CRAT links cholesterol metabolism to innate immune responses in the heart.

Chronic inflammation is associated with increased risk and poor prognosis of heart failure; however, the precise mechanism that provokes sustained inflammation in the failing heart remains elusive. Here we report that depletion of carnitine acetyltransferase (CRAT) promotes cholesterol catabolism through bile acid synthesis pathway in cardiomyocytes. Intracellular accumulation of bile acid or intermediate, 7α-hydroxyl-3-oxo-4-cholestenoic acid, induces mitochondrial DNA stress and triggers cGAS-STING-dependent type I interferon responses. Furthermore, type I interferon responses elicited by CRAT deficiency substantially increase AIM2 expression and AIM2-dependent inflammasome activation. Genetic deletion of cardiomyocyte CRAT in mice of both sexes results in myocardial inflammation and dilated cardiomyopathy, which can be reversed by combined depletion of caspase-1, cGAS or AIM2. Collectively, we identify a mechanism by which cardiac energy metabolism, cholesterol homeostasis and cardiomyocyte-intrinsic innate immune responses are interconnected via a CRAT-mediated bile acid synthesis pathway, which contributes to chronic myocardial inflammation and heart failure progression.

Evaluation of long-chain fatty acid respiration in neonatal mouse cardiomyocytes using SeaHorse instrument.

Metabolic switches play a critical role in the pathophysiology of cardiac diseases, including heart failure. Here, we describe an assay for long-chain fatty acid oxidation in neonatal mouse cardiomyocytes by using a SeaHorse Flux Analyzer (Agilent). This protocol is a simplified but robust adaptation of the standard protocol that enables metabolic measurements in cells isolated from transgenic mouse models, which can be timesaving and informative. Cell isolation and culture represent a critical point that may require bench optimization. For complete details on the use and execution of this protocol, please refer to Angelini et al. (2021).

PHDs/CPT1B/VDAC1 axis regulates long-chain fatty acid oxidation in cardiomyocytes.

Cardiac metabolism is a high-oxygen-consuming process, showing a preference for long-chain fatty acid (LCFA) as the fuel source under physiological conditions. However, a metabolic switch (favoring glucose instead of LCFA) is commonly reported in ischemic or late-stage failing hearts. The mechanism regulating this metabolic switch remains poorly understood. Here, we report that loss of PHD2/3, the cellular oxygen sensors, blocks LCFA mitochondria uptake and ß-oxidation in cardiomyocytes. In high-fat-fed mice, PHD2/3 deficiency improves glucose metabolism but exacerbates the cardiac defects. Mechanistically, we find that PHD2/3 bind to CPT1B, a key enzyme of mitochondrial LCFA uptake, promoting CPT1B-P295 hydroxylation. Further, we show that CPT1B-P295 hydroxylation is indispensable for its interaction with VDAC1 and LCFA ß-oxidation. Finally, we demonstrate that a CPT1B-P295A mutant constitutively binds to VDAC1 and rescues LCFA metabolism in PHD2/3-deficient cardiomyocytes. Together, our data identify an oxygen-sensitive regulatory axis involved in cardiac metabolism.

Endothelium-specific depletion of LRP1 improves glucose homeostasis through inducing osteocalcin.

The vascular endothelium is present within metabolic organs and actively regulates energy metabolism. Here we show osteocalcin, recognized as a bone-secreted metabolic hormone, is expressed in mouse primary endothelial cells isolated from heart, lung and liver. In human osteocalcin promoter-driven green fluorescent protein transgenic mice, green fluorescent protein signals are enriched in endothelial cells lining aorta, small vessels and capillaries and abundant in aorta, skeletal muscle and eye of adult mice. The depletion of lipoprotein receptor-related protein 1 induces osteocalcin through a Forkhead box O -dependent pathway in endothelial cells. Whereas depletion of osteocalcin abolishes the glucose-lowering effect of low-density lipoprotein receptor-related protein 1 depletion, osteocalcin treatment normalizes hyperglycemia in multiple mouse models. Mechanistically, osteocalcin receptor-G protein-coupled receptor family C group 6 member A and insulin-like-growth-factor-1 receptor are in the same complex with osteocalcin and required for osteocalcin-promoted insulin signaling pathway. Therefore, our results reveal an endocrine/paracrine role of endothelial cells in regulating insulin sensitivity, which may have therapeutic implications in treating diabetes and insulin resistance through manipulating vascular endothelium.

