In vitro analysis with human bone marrow stem cells on Ti-15Mo alloy for dental and orthopedic implants application

Aim: Nowadays, research on orthopedic and dental implants is focused on titanium alloys for their mechanical properties and corrosion resistance in the human body environment. Another important aspect to be investigated is their surface topography, which is very important to osseointegration. With laser beam irradiation for roughening the implants surface an easier control of the microtopography is achieved, and surface contamination is avoided. The aim of this study was to assess human bone marrow stem cells response to a newly developed titanium alloy, Ti-15Mo, with surface topography modified by laser beam irradiation. Materials and methods: A total of 10 Ti machined disks (control), 10 Ti-15Mo machined disks and 10 Ti-15Mo disks treated by laser beam-irradiation were prepared. To study how Ti-15Mo surface topografy can induce osteoblast differentiation in mesenchymal stem cells, the expression levels of bone related genes and mesenchymal stem cells marker were analyzed, using real time Reverse Transcription-Polymerase Chain Reaction. Results: In Test 1 (comparison between Ti-15Mo machined disks and Ti-machined disks) quantitative real-time RT-PCR showed a significant induction of ALPL, FOSL1 and SPP1, which increase 20% or more. In Test 2 (comparison between Ti-15Mo laser treated disks and Ti-machined disks) all investigated genes were up-regulated. By comparing Test 1 and Test 2 it was detected that COL1A1, COL3A1, FOSL1 and ENG sensibly increased their expression whereas RUNX2, ALPL and SPP1 expression remained substantially unchanged. Conclusion: The present study demonstrated that laser treated Ti-15Mo alloys are promising materials for implants application.

Caracterização de mutações no gene MED12, dos miRNAS mapeados em 7q22 e dos candidatos a regulação do gene HMGA2 em leiomiomas uterinos

Pós-graduação em Ciências Biológicas (Genética) - IBB

Expressão de citocinas na glândula mamária de cadelas e sua relação com a malignidade dos tumores mamários

Pós-graduação em Medicina Veterinária - FCAV

Low expression of sodium iodide symporter identifies aggressive thyroid tumors

A decreased radioiodine uptake is frequently detected in differentiated thyroid carcinomas (DTC) and is associated with high recurrence rate and reduced survival. We investigated the correlation between NIS mRNA expression levels in the primary tumor and patient outcome using a quantitative real-time RT-PCR method. NIS expression was decreased in 17 DTC (21.04 +/- 39.66 pg Eq) compared to four autoimune thyroid disease (180.51 +/- 92.63 pg Eq) and 14 normal tissues (75.71 +/- 66.98 pg Eq) (p < 0.0001). The 17 thyroid differentiated carcinoma patients were submitted to surgery complemented by radioiodine ablation and had at least 24 months of follow-up, under levothyroxine continued suppressive therapy. According to their outcome, we could characterize a group of papillary carcinoma patients with aggressive carcinomas, whose NIS mRNA levels were markedly lower than a group with non-aggressive carcinomas (0.62 +/- 0.79 versus 54.87 +/- 53.79: p < 0.005). We suggest that the quantification of NIS mRNA relative levels in the primary tumor may predict poor outcome. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

