Arteriovenous malformations (AVMs) are vascular anomalies where arteries and veins are directly connected through a complex, tangled web of abnormal arteries and veins instead of a normal capillary network. AVMs in the brain, lung, and visceral organs, including the liver and gastrointestinal tract, result in considerable morbidity and mortality. AVMs are the underlying cause of three major clinical symptoms of a genetic vascular dysplasia termed hereditary hemorrhagic telangiectasia (HHT), which is characterized by recurrent nosebleeds, mucocutaneous telangiectases, and visceral AVMs and caused by mutations in one of several genes, including activin receptor–like kinase 1 (ALK1). It remains unknown why and how selective blood vessels form AVMs, and there have been technical limitations to observing the initial stages of AVM formation. Here we present in vivo evidence that physiological or environmental factors such as wounds in addition to the genetic ablation are required for Alk1-deficient vessels to develop to AVMs in adult mice. Using the dorsal skinfold window chamber system, we have demonstrated for what we believe to be the first time the entire course of AVM formation in subdermal blood vessels by using intravital bright-field images, hyperspectral imaging, fluorescence recordings of direct arterial flow through the AV shunts, and vascular casting techniques. We believe our data provide novel insights into the pathogenetic mechanisms of HHT and potential therapeutic approaches.
Sung Ok Park, Mamta Wankhede, Young Jae Lee, Eun-Jung Choi, Naime Fliess, Se-Woon Choe, Seh-Hoon Oh, Glenn Walter, Mohan K. Raizada, Brian S. Sorg, S. Paul Oh
Protein S (ProS) is a blood anticoagulant encoded by the Pros1 gene, and ProS deficiencies are associated with venous thrombosis, stroke, and autoimmunity. These associations notwithstanding, the relative risk that reduced ProS expression confers in different disease settings has been difficult to assess without an animal model. We have now described a mouse model of ProS deficiency and shown that all Pros1–/– mice die in utero, from a fulminant coagulopathy and associated hemorrhages. Although ProS is known to act as a cofactor for activated Protein C (aPC), plasma from Pros1+/– heterozygous mice exhibited accelerated thrombin generation independent of aPC, and Pros1 mutants displayed defects in vessel development and function not seen in mice lacking protein C. Similar vascular defects appeared in mice in which Pros1 was conditionally deleted in vascular smooth muscle cells. Mutants in which Pros1 was deleted specifically in hepatocytes, which are thought to be the major source of ProS in the blood, were viable as adults and displayed less-severe coagulopathy without vascular dysgenesis. Finally, analysis of mutants in which Pros1 was deleted in endothelial cells indicated that these cells make a substantial contribution to circulating ProS. These results demonstrate that ProS is a pleiotropic anticoagulant with aPC-independent activities and highlight new roles for ProS in vascular development and homeostasis.
Tal Burstyn-Cohen, Mary Jo Heeb, Greg Lemke
Paracrine signaling from lung epithelium to the surrounding mesenchyme is important for lung SMC development and function and is a contributing factor in an array of pulmonary diseases such as bronchopulmonary dysplasia, pulmonary hypertension, and asthma. Wnt7b, which is exclusively expressed in the lung epithelium, is important for lung vascular smooth muscle integrity, but the underlying mechanism by which Wnt signaling regulates lung SMC development is unclear. In this report, we have demonstrated that Wnt7b regulates a program of mesenchymal differentiation in the mouse lung that is essential for SMC development. Genetic loss-of-function studies showed that Wnt7b and β-catenin were required for expression of Pdgfrα and Pdgfrβ and proliferation in pulmonary SMC precursors. In contrast, gain-of-function studies showed that activation of Wnt signaling increased the expression of both Pdgfrα and Pdgfrβ as well as the proliferation of SMC precursors. We further showed that the effect on Pdgfr expression was, in part, mediated by direct transcriptional regulation of the ECM protein tenascin C (Tnc), which was necessary and sufficient for Pdgfrα/β expression in lung explants. Moreover, this pathway was highly upregulated in a mouse model of asthma and in lung tissue from patients with pulmonary hypertension. Together, these data define a Wnt/Tnc/Pdgfr signaling axis that is critical for smooth muscle development and disease progression in the lung.
