bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5328: Intracellular Calcium Mediated Late-Phase 3 Early Afterdepolarization Induces Ventricular Fibrillation in Isolated Rabbit Hearts with Activated K
ATP
Channel
Authors: Tang Liang , Joung Boyoung , Song Juan , Ogawa Masahiro , Chen Peng-Sheng , Lin Shien-Fong
Published: July 2021

Introduction: The mechanism and importance of late-phase 3 early after depolarization (EAD) in ventricular arrhythmogenesis and the corresponding intracellular calcium dynamics are unknown.

Methods: We studied 22 Langendorff-perfused isolated normal rabbit hearts with and without K
ATP
channels opener, pinacidil (80 μM). In group I (n=10), we performed simultaneous dual optical mapping of transmembrane potential (V
m
) and intracellular calcium (Ca
i
) transient on left ventricular endocardium. In group II (n=6), glass microelectrode single cell recording of transmembrane potential (TMP) was performed. In group III (n=6), we examined the effects of BAPTA-AM, a calcium chelator, on late-phase 3 EAD and VF induction.

Results: Ventricular endocardium was paced at PCL between 60 ms and 350 ms for 29 beats. Pinacidil shortened both APD
90
(192±11 ms at baseline to 98±9 ms) and duration of Ca
i
at 50% of amplitude (DCaT
50
, 152±10 ms at baseline to 84±9 ms) at PCL of 250 ms. At pacing rate < 200 ms, the first post-pacing spontaneous beat showed persistent APD shortening and late-phase 3 EAD accompanied by large Ca
i
elevation. There was a positive correlation between the EAD amplitudes and the amplitude of Ca
i
elevation (r=0.899). At a critical Ca
i
elevation magnitude, the late-phase 3 EAD then induced triggered activity and VF in 10 episodes. The single cell TMP recording confirmed the optical mapping data of late-phase 3 EAD-induced VF. Infusion of BAPTA-AM significantly reduced the maximal Ca
i
amplitude in the mapped region (26.4±3.5% of the control; p<0.001). Due to the lowered Ca
i
level, neither EAD in the first post-pacing beat nor VF induction was observed after pacing train at any PCLs.

Conclusions: K
ATP
channel activation leads to shortened APD with correspondingly large Ca
i
elevation, resulting in late-phase 3 EAD in first spontaneous beat at the cessation of rapid pacing. Reducing intracellular calcium handling by BAPTA-AM prevented EAD and VF induction/reinitiation. Because acute myocardial ischemia activates the K
ATP
channel, the calcium mediated late-phase 3 EAD induced triggered activity may be a novel mechanism for VF and sudden death during acute myocardial ischemia.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 5346: Regulation of Cardiac Na
+
Current by Pyridine Nucleotides: A Possible Explanation of Glycerol-3-Phosphate Dehydrogenase-Linked Brugada Syndrome
Authors: Sanyal Shamarendra , Gurung Iman S , Pfahnl Arnold E , Shang Lijuan L , Iravanian Shahriar , Allen Jon , Liu Man , Grace Andrew , London Barry , Dudley Samuel C
Published: July 2021

