Meprinα transactivates the epidermal growth factor receptor (EGFR) via ligand shedding, thereby enhancing colorectal cancer cell proliferation and migration

Meprinα, an astacin-type metalloprotease is overexpressed in colorectal cancer cells and is secreted in a non-polarized fashion, leading to the accumulation of meprinα in the tumor stroma. The transition from normal colonocytes to colorectal cancer correlates with increased meprinα activity at primary tumor sites. A role for meprinα in invasion and metastatic dissemination is supported by its pro-angiogenic and pro-migratory activity. In the present study, we provide evidence for a meprinα-mediated transactivation of the EGFR signaling pathway and suggest that this mechanism is involved in colorectal cancer progression. Using alkaline phosphatase-tagged EGFR ligands and an ELISA assay, we demonstrate that meprinα is capable of shedding epidermal growth factor (EGF) and transforming growth factor-α (TGFα) from the plasma membrane. Shedding was abrogated using actinonin, an inhibitor for meprinα. The physiological effects of meprinα-mediated shedding of EGF and TGFα were investigated with human colorectal adenocarcinoma cells (Caco-2). Proteolytically active meprinα leads to an increase in EGFR and ERK1/2 phosphorylation and subsequently enhances cell proliferation and migration. In conclusion, the implication of meprinα in the EGFR/MAPK signaling pathway indicates a role of meprinα in colorectal cancer progression.

Vascular endothelial growth factor induces contralesional corticobulbar plasticity and functional neurological recovery in the ischemic brain

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor, which also has neuroprotective activity. In view of these dual actions on vessels and neurons, we were interested whether VEGF promotes long distance axonal plasticity in the ischemic brain. Herein, we show that VEGF promotes neurological stroke recovery in mice when delivered in a delayed way starting 3 days after middle cerebral artery occlusion. Using anterograde tract-tracing experiments that we combined with histochemical and molecular biological studies, we demonstrate that although VEGF promoted angiogenesis predominantly in the ischemic hemisphere, pronounced axonal sprouting was induced by VEGF in the contralesional, but not the ipsilesional corticobulbar system. Corticobulbar plasticity was accompanied by the deactivation of the matrix metalloproteinase MMP9 in the lesioned hemisphere and the transient downregulation of the axonal growth inhibitors NG2 proteoglycan and brevican and the guidance molecules ephrin B1/2 in the contralesional hemisphere. The regulation of matrix proteinases, growth inhibitors, and guidance molecules offers insights how brain plasticity is controlled in the ischemic brain.

Connective tissue growth factor level is increased in patients with liver cirrhosis but is not associated with complications or extent of liver injury

Connective tissue growth factor (CTGF) is a profibrotic protein whose systemic levels are increased in liver cirrhosis. Here, association of CTGF with stages of liver injury and complications of cirrhotic liver disease has been analyzed in patients with different aetiologies of hepatic injury. CTGF is significantly increased in portal venous serum (PVS), hepatic venous serum (HVS) and systemic venous serum (SVS) of 46 patients with liver cirrhosis compared to eight liver-healthy controls. In patients´ blood samples CTGF in HVS is about 6% higher than PVS levels indicating that CTGF produced in the liver is released to the circulation. CTGF is not associated with stages of liver cirrhosis defined by CHILD-PUGH or MELD score nor with secondary complications of portal hypertension (varices, ascites, spontaneous bacterial peritonitis). Transforming growth factor β (TGFβ) induces CTGF synthesis in hepatocytes and a positive association of systemic TGFβ1 and SVS and HVS CTGF is found. Three months after placing transjugular intrahepatic portosystemic shunt (TIPS) hepatic venous pressure gradient is reduced whereas CHILD-PUGH score, TGFβ1 and CTGF are not altered in serum of 15 patients. Current data show that the cirrhotic liver releases little CTGF but SVS, HVS and PVS CTGF levels are not associated with residual liver function and complications of cirrhosis.

