Ablation of lysophosphatidic acid receptor 1 attenuates hypertrophic cardiomyopathy in a mouse model.

Myocardial fibrosis is a key pathologic feature of hypertrophic cardiomyopathy (HCM). However, the fibrotic pathways activated by HCM-causing sarcomere protein gene mutations are poorly defined. Because lysophosphatidic acid is a mediator of fibrosis in multiple organs and diseases, we tested the role of the lysophosphatidic acid pathway in HCM. Lysphosphatidic acid receptor 1 (LPAR1), a cell surface receptor, is required for lysophosphatidic acid mediation of fibrosis. We bred HCM mice carrying a pathogenic myosin heavy-chain variant (403+/-) with Lpar1-ablated mice to create mice carrying both genetic changes (403+/- LPAR1 -/-) and assessed development of cardiac hypertrophy and fibrosis. Compared with 403+/- LPAR1WT, 403+/- LPAR1 -/- mice developed significantly less hypertrophy and fibrosis. Single-nucleus RNA sequencing of left ventricular tissue demonstrated that Lpar1 was predominantly expressed by lymphatic endothelial cells (LECs) and cardiac fibroblasts. Lpar1 ablation reduced the population of LECs, confirmed by immunofluorescence staining of the LEC markers Lyve1 and Ccl21a and, by in situ hybridization, for Reln and Ccl21a. Lpar1 ablation also altered the distribution of fibroblast cell states. FB1 and FB2 fibroblasts decreased while FB0 and FB3 fibroblasts increased. Our findings indicate that Lpar1 is expressed predominantly by LECs and fibroblasts in the heart and is required for development of hypertrophy and fibrosis in an HCM mouse model. LPAR1 antagonism, including agents in clinical trials for other fibrotic diseases, may be beneficial for HCM.

Augmentation of Endothelial S1PR1 Attenuates Postviral Pulmonary Fibrosis.

Respiratory viral infections are frequent causes of acute respiratory distress syndrome (ARDS), a disabling condition with a mortality of up to 46%. The pulmonary endothelium plays an important role in the development of ARDS as well as the pathogenesis of pulmonary fibrosis; however, the therapeutic potential to modulate endothelium-dependent signaling to prevent deleterious consequences has not been well explored. Here, we used a clinically relevant influenza A virus infection model, endothelial cell-specific transgenic gain-of-function and loss-of-function mice as well as pharmacologic approaches and in vitro modeling, to define the mechanism by which S1PR1 expression is dampened during influenza virus infection and determine whether therapeutic augmentation of S1PR1 has the potential to reduce long-term postviral fibrotic complications. We found that the influenza virus-induced inflammatory milieu promoted internalization of S1PR1, which was pharmacologically inhibited with paroxetine, an inhibitor of GRK2. Moreover, genetic overexpression or administration of paroxetine days after influenza virus infection was sufficient to reduce postviral pulmonary fibrosis. Taken together, our data suggest that endothelial S1PR1 signaling provides critical protection against long-term fibrotic complications after pulmonary viral infection. These findings support the development of antifibrotic strategies that augment S1PR1 expression in virus-induced ARDS to improve long-term patient outcomes.

Tailored Chemical Reactivity Probes for Systemic Imaging of Aldehydes in Fibroproliferative Diseases.

During fibroproliferation, protein-associated extracellular aldehydes are formed by the oxidation of lysine residues on extracellular matrix proteins to form the aldehyde allysine. Here we report three Mn(II)-based, small-molecule magnetic resonance probes that contain α-effect nucleophiles to target allysine in vivo and report on tissue fibrogenesis. We used a rational design approach to develop turn-on probes with a 4-fold increase in relaxivity upon targeting. The effects of aldehyde condensation rate and hydrolysis kinetics on the performance of the probes to detect tissue fibrogenesis non-invasively in mouse models were evaluated by a systemic aldehyde tracking approach. We showed that, for highly reversible ligations, off-rate was a stronger predictor of in vivo efficiency, enabling histologically validated, three-dimensional characterization of pulmonary fibrogenesis throughout the entire lung. The exclusive renal elimination of these probes allowed for rapid imaging of liver fibrosis. Reducing the hydrolysis rate by forming an oxime bond with allysine enabled delayed phase imaging of kidney fibrogenesis. The imaging efficacy of these probes, coupled with their rapid and complete elimination from the body, makes them strong candidates for clinical translation.

