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 angiogenesis: its emergence, its characteristics, and its significance.

This review shall familiarize the reader with the various aspects of intussusceptive angiogenesis (IA). The basic event in IA is the formation of transvascular tissue pillars. Depending on location, timing, and frequency of pillar emergence, the IA process has different outcomes. In capillaries, a primary IA function is to expand the capillary bed in size and complexity (intussusceptive microvascular growth). It represents an alternative to capillary sprouting. Highly ordered pillar formation in a developing capillary network leads to the formation of vascular trees (intussusceptive arborization). In small arteries and veins, pillar formation at the vessels' branching angles leads either to remodeling of the branching geometry or even to vascular pruning (intussusceptive branching remodeling). It appears essential that future angiogenic research considers always both phenomena, sprouting and intussusception. Vascularization of tissues, organs, and tumors rely heavily on both mechanisms; neglecting one or the other would obscure our understanding of the angiogenesis process.     

Intussusceptive lymphangiogenesis in vascular transformation of lymph node sinuses

Numerous lymphatic anastomosing channels in the lymph nodes are the most demonstrative finding of the rare lesion termed “vascular transformation of lymph node sinuses” (VTS). The mechanism of lymphatic vessel formation in VTS has not been studied. Vessel intussusception contributes to vascular expansion, and intraluminal pillars/posts, interstitial tissue structures or larger pillars (ITSs) and folds are the hallmarks of this process in blood vessels. The aim of this work is to assess whether these hallmarks of intussusception occur in VTS lymphatic vessels, indicating intussusceptive lymphangiogenesis. For this purpose, specimens of five cases of VTS were used for serial histological sections, immunohistochemistry and immunofluorescence in confocal microscopy, which enabled us to demonstrate the 3D image that defines the pillars. The studies showed a) meshworks of lymphatic vessels, which form complex loops, resembling sinuses of lymph nodes, b) presence of intralymphatic pillars, ITSs and folds, with a cover of lymphatic endothelial cells expressing podoplanin and a varying-sized connective core (e.g. collagen), and c) increase of vessel meshwork and linear arrangement, splitting and fusion of ITSs, pillars and folds, with remodelling and segmentation. In conclusion, the development of lymphatic vessel loops, ITSs, pillars and folds with segmentation in VTS supports intussusceptive lymphangiogenesis. This mechanism of intussusception is of interest because it participates in VTS histogenesis, contributes to general knowledge of intussusceptive lymphangiogenesis, which has received less attention than intussusception in blood vessels, and provides a basis for further studies in other lymphatic conditions.     

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

Summary 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 m 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.A poster about this work presented at the meeting of the Union of the Swiss Societies for Experimental Biology was awarded the Gian Töndùry-Price 1990 of the Swiss Society for Anatomy, Histology and Embryology     

Tumor recovery by angiogenic switch from sprouting to intussusceptive angiogenesis after treatment with PTK787/ZK222584 or ionizing radiation

Inhibitors of angiogenesis and radiation induce compensatory changes in the tumor vasculature both during and after treatment cessation. To assess the responses to irradiation and vascular endothelial growth factor-receptor tyrosine kinase inhibition (by the vascular endothelial growth factor tyrosine kinase inhibitor PTK787/ZK222854), mammary carcinoma allografts were investigated by vascular casting; electron, light, and confocal microscopy; and immunoblotting. Irradiation and anti-angiogenic therapy had similar effects on the tumor vasculature. Both treatments reduced tumor vascularization, particularly in the tumor medulla. After cessation of therapy, the tumor vasculature expanded predominantly by intussusception with a plexus composed of enlarged sinusoidal-like vessels containing multiple transluminal tissue pillars. Tumor revascularization originated from preserved α-smooth muscle actin-positive vessels in the tumor cortex. Quantification revealed that recovery was characterized by an angiogenic switch from sprouting to intussusception. Up-regulated α-smooth muscle actin-expression during recovery reflected the recruitment of α-smooth muscle actin-positive cells for intussusception as part of the angio-adaptive mechanism. Tumor recovery was associated with a dramatic decrease (by 30% to 40%) in the intratumoral microvascular density, probably as a result of intussusceptive pruning and, surprisingly, with only a minimal reduction of the total microvascular (exchange) area. Therefore, the vascular supply to the tumor was not severely compromised, as demonstrated by hypoxia-inducible factor-1α expression. Both irradiation and anti-angiogenic therapy cause a switch from sprouting to intussusceptive angiogenesis, representing an escape mechanism and accounting for the development of resistance, as well as rapid recovery, after cessation of therapy.     

