Simultaneous Pharmacologic Inhibition of Yes-Associated Protein 1 and Glutaminase 1 via Inhaled Poly(Lactic-co-Glycolic) Acid-Encapsulated Microparticles Improves Pulmonary Hypertension

Abstract

Background Pulmonary hypertension (PH) is a deadly disease characterized by vascular stiffness and altered cellular metabolism. Current treatments focus on vasodilation and not other root causes of pathogenesis. Previously, it was demonstrated that glutamine metabolism, as catalyzed by GLS1 (glutaminase 1) activity, is mechanoactivated by matrix stiffening and the transcriptional coactivators YAP1 (yes-associated protein 1) and transcriptional coactivator with PDZ-binding motif (TAZ), resulting in pulmonary vascular proliferation and PH. Pharmacologic inhibition of YAP1 (by verteporfin) or glutaminase (by CB-839) improved PH in vivo. However, systemic delivery of these agents, particularly YAP1 inhibitors, may have adverse chronic effects. Furthermore, simultaneous use of pharmacologic blockers may offer additive or synergistic benefits. Therefore, a strategy that delivers these drugs in combination to local lung tissue, thus avoiding systemic toxicity and driving more robust improvement, was investigated. Methods and Results We used poly(lactic-co-glycolic) acid polymer-based microparticles for delivery of verteporfin and CB-839 simultaneously to the lungs of rats suffering from monocrotaline-induced PH. Microparticles released these drugs in a sustained fashion and delivered their payload in the lungs for 7 days. When given orotracheally to the rats weekly for 3 weeks, microparticles carrying this drug combination improved hemodynamic (right ventricular systolic pressure and right ventricle/left ventricle+septum mass ratio), histologic (vascular remodeling), and molecular markers (vascular proliferation and stiffening) of PH. Importantly, only the combination of drug delivery, but neither verteporfin nor CB-839 alone, displayed significant improvement across all indexes of PH. Conclusions Simultaneous, lung-specific, and controlled release of drugs targeting YAP1 and GLS1 improved PH in rats, addressing unmet needs for the treatment of this deadly disease.

Keywords: mechanotransduction; metabolism; nanoparticle; pulmonary hypertension; therapy.

Figure 1. Local inhibition of YAP1/TAZ and glutaminase pathways for effective amelioration of pulmonary hypertension.

PLGA indicates poly(lactic‐co‐glycolic) acid; TAZ, transcriptional coactivator with PDZ‐binding motif; and YAP1, yes‐associated protein 1.

Figure 2. PLGA microparticles are within a size range for inhalation and release verteporfin and CB‐839 in a sustained manner.

(A) Scanning electron microscope images of CB‐839 alone encapsulated, verteporfin alone encapsulated, and CB‐839 with verteporfin encapsulated microparticles displayed smooth surface morphology. (B) Size distribution of the microparticles obtained from dynamic light scattering experiments demonstrated that the average microparticle size for all the microparticles was approximately 1 μm. (C) Release kinetics of CB‐839 from PLGA microparticles encapsulating CB‐839‐verteporfin or encapsulating CB‐839 only. (D) Release kinetics of verteporfin from PLGA microparticles encapsulating CB‐839‐verteporfin or encapsulating verteporfin only. Error bars represent SD. PLGA indicates poly(lactic‐co‐glycolic) acid.

Figure 3. PLGA microparticles deliver payload into the lungs of rats.

(A) Fluorescence image of the lungs of rats after intratracheal administration with PLGA microparticles encapsulating near infrared dye IR780 vs no dye, imaged on day 0 and day 7 after administration. (B) Confocal microscopy was performed on precision cut lung slices of rats after intratracheal administration with PLGA microparticles encapsulating rhodamine 6G dye vs no dye imaged on day 3 after administration. Lung slices were stained with antibody against α‐SMA (green) to identify muscularized pulmonary arterioles. Representative matched images are shown from each of N=3 rats/group to visualize α‐SMA (green) and rhodamine 6G dye (Rh; red; bars=50 μm). (C) Representative matched images are shown (as in B) to visualize α‐SMA (arterioles, green) and rhodamine 6G dye (Rh; red) in precision cut kidney slices of rats after intratracheal administration with PLGA microparticles encapsulating rhodamine 6G dye vs no dye imaged on day 3 after administration (bars=50 μm). α‐SMA indicates α‐smooth muscle actin; and PLGA, poly(lactic‐co‐glycolic) acid.

Figure 4. Simultaneous pharmacologic inhibition of GLS1 and YAP1/TAZ in monocrotaline‐exposed rats modulates YAP1‐specific gene expression and glutaminase activity in the lungs.

(A) Study design is shown for the induction of PH using monocrotaline via intraperitoneal injection followed by administration of microparticles orotracheally for treatments. (B–D) As assessed by reverse transcriptase–quantitative polymerase chain reaction of rat lung, YAP1‐dependent gene transcripts for CTGF (B), ANKRD1 (C), and CYR61 (D) were downregulated modestly by PLGA‐encapsulated CB‐839 and more robustly by inhaled PLGA‐encapsulated verteporfin. (E) As assessed by glutaminase activity assay of rat lung, glutaminase activity was robustly downregulated by inhaled PLGA‐encapsulated CB‐839 but not verteporfin alone. In all images, mean expression in untreated groups was assigned a fold change of 1, to which relevant samples were compared. In each group, n=3 (untreated), n=5 (blank microparticles), n=3 (saline), n=5 (verteporfin), n=5 (CB‐839), and n=6 (CB839+verteporfin). Error bars represent mean±SEM. By 1‐way ANOVA and post hoc Bonferroni testing, significantly different values are represented by *<0.05, *<0.01, ***<0.001, and ****<0.00001. ANKRD1 indicates ankyrin repeat domain 1; CTGF, connective tissue growth factor; CYR61, cysteine‐rich angiogenic inducer 61; GLS1, glutaminase 1; MCT, monocrotaline; PLGA, poly(lactic‐co‐glycolic) acid; TAZ, transcriptional coactivator with PDZ‐binding motif; and YAP1, yes‐associated protein 1.

