Microvascular Endothelial Cell Barrier Research Articles

The calcineurin-NFATc pathway modulates the lipid mediators in BAL fluid extracellular vesicles, thereby regulating microvascular endothelial cell barrier function.

Extracellular vesicles mediate intercellular communication by transporting biologically active macromolecules. Our prior studies have demonstrated that the nuclear factor of activated T cell cytoplasmic member 3 (NFATc3) is activated in mouse pulmonary macrophages in response to lipopolysaccharide (LPS). Inhibition of NFATc3 activation by a novel cell-permeable calcineurin peptide inhibitor CNI103 mitigated the development of acute lung injury (ALI) in LPS-treated mice. Although pro-inflammatory lipid mediators are known contributors to lung inflammation and injury, it remains unclear whether the calcineurin-NFATc pathway regulates extracellular vesicle (EV) lipid content and if this content contributes to ALI pathogenesis. In this study, EVs from mouse bronchoalveolar lavage fluid (BALF) were analyzed for their lipid mediators by liquid chromatography in conjunction with mass spectrometry (LC-MS/MS). Our data demonstrate that EVs from LPS-treated mice contained significantly higher levels of arachidonic acid (AA) metabolites, which were found in low levels by prior treatment with CNI103. The catalytic activity of lung tissue cytoplasmic phospholipase A2 (cPLA2) increased during ALI, correlating with an increased amount of arachidonic acid (AA) in the EVs. Furthermore, ALI is associated with increased expression of cPLA2, cyclooxygenase 2 (COX2), and lipoxygenases (5-LOX, 12-LOX, and 15-LOX) in lung tissue, and pretreatment with CNI103 inhibited the catalytic activity of cPLA2 and the expression of cPLA2, COX, and LOX transcripts. Furthermore, co-culture of mouse pulmonary microvascular endothelial cell (PMVEC) monolayer and NFAT-luciferase reporter macrophages with BALF EVs from LPS-treated mice increased the pulmonary microvascular endothelial cell (PMVEC) monolayer barrier permeability and luciferase activity in macrophages. However, EVs from CNI103-treated mice had no negative impact on PMVEC monolayer barrier integrity. In summary, BALF EVs from LPS-treated mice carry biologically active NFATc-dependent, AA-derived lipids that play a role in regulating PMVEC monolayer barrier function.

Investigation of the Role of Aging on Pulmonary Microvascular Endothelial Cell Barrier Function During Mechanical Ventilation

Elderly individuals have substantially elevated morbidity and mortality following pulmonary insult that require mechanical ventilation (MV) to sustain respiratory function. It is not known if an altered response to MV contributes to the worsened outcomes in the elderly. MV is associated with injury to pulmonary microvascular endothelial cells (PMVEC), leading to a compromised vascular barrier, and fluid and protein leakage within the tissue. However, the precise impact that age has on the vascular endothelium during MV is unknown. We hypothesized that aging augments PMVEC barrier dysfunction during MV. To address this hypothesis, young and aged mice were ventilated at a tidal volume of 20 mL/kg for 3 hours. To assess microvascular permeability, lungs were lavaged and abundance of the serum protein, immunoglobulin M, within the lavage fluid was measured. Lung tissue was dissociated into single cells and RNA was collected for single-cell RNA sequencing (scRNAseq). Subsequent analysis was performed using the Seurat package in R to give insight into the role of specific cell types, with a particular emphasis on microvascular endothelial cells. To directly assess molecular mechanisms directly in the cells, PMVEC were isolated from a separate cohort of non-ventilated young and aged mice and cultured in vitro. Cells were grown to confluency, then assessed for barrier integrity by Evans blue-labelled albumin leak across monolayers and immunofluorescence staining of cell junctional adherens and tight junction proteins, VE-cadherin and claudin-5. Compared to young, aged-ventilated animals had a significant increase in immunoglobulin M in their lavage fluid. Analysis of scRNAseq data revealed more robust activation and inflammation in the endothelial cells from aged animals following MV. Endothelial monolayers from aged animals in vitro exhibited a significant increase in albumin flux, which was associated with poor VE-cadherin and claudin-5 junction formation. In conclusion, PMVEC from aged mice exhibit compromised barrier function, leading to augmented permeability following MV. The age-dependent loss in barrier function appears to be due to an impaired ability to re-establish functional cell-cell junctions. These findings suggest that the aged vascular barrier is more susceptible to injury from MV, due to impaired PMVEC cell-cell junction integrity. Our results may highlight molecular pathways involved in predisposing aged individuals to worsened outcomes during MV, which can help in developing targeted therapeutics. Funded by Lawson Health Research Institute, CIHR, the Ontario Graduate Scholarship, and Western University. This is the full abstract presented at the American Physiology Summit 2024 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.

NSCLC Extracellular Vesicles Containing miR-374a-5p Promote Leptomeningeal Metastasis by Influencing Blood‒Brain Barrier Permeability.

