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.
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 has 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 greater than 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.
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.
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.
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 ?l (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 (?l (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.
Polymorphonuclear neutrophils (PMNs) are critical for the formation, maintenance, and resolution of bacterial abscesses. However, the mechanisms that regulate PMN survival and proliferation during the evolution of an abscess are not well defined. Using a mouse model of Staphylococcus aureus abscess formation within a cutaneous wound, combined with real-time imaging of genetically tagged PMNs, we observed that a high bacterial burden elicited a sustained mobilization of PMNs from the bone marrow to the infected wound, where their lifespan was markedly extended. A continuous rise in wound PMN number, which was not accounted for by trafficking from the bone marrow or by prolonged survival, was correlated with the homing of c-kit(+)-progenitor cells from the blood to the wound, where they proliferated and formed mature PMNs. Furthermore, by blocking their recruitment with an antibody to c-kit, which severely limited the proliferation of mature PMNs in the wound and shortened mouse survival, we confirmed that progenitor cells are not only important contributors to PMN expansion in the wound, but are also functionally important for immune protection. We conclude that the abscess environment provides a niche capable of regulating PMN survival and local proliferation of bone marrow-derived c-kit(+)-progenitor cells.
We apply positron emission tomography (PET) to elucidate changes in nanocarrier extravasation during the transition from premalignant to malignant cancer, providing insight into the use of imaging to characterize early cancerous lesions and the utility of nanoparticles in early disease.
Inhibition of phosphodiesterase 4 (PDE4) to increase endothelial cAMP and stabilize the endothelial barrier attenuates acute inflammatory increases in vascular permeability.We extended this approach to attenuate physiological increases in vascular permeability in response to atrial natriuretic peptide (ANP), which acts with the kidney to regulate plasma volume. We measured blood-to-tissue albumin clearance and changes in plasma volume in isoflurane-anaesthetized mice (C57BL/6J) pre-treated with rolipram (8 mg kg(-1) I.P., 30 min). Rolipram significantly reduced albumin permeability, measured using a dual-label fluorescence method, in skin and skeletal muscle compared with ANP alone (500 ng kg(-1) min(-1)). Skin and muscle tissue accounted for 70% of the reduction in whole body albumin clearance taking into account albumin clearance in gastrointestinal (GI) tissue, heart and kidney. The action of ANP and rolipram to modify albumin clearances in duodenum and jejunum could be accounted for by local increases in vascular perfusion to increase surface area for exchange. ANP increased haematocrit from 40.6% to 46.8%, corresponding to an average loss of 22% plasma fluid volume (227 ?l), and this was almost completely reversed with rolipram. Renal water excretion accounted for less than 30% of plasma fluid loss indicating that reduced albumin permeability and reduced filtration into vasodilated GI tissue were the predominant actions of PDE4 inhibition. Similar fluid retention was measured in mice with endothelial-restricted deletion of the guanylyl cyclase-A receptor for ANP. Stabilizing the endothelial barrier to offset ANP-induced increases in vascular permeability may be part of a strategy to maintain plasma volume.
Although several cultured endothelial cell studies indicate that sphingosine-1-phosphate (S1P), via GTPase Rac1 activation, enhances endothelial barriers, very few in situ studies have been published. We aimed to further investigate the mechanisms whereby S1P modulates both baseline and increased permeability in intact microvessels.
Multiple processes modulate net blood-to-tissue exchange in a microvascular unit in normal and pathophysiological conditions. These include mechanisms that control the number and type of microvessels perfused, the balance of adhesion and contractile forces that determine the conductance of the spaces between endothelial cells to water and solutes, the pressure and chemical potential gradients determining the driving forces through these conductive pathways, and the organization of barriers to macromolecules in the endothelial glycocalyx. Powerful methods are available to investigate these mechanisms at the levels of cultured endothelial monolayers, isolated microvessels, and the microvascular units within intact organs. Here we focus on current problems that limit the integration of our knowledge of mechanisms investigated in detail at the cellular level into a more complete understanding of modulation of blood-to-tissue exchange in whole organs when the endothelial barrier is exposed to acute and more long-term inflammatory conditions. First, we review updated methods, applicable in mouse models of vascular permeability regulation, to investigate both acute and long-term changes in permeability. Methods to distinguish tracer accumulation due to change in perfusion from real increases in extravascular accumulation are emphasized. The second part of the review compares normal and increased permeability in individually perfused venular microvessels and endothelial cell monolayers. The heterogeneity of endothelial cell phenotypes in the baseline state and after exposure to injury and inflammatory conditions is emphasized. Lastly, we review new approaches to investigation of the glycocalyx barrier properties in cultured endothelial monolayers and in whole-body investigations.