Loss of bone morphogenetic protein-binding endothelial regulator causes insulin resistance.

Accumulating evidence suggests that chronic inflammation of metabolic tissues plays a causal role in obesity-induced insulin resistance. Yet, how specific endothelial factors impact metabolic tissues remains undefined. Bone morphogenetic protein (BMP)-binding endothelial regulator (BMPER) adapts endothelial cells to inflammatory stress in diverse organ microenvironments. Here, we demonstrate that BMPER is a driver of insulin sensitivity. Both global and endothelial cell-specific inducible knockout of BMPER cause hyperinsulinemia, glucose intolerance and insulin resistance without increasing inflammation in metabolic tissues in mice. BMPER can directly activate insulin signaling, which requires its internalization and interaction with Niemann-Pick C1 (NPC1), an integral membrane protein that transports intracellular cholesterol. These results suggest that the endocrine function of the vascular endothelium maintains glucose homeostasis. Of potential translational significance, the delivery of BMPER recombinant protein or its overexpression alleviates insulin resistance and hyperglycemia in high-fat diet-fed mice and Leprdb/db (db/db) diabetic mice. We conclude that BMPER exhibits therapeutic potential for the treatment of diabetes.

Prolyl Hydroxylase Domain-2 Protein Regulates Lipopolysaccharide-Induced Vascular Inflammation.

Acute lung injury and its more severe form, acute respiratory distress syndrome, are life-threatening respiratory disorders. Overwhelming pulmonary inflammation and endothelium disruption are commonly observed. Endothelial cells (ECs) are well recognized as key regulators in leukocyte adhesion and migration in response to bacterial infection. Prolyl hydroxylase domain (PHD)-2 protein, a major PHD in ECs, plays a critical role in intracellular oxygen homeostasis, angiogenesis, and pulmonary hypertension. However, its role in endothelial inflammatory response is unclear. We investigated the role of PHD2 in ECs during endotoxin-induced lung inflammatory responses with EC-specific PHD2 inducible knockout mice. On lipopolysaccharide challenge, PHD2 depletion in ECs attenuates lipopolysaccharide-induced increases of lung vascular permeability, edema, and inflammatory cell infiltration. Moreover, EC-specific PHD2 inducible knockout mice exhibit improved adherens junction integrity and endothelial barrier function. Mechanistically, PHD2 knockdown induces vascular endothelial cadherin in mouse lung microvascular primary endothelial cells. Moreover, PHD2 knockdown can increase hypoxia-inducible factor/vascular endothelial protein tyrosine phosphatase signaling and reactive oxygen species-dependent p38 activation, leading to the induction of vascular endothelial cadherin. Data indicate that PHD2 depletion prevents the formation of leaky vessels and edema by regulating endothelial barrier function. It provides direct in vivo evidence to suggest that PHD2 plays a pivotal role in vascular inflammation. The inhibition of endothelial PHD2 activity may be a new therapeutic strategy for acute inflammatory diseases.

Correction: BMPER Promotes Epithelial-Mesenchymal Transition in the Developing Cardiac Cushions.

[This corrects the article DOI: 10.1371/journal.pone.0139209.].

Emerging Roles of Vascular Endothelium in Metabolic Homeostasis.

