bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4930: NADPH Oxidase 2 Regulates Stem/Progenitor Cell Mobilization via Modulating Hypoxic Gradient and Proteolytic Enzymes in Bone Marrow Following Hindlimb Ischemia
Authors: Urao Norifumi , Kaplan Nihal , McKinney Ronald , Fukai Tohru , Ushio-Fukai Masuko
Published: April 2024

Bone marrow (BM) microenvironment, which is regulated by hypoxia and proteolytic enzymes, is crucial for stem/progenitor cells mobilization during postnatal neovascularization. We demonstrated that NADPH oxidase2 (Nox2)-derived reactive oxygen species (ROS) in BM play a role in ischemia-induced stem/progenitor mobilization and revascularization. However, role of Nox2 in regulating BM microenvironment remains unknown. Here we show that hindlimb ischemia of mice increases Nox2 expression (2.2-fold) and ROS production (7.2-fold) in BM cells with peak at day 3, which is cotemporaneous with increase in circulating Sca-1
+
/Flk-1
+
endothelial progenitor cells (2.0-fold). These increases are inhibited in Nox2-deficient mice. Using hypoxia bioprobe, BM section shows that endosteum region is hypoxic in basal steady-state. After contralateral hindlimb ischemia, hypoxic areas are expanded with an increase in HIF-1alpha and Nox2 expression throughout the BM peaking at day 3 in wild type (WT) mice, which are markedly reduced in Nox2-deficient mice. In parallel, hindlimb ischemia increases the expansion of cKit
+
Lin
−
cells (1.6-fold) and angiogenic CXCR4
+
VEGFR1
+
Lin
low
progenitor cells (31%) in WT mice, which are inhibited in Nox2-deficient mice. This is due to increase in apoptotic (annexin V+) Lin- cells (2.0-fold) and decrease in proliferative (BrdU+) primitive stem/progenitor cKit
+
/Sca1
+
/Lin
−
cells (36%). Moreover, hindlimb ischemia increases Akt phosphorylation (2.2-fold), MT1-MMP (4.2-fold) expression, and decreases its membrane-anchored inhibitor, RECK (62%) in BM, which are inhibited in Nox2-deficient mice. In summary, Nox2-derived ROS in BM regulate expansion of stem/progenitors, thereby promoting local hypoxia that increases HIF-1alpha in response to hindlimb ischemia. They also activate proteolytic enzyme activity in BM, which may facilitate mobilizing stem/progenitor cells leading to reparative neovascularization.

This research has received full or partial funding support from the American Heart Association, Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).
Abstract 4941: Dietary Alpha-Linolenic Acid Supplementation Inhibits Thrombus Formation in vivo
Authors: HOLY Erik , Beer Jürg H , Forestier Marc , Akhmedov Alexander , Mocharla Pavani , Luscher Thomas F , Tanner Felix C
Published: April 2024

Background.
Considering the reduced availability of marine derived long-chain n-3 fatty acids (n-3 FA), the use of the plant-derived n-3 FA
α
-linolenic acid (ALA) may constitute an attractive cardioprotective alternative. However, despite promising epidemiological data, the effect of dietary ALA supplementation on arterial thrombus formation remains unknown.

Methods and Results.
8 week-old male C57Bl/6 mice were fed either a high ALA (7.3g%) or low ALA (0.03g%) diet for 2 weeks. Mice fed a high ALA diet showed delayed carotid artery thrombus formation in a mouse photochemical injury model as compared to mice on a low ALA diet (68±6 vs 40±4.2 minutes; n=7; p<0.05). High dietary ALA supplementation impaired platelet aggregation as determined by whole blood aggregometry in isolated murine platelets (n=5; p<0.005), without affecting PTT and aPTT clotting times (n=7; p=NS). Mice on the high ALA diet exhibited reduced vascular tissue factor (TF) expression and activity, as well as reduced vascular NF kappa B activity (n=7; p<0.05). In human endothelial, vascular smooth muscle, and peripheral blood mononuclear cells ALA (10 –30
μ
mol/l) inhibited tumor necrosis factor (TNF)-
α
and thrombin induced TF expression as well as surface activity (n=4; p<0.01). Real time PCR and analysis of TF promoter activation demonstrated that ALA mediated inhibition of endothelial TF expression occurred at the transcriptional level involving the MAP kinases p38, ERK and JNK1/2 (−62%,−55% and −28% versus TNF-
α
alone; n=3; p<0.05). In endothelial cells ALA impaired TNF-á induced I kappa B-á degradation and NF kappa B activation (n=3; p<0.05).

Conclusions.
A diet rich in plant-derived ALA inhibits arterial thrombus formation via impaired platelet aggregation and reduced TF expression. Hence, dietary ALA supplementation represents an attractive antithrombotic nutritional supplement.
Abstract 4955: Akt Cooperates with Membrane Type 1-Matrix Metalloproteinase (MT1-MMP) in TNF-Alpha-Induced Signaling Pathways of Endothelial Dysfunction and Haemostasis
Authors: Ohkawara Hiroshi , Ishibashi Toshiyuki , Ando Katuya , Sugimoto Koichi , Uekita Hironori , Sakamoto Nobuo , Takeishi Yasuchika
Published: April 2024

Background:
Akt is a critical molecule in several signal transduction pathways involved in vascular responses. Membrane type 1-matrix metalloproteinase (MT1-MMP), a membrane-anchored MMP, functions as a signaling molecule in addition to a proteolytic enzyme.

Hypothesis:
Akt cooperates with MT1-MMP in tumor necrosis factor (TNF)-alpha-induced signaling pathways of vascular responses including endothelial dysfunction and haemostasis.

