bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5511: Natural Regulatory T Cells Control Coronary Arteriolar Endothelial Dysfunction and Hypertension in Mice
Authors: Elmageed Zakaria T Abd , Nair Devika , Gonzalez-Villalobos Romer A , Chentoufi Aziz A , Partyka Megan , Belmadani Souad , Matrougui Khalid
Published: April 2024

Background and Purpose: Coronary arteriolar endothelial dysfunction and hypertension are the main cause for heart disease, and represent a major health problem. The cellular and molecular mechanisms that trigger this pathology are not well understood. Evidence from experimental and clinical research indicates a central role of immune cells and inflammation in cardiovascular diseases. In this study we determined that CD4+CD25+ regulatory T cells, which actively maintain immunological tolerance to self and nonself antigens, are key element in the control of blood pressure and coronary arteriolar endothelial dysfunction in angiotensin-II-dependent hypertensive mice model.

Methods and Results: Mice infused with angiotensin-II significantly increased blood pressure determined by telemetry, reduced the number of CD4+CD25+ regulatory T cells apoptosis-dependent mechanism as determined by flow cytometry, increased macrophages inflammatory cytokines release (increased local tumor necrosis factor alpha release), and induction of adhesion molecules (I-CAM and V-CAM) expression facilitating the infiltration of macrophages into coronary arterioles and heart; and induced coronary arteriolar endothelial dysfunction assessed with reduced relaxation in response to dose-response of acetylcholine using arteriograph. Interestingly, hypertensive mice injected with purified CD4+CD25+ regulatory T cells (3 times a week for two weeks) or treated with copolymer-I (which enhances the conversion of CD4+CD25− T cells to CD4+CD25+ RegTC through the induction of transcription factor Foxp3 in CD4+ T cells and Known as glatiramer acetate, novel medication for the treatment of human multiple sclerosis) significantly reduced blood pressure and macrophages inflammatory cytokines release, and improved coronary arteriolar endothelial function.

Conclusion: Our novel data strongly indicate that CD4+CD25+ regulatory T cells play a critical role in the regulation of blood pressure and coronary arteriolar endothelial function. These results provide new direction in the investigation of hypertension and coronary arteriolar disease and could lead to new therapeutic approaches that involve immune system modulation using CD4+CD25+ regulatory T cells.
Abstract 5517: Coronary Microvessel Spasm is a Frequent Cause of Myocardial Ischemia in Women With Angina-like Chest Pain
Authors: Ohba Keisuke , Sugiyama Seigo , Sugamura Koichi , Nozaki Toshimitsu , Matsuzawa Yasushi , Matsubara Junichi , Konishi Masaaki , Akiyama Eiichi , Sumida Hitoshi , Kaikita Koichi , Kojima Sunao , Nagayoshi Yasuhiro , Yamamuro Megumi , Izumiya Yasuhiro , Sakamoto Kenji , Hokimoto Seiji , Yamabe Hiroshige , Ogawa Hisao
Published: April 2024

Background:
Angina without coronary stenosis is frequent in women, indicating that non-obstructive coronary artery disease (NOCAD) including coronary spasm should be clinically examined. Coronary microvessel spasm (CMVS) as well as epicardial coronary artery spastic angina (eCSA) can cause NOCAD, however clinical significance and diagnostic criteria of CMVS have not been established.

Methods and Results:
239 consecutive patients suspected NOCAD (135 women and 104 men, age 64±11) were investigated by the intra-coronary acetylcholine-provocation test (ACh-test) with simultaneous measurement of cardiac lactic acid production (CLAP) and coronary blood flow volume (CBFV) calculated from average peak velocity using Doppler FloWire and coronary diameter by quantitative coronary angiography. eCSA was predominantly provoked in men [women 66 (48.9%) vs. men 75 (72.2%), p<0.001]. We diagnosed CMVS by our following criteria; occurrence of chest pain with ischemic electrocardiographic change, decrease in CBFV, and positive CLAP without epicardial spasm. CMVS was significantly frequent in women [women 30 (22.2%) vs. men 2 (1.9%), p<0.001]. 51 patients (21.3%) exhibited no evidence of myocardial ischemia by ACh-test [control; women 26 (19.3%) vs. men 25 (24.0%), p=0.37] and remaining 15 patients (6.3%) showed equivocal results. At baseline condition, CMVS patients showed significantly lower coronary flow values of diastolic to systolic velocity ratio compared to control (1.5±0.2 vs. 1.9±0.4, p<0.001). Women with CMVS exhibited significant decrease in CBFV (−50.7±24.0%, p<0.001) and increase in CLAP (31.8±30.7%, p<0.001) as compatible as eCSA by ACh-test (eCSA: CBFV; −45.2±40.7%, p=0.58, CLAP; 29.1±44.7%, p=0.74). We treated both patients with CMVS and eCSA by calcium-channel blockers. During 39 months follow-up, four cardiovascular events were documented in patients with eCSA, however, none in patients with CMVS.

Conclusion:
CMVS is the frequent cause of myocardial ischemia (22%) in women with chest pain and the medical treatment with calcium-channel blockers is effective. CMVS could be clinically identified as NOCAD by the diagnostic ACh-test with simultaneous measurement of CLAP and CBFV to provide optimal care for improving prognosis.
Abstract 5523: Contribution of Ox-LDL Induced Arginase Activation to Vascular Dysfunction in Cigarette Smoke
Authors: Sikka Gautam , Bhunia Anil K , Soucy Kevin , Kim Jae H , Bugaj Lukasz , Oh Young J , Bivalacqua Trinity J , Santhanam Lakshmi , Sussan Thomas E , Biswal Shyam , Berkowitz Dan E
Published: April 2024

Although Atherosclerotic Vascular Disease is a complex process resulting from numerous genetic and environmental factors, it is well recognized that oxidized low density lipoproteins (Ox-LDL) produces pro-atherogenic effects in endothelial cells by inducing the expression of adhesion molecules, apoptosis, superoxide anion formation, and impairing nitric oxide (NO) formation. Exposure to second hand smoke (SHS) further exacerbates this process by enhancing oxidation of LDL and increasing oxidant stress in the endothelium. Ox-LDL results in early endothelial arginase activation and later transcriptional upregulation with reciprocal decrease in NO and ED (endothelial dysfunction). We tested the hypothesis that ArgII and the lectin like OxLDL receptor (LOX) are critical in mediating the ED associated with SHS.

