Publications

Background

Mural cells synthesize and deposit laminin to the basement membrane. To investigate the function of mural cell-derived laminin, we generated a mutant mouse line lacking mural cell-derived laminin (termed PKO). In a previous study, we showed that the PKO mice were grossly normal under homeostatic condition, but developed blood-brain barrier (BBB) breakdown with advanced age (> 8 months), suggesting that these mutants are intrinsically weak. Based on these findings, we hypothesized that PKO mice have exacerbated injuries in pathological conditions.

Methods

Using collagenase-induced intracerebral hemorrhage (ICH) as an injury model, we examined various stroke outcomes, including hematoma volume, neurological function, neuronal death, BBB integrity, paracellular/transcellular transport, inflammatory cell infiltration, and brain water content, in PKO mice and their wildtype littermates at young age (6–8 weeks). In addition, transmission electron microscopy (TEM) analysis and an in vitro ICH model were used to investigate the underlying molecular mechanisms.

Results

Compared to age-matched wildtype littermates, PKO mice display aggravated stroke outcomes, including larger hematoma size, worse neurological function, increased neuronal cell death, enhanced BBB permeability, increased transcytosis, and elevated inflammatory cell infiltration. These mutants also exhibit high baseline brain water content independent of aquaporin-4 (AQP4). In addition, mural cell-derived laminin significantly reduced caveolin-1 without affecting tight junction proteins in the in vitro ICH model.

Conclusions

These results suggest that mural cell-derived laminin attenuates BBB damage in ICH via decreasing caveolin-1 and thus transcytosis, regulates brain water homeostasis, and plays a beneficial role in ICH.

Laminin-α5, a major component of the basal lamina, is predominantly synthesized by endothelial and mural cells (pericytes and vascular smooth muscle cells) in the CNS. Loss of laminin-α5 in either population fails to induce any abnormalities due to functional redundancy. Thus, the functional significance of laminin-α5 in neurovascular integrity remains unknown. Here, we hypothesize that ablation of laminin-α5 in both endothelial and mural cells increases neurovascular permeability.

Methods

The compound knockout mice were generated by crossing laminin-α5 floxed mice with Tie2-Cre and PDGFRβ-Cre, which target endothelial cells and mural cells, respectively. Neurovascular permeability in these mutants was determined with both exogenous and endogenous tracers. Endothelial paracellular and transcellular permeability was assessed by examining the expression of tight junction proteins and transcytosis-associated proteins. In addition, transmission electron microscopy (TEM) was used to visualize tight junction ultrastructure and endothelial caveolae vesicles. Defects in pericytes and astrocytes were investigated by examining pericyte coverage/contact and astrocyte polarity.

Results

Elevated neurovascular permeability was observed in the mutants. Subsequent studies found increased Caveolin-1 and decreased major facilitator superfamily domain-containing protein 2a (MFSD2A) expression, but unaltered Claudin-5 or zonula occludens-1 (ZO-1) expression. Consistent with these results, mutant mice exhibited increased endothelial caveolae vesicle number with intact tight junction structure under TEM. Additionally, pericyte coverage and contact were also decreased in the mutant mice, while astrocyte polarity was unaffected.

Conclusions

These results strongly indicate that endothelial and mural cell-derived laminin-α5 actively maintains neurovascular integrity via the transcellular rather than paracellular mechanism.

Laminin, a major component of the basal lamina in the CNS, is also expressed in oligodendrocytes (OLs). However, the function of OL-derived laminin remains largely unknown. Here, we performed loss-of-function studies using two OL-specific laminin-α5 conditional knockout mouse lines. Both mutants were grossly normal and displayed intact blood-brain barrier (BBB) integrity. In a mouse model of intracerebral hemorrhage (ICH), control mice and both mutants exhibited comparable hematoma size and neurological dysfunction. In addition, similar levels of hemoglobin and IgG leakage were detected in the mutant brains compared to the controls, indicating comparable BBB damage. Consistent with this finding, subsequent studies revealed no differences in tight junction protein (TJP) and caveolin-1 expression among control and knockout mice, suggesting that neither paracellular nor transcellular mechanism was affected in the mutants. Furthermore, compared to the controls, both mutant lines showed comparable oligodendrocyte number, oligodendrocyte proliferation rate, MBP/MAG levels, and SMI-32 expression, highlighting a minimal role of OL-derived laminin-α5 in OL biology. Together, these findings highlight a dispensable role of OL-derived laminin-α5 in both brain homeostasis and ICH pathogenesis.

