A crystallinity study of dental tissues and tartar by infrared spectroscopy.

In this paper we report a study of an important property of biomineralized phases, crystallinity, on the basis of previous results for synthetic apatite. Crystallinity is not only important for understanding biomineralization, it is also related to the maturation and mechanisms of growth of calcium phosphates in biological surroundings. We studied two kinds of sample, teeth as an example of biomineralized tissues and dental calculi (adhering) as an example of mineralization without participation of biological agents, except possibly bacteria. The investigation focused on study of ν(1)-ν(3) infrared absorption bands of PO(4)(3-) phosphates. We used ATR (attenuated total reflection) analysis to examine human dental tissues and tartar on several samples. The results confirm for the first time previous assumptions about the growth and maturation of dental calculi, i.e., crystallinity progresses from regions of high crystallinity to regions of lower crystallinity, and, in addition, its quantification with spatial resolution in the sample. A gradual pattern was observed in dental calculus. Another result from this study was that cementum and dentine had similar crystallinity, despite their different biological and mechanical functions.

Vascular endothelial growth factor (VEGF121) protects rats from renal infarction in thrombotic microangiopathy.

BACKGROUND: Renal thrombotic microangiopathy, typified by the hemolytic uremic syndrome, is associated with endothelial cell injury in which the presence of cortical necrosis, extensive glomerular involvement, and arterial occlusive lesions correlates with a poor clinical outcome. We hypothesized that the endothelial survival factor vascular endothelial growth factor (VEGF) may provide protection. METHOD: Severe, necrotizing, thrombotic microangiopathy was induced in rats by the renal artery perfusion of antiglomerular endothelial antibody, followed by the administration of VEGF or vehicle, and renal injury was evaluated. RESULTS: Control rats developed severe glomerular and tubulointerstitial injury with extensive renal necrosis. The administration of VEGF significantly reduced the necrosis, preserved the glomerular endothelium and arterioles, and reduced the number of apoptotic cells in glomeruli (at 4 hours) and in the tubulointerstitium (at 4 days). The prosurvival effect of VEGF for endothelium may relate in part to the ability of VEGF to protect endothelial cells from factor-induced apoptosis, as demonstrated for tumor necrosis factor-alpha (TNF-alpha), which was shown to be up-regulated through the course of this model of renal microangiopathy. Endothelial nitric oxide synthase expression was preserved in VEGF-treated rats compared with its marked decrease in the surviving glomeruli and interstitium of the antibody-treated rats that did not receive VEGF. CONCLUSIONS: VEGF protects against renal necrosis in this model of thrombotic microangiopathy. This protection may be mediated by maintaining endothelial nitric oxide production and/or preventing endothelial cell death.

VEGF(121)- and bFGF-induced increase in collateral blood flow requires normal nitric oxide production.

The angiogenic proteins basic fibroblast growth factor (bFGF; FGF-2) and vascular endothelial growth factor 121 (VEGF(121)) are each able to enhance the collateral-dependent blood flow after bilateral femoral artery ligation in rats. To study the effect of nitric oxide (NO) synthase (NOS) inhibition on bFGF- or VEGF(121)-induced blood flow expansion, the femoral arteries of male Sprague-Dawley rats were ligated bilaterally, and the animals were given tap water [non-N(G)-nitro-L-arginine methyl ester (L-NAME) group; n = 36] or water that contained L-NAME (L-NAME group; 2 mg/ml, n = 36). Animals from each group were further divided into three subgroups: vehicle (n = 12), bFGF (5 microg x kg(-1) x day(-1), n = 12), or VEGF(121) (10 microg x kg(-1) x day(-1), n = 12). Growth factors were delivered via intra-arterial infusion with osmotic pumps over days 1-14. On day 16, after a 2-day delay to permit clearance of bFGF and VEGF from the circulation, maximal collateral blood flow was determined by (85)Sr- and (141)Ce-labeled microspheres during treadmill running. L-NAME (approximately 137 mg x kg(-1) x day(-1)) for 18 days increased systemic blood pressure (approximately 26%, P<0.001). In the absence of L-NAME, collateral-dependent blood flows to the calf muscles were greater in the VEGF(121)- and bFGF-treated subgroups (85 +/- 4.5 and 80 +/- 2.9 ml x min(-1) x 100 g(-1), respectively) than in the vehicle subgroup (49 +/- 3.0 ml x min(-1) x 100 g(-1), P<0.001). In the presence of NOS inhibition by L-NAME, blood flows to the calf muscles were essentially equivalent among the three subgroups (54 +/- 3.0, 56 +/- 5.1, and 47 +/- 2.0 ml x min(-1) x 100 g(-1) in the bFGF-, VEGF(121)-, and vehicle-treated subgroups, respectively) and were not different from the blood flow in the non-L-NAME vehicle subgroup. Our results therefore indicate that normal NO production is essential for the enhanced vascular remodeling induced by exogenous bFGF or VEGF(121) in this rat model of experimental peripheral arterial insufficiency. These results imply that a blunted endothelial NO production could temper vascular remodeling in response to these angiogenic growth factors.

