Suppression of 3-deoxyglucosone and heparin-binding epidermal growth factor-like growth factor mRNA expression by an aldose reductase inhibitor in rat vascular smooth muscle cells

Reactive carbonyl compounds and oxidative stress have been recently shown to up-regulate the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent mitogen for vascular smooth muscle cells (SMCs) produced by SMC themselves. Because the polyol pathway has been reported to influence the formation of carbonyl compounds and the oxidative stress in various cells, we conducted this study to investigate whether the polyol pathway affects HB-EGF expression along with the generation of carbonyl compounds and the oxidative stress in SMCs. We found that, compared with those cultured with 5.5mM glucose, SMCs cultured with 40 mM glucose showed the accelerated thymidine incorporation, elevated levels of intracellular sorbitol, 3-deoxyglucosone (3-DG), advanced glycation end products (AGEs), and thiobarbituric acid-reactive substances (TBARS) along with the enhanced expression of HB-EGF mRNA. An aldose reductase inhibitor (ARI), SNK-860, significantly inhibited all of these abnormalities, while aminoguanidine suppressed 3-DG levels and HB-EGF mRNA expression independent of sorbitol levels. The results suggest that the polyol pathway may play a substantial role in SMC hyperplasia under hyperglycemic condition in part by affecting HB-EGF mRNA expression via the production of carbonyl compounds and oxidative stress.     

A role of heparin-binding epidermal growth factor-like growth factor in cardiac remodeling after myocardial infarction

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is known to induce cell growth in various cell types via transactivation of epidermal growth factor receptor (EGFR). To investigate the involvement of HB-EGF and EGFR in cardiac remodeling after myocardial infarction (MI), we examined the expressions of mRNA and protein in rat hearts 6 weeks after MI-induction. Where increased expressions of HB-EGF mRNA and protein were observed, infarcted myocardium was replaced by extracellular matrix and interstitial fibroblasts. EGFR mRNA and protein expression did not show significant changes in sham-operated heart tissues, non-infarcted region, and infarcted region. In vitro study demonstrated that HB-EGF mRNA was expressed mainly in cultured fibroblasts rather than in myocytes. We suggest that the interaction between HB-EGF and EGFR transactivation is closely related to the proliferation of cardiac fibroblasts and cardiac remodeling after MI in an autocrine, paracrine, and juxtacrine manner.     

High glucose and hyperosmolarity increase heparin-binding epidermal growth factor-like growth factor (HB-EGF) production in cultured human aortic endothelial cells

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been shown to be a potent smooth muscle cell (SMC) mitogen and chemoattractant, and might be a candidate factor for the progression of atherosclerosis. We have investigated the effects of high glucose and hyperosmolarity on HB-EGF production in cultured human aortic endothelial cells. Following the culture of the cells for 2 days with high concentrations of glucose or in the hyperosmolar conditions, we measured the content of HB-EGF and the rate of production in the cells using a semi-quantitative immunofluorescent technique and a metabolic radiolabelling method. With high glucose (16.6 mmol) and hyperosmolar conditions (glucose 5.5 mmol + mannitol 11.1 mmol or glucose 5.5 mmol + raffinose 11.1 mmol), the content of HB-EGF was significantly increased and the metabolic rate was also significantly increased (more than a twofold increase, compared to that of 5.5 mmol glucose). In conclusion, conditions of high glucose or hyperosmolarity increase HB-EGF production in human aortic endothelial cells. These results suggest that diabetic macroangiopathy might be attributed at least in part to HB-EGF-related vascular changes which may be induced by glucose.     

