Disrupted BMP-9 Signaling Impairs Pulmonary Vascular Integrity in Hepatopulmonary Syndrome.

Rationale: Hepatopulmonary syndrome (HPS) is a severe complication of liver diseases characterized by abnormal dilation of pulmonary vessels, resulting in impaired oxygenation. Recent research highlights the pivotal role of liver-produced BMP-9 (bone morphogenetic protein-9) in maintaining pulmonary vascular integrity. Objectives: This study aimed to investigate the involvement of BMP-9 in human and experimental HPS. Methods: Circulating BMP-9 levels were measured in 63 healthy control subjects and 203 patients with cirrhosis with or without HPS. Two animal models of portal hypertension were employed: common bile duct ligation with cirrhosis and long-term partial portal vein ligation without cirrhosis. Additionally, the therapeutic effect of low-dose BMP activator FK506 was investigated, and the pulmonary vascular phenotype of BMP-9-knockout rats was analyzed. Measurements and Main Results: Patients with HPS related to compensated cirrhosis exhibited lower levels of circulating BMP-9 compared with patients without HPS. Patients with severe cirrhosis exhibited consistently low levels of BMP-9. HPS characteristics were observed in animal models, including intrapulmonary vascular dilations and an increase in the alveolar-arterial gradient. HPS development in both rat models correlated with reduced intrahepatic BMP-9 expression, decreased circulating BMP-9 level and activity, and impaired pulmonary BMP-9 endothelial pathway. Daily treatment with FK506 for 2 weeks restored the BMP pathway in the lungs, alleviating intrapulmonary vascular dilations and improving gas exchange impairment. Furthermore, BMP-9-knockout rats displayed a pulmonary HPS phenotype, supporting its role in disease progression. Conclusions: The study findings suggest that portal hypertension-induced loss of BMP-9 signaling contributes to HPS development.

Serum and Pulmonary Expression Profiles of the Activin Signaling System in Pulmonary Arterial Hypertension.

BACKGROUND: Activins are novel therapeutic targets in pulmonary arterial hypertension (PAH). We therefore studied whether key members of the activin pathway could be used as PAH biomarkers. METHODS: Serum levels of activin A, activin B, α-subunit of inhibin A and B proteins, and the antagonists follistatin and follistatin-like 3 (FSTL3) were measured in controls and in patients with newly diagnosed idiopathic, heritable, or anorexigen-associated PAH (n=80) at baseline and 3 to 4 months after treatment initiation. The primary outcome was death or lung transplantation. Expression patterns of the inhibin subunits, follistatin, FSTL3, Bambi, Cripto, and the activin receptors type I (ALK), type II (ACTRII), and betaglycan were analyzed in PAH and control lung tissues. RESULTS: Death or lung transplantation occurred in 26 of 80 patients (32.5%) over a median follow-up of 69 (interquartile range, 50-81) months. Both baseline (hazard ratio, 1.001 [95% CI, 1.000-1.001]; P=0.037 and 1.263 [95% CI, 1.049-1.520]; P=0.014, respectively) and follow-up (hazard ratio, 1.003 [95% CI, 1.001-1.005]; P=0.001 and 1.365 [95% CI, 1.185-1.573]; P<0.001, respectively) serum levels of activin A and FSTL3 were associated with transplant-free survival in a model adjusted for age and sex. Thresholds determined by receiver operating characteristic analyses were 393 pg/mL for activin A and 16.6 ng/mL for FSTL3. When adjusted with New York Heart Association functional class, 6-minute walk distance, and N-terminal pro-B-type natriuretic peptide, the hazard ratios for transplant-free survival for baseline activin A <393 pg/mL and FSTL3 <16.6 ng/mL were, respectively, 0.14 (95% CI, 0.03-0.61; P=0.009) and 0.17 (95% CI, 0.06-0.45; P<0.001), and for follow-up measures, 0.23 (95% CI, 0.07-0.78; P=0.019) and 0.27 (95% CI, 0.09-0.78, P=0.015), respectively. Prognostic values of activin A and FSTL3 were confirmed in an independent external validation cohort. Histological analyses showed a nuclear accumulation of the phosphorylated form of Smad2/3, higher immunoreactivities for ACTRIIB, ALK2, ALK4, ALK5, ALK7, Cripto, and FSTL3 in vascular endothelial and smooth muscle layers, and lower immunostaining for inhibin-α and follistatin. CONCLUSIONS: These findings offer new insights into the activin signaling system in PAH and show that activin A and FSTL3 are prognostic biomarkers for PAH.

