Transforming growth factor-beta inhibits the expression of clock genes

Disturbances of sleep-wake rhythms are an important problem in Alzheimer's disease (AD). Circadian rhythms are regulated by clock genes. Transforming growth factor-beta (TGF-β) is overexpressed in neurons in AD and is the only cytokine that is increased in cerebrospinal fluid (CSF). Our data show that TGF-β2 inhibits the expression of the clock genes Period (Per)1, Per2, and Rev-erbα, and of the clock-controlled genes D-site albumin promoter binding protein (Dbp) and thyrotroph embryonic factor (Tef). However, our results showed that TGF-β2 did not alter the expression of brain and muscle Arnt-like protein-1 (Bmal1). The concentrations of TGF-β2 in the CSF of 2 of 16 AD patients and of 1 of 7 patients with mild cognitive impairment were in the dose range required to suppress the expression of clock genes. TGF-β2-induced dysregulation of clock genes may alter neuronal pathways, which may be causally related to abnormal sleep-wake rhythms in AD patients.

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.

First-trimester serum levels of soluble endoglin and soluble fms-like tyrosine kinase-1 as first-trimester markers for late-onset preeclampsia

OBJECTIVE: The aim of this investigation was to assess soluble endoglin (sEng) and soluble fms-like tyrosine kinase-1 (sFlt1) as first-trimester serum markers to predict preeclampsia. STUDY DESIGN: First-trimester sera were obtained from 46 women with subsequent late-onset preeclampsia and from 92 controls. sEng and sFlt1 concentrations were determined immunoanalytically. Correlation analysis with inhibin A and placental growth factor levels was performed. RESULTS: sEng and sFlt1 serum concentrations were higher in women with subsequent preeclampsia than in controls (mean +/- SD, sEng: 5.57 +/- 1.18 ng/mL vs 5.02 +/- 1.01 ng/mL, P = .009; sFlt1: 1764 +/- 757 pg/mL vs 1537 +/- 812 pg/mL, P = .036). Sensitivities and specificities for predicting preeclampsia were 63% and 57% for sEng and 64% and 56% for sFlt1, respectively. When sEng and inhibin A were combined, the sensitivity increased to 68%, whereas the specificity was 61%. CONCLUSION: sEng and sFlt1 are increased in the first trimester in women with subsequent late-onset preeclampsia and might therefore prove useful to predict preeclampsia.

Stem cell-related "self-renewal" signature and high epidermal growth factor receptor expression associated with resistance to concomitant chemoradiotherapy in glioblastoma

PURPOSE: Glioblastomas are notorious for resistance to therapy, which has been attributed to DNA-repair proficiency, a multitude of deregulated molecular pathways, and, more recently, to the particular biologic behavior of tumor stem-like cells. Here, we aimed to identify molecular profiles specific for treatment resistance to the current standard of care of concomitant chemoradiotherapy with the alkylating agent temozolomide. PATIENTS AND METHODS: Gene expression profiles of 80 glioblastomas were interrogated for associations with resistance to therapy. Patients were treated within clinical trials testing the addition of concomitant and adjuvant temozolomide to radiotherapy. RESULTS: An expression signature dominated by HOX genes, which comprises Prominin-1 (CD133), emerged as a predictor for poor survival in patients treated with concomitant chemoradiotherapy (n = 42; hazard ratio = 2.69; 95% CI, 1.38 to 5.26; P = .004). This association could be validated in an independent data set. Provocatively, the HOX cluster was reminiscent of a "self-renewal" signature (P = .008; Gene Set Enrichment Analysis) recently characterized in a mouse leukemia model. The HOX signature and EGFR expression were independent prognostic factors in multivariate analysis, adjusted for the O-6-methylguanine-DNA methyltransferase (MGMT) methylation status, a known predictive factor for benefit from temozolomide, and age. Better outcome was associated with gene clusters characterizing features of tumor-host interaction including tumor vascularization and cell adhesion, and innate immune response. CONCLUSION: This study provides first clinical evidence for the implication of a "glioma stem cell" or "self-renewal" phenotype in treatment resistance of glioblastoma. Biologic mechanisms identified here to be relevant for resistance will guide future targeted therapies and respective marker development for individualized treatment and patient selection.

