Keratinocyte growth factor protects against Pseudomonas aeruginosa-induced lung injury

We have previously reported that keratinocyte growth factor (KGF) attenuates alpha-naphthylthiourea-induced lung injury by upregulating alveolar fluid transport. The objective of this study was to determine the effect of KGF pretreatment in Pseudomonas aeruginosa pneumonia. A 5% bovine albumin solution with 1 microCi of (125)I-labeled human albumin was instilled into the air spaces 4 or 24 h after intratracheal instillation of P. aeruginosa, and the concentration of unlabeled and labeled proteins in the distal air spaces over 1 h was used as an index of net alveolar fluid clearance. Alveolocapillary barrier permeability was evaluated with an intravascular injection of 1 microCi of (131)I-albumin. In early pneumonia, KGF increased lung liquid clearance (LLC) compared with that in nonpretreated animals. In late pneumonia, LLC was significantly reduced in the absence of KGF but increased above the control value with KGF. KGF pretreatment increased the number of polymorphonuclear cells recovered in the bronchoalveolar lavage fluid and decreased bacterial pulmonary translocation. In conclusion, KGF restores normal alveolar epithelial fluid transport during the acute phase of P. aeruginosa pneumonia and LLC in early and late pneumonia. Host response is also improved as shown by the increase in the alveolar cellular response and the decrease in pulmonary translocation of bacteria.     

Activated epidermal growth factor receptor (ErbB1) protects the heart against stress-induced injury in mice

Acute, high-intensity stress induces necrotic lesions in the heart. We found that restraint-and-cold (4 degrees C) exposure (RCE) raises plasma lactate dehydrogenase (LDH), creatine kinase (CK), and transaminase activity in a time-dependent manner, with a peak value 7 h after stimulus cessation. At 24 h, signs of necrotic lesions were observed in paraffin sections stained with hematoxylineosin: focal accumulation of mononuclear cells in subendocardial areas of the left ventricle wall and focal hemorrhage in papillary muscles. In contrast, intermale fighting (IF) did not increase plasma CK activity, although LDH and transaminase activities did increase. In IF, no histological evidence of heart injury was observed. Because IF, but not RCE, increased plasma epidermal growth factor (EGF) concentration by approximately 1,000-fold, we hypothesized that EGF receptor (ErbB1) activation may protect the heart against stress-induced injury. To examine this hypothesis, we injected the ErbB1 tyrosine kinase inhibitor tyrphostin AG-1478 (25 mg/kg ip) immediately before mice were exposed to IF. After 3 h, plasma activities of LDH-1 and CK increased. Plasma enzyme activities were as low in control mice (injected with vehicle alone) as in nonfighting mice. In the last experiment, we injected EGF (0.25 mg/kg ip) 20 min before exposing mice to RCE. After 7 h, plasma LDH-1 and CK activities were significantly lower in these animals than in mice injected with vehicle. The effect required ErbB1 activation, because simultaneous administration of AG-1478 completely abolished the effect of exogenous EGF. We conclude that activated ErbB1, by endogenous or exogenous ligands, may protect the heart against stress-induced injury.     

How insulin-like growth factor I binds to a hybrid insulin receptor type 1 insulin-like growth factor receptor

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Ablation of tumor necrosis factor receptor type I (p55) alters oxygen-induced lung injury

