6-7: Angiogenesis

Angiogenesis

• Angiogenesis involves the development of blood vessels and depends on growth factors
• During early development, vasculogenesis first forms the vessels
• After vasculogenesis, angiogenesis drives most blood vessel growth during development and in disease

Folkman's Discovery

• In 1971, Folkman found that solid tumor growth needs angiogenesis
• Tumors secrete an unknown "factor" to boost blood supply
• Blocking this factor could cause tumors to die
• Fibroblast growth factor helped uncover angiotensin and VEGF

Factors Promoting Proliferation and Differentiation

• Endothelial cells
• Smooth muscle cells
• Fibroblasts

Extracellular Matrix and Lymphocytes

• Extracellular matrix production and modification support angiogenesis
• Lymphocytes are recruited to sites of inflammation
• Arteries or veins form and junctions/nearby vessels are stabilized

Angioblast Regulation

• Angioblast differentiation and endothelial transdifferentiation are regulated
• Adhesion, migration, proliferation, and apoptosis are involved in angiogenesis

VEGF Signaling

• Vascular endothelial growth factor (VEGF)
• VEGF-A, placenta growth factor (PGF), VEGF-B, VEGF-C and VEGF-D are 5 main proteins

Major Actions of VEGFs

• Stimulation of endothelial cell migration
• Mitosis of endothelial cells
• Matrix metalloproteinase activity
• Creation of blood vessel lumen
• Generation of new junctions (fenestrations)
• Inflammation and vasodilation

VEGFs and Receptor Tyrosine Kinases (RTKs)

• VEGFs bind with high affinity to RTKs VEGFR1-R3
• VEGFR2 is a main signalling VEGFR in blood vascular endothelial cells

VEGFs and Neuropilins

• VEGFs also bind with high affinity to neuropilin (NRP) family members NRP1 and NRP2, as well as heparan sulfate proteoglycans (HSPGs)
• Neuropilins and heparan sulfate proteoglycans are denoted as VEGF co-receptors

Multiprotein Complex Formation

• VEGFs' ability to simultaneously bind to transmembrane proteins initiates multiprotein complexes
• These complexes include receptors, co-receptors and non-VEGF-binding auxiliary proteins like integrins and ephrin B2

Vasculogenesis

• Occurs in the developing mammalian embryo
• Angioblasts differentiate into endothelial cells
• Assembly into a vascular labyrinth

Angiogenesis and Arteriogenesis

• Sprouting ensures expansion of the vascular network
• Angiogenesis leads to arteriogenesis
• In arteriogenesis, endothelial cell channels are covered by pericytes or vascular smooth muscle cells (VSMCs)
• VSMCs provide stability and control blood perfusion

Intussusception

• Pre-existing vessels split to form daughter vessels

Angiogenesis and Tumors

• During tumorigenesis vessel co-option occurs
• Tumor cells hijack the existing vasculature

Angiogenesis and Tumor Cells

• Tumor cells can line vessels, known as vascular mimicry
• Can result in angiogenesis and new blood vessel growth to feed tumour cells and masses
• Mimicry involves the secretion of VEGF

Angiogenesis and Tumor Stem Cells

• Cancer stem-like cells can generate tumor endothelium
• Repair of adult vessels or expansion of pathological vessels can be aided by bone-marrow-derived cells (BMDCs) and/or endothelial progenitor cells
• Progenitor cells are incorporated into the endothelial lining

Sequential Steps of Vessel Branching

• Involves cellular states of quiescence
• Activation
• Resolution

Oxygen Supply and Endothelial Cells

• Vessels supply oxygen
• Endothelial cells are equipped with oxygen sensors and hypoxia-inducible factors

Prolyl Hydroxylase and Hypoxia-Inducible Factors

• Prolyl hydroxylase domain 2 (PHD2)
• Hypoxia-inducible factor-2a (HIF-2a)
• Allow vessels to re-adjust their shape

Events in Angiogenesis

• Upon sensing an angiogenic signal (VEGF, VEGF-C, ANG-2, FGFs, chemokines) released by hypoxic, inflammatory, or tumor cells
• Pericytes first detach from the vessel wall
• Pericytes liberate themselves from the basement membrane via proteolytic degradation by matrix metalloproteinases (MMPs)

VEGF's Role in Permeability

• VEGF increases endothelial cell layer permeability
• Causes plasma proteins to extravasate
• Lay down a provisional extracellular matrix (ECM) scaffold

Integrin Signaling

• Endothelial cells migrate onto the ECM surface in response to integrin signalling
• Proteases liberate angiogenic molecules stored in the ECM (VEGF and FGF)
• The ECM is remodeled into an angio-competent milieu

Tip Cell Selection

• To build a perfused tube and prevent mass endothelial cell movement, an endothelial tip cell is selected
• Tip cell is guided factors like VEGF receptors, neuropilins (NRPs), and NOTCH ligands

