Requirement of β1 integrin for endothelium-dependent vasodilation and collateral formation in hindlimb ischemia

An acute increase in blood flow triggers flow-mediated dilation (FMD), which is mainly mediated by endothelial nitric oxide synthase (eNOS). A long-term increase in blood flow chronically enlarges the arterial lumen, a process called arteriogenesis. In several common human diseases, these processes are disrupted for as yet unknown reasons. Here, we asked whether β1 integrin, a mechanosensory protein in endothelial cells, is required for FMD and arteriogenesis in the ischemic hindlimb. Permanent ligation of the femoral artery in C57BL/6J mice enlarged pre-existing collateral arteries and increased numbers of arterioles in the thigh. In the lower leg, the numbers of capillaries increased. Notably, injection of β1 integrin-blocking antibody or tamoxifen-induced endothelial cell-specific deletion of the gene for β1 integrin (Itgb1) inhibited both arteriogenesis and angiogenesis. Using high frequency ultrasound, we demonstrated that β1 integrin-blocking antibody or endothelial cell-specific depletion of β1 integrin attenuated FMD of the femoral artery, and blocking of β1 integrin function did not further decrease FMD in eNOS-deficient mice. Our data suggest that endothelial β1 integrin is required for both acute and chronic widening of the arterial lumen in response to hindlimb ischemia, potentially via functional interaction with eNOS

Identification of ILK as a critical regulator of VEGFR3 signalling and lymphatic vascular growth

Vascular endothelial growth factor receptor-3 (VEGFR3) signalling promotes lymphangiogenesis. While there are many reported mechanisms of VEGFR3 activation, there is little understanding of how VEGFR3 signalling is attenuated to prevent lymphatic vascular overgrowth and ensure proper lymph vessel development. Here, we show that endothelial cell-specific depletion of integrin-linked kinase (ILK) in mouse embryos hyper-activates VEGFR3 signalling and leads to overgrowth of the jugular lymph sacs/primordial thoracic ducts, oedema and embryonic lethality. Lymphatic endothelial cell (LEC)-specific deletion of Ilk in adult mice initiates lymphatic vascular expansion in different organs, including cornea, skin and myocardium. Knockdown of ILK in human LECs triggers VEGFR3 tyrosine phosphorylation and proliferation. ILK is further found to impede interactions between VEGFR3 and β1 integrin in vitro and in vivo, and endothelial cell-specific deletion of an Itgb1 allele rescues the excessive lymphatic vascular growth observed upon ILK depletion. Finally, mechanical stimulation disrupts the assembly of ILK and β1 integrin, releasing the integrin to enable its interaction with VEGFR3. Our data suggest that ILK facilitates mechanically regulated VEGFR3 signalling via controlling its interaction with β1 integrin and thus ensures proper development of lymphatic vessels

Sphingolipid subtypes differentially control proinsulin processing and systemic glucose homeostasis

Impaired proinsulin-to-insulin processing in pancreatic β-cells is a key defective step in both type 1 diabetes and type 2 diabetes (T2D) (refs. $^{1}$$^{,}$$^{2}$), but the mechanisms involved remain to be defined. Altered metabolism of sphingolipids (SLs) has been linked to development of obesity, type 1 diabetes and T2D (refs. $^{3-8}$); nonetheless, the role of specific SL species in β-cell function and demise is unclear. Here we define the lipid signature of T2D-associated β-cell failure, including an imbalance of specific very-long-chain SLs and long-chain SLs. β-cell-specific ablation of CerS2, the enzyme necessary for generation of very-long-chain SLs, selectively reduces insulin content, impairs insulin secretion and disturbs systemic glucose tolerance in multiple complementary models. In contrast, ablation of long-chain-SL-synthesizing enzymes has no effect on insulin content. By quantitatively defining the SL-protein interactome, we reveal that CerS2 ablation affects SL binding to several endoplasmic reticulum-Golgi transport proteins, including Tmed2, which we define as an endogenous regulator of the essential proinsulin processing enzyme Pcsk1. Our study uncovers roles for specific SL subtypes and SL-binding proteins in β-cell function and T2D-associated β-cell failure

Ancestral Vascular Lumen Formation via Basal Cell Surfaces

The cardiovascular system of bilaterians developed from a common ancestor. However, no endothelial cells exist in invertebrates demonstrating that primitive cardiovascular tubes do not require this vertebrate-specific cell type in order to form. This raises the question of how cardiovascular tubes form in invertebrates? Here we discovered that in the invertebrate cephalochordate amphioxus, the basement membranes of endoderm and mesoderm line the lumen of the major vessels, namely aorta and heart. During amphioxus development a laminin-containing extracellular matrix (ECM) was found to fill the space between the basal cell surfaces of endoderm and mesoderm along their anterior-posterior (A-P) axes. Blood cells appear in this ECM-filled tubular space, coincident with the development of a vascular lumen. To get insight into the underlying cellular mechanism, we induced vessels in vitro with a cell polarity similar to the vessels of amphioxus. We show that basal cell surfaces can form a vascular lumen filled with ECM, and that phagocytotic blood cells can clear this luminal ECM to generate a patent vascular lumen. Therefore, our experiments suggest a mechanism of blood vessel formation via basal cell surfaces in amphioxus and possibly in other invertebrates that do not have any endothelial cells. In addition, a comparison between amphioxus and mouse shows that endothelial cells physically separate the basement membranes from the vascular lumen, suggesting that endothelial cells create cardiovascular tubes with a cell polarity of epithelial tubes in vertebrates and mammals

Interdependent development of blood vessels and organs.

