standardized-scalable-assay-to-study-perfused-3d-angiogenic-sprouting

Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current ...

Leiden University Medical Center

There is an increasing prevalence of chronic kidney disease (CKD), which associates with the development of interstitial fibrosis. Pericytes (perivascular fibroblasts) provide a major source of α-SMA-positive myofibroblasts that are responsible for the excessive deposition of extracellular matrix. In order to identify pericyte long non-coding RNAs (lncRNAs) that could serve as a target to decrease myofibroblast formation and counteract the progression of kidney fibrosis we employed two models of experimental kidney injury, one focused on kidney fibrosis (unilateral ureteral obstruction; UUO), and one focused on acute kidney injury that yields kidney fibrosis in the longer term (unilateral ischemia-reperfusion injury; IRI). This was performed in FoxD1-GC;tdTomato stromal cell reporter mice that allowed pericyte fate tracing. Tomato red-positive FoxD1-derivative cells of control and injured kidneys were FACS-sorted and used for lncRNA and mRNA profiling yielding a distinctive transcriptional signature of pericytes and myofibroblasts with 244 and 586 differentially expressed lncRNAs (>twofold,  < 0.05), in the UUO and IRI models, respectively. Next, we selected two differentially expressed and conserved lncRNAs, Rian (RNA imprinted and accumulated in nucleus) and Miat (Myocardial infarction associated transcript), and explored their potential regulatory role in myofibroblast formation through knockdown of their function with gapmers. While Miat was upregulated in myofibroblasts of UUO and IRI in mice, gapmer silencing of Miat attenuated myofibroblast formation as evidenced by decreased expression of , , , and , as well as decreased α-SMA and pro-collagen-1α1 protein levels. In contrast, silencing Rian, which was found to be downregulated in kidney myofibroblast after IRI and UUO, resulted in increased myofibroblast formation. In addition, we found microRNAs that were previously linked to Miat (miR-150) and Rian (14q32 miRNA cluster), to be dysregulated in the FoxD1-derivative cells, suggesting a possible interaction between miRNAs and these lncRNAs in myofibroblast formation. Taken together, lncRNAs play a regulatory role in myofibroblast formation, possibly through interacting with miRNA regulation, implicating that understanding their biology and their modulation may have the potential to counteract the development of renal fibrosis.

Long Non-coding RNAs Rian and Miat Mediate Myofibroblast Formation in Kidney Fibrosis.

Previously we identified plasma microRNA (miR) profiles that associate with markers of microvascular injury in diabetic nephropathy (DN) patients. However, miRs circulate in extracellular vesicles (EVs) or in association with high-density lipoprotein (HDL) or the RNA-binding protein Argonaute-2 (Ago-2). Given that the EV- and HDL- mediated miR transfer towards endothelial cells (ECs) regulates cellular quiescence and inflammation, we hypothesized that the distribution of miRs amongst carriers affects microvascular homeostasis in DN. Therefore, we determined the miR expression in EVs, HDL- and Ago-2 fractions isolated from EDTA-plasma of healthy controls, patients with diabetes mellitus +/- early DN (eGFR>30 ml/min/1.73 m2) and DN patients (eGFR<30 ml/min/1.73 m2). Consistent with our hypothesis, we observed alterations in miR-carrier distribution in plasma of DM and DN patients compared to healthy controls. Both miR-21 and miR-126 increased in EVs of DN patients, whereas miR-660 increased in the Ago-2 fraction and miR-132 decreased in the HDL fractions. Moreover, in vitro, differentially expressed miRs improved EC barrier formation (EV-miR-21) and rescued the angiogenic potential (HDL-miR-132) of ECs cultured in serum from DM and DN patients. In conclusion, miR measurement in EVs, HDL and Ago-2 may improve the biomarker sensitivity of these miRs for microvascular injury in DN while carrier-specific miRs can improve endothelial barrier formation (EV-miR-21/126) or exert a pro-angiogenic response (HDL-miR-132).

Article	Year
Long Non-coding RNAs Rian and Miat Mediate Myofibroblast Formation in Kidney Fibrosis. Frontiers in pharmacology\| PubMed ID: 30914951	2019
Diabetic Nephropathy Alters the Distribution of Circulating Angiogenic miRNAs Between Extracellular Vesicles, HDL and Ago-2. Diabetes\| PubMed ID: 31506344	2019

Wendy Stam

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