Transforming growth factor-beta2 upregulates sphingosine kinase-1 activity, which in turn attenuates the fibrotic response to TGF-beta2 by impeding CTGF expression

Transforming growth factor-beta2 (TGF-beta2) stimulates the expression of pro-fibrotic connective tissue growth factor (CTGF) during the course of renal disease. Because sphingosine kinase-1 (SK-1) activity is also upregulated by TGF-beta, we studied its effect on CTGF expression and on the development of renal fibrosis. When TGF-beta2 was added to an immortalized human podocyte cell line we found that it activated the promoter of SK-1, resulting in upregulation of its mRNA and protein expression. Further, depletion of SK-1 by small interfering RNA or its pharmacological inhibition led to accelerated CTGF expression in the podocytes. Over-expression of SK-1 reduced CTGF induction, an effect mediated by intracellular sphingosine-1-phosphate. In vivo, SK-1 expression was also increased in the podocytes of kidney sections of patients with diabetic nephropathy when compared to normal sections of kidney obtained from patients with renal cancer. Similarly, in a mouse model of streptozotocin-induced diabetic nephropathy, SK-1 and CTGF were upregulated in podocytes. In SK-1 deficient mice, exacerbation of disease was detected by increased albuminuria and CTGF expression when compared to wild-type mice. Thus, SK-1 activity has a protective role in the fibrotic process and its deletion or inhibition aggravates fibrotic disease.

Engineered fibrin matrices for functional display of cell membrane-bound growth factor-like activities: study of angiogenic signaling by ephrin-B2

With the rapid increase in approaches to pro- or anti-angiogenic therapy, new and effective methodologies for administration of cell-bound growth factors will be required. We sought to develop the natural hydrogel matrix fibrin as platform for extensive interactions and continuous signaling by the vascular morphogen ephrin-B2 that normally resides in the plasma membrane and requires multivalent presentation for ligation and activation of Eph receptors on apposing endothelial cell surfaces. Using fibrin and protein engineering technology to induce multivalent ligand presentation, a recombinant mutant ephrin-B2 receptor binding domain was covalently coupled to fibrin networks at variably high densities. The ability of fibrin-bound ephrin-B2 to act as ligand for endothelial cells was preserved, as demonstrated by a concomitant, dose-dependent increase of endothelial cell binding to engineered ephrin-B2-fibrin substrates in vitro. The therapeutic relevance of ephrin-B2-fibrin implant matrices was demonstrated by a local angiogenic response in the chick embryo chorioallontoic membrane evoked by the local and prolonged presentation of matrix-bound ephrin-B2 to tissue adjacing the implant. This new knowledge on biomimetic fibrin vehicles for precise local delivery of membrane-bound growth factor signals may help to elucidate specific biological growth factor function, and serve as starting point for development of new treatment strategies.

Paramecium tetraurelia chromatin assembly factor-1-like protein PtCAF-1 is involved in RNA-mediated control of DNA elimination

Genome-wide DNA remodelling in the ciliate Paramecium is ensured by RNA-mediated trans-nuclear crosstalk between the germline and the somatic genomes during sexual development. The rearrangements include elimination of transposable elements, minisatellites and tens of thousands non-coding elements called internally eliminated sequences (IESs). The trans-nuclear genome comparison process employs a distinct class of germline small RNAs (scnRNAs) that are compared against the parental somatic genome to select the germline-specific subset of scnRNAs that subsequently target DNA elimination in the progeny genome. Only a handful of proteins involved in this process have been identified so far and the mechanism of DNA targeting is unknown. Here we describe chromatin assembly factor-1-like protein (PtCAF-1), which we show is required for the survival of sexual progeny and localizes first in the parental and later in the newly developing macronucleus. Gene silencing shows that PtCAF-1 is required for the elimination of transposable elements and a subset of IESs. PTCAF-1 depletion also impairs the selection of germline-specific scnRNAs during development. We identify specific histone modifications appearing during Paramecium development which are strongly reduced in PTCAF-1 depleted cells. Our results demonstrate the importance of PtCAF-1 for the epigenetic trans-nuclear cross-talk mechanism.

