Rat Lung Microvascular Endothelial Cells Research Articles

Species-specific responses during Seoul orthohantavirus infection in human and rat lung microvascular endothelial cells.

Seoul orthohantavirus (SEOV) is a rat-borne zoonotic virus that is transmitted via inhalation of aerosolized infectious excreta, and can cause hemorrhagic fever with renal syndrome (HFRS) in humans worldwide. In rats, SEOV predominantly exists as a persistent infection in the absence of overt clinical signs. Lack of disease in rats is attributed to downregulation of pro-inflammatory and upregulation of regulatory host responses. As lung microvascular endothelial cells (LMECs) represent a primary target of infection in both human and rats, infections in these cells provide a unique opportunity to study the central role of LMECs in the dichotomy between pathogenicity in both species. In this study, host responses to SEOV infection in primary human and rat LMECs were directly compared on a transcriptional level. As infection of rat LMECs was more efficient than human LMECs, the majority of anti-viral defense responses were observed earlier in rat LMECs. Most prominently, SEOV-induced processes in both species included responses to cytokine stimulus, negative regulation of innate immune responses, responses to type I and II interferons, regulation of pattern recognition receptor signaling and MHC-I signaling. However, over time, in the rat LMECs, responses shifted from an anti-viral state towards a more immunotolerant state displayed by a PD-L1, B2M-, JAK2-focused interaction network aiding in negative regulation of cytotoxic CD8-positive T cell activation. This suggests a novel mechanism by which species-specific orthohantavirus-induced endothelium and T cell crosstalk may play a crucial role in the development of acute disease in humans and persistence in rodents.

In vitro model of pulmonary candidiasis for testing novel therapeutics

Invasive candidiasis is one of the most common causes of hospital‐acquired blood infections, with the most common cause being Candida albicans. These bloodstream infections often lead to long hospital stays and have a high mortality rate. Once C. albicansenters the bloodstream it can disseminate and invade many organs of the body, including the lungs, where it can inhibit function and cause further complications. Furthermore, C. albicansis commonly isolated from the lungs of cystic fibrosis patients and often causes coinfection in patients with severe COVID‐19. Current antifungal drugs exhibit high toxicity, poor water solubility, and are ineffective against some strains of C. albicans due to antifungal resistance, creating an urgent need for new antifungal therapeutics. Here, we have developed a customizable in vitroplatform to study the interaction between rat lung microvascular endothelial cells (ECs), C. albicansSC5314, and novel liposomes encapsulating the antifungal drug anidulafungin. Liposome formulations composed of hydrogenated soy phosphatidylcholine, phosphatidylglycerol, cholesterol, and α‐tocopherol were fabricated containing three different % wt/wt concentrations of anidulafungin (0.31, 1.55, and 3.10, or 1x, 5x and 10x, respectively). The minimum inhibitory concentrations (MICs) of the different liposome formulations for C. albicans were determined using a microdilution assay. After 24 hours of exposure, the MICs were found to be 12.50, 6.25, and 1.56 μg/mL for 1x, 5x, and 10x liposome formulations, respectively. Cell Counting Kit 8 was used to determine EC viability after 24‐hour treatment with the different liposome formulations. The 1x and 5x anidulafungin liposome formulations showed no significant decrease in viability up to the maximum liposome concentration tested (50 μg/mL), with the 10x formulation causing a slight but significant decrease in viability. The 1x and 5x anidulafungin liposomes were then assessed at 6.25 and 12.5 μg/mL in a co‐culture setting. C. albicans were seeded onto established EC monolayers and were allowed to form hyphae, which is the invasive form. After the two hours, the co‐cultures were treated with the antifungal liposomes. Treatment with the liposomes reduced hyphae formation and prevented C. albicansovergrowth within four hours of exposure, with the 5x anidulafungin liposome at 12.5 μg/mL showing the strongest change. These data suggest that these novel antifungal liposomes have the potential to treat pulmonary candidiasis without the toxic side effects associated with current antifungal drugs.

Autophagy, TERT, and mitochondrial dysfunction in hyperoxia.

