Exogenous laminin exhibits a unique vascular pattern in the brain via binding to dystroglycan and integrins

Abstract

Background: Unlike other proteins that exhibit a diffusion pattern after intracerebral injection, laminin displays a vascular pattern. It remains unclear if this unique vascular pattern is caused by laminin-receptor interaction or laminin self-assembly.

Methods: We compared the distribution of various wild-type laminin isoforms in the brain after intracerebral injection. To determine what causes the unique vascular pattern of laminin in the brain, laminin mutants with impaired receptor-binding and/or self-assembly activities and function-blocking antibodies to laminin receptors were used. In addition, the dynamics of laminin distribution and elimination were examined at multiple time points after intracerebral injection.

Results: We found that β2-containing laminins had higher affinity for the vessels compared to β1-containing laminins. In addition, laminin mutants lacking receptor-binding domains but not that lacking self-assembly capability showed substantially reduced vascular pattern. Consistent with this finding, dystroglycan (DAG1) function-blocking antibody significantly reduced the vascular pattern of wild-type laminin-111. Although failed to affect the vascular pattern when used alone, integrin-β1 function-blocking antibody further decreased the vascular pattern when combined with DAG1 antibody. EDTA, which impaired laminini-DAG1 interaction by chelating Ca²⁺, also attenuated the vascular pattern. Immunohistochemistry revealed that laminins were predominantly located in the perivascular space in capillaries and venules/veins but not arterioles/arteries. The time-course study showed that laminin mutants with impaired receptor-engaging activity were more efficiently eliminated from the brain compared to their wild-type counterparts. Concordantly, significantly higher levels of mutant laminins were detected in the cerebral-spinal fluid (CSF).

Conclusions: These findings suggest that intracerebrally injected laminins are enriched in the perivascular space in a receptor (DAG1/integrin)-dependent rather than self-assembly-dependent manner and eliminated from the brain mainly via the perivascular clearance system.

Keywords: Dystroglycan; Integrins; Laminin; Perivascular space; Vascular pattern.

Fig. 1

Exogenous laminins exhibit a vascular pattern in the brain via binding to their receptors rather than self-assembly. A Illustration of the structures of various wild-type and mutant laminins. B Representative low-magnification image showing the vascular pattern of intracerebrally injected laminins. C Representative images of Alexa-555 labeled laminins (red) and SMA (green) in the brain at 24 h after intracerebral injection. D Quantification of Alexa-555 fluorescent intensity in (C). n = 4, *p = 0.0286 compared to laminin-β1 counterparts by Mann–Whitney U test. ^#p = 0.0286 compared to their wild-type counterparts by Mann–Whitney U test. Data are represented as mean ± SD. SMA, α-smooth muscle actin

Fig. 2

Laminin receptors mediate the vascular pattern of intracerebrally injected laminins. A Representative images of Alexa-555 labeled laminin-111 (red) in the brain at 24 h after intracerebral injection in the presence of IgM control, ITGB1 function-blocking antibody, DAG1 function-blocking antibody, and both ITGB1 and DAG1 function-blocking antibodies. B Quantification of Alexa-555 fluorescent intensity in blood vessels in A. n = 4, *p = 0.0286 by Mann–Whitney U test. C Representative images of Alexa-555 labeled laminin-111 (red) in the brain at 24 h after intracerebral injection in the presence of CaCl₂ or EDTA. D Quantification of Alexa-555 fluorescent intensity in blood vessels in C. n = 4, *p = 0.0286 by Mann–Whitney U test. Data are represented as mean ± SD. DAG1, dystroglycan; ITGB1, integrin-β1; EDTA, ethylenediaminetetraacetic acid

Fig. 3

Exogenous laminin-111 is enriched in the perivascular space in cerebral vasculature. A Representative low-magnification images of Alexa-555 labeled laminin-111 (red) and CD31 (green) in the brain at 24 h after intracerebral injection. B Quantification of vessel length calculated with laminin-111 and CD31 signals. n = 4, *p = 0.0286 by Mann–Whitney U test. C Quantification of vessel area calculated with laminin-111 and CD31 signals. n = 4, *p = 0.0286 by Mann–Whitney U test. D Quantification of laminin-111 contact. n = 4. E Quantification of laminin-111 coverage. n = 4. F Representative high-magnification image of CD31 (green), Alexa-555 labeled laminin-111 (red), and AQP4 (blue) in the brain at 24 h after intracerebral injection. White arrows indicate two sites, where spatial profiles of fluorescence intensity were performed. G, H Spatial profiles of CD31 (green), laminin-111 (red), and AQP4 (blue) along white lines crossing representative capillaries in site 1 (G) and site 2 (H) in F. I Representative high-magnification images of CD31 (green), Alexa-555 labeled laminin-111 (red), and AQP4 (blue) showing the distribution of laminin-111 along the longitudinal axis of blood vessels. White arrows indicate the vascular pattern of laminin-111 at the abluminal side of endothelial cells. Data are represented as mean ± SD

Fig. 4

Exogenous laminin-111 and -111ΔLG1-5 are eliminated from the brain via the perivascular system. A Representative images of Alexa-555 labeled laminin-111 (red) and -111ΔLG1-5 (red) in the brain at various time points after intracerebral injection. B Quantification of total laminin levels at each time point in A. n = 4, *p = 0.0286 by Mann–Whitney U test. C Quantification of vessel-associated laminin levels at 12 and 24 h after intracerebral injection in A. n = 4, *p = 0.0286 by Mann–Whitney U test. D, E Quantification of Alexa-555 fluorescent intensity in the CSF at 15 min (D) and 24 h (E) after intracerebral injection. n = 5–6, *p = 0.0152 and **p = 0.0079 by Mann–Whitney U test. Data are represented as mean ± SD