Helium Conditioning Increases Cardiac Fibroblast Migration Which Effect Is Not Propagated via Soluble Factors or Extracellular Vesicles

Abstract

Helium inhalation induces cardioprotection against ischemia/reperfusion injury, the cellular mechanism of which remains not fully elucidated. Extracellular vesicles (EVs) are cell-derived, nano-sized membrane vesicles which play a role in cardioprotective mechanisms, but their function in helium conditioning (HeC) has not been studied so far. We hypothesized that HeC induces fibroblast-mediated cardioprotection via EVs. We isolated neonatal rat cardiac fibroblasts (NRCFs) and exposed them to glucose deprivation and HeC rendered by four cycles of 95% helium + 5% CO2 for 1 h, followed by 1 h under normoxic condition. After 40 h of HeC, NRCF activation was analyzed with a Western blot (WB) and migration assay. From the cell supernatant, medium extracellular vesicles (mEVs) were isolated with differential centrifugation and analyzed with WB and nanoparticle tracking analysis. The supernatant from HeC-treated NRCFs was transferred to naïve NRCFs or immortalized human umbilical vein endothelial cells (HUVEC-TERT2), and a migration and angiogenesis assay was performed. We found that HeC accelerated the migration of NRCFs and did not increase the expression of fibroblast activation markers. HeC tended to decrease mEV secretion of NRCFs, but the supernatant of HeC or the control NRCFs did not accelerate the migration of naïve NRCFs or affect the angiogenic potential of HUVEC-TERT2. In conclusion, HeC may contribute to cardioprotection by increasing fibroblast migration but not by releasing protective mEVs or soluble factors from cardiac fibroblasts.

Effect of hypercholesterolemia on circulating and cardiomyocyte-derived extracellular vesicles

“…After treatment of THP-1-ASC-GFP cells as described above, cells were lyzed in quiazol lysis reagent (Quiagen, Germantown, MD, USA) and stored at − 80 °C. Total RNA isolation, cDNA synthesis and qPCR measurements were performed as described earlier 86 . Measurements were performed on a LightCycler 480 Real-Time PCR System (Roche Diagnostics, Basel, Switzerland) using LightCycler ® RNA Master SYBR Green I reagent (Roche Diagnostics, Basel, Switzerland) with primers presented in Supplementary Table S2 , with the following protocol: Initiation: 95 °C 2 min; Amplification: (45x) 95 °C 5 s, 57 °C 10 s, 72 °C 20 s. Data were analyzed by ΔΔCt calculation method according to Schmittgen and Livak 2008 87 , using Hypoxanthine Phosphoribosyltransferase 1 (HPRT) as a housekeeping gene.…”

Saxagliptin Cardiotoxicity in Chronic Heart Failure: The Role of DPP4 in the Regulation of Neuropeptide Tone

“…Primary neonatal rat hearts were isolated from 1–3-day-old Wistar rats (~3–4 animal/24-well plate) as previously described [ 31 ]. Briefly, rats were disinfected with 70% ethanol and sacrificed by cervical dislocation.…”

“…Scratch assays were performed as reported previously [ 31 ]. Briefly, cells were seeded on 24-well plates, and were pretreated with 500 nM saxagliptin or its solvent for 1 h. After pretreatment, the cell monolayer was scratched with a 200 µL pipette tip in a 30° angle.…”

Noble Gases Therapy in Cardiocerebrovascular Diseases: The Novel Stars?

“…In contrast to the healthy Wistar Kyoto rats (WKRs), only a triple intervention of He conditioning can reduce cell damage after myocardial I/R in spontaneous hypertensive rats (SHR), suggesting the presence of a threshold in the hypertensive heart ( 54 ) ( Table 1 ). An in-vitro study indicated that He conditioning contributed to cardioprotection by increasing fibroblast migration, but not by releasing protective medium extracellular vesicles or soluble factors from the cardiac fibroblasts ( 88 ). Our recent study indicated that intraperitoneal injection of 99.999% He gas improved lipopolysaccharide (LPS)-induced left ventricular dysfunction and cavity enlargement in a dose-dependent manner, it is better at the dose of 1.0 ml/100 g ( 89 ).…”