Recently, glycerol-3-phosphate dehydrogenase 1-like (GPD1-L) gene mutations have been shown to reduce cardiac Na
+
current and cause Brugada Syndrome (BrS). The glycerol-3-phosphate dehydrogenase (GPD) family of genes is involved in nicotinamide adenine dinucleotides (NAD)-dependent energy metabolism, and GDP1-L has >80% amino acid homology with GPD. Therefore, we tested whether mutations in GPD1-L could be acting through NAD (H) to alter Na
+
current. Human embryonic kidney (HEK) cells stably expressing the human cardiac sodium channel (SCN5A) were used to assess the effects of wild-type (WT) and mutant (MT) GPD1-L on cellular NAD(H) and the effects of NAD(H) on Na
+
current. A mouse model of BrS was used to assess the effect of reduced NAD
+
on arrhythmic risk. MT GPD1-L raised cellular NADH level by 4.3 fold (p<0.01) and reduced Na
+
current by 69.9% (p<0.01). Extracellular NADH (300 μM) raised cellular NADH by 5.3 fold and decreased whole cell peak conductance by 71.4% (p<0.001). Extracellular NAD
+
(300 μM) raised conductance by 30.3% (p< 0.001). Fluorescent microscopy showed parallel changes in membrane-associated, GFP-tagged Na
+
channels. Intracellular application of NADH or NAD
+
resulted in an immediate change in Na
+
current of −55.5% (p < 0.01) and +66.6% (p < 0.01), respectively. External NAD
+
could prevent the reduction in Na
+
current caused by MT GPD1-L. Apocynin (100 μM), an NAD(P)H oxidase inhibitor, or the reducing agent, dithiothreitol (DTT), prevented the NADH-induced reduction in Na
+
current (p < 0.01). Application of 100 μM NAD
+
to a mouse model of BrS reduced the programmed electrical stimulation induced ventricular tachycardia. GPD1-L mutations may cause BrS through alterations in cellular NAD(H), and NAD
+
might represent a novel treatment for BrS.
Abstract 5362: Inhibition Of The Epidermal Growth Factor Receptor Transactivation By The Angiotensin II Type I Receptor Prevents Compensatory Cardiac Hypertrophy And Worsens Cardiac Function Following Myocardial Infarction
Authors: Galeotti Jonathan , Hsu Chiao Po , Zhai Peiyong , Sadoshima Junichi
Published: July 2021

The angiotensin II type I receptor (AT1R) has been shown to activate the epidermal growth factor receptor (EGFR). However, the downstream effects of this activation have not yet been elucidated in the heart. To examine the function of the AT1R-EGFR pathway, we have created an AT1R mutant in which amino acid 319 is mutated from tyrosine to phenylalanine (Y319F). The Y319F mutant lacks the ability to transactivate the EGFR. We generated transgenic lines overexpressing either wild type AT1R (Tg-WT) or Y319F (Tg-Y319F) only in the heart with similar levels of overexpression, and evaluated the contribution of the AT1R-EGFR pathway to cardiac responses against stress. Under baseline conditions at 3 months of age, cardiac function of Tg-WT and Tg-Y319F was not significantly different from non-transgenic mice (NTg) except that Tg-WT showed mild left ventricular hypertrophy. To examine the role of the AT1R-EGFR pathway under stress, we induced myocardial infarction (MI) by permanently ligating the left anterior descending coronary artery. Four weeks after MI, increases in heart weight/tibia length (Tg-WT, Tg-Y319F, NTg: 12.57, 10.11, 9.96, p<0.05 vs Tg-WT) and left ventricular myocyte cross sectional area (35, 5, 29% vs sham, p<0.05) were significantly attenuated in Tg-Y319F compared to Tg-WT. Fibrosis was also milder in Tg-Y319F than in Tg-WT (10.7% vs 22.9%). These results suggest that the AT1R-EGFR pathway plays an important role in mediating LV hypertrophy and fibrosis after MI. Interestingly, however, echocardiographic measurement showed that Tg-Y319F have impaired LV ejection fraction (Tg-WT, Tg-Y319F, NTg: 51, 19, 44%, p<0.05) and %fractional shortening (21.4, 6.7, 17.7%, p<0.05) compared with Tg-WT or NTg. Tg-Y319F showed an increase in mortality in days 0–28 following MI, when compared to Tg-WT and NTg (35, 67, 30%, p<0.5). Additionally, lung weight/tibia length was increased in Tg-Y319F (11.3, 24.7, 16.1, p<0.05) These results suggest that the lack of EGFR activation causes cardiac dysfunction after MI. In summary, transactivation of the EGFR following MI may play a compensatory role, thereby protecting the heart from further deterioration of cardiac function.

