Nanoparticle endothelial delivery of PGC-1α attenuates hypoxia-induced pulmonary hypertension by attenuating EndoMT-caused vascular wall remodeling

Fig. 1. PGC-1α expression was decreased in pulmonary artery endothelial cells in hypoxia-induced PAH mouse model. Wild-type (WT) mice were exposed to normoxia (n = 6) or hypoxia (10% O2, n = 6) for 4 weeks. A, Representative images of immunohistochemistry staining of PGC-1α in pulmonary arteries. Scale Bar: 100 μm. B–C, PGC-1α protein expression in the lung tissues, as detected by Western blot (B) and quantified by normalizing to GAPDH level. *p < 0.01 compared with the Control (Ctrl) group. D, PGC-1α expression in endothelial cells of normoxia (Ctrl) and hypoxia-treated lungs. Co-immunostaining of CD31 (red) with PGC-1α (green) was performed in mouse pulmonary arteries. Scale Bar: 100 μm. E, The percentage of PGC-1α + CD31+ cells relative to the total CD31+ cells as quantified from 10 sections of each artery. *p < 0.01 compared to the Ctrl group, n = 6. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2. PGC-1α overexpression in pulmonary artery endothelial cells via nanoparticle delivery in hypoxia-induced PAH mice. Nanoparticles containing empty vector or PGC-1α overexpression plasmid (OE: PGC-1α) was injected to mice via tail vein. After 7 days, mice were exposed to normoxia or hypoxia (10% O2) for 4 weeks. A, Immunohistochemistry (IHC) staining for PGC-1α (upper panel) and co-immunostaining of PGC-1α with CD31 (lower panels) of lung tissues. PGC-1α was downregulated in PAECs after hypoxia treatment. However, nanoparticle delivery of PGC-1α plasmid restored the PGC-1α expression. Scale bar: 100 μm. B, Relative PGC-1α levels in IHC staining as shown in A, as quantified by normalizing to the PGC-1α signal intensity in the normoxia group (Ctrl), which was set as 1. *P < 0.01 vs. Ctrl; #P < 0.01 vs. Vector group; n = 6. C, The percentage of PGC-1α+CD31+ cells relative to the total CD31+ cells, as quantified from 10 sections of each animals. D-E, Flow cytometry analyses. Lung tissues were digested, and PAECs were isolated by CD31 magnetic beads. Flow cytometry was performed to quantify PGC-1α+ PACEs (D), and the percentage of PGC-1α+ CD31+ cells relative to the total CD31+ cells…

Fig. 3. Overexpression of PGC-1α in pulmonary artery endothelial cells ameliorated hypoxia-induced PAH and vascular remodeling in lungs. Nanoparticles containing control vector or PGC1-a overexpression plasmid (OE:PGC-1α) was injected to mice via tail vein. Mice were exposed to normoxia or hypoxia (10% O2) for 4 weeks. A-B, Assessment of mean pulmonary aorta pressure (mPAP). *P < 0.01 vs. Ctrl; #P < 0.01 vs. Vector group; n = 6. C, Ratio of right ventricle to left ventricle + septum weight [RV/(LV + S)]. *P < 0.01 vs. Ctrl; #P < 0.01 vs. Vector group; n = 6. D-E, H &E (D) and elastic van Gieson staining (E) of the pulmonary arteries. Artery wall thickening of pulmonary arteries with different diameters are shown. Scale Bar: 100 μm. F, Quantification of pulmonary arterial muscularization in arteries with diameters 20–70 μm. N = non-muscularized vessels; P = partially muscularized vessels. F = fully muscularized vessels. *P < 0.01 vs. Ctrl; #P < 0.01 vs. the vector group for each vessel type, respectively; n = 6. G, Quantitative assessment of the wall thickness of the pulmonary arteries as described in Methods. *P < 0.01 vs. Ctrl; #P < 0.01 vs. Vector group; n = 6.

Fig. 4. PGC-1α overexpression attenuated hypoxia-induced endothelial-mesenchymal transition in vivo. Nanoparticles containing control vector (WT) or PGC-1α expression plasmid (OE:PGC1-a) were injected to mice via tail vein. Mice were exposed to normoxia (Ctrl) or hypoxia (10% O2) for 4 weeks. A, PGC-1α, mesenchymal markers N-Cadherin and vimentin, and endothelial cell markers VE-Cadherin and CD31 protein expression levels in the lung tissues, as detected by Western blotting. B–F, Quantification of PGC-1α (B), N-Cadherin (C), vimentin (D), VE-Cadherin (E) and CD31 (F) protein levels by normalizing to GAPDH, respectively. Shown are fold changes relative to the normoxia group. *P < 0.01 vs. Ctrl; #P < 0.01 vs. Vector group for each individual proteins, respectively; n = 6. G, Co-immunostaining of vWF with α-SMA or Vimentin in lung tissues. vWF co-localized with α-SMA and Vimentin in PAECs after hypoxia treatment (white arrowheads), which was diminished by nanoparticle delivery of PGC-1α. Scale bar: 40 μm. H–I, The percentage of vWF+ α-SMA + cells (H) and vWF + Vimentin + cells (I) relative to the total vWF + cells, as quantified from 10 sections of each lung arteries. *P < 0.01 vs. Ctrl; #P…