Leptomeningeal metastasis (LM) is a devastating complication of advanced non-small cell lung cancer (NSCLC). Its diagnosis and monitoring can be challenging. Recently, extracellular vesicle (EV) miRNAs have become a new noninvasive diagnostic biomarker. The purpose of this study was to examine the clinical value and role of EV miRNAs in NSCLC-LM. Next-generation sequencing analysis revealed that miRNAs with differential expression of EVs in sera of patients with NSCLC with LM and non-LM were detected to identify biological markers for the diagnosis of LM. Cellular and in vivo experiments were conducted to explore the pathogenesis of EV miRNA promoting LM in NSCLC. In the present study, we first demonstrated that the serum level of EV-associated miR-374a-5p in patients with LM of lung cancer was much higher than that in patients without LM and was correlated with the survival time of patients with LM. Further studies showed that EV miR-374a-5p efficiently destroys tight junctions and the integrity of the cerebral microvascular endothelial cell barrier, resulting in increased blood-brain barrier permeability. Mechanistically, miR-374a-5p regulates the distribution of ZO1 and occludin in endothelial cells by targeting γ-adducin, increasing vascular permeability and promoting LM. Implications: These results suggest that serum NSCLC-derived EV miR-374a-5p is involved in premetastatic niche formation by regulating the permeability of the blood-brain barrier to promote NSCLC-LM and can be used as a blood biomarker for the diagnosis and prognosis of NSCLC-LM.

Simultaneous Targeting of IL-1-Signaling and IL-6-Trans-Signaling Preserves Human Pulmonary Endothelial Barrier Function During a Cytokine Storm.

Systemic inflammatory diseases, such as sepsis and severe COVID-19, provoke acute respiratory distress syndrome in which the pathological hyperpermeability of the microvasculature, induced by uncontrolled inflammatory stimulation, causes pulmonary edema. Identifying the inflammatory mediators that induce human lung microvascular endothelial cell barrier dysfunction is essential to find the best anti-inflammatory treatments for critically ill acute respiratory distress syndrome patients. We have compared the responses of primary human lung microvascular endothelial cells to the main inflammatory mediators involved in cytokine storms induced by sepsis and SARS-CoV2 pulmonary infection and to sera from healthy donors and severely ill patients with sepsis. Endothelial barrier function was measured by electric cell-substrate impedance sensing, quantitative confocal microscopy, and Western blot. The human lung microvascular endothelial cell barrier was completely disrupted by IL (interleukin)-6 conjugated with soluble IL-6R (IL-6 receptor) and by IL-1β, moderately affected by TNF (tumor necrosis factor)-α and IFN (interferon)-γ and unaffected by other cytokines and chemokines, such as IL-6, IL-8, MCP (monocyte chemoattractant protein)-1 and MCP-3. The inhibition of IL-1 and IL-6R simultaneously, but not separately, significantly reduced endothelial hyperpermeability on exposing human lung microvascular endothelial cells to a cytokine storm consisting of 8 inflammatory mediators or to sera from patients with sepsis. Simultaneous inhibition of IL-1 and JAK (Janus kinase)-STAT (signal transducer and activator of transcription protein), a signaling node downstream IL-6 and IFN-γ, also prevented septic serum-induced endothelial barrier disruption. These findings strongly suggest a major role for both IL-6 trans-signaling and IL-1β signaling in the pathological increase in permeability of the human lung microvasculature and reveal combinatorial strategies that enable the gradual control of pulmonary endothelial barrier function in response to a cytokine storm.

Mechanisms of pulmonary microvascular endothelial cells barrier dysfunction induced by LPS: The roles of ceramides and the Txnip/NLRP3 inflammasome.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) are characterized by pulmonary microvascular endothelial cells (PMVECs) barrier dysfunction and proinflammatory cytokine influx into lung tissue, resulting in pulmonary oedema. Ceramide overproduction is an important mediator of pulmonary hyperinflammation and pulmonary oedema in Acute lung injury (ALI). Ceramides induce NLRP3 inflammasome activation are essential for the hyperinflammatory response. However, the roles and specific mechanisms of ceramide-induced NLRP3 inflammasome activation, proinflammatory cytokine manufacturing and PMVECs barrier dysfunction in ALI are unclear. Herein, pretreatment with the acid sphingomyelinase (ASMase) inhibitor imipramine (but not a neutral sphingomyelinase (NSMase) inhibitor or de novo pathway inhibitor) significantly inhibited ceramide early production in rats with lipopolysaccharide (LPS)-induced ALI; Furthermore, the Txnip/NLRP3 inflammasome activation, proinflammatory cytokine release, increased PMVECs permeability and lung injury were significantly decreased. Verapamil, a Txnip inhibitor, substantially inhibited Txnip/NLRP3 inflammasome activation, proinflammatory cytokine release, increased PMVECs permeability and lung injury in rats with C8-ceramide-induced ALI. In vitro, short hairpin RNA-mediated Txnip silencing significantly inhibited C8-ceramide-induced Txnip/NLRP3 inflammasome activation in NR8383 alveolar macrophages (AMs) and early secretion of the proinflammatory cytokines IL-1β (4-12h) as well as IL-6 and TNF-α at subsequent times (later than 12h). However, C8-ceramide significantly increased the early secretion (within 8h) of the proinflammatory cytokines IL-1β, IL-6 and TNF-α in a co-culture model of NR8383 AMs and PMVECs, and Txnip silencing of NR8383 AMs inhibited the secretion of pro-inflammatory cytokines and reduced cytoskeletal rearrangements, intercellular connection breakage and hyperpermeability in PMVECs. Overall, our results suggest that in LPS-induced ALI, ceramide-mediated Txnip/NLRP3 inflammasome activation in NR8383 AMs leads to early IL-1β release, subsequently inducing PMVECs injury and release of the proinflammatory cytokines IL-6 and TNF-α, ultimately leading to PMVECs barrier dysfunction and ALI.

