Lung Microvascular Endothelium Research Articles

Lipidated brush-PEG polymers as low molecular weight pulmonary drug delivery platforms

ABSTRACT Objectives Nanomedicines are being actively developed as inhalable drug delivery systems. However, there is a distinct utility in developing smaller polymeric systems that can bind albumin in the lungs. We therefore examined the pulmonary pharmacokinetic behavior of a series of lipidated brush-PEG (5 kDa) polymers conjugated to 1C2, 1C12 lipid or 2C12 lipids. Methods The pulmonary pharmacokinetics, patterns of lung clearance and safety of polymers were examined in rats. Permeability through monolayers of primary human alveolar epithelia, small airway epithelia and lung microvascular endothelium were also investigated, along with lung mucus penetration and cell uptake. Results Polymers showed similar pulmonary pharmacokinetic behavior and patterns of lung clearance, irrespective of lipid molecular weight and albumin binding capacity, with up to 30% of the dose absorbed from the lungs over 24 h. 1C12-PEG showed the greatest safety in the lungs. Based on its larger size, 2C12-PEG also showed the lowest mucus and cell membrane permeability of the three polymers. While albumin had no significant effect on membrane transport, the cell uptake of C12-conjugated PEGs were increased in alveolar epithelial cells. Conclusion Lipidated brush-PEG polymers composed of 1C12 lipid may provide a useful and novel alternative to large nanomaterials as inhalable drug delivery systems.

Fluorinated amphiphilic Poly(β-Amino ester) nanoparticle for highly efficient and specific delivery of nucleic acids to the Lung capillary endothelium

Endothelial cell dysfunction occurs in a variety of acute and chronic pulmonary diseases including pulmonary hypertension, viral and bacterial pneumonia, bronchopulmonary dysplasia, and congenital lung diseases such as alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). To correct endothelial dysfunction, there is a critical need for the development of nanoparticle systems that can deliver drugs and nucleic acids to endothelial cells with high efficiency and precision. While several nanoparticle delivery systems targeting endothelial cells have been recently developed, none of them are specific to lung endothelial cells without targeting other organs in the body. In the present study, we successfully solved this problem by developing non-toxic poly(β-amino) ester (PBAE) nanoparticles with specific structure design and fluorinated modification for high efficiency and specific delivery of nucleic acids to the pulmonary endothelial cells. After intravenous administration, the PBAE nanoparticles were capable of delivering non-integrating DNA plasmids to lung microvascular endothelial cells but not to other lung cell types. IVIS whole body imaging and flow cytometry demonstrated that DNA plasmid were functional in the lung endothelial cells but not in endothelial cells of other organs. Fluorination of PBAE was required for lung endothelial cell-specific targeting. Hematologic analysis and liver and kidney metabolic panels demonstrated the lack of toxicity in experimental mice. Thus, fluorinated PBAE nanoparticles can be an ideal vehicle for gene therapy targeting lung microvascular endothelium in pulmonary vascular disorders.

Abstract P2188: Covid-19 Plasma Exposure Causes Transcriptional, Morphological, And Functional Changes Within The Pulmonary Microvascular Endothelium

Introduction and Aims: While primarily a pulmonary disease, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can affect multiple organ systems, including the vasculature. Thus far studies have successfully characterised the whole organ compartment, however, our understanding of the mechanisms associated with COVID-19 infection and its impact on pulmonary microvasculature (pMV) remains incomplete. In human pulmonary microvascular endothelial cells (HPMVEC) treated with plasma from patients hospitalised with COVID-19, we aimed to evaluate transcriptional, morphological and functional changes, and to provide a mechanism for the excess mortality in patients with COVID-19 and how it may inform therapeutic advancement. Methods and Results: Plasma acquired from hospitalised patients with COVID-19 was compared to plasma from patients hospitalised without COVID-19 but with other acute illnesses, such as COPD. Exposure to plasma from patients with COVID-19 caused a significant functional decline in HPMVEC, with a decrease in both cell viability on the WST-1 cell-proliferation assay (n=75, p= 0.013) and cell-to-cell barrier function measured by electric cell-substrate impedance sensing (n=3, p = 0.033) . High content imaging using the cell painting image-based assay showed a phenotypic change in nuclear (n=4, p=0.019) and nucleic acid (n=4, p=0.022) morphology, indicative of a deterioration in core biological processes including protein transcription and metabolic output. Comparative deep RNA-sequencing of HPMVECs treated with COVID-19 plasma (n=5) confirmed upregulation of genes, notably IFIT5 and SFMBT1 . These genes are known to negatively regulate transcription and respond to viral infection, respectively, further demonstrating the phenotypic and transcriptional decline in the pulmonary microvasculature in patients with COVID-19. Conclusion and Impact: Our findings suggest that core biological processes are disrupted in the lung microvascular endothelium during severe COVID-19 disease contributing to aggravated disease. From a translational perspective, this knowledge could provide insight into possible treatment opportunities and prognostic tools that may directly target the dysregulated endothelium.

