Nitric oxide synthase and reduced arterial tone contribute to arteriovenous malformation.

Mechanisms underlying arteriovenous malformations (AVMs) are poorly understood. Using mice with endothelial cell (EC) expression of constitutively active Notch4 (Notch4*EC), we show decreased arteriolar tone in vivo during brain AVM initiation. Reduced vascular tone is a primary effect of Notch4*EC, as isolated pial arteries from asymptomatic mice exhibited reduced pressure-induced arterial tone ex vivo. The nitric oxide (NO) synthase (NOS) inhibitor NG-nitro-l-arginine (L-NNA) corrected vascular tone defects in both assays. L-NNA treatment or endothelial NOS (eNOS) gene deletion, either globally or specifically in ECs, attenuated AVM initiation, assessed by decreased AVM diameter and delayed time to moribund. Administering nitroxide antioxidant 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl also attenuated AVM initiation. Increased NOS-dependent production of hydrogen peroxide, but not NO, superoxide, or peroxynitrite was detected in isolated Notch4*EC brain vessels during AVM initiation. Our data suggest that eNOS is involved in Notch4*EC-mediated AVM formation by up-regulating hydrogen peroxide and reducing vascular tone, thereby permitting AVM initiation and progression.

Endothelial Rbpj deletion normalizes Notch4-induced brain arteriovenous malformation in mice.

Upregulation of Notch signaling is associated with brain arteriovenous malformation (bAVM), a disease that lacks pharmacological treatments. Tetracycline (tet)-regulatable endothelial expression of constitutively active Notch4 (Notch4*tetEC) from birth induced bAVMs in 100% of mice by P16. To test whether targeting downstream signaling, while sustaining the causal Notch4*tetEC expression, induces AVM normalization, we deleted Rbpj, a mediator of Notch signaling, in endothelium from P16, by combining tet-repressible Notch4*tetEC with tamoxifen-inducible Rbpj deletion. Established pathologies, including AV connection diameter, AV shunting, vessel tortuosity, intracerebral hemorrhage, tissue hypoxia, life expectancy, and arterial marker expression were improved, compared with Notch4*tetEC mice without Rbpj deletion. Similarly, Rbpj deletion from P21 induced advanced bAVM regression. After complete AVM normalization induced by repression of Notch4*tetEC, virtually no bAVM relapsed, despite Notch4*tetEC re-expression in adults. Thus, inhibition of endothelial Rbpj halted Notch4*tetEC bAVM progression, normalized bAVM abnormalities, and restored microcirculation, providing proof of concept for targeting a downstream mediator to treat AVM pathologies despite a sustained causal molecular lesion.

Monitoring of cell-cell communication and contact history in mammals.

Monitoring of cell-cell communication in multicellular organisms is fundamental to understanding diverse biological processes such as embryogenesis and tumorigenesis. To track cell-cell contacts in vivo, we developed an intercellular genetic technology to monitor cell-cell contact and to trace cell contact histories by permanently marking contacts between cells. In mice, we engineered an artificial Notch ligand into one cell (the sender cell) and an artificial receptor into another cell (the receiver cell). Contact between the sender and receiver cells triggered a synthetic Notch signaling that activated downstream transcriptional programs in the receiver cell, thereby transiently or permanently labeling it. In vivo cell-cell contact was observed during development, tissue homeostasis, and tumor growth. This technology may be useful for studying dynamic in vivo cell-cell contacts and cell fate plasticity.

[Combination with different anticholinergic eyedrops for the treatment of children myopia].

