Severe acute respiratory syndrome coronavirus 2, responsible for the severe acute respiratory syndrome known as COVID-19, has rapidly spread in almost every country and devastated the global economy and health care system. Lung injury is an early disease manifestation believed to be a major contributor to short- and long-term pathological consequences of COVID-19, and thus drug discovery aiming to ameliorate lung injury could be a potential strategy to treat COVID-19 patients. By inducing a severe acute respiratory syndrome–like pulmonary disease model through infecting A/J mice with murine hepatitis virus strain 1 (MHV-1), we show that i.v. administration of pazopanib ameliorates acute lung injuries without affecting MHV-1 replication. Pazopanib reduces cell apoptosis in MHV-1–infected lungs. Furthermore, we also identified that pazopanib has to be given no later than 48 h after the virus infection without compromising the therapeutic effect. Our study provides a potential treatment for coronavirus-induced lung injuries and support for further evaluation of pazopanib in COVID-19 patients.

The current COVID-19 pandemic is caused by a novel coronavirus, designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The disease causes the death of ∼1.2% of cases probably due to massive alveolar damage and progressive respiratory failure (1). Published pulmonary pathology and radiological reports of COVID-19 and previous SARS-CoV or Middle Eastern respiratory syndrome provide a clear association of the disease with acute respiratory distress syndrome (ARDS) (25).

ARDS is a severe form of acute lung injury (ALI), which is caused by direct or indirect insults to the lung (6, 7). In the case of COVID-19, lung injury is likely caused by both direct and indirect means. SARS-CoVs have been shown to cause necrosis of lung epithelial cells (direct insult) (2, 8). In addition, strong inflammatory responses elicited by the viral infection should also cause lung injury (indirect insult). Besides viral infection, there are many other causes for ALI/ARDS, including bacterial infection, gastric acid aspiration, and trauma. The incidence of ALI/ARDS is reported to be ∼200,000 per year in the United States (excluding COVID-19) with a mortality rate of ∼40% (6). Currently, there is no pharmacological intervention for the disease. Care of these conditions is largely dependent on supportive measures (9, 10). This probably contributes to the high fatality rate of COVID-19 and/or many of COVID-19–associated short-term and long-term morbidities as well as mortality.

Pazopanib is a multikinase inhibitor that acts as an angiogenesis inhibitor targeting vascular endothelial growth factor, platelet-derived growth factor, and c-Kit receptors, among others. By binding to its receptors, pazopanib crosses the cell membrane to interact with the intracellular domain of the tyrosine kinase receptors and later on to compete with ATP, where it inhibits downstream signaling pathways involved in tumor angiogenesis, growth, and metastasis (11, 12). Pazopanib was approved by the Food and Drug Administration (FDA) in 2012 for use in soft tissue sarcomas. We recently showed that pazopanib was also a high-affinity, substrate-specific inhibitor of serine/threonine protein kinases MAP3K2 and MAP3K3 for their phosphorylation of Nox2 subunit p47phox at Ser208 (13). Pazopanib treatment ameliorated lung injuries in both LPS (inflammation)-induced and acid (direct insult)-induced acute lung injury mouse models. Mechanistically, pazopanib acted through moderate increases in reactive oxygen species generation from neutrophils, which enhances pulmonary vasculature integrity and promotes lung epithelial cell survival and proliferation, leading to the increased pulmonary barrier function and resistance to ALI (13). Because there is an abundant presence of lung neutrophils in SARS-CoV–induced ARDS (25), a hallmark of ALI/ARDS (14), we postulate that pazopanib may be effective in curbing lung injury associated with the viral infection including COVID-19.

Intranasal infection of A/J mice with the murine coronavirus, murine hepatitis virus strain 1 (MHV-1), produced pulmonary pathological features of SARS. All MHV-1–infected A/J mice developed progressive interstitial edema, neutrophil/macrophage infiltrates, and hyaline membranes (15). In this study, using MHV-1–infected A/J mice as an animal model, we found that pazopanib, an FDA-approved drug, can reduce lung injuries caused by coronavirus infection, which provides a potential treatment for COVID-19.

Female A/J mice 6–8 wk of age (The Jackson Laboratory) were maintained in specific pathogen-free facilities with free access to sterile water. The mice were housed under 12-h light/12-h dark cycles. All experiments and animal use were performed with the approval of the Institutional Animal Care and Use Committee at Yale University.

MHV-1 was originally obtained from the American Type Culture Collection. MHV-1 stocks used in these studies were grown and titered in L2 Percy cells by Dr. Susan Compton at Yale University. Mice were infected with 5000 PFU intranasally for all experiments.

