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.

Apoptotic neutrophils release macrophage migration inhibitory factor upon stimulation with tumor necrosis factor-alpha

Macrophage migration inhibitory factor (MIF) is an important cytokine involved in the regulation of innate immunity and present at increased levels during inflammatory responses. Here we demonstrate that mature blood and tissue neutrophils constitutively express MIF as a cytosolic protein not associated with azurophil granules. Functionally active MIF, but not proteases stored in azurophil granules, was released from apoptotic neutrophils following short term tumor necrosis factor (TNF)-alpha stimulation in a caspase-dependent manner and prior to any detectable phagocytosis by monocyte-derived macrophages. Moreover, TNF-alpha-mediated MIF release was blocked by glyburide and propenicide, both inhibitors of ATP-binding cassette-type transporters, suggesting that this transporter system is activated during neutrophil apoptosis. Taken together, apoptotic mature neutrophils release MIF upon short term TNF-alpha stimulation. Therefore, apoptosis may not always occur without the induction of pro-inflammatory mechanisms.

The -308 tumour necrosis factor-alpha gene polymorphism predicts therapeutic response to TNFalpha-blockers in rheumatoid arthritis and spondyloarthritis patients

OBJECTIVE: To examine whether the G-to-A polymorphism at position -308 in the promoter of the tumour necrosis factor-alpha (TNFalpha) gene influences the therapeutic response to TNFalpha-blockers in patients with rheumatoid arthritis (RA), psoriatic arthritis (PsA) and ankylosing spondylitis (AS). METHODS: A total of 54 patients with RA, 10 with PsA and 22 with AS were genotyped by polymerase chain reaction for the -308 TNFalpha promoter polymorphism. They were treated with infliximab (n = 63), adalimumab (n = 10) or etanercept (n = 13). Clinical response was assessed after 24 weeks by the Disease Activity Score in 28 joints (DAS28) for RA and PsA, and the Bath Ankylosing Spondylitis Activity Index (BASDAI) for AS patients. RESULTS: All patients with the A/A genotype (n = 3, all RA) and two patients with the A/G genotype (AS) failed to respond to anti-TNF treatment. Irrespective of the underlying disease, moderate response (n = 44) was predominantly associated with the A/G genotype (A/G 18/22, G/G 4/22), whereas good response (n = 59) was exclusively seen in patients with the G/G genotype. The average improvement in the DAS28 score was 0.83 in the A/A, 1.50 in the A/G and 2.64 in the G/G group of RA and PsA patients (P < 0.0001). The BASDAI score in AS improved on average by 1.21 in the A/G and by 3.30 in the G/G group (P < 0.005). CONCLUSIONS: The data suggest that humans with a TNFalpha -308 G/G genotype are better responders to anti-TNFalpha treatment than those with A/A or A/G genotypes independent of the treated rheumatic disease (RA, PsA or AS).

Effects of probiotic bacteria in dogs with food responsive diarrhoea treated with an elimination diet

We evaluated whether a probiotic supplementation in dogs with food responsive diarrhoea (FRD) has beneficial effects on intestinal cytokine patterns and on microbiota. Twenty-one client-owned dogs with FRD were presented for clinically needed duodeno- and colonoscopy and were enrolled in a prospective placebo (PL)-controlled probiotic trial. Intestinal tissue samples and faeces were collected during endoscopy. Intestinal mRNA abundance of interleukin (IL)-5, -10, -12p40 and -13, tumour necrosis factor-alpha, transforming growth factor-beta1 and interferon (IFN)-gamma were analysed and numbers of Lactobacillus spp., Bifidobacterium spp., Enterococcus spp. and Enterobacteriaceae and supplemented probiotic bacteria were determined in faeces. The Canine Inflammatory Bowel Disease Activity Index, a scoring system comprising general attitude, appetite, faecal consistency, defecation frequency, and vomitus, decreased in all dogs (p < 0.0001). Duodenal IL-10 mRNA levels decreased (p = 0.1) and colonic IFN-gamma mRNA levels increased (p = 0.08) after probiotic treatment. Numbers of Enterobacteriaceae decreased in FRD dogs receiving probiotic cocktail (FRD(PC)) and FRD dogs fed PL (FRD(PL)) during treatment (p < 0.05), numbers of Lactobacillus spp. increased in FRD(PC after) when compared with FRD(PC before) (p < 0.1). One strain of PC was detected in five of eight FRD(PC) dogs after probiotic supplementation. In conclusion, all dogs clinically improved after treatment, but cytokine patterns were not associated with the clinical features irrespective of the dietary supplementation.

