bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5571: Induction of Macrophage Activation and Foam Cell Formation by High Glucose is Mediated by Rho-associated Coiled-coil Forming Kinase
Authors: Cheng Cheng-I , Cheng Shu-Meng , Li Yuxin , Zhou Qian , Liao James K
Published: April 2024

Background:
Vascular complications are major causes of morbidity and mortality in patients with diabetes mellitus. Although the precise mechanism by which diabetes leads to vascular disease remains unknown, hyperglycemia has been shown to accelerate atherogenesis, a process involving the uptake of modified low-density lipoproteins (LDL) by macrophages and formation of macrophage-derived foam cells. We have recently shown that deletion of Rho-associated coiled-coil forming kinase (ROCK)-1 in macrophages leads to decreased LDL uptake, foam cell formation, and atherosclerosis. Therefore, we hypothesize that high glucose may enhance atherogenesis by inducing uptake of LDL by macrophages and increasing foam cell formation through activation of ROCK.

Methods & Results:
To investigate the effects of high glucose on ROCK and macrophage function, we isolated bone marrow-derived macrophage (BMDM). Treatment with high glucose but not mannitol increased ROCK activity in BMDM in a dose-dependent manner, with initial increase observed after 8 hrs. Downstream metabolites of glucose such as sorbitol and L-glutamine, as well as intracellular oxidative stress and protein kinase C all induced ROCK activation in BMDM. Indeed, co-treatment with the ROCK inhibitor, fasudil (30 uM), decreased glucose-induced oxidative stress. High glucose increased BMDM adhesion and migration, which was inhibited by co-treatment with fasudil. Proliferation and phagocytic activity of BMDM as determined by [
3
H]-tritium incorporation and zymosan uptake, respectively, were increased in high glucose culture medium, but these effects of high glucose were not affected by fasudil. High glucose (4.5 g/L) increased the size of BMDM compared to low glucose (1.0 g/L), and led to more foam cell formation in the presence of 50 ug/ml of cholesterol. In addition, the expression of TNFalpha in BMDM was increased by 2.7-fold in high glucose medium, which was suppressed by treatment with fasudil.

Conclusion:
These findings indicate that high glucose induces macrophage activation and foam cell formation is mediated by increased ROCK activity. These results suggest that targeting ROCK may have therapeutic benefits in ameliorating atherosclerosis in diabetic patients.
Abstract 5576: Nitro-fatty Acids Reduce Atherosclerosis in ApoE Deficient Mice
Authors: Rudolph Tanja K , Rudolph Volker , Edreira Martin M , Cole Marsha , Bonacci Gustavo , Schopfer Francisco J , Woodcock Steven R , Groeger Alison , Khoo Nicholas K , Freeman Bruce A
Published: April 2024

Objective
Inflammatory processes and foam cell formation are key determinants in atherosclerosis. Electrophilic nitro-fatty acid derivatives, endogenously generated by nitric oxide- and nitrite-dependent redox reactions, mediate potent anti-inflammatory signaling actions via both receptor-dependent pathways and posttranslational modification. In vitro evidence supports that nitro-fatty acids inhibit vascular inflammatory signaling, but their in vivo actions in murine models of vascular disease remains to be elucidated.

Methods and Results
Herein, we demonstrate that subcutaneous administration of nitro-oleic acid (OA-NO
2
) to apolipoprotein E deficient mice as compared to vehicle or oleic acid (OA) potently reduced atherosclerotic lesion formation in the aortic valve area (lesion area OA-NO
2
: 0.37±0.03 mm
2
; vehicle: 0.54±0.02 mm
2
; OA: 0.57±0.01 mm
2
; p<0.001) as well as in the entire aorta (% of atherosclerotic surface area: OA-NO
2
: 11.3±2.5%; vehicle: 18.5±2.6%; OA: 21.1±3.5%; p<0.001). Immunostaining and gene expression analyses revealed that OA-NO
2
significantly inhibited adhesion molecule expression (VCAM and ICAM) and decreased infiltration of inflammatory cells (% of monocyte/macrophage positive area: OA-NO
2
: 23.9±4.3%; vehicle: 48.0±2.0%; OA: 45.7±1.5%; p<0.001; number of neutrophils/field: OA-NO
2
: 10.8±4.8; vehicle: 45.3±3.9; OA: 41.5±3.4; p<0.001). Both in vitro and in vivo models showed that OA-NO
2
reduced foam cell formation by attenuating oxidized LDL induced phosphorylation of signal transducer and activator of transcription-1 (STAT-1), a transcription factor recently linked to foam cell formation in atherosclerotic plaques. In addition, atherosclerotic lesions of OA-NO
2
-treated mice had a higher content of collagen (% of trichrome positive area: OA-NO
2
: 34.3±2.6%; vehicle: 18.3±2.1%; OA: 20.5±2.7%; p<0.001) and
α
-smooth muscle actin (% of
α
-SMA positive area: OA-NO
2
: 24.0±3.0%; vehicle: 11.4±0.7%; OA: 11.9±0.6%; p<0.001), suggesting greater plaque stability. Nitro-fatty acids did not influence metabolic profile.

Conclusions
These results reveal a promising therapeutic benefit in the treatment or prevention of atherosclerosis by supplementing endogenously occurring nitro-fatty acid.
Abstract 5580: Nanolytic Approach to Prompt Revascularization in Acute Ischemic Stroke
Authors: Marsh Jon , Grace Hu , Wallace Kirk D , Hockett Frank D , Abendschein Dana R , Gaffney Patrick J , Wickline Samuel A , Lanza Gregory M
Published: April 2024

Background:
Vascular-constrained fibrin-specific nanolytic agents could permit early revascularization in the setting of acute ischemic stroke. Development of fibrin-targeted nanolytics requires optimized surface co-presentation of homing ligands and fibrinolytic enzymes on nanoparticles without compromising bioactivity.

Objective
: The study optimized human urokinase coupling and anti-fibrin homing ligand density for targeted thrombolytic efficacy and demonstrated the concept in vivo.