Objective: To observe the efficacy and feasibility of a new therapy using a combination of different anticholinergic eyedrops in controlling myopia progression and axial prolongation in adolescents. Methods: Between July 2013 and June 2014, a total of 150 myopia adolescents aged 6-12 years were recruited at the clinic of Tongji Hospital in Shanghai. Participants were assigned in a 1∶2∶2 ratio to placebo group (no medication), combined treatment group (0.5% racanisodamine eyedrops were used twice a day during semesters, 1% atropine eyedrops were used before sleep during vacation) and atropine group (1% atropine eyedrops were used before sleep everyday). All subjects wore glasses. Visual acuity, best corrected visual acuity, cycloplegic refraction, corneal curvature, axial length, intraocular pressure, fundus and adverse events were recorded every 6 months during follow-up for 24 months. Results: At baseline, there was no significant difference in age,equivalent spherical mirror number and axial length among the three groups (all P>0.05). At the end of the second year,the mean myopia progression (changes of spherical equivalent) was -2.34 (-2.93,-1.75) D,-0.63 (-1.00,-0.50) D and -0.25 (-0.50,-0.06) D in placebo group, combined treatment group and atropine group, respectively (P<0.001), and there was statistically significant difference between each two groups (all P<0.001). The axial length change of each group were (1.51±0.23) mm, (0.69±0.30) mm and (0.31±0.30) mm, respectively (P<0.001), and there was statistically significant difference between each two groups (all P<0.001). Conclusion: Therapy using a combination of different anticholinergic eyedrops can effectively control the progression of myopia and axial prolongation in adolescents, and increase the compliance of children and the safety of drug use.

Mechanical Stretch Increases Expression of CXCL1 in Liver Sinusoidal Endothelial Cells to Recruit Neutrophils, Generate Sinusoidal Microthombi, and Promote Portal Hypertension.

BACKGROUND & AIMS: Mechanical forces contribute to portal hypertension (PHTN) and fibrogenesis. We investigated the mechanisms by which forces are transduced by liver sinusoidal endothelial cells (LSECs) into pressure and matrix changes. METHODS: We isolated primary LSECs from mice and induced mechanical stretch with a Flexcell device, to recapitulate the pulsatile forces induced by congestion, and performed microarray and RNA-sequencing analyses to identify gene expression patterns associated with stretch. We also performed studies with C57BL/6 mice (controls), mice with deletion of neutrophil elastase (NE-/-) or peptidyl arginine deiminase type IV (Pad4-/-) (enzymes that formation of neutrophil extracellular traps [NETs]), and mice with LSEC-specific deletion of Notch1 (Notch1iΔEC). We performed partial ligation of the suprahepatic inferior vena cava (pIVCL) to simulate congestive hepatopathy-induced portal hypertension in mice; some mice were given subcutaneous injections of sivelestat or underwent bile-duct ligation. Portal pressure was measured using a digital blood pressure analyzer and we performed intravital imaging of livers of mice. RESULTS: Expression of the neutrophil chemoattractant CXCL1 was up-regulated in primary LSECs exposed to mechanical stretch, compared with unexposed cells. Intravital imaging of livers in control mice revealed sinusoidal complexes of neutrophils and platelets and formation of NETs after pIVCL. NE-/- and Pad4-/- mice had lower portal pressure and livers had less fibrin compared with control mice after pIVCL and bile-duct ligation; neutrophil recruitment into sinusoidal lumen of liver might increase portal pressure by promoting sinusoid microthrombi. RNA-sequencing of LSECs identified proteins in mechanosensitive signaling pathways that are altered in response to mechanical stretch, including integrins, Notch1, and calcium signaling pathways. Mechanical stretch of LSECs increased expression of CXCL1 via integrin-dependent activation of transcription factors regulated by Notch and its interaction with the mechanosensitive piezo calcium channel. CONCLUSIONS: In studies of LSECs and knockout mice, we identified mechanosensitive angiocrine signals released by LSECs which promote PHTN by recruiting sinusoidal neutrophils and promoting formation of NETs and microthrombi. Strategies to target these pathways might be developed for treatment of PHTN. RNA-sequencing accession number: GSE119547.

Effectiveness and safety of drug-eluting stent implantation following rotational atherectomy for severe coronary arteries calcification in elderly patients / 中华老年医学杂志