Inhibition of TRPC4/5 Channel Is Effective in Protecting against Histamine-Induced Hyperpermeability by Blocking Ca2+ Influx in Lung Microvascular Endothelial Cells.

Dysfunction of lung microvascular endothelium is a major feature in the pathobiology of pulmonary edema and hypoxic respiratory failure. Histamine induces lung microvascular endothelial barrier disruption and hyperpermeability upon evoking intracellular Ca2+ ([Ca2+]i) dynamics via binding to its receptors. Transient receptor potential canonical (TRPC) channels are Ca2+-permeable channel and stimulated by the agonists of G-protein-coupled receptors (GPCR). Here, we assessed histamine induced [Ca2+]i increases in human lung microvascular endothelial cells (HLMVEC) by using live cell Ca2+ imaging. We found that histamine increased [Ca2+]i was maintained at a static elevated level after a transient peak. The elevated Ca2+ plateau was vanished when extracellular Ca2+ was removed, indicating Ca2+ influx from extracellular mediated the histamine-induced Ca2+ plateau. TRPC4/5 channels antagonists, ML204 (10 µM) and HC070 (1 µM), significantly inhibited the Ca2+ plateaus, which was not influenced by Pyr3 or larixyl, the antagonists of TRPC3/6. Furthermore, ML204 or HC070 effectively suppressed the permeability response to histamine in HLMVEC. Our results indicated that histamine-induced Ca2+ influx may be mediated by TRPC4/5 channels and the antagonist of the channel significantly improved histamine-induced HLMVEC dysfunction.

Effects of Hyperoxia and Hyperoxic Oscillations on the Proteome of Murine Lung Microvascular Endothelium.

Patients presenting with insufficient tissue oxygenation and impaired lung function as in acute respiratory distress syndrome (ARDS) frequently require mechanical ventilation with supplemental oxygen. Despite the lung being used to experiencing the highest partial pressure of oxygen during healthy breathing, the organ is susceptible to oxygen-induced injury at supraphysiological concentrations. Hyperoxia-induced lung injury (HALI) has been regarded as a second hit to pre-existing lung injury and ventilator-induced lung injury (VILI) attributed to oxidative stress. The injured lung has a tendency to form atelectasis, a cyclic collapse and reopening of alveoli. The affected lung areas experience oxygen conditions that oscillate between hyperoxia and hypoxia rather than remaining in a constant hyperoxic state. Mechanisms of HALI have been investigated in many animal models previously. These studies provided insights into the effects of hyperoxia on the whole organism. However, cell type-specific responses have not been dissected in detail, but are necessary for a complete mechanistic understanding of ongoing pathological processes. In our study, we investigated the effects of constant and intermittent hyperoxia on the lung endothelium from a mouse by an in vitro proteomic approach. We demonstrate that these oxygen conditions have characteristic effects on the pulmonary endothelial proteome that underlie the physiological (patho)mechanisms.

Complement contributions to COVID-19.

COVID-19 remains a major source of concern, particularly as new variants emerge and with recognition that patients may suffer long-term effects. Mechanisms underlying SARS-CoV-2 mediated organ damage and the associated vascular endotheliopathy remain poorly understood, hindering new drug development. Here, we highlight selected key concepts of how the complement system, a major component of innate immunity that is dysregulated in COVID-19, participates in the thromboinflammatory response and drives the vascular endotheliopathy. Recent studies have revealed mechanisms by which complement is activated directly by SARS-CoV-2, and how the system interfaces with other innate thromboinflammatory cellular and proteolytic pathways involving platelets, neutrophils, neutrophil extracellular traps and the coagulation and kallikrein-kinin systems. With this new information, multiple potential sites for therapeutic intervention are being uncovered and evaluated in the clinic. Infections with SARS-CoV-2 cause damage to the lung alveoli and microvascular endothelium via a process referred to as thromboinflammation. Although not alone in being dysregulated, complement is an early player, prominent in promoting the endotheliopathy and consequential organ damage, either directly and/or via the system's complex interplay with other cellular, molecular and biochemical pathways. Delineating these critical interactions is revealing novel and promising strategies for therapeutic intervention.