Ventilation with gases containing enhanced fractions of oxygen is the cornerstone of therapy for patients with hypoxia and acute respiratory distress syndrome. Yet, hyperoxia treatment increases free reactive oxygen species (ROS)-induced lung injury, which is reported to disrupt autophagy/mitophagy. Altered extranuclear activity of the catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), plays a protective role in ROS injury and autophagy in the systemic and coronary endothelium. We investigated interactions between autophagy/mitophagy and TERT that contribute to mitochondrial dysfunction and pulmonary injury in cultured rat lung microvascular endothelial cells (RLMVECs) exposed in vitro, and rat lungs exposed in vivo to hyperoxia for 48 h. Hyperoxia-induced mitochondrial damage in rat lungs [TOMM20, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)], which was paralleled by increased markers of inflammation [myeloperoxidase (MPO), IL-1β, TLR9], impaired autophagy signaling (Beclin-1, LC3B-II/1, and p62), and decreased the expression of TERT. Mitochondrial-specific autophagy (mitophagy) was not altered, as hyperoxia increased expression of Pink1 but not Parkin. Hyperoxia-induced mitochondrial damage (TOMM20) was more pronounced in rats that lack the catalytic subunit of TERT and resulted in a reduction in cellular proliferation rather than cell death in RLMVECs. Activation of TERT or autophagy individually offset mitochondrial damage (MTT). Combined activation/inhibition failed to alleviate hyperoxic-induced mitochondrial damage in vitro, whereas activation of autophagy in vivo decreased mitochondrial damage (MTT) in both wild type (WT) and rats lacking TERT. Functionally, activation of either TERT or autophagy preserved transendothelial membrane resistance. Altogether, these observations show that activation of autophagy/mitophagy and/or TERT mitigate loss of mitochondrial function and barrier integrity in hyperoxia.NEW & NOTEWORTHY In cultured pulmonary artery endothelial cells and in lungs exposed in vivo to hyperoxia, autophagy is activated, but clearance of autophagosomes is impaired in a manner that suggests cross talk between TERT and autophagy. Stimulation of autophagy prevents hyperoxia-induced decreases in mitochondrial metabolism and sustains monolayer resistance. Hyperoxia increases mitochondrial outer membrane (TOMM20) protein, decreases mitochondrial function, and reduces cellular proliferation without increasing cell death.

Mitochondrial Fission Mediated Cigarette Smoke-induced Pulmonary Endothelial Injury.

Cigarette smoke (CS) exposure increases the risk for acute respiratory distress syndrome in humans and promotes alveolar-capillary barrier permeability and acute lung injury in animal models. However, the underlying mechanisms are not well understood. Mitochondrial fusion and fission are essential for mitochondrial homeostasis in health and disease. In this study, we hypothesized that CS caused endothelial injury via an imbalance of mitochondrial fusion and fission and resultant mitochondrial oxidative stress and dysfunction. We noted that CS altered mitochondrial morphology by shortening mitochondrial networks and causing perinuclear accumulation of damaged mitochondria in primary rat lung microvascular endothelial cells. We also found that CS increased mitochondrial fission likely by decreasing Drp1-S637 and increasing FIS1, Drp1-S616 phosphorylation, mitochondrial translocation, and tetramerization and reduced mitochondrial fusion likely by decreasing Mfn2 in lung microvascular endothelial cells and mouse lungs. CS also caused aberrant mitophagy, increased mitochondrial oxidative stress, and reduced mitochondrial respiration. An inhibitor of mitochondrial fission and a mitochondria-specific antioxidant prevented CS-induced increased endothelial barrier dysfunction and apoptosis. Our data suggest that excessive mitochondrial fission and resultant oxidative stress are essential mediators of CS-induced endothelial injury and that inhibition of mitochondrial fission and mitochondria-specific antioxidants may be useful therapeutic strategies for CS-induced endothelial injury and associated pulmonary diseases.

Focal adhesion kinase and Src mediate microvascular hyperpermeability caused by fibrinogen- γC- terminal fragments.

