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Exploiting Human CD34+ Stem Cell–conditioned Medium for Tissue Repair

Despite the progress in our understanding of genes essential for stem cell regulation and development, little is known about the factors secreted by stem cells and their effect on tissue regeneration. In particular, the factors secreted by human CD34+ cells remain to be elucidated. We have approached this challenge by performing a cytokine/growth factor microarray analysis of secreted soluble factors in medium conditioned by adherent human CD34+ cells. Thirty-two abundantly secreted factors have been identified, all of which are associated with cell proliferation, survival, tissue repair, and wound healing. The cultured CD34+ cells expressed known stem cell genes such as Nanog, Oct4, Sox2, c-kit, and HoxB4. The conditioned medium containing the secreted factors prevented cell death in liver cells exposed to liver toxin in vitro via inhibition of the caspase-3 signaling pathway. More importantly, in vivo studies using animal models of liver damage demonstrated that injection of the conditioned medium could repair damaged liver tissue (significant reduction in the necroinflammatory activity), as well as enable the animals to survive. Thus, we demonstrate that medium conditioned by human CD34+ cells has the potential for therapeutic repair of damaged tissue in vivo.

INTRODUCTION
Somatic stem cells have the capacity for self-renewal and multilineage differentiation. [1][2][3] They reside in various tissue types including the bone marrow, brain, skin, muscle, pancreas, and liver. 4 Studies have shown that soluble factors secreted within the tissue microenvironment including cytokines, and growth factors are essential in stem cell regulation and development. [5][6][7][8][9][10] They are particularly important in maintaining the homeostasis of their microenvironment, in addition to maintaining a crosstalk with surrounding stromal cells (fibroblasts, endothelial cells, and macrophages). Not surprisingly, emerging evidence suggests that cytokines can also influence stem cell fate in vitro, as well as in vivo. 11 Although genes associated with stem cell regulation and development have been studied, 12 the impact of cytokines and growth factors associated with these regulatory events is less well characterized.
Human CD34 + cells are the most widely studied adult stem cells. 12,13 They reside in the bone marrow, as well as in other tissues and play an essential role in sustaining the formation of the blood and immune systems. ,12,14-16 More recently, their proposed ability to differentiate into multiple tissue lineages has been exploited for the treatment of various debilitating diseases. [17][18][19][20] Studies have shown that CD34 + cells are influenced by cytokines and growth factors, in addition to adhesion proteins such as integrins. 21 However, only a few studies currently show the benefit in vivo of factors secreted by human CD34 + cells on damaged tissue.
We have previously characterized a population of adherent CD34 + cells and based on morphology, flow cytometry, and gene expression analysis they expressed genes corresponding to lineages of tissue differentiation (liver, pancreas, heart, and nerve), as well as hematopoiesis. 18 Importantly, they also expressed stem cell genes including Oct4, Nanog, and Rex-1. In order to identify the factors secreted by these cells, we have approached this issue by performing a cytokine/growth factor microarray analysis of factors secreted by adherent CD34 + cells cultured in a defined serum-free medium. Several abundantly secreted factors were identified from the conditioned medium. Moreover, the conditioned medium prevented cell death in cells treated with a liver toxin in vitro, as well as demonstrating tissue repair properties in two independent animal models of liver damage. Our studies suggest that medium conditioned by human CD34 + cells may be exploited for therapeutic applications.

Adherent human CD34 + cells cultured in a defined serum-free medium
To better understand which cytokines and growth factors are secreted by human CD34 + cells in vitro, we have used a defined serum-free medium in which to grow these cells. 22 A pure population of isolated human CD34 + cells were used for the studies (Figure 1a,b). Adherent human CD34 + cells were grown in a defined serum-free medium containing interleukin (IL)-3, IL-6, and stem cell factor for 7 days as previously described. 22 The CD34 + cells initially form small clusters that gradually merge into a homogenous cell population (Figure 1c). We found that the cells were proliferating as determined by the cell cycle profile (Figure 1d) and by protein expression of proliferation markers, Cyclin D1 and PCNA (Figure 1e). These data suggest that the combination of the three cytokines in the serum-free medium is able to sustain CD34 + cell growth and proliferation.

Expression of cell surface and stem cell markers
We next used flow cytometry and immunostaining approaches to determine whether the cells grown in the defined medium still expressed known cell lineage and stem cell markers. Several important cell surface markers including CD34, c-kit, CD45, and ICAM3 were expressed (Figure 2a). In addition to the cell lineage markers, specific stem cell markers including Oct4, HoxB4, Nanog, and Sox2 were expressed positively, in addition to being coexpressed with CD34 (Figure 2b-e). Stem cell genes such as Oct4, Sox2 and Nanog are essential in stem cell regulation and self-renewal. 2 Collectively, these results suggest that the CD34 + cells cultured in the serum-free defined medium are able to maintain the expression of several important cell lineage and stem cell markers.

Cytokine expression profile of conditioned medium
In order to identify the secreted factors, the conditioned and control mediums were analyzed using a 174 cytokine antibody microarray chip for secreted soluble factors. Of the numerous factors represented on the array (Figure 3a), 32 were significantly abundant in the conditioned medium compared with the control medium (Figure 3b). Some of the identified cytokines and growth factors such as FGF-6, IL-8, IL-10, M-CSF, GM-CSF, and angiopoietin-2 are important in stem cell regulation, as well as in cell proliferation and tissue repair. 6,9,11 To gain further insight into how the secreted factors are interconnected with each other, we performed a bioinformatic analysis for proteinprotein interactions using a database called STRING (Search Tool for the Retrieval of Interacting Genes/Proteins). 23,24 This database examines multiple genes and proteins simultaneously for physical and functional interactions based on seven different criteria for known individual factors. Using this program, a predicted functional protein-protein interaction map was generated between the different secreted factors (Figure 3c). The protein-protein interaction map revealed that 17 out of the 32 secreted factors have some degree of multiple functional x axis shows CD34-Cy5 and the y axis shows lineage cocktail antibodies (LIN) conjugated to fluorescein isothiocyanate (FITC). The LIN cocktail of antibodies include: CD3, CD14, CD19, CD20, CD56, CD11b, CD10, CD235a, CD7, CD8a, and CD2. Gating was determined by setting a 1% above the isotype control antibody to be considered positive staining. (c) Phase-contrast bright field images of 7-day-cultured adherent CD34 + cells in the defined serum-free medium. Representative images from four independent experiments. Bar scale, 100 µm. (d) Cell cycle profile of 7 day culturing of adherent CD34 + cells and K562 cells was used as a positive control. (e) Immunoblots of cyclin D1 and PCNA proliferation markers.
Nanog antibody Oct4 antibody HoxB4 antibody Sox2 antibody C-kit antibody Molecular Therapy interactive partners. A closer analysis of these 17 factors for molecular and cellular functions revealed that several of them are important for cell proliferation, wound healing, and immune response ( Table 1). These molecules frequently have overlapping activities and can act in an autocrine or paracrine fashion. This is not surprising because a complex network of growth factors and cytokines are essential for cellular differentiation and tissue regeneration.

Conditioned medium Control medium
Array map    A total of 32 factors were scored positive. Densitometry was used to quantify the relative intensity of the spots to the control. (c) A protein-protein interaction network map for the 32 secreted factors was generated using the STRING program. The STRING program covers over 2.5 million proteins. The confidence score was set at the highest level, >90%.
• Wound healing and inflammatory response.
• Cell trafficking and differentiation.
• Wound healing and inflammatory response.
• Secretion of factors involved in tissue repair.

Conditioned medium prevents cell death in liver cells
The information from the protein-protein interaction map lead us to consider the possibility that the conditioned medium may have protective and wound healing properties based on what is already known about some of the secreted factors. As it is complex and technically challenging to test all of the 32 secreted factors individually or in various combinations, we decided to directly test the conditioned medium. We found that the conditioned medium can prevent cell death in liver cells treated with a liver toxin, thioacetamide (TAA) (Figure 4a). We found that liver cells treated with TAA displayed severe structural damages, whereas TAA-treated cells that received the conditioned medium maintained normal structural integrity, as well as cell viability (Figure 4a,b). As it is known that TAA activates the caspase-3 signaling pathway, 25 we tested for caspase-3 activity under these conditions. We observed a reduced level of caspase-3 activity in liver cells treated with the conditioned medium (Figure 4c). These results suggest that the conditioned medium contains secreted factors that may prevent cell death.