Hyperoxic lung injury, believed to be mediated by reactive oxygen species, inflammatory cell activation, and release of cytotoxic cytokines, complicates the care of many critically ill patients. The cytokine tumor necrosis factor (TNF)-alpha is induced in lungs exposed to high concentrations of oxygen; however, its contribution to hyperoxia-induced lung injury remains unclear. Both TNF-alpha treatment and blockade with anti-TNF antibodies increased survival in mice exposed to hyperoxia. In the current study, to determine if pulmonary oxygen toxicity is dependent on either of the TNF receptors, type I (TNFR-I) or type II (TNFR-II), TNFR-I or TNFR-II gene-ablated [(-/-)] mice and wild-type control mice (WT; C57BL/6) were studied in >95% oxygen. There was no difference in average length of survival, although early survival was better for TNFR-I(-/-) mice than for either TNFR-II(-/-) or WT mice. At 48 h of hyperoxia, slightly more alveolar septal thickening and peribronchiolar and periarteriolar edema were detected in WT than in TNFR-I(-/-) lungs. By 84 h of oxygen exposure, TNFR-I(-/-) mice demonstrated greater alveolar debris, inflammation, and edema than WT mice. TNFR-I was necessary for induction of cytokine interleukin (IL)-1beta, IL-1 receptor antagonist, chemokine macrophage inflammatory protein (MIP)-1beta, MIP-2, interferon-gamma-induced protein-10 (IP-10), and monocyte chemoattractant protein (MCP)-1 mRNA in response to intratracheal administration of recombinant murine TNF-alpha. However, IL-1beta, IL-6, macrophage migration inhibitory factor, MIP-1alpha, MIP-2, and MCP-1 mRNAs were comparably induced by hyperoxia in TNFR-I(-/-) and WT lungs. In contrast, mRNA for manganese superoxide dismutase and intercellular adhesion molecule-1 were induced by hyperoxia only in WT mice. Differences in early survival and toxicity suggest that pulmonary oxygen toxicity is in part mediated by TNFR-I. However, induction of specific cytokine and chemokine mRNA and lethality in response to severe hyperoxia was independent of TNFR-I expression. The current study supports the prediction that therapeutic efforts to block TNF-alpha receptor function will not protect against pulmonary oxygen toxicity.     

Mutants of human insulin-like growth factor I with reduced affinity for the type 1 insulin-like growth factor receptor

Four mutants of human insulin-like growth factor I (hIGF I) have been purified from the conditioned media of yeast transformed with an expression vector containing a synthetic gene for hIGF I altered by site-directed mutagenesis. hIGF I has the sequence Phe-23-Tyr-24-Phe-25 which is homologous to a region in the B-chain of insulin. [Phe23,Phe24,Tyr25]IGF I, in which the sequence is altered to exactly correspond to the homologous sequence in insulin, is equipotent to hIGF I at the types 1 and 2 IGF and insulin receptors. [Leu24]IGF I and [Ser24]IGF I have 32- and 16-fold less affinity than hIGF I at the human placental type 1 IGF receptor, respectively. These peptides are 10- and 2-fold less potent at the placental insulin receptor, respectively. [Leu24]IGF I and [Ser24]IGF I have similarly reduced affinities for the type 1 IGF receptor of rat A10 and mouse L cells. Thus, the importance of the interaction of residue 24 with the receptor is conserved in several species. In three cell-based assays, [Leu24]IGF I and [Ser24]IGF I are full agonists with reduced efficacy compared to hIGF I. Desoctapeptide [Leu24]IGF I, in which the loss of aromaticity at position 24 is combined with the deletion of the carboxyl-terminal D region of hIGF I, has 3-fold lower affinity than [Leu24]IGF I for the type 1 receptor and 2-fold higher affinity for the insulin receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tissue distribution and cancer growth inhibition of magnetic lipoplex-delivered type 1 insulin-like growth factor receptor shRNA in nude mice

The targeted delivery of therapeutic genes into specific tissues, as well as the determination of the biological fate and potential toxicity of nanoparticles, remains a highly relevant challenge for gene-based therapies. Type 1 insulin-like growth factor receptor (IGF-1R), an important oncogene, is frequently over-expressed in lung cancer and mediates cancer cell proliferation as well as tumor growth. In our previous studies, we have successfully applied gene delivery mediated by commercially available nanoparticles (CombiMAG) under a magnetic field, which suppresses IGF-1R expression in a non-small cell lung cancer cell line (A549) in vitro. In the present study, we aimed to investigate the biological distribution and target tumor suppression of magnetofection, as well as its potential toxicity via CombiMAG-carrying plasmids expressing green fluorescent protein (GFP) and short hairpin RNAs (shRNAs) targeting IGF-1R (pGFPshIGF-1Rs) in tumor-bearing mice. The peak expression in various organs appeared 48 h after transfection. Transgene expression via magnetofection was 3-fold improvement than via lipofection. On the 30th day after injection, the tumor size and weight of the CombiMAG-treated group (789.32 ± 39.43 mm(3), 105.5 ± 6.1 mg) were significantly decreased compared with those of the lipofection group (893.83 ± 31.23 mm(3), 164.5 ± 9.1 mg; P< 0.05), and the suppression rate was ～36%. After a 30-day observation, the injection of CombiMAG did not cause any apparent toxicity. Therefore, IGF-1R shRNA nanoparticles can be valuable and safe delivery agents for RNA interference therapy to tumors in vivo.     