ObjectivesWe previously reported microvascular leakage resulting from fibrinogen-γ chain C-terminal products (γC) occurred via a RhoA-dependent mechanism. The objective of this study was to further elucidate the signaling mechanism by which γC induces endothelial hyperpermeability. Since it is known that γC binds and activates endothelial αvβ3, a transmembrane integrin receptor involved in intracellular signaling mediated by the tyrosine kinases FAK and Src, we hypothesized that γC alters endothelial barrier function by activating the FAK-Src pathway leading to junction dissociation and RhoA driven cytoskeletal stress-fiber formation.Methods and resultsUsing intravital microscopy of rat mesenteric microvessels, we show increased extravasation of plasma protein (albumin) resulting from γC administration. In addition, capillary fluid filtration coefficient (Kfc) indicated γC-induced elevated lung vascular permeability. Furthermore, γC decreased transendothelial barrier resistance in a time-dependent and dose-related fashion in cultured rat lung microvascular endothelial cells (RLMVECs), accompanied by increased FAK/Src phosphorylation detection by western blot. Experiments with pharmacological inhibition or gene silencing of FAK showed significantly reduced γC-induced albumin and fluid leakage across microvessels, stress-fiber formation, VE-cadherin tyrosine phosphorylation, and improved γC-induced endothelial barrier dysfunction, indicating the involvement of FAK in γC mediated hyperpermeability. Comparable results were found when Src was targeted in a similar manner, however inhibition of FAK prevented Src activation, suggesting that FAK is upstream of Src in γC-mediated hyperpermeability. In addition, γC-induced cytoskeletal stress-fiber formation was attenuated during inhibition or silencing of these tyrosine kinases, concomitantly with RhoA inhibition.ConclusionThe FAK-Src pathway contributes to γC-induced microvascular barrier dysfunction, junction protein phosphorylation and disorganization in a manner that involves RhoA and stress-fiber formation.

Role of Mitochondrial Ca2+‐Activated Cl− Channel anoctamin‐1 and mitochondrial fusion protein OPA1 in pulmonary arterial hypertension

Activation of a Ca2+‐activated chloride channel, anoctamin‐1 (Ano1), is associated with enhanced cell proliferation in multiple cell types, such as endothelial cells (ECs). Recent proteomics studies indicate that Ano1 may interact with mitochondrial proteins in addition to plasma membrane (PM)‐localized proteins. However, the expression levels of Ano1 and its functions in other cellular compartments including mitochondria remain unclear. We previously showed that Ano1 is overexpressed in pulmonary artery ECs isolated from patients with idiopathic pulmonary arterial hypertension (PAH) and that Ano1 activation promotes apoptosis of pulmonary artery ECs from patients with idiopathic PAH, likely via depolarization of mitochondrial membrane potential and increased mitochondrial reactive oxygen species (ROS) generation. Therefore, we hypothesized that Ano1 exists not only at PM, but also in the mitochondria, which may contribute to a hyperproliferative phenotype of ECs under PAH. Here, we determined the mitochondrial expression of Ano1 and further investigated its role in the mitochondria. Subcellular localization of Ano1 was determined by immunostaining of Ano1 in Rat lung microvascular ECs (RLMVECs) and by live cell imaging of HEK293T cells expressing GFP‐tagged Ano1. The mitochondrial and PM localization of Ano1 was also confirmed by biochemical assays using protein fractionation from RLMVECs. Moreover, co‐immunoprecipitation assay showed that Ano1 interacts with a mitochondrial fusion protein OPA1 which is the critical regulator of mitochondrial bioenergetics and cristae formation and possesses a trans‐membrane domain at the inner mitochondrial membrane (IMM). These results indicate that 1) Ano1 expresses at the IMM in addition to the PM and 2) Ano1 and OPA1 interactions may be partly involved in the molecular mechanism underlying the hyperproliferation and apoptosis resistance of pulmonary artery ECs during PAH

Norepinephrine Induces Lung Microvascular Endothelial Cell Death by NADPH Oxidase-Dependent Activation of Caspase-3.

Norepinephrine (NE) is the naturally occurring adrenergic agonist that is released in response to hypotension, and it is routinely administered in clinical settings to treat moderate to severe hypotension that may occur during general anesthesia and shock states. Although NE has incontestable beneficial effects on blood pressure maintenance during hypotensive conditions, deleterious effects of NE on endothelial cell function may occur. In particular, the role of reactive oxygen species (ROS) and NADPH oxidase (Nox) on the deleterious effects of NE on endothelial cell function have not been fully elucidated. Therefore, we investigated the effects of NE on ROS production in rat lung microvascular endothelial cells (RLMEC) and its contribution to cell death. RLMEC were treated with NE (5 ng/mL) for 24 hours and ROS production was assessed by CellROX and DCFDA fluorescence. Nox activity was assessed by NADPH-stimulated ROS production in isolated membranes and phosphorylation of p47phox; cell death was assessed by flow cytometry and DNA fragmentation. Caspase activation was assessed by fluorescent microscopy. Nox1, Nox2, and Nox4 mRNA expression was assessed by real-time PCR. NE increased ROS production, Nox activity, p47phox phosphorylation, Nox2 and Nox4 mRNA content, caspase-3 activation, and RLMEC death. Phentolamine, an α1-adrenoreceptor antagonist, inhibited NE-induced ROS production and Nox activity and partly inhibited cell death while β-blockade had no effect. Apocynin and PEGSOD inhibited NE-induced caspase-3 activation and cell death while direct inhibition of caspase-3 abrogated NE-induced cell death. PEG-CAT inhibited NE-induced cell death but not caspase-3 activation. Collectively, these results indicate that NE induces RLMEC death via activation of Nox by α-adrenergic signaling and caspase-3-dependent pathways. NE has deleterious effects on RLMECs that may be important to its long-term therapeutic use.

