Yoichi Takami

Novel Autologous Cell Therapy in Ischemic Limb Disease Through Growth Factor Secretion by Cultured Adipose Tissue–Derived Stromal Cells

Objective—The delivery of autologous progenitor cells into ischemic tissue of patients is emerging as a novel therapeutic option. Here, we report the potential impact of cultured adipose tissue–derived cells (ADSC) on angiogenic cell therapy. Method and Results—ADSC were isolated from C57Bl/6 mouse inguinal adipose tissue and showed high expression of ScaI and CD44, but not c-kit, Lin, CD34, CD45, CD11b, and CD31, compatible with that of mesenchymal stem cells from bone marrow. In coculture conditions with ADSC and human aortic endothelial cells (ECs) under treatment with growth factors, ADSC significantly increased EC viability, migration and tube formation mainly through secretion of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). At 4 weeks after transplantation of ADSC into the ischemic mouse hindlimb, the angiogenic scores were improved in the ADSC-treated group, which were evaluated with blood flow by laser Doppler imaging (LDI) and capillary density by immunostaining with anti-CD31 antibody. However, injected ADSC did not correspond to CD31, von Willebrand factor, and &agr;-smooth muscle actin-positive cells in ischemic tissue. Conclusion—These adipose tissue–derived cells demonstrated potential as angiogenic cell therapy for ischemic disease, which appears to be mainly achieved by their ability to secrete angiogenic growth factors.

Ubiquitin Carboxyl-Terminal Hydrolase L1, a Novel Deubiquitinating Enzyme in the Vasculature, Attenuates NF-κB Activation

Objective— We identified a ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) gene, which encodes a deubiquitinating enzyme and is expressed in the vasculature, by functional screening of a human endothelial cell (EC) cDNA library. UCHL1 is expressed in neurons, and abnormalities in UCHL1 are responsible for inherited Parkinson’s disease via its effects on the ubiquitin-proteasome system. Therefore, the goal of present study was to clarify the role of the UCHL1 gene in vascular remodeling by evaluating nuclear factor-&kgr;B (NF-&kgr;B) inactivation in ECs and vascular smooth muscle cells (VSMCs). Methods and Results— From Northern blot and immunohistochemical analysis, the UCHL1 gene was endogenously expressed in vascular ECs, VSMCs, and brain tissue. Expression of UCHL1 was markedly increased in the neointima of the balloon-injured carotid artery and was also present in atherosclerotic lesions from human carotid arteries. Overexpression of the UCHL1 gene significantly attenuated tumor necrosis factor (TNF)-α–induced NF-&kgr;B activity in vascular cells and increased inhibitor of kappa B-α (I&kgr;B-α), possibly through the attenuation of I&kgr;B-α ubiquitination, leading to decreased neointima in the balloon-injured artery. In contrast, knockdown of UCHL1 by small interfering RNA resulted in increased NF-&kgr;B activity in VSMCs. Conclusions— These data suggest that UCHL1 may partially attenuate vascular remodeling through inhibition of NF-&kgr;B activity.

Loss of ACE2 Exaggerates High-Calorie Diet–Induced Insulin Resistance by Reduction of GLUT4 in Mice

ACE type 2 (ACE2) functions as a negative regulator of the renin-angiotensin system by cleaving angiotensin II (AII) into angiotensin 1–7 (A1–7). This study assessed the role of endogenous ACE2 in maintaining insulin sensitivity. Twelve-week-old male ACE2 knockout (ACE2KO) mice had normal insulin sensitivities when fed a standard diet. AII infusion or a high-fat, high-sucrose (HFHS) diet impaired glucose tolerance and insulin sensitivity more severely in ACE2KO mice than in their wild-type (WT) littermates. The strain difference in glucose tolerance was not eliminated by an AII receptor type 1 (AT1) blocker but was eradicated by A1–7 or an AT1 blocker combined with the A1–7 inhibitor (A779). The expression of GLUT4 and a transcriptional factor, myocyte enhancer factor (MEF) 2A, was dramatically reduced in the skeletal muscles of the standard diet–fed ACE2KO mice. The expression of GLUT4 and MEF2A was increased by A1–7 in ACE2KO mice and decreased by A779 in WT mice. A1–7 enhanced upregulation of MEF2A and GLUT4 during differentiation of myoblast cells. In conclusion, ACE2 protects against high-calorie diet–induced insulin resistance in mice. This mechanism may involve the transcriptional regulation of GLUT4 via an A1–7–dependent pathway.

