tbuckley/cabot-search huggingface dataset:
Abstract 5593: Toll-like Receptor 2/6 Stimulation Promotes Angiogenesis via Induction of Endothelial GM-CSF Expression
Authors: Karsten Grote, Harald Schuett, Gustavo Salguero, Peter Mühlradt, Bernhard Schieffer
Journal: Circulation (2009)

Background: Toll-like receptors (TLRs) are known as pathogen recognition receptors of the innate immunity, initiating inflammatory pathways to organize the immune defense. More recently, an involvement of TLRs in various physiological and pathological processes has been reported. Since many of these processes implicate angiogenesis, we here elucidated the role of a TLR2/6-dependent pathway on angiogenesis using the specific TLR2/6 agonist MALP-2, a common bacterial lipopeptide. Methods and Results: MALP-2 induced angiogenesis in vivo (Matrigel implants, hemoglobin content, H&amp;E staining, P&lt;0.01, n=4–8) and tube formation in vitro (Matrigel, P&lt;0.05, n=3–5) which could be inhibited with neutralizing antibodies against TLR2/6. Moreover, endothelial cells responded to MALP-2 with enhanced proliferation and migration (BrdU-incorporation, transwell, P&lt;0.01, n=5–7). Protein array revealed a strong secretion of granulocyte-macrophage colony stimulating factor (GM-CSF) from endothelial cells which was confirmed by ELISA (P&lt;0.01, n=4) and could be inhibited with antibodies against TLR2/6. By contrast, smooth muscle cells and fibroblasts did not secrete GM-CSF in response to MALP-2 (ELISA, n=3–6). In addition, GM-CSF release from isolated vascular segments following MALP-2 stimulation was completely prevented when the endothelium was removed mechanically (ELISA, P&lt;0.01, n=4). Capillaries generated by MALP-2 treatment contained erythrocytes as well as CD45 + cells (Matrigel implants, CD45 immunostaining, n=4). Following MALP-2 stimulation CD45 + cells showed likewise enhanced migration (transwell, P&lt;0.05, n=5) and GM-CSF release (ELISA, P&lt;0.01, n=5) however to a lower extent compared to endothelial cells. Finally, inhibition of GM-CSF by siRNA in vitro (Matrigel, P&lt;0.05, n=4) or by antibodies in vivo (Matrigel implants, P&lt;0.01, n=4–8) inhibited the MALP-2 induced angiogenesis. Conclusion: These results demonstrate a TLR2/6-dependent induction of angiogenesis by the bacterial lipopeptide MALP-2 which is mediated by GM-CSF. This might represent a general mechanism to enhance or restore blood flow and recruit immune cells for pathogen defense and tissue regeneration.
Abstract 5556: Immunoglobulin-like Cell Adhesion Molecule Necl-5 Regulates Vascular Endothelial Growth Factor-Induced Angiogenesis by Controlling the Interaction Between VEGFR2 and Integrin <i>α</i> v <i>β</i> 3
Authors: Mitsuo Kinugasa, Yoshiyuki Rikitake, Hisayuki Amano, Seimi Satomi-Kobayashi, Hideto Tawa, Fumie Kureha, Yusuke Kurogane, Yuichi Nagamatsu, Jun Miyoshi, Yoshimi Takai, Ken-ichi Hirata
Journal: Circulation (2009)

Background Peripheral arterial disease (PAD) is highly prevalent worldwide. PAD is a major cause of lower limb amputation, which is associated with a poor quality of life and prognosis. Vascular endothelial growth factor (VEGF) is a key pro-angiogenic cytokine proposed as therapeutic angiogenesis for PAD. However, more investigation is needed to better understand VEGF-induced angiogenesis and its application to PAD. The activity of VEGF receptor 2 (VEGFR2) is regulated by direct interactions with VEGF and other cell surface proteins, including integrin α v β 3. However, the regulation mechanism of VEGFR2 and integrin α v β 3 interaction has not been established. Nectin-like molecules (Necls) are immunoglobulin-like cell adhesion molecules (CAMs) that regulate cell movement, proliferation and survival. Here we show