Liver regeneration in heparin-binding EGF-like growth factor transgenic mice after partial hepatectomy

BACKGROUND & AIMS: Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the EGF family, is synthesized in the form of a membrane-anchored precursor (proHB-EGF), which subsequently is processed proteolytically to mature HB-EGF. This study describes the effects of HB-EGF on liver regeneration after 70% partial hepatectomy in proHB-EGF transgenic mice with liver-specific expression. Methods & Results: No significant differences in liver/body weight ratios and in bromodeoxyuridine (BrdU)-labeling index (the ratios of BrdU-positive hepatocyte nuclei) were found between adult transgenic and wild-type mice. However, in regenerating liver after partial hepatectomy, transgenic mice had higher liver/body weight ratios than wild-type mice and at 120 hours reached a level equal to that before partial hepatectomy. The BrdU-labeling index was about 5 times higher in the livers of transgenic mice compared with the wild type (51.5% vs. 10.2%, respectively; P < 0.01) at 48 hours after partial hepatectomy. Activation of microtubule-associated protein kinase after partial hepatectomy was higher and earlier in the transgenic mice as compared with the wild-type mice. Soluble HB-EGF was increased in the liver (at 8 min) after partial hepatectomy, indicating that the shedding of proHB-EGF occurred after partial hepatectomy. CONCLUSIONS: The transgenic expression of HB-EGF accelerates the proliferation of hepatocytes after partial hepatectomy, suggesting that HB-EGF functions as a hepatotrophic factor in vivo.     

Serum contains a platelet-derived transforming growth factor.

High concentrations of fetal bovine serum induced colony formation in soft agar by anchorage-dependent, nontransformed mouse AKR-2B and rat NRK cells. The colony-stimulating activity in fetal bovine serum was precipitated by 45% saturated ammonium sulfate and migrated in molecular sieve chromatography as a single peak of activity in the 10,000-15,000 molecular weight range. The colony-stimulating activity was heat and acid stable and was destroyed by trypsin and dithiothreitol, indicating the activity is due to a polypeptide that requires disulfide bonds for biological activity. No competition for binding to the epidermal growth factor receptor was associated with the colony-stimulating activity. Isoelectric focusing revealed activity in the pI 4-5 range. The colony-stimulating activity in serum appeared to be of platelet origin because platelet-poor plasma and platelet-poor plasma-derived serum contained little activity, whereas acid/ethanol extracts of bovine and human platelets had potent colony-stimulating activity. Chromatography of platelet extracts on Bio-Gel P-60 revealed peaks of AKR-2B colony-stimulating activity in the 12,000 and 20,000 molecular weight ranges. The other biological and chemical properties of the platelet colony-stimulating activity were the same as those for the serum activity. The data indicate the presence in serum of a platelet-derived growth factor(s) with properties similar to those of the transforming growth factors.     

Cytosolic calcium regulates epidermal growth factor endocytosis in rat pancreas and cultured fibroblasts.

Cholecystokinin octapeptide (CCK8), the COOH-terminal moiety of cholecystokinin (CCK), exerted a rapid inhibitory effect on total cell-associated 125I-labeled epidermal growth factor (125I-EGF) binding by decreasing the rate of EGF internalization in isolated rat pancreatic acini. Removal of CCK8 from incubation medium followed by extensive washing of acini did not abolish its inhibitory effect, indicating that its action was not readily reversible. Proglumide, a competitive antagonist of CCK8, blocked the inhibitory action of the secretagogue. Addition of CCK8 to cells previously exposed to 125I-EGF did not enhance the release of cell-associated 125I activity. CCK8 did not inhibit the binding of 125I-labeled insulin to pancreatic acini. Other pancreatic secretagogues that enhance digestive-enzyme release through Ca2+, including caerulein, bombesin, carbachol, gastrin, and the Ca2+ ionophore A23187, also inhibited cell-associated 125I-EGF radioactivity. Further, at 37 degrees C the ionophore A23187 inhibited specific 125I-EGF binding in human A-431 carcinoma cells, Swiss 3T3 cells, and Rat-1 fibroblasts, and this effect was abolished when 125I-EGF internalization was reduced by incubating cells at 4 degrees C. It is concluded that alterations in cellular Ca2+ in the pancreas and other cells lead to inhibition of EGF endocytosis.     

Expression of heparin-binding epidermal growth factor-like growth factor in pancreatic adenocarcinoma.

