Abstract 13667: Programming to S1PR1 + Endothelial Cell Population Promotes the Restoration of Vascular Integrity in vivo

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Zahid Akhter ◽  
Jagdish Chandra Joshi ◽  
Vijay Avin Balaji Ragunathrao ◽  
Richard L Proia ◽  
Asrar B Malik ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Lung Injury ◽  
Endothelial Permeability ◽  
Vascular Diseases ◽  
Mechanistic Study ◽  
Rna Seq ◽  
Vascular Integrity ◽  
Injured Lung ◽  
Sphingosine 1 Phosphate

Introduction: Increased endothelial permeability and failure to repair is the hallmark of several vascular diseases including acute lung injury (ALI). However, little is known about the intrinsic pathways that activate the endothelial cell (EC) regenerative programs facilitating thereby tissue repair. Studies have invoked a crucial role of sphingosine-1-phosphate (S1P) in resolving endothelial hyperpermeability through activation of the G-protein coupled receptor, sphingosine-1-phosphate receptor 1 (S1PR1). Hypothesis: We postulate that S1PR1 + EC serve as an endogenous means to prevent endothelial injury. Methods: Studies were made using EC-S1PR1 null mice and S1PR1-GFP reporter mice to trace the generation and characteristics of S1PR1 + EC by exploiting immuno-histochemical analysis and FACS. RNA-seq analysis was performed to identify the genetic signature of S1PR1 + EC. Combination of genetic and pharmacological strategies were included for mechanistic study. Transplantation of S1PR1 + EC and edema measurement was performed in EC-S1PR1 null mice. Results: We observed in a mouse model of endotoxemia that LPS via generation of S1P induced the programming of S1PR1 lo EC to S1PR1 + EC, comprising 80% of lung EC. Their generation preceded the vascular repair phase and these cells were required for reestablishing the endothelial barrier function. Thus, conditional deletion of S1PR1 in EC spontaneously increased lung vascular permeability. RNA-seq analysis of S1PR1 + EC showed enrichment of genes regulating S1P synthesis and transport, sphingosine kinase 1 (SPHK1) and SPNS2, respectively, as well as transcription factors EGR1 and STAT3. EGR1 and STAT3 were essential for transcribing SPHK1 and SPNS2, respectively to increase S1P concentration that served to amplify S1PR1 + EC transition. Transplantation of S1PR1 + EC into injured lung vasculature of EC-S1PR1 -/- mice restored endothelial integrity. Conclusions: Findings illustrate that generation of a specialized S1PR1 + EC population has the potential to activate key endothelial regenerative program mediating vascular endothelial repair raising the possibility of activating this pathway to restore vascular homeostasis in inflammatory lung injury.

scholarly journals Programming to S1PR1 + Endothelial Cells Promote Restoration of Vascular Integrity

2021 ◽  
Author(s):  
Md Zahid Akhter ◽  
Jagdish Chandra Joshi ◽  
Vijay Avin Balaji Ragunathrao ◽  
Mark Maienschein-Cline ◽  
Richard L Proia ◽  
...  
Keyword(s):  
Endothelial Permeability ◽  
Vascular Diseases ◽  
Vascular Integrity ◽  
Conditional Deletion ◽  
Reporter Mice ◽  
Endothelial Repair ◽  
Activation Of Transcription ◽  
Injured Lung ◽  
Sphingosine 1 Phosphate ◽  
Endothelial Integrity

