Determination of Solute Permeability of Microvascular Endothelial Cell Monolayers In Vitro.

The microvascular endothelium has a critical role in regulating the delivery of oxygen, nutrients, and water to the surrounding tissues. Under inflammatory conditions that accompany acute injury or disease, microvascular permeability becomes elevated. When microvascular hyperpermeability becomes uncontrolled or chronic, the excessive escape of plasma proteins into the surrounding tissue disrupts homeostasis and ultimately leads to organ dysfunction. Much remains to be learned about the mechanisms that control microvascular permeability. In addition to in vivo and isolated microvessel methods, the cultured endothelial cell monolayer protocol is an important tool that allows for understanding the specific, endothelial subcellular mechanisms that determine permeability of the endothelium to plasma proteins. In this chapter, two variations of the popular Transwell culture methodology to determine permeability to using fluorescently labeled tracers are presented. The strengths and weaknesses of this approach are also discussed.

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.

Edema and lymphatic clearance: molecular mechanisms and ongoing challenges.

Resolution of edema remains a significant clinical challenge. Conditions such as traumatic shock, sepsis, or diabetes often involve microvascular hyperpermeability, which leads to tissue and organ dysfunction. Lymphatic insufficiency due to genetic causes, surgical removal of lymph nodes, or infections, leads to varying degrees of tissue swelling that impair mobility and immune defenses. Treatment options are limited to management of edema as there are no specific therapeutics that have demonstrated significant success for ameliorating microvascular leakage or impaired lymphatic function. This review examines current knowledge about the physiological, cellular, and molecular mechanisms that control microvascular permeability and lymphatic clearance, the respective processes for interstitial fluid formation and removal. Clinical conditions featuring edema, along with potential future directions are discussed.

Endothelial mechanisms for inactivation of inflammation-induced hyperpermeability.

Microvascular hyperpermeability is a hallmark of inflammation. Many negative effects of hyperpermeability are due to its persistence beyond what is required for preserving organ function. Therefore, we propose that targeted therapeutic approaches focusing on mechanisms that terminate hyperpermeability would avoid the negative effects of prolonged hyperpermeability while retaining its short-term beneficial effects. We tested the hypothesis that inflammatory agonist signaling leads to hyperpermeability and initiates a delayed cascade of cAMP-dependent pathways that causes inactivation of hyperpermeability. We applied platelet-activating factor (PAF) and vascular endothelial growth factor (VEGF) to induce hyperpermeability. We used an Epac1 agonist to selectively stimulate exchange protein activated by cAMP (Epac1) and promote inactivation of hyperpermeability. Stimulation of Epac1 inactivated agonist-induced hyperpermeability in the mouse cremaster muscle and in human microvascular endothelial cells (HMVECs). PAF induced nitric oxide (NO) production and hyperpermeability within 1 min and NO-dependent increased cAMP concentration in about 15-20 min in HMVECs. PAF triggered phosphorylation of vasodilator-stimulated phosphoprotein (VASP) in a NO-dependent manner. Epac1 stimulation promoted cytosol-to-membrane eNOS translocation in HMVECs and in myocardial microvascular endothelial (MyEnd) cells from wild-type mice, but not in MyEnd cells from VASP knockout mice. We demonstrate that PAF and VEGF cause hyperpermeability and stimulate the cAMP/Epac1 pathway to inactivate agonist-induced endothelial/microvascular hyperpermeability. Inactivation involves VASP-assisted translocation of eNOS from the cytosol to the endothelial cell membrane. We demonstrate that hyperpermeability is a self-limiting process, whose timed inactivation is an intrinsic property of the microvascular endothelium that maintains vascular homeostasis in response to inflammatory conditions.NEW & NOTEWORTHY Termination of microvascular hyperpermeability has been so far accepted to be a passive result of the removal of the applied proinflammatory agonists. We provide in vivo and in vitro evidence that 1) inactivation of hyperpermeability is an actively regulated process, 2) proinflammatory agonists (PAF and VEGF) stimulate microvascular hyperpermeability and initiate endothelial mechanisms that terminate hyperpermeability, and 3) eNOS location-translocation is critical in the activation-inactivation cascade of endothelial hyperpermeability.

Lymphatic Clearance and Pump Function.

Lymphatic vessels have an active role in draining excess interstitial fluid from organs and serving as conduits for immune cell trafficking to lymph nodes. In the central circulation, the force needed to propel blood forward is generated by the heart. In contrast, lymphatic vessels rely on intrinsic vessel contractions in combination with extrinsic forces for lymph propulsion. The intrinsic pumping features phasic contractions generated by lymphatic smooth muscle. Periodic, bicuspid valves composed of endothelial cells prevent backflow of lymph. This work provides a brief overview of lymph transport, including initial lymph formation along with cellular and molecular mechanisms controlling lymphatic vessel pumping.

Impact of Goreisan components on rat mesenteric collecting lymphatic vessel pumping.

