Venular basement membranes ubiquitously express matrix protein low-expression regions: characterization in multiple tissues and remodeling during inflammation

Abstract

The venular basement membrane plays a critical role in maintaining the integrity of blood vessels and through its dense and highly organized network of matrix proteins also acts as a formidable barrier to macromolecules and emigrating leukocytes. Leukocytes can however penetrate the venular basement membrane at sites of inflammation, though the associated in vivo mechanisms are poorly understood. Using whole mount immunostained tissues and confocal microscopy, we demonstrate that the venular basement membrane of multiple organs expresses regions of low matrix protein (laminin-511 and type IV collagen) deposition that have been termed low-expression regions (LERs). In the multiple tissues analyzed (eg, cremaster muscle, skin, mesenteric tissue), LERs were directly aligned with gaps between adjacent pericytes and were more prevalent in small venules. As predicted by their permissive nature, LERs acted as "gates" for transmigrating neutrophils in all inflammatory reactions investigated (elicited by leukotriene B(4) [LTB(4)], CXCL1, tumor necrosis factor [TNF]alpha, endotoxin, and ischemia/reperfusion [I/R] injury), and this response was associated with an enhancement of the size of laminin-511 and type IV collagen LERs. Transmigrated neutrophils stained positively for laminins but not type IV collagen, suggesting that different mechanisms exist in remodeling of different basement membrane networks. Collectively the findings provide further insight into characteristics of specialized regions within venular basement membranes that are preferentially used and remodeled by transmigrating neutrophils.

Figure 1

Expression profiles of key venular basement membrane constituents in cremasteric venules. Unstimulated mouse cremaster muscles were collected, fixed, and whole-mount immunostained for the different components of the venular basement membrane and subsequently analyzed by confocal microscopy. The panels show representative three-dimensional images of half postcapillary venules (reconstructed using the Imaris software) immunostained for laminin-α5 chain (Lmα5, to detect the laminin-511 isoform), laminin-α4 chain (Lmα4, to detect the laminin-411 isoform), type IV collagen (Col IV), entactin/nidogen or perlecan (top panels), and with αSMA (middle panels) as a marker for pericytes. The images demonstrate the direct association between basement membrane LERs and αSMA-negative regions in the pericyte sheath (circles). In contrast, images from cremasteric venules that have been immunostained for perlecan and αSMA demonstrate the presence of perlecan LERs in the basement membrane with αSMA (pericyte) expressing regions. Histogram plots in the bottom panels represent intensity profiles of the basement membrane components of interest (green) and pericytes (red) along the indicated vessel segment (yellow dotted line on images) showing that LERs (closed arrows) co-localize with αSMA negative regions (gaps between pericytes) for all of the proteins investigated except for perlecan (open arrow). All of the images are representative of at least 4 vessels analyzed from 4 to 6 mice, Scale bar = 10 μm.

Figure 2

Detection and analysis of laminin-511 LERs in different vascular beds. Mouse cremaster muscle, skin, mesenteric tissue, peritoneal wall, and diaphragm (all unstimulated) were collected, fixed, and whole-mount immunostained for analysis by confocal microscopy. The panels show representative three-dimensional images of half postcapillary venules (reconstructed using the Imaris software) from the above tissues immunostained for laminin-α5 chain (Lmα5, left panels) and αSMA (right panels) to detect the venular basement membrane (laminin-511 isoform) and pericytes, respectively. The corresponding intensity profiles of the images with a spectrum color coding (blue indicating low-intensity sites and red indicating high-intensity sites) are also shown as insets. The images demonstrate the presence of low-expression regions (circles) in the venular basement membrane of all analyzed tissues, sites that are in line with αSMA negative regions (ie, gaps between pericytes). The images are representative of at least 4 vessels from more than 4 animals. Scale bar = 10 μm.

