bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5112: Contribution of Th17 Cells, a New Subset of CD4
+
T Cells, to Angiogenic Response to Hindlimb Ischemia
Authors: Hata Takeki , Takahashi Masafumi , Kawaguchi Masanori , Kashima Yu-ichiro , Izawa Atsushi , Hida Shigeaki , Iwakura Yo-ichiro , Taki Shinsuke , Ikeda U-ichi
Published: April 2024

Background:
Accumulating evidence suggests contribution of T cell to the collateral development in ischemic tissue; however, its mechanism remains unclear. Th17 cells are recently identified as a new subset of CD4
+
T cells and specifically secrete interleukin-17 (IL-17). Here, we investigated the role of CD4
+
T and Th17 cells in the induction of angiogenesis.

Methods and Results:
Unilateral hindlimb ischemia was produced in wild-type mice (WT, C57BL/6). Neutralization using anti-CD4 antibody resulted in complete depletion of CD4
+
T cells (flow cytometry) and a significant reduction of blood flow perfusion (laser Doppler perfusion imaging, LDPI: 0.61±0.08 vs. 0.41±0.11 p<0.05) in the ischemic limbs. Since IL-6, inducer of Th17 cells, was upregulated in the ischemic limbs, we next focused on Th17 cells. The expression of IL-17 and RORgt (Th17 regulator) was upregurated in the ischemic limbs, but not in the contralateral limbs. IL-17-deficient (IL-17
−/−
) mice also showed a significant decrease in angiogenesis, compared with WT mice (LDPI: 0.72±0.26 vs. 0.38±0.27, p<0.01), accompanied with significant reduction of capillary formation (CD31 immunostaining: 1223.3±267.3/mm
2
vs. 692.9±165.6/mm
2
, p<0.01). Immunostaining and flow cytometry showed decreased infiltration of neutrophils, monocyte/macrophages, and CD4
+
T cells in the ischemic limbs of IL-17
−/−
mice (p<0.05). The levels of angiogenic factors such as IL-1b and VEGF-A were also decreased in the ischemic limbs of IL-17
−/−
mice (p<0.05). To determine the contribution of bone marrow-derived Th17 cells, bone marrow-transplanted mice (BMT) were prepared. BMT
IL-17−/− to Wild
showed a significant decrease in angiogenic response, compared with BMT
Wild to Wild
(LDPI: 0.60±0.15 vs. 0.24±0.11, p<0.05). To assess the effect of Th17 cells on angiogenesis, spleen-derived CD4
+
T cells from IL-17
−/−
or WT mice were implanted into the ischemic limbs of athymic mice. Implantation of CD4
+
T cells from IL-17
−/−
mice showed a significant reduction of angiogenic response, compared with those from WT mice (LDPI: 0.71±0.17 vs. 0.51±0.13, p<0.05).

Conclusion:
CD4
+
Th17 cells contribute to the angiogenic response and provide new insights into the mechanism by which T cells promote collateral development.
Abstract 5114: Leptin Promotes Mobilization of Flk1-positive Vascular Progenitor Cells From the Bone Marrow and Enhances Neovascularization After Peripheral Ischemia
Authors: Schroeter Marco R , Stein Susanne , Heida Nana-Maria , Leifheit Maren , Hasenfuss Gerd , Konstantinides Stavros , Schaefer Katrin
Published: April 2024

Experimental evidence suggests that bone marrow-derived progenitor cells participate in the formation of new blood vessels and endothelial repair. We have previously shown that leptin enhances the recruitment and incorporation of human, culture-expanded endothelial progenitor cells (EPC) into neointimal lesions after vascular injury and promotes re-endothelialization. The aim of the present study was to examine the potential of circulating leptin to promote ischemia-induced neovascularization in vivo, and to determine the contribution of bone marrow-derived (endothelial) progenitor cells to this process. In wild-type mice, daily intraperitoneal injection of leptin over five days promoted neovascularization after hindlimb ischemia, as determined by the quantification of CD31-immunopositive capillary endothelial cells (P<0.01 vs. control). In vitro studies demonstrated that systemic administration of leptin increased the number of cultured, spleen-derived acLDL+, lectin+ EPC (P<0.01 vs. vehicle), and enhanced their potential to integrate into endothelial cell networks (P<0.001) or to adhere to endothelial cell sprouts (P<0.001). Analysis of mice after sublethal irradiation and transplantation of GFP-positive bone marrow revealed that bone marrow-derived cells are recruited to ischemic muscles and incorporate into CD31-positive capillaries. Although flow cytometry analysis revealed that leptin did not alter the number of sca1+, c-kit+ cells within the bone marrow or peripheral blood, leptin increased the number of circulating sca1+ or GFP+, Flk1+ cells, in contrast to vehicle or GCSF (P<0.05 for both). PCR analysis confirmed mRNA expression of the leptin receptor in bone marrow cells. Ongoing studies focussing on potential mechanisms mediating the effects of leptin on bone marrow cells indicate increased protein kinase B phosphorylation and MMP9 expression in response to leptin. Taken together, our studies suggest that the proangiogenic effects of leptin may involve the interaction with leptin receptor-positive cells within the bone marrow resulting in enhanced mobilization of Flk1+ vascular progenitors and neovascularization in response to ischemia.
Abstract 5121: Sustained Impairment of Endothelial Vasomotor Function Predicts Development of Renal Dysfunction in Patients With Chronic Ischemic Heart Disease
Authors: Takishima Isao , Kitta Yoshinobu , Hirano Mitsumasa , Nakamura Takamitsu , Ino Yuri , Makino Aritaka , Sano Keita , Saito Yukio , Kodama Yasushi , Obata Jun-ei , Kawabata Ken-ichi , Kugiyama Kiyotaka
Published: April 2024

Endothelial dysfunction in systemic arteries is associated with renal dysfunction. It is unclear whether reversibility of endothelial dysfunction in systemic arteries in response to risk factors reduction has prognostic value for future renal dysfunction. This study examined whether changes in endothelial vasomotor function in the brachial artery after optimized therapy for coronary artery disease (CAD) predict the development of renal dysfunction.

