Trophoblast–endothelium signaling involves angiogenesis and apoptosis in a dynamic bioprinted placenta model

Trophoblast invasion and remodeling of the maternal spiral arteries are required for pregnancy success. Aberrant endothelium–trophoblast crosstalk may lead to preeclampsia, a pregnancy complication that has serious effects on both the mother and the baby. However, our understanding of the mechanisms involved in this pathology remains elementary because the current in vitro models cannot describe trophoblast–endothelium interactions under dynamic culture. In this study, we developed a dynamic three-dimensional (3D) placenta model by bioprinting trophoblasts and an endothelialized lumen in a perfusion bioreactor. We found the 3D printed perfusion bioreactor system significantly augmented responses of endothelial cells by encouraging network formations and expressions of angiogenic markers, cluster of differentiation 31 (CD31), matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-9 (MMP9), and vascular endothelial growth factor A (VEGFA). Bioprinting favored colocalization of trophoblasts with endothelial cells, similar to in vivo observations. Additional analysis revealed that trophoblasts reduced the angiogenic responses by reducing network formation and motility rates while inducing apoptosis of endothelial cells. Moreover, the presence of endothelial cells appeared to inhibit trophoblast invasion rates. These results clearly demonstrated the utility and potential of bioprinting and perfusion bioreactor system to model trophoblast–endothelium interactions in vitro. Our bioprinted placenta model represents a crucial step to develop advanced research approach that will expand our understanding and treatment options of preeclampsia and other pregnancy-related pathologies.     

Mesenchymal stem cells and collagen patches for anterior cruciate ligament repair

AIM: To investigate collagen patches seeded with mesenchymal stem cells(MSCs) and/or tenocytes(TCs) with regards to their suitability for anterior cruciate ligament(ACL) repair. METHODS: Dynamic intraligamentary stabilization utilizes a dynamic screw system to keep ACL remnants in place and promote biological healing, supplemented by collagen patches. How these scaffolds interact with cells and what type of benefit they provide has not yet been investigated in detail. Primary ACL-derived TCs and human bone marrow derived MSCs were seeded onto two different types of 3D collagen scaffolds, Chondro-Gide?(CG) and Novocart?(NC). Cells were seeded onto the scaffolds and cultured for 7 d either as a pure populations or as "premix" containing a 1:1 ratio of TCs to MSCs. Additionally, as controls, cells were seeded in monolayers and in co-cultures on both sides of porous high-density membrane inserts(0.4 μm). We analyzed the patches by real time polymerase chain reaction, glycosaminoglycan(GAG), DNA and hydroxyproline(HYP) content. To determine cell spreading and adherence in the scaffolds microscopic imaging techniques, i.e., confocal laser scanning microscopy(c LSM) and scanning electron microscopy(SEM), were applied.RESULTS: CLSM and SEM imaging analysis confirmed cell adherence onto scaffolds. The metabolic cell activity revealed that patches promote adherence and proliferation of cells. The most dramatic increase in absolute metabolic cell activity was measured for CG samples seeded with tenocytes or a 1:1 cell premix. Analysis of DNA content and c LSM imaging also indicated MSCs were not proliferating as nicely as tenocytes on CG. The HYP to GAG ratio significantly changed for the premix group, resulting from a slightly lower GAG content, demonstrating that the cells are modifying the underlying matrix. Real-time quantitativepolymerase chain reaction data indicated that MSCs showed a trend of differentiation towards a more tenogenic-like phenotype after 7 d.CONCLUSION: CG and NC are both cyto-compatible with primary MSCs and TCs; TCs seemed to perform better on these collagen patches than MSCs.     

A physical map for the Amborella trichopoda genome sheds light on the evolution of angiosperm genome structure

Background

Recent phylogenetic analyses have identified Amborella trichopoda, an understory tree species endemic to the forests of New Caledonia, as sister to a clade including all other known flowering plant species. The Amborella genome is a unique reference for understanding the evolution of angiosperm genomes because it can serve as an outgroup to root comparative analyses. A physical map, BAC end sequences and sample shotgun sequences provide a first view of the 870 Mbp Amborella genome.

Results

Analysis of Amborella BAC ends sequenced from each contig suggests that the density of long terminal repeat retrotransposons is negatively correlated with that of protein coding genes. Syntenic, presumably ancestral, gene blocks were identified in comparisons of the Amborella BAC contigs and the sequenced Arabidopsis thaliana, Populus trichocarpa, Vitis vinifera and Oryza sativa genomes. Parsimony mapping of the loss of synteny corroborates previous analyses suggesting that the rate of structural change has been more rapid on lineages leading to Arabidopsis and Oryza compared with lineages leading to Populus and Vitis. The gamma paleohexiploidy event identified in the Arabidopsis, Populus and Vitis genomes is shown to have occurred after the divergence of all other known angiosperms from the lineage leading to Amborella.

Conclusions

When placed in the context of a physical map, BAC end sequences representing just 5.4% of the Amborella genome have facilitated reconstruction of gene blocks that existed in the last common ancestor of all flowering plants. The Amborella genome is an invaluable reference for inferences concerning the ancestral angiosperm and subsequent genome evolution.     

