Reversal of Hypoxic Pulmonary Hypertension by Hypoxia-Inducible Overexpression of Angiotensin-(1-7) in Pulmonary Endothelial Cells

Figures

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  Figure 1. HTSFcAng(1-7) Exhibited Specific Overexpression in Pulmonary Endothelial Cells and Hypoxic Inducibility in Hypoxia (A) Diagram of the constructed HTSFcAng(1-7) and HTSFc. (B) Effects of HTSFcAng(1-7) or HTSFc transfection on the expression of angiotensin-(1-7) in A549 cells, HEK293 cells, NIH 3T3 cells, pulmonary artery smooth muscle cells (PASMCs), pulmonary microendothelial cells (PMVECs), rat peripheral blood neutrophils, monocytes, NR8383 cells, and primary bone mesenchymal stem cells (MSCs). (C and D) Effect of oxygen concentration on the expression of angiotensin-(1-7) (C) and the increment rate of the expression of angiotensin-(1-7) (D) in PMVECs transfected with HTSFcAng(1-7). ∗p < 0.05. Data are represented as mean ± SD from three replicated experiments (B and C, respectively).

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  Figure 2. Effects of the Condition Medium from PMVECs Transfected with HTSFcAng(1-7) on Hypoxia-Induced Proliferation of Primary PASMCs PASMC proliferation was measured by the 3-(4, 5-dimethylthiazal-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay. (A) Effects of oxygen concentration on the proliferation of rat primary PASMCs. (B) Inhibition effects of the condition medium from PMVECs transfected with HTSFcAng(1-7) on the proliferation of PASMCs cultured in normoxia (20% O2) and in hypoxia (10%, 5%, and 1% O2). (C and D) Concentration (v/v)-dependent inhibition effects of the condition medium, from the PMVECs transfected with HTSFcAng(1-7), on the proliferation of PASMCs cultured in 10% O2 concentration. CM, condition medium. ∗p < 0.05. Data are represented as mean ± SD from three replicated experiments in each panel.

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  Figure 3. Effects of the Condition Medium from PMVECs Transfected with HTSFcAng(1-7) on the Constriction and Relaxation of the Isolated Pulmonary Artery Rings in Hypoxia (A) The representative curve for the effects of acute hypoxia on phenylephrine-precontracted pulmonary artery rings. Acute hypoxia resulted in vascular biphasic contractions: an initial transient contraction, then a transient relaxation (maximum dilation), and finally a delayed, sustained contraction (phase II constriction). (B–E) The representative curve for the effect of the condition medium, which was from PMVECs transfected with HTSFcAng(1-7) (B and C) or HTSFc (D and E), on the artery ring in hypoxia (% O2) with (C and E) or without (B and D Mas receptor antagonist A-779 (1 μM). (F and G) The summarized data of maximum vasodilation (F) and phase II vasoconstriction and phase II vasodilation (G). CM, condition medium. *p < 0.05 versus control and HTSFcAng(1-7). Data are represented as mean ± SD, n = 6.

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  Figure 4. Dynamic Changes of Hemodynamic and Pulmonary Vascular Remodeling during 4 Weeks of Hypoxia Exposure in Rats (A–E) The dynamic changes of the carotid pressure (A), the right ventricle systolic pressure (RVSP; B), the ratio of right ventricle weight to left ventricle plus septum weight (RV/(LV + S); C), the pulmonary artery percent wall thickness (WT%; D), and percent wall area (WA%; E) during normoxia or hypoxia exposure in rats. (F) Representative hematoxylin and eosin staining of pulmonary vessels. ∗p < 0.05 versus normoxia; n = 6 for RVSP and RV/(LV + S); n = 60 (muscular arteries) for WT% and WA% (for details, see Materials and Methods). Data are expressed as mean ± SD.

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  Figure 5. Therapeutic Effects of HTSFcAng(1-7) Administration on Hypobaric Hypoxia-Induced Pulmonary Hypertension in Rats (A–C) Administration of adeno-associated virus 9 containing HTSFcAng(1-7) had no significant effects on the mean carotid artery pressure (mCAP; A) but significantly reduced the hypoxia-increased right ventricle systolic pressure (RVSP; B) and the ratio of right ventricle weight to left ventricle plus septum weight (RV/(LV + S); C). (D) Representative hematoxylin and eosin staining of pulmonary vessels. (E and F) The summarized data of pulmonary artery percent wall thickness (WT%; E) and percent wall area (WA%; F). ∗p < 0.05. Data are expressed as mean ± SD, n = 6.

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  Figure 6. HTSFcAng(1-7) Increased the Expression of Angiotensin-(1-7) in the Lungs in Hypobaric Hypoxia-Induced Pulmonary Hypertension Rats (A) Dynamic changes of the expression of angiotensin-(1-7) in lungs during the development of hypoxic pulmonary hypertension rats. ∗p < 0.05 versus normoxia. (B) Effects of administration of adeno-associated virus 9 containing HTSFcAng(1-7) and HTSFc on the expression of angiotensin-(1-7) in lungs, livers, hearts, and kidneys. ∗p < 0.05 versus normoxia-corresponding organ; #p < 0.05 versus hypoxia lung. Data are expressed as mean ± SD, n = 6.

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  Figure 7. Angiotensin-(1-7) Colocalized with CD31 in the Lung Endothelial Cells in Hypobaric Hypoxia-Induced Pulmonary Hypertension Rats (A and B) Immunofluorescence costaining of angiotensin-(1-7) and CD31 (A) and angiotensin-(1-7) and CD11b (B) was measured in lungs in hypobaric hypoxia-induced pulmonary hypertension rats. Angiotensin-(1-7) is stained green; CD31 and CD11b are stained red. The nucleus was stained blue by DAPI. The images are acquired using a confocal laser microscope with the same exposure time during the same imaging session. Scale bars, 20 μM.