Identificação de genes candidatos ao desenvolvimento de Leiomiomas Uterinos

Uterine leiomyomas (UL) are benign smooth muscle tumors from myometrium, representing the major indication for hysterectomies and a significant public health problem. Several genetics alterations have been associated with its development and pathogenesis. Although the initial factors that lead to the development of uterine leiomyomas are unclear, there are several evidences showing that ovarian steroids, estrogen and progesterone, are associated with these tumors.Recent reports, however, show others genes related with distinct signalization pathways besides sex hormones, which may be or not activated by these hormones, are associated with uterine leiomyomas development. In this study, the AHR, ESR1, ESR2, PGR and WNT7A gene transcripts expression was evaluated using quantitative real time RT-PCR in 58 UL samples compared to five normal myometrium tissues to explore the hormonal molecular basis of these tumors and associate these expression pattern with clinical and pathological features. The present study showed AHR, ESR1, ESR2, PGR and WNT7A down expression in 60%, 43%, 52%, 60% e 40% of the tumors, respectively, and a significant AHR loss of expression compared with the controls samples (P=0.0130). The comparison between the genes expression values revealed a positive correlation between the AHR and ESR1 transcripts (P<0.0001; r=0.6911) and between the genes ESR1 and WNT7A (P<0.0001; r=0.5655). The expression pattern was compared with clinical and pathological features. The WNT7A gene presented a differential expression pattern between the proliferative and secretory phase of menstrual cycle (P=0.0373) and the AHR and ESR1 genes were differentially expressed between women in reproductive and menopause years (P=0.0267; P=0.0415, respectively). The ESR1 and WNT7A down regulation was statistically ...(Complete abstract click electronic access below)

Tumor microenvironmental genomic alterations in juvenile nasopharyngeal angiofibroma

Background To better characterize the pathophysiology of juvenile nasopharyngeal angiofibroma (JNA), endothelial and stromal cells were evaluated by genomic imbalances in association with transcript expression levels of genes mapped on these altered regions. Methods. High-resolution comparative genomic hybridization (HR-CGH) was used in laser-captured endothelial and stromal cells from 9 JNAs. Ten genes were evaluated by quantitative real-timereverse transcription polymerase chain reaction (qRT-PCR) in 15 cases. Results. Although gains were more frequently detected in endothelial cells, 57% of chromosomal alterations were common by both components. Gene expression analyses revealed a positive correlation between endothelial and stromal components for ASPM, CDH1, CTNNB1, FGF18, and SUPT16H. A significant difference was found for FGF18 and AURKB overexpression in stromal cells and AR down-expression in endothelial cells. Conclusions. A similar pattern of gene expression and chromosomal imbalances in both exponents would suggest a common mechanism of functional regulation. AURKB, FGF18, and SUPT16H were identified as potential molecular markers in JNA. (C) 2011 Wiley Periodicals, Inc. Head Neck 34: 485-492, 2012

Die Leber als Latenzort des murinen Cytomegalovirus: Identifizierung und Charakterisierung des latent infizierten Zelltyps

