Mucinous tubular and spindle cell carcinoma with a high nuclear grade and micropapillary pattern: A case report.

Mucinous tubular and spindle cell carcinoma (MTSCC) is rare in renal cell carcinoma (RCC) and usually demonstrates a low nuclear grade and a better prognosis compared with other RCCs. The authors present a case report of MTSCC containing an area of Fuhrman nuclear grade 3, in addition to an area with a micropapillary pattern. An 82-year-old man consulted a hospital due to macrohematuria, and a tumor in the right kidney was detected. The tumor was resected and histologically examined. The tumor consisted of various growth patterns: Elongated tubular structure, a papillary structure containing a micropapillary pattern and solid pattern with spindle cells. The tumor cells demonstrated Fuhrman nuclear grades 2 and 3. Invasion into the lymph vessel and metastasis into the regional lymph node were observed. Thus, the tumor was diagnosed as a high grade MTSCC. Five months following resection, a computed tomography scan suggested metastasis of the tumor into the para-aortic lymph nodes and liver, and the patient succumbed to brain metastasis. When MTSCC of kidney is observed, careful histological observation is important to avoid missing a high nuclear grade area.

A case of CD10-negative angioimmunoblastic T cell lymphoma with leukemic change and increased plasma cells mimicking plasma cell leukemia: A case report.

Angioimmunoblastic T cell lymphoma (AITL) is a peripheral T cell lymphoma, known to express CD3 and CD4, and, frequently, also CD10 and c-Maf-1. Hypergammaglobulinemia is not particularly rare in patients with AITL. However, AITL in conjunction with plasmacytosis in the peripheral blood is rare. The current report presents a case of CD10-negative AITL demonstrating leukemic change and plasmacytosis in the peripheral blood mimicking plasma cell leukemia. A 78-year-old male was admitted to hospital due to systemic lymph node enlargement, high serum IgG and IgA, and increased counts of plasmacytoid cells and lymphoid cells with atypical nuclei in the peripheral blood. Initially, plasma cell leukemia was suspected, due to the extreme increase in the number of plasma cells in the peripheral blood. However, the plasma cells did not show clonal expansion on examination by flow cytometry. Based on histological analyses, following a biopsy of an enlarged lymph node, the patient was diagnosed with AITL. This case suggests that when hypergammaglobulinemia and increases in B-lineage cells are observed, AITL should be considered in addition to disorders of B-lineage cells.

Activation of phosphatidylinositol 3-kinase ß by the platelet collagen receptors integrin α2ß1 and GPVI: The role of Pyk2 and c-Cbl.

Phosphatidylinositol 3-kinaseß (PI3Kß) plays a predominant role in integrin outside-in signaling and in platelet activation by GPVI engagement. We have shown that the tyrosine kinase Pyk2 mediates PI3Kß activation downstream of integrin αIIbß3, and promotes the phosphorylation of the PI3K-associated adaptor protein c-Cbl. In this study, we compared the functional correlation between Pyk2 and PI3Kß upon recruitment of the two main platelet collagen receptors, integrin α2ß1 and GPVI. PI3Kß-mediated phosphorylation of Akt was inhibited in Pyk2-deficient platelets adherent to monomeric collagen through integrin α2ß1, but occurred normally upon GPVI ligation. Integrin α2ß1 engagement led to Pyk2-independent association of c-Cbl with PI3K. However, c-Cbl was not phosphorylated in adherent platelets, and phosphorylation of Akt occurred normally in c-Cbl-deficient platelets, indicating that the c-Cbl is dispensable for Pyk2-mediated PI3Kß activation. Stimulation of platelets with CRP, a selective GPVI ligand, induced c-Cbl phosphorylation in the absence of Pyk2, but failed to promote its association with PI3K. Pyk2 activation was completely abrogated in PI3KßKD, but not in PI3KγKD platelets, and was strongly inhibited by Src kinases and phospholipase C inhibitors, and by BAPTA-AM. The absence of PI3Kß activity also hampered GPVI-induced tyrosine-phosphorylation and activation of PLCγ2, preventing intracellular Ca2+ increase and phosphorylation of pleckstrin. Moreover, GPVI-induced intracellular Ca2+ increase and pleckstrin phosphorylation were also strongly inhibited in human platelets treated with the PI3Kß inhibitor TGX-221. These results outline important differences in the regulation of PI3Kß by GPVI and integrin α2ß1 and suggest that inhibition of Pyk2 may target PI3Kß activation in a selective context of platelet stimulation.

