Publications

Lymphatic and blood microvascular networks play critical roles in the clearance of excess fluid from local tissue spaces. Given the importance of these dynamics in inflammation, tumor metastasis, and lymphedema, understanding the coordinated function and remodeling between lymphatic and blood vessels in adult tissues is necessary. Knowledge gaps exist because the functions of these two systems are typically considered separately. The objective of this review was to highlight the coordinated functional relationships between blood and lymphatic vessels in adult microvascular networks. Structural, functional, temporal, and spatial relationships will be framed in the context of maintaining tissue homeostasis, vessel permeability, and system remodeling. The integration across systems will emphasize the influence of the local environment on cellular and molecular dynamics involved in fluid flow from blood capillaries to initial lymphatic vessels in microvascular networks.

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.

Renal dysfunction is one of the most common complications of septic injury. One critical contributor to septic injury-induced renal dysfunction is renal vascular dysfunction. Protein palmitoylation serves as a novel regulator of vascular function. Here, we examined whether palmitoyl acyltransferase (PAT)-DHHC21 contributes to septic injury-induced renal dysfunction through regulating renal hemodynamics. Multispectral optoacoustic imaging showed that cecal ligation and puncture (CLP)-induced septic injury caused impaired renal excretion, which was improved in DHHC21 functional deficient (Zdhhc21dep/dep) mice. DHHC21 deficiency attenuated CLP-induced renal pathology, characterized by tissue structural damage and circulating injury markers. Importantly, DHHC21 loss-of-function led to better-preserved renal perfusion and oxygen saturation after CLP. The CLP-caused reduction in renal blood flow was also ameliorated in Zdhhc21dep/dep mice. Next, CLP promoted the palmitoylation of vascular α1-adrenergic receptor (α1AR) and the activation of its downstream effector ERK, which were blunted in Zdhhc21dep/dep mice. Vasoreactivity analysis revealed that renal arteries from Zdhhc21dep/dep mice displayed reduced constriction response to α1AR agonist phenylephrine compared to those from wild-type mice. Consistently, inhibiting PATs with 2-bromopalmitate caused a blunted vasoconstriction response to phenylephrine in small arteries isolated from human kidneys. Therefore, DHHC21 contributes to impaired renal perfusion and function during septic injury via promoting α1AR palmitoylation-associated vasoconstriction.

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.

Anemia is a frequent diagnosis in critically ill infants, but the clinical implications of severe anemia in these patients remain unclear. In this study, we examined preweaned mice to investigate the effects of severe anemia during early infancy on gut mucosal permeability. C57BL/6 mice were subjected to timed phlebotomy between postnatal days (P) 2-10 to induce severe anemia (hematocrits 20%-24%), and intestinal permeability was tracked longitudinally between P10 and P20 as intestine-to-plasma translocation of enteral macromolecules and bacterial translocation. Epithelial junctions were evaluated by electron microscopy, polymerase chain reactions, immunohistochemistry, and/or enzyme immunoassays on intestinal tissues, Caco-2 intestinal epithelial-like cells, and colonic organoids. Preweaned mouse pups showed an age-related susceptibility to severe anemia, with increased intestinal permeability to enteral macromolecules (dextran, ovalbumin, β-lactoglobulin) and luminal bacteria. Electron micrographs showed increased paracellular permeability and ultrastructural abnormalities of the adherens junctions. These findings were explained by the loss of E-cadherin in epithelial cells, which was caused by destabilization of the E-cadherin (Cdh1) mRNA because of microRNA let-7e-5p binding to the 3'-untranslated region. Severe anemia resulted in a disproportionate and persistent increase in intestinal permeability in preweaned mice because of the disruption of epithelial adherens junctions. These changes are mediated via microRNA let-7e-mediated depletion of Cdh1 mRNA.NEW & NOTEWORTHY This research article shows that newborn infants with severe anemia show an age-related susceptibility to developing increased intestinal permeability to ingested macromolecules. This abnormal permeability develops because of abnormalities in intestinal epithelial junctions caused by a deficiency of the molecule E-cadherin in epithelial cells. The deficiency of E-cadherin is caused by destabilization of its mRNA precursor because of increased expression and binding of another molecule, the microRNA let-7e-5p, to the E-cadherin mRNA.

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.