Methods:
This study included 214 patients with newly diagnosed chronic CAD who had an impaired flow-mediated dilation (FMD) of the brachial artery and chronic kidney disease (CKD) stage 1 or 2 at enrollment. After enrollment, all patients had individualized, optimized therapies including medications and life style changes based on AHA guidelines to reduce risk factors for CAD. Measurement of FMD was repeated at enrollment (1
st
test) and 6 months after therapies were initiated (2
nd
test). Thereafter, all patients continued their same medications and were prospectively followed for ≤ 36 months or until the progression to CKD stage 3. Impairment of FMD was defined as < 5.5% (mean minus 1 SD of FMD in 100 age- and sex-matched normal subjects).

Results:
FMD was persistently impaired (< 5.5%) in 114 (53.2%) patients at the 2
nd
test, whereas it was improved (FMD ≥ 5.5%) in the remaining 100 (46.7%) patients. During the follow-up period, the progression to CKD stage 3 occurred in 46 (40%) patients with persistently impaired FMD and in 21 (21%) patients with improved FMD (p < 0.01). Multivariate Cox proportional hazards analysis showed that persistent impairment of FMD was a significant predictor of the progression to CKD stage 3 (HR 2.04, 95% CI 1.1 - 3.9, p < 0.01) that was independent of 1
st
FMD, hypertension, diabetes, and age. Using a
c
-statistic, the predictive value of persistently impaired FMD was incremental over that of 1
st
FMD, hypertension, diabetes and age (area under the ROC curve; 0.80 and 0.71, respectively, p = 0.01).

Conclusions:
Persistent impairment of endothelial vasomotor function despite optimized therapy to reduce CAD risk factors is an independent predictor of the development of renal dysfunction. The periodic measurement of FMD is useful for risk stratification for future renal dysfunction.
Abstract 5134: Distribution of Dipyridamole in Blood Components Among Post-Stroke Patients Treated With Extended Release Formulation
Authors: Serebruany Victor L , Sabaeva Elena , Booze Christopher , Atar Oliver , Hanley Dan
Published: April 2024

Background and Purpose - Extended release dipyridamole (ERD) is widely used in patients after ischemic stroke; however, the ability of this antithrombotic agent to be stored in different blood cells has never been explored in post-stroke patients. We hypothesized that since ERD is known to be highly lipophylic, the drug may be present not only in plasma, but also accumulated in platelets, leukocytes, and erythrocytes.

Methods - Fifteen patients after documented ischemic stroke were treated with Aggrenox (ERD and low dose aspirin combination) BID for 30 days, and 12 of them completed the study. ERD concentrations in blood cells and platelet poor plasma were measured by spectrofluorimetry at Baseline, Day 14, and Day 30 after the initiation of therapy.

Results - The background level of spectrofluorometry readings differs slightly among the blood components (132–211 ng/mL) due to the differences in the preparation of samples and cell isolation techniques. As expected, 2 weeks of ERD therapy produced steady state plasma concentration of dipyridamole already at Day 14 (1680±542ng/mL), followed by a slight not significant decrease at one month (1619±408). Two weeks of therapy was sufficient to achieve a consistent dipyridamole accumulation in erythrocytes (361±43), but not in platelets (244±78), or leukocytes (275±49). In fact, while blood cells continued dipyridamole intake beyond 14 days period, and this increase (398±66) was significant (p=0.02) at thirty days.

Conclusion - Treatment with ERD in post-stroke patients resulted not only in the achieving of therapeutic plasma dipyridamole concentrations, but also deposition of the drug in erythrocytes and leukocytes, but not in platelets. If confirmed, these data will affect our better understanding of dipyridamole pleiotropy, and may expand future clinical indications of ERD formulation.
Abstract 5143: Pharmacological Inhibition of Phosphoinositide 3-kinase Gamma Suppresses Reparative Neovascularization and Impairs Recovery From Myocardial Infarction
Authors: Siragusa Mauro , Katare Rajesh G , Emanueli Costanza , Madeddu Paolo
Published: April 2024

Phosphoinositide 3-kinase gamma (PI3K
γ
) is primarily involved in G
i
-coupled receptor-mediated signaling controlling innate and adaptive immune response. Recently, PI3K
γ
inhibitors have been proposed as anti-inflammatory agents in a wide spectrum of pathologies, including cardiovascular disease. We have shown previously that PI3K
γ
plays a pivotal role in vasculogenesis and reparative angiogenesis. Here we aimed to determine whether inhibition of PI3K
γ
could be detrimental for recovery from myocardial infarction (MI). MI was induced in CD1 mice by permanent LAD ligation. The PI3K
γ
-selective inhibitor AS605240 (AS) or DMSO (control) was injected i.p. daily from 3 days before MI until either 3 or 14 days post-MI. In AS-treated infarcted hearts, echocardiographic measurements carried out at 14 days post-MI showed extensive wall thinning, reduced stroke volume (24.9±2.5 vs. 36.1±3.6
μ
l, P<0.05), ejection fraction (19.2±1.9 vs. 26.4±3.0%, P<0.05), fractional shortening (9.1±0.9 vs. 12.5±1.5%, P<0.05) and cardiac output (12.1±1.3 vs. 18.0±1.8 ml/min, P<0.05). Histological assessment of fibrosis revealed that infarct size was increased by 1.4 fold (P<0.01 vs. control). In control hearts at 14 days post-MI, the density of capillaries and arterioles in the peri-infarct area increased by 1.3 and 2.6 folds, respectively (P<0.01 vs. remote area for both comparisons). In contrast, neovascularization response in the peri-infarct area of AS-treated hearts was completely suppressed at both capillary and arteriole level. Interestingly, at 3 days post-MI the number of apoptotic microvascular endothelial cells and cardiomyocytes in the peri-infarct area was increased by 2.6 and 2 folds, respectively (P<0.01 vs. control for both comparisons). Furthermore, PI3K
γ
inhibition strongly reduced the number of CD45
+
MNCs infiltrating the interstitial space (15.8±6.9 vs. 106.9±28.2 cells/mm
2
, P<0.05) and surrounding arterioles (0.1±0.1 vs. 1.2±0.3 cells/arteriole, P<0.001) of the peri-infarct area at 3 days post-MI. Our results indicate that PI3K
γ
is a key modulator of post-infarct remodeling and reparative neovascularization. Caution is recommended when using PI3K
γ
inhibitors as anti-inflammatory drugs in patients with coronary heart disease.
Abstract 5150: The Difference in Awake Ambulatory Blood Pressure and Office Blood Pressure: A Meta-analysis
Authors: Ishikawa Joji , Ishikawa Yukiko , Schwartz Joseph E , Pickering Thomas G
Published: April 2024

