Integrated Transcriptomic and Proteomic Analysis of Primary Human Umbilical Vein Endothelial Cells

Abstract

Understanding the molecular profile of every human cell type is essential for understanding its role in normal physiology and disease. Technological advancements in DNA sequencing, mass spectrometry and computational methods allow us to carry out multi-Omics analyses although such approaches are not routine yet. Human umbilical vein endothelial cells (HUVECs) are a widely-used model system to study pathological and physiological processes associated with the cardiovascular system. In this study, we employed next generation sequencing and high-resolution mass spectrometry to profile the transcriptome and proteome of primary HUVECs. Analysis of 145 million paired-end reads from next generation sequencing confirmed expression of 12,186 protein-coding genes (FPKM≥0.1), 439 novel long non-coding RNAs and revealed 6,089 novel isoforms that were not annotated in GENCODE. Proteomics analysis identified 6,477 proteins including confirmation of N-termini for 1,091 proteins, isoforms for 149 proteins and 1,034 phosphosites. A database search to specifically identify other post-translational modifications provided evidence for a number of modification sites on 117 proteins which included ubiquitylation, lysine acetylation and mono, di- and tri-methylation events. Based on the data from this study and a survey of other databases, we provide evidence for 11 “missing proteins,” which are proteins for which there was insufficient or no protein level evidence. Peptides supporting missing protein and novel events were validated by comparison of MS/MS fragmentation patterns with synthetic peptides. Finally, we identified 245 variant peptides derived from 207 expressed proteins in addition to alternate translational start sites for seven proteins and evidence for novel proteoforms for five proteins resulting from alternative splicing. Overall, we believe that the integrated approach employed in this study is widely applicable to study any primary cell type for deeper molecular characterization.

Functionalized Vascular Structure in Bioengineered Liver Identified with Proteomics

“…Compared with other relevant HUVEC/liver proteomics studies with strong cation exchange chromatography, our lower protein extraction efficiency in recellularized scaffolds should be improved in future study. 25,26 Furthermore, coculture of endothelial cells, pericytes, and smooth muscle cells may be an important strategy to further improve angiogenesis, myogenesis, and vascular function.…”

Spire1 and Myosin Vc promote Ca2+-evoked externalization of von Willebrand factor in endothelial cells

“…However, the known interaction partners of Spire1—FMN1 and FMN2 [ 30 , 50 , 51 ]—appear not to be expressed in HUVEC in significant amounts at the RNA (Fig. S7) and protein level [ 16 , 52 ]. On the other hand, in invadosomes Spire1 seems to form a complex with mDia1 [ 34 ] and the human diaphanous homolog 1 was found to significantly associate with both Rab27a and Rab3b in HUVEC ( p < 0.05) [ 16 ].…”

SICA-mediated cytoadhesion of Plasmodium knowlesi-infected red blood cells to human umbilical vein endothelial cells

“…Thus, these molecules are receptor candidates of these cells for cytoadhesion of P. knowlesi -iRBCs. As for HUVECs, comprehensive proteomics analysis revealed that of the above mentioned 6 molecules the highest expressions observed were for PECAM1 (Peptide spectrum match (PSM) = 431), followed by ICAM1 (PSM = 59), and low expression of VCAM1 (PSM = 0.21); whereas CSPG4, P-selectin, and CD36 were not detected 23 . It would be of interest to examine if the first three molecules are receptors of SICA-HUVEC.…”