Loss of cAbl Tyrosine Kinase in Pulmonary Arterial Hypertension Causes Dysfunction of Vascular Endothelial Cells.

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease characterized by the dysfunction of pulmonary endothelial cells (ECs) and obstructive vascular remodeling. cAbl (non-receptor tyrosine kinase c-Abelson) plays central roles in regulating cell-cycle arrest, apoptosis, and senescence after cellular stress. We hypothesized that cAbl is downactivated in experimental and human PAH, thus leading to reduced DNA integrity and angiogenic capacity of pulmonary ECs from patients with PAH (PAH-ECs). We found cAbl and phosphorylated cAbl concentrations to be lower in the endothelium of remodeled pulmonary vessels in the lungs of patients with PAH than in control subjects. Similar observations were obtained for the lungs of Sugen + hypoxia and monocrotaline rats with established pulmonary hypertension. These in situ abnormalities were also replicated in vitro, with cultured PAH-ECs displaying lower cAbl expression and activity and an altered DNA damage response and capacity of tube formation. Downregulation of cAbl by RNA interference in control ECs or its inhibition with dasatinib resulted in genomic instability and the failure to form tubes, whereas upregulation of cAbl with 5-(1,3-diaryl-1H-pyrazol-4-yl) hydantoin reduced DNA damage and apoptosis in PAH-ECs. Finally, we establish the existence of cross-talk between cAbl and bone morphogenetic protein receptor type II. This work identifies the loss of cAbl signaling as a novel contributor to pulmonary EC dysfunction associated with PAH.

Effects of B Cell Depletion by CD19-Targeted Chimeric Antigen Receptor T Cells in a Murine Model of Systemic Sclerosis.

OBJECTIVE: Our goal was to study the tolerance and efficacy of two B cell depletion strategies, including one with CD19-targeted chimeric antigen receptor (CAR) T cells, in a preclinical model mimicking the severe lung damages observed in systemic sclerosis. METHODS: B cell depletion strategies were evaluated in the Fra-2 transgenic (Tg) mouse model. We considered a first group of 16 untreated mice, a second group of 15 mice receiving a single dose of anti-CD20 monoclonal antibody (mAb), and a third group of 8 mice receiving CD19-targeted CAR-T cells in combination with anti-CD20 monoclonal antibody. After six weeks of clinical evaluation, different validated markers of inflammation, lung fibrosis, and pulmonary vascular remodeling were assessed. RESULTS: CD19-targeted CAR-T cells infusion in combination with anti-CD20 mAb resulted in a deeper B cell depletion than anti-CD20 mAb alone in the peripheral blood and lesional lungs of Fra-2 Tg mice. CAR-T cell infusion worsened the clinical score and increased mortality in Fra-2 Tg mice. In line with the above findings, CAR-T cell infusion significantly increased lung collagen content, the histological fibrosis score, and right ventricular systolic pressure. CAR-T cells accumulated in lesional lungs and promoted T activation and inflammatory cytokine production. Treatment with anti-CD20 mAb in monotherapy had no impact on lung inflammation-driven fibrosis and pulmonary hypertension. CONCLUSION: B cell therapies failed to show efficacy in the Fra2 Tg mice. The exacerbated Fra-2 lung inflammatory burden stimulated accumulation and expansion of activated CD19-targeted CAR-T cells, secondarily inducing T cell activation and systemic inflammation, finally leading to disease worsening.

Central role of ubiquitin-specific protease 8 in leptin signaling pathway in pulmonary arterial hypertension.

BACKGROUND: Leptin receptor (ObR-b) is overexpressed in pulmonary artery smooth muscle cells (PA-SMCs) from patients with pulmonary arterial hypertension (PAH) and is implicated in both mechanisms that contribute to pulmonary vascular remodeling: hyperproliferation and inflammation. Our aim was to investigate the role of ubiquitin-specific peptidase 8 (USP8) in ObR-b overexpression in PAH. METHODS: We performed in situ and in vitro experiments in human lung specimens and isolated PA-SMCs combined with 2 different in vivo models in rodents and we generated a mouse with an inducible USP8 deletion specifically in smooth muscles. RESULTS: Our results showed an upregulation of USP8 in the smooth muscle layer of distal pulmonary arteries from patients with PAH, and upregulation of USP8 expression in PAH PA-SMCs, compared to controls. USP8 inhibition in PAH PA-SMCs significantly blocked both ObR-b protein expression level at the cell surface as well as ObR-b-dependant intracellular signaling pathway as shown by a significant decrease in pSTAT3 expression. USP8 was required for ObR-b activation in PA-SMCs and its inhibition prevented Ob-mediated cell proliferation through STAT3 pathway. USP8 inhibition by the chemical inhibitor DUBs-IN-2 protected against the development of experimental PH in the 2 established experimental models of PH. Targeting USP8 specifically in smooth muscle cells in a transgenic mouse model also protected against the development of experimental PH. CONCLUSIONS: Our findings highlight the role of USP8 in ObR-b overexpression and pulmonary vascular remodeling in PAH.