Mutants of human insulin-like growth factor II with altered affinities for the type 1 and type 2 insulin-like growth factor receptor.

With the aim to produce insulin-like growth factors (IGF) with enhanced specificity for the type 1 or type 2 IGF receptors, three mutants of IGF II have been prepared and expressed in NIH-3T3 cells. IGF II mutated at Tyr27 to Leu and Glu showed a 25- and 54-fold decrease in affinity for the type 1 IGF receptor and a 3.4- and 9.2-fold decrease in affinity for the type 2 IGF receptor. IGF II mutated at Phe48 to Glu showed a 18-fold decrease in affinity for the type 2 IGF receptor and a 2.8-fold decrease in affinity for the type 1 IGF receptor. These affinities were measured in radioreceptor assays using type 1 or 2 IGF receptor overexpressing cells. Data obtained on receptor cross-linking and thymidine incorporation assays confirmed the results of the radioreceptor assays. It is concluded that mutations of Tyr27 preferentially decrease binding to the type 1 IGF receptor and of Phe48 to the type 2 IGF receptor, either by the loss of a residue involved in receptor binding or by preferentially destabilizing the region involved in receptor binding.     

Keratinocyte growth factor protects against elastase-induced pulmonary emphysema in mice

Pulmonary emphysema is characterized by persistent inflammation and progressive alveolar destruction. The keratinocyte growth factor (KGF) favorably influences alveolar maintenance and repair and possesses anti-inflammatory properties. We aimed to determine whether exogenous KGF prevented or corrected elastase-induced pulmonary emphysema in vivo. Treatment with 5 mg x kg(-1) x day(-1) KGF before elastase instillation prevented pulmonary emphysema. This effect was associated with 1) a sharp reduction in bronchoalveolar lavage fluid total protein and inflammatory cell recruitment, 2) a reduction in the pulmonary expression of the chemokines CCL2 (or monocyte chemoattractant protein-1) and CXCL2 (or macrophage inflammatory protein-2alpha) and of the adhesion molecules ICAM-1 and VCAM-1, 3) a reduction in matrix metalloproteinase (MMP)-2 and MMP-9 activity at day 3, and 4) a major reduction in DNA damage detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) in alveolar cells at day 7. Treatment with KGF after elastase instillation had no effect on elastase-induced emphysema despite the conserved expression of the KGF receptor in the lungs of elastase-instilled animals as determined by immunohistochemistry. In vitro, KGF abolished the elastase-induced increase in CCL2, CXCL2, and ICAM-1 mRNA in the MLE-12 murine alveolar epithelial cell line. We conclude that KGF pretreatment protected against elastase-induced pulmonary inflammation, activation of MMPs, alveolar cell DNA damage, and subsequent emphysema in mice.     

