Two- and three-dimensional ultrastructural observations of angiogenesis in juvenile hemangioma

Two- and three-dimensional electron microscopic observations by a serial sectioning method revealed endothelial sprouts with intracytoplasmic vacuolization in rapidly growing human juvenile hemangioma. A large vacuole bounded by a single unit membrane was enclosed in the cytoplasm and on the inner aspect of the vacuolar membrane several short microvilli were demonstrated. These appearances have not been reported before. The presence of microvilli in the vacuole indicates that the endothelium has reached the point of differentiation when a vascular lumen forms. In the cytoplasm adjacent to the vacuolar membrane, a significant number of 7 to 10 nm microfilaments were identified. These intracytoplasmic microfilaments are assumed to play a mechanical role in the development of the cytoplasmic vacuole and/or the sprout. The formation of a vacuole observed in the endothelial sprout is similar to the findings of Sabin (1920) by light microscopy in endothelial sprouts in the blood island of the chick embryo. The active endothelial sprout in juvenile hemangiomas is considered to be at least partially responsible for the capillary proliferation and enlargement of the tumor.     

Absence of OX-43 antigen expression in invasive capillary sprouts: identification of a capillary sprout-specific endothelial phenotype

Endothelial cells exhibit a number of unique phenotypes, some of which are angiogenesis dependent. To identify a capillary sprout-specific endothelial phenotype, we labeled angiogenic rat mesentery tissue using a microvessel and capillary sprout marker (laminin), selected endothelial cell markers (CD31, tie-2, and BS-I lectin), and the OX-43 monoclonal antibody, which recognizes a 90-kDa membrane glycoprotein of unknown function. In tissues that were stimulated through wound healing and compound 48/80 application, double-immunolabeling experiments with an anti-laminin antibody revealed that the OX-43 antigen was expressed strongly in all microvessels. However, the OX-43 antigen was completely absent from a large percentage (>85%) of the capillary sprouts that were invading the avascular tissue space. In contrast, sprouts that were introverting back into the previously vascularized tissue retained high levels of OX-43 antigen expression. Double-labeling experiments with endothelial markers indicated that the OX-43 antigen was expressed by microvessel endothelium but was absent from virtually all invasive capillary sprout endothelial cells. We conclude that the absence of OX-43 antigen expression marks a novel, capillary sprout-specific, endothelial cell phenotype. Endothelial cells of this phenotype are particularly abundant in capillary sprouts that invade avascular tissue during angiogenesis.     

Guided migration as a novel mechanism of capillary network remodeling is regulated by basic fibroblast growth factor

 To investigate mechanisms of capillary network remodeling, we developed a serum-free angiogenesis in vitro system in three-dimensional fibrin matrices which allows the study of directional growth of endothelial sprouts, anastomosis, and remodeling (’pruning’) of the primitive plexus toward more elaborated capillary trees. To follow the movements of living endothelial cells by inverse-fluorescence microscopy, we cocultured unlabeled endothelial cells with endothelial cells labeled with the carbocyanine dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI). We show that elongation and retraction of neighboring capillary sprouts occurs simultaneously, resembling a tug-of-war by which endothelial cells are withdrawn from shortening sprouts to become incorporated in other sprouts nearby. For the first time, we directly demonstrate the long-suspected parallel sliding movement of endothelial cells. We show that cell migration persists within immature capillaries even after sprouts have merged to continuous capillary loops, leading to overlapping growth of opposing sprout tips. As a novel concept of capillary remodeling, we distinguish two types of endothelial cell migration: sprouting and guided migration. Sprouting is the de novo invasion of a matrix by endothelial cells, and guided migration is the locomotion of cells along preexistent capillary-like structures. We show that guided migration leads to remodeling of immature capillary networks and to the retraction of sprouts. We describe a method for quantification of sprouting versus guided migration in DiI-mosaic-labeled capillary networks, and we present evidence that endothelial cell-derived basic fibroblast growth factor serves as a chemotactic signal for other cells to migrate along a preestablished capillary-like structure. Accepted: 3 November 1997     

Chondrocytes inhibit endothelial sprout formation in vitro: evidence for involvement of a transforming growth factor-beta.

