RhoC maintains vascular homeostasis by regulating VEGF-induced signaling in endothelial cells.

Vasculogenesis and angiogenesis are controlled by vascular endothelial growth factor A (VEGF-A). Dysregulation of these physiological processes contributes to the pathologies of heart disease, cancer and stroke. Rho GTPase proteins play an integral role in VEGF-mediated formation and maintenance of blood vessels. The regulatory functions of RhoA and RhoB in vasculogenesis and angiogenesis are well defined, whereas the purpose of RhoC remains poorly understood. Here, we describe how RhoC promotes vascular homeostasis by modulating endothelial cell migration, proliferation and permeability. RhoC stimulates proliferation of human umbilical vein endothelial cells (HUVECs) by stabilizing nuclear ß-catenin, which promotes transcription of cyclin D1 and subsequently drives cell cycle progression. RhoC negatively regulates endothelial cell migration through MAPKs and downstream MLC2 signaling, and decreases vascular permeability through downregulation of the phospholipase Cγ (PLCγ)-Ca(2+)-eNOS cascade in HUVECs. Using a VEGF-inducible zebrafish (Danio rerio) model, we observed significantly increased vascular permeability in RhoC morpholino (MO)-injected zebrafish compared with control MO-injected zebrafish. Taken together, our findings suggest that RhoC is a key regulator of vascular homeostasis in endothelial cells.

Transcriptional inhibition of etv2 expression is essential for embryonic cardiac development.

E-twenty six variant 2 (Etv2) transcription factor participates in cardiac, vascular-endothelial and blood cell lineage specification decisions during embryonic development. Previous studies have identified genomic elements in the etv2 locus responsible for vascular endothelial cell specification. Using transgenic analysis in zebrafish, we report here an etv2 proximal promoter fragment that prevents transgene misexpression in myocardial progenitor cells. This inhibition of etv2 expression in the cardiac progenitor population is partly mediated by Scl and Nkx2.5, likely through direct binding to the etv2 promoter, and cis-regulatory elements located in the first and second introns. The results identify an etv2 cis-regulatory mechanism controlling cardiovascular fate choice implying that etv2 participates in a transcriptional network mediating developmental plasticity of endothelial progenitor cells during embryonic development.

Rap1 promotes VEGFR2 activation and angiogenesis by a mechanism involving integrin αvß3.

Vascular endothelial growth factor (VEGF) acting through VEGF receptor 2 (VEGFR2) on endothelial cells (ECs) is a key regulator of angiogenesis, a process essential for wound healing and tumor metastasis. Rap1a and Rap1b, 2 highly homologous small G proteins, are both required for angiogenesis in vivo and for normal EC responses to VEGF. Here we sought to determine the mechanism through which Rap1 promotes VEGF-mediated angiogenesis. Using lineage-restricted Rap1-knockout mice we show that Rap1-deficiency in endothelium leads to defective angiogenesis in vivo, in a dose-dependent manner. Using ECs obtained from Rap1-deficient mice we demonstrate that Rap1b promotes VEGF-VEGFR2 kinase activation and regulates integrin activation. Importantly, the Rap1b-dependent VEGF-VEGFR2 activation is in part mediated via integrin α(v)ß(3). Furthermore, in an in vivo model of zebrafish angiogenesis, we demonstrate that Rap1b is essential for the sprouting of intersomitic vessels, a process known to be dependent on VEGF signaling. Using 2 distinct pharmacologic VEGFR2 inhibitors we show that Rap1b and VEGFR2 act additively to control angiogenesis in vivo. We conclude that Rap1b promotes VEGF-mediated angiogenesis by promoting VEGFR2 activation in ECs via integrin α(v)ß(3). These results provide a novel insight into the role of Rap1 in VEGF signaling in ECs.

Nogo-B receptor is essential for angiogenesis in zebrafish via Akt pathway.

Our previous work has shown that axon guidance gene family Nogo-B and its receptor (NgBR) are essential for chemotaxis and morphogenesis of endothelial cells in vitro. To investigate NogoB-NgBR function in vivo, we cloned the zebrafish ortholog of both genes and studied loss of function in vivo using morpholino antisense technology. Zebrafish ortholog of Nogo-B is expressed in somite while expression of zebrafish NgBR is localized in intersomitic vessel (ISV) and axial dorsal aorta during embryonic development. NgBR or Nogo-B knockdown embryos show defects in ISV sprouting in the zebrafish trunk. Mechanistically, we found that NgBR knockdown not only abolished its ligand Nogo-B-stimulated endothelial cell migration but also reduced the vascular endothelial growth factor (VEGF)-stimulated phosphorylation of Akt and vascular endothelial growth factor-induced chemotaxis and morphogenesis of human umbilical vein endothelial cells. Further, constitutively activated Akt (myristoylated [myr]Akt) or human NgBR can rescue the NgBR knockdown umbilical vein endothelial cell migration defects in vitro or NgBR morpholino-caused ISV defects in vivo. These data place Akt at the downstream of NgBR in both Nogo-B- and VEGF-coordinated sprouting of ISVs. In summary, this study identifies the in vivo functional role for Nogo-B and its receptor (NgBR) in angiogenesis in zebrafish.

Nicotine Exacerbates TAAD Formation Induced by Smooth Muscle-Specific Deletion of the TGF-ß Receptor 2.

