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Abstract

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  • 1
    Integrin-mediated endothelial cell-extracellular matrix adhesion plays a critical role in maintaining the structural integrity of microvascular walls. The aim of this study was to evaluate the impact of specific integrin extracellular domain binding to matrix fibronectin and vitronectin on the barrier function of intact microvascular endothelium.
  • 2
    The apparent permeability coefficient of albumin was measured in isolated and perfused porcine coronary venules using a fluorescence ratioing technique with the aid of fluorescence microscopy. Inhibition of integrin binding to either fibronectin with GRGDdSP peptide or vitronectin with GPenGRGDSPCA peptide dose-dependently increased venular permeability by 2- to 3-fold. The effects were sustained for more than 60 min and were reversible upon clearance of the peptides. In contrast, the inactive control peptide GRADSP did not significantly affect venular permeability. Pretreatment of the venules with purified human fibronectin and vitronectin, respectively, prevented the hyperpermeability response to GRGDdSP and GPenGRGDSPCA.
  • 3
    GRGDSP, a peptide that inhibits integrin binding to both fibronectin and vitronectin, produced an even higher permeability (4.5-fold) in venules than GRGDdSP or GPenGRGDSPCA alone, and the effect was blunted in vessels preincubated with both fibronectin and vitronectin.
  • 4
    The results indicate the importance of integrin-matrix interaction in the physiological regulation of microvascular permeability. It is likely that both fibronectin and vitronectin binding to integrins contribute to the maintenance of endothelial barrier function in venules.

Endothelial cells lining the microvascular wall impose a semi-permeable barrier to the transvascular movement of blood components by forming cell-cell junctional connections and cell-substratum focal adhesions. The attachment of endothelial monolayers to extracellular matrices (ECMs) is mainly mediated by a family of transmembrane receptors designated as integrins (Burridge et al. 1988). These molecules, most of which are capable of recognizing the sequence Arg-Gly-Asp (RGD) located in many ECM proteins, serve as a bridge for the transmission of chemical signals and mechanical forces between the cytoskeletal elements of cells and the ECM (Luscinskas & Lawler, 1994). In the vasculature, this process is essential to the maintenance of normal structure as well as to the development of new blood vessels as seen in vasculargenesis, angiogenesis, inflammation and wound healing.

Multiple integrin receptors with distinctive combinations of α and β subunits have been identified on the surface of vascular endothelial cells (Albelda et al. 1989; Luscinskas & Lawler, 1994). Among them, α5β1 and αvβ3, which bind to the ECM proteins fibronectin and vitronectin, respectively, are the best characterized and appear to be critical in the establishment and stabilization of endothelial monolayers (Cheng & Kramer, 1989; Cheng et al. 1991; Dejana et al. 1988). Synthetic peptides that compete with ECM proteins for the integrins or antibodies directed against the α5β1 and αvβ3 integrins have been shown to cause endothelial cell detachment from fibronectin and vitronectin (Hayman et al. 1985; Dejana et al. 1990; Pierschbacher & Ruoslahti, 1987). Mutation of the murine α5 integrin by gene targeting in embryonic stem cells produces blood vessels that are distended and leaky (Yang et al. 1993).

Recent evidence showed that RGD peptides increased the permeability of endothelial monolayers (Curtis et al. 1995; Qiao et al. 1995), indicating the importance of integrin-ECM binding to the maintenance of endothelial barrier function. However, the conclusion was drawn based on in vitro studies of cultured cells, rendering difficulties in determining the involvement of this process in the physiological regulation of microvascular exchange. This is especially a concern in view of the fact that the integrin expression and organization are variable depending on the specific substrates used in culture (Dejana et al. 1988, 1990; Albelda et al. 1989). In this regard, measurements of macromolecular transport across endothelial monolayers grown on artificial substratum containing single ECM proteins may not fully reflect the barrier property of microvascular walls.

The aim of this study was to investigate the effect of RGD peptides on the barrier function of exchange microvessels. The endothelial permeability was measured in intact, isolated coronary venules, providing direct evidence for the role of integrin-ECM interaction in the regulation of microvascular permeability.

