Mesenchymal Stromal Cell Derived Membrane Particles Are Internalized by Macrophages and Endothelial Cells Through Receptor-Mediated Endocytosis and Phagocytosis

Abstract

Mesenchymal stromal cells (MSC) are a promising therapy for inflammatory diseases. However, MSC are large and become trapped in the lungs after intravenous infusion, where they have a short survival time. To steer MSC immunoregulatory therapy beyond the lungs, we generated nm-sized particles from MSC membranes (membrane particles, MP), which have immunomodulatory properties, and investigated their internalization and mode of interaction in macrophages subtypes and human umbilical vein endothelial cells (HUVEC) under control and inflammatory conditions. We found that macrophages and HUVEC take up MP in a dose, time, and temperature-dependent manner. Specific inhibitors for endocytotic pathways revealed that MP internalization depends on heparan sulfate proteoglycan-, dynamin-, and clathrin-mediated endocytosis but does not involve caveolin-mediated endocytosis. MP uptake also involved the actin cytoskeleton and phosphoinositide 3-kinase, which are implicated in macropinocytosis and phagocytosis. Anti-inflammatory M2 macrophages take up more MP than pro-inflammatory M1 macrophages. In contrast, inflammatory conditions did not affect the MP uptake by HUVEC. Moreover, MP induced both anti- and pro-inflammatory responses in macrophages and HUVEC by affecting gene expression and cell surface proteins. Our findings on the mechanisms of uptake of MP under different conditions help the development of target-cell specific MP therapy to modulate immune responses.

Sperm-like nanocarriers for ultrafast delivery of antisense DNA

“…Generally, according to different sizes of entrapped substances and uptake mechanisms, endocytosis can be divided into phagocytosis, macropinocytosis, and receptor-mediated endocytosis. 23 Since phagocytosis refers to the process of ingesting particulate matter larger than 1 μm in diameter, and the size of nano-sperms does not qualify, we explored the two other ways. First, to address whether nanosperms would enter cells through various receptor-mediated endocytosis processes, methyl-beta-cyclodextrin (MβCD, which inhibits clathrin-mediated endocytosis), chlorpromazine (CPM, which inhibits caveolae-mediated endocytosis), nystatin (NYS, which inhibits kinin-dependent endocytosis) and dynasore (DYN, which inhibits lipid raft-mediated endocytosis) were utilized, 24,25 and the fluorescence intensity changed to varying degrees.…”

A novel pathway of functional microRNA uptake and mitochondria delivery

“…The uptake of miRNAs in the presence of serum was further validated in different cell types including HEK293T, A549, U87MG, Min6 and SGC-7901, using fluorescent miRNAs (Extended Data, figure 1c). As disrupting cellular microfilament networks can block endocytosis and micropinocytosis (Al Soraj et al , 2012; da Costa Goncalves et al , 2021), we further analyze the uptake of 5’-Cy5-miR-29a in the presence of serum after treating Hela cells with cytochalasin B, rottlerin, dynarose, nocodazole or other cytoskeleton disrupting reagents. After 1 h incubation, we found that rottlerin and nocodazole, two macropinocytosis inhibitor (Hufnagel et al , 2009), strongly prevented cellular uptake of 5’-Cy5-miR-29a, whereas other reagents displayed no inhibition on 5’-Cy5-miR-29a uptake (Fig.…”

Microvesicles released from activated CD4+ T cells alter microvascular endothelial cell function

“…In this regard, recent data suggested that mesenchymal stromal cell‐derived membrane particles might be internalised by endothelial cells through receptor‐mediated endocytosis and phagocytosis. 26 Accordingly, membrane particle uptake by endothelial cells involved the actin cytoskeleton and phosphoinositide 3‐kinase, which are implicated in macropinocytosis and phagocytosis. In the present study, MV.Act induced an increase in the F/G‐actin ratio in HMVECs consistent with actin polymerisation and cytoskeleton rearrangement.…”