bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5224: CB1 Receptor Inhibition Leads to Decreased Vascular AT1 Receptor Expression, Inhibition of Oxidative Stress and Improved Endothelial Function
Authors: Tiyerili Vedat , Zimmer Sebastian , Jung Suzin , Wassmann Kerstin , Nickenig Georg , Wassmann Sven
Published: April 2024

Inhibition of the cannabinoid receptor CB1 (CB1-R) exerts numerous effects such as modulation of blood pressure, insulin sensitivity and lipid concentrations. However, direct vascular effects of CB1-R inhibition remain unclear.

Methods and Results:
CB1-R expression was validated in vascular smooth muscle cells (VSMC) and aortic tissue of mice (real-time RT-PCR). Treatment of cultured VSMC with the selective CB1-R antagonist rimonabant resulted in reduced angiotensin II (ang II)-mediated reactive oxygen species (ROS) production (DCF fluorescence) and NADPH oxidase activity (lucigenin-enhanced chemiluminescence). Because rimonabant inhibited ang II-induced but not basal ROS production, and ang II activates NADPH oxidase through ang II type-1 receptor (AT1-R) stimulation, we analyzed AT1-R expression in VSMC. CB1-R inhibition with rimonabant or AM251 led to down-regulation of AT1-R protein expression (Western blot), stimulation with the CB1-R agonist CP55,940 resulted in AT1-R up-regulation, and co-incubation with CP55,940 abolished the effect of rimonabant, indicating that AT1-R expression is directly regulated by the CB1-R. The expression of rac1 and MnSOD was not affected. VSMC showed no significant expression of the CB2-R under basal conditions (real-time PCR, Western blot), and CB2-R inhibition had no impact on AT1-R expression in VSMC. To investigate the relevance of CB1-R inhibition in vivo, apolipoprotein E-deficient (ApoE−/−) mice were treated with cholesterol-rich diet and rimonabant or vehicle for 7 weeks. Consistent with the in vitro findings, CB1-R inhibition decreased AT1-R expression (real-time RT-PCR, Western blot), NADPH oxidase activity, and ROS production (L-012 chemiluminescence) in the aortic wall. Importantly, CB1-R inhibition improved endothelium-dependent vasodilation of isolated aortic segments (organ chamber experiments), indicating a functional relevance in vivo.

Conclusions:
CB1-R inhibition leads to decreased vascular AT1-R expression, NADPH oxidase activity and ROS production in vitro and in vivo. This antioxidative effect is associated with improved endothelial function in ApoE−/− mice, indicating beneficial direct vascular effects of CB1-R inhibition.
Abstract 5251: H
2
O
2
-induced Protein Kinase G Dimerization and Vasodilation in Human Coronary Arteriole
Authors: Zhang David , Mendoza Suelhem , Zinkevich Natalya , Borbouse Lena , Gutterman David
Published: April 2024

Flow-induced dilation of human coronary arterioles (HCA) is mediated by a unique mechanism involving the release of H
2
O
2
from the mitochondria of endothelial cells and subsequent smooth muscle relaxation via K
+
-channel-dependent membrane hyperpolarization. The precise mechanisms by which H
2
O
2
induces smooth muscle hyperpolarization remain largely undefined. An important mechanism of action of H
2
O
2
involves the oxidation of key cysteine residues in its target proteins, including protein kinase G 1-alpha (PKG-1
α
). Here we hypothesize that H
2
O
2
dilates HCA through direct oxidation and activation of PKG-1
α
leading to the subsequent opening of large-conductance Ca
2+
-activated K
+
channels (BK
Ca
) in smooth muscle cells. In isolated HCA, H
2
O
2
(10
−6
−3×10
−4
M) induced dose-dependent dilations in both endothelium-intact and -denuded vessels (relaxations at 10
−4
M of 83.5±3.7% and 85.1±8.4%, respectively; n=4 – 6). The relaxations were largely abolished by iberiotoxin, a BK
Ca
blocker (3.4±2.1% and 19.0±10.2% in intact and denuded vessels, respectively). The PKG inhibitor Rp-8-Br-cGMP also markedly inhibited H
2
O
2
-induced dilations (4.5±6.0%; n=6), whereas the guanylyl cyclase inhibitor ODQ has no significant effects (75.6±8.4%; n=3). The expression of PKG-1
α
and BK
Ca
channels in HCA smooth muscles were confirmed by immunohistochemistry and Western blot analysis. In cultured human coronary arterial smooth muscle cells, H
2
O
2
induced dose-dependent dimerization of PKG-1
α
, which was subsequently reduced to the monomer forms by the reducing agent
β
-mecaptomethanol. We also examined flow-mediated dilation in HCA pretreated with iberiotoxin or Rp-8-Br-cGMP. Both agents markedly inhibited relaxation responses as compared to control (maximal relaxations of 36.0±3.8% vs. 69.2±6.8% in controls, and 30.1±4.8% vs. 57.8±3.0% in controls, respectively; n=4 –5). It is concluded that the activation of PKG-1
α
via H
2
O
2
-induced protein dimerization and subsequent opening of BK
Ca
channels serves as a novel mechanism of flow-induced H
2
O
2
-mediated relaxations in HCA. This signaling pathway may have implications for the pathogenesis and therapy of coronary disease.
Abstract 5275: STAT3-dependent Acute RANTES Production in Vascular Smooth Muscle Cells Modulates Inflammation Following Arterial Injury
Authors: Kovacic Jason C , Gupta Rohit , Lee Angela C , Ma Mingchao , Fang Fang , Tolbert Claire N , Walts Avram D , Beltran Leilani E , San Hong , Chen Guibin , Boehm Manfred
Published: April 2024

