Interaction Between the a3 Region of Factor VIII and the TIL'E' Domains of the von Willebrand Factor.

The von Willebrand factor (VWF) and coagulation factor VIII (FVIII) are intricately involved in hemostasis. A tight, noncovalent complex between VWF and FVIII prolongs the half-life of FVIII in plasma, and failure to form this complex leads to rapid clearance of FVIII and bleeding diatheses such as hemophilia A and von Willebrand disease (VWD) type 2N. High-resolution insight into the complex between VWF and FVIII has so far been strikingly lacking. This is particularly the case for the flexible a3 region of FVIII, which is imperative for high-affinity binding. Here, a structural and biophysical characterization of the interaction between VWF and FVIII is presented with focus on two of the domains that have been proven pivotal for mediating the interaction, namely the a3 region of FVIII and the TIL'E' domains of VWF. Binding between the FVIII a3 region and VWF TIL'E' was here observed using NMR spectroscopy, where chemical shift changes were localized to two ß-sheet regions on the edge of TIL'E' upon FVIII a3 region binding. Isothermal titration calorimetry and NMR spectroscopy were used to characterize the interaction between FVIII and TIL'E' as well as mutants of TIL'E', which further highlights the importance of the ß-sheet region of TIL'E' for high-affinity binding. Overall, the results presented provide new insight into the role the FVIII a3 region plays for complex formation between VWF and FVIII and the ß-sheet region of TIL'E' is shown to be important for FVIII binding. Thus, the results pave the way for further high-resolution insights into this imperative complex.

A novel B-domain O-glycoPEGylated FVIII (N8-GP) demonstrates full efficacy and prolonged effect in hemophilic mice models.

Frequent infusions of intravenous factor VIII (FVIII) are required to prevent bleeding associated with hemophilia A. To reduce the treatment burden, recombinant FVIII with a longer half-life was developed without changing the protein structure. FVIII-polyethylene glycol (PEG) conjugates were prepared using an enzymatic process coupling PEG (ranging from 10 to 80 kDa) selectively to a unique O-linked glycan in the FVIII B-domain. Binding to von Willebrand factor (VWF) was maintained for all conjugates. Upon cleavage by thrombin, the B-domain and the associated PEG were released, generating activated FVIII (FVIIIa) with the same primary structure and specific activity as native FVIIIa. In both FVIII- and VWF-deficient mice, the half-life was found to increase with the size of PEG. In vivo potency and efficacy of FVIII conjugated with a 40-kDa PEG (N8-GP) and unmodified FVIII were not different. N8-GP had a longer duration of effect in FVIII-deficient mouse models, approximately a twofold prolonged half-life in mice, rabbits, and cynomolgus monkeys; however, the prolongation was less pronounced in rats. Binding capacity of N8-GP on human monocyte-derived dendritic cells was reduced compared with unmodified FVIII, resulting in several-fold reduced cellular uptake. In conclusion, N8-GP has the potential to offer efficacious prevention and treatment of bleeds in hemophilia A at reduced dosing frequency.

Implications for the offspring of circulating factors involved in beta cell adaptation in pregnancy.

OBJECTIVE: Several studies have shown an increase in beta cell mass during pregnancy. Somatolactogenic hormones are known to stimulate the proliferation of existing beta cells in rodents whereas the mechanism in humans is still unclear. We hypothesize that in addition to somatolactogenic hormones there are other circulating factors involved in beta cell adaptation to pregnancy. This study aimed at screening for potential pregnancy-associated circulating beta cell growth factors. SAMPLES: Serum samples from nonpregnant and pregnant women. METHODS: The effect of serum from pregnant women on the proliferation of rat beta cells was studied using [3H]thymidine incorporation and 5-ethynyl-2'-deoxyuridine proliferation assays. In addition, serum from pregnant and nonpregnant women was fractionated by gel filtration and high performance liquid chromatography. The fractionated serum was screened for mitogenic activity in INS-1E cells. Proteins and peptides in mitogenic active serum fractions were identified by amino acid sequencing and mass spectrometry. MAIN OUTCOME MEASURES: Presence of circulating beta cell proliferating factors. RESULTS: Late gestational pregnancy serum significantly increased proliferation of rat beta cells compared with early pregnancy and nonpregnancy. The mitogenic active serum fractions contained proteins and peptides derived from kininogen-1, fibrinogen-α, α1-antitrypsin, apolipoprotein-A1, placental lactogen, angiotensinogen and serum albumin. CONCLUSION: Pregnancy serum is able to stimulate proliferation of rat beta cells. We have identified several circulating factors that may contribute to beta cell adaptation to pregnancy. Further studies are needed to elucidate their possible role in glucose homeostasis in the mother and her offspring.