Figure 5. Delivery of verteporfin and CB‐839 simultaneously in vivo improves hemodynamic manifestations of PH in monocrotaline‐exposed rats.

(A) Poly(lactic‐co‐glycolic) acid MP delivering verteporfin and CB‐839 and poly(lactic‐co‐glycolic) acid MP delivering verteporfin alone significantly decreased the Fulton index (RV/LV+S mass) compared with blank MP. (B) PLGA MP delivering verteporfin and CB‐839 significantly decreased RVSP compared with the control of blank MP, and this RVSP was not significantly different than the untreated rats (n=3–6; error bars represent mean±SEM. By 1‐way ANOVA and post hoc Bonferroni testing, significantly different values are represented by *<0.05, *<0.01, ***<0.001, and ****<0.00001. LV+S indicates left ventricle+septum; MP, microparticles; RV, right ventricle; and RVSP, right ventricular systolic pressure.

Figure 6. Simultaneous pharmacologic inhibition of GLS1 and YAP1/TAZ in monocrotaline‐exposed rats decreases pulmonary vascular cell proliferation and pulmonary vascular remodeling.

(A) Representative images of small pulmonary arterioles (<100 μm diameter) of the lungs (blue, nuclei; red, PCNA; green, α‐SMA; bar=30 μm). (B) The percentage of PCNA of α‐SMA–positive vascular cells in the CB‐839 and verteporfin combination group was significantly lower than negative controls of saline and blank MP and significantly different than single drug treatments alone (n=7–13; error bars represent mean±SEM). By 1‐way ANOVA and post hoc Bonferroni testing, significant P values were calculated as follows for CB‐839+verteporfin‐treated rodents: *P=0.0003 vs saline, P=0.0001 vs blank MP, P=0.0147 vs CB‐839, P=0.0041 vs verteporfin. Notably, P=0.8684 vs untreated. Other comparisons among monocrotaline‐PAH rat cohorts were not significant (P>0.05). (C) The wall thickness of pulmonary arterioles (diameter<100 μm) in the verteporfin+CB‐839 combination treatment group was significantly lower than either single drug treatment or negative controls of saline and blank MP; mean expression in the untreated group was assigned a fold change of 1, to which relevant samples were compared (n=11–16 vessels; error bars represent mean±SEM). By 1‐way ANOVA and post hoc Bonferroni testing, significant P values were calculated as follows for CB‐839+verteporfin‐treated rodents: *P=2.36E‐05 vs saline, P=2.40E‐05 vs blank MP, P=0.0009 vs CB‐839, P=0.0465 vs verteporfin. Notably, P=0.1241 vs untreated. For verteporfin‐treated rodents, significant P values included the following: $P=3.08E‐05 vs saline, P=0.0001 vs blank MP, P=0.017 vs CB‐839, P=0.0465 vs CB‐839+verteporfin, P=0.002 vs untreated. Other comparisons among monocrotaline‐PAH rat cohorts were not significant (P>0.05). α‐SMA indicates α‐smooth muscle actin; GLS1, glutaminase 1; MCT, monocrotaline; MP, microparticles; PCNA, proliferating cell nuclear antigen; TAZ, transcriptional coactivator with PDZ‐binding motif; and YAP1, yes‐associated protein 1.

Figure 7. Simultaneous pharmacologic inhibition of GLS1 and YAP1/TAZ in monocrotaline‐exposed rats decreases collagen deposition and collagen cross‐linking in pulmonary arterioles.

(A) Representative images of picrosirius red stain of lung tissues showing fibrillar collagen deposition (red, bright field) and cross‐linked fibrillar collagen assembly (using orthogonal polarized images, red is collagen type I and green is collagen type III [bar=50 μm]). (B) Quantification of the percent area of picrosirius red stain under nonpolarized light (represented as a.u.) showed that the CB‐839 and verteporfin combination significantly decreased pulmonary arteriolar collagen deposition compared with negative controls of saline and blank MP and was significantly different than either single drug treatment alone (n=6–10; error bars represent mean±SEM). By 1‐way ANOVA and post hoc Bonferroni testing, P values were calculated as follows for CB‐839+verteporfin‐treated rodents: *P=0.0003 vs saline, P=0.0438 vs blank MP, P=0.0036 vs CB‐839; P=0.0361 vs verteporfin. Notably, P=0.3312 vs untreated. Other comparisons among monocrotaline‐PAH rat cohorts were not significant (P>0.05). (C) Quantification of the percent area of picrosirius red stain under polarized light (represented as a.u.) demonstrated that the CB‐839 and verteporfin combination group significantly decreased pulmonary arteriolar cross‐linked collagen compared with negative controls of saline and blank MP and was significantly different than verteporfin‐alone treatment (n=6–10; error bars represent mean±SEM). By 1‐way ANOVA and post hoc Bonferroni testing, significant P values were calculated as follows for CB‐839+verteporfin‐treated rodents: *P=3.65E‐05 vs saline, P=0.0047 vs blank MP, P=0.012 vs verteporfin. Notably, P=0.1363 vs CB‐839 and P=0.095 vs untreated. Other comparisons among monocrotaline‐PAH rat cohorts were not significant (P>0.05). a.u. indicates arbitrary units; GLS1, glutaminase 1; MCT, monocrotaline; MP, microparticles; TAZ, transcriptional coactivator with PDZ‐binding motif; and YAP1, yes‐associated protein 1.