Rationale: Increased endothelial permeability and defective repair are the hallmarks of several vascular diseases including acute lung injury (ALI). However, little is known about the intrinsic pathways activating the endothelial cell (EC) regenerative programs. Objective: Studies have invoked a crucial role of sphingosine-1-phosphate (S1P) in resolving endothelial hyperpermeability through the activation of the G-protein coupled receptor, sphingosine-1-phosphate receptor 1 (S1PR1). Here we addressed mechanisms of generation of a population of S1PR1 + EC and their pivotal role in restoring endothelial integrity. Methods and Results: Studies were made using inducible EC-S1PR1 -/- (iEC-S1PR1 -/- ) mice and S1PR1-GFP reporter mice to trace the generation of S1PR1 + EC. We observed in a mouse model of endotoxemia that S1P generation induced the programming of S1PR1 lo to S1PR1 + EC, which eventually comprised 80% of the lung EC. The cell transition was required for reestablishing the endothelial junctional barrier. We observed that conditional deletion of S1PR1 in EC increased endothelial permeability. RNA-seq analysis of S1PR1 + EC showed enrichment of genes regulating S1P synthesis and transport, specifically sphingosine kinase 1 (SPHK1) and SPNS2. Activation of transcription factors EGR1 and STAT3 was required for transcribing SPHK1 and SPNS2, respectively and both served to increase S1P production and amplify S1PR1 + EC transition. Furthermore, transplantation of S1PR1 + EC population into injured lung vasculature restored endothelial integrity. Conclusions: Our findings show that generation of the S1PR1 + EC population activates the endothelial regenerative program to mediate endothelial repair. Results raise the possibility of harnessing this pathway to restore vascular homeostasis in inflammatory vascular injury states.

scholarly journals GRP78 is a novel receptor initiating a vascular barrier protective response to oxidized phospholipids

2014 ◽  
Vol 25 (13) ◽  
pp. 2006-2016 ◽  
Author(s):  
Anna A. Birukova ◽  
Patrick A. Singleton ◽  
Grzegorz Gawlak ◽  
Xinyong Tian ◽  
Tamara Mirzapoiazova ◽  
...  
Keyword(s):  
Acute Lung Injury ◽  
Endothelial Cell ◽  
Lung Injury ◽  
Ischemia Reperfusion ◽  
Cell Junctions ◽  
Endothelial Barrier ◽  
Oxidized Phospholipids ◽  
Vascular Integrity

Vascular integrity and the maintenance of blood vessel continuity are fundamental features of the circulatory system maintained through endothelial cell–cell junctions. Defects in the endothelial barrier become an initiating factor in several pathologies, including ischemia/reperfusion, tumor angiogenesis, pulmonary edema, sepsis, and acute lung injury. Better understanding of mechanisms stimulating endothelial barrier enhancement may provide novel therapeutic strategies. We previously reported that oxidized phospholipids (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine [OxPAPC]) promote endothelial cell (EC) barrier enhancement both in vitro and in vivo. This study examines the initiating mechanistic events triggered by OxPAPC to increase vascular integrity. Our data demonstrate that OxPAPC directly binds the cell membrane–localized chaperone protein, GRP78, associated with its cofactor, HTJ-1. OxPAPC binding to plasma membrane–localized GRP78 leads to GRP78 trafficking to caveolin-enriched microdomains (CEMs) on the cell surface and consequent activation of sphingosine 1-phosphate receptor 1, Src and Fyn tyrosine kinases, and Rac1 GTPase, processes essential for cytoskeletal reorganization and EC barrier enhancement. Using animal models of acute lung injury with vascular hyperpermeability, we observed that HTJ-1 knockdown blocked OxPAPC protection from interleukin-6 and ventilator-induced lung injury. Our data indicate for the first time an essential role of GRP78 and HTJ-1 in OxPAPC-mediated CEM dynamics and enhancement of vascular integrity.

Real-time differential labeling of blood, interstitium, and lymphatic and single-field analysis of vasculature dynamics in vivo

2012 ◽  
Vol 302 (10) ◽  
pp. C1460-C1468 ◽  
Author(s):  
Gor Sarkisyan ◽  
Stuart M. Cahalan ◽  
Pedro J. Gonzalez-Cabrera ◽  
Nora B. Leaf ◽  
Hugh Rosen
Keyword(s):  
Endothelial Cells ◽  
Real Time ◽  
Multiphoton Microscopy ◽  
Endothelial Permeability ◽  
Field Analysis ◽  
Lymphocyte Trafficking ◽  
Vascular Integrity ◽  
Sphingosine 1 Phosphate ◽  
Single Field