Die Untersuchungen der murinen Cytomegalovirus (mCMV) Infektion im BALB/c Mausmodell konzentrierten sich bislang auf die Lunge, da diese einen Hauptort der mCMV Latenz darstellt. Da latentes CMV auch häufig durch Lebertransplantationen übertragen wird, wurde in dieser Arbeit die Leber als ein weiteres medizinisch relevantes Organ der CMV Latenz und Reaktivierung untersucht. Um zunächst die zellulären Orte der mCMV Latenz in der Leber zu ermitteln, wurden verschiedengeschlechtliche Knochenmarktransplantationen (KMT) mit männlichen tdy-positiven Spendern und weiblichen, tdy-negativen Empfängern, mit anschließender mCMV Infektion durchgeführt, um latent infizierte Mäuse mit geschlechtschromosomalem Chimärismus zu generieren. Diese Chimären erlaubten eine Unterscheidung zwischen tdy-positiven Zellen hämatopoetischen Ursprungs und tdy-negativen stromalen und parenchymalen Gewebszellen. Die Separation von Leberzellen der Chimären mittels zentrifugaler Elutriation und anschließender DNA Quantifizierung viraler und zellulärer Genome durch eine quantitative real-time PCR ergab einen ersten Hinweis, dass Endothelzellen ein zellulärer Ort der mCMV Latenz sind. Die darauf folgende immunomagnetische Zelltrennung lokalisierte latente virale DNA in der CD31-positiven Zellfraktion. Die Koexpression von CD31 mit dem endothelzellspezifischen Oberflächenmarker ME-9F1 identifizierte die sinusoidalen Endothelzellen der Leber (LSEC) als die Zellen, die latente virale DNA beherbergen. In den zytofluorometrisch aufgereinigten CD31+/ME-9F1+ LSEC waren bei gleichzeitigem Rückgang der männlichen tdy Markergene virale Genome angereichert, was darauf hinwies, dass Zellen, die virale DNA enthalten, vom Knochenmark-Empfänger stammen. Durch zytofluorometrische Analysen isolierter LSEC konnte eine vom Spender abstammende Subpopulation MHCII+/CD11b+ LSEC identifiziert werden. Anschließende Quantifizierungen viraler DNA aus latent infizierten Mäusen detektierten eine Abnahme viraler Genome mit zunehmender Menge an tdy-positiven Zellen, was beweist, dass MHCII+/CD11b+ LSEC keinen Ort der mCMV Latenz darstellen. Die limiting dilution Untersuchungen der isolierten latent infizierten LSEC ergaben eine Frequenz von einer latent infizierten Zelle unter ~1,9x104 LSEC und eine Anzahl von 7 bis 19 viralen Genomen pro latent infizierter Zelle. Nach 24 Stunden Kultivierung der LSEC konnte mittels quantitativer real-time RT-PCR mit Gesamt-RNA aus LSEC ein Anstieg der Genexpression der immediate early Gene ie1 und ie3 sowie eine Induktion des early Gens e1 gezeigt werden. Eine Erhöhung der transkriptionellen Reaktivierung durch die Inkubation der LSEC mit unterschiedlichen HDAC Inhibitoren konnte allerdings nicht erzielt werden, da sowohl die Menge der isolierten RNA aus behandelten Kulturen, als auch die Anzahl viraler Transkripte im Vergleich zu den unbehandelten Kulturen erniedrigt war. Aufgrund der kurzen Lebensdauer isolierter LSEC in vitro konnte durch Kokultivierungen latent infizierter LSEC zusammen mit murinen embryonalen Fibroblasten keine Virusreaktivierung induziert werden. Im Gegensatz dazu wurden durch den Transfer gereinigter ME-9F1+/CD31+ LSEC aus latent infizierten Spendern in immunsupprimierte Empfänger virale Rekurrenzen in Lungenexplantatkulturen des Rezipienten detektiert. Damit konnten LSEC eindeutig als zellulärer Ort von mCMV Latenz und Reaktivierung in der Leber identifiziert werden.

Bedeutung der viralen IE4-Region für die Genexpression und Replikation des humanen Cytomegalovirus

