Pulmonary Vascular Leakage Research Articles

Murine alveolar macrophages rapidly accumulate intranasally administered SARS-CoV-2 Spike protein leading to neutrophil recruitment and damage

The trimeric SARS-CoV-2 Spike protein mediates viral attachment facilitating cell entry. Most COVID-19 vaccines direct mammalian cells to express the Spike protein or deliver it directly via inoculation to engender a protective immune response. The trafficking and cellular tropism of the Spike protein in vivo and its impact on immune cells remains incompletely elucidated. In this study, we inoculated mice intranasally, intravenously, and subcutaneously with fluorescently labeled recombinant SARS-CoV-2 Spike protein. Using flow cytometry and imaging techniques, we analyzed its localization, immune cell tropism, and acute functional impact. Intranasal administration led to rapid lung alveolar macrophage uptake, pulmonary vascular leakage, and neutrophil recruitment and damage. When injected near the inguinal lymph node medullary, but not subcapsular macrophages, captured the protein, while scrotal injection recruited and fragmented neutrophils. Widespread endothelial and liver Kupffer cell uptake followed intravenous administration. Human peripheral blood cells B cells, neutrophils, monocytes, and myeloid dendritic cells all efficiently bound Spike protein. Exposure to the Spike protein enhanced neutrophil NETosis and augmented human macrophage TNF-α (tumor necrosis factor-α) and IL-6 production. Human and murine immune cells employed C-type lectin receptors and Siglecs to help capture the Spike protein. This study highlights the potential toxicity of the SARS-CoV-2 Spike protein for mammalian cells and illustrates the central role for alveolar macrophage in pathogenic protein uptake.

Murine alveolar macrophages rapidly accumulate intranasally administered SARS-CoV-2 Spike protein leading to neutrophil recruitment and damage.

The trimeric SARS-CoV-2 Spike protein mediates viral attachment facilitating cell entry. Most COVID-19 vaccines direct mammalian cells to express the Spike protein or deliver it directly via inoculation to engender a protective immune response. The trafficking and cellular tropism of the Spike protein in vivo and its impact on immune cells remains incompletely elucidated. In this study, we inoculated mice intranasally, intravenously, and subcutaneously with fluorescently labeled recombinant SARS-CoV-2 Spike protein. Using flow cytometry and imaging techniques, we analyzed its localization, immune cell tropism, and acute functional impact. Intranasal administration led to rapid lung alveolar macrophage uptake, pulmonary vascular leakage, and neutrophil recruitment and damage. When injected near the inguinal lymph node medullary, but not subcapsular macrophages, captured the protein, while scrotal injection recruited and fragmented neutrophils. Widespread endothelial and liver Kupffer cell uptake followed intravenous administration. Human peripheral blood cells B cells, neutrophils, monocytes, and myeloid dendritic cells all efficiently bound Spike protein. Exposure to the Spike protein enhanced neutrophil NETosis and augmented human macrophage TNF-α (tumor necrosis factor-α) and IL-6 production. Human and murine immune cells employed C-type lectin receptors and Siglecs to help capture the Spike protein. This study highlights the potential toxicity of the SARS-CoV-2 Spike protein for mammalian cells and illustrates the central role for alveolar macrophage in pathogenic protein uptake.

The paracrine isthmin1 transcriptionally regulated by C/EBPβ exacerbates pulmonary vascular leakage in murine sepsis.

It is known that pulmonary vascular leakage, a key pathological feature of sepsis-induced lung injury, is largely regulated by perivascular cells. However, the underlying mechanisms have not been fully uncovered. In the present study, we aimed to evaluate the role of isthmin1, a secretory protein originating from alveolar epithelium, in the pulmonary vascular leakage during sepsis and to investigate the regulatory mechanisms of isthmin1 gene transcription. We observed an elevated isthmin1 gene expression in the pulmonary tissue of septic mice induced by cecal ligation and puncture (CLP), as well as in primary murine alveolar type II epithelial cells (ATII) exposed to lipopolysaccharide (LPS). Furthermore, we confirmed that isthmin1 derived from ATII contributes to pulmonary vascular leakage during sepsis. Specifically, adenovirus-mediated isthmin1 disruption in ATII led to a significant attenuation of the increased pulmonary microvascular endothelial cell (PMVEC) hyperpermeability in a PMVEC/ATII coculture system when exposed to LPS. In addition, adeno-associated virus 9 (AAV9)-mediated knockdown of isthmin1 in the alveolar epithelium of septic mice significantly attenuated pulmonary vascular leakage. Finally, mechanistic studies unveiled that nuclear transcription factor CCAAT/enhancer binding protein (C/EBP)β participates in isthmin1 gene activation by binding directly to the cis-regulatory element of isthmin1 locus and may contribute to isthmin1 upregulation during sepsis. Collectively, the present study highlighted the impact of the paracrine protein isthmin1, derived from ATII, on the exacerbation of pulmonary vascular permeability in sepsis and revealed a new regulatory mechanism for isthmin1 gene transcription.NEW & NOTEWORTHY This article addresses the role of the alveolar epithelial-secreted protein isthmin1 on the exacerbation of pulmonary vascular permeability in sepsis and identified nuclear factor CCAAT/enhancer binding protein (C/EBP)β as a new regulator of isthmin1 gene transcription. Targeting the C/EBPβ-isthmin1 regulatory axis on the alveolar side would be of great value in the treatment of pulmonary vascular leakage and lung injury induced by sepsis.

