Epac1 Is Crucial for Maintenance of Endothelial Barrier Function through A Mechanism Partly Independent of Rac1.

Epac1 (exchange protein activated by cAMP) stabilizes the endothelial barrier, but detailed studies are limited by the side effects of pharmacological Epac1 modulators and transient transfections. Here, we compare the key properties of barriers between endothelial cells derived from wild-type (WT) and Epac1-knockout (KO) mice myocardium. We found that KO cell layers, unlike WT layers, had low and cAMP-insensitive trans-endothelial resistance (TER). They also had fragmented VE-cadherin staining despite having augmented cAMP levels and increased protein expression of Rap1, Rac1, RhoA, and VE-cadherin. The simultaneous direct activation of Rac1 and RhoA by CN04 compensated Epac1 loss, since TER was increased. In KO-cells, inhibition of Rac1 activity had no additional effect on TER, suggesting that other mechanisms compensate the inhibition of the Rac1 function to preserve barrier properties. In summary, Epac1 is crucial for baseline and cAMP-mediated barrier stabilization through mechanisms that are at least partially independent of Rac1.

Understanding and extending the Starling principle.

The Starling Principle states that fluid movements between blood and tissues are determined by differences in hydrostatic and colloid osmotic (oncotic) pressures between plasma inside microvessels and fluid outside them. The Revised Starling Principle recognizes that, because microvessels are permeable to macromolecules, a balance of pressures cannot halt fluid exchange. In most tissues, steady oncotic pressure differences between plasma and interstitial fluid depend on low levels of steady filtration from plasma to tissues for which the Revised Principle provides the theory. Plasma volume is normally maintained by fluid losses from filtration being matched by fluid gains from lymph. Steady state fluid uptake into plasma only occurs in tissues such as intestinal mucosa and renal peri-tubular capillaries where a protein-free secretion of adjacent epithelia contributes significantly to interstitial fluid volume and keeps interstitial oncotic pressure low. Steady filtration rates in different tissues are disturbed locally by reflex changes in capillary pressure and perfusion. The rapid overall decline in capillary pressure after acute blood loss initiates rapid fluid uptake from tissue to plasma, that is, autotransfusion. Fluid uptake is transient, being rapid at first then attenuating but low levels may continue for more than an hour. The Revised Principle highlights the role of oncotic pressure of small volumes of interstitial fluid within a sub-compartment surrounding the microvessels rather than the tissue's mean interstitial fluid oncotic pressure. This maximizes oncotic pressure differences when capillary pressure are high and enhances initial absorption rates when pressures are low, accelerating short-term regulation of plasma volume.

Epac1 null mice have nephrogenic diabetes insipidus with deficient corticopapillary osmotic gradient and weaker collecting duct tight junctions.

AIM: The cAMP-mediator Epac1 (RapGef3) has high renal expression. Preliminary observations revealed increased diuresis in Epac1-/- mice. We hypothesized that Epac1 could restrict diuresis by promoting transcellular collecting duct (CD) water and urea transport or by stabilizing CD paracellular junctions to reduce osmolyte loss from the renal papillary interstitium. METHODS: In Epac1-/- and Wt C57BL/6J mice, renal papillae, dissected from snap-frozen kidneys, were assayed for the content of key osmolytes. Cell junctions were analysed by transmission electron microscopy. Urea transport integrity was evaluated by urea loading with 40% protein diet, endogenous vasopressin production was manipulated by intragastric water loading and moderate dehydration and vasopressin type 2 receptors were stimulated selectively by i.p.-injected desmopressin (dDAVP). Glomerular filtration rate (GFR) was estimated as [14 C]inulin clearance. The glomerular filtration barrier was evaluated by urinary albumin excretion and microvascular leakage by the renal content of time-spaced intravenously injected 125 I- and 131 I-labelled albumin. RESULTS: Epac1-/- mice had increased diuresis and increased free water clearance under antidiuretic conditions. They had shorter and less dense CD tight junction (TJs) and attenuated corticomedullary osmotic gradient. Epac1-/- mice had no increased protein diet-induced urea-dependent osmotic diuresis, and expressed Wt levels of aquaporin-2 (AQP-2) and urea transporter A1/3 (UT-A1/3). Epac1-/- mice had no urinary albumin leakage and unaltered renal microvascular albumin extravasation. Their GFR was moderately increased, unless when treated with furosemide. CONCLUSION: Our results conform to the hypothesis that Epac1-dependent mechanisms protect against diabetes insipidus by maintaining renal papillary osmolarity and the integrity of CD TJs.

