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A growing number of antiangiogenesis strategies have been investigated for the treatment of cancer and other angiogenesis-dependent diseases. One of the most promising strategies is to systemically administer one or more antiangiogenic proteins frequently enough to achieve a sufficient long-term steady state level of the protein(s) to achieve the maximum beneficial effect. However, the utility of this strategy is limited because of many technical difficulties, including obtaining both the quantity and quality of the protein(s) necessary for optimal therapeutic benefit. To overcome these difficulties, we hypothesized that a single administration of a replication-defective adenoviral vector expressing a secretable antiangiogenic protein could achieve an optimal long-term systemic concentration. We constructed a recombinant adenoviral vector, Av3mEndo, which encodes a secretable form of murine endostatin. We demonstrated secretion of endostatin from several cell lines transduced with Av3mEndo. Partially purified endostatin secreted from Av3mEndo-transduced mammalian cells was shown to potently inhibit endothelial cell migration in vitro. A single intravenous administration of Av3mEndo in mice was shown to result in (1) prolonged and elevated levels of circulating endostatin, (2) partial inhibition of VEGF-induced angiogenesis in a VEGF implant angiogenesis model, and (3) prolonged survival and in 25% of mice the complete prevention of tumor growth in a prophylactic human colon/liver metastasis xenograft murine model. These results support our contention that adenoviral vectormediated expression of an antiangiogenic protein(s) represents an attractive therapeutic approach to cancer and other angiogenesis-dependent diseases.

Epidermal growth factor (EGF) is trophic for varying regions of the developing gastrointestinal tract (GIT) of suckling rats. The presence of large amounts of EGF in milk from various species, combined with low production of EGF by suckling animals, led to speculation that milk is a major source of EGF for suckling rats. We report that short-term fasting (8 hr) of 12-day-old suckling rats resulted in a significant decrease in the levels of immunoreactive EGF (irEGF) in the GIT. Pups refed by lactating mothers for 1 to 4 hr exhibited an increase in irEGF to original levels, whereas pups fed a rat milk substitute by gastric gavage did not have an increase in irEGF content. The irEGF levels in the GIT of pups that were manually fed normal rat milk, or rat milk substitute supplemented with EGF, returned to the prefasted levels. Fasted suckling rats refed 2 ml of rat milk in 2 h exhibited significantly higher level of irEGF in the GIT than did those refed with 0.5 ml in 45 min. Since rat milk irEGF exists in three distinct forms (A, B, and C; C is equal to authentic submandibular gland EGF, the irEGF forms in the GIT were characterized by native polyacrylamide gel electrophoresis. In the stomach luminal contents of the fed suckling rats, only the larger form, Peak B, was observed. Both the luminal content and the mucosa scrapings of all other segments of all groups contained only Form D (comigrating with desarginyl EGF), a metabolic derivative of EGF. All forms were immunoreactive, exhibited receptor binding, and stimulated DNA synthesis in growth-arrested fibroblasts. The rapid changes in EGF within the GIT of suckling rats suggest the EGF can acutely modify some GIT functions of suckling rats.

