Local regulatory factors in regulation of ovarian function: Role of prorenin-renin-angiotensin-system

669-681<span style="font-size:14.0pt;line-height: 115%;font-family:" times="" new="" roman";mso-fareast-font-family:"times="" roman";="" mso-ansi-language:en-in;mso-fareast-language:en-in;mso-bidi-language:hi"="" lang="EN-IN">During reproductive life in the female, there is a continuous flow of growth, maturation and demise of ovarian follicles, unless pregnancy occurs. Although ovarian function is primarily controlled by the hypothalamus-pituitary-axis, there is no doubt that a hormonal microenvironment specific for each individual follicle is established, that finally determines whether a follicle ovulates and beeomes a corpus luteum or undergoes atresia. In this respeet, autocrine and paracrine ractors that act alone or modulate gonadotropins action arc of paramount importance. In this article, we want to introduce the ovarian prorenin -renin -angiotensin- system (PRAS) and summarize what is actually known about its involvement in ovarian physiology and pathology.</span

Homologous and lysophosphatidic acid-induced desensitization of the atrial natriuretic peptide receptor, guanylyl cyclase-A, in MA-10 leydig cells

The cardiac hormone atrial natriuretic peptide (ANP) signals via interaction with a plasma membrane receptor, which has guanylyl cyclase (GC) activity and is referred to as GC-A. Desensitization of GC-A is thought to represent a physiologically important regulatory mechanism, but the signaling pathways implicated and cell type-specific effects are still poorly understood. Here we demonstrate that sustained exposure to either ANP itself or the bioactive lipid lysophosphatidic acid (LPA) elicits GC-A desensitization in MA-10 Leydig cells. Both reactions show similar kinetics and evoke equal decreases (by 40%) in GC-A hormone responsiveness. Homologous (ANP induced) desensitization, in which cGMP is generated as second messenger, is blocked by distinct cAMP-dependent protein kinase [protein kinase A (PKA)] inhibitors, H 89, and Rp-8-CPT-cAMPs, providing evidence that PKA mediates the reaction. Accordingly, the ANP/cGMP-elicited effects are mimicked by a cAMP analog, 8-bromo-cAMP. The LPA-induced (heterologous) desensitization is not blocked by PKA inhibition, indicating a different signaling pathway. LPA, but not ANP, enhances ERK phosphorylation and induces cell rounding together with a dramatic reorganization of actin filaments. Consistent with the identification of LPA receptor (LPA2 and LPA3) gene expression, the findings are indicative of LPA receptor-mediated reactions. This study demonstrates for the first time coexistence of homologous and heterologous desensitization of GC-A in the same cell type, reveals that these reactions are mediated by different pathways, and identifies a novel cross talk between phospholipid and natriuretic peptide signaling. The morphoregulatory activities exerted by LPA suggest a crucial role for Leydig cell physiology

Radiation rescue: mesenchymal stromal cells protect from lethal irradiation.

BACKGROUND: Successful treatment of acute radiation syndromes relies on immediate supportive care. In patients with limited hematopoietic recovery potential, hematopoietic stem cell (HSC) transplantation is the only curative treatment option. Because of time consuming donor search and uncertain outcome we propose MSC treatment as an alternative treatment for severely radiation-affected individuals. METHODS AND FINDINGS: Mouse mesenchymal stromal cells (mMSCs) were expanded from bone marrow, retrovirally labeled with eGFP (bulk cultures) and cloned. Bulk and five selected clonal mMSCs populations were characterized in vitro for their multilineage differentiation potential and phenotype showing no contamination with hematopoietic cells. Lethally irradiated recipients were i.v. transplanted with bulk or clonal mMSCs. We found a long-term survival of recipients with fast hematopoietic recovery after the transplantation of MSCs exclusively without support by HSCs. Quantitative PCR based chimerism analysis detected eGFP-positive donor cells in peripheral blood immediately after injection and in lungs within 24 hours. However, no donor cells in any investigated tissue remained long-term. Despite the rapidly disappearing donor cells, microarray and quantitative RT-PCR gene expression analysis in the bone marrow of MSC-transplanted animals displayed enhanced regenerative features characterized by (i) decreased proinflammatory, ECM formation and adhesion properties and (ii) boosted anti-inflammation, detoxification, cell cycle and anti-oxidative stress control as compared to HSC-transplanted animals. CONCLUSIONS: Our data revealed that systemically administered MSCs provoke a protective mechanism counteracting the inflammatory events and also supporting detoxification and stress management after radiation exposure. Further our results suggest that MSCs, their release of trophic factors and their HSC-niche modulating activity rescue endogenous hematopoiesis thereby serving as fast and effective first-line treatment to combat radiation-induced hematopoietic failure