A new mechanism for pillar formation during tumor-induced intussusceptive angiogenesis: inverse sprouting

One of the hallmarks of intussusceptive angiogenesis is the development of intraluminal connective tissue pillars. The exact mechanism of pillar formation has not yet been elucidated. By using electron and confocal microscopy, we observed intraluminal nascent pillars that contain a collagen bundle covered by endothelial cells (ECs) in the vasculature of experimental tumors. We proposed a new mechanism for the development of these pillars. First, intraluminal endothelial bridges are formed. Second, localized dissolution of the basement membrane occurs and a bridging EC attaches to a collagen bundle in the underlying connective tissue. A pulling force is then exerted by the actin cytoskeleton of the ECs via specific attachment points, which contain vinculin, to the collagen bundle, resulting in suction and subsequent transport of the collagen bundle into and through the vessel lumen. Third, the pillar matures through the immigration of connective tissue cells and the deposition of new collagenous connective tissue. The proposed simple mechanism generates a connection between the processes of endothelial bridging and intussusceptive angiogenesis and identifies the source of the force behind pillar formation. Moreover, it ensures the rapid formation of pillars from pre-existing building blocks and the maintenance of EC polarity. To describe it, we coined the term inverse sprouting.     

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.     

Arterial branching in various parts of the cardiovascular system

Angiographic pictures of vascular beds in various parts of the cardiovascular system were analyzed to study the geometrical structure of arterial bifurcations. The sites of arterial bifurcations were enlarged individually, and measurements were made of the branching angles and branch diameters at each site. Results from various parts of the cardiovascular system of man, and some from rabbit and pig, were compared with each other. The measurements were also compared with “optimum” values of branching angles and branch diameters which have been predicted by various theoretical studies. In general the measurements were found to give support to the theoretical premise that branching angles and branch diameters in the cardiovascular system are dictated by certain optimality principles which aim to maximize the efficiency of the system in its fluid-conducting function. In some parts of the system, however, the measured angles and diameters were found to be decidedly lower than those predicted by theory.     

Three-dimensional arrangement of the vasa vasorum in explanted segments of the aged human great saphenous vein: scanning electron microscopy and three-dimensional morphometry of vascular corrosion casts

The vasa vasorum of skeletonized and nonskeletonized segments of five human great saphenous veins (GSVs), harvested during coronary bypass grafting, were cannulated, rinsed, and injected (casted) with the polymerizing resin Mercox-Cl-2B. After removal of the dry vascular tissue, the casts were examined using scanning electron microscopy. Stereopaired images (tilt angle, 6 degrees ) were taken, imported into a 3D morphometry system, and the 3D architecture of the vasa vasorum (arterial and venous vasa as well as capillaries) was studied qualitatively and quantitatively in terms of vasa diameters, intervascular and interbranching distances, and branching angles. Diameters of parent (d(0)) and large (d(1)) and small (d(2)) daughter vessels of arterial and venous bifurcations served to calculate asymmetry ratios (alpha) and area ratios (beta). Additionally, deviations of bifurcations and branching angles from optimal branches were calculated for selected arterial vasa. The arrangement of the vasa vasorum closely followed the longitudinally oriented connective tissue fibers in the adventitia and the circularly arranged smooth muscle cell layers within the outer layers of the media. Venous vasa by far outnumbered arterial vasa. Vasa vasorum changed their course several times in acute angles and revealed numerous circular constrictions, kinks, and outpouchings. Due to their spatial arrangement, the vasa vasorum are prone to tolerate vessel wall distension generated by acute increases in blood pressure or stretching of the vessel without severe impact on vessel functions. Preliminary comparisons of data from the bifurcations of cast arterial vasa vasorum, with calculated optimal bifurcations, do not yet give clear insights into the optimality principle(s) governing the design of arterial vasa vasorum bifurcations of the human GSVs.     

