Dyslipidemia is associated with a prothrombotic phenotype; however, the mechanisms responsible for enhanced platelet reactivity remain unclear. Proatherosclerotic lipid abnormalities are associated with both enhanced oxidant stress and the generation of biologically active oxidized lipids, including potential ligands for the scavenger receptor CD36, a major platelet glycoprotein. Using multiple mouse in vivo thrombosis models, we now demonstrate that genetic deletion of Cd36 protects mice from hyperlipidemia-associated enhanced platelet reactivity and the accompanying prothrombotic phenotype. Structurally defined oxidized choline glycerophospholipids that serve as high-affinity ligands for CD36 were at markedly increased levels in the plasma of hyperlipidemic mice and in the plasma of humans with low HDL levels, were able to bind platelets via CD36 and, at pathophysiological levels, promoted platelet activation via CD36. Thus, interactions of platelet CD36 with specific endogenous oxidized lipids play a crucial role in the well-known clinical associations between dyslipidemia, oxidant stress and a prothrombotic phenotype.

Akt kinases control essential cellular functions, including proliferation, apoptosis, metabolism and transcription, and have been proposed as promising targets for treatment of angiogenesis-dependent pathologies, such as cancer and ischemic injury. But their precise roles in neovascularization remain elusive. Here we show that Akt1 is the predominant isoform in vascular cells and describe the unexpected consequences of Akt1 knockout on vascular integrity and pathological angiogenesis. Angiogenic responses in three distinct in vivo models were enhanced in Akt1(-/-) mice; these enhanced responses were associated with impairment of blood vessel maturation and increased vascular permeability. Although impaired vascular maturation in Akt1(-/-) mice may be attributed to reduced activation of endothelial nitric oxide synthase (eNOS), the major phenotypic changes in vascular permeability and angiogenesis were linked to reduced expression of two endogenous vascular regulators, thrombospondins 1 (TSP-1) and 2 (TSP-2). Re-expression of TSP-1 and TSP-2 in mice transplanted with wild-type bone marrow corrected the angiogenic abnormalities in Akt1(-/-) mice. These findings establish a crucial role of an Akt-thrombospondin axis in angiogenesis.

Reciprocity of inflammation, oxidative stress and neovascularization is emerging as an important mechanism underlying numerous processes from tissue healing/remodeling to cancer progression1,2. Whereas the mechanism of hypoxia-driven angiogenesis is well understood3,4, the link between inflammation-induced oxidation and de novo blood vessel growth remains obscure. Here we show that the end products of lipid oxidation, ω-(2-carboxyethyl)pyrrole (CEP) and other related pyrroles5, are generated during inflammation and wound healing and accumulate at high levels in aging tissues and in highly vascularized tumors. The molecular patterns of carboxyalkylpyrroles are recognized by Toll-like receptor 2 (TLR2), but not TLR4 nor scavenger receptors on endothelial cells (ECs), leading to a VEGF-independent angiogenic response. CEP promoted angiogenesis in hind limb ischemia and wound healing models through TLR2 signaling in a MyD88-dependent manner. Neutralization of endogenous carboxyalkylpyrroles impaired wound healing and tissue re-vascularization and diminished tumor angiogenesis. Both TLR2 and MyD88 are required for CEP-induced stimulation of Rac1 and endothelial migration. Together, these findings establish a new function of TLR2 as a sensor of oxidation-associated molecular patterns, providing a key link connecting inflammation, oxidative stress, innate immunity and angiogenesis.

Despite the damaging effect on tissues at a high concentration, it has been gradually established that oxidative stress plays a positive role during angiogenesis. In adults, physiological or pathological angiogenesis is initiated by tissue demands for oxygen and nutrients, resulting in a hypoxia/reoxygenation cycle, which, in turn promotes the formation of reactive oxygen species (ROS). The ROS can be generated either endogenously, through mitochondrial electron transport chain reactions and nicotinamide adenine dinucleotide phosphate oxidase, or exogenously, resulting from exposure to environmental agents, such as ultraviolet or ionizing radiation. In many conditions, ROS promotes angiogenesis, either directly or via the generation of active oxidation products, including peroxidized lipids. The latter lipid metabolites are generated in excess during atherosclerosis, thereby linking atherogenic processes and pathological angiogenesis. Although the main mechanism of oxidative stress-induced angiogenesis involves hypoxia-inducible factor/vascular endothelial growth factor (VEGF) signaling, recent studies have identified several pathways that are VEGF-independent. This review aims to provide a summary of the past and present views on the role of oxidative stress as a mediator and modulator of angiogenesis, and to highlight newly identified mechanisms.

