Intussusceptive Microvascular Growth Research Articles

Vascular morphogenesis and remodeling in a human tumor xenograft: blood vessel formation and growth after ovariectomy and tumor implantation.

To determine mechanisms of blood vessel formation and growth in solid tumors, we used a model in which LS174T human colon adenocarcinomas are grown in the isolated ovarian pedicle of nude mice. Reconstruction of 3500 histological serial sections demonstrated that a new vascular network composed of venous-venous loops of varying sizes grows inside the tumor from the wall of the adjacent main vein. Loops elongate and remodel to establish complex loop systems. The mechanisms of loop formation and remodeling correspond to intussusceptive microvascular growth (IMG). In the tissue surrounding the tumor segmentation, another mechanism of IMG is prevalent in venous vessels. Comparison to vascular morphogenesis in the ovariectomized pedicle not only confirms the existence of corresponding mechanisms in both systems, but also reveals numerous sprouts that are superimposed onto loop systems and pathological deviations of loop formation, remodeling, and segmentation in the tumor. These pathological mechanisms interfere with vessel patency that likely cause heterogenous perfusion and hypoxia thus perpetuating angiogenesis. Blood vessel formation based on IMG was also detected in a large thrombus that completely occluded a part of an ovarian artery branch.

Intussusceptive arborization contributes to vascular tree formation in the chick chorio-allantoic membrane.

Various reports indicate that the process of intussusceptive microvascular growth (IMG) plays a crucial role in capillary network formation of the chorio-allantoic membrane (CAM). In the present study we demonstrate by methylmethacrylate (Mercox) casting and in vivo time-lapse observations that intussusception, i.e. insertion of transcapillary tissue pillars, is also strongly involved in vascular tree formation, a process we refer to as intussusceptive arborization (IAR). From day 7 to day 14 of incubation, several arterial and venous branching generations arise from the capillary plexus. The process is initiated by pillar formation in rows, which are demarcating future large vessels in the capillary meshwork. In a subsequent step the pillars undergo reshaping to form narrow tissue septa that successively merge, which results in the production of new generations of blood vessels. This is followed by growth and maturation of all vascular components. The process of IAR in the CAM is very active at days 10 and 11 of incubation and takes place in preferentially perfused capillary regions determining "dynamic areas". The process of intussusception may be preceded by endothelial division, but the transcapillary pillar formation itself occurs primarily by rearrangement and attenuation of the endothelial cells without local endothelial cell proliferation. We conclude that after the early sprouting phase, the process of intussusception is the basic mechanism of CAM vascularization. It leads to capillary network growth and expansion (IMG) and, at the same time to feed vessel formation with several branching generations (IAR).

Intussusceptive microvascular growth in the lung of larval Xenopus laevis Daudin: a light microscope, transmission electron microscope and SEM study of microvascular corrosion casts.

The remodeling of the uniform wide, plexus-like capillary bed of the lung of metamorphosing tadpoles of the South African clawed toad Xenopus laevis (Daudin) is studied from developmental stages 54 to 65 by scanning electron microscopy (SEM) of microvascular corrosion casts (VCCs), light microscopy (LM) and transmission electron microscopy (TEM). VCCs reveal that the remodeling of the existing uniform, plexus-like lung capillary bed into well-defined alveolar capillary meshworks starts in the caudal lung and then gradually proceeds cranially. Vascular remodeling is entirely by intussusceptive microvascular growth through insertion and enlargement of new and fusion of pre-existing capillary meshes. Analyses of lung tissue serial sections at the LM and TEM level confirm the presence of intracapillary cushions and tissue posts and correlate these structures in respect of size and location to the round to slit-like imprints and tiny "holes" found in VCCs. Additionally, SEM of VCCs give clear evidence that intussusceptive microvascular growth is also involved in the remodeling and maturation of alveolar arterioles and venules.

Intussusceptive angiogenesis: its role in embryonic vascular network formation.

