The specification of arteries and veins is an essential process in establishing and maintaining a functional blood vessel system. Incorrect arteriovenous specification disrupts embryonic development but has also been diagnosed in human syndromes such as hypotrichosislymphedema-telangiectasia, characterized by defects in blood and lymphatic vessels and associated with mutations in SOX18. Here we characterize the role of sox7 and sox18 during zebrafish vasculogenesis. Sox7 and sox18 are specifically expressed in the developing vasculature, and simultaneous loss of their function results in a severe loss of the arterial identity of the presumptive aorta which instead expresses venous markers, followed by dramatic arteriovenous shunt formations. Our study identifies members of the Sox family as key factors in specifying arteriovenous identity and will help to better understand hypotrichosis-lymphedema-telangiectasia and other diseases.A rteriovenous (AV) specification and differentiation are two critical events required for the progression of vascular development and function, as evidenced by diseases such as hypotrichosis-lymphedema-telangiectasia and hereditary hemorrhagic telangiectasia, which have been associated with mutations in SOX18 and endoglin/activin-like receptor kinase-1, respectively. 1-3 To study the process of AV specification, zebrafish embryos have proven particularly useful. Following the specification of arterial and venous cell types, 4 endothelial cells coalesce into cord-like midline structures and subsequently reshape into tubes. 5 However, although several signaling molecules and transcription factors 4,6 -8 have been implicated in these processes, we are still only beginning to understand their regulation. In an attempt to identify new factors involved in the regulation of AV specification and vasculogenesis, we analyzed the function of sox7 and sox18, which, together with the endodermally expressed sox17, form the Sox-F (Sry-related HMG box) family of DNA-binding proteins. 9 Members of the Sox-F family play crucial roles during the formation of definitive endoderm, 10 hematopoietic stem cell regulation, 11 and cardiovascular development. 12 Here we show temporal and spatial overlap of sox7 and sox18 expression and identify functionally redundant roles for these genes during vascular development in zebrafish embryos. Our results demonstrate a novel role for sox7 and sox18 in specifying the molecular identity of endothelial cells in their commitment to arterial tissues during vasculogenesis. Materials and Methods Zebrafish HusbandryZebrafish (Danio rerio) were raised as described. 13 Histological ProceduresIn situ hybridization and immuno-histochemistry were performed as described. 5,13 The riboprobes used are specified in the online data supplement, available at http://circres.ahajournals.org. Morpholino Injections and MicroangiographsProcedures are specified in the online data supplement. Results and DiscussionExpression Analysis for sox7 and sox18We first examined embryonic expres...

Cell therapy is a field of growing interest in the prevention of post acute myocardial infarction (AMI) heart failure. Stem cell retention upon local delivery to the heart, however, is still unsatisfactory. CellBeads were recently developed as a potential solution to this problem. CellBeads are 170-μm alginate microspheres that contain mesenchymal stem cells (MSCs) genetically modified to express glucagon-like peptide-1 (GLP-1) supplementary to inherent paracrine factors. GLP-1 is an incretin hormone that has both antiapoptotic and cardioprotective effects. Transplanting CellBeads in the post-AMI heart might induce cardiomyocyte salvage and ultimately abrogate adverse cardiac remodeling. We aimed to investigate the feasibility of intracoronary infusion of CellBeads in a large animal model of AMI. Four pigs were used in a pilot study to assess the maximal safe dose of CellBeads. In the remaining 21 animals, an AMI was induced by balloon occlusion of the left circumflex coronary artery for 90 min. During reperfusion, 60,000 CellBeads (n = 11), control beads (n = 4), or lactated Ringers' (n = 6) were infused. Animals were sacrificed after 2 or 7 days, and the hearts were excised for histological analyses. Intracoronary infusion did not permanently affect coronary flow in any of the groups. Histological analysis revealed CellBeads containing viable MSCs up to 7 days. Viability and activity of the MSCs was confirmed by qPCR analysis that showed expression of recombinant GLP-1 and human genes after 2 and 7 days. CellBeads reduced inflammatory infiltration by 29% (p = 0.001). In addition, they decreased the extent of apoptosis by 25% (p = 0.001) after 2 days. We show that intracoronary infusion of 5 million encapsulated MSCs is safe and feasible. Also, several parameters indicate that the cells have paracrine effects, suggesting a potential therapeutic benefit of this new approach.

Mechanical forces are translated into biochemical stimuli by mechanotransduction channels, such as the mechanically-activated cation channel Piezo2. Lung Piezo2 expression has recently been shown to be restricted to endothelial cells. Hence, we aimed to investigate the role of Piezo2 in regulation of pulmonary vascular function and structure as well as its contribution to development of pulmonary arterial hypertension (PAH). The expression of Piezo2 was significantly reduced in pulmonary microvascular endothelial cells (MVECs) from PAH patients, in lung tissue from mice with a Bmpr2+/R899X knock-in mutation commonly found in pulmonary hypertension patients, and in lung tissue of MCT and SuHx PAH rat models as well as from a swine model with pulmonary vein banding. In MVECs, Piezo2 expression was reduced in response to abnormal shear stress, hypoxia and TGFβ stimulation. Functional studies in MVECs exposed to shear stress illustrated that siRNA-mediated Piezo2 knockdown impaired endothelial alignment, calcium influx, phosphorylation of AKT and nitric oxide production. In addition, siPiezo2 reduced the expression of the endothelial marker PECAM-1 and increased expression of vascular smooth muscle markers ACTA2, SM22a and Calponin. Thus, Piezo2 acts as a mechanotransduction channel in pulmonary MVECs, stimulating shear-induced production of nitric oxide and is essentially involved in preventing endothelial to mesenchymal transition. Its blunted expression in pulmonary hypertension could impair the vasodilator capacity and stimulate vascular remodelling, indicating that Piezo2 might be an interesting therapeutic target to attenuate progression of the disease.

Pulmonary vein stenosis (PVS) causes a rare type of Pulmonary Hypertension (PH), by impacting the flow and pressure within the pulmonary vasculature resulting in endothelial dysfunction and metabolic changes. A prudent line of treatment in this type of PH would be targeted therapy in order to relieve the pressure and reverse the flow related changes. We used a swine model in order to mimic PH post PVS using pulmonary vein banding (PVB) of the lower lobes for 12 weeks to mimic the hemodynamic profile associated with PH and investigate the molecular alterations that provide an impetus for development of PH. Our current study aimed to employ unbiased proteomic and metabolomic analyses on both the upper and lower lobes of the swine lung to identify the regions with metabolic alterations. We detected changes in the upper lobes for the PVB animals mainly pertaining to fatty acid metabolism, ROS signaling and extracellular matrix remodeling, and small albeit significant changes in the lower lobes for purine metabolism.

Pulmonary vein stenosis (PVS) causes a rare type of pulmonary hypertension (PH) by impacting the flow and pressure within the pulmonary vasculature, resulting in endothelial dysfunction and metabolic changes. A prudent line of treatment in this type of PH would be targeted therapy to relieve the pressure and reverse the flow-related changes. We used a swine model in order to mimic PH after PVS using pulmonary vein banding (PVB) of the lower lobes for 12 weeks to mimic the hemodynamic profile associated with PH and investigated the molecular alterations that provide an impetus for the development of PH. Our current study aimed to employ unbiased proteomic and metabolomic analyses on both the upper and lower lobes of the swine lung to identify regions with metabolic alterations. We detected changes in the upper lobes for the PVB animals mainly pertaining to fatty acid metabolism, reactive oxygen species (ROS) signaling and extracellular matrix (ECM) remodeling and small, albeit, significant changes in the lower lobes for purine metabolism.