Aminoglycoside antibiotics act as nephrotoxic drugs, inducing a lysosomal phospholipidosis and necrotic lesions essentially in convoluted proximal tubules. Previous studies have demonstrated that tubular injury caused by these compounds elicits a process of renal tissue repair (tubular regeneration) involving an increase of cell turnover in tubular epithelium. The present study was performed in order to: (i) achieve further insight into the temporal relationship between aminoglycoside-induced phospholipidosis, tubular necrosis, and tubular regeneration; and (ii) approach the control of tubular regeneration after nephrotoxin-induced insult. To investigate the latter point, we examined by immunocytochemistry the intrarenal distribution of epidermal growth factor (EGF) during tubular regeneration. Five groups of female Sprague-Dawley rats (n = 5) were treated for 4 days with gentamicin i.p. at a daily dose of 50 mg/kg delivered in 2 injections per day. Sham-treated animals (n = 5) received an equivalent amount of vehicle (0.9% NaCl) according to the same protocol. Groups of treated rats, and controls, were terminated 16 h (day 1),4 days, 7 days, 14 days, and 21 days after the end of gentamicin administration. One hour prior to necropsy, each animal was given an i.p. injection of 40 mg 5-bromo-2'-deoxyuridine (BrdU)for the immunocytochemical demonstration ofS-phase cells, using an anti-BrdU monoclonal antibody. Renal tissue was processed for light microscopy analysis, namely: a computer-aided morphometry of lysosomes in proximal tubular cells, a single-blind evaluation of gentamicin-induced tubular injury, the measurement of cell proliferation by immunocyto-chemical detection of BrdU-labeled nuclei, the demonstration of EGF-like immunoreactive material in renal tissue by using anti-rat EGF antiserum and immunogold-silver staining. As revealed by the morphometry of lysosomes in proximal tubular epithelium, the degree of gentamicin-induced phospholipidosis was maximum at day 1 (relative area occupied by lysosomes was increased 25-fold over mean control value) and declined thereafter. In contrast, tubular necrosis reached a peak 4 days after the end of drug administration. In proximal tubular epithelium, the stimulation of cell turnover associated with tubular regeneration showed a peak at day 7 (15-fold the mean control value). Tubular regeneration was also accompanied by mild interstitial hyperplasia. Three weeks after treatment with gentamicin, morphological evidence of drug-induced injury had disappeared due to the tissue repair process, except for the occasional presence of small hyperplastic foci in renal cortex interstitium. In both treated animals and controls, EGF immuno-reactivity as revealed by immunocytochemical staining was associated with distal tubules (renal cortex and outer medulla). However, during tubular necrosis and regeneration there was a transitory reduction of EGF immunolabeling in distal tubules. Thus, the incidence of EGF-positive tubules, as determined by morphometry, decreased markedly to reach a trough by day 4. Afterwards, the distribution of EGF immunoreactivity resumed a normal appearance in parallel with the repair of tubular lesions. Altogether, these data show that aminoglycoside-induced phospholipidosis, tubular necrosis, and regeneration actually occur in succession, and suggest a possible involvement of EGF as an endogenous mediator during renal tubular regeneration.

Aromatic amino acid residues in epidermal growth factor (EGF) isolated from the rat have been investigated by proton n.m.r. and nuclear Overhauser methods at 500 MHz and by photochemically induced dynamic nuclear polarization (photo-c.i.d.n.p.) experiments at 360 MHz. Rat EGF contains six aromatic residues, i.e. one histidine and five tyrosine residues. pH titration data allow identification of the histidine imidazole ring protons, whereas two-dimensional n.m.r. correlated spectroscopy establishes connectivities between tyrosine ring (2,6) and (3,5) proton resonances. Photo-c.i.d.n.p. data give evidence for solvent exposure of the one histidine and the five tyrosine residues in rat EGF. Nuclear Overhauser experiments and pH titration data suggest proximity relationships among four of the tyrosine residues and the histidine residue. These data indicate the presence of a clustered, aromatic, structural domain on the protein surface and may provide a clue to the understanding of the functional structure of EGF.

ABSTRACTMutation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene by homologous recombination causes progressive pulmonary alveolar proteinosis (PAP) in GM-CSF-deficient mice (GM/). The present study tested whether adenovirus-mediated expression of GM-CSF alters the progression of PAP in GM/ mice. Adult mice were pretreated with an anti-T cell receptor (TCR) antibody to block T cell-mediated immune response, followed by intratracheal instillation of E1E3 replication-deficient adenovirus expressing mouse GM-CSF (Av1mGM). Mice were killed 1, 3, and 5 weeks after treatment to assess lungs for GM-CSF, surfactant protein B (SP-B), alveolar macrophage maturation, and type II cell proliferation. GM-CSF was detected in BAL fluid from GM/ mice 1 week after Av1mGM treatment, and GM-CSF mRNA was detected by RT-PCR through 5 weeks. Five weeks after Av1mGM treatment, PAP was improved and SP-B decreased as assessed by ELISA and immunostaining. Increased numbers of alveolar macrophages stained with -naphthyl acetate esterase (-NAE) following treatment with Av1mGM. Local expression of GM-CSF with a recombinant adenovirus ameliorated PAP in the GM/ mice in association with enhanced maturation of alveolar macrophages.