Role of Mitochondrial Expression of the Calcium-Activated Chloride Channel Anoctamin-1 in Pulmonary Artery Endothelial Cells

Activation of a Ca2+-activated chloride channel, anoctamin-1 (Ano1), is associated with enhanced cell proliferation in multiple cell types, such as endothelial cells (ECs). Recent proteomics studies indicate that Ano1 may interact with mitochondrial proteins in addition to plasma membrane (PM)-localized proteins. However, the expression levels of Ano1 and its functions in other cellular compartments including mitochondria remain unclear. We previously showed that Ano1 is overexpressed in pulmonary artery ECs isolated from patients with idiopathic pulmonary arterial hypertension (PAH) and that Ano1 activation promotes apoptosis of pulmonary artery ECs from patients with idiopathic PAH, likely via depolarization of mitochondrial membrane potential and increased mitochondrial reactive oxygen species (ROS) generation. Therefore, we hypothesized that Ano1 exists not only at PM, but also in the mitochondria, which may contribute to a hyperproliferative phenotype of ECs under PAH. Here, we determined the mitochondrial expression of Ano1 and further investigated its role in the mitochondria. Subcellular localization of Ano1 was determined by immunostaining of Ano1 in Rat lung microvascular ECs (RLMVECs) and by live cell imaging of HEK293T cells expressing GFP-tagged Ano1. The mitochondrial and PM localization of Ano1 was also confirmed by biochemical assays using protein fractionation from RLMVECs. Moreover, co-immunoprecipitation assay showed that Ano1 interacts with a mitochondrial fusion protein OPA1 which is the critical regulator of mitochondrial bioenergetics and cristae formation and possesses a trans-membrane domain at the inner mitochondrial membrane (IMM). These results indicate that 1) Ano1 expresses at the IMM in addition to the PM and 2) Ano1 and OPA1 interactions may be partly involved in the molecular mechanism underlying the hyperproliferation and apoptosis resistance of pulmonary artery ECs during PAH.

Alteration of the extracellular matrix and alpha-gal antigens in the rat lung scaffold reseeded using human vascular and adipogenic stromal cells.

Regenerated organs are expected to solve the problem of donor organ shortage in transplantation medicine. One approach to lung regeneration is to decellularize the organ and reseed it with selected cells. An advantage of the procedure is reduced immunogenicity, because all cells can be theoretically replaced by autologous cells. However, little is known regarding the extracellular matrix (ECM) damage during decellularization and ECM reconstruction process in the organ regeneration. We aimed to evaluate ECM damage and reconstruction of the decellularized-recellularized rat lung, including the removal of alpha-gal xenoantigens. Rat lungs were perfused with sodium dodecyl sulfate and Triton X-100 via the pulmonary artery, after which the decellularized scaffold was reseeded with rat or human endothelial cells and adipose-derived stem cell (ASCs). The ECM and alpha-gal antigen were evaluated using immunohistochemistry, western blotting, and a glycosaminoglycan assay. Alcian blue staining revealed increased production of proteoglycan following the addition of ASCs to the rat lung recellularized with rat lung microvascular endothelial cells. Glycosaminoglycan levels decreased in the decellularized lung and increased in the recellularized lung, especially in the ASC-treated group. Immunohistochemical expression of the alpha-gal protein was decreased to an undetectable level in the decellularized lung tissue and disappeared after recellularization with human cells. In western blot analysis, the bands of alpha-gal protein almost disappeared after recellularization with human cells. In conclusion, characteristics of the regenerated ECM might depend on the species and type of cells used for recellularization. Therefore, alpha-gal antigen might be eliminated after a prolonged culture, when using human cells.

