Brain Angiogenesis Research Articles

1255-P: The Role of BAI3 Adhesion G-Protein Coupled Receptor in Insulin Secretion and Glucose Tolerance

Insulin secretion from beta-cells of pancreatic islets is essential for regulating whole-blood glucose homeostasis. Impaired insulin secretion is an early event in the development of obesity-linked type 2 diabetes (T2D). However, underlying molecular mechanisms that cause a reduction in insulin secretion are not completely understood. We recently identified that brain angiogenesis inhibitor-3 (BAI3) adhesion G-protein coupled receptor is expressed in beta-cells, and when activated by its native ligand, complement-1q like-3 (C1ql3), blunts insulin secretion in response to cyclic adenosine monophosphate (cAMP) in an autocrine/paracrine manner. The expression of BAI3 is conserved in human islets, yet the role of islet BAI3 in whole-body glucose homeostasis remains uncharacterized. We generated C57BL6/J mice that are homozygous for the loss of the BAI3 gene by using CRISPR technology. Our data show that the BAI3-/- mice have reduced body weight and impaired glucose tolerance compared to the wild-type control (WT) mice. These mice have elevated in vivo insulin secretion after 10 min of glucose challenge and an increased ex vivo islet insulin secretion in response to stimulation by 40 mM KCL and 11 mM glucose with no effect observed at 2.8 mM glucose. Concomitantly, the insulin action is decreased in the BAI3-/- mice. These outcomes show that increased insulin secretion from beta-cells due to the loss of the BAI3 gene contributes to reduced insulin sensitivity and glucose tolerance. The expression of BAI3 is increased by obesity, indicating that the activation of BAI3 signaling may contribute to reduced insulin secretion, causing beta-cell dysfunction in T2D. In sum, we have established an important role of an understudied BAI3 GPCR signaling in inhibiting insulin secretion, potentially identifying an attractive target for novel T2D therapeutics. Disclosure H. Alsharif: None. M. Rahman: None. A. Pathak: None. J. E. Trombley: None. S. Bhatnagar: None. Funding National Institutes of Health (R01DK120684-01)

Developmental regulation of barrier- and non-barrier blood vessels in the CNS.

The blood-brain barrier (BBB) is essential for creating and maintaining tissue homeostasis in the central nervous system (CNS), which is key for proper neuronal function. In most vertebrates, the BBB is localized to microvascular endothelial cells that acquire barrier properties during angiogenesis of the neuroectoderm. Complex and continuous tight junctions, and the lack of fenestrae combined with low pinocytotic activity render the BBB endothelium a tight barrier for water-soluble molecules that may only enter the CNS via specific transporters. The differentiation of these unique endothelial properties during embryonic development is initiated by endothelial-specific flavours of the Wnt/β-catenin pathway in a precise spatiotemporal manner. In this review, we summarize the currently known cellular (neural precursor and endothelial cells) and molecular (VEGF and Wnt/β-catenin) mechanisms mediating brain angiogenesis and barrier formation. Moreover, we introduce more recently discovered crosstalk with cellular and acellular elements within the developing CNS such as the extracellular matrix. We discuss recent insights into the downstream molecular mechanisms of Wnt/β-catenin in particular, the recently identified target genes like Foxf2, Foxl2, Foxq1, Lef1, Ppard, Zfp551, Zic3, Sox17, Apcdd1 and Fgfbp1 that are involved in refining and maintaining barrier characteristics in the mature BBB endothelium. Additionally, we elute to recent insight into barrier heterogeneity and differential endothelial barrier properties within the CNS, focussing on the circumventricular organs as well as on the neurogenic niches in the subventricular zone and the hippocampus. Finally, open questions and future BBB research directions are highlighted in the context of taking benefit from understanding BBB development for strategies to modulate BBB function under pathological conditions.