Arsenic trioxide demonstrates efficacy in a mouse model of preclinical systemic sclerosis.

BACKGROUND: Uncontrolled T-cell activation plays a key role in systemic sclerosis (SSc). Arsenic trioxide (ATO) has immunological effects and has demonstrated potential in preclinical SSc models. In this study, we assessed the efficacy of ATO in Fra2 transgenic (Fra2TG) mice, which develop severe vascular remodeling of pulmonary arterioles and nonspecific interstitial pneumonia-like lung disease, closely resembling human SSc-associated pulmonary hypertension, therefore partially resembling to the SSc human disease. METHODS: The efficacy of ATO in Fra2TG mice was evaluated through histological scoring and determination of cell infiltration. Fibrotic changes in the lungs were assessed by measuring collagen content biochemically, using second harmonic generation to measure fibrillar collagen, and imaging via computed tomography. Cardiovascular effects were determined by measuring right ventricular systolic pressure and vessel remodeling. The mechanism of action of ATO was then investigated by analyzing lung cell infiltrates using flow cytometry and bulk RNA with sequencing techniques. RESULTS: After ATO treatment, the Ashcroft histological score was substantially decreased by 33% in ATO-treated mice compared to control mice. Other investigations of fibrotic markers showed a trend of reduction in various measurements of fibrosis, but the differences did not reach significance. Further cardiovascular investigations revealed convergent findings supporting a beneficial effect of ATO, with reduced right ventricular systolic pressure and medial wall thickness, and a significant decrease in the number of muscularized distal pulmonary arteries in ATO-treated Fra2TG mice compared to untreated Fra2TG mice. Additionally, inflammatory cell infiltration was also markedly reduced in lesioned lungs. A reduction in the frequency of CD4 + and T effector memory cells, and an increase in the percentage of CD4 + T naive cells in the lungs of ATO-treated Fra-2TG mice, was observed when compared to PBS group Fra-2Tg mice. RNA-seq analysis of ATO-treated mouse lungs revealed a downregulation of biological pathways associated with immune activity and inflammation, such as T-cell activation, regulation of leucocyte activation, leucocyte cell-cell adhesion, and regulation of lymphocyte activation. CONCLUSIONS: Our results suggest the clinical relevance of ATO treatment in SSc. Using the Fra2TG mouse model, we observed significant lung histological changes, a trend towards a decrease in various fibrotic makers, and a strong reduction in vascular remodeling. The mechanism of action of ATO appears to involve a marked counteraction of the immune activation characteristic of SSc, particularly T-cell involvement. These findings pave the way for further studies in SSc.

Inhibition of Soluble Epoxide Hydrolase Does Not Promote or Aggravate Pulmonary Hypertension in Rats.

Inhibitors of soluble epoxide hydrolase (sEH), which catalyzes the hydrolysis of various natural epoxides to their corresponding diols, present an opportunity for developing oral drugs for a range of human cardiovascular and inflammatory diseases, including, among others, diabetes and neuropathic pain. However, some evidence suggests that their administration may precipitate the development of pulmonary hypertension (PH). We thus evaluated the impact of chronic oral administration of the sEH inhibitor TPPU (N-[1-(1-Oxopropyl)-4-piperidinyl]-N'-[4-(trifluoromethoxy)phenyl]-urea) on hemodynamics, pulmonary vascular reactivity, and remodeling, as well as on right ventricular (RV) dimension and function at baseline and in the Sugen (SU5416) + hypoxia (SuHx) rat model of severe PH. Treatment with TPPU started 5 weeks after SU5416 injection for 3 weeks. No differences regarding the increase in pulmonary vascular resistance, remodeling, and inflammation, nor the abolishment of phenylephrine-induced pulmonary artery constriction, were noted in SuHx rats. In addition, TPPU did not modify the development of RV dysfunction, hypertrophy, and fibrosis in SuHx rats. Similarly, none of these parameters were affected by TPPU in normoxic rats. Complementary in vitro data demonstrated that TPPU reduced the proliferation of cultured human pulmonary artery-smooth muscle cells (PA-SMCs). This study demonstrates that inhibition of sEH does not induce nor aggravate the development of PH and RV dysfunction in SuHx rats. In contrast, a potential beneficial effect against pulmonary artery remodeling in humans is suggested.