This research has received full or partial funding support from the American Heart Association, AHA Founders Affiliate (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont).
Abstract 5393: Increased C-Reactive Protein Expression Exacerbates Left Ventricular Dysfunction and Remodeling after Myocardial Infarction
Authors: Takahashi Toshiyuki , Anzai Toshihisa , Kaneko Hidehiro , Anzai Atsushi , Mano Yoshinori , Maekawa Yuichiro , Ogawa Satoshi , Yoshikawa Tsutomu
Published: July 2021

We have previously reported that elevated serum C-reactive protein (CRP) level after acute myocardial infarction (MI) is associated with adverse outcomes including cardiac rupture, left ventricular (LV) remodeling and cardiac death. Recent experimental studies have shown that CRP per se has some biological properties including proinflammatory and proapoptotic effects, suggesting a pathogenetic role of CRP in the remodeling process after MI. We tested the hypothesis that increased CRP expression would exacerbate adverse LV remodeling after MI through some deleterious effects of CRP. Transgenic mice with human CRP expression (CRP-Tg) and their nontransgenic littermates (Control) underwent proximal ligation of the left coronary artery. Despite increased serum CRP level and cardiac CRP expression in CRP-Tg mice, there was no difference in phenotype between CRP-Tg and control mice before MI. Mortality at five weeks after MI was not different between groups (CRP-Tg: 49%, n=35; Control: 38%, n=40,
P
=0.28). Five weeks after MI, echocardiography showed that CRP-Tg mice had more LV dilation (LVEDD, CRP-Tg: 5.8 ± 0.1 mm, n=14; Control: 5.2 ± 0.1 mm, n=17,
P
=0.002) and worse LV function (EF, CRP-Tg: 13 ± 2%, n=14; Control: 19 ± 1%, n=17,
P
=0.01). Hemodynamic studies indicated that LV +dP/dt (CRP-Tg: 2,947 ± 480 mmHg/s, n=9; Control: 3,788 ± 656 mmHg/s, n=10,
P
=0.02) and -dP/dt (CRP-Tg: −2,230 ± 48 mmHg/s, n=9; Control: −2,890 ± 161 mmHg/s, n=10,
P
=0.003) were lower in the CRP-Tg group than in the Control group, although infarct size was comparable. Histological evaluation at one week after MI showed a higher rate of apoptosis in the border zone of infarcted hearts from CRP-Tg mice (CRP-Tg: 1,434 ± 322 per 10
5
nuclei; Control: 596 ± 112 per 10
5
nuclei, n=6 for each,
P
=0.03). Quantitative RT-PCR showed that angiotensin II type 1a receptor and interleukin-6 were upregulated in viable LV samples from CRP-Tg mice compared with controls. Increased CRP expression exacerbates LV dysfunction and remodeling after MI, associated with increased apoptotic rates, increased angiotensin II receptor expression and exaggerated inflammatory response.
Abstract 5416: Ablation of TRPV1 Exacerbates Pressure Overload-Induced Cardiac Hypertrophy
Authors: Zhong Beihua , Rubinstein Jack , Wang Donna H
Published: July 2021

Background

We have recently shown that the transient receptor potential vanilloid type 1 (TRPV1), a ligand-gated cation channel expressed in sensory nerves innervating the heart, plays an important role in protecting the heart from ischemia/reperfusion injury. However, the role of TRPV1 in pressure overload-induced hypertrophy of the heart is unknown.

Objective

-
This study tests the hypothesis that TRPV1 protects the heart from pressure overload-induced hypertrophy and functional deterioration using gene-targeted TRPV1-null mutant (TRPV1
−/−
) or wild-type (WT) mice.