CTGF expression is induced by TGF- beta in cardiac fibroblasts and cardiac myocytes: a potential role in heart fibrosis.

Connective tissue growth factor (CTGF) is a cysteine-rich protein induced by transforming growth factor beta (TGF- beta) in connective tissue cells. CTGF can trigger many of the cellular processes underlying fibrosis, such as cell proliferation, adhesion, migration and the synthesis of extracellular matrix; however, its role in acute and chronic cardiac injury is not fully understood. Here, we show that TGF- beta is a specific inducer of CTGF expression in both cardiac fibroblasts and cardiac myocytes. The activity of a CTGF promoter-based reporter construct correlated with endogenous CTGF expression, suggesting that TGF- beta induces CTGF expression most likely by activating its promoter. Upregulation of CTGF coincided with an increase in fibronectin, collagen type I and plasminogen activator inhibitor-1 production. Forskolin, a stimulator of cyclic AMP, blocked TGF- beta induced CTGF expression and reduced the basal level of CTGF, whereas an inhibitor that blocks the MAP kinase signaling pathway (PD 98059) significantly enhanced TGF- beta induced CTGF expression. Furthermore, we found that both TGF- beta and CTGF mRNAs were significantly elevated in the left ventricles and septa of rat hearts 2-16 weeks following myocardial infarction. This correlated well with concomitant increases in fibronectin, and type I and type III collagen mRNA levels in these animal hearts. Significant upregulation of CTGF was also detected in human heart samples derived from patients diagnosed with cardiac ischemia. Based on these findings, we propose that CTGF is an important mediator of TGF- beta signaling in the heart and abnormal expression of this gene could be used as a diagnostic marker for cardiac fibrosis.

Heparin blockade of thrombin-induced smooth muscle cell migration involves inhibition of epidermal growth factor (EGF) receptor transactivation by heparin-binding EGF-like growth factor.

Agonists of G protein-coupled receptors, such as thrombin, act in part by transactivating the epidermal growth factor (EGF) receptor (EGFR). Although at first a ligand-independent mechanism for EGFR transactivation was postulated, it has recently been shown that this transactivation by various G protein-coupled receptor agonists can involve heparin-binding EGF-like growth factor (HB-EGF). Because thrombin stimulation of vascular smooth muscle cell migration is blocked by heparin and because heparin can displace HB-EGF, we investigated the possibility that thrombin stimulation of smooth muscle cells (SMCs) depends on EGFR activation by HB-EGF. In rat SMCs, EGFR phosphorylation and extracellular signal-regulated kinase (ERK) activation in response to thrombin are inhibited not only by the EGFR inhibitor AG1478 and by EGFR blocking antibody but also by heparin and by neutralizing HB-EGF antibody. HB-EGF-dependent signaling induced by thrombin is inhibited by batimastat, which suggests a requirement for pro-HB-EGF shedding by a metalloproteinase. We further demonstrate that this novel pathway is required for the migration of rat and baboon SMCs in response to thrombin. We conclude from these data that the inhibitory effect of heparin on SMC migration induced by thrombin relies, at least in part, on a blockade of HB-EGF-mediated EGFR transactivation.