Methods/Results:
Wild type (C57BL/6J, WT), LOX
−/−
and ArgII
−/−
male mice (3– 4 months old, n=6) were exposed to SHS (6 hrs/Day, 5 Days/Wk for 2 Wks; 2R4F reference cigarettes) obtaining WT-SHS, LOX
−/−
SHS and ArgII
−/−
SHS groups and C (control mice) were exposed to room air. Aorta from WT-SHS demonstrated a 2.5 fold induction of arginase activity vs C. Strikingly LOX
−/−
mice showed decreased SHS induced arginase activation. Vascular reactivity studies performed in organ chambers demonstrated that aortic rings isolated from WT-SHS exhibited markedly attenuated endothelium-dependent vasorelaxation as compared to WT C (78.80%±8 vs 46.58%±5). LOX
−/−
SHS (68.60%±3) showed less impairment in EF (endothelial function). EF was completely protected in ArgII
−/−
SHS vessels. ArgII
−/−
SHS vs WT C (67.48%±7 vs 78.80%±8). PWV (m/s), index of central aortic stiffness, measured by high frequency Doppler was significantly elevated in WT SHS vs C (4.13±0.11 vs 3.4±0.06). LOX
−/−
SHS (3.6±0.15) and ArgII
−/−
SHS (3.529±0.2131) demonstrated no significant increase in vascular stiffness.

Conclusion:
The protection from injurious effects of smoking in LOX
−/−
and ArgII
−/−
mice demonstrates that the dysregulation of arginase may be a primary mechanism of smoking induced vascular injury, but lends further support to the coupling of Ox-LDL and activation of Arginase. Hence downstream Arginase activation may be a critical target in smoke induced vascular injury.
Abstract 5539: Tenascin-c Has a Protective Role During the Development of Abdominal Aortic Aneurysm
Authors: Kimura Taizo , Yoshimura Koichi , Aoki Hiroki , Ikeda Yasuhiro , Imanaka-Yoshida Kyoko , Yoshida Toshimichi , Hamano Kimikazu , Hiroe Michiaki , Aonuma Kazutaka , Matsuzaki Masunori
Published: April 2024

Abdominal aortic aneurysm (AAA) is an important healthcare problem for which non-surgical treatment is not available. Recently we reported that c-Jun N-terminal kinase-targeted pharmacotherapy can cause the regression of AAA by regulating inflammation and abnormal metabolism of extracellular matrix (ECM). To realize the pharmacotherapy, it is essential to develop the therapeutic biomarker that is involved in the pathogenesis and reflects the disease activity of AAA. We previously reported that tenascin-C (TN-C), an ECM protein, is an appropriate biomarker and can be used for the bioimaging of the inflammatory sites in several inflammatory diseases such as myocarditis. In this study, we tested if TN-C is a suitable biomarker for AAA. The tissue expression of TN-C was highest in human AAA with the diameter of 45–55 mm, the phase when AAA starts to progress rapidly. TN-C expression in individual aneurysms was highest at the transitional zone between normal and aneurysmal tissue where inflammatory process is most active. In mouse model of AAA induced by periaortic application of CaCl2, TN-C expression increased 4 to 6 weeks after the CaCl2 treatment that coincided with the rapid progression of AAA. Next, we examined if TN-C is involved in the pathogenesis of AAA by creating mouse model with periaortic application of CaCl2 followed by continuous infusion of angiotensin II in wild type (WT) and TN-C knockout (KO) mice. Surprisingly, 5 out of 15 TN-C KO mice developed large suprarenal aneurysms, while none of 14 WT mice did. Histologically, TN-C KO mice showed much more severe inflammation and destruction of elastic lamellae compared to WT. This may be because TN-C-deficient vascular smooth muscle cells express much higher cathepsin S, an elastolytic protease, and inflammatory mediators such as pentraxin 3. These results indicate that TN-C is expressed at the active lesion of AAA and critically involved in the pathogenesis by protecting aorta against tissue destruction. Because serum TN-C reflects the severity of inflammation and is also suitable for bioimaging in various inflammatory diseases, current study suggests that TN-C is an excellent candidate for the therapeutic biomarker and a potential therapeutic target for AAA.
Abstract 5554: FoxO1 Stimulates Autophagic Flux Through Upregulation of the Small GTP Binding Protein Rab7
Authors: Hariharan Nirmala , Sadoshima Junichi
Published: April 2024

We have shown previously that FoxO1, a member of the FoxO transcription factor family, is upregulated by nutrient starvation and cardiac unloading. FoxO1 in turn promotes autophagy, thereby mediating survival and regression of hypertrophy in cardiac myocytes. The current investigation addressed the molecular mechanism by which FoxO1 promotes autophagy in cardiac myocytes. In order to evaluate formation of both autophagosomes and autolysosomes, cultured cardiac myocytes were transduced with adenovirus harboring mRFP-GFP-LC3. Adenoviral mediated overexpression of FoxO1 increased accumulation of both GFP-LC3 dots (1.5 fold vs Ad-LacZ) and mRFP-LC3 dots (2 fold vs Ad-LacZ, p<0.01), which was further enhanced in the presence of protease inhibitors(PI) (1.5 fold vs Ad-LacZ+PI, p<0.01). FoxO1 overexpression caused greater increases in mRFP-LC3 dots than GFP-LC3 dots, suggesting that FoxO1 enhances autolysosome formation more strongly than autophagosome formation, thereby stimulating autophagic flux. Rab7 is a small GTP binding protein which plays an important role in mediating maturation of late autophagic vacuoles. mRNA expression of Rab7 is strongly upregulated in cardiac myocytes (4.7 fold, p<0.05) and hearts (2.5 fold) with FoxO1 overexpression. Overexpression of Rab7 was sufficient to stimulate autolysosome formation and autophagic flux in cultured cardiac myocytes, as evidenced by accumulation of LC3-II (2.8 fold, p<0.05) and mRFP-LC3 dots (2.3 fold, p<0.05), and decreased p62 (−40%, p<0.05). Lentivirus mediated knockdown of Rab7 (LV-sh-Rab7) attenuated FoxO1- induced autophagy, as indicated by LC3-II accumulation (Ad-Foxo1WT 1.9, Ad-Foxo1WT + LV-sh-Rab7 0.11 vs Ad-LacZ, p<0.05) and p62 expression levels (Ad-Foxo1WT 0.49, Ad-Foxo1WT + LV-sh-Rab7 0.84 vs Ad-LacZ, p<0.05), suggesting that Rab7 mediates FoxO1-induced autophagy in cardiac myocytes. In summary, FoxO1 stimulates autophagosome/autolysosome formation and autophagic flux in cardiac mycoytes, accompanied by upregulation of Rab7. Rab7 plays an essential role in mediating FoxO1-induced stimulation of autophagy, possibly by stimulating autophagosome -lysosomal fusion in cardiac myocytes.
Abstract 5614: In vivo Angiogenic and Anti-Apoptotic Signaling of Trx1 Against Acute Myocardial Infarction: An Insight With Transgenic Mice Overexpressing Trx1
Authors: Adluri RamSudheer , Samuel Samson Mathews , Thirunavukkarasu Mahesh , Muang-In Sataporn , Zhan Lijun , Akita Yuzo , Ho Ye-Shih , Maulik Nilanjana
Published: April 2024

