Heparin-binding EGF-like growth factor in human serum. Association with high blood cholesterol and heart hypertrophy

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) belongs to the EGF family of growth factors which are ligands of the ErbB receptors. Studies in animals suggest the role of HB-EGF in several pathogenic processes such as atherosclerosis and heart hypertrophy. Here, we set up an assay to measure HB-EGF in human serum. Our ELISA determined serum HB-EGF in the range of 0.03–3 nM. It did not cross-react with EGF or with transforming growth factor-α. The mean serum HB-EGF was 0.26 nM (confidence interval: 0.13–0.39) in women and 0.28 nM (confidence interval: 0.09–0.47) in men. In a cohort of 121 healthy volunteers, we identified nine individuals with high serum HB-EGF (above 0.47 nM). These individuals had higher left ventricle mass (determined by Colour Doppler echocardiography) and greater total and low density lipoprotein cholesterol than control. On the basis of our results, we propose that increased serum HB-EGF is associated with heart hypertrophy and elevated blood cholesterol.     

Analysis of the length response to a force step in smooth muscle from rabbit urinary bladder

Responses to isotonic quick release of AC-stimulated smooth muscle strips from rabbit urinary bladder were analysed. Releases were performed at the peak of contraction and at a preset tension level in the contraction and relaxation phase. In other expts. responses at 37 degrees C and 27 degrees C were compared. The length response always consisted of 3 parts: (1) elastic recoil, (2) rapid length change (isotonic transient), (3) steady length change. Qualitatively, phases (1)-(3) could be distinguished also in responses to isotonic quick stretch. The immediate elastic recoils, phase (1), were described by exponential stress-strain relations. Stiffness was found to be somewhat lower during relaxation than during contraction. No effect of temperature on the elastic recoil was seen. The initial velocity in phase (2) was 2-3 times greater than the velocity 100 ms after release. By means of computer analysis of the length records during phases (2) and (3) two decaying exponential processes with widely different time constants could be separated. The time constant of the faster process was of the order of 15-30 ms at 37 degrees C. It decreased with increasing force steps and with increasing temperature. The amount of shortening associated with this process was correlated with the size of the force step, reaching a maximum of about 1.2% of the muscle length. The shortening velocities in phase (3), measured 100 ms after release, were described by Hill's equation. Vmax in the rising part and at the peak of contraction were 0.7 and 0.6 L/s respectively at 37 degrees C. Lower values were found during relaxation and at 27 degrees C. We suggest that part of the elastic recoil in phase (1) occurs in structures associated with the individual cross-bridges, that phase (2) is dominated by a change in the distribution of conformations of bridges in the attached position and that the shortening rate in phase (3) is determined by the entire cycle of events during turnover of bridges after the muscle has adapted to the new load. Observations on the force response to length steps and on shifts from isometric to afterloaded isotonic contraction and vice versa are consistent with this interpretation.     

Heparin-binding EGF-like growth factor attenuates lung inflammation and injury in a murine model of pulmonary emphysema

Pulmonary inflammation and progressive lung destruction are the major causes of chronic obstructive pulmonary disease (COPD), resulting in emphysema and irreversible pulmonary dysfunction. Heparin-binding EGF-like growth factor (HB-EGF), is known to play a protective role in the process of various inflammatory diseases. However, its effect on COPD is poorly understood. This study was designed to determine the effect of HB-EGF on lung inflammation and injury in a murine model of pulmonary emphysema. HB-EGF promoted percent survival and body weight, attenuated lung injury, inflammatory cells, and cytokines infiltration, and prevented lung function decline. Additionally, treatment of rHB-EGF suppressed the nuclear translocation of nuclear factor κB (NF-κB)/p65, decreased TUNEL-positive cells and the expression of caspase 3, and increased the expression of PCNA, HB-EGF, and EGF receptor (EGFR). We conclude that HB-EGF attenuates lung inflammation and injury, probably through the activation of EGFR, followed by suppression of NF-ΚB signalling, promotion of cell proliferation, and inhibition of apoptosis.     