Heparin-binding EGF-like growth factor contributes to reduced glomerular filtration rate during glomerulonephritis in rats.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family, is expressed during inflammatory and pathological conditions. We have cloned the rat HB-EGF and followed the expression of HB-EGF in rat kidneys treated with anti- glomerular basement membrane (anti-GBM) antibody (Ab) to induce glomerulonephritis (GN). We observed glomerular HB-EGF mRNA and protein within 30 minutes of Ab administration and showed by in situ hybridization that glomerular HB-EGF mRNA expression was predominantly in mesangial and epithelial cells. Expression of HB-EGF correlated with the onset of decreased renal function in this model. To test the direct effect of HB-EGF on renal function, we infused the renal cortex with active rHB-EGF, prepared from transfected Drosophila melanogaster cells. This treatment induced a significant decrease in single nephron GFR (SNGFR), single nephron plasma flow, and glomerular ultrafiltration coefficient and an increase in the glomerular capillary hydrostatic pressure gradient. In addition, anti-HB-EGF Ab administered just before anti-GBM Ab blocked the fall in SNGFR and GFR at 90 minutes without any change in the glomerular histologic response. These studies suggest that HB-EGF expressed early in the anti-GBM Ab GN model contributes to the observed acute glomerular hemodynamic alterations.

Insulin-like growth factor-binding protein-3 induces fetalization in neonatal rat cardiomyocytes.

We employed cDNA microarrays representing 4000 distinct sequences to profile changes in gene expression in a rodent model of heart disease, namely, progression to heart failure after myocardial infarction. Differential gene expression in the left ventricle was examined at 4-week intervals over a 12-week period after coronary artery ligation in rats. Over this time course, insulin-like growth factor-binding protein-3 (IGFBP-3) was found to have a greater expression than in nondiseased tissues. We then employed quantitative real-time PCR to analyze gene expression in neonatal rat cardiac myocytes that had been treated with recombinantly expressed IGFBP-3 to examine a number of transcriptional responses designed to reflect the heart failure phenotype. The IGFBP-3 protein was shown to induce transcription of atrial natriuretic factor (ANF) and beta-myosin heavy chain (B-MHC). Analysis of conditioned media taken from IGFBP-3-treated cardiac myocyte cultures demonstrated an increase in ANF protein as well as in protein synthesis, as determined by metabolic incorporation of a radiolabeled amino acid. However, transcriptional changes of troponin-1, endothelin-1, or angiotensin-II by IGFBP-3 were not observed.

Production of heparin binding epidermal growth factor-like growth factor in the early phase of regeneration after acute renal injury. Isolation and localization of bioactive molecules.

We have recently reported that heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA is induced in the rat kidney after acute ischemic injury. The present studies were designed to investigate whether bioactive HB-EGF protein is also produced in response to renal injury induced by either ischemia/reperfusion or aminoglycosides. Heparin-binding proteins were purified from kidney homogenates by heparin affinity column chromatography using elution with a 0.2-2.0 M gradient of NaCl. A single peak of proteins that eluted at 1.0-1.2 M NaCl was detected in the postischemic kidney within 6 h of injury. This eluate fraction stimulated DNA synthesis in quiescent Balb/c3T3, RIE, and NRK-52E cell lines, all of which are responsive to the epidermal growth factor family of mitogenic proteins. The EGF receptor of A431 cells was also tyrosine phosphorylated by this eluate peak. Furthermore, immunoblotting with a polyclonal antibody against rat HB-EGF indicated that the eluate peak contained immunoreactive proteins of 22 and 29 kD mol wt, consistent with the reported sizes of the secreted form and membrane anchored form of HB-EGF, respectively. Immunohistochemical studies revealed that HB-EGF was produced predominantly in distal tubules in kidneys injured either by ischemia/reperfusion or aminoglycoside administration. We also found that during metanephric development immunoreactive HB-EGF was detected in the ureteric bud as early as E14.5 and persisted in structures arising from the ureteric bud throughout embryogenesis. These results suggest that in response to acute injury, HB-EGF is produced predominantly in distal tubules and that endogenous HB-EGF may be an important growth factor involved in renal epithelial cell repair, proliferation, and regeneration in the early stages of recovery after acute renal injury, as well as in nephrogenesis.

