Early inflammatory reactions in atherosclerosis are induced by proline-rich tyrosine kinase/reactive oxygen species-mediated release of tumor necrosis factor-alpha and subsequent activation of the p21Cip1/Ets-1/p300 system.

OBJECTIVE: Reactive oxygen species (ROS) are involved in the initial process of atherosclerosis, whereas it remains to be determined how atherogenic stimulus causes ROS-mediated proinflammatory reactions. Here, we focused on proline-rich tyrosine kinase (PYK2)-mediated ROS generation and examined how atherogenic stimulus causes early proinflammatory reactions. METHODS AND RESULTS: PYK2-deficient (knockout [KO]) (PYK2-KO) mice were crossbred with apolipoprotein E (ApoE)-deficient (PYK2-KO/ApoE-KO) mice. PYK2-KO/ApoE-KO mice and endothelial cells (EC) were used for the study. Aortic atherogenic lesions in PYK2-KO/ApoE-KO mice were markedly decreased (55% versus ApoE-KO) after 8 weeks of a Western diet. Aortic PYK2 was activated as early as 7 days after the Western diet, when inflammatory cells were not yet activated. Addition of the proatherogenic oxidized phospholipid lysophosphatidylcholine caused activation of endothelial PYK2. Lysophosphatidylcholine-activated PYK2 induced NADPH oxidase-mediated ROS generation and ROS-mediated synthesis of tumor necrosis factor-α (TNFα), vascular cell adhesion molecule-1 (VCAM-1), monocyte chemotactic protein-1 (MCP-1), and p21Cip1/Ets-1. Neutralizing anti-TNFα antibody or knockdown of p21Cip1/Ets-1 system blocked the induction of VCAM-1 and MCP-1. PYK2 deficiency abolished these ROS-mediated proinflammatory reactions. Further analysis revealed that PYK2/ROS-mediated p21Cip1/Ets-1 activation upregulated the transcription of the MCP-1 gene in collaboration with p300 transcription coactivator. CONCLUSIONS: PYK2 is a key tyrosine kinase activated by high cholesterol exposure, which causes ROS-mediated TNFα release and induces TNFα-dependent expression of proinflammatory molecules via the p21Cip1/Ets-1/p300 transcription system.

Endothelial FGF receptor signaling accelerates atherosclerosis.

Members of the fibroblast growth factor (FGF) family have been clinically applied to the treatment of ischemic diseases because of their strong angiogenic actions. Although tissue ischemia is predominantly caused by atherosclerosis, the roles of endothelial FGF receptors (FGF-Rs) in atherosclerosis remain obscure. We generated endothelial cell (EC)-targeted constitutively active FGF-R2-overexpressing mice, using the Tie2 promoter (Tie2-FGF-R2-Tg), and crossed them with apolipoprotein E (ApoE)-deficient mice (ApoE-KO) to generate Tie2-FGF-R2-Tg/ApoE-deficient mice (Tie2-FGF-R2-Tg/ApoE-KO). After being fed a Western diet for 8 wk, the Tie2-FGF-R2-Tg/ApoE-KO demonstrated 2.0-fold greater atherosclerotic lesion area on the luminal surfaces of the aortas than the ApoE-KO (P < 0.01). The level of p21(Cip1) protein, a cell cycle inhibitor, in the FGF-R2-overexpressing EC was 2.5-fold greater than that in the wild-type (WT) EC at the baseline (P < 0.01). FGF-R2 overexpression in the EC resulted in increased expression of VCAM-1 and ICAM-1, acceleration of apoptosis, and decreased proliferative activity, all of which were normalized by small interfering RNA (siRNA)-mediated knockdown of p21(Cip1) (75% reduction in protein level, P < 0.01). Furthermore, the expression of PDGF-B and Egr-1, a PDGF/p21(Cip1)-inducible transcription factor, in the aortic endothelium of Tie2-FGF-R2-Tg/ApoE-KO was significantly greater than that in ApoE-KO. The proliferation of vascular smooth muscle cells in the aortic media of Tie2-FGF-R2-Tg/ApoE-KO was 2.0-fold higher than that in ApoE-KO (P < 0.01). Thus our study reveals that endothelial FGF-R2 signaling aggravates atherosclerosis by promoting p21(Cip1)-mediated EC dysfunction and cautions against the use of FGF for therapeutic angiogenesis in the setting of atherosclerosis.