Spotted fever rickettsia-induced microvascular endothelial barrier dysfunction is delayed by the calcium channel blocker benidipine

The tick-borne bacterium Rickettsia parkeri is an obligate intracellular pathogen that belongs to spotted fever group rickettsia (SFGR). The SFG pathogens are characterized by their ability to infect and rapidly proliferate inside host vascular endothelial cells that eventually result in impairment of vascular endothelium barrier functions. Benidipine, a wide range dihydropyridine calcium channel blocker, is used to prevent and treat cardiovascular diseases. In this study, we tested whether benidipine has protective effects against rickettsia-induced microvascular endothelial cell barrier dysfunction in vitro. We utilized an in vitro vascular model consisting of transformed human brain microvascular endothelial cells (tHBMECs) and continuously monitored transendothelial electric resistance (TEER) across the cell monolayer. We found that during the late stages of infection when we observed TEER decrease and when there was a gradual increase of the cytoplasmic [Ca2+], benidipine prevented these rickettsia-induced effects. In contrast, nifedipine, another cardiovascular dihydropyridine channel blocker specific for L-type Ca2+ channels, did not prevent R. parkeri-induced drop of TEER. Additionally, neither drug was bactericidal. These data suggest that growth of R. parkeri inside endothelial cells is associated with impairment of endothelial cell monolayer integrity due to Ca2+ flooding through specific, benidipine-sensitive T- or N/Q-type Ca2+ channels but not through nifedipine-sensitive L-type Ca2+ channels. Further study will be required to discern the exact nature of the Ca2+ channels and Ca2+ transporting system(s) involved, any contributions of the pathogen toward this process, as well as the suitability of benidipine and new dihydropyridine derivatives as complimentary therapeutic drugs against Rickettsia-induced vascular failure.

Tetramethylpyrazine ameliorates acute lung injury by regulating the Rac1/LIMK1 signaling pathway.

Acute lung injury (ALI) is a respiratory disorder characterized by severe inflammation of the alveoli and lung parenchyma. Tetramethylpyrazine (TMP), the main active compound in Ligusticum chuanxiong Hort (LC), can protect against lipopolysaccharide (LPS)-induced ALI. Our study aimed to investigate how TMP protects the endothelial cell barrier in pulmonary capillaries. We administered TMP intraperitoneally at different doses and found that acute lung injury in mice was improved, but not in a dose-dependent manner. TMP toxicity was tested in vitro. We observed that LPS-induced cytoskeletal remodeling was inhibited by TMP. Murine ALI was induced as follows: For the 1st hit, LPS (2mg/kg) was injected intraperitoneally; after 16h, for the 2nd hit, LPS (4mg/kg) was instilled intratracheally. The mice in treatment groups had TMP or dexamethasone administered intraperitoneally 30min prior to the 1st hit and 30min past the 2nd hit. Mice were euthanized 24h after the last injecting. We measured protein and mRNA levels using enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase real-time PCR (RT-qPCR), respectively. The ultrastructural analysis was performed with transmission electron microscopy (TEM) and the cytoskeleton was observed by immunofluorescence. Immunohistochemistry and Western blotting were used to detect protein expression in the Rac1/LIMK1/ZO-1/occludin signal pathway. The results showed that TMP treatment decreased inflammatory cell infiltration and alleviated LPS-induced damage in lung tissue. Also, TMP significantly inhibited the Rac1/LIMK1/ZO-1/occludin signaling pathway. Our findings show that using TMP during sepsis can protect the pulmonary microvascular endothelial cell barrier and suppress inflammation. Therefore, TMP may have a promising therapeutic role in preventing acute lung injury from sepsis.

ALDH2 attenuates LPS-induced increase of brain microvascular endothelial cell permeability by promoting fusion and inhibiting fission of the mitochondria

To investigate the effect of aldehyde dehydrogenase 2 (ALDH2) on lipopolysaccharide (LPS)- induced damage of mouse brain microvascular endothelial barrier and explore the role of mitochondrial fusion and fission in maintaining the integrity of endothelial barrier. Mouse brain microvascular endothelial cells were treated with 1 μg/ mL LPS for 24 h with or without pretreatment with 20 μmol/mL Alda-1 (a ALDH2 agonist) for 1 h. The changes in cell viability were assessed using cell counting Kit-8 (CCK8) assay, and the cell permeability was evaluated using transendothelial cell resistance (TEER) and FITC-Dextran assay. The level of oxidative stress in the cells was assessed by detecting the levels of malondialdehyde (MDA) and superoxide dismutase (SOD), and the content of reactive oxygen species (ROS) was detected using a superoxide anion fluorescent probe (DHE). Western blotting was performed to detect the expressions of ALDH2, tight junction proteins ZO-1 and occludin, and mitochondrial fusion- and division-related proteins Mfn2, OPA1, Drp1 and Fis1. Compared with the untreated cells, the cells treated with LPS showed significantly decreased TEER, increased FITC-dextran leakage, MDA content and ROS production, decreased SOD activity expressions of ALDH2, ZO-1, occludin, Mfn2 and OPA1, and increased expressions of Drp1 and Fis1 (P < 0.05). Pretreatment with Alda-1 prior to LPS exposure strongly suppressed the increase of endothelial cell membrane permeability, reduced ROS production, increased the expressions of ALDH2, ZO-1, occludin, OPA1 and Mfn2, and lowered the expressions of Drp1 and Fis1 (P < 0.05). ALDH2 can alleviate LPS-induced damage of brain microvascular endothelial cell barrier by inhibiting the mitochondrial ROS production and promoting mitochondrial fusion and inhibiting mitochondrial fission.

