Alleviating experimental pulmonary hypertension via co-delivering FoxO1 stimulus and apoptosis activator to hyperproliferating pulmonary arteries

Abstract

Pulmonary hypertension (PH) is an insidious pulmonary vasculopathy with high mortality and morbidity and its underlying pathogenesis is still poorly delineated. The hyperproliferation and apoptosis resistance of pulmonary artery smooth muscle cells (PASMCs) contributes to pulmonary vascular remodeling in pulmonary hypertension, which is closely linked to the downregulation of fork-head box transcriptional factor O1 (FoxO1) and apoptotic protein caspase 3 (Cas-3). Here, PA-targeted co-delivery of a FoxO1 stimulus (paclitaxel, PTX) and Cas-3 was exploited to alleviate monocrotaline-induced pulmonary hypertension. The co-delivery system is prepared by loading the active protein on paclitaxel-crystal nanoparticles, followed by a glucuronic acid coating to target the glucose transporter-1 on the PASMCs. The co-loaded system (170 nm) circulates in the blood over time, accumulates in the lung, effectively targets the PAs, and profoundly regresses the remodeling of pulmonary arteries and improves hemodynamics, leading to a decrease in pulmonary arterial pressure and Fulton's index. Our mechanistic studies suggest that the targeted co-delivery system alleviates experimental pulmonary hypertension primarily via the regression of PASMC proliferation by inhibiting cell cycle progression and promoting apoptosis. Taken together, this targeted co-delivery approach offers a promising avenue to target PAs and cure the intractable vasculopathy in pulmonary hypertension.

Keywords: Caspase 3; Co-delivery; Fork-head box transcriptional factor O1; Pulmonary artery smooth muscle cells; Pulmonary hypertension.

Graphical abstract

Figure 1

Characterization of GlcA-NPplex. (A) Schematic illustration of the preparation process of GlcA-NPplex. (B) Native PAGE study of Cas-3 loaded in NPplex. (C) Influence of mass ratio of TA/Cas-3 on the particle size of NPplex (n = 3). (D) Particle size distribution of GlcA-NPlex measured by DLS. (E) TEM image of GlcA-NPlex. The scale bar is 200 nm. (F) Stability of GlcA-NPplex from optimized formulation stored in a 50% serum at 37 °C for 12 h (n = 3). In vitro release profile of (G) PTX and (H) Cas-3 from GlcA-NPplex in buffer solution at different pH conditions at 37 °C for 24 h (n = 3). The results are given as the mean ± SD.

Figure 2

PASMC-targeting and improved apoptosis. (A and B) Influence of GLUT-1 inhibitor on GlcA-NPplex uptake, (A) qualitative and (B) quantitative analysis of cellular uptake. PASMCs were pre-treated with the GLUT-1 inhibitor BAX-876 at a 2 nmol/L concentration for 0.5 h at 37 °C and then were incubated with the FITC-labeled nanoparticles at 5 μg/mL FITC at 37 °C for 3 h. The cells without BAX-876 pretreatment were used as a control. n = 3, ∗∗P < 0.01. The scale bar is 20 μm. (C) Quantified apoptosis following preparation treatment. FCM analysis of PASMC apoptosis was performed after 24-h incubation with 0.25 μg/mL PTX or 0.025 μg/mL Cas-3. Data are represented as mean ± SD. n = 3, ∗∗P < 0.01, ∗∗∗P < 0.001 compared to saline group; ^###P < 0.001 compared to GlcA-NPplex group.

Figure 3

Targeting the axis of lung-PA-PASMCs. (A) Ex vivo images displaying tissue distribution of DiR-labeled GlcA-NPplex at 24 h after administration via tail vein. Scale bar, 2 cm. (B) Quantification of nanoparticle distribution (Data are represented as mean ± SD. n = 3, ∗∗P < 0.01 compared to free DiR group). Scale bar, 50 μm. (C) Co-localization of IR783-labeled GlcA-NPplex with PA α-SMA in MCT-PH rats. (D) Quantified fluorescence intensity for PA-targeting of IR783-labeled preparations (n = 3, ∗∗P < 0.01). The preparation was dosed at 0.5 mg/kg IR783, according to the animal body weight. The lung was isolated at 4 h post-treatment, sectioned and marked with anti-α-SMA antibody. Yellow fluorescence implies the co-localization.