Figure 3

Size characteristics of laminin-511 and type IV collagen LERs. Whole mounted mouse tissues (cremaster muscle, skin of the ear, and mesenteric tissue) were immunostained for laminin-α5 chain or type IV collagen and αSMA to detect the venular basement membrane and pericytes, respectively, and were analyzed by confocal microscopy. A: The graphs show the area of gaps between adjacent pericytes and laminin-511 and type IV collagen LERs in postcapillary venules of the indicated tissues analyzed. The median is indicated by a dotted line. Significant differences are indicated by lines, *P < 0.05; **P < 0.01, and ***P < 0.001. B: The graphs show the frequency (%) of expression of different size laminin-511 LER areas (μm²) in the 3 different tissues analyzed. The percentage of LER areas within a range of 1 to 10 μm² (cremasteric muscle, skin, and mesentery) or 1 to 40 μm² (mesentery) are indicated above the double headed arrow. Results are expressed as mean ± SEM and were obtained by analyzing a total of 2059 LERs from at least 4 vessels per tissues; from at least 4 mice per group.

Figure 4

Characteristics of low expression regions. Whole mount mouse cremaster muscle, skin, and mesenteric tissue were immunostained for vessel wall components and the venular basement membrane characteristics were analyzed by confocal microscopy. A: The graphs show the number/unit area of gaps between adjacent pericytes (left panel), laminin-511 (Lmα5 chain, middle panel), and type IV collagen (right panel) LERs quantified in the venular basement membrane of the indicated tissues. Significant differences are indicated by lines, *P < 0.05; **P < 0.01, and ***P < 0.001. B: The graph shows a significant linear relationship between blood vessel diameter and the number/unit area of laminin-511 LERs in cremasteric, skin, and mesenteric venules. C: The graph shows the linear correlation between the size of LERs and the diameter of the postcapillary venules (each point representing one individual vessel) in the cremaster muscle. D: The confocal image is a three-dimensional reconstructed (Imaris) basement membrane from a half cremasteric postcapillary venule stained for laminin-α5 chain and presented using a spectrum color code (blue and red indicate low- and high-intensity regions, respectively). The selected venular segment exhibits LERs of different sizes (labeled from 1 to 3). The respective area of theses LERs is indicated on the image, and the corresponding intensity profile of the laminin-α5 staining along the dashed arrow is shown below the image (Bar, 10 μm). E: The graph shows the linear relationship between the mean size of LERs and the mean intensity of multiple venules (each point represents individual vessels) from cremasteric muscle (open circles) and skin (filled circles). Dotted curved lines in the correlation plots represent 95% confidence intervals. All of the results shown are from n = 2 to 4 vessels from 3 to 4 mice.

Figure 5

Neutrophil migration through cremasteric venular basement membrane low expression regions as elicited by multiple inflammatory reactions. To analyze the role and regulation of expression of LERs in neutrophil transmigration cremaster muscles were stimulated by intrascrotal injection of CXCL1 (500 ng) for 2 hours, TNFα (300 ng), or LPS (300 ng) for 4 hours. Tissues were subsequently immunostained for type IV collagen or Lmα5 chain (laminin-511 basement membrane marker), αSMA (pericyte marker), and MRP-14 (neutrophil marker) and analyzed by confocal microscopy. A: Representative three-dimensional images of half vessels were generated by Imaris software to observe neutrophil migration in stimulated venules. B: Representative latitudinal cross sections (1 μm thick) of venules shown in panel A were analyzed for localization of transmigrating neutrophils in relation to LERs and also the intensity profiles of LERs associated with neutrophils. The images show that transmigrating neutrophils migrate through gaps between pericytes and through LERs (filled arrows). The corresponding intensity profiles for Lmα5 immunostainning are shown below each image to demonstrate the loss of fluorescence and therefore of matrix protein content in LERs that are traversed by neutrophils. The open arrowheads show LERs that appear not to be remodeled by a neutrophil, as shown by a higher intensity of fluorescence as compared with a neutrophil-traversed LER. C: The images show a 1-μm cross section of cremasteric venule from LPS stimulated tissues immunostained for laminin-411 (Lmα4 chain), αSMA, and MRP-14 demonstrating that neutrophils can migrate through laminin-411 LERs (filled arrow). The corresponding intensity profile for the laminin-α4 immunostainning is shown below the images to demonstrate the loss of fluorescence in the LER traversed by a neutrophil as compared with a nontraversed LER (open arrows). All images are representative of 4 vessels obtained from 5 to 6 mice per group, bar = 10 μm.

