Lei Zhan

Soluble FLT1 Gene Therapy Alleviates Brain Arteriovenous Malformation Severity

Background and Purpose— Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. Current therapies are associated with high morbidities. Excessive vascular endothelial growth factor has been implicated in bAVM pathophysiology. Because soluble FLT1 binds to vascular endothelial growth factor with high affinity, we tested intravenous delivery of an adeno-associated viral vector serotype-9 expressing soluble FLT1 (AAV9-sFLT1) to alleviate the bAVM phenotype. Methods— Two mouse models were used. In model 1, bAVM was induced in R26CreER;Eng2f/2f mice through global Eng gene deletion and brain focal angiogenic stimulation; AAV2-sFLT02 (an AAV expressing a shorter form of sFLT1) was injected into the brain at the time of model induction, and AAV9-sFLT1, intravenously injected 8 weeks after. In model 2, SM22&agr;Cre;Eng2f/2f mice had a 90% occurrence of spontaneous bAVM at 5 weeks of age and 50% mortality at 6 weeks; AAV9-sFLT1 was intravenously delivered into 4- to 5-week-old mice. Tissue samples were collected 4 weeks after AAV9-sFLT1 delivery. Results— AAV2-sFLT02 inhibited bAVM formation, and AAV9-sFLT1 reduced abnormal vessels in model 1 (GFP versus sFLT1: 3.66±1.58/200 vessels versus 1.98±1.29, P<0.05). AAV9-sFLT1 reduced the occurrence of bAVM (GFP versus sFLT1: 100% versus 36%) and mortality (GFP versus sFLT1: 57% [12/22 mice] versus 24% [4/19 mice], P<0.05) in model 2. Kidney and liver function did not change significantly. Minor liver inflammation was found in 56% of AAV9-sFLT1–treated model 1 mice. Conclusions— By applying a regulated mechanism to restrict sFLT1 expression to bAVM, AAV9-sFLT1 can potentially be developed into a safer therapy to reduce the bAVM severity.

Bone Fracture Pre-Ischemic Stroke Exacerbates Ischemic Cerebral Injury in Mice.

Ischemic stroke is a devastating complication of bone fracture. Bone fracture shortly after stroke enhances stroke injury by augmenting inflammation. We hypothesize that bone fracture shortly before ischemic stroke also exacerbates ischemic cerebral injury. Tibia fracture was performed 6 or 24 hours before permanent middle cerebral artery occlusion (pMCAO) on C57BL/6J mice or Ccr2RFP/+Cx3cr1GFP/+ mice that have the RFP gene knocked into one allele of Ccr2 gene and GFP gene knocked into one allele of Cx3cr1 gene. Behavior was tested 3 days after pMCAO. Infarct volume, the number of CD68+ cells, apoptotic neurons, bone marrow-derived macrophages (RFP+), and microgila (GFP+) in the peri-infarct region were quantified. Compared to mice subjected to pMCAO only, bone fracture 6 or 24 hours before pMCAO increased behavioral deficits, the infarct volume, and the number of CD68+ cells and apoptotic neurons in the peri-infarct area. Both bone marrow-derived macrophages (CCR2+) and microglia (CX3CR1+) increased in the peri-infarct regions of mice subjected to bone fracture before pMCAO compared to stroke-only mice. The mice subjected to bone fracture 6 hours before pMCAO had more severe injury than mice that had bone fracture 24 hours before pMCAO. Our data showed that bone fracture shortly before stroke also increases neuroinflammation and exacerbates ischemic cerebral injury. Our findings suggest that inhibition of neuroinflammation or management of stroke risk factors before major bone surgery would be beneficial for patients who are likely to suffer from stroke.

Thalidomide Reduces Hemorrhage of Brain Arteriovenous Malformations in a Mouse Model

Background and Purpose— Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. Current treatments for bAVM are all associated with considerable risks. There is no safe method to prevent bAVM hemorrhage. Thalidomide reduces nose bleeding in patients with hereditary hemorrhagic telangiectasia, an inherited disorder characterized by vascular malformations. In this study, we tested whether thalidomide and its less toxic analog, lenalidomide, reduce bAVM hemorrhage using a mouse model. Methods— bAVMs were induced through induction of brain focal activin-like kinase 1 (Alk1, an AVM causative gene) gene deletion and angiogenesis in adult Alk1-floxed mice. Thalidomide was injected intraperitoneally twice per week for 6 weeks, starting either 2 or 8 weeks after AVM induction. Lenalidomide was injected intraperitoneally daily starting 8 weeks after AVM induction for 6 weeks. Brain samples were collected at the end of the treatments for morphology, mRNA, and protein analyses. The influence of Alk1 downregulation on PDGFB (platelet-derived growth factor B) expression was also studied on cultured human brain microvascular endothelial cells. The effect of PDGFB in mural cell recruitment in bAVM was explored by injection of a PDGFB overexpressing lentiviral vector to the mouse brain. Results— Thalidomide or lenalidomide treatment reduced the number of dysplastic vessels and hemorrhage and increased mural cell (vascular smooth muscle cells and pericytes) coverage in the bAVM lesion. Thalidomide reduced the burden of CD68+ cells and the expression of inflammatory cytokines in the bAVM lesions. PDGFB expression was reduced in ALK1-knockdown human brain microvascular endothelial cells and in mouse bAVM lesion. Thalidomide increased Pdgfb expression in bAVM lesion. Overexpression of PDGFB mimicked the effect of thalidomide. Conclusions— Thalidomide and lenalidomide improve mural cell coverage of bAVM vessels and reduce bAVM hemorrhage, which is likely through upregulation of Pdgfb expression.

Correction: Bone Fracture Pre-Ischemic Stroke Exacerbates Ischemic Cerebral Injury in Mice

[This corrects the article DOI: 10.1371/journal.pone.0153835.].

385. Intravenous Delivery of AAV9-CMV-sFLT1 Reduces the Severity of Brain Arteriovenous Malformation Without Causing Significant Side Effects in a Mouse Model

IV injection of AAV9-CMV-sFLT1 reduces bAVM severity, does not cause neuronal loss and lymphocyte infiltration in the brain, and does not significantly alter liver and kidney function. Since inflammatory cell infiltration has been detected in the liver of AAV9-treated mice, strategies for limiting liver gene transduction and expression should be developed for systemic delivery of AAV-sFLT1 for treating bAVM.