bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5327: Circulating Leukocyte Telomere Length Reflects the Burden of Oxidative Stress and Inflammation in Patients With Acute Myocardial Infarction
Authors: Saliques Sébastien , Zeller Marianne , Vergely Catherine , Amoureux Sebastien , Sicard Pierre , Wolf Jean-Eric , Lorin Julie , Lagrost Anne-Cécile , Berchoud Juliane , Javouhey-Donzel Anne , Cottin Yves , Teyssier Jean-Raymond , Rochette Luc
Published: April 2024

Objectives:
Recent data suggest that leukocyte telomere length (TL) may be a possible reliable marker in human-aging diseases and cardiovascular illness such as coronary artery disease (CAD) and heart failure. The main outcome of our study was to identify the possible correlation between TL and biomarkers of oxidative and inflammatory status. in patients with acute myocardial infarction (MI).

Materials and Methods
: The TL was determined prospectively by quantitative Polymerase Chain Reaction method from circulating leukocytes of consecutive patients admitted <24 h in intensive care unit for an acute MI, as well as leukocyte expression of
c-Fos,
gene involved in cellular proliferation and oxidative stress. Left ventricular ejection fraction (LVEF) was assessed by echocardiography <3 days after admission.

Results:
Among the 69 patients included in the study, mean age was 66±2 and 78% were male, and TL was 1.34±0.01. A negative correlation was found between leukocyte TL and age (r=−0.37, p=0.002),
cFos
gene expression (r=−0.46, p<0.001), logNT-proBNP (r=−0.30, p=0.019) and CRP (r=−0.29, p=0.018). In contrast, creatinine clearance (r= +0.30, p=0.02), and LVEF (r= +0.26, p=0.04) were positively correlated with TL. Leukocyte count was not related with TL (p=0.54). Age-adjusted TL was significantly reduced in male vs. women (p=0.001), and in patients with hypertension (p=0.023), family history of CAD (p=0.006), smokers (p<0.001), or clinical heart failure on admission (p=0.009). Backward multivariable linear regression analysis identified
cFos
(
β
=0.51, p<0.001), smoking (
β
=0.25, p=0.031) and prior CAD (
β
=−0.266, p=−0.011) as factors independently associated with LT (R
2
=0.43).

Conclusion
: Our study suggests that in patients with acute MI, circulating leukocyte TL reflects increased oxidative stress and inflammation, while the independent influence of age is limited. Considering the relevance of these findings, definitive question is if telomere attrition is a primary cause or a consequence of the development of the illness, encouraging further studies in this field.
Abstract 5328: Excessive Superoxide Production and Endothelial Dysfunction in Cerebral Arteries of Atherosclerotic Mice Are Due to Enhanced Activity of Nox2-containing NADPH-oxidase
Authors: Miller Alyson A , DeSilva Michael , Judkins Courtney P , Diep Henry , Drummond Grant R , Sobey Christopher G
Published: April 2024

We tested if enhanced superoxide (O2−) production by Nox2-containing NADPH oxidase leads to impaired endothelial function in cerebral arteries of high fat-fed apolipoprotein E-deficient (ApoE−/−) mice. All mice were fed a high fat diet from 5 wks of age for 7–14 wks. Intracranial cerebral arteries were isolated from C57Bl6/J wild-type (n=13), ApoE−/− (n=26), Nox2−/− (n=10) and novel Nox2−/−/ApoE−/− (n=23) mice. Basal O2− production by cerebral arteries was measured using L012 (100 umol/L)-enhanced chemiluminescence. Endothelial function was assessed in isolated cannulated middle cerebral arteries using a perfusion myograph via the vasoconstrictor response to the nitric oxide synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME; 100 umol/L). Plasma total cholesterol levels of wild-type (n=6), ApoE−/− (n=7) and Nox2−/−/ApoE−/− (n=8) mice were 6.1±0.2, 39.5±5.3, and 41.93±1.6 mg/L, respectively. En face oil red O staining showed that there were no atherosclerotic lesions in aortae of wild-type mice (n=13), whereas aortic lesions were prominent in ApoE−/− mice (~9% of the total aortic surface, n=13). By contrast, no lesions were observed in cerebral arteries from ApoE−/− mice. O2− production was elevated in cerebral arteries from ApoE−/− (35±5 × 103 counts/mg; n=14, P<0.05) versus wild-type (16±2 × 103 counts/mg; n=11) and Nox2−/− (10±2 ×103 counts/mg; n=6) mice. However, in Nox2−/−/ApoE−/− mice, cerebral artery O2− production was not elevated (11±1 ×103 counts/mg; n=17; P< 0.05). The magnitude of L-NAME-induced contractions of isolated middle cerebral arteries from ApoE−/− mice (Δ diameter =−13±4 %; n=6) was <50% of that in wild-type (−34±2 %; n=6) mice, whereas in Nox2−/−/ApoE−/− (−33±10 %; n=6) mice endothelial function was comparable to that in wild-types. By contrast, similar constrictor responses to high K+ (124 mM) were observed in wild-type (−59±4 %), ApoE−/− (−60±5 %), and Nox2−/−/ApoE−/− (−65±3 %) mice. In summary, excessive O2− production and endothelial dysfunction occur in cerebral arteries of atherosclerotic mice even in the absence of lesions. Furthermore, we report for the first time that these changes appear to be exclusively due to increased activity of Nox2-containing NADPH oxidase.
Abstract 5329: ROS-dependent Cysteine Sulfenic Acid Formation of IQGAP1 and ERK1/2 in VEGF-induced Endothelial Cell Proliferation and Migration
Authors: Kaplan Nihal , Urao Norifumi , Razvi Masooma , Nakamura Yoshimasa , Poole Leslie B , Fukai Tohru , Ushio-Fukai Masuko
Published: April 2024