Background
: Masked hypertension is associated with an increased risk of cardiovascular events, while white coat hypertension is considered to be a relatively lower risk condition; both are defined by the difference between awake ambulatory blood pressure (ABP) and office blood pressure (OBP). We review evidence on the relationship of this difference to age.

Method
: We performed a systematic review of papers in PubMed using the combination of terms “office and ambulatory or home blood pressure (HBP),” and selected articles in which the OBP and awake ABP (or HBP) were presented. A random effects model was used for the meta-analyses.

Results
: In the 18 studies of adults (N=8779), OBP was higher than awake ABP (the difference; 2.0±0.04/1.0±0.03 mmHg, both P<0.001); however, in the 5 studies of children and youth (N=1829), awake ABP was higher than OBP (7.9±0.12/7.9±0.10 mmHg, both P<0.001). In studies of general populations, OBP became higher than awake ABP after age 49.1/43.0 in women (N=5893) and age 44.7/44.8 in men (N=6776). In the studies comparing OBP and HBP (N=8079), OBP was higher than HBP at all ages. In the studies in which OBP, awake ABP, and HBP were all available, HBP was lower than awake ABP, and became similar to awake ABP around 65.2 years old (Figure

).

Conclusion
: Awake ABP tends to exceed OBP at younger ages while the reverse is true after age 50; in contrast OBP tends to exceed HBP at all ages. This suggests that the prevalence of masked hypertension will be lower, while that of white coat hypertension will be greater when defined by HBP instead of ABP. The determinants of the age-related differences between ABP (or HBP) and OBP merit further investigation.
Abstract 5162: Inhibition of Interleukin-6 Transsignaling Suppresses Atherosclerotic Lesion Development
Authors: Schuett Harald , Schley René , Salguero Gustavo , Tietge Uwe J , Waetzig Georg H , Seegert Dirk , Rose-John Stefan , Grote Karsten , Schieffer Bernhard
Published: April 2024

Introduction:
The inflammatory cytokine interleukin-6 (IL-6) is a central mediator in the pathophysiology of various acute and chronic inflammatory disorders e.g. sepsis, Crohn disease or rheumatoid arthritis. Of interest, mainly the IL-6 transsignaling which comprises binding of IL-6 to the soluble IL-6 receptor (sIL-6R) and subsequent signal transduction as an IL-6/sIL-6R complex via ubiquitary expressed gp130 plays a pivotal role in these processes. Since atherosclerosis is likewise characterized by chronic vascular inflammation we here investigated the impact of IL-6 transsignaling on atherosclerotic plaque development in a murine model. For this reason, we used the fusion protein sgp130Fc which competitively binds to the IL-6/sIL-6R complex and thus specifically inhibits the IL-6 transsignaling.

Methods and results:
Initial
in vitro
experiments confirmed that recombinant sgp130Fc was able to inhibit the IL-6-induced STAT-3 phosphorylation in primary murine aortic vascular smooth muscle cells and murine monocytic RAW 264.7 cells dose-dependently (VSMC maximal at 10
μ
g/ml; RAW maximal at 50
μ
g/ml, Western blot, P<0.05). IL-6 induced the migration and proliferation of VSMC
in vitro
which could be inhibited by sgp130Fc (transwell inserts, BrdU-incorporation, −30%, P<0.05). Migration of RAW cells was likewise suppressed when IL-6 transsignaling was abrogated by sgp130Fc pre-treatment (transwell inserts, −40%, P<0.05). Finally, male atherosclerosis-prone LDLR
−/−
mice on a cholesterol rich diet were treated with sgp130Fc (0.5 mg/kg/bw i.p., twice-weekly) or the respective vehicle for 8 and 16 weeks. Even though serum lipid levels were unchanged, sgp130Fc administration led to a dramatically decreased atherosclerotic plaque development (thoracoabdominal aorta −65%/aortic root −35%; oil red O staining, P<0.05, n=5–9) as well as a reduced monocyte invasion into the vessel wall (aortic root −35%; MOMA-2 immunostaining, P<0.05, n=5–9).

Conclusion:
We here demonstrate - for the first time - that inhibition of the IL-6 transsignaling by the pharmacological compound sgp130Fc suppresses pro-atherosclerotic processes and subsequently the development of atherosclerotic plaques.
Abstract 5166: Inhibition of Pro-inflammatory Cytokine Light Reduces Atherosclerotic Lesion Development in Apo E
−/−
Mice
Authors: Akhavanpoor Mohammad , Erbel Christian , Shankar Vijay , Celik Sultan , Lasitschka Felix , Katus Hugo A , Dengler Thomas J
Published: April 2024

