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Fibroblast Generated Extracellular Vesicles Induces Pro-Inflammatory and Metabolic Reprogramming in Bone Marrow Derived Macrophages
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A2090 - Fibroblast Generated Extracellular Vesicles Induces Pro-Inflammatory and Metabolic Reprogramming in Bone Marrow Derived Macrophages
Author Block: S. Kumar, S. Riddle, M. Li, H. Zhang, A. Flockton, B. A. McKeon, M. G. Frid, K. R. Stenmark; University of Colorado Denver, Aurora, CO, United States.
Background:
There is emerging evidence that perivascular inflammation characterized by macrophage accumulation and activation is causative to the vascular remodeling process associated with pulmonary hypertension (PH). Our group has recently reported that pulmonary artery adventitial fibroblasts from patients with PH and from calves with hypoxia-induced PH (PH-Fibs) can recruit, retain, and activate
macrophages. Extracellular vesicles (EV) are nanosized, membrane-bound vesicles released into the extracellular environment to mediate inter-cellular communication. EVs carry proteins, nucleic acids, lipids, signaling molecules and metabolites, which can modulate specific functions in recipient cells. Yet, if and how EV promotes macrophage recruitment and activation within this fibroblast-macrophage cell communication remain undefined.
Methods and Results:
Bovine PH-Fibs and control fibroblasts (CO-Fibs) were cultured upto 80% confluency; thereafter, media was replaced with fresh media with 10% exosome-depleted bovine calf serum and cultured under normoxic (21% O2) or hypoxic (1% O2) conditions for 48 hrs. Subsequently, conditioned media was harvested and EVs were isolated by traditional methods using serial centrifugation at low speed, followed by ultracentrifugation at 100,000g. Isolated EVs were analyzed using the Nanosight LM10 system equipped with a blue laser (405 nm). Wild type (wt) primary bone marrow derived macrophages (BMDMs) were exposed to EV from PH-Fibs and CO-Fibs under normoxic conditions.
EV from bovine PH-Fibs and CO-Fibs cultures were used to activate mouse BMDM. EV from PH-Fibs but not from CO-Fibs resulted in significant increases in IL-1b and IL-6 production in BMDM. Accordingly, next we compared of mRNA expression of some selected glycolytic pathway genes and we observed that levels of Glut1, Hk2, Pgk1, Pkm, Ldha and Mct1 expression were increased in mouse BMDMs treated with PH-Fib derived EV. In addition, BMDMs treated with EV from PH-Fibs displayed increased motility compared with EVs from CO-Fibs.
Conclusions: Extracellular vesicles produced by activated fibroblasts from pulmonary hypertensive vessels mediated macrophage polarization towards a pro-inflammatory phenotype. PH-Fibs derived EV induces metabolic reprograming of BMDM and also PH-Fibs derived EV enhance the motality of BMDM. This suggests that EV could be a critical player in the vascular remodeling process associated with Pulmonary Hypertension.
Author Block: S. Kumar, S. Riddle, M. Li, H. Zhang, A. Flockton, B. A. McKeon, M. G. Frid, K. R. Stenmark; University of Colorado Denver, Aurora, CO, United States.
Background:
There is emerging evidence that perivascular inflammation characterized by macrophage accumulation and activation is causative to the vascular remodeling process associated with pulmonary hypertension (PH). Our group has recently reported that pulmonary artery adventitial fibroblasts from patients with PH and from calves with hypoxia-induced PH (PH-Fibs) can recruit, retain, and activate
macrophages. Extracellular vesicles (EV) are nanosized, membrane-bound vesicles released into the extracellular environment to mediate inter-cellular communication. EVs carry proteins, nucleic acids, lipids, signaling molecules and metabolites, which can modulate specific functions in recipient cells. Yet, if and how EV promotes macrophage recruitment and activation within this fibroblast-macrophage cell communication remain undefined.
Methods and Results:
Bovine PH-Fibs and control fibroblasts (CO-Fibs) were cultured upto 80% confluency; thereafter, media was replaced with fresh media with 10% exosome-depleted bovine calf serum and cultured under normoxic (21% O2) or hypoxic (1% O2) conditions for 48 hrs. Subsequently, conditioned media was harvested and EVs were isolated by traditional methods using serial centrifugation at low speed, followed by ultracentrifugation at 100,000g. Isolated EVs were analyzed using the Nanosight LM10 system equipped with a blue laser (405 nm). Wild type (wt) primary bone marrow derived macrophages (BMDMs) were exposed to EV from PH-Fibs and CO-Fibs under normoxic conditions.
EV from bovine PH-Fibs and CO-Fibs cultures were used to activate mouse BMDM. EV from PH-Fibs but not from CO-Fibs resulted in significant increases in IL-1b and IL-6 production in BMDM. Accordingly, next we compared of mRNA expression of some selected glycolytic pathway genes and we observed that levels of Glut1, Hk2, Pgk1, Pkm, Ldha and Mct1 expression were increased in mouse BMDMs treated with PH-Fib derived EV. In addition, BMDMs treated with EV from PH-Fibs displayed increased motility compared with EVs from CO-Fibs.
Conclusions: Extracellular vesicles produced by activated fibroblasts from pulmonary hypertensive vessels mediated macrophage polarization towards a pro-inflammatory phenotype. PH-Fibs derived EV induces metabolic reprograming of BMDM and also PH-Fibs derived EV enhance the motality of BMDM. This suggests that EV could be a critical player in the vascular remodeling process associated with Pulmonary Hypertension.
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