Increased microvascular permeability in mice lacking Epac1 (Rapgef3).

AIM: Maintenance of the blood and extracellular volume requires tight control of endothelial macromolecule permeability, which is regulated by cAMP signalling. This study probes the role of the cAMP mediators rap guanine nucleotide exchange factor 3 and 4 (Epac1 and Epac2) for in vivo control of microvascular macromolecule permeability under basal conditions. METHODS: Epac1-/- and Epac2-/- C57BL/6J mice were produced and compared with wild-type mice for transvascular flux of radio-labelled albumin in skin, adipose tissue, intestine, heart and skeletal muscle. The transvascular leakage was also studied by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) using the MRI contrast agent Gadomer-17 as probe. RESULTS: Epac1-/- mice had constitutively increased transvascular macromolecule transport, indicating Epac1-dependent restriction of baseline permeability. In addition, Epac1-/- mice showed little or no enhancement of vascular permeability in response to atrial natriuretic peptide (ANP), whether probed with labelled albumin or Gadomer-17. Epac2-/- and wild-type mice had similar basal and ANP-stimulated clearances. Ultrastructure analysis revealed that Epac1-/- microvascular interendothelial junctions had constitutively less junctional complex. CONCLUSION: Epac1 exerts a tonic inhibition of in vivo basal microvascular permeability. The loss of this tonic action increases baseline permeability, presumably by reducing the interendothelial permeability resistance. Part of the action of ANP to increase permeability in wild-type microvessels may involve inhibition of the basal Epac1-dependent activity.

Multimodal approach to assess tumour vasculature and potential treatment effect with DCE-US and DCE-MRI quantification in CWR22 prostate tumour xenografts.

The aim of this study was to compare intratumoural heterogeneity and longitudinal changes assessed by dynamic contrast-enhanced ultrasound (DCE-US) and dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in prostate tumour xenografts. In vivo DCE-US and DCE-MRI were obtained 24 h pre- (day 0) and post- (day 2) radiation treatment with a single dose of 7.5 Gy. Characterization of the tumour vasculature was determined by Brix pharmacokinetic analysis of the time-intensity curves. Histogram analysis of voxels showed significant changes (p < 0.001) from day 0 to day 2 in both modalities for kep , the exchange rate constant from the extracellular extravascular space to the plasma, and kel , the elimination rate constant of the contrast. In addition, kep and kel values from DCE-US were significantly higher than those derived from DCE-MRI at day 0 (p < 0.0001) for both groups. At day 2, kel followed the same tendency for both groups, whereas kep showed this tendency only for the treated group in intermediate-enhancement regions. Regarding kep median values, longitudinal changes were not found for any modality. However, at day 2, kep linked to DCE-US was correlated to MVD in high-enhancement areas for the treated group (p = 0.05). In contrast, correlation to necrosis was detected for the control group in intermediate-enhancement areas (p < 0.1). Intratumoural heterogeneity and longitudinal changes in tumour vasculature were assessed for both modalities. Microvascular parameters derived from DCE-US seem to provide reliable biomarkers during radiotherapy as validated by histology. Furthermore, DCE-US could be a stand-alone or a complementary technique.

Image-based assessment of microvascular function and structure in collagen XV- and XVIII-deficient mice.

