To enable arrayed or pooled loss-of-function screens in a wide range of mammalian cell types, including primary and nondividing cells, we are developing lentiviral short hairpin RNA (shRNA) libraries targeting the human and murine genomes. The libraries currently contain 104,000 vectors, targeting each of 22,000 human and mouse genes with multiple sequence-verified constructs. To test the utility of the library for arrayed screens, we developed a screen based on high-content imaging to identify genes required for mitotic progression in human cancer cells and applied it to an arrayed set of 5,000 unique shRNA-expressing lentiviruses that target 1,028 human genes. The screen identified several known and approximately 100 candidate regulators of mitotic progression and proliferation; the availability of multiple shRNAs targeting the same gene facilitated functional validation of putative hits. This work provides a widely applicable resource for loss-of-function screens, as well as a roadmap for its application to biological discovery.

Clinical studies using bone marrowderived proangiogenic cells (PACs) have demonstrated modest improvements of function and/or perfusion of ischemic myocardium or skeletal muscle. Because the identities of these PACs and their functional ability to promote neovascularization remain poorly understood, it is possible that a subset of robust PACs exists but is obscured by the heteroge- IntroductionTherapeutic angiogenesis has emerged as an innovative strategy for the treatment of patients with cardiovascular ischemic disease states, such as myocardial ischemia or peripheral artery disease. Several clinical trials using autologous, bone marrow (BM)-derived mononuclear cells have demonstrated only modest improvements of function and/or perfusion of ischemic myocardium or limb in patients. [1][2][3][4][5][6] However, the identities of these BM-derived mononuclear cells and their functional ability to promote neovascularization remain poorly understood.Accumulating evidence has indicated that proangiogenic cells (PACs) may originate from the BM and are capable of being recruited to sites of ischemic injury, where they contribute to neovascularization and tissue repair through paracrine/autocrine mechanisms or by direct incorporation into the vessel wall. [7][8][9][10] Indeed, PAC function and/or number have been found to be markedly impaired in patients with coronary or peripheral artery disease. 11-17 Herein, we provide evidence that a hierarchy exists among mouse hematopoietic stem cell (HSC) progenitors in their ability to differentiate into PACs and promote neovascularization. Complementary functional studies revealed that common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) preferentially differentiate into PACs as opposed to megakaryocyte-erythrocyte progenitors (MEPs), HSCs, and common lymphoid progenitors. Moreover, only PACs derived from BM-derived CMPs and GMPs were able to enhance neovascularization in vivo. Taken together, these findings reveal a novel and unexpected hierarchy among BM-derived progenitors in their ability to promote neovascularization. Methods Isolation and in vitro culture of PACsProgenitor cells were isolated from C57BL/6 mice (n ϭ 8-10) BM by using multicolor FACSAria (BD Biosciences) as previously described. 18,19 Briefly, approximately 2 to 5 ϫ 10 5 BM-derived cells were prospectively isolated and purified as follows: HSCs (Lin Ϫ Sca1 ϩ c-kit ϩ ), CMPs (Lin Ϫ Sca1 Ϫ ckit ϩ CD34 ϩ Fc␥RII/II lo ), GMPs (Lin Ϫ Sca1 Ϫ c-kit ϩ CD34 ϩ Fc␥RII/III hi ), and MEPs (Lin Ϫ Sca1 Ϫ c-kit ϩ CD34 Ϫ Fc␥RII/III lo ; supplemental Figure 1A, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Subsequently, cells were incubated in endothelial growth media-2 (EGM-2; Lonza) for 8 to 10 days and subjected to flow cytometry to detect expression of vascular endothelial growth factor receptor-2 (VEGFR2), CD31, CD133, CD34, CD45, Tie2, von Willebrand factor, endothelial nitric oxide synthase, and VE-cadherin (eBioscience). Cells were a...