Toxicities of CAR T-cell therapy: a review of current literature

The chimeric antigen receptor (CAR) design, first invented by Zelig Eshhar, paved the way for the use of genetically modified T-cells in targeted therapy against cancer cells. Since then, it has gone through many generations, especially with the integration of co-stimulation in the second and third-generation CARs. However, it also mounts a hyperactive immune response named as cytokine release syndrome with the release of several cytokines eventually resulting in multiple end-organ toxicities. The severity of cytokine release syndrome depends upon certain factors such as the tumor burden, choice of co-stimulation, and degree of lymphodepletion, and can manifest as pulmonary edema, vascular leak, renal dysfunction, cardiac problems, hepatic failure, and coagulopathy. Many grading criteria have been used to define these clinical manifestations but they lack harmonization. Neurotoxicity has also been significantly associated with CAR T-cell therapy but it has not been studied much in previous literature. This review aims to provide a comprehensive account of the clinical manifestations, diagnosis, management, and treatment of CAR T-cell associated neurotoxicity.

First magnetic particle imaging to assess pulmonary vascular leakage in vivo in the acutely injured and fibrotic lung.

Increased pulmonary vascular permeability is a characteristic feature of lung injury. However, there are no established methods that allow the three-dimensional visualization and quantification of pulmonary vascular permeability in vivo. Evans blue extravasation test and total protein test of bronchoalveolar lavage fluid (BALF) are permeability assays commonly used in research settings. However, they lack the ability to identify the spatial and temporal heterogeneity of endothelial barrier disruption, which is typical in lung injuries. Magnetic resonance (MR) and near-infrared (NIR) imaging have been proposed to image pulmonary permeability, but suffer from limited sensitivity and penetration depth, respectively. In this study, we report the first use of magnetic particle imaging (MPI) to assess pulmonary vascular leakage noninvasively in vivo in mice. A dextran-coated superparamagnetic iron oxide (SPIO), synomag®, was employed as the imaging tracer, and pulmonary SPIO extravasation was imaged and quantified to evaluate the vascular leakage. Animal models of acute lung injury and pulmonary fibrosis (PF) were used to validate the proposed method. MPI sensitively detected the SPIO extravasation in both acutely injured and fibrotic lungs in vivo, which was confirmed by ex vivo imaging and Prussian blue staining. Moreover, 3D MPI illustrated the spatial heterogeneity of vascular leakage, which correlated well with CT findings. Based on the in vivo 3D MPI images, we defined the SPIO extravasation index (SEI) to quantify the vascular leakage. A significant increase in SEI was observed in the injured lungs, in consistent with the results obtained via ex vivo permeability assays. Overall, our results demonstrate that 3D quantitative MPI serves as a useful tool to examine pulmonary vascular integrity in vivo, which shows promise for future clinical translation.

Rap1 small GTPase is essential for maintaining pulmonary endothelial barrier function in mice.

Vascular permeability is dynamically but tightly controlled by vascular endothelial (VE)-cadherin-mediated endothelial cell-cell junctions to maintain homeostasis. Thus, impairments of VE-cadherin-mediated cell adhesions lead to hyperpermeability, promoting the development and progression of various disease processes. Notably, the lungs are a highly vulnerable organ wherein pulmonary inflammation and infection result in vascular leakage. Herein, we showed that Rap1, a small GTPase, plays an essential role for maintaining pulmonary endothelial barrier function in mice. Endothelial cell-specific Rap1a/Rap1b double knockout mice exhibited severe pulmonary edema. They also showed vascular leakage in the hearts, but not in the brains. En face analyses of the pulmonary arteries and 3D-immunofluorescence analyses of the lungs revealed that Rap1 potentiates VE-cadherin-mediated endothelial cell-cell junctions through dynamic actin cytoskeleton reorganization. Rap1 inhibits formation of cytoplasmic actin bundles perpendicularly binding VE-cadherin adhesions through inhibition of a Rho-ROCK pathway-induced activation of cytoplasmic nonmuscle myosin II (NM-II). Simultaneously, Rap1 induces junctional NM-II activation to create circumferential actin bundles, which anchor and stabilize VE-cadherin at cell-cell junctions. We also showed that the mice carrying only one allele of either Rap1a or Rap1b out of the two Rap1 genes are more vulnerable to lipopolysaccharide (LPS)-induced pulmonary vascular leakage than wild-type mice, while activation of Rap1 by administration of 007, an activator for Epac, attenuates LPS-induced increase in pulmonary endothelial permeability in wild-type mice. Thus, we demonstrate that Rap1 plays an essential role for maintaining pulmonary endothelial barrier functions under physiological conditions and provides protection against inflammation-induced pulmonary vascular leakage.