All viral infection studies were performed in a viral isolation room. Mice were anesthetized with ketamine/xylazine (100 and 10 mg/kg). Immediately, mice received an intranasal inoculation of 5000 PFU of MHV-1 in 20 μl of ice-cold DMEM. The virus (5000 PFU) was instilled into the nares, and mice were observed until the virus was inhaled. Mice were treated with pazopanib (3 mg/kg) or vehicle i.v. at the indicated time during MHV-1 infection. To measure pulmonary permeability, mice were injected with 100 μl of FITC-labeled albumin (10 mg/ml) via retro-orbital vein 2 h before sacrificing. Immediately after sacrifice, 1 ml of PBS was instilled into the lungs and retrieved via a tracheal catheter to obtain bronchoalveolar lavage (BAL). The FITC-albumin in the recovered fluid was measured by using a fluorescence plate reader.

Lung tissues with 1 ml of TRIzol snap-frozen at −80°C were thawed, and total RNAs were isolated from cells with a Direct-zol RNA kit (Zymo Research), as per the manufacturer’s instructions. cDNAs were synthesized from the RNAs with the iScript cDNA synthesis kit (Bio-Rad), according to the manufacturer’s protocol. Quantitative PCR was done with iTaq Universal SYBR Green supermix (Bio-Rad) (16). The primer sequences for virus load detection are 5′-GTCATGAGGCTATTCCTACTA-3′ and 5′-ATACACATCTTTGGTGGG-3′.

To prepare leukocytes in the BAL, 1 ml of PBS was instilled into the lungs and retrieved via a tracheal catheter to obtain BAL. Leukocytes in the BAL fluid were pelleted by centrifugation (500 × g for 5 min at 4°C). To prepare lung-infiltrating leukocytes, lungs were minced with scissors and incubated with digestion buffer (RPMI 1640 medium, 5% FBS, 1% penicillin-streptomycin, 25 mM HEPES, and 300 U of collagenase (Sigma-Aldrich, C0130) in a shaker for 1 h at 37°C. Dispersed cells were filtered through a 70-μm cell strainer to eliminate clumps and debris. After centrifugation for 5 min (500 × g) at 4°C, cell pellets were resuspended in RBC lysis buffer (Sigma-Aldrich, R7757) and incubated at room temperature for 5 min to remove erythrocytes. Cells were pelleted again and resuspended in PBS.

H&E staining of unlavaged lung tissues was performed at the Comparative Pathology Research Core at Yale School of Medicine. Briefly, mouse right ventricles were perfused with 10 ml of PBS and the lungs were inflated with 4% paraformaldehyde at a constant pressure of 25 cm H2O, and then fixed in 4% paraformaldehyde for 24 h at 4°C. The tissues were embedded in paraffin and cut into 5-μm-thick sections. Sections were stained with H&E, and images were taken with a Keyence BZ-X800 cell imaging microscope. ALI indices were quantified using H&E-stained lung sections as previously described (17). Five parameters (A, neutrophils in the alveolar space; B, neutrophils in the interstitial space; C, hyaline membranes; D, proteinaceous debris filling the airspaces; E, alveolar septal thickening) were evaluated. The lung injury score was calculated as [(20 × A) + (14 × B) + (7 × C) + (7 × D) + (2 × E)]/(number of fields × 100). Each datum is the average score of the five criteria from five fields, which were uniformly and randomly picked from one lung section of one mouse. Five mice were included in each group. Images were evaluated by two investigators who were blinded to the identity of the sections.

Flow cytometry was performed as previously described (18). Cells in single-cell suspension were fixed with 2% PFA (Santa Cruz, sc-281692). After being washed with a flow cytometry staining buffer (eBioscience, 00-4222-26), cells were stained with Abs for cell surface markers for 1 h on ice in the dark. The cells were then washed, pelleted, and resuspended in the flow cytometry staining buffer for flow cytometry analysis. The absolute number of cells was counted by using CountBright absolute counting beads (Invitrogen, C36950), according to the manufacturer’s instructions. Abs for flow cytometry are as follows: mouse CD45-BUV395 (BD Biosciences), mouse CD4-FITC (BioLegend), mouse CD8-Pacific Blue (BioLegend), mouse CD11b-Pacific Blue (BioLegend), mouse CD11b-allophycocyanin (BioLegend), mouse Ly6G-Pacific Blue (BioLegend), mouse Ly6C-FITC (BioLegend), mouse NK1.1-allophycocyanin (BioLegend), and mouse B220-PE (BioLegend), mouse F4/80-allophycocyanin (BioLegend). Major immune cell populations were determined by CD45+CD11b+Ly6G+ (neutrophils), CD45+CD11b+Ly6GLy6C+ (monocytes), CD45+CD4CD8B220NK1.1+ (NK cells), CD45+CD4+ (CD4 T cells), CD45+CD8+ (CD8 T cells), CD45+CD11b+F4/80+ (macrophages).