Single-cell transcriptomic profiling of lung endothelial cells identifies dynamic inflammatory and regenerative subpopulations.

Studies have demonstrated the phenotypic heterogeneity of vascular endothelial cells (ECs) within a vascular bed; however, little is known about how distinct endothelial subpopulations in a particular organ respond to an inflammatory stimulus. We performed single-cell RNA-Seq of 35,973 lung ECs obtained during baseline as well as postinjury time points after inflammatory lung injury induced by LPS. Seurat clustering and gene expression pathway analysis identified 2 major subpopulations in the lung microvascular endothelium, a subpopulation enriched for expression of immune response genes such as MHC genes (immuneEC) and another defined by increased expression of vascular development genes such as Sox17 (devEC). The presence of immuneEC and devEC subpopulations was also observed in nonhuman primate lungs infected with SARS-CoV-2 and murine lungs infected with H1N1 influenza virus. After the peak of inflammatory injury, we observed the emergence of a proliferative lung EC subpopulation. Overexpression of Sox17 prevented inflammatory activation in ECs. Thus, there appeared to be a "division of labor" within the lung microvascular endothelium in which some ECs showed propensity for inflammatory signaling and others for endothelial regeneration. These results provide underpinnings for the development of targeted therapies to limit inflammatory lung injury and promote regeneration.

Calcium-Permeable Channels and Endothelial Dysfunction in Acute Lung Injury.

The increased permeability of the lung microvascular endothelium is one critical initiation of acute lung injury (ALI). The disruption of vascular-endothelium integrity results in leakiness of the endothelial barrier and accumulation of protein-rich fluid in the alveoli. During ALI, increased endothelial-cell (EC) permeability is always companied by high frequency and amplitude of cytosolic Ca2+ oscillations. Mechanistically, cytosolic calcium oscillations include calcium release from internal stores and calcium entry via channels located in the cell membrane. Recently, numerous publications have shown substantial evidence that calcium-permeable channels play an important role in maintaining the integrity of the endothelium barrier function of the vessel wall in ALI. These novel endothelial signaling pathways are future targets for the treatment of lung injury. This short review focuses on the up-to-date research and provide insight into the contribution of calcium influx via ion channels to the disruption of lung microvascular endothelial-barrier function during ALI.

Abstract 2641: Development of a 3D in vitro model of the prostate tumor microenvironment

Abstract Prostate cancer (PCa) is the most common occurring cancer in men and the second-most leading cause of cancer-related deaths in the United States. To improve patient outcome, research tools which mimic the prostate tumor microenvironment (TME) and accurately predict drug response are urgently needed. However, current in vitro PCa models do not recapitulate the complexities of the prostate TME including three-dimensional (3D) orientation, perfusion, extracellular matrix (ECM), and the presence of multiple cell types such as tumor cells, fibroblasts, and endothelium. Further, many complex in vitro model systems fail to maintain the throughput required for robust drug screening. To address this need, here, we highlight the stepwise development of a 3D in vitro PCa model by optimizing the individual culture conditions for each cell type (PCa cells, fibroblasts (FB), endothelial cells (EC)) within MIMETAS' high throughput perfusable 3D cell culture platform, the OrganoPlate® 2-lane 96. All monocultures (PCa, fibroblast, endothelial cell) were viable and expressed respective phenotypic markers (PCa: AR and PSA; FB: vimentin; EC: CD-31, VE-cadherin) as detected by immunofluorescent staining and high-content imaging. To demonstrate the statistical retention of heterogeneity within PCa populations, PCa cells (MDA-PCa-2b) were pre-labeled with 4 different tracking dyes and clustered into multicellular aggregates using a multiwell, ultra-low attachment plate. After 3D encapsulation within a migration permissive hyaluronic acid (MP-HA) hydrogel, PCa clusters retained uniform size clusters and even distribution of the pre-labeled populations within each cluster. Additionally, use of this pre-clustering reduced cell debris in the encapsulated 3D cultures. 3D FB cultures, mimicking normal or reactive stroma, were established with bone stroma cell lines and primary human mesenchymal stem cells. The cells' phenotypic stretched morphology and migration through the ECM were tuned by tailoring the hydrogel crosslinking assessed further by measuring the permeability of 2,000 kDa FITC-dextran. EC (primary human lung microvascular endothelium) were seeded against MP-HA to form a tubule structure within the perfusion channel. Culture conditions were optimized to reduce the permeability of smaller molecules (4.4, 150 KDa Dextran) through EC barriers and further imaging revealed the formation of a 3D blood vessel-like structure within the perfusion channel. Studies are ongoing to combine all three cell types into a single model. This stepwise approach, for building a complex, 3D prostate tumor model, will enable the full incorporation of all cell types and ECM into a single model which will have the potential to better recapitulate the in vivo prostate TME and improve the predictivity of PCa in vitro models. Citation Format: Divya Iyer, Andrei Bonteanu, Jedidiah Z. Zhu, Peter Shepherd, Rick Kittles, Nora M. Navone, Daniel A. Harrington, Dwayne Dexter, Kristin M. Bircsak. Development of a 3D in vitro model of the prostate tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2641.

