Effect of Subacute Angiotensin II Elevations on Sphingolipid Levels and Lung Injury in Mice

Background: There is a paucity of therapies for acute lung injury (ALI) induced by respiratory viruses. A previously demonstrated key mechanism of ALI, particularly in the setting of severe acute respiratory syndrome coronavirus infections, has been ascribed to decreased cell surface angiotensin converting enzyme 2 (ACE2) leading to increased circulating levels of angiotensin II (Ang2). In turn, supraphysiological Ang2 levels trigger a cascade of events that culminates with endothelial injury in the systemic circulation via acid sphingomyelinase (ASMase) activation. ASMase has been implicated in several models of ALI, but its specific involvement in Ang2-induced ALI is unknown. ASMase hydrolyzes sphingomyelin to pro-apoptotic, edemagenic ceramide, which can be metabolized to endothelial-protective sphingosine-1-phosphate (S1P). Therefore, the ratio of ceramide/S1P can determine endothelial cell fate and lung vascular permeability. We hypothesized that ceramide levels are increased relative to S1P in mice with Ang2-induced ALI. Methods: Following a published protocol of Ang2-induced ALI (Wu et al, 2017), we delivered Ang2 via osmotic pumps (1 ug/kg/min, 7 days;Ang2-mice), using saline (sham) or untreated C57BL/6 mice as controls. We evaluated pulmonary function (FlexiVent);albumin, IgM (ELISA), and inflammatory cell abundance in bronchoalveolar lavage fluid (BALF);and lung parenchyma inflammation and fibrosis (Ashcroft score) on H/E-stained lungs. Sphingolipid levels in lungs and plasma were measured by tandem liquid chromatography/mass spectrometry. Results: Inspiratory capacity, lung compliance, and body weight all decreased in Ang2-mice (by 13-14%, p<0.05 each) compared to sham. Lung pressure-volume loops exhibited a right-shift in Ang2- vs. sham or untreated mice. There was no significant change in BALF albumin, IgM, or inflammatory cells, or in lung histology inflammation or fibrosis scores in Ang2-mice. Compared to sham, S1P levels were significantly increased in plasma and unlavaged lung in Ang2-mice, decreasing ceramide/S1P ratios (from 3.1 to 2.0, and 26 to 20, respectively, p<0.05 each). Conclusions: Sustained subacute systemic elevations of Ang2 increased lung stiffness, but did not cause severe ALI in mice. Lung and circulatory elevations of S1P but not ceramide may have protected against lung edema and inflammatory injury. Although the cause of increased lung stiffness in this model remains to be elucidated, it is notable that chronic (months) supraphysiological elevations of either Ang2 or S1P have been associated with lung fibrosis. In conclusion, a second-hit injury may be necessary to augment the susceptibility of murine lung to Ang2-induced endothelial damage and inflammation relevant to coronavirus.

PTPα Is a Regulator of Fibroproliferation during ARDS

Rationale: Acute Respiratory Distress Syndrome (ARDS) is a major healthcare issue resulting in high morbidity and mortality. Up to 25% of patients with ARDS will develop pathological fibrosis, termed Fibroproliferative ARDS (FP-ARDS). In this subset of patients, ongoing injury and dysregulated repair results in persistence of inflammatory infiltrates, myofibroblast differentiation, and dysregulated deposition of extracellular matrix. The consequences of this pathologic fibroproliferation include increased mortality, prolonged ventilator dependence, and diminished quality of life for survivors. This is especially relevant in the context of the COVID-19 pandemic, during which fibroproliferation following ARDS is an increasingly recognized entity. We demonstrate that a tyrosine phosphatase, PTPα, plays an important role in promoting fibroproliferation following ARDS. Methods: Mice deficient in PTPα and littermate controls were treated with intratracheal hydrochloric acid;histological, biochemical, and gene expression endpoint analyses were performed. NIH 3T3 (murine) fibroblasts or normal human lung fibroblasts (NLHFs) were stimulated with BAL fluid from these mice, or from ARDS patients and healthy controls, respectively. Human lung fibroblasts in which PTPα was deleted via CRISPR-cas9 targeted guide RNA were treated with exogenous TGF-β. Analysis of gene expression by qPCR was performed. Results: Mice deficient in PTPα demonstrated reduced fibrotic outcomes after lung injury. A fibrosis-focused qPCR array showed significant attenuation of key pro-fibrotic genes in PTPα-null mice, even at early timepoints during which inflammatory outcomes were unchanged. BAL fluid from PTPα-null mice treated with HCl induced less fibroproliferative gene expression in murine fibroblasts than BAL fluid from littermate controls. Human fibroblasts lacking PTPα (generated via CRISPR-cas9 deletion) showed reduced pro-fibrotic gene expression responses following stimulation with exogenous TGF-β. NHLFs stimulated with ARDS patient BAL fluid demonstrated increased pro-fibrotic gene expression as compared to BAL fluid from control patients. Conclusions: We conclude that absence of PTPα is protective in a model of FP-ARDS, through mechanisms that result in reduced TGF-β-dependent gene expression. The alveolar milieu of mice lacking PTPα has reduced fibroproliferative potential than those that express PTPα. Similarly, human fibroblasts that have been genetically modified to delete PTPα are less responsive to stimulation with TGF-β, suggesting that this pathway is relevant to human pathology. Since human ARDS BAL fluid induces the same changes in normal human lung fibroblasts that are attenuated by the absence of PTPα in experimental models, this suggests that targeting of this protein could be a strategy to alleviate pathologic fibroproliferation following lung injury.