LPS induces pulmonary microvascular endothelial cell barrier dysfunction by upregulating ceramide production

The specific role of ceramides in pulmonary microvascular endothelial cell (PMVEC) barrier dysfunction remains unclear. In the present study, pretreatment with pan-caspase inhibitors significantly reduced LPS-induced PMVEC apoptosis and helped to stimulate PMVEC barrier reconstruction after 12 h but had no effect on PMVEC barrier dysfunction in the first 8 h. Further studies showed that imipramine, an acid sphingomyelinase (ASMase) inhibitor, significantly inhibited LPS-induced barrier dysfunction, while an siRNA targeting serine palmityl transferase subunit 1 (SPTLC1) and the pharmacological inhibitor myriocin did not inhibit early acute barrier dysfunction but significantly inhibited PMVEC apoptosis and apoptosis-dependent delayed barrier dysfunction. In addition, LPS was shown to activate RhoA by inducing transient receptor potential channel 6 (TRPC6) overexpression and calcium influx through the ASMase/ceramide pathway, and activation of RhoA further induced the cytoskeletal rearrangement of PMVECs and destruction of intercellular junctions, ultimately leading to early acute PMVEC barrier dysfunction. However, regarding apoptosis-dependent delayed barrier dysfunction, the ceramide-induced de novo synthesis pathway in paracellular cells induced the apoptosis of PMVECs, in which Txnip overexpression inhibited Trx activity and subsequently activated ASK1 in the context of LPS-induced PMVEC apoptosis, acting upstream of the ceramide-induced activation of p38 MAPK and JNK. At the same time, in rats with LPS- or exogenous C8 ceramide-induced ALI, ceramide was demonstrated to play an important role in lung injury by inducing the Txnip/TRX/ASK1/P38 and JNK pathways. Thus, the Txnip/TRX/ASK1/p38 and JNK pathways might be involved in ceramide-mediated PMVEC apoptosis in LPS-induced ALI.

Roles of RAGE/ROCK1 Pathway in HMGB1-Induced Early Changes in Barrier Permeability of Human Pulmonary Microvascular Endothelial Cell.

BackgroundHigh mobility group box 1 (HMGB1) causes microvascular endothelial cell barrier dysfunction during acute lung injury (ALI) in sepsis, but the mechanisms have not been well understood. We studied the roles of RAGE and Rho kinase 1 (ROCK1) in HMGB1-induced human pulmonary endothelial barrier disruption.MethodsIn the present study, the recombinant human high mobility group box 1 (rhHMGB1) was used to stimulate human pulmonary microvascular endothelial cells (HPMECs). The endothelial cell (EC) barrier permeability was examined by detecting FITC-dextran flux. CCK-8 assay was used to detect cell viability under rhHMGB1 treatments. The expression of related molecules involved in RhoA/ROCK1 pathway, phosphorylation of myosin light chain (MLC), F-actin, VE-cadherin and ZO-1 of different treated groups were measured by pull-down assay, western blot and immunofluorescence. Furthermore, we studied the effects of Rho kinase inhibitor (Y-27632), ROCK1/2 siRNA, RAGE-specific blocker (FPS-ZM1) and RAGE siRNA on endothelial barrier properties to elucidate the related mechanisms.ResultsIn the present study, we demonstrated that rhHMGB1 induced EC barrier hyperpermeability in a dose-dependent and time-dependent manner by measuring FITC-dextran flux, a reflection of the loss of EC barrier integrity. Moreover, rhHMGB1 induced a dose-dependent and time-dependent increases in paracellular gap formation accompanied by the development of stress fiber rearrangement and disruption of VE-cadherin and ZO-1, a phenotypic change related to increased endothelial contractility and endothelial barrier permeability. Using inhibitors and siRNAs directed against RAGE and ROCK1/2, we systematically determined that RAGE mediated the rhHMGB1-induced stress fiber reorganization via RhoA/ROCK1 signaling activation and the subsequent MLC phosphorylation in ECs.ConclusionHMGB1 is capable of disrupting the endothelial barrier integrity. This study demonstrates that HMGB1 activates RhoA/ROCK1 pathway via RAGE, which phosphorylates MLC inducing stress fiber formation at short time, and HMGB1/RAGE reduces AJ/TJ expression at long term independently of RhoA/ROCK1 signaling pathway.