Unlavaged lungs were inflated and then fixed with 4% PFA for 4–6 h on a shaker at 4°C. They were then washed with PBS three times and perfused in 30% sucrose solution in PBS overnight at 4°C. They were subsequently mounted in OCT embedding compound and frozen first at −20°C and then at −80°C. Tissue sections were prepared at 8-μm thickness with a cryostat and mounted onto gelatin-coated histological slides, which were stored at −80°C. For immunostaining, slides were thawed to room temperature and fixed in pre-cooled acetone for 10 min, then rehydrated in PBS for 10 min. The slides were incubated in a blocking buffer (1% horse serum and 0.02% Tween 20 in PBS) for 1 h at room temperature, then incubated with anti-ABCA3 (Abcam, ab24751), anti-cleaved caspase-3 Abs (Cell Signaling Technology, 9661), anti-podoplanin (PDPN) (R&D Systems, AF3244), and anti-CD31 (R&D Systems, AF3628), which were diluted in the blocking buffer overnight at 4°C. The slides were then washed three times with PBS and incubated with a secondary Ab in the incubation buffer for 1 h at room temperature. After repeated washes, the slides were mounted with an anti-fade mounting medium containing DAPI (Thermo Fisher Scientific, P36931) and visualized with a Keyence BZ-X800 cell imaging microscope (19). Apoptotic cells were quantified by using ImageJ software. Lung apoptotic cells were assessed by normalizing the area of active caspase-3–positive signals to the area of DAPI signals. Lung apoptotic type I epithelial cells, type II epithelial cells, and endothelial cells were assessed by normalizing the area of active caspase-3 and cell marker double-positive signals to the area of cell marker–positive signals. Each datum represents one field from one section of one mouse. Five fields were evaluated for each section and three mice were included in each group.

Mice were anesthetized with ketamine/xylazine (100 and 10 mg/kg) for blood collection by retro-orbital bleeding. Serum was collected by centrifugation of the blood in BD Microtainer blood collection tubes (BD Biosciences, 365985) at 10,000 × g for 1 min. Serum was then transferred to the Eppendorf tube for liquid chromatography–mass spectrometry measurement.

Comparisons of means between two groups and multiple groups were tested by unpaired, two-tailed t test and one-way ANOVA test using Prism 9.2.0 software (GraphPad Software). For Kaplan–Meier survival analysis a log-rank test was used. Statistical tests used biological replicates. Statistical significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

To recapitulate pulmonary pathological manifestations of COVID-19 in mice, we infected A/J mice intranasally with 5000 PFU of MHV-1. Forty-eight hours after MHV-1 infection, we examined the lung vascular permeability, which indicated the severity of acute lung injury. BAL was collected from MHV-1–infected A/J mice 2 h after injection of FITC-albumin i.v. MHV-1 inoculation increased FITC-albumin extravasation into the BAL when compared with the control treatment, indicating that MHV-1 infection increased pulmonary permeability, a hallmark of ALI, in A/J mice (Fig. 1A). Neutrophils also infiltrated into BAL after MHV-1 infection, another hallmark of acute lung injury (Fig. 1B, Supplemental Fig. 1A). Additionally, H&E histological staining revealed that MHV-1 infection caused interstitial alveolar thickening, fluid accumulation in alveolar spaces (pulmonary edema), and heavy leukocyte infiltrates (Fig. 1C, 1D). Taken together, intranasal inoculation of MHV-1 induced lung injury 48 h after challenge in A/J mice, with characteristics very similar to those observed in human COVID-19, including interstitial pulmonary infiltrates, congestion, and pulmonary edema.

FIGURE 1.

Coronavirus MHV-1–induced lung injury in A/J mice. (A and B) A/J mice (8 wk old) were i.n. infected with 5000 PFU of MHV-1. BAL was collected 48 h after MHV-1 infection, followed by the measurements of pulmonary permeability (A) and the total number of neutrophils (B). Data were combined from two independent experiments with four or two mice per group per experiment; each datum represents one mouse. (C) Representative histology of injured lungs 48 h after MHV-1 infection. (D) Quantification of lung injuries. Each datum is the average score of five fields, which were uniformly and randomly picked from one lung section of one mouse. Five mice were included in each group. The samples were collected from two independent experiments with three or two mice per group per experiment. The samples were then combined for the following processing, staining, and evaluation. Data in (A), (B), and (D) are presented as means ± SEM. ***p < 0.001, by Student t test; ns, not significant.

FIGURE 1.

Coronavirus MHV-1–induced lung injury in A/J mice. (A and B) A/J mice (8 wk old) were i.n. infected with 5000 PFU of MHV-1. BAL was collected 48 h after MHV-1 infection, followed by the measurements of pulmonary permeability (A) and the total number of neutrophils (B). Data were combined from two independent experiments with four or two mice per group per experiment; each datum represents one mouse. (C) Representative histology of injured lungs 48 h after MHV-1 infection. (D) Quantification of lung injuries. Each datum is the average score of five fields, which were uniformly and randomly picked from one lung section of one mouse. Five mice were included in each group. The samples were collected from two independent experiments with three or two mice per group per experiment. The samples were then combined for the following processing, staining, and evaluation. Data in (A), (B), and (D) are presented as means ± SEM. ***p < 0.001, by Student t test; ns, not significant.