that Necl-5, a member of Necls that binds integrin α v β 3, regulates the interaction between VEGFR2 and integrin α v β 3 in vascular endothelial cells (ECs) and that this CAM plays an important role in angiogenesis. Methods and Results Immunohistochemistry revealed that Necl-5 was preferentially expressed in ECs including mouse hind limb capillary endothelium. Flow cytometry showed that Necl-5 was expressed in various cultured ECs. Although Necl-5-knockout mice displayed no obvious defect in vascular development, these mice exhibited impaired adaptive angiogenesis following hind limb ischemia. To clarify the regulation mechanism of angiogenesis by Necl-5, we knocked down Necl-5 by siRNA and studied the role of Necl-5 in angiogenesis in vitro . VEGF-induced tube formation on Matrigel, migration, and proliferation were significantly inhibited in Necl-5-knockdown ECs. Conversely, serum deprivation-induced apoptosis was enhanced in Necl-5-knockdown ECs. Knockdown of Necl-5 prevented VEGF-induced interaction of integrin α v β 3 with VEGFR2, and then suppressed the VEGFR2-mediated activation of Akt, a critical signal for angiogenesis and cell survival. Conclusions These findings indicate that Necl-5 regulates VEGF-induced angiogenesis by controlling the interaction between VEGFR2 and integrin α v β 3, and the VEGFR2-mediated signaling. Therapeutic intervention of Necl-5 may represent a novel strategy in the patients with PAD.
Abstract 5599: Secreted Frizzled Protein 2, a Cardioprotective Gene is Upregulated in Mesenchymal Stem Cells Following Engraftment in the Injured Myocardium
Authors: Claire Kavanagh, Garry P Duffy, Kaveh Mashayekhi, Timothy O’Brien, Frank Barry
Journal: Circulation (2009)

A variety of stem cells including Mesenchymal Stem Cells (MSCs) have been shown to have therapeutic potential in the treatment of myocardial damage. While the mechanism of action still remains unknown, MSCs have been shown to produce and secrete a broad variety of cytokines, chemokines and growth factors that may potentially be involved in cardiac repair. This has been confirmed by reports that human MSCs can exert favourable cardiac effects after myocardial infarction (MI) without persistence, engraftment, transdifferentation or cell fusion. Thus, the mechanisms responsible for their functional benefits appear to be attributable to paracrine effects. Secreted frizzled protein 2 (SFRP2) is expressed in MSCs following Akt gene transfer and has been shown to have paracrine protective effects. The objective of this study was to determine the effect of the ischemic environment on the engrafted MSC in order to understand the host - stem cell interaction. Using laser microdissection (LMD), the engrafted MSCs were retrieved from the injured myocardium 1 week post MI. To determine if SFRP2 was upregulated in our retrieved MSC population, RT PCR analysis of the retrieved MSCs Vs control MSCs was performed. SFRP2 was found to be 5,700 fold upregulated in MSCs exposed to the infarcted myocardium compared to control MSCs. Subsequent in vitro cardioprotection assays identified SFRP2 as a potent protector of cardiomyocytes during hypoxia treatment. Mitochondrial activity increased by 52% when neonatal rat cardiomyocytes were treated with SFRP2 recombinant protein during hypoxia treatment when compared to hypoxia alone. Caspase activity was also decreased in cardiomyocytes treated with SFRP2 during hypoxia. Cells also showed a healthy morphology and maintained beating after exposure to hypoxia. These data suggest that the injured host can induce MSCs to express cytoprotective factors such as SFRP2 and identify the host - stem cell interaction as an important determinant of the therapeutic effect of stem cell administration.