Our understanding of the role of the vascular endothelium has evolved over the past 2 decades, with the recognition that it is a dynamically regulated organ and that it plays a nodal role in a variety of physiological and pathological processes. Endothelial cells (ECs) are not only a barrier between the circulation and peripheral tissues, but also actively regulate vascular tone, blood flow, and platelet function. Dysregulation of ECs contributes to pathological conditions such as vascular inflammation, atherosclerosis, hypertension, cardiomyopathy, retinopathy, neuropathy, and cancer. The close anatomic relationship between vascular endothelium and highly vascularized metabolic organs/tissues suggests that the crosstalk between ECs and these organs is vital for both vascular and metabolic homeostasis. Numerous reports support that hyperlipidemia, hyperglycemia, and other metabolic stresses result in endothelial dysfunction and vascular complications. However, how ECs may regulate metabolic homeostasis remains poorly understood. Emerging data suggest that the vascular endothelium plays an unexpected role in the regulation of metabolic homeostasis and that endothelial dysregulation directly contributes to the development of metabolic disorders. Here, we review recent studies about the pivotal role of ECs in glucose and lipid homeostasis. In particular, we introduce the concept that the endothelium adjusts its barrier function to control the transendothelial transport of fatty acids, lipoproteins, LPLs (lipoprotein lipases), glucose, and insulin. In addition, we summarize reports that ECs communicate with metabolic cells through EC-secreted factors and we discuss how endothelial dysregulation contributes directly to the development of obesity, insulin resistance, dyslipidemia, diabetes mellitus, cognitive defects, and fatty liver disease.

Low-Density Lipoprotein Receptor-Related Protein-1 Signaling in Angiogenesis.

Low-density lipoprotein receptor-related protein-1 (LRP1) plays multifunctional roles in lipid homeostasis, signaling transduction, and endocytosis. It has been recognized as an endocytic receptor for many ligands and is involved in the signaling pathways of many growth factors or cytokines. Dysregulation of LRP1-dependent signaling events contributes to the development of pathophysiologic processes such as Alzheimer's disease, atherosclerosis, inflammation, and coagulation. Interestingly, recent studies have linked LRP1 with endothelial function and angiogenesis, which has been underappreciated for a long time. During zebrafish embryonic development, LRP1 is required for the formation of vascular network, especially for the venous development. LRP1 depletion in the mouse embryo proper leads to angiogenic defects and disruption of endothelial integrity. Moreover, in a mouse oxygen-induced retinopathy model, specific depletion of LRP1 in endothelial cells results in abnormal development of neovessels. These loss-of-function studies suggest that LRP1 plays a pivotal role in angiogenesis. The review addresses the recent advances in the roles of LRP1-dependent signaling during angiogenesis.

LRP1-Dependent BMPER Signaling Regulates Lipopolysaccharide-Induced Vascular Inflammation.

OBJECTIVE: Bacterial endotoxin (lipopolysaccharide)-mediated sepsis involves dysregulated systemic inflammation, which injures the lung and other organs, often fatally. Vascular endothelial cells act as both targets and mediators of lipopolysaccharide-induced inflammatory responses. Dysfunction of endothelium results in increases of proinflammatory cytokine production and permeability leakage. BMPER (bone morphogenetic protein-binding endothelial regulator), an extracellular modulator of bone morphogenetic protein signaling, has been identified as a vital component in chronic endothelial inflammatory responses and atherosclerosis. However, it is unclear whether BMPER also regulates inflammatory response in an acute setting such as sepsis. To address this question, we investigated the role of BMPER during lipopolysaccharide-induced acute lung injury. APPROACH AND RESULTS: Mice missing 1 allele of BMPER (BMPER+/- mice used in the place of BMPER-/- mice that die at birth) were used for lipopolysaccharide challenge. Lipopolysaccharide-induced pulmonary inflammation and injury was reduced in BMPER+/- mice as shown by several measures, including survival rate, infiltration of inflammatory cells, edema, and production of proinflammatory cytokines. Mechanistically, we have demonstrated that BMPER is required and sufficient for the activation of nuclear factor of activated T cells c1. This BMPER-induced nuclear factor of activated T cells activation is coordinated by multiple signaling pathways, including bone morphogenetic protein-independent low-density lipoprotein receptor-related protein 1-extracellular signal-regulated kinase activation, calcineurin signaling, and low-density lipoprotein receptor-related protein 1ß-mediated nuclear factor 45 nuclear export in response to BMPER treatment. CONCLUSIONS: We conclude that BMPER plays a pivotal role in pulmonary inflammatory response, which provides new therapeutic options against sepsis shock. The new signaling pathway initiated by BMPER/low-density lipoprotein receptor-related protein 1 axis broadens our understanding about BMPER's role in vascular homeostasis.