Ethan David Cohen, Kaori Ihida-Stansbury, Min Min Lu, Reynold A. Panettieri, Peter Lloyd Jones, Edward E. Morrisey
Masahiro Murakami, Loc T. Nguyen, Zhen W. Zhuang, Karen L. Moodie, Peter Carmeliet, Radu V. Stan, Michael Simons
Maintenance of vascular integrity is critical for homeostasis, and temporally and spatially regulated vascular leak is a central feature of inflammation. Sphingosine-1-phosphate (S1P) can regulate endothelial barrier function, but the sources of the S1P that provide this activity in vivo and its importance in modulating different inflammatory responses are unknown. We report here that mutant mice engineered to selectively lack S1P in plasma displayed increased vascular leak and impaired survival after anaphylaxis, administration of platelet-activating factor (PAF) or histamine, and exposure to related inflammatory challenges. Increased leak was associated with increased interendothelial cell gaps in venules and was reversed by transfusion with wild-type erythrocytes (which restored plasma S1P levels) and by acute treatment with an agonist for the S1P receptor 1 (S1pr1). S1pr1 agonist did not protect wild-type mice from PAF-induced leak, consistent with plasma S1P levels being sufficient for S1pr1 activation in wild-type mice. However, an agonist for another endothelial cell Gi-coupled receptor, Par2, did protect wild-type mice from PAF-induced vascular leak, and systemic treatment with pertussis toxin prevented rescue by Par2 agonist and sensitized wild-type mice to leak-inducing stimuli in a manner that resembled the loss of plasma S1P. Our results suggest that the blood communicates with blood vessels via plasma S1P to maintain vascular integrity and regulate vascular leak. This pathway prevents lethal responses to leak-inducing mediators in mouse models.
Eric Camerer, Jean B. Regard, Ivo Cornelissen, Yoga Srinivasan, Daniel N. Duong, Daniel Palmer, Trung H. Pham, Jinny S. Wong, Rajita Pappu, Shaun R. Coughlin
Cardiac atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) modulate blood pressure and volume by activation of the receptor guanylyl cyclase–A (GC-A) and subsequent intracellular cGMP formation. Here we report what we believe to be a novel function of these peptides as paracrine regulators of vascular regeneration. In mice with systemic deletion of the GC-A gene, vascular regeneration in response to critical hind limb ischemia was severely impaired. Similar attenuation of ischemic angiogenesis was observed in mice with conditional, endothelial cell–restricted GC-A deletion (here termed EC GC-A KO mice). In contrast, smooth muscle cell–restricted GC-A ablation did not affect ischemic neovascularization. Immunohistochemistry and RT-PCR revealed BNP expression in activated satellite cells within the ischemic muscle, suggesting that local BNP elicits protective endothelial effects. Since within the heart, BNP is mainly induced in cardiomyocytes by mechanical load, we investigated whether the natriuretic peptide/GC-A system also regulates angiogenesis accompanying load-induced cardiac hypertrophy. EC GC-A KO hearts showed diminished angiogenesis, mild fibrosis, and diastolic dysfunction. In vitro BNP/GC-A stimulated proliferation and migration of cultured microvascular endothelia by activating cGMP-dependent protein kinase I and phosphorylating vasodilator-stimulated phosphoprotein and p38 MAPK. We therefore conclude that BNP, produced by activated satellite cells within ischemic skeletal muscle or by cardiomyocytes in response to pressure load, regulates the regeneration of neighboring endothelia via GC-A. This paracrine communication might be critically involved in coordinating muscle regeneration/hypertrophy and angiogenesis.
Michaela Kuhn, Katharina Völker, Kristine Schwarz, Javier Carbajo-Lozoya, Ulrich Flögel, Christoph Jacoby, Jörg Stypmann, Martin van Eickels, Stepan Gambaryan, Michael Hartmann, Matthias Werner, Thomas Wieland, Jürgen Schrader, Hideo A. Baba
Although endothelial dysfunction, defined as abnormal vasoreactivity, is a common early finding in individuals with type 2 diabetes, the endothelium has not been known to regulate metabolism. As PPARγ, a transcriptional regulator of energy balance, is expressed in endothelial cells, we set out to investigate the role of endothelial cell PPARγ in metabolism using mice that lack PPARγ in the endothelium and BM (γEC/BM-KO). When γEC/BM-KO mice were fed a high-fat diet, they had decreased adiposity and increased insulin sensitivity compared with control mice, despite increased serum FFA and triglyceride (TG) levels. After fasting or olive oil gavage, γEC/BM-KO mice exhibited significant dyslipidemia and failed to respond to the FFA and TG lowering effects of the PPARγ agonist rosiglitazone. BM transplantation studies, which reconstituted hematopoietic PPARγ, established that these metabolic phenotypes were due to endothelial PPARγ deficiency. We further found that the impairment in TG-rich lipoprotein metabolism in γEC/BM-KO mice was associated with fatty acid–mediated lipoprotein lipase inhibition and changes in a PPARγ-regulated endothelial cell transcriptional program. Despite their metabolic improvements, high-fat diet–fed γEC/BM-KO mice had impaired vasoreactivity. Taken together, these data suggest that PPARγ in the endothelium integrates metabolic and vascular responses and may contribute to the effects of PPARγ agonists, thus expanding what endothelial function and dysfunction may entail.
