ABSTRACT
Nosebleeds and intracranial hemorrhage from brain arteriovenous malformations (bAVMs) are among the most devastating symptoms of patients with hereditary hemorrhagic telangiectasis (HHT). All available managements have limitations. We showed that intravenous delivery of soluble FMS-related tyrosine kinase 1 using an adeno-associated viral vector (AAV9-sFLT1) reduced bAVM severity of endoglin deficient mice. However, minor liver inflammation and growth arrest in young mice were observed. To identify AAV variants and delivery methods that can best transduce brain and nasal tissue with an optimal transduction profile, we compared 3 engineered AAV capsids (AAV.cc47, AAV.cc84 and AAV1RX) with AAV9. A single-stranded CBA promoter driven tdTomato transgene was packaged in these capsids and delivered intravenously (i.v.) or intranasally (i.n.) to wild-type mice. A CMV promoter driven Alk1 transgene was packaged into AAV.cc84 and delivered to PdgfbiCre;Alk1 f/f mice through i.v. injection followed by brain AVM induction. Transduced cells in different organs, vessel density and abnormal vessels in the bAVMs, and liver inflammation were analyzed histologically. Liver and kidney function were measured enzymatically. Compared to other viral vectors, AAV.cc84, after i.v. delivery, transduced a high percentage of brain ECs and few hepatocytes; whereas after i.n. delivery, AAV.cc84 transduced ECs and perivascular cells in the brain, and ECs, epithelial cells, and skeletal muscles in the nose with minimum hepatocyte transduction. No changes to liver or kidney function were detected. Delivery of AAV.cc84-Alk1 through i.v. to PdgfbiCre;Alk1 f/f mice reduced bAVM severity. In summary, we propose that AAV.cc84-Alk1 is a promising candidate for developing gene therapy in HHT patients.
ABSTRACT
Nosebleeds and intracranial hemorrhage from brain arteriovenous malformations (bAVMs) are among the most devastating symptoms of patients with hereditary hemorrhagic telangiectasis (HHT). All available managements have limitations. We showed that intravenous (i.v.) delivery of soluble Feline McDonough Sarcoma (FMS)-related tyrosine kinase 1 using an adeno-associated viral vector (AAV9-sFLT1) reduced bAVM severity of endoglin deficient mice. However, minor liver inflammation and growth arrest in young mice were observed. To identify AAV variants and delivery methods that can best transduce brain and nasal tissue with an optimal transduction profile, we compared 3 engineered AAV capsids (AAV.cc47, AAV.cc84, and AAV1RX) with AAV9. A single-stranded CBA promoter driven tdTomato transgene was packaged in these capsids and delivered i.v. or intranasally (i.n.) to wild-type mice. A CMV promoter driven Alk1 transgene was packaged into AAV.cc84 and delivered to PdgfbiCre;Alk1f/f mice through i.v. followed by bAVM induction. Transduced cells in organs, vessel density, abnormal vessels in the bAVMs, and liver inflammation were analyzed histologically. Liver and kidney function were measured enzymatically. Compared to other viral vectors, AAV.cc84, after i.v. delivery, transduced a high percentage of brain endothelial cells (ECs) and few hepatocytes; whereas after i.n. delivery, AAV.cc84 transduced ECs and perivascular cells in the brain, and ECs, epithelial cells, and muscles in the nose with minimum hepatocyte transduction. No changes to liver or kidney function were detected. The delivery of AAV.cc84-Alk1 through i.v. to PdgfbiCre;Alk1f/f mice reduced bAVM severity. In summary, we propose that AAV.cc84-Alk1 is a promising candidate for developing gene therapy in HHT patients.