Monogenic deficiency diseases provide unique opportunities to define the contributions of individual molecules to human physiology and to identify pathologies arising from their dysfunction. Here we describe a deficiency disease in two human siblings that presented with severe bleeding, frequent infections and osteopetrosis at an early age. These symptoms are consistent with but more severe than those reported for people with leukocyte adhesion deficiency III (LAD-III). Mechanistically, these symptoms arose from an inability to activate the integrins expressed on hematopoietic cells, including platelets and leukocytes. Immortalized lymphocyte cell lines isolated from the two individuals showed integrin activation defects. Several proteins previously implicated in integrin activation, including Ras-associated protein-1 (RAP1) 1 and calcium and diacylglycerol-regulated guanine nucleotide exchange factor-1 (CALDAG-GEF1) 2 , were present and functional in these cell lines. The genetic basis for this disease was traced to a point mutation in the coding region of the KINDLIN3 (official gene symbol FERMT3) gene 3 . When wild-type KINDLIN-3 was expressed in the immortalized lymphocytes, their integrins Correspondence should be addressed to T.V.B. (byzovat@ccf.org). 6 These authors contributed equally to this work.Note: Supplementary information is available on the Nature Medicine website. AUTHOR CONTRIBUTIONS N.L.M. identified the Kindlin-3 mutation, performed molecular biology and protein biochemistry studies and wrote the manuscript; L.Z. contributed to study design and experiments on primary leukocytes from subjects; J.C. performed assays with EGFP-Kindlin-3 rescue and siRNA-mediated KINDLIN3 knockdown and western blotting; A.C. performed microscopy studies and FACS analysis; O.R. performed cell culture work and molecular biology; Y.-Q.M. performed molecular biology and Kindlin-3-specific antibody preparation; E.A.P. performed platelet studies; M.T. performed neutrophil analysis; D.P.L. and A.I.C. performed osteogenesis assays; S.B.S. originally described the subjects, designed clinical studies and wrote the manuscript; E.F.P. designed the studies, interpreted the results and wrote the manuscript; T.V.B. performed experiments with platelets and leukocytes, designed the general strategy, interpreted data and wrote the manuscript. Kindlin-3 is one of the three-member kindlin family of intracellular proteins that are linked to the actin cytoskeleton 3 . The family is evolutionarily conserved with an ortholog, UNC-112, found in Caenorhabditis elegans 4 . Each kindlin contains a C-terminal FERM domain that is most similar to that of talin, another cytoskeletal protein involved in integrin regulation. Kindlins and talin bind to nonoverlapping sites in the cytoplasmic tails of integrins 5 . Kindler disease, associated with a deficiency of Kindlin-1, has multiple symptoms, including skin blistering and poikiloderma 6 . Kindlin-2 deficiency is embryonically lethal in zebrafish and mice but has not been described in hu...

