bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1035: Functional Polymorphism of the Matrix Metalloproteinase 1 Gene Promoter (-1607 1G/2G) Predicts Carotid Intra-plaque Hemorrhage in Patients Submitted to Endarterectomy
Authors: Santos Katia G. , Narvaes Luciane , Silvello Daiane , Albuquerque Luciano , Rohde Luis
Published: July 2021

Introduction:
Carotid intraplaque hemorrhage is a marker of atheroma instability. Matrix metalloproteinase (MMP)-1 and -3 are proteolytic enzymes that degrade extracellular matrix components, leading to structural instability of vulnerable plaques.

Hypothesis:
We hypothesize that funcional polymorphisms of the MMP-1 and MMP-3 genes could identify patients prone to atheroma bleeding in the carotid artery.

Methods
: Consecutive patients submitted to carotid endarterectomy underwent high-resolution magnetic resonance imaging (MRI) - to evaluate the presence of a hyperintense signal on the T1-weighted sequence - and histological analysis - to assess the presence of intraplaque bleeding (stained by hematoxylineosin and Mallory trichromic). Genotyping of MMP-1 polymorphisms at nucleotide -1607 (1G/2G) and of MMP-3 at nucleotide -1612 (5A/6A) was performed by polymerase chain reaction with restriction fragment length polymorphism analysis.

Results:
We evaluated 48 predominantly male (63%) and hypertensive (88%) patients (66 ± 9 years-old). Intraplaque hemorrhage was identified in 30 (62.5%) patients by histology and classified as acute in 17 (35.4%), recent in 11 (22.9%) and late in 2 (4.2%) patients. There was an excellent agreement between acute or recent hemorrhage on histology and the presence of a hyperintense signal at MRI (kappa coefficient of 0.91, 95% confidence interval of 0.75–1.00). MMP-1 genotypes were 1G/1G in 7 (15%) subjects, 1G/2G in 30 (62%) and 2G/2G in 11 (23%) patients. MMP-3 genotypes were 5A/5A in 6 (12%) subjects, 5A/6A in 30 (63%) and 6A/6A in 12 (25%) patients. No significant association between MRI findings and MMPs genotypes was observed. However, carriers of the 2G allele, responsible for a greater proteolyic activity of MMP-1, were more prevalent in plaques with signs of hemorrhage on hystology compared to patients without intraplaque bleeding (28 out 30 [93%] versus 13 out of 18 [72%], respectively, p = 0.045). No interaction was observed between MMP-3 genotypes and carotid intraplaque hemorrhage (all p values > 0.2).

Conclusions -
In conclusion, our data indicate that genetically mediated variability in MMP-1 activity may be involved in the events that lead to intraplaque hemorrhage.
Abstract 1060: Regulation of Protease Activated Receptor-1 signaling by G-protein coupled receptor kinase 3
Authors: Casey Liam M , Greenman Jason , Aguilar Frederick , Dunaevsky Olga , Blaxall Burns C
Published: July 2021

G protein-coupled receptors (GPCRs) play crucial roles in normal heart function and dysregulated GPCR signaling contributes to heart failure (HF). Protease-activated receptors (PARs) are one class of GPCR expressed in the heart. Emerging evidence demonstrates that excessive PAR-1 signaling induces cardiac dysfunction. An important component of PAR signaling is ERK1/2, which is phosphorylated in response to PAR stimulation and can influence myocyte hypertrophy and survival. Cytoplasmic pERK1/2 accumulation depends in part on scaffolding complexes that assemble with internalized GPCRs. Activation of an internalization-defective PAR mutant leads to enhanced nuclear pERK1/2 accumulation upon stimulation. The nuclear/cytoplasmic distribution of pERK1/2 may be a key factor in determining the cellular effects of PAR stimulation as elevated nuclear pERK in cardiomyocytes is suggested to promote survival and physiological hypertrophy. Phosphorylation of PARs by G-protein coupled receptor kinase 3 (GRK3) is thought to promote receptor internalization, potentially influencing overall pERK1/2 accumulation and subcellular distribution. We have used a dominant negative form of GRK3 lacking the kinase domain (GRK3ct) to test the hypothesis that GRK3 influences PAR1 internalization and ERK1/2 phosphorylation. By measuring cell surface receptor levels we demonstrate that GRK3ct interferes with PAR1 internalization. Immunofluorescent staining and cellular fractionation techniques further show that GRK3ct enhances nuclear accumulation of pERK1/2 in COS-7 cells and adult mouse cardiomyocytes. Furthermore we find that GRK3ct overexpression in neonatal rat cardiomyocytes increases PAR1-activation induced physiologic hypertrophy. In summary these results may explain recent unpublished reports that mice overexpressing GRK3ct in the heart are protected against ischemic injury, a heart failure model that involves pathologic PAR signaling. Thus we conclude that following ischemic injury, reducing PAR1 internalization via interfering with endogenous GRK3 activity or promoting nuclear pERK accumulation might improve cardiac recovery.
Abstract 1065: Class-II Histone Deacetylases Regulate Myofilament Calcium Sensitivity in the Mouse Heart
Authors: Smith Stephen H , Gupta Mahesh P , Samant Sadhana , Gupta Madhu , Shroff Sanjeev G
Published: July 2021