Heparin-binding EGF-like growth factor is a mitogen for rat alveolar type II cells.

Alveolar type II cells proliferate and differentiate into type I epithelial cells to restore the alveolar epithelium after lung injury. Since mitogens that bind the epidermal growth factor (EGF), EGF, receptor and transforming growth factor alpha (TGF alpha) have been shown to stimulate type II cell proliferation, studies were undertaken to determine whether the recently described protein, heparin-binding EGF-like growth factor (HB-EGF), was a mitogen for rat alveolar type II cells in primary culture. In addition, since HB-EGF is produced by macrophages, it was of interest to determine whether mitogenic activity for type II cells present in macrophage conditioned medium was due to HB-EGF. Rat and human recombinant HB-EGF stimulated thymidine incorporation into rat type II cells in a concentration-dependent manner up to 10-50 ng/ml then became inhibitory. The nuclear labeling index of type II cells increased from 2% to 16% with 10 ng/ml HB-EGF. However, HB-EGF induced only a small increase in cell number after 48 h and did not support low-density proliferation of alveolar type II cells. Conditioned medium from the human monocytic cell line, U937, stimulated type II cell DNA synthesis, and stimulatory activity could be partially purified by S-sepharose and heparin-sepharose chromatography. The growth-promoting activity from U937 cells that bound to heparin-sepharose was inhibited by a neutralizing antibody to human HB-EGF. Immunoblot analysis of active fractions also verified the presence of HB-EGF. However, the neutralizing antibody to rat HB-EGF did not inhibit mitogenic activity for type II cells found in rat bronchoalveolar lavage fluid. HB-EGF mRNA was found to be expressed in human alveolar macrophages to similar levels as differentiated U937 cells but was not detected in rat alveolar macrophages by Northern analysis of total mRNA. There was no difference in the level of HB-EGF mRNA expression in human alveolar macrophages from patients with interstitial lung disease compared with macrophages from normal subjects. The results demonstrate that HB-EGF is a mitogen for rat alveolar type II cells but appears to show species-specific differences with regard to its production by macrophages. Leslie, C. C., K. McCormick-Shannon, J. M. Shannon, B. Garrick, D. Damm, J. A. Abraham, and R. J. Mason. 1997. Heparin-binding EGF-like growth factor is a mitogen for rat alveolar type II cells. Am. J. Respir. Cell Mol. Biol. 16:379-387.

Involvement of wound-associated factors in rat brain astrocyte migratory response to axonal injury: in vitro simulation.

The poor ability of mammalian central nervous system (CNS) axons to regenerate has been attributed, in part, to astrocyte behavior after axonal injury. This behavior is manifested by the limited ability of astrocytes to migrate and thus repopulate the injury site. Here, the migratory behavior of astrocytes in response to injury of CNS axons in vivo was simulated in vitro using a scratch-wounded astrocytic monolayer and soluble substances derived from injured rat optic nerves. The soluble substances, applied to the scratch-wounded astrocytes, blocked their migration whereas some known wound-associated factors such as transforming growth factor-beta 1 (TGF-beta 1), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), and heparin-binding epidermal growth factor in combination with insulin-like growth factor-1 (HB-EGF + IGF-1) stimulated intensive migration with consequent closure of the wound. Migration was not dominated by proliferating cells. Both bFGF and HB-EGF + IGF-1, but not TGF-beta 1, could overcome the blocking effect of the optic nerve-derived substances on astrocyte migration. The induced migration appeared to involve proteoglycans. It is suggestive that appropriate choice of growth factors at the appropriate postinjury period may compensate for the endogenous deficiency in glial supportive factors and/or presence of glial inhibitory factors in the CNS.

Rapid induction of heparin-binding epidermal growth factor/diphtheria toxin receptor expression by Raf and Ras oncogenes.