Conditioned medium repairs liver damage in vivo
Based on the results from the in vitro studies (Figure 4) and information from Table 1, we next investigated the effect of the conditioned medium in a rat liver model. Specifically, we used a clinically relevant cirrhotic rat liver model which demonstrates features similar to human liver cirrhosis. 26 The induced cirrhotic rats were injected six times via tail vein with the conditioned medium and killed 3 days after the last injection. Important liver functions including aspartate aminotransferase, alanine transaminase, bilirubin, and albumin were measured. We found that animals treated with the conditioned medium performed much better for all the liver functions when compared with the control group (Figure 5a-d). More importantly, all the animals survived in the treated group (Figure 6a), suggesting that the conditioned medium has the capacity to extend the survival outcome of the treated animals. To validate this observation, we specifically determined the amount of necroinflammatory activity in the damaged liver tissue. The measurement of its activity reflects the degree of necrosis and inflammation in the liver. The staining revealed a reduced necroinflammatory area (Figure 6b,c) in the treated group (15.8 ± 2.93%) compared with the control group (24.8 ± 4.28%). As these results suggest that the conditioned medium may have the capacity to repair damaged liver tissue, we next determined whether the cells would also have a similar affect. Surprisingly, when 7-day-cultured cells were injected into rats that were pretreated with a well-established liver toxin, TAA, we found improved albumin and bilirubin liver functions (Supplementary Figure S1).

DISCUSSION
There is a high expectation that stem cells may deliver new therapeutic strategies for debilitating diseases such as neurodegenerative conditions, diabetes, cardiovascular disorders, and liver disease. 26 Tremendous effort has been invested in developing new stem cell-based therapeutic products using embryonic and adult stem cells for regenerative medicine. In addition to exploiting stem cells themselves as the deliverable therapeutic agent, the idea of exploiting the factors secreted by stem cells has been less characterized.
The approach we have taken, using a defined serum-free medium to culture human CD34 + cells, has enabled the identification of several enriched secreted factors. More importantly, our study is the first to demonstrate that the medium conditioned by adherent CD34 + cells are able to reach the damaged liver and repair tissue in a clinically relevant liver animal model. We found a significant reduction in the degree of necrosis and inflammation (necroinflammatory activity) in the liver treated with the conditioned medium, suggesting that the factors in the conditioned medium are capable of reducing the inflammatory response. We speculate that under these conditions, it is possible that the secreted factors in the conditioned medium may provide the necessary survival and repair factors to recruit various cell types and to assist in the tissue repair process. In accordance with this idea, studies have shown that factors secreted from stem cells may be associated with vascular repair and regeneration. 27,28 It is interesting to note that many of the identified factors secreted by human CD34 + cells are involved in cell survival and wound healing. For example, the protein-protein interaction map revealed four highly interactive networks of chemokines (CXCL1, CXCL5, CXCL6, and PPBP) that belong to CXC ligand (CXCL) chemokine family, which is well known to recruit neutrophils to the wound site and actively aid in the repair process. 29 The healing properties of this family have been expanded to include an efficient repair and regeneration of mice liver with partial hepatectomy. 29 There is growing evidence that this hepatic repair process is regulated by CXC chemokine receptor 2 (CXCR2), 30,31 which commonly binds to members of the CXCL chemokine family, 32,33 suggesting that the four chemokines may play a major role in repairing injured liver cells but further studies are needed.
In summary, we have shown proof-of-concept that factors secreted by human CD34 + cells in the conditioned medium have tissue repair properties. If this approach proves to be successful, the secreted factors may be developed into a clinical product that could be readily processed and stored for therapeutic applications.

MATERIALS AND METHODS
Cell culture. Normal rat liver cell line CRL-1439 was obtained from American Type Culture Collection (Rockville, MD) and cultured in RPMI supplemented with 10% fetal bovine serum.

Isolation and growth of adherent CD34 + cells.
Allogeneic human hematopoietic donor blood samples were obtained with informed patient consent and approved by the Hammersmith Hospital Research Ethics Committee. Samples of granulocyte-colony stimulating factor (G-CSF) mobilized peripheral blood progenitor cells were processed by leukapheresis at the Stem Cell Laboratory at the Hammersmith Hospital. Briefly, human mobilized peripheral blood samples were diluted in a ratio of 1:4 in Hanks' buffered saline solution (Invitrogen, Paisley, UK), the mononuclear cells were separated by centrifugation over a Lymphoprep (Axis-Shield, Dundee, Scotland) density gradient at 1800 rpm for 30 minutes. The mononuclear cell fraction at the interface was aspirated and washed twice with Hanks' buffered saline solution, and finally with magnetic cell sorting buffer (phosphate-buffered saline solution at pH 7.2 supplemented with 0.5% bovine serum albumin and 2 mmol/l EDTA). CD34 + cells were isolated using a CD34 + isolation kit (Miltenyi Biotec, Surrey, UK) according to the manufacturer's protocol. The purity of CD34 + cells were determined by FACS analysis and only samples that were >95% pure were used for the studies. Flow cytometry analysis of isolated CD34 + cells were confirmed by the following antibodies: CD34-PerCP-Cy5 (1:300); lineage cocktail antibodies CD3, CD14, CD19, CD20, CD56, CD11b, CD10, CD235a, CD7, CD8a, and CD2 conjugated to fluorescein isothiocyanate. Directly conjugated isotype-matched controls (1:300) (IgG1κ, IgG2a; BD Bioscience, Oxford, UK) were used as negative controls, while Fluorescence Minus One was used alternatively. Stained cells were analyzed using the BD LSRII flow cytometer with 488-530/30, 488-695/40, and 488-780/60 filters to view fluorescein isothiocyanate and PerCP-Cy5.5 respectively. At least 10,000 cells were collected for each test sample to ensure a sufficient number of positive stained cells. FlowJo 7.5 software was used for the presentation of the results. The isolated CD34 + cells were added to 24-well or 35-mm tissue-culture-treated dishes (Nunc) (Sigma-Aldrich Company, Dorset, UK) at a density of 2.5-5 × 10 5 cells in α-minimum essential medium medium to isolate the adherent CD34 + cells. After 30 minutes incubation, cells were rinsed and grown in a defined serum-free medium (CellGro; CellGenix GmbH, Freiburg, Germany) containing three cytokines: 250 ηg/ml of stem cell factor, 250 ηg/ml of IL-6, and 250 ηg/ml of IL-3 (Invitrogen or CellGenix GmbH) in 0.5% penicillin/streptomycin antibiotics. Cells were incubated at 37 °C in 5% CO 2 . 22 Approximately 1 ml of the defined serum medium was added when culturing in 35-mm culture plates and 500 µl per well when 24-well plates were used. Total viable cells were counted using the trypan exclusion assay.
Cytokine expression profiling. The isolated adherent CD34 + cells (35-mm dishes, Nunc) (Sigma-Aldrich Company) were cultured in the defined serum-free medium (5 plates per array). The control dishes only contained the defined serum-free medium. The conditioned medium was collected on the 3rd and 7th days. In order to prevent any cells remaining in the conditioned medium, the medium was centrifuged for 1 hour at 13,000 RPM at 0 °C. The conditioned media were pooled and concentrated to 2 ml using a Vivaspin-20 concentrator (Sigma-Aldrich Company). The concentrated medium (2.5-fold) was used for the RayBio human cytokine antibody array C-series 2000 (174 cytokines). Briefly, membranes were blocked (blocking buffer) followed by adding the conditioned medium or control medium. After 2 hours of incubation, membranes were repeatedly washed and then incubated with biotin-conjugated antibodies for 2 hours at room temperature. The membrane was washed followed by horse-radish peroxidase conjugated streptavidin detection for 1 hour followed by several washes. The membranes were processed followed by exposure to hyperfilm (GE Healthcare, Buckinghamshire, UK). The film was processed and scanned, and densitometric value of each spot on the array was measured using the UVP GelDoc system (UVP, Cambridge, UK). Cytokines that had 1.5-fold or more relative to the control were scored as positive. Two independent experiments were performed and analyzed. Each array contained duplicate spots of the cytokines or growth factors. Protein-protein interaction was generated using the STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) database. 23,24 Immunofluorescence microscopy, flow cytometry, cell cycle, and western blot. To analyze the expression of cell surface and intracellular markers, flow cytometry and immunofluorescence analysis were performed. Approximately 1 × 10 5 cells were stained and analyzed per sample. The expression of specific stem cell markers was performed on day-7-cultured cells. For cell surface markers, no permeabilization was performed before immunolabeling with the appropriate primary antibodies. For intracellular staining, cells were fixed and permeabilized using Fix & Perm kit (Invitrogen) or 0.2% Triton-X-100 in phosphate-buffered saline followed by labeling with appropriate primary antibodies. Each tube was washed and stained with the appropriate secondary antibody (1:500). Isotype-matched controls were used for each antigen stained. Analysis of the labeled cells was performed with a FACScalibur flow cytometer (BD Bioscience). Ten thousand cells were collected for each test sample to ensure a sufficient number of positive stained cells. For the immunofluorescence staining, after several washes in phosphate-buffered saline, coverslips were mounted on glass slides with Vectashield containing 4′6′-diamidino-2-phenylindole (Vector Laboratories, Peterborough, UK). Slides were visualized on a Leica DM4000 or Zeiss Axiovert 200 inverted fluorescence microscope. Antibodies used were anti-Nanog (1:50  . Appropriate isotype control antibodies were purchased from BD Biosciences. Gating was determined by setting a 1% above the isotype control antibody to be considered positive staining. For the double staining of CD34 and stem cell markers, the following antibodies were used: CD34-PE-Cy5, c-kit-PE), Nanog-AlexaFluor488, Oct4-AlexaFluor488, HoxB4-488, Sox2-Alexafluor488 and isotype-matched controls (IgG1κ and IgG2a,) were used as negative controls (BD Bioscience and Abcam). Stained cells were analyzed using the BD LSRII flow cytometer with 488-530/30, 488-575/26, and 488-695/40 filters to view Alexa488, PE and PE-Cy5 respectively. For the western blot, all cell extracts were prepared from CD34 + cells cultured in the defined growth medium. CD34 + cells not cultured in the growth medium were used as control cells. Equal protein concentration of 60 µg were separated by SDS-PAGE. Antibodies used were anti-CyclinD1 (1:100) (New England Biolab, Ipswich); anti-PCNA (1:100) (New England Biolab); and anti-β actin (1:300) (Sigma-Aldrich Company). Propidium iodide staining was used to analyze the cell cycle and K562 cell line was used as a positive control.
In vitro TAA studies. To test the CD34 + cells cultured conditioned medium, rat liver cells were seeded at 2.5 × 10 5 on 24-well plates. After 24 hour incubation, the cells were washed once with phosphate-buffered saline followed by α-minimum essential medium. The cells were then treated with liver toxin, TAA (0.2 µg/µl) (Sigma-Aldrich Company) or TAA with the conditioned medium (20 µl) in α-minimum essential medium. Standard culturing medium was used a positive control. The treated cells were incubated for 48 hours before performing cell viability and caspase-3 assays. Cell viability was performed with WST-1 kit (Roche, West Sussex, UK) and the caspase-3 activity was determined using Caspase-3 kit (Invitrogen). Cell morphological analysis was determined using an Olympus microscope.
In vivo animal studies. We used a clinically relevant cirrhotic animal model generated from a modified diethylnitrosamine feeding protocol. 26 Briefly, male Wistar rats (150-180 g) at 7 weeks of age were obtained from the Animal Center of National Taiwan University. All the experiments were conducted in accordance with the "Guide for the Care and Use of Laboratory Animals" prepared by the Institutional Animal Care and Use Committee of National Taiwan University. The animals were given diethylnitrosamine solution daily for 11 weeks, starting with 100 ppm in the first week. The average body weight of the animals was measured once a week and the concentration of diethylnitrosamine in their drinking water was adjusted in proportion to the body weight each week relative to that of the first week. After 11 weeks, the functional liver insufficiency was determined by the decreased bile flow as previously described. 26 Liver cirrhosis was determined by irreversible pathological change and collage accumulation in the liver after 9 weeks into the induction. For the in vivo study, 100 µl of the conditioned medium of CD34 + cells were injected via tail vein per cirrhotic rat in a 2-week period (a total of six injections per rat, eight rats per group). Control animals (n = 8) were injected with equal volume of control medium. The rats were killed 3 days after the last injection. Four-micrometer-thick liver sections were deparaffinized and rehydrated. For histological examination, the slides were stained with hematoxylin and eosin. Morphometric analysis was performed measuring the percentage of necrotic areas in 10 fields versus total section area at 200 magnification using the Digital Camera System HC-2500, Adobe Photoshop version 5.0J, and Image-Pro Plus version 3.0.1J (Media Cybernetics, Bethesda, MD).
For the in vivo study using the hepatotoxin TAA, rats were exposed to the TAA at 350 mg per Kg body weight, which induces liver damage. Male Wistar rats (Rattus norvegicus) were used in this study. Three groups were in the study: group 1 (n = 5, control with no TAA), group 2 (n = 7, TAA treated), and group 3 (n = 7, TAA treated followed by injection of cultured stem cells). The animals were treated with TAA for 2 consecutive days followed by immunosuppressant treatment (cyclosporine) at 0.5 mg/100 g body weight. The following day the rats were injected with 2 × 10 7 -cultured stem cells per rat. After 3 days of treatment, liver tissues and blood samples were taken from all the three groups for analysis. Liver functions were assayed by measuring bilirubin and albumin levels (BioAssay Systems, Hayward, CA). Hematoxylin and eosin staining were performed on rat liver tissues (5 µm sections). All animal experiments were carried out according to the institutional and Home Office guidelines.

