Congruence of vascular network remodeling and neuronal dispersion in the hippocampus of reelin-deficient mice.

In the hippocampus, neurons and fiber projections are strictly organized in layers and supplied with oxygen via a vascular network that also develops layer-specific characteristics in wild-type mice, as shown in the present study for the first time in a quantitative manner. By contrast, in the reeler mutant, well known for its neuronal migration defects due to the lack of the extracellular matrix protein reelin, emerging layer-specific characteristics of the vascular pattern were found to be remodeled during development of the dentate gyrus. Remarkably, in the first postnatal week, when a granule cell layer was still discernable in the reeler dentate gyrus, also the reeler vascular pattern resembled wild type. Thus, at postnatal day 6, unbranched microvessels traversed the granule cell layer and bifurcated when reaching the subgranular zone. Only after the first postnatal week vascular network remodeling in the reeler dentate gyrus became apparent, when the proportion of dispersed granule cells increased. Hence, vessel bifurcation frequency decreased in the maturing reeler dentate gyrus, but increased in wild type, resulting in significant differences (approx. 100%; p < 0.01) between adult wild type and reeler. Moreover, layer-specific vessel bifurcation frequencies disappeared in the maturing reeler dentate gyrus. Finally, a wild type-like vascular pattern was also found in the dentate gyrus of mice deficient for the reelin receptor very low density lipoprotein receptor (VLDLR), precluding a requirement of VLDLR for normal vascular pattern formation in the dentate gyrus. In sum, our findings show that vascular network remodeling in the reeler dentate gyrus is closely linked to the progression of granule cell dispersion.

The effect of scaffold composition on the early structural characteristics of chondrocytes and expression of adhesion molecules.

Previously we demonstrated that chondrocyte ECM synthesis and mitotic activity was dependent on scaffold composition when cultured on uncoated PCL scaffolds (pPCL) or PCL composites containing hyaluronan (PCL/HA), chitosan (PCL/CS), fibrin (PCL/F), or collagen type I (PCL/COL1). We hypothesized that initial cell contact with these biomaterials results in ultrastructural changes and alters CD44 and integrin beta1 expression. The current study was designed to investigate the early ultrastructural responses of chondrocytes on these scaffolds and expression of CD44 and integrin beta1. A common observation 1 h after seeding was the abundance of cell processes. Different types of cell processes occurred in different areas of the same cell and on different cells within the same composite. Chondrocytes seeded onto PCL/CS had the greatest cell surface enhancement. PCL/HA promoted CD44 expression and almost spherical cells with a low degree of surface enhancement. PCL/COL1 enabled continuing expression of integrin beta1 and CD44. In contrast, cells in PCL/CS, PCL/F and pPCL promoted elliptic cells with a higher degree of surface enhancement and no prolonged CD44 and integrin beta1 expression. A strong variability of cell surface processes indicated either reparative or degenerative adaptation to the artificial environment. Interestingly, we found initial integrin beta1 expression in all composite scaffolds, but not in pPCL although this promoted strong adhesiveness as indicated by the formation of stress fibers. In conclusion, chondrocytes respond to biomaterials early after implantation by altering ultrastructural characteristics and expression of CD44 and integrin beta1.

A computational model of intussusceptive microvascular growth and remodeling.

Non-sprouting angiogenesis, also known as intussusceptive angiogenesis (IA), is an important modality of blood vessel morphogenesis in growing tissues. We present a novel computational framework for simulation of IA to answer some of the questions concerning the underlying mechanisms of the remodeling process. The model relies on mechanical interactions between blood and tissue, includes the structural maturation of the vessel wall, and is controlled by stimulating or inhibiting chemical agents. The model provides a simple explanation for the formation of microvessels and bifurcations from capillaries via IA, allowing for both maintenance and avoidance of shunt vessels. Detailed hemodynamic and transport properties for oxygen, metabolites or growth factors can be predicted. The model is an in silico framework for testing certain conceptual ideas about the mechanisms of intussusceptive growth and remodeling, in particular those related to mechanical and transport phenomena.

