Hemorrhage-activated NRF2 in tumor-associated macrophages drives cancer growth, invasion, and immunotherapy resistance.

Microscopic hemorrhage is a common aspect of cancers, yet its potential role as an independent factor influencing both cancer progression and therapeutic response is largely ignored. Recognizing the essential function of macrophages in red blood cell disposal, we explored a pathway that connects intratumoral hemorrhage with the formation of cancer-promoting tumor-associated macrophages (TAMs). Using spatial transcriptomics, we found that NRF2-activated myeloid cells possessing characteristics of procancerous TAMs tend to cluster in perinecrotic hemorrhagic tumor regions. These cells resembled antiinflammatory erythrophagocytic macrophages. We identified heme, a red blood cell metabolite, as a pivotal microenvironmental factor steering macrophages toward protumorigenic activities. Single-cell RNA-Seq and functional assays of TAMs in 3D cell culture spheroids revealed how elevated intracellular heme signals via the transcription factor NRF2 to induce cancer-promoting TAMs. These TAMs stabilized epithelial-mesenchymal transition, enhancing cancer invasiveness and metastatic potential. Additionally, NRF2-activated macrophages exhibited resistance to reprogramming by IFN-γ and anti-CD40 antibodies, reducing their tumoricidal capacity. Furthermore, MC38 colon adenocarcinoma-bearing mice with NRF2 constitutively activated in leukocytes were resistant to anti-CD40 immunotherapy. Overall, our findings emphasize hemorrhage-activated NRF2 in TAMs as a driver of cancer progression, suggesting that targeting this pathway could offer new strategies to enhance cancer immunity and overcome therapy resistance.

Haptoglobin Treatment for Aneurysmal Subarachnoid Hemorrhage: Review and Expert Consensus on Clinical Translation.

Aneurysmal subarachnoid hemorrhage (aSAH) is a devastating form of stroke frequently affecting young to middle-aged adults, with an unmet need to improve outcome. This special report focusses on the development of intrathecal haptoglobin supplementation as a treatment by reviewing current knowledge and progress, arriving at a Delphi-based global consensus regarding the pathophysiological role of extracellular hemoglobin and research priorities for clinical translation of hemoglobin-scavenging therapeutics. After aneurysmal subarachnoid hemorrhage, erythrocyte lysis generates cell-free hemoglobin in the cerebrospinal fluid, which is a strong determinant of secondary brain injury and long-term clinical outcome. Haptoglobin is the body's first-line defense against cell-free hemoglobin by binding it irreversibly, preventing translocation of hemoglobin into the brain parenchyma and nitric oxide-sensitive functional compartments of cerebral arteries. In mouse and sheep models, intraventricular administration of haptoglobin reversed hemoglobin-induced clinical, histological, and biochemical features of human aneurysmal subarachnoid hemorrhage. Clinical translation of this strategy imposes unique challenges set by the novel mode of action and the anticipated need for intrathecal drug administration, necessitating early input from stakeholders. Practising clinicians (n=72) and scientific experts (n=28) from 5 continents participated in the Delphi study. Inflammation, microvascular spasm, initial intracranial pressure increase, and disruption of nitric oxide signaling were deemed the most important pathophysiological pathways determining outcome. Cell-free hemoglobin was thought to play an important role mostly in pathways related to iron toxicity, oxidative stress, nitric oxide, and inflammation. While useful, there was consensus that further preclinical work was not a priority, with most believing the field was ready for an early phase trial. The highest research priorities were related to confirming haptoglobin's anticipated safety, individualized versus standard dosing, timing of treatment, pharmacokinetics, pharmacodynamics, and outcome measure selection. These results highlight the need for early phase trials of intracranial haptoglobin for aneurysmal subarachnoid hemorrhage, and the value of early input from clinical disciplines on a global scale during the early stages of clinical translation.

Antibody-induced erythrophagocyte reprogramming of Kupffer cells prevents anti-CD40 cancer immunotherapy-associated liver toxicity.