The focal adhesion kinase Pyk2 links Ca2+ signalling to Src family kinase activation and protein tyrosine phosphorylation in thrombin-stimulated platelets.

In blood platelets, stimulation of G protein-coupled receptors (GPCRs) by thrombin triggers the activation of Src family kinases (SFKs), resulting in the tyrosine-phosphorylation of multiple substrates, but the mechanism underlying this process is still poorly understood. In the present study, we show that the time-dependent protein-tyrosine phosphorylation triggered by thrombin in human or murine platelets was totally suppressed only upon concomitant chelation of intracellular Ca(2+) and inhibition of SFKs. Thrombin-induced activation of SFKs was regulated by intracellular Ca(2+) and accordingly the Ca(2+) ionophore A23187 was sufficient to stimulate SFKs. A23187 also triggered the phosphorylation and activation of the Ca(2+)-dependent focal adhesion kinase Pyk2 and Pyk2 activation by thrombin was Ca(2+)-dependent. Stimulation of SFKs by thrombin or A23187 was strongly reduced in platelets from Pyk2 knockout (KO) mice, as was the overall pattern of protein-tyrosine phosphorylation. By immunoprecipitation experiments, we demonstrate that Lyn and Fyn, but not Src, were activated by Pyk2. Inhibition of SFKs by PP2 also reduced the phosphorylation of Pyk2 in thrombin or A23187-stimulated platelets. Analysis of KO mice demonstrated that Fyn, but not Lyn, was required for complete Pyk2 phosphorylation by thrombin. Finally, PP2 reduced aggregation of murine platelets to a level comparable to that of Pyk2-deficient platelets, but did not have further effects in the absence of Pyk2. These results indicate that in thrombin-stimulated platelets, stimulation of Pyk2 by intracellular Ca(2+) initiates SFK activation, establishing a positive loop that reinforces the Pyk2/SFK axis and allows the subsequent massive tyrosine phosphorylation of multiple substrates required for platelet aggregation.

A nuclear factor-κB inhibitor, dehydroxymethylepoxyquinomicin, ameliorates GVHD in allogeneic bone marrow transplantation.

GVHD is a crucial mortality factor in allogeneic bone marrow transplantation (ABMT). In this paper, we show that dehydroxymethylepoxyquinomicin (DHMEQ), a novel inhibitor of nuclear factor-κB, suppresses GVHD, resulting in an improved mortality rate in a mouse ABMT model. Bone marrow cells from C57BL/6 mice (B6 mice) were transplanted into lethally irradiated BALB/c mice. Two weeks later, spleen cells from B6 mice were transplanted into the irradiated BALB/c mice. From one week after the injection of spleen cells, when the mice started to show GVHD, the mice were also injected intraperitoneally daily with DHMEQ or vehicle only (DMSO) for 4 weeks. By 80 days after the ABMT, 6/14 of the vehicle-injected mice (43%) had died because of GVHD, whereas all DHMEQ-injected mice survived this observation period and developed milder GVHD than the vehicle-injected mice. When regulatory T cells were reduced by the injection of anti-folate receptor 4 (FR4) antibody, the effects of DHMEQ were reduced. These findings suggest that administration of DHMEQ could become a new strategy for preventing fatalities from GVHD.

Distinct role of Pyk2 in mediating thromboxane generation downstream of both G12/13 and integrin αIIbß3 in platelets.

