Sex steroids and the endothelium.

Gonadal steroids clearly influence the course of atherosclerotic cardiovascular disease in women. This observation has suggested that these hormones have beneficial effects on the physiology of the vascular wall. Increased arterial vascular caliber after estrogen treatment, decreased lipid levels in subjects receiving hormone replacement therapy, and the markedly decreased extent of atherosclerotic plaque formation in young women as compared with young men support a cardioprotective effect of ovarian steroids. Generally, it appears that the effects of 17beta-estradiol are particularly beneficial, and the mechanism of action is targeted largely to the endothelial cell. This review describes the evidence for positive effects of estrogens on endothelial cell biology and considers potential mechanisms for estrogen actions on endothelial cell signal transduction.

TGF-beta1 activates MAP kinase in human mesangial cells: a possible role in collagen expression.

BACKGROUND: Although the pathogenic relevance of transforming growth factor-beta (TGF-beta) to glomerular sclerosis has been established, the intracellular mechanisms by which TGF-beta induces extracellular matrix accumulation are not fully understood. We examined whether the mitogen-activated protein (MAP) kinase pathway is involved in TGF-beta1-induced collagen expression by cultured human mesangial cells. METHODS: The activation of MAP kinase pathways by TGF-beta1 was assessed by immunoblot with anti-phospho-ERK or -JNK antibodies and by transfection of plasmids expressing pathway-specific transcription activators fused to the DNA-binding domain of GAL4, as well as a GAL4 response element-luciferase reporter gene. The role of MAP kinase was assessed using biochemical inhibitors and transiently expressed dominant negative mutant constructs. The effects on TGF-beta1-induced alpha1(I) collagen expression were evaluated by Northern blot and by activation of a transiently transfected alpha1(I) promoter-luciferase reporter construct. RESULTS: ERK and JNK phosphorylation occurred 30 minutes and one hour, respectively, after TGF-beta1 treatment. A biochemical blockade of the ERK pathway inhibited TGF-beta1-induced alpha1(I) collagen expression. A dominant negative mutant of ERK1 but not of JNK decreased alpha1(I) gene promoter activation. Activation of the TGF-beta-responsive p3TP-Lux construct was partially inhibited by cotransfection of an ERK1 dominant negative mutant. CONCLUSION: These data indicate that MAP kinase pathways can be activated by TGF-beta1 in mesangial cells and that the ERK MAP kinase plays a role in TGF-beta-stimulated collagen I expression. Because we have shown previously that SMADs mediate TGF-beta1-stimulated collagen I expression, our findings raise the possibility of interactions between the MAP kinase and the SMAD pathways.

Angiostatin binds ATP synthase on the surface of human endothelial cells.

Angiostatin, a proteolytic fragment of plasminogen, is a potent antagonist of angiogenesis and an inhibitor of endothelial cell migration and proliferation. To determine whether the mechanism by which angiostatin inhibits endothelial cell migration and/or proliferation involves binding to cell surface plasminogen receptors, we isolated the binding proteins for plasminogen and angiostatin from human umbilical vein endothelial cells. Binding studies demonstrated that plasminogen and angiostatin bound in a concentration-dependent, saturable manner. Plasminogen binding was unaffected by a 100-fold molar excess of angiostatin, indicating the presence of a distinct angiostatin binding site. This finding was confirmed by ligand blot analysis of isolated human umbilical vein endothelial cell plasma membrane fractions, which demonstrated that plasminogen bound to a 44-kDa protein, whereas angiostatin bound to a 55-kDa species. Amino-terminal sequencing coupled with peptide mass fingerprinting and immunologic analyses identified the plasminogen binding protein as annexin II and the angiostatin binding protein as the alpha/beta-subunits of ATP synthase. The presence of this protein on the cell surface was confirmed by flow cytometry and immunofluorescence analysis. Angiostatin also bound to the recombinant alpha-subunit of human ATP synthase, and this binding was not inhibited by a 2,500-fold molar excess of plasminogen. Angiostatin's antiproliferative effect on endothelial cells was inhibited by as much as 90% in the presence of anti-alpha-subunit ATP synthase antibody. Binding of angiostatin to the alpha/beta-subunits of ATP synthase on the cell surface may mediate its antiangiogenic effects and the down-regulation of endothelial cell proliferation and migration.

Increased expression of extracellular matrix proteins and decreased expression of matrix proteases after serial passage of glomerular mesangial cells.