Abstract 63: Mutation of Platelet-Derived Growth Factor Receptor B Causes Cerebrovascular Malformation and Enhances Brain Arteriovenous Malformation Severity in Mice

Background and Purpose: Deletion of Endoglin (Eng), an arteriovenous malformation (AVM) causative gene causes AVM in the brain angiogenic region. Reduction of pericytes is correlate with brain AVM hemorrhage. The platelet-derived growth factor B and its receptor β (PDGF-B/PDGFRβ) play important roles in regulating pericyte recruitment during angiogenesis. We hypothesize that mutation of PDGFRβ causes cerebrovascular malformation and enhances bAVM severity in Eng mutant mice. Methods: Three mouse models were used: (1) Pdgfrβ F7 (F7) mice that have mutations disrupting Pdgfrβ signaling, (2) Pdgfb -icreER; Eng f /f mice that have Pdgfb promoter driving, tamoxifen (TM) inducible cre expression in ECs and an floxed Eng gene; and (3) Pdgfb -icreER; Eng f /f ;F7 +/- mice. Brain angiogenesis was induced by intra-brain injection of an adeno-associated viral vector expressing vascular endothelial growth factor (AAV-VEGF). Brain AVMs were induced in Pdgfb -icreER; Eng f /f and Pdgfb -icreER; Eng f /f ;F7 mice by TM induced EC Eng deletion and intra-brain AAV-VEGF injection. Brain AVM phenotypes were analyzed 8-weeks after model induction by latex vascular cast to detect arteriovenous shunts and macroscopic level of AVMs, immunostaining and Prussian blue staining to quantify dysplasia vessels and hemorrhage. Results: Compared to WT mice, F7 +/- and F7 +/+ mice have more dysplastic vessels and fewer vascular pericyte before and after AAV-VEGF injection. F7 +/- and F7 +/+ mice showed hemorrhage on the AAV-VEGF injection sites and AVM like vessels (a few arteriovenous shunts) in 16% F7 +/- and 66% of F7 +/+ mice. Pdgfb -icreER; Eng f /f mice showed dysplastic vessels and hemorrhage at AAV-VEGF injection sites. Compared to Pdgfb -icreER; Eng f /f mice, Pdgfb -icreER; Eng f /f ;F7 +/- had a higher penetrance of bAVM (71% vs 50%) and more dysplastic vessels. Conclusion: F7 mutation cause AVM like structure in mouse brain and exacerbates bAVM phenotype in Eng mutant mice.

Cyclooxygenase inhibition attenuates brain angiogenesis and independently decreases mouse survival under hypoxia.

Although cyclooxygenase (COX) role in cancer angiogenesis has been studied, little is known about its role in brain angioplasticity. In the present study, we chronically infused mice with ketorolac, a non‐specific COX inhibitor that does not cross the blood–brain barrier (BBB), under normoxia or 50% isobaric hypoxia (10% O2 by volume). Ketorolac increased mortality rate under hypoxia in a dose‐dependent manner. Using in vivo multiphoton microscopy, we demonstrated that chronic COX inhibition completely attenuated brain angiogenic response to hypoxia. Alterations in a number of angiogenic factors that were reported to be COX‐dependent in other models were assayed at 24‐hr and 10‐day hypoxia. Intriguingly, hypoxia‐inducible factor 1 was unaffected under COX inhibition, and vascular endothelial growth factor receptor type 2 (VEGFR2) and C‐X‐C chemokine receptor type 4 (CXCR4) were significantly but slightly decreased. However, a number of mitogen‐activated protein kinases (MAPKs) were significantly reduced upon COX inhibition. We conclude that additional, angiogenic factor‐independent mechanism might contribute to COX role in brain angioplasticity, probably including mitogenic COX effect on endothelium. Our data indicate that COX activity is critical for systemic adaptation to chronic hypoxia, and BBB COX is essential for hypoxia‐induced brain angioplasticity. These data also indicate a potential risk for using COX inhibitors under hypoxia conditions in clinics. Further studies are required to elucidate a complete mechanism for brain long‐term angiogenesis regulation through COX activity.

An early endothelial cell-specific requirement for Glut1 is revealed in Glut1 deficiency syndrome model mice.