Oxidative stress & alterations in thioredoxin-1 (Trx1) expression/activity & its interactions with several other proteins play a crucial role in cardiovascular complications after acute myocardial infarction (MI). Apart from its antioxidative capacity, Trx1 is known to possess antiapoptotic, growth stimulatory & angiogenic properties by modulating the expression/activity of its protein interacting partners thereby modifying the cell’s response to oxidative stress. However, the exact mechanism of the cardioprotective effect of overexpressing Trx1 has not been elucidated in acute MI. Therefore in the present study we have explored the angiogenic & cardioprotective potential of transgenic overexpression of Trx1 in mice in vivo MI model. The wild type (W) & the Trx1 transgenic (Tg+/+) mice were randomized into 4 groups:

Wild type Sham (WS),

WMI,

Tg+/+ Sham (TS)

Tg+/+ MI (TMI).

MI was induced by permanent occlusion of LAD coronary artery. Sham groups underwent the same time matched surgical procedure without LAD ligation. We observed significant, reduction in infarct size (49±2vs35±2% infarct size/area at risk) & increase in capillary (2483±112vs3012±178) & arteriolar (15±0.9vs27±1) density (counts/mm2) with a consequent improvement in the myocardial functional parameters such as decreased LVIDs (3±0.2vs2.8±0.2) & increased ejection fraction (40. ±3vs53±3), & fractional shortening (19±2vs27±1) as assessed by echocardiography in the TMI compared to WMI. Immunoblot analysis revealed increased expression of Trx1 (6.2 fold), angiogenic VEGF (1.7 fold), anti-apoptotic survivin (2.4 fold) & Bcl2 (3.2 fold) in the TMI compared to WMI. Our study also identifies the potential of Trx1 overexpression in inducing angiogenesis probably via VEGF, reducing cell death & ventricular remodeling & a possible novel treatment strategy where Trx1 overexpression might alleviate oxidative stress & related human heart failure in several pathological conditions. The results obtained & the proteins identified in our current study may prove to be potential therapeutic targets in the treatment of ischemic heart diseases.
Abstract 5634: Molecular Imaging of Bone Marrow Mononuclear Cell Survival and Homing in Murine Peripheral Artery Occlusive Disease
Authors: Van Der Bogt Koen E , Hellingman Alwine A , Lijkwan Maarten A , Bos Ernst J , de Vries Margreet R , Fischbein Michael P , Quax Paul H , Robbins Robert C , Hamming Jaap F , Wu Joseph C
Published: April 2024

Introduction

. Bone marrow mononuclear cell (BMMN) therapy has emerged as a potential treatment for peripheral artery obstructive disease (PAOD). However, much is unknown about cellular kinetics following transplantation. This study aimed to use molecular imaging to provide insight into cellular behavior following different transplantation methods.

Methods

. BMMN were isolated from male F6 transgenic mice (FVB background) that express firefly luciferase (Fluc) and green fluorescence protein (GFP). BMMNs were characterized by bioluminescence imaging (BLI). Male FVB mice (n=33) underwent ligation of the superficial femoral artery and were randomized into 4 groups to receive:

Single intramuscular (i.m.) injection of 2×10
6
MN;

4 weekly i.m. injections of 5×10
5
MN;

PBS control; and

5×10
6
MN intravenously (i.v.).

Cellular kinetics were measured by BLI. Paw perfusion was monitored by Laser Doppler Perfusion Imaging (LDPI).

Results.

Fluc expression correlated with cell number in all groups (r
2
=0.97). In vivo BLI revealed dismal cell survival within 4 weeks following both i.m. transplanted groups. Following i.v. transplantation, BLI showed early homing to the injured area as well as to liver, spleen, and bone marrow. LDPI showed no significant differences in paw perfusion among i.m. groups.

Conclusion.

This is the first study to assess kinetics of transplanted BMMN in PAOD using in vivo molecular imaging. BMMN survival after both i.m. and i.v. transplantation is short lived and BMMN do not preserve perfusion in this model.
Abstract 5635: Estradiol Promotes Neural Stem Cell-based Peripheral Nerve Repair by Enhancing Angiogenesis
Authors: Sekiguchi Haruki , Ii Masaaki , Jujo Kentaro , Thorne Tina , Scarpelli Andy , Clarke Trevor , Ito Aiko , Misener Sol , Klyachko Ekaterina , Hagiwara Nobuhisa , Losordo Douglas
Published: April 2024

Background:
Neural stem cells (NSCs) have been shown to have the potential to differentiate into endothelial cells (ECs) as well as neuronal cells (NCs). Estradiol (E2) is known to exhibit pro-angiogenic effects on ischemic tissue via EC activation. Based on these prior findings, we hypothesized that E2 could promote the therapeutic potential of NSC xplant for nerve repair via NSC differentiation into ECs and neovascularization.

