Heparin-binding EGF-like growth factor is up-regulated in the obstructed kidney in a cell- and region-specific manner and acts to inhibit apoptosis.

The expression of certain growth factors in the epidermal growth factor (EGF) family is altered in response to renal injury. Recent studies have demonstrated that heparin binding EGF-like growth factor (HB-EGF) expression may be cytoprotective in response to apoptotic signals. The purpose of this study was to investigate the potential role of HB-EGF in the upper urinary tract following unilateral ureteral obstruction. We present evidence that: i) ureteral obstruction induced cell-specific but transient activation of HB-EGF gene expression; ii) HB-EGF expression in renal epithelial cells increased under conditions where mechanical deformation, such as that caused by hydronephrotic distension, induces apoptosis, but HB-EGF expression did not increase in renal pelvis smooth muscle cells under identical conditions; and iii) enforced expression of HB-EGF served to protect renal epithelial cells from stretch-induced apoptosis. These results suggest a potential mechanism by which the kidney protects itself from apoptosis triggered by urinary tract obstruction.

AP-1 mediates stretch-induced expression of HB-EGF in bladder smooth muscle cells.

Mechanical induction of growth factor synthesis may mediate adaptive responses of smooth muscle cells (SMC) to increases in physical load. We previously demonstrated that cyclic mechanical stretch induces expression of the SMC, fibroblast, and epithelial cell mitogen heparin-binding epidermal growth factor-like growth factor (HB-EGF) in bladder SMC, an observation that suggests that this growth factor may be involved in compensatory bladder hypertrophy. In the present study we provide evidence that the activator protein-1 (AP-1) transcription factor plays a critical role in this mechanoinduction process. Rat bladder SMC were transiently transfected with a series of 5' deletion mutants of a promoter-reporter construct containing 1. 7 kb of the mouse HB-EGF promoter that was previously shown to be stretch responsive. The stretch-mediated increase in promoter activity was completely ablated with deletion of nucleotide positions -1301 to -881. Binding of AP-1, as evaluated by electrophoretic mobility shift assay, to a synthetic oligonucleotide containing an AP-1 binding site increased in response to stretch, and binding was inhibited by excess unlabeled DNA corresponding to nucleotides -993 to -973 from the HB-EGF promoter, a region that contains a previously recognized composite AP-1/Ets site. Stretch-induced promoter activity was significantly inhibited by site-directed mutagenesis of the AP-1 or Ets components of this site. Consistent with the promoter and gel-shift studies, curcumin, an inhibitor of AP-1 activation, suppressed the HB-EGF mRNA induction after stretch. Stretch also specifically increased mRNA levels for matrix metalloproteinase (MMP)-1, the promoter of which contains a functional AP-1 element, but not for MMP-2, the promoter of which does not contain an AP-1 element. The stretch response of the MMP-1 gene was also completely inhibited by curcumin. Collectively, these findings indicate that AP-1-mediated transcription plays an important role in the regulation of gene expression in bladder muscle in response to mechanical forces.

Direct in vivo gene transfer to urological organs.

PURPOSE: Patients with urological disorders may benefit from gene based therapy. We investigated the feasibility of delivering exogenous genes into urological tissues in vivo using direct in vivo electrotransfection. MATERIALS AND METHODS: Gene transfer to rat kidneys, testes and bladders was accomplished via direct local injection of pGL3/luciferase and beta-galactosidase reporter gene constructs, followed by an electrical pulse ranging from 55 to 115 msec at 100 V. Direct injection of deoxyribonucleic acid without an electrical pulse served as the control. The transfected and nontransfected organs were retrieved and analyzed by luciferase activity assay, histochemical and immunocytochemical staining for beta-galactosidase, and reverse transcription polymerase chain reaction with primers specific for beta-galactosidase messenger ribonucleic acid. RESULTS: There was significant luciferase activity 1, 3 and 5 days after direct in vivo electrotransfection in kidneys and testes, and after 3, 5, 7 and 10 days in bladders. Positive beta-galactosidase enzyme activity and beta-galactosidase immunoreactivity were observed in the transfected renal tubular cells, testicular interstitial and germ cells, and uroepithelial bladder layer. Reverse transcription-polymerase chain reaction products of the transfected organs were noted, indicating the successful transcription of messenger ribonucleic acid. CONCLUSIONS: This study demonstrates that direct in vivo electrotransfection is a feasible method of transient gene delivery into intact urological organs. Its apparent safety and relative simplicity suggest that direct in vivo electrotransfection may be useful clinically.

Human albumin solders for clinical application during laser tissue welding.

