Integrins are transmembrane heterodimeric receptors that contribute to diverse biological functions and play critical roles in many human diseases. Studies using integrin subunit knockout mice and inhibitory antibodies have identified important roles for nearly every integrin heterodimer and led to the development of a number of potentially useful therapeutics. One notable exception is the αvβ1 integrin. αv and β1 subunits are individually present in numerous dimer pairs, making it challenging to infer specific roles for αvβ1 by genetic inactivation of individual subunits, and αvβ1 complex-specific blocking antibodies do not yet exist. We therefore developed a potent and highly specific small-molecule inhibitor of αvβ1 to probe the function of this understudied integrin. We found that αvβ1, which is highly expressed on activated fibroblasts, directly binds to the latency-associated peptide of transforming growth factor-β1 (TGFβ1) and mediates TGFβ1 activation. Therapeutic delivery of this αvβ1 inhibitor attenuated bleomycin-induced pulmonary fibrosis and carbon tetrachloride-induced liver fibrosis, suggesting that drugs based on this lead compound could be broadly useful for treatment of diseases characterized by excessive tissue fibrosis.

Sleep deprivation can generate inflammatory responses in the central nervous system. In turn, this inflammation increases sleep drive, leading to a rebound in sleep duration. Microglia, the innate immune cells found exclusively in the CNS, have previously been found to release inflammatory signals and exhibit altered characteristics in response to sleep deprivation. Together, this suggests that microglia may be partially responsible for the brain's response to sleep deprivation through their inflammatory activity. In this study, we ablated microglia from the mouse brain and assessed resulting sleep, circadian, and sleep deprivation phenotypes. We find that microglia are dispensable for both homeostatic sleep and circadian function and the sleep rebound response to sleep deprivation. However, we uncover a phenomenon by which microglia appear to be essential for the protection of fear-conditioning memories formed during the recovery sleep period following a period of sleep deprivation. This phenomenon occurs potentially through the upregulation of synaptic-homeostasis related genes to protect nascent dendritic spines that may be otherwise removed or downscaled during recovery sleep. These findings further expand the list of known functions for microglia in synaptic modulation.

As the resident macrophages of the brain and spinal cord, microglia are crucial for the phagocytosis of infectious agents, apoptotic cells and synapses. During brain injury or infection, bone-marrow derived macrophages invade neural tissue, making it difficult to distinguish between invading macrophages and resident microglia. In addition to circulation-derived monocytes, other non-microglial central nervous system (CNS) macrophage subtypes include border-associated meningeal, perivascular and choroid plexus macrophages. Using immunofluorescent labeling, flow cytometry and Cre-dependent ribosomal immunoprecipitations, we describe P2ry12-CreER, a new tool for the genetic targeting of microglia. We use this new tool to track microglia during embryonic development and in the context of ischemic injury and neuroinflammation. Because of the specificity and robustness of microglial recombination with P2ry12-CreER, we believe that this new mouse line will be particularly useful for future studies of microglial function in development and disease.

The blood-brain barrier (BBB) is a term used to describe the unique properties of central nervous system (CNS) blood vessels. One important BBB property is the formation of a paracellular barrier made by tight junctions (TJs) between CNS endothelial cells (ECs). Here, we show that Lipolysis-stimulated lipoprotein receptor (LSR), a component of paracellular junctions at points in which three cell membranes meet, is greatly enriched in CNS ECs compared with ECs in other nonneural tissues. We demonstrate that LSR is specifically expressed at tricellular junctions and that its expression correlates with the onset of BBB formation during embryogenesis. We further demonstrate that the BBB does not seal during embryogenesis in Lsr knockout mice with a leakage to small molecules. Finally, in mouse models in which BBB was disrupted, including an experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis and a middle cerebral artery occlusion (MCAO) model of stroke, LSR was down-regulated, linking loss of LSR and pathological BBB leakage.

Several critical events dictate the successful establishment of nascent vasculature in yolk sac and in the developing embryos. These include aggregation of angioblasts to form the primitive vascular plexus, followed by the proliferation, differentiation, migration, and coalescence of endothelial cells. Although transforming growth factor-β (TGF-β) is known to regulate various aspects of vascular development, the signaling mechanism of TGF-β remains unclear. Here we show that homeodomain interacting protein kinases, HIPK1 and HIPK2, are transcriptional corepressors that regulate TGF-β-dependent angiogenesis during embryonic development. Loss of HIPK1 and HIPK2 leads to marked up-regulations of several potent angiogenic genes, including Mmp10 and Vegf, which result in excessive endothelial proliferation and poor adherens junction formation. This robust phenotype can be recapitulated by siRNA knockdown of Hipk1 and Hipk2 in human umbilical vein endothelial cells, as well as in endothelial cell-specific TGF-β type II receptor (TβRII) conditional mutants. The effects of HIPK proteins are mediated through its interaction with MEF2C, and this interaction can be further enhanced by TGF-β in a TAK1-dependent manner. Remarkably, TGF-β-TAK1 signaling activates HIPK2 by phosphorylating a highly conserved tyrosine residue Y-361 within the kinase domain. Point mutation in this tyrosine completely eliminates the effect of HIPK2 as a transcriptional corepressor in luciferase assays. Our results reveal a previously unrecognized role of HIPK proteins in connecting TGF-β signaling pathway with the transcriptional programs critical for angiogenesis in early embryonic development.