Das humane Cytomegalovirus (HCMV) ist ein fakultativ-pathogener Erreger, der bei Patienten mit geschwächter oder unausgereifter Immunabwehr schwerwiegende Erkrankungen hervorrufen kann. Wie alle Herpesviren zeigt das HCMV eine streng koordinierte Expression viraler Gene, die in eine „sehr frühe-“ (IE), „frühe “ (E) und „späte-“ (L) Phase unterteilt werden kann. Die Produkte der IE-Gene IE1 und IE2 sind für die Expression der frühen Gene und somit für die Initiation der viralen DNA-Replikation entscheidend. Sie greifen gleichzeitig in den zellulären Stoffwechsel ein und schaffen damit optimale Vorraussetzungen für die virale Vermehrung. Zu Beginn dieser Arbeit war bekannt, dass HCMV in lytisch infizierten Zellen ein abundantes IE-Transkript von 5 kb (IE4-RNA) exprimierte, dessen Funktion bislang unklar war. Ältere Publikationen deuteten darauf hin, dass die IE4-Genregion an der Transformation eukaryonter Zellen beteiligt sein könnte. Neuere Arbeiten zeigten, dass es sich bei diesem IE4-Transkript um ein metabolisch stabiles Intron handelt. Im Rahmen dieser Arbeit sollte zunächst geklärt werden, ob die IE4-Genregion ein Protein kodiert. In der Folge sollten mit viralen Deletionsmutanten Hinweise auf die biologische Funktion des IE4-Bereichs erarbeitet werden. Durch Northern Blot Analysen und cDNA-Klonierungsexperimente konnte eine Reihe neuer Spleiß-Varianten der IE4-RNA identifiziert werden. Durch Sequenzanalysen wurde gezeigt, dass diese Transkripte keine längeren offenen Leserahmen enthalten. Zusammen mit bereits publizierten Erkenntnissen, kann aus diesen Ergebnissen mit hoher Wahrscheinlichkeit geschlossen werden, dass die IE4 Region nicht für ein Protein kodiert. Zur Analyse der biologischen Funktion der IE4-Region wurde das DNA-Genom des HCMV gezielt mutagenisiert. Eine phänotypische Analyse der entsprechenden Viren mittels Reportergen-Tests und quantitativer RealTime RT-PCR zeigte, dass einige der Mutanten eine verringerte Expression früher Gene aufwiesen, die mit einer Beeinträchtigung ihrer Replikationsfähigkeit in Fibroblastenkulturen korrelierte. Dabei war die Ausbildung eines Phänotyps jedoch von dem genetischen Hintergrund des verwendeten viralen Ausgangsstammes abhängig. Auffällig war, dass phänotypische Veränderungen nur bei solchen Mutanten sichtbar wurden, die auf der Grundlage des Laborstammes Ad169 des HCMV generiert worden waren. Die nachfolgende Analyse der Ausgangsstämme ergab deutliche Unterschiede in der IE-Genexpression. Die Ergebnisse dieser Arbeit zeigen somit, dass die IE4-RNA mit hoher Wahrscheinlichkeit nicht für ein Protein kodiert, aber bei limitierender Expression der essentiellen Regulatoren IE1 und IE2 die frühe lytische Genexpression stimuliert. Die Ergebnisse dieser Arbeit stellen die Grundlage für nachfolgende Untersuchungen zur Aufklärung der molekularen Funktion der IE4-RNA im Rahmen der lytischen Infektion des HCMV dar.

Genetische und funktionelle Analyse von Vesikelmembranprotein-Mutanten in Maus und Caenorhabditis elegans

