Search

Brain arteriovenous malformation (bAVM) is the most common cause of intracranial hemorrhage (ICH), particularly in young patients. However, the exact cause of bAVM bleeding and rupture is not yet fully understood. In bAVMs, blood bypasses the entire capillary bed and directly flows from arteries to veins. The vessel walls in bAVMs have structural defects, which impair vascular integrity. Mural cells are essential structural and functional components of blood vessels and play a critical role in maintaining vascular integrity. Changes in mural cell number and coverage have been implicated in bAVMs. In this review, we discussed the roles of mural cells in bAVM pathogenesis. We focused on 1) the recent advances in human and animal studies of bAVMs; 2) the importance of mural cells in vascular integrity; 3) the regulatory signaling pathways that regulate mural cell function. More specifically, three signaling pathways that regulate mural cell-recruitment during vascular remodeling were discussed in detail, including the platelet-derived growth factor-B (PDGF-B)/PDGF receptor-β (PDGFR-β), EphrinB2/EphB4, and angiopoietins/tie2 signaling pathways.

Homozygous mutation in arteriovenous malformation (AVM) causative genes in a fraction of endothelial cells (ECs) causes brain AVMs (bAVMs). Heterozygous mutation of an AVM causative gene, endoglin, in bone marrow (BM) caused capillary dysplasia in mouse brain. We hypothesize that homozygous mutation of activin receptor-like kinase 1 (Alk1, another AVM causative gene) in BM derived ECs (BMDECs) is sufficient to cause bAVM in brain angiogenic region, Alk1(−) ECs can clonally expand in bAVM, and the burden of Alk1(−) ECs correlates with bAVM severity. The BMs of PdgfbiCreER;Alk1(2f/2f);Ai14 mice with EC-specific tamoxifeninducible Cre and Alk1 floxed alleles were transplanted into wild-type mice to analyze the role of BMDECs in bAVM development. PdgfbiCreER;Alk1(2f/2f);confetti(+/−) mice with Cre-regulated confetti transgene were used to study EC clonal expansion. BAVMs were induced by intra-brain injection of an adeno-associated viral vector expressing vascular endothelial growth factor followed with intra-peritoneal injection of tamoxifen. Wild-type mice transplanted with PdgfbiCreER;Alk1(2f/2f);Ai14 BM developed bAVMs after bAVM induction. Recombined BMDECs were detected in bAVMs. The presence of clusters of ECs expressing same confetti color in bAVMs suggests that Alk1(−) ECs have been clonally expanded. Increasing tamoxifen dose increased the number of Alk1(−) ECs and abnormal vessels in bAVMs of PdgfbiCreER;Alk1(2f/2f) mice. Our data indicated that homozygous mutation of Alk1 in BMDECs is sufficient to cause bAVM development in brain angiogenic region; clonal expansion of Alk1(−) ECs provides a likely mechanism for how a fraction of mutant ECs causes bAVM; and the burden of Alk1(−) ECs correlates with AVM severity.

Previous studies have shown that adoptive cell transfer (ACT) of regulatory T cells (Tregs) mitigate AAA growth in mice, however these studies have used a dissection AAA model and have not thoroughly studied Treg function in mice after ACT. Treg suppression of systemic or local inflammation has not been rigorously studied in our model. We therefore sought to evaluate the effects of Treg ACT in mice with a specific focus on Treg migration, colonization, and inflammatory changes within local tissue. Methods: AAA was induced in B57Bl/6 mice expressing the Thy 1.2 allele using a topical elastase + BAPN model (E-BAPN). Heat inactivated elastase (HiE-BAPN) was used as a control. On postoperative day (POD) 2, animals were treated with PBS (E-BAPN-PBS) injection or ACT of 2 million Tregs (E-BAPN-Tregs). CD4+ CD25+ FoxP3+ Tregs used for ACT were isolated from B57Bl/6 mice expressing the Thy 1.1 allele. Mice underwent surveillance ultrasounds (US) weekly to track AAA growth for 42 days. At specific time points mice were sacrificed and aortic diameter was recorded. Aortic tissue and regional lymph nodes were harvested for analysis. Results: Maximum aortic diameter (MAD) by both US and video microscopy was significantly higher in the AAA group (E-BAPN vs. HiE-BAPN; pThy1.1+ Tregs were detected in the tissue and regional aortic lymph nodes (LN) until day 42 with only a slight declination in the percent Thy1.1 + cells over time. Thy1.1+ Tregs retained phenotypic markers over time. E-BAPN-Treg mice had significantly altered inflammatory markers compared to E-BAPN-PBS mice including local macrophage and T-cell composition as well as inflammatory cytokines. Conclusion: Treg ACT suppress AAA growth in a topical elastase + BAPN mouse model through tissue and LN colonization and alteration of the local inflammatory milieu.