Methods and Results
: NSCs were isolated from the brains of newborn mice. After unilateral sciatic nerve crush injury, mice were randomly assigned to one of 4 groups: E2 (100
μ
g administered locally in biodegradable gel), Control (C) (vehicle/gel), NSC (10
5
cells), and combined E2/NSCs. The E2/NSC group exhibited the greatest nerve functional recovery in motor nerve conduction velocity (C: 33.15±5.23, NSC: 46.97±2.62, E2: 45.32±4.75, E2/NSC: 56.02±6.26 m/s), voltage amplitude (C: 2.7±1.48, NSC: 4.42±0.28, E2: 3.23±1.00, E2/NSC: 6.86±1.48 mV) and exercise tolerance (C: 376.8±68.0; NSC: 437.6±124.9; E2: 428.6±118.2; E2/NSC: maximum duration, 600 sec, same as noninjured) and also showed significantly greater intraneural vascularity and blood perfusion (Laser Doppler) (C: 3.23±1.38, NSC: 10.08±1.15, E2: 14.57±3.31, E2/NSC: 28.14±7.03 flux) compared to the other groups (P<0.05). Fluorescent immunohistology analyses showed that when E2 and DiI-labeled NSCs were locally transplanted in injured nerves, double-positive cells (DiI and EC marker CD31) were more frequently observed (E2/NSC: 6.33±1.53, NSC: 1.33±0.57/nerve, p<0.01), suggesting that E2 promoted NSC differentiation into ECs. The effect of E2 on NSC differentiation into the EC-lineage was further investigated in CD31-negative cells selected by FACS; 5.69% of initially CD31-negative NSCs expressed CD31 when cultured with E2 (10
−8
M) for 10 days, and blockade of the E2 receptor with ICI blocked CD31 expression (0%) in E2-treated NSCs.

Conclusions:
The combination of E2 and NSC transplantation cooperatively improved the functional recovery of injured peripheral nerves, at least in part, by E2 associated NSC differentiation into ECs. These findings provide a novel mechanistic insight not only in NSC biology but also regarding the biological effect of endogenous E2.
Abstract 5642 Thrombospondin-1, via CD47, Blocks Endothelial-Dependent Arterial Relaxation by Limiting Endothelial Nitric Oxide Synthase Activation
Authors: Isenberg Jeffrey S , Bauer Eileen , Qin Yan , Miller Thomas , Bandle Russell , Csanyi Gabor , Pagano Patrick J , Bauer Phillip M , Schnermann Jurgen B , Roberts David D
Published: April 2024

Introduction:
The secreted protein thrombospondin-1 (TSP1) is an important negative regulator of nitric oxide/cGMP signaling in vascular cells. TSP1, signaling through its necessary receptor CD47, limits activation of soluble guanylate cyclase and cGMP-dependent protein kinase. Extending these discoveries we recently defined a role for TSP1 in the acute control of blood pressure. However, it is not clear if the inhibitory role TSP-CD47 signaling has on blood pressure is secondary to a direct effect on arterial relaxation. To investigate this we tested the hypothesis that TSP1 directly limits arterial vasorelaxation by limiting activation of endothelial nitric oxide synthase (eNOS).

Methods:
TSP1-CD47 regulation of eNOS activation was studied in endothelial cells and arteries. Vasorelaxation was assessed ex vivo in arteries from wild type, TSP1 and CD47 null mice. Endothelial dependent changes in blood pressure were studied in transgenic mice.

Results:
TSP1 and CD47 null arteries demonstrated significantly greater dilation in response to physiologic activation of eNOS by acetylcholine compared to wild type vessels. Treating the luminal endothelial compartment of both wild type and TSP1 null arteries (but not CD47 null) with TSP1 inhibited vessel relaxation to acetylcholine and potentiated vasoconstriction to phenylephrine. TSP1 and a recombinant fragment of the signature domain that specifically binds CD47 potently blocked both acetylcholine-stimulated eNOS activation and changes in intracellular calcium in cultured endothelial cells. In vivo, acetylcholine-stimulated hypotention in TSP1 null mice was blocked by intravenous replacement of TSP1.

Conclusion:
Circulating TSP1, through CD47, limits endothelial dependent arterial relaxation and demonstrates a role for circulating TSP1 as a hypertensive agent supporting blood pressure through continuous modulation of both endogenous NO production and signaling.
Abstract 5647: Impact of Growth Factors, Shear Stress and Transcription Factor COUP-TFII on the Notch Pathway in Human Endothelial Cells
Authors: Morawietz Henning , Brunssen Coy , Gryczka Corina , Korten Slobodanka , Goettsch Winfried
Published: April 2024

Arterial and venous endothelial cells can differ in vascular function thus leading to vessel-specific diseases. The molecular mechanisms for differentiation and maintenance of the phenotype of arterial and venous endothelial cells are not well understood. Using primary cultures of human umbilical artery (HUAEC) or vein (HUVEC) endothelial cells we identified by DNA microarrays Hey2 as arterial and COUP-TFII as venous marker gene. Differential expression of these genes and preferential arterial expression of further genes of the Notch signalling pathway (Dll4, Notch4, Hey1, Hey2) were confirmed by RT- and real-time PCR in independent HUAEC and HUVEC preparations. Differential protein expression of Hey2 and COUP-TFII was confirmed as well. We further showed exclusive expression of Hey2 and COUP-TFII in HUAEC and HUVEC over several passages (up to passage 12). Next, we analyzed the impact of growth factors on the preferential arterial genes of the Notch pathway. In medium supplemented with different growth factors (IGF-1, hEGF-1, PDGF-BB, bFGF-2 or VEGF-A) only VEGF-A was able to induce expression of Dll4, Notch4 and Hey1 in venous endothelial cells. A further increase of the high arterial Dll-4 and Hey1 expression was achieved by VEGF-A incubation in HUAEC. This increased expression of Dll-4 and Hey-1 was prevented by VEGF receptor-2 (KDR) inhibitor SU5416. Inhibition of the Notch pathway by gamma-secretase inhibitor X (GSI-X) reduced Dll-4 and Hey-1 expression in venous and arterial endothelial cells without effecting Hey2 expression. In order to reduce arterial Hey2 expression we next applied shear stress (1 dyn/cm
2
or 30 dyn/cm
2
) to arterial endothelial cells. The main arterial transcription factor Hey2 was not changed. Next, we cloned full-length and terminal deletions of the human Hey2 promoter (−2132/+194; −1080/+194; −418/+194; −54/+194 bp). All fragments showed high basal activity (82-, 84-, 293-, 25-fold). Finally, double transfection with a COUP-TFII overexpression plasmid reduced Hey2 promoter activity of the −2132/+194 fragment by 50%. In conclusion, our data suggest an interaction of the venous marker COUP-TFII and the arterial marker Hey2 at the Hey2 promoter.
Abstract 5670: KLF5 Controls Macrophage Polarity in Chronic Inflammatory Diseases via S100 Proteins
Authors: Fujiu Katsuhito , Manabe Ichiro , Nagai Ryozo
Published: April 2024