Deficiency of S100A8/A9 attenuates pulmonary microvascular leakage in septic mice

BackgroundWe have reported a positive correlation between S100 calcium-binding protein (S100) A8/S100A9 and sepsis-induced lung damage before. However, limited knowledge exists concerning the biological role of S100A8/A9 in pulmonary vascular endothelial barrier dysfunction, as well as the diagnostic value of S100A8/A9 in sepsis.MethodsSepsis was induced in C57BL/6J mice and S100A9-knockout (KO) mice through the cecal ligation and puncture (CLP). Pulmonary vascular leakage was determined by measuring extravasated Evans blue (EB). Reverse transcription polymerase chain reaction and the histological score were used to evaluate inflammation and lung injury, respectively. Recombinant S100A8/A9 (rhS100A8/A9) was used to identify the effects of S100A8/A9 on endothelial barrier dysfunction in human umbilical vein endothelial cells (HUVECs). Additionally, the diagnostic value of S100A8/A9 in sepsis was assessed using receiver operating characteristic.ResultsS100A8/A9 expression was up-regulated in the lungs of CLP-operated mice. S100A9 KO significantly reversed CLP-induced hypothermia and hypotension, resulting in an improved survival rate. S100A9 KO also decreased the inflammatory response, EB leakage, and histological scores in the lungs of CLP-operated mice. Occludin and VE-cadherin expressions were decreased in the lungs of CLP-operated mice; However, S100A9 KO attenuated this decrease. Moreover, CLP-induced signal transducer and activator of transcription 3 (STAT3) and p38/extracellular signal-regulated kinase (ERK) signalling activation and apoptosis were mitigated by S100A9 KO in lungs. In addition, rhS100A8/A9 administration significantly decreased occludin and VE-cadherin expressions, increased the phosphorylated (p)-ERK/ERK, p-p38/p38, and B-cell leukaemia/lymphoma 2 protein (Bcl-2)-associated X protein/Bcl-2 ratios in HUVECs.ConclusionThe present study demonstrated S100A8/A9 aggravated sepsis-induced pulmonary inflammation, vascular permeability, and lung injury. This was achieved, at least partially, by activating the P38/STAT3/ERK signalling pathways. Moreover, S100A8/A9 showed the potential as a biomarker for sepsis diagnosis.

Targeting EB3-IP3R3 Interface with Cognate Peptide Protects from Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) is a lung disease characterized by acute onset of noncardiogenic pulmonary edema, hypoxemia, and respiratory insufficiency. The current treatment for ARDS is mainly supportive in nature, providing a critical need for targeted pharmacological management. We addressed this medical problem by developing a pharmacological treatment for pulmonary vascular leakage, a culprit of alveolar damage and lung inflammation. Our novel therapeutic target is the microtubule accessory factor EB3 (end binding protein 3), which contributes to pulmonary vascular leakage by amplifying pathological calcium signaling in endothelial cells in response to inflammatory stimuli. EB3 interacts with IP3R3 (inositol 1,4,5-trisphosphate receptor 3) and orchestrates calcium release from endoplasmic reticulum stores. Here, we designed and tested the therapeutic benefits of a 14-aa peptide named CIPRI (cognate IP3 receptor inhibitor), which disrupted EB3-IP3R3 interaction invitro and in lungs of mice challenged with endotoxin. Treatment with CIPRI or depletion of IP3R3 in lung microvascular endothelial monolayers mitigated calcium release from endoplasmic reticulum stores and prevented a disassembly of vascular endothelial cadherin junctions in response to the proinflammatory mediator α-thrombin. Furthermore, intravenous administration of CIPRI in mice mitigated inflammation-induced lung injury, blocked pulmonary microvascular leakage, prevented activation of NFAT (nuclear factor of activated T cells) signaling, and reduced production of proinflammatory cytokines in the lung tissue. CIPRI also improved survival of mice from endotoxemia and polymicrobial sepsis. Together, these data demonstrate that targeting EB3-IP3R3 interaction with a cognate peptide is a promising strategy to address hyperpermeability of microvessels in inflammatory lung diseases.