Methods and Results

Four weeks after transverse aortic constriction (
TAC
) in TRPV1
−/−
and WT mice,
echo
cardiography demonstrated that cardiac fractional shortening (FS) and ejection fraction (EF) were suppressed in a greater extent in TRPV1
−/−
than WT mice (FS: 14 ± 3% in TRPV1
−/−
vs. 18 ± 2% in WT mice,
P
<0.05; EF: 34 ± 6% in TRPV1
−/−
vs. 43 ± 3% mice,
P
<0.05), while there was no difference in FS and EF in TRPV1
−/−
and WT sham-operated mice. The ratio of
heart
weight to body weight (mg/g) was greater in TRPV1
−/−
than in WT mice (7.39±1.34 in TRPV1
−/−
vs. 6.26±1.07 in
WT
mice,
P
<0.05), and the increase in heart weigh was accompanied by an increase in cardiomyocyte cross-sectional areas in TRPV1
−/−
compared to WT mice (248±8.2 in TRPV1
−/−
vs 224±8.7 in WT μm
2
,
P
<0.05).
Immunohistochemistry study showed enhanced
cellular infiltration and fibrosis in TRPV1
−/−
compared to WT mice. Consistently, hydroxyproline assay showed that the collagen level was higher in TRPV1
−/−
than WT mice (1.7±0.3 in TRPV1
−/−
vs 1.3±0.4 μg/mg dry tissue in WT,
P
<0.05), and ELISA assay showed that interleukin-6 levels were higher in TRPV1
−/−
than in WT mice (14.2±4.6 in TRPV1
−/−
vs. 10.6±2.6 pg/mg protein in WT,
P
<0.05).

Conclusions

These data show that ablation of TRPV1 results in exaggerated cardiac hypertrophy, fibrosis, and dysfunction induced by pressure overload, which may result from enhanced inflammatory responses in the absence of TRPV1.
Abstract 5422: Effect of Age on Expression of AT
1
and AT
2
Receptors and ACE-2, Angiotensin (1–7), Ac-SDKP and Smad-2 Proteins After Acute Reperfused ST-Segment Elevation Myocardial Infarction
Authors: Palaniyappan Arivazhagan , Idikio Halliday , Jugdutt Bodh I
Published: July 2021

Recent evidence suggests that aging alters the expression of inflammatory cytokines, impairs healing and promotes adverse left ventricular (LV) remodeling after chronic reperfused ST-segment elevation myocardial infarction (RSTEMI). Whether aging alters the expression of angiotensin II type 1 (AT
1
) and type 2 (AT
2
) receptors, and angiotensin-converting-enzyme-2 (ACE-2), angiotensin (Ang) (1–7), N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) and Smad-2 proteins early after acute RSTEMI is not known. AT
2
receptors have been implicated in cardioprotection, ACE-2 and Ang (1–7) in the counter-regulatory arm of the renin-angiotensin-system (RAS) and Ac-SDKP in inflammation and collagen synthesis. We hypothesized that aging is associated with downregulation of AT
2
receptors and ACE-2, Ang (1–7), Ac-SDKP and Smad-2 proteins. We compared in-vivo LV remodeling and function (echocardiography/Doppler) and the ex-vivo molecular expression of AT
1
and AT
2
receptors, ACE-2, Ang (1–7) and Ac-SDKP after acute RSTEMI (90 min no-flow ischemia and 120 min reperfusion) in young (group 1, n=12) and old (group 2, n=12) dogs. Compared to group 1 controls, group 2 hearts showed more severe echocardiographic LV remodeling and dysfunction (with lower ejection fraction, larger volumes and more diastolic dysfunction, infarct expansion and thinning). In addition, group 2 hearts showed no change in AT
1
receptor protein and decrease in AT
2
receptor protein and ACE-2, Ang (1–7), Ac-SDKP and Smad-2 proteins in the reperfused ischemic zone. The findings suggest that aging is associated with changes in proteins in the regulatory as well as the counter-regulatory arm of the RAS during acute RSTEMI. The age-related downregulation of AT
2
receptors, ACE-2, Ang (1–7), Ac-SDKP and Smad-2 may contribute to the more severe LV remodeling and dysfunction after acute RSTEMI. Targeting these proteins early during reperfusion may improve outcome in acute RSTEMI.
Abstract 5447: Thromboxane A
2
Receptor Activation Attenuates Insulin Signaling and Angiogenesis by Upregulating Phosphatase PTEN in Cultured Endothelial Cells
Authors: Song Ping , Wang Shuangxi , Zhang Maio , Xu Jian , Wu Yong , Zou Ming-Hui
Published: July 2021

