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

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.

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

Circulation ◽

10.1161/circ.142.suppl_3.13667 ◽

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.

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Heterotypic inter-GPCR ß-arrestin coupling regulates lymphatic endothelial junctional architecture in murine lymph nodes

10.1101/435776 ◽

2018 ◽

Author(s):

Yu Hisano ◽

Mari Kono ◽

Eric Engelbrecht ◽

Koki Kawakami ◽

Keisuke Yanagida ◽

...

Keyword(s):

Endothelial Cells ◽

Lymph Nodes ◽

Cross Talk ◽

Transcriptional Activation ◽

Endothelial Permeability ◽

Lipid Mediator ◽

Protein Signaling ◽

A Genome ◽

Sphingosine 1 Phosphate ◽

A Site

AbstractLysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) activate G protein-coupled receptors (GPCRs) to regulate key pathobiological processes. Here we report a novel lipid mediator GPCR cross-talk mechanism that modulates lymphatic endothelial junctional architecture in lymph nodes. LPAR1 was identified as an inducer of S1PR1/ ß-arrestin coupling from a genome-wide CRISPR/ Cas9 transcriptional activation screen. LPAR1 activation induced S1PR1 ß-arrestin recruitment while suppressing Gαi protein signaling. Lymphatic endothelial cells from cortical and medullary sinuses of lymph nodes which express LPAR1 and S1PR1, exhibit porous junctional architecture and constitutive S1PR1 coupling to ß-arrestin which was suppressed by the LPAR1 antagonist AM095. In endothelial cells, LPAR1-activation increased trans-endothelial permeability and junctional remodeling from zipper-like structures to puncta of adhesion plaques that terminate at actin-rich stress fibers with abundant intercellular gaps. Cross-talk between LPA and S1P receptors regulates complex junctional architecture of lymphatic sinus endothelial cells, a site of high lymphocyte traffic and lymph flow.

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

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00173.2013 ◽

2013 ◽

Vol 305 (11) ◽

pp. L844-L855 ◽

Cited By ~ 47

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.

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Abstract 585: Endothelial Cells from Adipose Tissue of Obese Humans Display Mesenchymal Characteristics

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.585 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Bronson A Haynes ◽

Eric J Lehrer ◽

Giann J Bhatt ◽

Ryan W Huyck ◽

Ashley N James ◽

...

Keyword(s):

Endothelial Cells ◽

Adipose Tissue ◽

Prolonged Exposure ◽

Endothelial Permeability ◽

Fold Increase ◽

Atp Production ◽

Functional Changes ◽

Twist 1

The mechanisms underlying vascular dysfunction in adipose tissue (AT) in obesity are not clearly understood. Our hypothesis is that in response to pro-inflammatory cytokines (PIC) present in obese AT, endothelial cells (EC) can de-differentiate and acquire a mesenchymal-like phenotype (EndoMT) that leads to endothelial dysfunction. To test our hypothesis, we measured endothelial and mesenchymal markers of CD31 + CD34 + EC isolated from omental (OM) and subcutaneous (SC) AT of bariatric subjects (BAMVEC) using RT-PCR and western blot. Permeability and oxidative metabolism were determined by ECIS and Seahorse analyzer XF e 24, respectively. BAMVEC isolated from both OM and SC fat showed very low protein expression of vWF and VE-Cadherin (EC markers) and abundantly expressed αSMA and the EMT transcription factor twist-1. To determine effects of PIC on EndoMT, commercially available primary endothelial cells from AT (HAMVEC) were treated in vitro with PIC (2.5ng/mL TNFα, IFNγ and TGFβ) for 1, 3 or 6 days. We found progressive down-regulation by >2-fold (p<0.001) of the EC markers vWF, VE-Cadherin, and Occludin compared to controls. As early as 1 day of PIC treatment twist-1 (p<0.001) and snail1 (p<0.05) showed an increase by >2-fold. Similarly, OM and SC BAMVEC expressed >2-fold increase in the mesenchymal genes twist-1, FSP1, αSMA, and snail1 compared to untreated HAMVEC. Metabolically, BAMVEC had increased ATP production and maximal respiration compared to HAMVEC suggesting increased oxidative phosphorylation, a marker of mesenchymal-like cells. PIC stimulation of HAMVEC yielded significant increases in endothelial permeability and motility (p<0.001). Notably, there were no significant differences in any of the markers between OM and SC BAMVEC. These results show that EC in obese AT exhibit a mesenchymal-like phenotype which may account for functional changes such as increased permeability and migration and are not depot specific. Using primary EC from human AT we showed that prolonged exposure to PIC induces a phenotype similar to CD31+CD34+ EC from obese AT. This supports the concept that AT inflammation can promote EC de-differentiation in vivo and our in vitro model is suitable for future studies to uncover the relevant mechanisms.