FX06 to rescue SARS-CoV-2-induced acute respiratory distress syndrome: a randomized clinical trial

BackgroundVascular leakage is a major feature of acute respiratory distress syndrome (ARDS). We aimed to evaluate the efficacy of FX06, a drug under development that stabilizes interendothelial cell junctions, at reducing vascular leakage during SARS-CoV-2-induced ARDS.MethodsThis multicenter, double-blinded, randomized trial included adults with COVID-19-associated ARDS who had received invasive mechanical ventilation for < 5 days and were randomized to receive either intravenous FX06 (400 mg/d, for 5 days) or its vehicle as placebo. The primary endpoint was the lowering—from day 1 to day 7—of the transpulmonary thermodilution-derived extravascular lung-water index (EVLWi).ResultsTwenty-five patients were randomized to receive FX06 and 24 the placebo. Although EVLWi was elevated at baseline (median [IQR] 15.6 mL/kg [13.5; 18.5]), its declines from day 1 to day 7 were comparable for FX06 recipients and controls (respectively, − 1.9 [− 3.3; − 0.5] vs. − 0.8 [− 5.5; − 1.1] mL/kg; estimated effect − 0.8 [− 3.1; + 2.4], p = 0.51). Cardiac indexes, pulmonary vascular permeability indexes, and fluid balances were also comparable, as were PaO2/FiO2 ratios and durations of mechanical ventilation. Adverse event rates were similar for the 2 groups, although more FX06 recipients developed ventilator-associated pneumonia (16/25 (64%) vs. 6/24 (24%), p = 0.009).ConclusionsIn this unique-dosing–regimen study, FX06 did not lower SARS-CoV-2-induced pulmonary vascular leakage. Future investigations will need to evaluate its efficacy at earlier times during the disease or using other regimens.Trial registration NCT04618042. Registered 5 November 2020.

Inhibition of EB3 promotes acute lung injury resolution by promoting the regenerative FOXM1 pathway in endothelial cells

Acute respiratory distress syndrome (ARDS) is the acute onset of non-cardiogenic pulmonary edema, hypoxemia, and respiratory failure. ARDS is frequently caused by bacterial and viral lung infections including the recent SARS-CoV-2. Development of therapeutic approaches to combat ARDS have become an urgent unmet medical need. We address this medical problem by developing a pharmacological treatment for pulmonary vascular leakage, a culprit of alveolar damage and lung inflammation. Our novel therapeutic target is the microtubule accessory factor End Binding protein 3 (EB3), which contributes to pulmonary vascular leakage by amplifying pathological calcium signaling in endothelial cells. Using a structure-activity-relationship approach by Nuclear Magnetic Resonance, we have identified a drug candidate Vascular Therapeutics (VT)-109, an allosteric EB3 inhibitor with optimized physicochemical and biochemical properties. The VT-109 shows marked therapeutic benefits in treating lung injury and inflammation in various models of ARDS including high-volume mechanical ventilation, polymicrobial and SARS-CoV-2 infections. Transcriptome analysis of lung endothelial cells in mice challenged with endotoxin shows that VT-109 upregulates FOXM1 target genes suggesting that the treatment not only mitigates vascular injury but also promotes endothelial cell regeneration by activating the FOXM1 pathway. Our work demonstrates that VT-109 is a promising drug candidate for future clinical studies. Funding for this work is provided by the Congressionally Directed Medical Research Programs through the Peer Reviewed Medical Research Program (PRMRP) under award No. W81XWH-21-1-0639. Opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the Department of Defense. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The impact of aging on pulmonary microvascular endothelial cell barrier function

Elderly individuals have substantially elevated morbidity and mortality during conditions of lung injury. However, the precise underlying mechanisms leading to this elevated mortality are ill-defined. Lung injury is associated with damage to pulmonary microvascular endothelial cells (PMVEC) leading to a compromised vascular barrier, fluid and protein leakage into the tissue, and subsequent respiratory dysfunction. Cell-cell junctions, including adherens junctions and tight junctions, are crucial for the maintenance of the endothelial barrier and have been shown to be dysregulated in various disease models. Pilot data from our lab demonstrated increased pulmonary vascular leak in aged mice during lung injury compared to young mice. We hypothesized that aging contributes to PMVEC barrier dysfunction due to impaired cell-cell junction integrity.To address our hypothesis, PMVEC isolated from young and aged mice were cultured in vitro until a confluent monolayer was formed. Endothelial barrier integrity was assessed by Evans blue-labelled albumin flux across the monolayers, as well as immunofluorescence staining of the adherens junction protein, vascular endothelial (VE)-cadherin, and tight junction protein, claudin-5. Localization of leak in relation to junctional proteins was performed using the fluorescently labelled macromolecule, NeutrAvidin. Proteomics analysis was conducted to identify differentially enriched pathways in young and aged PMVEC.Endothelial monolayers from aged mice exhibited a significant increase in albumin leak associated with augmented VE-cadherin and claudin-5 disruption vs. endothelial monolayers form young mice. Furthermore, NeutrAvidin staining localized to paracellular regions with VE-cadherin discontinuity was increased in PMVEC monolayers from aged mice vs. young mice, suggesting that the junctional disruption in aged PMVEC was directly associated with leak. Proteomics analysis revealed pathways known to be implicated in endothelial barrier dysfunction, including regulation of oxidoreduction and protein processing and trafficking, that were altered in PMVEC from aged mice.In summary, PMVEC from aged mice exhibit compromised barrier function, which is directly associated with impaired cell-cell junction integrity. We will next validate and target protein pathways identified in our proteomics analysis that were dysregulated in aged PMVEC to potentially rescue age-induced barrier dysfunction. These findings may highlight molecular pathways involved in predisposing aged individuals to worsened outcomes during lung injury, which can help in the development of therapeutics. Funded by Lawson Health Research Institute, CIHR, and Western University This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