VEGF as a Key Orchestrator

• VEGF is the primary activator of tip cells
• VEGF leads vascular remodeling
• Several signaling molecules and pathways collaborate to create a new blood vessel

VEGF Receptors and Tyrosine Kinases

• VEGFs bind with high affinity to receptor tyrosine kinases (RTKs) VEGFR1–R3
• There are ~ 90 RTKs in the human genome

RTK Activation

• Signaling molecules bind to RTKs, cluster neighboring RTKs, and form cross-linked dimers
• Cross-linking activates RTKs through phosphorylation

Cross-Phosphorylation

• Each RTK in the dimer phosphorylates tyrosines on the other RTK
• Cytoplasmic tails of cross-phosphorylated RTKs serve as docking platforms for intracellular proteins in signal transduction

SH2 Domains

• Proteins with SH2 domains bind to phosphorylated tyrosines in RTK receptor tails
• Activated RTKs can bind multiple SH2-containing proteins, allowing simultaneous activation of several intracellular signaling pathways

RTKs Intrinsic Enzyme Activity

• RTKs possess intrinsic enzyme activity unlike other cell surface receptors
• Binding of a signaling molecule activates tyrosine kinase in the cytoplasmic tail
• Enzymatic reactions carry the signal to the nucleus, altering patterns of protein transcription

Clinical Strategies

• Blocking angiogenesis via drugs
• Interfering with ligand binding to the receptor
• Inhibiting PTK kinase activity
• Blocking secondary messengers

Bevacizumab

• An antibody-based biologic drug
• Treats colon cancer, lung cancer, glioblastoma, and renal-cell carcinoma; effective against macular degeneration
• Binds to VEGF, prevents it from binding to VEGF receptors

Pazopanib

• A potent and selective multi-targeted receptor tyrosine kinase (RTK) inhibitor
• Blocks tumor growth and inhibits angiogenesis
• Used to treat renal cancer

Sorafenib

• Used for renal cell carcinoma, hepatocellular carcinoma, and radioactive iodine resistant advanced thyroid carcinoma
• It is a RTK inhibitor
• Inhibits VEGFR cross-phosphorylation

BIBF 1120

• A combined VEGFR, FGFR, and PDGFR inhibitor
• Blocks PTK kinase activity by binding to the ATP binding site
• Under evaluation in clinical trials for non-small cell lung carcinoma and other cancers

Dimerization of RTKs

• Signaling by receptor tyrosine kinases (RTKs) requires dimerization

Receptor Positioning

• Precise positioning of monomeric receptor subunits in the dimer is needed for correct signaling

Role of Ligand Binding

• Ligand binding promotes positioning

Intracellular Domains

• Intracellular tyrosine kinase domains of the receptor complex are activated upon structural rearrangements
• VEGF binding to the receptor dimer

VEGF Receptor Constructs

• The extracellular domain (ECD) of VEGFR-2 has seven immunoglobulin-like domains
• A DNA expression construct for an ECD monomer, and an ECD dimer carrying a GCN4 coiled coil

Receptor-Ligand Complex Isolation

• Receptor proteins are incubated with excess VEGF
• Receptor-ligand complexes are separated from free ligand by gel-filtration chromatography

Static Light Scattering Analysis

• VEGFR-2 ECD complexes have Mr of 270 kDa
• VEGFR-2 ECD GCN4 complexes measure 290 kDa

Complex Formation

• VEGFR-2 ECDs form 2:1 complexes with ligand
• Good model system for dimerization

Electron Microscopy

• Used to gain structural information on VEGFR-2 and its complex with ligand

Unliganded Receptor

• Clearly monomeric
• Chain-like molecules appeared to adopt essentially random conformations

Predimerized Receptor

• Molecules twice as long as the monomeric receptor
• Equally random conformations

Complexes of Predimerized Receptor

• Predimerized receptor with ligand assumed better defined conformations

Class Averages Show

• Receptor chains always held together at both ends

Molecular Contact

• Molecules contact each other at the very tip, the position of immunoglobulin-like domain 7 and the C-terminal coiled-coil domain

Presence of Extra Density

• Receptors are bridged by extra density
• Density is assumed to represent VEGF ligand

VEGF's Role

• Binds immunoglobulin-like domains 2 and 3

Complexes with Monomeric Receptor

• Constructed using monomeric receptor construct
• Micrographs suggest lessened stability relative to complexes of predimerized receptor

C-Terminal Tip

• The interactions occur frequently at receptor's C-terminal
• domain 7 is thought to be represented

Immunoglobulin-Like Domain 7

• Substitutions of domain 7, which is located on the VEGF-2 membrane, show limited activity.
• Likely due to the transmembrane domain and cytoplasmic domain interactions.

VEGF binding increases likelihood

• VEGF must bind to domain 3 & 4 for a second receptor to bind