Abstract 5553: S100A1 Modulates Endothelial Nitric Oxide Availability by Co-ordinate Regulation of Nitric Oxide Synthase Phosphorylation and Gene Expression of Related Signaling Molecules
Authors: Krystyna Teichert-Kuliszewska, Jean-Francois Desjardins, Thomas G Parker
Journal: Circulation (2009)

Background: S100A1, a Ca 2+ -sensor protein in endothelial cell, regulates endothelial function. S100A1 knockout mice (KO) are hypertensive with impaired nitric oxide (NO) production and endothelial-dependent vasodilatation. We examine the impact of S100A1 on phosphorylation of Ser 1177 and Thr 495 , positive and negative regulatory residues, respectively, in endothelial nitric oxide synthase (eNOS) and on expression of diverse genes involved in eNOS signaling. Methods and Results: Endothelial cells (EC) were isolated and from pulmonary vasculature of wild-type (WT) and KO mice. WT EC exhibited higher proliferative rates than KO measured by WST-method, and expressed as absorbance/5000 cells/well (0.8923±0.091 vs. 0.268±0.055, p&lt;0.05, n=3) and higher NO production (2214.00±1016.17 vs. 566.75±243.79 nM, p&lt;0.05, n=6). Lower eNOS activity in KO was associated with higher and lower basal phosphorylation at Thr 495 and Ser 1177 , respectively. Bradykinin (100 nM) resulted in rapid (1 minute) 3-fold higher maximal phosphorylation of Ser 1177 in WT vs. KO with equivalent rapid dephosphorylation of Thr 495 in both cell types. By contrast, acetylcholine (10 μ M) at 30 seconds produced more robust dephosphorylation of Thr 495 in WT compared to KO, with comparable effects on Ser 1177 phosphorylation. In KO versus WT EC, PCR microarray analysis demonstrated greater than 2-fold increase in mRNA expression of caveolin-1, NADPH oxidase 1, glutathione peroxidase, and calmodulin-1. KO EC exhibited a 5-fold reduction in eNOS mRNA, confirmed by a reduction in eNOS protein (4.02±0.19 fold, n=3). Conclusion: Absence of S100A1 in endothelial cells results in impaired proliferation, decreased NO availability, and regulation of gene expression that contributes or, alternately, compensates for this phenotype. Lack of S100A1 also impairs co-ordinated eNOS phosphorylation at baseline, and in response to agonists, that augments NO production. Thus, S100A1 plays a multifactorial and novel role in controlling NO synthesis and the endothelial phenotype.
Abstract 5522: Improvement of Angiotensin-II-Induced Vascular Dysfunction and Oxidative Stress by the Organic Nitrate Pentaerithrityl Tetranitrate Depends on Heme Oxygenase-1-Induction
Authors: Philip Wenzel, Swenja Schuhmacher, Eberhard Schulz, Matthias Oelze, Jens Kamuf, Tommaso Gori, Marcus Hortmann, Aruni Bhatnagar, Andreas Daiber, Thomas Münzel
Journal: Circulation (2009)

Objective: Pentaerithrityl tetranitrate (PETN)-treatment is devoid of nitrate tolerance which has been attributed to the induction of the antioxidant enzyme heme oxygenase-1 (HO-1). With the present study we tested, whether chronic treatment with PETN can improve angiotensin-II (AT-II)-induced vascular oxidative stress and dysfunction. Methods and Results: In contrast to isosorbide mononitrate (ISMN, 75mg/kg/d/7d), co-treatment with PETN (15mg/kg/d/7d) improved impaired endothelial and smooth muscle function and normalized vascular and cardiac ROS production (mitochondria, NADPH oxidase activity and uncoupled endothelial nitric oxide synthase [eNOS]) as assessed by dihydroethidine (DHE)-staining, lucigenin-enhanced chemiluminescence and quantitative HPLC of DHE-oxidation products in AT-II (1mg/kg/d/7d) treated rats. The antioxidant features of PETN were recapulated in spontaneously hypertensive rats. In addition to increase in HO-1 protein expression, PETN but not ISMN augmented aortic protein levels of the tetrahydrobiopterin-synthesizing enzymes GTP-cyclohydrolase-I and dihydrofolate reductase in AT-II-treated rats, thereby preventing eNOS uncoupling. In AT-II-treated (0.1mg/kg/d/7d) HO-1+/− mice, PETN failed to be antioxidant, whereas HO-1 induction by hemin (25mg/kg) mimicked the PETN effect in AT-II-treated HO-1+/+ mice. Conclusions: PETN improves AT-II-induced vascular oxidative stress and dysfunction via induction of HO-1. Thus, PETN is the first organic nitrate to be beneficial in an experimental model of arterial hypertension.