BACKGROUND: Agonistic anti-CD40 monoclonal antibodies (mAbs) have emerged as promising immunotherapeutic compounds with impressive antitumor effects in mouse models. However, preclinical and clinical studies faced dose-limiting toxicities mediated by necroinflammatory liver disease. An effective prophylactic treatment for liver immune-related adverse events that does not suppress specific antitumor immunity remains to be found. METHODS: We used different mouse models and time-resolved single-cell RNA-sequencing to characterize the pathogenesis of anti-CD40 mAb induced liver toxicity. Subsequently, we developed an antibody-based treatment protocol to selectively target red blood cells (RBCs) for erythrophagocytosis in the liver, inducing an anti-inflammatory liver macrophage reprogramming. RESULTS: We discovered that CD40 signaling in Clec4f+ Kupffer cells is the non-redundant trigger of anti-CD40 mAb-induced liver toxicity. Taking advantage of the highly specific functionality of liver macrophages to clear antibody-tagged RBCs from the blood, we hypothesized that controlled erythrophagocytosis and the linked anti-inflammatory signaling by the endogenous metabolite heme could be exploited to reprogram liver macrophages selectively. Repeated low-dose administration of a recombinant murine Ter119 antibody directed RBCs for selective phagocytosis in the liver and skewed the phenotype of liver macrophages into a Hmoxhigh/Marcohigh/MHCIIlow anti-inflammatory phenotype. This unique mode of action prevented necroinflammatory liver disease following high-dose administration of anti-CD40 mAbs. In contrast, extrahepatic inflammation, antigen-specific immunity, and antitumor activity remained unaffected in Ter119 treated animals. CONCLUSIONS: Our study offers a targeted approach to uncouple CD40-augmented antitumor immunity in peripheral tissues from harmful inflammatoxicity in the liver.

Erythrophagocytes in hemolytic anemia, wound healing, and cancer.

Hemolysis is a ubiquitous pathology defined as premature red blood cell destruction within the circulation or local tissues. One of the most archetypal functions of macrophages is phagocytosis of damaged or extravasated red blood cells, preventing the extracellular release of toxic hemoglobin and heme. Upon erythrophagocytosis, spiking intracellular heme concentrations drive macrophage transformation into erythrophagocytes, leveraging antioxidative and iron recycling capacities to defend against hemolytic stress. This unique phenotype transformation is coordinated by a regulatory network comprising the transcription factors BACH1, SPI-C, NRF2, and ATF1. Erythrophagocytes negatively regulate inflammation and immunity and may modulate disease-specific outcomes in hemolytic anemia, wound healing, atherosclerosis, and cancer. In this opinion article, we outline the known and presumed functions of erythrophagocytes and their implications for therapeutic innovation and research.

The HeMoVal study protocol: a prospective international multicenter cohort study to validate cerebrospinal fluid hemoglobin as a monitoring biomarker for aneurysmal subarachnoid hemorrhage related secondary brain injury.

INTRODUCTION: Preclinical studies provided a strong rationale for a pathophysiological link between cell-free hemoglobin in the cerebrospinal fluid (CSF-Hb) and secondary brain injury after subarachnoid hemorrhage (SAH-SBI). In a single-center prospective observational clinical study, external ventricular drain (EVD) based CSF-Hb proved to be a promising biomarker to monitor for SAH-SBI. The primary objective of the HeMoVal study is to prospectively validate the association between EVD based CSF-Hb and SAH-SBI during the first 14 days post-SAH. Secondary objectives include the assessment of the discrimination ability of EVD based CSF-Hb for SAH-SBI and the definition of a clinically relevant range of EVD based CSF-Hb toxicity. In addition, lumbar drain (LD) based CSF-Hb will be assessed for its association with and discrimination ability for SAH-SBI. METHODS: HeMoVal is a prospective international multicenter observational cohort study. Adult patients admitted with aneurysmal subarachnoid hemorrhage (aSAH) are eligible. While all patients with aSAH are included, we target a sample size of 250 patients with EVD within the first 14 day after aSAH. Epidemiologic and disease-specific baseline measures are assessed at the time of study inclusion. In patients with EVD or LD, each day during the first 14 days post-SAH, 2 ml of CSF will be sampled in the morning, followed by assessment of the patients for SAH-SBI, co-interventions, and complications in the afternoon. After 3 months, a clinical follow-up will be performed. For statistical analysis, the cohort will be stratified into an EVD, LD and full cohort. The primary analysis will quantify the strength of association between EVD based CSF-Hb and SAH-SBI in the EVD cohort based on a generalized additive model. Secondary analyses include the strength of association between LD based CSF-Hb and SAH-SBI in the LD cohort based on a generalized additive model, as well as the discrimination ability of CSF-Hb for SAH-SBI based on receiver operating characteristic (ROC) analyses. DISCUSSION: We hypothesize that this study will validate the value of CSF-Hb as a biomarker to monitor for SAH-SBI. In addition, the results of this study will provide the potential base to define an intervention threshold for future studies targeting CSF-Hb toxicity after aSAH. STUDY REGISTRATION: ClinicalTrials.gov Identifier NCT04998370 . Date of registration: August 10, 2021.