Heparin-binding EGF-like growth factor (HB-EGF) decreases oxygen free radical production in vitro and in vivo

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been shown to protect intestinal epithelial cells from anoxia/reoxygenation in vitro, and to protect the intestines from ischemia/reperfusion (I/R) injury in vivo. The goal of the present study was to determine whether the cytoprotective effects of HB-EGF were due, in part, to its ability to decrease reactive oxygen species (ROS) production. Human whole blood, polymorphonuclear leukocytes, and monocytes, as well as rat intestinal epithelial cells, were exposed to stimuli designed to produce an oxidative burst in these cells. Treatment of the cells with HB-EGF led to a significant decrease in oxidative burst production. In vivo, total midgut I/R injury in rats led to increased ROS production, which was markedly decreased by HB-EGF treatment. Histochemically, I/R injury led to increased ROS production, which was significantly decreased with HB-EGF treatment. HB-EGF cytoprotection is due, in part, to its ability to decrease ROS production. Future studies will determine the mechanisms by which HB-EGF exerts these effects.     

Heparin-binding EGF-like growth factor protects intestinal stem cells from injury in a rat model of necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is an often catastrophic disease that typically affects premature newborns. Although the exact etiology of NEC is uncertain, the disease is associated with formula feeding, bacterial colonization of the gut, hypoxia and hypoperfusion. In light of the pathogenesis of NEC, the integrity and function of the intestinal mucosa has a major defensive role against the initiation of NEC. Various forms of intestinal injury, including NEC, injure the intestinal epithelial cell (IEC) lineages, including the intestinal stem cells (ISCs), thereby disrupting the normal homeostasis needed to maintain gut barrier function. In the current study, we examined the effects of heparin-binding EGF-like growth factor (HB-EGF) administration on enterocytes, goblet cells, neuroendocrine cells and ISCs in a newborn rat model of experimental NEC. We also examined the cytoprotective effects of HB-EGF on ISCs in in vitro cell cultures and in ex vivo crypt-villous organoid cultures. We found that HB-EGF protects all IEC lineages, including ISCs, from injury. We further found that HB-EGF protects isolated ISCs from hypoxic injury in vitro, and promotes ISC activation and survival, and the expansion of crypt transit-amplifying cells, in ex vivo crypt-villous organoid cultures. The protective effects of HB-EGF were dependent on EGF receptor activation, and were mediated via the MEK1/2 and PI3K signaling pathways. These results show that the intestinal cytoprotective effects of HB-EGF are mediated, at least in part, through its ability to protect ISCs from injury.     

Acute resistance exercise increases skeletal muscle angiogenic growth factor expression

AIMS: Both aerobic and resistance exercise training promote skeletal muscle angiogenesis. Acute aerobic exercise increases several pro-angiogenic pathways, the best characterized being increases in vascular endothelial growth factor (VEGF). We hypothesized that acute resistance exercise also increases skeletal muscle angiogenic growth factor [VEGF and angiopoietin (Ang)] expression. METHODS: Seven young, sedentary individuals had vastus lateralis muscle biopsies and blood drawn prior to and at 0, 2 and 4 h post-resistance exercise for the measurement of VEGF; VEGF receptor [KDR, Flt-1 and neuropilin 1 (Nrp1)]; Ang1 and Ang2; and the angiopoietin receptor--Tie2 expression. Resistance exercise consisted of progressive knee extensor (KE) exercise to determine one repetition maximum (1-RM) followed by three sets of 10 repetitions (3 x 10) of KE exercise at 60-80% of 1-RM. RESULTS: Resistance exercise significantly increased skeletal muscle VEGF mRNA and protein and plasma VEGF protein at 2 and 4 h. Resistance exercise increased KDR mRNA and Tie2 mRNA at 4 h and Nrp1 mRNA at 2 and 4 h. Skeletal muscle Flt-1, Ang1, Ang2 and Ang2/Ang1 ratio mRNA were not altered by resistance exercise. CONCLUSIONS: These findings suggest that acute resistance exercise increases skeletal muscle VEGF, VEGF receptor and angiopoietin receptor expression. The increases in muscle angiogenic growth factor expression in response to acute resistance exercise are similar in timing and magnitude with responses to acute aerobic exercise and are consistent with resistance exercise promoting muscle angiogenesis.     

