Peter H. Burri

Cell-Demanded Liberation of VEGF121 From Fibrin Implants Induces Local and Controlled Blood Vessel Growth

Abstract— Although vascular endothelial growth factor (VEGF) has been described as a potent angiogenic stimulus, its application in therapy remains difficult: blood vessels formed by exposure to VEGF tend to be malformed and leaky. In nature, the principal form of VEGF possesses a binding site for ECM components that maintain it in the immobilized state until released by local cellular enzymatic activity. In this study, we present an engineered variant form of VEGF, &agr;2PI1–8- VEGF121, that mimics this concept of matrix-binding and cell-mediated release by local cell–associated enzymatic activity, working in the surgically-relevant biological matrix fibrin. We show that matrix-conjugated &agr;2 PI1–8- VEGF121 is protected from clearance, contrary to native VEGF121 mixed into fibrin, which was completely released as a passive diffusive burst. Grafting studies on the embryonic chicken chorioallantoic membrane (CAM) and in adult mice were performed to assess and compare the quantity and quality of neovasculature induced in response to fibrin implants formulated with matrix-bound &agr;2 PI1–8- VEGF121 or native diffusible VEGF121. Our CAM measurements demonstrated that cell-demanded release of &agr;2 PI1–8- VEGF121 increases the formation of new arterial and venous branches, whereas exposure to passively released wild-type VEGF121 primarily induced chaotic changes within the capillary plexus. Specifically, our analyses at several levels, from endothelial cell morphology and endothelial interactions with periendothelial cells, to vessel branching and network organization, revealed that &agr;2 PI1–8- VEGF121 induces vessel formation more potently than native VEGF121 and that those vessels possess more normal morphologies at the light microscopic and ultrastructural level. Permeability studies in mice validated that vessels induced by &agr;2 PI1–8- VEGF121 do not leak. In conclusion, cell-demanded release of engineered VEGF121 from fibrin implants may present a therapeutically safe and practical modality to induce local angiogenesis.

Structural Aspects of Postnatal Lung Development – Alveolar Formation and Growth

The human lung is born with a fraction of the adult complement of alveoli. The postnatal stages of human lung development comprise an alveolar stage, a stage of microvascular maturation, and very likely a stage of late alveolarization. The characteristic structural features of the alveolar stage are well known; they are very alike in human and rat lungs. The bases for alveolar formation are represented by immature interairspace walls with two capillary layers with a central sheet of connective tissue. Interalveolar septa are formed by folding up of one of the two capillary layers. In the alveolar stage, alveolar formation occurs rapidly and is typically very conspicuous in both species; it has therefore been termed ‘bulk alveolarization’. During and after alveolarization the septa with double capillary networks are restructured to the mature form with a single network. This happens in the stage of microvascular maturation. After these steps the lung proceeds to a phase of growth during which capillary growth by intussusception plays an important role in supporting gas exchange. In view of reports that alveoli are added after the stage of microvascular maturation, the question arises whether the present concept of alveolar formation needs revision. On the basis of morphological and experimental findings we can state that mature lungs contain all the features needed for ‘late alveolarization’ by the classical septation process. Because of the high plasticity of the lung tissues, late alveolarization or some forms of compensatory alveolar formation may be considered for the human lung.

The postnatal development and growth of the human lung. II. Morphology.

The morphology of postnatal human lung development and growth has been investigated by light and by scanning and transmission electron microscopy in seven children dying from non-respiratory causes and aged between 26 days and 64 months. The findings are compared with those of adult human lungs and are discussed in relation to the postnatal lung development in other species, particularly rodents. Within the first 1 1/2 postnatal years lung parenchyma undergoes a substantial structural remodeling due to bulk alveolar formation and to the restructuring of septal morphology. At one month alveolar formation appears to be well under way: The human lung is comparable then to a rat lung aged one week. In the parenchyma, numerous short and blunt tissue ridges, so-called secondary septa, subdivide the peripheral airspaces into an increasing number of still very shallow alveoli. The parenchymal septa present during and after alveolization are immature: they contain a double capillary network with a central, highly cellular sheet of connective tissue. The septal maturation sets in a few months after birth and consists of a reduction in the interstitial tissue mass and a complex process of capillary remodeling. Both alveolization and parenchymal maturation progress rapidly: by 6 months the lung has taken a big step towards maturity. By 1 1/2 years most septa show the adult structure where a single capillary network interwoven with connective tissue strands stabilizes the interalveolar wall. After the septal restructuring, lung development is considered complete, and the lung enters a period of normal growth that lasts until adulthood. From our observations we conclude that postnatal human lung development is made of two overlapping stages: (a) the alveolar stage, which starts in late fetal life and lasts to about 1-1 1/2 years, and (b) a stage of microvascular maturation, thought to extend from the first months after birth to the age of 2-3 years.