Inflammation is a key component of arterial injury, with vascular smooth muscle cell (VSMC) proliferation and neointimal formation the final outcome of this process. However, the acute events that establish the blueprint for this vascular inflammatory response are incompletely understood. To address this deficiency we examined events immediately after femoral artery wire injury in mice and identified a marked acute recruitment of macrophages and T cells, with vessel-associated CD3
+
T cells increasing ~20-fold within 1 day of injury. Correspondingly RANTES, a T cell and macrophage chemo-attractant, was upregulated in vessels > 20-fold within 6 h of injury. RANTES production was modulated by p21
Cip1
; at 6 h local RANTES was 45-fold higher in
p21
−/−

than wild type (WT) mice, and acute T cell infiltration was > 2-fold greater in
p21
−/−

than WT mice at 1 day post injury. Immune-staining and reciprocal bone marrow (BM) transplantation between WT and
RANTES
−/−

mice identified medial VSMCs as the principal source of acute RANTES production. Unique to VSMCs, RANTES production was initiated by TNF
α
, but not IL-6 or gp130 (an IL-6 signal transducer).
RANTES
transcription was dependent on downstream binding of a complex formed between NF-
κ
B (p65 subunit) and STAT3 to NF-
κ
B binding sites in the
RANTES
promoter, with shRNA knockdown of either STAT3 or p65 in VSMCs markedly abrogating RANTES production. p21
Cip1
potentially modulated RANTES levels by both restraining STAT3 transcription and co-association with p65.
In vivo
, acute activation of NF-
κ
B and STAT3 in medial VSMCs was identified, with RANTES levels 6 h after injury in
TNFα
−/−

mice significantly reduced compared to WT controls. One day after injury,
RANTES
−/−

mice reconstituted with WT BM displayed a marked reduction in T cell and macrophage infiltration compared to identically reconstituted WT mice. Finally, we generated mice with smooth muscle cell-specific conditional STAT3-knockout (
STAT3

fl/fl

;SM22α-Cre
) and confirmed the STAT3-dependence of acute RANTES production by VSMCs. We conclude that early inflammatory cell recruitment following arterial injury is regulated by acute local RANTES production by medial VSMCs, which is in turn stimulated by TNF
α
and modulated by downstream NF-
κ
B-STAT3 signaling.
Abstract 5280: Comparing Epigenetic Modifications of Adult Progenitor Cells
Authors: Boeckel Jes-Niels , Koyanagi Masamichi , Ohtani Kisho , Mummery Christine , Momma Stefan , Zeiher Andreas M , Dimmeler Stefanie
Published: April 2024

Cell therapy is a promising option for treatment of cardiovascular diseases. Recent studies demonstrate that pluripotency can be induced in somatic cells by a combination of genes such as Oct4, Klf4, c-Myc and Sox2. To address whether adult progenitor cells might express some of the pluripotency genes and therefore might be more easily reprogrammable, we analyzed expression and accessibility of the promoters of pluripotency genes in adult progenitor cells. Oct4 mRNA was expressed at low level (0.2–0.5% compared to human embryonic stem cells (ESC)) in peripheral blood derived early endothelial progenitor cells (EPC), bone marrow-derived mesenchymal stem cells (MSC) and CD34+ cells, whereas neuronal stem cells (NSC) did not express Oct4. Klf4 mRNA was highly expressed in EPC, MSC and CD34+ cells but not in NSC. Interestingly, Sox2 was selectively expressed in NSC (71,65±13,01%) but was low in EPC, MSC and CD34+ (< 4% of hESC). To investigate whether epigenetic mechanisms account for the differential expression of the pluripotency genes, we analyzed promoter DNA methylation by bisulfite sequencing with 2 different primers. The Oct4 promoter showed high incidence of methylated CpG islands in Region A (EPC:82%; MSC:87%; CD34+ 71% compared to ESC: 5 %). Interestingly, the DNA methylation of the Nanog promoter in Region B was low in EPC (37%), MSC (8%), and ESC (9 %) in contrast to CD34+ cells (97%). At the level of histones, chromatine immunoprecipitation experiments demonstrated that the active marks, acetylated-histone H3 (H3Ac) and trimethylation of H3 lysine 4 (H3K4m3) were increased at the Nanog promoter in EPC and MSC. The Sox2 promoter showed a bivalent modification with active H3K4me3 and repressive H3K27m3 marks in EPC, MSC and CD34+. The Klf4 promoter exhibits high levels of active H3Ac and H3K4m3 but no repressive modification in EPC, MSC and CD34+ cells. These data indicate that adult progenitor cells express variable levels of the pluripotency genes. Modification of the epigenetic histone code, e.g. by histone demethylases, might facilitate the induction of pluripotency by remodeling the accessibility of the promoter regions. This may provide an attractive tool to reprogram adult progenitor cells to generate patient-specific stem cells.
Abstract 5305: Intracoronary Upregulation of Platelet P-selectin and Platelet-monocyte Aggregation Correlate With Coronary Lesion Stenosis Severity Despite Aspirin and Clopidogrel Use
Authors: Yong Andy S , Pennings Gabrielle , Chung Tommy , Brieger David , Ng Martin K , Lowe Harry C
Published: April 2024

Background:
In vitro
studies suggest that platelets are activated by shear stress as they travel through areas of significant coronary stenosis. However, it is unknown whether coronary stenoses cause platelet activation in the presence of dual anti-platelet therapy with aspirin and clopidogrel. We hypothesized that platelet P-selectin, a key regulator of platelet-leukocyte interactions and inflammation, is upregulated as platelets cross coronary plaques despite dual anti-platelet therapy, and this upregulation is directly associated with stenosis severity.

Methods:
Prior to PCI, blood was collected in sequence from the coronary sinus (CS, in left coronary target lesions), coronary artery distal (DA) and proximal (PA) to target lesion, and CS a second time immediately after intracoronary sampling. Quantitative coronary angiography was used to measure target lesion percentage area stenosis. Percentage platelet P-selectin (CD62P) expression, GPIIb/IIIa conformational change (PAC-1), and platelet-monocyte aggregate formation were quantified by flow cytometry.