Characterization of the gene encoding murine heparin-binding epidermal growth factor-like growth factor

The mouse gene (mHB-EGF) encoding heparin-binding epidermal growth factor-like growth factor was isolated from a mouse 129SVJ genomic library. DNA sequence analysis confirmed that the clone contained six exons (I–VI) and five introns (A–E), and spanned approx. 14 kb of DNA. PCR analysis showed that introns A–E of mHB-EGF are 203 bp, 2.5 kb, 5.5 kb, 825 bp and 272 bp in length, respectively. These results establish that mHB-EGF is similar in organization to human HB-EGF (hHB-EGF). However, DNA sequence analysis of introns A–E of mHB-EGF failed to show significant overall homology with those of hHB-EGF     

Stretch activates heparin-binding EGF-like growth factor expression in bladder smooth muscle cells

Cultured rat bladder smooth muscle cells (SMC) were grown oncollagen-coated silicone membranes and subjected to continuous cyclesof stretch-relaxation. Semiquantitative RT-PCR analysis revealed atime-dependent increase in heparin-binding epidermal growth factor(EGF)-like growth factor (HB-EGF) mRNA levels after stretch, withmaximal levels appearing after 4 h. Immunostaining for proHB-EGFrevealed higher levels of HB-EGF protein in the stretched than in thenonstretched SMC. The ANG II receptor type 1 antagonist losartanmarkedly suppressed stretch-activated HB-EGF expression. ANG II levelswere 3.3-fold higher in the stretch- than in thenon-stretch-conditioned media. Stretch stimulation of bladder SMC thathad been transiently transfected with an HB-EGF promoter-luciferaseexpression construct resulted in an 11-fold increase in reporteractivity. Mechanical stretch induced a 4.7-fold increase in tritiatedthymidine incorporation rate, and this was reduced by 25% in thepresence of losartan. We conclude that mechanical stretch activatesHB-EGF gene expression in bladder SMC and that this is mediated in partby autocrine ANG II secretion.

     

Secretion of a new growth factor, smooth muscle cell derived growth factor, distinct from platelet derived growth factor by cultured rabbit aortic smooth muscle cells

In attempts to determine the mechanism of proliferation of arterial smooth muscle cells (SMC) in intimal atheromatous lesions, autocrine secretion of growth factors by SMC has recently received much attention. Here we report a new growth factor named smooth muscle cell derived growth factor (SDGF). Cultured rabbit medial SMC secreted SDGF for 1 week during their incubation in serum-free media only after at least 4 passages. SDGF differed from platelet derived growth factor (PDGF) physicochemically, immunologically, and biologically. The properties of SDGF also seemed different from those of other known growth factors that stimulate the proliferation of mesenchymal cells.     

Secretion of an epidermal growth factor-like growth factor by epidermal growth factor-independent rat mammary carcinoma cells.

Rat mammary carcinoma (RMC) cells derived from serially transplantable mammary tumors are independent of epidermal growth factor (EGF) for long-term growth in serum-free medium. This phenotype is in contrast to that of normal mammary epithelial cells or cells derived from nontransplantable tumors that express an absolute requirement for EGF for growth in culture. The results of the experiments reported here indicate that EGF-independent RMC cells secrete a growth factor with potent EGF-like mitogenic activity. Conditioned media obtained from these cells can substitute for EGF for the growth of the EGF-dependent cell line MCF-10. This growth factor is neither EGF nor transforming growth factor alpha and does not compete with 125I-EGF for binding to EGF receptors. Phosphotyrosine Western blot analysis of lysates obtained from EGF-independent RMC cells revealed the presence of a 190 kilodalton (kDa) protein that was distinct from the EGF receptor. Similarly, growth of MCF-10 cells to confluence in serum-free medium supplemented with conditioned medium growth factor in place of EGF resulted in the disappearance of the EGF receptor band and appearance of the 190 kDa band in phosphotyrosine Western blots. The 190 kDa tyrosine-phosphorylated protein detected in cells stimulated by the conditioned medium factor is unlikely to be the c-erbB-2 protein, as indicated by negative results in immunoprecipitation experiments and in vitro kinase assays. In summary, EGF-independent RMC cells secrete a factor with potent EGF-like mitogenic activity. This suggests that an autocrine loop involving this growth factor mediates EGF independence in these cells.     

Preferential inhibition of elastin synthesis by epidermal growth factor in chick aortic smooth muscle cells.

Epidermal growth factor inhibits elastin synthesis in chick aortic smooth muscle cells. The inhibitory effect was dose-dependent with a ninety percent reduction at 100 ng/ml and time-dependent with at least 6h lag phase for the expression of the effect. In contrast, collagen synthesis remained constant. The degree of inhibition in elastin synthesis was parallel to the decrease in the steady-state levels of elastin mRNA. These results indicated that epidermal growth factor specifically inhibits elastin synthesis and will be a useful suppressor for elastogenesis under pathological conditions.     