The effects of PTK787/ZK222584, an inhibitor of VEGFR and PDGFRβ pathways, on intussusceptive angiogenesis and glomerular recovery from Thy1.1 nephritis

The aim of our study was to investigate the phenomenon of intussusceptive angiogenesis with a focus on its molecular regulation by vascular endothelial growth factor receptor (VEGFR)/platelet-derived growth factor receptor β (PDGFRβ) pathways and biological significance for glomerular recovery after acute injury. Glomerular healing by intussusception was examined in a particular setting of Thy1.1 nephritis, where the lysis of mesangial cells results in an initial collapse and successive rebuilding of glomerular capillary structure. Restoration of capillary structure after induction of Thy1.1 nephritis occurred by intussusceptive angiogenesis resulting in i) rapid expansion of the capillary plexus with reinstatement of the glomerular filtration surface and ii) restoration of the archetypical glomerular vascular pattern. Glomerular capillaries of nephritic rats after combined VEGFR2 and PDGFRβ inhibition by PTK787/ZK222584 (PTK/ZK) were tortuous and irregular. However, the onset of intussusceptive angiogenesis was influenced only after long-term PTK/ZK treatment, providing an important insight into differential molecular regulation between sprouting and intussusceptive angiogenesis. PTK/ZK treatment abolished α-smooth muscle actin and tensin expression by injured mesangial cells, impaired glomerular filtration of microspheres, and led to the reduction of glomerular volume and the presence of multiple hemorrhages detectable in the tubular system. Collectively, treatment of nephritic patients with PTK/ZK compound is not recommended.     

Spatial growth and pattern formation in the small intestine microvascular bed from larval to adult Xenopus laevis: a scanning electron microscope study of microvascular corrosion casts

The microvascular anatomy of the small intestine of metamorphosing tadpoles of the South African Clawed Toad, Xenopus laevis (Daudin) is studied from developmental stages 55 to 65 and in adults by scanning electron microscopy (SEM) of vascular corrosion casts (VCCs) and light microscopy. Up to stage 62, VCCs reveal a dense two-dimensional vascular network ensheating the intestinal tube, whose proximal portion forms a clockwise spiralling outer and its distal portion an anti-clockwise spiralling inner coil. Vessels of the intestinal network impose flat and run circularly to slightly obliquely. Locally, dense capillary plexus with small “holes” indicating ongoing intussusceptive microvascular growth (IMG) and vessel maturation, are present. The typhlosole, an invagination along the proximal portion of the small intestine, reveals a dense capillary bed with locally ongoing IMG. VCCs of stages 62/63 for the first time reveal a three-dimensional vascular bed with longitudinal intestinal folds of varying size and heights greatly enlarging the luminal exchange area of the intestinal tube. From stage 65 onwards, longitudinal intestinal folds undulate and, though smaller in size and less mature as indicated in VCCs by the presence of wider, sinus-like vessels with small “holes” interposed between, closely resemble the intestinal folds present in the small intestine of adult Xenopus. Our data suggest that maturation of the vascular pattern in the small intestine of X. laevis tadpoles takes place successively after stages 62–63, and growth during this period is preferentially by intussusception.     

Chorioallantoic membrane capillary bed: a useful target for studying angiogenesis and anti-angiogenesis in vivo.

