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Wound healing is a complex biologic process that involves chemotaxis and division of cells, neovascularization, synthesis of extracellular matrix proteins, and remodeling of scar. Peptide growth factors have been shown to regulate many of these processes in vitro, leading to the hypothesis that peptide growth factors also regulate important phases of wound healing in vivo. Part I of this two-part series presents an overview of the biochemical properties of five families of peptide growth factors that are thought to be involved in wound healing: epidermal growth factor (EGF), transforming growth factor-beta (TGF-beta), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), and fibroblast growth factor (FGF).

Rabbits undergoing full-thickness glaucoma filtering surgery were exposed for 5 minutes to one of three intraoperative treatments: (1) distilled water; (2) fluorouracil, 50 mg/mL; or (3) mitomycin C, 0.4 mg/mL. Tissue samples were taken from the subconjunctival and scleral tissues at the treated area and 90 degrees and 180 degrees from the center of the treated area and the adjacent cornea 2 mm from the limbus, 1 hour, 5 days, and 30 days postoperatively. The biopsy specimens were then placed in tissue culture media and the fibroblast outgrowths measured. Five-minute intraoperative treatments with fluorouracil resulted in a reversible delay of fibroblast outgrowths from treated subconjunctival and scleral tissues of just over 1 week in this model, whereas treatment with mitomycin C, 0.4 mg/mL, resulted in prolonged inhibition of at least 30 days. These effects were localized to the area treated. The many clinical implications of these findings are discussed.

Rabbits undergoing full thickness glaucoma filtering surgery were exposed to one of 4 treatments. Group 1 received intraoperative distilled water, group 2 received intraoperative mitomycin-c 0.2 mg/ml for 5 minutes, group 3 received 5 post operative injections of 5-Fluorouracil (5-FU) 5 mg in 0.1 ml, and group 4 received intraoperative 5-FU 50 mg/ml followed by 5 post operative injections of 5-FU. 30 days after the operation tissue biopsies were taken from the subconjunctival and scleral tissue at the treated area and 90 and 180 degrees from the treated area. The biopsies were then placed in tissue culture media and the outgrowths quantitated. The fibroblast outgrowths from all areas did not differ significantly from each other except for the outgrowths from the areas directly treated with mitomycin 0.2 mg/ml which were significantly smaller. In addition then cells were morphologically abnormal although there were foci of normal cells which appeared to be growing from localised areas in the tissue biopsies. The outgrowths from the areas 90 and 180 degrees from the treated area were normal. Intraoperative treatments with mitomycin-c result in long term inhibition of fibroblast proliferation limited to the treated area, when compared with intraoperative and postoperative treatment with 5-FU. Failure of filtration surgery in eyes treated with intraoperative mitomycin may in part be due to unaffected cells reproliferating.

Objective: Human placenta expresses receptors for transforming growth factor-α and epidermal growth factor throughout pregnancy. Experiments were performed to determine whether epidermal growth factor or transforming growth factor-α might be synthesized by placental cells and act through an autocrine mechanism to influence functioning of placental cells in vivo. Study design: Human placentas from early, mid, and late gestations were analyzed for transforming growth factor-α and epidermal growth factor messenger ribonucleic acid and proteins. Polyadenylic acid—positive ribonucleic acid was isolated from placentas from 10, 11, 13, 21, 32, 38, 39, and 40 weeks of gestation and analyzed by Northern analysis for hybridization with complementary deoxyribonucleic acid probes specific for epidermal growth factor or transforming growth factor-α. Levels of immunoreactive epidermal growth factor and transforming growth factor-α were measured by specific radioimmunoassays in pools of placentas from early, mid, and late gestations, and levels of epidermal growth factor and transforming growth factor-α receptor—active protein were measured by radioreceptor assay. Results: All placentas had a strong transforming growth factor-α hybridization band at 4.5 kb and a weak epidermal growth factor hybridization band at 5.2 kb. High levels of transforming growth factor-α immunoreactive protein (90 to 180 ng/mg protein) and low levels of immunoreactive epidermal growth factor (3 to 9 pg/mg protein) were detected in pools of placentas from early, mid, and late gestations. High levels of epidermal growth factor and transforming growth factor-α receptor-active protein (250 ng/mg protein) were also detected. Conclusion: Human placentas contain relatively high levels of immunoreactive and receptor-active transforming growth factor-α, as well as transforming growth factor-α messenger ribonucleic acid, throughout gestation. This finding suggests that transforming growth factor-α may act by an autocrine system to influence human placental cell function in vivo.

Transforming growth factor β (TGFβ) is an extremely potent chemoattractant for macrophages, mononuclear leukocytes, and fibroblasts. It also acts as a potent stimulant for collagen and fibronectin synthesis and inhibits epithelial cell growth. TGFβ plays an important role in healing many types of wounds, but its role in peritoneal adhesion formation is not known. These studies were performed to determine if TGFβ could affect postoperative wound healing in a rat model. In the first experiment, 20 rats were divided into two groups and received either 2 μg TGFβ or control diluent IP daily for 5 days after surgical injury to the uterine horns. The severity of the adhesions were graded 2 weeks postoperatively using a score of 0–3. The TGFβ group showed a higher adhesion score at 2 weeks compared to control, 2.9 ± 0.34 and 1.6 ± 0.61, respectively (P < 0.001). On H&E stained sections of the adhesions, there was an increase in the number of both inflammatory cells and fibroblasts in the TGFβ-treated animals. A comparison trial of bone-derived TGFβ (a gift from Collagen Corporation, Palo Alto, CA) versus recombinant TGFβ (a gift from Oncogen, Seattle, WA) versus control using the same protocol as above showed that both sources of TGFβ were more effective in promoting postoperative adhesions when compared to controls, and there was no difference between TGFβ groups, 3.0 ± 0 for both TGFβ groups, and 2.2 ± 0.91 for control (P < 0.001). TGFβ did not induce adhesions when given in the same dosage for 5 days ip in unoperated rats, although it did induce fibrosis if injected into the abdominal wall. These data suggest that TGFβ may play a role in stimulating fibrosis in postoperative fibrinous adhesions, but has no effect on intact mesothelium. Prevention of TGFβ production postoperatively may help prevent adhesion formation.