Collagen XV and XVIII are ubiquitous constituents of basement membranes. We aimed to study the physiological roles of these two components of the permeability barrier non-invasively in striated muscle in mice deficient in collagen XV or XVIII by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Structural information was obtained with transmission electron microscopy (TEM). MR data were analysed by two different analysis methods to quantify tissue perfusion and microcirculatory exchange parameters to rule out data analysis method-dependent results. Control mice (C57BL/6J Ola/Hsd strain) or mice lacking either collagen XV (Col15a1(-/-)) or XVIII (Col18a1(-/-)) were included in the study. MR images were acquired using a preclinical system using gadodiamide (Gd-DTPA-BMA, molecular weight 0.58 kDa) as a tracer. Exchange capacity (permeability (P)-surface area (S) product relative to blood flow (FB)) was increased in test mice compared to controls, but the contributions from P, S, and FB were different in these two phenotypes. FB was significantly increased in Col18a1(-/-), but slightly decreased in Col15a1(-/-). PS was significantly increased only in Col18a1(-/-) even though P was increased in both phenotypes suggesting S might also be reduced in Col15a1(-/-) mice. Immunohistochemistry and electron microscopy demonstrated alterations in capillary density and morphology in both knockout mouse strains in comparison to the control mice. Both collagen XV and XVIII are important for maintaining normal capillary permeability in the striated muscle. DCE-MRI and the perfusion analyses successfully determined microvascular haemodynamic parameters of genetically modified mice and gave results consistent with more invasive methods.

Lowered albumin extravasation rate in heart but not in other organs in beta3-integrin-deficient mice.

AIM: The vascular protein permeability is dependent on the integrity of the vascular wall. The heart capillaries in male mice lacking beta3 integrins have an immature phenotype. Previously, we have demonstrated a role for alphavbeta3 integrins in control of interstitial fluid pressure (Pif) and thereby in the fluid flux during inflammation. We wanted to explore a possible role for alphavbeta3 integrins in controlling capillary protein permeability during control situation and inflammation. METHODS: We performed double-tracer and microdialysis experiments on beta3-integrin-deficient mice and wild type control mice. We also measured blood pressure and heart rate in the two mice strains. RESULTS: We found reduced albumin extravasation (during 25 min) in the heart capillaries (0.053 +/- 0.003 vs. 0.087 +/- 0.009 mL g(-1) dw, P < 0.05), and an increased cardiac mass/body weight (5.3 x 10(-3) +/- 0.3 x 10(-3) vs. 3.8 x 10(-3) +/- 0.1 x 10(-3), P < 0.01) in the beta3-integrin-deficient mice (n = 6) compared with the controls (n = 6). Heart rate and blood pressure were the same in mice with and without beta3-integrins. No difference in permeability was found in other tissues studied, or under local inflammation. CONCLUSION: These results show a function for the alphavbeta3 integrin in the regulation of protein permeability, selective for the heart capillaries.

Hyperoxia retards growth and induces apoptosis, changes in vascular density and gene expression in transplanted gliomas in nude rats.

This study describes the biological effects of hyperoxic treatment on BT4C rat glioma xenografts in vivo with special reference to tumor growth, angiogenesis, apoptosis, general morphology and gene expression parameters. One group of tumor bearing animals was exposed to normobaric hyperoxia (1 bar, pO(2) = 1.0) and another group was exposed to hyperbaric hyperoxia (2 bar, pO(2) = 2.0), whereas animals housed under normal atmosphere (1 bar, pO(2) = 0.2) served as controls. All treatments were performed at day 1, 4 and 7 for 90 min. Treatment effects were determined by assessment of tumor growth, vascular morphology (immunostaining for von Willebrand factor), apoptosis by TUNEL staining and cell proliferation by Ki67 staining. Moreover, gene expression profiles were obtained and verified by real time quantitative PCR. Hyperoxic treatment caused a approximately 60% reduction in tumor growth compared to the control group after 9 days (p < 0.01). Light microscopy showed that the tumors exposed to hyperoxia contained large "empty spaces" within the tumor mass. Moreover, hyperoxia induced a significant increase in the fraction of apoptotic cells ( approximately 21%), with no significant change in cell proliferation. After 2 bar treatment, the mean vascular density was reduced in the central parts of the tumors compared to the control and 1 bar group. The vessel diameters were significantly reduced (11-24%) in both parts of the tumor tissue. Evidence of induced cell death and reduced angiogenesis was reflected by gene expression analyses.Increased pO(2)-levels in experimental gliomas, using normobaric and moderate hyperbaric oxygen therapy, caused a significant reduction in tumor growth. This process is characterized by enhanced cell death, reduced vascular density and changes in gene expression corresponding to these effects.