The chick embryo chorioallantoic membrane (CAM) is an extraembryonic membrane that is commonly used in vivo to study both angiogenesis and anti-angiogenesis. This review 1) summarizes the current knowledge about the structure of the CAM's capillary bed; 2) discusses the controversy about the existence of a single blood sinus or a capillary plexus underlying the chorionic epithelium; 3) describes a new model of the CAM vascular growth, namely the intussusceptive mode; 4) reports findings regarding the role played by endogenous fibroblast growth factor-2 in CAM vascularization; and 5) addresses the use and limitations of the CAM as a model for studying angiogenesis and anti-angiogenesis.     

脊髓内部动脉构筑的优化分析

4例新鲜足月胎儿尸体脊髓,经墨汁灌注,厚片透明后,选择Y型的动脉分支314个(颈段91个,胸段125个,腰段98个),同步测量动脉分支管径和夹角,对其进行了优化分析。从实测值和理论值的定性比较图中可以看出,动脉分支的管径和夹角的实测值和理论值是近乎一致的,同时又具有合理的生物学离散特点,从定量角度出发,如果规定实测值与理论值的相对误差低于10％时为优化状态,据此标准较大分支的管径处于优化状态的动脉分支有51％,较小分支的管径处于优化状态的动脉分支有50％,较大分支与母管夹角处于优化状态的有47％,较小分支与母管夹角处于优化状态的有28％,分支总角度处于优化状态的有49％,面积比处于优化状态的有49％。以上结果显示人脊髓内部动脉分支的几何形态也支持有关动脉分支的理论假设,即心血管系统动脉分支的管径和夹角被某些与心血管系统生理机能有关的优化原则所制约。     

CD105/Ki67 double immunostaining expression in liver metastasis from colon carcinoma

The liver is the most common and critical site for the development of colon cancer metastases. Tumor angiogenesis in liver metastasis from colon carcinoma is a controversial subject. Liver microenvironment, immunophenotypical and morphological particularities of hepatic vessels are only few aspects, which establish difficulties in quantification of tumor vascularisation from liver metastasis. The aim of this work is to study the distribution of CD105 positive vessels and the proliferation rate of endothelial cells from liver metastasis of colon carcinoma based on double immunostaining CD105/Ki67. In liver metastasis from well-differentiated adenocarcinoma we found a high number of CD105+/Ki67- vessels. On the other hand, in liver metastasis from poorly differentiated adenocarcinoma we noticed rare CD105+/Ki67+ vessels. It is hypothesized that neoangiogenesis of liver metastasis is performed through intussusceptive mechanism rather than sprouting and could be supported by the presence of kissing phenomenon, CD105 positive transcapillary pillars and the absence of endothelial cells proliferation in this vessels. We conclude that in liver metastasis principal mechanism of neovascularisation formation is based on intussusception.     

Intussusceptive microvascular growth in human glioma

Intussusceptive microvascular growth (IMG), which occurs by splitting of the existing vasculature by transluminal pillars or transendothelial bridges, has been demonstrated in several tumors such as colon and mammary carcinomas, melanoma and B-cell non-Hodgkin’s lymphomas. In this study, we have correlated in human glioma the extent of angiogenesis, evaluated as microvascular density, the immunoreactivity of tumor cells to vascular endothelial growth factor (VEGF), vessel diameter and IMG to the tumor stage. Results demonstrate for the first time a relationship in human glioma progression between angiogenesis, VEGF immunoreactivity of tumor cells, vessel diameter and the number of connections of intraluminal tissue folds with the opposite vascular wall, expression of IMG and suggest that IMG could be a mechanism of compensatory vascular growth occurring in human glioma. The advantages are that (1) blood vessels are generated more rapidly; (2) it is energetically and metabolically more economic; (3) the capillaries thereby formed are less leaky.     

Chorioallantoic membrane capillary bed: A useful target for studying angiogenesis and anti-angiogenesis in vivo

The chick embryo chorioallantoic membrane (CAM) is an extraembryonic membrane that is commonly used in vivo to study both angiogenesis and anti-angiogenesis. This review 1) summarizes the current knowledge about the structure of the CAM's capillary bed; 2) discusses the controversy about the existence of a single blood sinus or a capillary plexus underlying the chorionic epithelium; 3) describes a new model of the CAM vascular growth, namely the intussusceptive mode; 4) reports findings regarding the role played by endogenous fibroblast growth factor-2 in CAM vascularization; and 5) addresses the use and limitations of the CAM as a model for studying angiogenesis and anti-angiogenesis. Anat Rec 264:317–324, 2001. © 2001 Wiley-Liss, Inc.     