Paucity of the glucose transporter-1 (Glut1) protein resulting from haploinsufficiency of the SLC2A1 gene arrests cerebral angiogenesis and disrupts brain function to cause Glut1 deficiency syndrome (Glut1 DS). Restoring Glut1 to Glut1 DS model mice prevents disease, but the precise cellular sites of action of the transporter, its temporal requirements, and the mechanisms linking scarcity of the protein to brain cell dysfunction remain poorly understood. Here, we show that Glut1 functions in a cell-autonomous manner in the cerebral microvasculature to affect endothelial tip cells and, thus, brain angiogenesis. Moreover, brain endothelial cell–specific Glut1 depletion not only triggers a severe neuroinflammatory response in the Glut1 DS brain, but also reduces levels of brain-derived neurotrophic factor (BDNF) and causes overt disease. Reduced BDNF correlated with fewer neurons in the Glut1 DS brain. Controlled depletion of the protein demonstrated that brain pathology and disease severity was greatest when Glut1 scarcity was induced neonatally, during brain angiogenesis. Reducing Glut1 at later stages had mild or little effect. Our results suggest that targeting brain endothelial cells during early development is important to ensure proper brain angiogenesis, prevent neuroinflammation, maintain BDNF levels, and preserve neuron numbers. This requirement will be essential for any disease-modifying therapeutic strategy for Glut1 DS.

Myrtenol improves brain damage and promotes angiogenesis in rats with cerebral infarction by activating the ERK1/2 signalling pathway

Context Cerebral ischaemia/reperfusion (I/R) injury has a high disability and fatality worldwide. Myrtenol has protective effects on myocardial I/R injury through antioxidant and anti-apoptotic effects. Objective This study investigated the effect of myrtenol on cerebral ischaemia/reperfusion (I/R) injury and the underlying mechanism. Materials and methods Cerebral I/R injury was induced in adult Sprague-Dawley rats by middle cerebral artery occlusion (MCAO) for 90 min. MCAO rats were treated with or without myrtenol (10, 30, or 50 mg/kg/day) or/and U0126 (10 μL) intraperitoneally for 7 days. Results In the present study, myrtenol had no toxicity at concentrations up to 1.3 g/kg. Myrtenol treatment improved neurological function of MCAO rats, with significantly (p < 0.05) improved neurological deficits (4.31 ± 1.29 vs. 0.00) and reduced brain edoema (78.95 ± 2.27% vs. 85.48 ± 1.24%). Myrtenol extenuated brain tissue injury and neuronal apoptosis, with increased Bcl-2 expression (0.48-fold) and decreased Bax expression (2.02-fold) and caspase-3 activity (1.36-fold). Myrtenol promoted angiogenesis in the brain tissues of MCAO rats, which was reflected by increased VEGF (0.86-fold) and FGF2 (0.51-fold). Myrtenol promoted the phosphorylation of MEK1/2 (0.80-fold) and ERK1/2 (0.97-fold) in MCAO rats. U0126, the inhibitor of ERK1/2 pathway, reversed the protective effects of myrtenol on brain tissue damage and angiogenesis in MCAO rats. Discussion and conclusions Myrtenol reduced brain damage and angiogenesis through activating the ERK1/2 signalling pathway, which may provide a novel alternative strategy for preventing cerebral I/R injury. Further in vitro work detailing its mechanism-of-action for improving ischaemic cerebral infarction is needed.

H2S promotes developmental brain angiogenesis via the NOS/NO pathway in zebrafish

BackgroundHydrogen sulphide (H2S) is considered as the third member of the gasotransmitter family, along with nitric oxide (NO) and carbon monoxide. H2S has been reported to induce angiogenesis by promoting...

BMS-986020, a Specific LPA1 Antagonist, Provides Neuroprotection against Ischemic Stroke in Mice.

