The regulation of blood vessel formation is of fundamental importance to many physiological processes, and angiogenesis is a major area for novel therapeutic approaches to diseases from ischemia to cancer. A poorly understood clinical manifestation of pathological angiogenesis is angiodysplasia, vascular malformations that cause severe gastrointestinal bleeding. Angiodysplasia can be associated with von Willebrand disease (VWD), the most common bleeding disorder in man. VWD is caused by a defect or deficiency in von Willebrand factor (VWF), a glycoprotein essential for normal hemostasis that is involved in inflammation. We hypothesized that VWF regulates angiogenesis. Inhibition of VWF expression by short interfering RNA (siRNA) in endothelial cells (ECs) caused increased in vitro angiogenesis and increased vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2)-dependent proliferation and migration, coupled to decreased integrin ␣v␤3 levels and increased angiopoietin (Ang)-2 release. ECs expanded from blood- IntroductionAngiogenesis, the formation of new vessels from pre-existing ones, occurs physiologically in specific circumstances such as wound healing and the menstrual cycle. Dysregulated angiogenesis contributes to the pathogenesis of many disorders, including diabetes, cancer, and macular degeneration (reviewed in Carmeliet 1 ). Angiogenic factors such as vascular endothelial growth factor (VEGF) and the angiopoietins (Ang) orchestrate signaling pathways that promote endothelial cell (EC) migration, proliferation, and ultimately the formation of a new vessel. VEGF-A is a major regulator of angiogenesis (reviewed in Grothey and Galanis 2 ) and acts on ECs mainly through VEGF receptor-2 (VEGFR-2), a tyrosine kinase receptor (reviewed in Olsson 3 ), to promote endothelial proliferation, migration, and sprouting of tip cells in the early stages of angiogenesis (reviewed in Gerhardt 4 ). Ang-1 and Ang-2, which bind to the endothelial Tie-2 receptor, act in the later stages of blood vessel formation and are essential for the maturation of a stable vascular network and for the maintenance of endothelial integrity (reviewed in Thomas and Augustin 5 ). Ang-1 and Ang-2 were originally identified as agonist and antagonist of Tie-2 signaling, respectively, with Ang-1 supporting EC survival and endothelial integrity 6 and Ang-2 promoting blood vessel destabilization and regression. 7 However, recent data suggest a more complex picture that includes cross-talk between the VEGF and the Ang pathways. 8 Growth factor signaling pathways are influenced by surface adhesion molecules that mediate cell-cell or cell-matrix interactions, particularly by members of the integrin superfamily. The integrin that has received most attention in ECs is ␣v␤3 (reviewed in Hodivala-Dilke 9 ), which mediates binding to several extracellular matrix proteins and growth factor receptors including VEGFR-2, thus influencing VEGFR-2 signaling (reviewed in Somanath et al 10 ). ␣v␤3 plays a complex role in angiogenesis. Although the origina...

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Haploinsufficiency of ribosomal proteins (RPs) has been proposed to be the common basis for the anemia observed in Diamond-Blackfan anemia (DBA) and myelodysplastic syndrome with loss of chromosome 5q [del(5q) MDS]. We have modeled DBA and del(5q) MDS in zebrafish using antisense morpholinos to rps19 and rps14, respectively, and have demonstrated that, as in humans, haploinsufficient levels of these proteins lead to a profound anemia. To address the hypothesis that RP loss results in impaired mRNA translation, we treated Rps19 and Rps14-deficient embryos with the amino acid L-leucine, a known activator of mRNA translation. This resulted in a striking improvement of the anemia associated with RP loss. We confirmed our findings in primary human CD34 ؉ cells, after shRNA knockdown of RPS19 and RPS14. Furthermore, we showed that loss of Rps19 or Rps14 activates the mTOR pathway, and this is accentuated by L-leucine in both Rps19 and Rps14 morphants. This effect could be abrogated by rapamycin suggesting that mTOR signaling may be responsible for the improvement in anemia associated with L-leucine. Our studies support the rationale for ongoing clinical trials of L-leucine as a therapeutic agent for DBA, and potentially for patients with del(5q) MDS. (Blood. 2012; 120(11):2214-2224) IntroductionDiamond-Blackfan anemia (DBA; MIM# 105650) is a congenital bone marrow failure syndrome of childhood manifested as normochromic macrocytic anemia with absence or insufficient erythroid precursors in the bone marrow. 1,2 Twenty-five percent of DBA patients have mutations in the RPS19 gene, which encodes a component of the 40S ribosomal subunit. 3,4 A further 25% of DBA patients have been shown to have mutations in other ribosomal protein genes, 5 supporting the hypothesis that DBA is a disease of altered ribosome assembly or function. DBA shares a number of its clinical features with several other congenital syndromes that also carry heterozygous mutations affecting ribosome biogenesis, such as Shwachman-Diamond syndrome (SDS), cartilage-hair hypoplasia syndrome, and dyskeratosis congenita (DC) suggesting that all of these conditions share at least some common pathogenic mechanisms; they have thus been termed "ribosomopathies." 6 In addition, evidence suggests that the anemia associated with the 5q minus (5qϪ) syndrome (or myelodysplastic syndrome with loss of all or part of chromosome 5q [del(5q) MDS]), a distinct subtype of myelodysplastic syndrome results from somatic heterozygous loss of the ribosomal protein gene RPS14 in hematopoietic stem cells. 7,8 Efforts to understand why ribosomal protein haploinsufficiencies have such a specific and profound effect on erythroid development at the molecular level have focused on the activation and stabilization of p53 in response to ribosomal stress. 9 However, the precise mechanisms governing how p53 stabilization occurs in response to ribosomal protein haploinsufficiency have not been clearly defined. Furthermore, not all bone marrow samples from patients with del(5q) MDS or DBA sh...

A substantial number of individuals with bone marrow failure (BMF) present with one or more extra-hematopoietic abnormality. This suggests a constitutional or inherited basis, and yet many of them do not fit the diagnostic criteria of the known BMF syndromes. Through exome sequencing, we have now identified a subgroup of these individuals, defined by germline biallelic mutations in DNAJC21 (DNAJ homolog subfamily C member 21). They present with global BMF, and one individual developed a hematological cancer (acute myeloid leukemia) in childhood. We show that the encoded protein associates with rRNA and plays a highly conserved role in the maturation of the 60S ribosomal subunit. Lymphoblastoid cells obtained from an affected individual exhibit increased sensitivity to the transcriptional inhibitor actinomycin D and reduced amounts of rRNA. Characterization of mutations revealed impairment in interactions with cofactors (PA2G4, HSPA8, and ZNF622) involved in 60S maturation. DNAJC21 deficiency resulted in cytoplasmic accumulation of the 60S nuclear export factor PA2G4, aberrant ribosome profiles, and increased cell death. Collectively, these findings demonstrate that mutations in DNAJC21 cause a cancer-prone BMF syndrome due to corruption of early nuclear rRNA biogenesis and late cytoplasmic maturation of the 60S subunit.