The cardiovascular system is the first functional organ in the vertebrate embryo, and many organs start to develop adjacent to cells of the cardiovascular system. Endothelial cells (EC) form the inner cell lining of blood vessels and represent the major cell type that interacts with developing organs. On the one hand, EC provide organs with signals. These signals determine the location, differentiation and morphology of an organ. On the other hand, EC receive signals from the organ-specific cell types. Such signals give EC organ-specific features that the organ needs to interact with the circulatory system. This review provides the reader with specific examples of an interdependent development of organs and blood vessels

Islet dynamics, A glimpse at beta cell proliferation

Pancreatic islets consist of 60-80% beta cells, which secrete insulin, a hormone of profound importance in the regulation of carbohydrate, fat and protein metabolism. Beta cell death and/or dysfunction result in an insufficient amount of insulin that leads to high glucose levels in the blood, a metabolic disorder known as Diabetes mellitus. Many studies aiming to establish new therapeutic applications for this disorder are targeted at understanding and manipulating the mechanisms of beta cell proliferation and function. The present comprehensive review summarizes the advances in the field of beta cell renewal and focuses on three fundamental issues: (i) identification of the cellular origins of new beta cells in the adult, (ii) regulation of beta cell proliferation, and (iii) downstream signaling events controlling the cell cycle machinery. Although the source of new adult beta cells is still being debated, recent findings in mice show an important contribution of beta cell proliferation to adult beta cell mass. In conjunction with describing characterized beta cell mitogens and components of the beta cell cycle machinery, we discuss how manipulating the proliferative potential of beta cells could provide novel methods for expanding beta cell mass. Such an expansion could be achieved either through in vitro systems, where functional beta cells could be generated, propagated and further used for transplantation, or in vivo, through directed beta cell renewal from sources in the organism. Once established, these methods would have profound benefits for diabetic patients

Metabolism of Human Diseases: Organ Physiology and Pathophysiology

Metabolism of Human Diseases is directed at advanced students, doctors, and scientists from all categories of life sciences and medicine (e.g., biochemists, biologists, physiologists, pharmacologists, pharmacists, toxicologists, and physicians) with an interest in the metabolism and molecular mechanisms of human diseases, irrespective of their specialization. The book is divided into different parts, each related to a human organ or tissue. Each part begins with an overview chapter presenting the a natomic and physiological properties of the organ or tissue in question relevant for the subsequent disease chapters of the section. The overview introduces organ- or tissue-speci? c metabolism and signaling pathways as well as intra- and inter-organ communication (i.e., “inside-in,” inside-out,” and “outside-in” signaling). The disease chapters discuss pathomechanisms of the diseases with emphasis on metabolic alterations and affected signaling pathways. In addition, they brie? y introduce major treatments currently in use and in clinical trials as well as their in? uence on the patient’s metabolism

Quantitative assessment of angiogenesis and pericyte coverage in human cell-derived vascular sprouts

BACKGROUND: Pericytes, surrounding the endothelium, fulfill diverse functions that are crucial for vascular homeostasis. The loss of pericytes is associated with pathologies, such as diabetic retinopathy and Alzheimer’s disease. Thus, there exists a need for an experimental system that combines pharmacologic manipulation and quantification of pericyte coverage during sprouting angiogenesis. Here, we describe an in vitro angiogenesis assay that develops lumenized vascular sprouts composed of endothelial cells enveloped by pericytes, with the additional ability to comparatively screen the effect of multiple small molecules simultaneously. For automated analysis, we also present an ImageJ plugin tool we developed to quantify sprout morphology and pericyte coverage. METHODS: Human umbilical vein endothelial cells and human brain vascular pericytes were coated on microcarrier beads and embedded in fibrin gels in a 96-well plate to form lumenized vascular sprouts. After treatment with pharmacologic compounds, sprouts were fixed, stained, and imaged via optical z-sections over the area of each well. The maximum intensity projections of these images were stitched together to form montages of the wells, and those montages were processed and analyzed. RESULTS: Vascular sprouts formed within 4–12 days and contained a patent lumen surrounded by a layer of human endothelial cells and pericytes. Using our workflow and image analysis, pericyte coverage after treatment with various compounds was successfully quantified. CONCLUSIONS: Here we present a robust in vitro assay using primary human vascular cells that allows researchers to analyze the effects of multiple compounds on sprouting angiogenesis and pericyte coverage. Our ImageJ plugin offers automated evaluation across multiple different vascular parameters, such as sprout length, cell density, branch points, and pericyte coverage