HoxB5 induces endothelial sprouting in vitro and modifies intussusceptive angiogenesis in vivo involving angiopoietin-2.

AIMS: Homeobox (Hox) proteins are transcriptional regulators in embryonic patterning, cell differentiation, proliferation, and migration in vertebrates and invertebrates. A growing body of evidence suggests that Hox proteins are involved in endothelial cell regulation. We have shown earlier that HoxB5 upregulates vascular endothelial growth factor receptor-2 and thereby contributes to enhanced endothelial precursor cell differentiation. Here we aim to elucidate the role of HoxB5 in angiogenesis. METHODS AND RESULTS: Endothelial cell sprouting was investigated in the human umbilical vein endothelial cell spheroid assay. We investigated in vivo angiogenesis in the chick (Gallus gallus) chorioallantoic membrane assay. Expression profiling of proangiogenic factors was done by quantitative PCR. The angiopoietin-2 (Ang2) promoter and deletion fragments thereof were cloned into the pGL3 reporter system for analysis of transcriptional activity. We observed that HoxB5 enhances endothelial cell sprouting and modulates the expression of adhesion molecules in vitro. Accordingly, we observed a modification of vascular growth by HoxB5 in vivo. The HoxB5 effect is reminiscent of the effects of angiopoietins. We demonstrate that Ang2 is upregulated upon HoxB5 overexpression and that the HoxB5 effect is abolished by the angiopoietin antagonist soluble Tie-2. CONCLUSION: HoxB5 has an activating effect on Ang2 that is essential for endothelial cell sprouting and coordinated vascular growth.

Cell lineages and early patterns of embryonic CNS vascularization.

First steps of blood vessel formation and patterning in the central nervous system (CNS) of higher vertebrates are presented. Corresponding to the regional diversity of the embryonic CNS (unsegmented spinal cord vs segmented brain anlagen) and its surroundings (segmented trunk vs unsegmented head mesoderm, neural crest-derived mesenchyme), cells of different origins contribute to the endothelial and mural cell populations. The autonomous migratory potential of endothelial cells is guided by attractive and repulsive clues. Nevertheless, a common pattern in both spinal cord and forebrain vascularization appears, with primary ventral vascular sprouts supplying the periventricular vascular plexus of the neural tube, whereas dorsolateral sprouts appear later.

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.

Pericytes in the mature chorioallantoic membrane capillary plexus contain desmin and alpha-smooth muscle actin: relevance for non-sprouting angiogenesis.

The chicken chorioallantoic membrane (CAM) is a frequently used tissue for studying vascular growth and remodeling, notably non-sprouting angiogenesis by formation of transluminal pillars. Vascular pericytes have received increasing attention in the field of angiogenesis research and appear important for pillar growth. Our earlier observation that desmin (DES), but not alpha-smooth muscle actin (alphaSMA) was expressed in pericytes of the mature CAM capillary plexus after E12 was confirmed by others. However, in different species or vascular beds, either marker or both have been used to identify pericytes, raising the questions if (1) expression of these cytoskeletal proteins really was mutually exclusive; or (2) different types of pericytes existed in the same vascular bed. Using triple labeling with fluorochrome-conjugated markers Sambucus nigra agglutinin, DES or alphaSMA, and DNA-specific YoPro-1, we report here for the first time a delicate filamentous, circumferentially oriented alphaSMA pattern in periendothelial cells of the mature CAM capillary plexus, quite different from the coarser, axially oriented DES pattern. A new method for automatic classification of DNA-staining pattern was applied to compare nuclei of DES- or alphaSMA-positive cells. It predicted colocalisation of both proteins in most capillary pericytes, which was confirmed by double immunostaining for DES and alphaSMA. We conclude that (1) in contrast to published work, DES and alphaSMA are not mutually exclusive in most pericytes; (2) different types of pericytes may co-exist in the same vascular bed; (3) on average, one pericyte is associated with two transluminal pillars; (4) a novel imaging modality may be useful for cell identification in angiogenesis research and elsewhere.

