A microcarrier-based cocultivation system for the investigation of factors and cells involved in angiogenesis in three-dimensional fibrin matrices in vitro

Angiogenesis in situ includes coordinated interactions of various microvascular cell types, i.e., endothelial cells, pericytes and perivascular fibroblasts. To study the cellular interactions of microvascular cells in vitro, we have developed a microcarrier-based cocultivation system. The technical details of this method include seeding of endothelial cells on unstained cytodex-3 microcarriers and seeding of pericytes, fibroblasts or vascular smooth muscle cells on microcarriers which have been labeled by trypan blue staining. A mixture of both unstained and trypan blue-stained microcarriers was subsequently embedded in a three-dimensional fibrin clot. The growth characteristics of each cell type could be conveniently observed since the majority of cells left their supporting microcarriers in a horizontal direction to migrate into the transparent fibrin matrix. As differently stained microcarriers were randomly arranged in the fibrin matrix, the characteristic patterns of the microcarriers allowed location of particular points of interest at different developmental stages, facilitating the observation of cellular growth over the course of time. One further advantage of this microcarrier-based system is the possibility of reliably quantifying capillary growth by determination of average numbers of capillary-like formations per microcarrier. Thus, this model allows convenient evaluation of the effects of non-endothelial cells on angiogenesis in vitro. By using this coculture system, we demonstrate that endothelial capillary-like structures in vitro do not become stabilized by contacting vascular smooth muscle cells or pericytes during the initial stages of capillary formation.     

Pericyte involvement in capillary sprouting during angiogenesis in situ

Summary To investigate the participation of microvascular pericytes in the process of capillary sprouting, we examined whole-mount preparations of the rat mesentery by use of a double immunofluorescence approach. Angiogenesis was induced by intraperitoneal injections of either the mast cell-degranulating substance compound 48/80 or tumor cell-conditioned medium. Capillary sprouts were visualized by staining with rhodaminconjugated phalloidin and pericytes were simultaneosly stained by an antibody to the intermediate filament protein desmin. Developing pericytes were negative for the smooth-muscle isoform of -actin, bbut were clearly reactive for desmin. Pericytes appear to be involved in the carliest stages of capillary sprouting. Pericytes were regularly found lying at and in front of the advancing tips of endothelial sprouts. At many sites pericytes were seen to bridge the gap between the leading edges of opposing endothelial sprouts, which were apparently preparing to merge, suggesting that pericytic processes may serve as guiding structures aiding outgrowth of endothelial cells.     

The presence of pericytes and transitional cells in the vasculature of the human dental pulp: an ultrastructural study

The aim of this study was to determine the ultrastructural characteristics of the microvasculature of healthy human dental pulp, with particular reference to pericytes. Pulp tissue was taken from healthy impacted third molars following extraction. Eight teeth were obtained from 17- to 25-year-old patients and pulp tissue was processed for examination using standard techniques for transmission electron microscopy. The pulp was rich in capillaries composed of endothelial and peri-endothelial cells in a 4 : 1 ratio. Endothelial cells contained typical and abundant Weibel–Palade bodies. Three types of peri-endothelial cells were identified: pericytes, transitional cells and fibroblasts. Pericytes were embedded within the capillary basement membrane. Transitional cells were partly surrounded by basement membrane, but separated from the endothelium by collagen fibrils; fibroblasts were outside, but adjacent to the basement membrane and closely associated with collagen fibrils. Pericytes and transitional cells, but not peri-endothelial fibroblasts, contained low numbers of dense bodies similar to the endothelial Weibel–Palade bodies. Our observations are consistent with the hypothesis that, during normal tissue turnover, some pericytes may originate from endothelium and migrate away from the vessel wall to undergo transition to a fibroblastic phenotype.     