The role of phosphorylation of myofilaments in regulating cardiac muscle calcium sensitivity and force production is well established. Recently it was shown that trichostatin A (TSA), a class-I and II histone deacetylase (HDAC) inhibitor, improved cardiac contractile function
in vivo
. Because class-II HDACs are able to translocate to the cytoplasm from the nucleus, it is postulated that some HDACs may interact with contractile proteins and thereby regulate myofilament calcium sensitivity and/or force production. Papillary muscle strips from mouse hearts were skinned for 4 hours and then treated overnight with two structurally dissimilar HDAC inhibitors, TSA and MS275. Both HDAC inhibitors significantly increased myofilament calcium sensitivity as quantified by pCa
50
[Baseline (n=9): 5.75±0.04; TSA (n=6): 5.84±0.02; MS275 (n=7): 5.87±0.03;
P
<0.05
vs
. Baseline], without any significant changes in maximally activated force. Similar increase in calcium sensitivity was also observed when skinned fibers were incubated overnight in a buffer containing acetyl-CoA [acetyl-CoA (n=6): 5.84±0.04;
P
<0.05
vs
. Baseline]. These observations suggest that acetylation of myofilament proteins can regulate myofilament calcium sensitivity. Western blot analyses of skinned fibers revealed that HDAC4 and HDAC5 were specifically associated with myofilament proteins, and acetylation of several myofilament proteins was increased following TSA treatment. One of these acetylated proteins was identified as muscle LIM protein (MLP). Protein-protein interaction analyses and co-localization studies showed that MLP specifically binds to HDAC4. Functional studies with skinned fibers isolated from MLP knock out [MLP
−/−
] mice (C57BL6 strain) indicated that TSA-induced increase in calcium sensitivity was completely prevented [MLP
−/−
-Baseline (n=6): 5.60±0.03; MLP
−/−
-TSA (n=6): 5.61±0.02;
P
<NS] compared to wild-type (WT) mice of the same strain [WT-Baseline (n=6): 5.59±0.02; WT-TSA (n=6): 5.70±0.02;
P
<0.05]. These data demonstrate, for the first time, that class-II HDACs bind to cardiac myofilament proteins, with MLP being one of the binding partners, and play a role in regulating myofilament contractile function.
Abstract 1067: CamKII Regulates NFAT Nuclear Translocation in a Calcineurin Independent Manner
Authors: MacDonnell Scott M , Weisser-Thomas Jutta , Kubo Hajime , Chen Xiongwen , Berretta Remus , Jaleel Naser , Brown Joan H , Molkentin Jeffery D , Houser Steven R
Published: July 2021

Pathologic cardiac myocyte hypertrophy involves stress induced increases in intracellular Ca
2+
, which binds to calmodulin (CaM) and then activates both the phosphatase calcineurin (Cn) and CaM kinase (CaMKII). In hypertrophy, Cn dephosphorylates the cytoplasmic nuclear factor of activated T-cells (NFAT), inducing nuclear translocation where it activates hypertrophic target genes. While GSK3, p38, and JNK are kinases that have been shown to directly phosphorylate NFAT, resulting in nuclear export, the role of cytoplasmic CamKII in NFAT translocation is unknown. This study explored the hypothesis that cytoplasmic CamKII directly induces NFAT cytoplasmic localization which antagonizes Cn mediated nuclear NFAT translocation.

Methods:
Adenovirus gene delivery was used to express GFP-tagged NFATc3 (m.o.i - 100, 12h) in either cultured neonatal rat (NRVM) or in adult feline ventricular myocytes (AFVM). Myocytes were co-infected with either constitutively active (CamKII-CA) or dominant negative (CamKII-DN) mutants of cytoplasmic targeted CamKII
δc
(m.o.i - 100, 12 h). FK506 (2uM) or CsA (2uM) were used to inhibit Cn. The % of myocytes with nuclear translocated NFAT is reported.

Results:
In NRVM (and in AFVM), NFAT nuclear translocation (Control: 14.9±1%) was significantly increased (29±2%, p<0.05 vs control) by raising the bath [Ca
2+
] by 2X. This Ca
2+
mediated NFAT translocation was blocked by FK506 (3.8±1%) and CsA (7.3±2%, p<0.05 vs control). CamKII-DN (CaMKII inhibition) increased (44.9±3%, P<0.001 vs control) and CamKII-CA (CaMKII activation) decreased (6.8±2%, P<0.01 vs control) NFAT translocation. FK506 and CsA did not block NFAT translocation in CamKII-DN NRVM (FK506: 50.7±2, CsA: 46.1±2%). CamKII-DN also increased NFAT translocation (50.5±3% vs 24.8±2% in control, P<0.01) in AFVM. CsA treatment also failed to reduce CamKII-DN mediated NFAT translocation (59.6±9, P<0.01 vs control) in adult myocytes.

Conclusion:
These data show for the first time that activation of cytoplasmic CaMKII inhibits NFAT nuclear translocation in both neonatal and adult ventricular myocytes, possibly by direct NFAT phosphorylation. These effects are in parallel with but independent of Ca
2+
dependent Cn signaling.
Abstract 1082: Cytoskeletal Protein 4.1R Affects Repolarisation And Regulates Calcium Handling In The Heart
Authors: Terracciano Cesare M , Sohrabi Nadia , Siedlecka Urszula , Stagg Mark A , Soppa Gopal K , Lee Joon , Barton Paul J , Bennett Pauline , Pinder Jennifer C , Yacoub Magdi H
Published: July 2021

The 4.1 proteins are a family of multifunctional adaptor proteins that organise signalling/transport/cell adhesion molecules. They are capable of interaction with the spectrin-actin network thereby conferring mechanical stability to the cell membrane, with several ion transporters associated to this macromolecular complex. Protein 4.1R is expressed in the heart and upregulated in deteriorating human heart failure. However, no data exists on the roles of protein 4.1R in myocardial regulation and function. In particular, it is unknown whether this protein can influence cardiac contractility and/or electrophysiology. 4.1R-deficient mice (KO) were studied using echocardiography and ECG monitoring with radiotelemetry. Left ventricular dimensions were increased in KO mice (LV diameter - Dia (cm): WT = 0.43 ± 0.01 [6] (mean ± SEM [n]); KO = 0.49 ± 0.01 [6]; p < 0.01) - Sys cm): WT = 0.29 ± 0.01 [6]; KO = 0.34 ± 0.02 [6]; p < 0.05) with no changes in ejection fraction and fractional shortening. ECG analysis revealed reduced heart rate (RR interval (ms): WT = 113±5 [6]; KO = 139 ± 601 [6]; p < 0.01) accompanied by prolonged QT interval (corrected (ms): WT = 46 ± 2 [6]; KO = 52 ± 1 [6]; p < 0.05). The action potential duration (APD) measured in isolated ventricular myocytes was prolonged in KO (APD 90% at 1Hz (ms) WT = 146 ± 20 [21]; KO = 231 ± 29 [28]; p < 0.05). Ca transients, elicited by 1Hz field-stimulation and measured using the fluorescent indicator indo-1, were larger (amplitude (ratio units r.u.): WT = 0.07 ± 0.006 [22]; KO = 0.1 ± 0.006 [33]; p<0.05) and slower to decay in the KO group (time to 50% decline (ms): WT = 78 ± 3 [22]; KO = 91 ± 3 [33]; p < 0.05). This was associated with increased SR Ca content, (20 mM caffeine-induced indo-1 transient amplitude (r.u.): WT = 0.09 ± 0.01 [7]; KO = 0.13 ± 0.01 [16]; p < 0.05) and increased frequency of Ca sparks, measured by confocal microscopy using Fluo-4 (sparks/100μm/s. WT = 0.52 ± 0.08 [111]; KO = 1.21 ± 0.14 [124]; p < 0.001). We conclude that protein 4.1 R affects repolarisation of cardiac myocytes. This may have a role in bringing about QT prolongation in KO mice. The prolonged APD, together with effects on Ca handling proteins, may alter Ca regulation and cell contractility. The specific mechanisms controlling these effects are under investigation.
Abstract 1097: Very-low Dose Endotoxemia Induces High Density Lipoprotein Remodeling and Reduces Cholesterol Efflux in the Absence of a Clinical Inflammatory Response
Authors: Heffron Sean P , Mehta Nehal N , de la Llera-Moya Margarita , Terembula Karen , Hinkle Christine , Wolfe Megan , Reilly Muredach P
Published: July 2021