Atrial natriuretic peptide (ANP) via its guanylyl cyclase-A (GC-A) receptor participates in regulation of arterial blood pressure and vascular volume. Previous studies demonstrated that concerted renal diuretic/natriuretic and endothelial permeability effects of ANP cooperate in intravascular volume regulation. We show that the microvascular endothelial contribution to the hypovolaemic action of ANP can be measured by the magnitude of the ANP-induced increase in blood-to-tissue albumin transport, measured as plasma albumin clearance corrected for intravascular volume change, relative to the corresponding increase in ANP-induced renal water excretion. We used a two-tracer method with isotopically labelled albumin to measure clearances in skin and skeletal muscle of: (i) C57BL6 mice; (ii) mice with endothelium-restricted deletion of GC-A (floxed GC-A x tie2-Cre: endothelial cell (EC) GC-A knockout (KO)); and (iii) control littermates (floxed GC-A mice with normal GC-A expression levels). Comparison of albumin clearances in hypervolaemic EC GC-A KO mice with normovolaemic littermates demonstrated that skeletal muscle albumin clearance with ANP treatment accounts for at most 30% of whole body clearance required for ANP to regulate plasma volume. Skin microcirculation responded to ANP similarly. Measurements of permeability to a high molecular mass contrast agent (35 kD Gadomer) by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) enabled repeated measures in individual animals and confirmed small increases in muscle and skin microvascular permeability after ANP. These quantitative methods will enable further evaluation of the contribution of ANP-dependent microvascular beds (such as gastro-intestinal tract) to plasma volume regulation.
A clinical measure of endothelial glycocalyx structure would have great potential importance, because lesions of the glycocalyx may be the first changes to occur in diabetes and in a wide range of vascular diseases. A method recently described by Nieuwdorp et al. for estimating the volume of the luminal glycocalyx of the entire human vascular system would seem to be the first attempt to develop a measure of this kind. It is based on the tracer dilution principle, and this review considers the principles and conditions that underlie this method and the extent to which the conditions appear to have been fulfilled in this case. Our analysis raises two questions about 1) the estimation of the concentration of the tracer (dextran 40) at zero time and 2) the estimation of plasma volume, both of which can be answered by changes in experimental protocol. A third question, concerning the partition coefficient of the tracer between plasma and the fluid within the glycocalyx, cannot be answered at the present time, and until it has been resolved, glycocalyx volume cannot be estimated from the dilution of a macromolecular tracer.
Transport of macromolecules and transmigration of leukocytes across vascular endothelium are regulated by a tight molecular junction, but the mechanisms by which these two inflammatory events are differentially controlled in time and magnitude during aseptic cutaneous wounding remain elusive. A real-time fluorescence imaging technique was developed to simultaneously track influx of Alexa 680-labeled albumin and genetically tagged enhanced green fluorescent protein-neutrophils [polymorphonuclear neutrophils (PMN)] within the wound bed. Vascular permeability increased approximately threefold more rapidly than the rate of PMN influx, reaching a maximum at 12 h, on the order of approximately 0.15% per minute versus approximately 0.05% per minute for PMN influx, which peaked at 18 h. Systemic depletion of PMN with antibody blocked their extravasation to the wound but did not alter the increase in vascular permeability. In contrast, pretreatment with antiplatelet GPIb decreased permeability by 25% and PMN influx by 50%. Hyperpermeability stimulated by the endothelium-specific agonists VEGF or thrombin at 24 h postwounding was completely inhibited by blocking Rho-kinase-dependent signaling, whereas less inhibition was observed at 1 h and neutrophil influx was not perturbed. These data suggest that in aseptic wounds, the endothelium maintains a tight junctional barrier to protein leakage that is independent of neutrophil transmigration, partially dependent on circulating platelets, and associated with Rho-kinase-dependent signaling.
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.
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.
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