Heme-stress activated NRF2 skews fate trajectories of bone marrow cells from dendritic cells towards red pulp-like macrophages in hemolytic anemia.

Heme is an erythrocyte-derived toxin that drives disease progression in hemolytic anemias, such as sickle cell disease. During hemolysis, specialized bone marrow-derived macrophages with a high heme-metabolism capacity orchestrate disease adaptation by removing damaged erythrocytes and heme-protein complexes from the blood and supporting iron recycling for erythropoiesis. Since chronic heme-stress is noxious for macrophages, erythrophagocytes in the spleen are continuously replenished from bone marrow-derived progenitors. Here, we hypothesized that adaptation to heme stress progressively shifts differentiation trajectories of bone marrow progenitors to expand the capacity of heme-handling monocyte-derived macrophages at the expense of the homeostatic generation of dendritic cells, which emerge from shared myeloid precursors. This heme-induced redirection of differentiation trajectories may contribute to hemolysis-induced secondary immunodeficiency. We performed single-cell RNA-sequencing with directional RNA velocity analysis of GM-CSF-supplemented mouse bone marrow cultures to assess myeloid differentiation under heme stress. We found that heme-activated NRF2 signaling shifted the differentiation of bone marrow cells towards antioxidant, iron-recycling macrophages, suppressing the generation of dendritic cells in heme-exposed bone marrow cultures. Heme eliminated the capacity of GM-CSF-supplemented bone marrow cultures to activate antigen-specific CD4 T cells. The generation of functionally competent dendritic cells was restored by NRF2 loss. The heme-induced phenotype of macrophage expansion with concurrent dendritic cell depletion was reproduced in hemolytic mice with sickle cell disease and spherocytosis and associated with reduced dendritic cell functions in the spleen. Our data provide a novel mechanistic underpinning of hemolytic stress as a driver of hyposplenism-related secondary immunodeficiency.

Modular Platform for the Development of Recombinant Hemoglobin Scavenger Biotherapeutics.

Cell-free hemoglobin (Hb) is a driver of disease progression in conditions with intravascular or localized hemolysis. Genetic and acquired anemias or emergency medical conditions such as aneurysmal subarachnoid hemorrhage involve tissue Hb exposure. Haptoglobin (Hp) captures Hb in an irreversible protein complex and prevents its pathophysiological contributions to vascular nitric oxide depletion and tissue oxidation. Preclinical proof-of-concept studies suggest that human plasma-derived Hp is a promising therapeutic candidate for several Hb-driven diseases. Optimizing the efficacy and safety of Hb-targeting biotherapeutics may require structural and functional modifications for specific indications. Improved Hp variants could be designed to achieve the desired tissue distribution, metabolism, and elimination to target hemolytic disease states effectively. However, it is critical to ensure that these modifications maintain the function of Hp. Using transient mammalian gene expression of Hp combined with co-transfection of the pro-haptoglobin processing protease C1r-LP, we established a platform for generating recombinant Hp-variants. We designed an Hpß-scaffold, which was expressed in this system at high levels as a monomeric unit (mini-Hp) while maintaining the key protective functions of Hp. We then used this Hpß-scaffold as the basis to develop an initial proof-of-concept Hp fusion protein using human serum albumin as the fusion partner. Next, a hemopexin-Hp fusion protein with bispecific heme and Hb detoxification capacity was generated. Further, we developed a Hb scavenger devoid of CD163 scavenger receptor binding. The functions of these proteins were then characterized for Hb and heme-binding, binding of the Hp-Hb complexes with the clearance receptor CD163, antioxidant properties, and vascular nitric oxide sparing capacity. Our platform is designed to support the generation of innovative Hb scavenger biotherapeutics with novel modes of action and potentially improved formulation characteristics, function, and pharmacokinetics.