Using a quantitative in vitro model of spontaneous endothelial sprout formation, we have attempted to define physiological inhibitors of angiogenesis from hyaline cartilage, a tissue whose antiangiogenic properties have been well described. The model consists of embedding bovine microvascular endothelial cell aggregates into fibrin or collagen gels, which results in the formation of radially growing sprouts. When chondrocytes derived from the permanent cartilagenous region of the chick embryo sternum are cocultured with the endothelial cell aggregates, sprout formation is markedly inhibited. Addition of anti-TGF-beta antibodies to the cocultures significantly reduced the inhibitory effect of chondrocytes on sprout formation. Chondrocyte-conditioned medium or exogenously added TGF-beta 1 have a similar albeit transient inhibitory effect. Depletion of TGF-beta from chondrocyte conditioned medium with anti-TGF-beta antibodies and solid-phase protein-A significantly decreases the inhibition of sprout formation. These results demonstrate that a chondrocyte-derived TGF-beta-like molecule inhibits capillary sprout formation in vitro and suggest that the antiangiogenic properties of cartilage may at least in part, be mediated by TGF-beta.     

Continuous and discrete mathematical models of tumor-induced angiogenesis

Angiogenesis, the formation of blood vessels from a pre-existing vasculature, is a process whereby capillary sprouts are formed in response to externally supplied chemical stimuli. The sprouts then grow and develop, driven initially by endothelial-cell migration, and organize themselves into a dendritic structure. Subsequent cell proliferation near the sprout tip permits further extension of the capillary and ultimately completes the process. Angiogenesis occurs during embryogenesis, wound healing, arthritis and during the growth of solid tumors. In this paper we present both continuous and discrete mathematical models which describe the formation of the capillary sprout network in response to chemical stimuli (tumor angiogenic factors, TAF) supplied by a solid tumor. The models also take into account essential endothelial cell-extracellular matrix interactions via the inclusion of the matrix macromolecule fibronectin. The continuous model consists of a system of nonlinear partial differential equations describing the initial migratory response of endothelial cells to the TAF and the fibronectin. Numerical simulations of the system, using parameter values based on experimental data, are presented and compared qualitatively with in vivo experiments. We then use a discretized form of the partial differential equations to develop a biased random-walk model which enables us to track individual endothelial cells at the sprout tips and incorporate anastomosis, mitosis and branching explicitly into the model. The theoretical capillary networks generated by computer simulations of the discrete model are compared with the morphology of capillary networks observed in in vivo experiments.     

In vivo angiogenesis in adult rat skeletal muscle: early changes in capillary network architecture and ultrastructure

The individual structural stages in capillary growth have been identified during development and under pathological circumstances in adults (wound healing, tumors), but there are no data to indicate whether these steps are similar when angiogenesis is induced in a fully differentiated microvascular bed in normal, uninjured adult skeletal muscle. In this study changes in capillary ultrastructure were correlated with capillary density and network morphology to elucidate the sequelae of angiogenesis in adult rat extensor digitorum longus (EDL) muscle whose activity was increased by stimulation at 10rHz (8rh/day). This resulted in an increased capillary/fiber (C/F) ratio (based on staining for alkaline phosphatase) after 4rdays; by 7rdays C/F ratio was increased further, by approximately 50%. The ultrastructure of capillary endothelium in both the EDL and extensor hallucis proprius (EHP) was similar to control muscles after 2rdays of stimulation, whereas endothelial cells in some capillaries in muscle stimulated for 4rdays revealed signs of metabolic activation such as proliferation of organelles (Golgi apparatus, endoplasmic reticulum, ribosomes and mitochondria) and fewer pinocytic vesicles. Luminal surfaces were often irregular with numerous pseudopodial processes. Basement membranes were always present but amorphous regions were observed, particularly near pericyte processes. Unusually small capillary profiles, with either a slit-like lumen or with cisternae but no lumen, probably represented capillary sprouts. The interstitium contained increased collagenous and granular extracellular matrix surrounding capillaries, and numerous activated fibroblasts which were closely apposed to many capillaries. Capillary growth in EHP was also evaluated by confocal microscopy using whole mounts. The complex pattern of vessels underwent remodelling between 2 and 7rdays of stimulation, resulting in more tortuous capillaries with numerous sprouts and loops. These combined observations suggest that angiogenesis may occur by a combination of sprouting, intussusceptive growth and elongation; also, that activation of endothelial cells occurs at the same time as disturbance of basement membranes during the earliest phase of growth and remodelling of the capillary bed. These changes are postulated to occur in connection with increased shear stress and/or capillary wall tension, which have been demonstrated previously.     