Signaling from fibroblast growth factor receptor 2 in immature hematopoietic cells facilitates donor hematopoiesis after intra-bone marrow-bone marrow transplantation.

Fibroblast growth factor (FGF) and FGF receptor (FGFR) are expressed in various cells including endothelial progenitor cells and hematopoietic cells. The interaction between FGF and FGFR is associated with the proliferation, migration, and survival of these cells. In this report, we examined the effects of FGFR2 signaling on hematopoiesis in immature hematopoietic cells, using mutant mice in which a constitutively active form of FGFR2 mutant was caused to be overexpressed by the Tie2 promoter (FGFR2 Tg mice). Under normal conditions, hematopoiesis of FGFR2 Tg mice and wild type (Wt) mice do not differ significantly, except for the weight and cell numbers of the thymus. However, the c-kit(+)Sca-1(+)lineage⁻ bone marrow cells (BMCs) of FGFR2 Tg mice facilitate the formation of colony-forming units of culture. When these BMCs were transplanted into the recipient bone marrow (intra-bone marrow-bone marrow transplantation), there was better reconstitution of donor hematopoietic cells. In the in vitro experiment, the c-kit(+)Sca-1(+)lineage⁻ BMCs from FGFR2 Tg mice showed fewer apoptotic cells than those from Wt mice. These results suggest that the antiapoptotic effect of FGFR2 signaling facilitates the hematopoiesis of FGFR2 Tg mice.

Endothelium-targeted overexpression of constitutively active FGF receptor induces cardioprotection in mice myocardial infarction.

Fibroblast growth factor receptor (FGFR) is expressed in a variety of cells and is involved in their proliferation/migration/survival. To elucidate FGFR-mediated specific action of vascular endothelial cells (ECs) on myocardial ischemia, we generated endothelium-targeted transgenic mice overexpressing constitutively active FGFR2 using Tie2 promoter (FGFR2-Tg). Infarct size, vessel formation and blood perfusion were significantly improved 28 days after myocardial infarction (MI) in FGFR2-Tg, compared with wild-type mice. Aortic ECs isolated from FGFR-Tg showed a marked increase in migratory capacity and tube formation. These in vitro angiogenic activities were blocked by PI3-kinase inhibitor. Whereas, parameters obtained from echocardiography were already improved at three days after MI. Cardiomyocyte apoptosis at the ischemic border zone was decreased in FGFR2-Tg (32.1%, p < 0.05) and cardiac mRNA expression of FGF2 (basic FGF) was also up-regulated (142%, p < 0.05) at 3 days after MI. 1% oxygen-mediated apoptosis was significantly inhibited in FGFR2-Tg-ECs and this inhibition was abolished by PI3-kinase inhibitor. FGFR2-Tg-ECs exposed to 1% oxygen exhibited enhanced phosphorylation of 416-Tyr-Src, 473-Ser-Akt, and HIF1alpha accumulation. The production of FGF2 was enhanced 2.1-fold in FGFR-Tg-ECs under 1% oxygen via the Src/Akt/HIF1alpha pathway, which induced the peri-vessel migration of vascular smooth muscle cells (VSMCs) and anti-apoptotic effects on VSMCs and cardiomyocytes. FGF receptor signaling in ECs promoted migration, survival and autocrine production of FGF2, leading to reduced infarct size, which is associated with anti-apoptotic action in the early stage and with enhanced angiogenesis in the late stage after MI.