Antiquity and evolution of the MADS-box gene family controlling flower development in plants

MADS-box genes in plants control various aspects of development and reproductive processes including flower formation. To obtain some insight into the roles of these genes in morphological evolution, we investigated the origin and diversification of floral MADS-box genes by conducting molecular evolutionary genetics analyses. Our results suggest that the most recent common ancestor of today's floral MADS-box genes evolved roughly 650 MYA, much earlier than the Cambrian explosion. They also suggest that the functional classes T (SVP), B (and Bs), C, F (AGL20 or TM3), A, and G (AGL6) of floral MADS-box genes diverged sequentially in this order from the class E gene lineage. The divergence between the class G and E genes apparently occurred around the time of the angiosperm/gymnosperm split. Furthermore, the ancestors of three classes of genes (class T genes, class B/Bs genes, and the common ancestor of the other classes of genes) might have existed at the time of the Cambrian explosion. We also conducted a phylogenetic analysis of MADS-domain sequences from various species of plants and animals and presented a hypothetical scenario of the evolution of MADS-box genes in plants and animals, taking into account paleontological information. Our study supports the idea that there are two main evolutionary lineages (type I and type II) of MADS-box genes in plants and animals.     

Conservation and divergence in the AGAMOUS subfamily of MADS-box genes: evidence of independent sub- and neofunctionalization events

The MADS-box gene AGAMOUS (AG) plays a key role in determining floral meristem and organ identities. We identified three AG homologs, EScaAG1, EScaAG2, and EScaAGL11 from the basal eudicot Eschscholzia californica (California poppy). Phylogenetic analyses indicate that EScaAG1 and EScaAG2 are recent paralogs within the AG clade, independent of the duplication in ancestral core eudicots that gave rise to the euAG and PLENA (PLE) orthologs. EScaAGL11 is basal to core eudicot AGL11 orthologs in a clade representing an older duplication event after the divergence of the angiosperm and gymnosperm lineages. Detailed in situ hybridization experiments show that expression of EScaAG1 and EScaAG2 is similar to AG; however, both genes appear to be expressed earlier in floral development than described in the core eudicots. A thorough examination of available expression and functional data in a phylogenetic context for members of the AG and AGL11 clades reveals that gene expression has been quite variable throughout the evolutionary history of the AG subfamily and that ovule-specific expression might have evolved more than twice. Although sub- and neofunctionalization are inferred to have occurred following gene duplication, functional divergence among orthologs is evident, as is convergence, among paralogs sampled from different species. We propose that retention of multiple AG homologs in several paralogous lineages can be explained by the conservation of ancestral protein activity combined with evolutionarily labile regulation of expression in the AG and AGL11 clades such that the collective functions of the AG subfamily in stamen and carpel development are maintained following gene duplication.     

Expression pattern shifts following duplication indicative of subfunctionalization and neofunctionalization in regulatory genes of Arabidopsis

Gene duplication plays an important role in the evolution of diversity and novel function and is especially prevalent in the nuclear genomes of flowering plants. Duplicate genes may be maintained through subfunctionalization and neofunctionalization at the level of expression or coding sequence. In order to test the hypothesis that duplicated regulatory genes will be differentially expressed in a specific manner indicative of regulatory subfunctionalization and/or neofunctionalization, we examined expression pattern shifts in duplicated regulatory genes in Arabidopsis. A two-way analysis of variance was performed on expression data for 280 phylogenetically identified paralogous pairs. Expression data were extracted from global expression profiles for wild-type root, stem, leaf, developing inflorescence, nearly mature flower buds, and seedpod. Gene, organ, and gene by organ interaction (G x O) effects were examined. Results indicate that 85% of the paralogous pairs exhibited a significant G x O effect indicative of regulatory subfunctionalization and/or neofunctionalization. A significant G x O effect was associated with complementary expression patterns in 45% of pairwise comparisons. No association was detected between a G x O effect and a relaxed evolutionary constraint as detected by the ratio of nonsynonymous to synonymous substitutions. Ancestral gene expression patterns inferred across a Type II MADS-box gene phylogeny suggest several cases of regulatory neofunctionalization and organ-specific nonfunctionalization. Complete linkage clustering of gene expression levels across organs suggests that regulatory modules for each organ are independent or ancestral genes had limited expression. We propose a new classification, regulatory hypofunctionalization, for an overall decrease in expression level in one member of a paralogous pair while still having a significant G x O effect. We conclude that expression divergence specifically indicative of subfunctionalization and/or neofunctionalization contributes to the maintenance of most if not all duplicated regulatory genes in Arabidopsis and hypothesize that this results in increasing expression diversity or specificity of regulatory genes after each round of duplication.     

Alternate paths of evolution for the photosynthetic gene rbcL in four nonphotosynthetic species of Orobanche

We have determined the nucleotide sequence for the Rubisco large subunit from four holoparasitic species of Orobanche. Intact open reading frames are present in two species (O. corymbosa and O. fasciculata), whereas the remaining species (O. cernua and O. ramosa) have rbcL pseudogenes. Sequences for rbcL 5'-UTRs from species of Orobanche have few changes in the promoter and ribosome binding sites compared to photosynthetic higher plants. Comparison of rbcL 3'-UTR sequences for Nicotiana, Ipomoea, Cuscuta, and Orobanche reveal that nucleotide sequences from parasitic plants have regions capable of forming stem-loop structures, but 56–69 nt are deleted upstream of the stem-loop in the parasitic plants compared to their photosynthetic relatives. Although rbcL pseudogenes of O. cernua and O ramosa have many large and small deletions, few indels are shared in common, implying that their common ancestor probably had an intact rbcL reading frame. Intact rbcL reading frames in O. corymbosa and O. fasciculata retain a bias of synonymous over nonsynonymous substitutions and deduced protein sequences are consistent with potentially functional Rubisco large subunit proteins. A conservative model of random substitution processes in pseudogene sequences estimates that the probability is low (P<0.028) that these sequences would retain an open reading frame by chance. Species of Orobanche have either had recent photosynthetic ancestors, implying multiple independent losses of photosynthesis in this genus, or the rbcL gene may serve an unknown function in some nonphotosynthetic plants.