SPATIAL DISTRIBUTION AND DEVELOPMENT OF ROOT SPROUTS IN FAGUS GRANDIFOLIA (FAGACEAE)

Root sprouts around 31 Fagus grandifolia parent trees, some declining due to beech bark disease, were studied to describe the pattern of sprout distribution, the ecological importance of this pattern, and the relationship between sprout patterns and parent vigor. Microtome sections of roots were studied to determine the histological origin of sprouts. Sprout distribution was circular and usually centered on the parent tree. Most sprouts were within 8 m of the parent and remained attached to the parent root system even after the sprouts reached 10 yrs of age. Root sprouting in F. grandifolia may be effective in replacing dead parent trees, but the potential for clones to spread is limited. Spatial distribution of sprouts was mostly unaffected by tree vigor. Root sprouts originated from callus tissues associated with wounds on superficial woody roots.     

Incorporation of axonally transported glycoproteins into axolemma during nerve regeneration

The insertion of axonally transported fucosyl glycoproteins into the axolemma of regenerating nerve sprouts was examined in rat sciatic motor axons at intervals after nerve crush. [(3)H]Fucose was injected into the lumbar ventral horns and the nerves were removed at intervals between 1 and 14 d after labeling. To follow the fate of the “pulse- labeled” glycoproteins, we examined the nerves by correlative radiometric and EM radioautographic approaches. The results showed, first, that rapidly transported [(3)H]fucosyl glycoproteins were inserted into the axolemma of regenerating sprouts as well as parent axons. At 1 d after delivery, in addition to the substantial mobile fraction of radioactivity still undergoing bidirectional transport within the axon, a fraction of label was already associated with the axolemma. Insertion of labeled glycoproteins into the sprout axolemma appeared to occur all along the length of the regenerating sprouts, not just in sprout terminals. Once inserted, labeled glycoproteins did not undergo extensive redistribution, nor did they appear in sprout regions that formed (as a result of continued outgrowth) after their insertion. The amount of radioactivity in the regenerating nerves decreased with time, in part as a result of removal of transported label by retrograde transport. By 7-14 d after labeling, radioautography showed that almost all the remaining radioactivity was associated with axolemma. The regenerating sprouts retained increased amounts of labeled glycoproteins; 7 or 14 d after labeling, the regenerating sprouts had over twice as much of radioactivity as comparable lengths of control nerves or parent axons. One role of fast axonal transport in nerve regeneration is the contribution to the regenerating sprout of glycoproteins inserted into the axolemma; these membrane elements are added both during longitudinal outgrowth and during lateral growth and maturation of the sprout.     

Identification of vascular endothelial cells in murine omentum using the lectin, Dolichos biflorus agglutinin: possible applications in the study of angiogenesis

Summary The purpose of this study was to determine whether the plant lectin,Dolichos biflorus agglutinin (DBA), can be used to recognize capillary endothelial cells and their processes during angiogenesis. By means of a peroxidase conjugate of DBA, blood vessels were visualized in whole mounts and ultrathin sections of mouse omentum. A part of this mesentery normally comprises an avascular membrane that is approximately 30 µm in thickness. Changes in the vascular plexus bordering this membrane were induced by intraperitoneal injection of irradiated Landschutz cells. Vascular endothelial cells were precisely and intensely stained, and vasculogenic processes were reliably distinguished from those of other cells. This technique permitted observation of the structure and distribution of capillary sprouts, and their relationship to each other and to pre-existing blood vessels. It was discovered that filiform projections extend from sprout apices. These projections may fuse allowing adjacent sprouts to form a new capillary loop.     

Enhanced angiogenesis in obesity and in response to PPARgamma activators through adipocyte VEGF and ANGPTL4 production

PPARgamma activators such as rosiglitazone (RSG) stimulate adipocyte differentiation and increase subcutaneous adipose tissue mass. However, in addition to preadipocyte differentiation, adipose tissue expansion requires neovascularization to support increased adipocyte numbers. Paradoxically, endothelial cell growth and differentiation is potently inhibited by RSG in vitro, raising the question of how this drug can induce an increase in adipose tissue mass while inhibiting angiogenesis. We find that adipose tissue from mice treated with RSG have increased capillary density. To determine whether adipose tissue angiogenesis was stimulated by RSG, we developed a novel assay to study angiogenic sprout formation ex vivo. Angiogenic sprout formation from equally sized adipose tissue fragments, but not from aorta rings, was greatly increased by obesity and by TZD treatment in vivo. To define the mechanism involved in RSG-stimulated angiogenesis in adipose tissue, the expression of proangiogenic factors by adipocytes was examined. Expression of VEGFA and VEGFB, as well as of the angiopoietin-like factor-4 (ANGPTL4), was stimulated by in vivo treatment with RSG. To define the potential role of these factors, we analyzed their effects on endothelial cell growth and differentiation in vitro. We found that ANGPTL4 stimulates endothelial cell growth and tubule formation, albeit more weakly than VEGF. However, ANGPTL4 mitigates the growth inhibitory actions of RSG on endothelial cells in the presence or absence of VEGF. Thus, the interplay between VEGF and ANGPTL4 could lead to a net expansion of the adipose tissue capillary network, required for adipose tissue growth, in response to PPARgamma activators.     

