Transendothelial Pathway Research Articles

Intracellular Membrane Transport in Vascular Endothelial Cells.

The main component of blood and lymphatic vessels is the endothelium covering their luminal surface. It plays a significant role in many cardiovascular diseases. Tremendous progress has been made in deciphering of molecular mechanisms involved into intracellular transport. However, molecular machines are mostly characterized in vitro. It is important to adapt this knowledge to the situation existing in tissues and organs. Moreover, contradictions have accumulated within the field related to the function of endothelial cells (ECs) and their trans-endothelial pathways. This has induced necessity for the re-evaluation of several mechanisms related to the function of vascular ECs and intracellular transport and transcytosis there. Here, we analyze available data related to intracellular transport within ECs and re-examine several hypotheses about the role of different mechanisms in transcytosis across ECs. We propose a new classification of vascular endothelium and hypotheses related to the functional role of caveolae and mechanisms of lipid transport through ECs.

Exosomes form tunneling nanotubes (TUNTs) in the blood-brain barrier: a nano-anatomical perspective of barrier genesis.

The blood-brain barrier (BBB) is a robust interface between the blood and the central nervous system. Barrier type endothelium is able to limit paracellular (PC) movement, relegating molecular flux to the transendothelial pathways of brain endothelial cells (BECs). It is, therefore, apparent that any leakage via the PC shunts would effectively nullify the regulation of molecular flux across the transcellular pathways. The application of higher-resolution scanning electron microscopy (HR-SEM) illuminates the heterogenous, morphological profile that exists on the surface of BEC membranes and the relationship between these ultrastructures during the molecular construction of the PC space between adjacent BECs. In this study developing BEC monolayers were grown on mixed, cellulose esters insert membranes in a bicameral system. BEC monolayers were fixed in 2.5% glutaraldehyde, hydrated, critically dried, and sputter-coated, for imaging utilizing HR-SEM. This study, for the first time, showed membrane-bound exosomes were attached to the plasma membrane surfaces of the BECs. The exosomes were characterized as small membrane-bound, nano-sized exosomes (30–300 nm). Based on their membrane morphology and anatomical structure, exosomes appear to possess two distinct functions, namely: paracrine secretion and nanotube construction between adjacent BECs, during in vitro barrier genesis. The HR-SEM micrographs in conjunction with the Tipifarnib inhibition of exosome formation, suggests that brain capillary endothelial exosomes play a prominent role in the bilateral signaling, which contribute to the regulation of the permeability of the BBB. Given that blood-brain barrier permeability has been implicated in the progression of many neurodegenerative pathologies, the role of these exosomes and TUNTs posits the capacity of these structures to exacerbate neuropathologies that implicate BBB permeability. These findings could lead to the development of novel treatment interventions and moreover, the characterization of BBB exosomes may be a reliable target for identifying therapeutic biomarkers in neurodegenerative disease. Conversely, the presence of BBB exosomes raises a critical enterprise to target the exosome-induced nanotubes as a vehicle for transferring therapeutic treatments across the BBB.

Photoacoustic Force-Guided Precise and Fast Delivery of Nanomedicine with Boosted Therapeutic Efficacy.

Precise and efficient delivery of nanomedicine to the target site has remained as a major roadblock in advanced cancer treatment. Here, a novel photoacoustic force (PAF)‐guided nanotherapeutic system is reported based on a near‐infrared (NIR)‐absorbing semiconducting polymer (SP), showing significantly improved tumor accumulation and deep tissue penetration for enhanced phototherapeutic efficacy. The accumulation of nanoparticles in 4T1 tumor‐bearing mice induced by the PAF strategy displays a fivefold enhancement in comparison with that of the traditional passive targeting pathway, in a significantly shortened time (45 min vs 24 h) with an enhanced penetration depth in tumors. Additionally, a tumor‐bearing mouse model is rationally designed to unveil the mechanism, indicating that the nanoparticles enter solid tumors through enhanced transportation across blood vessel barriers via both inter‐endothelial gaps and active trans‐endothelial pathways. This process is specifically driven by PAF generated from the nanoparticles under NIR laser irradiation. The study thus demonstrates a new nanotherapeutic strategy with low dose, enhanced delivery efficiency in tumor, and boosted therapeutic efficacy, opening new doors for designing novel nanocarriers.