Stroke is a leading cause of death. Stroke survivors often suffer from long-term functional disability. This study demonstrated neuroprotective effects of BMS-986020 (BMS), a selective lysophosphatidic acid receptor 1 (LPA1) antagonist under clinical trials for lung fibrosis and psoriasis, against both acute and sub-acute injuries after ischemic stroke by employing a mouse model with transient middle cerebral artery occlusion (tMCAO). BMS administration immediately after reperfusion significantly attenuated acute brain injuries including brain infarction, neurological deficits, and cell apoptosis at day 1 after tMCAO. Neuroprotective effects of BMS were preserved even when administered at 3 h after reperfusion. Neuroprotection by BMS against acute injuries was associated with attenuation of microglial activation and lipid peroxidation in post-ischemic brains. Notably, repeated BMS administration daily for 14 days after tMCAO exerted long-term neuroprotection in tMCAO-challenged mice, as evidenced by significantly attenuated neurological deficits and improved survival rate. It also attenuated brain tissue loss and cell apoptosis in post-ischemic brains. Mechanistically, it significantly enhanced neurogenesis and angiogenesis in injured brains. A single administration of BMS provided similar long-term neuroprotection except survival rate. Collectively, BMS provided neuroprotection against both acute and sub-acute injuries of ischemic stroke, indicating that BMS might be an appealing therapeutic agent to treat ischemic stroke.

Inhibition of LPA5 Activity Provides Long-Term Neuroprotection in Mice with Brain Ischemic Stroke.

Stroke is a leading cause of long-term disability in ischemic survivors who are suffering from motor, cognitive, and memory impairment. Previously, we have reported suppressing LPA5 activity with its specific antagonist can attenuate acute brain injuries after ischemic stroke. However, it is unclear whether suppressing LPA5 activity can also attenuate chronic brain injuries after ischemic stroke. Here, we explored whether effects of LPA5 antagonist, TCLPA5, could persist a longer time after brain ischemic stroke using a mouse model challenged with tMCAO. TCLPA5 was administered to mice every day for 3 days, starting from the time immediately after reperfusion. TCLPA5 administration improved neurological function up to 21 days after tMCAO challenge. It also reduced brain tissue loss and cell apoptosis in mice at 21 days after tMCAO challenge. Such long-term neuroprotection of TCLPA5 was associated with enhanced neurogenesis and angiogenesis in post-ischemic brain, along with upregulated expression levels of vascular endothelial growth factor. Collectively, results of the current study indicates that suppressing LPA5 activity can provide long-term neuroprotection to mice with brain ischemic stroke.

Hurdle Aerobic Exercise Increases Angiogenesis and Neuroplasticity in the Hippocampus and Improves the Spatial Memory Ability of Middle-aged Mice

BACKGROUND: Complex aerobic exercise is believed to induce positive effects on neuron structure and cognitive function. Long-term and continual cognitive stimulation increases neuroplasticity by stimulating the synthesis of neuronal growth proteins and the formation of new synapses. Exercise also increases the ability of neurons to survive and improves brain vascularization. Further investigations should be conducted to explore what types of aerobic exercise are beneficial for cognitive function.&#x0D; AIM: This study investigated the effects of hurdle aerobic exercise on developmentally regulated brain protein-A (Drebrin-A) as a neuroplasticity indicator, and on vascular endothelial growth factor (VEGF) as an angiogenesis marker in the hippocampus.&#x0D; METHODS: This study was an experimental study with post-test only control group design. Thirty-three adult mice were divided into control, hurdle aerobic runner (HAR), and plain aerobic runner (PAR) groups (n = 11 for each group). Fiberglass running wheels were originally designed and modified to assemble hurdles inside with adjustable speed. Speed adaptation was intended to achieve aerobic intensity. The experiment was performed 5 times a week for 8 weeks. The Morris water maze test (MWMT) was used to assess spatial memory ability. One day after the last running exercise and final MWMT, the mice were sacrificed and the right side of the hippocampus was obtained for Drebrin-A analysis by enzyme-linked immunosorbent assay (ELISA). The entire right side brain tissue after hippocampus was removed then used for the neuroglobin ELISA assay. To analyze VEGF expression and calculation of blood vessel, the left side of the brain was prepared for hematoxylin eosin and immunohistochemistry staining. To assess the effect of exercise on vascular widening, the analysis of the slides was performed by calculating the percentage of blood vessels with diameters more than 15 μm. One-way ANOVA and Fisher’s least significant difference test was used for statistical analysis.&#x0D; RESULTS: There was a significant difference in the levels of Drebrin-A between the HAR and PAR groups. Both exercise groups had higher levels of Drebrin-A than the control group. HAR and PAR groups exhibited significantly higher percentages of blood vessels expressing VEGF in hippocampus compared to control. HAR and PAR groups had the higher percentages of larger vessels compare to control. There was no significant difference of neuroglobin levels among the three groups. Both the HAR and PAR groups exhibited better spatial memory than the control group.&#x0D; CONCLUSION: Both aerobic exercises induced positive effects on brain angiogenesis, while the intensity of aerobic exercises did not result in high hypoxic stress in the brain.

