The loss of cardiac SIRT3 decreases metabolic flexibility and proteostasis in an age-dependent manner.

SIRT3 is a longevity factor that acts as the primary deacetylase in mitochondria. Although ubiquitously expressed, previous global SIRT3 knockout studies have shown primarily a cardiac-specific phenotype. Here, we sought to determine how specifically knocking out SIRT3 in cardiomyocytes (SIRTcKO mice) temporally affects cardiac function and metabolism. Mice displayed an age-dependent increase in cardiac pathology, with 10-month-old mice exhibiting significant loss of systolic function, hypertrophy, and fibrosis. While mitochondrial function was maintained at 10 months, proteomics and metabolic phenotyping indicated SIRT3 hearts had increased reliance on glucose as an energy substrate. Additionally, there was a significant increase in branched-chain amino acids in SIRT3cKO hearts without concurrent increases in mTOR activity. Heavy water labeling experiments demonstrated that, by 3 months of age, there was an increase in protein synthesis that promoted hypertrophic growth with a potential loss of proteostasis in SIRT3cKO hearts. Cumulatively, these data show that the cardiomyocyte-specific loss of SIRT3 results in severe pathology with an accelerated aging phenotype.

Endothelial VWF is critical for the pathogenesis of vaso-occlusive episode in a mouse model of sickle cell disease.

Vaso-occlusive episode (VOE) is a common and critical complication of sickle cell disease (SCD). Its pathogenesis is incompletely understood. von Willebrand factor (VWF), a multimeric plasma hemostatic protein synthesized and secreted by endothelial cells and platelets, is increased during a VOE. However, whether and how VWF contributes to the pathogenesis of VOE is not fully understood. In this study, we found increased VWF levels during tumor necrosis factor (TNF)-induced VOE in a humanized mouse model of SCD. Deletion of endothelial VWF decreased hemolysis, vascular occlusion, and organ damage caused by TNF-induced VOE in SCD mice. Moreover, administering ADAMTS13, the VWF-cleaving plasma protease, reduced plasma VWF levels, decreased inflammation and vaso-occlusion, and alleviated organ damage during VOE. These data suggest that promoting VWF cleavage via ADAMTS13 may be an effective treatment for reducing hemolysis, inflammation, and vaso-occlusion during VOE.

Metabolic flexibility maintains proliferation and migration of FGFR signaling-deficient lymphatic endothelial cells.

Metabolic flexibility is the capacity of cells to alter fuel metabolism in response to changes in metabolic demand or nutrient availability. It is critical for maintaining cellular bioenergetics and is involved in the pathogenesis of cardiovascular disease and metabolic disorders. However, the regulation and function of metabolic flexibility in lymphatic endothelial cells (LECs) remain unclear. We have previously shown that glycolysis is the predominant metabolic pathway to generate ATP in LECs and that fibroblast growth factor receptor (FGFR) signaling controls lymphatic vessel formation by promoting glycolysis. Here, we found that chemical inhibition of FGFR activity or knockdown of FGFR1 induces substantial upregulation of fatty acid ß-oxidation (FAO) while reducing glycolysis and cellular ATP generation in LECs. Interestingly, such compensatory elevation was not observed in glucose oxidation and glutamine oxidation. Mechanistic studies show that FGFR blockade promotes the expression of carnitine palmitoyltransferase 1A (CPT1A), a rate-limiting enzyme of FAO; this is achieved by dampened extracellular signal-regulated protein kinase activation, which in turn upregulates the expression of the peroxisome proliferator-activated receptor alpha. Metabolic analysis further demonstrates that CPT1A depletion decreases total cellular ATP levels in FGFR1-deficient rather than wildtype LECs. This result suggests that FAO, which makes a negligible contribution to cellular energy under normal conditions, can partially compensate for energy deficiency caused by FGFR inhibition. Consequently, CPT1A silencing potentiates the effect of FGFR1 knockdown on impeding LEC proliferation and migration. Collectively, our study identified a key role of metabolic flexibility in modulating the effect of FGFR signaling on LEC growth.

Metabolic Analysis of Lymphatic Endothelial Cells.

Metabolism is pivotal for formation of the lymphatic vasculature. Understanding metabolism in lymphatic endothelial cells (LECs) requires quantitative characterization of specific metabolic pathways. Here we describe methods for using radioactive tracers to assess flux rates of glycolysis, fatty acid ß-oxidation, glucose oxidation, and glutamine oxidation. We also provide a detailed method for utilizing mass spectrometry (MS) to measure glycolytic intermediates and ATP.