Tetraspan vesicle membrane proteins (TVPs) sind konservierte, ubiquitär vorkommende Membranproteine synaptischer Vesikel und zytoplasmatischer Transportvesikel. Bei Säugetieren lassen sie sich in die Physine, Gyrine und SCAMPs (secretory carrier-associated membrane proteins) unterteilen, die im Nematoden C. elegans jeweils nur durch ein einzelnes Polypeptid vertreten sind (Synaptophysin-1 [SPH-1], Synaptogyrin-1 [SNG-1] und SCAMP-1 [SCM-1]). Obwohl den TVPs eine Beteiligung bei der Regulation des Vesikelzyklus zugesprochen wurde, sind Synaptophysin-1-Knockout-Mäuse und vollständig TVP-defiziente Würmer gesund und weisen nur geringgradige Veränderungen auf. In dieser Arbeit sollten daher zum einen genomweite komparative Transkriptomanalysen durchgeführt werden, um mögliche Kompensationsmechanismen in der Maus und C. elegans zu finden, zum anderen sollten mit Hilfe pharmakologischer Stressassays und genetischer Verfahren Schwachstellen und Redundanzen identifiziert werden. Erstaunlicherweise konnten durch Affymetrix GeneChip-Analysen der RNA in der Retina von Synaptophysin-1-/--Mäusen keine differenziell exprimierten Gene gefunden werden. Bei der Untersuchung der C. elegans-TVP-Dreifachmutante wurden hingegen 17 Gene mit erhöhter und 3 mit erniedrigter Transkription identifiziert. Die Befunde für 12 hochregulierte Gene wurden durch quantitative Real-Time RT-PCR bestätigt. Das am stärksten hochregulierte Gen arf-1.1 kodiert für eine GTPase, die vermutlich an der Regulation der Vesikelbildung beteiligt ist. Von den ebenso identifizierten Genen cdr-2, cdr-4 und pgp-9 ist bekannt, dass sie in Stresssituationen, z. B. in Gegenwart von Cadmium, verstärkt transkribiert werden. ugt-62 und ugt-19 kodieren für Glucuronosyltransferasen. Für arf-1.1, cdr-2, ugt-62 sowie für das Gen T16G1.6, das für eine coiled-coil-Domäne kodiert, wurden im Folgenden fluoreszierende Promoterkonstrukte hergestellt, um Koexpressionsmuster mit TVPs zu bestimmen. Es stellte sich heraus, dass alle vier Promoterkonstrukte im Darm zusammen mit SPH-1 und SCM-1 im Darm transkribiert werden. Mit fluoreszierenden Translationschimären konnte weiterhin gezeigt werden, dass ARF-1.1 und CDR-2 mit den Darm-spezifischen TVPs im apikalen Bereich der Darmzellen kolokalisieren. Um mehr über die Funktion von TVPs im Vesikelzyklus zu erfahren, wurden pharmakologische und genetische Analysen von Würmern durchgeführt, in denen die Expression des Neuronen-spezifischen SNG-1 verändert ist. Deletion oder Überexpression führte zu einer Resistenz gegenüber dem Acetylcholinesterase-Inhibitor Aldicarb und zu erhöhter Empfindlichkeit gegenüber dem GABA-Rezeptor-Antagonisten Pentylentetrazol. Auf genetischer Ebene zeigte sich, dass sng-1 synthetisch mit den Genen für Synaptotagmin-1, Endophilin A sowie Synaptojanin wirkt. Die beobachteten Effekte weisen auf alternative Funktionen in der synaptischen Übertragung hin und unterstützen zugleich die Hypothese, dass SNG-1 im synaptischen Vesikelzyklus eine wichtige Funktion erfüllt, die möglicherweise einem noch unbekannten redundanten Kompartiment-spezifischen Signalweg der synaptischen Transmission zuzuordnen ist.

Toll-like receptors in domestic animals

Toll-like receptors are pattern recognition receptors with which hosts recognize pathogen-associated molecular patterns (PAMP). This recognition process is translated rapidly into a meaningful defense reaction. This form of innate host defense is preserved in the animal kingdom: invertebrates heavily depend on it; higher vertebrates also have an adaptive immune system. Both adaptive and innate immune systems are intertwined in that the former also depends on an intact innate recognition and response system. Members of the TLR system cover recognition of parasitic, bacterial or viral germs. Due to the constraints imposed by the necessity to recognize PAMP and to interact with downstream signaling molecules, the TLR system is relatively conserved in evolution. Nevertheless, subtle species differences have been reported for several mammalian TLR members. Examples of this will be given. In all mammalian species investigated, part of the coding sequence is available for the most important TLR members, thus allowing study of expression of these TLR members in various tissues by reverse-transcription polymerase chain reaction in its classical (RT-PCR) and quantitative real time RT-PCR (qRT-PCR) form. In some species, the whole coding sequences of the most important or even all TLR members are known. This allows construction of cDNA and transfection of common host cells, thus permitting functional studies. Extensive investigations were devoted to the study of non-synonymous single nucleotide polymorphisms. In a few cases, expression of a given amino acid in the extracellular (ligand-binding) portion of TLR members could be associated with infectious diseases. This will be discussed below.

Evidence for dissociation of TLR mRNA expression and TLR agonist-mediated functions in bovine macrophages