Tobacco smoke is an established risk factor for thoracic aortic aneurysms and dissections (TAAD). However, little is known about its underlying mechanisms due to the lack of validated animal models. The present study developed a mouse model that may be utilized to investigate exacerbation of TAAD formation by mimetics of tobacco smoke. TAADs were created via inducible deletion of smooth muscle cell-specific Tgfbr2 receptors. Using this model, the first set of experiments evaluated the efficacy of nicotine salt (34.0 mg/kg/day), nicotine free base (NFB, 5.0 mg 90-day pellets), and cigarette smoke extract (0.1 ml/mouse/day). Compared with their respective control groups, only NFB pellets promoted TAAD dilation (23 ± 3% vs. 12 ± 2%, P = 0.014), and this efficacy was achieved at a cost of >50% acute mortality. Infusion of NFB with osmotic minipumps at extremely high, but nonlethal, doses (15.0 or 45.0 mg/kg/day) failed to accelerate TAAD dilation. Interestingly, costimulation with ß-aminopropionitrile (BAPN) promoted TAAD dilation and aortic rupture at dosages of 3.0 and 45.0 mg/kg/day, respectively, indicating that BAPN sensitizes the response of TAADs to NFB. In subsequent analyses, the detrimental effects of NFB were associated with clustering of macrophages, neutrophils, and T-cells in areas with structural destruction, enhanced matrix metalloproteinase- (MMP-) 2 production, and pathological angiogenesis with attenuated fibrosis in the adventitia. In conclusion, modeling nicotine exacerbation of TAAD formation requires optimization of chemical form, route of delivery, and dosage of the drug as well as the pathologic complexity of TAADs. Under the optimized conditions of the present study, chronic inflammation and adventitial mal-remodeling serve as critical pathways through which NFB exacerbates TAAD formation.

A validated mouse model capable of recapitulating the protective effects of female sex hormones on ascending aortic aneurysms and dissections (AADs).

Fewer females develop AADs (ascending aortic aneurysms and dissections) and the reasons for this protection remain poorly understood. The present study seeks to develop a mouse model that may be utilized to address this sexual dimorphism. Adult normolipidemic mice were challenged with BAPN (ß-aminopropionitrile), AngII (angiotensin II), or BAPN + AngII. An initial protocol optimization found that 0.2% BAPN in drinking water plus AngII-infusion at 1,000 ng kg-1  min-1 produced favorable rates of AAD rupture (~50%) and dilation (~40%) in 28 days. Using these dosages, further experiments revealed that BAPN is toxic to naïve mature aortas and it acted synergistically with AngII to promote aortic tears and dissections. BAPN + AngII provoked early infiltration of myeloid cells and subsequent recruitment of lymphoid cells to the aortic wall. AADs established with BAPN + AngII, but not AngII alone, continued to expand after the cessation of AngII-infusion. This indefinite growth precipitated a 61% increase in the AAD diameter in 56 days. More importantly, with the optimized protocol, significant differences in AAD dilation (p = .012) and medial degeneration (p = .036) were detected between male and female mice. Treatment of ovariectomized mice with estradiol protected AAD formation (p = .014). In summary, this study developed a powerful mouse AAD model that can be used to study the sexual dimorphism in AAD formation.

The homologous recombination component EEPD1 is required for genome stability in response to developmental stress of vertebrate embryogenesis.

Stressed replication forks can be conservatively repaired and restarted using homologous recombination (HR), initiated by nuclease cleavage of branched structures at stalled forks. We previously reported that the 5' nuclease EEPD1 is recruited to stressed replication forks, where it plays critical early roles in HR initiation by promoting fork cleavage and end resection. HR repair of stressed replication forks prevents their repair by non-homologous end-joining (NHEJ), which would cause genome instability. Rapid cell division during vertebrate embryonic development generates enormous pressure to maintain replication speed and accuracy. To determine the role of EEPD1 in maintaining replication fork integrity and genome stability during rapid cell division in embryonic development, we assessed the role of EEPD1 during zebrafish embryogenesis. We show here that when EEPD1 is depleted, zebrafish embryos fail to develop normally and have a marked increase in death rate. Zebrafish embryos depleted of EEPD1 are far more sensitive to replication stress caused by nucleotide depletion. We hypothesized that the HR defect with EEPD1 depletion would shift repair of stressed replication forks to unopposed NHEJ, causing chromosome abnormalities. Consistent with this, EEPD1 depletion results in nuclear defects including anaphase bridges and micronuclei in stressed zebrafish embryos, similar to BRCA1 deficiency. These results demonstrate that the newly characterized HR protein EEPD1 maintains genome stability during embryonic replication stress. These data also imply that the rapid cell cycle transit seen during embryonic development produces replication stress that requires HR to resolve.

Sucrose non-fermenting related kinase enzyme is essential for cardiac metabolism.

In this study, we have identified a novel member of the AMPK family, namely Sucrose non-fermenting related kinase (Snrk), that is responsible for maintaining cardiac metabolism in mammals. SNRK is expressed in the heart, and brain, and in cell types such as endothelial cells, smooth muscle cells and cardiomyocytes (CMs). Snrk knockout (KO) mice display enlarged hearts, and die at postnatal day 0. Microarray analysis of embryonic day 17.5 Snrk hearts, and blood profile of neonates display defect in lipid metabolic pathways. SNRK knockdown CMs showed altered phospho-acetyl-coA carboxylase and phospho-AMPK levels similar to global and endothelial conditional KO mouse. Finally, adult cardiac conditional KO mouse displays severe cardiac functional defects and lethality. Our results suggest that Snrk is essential for maintaining cardiac metabolic homeostasis, and shows an autonomous role for SNRK during mammalian development.