High-dose, short-term, anti-inflammatory treatment with dexamethasone reduces growth and augments the effects of 5-fluorouracil on dimethyl-alpha-benzanthracene-induced mammary tumors in rats.

OBJECTIVE: To evaluate the effects of dexamethasone (DXM) alone or in combination with 5-fluorouracil (5-FU) on dimethyl-alpha-benzanthracene (DMBA)-induced mammary tumors in rats. MATERIAL AND METHODS: Female Sprague-Dawley rats were divided into 4 groups receiving: 1) saline (controls), 2) DXM (3 mg/kg), 3) 5-FU (1.5 mg/kg) and 4) DXM and 5-FU combined. The drugs were given i.p. every day for 4 days. Interstitial fluid pressure (Pif) and tumor growth were determined in all tumors on days 1, 5 and 7 using the "wick-in-the needle" technique and by external size measurements, respectively. Vessel density and inflammatory cell infiltration of tumor tissue were analyzed by immunohistochemistry. RESULTS: DXM treatment significantly retarded tumor growth and reduced Pif. Treatment with a combination of DXM and 5-FU reduced tumor size significantly more than any of the agents alone (p<0.01-0.001). Enhanced uptake of 5-FU by DXM treatment was demonstrated by microdialysis. There were no differences in the density of CD31-positive vessels after DXM or 5-FU treatment, but inflammatory cell infiltration of tumor tissue was significantly reduced after DXM treatment. CONCLUSIONS: Our data suggest that DXM may be beneficial as an adjuvant to chemotherapy in the treatment of mammary cancer by increasing the uptake of 5-FU in the tumor.

Changes in plasma protein extravasation in rat skin during inflammatory challenges evaluated by microdialysis.

Docetaxel and prostaglandin E1 (PGE1) increase transcapillary albumin extravasation and reduce interstitial fluid pressure in the skin. In this study the microdialysate concentration (Cm) of 125I-labeled human serum albumin (125I-HSA) and different-sized endogenous plasma proteins (EPP) was compared to evaluate changes in transcapillary extravasation of plasma proteins. 125I-HSA was also used to estimate changes in the specific activity of albumin. Extravasation of 125I-HSA and EPP from plasma to interstitium in the rat skin was compared during continuous administration of docetaxel and PGE1 by using microdialysis in anesthetized rats. Also, 20 ml of Ringer solution (RS) were injected intravenously during 10 min in a separate group. Two hollow plasmapheresis fibers (3 cm, cut off 3,000 kDa), one acting as control, were placed subcutaneously on the back skin and perfused with RS (5 microl/min, 140 min, collected every 10 min). The size of the different EPP was estimated to be 73, 65, 56, 47, and 39 A, separated by a size-exclusion high-performance liquid chromatography column and quantified by UV detection (280 nm). Docetaxel (0.5 mg/ml, n = 5) increased Cm of 125I-HSA and EPP of sizes 73, 65, 56, and 39 A significantly (P < 0.05) compared with control. PGE1 (20 microg/ml, n = 6) increased Cm of 125I-HSA significantly (P < 0.05) but none of the different-sized EPP was increased compared with control. Intravenous RS (20 ml, n = 6) increased Cm of 125I-HSA and increased all the different-sized EPP significantly (P < 0.05) compared with control. Although the microdialysis method is able to monitor qualitative changes in capillary permeability, a quantitative determination of the capillary reflection coefficient or permeability-surface area product was not possible, because steady state between plasma and dialysate was not achieved during the measurement period. The different pattern of extravasation of EPP and 125I-HSA after docetaxel, PGE1, and RS indicates increased interstitial transport rate and/or increased capillary permeability after docetaxel and RS, whereas PGE1 seems to increase transcapillary fluid flux without altering the permeability.

