Haymo Kurz

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.

Neuroectodermal origin of brain pericytes and vascular smooth muscle cells

The origin of vascular pericytes (PCs) and smooth muscle cells (vSMCs) in the brain has hitherto remained an open question. In the present study, we used the quail‐chick chimerization technique to elucidate the lineage of cranial PCs/vSMCs. We transplanted complete halves of brain anlagen, or dorsal (presumptive neural crest [NC]) or ventral cranial neural tube. Additional experiments included transplantations of neuroectoderm into limb mesenchyme, and of head mesoderm or limb mesenchyme into paraxial head mesoderm. After interspecific transplantation of quail brain rudiment, graft‐derived vSMCs were found in the vessel walls of the grafted brain. Notably, transplanted ventral neural tube also gave rise to vSMCs. After grafting of quail head mesoderm, quail endothelial cells were found in the host brain, but no vSMCs of donor origin. Grafting of quail whole or ventral neural tube into the limb bud led to endowment of graft and host vessels with graft‐derived vSMCs. Quail limb bud mesenchyme contributed to vSMCs in the ectopic neural graft, but, when transplanted into paraxial head mesenchyme, it did not form intraneural vSMCs. After orthotopic transplantation of cranial NC, graft‐derived vSMCs were not only found in meninges and brain of the operated side, but also on the contralateral side. Our results show that 1) avian cranial neuroectoderm is able to differentiate into vSMCs of the brain; 2) this potential is not restricted to the prospective NC; and 3) neither cranial mesoderm nor cranially transplanted limb bud mesoderm can give rise to brain vSMC. J. Comp. Neurol. 442:78–88, 2002.

Structural and biophysical simulation of angiogenesis and vascular remodeling.

The purpose of this report is to introduce a new computer model for the simulation of microvascular growth and remodeling into arteries and veins that imitates angiogenesis and blood flow in real vascular plexuses. A C++ computer program was developed based on geometric and biophysical initial and boundary conditions. Geometry was defined on a two‐dimensional isometric grid by using defined sources and drains and elementary bifurcations that were able to proliferate or to regress under the influence of random and deterministic processes. Biophysics was defined by pressure, flow, and velocity distributions in the network by using the nodal‐admittance‐matrix‐method, and accounting for hemodynamic peculiarities like Fahraeus‐Lindqvist effect and exchange with extravascular tissue. The proposed model is the first to simulate interdigitation between the terminal branches of arterial and venous trees. This was achieved by inclusion of vessel regression and anastomosis in the capillary plexus and by remodeling in dependence from hemodynamics. The choice of regulatory properties influences the resulting vascular patterns. The model predicts interdigitating arteriovenous patterning if shear stress‐dependent but not pressure‐dependent remodeling was applied. By approximating the variability of natural vascular patterns, we hope to better understand homogeneity of transport, spatial distribution of hemodynamic properties and biomass allocation to the vascular wall or blood during development, or during evolution of circulatory systems.

VEGF Coordinates Interaction of Pericytes and Endothelial Cells During Vasculogenesis and Experimental Angiogenesis

Biological activities of vascular endothelial growth factor (VEGF) have been studied extensively in endothelial cells (ECs), but few data are available regarding its effects on pericytes. In murine embryoid body cultures, VEGF‐induced expression of desmin and α‐smooth muscle actin (α‐SMA) in CD‐31+ cells. The number of CD‐31+/desmin+ vascular chords increased with VEGF treatment time and peaked during a differentiation window between 6 and 9 days after plating. In vivo, VEGF‐induced elongation and migration of desmin‐positive pericytes and coverage of angiogenic capillaries, as revealed by analysis of Sambucus nigra lectin‐stained vascular beds of the chick chorioallantoic membrane. VEGF also caused significant decrease of intercapillary spaces, an indicator for intussusceptive vascular growth. These VEGF‐mediated effects point at a more intricate interaction between ECs and pericytes cells than previously demonstrated and suggest that pericytes may be derived from EC progenitors in vitro and not only stabilize capillaries but also participate in vascular remodeling in vivo. Developmental Dynamics 230:23–33, 2004.

Embryonic CNS macrophages and microglia do not stem from circulating, but from extravascular precursors

Invasion of mesoderm‐derived cells into the developing spinal cord and brain has been shown to produce early central nervous system (CNS) macrophage and microglia populations in avian embryos. A triplicate mode of entry has been proposed: through the endothelial wall of CNS blood vessels; from the ventricular cavities; and through the pial surface. Invasion of circulating blood cells (monocytes) has not yet been proved in embryonic CNS. This report demonstrates: 1) the use of chick‐quail blood chimeras by way of parabiosis (two embryos in one egg); 2) the use of QH1 monoclonal antibody for detection of quail cells circulating in chick blood vessels; 3) the presence of extravascular QH1‐positive cells (macrophages) in E7–10 CNS in parabiosis quail, and their absence in parabiosis chick. We conclude that avian macrophages/microglia precursors do not penetrate through the wall of embryonic CNS vessels. In combination with published results, this finding strongly supports the view that invasion of migratory macrophages from the pial surface and proliferation inside the CNS generate all microglia in avian embryos. GLIA 22:98–102, 1998.