Objective To investigate the effectiveness and safety of drug-eluting stent implantation following rotational atherectomy (RA)for severe coronary arteries calcification in elderly patients. Methods A total of 21 patients receiving RA and drug-eluting stent implantation were enrolled in this study in our cardiology department from Sep.2014 to Sep.2017. Twenty-one patients with 27 severe calcified lesions were treated with the stent implantation following RA . The primary endpoints of the study were the immediate operation success rate and the rate of major adverse cardiac and cerebral events (MACCE)at 6 month after surgery ,including angina recurrence ,need for target vessel revascularization ,myocardial infarction ,stent thrombosis and cardiovascular death. Results 14 patients(66.7% ,14/21)received RA by using 1.5 mm burr ,and 7 patients(33.3% ,7/21)by using both 1.25 mm and 1.5 mm burrs. The average ratio of burr to artery diameter was (0.5 ± 0.1). A total of 29 stents were successfully implanted in all patients (100% ,21/21 patients).None of the patients experienced any acute coronary artery rupture or other severe complications during percutaneous coronary intervention (PCI ) after RA. Two cases (2/21 ,9.5% ) suffered from slow flow ,and the coronary blood flow was restored to TIMI grade Ⅲ after treatment. The coronary blood flow in the other 19 cases(19/21 ,90.4% )was TIMI grade Ⅲ after RA.Intravascular ultrasonography (IVUS) showed that the stents were well adhered without stent rupture and intimal tear in 12 cases(12/21 , 57.1% ) ,and postdilation was performed in 9 cases(9/21 ,42.9% ).All patients were followed up for at least 6 months ,and target vessel revascularization and death were not found. Conclusions A drug-eluting stent implantation following rotational atherectomy is effective and safe for treating severe coronary arteries calcification in elderly coronary heart disease patients. The IVUS-guided rotational atherectomy combined with drug-eluting stent implantation can reduce the risk of MACCE ,such as under-expansion stent ,stent thrombosis ,myocardial infarction ,cardiovascular death ,and improve clinical outcomes in elderly patients with severe coronary arteries calcification.

Evaluation of autologous bone marrow mononuclear cells treatment for heart failure due to non-ischemic cardiomyopathy using PET myocardial perfusion/metabolic imaging / 中华核医学与分子影像杂志

Objective To evaluate the clinical improvements after autologous bone marrow mononuclear cells (BMMNCs) percutaneously injected into coronary artery in patients with heart failure due to non-ischemic cardiomyopathy using PET myocardial peffusion/metabolic imaging.Methods From February 2011 to October 2012,40 patients with heart failure due to non-ischemic cardiomyopathy were selected.The test group including 15 patients (13 males,2 females,average age (57.5±14.5) years) received the autologous BMMNCs intracoronary injection on the basis of drug treatment.The other 25 cases (21 males,4 females,average age (58.0±12.0) years) were taken as the control group and only received the drug treatment.All patients were followed up for 24 months,and the myocardial perfusion/metabolism imaging,echocardiography,brain natriuretic peptide (BNP) test,6-minute walking experiment were performed.The data were analyzed by two-sample t test.Results During the follow-up period,the test group had no ventricular arrhythmia and other serious complications,and the patients' symptoms had been improved.There was no change in myocardial perfusion after treatment of autologous BMMNCs,but the myocardial metabolic defect by volume reduced from (43.79± 17.99) cm3 to (28.19±9.27) cm3 (t =3.33,P＜0.01) 24 months after the treatment.The myocardial metabolic defect by volume at the baseline and after 24 months in the control group was (43.30±15.70) cm3,(48.51±15.77) cm3 respectively (t=1.01,P＞0.05).In the test group,the left ventricular end-diastolic diameter decreased from (64.0±8.0) mm to (59.0±7.0) mm 24 months after the treatment (t=2.04,P＜0.05),and the left ventricular ejection fraction was significantly higher than that before treatment:(45.0±4.0) ％ vs (27.0±6.0) ％ (t =10.81,P＜0.01).Conclusion PET myocardial perfusion/metabolic imaging can be used as tools in evaluating the therapeutic effect of autologous BMMNCs in patients with heart failure due to non-ischemic cardiomyopathy.

Abnormal arterial-venous fusions and fate specification in mouse embryos lacking blood flow.

The functions of blood flow in the morphogenesis of mammalian arteries and veins are not well understood. We examined the development of the dorsal aorta (DA) and the cardinal vein (CV) in Ncx1 -/- mutants, which lack blood flow due to a deficiency in a sodium calcium ion exchanger expressed specifically in the heart. The mutant DA and CV were abnormally connected. The endothelium of the Ncx1 -/- mutant DA lacked normal expression of the arterial markers ephrin-B2 and Connexin-40. Notch1 activation, known to promote arterial specification, was decreased in mutant DA endothelial cells (ECs), which ectopically expressed the venous marker Coup-TFII. These findings suggest that flow has essential functions in the DA by promoting arterial and suppressing venous marker expression. In contrast, flow plays a lesser role in the CV, because expression of arterial-venous markers in CV ECs was not as dramatically affected in Ncx1 -/- mutants. We propose a molecular mechanism by which blood flow mediates DA and CV morphogenesis, by regulating arterial-venous specification of DA ECs to ensure proper separation of the developing DA and CV.