The cellular events causing pathological extracellular matrix (ECM) accumulation in vivo are not well understood. Prolonged serial passage of several cell types in culture leads to increased production of extracellular matrix (ECM) proteins, but the mechanism for these putative fibrotic changes is not known. Here, human fetal glomerular mesangial cells were subjected to serial passage (P) in culture and the expression of ECM proteins, proteases and protease inhibitors was comprehensively evaluated. From P11 through P14, a series of phenotypic changes occurred. Steady-state expression of mRNA for alpha 1 chains of type III and type IV (but not type I) collagen, and for laminin beta 1 and gamma 1, increased 2- to 8-fold, while expression of mRNA for interstitial collagenase (MMP-1) and gelatinase A (MMP-2) virtually ceased. Expression of tissue-type plasminogen activator (tPA) mRNA also decreased markedly. Expression of mRNA for the tissue inhibitor of metalloproteinases (TIMP)-1, and of the smaller of two mRNA species for the PA inhibitor PAI-1, ceased by P14. There was a switch in expression of the two species of TIMP-2 mRNA: whereas the ratio of signal intensity comparing the 3.5 kb mRNA species to the 1.0 kb species was 5:1 up to P11, it was reversed (1:5) at P14 and later. Serial passage also led to changes in protein expression, with increased type IV collagen and laminin, but decreased interstitial collagenase and gelatinase A. The cells showed a progressive increase in staining for type IV collagen. These findings define the appearance of a matrix-accumulating phenotype in later-passage mesangial cells. Matrix expansion in vivo has been associated with increased transforming growth factor (TGF)-beta synthesis; the cells were found to show at least 5-fold increased expression of TGF-beta 1 mRNA from P8 to P16. However, treatment of P9 or P10 cells with graded doses of TGF-beta 1 increased expression of both collagen IV and gelatinase A mRNA and did not alter the ratio of signal intensity for TIMP-2 mRNA species. Thus, assumption of a matrix-accumulating phenotype by these cultured fetal glomerular mesangial cells is not accelerated by exogenous TGF-beta. These data describe an in vitro model of mesangial cell matrix turnover in which matrix accumulation could result from a concerted increase in ECM synthesis and decrease in ECM degradation.

Expression of an estrogen receptor by human coronary artery and umbilical vein endothelial cells.

BACKGROUND: Premenopausal women have much lower susceptibility to coronary artery disease than do men or postmenopausal women. It has been proposed that estrogen plays a role in cardioprotection, but little information is available regarding the mechanism by which estrogen may help to protect the vasculature. Here, we describe an estrogen receptor (ER) in human coronary artery and umbilical vein endothelial cells. METHODS AND RESULTS: Human umbilical vein endothelial cells and human coronary artery endothelial cells were cultured in hormone-free medium for 48 hours before experiments. Estradiol (3.7 nmol/L) added to cultures promoted proliferation by a mechanism that is inhibited by the specific ER antagonist ICI182,780. Estradiol-treated cells incorporated twice the [3H]thymidine of hormone-free cells; this increase was prevented by ICI182,780. Endothelial cells from both sources stained in a nuclear pattern with an ER-specific antibody. Ribonuclease protection assay detected mRNA for the ER. Ligand-binding studies estimated 2 x 10(4) to 8 x 10(4) receptors per cell and a Kd of approximately 5 nmol/L. Interaction of ERs with a consensus estrogen response element was shown by an electrophoretic mobility shift assay. In addition, an antibody against the ER supershifted the protein-DNA complex. CONCLUSIONS: These studies define the presence of an ER in human coronary artery and umbilical vein endothelial cells. They support the hypothesis that cardioprotective effects of estrogen are mediated, at least in part, through a classic steroid hormone receptor mechanism.

Multiple genetic alterations in hamster pancreatic ductal adenocarcinomas.

Pancreatic ductal adenocarcinomas induced in the Syrian golden hamster (SGH) by N-nitrosobis(2-oxopropyl)amine share many similarities with the human disease, including mutations of the K-ras oncogene. In vitro carcinogenesis studies with immortal SGH pancreatic duct cells indicate that neoplastic transformation in this system can occur without mutational inactivation of p53 suppressor gene. In this study we extend the genetic analysis of the in vivo SGH model to increase the number of cases analyzed for the status of K-ras and to determine further the spectrum of alterations involved; we have studied the status of the p53, DCC, and Rb-1 suppressor genes and the status of the mdm2 oncogene, which can involve p53 indirectly. The partial SGH-coding sequence of mdm2 and DCC was determined. K-ras mutation in the second position of codon 12 was present in 17 of 19 (90%) of tumors. Immunohistochemistry and single strand conformation polymorphism analysis showed no evidence of p53 mutation in 21 tumors. RNase protection assays showed overexpression of mdm2 in 5 of 19 (26%) tumors. Semiquantitative reverse transcription-PCR analysis showed a complete or partial loss of DCC expression in 10 of 19 (53%) neoplasms and of Rb-1 (42%) expression in 8 of 19 tumors when compared to matched controls. Deregulation of these genes appears to be significant in SGH pancreatic carcinogenesis as indicated by their frequencies. However, the fact that 6 tumors showed either only a K-ras mutation or the absence of alterations of the 5 genes analyzed indicates that additional as yet unstudied or unknown genes are also involved in SGH pancreatic duct carcinogenesis.

