Vascular permeability modulation at the cell, microvessel, or whole organ level: towards closing gaps in our knowledge

Abstract

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.

Figure 1

Measurement of 35 kDa Gadomer contrast agent apparent permeability coefficient in skin and muscle tissue of C57BL6 control mouse muscle and cheek during vehicle (saline) infusion. (A) MR Image (axial slice) of mouse head acquired 200 s after contrast agent injection via the tail vein. The regions of interest (ROIs) were carefully selected using anatomical references for muscle, skin, and vessels. (B) Shown is the subtracted image (image in A minus baseline image) recording the signal increase in tissue after injection of Gadomer, where cold colours indicate low signal enhancement and warm colours indicate high signal enhancement. Note the high signal intensity in large arteries showing that most of the high molecular weight Gadomer contrast agent is mainly in the vascular space. (C) Curve showing the signal intensity changes over time in an ROI used to estimate Gadomer permeability coefficient in the skin. As the contrast agent is injected, there is a step increase in tracer signal intensity above background as the vascular volume in the ROI is filled with the contrast agent. The tracer continues to accumulate in the ROI as it enters the extravascular space. The initial rate of tracer accumulation is estimated from the slope of the signal intensity over the first 100–150 s. An initial estimate of the vascular permeability is obtained from the magnitude of the initial slope and step. This initial estimate can be corrected for the fall in vascular tracer concentration (as measured from the signal intensity over an adjacent artery; see inset). (D) Signal intensity over time in an ROI over masseter muscle. Muscle permeability is less than in skin. The analysis to estimate vascular permeability is over an ROI containing no vessels larger than 100 µm diameter. Thus, assuming a mean plasma volume to exchange surface area of 4.4 × 10⁻⁴ cm, the vascular permeability coefficients in skin and muscle tissue were 4.6 ± 0.6 × 10⁻⁷ and 26 ± 3 × 10⁻⁷ cm/s, respectively. From Reference; used with permission from Wiley–Blackwell.

Figure 2

Measured parameters and diagram of the endothelial cleft. Diagrams illustrating measurement parameters used to construct mathematical model. (A) Area, A_c, and perimeter, p_c, measurements of individual cells seen in silver-stained whole mounts of venular microvessels were used to calculate cleft length per unit area, L_c. (B) From each electron microscopic image, the cleft depth, L, was measured along the contour between facing cells from the luminal cleft opening (x₀) to the abluminal cleft exit (x₂). The total distance from lumen to interstitium through the cleft is the sum of L₁, distance to the tight junction strand (x₁) from luminal cleft entrance, and L₂, distance from tight junction strand to the abluminal cleft exit. The cleft width is the distance between the outer leaflets of the cell membranes of the two facing cells. L_f, depth of the glycocalyx. (C) Oblique view of cleft segment reconstructed from serial sections illustrates the length of tight junction strand gaps, 2d, and the mean distance between strand gap centres, equal to the functional unit length, 2D. From Reference; used with permission from Wiley–Blackwell.

Figure 3

Increased cAMP inhibits PAF-induced changes in VE-cadherin and occludin in situ. Immunofluorescent localization of VE-cadherin (middle column) and occludin (left column) after perfusion with vehicle control (top row), PAF (middle row; 10 nM), and pre-treated with rolipram and forskolin to increase intracellular cAMP prior to PAF (bottom row). After control perfusion VE-cadherin appeared in a continuous peripheral ribbon with frequent broad areas, likely corresponding to regions of extensive (order of 1–2 µm) endothelial overlap, while occludin was largely restricted to a narrow line. After PAF there were numerous spikes (asterisks) of VE-cadherin label oriented transverse to the endothelial perimeter and there were frequent discontinuities (arrows) in the label. Breaks in the continuity of occludin corresponded to similar discontinuities in the VE-cadherin, confirming loss of the tight junction barrier at sites of adherens junction loss. Increased cAMP prevented rearrangement of both VE-cadherin and occludin and very strongly inhibited any increase in Lp (not shown). Modified from Reference.

Figure 4

(A) Comparison of thrombin response in inflamed and non-inflamed rat mesentery venules. In vessels not previously manipulated (Day 1) L_p measured during thrombin perfusion is not different from that measured during perfusion with vehicle control solution (n = 8). When L_p is measured 24 h after initial perfusion, thrombin stimulates a large L_p response (Day 2), approximately five times greater than Day 2 vehicle control L_p (n = 11; P < 0.05 *different from paired Day 2 vehicle control, ^†different from Day 1 thrombin, non-paired). (B) Response to PAF 24 h after initial perfusion in rat mesentery venules. Paired L_p measurements in response to PAF (10 nM) on the second day of a 2-day experiment (solid bar, Day 2) were not significantly different from the response measured initially in 6 venules (striped bar, Day 1). Corresponding vehicle control measurements are shown (open bars). (C) Rho-kinase inhibition reduces thrombin- and VEGF-stimulated vascular hyperpermeability in a mouse skin wound model. (C) Vascular permeability at 1 h and 24 h post-wounding and effect of Rho-kinase inhibition with Y-27632 on basal, thrombin-, and VEGF-induced vascular permeability. *P < 0.05 and **P < 0.01 between groups; n = 5–7 mice in each group. From Curry et al. for (A) and (B) and Kim et al. for (C), used with permission from the American Physiological Society.