Three-dimensional induced pluripotent stem-cell models of human brain angiogenesis

During brain development, chemical cues released by developing neurons, cellular signaling with pericytes, and mechanical cues within the brain extracellular matrix (ECM) promote angiogenesis of brain microvascular endothelial cells (BMECs). Angiogenesis is also associated with diseases of the brain due to pathological chemical, cellular, and mechanical signaling. Existing in vitro and in vivo models of brain angiogenesis have key limitations. Here, we develop a high-throughput in vitro blood-brain barrier (BBB) bead assay of brain angiogenesis utilizing 150 μm diameter beads coated with induced pluripotent stem-cell (iPSC)-derived human BMECs (dhBMECs). After embedding the beads within a 3D matrix, we introduce various chemical cues and extracellular matrix components to explore their effects on angiogenic behavior. Based on the results from the bead assay, we generate a multi-scale model of the human cerebrovasculature within perfusable three-dimensional tissue-engineered blood-brain barrier microvessels. A sprouting phenotype is optimized in confluent monolayers of dhBMECs using chemical treatment with vascular endothelial growth factor (VEGF) and wnt ligands, and the inclusion of pro-angiogenic ECM components. As a proof-of-principle that the bead angiogenesis assay can be applied to study pathological angiogenesis, we show that oxidative stress can exert concentration-dependent effects on angiogenesis. Finally, we demonstrate the formation of a hierarchical microvascular model of the human blood-brain barrier displaying key structural hallmarks. We develop two in vitro models of brain angiogenesis: the BBB bead assay and the tissue-engineered BBB microvessel model. These platforms provide a tool kit for studies of physiological and pathological brain angiogenesis, with key advantages over existing two-dimensional models.

Nicotine promotes angiogenesis in mouse brain after intracerebral hemorrhage

Here we examined the effect of nicotine on angiogenesis in the brain after intracerebral hemorrhage (ICH), as angiogenesis is considered to provide beneficial effects on brain tissues during recovery from injury after stroke. Nicotine was administered to C57BL/6 mice suffering from collagenase-induced ICH in the striatum, either by inclusion in drinking water or by daily intraperitoneal injection. Nicotine administration by both routes enhanced angiogenesis within the hematoma-affected regions, as revealed by increased CD31-immunopositive area at 7 and 14 d after ICH. Double immunofluorescence histochemistry against CD31 and proliferating cell nuclear antigen revealed that nicotine increased the number of newly generated vascular endothelial cells within the hematoma. In spite of enhanced angiogenesis, nicotine did not worsen vascular permeability after ICH, as assessed by Evans Blue extravasation. These effects of nicotine were accompanied by an increased number of surviving neurons in the hematoma at 7 d after ICH. Unexpectedly, nicotine did not increase expression of vascular endothelial growth factor mRNA in the brain and did not enhance recruitment of endothelial progenitor cells from the bone marrow. These results suggest that nicotine enhances angiogenesis in the brain after ICH, via mechanisms distinct from those involved in its action on angiogenesis in peripheral tissues.

Lack of Flvcr2 impairs brain angiogenesis without affecting the blood-brain barrier.