Platelet-derived growth factor regulates actin isoform expression and growth state in cultured rat aortic smooth muscle cells

The role of platelet-derived growth factor (PDGF) in the control of smooth muscle cell (SMC) differentiation was explored in vitro by examining its effects on expression of the smooth muscle (SM) specific contractile protein SM alpha actin in cultured rat aortic SMC. Quiescent, postconfluent SMC express maximal levels of alpha actin and responded to human platelet-derived growth factor (partially purified from platelets) by entering the cell cycle and undergoing approximately one synchronous round of DNA synthesis. Concomitantly, these cultures exhibited a marked reduction in alpha actin synthesis. Chronic treatment with PDGF (72 hours at 8 or 12 hour intervals) was associated with a transient increase in thymidine labeling index and a decrease in alpha actin expression. Interestingly, between 48 and 72 hours following initial treatment, thymidine labeling indices returned to near control levels while SM alpha actin expression remained depressed. This effect was reversible; fractional alpha actin synthesis increased immediately after PDGF removal. When subsequently stimulated with 10% fetal bovine serum (FBS), cells chronically pretreated with PDGF entered S phase approximately 4 hours earlier than cells pretreated with PDGF vehicle, consistent with the idea that the maintained suppression of alpha actin synthesis in SMC subjected to chronic PDGF treatment was associated with partial cell cycle transit. Chronic treatment with highly purified recombinant PDGF-BB elicited similar effects on alpha actin synthesis and partial cell cycle transit. Flow cytometric analysis of chronic PDGF-treated SMC demonstrated a 25% increase in forward angle light scatter, an index of cell size. These data implicate a possible role for PDGF in regulation of SMC differentiation and suggest a potentially important role for this mitogen in the phenotypic modulation accompanying SMC growth and in mediation of the cellular hypertrophy associated with cell cycle progression.     

Extracellular superoxide dismutase attenuates release of pulmonary hyaluronan from the extracellular matrix following bleomycin exposure

The major pulmonary antioxidant enzyme involved in the protection of the lung interstitium from oxidative stress is extracellular superoxide dismutase (EC-SOD). It has been previously shown that EC-SOD knock-out mice are more susceptible to bleomycin-induced lung injury, however, the molecular mechanism(s) remains unclear. We report here that bleomycin-induced lung damage, in EC-SOD KO mice, is associated with increased hyaluronan release into alveolar fluid. Analysis of hyaluronan synthase gene expression and hyaluronan molecular weight distribution suggested that elevated levels of hyaluronan in the alveolar fluid are mostly due to its release from the interstitium. Our results indicate that EC-SOD attenuates bleomycin-induced pulmonary injury, at least in part, by preventing superoxide-mediated release of hyaluronan into alveolar space.     

The effects of heparin-binding epidermal growth factor-like growth factor on preimplantation-embryo development and implantation in the rat.

This study examined the effects of heparin-binding epidermal growth factor-like growth factor (HB-EGF) on preimplantation-embryo development and initiation of implantation in the rat. In vitro studies showed that HB-EGF improved the development of 8-cell embryos to the blastocyst stage in a concentration-dependent manner, and the growth factor had no effect on the cell number of the blastocyst developed. Intraluminal injection of an anti-HB-EGF antiserum into the uterine horns at 0600 h on day 5 of pregnancy decreased the number of implantation sites (blue dye reaction) at 0200 h on day 6. Intraluminal injection of 20 microl of HB-EGF solution (10 or 100 ng/ml) into each uterine horn induced implantation in about half of the ovariectomized progesterone-treated delayed implanting rats, and the number of implantation sites per rat increased dose-dependently. These results suggest that HB-EGF is involved in the preimplantation-embryo development and initiation of implantation in the rat.     