We have used differential display PCR to search for mRNAs induced by delta Raf-1:ER, an estradiol-dependent form of Raf-1 kinase. Through this approach the gene encoding heparin-binding epidermal growth factor (HB-EGF) was identified as an immediate-early transcriptional target of oncogenic Raf kinases. Activation of delta Raf-1:ER and a conditional oncogenic form of B-Raf, delta B-RAF:ER, resulted in rapid and sustained induction of HB-EGF mRNA expression and secretion of mature HB-EGF from cells. Neutralizing anti-HB-EGF antisera prevented the delayed activation of the c-Jun amino-terminal kinases that is observed in cells transformed by delta Raf-1:ER. These results demonstrate that distinct signaling pathways can cross talk via the secretion of polypeptide growth factors. Furthermore, cells transformed by oncogenic Ras, which also induced HB-EGF expression, demonstrated a marked increase in sensitivity to the cytotoxic action of diphtheria toxin, for which the membrane anchored HB-EGF precursor acts as a cell-surface receptor.

Carboxyl-terminal truncation of leucine76 converts heparin-binding EGF-like growth factor from a heparin-enhancible to a heparin-suppressible growth factor.

Previous studies have indicated that heparin differentially regulates heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin (AR) mitogenic activity. To further explore this phenomenon, these mitogens were compared under identical cell culture conditions in two different assays. The results of our present investigation demonstrated that AR-mediated mitogenic activity in the murine AKR-2B fibroblast-like cell line was inhibited by heparin, while HB-EGF activity was enhanced. However, the absolute effect of heparin appeared to be cell type specific since HB-EGF mitogenic activity was not dramatically affected by coincubation with heparin when tested on human dermal fibroblasts. Several studies have indicated that mutation of a conserved leucine in the carboxyl-terminal region of both EGF and transforming growth factor-alpha results in decreased affinity for EGF receptors. Since this leucine is present in the analogous position of HB-EGF, but absent in AR, we examined the effect of deleting this residue by carboxyl-terminal truncation of HB-EGF. Analysis of recombinant forms of HB-EGF demonstrated that HB-EGF can be converted to a heparin-inhibited growth factor if the putative mature form of the protein is truncated by two residues (leucine76 and proline77) at the carboxyl terminus. Further analysis demonstrated that only leucine76 appears to be required for heparin-dependent enhancement of HB-EGF-mediated mitogenic activity, indicating that this amino acid may play a pivotal role in controlling the response of HB-EGF to heparin or related glycosaminoglycan sulfates. Our results also suggest that expression of different HB-EGF forms in vivo could result in the production of HB-EGFs with divergent responses to sulfated glycosaminoglycans and proteoglycans.

Purification and characterization of transmembrane forms of heparin-binding EGF-like growth factor.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), whose cDNA has a predicted 208-codon open reading frame, is synthesized as a membrane-spanning precursor that is processed to release mature mitogenic proteins of approximately 73-87 amino acids in length. Previous work has focused on the structural and biological properties of secreted HB-EGF. In this study, human recombinant transmembrane HB-EGF, produced by expression of HB-EGF1-208 cDNA in a baculovirus system, has been isolated, purified, and characterized structurally and biologically. Two isoforms of transmembrane HB-EGF (HB-EGFTM) were purified from membrane fractions of infected insect cells by a combination of heparin affinity chromatography and reversed-phase high performance liquid chromatography. The isoform designated as HB-EGFTM-1, a 21.5-kDa protein, yielded no N-terminal sequence, suggesting that it is N-terminally blocked. However, HB-EGFTM-II, a 24-kDa protein, was N-terminally sequenced and found to be initiated at Asp63 in the 208-amino acid residue primary translation product. This N terminus is the same as that determined for a 18-kDa isoform of secreted HB-EGF purified from the conditioned medium of insect cells expressing HB-EGF1-149 cDNA and is also identical to the N terminus of the longest form of secreted HB-EGF initially purified from human macrophage-like U-937 cell conditioned medium. HB-EGFTM-II cross-reacted on a Western blot with an antibody directed against the 16 C-terminal amino acids of the cytoplasmic tail of HB-EGF, indicating that it contains a putative transmembrane domain. HB-EGFTM-II was bioactive and stimulated the proliferation of BALB/c 3T3 cells and smooth muscle cells and the motility of smooth muscle cells, albeit with approximately 10-25% of the specific activity of secreted HB-EGF isoforms. We concluded that transmembrane HB-EGF is bioactive when isolated, consistent with the possibility of its functioning as a juxtacrine growth factor when still tethered to the cell.