Proline-rich tyrosine kinase 2 (Pyk2) is activated by various agonists in platelets. We evaluated the signaling mechanism and the functional role of Pyk2 in platelets by using pharmacological inhibitors and Pyk2-deficient platelets. We found that platelet aggregation and secretion in response to 2-methylthio-ADP (2-MeSADP) and AYPGKF were diminished in the presence of Pyk2 inhibitors or in Pyk2-deficient platelets, suggesting that Pyk2 plays a positive regulatory role in platelet functional responses. It has been shown that ADP-, but not thrombin-induced thromboxane (TxA2) generation depends on integrin signaling. Unlike ADP, thrombin activates G12/13 pathways, and G12/13 pathways can substitute for integrin signaling for TxA2 generation. We found that Pyk2 was activated downstream of both G12/13 and integrin-mediated pathways, and both 2-MeSADP- and AYPGKF-induced TxA2 generation was significantly diminished in Pyk2-deficient platelets. In addition, TxA2 generation induced by co-stimulation of Gi and Gz pathways, which is dependent on integrin signaling, was inhibited by blocking Pyk2. Furthermore, inhibition of 2-MeSADP-induced TxA2 generation by fibrinogen receptor antagonist was not rescued by co-stimulation of G12/13 pathways in the presence of Pyk2 inhibitor. We conclude that Pyk2 is a common signaling effector downstream of both G12/13 and integrin αIIbß3 signaling, which contributes to thromboxane generation.

A novel nuclear factor κB inhibitor, dehydroxymethylepoxyquinomicin, ameliorates puromycin aminonucleoside-induced nephrosis in mice.

BACKGROUND/AIMS: Minimal-change nephrotic syndrome (MCNS) is a kidney disease defined by selective proteinuria and hypoalbuminemia occurring in the absence of cellular glomerular infiltrates or immunoglobulin deposits. Recent observations suggest that nuclear factor κB (NF-κB) of podocyte is strongly associated with the development of proteinuria in MCNS. Dehydroxymethylepoxyquinomicin (DHMEQ) is a novel NF-κB inhibitor that potently inhibits DNA-binding activity of NF-κB, resulting in several therapeutic effects in various pathological conditions. We conducted this study to ask whether DHMEQ may ameliorate the nephrosis in mice induced by puromycin aminonucleoside (PAN), which is considered to be an animal model for MCNS. METHODS/RESULTS: Pretreatment with DHMEQ alleviated the proteinuria and reversed the serum abnormalities in mice nephrosis induced by 450 mg/kg of PAN. Increased serum interleukin-6 level in PAN-induced nephrosis was also completely suppressed by DHMEQ. Electron microscopic analyses of glo-meruli indicated that DHMEQ can inhibit the podocyte foot process effacement via blocking the translocation of podocyte NF-κB from cytoplasm to nucleus. CONCLUSIONS: These results suggest that DHMEQ can be a potential therapeutic agent for MCNS.

Neuroprotective effects of granulocyte colony-stimulating factor on ischemia-reperfusion injury of the retina.

PURPOSE: It has been reported that granulocyte colony-stimulating factor (G-CSF) provides neuroprotection in models in which neuronal cell death is induced. This research was designed to investigate the effects of G-CSF on neurodegeneration of the inner retinal layer in a rat model of ischemic reperfusion (I/R) injury. MATERIALS AND METHODS: Retinal ischemia was induced by increasing the intraocular pressure to 110 mm Hg for 45 min in the left eyes of the rats. A sham operation was carried out on the right eyes. G-CSF (100 µg/kg/day in 0.3 ml saline) or the same volume of saline was intraperitoneally injected just before the operation and continued for 4 consecutive days (a total of 5 consecutive days). Morphological examinations, including the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, were performed 7 days after I/R induction. The expression of phosphorylated AKT in the retina was examined by Western blot analysis and immunohistochemistry. RESULTS: Cell loss in the ganglion cell layer was more significantly reduced in the I/R-induced eyes of the G-CSF-injected rats than in the I/R-induced eyes of the saline-injected rats (20.3 vs. 6.6%). The inner retinal thickness ratios, such as the inner plexiform layer to the inner limiting membrane/outer nuclear layer and the inner nuclear layer/outer nuclear layer, were significantly better preserved in the I/R-induced eyes of the G-CSF-injected rats than in the I/R-induced eyes of the saline-injected rats. TUNEL assays showed fewer apoptotic cells in the retinal sections of the I/R-induced eyes of the G-CSF-injected rats. The phosphorylation of AKT (p-AKT/AKT) was upregulated in the retinas of the I/R-induced eyes of the G-CSF-injected rats. CONCLUSION: Our results demonstrated that systemic injection of G-CSF can protect retinal ganglion cells and inner retinal layers from I/R injury. The effects could be associated with the activation of AKT.