Introduction:
Chronic low-grade inflammation is thought to induce atherogenic changes in HDL function. Indeed, we have demonstrated HDL particle remodeling during experimental human endotoxemia (3ng/kg). However, this model is associated with marked clinical symptoms, and may not reflect the chronic low-grade inflammation characteristic of insulin resistant atherogenic states.

Hypothesis:
We hypothesized that very low-dose human endotoxemia (LPS; 0.6ng/kg) induces sub-clinical inflammation causing changes in HDL composition and function, without generating measurable clinical responses.

Methods:
Ten healthy, human volunteers (50% male, 90% Caucasian, mean age 22.7 ± 3.8) were randomized to separate 36-hour inpatient visits (placebo versus intravenous LPS 0.6ng/kg) in a double-masked, placebo-controlled, crossover study. Serial plasma and serum samples were collected for measurement of cytokines, acute phase reactants, lipids and lipoproteins.
Ex-vivo
3
H-cholesterol efflux from FU5AH cells (SR-BI model) to the HDL fraction from serum, serially isolated during placebo/endotoxemia, was examined (N=5). Repeated measures ANOVA was applied to the data.

Results:
There was no significant difference between placebo and LPS in the clinical measures of inflammatory response, including body temperature and heart rate. Relative to placebo, LPS produced a peak 20-fold increase in TNFα (p = 0.01) and a 15-fold increase in CRP (p <0.001). LPS had no effect on levels of HDL cholesterol, apoA-I, apoA-II or apoB lipoproteins. However, endotoxemia was associated with a significant decrease in plasma phospholipids (p = 0.03) and a 7-fold increase in HDL-associated SAA (p <0.001). Further, LPS induced a 40% reduction in cholesterol efflux to HDL by 12 hours post-LPS administration (p = 0.026) which recovered only slightly by 24 hours post-LPS.

Conclusions:
A very low-dose of endotoxin produces relatively low-grade innate immune responses, without clinical symptoms, but with significant changes in HDL composition and function. This study provides evidence that a modest inflammatory response, consistent with chronic inflammation in atherogenic states
in vivo
, induces atherogenic changes in HDL.
Abstract 1134: Crucial Role of Cu/Zn-SOD in the Synthesis of Endothelium-Derived Hyperpolarizing Factor (EDHF) during Reactive Hyperemia in Mouse Mesenteric Microcirculation in Vivo
Authors: Yada Toyotaka , Shimokawa Hiroaki , Morikawa Keiko , Takaki Aya , Shinozaki Yoshiro , Mori Hidezo , Goto Masami , Ogasawara Yasuo , Kajiya Fumihiko
Published: July 2021

Background:

We have recently demonstrated that endothelial Cu/Zn-superoxide dismutase (SOD) plays an important role as an EDHF synthase. In this study, we examined the possible role of Cu/Zn-SOD in the synthesis of EDHF/H
2
O
2
during reactive hyperemia (RH) in mouse mesenteric microcirculation in vivo.

Methods:

Mesenteric arterioles from wild-type (wild) and Cu/Zn-SOD
−/−
mice (SOD-KO) (n=5 each, 22– 60 μm) were continuously observed with a pencil-typed intravital microscope (×600 magnification) during acetylcholine (ACh)-induced vasodilatation and RH (reperfusion after 60 sec of mesenteric artery occlusion) under cyclooxygenase blockade (indomethacin) under the following 3 conditions; control, NO synthase inhibitor (L-NMMA) and L-NMMA+catalase (a specific decomposer of H
2
O
2
).

Results:

In mesenteric arterioles of wild-type mice, endothelium-dependent relaxations to ACh (34±4%) and vasodilatation during RH (40±5%) were resistant to L-NMMA (ACh 23±7% and RH 20±5%, both P<0.05), but were markedly inhibited by L-NMMA+catalase (ACh, 8±2% and RH, 4±3%, both P<0.01 vs. control). In mesenteric arterioles of SOD-KO mice, L-NMMA markedly inhibited the relaxations to ACh (30±4% to 8±2%, P<0.01) and vasodilatation during RH (40±5% to 5±2%, P<0.01), and the responses were not inhibited by the addition of catalase (ACh, 3±2% and RH, 4±2%, not significant vs. control). In the presence of L-NMMA, RH-induced increase in blood flow was 330±5% of baseline in the wild-type mice, whereas in the SOD-KO mice, the response was significantly reduced to 145±23% (P<0.01). In the mesenteric arterioles of SOD-KO mice, tempol, an SOD mimetic, significantly increased the ACh-induced vasodilation, which effect was abolished by catalase. Vascular H
2
O
2
production in response to ACh in mesenteric arterioles (fluorescent method) was significantly increased in wild-type mice but was markedly impaired in SOD-KO mice. By contrast, vascular NO production was significantly increased in wild-type mice and was unaltered in SOD-KO mice. Vascular level of BH
4
in the aorta was comparable between the two genotypes.