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Abstract 23: Microrna302-367 Sphingosine 1 Phosphate Receptor 1 Pathway Prevents Tumor Growth via Restricting Angiogenesis and Enhancing Vascular Stability

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.23 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Jinjiang Pi ◽

Ting Tao ◽

Tao Zhuang ◽

Huimin Sun ◽

Xiaoli Chen ◽

...

Keyword(s):

Endothelial Cells ◽

Tumor Growth ◽

Ex Vivo ◽

Downstream Target ◽

Sprouting Angiogenesis ◽

Pathological Angiogenesis ◽

Vascular Stability ◽

Sphingosine 1 Phosphate

Angiogenic hypersprouting and leaky immature vessels of pathological angiogenesis are essential for tumor growth. MicroRNAs have unique therapeutic advantages by targeting multiple pathways of tumor-associated angiogenesis, but the function of individual miRNAs in angiogenesis and tumors has not yet been fully evaluated. Here, we show that miR302-367 elevation in endothelial cells reduces retina sprouting angiogenesis and promotes vascular stability in vivo, ex vivo and in vitro. Erk1/2 are identified as direct targets of miR302-367, and down-regulation of Erk1/2 upon miR302-367 elevation in endothelial cells increases the expression of Klf2 and in turn S1pr1 and its downstream target VE-cadherin, suppressing angiogenesis and improving vascular stability. Conversely, both pharmacological blockade and genetic deletion of S1pr1 in endothelial cells reverse the anti-angiogenic and vascular stabilizing effect of miR302-367 in mice. Pathological angiogenesis in tumors shares features of developmental angiogenesis, and endothelial specific elevation of miR302-367 reduces tumor growth by restricting sprout angiogenesis and decreasing vascular permeability via the same Erk1/2-Klf2-S1pr1 pathways. In conclusion, miR302-367 regulation of an Erk1/2-Klf2-S1pr1 pathway in the endothelium advances our understanding of angiogenesis, meanwhile also provides opportunities for therapeutic intervention of tumor growth.

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Differential regulation of diverse physiological responses to VEGF in pulmonary endothelial cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.2001.281.6.l1500 ◽

2001 ◽

Vol 281 (6) ◽

pp. L1500-L1511 ◽

Cited By ~ 77

Author(s):

Patrice M. Becker ◽

Alexander D. Verin ◽

Mary Ann Booth ◽

Feng Liu ◽

Anna Birukova ◽

...

Keyword(s):

Endothelial Cells ◽

Fetal Liver ◽

Physiological Responses ◽

Src Kinase ◽

Endothelial Permeability ◽

Barrier Dysfunction ◽

Pulmonary Endothelial Cells ◽

Mitogen Activated Protein ◽

Lung Endothelial Cells

The mechanisms responsible for the divergent physiological responses of endothelial cells to vascular endothelial growth factor (VEGF) are incompletely understood. We hypothesized that VEGF elicits increased endothelial permeability and cell migration via differential activation of intracellular signal transduction pathways. To test this hypothesis, we established a model of VEGF-induced endothelial barrier dysfunction and chemotaxis with bovine pulmonary endothelial cells. We compared the effects of VEGF on transendothelial electrical resistance (TER), actin cytoskeletal remodeling, and chemotaxis of lung endothelial cells and then evaluated the role of the mitogen-activated protein kinases (MAPKs) p38 and extracellular signal-regulated kinase (ERK)1/2 in VEGF-mediated endothelial responses. The dose response of pulmonary arterial and lung microvascular endothelial cells to VEGF differed when barrier regulation and chemotaxis were evaluated. Inhibition of tyrosine kinase, phosphoinositol 3-kinase, or p38 MAPK significantly attenuated VEGF-mediated TER, F-actin remodeling, and chemotaxis. VEGF-mediated decreased TER was also significantly attenuated by inhibition of ERK1/2 MAPK but not by inhibition of fetal liver kinase-1 (flk-1) or Src kinase. In contrast, VEGF-mediated endothelial migration was not attenuated by ERK1/2 inhibition but was abolished by inhibition of either flk-1 or Src kinase. These data suggest potential mechanisms by which VEGF may differentially mediate physiological responses in vivo.