Antibody-activated trans-endothelial delivery of mesoporous organosilica nanomedicine augments tumor extravasation and anti-cancer immunotherapy

Tumor vasculature constitutes a formidable hurdle for the efficient delivery of cancer nanomedicine into tumors. The leverage of passive pathway through inter-endothelial gaps in tumor blood vessels might account for limited extravasation of nanomedicine into tumor microenvironment (TME). Herein, Annexin A1 antibody-installed mesoporous organosilica nanoplatforms carrying immunotherapeutics of anti-PD-L1 antibody (aPD-L1) and Indoximod are developed to target at caveolar Annexin-A1 protein of luminal endothelial cells and to trigger the active trans-endothelial transcytosis of nanomedicine mediated by caveolae. Such strategy enables rapid nanomedicine extravasation across tumor endothelium and relatively extensive accumulation in tumor interstitium. aPD-L1 and Indoximod release from aPD/IND@MON-aANN in a reduction-responsive manner and synergistically facilitate the intratumoral infiltration of cytotoxic T lymphocytes and reverse the immunosuppressive TME, thus demonstrating substantial anti-tumor efficacy in subcutaneous 4T1 breast tumors and remarkable anti-metastatic capacity to extend the survival of 4T1 tumor metastasis model. Moreover, aPD/IND@MON-aANN nanomedicine also exhibits distinct superiority over the combination therapy of free drugs to potently attenuate the progression of urethane-induced orthotopic lung cancers. Collectively, aPD/IND@MON-aANN nanoplatforms with boosted delivery efficiency via antibody-activated trans-endothelial pathway and enhanced immunotherapeutic efficacy provides perspectives for the development of cancer nanomedicines.

Presence of claudins mRNA in the brain: Selective modulation of expression by kindling epilepsy

In the central nervous system, the junctional types that establish and maintain tissue architecture include gap junctions, for cytoplasmic connectivity, and tight junctions, for paracellular and/or cell polarity barriers. Connexins are the integral membrane proteins of gap junctions, whereas occludin and members of the multigene family of claudins form tight junctions. In the brain, there are no transendothelial pathways, as continuous tight junctions are present between the endothelial cells. Thus, they provide a continuous cellular barrier between the blood and the insterstitial fluid. However, several brain pathologies, including epilepsy, are known to alter the permeability of the blood–brain barrier and to cause edema. Therefore, since claudins, as constitutive proteins of tight junctions are likely candidates for modulation under pathological states, we explored their normal pattern of expression in the brain and its modulation by seizures. We found that several members of this family are normally expressed in the hippocampus and cortex. Interestingly, claudin-7 is expressed in the hippocampus but not in the cortex. On the other hand, the expression of claudin-8 is selectively down-regulated in the hippocampus as kindling evolves. These results link for the first time the modulation of expression of a tight junction protein to abnormal neuronal synchronization that could probably be reflected in permeability changes of the blood–brain barrier or edema.

Transendothelial Pathways in Venular Microvessels Exposed to Agents Which Increase Permeability: The Gaps in Our Knowledge

Transendothelial Pathways in Venular Microvessels Exposed to Agents Which Increase Permeability: The Gaps in Our Knowledge

Transendothelial Pathways in Venular Microvessels Exposed to Agents Which Increase Permeability: The Gaps in Our Knowledge

Transendothelial Pathways in Venular Microvessels Exposed to Agents Which Increase Permeability: The Gaps in Our Knowledge

Transendothelial Pathways in Venular Microvessels Exposed to Agents Which Increase Permeability: The Gaps in Our Knowledge

Transendothelial Pathways in Venular Microvessels Exposed to Agents Which Increase Permeability: The Gaps in Our Knowledge

Transendothelial Pathways in Venular Microvessels Exposed to Agents Which Increase Permeability: The Gaps in Our Knowledge

Transendothelial Pathways in Venular Microvessels Exposed to Agents Which Increase Permeability: The Gaps in Our Knowledge

Permselectivity of Angiogenic Microvessels Following Alteration of the Endothelial Fiber Matrix by Oligosaccharides