Methods and Results:
Minimal thiolation (N-succinimidyl-S-acetylthioacetate, SATA) of urokinase (i.e., 1 to 3 SATA/molecule) diminished human clot dissolution time relative to the native molecule; greater degrees of thiolation (3 to 6 SATA/molecule) severely decreased activity. For dog clots, which digested slower than human clots, 1 to 3 SATA/urokinase did not further impair fibrinolysis rate. Concomitant coupling of thiolated urokinase and anti-fibrin antibody (50:5, 100:10, 200:20, and 400:40 ratio, respectively) to perfluorocarbon nanoparticles resulted in optimal clot dissolution for the 100:10 and 200:20 formulations. Increasing the surface urokinase-to-antibody ratio from 0:10 to 50:10, 100:10, 200:10, and 400:10 increased particle size from 273 nm (0:10) to 340 nm at the 50:10 and 100:10 enzyme levels, and to more than 400 nm with 200 urokinase molecules per particle. Acoustic microscopy (25MHz) was used to quantify binding of the fibrin-targeted PFC nanoparticles to acoustically transparent human clots. Fibrin targeting with 10 antibodies per particle provided minimal acoustic enhancement in comparison with the relatively increased (p<0.05) but equivalent signals obtained with 20 or 40 antibodies per particle. Dissolution of canine femoral clots, created by electrical injury, was monitored with low MI ultrasound under open circulation conditions following in situ targeting with the nanolytic system. Substantial subjective clot dissolution and fragmentation was observed in three animals over 60 minutes, demonstrating the nanolytic concept.

Conclusion:
Nanolytic agents can target fibrinolytic enzymes to lyse nascent thrombus, which may permit early revascularization in acute ischemic stroke with minimal hemorrhagic risk.
Abstract 5598: Notch Signaling in Intrinsic Stem Cell is a Prerequisite for Therapeutic Angiogenesis
Authors: Tateno Kaoru , Minamino Tohru , Komuro Issei
Published: April 2024

Cell transplantation became a prevalent method to induce therapeutic angiogenesis for patients with ischemic burden. However, its mechanism has been a matter of debate. We previously reported that therapeutic limb angiogenesis by peripheral blood mononuclear cells (PBMNC) transplantation was accomplished through enhancement of muscle regeneration process, which essentially coincides with massive production of angiogenic growth factors. Here, we show that Notch signaling in skeletal myoblasts is critical to initiate this therapeutic process. PBMNC instantly induced robust proliferation of interstitial cells in ischemic tissue. In sections, majority of these cells stained double-positive for myoblast markers and activated Notch-1 receptor. Production of cytokines and myogenic growth factors by host tissue, and subsequent recruitment of activated macrophages, were also observed as acute effects after PBMNC transplantation, and was shown to be crucial for adequate muscle regeneration and angiogenesis in the later phase. Such beneficial effects by PBMNC were abolished in myoblast null Pax7 knockout mice, indicating a critical role of intrinsic stem cells in therapeutic angiogenesis. Importantly, treatment of wild type ischemic host with a Notch inhibitor markedly reduced the ratio of myoblasts among the proliferating interstitial cells, resulting in severe impairment of both acute and late-phase effects by PBMNC transplantation. Notch-1 conditional knockout mice showed similar results after PBMNC transplantation, further confirming the importance of host Notch signaling. Interestingly, deletion of a Notch ligand Jagged-1 from the donor PBMNC resulted in marked attenuation of muscle regeneration and neovascularization. Conversely, over expression of Jagged-1 in the implanted cells significantly increased the efficacy of this treatment. Taken together, we conclude that activation of Notch signaling in myoblasts is crucial for tissue regeneration by cell therapy. Additionally, we provide a model showing that activation of intrinsic stem cells, through exogenous introduction of Notch ligands, could be a novel strategy to enhance tissue regeneration in a therapeutic context.
Abstract 5607: Risk of Re-hospitalization for Patients Using Clopidogrel With a Proton Pump Inhibitor
Authors: Stockl Karen M , Le Lisa , Zakharyan Armen , Harada Ann S , Solow Brian K , Addiego Joseph E , Ramsey Scott D
Published: April 2024

Background:
Recent pharmacodynamic and retrospective clinical analyses have suggested that proton pump inhibitors (PPIs) may modify the antiplatelet effects of clopidogrel.

Methods:
We conducted a retrospective cohort study of persons enrolled in a large multi-state health insurance plan with commercial and Medicare clients to evaluate adverse clinical outcomes for patients using clopidogrel plus PPI compared to clopidogrel alone. Using propensity scoring methodology, patients discharged from the hospital for myocardial infarction (MI) or coronary stent placement and treated with clopidogrel plus PPI were matched 1:1 with patients with similar cardiovascular risk factors treated with clopidogrel alone. Re-hospitalizations for MI or coronary stent placement were evaluated over a period of up to 360 days. A subanalysis was conducted to study the impact of pantoprazole, the most prescribed PPI.

Results:
Rates of re-hospitalization for MI or coronary stent placement were higher for clopidogrel plus PPI versus clopidogrel alone (Table

). Patients receiving clopidogrel plus PPI had 92% higher risk of re-hospitalization for MI and 53% higher risk of re-hospitalization for MI or coronary stent placement than patients receiving clopidogrel alone. Increased risk of re-hospitalization for MI or coronary stent placement was also observed for the subgroup of clopidogrel plus PPI patients receiving pantoprazole.

Conclusion:
Patients receiving clopidogrel plus PPI had significantly higher risk of re-hospitalization for MI or coronary stent placement than patients receiving clopidogrel alone. Prospective clinical trials and laboratory analyses of biochemical interactions are warranted to further evaluate the potential impact of PPIs on the efficacy of clopidogrel.