Lymph nodes are highly organized structures specialized for efficient regulation of adaptive immunity. The blood and lymphatic systems within a lymph node play essential roles by providing functionally distinct environments for lymphocyte entry and egress, respectively. Direct imaging and measurement of vascular microenvironments by intravital multiphoton microscopy provide anatomical and mechanistic insights into the essential events of lymphocyte trafficking. Lymphocytes, blood endothelial cells, and lymphatic endothelial cells express sphingosine 1-phosphate receptor 1, a key G protein-coupled receptor regulating cellular egress and a modulator of endothelial permeability. Here we report the development of a differential vascular labeling (DVL) technique in which a single intravenous injection of a fluorescent dextran, in combination with fluorescent semiconductor quantum dot particles, differentially labels multiple blood and lymphatic compartments in a manner dependent on the size of the fluorescent particle used. Thus DVL allows measurement of endothelial integrity in multiple vascular compartments and the affects or pharmacological manipulation in vascular integrity. In addition, this technique allows for real-time observation of lymphocyte trafficking across physiological barriers differentiated by DVL. Last, single-field fluid movement dynamics can be derived, allowing for the simultaneous determination of fluid flow rates in diverse blood and lymphatic compartments.

Abstract 73: Bone Morphogenetic Protein Activity Modulates Endothelial Barrier Function

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Thomas Helbing ◽  
Elena Ketterer ◽  
Bianca Engert ◽  
Jennifer Heinke ◽  
Sebastian Grundmann ◽  
...  
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Barrier Function ◽  
Endothelial Permeability ◽  
Bmp Signaling ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function

Introduction: Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome, are associated with high morbidity and mortality in patients. During the progression of ALI, the endothelial cell barrier of the pulmonary vasculature becomes compromised, leading to pulmonary edema, a characteristic feature of ALI. It is well-established that EC barrier dysfunction is initiated by cytoskeletal remodeling, which leads to disruption of cell-cell contacts and formation of paracellular gaps, allowing penetration of protein-rich fluid and inflammatory cells. Bone morphogenetic proteins (BMPs) are important players in endothelial dysfunction and inflammation but their effects on endothelial permeability in ALI have not been investigated until now. Methods and Results: As a first approach to assess the role of BMPs in acute lung injury we analysed BMP4 and BMPER expression in an infectious (LPS) and a non-infectious (bleomycin) mouse models of acute lung injury. In both models BMP4 and BMPER protein expression levels were reduced demonstrated by western blots, suggesting that BMPs are involved in progression ALI. To assess the role of BMPs on vascular leakage, a key feature of ALI, BMP activity in mice was inhibited by i.p. administration of LDN193189, a small molecule that blocks BMP signalling. After 3 days Evans blue dye (EVB) was administered i.v. and dye extravasation into the lungs was quantified as a marker for vascular leakage. Interestingly, LDN193189 significantly increased endothelial permeability compared to control lungs, indicating that BMP signaling is involved in maintenance of endothelial barrier function. To quantify effects of BMP inhibition on endothelial barrier function in vitro, HUVECs were seeded onto transwell filters and were exposed to LDN193189. After 3 days FITC-dextrane was added and passage into the lower chamber was quantified as a marker for endothelial barrier function. Thrombin served as a positive control. As expected from our in vivo experiments inhibition of BMP signaling by LDN193189 enhanced FITC-dextrane passage. To study specific effects of BMPs on endothelial barrier function, two protagonist of the BMP family, BMP2 and BMP4, or BMP modulator BMPER were tested in the transwell assay in vitro. Interestingly BMP4 and BMPER, but not BMP2, reduced FITC-dextrane passage demonstrating that BMP4 and BMPER improved endothelial barrier function. Vice versa, specific knock down of BMP4 or BMPER increased leakage in transwell assays. Im immuncytochemistry silencing of BMPER or BMP4 induced hyperpermeability as a consequence of a pro-inflammatory endothelial phenotype characterised by reduced cell-cell contacts and increased actin stress fiber formation. Additionally, the pro-inflammatory endothelial phenotype was confirmed by real-time revealing increased expression of adhesion molecules ICAM-1 or proinflammatory cytokines such as IL-6 and IL-8 in endothelial cells after BMPER or BMP4 knock down. Confirming these in vitro results BMPER +/- mice exhibit increased extravasation of EVB into the lungs, indicating that partial loss of BMPER impairs endothelial barrier function in vitro and in vivo. Conclusion: We identify BMPER and BMP4 as local regulators of vascular permeability. Both are protective for endothelial barrier function and may open new therapeutic avenues in the treatment of acute lung injury.