The endothelial glycocalyx, which is composed of integral and peripheral glycoconjugates, forms a fibrous matrix that confers macromolecular sieving properties on the microvascular wall. Changes in pore size within the matrix may regulate macromolecular access to the paracellular and/or vesicular transendothelial pathways. We tested the hypothesis that modifications of the endothelial glycocalyx might play a role in the ontogeny of endothelial permselectivity in proliferating microvessels of the chick chorioallantoic membrane (CAM). Accordingly, we evaluated the effects ofDolichos biflorusagglutinin (DBA) orArachis hypogaeaagglutinin (PNA) lectin binding, andN′N′diacetylchitobiose or hydroxyethyl starch polysaccharide (HES) incorporation on CAM endothelial restriction of FITC-dextrans 40 or 150 at Days 4.5 and 5.0 of development. Extravasation of FITC-dextrans was determined by recording their perivascular interstitial intensities. Following DBA, PNA, andN′N′diacetylchitobiose administration, interstitial accumulation of the tracers near first-order pre- and postcapillaries, and surrounding the capillaries, was similar to that of controls at both Days 4.5 and 5.0. At Day 4.5, pretreatment with HES significantly decreased extravasation of FITC-dextran 40. Thus, retention of HES molecules within the glycocalyx might tighten the matrix, and reduce access of dextran 40 to transendothelial pathways across the angiogenic microvessels.

Degranulation of mast cells in the chick chorioallantoic membrane does not increase macromolecular extravasation during normal angiogenesis.

To test the hypothesis that stimulation of mast cell degranulation during normal angiogenesis in the chick chorioallantoic membrane (CAM) serves to acutely activate macromolecular transendonelial pathways. Using shell-less cultures of 6-day-old chick embryos, efflux of fluorescein isothiocyanate (FITC)-dextran 150 from CAM microvessels was evaluated, after applications of compound 48/80 or exogenous histamine, by computer-assisted videodensitometric analyses. Real-time confocal imaging enabled optical differentiation of first-order pre- and postcapillaries from the capillary networks. Cytologic ultrastructure of the microvascular units was also evaluated. Although endogenous histamine was measurable and its concentration was increased after application of compound 48/80, segmental endothelia of the CAM microvascular units consistently restricted extravasation of FITC-dextran 150. Furthermore, intravascular injections of histamine also failed to induce interendothelial gap formation or macromolecular flux across the segmental CAM endothelia. These results are consistent with the interpretation that histamine-activated transendothelial pathways are inactive in the CAM at day 6. Whether such inactivity serves to retard facilitation of CAM angiogenesis by activated mast cells remains to be tested.

Mast Cell Activation Accelerates the Normal Rate of Angiogenesis in the Chick Chorioallantoic Membrane

Using shell-less cultures of chick embryos, the effect of mast cell degranulation on normal rates of angiogenesis in the chorioallantoic membrane (CAM) was recorded from Day 6 to Day 9. Based on the centripetal ordering method with capillaries as the initial point of reference, the first two orders of pre- and postcapillary microvessels were evaluated. After infusion of compound 48/80 into the allantoic cavity, the normal proliferation rate of order-1 pre- and postcapillaries was markedly increased. Increases of second-order vessel numerical densities (number/cm2) were less than those of the order-1 vessels. Thus, length densities (mean length × numerical density) were increased for the order-1 vessels only. During the accelerated rate of CAM angiogenesis, endothelial restriction of FITC–dextran 150 remained uniformly high. Hence, secreted mast cell products likely worked synergistically with endogenous CAM angiogenic factors between Days 6 and 9, without increasing rates of macromolecular extravasation. However, histamine injection into the allantoic cavity at Day 10, in conjunction with calcium supplementation of the shell-less cultures at Day 9, elevated the rate of FITC–dextran 150 extravasation. Whether differentiation of this transendothelial pathway is required for enhancement of CAM angiogenesis after Day 10 remains uncertain.