Evaluation of the metal binding sites in a recombinant coagulation factor VIII identifies two sites with unique metal binding properties.

Coagulation factor VIII is a glycosylated, non-covalent heterodimer consisting of a heavy chain (A1-A2-B domains) and a light chain (A3-C1-C2 domains). The association of the chains, and the stability and function of the dimer depend on the presence of metal ions. We applied X-ray fluorescence, X-ray crystallographic structure determination with anomalous signals at different wavelengths, and colorimetric measurements to evaluate the metal binding sites in a recombinant factor VIII molecule, turoctocog alfa. We identified a metal binding site in domain A3 dominated by Cu(+) binding and a site in domain A1 dominated by Zn(2+) binding.

The intestinotrophic peptide, GLP-2, counteracts the gastrointestinal atrophy in mice induced by the epidermal growth factor receptor inhibitor, erlotinib, and cisplatin.

PURPOSE: Erlotinib, an epidermal-growth-factor receptor inhibitor, belongs to a new generation of targeted cancer therapeutics. Gastrointestinal side-effects are common and have been markedly aggravated when erlotinib is combined with cytostatics. We examined the effects of erlotinib alone and combined with the cytostatic, cisplatin, on the gastrointestinal tract and examined whether glucagon-like peptide-2 (GLP-2), an intestinal hormone with potent intestinotrophic properties, might counteract the possible damaging effects of the treatments. EXPERIMENTAL DESIGN: Groups of ten mice were treated for 10 days with increasing doses of erlotinib alone or in combination with cisplatin and/or GLP-2. Weight and length of the gastrointestinal organs were determined and histological sections were analyzed with morphometric methods as well as BrdU- and ApopTag-staining to determine mitotic and apoptotic activity. RESULTS: Erlotinib was found to induce small-intestinal and colonic growth inhibition through an increased apoptotic activity but had no effect on mitotic activity. The combined treatment with cisplatin synergistically aggravated the intestinal growth inhibition. Erlotinib, and especially the combination therapy, increased the weight of the stomach contents considerably. Concomitant treatment with GLP-2 counteracted the intestinal mucosal atrophy induced both by erlotinib alone and combined with cisplatin through a reduction of the apoptotic activity. There was no influence on the mitotic activity. CONCLUSIONS: The findings demonstrate that the intestinal mucosal damage induced by erlotinib alone and in combination with cisplatin can be counteracted by GLP-2 treatment, which might suggest a role for GLP-2 in the treatment of the gastrointestinal side-effects caused by these cancer therapeutics.

The trefoil factor 1 participates in gastrointestinal cell differentiation by delaying G1-S phase transition and reducing apoptosis.

Trefoil factor (TFF)1 is synthesized and secreted by the normal stomach mucosa and by the gastrointestinal cells of injured tissues. The link between mouse TFF1 inactivation and the fully penetrant antropyloric tumor phenotype prompted the classification of TFF1 as a gastric tumor suppressor gene. Accordingly, altered expression, deletion, and/or mutations of the TFF1 gene are frequently observed in human gastric carcinomas. The present study was undertaken to address the nature of the cellular and molecular mechanisms targeted by TFF1 signalling. TFF1 effects were investigated in IEC18, HCT116, and AGS gastrointestinal cells treated with recombinant human TFF1, and in stably transfected HCT116 cells synthesizing constitutive or doxycycline-induced human TFF1. We observed that TFF1 triggers two types of cellular responses. On one hand, TFF1 lowers cell proliferation by delaying G1-S cell phase transition. This results from a TFF1-mediated increase in the levels of cyclin-dependent kinase inhibitors of both the INK4 and CIP subfamilies, leading to lower E2F transcriptional activity. On the other hand, TFF1 protects cells from chemical-, anchorage-free-, or Bad-induced apoptosis. In this process, TFF1 signalling targets the active form of caspase-9. Together, these results provide the first evidence of a dual antiproliferative and antiapoptotic role for TFF1. Similar paradoxical functions have been reported for tumor suppressor genes involved in cell differentiation, a function consistent with TFF1.