Endothelial LRP1 regulates metabolic responses by acting as a co-activator of PPARγ.

Low-density lipoprotein receptor-related protein 1 (LRP1) regulates lipid and glucose metabolism in liver and adipose tissue. It is also involved in central nervous system regulation of food intake and leptin signalling. Here we demonstrate that endothelial Lrp1 regulates systemic energy homeostasis. Mice with endothelial-specific Lrp1 deletion display improved glucose sensitivity and lipid profiles combined with increased oxygen consumption during high-fat-diet-induced obesity. We show that the intracellular domain of Lrp1 interacts with the nuclear receptor Pparγ, a central regulator of lipid and glucose metabolism, acting as its transcriptional co-activator in endothelial cells. Therefore, Lrp1 not only acts as an endocytic receptor but also directly participates in gene transcription. Our findings indicate an underappreciated functional role of endothelium in maintaining systemic energy homeostasis.

Dioxygen and Metabolism; Dangerous Liaisons in Cardiac Function and Disease.

The heart must consume a significant amount of energy to sustain its contractile activity. Although the fuel demands are huge, the stock remains very low. Thus, in order to supply its daily needs, the heart must have amazing adaptive abilities, which are dependent on dioxygen availability. However, in myriad cardiovascular diseases, "fuel" depletion and hypoxia are common features, leading cardiomyocytes to favor low-dioxygen-consuming glycolysis rather than oxidation of fatty acids. This metabolic switch makes it challenging to distinguish causes from consequences in cardiac pathologies. Finally, despite the progress achieved in the past few decades, medical treatments have not improved substantially, either. In such a situation, it seems clear that much remains to be learned about cardiac diseases. Therefore, in this review, we will discuss how reconciling dioxygen availability and cardiac metabolic adaptations may contribute to develop full and innovative strategies from bench to bedside.

LRP1 Regulates Retinal Angiogenesis by Inhibiting PARP-1 Activity and Endothelial Cell Proliferation.

OBJECTIVE: We recently demonstrated that low-density lipoprotein receptor-related protein 1 (LRP1) is required for cardiovascular development in zebrafish. However, what role LRP1 plays in angiogenesis remains to be determined. To better understand the role of LRP1 in endothelial cell function, we investigated how LRP1 regulates mouse retinal angiogenesis. APPROACH AND RESULTS: Depletion of LRP1 in endothelial cells results in increased retinal neovascularization in a mouse model of oxygen-induced retinopathy. Specifically, retinas in mice lacking endothelial LRP1 have more branching points and angiogenic sprouts at the leading edge of the newly formed vasculature. Increased endothelial proliferation as detected by Ki67 staining was observed in LRP1-deleted retinal endothelium in response to hypoxia. Using an array of biochemical and cell biology approaches, we demonstrate that poly(ADP-ribose) polymerase-1 (PARP-1) directly interacts with LRP1 in human retinal microvascular endothelial cells. This interaction between LRP1 and PARP-1 decreases under hypoxic condition. Moreover, LRP1 knockdown results in increased PARP-1 activity and subsequent phosphorylation of both retinoblastoma protein and cyclin-dependent kinase 2, which function to promote cell cycle progression and angiogenesis. CONCLUSIONS: Together, these data reveal a pivotal role for LRP1 in endothelial cell proliferation and retinal neovascularization induced by hypoxia. In addition, we demonstrate for the first time the interaction between LRP1 and PARP-1 and the LRP1-dependent regulation of PARP-1-signaling pathways. These data bring forth the possibility of novel therapeutic approaches for pathological angiogenesis.

The Role of Oxygen Sensors, Hydroxylases, and HIF in Cardiac Function and Disease.