METHODS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

Solutions and chemicals

An albumin-physiological salt solution (APSS) was used as a bathing solution while the microvessels were being dissected. It contained the following (mm): NaCl 145.0, KCl 4.7, CaCl2 2.0, MgSO4 1.17, NaH2PO4 1.2, glucose 5.0, pyruvate 2.0, EDTA 0.02 and Mops 3.0. After addition of 1 % bovine serum albumin, the solution was buffered to a pH of 7.40 at 4 °C and then filtered through a Millex-PF 0.22 μm filter unit (Millipore, Bedford, MA, USA). The APSS used to perfuse the vessels during permeability measurements had the same composition as above, but was buffered to a pH of 7.40 at 37 °C. The chemicals used to make the perfusate, including FITC-albumin, were purchased from Sigma (St Louis, MO, USA). Bovine serum albumin was obtained from United States Biochemical (Cleveland, OH, USA). The specific fibronectin-competing peptide GRGDdSP (Gly-Arg-Gly-Asp-d-Ser-Pro), the vitronectin-competing peptide GPenGRGDSPCA (Gly-Pen-Gly-Arg-Gly-Asp-Ser-Pro-Cys-Ala), and the fibronectin-vitronectin-competing peptide GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) were ordered from Gibco (Rockville, MD, USA). The specificity of the peptides has been demonstrated in previous studies (Pierschbacher & Ruoslahti, 1987). Purified human fibronectin and vitronectin were also from Gibco.

Animal preparation

Yorkshire pigs weighing 9-13 kg were anaesthetized with sodium pentobarbital (30 mg kg−1, i.v.) and treated with heparin (250 units kg−1, i.v.). Following a tracheotomy and intubation, the animal was ventilated with room air. A left thoracotomy was performed and the heart was electrically fibrillated, excised, and placed in 4 °C physiological saline. The pigs were killed through surgical removal of the heart.

The animals were housed and handled in accordance with the protocols approved by the Institutional Animal Care and Use Committee of the Texas A&M University and Scott and White Hospital.

Isolation and perfusion of coronary venules

The coronary sinus was cannulated, and 3 ml India ink-gelatin- physiological salt solution was infused to clearly define venular microvessels. This solution was prepared by adding 0.2 ml of India ink (Koh-I-Noor, Bloomsbury, NJ, USA) and 0.35 g of porcine skin gelatin to 10 ml of warm physiological salt solution and filtered through P8 filter paper (Fisher Scientific, Pittsburgh, PA, USA). Information regarding the validation and limitation of the ink-perfusion procedure has been provided in our previous publication (Yuan et al. 1993a). The method for isolating and cannulating coronary venules has also been described in detail in that study. Briefly, a suitable venule (length 0.8-1.2 mm, diameter 20-60 μm) was dissected from the surrounding myocardium in a dissecting chamber containing APSS at 4 °C with the aid of a Zeiss stereo dissecting microscope. The vessel was transferred to a cannulating chamber, which was mounted on a Zeiss axiovert microscope. The isolated vessel was cannulated with an inflow and outflow micropipette on each end and secured with 11-0 suture (Alcon, Fort Worth, TX, USA). A third, smaller pipette was inserted into the inflow pipette. The vessel was perfused with either APSS through the outer inflow pipette or APSS containing FITC-albumin through the inner inflow pipette. Each cannulating micropipette was connected to a reservoir and the vessel was perfused at a relatively constant intraluminal pressure and flow. The bath solution in the chamber was maintained at 37 °C and pH 7.4 throughout the experiments. The image of the vessel was projected onto a Hamamatsu charged coupled device-intensified camera and was displayed on a high resolution monochromatic video monitor and recorded onto a VHS video recorder. The diameter of the vessel was measured on-line with a video caliper (Microcirculation Research Institute, Texas A&M University, College Station, TX, USA).