Excessive microvascular permeability is a serious complication following hemorrhagic shock and resuscitation (HSR). S1P has been shown to ameliorate microvascular leakage in a model of combined alcohol intoxication and HSR. In the current study, we tested the hypothesis that S1P reduces HSR-induced microvascular leakage by preserving endothelial cell junctional structure and the endothelial glycocalyx through the protection of mitochondrial function. We used an established in vivo rat model of conscious HSR and assessed microvascular leakage, endothelial glycocalyx integrity, and mitochondrial function by intravital microscopy. Junctional integrity in the mesenteric microcirculation was assessed by confocal microscopy. Cultured rat intestinal microvascular endothelial cells monolayers were used to test the ability of S1P to protect against glycocalyx shedding and endothelial barrier dysfunction caused by direct disruption of mitochondrial integrity due to inhibition of mitochondrial complex III. The results show that in vivo, S1P protects against HSR-induced hyperpermeability, preserves the expression of adherens junctional proteins, and protects against glycocalyx degradation. S1P treatment during HSR also protects against mitochondrial membrane depolarization. S1P also protects against mitochondrial dysfunction-induced endothelial barrier dysfunction and glycocalyx degradation by acting through mitochondrial complex III. Taken together, our data indicate that S1P protects against HSR-induced mitochondrial dysfunction in endothelial cells, which in turn improves the structure of the endothelial glycocalyx after HSR and allows for better junctional integrity to the prevention of excess microvascular permeability.

OBJECTIVE: S1P has known endothelial barrier-protective properties, but whether this extends to the BBB is unclear. We hypothesized that alcohol-induced disruption of brain microvascular endothelial barrier function and junctional protein organization can be ameliorated by S1P treatment.

METHODS: Cultured primary HBMEC monolayers were used to characterize endothelial-specific mechanisms of BBB regulation. TER and apparent permeability coefficients for albumin, dextran-4 kDa, and sodium fluorescein were used as indices of barrier function. Junctional localization of Claudin-5, VE-cadherin, and β-catenin was determined by immunofluorescence confocal microscopy. S1P was applied following treatment with alcohol.

RESULTS: Alcohol significantly impaired HBMEC TER. Application of S1P after alcohol treatment resulted in a hastened recovery to the baseline HBMEC TER. Alcohol-treated HBMEC had a significantly higher mean permeability than control that was reversed by S1P. Alcohol caused the formation of gaps between cells. Treatment with S1P (after alcohol) increased junctional localization of VE-Cadherin, Claudin-5, and β-catenin.

CONCLUSIONS: Alcohol impairs the barrier function and junctional organization of HBMEC monolayers. S1P enhanced barrier function and restored junctions in the presence of alcohol, and thus may be useful for restoring BBB function during alcohol intoxication.

In the current study, the potential contributions of ryanodine receptors (RyRs) to intrinsic pumping and responsiveness to substance P (SP) were investigated in isolated rat mesenteric collecting lymphatic vessels. Responses to SP were characterized in lymphatic vessels in the absence or presence of pretreatment with nifedipine to block L-type Ca2+ channels, caffeine to block normal release and uptake of Ca2+ from the sarcoplasmic reticulum, ryanodine to block all RyR isoforms, or dantrolene to more selectively block RyR1 and RyR3. RyR expression and localization in lymphatics was also assessed by quantitative PCR and immunofluorescence confocal microscopy. The results show that SP normally elicits a significant increase in contraction frequency and a decrease in end-diastolic diameter. In the presence of nifedipine, phasic contractions stop, yet subsequent SP treatment still elicits a strong tonic contraction. Caffeine treatment gradually relaxes lymphatics, causing a loss of phasic contractions, and prevents subsequent SP-induced tonic contraction. Ryanodine also gradually diminishes phasic contractions but without causing vessel relaxation and significantly inhibits the SP-induced tonic contraction. Dantrolene treatment did not significantly impair lymphatic contractions nor the response to SP. The mRNA for all RyR isoforms is detectable in isolated lymphatics. RyR2 and RyR3 proteins are found predominantly in the collecting lymphatic smooth muscle layer. Collectively, the data suggest that SP-induced tonic contraction requires both extracellular Ca2+ plus Ca2+ release from internal stores and that RyRs play a role in the normal contractions and responsiveness to SP of rat mesenteric collecting lymphatics.NEW & NOTEWORTHY The mechanisms that govern contractions of lymphatic vessels remain unclear. Tonic contraction of lymphatic vessels caused by substance P was blocked by caffeine, which prevents normal uptake and release of Ca2+ from internal stores, but not nifedipine, which blocks L-type channel-mediated Ca2+ entry. Ryanodine, which also disrupts normal sarcoplasmic reticulum Ca2+ release and reuptake, significantly inhibited substance P-induced tonic contraction. Ryanodine receptors 2 and 3 were detected within the smooth muscle layer of collecting lymphatic vessels.