Statistical analysis.
Statistical analyses were performed using Prism software (version 3.0). Statistical significant differences were determined by Student's t-test or one-way analysis of variance test. Data are presented as mean ± SDs. Figure S1. Effect of the expanded CD34 + cells transplanted on damaged rat liver in vivo.


extfieldsofstudy: Medicine Biology

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AtCTF7 is required for establishment of sister chromatid cohesion and association of cohesin with chromatin during meiosis in Arabidopsis

Background The establishment of sister chromatid cohesion followed by its controlled release at the metaphase to anaphase transition is necessary for faithful segregation of chromosomes in mitosis and meiosis. Cohesion is established by the action of Ctf7/Eco1 on the cohesin complex during DNA replication following loading of cohesin onto chromatin by the Scc2-Scc4 complex. Ctf7 is also required for sister chromatid cohesion during repair of DNA double strand breaks. Ctf7 contains an acetyltransferase domain and a zinc finger motif and acetylates conserved lysine residues in the Smc3 subunit of cohesin. In Arabidopsis CTF7 is encoded by a single gene and mutations in AtCTF7 cause embryo lethality indicating that the gene is essential. Results To study the function of Ctf7 in plants and to determine its role in sister chromatid cohesion, we constructed a conditional allele of AtCTF7 in Arabidopsis using an inducible RNA interference (RNAi) strategy, so as to avoid the embryo lethality caused by mutations in AtCTF7. We found that induction of RNAi against AtCTF7 caused severe inhibition and defects in growth during vegetative and reproductive stages as well as sterility. AtCTF7-RNAi plants displayed chromosome fragmentation and loss of sister chromatid cohesion during meiosis. Immunostaining for the cohesion subunit AtSCC3 showed a marked reduction in association of cohesin with chromatin during meiosis in AtCTF7-RNAi plants. Conclusions We find that AtCTF7 is essential for sister chromatid cohesion during meiosis in Arabidopsis and is required for association of cohesin with chromatin in prophase of meiosis.

Background
Proper chromosome segregation during cell division requires that sister chromatids produced by DNA replication are held together until their controlled separation at anaphase. This function is accomplished by the cohesin complex, whose conserved core subunits consist of the Structural Maintenance of Chromosome (SMC) proteins Smc1 and Smc3, the Sister Chromatid Cohesion (SCC) protein Scc3, and the α-kleisin protein Scc1 [1]. According to the ring model of cohesin action, Smc1 and Smc3 interact to form a V shaped heterodimer, closed by Scc1 with the help of Scc3, to form a ring that is considered to entrap sister chromatids and hold them physically together [2,3]. Cohesion is released at anaphase by the cleavage of Scc1 by separase, a protease that is activated by the anaphase promoting complex/ cyclosome (APC/C) [3].
In Saccharomyces cerevisiae, cohesion is established by Ctf7/Eco1, after cohesin has been loaded on chromatin by the Scc2-Scc4 complex [4][5][6]. Ctf7 establishes cohesion during S phase, and interacts with components of the DNA replication machinery, including PCNA and RFC [5][6][7]. These results led to a model in which sister chromatid cohesion is established concomitantly with DNA replication [8]. Ctf7 encodes a zinc finger protein with an active acetyltransferase domain, and it was found that Ctf7 acetylation of Smc3 on conserved lysines, was critical for establishment of cohesion by counteracting the Wpl1-Pds5 complex in preventing establishment of cohesion [9][10][11][12][13][14]. Establishment of cohesion has been suggested to occur in concert with lagging strand synthesis [15], and Smc3 acetylation leading to establishment of functional cohesion occurs only in association with replication [16]. Recycling of the Smc3 subunit is aided by deacetylation by Hos1 following cleavage of Scc1 by separase to release cohesion at the metaphase to anaphase transition, and is important for establishment of cohesion [17][18][19]. The eso1-H17 mutant in Schizosaccharomyces pombe exhibits delayed mitosis as a result of activation of the spindle checkpoint, and defective segregation of chromosomes in mitosis [20]. In Drosophila, mutations in Deco result in altered distribution of cohesin at metaphase, and premature entry into anaphase [21]. In humans, mutations in ESCO2 cause Roberts syndrome which results from a deficiency of cohesion around the centromeres, and encompasses a number of developmental abnormalities as well as mental retardation and renal and cardiac dysfunction [22].
The machinery for establishment of cohesion is conserved in Arabidopsis, and homologues of Scc2 and Ctf7 have been identified and functionally characterized. Mutations in AtSCC2 and AtCTF7 result in embryo lethality, however AtCTF7 is dispensable for endosperm growth [23,24]. Interestingly, AtCTF7 was found to possess acetyltransferase activity in vitro, and could complement the yeast ctf7-203 mutant, suggesting conserved biochemical function with its yeast counterpart [23]. By using a conditional RNA interference (RNAi) approach, it has been demonstrated that AtSCC2 is required during meiosis for sister chromatid cohesion, chromosomal axis formation and synapsis between homologues [24]. The function of AtCTF7 in establishment of sister chromatid cohesion in planta remains to be shown.
Here, we used a conditional RNAi approach to examine the role of AtCTF7 in sister chromatid cohesion, and to analyze the effects of the loss of AtCTF7 during vegetative and reproductive development. We found that downregulation of AtCTF7 severely inhibited growth during vegetative and reproductive stages, and resulted in both male and female sterility. During meiosis, AtCTF7-RNAi lines displayed typical loss of cohesion phenotypes, including abnormal chromosome organization, impaired chromosome synapsis and DNA fragmentation. Consistent with an expected involvement of AtCTF7 in cohesion, we found that sister chromatid cohesion was lost at both chromosome arms and centromeres in AtCTF7-RNAi plants. Finally, we found that AtSCC3 localization on chromatin was compromised during meiosis in AtCTF7-RNAi plants, indicating that AtCTF7 is required for association of cohesin with chromosomes in Arabidopsis, a feature that appears to be similar to Drosophila, where Deco is required for Scc1 association with chromosomes during M phase [21]. Overall, our results establish an essential role for AtCTF7 in vegetative development and in sister chromatid cohesion during meiosis.

