Journal: Frontiers in Cell and Developmental Biology

Article Title: Platelet-Derived Extracellular Vesicles Increase Col8a1 Secretion and Vascular Stiffness in Intimal Injury

doi: 10.3389/fcell.2021.641763

Figure Lengend Snippet: PIP 3 /Akt signaling regulated the production of Col8a1. (A) IPA software was used to analyze related molecules that can participate in the regulation of “formation of extracellular matrix” and (B) the core signaling pathways in the cytoplasm and nucleus participated in this network. (C) Western blotting was used to measure the expression of phosphorylated Akt in VSMCs, and the results showed that Akt was significantly activated after pEV stimulation. (D–F) ELISA and western blotting were used to analyze molecules involved in the activation of Akt. Only the level of PIP 3 increased significantly in VSMCs after pEV stimulation, (D) while PDK1 (E) and mTORC (F) were unchanged. (G–J) The specific PIP 3 /Akt signaling inhibitor LY294002 was used to examine the effect of PIP 3 /Akt signaling on the production of Col8a1. (G) LY294002 significantly abrogated the expression of p-Akt induced by pEVs and blocked the mRNA (I) and protein expression of col8a1 (H,J) . The values are shown as the mean ± SD, * P < 0.05, ** P < 0.01 vs. control ( n = 4 biological replicates).

Article Snippet: The possible biological processes and functional classifications were obtained with Ingenuity Pathway Analysis (IPA) software (Content version: 57662101, Qiagen, Germany).

Techniques: Software, Protein-Protein interactions, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Activation Assay, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Platelet-Derived Extracellular Vesicles Increase Col8a1 Secretion and Vascular Stiffness in Intimal Injury

doi: 10.3389/fcell.2021.641763

Figure Lengend Snippet: miR-92a-3p upregulated the production of Col8a1 by inhibiting the expression of PTEN. (A) mRNA sequencing was used to analyze activated platelets and their secreted pEVs, and the top 30 miRs in pEVs are shown. (B) IPA software was used to analyze the downstream target molecules of the top 30 miRs in pEVs, and these molecules were mainly related to “cellular movement” and “cardiovascular system development.” (C) IPA software was used to analyze the core downstream network that intersected PI3K/AKT signaling and identified 7 key molecules involved in the Akt signaling pathway. (D) IPA software and a literature search were used to analyze miRs and their target mRNAs. Among the 30 miRs, 14 miRs targeted the same molecule, PTEN. (E,F) Real-time RT-PCR was used to measure miR-92a-3p expression in response to intimal injury or pEV stimulation in vitro . The results showed that in response to either pEV stimulation (E) or intimal injury (F) , miR-92a-3p expression increased significantly. (G) In the intimal injury model, FISH was used to measure miR-92a-3p expression in situ . The results validated that miR-92a-3p expression levels were significantly upregulated in VSMCs compared with the self-contralateral carotid artery. (H) The PTEN 3’UTR has a binding site for miR-92a-3p. (I) The qPCR results showed that the miR-92a-3p mimic or inhibitor significantly increased or decreased miR-92a-3p expression in VSMCs, respectively. (J) Western blotting indicated that in VSMCs, the miR-92a-3p mimic reduced the protein expression of PTEN, while the miR-92a-3p inhibitor increased PTEN expression compared with that of the control. (K) A dual luciferase reporter gene system was used to examine the luciferase activity in wild-type (WT) and mutant PTEN 3’UTRs in negative control and miR-92a-3p mimic-treated HEK-293T cells. miR-92a-3p significantly reduced the luciferase activity of the wild-type PTEN 3’UTR compared with the negative control in three culture replicates. (L) Western blotting indicated that in VSMCs, miR-92a-3p mimics increased the protein expression of Col8a1, which was mediated by PTEN. The values are shown as the mean ± SD, * P < 0.05, ** P < 0.01 vs. control ( n = 4 biological replicates).

Article Snippet: The possible biological processes and functional classifications were obtained with Ingenuity Pathway Analysis (IPA) software (Content version: 57662101, Qiagen, Germany).

Techniques: Expressing, Sequencing, Software, Quantitative RT-PCR, In Vitro, In Situ, Binding Assay, Western Blot, Control, Luciferase, Activity Assay, Mutagenesis, Negative Control