Activation of Anoctamin-1 Limits Pulmonary Endothelial Cell Proliferation via p38-Mitogen-activated Protein Kinase-Dependent Apoptosis.

Hyperproliferative endothelial cells (ECs) play an important role in the pathogenesis of pulmonary arterial hypertension (PAH). Anoctamin (Ano)-1, a calcium-activated chloride channel, can regulate cell proliferation and cell cycle in multiple cell types. However, the expression and function of Ano1 in the pulmonary endothelium is unknown. We examined whether Ano1 was expressed in pulmonary ECs and if altering Ano1 activity would affect EC survival. Expression and localization of Ano1 in rat lung microvascular ECs (RLMVECs) was assessed using immunoblot, immunofluorescence, and subcellular fractionation. Cell counts, flow cytometry, and caspase-3 activity were used to assess changes in cell number and apoptosis in response to the small molecule Ano1 activator, Eact. Changes in mitochondrial membrane potential and mitochondrial reactive oxygen species (mtROS) were assessed using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine, iodide (mitochondrial membrane potential dye) and mitochondrial ROS dye, respectively. Ano1 is expressed in RLMVECs and is enriched in the mitochondria. Activation of Ano1 with Eact reduced RLMVEC counts through increased apoptosis. Ano1 knockdown blocked the effects of Eact. Ano1 activation increased mtROS, reduced mitochondrial membrane potential, increased p38 phosphorylation, and induced release of apoptosis-inducing factor. mtROS inhibition attenuated Eact-mediated p38 phosphorylation. Pulmonary artery ECs isolated from patients with idiopathic PAH (IPAH) had higher expression of Ano1 and increased cell counts compared with control subjects. Eact treatment reduced cell counts in IPAH cells, which was associated with increased apoptosis. In summary, Ano1 is expressed in lung EC mitochondria. Activation of Ano1 promotes apoptosis of pulmonary ECs and human IPAH-pulmonary artery ECs, likely via increased mtROS and p38 phosphorylation, leading to apoptosis.

Efficient and Functional Endothelial Repopulation of Whole Lung Organ Scaffolds.

To date, efforts to generate engineered lung tissue capable of long-term function have been limited by incomplete barrier formation between air and blood and by thrombosis of the microvasculature upon exposure of blood to the collagens within the decellularized scaffold. Improved barrier function and resistance to thrombosis both depend upon the recapitulation of a confluent monolayer of functional endothelium throughout the pulmonary vasculature. This manuscript describes novel strategies to increase cell coverage of the vascular surface area, compared to previous reports in our lab and others, and reports robust production of multiple anticoagulant substances that will be key to long-term function in vivo once additional strides are made in improving barrier function. Rat lung microvascular endothelial cells were seeded into decellularized rat lungs by both the pulmonary artery and veins with the use of low-concentration cell suspensions, pulsatile, gravity-driven flow, and supraphysiological vascular pressures. Together, these strategies yielded 72.44 ± 10.52% endothelial cell nuclear coverage of the acellular matrix after 3-4 d of biomimetic bioreactor culture compared to that of the native rat lung. Immunofluorescence, Western blot, and PCR analysis of these lungs indicated robust expression of phenotypic markers such as CD31 and VE-Cadherin after time in culture. Endothelial-seeded lungs had CD31 gene expression of 0.074 ± 0.015 vs 0.021 ± 0.0023 for native lungs, p = 0.025, and VE-Cadherin gene expression of 0.93 ± 0.22 compared to that of the native lung at 0.13 ± 0.02, p = 0.023. Precursors to antithrombotic substances such as tissue plasminogen activator, prostacyclin synthase, and endothelial nitric oxide synthase were expressed at levels equal to or greater than those of the native lung. Engineered lungs reseeded with endothelial cells were implanted orthotopically and contained patent microvascular networks that had gas exchange function during mechanical ventilation on 100% O2 greater than that of decelluarized lungs. Taken together, these data suggest that these engineered constructs could be compatible with long-term function in vivo when utilized in future studies in tandem with improved barrier function.

