Christopher A. Mitchell

VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia

Vascular endothelial growth factor (VEGF-A) is a major regulator of blood vessel formation and function. It controls several processes in endothelial cells, such as proliferation, survival, and migration, but it is not known how these are coordinately regulated to result in more complex morphogenetic events, such as tubular sprouting, fusion, and network formation. We show here that VEGF-A controls angiogenic sprouting in the early postnatal retina by guiding filopodial extension from specialized endothelial cells situated at the tips of the vascular sprouts. The tip cells respond to VEGF-A only by guided migration; the proliferative response to VEGF-A occurs in the sprout stalks. These two cellular responses are both mediated by agonistic activity of VEGF-A on VEGF receptor 2. Whereas tip cell migration depends on a gradient of VEGF-A, proliferation is regulated by its concentration. Thus, vessel patterning during retinal angiogenesis depends on the balance between two different qualities of the extracellular VEGF-A distribution, which regulate distinct cellular responses in defined populations of endothelial cells.

Cellular differences in the regeneration of murine skeletal muscle : a quantitative histological study in SJL/J and BALB/c mice

SummarySkeletal muscle regeneration in SJL/J and BALB/c mice subjected to identical crush injuries is markedly different: in SJL/J mice myotubes almost completely replace damaged myofibres, whereas BALB/c mice develop fibrotic scar tissue and few myotubes. To determine the cellular changes which contribute to these differential responses to injury, samples of crushed tibialis anterior muscles taken from SJL/J and BALB/c mice between 1 and 10 days after injury were analysed by light and electron microscopy, and by autoradiography. Longitudinal muscle sections revealed about a 2-fold greater total mononuclear cell density in SJL/J than BALB/c mice at 2 to 3 days after injury. Electron micrographs identified a similar proportion of cell types at 3 days after injury. Autoradiographic studies showed that the proportions of replicating mononuclear cells in both strains were similar: therefore greater absolute numbers of cells (including muscle precursors and macrophages) were proliferating in SJL/J muscle. Removal of necrotic muscle debris in SJL/J mice was rapid and extensive, and by 6 to 8 days multinucleated myotubes occupied a large part of the lesion. By contrast, phagocytosis was less effective in BALB/c mice, myotube formation was minimal, and fibrotic tissue conspicuous. These data indicate that the increased mononuclear cell density, more efficient removal of necrotic muscle, together with a greater capacity for myotube formation in SJL/J mice, contribute to the more successful muscle regeneration seen after injury.

The Exogenous Administration of Basic Fibroblast Growth Factor to Regenerating Skeletal Muscle in Mice Does Not Enhance the Process of Regeneration

The effects, in vivo, of the exogenous administration of bFGF on myogenesis of regenerating skeletal muscle was assessed either morphometrically or autoradiographically in three separate models of muscle injury in mice: crush-injured, denervated, and dystrophic (mdx) muscles. The bFGF was administered at various doses and different time schedules, sometimes in combination with heparin, into injured tibialis anterior muscles of mice. Delivery of the bFGF was either by direct intramuscular injection or by the sustained release from 888polymers (Hydron or Elvax) implanted into the muscles. The bioactivity of bFGF was confirmed in vitro by measuring its ability to stimulate the proliferation of BALB/c-3T3 fibroblasts and muscle precursor cell lines. The ability of bFGF to stimulate angiogenesis in vivo was confirmed by the implantation of controlled-release polymers containing bFGF into the normally avascular cornea of rats. No measurable effect of bFGF was seen in any of the models of skeletal muscle injury under these experimental conditions, indicating that the availability of biologically active bFGF is not a limiting factor in the regeneration of skeletal muscle following injury.

The process of new plasmalemma formation in focally injured skeletal muscle fibers

The major ultrastructural events in murine skeletal muscle fibers were examined 3 to 24 hr after segmental injury induced by painting with aldehyde fixative. At 3 hr the viable stump of injured myofibers was separated from the necrotic segment by a zone of supercontracted myofibrils. No demarcating membrane was evident at this time, although occasionally collapsed segments of plasmalemma partially covered the viable stump. By 12 hr after injury myonuclei near the viable stump were centrally placed and numerous whorls of membrane material appeared in the vicinity of the Golgi apparatus. At this stage a convoluted, tortuous membrane exhibiting extensive interdigitations has sealed the structurally normal part of the injured fiber. The myoplasm immediately within this demarcating membrane possessed few myofilaments but numerous vesicles and tubules, several of which were continuous with the demarcating membrane; most degraded sarcoplasmic organelles remained external to the demarcating membrane and leukocytes were observed internalizing the debris. It appears that after segmental injury to skeletal muscle fibers, active production of new sarcoplasmic membranes occurs, which contributes to the formation of the part of the plasmalemma that demarcates the viable portion of the muscle fiber from the injured area.

Preconditioned 70S30C bioactive glass foams promote osteogenesis in vivo.