Anti-malarial drug artesunate protects against cigarette smoke-induced lung injury in mice

Cigarette smoking is the primary cause of chronic obstructive pulmonary disease (COPD), which is mediated by lung infiltration with inflammatory cells, enhanced oxidative stress, and tissue destruction. Anti-malarial drug artesunate has been shown to possess anti-inflammatory and anti-oxidative actions in mouse asthma models. We hypothesized that artesunate can protect against cigarette smoke-induced acute lung injury via its anti-inflammatory and anti-oxidative properties. Artesunate was given by oral gavage to BALB/c mice daily 2 h before 4% cigarette smoke exposure for 1 h over five consecutive days. Bronchoalveolar lavage (BAL) fluid and lungs were collected for analyses of cytokines, oxidative damage and antioxidant activities. Bronchial epithelial cell BEAS-2B was exposed to cigarette smoke extract (CSE) and used to study the mechanisms of action of artesunate. Artesunate suppressed cigarette smoke-induced increases in BAL fluid total and differential cell counts; levels of IL-1β, MCP-1, IP-10 and KC; and levels of oxidative biomarkers 8-isoprostane, 8-OHdG and 3-nitrotyrosine in a dose-dependent manner. Artesunate promoted anti-oxidant catalase activity and reduced NADPH oxidase 2 (NOX2) protein level in the lungs from cigarette smoke-exposed mice. In BEAS-2B cells, artesunate suppressed pro-inflammatory PI3 K/Akt and p44/42 MAPK signaling pathways, and increased nuclear Nrf2 accumulation in response to CSE. Artesunate possesses anti-inflammatory and anti-oxidative properties against cigarette smoke-induced lung injury, probably via inhibition of PI3K and p42/22 MAPK signaling pathways, augmentation of Nrf2 and catalase activities, and reduction of NOX2 level. Our data suggest that artesunate may have therapeutic potential for treating COPD.     

SCD1 deficiency protects mice against ethanol-induced liver injury

Stearoyl-CoA desaturase 1 (SCD1) is a delta-9 fatty acid desaturase that catalyzes the synthesis of mono-unsaturated fatty acids (MUFA). SCD1 is a critical control point regulating hepatic lipid synthesis and β-oxidation. Scd1 KO mice are resistant to the development of diet-induced non-alcoholic fatty liver disease (NAFLD). Using a chronic-binge protocol of ethanol-mediated liver injury, we aimed to determine if these KO mice are also resistant to the development of alcoholic fatty liver disease (AFLD).Mice fed a low-fat diet (especially low in MUFA) containing 5% ethanol for 10 days, followed by a single ethanol (5 g/kg) gavage, developed severe liver injury manifesting as hepatic steatosis. This was associated with an increase in de novo lipogenesis and inflammation. Using this model, we show that Scd1 KO mice are resistant to the development of AFLD. Scd1 KO mice do not show accumulation of hepatic triglycerides, activation of de novo lipogenesis nor elevation of cytokines or other pro-inflammatory markers. Incubating HepG2 cells with a SCD1 inhibitor induced a similar resistance to the effect of ethanol, confirming a role for SCD1 activity in mediating ethanol-induced hepatic injury.Taken together, our study shows that SCD1 is a key player in the development of AFLD and associated deleterious effects, and suggests SCD1 inhibition as a therapeutic option for the treatment of this hepatic disease.     

Matrix metalloproteinase-10 protects against acute kidney injury by augmenting epidermal growth factor receptor signaling

Matrix metalloproteinase-10 (MMP-10) is a zinc-dependent endopeptidase involved in regulating a wide range of biologic processes, such as apoptosis, cell proliferation, and tissue remodeling. However, the role of MMP-10 in the pathogenesis of acute kidney injury (AKI) is unknown. In this study, we show that MMP-10 was upregulated in the kidneys and predominantly localized in the tubular epithelium in various models of AKI induced by ischemia/reperfusion (IR) or cisplatin. Overexpression of exogenous MMP-10 ameliorated AKI, manifested by decreased serum creatinine, blood urea nitrogen, tubular injury and apoptosis, and increased tubular regeneration. Conversely, knockdown of endogenous MMP-10 expression aggravated kidney injury. Interestingly, alleviation of AKI by MMP-10 in vivo was associated with the activation of epidermal growth factor receptor (EGFR) and its downstream AKT and extracellular signal-regulated kinase-1 and 2 (ERK1/2) signaling. Blockade of EGFR signaling by erlotinib abolished the MMP-10-mediated renal protection after AKI. In vitro, MMP-10 potentiated EGFR activation and protected kidney tubular cells against apoptosis induced by hypoxia/reoxygenation or cisplatin. MMP-10 was colocalized with heparin-binding EGF-like growth factor (HB-EGF) in vivo and activated it by a process of proteolytical cleavage in vitro. These studies identify HB-EGF as a previously unrecognized substrate of MMP-10. Our findings also underscore that MMP-10 can protect against AKI by augmenting EGFR signaling, leading to promotion of tubular cell survival and proliferation after injury.Subject terms: Apoptosis, Cell growth     