Measurement of venular permeability

The permeability of the vessel was measured with a fluorescence ratioing technique (Huxley et al. 1987; Yuan et al. 1993a). Using an optical window of a video photometer positioned over the venules and adjacent space on the monitor, the fluorescence intensity from the window was measured and digitized on-line. For each measurement, the isolated venule was first perfused with APSS through the outer inflow pipette to establish a baseline intensity. The venular lumen was then rapidly filled with APSS containing FITC-albumin by switching the perfusion to the inner inflow pipette. This produced an initial step-increase, followed by a gradual increase, in fluorescence intensity. There was a step-decrease of intensity when the fluorescently labelled molecules were washed out of the vessel lumen by switching the perfusion back to the outer inflow pipette. The apparent solute permeability coefficient of albumin (Pa) was calculated using the equation Pa= (1/ΔIf)(dIf/dt)0(r/2), where ΔIf is the initial step increase in fluorescence intensity, (dIf/dt)0 is the initial rate of the gradual increase in intensity as the fluorescently labelled solutes diffuse out of the vessel into the extravascular space, and r is the venular radius.

In each experiment, the cannulated venule was perfused at a constant perfusion pressure of 20 cmH2O and flow velocity of 7 mm s−1. The preparation was equilibrated for 45-60 min after cannulation and the measurements were conducted at 36-37 °C and a pH of 7.35-7.45. Drugs were topically applied into the suffusion bath and the time course and dose dependence of RGD-induced changes in Pa were closely monitored.

Data analysis

In the intact vessel studies, Pa was measured two to three times for each venule at each experimental intervention and the values were averaged. For all experiments n is the number of vessels studied, with each vessel representing a separate animal. For each experimental condition, the values of Pa from different venules were averaged, normalized to the control values obtained before drug treatments, and reported as a percentage of control in mean ±s.e. For comparisons of the Pa values before and after treatment in the same vessels (e.g. repeated measurements of Pa in the time course study), Student's t test was applied to evaluate the significance of the difference. To compare the dose-responsive effects of RGD peptides and the inhibitory effects of fibronectin and vitronectin in different vessels, analysis of variance was used to evaluate the significance of intergroup differences. A value of P < 0.05 was considered significant for the comparisons.

RESULTS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

Time course of RGD-induced hyperpermeability

Topical administration of GRGDdSP (10−4m), GPenGRGDSPCA (10−4m), or GRGDSP (10−4m) caused a time-dependent increase in the apparent permeability coefficient of albumin in isolated and perfused coronary venules (Fig. 1). The hyperpermeability effect was seen as early as 5 min after adding the peptides and persisted for 1-3 h. The changes in barrier function were reversible as indicated by a Pa value near the control level after clearance of the peptides. The inactive control peptide GRADSP (10−4m) did not display any significant effect on venular permeability within the same time course.

image

Figure 1. Time course of changes in the apparent permeability coefficient of albumin (Pa) in isolated and perfused coronary venules during topical administration of the fibronectin-competing peptide GRGDdSP (10−4m, n= 9, the vitronectin-competing peptide GPenGRGDSPCA (10−4m, n= 6, the fibronectin/ vitronectin-competing peptide GRGDSP (10−4m, n= 14, and the inactive control peptide GRADSP (10−4m, n= 3. * Significant difference (P < 0.05vs. control values obtained at t= 0.

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Dose-responsive effects of RGD peptides

All three RGD peptides increased the permeability of coronary venules in a dose-dependent manner. Specifically, GRGDdSP, the inhibitor of fibronectin binding, elevated the Pa value by nearly 3-fold at 10−4m (Fig. 2). Similarly, a 2.5-fold increase in Pa was observed in vessels exposed to the inhibitor of vitronectin binding GPenGRGDSPCA at 10−3m (Fig. 3). Compared with GRGDdSP or GPenGRGDSPCA, the peptide that competes with both fibronectin and vitronectin binding, GRGDSP, exerted a more potent effect on the induction of venular hyperpermeability: it increased the Pa value by 4.5-fold at 10−4m (Fig. 4). This effect is more dramatic than the hyperpermeability response to histamine, which induced a 2- to 3-fold increase in the permeability of isolated coronary venules at 10−4m (Yuan et al. 1993b).

image

Figure 2. The permeability of isolated and perfused porcine coronary venules exposed to different doses of the fibronectin-competing peptide GRGDdSP for 20 min. * Significant difference (P < 0.05vs. basal value. Numbers in parentheses represent the number of vessels studied.

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image

Figure 3. The permeability of isolated and perfused porcine coronary venules exposed to different doses of the vitronectin-competing peptide GPenGRGDSPCA for 20 min. * Significant difference (P < 0.05vs. basal value. Numbers in parentheses represent the number of vessels studied.