Intussusceptive angiogenesis is a novel mode of blood vessel formation and remodeling, which occurs by internal division of the preexisting capillary plexus without sprouting. In this study, the process is demonstrated in developing chicken eye vasculature and in the chorioallantoic membrane by methylmethacrylate (Mercox) casting, transmission electron microscopy, and in vivo observation. In a first step of intussusceptive angiogenesis, the capillary plexus expands by insertion of numerous transcapillary tissue pillars, ie, by intussusceptive microvascular growth. In a subsequent step, a vascular tree arises from the primitive capillary plexus as a result of intussusceptive pillar formation and pillar fusions, a process we termed "intussusceptive arborization." On the basis of the morphological observations, a 4-step model for intussusceptive arborization is proposed, as follows: phase I, numerous circular pillars are formed in rows, thus demarcating future vessels; phase II, formation of narrow tissue septa by pillar reshaping and pillar fusions; phase III, delineation, segregation, growth, and extraction of the new vascular entity by merging of septa; and phase IV, formation of new branching generations by successively repeating the process, complemented by growth and maturation of all components. In contrast to sprouting, intussusceptive angiogenesis does not require intense local endothelial cell proliferation; it is implemented primarily by rearrangement and attenuation of the endothelial cell plates. In summary, transcapillary pillar formation, ie, intussusception, is a central and probably widespread process, which plays a role not only in capillary network growth and expansion (intussusceptive microvascular growth), but also in vascular plexus remodeling and tree formation (intussusceptive arborization).

Vasculogenesis and angiogenesis as mechanisms of vascular network formation, growth and remodeling.

Two distinct mechanisms, vasculogenesis and angiogenesis implement the formation of the vascular network in the embryo. Vasculogenesis gives rise to the heart and the first primitive vascular plexus inside the embryo and in its surrounding membranes, as the yolk sac circulation. Angiogenesis is responsible for the remodeling and expansion of this network. While vasculogenesis refers to in situ differentiation and growth of blood vessels from mesodermal derived hemangioblasts, angiogenesis comprises two different mechanisms: endothelial sprouting and intussusceptive microvascular growth (IMG). The sprouting process is based on endothelial cell migration, proliferation and tube formation. IMG divides existing vessel lumens by formation and insertion of tissue folds and columns of interstitial tissue into the vessel lumen. The latter are termed interstitial or inter-vascular tissue structures (ITSs) and tissue pillars or posts. Intussusception also includes the establishment of new vessels by in situ loop formation in the wall of large veins. The molecular regulation of these distinct mechanisms is discussed in respect to the most important positive regulators, vascular endothelial growth factor (VEGF) and its receptors flk-1 (KDR) and flt-1, the Angiopoietin/tie system and the ephrin-B/EpH-B system. The cellular mechanisms and the molecular regulation of angiogenesis in the pathological state are summarized and the differences of physiological and pathological angiogenesis elaborated.

Angiogenesis and angioarchitecture of transplanted fetal porcine islet-like cell clusters.

Normoglycemic, athymic nude mice were implanted with 3 microl (approximately 250) fetal, porcine islet-like cell clusters under the renal capsule. The angioarchitecture of the transplanted islets was studied by microvascular corrosion casts 3 or 52 weeks after implantation. Arterioles were few, and observed mainly in the older age group. This is likely to be due to the fact that the arterioles were derived from intrarenal blood vessels, i.e., they were not visible on the graft surface. Within the grafts nests of capillaries, probably supplying a single islet-like cell clusters, could be seen in both groups. Numerous capillary sprouts were seen within the graft after 3 weeks, and to a slighter extent also after 1 year. Moreover, especially in grafts examined 3 weeks, but also 52 weeks, after transplantation, holes were observed in dilated capillary segments, suggesting that intussusceptive microvascular growth occurred in parallel with angiogenesis. A well-developed microvasculature could be observed 52 weeks after transplantation, whereas the number of capillaries in the implant was less pronounced 3 weeks postimplantation. The efferent venules were located peripherally in the islets and drained immediately into larger veins, derived from capsular veins clearly seen on the surface of the graft. It is concluded that xenotransplanted islet-like cell clusters develop an autonomous microcirculation by stimulating angiogenesis from surrounding blood vessels. Our findings suggest that single islet-like cell clusters remain morphologically intact after transplantation, and probably function as single endocrine units rather than forming a single homogenous endocrine tissue. Furthermore, it seems as if a continuous reorganization of the vasculature, with an associated angiogenesis, occurs throughout the observation period.