BACKGROUND AND OBJECTIVE: Fifty percent human albumin solder significantly improves weld strength when compared to lower concentrations [Wright et al., ASLMS meeting, April, 1995]. We developed a method for preparing 50% human albumin that may be considered compatible for clinical applications. STUDY DESIGN/MATERIALS AND METHODS: Fifty percent human albumin solder was prepared from 25% commercially available human albumin using a lyophilization technique. Assessment of sterility, viscosity, pH, and peak absorption wavelength were performed. RESULTS: This report describes the methodology used to prepare a 50% human albumin solder that is compatible with clinical use. Maintenance of the structural integrity of the albumin was confirmed by polyacrylamide gel electrophoresis. CONCLUSION: This solder preparation can be used alone or with the addition of exogenous chromophores. The final product is sterile, incorporates viral free protocols, maintains high viscosity, and can be applied easily during open or laparoscopic procedures.

Culture of a prostatic cell line in basement membrane gels results in an enhancement of malignant properties and constitutive alterations in gene expression.

Interaction of a transformed rat prostate epithelial cell (NbMC-2) with basement membrane gels (Matrigel) has been evaluated using a long-term matrix culture system. NbMC-2 cells, and single-cell clonal derivatives, formed spheroidal multicellular structures (aggregates) on Matrigel surfaces and were weakly invasive or noninvasive during a 1 week period. During subsequent 2-4 week periods, invasive cells originating from the aggregates and exhibiting a fusiform morphology became evident and increased in number in the matrix cultures. This biphasic pattern of behavior did not occur on laminin, type I or type IV collagen, or fibronectin substrates, but it did occur on Matrigel in serum-free medium. Characterization of sublines enriched in fusiform cells indicated that they maintained their distinct morphology with continuous culture. Further, they exhibited significantly greater invasive potential, saturation density, and random motility (chemokinesis) than the parent cell line. Steady-state levels of fibronectin mRNA were highly elevated in the fusiform variants, demonstrating a constitutive alteration in patterns of gene expression coinciding with the altered morphology. These results indicate that clonal NbMC-2 cells differentiate at a reproducible frequency into a more aggressive cell type in response to culture in the basement membrane-like matrix. The altered phenotypic properties appear to be stable since they can be inherited by daughter cells and because they are evident in the absence of matrix. These observations suggest a cell-specific mechanism for promotion of malignant growth by matrix-mediated induction of novel cell properties.

Epidermal growth factor receptor-mediated autocrine and paracrine stimulation of human transitional cell carcinoma.

Stromal-epithelial interactions may play a key role in tumor growth and metastasis. We have established a model to study the cellular and molecular basis of this paracrine interaction both in vivo and in vitro using a human transitional cell carcinoma cell line (WH). s.c. coinoculation of 1 x 10(6) WH cells with 1 x 10(6) nontumorigenic fetal rat urogenital sinus mesenchymal (rUGM) cells in athymic mice accelerated carcinoma growth 20 times faster than isolated WH cell inoculations and 4 times faster than coinoculations of the same number of NIH-3T3 or human bladder fibroblasts. Characterization of these chimeric tumors with immunohistochemical and DNA dot-blot analyses documented their predominantly human component. To evaluate the underlying mechanisms involved in this paracrine-mediated in vivo tumor growth acceleration, Northern analyses for growth factors (GFs) and extracellular matrix (ECM) expression in the different cell lines, as well as in vitro mitogenic assays, were performed. Northern analysis revealed basic fibroblast growth factor, transforming growth factor alpha, and epidermal growth factor receptor expression by WH cells but not rUGM cells; ECM components (fibronectin and collagens I and IV) were expressed only in the fibroblast cell lines. Cell type-specific paracrine growth factors are produced by cultured stromal and epithelial cells with a 2-3-fold bidirectional increase in WH and rUGM cell growth when cultured with reciprocal cell-type conditioned medium. An autocrine growth loop was observed for WH but not rUGM cells. WH cell growth is stimulated in vitro by low concentrations of transforming growth factor alpha and epidermal growth factor, while rUGM cell growth is stimulated 3-fold by basic fibroblast growth factor. Antiepidermal growth factor receptor antibodies completely inhibited autocrine and paracrine pathways stimulating WH cell growth, while anti-basic fibroblast growth factor antibodies had no inhibitory effect. These observations suggest that autocrine and paracrine growth factor stimulation of WH bladder carcinoma cell growth is most likely mediated by an epidermal growth factor receptor-related pathway. The predominant expression of ECM by fibroblasts in this model suggests that stromal cell ECM components may modulate tumor cell growth and angiogenesis possibly through mechanisms involving cellular adhesion, chemotaxis, or growth factor action.