Importance of cocaine- and amphetamine-regulated transcript peptide in the central nucleus of amygdala in anxiogenic responses induced by ethanol withdrawal.

We studied the involvement of cocaine- and amphetamine-regulated transcript peptide (CART) in the central nucleus of amygdala (CeA), lateral bed nucleus of the stria terminalis (BNSTl) and nucleus accumbens shell (AcbSh) in generation of ethanol withdrawal symptoms, with particular focus on anxiety-like behavior using a social interaction test. Administration of CART (54-102) into the lateral ventricle (50 and 100 ng) and bilaterally in the CeA (10 and 20 ng) caused a significant reduction in social interaction, suggesting an anxiogenic action of the peptide. Chronic ethanol treatment for 15 days followed by withdrawal precipitated an anxiogenic response at 24 h that was attenuated by intracerebroventricular (5 mul) and intra-CeA (1 mul) administration of antibodies against CART (1 : 500 dilution). An immunocytochemistry protocol was employed to study the response of the endogenous CART system in the CeA following chronic ethanol withdrawal. At 0 h ethanol withdrawal, CART immunoreactivity was apparent in few fibers and the profile was similar to that in the pair-fed control rats. Twenty-four hours following ethanol withdrawal, a highly significant increase (P<0.001) in CART immunoreactivity was noticed in the CeA, which returned to normal 48 and 72 h post-withdrawal. Similar doses of CART or CART antibody injected bilaterally into the BNSTl or AcbSh produced no response in the social interaction test. Furthermore, the CART immunoreactivity profile did not change at the post-withdrawal time points in each of these brain sites. We suggest that CART may mediate the early signs of anxiety-like behavior induced by ethanol withdrawal within the neuroanatomical framework of the CeA.

Association of cocaine- and amphetamine-regulated transcript and neuropeptide Y in the forebrain and pituitary of the catfish, Clarias batrachus: a double immunofluorescent labeling study.

Cocaine- and amphetamine-regulated transcript (CART) and neuropeptide Y (NPY) are involved in the regulation of food intake, body weight, pituitary hormones, and reproduction. While CART and NPY occupy overlapping fields in the brain of mammals, little is known about the interaction between these peptide-containing systems in other vertebrates. We explored neuroanatomical associations between CART and NPY in the olfactory system, forebrain and pituitary of the catfish, Clarias batrachus, using double immunofluorescence method. NPY-containing fascicles from olfactory receptor neurons innervated the olfactory glomeruli and mitral cell layer in close association with CART-containing terminal fields. Distinct CART- or NPY-containing fibers were seen in the medial olfactory tract. In the dorsal telencephalon, CART- and NPY-immunoreactive axons were closely associated in area dorsalis telencephali/pars lateralis dorsalis (Dld), and posterioris (Dlp). In the ventral telencephalon, while most of the cells of nucleus entopeduncularis (NE) showed the presence of CART as well as NPY, a few cells with only NPY-immunoreactivity were observed. Similarly, a CART and NPY colocalized cell population was prominent in the preoptic area (POA); and a small population of cells with NPY-immunoreactivity was also evident. Other areas where CART and NPY were colocalized included fibers in the tuberal area, inferior lobe, neurohypophysis, proximal pars distalis and pars intermedia of the pituitary. No association between CART and NPY was observed in the thalamus and habenular ganglion. These results suggest that CART- and NPY-peptidergic systems may interact in NE, POA, tuberal area, certain telencephalic areas and pituitary and jointly process information relating to reproduction, feeding and neuroendocrine regulation.

Trefoil factor 3 in perinatal pancreas is increased by gestational low protein diet and associated with accelerated ß-cell maturation.