Vascular remodeling by intussusceptive angiogenesis

Intussusception (growth within itself) is an alternative to the sprouting mode of angiogenesis. The protrusion of opposing microvascular walls into the capillary lumen creates a contact zone between endothelial cells. The endothelial bilayer is perforated, intercellular contacts are reorganized, and a transluminal pillar with an interstitial core is formed, which is soon invaded by myofibroblasts and pericytes leading to its rapid enlargement by the deposition of collagen fibrils. Intussusception has been implicated in three processes of vascular growth and remodeling. (1) Intussusceptive microvascular growth permits rapid expansion of the capillary plexus, furnishing a large endothelial surface for metabolic exchange. (2) Intussusceptive arborization causes changes in the size, position, and form of preferentially perfused capillary segments, creating a hierarchical tree. (3) Intussusceptive branching remodeling (IBR) leads to modification of the branching geometry of supplying vessels, optimizing pre- and postcapillary flow properties. IBR can also lead to the removal of branches by pruning in response to changes in metabolic needs. None of the three modes requires the immediate proliferation of endothelial cells but rather the rearrangement and plastic remodeling of existing ones. Intussusception appears to be triggered immediately after the formation of the primitive capillary plexus by vasculogenesis or sprouting. The advantage of this mechanism of growth over sprouting is that blood vessels are generated more rapidly in an energetically and metabolically more economic manner, as extensive cell proliferation, basement membrane degradation, and invasion of the surrounding tissue are not required; the capillaries thereby formed are less leaky. This process occurs without disrupting organ function. Improvements in our understanding of the process should enable the development of novel pro- and anti-angiogenic therapeutic treatments.

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 CAMs 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.

Optimality in the developing vascular system: branching remodeling by means of intussusception as an efficient adaptation mechanism.

The theory of bifurcating vascular systems predicts vessel diameters that are related to optimality criteria like minimization of pumping energy or of building material. However, mechanisms for producing the postulated optimality have not been described so far, and quantitative data on bifurcation diameters during development are scarce. We used an embryonic vascular bed that rapidly grows and adapts to changing hemodynamic conditions, the chicken chorioallantoic membrane (CAM), and correlated vascular cast and tissue section morphology with in vivo time‐lapse video monitoring. The bifurcation exponent Δ and associated parameters were quantitatively assessed in arterial and venous microvessels ranging in diameter from 30 to 100 μm. We observed emergence of optimality by means of intussusception, i.e., formation of transvascular tissue pillars. In addition to intussusceptive microvascular growth (IMG = expansion of capillary networks) and intussusceptive arborization (IAR = formation of feeding vessels from capillaries) the observed intussusception at bifurcations represents a third variant of nonsprouting angiogenesis. We call it intussusceptive branching remodeling (IBR). IBR occurred in vessels of considerable diameter by means of two alternative mechanisms: either through pillars arising close to a bifurcation, which increased in girth until they merged with the connective tissue in the bifurcation angle; or through pillars arising at some distance from the bifurcation point, which then expanded by formation of ingrowing tissue folds until they became connected to the tissue of the bifurcation angle. Morphologic evidence suggests that IBR is a wide‐spread phenomenon, taking place also in lung, intestinal, kidney, eye, etc., vasculature. Irrespective of the mode followed, IBR led to a branching pattern close to the predicted optimum, Δ = 3.0. Significant differences were observed between Δ at arterial bifurcations (2.70 to 2.90) and Δ at venous bifurcations (2.93 to 3.75). IBR, by means of eccentric pillar formation and fusion, was also involved in vascular pruning. Experimental changes in CAM hemodynamics (by locally increasing blood flow) induced onset of IBR within less than 1 hr. Our study provides morphologic and quantitative evidence that a similar cellular machinery is used for all three variants of vascular intussusception, IMG, IAR, and IBR. It thus provides a mechanism of efficiently generating complex blood transport systems from limited genetic information. Differential quantitative outcome of IBR in arteries and veins, and the experimental induction of IBR strongly suggest that hemodynamic factors can instruct embryonic vascular remodeling toward optimality.

