A unique pulmonary microvascular endothelial cell niche revealed by Weibel-Palade bodies and Griffonia simplicifolia

Abstract

Pulmonary endothelium displays considerable heterogeneity along the vascular axis, from arteries to capillaries to veins. Griffonia simplicifolia is a lectin that recognizes pulmonary microvascular endothelium with preference over extra-alveolar endothelium in both arteries and veins, yet the precise vascular location where this phenotypic shift occurs is poorly resolved. We gelatin-filled the circulation and agarose-loaded the airways and then labeled the lung with Griffonia lectin to enable visualization of the endothelial transition zone. Endothelium in vessels with internal diameters less than 38 μm were uniformly Griffonia positive, whereas vessels with internal diameters greater than 60 μm were always Griffonia negative. Two populations of endothelium were identified in vessels ranging from 38 to 60 μm in diameter, including some that were positive and others that were negative for binding to G. simplicifolia. To better resolve this endothelial transition zone, we performed morphology studies to measure the distribution of Weibel-Palade bodies (WPbs), since WPbs are present in conduit vessel endothelium and absent in capillary endothelium. WPbs were found in endothelium with vascular dimensions as small as 18 μm in diameter but were not found in capillaries. Thus, we identify with precision that the endothelial phenotype transition from a cell that does not interact with Griffonia lectin to one that does occurs in blood vessels with internal diameters of approximately 38 μm, and we reveal an unappreciated vascular zone, between 18 and 38 μm in diameter, where endothelium both is Griffonia positive and possesses WPbs.

Keywords: P-selectin; heterogeneity; lectin; vascular; von Willebrand factor.

Figure 1

Alveolar capillary and extra-alveolar microvessel endothelium consistently exhibit positive surface binding of Griffonia simplicifolia. Fluorescence imaging of endothelial surface staining of Alexa Fluor 568–conjugated G. simplicifolia in alveolar capillaries and extra-alveolar microvessels. Representative projected images of all parts of Z-stacks, combining both fluorescence and differential interference contrast modes, taken at 0.2-μm intervals in <200-μm viable slices of a mouse lung. Note the appearance of Alexa Fluor 568 fluorescence on the alveolar septal capillary network as well as on the luminal surface of a small extra-alveolar vessel (EAV) in A (arrowheads), but not on the surface of a large EAV in B (arrows). Lung cell nuclei were visualized using 4′,6-diamidino-2-phenylindole (DAPI) staining (blue). Images were acquired at 21°C on a Nikon A1 confocal laser microscope with a ×60, 1.20 numerical aperture water immersion objective (replicated 3 times). Scale bars = 50 μm.

Figure 2

Summarized data from a total of 56 measurements of the diameter of the vessels where the binding of Griffonia simplicifolia was positive (plus sign; n = 20) or negative (minus sign; n = 36), obtained from mouse lungs (n = 3). Symbols, scatter plot (note that data obtained from each individual mouse are identified by different colors, i.e., green, blue, and black); red lines with error bars indicate median and interquartile range. The asterisk indicates P < 0.0001, per Student’s t test.

Figure 3

Heterogeneity of Weibel-Palade body (WPb) distribution in pulmonary vascular endothelium. Cross-section transmission electron micrographs of an extra-alveolar vessel and adjacent alveolar capillaries in a mouse lung. A, Low-magnification micrograph taken at the junction where an extra-alveolar vessel connects to an alveolar capillary (Cap). B, C, Higher powers of magnification of the boxed area in A, demonstrating a cluster of WPbs (arrowheads in C). Note that the endothelial cell exhibiting WPbs is at the extra-alveolar side of the junction.

Figure 4

Summary data displayed as a box-and-whisker diagram from a total of 90 measurements of the diameter of the vessels where the WPbs were (plus sign) or were not (minus sign) detected, obtained from mouse lungs (n = 5). Asterisk indicates P < 0.0001, per Student’s t test.