The endocrine pancreas expands markedly in the first postnatal days and the insulin producing ß-cells initiate a functional maturation preceded by a morphological change of the islets of Langerhans. Trefoil factor 3 (TFF3) is a secreted peptide expressed in intestinal epithelia, where it promotes migration, but its role in the pancreas is not characterized. The aim of this study was to examine the expression and function of TFF3 in perinatal rat pancreas, ex vivo cultured fetal rat pancreas and in the rat ß-cell line INS-1E. Control or gestational low-protein diet perinatal rat pancreas was harvested at embryonic day 20 (E20), day of birth (P0) and postnatal day 2 (P2). TFF3 mRNA was upregulated 4.5-fold at P0 vs. E20 and downregulated again at P2. In protein-undernourished pups induction of TFF3 at P0 was further increased to 9.7-fold and was increased at P2. TFF3 caused tyrosine phosphorylation of EGFR in INS-1E ß-cells, and purified recombinant TFF3 increased both attachment and spreading of INS-1E ß-cells. In ex vivo cultures of collagenase digested fetal rat pancreas, a model of perinatal ß-cell maturation, TFF3 increased cellular spreading as well as insulin mRNA levels. TFF3 also increased the expression of Pref1/Dlk1 that shares similarities in expression and regulation with TFF3. These results suggest that TFF3 may promote adhesion and spreading of cells to accelerate ß-cell maturation. This study indicates a functional role for TFF3 in pancreatic ß-cell maturation in the perinatal period, which is altered by low protein diet during gestation.

Immunohistochemical localization of cocaine- and amphetamine-regulated transcript peptide in the brain of the catfish, Clarias batrachus (Linn.).

The organization of cocaine- and amphetamine-regulated transcript peptide (CARTp, 54-102) immunoreactivity was investigated in the brain of the catfish, Clarias batrachus. CARTp-immunoreactivity was observed in several granule cells of the olfactory bulbs, in dot-like terminals around mitral cells, and in the fibers of the medial olfactory tracts. While several groups of discrete cells in the telencephalon showed CARTp-immunoreactivity, the immunostained fibers were widely distributed in the area dorsalis and ventralis telencephali. Immunoreactivity was seen in several periventricular and a few magnocellular neurons, and in a dense fiber network throughout the preoptic area. Varying degrees of immunoreactive fibers were seen in the periventricular region in the thalamus, hypothalamus, and pituitary. Some neurons in the nucleus preglomerulosus medialis and lateralis, central nucleus of the inferior lobes, nucleus lobobulbaris of the posterior tuberculum, and nucleus recessus posterioris showed distinct CARTp-immunoreactivity. Considerable immunoreactivity was seen in the optic tectum, rostral torus semicircularis, central pretectal area, and granule cells of the cerebellum. While only isolated immunoreactive cells were seen at three distinct sites in the metencephalon, a fiber network was seen in the facial and vagal lobes and periventricular and ventral regions of the medulla oblongata. The pattern of the CARTp distribution in the brain of C. batrachus suggests that it may play an important role in the processing of sensory information, the regulation of hormone secretion by hypophysial cell types, and motor and vegetative function. Finally, as in other animal species, CARTp seems to play a role in the processing of gustatory information.

Glucagon-like peptide-2 improves both acute and late experimental radiation enteritis in the rat.

PURPOSE: Acute and/or chronic radiation enteritis can develop after radiotherapy for pelvic cancers. Experimental and clinical observations have provided evidence of a role played by acute mucosal disruption in the appearance of late effects. The therapeutic potential of acute administration of glucagon-like peptide-2 (GLP-2) against acute and chronic intestinal injury was investigated in this study. METHODS AND MATERIALS: Intestinal segments were surgically exteriorized and exposed to 16.7 or 19 Gy X-rays. The rats were treated once daily with vehicle or a protease-resistant GLP-2 derivative for 14 days before irradiation, with or without 7 days of GLP-2 after treatment. Macroscopic and microscopic observations were made 2 and 15 weeks after radiation exposure. RESULTS: In the control animals, GLP-2 induced an increase in intestinal mucosal mass, along with an increase in villus height and crypt depth. GLP-2 administration before and after irradiation completely prevented the acute radiation-induced mucosal ulcerations observed after exposure to 16.7 Gy. GLP-2 treatment strikingly reduced the late radiation damage observed after 19 Gy irradiation. Microscopic observations revealed an improved organization of the intestinal wall and an efficient wound healing process, especially in the smooth muscle layers. CONCLUSION: GLP-2 has a clear therapeutic potential against both acute and chronic radiation enteritis. This therapeutic effect is mediated through an increased mucosal mass before tissue injury and the stimulation of still unknown mechanisms of tissue response to radiation damage. Although these preliminary results still need to be confirmed, GLP-2 might be a way to limit patient discomfort during radiotherapy and reduce the risk of consequential late effects.