Toll-like receptors are of key importance in the recognition of and response to infectious agents by cells of the innate immune system. TLR mRNA expression and TLR-mediated functions were determined in bovine macrophages (MPhi) infected with bovine viral diarrhea virus (BVDV) or stimulated with interferon-gamma (IFN-gamma) in order to see whether they are correlated under these conditions. As parameters quantitative real time RT-PCR (QRT-PCR) for TLR2, TLR3 and TLR4, NO and TNF production were measured. Triggering of bovine MPhi with bona fide TLR2 and TLR4 agonists (lipopolysaccharide, lipoteichoic acid, peptidoglycan, lipopetide) led to NO and TNF production but neither TLR3 nor TLR9 agonists (double-stranded RNA, CpG DNA) showed this effect. The mRNA expression of TLR2, TLR3 and TLR4 was neither influenced by MPhi costimulation with IFN-gamma nor by MPhi preinfection with BVDV nor by the ligands themselves. However, NO production induced by TLR2 or TLR4 agonists was strongly modulated either by IFN-gamma costimulation or BVDV preinfection. Thus costimulation of MPhi with IFN-gamma resulted in an increase of both NO synthesis and TNF expression by cells stimulated simultaneously by TLR2 or TLR4 agonists. Preinfection of bovine MPhi by BVDV resulted in upregulation of TLR2- and TLR4-mediated NO synthesis. Collectively, these data show that TLR-mediated functions may be modulated by viral infection or activation via IFN-gamma of MPhi whereas the mRNA concentrations of relevant TLR members were not significantly influenced. Thus, the amount of TLR2, TLR3 and TLR4 mRNA transcripts is stable at least under the conditions tested. More importantly, modulation of TLR-mediated responses was dissociated from mRNA expression of TLR members.

Nitric oxide synthase protein levels, not the mRNA, are downregulated in olfactory bulb interneurons of reeler mice

Homozygous mutations in the Reelin gene result in severe disruption of brain development. The histogenesis of layered regions, like the neocortex, hippocampus and the cerebellum, is most notably affected in mouse reeler mutants and similar traits are also present in mice lacking molecular components of the Reelin signalling pathway. Moreover, there is evidence for an additional role of Reelin in sustaining synaptic plasticity in adult networks. Nitric oxide is an important gaseous messenger that can modulate neuronal plasticity both in developing and mature synaptic networks and has been shown to facilitate synaptic changes in the hippocampus, cerebellum and olfactory bulb. We studied the distribution and content of neuronal nitric oxide synthase in the olfactory bulbs of reeler and wildtype mice. Immunocytochemistry reveals that Reelin and neuronal nitric oxide synthase containing interneurons are two distinct, non overlapping cell populations of the olfactory bulb. We show by in situ hybridization that both nitrergic and Reelin expressing cells represent only a subset of olfactory bulb GABAergic neurons. Immunoblots show that neuronal nitric oxide synthase protein content is decreased by two thirds in reeler mice causing a detectable loss of immunolabelled cells throughout the olfactory bulb of this strain. However, neuronal nitric oxide synthase mRNA levels, essayed by quantitative real-time RT-PCR, are unaffected in the reeler olfactory bulb. Thus, disruption of the Reelin signalling pathway may modify the turnover of neuronal nitric oxide synthase in the olfactory bulb and possibly affects nitric oxide functions in reeler mice.

Upregulation of toll-like receptors in chronic enteropathies in dogs

Background: Inflammatory bowel disease (IBD) is thought to result from a dysregulated interaction between the host immune system and commensal microflora. Toll-like receptors (TLRs) recognize microbe-associated molecular patterns (MAMPs), but their role in enteropathies in dogs is unknown. Hypothesis: That there is a dysregulation of TLRs recognizing bacterial MAMPs in dogs with IBD. Animals: Sixteen healthy beagles and 12 dogs with steroid-treated (ST) and 23 dogs with food-responsive (FR) diarrhea. Methods: Prospective, observational study. mRNA expression of canine TLR2, 4, and 9 was evaluated by quantitative real-time RT-PCR in duodenal and colonic biopsies obtained before and after standard therapy. Samples from control dogs were taken at necropsy, with additional biopsies of stomach, jejunum, ileum, and mesenteric lymph node in 6 dogs. Results: There were significant differences (P small intestine >/= colon). Before therapy, ST expressed more mRNA than control dogs for all 3 receptors (P < .05). There were no significant differences between pretreatment and posttreatment values, even though 32/35 dogs improved clinically. No associations were found when comparing receptor mRNA expression with either histology or clinical activity scores. Conclusions and Clinical Importance: Bacteria-responsive TLR2, 4, and 9 are upregulated in duodenal and colonic mucosa in IBD. This might lead to increased inflammation through interaction with the commensal flora. The absence of significant changes after therapy despite clinical improvement might point toward the existence of a genetic predisposition to IBD as described in human IBD.