Results
AtCTF7 is required for growth during vegetative and reproductive stages To examine the requirement for AtCTF7 during different stages of development we constructed a conditional allele using an inducible RNAi approach to deplete AtCTF7 mRNA. A hairpin RNAi construct for AtCTF7 was made and placed under control of the dexamethasoneinducible transactivator LhGR [25]. T1 transformants were grown and seeds collected. T2 plants were then grown and treated with dexamethasone either at the vegetative stage or after bolting. Treatment during the vegetative stage resulted in defective growth in both aerial and root tissues, accompanied by loss of greening ( Figure 1A and B, Additional file 1: Figure S1), indicating that AtCTF7 is required for plant growth during vegetative stages. Treatment with dexamethasone following bolting resulted in strong inhibition of growth of the inflorescence as well as sterility ( Figure 1C). Pollen was largely sterile and inviable following dexamethasone treatment ( Figure 1D-G), and lacked clearly defined sperm and vegetative nuclei ( Figure 1L and M, Additional file 1: Figure S2). Ovules in treated plants showed arrest in female gametogenesis starting at an early stage (1n) as well as missing embryo sacs ( Figure 1H-K). A proportion of ovules also showed defects in integument development ( Figure 1J). A total of 29 independent transformants were examined, out of which 5 lines showed strong growth defects and sterility following treatment with dexamethasone. Quantitative comparison of AtCTF7 expression indicated a reduction in treated compared to untreated control plants (Additional file 1: Figure S3), consistent with the growth defects being due to depletion of AtCTF7. Examination of AtCTF7 promoter activity using a P AtCTF7 nlsGUS reporter indicated that the AtCTF7 promoter is active in both the shoot and root meristematic regions ( Figure 2A and 2C), as well as in young buds and leaves (Figure 2A and 2B). Expression is reduced in older leaves and flowers ( Figure 2B). Within reproductive cells, expression was observed in pollen and in the female gametophyte ( Figure 2D and 2E), and is consistent with the defects in gametogenesis described above for AtCTF7-RNAi plants.
These results indicate that AtCTF7 is expressed in dividing cells and is required for normal development and growth during both vegetative and reproductive stages.

Knockdown of AtCTF7 results in defects in sister chromatid cohesion and chromosome organization during meiosis
To examine whether AtCTF7 is required in meiosis, chromosome spreads were carried out on male meiocytes from AtCTF7-RNAi plants ( Figure 3). Plants that had not been treated with dexamethasone showed normal pairing and synapsis, and normal organization of meiotic chromosomes ( Figure 3A-H). Early prophase stages were marked by the appearance of thin chromosome threads at leptotene, followed by zygotene and pachytene stages showing normal pairing and synapsis of chromosomes ( Figure 3A-D). Subsequent late prophase stages and meiosis I and II stages, were also seen to occur normally in untreated plants ( Figure 3E-H). In contrast, plants that were treated with dexamethasone showed severe defects in meiotic chromosome organization ( Figure 3I-L). The chromosomal defects were apparent early in prophase I for which the characteristic stages could not be clearly distinguished. Chromosomes appeared disorganized and had a patchy appearance with discontinuities. The synizetic knot did not form and mid to late prophase stages were marked by chromosomes appearing clumped and highly fragmented ( Figure 3I-K). Late prophase stages were characterized by the presence of condensed fragments of chromosomes which could exceed 20 in number indicating that fragmentation had taken place ( Figure 3L).
To examine the role of AtCTF7 in sister chromatid cohesion and pairing at the centromeric region, we carried out fluorescence in situ hybridization (FISH) using a centromere repeat probe that hybridizes to all the centromeres [24]. In untreated control plants, [8][9][10] centromere signals (mean = 9.5; n = 33) could be clearly detected at early prophase stages (leptotene) in meiocytes, whereas at zygotene and pachytene stages, 3-5 signals (mean = 3.1; n = 72) were observed ( Figure 4A-L). For dexamethasone treated plants, the number of signals at early prophase ranged between 9 and 20 (mean = 12.0; n = 64). The presence of greater than 10 signals at prophase I indicated loss of centromere cohesion in AtCTF7-RNAi plants ( Figure 4M-X). In addition there was variability in shape of the signal in AtCTF7-RNAi plants compared to untreated control plants which suggested defects in centromere organization. These results indicate that AtCTF7 is required for pairing, cohesion, and proper organization of centromeric regions. To examine arm cohesion we used a BAC probe specific for chromosome 4 ( Figure 5). In untreated plants, we observed two signals in early prophase, and a single signal in meiocytes at mid-prophase stages when chromosomes had fully synapsed ( Figure 5A-F), whereas in plants that were treated with dexamethasone, we observed more than two signals ( Figure 5G-L). Overall the results indicate that AtCTF7 is required for both centromere and arm cohesion.

AtCTF7-RNAi plants
In order to examine the role of AtCTF7 in sister chromatid cohesion and chromosome organization,we examined the localization of the cohesin subunit AtSCC3 [27], and the axial element protein ASY1 [28] during  male meiosis (Figure 6, Additional file 1: Figure S4). In untreated control plants, AtSSC3 immunostaining extended throughout the chromatin at early prophase stages, and marked chromosomal axes during zygotene and pachytene stages ( Figure 6A-P). In contrast, the level of AtSCC3 immunostaining, was greatly reduced in male meiocytes from AtCTF7-RNAi plants from early prophase onwards ( Figure 6Q-AF). ASY1 immunostaining in control plants is seen at early prophase and marks the chromosome axes at leptotene ( Figure 6C, G, K, and O). The staining pattern overlaps with that of AtSCC3 ( Figure 6D, H, L, P). In the case of AtCTF7-RNAi plants, association of ASY1 with chromatin was comparable to that of control plants ( Figure 6S, W, AA, AE). Thus, although ASY1 staining of chromatin shows significant overlap with that of AtSCC3, its association with chromatin appears to be independent of AtSCC3 which is known to precede ASY1, and is also consistent with analysis of the Atscc3-1 mutant [27].