VEGF receptor type 2 (VEGFR2) activation induces endothelial cell (EC) proliferation and migration. ROS-mediated protein modification involves oxidation of reactive cysteine residues to form a cysteine sulfenic acid (Cys-SOH) and subsequent oxidation products. However, role of Cys-SOH formation in VEGF signaling linked to angiogenesis is unknown. We previously identified IQGAP1, as a novel VEGFR2 binding protein, involved in regulating ROS-dependent EC migration and proliferation by recruiting Nox2 NADPH oxidase to activated VEGFR2 signaling complex. By using a newly-developed Cys-SOH trapping reagent we show that VEGF stimulation significantly increases protein-Cys-SOH formation in HUVECs. Immunofluorescence reveals that Cys-SOH modified proteins accumulate at the leading edge where they colocalize with NADPH oxidase, F-actin and IQGAP1 in actively migrating ECs. VEGF stimulation increases IQGAP1-Cys-SOH formation within 5min (60%) with a maximum increase at 15min (90%). VEGF-induced Cys-SOH formation of IQGAP1 is prevented by pretreatment with a GSH donor antioxidant, N-acetylcysteine. Knockdown of IQGAP1 by siRNA or trapping of Cys-SOH with dimedone, blocks wound-induced Cys-SOH formation at the leading edge and EC migration toward the injured site (80%), EC chemotaxis (90%) and matrigel angiogenesis (80%). These suggest linkage between IQGAP1-Cys-SOH formation and IQGAP1-mediated EC migration. In addition, dimedone pretreatment inhibits VEGF-stimulated EC proliferation. VEGF stimulation also induces oxidation of ERK1/2 upon 5 min (50%) and 15min (80%) stimulation. Trapping Cys-SOH by dimedone inhibits ERK phosphorylation (95%), which is associated with increasing IQGAP1/ERK complex (50%) after VEGF stimulation. Hind limb ischemia increases oxidation of tissue proteins including IQGAP1 (80%) and ERK1/2 (60%) at day 1 after injury, which is prevented in mice lacking Nox2. Thus, oxidation of IQGAP1 and ERK contributes to directional migration and proliferation of ECs and to neo-vascularization in ischemic tissue. The present study should provide insights into identifying and visualizing Cys-SOH formed proteins as important strategy for understanding mechanisms by which ROS regulate EC function such as angiogenesis.
Abstract 5330: CD36 Participates in a Signaling Pathway That Regulates Reactive Oxygen Species Formation
Authors: Li Wei , Febbraio Maria , Silverstein Roy
Published: April 2024

Previous studies showed that CD36 promotes reactive oxygen species (ROS) formation in mouse models of cerebral and cardiac ischemia. Using CD36
−/−
and wild type (WT) mice and cultured murine aortic vascular smooth muscle cells (VSMC), we examined the mechanisms by which CD36 regulates ROS formation and their potential functional significance. Two-Dimensional Difference Gel Electrophoresis and Mass Spectrometry analysis revealed significantly increased expression of Peroxiredoxin 2 (Prdx 2), an antioxidant enzyme known to detoxify ROS, in carotid arteries from CD36
−/−
mice. Immunohistochemistry confirmed the increase and revealed that expression was mainly in smooth muscle. Western blots showed increased Prdx2 expression in isolated VSMC from CD36
−/−
mice compared to WT, as well as increased levels of Nrf2, a transcription factor known to regulate anti-oxidant systems. Chromatin immunoprecipitation assays revealed that Nrf2 bound to the antioxidant responsive elements in the Prdx2 promoter and transfection of Nrf2 cDNA into HEK293 cells induced Prdx2 expression. In CD36
−/−
VSMC exposure to oxidant stress (H
2
O
2
) induced higher Prdx2 expression than in WT cells. Pharmacologic inhibition of Src kinases increased nuclear accumulation of Nrf2 in VSMC, suggesting that CD36 signaling through src kinases contributes to Nrf2 nuclear export or degradation. We previously showed that CD36−/− mice had prolonged thrombosis times after carotid artery injury induced by FeCl
3
. We therefore monitored ROS production in the vessel wall after injury using hydroethidine, a fluorescent superoxide probe. FeCl
3
dose dependently induced accumulation of ROS; however, the degree was significantly lower in CD36
−/−
than in WT mice. Direct injection of Edavarone, a free radical scavenger, significantly prolonged time to thrombosis in all mice but the effect was much less in CD36
−/−
animals than in WT. We also found fewer endothelial cell-derived microparticles in plasma of CD36
−/−
mice after FeCl
3
treatment, suggesting less endothelial injury. We conclude that CD36 mediated down-regulation of antioxidant systems in vascular cells may contribute to its pro-thrombotic and atherogenic effects.
Abstract 5420: Suppression of Aldosterone Mediates Left Ventricular Hypertrophy Regression in Patients With Hypertension
Authors: Pouleur Anne-Catherine , Uno Hajime , Prescott Margaret , Desai Akshay , Appelbaum Evan , Lukashevich Valentina , Smith Beverly A , Dahlöf Björn , Solomon Scott D
Published: April 2024

Background

. Although high circulating aldosterone levels stimulate myocardial fibrosis and left ventricular hypertrophy (LVH), it is less clear whether suppression of aldosterone is associated with LVH regression in hypertensive patients.

Methods.

The Aliskiren in Left Ventricular Hypertrophy (ALLAY) trial randomized 465 hypertensive overweight subjects to the direct renin inhibitor aliskiren 300mg, losartan 100mg or the combination and followed patients for 9 months. All patients were treated to standard blood pressure targets. LV mass index (LVMI) and LV wall thickness (LVWT) were assessed by cardiac magnetic resonance. A subset of 135 patients with plasma aldosterone (ALDO) measured at baseline and study end was analyzed.

Results.

At baseline, ALDO correlated poorly with systolic blood pressure (SBP) (r=0.2, p=0.02), LVMI (r=0.18, p=0.03) and LVWT (r=0.22, p=0.01). Mean blood pressure was 144±13/90±9mmHg, mean LVMI was 75±16 g/m
2
, and mean LVWT was 9.8±2.2mm. ALDO was reduced between baseline and end of the study in the aliskiren [244 (202–294) vs. 198 (161–243) pmol/L, p=0.03] and combination groups [209 (169–257) vs. 164 (138–196) pmol/L, p=0.0029], but not in the losartan group [223 (181–275) vs. 235 (199–278) pmol/L, p=0.63] [geometric means (95%CI)]. Reduction in ALDO was associated with regression of LVWT, even after adjustment for SBP reduction, treatment, baseline ALDO and baseline LVWT (p= 0.041).

