Korrektur der beta c-defizienten pulmonalen Alveolarproteinose (PAP) durch hämatopoetischen Stammzell-Gentransfer in einem beta c-defizienten Mausmodell

Die βc-defiziente Pulmonale Alveolarproteinose (βc-PAP), die durch das Fehlen der gemeinsamen β-Kette (βc) der IL-3-/IL-5-/GM-CSF-Rezeptoren charakterisiert ist, stellt eine monogene Erkrankung dar, die durch eine funktionelle Insuffizienz der pulmolaren Alveolarmakrophagen bedingt ist. Hämatopoetische Stammzell-Gentherapie könnte die defekte βc-Rezeptorkette in den Alveolarmakrophagen korrigieren und eine kurative Behandlungsoption darstellen. Ziel dieser Arbeit war es, in einem murinen mβc-„knock out“-Modell den Effekt des mβc-Gentransfers in vitro und in vivo zu untersuchen. Dazu wurde im ersten Schritt ein retroviraler Vektor konstruiert, der die cDNA der murinen-βc (mβc-)-Kette in Kombination mit der cDNA einer Punktmutante des Chemotherapie-Resistenzgens O6-Methylguanin-DNA-Methyltansferase (MGMTP140K) als selektierbaren Marker exprimiert. Für eine effiziente Transduktion hämatopoetischer Zellen war es dann notwendig, hochtitrige Viruspräparationen zu generieren. Dazu wurde eine neue stabile ekotrope Verpackungszelllinie hergestellt, die es ermöglichte, Viruspräparationen mit infektiösen Titern von bis zu 2 x 106 IE/ml zu produzieren. Mit diesen Viruspräparationen ließ sich eine Gentransfereffizienz von 13 - 35% in primären hämatopoetischen Zellen mβc-defizienter Mäuse erreichen. In vitro konnte durch den retroviralen Gentransfer der mβc-cDNA in hämatopoetische Vorläuferzellen von mβc-/--Mäusen eine Wiederherstellung der GM-CSF-Antwort erreicht werden, wie sich durch eine Koloniebildung von korrigierten KM-Zellen in Gegenwart von GM-CSF nachweisen ließ. Die GM-CSF-Sensitivität derartig „reparierter“ mβc-/--Zellen entsprach der von KM-Zellen aus Wildtypmäusen. In Wildtyp-KM-Zellen wurde zumindest in den Experimenten der vorliegenden Arbeit keine Veränderung der GM-CSF-Sensitivität nach zusätzlicher transgener mβc-Expression beobachtet. Zudem konnte in vitro eine funktionelle MGMTP140K-Expression nach Gentransfer durch eine erhöhte Resistenz transduzierter Vorläuferzellen in klonogenen Assays gegenüber einer kombinierten O6-Benzylguanin/Temozolomid (BG/TMZ) Behandlung beobachtet, sowie eine signifikante Anreicherung genetisch korrigierter mβc-/--Zellen nach BG/TMZ-Exposition gezeigt werden. In der Folge konnte in unserem murinen βc-PAP-Mausmodell auch in vivo die Wiederherstellung der GM-CSF-Sensitivität der hämatopoetischen Vorläuferzellen nachgewiesen werden. Vor allem aber wurde zum ersten Mal gezeigt, dass durch den mβc-Gentransfer eine Rückbildung der pulmonalen Erkrankung erreicht werden kann. Die Alveolarräume der genetisch korrigierten mβc-/--Mäuse wiesen eine nahezu vollständige Reduktion des akkumulierten PAS-positiven Proteinmaterials sowie der erkrankungstypischen peribronchioalveolären Lymphozyteninfiltrate auf. Eine zusätzliche BG/TMZ-Behandlung führte zu einer effektiven In-vivo-Anreicherung transduzierter Zellen in den korrigierten mβc-/--Mäusen, war jedoch in den Experimenten der vorliegenden Arbeit zur Behebung der pulmonalen Erkrankung nicht erforderlich. Zusammenfassend zeigen diese Daten, dass die genetische wie funktionelle Korrektur des Defekts in mβc-/--Zellen in vitro wie auch in vivo möglich ist, und die MGMTP140K-Expression bei Bedarf die Anreicherung genetisch korrigierter Zellen zulässt. Damit ist ein wichtiger erster Schritt in Richtung einer potentiellen klinischen Anwendung des retroviralen βc-Gentransfers als mögliche Behandlungsmethode der βc-PAP getan

A suppressive antagonism evidences Progesterone and Estrogen receptor pathway interaction with concomitant regulation of Hand2, Bmp2 and ERK during early decidualization