Methods for Three-Dimensional Geometric Characterization of the Arterial Vasculature

Complex vascular anatomy often affects endovascular procedural outcome. Accurate quantitative assessment of three-dimensional (3D) in-vivo arterial morphology is therefore vital for endovascular device design, and preoperative planning of percutaneous interventions. The aim of this work was to establish geometric parameters describing arterial branch origin, trajectory, and vessel curvature in 3D space that eliminate the errors implicit in planar measurements. 3D branching parameters at visceral and aortic bifurcation sites, as well as arterial tortuosity were determined from vessel centerlines derived from magnetic resonance angiography data for three subjects. Errors in coronal measurements of 3D branching angles for the right and left renal arteries were 3.1 ± 3.4° and 7.5 ± 3.7°, respectively. Distortion of the anterior visceral branching angles from sagittal measurements was less pronounced. Asymmetry in branching and planarity of the common iliac arteries was observed at aortic bifurcations. The renal arteries possessed considerably greater 3D curvature than the abdominal aorta and common iliac vessels with mean average values of 0.114 ± 0.015 and 0.070 ± 0.019 mm⁻¹ for the left and right, respectively. In conclusion, planar projections misrepresented branch trajectory, vessel length, and tortuosity proving the importance of 3D geometric characterization for possible applications in planning of endovascular interventional procedures and providing parameters for endovascular device design.     

Branching Pattern of the Cerebral Arterial Tree

Quantitative data on branching patterns of the human cerebral arterial tree are lacking in the 1.0–0.1 mm radius range. We aimed to collect quantitative data in this range, and to study if the cerebral artery tree complies with the principle of minimal work (Law of Murray). To enable easy quantification of branching patterns a semi-automatic method was employed to measure 1,294 bifurcations and 2,031 segments on 7 T-MRI scans of two corrosion casts embedded in a gel. Additionally, to measure segments with a radius smaller than 0.1 mm, 9.4 T-MRI was used on a small cast section to characterize 1,147 bifurcations and 1,150 segments. Besides MRI, traditional methods were employed. Seven hundred thirty-three bifurcations were manually measured on a corrosion cast and 1,808 bifurcations and 1,799 segment lengths were manually measured on a fresh dissected cerebral arterial tree. Data showed a large variation in branching pattern parameters (asymmetry-ratio, area-ratio, length-radius-ratio, tapering). Part of the variation may be explained by the variation in measurement techniques, number of measurements and location of measurement in the vascular tree. This study confirms that the cerebral arterial tree complies with the principle of minimum work. These data are essential in the future development of more accurate mathematical blood flow models. Anat Rec, 302:1434–1446, 2019. © 2018 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.     

Intussusceptive angiogenesis: pillars against the blood flow

Adaptation of vascular networks to functional demands needs vessel growth, vessel regression and vascular remodelling. Biomechanical forces resulting from blood flow play a key role in these processes. It is well-known that metabolic stimuli, mechanical forces and flow patterns can affect gene expression and remodelling of vascular networks in different ways. For instance, in the sprouting type of angiogenesis related to hypoxia, there is no blood flow in the rising capillary sprout. In contrast, it has been shown that an increase of wall shear stress initiates the splitting type of angiogenesis in skeletal muscle. Otherwise, during development, both sprouting and intussusception act in parallel in building the vascular network, although with differences in spatiotemporal distribution. Thereby, in addition to regulatory molecules, flow dynamics support the patterning and remodelling of the rising vascular tree. Herewith, we present an overview of angiogenic processes with respect to intussusceptive angiogenesis as related to local haemodynamics.