Conclusion

. In hypertensive patients with increased LVWT, regression of LVH was associated with suppression of ALDO, independently of the change in SBP, suggesting that ALDO, a known mediator of LVH, may be a particularly important target for suppression.
Abstract 5437: Pluripotent Stem Cells Induced From Adult Somatic Cells by Dedifferentiation Without Genetic Manipulation
Authors: Lee Choon Soo , Cho Hyun-Jai , Kwon Yoo-Wook , Paek Jae Seung , Lee Sun-Hee , Jo Sun-Young , Hur Jin , Lee Eun Ju , Kang Hyun-Jae , Oh Byung-Hee , Park Young-Bae , Kim Hyo-Soo
Published: April 2024

Background:
Reprogramming of somatic cells into pluripotent stem cells have opened the new-era in regenerative medicine. Viral transduction of defined factors successfully achieved pluripotency from mouse and human somatic cells. However, in the generation process of induced pluripotent stem (iPS) cells, genetic integration of certain factors may cause mutagenesis or tumorigenicity, which hampers further application for cell therapy.

Methods and Results:
We investigated that the transient cellular delivery of mouse ES cell-derived proteins enables dedifferentiation of mouse somatic cells into pluripotent stem cells without genetic manipulation. We found that transient delivery of ES cell-derived proteins using reversible permeabilisation can activate endogenous transcription factors related pluripotency. Dedifferentiated somatic cells are morphologically indistinguishable from the authentic ES cells. In the process, the methylation and histone modification status in the promoter regions of pluripotency-related genes are converted from somatic to ES-equivalent statues. Molecular biologically, the patterns of global gene expression, genomewide ChIP sequencing and proteomics using Itracq are very similar to those of protein donor ES cells. Functionally, dedifferentiated cells form embryoid body (EB) structures in suspension culture. On 7 days of re-differentiation, cells contract spontaneously and express cardiac troponin T and
α
-sarcomeric actinin, indicating the properties of cardiomyocytes. And also the certain portions of re-differentiated cells express ectoderm, mesoderm and endoderm proteins. Furthermore, dedifferentiated cells possess in vivo differentiation and developmental potentials including well-differentiated teratoma formation, contribution to chimeras and germline transmission.

Conclusions:
These results demonstrate that transient delivery of ES cell-derived proteins can induce dedifferentiation of adult somatic cells into pluripotent stem cells. Our results provide an alternative and safe strategy for dedifferentiation or reprogramming of adult somatic cells and may facilitate tailored- or patient-specific pluripotent stem cell-derived cell therapy.
Abstract 5453: Endothelial Dysfunction Plays Important Roles on Development of Acute Coronary Syndrome and Fatal Events
Authors: Murakami Tatsuaki , Moriuchi Ikuo
Published: April 2024

Background and hypothesis:
Endothelial dysfunction is thought to contribute to atherothrombogenic process, however we identify little about practical relationship between clinical evolution of acute coronary syndrome (ACS) and endothelial dysfunction. This study assessed hypothesis that endothelial dysfunction has clinical impact on development of ACS and the related prognosis.

Methods:
Endothelial dysfunction was graded by ultrasonic measured reactive changes in lumen diameter of right brachial artery following transient forearm occlusion for 5 minutes (FMD; flow-mediated endothelium-dependent vasodilation) in consecutive 518 patients with stable coronary artery disease. The enrolled patients were categorized into three groups according to the values of FMD, and their cardiovascular and other clinical events were prospectively followed-up for no less than 36 months.

Results:
For a mean follow-up period of 60 months with 100% follow-up, the patients with severe endothelial dysfunction (FMD<4%; Group-L, n=174), far more frequently developed ACS, compared to those in Group-M with mild endothelial dysfunction (4%≤FMD<8%, n=171) or Group-H with preserved endothelial function (FMD 8% or more, Group-H, n=173), [p<0.001, by Kaplan-Meier analysis], while the patients in group-H remained free from ACS. More than two thirds of the patients with fatal events belonged to Group-L. Cox proportional hazard model analysis showed that severe endothelial dysfunction was the most powerful predictor for future development of ACS (hazard ratio=5.77, 95%confidential interval; 2.52–13.22, p<0.001) and any fatal events (hazard ratio=5.87, 95%confidential interval; 1.26–72.25, p=0.009).

Conclusion:
These results certainly suggest endothelial dysfunction plays important roles on development of ACS or any fatal events in the near future and strategies based on practical status of endothelial function are required to prevent ACS or fatal clinical events.

Relationship Between Incidence of ACS or Fatal Events and Endothelial Dysfunction
Abstract 5458: VIA-3196, a Liver-directed Thyroid Beta Agonist for Treating Cardiometabolic Disease
Authors: Taub Rebecca , Grimsby Joseph , Larigan J. D , Pietranico Sherrie , Dvorozniak Mark , Conde-Knape Karin
Published: April 2024