Physiology of Angiogenesis

Angiogenesis is a key prerequisite for growth in all vertebrate embryos and in many tumors. Rapid growth requires efficient transport of oxygen and metabolites. Hence, for a better understanding of tissue growth, biophysical properties of vascular systems, in addition to their molecular mechanisms, need to be investigated. The purpose of this article is twofold: (1) to discuss the biophysics of growing and perfused vascular systems in general, emphasizing non-sprouting angiogenesis and remodeling of vascular plexuses; and (2) to report on cellular details of sprouting angiogenesis in the initially non-perfused embryonic brain and spinal cord. It is concluded that (1) evolutionary optimization of the circulatory system corresponds to highly conserved vascular patterns and angiogenetic mechanisms; (2) deterministic and random processes contribute to both extraembryonic and central nervous system vascularization; (3) endothelial cells interact with a variety of periendothelial cells during angiogenesis and remodeling; and that (4) mathematical models integrating molecular, morphological and biophysical expertise improve our understanding of normal and pathological angiogenesis and account for allometric relations.

Cell-laden and cell-free biopolymer hydrogel for the treatment of osteochondral defects in a sheep model.

The objective of the current study was to determine the suitability of cell-laden and cell-free alginate-gelatin biopolymer hydrogel for osteochondral restoration in a sheep model (n = 12). Four femoral defects per animal were filled with hydrogel (cHG) plus autologous chondrocytes (cHG + C) or periosteal cells (cHG + P) or gel only (cHG) or were left untreated (E). In situ solidification enabled instantaneous implant fixation. Sixteen weeks postoperatively, defect sites were processed for light microscopy and immunofluorescence. A modified Mankin and a semi-quantitative immunoreactivity score were used to evaluate histology and immunofluorescence, respectively. Defects after cHG + C were restored with smooth, hyaline-like neo-cartilage and trabecular subchondral bone. cHG + P and cHG treatments revealed slightly inferior regenerate morphology. Undifferentiated tissue was found in E. The histological score showed significant (p < 0.05) differences between all treatment groups. In conclusion, cHG induces satisfactory defect regeneration. Complete filling of the cavity in one step and subsequent rapid in situ solidification was feasible and facilitated graft fixation. Cell implantation might be beneficial, because cells seem to play a key role in histological outcome. Still, their contribution to the repair process remains unresolved because host cell influx takes place. The combination of alginate and gelatin, however, creates an environment capable of serving implanted and host cells for osteo-chondrogenic tissue regeneration.

Measuring fractal dimension and complexity — an alternative approach with an application

Fractal dimension has often been applied as a parameter of complexity, related to, for example, surface roughness, or for classifying textures or line patterns. Fractal dimension can be estimated statistically, if the pattern is known to be self‐similar. However, the fractal dimension of more general patterns cannot be estimated, even though the concept may be retained to characterize complexity. We here show that the usual statistical methods, e.g. the box counting method, are not appropriate to measure complexity. A recently developed approach, the extended counting method, whose properties are closer to what fractal dimension means, is considered here in more detail. The methods are applied to geometric and to blood vessel patterns. The weak assumptions about the structure, and the lower variance of the estimate, suggest that the extended counting method has beneficial properties for comparing complexity of naturally occurring patterns.

Lack of correlation between placenta and offspring size in mouse interspecific crosses.

The placenta plays a pivotal role in fetal growth control and is considered a major site of genetic conflict between maternal and paternal genomes within the conceptus and, in addition, the genome of the mother. Accordingly, placental development is a strictly controlled process, and both placental and fetal weights do not vary much in intraspecific crosses of laboratory mice (Mus musculus). In mouse interspecific crosses and backcrosses [(M. musculus×M. spretus) ×M. musculus], tremendous variation of placental, but not of fetal weight was observed. We have studied trophoblast cell type distribution and differentiation, and their effect on the associated placentas and fetuses in such backcrosses. Differentiation of spongious trophoblast, but not size of materno-fetal interface, correlated with fetal weight. Giant fetuses were observed only if less than one third of the spongiotrophoblast was formed by glycogen cells. Thus, placental efficiency was inversely related to the amount of glycogen cells. This influence of a trophoblast-derived cell type on fetal growth was not anticipated. We conclude that: (1) glycogen cells are able to negatively modulate fetal growth by an as yet unidentified mechanism; (2) correlation between fetal and placental weights is weak or absent in interspecific hybrids; (3) impaired control over placental and embryonic development in hybrids may contribute to post-mating isolation of species.

Ventral axial organs regulate expression of myotomal Fgf-8 that influences rib development

Fgf-8 encodes a secreted signaling molecule mediating key roles in embryonic patterning. This study analyzes the expression pattern, regulation, and function of this growth factor in the paraxial mesoderm of the avian embryo. In the mature somite, expression of Fgf-8 is restricted to a subpopulation of myotome cells, comprising most, but not all, epaxial and hypaxial muscle precursors. Following ablation of the notochord and floor plate, Fgf-8 expression is not activated in the somites, in either the epaxial or the hypaxial domain, while ablation of the dorsal neural tube does not affect Fgf-8 expression in paraxial mesoderm. Contrary to the view that hypaxial muscle precursors are independent of regulatory influences from axial structures, these findings provide the first evidence for a regulatory influence of ventral, but not dorsal axial structures on the hypaxial muscle domain. Sonic hedgehog can substitute for the ventral neural tube and notochord in the initiation of Fgf-8 expression in the myotome. It is also shown that Fgf-8 protein leads to an increase in sclerotomal cell proliferation and enhances rib cartilage development in mature somites, whereas inhibition of Fgf signaling by SU 5402 causes deletions in developing ribs. These observations demonstrate: (1) a regulatory influence of the ventral axial organs on the hypaxial muscle compartment; (2) regulation of epaxial and hypaxial expression of Fgf-8 by Sonic hedgehog; and (3) independent regulation of Fgf-8 and MyoD in the hypaxial myotome by ventral axial organs. It is postulated that the notochord and ventral neural tube influence hypaxial expression of Fgf-8 in the myotome and that, in turn, Fgf-8 has a functional role in rib formation.