Peptide growth factors produced by platelets, macrophages, epidermal, and dermal cells may play key roles in regulating healing of partial-thickness skin wounds. We examined the effects of recombinant transforming growth factor beta (TGF-beta) on cultures of epidermal and dermal cells in vitro and on healing of partial-thickness injuries in vivo. Increasing concentrations of TGF-beta (0.1, 1, and 10 ng/ml) progressively inhibited serum-stimulated DNA synthesis by up to 95% in cultures of adult human keratinocytes during 48 hr of exposure to TGF-beta. In contrast, TGF-beta (10 and 100 ng/ml) in serum-free media stimulated DNA synthesis by up to 80% compared to serum-free control cultures of adult human dermal fibroblasts. To evaluate the effects of TGF-beta on healing of partial-thickness injuries in vivo, wounds (20 x 20 x 0.6 mm) were created on the dorsal thoracolumbar region of adult pigs by an electrokeratome and were treated daily for 5 days after injury with vehicle or vehicle containing 0.1 or 1 microgram/ml TGF-beta and covered with occlusive dressing. Computerized planimetry of wound photographs demonstrated that TGF-beta treatment stimulated statistically significantly increases in the area of regenerated epidermis compared to wounds treated with saline vehicle on Days 3, 4, 5, and 7 after injury probably due to TGF-beta increasing the rate of epidermal cell migration. In addition, morphometry of biopsy specimens showed that TGF-beta treatment stimulated statistically significant increases in the cross-sectional depths of regenerated dermis compared to wounds treated with saline or Silvadene vehicles on Days 5, 6, and 8 after injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Wound healing is a localized process which involves inflammation, wound cell migration and mitosis, neovascularization, and regeneration of the extracellular matrix. Recent data suggest the actions of wound cells may be regulated by local production of peptide growth factors which influence wound cells through autocrine and paracrine mechanisms. Two peptide growth factors which may play important roles in normal wound healing in tissues such as skin, cornea, and gastrointestinal tract are the structurally related peptides epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha). EGF/TGF-alpha receptors are expressed by many types of cells including skin keratinocytes, fibroblasts, vascular endothelial cells, and epithelial cells of the GI tract. In addition, EGF or TGF-alpha are synthesized by several cells involved in wound healing including platelets, keratinocytes, and activated macrophages. Healing of a variety of wounds in animals and patients was enhanced by treatment with EGF or TGF-alpha. Epidermal regeneration of partial thickness burns on pigs or dermatome wounds on patients was accelerated with topical application of EGF or TGF-alpha, and EGF treatment accelerated healing of gastroduodenal ulcers. EGF also increased tensile strength of skin incisions in rats and corneal incisions in rabbits, cats, and primates. Additional research is needed to better define the roles of EGF, TGF-alpha and their receptor in normal wound healing, to determine if alterations have occurred in the EGF/TGF-alpha system in chronic wounds, and optimize vehicles for effective delivery of peptide growth factors to wounds.

PURPOSE. To generate data concerning changes in levels of protein expression associated with wound healing and bleb failure in a rat model of glaucoma filtration surgery (GFS), and to identify factors that may play a role in this process. METHODS. Of 36 Sprague-Dawley rats, GFS was performed on 27 by introducing a silicone cannula through a scleral tunnel under a conjunctival flap, resulting in aqueous-filtering blebs that failed over 8 to 13 days. The additional nine rats were used as the nonsurgical control. Nine blebs were harvested at each of days 0, 2, 5, and 12 and pooled, yielding three replicates of three blebs per time point. RNA was extracted, labeled, and hybridized to 230A rat GeneChip arrays (Affymetrix, Santa Clara, CA). RESULTS. Of the 15,924 probe sets/genes present on the array, 923 genes were indicated to have a significant treatment effect at P < 0.005. Eight gene expression clusters were identified that could be broadly classified into three basic patterns. These were an increase on day 2, a decrease on day 2, or an increase on either day 5 or 12. The greatest change occurred between days 0 and 2. The most heavily populated functional categories included growth factors, structural proteins, and matrix metalloproteinases. CONCLUSIONS. This study represents the first large-scale gene expression analysis after GFS. Expression patterns for known mediators of the bleb scarring process, including transforming growth factor-β, connective tissue growth factor, and matrix metalloproteinases, were confirmed, and a number of mediators not previously associated with this process were identified.

Purpose This study was undertaken to investigate the role of connective tissue growth factor (CTGF) in fibroblast-to-myofibroblast differentiation and fibroblast-mediated collagen matrix contraction in the presence of mechanical stress. Methods An in vitro three-dimensional contraction model of human corneal-fibroblast-seeded collagen lattices (FSCLs) in the presence of mechanical stress generated by attaching the lattices to the culture well was used to measure FSCL contraction. FSCLs were treated with CTGF; TGF-beta1; serum-free (SF) control medium; or TGF-beta1 plus antisense oligodeoxynucleotides to CTGF; TGF-beta1 plus scrambled-sequence oligodeoxynucleotide to CTGF; or TGF-beta antibody. Expression of alpha-smooth muscle actin (alpha-SMA) by fibroblasts in FSCLs was detected by immunostaining and confocal microscopy, whereas ELISA was used for the fibroblasts cultured on plastic. The conditioned media were analyzed by ELISA for CTGF production. Results Exogenous CTGF stimulated significantly less collagen matrix contraction and myofibroblast differentiation than TGF-beta1, but similar to that stimulated by SF. TGF-beta1 stimulated fibroblasts to express CTGF. CTGF antisense oligodeoxynucleotide inhibited TGF-beta1-stimulated myofibroblast differentiation and FSCL contraction. Exogenous CTGF circumvented the inhibitory effects of CTGF antisense on FSCL contraction. TGF-beta antibody significantly inhibited FSCL contraction and myofibroblast differentiation under mechanical stress and SF control conditions. Conclusions In the presence of mechanical stress, CTGF is necessary for TGF-beta1-stimulation of myofibroblast differentiation and subsequent collagen matrix contraction, but CTGF alone is not sufficient to induce myofibroblast differentiation and collagen matrix contraction. Thus, TGF-beta1 appears to regulate multiple genes that are essential for fibroblast-mediated contraction of stressed matrix, one of which is CTGF.