Thyroid hormone has beneficial metabolic benefits such as cholesterol, triglyceride and glucose lowering primarily through its action in the liver. However, in excess, TH causes adverse effects in heart and bone primarily mediated by the THR-alpha receptor. VIA-3196 is a novel THRbeta receptor selective (17/1 relative to TH) thyromimetic with high hepatic uptake and the possibility of providing metabolic benefits and avoiding adverse effects on the thyroid axis, heart and bone. In a rodent hypothyroid model, as compared with TH (T3), VIA-3196 showed no upregulation of a TH regulated gene in the heart even at high doses. In hypercholesterolemic rabbits, VIA- 3196 lowered non-HDL cholesterol alone or in synergy with atorvastatin up to 50% (p<0.001), increased the HDL/LDL ratio, and lowered plasma triglycerides 35% (p<0.05) alone and 50% (p<0.001) in combination with atorvastatin. In hypercholesterolemic rats, VIA-3196 lowered non-HDL cholesterol 73%, (p<0.005) and liver triglycerides 66% (p<0.0001), and did not decrease TSH, whereas T3 had little effect on cholesterol or liver triglycerides at doses that suppressed TSH. Steatosis is a major cause of liver insulin resistance (IR) and non-alcoholic steatohepatitis. In preclinical mouse models of IR, at doses as low as 0.3 mg/kg VIA-3196 reduced plasma cholesterol 40% (p<5×10
−6
) and liver triglycerides 51% (p<0.05). In mouse models of IR, VIA-3196 at 1 mg/kg improved insulin sensitivity as demonstrated by an insulin tolerance test (p<0.05), lowered glucose to a near normal level (p<0.05) similar to rosiglitazone, and unlike rosiglitazone, did not cause weight gain. Hepatic gene expression studies suggest that VIA-3196 may lower cholesterol through increasing cholesterol metabolism and biliary excretion (upregulation of Cyp7A1, ABCB11), perhaps increasing reverse cholesterol transport. Interestingly, VIA-3196 downregulated hepatic inflammatory genes CRP and Saa2 (serum amyloid a2) that are associated with cardiovascular risk. VIA-3196 demonstrates good pharmaceutical properties predictive of qd dosing in humans. VIA-3196 has the potential to provide benefit to patients with diabetes and dislipidemia and ultimately, reduce their risk from atherosclerotic cardiovascular disease.
Abstract 5459: Unexpected Acceleration of Atherosclerosis in Ldlr-null Mice Reconstituted With Group X Secretory Phospholipase A2-Deficient Bone Marrow
Authors: Ait-Oufella Hafid , Coatrieux Christelle , Ramkhelawon Bhama , Blanc-Brude Olivier , Sirvent Jérôme , Laurans Ludivine , Lambeau Gérard , Mallat Ziad
Published: April 2024

Group X (GX) secretory phospholipase A2 (sPLA2) has the most potent hydrolyzing activity toward phosphatidylcholine and elicits a marked release of arachidonic acid among several types of sPLA2. GX is expressed by vascular cells and inflammatory cells like macrophages and CD4+ T lymphocytes and is therefore believed to play a pathogenic role in atherosclerosis. We evaluated the effects of GX deficiency on systemic immune response and the development of atherosclerosis, a vascular disease sustained by lipid modifications and inflammation. GX KO mice showed increased spleen size confirmed by higher splenocyte number compared to C57BL6 controls (P<0.01). LPS/IFN-
γ
-stimulated GX-deficient splenocytes produced more IL-12 than control splenocytes (3380 vs 1599 pg/ml, P<0.05), with no difference in IL-10. Purified splenic CD4+ T cells from GX KO mice showed enhanced proliferation and production of the Th1 cytokine IFN-
γ
(5575 vs 1443 pg/ml, respectively, p<0.05) compared with controls. GX-deficient blood mononuclear cells showed enhanced adhesion on activated HUVECs (8.8 vs 3.2 cell/field, P<0.01) and on LDLr KO carotid arteries (4.2 vs 6.4 cell/mm, P<0.01) ex vivo, compared to control cells. When challenged in vitro to test cell death susceptibility to a variety of apoptotic stimuli, bone marrow-derived GX-deficient macrophages displayed a significant increase of apoptosis compared to control cells. Finally, LDLr KO mice were lethally irradiated and reconstituted with either GX KO or wild type bone marrow. After 8 weeks of fat diet, mice reconstituted with GX KO bone marrow showed a ~100% increase of plaque size in the aortic sinus compared to mice with WT bone marrow (294 254
μ
m2 vs 142 304
μ
m2, respectively, P=0.001), despite similar plasma cholesterol level. This was associated with a more advanced plaque phenotype and marked accumulation of TUNEL-positive apoptotic cells (2.4 % vs 0.08 P<0.001). Immunologic analysis of splenocytes and splenic CD4+ T cells from GX/LDLr chimeric mice showed increased immune cell activation and Th1-related cytokine production. In conclusion, GX sPLA2 deficiency increases innate and adaptive immune responses, enhances macrophage susceptibility to apoptosis and accelerates atherosclerosis in mice.
Abstract 5470: Cardiomyocyte-specific Overexpression of Nox4 Attenuates Adverse Cardiac Remodeling After Myocardial Infarction
Authors: Zhang Min , Looi Yee H , Brewer Alison C , Shah Ajay M
Published: April 2024

Background.
Reactive oxygen species (ROS) production is implicated in the development of cardiac remodeling after myocardial infarction (MI). NADPH oxidases are major sources of cardiovascular ROS, with cardiomyocytes expressing both Nox2 and Nox4 isoforms. We previously showed that Nox2 contributes significantly to the processes underlying adverse cardiac remodeling and contractile dysfunction post-MI, but the effect of Nox4 remains unclear. The aim of this study was to investigate the role of Nox4 in cardiac remodelling after MI.

Methods and Results.
We generated transgenic mice (TG) with cardiomyocyte-specific overexpression of Nox4, expressing the mouse full-length Nox4 cDNA under control of the
α
-MHC promoter. TG were backcrossed onto a C57BL6/J background and wild-type littermates (WT) used as controls. Nox4 TG mice were grossly similar to WT in terms of body weight, growth, activity, fertility and basal cardiac function. TG displayed heart-specific expression of the Nox4 transgene, had significantly higher Nox4 protein expression, over 60% higher heart NADPH oxidase activity and 42% increased hydrogen peroxide production (all P<0.05). Compared with WT, p22
phox
protein expression was increased by 2.4-fold, which may be related to an increased protein stability. There was no change in protein expression level of Nox2 or eNOS. MI was achieved by permanent left coronary ligation for 4 weeks. Compared with WT/MI, TG/MI groups displayed significantly high survival rate (72 % vs 95%) and less hypertrophy at 4 weeks post MI in terms of heart/body weight ratio (6.95±0.16 vs 6.44±0.14, p<0.01), although the infarct size was similar (38.3±2.0 vs 39.9±2.8%). TG mice also exhibited less apoptosis and interstitial cardiac fibrosis (0.52±0.03 vs 0.65±0.05%, p<0.05), and lower increases in expression of fibronectin and procollagen III mRNA. Both echocardiography and cardiac catheterization demonstrated less left ventricular cavity dilatation and a preservation of cardiac function in TG than WT mice.

