The ability to differentiate human pluripotent stem cells into endothelial cells with properties of cord-blood endothelial colony–forming cells (CB-ECFCs) may enable the derivation of clinically relevant numbers of highly proliferative blood vessel–forming cells to restore endothelial function in patients with vascular disease. We describe a protocol to convert human induced pluripotent stem cells (hiPSCs) or embryonic stem cells (hESCs) into cells similar to CB-ECFCs at an efficiency of >108 ECFCs produced from each starting pluripotent stem cell. The CB-ECFC-like cells display a stable endothelial phenotype with high clonal proliferative potential and the capacity to form human vessels in mice and to repair the ischemic mouse retina and limb, and they lack teratoma formation potential. We identify Neuropilin-1 (NRP-1)-mediated activation of KDR signaling through VEGF165 as a critical mechanism for the emergence and maintenance of CB-ECFC-like cells.

This study, using mouse embryonic fibroblast (MEF) cells derived from ROCK1−/− and ROCK2−/− mice, is designed to dissect roles for ROCK1 and ROCK2 in regulating actin cytoskeleton reorganization induced by doxorubicin, a chemotherapeutic drug. ROCK1−/− MEFs exhibited improved actin cytoskeleton stability characterized by attenuated periphery actomyosin ring formation and preserved central stress fibers, associated with decreased myosin light chain 2 (MLC2) phosphorylation but preserved cofilin phosphorylation. These effects resulted in a significant reduction in cell shrinkage, detachment, and predetachment apoptosis. In contrast, ROCK2−/− MEFs showed increased periphery membrane folding and impaired cell adhesion, associated with reduced phosphorylation of both MLC2 and cofilin. Treatment with inhibitor of myosin (blebbistatin), inhibitor of actin polymerization (cytochalasin D), and ROCK pan-inhibitor (Y27632) confirmed the contributions of actomyosin contraction and stress fiber instability to stress-induced actin cytoskeleton reorganization. These results support a novel concept that ROCK1 is involved in destabilizing actin cytoskeleton through regulating MLC2 phosphorylation and peripheral actomyosin contraction, whereas ROCK2 is required for stabilizing actin cytoskeleton through regulating cofilin phosphorylation. Consequently, ROCK1 and ROCK2 can be functional different in regulating stress-induced stress fiber disassembly and cell detachment.

Summary The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. We found that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. A broadly applicable, highly integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora A kinase (AURKA), which has not been studied extensively in megakaryocytes. Moreover, we discovered that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in AMKL blasts and displayed potent anti-AMKL activity in vivo. This research provides the rationale to support clinical trials of MLN8237 and other inducers of polyploidization in AMKL. Finally, we have identified five networks of kinases that regulate the switch to polyploidy.

Summary We show constitutive activation of Rho kinase (ROCK) in cells bearing oncogenic forms of KIT, FLT3 and BCR-ABL, which is dependent on PI3K and Rho GTPase. Genetic or pharmacologic inhibition of ROCK in oncogene bearing cells impaired their growth as well as the growth of acute myeloid leukemia patient derived blasts and prolonged the life span of mice bearing myeloproliferative disease. Downstream from ROCK, rapid dephosphorylation or loss of expression of myosin light chain resulted in enhanced apoptosis, reduced growth and loss of actin polymerization in oncogene bearing cells leading to significantly prolonged life span of leukemic mice. In summary, we describe a pathway involving PI3K/Rho/ROCK/MLC which may contribute to myeloproliferative disease and/or acute myeloid leukemia in humans.

Rho kinases belong to a family of serine/ threonine kinases whose role in recruitment and migration of inflammatory cells is poorly understood. We show that deficiency of ROCK1 results in increased recruitment and migration of macrophages and neutrophils in vitro and in vivo. Enhanced migration resulting from ROCK1 deficiency is observed despite normal expression of ROCK2 and a significant reduction in overall ROCK activity. ROCK1 directly binds PTEN in response to receptor activation and is essential for PTEN phosphorylation and stability. In the absence of ROCK1, PTEN phosphorylation, stability, and its activity are significantly impaired. Consequently IntroductionNeutrophils and macrophages are a major cellular component of the innate immune response that are rapidly recruited in large numbers to sites of infection. [1][2][3] In response to inflammation, neutrophils and macrophages migrate from blood to infected sites in various tissues and protect the host by destroying invading bacterial and fungal pathogens. This process is thought to involve chemokines, cytokines, extracellular matrix proteins, and members of the ␤1 integrin family, including ␣4␤1and ␣5␤1. Importantly, interfering with the function of ␤1 integrins impairs the ability of macrophages to be recruited to the sites of inflammation. 4,5 Although it is clear that cytokines such as macrophage colony stimulating factor (M-CSF), chemokines such as monocyte chemotactic protein-1 (MCP-1), and ␤1 integrins play a significant role in regulating adhesion and migration of macrophages and neutrophils, the signaling pathways responsible for coordinating these processes downstream from these molecules are poorly defined.Recent studies have implicated phosphatidylinositol 3,4,5-trisphosphate (PIP3) in regulating several aspects of cytoskeletonbased functions, including adhesion and migration in response to activation of a variety of cell surface receptors. 6,7 In macrophages and neutrophils, PIP3 regulates adhesion, migration, and polarization as a consequence of activation of the enzyme phosphatidylinositol-3 kinase (PI3K). End products of PI3K are partly regulated by phosphatase and tensin homolog deleted on chromosome 10 (PTEN). PTEN is a tumor suppressor gene that is often mutated in several tumors. 8 PTEN inactivates PI3K by dephosphorylating PIP3 to PIP2. 9 The structure of PTEN involves multiple domains, including the phosphatase domain (C2), PDZ binding domain, as well as various phosphorylation sites implicated in regulating the activity/stability of PTEN. Although it is widely accepted that deletion or mutation of PTEN can contribute to tumor formation, recent studies suggest that modulation in the levels of PTEN expression may also contribute to tumorigenesis. 10 To this end, studies have shown that PTEN protein expression is reduced in a significant number of breast cancers. 11 Although the precise reason behind reduced PTEN protein levels in these cancers is poorly understood, alteration in the transcription of PTEN as well as changes in the ac...

