Intussusceptive angiogenesis and its role in vascular morphogenesis, patterning, and remodeling
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- Makanya, A.N., Hlushchuk, R. & Djonov, V.G. Angiogenesis (2009) 12: 113. doi:10.1007/s10456-009-9129-5
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New blood vessels arise initially as blood islands in the process known as vasculogenesis or as new capillary segments produced through angiogenesis. Angiogenesis itself encompasses two broad processes, namely sprouting (SA) and intussusceptive (IA) angiogenesis. Primordial capillary plexuses expand through both SA and IA, but subsequent growth and remodeling are achieved through IA. The latter process proceeds through transluminal tissue pillar formation and subsequent vascular splitting, and the direction taken by the pillars delineates IA into overt phases, namely: intussusceptive microvascular growth, intussusceptive arborization, and intussusceptive branching remodeling. Intussusceptive microvascular growth circumscribes the process of initiation of pillar formation and their subsequent expansion with the result that the capillary surface area is greatly enhanced. In contrast, intussusceptive arborization entails formation of serried pillars that remodel the disorganized vascular meshwork into the typical tree-like arrangement. Optimization of local vascular branching geometry occurs through intussusceptive branching remodeling so that the vasculature is remodeled to meet the local demand. In addition, IA is important in creation of the local organ-specific angioarchitecture. While hemodynamic forces have proven direct effects on IA, with increase in blood flow resulting in initiation of pillars, the preponderant mechanisms are unclear. Molecular control of IA has so far not been unequivocally elucidated but interplay among several factors is probably involved. Future investigations are strongly encouraged to focus on interactions among angiogenic growth factors, angiopoetins, and related receptors.
KeywordsIntussusceptive angiogenesisVascular growthVascular morphogenesisSproutingVascular remodeling
Definition of intussusceptive angiogenesis
Intussusceptive angiogenesis defines the process in which transluminal tissue pillars develop within capillaries, small arteries, and veins and subsequently fuse, thus delineating new vascular entities or resulting in vessel remodeling. The concept of intussusceptive angiogenesis was first floated by Caduff et al.  when they encountered several tiny holes in the vascular casts of developing pulmonary vessels. They postulated the holes to represent spaces for tissue posts that had been inserted into the vascular lumina and that were digested away during tissue corrosion. These authors coined the name ‘intussusceptional angiogenesis’ to describe this process of ‘in-itself’ vascular growth. This terminology was modified to intussusceptive angiogenesis by Burri and Tarek  who demonstrated the tissue posts to be pillars in the vascular lumina of developing vessels by serial sectioning. Such pillars were likened to slipping of a piece of tissue into another one and presence of such pillars is, therefore, the quintessence of intussusceptive angiogenesis .
Mechanisms of intussusceptive angiogenesis
In the chick chorioallantoic membrane (CAM), it has been demonstrated that endothelial cell proliferation declines between days 10 and 11 of incubation, when intussusceptive angiogenesis reaches its peak . Further evidence for the non-proliferative nature of IA was adduced in the developing rat lung when the capillary volume and surface area were shown to increase 35- and 20-fold, respectively [5, 6], with the virtual absence of endothelial cell proliferation . During IA vascular expansion entails thinning and spreading of the existing endothelial cell population , a phenomenon that is also demonstrated in the alveolar epithelial cells of the developing marsupial lung . In the CAM, average endothelial cell thickness is reduced by >50% during IA , plausibly by thinning and spreading of the cells as a result of redistribution of the cytoplasm and cell organelles.
Unambiguous identification of pillars requires specific three-dimensional visualization techniques or even a combination of such techniques. Intravascular casting coupled with serial sectioning for light or transmission electron microscopy and demonstration of pillars or use of confocal laser scanning microscopy avail indubitable evidence for the presence of transluminal pillars [11–13]. A combination of morphological evidence with in vivo observations helps to match time-course events of intussusception with structural alterations (Fig. 1).
Unequivocal evidence for intussusceptive angiogenesis was demonstrated in vivo in developing CAM vessels where pillars were seen to appear as dark spots on blood vessels (Fig. 1). Procurement of the vessel region and subsequent serial sectioning revealed the dark spots to be tissue pillars (Fig. 1) with the archetypical structural characteristics [12, 14]. Three-dimensional methods, such as magnetic resonance imaging (MRI), micro-computer tomography, angiography, and ultrasonography, lack the 1 μm minimum resolution requisite for discerning pillars. Presence of septal tissue between two capillary lumina in single two-dimensional sections does not necessarily represent pillars (however small the septum may be), because such could be just two distinct but closely associated capillaries.