Platelet activating factor (PAF) increases plasma protein extravasation and induces lowering of interstitial fluid pressure (P) in rat skin.

AIM: To investigate the ability of the microdialysis technique to measure capillary selectivity of different sized plasma proteins induced by local administration of platelet activating factor (PAF). METHODS: We used hollow plasmapheresis fibres with 3 cm membrane (cut off 3000 kDa) placed on the back of anaesthetized rats. RESULTS: Platelet activating factor (50 microg mL(-1)) administered locally via the fibre, increased extravasation of radiolabelled 125I-HSA from plasma to the microdialysis fibre by approximately 900% compared both to baseline and the control fibre within 70 min (n = 6, P < 0.05). The extravasation in the control fibre did not change over time. HPLC measurement of plasma proteins in the microdialysis perfusate also demonstrated decreased capillary selectivity for proteins in the diameter range of 73 A, 56 A and 39 A after local administration of PAF (n = 6, P < 0.05). PAF also significantly lowered interstitial fluid (P(if)) pressure after subcutaneous administration (50 microg mL(-1)). Mean arterial pressure (MAP) after intravenous injection of PAF (0.4 microg kg(-1)) fell instantly by about 50 mmHg, and stabilized at 50 mmHg after 15 min (n = 6). MAP was unaltered when PAF was given through the microdialysis fibre (n = 4). Both total tissue water (TTW) and extravasation of albumin, measured as the plasma-to-tissue clearance (E-alb) showed a significant increase after PAF (n = 7, P < 0.05). CONCLUSIONS: The present study demonstrates that PAF induces plasma protein extravasation and decrease capillary selectivity of different sized plasma proteins. It also increases transcapillary fluid flux, and lowers P(if), indicating a role for PAF in the interstitium for generation of transcapillary transport of water and large molecules followed by formation of oedema.

Effect of the cytoskeletal fixation agent phalloidin on transcapillary albumin transport and interstitial fluid pressure following subdermal prostaglandin E1 administration in the rat.

AIM: Lowering of interstitial fluid pressure (Pif) facilitates fluid filtration across the capillary membrane and oedema formation in acute inflammation. The cellular mechanism behind this lowering of Pif involves beta1-integrins mediating contact between dermal cells and the extracellular matrix fibres, and also the cell cytoskeleton as disruption of actin filaments using cytochalasin-D induced a lowering of Pif and oedema formation. Fixation of actin with phalloidin attenuates oedema formation and abolishes lowering of Pif in anaphylaxis in the rat. The objective of this study was to determine whether phalloidin modifies lowering of Pif and albumin extravasation in rat skin also after prostaglandin E1 (PGE1). METHODS: Pif was measured using micropipettes connected to a servo-controlled counterpressure system. Microvascular permeability was estimated as the albumin extravasation (Ealb) using radiolabelled human serum albumin. RESULTS: Subdermal injection of PGE1 (0.85 mg mL-1) lowered Pif from -0.8 +/- 0.8 mmHg (SD) in control to -3.5 +/- 0.9 mmHg (P < 0.05) within 30 min. Pre-treatment with phalloidin (500 microg kg-1) before PGE1 resulted in Pif of -1.7 +/- 1.0 mmHg (P < 0.05 compared with PGE1). Ealb after subdermal saline was 0.07 +/- 0.04 mL g-1 DW and increased to 0.32 +/- 0.32 mL g-1 DW with PGE1 (P < 0.05) but was unaffected by pre-treatment with phalloidin given before PGE1 0.32 +/- 0.35 mL g-1 DW (P > 0.05 compared with PGE1 alone). CONCLUSION: These results are consistent with the concept that the cytoskeleton actin filaments participate in control of Pif.

Continuous measurements of plasma protein extravasation with microdialysis after various inflammatory challenges in rat and mouse skin.