Atherosclerotic plaque typically contains infiltrates of activated macrophages and T cells. We previously showed higher levels of LIGHT in human carotid artery plaque tissue samples of symptomatic (TIA, Stroke) compared to asymptomatic patients. Effects of inhibition of the pro-inflammatory cytokine LIGHT on atherosclerosis development was studied in a rodent model. 30 male apoE
−/−
mice fed a western diet were treated either with an adenoviral vector, containing the gene sequence for a fusion protein of the extracellular part of the LIGHT receptor molecule TR2 linked with the Fc part of human IgG1 (AdmTR2hIgG), acting as a specific blocking agent against LIGHT (3×10
9
infection units, tail vein injection) (n=10) or an adenoviral vector containing GFP-gene sequence only (AdGFP, sham) (n=10) or mice receiving sodium chloride (untreated group) (n=10). After 8 weeks aortic root was embedded in OCT and serially cryosectioned in 5
μ
m intervals. Sections every 75
μ
m were stained with Oil RedO, the lesion area was quantified using PC-based image analysis. Inhibition of LIGHT by AdmTR2hIgG markedly reduced atherosclerotic plaque volume (p=0.001) and maximum stenosis (p=0.001) compared to the untreated group or AdGFP treated mice (sham). Analysis of peripheral blood and spleen cells revealed no significant alterations in composition of immune cell compartments or cellular activation status. Immunohistochemistry revealed significant reduction of cellular infiltration by T cells and macrophages as well as T cell- and macrophage density (cell number/lesion area) in atherosclerotic lesions in AdmTR2hIgG compared to untreated/sham mice. Functional blockade of the pro-inflammatory cytokine LIGHT reduced atherosclerotic lesion development in apoE
−/−
mice without major derangement of peripheral cellular immunity. Our findings suggest a relevant role for LIGHT in atherosclerosis warranting further study of pathogenic mechanisms and therapeutic potential.
Abstract 5167: Neutrophil Infiltration of Human Carotid Plaques Correlates With the Characteristics of Rupture-prone Lesions
Authors: Ionita Mihaela G , Vink Aryan , van den Borne Pleunie , van der Kraak Petra , de Vries JP , Moll Frans , Pasterkamp Gerard , de Kleijn Dominique
Published: April 2024

Aim

: Atherosclerosis is a chronic, complex inflammatory process and is the underlying cause of stroke and myocardial infarction due to rupture of the atherosclerotic plaque leading to acute occlusion of the artery in the brain or heart. Little is known about neutrophil infiltration in human carotid atherosclerotic plaques and its association with plaque morhology has not been described yet. This, despite accumulating evidence suggesting a link between systemic inflammation and atherosclerosis. To study this, we scored the number of plaque neutrophils and related this to plaque morphology and inflammatory status.

Methods and Results

: Carotid atherectomy specimens were obtained from 340 patients and stained for the neutrophil marker CD66b. CD66b-positive neutrophils were counted and expressed as number per square millimeter of tissue. Higher neutrophil numbers were found in plaques with a high lipid core, high macrophage numbers and low collagen and smooth muscle cell numbers. Plaques with high neutrophil numbers contained significant higher interleukin-8 (p=0.001), matrix metalloprotease (MMP)-8 (p=0.039) and MMP-9 (p=0.002) concentrations. The number of new capillaries within plaques was significantly (p=0.045) correlated with high neutrophil numbers.

Conclusion

: This shows for the first time that Neutrophil-numbers are strongly associated with the histopathological features and the inflammatory status of rupture-prone atherosclerotic lesions, suggesting a possible role for neutrophils in plaque destabilization.
Abstract 5171: High Levels of Myeloid-related Protein 14 in Human Atherosclerotic Plaques Correlate With the Characteristics of Rupture-prone Lesions
Authors: Ionita Mihaela G , Vink Aryan , Dijke Esme , Laman Jon , Peeters Wouter , de Vries Jean Paul , Moll Frans , Pasterkamp Gerard , de Kleijn Dominique
Published: April 2024

Objective

: Atherosclerotic plaque rupture can lead to severe complications such as myocardial infarction and stroke. Myeloid related protein (Mrp)-14, Mrp-8 and Mrp-8/14 complex are inflammatory markers associated with myocardial infarction. It is, however, unknown if Mrps are associated with a rupture-prone plaque phenotype. In this study, we determined the association between Mrp-14, -8, -8/14 plaque levels and plaque characteristics.

Methods and Results

: In 186 human carotid plaques, levels of Mrp-14, -8 and -8/14 were quantified using ELISA. High levels of Mrp-14 were found in lesions with a large lipid core, high macrophage staining and low smooth muscle cell and collagen amount. Plaques with high levels of Mrp-14 contained high interleukin (IL)-6, IL-8, matrix metalloprotease (MMP)-8, MMP-9 and low MMP-2 concentrations. Mrp-8 and Mrp-8/14 showed a similar trend. Within plaques, a subset of non-foam macrophages expressed Mrp-8 and Mrp-14 and the percentage of Mrp-positive macrophages was higher in rupture-prone lesions compared to stable ones.
In vitro
, this subset of macrophages does not acquire a foamy phenotype when fed with human oxidized low density lipoprotein (oxLDL). In contrast, the vast majority of macrophages lacking Mrp-8/14 membrane expression accumulated lipid droplets in the cytoplasm when fed with oxLDL.

Conclusion

: High levels of Mrp-14 and to a lesser extent also Mrp-8 and Mrp-8/14, expressed by a subset of non-foam macrophages in human plaques, are strongly associated with both histopathological features and the inflammatory status of rupture prone lesions. These results identify Mrps as possible imaging markers to detect the hidden rupture-prone plaque.
Abstract 5208: S-glutathiolation Uncouples Endothelial Nitric Oxide Synthase: A Novel Mechanism of Redox Signaling and Vascular Regulation
Authors: Chen Chun-An , Wang Tse-Yao , Varadharaj Saradhadevi , Reyes Levy , Talukder M.A. Hassan , Chen Yeong-Renn , Druhan Lawrence J , Zweier Jay L
Published: April 2024