Significance All lymphoid cells are considered to be products of hematopoietic stem cells (HSCs); however, it has been suggested, but not proven, that innate immune B-1 progenitor cells develop independently of HSCs in the fetal liver. B-1 cells, especially B-1a cells, are not replaced by adult bone marrow transplantation. Thus, it is critical to understand the origin and mechanisms required to sustain these cells in vivo because B-1 cells play important roles in the first line of defense against microbial infection and in preventing organ damage in autoimmune patients and infections in some patients after bone-marrow transplantation. We demonstrate that B-1 progenitor cells can develop independently of HSCs in the fetal liver and that their development relies critically on the expression of core-binding factor beta.

Neurofibromatosis type 1 (NF1) results from mutations in the NF1 tumor suppressor gene, which encodes the protein neurofibromin. NF1 patients display diverse clinical manifestations, including vascular disease, which results from neointima formation and vessel occlusion. However, the pathogenesis of NF1 vascular disease remains unclear. Vessel wall homeostasis is maintained by complex interactions between vascular and bone marrow-derived cells (BMDCs), and neurofibromin regulates the function of each cell type. Therefore, utilizing cre/lox techniques and hematopoietic stem cell transplantation to delete 1 allele of Nf1 in endothelial cells, vascular smooth muscle cells, and BMDCs alone, we determined which cell lineage is critical for neointima formation in vivo in mice. Here we demonstrate that heterozygous inactivation of Nf1 in BMDCs alone was necessary and sufficient for neointima formation after vascular injury and provide evidence of vascular inflammation in Nf1 +/-mice. Further, analysis of peripheral blood from NF1 patients without overt vascular disease revealed increased concentrations of inflammatory cells and cytokines previously linked to vascular inflammation and vasoocclusive disease. These data provide genetic and cellular evidence of vascular inflammation in NF1 patients and Nf1 +/-mice and provide a framework for understanding the pathogenesis of NF1 vasculopathy and potential therapeutic and diagnostic interventions. IntroductionNeurofibromatosis type 1 (NF1) is an autosomal dominant disorder that results from mutations in the tumor suppressor gene NF1 (1). Neurofibromin, the protein product of NF1, functions as a p21 Ras (Ras) GTPase-activating protein (GAP) to negatively regulate Ras activity (2). More than 240 different mutations have been described within the NF1 gene, all of which result in little or no protein product (3). While loss of heterozygosity has been described in primary tumor samples (4), the germline mutations that cause NF1 affect only 1 copy of the NF1 gene. Haploinsufficiency of NF1 results in disease with complete penetrance and a range of clinical complications.The most common clinical manifestations of NF1 include dermal and plexiform neurofibromas, learning deficits, and skeletal abnormalities. Vascular disease associated with NF1 is an underrecognized complication that results in increased morbidity and mortality, particularly among younger patients (5, 6). In 2001, an analysis of 3,253 death certificates of persons with NF1 indicated that the median age of death for NF1 patients was 15 years lower that of the general population (6). In this report, a diagnosis suggestive of NF1 vasculopathy was listed 7.2 times more often than expected among NF1 patients less than 30 years old at time of death and 2.2 times more often than expected among patients 30-40 years old at the time of death (6). Another study demonstrated that 2.5% of children with NF1 who had undergone brain MRI were found to have cerebrovascular system abnormalities including narrowed vessels, moyamoya, va...

SUMMARY Oncogenic mutations of FLT3 and KIT receptors are associated with poor survival in patients with acute myeloid leukemia (AML) and myeloproliferative neoplasms (MPN) and currently available drugs are largely ineffective. Although Stat5 has been implicated in regulating several myeloid and lymphoid malignancies, how precisely Stat5 regulates leukemogenesis, including its nuclear translocation to induce gene transcription is poorly understood. In leukemic cells, we show constitutive activation of focal adhesion kinase (FAK), whose inhibition represses leukemogenesis. Downstream of FAK, activation of Rac1 is regulated by RacGEF Tiam1, whose inhibition prolongs the survival of leukemic mice. Inhibition of the Rac1 effector PAK1 prolongs the survival of leukemic mice in part by inhibiting the nuclear translocation of Stat5. These results reveal a leukemic pathway involving FAK/Tiam1/Rac1/PAK1 and demonstrate an essential role for these signaling molecules in regulating the nuclear translocation of Stat5 in leukemogenesis.