Abstract 14741: Autologous Cd34+ Cells (clbs119) for Repair of Covid-19 Induced Microvascular Lung Damage: Rationale and Study Design

Early evidence from COVID-19 patients and autopsy series indicates that moderate to severe cases are accompanied by damage to the pulmonary endothelium and microcirculation. These data indicate that SARS-CoV-2 infection is particularly avid for lung microvascular endothelium, reflecting endothelial cell infection, inflammation, and focal thrombi occluding the pulmonary circulation leading to progressive fibrosis reminiscent of endothelial-mesenchymal transition. This appears to be a potential mechanism by which COVID-19 causes progressive fibrotic changes, impairment in the pulmonary vasculature, and loss of lung function that persists after acute recovery. Early data from the SARS-CoV-1 epidemic indicated that CD34+ cells in the lung could also be a target of infection and that destruction of lung CD34+ progenitors could account for persistence of pulmonary manifestations. Preclinical studies reveal that restoration of microvasculature in the lung can trigger and sustain regeneration of lung tissue. The microvascular repair function of CD34+ has been documented in preclinical models and multiple human trials in ischemic tissue repair. Accordingly, we designed a clinical trial to evaluate autologous CD34+ cells (CLBS119) for amelioration/repair of COVID-19 lung damage. Subjects must be hospitalized for COVID-19 and have ongoing pulmonary involvement based on room air hypoxia. The study will include approximately 50% of subjects who cannot be liberated from ventilatory support. CD34+ cells will be mobilized with plerixafor and selected from the mononuclear fraction following apheresis. Doses up to 500 x 10 6 cells will be administered IV. Subjects will receive a single administration of cells. Key measures will include assessment of oxygenation, need for supplemental oxygen, pulmonary function assessments, radiographic assessments, and biomarkers. Subjects will be followed for 6 months following administration. Interim findings will be presented.

Oxidized Hemoglobin Causes Human Lung Microvascular Endothelial Barrier Dysfunction

Cell‐free hemoglobin is released into the circulation during conditions in which red blood cell lysis occurs such as hemolytic disorders and sepsis. In septic patients, increased levels of circulating cell‐free hemoglobin are independently associated with mortality and contribute to organ dysfunction, including acute respiratory distress syndrome (ARDS). Furthermore, during inflammatory conditions in which reactive oxygen species are high, cell‐free hemoglobin can be converted to higher oxidation states which enhances its damaging capacity. We previously showed that hemoglobin contributes to tissue edema in an ex vivo perfused human lung and microvascular endothelial barrier dysfunction in vitro; hemoprotein reductants (ascorbate and acetaminophen) protected against this phenomenon. Therefore, we hypothesized that higher oxidation states of hemoglobin are more injurious to the barrier function of lung microvascular endothelium. Utilizing primary human lung microvascular endothelial cells (HLMVEC), we found that stimulation with 0.5 mg/mL ferric (Hb3+; −380.2 Ω, p<0.001 vs. control) but not ferrous (Hb2+; +278.3 Ω, p=0.9893 vs. control) hemoglobin resulted in a drop in transendothelial electrical resistance (Figure ). The barrier‐disrupting effects of Hb3+ were reduced with haptoglobin (binds free hemoglobin; +208.5 Ω, p<0.0001 vs. Hb3+) and hemopexin (binds free heme), but not the iron chelator deferoxamine. The mechanism of Hb3+‐mediated microvascular endothelial barrier dysfunction involved disruption of adherens junction protein VE‐cadherin at cell‐cell borders and rearrangement of the actin cytoskeleton with intercellular gap formation. These data suggest that oxidized cell‐free hemoglobin causes microvascular endothelial barrier dysfunction to a greater degree than its non‐oxidized form.Support or Funding InformationThis work is funded by NIH HL135849, HL103836, and HL094296.Figure 1

Mechanosensing and Mechanoregulation of Endothelial Cell Functions.