Discussion
Ctf7/Eco1 proteins have been shown to control establishment of cohesion in yeast, Drosophila, and mammals [5,6,20,21,29]. In the case of plants, the Arabidopsis homolog of Ctf7 (AtCTF7) has been shown to be required for embryo development but not required for development of the endosperm [23], leaving open the possibility of a Ctf7-independent mechanism for sister chromatid segregation operating in meiosis. Evidence for a Ctf7-independent mechanism for sister chromatid segregation in yeast is based on the viability of an eco1Δ wpl1Δstrain [30]. A dosage dependent role for Ctf7 in meiosis in yeast has been suggested based on haploinsufficiency during sporulation [15]. Establishment of sister chromatid cohesion in meiosis in yeast may therefore be more sensitive to reduced dosage of Ctf7 than in mitosis. Alternatively, acetylation of other proteins by Ctf7 during meiosis may also be involved [31]. A role for Ctf7 in meiosis is also suggested from an examination of the localization and regulation of murine ESCO2 [32], however a requirement for Ctf7 in sister chromatid cohesion specifically during meiosis remains to be established. In this study we have shown using Arabidopsis as a model, that AtCTF7 is also required for sister chromatid cohesion in meiosis.
The conditional RNAi approach to examine the function of AtCTF7 in plants revealed defects in sister chromatid cohesion in meiosis. The establishment of sister chromatid cohesion in both arm and centromeric regions during meiosis was dependent upon AtCTF7. The meiotic phenotypes comprised defects early in prophase which presented as discontinuities in the thread-like appearance characteristic of leptotene and zygotene stages. In the most severe cases, the thread-like structure was largely absent and the chromatin appeared highly fragmented. At later prophase stages, the fragmented phenotype was further apparent by the presence of a large number of separated and condensed chromatin fragments. The results are consistent with a failure (in meiosis) to repair double strand breaks for which Ctf7 is known to be required [33]. The fragmentation phenotype is similar to that observed for Atrec8 and Atmnd1 mutants, which are defective in repair of meiotic double strand breaks [34,35]. However, since the RNAi strategy employed is not specific to meiosis, the possibility that the fragmentation phenotype may also be influenced by depletion of AtCTF7 earlier during mitosis in the progenitor cells of the meiocytes is not ruled out.
Arabidopsis mutants defective in both cohesion and formation of meiotic double strand breaks do not display such fragmentation phenotypes [27,36].
We observed severe defects in vegetative as well as reproductive growth and development, pointing to a role for AtCTF7 throughout the plant life cycle, and extending previous work showing an essential requirement for AtCTF7 in embryo development [23]. A P AtCTF7 nlsGUS reporter was strongly expressed in root and shoot meristems, and in young buds and leaves. In young developing leaves, a polarity in expression was observed with GUS staining confined to the basal part of the leaf and absent towards the distal portion. The gradient of expression is similar to that for the cell division marker CycB1;1::GUS [37]. Expression declined in older buds and was not observed in expanded rosette leaves. The expression of AtCTF7 is thus seen to occur in tissues that are undergoing active cell division, consistent with the known involvement of Ctf7 in promoting establishment of cohesion in conjunction with DNA replication [8]. Within reproductive cells, expression was observed in pollen and in the female gametophyte. The sterile phenotype we observed is likely to be accounted for mainly by the defects in meiosis as well as a possible contribution from a gametophytic component.
Establishment of cohesion by Ctf7 involves acetylation of conserved lysine residues in the Smc3 subunit of cohesion which inhibits the action of the Wpl1-Pds5 complex in preventing establishment of cohesion [9,10,[12][13][14]. In yeast, Drosophila, and human cells, Ctf7/Eco1 is required for the establishment of cohesion but not for association of cohesin with chromatin in interphase [5,21,29]. In Drosophila, a deco mutant shows reduced staining for the cohesin subunit Scc1 at prometaphase of mitosis [21]. The strong reduction in association of AtSCC3 with chromatin in early meiotic  prophase as revealed by immunostaining of meiocytes in AtCTF7-RNAi plants is similar to what has been observed for the deco mutant in prometaphase of mitosis, and suggests conservation of Ctf7/Eco1 function in plants.

Conclusions
In conclusion, our findings show that AtCTF7 is required for establishment of sister chromatid cohesion during meiosis in Arabidopsis, and that continued association of cohesin with chromatin in meiosis depends on AtCTF7.

Plant materials and growth conditions
The Arabidopsis thaliana strains used were of the Columbia ecotype (Col-0). Plants were grown as described in [38]. To generate transgenic Arabidopsis, constructs were mobilized into Agrobacterium tumefaciens strain AGL-1 using triparental mating, and transformed into Arabidopsis by vacuum infiltration as described in [39]. P AtCTF7 nlsGUS was transformed into wild-type Col, and AtCTF7-RNAi was transformed into a line carrying a P CaMV35S LhGR-N transgene [25]. Transgenic plants were selected on MS media, containing 120 μg/ml gentamycin (Sigma-Aldrich) for AtCTF7-RNAi transformants, and 50 μg/ml kanamycin for P AtCTF7 nlsGUS transformants, and were further confirmed by PCR.

Cloning procedures
The AtCTF7 promoter, comprising 646 bp upstream of the ATG and 45 bp from AtCTF7 coding sequence, was amplified by PCR using primers Fctf7gusHindIII and Rctf7gusnlsBamH1, and cloned as a BamH1-HindIII fragment in frame with a nlsGUS tag in the pBI101.2 binary vector. For the RNAi construct, 658 bp fragments were amplified by PCR using primer pairs F1rnaiXba1 and R1rnaiBamH1, and F1rnaiXho1 and R1rnaiEcoR1, and cloned as Xba1-BamH1 and Xho1-EcoR1 fragments in opposite directions in pKANNiBAL [40]. The RNAi cassette was excised as a XhoI-BamH1 fragment and cloned into the binary vector pZP222-6xPOP described in [25].

Dexamethasone treatment
Transgenic seeds were germinated on MS plates, and grown for 7 days after which they were transferred on MS plate containing 20 μM dexamethasone (Sigma). Seedlings were analyzed for phenotypes 7 days after transfer on dexamethasone plates. Treatment of adult plants after bolting was carried out by inclusion of 20 μM dexamethasone in the watering solution which was delivered by subirrigation. Samples for meiotic analysis were collected for analysis 5 days after the start of dexamethasone treatment.

RNA isolation and quantitative RT-PCR
Total RNA was isolated using Trizol (Invitrogen) following the manufacturer's protocol. cDNA synthesis was performed using Reverse Transcription System (Invitrogen SuperScript II) and oligo(dT) primers. Real Time PCR reactions were performed using SYBR Green PCR master mix (Applied Biosystems). GAPC was used as the internal normalization control. PCR was performed on the ABI Prism 7900 HT Sequence Detection System (Applied Biosystems) in a 384 well reaction plate according to the manufacturer's recommendations. Primers used were Ctf7qRTF and Ctf7qRTR for AtCTF7, and GAPRTF and GAPRTR for GAPC (Additional file 1: Table S1). Cycling parameters consisted of 2 minutes incubation at 50°C, 10 minutes at 95°C, and 40 cycles of 95°C for 15 seconds, 57°C for 30 seconds and 67°C for 30 seconds. The PCR reaction was performed in triplicate for each RNA sample, and the experiment was carried out on two different biological samples representing the same RNAi line. Specificity of the amplifications was verified at the end of each PCR run using ABI prism dissociation curve analysis software. Results from the ABI Prism 7900 HT Sequence Detection System were analyzed further using Microsoft Excel. Relative amounts of mRNA were calculated from threshold points (Ct values) located in the log-linear range of real time PCR amplification plots using the 2-ΔCt method. Standard deviations in Additional file 1: Figure S1 are for variation across biological samples.

Cytological procedures
Whole mount analysis of ovules was done after fixing and clearing the inflorescence in methyl benzoate as described previously [38]. Scanning electron microscopic (SEM) analysis of pollen was carried out using a Hitachi scanning electron microscope (model 3400 N, http://www.hitachi-hitec.com). Pollen viability was examined using Alexander staining [41]. For DAPI analysis of pollen, anthers were squashed and stained with DAPI (1 μg/ml). Meiotic chromosome spreads were carried out as described in [42], with minor modifications [43]. Observations were made on a Zeiss Axioplan 2 imaging microscope, using a Plan Apochromat 63 × oil immersion objective. Tissue from P AtCTF7 nlsGUS transgenic plants was stained for GUS activity as described in [38].
For FISH, chromosome spreads were carried out as described above, and FISH analysis was carried out according to the method described in [44], with minor modifications [35]. The 180-bp centromeric pAL1 repeat was used to detect centromere sequences [45]. A plasmid harboring two copies of the pAL1 repeat was subjected to PCR in the presence of Cy3-dATP (GE Healthcare), using PAL forward and reverse primers (Additional file 1: Table S1). BAC clones T19F6 and T22A6 from chromosome 4 were used as probes to monitor arm cohesion after being subjected to nick translation and labeling by Cy3-dATP (Roche). Slides were observed under a Zeiss Axioplan 2 imaging microscope equipped with a Plan Apochromat 63× oil immersion objective, using an excitation (Cy3, 550 nm) and long-pass emission (Cy3, 570 nm) filter.
For immunostaining, inflorescences were fixed as described in [46]. Young buds were dissected out and washed with 10 mM Citrate Buffer pH4.5 (1× CB), followed by digestion with a cell wall digesting enzyme mix containing 0.3% cellulase, 0.3% pectolyase, 0.4% cytohelicase (all Sigma) in 1× CB, and incubated for 30 min at 37°C. The enzyme mix was replaced with 1× PBS, and anthers were dissected out from buds on a slide and squashed using a 22×22 mm coverslip. The slide was snap-frozen by dipping in liquid nitrogen and the coverslip was immediately removed. Slides were then dried and dipped briefly in molten 1% gelatin, 1% agarose solution to cover the cells with a thin layer of gelatin-agarose and dried. Slides were rehydrated in 1× PBS, and digested with the enzyme mix described above for 30 min at 37°C. This was followed by permeabilization of the cells in 1× PBS, 1% Triton-X100, for 30 min and washing of the slides 2-3 times in 1× PBS containing 0.1% Triton-X100. Immunostaining was performed as described in [24], using ASY1 antibody at a 1:1000 dilution, and AtSCC3 antibody at 1:200 dilution. All secondary antibodies were used at a dilution of 1:100. Slides were mounted in 1ug/ml DAPI in Vectashield (VectorLabs). Cells were imaged using a Zeiss Axio Imager.Z1 microscope equipped with an apotome module, using a Plan-Apochromat 63× oilimmersion objective.
While this manuscript was under review, a related study appeared online by Bolanos-Villegas et al., on the role of AtCTF7 in DNA repair, mitosis, and meiosis [47].