Results:
In total, 15 consecutive patients pre-treated with dual anti-platelet therapy undergoing elective percutaneous coronary intervention (PCI) to a single target lesion were recruited. Mean age was 67.5y, and 88% were male. Mean percentage lesion area stenosis was 80.8±8.2%. Percentage area stenosis correlated with trans-lesion gradients (DA-PA) for CD62P (r=0.62, p=0.01) and platelet-monocyte aggregation (r=0.76, p<0.01), but not for PAC-1 (r=0.03, p=0.90). CS samples were significantly more activated than PA for CD62P (p=0.03) but CS samples and trans-cardiac gradients (CS-PA) did not correlate with lesion stenosis. There were no significant differences between first and second CS samples, indicating no artificial activation caused by sampling.

Conclusions:
We provide evidence of a direct relationship between coronary stenosis severity and intracoronary platelet P-selectin expression and platelet-monocyte aggregation despite dual anti-platelet therapy. Upregulation of P-selectin by shear stress and/or other mechanical factors under conditions where IIb/IIIa is not, appears to be resistant to current conventional anti-platelet therapy.
Abstract 5311: Proteolysis-resistant Collagen Drives Vascular Smooth Muscle Cell Senescence
Authors: Vafaie Faran , O’Neil Caroline , Watson Alanna , Holdsworth David , Pickering J. Geoffrey
Published: April 2024

Senescence of vascular smooth muscle cells (SMCs) has recently been identified as a feature of atherosclerosis. However, the factors that lead to premature SMC senescence are not well understood. Plaque SMCs reside within a milieu of type I collagen fibrils that, over time, can undergo progressive intermolecular cross-linking rendering the collagen resistant to proteol-ysis. We hypothesized that vascular SMC longevity depends on the extent to which the surrounding type I collagen can be proteolytically edited. To test this, we studied mice with a targeted mutation of the 3/4 −1/4 collagenase cleavage site in type I collagen (Col1a1r/r). Mice harboring the Col1a1r/r mutation appeared normal up to 5 months of age but subsequently developed features of premature aging, including shortened lifespan (p<0.01), impaired weight gain (p<0.01), decreased adipose tissue content (p<0.01), and increased kyphosis (p<0.05), the latter two attributes quantified by micro CT imaging. Furthermore, SMCs in the aortic wall of aged, Col1a1r/r mice showed increased accumulation of reactive oxygen species, as assessed by hydroethidine incubation and nuclear fluorescence. To determine if collagenase-resistant collagen directly impacted SMC lifespan, primary human SMCs were cultured on collagen harvested from the tails of wild-type or Col1a1r/r mice. SMCs on mutant collagen displayed a 67% decrease in cumulative population doublings (p<0.01), which was associated with increased senescence-associated beta-galactosidase (SA-β-gal) activity (p<0.05) and increased p21 expression (p<0.05). Furthermore, stress-induced premature senescence, initiated by serum-deprivation and assessed by SA-β-gal activity and p21 expression, was also amplified in cells cultured on collagenase-resistant collagen.

Conclusions: Collagenase-resistant type I collagen induces a premature aging-like syndrome in mice and accelerates both replicative and stress-induced senescence of vascular SMCs. These findings identify a novel, extracellular driver of vascular cell senescence that may underlie plaque instability in aging arteries.
Abstract 5316: The Regulating Role of MicroRNAs for Smooth Muscle Cell Function
Authors: Bielenberg Wiebke , Prock Andre , Tillmanns Harald , Sedding Daniel
Published: April 2024

Background:

MicroRNAs (miRNAs) comprise key regulators for cellular function. Maturation and expression of miRNAs is tightly controlled by the two processing enzymes Dicer and Drosha. Up to now, the role of miRNAs for vascular smooth muscle cell (VSMC) function and the development of neointimal lesions remains elusive.

Methods and Results:

Following wire-induced injury of the femoral artery of C57BL/6N mice, Dicer and Drosha mRNA expression was significantly downregulated at day 10 and 21. Microarray analysis of miRNA expression at these time points revealed a significant downregulation of miRNAs implicated to regulate key molecules of the cell cycle maschinery. Indeed, siRNA-mediated knock down of Dicer or Drosha in human coronary SMC significantly augmented the growth factor-induced expression of CDK4, CDK6, SKP2, accelerated cell cycle progression as determined by FACS analysis and increased VSMC proliferation as determined by BrdU incorporation as well as total cell count. Interestingly, knock down of Dicer and Drosha also augmented migration, whereas there was no effect on apoptosis. Since apoptotic (H
2
O
2
) or inflammatory stimulation had no effect on dicer and Drosha expression, mitogenic stimulation (FCS) resulted in a robust downregulation of Dicer and Drosha expression. In vivo, the knock down of Dicer and Drosha after wire injury led to increased proliferation of neointimal cells, resulting in a significantly enhanced neointima formation. Further in silico analysis revealed similar and phylogenetically well conserved promoter sets in both promoter regions and the FoxO transcription factor FoxO1a was identified as transcriptional regulator of Dicer and Drosha under mitogenic conditions as determined by ChIP-analysis and siRNA-mediated knock down or overexpression of FoxO1a.