This study describes the use of microdialysis technique for continuous measurement of plasma protein extravasation (PPE) in rat and mouse skin with drug application either intravenously or via the microdialysis fiber. Hollow plasmapheresis fibers (3-cm length, 0.4-mm diameter, cutoff 3,000 kDa) were placed subcutaneously on the back of anesthetized mice and rats. Intravenous injection of dextran (Macrodex, 60 mg/ml) increased PPE by 355% from baseline within 30 min in rats with ligated kidneys (n = 6; P < 0.05) but not in animals with intact kidneys. Phalloidin (500 microg/kg iv 40 min before dextran, n = 6; P < 0.05) did not change the response to dextran in either group. Animals receiving PGE1, compound 48/80 (mice), paclitaxel, docetaxel, and cremophor EL via the microdialysis fiber were also provided with a control fiber receiving vehicle. Both rats and mice had constant PPE in the control fiber, and there was no change in PPE in the NaCl-treated groups (rats, n = 4; mice, n = 6). Application via the fiber of PGE1 (20 microg/ml), compound 48/80 (mice; 4 mg/ml), and docetaxel (0.5 mg/ml) increased PPE compared with baseline within 60 min by 139% (n = 6; P < 0.05), 273% (n = 6; P < 0.05), and 325% (n = 5; P < 0.05), respectively. Phalloidin alone did not increase PPE (n = 5; P < 0.05). Pretreatment with phalloidin did not inhibit the increase after PGE1 or compound 48/80 but inhibited that after docetaxel (n = 6). Paclitaxel (0.6 mg/ml, n = 5) or vehicle (Cremophor) (n = 5) gave no increase in PPE. The results demonstrate that microdialysis can be used to continuously measure changes in PPE after inflammatory challenges in skin of rats and mice.

Preliminary model of fluid and solute distribution and transport during hemorrhage.

The distribution and transport of fluid, ions, and other solutes (plasma proteins and glucose) are described in a mathematical model of unresuscitated hemorrhage. The model is based on balances of each material in both the circulation and its red blood cells, as well as in a whole-body tissue compartment along with its cells. Exchange between these four compartments occurs by a number of different mechanisms. The hemorrhage model has as its basis a validated model, due to Gyenge et al., of fluid and solute exchange in the whole body of a standard human. Hypothetical but physiologically based features such as glucose and small ion releases along with cell membrane changes are incorporated into the hemorrhage model to describe the system behavior, particularly during larger hemorrhages. Moderate (10%-30% blood volume loss) and large (> 30% blood loss) hemorrhage dynamics are simulated and compared with available data. The model predictions compare well with the available information for both types of hemorrhages and provide a reasonable description of the progression of a large hemorrhage from the compensatory phase through vascular collapse.

Fluid pressure in human dermal fibroblast aggregates measured with micropipettes.

Previous studies indicated that connective tissue cells in dermis are involved in control of interstitial fluid pressure (Pif). We wanted to develop and characterize an in vitro model representative of loose connective tissue to study dynamic changes in fluid pressure (Pf) over a time course of a few minutes. Pf was measured with micropipettes in human dermal fibroblast cell aggregates of varying size (<100- and >100-microm diameter) and age (days 1-4) kept at different temperatures (approximately 15, 25, and 35 degrees C). Pressures were measured at different depths of micropipette penetration and after treatment with prostaglandin E1 isopropyl ester (PGE1), latanoprost (PGF2alpha), and ouabain. Pf was positive (more than +2 mmHg) during control conditions and increased with increasing aggregate size (day 2), age (day 4 vs. day 1), temperature, and depth of micropipette penetration. Pf decreased from 2.9 to 2.0 mmHg during the first 10 min after application of 10 microl of 1 mM PGE1 (P < 0.001). Pf increased from 3.0 to 4.8 mmHg (P < 0.01) after administration of 10 microl of 1.4 microM ouabain and from 3.1 to 4.4 mmHg after addition of 5 microl of 1.42 mM PGF2alpha (P > 0.05). In conclusion, we have developed and validated a new in vitro method for studying fluid pressure in loose connective tissue elements with the advantage of allowing reliable and rapid screening of substances that have a potential to modify Pf and studying in more detail specific cell types involved in control of Pf. This study also provides evidence that fibroblasts in the connective tissue can actively modulate Pf.