Heparin-binding EGF-like growth factor is expressed by mesangial cells and is involved in mesangial proliferation in glomerulonephritis.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a new member of the EGF family, is mitogenic for several types of cells, through binding to cell surface heparan sulphate proteoglycans. This study has attempted to delineate HB-EGF expression by mesangial cells and to identify its role in experimental and human glomerulonephritis. Rat mesangial cells, cultured in the presence of phorbol acetate, hydrogen peroxide, interleukin-1beta, and tumour necrosis factor-alpha, expressed HB-EGF mRNA. Recombinant HB-EGF stimulated rat mesangial cells to proliferate and to express types I and III collagen. In the rat anti-Thy-1.1 nephritis, glomerular HB-EGF mRNA was up-regulated and peaked at days 5-7; its expression at the protein level in the glomerulus was prominent at days 5-10. By immunofluorescence, HB-EGF was positive predominantly in the mesangial area of renal tissues from 23 of 45 patients with various types of human glomerulonephritis, showing a significant correlation with the grade of mesangial proliferation; there was no staining in tissues from patients with minimal change nephrotic syndrome and normal kidney tissues. These data provide the evidence that HB-EGF is synthesized and expressed by mesangial cells and stimulates mesangial cell proliferation and collagen synthesis in vitro. HB-EGF is a potential mediator in mesangial cell proliferation and matrix expansion in experimental and human glomerulonephritis.     

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.     

Induction of smooth muscle cell-like phenotype in marrow-derived cells among regenerating urinary bladder smooth muscle cells

Tissue regeneration on acellular matrix grafts has great potential for therapeutic organ reconstruction. However, hollow organs such as the bladder require smooth muscle cell regeneration, the mechanisms of which are not well defined. We investigated the mechanisms by which bone marrow cells participate in smooth muscle formation during urinary bladder regeneration, using in vivo and in vitro model systems. In vivo bone marrow cells expressing green fluorescent protein were transplanted into lethally irradiated rats. Eight weeks following transplantation, bladder domes of the rats were replaced with bladder acellular matrix grafts. Two weeks after operation transplanted marrow cells repopulated the graft, as evidenced by detection of fluorescent staining. By 12 weeks they reconstituted the smooth muscle layer, with native smooth muscle cells (SMC) infiltrating the graft. In vitro, the differential effects of distinct growth factor environments created by either bladder urothelial cells or bladder SMC on phenotypic changes of marrow cells were examined. First, supernatants of cultured bladder cells were used as conditioned media for marrow cells. Second, these conditions were reconstituted with exogenous growth factors. In each case, a growth factor milieu characteristic of SMC induced an SMC-like phenotype in marrow cells, whereas that of urothelial cells failed. These findings suggest that marrow cells differentiate into smooth muscle on acellular matrix grafts in response to the environment created by SMC.     

Contractile effects of endothelin-I and localization of endothelin binding sites in rabbit lower urinary tract smooth muscle