Introduction: Shear stress stands as a potent modulator of vascular remodeling with reduced shear leading to enhanced intimal hyperplasia within vein grafts. This shear-regulated intimal growth results from changes in cell proliferation and MMP-2 activation, however the mediators through which alterations in shear lead to remote changes in the developing neointima have not been well characterized. CTGF, acting as a downstream effecter of TGF-β, has a pivotal role in the regulation of matrix reorganization and cell homeostasis during pathologic stromal scar formation. We hypothesize CTGF to have a significant role the accelerated intimal growth seen in reduced shear conditions. Methods: Following bilateral carotid, vein graft placement within the rabbit neck (n = 9), partial distal branch ligation was performed on the right to create a flow differential between the grafts. Grafts were harvested at 28 days to evaluate morphology and CTGF gene and protein expression, using PCR (Taqman) and ELISA techniques. Immunohistochemistry was used to localize CTGF within the wall. Results: Unilateral ligation led to a significant disparity in wall shear (5.6 ± 1.1 vs. 1.2 ± 0.4 dynes/cm2, p = 0.01). Grafts demonstrated a marked difference in remodeling, with decreased intimal area under high shear conditions (21 ± 4 vs. 89 ± 8 mm2, p = 0.001). A significant interaction between CTGF and the imposed hemodynamic environment was observed, with elevations in shear stress leading to marked up-regulation of CTGF. Immunohistochemistry for CTGF revealed localization of protein almost exclusively to the endothelium. Conclusions: Elevated shear stress leads to an increase in endothelial CTGF production during early graft remodeling, suggesting CTGF to be an important mediator of intimal hyperplasia. Intervention in CTGF signaling may offer a therapeutic strategy to reduce vein graft failure. Download high-res image (131KB) Download full-size image

Objective To assess the potential role of matrix metalloproteinases (MMPs) in the pathogenesis of pterygia by comparing the immunolocalization patterns of MMPs in altered limbal basal stem cells, activated fibroblasts, and areas of elastotic degeneration adjacent to the pterygia. Methods Nine primary and 1 recurrent pterygia along with normal superior limbal-conjunctival tissue and cornea were immunostained with mouse monoclonal antibodies specific for MMP-1, MMP-2, MMP-3, MMP-9, membrane type 1 (MT1)–MMP (MMP-14), and membrane type 2–MMP (MMP-15). Results Normal conjunctival, limbal, and corneal cells lacked significant immunostaining except for cell surface MT1-MMP. In contrast, altered limbal basal epithelial cells of the 9 primary and 1 recurrent pterygia immunostained for all 6 MMPs. Activated and altered fibroblasts associated with the pterygia immunostained primarily for MMP-1. In contrast, stromal areas of elastotic degeneration(pingueculae) showed variable immunostaining of MMPs. Conclusions Altered limbal basal epithelial cells (pterygium cells) immunostained for multiple types of MMPs in contrast to normal conjunctival, limbal, and corneal cells. The pterygium cells invading over Bowman's layer produce elevated MMP-1, MMP-2, and MMP-9 expression, which probably are the main MMPs responsible for the dissolution of Bowman's layer. Pterygium cells may also cause activation of fibroblasts at the head of the pterygium, leading to the initial cleavage of fibrillar collagen in Bowman's layer by the production of MMP-1. Altered fibroblasts in areas of elastotic degeneration (pingueculae) trailing behind the pterygium constitute a second type of tumor, which is noninvasive. Clinical Relevance These data of altered MMP expression support the concept that altered basal limbal epithelial cells play a key role in the formation and migration of a pterygium.

Proliferating human Tenon's capsule fibroblasts were exposed for 5 minutes to a wide range of concentrations of fluorouracil, floxuridine, and mitomycin. High concentrations of all three agents had prolonged effects on cell proliferation and morphologic characteristics compared with untreated control cells up to 36 days. The highest concentrations of both floxuridine (15,000 micrograms/mL) and mitomycin (1000 micrograms/mL) had an apparent cidal effect, reducing cell numbers below initial cell density. In contrast, although the highest concentration of fluorouracil (25,000 micrograms/mL) inhibited cell proliferation by more than 50% relative to the untreated control cells at 36 days, the cell numbers still increased fourfold compared with the initial cell density. These results demonstrate that 5-minute treatments with high concentrations of these drugs have prolonged effects on the proliferation of human Tenon's capsule fibroblasts in vitro. Single-dose regimens using high concentrations of these drugs at the time of operation may achieve results similar to those of protocols that involve repeated applications.

Specific binding of iodine 125-labeled epidermal growth factor was measured in membrane homogenates of third-trimester and term human placentas of male and female fetuses. All placentas (n = 35) generated curvilinear Scatchard plots, and all placentas had similar equilibrium dissociation constants for both high-affinity (210 pmol/L) and low-affinity (830 pmol/L) binding sites. Mean maximum number of available binding sites of term placentas from male fetuses (330 +/- 110 fmol/mg protein; n = 11) was significantly higher (p less than 0.001) than that of female fetuses (170 +/- 80 fmol/mg protein, n = 13). Occupation of epidermal growth factor receptors by endogenous epidermal growth factor could not account for the difference in levels of epidermal growth factor binding. Kendall's rank order correlation test demonstrated a significant positive correlation of the gestational age in weeks with the maximum number of epidermal growth factor receptors in placentas from male fetuses (p less than 0.002; T = 0.527; n = 17) and female fetuses (p less than 0.01; T = 0.404; n = 18). These results indicate that there is a sexual dimorphism in the level of epidermal growth factor receptors in placentas of male and female fetuses during the third trimester and that the level of epidermal growth factor receptors increases during the third trimester for placentas of both male and female fetuses.