Recent advances in understanding lymphangiogenesis and metabolism.

The blood and lymphatic vasculatures are vital to the maintenance of homeostasis. The interaction between two vascular networks throughout the body is precisely controlled to enable oxygen and nutrient delivery, removal of carbon dioxide and metabolic waste, drainage of interstitial fluid, transport of immune cells, and other key activities. Recent years have seen an explosion of information dealing with the development and function of the lymphatic system. The growth of lymphatic vessels, termed lymphangiogenesis, is a high-energy requirement process that involves sprouting, proliferation, migration, and remodeling of lymphatic endothelial cells and capillaries. Although there has been substantial progress in identifying growth factors and their downstream signaling pathways that control lymphangiogenesis, the role of metabolic processes during lymphangiogenesis and their links to growth factor signaling are poorly understood. In this review, we will discuss recent work that has provided new insights into lymphatic metabolism and its role in lymphangiogenesis.

Endothelial Metabolic Control of Lymphangiogenesis.

Lymphangiogenesis is an important developmental process that is critical to regulation of fluid homeostasis, immune surveillance and response as well as pathogenesis of a number of diseases, among them cancer, inflammation, and heart failure. Specification, formation, and maturation of lymphatic blood vessels involves an interplay between a series of events orchestrated by various transcription factors that determine expression of key genes involved in lymphangiogenesis. These are traditionally thought to be under control of several key growth factors including vascular growth factor-C (VEGF-C) and fibroblast growth factors (FGFs). Recent insights into VEGF and FGF signaling point to their role in control of endothelial metabolic processes such as glycolysis and fatty acid oxidation that, in turn, play a major role in regulation of lymphangiogenesis. These advances have significantly increased our understanding of lymphatic biology and opened new therapeutic vistas. Here we review our current understanding of metabolic controls in the lymphatic vasculature.

FGF-dependent metabolic control of vascular development.

Blood and lymphatic vasculatures are intimately involved in tissue oxygenation and fluid homeostasis maintenance. Assembly of these vascular networks involves sprouting, migration and proliferation of endothelial cells. Recent studies have suggested that changes in cellular metabolism are important to these processes. Although much is known about vascular endothelial growth factor (VEGF)-dependent regulation of vascular development and metabolism, little is understood about the role of fibroblast growth factors (FGFs) in this context. Here we identify FGF receptor (FGFR) signalling as a critical regulator of vascular development. This is achieved by FGF-dependent control of c-MYC (MYC) expression that, in turn, regulates expression of the glycolytic enzyme hexokinase 2 (HK2). A decrease in HK2 levels in the absence of FGF signalling inputs results in decreased glycolysis, leading to impaired endothelial cell proliferation and migration. Pan-endothelial- and lymphatic-specific Hk2 knockouts phenocopy blood and/or lymphatic vascular defects seen in Fgfr1/Fgfr3 double mutant mice, while HK2 overexpression partly rescues the defects caused by suppression of FGF signalling. Thus, FGF-dependent regulation of endothelial glycolysis is a pivotal process in developmental and adult vascular growth and development.

Lymphatic fate specification: an ERK-controlled transcriptional program.

Lymphatic vessels are intimately involved in the regulation of water and solute homeostasis by returning interstitial fluid back to the venous circulation and play an equally important role in immune responses by providing avenues for immune cell transport. Defects in the lymphatic vasculature result in a number of pathological conditions, including lymphedema and lymphangiectasia. Knowledge of molecular mechanisms underlying lymphatic development and maintenance is therefore critical for understanding, prevention and treatment of lymphatic circulation-related diseases. Research in the past two decades has uncovered several key transcriptional factors (Prox1, Sox18 and Coup-TFII) controlling lymphatic fate specification. Most recently, ERK signaling has emerged as a critical regulator of this transcriptional program. This review summarizes our current understanding of lymphatic fate determination and its transcriptional controls.

Endothelial nuclear factor-κB-dependent regulation of arteriogenesis and branching.