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Our previous study showed that pazopanib ameliorated acute lung injury by protecting pulmonary barrier functions in LPS and acid-induced mouse ALI models (13). To test whether pazopanib can protect against MHV-1–induced lung injury, A/J mice were treated with pazopanib during MHV-1 infection. A pharmacokinetic study revealed a short half-life (5.9 h) of pazopanib in the blood, suggesting that pazopanib was cleared quickly after i.v. delivery to mice (Supplemental Fig. 1B). Based on this characteristic feature, pazopanib may need to be administered to mice multiple times with a short interval. First, we tried a three-dose regimen of pazopanib treatment. The first dose was injected i.v. at 6 h after MHV-1 inoculation. The second and third doses were delivered at 15 and 26 h after the first dose. In this scenario, pazopanib significantly reduced pulmonary permeability versus vehicle control (Fig. 2A, 2B). Next, we examined whether two doses of pazopanib can also ameliorate lung injury. In this scenario, two doses of pazopanib were given at 24 and 33 h after MHV-1 inoculation, and a significant reduction of pulmonary permeability was observed as well (Fig. 2C, 2D). Next, we proceeded to evaluate a one-dose regimen (24 h after virus inoculation) of pazopanib treatment. Although the one-dose regimen had a lower concentration of pazopanib in the blood (Supplemental Fig. 1C), it still showed a significant reduction in lung permeability (Fig. 2E, 2F). Histological assessment of the lung further demonstrated that treatment with pazopanib reduced MHV-1–induced lung pathology (Fig. 2G, 2H, Supplemental Fig. 1D, 1E). Unlike the role of pazopanib in suppressing acute lung injury, liver and kidney injury induced by MHV-1 were not ameliorated by pazopanib treatment (Supplemental Fig. 1F). To examine whether the effect of pazopanib on suppressing acute lung injury was due to alteration of virus replication, we determined the viral loads in the lungs from the MHV-1–infected mice with or without pazopanib treatment. Pazopanib did not affect viral loads regardless of the treatment modalities (Supplemental Fig. 1G). Major immune cell populations in the BAL and lung were also examined after pazopanib treatment. Pazopanib did not affect neutrophil infiltration into BAL and lung (Supplemental Fig. 2A, 2B). The infiltration of CD4 T cells, CD8 T cells, or macrophages in the lung was not affected by pazopanib either (Supplemental Fig. 2B, 2C). However, pazopanib treatment appeared to increase NK cell presence and CD8 expression in the lungs (Supplemental Fig. 2D, 2E). These results together indicated that i.v. delivery of pazopanib suppressed MHV-1–induced acute lung injury in mice without affecting MHV-1 replication in the lungs.

FIGURE 2.

Pazopanib treatment can reduce MHV-1–induced lung injury. (A, C, and E) Schematic diagram of the different regimens of pazopanib treatment. (B, D, and F) BAL was collected 48 h after MHV-1 infection and pulmonary permeability was examined. Data in (B) were combined from two independent experiments. The vehicle group included four and two mice per experiment. The pazopanib group included five or one mouse per experiment; each datum represents one mouse. Data in (D) were combined from two independent experiments with four or two mice per group per experiment; each datum represents one mouse. Data in (F) were combined from three independent experiments; in each independent experiment, each group contained two or three mice, and each datum represents one mouse. (G and H). A/J mice (8 wk old) were intranasally infected with medium or 5000 PFU of MHV-1. Pazopanib or vehicle was delivered 24 h after MHV-1 inoculation. At 48 h postinfection, mice were sacrificed and lungs were collected for histology analysis. Representative lung histology images (H&E staining) are shown in (G). Quantification of lung injuries in (H). Each datum is the average score of five fields, which were uniformly and randomly picked from one lung section of one mouse. Five mice were included in each group. The samples were collected from two independent experiments with three or two mice per group per experiment. The samples were then combined for the following processing, staining, and evaluation. Data in (B), (D), and (F) are presented as means ± SEM (Student t test), and data in (H) are presented as means ± SEM (one-way ANOVA). **p < 0.01, ***p < 0.001; ns, not significant.

FIGURE 2.

Pazopanib treatment can reduce MHV-1–induced lung injury. (A, C, and E) Schematic diagram of the different regimens of pazopanib treatment. (B, D, and F) BAL was collected 48 h after MHV-1 infection and pulmonary permeability was examined. Data in (B) were combined from two independent experiments. The vehicle group included four and two mice per experiment. The pazopanib group included five or one mouse per experiment; each datum represents one mouse. Data in (D) were combined from two independent experiments with four or two mice per group per experiment; each datum represents one mouse. Data in (F) were combined from three independent experiments; in each independent experiment, each group contained two or three mice, and each datum represents one mouse. (G and H). A/J mice (8 wk old) were intranasally infected with medium or 5000 PFU of MHV-1. Pazopanib or vehicle was delivered 24 h after MHV-1 inoculation. At 48 h postinfection, mice were sacrificed and lungs were collected for histology analysis. Representative lung histology images (H&E staining) are shown in (G). Quantification of lung injuries in (H). Each datum is the average score of five fields, which were uniformly and randomly picked from one lung section of one mouse. Five mice were included in each group. The samples were collected from two independent experiments with three or two mice per group per experiment. The samples were then combined for the following processing, staining, and evaluation. Data in (B), (D), and (F) are presented as means ± SEM (Student t test), and data in (H) are presented as means ± SEM (one-way ANOVA). **p < 0.01, ***p < 0.001; ns, not significant.

