Blog

By Zeinab Y. Motawe, Salma S. Abdelmaboud, and Jerome W. Breslin

Excerpt from the article published in Lymphatic Research and Biology.Jun 2021.231-239. http://doi.org/10.1089/lrb.2020.0060

Editor’s Highlights

• The lymphatic system has an important role in fluid homeostasis, lymph transport, lipid trafficking, and immunity.
• Lymphatic function starts in the vast networks of lymphatic capillaries (also termed initial lymphatic vessels), which are blind-ended vessels that absorb interstitial fluid and selectively allow entry of immune cells to form lymph.
• Lymphatic capillaries are composed of a single, nonfenestrated endothelial cell layer, which is well suited for the uptake of fluid, macromolecules, and cells.
• Lymphatic endothelial cells (LECs) compose a barrier between the lymph and interstitial compartments which is important for lymphatic homeostasis.
• Lymphatic barrier dysfunction is thought to contribute to the overall pathology of lymphatic diseases, such as lymphedema.
• The Sigma-1 receptor (σ1) is expressed in rat mesenteric collecting lymphatic vessels.
• σ1 is an important modulator of lymphatic endothelial barrier function. In addition, sigma-1 activation elicits a shift toward more glycolytic ATP production upon its activation.
• These findings suggest a novel role of σ1 in lymphatic endothelial bioenergetics and barrier function and expand the current known uses and applications of σ1 agonists.

Abstract

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)-1β-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-1β-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.

Introduction

The lymphatic system has an important role in fluid homeostasis, lymph transport, lipid trafficking, and immunity.¹Lymphatic dysfunction can result in insufficient clearance of interstitial fluid from tissues, resulting in lymphedema.^2,3Lymphatic function starts in the vast networks of lymphatic capillaries (also termed initial lymphatic vessels), which are blind-ended vessels that absorb interstitial fluid and selectively allow entry of immune cells to form lymph. The initially formed lymph is then propelled through downstream precollector vessels and collecting lymphatic vessels that carry lymph to lymph nodes and eventually back to the central circulation.^4,5

Lymphatic capillaries are composed of a single, nonfenestrated endothelial cell layer, which is well suited for the uptake of fluid, macromolecules, and cells. On the contrary, collecting lymphatic vessels include an endothelial layer, a smooth muscle layer, and periodic one-way valves, collectively to serve the purpose of pumping lymph fluid forward through the network. Lymphatic endothelial cells (LECs) share some common properties with the endothelium of blood vessels, and also differ at the ultrustructural level. For example, in lymphatic capillaries, the basement membrane is incomplete and there are no supporting pericytes.⁶ In addition, LECs express some markers that are seen in blood endothelial cells, such as CD31, CD34, and VEGFR-2, but also have expression markers not typically found on blood endothelial cells, including podoplanin, VEGFR-3, PROX-1, and LYVE-1.^7,8

LECs compose a barrier between the lymph and interstitial compartments which is important for lymphatic homeostasis.⁹Although the permeability and special configuration of endothelial junctions of the lymphatic capillary endothelium plays an important role in lymph formation,¹⁰ the permeability of collecting lymphatics is thought to be more restrictive, to prevent leakage of lymph components into the surrounding tissue. However, certain compounds such as atrial and brain natriuretic peptides can increase the permeability of collecting lymphatics to albumin, and inflammatory conditions during severe diabetes also can greatly enhance collecting lymphatic permeability.^11,12 Alcohol intoxication has also been reported to increase mesenteric collecting lymphatic permeability in rats, probably owing to local immune-metabolic dysregulation.¹³Moreover, when used as a model of the lymphatic endothelial barrier, cultured LEC monolayers also display decreased barrier function in response to stimuli such as tumor necrosis factor α, interleukin (IL)-6, IL-1β, interferon γ (IFN-γ) and LPS that in combination with reduction in the expression of VE-cadherin and an increase in scavenging of β-catenin.⁹ Other inflammatory mediators, such as histamine and thrombin, as well as the growth factor VEGF-C, have also been shown to transiently disrupt the barrier function of cultured lymphatic endothelial monolayers.^14,15 In contrast, LECs are exposed to increases in shear stress displayed a transient enhancement of lymphatic barrier function through a mechanism involving Rac1-indcued cytoskeletal changes.¹⁶ Collectively, these findings suggest that the lymphatic barrier can be modulated by changes in local inflammatory conditions and by mechanical stimuli.