Vascular development of the central nervous system and blood-brain barrier (BBB) induction are closely linked processes. The role of factors that promote endothelial sprouting and vascular leak, such as vascular endothelial growth factor A, are well described, but the factors that suppress angiogenic sprouting and their impact on the BBB are poorly understood. Here, we show that integrin αVβ8 activates angiosuppressive TGFβ gradients in the brain, which inhibit endothelial cell sprouting. Loss of αVβ8 in the brain or downstream TGFβ1-TGFBR2-ALK5-Smad3 signaling in endothelial cells increases vascular sprouting, branching and proliferation, leading to vascular dysplasia and hemorrhage. Importantly, BBB function in Itgb8 mutants is intact during early stages of vascular dysgenesis before hemorrhage. By contrast, Pdgfb(ret/ret) mice, which exhibit severe BBB disruption and vascular leak due to pericyte deficiency, have comparatively normal vascular morphogenesis and do not exhibit brain hemorrhage. Our data therefore suggest that abnormal vascular sprouting and patterning, not BBB dysfunction, underlie developmental cerebral hemorrhage.

Fibrosis is a common pathological response to inflammation in many peripheral tissues and can prevent tissue regeneration and repair. Here, we identified persistent fibrotic scarring in the CNS following immune cell infiltration in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Using lineage tracing and single-cell sequencing in EAE, we determined that the majority of the fibrotic scar is derived from proliferative CNS fibroblasts, not pericytes or infiltrating bone marrow-derived cells. Ablating proliferating fibrotic cells using cell-specific expression of herpes thymidine kinase led to an increase in oligodendrocyte lineage cells within the inflammatory lesions and a reduction in motor disability. We further identified that interferon-gamma pathway genes are enriched in CNS fibrotic cells, and the fibrotic cell-specific deletion of Ifngr1 resulted in reduced fibrotic scarring in EAE. These data delineate a framework for understanding the CNS fibrotic response.

Microglia play a pivotal role in the coordination of brain development and have emerged as a critical determinant in the progression of neurodegenerative diseases; however, the role of microglia in the onset and progression of neurodevelopmental disorders is less clear. Here we show that conditional deletion of αVβ8 from the central nervous system (Itgb8ΔCNS mice) blocks microglia in their normal stepwise development from immature precursors to mature microglia. These "dysmature" microglia appear to result from reduced TGFβ signaling during a critical perinatal window, are distinct from microglia with induced reduction in TGFβ signaling during adulthood, and directly cause a unique neurodevelopmental syndrome characterized by oligodendrocyte maturational arrest, interneuron loss, and spastic neuromotor dysfunction. Consistent with this, early (but not late) microglia depletion completely reverses this phenotype. Together, these data identify novel roles for αVβ8 and TGFβ signaling in coordinating microgliogenesis with brain development and implicate abnormally programmed microglia or their products in human neurodevelopmental disorders that share this neuropathology.

Myofibroblasts are the major source of extracellular matrix components that accumulate during tissue fibrosis, and hepatic stellate cells (HSCs) are believed to be the major source of myofibroblasts in the liver. To date, robust systems to genetically manipulate these cells have not been developed. We report that Cre under control of the promoter of Pdgfrb (Pdgfrb-Cre) inactivates loxP-flanked genes in mouse HSCs with high efficiency. We used this system to delete the gene encoding α(v) integrin subunit because various α(v)-containing integrins have been suggested as central mediators of fibrosis in multiple organs. Such depletion protected mice from carbon tetrachloride-induced hepatic fibrosis, whereas global loss of β₃, β₅ or β₆ integrins or conditional loss of β₈ integrins in HSCs did not. We also found that Pdgfrb-Cre effectively targeted myofibroblasts in multiple organs, and depletion of the α(v) integrin subunit using this system was protective in other models of organ fibrosis, including pulmonary and renal fibrosis. Pharmacological blockade of α(v)-containing integrins by a small molecule (CWHM 12) attenuated both liver and lung fibrosis, including in a therapeutic manner. These data identify a core pathway that regulates fibrosis and suggest that pharmacological targeting of all α(v) integrins may have clinical utility in the treatment of patients with a broad range of fibrotic diseases.