Chronic kidney disease is a major risk factor for atherosclerotic cardiovascular disease. Chronic inflammation has been shown to be involved in both diseases. Therefore, the molecular mechanisms underlying them are likely to be closely linked with each other. We have previously demonstrated that the transcription factor KLF5 is involved in cardiovascular remodeling. In the present study we addressed if KLF5 also plays a role in chronic kidney disease. KLF5 expression levels were increased in various kidney disease models including unilateral ureteral obstruction (UUO) and
KLF5
+/− mice ameliorated renal pathology induced by UUO. Genome-wide analyses of potential KLF5 target genes using ChIP-seq and microarrays identified S100A8 and S100A9. S100A8 and A9 are secretory proteins and form a heterodimer. S100A8/A9 induced macrophage accumulation in Matrigel plugs and direct injection of S100A8/A9 into kidneys resulted in infiltration of M1 classically activated macrophages. In
KLF5
+/− mice S100A8/A9 expression was reduced and M1 macrophage infiltration was markedly suppressed in UUO. Interestingly, UUO induced infiltration of M2 alternatively activated macrophages during later stages of disease development. In
KLF5
+/− mice macrophage activation was shifted to the M2 polarity. M2 macrophages produced anti-inflammatory and pro-fibrotic cytokines, including IL-10 and TGFb1. This shift in macrophage polarity is likely to be primarily accountable for the UUO phenotype of
KLF5
+/−
,
in which tissue inflammation, apoptosis and destruction were ameliorated during early stages, while fibrosis was increased in later time points. Taken together, KLF5 appears to regulate tissue remodeling by controlling the macrophage polarity via inflammatory mediators including S100A8/A9. Because KLF5 is also essential for cardiovascular remodeling, the KLF5-S100 axis may play an important role in chronic inflammation in various chronic diseases, including cardiovascular, renal and metabolic diseases.
Abstract 5680: Serum and Glucocorticoid-Inducible Kinase 1 Regulates Sirtuin 3, the Mitochondrial Deacetylase via Phosphorylation and Sumoylation Mechanisms in Rat Aortic Smooth Muscle Cells
Authors: Zhong Wei , Thompson Winston E , Francis-David Sharon
Published: April 2024

Serum and glucocorticoid-inducible kinase 1 (SGK1) is a serine/threonine kinase whose activity and expression is up-regulated in vascular neointimal lesions in association with restenosis. Our previous studies show that vascular smooth muscle cell (VSMC) proliferation is augmented upon activation of SGK1. However, the molecular mechanism(s) was not fully examined. Thus, to identify proteins that participate in SGK1-mediated stimulation of VSMC proliferation, we performed a kinase substrate protein array with activated SGK1. We identified sirtuin3 (Sirt3), a class III histone deacetylase as a novel putative substrate for SGK1 in this screen. Preliminary studies from two-dimensional SDS-PAGE indicate that expression of active SGK1 (S422D-SGK1) is associated with a shift in Sirt3 to a more acidic profile, consistent with an increase in phosphorylation. Moreover, S422D-SGK1 expression resulted in a 2- and 1.6-fold increase in Sirt3 mRNA and protein; respectively, compared to empty vector or a kinase-defective SGK1 mutant (S422A-SGK1). To determine whether Sirt3 expression is regulated by growth factors, we performed timecourse studies with PDGF-BB, a potent inducer of VSMC proliferation as well as SGK1. Quantitative real-time PCR analysis, revealed a modest reduction in Sirt3 mRNA (~25%) after 4 hours of stimulation. However, under the same conditions, PDGF-BB augments cytoplasmic Sirt3 protein expression more than 32-fold, suggesting a posttranslational method of regulation. Notably, sumoylation is known to regulate the activity, stability or subcellular distribution of its substrates and our analysis indicates that Sirt3 contains the core SUMO consensus motif. Thus, we assessed whether Sirt3 can be sumoylated and tested the hypothesis that PDGF acting through SGK1 signaling modulates sumoylation of Sirt3 in VSMC. Indeed, results obtained from an
in vitro
sumoylation assay indicate that Sirt3 is a target for SUMO-1. Lastly, we found a higher level of SUMO-1 conjugation in Sirt3 immunoprecipitates from VSMC expressing S422D-SGK1 compared to empty vector- or S422A-SGK1-expressing cells. Altogether, our findings implicate Sirt3 as novel substrate and potential mediator of the growth effects of SGK1 in VSMC.
Abstract 5766: Reduction of Oxidized Stress by High-Dose N-Acetylcysteine versus Placebo in Patients With ST-Elevation Myocardial Infarction Undergoing Primary Percutaneous Coronary Intervention
Authors: Thiele Holger , Eitel Ingo , Fuernau Georg , Adams Volker , Gutberlet Matthias , Schuler Gerhard
Published: April 2024

Background
STEMI patients undergoing primary PCI develop oxidative stress which might have an impact on reperfusion injury. High-dose N-Acetylcysteine (N-ACC) might prevent oxidative stress with subsequent reduction of reperfusion injury and also the incidence of contrast-induced nephropathy (CIN). Aim of this randomized, controlled, single-blind trial was to assess the effects of N-ACC on oxidative stress, reperfusion injury and CIN in patients undergoing primary PCI.

Methods
Two hundred-fifty patients undergoing primary PCI were randomized to either N-ACC (2×1200 mg/d for 48 hours) or placebo plus optimal hydration. The two primary endpoints were:

occurrence of CIN defined as an increase in the serum creatinine concentration of >25% from the baseline value within 72 h;

Myocardial salvage measured by T2-weighted STIR-images and delayed enhancement MRI at day 2–4 after primary PCI. Secondary endpoints were oxidative stress, infarct size, microvascular obstruction, ST-resolution at 90 minutes and occurrence of MACE at 30 day follow-up.