Myofibroblast-specific inhibition of the Rho kinase-MRTF-SRF pathway using nanotechnology for the prevention of pulmonary fibrosis.

Pulmonary fibrosis is characterized by the accumulation of myofibroblasts in the lung and progressive tissue scarring. Fibroblasts exist across a spectrum of states, from quiescence in health to activated myofibroblasts in the setting of injury. Highly activated myofibroblasts have a critical role in the establishment of fibrosis as the predominant source of type 1 collagen and profibrotic mediators. Myofibroblasts are also highly contractile cells and can alter lung biomechanical properties through tissue contraction. Inhibiting signaling pathways involved in myofibroblast activation could therefore have significant therapeutic value. One of the ways myofibroblast activation occurs is through activation of the Rho/myocardin-related transcription factor (MRTF)/serum response factor (SRF) pathway, which signals through intracellular actin polymerization. However, concerns surrounding the pleiotropic and ubiquitous nature of these signaling pathways have limited the translation of inhibitory drugs. Herein, we demonstrate a novel therapeutic antifibrotic strategy using myofibroblast-targeted nanoparticles containing a MTRF/SRF pathway inhibitor (CCG-1423), which has been shown to block myofibroblast activation in vitro. Myofibroblasts were preferentially targeted via the angiotensin 2 receptor, which has been shown to be selectively upregulated in animal and human studies. These nanoparticles were nontoxic and accumulated in lung myofibroblasts in the bleomycin-induced mouse model of pulmonary fibrosis, reducing the number of these activated cells and their production of profibrotic mediators. Ultimately, in a murine model of lung fibrosis, a single injection of these drugs containing targeted nanoagents reduced fibrosis as compared with control mice. This approach has the potential to deliver personalized therapy by precisely targeting signaling pathways in a cell-specific manner, allowing increased efficacy with reduced deleterious off-target effects.

Endothelial-Specific Loss of Sphingosine-1-Phosphate Receptor 1 Increases Vascular Permeability and Exacerbates Bleomycin-induced Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease which leads to significant morbidity and mortality from respiratory failure. The two drugs currently approved for clinical use slow the rate of decline in lung function but have not been shown to halt disease progression or reverse established fibrosis. Thus, new therapeutic targets are needed. Endothelial injury and the resultant vascular permeability are critical components in the response to tissue injury and are present in patients with IPF. However, it remains unclear how vascular permeability affects lung repair and fibrosis following injury. Lipid mediators such as sphingosine-1-phosphate (S1P) are known to regulate multiple homeostatic processes in the lung including vascular permeability. We demonstrate that endothelial cell-(EC) specific deletion of the S1P receptor 1 (S1PR1) in mice (EC-S1pr1-/-) results in increased lung vascular permeability at baseline. Following a low-dose intratracheal bleomycin challenge, EC-S1pr1-/- mice had increased and persistent vascular permeability compared with wild-type mice, which was strongly correlated with the amount and localization of resulting pulmonary fibrosis. EC-S1pr1-/- mice also had increased immune cell infiltration and activation of the coagulation cascade within the lung. However, increased circulating S1P ligand in ApoM-overexpressing mice was insufficient to protect against bleomycin-induced pulmonary fibrosis. Overall, these data demonstrate that endothelial cell S1PR1 controls vascular permeability in the lung, is associated with changes in immune cell infiltration and extravascular coagulation, and modulates the fibrotic response to lung injury.

Vascular permeability in the fibrotic lung.