Epac1-/- mice have elevated baseline permeability and do not respond to histamine as measured with dynamic contrast-enhanced magnetic resonance imaging with contrast agents of different molecular weights.

AIM: Epac1-/- mice, but not Epac2-/- mice have elevated baseline permeability to albumin. This study extends the investigations of how Epac-dependent pathways modulate transvascular exchange in response to the classical inflammatory agent histamine. It also evaluates the limitations of models of blood-to-tissue exchange in transgenic mice in DCE-MRI measurements. METHODS: We measured DCE-MRI signal intensity in masseter muscle of wt and Epac1-/- mice with established approaches from capillary physiology to determine how changes in blood flow and vascular permeability contribute to overall changes of microvascular flux. We used two tracers, the high molecular weight tracer (Gadomer-17, MW 17 kDa, apparent MW 30-35 kDa) is expected to be primarily limited by diffusion and therefore less dependent on changes in blood flow and the low molecular weight tracer (Dotarem (MW 0.56 kDa) whose transvascular exchange is determined by both blood flow and permeability. Paired experiments in each animal combined with analytical methods provided an internally consistent description of microvascular transport. RESULTS: Epac1-/- mice had elevated baseline permeability relative to wt control mice for Dotarem and Gadomer-17. In contrast to wt mice, Epac1-/- mice failed to increase transvascular permeability in response to histamine. Dotarem underestimated blood flow and vascular volume and Gadomer-17 has limited sensitivity in extravascular accumulation. CONCLUSION: The study suggests that the normal barrier loosening effect of histamine in venular microvessels do not function when the normal barrier tightening effect of Epac1 is already compromised. The study also demonstrated that the numerical analysis of DCE-MRI data with tracers of different molecular weight has significant limitations.

The Molecular Structure of the Endothelial Glycocalyx Layer (EGL) and Surface Layers (ESL) Modulation of Transvascular Exchange.

There has been rapid progress over the past decade to extend the concept that a quasiperiodic inner endothelial glycocalyx layer (EGL, <300 nm thick, with key components associated with the endothelial cell membrane) forms the primary molecular filter between circulating blood and the body tissues. The EGL is common to both continuous and fenestrated microvessels. The revised Starling Principle describing steady-state fluid exchange across the EGL describes new ways to understand transvascular exchange of water and plasma proteins in microvessels in both normal and disturbed states such as hemorrhage and fluid replacement during surgery. At the same time, direct optical observations describe endothelial surface layers (ESLs) with porous outer layers that extend 1-2 µm beyond the EGL. Preliminary analyses of water and plasma protein transport through barriers formed by a thick ESL in series with the EGL indicate that such two-layer structures can have permeability properties that are not consistent with measured water and plasma exchange in microvessels. Such multilayer models provide a basis for future detailed evaluations of both transports across endothelial surface layers and the methods to image components of both the EGL and the ESL. Furthermore changes in the thickness and distribution of thick ESLs in vessels with diameters larger than 50 µm may not reflect functional changes in the inner glycocalyx layer.

Dynamic contrast enhanced MRI detects changes in vascular transport rate constants following treatment with thermally-sensitive liposomal doxorubicin.