Prostaglandin D2 strengthens human endothelial barrier by activation of E-type receptor 4

Life-threatening inflammatory conditions such as acute respiratory distress syndrome or sepsis often go hand in hand with severe vascular leakage. During inflammation, endothelial cell integrity and intact barrier function are crucial to limit leukocyte and plasma extravasation. Prostaglandin D2 (PGD2) is a potent inflammatory lipid mediator with vasoactive properties. Previous studies suggest that PGD2 is involved in the regulation of endothelial barrier function; however, it is unclear whether this is also true for primary human pulmonary microvascular endothelial cells. Furthermore, as PGD2 is a highly promiscuous ligand, we set out to determine which receptors are important in human pulmonary endothelial cells. In the current study, we found that PGD2 and the DP1 agonist BW245c potently strengthened pulmonary and dermal microvascular endothelial cell barrier function and protected against thrombin-induced barrier disruption. Yet surprisingly, these effects were mediated only to a negligible extent via DP1 receptor activation. In contrast, we observed that the EP4 receptor was most important and mediated the barrier enhancement by PGD2 and BW245c. Stimulation with PGE2 or PGD2 reduced AKT phosphorylation which could be reversed by prior blockade of EP4 receptors. These data demonstrate a novel mechanism by which PGD2 may modulate inflammation and emphasizes the role of EP4 receptors in human endothelial cell function.

&lt;p&gt;The role of tissue inhibitors of metalloproteinases in microvascular endothelial cell barrier dysfunction during sepsis&lt;/p&gt;

&lt;p&gt;The role of tissue inhibitors of metalloproteinases in microvascular endothelial cell barrier dysfunction during sepsis&lt;/p&gt;

Differential Mechanisms of Septic Human Pulmonary Microvascular Endothelial Cell Barrier Dysfunction Depending on the Presence of Neutrophils.

Sepsis is characterized by injury of pulmonary microvascular endothelial cells (PMVEC) leading to barrier dysfunction. Multiple mechanisms promote septic PMVEC barrier dysfunction, including interaction with circulating leukocytes and PMVEC apoptotic death. Our previous work demonstrated a strong correlation between septic neutrophil (PMN)-dependent PMVEC apoptosis and pulmonary microvascular albumin leak in septic mice in vivo; however, this remains uncertain in human PMVEC. Thus, we hypothesize that human PMVEC apoptosis is required for loss of PMVEC barrier function under septic conditions in vitro. To assess this hypothesis, human PMVECs cultured alone or in coculture with PMN were stimulated with PBS or cytomix (equimolar interferon γ, tumor necrosis factor α, and interleukin 1β) in the absence or presence of a pan-caspase inhibitor, Q-VD, or specific caspase inhibitors. PMVEC barrier function was assessed by transendothelial electrical resistance (TEER), as well as fluoroisothiocyanate-labeled dextran and Evans blue-labeled albumin flux across PMVEC monolayers. PMVEC apoptosis was identified by (1) loss of cell membrane polarity (Annexin V), (2) caspase activation (FLICA), and (3) DNA fragmentation [terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)]. Septic stimulation of human PMVECs cultured alone resulted in loss of barrier function (decreased TEER and increased macromolecular flux) associated with increased apoptosis (increased Annexin V, FLICA, and TUNEL staining). In addition, treatment of septic PMVEC cultured alone with Q-VD decreased PMVEC apoptosis and prevented septic PMVEC barrier dysfunction. In septic PMN–PMVEC cocultures, there was greater trans-PMVEC macromolecular flux (both dextran and albumin) vs. PMVEC cultured alone. PMN presence also augmented septic PMVEC caspase activation (FLICA staining) vs. PMVEC cultured alone but did not affect septic PMVEC apoptosis. Importantly, pan-caspase inhibition (Q-VD treatment) completely attenuated septic PMN-dependent PMVEC barrier dysfunction. Moreover, inhibition of caspase 3, 8, or 9 in PMN–PMVEC cocultures also reduced septic PMVEC barrier dysfunction whereas inhibition of caspase 1 had no effect. Our data demonstrate that human PMVEC barrier dysfunction under septic conditions in vitro (cytomix stimulation) is clearly caspase-dependent, but the mechanism differs depending on the presence of PMN. In isolated PMVEC, apoptosis contributes to septic barrier dysfunction, whereas PMN presence enhances caspase-dependent septic PMVEC barrier dysfunction independently of PMVEC apoptosis.

Activation of A2b adenosine receptor decreases lipopolysaccharide-induced pulmonary microvascular permeability