Methods and Results:
TNF-alpha (10 ng/mL) induced a transient increase in Akt phosphorylation within 15 minutes, followed by the profound decrease of Akt phosphorylation and the increase in MT1-MMP activity within 60 minutes, in cultured human aortic endothelial cells (ECs). To demonstrate the role of MT1-MMP for Akt signaling pathway in TNF-alpha-stimulated ECs, we used siRNA to knockdown MT1-MMP protein in ECs. Silencing of MT1-MMP reversed TNF-alpha-triggered transient upregulation of Akt phosohorylation within 15 minutes and the downregulation within 60 minutes, suggesting that MT1
-
MMP may be a key regulator of Akt phosphortlation in TNF-alpha stimulated ECs. In the downstream events, TNF-alpha increased tissue factor (TF) expression through NF-kappa B activation and decreased thrombomoduline (TM) expression. Inhibition of Akt by Akt inhibitor X markedly enhanced TNF-alpha-induced upregulation of TF expression and downregulation of TM expression. Silencing of MT1-MMP by siRNA also reversed the changed expression of TF and TM induced by TNF-alpha. These results demonstrated that MT1-MMP-mediated Akt phosphorylation is a critical mechanism in modulation of TNF-alpha-induced signaling pathways and downstream events. Furthermore, we found that pioglitazone, PPAR gamma agonist, suppressed TNF-alpha-induced downregulation of Akt phosphorylation and reversed the upregulated TF expression and upregulated TM expression by TNF-alpha in ECs. This supports a mechanism for pleiotropic effect of pioglitazone on cardiovascular disease.

Conclusions:
We show new evidence that Akt is a key modifier for TNF-alpha-induced signaling pathways in cooperation with MT1-MMP for modulation of endothelial dysfunction and haemostasis.
Abstract 4958: A Novel Mouse Model of Thoracic Aortic Aneurysm Revealed the Protective Property of TNF-alpha Against the Rupture of Aneurysm
Authors: Iwata Hiroshi , Manabe Ichiro , Nagai Ryozo
Published: April 2024

Despite high clinical frequencies, precise mechanism in development of aortic aneurysm and dissection remains largely unknown. We recently developed a novel mouse model of constantly producing thoracic aortic aneurysm. A nylon line (0.486mm in diameter) was inserted since the side-branch of femoral artery until the end reached the mid of thoracic aorta. The line was tied and placed in the artery. In wild-type mice, survival rate at 4 weeks after the procedure was 67% (28/42) and thoracic aorta aneurysm developed in 85% (26/28). Massive thoracic hemorrhage indicating aortic rupture was observed in 74% of dead mice (10/14). Proinflammatory genes such as TNF-alpha, MCP1 and IL6 were highly expressed in the aneurysm. Among them, TNF-alpha was constantly and highly expressed shortly (1 day) after the procedure, whereas peak transcriptional levels of other genes were observed at 2 weeks. As the unique temporal gene-expression pattern was observed, we performed this injury model to TNF-alpha-mutant mice. Surprisingly, Kaplan-Mayer analysis showed that survival rate in TNF-alpha-mutant mice was significantly lower in comparison with wild-type mice. 13 of 16 of mice (81%) died within 7 days and survival rate at 4 weeks was 0% (0/16). Histological analysis showed apparent hypocellularity around the injured aorta in TNF-alpha-mutant mice. These results suggest that TNF-alpha might play the role not only in accelerating vascular remodeling, but also in salvage from the aortic rupture in severe injured aorta by accumulating supporting cells.
Abstract 4964: Tnf-
α
Antagonists Improve Arterial Stiffness in Patients With Inflammatory Arthropathies: Results From a One Year Controlled Study
Authors: Angel Kristin , Provan Sella , Kvien Tore K , Atar Dan
Published: April 2024

Introduction:
The chronic inflammatory state of rheumatoid arthritis (RA), ankylosing spondylitis (AS) and psoriatic arthritis (PsA) contributes to the accelerated atherosclerosis associated with these diseases.

Hypothesis:
We assessed the hypothesis that one year treatment with TNF-
α
antagonists would improve arterial stiffness in patients with RA, AS and PsA.

Methods:
A total of 52 patients with RA, AS or PsA and clinical indication for anti-TNF-
α
therapy were included. 36 patients started with anti-TNF-
α
therapy and were compared with a non-treatment group of 16 patients. Aortic pulse wave velocity (aPWV) and augmentation index (AIx) were measured at baseline and after one year (Sphygmocor). Each visit also included blood samples and assessment of disease activity.

Results:
Values are given as mean± SD. Age of the patients in the treatment/control group was 46.2±12.2/49.0±14.1 years (p=0.21), 42.9/50.0 % (p=0.63) were females and disease duration was 11.0±9.6/11.6±10.1 years (p=0.56). Patients who started anti-TNF-
α
therapy had a significant decrease in aPWV (−0.53±0.13 m/s) whereas the patients in the control group had no change (+0.08±0.10 m/s, p=0.001 for between groups changes). Between groups differences for AIx were not observed (change 0.1±6.1 % and 0.2±5.7 %, p=0.95). There was no change in blood pressures, lipid levels or kidney function in any of the groups. A significant reduction in CRP and DAS28 (RA patients) was observed in the treatment group, but we did not find any significant bivariate correlations between the changes in aPWV and CRP (
ρ
=0.11, p=0.55) or between changes in aPWV and DAS28 in the RA group (
ρ
= −0.21, p=0.44).

Conclusion:
The present study shows for the first time in a comparative design that long term anti-TNF-
α
therapy improves aPWV in patients with RA, AS and PsA. This finding supports the idea that atherosclerosis is partly driven by inflammation, and that this process is amenable to pharmacologic intervention.

Baseline value and change in key variables
Abstract 5016: Circulating MicroRNA: A Novel Potential Biomarker for Early Detection of Acute Myocardial Infarction
Authors: Wang Guo-Kun , Zhu Jia-Qi , Zhang Jun-Tao , Li Qing , Li Yue , Qin Yong-Wen , Jing Qing
Published: April 2024

Background:
MicroRNA (miRNA), a class of 19~25-nucletide noncoding RNA, is present in the plasma or serum of humans and other animals, and has been increasingly reported to be the potential biomarkers for several diseases including cancers and liver injury. The objective of this study is to determine the potential of cardiac-specific miRNAs to serve as early-detectable biomarkers in the plasma for acute myocardial infarction (AMI).