Superoxide (·O
2
−
) and secondary oxidants are critical mediators of cellular signaling, proliferation and apoptosis. In response to oxidative stress, protein thiols undergo S-glutathiolation forming mixed disulfides and this modification plays an important role as an adaptive response to oxidative injury in cells, controlling cellular signaling in a manner similar to protein phosphorylation. We investigated the effects of S-glutathiolation on the regulation of eNOS function
in vitro
. We observe that S-glutathiolation greatly reduces NO synthase activity (70% ± 1%), and this loss of activity was reversed by DTT. In contrast, only a 56%±0.9% decrease in NADPH consumption was seen that was only partially inhibited by L-NAME or the calcium chelator EGTA, implicating an increase in eNOS derived ·O
2
−
. Using EPR spin-trapping to directly measure eNOS derived ·O
2
−
, we proved that S-glutathiolation greatly increased ·O
2
−
generation from the enzyme. S-glutathiolation of human eNOS increased ·O
2
−
generation >5-fold and L-NAME and EGTA only partially blocked this ·O
2
−
generation. Thus, S-glutathiolation uncouples eNOS greatly increasing ·O
2
−
generation and the partial or complete lack of inhibition of this by L-NAME and EGTA indicates that the observed ·O
2
−
is largely derived from the reductase domain. Mass spectrometric analysis and molecular modeling revealed that two sights of S-glutathiolation in eNOS are two highly conserved cysteines found at the interface between the FMN and FAD-binding domains, C
689
and C
908
. The modification of these two cysteins appears to interrupt electron transfer to the oxygenase domain, leading to electron leakage. Furthermore, we observe that BCNU and H
2
O
2
- derived oxidative stress induces S-glutathiolation of eNOS in endothelial cells, with measured loss of NO and gain of ·O
2
−
generation, and we show that this process impairs endothelium-dependent vasodilation. Additionally, we observe that in hypertensive vessels eNOS-S-glutathiolation is increased with impaired endothelium-dependent vasodilation that is restored by thiol-specific reducing agents. Thus, S-glutathiolation of eNOS serves as a pivotal switch that provides redox regulation of endothelial function and vascular tone.

This research has received full or partial funding support from the American Heart Association, Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).
Abstract 5220: Cannabinoid Receptor Activation Protects Coronary Endothelium Against Reperfusion Induced Intercellular Gap Formation in a Cellular Model of Ischemia and Reperfusion
Authors: Kasseckert Sascha A , Micoogullari Mustafa , Piper Hans M , Schlüter Klaus-Dieter , Abdallah Yaser
Published: April 2024

Ischemia and reperfusion provokes barrier failure of the coronary microvasculature through activation of the endothelial contractile machinery and formation of intercellular gaps, leading to myocardial edema development that jeopardizes functional recovery of the heart. In vivo studies have shown that cannabinoid receptor (CBR) activation protects reperfused hearts against reperfusion injury in terms of infarct size reduction and functional recovery. The aim of the present study is to investigate whether CBR activation with the CBR agonist R1-Methanandamide (R1M, 10
μ
M) can protect coronary endothelium against reperfusion induced intercellular gap formation. Cultured rat coronary endothelial monolayers were subjected to conditions of simulated ischemia (ANoxia 40min; pH6.4; no glucose) and reperfusion (NORMoxia 40min; pH7.4; with glucose). Stimulation of cannabinoid receptors during reperfusion significantly reduced reperfusion-induced intercellular gap formation (gaps[a.U.]:control:283±21; R1M:172±10*; n=12–16; *p<0.05 vs. control). CB1 receptor blocking with AM251 [250nM] during reperfusion and R1M application completely abolished the R1M mediated protection (gaps[a.U.]: R1M+AM251: 269±32#; n=10; #p<0.05 vs. R1M). Whereas the protective effect of R1M application during reperfusion could be further enhanced if AM630 [300nM], a CB2 receptor antagonist, was added to the reperfusion medium simultaneously to R1M application (gaps[a.U.]: R1M+AM630:132±23#; n=12; #p<0.05 vs. R1M). Inhibition of nitric oxide synthases (NOS) through application of L-NAME [200
μ
M] during reperfusion significantly blunts the protection against reperfusion induced gap formation (gaps[a.U.]:control:305±29; R1M:221±21*; R1M+L-NAME:270±27#; n=6 – 8; *p<0.05 vs. control; #p<0.05 vs. R1M). In conclusion, activation of endothelial cannabinoid receptors protects the coronary endothelium against reperfusion-induced gap formation. This protection is mediated through CB1 receptor mediated NOS activation, whereas simultaneous CB2 receptor activation diminishes this protective effect.
Abstract 5222: Genetic Deficiency of Arginase II Reduces Endothelial Dysfunction and Atherosclerosis in ApoE-Knockout Mice
Authors: Oh Young Jun , Kim Jae Hyung , Ryoo Sungwoo , Sikka Gautam , Berkowitz Daniel
Published: April 2024

Introduction:
Arginase II is known to be one of the key mediator in vascular pathophysiology of cholesterol-medicated endothelial dysfunction by reciprocal regulation of NOS and NO production through substrate competition. We aimed to investigate whether the transgenic Arginase II inhibition could reduce the endothelial dysfunction and atherosclerosis in apolipoprotein E-knockout (ApoE-KO) mice.

Methods and Results:
ApoE-KO mice were bred on an arginase II KO (ArgII-KO) background. Mice that were homozygous deleted at both alleles were used in the study. Male 8 – 12 wk old Arg-KO mice, ApoE-KO mice, Arg/ApoE-KO (Dbl-KO) mice fed high cholesterol diet for 16 weeks, and age-matched wild type (WT) mice fed normal diet were enrolled. Arginase activity was significantly higher in the aorta of ApoE-KO mice than compared to WT mice (1612.2±445.9 pmol Urea/mg protein/min vs. 836.3±12.2 pmol Urea/mg protein/min, n=5,
p
< 0.01), but not detected in Arg-KO and Dbl-KO mice. We measured NO an ROS in vascular strips pinned down endothelial side up using the fluorescent probes DAF-FM diacetate and DHE. Dbl-KO mice showed significantly higher NO production (0.0737±0.0056 intensity/sec vs. 0.0256±0.0029 intensity/sec, n=6,
p
< 0.05) and lower ROS production than ApoE-KO mice (0.0676±0.0065 intensity/sec vs. 0.1147±0.01619 intensity/sec, n=6,
p
< 0.05). We tested endothelial dependent vasorelaxation in aortic rings in organ chambers. The vasorelaxation response to acetylcholine (Ach) in Dbl-KO mice was greater than in ApoE-KO mice (log EC
50
, −5.68 vs. −5.13,
p
<0.05), although it was less than in Arg-KO and WT mice. N
G
-nitro-L-arginine methyl ester (L-NAME) incubation didn’t only impair Ach response, but also abolished the inter-group difference. The aortic atheromatous plaque burden as measured by Sudan IV staining was significantly reduced in Dbl-KO group compared to ApoE-KO group (38.4±3.8% vs. 52.0±2.8%, n=6,
p
<0.01), No significant plaque was detectable in Arg-KO and WT mice.

