Although it is well established that tumors initiate an angiogenic switch, the molecular basis of this process remains incompletely understood. Here we show that the miRNA miR-132 acts as an angiogenic switch by targeting p120RasGAP in the endothelium and thereby inducing neovascularization. We identified miR-132 as a highly upregulated miRNA in a human embryonic stem cell model of vasculogenesis and found that miR-132 was highly expressed in the endothelium of human tumors and hemangiomas but was undetectable in normal endothelium. Ectopic expression of miR-132 in endothelial cells in vitro increased their proliferation and tube-forming capacity, whereas intraocular injection of an antagomir targeting miR-132, anti–miR-132, reduced postnatal retinal vascular development in mice. Among the top-ranking predicted targets of miR-132 was p120RasGAP, which we found to be expressed in normal but not tumor endothelium. Endothelial expression of miR-132 suppressed p120RasGAP expression and increased Ras activity, whereas a miRNA-resistant version of p120RasGAP reversed the vascular response induced by miR-132. Notably, administration of anti–miR-132 inhibited angiogenesis in wild-type mice but not in mice with an inducible deletion of Rasa1 (encoding p120RasGAP). Finally, vessel-targeted nanoparticle delivery1 of anti–miR-132 restored p120RasGAP expression in the tumor endothelium, suppressed angiogenesis and decreased tumor burden in an orthotopic xenograft mouse model of human breast carcinoma. We conclude that miR-132 acts as an angiogenic switch by suppressing endothelial p120RasGAP expression, leading to Ras activation and the induction of neovascularization, whereas the application of anti–miR-132 inhibits neovascularization by maintaining vessels in the resting state.

RASA1 (also known as p120 RasGAP) is a Ras GTPase-activating protein that functions as a regulator of blood vessel growth in adult mice and humans. In humans, RASA1 mutations cause capillary malformation-arteriovenous malformation (CM-AVM); whether it also functions as a regulator of the lymphatic vasculature is unknown. We investigated this issue using mice in which Rasa1 could be inducibly deleted by administration of tamoxifen. Systemic loss of RASA1 resulted in a lymphatic vessel disorder characterized by extensive lymphatic vessel hyperplasia and leakage and early lethality caused by chylothorax (lymphatic fluid accumulation in the pleural cavity). Lymphatic vessel hyperplasia was a consequence of increased proliferation of lymphatic endothelial cells (LECs) and was also observed in mice in which induced deletion of Rasa1 was restricted to LECs. RASA1-deficient LECs showed evidence of constitutive activation of Ras in situ. Furthermore, in isolated RASA1-deficient LECs, activation of the Ras signaling pathway was prolonged and cellular proliferation was enhanced after ligand binding to different growth factor receptors, including VEGFR-3. Blockade of VEGFR-3 was sufficient to inhibit the development of lymphatic vessel hyperplasia after loss of RASA1 in vivo. These findings reveal a role for RASA1 as a physiological negative regulator of LEC growth that maintains the lymphatic vasculature in a quiescent functional state through its ability to inhibit Ras signal transduction initiated through LEC-expressed growth factor receptors such as VEGFR-3.