These studies were undertaken to determine whether term human placental microvillus plasma membranes, which are exposed to maternal blood, and basolateral plasma membranes, which are in close proximity to fetal blood capillaries, contain receptors for epidermal growth factor (EGF). These two highly purified membranes bound 125I-EGF with similar affinity (apparent dissociation constants, 0.07-0.12 nM, but the total number of available receptors was greater in microvillus (8.2 pmol/mg protein) compared to basolateral (4.9 pmol/mg protein) plasma membranes. Detailed characterization of 125I-EGF binding to these membranes revealed numerous similarities as well as differences. The two membranes contained two major (155 and 140 kDa) and at least three minor (115, 175, and 210 kDa) specific 125I-EGF binding proteins. The 115-kDa protein was only found in basolateral plasma membranes. The 155-kDa protein was predominantly labeled in microvillus, whereas the 140-kDa protein was labeled predominantly in basolateral plasma membranes. The addition of protease inhibitors did not alter the multiple 125I-EGF binding proteins pattern found in these membranes. EGF stimulated phosphorylation of 140- and 155-kDa proteins in both microvillus and basolateral plasma membranes. However, the 155-kDa protein was phosphorylated to a greater extent in microvillus, whereas both 140- and 155-kDa proteins were phosphorylated equally in basolateral plasma membranes. Light and electron microscope autoradiographic studies revealed that 125I-EGF preferentially associated with microvillus plasma membranes. The data demonstrates the presence of EGF receptors in outer cell membranes of syncytiotrophoblasts and suggests that maternal EGF may influence syncytiotrophoblast function by binding to receptors in microvillus plasma membranes, while fetal EGF may also influence syncytiotrophoblast function but via receptors in basolateral plasma membranes.

Treatment of rabbit corneal wounds with topical corticosteroid retards both epithelial regeneration and healing of penetrating stromal wounds. Currently, no clinical agent is available which accelerates the rate of stromal wound healing. Epidermal growth factor (EGF, 0·5 mg ml−1), fibroblast growth factor (FGF, 20 μg ml−1), and insulin (0·5 mg ml−1) were tested for their ability to accelerate healing of totally penetrating wounds in rabbit corneas when the hormones were administered alone or in combination with dexamethasone (1 mg ml−1). After 5 days of treatment with eye drops, the tensile strengths of corneal wounds treated with EGF (54±4 g mm−1) or treated with EGF and dexamethasone (32±9 g mm−1) were significantly higher than the tensile strengths of corneal wounds treated with only saline vehicle (3±1 g mm−1) or dexamethasone (1±0 g mm−1) (P < 0·001). The combination of dexamethasone with EGF signficantly (P < 0·025) reduced the strength of corneal wounds compared to treatment with EGF alone. Similarly, the tensile strength of corneal wounds after 5 days of insulin treatment alone (28±8 g mm−1) or in combination with dexamethasone (25±7 g mm−1) was significantly increased compared with saline- or dexamethasone-treated corneas (P < 0·001). In the absence of dexamethasone, EGF increased the tensile strength of corneal wounds significantly better than insulin (P < 0·01). However, when EGF or insulin were given in combination with dexamethasone there was no significant difference between the tensile strength produced by the peptide hormones. In comparison to the tensile strength of corneal wounds treated by EGF or insulin, treatment with FGF alone (5±4 g mm−1) or in combination with dexamethasone (2±1 g mm−1) produced poor wound healing. The in vitro actions of EGF or FGF alone or in combination with dexamethasone were tested for ability to stimulate [3H]-thymidine incorporation into pure cultures of human corneal fibroblasts (HCF) in defined culture medium. EGF (5 mm) or FGF (100 ng ml−1) alone stimulated [3H]-thymidine incorporation approximately 2·5-fold compared to control cultures, whereas in combination with dexamethasone (10 nm), the stimulatory action of FGF, but not EGF, was abolished. Dose-response curves indicated that HCF in culture were very sensitive to EGF, insulin, and FGF with maximum stimulation of [3H]-thymidine incorporation occurring at approximately 1 nm for EGF and insulin and at 100 μg ml−1 for FGF. Binding of 125I-EGF to HCF reached maximum after 2 hr at 37°C and was specific, saturable, and of high affinity (half saturation at 1 nm). These results indicate that EGF alone or in combination with dexamethasone is a potent accelerator of rabbit corneal stromal wound healing in vivo, and that EGF directly stimulates DNA synthesis of HCF in vitro presumably through specific high affinity receptors on corneal fibroblasts. EGF alone or in combination with corticosteroids may be useful in promoting healing in patients with corneal lacerations.

