Loss of Macrophage mTORC2 Drives Atherosclerosis via FoxO1 and IL-1ß Signaling.

BACKGROUND: The mTOR (mechanistic target of rapamycin) pathway is a complex signaling cascade that regulates cellular growth, proliferation, metabolism, and survival. Although activation of mTOR signaling has been linked to atherosclerosis, its direct role in lesion progression and in plaque macrophages remains poorly understood. We previously demonstrated that mTORC1 (mTOR complex 1) activation promotes atherogenesis through inhibition of autophagy and increased apoptosis in macrophages. METHODS: Using macrophage-specific Rictor- and mTOR-deficient mice, we now dissect the distinct functions of mTORC2 pathways in atherogenesis. RESULTS: In contrast to the atheroprotective effect seen with blockade of macrophage mTORC1, macrophage-specific mTORC2-deficient mice exhibit an atherogenic phenotype, with larger, more complex lesions and increased cell death. In cultured macrophages, we show that mTORC2 signaling inhibits the FoxO1 (forkhead box protein O1) transcription factor, leading to suppression of proinflammatory pathways, especially the inflammasome/IL (interleukin)-1ß response, a key mediator of vascular inflammation and atherosclerosis. In addition, administration of FoxO1 inhibitors efficiently rescued the proinflammatory response caused by mTORC2 deficiency both in vitro and in vivo. Interestingly, collective deletion of macrophage mTOR, which ablates mTORC1- and mTORC2-dependent pathways, leads to minimal change in plaque size or complexity, reflecting the balanced yet opposing roles of these signaling arms. CONCLUSIONS: Our data provide the first mechanistic details of macrophage mTOR signaling in atherosclerosis and suggest that therapeutic measures aimed at modulating mTOR need to account for its dichotomous functions.

Deficits in Col5a2 Expression Result in Novel Skin and Adipose Abnormalities and Predisposition to Aortic Aneurysms and Dissections.

Classic Ehlers-Danlos syndrome (cEDS) is characterized by fragile, hyperextensible skin and hypermobile joints. cEDS can be caused by heterozygosity for missense mutations in genes COL5A2 and COL5A1, which encode the α2(V) and α1(V) chains, respectively, of collagen V, and is most often caused by COL5A1 null alleles. However, COL5A2 null alleles have yet to be associated with cEDS or other human pathologies. We previously showed that mice homozygous null for the α2(V) gene Col5a2 are early embryonic lethal, whereas haploinsufficiency caused aberrancies of adult skin, but not a frank cEDS-like phenotype, as skin hyperextensibility at low strain and dermal cauliflower-contoured collagen fibril aggregates, two cEDS hallmarks, were absent. Herein, we show that ubiquitous postnatal Col5a2 knockdown results in pathognomonic dermal cauliflower-contoured collagen fibril aggregates, but absence of skin hyperextensibility, demonstrating these cEDS hallmarks to arise separately from loss of collagen V roles in control of collagen fibril growth and nucleation events, respectively. Col5a2 knockdown also led to loss of dermal white adipose tissue (WAT) and markedly decreased abdominal WAT that was characterized by miniadipocytes and increased collagen deposition, suggesting α2(V) to be important to WAT development/maintenance. More important, Col5a2 haploinsufficiency markedly increased the incidence and severity of abdominal aortic aneurysms, and caused aortic arch ruptures and dissections, indicating that α2(V) chain deficits may play roles in these pathologies in humans.

Mucosal Administration of Collagen V Ameliorates the Atherosclerotic Plaque Burden by Inducing Interleukin 35-dependent Tolerance.

We have shown previously that collagen V (col(V)) autoimmunity is a consistent feature of atherosclerosis in human coronary artery disease and in the Apoe(-/-) mouse model. We have also shown sensitization of Apoe(-/-) mice with col(V) to markedly increase the atherosclerotic burden, providing evidence of a causative role for col(V) autoimmunity in atherosclerotic pathogenesis. Here we sought to determine whether induction of immune tolerance to col(V) might ameliorate atherosclerosis, providing further evidence for a causal role for col(V) autoimmunity in atherogenesis and providing insights into the potential for immunomodulatory therapeutic interventions. Mucosal inoculation successfully induced immune tolerance to col(V) with an accompanying reduction in plaque burden in Ldlr(-/-) mice on a high-cholesterol diet. The results therefore demonstrate that inoculation with col(V) can successfully ameliorate the atherosclerotic burden, suggesting novel approaches for therapeutic interventions. Surprisingly, tolerance and reduced atherosclerotic burden were both dependent on the recently described IL-35 and not on IL-10, the immunosuppressive cytokine usually studied in the context of induced tolerance and amelioration of atherosclerotic symptoms. In addition to the above, using recombinant protein fragments, we were able to localize two epitopes of the α1(V) chain involved in col(V) autoimmunity in atherosclerotic Ldlr(-/-) mice, suggesting future courses of experimentation for the characterization of such epitopes.