Differential regulation of metzincins in experimental chronic renal allograft rejection: potential markers and novel therapeutic targets

Chronic renal allograft rejection is characterized by alterations in the extracellular matrix compartment and in the proliferation of various cell types. These features are controlled, in part by the metzincin superfamily of metallo-endopeptidases, including matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase (ADAM) and meprin. Therefore, we investigated the regulation of metzincins in the established Fisher to Lewis rat kidney transplant model. Studies were performed using frozen homogenates and paraffin sections of rat kidneys at day 0 (healthy controls) and during periods of chronic rejection at day +60 and day +100 following transplantation. The messenger RNA (mRNA) expression was examined by Affymetrix Rat Expression Array 230A GeneChip and by real-time Taqman polymerase chain reaction analyses. Protein expression was studied by zymography, Western blot analyses, and immunohistology. mRNA levels of MMPs (MMP-2/-11/-12/-14), of their inhibitors (tissue inhibitors of metalloproteinase (TIMP)-1/-2), ADAM-17 and transforming growth factor (TGF)-beta1 significantly increased during chronic renal allograft rejection. MMP-2 activity and immunohistological staining were augmented accordingly. The most important mRNA elevation was observed in the case of MMP-12. As expected, Western blot analyses also demonstrated increased production of MMP-12, MMP-14, and TIMP-2 (in the latter two cases as individual proteins and as complexes). In contrast, mRNA levels of MMP-9/-24 and meprin alpha/beta had decreased. Accordingly, MMP-9 protein levels and meprin alpha/beta synthesis and activity were downregulated significantly. Members of metzincin families (MMP, ADAM, and meprin) and of TIMPs are differentially regulated in chronic renal allograft rejection. Thus, an altered pattern of metzincins may represent novel diagnostic markers and possibly may provide novel targets for future therapeutic interventions.

Tumor necrosis factor-alpha: alternative role as an inhibitor of osteoclast formation in vitro

TNFalpha is known to stimulate the development and activity of osteoclasts and of bone resorption. The cytokine was found to mediate bone loss in conjunction with inflammatory diseases such as rheumatoid arthritis or chronic aseptic inflammation induced by wear particles from implants and was suggested to be a prerequisite for the loss of bone mass under estrogen deficiency. In the present study, the regulation of osteoclastogenesis by TNFalpha was investigated in co-cultures of osteoblasts and bone marrow or spleen cells and in cultures of bone marrow and spleen cells grown with CSF-1 and RANKL. Low concentrations of TNFalpha (1 ng/ml) caused a >90% decrease in the number of osteoclasts in co-cultures, but did not affect the development of osteoclasts from bone marrow cells. In cultures with p55TNFR(-/-) osteoblasts and wt BMC, the inhibitory effect was abrogated and TNFalpha induced an increase in the number of osteoclasts in a dose-dependent manner. Osteoblasts were found to release the inhibitory factor(s) into the culture supernatant after simultaneous treatment with 1,25(OH)(2)D(3) and TNFalpha, this activity, but not its release, being resistant to treatment with anti-TNFalpha antibodies. Dexamethasone blocked the secretion of the TNFalpha-dependent inhibitor by osteoblasts, while stimulating the development of osteoclasts. The data suggest that the effects of TNFalpha on the differentiation of osteoclast lineage cells and on bone metabolism may be more complex than hitherto assumed and that these effects may play a role in vivo during therapies for inflammatory diseases.

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.

Effects of Delta12-prostaglandin J2 on bone regeneration and growth factor expression in rats