We generated mutants of the transporter associated with antigen-processing subunits TAP1 and TAP2 that were altered at the conserved lysine residue in the Walker A motifs of the nucleotide binding domains (NBD). In other ATP binding cassette transporters, mutations of the lysine have been shown to reduce or abrogate the ATP hydrolysis activity and in some cases impair nucleotide binding. Mutants TAP1(K544M) and TAP2(K509M) were expressed in insect cells, and the effects of the mutations on nucleotide binding, peptide binding, and peptide translocation were assessed. The mutant TAP1 subunit is significantly impaired for nucleotide binding relative to wild type TAP1. The identical mutation in TAP2 does not significantly impair nucleotide binding relative to wild type TAP2. Using fluorescence quenching assays to measure the binding of fluorescent peptides, we show that both mutants, in combination with their wild type partners, can bind peptides. Since the mutant TAP1 is significantly impaired for nucleotide binding, these results indicate that nucleotide binding to TAP1 is not a requirement for peptide binding to TAP complexes. Peptide translocation is undetectable for TAP1⅐TAP2(K509M) complexes, but low levels of translocation are detectable with TAP1(K544M)⅐TAP2 complexes. These results suggest an impairment in nucleotide hydrolysis by TAP complexes containing either mutant TAP subunit and indicate that the presence of one intact TAP NBD is insufficient for efficient catalysis of peptide translocation. Taken together, these results also suggest the possibility of distinct functions for TAP1 and TAP2 NBD during a single translocation cycle.The transporter associated with antigen processing (TAP) 1 is a critical component of the major histocompatibility complex (MHC) class I antigen presentation (1-3). TAP functions to translocate peptides from the cytosol to the ER. Binding of peptides to newly synthesized MHC class I molecules in the ER stabilizes the MHC class I heterodimer and allows transit of MHC class I-peptide complexes to the cell surface for immune surveillance by T cells (4). Two structurally related subunits of the TAP transporter, TAP1 and TAP2, form a complex on the ER membrane that is necessary and sufficient for peptide translocation from the cytosol into the ER. The cytosolic face of TAP1⅐TAP2 complexes contains a binding site for peptides (5), which can function in the sequestration of peptides derived from proteasomal proteolysis. A recently discovered protein called tapasin is associated with the TAP1⅐TAP2 complex (6, 7). Tapasin has been shown to enhance the expression level of TAP1 and increase peptide transport by TAP complexes (8). However, tapasin is not required for peptide binding by TAP1⅐TAP2 complexes or for translocation per se, since TAP1⅐TAP2 complexes expressed heterologously in insect cells can bind and transport peptides (9).TAP is a member of the ATP-binding cassette (ABC) family of transmembrane transport proteins (10). Members of this family transport various substrates acro...

Mutations in gene RASA1 have been historically associated with capillary malformation-arteriovenous malformation, but sporadic reports of lymphatic involvement have yet to be investigated in detail. To investigate the impact of RASA1 mutations in the lymphatic system, we performed investigational near-infrared fluorescence lymphatic imaging and confirmatory radiographic lymphangiography in a Parkes-Weber syndrome (PKWS) patient with suspected RASA1 mutations and correlated the lymphatic abnormalities against that imaged in an inducible Rasa1 knockout mouse. Whole-exome sequencing (WES) analysis and validation by Sanger sequencing of DNA from the patient and unaffected biological parents enabled us to identify an early-frameshift deletion in RASA1 that was shared with the father, who possessed a capillary stain but otherwise no overt disease phenotype. Abnormal lymphatic vasculature was imaged in both affected and unaffected legs of the PKWS subject that transported injected indocyanine green dye to the inguinal lymph node and drained atypically into the abdomen and into dermal lymphocele-like vesicles on the groin. Dermal lymphatic hyperplasia and dilated vessels were observed in Rasa1-deficient mice, with subsequent development of chylous ascites. WES analyses did not identify potential gene modifiers that could explain the variability of penetrance between father and son. Nonetheless, we conclude that the RASA1 mutation is responsible for the aberrant lymphatic architecture and functional abnormalities, as visualized in the PKWS subject and in the animal model. Our unique method to combine investigatory near-infrared fluorescence lymphatic imaging and WES for accurate phenoptyping and unbiased genotyping allows the study of molecular mechanisms of lymphatic involvement of hemovascular disorders.CM-AVM | lymphatics | near-infrared fluorescence imaging | indocyanine green imaging T he gene RASA1 encodes for protein p120 Ras GTPaseactivating protein (p120 RasGAP or RASA1) that regulates Ras activation and blood vessel growth in humans. Germ-line mutations in RASA1 cause the autosomal dominant vascular disorder capillary malformation-arteriovenous malformation (CM-AVM), with high penetrance and variable expressivity [Online Mendelian Inheritance in Man (OMIM) no. 608354] (1-5). CMs are observed in all patients with RASA1 mutations and present as single or multiple small pink cutaneous lesions. Fast-flow lesions that include AVMs, arteriovenous fistulas, and Parkes-Weber syndrome (PKWS) (OMIM no. 608355) are observed in one-third of cases. PKWS caused by RASA1 mutation typically presents with symmetrical overgrowth of the involved extremity with large cutaneous CM and diffuse microshunting causing cardiac volume overload. Recent reports of upper-and lower-extremity lymphedema (6, 7), together with the finding that a small number of CM-AVM patients develop chylothorax and chylous ascites (5), indicate that lymphatic malformation may be an additional phenotype associated with mutations in RASA1. In mice, deletion of ...

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