In isolated rabbit bladder and urethral smooth muscle, endothelin-1 caused concentration-related, slowly developing contractions that were difficult to wash out. Relative to contractions induced by K⁺ (124 mM), contractions in bladder preparations reached a higher amplitude than in urethral preparations. There was a marked tachyphylaxis to the effects of the peptide. The endothelin-l-induced contractions were not significantly affected by phentolamine or indomethacin in the urethra, or by scopolamine or indomethacin in the bladder. Incubation for 30 min in a Ca²⁺-free solution abolished the endothelin-l-induced contractions. Nifedipine did not affect the actions of endothelin-1 in the urethra but had a marked inhibitory action on its effects in the bladder. In the presence of endothelin-1, Ca²⁺-induced contractions were significantly blocked by nifedipine in the bladder but not in the urethra. Urethral preparations at resting tension responded to electrical stimulation by tetrodotoxin-sensitive, frequency-dependent contractions sensitive to α-adrenoceptor blockade. Pretreatment with endothelin-1 (10^-9′ M) produced a significant increase in the nerve-induced contractions but had no significant effect on contractions induced by exogenous noradrenaline. Endothelin-1 did not affect spontaneous or stimulation-induced efflux of ³H-labelled noradrenaline in urethral smooth muscle. Preparations contracted by endothelin-1 were frequency-dependently relaxed by electrical stimulation. The peptide had no significant effect on the responses induced by electrical stimulation in the bladder preparations. In both bladder and urethra, [¹²⁵]endothelin-l binding sites were found mainly in the outer longitudinal muscle layer, in vessels and in the submucosa. The highest density of binding sites appeared to be in vessels and the outer muscle layer in both types of muscle. The results suggest that in the rabbit both bladder and urethral smooth muscle contain binding sites for endothelin. The peptide has contractant effects dependent on extracellular calcium in both types of tissue, but voltage-operated calcium channels seem to involved in activation only of bladder smooth muscle. The functional importance of endothelin-1 in the rabbit lower urinary tract remains to be elucidated.     

Contractile effects of endothelin-1 and localization of endothelin binding sites in rabbit lower urinary tract smooth muscle

In isolated rabbit bladder and urethral smooth muscle, endothelin-1 caused concentration-related, slowly developing contractions that were difficult to wash out. Relative to contractions induced by K+ (124 mM), contractions in bladder preparations reached a higher amplitude than in urethral preparations. There was a marked tachyphylaxis to the effects of the peptide. The endothelin-1-induced contractions were not significantly affected by phentolamine or indomethacin in the urethra, or by scopolamine or indomethacin in the bladder. Incubation for 30 min in a Ca2(+)-free solution abolished the endothelin-1-induced contractions. Nifedipine did not affect the actions of endothelin-1 in the urethra but had a marked inhibitory action on its effects in the bladder. In the presence of endothelin-1, Ca2(+)-induced contractions were significantly blocked by nifedipine in the bladder but not in the urethra. Urethral preparations at resting tension responded to electrical stimulation by tetrodotoxin-sensitive, frequency-dependent contractions sensitive to alpha-adrenoceptor blockade. Pretreatment with endothelin-1 (10(-9) M) produced a significant increase in the nerve-induced contractions but had no significant effect on contractions induced by exogenous noradrenaline. Endothelin-1 did not affect spontaneous or stimulation-induced efflux of 3H-labelled noradrenaline in urethral smooth muscle. Preparations contracted by endothelin-1 were frequency-dependently relaxed by electrical stimulation. The peptide had no significant effect on the responses induced by electrical stimulation in the bladder preparations. In both bladder and urethra, [125]endothelin-1 binding sites were found mainly in the outer longitudinal muscle layer, in vessels and in the submucosa. The highest density of binding sites appeared to be in vessels and the outer muscle layer in both types of muscle. The results suggest that in the rabbit both bladder and urethral smooth muscle contain binding sites for endothelin. The peptide has contractant effects dependent on extracellular calcium in both types of tissue, but voltage-operated calcium channels seem to involved in activation only of bladder smooth muscle. The functional importance of endothelin-1 in the rabbit lower urinary tract remains to be elucidated.     

