Vascular Buds Research Articles

Tissue-Specific Activity of Two Manganese Superoxide Dismutase Promoters in Transgenic Tobacco

In eukaryotes, manganese superoxide dismutase is a nuclear-encoded protein that scavenges superoxide radicals in the mitochondrial matrix. We have isolated two manganese superoxide dismutase genes from Nicotiana plumbaginifolia L. and fused the 5' upstream regulatory region of these genes to the beta-glucuronidase reporter gene. The two gene fusions displayed a differential tissue specificity in transgenic tobacco (Nicotiana tabacum). Promoter activity of the SodA1 gene fusion was found in the pollen, middle layer, and stomium of anthers, but was usually undetectable in vegetative organs of mature plants. The SodA2 gene fusion was expressed in the leaves, stems, roots, and flowers. SodA2 promoter activity was most prominent in the vascular bundles, stomata, axillary buds, pericycle, stomium, and pollen. Histochemical analysis of succinate dehydrogenase activity suggested that the spatial expression of the two gene fusions is generally correlated with mitochondrial respiratory activity.

Morphologic differences of the vascular buds in the vertebral endplate: scanning electron microscopic study.

Vascular buds in rabbit vertebral endplates were examined by scanning electron microscopy of corrosion casts. To examine morphologic differences between vascular buds in two regions of the vertebral endplate (inner anular and nucleus pulposar). Vascular buds are specific structures present at the vertebral endplate that are important as nourishing channels. There is a significant difference in permeability between the lateral portion (inner anular) and the central portion (nucleus pulposar) of the endplate, the latter usually being permeable and the former being impermeable. Morphologic differences between vascular buds in the two regions have not been investigated previously. Eight 20-week-old rabbits were used. Vascular buds in rabbit vertebral endplates were examined by scanning electron microscopy of corrosion casts. The vascular buds in the region of the inner anulus form simple loops, but those in the area near the nucleus pulposus exhibit swollen and complex coil-like loops. Although they differ structurally, the average number of vascular buds per area does not vary between the two regions. We suggest that the morphologic difference between the vascular buds in the two regions (inner anular and nucleus pulposar) plays a principal role in permeability at the endplate.

Scanning electron microscopic observations of the vascular structure of vertebral end-plates in rabbits.

We used scanning electron microscopy to examine the microcirculation in the vertebral end-plates of rabbits. The arteriola arborizes and swells at the end-plate and then coils to make a microvessel loop. This loop is the structure we refer to as a vascular bud. We suggest that these loop structures of the capillaries play a major role in the nutrition of the intervertebral discs.

Peculiar structures of the blood vessels forming the secondary ossification center in the rat femoral heads.

To investigate the development of the blood vessels forming the secondary ossification center in the femoral heads of growing rats, we observed the specimens of the proximal femurs having vascular casts using both an optical and a scanning electron microscopy (SEM). Histologically, the vascular invasion occurred from the lateral part of the cartilaginous epiphysis, prior to the appearance of the ossification center. The vascular casts were prominent within the cellular spaces consisting of a few hypertrophic chondrocytes. The SEM study suggested that the vascular buds had peculiar structures and invaded the cartilaginous epiphysis by breaking through the cellular spaces. The blood vessels budded out from the arch of the lateral epiphyseal vessels toward the femoral heads. The sizes of the large-caliber vascular buds of about 50 microns in diameter were similar to those of the cellular spaces, and they penetrated into the epiphysis by repeated merging and branching. Many deep impressions were noted on the surface of the vascular buds, and the tips showed the peculiar shape similar to a suction cup.

Intense vascular sprouting from rat femoral vein induced by prostaglandins E1 and E2.

The formation of new capillaries from the rat femoral vein was specifically explored to assess whether venous vessels of this caliber may participate in the process of angiogenesis. Prostaglandins of the E series (PGE1 and PGE2) were administered into the soft connective tissue surrounding the rat femoral vessels as angiogenic inducers. In these conditions, between 2 and 7 days, a great number of new capillaries were observed in the media of the femoral vein, arising from the endothelial cells (EC) in the intima. The events of the capillary growth from the femoral vein included EC activation, local degradation of the basal membrane followed by migration and proliferation of EC, solid sprout formation with posterior canalization, development of a new basal membrane, and appearance of pericytes around the new capillary. Although numerous vascular buds were also observed arising from the small venules and capillaries in the periadventitial tissues, they were separated at first from those in the media of the femoral vein by the venous adventitia. Later, connections were observed between both newly formed microcirculations. The present study shows the capacity of PGE1 and PGE2 in the extravascular position of inducing capillary sprouting from veins. Furthermore, the observations provide greater evidence that vessels with characteristics similar to those of the rat femoral vein may contribute to angiogenesis, on occasion with an intense neovascularization. This fact may be of interest for the establishment of a functional circulation after angiogenesis by anastomoses of the new capillaries with those arising from pre-existing vessels of greater caliber than the venules.