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image

Figure 4. The permeability of isolated and perfused porcine coronary venules exposed to different doses of the fibronectin/vitronectin-competing peptide GRGDSP for 20 min. * Significant difference (P < 0.05vs. basal value. Numbers in parentheses represent the number of vessels studied.

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Effects of matrix proteins

Treatment of the venules with fibronectin, vitronectin, or a combination of fibronectin and vitronectin did not significantly alter the basal permeability but prevented the hyperpermeability response to GRGDdSP, GPenGRGDSPCA, or GRGDSP, respectively. As shown in Fig. 5, after incubation of the venules with fibronectin (25 μg ml−1, 20 min), administration of GRGDdSP (10−4m) only increased the permeability to 127.80 ± 9.05 % of control (P > 0.05 vs. Basal). In venules treated with vitronectin (5 μg ml−1, 20 min), the permeability was 110.23 ± 8.01 % of control after GPenGRGDSPCA at 10−4m (P > 0.05 vs. control). Furthermore, pre-incubation with both fibronectin (25 μg ml−1) and vitronectin (5 μg ml−1) for 20 min did not alter the basal Pa but blocked the GRGDSP-induced increase in venular permeability.

image

Figure 5. Pretreatment of isolated coronary venules with fibronectin (Fn, 25 μg ml−1, 20 min) and vitronectin (Vn, 5 μg ml−1, 20 min) prevents the hyperpermeability responses to GRGDdSP (10−4m, 20 min, n= 8, GPenGRGDSPCA (10−4m, 20 min, n= 4, and GRGDSP (10−4m, 20 min, n= 4. * Significant difference (P < 0.05vs. Basal value. § Significant difference (P < 0.05vs. GRGDdSP or GPenGRGDSPCA.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

The prevailing view of endothelial barrier mechanics envisions a cytoskeletal contractile force counterbalanced by tethering forces exerted by cell-cell and cell-matrix adhesions. Numerous studies clearly demonstrate the hyperpermeability-inducing actions of increased cytoskeletal activation and inhibition of interendothelial cell adhesion (Garcia & Schaphorst, 1995; Lum & Malik, 1994; Yuan et al. 1997; Tinsley et al. 1999). A major objective of this study was to examine the role of normal cell-matrix tethering forces in the maintenance of basal venular permeability to albumin. Elimination of either fibronectin or vitronectin tethering force alone caused a sustained increase in venular permeability of a magnitude comparable to or greater than the maximum permeabilities achieved with histamine (i.e. 2- to 3-fold increase; Yuan et al. 1993b). Simultaneous blockage of fibronectin and vitronectin adhesion resulted in the most intense hyperpermeability response (i.e. 4.5-fold increase) we have observed in the isolated porcine coronary venule preparation. Another interesting difference in the responses elicited by histamine and vascular endothelial growth factor (VEGF) and by RGD analogues centres on the time course of albumin extravasation. Histamine and VEGF initiate spikes in permeability that peak in the range 3-10 min and return towards normal over the next 15-20 min (Yuan et al. 1993b; Wu et al. 1996, 1999). By contrast, the RGD peptides produce a sustained increase in permeability that wanes slowly over a 60 min period. The time course of the response is more consistent with competitive binding of tethering sites than with outside-in signalling of kinases associated with focal adhesion complexes.

The contribution of integrin-ECM binding to the maintenance of normal endothelial barrier function is evidenced by previous studies showing that antibodies directed against the fibronectin receptor α5β1 caused a dose-dependent inhibition of endothelial cell adhesion to substratum and a dose-dependent increase in the transendothelial flux of horseradish peroxidase (Lum & Malik, 1994). In cultured bovine pulmonary microvascular endothelial cell monolayers, treatment with antibody to α5β1 integrin or GRGDSP peptide, which competes with fibronectin and vitronectin for binding to integrins, produced a dramatic increase in endothelial hydraulic conductivity as well as albumin permeability (Curtis et al. 1995; Qiao et al. 1995). Co-incubation of soluble human fibronectin with the RGD peptide or the α5β1 integrin antibody prevented the hyperpermeability response (Curtis et al. 1995). While most of the previous measurements of endothelial permeability were performed on cultured cells, their relevance to the in vivo system has not been established.