Ischemic heart disease is the leading cause of death worldwide. Oxygen-sensing proteins are critical components of the physiological response to hypoxia and reperfusion injury, but the role of oxygen and oxygen-mediated effects is complex in that they can be cardioprotective or deleterious to the cardiac tissue. Over 200 oxygen-sensing proteins mediate the effects of oxygen tension and use oxygen as a substrate for posttranslational modification of other proteins. Hydroxylases are an essential component of these oxygen-sensing proteins. While a major role of hydroxylases is regulating the transcription factor HIF, we investigate the increasing scope of hydroxylase substrates. This review discusses the importance of oxygen-mediated effects in the heart as well as how the field of oxygen-sensing proteins is expanding, providing a more complete picture into how these enzymes play a multifaceted role in cardiac function and disease. We also review how oxygen-sensing proteins and hydroxylase function could prove to be invaluable in drug design and therapeutic targets for heart disease.

BMPER Promotes Epithelial-Mesenchymal Transition in the Developing Cardiac Cushions.

Formation of the cardiac valves is an essential component of cardiovascular development. Consistent with the role of the bone morphogenetic protein (BMP) signaling pathway in cardiac valve formation, embryos that are deficient for the BMP regulator BMPER (BMP-binding endothelial regulator) display the cardiac valve anomaly mitral valve prolapse. However, how BMPER deficiency leads to this defect is unknown. Based on its expression pattern in the developing cardiac cushions, we hypothesized that BMPER regulates BMP2-mediated signaling, leading to fine-tuned epithelial-mesenchymal transition (EMT) and extracellular matrix deposition. In the BMPER-/- embryo, EMT is dysregulated in the atrioventricular and outflow tract cushions compared with their wild-type counterparts, as indicated by a significant increase of Sox9-positive cells during cushion formation. However, proliferation is not impaired in the developing BMPER-/- valves. In vitro data show that BMPER directly binds BMP2. In cultured endothelial cells, BMPER blocks BMP2-induced Smad activation in a dose-dependent manner. In addition, BMP2 increases the Sox9 protein level, and this increase is inhibited by co-treatment with BMPER. Consistently, in the BMPER-/- embryos, semi-quantitative analysis of Smad activation shows that the canonical BMP pathway is significantly more active in the atrioventricular cushions during EMT. These results indicate that BMPER negatively regulates BMP-induced Smad and Sox9 activity during valve development. Together, these results identify BMPER as a regulator of BMP2-induced cardiac valve development and will contribute to our understanding of valvular defects.

PHD2/3-dependent hydroxylation tunes cardiac response to ß-adrenergic stress via phospholamban.

Ischemic heart disease is the leading cause of heart failure. Both clinical trials and experimental animal studies demonstrate that chronic hypoxia can induce contractile dysfunction even before substantial ventricular damage, implicating a direct role of oxygen in the regulation of cardiac contractile function. Prolyl hydroxylase domain (PHD) proteins are well recognized as oxygen sensors and mediate a wide variety of cellular events by hydroxylating a growing list of protein substrates. Both PHD2 and PHD3 are highly expressed in the heart, yet their functional roles in modulating contractile function remain incompletely understood. Here, we report that combined deletion of Phd2 and Phd3 dramatically decreased expression of phospholamban (PLN), resulted in sustained activation of calcium/calmodulin-activated kinase II (CaMKII), and sensitized mice to chronic ß-adrenergic stress-induced myocardial injury. We have provided evidence that thyroid hormone receptor-α (TR-α), a transcriptional regulator of PLN, interacts with PHD2 and PHD3 and is hydroxylated at 2 proline residues. Inhibition of PHDs increased the interaction between TR-α and nuclear receptor corepressor 2 (NCOR2) and suppressed Pln transcription. Together, these observations provide mechanistic insight into how oxygen directly modulates cardiac contractility and suggest that cardiac function could be modulated therapeutically by tuning PHD enzymatic activity.

Depletion of PHD3 protects heart from ischemia/reperfusion injury by inhibiting cardiomyocyte apoptosis.