Conclusion:

These data indicate that the key miRNA-processing enzymes Dicer and Drosha are down regulated in neointimal lesions, most likely by mitogenic stimuli via the Foxo transcription factor FoxO1a. Moreover, altered expression of these enzymes and thus of miRNAs seems to regulate VSMC function, especially proliferation and migration, during the development of vascular proliferative diseases.
Abstract 5317: Bone Morphogenetic Protein Recruits a Peroxisome Proliferator-Activated Receptor
γ
and
β
-catenin Transcription Factor Complex to Regulate Apelin Expression in Pulmonary Endothelial Cells
Authors: Alastalo Tero-Pekka , Li Molong , de Jesus Perez Vinicio , Sawada Hirofumi , Rabinovitch Marlene
Published: April 2024

Our previous studies on the pathobiology of pulmonary arterial hypertension (PAH) demonstrated that bone morphogenetic protein receptor II (BMPRII) mediated signaling plays a key role in pulmonary arterial (PA) endothelial cell (EC) survival and proliferation via activation of beta-catenin (
β
C). We have also shown that a
β
C interacting nuclear receptor, peroxisome proliferator-activated receptor
γ
(PPAR
γ
), is a mediator of BMPRII signaling in PA smooth muscle cells (SMCs). A mouse model with targeted disruption of PPAR
γ
in EC shows PAH. The downstream gene targets of BMPRII signaling via PPAR
γ
in EC are, however, unknown. We hypothesized that BMPRII induces a PPAR
γ
-βC transcription factor complex that regulates genes necessary for PAEC homeostasis. We first performed co-immunoprecipitation (IP) and confocal microscopy to demonstrate a relationship between formation of the PPAR
γ
-βC complex and EC survival. We identified target genes for PPAR
γ
-
β
C complex in PAEC by comparing those on whole genome wide ChIP-Chip analyses with those altered on expression profiling microarrays after silencing either BMPRII or βC by siRNA. PPAR
γ
-
β
C complex targets were then analyzed by western immunoblotting and qPCR in PAEC from idiopatic PAH (IPAH) vs. control patients, and in PPAR
γ
deficient vs. wild type mice. By IP and confocal microscopy we demonstrated that PPAR
γ
interacts, and co-localizes in the nucleus, with
β
C in PAEC. This interaction was increased 3 fold upon BMP2 stimulation. Furthermore, ChIP-Chip analysis revealed
β
C co-occupancy in over 70% of PPAR
γ
bound promoters. Microarray analyses confirmed significant changes in the expression of 35 of those genes after silencing of BMPRII and
β
C in PAEC. We then focused on apelin as a ChIP-Chip identified target important in endothelial homeostasis. Loss-of either BMPRII or
β
C down-regulated apelin > 1.5 fold, and apelin mRNA and protein were decreased in PAEC of IPAH vs. control patients (60% and 35% respectively) and in PPAR
γ
deficient vs. WT mice (P < 0.05 for all). We conclude that BMP signaling induces a PPAR
γ
-
β
C interaction that promotes EC homeostasis by regulating critical targets such as apelin. We speculate that down-regulation of apelin may be important in the pathobiology of PAH.
Abstract 5413: G Protein-Coupled Receptor Type 4 Gene Variants and Response to Antihypertensive Medication
Authors: Sanada Hironobu , Yatabe Junichi , Yatabe Midori Sasaki , Yokokawa Hirohide , Williams Scott , Bartlett Jacquelaine , Wang Zheng , Felder Robin , Jose Pedro A
Published: April 2024

Renal dopamine counteracts the prohypertensive and sodium retaining actions of the renin-angiotensin system (RAS). In genetic hypertension, the ability of dopamine to counteract the actions of the RAS is impaired by an increased constitutive activity of genetic variants of G protein-coupled receptor kinase type 4 (GRK4) that desensitize DRD1 dopamine receptors. Selective renal silencing of Grk4 improves DRD1 function and ameliorates the hypertension in spontaneously hypertensive rats. GRK4
γ
wild-type transgenic mice are normotensive and salt-resistant; GRK4
γ
142V transgenic mice are hypertensive while Grk4
γ
486V transgenic mice are salt-sensitive. Renal Grk4 and angiotensin type 1 receptor (AT1R) interact to regulate blood pressure (BP) in rats and GRK4
γ
142V transgenic mice. We tested the association between essential hypertension in 1102 Japanese (616 cases and 486 controls) and single nucleotide polymorphisms (SNPs)/markers in the genes encoding GRK4, ACE, angiotensinogen, AT1R, aldosterone synthase, plasminogen activator inhibitor-1, DRD1, and GNB3. In addition, GRK4 genotype and the response to angiotensin receptor blockers (ARBs) without or with diuretic therapy in 161 Japanese with mild essential hypertension (systolic[SBP]/diastolic [DBP]=148.5±3.3/92.9±2.9 mmHg, age 55±10 yrs) were examined. All three GRK4 SNPs (R65L, A142V, and A486V) associated with hypertension (allelic and genotypic tests, P<4×10
−8
for all three markers). The GRK4 three variant haplotype odds ratio for hypertension was 2.49(P<4×10
−14
). In the responders to ARBs (n=67) BP decreased to 132.8±3.7/84.7±3.1 mmHg and was highly associated with the GRK4142V (P<0.003). The absolute decrease in SBP and DBP in response to ARB was also associated with GRK4142V (P<0.007). The addition of a diuretic (bumetanide) to the non-responders (n=94) decreased BP (127.8±5.0/84.4±2.7, n=47) which was associated with GRK4A486V (P<0.005). The GRK4 variant 142V may be both strongly predisposing to essential hypertension and predictive of the initial response to ARB monotherapy while the GRK4 variant 486V may be predictive of the response to dual therapy with an ARB and a diuretic. Therefore, pharmacogenetics of GRK4 is important in guiding the therapy for hypertension.
Abstract 5416: Exaggerated Mortality Risk in Patients With Severe Left Ventricular Hypertrophy
Authors: Doumas Michael , Papademetriou Vasilios , Kukar Nina , Faselis Charles , Tsioufis Costas , Kokkinos Peter
Published: April 2024

Introduction:
Mild to moderate left ventricular hypertrophy (LVH) has been identified as an independent predictor of increased mortality, but information in patients with severe or very severe LVH is scarce. Thus, we assessed the mortality risk associated with the severity of LVH in a large cohort of male veterans.