Methods and Results:
With miRNA microarray analysis, we observed the expression profile of miRNAs from the plasma of 5 healthy people. In the plasma, several miRNAs including miR-451 and miR-16 were presented with relative high levels, and the muscle-specific miRNAs, miR-1 and miR-133a were detected with low abundance. However, miR-499 and miR-208, specifically expressed in heart, were undetectable in the plasma. The above results with 6 miRNAs were additionally confirmed by real-time PCR analysis. Interestingly, the levels of miR-1, miR-133a, miR-499, and miR-208, but not miR-451 and miR-16 were significantly increased in the plasma from the rats with coronary artery ligation for 3 hours, compared to those from sham-operated controls (n=6 in both groups). Among them, the elevation of miR-208 was most remarkable. The level of miR-208 in plasma was then examined at various time points (0, 1, 3, 6, 12, 24 hours after coronary artery occlusion). Real-time PCR analysis showed that the amount of miR-208 in plasma became detectable at as early as 1 hour, peaked at 3~6 hours, and declined after 12 hours. Next, the levels of these 6 miRNAs in plasma from 18 patients with AMI, 12 patients with non-AMI CHD and 15 patients with non-CHD were evaluated. The levels of miR-1, miR-133a, miR-499 and miR-208 were substantially higher in AMI group than those in the two other groups. Additionally, the plasma levels of these 4 miRNAs from 5 of 18 AMI patients were decreased during the follow-up investigation.

Conclusions:
Our results that the levels of miR-1, miR-133a, miR-499 and miR-208 increase in plasma from both AMI rats and patients reveal for the first time that they may serve as potential biomarkers for AMI diagnosis. Importantly, miR-208, increased rapidly at the early stage of AMI, may improve the accuracy in early diagnosis of myocardial damage.
Abstract 5018: Soluble fms-like Tyrosine Kinase (sFLT1): A Biomarker of Endothelial Origin in Acute ST-segment Elevation Myocardial Infarction (STEMI)
Authors: Kapur Navin K , Yunis Adil A , Nguyen Tuan A , Parpos Peter , Krishnamurthy Barath , Cross Brian J , Aronovitz Mark , Weintraub Andrew , Kimmelstiel Carey , Jaffe Iris Z , Karas Richard H , Mendelsohn Michael E
Published: April 2024

In STEMI, early recognition improves clinical outcomes. Current biomarkers of myocardial necrosis are limited by poor sensitivity and specificity during the acute phase of coronary occlusion. sFLT1 is an endothelial-derived, hypoxia-inducible protein. We hypothesized that vascular occlusion induces early expression of sFLT1 in STEMI.

Methods & Results:
In a prospective, observational study, serum sFLT1 levels were measured by ELISA in 30 patients with STEMI referred for emergent catheterization. Compared to 20 healthy, age-matched controls, sFLT1 levels were markedly increased at the time of presentation (11.7±2 vs 210.5±80 pg/mL, p<0.0001), then substantially reduced within 24 hours of reperfusion (22.3±15 pg/mL, p<0.0001). On admission, 100% of STEMI patients had elevated sFLT-1 levels (>50pg/mL), whereas 73% and 64% had levels of CK-MB >5ng/mL and Troponin-I >1.0 ng/mL respectively. Two patients undergoing alcohol septal ablation were studied as positive controls. sFLT1 levels increased within 40 minutes of occlusion (27.8 vs 216.2 pg/mL, pre- vs post-ablation, p<0.03). We also studied sFLT1 expression in a mouse model of myocardial infarction (MI). After 1 hour of left coronary ligation in 10-week old male wild-type mice (n=6/group), left ventricular sFLT1 mRNA levels were increased (p<0.01 MI vs. sham), while aortic and lung mRNA expression were unchanged. Serum sFLT1 levels were also elevated in MI (170±71 vs 541±107pg/mL, sham vs. MI, p<0.002). To explore the cellular origin of sFLT1, human coronary endothelial (HCE) and smooth muscle cells (HSMC) were rendered hypoxic. Within 1 hour, sFLT1 mRNA expression in HCE increased by 173±35% (p=0.03). By 12 hours, sFLT1 mRNA and protein expression in hypoxic HCE increased by 313±15% (p<0.01) and 256±17% (p<0.001) respectively. To simulate the effects of reperfusion on sFLT1 expression, HCE were exposed to 12 hours of hypoxia, then 6 hours of normoxia. After normoxic rescue, sFLT1 protein expression decreased by 36% (p<0.01, vs hypoxic HCE). Hypoxia failed to induce sFLT1 mRNA or protein expression in HSMC.

Conclusion:
sFLT1 is a highly sensitive biomarker of vascular occlusion released by the endothelium during the acute phase of STEMI and may be an important new marker for acute ischemic syndromes.

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 5021: Copeptin Improves Early Diagnosis of Acute Myocardial Infarction
Authors: Keller Till , Tzikas Stergios , Zeller Tanja , Czyz Ewa , Lillpopp Lars , Roth Alexander , Sinning Christoph , Wild Philipp S , Lubos Edith , Peetz Dirk , Kunde Jan , Hartmann Oliver , Bergmann Andreas , Post Felix , Lackner Karl J , Genth-Zotz Sabine , Nicaud Viviane , Tiret Laurence , Münzel Thomas , Blankenberg Stefan
Published: April 2024

Introduction: Early diagnosisof myocardial infarction in chest pain patients is crucial to identify patients at risk and to maintain a fast treatment initiation.

Background: Aim of the current investigation is to test whether determination of copeptin, an indirect marker for arginin-vasopressin, adds diagnostic information to conventional troponin in early evaluation of patients with suspected myocardial infarction.

Methods: Between 06/2006 and 03/2008 patients with suspected acute coronary syndrome were enrolled consecutively. Copeptin, troponin T, myoglobin and creatine kinase-MB were determined at admission and after 3 and 6 hours.

Results: Of 1293 (66.1% male) enrolled patients 244 (18.9%) had the discharge diagnosis of acute myocardial infarction, 211 (16.3%) presented with unstable angina and in 838 (64.8%) patients an acute coronary syndrome could be excluded. Combined measurement of copeptin and troponin T at admission improved the c-statistic from 0.85 for troponin T alone to 0.94 in the overall population and from 0.77 to 0.91 in patients presenting within 3 hours after chest pain onset. In this group the combination provided an excellent negative predictive value of 91.1%.