Temperature-sensitive liposomal formulations of chemotherapeutics, such as doxorubicin, can achieve locally high drug concentrations within a tumor and tumor vasculature while maintaining low systemic toxicity. Further, doxorubicin delivery by temperature-sensitive liposomes can reliably cure local cancer in mouse models. Histological sections of treated tumors have detected red blood cell extravasation within tumors treated with temperature-sensitive doxorubicin and ultrasound hyperthermia. We hypothesize that the local release of drug into the tumor vasculature and resulting high drug concentration can alter vascular transport rate constants along with having direct tumoricidal effects. Dynamic contrast enhanced MRI (DCE-MRI) coupled with a pharmacokinetic model can detect and quantify changes in such vascular transport rate constants. Here, we set out to determine whether changes in rate constants resulting from intravascular drug release were detectable by MRI. We found that the accumulation of gadoteridol was enhanced in tumors treated with temperature-sensitive liposomal doxorubicin and ultrasound hyperthermia. While the initial uptake rate of the small molecule tracer was slower (k1=0.0478±0.011s-1 versus 0.116±0.047s-1) in treated compared to untreated tumors, the tracer was retained after treatment due to a larger reduction in the rate of clearance (k2=0.291±0.030s-1 versus 0.747±0.24s-1). While DCE-MRI assesses a combination of blood flow and permeability, ultrasound imaging of microvascular flow rate is sensitive only to changes in vascular flow rate; based on this technique, blood flow was not significantly altered 30min after treatment. In summary, DCE-MRI provides a means to detect changes that are associated with treatment by thermally-activated particles and such changes can be exploited to enhance local delivery.

Vector-free intracellular delivery by reversible permeabilization.

Despite advances in intracellular delivery technologies, efficient methods are still required that are vector-free, can address a wide range of cargo types and can be applied to cells that are difficult to transfect whilst maintaining cell viability. We have developed a novel vector-free method that uses reversible permeabilization to achieve rapid intracellular delivery of cargos with varying composition, properties and size. A permeabilizing delivery solution was developed that contains a low level of ethanol as the permeabilizing agent. Reversal of cell permeabilization is achieved by temporally and volumetrically controlling the contact of the target cells with this solution. Cells are seeded in conventional multi-well plates. Following removal of the supernatant, the cargo is mixed with the delivery solution and applied directly to the cells using an atomizer. After a short incubation period, permeabilization is halted by incubating the cells in a phosphate buffer saline solution that dilutes the ethanol and is non-toxic to the permeabilized cells. Normal culture medium is then added. The procedure lasts less than 5 min. With this method, proteins, mRNA, plasmid DNA and other molecules have been delivered to a variety of cell types, including primary cells, with low toxicity and cargo functionality has been confirmed in proof-of-principle studies. Co-delivery of different cargo types has also been demonstrated. Importantly, delivery occurs by diffusion directly into the cytoplasm in an endocytic-independent manner. Unlike some other vector-free methods, adherent cells are addressed in situ without the need for detachment from their substratum. The method has also been adapted to address suspension cells. This delivery method is gentle yet highly reproducible, compatible with high throughput and automated cell-based assays and has the potential to enable a broad range of research, drug discovery and clinical applications.

The role of atrial natriuretic peptide to attenuate inflammation in a mouse skin wound and individually perfused rat mesenteric microvessels.

We tested the hypothesis that the anti-inflammatory actions of atrial natriuretic peptide (ANP) result from the modulation of leukocyte adhesion to inflamed endothelium and not solely ANP ligation of endothelial receptors to stabilize endothelial barrier function. We measured vascular permeability to albumin and accumulation of fluorescent neutrophils in a full-thickness skin wound on the flank of LysM-EGFP mice 24 h after formation. Vascular permeability in individually perfused rat mesenteric microvessels was also measured after leukocytes were washed out of the vessel lumen. Thrombin increased albumin permeability and increased the accumulation of neutrophils. The thrombin-induced inflammatory responses were attenuated by pretreating the wound with ANP (30 min). During pretreatment ANP did not lower permeability, but transiently increased baseline albumin permeability concomitant with the reduction in neutrophil accumulation. ANP did not attenuate acute increases in permeability to histamine and bradykinin in individually perfused rat microvessels. The hypothesis that anti-inflammatory actions of ANP depend solely on endothelial responses that stabilize the endothelial barrier is not supported by our results in either individually perfused microvessels in the absence of circulating leukocytes or the more chronic skin wound model. Our results conform to the alternate hypothesis that ANP modulates the interaction of leukocytes with the inflamed microvascular wall of the 24 h wound. Taken together with our previous observations that ANP reduces deformability of neutrophils and their strength of attachment, rolling, and transvascular migration, these observations provide the basis for additional investigations of ANP as an anti-inflammatory agent to modulate leukocyte-endothelial cell interactions.