Introduction: Regulatory T cells (Tregs) suppress inflammation in atherosclerosis, and therefore have the therapeutic potential to reduce the risk of MI and stroke. However, there is currently no method to generate antigen specific Tregs that target atherosclerosis. We therefore engineered Tregs that express a Chimeric Antigen Receptor (CAR) targeting malonaldehyde-modified LDL (MDA-LDL), the most common form of oxidized LDL and a key molecular component of atherosclerosis. Methods: Novel single chain variable fragments (scFv) were synthesized using sequences from antibodies targeting human MDA-LDL. Oxidized-LDL specific CARs (ox-CARs) were subsequently engineered by fusing each scFv to an IgG4 hinge, CD28 transmembrane and CD28/CD3z cytoplasmic domains. CD4 + CD25 + CD127 low/- Tregs were purified from human blood via FACS and lentivirally transduced to express the novel ox-CARs (ox-CAR-Tregs). Human atherosclerotic plaques were obtained from patients undergoing carotid endarterectomy (CEA). Autologous ox-CAR-Tregs were analyzed for activation after ex-vivo co-culture with CEA samples. Results: A rationally designed panel of 42 ox-CARs were engineered using scFv derived from 12 antibodies targeting MDA-LDL. We first assessed CAR expression and activation in Jurkat T cells to identify promising ox-CAR variants for further evaluation in human Tregs. After culture in the presence of MDA-LDL, six ox-CAR-Treg variants consistently showed significant activation, compared to controls, based on CD71 expression, cytokine expression and proliferation in the absence of CD3/28 stimulation. Human atherosclerotic samples were identified to have substantial amounts of MDA-LDL epitopes using IHC. Autologous ox-CAR-Tregs showed a dose-dependent increase in CD71 expression after ex-vivo co-culture with atherosclerotic plaque. Conclusion: An optimized CAR targeting MDA-LDL activates Tregs when cultured with human atherosclerotic plaque ex-vivo.

Aim: To test if the impairment of mononuclear cell (MNC) migration in patients with hereditary hemorrhagic telangiectasia (HHT) is due to the reduction of the endoglin (ENG) receptor on the cell surface and oxidative stress. Method: MNCs of HHT patients and normal controls were subjected to migration assay. Fractions of MNCs were pre-incubated with antibodies specific to HHT causative genes ENG [hereditary hemorrhagic telangiectasia type 1 (HHT1)] or activin receptor-like kinase 1 [ALK1, hereditary hemorrhagic telangiectasia type 2 (HHT2)], AMD3100 or Diprotin-A to block ENG, ALK1 C-X-C chemokine receptor 4 (CXCR4) or CD26 (increased in HHT1 MNCs) before migration assay. The MNCs were allowed to migrate toward stromal cell-derived factor-1alpha (SDF-1alpha) for 18 h. The expression of CXCR4, CD26, superoxide dismutase 1 (SOD1) and glutathione peroxidase 1 (GPX1) in MNCs and nitric oxide levels in the plasma were analyzed. Results: Compared to the controls, fewer HHT1 MNCs and similar number of HHT2 MNCs migrated toward SDF-1. Diprotin-A pre-treatment improved HHT1 MNC-migration, but had no effect on normal and HHT2 MNCs. Pre-incubation with an anti-ENG antibody reduced the migration of normal MNCs. Diprotin-A did not improve the migration of ENG antibody pre-treated MNCs. Anti-ALK1 antibody had no effect on MNC-migration. AMD3100 treatment reduced normal and HHT MNC-migration. ENG mRNA level was reduced in HHT1 and HHT2 MNCs. ALK1 mRNA was reduced in HHT2 MNCs only. CD26 expression was higher in HHT1 MNCs. Pre-treatment of MNCs with anti-ENG or anti-ALK1 antibody had no effect on CD26 and CXCR4 expression. The expression of antioxidant enzymes, SOD1, was reduced in HHT1 MNCs, which was accompanied with an increase of ROS in HHT MNCs and nitric oxide in HHT1 plasma. Conclusions: Reduction of ENG receptor on MNC surface reduced monocyte migration toward SDF-1alpha independent of CD26 expression. Increased oxidative stress could alter HHT MNC migration behavior.