TIE1 and TIE2 Receptor Tyrosine Kinases Inversely Regulate Embryonic Angiogenesis by the Mechanism of Intussusceptive Microvascular Growth

As shown previously, TIE1 and TIE2 receptor tyrosine kinases are specifically expressed in endothelial cells during embryonic angiogenesis. A detailed analysis of the vascular malformations of homozygous mice for a targeting mutation of both receptors was performed at the histological and cellular level. The data demonstrate that the TIE1 and TIE2 receptor inversely and concomitantly mediate interactions between endothelial cells with their extracellular matrix and with surrounding mesenchymal cells. These interactions are obviously crucial for normal endothelial cell motility and/or attachment and also for recruitment of periendothelial cells. The analysis of the TIE2-deficient embryos demonstrates how these cell/cell- and cell/matrix interactions subsequently influence the formation of normally structured tissue folds that divide the vessel lumen. They are also essential for the formation of vessel loops that compose a new vascular network and for the development of the ventricle in the heart. Fold and loop formation follow the principles of intussusceptive microvascular growth. The localization of the cardiovascular malformations corresponds to the temporal and spatial expression pattern of the TIE2 receptor. Angiopoietin-1, a ligand that activates the TIE2 receptor, is expressed in mesenchymal cells surrounding the endothelium. This local relationship is indicative of a paracrine regulation.

Quantitative Study of Intussusceptive Capillary Growth in the Chorioallantoic Membrane (CAM) of the Chicken Embryo

In an attempt to sort out the respective contributions of sprouting and intussusceptive microvascular growth (IMG) during chicken chorioallantoic membrane (CAM) development, we analyzed the morphology and the quantitative growth of the capillary bed of the CAM by light microscopy. By perfusing the CAM microvasculature with highly concentrated colloidal gold particles, the capillaries could be unambiguously distinguished from the surrounding unlabelled tissue. This allowed us to identify, count and measure the intercapillary tissue profiles. By means of morphometric analysis we could show that CAM angiogenesis undergoes three phases of development. In an early phase, from Day 5 to Day 7, the major mechanism of capillary network growth is sprouting. In an intermediate phase, from Day 8 to Day 12, IMG is prevailing, and at Days 13 and 14, CAM structure is undergoing expansion with only a small increase in complexity. These findings are important in view of experimental protocols using the CAM as a model for testing angiogenetic factors. Indeed, care has to be taken not to misinterpret normal age-dependent alterations of the CAM vascular architecture as specific responses to tested agents.

Implementation of Intussusceptive Microvascular Growth in the Chicken Chorioallantoic Membrane (CAM): 2. Pillar Formation by Capillary Fusion

Intussusceptive microvascular growth (IMG) is a new mechanism of capillary growth: The vascular network expands by insertion of newly formed columns of interstitial tissue (interstitial tissue structures) into the vascular lumen called tissue pillars or posts (diameter: 0.5–2.5 μm). IMG has so far been described during organ development and growth and in tumor angiogenesis. Different modes of its implementation could be demonstrated in the rat lung and the chicken chorioallantoic membrane (CAM). In the present investigation a further mechanism of IMG is reported in the chicken CAM: tissue pillars form by splitting of larger interstitial tissue structures and intercapillary walls located between neighboring capillary segments which will consecutively fuse. Splitting is dependent on the existence of a pillar's core composed of a bundle of collagen fibrils ensheathed by extensions of endothelial-like cells inside these structures. Pillar cores thus represent the smallest unit of interstitial tissue around which the vascular lumen might expand. This mode of IMG is obviously connected to physiological remodeling of the capillary network and appears to be dominant during later stages of CAM development.

Microvascular growth in the chicken chorio-allantoic membrane.

The chorio-allantoic membrane (CAM) plays a vital role providing the chicken embryo with O2. Its growth and in particular the growth of its capillary network has important implications for the development of the embryo. And yet, it still remains to be determined to what extent the capillary growth is affected on the one hand via vessel sprouting, and on the other hand by the insertion of transcapillary tissue pillars (posts) to form new intercapillary meshes (intussusceptive microvascular growth, IMG).