Additional file
Additional file 1: Figure S1. Effect of dexamethasone treatment on wild type. Figure S2. Microscopic analysis of pollen in AtCTF7-RNAi. Figure S3. Quantitative reverse transcription PCR (q-RT-PCR) of AtCTF7 in AtCTF7-RNAi. Figure S4. Raw pictures of AtSCC3 and ASY1 immunostaining on untreated and treated AtCTF7-RNAi line. Table S1. List of primers used.


s2fieldsofstudy: Biology Medicine

text:
Impact of AT2-receptor stimulation on vascular biology, kidney function, and blood pressure

The angiotensin type 2 receptor (AT2R) and the receptor MAS are receptors within the renin–angiotensin system, which mediate tissue-protective actions such as anti-inflammation, antifibrosis, and antiapoptosis. In recent years, several programs have been launched in order to develop drugs that act as agonists on the AT2R or MAS to take therapeutic advantage of the protective and regenerative properties of these receptors. This review article will focus on recent data obtained in preclinical animal and in vitro models with new AT2R-agonistic molecules (Compound 21 and β-amino acid substituted angiotensin II) and with relevance for blood pressure (BP) regulation or hypertensive end-organ damage. These data will include studies on vasodilation/vasoconstriction in isolated resistance arteries ex vivo, studies on kidney function, studies on vascular remodeling, and studies that measured the net effect of AT2R stimulation on BP in vivo. Current data indicate that although AT2R stimulation causes vasodilation ex vivo and promotes natriuresis, it does not alter BP levels in vivo acutely – at least as long as there is no additional low-dose blockade of AT1R. However, AT2R stimulation alone is able to attenuate hypertension-induced vascular remodeling and reduce arterial stiffening, which in more chronic settings and together with the natriuretic effect may result in modest lowering of BP. We conclude from these preclinical data that AT2R agonists are not suitable for antihypertensive monotherapy, but that this new future drug class may be beneficial in combination with established antihypertensives for the treatment of hypertension with improved protection from end-organ damage.

Introduction
The renin-angiotensin system (RAS) is essentially involved in the control of blood pressure (BP) and body volume. 1 Angiotensin II (Ang II), acting via the angiotensin type 1 receptor (AT1R), causes vasoconstriction as well as sodium and water retention. 2 Furthermore, it is involved in the pathogenesis of hypertensive and diabetic end-organ damage by promoting inflammation and fibrosis. Pharmacological interference with the RAS by direct renin inhibitors, angiotensin-converting enzyme (ACE) inhibitors, or AT1R blockers (ARB) is a common therapeutic approach for the treatment of hypertension. 3 In addition, ACE inhibitors and ARBs are standard treatment for heart failure and diabetic nephropathy.
In the recent past, research on potential new drug targets within the RAS and the development of respective novel drugs have gained significant momentum. [4][5][6] These current efforts mainly aim at making therapeutic use of the so-called protective RAS,

154
Danyel et al which comprises the AT2R and the ACE2-angiotensin-(1-7) (Ang- [1][2][3][4][5][6][7])-MAS axis. Both the AT2R and the receptor MAS mediate a broad array of tissue-protective effects, including anti-inflammation, antifibrosis, antiapoptosis, neuroprotection, favorable metabolic effects, and vasodilation. In particular, their ability to counteract vasoconstriction, inflammation, and fibrosis makes the AT2R and MAS potential drug targets for the treatment of hypertension and related end-organ damage. Interestingly, there seems to be a positive feedback loop within the protective RAS because, as has been published very recently, the expression of ACE2, Ang-(1-7), and MAS, as well as ACE2 activity, were increased in the kidneys of obese Zucker rats treated for 2 weeks with the AT2R agonist CGP 42112A. 7 Regarding the AT2R, there are currently three types of new agonistic molecules with the potential for drug development: 1) the nonpeptide small molecule agonist Compound 21 (C21; Vicore Pharma, Gothenburg, Sweden, www.vicorepharma.com), 8 2) the cyclic Ang II derivative (one amino acid exchanged for another, unknown [unpublished] amino acid) LP2-3 (Lanthio Pharma, Groningen, the Netherlands, www.lanthiopep.nl), 9 and 3) a group of Ang II derivatives in which individual amino acids in the sequence of native Ang II are substituted by the respective β-amino acid. 10 C21 is currently in the final stage of preclinical development and is expected to enter clinical testing in 2014, the status of LP2-3 is unknown, and the β-amino acid substituted molecules are currently used only for academic purposes.
This review article will discuss physiology and potential therapeutic use of the AT2R with a focus on its role in BP regulation and hypertensive end-organ damage.

Vasodilation and blood pressure
Stimulation of the AT2R has been shown to act in a vasodilatory way in various species and multiple vascular beds such as mesenteric, [11][12][13][14][15] renal, [16][17][18] coronary, 19 cerebral, 20 cutaneous, 21 and uterine arteries. 22,23 In addition, AT2R knockout mice exhibit higher basal BP levels than wild-type mice, and they react with a stronger increase in BP to infusion of Ang II. 24,25 Conversely, in mice overexpressing AT2Rs in the vasculature, the pressor response to Ang II is markedly impaired. 26 Vasodilation was also shown for more recently developed AT2R agonists such as the first nonpeptide agonist C21 8 or for the new peptide agonists generated by substituting individual amino acids in the sequence of native Ang II by the respective β-amino acid (Table 1). 9 Vasodilation in response to these new ligands at concentrations between 10 −11 and 10 −6 M was observed in aorta from normotensive mice or hypertensive rats and in mouse mesenteric arteries in an AT2R-dependent manner, because these effects could be blocked by the AT2R antagonist PD 123319. 10,27 Vasodilation in response to C21 was also reported by Verdonk et al 28 in coronary, iliac, and mesenteric arteries of rats and mice; however, only at concentrations between 10 −6 and 10 −3 M. This vasodilatory response of C21 was unrelated to the AT2R because it could not be blocked by the AT2R antagonist PD 123319, which with 1 µmol/L, however, was underdosed for most tested concentrations of C21, and because it was absent in AT2R-deficient mice. The mechanism of AT2R-unrelated vasodilation in response to C21 is still unknown but may involve blockade of calcium transport into the cell. Vasodilation caused by very high concentrations of C21 was preceded by a short vasoconstriction, which was obviously due to AT1R stimulation, because it could be blocked by AT1R antagonists. 28 This observation was not surprising, as Bosnyak et al 27 had previously described that C21 stimulates the AT1R at very high concentrations, resulting in a rise in BP. In general, most small molecule drugs lose specificity at concentrations .1 µM or even lower. Well-known examples are β2-receptor mimetics used for the treatment of asthma, or β1-receptor blockers used for the treatment of hypertension, both of which bind to the respective other receptor subtype at high concentrations. For example, the Ki of the β2-receptor mimetic salmeterol, which possesses the highest selectivity of this drug class, is 24.6 nM for the β2-receptor and 1,600 nM for the β1-receptor; the selective β1-receptor blocker metoprolol has a Ki of 47 nM for the β1-receptor and of 2.960 nM for the β2-receptor. 29,30 With a Ki for the AT2R of 0.4 nM (2 nM for the human receptor) and .10,000 mM for the AT1R, the selectivity of C21 is not any worse than selectivity of so-called selective β-blockers or β2-receptor mimetics.
It seems logical that a hormone or drug that is able to cause vasodilation in isolated blood vessels would also be able to lower BP in animals and/or humans, as the vascular tone in resistance arteries is a major determinant of systolic BP. However, for the AT2R, this seems not to be the case ( Table 2). Several studies using peptide or nonpeptide AT2R agonists, including the new molecules discussed previously, demonstrated that an AT2Rmediated decrease in BP occurred only when there was a concomitant low-dose blockade of AT1Rs. For example, this was shown by the Carey et al 31

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AT2-receptor function in vasculature and kidney description of design and synthesis of C21. 8 In this study, C21 caused a fall in BP of up to 25 mmHg. However, it has to be noted that anesthesia probably caused unphysiological effects of this pharmacological intervention with the RAS, which is activated by anesthesia. 33 The observation that AT2R stimulation lowers BP only in the presence of low-level AT1R blockade indicates that in vivo a continuous angiotensinergic tone mediated via the AT1R seems dominant over any vasodilatory effect of AT2Rs. Consequently, AT2R agonists will most likely not become antihypertensive drugs suitable for monotherapy. However, due to their tissueprotective effects discussed as follows, the combination of established antihypertensives with AT2R agonists may result in better long-term prevention of hypertensive end-organ damage. Moreover, long-term therapy with AT2R agonists may have a modest BP-lowering effect due to structural changes of the vessel walls (see paragraph about "Vascular remodeling" below) and due to a recently described diuretic effect (see paragraph about "AT2 receptor activation in renal physiology and disease" below). 34-36 AT2R stimulation may further act antihypertensive by CNS related mechanisms that become apparent only if C21, which crosses the blood-brain barrier only very poorly, is applied intracerebroventricularly. 37 Normal pregnancy and preeclampsia are conditions during which expression of components of the RAS is altered in a way that the peripheral and tissue RAS are activated. 38 In preeclampsia in patients or in respective animal models, the ratio of expression of AT1R and AT2R changes is favor of the AT1R in placental and uterine tissue and arteries. [39][40][41] The AT2R has been described to act in a vasodilatory manner and to counteract the enhanced vasoconstrictive effect of Ang II via the AT1R during pregnancy. 22 Thus, it can