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The one-dose regimen of pazopanib treatment at 24 h after virus inoculation provided significant protection from coronavirus-induced acute lung injury. In the real world, COVID-19 patients may not know that they have been infected with the virus until the onset of the symptoms. To find out what is the best time for starting pazopanib treatment, two more single-dose regimens, 48 or 72 h after virus inoculation, were tested in the mouse model. Significant improvement was observed in the single-dose regimen at 48 h postinfection (Fig. 3A, 3B), but not in the regimen at 72 h postinfection (Fig. 3C, 3D). Because these mice had developed the severe progressive pulmonary disease at 48 h, the findings suggest that pazopanib treatment may be more effective when administrated early in the disease.

FIGURE 3.

Pazopanib has to be given no later than 48 h after MHV-1 infection. (A and C) Schematic diagram of the different regimens of pazopanib treatment. (B and D) BAL was collected at the indicated time points after MHV-1 infection and pulmonary permeability was examined. Data in (B) were combined from two independent experiments: in the first independent experiment, each group (from left to right) contained two, four, and three mice, and in the second independent experiment, each group (from left to right) contained two, two, and three mice; each datum represents one mouse. Data in (D) were combined from two independent experiments: in the first independent experiment, each group (from left to right) contained two, four, and three mice, and in the second independent experiment, each group (from left to right) contained two mice; each datum represents one mouse. Data in (B) and (D) are presented as means ± SEM (one-way ANOVA). **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant.

FIGURE 3.

Pazopanib has to be given no later than 48 h after MHV-1 infection. (A and C) Schematic diagram of the different regimens of pazopanib treatment. (B and D) BAL was collected at the indicated time points after MHV-1 infection and pulmonary permeability was examined. Data in (B) were combined from two independent experiments: in the first independent experiment, each group (from left to right) contained two, four, and three mice, and in the second independent experiment, each group (from left to right) contained two, two, and three mice; each datum represents one mouse. Data in (D) were combined from two independent experiments: in the first independent experiment, each group (from left to right) contained two, four, and three mice, and in the second independent experiment, each group (from left to right) contained two mice; each datum represents one mouse. Data in (B) and (D) are presented as means ± SEM (one-way ANOVA). **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant.

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Our previous study showed that pazopanib could protect lung epithelial cells from apoptosis (13). To determine whether pazopanib also has the same effect during MHV-1 infection, we examined the active caspase-3 expression in the lung tissues at 48 h after MHV-1 inoculation. MHV-1 infection markedly induced the expression of active caspase-3 in the lungs, which was significantly reduced by pazopanib treatment (Fig. 4A, 4B). Next, we costained active caspase-3 with PDPN (type I epithelial cell marker), ABCA3 (type II epithelial cell marker), and CD31 (endothelial cell marker), respectively. Active caspase-3 was found in all three cell types, but it was mainly colocalized with ABCA3. Pazopanib treatment reduced active caspase-3 in both epithelial cells and endothelial cells (Fig. 4C–H). These results suggested that pazopanib could inhibit apoptosis of lung cells and prolong their survival.

FIGURE 4.

Pazopanib treatment reduced lung cells apoptosis. A/J mice (8 wk old) were intranasally infected with medium or 5000 PFU of MHV-1. Pazopanib or vehicle was delivered 24 h after MHV-1 inoculation. (AH) At 48 h postinfection, mice were sacrificed and lung sections were stained with anti-active caspase-3 (Casp-3) and DAPI (A and B), anti-active Casp-3, ABCA3, and DAPI (C and D), anti-active caspase-3 (Casp-3), PDPN, and DAPI (E and F), and anti-active Casp-3, CD31, and DAPI (G and H) as indicated in the panels. Representative images are shown in (A), (C), (E), and (G). Quantifications are shown in (B), (D), (F), and (H). Each datum represents one field from one section of one mouse. Five fields were evaluated for each section and three mice were included in each group. The samples were collected from two independent experiments with two or one mouse per group per experiment. The samples were then combined for the following processing, staining, and evaluation. Data in (B) are presented as means ± SEM (one-way ANOVA), and data in (D), (F), and (H) are presented as means ± SEM (Student t test). ***p < 0.001, ****p < 0.0001 (B, D, F, and H); n = 15.

FIGURE 4.