Rates and Risk of Hospitalization for MI or Coronary Stent Placement
Abstract 5644: Adenosine Diphosphate Signaling in Vascular Endothelial Cells: A Novel Role for AMP-activated Protein Kinase in eNOS Regulation
Authors: Hess Connie N , Kou Ruqin , Li Gordon K , Michel Thomas
Published: April 2024

Adenosine diphosphate (ADP) responses underlie therapeutic approaches to many cardiovascular diseases, yet the pathways of ADP signaling in endothelial cells remain incompletely understood. In cultured aortic endothelial cells, we found that ADP rapidly promotes phosphorylation of eNOS at Ser
1179
(4.7±0.7-fold increase; p<0.05; n=4) and Ser
635
(4.4±0.5-fold increase; p<0.01; n=3), as well as dephosphorylation of eNOS at Ser
116
(51±2% decrease; p<0.01; n=4), with an EC
50
of ~15
μ
M. eNOS enzyme activity is stimulated by both ADP (3.2±0.1-fold increase; p<0.05; n=6) and ATP (3.0±0.1-fold increase; p<0.05; n=4). However, only ADP but not ATP signaling is inhibited by the P2Y1 receptor antagonist MRS 2179 and by siRNA-mediated knockdown of P2Y1. These data distinguish ADP from ATP responses. We analyzed signaling pathways involving PI3K/Akt, ERK1/2, Src, phospholipase C, and calcium/calmodulin-dependent kinase kinase β (CaMKKβ), using the inhibitors wortmannin, PD90859, PP2, U-73122, and STO-609, respectively. None of these inhibitors alters ADP-modulated eNOS phosphorylation. siRNA-mediated knockdown of AMP-activated protein kinase (AMPK) does not affect ADP-dependent eNOS Ser
1179
phosphorylation but significantly inhibits eNOS Ser
635
phosphorylation and eNOS activity (52±2% decrease; p<0.01; n=5). Importantly, the AMPK enzyme inhibitor compound C has no effect on ADP-induced eNOS activity, despite completely blocking AMPK activity. Similarly, CaMKKβ knockdown suppresses ADP-stimulated eNOS activity, while complete inhibition of CaMKKβ kinase activity using STO-609 has no effect on ADP signaling to eNOS. ADP activates the small GTPase Rac1 and promotes endothelial cell migration (2.5±0.9-fold increase; p<0.001; n=4). ADP-promoted cell migration is abrogated by siRNA-mediated knockdown of Rac1. Notably, siRNA knockdown of Rac1 blocks ADP-mediated eNOS Ser
1179
and Ser
635
phosphorylation (43±8% decrease; p<0.05; n=4) as well as eNOS activation (55±14% decrease; p<0.05; n=4). These data suggest that the expression- but not the kinase activity- of AMPK and CaMKKβ is necessary for ADP signaling to eNOS. These findings also establish a novel role for Rac1 in ADP regulation of eNOS activity and cell migration in endothelial cells.
Abstract 5660: Essential Role of Non-muscle Myosin Light Chain Kinase in Hypoxia-induced Murine Pulmonary Hypertension
Authors: Moreno-Vinasco Liliana , Mirzapoiazova Tamara , Zaidi Rafe , Sammani Saad , Machado Roberto , Garcia Joe G
Published: April 2024

Introduction: The
pulmonary arterial hypertension (PAH) phenotype shares several pathways involved in cancer pathobiology. We previously demonstrated that the multi-kinase inhibitor, sorafenib, ameliorates rodent PAH
(Moreno-Vinasco et al 2008)
with genomic signatures which strongly implicated the endothelial cell cytoskeleton in the development of PAH vascular remodeling including non-muscle caldesmon, Rho-kinase and both smooth muscle- and non-muscle myosin light chain kinase (nmMLCK) isoforms. We hypothesized that the acto-myosin machinery present within lung endothelium are critical to the development of PAH in a murine model of hypoxia-induced pulmonary hypertension.

Methods:
Wild type (WT) and genetically engineered mice with targeted deletion of the nmMLCK allele were exposed to normoxia, or to hypoxia and SU5416, a VEGFR-1,2 inhibitor that induces severe PH (3.5 weeks). WT and nmMLCK KO mice were assessed for hemodynamics and histopathology.

Results:
Hypoxia-SU5416 WT mice developed pulmonary artery phenotype with significant increases in diphosphorylated MLC levels (2-fold, no change in total MLC), increased mean right ventricular systolic pressures (RVSP) and hypertrophy (RV/LV+S) and increased hematocrit when compared to WT-normoxic controls. However, in response to hypoxia/SU, nmMLCK-KO mice displayed reduced pMLC, significant decreases in right ventricular hypertrophy (27.3% decrease), in RVSP (25% decrease) without changes in hematocrit. Histopathology indicates reduced peri-vascular infiltration and remodeling in the nmMLCK KO mice compared to WT controls.

Conclusions:
Our results are consistent with the notion that myosin light chains (MLC) are a canonical and downstream mediator of mechanical events underlying the actin-myosin interactions in lung endothelium during PH remodeling and nmMLCK is a potential therapeutic target in PAH-mediated remodeling.
Abstract 5663: Imatinib Inhibits Proliferation and Migration and Induces Apoptosis in Pulmonary Artery Smooth Muscle Cells From Patients With Idiopathic Pulmonary Arterial Hypertension
Authors: Nakamura Kazufumi , Akagi Satoshi , Matsubara Hiromi , Ogawa Aiko , Miura Aya , Miura Daiji , Nishii Nobuhiro , Nagase Satoshi , Morita Hiroshi , Hata Yoshiki , Kusano Kengo F , Ito Hiroshi
Published: April 2024

Background
: Idiopathic pulmonary arterial hypertension (IPAH) is characterized by inappropriate increase of pulmonary artery smooth muscle cells (PASMCs). We assessed the inhibitory effects of imatinib, a platelet-derived growth factor (PDGF)-receptor tyrosine kinase inhibitor, on PDGF-induced proliferation and migration of PASMCs obtained from patients with IPAH, and investigated whether imatinib induces apoptosis of PASMCs.