Vascular endothelial cells (ECs) form a semiselective barrier for macromolecules and cell elements regulated by dynamic interactions between cytoskeletal elements and cell adhesion complexes. ECs also participate in many other vital processes including innate immune reactions, vascular repair, secretion, and metabolism of bioactive molecules. Moreover, vascular ECs represent a unique cell type exposed to continuous, time-dependent mechanical forces: different patterns of shear stress imposed by blood flow in macrovasculature and by rolling blood cells in the microvasculature; circumferential cyclic stretch experienced by the arterial vascular bed caused by heart propulsions; mechanical stretch of lung microvascular endothelium at different magnitudes due to spontaneous respiration or mechanical ventilation in critically ill patients. Accumulating evidence suggests that vascular ECs contain mechanosensory complexes, which rapidly react to changes in mechanical loading, process the signal, and develop context-specific adaptive responses to rebalance the cell homeostatic state. The significance of the interactions between specific mechanical forces in the EC microenvironment together with circulating bioactive molecules in the progression and resolution of vascular pathologies including vascular injury, atherosclerosis, pulmonary edema, and acute respiratory distress syndrome has been only recently recognized. This review will summarize the current understanding of EC mechanosensory mechanisms, modulation of EC responses to humoral factors by surrounding mechanical forces (particularly the cyclic stretch), and discuss recent findings of magnitude-specific regulation of EC functions by transcriptional, posttranscriptional and epigenetic mechanisms using -omics approaches. We also discuss ongoing challenges and future opportunities in developing new therapies targeting dysregulated mechanosensing mechanisms to treat vascular diseases. © 2019 American Physiological Society. Compr Physiol 9:873-904, 2019.

Connexin 40 regulates lung endothelial permeability in acute lung injury via the ROCK1-MYPT1- MLC20 pathway.

Increased pulmonary vascular permeability is a hallmark of acute lung injury (ALI). Connexin 40 (Cx40) is a gap junctional protein abundantly present in the lung microvascular endothelium. Yet, the role of Cx40 in the regulation of lung vascular permeability and its underlying mechanisms are unclear. Here, we tested the hypothesis that Cx40 participates in regulation of lung endothelial permeability via a mechanism involving a Rho-associated protein kinase (ROCK) dependent regulation of myosin light chain (MLC). In murine models of intratracheal acid- or LPS-induced lung injury, genetic deficiency of Cx40 attenuated key features of ALI including vascular barrier failure. In human pulmonary microvascular endothelial cells (PMVECs), thrombin-induced loss of transendothelial electrical resistance was attenuated by a Cx40-inhibiting mimetic peptide (40GAP27), Cx40-specific shRNA, or ROCK inhibitor Y27632. In isolated perfused mouse lungs, platelet-activating factor-induced lung weight gain was abrogated by gap junction blocker carbenoxolone, 40GAP27, Y27632, or genetic deficiency of Cx40. Phosphorylation of MLC20 increased drastically in both LPS-treated PMVECs and HCl-treated mouse lungs. Expression of ROCK1 was increased in both LPS-treated PMVECs and HCl-treated mouse lungs, and paralleled by phosphorylation of MLC20. Coimmunoprecipitation experiments revealed protein-protein interaction between ROCK1 and Cx40. LPS-induced upregulation of ROCK1 and phosphorylation of MLC20 were blocked by knockdown of Cx40. LPS caused phosphorylation of myosin phosphatase targeting subunit 1, which could be abrogated by Y27632 or Cx40-shRNA. Our findings reveal a role of Cx40 in regulation of ROCK1 and MLC20 that contributes critically to lung vascular barrier failure in ALI.

Modulation of cellular bioenergetics by CO-releasing molecules and NO-donors inhibits the interaction of cancer cells with human lung microvascular endothelial cells.