scholarly journals Metformin-stimulated AMPK-α1 promotes microvascular repair in acute lung injury

2013 ◽  
Vol 305 (11) ◽  
pp. L844-L855 ◽  
Author(s):  
Ming-Yuan Jian ◽  
Mikhail F. Alexeyev ◽  
Paul E. Wolkowicz ◽  
Jaroslaw W. Zmijewski ◽  
Judy R. Creighton
Keyword(s):  
Endothelial Cells ◽  
Acute Lung Injury ◽  
Lung Injury ◽  
Ex Vivo ◽  
Endothelial Permeability ◽  
Beneficial Effects ◽  
Isolated Lung

Acute lung injury secondary to sepsis is a leading cause of mortality in sepsis-related death. Present therapies are not effective in reversing endothelial cell dysfunction, which plays a key role in increased vascular permeability and compromised lung function. AMP-activated protein kinase (AMPK) is a molecular sensor important for detection and mediation of cellular adaptations to vascular disruptive stimuli. In this study, we sought to determine the role of AMPK in resolving increased endothelial permeability in the sepsis-injured lung. AMPK function was determined in vivo using a rat model of endotoxin-induced lung injury, ex vivo using the isolated lung, and in vitro using cultured rat pulmonary microvascular endothelial cells (PMVECs). AMPK stimulation using N1-(α-d-ribofuranosyl)-5-aminoimidizole-4-carboxamide or metformin decreased the LPS-induced increase in permeability, as determined by filtration coefficient ( Kf) measurements, and resolved edema as indicated by decreased wet-to-dry ratios. The role of AMPK in the endothelial response to LPS was determined by shRNA designed to decrease expression of the AMPK-α1 isoform in capillary endothelial cells. Permeability, wounding, and barrier resistance assays using PMVECs identified AMPK-α1 as the molecule responsible for the beneficial effects of AMPK in the lung. Our findings provide novel evidence for AMPK-α1 as a vascular repair mechanism important in the pulmonary response to sepsis and identify a role for metformin treatment in the management of capillary injury.

scholarly journals Substrate stiffness-dependent exacerbation of endothelial permeability and inflammation: mechanisms and potential implications in ALI and PH (2017 Grover Conference Series)

2018 ◽  
Vol 8 (2) ◽  
pp. 204589401877304 ◽  
Author(s):  
Pratap Karki ◽  
Anna A. Birukova
Keyword(s):  
Lung Injury ◽  
Inflammatory Responses ◽  
Mechanical Strain ◽  
Endothelial Permeability ◽  
Endothelial Barrier ◽  
In Vivo Studies ◽  
Potential Implication ◽  
Vascular Stiffening ◽  
Life Threatening