VEGF121Induces Proliferation of Vascular Endothelial Cells and Expression offlk-1without Affecting Lymphatic Vessels of the Chorioallantoic Membrane

We have studied the effect of VEGF121homodimer and VEGF121/165heterodimer on the chorioallantoic membrane (CAM) of 13-day-old chick embryos. The factors were applied in doses of 2–4 μg and the effects were evaluated macroscopically after 2 and 3 days. Histological studies were performed on semi- and ultrathin sections. Proliferation was studied according to the BrdU–anti-BrdU method on whole mounts and sections. The labeling density was quantified in whole mounts. The fractal dimension,D,of the vascular tree was assessed as a value for vascular bifurcation density. Both forms of VEGF induce brush-like vessel formation in the precapillary region. New capillaries are found in the stroma of the CAM, which normally does not contain capillaries. Our results show that VEGF121is a specific endothelial cell mitogen. A fourfold increase of BrdU-labeled endothelial cells is found after VEGF121application. The fractal dimension of the vascular tree increases from 1.26 in the controls to 1.44 (VEGF121) and 1.41 (VEGF121/165). The endothelial cells of the newly formed capillaries possess many mitochondria and micropinocytotic vesicles, but no fenestrations. These capillaries are obviously formed by intussusceptive microvascular growth. Signs of sprouting are almost absent. An effect on the lymphatic vessels of the CAM is not detectable. Compared to VEGF165and VEGF121/165, VEGF121diffuses over a slightly greater distance. Usingin situhybridization, VEGF receptor-2 (flk-1/Quek1) and the homologousflt-4(Quek2) receptor were studied in the CAM of normal quail embryos and after VEGF121application on the CAM of 11-day-old quail embryos. During normal development,flk-1expression becomes restricted to vascular endothelial cells of large vessels in the stroma of the CAM. VEGF121application induces expression offlk-1in capillaries that normally do not express the receptor. In the normal development of the CAM,flt-4becomes restricted to endothelial cells of vessels that appear to be lymphatic vessels. Application of VEGF121does not alterflt-4expression.

Intussusceptive Microvascular Growth in a Human Colon Adenocarcinoma Xenograft: A Novel Mechanism of Tumor Angiogenesis

Intussusceptive microvascular growth refers to vascular network formation by insertion of interstitial tissue columns, called tissue pillars or posts, into the vascular lumen and subsequent growth of these columns, resulting in partitioning of the vessel lumen. While intussusception has been reported in normal developing organs, its existence in solid tumors has not been previously documented. By observing the growth of the human colon adenocarcinoma (LS174T)in vivofor a period of 6 weeks, we demonstrate that intussusception is an important mechanism of tumor angiogenesis. At the leading edge of the tumor, vascular growth was found to occur by both intussusception and endothelial sprouting. In the stabilized regions, intussusception led to network remodeling and occlusion of vascular segments. The formation of some tissue pillars appears to depend on intravascular blood-flow patterns or changes in intravascular shear stress. The rapid vascular remodeling by intussusception could possibly contribute to intermittent blood flow in tumors.

Implementation of Intussusceptive Microvascular Growth in the Chicken Chorioallantoic Membrane (CAM):: 1. Pillar Formation by Folding of the Capillary Wall

Intussusceptive microvascular growth is a new mode of capillary network growth originally described in the lungs of rabbits and rats. It constitutes an alternative to endothelial sprouting. The capillary network grows by insertion of new intercapillary meshes with dimensions around 1.5 μm called tissue pillars or posts. In a recent investigation, growth by intussusception was demonstrated in the chicken chorioallantoic membrane (CAM). In the present study the first of several modes of its implementation can now be presented in the CAM byin vivovideo microscopy and analyses of light and electron microscopic serial sections: Cores of tissue pillars containing collagen fibrils ensheathed by extensions of endothelial-like cells will form within the tips of vertically running tissue folds that project into the capillary lumen. Due to retraction of tissue toward the intercapillary space the fold is thinning. Finally, the pillar's core is connected to its fold by a very slender extension of a single endothelial cell. Cell membrane fusion within that slender membrane-like structure causes subsequent separation of the pillar from its fold throughout an increasing vertical distance. This mechanism allows for expansion of the capillary network into the surrounding tissue, leaving behind tissue pillars as remnants of folds.