Fig. 5. Overexpression of PGC-1α reduced pulmonary artery stiffness. Nanoparticles containing empty vector and PGC-1α expression plasmid (OE:PGC1-a) were injected to mice via tail vein. Mice were exposed to normoxia (Ctrl) or hypoxia (10% O2) for 4 weeks. A, The pulse-wave Doppler recordings. The pulse wave velocity (PWV) was increased in hypoxia-treated mouse pulmonary arteries, which was attenuated by endothelial overexpression of PGC-1α. B, PWVs were shown as millimeter/millisecond. *P < 0.01 vs. Ctrl; #P < 0.01 vs. Vector group; n = 5. C, Picrosirius red staining of pulmonary arteries with various diameters as indicated. Scale Bar: 100 μm D, Artery distensibility measurement. The pulmonary arteries from the left lobe of 7-8-week old male mice were isolated, and third order branches (<200 μm) were selected for pressure myography. The diameter alterations under the indicated pressures were recorded. *P < 0.01 vs. Ctrl; #P < 0.01 vs. Vector group with each individual pressure; n = 6. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6. Overexpression of PGC-1α attenuated TGF-β-induced EndoMT and disruption of endothelial monolayer integrity. Human pulmonary artery endothelial cells (PAECs) were transfected with empty vector (Ctrl) or PGC-1α expression plasmid and then treated with vehicle or TGF-β (5 ng/ml) for 3 days. A-B, PGC-1α, N-Cadherin, Vimentin, VE-Cadherin, CD31 protein expression, as detected by Western blotting (A) and quantified by normalizing to GAPDH (B), respectively. *P < 0.05 vs. vehicle-treated cells; #P < 0.01 vs. Ctrl vector-transfected cells with TGF-β for each individual proteins; n = 3. C,Trans-Endothelial Electrical Resistance (TEER) measurement. TEER was measured continuously in PAECs with or without TGF-β treatment. After baseline TEER was achieved, TGF-β were added. 3 days later, the resistance was measured at 4000 Hz in 20 min intervals and normalized to the baseline level. *P < 0.05 vs. vehicle-treated cells; #P < 0.05 vs. Ctrl vector-transfected cells with TGF-β; n = 3. D, PAEC permeability assay. PAECs were plated on Transwell inserts and cultured until confluence. Vehicle or PBS along with fluorescein isothiocyanate (FITC)-dextran was added to the bottom chamber and incubated for…

Fig. 7. Overexpression of PGC-1α improved PAEC function and inhibits calcium release in smooth muscle cells (SMCs). Nanoparticles containing control vector or PGC1-α expression plasmid (OE:PGC1-α) were injected to mice via tail vein. Mice were exposed to normoxia (Ctrl) or hypoxia (10% O2) for 4 weeks. A-B, Endothelial nitric oxide synthase (eNOS) expression in lung tissues, as detected by Western blotting (A) and normalized to GAPDH. *P < 0.01 vs. Ctrl; #P < 0.01 vs. Vector group; n = 6. C-D, eNOS protein expression. Human PAECs were transfected with empty vector (Ctrl) or PGC-1α expression plasmid and then treated with vehicle or TGF-β (5 ng/ml) for 3 days. eNOS expression was detected by Western blotting (C) and normalized to GAPDH (D). *P < 0.05 vs. vehicle-treated cells; #P < 0.01 vs. Ctrl vector-transfected cells with TGF-β; n = 6. E, Nitric oxide (NO) release assay. PAECs were treated with nanoparticles containing empty Vector (Ctrl) or PGC-1α expression plasmid (PGC-1α). One day later, cells were treated with vehicle or TGF-β for 20 min. NO generated by PAECs was measured using a Nitric Oxide Assay Kit (Abcam, ab65327). The relative NO levels were quantified by normalizing to the…

Fig. 8. PGC-1α was decreased in pulmonary artery endothelial cells of patients with idiopathic pulmonary arterial hypertension (IPAH). A, Representative images of immunohistochemistry staining of PGC-1α in pulmonary arteries. Scale Bar: 200 μm. B–C, PGC-1α protein expression in healthy and IPAH lungs, as detected by Western blotting (B) and quantified by normalizing to GAPDH level (C). *P < 0.01 vs. Healthy lungs, n = 6. D, PGC-1α expression in human pulmonary artery endothelium, as detected by co-immunostaining of PGC-1α (green) with CD31 (red) in lung tissues. Scale Bar: 100 μm. E, The percentages of PGC-1α+ CD31+ cells relative to the total CD31+ cells, which was quantified from 10 sections of each patients. *P < 0.01 vs. Healthy lungs, n = 6. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)