Interactions between cancer cells and the endothelium play a crucial role during metastasis. Here we examined the effects of a carbon monoxide-releasing molecule (CORM-401) and a nitric oxide donor (PAPA NONOate) given alone or in combination on breast cancer cell adhesion and transmigration across the lung microvascular endothelium. We further explored whether the effects of CO and NO on cancer-endothelial cells interactions are linked with changes in cellular bioenergetics in breast cancer or endothelial cells. We found that CORM-401 and PAPA NONOate alone or in combination markedly decreased transmigration of breast cancer cells across human lung microvascular endothelial cells (hLMVEC), while cancer cell adhesion to the endothelium was diminished only by a combination of the two compounds. In hLMVECs, CORM-401 decreased glycolysis and stimulated mitochondrial respiration, while in breast cancer cells CORM-401 decreased both glycolysis and mitochondrial respiration. In contrast, PAPA NONOate decreased mitochondrial respiration and slightly stimulated glycolysis in both cell lines. When both donors were given together, mitochondrial respiration and glycolysis were both profoundly inhibited, and cancer-endothelial cells interactions were additively suppressed. Intercellular adhesion molecule-1 (ICAM-1), involved in breast cancer cell adhesion to hLMVECs, was downregulated by CORM-401 and PAPA NONOate, when applied alone, while a combination of both compounds did not cause any enhancement of ICAM-1 downregulation. In conclusion, our findings demonstrate that CO and NO differently affect cellular bioenergetics of cancer and endothelial cells and suggest that this phenomenon may contribute to additive anti-adhesive and anti-transmigratory effects of CO and NO. Pharmacological attenuation of metabolism represents a novel, effective way to prevent cancer cell interactions with the endothelium, that is an energy-demanding process.

Experimental Lung Injury Reduces Krüppel-like Factor 2 to Increase Endothelial Permeability via Regulation of RAPGEF3-Rac1 Signaling.

Acute respiratory distress syndrome (ARDS) is caused by widespread endothelial barrier disruption and uncontrolled cytokine storm. Genome-wide association studies (GWAS) have linked multiple genes to ARDS. Although mechanosensitive transcription factor Krüppel-like factor 2 (KLF2) is a major regulator of endothelial function, its role in regulating pulmonary vascular integrity in lung injury and ARDS-associated GWAS genes remains poorly understood. To examine KLF2 expression in multiple animal models of acute lung injury and further elucidate the KLF2-mediated pathways involved in endothelial barrier disruption and cytokine storm in experimental lung injury. Animal and in vitro models of acute lung injury were used to characterize KLF2 expression and its downstream effects responding to influenza A virus (A/WSN/33 [H1N1]), tumor necrosis factor-α, LPS, mechanical stretch/ventilation, or microvascular flow. KLF2 manipulation, permeability measurements, small GTPase activity, luciferase assays, chromatin immunoprecipitation assays, and network analyses were used to determine the mechanistic roles of KLF2 in regulating endothelial monolayer integrity, ARDS-associated GWAS genes, and lung pathophysiology. KLF2 is significantly reduced in several animal models of acute lung injury. Microvascular endothelial KLF2 is significantly induced by capillary flow but reduced by pathologic cyclic stretch and inflammatory stimuli. KLF2 is a novel activator of small GTPase Ras-related C3 botulinum toxin substrate 1 by transcriptionally controlling Rap guanine nucleotide exchange factor 3/exchange factor directly activated by cyclic adenosine monophosphate, which maintains vascular integrity. KLF2 regulates multiple ARDS GWAS genes related to cytokine storm, oxidation, and coagulation in lung microvascular endothelium. KLF2 overexpression ameliorates LPS-induced lung injury in mice. Disruption of endothelial KLF2 results in dysregulation of lung microvascular homeostasis and contributes to lung pathology in ARDS.

P3.01-050 Isolation and Characterization of Lymphatic Endothelial Cells from Neoplastic and Normal Human Lung: Topic: Functional Biology in Lung Cancer