Methods and Results
: PASMCs were obtained from 7 patients with IPAH undergoing lung transplantation. PDGF (10 ng/mL) stimulation caused a higher growth rate of PASMCs from patients with IPAH than that of normal control PASMCs (n=6) as assessed by
3
H-thymidine incorporation (normal: 179±45 vs. IPAH: 310±31 % of counts before PDGF,
P
<0.05). After treatment with imatinib (0.1 and 1
μ
g/mL), PDGF-induced cell proliferation of PASMCs from IPAH patients decreased by 51% and 90 % respectively (PDGF vs PDGF+imatinib (0.1 and 1
μ
g/mL):
P
<0.001). Western blot analysis revealed that imatinib (1
μ
g/mL) increased the expression of cyclin-dependent kinase inhibitor p27 in IPAH-PASMCs compared with treatment with PDGF (53%; PDGF vs. PDGF+imatinib,
P
<0.005). A time-lapse system revealed that PDGF increased the migration distance of IPAH-PASMCs compared to that of normal PASMCs (
P
<0.0001), and imatinib (1
μ
g/mL) inhibited PDGF-induced migration (
P
<0.0001). TUNEL assay revealed that imatinib (1
μ
g/mL) alone did not induce apoptosis in IPAH-PASMCs, but combination of imatinib and PDGF increased apoptotic cells (Figure

).

Conclusions:
Inhibition of PDGF signaling by imatinib may become a useful molecular-targeted therapy for IPAH.

Figure.

Effect of imatinib on apoptosis of PASMICs in Tunnel assay.
Abstract 5761: Anti-thrombotic and Anti-platelet Therapies for Acute Myocardial Infarction in Dialysis Patients: A Collaborative Project of the United States Renal Data System and National Registry of Myocardial Infarction
Authors: Shroff Gautam R , Frederick Paul , Herzog Charles A
Published: April 2024

Background:
Dialysis pts sustain high mortality rates from acute myocardial infarction (AMI) but were excluded from trials evaluating therapeutic strategies in AMI.

Aims:
To compare the utilization of anti-thrombotic (AT) and anti-platelet (AP) therapies during AMI in dialysis vs. non-dialysis pts and the relation between outcomes of dialysis pts and the use of AT and AP therapies.

Methods:
Retrospective cohort study using cross-matched data from the US Renal Data System database and 3rd National Registry of Myocardial Infarction for AMI hospitalizations between 04/01/1998 and 06/30/2000. A cohort of 3049 matching dialysis pts in both databases were identified; pts transferred in or out of the index hospital were excluded, leaving 2390 dialysis and 304096 non-dialysis pts for analysis.

Results:
Table 1

describes utilization of AT and AP therapies in dialysis pts with AMI. Table 2

details likelihood of adverse outcomes relative to the use of AT and AP agents.

Conclusions:
AT and AP therapies are underutilized in dialysis pts with AMI. Aspirin use is associated with marked reduction in mortality and major cardiac events in both dialysis and non-dialysis pts. In contrast to non-dialysis patients, GP IIb/IIIa antagonist use is associated with a 3 fold increase in major bleeding in dialysis pts without any reduction in risk of cardiac events; and UF and LMW heparin use are also not associated with any reduction in mortality and cardiac events. Optimal AT and AP strategies in dialysis pts need to be further examined in prospective studies.
Abstract 5778: Protein Phosphatase 1 Signalling Pathway is Involved in the Control of Vascular Smooth Muscle Cells Proliferation
Authors: Hadri Lahouraria , Karakikes Ioannis , Merlet Elise , Lompré Anne-Marie , Kranias Evangelia , Hajjar Roger J , Lipskaia Larissa
Published: April 2024

Protein phosphatase 1 (PP1), the main regulator of the transcription factor CREB, is strongly involved in the control of cell proliferation via p53 and p21. PP1 is regulated by phosphatase inhibitor-1 (I-1), which is activated by PKA phosphorylation and inhibited by protein phosphatase PP2A and PP2B (calcineurin). I-1 is expressed in vascular smooth muscle cells (VSMC), but does not appear to play a significant role in contractile or relaxant response We tested whether the I-1/PP1 pathway is involved in the control of VSMC proliferation Confocal immunofluorescence, Western blot analysis and real time PCR demonstrated that I-1 protein was expressed in the media layer of healthy human coronary and mammary arteries and down regulated in atherosclerotic vessels and proliferating human VSMC (1000 fold lower of mRNA). By contrast, PP1 expression was increased in proliferating human VSMC. Inhibition of I-1/PP1 signalling by siRNA PP1 or by adenoviral gene transfer of a constitutively active I-1 (I-1c) inhibited VSMC proliferation and migration in vitro. Promoter-reporter assay showed that in vitro I-1c gene transfer increased activity of CREB. Western blot analysis demonstrated that I-1c and siRNA PP1 increased phosphorylation of CREB, expression of p21, p53 and led to down regulation of cell cycle controlling proteins cyclin D1 and pRB. To assess the potential effect of I-1c gene transfer in the treatment of restenosis, we used a rat carotid artery injury model. I-1 was expressed in normal carotid artery but it was non-detectable two weeks after injury, which correlated with vascular remodelling during neointimal proliferation. Adenoviral gene transfer of I-1c prevented neointimal proliferation in the carotid injury model. In conclusion, the I-1/PP1 signalling pathway is involved in the control of VSMC proliferation and migration via the transcription factor CREB.
Abstract 5785: A Novel Function of Regulator of G-protein Signaling 2: Stabilization of Signal Transducer and Activator of Transcription 3 Phosphorylation
Authors: Yue Hong , Desnoyer Russell , Li Wei , Karnik Sadashiva
Published: April 2024

Background: Regulator of G protein signaling proteins (RGS) are known to regulate G protein coupled receptor (GPCR) signaling at the plasma membrane through their GTPase-activating protein activity. We have previously identified RGS2 as a downstream target of unphosphorylated Signal Transducer and Activator of Transcription 3 (U-STAT3). U-STAT3 mediated Angiotensin II (AngII) type 1 receptor (AT1R), a GPCR, induced cardiac hypertrophy but the mechanism is unclear.

Hypothesis: RGS2 is the mediator of U-STAT3 regulated Ang II/AT1R signaling.