OBJECTIVEA high level of vascular endothelial growth factor (VEGF) has been implicated in brain arteriovenous malformation (bAVM) bleeding and rupture. However, direct evidence is missing. In this study the authors used a mouse bAVM model to test the hypothesis that elevation of focal VEGF levels in bAVMs exacerbates the severity of bAVM hemorrhage.METHODSBrain AVMs were induced in adult mice in which activin receptor–like kinase 1 (Alk1, a gene that causes AVM) gene exons 4–6 were floxed by intrabasal ganglia injection of an adenoviral vector expressing Cre recombinase to induce Alk1 mutation and an adeno-associated viral vector expressing human VEGF (AAV-VEGF) to induce angiogenesis. Two doses of AAV-VEGF (5 × 109 [high] or 2 × 109 [low]) viral genomes were used. In addition, the common carotid artery and external jugular vein were anastomosed in a group of mice treated with low-dose AAV-VEGF 6 weeks after the model induction to induce cerebral venous hypertension (VH), because VH increases the VEGF level in the brain. Brain samples were collected 8 weeks after the model induction. Hemorrhages in the bAVM lesions were quantified on brain sections stained with Prussian blue, which detects iron deposition. VEGF levels were quantified in bAVM tissue by enzyme-linked immunosorbent assay.RESULTSCompared to mice injected with a low dose of AAV-VEGF, the mice injected with a high dose had higher levels of VEGF (p = 0.003) and larger Prussian blue–positive areas in the bAVM lesion at 8 or 9 weeks after model induction (p = 0.002). VH increased bAVM hemorrhage in the low-dose AAV-VEGF group. The overall mortality in the high-dose AAV-VEGF group was 26.7%, whereas no mouse died in the low-dose AAV-VEGF group without VH. In contrast, VH caused a mortality of 50% in the low-dose AAV-VEGF group.CONCLUSIONSUsing mouse bAVM models, the authors provided direct evidence that elevation of the VEGF level increases bAVM hemorrhage and mouse mortality.

Background and Purpose: Homozygous mutation in arteriovenous malformation (AVM) causative genes in a fraction of endothelial cells (ECs) causes brain AVMs (bAVMs). Heterozygous mutation of endoglin, an AVM causative gene, in bone marrow (BM) caused capillary dysplasia in mouse brain. We tested if (1) homozygous mutation of activin receptor-like kinase 1 ( Alk1, another AVM causative gene) in BM derived ECs (BMDECs) can cause bAVM in brain angiogenic region, (2) Alk1 - ECs clonally expand in bAVM, and (3) the burden of Alk1 - ECs correlates with bAVM severity. Methods: The BMs of Pdgfb iCreER; Alk1 2 f/2f ;Ai14 mice with EC-specific tamoxifen-inducible Cre and Alk1 floxed alleles were transplanted into wild-type mice to analyze the role of BMDECs in bAVM development. Pdgfb iCreER; Alk1 2 f/2f ;confetti +/- mice with Cre-regulated confetti transgene were used to study EC clonal expansion. Brain AVMs were induced by intra-brain injection of an adeno-associated viral vector expressing vascular endothelial growth factor followed by intra-peritoneal injection of tamoxifen. Results: Wild-type mice transplanted with Pdgfb iCreER; Alk1 2 f/2f ;Ai14 BM developed bAVMs after bAVM induction. Recombined BMDECs were detected in bAVMs. The presence of clusters of ECs expressing same confetti color in bAVMs suggests that Alk1 - ECs had been clonally expanded. Increasing tamoxifen dose increased the number of Alk1 - ECs and abnormal vessels in bAVMs of Pdgfb iCreER; Alk1 2 f/2f mice. Conclusion: Homozygous mutation of Alk1 in BMDECs is sufficient to cause bAVM development in brain angiogenic region; clonal expansion of Alk1 - ECs provides a likely mechanism for how a fraction of mutant ECs causes bAVM; and the burden of Alk1 - ECs correlates with AVM severity.