Idiopathic pulmonary fibrosis (IPF) is thought to result from aberrant tissue repair processes in response to chronic or repetitive lung injury. The origin and nature of the injury, as well as its cellular and molecular targets, are likely heterogeneous, which complicates accurate pre-clinical modelling of the disease and makes therapeutic targeting a challenge. Efforts are underway to identify central pathways in fibrogenesis which may allow targeting of aberrant repair processes regardless of the initial injury stimulus. Dysregulated endothelial permeability and vascular leak have long been studied for their role in acute lung injury and repair. Evidence that these processes are of importance to the pathogenesis of fibrotic lung disease is growing. Endothelial permeability is increased in non-fibrosing lung diseases, but it resolves in a self-limited fashion in conditions such as bacterial pneumonia and acute respiratory distress syndrome. In progressive fibrosing diseases such as IPF, permeability appears to persist, however, and may also predict mortality. In this hypothesis-generating review, we summarise available data on the role of endothelial permeability in IPF and focus on the deleterious consequences of sustained endothelial hyperpermeability in response to and during pulmonary inflammation and fibrosis. We propose that persistent permeability and vascular leak in the lung have the potential to establish and amplify the pro-fibrotic environment. Therapeutic interventions aimed at recognising and "plugging" the leak may therefore be of significant benefit for preventing the transition from lung injury to fibrosis and should be areas for future research.

The Rho Kinase Isoforms ROCK1 and ROCK2 Each Contribute to the Development of Experimental Pulmonary Fibrosis.

Pulmonary fibrosis is thought to result from dysregulated wound repair after repetitive lung injury. Many cellular responses to injury involve rearrangements of the actin cytoskeleton mediated by the two isoforms of the Rho-associated coiled-coil-forming protein kinase (ROCK), ROCK1 and ROCK2. In addition, profibrotic mediators such as transforming growth factor-ß, thrombin, and lysophosphatidic acid act through receptors that activate ROCK. Inhibition of ROCK activation may be a potent therapeutic strategy for human pulmonary fibrosis. Pharmacological inhibition of ROCK using nonselective ROCK inhibitors has been shown to prevent fibrosis in animal models; however, the specific roles of each ROCK isoform are poorly understood. Furthermore, the pleiotropic effects of this kinase have raised concerns about on-target adverse effects of ROCK inhibition such as hypotension. Selective inhibition of one isoform might be a better-tolerated strategy. In the present study, we used a genetic approach to determine the roles of ROCK1 and ROCK2 in a mouse model of bleomycin-induced pulmonary fibrosis. Using ROCK1- or ROCK2-haploinsufficient mice, we found that reduced expression of either ROCK1 or ROCK2 was sufficient to protect them from bleomycin-induced pulmonary fibrosis. In addition, we found that both isoforms contribute to the profibrotic responses of epithelial cells, endothelial cells, and fibroblasts. Interestingly, ROCK1- and ROCK2-haploinsufficient mice exhibited similar protection from bleomycin-induced vascular leak, myofibroblast differentiation, and fibrosis; however, ROCK1-haploinsufficient mice demonstrated greater attenuation of epithelial cell apoptosis. These findings suggest that selective inhibition of either ROCK isoform has the potential to be an effective therapeutic strategy for pulmonary fibrosis.

The Rho kinases: critical mediators of multiple profibrotic processes and rational targets for new therapies for pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by progressive lung scarring, short median survival, and limited therapeutic options, creating great need for new pharmacologic therapies. IPF is thought to result from repetitive environmental injury to the lung epithelium, in the context of aberrant host wound healing responses. Tissue responses to injury fundamentally involve reorganization of the actin cytoskeleton of participating cells, including epithelial cells, fibroblasts, endothelial cells, and macrophages. Actin filament assembly and actomyosin contraction are directed by the Rho-associated coiled-coil forming protein kinase (ROCK) family of serine/threonine kinases (ROCK1 and ROCK2). As would therefore be expected, lung ROCK activation has been demonstrated in humans with IPF and in animal models of this disease. ROCK inhibitors can prevent fibrosis in these models, and more importantly, induce the regression of already established fibrosis. Here we review ROCK structure and function, upstream activators and downstream targets of ROCKs in pulmonary fibrosis, contributions of ROCKs to profibrotic cellular responses to lung injury, ROCK inhibitors and their efficacy in animal models of pulmonary fibrosis, and potential toxicities of ROCK inhibitors in humans, as well as involvement of ROCKs in fibrosis in other organs. As we discuss, ROCK activation is required for multiple profibrotic responses, in the lung and multiple other organs, suggesting ROCK participation in fundamental pathways that contribute to the pathogenesis of a broad array of fibrotic diseases. Multiple lines of evidence therefore indicate that ROCK inhibition has great potential to be a powerful therapeutic tool in the treatment of fibrosis, both in the lung and beyond.

