular vesicles produced by macrophages derived from the bone marrow of Wildtype (WT-BMDM-EV) and ApoE deficient (EKO-BMDM-EV) mice, we uncovered a critical role for apoE expression in regulating their cell signaling properties. WT-BMDM-EV communicated anti-inflammatory properties to recipient myeloid cells by increasing cellular levels of apoE and miR-146a-5p, that reduced NF-κB signalling. They also downregulated cellular levels of miR-142a-3p, resulting in increased levels of its target carnitine palmitoyl transferase 1A (CPT1A) which improved fatty acid oxidation (FAO) and oxidative phosphorylation (OxPHOS) in recipient cells. Such favorable metabolic polarization enhanced cell-surface MerTK levels and the phagocytic uptake of apoptotic cells. In contrast, EKO-BMDM-EV exerted opposite effects by reducing cellular levels of apoE and miR-146a-5p, which increased NF-κB-driven GLUT1-mediated glucose uptake, aerobic glycolysis, and oxidative stress. Furthermore, EKO-BMDM-EV increased cellular miR-142a-3p levels, which reduced CPT1A levels and impaired FAO and OxPHOS in recipient myeloid cells. When cultured with naïve CD4+ T lymphocytes, EKO-BMDM-EV drove their activation and proliferation, and fostered their transition to a Th1 phenotype. While infusions of WT-BMDM-EV into hyperlipidemic mice resolved inflammation, infusions of EKO-BMDM-EV increased hematopoiesis and drove inflammatory responses in myeloid cells and T lymphocytes. ApoE-dependent immunometabolic signaling by macrophage extracellular vesicles was dependent on transcriptional axes controlled by miR-146a-5p and miR-142a-3p that could be reproduced by infusing miR-146a mimics & miR-142a antagonists into hyperlipidemic apoE-deficient mice. Together, our findings unveil a novel property for apoE expression in macrophages that modulates the immunometabolic regulatory properties of their secreted extracellular vesicles.

Polarized macrophages regulate fibro/adipogenic progenitor (FAP) adipogenesis through exosomes.

BACKGROUND: Macrophage polarization has been observed in the process of muscle injuries including rotator cuff (RC) muscle atrophy and fatty infiltration after large tendon tears. In our previous study, we showed that fibrogenesis and white adipogenesis of muscle residential fibro/adipogenic progenitors (FAPs) cause fibrosis and fatty infiltration and that brown/beige adipogenesis of FAPs promotes rotator cuff muscle regeneration. However, how polarized macrophages and their exosomes regulate FAP differentiation remains unknown. METHODS: We cultured FAPs with M0, M1, and M2 macrophages or 2 × 109 exosomes derived from M0, M1 and M2 with and without GW4869, an exosome inhibitor. In vivo, M0, M1, and M2 macrophages were transplanted or purified macrophage exosomes (M0, M1, M2) were injected into supraspinatus muscle (SS) after massive tendon tears in mice (n = 6). SS were harvested at six weeks after surgery to evaluate the level of muscle atrophy and fatty infiltration. RESULTS: Our results showed that M2 rather than M0 or M1 macrophages stimulates brown/beige fat differentiation of FAPs. However, the effect of GW4869, the exosome inhibitor, diminished this effect. M2 exosomes also promoted FAP Beige differentiation in vitro. The transplantation of M2 macrophages reduced supraspinatus muscle atrophy and fatty infiltration. In vivo injections of M2 exosomes significantly reduced muscle atrophy and fatty infiltration in supraspinatus muscle. CONCLUSION: Results from our study demonstrated that polarized macrophages directly regulated FAP differentiation through their exosomes and M2 macrophage-derived exosomes may serve as a novel treatment option for RC muscle atrophy and fatty infiltration.