Pazopanib treatment reduced lung cells apoptosis. A/J mice (8 wk old) were intranasally infected with medium or 5000 PFU of MHV-1. Pazopanib or vehicle was delivered 24 h after MHV-1 inoculation. (AH) At 48 h postinfection, mice were sacrificed and lung sections were stained with anti-active caspase-3 (Casp-3) and DAPI (A and B), anti-active Casp-3, ABCA3, and DAPI (C and D), anti-active caspase-3 (Casp-3), PDPN, and DAPI (E and F), and anti-active Casp-3, CD31, and DAPI (G and H) as indicated in the panels. Representative images are shown in (A), (C), (E), and (G). Quantifications are shown in (B), (D), (F), and (H). Each datum represents one field from one section of one mouse. Five fields were evaluated for each section and three mice were included in each group. The samples were collected from two independent experiments with two or one mouse per group per experiment. The samples were then combined for the following processing, staining, and evaluation. Data in (B) are presented as means ± SEM (one-way ANOVA), and data in (D), (F), and (H) are presented as means ± SEM (Student t test). ***p < 0.001, ****p < 0.0001 (B, D, F, and H); n = 15.

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To test whether pazopanib can reduce mortality induced by MHV-1 infection, A/J mice were infected with MHV-1, and pazopanib was i.v. delivered to mice once daily beginning 24 h after virus inoculation. The last dose was given at 120 h after inoculation. Mice started to die on day 5; however, pazopanib treatment did not change the survival rate or body weight loss after MHV-1 inoculation (Fig. 5A, 5B). These results suggested that pazopanib alone cannot extend the survival of A/J mice after MHV-1 inoculation.

FIGURE 5.

Pazopanib treatment does not affect survival of mice in MHV-1–induced lung injury model. (A and B) The effect of pazopanib on mortality (A) and weight change (B) in mice was examined in the MHV-1–induced ALI model. Experiments for (A) and (B) were conducted for twice with similar results (the first independent experiment contained five mice in the vehicle control group [vehicle] and six mice in the pazopanib treatment group [Pazo], and the second independent experiment contained seven mice per group). Data from the first independent experiment are shown in (A) and (B). Data in (A) are presented using a Mantel–Cox log-rank test. ns, not significant.

FIGURE 5.

Pazopanib treatment does not affect survival of mice in MHV-1–induced lung injury model. (A and B) The effect of pazopanib on mortality (A) and weight change (B) in mice was examined in the MHV-1–induced ALI model. Experiments for (A) and (B) were conducted for twice with similar results (the first independent experiment contained five mice in the vehicle control group [vehicle] and six mice in the pazopanib treatment group [Pazo], and the second independent experiment contained seven mice per group). Data from the first independent experiment are shown in (A) and (B). Data in (A) are presented using a Mantel–Cox log-rank test. ns, not significant.

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In this study, we demonstrated that i.v. administration of pazopanib can significantly reduce lung injury in coronavirus MHV-1–infected A/J mice. Pazopanib is an FDA-approved anticancer drug, which was identified as inhibiting multiple tyrosine kinase receptors associated with angiogenesis and tumor cell proliferation. Our previous study showed that pazopanib was a potent inhibitor for MAP3K2/3-mediated phosphorylation of p47phox at Ser208 and acts largely through this MAP3K2/3-p47phox pathway to ameliorate lung injuries and extend survival in mouse LPS- and HCl-induced ALI models (13). COVID-19, caused by SARS-CoV-2, has become a pandemic for almost 2 y. Most patients with COVID-19 exhibit mild to moderate symptoms, but ∼20% develop ALI/ARDS (20). Currently, there is no approved drug for treating this SARS-CoV-2–induced ALI/ARDS. Pazopanib has been approved by FDA in the clinic for years and is well tolerated even for long-term use (21). Thus, our current study demonstrates that pazopanib has a promising potential for the treatment of ALI/ARDS in COVID patients.

The mechanism of action of pazopanib described in our previous study is through a paracrine mechanism. The moderate elevation of extracellular H2O2 released from neutrophils triggers the pulmonary microenvironment remodeling through crosstalk and interactions of different lung cell types, leading to the protection of lungs from acute injuries (13). Neutrophils are also abundantly recruited to the lungs in SARS-CoV-2–induced ALI/ARDS (4, 5). In autopsy samples from the lungs of COVID-19 patients, neutrophil infiltration was observed in pulmonary capillaries, and extravasation of neutrophils into the alveolar space was noted as well (22). Thus, we believe that pazopanib ameliorates coronavirus-induced lung injuries through the same mechanism. In the present study, we found that neutrophils were accumulated in the lungs of MHV-1–infected mice. Consistent with our previous observations in the other mouse ALI models, pazopanib treatment did not alter neutrophil infiltration into the lungs in this MHV-1 infection model. Our histology results showed that pazopanib treatment substantially inhibits the apoptosis of pulmonary cells. Our previous study has already demonstrated that pazopanib cannot affect pulmonary cells by itself (13), and therefore these results suggested that pazopanib-treated neutrophils impact pulmonary cells to favor enhancement of barrier functions via a paracrine mechanism in coronavirus-induced ALI. As neutrophil presence is only dominant at the early stage of viral infection, it may help to explain why pazopanib is more efficacious at the early stage of the disease.