Transient Expression of Iron Transport Proteins in the Capillary of the Developing Rat Brain

Iron is essential for normal brain function and its uptake in the developing rat brain peaks during the first two weeks after birth, prior to the formation of the blood–brain barrier (BBB). The first step of iron transport from the blood to the brain is transferrin receptor (TfR)-mediated endocytosis in the capillary endothelial cells. However, the subsequent step from the endothelium into interstitium has not been fully described. The goal of this study was to examine the expression of iron transport proteins by immunodetection and RT–PCR in the developing rat brain. Tf and TfR are transiently expressed in perivascular NG2+ cells of the capillary wall during the early postnatal weeks in the rat brain. However, MTP-1 and hephaestin were expressed in endothelial cells, but not in the NG2+ perivascular cells. Immunoblot analysis for these iron transfer proteins in the developing brain generally confirmed the immunochemical findings. Furthermore, the expression of Tf and TfR in the blood vessels precedes its expression in oligodendrocytes, the main iron-storing cells in the vertebrate brain. RT–PCR analysis for the primary culture of endothelial cells and pericytes revealed that Tf and TfR were highly expressed in the pericytes while MTP-1 and hephaestin were expressed in the endothelial cells. The specific expression of Tf and TfR in brain perivascular cells and MTP-1 and hephaestin in endothelial cells suggest the possibility that trafficking of elemental iron through perivascular cells may be instrumental in the distribution of iron in the developing central nervous system.     

Light- and electron-microscopic study on the maturation of the primary portal plexus during the perinatal period in rats

The maturation of the capillaries of the primary portal plexus in rats during the perinatal period has been studied light- and electron-microscopically. The number of capillaries covering the median eminence and of those invading the nervous tissue (capillary loops) increases significantly with age. Capillary loops were observed as early as the 18th fetal day. The mitotic divisions of the endothelial cells within the preexisting capillaries seem to be the main reason for the vascular growth. Immature capillaries with a characteristic narrow lumen are surrounded by a fuzzy basal lamina; their wall is formed by a generally expanded endothelium with rather sparse organelles and inclusions, and by minute flattened areas. The maturation of the capillary results in a progressive spread of flattened endothelium followed by an enlargment of the capillary lumen. Moreover, a rising concentration of organelles and inclusions, relatively numerous luminal microvilli, and a dense and uniform basal lamina become noticeable as capillary differentiation proceeds. These data are thought to reflect the progressive increase in the metabolic activity of the endothelium as well as the establishment of capillary patency during the perinatal period of rats.     

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.     

Contact-dependent inhibition of angiogenesis by cardiac fibroblasts in three-dimensional fibrin gels in vitro: implications for microvascular network remodeling and coronary collateral formation

Angiogenesis and coronary artery collateral formation can improve blood flow and thereby prevent myocardial ischemia. The role of perivascular fibroblasts in neovascularization remains incompletely understood. Here we investigated the effects of epicardial and myocardial fibroblasts on angiogenesis in vitro by using a serum-free microcarrier-based fibrin gel angiogenesis system. To clearly distinguish between different cell types, we either stained endothelial cells or fibroblasts in the living with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine-perchlorate (DiI). In cocultures, low numbers of heart fibroblasts stimulated endothelial sprouting, and capillary growth was also induced by fibroblast-conditioned media, indicating a paracrine mechanism. Capillary formation was decreased by increasing the density of fibroblasts in the cocultures, indicating contact-dependent inhibition. Using time-lapse studies, it turned out that close contacts between fibroblasts and endothelial cells resulted in rapid retraction of endothelial cells or, rarely, in cell death. Depending on the local ratio of fibroblasts to endothelial cell numbers, fibroblasts determined the location of capillary growth and the size of developing capillaries and thereby contributed to capillary network remodeling. In contrast to primary heart fibroblasts, NIH 3T3 fibroblasts did not display contact-dependent inhibition of endothelial sprouts. NIH fibroblasts were frequently seen in close association with endothelial capillaries, resembling pericytes. Contact-dependent inhibition of angiogenesis by epicardial fibroblasts could not be reversed by addition of neutralizing anti-TGF-β1 antibodies, by addition of serum, of medium conditioned by hypoxic tumor cells or myocardium, by various cytokines or by growing cocultures under hypoxic conditions. Our results implicate a pivotal role of periendothelial mesenchymal cells for the regulation of microvascular network remodeling and collateral formation. Received: 15 September 1997 / Accepted: 6 April 1998     

Changes in distribution of extracellular matrix proteins during wound repair in corneal endothelium

The distribution of fibronectin (FN) and laminin (LM) in non-injured and injured rat corneal endothelium in vivo was investigated by light microscopy using immunoperoxidase cytochemistry. In non-injured tissues, both FN and LM have distinct pericellular staining patterns and exhibit some diffuse cytoplasmic staining. After a circular freeze injury, cells migrating into the wound area at 24 hr lack the characteristic pericellular staining observed in non-injured cells but show cytoplasmic staining for both extracellular matrix glycoproteins. Endothelial cells on the periphery of such preparations do not partake in wound repair and retain their pericellular staining patterns. Forty-eight hours after injury, cells have filled in the wound area but are disorganized. They display intracellular FN and LM staining but do not demonstrate any pericellular staining. When observed 10 days after injury, a uniform monolayer has formed but neither FN nor LM is detected pericellularly. By 14 days post injury, endothelial cells in the wound area display pericellular FN patterns but not LM patterns. This may reflect differences in the function of each glycoprotein in maintaining the attachment of the endothelium to Descemet's membrane.     