BACKGROUND: Previous studies demonstrated that heparin-binding epidermal growth factor-like growth factor (HB-EGF) contributes to carcinogenesis and carcinoma progression. In this study, we investigated its expression in human pancreatic adenocarcinoma. METHODS: We immunohistochemically investigated the expression of HB-EGF in 40 cases of pancreatic adenocarcinoma. RESULTS: HB-EGF was only occasionally and faintly expressed in normal and hyperplastic pancreas duct epithelia. In pancreatic adenocarcinoma, 22 (55.0%) of the 40 cases were classified as positive for HB-EGF. Its expression was more frequently observed in cases with a low Ki-67 labeling index, well differentiated. early stage, small size, without lymph node metastasis and low EGF-R expression. CONCLUSION: These results suggest that HB-EGF mainly plays a role in early phase of the progression of pancreatic adenocarcinoma.     

Receptor- and heparin-binding domains of basic fibroblast growth factor.

Two functional domains in the primary structure of basic fibroblast growth factor (FGF) have been identified on the basis of their ability to interact with the FGF receptor, bind radiolabeled heparin, and modulate the cellular response to FGF. Peptides derived from these two functional domains can act as partial agonists and antagonists in biological assays of FGF activity. Peptides related to the sequences of FGF-(24-68)-NH2 and FGF-(106-115)-NH2 inhibit thymidine incorporation into 3T3 fibroblasts when they are stimulated by FGF but have no effect when the cells are treated with either platelet-derived growth factor or epidermal growth factor. They also possess partial agonist activity and can stimulate DNA synthesis when tested in the absence of exogenous FGF. The active peptides have no effect on the binding of epidermal growth factor to its receptor on A431 cells and they can modulate the effects of FGF, but not fibronectin, on endothelial cell adhesion. The results suggest the possibility of designing specific analogs of FGF that are capable of inhibiting the biological effects of FGF.     

Platelet-derived growth factor stimulates low density lipoprotein receptor activity in cultured human fibroblasts.

Human platelets contain a mitogen, the platelet-derived growth factor (PDGF), that stimulates the proliferation of a variety of cell types in culture and that may play a role in atherogenesis. Studies were conducted to explore the effects of PDGF on low density lipoprotein (LDL) receptor activity of cultured human fibroblasts. The PDGF utilized in these studies was partially purified from human platelet-rich plama by ion exchange chromatography and gel filtration. LDL receptor activity was assessed by both specific binding of 125I-labeled LDL to the fibroblast's surface at 4 degrees C, and the incorporation of [14C]oleate into cholesteryl esters. Exposure of normal human fibroblasts to increasing amounts of PDGF (0.1-10 microgram/ml) for 48 hr caused a dose-related increase in 125I-labeled LDL binding to a maximum of approximately 300%. In the presence of added LDL, this increase in LDL binding was not seen. Cholesterol esterifiction was also stimulated following a 48-hr exposure to PDGF. Following a conditioning period in LDL- and PDGF-depleted medium, cholesterol esterification was greatly increased during a 48-hr exposure to LDL alone; a smaller but significant increase occurred with PDGF alone. However, both PDGF and LDL were required to return the level of esterification to that observed with whole human serum. Fibroblasts from a patient with homozygous familial hypercholesterolemia, which lack the LDL receptor, also showed a significant increase in cholesteol esterification with PDGF alone, whereas LDL had no effect. These studies demonstrate that PDGF can stimulate the LDL receptor activity in cultured human fibroblasts. The effect on other related activities of the LDL receptor system and the mechanism involved remain to be defined.     

Appearance of heparin-binding EGF-like growth factor in wound fluid as a response to injury.

Wound fluid was obtained from porcine partial-thickness excisional wounds and analyzed for heparin-binding growth factors. Two heparin-binding growth factor activities were detected, a relatively minor one that was eluted from a heparin affinity column with 0.65 M NaCl and a major one that was eluted with 1.1 M NaCl. These activities were not present in wound fluid 1 hr after injury but appeared 1 day after injury, were maximal 2-3 days after injury, and were not detectable by 8 days after injury. The heparin-binding growth factor eluted with 0.65 M NaCl was identified as a platelet-derived growth factor (PDGF)-like activity by the use of specific anti-PDGF neutralizing antibodies. The heparin-binding growth factor eluted with 1.1 M NaCl was shown to be structurally related to heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) by several criteria, including binding to heparin affinity columns and elution with 1.1 M NaCl, competition with the binding of 125I-EGF to the EGF receptor, triggering phosphorylation of the EGF receptor, immunodetection on a Western blot, and stimulation of fibroblast and keratinocyte growth. It was concluded that HB-EGF is a major growth factor component of wound fluid and, since it is mitogenic for fibroblasts and keratinocytes, that it might play an important role in wound healing.     