Norepinephrine Induces Endothelial Albumin Uptake via ROS and Src‐Dependent Mechanisms

Hypertensive pulmonary edema often occurs acutely and is typically associated with normal left ventricular performance. Factors other than simple Starling forces may contribute to the molecular mechanisms by which increased blood pressure leads to acute endothelial barrier failure and lung edema.MethodsTo mimic an adrenergically‐mediated hypertensive crisis, we administered norepinephrine (NE) to rats and assessed indices of edema development. Rats were anesthetized with isoflurane and mechanically‐ventilated; subjects received an infusion of NE to target a MAP = 150mmHg. Lung wet/dry ratio, ΔPaO2, plasma hematocrit, lung MPO activity, BAL protein and cell analysis were assessed. Inhibition of nitric oxide synthase (NOS) by L‐NAME was used to test the contribution of endothelial mechanotransduction on changes in permeability. In parallel studies, rat lung microvascular endothelial cells were stimulated with norepinephrine in the presence or absence of propranolol (10−5M, β‐adrenoceptor antagonist), phentolamine (10−5M, α‐adrenoceptor antagonist), PP2 (10−5M, Src inhibitor), apocynin (3×10−5M, ROS scavenger), or L‐NIO (10−4M, NOS inhibitor). Reactive oxygen species (ROS) production was determined by DHE fluorescence. Albumin uptake was assessed by ELISA and albumin transport was measured by fluorimetry.ResultsNE‐treated rats had a decrease in PaO2, an increase in lung albumin content, MPO activity and wet/dry ratio. BAL fluid had increased protein content and number of macrophages indicative of inflammation. L‐NAME significantly attenuated NE‐induced pulmonary edema. In vitro, albumin uptake and transport was increased by NE in a time‐dependent manner. ROS production was augmented by NE. Phentolamine, PP2 and apocynin decreased NE‐induced albumin uptake and ROS generation, effects not altered by L‐NIO.SummaryWe describe a novel pathway whereby norepinephrine stimulates albumin uptake and potentially endothelial permeability which might be important in hypertensive pulmonary edema.Support or Funding InformationDepartment of Anesthesiology, University of Illinois at Chicago

Enhanced Re-Endothelialization of Decellularized Rat Lungs.

Decellularized lung tissue has been recognized as a potential platform to engineer whole lung organs suitable for transplantation or for modeling a variety of lung diseases. However, many technical hurdles remain before this potential may be fully realized. Inability to efficiently re-endothelialize the pulmonary vasculature with a functional endothelium appears to be the primary cause of failure of recellularized lung scaffolds in early transplant studies. Here, we present an optimized approach for enhanced re-endothelialization of decellularized rodent lung scaffolds with rat lung microvascular endothelial cells (ECs). This was achieved by adjusting the posture of the lung to a supine position during cell seeding through the pulmonary artery. The supine position allowed for significantly more homogeneous seeding and better cell retention in the apex regions of all lobes than the traditional upright position, especially in the right upper and left lobes. Additionally, the supine position allowed for greater cell retention within large diameter vessels (proximal 100-5000 μm) than the upright position, with little to no difference in the small diameter distal vessels. EC adhesion in the proximal regions of the pulmonary vasculature in the decellularized lung was dependent on the binding of EC integrins, specifically α1β1, α2β1, and α5β1 integrins to, respectively, collagen type-I, type-IV, and fibronectin in the residual extracellular matrix. Following in vitro maturation of the seeded constructs under perfusion culture, the seeded ECs spread along the vascular wall, leading to a partial reestablishment of endothelial barrier function as inferred from a custom-designed leakage assay. Our results suggest that attention to cellular distribution within the whole organ is of paramount importance for restoring proper vascular function.

Tissue inhibitor of metalloproteinase-2 inhibits burn-induced derangements and hyperpermeability in microvascular endothelial cells

BackgroundBurns induce microvascular hyperpermeability. We hypothesize that this occurs partly through an imbalance between matrix metalloproteinases (MMPs) and endogenous MMP inhibitors such as tissue inhibitors of metalloproteinases (TIMPs), and that such derangements can be attenuated with the use of TIMP-2. MethodRats underwent either sham or burn: serum and tissue were collected. Western blot was used to examine MMP-9 and TIMP-2 levels and MMP activity was assayed from lung tissue. Rat lung microvascular endothelial cells were used to assess monolayer permeability and evaluate the adherens junction proteins β-catenin, vascular endothelial cadherin and filamentous actin after exposure to burn serum ± TIMP-2. ResultsLung tissue from burn animals showed increased MMP activity, decreased levels of TIMP-2, and no difference in levels of active MMP-9 in burn vs control groups. Burn serum increased monolayer permeability, damaged adherens junction proteins, and incited actin stress fiber formation; TIMP-2 attenuated these derangements. ConclusionsBurns may lower TIMP-2 levels and increase MMP activity and that TIMP-2 application in vitro may attenuate burn-induced hyperpermeability and decreases damage to endothelial structural proteins. These links warrant further investigation.