Acute tubular necrosis induced by aminoglycoside antibiotics and various other nephrotoxins is followed by a regenerative process which leads to the restoration of damaged tubules. Several lines of evidence indicate that tubular regeneration is mediated by polypeptide growth factors such as epidermal growth factor (EGF). Previous studies devoted to cisplatin nephrotoxicity have shown that this agent causes tubular cystic degeneration possibly related to an impairment of renal tissue repair. Thus, we examined on a comparative basis the time course of the regenerative response subsequent to tubular damage induced by tobramycin or cisplatin, particular attention being paid to renal EGF and its receptor. Female Sprague-Dawley rats (160-180 g body weight) were treated during 4 consecutive days with daily doses of 200 mg/kg tobramycin i.p. (BID) or 2 mg/kg cisplatin (once a day). Sham-treated rats were given 0.9% NaCl i .p. following the same protocol. Groups of experimental animals (n = 5-10) were terminated at increasing time intervals (1, 4, 7,14, 21, 60 days) after cessation of treatment. One hour prior to sacrifice, each individual received i.p. 200 mg/kg 5-bromo-2'-deoxyuridine (BrdU)for the immunohisto-chemical demonstration of cell proliferation. Blood was collected at the time of sacrifice in order to assess glomerular filtration rate by measuring serum creatinine and BUN levels. Kidneys were analyzed with respect to total EGF determined by RIA in renal tissue homogenates, and soluble EGF was assayed in extracts prepared by centrifugation. Renal tissue was processed for the immunohistochemical detection of S-phase cells, of EGF, of EGF receptors, and of the intermediate filament vimentin, the latter being used as a marker of epithelium dedifferentiation. In absence of nephrotoxic alterations, EGF was immunolocalized in distal tubules, whereas EGF receptor immunostaining was seen in proximal tubules cells. Vimentin immunostaining was confined to glomeruli and blood vessels. Tobramycin and cisplatin caused acute tubular necrosis in proximal convoluted tubules and proximal straight tubules, respectively. Tissue damage was accompanied by renal dysfunction reflected by an elevation of serum creatinine and BUN levels. Tubular necrosis was followed by a proliferative response indicative of tubular regeneration. Regenerative hyper-plasia was associated with a reduction of total immunoreactive EGF due to a decrease of tissue-bound proEGF. Tubules undergoing regenerative repair were characterized by a disappearance of EGF receptors and the presence of immunoreactive vimentin. In tobramycin-treated rats, renal dysfunction lasted for 4-7 days and was fully reversible, as indicated by the return of serum markers to normal values. Accordingly, tubular regeneration had led to the restoration of tubular epithelium in approximately 7-14 days. Moreover, the reduction of proEGF in renal tissue, and the loss of EGF receptors and vimentin expression in regenerating tubules occurred as a sequence of transient events so that kidneys had resumed a normal appearance by 14 days. In contrast with what was observed after exposure to tobramycin, cisplatin administration induced a protracted renal impairment probably related to a defective tissue repair. Kidneys of treated animals displayed a persistent depletion of immunoreactive proEGF. In addition, cystic tubules developing in the long term after exposure to cisplatin remained devoid of EGF receptors and exhibited continuous vimentin expression. The current study suggests that renal tubules damaged by cisplatin cannot undergo normal regeneration and probably remain in a dedifferentiated state.

Taxol has the following effects on myogenic cultures: (a) it blocks cell replication of presumptive myoblasts and fibroblasts. (b) It induces the aggregation of microtubules into sheets or massive cables in presumptive myoblasts and fibroblasts, but not in postmitotic, mononucleated myoblasts. (c) It induces normally elongated postmitotic myoblasts to form stubby, star-shaped cells. (d) It reversibly blocks the fusion of the star-shaped myoblasts into multinucleated myotubes. (e) It augments the number of microtubules in postmitotic myoblasts, and these are assembled into interdigitating arrays of microtubules and myosin filaments. (f) Actin filaments are largely excluded from these interdigitating microtubule-myosin complexes. (g) The myosin filaments in the interdigitating microtubule-myosin arrays are aligned laterally, forming A-bands approximately 1.5 micrometers long.

In order to obtain information on the possible role of epidermal growth factor (EGF) in rat prenatal development, we tested the effects of a neutralizing antiserum to rat EGF and of recombinant human EGF on the growth and development of transplanted rat embryos and fetal structures. Ten-day embryos or 16-day fetal intestines (ileum and jejunum) or paws were transplanted under the capsule of both kidneys of young adult syngeneic host rats. Osmotic minipumps were used to infuse antiserum to rat EGF or normal rabbit serum (NRS) into the renal artery of the right kidney to ascertain direct effects on development. The transplants on the contralateral side served as internal controls. Infusion of the NRS did not affect growth of any of the fetal structures or of the embryo transplants. The antiserum to rEGF did not affect growth of the fetal ileum or paw transplants, but it inhibited growth of the fetal jejunum by 38%, and suppressed differentiation of hair follicles in the paws by ∼90%. Tissue differentiation in the two segments of the intestine was unaffected by the antiserum. By contrast, growth of embryo transplants was stimulated by approximately 60% by the anti-EGF serum. Infusion of antiserum did not affect the growth of the kidneys upon which the transplants were grown, and infusion of different doses of recombinant human EGF had no effect on growth of embryo transplants.Our data suggest that EGF may function as a negative growth regulator during the embryonic period, but it becomes a growth stimulator for specific tissues during the fetal period. These results are consistent with our previous findings with basic fibroblast and insulin-like growth factors in illustrating that the regulatory functions of growth factors may be more general during embryogenesis but they become more organ or tissue specific in the fetal period of development.