Transforming growth factor-beta as a differentiating factor for cultured smooth muscle cells.

The aim of the present study was to determine whether the development of supercontractile smooth muscle cells, contributing to the nonspecific hyperreactivity of airways in asthmatic patients, is due to transforming growth factor (TGF)-beta. In cultured smooth muscle cells starved by removal of 10% foetal bovine serum for 7 days, growth arrest was seen; 30% became elongated and demonstrated super contractility. Study of conditioned medium suggested that the differentiating factor was TGF-beta. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on conditioned medium from the arrested cells. Two protein bands were identified as matrix metalloproteinase (MMP)-2 and TGF-beta1. To determine second messenger signalling by SMAD2, Western blotting and confocal microscopy were employed. Conditioned medium from arrested cultures showed the presence of MMP-2 and TGF-beta1, as revealed by SDS-PAGE; 68- and 25-kDa bands were seen. Differentiation was confirmed by upregulation of marker proteins, smooth muscle type myosin heavy chain and myosin light chain kinase. Confirmation was obtained by downregulating these proteins with decorin treatment, which reduces the levels of active TGF-beta and an adenoviral dominant-negative vector coding for a mutated type II TGF-beta-receptor. Activation of second messenger signalling was demonstrated immunocytochemically by the presence of phosphorylated SMAD2 and SMAD4. Transforming growth factor-beta is likely to be the differentiating factor responsible for the development of these supercontractile smooth muscle cells. The development of such cells in vivo after cessation of an asthmatic attack could contribute to the nonspecific hyperreactivity of airways seen in patients.     

Tissue distribution of hepatopoietin-A: a heparin-binding polypeptide growth factor for hepatocytes.

Hepatopoietin-A (HPTA) is a heparin-binding polypeptide growth factor which consists of a heavy and a light polypeptide chain with molecular weights of 70,000 and 35,000, respectively. It stimulates DNA synthesis in primary cultures of normal rat hepatocytes in serum-free medium. The complete purification and characterization of HPTA from rabbit serum were reported by us elsewhere. Recently we have determined the amino-terminal amino acid sequence of the rabbit HPTA light chain up to 24 residues and have shown that the sequence is not homologous with other known sequences. [N.B. Human hepatocyte growth factor, recently sequenced by two other groups, is the same molecular species as HPTA.] In the present paper we report the production of a neutralizing polyclonal antiserum raised in chicken against purified rabbit HPTA. This antiserum does not inhibit the mitogenic effect of other potent inducers of hepatocyte DNA synthesis (epidermal growth factor or acidic fibroblast growth factor), nor does it interact with these growth factors in an enzyme-linked immunosorbent assay (ELISA). The antibody recognizes HPTA, as was determined by Western immunoblotting. Since the tissue origin of HPTA is not known, this anti-HPTA antiserum was used to investigate the tissue distribution of HPTA in rabbits by immunohistostaining methods. Acinar cells of the pancreas, neurons of the brain, C cells of the thyroid, ductal cells of the salivary glands, and Brunners glands of the duodenum stained with anti-HPTA antibody. Liver, spleen, thymus, and kidney do not seem to contain appreciable amounts of HPTA. We confirmed these findings by extracting and purifying active HPTA from the stained tissues listed above. The anti-HPTA antibody recognizes HPTA purified from different tissues, as was determined by ELISA, Western immunoblotting, and immunoneutralization experiments.     

Platelet alpha granules contain a growth factor for fibroblasts.

Platelets contain a polypeptide growth factor that stimulates the replication of normal connective tissue cells; this platelet-derived growth factor (PDGF) is released during the clotting process. Human platelets from normal volunteers were disrupted by nitrogen cavitation, and the subcellular organelles were fractionated by ultracentrifugation through a 30%--60% sucrose gradient. Electron microscopy revealed that fraction 7 (density 1.23 g/liter) contained the largest number of alpha granules. The specific activity of platelet fibrinogen, an alpha-granule marker, was also highest in this fraction. The subcellular fractions were assay for the presence of PDGF and for beta-thromboglobulin. PDGF was assayed quantitatively by the stimulation of DNA synthesis in confluent growth-arrested BALB/c-3T3 cells, whereas the concentration of beta-thromboglobulin was determined by radioimmunoassay. The highest concentrations of both PDGF and beta-thromboglobulin were found in the alpha-granule fraction. In contrast, beta-glucuronidase, a lysosomal enzyme, was more diffusely distributed and had its highest specific activity in fractions of lower density than those for PDGS, beta-thromboglobulin, or fibrinogen. The data demonstrate that the alpha granules of platelets provide a unique delivery system for PDGF, a polypeptide hormone with growth-promoting activity for connective tissue cells.     