Conclusions.
In contrast to Nox2, which contributes to cardiac dilation, contractile dysfunction and fibrosis, cardiomyocyte Nox4 was found to exert protective effects against adverse remodelling post-MI.
Abstract 5471: NAD(P)H Oxidase Nox4 in Vascular Endothelial and Smooth Muscle Cells May Be Involved in the Regulation of Basal Blood Pressure
Authors: Kuroda Junya , Ago Tetsuro , Sadoshima Junichi
Published: April 2024

Reactive oxygen species (ROS) play an important role in mediating changes in blood pressure, vascular remodeling, and arteriosclerosis. All vascular cell types including vascular endothelial cells (ECs) and smooth muscle cells (SMCs) produce ROS, primarily through NAD(P)H oxidase (Nox) family enzymes, including Nox1, Nox2 and Nox4. Nox4 is predominantly expressed in ECs and SMCs and its expression in the vasculature is upregulated by aging. In order to elucidate the function of Nox4 in blood vessels, we established transgenic mice with EC- and SMC-specific overexpression of wild-type Nox4 (EC-Nox4, SMC-Nox4) and dominant negative Nox4 (EC-DN-Nox4, SMC-DN-Nox4). EC- and SMC-specific overexpression was achieved with SM22
α
and Tie2 promoters, respectively. Enhanced expression of Nox4 or DN-Nox4 in ECs or SMCs was confirmed by immunostaining of blood vessels with a specific anti-Nox4 antibody. Superoxide producing activity in blood vessels was enhanced in EC-Nox4 (129.5±15.2%) and SMC-Nox4 (142.2±12.9%), and attenuated in EC-DN-Nox4 (30.6±4.9%) and SMC-DN-Nox4 (49.4±10.2%) compared to non-transgenic controls, as evaluated by dihydroethidium staining. At 3 months of age, EC-Nox4 exhibited significantly lower systolic blood pressure than WT (96.3±5.5 vs. 110.3±5.0 mmHg, p < 0.05) as determined by the tail-cuff method. Conversely, SMC-Nox4 exhibited significantly higher systolic blood pressure than WT (122.7±3.2 vs. 107.3±3.9 mmHg, p < 0.05). Blood pressure in EC-DN-Nox4 or SMC-DN-Nox4 was not statistically different from WT. At 3 months of age, none of the transgenic mouse lines exhibited obvious histopathological changes in vasculature, including aortas and arterioles in hearts and kidneys. Elevation of blood pressure induced by continuous infusion of angiotensin II (Ang II) (0.6 ng/g/min) in EC-Nox4 or SMC-Nox4 was not significantly different from WT at day 3, 7 and 14. Taken together, these results suggest that upregulation of Nox4 in SMCs and ECs increases superoxide production, thereby increasing or decreasing, respectively, blood pressure at baseline, without affecting Ang II-induced elevation in blood pressure.
Abstract 5484: Activation of Endothelial NF-
κ
B Signaling Promotes Coagulation by Inhibiting Protein C Anticoagulant Mechanism in Endotoxemic Mice
Authors: Song Dongmei , Ye Xiaobing , Liu Gang , Liu Shu Fang
Published: April 2024

Although the role of systemic activation of NF-
κ
B pathway in septic coagulation has been well documented, little is known about the mechanistic contribution of endothelial intrinsic NF-
κ
B signaling in this pathological process. This study defined the causative role of endothelial NF-
κ
B signaling in septic coagulation. Wild type (WT) and transgenic mice (TG) that conditionally overexpress a mutant I-
κ
B
β
, an inhibitor of NF-
κ
B, selectively on endothelium, were injected with saline (1 ml/kg, i.p.) or
E Coli
LPS (5 mg/kg, i.p.). Tissue fibrin deposition, plasma markers of coagulation, plasma levels of protein C and plasminogen activator inhibitor-1 (PAI-1) were measured at 6 hours after LPS injection. Immunohistochemical staining of frozen tissue sections of heart, liver and kidney from WT-LPS mice showed a widespread tissue fibrin deposition. Fibrin deposition was not evident in tissue sections of the three organs from TG-LPS mice. Compared with WT-Con and TG-Con mice, WT-LPS mice showed a 79% reduction in plasma fibrinogen level, which was restored to control level in TG-LPS mice. WT-LPS displayed 13.7- and 3.2-fold increase in plasma levels of thrombin-antithrombin and D-dimer, which were reduced by approximately 69% and 37% in TG-LPS mice. Plasma levels of total and activated protein C (mO.D. units) for WT-Con, TG-Con, WT-LPS and TG-LPS groups were: 73.4±2.4, 72.0±3.2, 26.0±6.2 and 58.5±4.7 (p < 0.01, WT-LPS vs other 3 groups), and 45.6±1.6, 43.0±1.2, 2.8±1.8 and 29.9±4.1 (p < 0.01, WT-LPS vs other 3 groups). Plasma level of PAI-1 (ng/ml) for WT-Con, TG-Con, WT-LPS and TG-LPS groups was 0.06±0.05, 0.0±0.0, 31.3±4.8 and 18.1±3.3 (p < 0.05, WT-LPS vs other 3 groups). These results indicate that endothelial intrinsic NF-
κ
B signaling plays a pivotal role in the activation of coagulation in endotoxemic mice, and suggest that activation of endothelial NF-
κ
B pathway promotes coagulation by impairing the protein C anticoagulant mechanism and by inhibiting the fibrinolytic system.