The interplay of cyclic stretch and vascular endothelial growth factor in regulating the initial steps for angiogenesis

Angiogenesis is regulated by chemical and mechanical factors in vivo. The regulatory role of mechanical factors and how chemical and mechanical angiogenic regulators work in concert remains to be explored. We investigated the effect of cyclic uniaxial stretch (20%, 1 Hz), with and without the stimulation of vascular endothelial growth factor (VEGF), on sprouting angiogenesis by employing a stretchable three‐dimensional cell culture model. When compared to static controls, stretch alone significantly increased the density of endothelial sprouts, and these sprouts aligned perpendicular to the direction of stretch. The Rho‐associated kinase (ROCK) inhibitor Y27632 suppressed stretch‐induced sprouting angiogenesis and associated sprout alignment. While VEGF is a potent angiogenic stimulus through ROCK‐dependent pathways, the combination of VEGF and stretch did not have an additive effect on angiogenesis. In the presence of VEGF stimulation, the ROCK inhibitor suppressed stretch‐induced sprout alignment but did not affect stretch‐induced sprout density; in contrast, the receptor tyrosine kinase (RTK) inhibitor sunitinib had no effect on stretch‐induced alignment but trended toward suppressed stretch‐induced sprout density. Our results suggest that the formation of sprouts and their directionality do not have completely identical regulatory pathways, and thus it is possible to separately manipulate the number and pattern of new sprouts. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:248–257, 2015     

Ca2+-ATPase cytochemistry in the spinal cord microvasculature of prenatal rats

Summary Membrane-bound Ca²⁺-ATPase activity was localized cytochemically in the blood vessels of the spinal cord of rat embryos to obtain a better understanding of the membrane activities of vascular cells.The cytochemical method revealed a growth of the parenchymal vasculature. In the parenchyma, reaction product was dense over the entire plasma membrane of voluminous endothelial cells provided with large nuclei and enriched cytoplasmic organelles, suggesting that the endothelial cells may be of a vascular sprout. The parenchymal vessels with a wide lumen were frequently associated with pericytes, and the Ca²⁺-ATPase activity was diminished in intensity on the luminal surface of the flattened endothelial cells. On the other hand, the endothelium of extraparenchymal capillaries exhibited Ca²⁺-ATPase activity primarily on the luminal surface of the plasma membrane. Quercetin, a Ca²⁺-transporting ATPase inhibitor, considerably decreased the abluminal activity in the voluminous endothelial cells with slit-like vascular lumen and the luminal activity of functioning capillary endothelium as well. Thus, a dual activity of Ca²⁺-ATPase, postulating for the activities of Ca²⁺-transporting ATPase and ecto-ATPase, was closely correlated with the maturation processes of the capillary endothelium.     

A maternal influence on the conditioning to plant cues of Aphidius colemani Viereck, parasitizing the aphid Myzus persicae Sulzer

Abstract. The offspring of parasitoids, Aphidius colemani Viereck, reared on Brussels sprouts and emerging from Myzus persicae Sulzer on a fully defined artificial diet, show no preferences in a four-way olfactometer, either for the odour of the diet, the odour of Brussels sprouts, or the odour of two other crucifers (cabbage and Chinese cabbage). A similar lack of odour preferences is shown when the host aphids are exposed for parasitization (for 48 h) on cabbage, Chinese cabbage or wheat. However, if parasitization occurs on Brussels sprouts, a weak but statistically highly significant response to Brussels sprout odour is observed. Although as many as 30–35% of the parasitoids show no response to any odour, another 35% respond positively to the odour of Brussels sprout compared with responses to the odours of cabbage, Chinese cabbage or wheat of only approximately 10%. An analagous result is obtained when the parent parasitoids are reared on cabbage. In this case, significant positive responses of their offspring to cabbage odour occur only if the 48-h parasitization has occurred also on cabbage. However, with parasitoids from Brussels sprouts parasitizing the aphids for 48 h also on Brussels sprouts, the offspring subsequently emerging from pupae excised from the mummies show no preference for Brussels sprout odour. Thus, although the Brussels sprout cue had been experienced early in the development of the parasitoids, they only become conditioned to it when emerging from the mummy. Both male and female parasitoids respond very similarly in all experiments. It is proposed that the chemical cue (probably glucosinolates in these experiments) is most likely in the silk surrounding the parasitoid pupa, and that the mother may leave the chemical in or around the egg at oviposition, inducing chemical defences in her offspring to the secondary plant compounds that the offspring are likely to encounter.     