Pazopanib extends the survival of HCl- or LPS-induced ALI mice in our previous study; however, the survival is not significantly changed in this coronavirus-induced ALI model. This is probably because HCl or LPS only induces a transient insult to the lung, whereas coronavirus MHV-1 can proliferate in the lung and cause a continuous and less confined insult. Multiple organs, including livers, have been observed to be affected by MHV-1 infection (15), and our data indicated that pazopanib failed to reduce the liver and kidney injury induced by MHV-1 (Supplemental Fig. 1F). Given that pazopanib cannot inhibit virus proliferation (Supplemental Fig. 1G), the protective effect of pazopanib might be overridden as more and more viruses are amplifying. Future studies may be needed to investigate whether combining pazopanib with other antivirus drugs could give us better results or even extend survival in the coronavirus-induced ALI model.

This work was supported by National Institutes of Health/National Heart, Lung, and Blood Institute Grants R01HL145152 (to W.T.), R35HL135805 (to D.W.), and a Sponsor Research Grant fromQx Therapeutics, Inc.

W.T. and D.W. designed the experiments; Y.L., Q.Y., Q.W., S.C., and W.T. performed the experiments; W.T., D.W., Y.L., Q.Y., and Q.W. analyzed the data; W.T., D.W., and Y.L. wrote the manuscript; and all authors reviewed and approved the manuscript.

The online version of this article contains supplemental material.

Abbreviations used in this article:

     
  • ALI

    acute lung injury

  •  
  • ARDS

    acute respiratory distress syndrome

  •  
  • BAL

    bronchoalveolar lavage

  •  
  • FDA

    Food and Drug Administration

  •  
  • MHV-1

    murine hepatitis virus strain 1

  •  
  • PDPN

    podoplanin

  •  
  • SARS-CoV-2

    severe acute respiratory syndrome coronavirus 2

1.
Maitz
T.
,
D.
Parfianowicz
,
A.
Vojtek
,
Y.
Rajeswaran
,
A. V.
Vyas
,
R.
Gupta
.
2022
.
COVID-19 cardiovascular connection: a review of cardiac manifestations in COVID-19 infection and treatment modalities.
Curr. Probl. Cardiol.
DOI: 10.1016/j.cpcardiol.2022.101186
.
2.
Ding
Y.
,
H.
Wang
,
H.
Shen
,
Z.
Li
,
J.
Geng
,
H.
Han
,
J.
Cai
,
X.
Li
,
W.
Kang
,
D.
Weng
, et al
2003
.
The clinical pathology of severe acute respiratory syndrome (SARS): a report from China.
J. Pathol.
200
:
282
289
.
3.
Ng
D. L.
,
F.
Al Hosani
,
M. K.
Keating
,
S. I.
Gerber
,
T. L.
Jones
,
M. G.
Metcalfe
,
S.
Tong
,
Y.
Tao
,
N. N.
Alami
,
L. M.
Haynes
, et al
2016
.
Clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of Middle East respiratory syndrome coronavirus infection in the United Arab Emirates, April 2014.
Am. J. Pathol.
186
:
652
658
.
4.
Tian
S.
,
W.
Hu
,
L.
Niu
,
H.
Liu
,
H.
Xu
,
S. Y.
Xiao
.
2020
.
Pulmonary pathology of early-phase 2019 novel coronavirus (COVID-19) pneumonia in two patients with lung cancer.
J. Thorac. Oncol.
15
:
700
704
.
5.
Xu
Z.
,
L.
Shi
,
Y.
Wang
,
J.
Zhang
,
L.
Huang
,
C.
Zhang
,
S.
Liu
,
P.
Zhao
,
H.
Liu
,
L.
Zhu
, et al
2020
.
Pathological findings of COVID-19 associated with acute respiratory distress syndrome.
Lancet Respir. Med.
8
:
420
422
.
6.
Johnson
E. R.
,
M. A.
Matthay
.
2010
.
Acute lung injury: epidemiology, pathogenesis, and treatment.
J. Aerosol Med. Pulm. Drug Deliv.
23
:
243
252
.
7.
Máca
J.
,
O.
Jor
,
M.
Holub
,
P.
Sklienka
,
F.
Burša
,
M.
Burda
,
V.
Janout
,
P.
Ševčík
.
2017
.
Past and present ARDS mortality rates: a systematic review.
Respir. Care
62
:
113
122
.
8.
Li
S.
,
Y.
Zhang
,
Z.
Guan
,
H.
Li
,
M.
Ye
,
X.
Chen
,
J.
Shen
,
Y.
Zhou
,
Z. L.
Shi
,
P.
Zhou
,
K.
Peng
.
2020
.
SARS-CoV-2 triggers inflammatory responses and cell death through caspase-8 activation.
Signal Transduct. Target. Ther.
5
:
235
.
9.
Brower
R. G.
,
M. A.
Matthay
,
A.
Morris
,
D.
Schoenfeld
,
B. T.
Thompson
,
A.
Wheeler
;
Acute Respiratory Distress Syndrome Network
.
2000
.
Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome.
N. Engl. J. Med.
342
:
1301
1308
.
10.
Weinert
C. R.
,
C. R.
Gross
,
W. A.
Marinelli
.
2003
.
Impact of randomized trial results on acute lung injury ventilator therapy in teaching hospitals.
Am. J. Respir. Crit. Care Med.
167
:
1304
1309
.
11.
Halim
N. A.
,
R. E.
Sayed
,
I. A.
Alameh
,
J.
Khoury
,
C. E.
Nakib
,
M. B.
Zerdan
,
M.
Charafeddine
,
F.
Farhat
,
F. E.
Karak
,
H. I.
Assi
.
2021
.
Safety and efficacy of pazopanib as a second-line treatment and beyond for soft tissue sarcomas: a real-life tertiary-center experience in the MENA region.
Cancer Treat. Res. Commun.
26
:
100275
.
12.
Sloan
B.
,
N. S.
Scheinfeld
.
2008
.
Pazopanib, a VEGF receptor tyrosine kinase inhibitor for cancer therapy.
Curr. Opin. Investig. Drugs
9
:
1324
1335
.
13.
Yuan
Q.
,
A.
Basit
,
W.
Liang
,
R.
Qu
,
Y.
Luan
,
C.
Ren
,
A.
Li
,
X.
Xu
,
X.
Liu
,
C.
Yang
, et al
2021
.
Pazopanib ameliorates acute lung injuries via inhibition of MAP3K2 and MAP3K3.
Sci. Transl. Med.
13
:
eabc2499
.
14.
Matthay
M. A.
,
R. L.
Zemans
.
2011
.
The acute respiratory distress syndrome: pathogenesis and treatment.
Annu. Rev. Pathol.
6
:
147
163
.
15.
De Albuquerque
N.
,
E.
Baig
,
X.
Ma
,
J.
Zhang
,
W.
He
,
A.
Rowe
,
M.
Habal
,
M.
Liu
,
I.
Shalev
,
G. P.
Downey
, et al
2006
.
Murine hepatitis virus strain 1 produces a clinically relevant model of severe acute respiratory syndrome in A/J mice.
J. Virol.
80
:
10382
10394
.
16.
Chen
Y.
,
L.
Wang
,
J.
Jin
,
Y.
Luan
,
C.
Chen
,
Y.
Li
,
H.
Chu
,
X.
Wang
,
G.
Liao
,
Y.
Yu
, et al
2017
.
p38 inhibition provides anti-DNA virus immunity by regulation of USP21 phosphorylation and STING activation.
J. Exp. Med.
214
:
991
1010
.
17.
Matute-Bello
G.
,
G.
Downey
,
B. B.
Moore
,
S. D.
Groshong
,
M. A.
Matthay
,
A. S.
Slutsky
,
W. M.
Kuebler
;
Acute Lung Injury in Animals Study Group
.
2011
.
An official American Thoracic Society workshop report: features and measurements of experimental acute lung injury in animals.
Am. J. Respir. Cell Mol. Biol.
44
:
725
738
.
18.
Sun
J.
,
Y.
Luan
,
D.
Xiang
,
X.
Tan
,
H.
Chen
,
Q.
Deng
,
J.
Zhang
,
M.
Chen
,
H.
Huang
,
W.
Wang
, et al
2016
.
The 11S proteasome subunit PSME3 is a positive feedforward regulator of NF-κB and important for host defense against bacterial pathogens.
Cell Rep.
14
:
737
749
.
19.
Qiu
B.
,
Y.
Xu
,
J.
Wang
,
M.
Liu
,
L.
Dou
,
R.
Deng
,
C.
Wang
,
K. E.
Williams
,
R. B.
Stewart
,
Z.
Xie
, et al
2019
.
Loss of FKBP5 affects neuron synaptic plasticity: an electrophysiology insight.
Neuroscience
402
:
23
36
.
20.
Cao
X.
2020
.
COVID-19: immunopathology and its implications for therapy.
Nat. Rev. Immunol.
20
:
269
270
.
21.
Zivi
A.
,
L.
Cerbone
,
F.
Recine
,
C. N.
Sternberg
.
2012
.
Safety and tolerability of pazopanib in the treatment of renal cell carcinoma.
Expert Opin. Drug Saf.
11
:
851
859
.
22.
Barnes
B. J.
,
J. M.
Adrover
,
A.
Baxter-Stoltzfus
,
A.
Borczuk
,
J.
Cools-Lartigue
,
J. M.
Crawford
,
J.
Daßler-Plenker
,
P.
Guerci
,
C.
Huynh
,
J. S.
Knight
, et al
2020
.
Targeting potential drivers of COVID-19: Neutrophil extracellular traps.
J. Exp. Med.
217
:
e20200652
.

D.W. is a co-founder and serves as a scientific advisory board member of Qx Therapeutics, which licensed the intellectual properties described in this study from Yale University. D.W., W.T., and Q.Y. are inventors of the patent application “Compounds, compositions and methods of treating or preventing acute lung injury” (no. 62/486,232). The other authors have no financial conflicts of interest.

Supplementary data