Results
The median volume of contrast agent during PCI was 190 ml (IQR 130, 250 ml) in the N-ACC and 180 (IQR 143; 228 ml) in the placebo group (p= n.s.). Baseline creatinine and creatinine clearance were 88 vs 86
μ
mol/l and 90 vs 95 ml/min, respectively. Oxidized low-density lipoprotein and advance oxidative protein products as marker for oxidative stress were reduced by 20% in the N-ACC group (p<0.05), whereas no change was evident in placebo. The primary endpoint CIN occurred in 14% in the N-ACC group and in 18% in the placebo group (p=n.s.). The primary endpoint reperfusion injury measured by myocardial salvage was also not different between both treatment groups (25.4%; IQR 14.1; 38.1 versus 22.5%; IQR 16.8; 36.5; p=n.s.). In addition, no differences in infarct size and microvascular obstruction as well as in ST-segment resolution were observed. The MACE rate after N-ACC was similar to placebo (19.4% versus 19.4%, p=n.s.).

Conclusion
High-dose N-ACC reduces oxidative stress. However, it does not provide an additional clinical benefit to placebo with respect to CIN and prevention of myocardial reperfusion injury in patients undergoing primary PCI with moderate doses of contrast medium and optimal hydration.
Abstract 5776: Deletion of the Systemic Gp130-dependent Acute Phase Response Inhibits Neointima Formation Following Carotid Artery Ligation
Authors: Grote Karsten , Schuett Harald , Salguero Gustavo , Tallone Ezequiel , Drexler Helmut , Schieffer Bernhard
Published: April 2024

Background:
Inflammation and vascular remodeling are hallmarks of atherosclerosis, hypertension and restenosis following angioplasty. Recently we demonstrated a crucial role of the hepatocyte gp130-dependent systemic acute phase response for atherogenesis (JEM 2007). Now we have elucidated the role of the hepatocyte gp130-dependent systemic acute phase response for vascular remodeling following experimental carotid artery ligation.

Methods and Results:
Mice with a hepatocyte-specific gp130-k.o. on an apoE
−/−
background (gp130
−
) were compared to control mice (gp130
flox
). Vascular remodeling was induced by permanent ligation of the left common carotid artery. This activated the systemic acute phase reaction in gp130
flox
mice as measured by acute phase serum amyloid A (SAA1) plasma levels and vascular SAA1 content and was completely abrogated in gp130
−
mice (ELISA, SAA1 immunostaining, P<0.05, n=5). Morphometric analysis of the carotid artery revealed severe neointima formation and media thickening 28 days post ligation in gp130
flox
mice which was inhibited in gp130
−
mice (P<0.01, n=6). Carotid segments from gp130
−
showed less smooth muscle cells (SMCs), proliferation and monocyte recruitment (
α
-SMC actin, Ki-67, MOMA-2 immunostaining, P<0.01, n=6). To evaluate the impact of the gp130-dependent systemic acute phase response on SMCs, hepatocytes from gp130
flox
and gp130
−
mice were stimulated with the gp130-activating interleukin (IL)-6. IL-6-induced secretion of SAA1 was completely abolished in gp130
−
hepatocytes (ELISA, P<0.01, n=4). Moreover, when stimulated with supernatants from gp130
−
hepatocytes, SMCs showed significantly less migration and proliferation compared to supernatants from gp130
flox
hepatocytes (transwell, scratch assay, BrdU-incorporation, P<0.01, n=4). Finally, recombinant SAA1 induced SMC migration and proliferation (transwell, scratch assay, BrdU-incorporation, P<0.01, n=3– 4) and SAA1-injection following carotid artery ligation restored vascular remodeling in gp130
−
mice (P<0.01, n=3–5).

Conclusion
: These results imply a critical role of the gp130-dependent systemic acute phase response for vascular inflammation, SMC migration and proliferation and subsequently for vascular remodeling.
Abstract 5782: Increased Cardiomyocyte Elasticity in the Transverse Direction in Hypertrophied Rat Hearts Induced by Chronic
β
-adrenergic Stimulation
Authors: Nakamura Kazufumi , Miura Daiji , Yoshikawa Wakako S , Hashimoto Ken , Kataoka Noriyuki , Shiizu Juichiro , Nishii Nobuhiro , Nagase Satoshi , Morita Hiroshi , Hata Yoshiki , Kusano Kengo F , Ito Hiroshi
Published: April 2024

Background:
Cardiomyocyte stiffness in longitudinal direction is increased in patients with diastolic heart failure. However, stiffness in transverse direction has not been elucidated. We hypothesized that cardiomyocyte elasticity in transverse direction is also increased in hypertrophied hearts. We measured the elasticity of a single cardiomyocyte by use of an atomic force microscope (AFM) in isoproterenol (ISO)-induced hypertrophied rat hearts.

Methods and Results:
Male Wistar rats (9 weeks old) received a vehicle (control), ISO (2.4 mg · kg
−1
· day
−1
) or ISO +
β
1-blocker metoprolol (MET) (24 mg · kg
−1
· day
−1
) subcutaneously (n=5 in each group). After 7 days, compared with those in control and ISO+MET groups, ISO administration had increased left ventricular (LV) wall thickness (P<0.05), and decreased LV diastolic function as assessed by increased LV end-diastolic pressure (ISO: 15.3±2.0 vs control: 4.8±0.5 or ISO + MET: 6.6±0.8 mmHg,
P
<0.05). Elasticity of 10 living cardiomyocytes from LV free wall was measured by parabolic force curves of cantilever deflection/indentation obtained by AFM (Fig A

). Elasticity of cardiomyocytes was significantly higher in ISO group than in control and ISO + MET groups (Fig B

). Next, we added butanedione monoxime (BDM) (20 mM), an inhibitor of actin-myosin interaction, and blebbistatin (10
μ
M), a specific myosin II inhibitor, to culture medium. BDM and blebbistatin significantly reduced the elasticity of cardiomyocytes in ISO group (Fig B

).