Methods:
From January of 1993 to March 2009, we identified 11,578 patients (mean age: 68±14) with a technically interpretable baseline echocardiogram at the Veterans Affairs Medical Center, Washington, DC. We calculated left ventricular mass index (LVMI) and classified patients as follows: No-LVH (LVMI <125 g/m
2
; n=6,835); Mild-Moderate-LVH (LVMI 125–160 g/m
2
; n=2,673); Severe-LVH (LVMI 161–200 g/m
2
; n=1,359); and Very-Severe-LVH (LVMI >200 g/m
2
; n=711). We then assessed mortality rate for a mean follow-up period of 5.8±4.5 years.

Results
: Echocardiographic parameters are presented in table

. During the follow-up period we observed a total of 3,950 deaths for an annual mortality rate of 5.85% in the entire cohort. The annual mortality rate was 4.87%; 6.53%; 8%; and 11% for the No-LVH; Mild-Moderate-LVH; Severe-LVH; and Very-Severe-LVH groups respectively. Cox proportional hazard analysis, adjusted for age, heart failure, and diabetes mellitus, revealed a 10% increase in mortality risk for every 20 g/m
2
increase in LVMI. When compared to the No-LVH group, the mortality risk was 29% higher for the Mild-Moderate-LVH (CI: 1.2–1.4); 57% higher for the Severe-LVH (CI: 1.4–1.7) and 115% higher for the Very-Severe-LVH (CI: 1.9–2.4).

Conclusions:

LVMI is directly associated with increased mortality;

Severe and very severe LVH are associated with exaggerated mortality risk and

Approaches to prevent or reverse LVH are urgently needed.
Abstract 5417: Peripheral Blood Monocytes Are Activated in Patients With Well Controlled Hypertension: Implications for the Understanding of the Residual Cardiovascular Risk
Authors: Czesnikiewicz-Guzik Marta , Bzowska Malgorzata , Jasiewicz-Honkisz Barbara , Korbut Richard , Naruszewicz Marek , Pryjma Juliusz , Guzik Tomasz J
Published: April 2024

Hypertension is associated with significant risk of cardiovascular events which remains increased even in the setting of well controlled hypertension. This residual risk may be related to inflammation persisting in spite of sufficient blood pressure control. We have recently reported that monocytes show pro-inflammatory and pro-atherosclerotic phenotype (co-expression of the Fc-gamma receptor-CD16+ and LPS receptor-CD14+) in relation to atherosclerosis risk factors and that hypertension is the strongest independent predictor of CD16+CD14+ monocytes. It is thus possible that monocytes remain activated in spite of appropriate blood pressure control and thus may be in part responsible for residual risk. We studied 112 hypertensive subjects and 25 control non-hypertensive individuals matched for major risk factors. Hypertension was well controlled (<140/90) in 52 and poorly controlled in 60 pts. Cytokine production was assessed by ELISA.

Results: The prevalence of CD14+CD16+ monocytes was significantly positively correlated with systolic and diastolic blood pressure (R=0.3; p<0.01). Both subpopulations CD14(high)CD16+ (R=0.4; p<0.01) and to the lesser extent CD14(dim)CD16+ (R=0.2; p<0.05) showed relationships to systolic but only the CD14(high)CD16+ to diastolic blood pressure. The percentage of total CD14+CD16+ and CD14(high)CD16+ monocytes were highest in subjects whose blood pressure was poorly controlled. A significant difference was also observed between normotensives and hypertensive subjects with well controlled blood pressure. Further analysis of monocyte subpopulations showed that levels of pro-atherosclerotic CD14(high)CD16+ monocytes, revealed a strong relationship to hypertension and greatest difference between well controlled hypertension and normotensives (6±0.2 vs 9±0.4 p<0.01). These monocytes showed significantly higher expression of activation markers HLA-DR and CD45RA and TNF-a production in patients with hypertension, even in the absence of high blood pressure at the time of the study. These results provide a novel marker of inflammation in hypertension which remains increased even in the setting of well controlled disease providing an inflammatory mechanism for the residual risk.
Abstract 5418: Structural Alterations in Subcutaneous Small Resistance Arteries May Predict Changes of the Renal Function in Hypertensive Patients
Authors: Boari Gianluca E , Rizzoni Damiano , Porteri Enzo , De Ciuceis Carolina , Mazza Monica , Brignani Alida , Rosei Claudia Agabiti , Caimi Luigi , Rosei Enrico Agabiti
Published: April 2024