Increasing evidence indicates that elevated levels of endogenous thromboxane receptor (TPr) agonists including thromboxane (Tx) A
2
, prostaglandin H
2
, and 8-iso-prostaglandin F
2a
contribute to insulin resistance, endothelial dysfunction, and accelerated atherosclerosis in diabetes. However, the mechanisms by which TPr triggers insulin resistance in endothelial cells remaine poorly defined. Here we show how TPr activation impairs insulin signaling and angiogenesis in endothelial cells. Exposure of cultured human umbilical vein endothelial cells (HUVEC) to a structurally related TxA
2
mimetic (IBOP, U46619, or carboxy-TxA
2
) for 3 or 16h significantly inhibited both basal- and insulin-stimulated Akt-Ser473 phosphorylation, Akt activity, the serine 1177 phosphorylation of endothelial nitric oxide synthase (eNOS-Ser1177), and angiogenesis. Concomitantly, IBOP significantly increased the levels of Phosphatase and TENsin homolog deleted on chromosome 10 (PTEN) and PTEN phosphorylation at Ser380/Thr382/383. Further, Y27632, a Rho kinase (ROCK) inhibitor, abolished the effects of IBOP on the phosphorylation of PTEN, Akt and eNOS, and the PTEN level. Similarly, inhibition of PTEN by small interference RNA (siRNA), like the selective TPr antagonist, SQ29548, or knockdown of TPr by siRNA, abolished IBOP-induced inhibition on both the phosphorylation of the Akt-eNOS axis and angiogenesis. Furthermore, IBOP significantly increased the Ser428 phosphorylation of LKB1, a known upstream kinase for PTEN. Finally, suppression of LKB1 abolished IBOP-induced PTEN phosphorylation and PTEN accumulation in HUVEC. We conclude that TPr suppresses insulin signaling and angiogenesis by upregulating RhoA mediated but LKB1-dependent PTEN phosphorylation in endothelial cells.

This research has received full or partial funding support from the American Heart Association, AHA South Central Affiliate (Arkansas, New Mexico, Oklahoma & Texas).
Abstract 5452: O-Linked-n-Acetylglucosamine Formation Reduces Nitric Oxide-Dependent Dilation in Arterioles Exposed to High Glucose Concentrations
Authors: Beleznai Timea , Feher Attila , Rutkai Ibolya , Edes Istvan , Bagi Zsolt
Published: July 2021

Hyperglycemia is associated with serious microvascular complications in diabetes mellitus. We hypothesized that under high glucose concentrations, activation of the hexosamine pathway leads to protein O-linked-N-acetylglucosamine (O-GlcNAc) formation which interferes with nitric oxide (NO)-dependent arteriolar dilation. Thus, diameter changes of skeletal muscle arterioles (diameter: ~ 160 μm) isolated from healthy, male Wistar rats were investigated after exposure to normal glucose (NG, 5.5 mM) or high glucose (HG, 30 mM for 2 hours) concentrations. In arterioles exposed to HG concentration, dilations to histamine were markedly reduced compared to those of under NG condition (max: −6±6% and 69±9%, respectively). Inhibition of NO synthesis with L-NAME reduced histamine-induced dilations in NG arterioles, but it had no effect on microvessels exposed to HG. Dilations to the NO donor, sodium nitroprusside were similar in the two groups of vessels. In the presence of the inhibitor of hexosamine pathway, azaserine histamine-induced dilations were significantly augmented and diminished by additional administration of L-NAME in arterioles exposed to HG concentrations (max: 67±2%). Moreover, under NG condition, exposure of vessels to glucosamine (5 mM, for 2 hours) resulted in reduced histamine-induced arteriolar dilations (max: 26±3%). Upon HG and glucosamine treatment an enhanced O-GlcNAc formation of endothelial NO synthase (eNOS) was detected by Western blots and immunoprecipitation. These findings indicate that high glucose concentration may lead to O-GlcNAc formation of eNOS, which impairs histamine-induced, NO-mediated arteriolar dilations. We propose that interfering with the hexosamine pathway may prevent microvascular complications in diabetes mellitus. On the other hand, our data suggest caveat for glucosamine supplementation to treat osteoarthritis, since it may impair microvascular function.
Abstract 5460: Promoting Effect of
188
Re Radiation on the L-arginine/nitric Oxide Pathway in the Balloon Injured Rat Abdominal Aortic Arteries
Authors: Zhong Guangzhen , Yang Xinchun , Chen Pengrong , Tang Chaoshu
Published: July 2021