Conclusions
: Cardiomyocyte elasticity in transverse direction was increased in hearts with ISO-induced hypertrophy along with diastolic dysfunction. This may be caused by incomplete relaxation.
Abstract 5784: Altered Receptor Distribution and Expression Causing Vascular Myocytes Insensitivity to
α
1
Adrenergic Activation
Authors: Ren Shuang , Fan Linlin , Zhou Hong , He Junqi , Luo Dali
Published: April 2024

Alpha
1D
-adrenergic receptor (
α
1D
-AR) plays a critical role in controlling peripheral vascular resistance and blood pressure. However, unlike
α
1A
- and
α
1B
-AR subtypes that primarily locate at the plasma membrane and function efficiently,
α
1D
-ARs are accumulated within intracellular compartments and poorly functioned in vitro. Using fluo4, BODIPY prazosin and subtype-specific antibodies to evaluate the native
α
1
AR regulated Ca
2+
signaling and their subcellular distribution in rat aortic and cardiac myocytes, we tested the hypothesis that
α
1D
-ARs are altered during cell isolation or/and culturing procedures, causing the responding discordance between tissue and cells. In aortic rings and freshly isolated myocytes
α
1
AR agonist phenylephrine (PE, 10
−5
M) induced vasoconstriction, and intracellular Ca
2+
increase and cell shortening, which could be abolished by a specific antagonist of
α
1D
-AR BMY7378 (10
−6
M), respectively. The cellular response to PE, however, was decreased to 55.2±4.9% in 1-day-and 0% in ≥2-day-cultured aortic myocytes (n= 40, and 238), but remained in all the cultured neonatal rat cardiomyocytes (n= 267). Similarly as the
α
1A
-ARs in cultured cardiomyocytes,
α
1D
-ARs distributed both intracellularly and at the cell membrane in freshly dispersed aortic myocytes, but mostly redistributed intracellularly in cultured aortic myocytes. Treating the culture medium with 2% charcoal/dextran (C/D) for 48 h caused restorations of the cell surface
α
1D
-AR distribution in 34.67±4.1% (n=84) and partial Ca
2+
signaling response to PE in 29.5±0.2% (n=81, p<0.05 vs. no C/D) cultured cells, suggesting an essential requirement for membrane expressed
α
1
ARs for their efficient intracellular coupling. Additionally, the
α
1D
-AR expression in the cultured aortic myocytes was reduced relative to the tissue. These results indicate that
α
1D
-ARs can primarily express at the cell membrane in native system but occur internalized into the cytoplasm and declined in expression in culture condition, resulting in the intracellular uncoupling to
α
1
adrenergic activation in vitro. Charcoal/dextran in culture medium partially prevents this receptor subtype redistribution.
Abstract 5795: Impact of the Modified Henry Ford Warfarin Maintenance Dosing Algorithm on Quality of Anticoagulation at a Specialist Anticoagulation Clinic
Authors: Nieuwlaat Robby , Kim Yang-Ki , Connolly Stuart J , Schulman Sam , Hirsh Jack , Meijer Karina , Raju Nina , Kaatz Scott , Eikelboom John W
Published: April 2024

Purpose:
The efficacy of warfarin depends on the time that the international normalized ratio (INR) is in the target therapeutic range. The time-in-therapeutic range (TTR) is a measure of anticoagulation quality, and should be maximized. The objective of our before-after study was to determine whether routine use of a simple manual warfarin dosing algorithm compared with ‘expertise-based’ dosing can improve the TTR for warfarin.

Methods:
The study was performed at a single anticoagulation clinic in Hamilton, Canada. In the ‘before’ phase (August, 2006 until September, 2007) we retrospectively calculated in patients on warfarin:

agreement between warfarin dosing and the modified Henry Ford dosing algorithm and

TTR, using Rosendaal’s linear interpolation method. In this period, warfarin was managed by experienced anticoagulation clinic physicians.

In the ‘after’ phase (July until December, 2008) we prospectively calculated the same parameters. Differences between ‘before’ and ‘after’ were tested with the independent t-test for continuous and chi-square for dichotomous variables.

Results:
We included 873 patients in the ‘before’ phase and 1,088 patients in the ‘after’ phase. Before introduction of the algorithm, 71% of warfarin dose adjustments were consistent with the algorithm in patients targeting an INR of 2–3 and 56% for those targeting INR 2.5–3.5, whereas after algorithm introduction agreement increased to 90% and 81%, respectively. Introduction of the dosing algorithm significantly increased the TTR in patients targeting an INR of 2–3 from 67.2% to 73.2% (p<0.001) and in those targeting an INR of 2.5–3.5 from 49.8% to 63.8% (p<0.001). This improvement of TTR was due to a decreased proportion of subtherapeutic INRs, from 18 to 13% (p<0.01) with target INR 2–3 and from 41 to 25% (p<0.01) with target INR 2.5–3.5, while the proportion of supratherapeutic INRs did not significantly change.

Conclusion:
Introduction of the modified Henry Ford manual warfarin maintenance dosing algorithm in place of expertise-based dosing significantly improved the mean TTR in our tertiary care anticoagulation clinic. This widely applicable algorithm could be an inexpensive evidence-based method to improve warfarin control and thereby patient outcomes.
Abstract 5811: Dual Function of Bone Morphogenetic Protein-2 in the Progression of Atherosclerotic Lesion and Atherosclerotic Intimal Calcification in vivo
Authors: Nakagawa Yusuke , Ikeda Koji , Nakano Ritsuko , Uraoka Maki , Koide Masahiro , Akakabe Yoshiki , Matsubara Hiroaki
Published: April 2024