To explore the role of the A2b adenosine receptor (Adora2b) in lipopolysaccharide (LPS)-induced injury of human pulmonary microvascular endothelial cells (HPMECs), and its mechanism. HPMECs were cultured in vitro. The LPS dose-effect experiment, time-effect experiment and the Adora2b agonist/antagonist intervention experiment were performed respectively. (1) Dose-effect and time-effect experiments: HPMECs were stimulated with 1, 10, 100, 1 000 μg/L LPS for 24 hours, or 100 μg/L LPS for 4, 8, 12, 16, 24 hours. Cell viability was measured by cell counting kit-8 (CCK8). The protein and mRNA expressions of Adora2b were determined by Western Blot and real-time reverse transcription-polymerase chain reaction (RT-PCR) respectively. (2) Adora2b agonist/antagonist intervention experiment: serum-starved HPMECs were pretreated with Adora2b specific agonist BAY60-6583 (0.1, 1, 10 μmol/L) or Adora2b specific antagonist PSB1115 (1 μmol/L) for 1 hour, respectively, and then incubated with 100 μg/L of LPS for 24 hours. The HPMECs without treatment were served as blank control group, and those treated with LPS, BAY60-6583 or PSB1115 alone were served as single challenge groups. The monolayer permeability of HPMECs was determined by fluorescein isothiocyanate (FITC)-dextran. Cell cycle was analyzed by flow cytometry. The mRNA expressions of VE-cadherin, occludin, vascular endothelial growth factor (VEGF) and angiopoietin-1 (ANGPT1) were determined by RT-PCR. (1) Dose-effect and time-effect experiments: LPS induced the decreased cell viability of HPMECs in dose and time-dependent manner. Compared with the control group, the protein expression of Adora2b was sharply up-regulated after 100 μg/L or 1 000 μg/L LPS stimulation. Meanwhile, LPS was shown to cause a dose and time-dependent induction of Adora2b transcript level. (2) Adora2b agonist/antagonist intervention experiments: compared with the control group, the monolayer permeability of HPMECs was rapidly enhanced after LPS treatment, and lower cell viability and proliferation, as well as the expression of cell junction and angiogenic factors were downregulated. Compared with LPS group, 0.1, 1, 10 μmol/L BAY 60-6583 pretreatment could decrease the endothelial cell barrier leakage in a dose-dependent manner [Pd: (203.06±15.24)%, (164.15±17.82)%, (125.69±10.38)% vs. (218.53±12.05)%], and promote cell proliferation of HPMECs [the proportion of S and G2 phases: (24.36±1.40)%, (32.37±0.95)%, (40.05±2.99)% vs. (18.83±0.73)%]. Pretreatment of 10 μmol/L BAY60-6583 also upregulated the mRNA expressions of cell junction and angiogenic factors [VE-cadherin (2-ΔΔCt): 2.17±0.23 vs. 0.56±0.10, occludin (2-ΔΔCt): 5.32±0.28 vs. 0.48±0.11, VEGF (2-ΔΔCt): 4.44±0.34 vs. 0.58±0.09, ANGPT-1 (2-ΔΔCt): 5.98±0.73 vs. 0.66±0.10, all P < 0.01]. PSB1115 pretreatment aggravated injury of microvascular endothelial cells after LPS incubation, with lower cell viability, slower proliferation and less expression of VEGF and ANGPT1. There was no influence of BAY 60-6583 or PSB1115 single treatment on cell viability, cell cycle and the expression of angiogenic factors in HPMECs. In vitro studies of cultured HPMECs exposed to LPS are identified as dose and time-dependent induction of Adora2b transcript and corresponding protein induction. Activation of Adora2b attenuates LPS-induced pulmonary microvascular endothelial cell barrier enhancement by regulating intercellular junction and promoting angiogenesis, suggesting Adora2b as potential therapeutic target in the treatment of LPS-induced forms of acute lung injury.

Abstract B41: Tumor endothelial cells as a targetable gateway that modulates access of drugs to cancer cells

Abstract Introduction: Based upon our demonstration that the human endothelial cell (EC) lining of the tumor vasculature of human prostate adenocarcinoma is preserved in primary xenografts of intact surgical remnant tissue, this study sought to determine the consequences of targeted perturbation of this key component of the tumor microenvironment on access of chemotherapeutic agents to prostate cancer cells (CaP). Methods: Primary xenografts of intact CaP tissue were employed to investigate multiple aspects of the response of the human ECs to iatrogenic modulation, including changes in gene expression, changes in cell-cell interaction, and in the function of human tissue/tumor microvascular EC-mediated, blood–tissue barrier. SCID mice were castrated and implanted with a device for sustained delivery of testosterone (T) to establish “humanized” mice. Freshly procured surgical specimens of human CaP or benign human prostate were transplanted onto the “humanized” mice within 3-7 days. Xenografts were allowed 30 days to engraft before the integrity of the human microvascular EC barrier was perturbed by removal of the T delivery device, T-deprivation (T-D). T-D induced apoptosis of the AR-expressing human ECs was maximal on Day 3, resulting in loss of 40-60% of human ECs. Significantly, the microvascular compartment re-endothelialized by Day 15 after T-D, in spite of the absence of T, providing a unique model for analysis of the role of the repaired human EC compartment in “acquired resistance.” After various times/treatments, xenografts were harvested, enzymatically disaggregated, and EC and CaP cells isolated using cell type-specific surface marker antibody-conjugated magnetic beads. Results: Enhanced access to the xenograft tissue of imaging markers in circulation during the interval of the compromised EC barrier, and after re-endothelialization, was demonstrated by: MRI (contrast dyes), MS (nanoparticles), immunohistochemistry (IHC) (lectins), and photo-acoustic imaging (hemoglobin). RNA-Seq of EC and epithelial/CaP populations characterized similarities/differences in gene expression profiles between cells recovered from fresh tissue versus established xenografts, and the effects of T-D in xenografts. Of ~2000 genes identified in the literature as AR-regulated/interacting, few were affected in either ECs or epithelial cells by T-D, with the notable exception of KLK2, KLK3, KLK4, MSMB, and ORM2. Expression profiles of membrane transporters and pumps for cellular uptake and efflux of chemotherapeutic agents were different between fresh tissue and established xenograft. Cisplatin (Cis-Pt) was investigated because it is excluded effectively by the membrane pumps/transporters in human prostate ECs, and it effectively kills slowly/nonproliferating cancer cells. IHC analysis using an antibody specific for Cis-Pt-induced DNA dimers demonstrated significantly greater levels of Pt-DNA adducts in xenografts treated during the T-D induced “window.” Quantitative analysis by Cytof technology of adduction in ECs and CaP cells isolated from xenografts is under way. Conclusions: Transient destruction of the EC barrier by T-D provides a therapeutically important “window” during which chemotherapeutic agents that are effectively excluded by the intact prostate EC barrier flood the CaP tissue microenvironment, providing therapeutically significant levels at reduced systemic dosages. Current analyses are focused on characterization of genes that respond to T-D, particularly differential characterization of gene expression profiles in the tumor ECs that re-endothelialize CaP xenografts after T-D. This model provides a unique tool for analysis of the roles of targeted microvascular damage in development of organ-specific therapies, and of the evolution of the EC compartment in response to iatrogenic intervention in “acquired resistance.” Citation Format: Michael J. Greene, Gary J. Smith, Yue Wu. Tumor endothelial cells as a targetable gateway that modulates access of drugs to cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B41.