Morphological, behavioral, and physiological masculinization of adult female mice that developed in utero between two male fetuses (2M females) has been previously attributed to the significantly higher concentration of testosterone in their fetal blood and amniotic fluid than that in female mice which had not been contiguous to males in utero (0M females). Serum testosterone levels of adult 2M and 0M females are not significantly different. To determine whether exposure of fetuses to different levels of testosterone during prenatal development alters adult biochemical parameters of a system responsive to testosterone, the level of epidermal growth factor (EGF) was measured by radioreceptor assay in the submandibular glands of adult CF-1 mice of known intrauterine position. The concentration of EGF was significantly higher (P less than 0.05) in the glands of 2M females (mean +/- SEM, 0.36 +/- 0.14 nmol/mg dry wt of tissue; n = 6) than that in 0M females (0.05 +/- 0.00 nmol/mg dry wt; n = 8). In contrast, EGF concentration did not differ significantly between the glands of 2M and 0M males (0.51 +/- 0.01 and 1.10 +/- 0.42 nmol/mg dry wt, respectively). EGF levels were also determined in the submandibular glands from adult animals of unknown intrauterine position which were gonadectomized and then treated with testosterone and estradiol. The concentrations of EGF in the glands of gonadectomized males and females were similar (0.13 +/- 0.01 and 0.23 +/- 0.09 nmol/mg dry wt, respectively). However, there was a significant difference in response to hormonal administration between males and females. The response of females exceeded that of males at 400 and 800 micrograms testosterone/day. These results suggest that the hormonal environment of a fetus, specifically modification of the fetal environment by the production of hormones from adjacent fetuses, is a major factor in the adult expression of testosterone-responsive proteins such as EGF.

A procedure for dissociating the rabbit aorta into single, functional smooth muscle cells is described. After removal of adventitia and intima, slices of media were incubated with purified collagenase, elastase, and soybean trypsin inhibitor in a Krebs-Ringer buffer modified with Hepes, amino acids, and a [Ca2+] of 0.2 mM. After enzymatic digestion and mechanical shear, the yield of dispersed cells was approximately 25% based on DNA recovered. Greater than 95% of the cells excluded trypan blue and approximately 80-90% adhered to tissue culture dishes. By phase contrast microscopy, most of the cells were elongate and approximately 10 micron X 30 micron in size. The remainder were either spherical or highly crenated and contracted. Electron microscopy of the cells showed that immediately after dissociation greater than 95% could be identified as smooth muscle, though most had undergone some degree of structural change compared to cells in situ. Depending on the preparation, from 5 to 50% of these cells contracted in response to agonists. Cells shortened by 10-15% and developed numerous evaginations when stimulated by angiotensin II norepinephrine, or carbamylcholine. Cells relaxed after washout of agonists and could subsequently be restimulated. Specific inhibitors of each of the agonists blocked the contractile response. Dispersed cells cultured for 1-5 days contracted in even higher numbers than the freshly prepared cells, suggesting restoration of hormone binding and/or contractile function in culture. This preparation provides a system in which the physiology of individual vascular smooth muscle cells may be studied.

The presence of receptors for epidermal growth factor (EGF) in nonpregnant uteri and the elevation of EGF levels in blood during early pregnancy suggest that EGF and its receptor may play important roles in the early stages of pregnancy. We determined the distribution of EGF receptors in uteri of nonpregnant and pregnant mice during the late preimplantation period (days 4.5-5.0 of pregnancy) using radioautograph and quantitative binding techniques. Radioautography of [125I]EGF binding to cornua from nonpregnant mice showed low levels of specific binding evenly distributed throughout the cornua. In contrast, radioautographs of cornua from pregnant mice showed bands of elevated binding encircling the lumen at sites of preimplantation. Results from radioautography were supported by quantitative analysis of [125I]EGF binding to uterine homogenates from nonpregnant and pregnant mice. Binding of [125I]EGF to uterine membranes was highly specific and time dependent. The average level of specific EGF binding calculated from Scatchard plots of nonpregnant uteri (27 +/- 13 fmol/mg protein) was significantly (P less than 0.05) lower than that in pregnant superovulated uteri (106 +/- 67 fmol/mg protein). Furthermore, specific binding of EGF was significantly (P less than 0.05) higher in preimplantation sites than in the intervening nonimplantation regions from the same uteri (42 +/- 6 vs. 29 +/- 4 fmol/mg protein, respectively). Differences in EGF binding appear to be due to changes in the number of EGF receptors, since half-displacement values (1 nM) were similar in all samples. These results demonstrate that alterations of EGF receptor levels occur at sites where implantation will occur in mouse uteri and support the concept that the transforming growth factor-alpha/EGF receptor and its ligands are involved in implantation of concepti.

Human epidermal growth factor (EGF) concentrations were measured by a specific solid phase RIA in random urine samples collected throughout the menstrual cycle of normal menstruating women (n = 8), women with tubal sterilization (n = 6), women taking a low dose oral contraceptive (n = 5), and women throughout pregnancy (n = 52) and delivery (n = 35). There were no differences in EGF concentrations between the proliferative and secretory phases of the menstrual cycle (P greater than 0.05). Normal menstruating women had higher urinary EGF concentrations [mean +/- SE, 37.2 +/- 6.0 micrograms/g creatinine (4.23 +/- 0.68 ng/mumol)] than women with tubal sterilization [32.7 +/- 4.0 (3.71 +/- 0.45)] or women taking a low dose oral contraceptive [19.5 +/- 6.0 (2.21 +/- 0.68)], but the differences were not significant (P greater than 0.05). During pregnancy, urinary EGF concentrations increased linearly from 6-20 weeks gestation (r = 0.76; P less than 0.001), then declined toward term (r = -0.71; P less than 0.001). EGF concentrations in early pregnancy (less than 12 weeks) or at term did not differ significantly from those in normal menstruating women (P greater than 0.05). For women delivering normal, appropriate for gestational age (AGA) infants, there was no correlation between urinary EGF concentrations and fetal weight or sex (P greater than 0.05). Urinary EGF concentrations in women delivering normal AGA infants [52.7 +/- 2.5 (5.98 +/- 0.28); n = 16] did not differ significantly (P greater than 0.05) from those in women with class A/B diabetes [41.9 +/- 2.8 (4.76 +/- 0.31); n = 6] or women delivering twins [45.6 +/- 2.6 (5.18 +/- 0.29); n = 8] with a greater fetoplacental mass. However, women delivering an intrauterine growth-retarded fetus with decreased fetoplacental mass had lower urinary EGF concentrations (24.9 +/- 2.2 (2.83 +/- 0.25); n = 5] than women with normal AGA infants (P less than 0.01). The significance of the rise in the urinary EGF concentration late in the second trimester and lower urinary EGF concentrations in women delivering intrauterine growth-retarded infants is not known, but may reflect an important physiological role for EGF in fetal-maternal hormonal interaction and development.