Targeting the insulin-like growth factor-1 receptor in human cancer

The insulin-like growth factor (IGF) signaling system plays a crucial role in human cancer and the IGF-1 receptor (IGF-1R) is an attractive drug target against which a variety of novel anti-tumor agents are being developed. Deregulation of the IGF signaling pathway frequently occurs in human cancer and involves the establishment of autocrine loops comprising IGF-1 or IGF-2 and/or IGF-1R over-expression. Epidemiologic studies have documented a link between elevated IGF levels and the development of solid tumors, such as breast, colon, and prostate cancer. Anti-cancer strategies targeting the IGF signaling system involve two main approaches, namely neutralizing antibodies and small molecule inhibitors of the IGF-1R kinase activity. There are numerous reports describing anti-tumor activity of these agents in pre-clinical models of major human cancers. In addition, multiple clinical trials have started to evaluate the safety and efficacy of selected IGF-1R inhibitors, in combination with standard chemotherapeutic regimens or other targeted agents in cancer patients. In this mini review, I will discuss the role of the IGF signaling system in human cancer and the main strategies which have been so far evaluated to target the IGF-1R.

Vascular endothelial growth factor-A and aldosterone: relevance to normal pregnancy and preeclampsia

Aldosterone levels are markedly elevated during normal pregnancy but fall even though volume contracts when preeclampsia occurs. The level of aldosterone in either condition cannot be explained solely by the activity of the renin-angiotensin II system. In normal gestation, vascular endothelial growth factor (VEGF) is thought to maintain vascular health, but its role in adrenal hormone production is unknown. We hypothesized that the role of VEGF in the adrenal gland is to maintain vascular health and regulate aldosterone production. Here, we demonstrate that supernatant of endothelial cells grown in the presence of VEGF enhanced aldosterone synthase activity in human adrenocortical cells. VEGF either alone or combined with angiotensin II increased aldosterone production in adrenal cells. These data suggest that endothelial cell-dependent and independent activation of aldosterone is regulated by VEGF. In contrast to angiotensin II, VEGF did not upregulate the steroidogenic acute regulatory protein. Consistent with this observation, angiotensin II stimulated both aldosterone and cortisol synthesis from progesterone, whereas VEGF stimulated selectively aldosterone production. In rats, overexpression of soluble fms-like tyrosine kinase-1, an endogenous VEGF inhibitor, led to adrenocortical capillary rarefaction and fall in aldosterone concentrations that correlated inversely with soluble fms-like tyrosine kinase-1 levels. These findings may explain why aldosterone increases so markedly during normal gestation and why preeclampsia, a condition characterized by high soluble fms-like tyrosine kinase-1, is associated with inappropriately low aldosterone levels in spite of relatively lower plasma volumes.

Fgfrl1, a fibroblast growth factor receptor-like gene, is found in the cephalochordate Branchiostoma floridae but not in the urochordate Ciona intestinalis

FGFRL1 is a novel member of the fibroblast growth factor receptor family that controls the formation of musculoskeletal tissues. Some vertebrates, including man, cow, dog, mouse, rat and chicken, possess a single copy the FGFRL1 gene. Teleostean fish have two copies, fgfrl1a and fgfrl1b, because they have undergone a whole genome duplication. Vertebrates belong to the chordates, a phylum that also includes the subphyla of the cephalochordates (e.g. Branchiostoma floridae) and urochordates (tunicates, e.g. Ciona intestinalis). We therefore investigated whether other chordates might also possess an FGFRL1 related gene. In fact, a homologous gene was found in B. floridae (amphioxus). The corresponding protein showed 60% sequence identity with the human protein and all sequence motifs identified in the vertebrate proteins were also conserved in amphioxus Fgfrl1. In contrast, the genome of the urochordate C. intestinalis and those from more distantly related invertebrates including the insect Drosophila melanogaster and the nematode Caenorhabditis elegans did not appear to contain any related sequences. Thus, the FGFRL1 gene might have evolved just before branching of the vertebrate lineage from the other chordates.