Conclusions:

These results indicate that endothelial Cu/Zn-SOD plays an important role as an EDHF synthase during RH in mouse mesenteric microcirculation in vivo.
Abstract 1137: Switch From Nitric Oxide To Hydrogen Peroxide Mediated Dilation In Coronary Arterioles Of Obese Rats
Authors: Focardi Marta , Ziche Marina , Marzilli Mario
Published: July 2021

Background. Obesity is associated with coronary endothelial dysfunction, an early marker of atherosclerosis; but impaired endothelial function in obese Zucker rats appears not to be mediated by NO production.

Hypothesis: We tested the hypothesis that endothelial derived hyperpolarizing factor (EDHF) is responsible for endothelial dilation in obese rats.

Methods: We used 4 groups of male Zucker rats: lean and obese on normal and low carb diets. Rats were fed
ad libitum
for 3 weeks; caloric intake and weight gain were similar. Coronary arterioles were cannulated and pressurized for diameter measurements. Vasodilation to acetylcholine (Ach) and flow was determined before and after incubation with L-NAME (10 μm), an inhibitor of NO synthase, plus the cyclooxygenase inhibitor indomethacin (Indo; 10 μm), after tetraethylammonium (TEA 1 mM), a non-selective K
+
channel antagonist, and after catalase, a H
2
O
2
scavenger (CAT; 500 U/ml).

Results Acetylcholine-induced vasodilation was impaired in obese rats compared to lean (41%; n=5
vs
87%; n=3; p<0.05) and did not change after L-NAME+Indo incubation in obese both on normal (47 ± 19%; n = 4
vs
. 40 ± 13%; n = 4;
p
=ns) and low carb diet (51 ± 1%; n = 3
vs
. 59 ± 6%; n = 3;
p
=ns). Flow-induced dilation was impaired in obese rats compared to lean (38%;n=5
vs
78%; n=2;p<0.05) and this dilation was not affected by L-NAME+Indo incubation in obese both on normal (45±12%; n=4
vs
48±10% n=4;
p
=ns) and low carb diet (53±14%; n=3
vs
61±12% n=3;
p
=ns). Importantly, TEA incubation decreased Ach dilation both in obese on normal (41±11%; n=5
vs
13±7% n=5;
p<
0.05) and low carb diet (76±25%; n=3
vs
22±18% n=3;
p<
0.05). Similar results were obtained during flow-induced dilation in obese both on normal (37±17%; n=5
vs
14±7% n=5;
p<
0.05) and low carb diet (54±14%; n=3
vs
15±3% n=3;
p<
0.05). Catalase administration blocked both FID and Ach dilation in obese both on normal (FID 45±15%
vs
5±3% n=3;
p<
0.05; Ach 39±14%
vs
12±5% n=3; p<0.05) and low carb diet (FID 52±19%
vs
18±7% n=2;
p<
0.05; Ach 75±35%
vs
15±2% n=2;
p<
0.05).

Conclusions: Endothelial dependent vasodilation of coronary arterioles in obese Zucker rats is mediated by H
2
O
2
and increases in endothelial function in obese rats on a low carb diet is mediated by increased H
2
O
2
-induced dilation.
Abstract 219: Histidine Substituted Cardiac Troponin I (a164h) Improves Survival And Protects Cardiac Contractility During Acute Hypoxia In The Aged Murine Heart.
Authors: Palpant Nathan , Day Sharlene , Converso Kimber , Metzger Joseph
Published: July 2021

Contractile dysfunction associated with ischemia is a significant cause of morbidity and mortality particularly in the elderly. Strategies designed to protect the aged heart from ischemia-mediated pump failure are needed. We have generated transgenic (Tg) mice expressing a modified form of adult cardiac troponin I, the Ca
++
-activated molecular switch of the myofilament. Consonant with the fetal isoform, this transgene encodes a histidine substitution (A164H) in the critical switch domain of TnI thus increasing myofilament calcium sensitivity in a pH-dependent manner. We hypothesized that aged mice (24 months), intrinsically susceptible to myocardial dysfunction, would retain improved cardiac contractility at baseline and during an acute hypoxic challenge by means of myofilament-mediated calcium sensitization.

Methods/Results:
At baseline, by echocardiography, Tg hearts had increased systolic function, with a 26% higher mean ejection fraction compared to nontransgenic (Ntg) mice: 75 ± 3% [Tg: n = 13] vs. 63 ± 4% [Ntg: n = 12], P < 0.05, with no differences in diastolic function between the groups. To study the effects of acute hypoxia on cardiac hemodynamics mice underwent microconductance Millar catheterization while ventilated with 12% oxygen. Aged Tg mice had improved survival compared to Ntg mice: time to pump failure (65% of baseline peak systolic pressure) 11.59 ± 1.25 min. [Tg: n = 3] vs. 4.11 ± 1.90 min. [Ntg: n = 3], P < 0.05. After four minutes of hypoxia, Tg mice had markedly improved cardiac contractility compared to Ntg mice with increased stroke volume (30.05 ± 4.49 uL [Tg] vs. 13.23 ± 3.21 uL [Ntg], P < 0.05), end systolic pressure (106.09 ± 11.81 mmHg [Tg] vs. 64.49 ± 4.05 mmHg [Ntg], P < 0.05) and rate of positive left ventricular pressure development (12958.66 ± 2544.68 mmHg/sec [Tg] vs. 5717.00 ± 745.67 mmHg/sec [Ntg], P = 0.05).