Biosynthesis and processing by phorbol ester of the cells surface-associated precursor form of heparin-binding EGF-like growth factor.

Human MDA MB 231 cells were found to synthesize mostly the cell surface-associated precursor form of heparin-binding EGF-like growth factor (HB-EGF), a 27-kDa protein. Evidence for this form of HB-EGF included increased fluorescence intensity when cells were analyzed by flow cytometry using anti-HB-EGF antibodies, lack of HB-EGF in conditioned medium, and sensitivity to diphtheria toxin, for which HB-EGF is the receptor. Phorbol ester treatment of cells resulted, within 30 minutes, in loss of cell surface 27 kDA HB-EGF, lack of interaction with anti-HB-EGF antibodies, accumulation of active 21 kDa HB-EGF in conditioned medium, and the acquisition of diphtheria toxin resistance. It was concluded that cell surface-associated HB-EGF is the precursor of a bioactive growth factor, is biologically active as the receptor for diphtheria toxin, and is susceptible to rapid processing.

Differential regulation of heparin-binding epidermal growth factor-like growth factor in the adult ovariectomized mouse uterus by progesterone and estrogen.

Expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) was studied in the adult ovariectomized mouse uterus in response to progesterone (P4) and/or 17 beta-estradiol (E2) using Northern blotting, in situ hybridization, and immunohistochemistry. A 2.5-kilobase transcript of HB-EGF messenger RNA (mRNA) was detected in total uterine RNA samples. Although low levels of this mRNA were detected in uterine samples of oil-treated ovariectomized mice (control), an injection of E2 promptly up-regulated the levels. The mRNA levels peaked at 2 h and returned to basal levels after 12 h. Injection of P4 alone did not influence the basal levels; however, coinjection of E2 with P4 caused a rapid, but transient, up-regulation of the mRNA. The levels peaked between 2-4 h and declined 6 h after the hormone injections. Coinjection of E2 with P4 after 1 day of P4 priming also resulted in peak levels of HB-EGF mRNA at 2 h; however, the levels were not sustained thereafter. Because P4 and E2 differentially regulate heterogeneous uterine cell types, in situ hybridization was performed to determine cell-specific expression of HB-EGF mRNA in the ovariectomized uterus before and after steroid treatments. In the oil-treated uterine sections, very low levels of autoradiographic signals were observed in the luminal epithelium. In contrast, an injection of E2 resulted in a marked accumulation of HB-EGF mRNA primarily in uterine epithelial cells within 2 h. Although specific hybridization signals could not be detected in any uterine cell types after P4 treatment, combined treatment with P4 and E2 resulted in an accumulation of HB-EGF mRNA in stromal cells. To determine whether uterine HB-EGF mRNA was translated, cellular distribution of HB-EGF protein was investigated by immunohistochemistry. In oil-treated uterine sections, an overall weak immunostaining was noted, whereas no staining could be detected in uterine sections after P4 treatment. In contrast, positive immunostaining was noted in epithelial cells after E2 treatment. Coinjection of P4 with E2 caused immunostaining in the stroma. These results are consistent with those of in situ hybridization. The present investigation establishes that in the adult ovariectomized mouse uterus, E2 regulates HB-EGF expression in the epithelium, whereas expression of HB-EGF in the stroma is regulated by P4 and E2.

Heparin-binding EGF-like growth factor gene is induced in the mouse uterus temporally by the blastocyst solely at the site of its apposition: a possible ligand for interaction with blastocyst EGF-receptor in implantation.