Tibia fracture (BF) enhances stroke injury and post-stroke memory dysfunction in mouse. Reduction of neuroinflammation by activation of α-7 nicotinic acetylcholine receptor (α-7 nAchR) reduced acute neuronal injury and sensorimotor dysfunction in mice with BF 1-day after stroke. We hypothesize that reduction of neuroinflammation by activation of α-7 nAchR improves long-term memory function of mice with BF 6-h before stroke. The mice were randomly assigned to saline, PHA-568487 (α-7 nAchR agonist) and methyllycaconitine (antagonist) treatment groups. The sensorimotor function was tested by adhesive removal and corner tests at 3 days, the memory function was tested by Y-maze test weekly for 8 weeks and novel objective recognition test at 8 weeks post-injuries. We found PHA-568487 treatment reduced, methyllycaconitine increased the number of CD68+ cells in the peri-infarct and hippocampal regions, neuronal injury in the infarct region, sensorimotor and long-term memory dysfunctions. PHA-568487 treatment also reduced, while methyllycaconitine treatment increased atrophy of hippocampal granule cell layer and white matter damage in the striatum. In addition, PHA-568487 treatment increased neuron proliferation in granule cell layer. Our data indicated that reduction of neuroinflammation through activation of α-7 nAchR decreased neuronal damage, sensorimotor and long-term memory dysfunction of mice with BF shortly before stroke.

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.

Previous reports suggest that diabetes may differentially affect the vascular beds of females and males. However, there is insufficient evidence to establish the timeline of the vascular dysfunction in diabetes, specifically in relation to sex. Here, we determined whether mesenteric arterial function is altered in UC Davis Type-2 Diabetes Mellitus (UCD-T2DM) rats and if this occurs as early as the pre-diabetic stage of the disease. Specifically, we investigated whether vascular dysfunction differs between pre-diabetic or diabetic status and if this varies by sex. We measured the responses to endothelium-dependent and -independent vasorelaxant as well as vasoconstrictor agents and explored the potential mechanisms involved in sex-specific development of arterial dysfunction in UCD-T2DM rats. In addition, indices of insulin sensitivity were assessed. We report the reduced insulin sensitivity in pre-diabetic males and diabetic females. Vascular relaxation to acetylcholine was impaired to a greater extent in mesenteric artery from males in the pre-diabetic stage than in their female counterparts. In contrast, the arteries from females with diabetes exhibited a greater impairment to acetylcholine compared with diabetic males. Additionally, the sensitivity of mesenteric artery to contractile agents in females, but not in males, after the onset of diabetes was increased. Our data suggest that the reduced insulin sensitivity through AKT may predispose vessels to injury in the pre-diabetic stage in males. On the other hand, reduced insulin sensitivity as well as enhanced responsiveness to contractile agents may predispose arteries to injury in the diabetic stage in females.

RationaleMutation in human arteriovenous malformation (AVM) causative genes in a fraction of endothelial cells (ECs) causes AVMs in mice. It is unclear how a small number of mutant ECs can lead to AVM formation.ObjectiveTo understand how a fraction of mutant ECs causes AVM, we tested the following hypotheses: (1) activin receptor-like kinase 1 (Alk1 or Acvlr1) mutant brain ECs undergo clonal expansion upon angiogenic stimulation, (2) Alk1 mutant ECs display growth advantage, (3) the burden of Alk1 mutant ECs correlates with AVM severity, and (4) Alk1 mutant bone marrow (BM) derived ECs alone is sufficient to cause AVM.Methods and ResultsWe used PdgfbiCreER;Alk1f/f;confetti+/− mice which express an EC-specific tamoxifen (TM)-inducible Cre recombinase, a Cre-regulated confetti transgene, and Alk1 floxed alleles. Brain AVMs were induced by direct brain injection of an adeno-associated viral vector expressing vascular endothelial growth factor (AAV-VEGF) followed with intra-peritoneal injection of TM two weeks later. Color-predominance of confetti reporter in AVMs compared to control brain ECs suggested that clonal expansion was associated with AVM development. We treated PdgfbiCreER;Alk1f/f with different doses of TM to create a mosaic of wild-type (WT) and mutant ECs and found that equal numbers of Alk1+ and Alk1− ECs were proliferating. Increase of TM dose increased the number of Alk1− ECs, the abnormal vessels in brain AVMs, the number of arteriovenous shunts in the intestines, and mouse mortality. To test if mutation of Alk1 in BM-derived ECs can cause brain AVM, we transplanted WT mice with BM of PdgfbiCreER;Alk1f/f mice. After AAV-VEGF and TM treatment, these mice developed AVMs in their brains and arteriovenous shunts in their intestines.ConclusionClonal expansion of Alk1 mutant ECs could partly explain why a fraction of mutant ECs causes AVM. Mutation of AVM causal genes in BM-derived ECs is sufficient to cause AVM formation.

