Yeeran

Alleviation of Experimental Pulmonary Arterial Hypertension in a Hyperproliferative State via Co-Delivery of a FoxO1 Stimulator and an Apoptosis Activator

I. Research Background
Pulmonary hypertension (PH) carries a dire prognosis, with a 5‑year survival rate of merely 20%–60%. The hallmark pathology is excessive proliferation and apoptosis resistance of pulmonary arterial smooth muscle cells (PASMCs), culminating in vascular remodeling, elevated pulmonary vascular resistance, and right ventricular hypertrophy and failure. Current pharmacotherapies—including PDE5 inhibitors and endothelin receptor antagonists—act solely as vasodilators and fail to reverse smooth muscle hyperproliferation, resulting in suboptimal outcomes.

Mechanistically, FoxO1 is a proliferation‑suppressing transcription factor; in PH‑affected PASMCs, its phosphorylation is increased while total protein levels are reduced, accompanied by upregulation of cyclin D1, which drives rampant proliferation. Paclitaxel (PTX) can upregulate FoxO1 and curtail its phosphorylation. Concurrently, caspase‑3 (Cas‑3), a key apoptosis effector, is markedly downregulated in PH, blunting PASMC apoptosis. While single‑agent strategies are ineffective, combined elevation of FoxO1 and replenishment of active Cas‑3 may synergistically inhibit proliferation and trigger apoptosis. Nevertheless, free proteins are rapidly degraded in vivo and lack pulmonary vascular specificity.

II. Nanodelivery System Design
To address these hurdles, we developed a GlcA‑NPplex targeted co‑delivery nanocomplex through a three‑step construction:

MPN‑coated PTX nanocrystals: Prepared via anti‑solvent ultrasonication and stabilized by a tannic acid–Fe³⁺ metal‑phenolic network (MPN). The PTX loading reached 27.8%, outperforming liposomal and polymeric systems. The MPN shell binds Cas‑3 through hydrogen bonding and π–π stacking, preserving its native conformation.

Activated Cas‑3 loading: At an optimized TA/Cas‑3 mass ratio of 20:1, the encapsulation efficiency was 68%, with electrophoresis confirming complete carrier binding and no free protein leakage.

Glucuronic acid (GlcA) surface modification: GlcA enables specific recognition of GLUT‑1, which is overexpressed on PASMCs, thus affording targeted enrichment in diseased pulmonary arteries. The resulting rod‑shaped nanoparticles (~170 nm) exhibit excellent serum stability and pH‑responsive release—rapidly liberating PTX and Cas‑3 in the acidic endolysosomal compartment while remaining stable in the neutral bloodstream, thereby minimizing off‑target toxicity.

III. Formulation Groups and Experimental Assignments

GlcA‑NPs: GlcA‑modified MPN nanoparticles loaded with PTX alone (without Cas‑3), serving as the single‑drug nanocarrier control.

GlcA‑NPplex: GlcA‑modified co‑loaded nanocomplex carrying both PTX and activated Cas‑3, representing the core experimental group for evaluating synergistic therapeutic efficacy.

PTX/Cas‑3 physical mixture: Unencapsulated free PTX plus free activated Cas‑3, used as the free dual‑drug control to benchmark the nano‑delivery advantage.

Free PTX and free Cas‑3 groups: Individual free agents to establish baseline single‑drug effects.

CON (healthy rats) and MCT model (monocrotaline‑induced PH rats, saline‑treated) groups served as negative controls.

IV.  Experimental Results
In vivo fluorescence imaging revealed that GlcA‑NPplex accumulation in the lungs was twice that of free dye. Co‑localization with α‑SMA confirmed that the nanoparticles precisely targeted the hyperplastic medial layer of pulmonary arteries, with negligible retention in normal tissues, underscoring their marked targeting specificity.

Hemodynamic and Right Ventricular Functional Improvement

In the MCT-induced model group, rats exhibited elevated mean pulmonary arterial pressure (mPAP), increased Fulton index (right ventricular hypertrophy index, RV/(LV+S)), shortened pulmonary acceleration time (PAAT), enlarged right ventricular internal diameter (RVID), and decreased tricuspid annular plane systolic excursion (TAPSE), indicating impaired right ventricular systolic function.

Following treatment with GlcA-NPplex, significant improvements were observed:

.mPAP decreased by 18.4%, and the right ventricular hypertrophy index was reduced by 21%.

.PAAT was prolonged, pulmonary vascular resistance (PVR) dropped by 31%, TAPSE increased by 51.4%, and cardiac output (CO) rose by 21.9%.

.In contrast, PTX-loaded nanoparticles alone or free drugs produced only modest improvements, confirming that the co-delivery system yielded the most favorable therapeutic outcomes.

Therapeutic Effects of Different Formulations on MCT‑Induced Pulmonary Hypertension and Right Ventricular Systolic Dysfunction in Rats

(A)  Schematic diagram of the animal experimental timeline.
(B)  Pulmonary arterial pressure was directly reflected by mean pulmonary arterial pressure (mPAP) on day 35.
(C)  The Fulton index [RV/(LV+S)] indicated the degree of right ventricular hypertrophy on day 35. Echocardiographic examinations were performed in MCT‑PH rats receiving GlcA‑NPplex or GlcA‑NPs on day 33.
(D)  Representative images of pulmonary artery blood flow spectral patterns, PAAT, RVID, and TAPSE.
(E–J)  Quantitative analyses of echocardiographic parameters, including PAAT, pulmonary vascular resistance (PVR), RVID, TAPSE, cardiac output (CO), and left ventricular ejection fraction (LVEF %). PAAT, PVR, RVID, and TAPSE reflect right ventricular function; elevated mPAP accelerates pulmonary arterial blood flow and shortens the acceleration time (i.e., PAAT). As pulmonary hypertension progresses, right ventricular afterload increases and RVID enlarges. TAPSE reflects right ventricular systolic function; CO represents cardiac output function; and LVEF reflects left ventricular systolic function.