Dispersed guinea pig pancreatic acinar cells have been used to investigate several aspects of stimulus-secretion coupling but possess the disadvantage that they are less sensitive and less responsive to secretagogues than in vitro preparations of intact pancreatic tissue (lobules). To overcome the poor responsiveness of isolated acinar cells, we have developed a new procedure for preparing dispersed, intact pancreatic acini whose sensitivity to secretagogues and morphological characteristics are similar to those of pancreatic lobules. Dispersed acini can be manipulated as suspensions of cells and full access of macromolecular probes to apical and basolateral plasmalemmal domains is obtained. Acini were prepared in good yields (~70% on a DNA basis) using only purified collagenase and mild mechanical shear in medium containing 2.0 mM Ca 2+ . Morphologically, acinar cells in the preparations retained intact junctional complexes, asymmetrical distribution of intramembranous particles between apical and basolateral plasmalemmal domains, and polarized distribution of intracellular organelles as found in intact pancreas. Dose-response curves of acini and mechanically prepared lobules to caerulein, carbachol, and bombesin were similar though acini were more sensitive to the C-terminal octapeptide of cholecystokinin. Net stimulated secretory protein discharge was ~36% over 2 h. Crude collagenase was purified for use in preparation of acini by Sephadex G-75 column chromatography which resolved collagenase from clostripain and a non-sulfhydryl-requiring protease. The purified collagenase contained at least four proteins with molecular weights between 85 000 and 110 000. Collagenase with 0.9 units of protease per unit of collagenase yielded unresponsive acini. Acini incubated with crude collagenase, chymotrypsin, or the non-sulfhydryl-requiring protease showed depressed secretory response to caerulein. Freeze-fracture electron microscopy of protease-treated acini indicated that the intramembranous particles aggregated and that many of the tight junctions had undergone a proliferation of non-cross-linked sealing strands which extended far down the basolateral plasma membrane and encircled gap junctions. Acini incubated with purified collagenase or with a clostripain-containing fraction from the Sephadex G-75 column appeared unaltered. This procedure produces acini which are morphologically and biochemically similar to the in situ pancreas and overcomes the poor response to secretagogues by isolated pancreatic acinar cells.

Epidermal regeneration depends on mitosis and migration of keratinocytes. Epidermal growth factor is known to stimulate growth of keratinocytes in vitro, thus it might be expected to promote wound healing. The results of this study show that topical application of biosynthetic human epidermal growth factor accelerates epidermal regeneration in split-thickness wounds and partial-thickness burns. The significant enhancement of epidermal regeneration suggests the potential for clinical use of epidermal growth factor for accelerating healing of burns, wounds from trauma, diabetic ulcers, skin graft donor sites, and others.

It has been suggested that choline and/or phosphatidylcholine may be beneficial in improving the memory deficits associated with aging and Alzheimer's disease. The effects of long-term choline or phosphatidylcholine treatment on cholinergic receptors in the brain have been investigated. Mice were maintained on one of four diets from 50 days of age until sacrificed at 20-24 months. [3H]-QNB binding in the cortex, hippocampus and striatum was specific, saturable and of high affinity. Animals treated with a phosphatidylcholine-enriched diet displayed a down-regulation of muscarinic receptors in the cortex and hippocampus, reflected by a decrease in Bmax. There were no significant differences in the binding affinities among the treatment groups. Choline levels were unaffected by the various diets, however phosphatidylcholine treatment resulted in an increase in phosphatidylcholine in the cortex and somewhat in the hippocampus. This study indicates that choline and phosphatidylcholine have different effects after long-term dietary administration. Phosphatidylcholine treatment results in a down-regulation of muscarinic receptors in certain brain areas which appears to be related to an increase in phosphatidylcholine concentration. Any potentially beneficial effects derived from chronic phosphatidylcholine treatment must overcome the apparent down-regulation of muscarinic receptors which may occur.

1. 1. Antisera to rat α-lactalbumin immediately displayed precipitation bands to human, bovine, pig and goat α-lactalbumin, whereas antisera to human α-lactalbumin displayed similar cross reactivities only at a later time. 2. 2. Cross reactivity of the antisera was determined by radioimmune assays to rat and human α-lactalbumin.

Objective While shear stress closely regulates vascular remodeling, the mediators of this process have been only partially elucidated. The current study examined the role of the gelatinases in flow-mediated vein graft intimal hyperplasia. We hypothesized that matrix metalloproteinase (MMP)–2 and MMP-9 expression and protein levels, relative to tissue inhibitors of metalloproteinase (TIMP)–1 and TIMP-2, are upregulated in a flow-dependent manner during vein graft arterialization. Methods Bilateral common carotid interposition vein grafting was performed in rabbits. Reduction in flow was achieved through unilateral ligation of the internal carotid artery and three of four branches of the external carotid artery. At 28 days grafts were harvested and analyzed for intimal area; MMP-2 and MMP-9, and TIMP-1 and TIMP-2 messenger RNA content, via quantitative reverse transcription polymerase chain reaction; and MMP-2 and MMP-9, and TIMP-1 and TIMP-2 protein concentrations, via both bioactivity assay and zymography. Results Branch ligation resulted in a 10-fold difference in mean flow rate and accelerated development of intimal hyperplasia in a low-flow environment. Exposure of the vein graft to arterial hemodynamics induced a marked rise in MMP-9 mRNA levels, whereas only a modest increase in MMP-2 mRNA was observed. MMP-2 protein was 50 to 100 times more abundant than MMP-9, and was significantly upregulated in grafts that demonstrated enhanced intimal thickening. Immunohistochemistry demonstrated that MMP-2 was located throughout the myointima, whereas MMP-9 was localized almost exclusively to the region of endothelium. No differences in TIMP-1 and TIMP-2 mRNA or protein levels were detected between high-flow and low-flow grafts. Conclusion MMP-2 is the predominate gelatinase that regulates early vein graft remodeling. Despite a marked increase in MMP-9 gene expression, development of intimal hyperplasia after a reduction in wall shear rate correlates with an increase in MMP-2 protein levels. These data suggest differential regulatory mechanisms for proteases within the remodeling vein graft wall. Modulation of extracellular matrix biologic features may offer therapeutic strategies for the prevention of vein graft failure.