Caspase‐Dependent Septic Pulmonary Microvascular Endothelial Cell Barrier Dysfunction is Associated with Vascular Endothelial‐Cadherin Disruption

BackgroundSepsis often causes dysfunction of pulmonary microvascular endothelial cells (PMVEC) leading to severe pulmonary edema. We previously reported in a murine model of sepsis that lung injury, including PMVEC barrier dysfunction, was strongly caspase‐dependent, and associated with PMVEC apoptosis. The mechanism through which caspases mediate PMVEC barrier function, however, was not determined. The classic role of caspases is the initiation and execution of apoptosis; however, caspases have important biological functions other than apoptosis. Indeed, caspases can cleave multiple targets, including proteins associated with cell‐cell junctions.ObjectiveTo identify the mechanisms through which caspases regulate septic PMVEC barrier dysfunction.HypothesisPMVEC barrier dysfunction in sepsis is mediated by caspase‐dependent disruption of inter‐PMVEC junctions.MethodsHuman PMVEC cultured alone or in co‐culture with neutrophils (PMN) were stimulated with cytomix (equimolar tumour necrosis factor α, interleukin 1β and interferon γ to mimic septic conditions). PMVEC barrier function was assessed by flux of Evans blue labelled‐albumin in a transwell assay. Specific microscopic localization of leak was determined by imaging paracellular fluorescein isothiocyanate labelled‐avidin leak across PMVEC monolayers in biotin‐coated wells. PMVEC apoptosis was examined using 3 markers: caspase activation (FLICA staining), loss of cell membrane polarity (Annexin V staining), and DNA fragmentation (terminal deoxynucleotidyl transferase dUTP nick end labelling [TUNEL] staining). PMVEC cell‐cell junctions were examined using immunofluorescence with antibodies against vascular endothelial (VE)‐cadherin.ResultsWe found that co‐culture of PMVEC with PMN significantly disrupts PMVEC barrier function under both basal and septic conditions vs. PMVEC cultured alone. Compared to cytomix‐treated PMVEC alone, the presence of PMN also significantly enhanced septic PMVEC caspase activation (FLICA staining), but this increase did not appear to be associated with any change in PMVEC apoptosis (Annexin V or TUNEL staining) vs. PMVEC cultured alone. We also found that microscopic paracellular leak co‐localized around cells positive for caspase activity, and was highly associated with disruption of circumferential PMVEC surface VE‐cadherin staining (97.1%). Moreover, PMVEC treatment with Q‐VD, a pan‐caspase inhibitor, rescued the septic barrier dysfunction, microscopic leak, and VE‐cadherin disruption.ConclusionOur data demonstrates that neutrophil presence enhances caspase‐dependent septic PMVEC barrier dysfunction independently of PMVEC apoptosis partly through disruption of VE‐cadherin.Support or Funding InformationThis work was supported by research funding from the Ontario Thoracic Society (S. Gill), the Program of Experimental Medicine (S. Gill and S. Mehta), and the Heart &amp; Stroke Foundation of Ontario/Canada grants #G‐16‐00014621 (S. Mehta and S. Gill).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hyperglycemia via activation of thromboxane A2 receptor impairs the integrity and function of blood-brain barrier in microvascular endothelial cells.

Diabetes is one of high risk factors for cardio- and cerebra-vascular diseases, including stroke, atherosclerosis and hypertension. This study was conducted to elucidate whether and how thromboxane receptor (TPr) activation contributes to blood-brain barrier (BBB) dysfunction in diabetes. Human brain microvascular endothelial cells (HBMECs) were cultured. The levels of phosphorylated endothelial nitric oxide synthase (eNOS) at Ser1177 (p-eNOS) and Akt at Ser473 (p-Akt) were assayed by western blot. Exposure of HBMECs to either high glucose (HG) or thromboxane A2 (TxA2) mimetic U46619, significantly reduced p-eNOS and p-Akt. These effects were abolished by pharmacological or genetic inhibitors of TPr. HG/U46619-induced suppressions of eNOS and Akt phosphorylation were accompanied by upregulation of PTEN and Ser380/Thr382/383 PTEN phosphorylation. PTEN-specific siRNA restored Akt-eNOS signaling in the face of TPr activation or HG. The small GTPase, Rho, was also activated by HG stimulation, and pretreatment of HBMECs with Y27632, a Rho-associated kinase (ROCK) inhibitor, rescued HG-impaired Akt-eNOS signaling. In STZ-injected rats, we found that hyperglycemia dramatically increased the levels of PTEN and PTEN-Ser380/Thr382/383 phosphorylation, reduced both levels of p-eNOS and p-Akt, and disrupted BBB function assayed by Evans blue staining, which were abolished by SQ29548 treatment. We conclude that hyperglycemia activates thromboxane A2 receptor to impair the integrity and function of blood-brain barrier via the ROCK-PTEN-Akt-eNOS pathway.