Expression of FGFRL1, a novel fibroblast growth factor receptor, during embryonic development

FGFRL1 is a novel member of the fibroblast growth factor receptor (FGFR) family. To investigate its expression during mammalian embryonic development, we have used the mouse system. Expression of Fgfrl1 is very low in mouse embryos of day 6 but steadily increases until birth. As demonstrated by in situ hybridization of 16-day-old embryos, the Fgfrl1 mRNA occurs in cartilaginous structures such as the primordia of bones and the permanent cartilage of the trachea, the ribs and the nose. In addition, some muscle types, including the muscles of the tongue and the diaphragm, express Fgfrl1 at relatively high level. In contrast, the heart and the skeletal muscles of the limbs, as well as many other organs (brain, lung, liver, kidney, gut) express Fgfrl1 only at basal level. It is conceivable that Fgfrl1 interacts with other Fgfrs, which are expressed in cartilage and muscle, to modulate FGF signaling.

The immunomodulator FTY720 and its phosphorylated derivative activate the Smad signalling cascade and upregulate connective tissue growth factor and collagen type IV expression in renal mesangial cells

1.--The immunomodulating agent FTY720 is a substrate for the sphingosine kinase and the phosphorylated form is able to bind to sphingosine 1-phosphate (S1P) receptors. In this study, we show that exposure of renal mesangial cells to phospho-FTY720 leads to a rapid and transient activation of several protein kinase cascades, including the mitogen- and stress-activated protein kinases. The nonphosphorylated FTY720 also increased MAPK phosphorylation, but with a reduced potency and a more delayed time course. In addition, phospho-FTY720 and FTY720 are able to increase phosphorylation of Smad proteins which are classical members of the transforming growth factor-beta (TGF-beta) signalling device, thus suggesting a crosstalk between FTY720 and TGF-beta signalling. 2.--Pretreatment with the S1P(3) receptor antagonist suramin inhibits FTY720 and phospho-FTY720-induced Smad phosphorylation, whereas pertussis toxin pretreatment, which blocks G(i/0) proteins, has no effect on Smad phosphorylation. 3.--Since TGF-beta is a potent profibrotic cytokine in mesangial cells and upregulates the connective tissue growth factor (CTGF) and collagen as important hallmarks in the fibrotic sequelae, we investigated whether FTY720 and phospho-FTY720 are able to mimic these effects of TGF-beta. Indeed, FTY720 and phospho-FTY720 markedly upregulate CTGF and collagen type IV protein expressions. In addition, the tissue inhibitor of metalloproteinase-1 is transcriptionally activated by FTY720, whereas cytokine-induced matrix metalloproteinase-9 is down-regulated by FTY720. 4.--Depletion of the TGF-beta receptor type II by the siRNA transfection technique blocks not only Smad phosphorylation but also CTGF upregulation. Similarly, Smad-4 depletion by siRNA transfection also abrogates CTGF upregulation induced by FTY720 and phospho-FTY720. 5.--In summary, our data show that FTY720 and phospho-FTY720 not only activate the Smad signalling cascade in mesangial cells, but also upregulate the expression of CTGF and collagen. These findings suggest that FTY720 may have additional effects besides the established immunomodulatory action and, importantly, a profibrotic activity has to be considered in future experimental approaches.

Gene transfer of hepatocyte growth factor by electroporation reduces bleomycin-induced lung fibrosis