Methods and Results: In AT1R overexpressing HEK293 cells and HL-1 cells (a mouse cardiomyocyte cell line), Ang II stimulation induced RGS2 expression in parallel with U-STAT3. The localization, distribution and expression of RGS2 were changed upon Ang II stimulation. RGS2 was mainly localized in the nucleus, which was also confirmed in human vascular smooth muscle cells. Gene transfection of RGS2 dose-dependently increased phosphorylated STAT3 (P-STAT3), and maintained a high level of P-STAT3 up to 24 hrs. Co-immunoprecipitation assay identified a complex formed between RGS2 and P-STAT3, suggesting that RGS2 plays a role in maintaining P-STAT3 stability. Treatment with AT1R antagonist, losartan, decreased RGS2/P-STAT3 interaction, suggesting that this interaction is dependent on AT1R activation. On the other hand, in cells overexpressing AT1R and Y705F mutant STAT3, which cannot be phosphorylated on residue 705 (U-STAT3), chromatin immunoprecipitation assay demonstrated that Y705F mutant STAT3 binds to a consensus GAS motif located in the RGS2 promoter, providing direct evidence that RGS2 is a downstream target of U-STAT3. siRNA-mediated gene knockdown of STAT3 consistently caused decreased RGS2 expression in AT1R overexpressing cells.

Conclusions: Chronic activation of AT1R induced U-STAT3 overexpression and subsequently induced overexpression of RGS2, which interacted with and stabilized P-STAT3. P-STAT3 further increases the transcription of stat3 gene, forming a positive feedback loop. We reported for the first time a novel function of RGS2, stabilization of P-STAT3, in mediating U-STAT3 regulated Ang II/AT1R signaling. Further studies are now underway utilizing engineered mice model.

This research has received full or partial funding support from the American Heart Association, Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).
Abstract 5787: Discovery and Characterisation of Linear and Cyclic Peptide Agonists of Apelins
Authors: Davenport Anthony P , Pitkin Sarah L , Maguire Janet J , Macaluso Maximilian , Glen Robert C
Published: April 2024

The recently discovered apelin family of peptides mediate their actions by a single G-protein coupled receptor, APJ. We have previously shown apelins have three major actions in the human cardiovascular system: endothelium-dependent vasodilatation; direct vasoconstriction by interacting with smooth muscle APJ receptor and increased cardiac contractility by action on cardiac myocytes. [Pyr
1
]apelin-13 was identified as the predominant isoform in human heart. Our aim was to discover shorter sequences of [Pyr
1
]apelin-13 retaining agonist activity and use a computational ligand based strategy to design cyclic peptides. Over 30 compounds were synthesised and tested in a competition binding assay against [Glp65,Nle75,Tyr77] [
125
I]Apelin-13. Functional assays measured the vaso-constrictor action in endothelium denuded human saphenous veins in oxygenated Krebs’ solution (37°C) in organ baths for isometric tension recordings. Cumulative concentration response curves were constructed to the endogenous and synthetic analogues. Responses were expressed as percentage of the terminal KCl (0.1M) constriction. Data are expressed as mean ± S.E. Mean and n-values are the number of patients from whom tissues were obtained. One micromolar concentrations of the predicted cleavage product of the angiotensin converting enzyme 2 (ACE-2), apelin-13(1–12), inhibiting radiolabelled apelin binding by 60.0±7.0% (n=3). The most potent cyclic analogue identified, MM07 inhibited binding by 42.2±4.8% (n=3) compared with 76.0±4.5% inhibition by (Pyr
1
)apelin-13. Both compounds potently constricted endothelium denuded saphenous vein (Apelin-13(1–12): pD
2
9.07±0.40, E
max
29.30±9.43% KCl, n=5; MM07: pD
2
10.53±0.24, E
max
21.80±5.72% KCl, n=3) with a comparable potency and efficacy to (Pyr
1
)apelin-13 (pD2 (pD
2
= 8.8±0.3, E
MAX
= 26±4%, n=15). These results show unexpectedly that the cleavage product of ACE-2 retains full agonist activity. The novel cyclic peptide retains potency and can be used as a pharmacological tool to characterise the apelin system and in the design of selective antagonists.
Abstract 5790: High Levels of Homocysteine and D-dimer Are Associated With Faster Functional Decline Among Men and Women With Lower Extremity Peripheral Arterial Disease
Authors: McDermott Mary M , Liu Kiang , Ferrucci Luigi , Tian Lu , Guralnik Jack , Tao Huimin , Ridker Paul , Criqui Michael
Published: April 2024

BACKGROUND.
We assessed associations of baseline biomarker levels with functional decline among participants with lower extremity peripheral arterial disease (PAD).

METHODS.
Participants were 352 men and women with an ankle brachial index (ABI) < 0.90 who attended a baseline and up to three annual follow-up visits. Baseline levels of homocysteine, C-reactive protein (CRP), interleukin-6 (IL-6), and D-dimer were obtained. Participants performed a six-minute walk test and the short physical performance battery (SPPB) at baseline and annually. The SPPB is a composite measure of lower extremity functioning scored on a 0 –12 scale (12= best). Outcomes were average annual decline in six-minute walk distance, average annual decline in the SPPB, and mobility loss. Mobility loss was defined as becoming unable to walk 1/4 mile or walk up and down one flight of stairs without assistance, among those without baseline mobility limitations. Results adjust for age, sex, race, body mass index, ABI, smoking, comorbidities, and other potential confounders.

RESULTS
. The 352 participants were average age 75±8 and 47% were women. The average ABI was 0.63 ± 0.16 and 33% had diabetes mellitus. Average baseline six-minute walk distance and SPPB scores were 1,101 feet ± 432 and 9 ± 3, respectively. Adjusting for confounders, higher CRP levels were associated with more rapid decline in six-minute walk performance (1
st
(lowest) Tertile -50 feet/year; 2
nd
Tertile -44 feet/year; 3
rd
(highest) Tertile -65 feet/year, p trend value = 0.04). Higher CRP levels were also associated with more rapid decline in the SPPB (1
st
(lowest) Tertile 0.3/year; 2
nd
Tertile -0.3/year; 3
rd
(highest) Tertile -0.5/year, p trend = 0.02). Similarly, higher levels of homocysteine were associated with more rapid decline in the SPPB (1
st
Tertile 0.3/year; 2
nd
Tertile -0.4/year; 3
rd
Tertile -0.5/year, p trend = 0.03). Higher levels of homocysteine were also associated with higher rates of mobility loss, p trend = 0.04). IL-6 and D-dimer were not significantly associated with declining functional performance.