Effect of Cordyceps sinensis on the Treatment of Experimental Autoimmune Encephalomyelitis: A Pilot Study on Mice Model.

BACKGROUND: As a traditional Chinese medicine, Cordyceps sinensis (CS) possesses a variety of immunoregulatory properties. This study aimed to explore the therapeutic potential of CS in a mice model of multiple sclerosis (MS)-experimental autoimmune encephalomyelitis (EAE). METHODS: Female C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein35-55to induce EAE, followed by an instant intragastric feeding with a low dosage of CS (low-CS group, n = 5), high dosage of CS (high-CS group, n = 5), or the same volume of normal saline (control group, n = 5). All the mice were observed for clinical assessment. Over the 30 days of CS treatment, flow cytometry was used to detect the frequency of helper T-cell (Th) subsets, Th1 and Th17, and CD4+ CD25+ regulatory T cells in the spleen and lymph nodes. Meanwhile, pathological changes in brain were determined using both hematoxylin-eosin and luxol fast blue staining. Data were analyzed using the one-way analysis of variance (ANOVA). RESULTS: Over the 15 and 30 days of CS treatment, the clinical assessment for EAE demonstrated that both high-CS group (2.51 ± 0.31 and 2.26 ± 0.39 scores, respectively) and low-CS group (2.99 ± 0.40 and 2.69 ± 0.46, respectively) had lower disease severity scores than those of control group (3.57 ± 0.53 and 3.29 ± 0.53, all P < 0.01, respectively). Meanwhile, after 15 and 30 days, the high-CS group (19.18 ± 1.34 g and 20.41 ± 1.56 g, respectively) and low-CS group (18.07 ± 1.18 g and 19.48 ± 1.69 g, respectively) had a lower body weight, as compared with control group (16.85 ± 1.15 g and 18.22 ± 1.63 g, all P < 0.01, respectively). At 30 days post-CS treatment, there was a lower Th1 frequency in the lymph nodes (2.85 ± 1.54% and 2.77 ± 1.07% vs. 5.35 ± 1.34%, respectively; P < 0.05) and spleens (3.96 ± 1.09% and 3.09 ± 0.84% vs. 5.07 ± 1.50%, respectively; P < 0.05) and less inflammatory infiltration and demyelination in the brain of CS-treated mice than that of control group. CONCLUSIONS: Our preliminary study demonstrated that CS efficiently alleviated EAE severity and EAE-related pathology damage and decreased the number of Th1s in the periphery, indicating its effectiveness in the treatment of murine EAE. Thus, our findings strongly support the therapeutic potential of this agent as a new traditional Chinese medicine approach in MS treatment.

Establishment of an individualized three-dimensional finite element model of type A coronary artery lesions / 中国组织工程研究

BACKGROUND:A cardiac model can be established by finite element analysis based on patient's MRI imaging data.The established model can be used to evaluate the rheological changes of the coronary artery by liquid-solid coupling.OBJECTIVE:To establish finite element models of the heart and coronary artery in patients with type A coronary artery disease using finite element analysis software,followed by three-dimensional (3D) printing,thereby providing a scientific basis for further simulation of interventional surgery.METHODS:Three patients with type A coronary artery lesions underwent MRI scanning from the aortic arch to the apex.The MRI images were then imported into the Mimics 17.0 software in Dicom format,and a complete cardiac model involving the coronary arteries was established by modeling and geometry cleanup.The 3D model was imported into Geomagic Studio 11.0 software,and was further processed.Finally,the 3D model was imported into ANSYS14.0 finite element analysis software.The finite element model with biofunction was established by attaching the material properties,followed by 3D printing on a 3D printer.RESULTS AND CONCLUSION:The 3D finite element model of type A coronary artery lesion was established successfully in three cases.The established heart model in each case presented with grid-based hexahedral solid elements.The number of solid elements was 24 532,25 771,and 24 330,respectively.In the meanwhile,the model of each coronary branch was established:the number of element at the right coronary artery was 3 320,3 518,and 3 310;the number of elements at the circumflex branch was 1 148,1 176,and 1 164;and the number of elements at the anterior descending coronary artery was 1 025,1 049,and 1 068,respectively.Afterwards,the 3D printing was performed successfully.These results suggest that the established 3D finite element model of the heart with coronary arteries,after 3D printing,displays the right coronary artery,anterior descending artery,circumflex artery and coronary sinus clearly,which paves ways for interventional simulation.Most importantly,it lays a solid foundation for the study on the blood-vessel dual-directional coupling,which is expected to be a new scientific method for rheological research.