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BMX Represses Thrombin-PAR1–Mediated Endothelial Permeability and Vascular Leakage During Early Sepsis

Circulation Research ◽

10.1161/circresaha.119.315769 ◽

2020 ◽

Vol 126 (4) ◽

pp. 471-485 ◽

Cited By ~ 5

Author(s):

Zhao Li ◽

Mingzhu Yin ◽

Haifeng Zhang ◽

Weiming Ni ◽

Richard W. Pierce ◽

...

Keyword(s):

Endothelial Cells ◽

Cecal Ligation ◽

Endothelial Permeability ◽

Vascular Leakage ◽

Lung Epithelial Cells ◽

Negative Regulator ◽

Cecal Ligation And Puncture ◽

Early Sepsis

Rationale: BMX (bone marrow kinase on the X chromosome) is highly expressed in the arterial endothelium from the embryonic stage to the adult stage in mice. It is also expressed in microvessels and the lymphatics in response to pathological stimuli. However, its role in endothelial permeability and sepsis remains unknown. Objective: We aimed to delineate the function of BMX in thrombin-mediated endothelial permeability and the vascular leakage that occurs with sepsis in cecal ligation and puncture models. Methods and Results: The cecal ligation and puncture model was applied to WT (wild type) and BMX-KO (BMX global knockout) mice to induce sepsis. Meanwhile, the electric cell-substrate impedance sensing assay was used to detect transendothelial electrical resistance in vitro and, the modified Miles assay was used to evaluate vascular leakage in vivo. We showed that BMX loss caused lung injury and inflammation in early cecal ligation and puncture–induced sepsis. Disruption of BMX increased thrombin-mediated permeability in mice and cultured endothelial cells by 2- to 3-fold. The expression of BMX in macrophages, neutrophils, platelets, and lung epithelial cells was undetectable compared with that in endothelial cells, indicating that endothelium dysfunction, rather than leukocyte and platelet dysfunction, was involved in vascular permeability and sepsis. Mechanistically, biochemical and cellular analyses demonstrated that BMX specifically repressed thrombin-PAR1 (protease-activated receptor-1) signaling in endothelial cells by directly phosphorylating PAR1 and promoting its internalization and deactivation. Importantly, pretreatment with the selective PAR1 antagonist SCH79797 rescued BMX loss-mediated endothelial permeability and pulmonary leakage in early cecal ligation and puncture–induced sepsis. Conclusions: Acting as a negative regulator of PAR1, BMX promotes PAR1 internalization and signal inactivation through PAR1 phosphorylation. Moreover, BMX-mediated PAR1 internalization attenuates endothelial permeability to protect vascular leakage during early sepsis.

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ANGPTL4 modulates vascular junction integrity by integrin signaling and disruption of intercellular VE-cadherin and claudin-5 clusters

Blood ◽

10.1182/blood-2011-01-328716 ◽

2011 ◽

Vol 118 (14) ◽

pp. 3990-4002 ◽

Cited By ~ 128

Author(s):

Royston-Luke Huang ◽

Ziqiang Teo ◽

Han Chung Chong ◽

Pengcheng Zhu ◽

Ming Jie Tan ◽

...

Keyword(s):