Vascular remodeling
Chronically elevated BP causes an inflammatory response followed by excess synthesis and accumulation of extracellular matrix mainly in the left cardiac ventricle, the kidneys, and the vascular wall. This fibrotic response to hypertension leads to deteriorated organ function and manifests as heart failure, renal disease, or vascular stiffening, the latter two of which reinforce the development of hypertension, thus establishing a vicious circle. 43 Early studies on the role of AT2R stimulation on vascular remodeling could show that the beneficial effects of ARBs were, at least in part, due to indirect AT2R stimulation (use of ARBs leads to an increase in renin release and thus a rise in angiotensin II levels, which, in turn, stimulates the unopposed AT2Rs; ARB-induced Ang II levels are, however, much lower than pharmacological AT2 agonist levels), as these effects could be reversed by blockade of AT2Rs. 44,45 Further indirect evidence for a favorable role of AT2Rs in vascular remodeling is coming from studies in AT2Rdeficient mice, which responded with augmented vascular hypertrophy of coronary, aortic, and femoral arteries to chronically elevated BP. [46][47][48] The impact of direct AT2R stimulation on hypertensioninduced vascular remodeling was studied recently in two studies in which C21 was applied orally to rats (Table 3). 49,50   The authors of these studies used different models of hypertension: ie, stroke-prone spontaneously hypertensive rats (SHR-SP) in one study, 50 and inhibition of endothelial nitric oxide synthase by application of Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) in the other study. 49 In both studies, the effect of AT2R stimulation was compared with the effect of an ARB alone or the combination of both. The main finding of both studies was that treatment with an AT2R agonist widely prevented the development of vascular hypertrophy and fibrosis. Remarkably, this reversal of hypertension-induced pathology was achieved without any significant effect on BP: ie, although BP stayed elevated in C21-treated rats, vascular remodeling was almost completely prevented. 49,50 The preventive effect of AT2R stimulation on vascular remodeling was shown in both studies to be related to a decrease in collagen deposition. Moreover, in our study in L-NAME-induced hypertension in rats, we measured pulse wave velocity, a marker for arterial stiffness and independent predictor of cardiovascular risk in patients, and we could again show that this indicator of vascular remodeling, which was increased in vehicle-treated hypertensive animals, was significantly attenuated in C21-treated, (still) hypertensive animals. 49 BP in our study was not significantly changed by treatment with C21, but there was a trend toward a reduction in BP, which may have been a result of reduced arterial stiffness. 49

Kidney function AT2-receptor deficiency in renal disease and injury
Considering the ubiquitous expression of the AT2R in fetal kidney, it may be surprising that AT2R knockout mice show no renal abnormalities in histology. 24,51 However, obvious differences in disease progression in AT2R knockout mice when compared with wild-type mice were revealed in the renal ablation model of renal injury. 51 Aggravated glomerular damage and impairment of renal function were shown in AT2R-deficient mice compared with wild-type mice, subsequently culminating in higher overall mortality. Moreover, albuminuria in knockout mice was pronounced and renal macrophage infiltration of glomerulus and interstitium increased compared with wild-type mice. Benndorf et al 51 ruled out systolic BP differences, podocyte or basal membrane damage, or upregulation of AT1Rs as possible causes for enhanced renal injury. Nonetheless, they elucidated one of the possible underlying mechanisms of disease progression in AT2R-deficient mice by showing a significant upregulation of asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase, in the knockout group, suggesting that the impairment in synthesis of nitric oxide may account for the aggravation of glomerular damage observed.
In accordance with these results, aggravated renal injury was observed in unilateral ureteral obstruction of AT2R-deficient mice, comprising severe interstitial fibrosis and greater abundance of fibroblasts and myofibroblasts in ipsilateral kidneys. 52 Moreover, in a model of type 1 diabetes in mice, AT2R knockout animals exhibited accelerated development of diabetic nephropathy. 53 Extracellular matrix (ECM) protein accumulation was measured by quantification of periodic acid-Schiff and Masson trichrome staining, as well as realtime quantitative polymerase chain reaction analysis of renal collagen IV messenger ribonucleic acid expression. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay was performed to semiquantify tubular apoptosis between groups. ECM accumulation and renal expression of collagen IV were significantly enhanced in nondiabetic AT2R knockout mice compared with nondiabetic controls. Additionally, tubular apoptosis was significantly increased in nondiabetic AT2R knockout mice. Accordingly, similar observations of increased ECM production and tubular apoptosis in AT2R-deficient animals were made in diabetic knockout animals when compared with wild-type mice, although values reached significance only for Masson trichrome staining arbitrary units. Chang et al 53 proposed that an increase in Heme oxygenase 1 expression, observed in proximal tubule cells of AT2R knockout mice, enhances oxidative stress. Moreover, an elevated ACE/ACE2 ratio in knockout animals could account for aggravation of renal injury during diabetic nephropathy.
It is important to note that deterioration of renal injury in AT2R-deficient mice may result from a persistent but yet unnoticed impairment in renal function that evolved during fetal development, due to the absence of the AT2R, which is usually expressed in high density during fetal life, and may not represent missing counter-regulatory actions of the receptor itself in the adult organism.

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Danyel et al summarized and will not be further discussed in this review. 54,55 Interpretation of results, however, is confounded by possible antagonistic effects of CGP 42112 and by agonistic effects of PD 123319 on the AT1R or even the AT2R, depending on timing and dosage. 28,56 Since C21 became available for experimental research, data about the role of the AT2R in kidney function have become more consistent and unequivocally strengthen the idea of a protective role of the AT2R in renal disease and of a natriuretic effect of AT2R stimulation (Table 4).
In Sprague Dawley rats, graded infusion of C21 (peak dosage: 300 ng/kg/min) significantly enhanced renal blood flow by simultaneously reducing renal vascular resistance in both female and male animals. 35 In addition, urine flow, urinary sodium excretion, and fractional sodium excretion showed a significant increase when compared with vehicletreated groups. All effects observed were abolished by concomitant administration of PD 123319. Interestingly, glomerular filtration rate remained stable in the C21 group, albeit the presence of renal vasodilation suggesting that the C21-induced increase in natriuresis is altered due to an effect on tubular function, but not due to hemodynamic effects. All effects were BP independent, as there was no statistically significant difference in BP between vehicle-and C21-treated animals. However, there was a small but significant difference between the C21-and the C21 plus PD 123319-treated animals (BP in PD 123319-treated animals being lower), but from these data it cannot be decided whether this difference was due to an effect of C21 or of PD 123319 or of both. 35 An AT2R-dependent but BP-independent natriuretic effect of C21 was further described recently in obese Zucker rats. 36 In analogy to the study by Hillard et al, 35 glomerular filtration rate remained unchanged in these animals, speaking again for a direct, tubular effect of C21.
In the two-kidney, one-clip rat model of hypertension (2K1C), Matavelli et al 57 evaluated the effects of AT2R stimulation in early renal inflammation. In 2K1C rats, kidney perfusion is reduced by unilateral clipping of the renal artery. Inflammatory markers of the subsequently developing ischemia were determined by in vivo recovery levels of renal intestinal fluid. Animals received vehicle, C21 (0.3 mg/kg/day, intraperitoneally), PD 123319 (10 mg/kg/day, osmotic minipump), or C21 plus PD 123319 over the 4-day course of the study. Unilateral stricture of the renal artery led to a significant increase in AT2R protein expression, which was even further enhanced in the C21 group but abolished by administration of PD 123319. Expression of tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β1, and interleukin (IL)-6 messenger ribonucleic acid and their renal interstitial fluid (RIF) recovery rates were significantly elevated in 2K1C compared with sham-operated animals. In contrast, nitric oxide and cyclic guanosine monophosphate RIF recovery rates were significantly reduced. Treatment with C21 reduced TNFα, TGFβ1, and IL-6 expression and, moreover, increased RIF recovery rates for nitric oxide and cyclic guanosine monophosphate. Extensive inflammatory cell infiltration observed by histological (hematoxylin/eosin) staining of 2K1C renal cortex and medulla was significantly reduced with C21 treatment when compared with the vehicle group. These effects were only partially inhibited by the AT2R antagonist PD 123319. 57 SHR-SPs represent an animal model of hypertension, characterized by the progressive development of renal damage and brain abnormality against the background of elevated Ang II blood levels. Oral treatment with C21 (peak dosage group: 10 mg/kg, suspended release from 0.5% sodium carboxymethylcellulose) significantly delayed the development of proteinuria and prevented the accumulation of high-molecular-weight proteins, which present markers of renal inflammation, in 24-hour urine electrophoresis. 58 Although plasma renin activity increased significantly in the vehicle group, plasma renin activity remained at basal levels in animals treated with C21. Finally, analysis of kidney histopathology revealed reduced renal lesions (attenuation of vascular lesions, tubular damage, luminal cast formation, glomerular sclerosis, inflammatory infiltrates) and attenuated renal macrophage infiltration in rats treated with C21.