Lowering of interstitial fluid pressure in rat trachea after substance P alone and in combination with calcitonin gene-related peptide.

UNLABELLED: Neurogenic inflammation is mediated following a release of sensory neuropeptides including calcitonin gene-related peptide (CGRP) and substance P (SP). The release of peptides can be mediated chemically with capsaicin, or electrically by stimulation of the vagal nerve, both inducing vasodilation, plasma protein extravasation and lowering of interstitial fluid pressure (Pif) which will contribute to the enhancement of oedema formation. AIM: Lowering of Pif has previously been demonstrated following intravenous (i.v.) treatment with CGRP, but it was not possible to demonstrate that SP had this effect under the same condition. METHODS: Micropuncture measurements of Pif in the submucosa, without opening of the trachea, was conducted on rats anaesthetized with pentobarbital sodium (50 mg kg-1) and cardiac arrest was induced with i.v. KCl. RESULTS: Pif in vehicle-treated animal averaged -1.7 +/- 0.4 (SD) mmHg (n = 9). Intravenous injection of SP induced significant lowering of Pif compared with control, both at low dose (0.47 nmol kg-1 body weight) with 1 min distribution time (P < 0.007, -4.2 +/- 2.3 mmHg) and at high dose with seconds of distribution time (P < 0.03, -4.2 +/- 1.6 mmHg). The same response was observed after treatment with SP co-injected with CGRP. CONCLUSIONS: Substance P alone or in combination with CGRP is able to induce a rapid lowering of Pif showing that this peptide is a potent agent in increasing the hydrostatic driving pressure initially transporting fluid into the tissue during an acute inflammatory reaction.

New and active role of the interstitium in control of interstitial fluid pressure: potential therapeutic consequences.

Here we present recent data indicating that the present view of the interstitium as a passive fluid reservoir has to be revised. The connective tissue cells and extracellular matrix have a role in the control of P(if) and a fundamental role in the rapid development of edema in burns and in the initial swelling in inflammation by generating a lowering of interstitial fluid pressure. In this process, the beta1-integrin system seems to provide a common pathway by which the cells can lower as well as raise P(if). Inflammatory swelling can be reversed by endo- and exogenous substances, thereby suggesting that the connective tissue can serve as a novel target for pharmacological intervention. Furthermore, the new knowledge in interstitial physiology on means to reduce interstitial fluid pressure may be of importance for drug delivery into solid tumors, where a high P(if) limits the uptake of therapeutic agents.

PGE1 induced transcapillary transport of 51Cr-EDTA in rat skin measured by microdialysis.

Interstitial fluid pressure (P(if)) is a key determinant in increasing the transcapillary driving pressure, pulling fluid from the microcirculation into the interstitial space at the onset of acute inflammatory reactions and the oedema formation associated with these. Prostaglandin E1 (PGE1) induces lowering of P(if) in rat skin which increases transcapillary transport of 51Cr-EDTA into the center of a tumor as measured by microdialysis. The aim of this study was twofold: First, to evaluate and develop the microdialysis technique thoroughly with regard to its suitability for investigating transcapillary water transport in rat skin using 51Cr-EDTA as a tracer. Secondly, to evaluate the effect of PGE1 on transcapillary transport of 51Cr-EDTA. This study demonstrates that PGE1 increases transcapillary transport of 51Cr-EDTA into skin interstitium. There were no significant differences between the experimental probe and the control probe when calculations from the entire experiment (90 min) were compared. On the other hand, significant differences were observed by examining the experiment in smaller time intervals. PGE1 increased transcapillary transport of 51Cr-EDTA during the first 15 min when administered through the microdialysis probe. This observation suggests that increased blood flow and/or permeability-surface area product are responsible for raising the transcapillary transport of 51Cr-EDTA, i.e. the transport is diffusion limited. Administration of PGE1 through the probe rather than around the probe resulted in less scatter between experiments than when PGE1 was injected subcutaneously around the probe.