Conclusions: In triage of chest pain patients determination of copeptin in addition to conventional troponin T improves diagnostic performance, especially early after chest pain onset. Combined determination of troponin T and copeptin provides an excellent negative predictive value virtually independent of chest pain onset time and therefore allows an early and safe rule-out of myocardial infarction.
Abstract 5037: Phosphorylation of eNOS Corrects Diabetic Vascular Dysfunction and Improves Outcome to Stroke
Authors: Li Qian , Atochin Dmitriy , Kashiwagi Satoshi , Earle John , Wang Annie , Sessa William , Huang Paul
Published: April 2024

Background: While diabetes is associated with diminished vascular NO levels, the precise mechanisms of diabetic endothelial dysfunction are not known. We hypothesized that deficient eNOS S1179 phosphorylation plays a key role in diabetic vascular abnormalities, and that increasing S1179 phosphorylation may improve endothelial function. To test this hypothesis, we created eNOS knock-in mice that carry a S1179D mutation in the eNOS gene, resulting in the generation of a phosphomimetic form of eNOS with increased enzymatic activity and NO generation. We bred these animals to db/db mice to obtain S1179D-db/db mice to test whether modulation of the S1179 phosphorylation site could overcome diabetic vascular dysfunction, and whether this could affect stroke size in vivo.

Experimental Procedures: Adult male mice were anesthetized by 30 % oxygen, 70 % nitrous oxide, and 1.5 % isoflurane. Body temperature was maintained at 36–37°C. Vessel reactivity studies were performed on isolated pressurized carotid arteries. For stroke studies we used the middle cerebral artery occlusion with subsequent reperfusion by filament with Laser Doppler cortical blood flow monitoring. Mice were examined for neurologic deficits 24 hours after stroke. Infarct sizes were determined by the indirect method by 2,3,5-triphenyltetrazolium chloride staining. Statistical analysis was performed with t-test.

Results: Db/db mice showed normal levels of total eNOS, while S1179 phospho-eNOS was diminished. Db/db mice are hypertensive compared with wild-type (WT) mice (116±4 vs 93±3 mm Hg, mean± SEM). The eNOS S1179D mutation normalizes the blood pressure of db/db mice (92±5 mmHg). Carotid artery vasodilation to ACh was impaired in db/db mice (EC50 43.4±6.1 nM) compared with WT mice (16.3±2.9 nM) and S1179D-db/db mice (26.6±4.2 nM). Db/db mice show a larger stroke (100±7 mm3) than do WT (65±7 mm3) and S1179D-db/db mice (80±4 mm3, P<0.05). This was associated with a functional improvement in the neurologic deficit.

Conclusion: Our results establish eNOS phosphorylation as an important cause of vascular dysfunction in db/db mice, and suggest eNOS phosphorylation as a novel target for the treatment and prevention of diabetic vascular disease and stroke.

This research has received full or partial funding support from the American Heart Association, National Center.
Abstract 5045: Muscle-Type Capillary Endothelial Cells Actively Transport Fatty Acids via Intracellular Fatty Acid Binding Protein-4 and -5
Authors: Iso Tatsuya , Maeda Kazuhisa , Hanaoka Hirofumi , Suga Toshihiro , Matsui Hiroki , Goto Kosaku , Arai Masashi , Endo Keigo , Hotamisligil Gohkan S , Kurabayashi Masahiko
Published: April 2024

Endothelial cells (EC) of muscle-type continuous capillary act as functional interface between the blood plasma and the tissue fluid. They have been considered to perform transendothelial exchange for many substances to transport them into interstitial compartment. Fatty acids are major source of energy metabolism in various organs including heart, skeletal muscle and adipose tissue, but it is uncertain how they can reach interstitial space through endothelial layer of capillary. Fatty acid binding proteins (FABP) are cytosolic fatty acid chaperones whose biological role is not fully understood. FABP4, also known as aP2, is believed to be exclusively expressed in adipocytes and macrophages while FABP5, also called mal1, is detected more widely. Thus far, we have reported that disrupted function of FABP4 and FABP5 in mice (double KO mice) exhibited a striking phenotype with strong protection from diet-induced obesity, insulin resistance and type 2 diabetes. Here we report an unexpected function of the FABPs in capillary EC. We first re-examined tissue distribution of the FABPs by immunohistochemistry. We found that in addition to highly expressing organs already reported, both FABP4/5 were strongly expressed in capillary EC, but not in any arteries, in heart, skeletal muscle, and adipose tissue. In mice deficient for either FABP4 or FABP5, or both, their expression was absent in capillary EC of the organs. Of interest, FABP5 expression was markedly enhanced in capillary EC in mice lacking FABP4, suggesting compensatory induction of FABP5. The tissue distribution of FABPs, along with the phenotype of the double KO mice, raised an intriguing possibility that capillary EC actively transport fatty acids from capillary lumen to interstitial compartment via intracellular FABP4 and FABP5. Consistent with this hypothesis, fatty acid metabolism was markedly reduced while glucose consumption was reciprocally and dramatically elevated in the heart (16.9 folds) and the skeletal muscle (3.7 folds) in the double KO mice, as estimated in vivo by uptake of fatty acid analog,
125
I-BMIPP, and glucose analog,
18
F-FDG. Considering the highest impacts of the FABPs in preventing the metabolic syndrome, this novel pathway may lend a new approach to treat the type 2 diabetes.
Abstract 5051: GATA-4 Promotes Mesenchymal Stem Cells Transdifferentiation Into Cardiomyocyte via Insulin Growth Factor Binding Protein 4 Upregulation
Authors: Li Hongxia , Zuo Shi , He Zhisong , Wang Yigang , Ashraf Muhammad , Xu Meifeng
Published: April 2024

It has been reported that GATA-4 stimulates differentiation of beating cardiomyocytes from stem cell. However, the underlying mechanism remains unknown. We hypothesize that GATA-4 enhances MSC mediated myogenesis via upregulating insulin growth factor binding protein 4 (IGFBP-4).