Progesterone receptor and estrogen receptor participate in growth and differentiation of the different rat decidual regions. Steroid hormone receptor antagonists were used to study steroid regulation of decidualization. Here we describe a suppressive interaction between progesterone receptor (onapristone) and estrogen receptor (ICI182780) antagonists and their relation to a rescue phenomenon with concomitant regulation of Hand2, Bmp2 and p-ERK1/2 during the early decidualization steps. Phenotypes of decidua development produced by antagonist treatments were characterized by morphology, proliferation, differentiation, angiogenesis and expression of signaling molecules. We found that suppression of progesterone receptor activity by onapristone treatment resulted in resorption of the implantation sites with concomitant decrease in progesterone and estrogen receptors, PCNA, KI67 antigen, DESMIN, CCND3, CX43, Prl8a2, and signaling players such as transcription factor Hand2, Bmp2 mRNAs and p-ERK1/2. Moreover, FGF-2 and Vegfa increased as a consequence of onapristone treatment. Implantation sites from antagonist of estrogen receptor treated rats developed all decidual regions, but showed an anomalous blood vessel formation at the mesometrial part of the decidua. The deleterious effect of onapristone was partially counteracted by the impairment of estrogen receptor activity with rescue of expression levels of hormone steroid receptors, proliferation and differentiation markers, and the induction of a probably compensatory increase in signaling molecules Hand2, Bmp2 and ERK1/2 activation compared to oil treated controls. This novel drug interaction during decidualization could be applied to pathological endometrial cell proliferation processes to improve therapies using steroid hormone receptor targets.Fil: Mestre Citrinovitz, Ana Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Kleff, Veronika. Universaetsklinikum Duisburg-Essen. Institut für Anatomie; AlemaniaFil: Vallejo, Griselda. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Winterhager, Elke . Universaetsklinikum Duisburg-Essen. Institut Für Molekularbiologie; AlemaniaFil: Saragüeta, Patricia Esther. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentin

Nestin(+) Tissue-Resident Multipotent Stem Cells Contribute to Tumor Progression by Differentiating into Pericytes and Smooth Muscle Cells Resulting in Blood Vessel Remodeling

Tumor vessels with resistance to anti-angiogenic therapy are characterized by the normalization of the vascular structures through integration of mature pericytes and smooth muscle cells (SMC) into the vessel wall, a process termed vessel stabilization. Unfortunately, stabilization-associated vascular remodeling can result in reduced sensitivity to subsequent anti-angiogenic therapy. We show here that blockade of VEGF by bevacizumab induces stabilization of angiogenic tumor blood vessels in human tumor specimen by recruiting Nestin-positive cells, whereas mature vessels down-regulated Nestin-expression. Using xenograft tumors growing on bone-marrow (BM) chimera of C57Bl/6 wildtype and Nestin-GFP transgenic mice, we show for first time that Nestin(+) cells inducing the maturation of tumor vessels do not originate from the BM but presumably reside within the adventitia of adult blood vessels. Complementary ex vivo experiments using explants of murine aortas revealed that Nestin(+) multipotent stem cells (MPSCs) are mobilized from their niche and differentiated into pericytes and SMC through the influence of tumor-cell-secreted factors. We conclude that tissue-resident Nestin(+) cells are more relevant than BM-derived cells for vessel stabilization and therefore have to be considered in future strategies for anti-angiogenic therapy. The identification of proteins mediating recruitment or differentiation of local Nestin(+) cells with potential stem cell character to angiogenic blood vessels may allow the definition of new therapeutic targets to reduce tumor resistance against anti-angiogenic drugs

Vascular Wall-Resident CD44+ Multipotent Stem Cells Give Rise to Pericytes and Smooth Muscle Cells and Contribute to New Vessel Maturation

Here, we identify CD44(+)CD90(+)CD73(+)CD34(−)CD45(−) cells within the adult human arterial adventitia with properties of multipotency which were named vascular wall-resident multipotent stem cells (VW-MPSCs). VW-MPSCs exhibit typical mesenchymal stem cell characteristics including cell surface markers in immunostaining and flow cytometric analyses, and differentiation into adipocytes, chondrocytes and osteocytes under culture conditions. Particularly, TGFß1 stimulation up-regulates smooth muscle cell markers in VW-MPSCs. Using fluorescent cell labelling and co-localisation studies we show that VW-MPSCs differentiate to pericytes/smooth muscle cells which cover the wall of newly formed endothelial capillary-like structures in vitro. Co-implantation of EGFP-labelled VW-MPSCs and human umbilical vein endothelial cells into SCID mice subcutaneously via Matrigel results in new vessels formation which were covered by pericyte- or smooth muscle-like cells generated from implanted VW-MPSCs. Our results suggest that VW-MPSCs are of relevance for vascular morphogenesis, repair and self-renewal of vascular wall cells and for local capacity of neovascularization in disease processes