Suckling rats exhibit age-dependent differences in epidermal growth factor (EGF) levels in several organs. The present studies evaluated the effects of two hormones known for their maturative effect on suckling rats, cortisone and tri-iodothyronine (T3), on immunoreactive EGF levels in specific organs. Suckling rats were administered cortisone (5 mg/100 g body weight per day) or T3(50 μg/100 g body weight per day) on days 8, 9, 10 and 11 after birth, and killed on day 12. Submandibular glands, kidneys, pancreas, liver and gastrointestinal tract mucosa and lumen were assayed for immunoreactive EGF by a speciesspecific radioimmunoassay. Low levels of EGF in the submandibular glands were increased slightly by both T3and cortisone treatment. Cortisone evoked a tenfold increase in EGF in the pancreas, but had no effect on levels in the kidney or liver. In contrast, T3evoked a sixfold increase in the EGF level in the kidney, but had no effect on levels in the pancreas or liver. Hormonal administration had no effect on EGF levels in the stomach. Within the intestinal tract, cortisone had no effect on the luminal EGF content of the duodenum, jejunum or ileum, but caused a decrease in the midjejunum. T3evoked a decrease in the luminal EGF content of the ileum. The effect of cortisone on mucosal EGF content varied between regions; an increase was seen in the duodenum with a decrease in the midjejunum and ileum. T3administration resulted in a significant decrease in EGF only in the mucosa of the ileum. The EGF-degradative capacity of the luminal contents of the jejunum and ileum, as studied in vitro, were increased by treatment with both T3and cortisone. However, no direct correlation was observed between alterations of the EGF content in the lumen or mucosa and the increased degradative capacity. We therefore conclude that modulation of EGF levels within the intestine cannot be explained exclusively by alterations in the degradation rates. The results indicate that both adrenal and thyroid glands may play an important role in the modulation of EGF levels in the developing rat.Journal of Endocrinology(1991) 131,95–100

This study evaluates the effect of dietary wheat bran on the levels of immunoreactive epidermal growth factor (EGF) in the gastrointestinal mucosa of Fischer 344 rats. Male rats were fed either a fiber-free diet or a diet containing 10% wheat bran (nine animals per group) for a period of 5 wk. The gastrointestinal tract of each animal was divided into four segments: proximal, middle and distal small intestine, and colon. Mucosa was removed by scraping, EGF was extracted by homogenization and the extracts were analyzed for immunoreactive rat EGF using a homologous RIA. Levels of immunoreactive EGF in all regions of the small intestine of Fischer rats were comparable to our previous measurements in Sprague-Dawley rats, and these levels were unaffected by diet. In contrast, the EGF levels in the colon of the Fischer rats were approximately fivefold greater than those of the Sprague-Dawley rats. These higher levels of immunoreactive EGF in the colon decreased 63% with the addition of 10% wheat bran to the diet (P< 0.02). These results represent the first demonstration of dietary fiber modulating the content of EGF in the gastrointestinal tract.