Vascular protective effects of ezetimibe in ApoE-deficient mice

One of the major risk factors for ischemic disease is hyperlipidemia, which is mainly regulated by endogenous cholesterol synthesis in the liver and dietary absorption in the small intestine. In this study, we evaluated the vascular protective effects of a potent cholesterol absorption inhibitor, ezetimibe. ApoE-deficient mice were fed a chow or high-fat diet with or without ezetimibe (5mg/(kgday)) for 3 months. Co-treatment with ezetimibe significantly reduced plasma cholesterol (by 76%; from 1592 to 381mg/dL) and LDL cholesterol (by 78%; from 1515 to 319mg/dL), and increased HDL cholesterol (by 187%; from 16 to 46mg/dL) in high-fat diet mice. Consistently, a marked inhibitory effect of ezetimibe on the development of lipid-rich plaque was observed, as assessed by oil red O staining. Of importance, treatment with ezetimibe significantly improved endothelial dysfunction as assessed by the vasodilator response to acetylcholine, accompanied by inhibition of interleukin-6 mRNA and an increase in endothelial nitric oxide synthase (eNOS) mRNA in the aorta. Ezetimibe also suppressed oxidative stress and the ubiquitination-proteasome system in the aorta. Although changes in body weight and several tissue weights were similar in the groups with and without ezetimibe administration, only liver weight was significantly decreased in the ezetimibe-treated group. Interestingly, ezetimibe markedly inhibited lipid accumulation in the liver. Furthermore, ezetimibe increased the mRNA expression of 3-hydroxy-3-methylglutaryl co-enzyme A (HMG-CoA) synthase as a counteraction in the liver, but not in the aorta. Overall, ezetimibe significantly prevented atherosclerosis through not only lipid-lowering effects, but also other direct and/or indirect vascular protective actions in ApoE-deficient mice.

Zyxin Mediates Actin Fiber Reorganization in Epithelial–Mesenchymal Transition and Contributes to Endocardial Morphogenesis

Epithelial-mesenchymal transition (EMT) confers destabilization of cell-cell adhesion and cell motility required for morphogenesis or cancer metastasis. Here we report that zyxin, a focal adhesion-associated LIM protein, is essential for actin reorganization for cell migration in TGF-beta1-induced EMT in normal murine mammary gland (NMuMG) cells. TGF-beta1 induced the relocation of zyxin from focal adhesions to actin fibers. In addition, TGF-beta1 up-regulated zyxin via a transcription factor, Twist1. Depletion of either zyxin or Twist1 abrogated the TGF-beta1-dependent EMT, including enhanced cell motility and actin reorganization, indicating the TGF-beta1-Twist1-zyxin signal for EMT. Both zyxin and Twist1 were predominantly expressed in the cardiac atrioventricular canal (AVC) that undergoes EMT during heart development. We further performed ex vivo AVC explant assay and revealed that zyxin was required for the reorganization of actin fibers and migration of the endocardial cells. Thus, zyxin reorganizes actin fibers and enhances cell motility in response to TGF-beta1, thereby regulating EMT.