Method:
MSC were isolated from rat bone marrow and transfected with GATA-4 (MSC
GATA-4
) using Clontech pMSCV retroviral expression system. MSC transfected with GFP (MSC
GFP
) was used as control. Cardiomyocytes (CM) were obtained from neonatal rat ventricles. The transdifferentiation of MSC was directly studied by co-culture MSC with CM, and confirmed by immunostaining and quantitative PCR.

Results:
GATA-4 not only remarkably upregulated the expression of myogenic genes (Table

), but also significantly increased the expression of IGFBP-4 (9.21±0.60 fold in MSC
GATA-4
) compared to those in MSC
GFP
. After co-culture with CM for 7 days, some MSC were tested positive for
α
-actinin (Fig. A

). The percentage of
α
-actinin positive green cells was significantly higher in MSC
GATA-4
(34.9%) than that in MSC
GFP
(8.6%). The expression of cardiac markers, including BNP, Nkx2.5 and Islet-1 was significantly increased in MSC
GATA-4
(Fig. B

). Transdifferentiated MSC also showed action potential which was similar to the native CM. To determine whether IGFBP-4 enhanced MSC mediated myogenesis, IGFBP-4 neutralizing antibody (20~40
μ
g/ml) was added into culture medium, which attenuated MSC
GATA-4
differentiatiation into cardiomyocyte.

Conclusion:
GATA-4 promotes MSC differentiation into myocardial phenotypes partially via upregulating IGFBP-4.

Table 1.

The expression of cardiac genes in MSC

GATA-4

(microarray analysis)
Abstract 5052: Attenuation of Ventricular Remodeling and Improvement of Left Ventricular Systolic Function by Injection of Human Fetal Cardiomyocyte Progenitor Cells After Acute Myocardial Infarction
Authors: den Haan Melina C , Grauss Robert W , Goumans Marie-Jose , Smits Anke M , Pijnappels Daniel A , Steendijk Paul , Gittenberger-de Groot Adriana C , van der Laarse Arnoud , de Vries Antoine A , Schalij Martin J , Doevendans Pieter A , Atsma Douwe E
Published: April 2024

Introduction:
The human cardiomyocyte progenitor cell (hCMPC) is part of a population of stem cells located in the human heart. Isolates of hCMPCs from human fetal hearts showed differentiation into functional cardiomyocytes in vitro. In the present study we investigated the potential of hCMPCs to restore ischemic myocardium and improve left ventricular (LV) function in a myocardial infarction (MI) model in mice.

Methods:
MI was induced in immune-compromised NOD/scid mice, immediately followed by injection of hCMPCs labeled with eGFP (hCMPC group) or vehicle only (control group). From sham operated mice we obtained reference values. Cardiac performance was measured 2 and 14 days after MI using 9.4 Tesla MRI. LV pressure-volume measurements were performed at day 15 followed by extensive immunohistological analysis.

Results:
Animals injected with hCMPCs after MI demonstrated a significantly higher LV ejection fraction, a lower LV end-systolic volume and a smaller ô (relaxation time constant), compared to animals from the control group (by 17%, by 22% and by 24% respectively, P<0.05). A trend towards a lower LV end-diastolic volume was also observed, although this did not reach significance (P=0.14). Both the infarct zone and border zone of the hCMCP group showed a significant increase in total blood vessel density (6.67%±0.85% and 9.13%±0.46%) compared to control (3.35%±1.13% and 4.31%±1.08%, P<0.01 in either zone). The infarct and border zone of the hearts injected with hCMPCs also had a diminished collagen type III density (1.07±0.06 and 1.11±0.07), compared to the control group (1.45±0.05 and 1.51±0.08, P<0.01 in either zone). Furthermore, nuclear PCNA expression indicated an increased rate of mitosis in the infarct (37.76%±2.23%) and border zone (48.84%±2.07%) compared to the infarct (25.48%±4.83%, P<0.01) and border zone (32.03%±1.03%, P<0.01) of the control group.

Conclusions:
Fetal hCMPCs engraft into infarcted myocardium where they improve LV systolic function and attenuate ventricular remodeling. Increased blood vessel density, elevated mitosis rate and decreased collagen III deposition may be involved in this process. These results indicate that hCMPCs are promising candidates for cell-based therapy.
Abstract 5128: Incidence of Stroke Among Patients With Paroxysmal Supraventricular Tachycardia
Authors: Brembilla-Perrot Béatrice , Zinzius Pierre Yves , Aliot Etienne , Blangy Hugues , Groben Laurent , Muresan Lucian , Magalhaes Sonia , Beurrier Daniel
Published: April 2024

Background
: Atrial fibrillation (AF) is a major cause of embolic event, but the significance of other arrhythmias remains unknown. The purpose of study was to assess the significance of unexplained stroke in patients who had apparently only supraventricular tachycardia (SVT).

Population
: Unexplained stroke was noted in 27 patients (group I) admitted for palpitations and electrophysiological study (EPS); they were issued from a population of 1007 patients with normal ECG in sinus rhythm, palpitations or tachycardia and with inducible SVT at EPS; 980 pts had no history of stroke (group II).

Methods
: EPS consisted of atrial pacing and programmed atrial stimulation in control state and if necessary after infusion of isoproterenol. Clinical and electrophysiological data of both groups were studied. Patients with stroke had a normal carotid ultrasound study and transcranial Doppler ultrasonography.

Results
: Patients with unexplained stroke represent 2.6 % of population with SVT. They were older than remaining patients without stroke (65±10 vs 50±19 years) (0.001). They have more frequently associated heart disease (8/27, 30 % vs 37/980, 4 %) (p < 0.001). Male gender was more frequent in group I (16/27, 59 %) than in group II (361/980, 37 %) (p< 0.05). Typical AV node re-entrant tachycardia was noted in 17 group I patients (65 %) and 695 group II patients (68 %) (NS); AV re-entrant tachycardia associated with a concealed accessory pathway was noted in 2 group I patients (7 %) and 169 group II patients (17 %) (NS); atypical AV node re-entrant tachycardia was more frequent in group I (7/27, 26 %) than in group II (115/980, 12 %) (p<0.05). AF induction was more frequent in group I (7/27, 26 %) than in group II (36/980, 4 %) (p< 0.001). Ablation was performed in 6 group I patients; one of them died from another stroke; another one died suddenly; another one developed a permanent AF.