PIMT is a novel and potent suppressor of endothelial activation.

Proinflammatory agonists provoke the expression of cell surface adhesion molecules on endothelium in order to facilitate leukocyte infiltration into tissues. Rigorous control over this process is important to prevent unwanted inflammation and organ damage. Protein L-isoaspartyl O-methyltransferase (PIMT) converts isoaspartyl residues to conventional methylated forms in cells undergoing stress-induced protein damage. The purpose of this study was to determine the role of PIMT in vascular homeostasis. PIMT is abundantly expressed in mouse lung endothelium and PIMT deficiency in mice exacerbated pulmonary inflammation and vascular leakage to LPS(lipopolysaccharide). Furthermore, we found that PIMT inhibited LPS-induced toll-like receptor signaling through its interaction with TNF receptor-associated factor 6 (TRAF6) and its ability to methylate asparagine residues in the coiled-coil domain. This interaction was found to inhibit TRAF6 oligomerization and autoubiquitination, which prevented NF-κB transactivation and subsequent expression of endothelial adhesion molecules. Separately, PIMT also suppressed ICAM-1 expression by inhibiting its N-glycosylation, causing effects on protein stability that ultimately translated into reduced EC(endothelial cell)-leukocyte interactions. Our study has identified PIMT as a novel and potent suppressor of endothelial activation. Taken together, these findings suggest that therapeutic targeting of PIMT may be effective in limiting organ injury in inflammatory vascular diseases.

SOX17 Enhancer Variants Disrupt Transcription Factor Binding And Enhancer Inactivity Drives Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a rare disease characterized by remodeling of the pulmonary arteries, increased vascular resistance, and right-sided heart failure. Genome-wide association studies of idiopathic/heritable PAH established novel genetic risk variants, including conserved enhancers upstream of transcription factor (TF) SOX17 containing 2 independent signals. SOX17 is an important TF in embryonic development and in the homeostasis of pulmonary artery endothelial cells (hPAEC) in the adult. Rare pathogenic mutations in SOX17 cause heritable PAH. We hypothesized that PAH risk alleles in an enhancer region impair TF-binding upstream of SOX17, which in turn reduces SOX17 expression and contributes to disturbed endothelial cell function and PAH development. CRISPR manipulation and siRNA were used to modulate SOX17 expression. Electromobility shift assays were used to confirm in silico-predicted TF differential binding to the SOX17 variants. Functional assays in hPAECs were used to establish the biological consequences of SOX17 loss. In silico analysis with the connectivity map was used to predict compounds that rescue disturbed SOX17 signaling. Mice with deletion of the SOX17-signal 1 enhancer region (SOX17-4593/enhKO) were phenotyped in response to chronic hypoxia and SU5416/hypoxia. CRISPR inhibition of SOX17-signal 2 and deletion of SOX17-signal 1 specifically decreased SOX17 expression. Electromobility shift assays demonstrated differential binding of hPAEC nuclear proteins to the risk and nonrisk alleles from both SOX17 signals. Candidate TFs HOXA5 and ROR-α were identified through in silico analysis and antibody electromobility shift assays. Analysis of the hPAEC transcriptomes revealed alteration of PAH-relevant pathways on SOX17 silencing, including extracellular matrix regulation. SOX17 silencing in hPAECs resulted in increased apoptosis, proliferation, and disturbance of barrier function. With the use of the connectivity map, compounds were identified that reversed the SOX17-dysfunction transcriptomic signatures in hPAECs. SOX17 enhancer knockout in mice reduced lung SOX17 expression, resulting in more severe pulmonary vascular leak and hypoxia or SU5416/hypoxia-induced pulmonary hypertension. Common PAH risk variants upstream of the SOX17 promoter reduce endothelial SOX17 expression, at least in part, through differential binding of HOXA5 and ROR-α. Reduced SOX17 expression results in disturbed hPAEC function and PAH. Existing drug compounds can reverse the disturbed SOX17 pulmonary endothelial transcriptomic signature.