AMPK activation by metformin protects against pulmonary hypertension in rats and relaxes isolated human pulmonary artery.

Pulmonary hypertension (PH) is associated with pulmonary vasoconstriction and endothelial dysfunction leading to impaired nitric oxide (NO) and prostacyclin (PGI2) pathways. Metformin, the first line treatment for type 2 diabetes and AMP-activated protein kinase (AMPK) activator, has been recently highlighted as a potential PH treatment. AMPK activation has been reported to improve endothelial function by enhancing endothelial NO synthase (eNOS) activity and to have relaxant effects in blood vessels. In this study, we examined the effect of metformin treatment on PH as well as on NO and PGI2 pathways in monocrotaline (MCT)-injected rats with established PH. Moreover, we investigated the anti-contractile effects of AMPK activators on endothelium-denuded human pulmonary arteries (HPA) from Non-PH and Group 3 PH patients (due to lung diseases and/or hypoxia). Furthermore, we explored the interaction between treprostinil and the AMPK/eNOS pathway. Our results showed that metformin protected against PH progression in MCT rats where it reduced the mean pulmonary artery pressure, pulmonary vascular remodeling and right ventricular hypertrophy and fibrosis compared to vehicle-treated MCT rats. The protective effects on rat lungs were mediated in part by increasing eNOS activity and protein kinase G-1 expression but not through the PGI2 pathway. In addition, incubation with AMPK activators reduced the phenylephrine-induced contraction of endothelium-denuded HPA from Non-PH and PH patients. Finally, treprostinil also augmented eNOS activity in HPA smooth muscle cells. In conclusion, we found that AMPK activation can enhance the NO pathway, attenuate vasoconstriction by direct effects on smooth muscles, and reverse established MCT-induced PH in rats.

Mineralocorticoid Receptor Antagonism by Finerenone Attenuates Established Pulmonary Hypertension in Rats.

BACKGROUND: We studied the ability of the nonsteroidal MR (mineralocorticoid receptor) antagonist finerenone to attenuate vascular remodeling and pulmonary hypertension using two complementary preclinical models (the monocrotaline and sugen/hypoxia rat models) of severe pulmonary hypertension. METHODS: We first examined the distribution pattern of MR in the lungs of patients with pulmonary arterial hypertension (PAH) and in monocrotaline and sugen/hypoxia rat lungs. Subsequent studies were performed to explore the effect of MR inhibition on proliferation of pulmonary artery smooth muscle cells derived from patients with idiopathic PAH. To validate the functional importance of MR activation in the pulmonary vascular remodeling characteristic of pulmonary hypertension, mice overexpressing human MR (hMR+) were studied, and curative treatments with finerenone (1 mg/kg per day by gavage), started 2 weeks after monocrotaline injection or 5 weeks after Sugen injection were realized. RESULTS: We demonstrated that MR is overexpressed in experimental and human PAH and that its inhibition following small interfering RNA-mediated MR silencing or finerenone treatment attenuates proliferation of pulmonary artery smooth muscle cells derived from patients with idiopathic PAH. In addition, we obtained evidence that hMR+ mice display increased right ventricular systolic pressure, right ventricular hypertrophy, and remodeling of pulmonary arterioles. Consistent with these observations, curative treatments with finerenone partially reversed established pulmonary hypertension, reducing total pulmonary vascular resistance and vascular remodeling. Finally, we found that continued finerenone treatment decreases inflammatory cell infiltration and vascular cell proliferation in monocrotaline and sugen/hypoxia rat lungs. CONCLUSIONS: Finerenone treatment appears to be a potential therapy for PAH worthy of investigation and evaluation for clinical use in conjunction with current PAH treatments.

Acazicolcept (ALPN-101), a dual ICOS/CD28 antagonist, demonstrates efficacy in systemic sclerosis preclinical mouse models.