Conclusions
: Unexplained stroke was a rare event in patients with paroxysmal SVT, noted in 2.6 % of this population. Old age, male gender, associated heart disease, and induction of atrial fibrillation were significant factors associated with the history of stroke in patients with SVT. They have a risk of adverse events during the follow-up.
Abstract 5132: Serum Pentraxin 3, Matrix Metalloproteinase 9 and Intraplaque Hemorrhage in Carotid Atherosclerosis
Authors: Moriguchi Emilio , Narvaes Luciane , Silvello Daiane , Albuquerque Luciano , Meurer Luise , Santos Kátia , Rohde Luis
Published: April 2024

Background - Carotid intraplaque hemorrhage (IPH) is a marker of atheroma instability. In the present study, we evaluated the potential association between serum levels of pentraxin 3 (PTX3, a novel marker of vascular inflammation) and matrix metalloproteinases (MMPs) with
in vivo
assessment of IPH by high-resolution magnetic resonance imaging (MRI).

Methods and Results - Consecutive patients submitted to carotid endarterectomy underwent high-resolution MRI; independent evaluation of neurological symptoms; histological analysis and measurement of serum PTX3, MMP-3 and MMP-9. IPH was determined non-invasively by the presence of a hyperintense signal on MRI. Eighty-two predominantly male (65%) and hypertensive (89%) patients (67±9 years-old) were studied. MR angiography identified 19(23%) patients with 50 – 69% stenosis, 23(28%) with 70 –90% stenosis, and 40(49%) with > 90% stenosis. High-resolution MRI depicted a hyperintense signal suggestive of intraplaque bleeding in 48 (58.5%) subjects. No usual clinical characteristic was predictive of the hyperintense signal on MRI (all p values >0.10). Serum PTX3 and MMPs levels were similar in different degrees of carotid stenosis assessed by MR angiography, but were significantly augmented in patients with signs of IPH on MRI and on histological analysis (all p values <0.05). We observed a strong positive association between PTX3 and MMP-9 levels (r=0.76; p<0.0001) and a weak to moderate association between hs-C-reactive protein and PTX3 (r=0.40; p=0.0002) and MMP-9 (r=0.35; p=0.001). In a multivariate logistic regression model adjusted for potential blood-derived markers, clinical neurological instability was the only variable independently associated to MRI-based IPH.

Conclusions - MRI-based IPH identified neurologically unstable patients, with increased levels of PTX3 and MMP-9, regardless of the degree of carotid luminal stenosis.
Abstract 5156: Plasma Des-acyl Ghrelin, but Not Plasma HMW Adiponectin, is a Useful Cardiometabolic Marker for Predicting Atherosclerosis in Elderly Hypertensive Patients
Authors: Yano Yuichiro , Toshinai Koji , Inokuchi Takashi , Shimada Kazuyuki , Kario Kazuomi , Nakazato Masamitsu
Published: April 2024

Objective:
The coming obesity epidemic in elderly persons necessitates the establishment of new and easy-to-use cardiometabolic markers to identify individuals most likely to develop atherosclerosis among elderly hypertensive patients.

Methods:
We measured plasma high-molecular-weight (HMW) adiponectin and des-acyl ghrelin levels, and carotid artery intima-media thickness (cIMT) in 263 elderly hypertensive patients (mean 72.6 years; 37% men). Other cardiometabolic markers, including lipid and glucose metabolites, inflammation (high-sensitivity C-reactive protein: hs-CRP), and hemostasis (plasminogen activator inhibitor-1: PAI-1), were also measured.

Results and conclusion:
Both HMW adiponectin and des-acyl ghrelin levels were inversely correlated with obesity (both
P
<0.01). The HMW adiponectin level was favorably associated with glucose and lipid metabolites, PAI-1(all
P
<0.05), and hs-CRP (
P
<0.07) after adjustment for age, sex, and BMI; however, it had no correlations with cIMT. In contrast, although there were no correlations between des-acyl ghrelin and any cardiometabolic markers, except for a positive association with the nitrite/nitrate (NOx) level (
P
=0.002), des-acyl ghrelin had a significant inverse correlation with cIMT (
P
=0.003). A multivariable regression analysis showed that des-acyl ghrelin, but not HMW adiponectin, was significantly associated with cIMT after adjusting for age, obesity, sex, smoking, 24-hour BP, and other cardiometabolic factors (
β
=−0.178, P=0.001). Moreover, receiver-operator curves to predict a high level of cIMT (above median) demonstrated that des-acyl ghrelin was the best predictor (area under the curve=0.622,
P
=0.001). The increased risk of cIMT among those with abdominal obesity compared with non-obesity (0.833±0.185mm vs. 0.782±0.163mm,
P
=0.019) was explained by the elevated 24-hour BP and reduced des-acyl ghrelin level, but not by other cardiometabolic parameters. These associations were unchanged after adding NOx to the model. In conclusion, the des-acyl ghrelin level is a useful cardiometabolic marker for predicting atherosclerosis in elderly hypertensive patients, and the pathologic pathway linking these factors is independent of its NO bioactivity.
Abstract 5163: Genetic Deletion of OX40 Ligand Suppresses the Development of Atherosclerosis in Apolipoprotein E-Deficient Mice
Authors: Nakano Makoto , Fukumoto Yoshihiro , Satoh Kimio , Ito Yoshitaka , Kagaya Yutaka , Ishii Naoto , Sugamura Kazuo , Shimokawa Hiroaki
Published: April 2024

Background:

Atherosclerotic lesions are characterized by infiltration of inflammatory cells, for which immunological mechanism may be involved. Furthermore, it has been previously demonstrated that immune system is involved in post-ischemic angiogenesis and that atherosclerosis is associated with enhanced vasa vasorum formation. Both soluble and contact-dependent mediators from T-cells play a crucial role in the development of atherosclerosis. OX40, a membrane-bound molecule of the tumor-necrosis-factor-receptor superfamily, is expressed in activated T-cells, while OX40 ligand (OX40L) is expressed in activated macrophages and endothelial cells. In this study, we examined whether OX40/OX40L system is involved in the pathogenesis of atherosclerosis, especially in terms of vasa vasorum formation.