Autotaxin activity increases locally following lung injury, but is not required for pulmonary lysophosphatidic acid production or fibrosis.

Lysophosphatidic acid (LPA) is an important mediator of pulmonary fibrosis. In blood and multiple tumor types, autotaxin produces LPA from lysophosphatidylcholine (LPC) via lysophospholipase D activity, but alternative enzymatic pathways also exist for LPA production. We examined the role of autotaxin (ATX) in pulmonary LPA production during fibrogenesis in a bleomycin mouse model. We found that bleomycin injury increases the bronchoalveolar lavage (BAL) fluid levels of ATX protein 17-fold. However, the LPA and LPC species that increase in BAL of bleomycin-injured mice were discordant, inconsistent with a substrate-product relationship between LPC and LPA in pulmonary fibrosis. LPA species with longer chain polyunsaturated acyl groups predominated in BAL fluid after bleomycin injury, with 22:5 and 22:6 species accounting for 55 and 16% of the total, whereas the predominant BAL LPC species contained shorter chain, saturated acyl groups, with 16:0 and 18:0 species accounting for 56 and 14% of the total. Further, administration of the potent ATX inhibitor PAT-048 to bleomycin-challenged mice markedly decreased ATX activity systemically and in the lung, without effect on pulmonary LPA or fibrosis. Therefore, alternative ATX-independent pathways are likely responsible for local generation of LPA in the injured lung. These pathways will require identification to therapeutically target LPA production in pulmonary fibrosis.-Black, K. E., Berdyshev, E., Bain, G., Castelino, F. V., Shea, B. S., Probst, C. K., Fontaine, B. A., Bronova, I., Goulet, L., Lagares, D., Ahluwalia, N., Knipe, R. S., Natarajan, V., Tager, A. M. Autotaxin activity increases locally following lung injury, but is not required for pulmonary lysophosphatidic acid production or fibrosis.

Lactate transport inhibition therapeutically reprograms fibroblast metabolism in experimental pulmonary fibrosis.

Myofibroblast differentiation, essential for driving extracellular matrix synthesis in pulmonary fibrosis, requires increased glycolysis. While glycolytic cells must export lactate, the contributions of lactate transporters to myofibroblast differentiation are unknown. In this study, we investigated how MCT1 and MCT4, key lactate transporters, influence myofibroblast differentiation and experimental pulmonary fibrosis. Our findings reveal that inhibiting MCT1 or MCT4 reduces TGFß-stimulated pulmonary myofibroblast differentiation in vitro and decreases bleomycin-induced pulmonary fibrosis in vivo. Through comprehensive metabolic analyses, including bioenergetics, stable isotope tracing, metabolomics, and imaging mass spectrometry in both cells and mice, we demonstrate that inhibiting lactate transport enhances oxidative phosphorylation, reduces reactive oxygen species production, and diminishes glucose metabolite incorporation into fibrotic lung regions. Furthermore, we introduce VB253, a novel MCT4 inhibitor, which ameliorates pulmonary fibrosis in both young and aged mice, with comparable efficacy to established antifibrotic therapies. These results underscore the necessity of lactate transport for myofibroblast differentiation, identify MCT1 and MCT4 as promising pharmacologic targets in pulmonary fibrosis, and support further evaluation of lactate transport inhibitors for patients for whom limited therapeutic options currently exist.

Optimization of an Allysine-Targeted PET Probe for Quantifying Fibrogenesis in a Mouse Model of Pulmonary Fibrosis.

