Semaphorin 3C exacerbates liver fibrosis.

BACKGROUND AND AIMS: Chronic liver disease is a growing epidemic, leading to fibrosis and cirrhosis. TGF-ß is the pivotal profibrogenic cytokine that activates HSC, yet other molecules can modulate TGF-ß signaling during liver fibrosis. Expression of the axon guidance molecules semaphorins (SEMAs), which signal through plexins and neuropilins (NRPs), have been associated with liver fibrosis in HBV-induced chronic hepatitis. This study aims at determining their function in the regulation of HSCs. APPROACH AND RESULTS: We analyzed publicly available patient databases and liver biopsies. We used transgenic mice, in which genes are deleted only in activated HSCs to perform ex vivo analysis and animal models. SEMA3C is the most enriched member of the semaphorin family in liver samples from patients with cirrhosis. Higher expression of SEMA3C in patients with NASH, alcoholic hepatitis, or HBV-induced hepatitis discriminates those with a more profibrotic transcriptomic profile. SEMA3C expression is also elevated in different mouse models of liver fibrosis and in isolated HSCs on activation. In keeping with this, deletion of SEMA3C in activated HSCs reduces myofibroblast marker expression. Conversely, SEMA3C overexpression exacerbates TGF-ß-mediated myofibroblast activation, as shown by increased SMAD2 phosphorylation and target gene expression. Among SEMA3C receptors, only NRP2 expression is maintained on activation of isolated HSCs. Interestingly, lack of NRP2 in those cells reduces myofibroblast marker expression. Finally, deletion of either SEMA3C or NRP2, specifically in activated HSCs, reduces liver fibrosis in mice. CONCLUSION: SEMA3C is a novel marker for activated HSCs that plays a fundamental role in the acquisition of the myofibroblastic phenotype and liver fibrosis.

Notch Signaling in the Vasculature: Angiogenesis and Angiocrine Functions.

Formation of a functional blood vessel network is a complex process tightly controlled by pro- and antiangiogenic signals released within the local microenvironment or delivered through the bloodstream. Endothelial cells precisely integrate such temporal and spatial changes in extracellular signals and generate an orchestrated response by modulating signaling transduction, gene expression, and metabolism. A key regulator in vessel formation is Notch signaling, which controls endothelial cell specification, proliferation, migration, adhesion, and arteriovenous differentiation. This review summarizes the molecular biology of endothelial Notch signaling and how it controls angiogenesis and maintenance of the established, quiescent vasculature. In addition, recent progress in the understanding of Notch signaling in endothelial cells for controlling organ homeostasis by transcriptional regulation of angiocrine factors and its relevance to disease will be discussed.

Ketone body oxidation increases cardiac endothelial cell proliferation.

Blood vessel formation is dependent on metabolic adaption in endothelial cells. Glucose and fatty acids are essential substrates for ATP and biomass production; however, the metabolism of other substrates remains poorly understood. Ketone bodies are important nutrients for cardiomyocytes during starvation or consumption of carbohydrate-restrictive diets. This raises the question whether cardiac endothelial cells would not only transport ketone bodies but also consume some of these to achieve their metabolic needs. Here, we report that cardiac endothelial cells are able to oxidize ketone bodies and that this enhances cell proliferation, migration, and vessel sprouting. Mechanistically, this requires succinyl-CoA:3-oxoacid-CoA transferase, a key enzyme of ketone body oxidation. Targeted metabolite profiling revealed that carbon from ketone bodies got incorporated into tricarboxylic acid cycle intermediates as well as other metabolites fueling biomass production. Elevation of ketone body levels by a high-fat, low-carbohydrate ketogenic diet transiently increased endothelial cell proliferation in mouse hearts. Notably, in a mouse model of heart hypertrophy, ketogenic diet prevented blood vessel rarefication. This suggests a potential beneficial role of dietary intervention in heart diseases.

The Endothelium: An Active Regulator of Lipid and Glucose Homeostasis.

The vascular endothelium serves as a dynamic barrier that separates blood from interstitia. Endothelial cells (ECs) respond rapidly to changes in the circulation and actively regulate vessel tone, permeability, and platelet functions. ECs also secrete angiocrine factors that dictate the function of adjacent parenchymal cells in an organ-specific manner. Endothelial dysfunction is considered as a hallmark of metabolic diseases. However, there is emerging evidence that ECs modulate the transfer of nutrients and hormones to parenchymal cells in response to alterations in metabolic profile. As such, a causal role for ECs in systemic metabolic dysregulation can be envisaged. This review summarizes recent progress in the understanding of regulated fatty acid, glucose, and insulin transport across the endothelium and discusses its pathophysiological implications.

Notch Signaling in Familial Cerebral Cavernous Malformations and Immunohistochemical Detection of Cleaved Notch1 Intracellular Domain.

