Gregg D. Phillips

Wound healing angiogenesis : indirect stimulation by basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) was tested for its ability to stimulate angiogenesis in vivo using the rabbit corneal assay. Basic FGF (50-1,000 ng) was incorporated into 10% Hydron, and 50-500 ng of bFGF were incorporated into 10% Elvax. Human serum albumin (HSA) (10 ng) and 50 ng of transforming growth factor-beta (TGF-beta) served as negative and positive controls. Pellets of the polymers containing test compounds were implanted in the rabbit cornea, examined daily, and after 7 days corneal angiogenesis was scored on a graded scale [(-) for no response and +4 for a maximum response]. Histologic analysis of the corneas was performed on days 2 and 7. Basic FGF (50-500 ng) in Hydron failed to stimulate significant angiogenesis, though it did induce angiogenesis accompanied by inflammation at the 1,000-ng dose. Basic FGF in Elvax elicited inflammation-associated +3 to +4 responses at all doses tested. New blood vessels did not form in response to HSA in Hydron or Elvax, while TGF-beta induced +4 angiogenesis accompanied by vigorous inflammation. In vivo release kinetics for bFGF in Hydron and Elvax were compared, and the release of bioactive bFGF from Hydron and Elvax was demonstrated in vitro. These results suggest that the bFGF and Elvax combination incites an inflammatory response which stimulates indirect angiogenesis, while the same concentrations of bFGF delivered in Hydron produced no inflammation or angiogenesis. Although bFGF alone is a potent mitogen for endothelial cells, it does not appear to directly stimulate in vivo angiogenesis.

Migration of myogenic cells in the rat extensor digitorum longus muscle studied with a split autograft model.

SummaryThe ability of myogenic cells to migrate perpendicular to the long axis of freely autografted muscles was examined. Rat extensor digitorum longus muscles were divided, and one half was devitalized by repeated freezing in liquid nitrogen while the other half was kept viable in physiologic saline. The halves were reunited with sutures and grafted back into the original muscle bed. At intervals between 5 and 25 days the grafts were removed and examined histologically for the presence of myotubes within the devitalized region. Myotubes were first seen in the devitalized half 10 days postgrafting with the maximum number of myotubes observed after 12 to 15 days. These results indicate that myogenic cells are capable of migration perpendicular to the long axis of the muscle fibers in an autograft.

PDGF-BB induced chemotaxis is impaired in aged capillary endothelial cells

The object of this study was to determine if the decreased angiogenesis in the healing wounds of the aged is due to the failure of endothelial cells to respond to locally produced growth factors. Endothelial cells isolated from wound sponges implanted in aged (24-month-old) and young (2-month-old) rats were tested for their chemotactic response to the BB isoform of platelet-derived growth factor (PDGF-BB). A similar number of cells isolated from both old and young rats stained positive (75-90%) for acetylated-LDL uptake, and the same number of viable cells was used in the chemotaxis assay. Endothelial cells from both old and young rats migrated in a dose-dependent (0.1-3.0 ng/ml) manner in response to PDGF-BB. At all concentrations tested, PDGF-BB elicited the migration of more endothelial cells from the young rats. The difference between the number of young and old cells that responded to PDGF-BB was statistically significant at the 1.0 ng/ml and 3.0 ng/ml concentrations. These results suggest that the impaired angiogenic response in the healing of wounds of the aged is due to altered endothelial cell reaction to the growth factors in the wound microenvironment.

Inhibition by Methylprednisolone Acetate Suggests an Indirect Mechanism for TGF-B Induced Angiogenesis

Angiogenesis induced by transforming growth factor beta (TGFB) implanted in the rabbit cornea is accompanied by an influx of inflammatory cells. To determine if the inflammatory cells are the mediators of the neovascularization, they were depleted by local administration of methylprednisolone acetate (MPA). Subconjunctival injections of 16 mg of MPA immediately following implantation of 50 ng of TGFB in the cornea prevented the inflammation and subsequent formation of capillaries. If the injections of MPA were delayed by 48 hr and the inflammatory cells were allowed to enter the cornea, angiogenesis occurred, demonstrating that MPA had no adverse effects on the ability of endothelial cells to form capillaries. These results confirm the hypothesis that TGFB induces angiogenesis indirectly by recruiting inflammatory cells capable of stimulating direct angiogenesis.