Efficacy of CU06-1004 via regulation of inflammation and endothelial permeability in LPS-induced acute lung injury

BackgroundAcute lung injury (ALI) is a life-threatening condition that fundamentally results from inflammation and edema in the lung. There are no effective treatments available for clinical use. Previously, we found that as a leakage blocker CU06-1004 prevents endothelial barrier disruption and enhances endothelial cell survival under inflammatory conditions. In this study, we aimed to elucidate the effect of CU06-1004 in terms of prevention of inflammation and endothelial dysfunction in an ALI mouse model.MethodsAn ALI model was established that included intraperitoneal administration of LPS. Following LPS administration, survival rates and lung wet/dry ratios were assessed. Histological analysis was performed using hematoxylin and eosin staining. Scanning electron microscopy was used to examine alveolar and capillary morphology. Cytokines such as IL-1β, IL-6, and TNF-α were analyzed using an ELISA assay of bronchoalveolar lavage fluid (BALF) and serum. Neutrophil infiltration was observed in BALF using Wright-Giemsa staining, and myeloperoxidase (MPO) activity was assessed. Pulmonary vascular leakage was confirmed using Evans-blue dye, and the expression of junctional proteins was evaluated using immunofluorescent staining. Expression of adhesion molecules was observed using immunofluorescence staining. NF-κB activation was determined using immunohistochemistry and western blot analysis.ResultsSurvival rates and pulmonary edema were ameliorated with CU06-1004 treatment. Administration of CU06-1004 normalized histopathological changes induced by LPS, and alveolar-capillary wall thickening was reduced. Compared with the LPS-challenged group, after CU06-1004 treatment, the infiltration of immune cells was decreased in the BALF, and MPO activity in lung tissue was reduced. Similarly, in the CU06-1004 treatment group, pro-inflammatory cytokines were significantly inhibited in both BALF and serum. Evans-blue leakage was reduced, and the expression of junctional proteins was recovered in the CU06-1004 group. Adhesion molecules were downregulated and NF-κB activation was inhibited after CU06-1004 treatment.ConclusionsThese results suggested that CU06-1004 had a therapeutic effect against LPS-induced ALI via alleviation of the inflammatory response and protection of vascular integrity.

Simultaneous attenuation of hyperglycemic memory-induced retinal, pulmonary, and glomerular dysfunctions by proinsulin C-peptide in diabetes

BackgroundHyperglycemic memory (HGM) is a pivotal phenomenon in the development of diabetic complications. Although coincident diabetic complications are reported, research on their development and treatment is limited. Thus, we investigated whether C-peptide can simultaneously inhibit HGM-induced retinal, pulmonary, and glomerular dysfunctions in diabetic mice supplemented with insulin.MethodsInsulin-treated diabetic mice were supplemented with human C-peptide by subcutaneous implantation of K9-C-peptide depots for 4 weeks, and reactive oxygen species (ROS) generation, transglutaminase (TGase) activity, and vascular leakage were examined in the retina, lung, and kidney.ResultsWe found hyperglycemia-induced persistent ROS generation and TGase activation after blood glucose normalization in the retina, lung, and kidney of insulin-supplemented diabetic mice. These pathological events were inhibited by systemic supplementation of human C-peptide via subcutaneous implantation of a thermosensitive biopolymer-conjugated C-peptide depot. ROS generation and TGase activation were in a vicious cycle after glucose normalization, and C-peptide suppressed the vicious cycle and subsequent endothelial permeability in human retinal endothelial cells. Moreover, C-peptide supplementation ameliorated HGM-induced retinal vascular leakage and neurodegeneration, pulmonary vascular leakage and fibrosis, and glomerular adherens junction disruption and vascular leakage.ConclusionsOverall, our findings demonstrate that C-peptide supplementation simultaneously attenuates vascular and neuronal dysfunctions in the retina, lung, and glomerulus of insulin-supplemented diabetic mice.

Blocking RAGE expression after injury reduces inflammation in mouse model of acute lung injury