Figure 6

Neutrophil migration is associated with the remodeling of venular basement membrane laminin-511 and type IV collagen LERs. A: Mouse cremasteric muscles were stimulated with LTB₄ (10⁻⁷ mol/L) for 2 hours, CXCL1 (500 ng) for 2 hours, or TNFα (300 ng) for 4 hours. Tissues were subsequently fixed, whole-mount immunostained for venular basement membrane (Laminin-α5 chain or type IV collagen) and neutrophils (MRP-14), and visualized by confocal microscopy. The panels show neutrophil migration into tissues (left panel), size of laminin-511 LER (Lmα5, middle panel), and size of type IV collagen LERs (Col IV, right panel), under different inflammatory scenarios. B: Mouse cremasteric muscle was subjected to ischemia (30 minutes)/reperfusion (120 minutes) injury and as described above, the neutrophil response (left panel) and the remodeling of both laminin-511 and type IV collagen LERs (middle and right panels, respectively) were quantified. C: Mouse ears were injected intradermally with TNFα for 4 hours, dissected away, fixed, and after immunostaining analyzed for neutrophil transmigration (left panel) and basement membrane remodeling (right panel) as detailed above. All results were obtained from 4 vessels (5 to 6 mice per group), and values are expressed as mean ± SEM. Significant differences in responses in stimulated tissues as compared with control conditions are indicated, *P < 0.05; **P < 0.01, and ***P < 0.001.

Figure 7

LPS induces a time-dependent remodeling of laminin-511 LER that is associated with neutrophil but not monocyte migration through venular walls. Cremasteric muscles of CX3CR1^gfp/+ mice were stimulated with LPS (300 ng), and tissues were collected at different time points after induction of inflammation (2, 4, 6, and 24 hours), fixed, and whole-mount immunostained for venular basement membrane (laminin-α5 chain) and neutrophils (MRP-14), and visualized by confocal microscopy. The size of LERs (A), neutrophils (B), and monocyte (C) response over time was quantified. Localization of leukocytes within the venular wall (left panels) and in the extravascular tissue (right panels) were also quantified. All results were obtained from at least 4 vessels (5 to 6 mice per group), and values are expressed as mean ± SEM. Significant differences in responses in stimulated tissues as compared with control conditions are indicated, *P < 0.05; **P < 0.01, and ***P < 0.001. Significant differences between two different time points are indicated by lines, *P < 0.05; **P < 0.01, and ***P < 0.001.

Figure 8

Transmigrated neutrophils are laminin-511– and laminin-411– but not collagen IV–positive. LTB₄- and LPS-stimulated cremaster muscles were fixed and whole-mount immunostained for molecules of interest and subsequently analyzed by confocal microscopy. The images shown are three-dimensional reconstructions of cremasteric venules after LTB₄ (2 hours, top panels) or LPS (4 hours, bottom panels) stimulation immunostained for neutrophils (MRP-14) and for the basement membrane components laminin-α5 chain of the laminin-511 isoform (Lmα5; A), laminin-α4 chain for the laminin-411 isoform (Lmα4; B) or type IV collagen (Col IV; C). The images show that transmigrated neutrophils stain positively for laminin-α5 and laminin–α4 chains (filled arrows) but are negative for type IV collagen (open arrowheads) on their cell surface. In the middle panels an opacity filter was used for the fluorescence intensity of the neutrophils (MRP-14) to make the cells semitransparent so that detection of immunostained basement membrane components on the neutrophils is easier to view. All images were obtained from at least 4 vessels (4 to 6 mice per group). Bar = 10 μm.

Figure 9

The heterogeneous expression profile of venular basement membrane components is governed by the pericyte coverage of venular walls. The schematic diagram illustrates the phenomenon that characteristics of the venular basement membrane are governed by the fact that this structure is generated by both cellular components of venules (ie, endothelial cells [ECs] and pericytes). Specifically, because unlike the confluent layer of endothelial cells pericytes are expressed in a loose and “net-like” manner around ECs, the “patchy” expression profile of pericytes results in the heterogeneous profile of key basement membrane components. Collectively, the morphology and expression profile of the two cellular components of venules, both of which contribute to the generation of venular basement membrane components, govern the structural properties of the venular basement membrane, in particular the existence and characteristics of LERs that are commonly localized within gaps between adjacent pericytes (circles).