Indication of Horizontal DNA Gene Transfer by Extracellular Vesicles

<div><p>The biological relevance of extracellular vesicles (EV) in intercellular communication has been well established. Thus far, proteins and RNA were described as main cargo. Here, we show that EV released from human bone marrow derived mesenchymal stromal cells (BM-hMSC) also carry high-molecular DNA in addition. Extensive EV characterization revealed this DNA mainly associated with the outer EV membrane and to a smaller degree also inside the EV. Our EV purification protocol secured that DNA is not derived from apoptotic or necrotic cells. To analyze the relevance of EV-associated DNA we lentivirally transduced <i>Arabidopsis thaliana</i>-DNA (<i>A</i>.<i>t</i>.-DNA) as indicator into BM-hMSC and generated EV. Using quantitative polymerase chain reaction (qPCR) techniques we detected high copy numbers of <i>A</i>.<i>t</i>.-DNA in EV. In recipient hMSC incubated with tagged EV for two weeks we identified <i>A</i>.<i>t</i>.-DNA transferred to recipient cells. Investigation of recipient cell DNA using quantitative PCR and verification of PCR-products by sequencing suggested stable integration of <i>A</i>.<i>t</i>.-DNA. In conclusion, for the first time our proof-of-principle experiments point to horizontal DNA transfer into recipient cells via EV. Based on our results we assume that eukaryotic cells are able to exchange genetic information in form of DNA extending the known cargo of EV by genomic DNA. This mechanism might be of relevance in cancer but also during cell evolution and development.</p></div

Detection of <i>A</i>.<i>t</i>.-DNA in EV-recipient cells with SYBR Green-based qPCR, TOPO^® TA Cloning and sequencing.

<p>Shown is the total number of primary PCR replicates carried out with 1 μg DNA/PCR using complete isolated DNA of each single tissue flask, therefrom resulting nested PCR replicates, the number of positive samples in SYBR Green nested PCR, the No. of colonies which underwent sequencing and No. of colonies with the correct sequence (sequencing positive). In total, three different EV preparations were applied in this experiment. For each EV sample, 2x T25 of recipient cells were incubated with unmanipulated EV (-) and 1x T25 with EV after DNaseI (+) treatment. The nomenclature C(-)16 stands for: EV sample C without DNase treatment C(-), qPCR No. 16 carried out with 1 μg DNA per reaction.</p

<i>Arabidopsis thaliana (A</i>.<i>t</i>.<i>)</i> virus production and transfer.

<p>(a) <i>A</i>.<i>t</i>.-DNA was cloned into the LeGO-V2-wpre plasmid vector containing Venus-fluorescence protein for detection. Primers for subsequent primary and nested <i>A</i>.<i>t</i>.-PCR shown with arrows were located within the <i>A</i>.<i>t</i>.-sequence giving rise to products of 387 bp and 106 bp respectively. (b, c) hMSC were transduced with LeGO-V2-wpre-<i>A</i>.<i>t</i>. virus supernatant. Shown is a hMSC culture 8 days after transduction (x40) detecting green cells (b) in a near confluent culture (c, phase contrast). (d, e) Recipient hMSC were incubated for 2 weeks with EV purified from hMSC-<i>A</i>.<i>t</i>. culture supernatant. Shown are Venus-positive cells (d) in the recipient culture after incubation with EV without (3 left images) or with DNase digestion (most right image) and their respective phase contrast pictures (e) (magnification x200).</p

Detection of <i>A</i>.<i>t</i>.-sequences in recipient cells using SYBR Green-based qPCR.

<p>(a) Standard dilutions of <i>A</i>.<i>t</i>.-DNA in duplicates with 10.000–10 copies/PCR reaction show a linear dependency whereas 1 copy/PCR was located below the detection limit. (b) Three to four replicates of DNA isolations from EV without DNase treatment (G(-); EV from harvest G without DNAse treatment) showed high abundant <i>A</i>.<i>t</i>.-sequences with Ct = 16 whereas those with DNase treatment (G(+)) showed much lower <i>A</i>.<i>t</i>.-DNA amounts with Ct near the detection limit. As comparison, positive standard with 100 copies/PCR was plotted. (c) Several replicates of the sample C(-)16 (EV from harvest C without DNase treatment, PCR run No. 16 carried out with 1μg DNA per reaction) were detected with Ct of ≥ 33. As comparison, positive standard with 10 copies/PCR was plotted. (d) Melting temperatures (Tm) of samples in (c) show the replicates with one high and several lower peaks with the correct Tm. The blue curves correspond to the positive standard of 10 copies/PCR reaction. Two exemplary arrows for sample C(-)16 in (c) and (d) point to lime and red colored probes with high and low Tm peaks, respectively.</p

Characterization of EV.

<p>Human MSC were cultured on ibidi μ-slides, fixed and analyzed using electron microscopy. Shown is a part of cell membrane of a hMSC releasing EV (a). After ultracentrifugation of the supernatant, EV were resuspended in small volumes, sucked into carbo-tubes, fixed and analyzed using electron microscopy. Patches (b) and single EV (c) of 50–1,000 nm were detected. Ten μg of characteristic EV-proteins (CD81, HSP70, CD9 and CD63, GAPDH as housekeeper) were analyzed by Western blot (d). For quantification of EV using flow cytometry, size beads ranging from 0.2–2 μm were used to define the EV analysis area P1 (e) and impurities of 0.1μm filtered PBS in P1 (f). Purified EV in P1 were quantified using counting beads excluding the particles contained in filtered PBS. Total EV amounts per harvest (samples A-N from three individual donors) blotted against the protein content of each EV harvest revealed interindividual differences in protein cargo but reproducibility within one donor culture after repeated EV harvests (g). To investigate the underestimation of EV due to “swarm detection” in flow cytometry, 6 EV harvests were measured with NanoSight revealing ca. 1,000 fold higher concentration (401 ± 290) with a mode size of 146 ± 7.7 nm (h).</p