Hematoencephalic barrier. Ultrastructure and histophysiology of the endothelium capillary of the neuronal nuclei of the mesencephalon.

The ultrastructure of the dorsal periaqueductal nucleus capillaries of the mesencephalon in the cat was studied under the electron microscope in relation to the hematoencephalic barrier, and its four structural levels: 1. Endothelium; 2. Basal membrane; 3, Pericytes; and 4. Glial prolongations. An analysis was performed of what occurs in these four components (in a non-experimental histophysiological state, and without manipulation by markers) in the thinnest capillaries of the centre of the mesencephalic neuronal nucleus. Special attention was placed on the first diffusion barrier formed by the endothelium capillary as the intimate guardian of the Central Nervous System (C.N.S.) neurons. The C.N.S. capillaries are formed from the continuous endothelium, with no fenestrations, and hermetic joining complexes, without pinocytosis vesicles on both sides of the plasmatic membrane (adluminal and external), and surrounded by a continuous basal membrane. The non-fenestrated capillaries of the C.N.S. are less permeable than those with similar characteristics located in other areas. In the C.N.S. these capillaries form a selective physiological barrier which determines the size of the molecules that are permitted to cross the capillary wall. It is suggested that the electron-dense globules found in the endothelium cytoplasm may be molecules assimilated from the blood, which might represent the first level or step to the selective diffusion entrusted to the hematoencephalic barrier. It is also suggested that the elongated electron-dense particles found in the endothelium cytoplasm and basal membrane may be macromolecules which are normally retained for an active defensive function. They would represent the first and second level or steps of the retention performed by the hematoencephalic barrier which blocks their passage to the confined space of the perivascular capillary.     

Isolation of villous microvessels from the human placenta

A method for isolating the microvessels of the human placental villi has been developed in order to culture perivascular cells. It consists of an initial selection of the villi by serial sieving. The villi retained by the 75 μm sieve were digested by collagenase-dispase. A Percoll gradient permitted the isolation of microvessels still surrounded by stromal fibres and cells. Another digestion by collagenase-dispase eliminated the contaminant elements and allowed, after a new Percoll gradient, microvessels with endothelium, basement membrane and a few perivascular cells to be obtained. Each step of the isolation of microvessels was monitored by light or electron microscopy. Our study confirms the isolation of microvessels embedded in their basement membrane and the preservation of endothelial and perivascular cells after digestion. This method, which has permitted the culture of placental endothelial cells and pericytes, appears of interest for studying microvascular angiogenesis and permeability.     

A scanning electron microscopy of the interstitial tissue of the boar testis

In this study, the architecture of the interstitial tissue of the boar testis was examined by using scanning and transmission electron microscopes. The boar testis was remarkable for the abundance of interstitial tissue, and Leydig cells having many microvilli in their surface were almost round in shape. Both bundles of collagen fibers and networks of reticular fibers were observed around the Leydig cells. The capillary in the interstitial tissue of the boar was a muscle type, and both pericytes and collagen fibers were observed around the capillaries. The lymphatic capillary was poorly developed in the interstitial tissues of the boar testis. Endothelial cells were the only component of the capillary wall, and anchoring filaments were often observed on the abluminal surface of the endothelium.     

Changes in the subendothelial compartment during the maturation process of cerebral microvessels

Basement lamina and pericytes of growing blood microvessels were analyzed in the chick embryo optic tectum, from the 8th incubation day to hatching. Formation of the basement lamina and morphological changes of the pericytes take place in a short range of time, but late in the embryonic life, when also the blood brain barrier (bbb) devices are developing. The spatial and temporal coincidence between basement lamina formation, endothelium tight junction differentiation, and perivascular arrangement of the astrocytic glia, indicates that these events are correlated and corroborates the hypothesis that the glia needs an extracellular matrix to induce the junctional system maturation in the neural endothelia. Pericytes are irregular in shape during the early neural angiogenesis and smooth and flattened later, as the basement lamina synthesis is taking place; these cells represent a second line of barrier beyond the endothelium when the bbb is immature, owing to their phagocytic and digestive properties.     

