Mu, J. | Search bioRxiv

bioRxiv2022

Expanding adult tubular microvessels on stiff substrates with endothelial cells and pericytes from the same tissue

Microvessels are essential for tissue engineering and regeneration. In current methods, endothelial cells are usually cultured in commercially available media and form a monolayer of cell sheets on stiff substrates and a tubular structure when cultured with soft hydrogels. To mimic the microvessels in vivo, researchers usually coculture the endothelial cells and pericytes from different adult tissues or derived from pluripotent stem cells in a three-dimensional hydrogel. However, there is a challenge for these models to reflect tissue-specific characteristics due to the vascular heterogeneity throughout the body. Here, we established a culture model for expanding adult tubular microvessels on stiff substrates with endothelial cells and pericytes derived from the same tissue. We isolated microvessels from adult rat subcutaneous soft connective tissue and cultured them on regular plastic dishes. We performed a series of screenings and formulated a custom-made medium (Medium-X), containing mainly antioxidants and three small molecules, Chir99021, A83-01, and Y27632. Medium-X significantly promoted adult microvessel growth while maintaining their characteristic tubular morphology up to 8 weeks in vitro, contrary to the monolayer of endothelial cell sheets in the commercially available medium EGM2MV. Transcriptomic analysis showed that Medium-X maintained the tubular morphology of microvessels by promoting angiogenesis and vascular remodeling while suppressing oxidation and lipid metabolic pathways. The model presented in this study can be applied to other organs for expanding organ-specific microvessels for tissue engineering and vascular regeneration.

bioengineering↗

bioRxiv2021

5-methylcytosine modification by Plasmodium NSUN2 stabilizes mRNA and mediates the development of gametocytes

5-methylcytosine (m5C) is an important epitranscriptomic modification involved in mRNA stability and translation efficiency in various biological processes. However, it remains unclear if m5C modification contributes to the dynamic regulation of the transcriptome during the developmental cycles of Plasmodium parasites. Here, we characterize the landscape of m5C mRNA modifications at single nucleotide resolution in the asexual replication stages and gametocyte sexual stages of rodent (P. yoelii) and human (P. falciparum) malaria parasites. While different representations of m5C-modified mRNAs are associated with the different stages, the abundance of the m5C marker is strikingly enhanced in the transcriptomes of gametocytes. Our results show that m5C modifications confer stability to the Plasmodium transcripts and that a Plasmodium ortholog of NSUN2 is a major mRNA m5C methyltransferase in malaria parasites. Upon knock-out of P. yoelii nsun2 (pynsun2), marked reductions of m5C modification were observed in a panel of gametocytogenesis-associated transcripts. These reductions correlated with impaired gametocyte production in rodent and human malaria parasites. Restoration of the nsun2 gene in the knock-out parasites rescued the gametocyte production phenotype as well as m5C modification of the gametocytogenesis-associated transcripts. Together with the mRNA m5C profiles for two species of Plasmodium, our findings demonstrate a major role for NSUN2-mediated m5C modifications in mRNA transcript stability and sexual differentiation in malaria parasites. SignificanceModifications of RNA including methylations of cytosine (m5C) and adenosine (m6A) have important roles in RNA metabolism, cellular responses to stress, and biological processes of differentiation and development. Here, we report on the profiles of m5C mRNA modifications in malaria parasites that infect rodents (Plasmodium yoelii) and humans (Plasmodium falciparum). These parasites have genes that encode homologs of human and plant NSUN2 methyltransferases (m5C "writers"). We show that one of these homologs, termed PyNSUN2, stabilizes mRNA transcripts in P. yoelii and mediates m5C-associated development of the parasite sexual stages (gametocytes). Further research on m5C and other epitranscriptomic modifications may yield new insights into molecular pathways of gametocyte development and mosquito infectivity that can be exploited to interrupt malaria transmission.

microbiology↗