Sphingosine-1-phosphate Maintains Normal Vascular Permeability by Preserving Endothelial Surface Glycocalyx in Intact Microvessels.

OBJECTIVE: S1P was found to protect the ESG by inhibiting MMP activity-dependent shedding of ESG in cultured endothelial cell studies. We aimed to further test that S1P contributes to the maintenance of normal vascular permeability by protecting the ESG in intact microvessels. METHODS: We quantified the ESG in post-capillary venules of rat mesentery and measured the vascular permeability to albumin in the presence and absence of 1 µM S1P. We also measured permeability to albumin in the presence of MMP inhibitors and compared the measured permeability with those predicted by a transport model for the inter-endothelial cleft. RESULTS: We found that in the absence of S1P, the fluorescence intensity of the FITC-anti-HS-labeled ESG was ~10% of that in the presence of S1P, whereas the measured permeability to albumin was ~6.5-fold of that in the presence of S1P. Similar results were observed with MMP inhibition. The predictions by the mathematical model further confirmed that S1P maintains microvascular permeability by preserving ESG. CONCLUSIONS: Our results show that S1P contributes to the maintenance of normal vascular permeability by protecting the ESG in intact microvessels, consistent with parallel observation in cultured endothelial monolayers.

Atrial natriuretic peptide down-regulates neutrophil recruitment on inflamed endothelium by reducing cell deformability and resistance to detachment force.

BACKGROUND: Recombinant atrial natriuretic peptide (ANP) is administered in patients with acute heart failure in Japan to improve renal function and hemodynamics, but its anti-inflammatory effect on activated leukocytes may also contribute to its therapeutic efficacy. OBJECTIVE: Examine unconventional role of ANP in neutrophil adhesion to inflamed endothelium. METHODS: Human neutrophils were perfused over endothelial monolayers in a microfluidic lab-chip assay. Cell rheology was assessed by micropipette aspiration to assess changes in cortical tension and viscosity. Fluorescence microscopy was applied to measure adhesive contact area and ß2-integrin focal bond formation. RESULTS: ANP inhibited neutrophil rolling and firm adhesion without influencing the upregulation of cellular adhesion molecules on endothelium or the regulation of high affinity CD18 and shedding of L-selectin during neutrophil activation. Exposed to fluid shear, integrin mediated arrest was disrupted with ANP treatment, which elicited formation of long tethers and diminished cell spreading and contact. This correlated with a ∼40% increase in neutrophil viscosity and a reduction in the adhesive footprint. CONCLUSIONS: A decrease in cell deformation and neutrophil flattening with ANP results in fewer integrin bond clusters, which translates to higher tensile forces and impaired adhesion strengthening and cell detachment.

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery.

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

Mechanosensing at the vascular interface.

Mammals are endowed with a complex set of mechanisms that sense mechanical forces imparted by blood flow to endothelial cells (ECs), smooth muscle cells, and circulating blood cells to elicit biochemical responses through a process referred to as mechanotransduction. These biochemical responses are critical for a host of other responses, including regulation of blood pressure, control of vascular permeability for maintaining adequate perfusion of tissues, and control of leukocyte recruitment during immunosurveillance and inflammation. This review focuses on the role of the endothelial surface proteoglycan/glycoprotein layer-the glycocalyx (GCX)-that lines all blood vessel walls and is an agent in mechanotransduction and the modulation of blood cell interactions with the EC surface. We first discuss the biochemical composition and ultrastructure of the GCX, highlighting recent developments that reveal gaps in our understanding of the relationship between composition and spatial organization. We then consider the roles of the GCX in mechanotransduction and in vascular permeability control and review the prominent interaction of plasma-borne sphingosine-1 phosphate (S1P), which has been shown to regulate both the composition of the GCX and the endothelial junctions. Finally, we consider the association of GCX degradation with inflammation and vascular disease and end with a final section on future research directions.

A physiological perspective on the use of imaging to assess the in vivo delivery of therapeutics.