Conclusion:
The genetic inhibition of Arginase II enhanced NO production, reduced ROS produnction restored endothelial function and lowered atheromatous plaque burden. Therefore, Arginase II represents a crtical novel target for atherosclerosis therapy.
Abstract 5225: Coronary Vasospasm Induced in Transgenic Mice With Variant Phospholipase C-
δ
1 (R257H)
Authors: Shibutani Shuji , Osanai Tomohiro , Ashitate Toshihiro , Echizen Takashi , Tanaka Makoto , Tomita Hirohumi , Miwa Takeshi , Matsubara Hiroaki , Okumura Ken
Published: April 2024

We previously showed that the activity of phospholipase C (PLC)-
δ
1, a key enzyme for Ca
2+
signaling in the coronary artery, was enhanced by 3 times in patients with coronary spastic angina (CSA) compared with control subjects, and it was positively correlated with coronary vasomotility. We further showed that structural mutation at 864G-A variant with the amino acid replacement of arginine 257 by histidine (R257H), was present in 10% of CSA patients and was related to enhanced PLC-
δ
1 activity. However, no evidence was documented whether this variant directly and causatively contributes to the enhanced coronary vasomotility. We therefore examined the direct role of structural mutant PLC-
δ
1 in the genesis of coronary spasm by generating the R257H variant PLC-
δ
1 overexpressing transgenic (TG) mice. In TG mice, the introduced gene of human R257H variant PLC-
δ
1 was expressed in overall vessels including the coronary arteries, and upregulated by 2 times compared with wild type (WT) littermate mice. Baseline blood pressure, heart rate, and body weight were similar between 20-week-old TG and WT mice. Ergonovine at 15, 50, and 80 mg/kg was injected through the carotid vein additively, and ECG was monitored continuously. The incidence of ST segment transient elevation was higher in TG mice than in WT after injection of ergonovine at 15 mg/kg (6/18 in TG vs 1/22 in WT, P<0.05), 50 mg/kg (18/18 in TG vs 3/22 in WT, P<0.01), and 80 mg/kg (18/18 in TG vs 11/22 in WT, P<0.01). Concomitantly with the ST segment elevation, AV block or sinus bradycardia was observed. To further examine the morphological changes in the coronary artery, the microvascular filling technique was used in mice untreated with ergonovine. In TG mice, the decrease in coronary arteriole diameter at baseline was documented in the widespread area, although no change was found in WT mice. These suggest that overexpression of R257H variant PLC-
δ
1 in the coronary artery causes coronary spasm at baseline and after ergonovine. The variant of PLC-
δ
1 appears to be a therapeutic target for coronary spasm in CSA patients.
Abstract 5254: The Role of Caveolae on the Inhibition of Coronary Arterial BK Channels by Angiotensin II in Control and Diabetic Rats
Authors: Lu Tong , Zhang Daimin , Wang Xiao-Li , He Tongrong , Katusic Zvonimir S , Lee Hon-Chi
Published: April 2024

Background:
We have found that the vascular large conductance Ca
2+
-activated K
+
channels (BK) are targeted to caveolae. The goal of this study is to determine the role of caveolae in BK regulation by angiotensin II (AII) in normal and diabetic rats.

Results:
In coronary arterial smooth muscle cells (CASMC) from control rats, AII (2
μ
M) inhibited BK by 40.5% from 227±26 pA/pF to 135±14 pA/pF (HP −60 mV, TP 150 mV, n=7, p<0.05). However, AII had no effects on BK in CASMC transfected with caveolin-1 siRNA (vs 50% inhibition in cells transfected with control siRNA) or in cells from Cav1−/− mice, suggesting the AII effects are caveolae-dependent. Density gradient analysis of rat vessel homogenates showed that caveolin-1, BK, Gq, cSrc, and NOX-1 all reside in the caveolae-rich fraction, where AT
1
R was absent unless the animal was pre-treated with AII, suggesting receptor translocation upon agonist activation. However, CASMC from streptozotocin-induced diabetic rats showed significant reduction in BK (61.5±10.6 pA/pF) with no response to AII (61.2±15.7 pA/pF, n=8). Vascular BK, caveolin-1, AT
1
R, cSrc, and NOX-1 expression in caveolae were all upregulated by 1.6- to 3.1-fold in diabetes. Density gradient analysis of diabetic vessels showed the presence of AT
1
R in the caveolae-rich fraction without AII pretreatment, suggesting that the AT
1
R signaling pathway might be constitutively active in diabetes. This was accompanied by a 2-fold increase in BK tyrosine phosphorylation and BK tyrosine nitration. The AII effects on BK function in diabetic CASMC were abolished by pretreatment (1 hr) with PKC inhibitor or by LavA (cSrc inhibitor) and can be mimicked by incubation (2 hr) of control CASMC with peroxynitrite.