PHD3, a member of a family of Prolyl-4 Hydroxylase Domain (PHD) proteins, has long been considered a pro-apoptotic protein. Although the pro-apoptotic effect of PHD3 requires its prolyl hydroxylase activity, it may be independent of HIF-1α, the common substrate of PHDs. PHD3 is highly expressed in the heart, however, its role in cardiomyocyte apoptosis remains unclear. This study was undertaken to determine whether inhibition or depletion of PHD3 inhibits cardiomyocyte apoptosis and attenuates myocardial injury induced by ischemia-reperfusion (I/R). PHD3 knockout mice and littermate controls were subjected to left anterior descending (LAD) coronary artery ligation for 40 min followed by reperfusion. Histochemical analysis using Evan's Blue, triphenyl-tetrazolium chloride and TUNEL staining, demonstrated that myocardial injury and cardiomyocyte apoptosis induced I/R injury were significantly attenuated in PHD3 knockout mice. PHD3 knockout mice exhibited no changes in HIF-1α protein level, the expression of some HIF target genes or the myocardium capillary density at physiological condition. However, depletion of PHD3 further enhanced the induction of HIF-1α protein at hypoxic condition and increased expression of HIF-1α inhibited cardiomyocyte apoptosis induced by hypoxia. In addition, it has been demonstrated that PHD3 plays an important role in ATR/Chk1/p53 pathway. Consistently, a prolyl hydroxylase inhibitor or depletion of PHD3 significantly inhibits the activation of Chk1 and p53 in cardiomyocytes and the subsequent apoptosis induced by doxorubicin, hydrogen peroxide or hypoxia/reoxygenation. Taken together, these data suggest that depletion of PHD3 leads to increased stabilization of HIF-1α and inhibition of DNA damage response, both of which may contribute to the cardioprotective effect seen with depletion of PHD3.

Role of cAMP-phosphodiesterase 1C signaling in regulating growth factor receptor stability, vascular smooth muscle cell growth, migration, and neointimal hyperplasia.

RATIONALE: Neointimal hyperplasia characterized by abnormal accumulation of vascular smooth muscle cells (SMCs) is a hallmark of occlusive disorders such as atherosclerosis, postangioplasty restenosis, vein graft stenosis, and allograft vasculopathy. Cyclic nucleotides are vital in SMC proliferation and migration, which are regulated by cyclic nucleotide phosphodiesterases (PDEs). OBJECTIVE: Our goal is to understand the regulation and function of PDEs in SMC pathogenesis of vascular diseases. METHODS AND RESULTS: We performed screening for genes differentially expressed in normal contractile versus proliferating synthetic SMCs. We observed that PDE1C expression was low in contractile SMCs but drastically elevated in synthetic SMCs in vitro and in various mouse vascular injury models in vivo. In addition, PDE1C was highly induced in neointimal SMCs of human coronary arteries. More importantly, injury-induced neointimal formation was significantly attenuated by PDE1C deficiency or PDE1 inhibition in vivo. PDE1 inhibition suppressed vascular remodeling of human saphenous vein explants ex vivo. In cultured SMCs, PDE1C deficiency or PDE1 inhibition attenuated SMC proliferation and migration. Mechanistic studies revealed that PDE1C plays a critical role in regulating the stability of growth factor receptors, such as PDGF receptor ß (PDGFRß) known to be important in pathological vascular remodeling. PDE1C interacts with low-density lipoprotein receptor-related protein-1 and PDGFRß, thus regulating PDGFRß endocytosis and lysosome-dependent degradation in an low-density lipoprotein receptor-related protein-1-dependent manner. A transmembrane adenylyl cyclase cAMP-dependent protein kinase cascade modulated by PDE1C is critical in regulating PDGFRß degradation. CONCLUSIONS: These findings demonstrated that PDE1C is an important regulator of SMC proliferation, migration, and neointimal hyperplasia, in part through modulating endosome/lysosome-dependent PDGFRß protein degradation via low-density lipoprotein receptor-related protein-1.