CONCLUSIONS
. This study demonstrates, for the first time, that higher baseline levels of CRP and homocysteine are associated with faster rates of decline in functional performance among men and women with PAD.
Abstract 5798: Nanoparticle-mediated Endothelial Cell-selective Delivery of Pioglitazone Improves Therapeutic Efficacy of Ischemic Neovascularization
Authors: Nagahama Ryoji , Matoba Tetsuya , Nakano Kaku , Hara Kaori , Tsujimoto Hiroyuki , Sunagawa Kenji , Egashira Kensuke
Published: April 2024

Background: Recent studies suggest that a peroxisome proliferator activated receptor-
γ
(PPAR
γ
) agonist pioglitazone reduces the incidence of ischemic cardiovascular events. However, it is unknown if pioglitazone induces therapeutic neovascularization after induction of ischemia. Moreover, novel drug delivery system may alleviate potential adverse effects of systemic administration of PPAR
γ
agonists. We recently reported that intramuscular injection of biodegradable polymeric NP resulted in cell-selective delivery of NP into vascular endothelial cells of ischemic muscles for 2 weeks post-injection in a murine model of hindlimb ischemia. Hence, we tested the hypothesis that nanoparticle (NP)-mediated endothelial cell selective delivery of pioglitazone is an effective therapy to induce ischemic neovascularization in vivo.

Methods and Results: In a murine model of hindlimb ischemia, systemic oral administration of pioglitazone at a dose of 1000
μ
g/kg per day, but not other lower doses, enhanced recovery of blood perfusion to the ischemic limb, increased angiogenesis and arteriogenesis 3 weeks after induction of ischemia (Figure A

). In contrast, single intramuscular injection of NP incorporated with pioglitazone at 1 or 10
μ
g/kg into ischemic muscles immediately after induction of ischemia significantly enhanced ischemic neovascularization at 3 weeks post-ischemia (Figure B

). Intramuscular injection of PBS, FITC-NP, or pioglitazone only at 1–10
μ
g/kg showed no effects on blood perfusion to the ischemic limb.

Conclusion: NP-mediated endothelial cell-selective delivery of pioglitazone may effectively induce therapeutic neovascularization.

A. Effects of Systemic Oral Administrate of Pioglitazone for 21 days

B. Effects of Single Intramuscular Injection of Pioglitazone-NP
Abstract 5803: Interaction of Docking Protein Gab1 With Tyrosine Phosphatase SHP2 Regulates Hepatocyte Growth Factor-dependent ERK5 Activation and Upregulation of KLF2 in the Vascular Endothelium
Authors: Shioyama Wataru , Nakaoka Yoshikazu , Higuchi Kaori , Minami Takashi , Hamakubo Takao , Arita Yoh , Yamamoto Kaori , Kidoya Hiroyasu , Naito Hisamichi , Takakura Nobuyuki , Fujio Yasushi , Yamauchi-Takihara Keiko , Hirano Toshio , Kodama Tatsuhiko , Mochizuki Naoki , Komuro Issei
Published: April 2024

Background
: Docking protein Gab1 has been reported to have crucial roles for activation of both mitogen-acitvated protein kinase and AKT downstream of various receptor tyrosine kinases. In the last scientific session, we reported that endothelium-specific Gab1 knockout mice (Gab1ECKO) displayed severe limb necrosis after operatively-induced hindlimb ischemia (HLI) due to the defect of angiogenic response. We also reported that Gab1 was essential for activation of both extracellular signal-regulated kinase 1/2 (ERK1/2) via tyrosine phosphatase SHP2 and AKT via PI3-kinase p85 subunit in human umbilical vein endothelial cells (HUVECs) in response to hepatocyte growth factor (HGF), respectively. Here, we investigated the downstream target of the HGF/Met/Gab1-dependent signaling pathway responsible for the maintenance of endothelium.

Methods and Results
: We examined the effect of adenovirus-mediated overexpression of
β
-galactosidase (
β
-gal), wild-type Gab1 (Gab1
WT
), or mutated Gab1 which can’t bind with SHP2 (Gab1
ΔSHP2
) or p85 (Gab1
Δp85
) on the activation of downstream signaling pathways in HUVECs. HGF-induced activation of both ERK1/2 and ERK5 was enhanced in the HUVECs expressing Gab1
WT
or Gab1
Δp85
, but abrogated in those expressing Gab1
ΔSHP2
, compared with control cells expressing
β
-gal. HGF-induced AKT activation was specifically attenuated in cells expressing Gab1
Δp85
, compared with other three groups. Using DNA microarray analysis, we found that the stimulation with HGF upregulated the expression of Kruppel-like factor 2 (KLF2), a crucial transcriptional factor for endothelial maintenance, and thrombomodulin, a well-known target gene of KLF2, through interaction of Gab1 with SHP2 in HUVECs. Next, we examined the involvement of MEK5-ERK5 pathway for the KLF2 gene expression. HGF-dependent upregulation of KLF2 was almost abrogated by the adenoviral overexpression of either dominant-negative MEK5 or dominant-negative ERK5 in HUVECs. Furthermore, the expression of KLF2 and TM in the endothelium was significantly decreased in the Gab1ECKO, compared with control after HLI.