Electron microscopical studies on the genesis of white adipocytes: differentiation of immature pericytes into adipocytes in transplanted preadipose tissue.

In order to clarify the relationships between perivascular cells, capillaries and fat cells, with a special reference to the origin of fat cells, we have made a light and electron microscopical study on the developing epididymal adipose tissue of newborn to 5-week-old rats, and also on the differentiating, transplanted epididymal preadipose tissue from 6-day-old rats. Development of epididymal preadipose tissue progressed rapidly 6 or 7 days after birth. The preadipose tissue on the 5th day after transplantation consisted of differentiated areas with many mature fat cells, and of undifferentiated areas in which these cells were scanty. In the differentiated areas of developing epididymal preadipose tissue, both in situ and transplanted, many fat cells seemed to develop in the area immediately adjacent to growing capillaries, but cells intermediate between perivascular cells and preadipocytes were seldom observed. However, in undifferentiated areas of transplanted tissue, we found ultrastructural evidence that immature pericytes of capillaries can differentiate into preadipocytes.     

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.     

The microvascular pattern and perivascular linings of the area postrema

Summary The microvasculature and perivascular linings of the area postrema (A.P.) were studied electron microscopically with the ultrathin section and freeze-etching techniques. Special attention was given to the intercellular contacts of the different cellular entities. Two types of microvascular segments were identified. The endothelium of these vascular segments reveals fenestrations and a high pinocytotic activity. There are no significant differences in the frequency and distribution of the endothelial openings between both types of capillaries. The endothelium of the blood vessels, however, is joined by different types of tight junctions. Focal tight junctions occur between pericytes and the endothelium, and between leptomeningeal cellular elements in the perivascular space. The cell membrane of the perivascular glia shows intramembrane particles which are either distributed at random or organized in the form of membrane-associated orthogonal particle complexes (MOPC, Dermietzel, 1974). The significance of these findings is discussed with respect to the modified blood-brain barrier mechanism in the A.P.Supported by a grant of the Deutsche Forschungsgemeinschaft (SFB 114 Bionach) to R. DermietzelThe authors are indebted to Mrs. D. Schünke and Mr. R. Eichner for technical assistance and Mr. A. Stapper for preparing the diagram     

Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse.

Development of a vascular system involves the assembly of two principal cell types - endothelial cells and vascular smooth muscle cells/pericytes (vSMC/PC) - into many different types of blood vessels. Most, if not all, vessels begin as endothelial tubes that subsequently acquire a vSMC/PC coating. We have previously shown that PDGF-B is critically involved in the recruitment of pericytes to brain capillaries and to the kidney glomerular capillary tuft. Here, we used desmin and alpha-smooth muscle actin (ASMA) as markers to analyze vSMC/PC development in PDGF-B-/- and PDGFR-beta-/- embryos. Both mutants showed a site-specific reduction of desmin-positive pericytes and ASMA-positive vSMC. We found that endothelial expression of PDGF-B was restricted to immature capillary endothelial cells and to the endothelium of growing arteries. BrdU labeling showed that PDGFR-beta-positive vSMC/PC progenitors normally proliferate at sites of endothelial PDGF-B expression. In PDGF-B-/- embryos, limb arterial vSMC showed a reduced BrdU-labeling index. This suggests a role of PDGF-B in vSMC/PC cell proliferation during vascular growth. Two modes of vSMC recruitment to newly formed vessels have previously been suggested: (1) de novo formation of vSMC by induction of undifferentiated perivascular mesenchymal cells, and (2) co-migration of vSMC from a preexisting pool of vSMC. Our data support both modes of vSMC/PC development and lead to a model in which PDGFR-beta-positive vSMC/PC progenitors initially form around certain vessels by PDGF-B-independent induction. Subsequent angiogenic sprouting and vessel enlargement involves PDGF-B-dependent vSMC/PC progenitor co-migration and proliferation, and/or PDGF-B-independent new induction of vSMC/PC, depending on tissue context.     