Macrophages play a unique role in the plaque calcification by enhancing the osteogenic signals exerted by vascular smooth muscle cells.

Vascular calcification is a major risk factor for the cardiovascular disease, yet its underlying molecular mechanisms remain to be elucidated. Recently, we identified that osteogenic signals via bone morphogenetic protein (BMP)-2 exerted by vascular smooth muscle cells (VSMCs) play a crucial role in the formation of atherosclerotic plaque calcification. Here we report a synergistic interaction between macrophages and VSMCs with respect to plaque calcification. Treatment with conditioned medium (CM) of macrophages dramatically enhanced BMP-2 expression in VSMCs, while it substantially reduced the expression of matrix Gla-protein (MGP) that inhibits the BMP-2 osteogenic signaling. As a result, macrophages significantly accelerated the osteoblastic differentiation of C2C12 cells induced by VSMC-CM. In contrast, macrophage-CM did not enhance the osteoblastic gene expressions in VSMCs, indicating that macrophages unlikely induced the osteoblastic trans-differentiation of VSMCs. We then examined the effect of recombinant TNF-α and IL-1ß on the VSMC-derived osteogenic signals. Similar to the macrophage-CM, both cytokines enhanced BMP-2 expression and reduced MGP expression in VSMCs. Nevertheless, only the neutralization of TNF-α but not IL-1ß attenuated the effect of macrophage-CM on the expression of these genes in VSMCs, due to the very low concentration of IL-1ß in the macrophage-CM. On the other hand, VSMCs significantly enhanced IL-1ß expression in macrophages, which might in turn accelerate the VSMC-mediated osteogenic signals. Together, we identified a unique role of macrophages in the formation of plaque calcification in coordination with VSMCs. This interaction between macrophages and VSMCs is a potential therapeutic target to treat and prevent the atherosclerotic plaque calcification.

p53-TIGAR axis attenuates mitophagy to exacerbate cardiac damage after ischemia.

Inhibition of tumor suppressor p53 is cardioprotective against ischemic injury and provides resistance to subsequent cardiac remodeling. We investigated p53-mediated expansion of ischemic damage with a focus on mitochondrial integrity in association with autophagy and apoptosis. p53(-/-) heart showed that autophagic flux was promoted under ischemia without a change in cardiac tissue ATP content. Electron micrographs revealed that ischemic border zone in p53(-/-) mice had 5-fold greater numbers of autophagic vacuoles containing mitochondria, indicating the occurrence of mitophagy, with an apparent reduction of abnormal mitochondria compared with those in WT mice. Analysis of autophagic mediators acting downstream of p53 revealed that TIGAR (TP53-induced glycolysis and apoptosis regulator) was exclusively up-regulated in ischemic myocardium. TIGAR(-/-) mice exhibited the promotion of mitophagy followed by decrease of abnormal mitochondria and resistance to ischemic injury, consistent with the phenotype of p53(-/-) mice. In p53(-/-) and TIGAR(-/-) ischemic myocardium, ROS production was elevated and followed by Bnip3 activation which is an initiator of mitophagy. Furthermore, the activation of Bnip3 and mitophagy due to p53/TIGAR inhibition were reversed with antioxidant N-acetyl-cysteine, indicating that this adaptive response requires ROS signal. Inhibition of mitophagy using chloroquine in p53(-/-) or TIGAR(-/-) mice exacerbated accumulation of damaged mitochondria to the level of wild-type mice and attenuated cardioprotective action. These findings indicate that p53/TIGAR-mediated inhibition of myocyte mitophagy is responsible for impairment of mitochondrial integrity and subsequent apoptosis, the process of which is closely involved in p53-mediated ventricular remodeling after myocardial infarction.

