352 - Therapeutic Effects of Extracellular Vesicles Derived from VEGF-Overexpressing Mesenchymal Stromal Cells in Neonatal Hyperoxic Lung Injury

Publication Number: 1336.352

Yea Jin Lee, Samsung Medical Center, yongin-si, Kyonggi-do, Republic of Korea; Misun Yang, Samsung Medical Center, seoul, Seoul-t'ukpyolsi, Republic of Korea; Se In Sung, Samsung Medical Center, seoul, Seoul-t'ukpyolsi, Republic of Korea; So Yoon Ahn, Samsung Medical Center, seoul, Seoul-t'ukpyolsi, Republic of Korea; Yun Sil Chang, samsung medical center, Gangnam-gu, Seoul-t'ukpyolsi, Republic of Korea

Background: We previously demonstrated that vascular endothelial growth factor(VEGF) plays a key role in mediating the therapeutic effects of mesenchymal stromal cells(MSCs), particularly in promoting tissue repair and angiogenesis in hyperoxia-induced lung injury.

Objective: This study aimed to investigate the therapeutic efficacy of extracellular vesicles derived from VEGF-overexpressing mesenchymal stromal cells in a neonatal rat model of hyperoxia-induced lung injury and right ventricular hypertrophy.

Design/Methods: VEGF-MSCs were generated from TERT-immortalized human Wharton's jelly-derived MSCs, and their EVs were collected using tangential flow filtration. Non-transduced MSCs were used as controls. In the in vivo model, newborn Sprague-Dawley rat pups were exposed to normoxia(21% O₂) or hyperoxia(90% O₂) from postnatal day(P) 1 to P14. VEGF overexpressed EVs(VEGF-EVs) or naïve EVs(1×10⁹ particles/50 µL) were administered intratracheally at P5. At P14, alveolarization, cell death, angiogenesis, and inflammation were evaluated in paraffin-embedded lung tissues. In heart tissues, right ventricular hypertrophy(RVH) was assessed by measuring the thickness of the right ventricle(RV), left ventricle(LV), and interventricular septum(IVS) to calculate RV/LV and RV/(LV+IVS) ratios.

Results: In hyperoxic rat lungs, angiogenesis indicated by vWF staining intensity was significantly decreased. However, naïve EV and VEGF-EV improved this hyperoxia induced impaired angiogenesis. Moreover, VEGF-EV displayed superior angiogenic effects than naïve EV. Hyperoxia induced activation of alveolar macrophages which evidenced by ED-1 staining was significantly attenuated by naïve EV and further improved by VEGF-EV. The mean alveolar volume, mean linear intercept, and the number of TUNEL-positive cells were significantly increased in hyperoxia compared with normoxia, but these findings were significantly improved in both VEGF-EV and naïve EV, without significant difference between two treatments. The hyperoxia significantly increased RV/LV and RV/(LV+IVS) ratios, indicating RVH. These ratios were significantly decreased in both VEGF-EV and naïve EV, without significant difference between two treatments.

Conclusion(s): In a hyperoxia-induced BPD newborn rat model, VEGF- EVs demonstrated superior therapeutic efficacy in enhancing angiogenesis and attenuating pulmonary inflammation compared to naïve EVs. However, VEGF-EVs did not further improve alveolarization, reduce lung cell death, or alleviate right ventricular hypertrophy relative to control EVs. Therefore, additional studies investigating the dose-dependent effects may be warranted.

Figure 1. Therapeutic effect of VEGF-EVs on pulmonary morphology in neonatal rats with hyperoxic lung injury
(a) Representative figures of H&E stained lung sections. (b) Mean alveolar volume (MAV), (c) Mean linear intercept (MLI). (d) Representative figures of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL), von Willebrand factor (vWF) and ED1 stained lung sections. The number of (e) terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL)-positive cells, and (f) von Willebrand factor (vWF) -positive vessels and (g) ED1-positive cells per high power field (HPF). Data are shown as mean ± SEM. n = 10, 19, 19 and 17 in normoxic control group (NC), hyperoxic control group (HC), hyperoxia with control EVs administration (H-CE) and hyperoxia with VEGF-EVs administration (H-VE), respectively. * p < 0.05 compared to NC group, # p < 0.05 compared to HC group.

Figure 2. Effects of VEGF-EVs on right ventricular hypertrophy (RVH) in neonatal rats with hyperoxic lung injury.
(a) Respresentative figures of H&E stained heart serial sections. (b) right ventricular (RV) to left ventricular (LV) ratio, and (c) Fulton's index [RV/(LV+inter ventricular septa (IVS))], reflecting right ventricular hypertrophy. Data are shown as mean ± SEM. n = 12, 7, 8 and 7 in NC, HC, H-CE and H-VE, respectively. * p < 0.05 compared to NC group, # p < 0.05 compared to HC group.