Our goal is to provide a physiological perspective on the use of imaging to optimize and monitor the accumulation of nanotherapeutics within target tissues, with an emphasis on evaluating the pharmacokinetics of organic particles. Positron emission tomography (PET), magnetic resonance imaging (MRI) and ultrasound technologies, as well as methods to label nanotherapeutic constructs, have created tremendous opportunities for preclinical optimization of therapeutics and for personalized treatments in challenging disease states. Within the methodology summarized here, the accumulation of the construct is estimated directly from the image intensity. Particle extravasation is then estimated based on classical physiological measures. Specifically, the transport of nanotherapeutics is described using the concept of apparent permeability, which is defined as the net flux of solute across a blood vessel wall per unit surface area of the blood vessel and per unit solute concentration difference across the blood vessel wall. The apparent permeability to small molecule MRI constructs is accurately shown to be far larger than that estimated for proteins such as albumin or nanoconstructs such as liposomes. Further, the quantitative measurements of vascular permeability are shown to facilitate detection of the transition from a pre-malignant to a malignant cancer and to quantify the delivery enhancement resulting from interventions such as ultrasound. While PET-based estimates facilitate quantitative comparisons of many constructs, high field MRI proves useful in the visualization of model drugs within small lesions and in the evaluation of the release and intracellular trafficking of nanoparticles and cargo.

Sphingosine-1-phosphate protects endothelial glycocalyx by inhibiting syndecan-1 shedding.

Endothelial cells (ECs) are covered by a surface glycocalyx layer that forms part of the barrier and mechanosensing functions of the blood-tissue interface. Removal of albumin in bathing media induces collapse or shedding of the glycocalyx. The electrostatic interaction between arginine residues on albumin, and negatively charged glycosaminoglycans (GAGs) in the glycocalyx have been hypothesized to stabilize the glycocalyx structure. Because albumin is one of the primary carriers of the phospholipid sphingosine-1-phosphate (S1P), we evaluated the alternate hypothesis that S1P, acting via S1P1 receptors, plays the primary role in stabilizing the endothelial glycocalyx. Using confocal microscopy on rat fat-pad ECs, we demonstrated that heparan sulfate (HS), chondroitin sulfate (CS), and ectodomain of syndecan-1 were shed from the endothelial cell surface after removal of plasma protein but were retained in the presence of S1P at concentrations of >100 nM. S1P1 receptor antagonism abolished the protection of the glycocalyx by S1P and plasma proteins. S1P reduced GAGs released after removal of plasma protein. The mechanism of protection from loss of glycocalyx components by S1P-dependent pathways was shown to be suppression of metalloproteinase (MMP) activity. General inhibition of MMPs protected against loss of CS and syndecan-1. Specific inhibition of MMP-9 and MMP-13 protected against CS loss. We conclude that S1P plays a critical role in protecting the glycocalyx via S1P1 and inhibits the protease activity-dependent shedding of CS, HS, and the syndecan-1 ectodomain. Our results provide new insight into the role for S1P in protecting the glycocalyx and maintaining vascular homeostasis.

The cargo of CRPPR-conjugated liposomes crosses the intact murine cardiac endothelium.

Ligand-conjugated liposomes and other nano-sized constructs are attractive drug carriers due to their extended plasma circulation; however, limited data are available as to whether their cargo can traverse the endothelium of solid organs. To determine whether the cargo of endothelially targeted liposomes is internalized by endothelial cells and transported into tissue, and to evaluate whether such liposomes can accumulate in models of cardiovascular disease, we tracked the fate of the cargo (a hydrophilic fluorescent dye) and shell (conjugated with a radioisotope) of a heart-homing liposome (CRPPR-conjugated). The ex vivo heart was imaged with confocal microscopy and the in vivo heart with positron emission tomography in sham-treated mice and models of ischemia/reperfusion (I/R) and myocardial infarction (MI). Within 30 min of injection of 20mg/kg CRPPR liposomes, fluorescence increased by 47 fold in the tissue surrounding the vascular lumen, as compared with non-targeted liposomes. Both the accumulation on the endothelium and the interstitial fluorescence saturated at an injected dose of 20mg/kg. In both I/R and MI models, CRPPR liposomes accumulated in diseased sites, although less than in surrounding healthy tissue. The accumulation in the diseased sites increased with time post-injury: the ratio of accumulated radioactivity in the diseased and healthy cardiac tissue increased from 0.20±0.04, to 0.58±0.12 and 0.61±0.19 for 1, 7, and 99 days post-MI, indicating the potential for adequate delivery and therapeutic efficacy if the targeted particles are injected at 7 or more days post-MI. In summary, CRPPR- liposomes accumulated in normal and diseased hearts, and the cargo accumulated in the tissue within minutes and remained detectable after 24 h.