PURPOSE: Idiopathic pulmonary fibrosis (IPF) is a destructive lung disease with a poor prognosis, an unpredictable clinical course, and inadequate therapies. There are currently no measures of disease activity to guide clinicians making treatment decisions. The aim of this study was to develop a PET probe to identify lung fibrogenesis using a pre-clinical model of pulmonary fibrosis, with potential for translation into clinical use to predict disease progression and inform treatment decisions. METHODS: Eight novel allysine-targeting chelators, PIF-1, PIF-2, …, PIF-8, with different aldehyde-reactive moieties were designed, synthesized, and radiolabeled with gallium-68 or copper-64. PET probe performance was assessed in C57BL/6J male mice 2 weeks after intratracheal bleomycin challenge and in naïve mice by dynamic PET/MR imaging and with biodistribution at 90 min post injection. Lung hydroxyproline and allysine were quantified ex vivo and histological staining for fibrosis and aldehyde was performed. RESULTS: In vivo screening of probes identified 68GaPIF-3 and 68GaPIF-7 as probes with high uptake in injured lung, high uptake in injured lung versus normal lung, and high uptake in injured lung versus adjacent liver and heart tissue. A crossover, intra-animal PET/MR imaging study of 68GaPIF-3 and 68GaPIF-7 confirmed 68GaPIF-7 as the superior probe. Specificity for fibrogenesis was confirmed in a crossover, intra-animal PET/MR imaging study with 68GaPIF-7 and a non-binding control compound, 68GaPIF-Ctrl. Substituting copper-64 for gallium-68 did not affect lung uptake or specificity indicating that either isotope could be used. CONCLUSION: A series of allysine-reactive PET probes with variations in the aldehyde-reactive moiety were evaluated in a pre-clinical model of lung fibrosis. The hydrazine-bearing probe, 68GaPIF-7, exhibited the highest uptake in fibrogenic lung, low uptake in surrounding liver or heart tissue, and low lung uptake in healthy mice and should be considered for further clinical translation.

Integrated Immunopeptidomic and Proteomic Analysis of COVID-19 lung biopsies.

Introduction: Severe respiratory illness is the most prominent manifestation of patients infected with SARS-CoV-2, and yet the molecular mechanisms underlying severe lung disease in COVID-19 affected patients still require elucidation. Human leukocyte antigen class I (HLA-I) expression is crucial for antigen presentation and the host's response to SARS-CoV-2. Methods: To gain insights into the immune response and molecular pathways involved in severe lung disease, we performed immunopeptidomic and proteomic analyses of lung tissues recovered at four COVID-19 autopsy and six non-COVID-19 transplants. Results: We found signals of tissue injury and regeneration in lung fibroblast and alveolar type I/II cells, resulting in the production of highly immunogenic self-antigens within the lungs of COVID-19 patients. We also identified immune activation of the M2c macrophage as the primary source of HLA-I presentation and immunogenicity in this context. Additionally, we identified 28 lung signatures that can serve as early plasma markers for predicting infection and severe COVID-19 disease. These protein signatures were predominantly expressed in macrophages and epithelial cells and were associated with complement and coagulation cascades. Discussion: Our findings emphasize the significant role of macrophage-mediated immunity in the development of severe lung disease in COVID-19 patients.

The Vasculature in Pulmonary Fibrosis.

Purpose of Review: The current paradigm of idiopathic pulmonary fibrosis (IPF) pathogenesis involves recurrent injury to a sensitive alveolar epithelium followed by impaired repair responses marked by fibroblast activation and deposition of extracellular matrix. Multiple cell types are involved in this response with potential roles suggested by advances in single-cell RNA sequencing and lung developmental biology. Notably, recent work has better characterized the cell types present in the pulmonary endothelium and identified vascular changes in patients with IPF. Recent Findings: Lung tissue from patients with IPF has been examined at single-cell resolution, revealing reductions in lung capillary cells and expansion of a population of vascular cells expressing markers associated with bronchial endothelium. In addition, pre-clinical models have demonstrated a fundamental role for aging and vascular permeability in the development of pulmonary fibrosis. Summary: Mounting evidence suggests that the endothelium undergoes changes in the context of fibrosis, and these changes may contribute to the development and/or progression of pulmonary fibrosis. Additional studies will be needed to further define the functional role of these vascular changes.