Conclusion:
An alteration in myofilament calcium sensitivity via a pH-responsive histidine button in cardiac troponin I augments baseline heart function in Tg mice over their lifetime. During acute hypoxia, cTnI A164H improves survival in aged mice by maintaining cardiac contractility, and thus holds promise for the design of gene therapeutics to treat pump failure associated with acute ischemic events in the elderly.
Abstract 1145: Functional Heterogeneity of Angiopoietin-1, -2 on Endothelial Progenitor Cell Fate, Assessed by Single Cell culture of Cord Blood CD133+cells for Endothelial Lineage Commitment Assay
Authors: Masuda Haruchika , Eguchi Masamichi , Ito Rie , Kobori Michiru , Kato Shunichi , Asahara Takayuki
Published: July 2021

Background; Angiopoietin-1, -2 (Ang-1, Ang-2) has been demonstrated to play a pivotal role in angio-vasculogenesis, i.e. Ang-1 for vascular maturation or Ang-2 for vascular sprouting. However, the developmental mechanism of endothelial progenitor cell (EPC) fate oriented by angiopoietins for vasculogenesis remains to be elucidated. Here, we report the assessment of primitive EPC fate determined by Ang-1,-2, using in vitro endothelial lineage commitment assay of single cord blood (CB)-CD133+ cells. Methods; Single CB-CD133+cells isolated by MACS were cultured in a suspension manner for 7 days in liquid culture containing TPO, SCF, IL-6, Flt-3 ligand, and VEGF with either of Ang-1 or Ang-2 at 200 ng/ml each. The cultured cells from single CB-CD133+cells were applied to vasculogenic methylcellulose culture for EPC colony forming assay (EPC-CFA). Results; EPC-CFA disclosed the analytic potential on the degree of differentiation and commitment of single CB-CD133+cells by the assessment of two types of EPC colonies with the individual cellular sizes, i.e. primitive CFU-small and/or definitive CFU-large EPCs produced by single CB-CD133+cell. Whole EPC colony producing potential was upregulated by both Ang-1 and Ang-2 (ratio of total CFU-EPC number; 1.49 fold in Ang-1, 1.39 fold in Ang-2 vs control, p < 0.05). Both the commitment and differentiation frequencies of EPC from CB-CD133+ cell were promoted by Ang-1, inversely the latter inhibited by Ang-2 (commitment= frequency of single CB-CD133+cell producing any CFU-EPC; 1.5 fold in Ang-1: differentiation= frequency ratio of single CB-CD133+cell producing definitive CFU-EPC/single CB-CD133+cell producing any CFU-EPC; 2 fold in Ang-1, 0.27 fold in Ang-2 vs control, p < 0.01). On the other hand, the expansion potential of primitive CFU-EPC from single CB-CD133+ cell was augmented by Ang-2, although downregulated by Ang-1 (expansion= number of primitive CFU-EPC/ single CB-CD133+cell producing any CFU-EPC; 0.78 fold in Ang-1, 1.6 fold in Ang-2 vs control, p< 0.05). Conclusion; A novel in vitro colony assay system to survey EPC fate of single CB-CD133+cells precisely provides the evidences of angiopoietin-1 mechanism for differentiation into definitive EPC and angiopoietin-2 for procurance of primitive.
Abstract 1164: Mast Cells Contribute to Pathogenesis in the Progression of Abdominal Aortic Aneuyrsm
Authors: Tsuruda Toshihiro , Kato Johji , Hatakeyama Kinta , Nakamura Kunihide , Imamura Takuroh , Onitsuka Toshio , Asada Yujiro , Kitamura Kazuo
Published: July 2021

Abdominal aortic aneurysm (AAA) is histologically characterized by medial degeneration and various degrees of chronic adventitial inflammation, while the mechanisms for progression of aneurysm are poorly understood. In the present study, we carried out histological study of AAA tissues of patients (75±1 years, Mean±SEM), and interventional animal and cell culture experiments to investigate the role of mast cells in the pathogenesis of AAA. The number of mast cells determined by positive staining for tryptase, a major neutral protease of human mast cell secretory granules, was found to increase in the outer media or adventitia of human AAA, compared to those in the control aorta (2.1±1.1/mm
2
(n=57) vs. 0.6±0.4/mm
2
(n=22), p<0.01), showing a positive correlation (r=0.442, p<0.01) between the cell number and the AAA diameter. Western blot demonstrated that the protein expressions of tryptase, stem cell factor (a ligand for c-kit) and the phosphorylation of c-kit were significantly (p<0.01) increased in the AAA tissues, compared with the control aorta. In the animal experiments, diameter of abdominal aorta of the control (+/+) rats was increased up to 55% at 14 days following peri-aortic application of 0.4 mol/L calcium chloride, but only a 13% increase in the mast cell-deficient mutant Ws/Ws rats. The AAA formation was accompanied by accumulation of mast cells, T-lymphocytes, interferon-γ positive cells and by activated matrix metalloproteinases (MMP)-2 and -9, reduced elastin levels and augmented angiogenesis in the aortic tissue, but these changes were much less in the Ws/Ws rats than in the control (+/+) rats. The pharmacological intervention with the mast cell stabilizer tranilast (400 mg/kg/day) for 14 days significantly (p<0.01) attenuated the AAA development in the control (+/+) rats. Co-culturing the mast cell line HMC-1 with the monocyte/macrophage cell line U937 significantly (p<0.01) augmented MMP-9 activity. Furthermore, 100 and 300 μmol/L of tranilast significantly (p<0.01) decreased MMP-9 activity in those cells. Collectively, these data suggest that mast cells may play important roles in the progression of AAA.
Abstract 1170: SHP2-dependent Dephosphorylation of p190A Rho GAP Induces RhoA Activation by Angiotensin II in Vascular Smooth Muscle Cells
Authors: Rolli-Derkinderen Malvyne , Brégeon Jérémy , Parsons Sarah J , Pacaud Pierre , Loirand Gervaise
Published: July 2021