Conclusion:
AT
1
R-BK-enzyme complex in the caveolae microdomain is critical in vascular BK regulation. Enhanced AII-signaling facilitates BK post-translational modification, resulting in BK dysfunction in diabetes.
Abstract 5256: Increased Endothelin-A Receptor-Protein Expression/Localization and Contractile Response to Endothelin-1 of Coronary Microvasculature From Diabetic Patients
Authors: Liu Yuhong , Feng Jun , Khabbaz Kamal , Hagberg Robert , Matyal Robina , Sellke Frank
Published: April 2024

Objectives:
Hyperglycemia is a potent stimulus for endothelin-1 (ET-1) production. Increased ET-1 exposure of coronary vasculature and myocytes in diabetics can hamper blood flow delivery to the vulnerable myocardium and exacerbate contractile function. In the present study, we investigated the protein expression/localization of ET-A receptors, the contractile response to ET-1 and the inhibition of ET-A receptors in the human coronary microvasculature in diabetic and case-matched non-diabetic patients.

Methods:
Right discarded atrial tissues were harvested pre-CPB from 30 patients with and without diabetics undergoing cardiac surgery. Coronary arterioles (90 – 180 micrometer in diameter) were dissected from the harvested tissue and placed in an organ bath containing Krebs solution. Microvascular constriction was assessed by videomicroscopy in response to ET-1, with and without an endothelin-A (ET-A) receptor antagonist. The expression and localization of ET-A receptors were also examined using immunoblot and confocal immunofluorescence microscopy.

Results:
ET-1 caused a dose-dependent contractile response of coronary arterioles from diabetics and non-diabetics. The contractile response of diabetic arterioles to ET-1 (10
−9
M) was significantly increased compared with those of non-diabetics (15±3% vs. 8±2%, P<0.05). The contractile response of microvessels from diabetics and non-diabetics to ET-1 was significantly inhibited in the presence of the ET-A receptor antagonist BQ123 (10
−7
M, P<0.05, respectively). The protein expression of ET-A receptors was significantly elevated from 1 fold of non-diabetics to 2.5 folds of diabetics (P<0.01). Immunofluorescent staining displayed stronger ET-A receptors signals of smooth muscle from diabetics than those of non-diabetics.

Conclusion:
Hyperglycemia up-regulates ET-A-receptor protein expression/localization and increases the contractile response to ET-1 in the diabetic coronary microvasculature. The contractile response to ET-1 is via activation of ET-A receptors in diabetics since blockade of ET-A receptors prevented ET-1-induced vasoconstriction. These findings support the notion that diabetic patients face an increased ET over-stimulation.
Abstract 5264: Recombinant Human Angiotensin Converting Enzyme 2 Normalizes Blood Pressure and Reduces the Progression of Diabetic Nephropathy
Authors: Oudit Gavin Y , Liu George C , Zhong Jiu-Chang , Schuster Manfred , Penninger Josef M , Herzenberg Andrew M , Kassiri Zamaneh , Scholey James W
Published: April 2024

Background: Diabetes nephropathy is one of the most common causes of end-stage renal failure. Inhibition of ACE2 function exacerbates diabetic kidney injury while renal ACE2 is downregulated in diabetic nephropathy. We hypothesize that recombinant human ACE2 (rhACE2) will slow the progression of diabetic kidney injury.

Methods and Results: Male 12-week old diabetic Akita mice (
Ins2
WT/C96Y
) and controls (
Ins2
WT/WT
) were injected daily with rhACE2 (2 mg/kg, i.p.) or placebo for four consecutive weeks. In Akita
Ins2
WT/C96Y
mice injected with hrACE2, plasma ACE2 activity showed a marked increase resulting in significant reduction in urinary albumin excretion (118±21 vs 224±16
μ
g/24 hrs; n=6~8; p<0.05). Treatment with rhACE2 decreased renal cortical angiotensin (Ang) II (12.1±1.9 vs 22.4±3.2 pg/mg; p<0.05) and increased Ang (1–7) (14.2±2.1 vs 8.8±1.9 pg/mg; p<0.05) levels, respectively, independent of changes in the expression of
Ace
and
Ace2
while NADPH oxidase activity in renal cortical tissue from Akita mice was reduced by treatment with rhACE2 (364±45 vs 1720±277 RLU/sec/mg protein; n=6, p<0.05). These effects resulted in normalization of the increased extracellular matrix gene expression (
α
-smooth muscle actin, collagen III and fibronectin), reduction in glomerular volume and basement membrane thickness in the diabetic Akita mice. Activation of protein kinase C (PKC)á and PKCâ was reduced by 60 –70% in Akita diabetic mice treated with rhACE2. Ang II and high glucose-induced reactive oxygen species generation was evaluated using lucigenin-based NADPH oxidase activity and DHE fluorescence in cultured rat mesangial cells was suppressed by 25 and 250 ng/ml of rhACE2 in a dose-dependent manner. The mild hypertension in Akita
Ins2
WT/C96Y
mice was normalized by treatment with rhACE2 (122.3±2.1 vs 108.4±1.9 mmHg; n=6, p<0.05) while the beneficial effects seen with rhACE2 occurred in the absence of hyperkalemia or acute renal failure and without a differential impact on hyperglycemia in the Akita diabetic model.

Conclusions: We conclude that biologic antagonism of the RAS using rhACE2 can slow the progression of diabetic kidney injury and ACE2 may prove to be a useful adjunctive therapy for diabetic kidney disease.
Abstract 5277: Central Role of Early Endosomal Antigen 1 (EEA1) in Organization of Angiotensin II Signaling Leading to Akt Activation in Early Endosome
Authors: Nazarewicz Rafal R , Salazar Gloria , Patrushev Nikolay , Hilenski Lula , Martin Alejandra S , Xiong Shiqin , Ushio-Fukai Masuko , Alexander R Wayne
Published: April 2024