Existing Crisis Standards of Care Triage Protocols May Not Significantly Differentiate Between Patients With Coronavirus Disease 2019 Who Require Intensive Care.

OBJECTIVES: To determine how several existing crisis standards of care triage protocols would have distinguished between patients with coronavirus disease 2019 requiring intensive care. DESIGN: Retrospective cohort study. SETTING: Single urban academic medical center. PATIENTS: One-hundred twenty patients with coronavirus disease 2019 who required intensive care and mechanical ventilation. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The characteristics of each patient at the time of ICU triage were used to determine how patients would have been prioritized using four crisis standards of care protocols. The vast majority of patients in the cohort would have been in the highest priority group using a triage protocol focusing on Sequential Organ Failure Assessment alone. Prioritization based on Sequential Organ Failure Assessment and 1-year life expectancy would have resulted in only slightly more differentiation between patients. Prioritization based on Sequential Organ Failure Assessment and 5-year life expectancy would have added significant additional differentiation depending on how priority groups were defined. CONCLUSIONS: There is considerable controversy regarding the use of criteria other than prognosis for short-term survival in initial allocation of critical care resources under crisis standards of care triage protocols. To the extent that initial triage protocols would not create sufficient differentiation between patients, effectively resulting in a first-come, first-served initial allocation of resources, it is important to focus on how resources would be reallocated in the event of ongoing scarcity.

Exercise performance in patients with post-acute sequelae of SARS-CoV-2 infection compared to patients with unexplained dyspnea.

BACKGROUND: Dyspnea and exercise intolerance are commonly reported post-acute sequelae of SARS-CoV-2 infection (PASC), but routine diagnostic testing is often normal. Cardiopulmonary exercise testing (CPET) offers comprehensive assessment of dyspnea to characterize pulmonary PASC. METHODS: We performed a retrospective cohort study of CPET performed on patients reporting dyspnea and/or exercise intolerance following confirmed Covid-19 between August 1, 2020 and March 1, 2021, and compared them to age- and sex-matched patients with unexplained dyspnea referred for CPET at the same center in the pre-Covid-19 era. FINDINGS: Compared to matched unexplained dyspnea comparators, PASC patients shared similar medical comorbidities and subjective dyspnea at referral (mMRC score 1.6 ± 0.9 vs. 1.4 ± 0.9, P = 0.5). Fifteen (83.3%) PASC patients underwent high resolution computed tomography of the chest, of which half (46.7%) were normal, and 17 (94.4%) patients had pulmonary function testing, of which the majority (76.5%) were normal. All patients underwent CPET, and 12 (67%) had normal findings. Compared to matched comparators, PASC patients had similar peak oxygen consumption, oxygen consumption at ventilatory anaerobic threshold, and ventilatory efficiency measured by the minute ventilation to carbon dioxide production (VE/VCO2) slope. INTERPRETATION: Despite prominent dyspnea, physiological abnormalities on CPET were mild across a range of initial Covid-19 severity and similar to matched comparators referred for dyspnea without antecedent SARS-CoV-2. FUNDING: The project was supported by the NHLBI (R01HL131029, R01HL151841, U10HL110337, T32HL116275) and a KL2 award (5KL2TR002542-02) from Harvard Catalyst.

Peroxidase Sensitive Amplifiable Probe for Molecular Magnetic Resonance Imaging of Pulmonary Inflammation.

An amplifiable magnetic resonance imaging (MRI) probe that combines the stability of the macrocyclic Gd-DOTAGA core with a peroxidase-reactive 5-hydroxytryptamide (5-HT) moiety is reported. The incubation of the complex under enzymatic oxidative conditions led to a 1.7-fold increase in r1 at 1.4 T that was attributed to an oligomerization of the probe upon oxidation. This probe, Gd-5-HT-DOTAGA, provided specific detection of lung inflammation by MRI in bleomycin-injured mice.