Characterization of a ferredoxin:NADP+ oxidoreductase from a nonphotosynthetic plant tissue

A flavoprotein with properties similar to those of ferredoxin:NADP+ oxidoreductases found in the leaves of higher plants has been purified to apparent homogeneity from bean sprouts, a nonphotosynthetic plant tissue. The absorbance and circular dichroism spectra of the bean sprout protein are similar to those of spinach leaf ferredoxin:NADP+ oxidoreductase and an antibody raised against the spinach enzyme recognized the bean sprout enzyme. The bean sprout enzyme catalyzed ferredoxin-dependent electron transfer from NADPH to equine cytochrome c at a high rate but, unlike the spinach enzyme, exhibited little NADPH to 2,6-dichlorophenol indophenol diaphorase activity. The bean sprout enzyme forms a 1:1 electrostatically stabilized complex with ferredoxins isolated from either bean sprouts or spinach leaves.     

Immunohistochemical identification of an extracellular matrix scaffold that microguides capillary sprouting in vivo.

To gain insight into how a naturally occurring scaffold composed of extracellular matrix (ECM) proteins provides directional guidance for capillary sprouting, we examined angiogenesis in whole-mount specimens of rat mesentery. Angiogenesis was studied in response to normal maturation, the injection of a mast cell degranulating substance (compound 48/80), and mild wounding. Confocal microscopy of specimens immunolabeled for elastin revealed a network of crosslinked elastic fibers with a density of 140.8 +/- 37 mm of fiber/mm(2) tissue. Fiber diameters ranged from 180 to 1400 nm, with a mean value of 710 +/- 330 nm. Capillary sprouts contained CD31- and OX-43-positive endothelial cells as well as desmin-positive pericytes. During normal maturation, leading endothelial cells and pericytes were in contact and aligned with an elastic fiber in approximately 80-90% of all sprouts. In wounding and compound 48/80-treated specimens, in which angiogenesis was markedly increased, leading endothelial cells remained in contact and aligned with elastic fibers in approximately 60-80% of all sprouts. These observations indicate that elastic fibers are used for endothelial and pericyte migration during capillary sprouting in rat mesentery. The composition of this elastic fiber matrix may provide important clues for the development of tissue-engineered scaffolds that support and directionally guide angiogenesis.     

Level of endothelial cell apoptosis required for a significant decrease in microvessel density

Endothelial cell apoptosis plays a critical role in the disruption of blood vessels mediated by natural inhibitors of angiogenesis and by anti-vascular drugs. However, the proportion of endothelial cells required to mediate a significant decrease in microvessel density is unknown. A system based on an inducible caspase (iCaspase-9) offers a unique opportunity to address this question. The dimerizer drug AP20187 induces apoptosis of human dermal microvascular endothelial cells stably transduced with iCaspase-9 (HDMEC-iCaspase-9), but not control cells (HDMEC-LXSN). Here, we generated blood vessels containing several HDMEC-iCaspase-9:HDMEC-LXSN ratios, and developed a mathematical modeling involving a system of differential equations to evaluate experimentally inaccessible ratios. A significant decrease in capillary sprouts was observed when at least 17% of the endothelial cells underwent apoptosis in vitro. Exposure to vascular endothelial growth factor (VEGF(165)) did not prevent apoptosis of HDMEC-iCaspase-9, but increased the apoptotic requirement for sprout disruption. In vivo experiments showed the requirement of at least 22% apoptotic endothelial cells for a significant decrease in microvascular density. The combined use of biological experimentation with mathematical modeling allowed us to conclude that apoptosis of a relatively small proportion of endothelial cells is sufficient to mediate a significant decrease in microvessel density.     