Epidermal growth factor protects the heart against low-flow ischemia-induced injury

The role of ErbB4 and ErbB2 in the heart of adult mammals is well established. The heart also expresses ErbB1 (the epidermal growth factor (EGF) receptor), but this receptor has received less attention. We studied the effect of EGF on the response of isolated mouse heart to low-flow ischemia and reperfusion. Reducing perfusate flow to 10% for 30 min resulted in an increase in anaerobic metabolism and the leakage of lactate dehydrogenase during reperfusion. In addition, left ventricle +dP/dt and developed pressure were depressed (20–25%) during reperfusion. The addition of EGF 5 min before and throughout the ischemic period prevented the increase in anaerobic metabolism and the leakage of intracellular lactate dehydrogenase during reperfusion. EGF improved both +dP/dt and developed pressure during ischemia and prevented the decrease in dP/dt during reperfusion. To determine whether the effect of EGF on cell integrity depends on its effect on contractility, we studied nonbeating isolated myocytes. In these cells, anoxia and reoxygenation reduced cell viability by nearly 25%. EGF prevented such a decrease. Our results indicate that, like ErbB4 and ErbB2, ErbB1 also has an important role in the heart of adult animals.     

Transforming growth factor alpha protects against Fas-mediated liver apoptosis in mice

The Fas/Fas ligand interaction plays a crucial role in various liver diseases, and administration of agonistic anti-Fas antibody to mice causes massive hepatic apoptosis and fulminant hepatic failure. Several growth factors have recently been found to function in preventing apoptosis. In this study, we demonstrated that overexpression of transforming growth factor alpha (TGFalpha) has a dramatic protective effect on Fas-mediated hepatic apoptosis at the biochemical and histological levels. Moreover, 85.7% (six out of seven) of TGFalpha transgenic mice survived the lethal liver damage, whereas all wild-type mice died. Expression of Bcl-xL, an anti-apoptotic protein, was greatly increased in the transgenic mice. Taken together, our findings suggest that TGFalpha protects against Fas-mediated liver apoptosis in vivo and up-regulation of Bcl-xL may participate in protective effect of TGFalpha.     

Alanine scanning mutagenesis of a type 1 insulin-like growth factor receptor ligand binding site

The high resolution crystal structure of an N-terminal fragment of the IGF-I receptor, has been reported. While this fragment is itself devoid of ligand binding activity, mutational analysis has indicated that its N terminus (L1, amino acids 1-150) and the C terminus of its cysteine-rich domain (amino acids 190-300) contain ligand binding determinants. Mutational analysis also suggests that amino acids 692-702 from the C terminus of the alpha subunit are critical for ligand binding. A fusion protein, formed from these fragments, binds IGF-I with an affinity similar to that of the whole extracellular domain, suggesting that these are the minimal structural elements of the IGF-I binding site. To further characterize the binding site, we have performed structure directed and alanine-scanning mutagenesis of L1, the cysteine-rich domain and amino acids 692-702. Alanine mutants of residues in these regions were transiently expressed as secreted recombinant receptors and their affinity was determined. In L1 alanine mutants of Asp(8), Asn(11), Tyr(28), His(30), Leu(33), Leu(56), Phe(58), Arg(59), and Trp(79) produced a 2- to 10-fold decrease in affinity and alanine mutation of Phe(90) resulted in a 23-fold decrease in affinity. In the cysteine-rich domain, mutation of Arg(240), Phe(241), Glu(242), and Phe(251) produced a 2- to 10-fold decrease in affinity. In the region between amino acids 692 and 702, alanine mutation of Phe(701) produced a receptor devoid of binding activity and alanine mutations of Phe(693), Glu(693), Asn(694), Leu(696), His(697), Asn(698), and Ile(700) exhibited decreases in affinity ranging from 10- to 30-fold. With the exception of Trp(79), the disruptive mutants in L1 form a discrete epitope on the surface of the receptor. Those in the cysteine-rich domain essential for intact affinity also form a discrete epitope together with Trp(79).     