Endothelial juxtaposition of distinct adult stem cells activates angiogenesis signaling molecules in endothelial cells.

Efficacy of therapeutic angiogenesis needs a comprehensive understanding of endothelial cell (EC) function and biological factors and cells that interplay with ECs. Stem cells are considered the key components of pro- and anti-angiogenic milieu in a wide variety of physiopathological states, and interactions of EC-stem cells have been the subject of controversy in recent years. In this study, the potential effects of three tissue-specific adult stem cells, namely rat marrow-derived mesenchymal stem cells (rBMSCs), rat adipose-derived stem cells (rADSCs) and rat muscle-derived satellite cells (rSCs), on the endothelial activation of key angiogenic signaling molecules, including VEGF, Ang-2, VEGFR-2, Tie-2, and Tie2-pho, were investigated. Human umbilical vein endothelial cells (HUVECs) and rat lung microvascular endothelial cells (RLMECs) were cocultured with the stem cells or incubated with the stem cell-derived conditioned media on Matrigel. Following HUVEC-stem cell coculture, CD31-positive ECs were flow sorted and subjected to western blotting to analyze potential changes in the expression of the pro-angiogenic signaling molecules. Elongation and co-alignment of the stem cells were seen along the EC tubes in the EC-stem cell cocultures on Matrigel, with cell-to-cell dye communication in the EC-rBMSC cocultures. Moreover, rBMSCs and rADSCs significantly improved endothelial tubulogenesis in both juxtacrine and paracrine manners. These two latter stem cells dynamically up-regulated VEGF, Ang-2, VREGR-2, and Tie-2 but down-regulated Tie2-pho and the Tie2-pho/Tie-2 ratio in HUVECs. Induction of pro-angiogenic signaling in ECs by marrow- and adipose-derived MSCs further indicates the significance of stem cell milieu in angiogenesis dynamics.

Propofol inhibits burn injury-induced hyperpermeability through an apoptotic signal pathway in microvascular endothelial cells.

Recent studies have revealed that an intrinsic apoptotic signaling cascade is involved in vascular hyperpermeability and endothelial barrier dysfunction. Propofol (2,6-diisopropylphenol) has also been reported to inhibit apoptotic signaling by regulating mitochondrial permeability transition pore (mPTP) opening and caspase-3 activation. Here, we investigated whether propofol could alleviate burn serum-induced endothelial hyperpermeability through the inhibition of the intrinsic apoptotic signaling cascade. Rat lung microvascular endothelial cells (RLMVECs) were pretreated with propofol at various concentrations, followed by stimulation with burn serum, obtained from burn-injury rats. Monolayer permeability was determined by transendothelial electrical resistance. Mitochondrial release of cytochrome C was measured by ELISA. Bax and Bcl-2 expression and mitochondrial release of second mitochondrial-derived activator of caspases (smac) were detected by Western blotting. Caspase-3 activity was assessed by fluorometric assay; mitochondrial membrane potential (Δψm) was determined with JC-1 (a potential-sensitive fluorescent dye). Intracellular ATP content was assayed using a commercial kit, and reactive oxygen species (ROS) were measured by dichlorodihydrofluorescein diacetate (DCFH-DA). Burn serum significantly increased monolayer permeability (P<0.05), and this effect could be inhibited by propofol (P<0.05). Compared with a sham treatment group, intrinsic apoptotic signaling activation - indicated by Bax overexpression, Bcl-2 downregulation, Δψm reduction, decreased intracellular ATP level, increased cytosolic cytochrome C and smac, and caspase-3 activation - was observed in the vehicle group. Propofol not only attenuated these alterations (P<0.05 for all), but also significantly decreased burn-induced ROS production (P<0.05). Propofol attenuated burn-induced RLMVEC monolayer hyperpermeability by regulating the intrinsic apoptotic signaling pathway.