The effectiveness and safety of paclitaxel-coated balloons in elderly patients with coronary in-stent restenosis / 中华老年医学杂志

Objective To assess the effectiveness and safety of paclitaxel-coated balloons for in-stent restenosis in patients aged 65 years and over.Methods Sixty elderly patients(≥65 years old)with in-stent restenosis were enrolled at the Department of Cardiology,the First Affiliated Hospital of Inner Mongolian Medical University.Based on different treatment methods for in-stent restenosis,patients were divided into the drug-eluting balloon(DEB,n=32)group and the drug-eluting stent(DES,n=28)group.The primary end point was late luminal loss,determined by angiography.Secondary end points included rates of restenosis and major adverse cardiac events (MACEs).Results Quantitative coronary angiography revealed no significant differences in baseline data At 3 months after treatment,the rate of MACEs was 28.6％ in the DES group and 12.5％ in the DEB group(P＜0.05).At 6 months after treatment,angiography showed that the (x)±s of insegment late luminal loss was(0.21±0.04)mm in the DES group versus(0.12±0.06)mm in the DEB group(P ＜0.05).Furthermore,7 of 28 patients (25 ％) in the DES group had restenosis,compared with 4 of 32 patients (12.5 ％)in the DEB group(P =0.03).Conclusions Paclitaxel-coated balloons for coronary in-stent restenosis in patients aged 65 years or over can significantly reduce the incidence of restenosis and lower the rate of MACEs.The procedure is safe with no serious complications,eliminates the need for additional stent implantation,and should be further assessed in future clinical trials.

The effectiveness and safety of paclitaxel-coated balloons in elderly patients with coronary in-stent restenosis / 中华老年医学杂志

Objective To assess the effectiveness and safety of paclitaxel-coated balloons for in-stent restenosis in patients aged 65 years and over.Methods Sixty elderly patients(≥65 years old)with in-stent restenosis were enrolled at the Department of Cardiology,the First Affiliated Hospital of Inner Mongolian Medical University.Based on different treatment methods for in-stent restenosis,patients were divided into the drug-eluting balloon(DEB,n=32)group and the drug-eluting stent(DES,n=28)group.The primary end point was late luminal loss,determined by angiography.Secondary end points included rates of restenosis and major adverse cardiac events (MACEs).Results Quantitative coronary angiography revealed no significant differences in baseline data At 3 months after treatment,the rate of MACEs was 28.6％ in the DES group and 12.5％ in the DEB group(P＜0.05).At 6 months after treatment,angiography showed that the (x)±s of insegment late luminal loss was(0.21±0.04)mm in the DES group versus(0.12±0.06)mm in the DEB group(P ＜0.05).Furthermore,7 of 28 patients (25 ％) in the DES group had restenosis,compared with 4 of 32 patients (12.5 ％)in the DEB group(P =0.03).Conclusions Paclitaxel-coated balloons for coronary in-stent restenosis in patients aged 65 years or over can significantly reduce the incidence of restenosis and lower the rate of MACEs.The procedure is safe with no serious complications,eliminates the need for additional stent implantation,and should be further assessed in future clinical trials.

Endothelial notch signaling is essential to prevent hepatic vascular malformations in mice.