Akt activation is a major output of Angiotensin II (AngII) signaling. Abundant evidence indicates that AngII signaling occurs in multiple signaling compartments. The mechanisms organizing post-membrane events are incompletely understood. Akt activation requires EGFR transacti-vation at the cell membrane and also requires the endocytotic machinery. We hypothesized that Akt activation appears within the endosomal compartment. In vascular smooth muscle cells, inhibition of endocytosis by phenylarsine oxide (10
μ
M), concanavalin A (0.3 mg/ml) or overexpression of dynamin K44A mutant, reduce by 90% AngII-induced Akt phosphorylation at Thr308 and Ser473. Cell fractionation shows that Akt phosphorylation occurs mainly in early endosomal fractions after 3 min of AngII (0.1
μ
M). We posited that the early endosomal marker and scaffolding protein, early endosome antigen 1 (EEA1), is crucial for organizing early endosomal AngII-Akt signaling. Cell fractionation, immunostaining and co-immunoprecipitation studies show that AngII recruits EEA1, AT1R, EGFR, PDK1, PAK1, PKC-alpha and Akt into early endosomes to form a signaling complex. We demonstrate that PAK1 and PKC-alpha are essential components of the EEA1-binding complex required for Akt activation. siRNA knockdown of EEA1 decreases AngII-induced ( 0.1
μ
M, 3 and 5 min) PDK1, PKC-alpha and Akt phosphorylation by 90%. AngII-induced (0.1
μ
M, 5 min) EGFR transactivation or EGFR degradation after 30 and 60 min of EGF stimulation (50 ng/ml) was unaffected. Further, downregulation of EEA1 prevents Akt activation by mislocation of critical components of the signaling complex. Taken together, we demonstrate a novel function for EEA1 itself as a central organizer of AngII-Akt signaling in early endosomes.
Abstract 5313: Reduction of S100A12 in Marfan Syndrome Aortic Smooth Muscle Cells Attenuates Inflammatory and Apoptotic Pathways
Authors: Bowman Marion Hofmann , Gawdzik Joseph , Nicolarsen Jeremy , Raman Jai , Dellefave Lisa , McNally Elizabeth
Published: April 2024

Marfan syndrome (MFS) is associated with thoracic aortic aneurysms. S100A12 has been associated with initiation of inflammatory pathways via activation of the receptor for advance glycation endproducts (RAGE) and is up regulated in smooth muscle cells of ruptured human coronary artery plaques. We tested the hypothesis that S100A12 is expressed in SMC in human aortic aneurysms of MFS, and that downregulation of S100A12 may suppress inflammatory and apoptosis pathways.

Methods: Aortic tissue from a patient undergoing surgery for MFS aneurysm and control aortic tissue were compared and several cell lines of primary SMC’s were established. SMC that carry a mutation in the FBN-1 gene (Arg529Term) and control SMC were each transfected with siRNA-S100A12-GFP and siRNA-control-GFP. GFP positve SMC were isolated by FACS, followed by qRT-RCR using arrays for inflammation and apoptosis pathways (RT2 profiler PCR Array, SA Biosciences).

Results:

Immunohistochemistry of aortic tissue from MFS revealed expression of S100A12 in SMC, but not in SMC of control aortic tissue. S100A12 was expressed strongest in SMC near the focal areas of medial degeneration.

Cultured SMC that carry the FBN-1 mutation strongly expressed S100A12 protein. S1090A12 expression was completely abolished by transfection with siRNA-S100A12-GFP construct but not with siRNA control-GFP construct.

S100A12 reduction in FBN-1 mutant SMC suppressed at least two fold the mRNA of 80% of genes on the inflammation array and of 91% of the genes on the apoptosis array.

The death receptor Fas, caspase 10 and effector caspases 3 and their substrate DFFA were probed using semi-quantitative immunoblotting. FBN-1 mutant SMC showed approximately 2-fold increase for Fas, and two fold increase in Casp3 and DFFA compared to control SMC. Treatment with Fas CH11 activating monoclonal antibody induced Casp3 approximately 12 fold in FBN-1 mutant SMC compared to a two fold induction in control SMC (p< 0.001). Reduction of S100A12 in FBN-1 mutant SMC reversed this pattern with s significant reduction in Fas, caspase 10 & 3, and DFFB. Summary: S100A12 is upregulated in SMC in thoracic aneurysms and reduction of S100A12 reduces Fas-mediated apoptosis in aortic SMC in MFS.

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 5419: Hypokalemia Blunts Left Ventricular Mass Regression in Hypertensive Patients During Losartan- or Atenolol-Based Treatment: The LIFE Echo-substudy
Authors: Chinali Marcello , Aurigemma Gerard P , Okin Peter M , Gerdts Eva , Wachtell Kristian , Kjeldsen Sverre E , Julius Stevo , Dahlöf Björn , de Simone Giovanni , Devereux Richard B
Published: April 2024

Background: It has been reported that hypokalemia (HypoK) is associated with persistent ECG left ventricular (LV) hypertrophy in treated hypertensive patients. The aim of the present study was to assess the impact of HypoK on echocardiographic LV mass regression during aggressive antihypertensive treatment.

Methods: We analyzed data from 863 patients in the LIFE echo substudy (age 66±7 years, 41% women) with available serum K
+
levels. Patients were dichotomized according to presence of HypoK (i.e. <3.90mmol/L, lowest quartile). LV mass regression after one year of treatment was compared among groups.

Results: Patients with HypoK had similar mean age and gender distribution to patients with normal K
+
(all p=ns), but higher systolic and diastolic blood pressure (BP) and heart rate (all p<0.05). After one year, similar reduction among groups in both systolic and diastolic BP (p=ns) was found. In multivariate analysis controlling for differences in age, gender, baseline LV mass, hydroclorothiazide use before or during the study, baseline BP, and BP change, patients with HypoK had a significantly lower regression of LV mass as compared to the normal K
+
group (p<0.001). Furthermore, while in patients randomized to Losartan (n=434) reduction in LV mass was not significantly different in the absence or presence of HypoK (p=ns), in the Atenolol group HypoK was associated with a nearly 60% lower LV mass reduction (Figure

; p<0.001).

Conclusions: Despite similar blood pressure reduction with treatment, patients with HypoK have a lower LV mass regression. While Losartan is able to offset the negative impact of HypoK, its presence markedly affects LV mass reduction on Atenolol-based therapy.

Figure.

Left ventricular mass reduction after one year of anti-hypertensive treatment according to serum K

+

levels and treatment arm.