Endothelial permeability under physiological and pathological conditions

Due to their special position at the tissue—blood interface, the endothelial cells are endowed with two mechanisms for handling plasma molecules: one, receptor-mediated endocytosis, which supplies nutrients for the cell's own metabolic needs; the other, direct transendothelial transport of material from plasma to the intima of arteries. Under normal physiological conditions, the latter transendothelial pathway is far more important than the endocytic one. Routes for exchange of both small and large plasma molecules include the clefts between adjacent endothelial cells (paracellular) and plasmalemmal vesicles. Paracellular transport is restricted to molecules smaller than 4 nm in diameter, and is the pathway for water filtration. Larger molecules, as large as 30 nm in diameter, are transported through plasmalemmal vesicles. Changes in endothelial permeability occur during inflammation, hypertension or diabetes.

Fine structural features of the cerebral microvasculature in hydrocephalic human infants: correlated clinical observations.

Four of 30 human cerebral cortex biopsies from infants ranging from four days to about ten years treated for hydrocephalus by shunt operations are described paying special attention to the vascular structures. The biopsy specimens were studied in semi-thin and ultrathin sections. Attention is drawn to the role of pinocytotic vesicles found in capillaries and smaller vessels as a possible transcellular route for the hydrocephalic oedema resolution. No intercellular dehiscences or the so called blisters were observed. With the passage of time, the number of membrane bound vesicles increased and arrays of pinocytotic vesicles were discernible both on the abluminal as well as luminal aspect of the capillary wall. Additionally, larger vacuoles containing electron-dense material, apparently undergoing autolysis, were also detected. The basal lamina was of uneven thickness and at places duplicated. Hypertrophic pericytes exhibited remarkable oedamatous changes, increased vesicular or vascular transport, demonstrating pericyte brain-barrier dysfunction. Swelling of the astrocytic end-feet bordering the capillaries was remarkable. These findings indicate that the CSF or oedema fluid is absorbed into the vascular system via a transendothelial pathway.

Theoretical modeling of fluid transport through endothelial junctions.

This paper will review some recent theoretical models of the fluid transport through an endothelial cell layer from the standpoint of hydrodynamic theories based on ultrastructural information. These theories attempt to use realistic models of the observed morphology of endothelial cell junctions and realistic estimates of the forces, pressures, and flows that can take place in narrow slits and porous media. The classical system of transendothelial pathways consisting of a small pore system, medium size pores and a few large pores for the passage of macromolecules is well supported by experimental data on filtration measured macroscopically. Excellent summaries of this theory and data are available (eg. Curry, 1984; Michel, 1984; Taylor and Granger,1984).1,2,3 However, the possible ultrastructural features of endothelial cells which correspond to the various sizes of pores continue to be refined by electron microscopic and other studies. One of the most striking developments is the demonstration that most vesicles of endothelial cells are attached to cell walls.4,5 This has led to a concept of a lesser role of vesicular transport and a greater emphasis on the filtration through the endothelial cell junctions.6,7 In these more recent theories, the classical system of pores is replaced by a system of narrow slits which realistically represent the endothelial cleft and tight junctions, some wider tortuous gaps between protein junctional strands, and transiently open junctions which are associated with the replacement phase of dying endothelial cells.8

Passage of ions and dextran molecules across the rete mirabile of the eel. The effects of charge.

The countercurrent-perfused rete mirabile of the eel is a preparation in which capillary permeability values can be determined accurately in broadscale fashion. To provide insight into charge effects on transendothelial passage, the permeability values of small inorganic cations (labeled sodium and rubidium, and stable potassium) and anions (labeled chloride, iodide, sulfate, and ferrocyanide) were compared to those expected for neutral solutes with approximately matched diffusion coefficients, and that of a neutral dextran fraction was compared to that of a negatively charged dextran sulfate with a similar diffusion coefficient. In the small ion experiments, the labeled iodide values were unexpectedly high, apparently due to the contamination of the labeled iodide solution with I-3. The permeabilities of the rest of the ions clustered at a level about 0.5 of the values which would have been expected for neutral solutes with similar diffusion coefficients. The decrement was interpreted to reflect the presence of both positive and negative charges along the transendothelial pathway, which effectively decrease the dimensions of the limiting part of the pathway for the charged microions relative to that accessible to comparable nonelectrolytes. The larger negatively charged dextran sulfate was also reduced in its passage, in comparison with its matching neutral dextran; this was taken to indicate the presence of a larger scale average net negative charge along its pathway. The data indicate the presence of a staggering of positive and negative charges along the transendothelial pathway accessible to the microions, and a net negative charge in the more restricted part of the pathway available to the dextrans.