The Balance Between Metalloproteinases and TIMPs: Critical Regulator of Microvascular Endothelial Cell Function in Health and Disease.

Endothelial cells (EC), especially the microvascular EC (MVEC), have critical functions in health and disease. For example, healthy MVEC provide a barrier between the fluid and protein found within the blood, and the surrounding tissue. Following tissue injury or infection, the microvascular barrier is often disrupted due to activation and dysfunction of the MVEC. Multiple mechanisms promote MVEC activation and dysfunction, including stimulation by cytokines, mechanical interaction with activated leukocytes, and exposure to harmful leukocyte-derived molecules, which collectively result in a loss of MVEC barrier function. However, MVEC activation is also critical to facilitate recruitment of inflammatory cells, such as neutrophils (PMNs) and monocytes, into the injured or infected tissue. Metalloproteinases, including the matrix metalloproteinases (MMPs) and the closely related, a disintegrin and metalloproteinases (ADAMs), have been implicated in regulating both MVEC barrier function, through cleavage of adherens and tight junctions proteins between adjacent MVEC and through degradation of the extracellular matrix, as well as PMN-MVEC interaction, through shedding of cell surface PMN receptors. Moreover, the tissue inhibitors of metalloproteinases (TIMPs), which collectively inhibit most MMPs and ADAMs, are critical regulators of MVEC activation and dysfunction through their ability to inhibit metalloproteinases and thereby promote MVEC stability. However, TIMPs have been also found to modulate MVEC function through metalloproteinase-independent mechanisms, such as regulation of vascular endothelial growth factor signaling. This chapter is focused on examining the role of the metalloproteinases and TIMPs in regulation of MVEC function in both health and disease.

Cardiotoxic drugs Herceptin and doxorubicin inhibit cardiac microvascular endothelial cell barrier formation resulting in increased drug permeability.

ABSTRACTCardiotoxicity induced by anti-cancer therapeutics is a severe, and potentially fatal, adverse reaction of the heart in response to certain drugs. Current in vitro approaches to assess cardiotoxicity have focused on analysing cardiomyocytes. More recently it has become apparent that non-cardiomyocyte cells of the heart can potentially contribute to cardiotoxicity. Herceptin and doxorubicin are known to induce cardiotoxicity in the clinic. The effect of these drugs on the endothelial tight junction barrier was tested by analysing tight junction formation and zona occludens-1 (ZO-1) levels, revealing that Herceptin and doxorubicin are able to induce barrier perturbment and decrease barrier function in human cardiac microvascular endothelial cells (HCMECs) leading to increased permeability. Herceptin treatment had no effect on the tight junction barrier function in human dermal and human brain microvascular endothelial cells. HCMECs showed detectable levels of HER2 compared with the other endothelial cells suggesting that Herceptin binding to HER2 in these cells may interfere with tight junction formation. Our data suggests that doxorubicin and Herceptin can affect tight junction formation in the cardiac microvasculature leading to increased drug permeability and adverse effects on the cardiac myocytes.

Growth-factor reduced Matrigel source influences stem cell derived brain microvascular endothelial cell barrier properties.

BackgroundPatient-derived induced pluripotent stem cells (iPSCs) are an innovative source as an in vitro model for neurological diseases. Recent studies have demonstrated the differentiation of brain microvascular endothelial cells (BMECs) from various stem cell sources, including iPSC lines. However, the impact of the culturing conditions used to maintain such stem cell pluripotency on their ability to differentiate into BMECs remains undocumented. In this study, we investigated the effect of different sources of Matrigel and stem cell maintenance medium on BMEC differentiation efficiency.MethodsThe IMR90-c4 iPSC line was maintained on mTeSR1 or in essential-8 (E-8) medium on growth factor-reduced (GFR) Matrigel from three different manufacturers. Cells were differentiated into BMECs following published protocols. The phenotype of BMEC monolayers was assessed by immunocytochemistry. Barrier function was assessed by transendothelial electrical resistance (TEER) and permeability to sodium fluorescein, whereas the presence of drug efflux pumps was assessed by uptake assay using fluorescent substrates.ResultsStem cell maintenance medium had little effect on the yield and barrier phenotype of IMR90-derived BMECs. The source of GFR-Matrigel used for the differentiation process significantly impacted the ability of IMR90-derived BMECs to form tight monolayers, as measured by TEER and fluorescein permeability. However, the Matrigel source had minimal effect on BMEC phenotype and drug efflux pump activity.ConclusionThis study supports the ability to differentiate BMECs from iPSCs grown in mTeSR1 or E-8 medium and also suggests that the origin of GFR-Matrigel has a marked inpact on BMEC barrier properties.