UNLABELLED: Liver vasculature is crucial for adequate hepatic functions. Global deletion of Notch signaling in mice results in liver vascular pathologies. However, whether Notch in endothelium is essential for hepatic vascular structure and function remains unknown. To uncover the function of endothelial Notch in the liver, we deleted Rbpj, a transcription factor mediating all canonical Notch signaling, or Notch1 from the endothelium of postnatal mice. We investigated the hepatic vascular defects in these mutants. The liver was severely affected within 2 weeks of endothelial deletion of Rbpj from birth. Two-week old mutant mice had enlarged vessels on the liver surface, abnormal vascular architecture, and dilated sinusoids. Vascular casting and fluorosphere passage experiments indicated the presence of porto-systemic shunts. These mutant mice presented with severely necrotic liver parenchyma and significantly larger hypoxic areas, likely resulting from vascular shunts. We also found elevated levels of VEGF receptor 3 together with reduced levels of ephrin-B2, suggesting a possible contribution of these factors to the generation of hepatic vascular abnormalities. Deletion of Rbpj from the adult endothelium also led to dilated sinusoids, vascular shunts, and necrosis, albeit milder than that observed in mice with deletion from birth. Similar to deletion of Rbpj, loss of endothelial Notch1 from birth led to similar hepatic vascular malformations within 2 weeks. CONCLUSIONS: Endothelial Notch signaling is essential for the development and maintenance of proper hepatic vascular architecture and function. These findings may elucidate the molecular pathogenesis of hepatic vascular malformation and the safety of therapeutics inhibiting Notch. (Hepatology 2016;64:1302-1316).

ASP2408 and ASP2409, novel CTLA4-Ig variants with CD86-selective ligand binding activity and improved immunosuppressive potency, created by directed evolution.

The CTLA4-Ig therapeutics abatacept and belatacept inhibit CD28-mediated T cell activation by binding CD80 (B7-1) and CD86 (B7-2) co-stimulatory ligands. Both compounds preferentially bind CD80, yet CD86 has been implicated as the dominant co-stimulatory ligand. Using directed evolution methods, novel CTLA4-Ig variants were created with selective CD86 binding affinity, a property that confers increased immunosuppressive potency and potentially improved efficacy and safety profiles. Relative to abatacept (wild-type CTLA4-Ig), ASP2408 and ASP2409 have 83-fold and 220-fold enhanced binding affinity to CD86 while retaining 1.5-fold and 5.6-fold enhanced binding affinity to CD80, respectively. Improvements in CD86 binding affinity correlates with increased immunosuppressive potencyin vitroandin vivo Our results highlight the power of directed evolution methods to obtain non-intuitive protein engineering solutions and represent the first examples of CD86-selective CTLA4-Ig compounds that have entered clinical trials.

Effect of GBOT on blood lipid and blood glucose metabolism in rats with atherosclerosis.

We observed the variation in in vivo blood lipid and blood glucose metabolism in rats with atherosclerosis after 5-(3,4-dihydroxy-phenyl)-1-piperidin-1-yl-penta-2,4-dien-1-one (GBOT) administration. Wistar rats aged 10 weeks received a high-fat diet to establish the atherosclerosis model. Metabolic indices related to blood lipid and blood glucose were measured before modeling and at 4 and 8 weeks after modeling. Liver fat levels in rats were measured at 8 weeks to analyze the relationship between liver fat and blood lipid levels. We examined the mechanism of blood lipid reduction. The levels of serum triglycerides, total cholesterol, and very-low-density lipoprotein cholesterol in rats in the control group were significantly decreased (P < 0.05) compared with those in the 4-week control group at 4 weeks and decreased significantly and continuously until the 8th week (P < 0.05). Compared with the 8-week control group, the blood glucose level in rats in the 8-week experimental group decreased significantly (P < 0.05), and the level of insulin sensitivity index decreased significantly (P < 0.05). Compared with the control group, triglyceride and total cholesterol levels per unit mass in rat liver tissue in the 8-week experimental group decreased significantly (P < 0.05). Western blotting indicated that GBOT significantly increased the expression of lecithin-cholesterol acyltransferase, low-density lipoprotein receptor, and cholesterol 7 alpha-hydroxylase proteins. GBOT can significantly decrease the levels of blood lipid and blood glucose in rat models of atherosclerosis, and its mechanism may be associated with the promotion of expression of lecithin-cholesterol acyltransferase, low-density lipoprotein receptor, and cholesterol 7 alpha-hydroxylase proteins.

Constitutively active Notch4 receptor elicits brain arteriovenous malformations through enlargement of capillary-like vessels.