Method and Results:

We first demonstrated that endothelial cells of the deep intima in human coronary atheroma were immunopositive for OX40L (n=5). We then examined whether or not the OX40/OX40L system influences the development of atherosclerosis in apolipoprotein E-deficient (ApoE
−/−
) mice. ApoE
−/−
mice and ApoE
−/−
/OX40L-double-deficient (ApoE
−/−
/OX40L
−/−
) mice were fed on a high-fat diet starting at 4 weeks of age. After 8 weeks, aortic en face and aortic root analysis with oil-red O staining demonstrated that the extent of aortic atheroma was significantly less in ApoE
−/−
/OX40L
−/−
mice as compared with ApoE
−/−
mice (9.6±2.2% vs. 13.6±3.5%, P<0.01, 8.1±4.3% vs. 12.7±7.4%, P<0.05, n=16, respectively). To elucidate the role of OX40L in vasa vasorum formation for atherogenesis, we examined adventitial vascularity by immunostaining and confirmed that the number of blood vessels in adventitia was significantly less in ApoE
−/−
/OX40L
−/−
mice as compared with ApoE
−/−
mice (16.0±3.2 vs. 33.5±16.1 vessels/HPF, P<0.01, n=8). Furthermore, capillary formation in implanted matrigel was significantly less in OX40L
−/−
mice as compared with WT mice.

Conclusions:

These results indicate that the OX40/OX40L system plays an important role in neovascularization of atheroma, which may promote the atherosclerotic process, suggesting that the OX40/OX40L system could be a new therapeutic target for the treatment of atherosclerosis.
Abstract 5192: G Protein-coupled Receptor Kinase-5 Attenuates Atherosclerosis by Reducing PDGF-induced Smooth Muscle Cell Proliferation and Migration
Authors: Wu Jiao-Hui , Cai Xinjiang , Zhang Lisheng , Peppel Karsten , Sharma Krishn , Zidar David A , Brian Leigh , Freedman Neil J
Published: April 2024

G protein-coupled receptor kinase-5 (GRK5) is a Ser/Thr kinase that up-regulates in atherosclerotic arteries; it phosphorylates and desensitizes many receptors important to atherogenesis, including heptahelical receptors and the platelet-derived growth factor receptor-
β
(PDGFR
β
). We therefore tested the hypothesis that GRK5 reduces atherogenesis, by comparing atherosclerosis in male C57BL/6-congenic
apoe
−/−
/
grk5
−/−
and
apoe
−/−
mice, fed a Western diet for 12 wk from age 5 wk, with equivalent serum cholesterols and blood pressures. Aortic atherosclerosis (en face) was 50% greater in
grk5
−/−
(n=20) than in
grk5
+/+
(n=18) mice: Sudanophilic lesions covered 10.6±0.8% versus 7.0±0.5% of aortic area, respectively (
p
<0.001). To test whether this difference could result from GRK5-mediated regulation of arterial smooth muscle cells (SMCs) in vivo, we denuded the common carotid endothelium with a 0.36-mm wire in 25-wk-old male B6-congenic WT and
grk5
KO mice, using a procedure we previously showed (by bone marrow transplant) produces neointimal hyperplasia comprising SMCs derived only from arterial media. Before injury, GRK5 KO and WT carotids had identical luminal and medial areas. However, 4 wk after injury, neointimal area was 350% greater in GRK5 KO (0.06±0.01 mm
2
) than in WT mice (0.017±0.007 mm
2
) (n=5/group, p<0.02), but medial areas were equivalent. Two wk after injury, medial SMCs showed 80±20% greater PDGFR
β
activation per cell in GRK5 KO than in WT arteries (n=3/group, p<0.02), judged by quantitative immunofluorescence microscopy for PDGFR
β
-phospho-Tyr
1021
; total PDGFR
β
levels were equivalent in KO and WT arteries. In vitro, SMC proliferation evoked by PDGF or 2.5% FBS was greater in GRK5 KO than in WT SMCs (3 lines/genotype, p<0.05), by 80±30% and 33±8%, respectively (assessed by SMC counting). Migration (in modified Boyden chambers) was also greater in GRK5 KO than WT SMCs, by 70±20%, 160±20% and 200±30%, respectively, in response to PDGF, 10% FBS, and activated macrophages (p<0.05), despite equivalent GRK5 KO and WT CD4
+
lymphocyte migration. We conclude that GRK5 attenuates atherosclerosis, at least in part, by reducing SMC migration and proliferation evoked through the PDGFR
β
, and perhaps other SMC receptors activated by macrophages.
Abstract 5212: Resolution of Pathological Q-waves is Associated With Improved Outcomes After Primary PCI for Acute Myocardial Infarction
Authors: Baran David A , Farkouh Michael E , Reiffel James , Dressler Ovidiu , Nikolsky Eugenia , Mehran Roxana , Lansky Alexandra J , Stone Gregg W
Published: April 2024

Background:
The presence of pathological Q-waves in patients presenting with ST-elevation myocardial infarction (STEMI) independently predicts 30-day mortality. A question that remains is whether new Q-waves which resolve within 30 days are predictive of an improved prognosis.

Methods:
3,189 patients with STEMI enrolled in the prospective, randomized Harmonizing Outcomes with Revascularization and Stents in Acute Myocardial Infarction (HORIZONS-AMI) trial were evaluated for the presence of new and persistent Q-waves by serial electrocardiography. ECGs were performed at baseline, 60 minutes post PCI, at hospital discharge and at 30 days, and were interpreted by independent cardiologists at an academic ECG core laboratory. Net Adverse Clinical Events (NACE) was defined as Major Adverse Cardiovascular Events (MACE (death, reinfarction, stroke or repeat revascularization) or major bleeding unrelated to CABG.