Pituitary fibroblast growth factor as a stimulator of growth in cultured rabbit articular chondrocytes.

Growth promoting activity for rabbit chondrocytes has been described as a contaminant of partially pruified TSH and LH prepared from bovine and ovine pituitaries. We have investigated fibroblast frowth factor (FGF), a small growth promoting peptide isolated from bovine pituitary tissue, in a rabbit chondrocyte system. The results suggest to us that FGF is the factor or one of the factors responsible for chondrocyte growth stimulating activity previously described in the pituitary hormone preparations. DNA synthesis in these cells is stimulated by FGF at final medium concentrations of 10(-9) g/ml. Bovine NIH-LH is not stimulatory below concentrations of 10(-7) g/ml. FGF also stimulates cell growth in the presence of 10% fetal bovine serum. Dexamethasone, at concentrations of 10(-5) to 10(-9) g/ml exerts a synergistic effect with FGF on both DNA synthesis and cell growth. Over a concentration range of 10(-6) to 10(-9) g/ml, FGF does not stimulate synthesis of sulfated mucopolysaccharides.     

Differential expression of heparin-binding epidermal growth factor-like growth factor in the rat ovary

We investigated the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) and its receptors in the rat ovary to define the role of HB-EGF in the ovarian function. The expression pattern of HB-EGF mRNA and protein were studied by semi-quantitative RT-PCR and immuno-histochemistry using an antibody that was specifically stained for the precursor form of HB-EGF in naturally cycling rats and immature pseudo-pregnant rat models. The immuno-histochemical study showed that in naturally cycling rats, HB-EGF was expressed in most granulosa cells of early follicles and all the developing follicles but not in preovulatory follicles. This was supported by the semi-quantitative RT-PCR results in that the lowest level of HB-EGF mRNA during the estrous cycle was found in the evening of proestrous when the HB-EGF negative preovulatory follicles were most prominent. The results suggest that HB-EGF might be a mitogen for granulosa cells and down regulation of its expression may be necessary for the final maturation of follicles. In corpora lutea, luteal cells of older generation stained stronger than those of younger generation. Moreover, luteal cells of late luteal phase stained stronger than those of the mid and early luteal phases in the immature pseudo-pregnant rat models, indicating that the precursor form may be associated with death of luteal cells. Finally, of the two cognate receptors for HB-EGF, erbB1 was expressed in the rat ovary, but erbB4 was specifically not expressed in this organ. The spatial and temporal pattern of HB-EGF expression suggest that HB-EGF may an important local regulator of ovarian function and structure.     

Regulation of insulin-like growth factor I messenger ribonucleic acid levels by serum in cultured rat fibroblasts.

Fibroblasts represent one of the in vivo sites of extrahepatic insulin-like growth factor I (IGF-I) production. In this study, cultured fibroblasts prepared from the skin of neonatal rats were used as a model to assess the role of serum in regulating IGF-I messenger RNA (mRNA) levels. IGF-I mRNA, as demonstrated by Northern blot analysis, was present in the cultured fibroblasts, and serum free media which was conditioned by fibroblasts for 20 h contained 108 pg/ml of immunoreactive IGF-I. Fetal calf serum (FCS) decreased steady state IGF-I mRNA levels, as measured by solution hybridization/RNase protection assay, in fibroblasts in a time- and dose-dependent fashion. Incubation of fibroblasts for 18 h in the presence of 0.3%, 0.6%, or 1% FCS decreased IGF-I mRNA levels to 76%, 56%, and 46% of the levels present in control cells which were maintained in serum free media with 0.25% BSA. Maximal inhibition to approximately 20% of control levels was seen with 4-10% FCS. In contrast, basic fibroblast growth factor and beta-actin mRNA levels increased 2- and 4-fold, respectively, with increasing concentrations of FCS. Treatment of the cells with 10 micrograms/ml cycloheximide resulted in partial abrogation of the inhibitory effect of FCS while protein synthesis in the cells was decreased to 6% of control levels. The addition of 2 micrograms/ml of insulin or 15-100 ng/ml of IGF-I to the fibroblasts did not reproduce the inhibitory effect of FCS. Finally, the inhibitory factor(s) present in the FCS was partially removed/inactivated by charcoal stripping or heat inactivating the serum, but delipidation of the FCS by chloroform extraction had no effect on the inhibitory effect of FCS. In summary, FCS contains a factor(s) that decreases IGF-I mRNA levels in cultured fibroblasts in a time- and dose-dependent fashion. The partial abrogation of the inhibitory effect of FCS with cycloheximide treatment suggests that this effect is at least partially dependent upon new protein synthesis. Furthermore, the studies using delipidated, heat-inactivated, and charcoal-stripped serum suggest that the inhibitory factor(s) is a peptide.     