This study provides direct evidence confirming the role of integrin-ECM adhesion in the regulation of endothelial barrier function in intact microvessels. The permeability property of the endothelium was quantified using the isolated and perfused venule model, producing information that is relevant to the physiological mechanisms controlling microvascular exchange. The rapid onset and magnitude of the hyperpermeability elicited by inhibition of integrin-ECM interaction is consistent with modulation of cell-matrix tethering forces as an important mechanism underlying physiological regulation of venular permeability by chemical factors. For example, autacoids and growth factors are capable of altering focal adhesion kinase activity (Abedi & Zachary, 1997; Yuan et al. 1998), which in turn can alter the extent of integrin binding to components of the ECM. Since we show that elimination of integrin tethering to fibronectin and vitronectin produces permeability increases substantially in excess of those observed with autacoids and growth factors, then modest alterations in these cell-matrix binding forces may underlie physiological changes in barrier function initiated by these substances.

The current findings also have important implications for pathological alterations in venular permeability. Tissue injury or activation of interstitial neutrophils, mast cells and fibroblasts usually results in the release of enzymes capable of exposing cryptic RGD fragments from collagen, vitronectin and fibronectin (Davis et al. 2000). These fragments could then diffuse to the extravascular surface of venular endothelial cells, where they bind to specific integrins and prevent association with insoluble, tethered ligands in the basement membrane. With reduced tethering force at the cell-matrix interface, the endothelial cell rounds up and more tension is placed on the intercellular junction. The final consequence would be a large, sustained increase in venular permeability.

Two possible mechanisms have been considered to be responsible for RGD peptide-induced endothelial barrier dysfunction. First, the inhibition of integrin binding of ECM proteins may result in intercellular gap formation (Lampugnai et al. 1991; Qiao et al. 1995). In this regard, some members of the integrin family have been identified to be located at endothelial cell-cell borders (Lampugnai et al. 1991). It is suggested that these integrins collaborate with other intercellular molecules to form lateral junctions and that disruption of the junctional connection is responsible for the permeation of macromolecules across confluent endothelial monolayers (Lampugnai et al. 1991). Second, the competition between RGD peptides and ECM proteins for integrins may cause endothelial detachment from the substratum (Hayman et al. 1985; Dejana et al. 1990). Based on our data, we favour the hypothesis that the RGD peptides produced a weak adhesion of the endothelial lining to the ECM, rather than a permanent disruption of the endothelium, because the venular barrier function recovered soon after the peptides were cleared off the vessels. The weakened adhesive force might promote endothelial cell retraction and even shrinkage, thus facilitating transendothelial flux of macromolecules (Dejana et al. 1990; Kajimura & Curry, 1999). This is supported by fluorescence microscopic observations that RGD peptides loosened, but did not detach, endothelial cells from the ECM (Qiao et al. 1995). However, we cannot exclude the possibility that the preventive effect of fibronectin and vitronectin on the RGD-induced hyperpermeability response was due to their binding in solution rather than interacting with the matrix proteins.

The reagents used in the current study are specific for fibronectin or vitronectin and a third peptide was used to examine the effect of blocking the two specific sites simultaneously (Pierschbacher & Ruoslahti, 1987). The additive nature of the simultaneous blockade of the two binding sites supports the specificity of the reactions. At 10−4 to 10−3m, these peptides elicited a 2- to 4.5-fold increase in venular permeability. By contrast, the same concentration of GRGDTP peptide (Gly-Arg-Gly-Asp-Thr-Pro) showed little or no effect on baseline permeability of frog mesenteric microvessels in the study by Kajimura & Curry (1999). The different basal effects of this RGD peptide and the three peptides investigated in the present study may be the consequence of heterogeneous responses of microvessels in different animal species and tissues.

In summary, this study reports that RGD peptides that compete with the specific binding of integrins to fibronectin and vitronectin increase the permeability of intact, isolated venules. We suggest that the microvascular endothelial barrier function is maintained and regulated by a complex interaction between the integrins and ECM components involving fibronectin and vitronectin.

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Acknowledgements

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

This work was supported by the National Heart, Lung, and Blood Institute grant HL21498. We thank Ms Wenjuan Xu for her excellent technical support.