Extracellular vesicle cross-talk between pulmonary artery smooth muscle cells and endothelium during excessive TGF-β signalling: implications for PAH vascular remodelling

Abstract

Background: Excessive TGF-β signalling has been shown to underlie pulmonary hypertension (PAH). Human pulmonary artery smooth muscle cells (HPASMCs) can release extracellular vesicles (EVs) but their contents and significance have not yet been studied. Here, we aimed to analyse the contents and biological relevance of HPASMC-EVs and their transport to human pulmonary arterial endothelial cells (HPAECs), as well as the potential alteration of these under pathological conditions. Methods: We used low-input RNA-Seq to analyse the RNA cargoes sorted into released HPASMC-EVs under basal conditions. We additionally analysed the effects of excessive TGF-β signalling, using TGF-β1 and BMP4, in the transcriptome of HPASMCs and their EVs. We then, for the first time, optimised Cre-loxP technology for its use with primary cells in vitro, directly visualising HPASMC-to-HPAEC communication and protein markers on cells taking up EVs. Furthermore we could analyse alteration of this transport with excessive TGF-β signalling, as well as by other cytokines involved in PAH: IL-1β, TNF-α and VEGFA. Results: We were able to detect transcripts from 2417 genes in HPASMC-EVs. Surprisingly, among the 759 enriched in HPASMC-EVs compared to their donor cells, we found Zeb1 and 2 TGF-β superfamily ligands, GDF11 and TGF-β3. Moreover, we identified 90 genes differentially expressed in EVs from cells treated with TGF-β1 compared to EVs in basal conditions, including a subset involved in actin and ECM remodelling, among which were bHLHE40 and palladin. Finally, using Cre-loxP technology we showed cell-to-cell transfer and translation of HPASMC-EV Cre mRNA from HPASMC to HPAECs, effectively evidencing communication via EVs. Furthermore, we found increased number of smooth-muscle actin positive cells on HPAECs that took up HPASMC-EVs. The uptake and translation of mRNA was also higher in activated HPAECs, when stimulated with TGF-β1 or IL-1β.

PERK Inhibition Promotes Post-angioplasty Re-endothelialization via Modulating SMC Phenotype Changes

“…42 Similarly, when switching to an inflammatory phenotype, SMC could secrete a range of pro-inflammatory factors (e.g., BMPs, CXCLs, ready-to-translate mRNAs) to damage EC homing, regeneration, and barrier function. 20,41,[43][44][45][46] In line with the most recent report regarding the negative impact of SMCderived CXCL10 on re-endothelialization, our in vitro and in vivo data implicated that CXCL10 was possibly one of the paracrine signaling factors that transmitted the impact of PERK from SMC onto EC. 20 EV-mediated paracrine regulation represents a novel SMC-EC interaction mechanism.…”

“…Baker AH's group initiated a series of studies on characterizing the coding and noncoding RNA cargos in EVs derived from pulmonary SMC, which led to the identification of several key candidates (e.g., miR-143, SMILR, TGFB3 mRNA) with profound influence to adjacent vascular cells. 39,45,53 Kapustin et al 47,54 provided the first proteomic analysis of EVs from phenotypically converted SMC and their implications in vascular calcification. In a recent report, Langley et al 55 examined the secretomic profiles from lipid-loaded human SMC as well as the ECM proteomic signatures in symptomatic carotid plaques.…”

Extracellular Endothelial Cell-Derived Vesicles: Emerging Role in Cardiac and Vascular Remodeling in Heart Failure

“…Therefore, numerous metabolic risk factors, such as resistance to insulin, hyperglycemia, abdominal obesity, and hyperuricemia, are considered to be early triggers for the mobilization of endothelial progenitor cells from bone marrow and peripheral tissue. These factors can also influence the transformation of several cells, such as fibroblasts and smooth muscle cells of vasculature into cells with endothelial cells' phenotype (83)(84)(85)(86). This process is under strong epigenetic control and circulating EVs originated from activated and apoptotic endothelial cells and their precursors are able to regulate the repair of tissues such as endothelium and vasculature myocardium through attraction of cells with high innate ability to post-natal transformation (87,88).…”

Extracellular Vesicle miRNAs in the Promotion of Cardiac Neovascularisation

“…Zomer et al ( 2015 ), previously showed that in this system, only Cre mRNA and not Cre protein is transferred into EVs. By performing qRT-PCR de la Cuesta et al ( 2019 ) confirmed efficient loading of Cre mRNA into HPASMC-EVs. Using this system, the authors showed that HPASMC-EV-mediated Cre recombination resulted in a colour switch from red to green in reporter + HPAECs, effectively evidencing communication via EVs.…”

“…Recently, Cre-loxP system was also introduced as a very promising strategy to study EV uptake by cancer cells (Zomer et al, 2016 ). Adapting this system, de la Cuesta et al ( 2019 ), visualised direct transfer of human pulmonary artery smooth muscle cells (HPASMCs) EVs to human pulmonary arterial endothelial cells (HPAECs). In particular, donor HPASMCs were transduced with a Cre recombinase lentiviral vector and HPAECs with a reporter lentiviral vector, that carried DsRed and eGFP separated by a loxP site.…”