Angiogenic activity in damaged skeletal muscle

Abstract After a muscle is damaged, blood vessels spontaneously grow into the injured region as the muscle fibers regenerate. The stimulus for this vascular ingrowth is currently unknown. We hypothesized that the damaged muscle releases a factor(s) capable of stimulating this revascularization. To test this theory, extracts were prepared from rabbit hind limb muscles and incorporated into Hydron, a slow-release polymer. Pellets of the extract containing Hydron were implanted between the layers of the rabbit corneal stroma as an assay for angiogenic activity. The normally avascular corneas were examined 7 days after surgery for the presence of new blood vessels. Skeletal muscle-derived extract from rabbits elicited positive angiogenic responses in a dose-dependent manner. Four hundred to 500 μg of the skeletal muscle-derived extract were required to produce maximum vessel ingrowth. The control, Dulbeccos phosphate-buffered saline in Hydron, failed to stimulate neovascularization.

Age-related alterations in the morphology of femoral artery vasa vasorum in the rat

The objective of this study was to explore any age-related morphological changes in the vasa vasorum of the rat femoral artery. Vascular corrosion casts were prepared from 2, 12 and 24-month-old rats. Examination of the casts with the scanning electron microscope revealed dramatic differences in the appearance of the vessels of young and aged rats. The vasa vasorum of 2-month-old rats consisted of a dense network of capillaries. These vessels were dramatically reduced in number by 12 months, and even fewer capillaries were present at 24 months. This reduction in capillary density is consistent with the observed age-related decreases in oxygen tension and may explain why the aged are more prone to atherosclerosis.

Regulation of cutaneous wound healing by growth factors

Abstract This paper reviews the roles of growth factors in the events of cutaneous wound healing. General categories of growth factors (mitogens, chemoattractants, angiogenesis factors and angiogenesis inhibitors) are defined, and the effects of specific growth factors (platelet-derived growth factor, basic and acid fibroblast growth factors, epidermal growth factor and transforming growth factor beta) on granulation tissue formation, epithelialization, angiogenesis, wound breaking strength, and wound contraction are discussed. In addition, animal and human models used in studying the interactions between growth factors and the cells and tissues associated with cutaneous wound healing are described.

An angiogenic extract from skeletal muscle stimulates monocyte and endothelial cell chemotaxis in vitro

Abstract The purpose of this study was to determine whether the extraction of skeletal muscle with a combination of ethanol and hydrochloric acid yields a product capable of stimulating angiogenesis. The resulting extract stimulated inflammation in the rabbit corneal assay, which was followed by capillary formation. In order to determine whether the observed angiogenesis was stimulated by a factor(s) acting directly on the endothelial cells versus a factor(s) recruiting macrophages that in turn release factors acting on endothelial cells, the muscle extract was tested for endothelial cell and monocyte chemotaxis activity in vitro. The muscle extract stimulated significant endothelial cell chemotaxis at concentrations between 94 and 750 μg of protein/ml and significant monocyte chemotaxis at concentrations between 8 and 75 μg of protein/ml. Polyacrylamide gel electrophoresis suggests that basic fibroblast growth factor and transforming growth factor-β may be present in this acid/ethanol extract of skeletal muscle.

Tumor necrosis factor alpha (rhTNF) fails to stimulate angiogenesis in the rabbit cornea

The objective of this study was to thoroughly examine the in vivo angiogenesis activity of human recombinant tumor necrosis factor alpha (rhTNF).

Proliferation of wound derived capillary endothelial cells: young versus aged

The objective of this study was to compare the proliferative potential of wound derived capillary endothelial cells (WCEC) from aged and young rats. Endothelial cells were isolated from subcutaneously implanted sponges in 2- and 24-month-old rats. The identity of the cells as endothelial was confirmed by staining for Ac-LDL uptake. Aged and young WCEC (20,000/well) were stimulated with increasing concentrations of fetal calf serum (0, 2.5, 5, 10 and 15%). The increase in cell number was determined with a Coulter counter. At all serum concentrations, the proliferative capacity of WCEC from aged rats was significantly higher than that of WCEC from young rats.