Transforming growth factor β2 (TGF-β2)-induced connective tissue growth factor (CTGF) expression requires sphingosine 1-phosphate receptor 5 (S1P5) in human mesangial cells

Transforming growth factor β2 (TGF-β2) is well known to stimulate the expression of pro-fibrotic connective tissue growth factor (CTGF) in several cell types including human mesangial cells. The present study demonstrates that TGF-β2 enhances sphingosine 1-phosphate receptor 5 (S1P5) mRNA and protein expression in a time and concentration dependent manner. Pharmacological and siRNA approaches reveal that this upregulation is mediated via activation of classical TGF-β downstream effectors, Smad and mitogen-activated protein kinases. Most notably, inhibition of Gi with pertussis toxin and downregulation of S1P5 by siRNA block TGF-β2-stimulated upregulation of CTGF, demonstrating that Gi coupled S1P5 is necessary for TGF-β2-triggered expression of CTGF in human mesangial cells. Overall, these findings indicate that TGF-β2 dependent upregulation of S1P5 is required for the induction of pro-fibrotic CTGF by TGF-β. Targeting S1P5 might be an attractive novel approach to treat renal fibrotic diseases.

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

Erratum to: Acta Neuropathol (2012) 123:273–284. DOI 10.1007/s00401‑011‑0914‑z. The authors would like to correct Fig. 3 of the original manuscript, since the image in Fig. 3b does not correspond to a VEGF treated animal. Corrected Fig. 3 is shown below. We apologize for this mistake.

Invited review: Growth-promoting effects of colostrum in calves based on interaction with intestinal cell surface receptors and receptor-like transporters

The postnatal development and maturation of the gastrointestinal (GI) tract of neonatal calves is crucial for their survival. Major morphological and functional changes in the calf's GI tract initiated by colostrum bioactive substances promote the establishment of intestinal digestion and absorption of food. It is generally accepted that colostrum intake provokes the maturation of organs and systems in young calves, illustrating the significance of the cow-to-calf connection at birth. These postnatal adaptive changes of the GI tissues in neonatal calves are especially induced by the action of bioactive substances such as insulin-like growth factors, hormones, or cholesterol carriers abundantly present in colostrum. These substances interact with specific cell-surface receptors or receptor-like transporters expressed in the GI wall of neonatal calves to elicit their biological effects. Therefore, the abundance and activity of cell surface receptors and receptor-like transporters binding colostral bioactive substances are a key aspect determining the effects of the cow-to-calf connection at birth. The present review compiles the information describing the effects of colostrum feeding on selected serum metabolic and endocrine traits in neonatal calves. In this context, the current paper discusses specifically the consequences of colostrum feeding on the GI expression and activity of cell-receptors and receptor-like transporters binding growth hormone, insulin-like growth factors, insulin, or cholesterol acceptors in neonatal calves.

A growth factor-induced, spatially organizing cytoskeletal module enables rapid and persistent fibroblast migration

Directional migration requires robust front/back polarity. We find that fibroblasts treated with platelet-derived growth factor (PDGF) and prepolarized by plating on a fibronectin line substrate exhibit persistent migration for hours. This does not occur in the absence of PDGF or on uniformly coated fibronectin substrates. Persistent migration arises from establishment of two functional modules at cell front and back. At the front, formation of a zone containing podosome-like structures (PLS) dynamically correlates with low RhoA and myosin activity and absence of a contractile lamella. At the back, myosin contractility specifically controls tail retraction with minimal crosstalk to the front module. The PLS zone is maintained in a dynamic steady state that preserves size and position relative to the cell front, allowing for long-term coordination of front and back modules. We propose that front/back uncoupling achieved by the PLS zone is crucial for persistent migration in the absence of directional cues.

Effect of fibroblast growth factor NV1FGF on amputation and death: a randomised placebo-controlled trial of gene therapy in critical limb ischaemia

Patients with critical limb ischaemia have a high rate of amputation and mortality. We tested the hypothesis that non-viral 1 fibroblast growth factor (NV1FGF) would improve amputation-free survival.