The maintenance of endothelial barrier integrity is absolutely essential to prevent the vascular leak associated with pneumonia, pulmonary edema resulting from inhalation of toxins, acute elevation to high altitude, traumatic and septic lung injury, acute lung injury (ALI), and its life-threatening complication, acute respiratory distress syndrome (ARDS). In addition to the long-known edemagenic and inflammatory agonists, emerging evidences suggest that factors of endothelial cell (EC) mechanical microenvironment such as blood flow, mechanical strain of the vessel, or extracellular matrix stiffness also play an essential role in the control of endothelial permeability and inflammation. Recent studies from our group and others have demonstrated that substrate stiffening causes endothelial barrier disruption and renders EC more susceptible to agonist-induced cytoskeletal rearrangement and inflammation. Further in vivo studies have provided direct evidence that proinflammatory stimuli increase lung microvascular stiffness which in turn exacerbates endothelial permeability and inflammation and perpetuates a vicious circle of lung inflammation. Accumulating evidence suggests a key role for RhoA GTPases signaling in stiffness-dependent mechanotransduction mechanisms defining EC permeability and inflammatory responses. Vascular stiffening is also known to be a key contributor to other cardiovascular diseases such as arterial pulmonary hypertension (PH), although the precise role of stiffness in the development and progression of PH remains to be elucidated. This review summarizes the current understanding of stiffness-dependent regulation of pulmonary EC permeability and inflammation, and discusses potential implication of pulmonary vascular stiffness alterations at macro- and microscale in development and modulation of ALI and PH.

Evidence for reprogramming of monocytes into reparative alveolar macrophages in vivo by targeting PDE4b

2021 ◽  
Author(s):  
Ian Rochford ◽  
Jagdish Chandra Joshi ◽  
Rayees Sheikh ◽  
Mumtaz Anwar ◽  
Md Zahid Akhter ◽  
...  
Keyword(s):  
Lung Injury ◽  
Alveolar Macrophages ◽  
Lung Edema ◽  
Rna Seq ◽  
Activation Of Transcription ◽  
The Impact ◽  
Camp Levels ◽  
Phosphodiesterase 4B ◽  
Lung Vascular Permeability

Increased lung vascular permeability and neutrophilic inflammation are hallmarks of acute lung injury. Alveolar macrophages (AMϕ), the predominant sentinel cell type in the airspace, die in massive numbers while fending off pathogens. Recent studies indicate that the AMϕ pool is replenished by airspace-recruited monocytes, but the mechanisms instructing the conversion of recruited monocytes into reparative AMϕ remain elusive. Cyclic AMP (cAMP) is a vascular barrier protective and immunosuppressive second messenger in the lung. Here, we subjected mice expressing GFP under the control of the Lysozyme-M promoter (LysM-GFP mice) to the LPS model of rapidly resolving lung injury to address the impact of mechanisms determining cAMP levels in AMϕ and regulation of mobilization of the reparative AMϕ-pool. RNA-seq analysis of flow-sorted Mϕ identified phosphodiesterase 4b (PDE4b) as the top LPS-responsive cAMP-regulating gene. We observed that PDE4b expression markedly increased at the time of peak injury (4 h) and then decreased to below the basal level during the resolution phase (24 h). Activation of transcription factor NFATc2 was required for transcription of PDE4b in Mϕ. Inhibition of PDE4 activity at the time of peak injury, using i.t. rolipram, increased cAMP levels, augmented the reparative AMϕ pool, and resolved lung injury. This response was not seen following conditional depletion of monocytes, thus establishing airspace-recruited PDE4b-sensitive monocytes as the source of reparative AMϕ. Interestingly, adoptive transfer of rolipram-educated AMϕ into injured mice resolved lung edema. We propose suppression of PDE4b as an effective approach to promote reparative AMϕ generation from monocytes for lung repair.

Immunotargeting of glucose oxidase to endothelium in vivo causes oxidative vascular injury in the lungs

2000 ◽  
Vol 278 (4) ◽  
pp. L794-L805 ◽  
Author(s):  
Melpo Christofidou-Solomidou ◽  
Giuseppe G. Pietra ◽  
Charalambos C. Solomides ◽  
Evgenia Arguiris ◽  
David Harshaw ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Lung Injury ◽  
Glucose Oxidase ◽  
Intravenous Injection ◽  
Vascular Injury ◽  
Target Cells ◽  
Pulmonary Endothelium ◽  
Endothelial Cell Surface ◽  
Novel Approach