Introduction: Tibia fracture (BF) enhances stroke injury and when occurring 6 hrs before stroke (BF6+Stroke) causes long-lasting cognitive dysfunction in mouse. Activation of α-7 nicotinic acetylcholine receptor (α-7 nAchR) reduced neuroinflammation, neuronal injury and sensorimotor dysfunction in mice with BF one day after stroke (Stroke+1BF). Hypothesis: Activation of α-7 nAchR improves long-term cognitive function of BF6+Stroke mice. Methods: BF6+Stroke mice were randomly assigned to saline, PHA-568487 (α-7 nAchR agonist) and MLA (α-7 nAchR antagonist) treatment groups. The sensorimotor function were tested by adhesive removal and corner tests at 3 days, the cognitive function was tested by Y-maze weekly for 8 weeks and Novel Objective Recognition (NOR) at 8 weeks post-injuries. The neuronal damage, neuroinflammaiton, neurogenesis were analyzed 3 and/or 8 weeks post-injuries. Results: Similar to Stroke+1BF mice, PHA reduced and MLA enhanced neuronal injury, neuroinflammation, and sensorimotor dysfunction of BF6+Stroke mice. Further, PHA reduced and MLA enhanced their long-term cognitive dysfunction. In Y maze test, all mice made fewer alternations 1-week post-surgeries than baseline; PHA group recovered to baseline at week 5 post-surgeries; saline and MLA groups continuously made fewer alternations throughout the 8-weeks. In NOR test, PHA group spent more time, MLA group spent less time than saline group on novel objects. Injection of BrdU in the 2 nd week post-surgeries labeled more neurons in the contralateral than in the ipsilateral dentate gyrus in all groups; PHA group had the most, MLA group had the least BrdU + neurons. Injection BrdU in the 7th week post-surgeries did not labeled any neuron. Conclusion: Activation of α-7 nAchR decreased neuronal damage and neuroinflammation, increased neurogenesis at the dentate gyrus of BF6+Stroke mice; and improved their sensorimotor and long-term cognitive function.

Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. The pathogenesis of bAVM has not been fully understood. Animal models are important tools for dissecting bAVM pathogenesis and testing new therapies. We have developed several mouse bAVM models using genetically modified mice. However, due to the body size, mouse bAVM models have some limitations. Recent studies identified somatic mutations in sporadic human bAVM. To develop a feasible tool to create sporadic bAVM in rodent and animals larger than rodent, we made tests using the CRISPR/Cas9 technique to induce somatic gene mutations in mouse brain in situ. Two sequence-specific guide RNAs (sgRNAs) targeting mouse Alk1 exons 4 and 5 were cloned into pAd-Alk1e4sgRNA + e5sgRNA-Cas9 plasmid. These sgRNAs were capable to generate mutations in Alk1 gene in mouse cell lines. After packaged into adenovirus, Ad-Alk1e4sgRNA + e5sgRNA-Cas9 was co-injected with an adeno-associated viral vector expressing vascular endothelial growth factor (AAV-VEGF) into the brains of wild-type C57BL/6J mice. Eight weeks after viral injection, bAVMs were detected in 10 of 12 mice. Compared to the control (Ad-GFP/AAV-VEGF-injected) brain, 13% of Alk1 alleles were mutated and Alk1 expression was reduced by 26% in the Ad-Alk1e4sgRNA + e5sgRNA-Cas9/AAV-VEGF-injected brains. Around the Ad-Alk1e4sgRNA + e5sgRNA-Cas9/AAV-VEGF injected site, Alk1-null endothelial cells were detected. Our data demonstrated that CRISPR/Cas9 is a feasible tool for generating bAVM model in animals.

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.