First appearance, distribution, and origin of macrophages in the early development of the avian central nervous system

A phagocytic cell system of hemopoietic origin exists in the early avian embryo (Cuadros, Coltey, Nieto, and Martin: Development 115:157-168, '92). In this study we investigated the presence of cells belonging to this system in the central nervous system (CNS) of chick and quail embryos by using both histochemical staining for acid phosphatase and immunolabelling with antibodies recognizing cells of quail hemangioblastic lineage. The origin of these cells was traced in interspecific chick-quail yolk sac chimeras. Hemopoietic cells were detected within the CNS from developmental stage HH15 on, and steadily increased in number at subsequent stages. Analysis of yolk sac chimeras revealed that most of these cells were of yolk sac origin, although some hemopoietic cells of intramebryonic origin were also found in the CNS. Immunocytochemical, histochemical, and ultrastructural characterization allowed us to identify hemopoietic cells in the CNS as macrophages. These cells were consistently found in the brain vesicles and spinal cord, appearing (1) between undifferentiated neuroepithelial cells at dorsal levels of the CNS; (2) in areas of cell death; (3) in the marginal layer in close relationship with developing axons; (4) in large extracellular spaces in the subventricular layer; (5) on vascular buds growing through the marginal and subventricular layers; and (6) in the ventricular lumen. Macrophages in different locations varied in morphology and ultrastructure, suggesting that in addition to their involvement in phagocytosis, they play a role in other processes in the developing CNS, such as axonal growth and vascular development. The first macrophages migrate to the CNS independently of its vascularization, apparently traversing the pial basal lamina to reach the nervous parenchyma. Other macrophages may enter the CNS together with vascular buds at subsequent stages during CNS vascularization.

Pericytes as a Supplementary Source of Osteoblasts in Periosteal Osteogenesis

In the adult rat femur, lifting a periosteum strip with microscopic bone flakes on its deep surface, if performed without damaging the surrounding microcirculation, rapidly leads to new bone formation and angiogenesis. Using vascular labeling, the pericytes and endothelial cells (ECs) were labeled with monastral blue (MB) in the preformed, preexisting postcapillary venules of the periosteal microcirculation. MB was detectable by light and electron microscopy and it persisted in some of the daughter cells. Between one and 21 hours, the MB labeling was restricted to the pericytes and ECs of postcapillary venules. Immediately afterward, both pericytes and ECs of these vessels were activated and continued to show MB. The phenomenon of pericyte activation includes enlargement, disruption of their basal lamina, separation from the walls of the preformed vessels, and the presence of mitotic figures. At this stage, activated pericytes with MB in their cytoplasm, fibroblast-like cells, and transitional cell forms between them were seen in interstitial areas. After 27 hours, vascular buds appeared and MB was detected in some ECs and pericytes. Between three and six days, when bone-tissue development was observed, some osteoblasts were MB labeled. Previous findings support the hypothesis that when the periosteum is activated, the process of bone formation from cells already present in the periosteum is augmented by proliferation and differentiation of pericytes, which contribute a supplementary population of osteoprogenitor cells.

Retinal and Vitreal Neovascularization in Retinopathy of Prematurity

The angioarchitecture of vitreal and retinal neovascularizations produced experimentally in the eyes of kittens aged 2 to 9 weeks was studied with scanning electron microscopy. Various forms of new retinal and vitreal vessels were observed depending on topographic locations. Intraretinal neovascularization was observed at the retinal periphery as it grew toward the avascular zone in forms of short vascular buds, aneurysmal outgrowths, and neovascular loops. Posterior or to this frond of neovascularization, intertwining intraretinal telangiectasia was observed. At the posterior pole, capillaries with microaneurysms extended posteriorly toward the deeper layers of the retina from the vascular trunks at the nerve fiber layer. Vitreal neovascularization broke through the internal limiting membrane and exhibited aneurysmal outgrowths, clusters of glomerular swellings, and sinusoidal vascular channels. At the optic disc, vitreous neovascularization took the form of aneurysmal outgrowths and long vascular buds. Vitreal neovascularization showed different characteristics from the intraretinal neovascularization. We hypothesize that the topographic variation of the angioarchitecture of retinal and vitreal neovascularizations depends on the maturity of the vessels and might be related to the hemodynamics at each site.