Bioactive glass scaffolds (70S30C; 70% SiO2 and 30% CaO) produced by a sol-gel foaming process are thought to be suitable matrices for bone tissue regeneration. Previous in vitro data showed bone matrix production and active remodelling in the presence of osteogenic cells. Here we report their ability to act as scaffolds for in vivo bone regeneration in a rat tibial defect model, but only when preconditioned. Pretreatment methods (dry, pre-wetted or preconditioned without blood) for the 70S30C scaffolds were compared against commercial synthetic bone grafts (NovaBone® and Actifuse®). Poor bone ingrowth was found for both dry and wetted sol-gel foams, associated with rapid increase in pH within the scaffolds. Bone ingrowth was quantified through histology and novel micro-CT image analysis. The percentage bone ingrowth into dry, wetted and preconditioned 70S30C scaffolds at 11 weeks were 10±1%, 21±2% and 39±4%, respectively. Only the preconditioned sample showed above 60% material-bone contact, which was similar to that in NovaBone and Actifuse. Unlike the commercial products, preconditioned 70S30C scaffolds degraded and were replaced with new bone. The results suggest that bioactive glass compositions should be redesigned if sol-gel scaffolds are to be used without preconditioning to avoid excess calcium release.

bFGF enhances the development of the collateral circulation after acute arterial occlusion

An adequate collateral circulation is crucial to tissue survival subsequent to proximal major arterial occlusion. The precise mechanism of collateral blood vessel development and the biochemical mediators involved in this process are unknown. To evaluate the influence of a number of agents on the development of the collateral circulation, we developed a rat model of severe hind limb ischaemia. The recovery of blood flow after acute arterial occlusion was increased by exogenous basic fibroblast growth factor and heparin, and decreased by protamine. Erucamide (cis-13-docosenamide), an angiogenic lipid, had no effect on collateral blood flow. These results indicate that basic fibroblast growth factor and heparin are potential therapeutic agents in the treatment of peripheral vascular disease.

Fusion between myogenic cells in Vivo: An ultrastructural study in regenerating murine skeletal muscle

Fusion of myogenic cells in adult murine skeletal muscle regenerating in vivo was examined at the ultrastructural level. Fusion of myoblast to myoblast, myoblast to myotube, and myotube to myotube was observed by 4 to 5 days after injury. Fusion between myogenic cells (myoblasts or myotubes) lacking a definitive glycocalyx or external lamina (basal lamina) occurred at multiple sites. It was defined by zones of cytoplasmic confluence between apposed cells at sites where contiguous segments of the cell membranes were interrupted while their edges had united resulting in linear continuity; vesicles of varying dimensions were frequent in these areas of fusion. Myoblasts were seen invaginating the surface of myofibres and again vesicles were seen in abundance in such regions. Cilia were often observed at this junctional zone suggesting that they might play a role in fusion. In the one example of probable fusion between a myotube and a myofibre, only a single area of cytoplasmic continuity was apparent.

Induction of Intrauterine Growth Restriction by Reducing Placental Vascular Growth with the Angioinhibin TNP-470

Abstract The placenta is a specialized vascular interface between the maternal and fetal circulations that increases in size to accommodate the nutritional and metabolic demands of the growing fetus. Vascular proliferation and expansion are critical components of placental development and, consequently, interference with vascular growth has the potential to severely restrict concurrent development of both the placenta and fetus. In this study, we describe the effects of an antiangiogenic agent, TNP-470, on placental vascular development and the induction of a form of intrauterine growth restriction (IUGR) in mice. Administration of TNP-470 to dams in the second half of pregnancy resulted in a smaller maternal weight gain accompanied by decreased placental and fetal sizes in comparison with control animals. Total numbers of fetuses per litter were not affected significantly. Stereological analysis of placentas revealed no changes in the combined lengths of vessels. However, the mean cross-sectional areas of maternal and fetal vessels in the labyrinth of TNP-470-treated mice were reduced at Embryonic Day 13.5 (E13.5) but not at E18.5. Further analysis showed reduced placental endothelial proliferation at E13.5 and E18.5 in TNP-470-treated animals. No other structural or morphometric differences in placentas were detected between TNP-470-treated and control mice at E18.5. This study provides conclusive evidence that administration of TNP-470 interferes with placental vascular proliferation and vessel caliber and results in a reproducible model of IUGR.

Dynamics of angiogenesis during wound healing: a coupled in vivo and in silico study.

Please cite this paper as: Machado, Watson, Devlin, Chaplain, McDougall and Mitchell (2011). Dynamics of Angiogenesis During Wound Healing: A Coupled In Vivo and In Silico Study. Microcirculation 18(3), 183–197.

Bioactive glass foam scaffolds are remodelled by osteoclasts and support the formation of mineralized matrix and vascular networks in vitro.

Remodelling of scaffolds and new bone formation is critical for effective bone regeneration. Herein is reported the first demonstration of resorption pits due to osteoclast activity on the surface of sol-gel bioactive glass foam scaffolds. Bioactive glass foam scaffolds are known to have osteogenic potential and suitable pore networks for bone regeneration. Degradation of the scaffolds is known to be initially solution mediated, but for effective bone regeneration, remodelling of the scaffold by osteoclasts and vascularisation of the scaffold is necessary. The culture of C7 macrophages on a bioactive glass scaffold induces the cells to differentiate into (TRAP(+ve) ) osteoclasts. They then form distinctive resorption pits within 3 weeks, while MC3T3-E1 pre-osteoblasts deposit mineralized osteoid on their surfaces in co-culture. The scaffolds are of the 70S30C (70 mol% SiO2 , 30 mol% CaO) composition, with modal pore and interconnect diameters of 373 μm and 172 μm respectively (quantified by X-ray micro-tomography and 3D image analysis). The release of soluble silica and calcium ions from 70S30C scaffolds induces an increase in osteoblast numbers as determined via the MTT assay. Scaffolds also support growth of endothelial cells on their surface and tube formation (characteristic of functional microvasculature) following 4 days in culture. This data supports the hypothesis that 70S30C bioactive glass scaffolds promote the differentiation of the 3 main cell types involved in vascularized bone regeneration.