Intussusceptive angiogenesis––the alternative to capillary sprouting

In contrast to sprouting angiogenesis, which is a well established mode of new blood vessel formation, intussusceptive angiogenesis (IA) is a relatively new concept in vascular biology. It was first discovered in the lung as a means of capillary network growth (intussusceptive microvascular growth). The mechanism consists in the repeated insertion of new slender transcapillary tissue pillars, which subsequently increase in size, thus allowing the capillary network to grow in itself (i.e., by intussusception). It could be shown that IA was present in all organs and species investigated so far, so that it appears to be an ubiquitous phenomenon in vertebrates at least. It was not a surprise therefore to find that IA also played a role in tumour vascularisation. Morphological analysis has yet brought evidence for 6 different modes of pillar formation. They all have in common that, at one time, two endothelial leaflets (e.g. of opposite capillary walls) come into close contact, form new junctional complexes, then thin out to finally give way to the invading interstitial tissue, particularly to fibroblasts, myofibroblasts and pericytes. Once such a transcapillary pillar is formed, it can subsequently grow to the size of a normal intercapillary mesh. The addition of collagen fibrils to the pillar core will stabilize the pillar mechanically. Recent observations allowed to extend the IA concept further: The same structural mechanism of intussusceptive pillar formation was shown to contribute also to the formation of vascular trees (arborisation) and to be involved in vascular remodeling. Although numerous growth factors and receptors have already been suggested as being active in IA, very few hard facts are at present available which would allow to get a comprehensive view of IA regulation.

A simple tool for stereological assessment of digital images: the STEPanizer

STEPanizer is an easy‐to‐use computer‐based software tool for the stereological assessment of digitally captured images from all kinds of microscopical (LM, TEM, LSM) and macroscopical (radiology, tomography) imaging modalities. The program design focuses on providing the user a defined workflow adapted to most basic stereological tasks. The software is compact, that is user friendly without being bulky. STEPanizer comprises the creation of test systems, the appropriate display of digital images with superimposed test systems, a scaling facility, a counting module and an export function for the transfer of results to spreadsheet programs. Here we describe the major workflow of the tool illustrating the application on two examples from transmission electron microscopy and light microscopy, respectively.

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

SummaryThe 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 μm 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.

Beeinflussung einer spezifischen cytoplasmatischen Organelle von Endothelzellen durch Adrenalin

SummaryEndothelial cells of vertebrate blood vessels contain specific cytoplasmic organelles, distinguishable from other dense bodies by their rod shape and internal tubular substructure. The following observations led to the hypothesis that these organelles could correspond to a procoagulative substance previously observed in arterial walls. a) These organelles have some resemblance to α-granules of thrombocytes which proved to be procoagulative elements. b) Intima and endothelium of human aorta contain thromboplastic substances. c) Large blood vessels contain more organelles, a fact which could be explained as an indication of their blood-directed function. d) The epinephrine perfused rabbit aorta delivers a coagulation activating substance into the perfusate. To check this hypothesis strips of aortic wall of five rats were incubated in 0.5 μg-% epinephrine solution for 20 sec and as controls in Ringer solution. By comparison some material was directly fixed by immersion in 1% OsO4.The volume-density of organelles in endothelial cytoplasm was determined by means of stereologic methods.On the average the volume-density decreased from 0.93% in immediatly fixed, to 0.83% in Ringer incubated, and to 0.53% in epinephrine incubated tissue samples. The diminution after Ringer incubation can be explained by swelling of cytoplasm. The loss of some 40% of organelles between Ringer and epinephrine incubated material is statistically highly significant. Evidence is presented that the organelles are expulsed towards the vessel lumen.These results give support to the hypothesis that these organelles could contain a procoagulative substance of aortic endothelium postulated by several authors. Further experiments are needed to prove this relationship.ZusammenfassungDie Blutgefäßendothelien der Wirbeltiere enthalten eine spezifische cytoplasmatische Organelle, der nach einer schon früher geäußerten Hypothese eine Funktion im Rahmen der Blutgerinnung zukommen könnte. Da Adrenalin fähig sein soll, aus der Aortenwand eine gerinnungsaktive Substanz freizusetzen, wurde in vitro an 5 Rattenaorten die Wirkung einer „physiologischen“ Adrenalinlösung auf die endothelspezifische Organelle geprüft. Als Kontrolle dienten direkt fixierte, sowie in Ringerlösung inkubierte Aortenringe des gleichen Tieres. Die durch die Inkubation hervorgerufenen morphologischen Veränderungen der Gefäßintima werden beschrieben und besprochen.Mit stereologischen Methoden wurde die Volumendichte der Endothelkörperchen in den Kontroll- und Testgruppen ermittelt und miteinander verglichen.Adrenalin vermag binnen 20 sec die Volumendichte der Organellen in Endothelcytoplasma auf ca. 60% des Kontrollwertes zu verringern. Diese Abnahme ist statistisch signifikant (P < 0,001). Die oft beobachteten engen topographischen Beziehungen der Organellen mit der Zellmembran lassen an eine Stoffabgabe ins Gefäßlumen denken.Die Resultate stützen die Hypothese, daß diese Organellen eine von verschiedenen Seiten postulierte, gerinnungsaktive Substanz enthalten könnten.