Morphological analysis of neovascularization at early stages of rat splenic autografts in comparison with tumor angiogenesis

This study was undertaken to reveal the neovascularization at early stages of splenic autografts three-dimensionally, to illustrate the differences between it and tumor angiogenesis, and to establish its origin. Early vascular formation after transplantation of the rat spleen or Walker tumor into the major omentum was examined by using a video macroscope, vascular casting methods and the organ culture technique. A complex vascular network layer (vascular cortex) was first formed beneath the capsule of an autograft; later, vascular buds grew from this network toward the necrotic center. They anastomosed and changed into a form resembling withered twigs (vascular medulla). Tumor angiogenesis did not present such morphological features and was characterized by capillary loop formation with a columnar vertex resembling an "inverted V". This fundamental structure did not change throughout angiogenesis except for dilation and irregularity of vascular diameter. The organ culture technique demonstrated that the preliminary vasculature was formed in splenic autografts by regeneration of preexisting vessels in the graft and not by invading capillaries. Transmission electron microscopy showed that the cells present had characteristics of sinus endothelial cells. These results suggest that preexisting sinus endothelial cells rearrange themselves after devascularization and reconstruct a new vasculature that anastomoses with the penetrating capillaries. This mechanism establishes vascular circulation at an early stage, and accelerates regeneration of the splenic autograft before complete necrosis.

The Influence of Calving and Puerperium on Clinical and Histopathologic Changes Associated with Laminitis and Sole Ulceration in a Group of First‐Carving Cows

The left hind and front claws of 43 heifers having recently calved were studied for signs of laminitis and sole ulceration. The left hind and front claws of 8 non-pregnant heifers served as controls. Thirty claws were selected for histologic examination. No influence of parturition and puerperium was found on signs of laminitis in both clinical and histologic investigations. A remarkable histologic finding was the occurrence of numerous vascular buds in the solar corium. It is suggested that the vascular buds may give rise to the glomus-like vascular clusters, which are commonly found in older cattle.

脱歯創の骨性治癒における血管構築の変化について

Successive changes in the vascular pattern during the osseous healing of extraction wounds were investigated by studying microvascular casts under a scanning electron microscope. The casts were prepared utilizing the plastic injection method, after the extraction of the upper four incisors of the Japanese monkey (Macaca fuscata). Five days after extraction, vascular buds have sprouted from a pre-existing blood vessel on the alveolar wall into the blood clot, and leakage of the plastic injected was found from the tips of these buds. One week after extraction, newly-formed vessels have extended widely to the socket center, and dilated vessels have arborized towards the socket opening. Two weeks after extraction, the socket was filled with thick, newly-formed vessels. In the socket fundus, the woven bone was formed between irregular vascular networks, but was arranged different in the socket wall. Four weeks after extraction, the new bone forming on the socket wall became thickened and converted to a lamella-like bone. Inside it the woven bone was raised from the fundus, and blood vessels leaving it were decreased in their thickness and passed toward the socket center. Five weeks after extraction, the new bone structures came up to the level of the socket opening, the surface of which appeared as a shallow concavity (pivot), from which vascular bundles were directed to the socket opening. A beginning of the bone-remodeling was seen in osseous trabeculae in the socket fundus. Six weeks after extraction, almost all of the socket became filled with new trabeculae, between which, fine vascular networks were sorted out and communicated with the periosteal vascular network beyond the socket margin. The interalveolar septum between the extraction sockets was thickened by deposition of the lamella-like bone to be remodeled to a cancellous bone. It can be said that microvascular patterns formed through all stages of the osseous healing of the extraction wounds contributed to a woven bone formation and its development.