Epidermal regeneration following middermal injuries to skin requires both proliferation and migration of keratinocytes. Epidermal growth factor (EGF) stimulates the proliferation of keratinocytes in culture, and topical administration of EGF accelerates epidermal regeneration of partial thickness burns or split-thickness incisions in vivo. Transforming growth factor-alpha (TGF-alpha) and vaccinia growth factor (VGF) have substantial sequence homology with EGF, and all appear to bind to the same receptor protein. Whether TGF-alpha or VGF can affect epidermal wound healing in vivo is not known. The present studies show that topical administration of TGF-alpha or VGF in antibiotic cream to partial thickness burns (second degree) accelerated epidermal regeneration in comparison with untreated or vehicle-treated burns. Low levels of both TGF-alpha and VGF (0.1 microgram per milliliter) appeared to be more effective than EGF in stimulating epidermal regeneration. Regenerated epithelium from burns treated with TGF-alpha or VGF appeared normal histologically. This finding suggests that topical application of selected growth factors may be useful in accelerating healing of partial thickness injuries.

Transforming growth factor-alpha (TGF-alpha) is a single chain polypeptide hormone of 50 amino acids that stimulates growth of some human cancer cells via an autocrine mechanism. The domain(s) of TGF-alpha that bind and activate its receptor have not been reported. Hydrophilicity plots of TGF-alpha indicate three discrete sequences that are theoretically exposed on the hormone's surface and thus potentially able to interact with the TGF-alpha receptor. Fragments of TGF-alpha encompassing these hydrophilic domains were prepared by using solid-phase peptide synthesis (SPPS) techniques and purified by use of high performance liquid chromotography (HPLC). Assessment of biological activity of the TGF-alpha fragments indicated that none of the fragments significantly inhibited binding of EGF to the receptor, stimulated DNA synthesis of cells, inhibited EGF-induced DNA synthesis of cells, stimulated growth of cells in soft agar, or induced phosphorylation of the receptor or p35 protein. These results indicate that the receptor binding domain of TGF-alpha is not totally encompassed by any of the separate fragments tested and probably is formed by multiple separate regions of TGF-alpha.

Experimental studies in animals have demonstrated that the topical application of epidermal growth factor accelerates the rate of epidermal regeneration of partial-thickness wounds and second-degree burns. We conducted a prospective, randomized, double-blind clinical trial using skin-graft-donor sites to determine whether epidermal growth factor would accelerate the rate of epidermal regeneration in humans. Paired donor sites were created in 12 patients who required skin grafting for either burns or reconstructive surgery. One donor site from each patient was treated topically with silver sulfadiazine cream, and one was treated with silver sulfadiazine cream containing epidermal growth factor (10 micrograms per milliliter). The donor sites were photographed daily, and healing was measured with the use of planimetric analysis. The donor sites treated with silver sulfadiazine containing epidermal growth factor had an accelerated rate of epidermal regeneration in all 12 patients as compared with that in the paired donor sites treated with silver sulfadiazine alone. Treatment with epidermal growth factor significantly decreased the average length of time to 25 percent and 50 percent healing by approximately one day and that to 75 percent and 100 percent healing by approximately 1.5 days (P less than 0.02). Histologic evaluation of punch-biopsy specimens taken from the centers of donor sites three days after the onset of healing supported these results. We conclude that epidermal growth factor accelerates the rate of healing of partial-thickness skin wounds. Further studies are required to determine the clinical importance of this finding.

There is no published data regarding whether human uterine tissues and leiomyomas contain binding sites for epidermal growth factor (EGF). The present study was undertaken with 33 myometria, 13 leiomyomas, and 4 endometria. All myometria (fundus) and leiomyomas and 3 of 4 endometria specifically bound 125I-labeled mouse EGF (myometria: mean, 1.1; range, 0.1-3.9 fmol/mg protein; leiomyomas: mean, 1.1; range, 0.2-2.6 fmol/mg protein; endometria: mean, 1.0; range, 0.0-3.1 fmol/mg protein). [125I]EGF binding to myometrium was ligand specific in that only unlabeled EGF, but not unlabeled insulin, hCG, human PRL, prostaglandin E1, or prostaglandin F2 alpha, competed with [125I]EGF for binding. The apparent dissociation constants and specific binding capacities for myometrium and leiomyoma were: 0.7 nM, 1.9 fmol/mg protein; and 0.1 and 3.7 nM, 0.1 and 4.4 fmol/mg protein, respectively. While smooth muscle content decreased from the fundus to the cervical end of uteri, [125I]EGF binding did not correspondingly decrease (r = 0.5; n = 4). The binding of [125I]EGF to myometrium did not vary with the phase of the menstrual cycle or the patients' diagnosis before hysterectomy (P greater than 0.1). In summary, these results demonstrate that human uteri and leiomyomas contain specific, high affinity binding sites for EGF, but the binding neither exhibited topographical changes nor varied with the phase of the menstrual cycle or benign pathological state of the tissue.