Fowler syndrome is a rare autosomal recessive brain vascular disorder caused by mutation in FLVCR2 in humans. The disease occurs during a critical period of brain vascular development, is characterized by glomeruloid vasculopathy and hydrocephalus, and is almost invariably prenatally fatal. Here, we sought to gain insights into the process of brain vascularization and the pathogenesis of Fowler syndrome by inactivating Flvcr2 in mice. We showed that Flvcr2 was necessary for angiogenic sprouting in the brain, but surprisingly dispensable for maintaining the blood-brain barrier. Endothelial cells lacking Flvcr2 had altered expression of angiogenic factors, failed to adopt tip cell properties, and displayed reduced sprouting, leading to vascular malformations similar to those seen in humans with Fowler syndrome. Brain hypovascularization was associated with hypoxia and tissue infarction, ultimately causing hydrocephalus and death of mutant animals. Strikingly, despite severe vascular anomalies and brain tissue infarction, the blood-brain barrier was maintained in Flvcr2 mutant mice. Our Fowler syndrome model therefore defined the pathobiology of this disease and provided new insights into brain angiogenesis by showing uncoupling of vessel morphogenesis and blood-brain barrier formation.

Importance of the Role of ω-3 and ω-6 Polyunsaturated Fatty Acids in the Progression of Brain Cancer.

Brain cancer is one of the most malignant types of cancer in both children and adults. Brain cancer patients tend to have a poor prognosis and a high rate of mortality. Additionally, 20–40% of all other types of cancer can develop brain metastasis. Numerous pieces of evidence suggest that omega-3-polyunsaturated fatty acids (ω-PUFAs) could potentially be used in the prevention and therapy of several types of cancer. PUFAs and oxylipins are fundamental in preserving physiological events in the nervous system; it is, therefore, necessary to maintain a certain ratio of ω-3 to ω-6 for normal nervous system function. Alterations in PUFAs signaling are involved in the development of various pathologies of the nervous system, including cancer. It is well established that an omega-6-polyunsaturated fatty acid (ω-6 PUFA)-rich diet has a pro-tumoral effect, whereas the consumption of an ω-3 rich diet has an anti-tumoral effect. This review aims to offer a better understanding of brain cancer and PUFAs and to discuss the role and impact of PUFAs on the development of different types of brain cancer. Considering the difficulty of antitumor drugs in crossing the blood–brain barrier, the therapeutic role of ω-3/ω-6 PUFAs against brain cancer would be a good alternative to consider. We highlight our current understanding of the role of PUFAs and its metabolites (oxylipins) in different brain tumors, proliferation, apoptosis, invasion, angiogenesis, and immunosuppression by focusing on recent research in vitro and in vivo.

PS1 FAD mutants decrease ephrinB2-regulated angiogenic functions, ischemia-induced brain neovascularization and neuronal survival.

Microvascular pathology and ischemic lesions contribute substantially to neuronal dysfunction and loss that lead to Alzheimer disease (AD). To facilitate recovery, the brain stimulates neovascularization of damaged tissue via sprouting angiogenesis, a process regulated by endothelial cell (EC) sprouting and the EphB4/ephrinB2 system. Here, we show that in cultures of brain ECs, EphB4 stimulates the VE-cadherin/Rok-α angiogenic complexes known to mediate sprouting angiogenesis. Importantly, brain EC cultures expressing PS1 FAD mutants decrease the EphB4-stimulated γ-secretase cleavage of ephrinB2 and reduce production of the angiogenic peptide ephrinB2/CTF2, the VE-cadherin angiogenic complexes and EC sprouting and tube formation. These data suggest that FAD mutants may attenuate ischemia-induced brain angiogenesis. Supporting this hypothesis, ischemia-induced VE-cadherin angiogenic complexes, levels of neoangiogenesis marker Endoglin, vascular density, and cerebral blood flow recovery, are all decreased in brains of mouse models expressing PS1 FAD mutants. Ischemia-induced brain neuronal death and cognitive deficits also increase in these mice. Furthermore, a small peptide comprising the C-terminal sequence of peptide ephrinB2/CTF2 rescues angiogenic functions of brain ECs expressing PS1 FAD mutants. Together, our data show that PS1 FAD mutations impede the EphB4/ephrinB2-mediated angiogenic functions of ECs and impair brain neovascularization, neuronal survival and cognitive recovery following ischemia. Furthermore, our data reveal a novel brain angiogenic mechanism targeted by PS1 FAD mutants and a potential therapeutic target for ischemia-induced neurodegeneration. Importantly, FAD mutant effects occur in absence of neuropathological hallmarks of AD, supporting that such hallmarks may form downstream of mutant effects on neoangiogenesis and neuronal survival.