Heparin-binding EGF-like growth factor (HB-EGF) is a newly discovered member of the EGF family of growth factors. HB-EGF can bind to two loci on cell surfaces, heparan sulphate proteoglycans and EGF-receptor (EGF-R), and either one or both of these interactions could play a role in cell-cell interactions. In the rodent, increased endometrial vascular permeability at the site of blastocyst apposition is considered to be an earliest discernible prerequisite event in the process of implantation and this event coincides with the initial attachment reaction between the blastocyst trophectoderm and uterine luminal epithelium. This investigation demonstrates that the HB-EGF gene is expressed in the mouse uterine luminal epithelium surrounding the blastocyst 6-7 hours before the attachment reaction that occurs at 2200-2300 hours on day 4 of pregnancy. It was further demonstrated that this gene is not expressed in the luminal epithelium at the site of the blastocyst apposition during the progesterone-maintained delayed implantation, but is readily induced in the luminal epithelium surrounding an activated blastocyst after termination of the delay by an estrogen injection. In vitro studies showed that HB-EGF induced blastocyst EGF-R autophosphorylation, and promoted blastocyst growth, zona-hatching and trophoblast outgrowth. These results suggest possible interactions between the uterine HB-EGF and blastocyst EGF-R very early in the process of implantation, earlier than any other embryo-uterine interactions defined to date at the molecular level.

Lysophosphatidylcholine upregulates the level of heparin-binding epidermal growth factor-like growth factor mRNA in human monocytes.

Lysophosphatidylcholine is increased in the plasma of hypercholesterolemic patients, is a component of oxidatively modified low-density lipoprotein, and, as such, may play an important role in atherosclerosis. Here we demonstrate that in human monocytes, lysophosphatidylcholine increases the level of mRNA encoding the heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent smooth muscle mitogen. Lysophosphatidylcholine treatment also enhances the release of heparin-binding mitogenic activity by these cells in culture. The anti-inflammatory glucocorticoid dexamethasone inhibits the upregulation of HB-EGF mRNA induced by either lysophosphatidylcholine or bacterial lipopolysaccharide in cultured monocytes. However, the responses induced by lysophosphatidylcholine and by lipopolysaccharide differ in their kinetics. In addition, the response to lysophosphatidylcholine is resistant to the action of cycloheximide, whereas the response to lipopolysaccharide is not, suggesting that the activation mechanisms induced by these two stimuli are different. Since a nuclear run-on assay showed no effect of lysophosphatidylcholine on the transcription of the HB-EGF gene, we speculate that lysophosphatidylcholine may increase the level of HB-EGF mRNA by altering the processing or degradation of primary or mature transcripts. Lysophosphatidylcholine enhancement of monocyte production of HB-EGF may represent an important result of the interactions among oxidized low-density lipoprotein and monocyte-derived macrophages and may play a role in initiation of smooth muscle proliferation in atherogenesis.

Heparin-binding EGF-like growth factor synthesis by smooth muscle cells.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been previously demonstrated to be a potent mitogen for smooth muscle cells. Evidence is now presented that these cells synthesize HB-EGF as well. Cultured fetal human vascular smooth muscle cells express 2.5-kb HB-EGF mRNA. These cells also release an HB-EGF-like activity that (i) stimulates smooth muscle cell and BALB/c 3T3 cell but not endothelial cell proliferation; (ii) binds to TSK heparin affinity columns and is eluted with 0.9-1.2 M NaCl, and (iii) triggers phosphorylation of a protein with the same molecular weight as the 170-kD EGF receptor. In addition, 125I-HB-EGF can be cross-linked to the EGF receptor on fetal human vascular smooth muscle cells. These results suggest that smooth muscle cells can both synthesize and respond to HB-EGF, and that HB-EGF may therefore be involved in autocrine regulation of these cells.

Heparin-binding EGF-like growth factor is a potent mitogen for rat hepatocytes.

We examined the hepatotrophic activity of heparin-binding EGF-like growth factor (HB-EGF), a recently identified potent mitogen for vascular smooth muscle cells and fibroblasts. HB-EGF stimulated DNA synthesis of rat hepatocytes in primary culture in a dose-dependent manner up to 30 ng/ml. The maximal stimulation by HB-EGF represented more than 80% of that induced by HGF. In normal rat liver, the transcript of HB-EGF gene was detected in the non-parenchymal cells and very low level in the hepatocytes. In the regenerating liver on the 3rd day after 70% hepatectomy, the HB-EGF mRNA increased in the non-parenchymal cells, suggesting that HB-EGF may contribute to liver regeneration through a paracrine mechanism.