Abstract-The functional responses of endothelial cells are dependent on signaling from peptide growth factors and the cellular adhesion receptors, integrins. These include cell adhesion, migration, and proliferation, which, in turn, are essential for more complex processes such as formation of the endothelial tube network during angiogenesis. This study identifies the molecular requirements for the cross-activation between ␤ 3 integrin and tyrosine kinase receptor 2 for vascular endothelial growth factor (VEGF) receptor (VEGFR-2) on endothelium. The relationship between VEGFR-2 and ␤ 3 integrin appears to be synergistic, because VEGFR-2 activation induces ␤ 3 integrin tyrosine phosphorylation, which, in turn, is crucial for VEGF-induced tyrosine phosphorylation of VEGFR-2. We demonstrate here that adhesionand growth factor-induced ␤ 3 integrin tyrosine phosphorylation are directly mediated by c-Src. VEGF-stimulated recruitment and activation of c-Src and subsequent ␤ 3 integrin tyrosine phosphorylation are critical for interaction between VEGFR-2 and ␤ 3 integrin. Moreover, c-Src mediates growth factor-induced ␤ 3 integrin activation, ligand binding, ␤ 3 integrin-dependent cell adhesion, directional migration of endothelial cells, and initiation of angiogenic programming in endothelial cells. Thus, the present study determines the molecular mechanisms and consequences of the synergism between 2 cell surface receptor systems, growth factor receptor and integrins, and opens new avenues for the development of pro-and antiangiogenic strategies. Key Words: angiogenesis Ⅲ endothelial cell Ⅲ ␤ 3 integrin signaling Ⅲ vascular endothelial growth factor receptor Ⅲ extracellular matrix proteins A ngiogenesis, the process of new blood vessel formation from preexisting vasculature, plays critical roles in tissue regeneration, postischemic tissue repair on myocardial infarction and stroke, and in the pathogenesis of cancer, rheumatoid arthritis, and diabetic microvascular disease. 1 Angiogenesis is triggered by angiogenic growth factors and their receptors in coordination with extracellular matrix (ECM) receptors known as integrins. 2 On integrin engagement, ECM triggers activation of numerous intracellular signaling pathways essential for endothelial cell (EC) survival, proliferation, migration, morphogenesis, and organization of ECs into blood vessels. 3 There are several manifestations of a tightly collaborative relationship between integrins and receptors for growth factors. 4,5 On ECs, engagement of ␣ v ␤ 3 integrin promotes phosphorylation and activation of vascular endothelial growth factor (VEGF) receptor (VEGFR)-2, thereby augmenting the mitogenic activity of VEGFs. 6 Among several integrins on ECs, ␣ v ␤ 3 is the most abundant and influential receptor regulating angiogenesis. 7 The upregulation of ␣ v ␤ 3 during angiogenesis suggests that this integrin might play a crucial role during this process. Indeed, antagonists of ␣ v ␤ 3 , including blocking monoclonal antibody (LM609) and RGD cyclic peptides, were shown ...

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We investigated the role of Akt-1, one of the major downstream effectors of phosphoinositide 3-kinase (PI3K), in platelet function using mice in which the gene for Akt-1 had been inactivated. Using ex vivo techniques, we showed that Akt-1-deficient mice exhibited impaired platelet aggregation and spreading in response to various agonists. These differences were most apparent in platelets activated with low concentrations of thrombin. Although Akt-1 is not the predominant Akt isoform in mouse platelets, its absence diminished the amount of total phospho-Akt and inhibited increases in intracellular Ca 2؉ concentration in response to thrombin. Moreover, thrombin-induced platelet ␣-granule release as well as release of adenosine triphosphate from dense granules was also defective in Akt-1-null platelets. Although the absence of Akt-1 did not influence expression of the major platelet receptors for thrombin and collagen, fibrinogen binding in response to these agonists was significantly reduced. As a consequence of impaired ␣ IIb ␤ 3 activation and platelet aggregation, Akt-1 null mice showed significantly longer bleeding times than wild-type mice. IntroductionUnder normal conditions, platelets circulate freely in the blood without interacting with each other or the vessel wall. On vascular injury, subendothelial matrix proteins, including collagens, or soluble agonists trigger platelet activation. The hallmark of platelet activation is the transformation of the major platelet glycoprotein, ␣ IIb ␤ 3 , from its resting to active state, which serves as a fibrinogen receptor, thereby mediating platelet aggregation. One of the most potent platelet agonists, thrombin, acts via a dual system of G protein-coupled protease-activated receptors, PAR3 and PAR4. Both of these receptors are required for optimal thrombin-induced aggregation and secretion. 1 The majority of platelet agonists, including thrombin and collagen, activate phosphoinositide 3-kinase (PI3K) in platelets. Inhibitors of PI3K (wortmannin and LY294002) block fibrinogen binding and platelet aggregation induced by thrombin and collagen, indicating a role for PI3K in ␣ IIb ␤ 3 activation. 2 Platelets contain 2 major forms of PI3K, p85/p110 PI3K, composed of a p110 catalytic and p85 regulatory subunit and PI3K␥, composed of a p110␥ catalytic and p101 regulatory subunit. Both forms of PI3K are involved in the inside-out signaling that activates ␣ IIb ␤ 3 and induces platelet aggregation. 2 Recent studies demonstrated that a deficiency in the p85␣ regulatory subunit in mice leads to a significant reduction of collagen-induced platelet aggregation, particularly at low doses of stimulus. . 3 The absence of PI3K␥ activity, indicated by the lack of Akt phosphorylation, leads to impaired platelet aggregation in response to adenosine diphosphate (ADP) and protects against thrombosis. 4 PI3Ks generate phosphoinositide products that target the Tec family tyrosine kinases, serine/threonine protein kinases such as Akt, guanosine diphosphate/guanosine triphosphate exchange f...