Melatonin inhibits thermal injury-induced hyperpermeability in microvascular endothelial cells.

Burns induce systemic inflammatory reactions and vascular hyperpermeability. Breakdown of endothelial cell adherens junctions is integral in this process, and reactive oxygen species (ROS) and proteolytic enzymes such as matrix metalloproteinase-9 (MMP-9) play pivotal roles therein. Outside trauma, melatonin has shown to exhibit anti-MMP activity and to be a powerful antioxidant. Consequently, we hypothesized that burn-induced junctional damage and hyperpermeability could be attenuated with melatonin. Sprague-Dawley rats were assigned to sham or burn groups. Fluorescein isothiocyanate-bovine albumin was administered intravenously. Venules were examined with intravital microscopy; fluorescence intensities were measured intravascularly and extravascularly. Serum was collected. Rat lung microvascular endothelial cells were grown as monolayers and divided into four groups: sham serum and burn serum with and without melatonin pretreatment. Fluorescein isothiocyanate-bovine albumin flux was measured. Immunofluorescence for adherens junction proteins and staining for actin were performed, and images were captured. Cells were grown on 96 well plates, and ROS species generation following application of burn and sham serum was analyzed with and without melatonin. Statistical analysis was conducted with the Student's t test. Intravital microscopy data revealed an increase in vascular hyperpermeability following burn (p < 0.05). Monolayer permeability was increased with burn serum (p < 0.05); this was attenuated with melatonin (p < 0.05). Immunofluorescence showed damage of rat lung microvascular endothelial cell adherens junctions with burn serum exposure, and melatonin restored integrity. Rhodamine phalloidin staining showed filamentous actin stress fiber formation after burn serum application, and melatonin decreased this. Burn serum significantly increased ROS species generation (p < 0.05), and melatonin negated this (p < 0.05). Burns damage endothelial adherens junctions and induce microvascular hyperpermeability; melatonin attenuates this process. This insight into the mechanisms of burn-induced fluid leak suggests the role of ROS and MMP-9 but more importantly hints at the possibility of new treatments to combat vascular hyperpermeability in burns.

Tumor necrosis factor-α-induced microvascular endothelial cell hyperpermeability: role of intrinsic apoptotic signaling.

Tumor necrosis factor-α (TNF-α), a pro-apoptotic cytokine, is involved in vascular hyperpermeability, tissue edema, and inflammation. We hypothesized that TNF-α induces microvascular hyperpermeability through the mitochondria-mediated intrinsic apoptotic signaling pathway. Rat lung microvascular endothelial cells grown on Transwell inserts, chamber slides, or dishes were treated with recombinant TNF-α (10 ng/ml) in the presence or absence of a caspase-3 inhibitor, Z-DEVD-FMK (100 μM). Fluorescein isothiocyanate (FITC)-albumin (5 mg/ml) was used as a marker of monolayer permeability. Mitochondrial reactive oxygen species (ROS) was determined using dihydrorhodamine 123 and mitochondrial transmembrane potential using JC-1. The adherens junction integrity and actin cytoskeletal organization were studied using β-catenin immunofluorescence and rhodamine phalloidin, respectively. Caspase-3 activity was measured fluorometrically. The pretreatment with Z-DEVD-FMK (100 μM) attenuated TNF-α-induced (10 ng/ml) disruption of the adherens junctions, actin stress fiber formation, increased caspase-3 activity, and monolayer hyperpermeability (p < 0.05). TNF-α (10 ng/ml) treatment resulted in increased mitochondrial ROS formation and decreased mitochondrial transmembrane potential. Intrinsic apoptotic signaling-mediated caspase-3 activation plays an important role in regulating TNF-α-induced endothelial cell hyperpermeability.

2D-Visualisierung des zellulären Sauerstoff verbrauchs in Mikrofluidiksystemen

A new imaging system allows visualizing oxygen distributions in 2D. Combining sensor foils with microfluidic devices enables online monitoring of cellular oxygen consumption in whole chip areas. Furthermore, suitable device materials depending on application and cell line can be determined. Numerically simulated oxygen consumption of rat lung microvascular endothelial cells and rat hepatocytes was experimentally validated.

Hypoxia Modulates the Cellular Signaling in Cultured Rat Lung Microvascular Endothelial Cells

Hypoxia Modulates the Cellular Signaling in Cultured Rat Lung Microvascular Endothelial Cells