Cerebrospinal fluid hemoglobin drives subarachnoid hemorrhage-related secondary brain injury.

Secondary brain injury after aneurysmal subarachnoid hemorrhage (SAH-SBI) contributes to poor outcomes in patients after rupture of an intracranial aneurysm. The lack of diagnostic biomarkers and novel drug targets represent an unmet need. The aim of this study was to investigate the clinical and pathophysiological association between cerebrospinal fluid hemoglobin (CSF-Hb) and SAH-SBI. In a cohort of 47 patients, we collected daily CSF-samples within 14 days after aneurysm rupture. There was very strong evidence for a positive association between spectrophotometrically determined CSF-Hb and SAH-SBI. The accuracy of CSF-Hb to monitor for SAH-SBI markedly exceeded that of established methods (AUC: 0.89 [0.85-0.92]). Temporal proteome analysis revealed erythrolysis accompanied by an adaptive macrophage response as the two dominant biological processes in the CSF-space after aneurysm rupture. Ex-vivo experiments on the vasoconstrictive and oxidative potential of Hb revealed critical inflection points overlapping CSF-Hb thresholds in patients with SAH-SBI. Selective depletion and in-solution neutralization by haptoglobin or hemopexin efficiently attenuated the vasoconstrictive and lipid peroxidation activities of CSF-Hb. Collectively, the clinical association between high CSF-Hb levels and SAH-SBI, the underlying pathophysiological rationale, and the favorable effects of haptoglobin and hemopexin in ex-vivo experiments position CSF-Hb as a highly attractive biomarker and potential drug target.

Acute Hemolysis and Heme Suppress Anti-CD40 Antibody-Induced Necro-Inflammatory Liver Disease.

Clearance of red blood cells and hemoproteins is a key metabolic function of macrophages during hemolytic disorders and following tissue injury. Through this archetypical phagocytic function, heme is detoxified and iron is recycled to support erythropoiesis. Reciprocal interaction of heme metabolism and inflammatory macrophage functions may modify disease outcomes in a broad range of clinical conditions. We hypothesized that acute hemolysis and heme induce acute anti-inflammatory signals in liver macrophages. Using a macrophage-driven model of sterile liver inflammation, we showed that phenylhydrazine (PHZ)-mediated acute erythrophagocytosis blocked the anti-CD40 antibody-induced pathway of macrophage activation. This process attenuated the inflammatory cytokine release syndrome and necrotizing hepatitis induced by anti-CD40 antibody treatment of mice. We further established that administration of heme-albumin complexes specifically delivered heme to liver macrophages and replicated the anti-inflammatory effect of hemolysis. The anti-inflammatory heme-signal was induced in macrophages by an increased intracellular concentration of the porphyrin independently of iron. Overall, our work suggests that induction of heme-signaling strongly suppresses inflammatory macrophage function, providing protection against sterile liver inflammation.

Agonistic Anti-CD40 Antibody Triggers an Acute Liver Crisis With Systemic Inflammation in Humanized Sickle Cell Disease Mice.