Heparin-binding EGF-like growth factor (HB-EGF) overexpression in transgenic mice downregulates insulin-like growth factor binding protein (IGFBP)-3 and -4 mRNA

An in vivo approach was taken to assess the biological significance of heparin-binding EGF-like growth factor (HB-EGF) using transgenic mice. Transgenic mice were generated using the pIRES-EGFP vector expressing a bicistronic mRNA containing both human HB-EGF (hHB-EGF) and enhanced green fluorescent protein (EGFP) coding sequences under the regulation of the cytomegalovirus immediate–early (CMV-IE) promoter. As a marker for transgene expression, EGFP fluorescence in 5?μm tissue sections was evaluated. To confirm HB-EGF expression in EGFP-containing tissues, HB-EGF mRNA was analyzed by RT-PCR and Northern blot analysis. Protein levels of HB-EGF and insulin-like growth factor binding protein-3 (IGFBP-3), a molecule that stabilizes IGFs, which in turn helps to promote growth, were analyzed by Western blot. Also, the weights of transgenic mice were compared with the weights of wild type non-transgenic littermates over a 10-week period. EGFP fluorescence, RT-PCR and Northern analysis of a variety of tissues from hHB-EGF transgenic mice indicate recombinant EGFP/hHB-EGF mRNA expression in kidney, liver, lung and stomach. Western blot analysis confirmed that HB-EGF protein levels were greater in these tissues from hHB-EGF transgenic mice compared to wild type non-transgenic littermates. IGFBP-3 protein was absent in serum of transgenic mice prior to the onset of puberty, but indistinguishable from wild type non-transgenic mice after puberty. Furthermore, IGFBP-3 and IGFBP-4 mRNA were downregulated in the kidney, but not liver or lung of the transgenic mice. In accordance with reduced IGFBP-3 and -4 levels, hHB-EGF transgenic mice exhibited a 20% decrease in weight prior to 6 weeks of age compared to wild type non-transgenic littermates. Our laboratory has generated a biologically functional transgenic mouse model exhibiting increased expression of hHB-EGF in kidney, liver, lung and stomach. Overexpression of hHB-EGF affected the growth rate of these transgenic mice possibly through a pathway involving IGFBP-3 and IGFBP-4.     

Heparin-binding EGF-like growth factor (HB-EGF) overexpression in transgenic mice downregulates insulin-like growth factor binding protein (IGFBP)-3 and -4 mRNA

An in vivo approach was taken to assess the biological significance of heparin-binding EGF-like growth factor (HB-EGF) using transgenic mice. Transgenic mice were generated using the pIRES-EGFP vector expressing a bicistronic mRNA containing both human HB-EGF (hHB-EGF) and enhanced green fluorescent protein (EGFP) coding sequences under the regulation of the cytomegalovirus immediate-early (CMV-IE) promoter. As a marker for transgene expression, EGFP fluorescence in 5 microm tissue sections was evaluated. To confirm HB-EGF expression in EGFP-containing tissues, HB-EGF mRNA was analyzed by RT-PCR and Northern blot analysis. Protein levels of HB-EGF and insulin-like growth factor binding protein-3 (IGFBP-3), a molecule that stabilizes IGFs, which in turn helps to promote growth, were analyzed by Western blot. Also, the weights of transgenic mice were compared with the weights of wild type non-transgenic littermates over a 10-week period. EGFP fluorescence, RT-PCR and Northern analysis of a variety of tissues from hHB-EGF transgenic mice indicate recombinant EGFP/hHB-EGF mRNA expression in kidney, liver, lung and stomach. Western blot analysis confirmed that HB-EGF protein levels were greater in these tissues from hHB-EGF transgenic mice compared to wild type non-transgenic littermates. IGFBP-3 protein was absent in serum of transgenic mice prior to the onset of puberty, but indistinguishable from wild type non-transgenic mice after puberty. Furthermore, IGFBP-3 and IGFBP-4 mRNA were downregulated in the kidney, but not liver or lung of the transgenic mice. In accordance with reduced IGFBP-3 and -4 levels, hHB-EGF transgenic mice exhibited a 20% decrease in weight prior to 6 weeks of age compared to wild type non-transgenic littermates. Our laboratory has generated a biologically functional transgenic mouse model exhibiting increased expression of hHB-EGF in kidney, liver, lung and stomach. Overexpression of hHB-EGF affected the growth rate of these transgenic mice possibly through a pathway involving IGFBP-3 and IGFBP-4.     