Constitutive expression of insulin-like growth factor 1 and insulin-like growth factor 1 receptor abrogates all requirements for exogenous growth factors.

BALB/c3T3 cells are exquisitely growth regulated and require both platelet-derived growth factor and insulin-like growth factor-1 (IGF-1) for optimal proliferation. BALB/c3T3 cells that constitutively express IGF-1 and elevated levels of IGF-1 receptor (IGF-1R) are capable of growth in serum-free medium without the addition of any exogenous growth factors. BALB/c3T3 cells overexpressing only the IGF-1R plasmid required IGF-1 or insulin for serum-free growth. Antisense oligodeoxynucleotides complementary to IGF-1R mRNA inhibited IGF-1-mediated cell growth. Under these conditions, neither the epidermal growth factor receptor nor phospholipase C gamma 1 was autophosphorylated. These findings indicate that constitutive expression of IGF-1 and IGF-1R allows 3T3 cells to grow in serum-free medium without addition of those exogenous growth factors that are required by the parent cell line.     

Selective modulation of type 1 insulin-like growth factor receptor signaling and functions by beta1 integrins

We show here that beta1 integrins selectively modulate insulin-like growth factor type I receptor (IGF-IR) signaling in response to IGF stimulation. The beta1A integrin forms a complex with the IGF-IR and insulin receptor substrate-1 (IRS-1); this complex does not promote IGF-I mediated cell adhesion to laminin (LN), although it does support IGF-mediated cell proliferation. In contrast, beta1C, an integrin cytoplasmic variant, increases cell adhesion to LN in response to IGF-I and its down-regulation by a ribozyme prevents IGF-mediated adhesion to LN. Moreover, beta1C completely prevents IGF-mediated cell proliferation and tumor growth by inhibiting IGF-IR auto-phosphorylation in response to IGF-I stimulation. Evidence is provided that the beta1 cytodomain plays an important role in mediating beta1 integrin association with either IRS-1 or Grb2-associated binder1 (Gab1)/SH2-containing protein-tyrosine phosphate 2 (Shp2), downstream effectors of IGF-IR: specifically, beta1A associates with IRS-1 and beta1C with Gab1/Shp2. This study unravels a novel mechanism mediated by the integrin cytoplasmic domain that differentially regulates cell adhesion to LN and cell proliferation in response to IGF.     

Preprotachykinin-A gene deletion protects mice against acute pancreatitis and associated lung injury

Impaired lung function in severe acute pancreatitis is the primary cause of morbidity and mortality in this condition. Preprotachykinin-A (PPT-A) gene products substance P and neurokinin (NK)-A have been shown to play important roles in neurogenic inflammation. Substance P acts primarily (but not exclusively) via the NK1 receptor. NKA acts primarily via the NK2 receptor. Earlier work has shown that knockout mice deficient in NK1 receptors are protected against acute pancreatitis and associated lung injury. NK1 receptors, however, bind other peptides in addition to substance P, not all of which are derived from the PPT-A gene. To examine the role of PPT-A gene products in acute pancreatitis, the effect of PPT-A gene deletion on the severity of acute pancreatitis and the associated lung injury was investigated. Deletion of PPT-A almost completely protected against acute pancreatitis-associated lung injury, with a partial protection against local pancreatic damage. These results show that PPT-A gene products are critical proinflammatory mediators in acute pancreatitis and the associated lung injury.