Homozygosity and Heterozygosity for Null Col5a2 Alleles Produce Embryonic Lethality and a Novel Classic Ehlers-Danlos Syndrome-Related Phenotype.

Null alleles for the COL5A1 gene and missense mutations for COL5A1 or the COL5A2 gene underlie cases of classic Ehlers-Danlos syndrome, characterized by fragile, hyperextensible skin and hypermobile joints. However, no classic Ehlers-Danlos syndrome case has yet been associated with COL5A2 null alleles, and phenotypes that might result from such alleles are unknown. We describe mice with null alleles for the Col5a2. Col5a2(-/-) homozygosity is embryonic lethal at approximately 12 days post conception. Unlike previously described mice null for Col5a1, which die at 10.5 days post conception and virtually lack collagen fibrils, Col5a2(-/-) embryos have readily detectable collagen fibrils, thicker than in wild-type controls. Differences in Col5a2(-/-) and Col5a1(-/-) fibril formation and embryonic survival suggest that α1(V)3 homotrimers, a rare collagen V isoform that occurs in the absence of sufficient levels of α2(V) chains, serve functional roles that partially compensate for loss of the most common collagen V isoform. Col5a2(+/-) adults have skin with marked hyperextensibility and reduced tensile strength at high strain but not at low strain. Col5a2(+/-) adults also have aortas with increased compliance and reduced tensile strength. Results thus suggest that COL5A2(+/-) humans, although unlikely to present with frank classic Ehlers-Danlos syndrome, are likely to have fragile connective tissues with increased susceptibility to trauma and certain chronic pathologic conditions.

The Pathobiology of Myocardial Recovery and Remission: From Animal Models to Clinical Observations in Heart Failure Patients.

Heart failure with reduced left ventricular (LV) ejection fraction (HFrEF) is a morbid and life-threatening disease, arising secondary to abnormalities of cardiac structure and function that lead to adverse LV remodeling. Implementation of medical and device therapies results in significant improvements in patient outcomes that are associated with reverse LV remodeling and improved LV ejection fraction. This review provides an overview of the pathobiology of reverse LV remodeling in animal models and in HFrEF patients. We emphasize the differences between myocardial recovery and remission as well as the fragile nature of maintaining a state of myocardial remission.

Identification of a leucine-mediated threshold effect governing macrophage mTOR signalling and cardiovascular risk.

High protein intake is common in western societies and is often promoted as part of a healthy lifestyle; however, amino-acid-mediated mammalian target of rapamycin (mTOR) signalling in macrophages has been implicated in the pathogenesis of ischaemic cardiovascular disease. In a series of clinical studies on male and female participants ( NCT03946774 and NCT03994367 ) that involved graded amounts of protein ingestion together with detailed plasma amino acid analysis and human monocyte/macrophage experiments, we identify leucine as the key activator of mTOR signalling in macrophages. We describe a threshold effect of high protein intake and circulating leucine on monocytes/macrophages wherein only protein in excess of ∼25 g per meal induces mTOR activation and functional effects. By designing specific diets modified in protein and leucine content representative of the intake in the general population, we confirm this threshold effect in mouse models and find ingestion of protein in excess of ∼22% of dietary energy requirements drives atherosclerosis in male mice. These data demonstrate a mechanistic basis for the adverse impact of excessive dietary protein on cardiovascular risk.

Comprehensive mass spectrometric mapping of the hydroxylated amino acid residues of the α1(V) collagen chain.

BACKGROUND: α1(V) is an extensively modified collagen chain important in disease. RESULTS: Comprehensive mapping of α1(V) post-translational modifications reveals unexpectedly large numbers of X-position hydroxyprolines in Gly-X-Y amino acid triplets. CONCLUSION: The unexpected abundance of X-position hydroxyprolines suggests a mechanism for differential modification of collagen properties. SIGNIFICANCE: Positions, numbers, and occupancy of modified sites can provide insights into α1(V) biological properties. Aberrant expression of the type V collagen α1(V) chain can underlie the connective tissue disorder classic Ehlers-Danlos syndrome, and autoimmune responses against the α1(V) chain are linked to lung transplant rejection and atherosclerosis. The α1(V) collagenous COL1 domain is thought to contain greater numbers of post-translational modifications (PTMs) than do similar domains of other fibrillar collagen chains, PTMs consisting of hydroxylated prolines and lysines, the latter of which can be glycosylated. These types of PTMs can contribute to epitopes that underlie immune responses against collagens, and the high level of PTMs may contribute to the unique biological properties of the α1(V) chain. Here we use high resolution mass spectrometry to map such PTMs in bovine placental α1(V) and human recombinant pro-α1(V) procollagen chains. Findings include the locations of those PTMs that vary and those PTMs that are invariant between these α1(V) chains from widely divergent sources. Notably, an unexpectedly large number of hydroxyproline residues were mapped to the X-positions of Gly-X-Y triplets, contrary to expectations based on previous amino acid analyses of hydrolyzed α1(V) chains from various tissues. We attribute this difference to the ability of tandem mass spectrometry coupled to nanoflow chromatographic separations to detect lower-level PTM combinations with superior sensitivity and specificity. The data are consistent with the presence of a relatively large number of 3-hydroxyproline sites with less than 100% occupancy, suggesting a previously unknown mechanism for the differential modification of α1(V) chain and type V collagen properties.