Abstract 5503: Sonothrombolysis in a Rabbit Model of Acute Insoluble Ischemic Stroke
Authors: Rene Flores, John D Lowery, Leah J Hennings, Aliza T Brown, Robert D Skinner, William C Culp
Journal: Circulation (2009)

Introduction: Evidence suggests that low-frequency ultrasound (US) improves tissue perfusion in the rabbit ischemic limb and enhances myocardial perfusion even without removing the arterial obstruction. We evaluated US-facilitated thrombolysis, or sonothrombolysis, with microbubbles (MB) and tissue plasminogen activator (tPA) for decreases in infarct volume in rabbits with insoluble middle cerebral artery (MCA) occlusions. Hypothesis: Sonothrombolysis with MB or tPA is effective at reducing infarcts without lysis of arterial obstruction. Methods: New Zealand White rabbits (n = 34; 5.2±0.07 kg) received angiography and three embolic spheres (diameter = 700 to 900 μ m) were injected into the internal carotid artery occluding the MCA. Rabbits were randomly assigned to one of four groups. Control (n = 10) rabbits were embolized but received no US, tPA, or MB. Other groups were tPA (n = 7) without US, tPA+US (n = 9), and MB+US (n = 8). Treatment was initiated one hour following angiographic verification of occlusion. Rabbits with US received pulsed wave US (1 MHz at 0.8 W/cm 2 ) for 1 hour and rabbits with tPA received intravenous tPA (0.9 mg/kg) over 1 hour. Rabbits with MB received intravenous MB (0.16 mg/kg) over 30 minutes. Rabbits were sacrificed at 24 hours and infarct volume was determined using vital stains on brain sections. Results: Percent infarct volume averaged 3.4±0.9%, 2.0±1.1%, 1.8±1.0%, and 0.92±0.9% for control, tPA, MB+US, and tPA+US rabbits, respectively; infarct volume tended (P = 0.066) to be decreased for rabbits treated with tPA+US compared to controls. Infarct volume did not differ (P &gt; 0.26) between controls and rabbits treated with tPA and MB+US. Conclusions: Combined treatment with tPA and US shows a trend towards decreasing brain infarct volume in rabbits with permanent MCA occlusion, but not to the extent found in muscle or myocardium at lower frequencies.
Abstract 5555: Shear Stress Induced Activation of MAPK Pathway and GTPases of Ras and Rho Families in a Three-layered Vascular Substitute
Authors: Caroline Basoni, Marie-Christine Fiola, Valerie Cattan, Dan Lacroix, Francois A Auger
Journal: Circulation (2009)

The use of a three dimensional vascular substitute to study cell physiology is a helpful tool to understand endothelial responses when submitted to shear stress. Our group has pioneered such substitute by self-assembly, from the secretion of its own biological biomaterial, in which flow culture conditions help mimic vascular physiology. The characterization of this vascular substitute is important for clinical and research applications. In this study, we considered the cellular responses of endothelial cells in the presence of fluidic forces, and biomechanical properties of our substitute. The models were maintained in a hemodynamic bioreactor where a peristaltic pump created pulsatile flow within the lumen of the vascular model to generate shear stress and lateral pressure forces. Immunofluorescence observations revealed that endothelial cells, known to be shear stress sensors, expressed functional markers such as PECAM, VE-Cadherin and von Willebrand factor. Those observations also showed that endothelial cells aligned themselves in the direction of the flow as they do in native blood vessels. Western Blot analysis of phosphorylated MAPK (Erk, P38MAPk and JNK) and GTPases of Ras and Rho families (Rac and Rap1) indicated that endothelial cells present in our construct can trigger signalization pathways known to be involved in mechanotransduction. In the same way, results of Quantitative RT-PCR show expression of α v β 3 and α 5 β 1 integrins, cell-matrix adhesion molecules usually found on endothelial cells. Burst pressure and other biomechanical properties were measured on specialized devices and yielded values similar to those measured on native blood vessels. This indicates a maturation of the extracellular matrix in the adventitia and media layers. Taken together, those results indicate that the endothelium is functional and that our substitute show similar characteristics to native blood vessels. Long-term functionality study is currently under progress in term of vessel replacement.