Extracellular matrix and androgen receptor expression associated with spontaneous transformation of rat prostate fibroblasts.

Spontaneous transformation in continuous culture of the androgen-sensitive rat prostate fibroblast cell line, NbF-1, resulted in an aggressively tumorigenic nonmetastatic phenotype that coincided with few gross chromosome abnormalities. This study identified transformation-associated alterations in extracellular matrix and androgen receptor expression in the NbF-1 cell line. Substantial levels of procollagens I, III, and IV and fibronectin mRNAs were detected in nontumorigenic NbF-1 cells. Laminin B1 and B2 mRNAs were also detectable, but at lower levels. Expression of all six extracellular matrix mRNAs was nonuniformly lower in tumorigenic NbF-1 cells. This decrease in expression was greatest for alpha 2 procollagen IV mRNA, which was reduced 17-fold. Proteoglycans and glycosaminoglycans synthesized by the NbF-1 cultures were also characterized. The NbF-1 cell line expressed chondroitin sulfate proteoglycans predominantly, and expression was reduced 5- to 10-fold in tumorigenic cultures. In contrast to the extensive alterations in the extracellular matrix, measurement of high-affinity androgen binding and androgen receptor mRNA levels showed substantial expression of androgen receptors in both NbF-1 cultures. Cultures of early and late passage NbF-1 cells demonstrated a mitogenic response to dihydrotestosterone. These data indicate (a) that alterations in expression of extracellular matrix components may represent early markers for tumorigenic transformation in prostatic mesenchymal cells and (b) that these changes can occur without disrupting androgen receptor expression and androgen sensitivity.

Regulation of gene expression in rat prostate by androgen and beta-adrenergic receptor pathways.

Denervation of rat ventral prostate has been accomplished by excising prostatic tissue fragments and implanting them under the renal capsules of intact syngeneic rats. This resulted in a substantial reduction of expression of a major organ-specific secretory protein, prostatic binding protein (PBP). The depressed level of PBP and its subunits and mRNAs could be restored, however, to as much as 80% of control levels by the administration of a pharmacological dose of exogenous androgen, testosterone propionate (TP), and/or a beta-adrenergic agonist, isoproterenol (ISO). Furthermore, compared to ascorbate-treated controls, TP and ISO increased the synthesis of total cellular protein and PBP by the prostatic renal implants. TP and/or ISO also remodelled the luminal epithelial structure and elevated secretory functions. ISO alone had no effect, however, in castrated animals, indicating that androgen plays a dominant role in the restoration of tissue PBP content. Concomitant to increased PBP content and remodelling of prostatic histomorphology, androgen was also found to raise the depressed levels of beta 2-adrenergic and androgen receptors in the prostatic isografts maintained in intact hosts. In contrast, although an established rat prostatic epithelial cell line (NbE-1) contains high affinity androgen receptor, androgen failed to restore beta-adrenergic receptor as well as PBP content in this cultured cell line. These results, taken together, suggest that a tight coupling between androgen receptor and beta 2-adrenergic receptor pathways may be a prerequisite for PBP expression and functional differentiation in the rat ventral prostate gland.

Proglumide inhibition of trophic action of pentagastrin.

Proglumide, a glutaramic acid derivative, inhibits the acid secretory effects of gastrin and is said to be a specific gastrin receptor blocking agent. In the current study we have shown that proglumide inhibits the binding of gastrin to its receptor in rat oxyntic gland mucosa and tested whether this receptor is also responsible for the trophic effect of gastrin. In the first study 400 mg/kg proglumide injected with 250 micrograms/kg pentagastrin every 8 h for 48 h totally prevented the trophic effects of pentagastrin in rat oxyntic gland mucosa. Parameters measured included DNA synthesis and DNA, RNA, and protein content. In a second study the lowest maximally effective dose of proglumide was determined to be 100 mg/kg. The trophic effect of pentagastrin was inhibited in duodenal mucosa, colonic mucosa, and pancreas as well as oxyntic gland mucosa. These data demonstrate that proglumide blocks the trophic action of exogenous gastrin and suggest that the trophic effect of gastrin is mediated by the gastrin receptor.

Regulation of antral gastrin content.