Tissue localization of human trefoil factors 1, 2, and 3.

Trefoil factors (TTFs) are small, compact proteins coexpressed with mucins in the gastrointestinal tract. Three trefoil factors are known in mammals: TFF1, TFF2, and TFF3. They are implicated to play diverse roles in maintenance and repair of the gastrointestinal channel. We compared the expression pattern of the three trefoil factors analyzing mRNA from a panel of 20 human tissues by conventional reverse transcriptase (RT) PCR and, in addition, by real-time PCR. These findings were supported by immunohistochemical analysis of paraffin-embedded human tissues using rabbit polyclonal antibodies raised against these factors. TFF1 showed highest expression in the stomach and colon, whereas TFF2 and TFF3 showed highest expression in stomach and colon, respectively. All three TFFs were found in the ducts of pancreas. Whereas TFF2 was found to be restricted to these two tissues, the structurally more closely related TFF1 and TFF3 showed a more general tissue distribution and were found to colocalize on an array of mucosal surfaces. This is the first thorough parallel description of the tissue distribution of TFFs in normal tissues, and it provides a baseline for similar analysis in diseased tissues.

Pharmacokinetics of trefoil peptides and their stability in gastrointestinal contents.

Trefoil factor family (TFF) peptides are considered promising for therapeutic use in gastrointestinal diseases, and there is a need to explore the fate of injected TFF and the stability of the peptides in the gastrointestinal tract. We studied the pharmacokinetics of intravenously (i.v.) administered hTFF2 in mice and rats and of hTFF3 administered i.v., intramuscularly, intraperitoneally, and subcutaneously in mice, and estimated by ELISA the decay of the peptides added to rat and human gastrointestinal contents. We found that i.v. injected hTFF2 and hTFF3 were cleared from the circulation within 2-3h, exhibiting comparable pharmacokinetic profiles. In contents from the rat stomach, hTFF levels remained unchanged for up to 6 days. In the small and large intestine of rats, the hTFF levels decreased markedly after 4 and 1h, respectively. In small intestinal contents from humans, the levels remained stable for more than 24h. We conclude that systemically administered hTFF2 and hTFF3 are rapidly eliminated from the circulation and that the stability of hTFF2 and hTFF3 in GI contents appeared higher in the gastric and small intestinal milieu than in the large intestine and feces, suggesting a higher stability toward gastric acid and digestive enzymes than toward microbial degradation.

Trefoil factors are expressed in human and rat endocrine pancreas: differential regulation by growth hormone.

Trefoil factors (TFFs) 1, 2, and 3 are expressed in mucosal epithelia. TFFs are particular abundant in the intestine in which they play a crucial role in maintenance and restitution of the epithelium. Because pancreas developmentally arises from the primitive foregut, we explored the expression of TFFs in the pancreas in man and rat. Immunocytochemical staining of adult human pancreas showed abundant TFF3 immunoreactivity in pancreatic islets and some duct cells, whereas weak TFF1 and no TFF2 staining were detected. In the islets TFF3 localized to most insulin and some glucagon and pancreatic polypeptide-producing cells. TFF3 immunoreactivity was colocalized with insulin and glucagon in distinct cell clusters in human fetal pancreas at wk 14 and in the newborn rat pancreas. In isolated human and rat islets, TFF3 and TFF1 mRNA was identified by RT-PCR, and TFF3 protein was detected in human pancreas and islets by ELISA. Exposure of neonatal rat islets or insulinoma cells to GH, a known beta-cell growth factor, resulted in markedly increased TFF3 but decreased TFF1 mRNA levels. The effect of GH on TFF3 expression was confirmed by Western blot. Culture of neonatal rat islets in the presence of TFF3 resulted in attachment and migration of the islet cells, but no effects on proliferation, insulin secretion or cytokine-induced apoptosis were seen. These data demonstrate expression of TFFs in the endocrine pancreas, but their possible functions remain unknown.

Naturally occurring glucagon-like peptide-2 (GLP-2) receptors in human intestinal cell lines.