We have previously demonstrated that structural alteration of subcutaneous small resistance arteries of hypertensive patients, as indicated by an increased media to lumen (M/L) ratio, has a strong prognostic significance. Among the predictors of M/L indicators of renal function are those more closely correlated. Aim of the present study was to assess whether M/L ratio can predict subsequent changes in renal function in hypertensive patients. Sixty patients (33 males; 27 females; 15 normotensives and 45 hypertensives) underwent a biopsy of subcutaneous fat at a mean age of 56 years. Resistance-sized arteries were dissected and mounted on a wire myograph according to Mulvany-Halpern technique, and M/L was measured. Patients were re-evaluated after a mean time period of 8.5 years; serum creatinine, blood urinary nitrogen (BUN) and uric acid were measured, and glomerular filtration rate (GFR) was calculated according to MDRD formula. At baseline, we observed significant correlations between M/L and serum creatinine (r=0.32; p=0.013), GFR (r= −0.26; p=0.047), BUN (r=0.37; p=0.009), systolic (r=0.45, p<0.001), diastolic (r=0.29, p=0.02), mean blood pressure (r=0.38, p=0.002) and pulse pressure (r=0.40, p=0.002). In addition, we observed significant correlations between M/L and creatinine at follow up (r=0.57; p<0.001), % changes in serum creatinine (r=0.46; p<0.001), GFR at follow up (r= −0.43; p<0.001); % changes in GFR (as ml/min r= −0.33; p=0.008), as well as yearly changes (r= −0.34, p=0.007), BUN at follow up, (r=0.55; p<0.001), as well as uric acid at follow up (r=0.35; p=0.026). A multivariate analysis in which all common cardiovascular risk factors were included, showed that M/L is the most potent predictor of changes in renal function. In conclusion, our data suggest that structural alterations of subcutaneous small resistance arteries (which may be present also in other vascular beds), may predict the time-course of changes of renal function during a follow-up period of about 9 years.
Abstract 5449: Diagnostic Performance of Perfusion Cardiovascular Magnetic Resonance Compared With Gated Myocardial Perfusion SPECT in Patients With Known or Suspected Coronary Artery Disease
Authors: Bucciarelli-Ducci Chiara , Lyne Jonathan , Reyes Eliana , Assomull Ravi , Petryka Joanna , Watkins Stuart , Gatehouse Peter , Roughton Micheal , Symmonds Karen , Asimacopoulos Eleni , Prasad Sanjay , Underwood S Richard , Pennell Dudley
Published: April 2024

Background:
Perfusion cardiac magnetic resonance (CMR) is emerging a clinically valuable imaging technique in patients with known or suspected coronary artery disease (CAD) and it has some potential advantages over myocardial perfusion scintigraphy.

Aim
: To evaluate the diagnostic performance of adenosine perfusion CMR vs gated single-photon emission computed tomography (SPECT) compared with the gold-standard of quantitative coronary angiography (QCA) in patients with known or suspected CAD.

Methods
: Ninety-five patients scheduled for coronary angiography underwent both adenosine perfusion CMR and adenosine technetium-99m-tetrofosmin SPECT. Stress CMR perfusion imaging was performed with a hybrid-EPI sequence after 4 minutes of 140
μ
g/kg/min adenosine and 0.1mmol/kg of gadolinium, and followed by late enhancement imaging. Tc-99m SPECT was performed with a 1day stress-rest protocol. CAD was defined as diameter stenosis ≥50% on QCA. Two blinded observers per modality analyzed the images both qualitatively (presence/absence of inducible ischemia) and quantitatively: myocardial perfusion reserve index (MPRI) by CMR and sum difference score (SDS) by SPECT. A MPRI ≤2 by CMR was considered abnormal. The comparison of CMR vs SPECT was based on receiver operating characteristic (ROC) analysis.

Results
: Analysis of all patients showed that perfusion CMR had similar diagnostic performance to SPECT: area under ROC curve (AUC): 0.74±0.050 vs 0.683±0.052 for SPECT, p=0.29 based on qualitative assessment and 0.675±0.054 vs 0.583±0.024 for SPECT, p=0.10 based on MPRI and SDS. Similar results were observed in the subgroup of patient
with
previous myocardial infarction. (n=34, 38%-defined as the presence of late myocardial enhancement by CMR). However, in the subgroup of patients
without
previous myocardial infarction, CMR MPRI had significantly better diagnostic performance than SPECT SDS: AUC 0.695±0.063 vs 0.552±0.029, respectively, p=0.027.

Conclusions
: In patients with known or suspected CAD, perfusion CMR has a similar diagnostic performance to Tc-99m gated SPECT and has particular advantage in patients without previous infarction. CMR represents a valuable alternative imaging modality to assess CAD.
Abstract 5474: Crucial Role of Nrf3 in Smooth Muscle Cell Differentiation From Stem Cells and Vessel Injury-Induced Neointima Formation
Authors: Pepe Anna E , Xiao Qingzhong , Zampetaki Anna , Hu Yanhua , Xu Qingbo
Published: April 2024

Nuclear factor erythroid 2-related factor 3 (Nrf3), a member of the cap’n’collar family of transcription factors that binds to the DNA-antioxidant responsive elements, have been reported to be involved in muscle precursor migration during early embryo development. The present study aimed to investigate the functional role of Nrf3 in stem cell differentiation into smooth muscle cells (SMCs). When embryonic stem cells were cultivated on collagen IV-coated plates, Nrf3 was up-regulated significantly following 1–6 days of cell differentiation. Knockdown of Nrf3 resulted in down-regulation of smooth muscle specific markers including smooth muscle actin, SM22, calponin and smooth muscle myosin heavy chain, while enforced expression of Nrf3 enhanced SMC differentiation in a dose-dependent manner. SMC-specific transcription factor myocardin, but not serum response factor, was significant upregulated by Nrf3 overexpression. Strikingly, the binding of serum response factor and myocardin to the promoter of smooth muscle differentiation genes was dramatic increased by Nrf3 overexpres-sion, and Nrf3 can direct bind to the smooth muscle gene promoters as demonstrated by chromatin immunoprecipitation (CHIP) assay. Moreover, NADPH-derived ROS production during SMC differentiation was further enhanced by Nrf3 overexpression through upregualtion of NADPH oxidase (NOX4) and inhibition of anti-oxidant signalling pathway. Furthermore, overexpression of Nrf3 resulted in increase of plasma phospholipase A2 (Pla2g7) production, while knockdown Nrf3 ablated the Pla2g7 gene expression and protein activity. Additionally, both Nrf3 and Pla2g7 were up-regulated in adult SMC phenotype swiching from proliferating to contractile phenotype upon serum starvation. Importantly, in vivo knockdown of Nrf3 in the vessel wall significant increased wire-injury-induced neointima formation in ApoE-deficient mice. Our findings demonstrated for the first time that Nrf3 has a crucial role in SMC differentiation from stem cells and important protective function in vessel injury-induced neointima formation, indicating that they could be potential new therapeutic targets for intervention of SMC proliferative-related vascular diseases.
Abstract 5483: The Inflammatory Protein Complement C3 is a Novel Substrate for Factor XIII and Impairs Fibrin Clot Lysis
Authors: Carter Angela M , Schroeder Verena , Polkinghorne Victoria R , Smith Kerrie A , Somani Riyaz , Standeven Kristina F , Grant Peter J
Published: April 2024

Introduction: Inflammation is fundamentally important in the pathogenesis of cardiovascular disease (CVD) and increasing evidence supports a functional role for complement C3. Using a functional proteomics approach we have identified zymogen and activated C3 in perfused solubilized clots, suggesting C3 influences thrombus formation.