BACKGROUND: Arteriogenesis and collateral formation are complex processes requiring integration of multiple inputs to coordinate vessel branching, growth, maturation, and network size. Factors regulating these processes have not been determined. METHODS AND RESULTS: We used an inhibitor of NFκB activation (IκBαSR) under control of an endothelial-specific inducible promoter to selectively suppress endothelial nuclear factor-κB activation during development, in the adult vasculature, or in vitro. Inhibition of nuclear factor-κB activation resulted in formation of an excessively branched arterial network that was composed of immature vessels and provided poor distal tissue perfusion. Molecular analysis demonstrated reduced adhesion molecule expression leading to decreased monocyte influx, reduced hypoxia-inducible factor-1α levels, and a marked decrease in δ-like ligand 4 expression with a consequent decrease in Notch signaling. The latter was the principal cause of increased vascular branching as treatment with Jagged-1 peptide reduced the size of the arterial network to baseline levels. CONCLUSIONS: These findings identify nuclear factor-κB as a key regulator of adult and developmental arteriogenesis and collateral formation. Nuclear factor-κB achieves this by regulating hypoxia-inducible factor-1α-dependent expression of vascular endothelial growth factor-A and platelet-derived growth factor-BB, which are necessary for the development and maturation of the arterial collateral network, and by regulating δ-like ligand 4 expression, which in turn determines the size and complexity of the network.

Transient receptor potential canonical channels in angiogenesis and axon guidance.

Wiring of vascular and neural networks requires precise guidance of growing blood vessels and axons, respectively, to reach their targets during development. Both of the processes share common molecular signaling pathways. Transient receptor potential canonical (TRPC) channels are calcium-permeable cation channels and gated via receptor- or store-operated mechanisms. Recent studies have revealed the requirement of TRPC channels in mediating guidance cue-induced calcium influx and their essential roles in regulating axon navigation and angiogenesis. Dissecting TRPC functions in these physiological processes may provide therapeutic implications for suppressing pathological angiogenesis and improving nerve regeneration.

TRPC1 is essential for in vivo angiogenesis in zebrafish.

RATIONALE: Wiring vascular and neural networks are known to share common molecular signaling pathways. Activation of transient receptor potential type C channels (TRPCs) has recently been shown to underlie chemotropic guidance of neural axons. It is thus of interest to examine whether TRPCs are also involved in vascular development. OBJECTIVE: To determine the role of TRPC1 in angiogenesis in vivo during zebrafish development. METHODS AND RESULTS: Knockdown of zebrafish trpc1 by antisense morpholino oligonucleotides severely disrupted angiogenic sprouting of intersegmental vessels (ISVs) in zebrafish larvae. This angiogenic defect was prevented by overexpression of a morpholino oligonucleotide-resistant form of zebrafish trpc1 mRNA. Cell transplantation analysis showed that this requirement of Trpc1 for ISV growth was endothelial cell-autonomous. In vivo time-lapse imaging further revealed that the angiogenic defect was attributable to impairment of filopodia extension, migration, and proliferation of ISV tip cells. Furthermore, Trpc1 acted synergistically with vascular endothelial growth factor A (Vegf-a) in controlling ISV growth, and appeared to be downstream to Vegf-a in controlling angiogenesis, as evidence by the findings that Trpc1 was required for Vegf-a-induced ectopic angiogenesis of subintestinal veins and phosphorylation of extracellular signal-regulated kinase. CONCLUSIONS: These results provide the first in vivo evidence that TRPC1 is essential for angiogenesis, reminiscent of the role of TRPCs in axon guidance. It implicates that TRPC1 may represent a potential target for treating pathological angiogenesis.

Deficiency of smarcal1 causes cell cycle arrest and developmental abnormalities in zebrafish.

Mutations in SMARCAL1 cause Schimke Immuno-Osseous Dysplasia (SIOD), an autosomal recessive multisystem developmental disease characterized by growth retardation, T-cell deficiency, bone marrow failure, anemia and renal failure. SMARCAL1 encodes an ATP-driven annealing helicase. However, the biological function of SMARCAL1 and the molecular basis of SIOD remain largely unclear. In this work, we cloned the zebrafish homologue of the human SMARCAL1 gene and found that smarcal1 regulated cell cycle progression. Morpholino knockdown of smarcal1 in zebrafish recapitulated developmental abnormalities in SIOD patients, including growth retardation, craniofacial abnormality, and haematopoietic and vascular defects. Lack of smarcal1 caused G0/G1 cell cycle arrest and induced cell apoptosis. Furthermore, using Electrophoretic Mobility Shift Assay and reporter assay, we found that SMARCAL1 was transcriptionally inhibited by E2F6, an important cell cycle regulator. Over-expression of E2F6 in zebrafish embryos reduced the expression of smarcal1 mRNA and induced developmental defects similar to those in smarcal1 morphants. These results suggest that SIOD may be caused by defects in cell cycle regulation. Our study provides a model of SIOD and reveals its cellular and molecular bases.