(Supported by NIH R01GM063907).
Abstract 5493: Genetic or Acquired CYP2C19 Enzyme Deficit is Associated With an Excess of Cardiovascular Risk in Clopidogrel-treated Patients
Authors: Hulot Jean-sebastien , Collet Jean-Philippe , Silvain Johanne , Pena Ana , Bellemain-Appaix Anne , Barthélémy Olivier , Anzaha Ghalia , Beygui Farzin , Montalescot Gilles
Published: April 2024

Introduction: Metabolic activation by CYP2C19 has emerged as a crucial determinant of clopidogrel responsiveness and efficacy. An increased risk of cardio-vascular events has been associated with the CYP2C19*2 allele, which causes loss-of-function, and with the use of proton pump inhibitors (PPI), which may also inhibit the CYP2C19, in clopidogrel-treated patients. We performed a meta-analysis to further estimate the relative effect in both sub-groups.

Methods: Sixteen published or presented studies evaluating the association between the loss-of-function CYP2C19*2 genetic variant (8 studies, 9427 patients) or the use of PPI (8 studies, 35108 patients) and outcomes in patients treated with clopidogrel were included. Data on the occurrence of ischemic events (death, recurrent myocardial infarction, urgent revascularization) were extracted and combined using a fixed-effect model.

Results: Of the 9427 patients, carriers of the loss-of-function CYP2C19*2 allele (28%, n=2674) displayed a 30% increase in the risk in any ischemic events compared to non carriers (10.4% vs 8.3%) (OR, 1.31; 95% CI, 1.12–1.53; p<0.001). This single gene variant (CYP2C19*2) was also associated with an excess of cardiovascular mortality (1.8% vs 1.0%)(OR 1.79; 95% CI: 1.10–2.91, p<0.019)(n=6225). PPI users (43%, n=15197) displayed an increased risk of ischemic events compared to non users (25.5% vs 19.4%) (OR, 1.48; 95% CI, 1.40–1.57; p<0.001)(n=34496). This metanalysis also showed an excess of mortality (16.9% vs 11.5%)(OR 1.20; 95% CI: 1.08–1.33, p<0.001)(n=15157) in PPI users compared to non users. When combining all data, CYP2C19 alteration due to the deficient allele or drug interaction has a 50% increased risk of ischemic events occurrence in clopidogrel treated patients (OR 1.49; 95% CI: 1.42–1.57, p<0.001).

Conclusions: CYP2C19*2 carriers and/or PPI users are patients exposed to a significantly increased risk of cardiovascular events when they are treated with clopidogrel.
Abstract 5516: Cardioprotective Effects of Hydrogen Peroxide and Erythropoietin During Acute Coronary Occlusion in Canine Coronary Native Collateral Microvessels in vivo
Authors: Yada Toyotaka , Shimokawa Hiroaki , Hiramatsu Osamu , Satoh Minoru , Kashihara Naoki , Shinozaki Yoshiro , Mori Hidezo , Takaki Aya , Goto Masami , Nakamoto Hiroshi , Ogasawara Yasuo , Kajiya Fumihiko
Published: April 2024

Background:

We have previously demonstrated that endothelium-derived hydrogen peroxide (H
2
O
2
) is an endothelium-derived hyperpolarizing factor (EDHF) in canine coronary autoregulation in vivo. The aim of this study was to examine the role of H
2
O
2
during acute coronary occlusion with recombinant human erythropoietin (EPO) in the collateral vasodilatation of coronary microcirculation in vivo.

Methods and Results:

Canine subepicardial native collateral coronary small arteries (CSA, ≥100
μ
m) and arterioles (CA, <100
μ
m) were observed by an intravital microscope under cyclooxygenase blockade. Experiments were performed after LAD occlusion (90 min) under the following 8 conditions (n=5, each); control, low-dose EPO (100 IU/kg, L-EPO), high-dose EPO (1000 IU/kg, H-EPO), H-EPO+catalase (a H
2
O
2
decomposer), H-EPO+NO synthase inhibitor (L-NMMA), H-EPO+L-NMMA+catalase, H-EPO+L-NMMA+tetraethylammonium (TEA, a K
Ca
channels blocker) and H-EPO+ wortmannin (PI3K inhibitor). Ischemia caused a significant collateral arteriolar vasodilatation of control, which was significantly inhibited by catalase. After H-EPO, the vasodilatation was significantly increased in both-sized arteries (CSA 6±1%, CA 22±4%) compared with control (CSA 1±1%, CA 16±3%) or L-EPO (CSA 2±1%, CA 18±3%) associated with increase in collateral blood flow (microsphere technique). H-EPO+catalase significantly decreased the vasodilatation in CA (12±2%). The vasodilatation was markedly attenuated after H-EPO+L-NMMA+catalase (CSA −4±1%, CA 6±1%), H-EPO+L-NMMA+TEA (CSA −5±1%, CA 6±1%) and H-EPO+ wortmannin (CSA −5±1%, CA 6±1%). In the control group, expression of Akt phosphorylation in the ischemic LAD area was significantly decreased compared with the H-EPO group. In H-EPO+L-NMMA+catalase or H-EPO+wortmannin group, expression of Akt phosphorylation was further decreased compared with H-EPO or L-NMMA+H-EPO group. Plasma 8-hydroxydeoxyguanosine from coronary sinus as a myocardial oxidative stress marker (HPLC-ECD method) significantly decreased in H-EPO compared with control.

Conclusions:

H
2
O
2
and EPO play an important collaborative role in native collateral vasodilatation in response to acute coronary occlusion in canine coronary microcirculation in vivo.
Abstract 5518: Abnormal Coronary Flow Reserve is Related to Impairment of Left Ventricular Function During Adenosine Stress Echocardiography in Untreated Hypertensive Patients
Authors: Ikonomidis Ignatios , Tzortzis Stavros , Lekakis John , Triantafyllidi Helen , Papadopoulos Costas , Tsitlakidis Costas , Paraskevaidis Ioannis , Kremastinos Dimitrios T
Published: April 2024

Objective: Coronary microcirculation is disturbed in hypertensive patients. We investigated the effects of coronary flow reserve (CFR) on the response of Left Ventricular (LV) systolic and diastolic function as assessed by Tissue Doppler Imaging during adenosine stress echocardiography in never-treated hypertensive patients.