BackgroundReceptor for Advanced Glycated Endproducts (RAGE) plays a major role in the inflammatory response to infectious and toxin induced acute lung injury. We tested the hypothesis that a RAGE blocking antibody when administered after the onset of injury can reduce lung inflammation compared to control antibody.MethodsMale and female C57BL/6 (WT) mice were used. Forty-six received lipopolysaccharide (LPS) and 26 PBS by nasal instillation on day one, repeated on day three. On day 2, 36 mice receiving LPS were divided into two groups of 18, one treated with 200 μg of non-immune isotype control IgG and the second group treated with 200 μg of anti-RAGE Ab, each dose divided between IV and IP. Ten of the 46 were not treated. On day 4, before euthanasia, mice were injected with fluorescein isothiocyanate (FITC) labelled albumen. BALF and serum samples were collected as well as lung tissue for immunohistochemistry (IHC). BALF was analyzed for cell (leukocyte) counts, for FITC BALF/serum ratios indicating pulmonary vascular leak, and for cytokines/chemokines using bead based multiplex assays. Quantitative IHC was performed for MPO and RAGE.ResultsTen LPS mice showed minimal inflammation by all measures indicating poor delivery of LPS and were excluded from analysis leaving n = 11 in the LPS + IgG group and n = 12 in the LPS + anti-RAGE group. BALF cell counts were low in the PBS administered mice (4.9 ± 2.1 × 105/ml) and high in the LPS injured untreated mice (109 ± 34) and in the LPS + IgG mice (91 ± 54) while in comparison, LPS + anti-RAGE ab mice counts were significantly lower (51.3 ± 18 vs. LPS + IgG, P = 0.03). The BALF/serum FITC ratios were lower for the LPS + anti-RAGE mice than for the LPS + IgG mice indicating less capillary leakiness. Quantitative IHC RAGE staining was lower in the LPS + anti-RAGE ab mice than in the LPS + IgG treated mice (P = 0.02).ConclusionsThese results describe a four-day LPS protocol to sustain lung injury and allow for treatment and suggests that treatment aimed at blocking RAGE when given after onset of injury can reduce lung inflammation.

Neutrophils drive pulmonary vascular leakage in MHV-1 infection of susceptible A/J mice.

Lung inflammation, neutrophil infiltration, and pulmonary vascular leakage are pathological hallmarks of acute respiratory distress syndrome (ARDS) which can lethally complicate respiratory viral infections. Despite similar comorbidities, however, infections in some patients may be asymptomatic while others develop ARDS as seen with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections for example. In this study, we infected resistant C57BL/6 and susceptible A/J strains of mice with pulmonary administration of murine hepatitis virus strain 1 (MHV-1) to determine mechanisms underlying susceptibility to pulmonary vascular leakage in a respiratory coronavirus infection model. A/J animals displayed increased lung injury parameters, pulmonary neutrophil influx, and deficient recruitment of other leukocytes early in the infection. Moreover, under basal conditions, A/J neutrophils overexpressed primary granule protein genes for myeloperoxidase and multiple serine proteases. During infection, myeloperoxidase and elastase protein were released in the bronchoalveolar spaces at higher concentrations compared to C57BL/6 mice. In contrast, genes from other granule types were not differentially expressed between these 2 strains. We found that depletion of neutrophils led to mitigation of lung injury in infected A/J mice while having no effect in the C57BL/6 mice, demonstrating that an altered neutrophil phenotype and recruitment profile is a major driver of lung immunopathology in susceptible mice. These results suggest that host susceptibility to pulmonary coronaviral infections may be governed in part by underlying differences in neutrophil phenotypes, which can vary between mice strains, through mechanisms involving primary granule proteins as mediators of neutrophil-driven lung injury.

Targeting VEGF-A/VEGFR2 Y949 Signaling-Mediated Vascular Permeability Alleviates Hypoxic Pulmonary Hypertension.

Pulmonary hypertension (PH) is associated with increased expression of VEGF-A (vascular endothelial growth factor A) and its receptor, VEGFR2 (vascular endothelial growth factor 2), but whether and how activation of VEGF-A signal participates in the pathogenesis of PH is unclear. VEGF-A/VEGFR2 signal activation and VEGFR2 Y949-dependent vascular leak were investigated in lung samples from patients with PH and mice exposed to hypoxia. To study their mechanistic roles in hypoxic PH, we examined right ventricle systolic pressure, right ventricular hypertrophy, and pulmonary vasculopathy in mutant mice carrying knock-in of phenylalanine that replaced the tyrosine at residual 949 of VEGFR2 (Vefgr2Y949F) and mice with conditional endothelial deletion of Vegfr2 after chronic hypoxia exposure. We show that PH leads to excessive pulmonary vascular leak in both patients and hypoxic mice, and this is because of an overactivated VEGF-A/VEGFR2 Y949 signaling axis. In the context of hypoxic PH, activation of Yes1 and c-Src and subsequent VE-cadherin phosphorylation in endothelial cells are involved in VEGFR2 Y949-induced vascular permeability. Abolishing VEGFR2 Y949 signaling by Vefgr2Y949F point mutation was sufficient to prevent pulmonary vascular permeability and inhibit macrophage infiltration and Rac1 activation in smooth muscle cells under hypoxia exposure, thereby leading to alleviated PH manifestations, including muscularization of distal pulmonary arterioles, elevated right ventricle systolic pressure, and right ventricular hypertrophy. It is important that we found that VEGFR2 Y949 signaling in myeloid cells including macrophages was trivial and dispensable for hypoxia-induced vascular abnormalities and PH. In contrast with selective blockage of VEGFR2 Y949 signaling, disruption of the entire VEGFR2 signaling by conditional endothelial deletion of Vegfr2 promotes the development of PH. Our results support the notion that VEGF-A/VEGFR2 Y949-dependent vascular permeability is an important determinant in the pathogenesis of PH and might serve as an attractive therapeutic target pathway for this disease.