Fetal microcirculation of abnormal human placenta II. Scanning electron microscopy of placental vascular casts from fetus with severe erythroblastosis fetalis

The latex injection—corrosion cast technique coupled with scanning electron microscopy was applied to the study of severe Rh sensitization of the placenta. The main observed ultrastructural changes in fetal vasculature in this type of placenta were very similar to those of the small for gestational age placenta; large-sized vessels exhibited less branching than those of the normal placenta, and the capillary network showed numerous vascular buds and “H”-shaped anastomoses. In addition, in Rh-sensitized placentas, the capillary network was remarkably sparse, and the cast also revealed smooth-surfaced veins with distinct U shapes undulating through the placental stroma. These U-shaped veins may be the result of umbilical flow impedance with subsequent umbilical venous hypertension, which stretches the veins into undulating form.

Laser subtotal meniscectomy in rabbits.

This study investigates the healing potential of the peripheral rim of rabbit meniscus following laser subtotal meniscectomy. Thirty animals were operated with 23 bilateral meniscectomies and 7 sham controls. When sacrificed at intervals up to 6 months, the harvested peripheral rims demonstrated a devitalized layer of fibrocartilage averaging 17 micron in thickness, indicating an extremely superficial thermal effect of the laser energy utilizing power densities of 20 to 38 W. Viable chondrocytes and minimal alteration of collagen fibril structure and orientation were noted within 20 to 50 micron of the meniscus free margin. By 2 weeks, a proliferation of vascular buds near the free margin of the meniscus was observed with an increase in ground substance production and chondrocyte population. By 10 weeks, the free margin was completely sealed and was composed of logically oriented collagen fibers and only slightly increased numbers of chondrocytes. It was concluded that the healing potential of rabbit meniscus is unimpaired following laser resection of the central margin, and that the repair process is mediated by a proliferation of chondrocytes, supported in part by neovascularity within the substance of the meniscus.

Nutritional Pathways of the Intervertebral Disc

The pathways of material transfer to the intervertebral disc were studied in adult dogs by measuring diffusion of hydrogen molecules in the nucleus pulposus before and after disruption of the route through the annulus fibrosus and before and after disruption of the end-plate route. The disruption of the end-plate route, even if the separation of the bone-disc interfaces was only in the central two-thirds of one side, caused significantly greater decrease in the rate of hydrogen washout than the disruption of the annulus route. Hitologically, the bone-cartilage interface was frequently perforated by marrow cavity and vascular buds. These findings suggest that the end-plate route is a major pathway for material transfer to the intervertebral disc.

ANGIOLYMPHOID HYPERPLASIA WITH EOSINOPHILS OF THE SKIN

A clinical, light and electron microscopic study of four patients with angiolymphoid hyperplasia with eosinophilia of the skin is presented. Light-microscopically, the lesions were characterized by the small size and superficial location, the proliferation of mostly delicate vessels with prominent endothelial hyperplasia and cellular areas without distinct vessel structures, but with infiltrates of eosinophilic leukocytes, lymphocytes and plasma cells. Ultrastructurally, a marked proliferation of both endothelial cells and pericytes could be demonstrated and the solid cellular areas were shown to be composed of solid vascular buds. A multi-layered basal lamina was enclosing pericytes and external surfaces of endothelial cells. The differential diagnosis is discussed and it is emphasized that this lesion may be misinterpreted as angiosarcoma.

Electron microscopical aspects of acid compartments of the ground substance and of collagen in different cases of intravitreal tissue proliferation.

It is demonstrated that 'activated' glial cells or NPE cells are capable of producing small collagen fibrils of the vitreous type as well as basal lamina material. In contrast to this MRPE cells produce a different type of collagen, exhibiting a larger periodicity and larger diameters. Granular and fibrillar polyanionic materials are produced by the MRPE cells as well as by proliferating endothelial cells. The latter substances combined with the newly formed collagen of the MRPE cells in regular distances of about 460-480 A. The most characteristic feature of very young vascular buds is a multilaminar basal lamina, which is established long before a vascular lumen is formed. Collagen fibrils are absent in the early stages of neovascularization; however, they are abundant in more advanced stages. These observations may be helpful in situations where a reliable diagnosis in respect to the origin of a certain cell population is impossible because typical cell characteristics are absent or poorly developed.

Subchondral route for nutrition to articular cartilage in the rabbit. Measurement of diffusion with hydrogen gas in vivo.