Methods: We studied 43 patients (mean age: 53±12 years), with untreated arterial hypertension (clinic blood pressure >140/90 mmHg and >125/80 mmHg in 24hour ambulatory BP monitoring) by adenosine stress echocardiography and 22 healthy controls matched for sex, age and atherosclerotic factors. We measured:

time integral (VTI-Vd) of diastolic (Vd) coronary blood flow velocity by transthoracic Doppler echocardiography of the LAD

Ratio of VTI-Vd after adenosine infusion (140
μ
g/kg/min) to VTI-Vd at baseline to assess CFR

E and A doppler, Sm, Em, Am lateral mitral annulus velocities with TDI, Em/Am ratio and E/Em ratio before and during adenosine infusion to assess LV systolic and diastolic function,

the percent % changes of the measured indices between baseline and peak adenosine infusion.

Results: All patients had normal baseline ejection fraction. Compared to controls, hypertensives had lower CFR (2.3±0.7 vs. 2.9±0.9, p=0.03). At baseline, hypertensives compared to controls, had lower Em/Am ratio (0.9±0.3 vs. 1.3±0.4, p=0.002), Sm (8.7±1.7 vs. 9.7±2.6, p=0.07), Em (10.1±2.8 vs 12.1±3.5, p=0.03) and higher Am (11.1±2.5 vs. 9.5±2.0, p=0.03). At peak adenosine infusion there were an increase in Sm, Em and Am in all patients (p<0.05). Furthermore a decreasing CFR was associated with a decreasing Em (r= 0.46, p=0.05), Em/Am (r=0.49, p=0.048) and increasing E/Em (r=−0.4, p=0.04). A reduced CFR was related with a smaller % increase of Sm (r= −0.40, p=0.049), % increase of Em (r=− 0.5, p=0.033) and higher % increase of E/Em (r=−.42, p=0.04) at peak stress. Patients with CFR<2.5 (n=28), compared to those with CFR≥2.5 had lower % change of Sm (27±20% vs. 51±34% p=0.04), % change of Em (8±3% vs. 46 ±3%, p=0.05) and higher % increase of E/Em (24±3% vs −1±2%, p=0.04).

Conclusions: An abnormal response of LV function during adenosine stress echocardiography is related with impaired CFR in untreated hypertensive patients.
Abstract 5520: Upregulation of Arginase I Reduces Nitric Oxide-mediated Coronary Arteriolar Dilation in Patients With Type 2 Diabetes
Authors: Bagi Zsolt , Beleznai Timea , Feher Attila , Kaley Gabor
Published: April 2024

We hypothesized that increased expression of arginase I reduces nitric oxide (NO)-mediated coronary arteriolar dilations in patients with type 2 diabetes (2-DM). Arterioles were dissected from right atrial appendages from patients with (N=17) or without (N=14) 2-DM. Coronary arterioles (123±16
μ
m) were cannulated, pressurized (80 mmHg) and changes in diameter were measured with videomicroscopy. After spontaneous tone developed, acetylcholine (ACh, 1 nM-1
μ
M) elicited dilations of coronary arterioles from patients without 2-DM (max. 82±5%), whereas it induced constrictions of arterioles from 2-DM patients (−41±9%). Dilations to the NO donor sodium nitroprusside were not different in the two groups. Inhibition of NO synthesis with L-NAME reduced coronary dilations in subjects without 2-DM, but did not affect constrictions to ACh in 2-DM patients. In vitro application of L-arginine (3 mM, 30 mins) restored coronary dilations to ACh in 2-DM patients (from −40±9 to 39±6%). Expression of arginase I, as detected by Western immunoblots and immunohistochemistry, was significantly increased in coronary arterioles of 2-DM patients. In conclusion, this study is the first to show that in coronary arterioles of patients with 2-DM arginase I expression is enhanced, which diminishes NO-mediated arteriolar dilation. Our findings allow for the feasibility that L-arginine supplementation may improve coronary endothelial function in patients with type 2 diabetes.Grants: AHA 0735540T and NIH NHLBI 43023.

This research has received full or partial funding support from the American Heart Association, Founders Affiliate (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont).
Abstract 5525: Caveolin-1 Downregulation Reduces NADPH Oxidase Assembly and Reverses eNOS Uncoupling
Authors: Lobysheva Irina , Rath Geraldine , Sekkali Belaid , Feron Olivier , Dessy Chantal , Gallez Bernard , Balligand Jean-Luc
Published: April 2024

Activation of the renin-angiotensin system generates endothelial dysfunction through increased oxidant stress. We examined the spatial co-localization of eNOS and NADPH oxidase in endothelial caveolae and the role of caveolin-1 (cav1) on eNOS activation and uncoupling in response to Angiotensin II (AII) in vitro and in vivo. NO and O2- production at the membrane of BAECs and HUVECs was measured by EPR spin-trapping (Fe(II)-[DETC]2 and DMPO, respectively). AII increased NO to 158±12% and O2- to 209±5% of control (all from min. 3 exp; P<0.05). AII-stimulated NO production was sensitive to inhibition of NADPH oxidase assembly (with apocynin, to 64±11% of ctl; P<0.05), as well as of the redox-sensitive PI3 kinase (with LY294002), cSrc (with PP2) and cAbl kinases (by siRNA targeting; all P<0.05). Reciprocally, the NOS inhibitor, L-NAME inhibited AII-stimulated O2- (by 47±11%;P<0.05), indicating eNOS uncoupling, as confirmed by increased eNOS monomer/dimer ratio. To resolve the role of eNOS and NADPH compartmentation for uncoupling, endothelial cell extracts were separated by isopycnic ultracentrifugation. Upon AII stimulation, p47phox, a NADPH subunit, was redistributed with eNOS to cav1-enriched fractions. siRNA downregulation of cav1 by ~50% while preserving eNOS confinement, inhibited AII-stimulated p47phox translocation, O2 - production (by 2.5-fold) in cav-1-enriched fractions and abrogated the L-NAME-sensitive O2 - production, indicating reversal of eNOS uncoupling. To test similar effects of moderate cav1 depletion in vivo, heterozygote cav-1+/− mice (and WT) were treated with AII infusion (osmotic minipump), their blood pressure recorded by telemetry and plasma Hb-NO measured by ESR. While AII infusion produced hypertension and decreased Hb-NO in WT (to 55±15% of ctl; p<0.05), cav-1+/− mice were fully preserved. We conclude that, in endothelial cells, co-localization of NADPH oxidase and eNOS in caveolae-enriched fractions activates eNOS through ROS-sensitive PI3K and cSrc/cAbl kinases but produces eNOS uncoupling in response to AII. Moderate downregulation of cav-1 prevents NADPH assembly and eNOS uncoupling, while preserving NO production, underlining the interest for cav-1 modulation to treat endothelial dysfunction.
Abstract 5540: Clarithromycin Attenuates Periodontal Bacteria-Induced Abdominal Aortic Aneurysms With Altered Expression of Matrix Metalloproteinases
Authors: Aoyama Norio , Suzuki Jun-Ichi , Ogawa Masahito , Izumi Yuichi , Hirata Yasunobu , Isobe Mitsuaki
Published: April 2024