Vascular immunotargeting is a novel approach for site-selective drug delivery to endothelium. To validate the strategy, we conjugated glucose oxidase (GOX) via streptavidin with antibodies to the endothelial cell surface antigen platelet endothelial cell adhesion molecule (PECAM). Previous work documented that 1) anti-PECAM-streptavidin carrier accumulates in the lungs after intravenous injection in animals and 2) anti-PECAM-GOX binds to, enters, and kills endothelium via intracellular H2O2 generation in cell culture. In the present work, we studied the targeting and effect of anti-PECAM-GOX in animals. Anti-PECAM-GOX, but not IgG-GOX, accumulated in the isolated rat lungs, produced H2O2, and caused endothelial injury manifested by a fourfold elevation of angiotensin-converting enzyme activity in the perfusate. In intact mice, anti-PECAM-GOX accumulated in the lungs (27 ± 9 vs. 2.4 ± 0.3% injected dose/g for IgG-GOX) and caused severe lung injury and 95% lethality within hours after intravenous injection. Endothelial disruption and blebbing, elevated lung wet-to-dry ratio, and interstitial and alveolar edema indicated that anti-PECAM-GOX damaged pulmonary endothelium. The vascular injury in the lungs was associated with positive immunostaining for iPF2α-III isoprostane, a marker for oxidative stress. In contrast, IgG-GOX caused a minor lung injury and little (5%) lethality. Anti-PECAM conjugated with inert proteins induced no death or lung injury. None of the conjugates caused major injury to other internal organs. These results indicate that an immunotargeting strategy can deliver an active enzyme to selected target cells in intact animals. Anti-PECAM-GOX provides a novel model of oxidative injury to the pulmonary endothelium in vivo.

Abstract 282: Endothelial-specific Deletion of Inhibitor of Kappa-B Kinase-β (IKK-β)

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Noboru Ashida ◽  
Sucharita SenBanerjee ◽  
Shohta Kodama ◽  
Joel A Spencer ◽  
Parisa Zamiri ◽  
...  
Keyword(s):  
Cell Survival ◽  
Endothelial Permeability ◽  
Vascular Diseases ◽  
Blue Dye ◽  
Physical Interaction ◽  
Serine Threonine Kinase ◽  
Akt Phosphorylation ◽  
Specific Deletion

The serine-threonine kinase, IKK-β, is required for classical activation of the NF-κB family of transcription factors which are central regulators of inflammation and cell survival that are activated in a variety of vascular diseases. [Methods and Results]: To examine the role of IKK-β and NF-κB in endothelial cells (EC), we generated mice with EC-specific deletion of IKK-β (IKK-β ΔEC ) on an ApoE −/− background using Tie2-Cre transgenic and IKK-β flox/flox mice. IKK-β ΔEC were born at less than the expected Mendelian frequency (7/48 vs 24/48 expected, p<0.0001) and were ~25% smaller than Cre-negative littermates (p<0.003). Vascular permeability assessed by in vivo microscopy of ear capillaries after intravenous injection of Evans blue dye was substantially increased (p<0.04). Several IKK-β ΔEC phenotypes were similar to Akt1 −/− mice, prompting us to examine Akt1 in the IKK-β ΔEC mice. Interestingly, phospho-Akt was markedly reduced in vascular endothelium from IKK-β ΔEC mice. In vitro studies of primary cultured EC demonstrated that deletion of IKK-β inhibited phosphorylation of Akt and increased permeability, while overexpression of either wild-type (WT) or kinase-deficient (KD) IKK-β enhanced Akt phosphorylation. Immunoprecipitation experiments demonstrated IGF-I-enhanced physical interaction of IKK-β with Akt. Moreover, IKK-β deletion inhibited Akt trafficking to lipid rafts rich in caveolae, an essential step in its activation. Expression of either WT or KD IKK-β in IKK-β-deleted EC in vitro restored normal Akt trafficking and reduced endothelial permeability. These results are in contrast to those reported with deletion of IKK-β in hepatocytes which have relatively few caveolae. [Conclusion] These data demonstrate that IKK-β modulates Akt subcellular trafficking and activation in EC through a kinase-independent mechanism. Understanding interactions between these signaling pathways may provide important insights in conditions marked by disregulated inflammation and/or cell survival including atherosclerosis and ischemic-injury.

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