Endothelial bioenergetics is an important factor in endothelial function, but is understudied in the lymphatic endothelium, which is exposed to a distinct metabolic environment. Lymph is rich in nutrients, although it is a heavily modified plasma filtrate that includes waste products from tissues.¹⁷ LECs are exposed to high concentrations of glucose (4–6 mM), protein (8–32 g/L), and triglycerides (8–40 mg/dL).¹⁸ In contrast, oxygen concentration in the lymph is relatively low, with partial pressures of oxygen (p_O2) at 15–42 mmHg, compared with 80–100 mmHg in the arterial blood.¹⁹ This environment determines the specific metabolic conditions under which the lymphatic endothelium can function.

It has been well documented that endothelial cells rely more on glycolysis for energy production, with 99% of glucose converted anaerobically to lactate and only 0.04% undergoing oxidative phosphorylation.²⁰ Similar to blood endothelium, LECs have also been reported to take up massive amounts of glucose compared with other cell types and to use glycolysis as the primary source of ATP (∼85%) with mitochondrial glucose oxidation almost completely inactive.²¹ This preference for glycolysis can enable ATP generation in peripheral regions of the cell, in very close proximity to cell locomotion sites such as lamellipodia and filopodia, facilitating coupling ATP production and usage, and sparing the need to transfer ATP from more central-located mitochondria.^22,23

Recently, sigma receptor-1 (σ1) was found to be expressed in lymphatic vessels, and its activation by the σ1-agonist afobazole was shown to cause lymphatic endothelial production of NO and relaxation of isolated rat lymphatic vessels.²⁴ σ1 is a chaperone protein associated with the endoplasmic reticulum-mitochondrial-associated membrane (ER-MAM) that has been shown to be neuroprotective and cardioprotective in various disease models.^25,26 However, little is known about other potential functions that σ1 has in the lymphatic endothelium. We recently demonstrated that σ1 receptor activation enhances barrier function of human umbilical vein endothelial cell monolayers, promotes greater glycolytic activity, and counteracts mitochondrial dysfunction-induced transendothelial electrical resistance (TER) drop in these cells.²⁷ In this study, we report new findings from our study in which we investigated the contribution of σ1 to lymphatic endothelial barrier function. We hypothesized that σ1 promotes lymphatic endothelial barrier integrity under normal and inflammatory conditions. In addition, considering the localization of σ1 at the ER-MAM, we investigated how σ1 affects endothelial bioenergetics, hypothesizing that σ1 activation promotes glycolysis, which has been previously documented to occur in close proximity to endothelial junctions.^21,28

…

Results

PRE-084 enhances TER of HDLEC monolayers

To assess the impact of σ1 activation on lymphatic endothelial barrier function, confluent HDLEC monolayers were treated with the selective σ1 agonist PRE-084 at concentrations ranging from 1 to 200 μM (Fig. 1A). Selection of this wide range of concentrations was based upon what was used in previous literature.³² Several of the concentrations of PRE-084 tested caused an apparent enhancement of TER compared with the control group, that plateaued at ∼8 hours after the its addition, and was largely sustained until the end of the experiment at 18 hours (Fig. 1A). The response was concentration dependent, with concentrations of 50 μM and higher eliciting a significant increase in HDLEC monolayer TER compared with control at 8 hours (Fig. 1B). We also compared the response at 18 hours and found significant elevations in TER in response to concentrations of 100 μM and higher of PRE-084 showing significant elevations (Fig. 1C). Based on these findings, we utilized the 100 μM concentration of PRE-084 subsequent experiments.