Effects of normobaric hyperoxia on water content in different organs in rats.

Pulmonary oxygen toxicity is a dose-dependent effect on alveolar epithelial and endothelial cells resulting in pulmonary oedema. Any concomitant effects on systemic capillary endothelium would be expected to result in capillary leakage and an increase in the tissues' water content. Total tissue water (TTW) in different organs was therefore studied in freely moving rats exposed to 100% O2 at normobaric pressure for 24 or 48 h, and compared to air-breathing control rats. The TTW for the following tissues was measured: Trachea, left bronchus, left lung, left and right ventricle, left kidney, skin (left paw-hindlimb), skin (back of the rat), left brain, left eye and thigh muscle left side. There was a significant increase in TTW of the lung accompanied by pleural effusion after 48 h of oxygen exposure as expected in all exposed animals. There was a small increase in TTW of the paw only, and a small decrease or no change in other tissues after 24 and 48 h of exposure. We conclude that there is no evidence of systemic capillary dysfunction as measured by tissue water content after exposure to hyperoxia in a dosage causing pulmonary oedema.

Interstitial fluid pressure surrounding rat mesenteric venules during changes in fluid filtration.

The interstitial fluid pressure (P(isf)) has been measured in the exposed superfused mesenteries of anaesthetised rats using the micropipette servo-null technique. When mesenteries were superfused with Ringer-Locke solutions, P(isf) was close to atmospheric pressure with mean +/- S.E.M. values of -0.46 +/- 0.14 cmH(2)O (n = 22). Superfusing with paraffin oil did not alter P(isf) significantly, but P(isf) could be lowered considerably by removing fluid from the upper surface of the mesentery. Measurements of P(isf) were also made in the tissues immediately outside mesenteric venules as the pressure inside these vessels and the filtration of fluid through their walls was varied. No significant changes in perivascular P(isf) could be detected even though the intravascular pressure varied from 20 to 70 cmH(2)O. Addition of histamine or the mast cell degranulating agent compound 48/80 to the superfusate had no significant effect on P(isf). The findings are relevant to experiments on the permeability of single perfused mesenteric microvessels. They strengthen the assumption, which is made in these studies, that P(isf) is close to atmospheric pressure and does not change significantly with changes in the filtration and reabsorption of fluid through the vessel walls. Experimental Physiology (2001) 86.1, 33-38.

Effects of lactoferrin on rat dermal interstitial fluid pressure (Pif) and in vitro endothelial barrier function.

We recently demonstrated that intravenous (i.v.) injection of the iron-binding protein lactoferrin (Lf) followed by antilactoferrin (aLf) antibodies or iron-saturated Lf alone increased albumin extravasation in vivo in several tissues including skin. Increased driving pressure for blood-tissue exchange or direct effects of Lf on the endothelial barrier are possible mechanisms. We therefore, firstly, measured interstitial fluid pressure (Pif) in dermis of rats given 1 mg Lf i.v. followed 30 min later by aLf or saline and circulatory arrest 1 or 5 min thereafter and compared with controls. Secondly, transmonolayer passage of Evans blue labelled albumin (EB-albumin) was evaluated in porcine pulmonary artery endothelial cells exposed to iron-free or iron-saturated Lf (both 100 microg mL-1) in the absence and presence of 0.5 mM hydrogen peroxide. Pif increased significantly at 11-30 min following Lf to +2.1 +/- 0.3 and +1.7 +/- 0.2 mmHg at 11-20 and 21-30 min, respectively, compared with +0.1 +/- 0.2 mmHg before Lf (P < 0.05, n=25). Endothelial transmonolayer passage of EB-albumin during 3 h was not affected by iron-free or iron-saturated Lf neither in the absence nor presence of hydrogen peroxide that increased passage 3.5 times compared with controls. In conclusion, Lf-induced increase in albumin extravasation in rat skin is not explained by changes in Pif (because Lf raised Pif significantly) or direct effects of Lf on the endothelial barrier.

