Sigma-1 receptor activation-induced glycolytic ATP production and endothelial barrier enhancement.

OBJECTIVE: We tested the hypothesis that σ1 modulates endothelial barrier function due to its influence on endothelial bioenergetics. METHODS: Cultured HUVEC monolayers were used to model the endothelial barrier. ECIS, Transwell assays, and immunofluorescence labeling of junctional proteins were used to evaluate endothelial barrier function. Endothelial cell bioenergetics was determined using extracellular flux analysis and direct ATP level measurements. The endothelial-specific contribution of σ1 was tested using the σ1-selective agonist, PRE-084, and with targeted knockdown of σ1 expression using siRNA. RESULTS: Activation of σ1 with PRE-084 significantly enhanced endothelial barrier function and decreased permeability to albumin and dextran. Knockdown of σ1 with siRNA reduced barrier function and abolished PRE-084-induced endothelial barrier enhancement. PRE-084 upregulated endothelial glycolysis and glycolytic ATP production, but this response was abolished by siRNA-mediated knockdown of σ1 expression. PRE-084 also reduced the degree of endothelial barrier dysfunction caused by the mitochondrial oxidative phosphorylation uncoupler CCCP. CONCLUSION: Activation of σ1 enhances endothelial barrier function and modulates the ratio of glycolytic versus mitochondrial ATP production. These novel findings suggest that endothelial σ1 may prove beneficial as a novel therapeutic target for reducing microvascular hyperpermeability and counteracting mitochondrial dysfunction.

Endothelial Protrusions in Junctional Integrity and Barrier Function.

Endothelial cells of the microcirculation form a semi-permeable diffusion barrier between the blood and tissues. This permeability of the endothelium, particularly in the capillaries and postcapillary venules, is a normal physiological function needed for blood-tissue exchange in the microcirculation. During inflammation, microvascular permeability increases dramatically and can lead to tissue edema, which in turn can lead to dysfunction of tissues and organs. The molecular mechanisms that control the barrier function of endothelial cells have been under investigation for several decades and remain an important topic due to the potential for discovery of novel therapeutic strategies to reduce edema. This review highlights current knowledge of the cellular and molecular mechanisms that lead to endothelial hyperpermeability during inflammatory conditions associated with injury and disease. This includes a discussion of recent findings demonstrating temporal protrusions by endothelial cells that may contribute to intercellular junction integrity between endothelial cells and affect the diffusion distance for solutes via the paracellular pathway.

Imaging and Analysis of the Dynamics of Filamentous Actin Structures in Live Endothelial Cells.

The ability to view and record the movements of subcellular structures is a powerful tool that has accelerated the discovery and understanding of signaling mechanisms that control microvascular functions such as the control of endothelial permeability. Advances in molecular biology over the past few decades have facilitated the generation of fusion proteins in which fluorescent reporters based upon the structure of green fluorescent protein can be linked to proteins found in human endothelial cells, such as VE-cadherin or ß-actin. These fusion proteins have been found to incorporate into structures alongside their native protein counterparts, allowing the dynamic visualization of how these subcellular structures are modified when cells are challenged with stimuli such as inflammatory mediators. The result of such studies has been a much more advanced view of the complex mechanisms by which endothelial cells maintain barrier properties than previously obtained by only viewing fixed cells labeled by immunofluorescence. Here, we describe our protocols that we have used to view the dynamics of actin filaments using time-lapse microscopy to record endothelial cells expressing GFP-actin and the analysis tools available to quantify dynamics of subcellular structures.

Evaluation of Glycolysis and Mitochondrial Function in Endothelial Cells Using the Seahorse Analyzer.

Endothelial bioenergetics have emerged as a key regulator of endothelial barrier function. Glycolytic parameters have been linked to barrier enhancement, and interruption with mitochondrial complexes was shown to disrupt endothelial barrier. Therefore, a new technology that has been introduced to assess bioenergetics and metabolism has also made it possible to determine roles of specific energy production pathways in endothelial health. The Seahorse extracellular flux analysis by Agilent technologies is a state of the art tool that has been more frequently used to evaluate bioenergetics of endothelial cells. This chapter includes details about different assays that can be used to study endothelial cells using the Seahorse analyzer and how interpretation of the results can provide novel insight about endothelial metabolism.

Female and male obese Zucker rats display differential inflammatory mediator and long non-coding RNA profiles.