ABSTRACT
BACKGROUND: Although many molecules have been investigated as biomarkers for spinal cord injury (SCI) or ischemic stroke, none of them are specifically induced in central nervous system (CNS) neurons following injuries with low baseline expression. However, neuronal injury constitutes a major pathology associated with SCI or stroke and strongly correlates with neurological outcomes. Biomarkers characterized by low baseline expression and specific induction in neurons post-injury are likely to better correlate with injury severity and recovery, demonstrating higher sensitivity and specificity for CNS injuries compared to non-neuronal markers or pan-neuronal markers with constitutive expressions. METHODS: In animal studies, young adult wildtype and global Atf3 knockout mice underwent unilateral cervical 5 (C5) SCI or permanent distal middle cerebral artery occlusion (pMCAO). Gene expression was assessed using RNA-sequencing and qRT-PCR, while protein expression was detected through immunostaining. Serum ATF3 levels in animal models and clinical human samples were measured using commercially available enzyme-linked immune-sorbent assay (ELISA) kits. RESULTS: Activating transcription factor 3 (ATF3), a molecular marker for injured dorsal root ganglion sensory neurons in the peripheral nervous system, was not expressed in spinal cord or cortex of naïve mice but was induced specifically in neurons of the spinal cord or cortex within 1 day after SCI or ischemic stroke, respectively. Additionally, ATF3 protein levels in mouse blood significantly increased 1 day after SCI or ischemic stroke. Importantly, ATF3 protein levels in human serum were elevated in clinical patients within 24 hours after SCI or ischemic stroke. Moreover, Atf3 knockout mice, compared to the wildtype mice, exhibited worse neurological outcomes and larger damage regions after SCI or ischemic stroke, indicating that ATF3 has a neuroprotective function. CONCLUSIONS: ATF3 is an easily measurable, neuron-specific biomarker for clinical SCI and ischemic stroke, with neuroprotective properties. HIGHLIGHTS: ATF3 was induced specifically in neurons of the spinal cord or cortex within 1 day after SCI or ischemic stroke, respectively. Serum ATF3 protein levels are elevated in clinical patients within 24 hours after SCI or ischemic stroke. ATF3 exhibits neuroprotective properties, as evidenced by the worse neurological outcomes and larger damage regions observed in Atf3 knockout mice compared to wildtype mice following SCI or ischemic stroke.
Subject(s)
Activating Transcription Factor 3 , Biomarkers , Ischemic Stroke , Neurons , Spinal Cord Injuries , Animals , Female , Humans , Male , Mice , Activating Transcription Factor 3/metabolism , Activating Transcription Factor 3/genetics , Biomarkers/metabolism , Biomarkers/blood , Disease Models, Animal , Ischemic Stroke/metabolism , Ischemic Stroke/genetics , Ischemic Stroke/blood , Mice, Knockout , Neurons/metabolism , Spinal Cord Injuries/metabolism , Spinal Cord Injuries/genetics , Spinal Cord Injuries/complicationsABSTRACT
Myeloid immune cells are abundant in both ruptured and unruptured brain arteriovenous malformations (bAVMs). The role of central nervous system (CNS) resident and circulating monocyte-derived macrophages in bAVM pathogenesis has not been fully understood. We hypothesize that CNS resident macrophages enhance bAVM development and hemorrhage. RNA sequencing using cultured endothelial cells (ECs) and mouse bAVM samples revealed that downregulation of two bAVM causative genes, activin-like kinase 1 (ALK1) or endoglin, increased inflammation and innate immune signaling. To understand the role of CNS resident macrophages in bAVM development and hemorrhage, we administrated a colony-stimulating factor 1 receptor inhibitor to bAVM mice with brain focal Alk1 deletion. Transient depletion of CNS resident macrophages at an early stage of bAVM development mitigated the phenotype severity of bAVM, including a prolonged inhibition of angiogenesis, dysplastic vasculature formation, and infiltration of CNS resident and circulating monocyte-derived macrophages during bAVM development. Transient depletion of CNS resident macrophages increased EC tight junction protein expression, reduced the number of dysplasia vessels and severe hemorrhage in established bAVMs. Thus, EC AVM causative gene mutation can activate CNS resident macrophages promoting bAVM progression. CNS resident macrophage could be a therapeutic target to mitigate the development and severity of bAVMs.