Results:
There were 2358 patients with new Q-waves either at baseline or which developed during hospitalization. Patients were classified for the presence of Q-wave resolution within 30 days (n=225) (Group A) or presence of persistent Q-waves (n=2133) (Group B). Group B patients were more likely to have a lower baseline left ventricular ejection fraction and a longer duration of symptom to balloon inflation time, and were less likely to have successful reperfusion as measured by post PCI infarct artery epicardial TIMI flow. Group B also had a significantly higher rate of NACE at 30-days (Table

).

Conclusions:
In patients with STEMI undergoing primary PCI, resolution of new Q-waves present during hospitalization is associated with a higher baseline left ventricular ejection fraction, faster and more effective reperfusion, and an improved short-term prognosis.

TABLE
Abstract 5265: Alternatively Spliced B-Type Natriuretic Peptide (ASBNP) is a Renal Selective Natriuretic and Diuretic Peptide Without Hypotensive Properties
Authors: Boerrigter Guido , Chen Horng H , Pan Shuchong , Simari Robert D , Burnett John C
Published: April 2024

Background:
B-type natriuretic peptide (BNP1–32) is a 32- amino acid cardiac peptide secreted in response to cardiac stress and plays an important compensatory role in cardiorenal homeostasis. Studies have recently reported that a mRNA encoding an alternatively spliced variant of BNP (ASBNP) exists in the human heart and like BNP1–32 is increased in the failing heart. While ASBNP and BNP1–32 have identical ring structures and amino termini, ASBNP and BNP1–32 differ in their carboxy terminus (34 vs. 6 amino acids, respectively). Studies have also reported that ASBNP activates cGMP, the second messenger of BNP1–32, in isolated glomeruli but not in endothelial cells. Also, unlike BNP1–32 ASBNP does not vasorelax isolated arteries or veins. The cardiorenal actions
in vivo
of ASBNP are unknown. We hypothesized that ASBNP
in vivo
would have selective renal actions yet as suggested by studies in isolated vessels would be devoid of blood pressure effects.

Methods:
We therefore determined for the first time the cardiorenal actions of three subsequent intravenous doses of ASBNP (10, 50, and 150 pmol/kg/min) in 5 healthy canines in an acute study under anesthesia in 30-minute clearances at baseline, during, and following infusion. Values provided are mean±SEM for baseline
vs.
150 pmol/kg/min. *p<0.05.

Results:
ASBNP dose-dependently increased plasma levels of cGMP (8.6±1.0
vs.
11.5±1.3* pmol/mL), and the same was true for urinary cGMP excretion (878±191
vs.
2028±551* pmol/min). Mean arterial pressure (p=0.83) and heart rate (p=0.43) remained unchanged. Urine flow increased (0.38±0.05
vs.
1.32±0.20* mL/min) as did urinary sodium excretion (38±15
vs.
133±38*
μ
Eq/min). Renal blood flow (p=0.51) and glomerular filtration rate (p=0.99) remained unchanged. BNP-immunoreactivity as measured by the Biosite BNP assay increased (5±4
vs.
3807±573* pg/mL).

Conclusion:
ASBNP, an alternatively spliced form of BNP produced in the human heart, activates cGMP and has diuretic and natriuretic actions but is without blood pressure lowering actions. Thus, ASBNP represents a novel renal selective, non-hypotensive, natriuretic, and diuretic peptide.
Abstract 5266: A Novel Technique for Assessing the Renal Microvasculature in Chronic Kidney Disease
Authors: Kuliszewski Michael A , Rudenko Dmitriy , Connelly Kim A , Yuen Darren , Trogadis Judy , Advani Andrew , Gilbert Richard E , Leong-Poi Howard
Published: April 2024

A variety of factors have been identified as playing an important role in the progression of chronic kidney disease (CKD) to end-stage renal failure. Microvascular loss plays a critical role, being pathologically linked to the development of glomerular sclerosis and tubulointerstitial fibrosis. Thus, we have developed a new and novel post-mortem fixation technique, fluorescent microangiography (FMA), for assessing the microvasculature in animal models of CKD.

Methods:
8 week old make Fischer 344 rats underwent subtotal nephrectomy (n=10) (SNX), or sham surgery (n=10). Systolic blood pressure (SBP), serum creatinine, and proteinuria were assessed at baseline and 8 weeks follwing surgery. At sacrifice rats were subjected to FMA. (10% solution of 0.02
μ
m fluorescent microspheres) Thick (200
μ
m) sections of fixed tissue can then be visualized under confocal microscopy. Stacks of images are collected and 3D reconstruction and visualization carried out in order to quantify the density, size and shape of blood vessels. An automated tracing algorithm was used to connect the tubular microvessels in 3 dimensions, and a solid 3D model was created (Neurolucida).

Results
: SNX animals showed increased in SBP (192±36 versus 123±4 mm Hg in controls; p < 0.0001), serum creatinine (65±12 versus 32±4
μ
M in controls; p < 0.001) and urinary protein (84±26 versus 13±1 mg/d in controls; p < 0.05). FMA analysis demonstrated glomerular hypertrophy (average glomerular diameter [185±7 versus 136±4
μ
M in controls; p < 0.01]) with microvascular loss in SNX animals (3.02E-03±2.20E-04 versus 4.45E-03±2.29E-04
μ
M in controls; p < 0.01, see figure below). SNX increased total vessel length (9809±627 versus 5879±393
μ
M in controls; p < 0.05), average vessel diameter (5.59±0.15 versus 5.24±0.18
μ
M in controls; p < 0.05).

Conclusion:
Fluorescent microangiography (FMA), with a spatial resolution of less than 1
μ
M is a practical and quantifiable method for visualizing the microvasculature of the kidney that allows for direct measurements of volume, diameter, length and vascular density. In addition FMA provides visualization of the phenotypic changes in the renal microvasculature associated with CKD.