Five groups of rats were fasted for 3 days and injected with either NaCl or 5, 10, 20, or 40 micrograms/kg bombesin every 8 h. The animals were killed, and their serum and antral gastrin levels were compared with those of normally fed rats. Fasting reduced serum gastrin to 14% of control; antral gastrin was reduced to 21% of control. All doses of bombesin significantly increased serum gastrin in fasted rats, and 20 and 40 micrograms/kg significantly increased antral gastrin. A group of normally fed rats was also compared with one fed a liquid diet for 7 days. Half of each of these was injected with 20 micrograms/kg bombesin (3 times/day) and the other half with NaCl. Bombesin significantly increased serum and antral gastrin in the rats fed solid food. The liquid diet lowered serum and antral gastrin to 17 and 59% of control values, respectively. Bombesin injection totally prevented these decreases. These data indicate that food in the gastrointestinal tract is not required for either gastrin release or synthesis. Furthermore, the data suggest that gastrin synthesis is regulated primarily by gastrin release or by direct stimulation by bombesin rather than by specific food products.

Stimulation of pancreatic growth by intraduodenal amino acids and HCl.

Exogenous secretin and cholecystokinin (CCK) have been shown to stimulate the growth of the exocrine pancreas. To conclude that a hormone produces an effect under normal physiological conditions, one must demonstrate that the endogenous hormone also produces the action in question. To release endogenous secretin 0.01 N HCl was continuously infused into the duodenums of eight rats for 5 days. Sixteen animals were prepared with catheters inserted 1 cm below the pylorus. The other eight animals were infused with PO4 buffer, pH 7.5. The infusion rate for both groups was 2 ml/h. Animals were killed at the end of 5 days, and the oxyntic gland mucosa and pancreas were examined. Pancreatic weights, DNA synthesis, DNa, RNA, and protein content were significantly increased in rats receiving acid. An identical experiment was set up to release endogenous CCK. In this experiment eight rats received a solution containing 50 mM phenylalanine and 50 mM tryptophan. The controls received the caloric equivalent in glucose. At the end of 1 wk the pancreases of the rats infused with amino acids averaged 405 (P < 0.001) heavier than the controls. Similar increases occurred in DNA, RNA, and protein content. These studies suggest that endogenous secretin and CCK can be released in amounts sufficient to stimulate growth.

Stimulation of rat oxyntic gland mucosal growth by epidermal growth factor.

Epidermal growth factor (EGF) inhibits gastric acid secretion, but its effects on the growth of gut mucosa have not been examined. Fasted male rats were given six injections of EGF (10 micrograms/kg) over a 48-h period. The animals were killed and the incorporation of [3H]thymidine into various tissues was examined and compared to rats treated with NaCl, pentagastrin, and EGF plus secretin. EGF and pentagastrin significantly increased DNA synthesis of the oxyntic gland mucosa. Pentagastrin, but not EGF, stimulated DNA synthesis of duodenal and colonic mucosa as well. Neither peptide altered skin DNA synthesis. Secretin inhibited the effects of pentagastrin but not EGF. Chronic administration of EGF (5 days) caused significant increases in oxyntic gland mucosal DNA, RNA, and protein content. These results not only demonstrate that EGF is a trophic agent for the oxyntic gland mucosa but lend further support to the hypothesis that acid secretion and mitogenesis are the result of two separate mechanisms.

Stimulation of DNA synthesis by big and little gastrin (G-34 and G-17).

Fasted rats were injected six times over a 48-hr period with G-17 I, G-17 II, G-34 II in doses doubling from 3.38 nmoles per kg to 54 nmoles per kg. Twelve rats were studied at each dose. NaCl-injected animals were used as controls. The rats were killed and the in vitro incorporation of [3H]thymidine into DNA as well as the total DNA content of the mucosa of the duodenum and oxyntic gland area of the stomach were determined. All gastrins, as well as pentagastrin, stimulated DNA synthesis. Peak stimulation occurred at 13.5 nmoles per kg for G-17 I and G-17 II and at 6.75 nmoles per kg for G-34 II. Pentagastrin's peak trophic effect was produced by 325 nmoles per kg. In order to compare the efficacies of the various types of gastrins, 60 rats were divided into groups of 12. One group received saline and the other four groups received the maximally effective dose of one of the four types of gastrins. The responses to each of the gastrins were not significantly different, and DNA synthesis was doubled in each tissue. Total DNA content increased slightly, but significantly, in response to each gastrin. Several conclusions can be drawn from these data: (1) G-17 and G-34 possess trophic activity; (2) the efficacies of pentagastrin, G-17 I, G-17 II, and G-34 II for the stimulation of DNA synthesis are not significantly different; (3) sulfation of G-17 has no significant effect on its trophic activity; (4) based on the effects of exogenous molar doses and their respective half-lives, both G-17 and G-34 would be expected to contribute to the trophic effect of endogenous gastrin; and (5) G-17 and G-34 are at least as effective in stimulating DNA synthesis as they are in stimulating gastric acid secretion.