Hypothesis for How Hyperoxic Therapy May Facilitate Effective Biologic “Correction” of Autism Spectrum Disorders

The existence of morphological and physiological abnormalities in the autism brain was recognized in the 1960s. Today, in view of many such extensive pathological findings, it has been stated and reiterated that autism spectrum disorder (ASD) has a biological rather than a psychological basis. Despite this, other than pharmacologic agents that address limited aspects of symptoms, development of therapies for ASD has been focused primarily on psychosocial measures based on principles of applied behavioral analysis. With this latter approach, however, a generally accepted intervention or group of interventions which consistently and comprehensively address the needs of most individuals with autism in a cost-effective and time-efficient manner has not yet been established. While examining reports of another medical intervention, hyperbaric oxygen therapy with inspired oxygen partial pressures typically ranging from 0.31 to 1.5 atm. for neurological conditions such as autism, we wondered if increased oxygen partial pressure, alone, without the encumbrance, risk, and cost of hyperbaric exposure would produce useful clinical outcomes. In five pilot cases, therefore, a normobaric hyperoxic treatment we have called Microbaric® Oxygen Therapy (MBO2) was administered to preteen and teenage boys with ASD. All results were positive, some remarkably so, and addressed the full scope of the subject’s symptoms. Also, follow-up for over seven years in several cases suggests these outcomes are permanent. To help encourage controlled research on MBO2 for ASD, we have laid out a hypothetical basis for its efficacy using our own pilot study outcomes and observations together with relevant information from the scientific literature, the most important of which we believe relates to regional brain hypoperfusion, neuroinflammation, and angiogenesis. In investigating research on angiogenesis and autism, we found a 2016 publication concerning autism postmortem brains which identified persistent splitting angiogenesis, as opposed to sprouting angiogenesis, in some regions of all donated autism brains and in none of the age-matched control brains. With this finding and the established effects of hyperoxia on angiogenesis and inflammation, we have developed a hypothesis that potentially accounts for regional cerebral hypoperfusion; delayed, progressive onset of ASD over the early years of life; increased perfusion in hypoperfused regions of the brain following hyperoxic therapy; the broad range of oxygen partial pressures that seem to have impact; permanent reduction in the symptoms of autism resulting from MBO2; and perhaps, a time-efficient and cost-effective method of treating autism that will help upgrade the long-term prognosis for affected individuals.

Expanding the clinical spectrum of Fowler syndrome: Three siblings with survival into adulthood and systematic review of the literature.

Proliferative vasculopathy and hydranencephaly-hydrocephaly syndrome (PVHH, OMIM 225790), also known as Fowler syndrome, is a rare autosomal recessive disorder of brain angiogenesis. PVHH has long been considered to be prenatally lethal. We evaluated the phenotypes of the first three siblings with survival into adulthood, performed a systematic review of the Fowler syndrome literature and delineated genotype-phenotype correlations using a scoring system to rate the severity of the disease. Thirty articles were included, describing 69 individual patients. To date, including our clinical reports, 72 patients have been described with Fowler syndrome. Only 6/72 (8%) survived beyond birth. Although our three patients carry the same mutations (c.327T>A-p.Asn109Lys and c.887C>T-p.Ser296Leu) in FLVCR2, only two of them presented with the same cerebral features, ventriculomegaly and cerebral calcifications, as affected fetuses. The third sibling has a surprisingly milder clinical and radiological phenotype, suggesting intrafamilial variability. Although no clear phenotype-genotype correlation exists, some variants appear to be associated with a less severe phenotype compatible with life. As such, it is important to consider Fowler syndrome in patients with gross ventriculomegaly, cortical malformations and/or cerebral calcifications on brain imaging.

