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TECHNICAL PAPERS: Cell

# Structural Mechanisms in the Abolishment of VEGF-induced Microvascular Hyperpermeability by cAMP

[+] Author and Article Information
Bingmei M. Fu1

Department of Biomedical Engineering, The City College of the City University of New York, 138th St. at Convent Ave., New York, NY 10031 and Department of Mechanical Engineering, University of Nevada, Las Vegas, 4505 Maryland Parkway, Box 454027, Las Vegas, NV 89154fu@ccny.cuny.edu

Shang Shen, Bin Chen

Department of Mechanical Engineering, University of Nevada, Las Vegas, 4505 Maryland Parkway, Box 454027, Las Vegas, NV 89154

1

Corresponding author.

J Biomech Eng 128(3), 317-328 (Nov 16, 2005) (12 pages) doi:10.1115/1.2187047 History: Received September 01, 2005; Revised November 16, 2005

## Abstract

To investigate the structural mechanisms by which elevation of the intraendothelial cAMP levels abolishes or attenuates the transient increase in microvascular permeability by vascular endothelial growth factor (VEGF), we examined cAMP effect on VEGF-induced hyperpermeability to small solute sodium fluorescein (Stokes $radius=0.45nm$) $Psodiumfluorescein$, intermediate-sized solute $α$-lactalbumin (Stokes $radius=2.01nm$) $Pα-lactalbumin$, and large solute albumin (BSA, Stokes $radius=3.5nm$) $PBSA$ on individually perfused microvessels of frog mesenteries. After $20min$ pretreatment of $2mM$ cAMP analog, 8-bromo-cAMP, the initial increase by $1nM$ VEGF was completely abolished in $Psodiumfluorescein$ (from a peak increase of $2.6±0.37$ times control with VEGF alone to $0.96±0.07$ times control with VEGF and cAMP), in $Pα-lactalbumin$ (from a peak increase of $2.7±0.33$ times control with VEGF alone to $0.76±0.07$ times control with VEGF and cAMP), and in $PBSA$ (from a peak increase of $6.5±1.0$ times control with VEGF alone to $0.97±0.08$ times control with VEGF and cAMP). Based on these measured data, the prediction from our mathematical models suggested that the increase in the number of tight junction strands in the cleft between endothelial cells forming the microvessel wall is one of the mechanisms for the abolishment of VEGF-induced hyperpermeability by cAMP.

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Copyright © 2006 by American Society of Mechanical Engineers
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## Figures

Figure 1

Paired measurements of apparent solute permeability (P) in individual frog mesenteric microvessels are shown as a function of time. 엯 matched control groups; ● test groups. In the matched control group, baseline P was first measured with Ringer perfusate containing 1% BSA (bovine serum albumin, 10mg∕ml), then P was measured in a sham experiment of reperfusion with control solution for ∼20min, and finally the P was measured under the treatment of 1nM VEGF for ∼5min. In the test group, baseline P was first measured with perfusate containing 1% BSA, then P was measured in the test experiment of reperfusion with the same solution also containing 2mM 8-bromo-cAMP for ∼20min, and finally the P was measured under the treatment of 1nM VEGF and 2mM 8-bromo-cAMP for ∼5min. (a) measurement for Psodiumfluorescein; (b) measurement for Pα-lactalbumin; (c) measurement for PBSA.

Figure 3

(a) plane view of the model for the interendothelial cleft in frog mesenteric microvessels (revised from Ref. 38). Junction strand with periodic breaks lies parallel to luminal front. L is the total depth of the cleft (∼400nm), Ljun is the thickness of the junction strand (∼10nm), and L1 and L3 are depths between the junction strand and luminal and abluminal fronts, respectively. Lf(∼100nm) is the thickness of fiber matrix at cleft entrance. Distance between two adjacent breaks in junction strand is 2D(∼2500nm), and 2d(∼150nm) is the width of large junction breaks. At the entrance of the cleft on the luminal side, surface glycocalyx structures are represented by a periodic square array of cylindrical fibers. Radius of these fibers is a, and gap spacing between fibers is Δ. (b) Three-dimensional sketch. There are two types of pores in the junction strand. Large breaks: 2d×2B=150×20nm; small continuous slit: 2bs≈1.5nm. Fu and Shen (7) suggested that 2B is increased to 50nm, 2.5-fold of 20nm under normal conditions, when the permeability is transiently (at ∼30s) increased by 1nM VEGF.

Figure 4

The model for explaining cAMP effect on microvessel permeability (revised from Ref. 32). (a) plane view. L1 and L2 are distances between the first and second junction strands and the luminal front. The relative locations of the junction strands and junction pores are arbitrary. (b) Three-dimensional sketch. There are two junction strands in the cleft under cAMP influence compared to one strand under normal conditions. The prediction from Ref. 32 confirmed the electron microscopic observation that elevation of intracellular cAMP increases the number of tight junction strands.

Figure 5

Model predictions for (a) increasing the cleft width 2B; (b) decreasing the surface glycocalyx thickness Lf; (c) increasing the junctional pore size; and (d) increasing the number of junctional pores, when there are two junction strands in the cleft. The two junction strands are located in between 25nm from the cleft entrance and 25nm from the cleft exit. The large pores in the two junction strands can be completely lined up or off lined. The plot shows the integrated results for all the locations (32). The control values are when there is one strand in the middle of the cleft and 2B=20nm.

Figure 6

Comparison of experimental data with the model predictions under various conditions for diffusive permeability Pd of frog mesenteric microvessels to (a) sodium fluorescein, (b) α-lactalbumin, and (c) BSA (bovine serum albumin). Under VEGF treatment, the model prediction is based on increasing the width of the interendothelial cleft by 2.5-fold and reducing the thickness of the glycocalyx layer by half (7); under cAMP treatment, the model prediction is based on increasing the number of junction strands in the cleft (32); and under cAMP/VEGF treatment, the model prediction is based on increasing the number of the junction strands in the cleft from 1 to 2, reducing the increase in the cleft width from 2.5- to 1.5-fold and restoration of the glycocalyx layer.

Figure 2

Effect of cAMP on VEGF-induced hyperpermeability in frog mesenteric microvessels for (a) sodium fluorescein, (b) α-lactalbumin, and (c) BSA (bovine serum albumin). Mean±SEP relative to baseline plotted as a function of time. In the matched control group (엯, BSA-BSA-VEGF), baseline P was first measured with Ringer perfusate containing 1% BSA (bovine serum albumin, 10mg∕ml), then P was measured in a sham experiment of reperfusion with control solution for ∼20min, and finally the P was measured under the treatment of 1nM VEGF for ∼5min. In the test group (●, BSA-cAMP-VEGF), baseline P was first measured with perfusate containing 1% BSA, then P was measured in the test experiment of reperfusion with the same solution also containing 2mM 8-bromo-cAMP for ∼20min, and finally the P was measured under the treatment of 1nM VEGF and 2mM 8-bromo-cAMP for ∼5min. p∗<0.05 compared with the baseline.

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