Cytochalasin D induces edema formation and lowering of interstitial fluid pressure in rat dermis.

The increased capillary fluid filtration required to create a rapid edema formation in acute inflammation can be generated by lowering the interstitial fluid pressure (P(IF)). The lowering of P(IF) appears to involve dynamic beta(1)-integrin-mediated interactions between dermal cells and extracellular matrix fibers. The present study specifically investigates the role of the cell cytoskeleton, i.e., the contractile apparatus of cells, in controlling P(IF) in rat skin as the integrins are linked to both the cytoskeleton and the extracellular matrix. P(IF) was measured using a micropuncture technique in the dorsal skin of the hind paw at a depth of 0.2--0.5 mm and following the induction of circulatory arrest with the intravenous injection of KCl in pentobarbital anesthesia. This procedure prevented the transcapillary flux of fluid and protein leading to edema formation in acute inflammation, which in turn can increase the P(IF) and therefore potentially mask a decrease of P(IF). Control P(IF) (n = 42) averaged -0.8 +/- 0.5 (means +/- SD) mmHg. In the first group of experiments, subdermal injection of 2 microl cytochalasin D, a microfilament-disrupting drug, lowered P(IF) to an average of -2.8 +/- 0.7 mmHg within 40 min postinjection (P < 0.05 compared with control). Subdermal injection of vehicle (10% DMSO in PBS or PBS alone) did not change the P(IF) (P > 0.05). Lowering of the P(IF) was not observed after the injection of colchicine or nocodazole, which specifically disrupts microtubuli in cultured cells. In the second group of experiments, 2 microl of cytochalasin D injected subdermally into rats with intact circulation increased the total tissue water (TTW) and albumin extravasation rate (E(ALB)) by 0.7 +/- 0.2 and 0.4 +/- 0.3 ml/g dry wt, respectively (P < 0.05 compared with vehicle). Nocodazole and colchicine did not significantly alter the TTW or E(ALB) compared with the vehicle (P > 0.05). Taken together, these findings strongly suggest that the connective tissue cells can participate in control of P(IF) via the actin filament system. In addition, the observation that subdermal injection of cytochalasin D lowered P(IF) indicates that a dynamic assembly and disassembly of actin filaments also occurs in the cells of dermal tissues in vivo.

Inhibition of platelet-derived growth factor receptors reduces interstitial hypertension and increases transcapillary transport in tumors.

Most solid malignancies display interstitial hypertension and a poor uptake of anticancer drugs. Platelet-derived growth factor (PDGF) and the cognate tyrosine kinase receptors are expressed in many tumors. Signaling through PDGFbeta receptors was shown recently to increase interstitial fluid pressure (IFP) in dermis after anaphylaxis-induced lowering of IFP. In this study, we show that treatment with the selective PDGF receptor kinase inhibitor, STI571, formerly known as CGP57148B, decreased the interstitial hypertension and increased capillary-to-interstitium transport of 51Cr-EDTA in s.c. growing rat PROb colonic carcinomas. Furthermore, treatment with an antagonistic PDGF-B oligonucleotide aptamer decreased interstitial hypertension in these tumors. PDGFbeta receptors were expressed in blood vessels and stromal cells but not in the tumor cells of PROb colonic carcinomas. Our study indicates a previously unrecognized role of PDGF receptors in tumor biology, although similar effects of PDGF on IFP have been demonstrated previously in the dermis. The data suggest interference with PDGF receptors, or their ligands, as a novel strategy to increase drug uptake and therapeutic effectiveness of cancer chemotherapy.