Cerebral cavernous malformations (CCMs) are a cause of stroke and seizure for which no effective medical therapies yet exist. CCMs arise from the loss of an adaptor complex that negatively regulates MEKK3–KLF2/4 signalling in brain endothelial cells, but upstream activators of this disease pathway have yet to be identified. Here we identify endothelial Toll-like receptor 4 (TLR4) and the gut microbiome as critical stimulants of CCM formation. Activation of TLR4 by Gram-negative bacteria or lipopolysaccharide accelerates CCM formation, and genetic or pharmacologic blockade of TLR4 signalling prevents CCM formation in mice. Polymorphisms that increase expression of the TLR4 gene or the gene encoding its co-receptor CD14 are associated with higher CCM lesion burden in humans. Germ-free mice are protected from CCM formation, and a single course of antibiotics permanently alters CCM susceptibility in mice. These studies identify unexpected roles for the microbiome and innate immune signalling in the pathogenesis of a cerebrovascular disease, as well as strategies for its treatment.

Cerebral cavernous malformation (CCM) is a common cerebrovascular disease that can occur sporadically or be inherited. They are major causes of stroke, cerebral hemorrhage, and neurological deficits in the younger population. Loss-of-function mutations in three genes, CCM1, CCM2, and CCM3, have been identified as the cause of human CCMs. Currently, no drug is available to treat CCM disease. Hyperactive mitogen-activated protein kinase kinase Kinase 3 (MEKK3) kinase signaling as a consequence of loss of CCM genes is an underlying cause of CCM lesion development. Using a U.S. Food and Drug Administration–approved kinase inhibitor library combined with virtual modeling and biochemical and cellular assays, we have identified a clinically approved small compound, ponatinib, that is capable of inhibiting MEKK3 activity and normalizing expression of downstream kruppel-like factor (KLF) target genes. Treatment with this compound in neonatal mouse models of CCM can prevent the formation of new CCM lesions and reduce the growth of already formed lesions. At the ultracellular level, ponatinib can normalize the flattening and disorganization of the endothelium caused by CCM deficiency. Collectively, our study demonstrates ponatinib as a novel compound that may prevent CCM initiation and progression in mouse models through inhibition of MEKK3-KLF signaling.

Aberrant tumor blood vessels are prone to propel the malignant progression of tumors, and targeting abnormal metabolism of tumor endothelial cells emerges as a promising option to achieve vascular normalization and antagonize tumor progression. Herein, we demonstrated that salvianic acid A (SAA) played a pivotal role in contributing to vascular normalization in the tumor-bearing mice, thereby improving delivery and effectiveness of the chemotherapeutic agent. SAA was capable of inhibiting glycolysis and strengthening endothelial junctions in the human umbilical vein endothelial cells (HUVECs) exposed to hypoxia. Mechanistically, SAA was inclined to directly bind to the glycolytic enzyme PKM2, leading to a dramatic decrease in endothelial glycolysis. More importantly, SAA improved the endothelial integrity via activating the β-Catenin/Claudin-5 signaling axis in a PKM2-dependent manner. Our findings suggest that SAA may serve as a potent agent for inducing tumor vascular normalization.

Cerebral cavernous malformations (CCMs) form following loss of the CCM protein complex in brain endothelial cells due to increased endothelial MEKK3 signaling and KLF2/4 transcription factor expression, but the downstream events that drive lesion formation remain undefined. Recent studies have revealed that CCM lesions expand by incorporating neighboring wild-type endothelial cells, indicative of a cell nonautonomous mechanism. Here we find that endothelial loss of ADAMTS5 reduced CCM formation in the neonatal mouse model. Conversely, endothelial gain of ADAMTS5 conferred early lesion genesis in the absence of increased KLF2/4 expression and synergized with KRIT1 loss of function to create large malformations. Lowering versican expression reduced CCM burden, indicating that versican is the relevant ADAMTS5 substrate and that lesion formation requires proteolysis but not loss of this extracellular matrix protein. These findings identify endothelial secretion of ADAMTS5 and cleavage of versican as downstream mechanisms of CCM pathogenesis and provide a basis for the participation of wild-type endothelial cells in lesion formation.