Role and regulation of phosphatidylinositol 3-kinase ß in platelet integrin α2ß1 signaling.

Integrin α2ß1-mediated adhesion of human platelets to monomeric type I collagen or to the GFOGER peptide caused a time-dependent activation of PI3K and Akt phosphorylation. This process was abrogated by pharmacologic inhibition of PI3Kß, but not of PI3Kγ or PI3Kα. Moreover, Akt phosphorylation was undetectable in murine platelets expressing a kinase-dead mutant of PI3Kß (PI3Kß(KD)), but occurred normally in PI3Kγ(KD) platelets. Integrin α2ß1 failed to stimulate PI3Kß in platelets from phospholipase Cγ2 (PLCγ2)-knockout mice, and we found that intracellular Ca(2+) linked PLCγ2 to PI3Kß activation. Integrin α2ß1 also caused a time-dependent stimulation of the focal kinase Pyk2 downstream of PLCγ2 and intracellular Ca(2+). Whereas activation of Pyk2 occurred normally in PI3Kß(KD) platelets, stimulation of PI3Kß was strongly reduced in Pyk2-knockout mice. Neither Pyk2 nor PI3Kß was required for α2ß1-mediated adhesion and spreading. However, activation of Rap1b and inside-out stimulation of integrin αIIbß3 were reduced after inhibition of PI3Kß and were significantly impaired in Pyk2-deficient platelets. Finally, both PI3Kß and Pyk2 significantly contributed to thrombus formation under flow. These results demonstrate that Pyk2 regulates PI3Kß downstream of integrin α2ß1, and document a novel role for Pyk2 and PI3Kß in integrin α2ß1 promoted inside-out activation of integrin αIIbß3 and thrombus formation.

p53 promotes cardiac dysfunction in diabetic mellitus caused by excessive mitochondrial respiration-mediated reactive oxygen species generation and lipid accumulation.

BACKGROUND: Diabetic cardiomyopathy is characterized by energetic dysregulation caused by glucotoxicity, lipotoxicity, and mitochondrial alterations. p53 and its downstream mitochondrial assembly protein, synthesis of cytochrome c oxidase 2 (SCO2), are important regulators of mitochondrial respiration, whereas the involvement in diabetic cardiomyopathy remains to be determined. METHODS AND RESULTS: The role of p53 and SCO2 in energy metabolism was examined in both type I (streptozotocin [STZ] administration) and type II diabetic (db/db) mice. Cardiac expressions of p53 and SCO2 in 4-week STZ diabetic mice were upregulated (185% and 152% versus controls, respectively, P<0.01), with a marked decrease in cardiac performance. Mitochondrial oxygen consumption was increased (136% versus control, P<0.01) in parallel with augmentation of mitochondrial cytochrome c oxidase (complex IV) activity. Reactive oxygen species (ROS)-damaged myocytes and lipid accumulation were increased in association with membrane-localization of fatty acid translocase protein FAT/CD36. Antioxidant tempol reduced the increased expressions of p53 and SCO2 in STZ-diabetic hearts and normalized alterations in mitochondrial oxygen consumption, lipid accumulation, and cardiac dysfunction. Similar results were observed in db/db mice, whereas in p53-deficient or SCO2-deficient diabetic mice, the cardiac and metabolic abnormalities were prevented. Overexpression of SCO2 in cardiac myocytes increased mitochondrial ROS and fatty acid accumulation, whereas knockdown of SCO2 ameliorated them. CONCLUSIONS: Myocardial p53/SCO2 signal is activated by diabetes-mediated ROS generation to increase mitochondrial oxygen consumption, resulting in excessive generation of mitochondria-derived ROS and lipid accumulation in association with cardiac dysfunction.

The kinase Pyk2 is involved in renal fibrosis by means of mechanical stretch-induced growth factor expression in renal tubules.