Distinct Inflammatory Milieu in Patients With Right Heart Failure.

BACKGROUND: Right heart failure (RHF) is associated with worse clinical outcomes. In addition to hemodynamic perturbations, the syndrome of RHF involves liver congestion and dysfunction. The mechanisms that underlie heart-liver interactions are poorly understood and may involve secreted factors. As a first step to understand the cardiohepatic axis, we sought to elucidate the circulating inflammatory milieu in patients with RHF. METHODS: Blood samples were collected from the inferior vena cava and hepatic veins during right heart catheterization from 3 groups of patients: (1) controls with normal cardiac function, (2) patients with heart failure who did not meet all criteria of RHF, and (3) patients who met prespecified criteria for RHF defined by hemodynamic and echocardiographic parameters. We performed a multiplex protein assay to survey levels of several circulating markers and analyzed their association with mortality and the need for a left ventricular assist device or heart transplant. Finally, we leveraged publicly available single-cell RNA sequencing data and performed tissue imaging to evaluate the expression of these factors in the liver. RESULTS: In this study, RHF was associated with elevated levels of a subset of cytokines/chemokines/growth factors compared with controls. In particular, soluble CD163 (cluster of differentiation 163) and CXCL12 (chemokine [C-X-C motif] ligand 12) were higher in RHF and predicted left ventricular assist device/transplant-free survival in an independent validation cohort. Furthermore, single-cell RNA sequencing and immunohistochemistry of human liver biopsies suggest that these factors are expressed by Kupffer cells and may be liver derived. CONCLUSIONS: RHF is associated with a distinct circulating inflammatory profile. Soluble CD163 and CXCL12 are novel biomarkers that can prognosticate patient outcomes. Future studies to define how these molecules influence heart failure phenotypes and disease progression may lead to new approaches to the management of patients with RHF.

A Distinct Inflammatory Milieu in Patients with Right Heart Failure.

Background: Right heart failure (RHF) is associated with worse clinical outcomes. In addition to hemodynamic perturbations, the syndrome of RHF involves liver congestion and dysfunction. The mechanisms that underlie heart-liver interactions are poorly understood and may involve secreted factors. As a first step to understand the cardiohepatic axis, we sought to elucidate the circulating inflammatory milieu in patients with RHF. Methods: Blood samples were collected from the IVC and hepatic veins during right heart catheterization from 3 groups of patients: 1) controls with normal cardiac function, 2) patients with heart failure (HF) who did not meet all criteria of RHF, and 3) patients who met prespecified criteria for RHF defined by hemodynamic and echocardiographic parameters. We performed multiplex protein assay to survey levels of several circulating markers and analyzed their association with mortality and need for left ventricular assist device or heart transplant. Finally, we leveraged publicly available single cell RNA sequencing (scRNAseq) data and performed tissue imaging to evaluate expression of these factors in the liver. Results: In this study of 43 patients, RHF was associated with elevated levels of a subset of cytokines/chemokines/growth factors compared to controls. In particular, soluble CD163 (sCD163) and CXCL12 were higher in RHF and predicted survival in an independent validation cohort. Furthermore, scRNAseq and immunohistochemistry of human liver biopsies suggest that these factors are expressed by Kupffer cells and may be liver derived. Conclusions: RHF is associated with a distinct circulating inflammatory profile. sCD163 and CXCL12 are novel biomarkers that can prognosticate patient outcomes. Future studies to define how these molecules influence HF phenotypes and disease progression may lead to new approaches to management of patients with RHF.

Use of acidic nanoparticles to rescue macrophage lysosomal dysfunction in atherosclerosis.

