Normal tissue function depends on adequate supply of oxygen through blood vessels. Understanding how blood vessels form has become a principal, yet challenging, objective of the last decade. Unraveling these mechanisms would offer therapeutic options to ameliorate or perhaps even cure disorders that are now leading causes of mortality. In this millenium, we will be required to answer such questions as: Will it be possible to treat ischemic heart disease by stimulating myocardial angiogenesis, and will it be feasible to cure cancer or inflammatory disorders by suppressing excessive vessel growth? Unfortunately, research on angiogenesis has for too long remained descriptive, mainly because the molecular 'players' were not identified. The recent discovery of candidates able to stimulate or inhibit endothelial cells has stirred a growing interest in using these molecules for therapeutic applications. This overview provides an update on the present understanding of the basic molecular mechanisms of how endothelial and smooth muscle cells interact with each other to form blood vessels, as a basis for design of future (anti)-angiogenic treatments.
Development of an endothelium-lined vasculature
Blood vessels in the embryo form through vasculogenesis; that is, through in situ differentiation of undifferentiated precursor cells (angioblasts) to endothelial cells that assemble into a vascular labyrinth 1 (Fig. ). Historically, the term angiogenesis was first used to describe the growth of endothelial sprouts from preexisting postcapillary venules (Fig. ). More recently, this term has been used to generally denote the growth and remodeling process of the primitive network into a complex network. This involves the enlargement of venules, which sprout or become divided by pillars of periendothelial cells (intussusception) or by transendothelial cell bridges, which then split into individual capillaries (Fig. ). New vessels in the adult arise mainly through angiogenesis, although vasculogenesis also may occur (Fig. ). Because vasculogenesis only leads to an immature, poorly functional vasculature, angiogenesis is a therapeutic goal. As the cellular and molecular mechanisms of angiogenesis differ in various tissues (vessels in psoriatic skin enlarge, but they sprout in ischemic retina), the therapeutic stimulation or inhibition of angiogenesis should be adjusted to the target tissue.
Smooth muscle-endothelial cell interactions
Although endothelial cells have attracted most attention, they alone can initiate, but not complete, angiogenesis; periendothelial cells are essential for vascular maturation (Fig. ). During 'vasular myogenesis', mural cells stabilize nascent vessels by inhibiting endothelial proliferation and migration, and by stimulating production of extracellular matrix (Fig. ). They thereby provide hemostatic control and protect new endothelium-lined vessels against rupture or regression. Indeed, vessels regress more easily as long as they are not covered by smooth muscle cells 2 ; the loss of pericytes around retinal ves-sels in diabetic patients causes aneurysmal dilatation, bleeding and blindness.During the subsequent arteriogenesis, vessels become covered by a muscular coat, thereby endowing blood vessels with viscoelastic and vasomotor properties, necessary to accommodate the changing needs in tissue perfusion (Fig. ). Periendothelial cells also assist endothelial cells in acquiring specialized functions in different vascular beds 3 . Arteriogenesis is recapitulated during the pathological enlargement of preexisting collateral vessels (Fig. ). Therefore, strategies to promote sustainable and functional new blood vessels should not be restricted to the induction of capillary angiogenesis, but should include the stimulation of arteriogenesis. Likewise, the therapeutic regression of 'muscularized' vessels may require strategies other than the inhibition of endothelium-lined vessels.
Vasculogenesis: the formation of a primitive network
Endothelial and hematopoietic cells share a common progenitor (the hemangioblast). In the yolk sac, hemangioblasts form aggregates in which the inner cells develop into hematopoietic precursors and the outer population into endothelial cells (Fig. ). Angioblasts may migrate extensively before in situ differentiation and plexus formation. Vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR) 2 and basic fibroblast growth factor (bFGF) influence angioblast differentiation 4-7 , whereas VEGFR1 suppresses hemangioblast commitment 8 . The molecular mechanisms of how transforming growth factor (TGF)-ฮฒ1 and TGF-ฮฒ receptor 2 affect vasculogenesis remain mostly undetermined 9 . Molecules mediating interactions between endothelial cells and matrix macromolecules, fibronectin or matrix receptors (ฮฑ 5 integrin), also affect vasculogenesis. The ฮฑ v ฮฒ 3 integrin mediates vasculogenesis in avian but not in murine embryo .Little is known about the mechanisms governing endothelial cell fate: Ets-1, Hex, Vezf1, Hox and GATA family members, basic helix-loop-helix factors and their inhibitors of differentiation may be involved 11 . Such molecules may be of therapeutic value, as they could determine the 'decision' of endothelial cells to become angiogenic during pathological conditions (called 'angiogenic switch') . The fate of endothelial cells to become integrated into arteries or veins is mediated by the bHLH transcription factor gridlock at the angioblast stage, and, subsequently, by members of the ephrin family, signals that are also involved in guidance of axons and repulsion of neurons 14 . It was once believed that endothelial precursors only exist during embryonic life. However, endothelial precursor cells have been identified in bone marrow and in peripheral blood in adults. VEGF, granulocyte-monocyte colony-stimulating factor, bFGF and insulin-like growth factor (IGF)-1 stimulate their differentiation and mobilization . Such precursors colonize angiogenic sites and vascular prostheses in the adult and may hold promise for future therapy (Fig. ).
PETER CARMELIET
Endothelial and smooth muscle cells interact with each other to form new blood vessels. In this review, the cellular and molecular mechanisms underlying the formation of endothelium-lined channels (angiogenesis) and their maturation via recruitment of smooth muscle cells (arteriogenesis) during physiological and pathological conditions are summarized, alongside with possible therapeutic applications.
Mechanisms of angiogenesis and arteriogenesis