Sickle cell disease (SCD) is an inherited hemolytic disorder, defined by a point mutation in the ß-globin gene. Stress conditions such as infection, inflammation, dehydration, and hypoxia trigger erythrocyte sickling. Sickled red blood cells (RBCs) hemolyze more rapidly, show impaired deformability, and increased adhesive properties to the endothelium. In a proinflammatory, pro-coagulative environment with preexisting endothelial dysfunction, sickled RBCs promote vascular occlusion. Hepatobiliary involvement related to the sickling process, such as an acute sickle hepatic crisis, is observed in about 10% of acute sickle cell crisis incidents. In mice, ligation of CD40 with an agonistic antibody leads to a macrophage activation in the liver, triggering a sequence of systemic inflammation, endothelial cell activation, thrombosis, and focal ischemia. We found that anti-CD40 antibody injection in sickle cell mice induces a systemic inflammatory and hemodynamic response with accelerated hemolysis, extensive vaso-occlusion, and large ischemic infarctions in the liver mimicking an acute hepatic crisis. Administration of the tumor necrosis factor-α (TNF-α) blocker, etanercept, and the heme scavenger protein, hemopexin attenuated end-organ damage. These data collectively suggest that anti-CD40 administration offers a novel acute liver crisis model in humanized sickle mice, allowing for evaluation of therapeutic proof-of-concept.

Hemolysis transforms liver macrophages into antiinflammatory erythrophagocytes.

During hemolysis, macrophages in the liver phagocytose damaged erythrocytes to prevent the toxic effects of cell-free hemoglobin and heme. It remains unclear how this homeostatic process modulates phagocyte functions in inflammatory diseases. Using a genetic mouse model of spherocytosis and single-cell RNA sequencing, we found that erythrophagocytosis skewed liver macrophages into an antiinflammatory phenotype that we defined as MarcohiHmoxhiMHC class IIlo erythrophagocytes. This phenotype transformation profoundly mitigated disease expression in a model of an anti-CD40-induced hyperinflammatory syndrome with necrotic hepatitis and in a nonalcoholic steatohepatitis model, representing 2 macrophage-driven sterile inflammatory diseases. We reproduced the antiinflammatory erythrophagocyte transformation in vitro by heme exposure of mouse and human macrophages, yielding a distinctive transcriptional signature that segregated heme-polarized from M1- and M2-polarized cells. Mapping transposase-accessible chromatin in single cells by sequencing defined the transcription factor NFE2L2/NRF2 as a critical driver of erythrophagocytes, and Nfe2l2/Nrf2 deficiency restored heme-suppressed inflammation. Our findings point to a pathway that regulates macrophage functions to link erythrocyte homeostasis with innate immunity.

Haptoglobin Therapeutics and Compartmentalization of Cell-Free Hemoglobin Toxicity.

Hemolysis and accumulation of cell-free hemoglobin (Hb) in the circulation or in confined tissue compartments such as the subarachnoid space is an important driver of disease. Haptoglobin is the Hb binding and clearance protein in human plasma and an efficient antagonist of Hb toxicity resulting from physiological red blood cell turnover. However, endogenous concentrations of haptoglobin are insufficient to provide protection against Hb-driven disease processes in conditions such as sickle cell anemia, sepsis, transfusion reactions, medical-device associated hemolysis, or after a subarachnoid hemorrhage. As a result, there is increasing interest in developing haptoglobin therapeutics to target 'toxic' cell-free Hb exposures. Here, we discuss key concepts of Hb toxicity and provide a perspective on the use of haptoglobin as a therapeutic protein.

Line-selective macrophage activation with an anti-CD40 antibody drives a hemophagocytic syndrome in mice.

Hemophagocytic syndromes comprise a cluster of hyperinflammatory disorders, including hemophagocytic lymphohistiocytosis and macrophage activation syndrome. Overwhelming macrophage activation has long been considered a final common pathway in the pathophysiology of hemophagocytic syndromes leading to the characteristic cytokine storm, laboratory abnormalities, and organ injuries that define the clinical spectrum of the disease. So far, it is unknown whether primary macrophage activation alone can induce the disease phenotype. In this study, we established a novel mouse model of a hemophagocytic syndrome by treating mice with an agonistic anti-CD40 antibody (Ab). The response in wild-type mice is characterized by a cytokine storm, associated with hyperferritinemia, high soluble CD25, erythrophagocytosis, secondary endothelial activation with multiple organ vaso-occlusion, necrotizing hepatitis, and variable cytopenias. The disease is dependent on a tumor necrosis factor-α-interferon-γ-driven amplification loop. After macrophage depletion with clodronate liposomes or in mice with a macrophage-selective deletion of the CD40 gene (CD40flox/flox/LysMCre), the disease was abolished. These data provide a new preclinical model of a hemophagocytic syndrome and reinforce the key pathophysiological role of macrophages.