Differences between daytime and nighttime blood pressure variability regarding systemic atherosclerotic change and renal function

Recently, new parameters related to hypertension, such as variability in blood pressure and ambulatory arterial stiffness index (AASI), were demonstrated to correlate with arteriosclerotic change. In this study, we investigated the correlation between circadian variability in blood pressure/AASI and renal function. We also investigated differences in the clinical impact of 24 h, daytime and nighttime blood pressure variability on renal and systemic atherosclerotic changes. We analyzed data from 120 patients who underwent renal Doppler ultrasonography (RDU) and ambulatory blood pressure monitoring (ABPM) at our hospital ward, and investigated the correlation between circadian variability in blood pressure/AASI and renal function, including resistive index (RI) evaluated with RDU, which is thought to be a good indicator of renal vascular resistance. Subjects with higher circadian variability in systolic blood pressure (SBP) had significantly higher RI. Daytime variability in SBP correlated more strongly with RI than nighttime variability. Meanwhile, only nighttime variability, but not daytime variability, in SBP was related to carotid atherosclerosis. Similarly, AASI was significantly correlated with RI. Circadian variability in SBP and AASI were both significantly correlated with renal function. Daytime SBP s.d. was especially more strongly correlated with renal vascular resistance, and nighttime SBP s.d. was significantly correlated with intima-media thickness (IMT) and plaque score. These results indicate that evaluating both daytime and nighttime blood pressure variability enables an assessment of pathological conditions in hypertensive patients to prevent cardiovascular diseases.

FHL-2 Suppresses VEGF-Induced Phosphatidylinositol 3-Kinase/Akt Activation via Interaction With Sphingosine Kinase-1

Objective—In the functional screening of a human heart cDNA library to identify a novel antiangiogenic factor, the prime candidate gene was “four-and-a-half LIM only protein-2” (FHL-2). The goal of this study is to clear the mechanism of antiangiogenic signaling of FHL-2 in endothelial cells (ECs). Methods and Results—Overexpressed FHL-2 strongly inhibited vascular endothelial growth factor (VEGF)-induced EC migration. In the angiogenic signaling, we focused on sphingosine kinase-1 (SK1), which produces sphingosine-1-phosphate (S1P), a bioactive sphingolipid, as a potent angiogenic mediator in ECs. Immunoprecipitation and immunostaining analysis showed that FHL-2 might bind to SK1. Importantly, overexpression of FHL-2 in ECs inhibited VEGF-induced SK1 activity, phosphatidylinositol 3-kinase activity, and phosphorylation of Akt and eNOS. In contrast, overexpression of FHL-2 had no effect on S1P-induced Akt phosphorylation. Interestingly, VEGF stimulation decreased the binding of FHL-2 and SK1. Depletion of FHL-2 by siRNA increased EC migration accompanied with SK1 and Akt activation, and increased the expression of VEGF receptor-2 which further enhanced VEGF signaling. Furthermore, injection of FHL-2 mRNA into Xenopus embryos resulted in inhibition of vascular network development, assessed by in situ hybridization with endothelial markers. Conclusions—FHL-2 may regulate phosphatidylinositol 3-kinase/Akt via direct suppression of the SK1-S1P pathway in ECs.

Potential Role of CYLD (Cylindromatosis) as a Deubiquitinating Enzyme in Vascular Cells

Data from several studies suggest that the ubiquitin-proteasome system may play a role in the progression of atherosclerosis. Here, we examined the potential role of the deubiquitinating enzyme CYLD (cylindromatosis), mutation of which has been reported to cause familial cylindromatosis. Northern blot analysis revealed expression of CYLD mRNA in the aorta, as well as in cultured human aortic endothelial cells (ECs) and vascular smooth muscle cells. Treatment with recombinant tumor necrosis factor (TNF)-alpha significantly increased CYLD expression in ECs and vascular smooth muscle cells. Immunostaining showed CYLD expression in atherosclerotic lesions from human carotid arteries and up-regulation of CYLD expression in the neointima of rat carotid arteries after balloon injury. Overexpression of CYLD in ECs resulted in inhibition of TNF-alpha-induced nuclear factor-kappaB activity through deubiquitination of TNFR-associated factor 2 (TRAF2), whereas overexpression of catalytically inactive CYLD had no effect. CYLD overexpression also inhibited expression of cyclin D1 and activation of the E2F pathway through deubiquitination of the upstream molecule Bcl-3 and inhibition of its translocation into the nucleus. Overexpressed CYLD also significantly inhibited cell viability. Furthermore, overexpression of CYLD in rat balloon-injured carotid artery attenuated neointimal formation through inactivation of nuclear factor-kappaB and E2F. In conclusion, these data demonstrate that the deubiquitinating enzyme CYLD may inhibit inflammation and proliferation in vascular cells and may represent a novel target for the treatment or prevention of atherosclerosis.