Pharmacokinetics and distribution of heparin-binding growth factor I (endothelial cell growth factor) in the rat

Heparin-binding growth factor I (HBGF I), previously designated as endothelial cell growth factor, is a potent mitogen for endothelial cells in vitro, which may prove useful for promoting endothelial regeneration in vivo. Analysis of the pharmacokinetics and organ distribution of HBGF I is necessary before use of HBGF I as a pharmacological agent. Consequently, pharmacological studies were carried out with [125I]HBGF I in the rat. Intravenous injections of HBGF I were given with or without heparin (2.5 units/ng HBGF I). Blood concentrations of HBGF I decreased by one half 17 seconds after HBGF I bolus. This time was prolonged to 60 seconds when HBGF I was injected with heparin. The elimination half-life of HBGF I was 14 minutes in the presence of heparin. The highest concentrations of HBGF I following intravenous bolus were found in kidney, liver, and spleen, and the lowest in fat and brain. Heparin increased HBGF I concentrations in blood and all organs measured except kidney, which was significantly decreased (p less than 0.01). Intact HBGF I was recoverable from blood 5 minutes following intravenous administration. HBGF I underwent near-complete proteolytic digestion after more prolonged ex vivo incubation with rat plasma, but HBGF I was protected from proteolysis when incubations were conducted in the presence of heparin. Thus, it is feasible that HBGF I can be administered as a pharmacological agent in the presence of heparin. Further studies assessing acceleration of in vivo endothelial growth using HBGF I with heparin appear warranted.     

Endogenous cholecystokinin, the major factor responsible for dietary protein-induced pancreatic growth.

This study was undertaken to clarify the role of endogenous cholecystokinin (CCK) in induction of pancreatic growth stimulated by a high protein diet. Rats with i.v. jugular cannulae in place and kept in Bollman cages were adapted to 5% casein diet for 9 days and switched to 70% casein for 2 days. MK-329, a CCK receptor antagonist, and SMS 201-995, a somatostatin agonist, were continuously infused at 0.5 mg/kg/h and 5 micrograms/kg/h, respectively, starting at the onset of feeding 70% casein. The 5 and 70% casein control groups were infused with saline. Feeding 70% casein significantly stimulated pancreatic hyperplasia and tissue hypertrophy. MK-329 and SMS 201-995 totally prevented 70% casein-induced increases in pancreatic weight and total RNA and DNA contents. The results indicate that endogenous CCK is the major factor responsible for pancreatic growth induced by a high protein diet.     

T lymphocytes synthesize and export heparin-binding epidermal growth factor-like growth factor and basic fibroblast growth factor, mitogens for vascular cells and fibroblasts: differential production and release by CD4+ and CD8+ T cells.

T lymphocytes infiltrate wounds, tumors, and atherosclerotic plaques, pathophysiological processes characterized by the migration and proliferation of vascular cells and fibroblasts. Although T lymphocytes are known to produce cytokines for inflammatory cells, it has not been demonstrated that they synthesize growth factors that are mitogenic for vascular cells and fibroblasts. We demonstrate that cultured T lymphocytes isolated from normal human peripheral blood synthesize and export two well-characterized growth factors, heparin-binding epidermal growth factor-like growth factor (HB-EGF) and basic fibroblast growth factor (bFGF). This conclusion is based on mRNA expression analysis, heparin-affinity chromatography profiles, target-cell specificity, and functional inhibition by specific neutralizing antibodies. Atypically, a substantial amount of T-cell-derived bFGF-like activity appears to be constitutively released into conditioned medium, almost as much as is associated with T-cell lysates. bFGF is synthesized and exported by purified CD4+ and CD8+ T cells, whereas HB-EGF is synthesized and exported primarily by CD4+ T cells. The T-cell-derived HB-EGF and bFGF activities are potent mitogens for fibroblasts and smooth muscle cells, and the bFGF-like activity is also mitogenic for endothelial cells. These results suggest that T lymphocytes may play key roles in mediating smooth muscle hyperplasia associated with atherosclerosis and in angiogenesis associated with wound healing and tumor growth by acting locally to deliver vascular-cell growth factors to tissues.