Abnormal alveolar wound repair contributes to the development of pulmonary fibrosis after lung injury. Hepatocyte growth factor (HGF) is a potent mitogenic factor for alveolar epithelial cells and may therefore improve alveolar epithelial repair in vitro and in vivo. We hypothesized that HGF could increase alveolar epithelial repair in vitro and improve pulmonary fibrosis in vivo. Alveolar wound repair in vitro was determined using an epithelial wound repair model with HGF-transfected A549 alveolar epithelial cells. Electroporation-mediated, nonviral gene transfer of HGF in vivo was performed 7 days after bleomycin-induced lung injury in the rat. Alveolar epithelial repair in vitro was increased after transfection of wounded epithelial monolayers with a plasmid encoding human HGF, pCikhHGF [human HGF (hHGF) gene expressed from the cytomegalovirus (CMV) immediate-early promoter and enhancer] compared with medium control. Electroporation-mediated in vivo HGF gene transfer using pCikhHGF 7 days after intratracheal bleomycin reduced pulmonary fibrosis as assessed by histology and hydroxyproline determination 14 days after bleomycin compared with controls treated with the same vector not containing the HGF sequence (pCik). Lung epithelial cell proliferation was increased and apoptosis reduced in hHGF-treated lungs compared with controls, suggesting increased alveolar epithelial repair in vivo. In addition, profibrotic transforming growth factor-beta1 (TGF-beta1) was decreased in hHGF-treated lungs, indicating an involvement of TGF-beta1 in hHGF-induced reduction of lung fibrosis. In conclusion, electroporation-mediated gene transfer of hHGF decreases bleomycin-induced pulmonary fibrosis, possibly by increasing alveolar epithelial cell proliferation and reducing apoptosis, resulting in improved alveolar wound repair.

Effects of local continuous release of brain derived neurotrophic factor (BDNF) on peripheral nerve regeneration in a rat model

The purpose of this study was to evaluate the effect of continuously released BDNF on peripheral nerve regeneration in a rat model. Initial in vitro evaluation of calcium alginate prolonged-release-capsules (PRC) proved a consistent release of BDNF for a minimum of 8 weeks. In vivo, a worst case scenario was created by surgical removal of a 20-mm section of the sciatic nerve of the rat. Twenty-four autologous fascia tubes were filled with calcium alginate spheres and sutured to the epineurium of both nerve ends. The animals were divided into 3 groups. In group 1, the fascial tube contained plain calcium alginate spheres. In groups 2 and 3, the fascial tube contained calcium alginate spheres with BDNF alone or BDNF stabilized with bovine serum albumin, respectively. The autocannibalization of the operated extremity was clinically assessed and documented in 12 additional rats. The regeneration was evaluated histologically at 4 weeks and 10 weeks in a blinded manner. The length of nerve fibers and the numbers of axons formed in the tube was measured. Over a 10-week period, axons have grown significantly faster in groups 2 and 3 with continuously released BDNF compared to the control. The rats treated with BDNF (groups 2 and 3) demonstrated significantly less autocannibalization than the control group (group 1). These results suggest that BDNF may not only stimulate faster peripheral nerve regeneration provided there is an ideal, biodegradable continuous delivery system but that it significantly reduces the neuropathic pain in the rat model.

An in vivo study of a growth-factor enhanced, cell free, two-layered collagen-tricalcium phosphate in deep osteochondral defects

Focal osteochondral defects are still a challenging problem in joint surgery. We have developed a two-layered implant consisting of a basal porous beta-tricalcium phosphate (TCP) for bone reconstruction and a superficial fibrous collagen type I/III layer for cartilage regeneration. Fifty-four osteochondral defects in the trochlear groove of 27 Göttinger Minipigs were created and either left untreated, treated with the implant alone, or the implant augmented with an additional growth factor mixture, which was assumed to stimulate cell and tissue differentiation. Follow-up was 6, 12 and 52 weeks with n=6 for each group. The repair tissue was evaluated for its gross appearance and biomechanical properties. Histological sections were semi-quantitatively scored for their histomorphological structure. Treatment with the two-layered implant improved defect filling and subchondral bone repair at 6 and 12 weeks follow-up. The TCP was replaced by cancellous bone at 52 weeks. Cartilage repair tissue mainly consisted of fibrocartilage and showed a moderate cell density up to the joint surface. Growth factor treatment improved the mechanical and histomorphological properties of the cartilage repair tissue at 12, but not at 52 weeks postoperatively. In conclusion, the two-layered collagen-TCP implant augmented with chondroinductive growth factors seems a promising new option for the treatment of deep osteochondral defects in joint surgery.