Boris Strilic

The Molecular Basis of Vascular Lumen Formation in the Developing Mouse Aorta

In vertebrates, endothelial cells (ECs) form blood vessels in every tissue. Here, we investigated vascular lumen formation in the developing aorta, the first and largest arterial blood vessel in all vertebrates. Comprehensive imaging, pharmacological manipulation, and genetic approaches reveal that, in mouse embryos, the aortic lumen develops extracellularly between adjacent ECs. We show that ECs adhere to each other, and that CD34-sialomucins, Moesin, F-actin, and non-muscle Myosin II localize at the endothelial cell-cell contact to define the luminal cell surface. Resultant changes in EC shape lead to lumen formation. Importantly, VE-Cadherin and VEGF-A act at different steps. VE-Cadherin is required for localizing CD34-sialomucins to the endothelial cell-cell contact, a prerequisite to Moesin and F-actin recruitment. In contrast, VEGF-A is required for F-actin-nm-Myosin II interactions and EC shape change. Based on these data, we propose a molecular mechanism of in vivo vascular lumen formation in developing blood vessels.

Platelet-Derived Nucleotides Promote Tumor-Cell Transendothelial Migration and Metastasis via P2Y2 Receptor

Tumor cells can activate platelets, which in turn facilitate tumor cell survival and dissemination. The exact mechanisms by which platelets promote metastasis have remained unclear. Here, we show that adenine nucleotides released from tumor cell-activated platelets induce opening of the endothelial barrier to allow transendothelial migration of tumor cells and thereby promote cancer cell extravasation. We identified the endothelial P2Y2 receptor, which is activated by ATP, as the primary mediator of this effect. Mice deficient in P2Y2 or lacking ATP secretion from platelets show strongly reduced tumor cell metastasis. These findings demonstrate a mechanism by which platelets promote cancer cell metastasis and suggest the P2Y2 receptor and its endothelial downstream signaling mechanisms as a target for antimetastatic therapies.

Mechanoinduction of lymph vessel expansion

In the mammalian embryo, few mechanical signals have been identified to influence organ development and function. Here, we report that an increase in the volume of interstitial or extracellular fluid mechanically induces growth of an organ system, that is, the lymphatic vasculature. We first demonstrate that lymph vessel expansion in the developing mouse embryo correlates with a peak in interstitial fluid pressure and lymphatic endothelial cell (LEC) elongation. In ‘loss‐of‐fluid’ experiments, we then show that aspiration of interstitial fluid reduces the length of LECs, decreases tyrosine phosphorylation of vascular endothelial growth factor receptor‐3 (VEGFR3), and inhibits LEC proliferation. Conversely, in ‘gain‐of‐fluid’ experiments, increasing the amount of interstitial fluid elongates the LECs, and increases both VEGFR3 phosphorylation and LEC proliferation. Finally, we provide genetic evidence that β1 integrins are required for the proliferative response of LECs to both fluid accumulation and cell stretching and, therefore, are necessary for lymphatic vessel expansion and fluid drainage. Thus, we propose a new and physiologically relevant mode of VEGFR3 activation, which is based on mechanotransduction and is essential for normal development and fluid homeostasis in a mammalian embryo.

Tumour-cell-induced endothelial cell necroptosis via death receptor 6 promotes metastasis

Metastasis is the leading cause of cancer-related death in humans. It is a complex multistep process during which individual tumour cells spread primarily through the circulatory system to colonize distant organs. Once in the circulation, tumour cells remain vulnerable, and their metastatic potential largely depends on a rapid and efficient way to escape from the blood stream by passing the endothelial barrier. Evidence has been provided that tumour cell extravasation resembles leukocyte transendothelial migration. However, it remains unclear how tumour cells interact with endothelial cells during extravasation and how these processes are regulated on a molecular level. Here we show that human and murine tumour cells induce programmed necrosis (necroptosis) of endothelial cells, which promotes tumour cell extravasation and metastasis. Treatment of mice with the receptor-interacting serine/threonine-protein kinase 1 (RIPK1)-inhibitor necrostatin-1 or endothelial-cell-specific deletion of RIPK3 reduced tumour-cell-induced endothelial necroptosis, tumour cell extravasation and metastasis. In contrast, pharmacological caspase inhibition or endothelial-cell-specific loss of caspase-8 promoted these processes. We furthermore show in vitro and in vivo that tumour-cell-induced endothelial necroptosis leading to extravasation and metastasis requires amyloid precursor protein expressed by tumour cells and its receptor, death receptor 6 (DR6), on endothelial cells as the primary mediators of these effects. Our data identify a new mechanism underlying tumour cell extravasation and metastasis, and suggest endothelial DR6-mediated necroptotic signalling pathways as targets for anti-metastatic therapies.