σ1 is important for maintenance of baseline lymphatic endothelial TER

We next investigated whether σ1 is required for normal baseline TER of HDLEC monolayers. We used transfection of SIGMAR1-selective siRNA to knock down protein expression of σ1, with transfection of nontargeting RNA serving as control. The transfected cell suspension was inoculated onto the ECIS electrodes and recording of TER started immediately after this step, and quickly rose as the cells initially attached to the substratum and began to spread out and form connections (Fig. 2A). A drop in the mean TER occurred when the media was changed at 2 hours to remove any remaining transfection solution, and reflects the response of the cells to the brief removal of media followed by recovery and adaptation to the fresh media. TER continuing to rise after this media change (2–12 hours) reflects further attachment and maturation of the junctions. A steady-state TER was observed from ∼12–24 hours reflecting that the cells had reached confluency. At 24 hours, media was changed again to replenish nutrients, and again a drop and recovery of the TER occurred, reflecting the response of the cells to the brief removal and then restoration of fluid. This pattern is consistent with previously published patterns for endothelial attachment on ECIS electrodes.^33,34 The two traces split starting ∼40 hours with the nontransfected control group’s TER kept increasing reflecting junctional maturation but the σ1 knockdown group failed to show the same increases. We observed that the TER decreased in cells treated with the SIGMAR1 siRNA, starting at ∼48 hours after the transfection, with an ongoing drop in TER until the end of the experiment at 70 hours (Fig. 2A). Comparison of the difference in TER between the two groups at the 70-hour time point showed it was significant, with the σ1 knockdown group displaying an almost 50% decrease in barrier function in comparison with the nontargeting RNA-transfected cells (Fig. 2B). We also verified that the SIGMAR1 siRNA diminished σ1 protein level expression by western blotting at 70 hours post-transfection (Fig. 2C). Of note, knockdown of σ1 did not affect the expression of σ2 receptor protein, and the lymphatic endothelial marker PROX-1 also appeared unchanged compared with the control (Fig. 2C)

PRE-084 enhances glycolysis and decreases OCR in HDLEC

Owing to the location of σ1 receptor at the ER-mitochondrial associated membrane,³⁵ and the previously reported link between σ1 and bioenergetics in other models,^26,27 we examined whether PRE-084 may affect bioenergetics in HDLEC monolayers. Because glycolysis is considered the predominant energy production pathway in endothelial cells,^20,21 we conducted a glycolytic rate assay on cells pretreated with PRE-084 for 18 hours (Fig. 3). Cells that were pretreated with PRE-084 showed an apparent elevation of glycolytic proton efflux rate (glycoPER) during the time course of the assay (Fig. 3B). We observed a significant enhancement of basal glycolysis (Fig. 3C), compensatory glycolysis (Fig. 3D), basal PER (Fig. 3E), and % PER from glycolysis (Fig. 3F) in cells treated with PRE-084. Of interest, PRE-084 also showed an apparent decrease in OCR when compared with control cells (Fig. 4A), and a significantly lower mitoOCR/glycoPER ratio (Fig. 4B). These findings suggest that σ1 activation can shift LEC energy production, enhancing a greater degree of glycolysis (Fig. 4B).

PRE-084 attenuated IL-1β-induced TER drop in HDLEC

We next wanted to validate whether the potential barrier-enhancing properties of the σ1 agonist PRE-084 may also serve to help restore the barrier dysfunction caused by the inflammatory mediator IL-1β. Cells were treated with 15 ng/mL IL-1β or vehicle, 5 minutes after pretreatment with either 100 μM PRE-084 or a vehicle control. IL-1β caused a noticeable drop in HDLEC monolayer TER over time in comparison with the control trace, but this was not the case when cells were pretreated with PRE-084 (Fig. 5A). A direct comparison of the TER at the 8-hour time point shows that PRE-084 attenuated the IL-1β-induced decreased in TER (Fig. 5B). Of note, PRE-084 alone caused enhancement of TER in comparison with control (Fig. 5B). The results suggest a possible anti-inflammatory role of σ1 agonism in lymphatic endothelium.

Discussion and Conclusions

Recently, we showed for the first time that σ1 is expressed in rat mesenteric collecting lymphatic vessels and that the sigma receptor agonist afobazole can elicit increased production of NO from LECs.²⁴ In this study, we identify another, significant role of σ1 in LECs, namely promotion of barrier function. Depletion of σ1 with selective siRNA knockdown significantly impaired the barrier function of cultured HDLEC monolayers, whereas the selective σ1 agonist PRE-084 enhanced barrier function in a concentration-dependent manner. These findings are similar to those we reported with vascular endothelial cells,²⁷ although the magnitude of the PRE-084-induced barrier enhancement was much greater in HDLEC. Moreover, we present that in LECs that PRE-084 also significantly shifts the production of ATP toward a more glycolytic state. Although LECs would already be expected to be in a relatively low-oxygen environment,¹⁹ this shift could potentially allow more oxygen to be available for local tissues.