The ability of surgeons to accelerate wound healing through pharmacologic intervention is limited. The effects of locally applied, biosynthetic human epidermal growth factor (EGF) and transforming growth factor-beta (TGF-beta) on tensile strength of experimental incisions were investigated. A single dose of EGF in saline failed to increase tensile strength over controls. Thus, EGF was incorporated into multilamellar liposomes, which prolonged the exposure of incisions to EGF (p less than 0.001). A single dose of EGF in multilamellar liposomes produced a 200% increase in wound tensile strength over controls between 7 and 14 days (p less than 0.05). Light and electron microscopy of the wounds revealed increased collagen formation and fibroblast proliferation. A single dose of TGB-beta in a collagen vehicle stimulated a 51% increase in wound tensile strength at 9 days (p less than 0.01). We conclude that addition of EGF and TGF-beta in appropriate vehicles stimulates early transient increases in wound tensile strength in normal rats.

The present study demonstrates that human fetal membranes bind 125I-epidermal growth factor (125I-EGF), with chorion binding more than amnion. The chorion binding was also higher than that by decidua, but lower than in placenta. The lower binding by chorion compared to placenta was entirely attributable to a lower number of available EGF receptors and not to lower affinity. Chorion and placenta , but not amnion or decidua, from Cesarean section bound significantly more 125I-EGF when compared to tissues obtained after vaginal delivery. The binding of 125I-EGF to chorion exhibited dependency on time, temperature of incubation, pH of the incubation media, and amount of chorion protein. The 125I-EGF was not degraded during the binding reaction with chorion. The binding was specific in that unlabeled EGF inhibited 125I-EGF binding in a dose-dependent manner, whereas very high concentrations of other unlabeled hormones and growth factors had minimal effects on 125I-EGF binding. When some of these other hormones were tested for binding as tracers (125I-hCG, [3H]prostaglandin E1 and F2 alpha), neither chorion, amnion, decidua, nor placenta specifically bound these ligands. The 125I-EGF specific binding to chorion was saturable and a Scatchard plot of this data was curvilinear, which appears to be due to negative cooperativity. The apparent dissociation constant calculated from the initial slope of the plot was 0.20 nM, which is in excellent agreement with the concentration of unlabeled EGF required for half maximal inhibition of 125I-EGF binding, 0.26 nM. Autoradiography at the light microscope level revealed the presence of silver grains in amnion and chorion only when excess unlabeled EGF addition was withheld. The quantification of grains revealed the presence of a significantly (P less than 0.01) greater number of grains in chorion than in amnion, supporting the conclusion of the binding data. The physiological significance of EGF binding to fetal membranes and decidua is not known, but the considerable amount of EGF binding reported here suggests that they are target tissues for EGF.

Specific binding sites for [Asp1,Ile5]angiotensin II (angiotensin) have been demonstrated in homogenates and subcellular fractions of aortic medial smooth muscle cells, but the localization of the angiotensin receptor responsible for contraction has not been determined [Devynck, M. A., & Meyer, P. (1976) Am. J. Med. 61, 758-767]. To establish the location of this receptor, we have prepared a membrane-impermeable analogue of angiotensin by acylating its N-terminal amino group with the N-hydroxysuccinimide ester of succinylated ferritin. This angiotensin-ferritin conjugate possessed the same intrinsic activity as angiotensin but was approximately 200 times less potent in inducing contraction in rabbit aortic strips. The stability of the conjugate was investigated, and approximately 5% of the contractile activity of the angiotensin-ferritin conjugate was attributable to low molecular weight components that were present before or after exposure to aortic strips. The time required for aortic strips to reach a plateau of contraction in response to angiotensin-ferritin was significantly longer than that required by free angiotensin to produce the same level of contraction. With enzymatically dispersed aortic smooth muscle cells, however, the time taken to produce contractions by both angiotensin and angiotensin-ferritin was indistinguishable. [Sar1,-Ala8]angiotensin II, a competitive inhibitor of angiotensin, completely suppressed contractions induced by angiotensin or angiotensin-ferritin in aortic strips or dispersed aortic smooth muscle cells. These results suggest that angiotensin need not directly penetrate the plasma membrane to cause contraction and imply that the angiotensin receptor responsible for initiating contraction of aortic smooth muscle cells is located on the plasma membrane.

Direct competition experiments with delta 9 -tetrahydrocannibinol (delta 9-THC) and estradiol in binding assays with rat uterine cytosol estrogen receptors showed that delta 9-THC was a weak, but nevertheless significant, competitor for binding to cytoplasmic estrogen receptors. These data support, at the molecular level, the observations that delta 9-THC has a weak estrogenic activity (at least the ability to bind to estrogen receptors). Moreover, estrogen-like binding suggests that delta 9-THC, acting at the level of estrogen receptor, causes a primary estrogenic effect rather than an indirect or secondary phenomenon.

Transforming growth factor alpha (TGFα) is a peptide growth factor which is highly homologous to epidermal growth factor (EGF) and shares the epidermal growth factor receptor (EGF-R) to affect its action. These growth factors may play a role in the normal physiology of the human reproductive system.

Regeneration of corneal epithelium following injury is essential for visual rehabilitation. A limited number of approaches are available for treating patients who fail to heal epithelial injuries adequately. The presence of specific receptors for epidermal growth factor (EGF) on epithelial cells suggests that this potent mitogen may play a role in normal epithelial wound healing. Topical application of biosynthetic human EGF significantly accelerated epithelial regeneration in primates following epikeratophakia surgery. Epidermal growth factor alone and with fibronectin accelerated epithelial regeneration of rabbits following mild alkali burns. Since prolonged exposure of cells to EGF is necessary to induce mitosis, the dynamics of EGF in the eye and with various lenses was studied. When applied in methylcellulose-based eye drops, 90% of the EGF was lost from tear film within 10 min, while a small amount (10%) remained associated with conjuctival tissue. Soft contact lenses or epikeratophakia lenticles took up substantial amounts of EGF (50 micrograms) and released 85% of the EGF within 24 h, with a half-life of 4 h in vitro. Epidermal growth factor did not diffuse through corneas or lenticles and did not promote epithelial downgrowth along sutures in primate corneas. These results suggest that biosynthetic growth factors may be useful in the treatment of some epithelial injuries.