In vitro carcinogenesis of hamster pancreatic duct cells: cellular and molecular alterations.

Neoplastic transformation of Syrian golden hamster (SGH) pancreatic duct cells was induced by in vitro treatment with the direct-acting carcinogens N-methylnitrosourea (MNU) and N-(2-hydroxypropyl)nitrosourea (HPNU), with subsequent selection by sustained culture in serum- and epidermal growth factor (EGF)-deprived medium. The present study examines the efficacy of serum and EGF deprivation as a selection pressure and the effect of the carcinogen dose, frequency and interval of exposure on tumorigenesis and K-ras mutation. Selection of carcinogen-initiated duct cells by serum and EGF deprivation is highly reproducible and effective, increasing the incidence of tumors from 26 to 93% for MNU or from 0 to 100% for HPNU. SGH pancreatic duct cells exposed to 0.5 mM MNU for 13 weeks (long-treatment schedule) produced K-ras mutations at codon 12 in six of six tumors. However, when cells were exposed to 0.125, 0.25 or 0.5 mM MNU daily for 5 days (short-treatment schedule), mutations of K-ras at codon 13 were identified in four of 16 tumors, the remaining 12 showing no mutations. Duct cells exposed to 0.5 mM HPNU by the short-treatment schedule produced K-ras mutations in codon 13 in six of six tumors, as contrasted to 12 tumors that developed from cells exposed to 0.125 or 0.25 mM HPNU, which all contained K-ras codon 12 mutations. The current experiments demonstrate that K-ras mutation in pancreatic carcinogenesis in vitro by MNU or HPNU can be modified by the nature and dose of the carcinogen as well as the frequency and duration of exposure.

Genomic p53 mutation in a chemically induced hamster pancreatic ductal adenocarcinoma.

This study investigates possible alterations in exons 5 through 8 of the p53 gene and altered p53 protein expression in the Syrian golden hamster model of pancreatic ductal adenocarcinoma. In the Syrian hamster p53 sequence, 1469 base pairs are presented for the genomic regions surrounding exons 5 through 8, along with the primer sequences specific for the enzymatic amplification of the individual exons. Single-stranded conformation polymorphism was analyzed on the products obtained by enzymatic amplification of hamster genomic DNA extracted from 2 transplantable pancreatic ductal adenocarcinomas, 6 groups of N-methylnitrosourea (MNU)-treated pancreatic duct cells, and 17 MNU-induced pancreatic ductal adenocarcinomas. The two transplantable adenocarcinomas were a well-differentiated ductal adenocarcinoma established from a N-nitrosobis(2-oxopropyl)amine-induced tumor and a poorly differentiated ductal adenocarcinoma established from a spontaneous tumor. An altered mobility indicated a conformational change in the first part of exon 5 in the solid form of the well-differentiated ductal adenocarcinoma. Direct sequencing of the amplified product revealed an A-->C transversion in codon 135, which corresponds to codon 132 in the human p53 gene. A conformational change in exon 7 was observed in 1 of 6 MNU-treated cell samples, and none of the 17 resultant tumors. Direct sequencing confirmed a deletion of one C of the three in codon 263, which generates a frameshift mutation. No conformational change was observed in any other products. Positive staining with PAb240 or DO7 antibodies against human p53 or with an antibody generated in our laboratory against the hamster p53 fusion protein was observed only in the solid form of well-differentiated ductal adenocarcinoma and in rare cells scattered in 4 of 28 MNU-induced tumors analyzed. This study provides a system to analyze p53 gene alterations in the hamster and is the initial report of a specific p53 mutation in a hamster pancreatic ductal adenocarcinoma.

Stimulation of DNA synthesis in pancreatic duct cells by gastrointestinal hormones: interaction with other growth factors.