Dysfunction in the macrophage lysosomal system including reduced acidity and diminished degradative capacity is a hallmark of atherosclerosis, leading to blunted clearance of excess cellular debris and lipids in plaques and contributing to lesion progression. Devising strategies to rescue this macrophage lysosomal dysfunction is a novel therapeutic measure. Nanoparticles have emerged as an effective platform to both target specific tissues and serve as drug delivery vehicles. In most cases, administered nanoparticles are taken up non-selectively by the mononuclear phagocyte system including monocytes/macrophages leading to the undesirable degradation of cargo in lysosomes. We took advantage of this default route to target macrophage lysosomes to rectify their acidity in disease states such as atherosclerosis. Herein, we develop and test two commonly used acidic nanoparticles, poly-lactide-co-glycolic acid (PLGA) and polylactic acid (PLA), both in vitro and in vivo. Our results in cultured macrophages indicate that the PLGA-based nanoparticles are the most effective at trafficking to and enhancing acidification of lysosomes. PLGA nanoparticles also provide functional benefits including enhanced lysosomal degradation, promotion of macroautophagy/autophagy and protein aggregate removal, and reduced apoptosis and inflammasome activation. We demonstrate the utility of this system in vivo, showing nanoparticle accumulation in, and lysosomal acidification of, macrophages in atherosclerotic plaques. Long-term administration of PLGA nanoparticles results in significant reductions in surrogates of plaque complexity with reduced apoptosis, necrotic core formation, and cytotoxic protein aggregates and increased fibrous cap formation. Taken together, our data support the use of acidic nanoparticles to rescue macrophage lysosomal dysfunction in the treatment of atherosclerosis.Abbreviations: BCA: brachiocephalic arteries; FACS: fluorescence activated cell sorting; FITC: fluorescein-5-isothiocyanatel; IL1B: interleukin 1 beta; LAMP: lysosomal associated membrane protein; LIPA/LAL: lipase A, lysosomal acid type; LSDs: lysosomal storage disorders; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFI: mean fluorescence intensity; MPS: mononuclear phagocyte system; PEGHDE: polyethylene glycol hexadecyl ether; PLA: polylactic acid; PLGA: poly-lactide-co-glycolic acid; SQSTM1/p62: sequestosome 1.

Steatosis drives monocyte-derived macrophage accumulation in human metabolic dysfunction-associated fatty liver disease.

Background & Aims: Metabolic dysfunction-associated fatty liver disease (MAFLD) is a common complication of obesity with a hallmark feature of hepatic steatosis. Recent data from animal models of MAFLD have demonstrated substantial changes in macrophage composition in the fatty liver. In humans, the relationship between liver macrophage heterogeneity and liver steatosis is less clear. Methods: Liver tissue from 21 participants was collected at time of bariatric surgery and analysed using flow cytometry, immunofluorescence, and H&E microscopy. Single-cell RNA sequencing was also conducted on a subset of samples (n = 3). Intrahepatic triglyceride content was assessed via MRI and tissue histology. Mouse models of hepatic steatosis were used to investigate observations made from human liver tissue. Results: We observed variable degrees of liver steatosis with minimal fibrosis in our participants. Single-cell RNA sequencing revealed four macrophage clusters that exist in the human fatty liver encompassing Kupffer cells and monocyte-derived macrophages (MdMs). The genes expressed in these macrophage subsets were similar to those observed in mouse models of MAFLD. Hepatic CD14+ monocyte/macrophage number correlated with the degree of steatosis. Using mouse models of early liver steatosis, we demonstrate that recruitment of MdMs precedes Kupffer cell loss and liver damage. Electron microscopy of isolated macrophages revealed increased lipid accumulation in MdMs, and ex vivo lipid transfer experiments suggested that MdMs may serve a distinct role in lipid uptake during MAFLD. Conclusions: The human liver in MAFLD contains macrophage subsets that align well with those that appear in mouse models of fatty liver disease. Recruited myeloid cells correlate well with the degree of liver steatosis in humans. MdMs appear to participate in lipid uptake during early stages of MALFD. Impact and implications: Metabolic dysfunction associated fatty liver disease (MAFLD) is extremely common; however, the early inflammatory responses that occur in human disease are not well understood. In this study, we investigated macrophage heterogeneity in human livers during early MAFLD and demonstrated that similar shifts in macrophage subsets occur in human disease that are similar to those seen in preclinical models. These findings are important as they establish a translational link between mouse and human models of disease, which is important for the development and testing of new therapeutic approaches for MAFLD.

Evaluation of mTORC1 signaling in mouse atherosclerotic macrophages by flow cytometry and immunofluorescence.

Previous studies have demonstrated that a high-protein diet leads to increased atherosclerosis in mice, and that this adverse effect is caused by activation of macrophage mTORC1 signaling. Here, we provide a detailed protocol for the evaluation of diet-induced mTORC1 signaling in plaque macrophages in atherosclerosis-prone apolipoprotein E (ApoE) knockout (KO) mice. This protocol includes flow cytometry and immunofluorescence analysis of atherosclerotic macrophages that can be used to study the atherogenic potential of a variety of mTORC1 modulators. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2020).