Evidence for intussusceptive capillary growth in the chicken chorio-allantoic membrane (CAM)

The aim of our investigations was to test whether the chicken chorio-allantoic membrane (CAM) could be an adequate in vivo model for a new mode of capillary growth, originally described in the rat lung and termed intussusceptive microvascular growth. According to that concept the capillary system does not grow by sprouting of vessels, but expands by insertion of transcapillary tissue pillars or posts which form new intercapillary meshes. In the present study, we observed slender transcapillary tissue pillars with diameters around 1 microns in the CAM by in vivo microscopy, and analyzed their ultrastructure by transmission electron microscopic investigation of serial sections. The pillars corresponded in size to those previously described in rat lung microvasculature. On day 7, the pillar core contained endothelial-, endothelial-like cells and collagen fibers, and on day 12 additionally chorionic epithelial cells. As a hypothesis we propose that slender cytoplasmic extensions of endothelial cells, heavily interdigitated in the post area and often projecting into the vascular lumen, could initiate the first step of pillar formation, i.e., interconnect opposite capillary walls. During both stages of development endothelial-like cells were observed in close relationship with the pillars. These cells seem to be relevant for tissue post completion and growth, as they were found to invade the core of the pillars. From the localization of the interendothelial junctions in the post region, a certain similarity to the concept proposed for the lung can be found. The observations confirm that the CAM is a very suitable material for the in vivo investigation of intussusceptive capillary growth.

Intussusceptive microvascular growth, a new mechanism of capillary network formation.

Growth by intussusception is defined as growth by deposition of new particles or pieces of formative material among those already embodied in a tissue or structure. In the context of capillary growth the term stands for the extension of the capillary system by the insertion of new capillary meshes within the existing network. New meshes arise as slender transcapillary tissue pillars. These are formed initially by a circumscribed fusion of opposite endothelial leaflets. Following reorganization of the junctional complexes, the pillar is invaded by interstitial tissue. This means that for the formation of new capillaries no sprouting of endothelial cords or tubules is required. The mechanism is described in the growing lung, but may occur in other tissues too. There may be a chance for in vivo observation of the process in the capillaries of the chorio-allantoic membrane of the chicken.

Intussusceptive Microvascular Growth: A Common Alternative to Capillary Sprouting.

Intussusceptive capillary growth represents a new principle for microvascular growth as described in the lungs of growing rats. According to this concept, the capillary network expands by the formation of slender transcapillary tissue pillars, which give rise to new vascular meshes. The process was first observed in Mercox casts of the lung microvasculature, which revealed the existence of multiple tiny holes with diameters around 1.5 microns. Consecutive transmission electron microscopic investigation of serial sections demonstrated that the holes corresponded to slender tissue pillars (Burri and Tarek, 1990). The corrosion cast technique thus appears to be an adequate screening method for intussusceptive growth. In the present investigation, Mercox casts of various vascular systems, namely, those of the eye, submandibular gland, heart, liver, stomach, small and large intestine, trachea, kidney, uterus and ovary were prepared from rats aged between 4 and 9 weeks in order to screen them for the existence of the typical tiny holes representing tissue pillars. In all organs investigated, these structures were observed in various locations to a variable degree. They were mainly encountered within dilated vascular segments or at triple or quadruple branching points of the circulation. Even in capillary networks with a three-dimensional arrangement could these pillars be detected. Intussusception thus appears to be a principle of growth appertaining to many vascular systems.

A novel mechanism of capillary growth in the rat pulmonary microcirculation

Postnatally, the rat lung parenchyma undergoes impressive growth. Within four months of birth, lung volume and alveolar and capillary surface areas increase over 20-fold and capillary volume 35-fold. Investigation of methacrylate casts of the pulmonary microvasculature revealed that, with age, lung capillaries were not only growing in surface and volume but also increasing their network density. We proposed that the capillary bed grows by formation of slender intravascular tissue pillars and termed this type of growth intussusceptive microvascular growth (Caduff et al., Anat. Rec., 216:154-164, 1986). The aim of this investigation was to detect the presence and to analyze the ultrastructure of slender tissue posts (diameter 1-2.5 microns) extending across the capillary lumina in serial electron microscopic sections of rat lung parenchyma (age 44 days). Computer-assisted three-dimensional reconstruction of the capillary lumen confirmed that tissue posts were matching the holes previously observed in casts. Post ultrastructure varied with size from a simple area of interendothelial contact to tissue pillars with a core of interstitial tissue. Based on the changing morphology of the pillars, a hypothesis for their development can be proposed: phase I, creation of a zone of contact between opposite capillary walls (formation of an interendothelial bridge); phase II, reorganization of the intercellular junctions of the endothelium, with central perforation of the capillary layer; phase III, formation of an interstitial post core, with successive invasion by cytoplasmic extensions of myofibroblasts, pericytes, and finally interstitial fibers; and phase IV, growth of the slender pillar to a normal full size capillary mesh. These findings support the new concept of intussusceptive growth of the lung capillary system.