Background: Matrix metalloproteinase (MMP) activity is known to be up-regulated in the abdominal aortic aneurysm (AAA). Periodontopathic bacteria were detected at a high rate of specimens of aortic walls from AAA patients. A major macrolide antibiotic, clarithromycin (CAM) has many biological functions including MMP regulation. However, little is known about the effect of CAM in AAA via MMPs. Thus, our purpose was to clarify the role of MMPs regulated by CAM in the progression of periodontopathic bacteria-induced AAA.

Methods and results: AAA was produced by periaortic application of 0.25M CaCl2 and subcutaneous infection of Porphyromonas gingivalis (Pg). We administered CAM (100mg/kg) to mice with AAA once a day for 28 days after CaCl2 application. After the infection with Pg, the plasma level of anti-Pg immunoglobulin-G was increased 2.1-fold (once infected) to 4.0-fold (repeatedly-infected) than the native. Four weeks after application of CaCl2, vehicle-treated mice showed the significant increase in aortic diameter (71±5%). However, CAM treatment significantly suppressed AAA development (12±1%). The CAM-treated mice showed the suppressed plasma level of MMP-9 (71±44 ng/ml) compared to control mice (91±22 ng/ml) using enzyme-linked immunosorbent assay (ELISA). As a compensatory action, the CAM-treated mice also exhibited less plasma level of tissue inhibitor of metalloproteinase (TIMP)-1 (1.96 ng/ml) than control (2.29 ng/ml) by ELISA.

Conclusion: These findings demonstrated that CAM prevented Pg-induced AAA development through the suppression of macrophage derived MMP production. Therefore, CAM could be clinically applicable for prevention of AAA development.
Abstract 5550: C-reactive Protein (CRP) Causes Insulin Resistance in Mice Through Fc
γ
RIIB-mediated Inhibition of Insulin Signaling
Authors: Tanigaki Keiji , Mineo Chieko , Longoria Christopher , Vongpatanasin Wanpen , Shaul Philip W
Published: April 2024

Insulin promotes the cardiovascular protective functions of the endothelium including NO production by activating endothelial NO synthase (eNOS). Insulin activation of eNOS increases skeletal muscle blood flow and thereby augments glucose uptake. C-reactive protein (CRP), which is modestly elevated in chronic inflammatory conditions including obesity, is positively correlated with type 2 diabetes. We recently showed in cultured endothelium that CRP antagonizes insulin signaling to eNOS by inhibiting the phosphorylation of Akt and eNOS, and that this occurs by CRP binding to the inhibitory IgG receptor Fc
γ
receptor IIB (Fc
γ
RIIB). In this study we determined if the antagonism of insulin signaling by CRP causes insulin resistance in mice. We found that CRP transgenic mice (TG-CRP) with CRP levels of 14±8
μ
g/ml were hyperinsulinemic, hyperglycemic, and hyperleptinemic. In addition, TG-CRP displayed impaired glucose tolerance and impaired insulin tolerance, and skeletal muscle glucose uptake in vivo was attenuated by 57%. There was no difference in energy expenditure or food intake, but TG-CRP weighed 4% less and had 62% greater fat content than controls. The role of Fc
γ
RIIB was determined in littermates from matings of TG-CRP with Fc
γ
RIIB
−/−
mice. CRP was <1
μ
g/mL in mice lacking CRP transgene and 13±2 and 10±3
μ
g/ml in Fc
γ
RIIB
+/+
TG-CRP and Fc
γ
RIIB
−/−
;TG-CRP, respectively. Compared to Fc
γ
RIIB
+/+
, fasting insulin was markedly increased in Fc
γ
RIIB
+/+
;TG-CRP; in contrast, it was normal in Fc
γ
RIIB
−/−
;TG-CRP. Whereas Fc
γ
RIIB
+/+
;TG-CRP were insulin resistant, Fc
γ
RIIB
−/−
;TG-CRP were not. Immunohistochemistry with anti-mouse Fc
γ
RIIB/Fc
γ
RIII Ab in Fc
γ
RIII
−/−
mice detected Fc
γ
RIIB protein in skeletal muscle microvascular endothelium, whereas endothelium in Fc
γ
RIIB
−/−
was negative. Fc
γ
RIIB protein was not detected in skeletal muscle cells or adipose. These findings indicate that CRP causes insulin resistance in mice that is due to impaired skeletal muscle glucose uptake and is mediated by Fc
γ
RIIB that is most likely in endothelium. Future therapies targeting Fc
γ
RIIB may potentially normalize both the endothelial dysfunction and insulin resistance characteristic of obesity and other chronic inflammatory conditions.
Abstract 5555: Shear Stress Induced Activation of MAPK Pathway and GTPases of Ras and Rho Families in a Three-layered Vascular Substitute
Authors: Basoni Caroline , Fiola Marie-Christine , Cattan Valerie , Lacroix Dan , Auger Francois A
Published: April 2024

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.