Figure 4

The curative effect of different formulations on the pulmonary hypertension and depressed right ventricle contractility of rats induced by MCT. (A) Schematic flowcharter of animal experiment. (B) Pulmonary arterial pressure was directly reflected by mPAP on Day 35. (C) Fulton's index [RV/(LV + S)] suggested the degree of right ventricular hypertrophy on Day 35. Echocardiographic examination was conducted in MCT-PH rats receiving GlcA-NPplex or GlcA-NPs on Day 33. (D) The representative images of pulmonary arterial blood flow spectral pattern, PAAT, RVID, and TAPSE. (E–J) Quantifying the echocardiographic parameters comprising PAAT, PVR, RVID, TAPSE, CO, LVEF (%). PAAT, PVR, RVID and TAPSE demonstrate RV function. Increasing the mPAP accelerates the PA blood flow and shorts the acceleration time or PAAT. With the PH development, the RV afterload rises and the RVID increases. TAPSE indicates RV systolic function. CO represents cardiac ejection function, while LVEF reflected the systolic function of left ventricle. Data are represented as mean ± SD. ∗P < 0.05, ∗∗∗P < 0.001 compared to MCT group; ns, no significance. n = 6, unless indicated otherwise. CON: controlled rats (normal rats) received intravenous injections of 0.9% saline; MCT: rats received MCT treatment. The MCT-induced rats were dosed with preparations at 0.4 mg/kg PTX and 0.04 mg/kg Cas-3 via the tail vein. PAAT: pulmonary arterial accelerating time; PVR: pulmonary vascular resistance; RVID: right ventricle internal diameter; TAPSE: tricuspid annular plane systolic excursion; CO: cardiac output; LVEF: left ventricle ejection fraction.

Figure 5

Efficacy of different formulations on the PA-remodeling regression and PASMC-hyperproliferation inhibition in MCT-PH rats. (A) H&E staining of the lung sections and (B) immunohistochemical detection of α-SMA expression. (C) Ki67 expression determined by immunohistochemistry. Semi-quantitative analysis of (D) PA medial thickness, (E) muscularization of pre-acinar arterioles, and (F) α-SMA expression relative to per artery in PAs. Semi-quantitative analysis of (G) Ki67 positive cells relative to PASMCs. Data are represented as mean ± SD. n = 4–6, ∗∗P < 0.01, ∗∗∗P < 0.001 compared to MCT group; ^#P < 0.05, ^##P < 0.01 compared to GlcA-NPplex group; ns, no significance. Scale bar, 50 μm. CON: controlled rats (normal rats) received intravenous injections of 0.9% saline; MCT: rats received MCT treatment. The MCT-induced rats were dosed with preparations at 0.4 mg/kg PTX and 0.04 mg/kg Cas-3 via the tail vein.

Figure 6

Upregulated FoxO1 expression in the media tunica of PAs in MCT-PH rats. (A) The representative immunofluorescent images displaying the expression and location of FoxO1 in the pulmonary vessels. α-Smooth muscle actin (α-SMA, Red), FoxO1 (Green), and the merge of α-SMA and FoxO1 (Yellow), with nucleus stain (DAPI). The scale bar is 50 μm. (B) Quantification of FoxO1 positive cells in the media tunica. (C) The expression of FoxO1 and phosphorylated FoxO1 in the lungs of rats examined by Western blotting. The representative changes of FoxO1 and phosphorylation status was shown with the same result in three separable experiments. (D and E) Semi-quantitative analysis. Data are represented as mean ± SD, n = 4. ∗∗P < 0.01, ∗∗∗P < 0.001 compared to MCT ; ^#P < 0.05 compared to GlcA-NPplex group; ns, no significance. Scale bar, 50 μm. CON: controlled rats (normal rats) received intravenous injections of 0.9% saline; MCT: rats received MCT treatment. The MCT-induced rats were dosed with preparations at 0.4 mg/kg PTX and 0.04 mg/kg Cas-3 via the tail vein.

Figure 7

Upregulated FoxO1 expression in the lung tissue and pathways of regressing the proliferated PAs in MCT-PH rats. (A) The expression of FoxO1, caspase 3, and cyclin D1 examined with the immunohistochemical assay. Scale bar, 50 μm. Quantified assay of (B) FoxO1-, (C) cyclin D1- (D) Cas-3-positive cells in the media tunica. Data are represented as mean ± SD. n = 6, ∗∗∗P < 0.001 compared to MCT group; ^###P < 0.001 compared to GlcA-NPplex group; ns, no significance. CON: controlled rats (normal rats) received intravenous injections of 0.9% saline; MCT: rats received MCT treatment; PTX/Cas-3: combination of PTX and Cas-3. The MCT-induced rats were dosed with preparations at 0.4 mg/kg PTX and 0.04 mg/kg Cas-3 via the tail vein.