Arteriovenous (AV) malformation (AVM) is a devastating condition characterized by focal lesions of enlarged, tangled vessels that shunt blood from arteries directly to veins. AVMs can form anywhere in the body and can cause debilitating ischemia and life-threatening hemorrhagic stroke. The mechanisms that underlie AVM formation remain poorly understood. Here, we examined the cellular and hemodynamic changes at the earliest stages of brain AVM formation by time-lapse two-photon imaging through cranial windows of mice expressing constitutively active Notch4 (Notch4*). AVMs arose from enlargement of preexisting microvessels with capillary diameter and blood flow and no smooth muscle cell coverage. AV shunting began promptly after Notch4* expression in endothelial cells (ECs), accompanied by increased individual EC areas, rather than increased EC number or proliferation. Alterations in Notch signaling in ECs of all vessels, but not arteries alone, affected AVM formation, suggesting that Notch functions in the microvasculature and/or veins to induce AVM. Increased Notch signaling interfered with the normal biological control of hemodynamics, permitting a positive feedback loop of increasing blood flow and vessel diameter and driving focal AVM growth from AV connections with higher blood velocity at the expense of adjacent AV connections with lower velocity. Endothelial expression of constitutively active Notch1 also led to brain AVMs in mice. Our data shed light on cellular and hemodynamic mechanisms underlying AVM pathogenesis elicited by increased Notch signaling in the endothelium.

Deletion of Rbpj from postnatal endothelium leads to abnormal arteriovenous shunting in mice.

Arteriovenous malformations (AVMs) are tortuous vessels characterized by arteriovenous (AV) shunts, which displace capillaries and shunt blood directly from artery to vein. Notch signaling regulates embryonic AV specification by promoting arterial, as opposed to venous, endothelial cell (EC) fate. To understand the essential role of endothelial Notch signaling in postnatal AV organization, we used inducible Cre-loxP recombination to delete Rbpj, a mediator of canonical Notch signaling, from postnatal ECs in mice. Deletion of endothelial Rbpj from birth resulted in features of AVMs by P14, including abnormal AV shunting and tortuous vessels in the brain, intestine and heart. We further analyzed brain AVMs, as they pose particular health risks. Consistent with AVM pathology, we found cerebral hemorrhage, hypoxia and necrosis, and neurological deficits. AV shunts originated from capillaries (and possibly venules), with the earliest detectable morphological abnormalities in AV connections by P8. Prior to AV shunt formation, alterations in EC gene expression were detected, including decreased Efnb2 and increased Pai1, which encodes a downstream effector of TGFß signaling. After AV shunts had formed, whole-mount immunostaining showed decreased Efnb2 and increased Ephb4 expression within AV shunts, suggesting that ECs were reprogrammed from arterial to venous identity. Deletion of Rbpj from adult ECs led to tortuosities in gastrointestinal, uterine and skin vascular beds, but had mild effects in the brain. Our results demonstrate a temporal requirement for Rbpj in postnatal ECs to maintain proper artery, capillary and vein organization and to prevent abnormal AV shunting and AVM pathogenesis.

Endothelial ephrin-B2 is essential for arterial vasodilation in mice.

OBJECTIVE: The cell surface protein ephrin-B2 is expressed in arterial and not venous ECs throughout development and adulthood. Endothelial ephrin-B2 is required for vascular development and angiogenesis, but its role in established arteries is currently unknown. We investigated the physiological role of ephrin-B2 signaling in adult endothelium. METHODS: We generated adult conditional knockout mice lacking the Efnb2 gene specifically in ECs and evaluated the vasodilation responses to blood flow increase and ACh in the cremaster muscle preparation by intravital microscope and in carotid artery by in vivo ultrasound. RESULTS: We found that the Efnb2 conditional knockout mice were defective in acute arterial dilation. Vasodilation was impaired in cremaster arterioles in response to either increased flow or ACh, and in the carotid arteries in response to increased flow. Levels of cGMP, an effector of NO, were diminished in mutant arteries following ACh stimulation. GSNO, a donor for the vasodilator NO, alleviated the vasodilatory defects in the mutants. Immunostaining showed that a subset of ephrin-B2 proteins colocalized with caveolin-1, a negative regulator of eNOS. CONCLUSIONS: Our data suggest that endothelial ephrin-B2 is required for endothelial-dependent arterial dilation and NO signaling in adult endothelium.