OBJECTIVES: Cyclopentenone prostaglandins have been shown to promote osteoblast differentiation in vitro. The aim of this study was to examine in a rat model the effects of local delivery of Delta(12)-prostaglandin J(2) (Delta(12)-PGJ(2)) on new bone formation and growth factor expression in (i) cortical defects and (ii) around titanium implants. MATERIAL AND METHODS: Standardized transcortical defects were prepared bilaterally in the femur of 28 male Wistar rats. Ten microliters of Delta(12)-PGJ(2) at 4 concentrations (10(-9), 10(-7), 10(-5) and 10(-3) mol/l) in a collagen vehicle were delivered inside a half-cylindrical titanium chamber fixed over the defect. Contralateral defects served as vehicle controls. Ten days after surgery, the amount of new bone formation in the cortical defect area was determined by histomorphometry and expression of platelet-derived growth factor (PDGF)-A and -B, insulin-like growth factor (IGF)-I/II, bone morphogenetic protein (BMP)-2 and -6 was examined by immunohistochemistry. In an additional six rats, 24 titanium implants were inserted into the femur. Five microliters of carboxymethylcellulose alone (control) or with Delta(12)-PGJ(2) (10(-5) and 10(-3) mol/l) were delivered into surgically prepared beds prior to implant installation. RESULTS: Delta(12)-PGJ(2) (10(-5) and 10(-3) mol/l) significantly enhanced new bone formation (33%, P<0.05) compared with control cortical defects. Delivery of Delta(12)-PGJ(2) at 10(-3) mol/l significantly increased PDGF-A and -B and BMP-2 and -6 protein expression (P<0.05) compared with control defects. No significant difference was found in IGF-I/II expression compared with controls. Administration of Delta(12)-PGJ(2) also significantly increased endosteal new bone formation around implants compared with controls. CONCLUSION: Local delivery of Delta(12)-PGJ(2) promoted new bone formation in the cortical defect area and around titanium implants. Enhanced expression of BMP-2 and -6 as well as PDGF-A and -B may be involved in Delta(12)-PGJ(2)-induced new bone formation.

Fluorescent in situ hybridization mapping of the epidermal growth factor receptor gene in donkey

The physical localization of the epidermal growth factor receptor (EGFR) gene was performed on donkey chromosomes. Bacterial artificial chromosome DNA containing the equine EGFR gene was used to map this gene by fluorescent in situ hybridization on donkey metaphase chromosomes. The gene was mapped on donkey 1q21.1 region.

Angiopoietin-1 and angiopoietin-2 share the same binding domains in the Tie-2 receptor involving the first Ig-like loop and the epidermal growth factor-like repeats

Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) have been identified as ligands with different effector functions of the vascular assembly and maturation-mediating receptor tyrosine kinase Tie-2. To understand the molecular interactions of the angiopoietins with their receptor, we have studied the binding of Ang-1 and Ang-2 to the Tie-2 receptor. Enzyme-linked immunosorbent assay-based competition assays and co-immunoprecipitation experiments analyzing the binding of Ang-1 and Ang-2 to truncation mutants of the extracellular domain of Tie-2 showed that the first Ig-like loop of Tie-2 in combination with the epidermal growth factor (EGF)-like repeats (amino acids 1-360) is required for angiopoietin binding. The first Ig-like domain or the EGF-like repeats alone are not capable of binding Ang-1 and Ang-2. Concomitantly, we made the surprising finding that Tie-2 exon-2 knockout mice do express a mutated Tie-2 protein that lacks 104 amino acids of the first Ig-like domain. This mutant Tie-2 receptor is functionally inactive as shown by the lack of ligand binding and receptor phosphorylation. Collectively, the data show that the first 104 amino acids of the Tie-2 receptor are essential but not sufficient for angiopoietin binding. Conversely, the first 360 amino acids (Ig-like domain plus EGF-like repeats) of the Tie-2 receptor are necessary and sufficient to bind both Ang-1 and Ang-2, which suggests that differential receptor binding is not likely to be responsible for the different functions of Ang-1 and Ang-2.

Epidermal growth factor receptor inhibitors plus chemotherapy in non-small-cell lung cancer: biologic rationale for combination strategies

Chemotherapy continues to play an essential role in the treatment of most stages of non-small-cell lung cancer (NSCLC). In fact, within the past 5 years, this role has greatly expanded into adjuvant therapy for early-stage resected disease. Likewise, agents targeting the epidermal growth factor receptor (EGFR), particularly the tyrosine kinase inhibitors gefitinib and erlotinib, have proven to be clinically active in patients with advanced-stage NSCLC. Because of these findings, it is logical to expect that combinations of these 2 classes of antineoplastic agents would prove more efficacious than either one alone. Yet 4 large randomized phase III trials of chemotherapy with or without an EGFR tyrosine kinase inhibitor in unselected patients with advanced-stage NSCLC, altogether totaling > 4000 patients, did not demonstrate improvement in clinical outcomes with the combination. Whether these negative results will be reproduced in ongoing combination studies of chemotherapy plus monoclonal antibodies directed against EGFR remain to be determined. Herein, we review recent preclinical and clinical data addressing this topic and explore the biologic rationale for developing new combination strategies based on patient selection by molecular and clinical factors, or by pharmacodynamic parameters.