The cardiovascular complications of obesity have prompted interest in dietary interventions to reduce weight, including low-carbohydrate diets that are generally high in protein and fat. However, little is known about the long-term effects of these diets on vascular health. We examined the cardiovascular effects of a low-carbohydrate, high-protein diet (LCHP) in the ApoE ؊/؊ mouse model of atherosclerosis and in a model of ischemia-induced neovascularization. Mice on a LCHP were compared with mice maintained on either the standard chow diet (SC) or the Western diet (WD) which contains comparable fat and cholesterol to the LCHP. LCHP-fed mice developed more aortic atherosclerosis and had an impaired ability to generate new vessels in response to tissue ischemia. These changes were not explained by alterations in serum cholesterol, inflammatory mediators or infiltrates, or oxidative stress. The LCHP diet substantially reduced the number of bone marrow and peripheral blood endothelial progenitor cells (EPCs), a marker of vascular regenerative capacity. EPCs from mice on a LCHP diet also manifest lower levels of activated (phosphorylated) Akt, a serine-threonine kinase important in EPC mobilization, proliferation, and survival. Taken together, these data demonstrate that in animal models LCHP diets have adverse vascular effects not reflected in serum markers and that nonlipid macronutrients can modulate vascular progenitor cells and pathophysiology.atherosclerosis ͉ dietary interventions ͉ progenitor cells ͉ neovascularization V ascular disease remains a dominant cause of morbidity and mortality throughout much of the world. The most common form of vascular disease is atherosclerosis, a chronic disorder marked by accumulation of lipid and fibrous material in the vessel wall that can culminate in ischemic tissue injury (1). Atherosclerosis is thought to form as an inflammatory response to a variety of stimuli, including serum lipids that induce endothelial dysfunction and lead to vascular recruitment of leukocytes (2). Similar lesions have been generated in a variety of animal models by increasing dietary fat and cholesterol (3, 4). Recent work has raised the possibility that endothelial progenitor cells (EPC) may help restore normal vascular function (5, 6). Consistent with this hypothesis, clinical studies suggest EPC number correlates with brachial artery reactivity (7) and inversely with prospectively assessed cardiovascular risk (8). However, the precise role of EPCs in atherogenesis remains poorly defined. In contrast, EPCs are more clearly implicated in enhancing neovascularization in response to tissue ischemia in adults (6, 9), a key component of the healing process after such injury.The growing epidemic of obesity and concerns over its complications including atherosclerotic vascular disease have prompted interest in interventions such as low-carbohydrate diets. Typically, a reduction in dietary carbohydrate is accompanied by an increase in dietary fat and protein, which proponents suggest could have saluta...

Emerging evidence demonstrates that proangiogenic cells (PACs) originate from the BM and are capable of being recruited to sites of ischemic injury where they contribute to neovascularization. We previously determined that among hematopoietic progenitor stem cells, common myeloid progenitors (CMPs) and granulocyte-macrophage progenitor cells (GMPs) differentiate into PACs and possess robust angiogenic activity under ischemic conditions. Herein, we report that a TGF-␤1-responsive Krü ppellike factor, KLF10, is strongly expressed in PACs derived from CMPs and GMPs, ϳ 60-fold higher than in progenitors lacking PAC markers. KLF10 ؊/؊ mice present with marked defects in PAC differentiation, function, TGF-␤ responsiveness, and impaired blood flow recovery after hindlimb ischemia, an effect rescued by wild-type PACs, but not KLF10 ؊/؊ PACs. Overexpression studies revealed that KLF10 could rescue PAC formation from TGF-␤1 ؉/؊ CMPs and GMPs. Mechanistically, KLF10 targets the VEGFR2 promoter in PACs which may underlie the observed effects. These findings may be clinically relevant because KLF10 expression was also found to be significantly reduced in PACs from patients with peripheral artery disease. Collectively, these observations identify TGF-␤1 signaling and KLF10 as key regulators of functional PACs derived from CMPs and GMPs and may provide a therapeutic target during cardiovascular ischemic states. (Blood. 2011;118(24):6450-6460) IntroductionAccumulating evidence suggests that in healthy persons, circulating endothelial progenitor cells, broadly defined as proangiogenic cells (PACs), represent a population of BM-derived stem and progenitor cells responsible for repairing injured tissue and initiating neovasculogenesis. 1,2 Potentiation of PAC mobilization, homing, or adhesion has been shown to ameliorate the development of ischemic injury in animal models. 1,2 In addition, blockade of proangiogenic cytokines or their signaling pathways is believed to alter PAC function and to lead to impaired angiogenesis in response to vascular injury and in end-organ ischemia. 1,2 Indeed, reduced levels of circulating PACs and diminished PAC function have been reported and found to correlate with a wide spectrum of atherosclerotic vascular diseases, including peripheral artery disease (PAD). [3][4][5] Several early phase 1/2 trials have been conducted to assess the efficacy of cell-based therapies to treat patients with PAD but have yielded mixed results. 1,2,6-9 Identification of specific PAC subtypes that are endowed with superior capacity to promote neovascularization may represent a particularly efficacious therapeutic strategy. We have demonstrated that among hematopoietic progenitor stem cells, the common myeloid progenitors (CMPs) and granulocytemacrophage progenitors (GMPs) constitute a population of BMderived cells that preferentially differentiate into PACs and possess robust angiogenic activity under ischemic conditions in vivo. 10 However, the signaling pathways and downstream factors that mediate these proangiogenic...