BACKGROUND: Goreisan is a traditional herbal formulation with diuretic properties tested as a clinical therapeutic to alleviate lymphedema in Japan. The present study aimed to determine how Goreisan and its five different components affect lymphatic pump function. METHODS: Mesenteric collecting lymphatics were isolated from anesthetized Sprague-Dawley rats and mounted on resistance-matched glass micropipettes in a 37°C physiological salt solution bath for studies. Diameter was continuously measured to obtain the following lymphatic pump parameters: contraction frequency (CF), end diastolic diameter (EDD), and end systolic diameter (ESD), contraction amplitude (AMP), ejection fraction (EF), and fractional pump flow (FPF). Goreisan and each of its components (Cinnamomi Cortex, Atractylodis Rhizoma, Alismatis Rhizoma, Polyporus, and Poria) were applied to the bath at concentrations of 1-30 µg/mL. RESULTS: The results show that while Goreisan causes no significant changes to lymphatic pumping, Alismatis Rhizoma and Polyporus each significantly reduce CF and FPF. In addition, rats that received oral administration of Goreisan and Alismatis Rhizoma for 1 week had elevated expression of VEGFR-3 in their mesenteric collecting lymphatics. CONCLUSIONS: Collectively, the results suggest that some components of Goreisan have a direct, rapid impact on lymphatic pumping. These findings provide new insights but also raise new questions about the therapeutic potential of Goreisan in patients with secondary lymphedema.

The Microvascular-Lymphatic Interface and Tissue Homeostasis: Critical Questions That Challenge Current Understanding.

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.

DHHC21 deficiency attenuates renal dysfunction during septic injury.

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.

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.

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.

Severe neonatal anemia increases intestinal permeability by disrupting epithelial adherens junctions.

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, ß-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.

Evidence of functional ryanodine receptors in rat mesenteric collecting lymphatic vessels.

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.

Nociceptive pulmonary-cardiac reflexes are altered in the spontaneously hypertensive rat.

KEY POINTS: We investigated the cardiovascular and respiratory responses of the normotensive Wistar-Kyoto (WKY) rat and the spontaneously hypertensive (SH) rat to inhalation and intravenous injection of the noxious stimuli allyl isothiocyanate (AITC). AITC inhalation evoked atropine-sensitive bradycardia in conscious WKY rats, and evoked atropine-sensitive bradycardia and atenolol-sensitive tachycardia with premature ventricular contractions (PVCs) in conscious SH rats. Intravenous injection of AITC evoked bradycardia but no tachycardia/PVCs in conscious SHs, while inhalation and injection of AITC caused similar bradypnoea in conscious SH and WKY rats. Anaesthesia (inhaled isoflurane) inhibited the cardiac reflexes evoked by inhaled AITC but not injected AITC. Data indicate the presence of a de novo nociceptive pulmonary-cardiac reflex triggering sympathoexcitation in SH rats, and this reflex is dependent on vagal afferents but is not due to steady state blood pressure or due to remodelling of vagal efferent function. ABSTRACT: Inhalation of noxious irritants/pollutants activates airway nociceptive afferents resulting in reflex bradycardia in healthy animals. Nevertheless, noxious pollutants evoke sympathoexcitation (tachycardia, hypertension) in cardiovascular disease patients. We hypothesize that cardiovascular disease alters nociceptive pulmonary-cardiac reflexes. Here, we studied reflex responses to irritants in normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive (SH) rats. Inhaled allyl isothiocyanate (AITC) evoked atropine-sensitive bradycardia with atrial-ventricular (AV) block in conscious WKY rats, thus indicating a parasympathetic reflex. Conversely, inhaled AITC in conscious SH rats evoked complex brady-tachycardia with both AV block and premature ventricular contractions (PVCs). Atropine abolished the bradycardia and AV block, but the atropine-insensitive tachycardia and PVCs were abolished by the ß1 -adrenoceptor antagonist atenolol. The aberrant AITC-evoked reflex in SH rats was not reduced by acute blood pressure reduction by captopril. Surprisingly, intravenous AITC only evoked bradycardia in conscious SH and WKY rats. Furthermore, anaesthesia reduced the cardiac reflexes evoked by inhaled but not injected AITC. Nevertheless, anaesthesia had little effect on AITC-evoked respiratory reflexes. Such data suggest distinct differences in nociceptive reflex pathways dependent on cardiovascular disease, administration route and downstream effector. AITC-evoked tachycardia in decerebrate SH rats was abolished by vagotomy. Finally, there was no difference in the cardiac responses of WKY and SH rats to vagal efferent electrical stimulation. Our data suggest that AITC inhalation in SH rats evokes de novo adrenergic reflexes following vagal afferent activation. This aberrant reflex is independent of steady state hypertension and is not evoked by intravenous AITC. We conclude that pre-existing hypertension aberrantly shifts nociceptive pulmonary-cardiac reflexes towards sympathoexcitation.

Sphingosine-1-Phosphate Reduces Hemorrhagic Shock and Resuscitation-Induced Microvascular Leakage by Protecting Endothelial Mitochondrial Integrity.

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.

Sphingosine-1-phosphate protects against brain microvascular endothelial junctional protein disorganization and barrier dysfunction caused by alcohol.

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 ß-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 ß-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.

Lymphatic Vessel Network Structure and Physiology.

The lymphatic system is comprised of a network of vessels interrelated with lymphoid tissue, which has the holistic function to maintain the local physiologic environment for every cell in all tissues of the body. The lymphatic system maintains extracellular fluid homeostasis favorable for optimal tissue function, removing substances that arise due to metabolism or cell death, and optimizing immunity against bacteria, viruses, parasites, and other antigens. This article provides a comprehensive review of important findings over the past century along with recent advances in the understanding of the anatomy and physiology of lymphatic vessels, including tissue/organ specificity, development, mechanisms of lymph formation and transport, lymphangiogenesis, and the roles of lymphatics in disease. © 2019 American Physiological Society. Compr Physiol 9:207-299, 2019.

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.