The phorbol ester TPA induces the sequential disassembly of myofibrils. First the alpha-actin thin filaments are disrupted and then, hours later, the myosin heavy chain (MHC) thick filaments. TPA does not induce the disassembly of the beta- and gamma-actin thin filaments of stress fibers in presumptive myoblasts or fibroblasts, nor does it block the reemergence of stress fibers in 72-h myosacs that have been depleted of all myofibrillar molecules. There are differences in where, when, and how myofibrillar alpha-actin and MHC are degraded and eliminated from TPA-myosacs. Though the anisodiametric myotubes have begun to retract into isodiametric myosacs after 5 h in TPA, staining with anti-MHC reveals normal tandem A bands. In contrast, staining with mAb to muscle actin fails to reveal tandem I bands. Instead, both mAb to muscle actin and rhophalloidin brilliantly stain numerous disk-like bodies approximately 3.0 micron in diameter. These muscle actin bodies do not fuse with one another, nor do they costain with anti-MHC. All muscle actin bodies and/or molecules disappear in 36-h myosacs. The collapse of A bands is first initiated in 10-h myosacs. Their loss correlates with the appearance of immense, amorphous MHC patches. MHC patches range from a few micrometers to over 60 micron in size. They do not costain with antimuscle actin or rho-phalloidin. While diminishing in number and fluorescence intensity, MHC aggregates are present in 30% of the 72-h myosacs. Myosacs removed from TPA rapidly elongate, and after 48 h display normal newly assembled myofibrils. TPA reversibly blocks incorporation of [35S]methionine into myofibrillar alpha-actin, MHC, myosin light chains 1 and 2, the tropomyosins, and troponin C. It does not block the synthesis of beta- or gamma-actins, the nonmyofibrillar MHC or light chains, tubulin, vimentin, desmin, or most household molecules.

To determine the relationship between thin filaments, Z-bands, microtubules, intermediate filaments (IFs), T-tubules, and sarcoplasmic reticulum (SR) during myofibrillogenesis, myotubes were selectively depleted of their myofibrils with 12-tetradecanoylphorbol 13-acetate (TPA) and then were allowed to regenerate in (i) normal medium, (ii) taxol, and (iii) Colcemid. Myofibrils assembled in normal medium formed typical A-, I-, Z-, M-, and H-bands and associated IFs, T-tubules, and SR. Myofibrils assembled in taxol formed "A-bands" of aligned thick filaments interdigitating with long microtubules and "I-bands" consisting only of microtubules. These unprecedented sarcomeres lacked thin filaments, Z-bands, and associated IFs and SR. "Solitary A-bands," consisting exclusively of laterally aligned bipolar thick filaments 1.6 microM in length without either thin filaments or microtubules, were observed. Myofibrils assembled in Colcemid formed all myofibrillar components in the absence of microtubules but these did not achieve rigorous lateral alignment. Colcemid and taxol induced the formation of patchy Z-bands that invariably served as insertion sites for thin filaments, irrespective of the presence or absence of adjacent thick filaments. Z-bands may function as actin-organizing centers for each sarcomere.

We have isolated and characterized cDNA clones from chicken cDNA libraries derived from skeletal muscle, body wall, and cultured fibroblasts. A clone isolated from a skeletal muscle cDNA library contains the complete protein-coding sequence of the 284-amino-acid skeletal muscle beta-tropomyosin together with 72 bases of 5' untranslated sequence and nearly the entire 3' untranslated region (about 660 bases), lacking only the last 4 bases and the poly(A) tail. A second clone, isolated from the fibroblast cDNA library, contains the complete protein-coding sequence of a 248-amino-acid fibroblast tropomyosin together with 77 bases of 5' untranslated sequence and 235 bases of 3' untranslated sequence through the poly(A) tract. The derived amino acid sequence from this clone exhibits only 82% homology with rat fibroblast tropomyosin 4 and 80% homology with human fibroblast tropomyosin TM30nm, indicating that this clone encodes a third 248-amino-acid tropomyosin isoform class. The protein product of this mRNA is fibroblast tropomyosin 3b, one of two low-molecular-weight isoforms expressed in chicken fibroblast cultures. Comparing the sequences of the skeletal muscle and fibroblast cDNAs with a previously characterized clone which encodes the smooth muscle alpha-tropomyosin reveals two regions of absolute homology, suggesting that these three clones were derived from the same gene by alternative RNA splicing.

Brains of adult male rats were dissected into five distinct regions: brainstem, cerebellum, hippocampus, diencephalon, and telencephalon. Epidermal growth factor-like immunoreactivity was isolated and characterized by radioimmunoassay and nondenaturing Polyacrylamide gel electrophoresis. Radioimmunoassay indicated levels of standard rat epidermal growth factor equivalents ranging from 0.99 to 0.33 ng/g wet weight of brain tissue. Competition curves were not parallel to those generated with standard rat epidermal growth factor, indicating a lack of structural identity between the immunoreactive material in the brain and standard rat epidermal growth factor. Extracts of submandibular gland and blood did, however, produce parallel competition curves. Electrophoresis indicated the presence of multiple bands of immunoreactive material in each of the regions of the brain. The major bands of activity migrated to positions distinct from that of standard rat epidermal growth factor. This is the first demonstration of multiple forms of epidermal growth factor-like immunoreactive material in the central nervous system.