Morphology and function of ovine articular cartilage chondrocytes in 3-d hydrogel culture.

Different cell- and biomaterial-based tissue engineering techniques are under investigation to restore damaged tissue. Strategies that use chondrogenic cells or tissues in combination with bioresorbable delivery materials are considered to be suitable to regenerate bio-artificial cartilage. Three-dimensional (3-D) cell embedding techniques can provide anchorage-independent cell growth and homogenous spatial cell arrangement, which play a key role in the maintenance of the characteristic phenotype and thus the formation of differentiated tissue. We developed a new injectable high water content (90%) hydrogel formulation with 5% sodium alginic acid and 5% gelatin as a temporary supportive intercellular matrix for 3-D cell culture. The objective was to determine whether the in vitro hydrogel culture of chondrocytes could preserve hyaline characteristics and thus could provide cartilage regeneration in vitro. Chondrocytes harvested from knee joints of skeletally mature sheep were cultured 3-D in hydrogel (7 x 10(6) cells/ml, 2.8-mul beads) for up to 10 weeks. Cell morphology and viability were evaluated with light microscopy, and proliferative activity was assessed with antibromodeoxyuridine immunofluorescence. Expression of collagens type I (COL1) and II (COL2), cartilage proteoglycans (PG) and hyaluronan synthases (HAS) were studied immunohistochemically. We observed that up to 36% of chondrocytes proliferated, while almost 100% presented a differentiated spheroidal phenotype. After an initial decrease at 2 weeks, cell density recovered to 85% of the initial absolute value at 10 weeks. Expression of hyaline matrix molecules resembled the in vivo pattern with increasing spatial deposition of PG and COL2. The proportion of PG-positive cells increased from initially 13 to 53% after 10 weeks, in contrast to consistently 100% COL2-positive cells. We conclude that 3-D hydrogel culture, even without mechanical stimulation or growth factor application, can keep chondrocytes in a differentiated state and provides a chondrogenic cell environment for in vitro cartilage regeneration for at least 10 weeks. Moreover, this hydrogel appears to be a suitable cell delivery material for subsequent in vivo implantation.

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 alpha-smooth muscle actin (alpha-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.

Morphogenesis of embryonic CNS vessels.

This chapter focuses on the morphology of blood vessel formation in and around the early central nervous system (CNS, i.e., brain and spinal cord) of avian embryos. We discuss cell lineages, proliferation and interactions of endothelial cells, pericytes and smooth muscle cells, and macrophages. Due to space limitations, we can not review the molecular control of CNS angiogenesis, but refer the reader to other chapters in this book and to recent publications on the assembly of the vasculature (1,2).

Three different fates of cells migrating from somites into the limb bud.

Cells from the ventrolateral dermomyotomal lips at limb levels undergo epithelio-mesenchymal transition and migrate as individual and undifferentiated cells into the limb buds. The cells give rise to myocytes and blood vascular endothelial cells (BECs) in the limb. Using vascular endothelial growth factor receptor-3 (VEGFR-3) as a marker, it has also been shown that the somites contribute to endothelial cells of lymphatic vessels in the limbs, but it is unknown where the lymphangiogenic precursors are located within the somite. In this study we used the transcription factor Prox1 as a lymphatic marker and investigated whether cells in the dorso-lateral quarter of the somite differentiate into lymphatic endothelial cells (LECs) of the limbs. To label the migrating cells, the dorso-lateral part of an epithelial brachial somite was grafted homotopically from quail into chick embryos at HH stages 13-14. The chick hosts were incubated until day 10-11 of development. The quail cell nuclei were identified with QCPN (anti-quail) antibodies. Cell differentiation was analysed by immunohistochemical staining with QH1, anti-desmin and anti-Prox1 antibodies, and by in situ hybridisation with Prox1 probes. Our results confirm that quail cell nuclei are incorporated into the myotubes of the limb muscles. Quail cells are found in the endothelium of limb blood vessels and lymphatics, predominantly the dermal lymphatics. This indicates that superficial lymphatics develop independently from the deep ones and shows that cells migrating from the lateral somitic edge into the limb buds differentiate into three cell populations: myocytes, BECs and LECs.