Ultrasound increases nanoparticle delivery by reducing intratumoral pressure and increasing transport in epithelial and epithelial-mesenchymal transition tumors.

Acquisition of the epithelial-mesenchymal transition (EMT) tumor phenotype is associated with impaired chemotherapeutic delivery and a poor prognosis. In this study, we investigated the application of therapeutic ultrasound methods available in the clinic to increase nanotherapeutic particle accumulation in epithelial and EMT tumors by labeling particles with a positron emission tomography tracer. Epithelial tumors were highly vascularized with tight cell-cell junctions, compared with EMT tumors where cells displayed an irregular, elongated shape with loosened cell-cell adhesions and a reduction in E-cadherin and cytokeratins 8/18 and 19. Without ultrasound, the accumulation of liposomal nanoparticles administered to tumors in vivo was approximately 1.5 times greater in epithelial tumors than EMT tumors. When ultrasound was applied, both nanoaccumulation and apparent tumor permeability were increased in both settings. Notably, ultrasound effects differed with thermal and mechanical indices, such that increasing the thermal ultrasound dose increased nanoaccumulation in EMT tumors. Taken together, our results illustrate how ultrasound can be used to enhance nanoparticle accumulation in tumors by reducing their intratumoral pressure and increasing their vascular permeability.

Single-channel blind estimation of arterial input function and tissue impulse response in DCE-MRI.

Multipass dynamic MRI and pharmacokinetic modeling are used to estimate perfusion parameters of leaky capillaries. Curve fitting and nonblind deconvolution are the established methods to derive the perfusion estimates from the observed arterial input function (AIF) and tissue tracer concentration function. These nonblind methods are sensitive to errors in the AIF, measured in some nearby artery or estimated by multichannel blind deconvolution. Here, a single-channel blind deconvolution algorithm is presented, which only uses a single tissue tracer concentration function to estimate the corresponding AIF and tissue impulse response function. That way, many errors affecting these functions are reduced. The validity of the algorithm is supported by simulations and tests on real data from mouse. The corresponding nonblind and multichannel methods are also presented.

Phosphodiesterase 4 inhibition attenuates plasma volume loss and transvascular exchange in volume-expanded mice.

We tested the hypothesis that inhibition of phosphodiesterase 4 (PDE4) with rolipram to increase vascular endothelial cAMP and stabilize the endothelial barrier would attenuate the action of endogenous atrial natriuretic peptide (ANP) to increase vascular permeability to the plasma protein albumin after an acute plasma volume expansion. After rolipram pretreatment (8 mg (kg body wt)(-1), intraperitoneal, 30 min) more than 95% of the peak increase in plasma volume after volume expansion (4.5% bovine serum albumin, 114 µl (g body wt)(-1) h(-1), 15 min) remained in the vascular space 75 min after the end of infusion, whereas only 67% of the fluid was retained in volume-expanded animals with no rolipram pretreatment. Rolipram significantly decreased 30 min fluorescently labelled albumin clearance (µl (g dry wt)(-1)) relative to untreated volume-expanded controls in skin (e.g. back, 10.4 ± 1.6 vs. 19.5 ± 3.6, P = 0.04), muscle (e.g. hamstring, 15.0 ± 1.9 vs. 20.8 ± 1.4, P = 0.04) and in colon, caecum, and rectum (average reduction close to 50%). The mass of muscle and skin tissue accounted for 70% of volume-expansion-dependent albumin shifts from plasma to interstitium. The results are consistent with observations that the PDE4 inhibitor rolipram attenuates ANP-induced increases in vascular permeability after infusion of exogenous ANP and observations of elevated central venous pressure after a similar volume expansion in mice with selective deletion of the endothelial ANP receptor. These observations may form the basis for new strategies to retain intravenous fluid containing macromolecules.