TDO2‐augmented fibroblasts secrete EVs enriched in immunomodulatory Y‐derived small RNA

Figures

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  FIGURE 1. Concentrated conditioned media from TDO2‐transduced fibroblasts are therapeutic in a 3‐week mouse model of myocardial infarction. (a) Three‐week murine model of myocardial infarction. (b) Improved ejection fraction after 3 weeks when treated with TDO2‐CCM (n = 7–9 per group). (c) Cardiac Troponin I levels were significantly decreased at 24 h in animals treated with TDO2‐CCM, (n = 4 per group). (d,e) End diastolic and systolic volumes tended to be less in TDO2‐CCM treated hearts (n = 7–9 per group) though differences were not significant. (f) Masson's trichrome staining of left ventricles after 3 weeks (representative images; arrows point to scar tissue) indicates reduction in scar formation in TDO‐CCM treated hearts (scale bar: 2 mm). b–e were analyzed using one‐way ANOVA with Sidak's multiple comparison test. Error bars represent standard deviation,*p < 0.05, **p < 0.01, ***p < 0.001. biological replicates. (c–g) Analyzed using one‐way ANOVA with Sidak's multiple comparison test. Error bars represent standard deviation,*p < 0.05, **p < 0.01, ***p < 0.001

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  FIGURE 2. Sequencing of mouse cardiac tissues 24‐h post myocardial infarction and treatment reveals inflammatory regulation with TDO2‐EVs treatment. (a) Heat map of the top differentially expressed genes in left ventricle (partially including the infarcted region). (b) Volcano plot depicting the differential expression of genes when treated with TDO2‐CCM versus HDF‐CCM. Cyclin‐dependent kinase inhibitors 1a‐c and il6, il1b, il23a and nlrp3 genes highlighted. (c–g) qPCR validation of differential expressed genes involved in the cdkn1a/p21 cell senescence/inflammasome pathway, n = 3 biological replicates. (g) qPCR validation of cdkn1a, cdkn1b and cdkn1c expression in the infarcted region of the left ventricle 24‐h post‐MI, n = 3

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  FIGURE 3. NT4 is a short non‐coding RNA fragment with immunomodulatory function derived from human Y RNA‐4 (hY4). (a) Schematic of the hY4 sequence and functional regions. (b) Sequence of NT4 and its position within the hY4 fragment EV‐YF1. (c) qPCR showing NT4 enrichment in CCM of fibroblasts transduced with TDO2‐activating lentivirus (TDO2‐CCM) compared to those from fibroblasts transduced with a lentivirus containing a control vector (VC‐CCM). (d) QPCR of NT4 from SEC‐isolated EVs compared to the EV‐depleted fraction. (e) Absolute quantification (NT4 copies/103 particles) of TDO2‐CCM, VC‐CCM, SEC‐isolated TDO2‐EVs and the EV‐depleted fraction. (f) RNAse protection assay demonstrating NT4 is encapsulated inside TDO2‐EVs. (g) Broad expression of the monocyte marker, CD68, in bone marrow‐derived macrophages. (h) Equivalent uptake of TDO2‐ and HDF‐EVs (EV ceramide dye; red, phalloidin: green and nuclei: blue) by bone marrow derived macrophages at 30, 60 and 180 min post exposure as measured by internalisation of red signals inside macrophages and, (i) area under the curve measurement of cumulative signal at all three time points. (j) Representative image of TDO2‐ and HDF‐EVs (EV ceramide…

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  FIGURE 4. Inflammatory activation and recruitment in bone marrow derived macrophages is regulated by pre‐treatment with TDO2‐EV or NT4. (a–d) Protein array demonstrating that pre‐treatment of LPS‐activated macrophages with TDO2‐CM or NT4 resulted in decreased levels of the inflammatory mediators IL‐1b (a), TNFα (b) and the chemokines CXCL1 (c), CXCL2 (d) and CXCL3 (e). (f) The pro‐angiogenic mediator VEGF was also significantly upregulated in macrophages pre‐treated with TDO2‐EV or NT4. Depletion of NT4 in TDO2‐CCM diminished or entirely abrogated those effects (a–f; all statistical analyses are done in comparison to the vehicle group). (g) Schematic of the rat model of ischemic cardiac injury and reperfusion. (h) Serum levels of Cardiac Troponin I were significantly decreased in NT4 and TDO2‐EV treated animals compared to those receiving no treatment, n = 4. (i) Quantification of the percentage of infarct normalised to section weight, n = 5 animals/group. (j) Representative images of TTC staining (infarct region outlined in black). The white‐stained tissue is indicative of infarcted (dead and dying) tissue