Although clinical trials with GLP-2 receptor agonists are currently ongoing, the mechanisms behind GLP-2-induced intestinal epithelial growth remain to be understood. To approach the GLP-2 mechanism of action this study aimed to identify intestinal cell lines endogenously expressing the GLP-2 receptor. Here we report the first identification of a cell line endogenously expressing functional GLP-2 receptors. The human intestinal epithelial cell line, FHC, expressed GLP-2 receptor encoding mRNA (RT-PCR) and GLP-2 receptor protein (Western blot). In cultured FHC cells, GLP-2 induced concentration dependent cAMP accumulation (pEC(50)=9.7+/-0.04 (mean+/-S.E.M., n=4)). In addition, a naturally occurring human intestinal fibroblast cell line, 18Co, endogenously expressing GLP-2 receptor encoding mRNA (RT-PCR) and protein (Western blot) was identified. No receptor functionality (binding or G-protein signalling) could be demonstrated in 18Co cells. The identified gut-relevant cell lines provide tools for future clarification of the mechanisms underlying GLP-2-induced epithelial growth.

Molecular design and downstream processing of turoctocog alfa (NovoEight), a B-domain truncated factor VIII molecule.

Turoctocog alfa (NovoEight) is a third-generation recombinant factor VIII (rFVIII) with a truncated B-domain that is manufactured in Chinese hamster ovary cells. No human or animal-derived materials are used in the process. The aim of this study is to describe the molecular design and purification process for turoctocog alfa. A five-step purification process is applied to turoctocog alfa: protein capture on mixed-mode resin; immunoaffinity chromatography using a unique, recombinantly produced anti-FVIII mAb; anion exchange chromatography; nanofiltration and size exclusion chromatography. This process enabled reduction of impurities such as host cell proteins (HCPs) and high molecular weight proteins (HMWPs) to a very low level. The immunoaffinity step is very important for the removal of FVIII-related degradation products. Manufacturing scale data shown in this article confirmed the robustness of the purification process and a reliable and consistent reduction of the impurities. The contribution of each step to the final product purity is described and shown for three manufacturing batches. Turoctocog alfa, a third-generation B-domain truncated rFVIII product is manufactured in Chinese hamster ovary cells without the use of animal or human-derived proteins. The five-step purification process results in a homogenous, highly purified rFVIII product.

Selective abrogation of the proinvasive activity of the trefoil peptides pS2 and spasmolytic polypeptide by disruption of the EGF receptor signaling pathways in kidney and colonic cancer cells.

Trefoil peptides (TFFs) are now considered as scatter factors, proinvasive and angiogenic agents acting through cyclooxygenase-2 (COX-2)- and thromboxane A2 receptor (TXA2-R)-dependent signaling pathways. As expression and activation levels of the epidermal growth factor receptor (EGFR) predict the metastatic potential of human colorectal cancers, the purpose of this study was to establish whether the EGF receptor tyrosine kinase (EGFR-TK) contributes to cellular invasion induced by TFFs in kidney and colonic cancer cells. Both the dominant negative form of the EGFR (HER-CD533) and the EGFR-TK inhibitor ZD1839 (Iressa) abrogated cellular invasion induced by pS2, spasmolytic polypeptide (SP) and the src oncogene, but not by ITF and the TXA2-R. Similarly, EGFR-TK inhibition by ZD1839 reversed the invasive phenotype promoted by the constitutively activated form of the EGFR (EGFRvIII) and the EGFR agonists transforming growth factor alpha (TGFalpha), amphiregulin and EGF. We also provide evidence that TFFs, EGFRvIII, and TGFalpha trigger common proinvasive pathways using the PI3'-kinase and Rho/Rho- kinase cascades. These findings identify the EGFR-TK as a key signaling element for pS2- and SP-mediated cellular invasion. It is concluded that although pS2, SP and ITF belong to the same family of inflammation- and cancer-associated regulatory peptides, they do not control identical signaling networks.

Trefoil peptides as proangiogenic factors in vivo and in vitro: implication of cyclooxygenase-2 and EGF receptor signaling.