Summary and conclusion
AT2R stimulation elicits effects on several physiological mechanisms, which contribute to the regulation of BP. Specifically, AT2R agonists induce vasodilation in isolated vessels ex vivo and they enhance natriuresis in vivo. Furthermore, in hypertensive rats, AT2R stimulation attenuates arterial stiffening. Nevertheless, in vivo BP is not altered acutely and only modestly chronically, the latter usually not reaching statistical significance. The lack of acute effect is probably due to some kind of counter-regulatory mechanism(s), which has not been characterized yet, but may involve activation of the "classic" RAS (increased levels of Ang II acting on the AT1R) or of the sympathetic nervous system. The trend toward a reduction in BP in more chronic settings seems likely due to secondary mechanisms such as natriuresis leading to volume reduction or attenuated vascular remodeling, resulting in lower peripheral resistance. According to these reviewed data from preclinical animal or in vitro/ex vivo studies, it is likely that AT2R agonists will be unsuitable as antihypertensive monotherapeutics in clinical use. However, they may enhance the effectiveness of other, established antihypertensives, and they may provide additive benefit in respect of protection from hypertensive end-organ damage. However, these assumptions need to be verified in future clinical studies.

Disclosure
U Muscha Steckelings received modest research support from Vicore Pharma. The other authors report no conflicts of interest in this work.


extfieldsofstudy: Biology Medicine

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Defending Sperm Function

ß-defensins are a large family of cationic peptides with antimicrobial activities in vitro against Gram-positive and Gramnegative bacteria, fungi, and enveloped viruses [1]. As such, they are thought to be an important component of the innate immune system and are expressed by cells such as phagocytes and epithelia that are involved in host defence against microbial infections. The defensins form pores within the target membranes allowing the movement of small molecules across the plasma membrane and causing decreased bacterial viability. Several ß-defensins are expressed by epithelial cells of the caput epididymis where they may have a role in innate immunity of the urogenital system [2] or protecting sperm from immunorecognition [3]. To date, however, the exact role of ß-defensins in contributing to innate immunity in vivo has not been unequivocally established. It is possible that ß-defensins may have other important physiological functions beyond their antimicrobial activities. Indeed, a mutation in the Defb103 defensin gene causes a dominant black coat colour in dogs [4]. The paper in this issue of PLOS Genetics by Yu S. Zhou and colleagues contributes to our understanding of the function of ß-defensins in vivo by showing that they also have a role in sperm function [5]. To my knowledge, this is the first unequivocal demonstration of a ßdefensin phenotype in transgenic knockout mice.

A New Role for ß-defensins ß-defensins are a large family of cationic peptides with antimicrobial activities in vitro against Gram-positive and Gramnegative bacteria, fungi, and enveloped viruses [1]. As such, they are thought to be an important component of the innate immune system and are expressed by cells such as phagocytes and epithelia that are involved in host defence against microbial infections. The defensins form pores within the target membranes allowing the movement of small molecules across the plasma membrane and causing decreased bacterial viability. Several ß-defensins are expressed by epithelial cells of the caput epididymis where they may have a role in innate immunity of the urogenital system [2] or protecting sperm from immunorecognition [3]. To date, however, the exact role of ß-defensins in contributing to innate immunity in vivo has not been unequivocally established. It is possible that ß-defensins may have other important physiological functions beyond their antimicrobial activities. Indeed, a mutation in the Defb103 defensin gene causes a dominant black coat colour in dogs [4]. The paper in this issue of PLOS Genetics by Yu S. Zhou and colleagues contributes to our understanding of the function of ß-defensins in vivo by showing that they also have a role in sperm function [5]. To my knowledge, this is the first unequivocal demonstration of a ßdefensin phenotype in transgenic knockout mice.

Events at Fertilization
Spermatozoa show exquisite structural adaptations for their destiny of egg fertilization. The genetic material is tightly compacted into a streamlined head, the mid-piece is packed with mitochondrial powerhouses, and a long tail provides efficient propulsion. The head is covered by the acrosome, a vesicular cap derived from the Golgi that contains enzymes required for fertilization. Freshly ejaculated sperm are motile but not capable of fertilization, as they need to undergo a process called capacitation, which involves biochemical and metabolic changes. These changes include removal of choles-terol from the sperm membranes. Capacitation allows the sperm to bind to the ZP3 protein, a component of the outer zona pellucida layer of the egg. Binding to ZP3 activates T-type and TRPC2 membrane channels that allow Ca 2+ entry into the sperm to enable acrosome exocytosis. In addition, a cation channel (CatSper) found in the tail region of the sperm that has a promiscuous activation profile can also allow Ca 2+ entry and may also play a role in facilitating the acrosome reaction. The acrosome reaction is an essential step to allow the sperm to fuse with the plasma membrane of the egg to complete fertilization. Fertilization causes release of Ca 2+ from intracellular stores in the egg, which induces exit from meiotic arrest and exocytosis of cortical granules to prevent multiple sperm entry.

ß-defensins Act as a Brake to Sperm Activation
In the article by Zhou et al. [5], the authors have identified that some ßdefensins are essential for normal sperm function and male fertility in the mouse. There are at least 50 ß-defensins genes in the mouse genome with a cluster of 31 located on chromosome 8. Analysis of the function of individual genes in vivo would be a Herculean task and might be compromised by functional redundancy such as in mice deficient for Defb1, which show only mild defects [6]. To overcome this problem, the authors took the pragmatic approach of deleting several genes at once. Deletion of a cluster of nine ßdefensin genes (Defb1, Defb50, Defb2, Defb10, Defb9, Defb11, Defb15, Defb35, and Defb13) produced mutant offspring with no obvious gross phenotype or increased inflammatory profiles under normal animal housing conditions. Mutant female mice were fertile, but males were infertile even though histological analysis showed normal spermatogenesis and sperm numbers in the epididymides.
The cause of the infertility was related to multiple defects in the sperm that would compromise their fertilization ability. The sperm showed increased fragility and decreased motility, probably caused by a disrupted microtubule structure in the tail. Around 20% of sperm isolated from the cauda (tail) part of the epididymis showed precocious sperm capacitation, as judged by the appearance of zonadhesin protein on the surface of the sperm. Twenty to twenty-eight percent of the sperm also showed a premature acrosome reaction which normally only occurs when sperm bind to the ZP3 protein. Consistent with the premature acrosome reaction, the mutant sperm showed increased levels of intracellular Ca 2+ compared to normal sperm.

Unanswered Questions
The data clearly identify a novel function for ß-defensins in sperm activation and male fertility. It is not yet known, however, whether these ß-defensins play a similar role in humans. Five of the nine ßdefensin genes that were deleted in the mouse have no human orthologues, so it is possible that these effects may be restricted to the mouse. Alternatively, the four genes that are conserved in both humans and mice (DEFB1/Defb1, DEFB106/Defb15, DEFB105Defb35, and DEFB107/Defb13) are all expressed in the human testes based on RNAseq expression profiling, so if any of these genes are responsible for the phenotype then they may show functional conservation between humans and mice.
It is also not known what the individual contribution of each of the deleted ßdefensin genes might be towards the phenotype. It is possible that the phenotype might be caused by the loss of only one of the nine ß-defensins genes. Analysis of the expression profile of each of these genes in the epididymis may provide a clue as to which is the most important. For example, the Defb11 gene is specific to the mouse and shows expression in epididymal epithelia. It might be possible to evaluate the effect of each ß-defensin protein by adding it to mutant sperm and testing whether it can prevent premature capacitation.
Another important question is to understand the mechanism by which these ß-defensins prevent premature activation of the sperm. The usual sequence of events in the sperm prior to fertilization is shown in Figure 1. Normally Ca 2+ entry occurs after binding to the egg ZP3 protein although some Ca 2+ can also enter by promiscuous activation of CatSper channels. The authors speculate that the ß-defensins might inhibit Ca 2+ channel activity in a way similar to the SPINK3 protein found in seminal fluid. The cationic nature of the ß-defensins will facilitate binding to the sperm plasma membrane where they would be well-placed to interfere with Ca 2+ channel opening. In addition, the ß-defensins may prevent premature sperm capacitation by inhibiting cholesterol removal from the sperm membranes or preventing hyperpolarization. Biochemical and electrophysiological studies should be able to test these hypotheses and provide a better insight into how ß-defensins modulate sperm activation. Figure 1. Events at egg fertilization and the possible roles of ß-defensin proteins in regulating this process. Sperm capacitation is required for egg fertilization and involves several cellular changes including loss of cholesterol from the sperm head and increased membrane hyperpolarization. ß-defensin proteins may inhibit these events to help prevent premature sperm capacitation. In addition, ß-defensins might also block Ca 2+ entry, which is required for the acrosome reaction. doi:10.1371/journal.pgen.1003889.g001