Hypothesis: In this study we assessed the hypothesis that complement C3 interacts with coagulation and fibrinolytic factors to influence thrombosis.

Methods and Results: Analysis of 123 paired plasma and serum samples revealed that 24.8% (SD 7.6) of plasma C3 was incorporated into the clot. To explore the mechanism of incorporation we investigated binding interactions between C3, fibrin and FXIII using ELISA based binding assays and surface plasmon resonance and found that C3 bound to immobilized fibrinogen and FXIII in a concentration dependent manner. Further analysis of the interactions between C3 and FXIII demonstrated that activated FXIII (FXIIIa) incorporated a known FXIIIa substrate, 5-(biotinamido)pentylamine, into immobilized C3 in a concentration dependent manner and FXIIIa also induced the formation of high-molecular weight C3 polymers, indicating that C3 is a FXIII substrate. Furthermore, FXIIIa incorporated plasma C3 into immobilized fibrin and Western blot analysis of clots formed from purified fibrinogen and C3 indicated high molecular weight bands that cross-reacted with specific antibodies against C3 and fibrinogen, suggesting that C3 is cross-linked to fibrin alpha chains. In turbidimetric assays using purified proteins C3 prolonged fibrin clot lysis times in a concentration dependent manner and Western blot analysis of lysed clots indicated that C3 was cleaved by plasmin, suggesting impaired fibrinolysis is due to substrate competition for plasmin. Increased plasma C3 concentration was significantly associated with increased ex vivo plasma clot density and prolonged fibrinolysis times, after adjusting for fibrinogen concentrations, to support our in vitro results. In conclusion, taken together these data indicate that complement C3 influences key processes involved in fibrin formation and fibrinolysis supporting a role for C3 in cardiovascular thrombosis.
Abstract 5487: Randomized Comparison of Prehospital Initiated Facilitated PCI versus Primary PCI in Acute Myocardial Infarction With < 3 h After Symptom Onset
Authors: Thiele Holger , Eitel Ingo , Meinberg Claudia , Plock Gerd , Pfeiffer Dietrich , Hartmann Andreas , Schuler Gerhard
Published: April 2024

Background:

Facilitated PCI with fibrinolysis did not show a benefit in comparison to primary PCI in recently published trials. However, a subgroup of patients with high-risk STEMI presenting early after symptom onset, treated with optimal antiplatelet co-medication, and with long transfer times might benefit from a fibrinolytic-based facilitated PCI. Aim of this multicenter trial was therefore to assess the merits of facilitated PCI versus primary PCI in a STEMI network with long transfer distances.

Methods:

Patients with STEMI (< 3 h after symptom onset) were randomized to either prehospital initiated facilitated PCI using tenecteplase (group A; n=80) or primary PCI (group B; n=80). Optimal prehospital co-medication consisted of 600 mg clopidogrel loading-dose plus aspirin. Glycoprotein IIb/IIIa-inhibitors were given liberally during PCI. The primary endpoint was infarct size assessed by MRI. Secondary endpoints were microvascular obstruction and myocardial salvage assessed by MRI, ST-segment resolution at 90 min, and a composite of death, re-MI, and congestive heart failure at 30 day follow-up. Safety was assessed by major bleeding and any stroke.

Results:

The median time from symptom-onset to randomization was 64 min (IQR 42; 103) in group A versus 55 min in group B (IQR 27; 91; p=0.26). The median symptom-onset to balloon time was 157 min (IQR 121; 224) versus 131 min (IQR 108; 177; p=0.03). Despite better preinterventional TIMI-flow in group A (76% versus 28% TIMI 2/3; p<0.001) the infarct size was similar in group A versus group B (14.1% of left ventricle [IQR 5.3; 26.7] versus 15.1% [IQR 7.5; 23.3]; p=0.75). There was also no difference in microvascular obstruction, myocardial salvage (p=0.65 and 0.71) and ST-segment resolution (p=0.42). For the combined clinical endpoint there was trend towards higher event rates in group A (18.9% versus 8.1%; p=0.09, relative risk 2.33, 95% confidence interval, 0.98–5.63) without a difference in major bleeding and stroke (8.2% versus 5.5%; p=0.74)

Conclusion:

In patients with STEMI presenting early after symptom onset a fibrinolytic-based facilitated PCI approach with optimal antiplatelet comedication does not offer a benefit over primary PCI with respect to infarct size and tissue perfusion.
Abstract 5489: Provisional (bail-out) Use versus Routine Use of Glycoprotein 2b/3a Blockers in Stemi: A Sub-analysis From the On-time 2 Trial
Authors: Hermanides Rik , Heestermans Ton , ten Berg Jurrien , Gosselink Marcel , Ottervanger Jan Paul , van Houwelingen Gert , Kolkman Evelien , Stella Pieter , Dill Thorsten , Hamm Christian , van ‘t Hof Arnoud W
Published: April 2024

Background:
In daily practice, in patients (pts) with acute ST-elevation myocardial infarction, glycoprotein 2b/3a blockers use is often limited to the provisional use in case of complications after PCI. However, it is unknown whether this strategy is of any value as compared to the routine use of these agents before PCI.