Patients with brain arteriovenous malformation (bAVM) are at risk of intracranial hemorrhage (ICH). Overall, bAVM accounts for 25% of hemorrhagic strokes in adults

Background and purpose: High level of VEGF has been implicated in bAVM bleeding and rupture through analyzing the VEGF levels in patients’ blood and in surgically resected bAVM specimens. However, the direct evidence is missing. Using a mouse bAVM model, we tested the hypothesis that elevation of focal VEGF level exacerbates the severity of bAVM hemorrhage. Methods: Brain AVMs were induced in adult mice that have Alk1 (an AVM causal gene) exons 4-6 floxed by injection of an adenoviral vector expressing Cre-recombinase (Ad-Cre) and an adeno-associated viral vector expressing human VEGF (AAV-VEGF) into the basal ganglia. Two doses of AAV-VEGF, 5x10 9 (high) or 2x10 9 (low) viral genomes were used. In addition, the common carotid artery and jugular vein were anastomosis in a group of mice treated with low dose AAV-VEGF 6 weeks after the model induction to induce venous hypertension (VH), because VH increases VEGF level in the brain. Hemorrhages in the bAVM lesions were quantified on brain sections stained with Prussian blue, which detect iron deposition 8 or 9 weeks after model induction. VEGF levels were quantified by ELISA. Results: Compared to mice injected with low dose AAV-VEGF, mice injected with high dose of AAV-VEGF had a higher level of VEGF, larger Prussian blue positive areas in the bAVM lesion at 8 and 9-weeks after model induction (P Conclusion: Using mouse bAVM models, we demonstrated that the evaluation of VEGF level through viral vector mediated overexpression of VEGF or create VH increases bAVM hemorrhage and mouse mortality.

We have recently shown that type of supplemented simple sugar, not merely calorie intake, determines adverse effects on metabolism and aortic endothelial function in female rats. The aim of the current study was to investigate and compare the effects of high consumption of glucose or fructose on mesenteric arterial reactivity and systolic blood pressure (SBP). Sprague-Dawley female rats were supplemented with 20% w/v glucose or fructose in drinking water for 8 weeks. Here, we show that both sugars alter insulin signaling in mesenteric arteries (MA), assessed by a reduction in phosphorylated Akt, and increase in SBP. Furthermore, ingestion of glucose or fructose enhances inducible nitric oxide synthase (iNOS) expression and contractile responses to endothelin and phenylephrine in MA of rats. The endothelium-dependent vasodilation to acetylcholine and bradykinin as well as the relaxation responses to the nitric oxide donor sodium nitroprusside are impaired in MA of fructose- but not glucose-supplemented rats. In contrast, only glucose supplementation increases the expression of phosphorylated endothelial NOS (eNOS) in MA of rats. In conclusion, this study reveals that supplementation with fructose or glucose in liquid form enhances vasocontractile responses and increases iNOS expression in MA, effects which are accompanied by increased SBP in those groups. On the other hand, the preserved vasodilatory responses in MA from glucose-supplemented rats could be attributed to the enhanced level of phosphorylated eNOS expression in this group. Keywords: Rat mesenteric arteries; Fructose; Glucose; Endothelial dysfunction; Blood pressure

UC Davis type-2 diabetes mellitus (UCD-T2DM) model is a novel and validated model of type-2 diabetes exhibiting etiology very similar to clinical T2DM seen in humans. This model is characterized by...

Hyperglycemia affects male and female vascular beds differently. We have previously shown that 1 week after the induction of diabetes with streptozotocin (STZ), male and female rats exhibit differences in aortic endothelial function. To examine this phenomenon further, aortic responses were studied in male and female rats 8 weeks after the induction of diabetes (intermediate stage). Endothelium-dependent vasodilation (EDV) to acetylcholine (ACh) was measured in phenylephrine (PE) pre-contracted rat aortic rings. Concentration response curves to PE were generated before and after L-NAME, a nitric oxide synthase (NOS) inhibitor. Furthermore, mRNA expression of endothelial nitric oxide synthase (eNOS) and NADPH oxidase subunit (Nox1) were determined. At 8 weeks, diabetes impaired EDV to a greater extent in female than male aortae. Furthermore, the responsiveness to PE was significantly enhanced only in female diabetic rats, and basal NO, as indicated by the potentiation of the response to PE after L-NAME, was reduced in female diabetic rat aortae to the same levels as in males. In addition, eNOS mRNA expression was decreased, while the Nox1 expression was significantly enhanced in diabetic female rats. These results suggest that aortic function in female diabetic rats after 8 weeks exhibits a more prominent impairment and that NO may be involved.