Hyaluronan synthases in normal and regenerating joint cartilage.

Repair of full thickness joint cartilage defects is within reach of routine clinical practice. The quality of regenerating hyaline cartilage, however, is difficult to assess. Synthesis of an extracellular matrix with high hyaluronan content is crucial for its metabolic and functional properties. We studied hyaluronan synthase (HAS) expression in knee joints of adult sheep as a novel cellular marker for chondrocyte function. Six house-bred Merino sheep (age 4-6 years) underwent two-stage surgery of their femoral condyles for autologous chondrocyte transplantation (ACT). First, cells were isolated from biopsies and expanded in vitro for 2 weeks using standard culture techniques. In a second session, three defects were made and either left untreated, covered with periosteal flap alone, or in combination with chondrocyte suspensions injected under the flaps. After 16 weeks, biopsies were taken from the operated knees at the defect sites and from the untreated condyle. Specimens were processed for safranin O and electron microscopy, and for immunofluorescence using three different polyclonal anti-HAS antibodies recognizing one or all of the three known mammalian HAS. Control and regenerating tissues were compared regarding their morphology and the expression of HAS, in relation to collagens type I and II, and adult cartilage proteoglycan core protein. In comparison with untreated defects or with periosteal flap alone, ACT provided a neocartilage with better-differentiated morphology. In healthy joint cartilage, about 50% of the chondrocytes expressed HAS, independent of antibody. Following ACT, a higher density of chondrocytes was observed, of which more than 75% expressed HAS, whereas the regenerates without ACT showed a lower density of HAS-expressing cells. We propose to use HAS immunofluorescence as an additional marker of matrix synthesis by chondrocytes and joint cartilage regeneration.

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.

Angiogenesis and vascular remodeling by intussusception: from form to function.

During most instances of angiogenesis, not only are the capillaries or terminal vessels generated and modified, but the supplying vascular system is subjected to remodeling as well. Intussusception, i.e., transluminal pillar formation, is one essential mechanism for growth, arborization, bifurcation remodeling, and pruning. Complex and efficient vascular beds can thus be generated by local interactions between vascular cells and hemodynamic conditions.

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 delta and associated parameters were quantitatively assessed in arterial and venous microvessels ranging in diameter from 30 to 100 microm. 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, delta = 3.0. Significant differences were observed between delta at arterial bifurcations (2.70 to 2.90) and delta 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.

Pericytes in experimental MDA-MB231 tumor angiogenesis.

The role of pericytes (PCs) during embryonic or tumor angiogenesis is a matter of debate. We studied the expression of cytoskeletal, membrane, and matrix markers in experimental tumors of the human mammary ductal adenoma MDA-MB231 cell line that were grown on avian chorioallantoic membranes (CAMs) from incubation day 10 to 18 (chick) or 8 to 15 (quail). The expression patterns of alpha-smooth muscle actin (alphaSMA) and desmin, of adhesion molecules beta1 integrin and neurothelin, and of fibronectin and laminin were analyzed with conventional and confocal laser scanning microscopy. The CAM arterial wall showed strong alphaSMA signal in all smooth muscle cell layers but the innermost layer, which was desmin positive. Ramified alphaSMA-negative cells with delicate desmin staining accompanied most minor vessels and were also seen basal to the capillary plexus indicating the presence of PCs. In the tumor nodules, a diffuse alphaSMA signal without definite relationship to vascular structures was detected. Strongly desmin-positive, alphaSMA-negative cells were frequent in the zone of contact to the CAM in small nodules, and were scattered in larger tumors. In some regions they were associated with microvessels, and in others appeared detaching from endothelial cells (ECs) or as single migrating cells. We conclude that: (a) the CAM tumor angiogenesis assay is useful for studying PC/EC interactions, (b) PCs are recruited from the CAM into experimental tumor nodules, (c) variability of vasculature in MDA-MB231 tumors may be due to variable PC/EC interactions, and (d) alphaSMA should be used with caution as a general PC marker.

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.