Perivascular cells expressing annexin A5 define a novel mesenchymal stem cell-like population with the capacity to differentiate into multiple mesenchymal lineages

The annexin A5 gene (Anxa5) was recently found to be expressed in the developing and adult vascular system as well as the skeletal system. In this paper, the expression of an Anxa5-lacZ fusion gene was used to define the onset of expression in the vasculature and to characterize these Anxa5-lacZ-expressing vasculature-associated cells. After blastocyst implantation, Anxa5-lacZ-positive cells were first detected in extra-embryonic tissues and in angioblast progenitors forming the primary vascular plexus. Later, expression is highly restricted to perivascular cells in most blood vessels resembling pericytes or vascular smooth muscle cells. Viable Anxa5-lacZ+ perivascular cells were isolated from embryos as well as adult brain meninges by specific staining with fluorescent X-gal substrates and cell-sorting. These purified lacZ+ cells specifically express known markers of pericytes, but also markers characteristic for stem cell populations. In vitro and in vivo differentiation experiments show that this cell pool expresses early markers of chondrogenesis, is capable of forming a calcified matrix and differentiates into adipocytes. Hence, Anxa5 expression in perivascular cells from mouse defines a novel population of cells with a distinct developmental potential.     

Über den granulofilamentären Umbau von Sekretgranula im schleimbildenden Epithel der Endocervix der Frau

Summary Glio-vascular relationships were studied in the developing rat cervical spinal cord with electron microscopy. Capillaries were first evident on the eleventh prenatal day and were surrounded by undifferentiated cells and neuroblasts separated from the capillary wall by a perivascular space. This space persisted until day thirteen, when basement membranes began to appear. Small cellular processes containing glycogen were visible adjacent to the capillary basement membrane beginning at day fifteen and became more prominent as development proceeded. The origin of these cellular processes was not apparent, but they were most likely collaterals of epithelial elements. In prenatal animals neuroblasts or neurons were in direct contact with the capillary basement membrane. Cells resembling adult astrocytes were first observed during the nineteenth day. Astrocytic processes completely invested the capillaries, separating them from neurons in the spinal cord of all postnatal animals.The author wishes to acknowledge the helpful guidance of Dr. Edith A. Maynard during the course of this study.     

Expression of Transferrin Binding Protein in the Capillaries of the Brain in the Developing Chick Embryo

Transferrin-binding protein (TfBP) has been shown to be a novel protein, structurally related to the chicken heat shock protein 108. The physiological function of this protein, however, has not yet been established. Antiserum to TfBP selectively stains transferrin- and iron-rich oligodendrocytes and choroidal epithelium in the adult and embryonic chick brain, suggesting a role for this protein in transferrin and iron storage in these cells. In this study, we further demonstrate TfBP-immunoreactivity (IR) in the blood vessels of the embryonic chick central nervous system. A strong TfBP-IR was present in blood vessels from E6, declined from E10 and was absent by E18. Thus, the expression of the TfBP in the blood vessels precedes its expression in the oligodendrocytes. At the subcellular level, TfBP-IR was confined to the cytoplasm of capillary pericytes while the Tf-receptor IR was associated with the capillary endothelium of the brain. The up-regulated expression of TfBP, together with the Tf-receptor of the brain capillaries, suggests that pericytes may be associated with the high iron uptake required for the metabolic demands of the developing brain. D. W. Kim and H. N. Lee contributed equally to this work.     

Extracellular perivascular connective tissue space in the medial terminal nucleus of the accessory optic system in rats

Summary Two types of capillaries were found in the medial terminal nucleus of the rat accessory optic system. Type one capillaries are surrounded by glial processes and lack a perivascular space, whereas the type two capillaries and arterioles show a distinct extracellular perivascular space often filled with collagen fibrils. An internal as well as an external basal lamina lines these spaces. No fenestration of the endothelium was observed.This study was supported by N. I. H. Grant RR-00165 to Yerkes Regional Primate Research Center, and N. I. H. Grants R01-EY 00565-03 and R01-EY 00638-02 to J. Tigges. The excellent technical assistance of Mrs. G. L. Luttrell is gratefully acknowledged. We thank Dr. W. K. O'Steen for providing the rats and Mr. F. H. Kiernan for photographic help. Special thanks are due to Mrs. B. A. Olberding, in charge of the maintenance of the electron microscope.