Takeshi Kanda, Jonathan D. Brown, Gabriela Orasanu, Silke Vogel, Frank J. Gonzalez, Juliano Sartoretto, Thomas Michel, Jorge Plutzky
Vascular proliferative diseases are characterized by VSMC proliferation and migration. Kinase interacting with stathmin (KIS) targets 2 key regulators of cell proliferation and migration, the cyclin-dependent kinase inhibitor p27Kip1 and the microtubule-destabilizing protein stathmin. Phosphorylation of p27Kip1 by KIS leads to cell-cycle progression, whereas the target sequence and the physiological relevance of KIS-mediated stathmin phosphorylation in VSMCs are unknown. Here we demonstrated that vascular wound repair in KIS–/– mice resulted in accelerated formation of neointima, which is composed predominantly of VSMCs. Deletion of KIS increased VSMC migratory activity and cytoplasmic tubulin destabilizing activity, but abolished VSMC proliferation through the delayed nuclear export and degradation of p27Kip1. This promigratory phenotype resulted from increased stathmin protein levels, caused by a lack of KIS-mediated stathmin phosphorylation at serine 38 and diminished stathmin protein degradation. Downregulation of stathmin in KIS–/– VSMCs fully restored the phenotype, and stathmin-deficient mice demonstrated reduced lesion formation in response to vascular injury. These data suggest that KIS protects against excessive neointima formation by opposing stathmin-mediated VSMC migration and that VSMC migration represents a major mechanism of vascular wound repair, constituting a relevant target and mechanism for therapeutic interventions.
Thomas H. Langenickel, Michelle Olive, Manfred Boehm, Hong San, Martin F. Crook, Elizabeth G. Nabel
ASK1-interacting protein-1 (AIP1), a recently identified member of the Ras GTPase-activating protein family, is highly expressed in vascular ECs and regulates EC apoptosis in vitro. However, its function in vivo has not been established. To study this, we generated AIP1-deficient mice (KO mice). Although these mice showed no obvious defects in vascular development, they exhibited dramatically enhanced angiogenesis in 2 models of inflammatory angiogenesis. In one of these models, the enhanced angiogenesis observed in the KO mice was associated with increased VEGF-VEGFR2 signaling. Consistent with this, VEGF-induced ear, cornea, and retina neovascularization were greatly augmented in KO mice and the enhanced retinal angiogenesis was markedly diminished by overexpression of AIP1. In vitro, VEGF-induced EC migration was inhibited by AIP1 overexpression, whereas it was augmented by both AIP1 knockout and knockdown, with the enhanced EC migration caused by AIP1 knockdown being associated with increased VEGFR2 signaling. We present mechanistic data that suggest AIP1 is recruited to the VEGFR2-PI3K complex, binding to both VEGFR2 and PI3K p85, at a late phase of the VEGF response, and that this leads to inhibition of VEGFR2 signaling. Taken together, our data demonstrate that AIP1 functions as an endogenous inhibitor in VEGFR2-mediated adaptive angiogenesis in mice.
Haifeng Zhang, Yun He, Shengchuan Dai, Zhe Xu, Yan Luo, Ting Wan, Dianhong Luo, Dennis Jones, Shibo Tang, Hong Chen, William C. Sessa, Wang Min
Mucin-type O-glycans (O-glycans) are highly expressed in vascular ECs. However, it is not known whether they are important for vascular development. To investigate the roles of EC O-glycans, we generated mice lacking T-synthase, a glycosyltransferase encoded by the gene C1galt1 that is critical for the biosynthesis of core 1–derived O-glycans, in ECs and hematopoietic cells (termed here EHC T-syn–/– mice). EHC T-syn–/– mice exhibited embryonic and neonatal lethality associated with disorganized and blood-filled lymphatic vessels. Bone marrow transplantation and EC C1galt1 transgene rescue demonstrated that lymphangiogenesis specifically requires EC O-glycans, and intestinal lymphatic microvessels in EHC T-syn–/– mice expressed a mosaic of blood and lymphatic EC markers. The level of O-glycoprotein podoplanin was significantly reduced in EHC T-syn–/– lymphatics, and podoplanin-deficient mice developed blood-filled lymphatics resembling EHC T-syn–/– defects. In addition, postnatal inactivation of C1galt1 caused blood/lymphatic vessel misconnections that were similar to the vascular defects in the EHC T-syn–/– mice. One consequence of eliminating T-synthase in ECs and hematopoietic cells was that the EHC T-syn–/– pups developed fatty liver disease, because of direct chylomicron deposition via misconnected portal vein and intestinal lymphatic systems. Our studies therefore demonstrate that EC O-glycans control the separation of blood and lymphatic vessels during embryonic and postnatal development, in part by regulating podoplanin expression.
Jianxin Fu, Holger Gerhardt, J. Michael McDaniel, Baoyun Xia, Xiaowei Liu, Lacramioara Ivanciu, Annelii Ny, Karlien Hermans, Robert Silasi-Mansat, Samuel McGee, Emma Nye, Tongzhong Ju, Maria I. Ramirez, Peter Carmeliet, Richard D. Cummings, Florea Lupu, Lijun Xia