Immunoelectron microscopic study of plasma protein pathways through the different segments of the rat intestinal vasculature

Nineteen Brown-Norway (BN) rats received intravenous injections of sheep anti-peroxidase (HRP) antibodies. Four BN rats were immunized to HRP. The anti-HRP antibodies were used to trace permeability pathways of large physiological molecules across different vessels of the small intestine. This organ was chosen because of the possibility of convenient "in situ" fixation and for the diversity of vessel types it contains. It was shown that: 1) There were no obvious transendothelial pathways in arteries with an elastic lamina. 2) The antibodies readily crossed fenestrated capillaries through the fenestrae. 3) There were two possible pathways through muscle capillaries and pericytic venules, namely transcytoplasmic vesicular "cactus-like" channels and interendothelial junctions. 4) Interendothelial permeability was a possible factor in veins with an elastic lamina. 5) Lymphatics were readily permeable through intercellular junctions and cytoplasmic vesicles.

Estimation of capillary permeability of inulin, sucrose and mannitol in rat brain cortex.

The present study analyzed the brain uptake of the differently sized hydrophilic molecules 14C-inulin, 14C-sucrose and 14C-mannitol. constant tracer concentrations were maintained in blood plasma after renal ligation. Accumulation of the indicators was measured in brain cortex during the approach to steady-state. At the conclusion of infusion periods of 5, 10, 15, 30 and 60 min, samples of cerebral cortex were analyzed for radioactivity. Fractions attributable to blood in the tissue were subtracted and time-dependent apparent distribution volumes of the indicators in the tissue were estimated. The brain level did not rise to more than 1--2% of that in the plasma for inulin and sucrose and to 6--7% for mannitol. The explanation for this could be a combination of restricted penetration from blood into brain and a sink effect of cerebrospinal fluid (csf). to determine the removal of the indicators into csf, ventriculo-cisternal perfusion was performed during the period of tracer uptake in the the tissue; the rate of passage of the indicators into perfusion fluid was found to be negligible in comparison to the rates of uptake in the tissue. As diffusion in the extracellular space could limit uptake rates from blood to brain, estimates of diffusion limited half-times were also made. Calculations showed that the major hindrance to indicator uptake in the tissue is located in the wall of the brain capillaries; thus the uptake data permit calculation of brain capillary permeability. The half-times for the distribution of the indicators in their equilibrated tissue distribution spaces were used to estimate brain capillary permeability. By comparison with the aqueous diffusion coefficients of the indicators it is concluded that the substances in the molecular weight range of 182 to 5 500 daltons are subjected to restricted diffusion during the passage of the blood-brain barrier and that in addition non-discriminative transendothelial pathways are available.

Lymphatic transport pathways during volume expansion

Morphological evidence on lymphatic transport was derived qualitatively and quantitatively in control and volume-expanded rats in order to identify any changes in transendothelial pathways which accompany enhanced lymph formation. Volume expansion was induced by intravenous mannitol and by intraperitoneal hypertonic or hypotonic saline (10% of body wt). Horseradish peroxidase and ferritin were used as macromolecular tracers. Volume expansion had little effect upon intraendothelial cytoplasmic components. Intracytoplasmic vesicles contained tracer molecules in control and experimental groups and quantitative analysis revealed only one significant difference between the groups—a reduction in vesicular volume and numerical densities in animals receiving mannitol. The major changes between control and experimental animals were in the presence of attenuated areas of cytoplasm, widening of intercellular pathways, and the presence of open regions in the expanded group. In these respects there was little qualitative difference among the three experimental groups, although some quantitative differences were recorded. It was concluded that vesicular transport plays a significant role in translymphatic protein movement and that when the interstitial fluid load to lymphatics is increased the excess fluid preferentially enters between adjacent endothelial cells or diffuses across areas of extreme attenuation. Open regions or junctions were considered to play little if any role in lymph formation under control conditions, but to achieve increasing importance with increases in fluid movement.