Cell culture models may be crucial to study lung microvascular endothelium and its role in cancer progression and treatment. In addition, a high heterogeneity among endothelial cells from various districts has been demonstrated, with greater difference on the lymphatic circulatory system. Organ-specific endothelial cells are essential to elucidate signaling pathways involved in the pathogenetic mechanisms of neoplastic lung diseases and to provide novel approaches to reach the goal of a true personalized therapy. The aim of the present study was to isolate and characterize lymphatic endothelial cells from neoplastic and healthy human lung. A simple and inexpensive method, requiring minimal equipment and accessories was utilized to harvest, isolate and expand lymphatic endothelial cells from human lung (Lu-LECs). Specifically, samples from lung cancer (T) and spared distal lung (D) of 46 patients undergoing lobectomy or pneumonectomy for NSCLC, were processed. To obtain a pure population of Lu-LEC, a two-step purification tool based on sorting with monoclonal antibody to CD31 and podoplanin coated paramagnetic beads was employed. Immunohistochemical analysis on harvested pulmonary tissues was performed to assess the presence or absence of neoplastic cells and to identify blood and lymphatic vasculature. The purity of cultured endothelial cells was ascertained by morphologic criteria including TEM analysis, immunocytochemistry, flow cytometry and functional assays. T and D Lu-LECs were positive for CD31. Moreover, to define the lymphatic phenotype, we examined the specific markers podoplanin, LYVE-1 and Prox-1. No significant immunophenotypic differences between D and T Lu-LEC were detected by FACS. Although T Lu-LEC were larger than D Lu-LEC, morphologic features as cytoplasmic microvescicles, Weibel-Palade Bodies and aggregate of parallel intermediate filaments were equally observed. Cells were characterized in vitro for the ability to express several receptor tyrosine kinases (RTKs) implicated in cell survival and proliferation and in the development and progression of cancer. Cultured lymphatic endothelial cells variably expressed VEGFR-2, VEGFR-3, PDGFR-beta, c-met, and IGF-1R according to D or T origin. Moreover, FGFR-1 and EGFR-1 were present in a large fraction of T and D Lu-LEC. Matrigel assay documented that T Lu-LEC more efficiently organized in tubular structures at early time point when compared to D counterpart. Conversely, wound healing assay revealed that D Lu-LEC had a superior migratory ability. Primary lines of LECs from the human lung have been consistently obtained and may represent an important tool to study NSLCL microenvironment, lymphoangiogenesis and anti-cancer therapy.

The NEU1-selective sialidase inhibitor, C9-butyl-amide-DANA, blocks sialidase activity and NEU1-mediated bioactivities in human lung in vitro and murine lung in vivo.

Neuraminidase-1 (NEU1) is the predominant sialidase expressed in human airway epithelia and lung microvascular endothelia where it mediates multiple biological processes. We tested whether the NEU1-selective sialidase inhibitor, C9-butyl-amide-2-deoxy-2,3-dehydro-N-acetylneuraminic acid (C9-BA-DANA), inhibits one or more established NEU1-mediated bioactivities in human lung cells. We established the IC50 values of C9-BA-DANA for total sialidase activity in human airway epithelia, lung microvascular endothelia and lung fibroblasts to be 3.74 µM, 13.0 µM and 4.82 µM, respectively. In human airway epithelia, C9-BA-DANA dose-dependently inhibited flagellin-induced, NEU1-mediated mucin-1 ectodomain desialylation, adhesiveness for Pseudomonas aeruginosa and shedding. In lung microvascular endothelia, C9-BA-DANA reversed NEU1-driven restraint of cell migration into a wound and disruption of capillary-like tube formation. NEU1 and its chaperone/transport protein, protective protein/cathepsin A (PPCA), were differentially expressed in these same cells. Normalized NEU1 protein expression correlated with total sialidase activity whereas PPCA expression did not. In contrast to eukaryotic sialidases, C9-BA-DANA exerted far less inhibitory activity for three selected bacterial neuraminidases (IC50>800 µM). Structural modeling of the four human sialidases and three bacterial neuraminidases revealed a loop between the seventh and eighth strands of the β-propeller fold, that in NEU1, was substantially shorter than that seen in the six other enzymes. Predicted steric hindrance between this loop and C9-BA-DANA could explain its selectivity for NEU1. Finally, pretreatment of mice with C9-BA-DANA completely protected against flagellin-induced increases in lung sialidase activity. Our combined data indicate that C9-BA-DANA inhibits endogenous and ectopically expressed sialidase activity and established NEU1-mediated bioactivities in human airway epithelia, lung microvascular endothelia, and fibroblasts in vitro and murine lungs in vivo.

Quantification of Breast Cancer Cell Transmigration across Brain and Lung Microvascular Endothelium

Extravasation is a crucial step during tumor metastasis. Breast cancer cells prefer to extravasate into the lungs and brain. However, where and how tumor cells transmigrate through these organ-specific endothelial barriers to complete their extravasation remains unclear. Using an in vitro transwell model, we performed a transmigration assay of malignant breast tumor cells (MDA-MB-231) through lung or brain microvascular endothelial monolayers under control and pathological conditions. The locations and rates of tumor cell transmigration as well as the changes in structural components of endothelial monolayers were investigated by confocal microscopy. Endothelial monolayer permeability to albumin Palbumin was also quantified under the same conditions. Our results show: 1) More than 95% of transmigration occurs at the joints of endothelial cells instead of cell bodies; 2) Tumor cell adhesion and transmigration degrade endothelial surface glycocalyx and disrupt junctional proteins between adjacent endothelial cells, consequently increasing Palbumin; 3) The transmigration rate of breast tumor cells is higher through the lung endothelial monolayer with higher Palbumin; 4) Both Palbumin and tumor transmigration rates are increased by vascular endothelial growth factor (VEGF) and an inflammatory agent, lipopolysaccharides (LPS) treatments. These results suggest that reinforcing endothelial structural integrity, e.g. preserving surface glycocalyx and junction proteins, is an effective approach for inhibiting tumor extravasation. Supported by NIH SC1CA153325-01.