Pancreatic duct cells of the Syrian hamster were grown as monolayers on thin layers of type I collagen coated onto microporous membranes. The effects of a number of potential trophic factors were tested by their ability to increase [3H]thymidine incorporation into cellular DNA. To measure the effect of growth factors, cells were subjected to a period of growth factor depletion to induce a state of partial quiescence in DNA synthesis. Cells responded with a significant increase in thymidine incorporation after the addition of epidermal growth factor (EGF) alone or a growth factor mixture containing EGF plus insulin, transferrin, selenium, linoleic acid, bovine pituitary extract, triiodothyronine, and dexamethasone. When the serum substitute, Nu Serum IV (5%, vol/vol), was added to this mixture, addition of several gastrointestinal (GI) hormones including secretin, vasoactive intestinal polypeptide (VIP), bombesin, and gastrin caused significant increases in thymidine incorporation at concentrations of 0.01-1 microM. At 1 microM, these hormones stimulated DNA synthesis relative to their respective control in the order secretin (178%) greater than bombesin (153%) greater than VIP (138%) greater than gastrin (126%). Cholecystokinin octapeptide, a known trophic factor for pancreatic acinar cells, did not cause significant increases in thymidine incorporation in cultured duct cells. These results suggest that pancreatic duct cells possess receptors for a number of GI hormones and respond to the trophic effects of hormones known to stimulate pancreatic growth in vivo.

Characterization of differentiated Syrian golden hamster pancreatic duct cells maintained in extended monolayer culture.

Epithelial cells isolated from fragments of hamster pancreas interlobular ducts were freed of fibroblast contamination by plating them on air-dried collagen, maintaining them in serum-free Dulbecco's modified Eagle's (DME):F12 medium supplemented with growth factors, and selecting fibroblast-free aggregates of duct cells with cloning cylinders. Duct epithelial cells plated on rat type I collagen gel and maintained in DME:F12 supplemented with Nu Serum IV, bovine pituitary extract, epidermal growth factor, 3,3',5-triiodothyronine, dexamethasone, and insulin, transferrin, selenium, and linoleic acid conjugated to bovine serum albumin (ITS+), showed optimal growth as monolayers with a doubling time of about 20 h and were propagated for as long as 26 wk. Early passage cells consisted of cuboidal cells with microvilli on their apical surface, complex basolateral membranes, numerous elongated mitochondria, and both free and membrane-bound ribosomes. Cells grown as monolayers for 3 mo. were more flattened and contained fewer apical microvilli, mitochondria, and profiles of rough surfaced endoplasmic reticulum; in addition, there were numerous autophagic vacuoles. Functional characteristics of differentiated pancreatic duct cells which were maintained during extended monolayer culture included intracellular levels of carbonic anhydrase and their capacity to generate cyclic AMP (cAMP) after stimulation by 1 X 10(-6) M secretin. From 5 to 7 wk in culture, levels of carbonic anhydrase remained stable but after 25 to 26 wk decreased by 1.9-fold. At 5 to 7 wk of culture, cyclic AMP increased 8.7-fold over basal levels after secretin stimulation. Although pancreatic duct cells cultured for 25 to 26 wk showed lower basal levels of cAMP, they were still capable of generating significant levels of cAMP after exposure to secretin with a 7.0-fold increase, indicating that secretin receptors and the adenyl cyclase system were both present and functional. These experiments document that pancreatic duct monolayer cultures can be maintained in a differentiated state for up to 6 mo. and suggest that this culture system may be useful for in vitro physiologic and pathologic studies.

Compensation for idiotype suppression. I. Acquirement of ability to compensate for TEPC-15 idiotype suppression in mice during the early neonatal period.

Neonatal injection of BALB/c mice with antibodies specific for the idiotype of TEPC-15 myeloma protein (T15id), which is serologically identical to the major idiotype of anti-phosphorylcholine (PC) antibody, renders the recipients completely unresponsive to PC. C57BL/6, (BALB/c X C57BL/6)F1 and C.B20 mice, similarly treated with anti-T15id antibody, also displayed tolerance to PC although they were relatively more resistant (8-13%) than BALB/c mice (less than 2% control response). When anti-T15id antibody was injected into 15-day-old neonates, the resistance to the tolerance in C57BL/6 and C.B20 mice was much more apparent (up to 80% of the control response) in contrast to that in BALB/c mice, which was not significant. Adoptive transfer of spleen cells from idiotypically suppressed BALB/c mice into 20-day-old, normal C. B20 mice resulted in suppression of T15id but not in tolerance to PC, due to increased production of non-T15id-bearing anti-PC antibody. These results suggest that the ability of clonal compensation for T15id suppression is acquired during early life (2-10 days), under the influence of gene(s) associated or linked with the Igh.