The Interplay between Aquaporin-1 and the Hypoxia-Inducible Factor 1α in a Lipopolysaccharide-Induced Lung Injury Model in Human Pulmonary Microvascular Endothelial Cells.

Aquaporin-1 (AQP1), a water channel, and the hypoxia-inducible factor 1α (HIF1A) are implicated in acute lung injury responses, modulating among others pulmonary vascular leakage. We hypothesized that the AQP1 and HIF1A systems interact, affecting mRNA, protein levels and function of AQP1 in human pulmonary microvascular endothelial cells (HPMECs) exposed to lipopolysaccharide (LPS). Moreover, the role of AQP1 in apoptosis and wound healing progression was examined. Both AQP1 mRNA and protein expression levels were higher in HPMECs exposed to LPS compared to untreated HPMECs. However, in the LPS-exposed HIF1A-silenced cells, the mRNA and protein expression levels of AQP1 remained unaltered. In the permeability experiments, a statistically significant volume increase was observed at the 360 s time-point in the LPS-exposed HPMECs, while LPS-exposed HIF1A-silenced HPMECs did not exhibit cell swelling, implying a dysfunctional AQP1. AQP1 did not seem to affect cell apoptosis yet could interfere with endothelial migration and/or proliferation. Based on our results, it seems that HIF1A silencing negatively affects AQP1 mRNA and protein expression, as well as AQP1 function, in the setting of lung injury.

Ablation of PDE4A Exacerbates Pseudomonas aeruginosa‐Induced Acute Lung Injury in Mice

Type 4 cyclic nucleotide phosphodiesterases (PDE4s) comprise a family of four isoenzymes, PDE4A to D, that hydrolyze the second messenger cAMP. Three of these enzymes, PDE4A, PDE4B and PDE4D, are widely expressed throughout pulmonary and immune cell types, and their concurrent inhibition with non/PAN‐selective PDE4 inhibitors is well‐known to exert potent anti‐inflammatory benefits. To determine if PDE4 inhibition may represent a therapeutic approach for acute lung injury resulting from severe pneumonia, we explored the effect of genetic ablation of individual PDE4 subtypes, or their concurrent inhibition with PAN‐PDE4 inhibitors, in a model of acute intra‐tracheal Pseudomonas aeruginosa (PA) infection in mice. We have shown recently that selective ablation of PDE4B is sufficient to protect mice from lung injury in this model by reducing pulmonary and systemic levels of pro‐inflammatory cytokines, as well as pulmonary vascular leakage and mortality. With the present study we explored whether selective inactivation of PDE4A or treatment with PAN‐PDE4 inhibitors may provide additional therapeutic benefits.MethodsMice deficient in PDE4A (PDE4A‐KO) and their wildtype (WT) littermate controls were infected intratracheally with the PA lab strain PA01 and subjected 16 h later to bronchoalveolar lavage (BAL), cardiac blood draws, and tissue extractions. Alternatively, vascular/alveolar leakage was assessed by measuring Evans Blue Dye extravasation. To test the effect of PAN‐PDE4 inhibition, mice were treated with the PDE4 inhibitor Piclamilast (5 mg/kg, i.p.) at 2 h prior to PA01 infection.ResultsCompared to WT controls, PDE4A‐KO mice displayed a hyper‐inflammatory phenotype characterized by increased levels of pro‐inflammatory cytokines, immune cells (neutrophils) in BAL fluid, and vascular leakage. Despite these hyperinflammatory responses, the bacterial load in lungs was also increased in PDE4A‐KO mice compared to WT controls. Treatment with a PAN‐PDE4 inhibitor produced incongruent effects, with an increased bacterial load mirroring the effect of PDE4A ablation, whereas reduced levels of inflammatory cytokines such as TNFα mimicked the effect of PDE4B ablation.ConclusionsAblation of PDE4A produces detrimental effects in this model of acute PA‐lung infection. Current studies aim to determine whether an impaired bacterial killing, and the resulting increase in bacterial load, are responsible for the hyperinflammation, or whether these are independent effects of PDE4A ablation. PAN‐PDE4 inhibition mimics some of the beneficial effects of selective PDE4B ablation, but also some of the detrimental effects of PDE4A ablation. These findings suggest that selective inactivation of PDE4B may be a more effective therapeutic approach, compared to PAN‐PDE4 inhibition, in settings of acute PA‐lung infection.