Subject(s)
Intracranial Arteriovenous Malformations , Macrophages , Monocytes , Neovascularization, Pathologic , Animals , Intracranial Arteriovenous Malformations/pathology , Intracranial Arteriovenous Malformations/metabolism , Intracranial Arteriovenous Malformations/genetics , Monocytes/metabolism , Macrophages/metabolism , Mice , Neovascularization, Pathologic/metabolism , Activin Receptors, Type II/metabolism , Activin Receptors, Type II/genetics , Humans , Endothelial Cells/metabolism , Endothelial Cells/pathology , Male , Mice, Knockout , Angiogenesis , EndoglinABSTRACT
Background: The increase in the collagen I (COL I)/COL III ratio enhances vessel wall stiffness and renders vessels less resistant to blood flow and pressure changes. Activated microglia enhance inflammation-induced fibrosis. Hypotheses: The COL I/COL III ratio in human and mouse brain arteriovenous malformations (bAVMs) is associated with bAVM hemorrhage, and the depletion of microglia decreases the COL I/COL III ratio and hemorrhage. Method: COL I, COL III, and hemorrhages were analyzed in 12 human bAVMs and 6 control brains, and mouse bAVMs induced in three mouse lines with activin receptor-like kinase 1 (n = 7) or endoglin (n = 7) deleted in the endothelial cells or brain focally (n = 5). The controls for the mouse study were no-gene-deleted litter mates. Mouse bAVMs were used to test the relationships between the Col I/Col III ratio and hemorrhage and whether the transient depletion of microglia reduces the Col I/Col III ratio and hemorrhage. Results: The COL I/COL III ratio was higher in the human and mouse bAVMs than in controls. The microhemorrhage in mouse bAVMs was positively correlated with the Col I/Col III ratio. Transient depletion of microglia reduced the Col I/Col III ratio and microhemorrhage. Conclusions: The COL I/COL III ratio in the bAVMs was associated with bAVM hemorrhage. The depletion of microglia reduced the bAVM Col I/Col III ratio and hemorrhage.
Subject(s)
Arteriovenous Malformations , Endothelial Cells , Humans , Animals , Mice , Brain , Hemorrhage/complications , Collagen Type IABSTRACT
Myeloid immune cells present abundantly in both ruptured and unruptured brain arteriovenous malformations (bAVMs). The role of central nervous system (CNS) resident and circulating monocyte-derived macrophages in bAVM pathogenesis has not been fully understood. RNA sequencing using cultured cells and bAVM samples revealed that downregulation of activin-like kinase 1 (ALK1) or endoglin (two bAVM causative genes) increased pro-angiogenic, endothelial inflammation and innate immune signaling, which provided endogenous underpinnings of the active inflammation in bAVM. To further understand the role of CNS resident macrophages in bAVM development and hemorrhage, we administrated a colony-stimulating factor 1 receptor (CSF1R) inhibitor to bAVM mice with endothelial Alk1 deletion. Transient depletion of CNS resident macrophages at early stage of bAVM development remarkably mitigated the subsequent phenotype severity of bAVM. This therapeutic effect exhibited a prolonged inhibition of angiogenesis, dysplastic vasculature formation, and infiltration of CNS resident and circulating monocyte-derived macrophages during bAVM development. Transient depletion of CNS resident macrophages also reduced the dysplasia vessels and improved the integrity of endothelial tight junctions in established bAVMs. Administration of CSF1R inhibitor also prevented severe hemorrhage of bAVMs. Thus, endothelial AVM causative gene mutation can activate CNS resident macrophages promoting bAVM progression. CNS resident macrophages could be specific targets to mitigate the development and severity of bAVMs.
ABSTRACT
We have previously demonstrated that deletion of activin receptor-like kinase 1 (Alk1) or endoglin in a fraction of endothelial cells (ECs) induces brain arteriovenous malformations (bAVMs) in adult mice upon angiogenic stimulation. Here, we addressed three related questions: (1) could Alk1- mutant bone marrow (BM)-derived ECs (BMDECs) cause bAVMs? (2) is Alk1- ECs clonally expended during bAVM development? and (3) is the number of mutant ECs correlates to bAVM severity? For the first question, we transplanted BM from PdgfbiCreER;Alk12f/2f mice (EC-specific tamoxifen-inducible Cre with Alk1-floxed alleles) into wild-type mice, and then induced bAVMs by intra-brain injection of an adeno-associated viral vector expressing vascular endothelial growth factor and intra-peritoneal injection of tamoxifen. For the second question, clonal expansion was analyzed using PdgfbiCreER;Alk12f/2f;confetti+/- mice. For the third question, we titrated tamoxifen to limit Alk1 deletion and compared the severity of bAVM in mice treated with low and high tamoxifen doses. We found that wild-type mice with PdgfbiCreER;Alk12f/2f BM developed bAVMs upon VEGF stimulation and Alk1 gene deletion in BMDECs. We also observed clusters of ECs expressing the same confetti color within bAVMs and significant proliferation of Alk1- ECs at early stage of bAVM development, suggesting that Alk1- ECs clonally expanded by local proliferation. Tamoxifen dose titration revealed a direct correlation between the number of Alk1- ECs and the burden of dysplastic vessels in bAVMs. These results provide novel insights for the understanding of the mechanism by which a small fraction of Alk1 or endoglin mutant ECs contribute to development of bAVMs.