AIMS: The goal of this study was to identify mediators in peri-lymphatic adipose tissue (PLAT) that are altered in obese versus lean Zucker rats, with focus on potential sex differences MAIN METHODS: Mesenteric PLAT was analyzed with protein and lncRNA arrays. Additional RT-PCR confirmation was performed with epididymal/ovarian fat. KEY FINDINGS: MCP-1, TCK-1, Galectin-1, Galectin-3, and neuropilin-1 were elevated in PLAT from obese rats of both sexes. However, 11 additional proteins were elevated only in obese males while 24 different proteins were elevated in obese females. Profiling of lncRNAs revealed lean males have elevated levels of NEAT1, MALAT1 and GAS5 compared to lean females. NEAT1, MALAT1, and GAS5 were significantly reduced with obesity in males but not in females. Another lncRNA, HOTAIR, was higher in lean females compared to males, and its levels in females were reduced with obesity. Obese rats of both sexes had similar histologic findings of mesenteric macrophage crown-like structures and hepatocyte fat accumulation. SIGNIFICANCE: While obese male and female Zucker rats both have increased inflammation, they have distinct signals. Future studies of the proteome and lncRNA landscape of obese males vs. females in various animal models and in human subjects are warranted to better guide development of therapeutics for obesity-induced inflammation.

Involvement of Sigma Receptor-1 in Lymphatic Endothelial Barrier Integrity and Bioenergetic Regulation.

Background: Lymphatic endothelium plays significant roles in lymph transport and maintaining a barrier between the lymph and interstitial compartments. Lymphatic endothelial dysfunction is suspected to be a key factor in the pathogenesis of lymphatic diseases such as lymphedema. Sigma receptor-1 (σ1) was recently identified to promote endothelial-dependent production of nitric oxide and relaxation of collecting lymphatic vessels. In this study, we investigated the potential role of σ1 in lymphatic endothelial barrier function. Methods and Results: Cultured adult human dermal lymphatic endothelial cells (HDLEC) were grown into confluent monolayers. Transendothelial electrical resistance (TER) served as an index of barrier function. Glycolytic rate of HDLEC was determined with the Agilent Seahorse system. The σ1-selective agonist PRE-084 was used to test the impact of σ1 on HDLEC monolayer barrier function and endothelial bioenergetics, whereas the contribution of basal σ1 activity was assessed with small interfering RNA (siRNA)-mediated knockdown of σ1 expression. The ability of σ1 activation to counteract interleukin (IL)-1ß-induced barrier dysfunction was also tested. The results show that PRE-084 increases HDLEC TER in a concentration-dependent manner, whereas reducing σ1 expression with siRNA decreases HDLEC TER. PRE-084 also enhances glycolytic rate parameters in HDLEC. Moreover, PRE-084 treatment partially counteracts IL-1ß-induced HDLEC monolayer barrier dysfunction. Conclusions: Collectively, the results suggest that σ1 contributes to basal lymphatic endothelial barrier function, potentially through its ability to enhance glycolytic energy production. Our work also highlights the therapeutic potential of σ1 agonists for preventing lymphatic barrier dysfunction caused by inflammatory mediators.

PRE-084 as a tool to uncover potential therapeutic applications for selective sigma-1 receptor activation.

The discovery of a highly selective putative sigma-1 (σ1) receptor agonist, PRE-084, has revealed the numerous potential uses of this receptor subtype as a therapeutic target. While much work has been devoted to determining the role of σ1 receptors in normal and pathophysiological states in the nervous system, recent work suggests that σ1 receptors may be important for modulating functions of other tissues. These discoveries have provided novel insights into σ1 receptor structure, function, and importance in multiple intracellular signaling mechanisms. These discoveries were made possible by σ1 receptor-selective agonists such as PRE-084. The chemical properties and pharmacological actions of PRE-084 will be reviewed here, along with the expanding list of potential therapeutic applications for selective activation of σ1 receptors.

VE-Cadherin Is Required for Lymphatic Valve Formation and Maintenance.

The lymphatic vasculature requires intraluminal valves to maintain forward lymph flow. Lymphatic valves form and are constantly maintained by oscillatory fluid flow throughout life, yet the earliest steps of how lymphatic endothelial cells are able to respond to fluid shear stress remain unknown. Here, we show that the adherens junction protein VE-cadherin is required for the upregulation of valve-specific transcription factors. Conditional deletion of VE-cadherin in vivo prevented valve formation in the embryo and caused postnatal regression of nearly all lymphatic valves in multiple tissues. Since VE-cadherin is known to signal through ß-catenin and the VEGFR/AKT pathway, each pathway was probed. Expression of a constitutively active ß-catenin mutant or direct pharmacologic activation of AKT in vivo significantly rescued valve regression in the VE-cadherin-deficient lymphatic vessels. In conclusion, VE-cadherin-dependent signaling is required for lymphatic valve formation and maintenance and therapies to augment downstream pathways hold potential to treat lymphedema in patients.