Phases and phenotypes of intussusceptive angiogenesis
Intussusceptive angiogenesis may be divided into three major phases depending on the outcomes or phenotypes accomplished at the end of the processes [11, 14–17]. Despite these disparate phases, the fundamental denominator to all of them is the formation of tissue pillars, the differences being inherent in the direction and arrangement of such pillars and hence the outcome accomplished. The three phases include intussusceptive microvascular growth (IMG), intussusceptive arborization (IAR), and intussusceptive branching remodeling (IBR). In developing organs, the three phases have been seen to initially occur in tandem, becoming contemporaneous later in development. IMG inaugurates the primordial capillary network expansion while IAR adapts the vasculature into the typical vascular tree pattern [11, 12, 18, 19], IBR finally remodels the vasculature to optimum local perfusion requirements. Normally, IA supplants sprouting angiogenesis after the establishment of the basic plexus but remodeling of the vasculature is achieved through IBR [9, 14, 18].
Another type of pruning achieved through leukocytes has been demonstrated in the retina . Leukocytes adhere to the vessels and this leads to Fas-ligand-mediated endothelial cell apoptosis. Whether this has any relation to pillar formation is not clear. Generally, reduction of vascular branches has been referred to as vascular pruning, but it is only on few occasions that IPR per se has been demonstrated [14, 31].
Temporospatial distribution of intussusceptive and sprouting angiogenesis
It is noteworthy that IA occurs only on pre-existing vasculature, formed either through sprouting angiogenesis or through vasculogenesis. As has been shown in several studies, IA is preceded by SA and forms an important part of the vascular remodeling. In the much studied CAM, blood vessels grow by SA in its first phase of development (E5–E7) and in the second phase (E8–E12) they grow mainly by IMG while the final phase grows without a substantial increase in vascular complexity . It was further shown that expansion of the CAM vasculature during the second phase was via IAR while IBR remodeled the vessels during the maturation phase [14, 17].
In the growing rat, the mammary vasculature during the pubertal, adult virgin, and early pregnancy stages grows by sprouting angiogenesis, after which this process gives way to massive intussusceptive angiogenesis . In the ephemeral ovarian follicles, sprouting angiogenesis characterizes the initial phase of development and this is soon supplanted by IA, which expands and remodels the vasculature towards the time of follicular maturation . Similarly, in the evanescent avian mesonephros, SA precedes and is supervened by IA. However, IA is short-lived, starting at E7, soon after the primitive mesonephric plexus is established and continuing to E11, whence obvious signs of vascular degeneration preponderate . In the chick embryo mesonephros, vascular development has a cranio-caudal orientation; sprouting establishes a denser network cranially by stages 29 (E6) and 30 (E7). IA gradually starts to replace SA so that the cranial aspect of the mesonephric vasculature casts has many pillar holes while the caudal part has many sprouts . This shows that the two processes can be contemporaneous in the same organ, albeit spatially discrete. The subsequent phases of vascular remodeling fail to inaugurate in the mesonephros since it starts to degenerate .
In the non-evanescent avian metanephros, SA is the preponderant mode of vessel development up to E13 when this is supervened by IA . Subsequently, the vasculature is expanded through IMG and IAR accomplishes the vascular tree pattern . Characteristic steps of IBR delineate individual glomeruli from large supplying vessels. This is accomplished through formation of pillars in rows along the longitudinal median axis of large vessels destined to form glomeruli. Fusion of such pillars results in two vessels each of which proceeds to form a new glomerulus .
The effect of IA does not become apparent in the embryonic avian lung until about the 15th day of incubation, when there is an angiogenic switch with an upsurge in the number of transluminal pillars . Initially, the vessels develop by sprouting angiogenesis, guided by the air conduits that develop ahead of the vasculature but as soon as the basic network is established, IA starts to delineate the fine supplying and draining vessels at the arterial and venous sides, respectively . In addition, IA is responsible for remodeling the fine capillaries that interlace with air capillaries to form the thin blood–gas barrier characteristic of the avian lung .
In pathological conditions that are characterized by excessive angiogenesis such as psoriasis, rheumatic disease, retinopathy and tumorigenesis, IA is suspected as one of the participating processes but this far overwhelming evidence has only been adduced for tumorigenesis [35, 36]. In a recent study, it has been shown that radiotherapy of tumors or treatment with an inhibitor of VEGF tyrosine kinase results in transient reduction in tumor growth rate with decreased tumor vascularization followed by post-therapy relapse with extensive IA . The switch from SA to IA is a part of the angioadaptive mechanism responsible for the tumor recovery in the early phase after anti-angiogenic treatment or radiotherapy .
Control of intussusceptive angiogenesis
Intussusceptive angiogenesis occurs during embryonic development, in physiological adaptations as may be seen in exercised muscles and also in pathological situations such as tumorigenesis (for details see reviews [3, 8, 15]). The stimuli for this type of angiogenesis are either physiological, such as alterations in blood flow, as occurs during exercise, or may be local biochemical alterations, as may be envisaged in increased expression of genes that transcribe molecules specific for endothelial cell growth. No specific roles of any such molecules, however, have been identified but a few have been seen to be up-regulated during IA.