A novel sensitive assay for detection of a biomarker of pericyte injury in cerebrospinal fluid.

Blood-brain barrier (BBB) breakdown and loss of brain capillary pericytes contributes to cognitive impairment. Pericytes express platelet-derived growth factor receptor-β (PDGFRβ) that regulates brain angiogenesis and blood vessel stability. Elevated soluble PDGFRβ (sPDGFRβ) levels in cerebrospinal fluid (CSF) indicate pericyte injury and BBB breakdown, which is an early biomarker of human cognitive dysfunction. A combination of reagents and conditions were tested, optimized, and validated on the Meso Scale Discovery electrochemiluminescence platform to develop a new sPDGFRβ immunoassay that was used to measure sPDGFRβ in human CSF from 147 individuals. We developed standard operating procedures for a highly sensitive and reproducible sPDGFRβ immunoassay with a dynamic range from 100 to 26,000 pg/mL, and confirmed elevated CSF sPDGFRβ levels in individuals with cognitive dysfunction. This assay could be applied at different laboratories to study brain pericytes and microvascular damage in relation to cognition in disorders associated with neurovascular and cognitive dysfunction.

Brain Angiogenesis Induced by Nonviral Gene Therapy with Potential Therapeutic Benefits for Central Nervous System Diseases.

Gene therapy employing nanocarriers represents a promising strategy to treat central nervous system (CNS) diseases, where brain microvasculature is frequently compromised. Vascular endothelial growth factor (VEGF) is a key angiogenic molecule; however, its in vivo administration to the CNS by nonviral gene therapy has not been conducted. Hence, we prepared and physicochemically characterized four cationic niosome formulations (1-4), which were combined with pVEGF-GFP to explore their capacity to transfer the VEGF gene to CNS cells and achieve angiogenesis in the brain. Experiments in primary neuronal cells showed successful and safe transfection with niosome 4, producing double levels of biologically active VEGF in comparison to the rest of the formulations. Intracortical administration of niosome 4 based nioplexes in mouse brain validated the ability of this nonviral vector to deliver the VEGF gene to CNS cells, inducing brain angiogenesis and emerging as a promising therapeutic approach for the treatment of CNS diseases.

Teratogenic effects of 1.5% EtOH in Cranial Blood Vessel development and Neurogenesis in Zebrafish larvae at 72hpf

Fetal alcohol spectrum disorder, which is observed in children who have been exposed to alcohol in utero, is a leading cause of neurodevelopmental disability. Studies in rodents have shown that prenatal ethanol exposure results in decreased blood flow from the umbilical artery to the fetal brain, compromising neuron formation and angiogenesis in the maturing brain. Ethanol (EtOH) has shown to disrupt coronary artery formation in the embryonic zebrafish, but the effects of ethanol on the developing cerebral blood vessels and neurogenesis in zebrafish larvae remains poorly understood. We exposed zebrafish larvae to 1.5% ethanol at 4 hours post fertilization (hpf) for 24 hours and imaged the brain at 72 hpf. We used Tg(fli1:EGFP) to image cerebral blood vessels and Tg(ETvmat2:GFP) to visualize monoaminergic neurons using confocal microscopy. Morphological data collected at 72hpf depicted significantly higher rates of craniofacial deformities, pericardial edema, and severe axial malformations in 1.5% EtOH‐treated embryos in comparison to control. Our preliminary results suggest that EtOH at 1.5% leads to reduced number of cranial vessels and altered vessel branching, and decreased density of telencephalic and hindbrain neurons. These findings lead to questions regarding molecular and vascular disparities in affected regions of the developing brain.Support or Funding InformationI am currently seeking for funds (nonmember student registration fee) for our undergraduate students to attend and present this poster at the conference.