Unilateral ureteral obstruction is a well-established experimental model of progressive renal fibrosis. We tested whether mechanical stretch and subsequent renal tubular distension might lead to renal fibrosis by first studying renal tubular epithelial cells in culture. We found that mechanical stretch induced reactive oxygen species that in turn activated the cytoplasmic proline-rich tyrosine kinase-2 (Pyk2). This kinase is abundantly expressed in tubular epithelial cells where it is activated by several stimuli. Using mice with deletion of Pyk2 we found that the expression of transforming growth factor-ß1 induced by mechanical stretch in renal tubular epithelial cells was significantly reduced. The expression of connective tissue growth factor was also reduced in the Pyk2(-/-) mice. We also found that expression of connective tissue growth factor was independent of transforming growth factor-ß1, but dependent on the Rho-associated coiled-coil forming protein kinase pathway. Thus, Pyk2 may be an important initiating factor in renal fibrosis and might be a new therapeutic target for ameliorating renal fibrosis.

Loss of bcl-2 during the senescence exacerbates the impaired angiogenic functions in endothelial cells by deteriorating the mitochondrial redox state.

Ageing is an important risk factor for ischemic cardiovascular diseases, although its underlying molecular mechanisms remain to be elucidated. Here, we report a crucial role of Bcl-2 in the impaired angiogenic functions in senescent endothelial cells (ECs) by modulating the mitochondrial redox state. Cellular senescence impaired angiogenic functions in ECs without attenuating the mitogen-activated protein kinase or Akt signaling, and vascular endothelial growth factor receptor 2 or Tie-2 expressions. We identified that Bcl-2 expression was markedly reduced in 3 independent models for senescent ECs, and pharmacological inhibition, as well as small interfering RNA-mediated gene silencing of Bcl-2, significantly impaired the angiogenic functions in young ECs. Bcl-2 has an antioxidative role by locating the glutathione at mitochondria, and we found that mitochondrial oxidative stress was significantly augmented in senescent ECs, in association with reduced mitochondria-associated glutathione. Transfection of Bcl-2 in senescent ECs significantly reduced the mitochondrial oxidative stress, restored the mitochondrial membrane potential, and improved the angiogenic capacity. Furthermore, gene transfer of Bcl-2 using adenovirus significantly improved the in vivo angiogenesis in the Matrigel plugs implanted into aged mice, whereas the Bcl-2 inhibitor reduced the angiogenesis in the Matrigel plugs implanted into young mice. Together, Bcl-2 plays a crucial role in the regulation of the mitochondrial redox state in ECs, and, thus, loss of Bcl-2 during the senescence exacerbates the impaired angiogenesis by augmenting the mitochondrial oxidative stress.

Apoptosis regulator through modulating IAP expression (ARIA) controls the PI3K/Akt pathway in endothelial and endothelial progenitor cells.

Endothelial and endothelial progenitor cells (ECs and EPCs) play a fundamental role in angiogenesis that is essential for numerous physiological and pathological processes. The phosphatase and tensin homolog (PTEN)/ phosphoinositide 3-kinase (PI3K) pathway has been implicated in angiogenesis, but the mechanism in the regulation of this pathway in ECs and EPCs is poorly understood. Here we show that ARIA (apoptosis regulator through modulating IAP expression), a transmembrane protein that we recently identified, regulates the PTEN/PI3K pathway in ECs and EPCs and controls developmental and postnatal angiogenesis in vivo. We found that ARIA is abundantly expressed in EPCs and regulates their angiogenic functions by modulating PI3K/Akt/endothelial nitric oxide synthase (eNOS) signaling. Genetic deletion of ARIA caused nonfatal bleeding during embryogenesis, in association with increased small vessel density and altered expression of various vascular growth factors including angiopoietins and VEGF receptors. Postnatal neovascularization induced by critical limb ischemia was substantially enhanced in ARIA-null mice, in conjunction with more bone marrow (BM)-derived ECs detected in ischemic muscles. Administration of PI3K or NO synthase inhibitor completely abolished the enhanced neovascularization in ARIA(-/-) mice. Mechanistically, we identified that ARIA interacts with PTEN at the intracellular domain independently of the PTEN phosphorylation in its C-terminal tail. Overexpressed ARIA increased PTEN in the membrane fraction, whereas ARIA-silencing reduced the membrane-associated PTEN, resulting in modified PI3K/Akt signaling. Taken together, our findings establish a previously undescribed mode of regulation of the PTEN/PI3K/Akt pathway by ARIA, and reveal a unique mechanism in the control of angiogenesis. These functions of ARIA might offer a unique therapeutic potential.