Intake of high fructose, especially in soft drinks, has shown to contribute to variety of metabolic disorders such as diabetes and obesity. The objective of this study was to compare the effects of...

Little is known about interaction of sex and diabetes in vasculature. Our study investigates the effects of type 2 diabetes on endothelium-dependent and -independent relaxations. Also, if there are sex-based changes in relative contributions of endothelium derived relaxing factors (EDRFs) in modulating vascular reactivity of mesenteric arteries (MA) from ZDF rats. Relaxation responses to acetylcholine (ACh) in MA pre-contracted with phenylephrine (PE) were obtained before and after pretreatment with indomethacin (cyclooxygenase inhibitor), L-NAME (nitric oxide synthase inhibitor) or barium chloride (Kir blocker) plus ouabain (Na+-K+-ATPase inhibitor). Vascular responses to sodium nitroprusside (SNP) were also measured in MA. ACh-induced relaxations were significantly impaired in MA of diabetic rats, regardless of sex. In diabetic females, the relative importance of endothelium derived hyperpolarizing factor (EDHF) in relaxation to ACh was reduced, while in diabetic males, role of nitric oxide (NO) was red...

Little is known about the interaction between diabetes and sex in vasculature. This study was designed to investigate whether there were sex differences in rat aortic endothelium-dependent vasodilation (EDV) in Zucker diabetic fatty (ZDF) rats, and the potential role of superoxide. EDV to acetylcholine (ACh) was measured in aortic rings pre-contracted with phenylephrine before and after pretreatment with apocynin (100 μM), a NADPH oxidase (Nox) inhibitor. In addition, the level of Nox (a potent source of superoxide) and PKCβ mRNA expression were determined using real-time RT-PCR. ACh-induced relaxations were significantly greater in female lean rats compared with male lean rats. Accordingly, male lean rats had higher PKCβI expression level than female lean rats. Diabetes significantly impaired EDV in aortic rings from female ZDF rats, however, potentiated the relaxation in males. Pre-incubation of aortic rings with apocynin increased EDV only in diabetic female group, suggesting impairment of EDV in femal...

High consumption of simple sugars causes adverse cardiometabolic effects. We investigated the mechanisms underlying the metabolic and vascular effects of glucose or fructose intake and determined whether these effects are exclusively related to increased calorie consumption. Female Sprague-Dawley rats were supplemented with 20% wt/vol glucose or fructose for 2 mo, and plasma analytes and aortic response to vasodilator and vasoconstrictor agents were determined. Expression of molecules associated with lipid metabolism, insulin signaling, and vascular response were evaluated in hepatic and/or aortic tissues. Caloric intake was increased in both sugar-supplemented groups vs. control and in glucose- vs. fructose-supplemented rats. Hepatic lipogenesis was induced in both groups. Plasma triglycerides were increased only in the fructose group, together with decreased expression of carnitine palmitoyltransferase-1A and increased microsomal triglyceride transfer protein expression in the liver. Plasma adiponectin and peroxisome proliferator-activated receptor (PPAR)-α expression was increased only by glucose supplementation. Insulin signaling in liver and aorta was impaired in both sugar-supplemented groups, but the effect was more pronounced in the fructose group. Fructose supplementation attenuated aortic relaxation response to a nitric oxide (NO) donor, whereas glucose potentiated it. Phenylephrine-induced maximal contractions were reduced in the glucose group, which could be related to increased endothelial NO synthase (eNOS) phosphorylation and subsequent elevated basal NO in the glucose group. In conclusion, despite higher caloric intake in glucose-supplemented rats, fructose caused worse metabolic and vascular responses. This may be because of the elevated adiponectin level and the subsequent enhancement of PPARα and eNOS phosphorylation in glucose-supplemented rats. NEW & NOTEWORTHY This is the first study comparing the effects of glucose and fructose consumption on metabolic factors and aortic function in female rats. Our results show that, although total caloric consumption was higher in glucose-supplemented rats, fructose ingestion had a greater impact in inducing metabolic and aortic dysfunction.