Short form of the heparin-binding EGF-like growth factor: Communication II

The structure of the green monkey Chlorocebus aethiops heparin-binding EGF-like growth factor (HB-EGF) gene was compared with that of the corresponding human gene. Exon 3a, characteristic of the short form of HB-EGF (SF-HB-EGF), was mapped between exons 3 and 4, approximately 700 bp away from the latter. In several human and simian cell lines, most of the SF-HB-EGF mRNA proved to lack exons 4 and 5, specific to the HB-EGF mRNA. In contrast to the HB-EGF mRNA, the SF-HB-EGF mRNA occurred predominantly in the P, rather than L, form, which codes for a protein with a different propeptide structure. Labeled SF-HB-EGF competed with HB-EGF and EGF for binding to the surface of A431 cells, suggesting its interaction with the specific EGF receptor. The results indicate that SF-HB-EGF plays a specific role in cell signaling.     

人SOD1基因在大鼠血管平滑肌细胞的表达及对抗氧自由基损伤的作用

本实验构建含人铜锌超氧化物歧化酶（ｈＳＯＤ１）基因的逆转录病毒载体,将其导入离体培养的鼠血管平滑肌细胞,观察ｈＳＯＤ１基因表达及其抗氧自由基损害作用,结果表明：（１）载体构建策略和方法正确,ｈＳＯＤ１基因可在靶细胞中高效稳定表达;（２）转化ｈＳＯＤ１的ＶＳＭＣｓ可对抗大剂量氧自由基对细胞的直接损伤作用;（３）小剂量氧自由基刺激ＶＳＭＣｓ增殖,而转化ｈＳＯＤ１的ＶＳＭＣｓ增殖反应受到抑制,本研究结果     

Immunohistochemical localization of heparin-binding epidermal growth factor-like growth factor in normal skin and skin cancers

Summary Heparin-binding epidermal growth factor (EGF)-like growth factor is a 22-kDa glycoprotein that was originally identified as a secreted product of cultured human macrophages. Although the growth factor mRNA has been identified in various cells and tissues, the tissue distribution of the protein itself has rarely been demonstrated. In this study, the EGF-like growth factor was detected immunohistochemically in a variety of human skin samples by indirect immunofluorescence using a polyclonal rabbit antiserum raised against residues 26–41 of mature heparin-binding EGF. The keratinocytes of a variety of epithelium-derived structures demonstrated reproducible, specific staining for the EGF. In normal tissues, this staining was prominent in the basal cells of the epidermis and in the epithelial cells lining epidermal appendages such as hair follicles, sebaceous sweat glands and eccrine sweat glands. In addition, specific staining was detected in skin cancers derived from the basal epithelial cell layer, including basal and squamous cell carcinomas of the skin, with no staining detected in melanoma specimens. Immunoreactive heparin-binding EGF was characteristically associated with the surface of cells. With minor exceptions, the immunoreactive sites are identical to the known EGF receptor distribution in the skin, and suggest that keratinocyte-derived heparin-binding EGF may act in concert with other EGF family members in processes such as skin morphogenesis and wound repair, as well as in the development of skin cancers This revised version was published online in November 2006 with corrections to the Cover Date.     

Extracellular calcium sensing in rat aortic vascular smooth muscle cells

Extracellular calcium (Ca(2+)(o)) can act as a first messenger in many cell types through a G protein-coupled receptor, calcium-sensing receptor (CaR). It is still debated whether the CaR is expressed in vascular smooth muscle cells (VSMCs). Here, we report the expression of CaR mRNA and protein in rat aortic VSMCs and show that Ca(2+)(o) stimulates proliferation of the cells. The effects of Ca(2+)(o) were attenuated by pre-treatment with MAPK kinase 1 (MEK1) inhibitor, as well as an allosteric modulator, NPS 2390. Furthermore, stimulation of the VSMCs with Ca(2+)(o)-induced phosphorylation of ERK1/2, but surprisingly did not cause inositol phosphate accumulation. We were not able to conclusively state that the CaR mediates Ca(2+)(o)-induced cell proliferation. Rather, an additional calcium-sensing mechanism may exist. Our findings may be of importance with regard to atherosclerosis, an inflammatory disease characterized by abnormal proliferation of VSMCs and high local levels of calcium.