Abbreviations: PAAT, pulmonary acceleration time; PVR, pulmonary vascular resistance; RVID, right ventricular internal diameter; TAPSE, tricuspid annular plane systolic excursion; CO, cardiac output; LVEF, left ventricular ejection fraction.

Reversal of Pulmonary Vascular Remodeling

This section quantitatively evaluated the interventional effects of various formulations on pulmonary artery remodeling and smooth muscle proliferation in MCT‑induced PH rats through pathological staining. The experimental groups included: CON (normal control), MCT model, free PTX, free Cas‑3, free PTX/Cas‑3 physical mixture, GlcA‑NPs (single‑drug nanocarrier), and GlcA‑NPplex (dual‑drug co‑delivery nanocarrier).

Figure 5A/D (H&E staining and medial wall thickness quantification): The pulmonary arterial medial thickness in the MCT group was approximately three times that of the normal control group. Among the treatments, free Cas‑3 alone showed no improvement, while all PTX‑containing formulations attenuated vascular wall thickening. Notably, GlcA‑NPplex exerted the most robust effect, reducing medial thickening by 34% compared to the model group (P < 0.001).

Figure 5E (Quantification of distal vascular muscularization): The MCT model exhibited extensive abnormal muscularization of distal small pulmonary arteries. Treatment with GlcA‑NPplex reduced the muscularization rate by 36% (P < 0.01), significantly reversing pathological muscularization.

Figure 5B/F (Immunohistochemical quantification of α‑SMA): α‑SMA serves as a marker of smooth muscle proliferation. Its expression was markedly elevated in the MCT group. GlcA‑NPplex treatment substantially downregulated α‑SMA expression (P < 0.001), effectively inhibiting abnormal smooth muscle hyperplasia.

Figure 5C/G (Ki67 proliferation index): Ki67 reflects cellular proliferative activity. The MCT group showed a significant increase in Ki67‑positive cells. All PTX‑containing formulations reduced the number of proliferating cells, with GlcA‑NPplex demonstrating the strongest inhibitory effect—reducing Ki67‑positive cells by half compared to the model group (P < 0.001).

Figure X. Effects of various formulations on pulmonary artery remodeling regression and suppression of excessive PASMC proliferation in MCT‑PH rats.

In Vivo Molecular Pathway Validation

In the PH model, two dysregulated pathways were identified: inactivation of the FoxO1 pathway and blockade of the apoptotic pathway.

1. FoxO1 pathway: In MCT rats, pulmonary vascular tissues exhibited low total FoxO1 levels and increased levels of inactivated p‑FoxO1 (phosphorylated FoxO1). Treatment with GlcA‑NPs alone produced only a modest elevation in FoxO levels, whereas GlcA‑NPplex significantly upregulated functional FoxO1 and reduced its phosphorylated (inactive) form.

2. Downstream proliferation and apoptosis-related proteins: The model group showed high expression of the pro‑mitotic protein Cyclin D1, while activated Cleaved Caspase‑3 was nearly absent. GlcA‑NPplex treatment downregulated Cyclin D1 to arrest the cell cycle and, concurrently, markedly elevated activated Caspase‑3 to induce apoptosis of hyperplastic smooth muscle cells.

Mechanistic summary: GlcA‑NPs activate only the FoxO1 pathway as a single intervention; in contrast, GlcA‑NPplex achieves a dual action—"inhibiting smooth muscle proliferation + inducing apoptosis of aberrant cells"—with the two pathways synergistically contributing to anti‑vascular remodeling effects. This synergistic effect was not achieved by free drug combinations.

Upregulation of FoxO1 expression in the medial layer of pulmonary arteries (PAs) in MCT‑PH rats

Upregulation of FoxO1 Expression and Regression of Proliferative Alveolar Epithelial Cells in the Lung Tissue of MCT‑PH Rats

V. Conclusion and Limitations

In this study, we constructed a GlcA‑modified MPN‑based co‑delivery nanosystem that targets hyperplastic pulmonary arterial smooth muscle cells. Through PTX‑mediated upregulation of FoxO1 to arrest the cell cycle and exogenous activated Caspase‑3 to initiate smooth muscle apoptosis, this system synergistically reverses pulmonary vascular remodeling, reduces pulmonary arterial pressure, and restores right ventricular function. This work provides a novel targeted nanotherapeutic strategy for pulmonary hypertension.

However, several limitations should be acknowledged. First, the therapeutic efficacy was evaluated only in a single MCT‑induced PH animal model, without verification in hypoxia‑induced or connective tissue disease‑associated PH subtypes. Second, long‑term repeated‑dose toxicity data are lacking. Third, no validation has been performed in human tissues or primary cells. Therefore, additional experimental evidence is still required to support clinical translation.

Publication Information

Authors:  Bingbing Li,  Chao Teng,  Huiling Yu,  Xiaohong Jiang, Xuyang Xing, Qi Jiang,  Chenshi Lin,  Zongmin Zhao, Ruifeng Zhang,  Wei He

Publisher:   Acta Pharmaceutica Sinica B，2023, Vol.6
doi:  https://doi.org/10.1016/j.apsb.2022.12.002.

Original Link:  https://www.sciencedirect.com/science/article/pii/S2211383522004968?via%3Dihub