Abstract 5534: Critical Role of CuZnSOD in Regulating Renal Microcirculation and in Angiotensin II-Induced Hypertension
Authors: Zufu Ma, Andreas Steege, Andreas Patzak, En Yin Lai, Ulf J Eriksson, Jon Lundberg, Erik G Persson, Mattias Carlström
Journal: Circulation (2009)

Superoxide (O 2 − ), main reactive oxygen species in the vasculature, plays a major role in both renal hemodynamic and blood pressure control. O 2 − levels are limited by superoxide dismutase (SOD) isoforms. Their functional significance in renal and systemic hemodynamics is not clear. The role of SOD1 in afferent arteriolar responsiveness and in angiotensin II (Ang II)-induced hypertension was investigated in SOD1-deficient (SOD1-ko), SOD1-transgenic (SOD1-tg) mice and in littermate controls (wild-type). Arteriolar constrictions to Ang II (10 −14 –10 −6 mol/l) were weaker in SOD1-tg (−14%) and stronger in SOD1-ko (−89%) compared with wild-types (−41%). Unspecific nitric oxide synthase (NOS) inhibition with N ω -Nitro-L-arginine methyl ester hydrochloride (L-NAME; 10 −4 mol/l) reduced basal diameters in wild-types by −8%, in SOD1-ko by −2%, and in SOD1-tg by −38%. Simultaneous application of L-NAME and Ang II caused a similar response in all groups. SOD-mimetic (Tempol; 10 −4 mol/l) had no significant effect in wild-types or SOD1-tg, but caused vasodilatation in SOD1-ko (11%). Simultaneous application of tempol and Ang II attenuated the contractile response only in SOD1-ko (−46%), compared with Ang II alone. Basal blood pressures were similar; however the hypertensive response to chronic Ang II infusion was exaggerated in SOD1-ko (27%) compared with wild-types (9%). The different SOD1-levels were not associated with changes in nitrate/nitrite (NOx) excretion or in renal mRNA expression for NOS-, NADPH oxidase-, SOD-isoforms, or Ang II receptors. In conclusion, SOD1 plays an important role in the control of afferent arteriolar tone, mainly by modulating the NO bioavailability. SOD1-deficiency aggravates Ang II-induced hypertension, which underscores the importance of oxidative stress in this model of hypertension.
Abstract 5507: Metabolic Regulation in Coronary Vascular Tone: Various Vasodilative Substances in Myocardial Ischemia
Authors: Takashi Kaneshiro, Shuichi Saitoh, Hirohumi Machii, Osamu Yamaguchi, Toshiyuki Ishibashi, Yasuchika Takeishi
Journal: Circulation (2009)

Metabolic regulation plays an important role in modifying coronary vascular tone. We hypothesized that H 2 O 2 , purinergic components, and angiotensin, produced by cardiac myocytes control coronary vascular tone in proportion to ischemia. To test this hypothesis, we measured changes in the diameter of isolated, pressurized coronary arterioles (65±5 μ m, n=42) in response to supernatant collected from isolated paced (400 bpm) cardiac myocytes in rat. Changes in diameter of arterioles were determined under control conditions following treatment of the isolated arterioles with A1, A2 antagonist, 8-PSPT (50 μ M), P2Y1 antagonist, MRS-2179 (100 μ M), or angiotensin II type-1 receptor antagonist, olmesartan (1 μ M). In a supernatant from 120 min pacing myocytes, increase of lactate level and decrease of %O 2 concentration were observed compared to unpacing (112±8.2 mg/dl vs. 2±0.4 mg/dl, 2±1% vs. 48±5%, P&lt;0.01, respectively). A supernatant (500 μ l to a 2 ml bath) from myocytes paced for 20, 60 and 120 min caused a graded vasodilation(14.1±0.4 %, 20.2±1.6 %, 53.8±6.2 %, respectively). After a 20 min pacing, catalase (10, 000 U) eliminated vasodilation (0.9±0.8 %, P&lt;0.01). However, 8-PSPT, MRS-2179 or olmesartan did not affect the vasodilative property. Following a 60 min pacing, catalase converted vasodilation to vasoconstriction (−15.1±1.0%, P&lt;0.01). This converted vasoconstriction was completely eliminated with olmesartan (−0.6±0.4%, P&lt;0.01). 