Endothelial Cells ◽

Integrin Signaling ◽

Wild Type ◽

Integrin Α5β1 ◽

Vascular Integrity ◽

Vascular Disruption ◽

Binding Partners ◽

Adhesive Proteins

Abstract Vascular disruption induced by interactions between tumor-secreted permeability factors and adhesive proteins on endothelial cells facilitates metastasis. The role of tumor-secreted C-terminal fibrinogen-like domain of angiopoietin-like 4 (cANGPTL4) in vascular leakiness and metastasis is controversial because of the lack of understanding of how cANGPTL4 modulates vascular integrity. Here, we show that cANGPTL4 instigated the disruption of endothelial continuity by directly interacting with 3 novel binding partners, integrin α5β1, VE-cadherin, and claudin-5, in a temporally sequential manner, thus facilitating metastasis. We showed that cANGPTL4 binds and activates integrin α5β1-mediated Rac1/PAK signaling to weaken cell–cell contacts. cANGPTL4 subsequently associated with and declustered VE-cadherin and claudin-5, leading to endothelial disruption. Interfering with the formation of these cANGPTL4 complexes delayed vascular disruption. In vivo vascular permeability and metastatic assays performed using ANGPTL4-knockout and wild-type mice injected with either control or ANGPTL4-knockdown tumors confirmed that cANGPTL4 induced vascular leakiness and facilitated lung metastasis in mice. Thus, our findings elucidate how cANGPTL4 induces endothelial disruption. Our findings have direct implications for targeting cANGPTL4 to treat cancer and other vascular pathologies.

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Efficient Barrier Protective Signaling by Activated Protein C Is Mechanistically Linked to Protein C Activation on Endothelial Cells.

Blood ◽

10.1182/blood.v106.11.28.28 ◽

2005 ◽

Vol 106 (11) ◽

pp. 28-28

Author(s):

Clemens Feistritzer ◽

Laurent O. Mosnier ◽

Enrico Di Cera ◽

John H. Griffin ◽

Matthias Riewald

Keyword(s):

Endothelial Cells ◽

Protein C ◽

Activated Protein C ◽

Endothelial Permeability ◽

Average Concentration ◽

Protective Effects ◽

Activation Pathway ◽

Protective Signaling ◽

Cell Medium

Abstract Protein C (PC) is activated by thrombomodulin-bound thrombin on the endothelial cell surface and activated protein C (APC) inhibits blood coagulation in a negative feedback loop. Endothelial PC receptor (EPCR) can bind PC/APC and activation of EPCR-bound PC is enhanced. Exogenous APC has barrier protective effects on endothelial cells that depend on EPCR binding and protease activated receptor-1 (PAR1) cleavage and that may contribute to the anti-inflammatory effects of APC. Plasma APC concentrations in vivo are low compared to the substrate PC and in order to induce protective signaling exogenous APC has to compete with PC for EPCR binding. In this study we investigated whether the endogenous PC activation pathway may be linked to efficient protective responses analyzing endothelial barrier permeability in a dual chamber system. When endothelial EA.hy926 cells were incubated for 3 h in the presence of 80 nM purified PC and different concentrations of thrombin a dose-dependent linear increase of APC activity in the cell medium was observed over time. APC generation was detectable upon incubation with 20 pM thrombin or higher and a significant barrier protective response to 20 pM thrombin was found only in the presence of PC. 40 pM thrombin enhanced barrier integrity in the presence and absence of PC, consistent with our previous results. To exclude direct thrombin effects on endothelial permeability and to compare protective effects of exogenous and endogenously generated APC, we used the anticoagulant double mutant thrombin W215A/E217A (WE). WE was about 10 times less active than wildtype thrombin for PC activation in our system. However, PAR1-dependent induction of MAP kinase phosphorylation required more than 1000-fold higher concentrations of the thrombin mutant. Thus, 1–10 nM WE leads to APC generation without directly inducing PAR1-dependent signaling. When cells were incubated with various concentrations of exogenous APC or WE+80 nM PC, barrier protective effects of 5 nM exogenous APC and 2 nM WE+80 nM PC (1.3 nM APC generated after 3 h) were similar. Because APC is generated at a constant rate during the incubation period, the average concentration of generated APC in the cell medium was only about 0.65 nM, suggesting that signaling by endogenously generated APC was significantly more efficient. To conclusively demonstrate that protective effects in response to WE are mediated by APC generation, we used recombinant zymogen wildtype PC and a PC variant with a substitution of the active site serine with alanine (PC S360A). Cells were incubated with control or 80 nM wildtype PC and PC S360A, in the presence or absence of WE (4 nM) and exogenous APC (3.3 nM). WE induced protective signaling only in the presence of wildtype PC but not PC S360A. Barrier protective effects of exogenous APC were blocked by both wildtype PC and PC S360A, consistent with their expected role as competitive inhibitors for APC binding to EPCR. These data demonstrate that efficient barrier enhancement by APC is indeed mechanistically coupled to the PC activation pathway. Signaling by endogenously generated APC may play an important role in the regulation of inflammation.

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