Prolonged amelioration of acute lung allograft rejection by sequential overexpression of human interleukin-10 and hepatocyte growth factor in rats

The effect of prolonged electroporation-mediated human interleukin-10 (hIL-10) overexpression 24 hours before transplantation, combined with sequential human hepatocyte growth factor (HGF) overexpression into skeletal muscle on day 5, on rat lung allograft rejection was evaluated. Left lung allotransplantation was performed from Brown-Norway to Fischer-F344 rats. Gene transfer into skeletal muscle was enhanced by electroporation. Three groups were studied: group I animals (n = 5) received 2.5 μg pCIK-hIL-10 (hIL-10/CMV [cytomegalovirus] early promoter enhancer) on day -1 and 80 μg pCIK-HGF (HGF/CMV early promoter enhancer) on day 5. Group II animals (n = 4) received 2.5 μg pCIK-hIL-10 and pUbC-hIL-10 (hIL-10/pUbC promoter) on day -1. Control group III animals (n = 4) were treated by sham electroporation on days -1 and 5. All animals received daily nontherapeutic intraperitoneal dose of cyclosporin A (2.5 mg/kg) and were sacrificed on day 15. Graft oxygenation and allograft rejection were evaluated. Significant differences were found between study groups in graft oxygenation (Pao(2)) (P = .0028; group I vs. groups II and III, P < .01 each). Pao(2) was low in group II (31 ± 1 mm Hg) and in group III controls (34 ± 10 mm Hg), without statistically significant difference between these 2 groups (P = .54). In contrast, in group I, Pao(2) of recipients sequentially transduced with IL-10 and HGF plasmids was much improved, with 112 ± 39 mm Hg (vs. groups II and III; P < .01 each), paralleled by reduced vascular and bronchial rejection (group I vs. groups II and III, P < .021 each). Sequential overexpression of anti-inflammatory cytokine IL-10, followed by sequential and overlapping HGF overexpression on day 5, preserves lung function and reduces acute lung allograft rejection up to day 15 post transplant as compared to prolonged IL-10 overexpression alone.

Differential effects of dexamethasone on the chondrogenesis of mesenchymal stromal cells: influence of microenvironment, tissue origin and growth factor

Mesenchymal stromal cells (MSCs), which reside within various tissues, are utilized in the engineering of cartilage tissue. Dexamethasone (DEX)--a synthetic glucocorticoid--is almost invariably applied to potentiate the growth-factor-induced chondrogenesis of MSCs in vitro, albeit that this effect has been experimentally demonstrated only for transforming-growth-factor-beta (TGF-β)-stimulated bone-marrow-derived MSCs. Clinically, systemic glucocorticoid therapy is associated with untoward side effects (e.g., bone loss and increased susceptibility to infection). Hence, the use of these agents should be avoided or limited. We hypothesize that the influence of DEX on the chondrogenesis of MSCs depends upon their tissue origin and microenvironment [absence or presence of an extracellular matrix (ECM)], as well as upon the nature of the growth factor. We investigated its effects upon the TGF-β1- and bone-morphogenetic-protein 2 (BMP-2)-induced chondrogenesis of MSCs as a function of tissue source (bone marrow vs. synovium) and microenvironment [cell aggregates (no ECM) vs. explants (presence of a natural ECM)]. In aggregates of bone-marrow-derived MSCs, DEX enhanced TGF-β1-induced chondrogenesis by an up-regulation of cartilaginous genes, but had little influence on the BMP-2-induced response. In aggregates of synovial MSCs, DEX exerted no remarkable effect on either TGF-β1- or BMP-2-induced chondrogenesis. In synovial explants, DEX inhibited BMP-2-induced chondrogenesis almost completely, but had little impact on the TGF-β1-induced response. Our data reveal that steroids are not indispensable for the chondrogenesis of MSCs in vitro. Their influence is context dependent (tissue source of the MSCs, their microenvironment and the nature of the growth-factor). This finding has important implications for MSC based approaches to cartilage repair.