Conclusion
: Interaction of Gab1 with SHP2 is essential for both activation of ERK5 and upregulation of KLF2 in response to HGF in the endothelium.
Abstract 5804: Embryonic Stem Cell-Derived Endothelial Cells Engraft Into the Ischemic Hindlimb and Restore Perfusion
Authors: Huang Ngan F , Niiyama Hiroshi , Peter Christoph , De Abhiiit , Natkunam Yasodha , Fleissner Felix , Li Zongjin , Rollins Mark D , Wu Joseph C , Gambhir Sanjiv S , Cooke John P
Published: April 2024

Peripheral arterial disease (PAD), which affects about 10 million Americans, is typically due to atherosclerotic arterial occlusive disease of the limbs and is associated with dysfunctional vascular endothelium. Cell-based therapy using endothelial cells derived from embryonic stem cells (ESC-ECs) is a promising approach for regenerating the endothelium. However, studies that characterize the localization and engraftment of embryonic stem cells (ESCs) and ESC-ECs into the ischemic limb are limited. In this study, we examined the survival, localization, and functional effects of exogenously administered ESC-ECs for repair of hindlimb ischemia, which is a murine model of PAD. Murine ESCs or ESC-ECs were stably transduced with a construct for bioluminescence imaging (BLI) and fluorescent detection. In a syngenic murine model of hindlimb ischemia, ESCs or ESC-ECs were delivered by intramuscular (IM), intra-femoral artery (IA), or intra-femoral vein (IV) injections (n=5 in each group). For 2 weeks, cell survival and localization were tracked by BLI and confirmed by gene expression, and functional improvement was assessed by laser Doppler blood perfusion. BLI results showed ESC persisting in the ischemic limb after IM or IA, but not after IV administration. Regardless of the route of administration, ESCs were detected outside of the hindlimb circulation in the spleen or lungs. ESCs did not improve limb perfusion, and induced teratomas. In contrast, ESC-ECs localized to the ischemic limb in all cases as assessed by BLI. Immunohistochemical studies confirmed the engraftment of ESC-ECs into the limb vasculature after 2 weeks. Intriguingly, ESC-ECs were not detected in the spleen or lungs after 2 weeks, even after IV administration. Furthermore, ESC-ECs improved limb perfusion. In comparison to parental ESCs, ESC-ECs preferentially localized in the ischemic hindlimb, even after IV delivery. ESC-EC localization was temporally related to the improvement in hindlimb perfusion, which was associated with engraftment of ESC-ECs into the ischemic microvasculature.

This research has received full or partial funding support from the American Heart Association, Western States Affiliate (California, Nevada & Utah).
Abstract 16636: The Procardiomyogenic Effect of miR499 is Synergistically Enhanced by miR133
Authors: Cervio Elisabetta , Barile Lucio , Pisano Federica , Altomare Claudia , Malpasso Giuseppe , Ciuffreda Maria Chiara , Zaza Antonio , Gnecchi Massimiliano
Published: April 2024

Background:
it has been described that miR499, 1 and 133 are involved in cardiac development and stem cell differentiation. In particular, miR499 enhances the differentiation of embryonic and adult stem cells into cardiomyocytes (CMC). However, it is currently unknown if different miR may act synergistically to improve differentiation efficiency.

Methods:
we used mouse P19 cells as cardiogenic differentiation model. P19 cells were transduced with a viral vector expressing EGFP under the control of a cTnI promoter. miR499 was transiently overexpressed in P19 cells individually or together with miR1 or miR133. Cardiac differentiation was quantified by: 1) count of spontaneous beating areas; 2) qPCR of the cardiac markers Nkx2.5, GATA4, MEF2c, MLC2v, Cx43 and cTnT; 3) Western Blot and ICC of cTnT and Cx43; 4) measure of inducibility of RyR-mediated Ca++ transients in P19 exposed to caffeine; 5) recording of mechanical activity after Ca++ depletion and in the presence of nifedipine or ryanodine.

Results:
the overexpression of miR499 alone increased the number of beating CMC by 1.7 fold compared with P19 CTRL (p<0.05). The association of miR499+133, but not miR499+1, further increased the number of beating CMC (2.02 fold vs. P19 CTRL, p<0.01). qPCR showed that the combination of miR499+133 increase the expression of GATA4, Nkx2.5, Cx43 and cTnT compared with P19 CTRL (p<0.01) and MEF2c and MLC2v compared with miR499 alone (p<0.05). Western Blot and ICC for Cx43 and cTnT confirmed these findings. Caffeine responsiveness was increased by 2.6 fold by miR499+133 compared with P19 CTRL (32.5 vs. 12.5%, p<0.05) and was directly correlated with the activation of the cTnI promoter. Cyclic contractions were reversibly abolished by extracellular Ca++ depletion and by both nifedipine and ryanodine. Finally, the overexpression of miR499+133 is associated with an earlier activation of cTnI promoter.

Conclusions:
our results show that concomitant overexpression of miR499 and miR133 significantly enhance cardiac differentiation. Most importantly the functional studies have demonstrated that the areas spontaneously contracting possess electrophysiological and pharmacological characteristics compatible with true cardiac excitation-contraction coupling.
Abstract 16640: Foxo1 Impairs Whereas Statin Protects Endothelial Function in Hyperglycemic Conditions Through Reciprocal Regulation of Kruppel-like Factor 2
Authors: Lee Hae-Young , Youn Seock-Won , Cho Hyun-Jai , Kwon Yoo-Wook , Lee Sae-Won , Park Young-Bae , Oh Byung-Hee , Kim Hyo-Soo
Published: April 2024