The route of nutrients going to articular cartilage was studied by determining the diffusion of hydrogen molecules from the subchondral circulation to the articular cartilage in rabbits. In all immature animals there was diffusion of hydrogen from subchondral bone into articular cartilage, while in the older immature animals the results were variable. None of the mature animals showed any diffusion of hydrogen into articular cartilage. The rate of diffusion of hydrogen was significantly lower in the articular cartilage than in the subchondral bone in the immature animals while the concentrations of hydrogen in the articular cartilage were only fractions of those in the subchondral bone at the same instant. Histologically, the deep layers of immature cartilage are penetrated extensively by vascular buds from the ossified portion of the epiphysis, while in adults the articular cartilage is separated from subchondral vascular spaces by an end-plate of bone. Blood vessels penetrating into the basilar layer of articular cartilage in immature animals appear to play an important role in the nutrition of articular cartilage coming from the subchondral region.

Treatment of Dermal and Decubitus Ulcers

Under optimal conditions, wound healing proceeds naturally through three stages. Firstly, in the exudative phase which begins at the time of injury, a vascular response in the form of increased capillary permeability allows phagocytes to move into the area, liberating lysosomal enzymes which devitalise bacteria and facilitate enzymatic digestion, bacterial decomposition or phagocytosis to separate and remove all non-viable material. Four or five days after the injury, the second stage, reparation and proliferation, begins with the formation of fresh granulation tissue. These vascular buds and epithelial tufts resist further bacteria growth . Fibrosis and continued formation of granulation tissue gradually reduce the size of the wound so that in the final stage, epithelisation and contraction, new epithelium forms and joins to complete the healing process. However, tissue repair can be retarded by systemic factors such as circulatory deficiencies, and by local factors such as the presence of bacterial infection and surface debris. Treatment of dermal and decubitus ulcers is directed toward providing the optimal conditions required for this natural healing process to progress, and consists primarily of arresting or reversing the systemic or local conditions which caused the ulcer, controlling the infection, and clearing wound debris.

Determination of polarity and bilateral asymmetry in palleal and vascular buds of the ascidian Botryllus schlosseri

Reversal of the bilateral asymmetry of the zooids was induced in a series of colonies of Botryllus schlosseri. Palleal buds from colonies with normal or reversed bilateral asymmetry were isolated in the early stages from the parental zooids and cultured in the vascularized tunic of the same colony or of another colony with opposite asymmetry. Vascular budding was induced in colonies with either type of asymmetry. The bud polarity was shown to depend on the vascularization; the test vessel entering the isolated palleal bud always causes the entrance point to become the posterior end of the developing zooid. On the contrary, the bilateral asymmetric type is predetermined in the bud primordium; the isolated palleal buds develop the type of asymmetry of their parents, even when grafted in the test of a colony with opposite asymmetry. Since the same was also true of the vascular buds, it is concluded that the information for the kind of bilateral asymmetry to be developed is conveyed by the epidermal envelope of the bud. The epidermis of the parental zooids influences the palleal buds, whereas the wall of the test vessels, epidermal extrusions of the zooids, influences the vascular buds.

Origin of hemopoietic stem cells in embryonic bursa of Fabricius and bone marrow studied through interspecific chimeras.

The histogenesis of the bursa of Fabricius and of bone marrow was studied by a biological cell marking technique based on differences in the nuclear structure of two species of birds, Japanese quail (Coturnix coturnix japonica) and chick (Gallus gallus). In quail cells the nucleus contains a large amount of heterochromatin associated with the nucleolus. That makes it possible to distinguish them from chick cells after Feulgen-Rossenbeck staining and by electron microscopy. By grafting bursal rudiments and limb buds of quail into chick and inversely it was possible to demonstrate that the whole hemopoietic population of the bursa of Fabricius and of bone marrow is derived from bloodborne extrinsic stem cells. Neither endoderm nor mesoderm of the bursal rudiments is capable of differentiating into lymphoid cells. Combinations of quail bursal endoderm with chick homologous mesenchyme showed that the reticular cells of the follicles are the only endodermal derivatives of the bursa. The mesenchymal bursal component gives rise to the interfollicular connective cells. The contribution to bone marrow histogenesis of cells of vascular and blood origin, on one hand, and of the elements of the cartilaginous model, on the other hand, was analyzed. It appeared that osteoblasts, osteocytes, and stromal cells of marrow are derived from the perichondrium. In contrast, the endothelium of the vascular buds and the hemopoietic cells which invade the diaphysal cartilage during the endochondral ossification process do not belong to the mesenchymal bone primordium but have a fully extrinsic origin.