An excessively leaky lymphatic vessel network would be expected to exacerbate inflammatory conditions. Lymphedema has a well-described inflammatory component that contributes to alteration of subcutaneous tissue, including alterations in fat content and the development of fibrosis.^36–38 Because of the significant contribution of inflammation in the pathology of lymphedema,³⁹ counteracting inflammation has been a major focus as a potential therapeutic target. Leukotriene B4 antagonism was shown to ameliorate experimental lymphedema.⁴⁰ In addition, blockade of transforming growth factor-β1 accelerates lymphatic regeneration during wound repair.⁴¹

Of note, LECs actively participate in the phenotypic consequences of a deranged lymphangiogenesis relating to tissue fluid accumulation in the pathogenesis of lymphedema.⁴² Lymphatic barrier dysfunction is thought to contribute to the overall pathology of lymphatic diseases.⁴³ Although the impact of inflammation or mechanical stimuli on lymphatic endothelial permeability has been studied,^9,11,12,16 discovering barrier enhancing agents for lymphatic endothelium has not been fully addressed. Our current findings show for the first time that PRE-084 can enhance TER of LECs. This was consistent with our recent findings that σ1 activation reduced solute permeability and promoted greater junctional integrity in vascular endothelium,²⁷ suggesting a potential positive effect of σ1 activation in lymphatic endothelial barrier enhancement. The specific contribution of σ1 to lymphatic endothelial barrier was shown using a model of σ1 knockdown in LECs, which showed deficient TER compared with controls, indicating the significance of σ1 in lymphatic barrier maintenance.

Of note, because the currently available instruments for assessing metabolic functions are limited to using static media conditions, all the experiments carried out to assess the contribution of σ1 in lymphatic barrier function were also performed under conditions in which the media was static and the cells were not exposed to shear stress. We do know from a previous study that shear stress changes can significantly alter barrier function of LEC monolayers.¹⁶ In addition, we have shown previously that the σ receptor agonist afobazole can elicit NO production in LECs and that blocking NO production inhibits the ability of afobazole to cause relaxation of isolated collecting lymphatic vessels.²⁴ The potential role of σ1 in shear stress sending by LECs is currently unknown and represents an additional direction of future investigation.

Lymphatic dysfunction is also related to inflammation and accumulation of inflammatory mediators such as IFN-γ, IL-1, IL-13, IL-4, IL-6, TGF-β1, and VEGF-C.⁴⁴ Cromer et al. have studied the effects of inflammatory cytokines on lymphatic endothelial barrier.⁹ Consistent with their finding that IL-1β caused lymphatic endothelial dysfunction and increased permeability in vitro,⁹ we found that IL-1β significantly decreased TER in HDLEC monolayer, plus we showed the novel finding that IL-1β-induced barrier dysfunction is attenuated by PRE-084 (Fig. 5). This finding suggests a possible LEC-specific anti-inflammatory role that can be beneficial in cases such as lymphedema.

Of interest, in the development of a mouse model to study the contribution of excessive lymphatic permeability in vivo, Yang et al. discovered that VE-cadherin signaling is also essential for lymphatic valve formation and maintenance.³ Hence, lymphatic endothelial monolayers represent an important tool for evaluating barrier function in the absence of confounding factors like insufficiently developed luminal valves.

Lymphatic dysfunction is also characterized by oxidative stress and metabolic impairments such as accumulation of reactive oxygen species.^45–47 Our results suggest the ability of PRE-084 to enhance glycolysis in favor of mitochondrial oxygen consumption for the production of ATP. This shift could spare oxygen, limit ROS formation, and also contribute to barrier enhancement.^47,48 The current results expand upon our previous findings that PRE-084 enhances glycolysis in vascular endothelium.²⁷ The ability of PRE-084 to enhance glycolytic production of ATP appears to be conserved among endothelial cells from both lymphatic and blood vessel sources.

Collectively, our results suggest that σ1 is an important modulator of lymphatic endothelial barrier function. In addition, σ1 activation elicits a shift toward more glycolytic ATP production upon its activation. These findings suggest a novel role of σ1 in lymphatic endothelial bioenergetics and barrier function and expand the current known uses and applications of σ1 agonists.³²