Barbara Garmy-Susini

Integrins in angiogenesis and lymphangiogenesis

Blood vessels promote tumour growth, and both blood and lymphatic vessels facilitate tumour metastasis by serving as conduits for the transport of tumour cells to new sites. Angiogenesis and lymphangiogenesis are regulated by integrins, which are members of a family of cell surface receptors whose ligands are extracellular matrix proteins and immunoglobulin superfamily molecules. Select integrins promote endothelial cell migration and survival during angiogenesis and lymphangiogenesis, whereas other integrins promote pro-angiogenic macrophage trafficking to tumours. Several integrin-targeted therapeutic agents are currently in clinical trials for cancer therapy. Here, we review the evidence implicating integrins as a family of fundamental regulators of angiogenesis and lymphangiogenesis.

Integrin α4β1–VCAM-1–mediated adhesion between endothelial and mural cells is required for blood vessel maturation

Neovascularization depends on vascular cell proliferation and on the stabilization of vessels by association of vascular smooth muscle-like pericytes with ECs. Here we show that integrin alpha4beta1 (VLA-4) and VCAM-1 promote close intercellular adhesion between ECs and pericytes and that this interaction is required for blood vessel formation. Integrin alpha4beta1 is expressed by proliferating but not quiescent ECs, while its ligand VCAM-1 is expressed by proliferating but not quiescent mural cells. Antagonists of this integrin-ligand pair block the adhesion of mural cells to proliferating endothelia in vitro and in vivo, thereby inducing apoptosis of ECs and pericytes and inhibiting neovascularization. These studies indicate that integrin alpha4beta1 and VCAM-1 facilitate a critical cell-cell adhesion event required for survival of endothelial and mural cells during vascularization.

Integrin α4β1 Signaling Is Required for Lymphangiogenesis and Tumor Metastasis

Recent studies have shown that lymphangiogenesis or the growth of lymphatic vessels at the periphery of tumors promotes tumor metastasis to lymph nodes. We show here that the fibronectin-binding integrin alpha4beta1 and its ligand fibronectin are novel functional markers of proliferative lymphatic endothelium. Tumors and lymphangiogenic growth factors, such as vascular endothelial growth factor-C (VEGF-C) and VEGF-A, induce lymphatic vessel expression of integrin alpha4beta1. Integrin alpha4beta1 then promotes growth factor and tumor-induced lymphangiogenesis, as genetic loss of integrin alpha4beta1 expression in Tie2Cre+ alpha4(loxp/loxp) mice or genetic loss of alpha4 signaling in alpha4Y991A knock-in mice blocks growth factor and tumor-induced lymphangiogenesis, as well as tumor metastasis to lymph nodes. In addition, antagonists of integrin alpha4beta1 suppress lymphangiogenesis and tumor metastasis. Our studies show that integrin alpha4beta1 and the signals it transduces regulate the adhesion, migration, invasion, and survival of proliferating lymphatic endothelial cells. As suppression of alpha4beta1 expression, signal transduction, or function in tumor lymphatic endothelium not only inhibits tumor lymphangiogenesis but also prevents metastatic disease, these results show that integrin alpha4beta1-mediated tumor lymphangiogenesis promotes metastasis and is a useful target for the suppression of metastatic disease.

Integrin α4β1 Promotes Monocyte Trafficking and Angiogenesis in Tumors

Monocytes and macrophages extensively colonize solid tumors, where they are thought to promote tumor angiogenesis. Here, we show that integrin α4β1 (VLA4) promotes the invasion of tumors by myeloid cells and subsequent neovascularization. Antagonists of integrin α4β1, but not of other integrins, blocked the adhesion of monocytes to endothelium in vitro and in vivo as well as their extravasation into tumor tissue. These antagonists prevented monocyte stimulation of angiogenesis in vivo, macrophage colonization of tumors, and tumor angiogenesis. These studies indicate the usefulness of antagonists of integrin α4β1 in suppressing macrophage colonization of tumors and subsequent tumor angiogenesis. These studies further indicate that suppression of myeloid cell homing to tumors could be a useful supplementary approach to suppress tumor angiogenesis and growth. (Cancer Res 2006; 66(4):2146-52)

Roles of integrins in tumor angiogenesis and lymphangiogenesis.