Relation between cell length and force production in urinary bladder smooth muscle

Guinea-pig and rabbit urinary bladders were fixed in glutaraldehyde at different volumes. Strips were dissected out, embedded and cut for phase contrast and electron microscopy. Muscle wall thickness decreased with increased bladder volume as did the radial number of muscle cells. Cell length, measured by a morphometric method increased linearly with bladder radius, indicating that no slippage between the muscle cells occurred. Number of cells per mm² cross sectional area increased linearly with bladder radius. Volume-active force relations were obtained by pelvic nerve stimulations of guinea pig bladders filled to different volumes. Maximum pressure was obtained at 0.15 ml bladder volume, and maximum wall tension at a volume of 2.5 ml which corresponds to a cell length of 400 μm and a cell packing density of about 107000×mm^-2. Estimation of the length-active tension curve for the average muscle cell in the guinea-pig bladder indicated a maximum active force of 5.5 μN/cell. Maximum active force per cm² muscle bundle was calculated to be about 59 N. No compensation for extracellular space and nonmuscular tissue within the muscle bundle was made.     

The force recovery following repeated quick releases applied to pig urinary bladder smooth muscle

Summary A method for measuring several quick-releases during one contraction of a pig urinary bladder smooth muscle preparation was developed. The force recovery following quick release in this muscle type was studied by fitting a multiexponential model to 926 responses measured during the first 700 ms after release, both in the stimulated and in the unstimulated muscle. It was concluded that the force recovery in this observation window was biexponential and that the two time constants result from two fundamentally different processes. The slower time constant in the order of 0.45 s was ascribed to crossbridge cycling, and this hypothesis was supported by the considerable dependence of the amplitude associated with this time constant on the stimulus condition of the muscle. The faster time constant in the order of 0.032 s was found to be largely independent of the degree of stimulation of the muscle and was ascribed to a passive, viscoelastic process.     

Vasopressin V1-antagonist increases the hydroosmotic response to arginine vasopressin in frog urinary bladder

V₁-antagonist, [Mca¹, D-Phe², Sar⁷] arginine vasopressin, at a concentration 0.5 nM to 5 nM increased hydroosmotic effect of 5 nM arginine vasopressin in frog urinary bladder. 1 nM V₁-antagonist [Mca¹,O-Me-Tyr²] arginine vasopressin does not changed hydroosmotic effect of 0.1 mM dibutyryl cAMP. We suggest that — both V₁- and V₂-receptors are present on epithelial cells of frog urinary bladder and that V₁-receptors are involved in the modulation or the hydroosmotic effect of arginine vasopressin — mediated by V₂-receptors.     

Expression of vascular endothelial growth factor in the lower urinary tract in rats after castration and estrogen administration

OBJECTIVE: To evaluate quantitatively, by means of immune histochemistry, the expression of the vascular endothelial growth factor (VEGF) in the bladder, vesicourethral junction, and urethra in normal, castrated adult rats and under estrogen administration. DESIGN: Sixty adult virgin rats (Rattus norvergicus albinus, Rodentia, Mammalia) from the CEDEME-UNIFESP Animal House were used. Rats were divided into three groups. Group I comprised noncastrated rats, group II comprised oophorectomized rats, and group III comprised castrated rats administered 17beta-estradiol in the form of subcutaneous implants at the dose of 0.18 mg/implant for 30 days. After performing standard immunohistochemistry procedures, the intensity of the dark-brown color was used as the cytoplasmic protein expression of VEGF. Cells without this coloration or weakly stained were considered negative. percentile of VEGF expression was obtained by counting 1,000 cells per slide and establishing the ratio between positive and total cells. RESULTS: The VEGF expression was uniform and similar along the urinary tract in group I. After castration, protein expression was almost absent in the bladder and was low in the vesicourethral junction and urethra. With estrogen replacement, very little of the expression was recovered in the bladder, and the reaction became evident in the vesicourethral junction and urethral sections. CONCLUSIONS: The present study implies a potential relationship between VEGF and urinary tract physiology. The results suggest that there are quantitative differences in VEGF expression in these tissues depending on steroid hormone status.