Agents used for chemoprevention of prostate cancer may influence PSA secretion independently of cell growth in the LNCaP model of human prostate cancer progression

BACKGROUND: The aim of this study was to evaluate the inhibitory growth effects of different potential chemopreventive agents in vitro and to determine their influence on PSA mRNA and protein expression with an established screening platform. METHODS: LNCaP and C4-2 cells were incubated with genistein, seleno-L-methionine, lycopene, DL-alpha-tocopherol, and trans-beta-carotene at three different concentrations and cell growth was determined by the MTT assay. PSA mRNA expression was assessed by quantitative real-time RT-PCR and secreted PSA protein levels were quantified by the microparticle enzyme immunoassay. RESULTS: Genistein, seleno-l-methionine and lycopene inhibited LNCaP cell growth, and the proliferation of C4-2 cells was suppressed by seleno-L-methionine and lycopene. PSA mRNA expression was downregulated by genistein in LNCaP but not C4-2 cells. No other compound tested altered PSA mRNA expression. PSA protein expression was downregulated by genistein, seleno-L-methionine, DL-alpha-tocopherol in LNCaP cells. In C4-2 cells only genistein significantly reduced the secretion of PSA protein. CONCLUSIONS: In the LNCaP progression model PSA expression depends on the compound, its concentration and on the hormonal dependence of the cell line used and does not necessarily reflect cell growth or death. Before potential substances are evaluated in clinical trials using PSA as a surrogate end point marker, their effect on PSA mRNA and protein expression has to be considered to correctly assess treatment response by PSA.

Vascular endothelial growth factors, angiogenesis, and survival in human ileal enterochromaffin cell carcinoids

BACKGROUND AND AIMS: Well-differentiated neuro-endocrine ileal carcinoids are composed of serotonin-producing enterochromaffin (EC) cells. Life expectancy is determined by metastatic spread to the liver because medical treatment options are still very limited. Selective inhibition of angiogenesis or lymphangiogenesis might prevent tumour growth and metastatic spread. We examined the role of the vascular endothelial growth factors (VEGFs) A, B, C, D, and their receptors (VEGFRs) 1, 2, 3 in angiogenesis and lymphangiogenesis of ileal EC cell carcinoids with and without liver metastases. METHODS: The expression of various VEGFs and VEGFRs was determined by quantitative real-time RT-PCR in healthy mucosa, primary tumour, lymph node metastases and liver metastases of 25 patients with ileal EC cell carcinoids. Microvessel density (MVD) was determined by CD-31 staining in primary tumours and lymphatic vessel density (LVD) by LYVE-1 staining. VEGF expression levels, MVD, LVD, and patients' survival time were correlated using logistic regression and Kaplan-Meier survival analysis. RESULTS: VEGF-A was highly expressed with no difference between normal mucosa and tumours. VEGF-B and -D as well as VEGFR-1 and -2 expression levels were significantly increased in the tumours when compared to normal mucosa. Patients with liver metastasis, however, had a significantly lower expression of the factors A, B, and C and the receptors 2 and 3. MVD in primary tumours positively correlated with the expression of VEGF ligands and their receptors, except for VEGF-D. LVD did not correlate with any VEGF ligand or receptor. Interestingly, low expression levels of VEGF-B were associated with poor survival. CONCLUSION: Patients with more aggressive metastatic spreading had relatively decreased expression levels of VEGF ligands and receptors. Thus, anti-angiogenic therapy may not be a suitable target in metastatic ileal EC cell carcinoids.