The lifelong dedication of Dr. Judah Folkman to understand how tumors co-opt vasculature to promote tumor growth and spread resulted in the development of an astounding body of knowledge and development of new clinical therapeutics for cancer. Angiogenesis is a critical point in the development and dissemination of most human tumors. Tumor-associated lymphangiogenesis also plays an important role in mediating tumor spread to lymph nodes. The molecular regulations of these processes are complex, and many key molecular families have been implicated in the regulation of angiogenesis and lymphangiogenesis. By regulating cell-cell and cell-matrix contacts, integrins participate in blood and lymphatic vessel growth by promoting endothelial cell migration and survival. Understanding the underlying mechanisms by which integrins promote tumor-associated blood and lymphatic vessel development might provide important modalities for the therapeutic intervention of metastatic spread. This review focuses on the role of integrins in angiogenesis and lymphangiogenesis. Integrins represent potential targets for pharmacological agents and open new avenues for the control of metastatic spread in the treatment of malignancies. This article is dedicated to the memory of Dr. Judah Folkman, an amazing and caring teacher, scientist, physician, and friend.

Fluorescent LYVE-1 antibody to image dynamically lymphatic trafficking of cancer cells in vivo.

BACKGROUND The lymphatic system is a major route for cancer cell dissemination, and a potential target for antitumor therapy. Despite ongoing interest in this area of research, the real-time behavior of cancer cells trafficking in the lymphatic system is poorly understood due to lack of appropriate tools to image this process. MATERIALS AND METHODS We have used monoclonal-antibody and fluorescence technology to color-code lymphatic vessels and the cancer cells inside them in a living animal. Monoclonal anti-mouse LYVE-1 antibody was conjugated to a green fluorophore and delivered to the lymphatic system of a nude mouse, allowing imaging of mouse lymphatics. Tumor cells engineered to express red fluorescent protein were then imaged traveling within the labeled lymphatics in real time. RESULTS AlexaFluor-labeled monoclonal anti-mouse LYVE-1 created a durable signal with clear delineation of lymphatic architecture. The duration of fluorescent signal after conjugated LYVE-1 delivery was far superior to that of fluorescein isothiocyanate-dextran or control fluorophore-conjugated IgG. Tumor cells engineered to express red fluorescent protein delivered to the inguinal lymph node enabled real-time tracking of tumor cell movement within the green fluorescent-labeled lymphatic vessels. CONCLUSIONS This technology offers a powerful tool for the in vivo study of real-time trafficking of tumor cells within lymphatic vessels, for the deposition of the tumor cells in lymph nodes, as well as for screening of potential antitumor lymphatic therapies.

Methods to study lymphatic vessel integrins.

The lymphatic system plays a key role in the drainage of fluids and proteins from tissues and in the trafficking of immune cells throughout the body. Comprised of a network of capillaries, collecting vessels, and lymph nodes, the lymphatic system plays a role in the metastasis of tumor cells to distant parts of the body. Tumors induce lymphangiogenesis, the growth of new lymphatic vessels, in the peritumoral space and also within tumors and lymph nodes. Tumor lymphangiogenesis has been shown to play a role in promoting tumor metastasis. As mediators of lymphatic endothelial cell adhesion, migration, and survival, integrins play key roles in the regulation of lymphangiogenesis. Recent studies indicate that select integrins promote lymphangiogenesis during development and disease and that inhibitors or loss of expression of these integrins can block lymphangiogenesis. In this report, we describe methods to isolate and culture murine and human lymphatic endothelial cells as well as methods to analyze the expression of integrins on these cells. We also show how to assess integrin-mediated adhesion, migration, and tube formation in vitro. We demonstrate how to evaluate integrin function during lymphangiogenesis in a variety of animal models in vivo. Additionally, we show how to study lymphangiogenesis using intravital microscopy.

A PKA-Csk-pp60Src signaling pathway regulates the switch between endothelial cell invasion and cell-cell adhesion during vascular sprouting

Angiogenesis is controlled by signals that stimulate motility in endothelial cells at the tips of vascular sprouts while maintaining cell-cell adhesion in the stalks of angiogenic sprouts. We show here that Gs-linked G protein-coupled receptor activation of cAMP-dependent protein kinase (PKA) plays an important role in regulating the switch between endothelial cell adhesion and migration by activating C-terminal Src kinase, leading to inhibition of pp60Src. Activated PKA blocks pp60Src-dependent vascular endot helial-cadherin phosphorylation, thereby stimulating cell-cell adhesion while suppressing endothelial cell polarization, motility, angiogenesis, and vascular permeability. Similar to the actions of Notch and Dll4, PKA activation blocks sprouting in newly forming embryonic blood vessels, while PKA inhibition promotes excessive sprouting in these vessels. These findings demonstrate that G protein-coupled receptors and PKA regulate vascular sprouting during angiogenesis by controlling endothelial cell migration and cell-cell adhesion through their actions on pp60Src.