Methods:
The On-TIME 2 trial compared routine pre-hospital initiation of high dose Tirofiban (HDT) versus provisional use of HDT in a randomized placebo controlled design. Thrombotic bail-out use of study medication was pre-defined and part of the combined clinical end-point. Within the pts who received blinded bail-out treatment, we compared pts who were pre-treated with placebo and received bail-out HDT (Tirofiban bail-out) versus those who were pre-treated with HDT and received bail-out placebo (Placebo bail-out).

Results:
Blinded bail-out use of study medication was used in 24% (237/980) of pts, with a significant higher use in pts pre-treated with placebo (29% (140/492) versus 20% (97/488), p=0.002). The indication for bail-out use of study medication was mostly distal embolisation or no-reflow after PCI and did not differ between the groups (73.6% vs 75.3% p=NS). As expected, bail-out use of study medication was associated with more residual ST deviation (5.5±7.2 vs 3.7±4.8mm), larger infarct size (median (IQR): 1750 (674–3117) vs 948 (379–2278) IU), and worse outcome (MACE 12.2% vs 5.6%, p<0.01 for all comparisons) as compared to no-bail-out treatment, table

.

Conclusion:
Routine pre-hospital treatment with HDT significantly reduced the use of blinded bail-out study medication. Bail-out study medication was associated with larger infarct size and poor outcome, especially in those pts pre-treated with placebo. Therefore, routine pre-treatment with HDT is preferred over provisional use of HDT in pts with STEMI. It is highly questioned whether provisional use of glycoprotein
2b3a
blockers in case of complications after PCI is effective at all.

Table
Abstract 5502: Ultrasound-Controlled Therapeutic Gas Delivery Provides Enhanced Neuroprotection Following Hypoxic-Ischemia
Authors: Kim Hyunggun , Britton George L , Holland Christy K , McPherson David D , Huang Shao-Ling
Published: April 2024

Background: Neurologic damage following ischemic insult is a major problem for stroke survivors. Xenon (Xe) exerts favorable neuroprotective properties with few side effects. This study describes a unique application of ultrasound for controlled release of Xe from liposomes (Xe-ELIP) for targeted therapeutic gas delivery and neuroprotection.

Method: Xe-ELIP were created by a pressurization-freezing method. One-MHz continuous ultrasound wave with pressure amplitudes of 0.16, 0.22 and 0.33 MPa was used to trigger Xe release from Xe-ELIP, and endothelial cell permeability and cell viability were measured. Male Sprague-Dawley rats underwent right middle cerebral artery occlusion for 2 hours (n=28). In the treatment group (n=8), Xe-ELIP were administered through the right common carotid artery. Continuous ultrasound (0.22 MPa) was applied over the common carotid artery during the Xe-ELIP administration for 4 minutes. Infarct size as well as behavioral outcomes were determined 3 days after treatments.

Results: Ultrasound application in the presence of ELIP resulted in a temporary (5 minutes) enhancement of cell permeability. The amount of released Xe depended on the ultrasound pressure amplitude. Xe-ELIP in combination with ultrasound exposure (0.22 MPa) enhanced neuroprotection by decreasing infarct volume by 88% vs the non-ultrasound Xe-ELIP group (64%). Behavioral tests mirrored neurological results.

Conclusions: One MHz continuous ultrasound can be used to release therapeutic gas from encapsulated liposomes. Administration of Xe-ELIP with ultrasound exposure over the carotid artery after a cerebral ischemic insult provides enhanced neuroprotection.

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 5510: Inflammation is Related to Coronary Flow Reserve Detected by Positron Emission Tomography in Healthy Twins
Authors: Vaccarino Viola , Khan Durreshahwar , Votaw John , Faber Tracy , Veledar Emir , Murrah Nancy V , Jones Linda R , Goldberg Jack , Raggi Paolo , Quyyumi Arshed A , Bremner J D
Published: April 2024

Introduction.
Coronary microvascular dysfunction is an early precursor of CAD and is thought to result from endothelial cell activation and inflammation. Coronary flow reserve (CFR) is an index of microvascular dysfunction, but limited and conflicting data exist on the relationship between CFR and inflammation.

Methods.
We examined 292 healthy male twins selected from the Vietnam Era Twin Registry. Plasma biomarkers of inflammation and endothelial cell activation were assessed and included C-reactive protein (CRP), interleukin-6 (IL-6), total white blood cell count (WBC), sVCAM-1 and sICAM-1. We performed myocardial perfusion imaging and blood flow quantitation with [13N] ammonia positron emission tomography at rest and after adenosine stress. CFR was measured as the ratio of maximum flow to baseline flow at rest averaged across 20 myocardial regions; abnormal CFR was defined as <2.5. A summed stress score for visible perfusion defects was also calculated. Traditional CAD risk factors were also measured. Mixed-effect and GEE models were used to conduct matched-pair analyses.

Results.
The twins had a mean age of 54 and 57% were monozygotic. In the overall sample, all biomarkers except sVCAM-1 were significantly correlated with CFR. In contrast, only IL-6 was correlated with the summed stress score of visible defects. Of 146 twin pairs, 138 were discordant for CFR (using no cutpoint) and 56 pairs were discordant for CFR <2.5. Values of all biomarkers were higher in twins with lower CFR than their brothers with higher CFR (Table

). The associations persisted after adjusting for summed stress score and CAD risk factors; after stratifying by zygosity results were similar.

Conclusions.
Systemic inflammation and endothelial cell activation are associated with coronary microvascular dysfunction, independent of CAD risk factors. Our results using a highly controlled twin design highlight the importance of inflammatory processes in the early phases of CAD.

Percent Higher Biomarker Level in Twin with Lower CFR Compared to Brother with Higher CFR

This research has received full or partial funding support from the American Heart Association, National Center.