Angiotensin II (Ang II) is a major regulator of blood pressure, that essentially acts through activation of Ang II type 1 receptor (AT1R) of vascular smooth muscle cells (VSMC). AT1R receptor activates numerous intracellular signaling pathways, including the small G protein RhoA known to control VSMC proliferation, migration, differentiation and contraction. Nevertheless, the mechanisms leading to RhoA activation by AT1R are unknown. RhoA activation can result from activation of RhoA exchange factor, that replaces bound GDP by GTP and/or inhibition of Rho GTPase-activating-protein (GAP) that hydrolyzes GTP to GDP. Here we assess the involvement of the p190A Rho GAP in RhoA activation induced by Ang II. The introduction of small interfering RNA (siRNA) targeting p190A in VSMC from rat aorta increased basal RhoA-Rho kinase pathway activity (790 ± 11% of control, n = 3). Moreover p190A-siRNA abolished the early activation of RhoA-Rho kinase pathway induced after 5 min of AngII (0.1 μM) stimulation but not the delayed RhoA activation induced after 60 min. We then measured p190A tyrosine phosphorylation known to reflect its activity. In resting VSMC, p190A was basally phosphorylated. In the presence of the AT2R inhibitor PD123319 (1 μM), activation of AT1R induced p190A dephosphorylation that was maximal at 5 min of Ang II stimulation (26 ± 5% of control, n = 4). The activation of AT2R by Ang II in the presence of losartan (1 μM) had no effect, neither on RhoA activation nor on p190A phosphorylation. Expression of a p190A phosphomimetic mutant decreased the basal activity of RhoA-Rho kinase pathway. In contrast expression of catalytically inactive or phosphoresistant p190A mutants increased the basal activity of RhoA-Rho kinase pathway and inhibited RhoA activation by Ang II. Moreover, using siRNA, we show that the tyrosine phosphatase SHP2, known to be activated by AT1R stimulation, was necessary for Ang II-mediated p190A dephosphorylation and RhoA activation. Our work demonstrates that RhoA/Rho kinase activity is controlled by the p190A Rho GAP in VSMC. Activity of p190A is basally high and maintains a low level of RhoA-Rho kinase activity in resting VSMC. Dephosphorylation of p190A through SHP2-dependent process is required for AT1R-induced RhoA activation.
Abstract 1175: Critical Role of Bone Marrow ASC (Apoptosis-associated Speck-like Protein Containing a CARD) in Neointimal Formation After Vascular Injury in Mice
Authors: Yajima Noriyuki , Takahashi Masafumi , Shiba Yuji , Ise Hirohiko , Takahashi Yasuko , Sagara Junji , Taniguchi Shunichiro , Ikeda Uichi
Published: July 2021

Background:
ASC is an adaptor protein that forms inflammasome complex and regulates caspase-1-dependent interleukin (IL)-1β and IL-18 generation; however, the role of ASC in the pathophysiology of cardiovascular diseases remains undefined. Here, we investigated the contribution of ASC to neointimal formation after vascular injury in ASC-deficient (ASC
−/−
) mice.

Methods and Results:
Wire-mediated vascular injury was produced in the femoral artery of ASC
−/−
and wild-type (WT: C57BL/6) mice. Immunohistochemical analysis revealed that ASC was markedly expressed in the infiltrated macrophages in the neointima. Histological analysis revealed that neointimal formation was significantly reduced in ASC
−/−
mice as compared to that in the WT mice (I/M ratio, p = 0.007). IL-1β and IL-18 were clearly expressed in the neointimal lesion of the WT mice but showed decreased expression in the lesion of ASC
−/−
mice. However, TUNEL staining showed no significant difference in apoptotic cells of the media between these mice. Further, no significant differences were observed in early reendothelialization (CD31) and the number of macrophages (F4/80) and smooth muscle cells (SMCs: αSMA) per unit of the neointima. Flow cytometry analysis also showed no difference in the number of peripheral CD34
+
/Flk-1
+
cells (EPCs) after injury. To determine the contribution of bone marrow cells, we developed 3 types of bone marrow transplantation (BMT
Wild+Wild
, BMT
Wild+ASC−/−
, and BMT
ASC−/−
→ Wild
) mice. The neointimal formation in BMT
ASC−/−+Wild
mice mice was remarkably reduced as compared to that in BMT
Wild+Wild
(I/M ratio, p = 0.0003) and BMT
Wild+ASC−/−
mice (p = 0.015), indicating that bone marrow ASC is critical for neointimal formation. In vitro experiments showed that the lipopolysaccharide-induced production of inflammatory cytokines such as IL-1β, IL-6, MCP-1, and IFN-γ, but not TNF-α, in ASC
−/−
bone marrow cells was significantly decreased. Further, serum-stimulated proliferation activity and MAP kinase (ERK1/2 and p38) activation of ASC
−/−
SMCs were not impaired.

Conclusions:
These findings suggest that bone marrow-derived ASC is critical for the neointimal formation after vascular injury and identify ASC as a novel therapeutic target for cardiovascular diseases.
Abstract 1196: Vascular Endothelial Cells specific Endothelin-1 mediates Early Inflammation in Murine Model of Neointimal Formation
Authors: Anggrahini Dyah Wulan , Adiarto Suko , Iwasa Naoko , Nakayama Kazuhiko , Widyantoro Bambang , Nonaka Hidemi , Miyagawa Kazuya , Kisanuki Yaz Y. , Yanagisawa Masashi , Emoto Noriaki
Published: July 2021

Complex mechanism of the chronic inflammatory process in vascular injury and atherosclerosis involves various types of cells and cytokines. Previous
in vitro
studies suggest that Endothelin-1 (ET-1) is produced by those cells, induces expression of several adhesion molecules and mediates the leukocyte-endothelial interaction. In the present study, we sought to determine whether local endothelial ET-1 plays a direct effect on early inflammation and neointimal formation as a response to vascular injury
in vivo
. Using Cre-loxP system and Tie-2 Cre promoter, Vascular Endothelial ET-1 Knockout (VEETKO) mice was generated. The mice showed 60% reduction of ET-1 mRNA level at major organs and undetected serum ET-1 level. To induce vascular inflammation, complete ligation of the left common carotid artery was performed in 12-weeks-old male VEETKO (n=35) and Wild Type (WT) littermates (n=34). Inhibition of vascular endothelial ET-1 prevented the 4-fold increase in ET-1 mRNA level of carotid artery after ligation, in contrast to that of WT mice. This prevention resulted in lower vascular inflammation revealed by lower mRNA and protein level of PECAM-1, ICAM-1, and MCP-1 in VEETKO mice. The lack of ET-1 in endothelium further inhibited the adhesion of inflammatory cells at early days after ligation as compared to that adhered in endothelium of WT mice (cell numbers, 20.7±4.9 vs. 54.6±3.9 respectively, p<0.05, VEEETKO n=8/WT n=10). Observation at 4 weeks after ligation noted lower neointimal formation in VEETKO mice (n=11) as compared to WT littermates (n=12) (neointimal/medial ratio, 0.2±0.03 vs. 0.8±0.25 respectively, p<0.05). Changes in carotid flow gave no hemodynamical effect as sistolic blood pressure maintained significantly lower in VEETKO mice as compared to WT mice (105.7±1.4 vs. 115.9±1.6 mmHg before ligation, p<0.05 and 105.7±2.4 vs.119.08±1.08 mmHg after ligation, p<0.05 n=20 each). In conclusion, these data demonstrated that lack of vascular endothelial ET-1 reduced inflammatory response of the vascular wall followed by inhibition of neointimal formation confirming the role of local endothelial ET-1 in mediating inflammation in vascular injury, in addition to its potent vasoconstrictor and proliferating effect.
Abstract 235: Hypoxia Enhanced Delivery of Mitochondria-Targeted Catalase Protects Choroid Retinal Microvascular Endothelial Cells from Oxidative Stress
Authors: Dougherty Christopher J , Prentice Howard , Dorey Kathleen , Webster Keith A , Blanks Janet C
Published: July 2021