Kruppel-like factor 2 is a transcriptional regulator of chronic and acute inflammation.

Although myeloid cell activation is requisite for an optimal innate immune response, this process must be tightly controlled to prevent collateral host tissue damage. Kruppel-like factor 2 (KLF2) is a potent regulator of myeloid cell proinflammatory activation. As an approximately 30% to 50% reduction in KLF2 levels has been observed in human subjects with acute or chronic inflammatory disorders, we studied the biological response to inflammation in KLF2(+/-) mice. Herein, we show that partial deficiency of KLF2 modulates the in vivo response to acute (sepsis) and subacute (skin) inflammatory challenge. Mechanistically, we link the anti-inflammatory effects of KLF2 to the inhibition of NF-κB transcriptional activity. Collectively, the observations provide biologically relevant insights into KLF2-mediated modulation of these inflammatory processes that could potentially be manipulated for therapeutic gain.

Oxidized LDL (oxLDL) activates the angiotensin II type 1 receptor by binding to the lectin-like oxLDL receptor

The angiotensin II type 1 receptor (AT1) is a 7‐transmembrane domain GPCR that when activated by its ligand angiotensin II, generates signaling events promoting vascular dysfunction and the development of cardiovascular disease. Here, we show that the single‐transmembrane oxidized LDL (oxLDL) receptor (LOX‐1) resides in proximity to AT1 on cell‐surface membranes and that binding of oxLDL to LOX‐1 can allosterically activate AT1‐dependent signaling events. oxLDL‐induced signaling events in human vascular endothelial cells were abolished by knockdown of AT1 and inhibited by AT1 blockade (ARB). oxLDL increased cytosolic G protein by 350% in Chinese hamster ovary (CHO) cells with genetically induced expression of AT1 and LOX‐1, whereas little increase was observed in CHO cells expressing only LOX‐1. Immunoprecipitation and in situ proximity ligation assay (PLA) assays in CHO cells revealed the presence of cell‐surface complexes involving LOX‐1 and AT1. Chimeric analysis showed that oxLDLinduced AT1 signaling events are mediated via interactions between the intracellular domain of LOX‐1 and AT1 that activate AT1. oxLDL induced impairment of endothelium‐dependent vascular relaxation of vascular ring from mouse thoracic aorta was abolished by ARB or genetic deletion of AT1. These findings reveal a novel pathway for AT1 activation and suggest a new mechanism whereby oxLDL may be promoting risk for cardiovascular disease.—Yamamoto, K., Kakino, A., Takeshita, H., Hayashi, N., Li, L., Nakano, A., Hanasaki‐Yamamoto, H., Fujita, Y., Imaizumi, Y., Toyama‐Yokoyama, S., Nakama, C., Kawai, T., Takeda, M., Hongyo, K., Oguro, R., Maekawa, Y., Itoh, N., Takami, Y., Onishi, M., Takeya, Y., Sugimoto, K., Kamide, K., Nakagami, H., Ohishi, M., Kurtz, T. W., Sawamura, T., Rakugi, H. Oxidized LDL (oxLDL) activates the angiotensin II type 1 receptor by binding to the lectin‐like oxLDL receptor. FASEB J. 29, 3342‐3356 (2015). www.fasebj.org