BACKGROUND: Uncontrolled immune response with T cell activation has a key role in the pathogenesis of systemic sclerosis (SSc), a disorder that is characterized by generalized fibrosis affecting particularly the lungs and skin. Costimulatory molecules are key players during immune activation, and recent evidence supports a role of CD28 and ICOS in the development of fibrosis. We herein investigated the efficacy of acazicolcept (ALPN-101), a dual ICOS/CD28 antagonist, in two complementary SSc-related mouse models recapitulating skin fibrosis, interstitial lung disease, and pulmonary hypertension. METHODS: Expression of circulating soluble ICOS and skin-expressed ICOS was investigated in SSc patients. Thereafter, acazicolcept was evaluated in the hypochlorous acid (HOCL)-induced dermal fibrosis mouse model and in the Fra-2 transgenic (Tg) mouse model. In each model, mice received 400 µg of acazicolcept or a molar-matched dose of an Fc control protein twice a week for 6 weeks. After 6 weeks, skin and lung were evaluated. RESULTS: ICOS was significantly increased in the sera from SSc patients and in SSc skin biopsies as compared to samples from healthy controls. Similar body weight changes were observed between Fc control and acazicolcept groups in both HOCL and Fra-2 Tg mice suggesting a good tolerance of acazicolcept treatment. In mice challenged with HOCL, acazicolcept induced a significant decrease in dermal thickness, collagen content, myofibroblast number, and inflammatory infiltrates characterized by B cells, T cells, neutrophils, and macrophages. In the Fra-2 Tg mouse model, acazicolcept treatment reduced lung collagen content, fibrillar collagen, histological fibrosis score, and right ventricular systolic pressure (RVSP). A reduction in frequency of CD4+ and T effector memory cells and an increase in the percentage of CD4+ T naïve cells in spleen and lung of acazicolcept-treated Fra-2 Tg mice was observed as compared to Fc control-treated Fra-2 Tg mice. Moreover, acazicolcept reduced CD69 and PD-1 expression on CD4+ T cells from the spleen and the lung. Target engagement by acazicolcept was demonstrated by blockade of CD28 and ICOS detection by flow cytometry in treated mice. CONCLUSIONS: Our results confirm the importance of costimulatory molecules in inflammatory-driven fibrosis. Our data highlight a key role of ICOS and CD28 in SSc. Using complementary models, we demonstrated that dual ICOS/CD28 blockade by acazicolcept decreased dermal and pulmonary fibrosis and alleviated pulmonary hypertension. These results pave the way for subsequent research on ICOS/CD28-targeted therapies.

Driving Role of Interleukin-2-Related Regulatory CD4+ T Cell Deficiency in the Development of Lung Fibrosis and Vascular Remodeling in a Mouse Model of Systemic Sclerosis.

OBJECTIVE: Systemic sclerosis (SSc) is a debilitating autoimmune disease characterized by severe lung outcomes resulting in reduced life expectancy. Fra-2-transgenic mice offer the opportunity to decipher the relationships between the immune system and lung fibrosis. This study was undertaken to investigate whether the Fra-2-transgenic mouse lung phenotype may result from an imbalance between the effector and regulatory arms in the CD4+ T cell compartment. METHODS: We first used multicolor flow cytometry to extensively characterize homeostasis and the phenotype of peripheral CD4+ T cells from Fra-2-transgenic mice and control mice. We then tested different treatments for their effectiveness in restoring CD4+ Treg cell homeostasis, including adoptive transfer of Treg cells and treatment with low-dose interleukin-2 (IL-2). RESULTS: Fra-2-transgenic mice demonstrated a marked decrease in the proportion and absolute number of peripheral Treg cells that preceded accumulation of activated, T helper cell type 2-polarized, CD4+ T cells. This defect in Treg cell homeostasis was derived from a combination of mechanisms including impaired generation of these cells in both the thymus and the periphery. The impaired ability of peripheral conventional CD4+ T cells to produce IL-2 may greatly contribute to Treg cell deficiency in Fra-2-transgenic mice. Notably, adoptive transfer of Treg cells, low-dose IL-2 therapy, or combination therapy changed the phenotype of Fra-2-transgenic mice, resulting in a significant reduction in pulmonary parenchymal fibrosis and vascular remodeling in the lungs. CONCLUSION: Immunotherapies for restoring Treg cell homeostasis could be relevant in SSc. An intervention based on low-dose IL-2 injections, as is already proposed in other autoimmune diseases, could be the most suitable treatment modality for restoring Treg cell homeostasis for future research.