Loss of pericytes is a critical event early in the progression of microvascular dysfunction in diabetic retinopathy. Pericyte loss may be linked to high glucose mediated reactive oxygen species generation, blocking N-cadherin trafficking to the endothelial cell surface preventing pericyte recruitment and vessel stabilization. Hydrogen peroxide has been identified as a major free radical produced during high glucose exposure in endothelial cells. The goal of this research is to determine if tissue-specific hypoxia-regulated expression of a mitochondria-targeted catalase can prevent or limit RF/6A microvascular endothelial cell apoptosis and decrease vascular permeability by limiting cellular oxidative stress. For the development of tissue-specific and hypoxia-enhanced expression vectors, promoters were constructed with nine tandem combinations of HREs. This 9x HRE oligomer enhancer was inserted together into a pGL3 firefly luciferase plasmid with the Tie2(
short
) promoter for endothelial-specific expression. The 9xHRE-Tie2(
sh
) promoter construct was highly selective for RF/6A cells producing a basal amount of mitochondria-targeted catalase equivalent to the Tie2(
short
) promoter alone. In response to hypoxia (
pO

2

=
1%
), the
9xHRE-Tie2(

short

) promoter showed a 21-fold hypoxia-inducible activation similar in strength to the CMV promoter
, measured by dual luciferase assay. The hybrid promoters were incorporated into a replication deficient AAV delivery system for apoptosis and cell culture based endothelial permeability assays. In preliminary assays using RF/6A microvascular endothelial cells, apoptosis was reduced by 58% and permeability was reduced by 46%. The results suggest that mitochondria-targeted catalase protects RF/6A microvascular endothelial cells from apoptosis and reduces endothelial permeability in a high-glucose, low-oxygen environment.
Abstract 1287: TRPV4-Deficient Mice Exhibit Impaired Endothelium-Dependent Relaxation Induced by Acetylcholine
in Vitro and in Vivo
Authors: Zhang David , Mendoza Suelhem , Bubolz Aaron , Suzuki Makoto , Gutterman David
Published: July 2021

Agonist-induced Ca
2+
entry in endothelial cells is important for the synthesis and release of vasoactive factors, although mechanisms of Ca
2+
entry remain largely unknown. Emerging evidence suggests that the transient receptor potential vanilloid 4 (TRPV4) channel, a Ca
2+
-permeant TRP channel, is expressed in endothelial cells and may be involved in the regulation of vascular tone. Here we investigated the potential role of TRPV4 channels in acetylcholine-induced vasodilation
in vitro and in vivo
using the TRPV4 knockout (TRPV4
−/−
) mice model. Carotid arteries were isolated and preconstricted with the thromboxane A2 mimetic U46619. Concentration-dependent relaxations to acetylcholine (10
−9
–10
−5
M) were markedly reduced in carotids of TRPV4
−/−
vs. wild-type (WT) mice (maximal relaxations of 31±12% vs 53±4%, respectively; n=4 mice). There was no significant change in the ED50 for Ach. In both WT and TRPV4
−/−
, acetylcholine-induced relaxations were blocked and converted to constrictions by the NO synthase inhibitor L-NAME (maximal relaxations of −25±6% and −24±7%, respectively). There was no difference in papaverine-induced relaxations between WT and TRPV4
−/−
mice (maximal relaxations of 93±3% vs. 90±3%, respectively). U46619 caused similar contractions in carotid arteries from those mice. We also compared
in vivo
vasodilator effects of acetylcholine by measuring changes in blood pressure in those animals. Intravenous administration of acetylcholine (15 ng/gm bolus) decreased blood pressure by 32±6 mmHg in WT mice (from 90±15 to 57±10 mmHg; n=6), whereas blood pressure was reduced by only 10 mmHg in TRPV4
−/−
mice (from 67±6 to 56±4 mmHg; n=12). Acetylcholine caused similar reductions in heart rate in WT and TRPV4
−/−
mice, with mean changes of 365±57 and 292±40 beats/min, respectively. We conclude that the endothelium-dependent vasodilator response to acetylcholine is reduced both
in vitro
and
in vivo
in TRPV4
−/−
mice, and these findings may provide novel insight into the mechanisms of Ca
2+
entry evoked by chemical agonists in endothelial cells. The paradoxically lower baseline blood pressure in TRPV4
−/−
mice requires further investigation.
Abstract 1295: The Vasomotor Effects of Apelin in vivo in Man
Authors: Japp Alan G , Cruden Nicholas L , Amer David A , Goudie Ewan B , Johnston Neil R , Webb David J , Megson Ian L , Flapan Andrew D , Newby David E
Published: July 2021

BACKGROUND In preclinical models, apelin, the endogenous ligand for the G protein-coupled APJ receptor, lowers systemic venous tone, causes nitric oxide (NO)-mediated arterial vasodilatation and is the most potent inotrope yet studied. We aimed to establish the in vivo vasomotor effects of apelin in man.