Microglia play a dual role in stroke depending on their pro-inflammatory and anti-inflammatory polarization. A study in PLOS Biology identifies a new mechanism, through which the transcription factor NR4A1 negatively regulates TNF expression in microglia.

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.

The function of fibroblasts in intracerebral hemorrhage (ICH) remains elusive. By targeting Col1α1, a fibroblast-specific marker, we generate mice with ablated Col1α1⁺ fibroblasts. These mutants show exacerbated blood-brain barrier (BBB) damage, enlarged injury volume, and worse neurological function, highlighting a beneficial role of Col1α1⁺ fibroblasts in ICH. Echoing these findings, fibroblasts significantly decrease endothelial permeability in an in vitro ICH model. Next, we demonstrate that fibroblasts promote BBB integrity in ICH mainly via up-regulating tight junction proteins without affecting transcytosis-associated proteins, indicating a paracellular rather than transcellular mechanism. A subsequent mechanistic study reveals that the BBB-protective effect of fibroblasts is partially mediated by TIMP metallopeptidase inhibitor 2 (TIMP2). Furthermore, we find that exogenous TIMP2 attenuates BBB disruption in these mutants after ICH. These results suggest that Col1α1⁺ fibroblasts repair BBB damage in ICH via the paracellular pathway in a TIMP2-dependent manner, and that Col1α1⁺ fibroblasts and TIMP2 may be targeted in ICH treatment.

Laminin, a major component of the basal lamina (BL), is a heterotrimeric protein with many isoforms. In the CNS, laminin is expressed by almost all cell types, yet different cells synthesize distinct laminin isoforms. By binding to its receptors, laminin exerts a wide variety of important functions. However, due to the reciprocal and cell-specific expression of laminin in different cells at the neurovascular unit, its functions in blood-brain barrier (BBB) maintenance and BBB repair after injury are not fully understood. In this review, we focus on the expression and functions of laminin and its receptors in the neurovascular unit under both physiological and pathological conditions. We first briefly introduce the structures of laminin and its receptors. Next, the expression and functions of laminin and its receptors in the CNS are summarized in a cell-specific manner. Finally, we identify the knowledge gap in the field and discuss key questions that need to be answered in the future. Our goal is to provide a comprehensive overview on cell-specific expression of laminin and its receptors in the CNS and their functions on BBB integrity.

Pericytes are multipotent perivascular cells that play important roles in CNS injury. However, controversial findings exist on how pericytes change and whether they differentiated into microglia-like cells after ischemic stroke. This discrepancy is mainly due to the lack of pericyte-specific markers: the “pericyte” population identified in previous studies contained vascular smooth muscle cells (vSMCs) and/or fibroblasts. Therefore, it remains unclear which cell type differentiates into microglia-like cells after stroke. In this study, lineage-tracing technique was used to mark α-smooth muscle actin (SMA)^{low/undetectable} pericytes, vSMCs, and fibroblasts, and their fates were analyzed after ischemic stroke. We found that SMA^{low/undetectable} pericytes and fibroblasts but not vSMCs substantially proliferated at the subacute phase after injury, and that SMA^{low/undetectable} pericyte but not vSMCs or fibroblasts differentiated into Iba1⁺ cells after ischemic stroke. Further imaging flow cytometry analysis revealed that SMA^{low/undetectable} pericytes differentiated into both microglia and macrophages at day 7 after stroke. These results demonstrate that SMA^{low/undetectable} pericytes rather than vSMCs or fibroblasts differentiate into both microglia-like and macrophage-like cells after stroke, suggesting that these pericytes may be targeted in the treatment of ischemic stroke.

Pericytes are a heterogenous population that plays multiple important roles in both physiological and pathological conditions. Although many markers and transgenic mouse lines have been used to identify pericytes, these tools all have limitations. For example, many of them are not pericyte-specific and none of them are able to distinguish different subtypes of pericyte. Here, we summarize commonly used pericyte markers and transgenic mouse lines, compare their unique features and limitations, and discuss key points to consider when using these tools or interpreting data generated by using them. Identifying/developing pericyte-specific and subtype-specific markers/tools will fill the gap of knowledge and substantially move the field forward.