P2Y2 and Gq/G11 control blood pressure by mediating endothelial mechanotransduction

Elevated blood pressure is a key risk factor for developing cardiovascular diseases. Blood pressure is largely determined by vasodilatory mediators, such as nitric oxide (NO), that are released from the endothelium in response to fluid shear stress exerted by the flowing blood. Previous work has identified several mechanotransduction signaling processes that are involved in fluid shear stress-induced endothelial effects, but how fluid shear stress initiates the response is poorly understood. Here, we evaluated human and bovine endothelial cells and found that the purinergic receptor P2Y2 and the G proteins Gq/G11 mediate fluid shear stress-induced endothelial responses, including [Ca2+]i transients, activation of the endothelial NO synthase (eNOS), phosphorylation of PECAM-1 and VEGFR-2, as well as activation of SRC and AKT. In response to fluid shear stress, endothelial cells released ATP, which activates the purinergic P2Y2 receptor. Mice with induced endothelium-specific P2Y2 or Gq/G11 deficiency lacked flow-induced vasodilation and developed hypertension that was accompanied by reduced eNOS activation. Together, our data identify P2Y2 and Gq/G11 as a critical endothelial mechanosignaling pathway that is upstream of previously described mechanotransduction processes and demonstrate that P2Y2 and Gq/G11 are required for basal endothelial NO formation, vascular tone, and blood pressure.

Resolving cell-cell junctions: lumen formation in blood vessels.

Formation of a patent vascular lumen is essential for the transport of oxygen, nutrients and waste products to and from tissues. No matter whether the blood vessel arises from vasculogenesis or angiogenesis, endothelial cells (EC) first have to form a cord, which subsequently lumenizes, in order to generate a functional vessel. During these processes, cellular junctions rearrange between adjacent ECs and are involved in EC polarization as a prerequisite for lumen formation. Here we review the role of EC junctions in vascular lumen formation within different vascular beds.

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.

Formation of cardiovascular tubes in invertebrates and vertebrates

The cardiovascular system developed early in evolution and is pivotal for the transport of oxygen, nutrients, and waste products within the organism. It is composed of hollow tubular structures and has a high level of complexity in vertebrates. This complexity is, at least in part, due to the endothelial cell lining of vertebrate blood vessels. However, vascular lumen formation by endothelial cells is still controversially discussed. For example, it has been suggested that the lumen mainly forms via coalescence of large intracellular vacuoles generated by pinocytosis. Alternatively, it was proposed that the vascular lumen initiates extracellularly between adjacent apical endothelial cell surfaces. Here we discuss invertebrate and vertebrate cardiovascular lumen formation and highlight the possible modes of blood vessel formation. Finally, we point to the importance of a better understanding of vascular lumen formation for treating human pathologies, including cancer and coronary heart disease.

Transforming Growth Factor-β–Activated Kinase 1 Regulates Angiogenesis via AMP-Activated Protein Kinase-α1 and Redox Balance in Endothelial Cells

Objective—Transforming growth factor-&bgr;–activated kinase 1 (TAK1) is a mitogen-activated protein 3-kinase and an AMP-activated protein kinase (AMPK) kinase in some cell types. Although TAK1−/− mice display defects in developmental vasculogenesis, the role of TAK1 in endothelial cells has not been investigated in detail. Approach and Results—TAK1 downregulation (small interfering RNA) in human endothelial cells attenuated proliferation without inducing apoptosis and diminished endothelial cell migration, as well as tube formation. Cytokine- and vascular endothelial growth factor (VEGF)–induced endothelial cell sprouting in a modified spheroid assay were abrogated by TAK1 downregulation. Moreover, VEGF–induced endothelial sprouting was impaired in aortic rings from mice lacking TAK1 in endothelial cells (TAK&Dgr;EC). TAK1 inhibition and downregulation also inhibited VEGF–stimulated phosphorylation of several kinases, including AMPK. Proteomic analyses revealed that superoxide dismutase 2 (SOD2) expression was reduced in TAK1-deficient endothelial cells, resulting in attenuated hydrogen peroxide production but increased mitochondrial superoxide production. Endothelial cell SOD2 expression was also attenuated by AMPK inhibition and in endothelial cells from AMPK&agr;1−/− mice but was unaffected by inhibitors of c-Jun N-terminal kinase, p38, extracellular signal–regulated kinase 1/2, or phosphatidylinositol 3-kinase/Akt. Moreover, the impaired endothelial sprouting from TAK&Dgr;EC aortic rings was abrogated in the presence of polyethylene glycol-SOD, and tube formation was normalized by the overexpression of SOD2. A similar rescue of angiogenesis was observed in polyethylene glycol-SOD–treated aortic rings from mice with endothelial cell–specific deletion of the AMPK&agr;1. Conclusions—These results establish TAK1 as an AMPK&agr;1 kinase that regulates vascular endothelial growth factor–induced and cytokine-induced angiogenesis by modulating SOD2 expression and the superoxide anion:hydrogen peroxide balance.

Intravascular Survival and Extravasation of Tumor Cells

Most metastasizing tumor cells reach distant sites by entering the circulatory system. Within the bloodstream, they are exposed to severe stress due to loss of adhesion to extracellular matrix, hemodynamic shear forces, and attacks of the immune system, and only a few cells manage to extravasate and to form metastases. We review the current understanding of the cellular and molecular mechanisms that allow tumor cells to survive in the intravascular environment and that mediate and promote tumor cell extravasation. As these processes are critical for the metastatic spread of tumor cells, we discuss implications for potential therapeutic approaches and future research.