Hox genes are involved in vascular wall-resident multipotent stem cell differentiation into smooth muscle cells

Human vascular wall-resident CD44+ multipotent stem cells (VW-MPSCs) within the vascular adventitia are capable to differentiate into pericytes and smooth muscle cells (SMC). This study demonstrates HOX-dependent differentiation of CD44(+) VW-MPSCs into SMC that involves epigenetic modification of transgelin as a down-stream regulated gene. First, HOXB7, HOXC6 and HOXC8 were identified to be differentially expressed in VW-MPSCs as compared to terminal differentiated human aortic SMC, endothelial cells and undifferentiated pluripotent embryonic stem cells. Silencing these HOX genes in VW-MPSCs significantly reduced their sprouting capacity and increased expression of the SMC markers transgelin and calponin and the histone gene histone H1. Furthermore, the methylation pattern of the TAGLN promoter was altered. In summary, our findings suggest a role for certain HOX genes in regulating differentiation of human VW-MPSC into SMCs that involves epigenetic mechanisms. This is critical for understanding VW-MPSC-dependent vascular disease processes such as neointima formation and tumor vascularization

Vascular wall-resident stem cells

New vessels in the adult have been considered to be formed not only by angiogenesis, but also by postnatal vasculogenesis via endothelial progenitor cells (EPCs). However, it is still a matter of debate as to what extent the EPCs contribute to new vessel formation in the adult. While the role of the circulating and bone marrow-derived EPCs has intensively been studied, the contribution of the vascular wall itself was neglected for a long time. Evidence published in the last few years strongly suggests the existence of different stem and progenitor cell types in the vascular wall. Particularly, the presence of EPCs and smooth muscle progenitor cells (SMPCs) in distinct zones of the vascular wall supports the hypothesis that not only BM- or C-EPCs, but also vascular wall-resident stem cells (VW-SCs) might contribute to new vessel formation and vascular wall morphogenesis. However, the differentiation potential of the VW-SCs, e.g. whether a VW-SC is able to give rise to a complete hierarchy of vascular progenitors still remains to be studied. This review will provide a survey about the VW-SCs and their potential impact in vascular biolog

Ein UV-Test zur Messung der α-Amylase-Aktivität in Serum und Urin

Peer Reviewe

Scheme depicting interaction between PR and ER pathways in decidualization.

<p>Individual antagonist treatments evidenced PR and ER gene regulation pathways, meanwhile combined antagonist treatment highlights the existence of P and E receptors pathways interaction. ONA +ICI treatment restores ISs development by the activation of ¨compensatory pathways¨. This failsafe road could include regulation of <i>Hand2</i>, <i>Bmp2</i> expression, ERK activation and/or unidentified signaling molecules (X). Arrows heads, positive regulation; blunt heads, negative regulation; point head; undefined regulation; black fill line, interactions evidenced in this work; red dot line, pathways proposed.</p

Effects of antagonist treatments on differentiation and vascularization.

<p>Samples from 7 dpc Oil, ONA, ICI or ONA+ICI treated rats were analyzed for protein and mRNA expression of differentiation and vascularization markers. A) Differentiation markers: protein levels of CCND3, DESMIN, CX43 and mRNA levels of <i>Prl8a2</i>. B) Vascularization markers: protein levels of FGF-2 and mRNA levels of <i>Vegfa</i>. Each treatment protein levels relative to GAPDH or ERK2, or mRNA levels relative to <i>β-Actin</i>, were divided by the corresponding Oil-treated value. Data represent mean fold change ± SEM from at least three independent rats/treatment. Insets show pictures of a representative western blot. *, P < 0.05; **, P < 0.01; ***, P < 0.001.</p

Effects of antagonist treatments on proliferation.

<p>Samples from 7 dpc Oil, ONA, ICI or ONA+ICI treated rats were analyzed for KI67 protein expression. A) Details of KI67 immunostaining of M, J and AM zones from a representative 7 dpc IS from Oil, ICI and ONA+ICI treated rats; and the border (b) and central (c) zones from a representative ONA-treated IS. Mean percentages ± SEM of positive KI67 cells are shown under each picture. B) KI67 quantification. Each point in the graph represents the percentage of the proliferative cells per field. Three fields per IS area were count and a minimum of 3 ISs were analyzed for each treatment. Fig 2A data represent mean fold change ± SEM from at least three independent rats/treatment, a minimum of 1 IS/rat was analyzed. *, P < 0.05; **, P < 0.001. O+I, ONA+ICI; M, mesometrial decidua; J, junctional zone; AM, antimesometrial decidua; b, border area of resorpted IS; c, center area of resorpted IS. Magnification bars = 400 μm; magnification bar = 20 μm.</p