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.

p53 and TIGAR regulate cardiac myocyte energy homeostasis under hypoxic stress.

Bioenergetic homeostasis is altered in heart failure and may play an important role in pathogenesis. p53 has been implicated in heart failure, and although its role in regulating tumorigenesis is well characterized, its activities on cellular metabolism are just beginning to be understood. We investigated the role of p53 and its transcriptional target gene TP53-induced glycolysis and apoptosis regulator (TIGAR) in myocardial energy metabolism under conditions simulating ischemia that can lead to heart failure. Expression of p53 and TIGAR was markedly upregulated after myocardial infarction, and apoptotic myocytes were decreased by 42% in p53-deficient mouse hearts compared with those in wild-type mice. To examine the effect of p53 on energy metabolism, cardiac myocytes were exposed to hypoxia. Hypoxia induced p53 and TIGAR expression in a p53-dependent manner. Knockdown of p53 or TIGAR increased glycolysis with elevated fructose-2,6-bisphosphate levels and reduced myocyte apoptosis. Hypoxic stress decreased phosphocreatine content and the mitochondrial membrane potential of myocytes without changes in ATP content, the effects of which were prevented by the knockdown of TIGAR. Inhibition of glycolysis by 2-deoxyglucose blocked these bioenergetic effects and TIGAR siRNA-mediated prevention of apoptosis, and, in contrast, overexpression of TIGAR reduced glucose utilization and increased apoptosis. Our data demonstrate that p53 and TIGAR inhibit glycolysis in hypoxic myocytes and that inhibition of glycolysis is closely involved in apoptosis, suggesting that p53 and TIGAR are significant mediators of cellular energy homeostasis and cell death under ischemic stress.

Paracrine osteogenic signals via bone morphogenetic protein-2 accelerate the atherosclerotic intimal calcification in vivo.

OBJECTIVE: Vascular calcification is an important risk factor for cardiovascular diseases. Here, we investigated a role of dedifferentiated vascular smooth muscle cells (VSMCs) in the atherosclerotic intimal calcification. METHODS AND RESULTS: We prepared human cultured VSMCs in either redifferentiatiated or dedifferentiated state and analyzed the gene expressions of bone-calcification regulatory factors. Expression of bone morphogenetic protein-2 (BMP-2), a potent initiator for osteoblast differentiation, was significantly enhanced in dedifferentiated VSMCs. Furthermore, endogenous BMP-2 antagonists, such as noggin, chordin, and matrix gamma-carboxyglutamic acid protein, were all downregulated in the dedifferentiated VSMCs. Conditioned medium from dedifferentiated VSMCs, but not from redifferentiated VSMCs, stimulated the osteoblastic differentiation of the mesenchymal progenitor C2C12 cells, which was abolished by BMP-2 knockdown. In atherosclerotic intima from apolipoprotein (apo)E-deficient mice, αSM-actin-positive cells, presumably dedifferentiated VSMCs, expressed BMP-2. We generated BMP-2-transgenic mice using αSM-actin promoter and crossed them with apoE-deficient mice (BMP-2-transgenic/apoE-knockout). Significantly accelerated atherosclerotic intimal calcification was detected in BMP-2-transgenic/apoE-knockout mice, although serum lipid concentration and atherosclerotic plaque size were not different from those in apoE-knockout mice. Enhanced calcification appeared to be associated with the frequent emergence of osteoblast-like cells in atherosclerotic intima in BMP-2-transgenic/apoE-knockout mice. CONCLUSIONS: Our findings collectively demonstrate an important role of dedifferentiated VSMCs in the pathophysiology of atherosclerotic calcification through activating paracrine BMP-2 osteogenic signals.

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.