Determining the Optimal Normalization Factor of Different Target Arteries for ex vivo Vascular Function Experiments: A New Standardized Procedure.

The standardization of resistance vessel preparation is crucial to compare physiologic vascular reactivity under different experimental conditions. Here, we describe a generalizable experimental setup for ex vivo vascular function experiments and their mathematical basis. Porcine basilar arteries and chicken common carotid arteries were isolated post mortem via standardized surgical approaches. The inner circumference of these vessels with a passive wall tension corresponding to 100 mm Hg (IC100) as well as the circumference at which the active force production of the vessel is maximal (IC1) were determined systematically. The IC1/IC100 ratio (also referred to as factor k), a value that is believed to be constant for a defined vessel type in one species, was calculated by a novel mathematical approach. Here, we present an easy-to-use toolbox for the systematic and computer-based calculation of factor k and simplified optimal pre-stretching of any vascular segments for wire myography experiments.

Haptoglobin administration into the subarachnoid space prevents hemoglobin-induced cerebral vasospasm.

Delayed ischemic neurological deficit (DIND) is a major driver of adverse outcomes in patients with aneurysmal subarachnoid hemorrhage (aSAH), defining an unmet need for therapeutic development. Cell-free hemoglobin that is released from erythrocytes into the cerebrospinal fluid (CSF) is suggested to cause vasoconstriction and neuronal toxicity, and correlates with the occurrence of DIND. Cell-free hemoglobin in the CSF of patients with aSAH disrupted dilatory NO signaling ex vivo in cerebral arteries, which shifted vascular tone balance from dilation to constriction. We found that selective removal of hemoglobin from patient CSF with a haptoglobin-affinity column or its sequestration in a soluble hemoglobin-haptoglobin complex was sufficient to restore physiological vascular responses. In a sheep model, administration of haptoglobin into the CSF inhibited hemoglobin-induced cerebral vasospasm and preserved vascular NO signaling. We identified 2 pathways of hemoglobin delocalization from CSF into the brain parenchyma and into the NO-sensitive compartment of small cerebral arteries. Both pathways were critical for hemoglobin toxicity and were interrupted by the large hemoglobin-haptoglobin complex that inhibited spatial requirements for hemoglobin reactions with NO in tissues. Collectively, our data show that compartmentalization of hemoglobin by haptoglobin provides a novel framework for innovation aimed at reducing hemoglobin-driven neurological damage after subarachnoid bleeding.

Revisiting the putative role of heme as a trigger of inflammation.

Activation of the innate immune system by free heme has been proposed as one of the principal consequences of cell-free hemoglobin (Hb) exposure. Nonetheless, in the absence of infection, heme exposures within a hematoma, during hemolysis, or upon systemic administration of Hb (eg, as a Hb-based oxygen carrier) are typically not accompanied by uncontrolled inflammation, challenging the assumption that heme is a major proinflammatory mediator in vivo. Because of its hydrophobic nature, heme liberated from oxidized hemoglobin is rapidly transferred to alternative protein-binding sites (eg, albumin) or to hydrophobic lipid compartments minimizing protein-free heme under in vivo equilibrium conditions. We demonstrate that the capacity of heme to activate human neutrophil granulocytes strictly depends on the availability of non protein-associated heme. In human endothelial cells as well as in mouse macrophage cell cultures and in mouse models of local and systemic heme exposure, protein-associated heme or Hb do not induce inflammatory gene expression over a broad range of exposure conditions. Only experiments in protein-free culture medium demonstrated a weak capacity of heme-solutions to induce toll-like receptor-(TLR4) dependent TNF-alpha expression in macrophages. Our data suggests that the equilibrium-state of free and protein-associated heme critically determines the proinflammatory capacity of the metallo-porphyrin. Based on these data it appears unlikely that inflammation-promoting equilibrium conditions could ever occur in vivo.

Basal mTORC2 activity and expression of its components display diurnal variation in mouse perivascular adipose tissue.