Blood flow within vessels results in stress, referred to as shear stress. Shear stress may be laminar, thus acting tangentially or parallel to the endothelial surface, or otherwise is oscillatory, also regarded as turbulent (see review by Davies ). Shear stress, defined as the force per unit area of the endothelial luminal surface imparted by flowing blood in capillaries, acts tangentially on the vascular wall. Enhanced laminar shear has been shown to preserve integrity of the walls of vessels, in part through Ets-1 dependent induction of protease inhibitors . In addition, laminar shear stress is known to inhibit tubule formation and migration of endothelial cells by an angiopoietin-2 (Ang-2)-dependent mechanism, which entails down-regulation of Ang-2  and is associated with IA. Laminar shear stress also increases endothelial actin filament bundles , but whether these fibers are important in the endothelial cell movements during pillar formation is unclear. In contrast, turbulent shear stress [41, 42] results in angiogenesis and remodeling of the vessels with an increase in cell proliferation and migration, a process characteristic of sprouting angiogenesis. Oscillatory shear stress results in the production of Ang-2 in endothelial cells and plays a critical role in migration and tubule formation and may be a culprit in diseases with disturbed flow and angiogenesis .
The role of hemodynamics in control of IA was demonstrated by clamping of one of the dichotomous branches of an artery in the developing CAM microvasculature . Increase in blood flow and pressure in the cognate artery resulted in an almost immediate effect on branching morphology with pillars beginning to appear in 15–30 min of clamping and a concomitant reduction in branching angles by about 20% after 40 min . This indicates that alterations in hemodynamics result in an immediate vascular adaptation. Egginton et al.  reported that capillary growth in muscles with increased blood flow occurred through intraluminal splitting, with no sprouting, a mechanism typical of IA. Notably, in the latter case, there was absence of endothelial cell proliferation or breakdown of the basement membrane. Indeed, increase in flow velocity increases shear stress.
Mechanical stretch of vessels may result in expansion, as may occur during increased flow probably providing the substrate for intussusceptive microvascular augmentation. Indeed, capillary network remodeling occurs in response to the mechanical forces of increased shear stress and cell stretch [24, 44]. Stretch behaves much more like oscillatory shear stress and results in up-regulation of MMPs and VEGF with consequent sprouting angiogenesis .
Investigations into molecular control of IA need to take into consideration the mechanisms that entail pillar formation, the archetype of this process. The primeval indicators of pillar initiation are intraluminal endothelial protrusions, discerned as minute shallow depressions on intravascular casts or projections into the vessel lumen in serial sections. Such protrusions are followed by endothelial cell contacts, reorganization of endothelial cell junctions and invasions of the pillar core by myofibroblasts and pericytes, which lay down collagen fibrils [2, 3]. The most important growth factor in angiogenesis is VEGF and is known to support both sprouting and intussusceptive angiogenesis [18, 19, 22]. Other endothelial growth factors implicated in IA include bFGF [18, 19] and PDGF-B .
It has been shown that bFGF up-regulates PDGFR-α and -β expression levels in the newly formed blood vessels and PDGF-AB and -BB act through PDGFR-β to enhance vessel stability . The factors that initiate transluminal pillars are unknown. However, PDGF and Ang-2 are both known to be important in pericyte recruitment [54–56] and may therefore play a role in pillar formation. In knock-out mice lacking angiopoetin-1 (Ang-1) and Tie-2, vessel growth is arrested at an early stage of development and further remodeling does not occur . On injection of a monoclonal antibody against PDGFR-β, the receptor for PDGF-B, in murine neonates completely blocks mural cell recruitment in the developing retinal vessels . PDGF-B promotes pericyte recruitment by stimulating both proliferation and migration of such cells . Angiopoietin-1 cannot initiate angiogenesis; it is constitutively expressed throughout the body and promotes vascular remodeling, maturation, and stabilization of vessels via its Tie-2 receptor . Over-expression of VEGF simultaneously with Ang-1 or Ang-2 results in large vessels and small holes in the casts of capillaries , a sign of intussusceptive angiogenesis. Molecules specifically associated with cell migration such as neuropilin, restin, and midkine are down-regulated during the phase of intussusceptive angiogenesis . To date, no direct evidence linking a specific molecule to intussusceptive angiogenesis has been adduced. However, a synergism between VEGF and Ang-1 is highly suspect. Transgenic mice over-expressing VEGF in the skin have numerous tortuous and leaky capillaries whereas those with Ang-1 over-expression have enlarged but less leaky vessels [60–62]. VEGF is essential for early blood vessel formation and mice deficient in VEGF gene die early during embryogenesis with severe defects in blood vessel formation [63, 64]. In contrast, Ang-1 is necessary for later stages of vessel development, and mice with deficiency die of problems associated with vessel remodeling and maturation . Future studies on molecular control of intussusceptive angiogenesis are persuaded to focus on the interactions between angiogenic growth factors (especially VEGF), angiopoetins and their cognate receptors.
The figures in this article were reproduced and/or modified with the kind permission from the relevant copyright holders. These studies were supported by a Swiss National Science foundation grant to VD.