8-PSPT or MRS-2179 did not affect the vasoactive property. Upon a 120 min pacing, catalase partially reduced vasodilation (37.2±3.8 %, P&lt;0.05). The remaining vasodilation was converted to vasoconstriction with 8-PSPT + MRS-2179 (−23.4±3.0 %, P&lt;0.01), and this vasoconstriction was completely eliminated with olmesartan (−0.5±0.9%, P&lt;0.01). The SOD activity with an ESR spectrometer attenuated 16.2±1.4 %, and H 2 O 2 concentration in myocytes supernatant were decreased in 120 min pacing compared to those in 60 min pacing (22±0.8 to 31±1.4 μ M, P&lt;0.05). ADP level in myocytes increased in 120 min pacing [8.2±0.8 μ mol/g, P&lt;0.01 vs. unpacing (not detected)]. These results suggest that main vasodilative metabolic modulators released from myocytes replaced from H 2 O 2 to purinergic components in proportion to ischemia.
Abstract 5535: Vascular Insulin Resistance in Prehypertensive Rats Contributes to the Development of Hypertension: Role of PI3 Kinase/Akt/Enos Signaling
Authors: Haifeng Zhang, Rong Li, Wenqing Wang, Xiaoming Wang, Chen Huang, Feng Gao
Journal: Circulation (2009)

Objective: It is well known that systemic insulin resistance (IR) is closely associated with metabolic syndrome including type 2 diabetes and hypertension. However, it remains unclear whether vascular IR acts as an early etiologic factor for the development of hypertension. The present study aimed to test the hypothesis that vascular IR in the conduit arteries contributes to the pathogenesis of hypertension in hypertension-prone normotensive rats, and to further investigate the underlying mechanism. Methods: Male spontaneously hypertensive rats (SHRs) aged 5-weeks (young) and 15-weeks (adult) were studied and vascular IR was assessed as the function of isolated aortic vasodilatory response to insulin in vitro . Results: Compared with Wistar-Kyoto (WKY) rats, adult SHRs exhibited significant hypertension (systolic blood pressure, 173±4 vs. 121±4 mmHg in adult WKY, n = 8, P &lt;0.01) with significantly decreased aortic vasodilatation to insulin (38.7±4.4% vs. 70.9±3.6%, n = 8 –10 aortic segments/group from 5–7 rats, P &lt;0.01), whereas young SHRs had normal blood pressure (121±2 vs. 114±4 mmHg in young WKY, P &gt;0.05) but exhibited similar vascular IR (42.3±5.2% vs. 68.5±5.9%, P &lt;0.01). Both young and adult SHRs showed significant downregulated expression of PI3-Kinase and decreased insulin-stimulated phosphorylations of Akt and eNOS in vascular tissues (n = 3, P &lt;0.01). More importantly, treatment with rosiglitazone (RSG), an insulin sensitizer, for 2 wks increased vascular PPAR γ expression and restored PI3-Kinase/Akt/eNOS-mediated signaling pathway induced by insulin, thus improved aortic vasodilatory response to insulin in young but not in adult SHRs ( P &lt;0.05). Conclusion: Vascular IR, characterized by the impairment of PI3-Kinase/Akt/eNOS signaling in vascular endothelium, may play a role in endothelial dysfunction and subsequent development of hypertension in normotensive young SHRs.