In adipose tissue mTOR complex 2 (mTORC2) contributes to the regulation of glucose/lipid metabolism and inflammatory molecule expression. Both processes display diurnal variations during the course of the day. RICTOR and mSIN1 are unique and essential components of mTORC2, which is activated by growth factors including insulin. To assess whether mTORC2 components display diurnal variations, we analyzed steady state mRNA expression levels of Rictor, mSin1, and mTor in various adipose tissues during a 24 h period. Diurnally regulated expression of Rictor was detected in brown adipose tissues displaying highest mRNA expression levels at the beginning of the 12 h light period (zeitgeber time 2, ZT2). Gene expression patterns of mSin1 and mTor displayed a similar diurnal regulation as Rictor in PVAT while smaller changes were detected for these genes in aorta during the course of the day. Basal mTORC2 activity was measured by phosphorylation of protein kinase C (PKC) α at serine 657 was higher at ZT14 as compared with ZT2 in PVAT. In line, gene expression of inflammatory molecules nitric oxide synthase 2 and tumor necrosis factor α was lower at ZT 14 compared to ZT2. Our findings provide evidence for a diurnal regulation of expression of mTORC2 components and activity. Hence, mTORC2 is possibly an integral part of diurnally regulated signaling pathways in PVAT and possibly in other adipose tissues.

Endothelial Rictor is crucial for midgestational development and sustained and extensive FGF2-induced neovascularization in the adult.

To explore the general requirement of endothelial mTORC2 during embryonic and adolescent development, we knocked out the essential mTORC2 component Rictor in the mouse endothelium in the embryo, during adolescence and in endothelial cells in vitro. During embryonic development, Rictor knockout resulted in growth retardation and lethality around embryonic day 12. We detected reduced peripheral vascularization and delayed ossification of developing fingers, toes and vertebrae during this confined midgestational period. Rictor knockout did not affect viability, weight gain, and vascular development during further adolescence. However during this period, Rictor knockout prevented skin capillaries to gain larger and heterogeneously sized diameters and remodeling into tortuous vessels in response to FGF2. Rictor knockout strongly reduced extensive FGF2-induced neovascularization and prevented hemorrhage in FGF2-loaded matrigel plugs. Rictor knockout also disabled the formation of capillary-like networks by FGF2-stimulated mouse aortic endothelial cells in vitro. Low RICTOR expression was detected in quiescent, confluent mouse aortic endothelial cells, whereas high doses of FGF2 induced high RICTOR expression that was associated with strong mTORC2-specific protein kinase Cα and AKT phosphorylation. We demonstrate that the endothelial FGF-RICTOR axis is not required during endothelial quiescence, but crucial for midgestational development and sustained and extensive neovascularization in the adult.

Deletion of Rictor in brain and fat alters peripheral clock gene expression and increases blood pressure.

The mammalian target of rapamycin complex 2 (mTORC2) contains the essential protein RICTOR and is activated by growth factors. mTORC2 in adipose tissue contributes to the regulation of glucose and lipid metabolism. In the perivascular adipose tissue, mTORC2 ensures normal vascular reactivity by controlling expression of inflammatory molecules. To assess whether RICTOR/mTORC2 contributes to blood pressure regulation, we applied a radiotelemetry approach in control and Rictor knockout (Rictor(aP2KO)) mice generated using adipocyte protein-2 gene promoter-driven CRE recombinase expression to delete Rictor. The 24-hour mean arterial pressure was increased in Rictor(aP2KO) mice, and the physiological decline in mean arterial pressure during the dark period was impaired. In parallel, heart rate and locomotor activity were elevated during the dark period with a pattern similar to blood pressure changes. This phenotype was associated with mild cardiomyocyte hypertrophy, decreased cardiac natriuretic peptides, and their receptor expression in adipocytes. Moreover, clock gene expression was reduced or phase-shifted in perivascular adipose tissue. No differences in clock gene expression were observed in the master clock suprachiasmatic nucleus, although Rictor gene expression was also lower in brain of Rictor(aP2KO) mice. Thus, this study highlights the importance of RICTOR/mTORC2 for interactions between vasculature, adipocytes, and brain to tune physiological outcomes, such as blood pressure and locomotor activity.