Endothelial dysfunction is an initial critical factor in the pathogenesis of diabetic vascular complications. Krüppel-like factor 2
(
KLF2) is implicated as a key molecule maintaining endothelial function. We investigated the role of a forkhead transcription factor, FOXO1, and HMG-CoA reductase inhibitor in KLF2 regulation in hyperglycemic conditions. FOXO1 expression was increased by 190% (p<0.05), whereas KLF2 and eNOS expression were decreased by 60% (p<0.05) and by 70% (p<0.05) in human umbilical vein endothelial cells incubated in 30 mM glucose for 24 h, all of which were completely reversed by FOXO1 siRNA. Adenovirus-mediated overexpression of FOXO1 decreased the expression of KLF2 and eNOS by 49% and 69% (p<0.05). Atorvastatin (1 μM) inhibited FOXO1 not only by increasing phosphorylation by 170% (p<0.05) but also by degrading FOXO1 protein by 70% (p<0.05). Atorvastatin also restored KLF2 expression suppressed in high glucose conditions similar to the control level (92.3 ± 3.4%, p=0.1), which was totally blocked by co-incubation with mevalonate. A chromatin immunoprecipitation analysis demonstrated that glucose and atorvastatin reciprocally regulated KLF2 at transcription level by modulating binding of FOXO1 to its promoter regions at -609 and -754bp.
In vivo
experiment with the OLETF rats, the animal models of type 2 diabetes, FOXO1 in the endothelial lining of the thoracic aorta was mainly located in the nucleus and KLF2 (41.5±3.9%, p<0.05) and eNOS (62.5 ± 1.8%, p<0.05) expressions were significantly decreased compared with the control LETO rats, which were reversed by atorvastatin administered with 5 mg/kg/day for > 4 weeks (KLF2 expression by 157.3 ± 37% and eNOS expression by 112.5 ± 27% of the control values). The endothelium-dependent vasodilation with acetylcholine were significantly decreased in OLETF rats (23.4 ± 22.8% of the LETO rats, p<0.05), which was significantly improved both by atorvastatin (67.3 ± 0.5%, p<0.05) and by adenovirus-mediated overexpression of KLF2 (61.4 ± 15.4%, p<0.05). In conclusion, KLF2 suppression by FOXO1 may be a plausible mechanism of endothelial dysfunction in diabetes. The findings of this study suggest implication of intensive statin treatment in preventing diabetic vasculopathy.
Abstract 16666: Osteopontin - A Novel Biomarker for Left Ventricular Hypertrophy and Diastolic Dysfunction
Authors: Lutz Matthias , Rosenberg Mark , Markeeva Evgenia , Heger Thomas , Katus Hugo A , Frey Norbert
Published: April 2024

Background and Hypothesis:
Osteopontin (OPN) is an extracellular matrix protein that plays an important role in myocardial remodeling. Moreover, our group has previously shown that OPN plasma levels are not only elevated in chronic systolic left heart failure but also predict an adverse prognosis. We now tested the hypothesis whether OPN plasma levels are of diagnostic value in patients with left ventricular hypertrophy (LVH) and diastolic dysfunction due to pressure overload caused by arterial hypertension.

Methods:
After exclusion of significant coronary artery disease we included 92 patients with cardiac hypertrophy and hypertensive heart disease, 34 healthy subjects served as controls. OPN plasma levels were determined by ELISA and correlated with clinical symptoms and echocardiographic as well as hemodynamic parameters of LVH and diastolic dysfunction.

Results:
Median OPN was significantly elevated in patients with LVH compared to healthy controls (682.2 ng/ml vs. 296.1 ng/ml; p 16 mmHg: 920.3 ng/ml vs. LVEDP ≤ 16 mmHg: 777.8 ng/ml; p<0.01). Additionally, OPN levels showed a positive correlation with LVEDP (r=0.32; p<0.05). Furthermore, higher OPN plasma levels were also associated with progressive enlargement of left atrial size (r=0.53; p<0.01). Consistently, OPN levels were higher in patients with atrial fibrillation (AF) compared to subjects with sinus rhythm (871.0 ng/ml vs. 627.9 ng/ml; p<0.05).

Conclusion:
Our data show for the first time that OPN plasma levels are elevated in patients with LVH and diastolic dysfunction. Moreover, increased OPN plasma levels appear to correlate with advanced cardiac remodeling and atrial fibrillation, a known trigger of morbidity and mortality in this patient population. Hence, OPN might be useful as a non-invasive biomarker in patients with LVH and diastolic dysfunction.
Abstract 16674: Using Novel Emergency Medical Services Data-Linkage to Evaluate the Timing and Quality of Pre-Hospital ST-Elevation Myocardial Infarction Care
Authors: Fosbol Emil L , Granger Chris , Lin Li , Lytle Barbara L , Mears Greg , Jollis James G , Peterson Eric D , Glickman Seth
Published: April 2024

Background:
Current national guidelines state that STEMI patients should receive primary percutaneous coronary intervention (PCI) within 90 minutes of “first medical contact” (FMC). Lacking access to emergency medical services (EMS) data, many hospitals are unprepared to implement this goal. Our objective was to utilize a novel statewide hospital/EMS data linkage to describe pre-hospital delays in care and to examine adherence to the 90 min rule.

Methods:
To assess pre-hospital patterns of care in North Carolina (NC), we linked data from the ACTION-GWTG registry and the statewide EMS Pre-hospital Medical Information System (PreMIS) from 06/2008 to 09/2010. We calculated the discrete elements of pre-hospital delay from the time of 911-activation to the time of hospital arrival using PreMIS data and in turn compared time from FMC to hospital arrival as recorded in PreMIS versus ACTION.

Results:
Analyses included 21 ACTION hospitals and 178 EMS agencies across NC. Of 6010 linked STEMI patients, 3077 (51%) arrived directly to a PCI capable hospital by EMS. Median age was 60 (IQR 52-71) and 69% were men. Calculated pre-hospital times are shown in the Table. Median total pre-hospital time interval was 42 min (IQR 33-53). Comparing PreMIS to ACTION data, time from FMC to hospital arrival was 30 min (IQR 23-40) vs. 34 min (IQR 26-45) with median difference of 3 minutes (IQR 0-7, p <.0001 by signed rank test). Among patients with available data in both data sources (n= 2201), only 53% achieved the metric of PCI within 90 min of FMC by PreMIS and 49% by the ACTION data with 12% discordance between ACTION and PreMIS (p <.0001 by McNemar's test).

Conclusions:
This work demonstrates that it is possible to link nationally representative hospital and EMS data for assessing the quality of pre-hospital STEMI care and further shortening treatment times. Pre-hospital data may also permit more accurate determination of first medical contact time than hospital data sources.