S99 Effects Of Differential Tnf Receptor Signalling In Modulating Neutrophil-endothelial Interactions In The Pulmonary Microvasculature

Neutrophil recruitment into the bronchoalveolar space is central to the pathogenesis of acute respiratory distress syndrome injury (ARDS), and occurs via interaction with the lung microvascular endothelium. Tumour Necrosis Factor (TNF) is a key mediator in these processes, activating endothelial cells and inducing changes in microvascular permeability, as well as priming neutrophils (a pre-requisite for neutrophil-mediated tissue damage) and modulating neutrophil lifespan. TNF signals through two cell surface receptors, TNFR1 and TNFR2 initiating distinct signalling pathways and cellular responses. In a human in vivo model of ARDS, selective TNFR1 antagonism attenuated pulmonary inflammation (O’Kane et al , Thorax 2013; 63:A50). Using TNF receptor specific muteins and a novel highly selective TNFR1 antagonist, we investigated the role of differential TNFR signalling on neutrophil-pulmonary microvascular endothelial cell interactions. TNF-induced alterations in the expression of the neutrophil cell surface molecules CD11b, CD62L, TNFR1 and TNFR2 were all modulated via TNFR1. TNFR1 was also the dominant receptor mediating reactive oxygen species generation by TNF-primed, fMLP-stimulated neutrophils. We further examined the role of TNF receptors in modulating neutrophil apoptosis; whilst engagement of both TNFR1 and 2 was required to induce early neutrophil apoptosis, TNFR1 antagonism reversed TNF-induced late survival to constitutive levels of apoptosis. TNFR1 antagonsim of human pulmonary microvascular endothelial monolayers significantly reduced TNF-induced production of IL-1beta, IL-6 and IL-8 (p Collectively, these results suggest that TNFR1 regulates multiple components of neutrophil-endothelial interactions. Selective TNFR1 antagonism may offer a novel therapeutic approach in ARDS; phase II clinical trials of this therapy are scheduled.

α1-Acid glycoprotein disrupts capillary-like tube formation of human lung microvascular endothelia

ABSTRACTPurpose: The acute phase protein, α1-acid glycoprotein, is expressed in the lung, and influences endothelial cell function. We asked whether it might regulate angiogenesis in human lung microvascular endothelia. Materials and Methods: α1-acid glycoprotein was isolated from human serum by HPLC ion exchange chromatography. Its effects on endothelial cell functions including capillary-like tube formation on Matrigel, migration in a wounding assay, chemotaxis in a modified Boyden chamber, adhesion, and transendothelial flux of the permeability tracer, 14C-albumin, were tested. Results: α1-acid glycoprotein dose-dependently inhibited capillary-like tube formation without loss of cell viability. At ≥0.50 mg/mL, it inhibited tube formation >70%, and at 0.75 mg/mL, >97%. α1-acid glycoprotein dose- and time-dependently restrained EC migration into a wound as early as 2 hours, and in washout studies, did so reversibly. It was inhibitory against vascular endothelial growth factor-A and fibroblast growth factor-2-driven migration but failed to inhibit chemotactic responsiveness. When α1-acid glycoprotein was added to preformed tubes, it provoked their almost immediate disassembly. As early as 15 minutes, it induced tube network collapse without endothelial cell–cell disruption. It exerted a biphasic effect on cell adhesion to the Matrigel substrate. At lower concentrations (0.05–0.25 mg/mL), it increased cell adhesion, whereas at higher concentrations (≥0.75 mg/mL) decreased adhesion. In contrast, it had no effect on transendothelial 14C-albumin flux. Conclusion: α1-acid glycoprotein, at concentrations found under physiological conditions, rapidly inhibits endothelial cell capillary-like tube formation that may be explained through diminished cell adhesion to the underlying matrix and/or reversibly decreased cell migration.