Vascular calcification is one of the most important risk factor for the cardiovascular diseases as well as a predictor of all-cause death. Recent findings suggest that bone calcification regulatory factors, locally expressed in blood vessels, play unique roles in the formation of vascular calcification. However, detailed molecular mechanisms, that regulate the intimal calcification associated with atherosclerosis, remain unclear. We first investigated the potential role of de-differentiated VSMCs in the formation of atherosclerotic intimal calcification. In vitro studies revealed that bone morphogenetic protein-2 (BMP-2), a potent initiator for osteoblastic differentiation was markedly up-regulated in the de-differentiated VSMCs comparing to the differentiated VSMCs. We therefore generated the BMP-2 transgenic mice in which BMP-2 expression was driven by
α
SMA-promoter, and mated them with ApoE-knockout mice to obtained BMP-2-Tg/ApoE-KO mice. After 8 weeks of high fat diet, atherosclerotic intimal calcification was not detectable even in BMP-2-Tg/ApoE-KO mice. Somewhat unexpectedly, atherosclerosis was significantly attenuated in BMP-2-Tg/ApoE-KO mice as compared to ApoE-KO mice. The number of
α
SMA-positive VSMCs and macrophages in the atherosclerotic intima was significantly reduced in BMP-2-Tg/ApoE-KO. Also, we found that BMP-2 inhibits the uptake of acetylated LDL in macrophage, and protects endothelial cells from apoptosis in vitro. Of note, when fed with high fat diet for 30 weeks, atherosclerotic intimal calcification was greater in BMP-2-Tg/ApoE-KO than in ApoE-KO mice, whereas the size of atherosclerotic lesion was not different between them. In conclusion, we revealed the dual function of BMP-2 in the atherosclerosis

anti-atherogenic function in the early atherosclerotic lesion, presumably through

reducing the proliferation of VSMCs

inhibiting the lipid-uptake in macrophage

protecting endothelial cells from apoptosis, and

pro-calcification function in the advanced atherosclerotic intima.
Abstract 5813: Hyperhomocysteinemia Enhances Bone Marrow-derived and Vessel Resident Ly6C Inflammatory Monocyte Differentiation Contributing to Atherogenesis
Authors: Zhang Daqing , Jiang Xiaohua , Moore Jodene K , Berretta Remus M , Schafer Andrew I , Houser Steven R , Durante William , Yang Xiaofeng , Wang Hong
Published: April 2024

Background:
We recently reported that hyperhomocysteinemia (HHcy) enriched circulating Ly6C monocytes (MCs) independent of hyperlipidemia. Here we studied the effect and mechanism of HHcy on Ly6C MC function and differentiation in aorta.

Methods and Results:
We generated a mouse model with cystathionine
β
-synthase (CBS) and LDLr deficiencies.
LDLr
−

/

−
CBS
−

/+

mice (8wk) received BM transplantation from EGFP mice. The chimeric mice were fed a high fat (HF) diet as control (Hcy, 10.1
μ
M), HF/high methionine (HM) diet to induce severe HHcy (275.6
μ
M), or HF/HM/high vitamin B-6/12 and folic acid diet as Hcy-lowering therapy (39.0
μ
M) for another 8wk. Morphological analysis exhibited that severe HHcy accelerated atherosclerosis and increased BM-derived Ly6C MC accumulation in the lesion. FACS analysis on whole aortic cell suspension showed that severe HHcy increased BM-derived and resident putative MC (CD11b
+
Ly6G
−
) to 4872 and 3761 from 1332 and 599 cells/aorta in the control. The putative MC was further divided into 4 distinct subsets, severe HHcy, compared with the control, increased BM-derived and resident Ly6C
hi
MC (Ly6C
hi
F4/80
−
, 816 vs 43 and 842 vs 115), differentiating MC (Ly6C
hi
F4/80
+
, 558 vs 43 and 784 vs 28), differentiated macrophage (Ly6C
low
F4/80
−
, 1874 vs 766 and 1095 vs 92), and Ly6C
low
MC (Ly6C
low
F4/80
−
, 1624 vs 480 and 1040 vs 365) in aorta. Plasma Hcy levels were significantly correlated with plasma TNF
α
(r=0.650), IL-6 (r=0.594) and MCP-1 (r=0.591) levels. Hcy-lowering therapy significantly reduced atherosclerotic lesion, plasma inflammatory cytokine levels and aortic MC differentiation. In vitro, L-Hcy (100 –500
μ
M) enhanced IFN
γ
-induced Ly6C
hi+mid
MC differentiation, which was abolished by Tiron, SOD/catalase, and NADPH oxidase inhibitor apocynin. L-Hcy (500
μ
M) increased the TNFa/IL-6 cytokine-producing Ly6C
hi+mid
MC by 3-fold in IFN
γ
-treated cells and significantly enhanced transendothelial migration of IFN
γ
-treated THP1 cells.

Conclusions:
Severe HHcy induces systemic inflammation and promotes BM-derived and vessel resident Ly6C inflammatory MC differentiation leading to exacerbated atherosclerosis. NADPH oxidase mediated oxidative stress participates in Ly6C inflammatory MC differentiation induced by Hcy.
Abstract 5816: The Human Apoa-1 Transgene Suppresses Myeloid Proliferation and Accelerated Atherosclerosis in Ldlr Deficient Mice Reconstituted With ABC Transporter Deficient Bone Marrow
Authors: Yvan-Charvet Laurent , Pagler Tamara , Gautier Emmanuel , Avagyan Serine , Siry Read , Snoeck Hans , Randolph Gwendalyn , Tall Alan R
Published: April 2024

Overexpression or infusions of apoA-1 or HDL lead to reduced atherosclerosis. A major mechanism to explain this effect is thought to be cholesterol efflux from arterial wall macrophage foam cells via ABC transporters. In this study we have tested this hypothesis by transplanting bone marrow deficient in ABCA1 and ABCG1 into atherogenic diet-fed Ldlr deficient mice with or without expression of a human apoA-1 transgene. Unexpectedly, the apoA-1 transgene dramatically suppressed the accelerated atherosclerosis as well as leukocytosis, monocytosis and myeloid cell infiltration of organs in mice receiving double KO bone marrow. Double KO bone marrow showed a 5-fold expansion of hematopoietic stem cells, increased cycling of stem cells, increased myeloid progenitor cells and increased proliferation of bone marrow myeloid cells in response to growth factors. Double KO bone marrow myeloid cells showed increased association of ras with the plasma membrane possibly reflecting increased cholesterol content on the inner leaflet of the plasma membrane and increased ERK signaling in response to IL-3 or GM-CSF treatments. HDL promoted cholesterol efflux and decreased Ras membrane association and ERK signaling and inhibited proliferation of both WT and double KO bone marrow myeloid cells. The findings indicate a novel mechanism in which HDL may suppress atherosclerosis by decreasing myeloid cell proliferation and consequent monocytosis.