METHODS Dorsal hand vein diameter was measured by the Aellig technique in 6 subjects during local intravenous infusions (0.1–3 nmol/min) of two apelin agonists, apelin-36 and (Pyr1)apelin-13, and sodium nitroprusside (SNP; 0.6 nmol/min) in the presence and absence of norepinephrine preconstriction. Forearm blood flow (FBF) responses to intrabrachial infusions of apelin-36 and (Pyr1)apelin-13 (0.1–30 nmol/min) were measured by venous occlusion plethysmography in 8 subjects on 4 occasions. In a 2×2 factorial design, a further 8 subjects received an intrabrachial infusion of (Pyr1)apelin-13 (0.3–3 nmol/min) in the presence or absence of the NO clamp (NO synthase inhibitor, L-N-monomethylarginine (8 micromol/min), co-infused with SNP (1–128 ng/min) to maintain basal flow and clamp endogenous NO production), and following a single oral dose of aspirin (600 mg) or matched placebo.

RESULTS Although SNP caused venodilatation (P<0.0001), apelin-36 and (Pyr1)apelin-13 had no effect on dorsal hand vein diameter in the presence or absence of norepinephrine (P=0.2). Both apelin-36 and (Pyr1)apelin-13 caused reproducible dose-dependent vasodilatation in forearm resistance vessels (P<0.0001): maximal increase of 198+/−27% and 90+/−7% respectively. Vasodilatation had a rapid onset (within 6 min) and slow offset that remained 36 min post-infusion (P<0.01). Plasma apelin concentrations and non-infused FBF (P<0.05) were increased at doses >3 nmol/min, consistent with systemic spillover. (Pyr1)apelin-13-mediated vasodilatation was attenuated by the NO clamp (AUC reduced by 64%; P=0.004) but was unaffected by aspirin (P=0.7).

CONCLUSION Although having no apparent effect on venous tone, apelin causes a rapid onset and sustained NO-dependent arterial vasodilatation in vivo in man. The apelin-APJ system merits further clinical investigation to determine its role in cardiovascular homeostasis and its therapeutic potential in heart failure.
Abstract 237: The Apolipoprotein A-I Mimetic Peptide D-4F Acts As A Scavenger For Myeloperoxidase-Derived Hypochlorous Acid
Authors: Wake Amanda , Datta Geeta , Mahtani Harry , Palgunachari Mayakonda , Parks Dale , Mishra Vinod , Anantharamaiah Gm , White C. Roger
Published: July 2021

Background:
Myeloperoxidase (MPO) and its product hypochlorous acid (HOCl) are proatherogenic molecules that functionally modify lipoproteins and impair endothelial cell function. MPO- and HOCl-induced changes in HDL function are thought to occur via modifications at the level of apolipoprotein (apo) A-I.

Objective
: We tested the hypothesis that the apo A-I mimetic peptide D-4F, which possesses HDL-like properties, prevents endothelial dysfunction by acting as a reactive substrate for HOCl.

Methods and Results:
Rat aortic ring segments were isolated and suspended in an isolated tissue bath and exposed to HOCl (10μM, 30min, n=7). Acetylcholine (Ach)-induced relaxation was inhibited by 51% compared to saline-treated controls (n=8). Concurrent exposure to HOCl and D-4F (10μM, n=9) significantly improved the response to Ach (15% inhibition vs control). Incubation of tissues with D-4F alone (n=9) did not impair endothelial function. Since increased superoxide formation has been implicated in HOCl-induced endothelial dysfunction, we tested whether D-4F influences formation of this oxidant. Electroparamagnetic resonance spectroscopy, in conjunction with BMPO (25mM) spin trapping, was used to determine whether D-4F reduces superoxide generated by xanthine (1mM)/xanthine oxidase (15mU). There was no difference in BMPO resonance spectra in the presence and absence of D-4F at concentrations up to 300 μM. In subsequent studies, effects of HOCl on structural properties of D-4F were assessed. Fluorescence spectroscopy revealed a significant reduction in tryptophan fluorescence of D-4F (20μM) in the presence of HOCl (10μM). Time of flight mass spectrometry was used to characterize reaction products formed by HOCl and D-4F. MS analysis of D-4F (20μM) alone yielded a product with the expected m/z ratio of 2,310. Addition of 10μM HOCl to D-4F yielded a new product with a net gain of 16 mass units. MS/MS analysis confirmed the addition of this mass to the tryptophan residue, reflecting oxidation of this residue.

Conclusion:
While D-4F was previously shown to inhibit lesion formation and inflammation in experimental models of atherosclerosis, results of the current studies suggest that D-4F may also serve as a scavenger of MPO-derived HOCl.
Abstract 1320: Reduced Fetuin-A Plasma Level is Associated with Faster Stenosis Progression and Increased Valvular Calcification in the Elderly Patients with Aortic Stenosis
Authors: Mohty Dania , Pibarot Philippe , Despres Jean-Pierre , Cartier Amelie , Arsenault Benoit , Mathieu Patrick
Published: July 2021

Background:
Fetuin A is a circulating calcium-regulatory glycoprotein that inhibits ectopic and vascular calcification. Aortic stenosis (AS) is a disease process involving an active calcification of the aortic valve (AV). Its prevalence increases markedly with aging. We examined the association between plasma level of Fetuin A and disease progression in AS

Methods and Results:
114 patients operated for AS, were divided into 2 groups according to their age: the young and middle-age group included the first (25–65 y-o) and second (66–74 y-o) tertiles and the elderly group included the last tertile (range 75–88 y-o). Plasma Fetuin-A level was determined by Elisa method and calcium content of the AV by a chelator quantitative method respectively. The annualized progression rate of the disease was calculated for the subset of patients in whom at least 2 transthoracic echocardiography separated by at least 6 months were available pre-operatively. In the elderly group, the preoperative progression rate of the maximum transvalvular gradient was 2-fold faster in patients with decreased plasma Fetuin-A level <0.36 ng/ml (median value) when compared to those with higher level of Fetuin-A (10±4 vs. 5±3 mmHg, p=0.01). Moreover, there was a significant correlation between calcium content of the AV explanted at the time of surgery and Fetuin-A plasma level (R=0.37, p<0.01). After adjusting for age, gender, bicuspid AV, creatinine, Fetuin-A remained significantly associated with AV calcification (R
2
=0.26, p=0.02). There was no correlation between Fetuin-A level and AV calcium content or AS progression in the younger patients group (R=0.06, p=0.62 and R=0.05, p=0.76, respectively)

Conclusion:
In the elderly patients, decreased level of Fetuin-A may contribute to enhanced valvular calcification and accelerated stenosis progression