Cerebral cavernous malformations (CCM) or cavernomas are slow-flow capillary vascular malformations with a mulberry-like appearance, which are predominantly located in the central nervous system. CCM can occur in a sporadic or a familial form. The latter is inherited in an autosomal dominant manner, and in the majority of the fragile lesions, mutations in the genes CCM1 (KRIT1), CCM2 (OSM), or CCM3 (PDCD10) can be detected. Loss of these genes leads to numerous alterations in endothelial cell signaling resulting in a disturbed vessel architecture and function. Lower activity of Notch signaling occurs upon loss of CCM1, CCM3, or the CCM1-interacting protein ICAP1 in cell culture and animal models. Notch signaling in endothelial cells is an essential regulator of angiogenesis, arterial-venous differentiation, vascular permeability and stability, mural cell recruitment, and flux of metabolites across the vessel wall. The purpose of this chapter is to briefly summarize the current understanding of Notch signaling in familial CCM and to provide a protocol for detecting cleaved Notch1 receptor proteins on paraformaldehyde-fixed paraffin-embedded mouse tissue.

Endothelial Notch signaling controls insulin transport in muscle.

The role of the endothelium is not just limited to acting as an inert barrier for facilitating blood transport. Endothelial cells (ECs), through expression of a repertoire of angiocrine molecules, regulate metabolic demands in an organ-specific manner. Insulin flux across the endothelium to muscle cells is a rate-limiting process influencing insulin-mediated lowering of blood glucose. Here, we demonstrate that Notch signaling in ECs regulates insulin transport to muscle. Notch signaling activity was higher in ECs isolated from obese mice compared to non-obese. Sustained Notch signaling in ECs lowered insulin sensitivity and increased blood glucose levels. On the contrary, EC-specific inhibition of Notch signaling increased insulin sensitivity and improved glucose tolerance and glucose uptake in muscle in a high-fat diet-induced insulin resistance model. This was associated with increased transcription of Cav1, Cav2, and Cavin1, higher number of caveolae in ECs, and insulin uptake rates, as well as increased microvessel density. These data imply that Notch signaling in the endothelium actively controls insulin sensitivity and glucose homeostasis and may therefore represent a therapeutic target for diabetes.

Endothelial Notch signalling limits angiogenesis via control of artery formation.

Angiogenic sprouting needs to be tightly controlled. It has been suggested that the Notch ligand dll4 expressed in leading tip cells restricts angiogenesis by activating Notch signalling in trailing stalk cells. Here, we show using live imaging in zebrafish that activation of Notch signalling is rather required in tip cells. Notch activation initially triggers expression of the chemokine receptor cxcr4a. This allows for proper tip cell migration and connection to the pre-existing arterial circulation, ultimately establishing functional arterial-venous blood flow patterns. Subsequently, Notch signalling reduces cxcr4a expression, thereby preventing excessive blood vessel growth. Finally, we find that Notch signalling is dispensable for limiting blood vessel growth during venous plexus formation that does not generate arteries. Together, these findings link the role of Notch signalling in limiting angiogenesis to its role during artery formation and provide a framework for our understanding of the mechanisms underlying blood vessel network expansion and maturation.

The same but different: signaling pathways in control of endothelial cell migration.

The coordinated migration of endothelial cells (ECs) plays a pivotal role not only in the assembly of the embryonic vasculature, but also during various physiological and pathological processes, such as tissue regeneration and wound healing. Recent reports studying EC migration in distinct vascular beds have revealed common principles, but also surprising differences, in the molecules ECs use to ensure proper migratory behaviors. In addition to genetic cues, hemodynamic forces in perfused blood vessels also affect EC migration, thereby contributing to blood vessel remodeling. In this review, we will discuss the distinct molecules guiding EC migration in different tissues and highlight their modes of action.

Arteries are formed by vein-derived endothelial tip cells.

Tissue vascularization entails the formation of a blood vessel plexus, which remodels into arteries and veins. Here we show, by using time-lapse imaging of zebrafish fin regeneration and genetic lineage tracing of endothelial cells in the mouse retina, that vein-derived endothelial tip cells contribute to emerging arteries. Our movies uncover that arterial-fated tip cells change migration direction and migrate backwards within the expanding vascular plexus. This behaviour critically depends on chemokine receptor cxcr4a function. We show that the relevant Cxcr4a ligand Cxcl12a selectively accumulates in newly forming bone tissue even when ubiquitously overexpressed, pointing towards a tissue-intrinsic mode of chemokine gradient formation. Furthermore, we find that cxcr4a mutant cells can contribute to developing arteries when in association with wild-type cells, suggesting collective migration of endothelial cells. Together, our findings reveal specific cell migratory behaviours in the developing blood vessel plexus and uncover a conserved mode of artery formation.