Regulation of blood vessel sprouting

Blood vessels are essential conduits of nutrients and oxygen throughout the body. The formation of these vessels involves angiogenic sprouting, a complex process entailing highly integrated cell behaviors and signaling pathways. In this review, we discuss how endothelial cells initiate a vessel sprout through interactions with their environment and with one another, particularly through lateral inhibition. We review the composition of the local environment, which contains an initial set of guidance cues to facilitate the proper outward migration of the sprout as it emerges from a parent vessel. The long-range guidance and sprout stability cues provided by soluble molecules, extracellular matrix components, and interactions with other cell types are also discussed. We also examine emerging evidence for mechanisms that govern sprout fusion with its target and lumen formation.     

The inhibition of potato sprout growth by light.

When potato seed tubers (Solanum tuberosum cv. Pentland Javelin) were stored in darkness or diffuse daylight at 9°C and transferred at intervals to conditions suitable for sprouting, their capacity for sprout growth was unaffected by the presence or absence of light during previous storage. When similar tubers were stored at 10°C, 18°C or 25°C, sprout growth commenced earliest at 25°C, but the date was unaffected by fluorescent light. It was concluded that light did not affect the length of the dormant period, but only the rate of sprout elongation after that period had ceased. When tubers with growing sprouts at 10°C or 18°C were transferred from darkness into fluorescent light, sprout growth virtually ceased. Transfer from light into darkness resulted in immediate sprout growth, at a rate comparable with tubers stored continuously in the dark. Tubers of three Peruvian cultivars, stored in farm-scale diffuse-daylight stores, grew progressively shorter sprouts with increasing daily exposure to light from 30 min to 12 h. Storage of cv. Wilja under 21 Wm^-2 (total) of white fluorescent light for 10 h per day maintained the sprouts at the same length as ten times this light intensity for 1 h per day. In a subsequent experiment with cv. Bintje the 10 h, low-intensity light regime gave slightly shorter sprouts. It appeared that the total light energy falling on the tubers was the dominant factor controlling sprout growth.     

Initiation and pattern of angiogenesis in wound healing in the rat.

The object of this study was to examine the initiation and pattern of capillary growth associated with wound healing. Collagen sponges were implanted subcutaneously in the hind limbs of adult male rats to stimulate the formation of granulation tissue. Blood vessels of the hind limbs of euthanized rats were perfused with Mercox (an acrylic monomer) via the abdominal aorta at selected periods of time following sponge implantation. When the perfusate was completely cured, the sponge and parajacent tissues were excised and subsequently macerated by alternating immersion in 40% KOH and distilled water. Cast replicas of the vascular lumina were coated with gold and imaged by scanning electron microscopy. At 6 hr, punctate depressions at the periphery of the replicas of vein and venule lumina were noted. The depressions represented sites of leukocyte margination. By 24 hr, the depressions increased numerically, indicating a great increase in the sites of leukocyte margination. The number of these depressions decreased by 48 hr. Concomitantly, the depressions representing endothelial cell nuclei became more pronounced, indicating nuclear hypertrophy of these cells. In addition, capillary bud formation was initiated. At 72 hr, capillary buds were quite apparent and arose solely from venules. Between 7 and 14 days, replicas of capillary lumina were longer and formed an elaborate network, presumably by end-to-end, side-to-side, and end-to-side anastomoses. The network was formed circumferential to the sponge and then capillary sprouts entered the sponge's interstitial spaces.     

Angiogenic activity of porcine granulosa cells co-cultured with endothelial cells in a microcarrier-based three-dimensional fibrin gel.

To verify the possible role played by pig granulosa cells in the ovarian angiogenic process, we have developed a reliable in vitro system which allows the evaluation of endothelial sprouting and capillary growth in three-dimensional matrices. Granulosa cells collected from porcine follicles of different size were co-cultured with porcine aortic endothelial cells (PAEC) in a microcarrier-based fibrin gel system; after 2 and 5 days of co-culture, we determined the number and length of all endothelial sprouts; moreover, these parameters were quantified only in capillary-like structures, which were defined as continuous multicellular sprouts at least 200 microm long. In granulosa cells- PAEC co-cultures we observed an increase of angiogenic activity as compared to controls (PAEC alone). Granulosa cells from follicles of different size regulate angiogenesis differently: cells from the small follicle group significantly enhanced endothelial sprouting, while those from the large follicle group favoured mainly capillary elongation. Our observations seem therefore to suggest that the development and growth of thecal vascular bed is controlled by paracrine factors of granulosa cell origin that may induce the formation of a primitive capillary plexus during the early phases of antral follicle growth, which will be remodelled in more advanced phases of follicular development.