We previously established that the trefoil peptides (TFFs) pS2, spasmolytic polypeptide, and intestinal trefoil factor are involved in cellular scattering and invasion in kidney and colonic cancer cells. Using the chorioallantoic membrane (CAM) assay and the formation of tube-like structures by human umbilical vein endothelial cells (HUVEC) plated on the Matrigel matrix substratum, we report here that TFFs are proangiogenic factors. Angiogenic activity of TFFs is comparable to that induced by vascular endothelial growth factor, leptin, and transforming growth factor-alpha. Stimulation of angiogenesis by pS2 in the CAM assay is blocked by pharmacological inhibitors of cyclooxygenase COX-2 (NS-398) and epidermal growth factor receptor (EGF-R) tyrosine kinase (ZD1839), but is independent of KDR/Flk-1 and thromboxane A2 receptors. In contrast, the morphogenic switch induced by pS2 in HUVEC cells could be inhibited by the specific KDR heptapeptide antagonist ATWLPPR and by inhibitors of COX-2 and EGF-R signaling. These results implicate TFFs in the formation of new blood vessels during normal and pathophysiological processes linked to wound healing, inflammation, and cancer progression in the digestive mucosa and other human solid tumors associated with aberrant expression of TFFs.

Luminal and parenteral TFF2 and TFF3 dimer and monomer in two models of experimental colitis in the rat.

BACKGROUND: Peptides of the trefoil factor family (TFF1, TFF2 and TFF3) are cosecreted with mucus from mucus-producing cells in most organ systems and are believed to interact with mucus to form high-viscosity stable gel complexes. In the gastrointestinal tract, they sustain the mucosal barrier, and both injected and orally administered TFF peptide have protective and healing functions in the gastric mucosa. AIM: To investigate the possible treatment effect of luminally and parenterally administered TFF peptides in experimental colitis in rats. METHODS: Colitis was induced by administration of 5% dextran sodium sulphate in the drinking water or by one intraperitoneal injection of mitomycin C, 3.75 mg/kg. TFF peptides were administered as subcutaneous injections or directly into the lumen via a catheter placed in the proximal colon. Treatments were saline, TFF2, TFF3 monomer or TFF3 dimer 5 mg/kg twice per day throughout the study [dextran sulphate sodium (DSS)] or from day 4 to 7 (mitomycin C). Colitis severity was scored in a stereomicroscope and histologically. RESULTS: Luminal treatment with TFF3 in its dimeric form significantly improved the colitis score in both colitis models, whereas TFF2 had positive effect only in DSS-induced colitis. The TFF3 monomer was without any effects in both models. Treatment effect was most pronounced in the middle part of the colon, closest to the tip of the catheter. Injected TFF peptides, especially the TFF3 monomer, aggravated the colitis score in both colitis models. CONCLUSIONS: Intracolonic administration of TFF3 dimer and TFF2 improves experimentally induced colitis in rats. The TFF3 monomer has no effect. Parenteral administration of TFF peptides aggravates the colitis especially the TFF3 monomer.

Trefoil peptide TFF2 treatment reduces VCAM-1 expression and leukocyte recruitment in experimental intestinal inflammation.

There is evidence for a beneficial effect of trefoil peptides in animal models of gastric damage and intestinal inflammation, but the optimal treatment strategy and the mechanistic basis have not been explored thoroughly. It has been suggested that these proteins may modulate the inflammatory response. The aims of this study were to compare the protective and curative value of systemic and topical trefoil factor family (TFF)2 administration in dextran sulfate sodium-induced experimental colitis and to investigate the relationship between the therapeutic effects of TFF2 and modulation of leukocyte recruitment and expression of cell adhesion molecules. Clinical and morphologic severity of colitis was evaluated at the end of the study (Day 10). Leukocyte-endothelial cell interactions were determined in colonic venules by fluorescence intravital microscopy. The expression of cell adhesion molecules vascular cell adhesion molecule 1 (VCAM-1) and mucosal addressin cell adhesion molecule 1 (MAdCAM-1) was measured by the dual radiolabeled monoclonal antibody technique. Pretreatment with TFF2 by subcutaneous or intracolonic (ic) route ameliorated the clinical course of colitis, and the luminal route had a significantly superior effect. This beneficial effect was correlated with significant reductions in endothelial VCAM-1 but not MAdCAM-1 expression and leukocyte adhesion to intestinal venules, which returned to levels similar to those of controls. In established colitis, ic TFF2 treatment did not modify the severity of colonic lesions. In conclusion, TFF2 is useful in the treatment of colitis, and topical administration is superior to the systemic route. Reduction in adhesion molecule expression and leukocyte recruitment into the inflamed intestine contributes to the beneficial effect of this treatment.