This study examined the effect of
188
Re radiation on L-Arginine(L-Arg)/NO pathway in the injured rat abdominal aortic artery and investigated the mechanism involved. Forty-three rats were randomly divided into 4 groups: control (n=10), 14 days after balloon injury without treatment (14B, n=10), 14 days after balloon injury plus radiation (14BR, n=13), radiation only (SR, n=10). Vascular lesions were created in the abdominal aortic arteries by overstretch balloon injury. Arteries were then left untreated or were treated with intra-aortic beta-radiation using
188
Re balloon to deliver 20Gy at 2 mm from the center of the source. Intima/medium(I/M) ratio of the artery were evaluated. Plasma NO, aortic NO generation, L-Arg transport, iNOS activity, iNOS mRNA and protein expression were studied. We found that I/M ratio in 14B group was higher than control (0.558±0.175 vs0.063±0.010, P<0.01), and higher than that in 14BR group (0.080±0.021, P<0.01). Plasma NO in each group was not significantly different with each other. NO generated by aortic artery in 14B group were 177% (P<0.01) higher than control (8.65±2.47μmol/mg protein) and 17% (P<0.05) lower than that in 14BR group. iNOS activity in the 14B group was 44% (P<0.01) higher than control(180±22 pmol.mg protein
−1
.min
−1
) and 11%(P<0.05) lower than that in 14BR group. Quantitative RT-PCR showed that iNOS mRNA in 14B group was 290% (P<0.01) higher than control and 19%(P<0.05) lower than that in 14BR group. Immunohistochemistry results showed no iNOS protein expression in control. iNOS protein were similarly expressed in the 14B and 14BR groups (P>0.05). However iNOS protein was strongly expressed in the SR group. Incubated in the KH solution with L-Arg(100μmol/L), L-Arg transport efficacy in 14B group was 248% (P<0.01)higher than control(1.04±0.15pmol.g protein
−1
.min
−1
), and 39%(P<0.01) higher than that in 14BR group. In summary, we found
188
Re inhibited neointima hyperplasia and promoted NO generation, iNOS activity and iNOS mRNA expression in the injured artery. However, it decreased L-Arg transport and had no significant influence on iNOS protein expression in the injured artery. Therefore, effect of radiation on the L-Arg/NO pathway explains partly the mechanism of intracoronary radiation.
Abstract 5481: Expression of Human ABCA1 in Endothelium Protects Against Diet-Induced Atherosclerosis in Mice
Authors: Vaisman Boris L , Knapper Catherine L , Demosky Stephen J , Stonik John A , Amar Marcelo J , Remaley Alan T
Published: July 2021

Cholesterol homeostasis in endothelium plays significant role in protecting against development of atherosclerosis and in supporting normal function of the vasculature. ABCA1 transporter is one of major players in reverse cholesterol transport responsible for biogenesis of HDL and cholesterol efflux from cells to lipid-poor apoA1 particles. To evaluate the contribution of endothelial-expressed ABCA1 in HDL generation and in processes of diet induced atherosclerosis, we expressed hABCA1 in endothelial cells with the endothelial specific Tie2 promoter. In isolated lung endothelial cells Tie2hABCA1 was expressed at the level 10+/−3% of expression of mouse ABCA1. No hABCA1 was found to be expressed in peritoneal macrophages. Expression of hABCA1 did not alter the pattern of expression of mouse ABCA1, SR-B1 and ABCG1 in aorta, heart, kidney, liver, lung, muscle, spleen and testis. Despite the modest level of expression of hABCA1 in endothelium, it had a significant impact on plasma levels of total cholesterol (TC) and HDL-C. In females, TC was increased in comparison with normal siblings from 85±3 to 101±3; HDL-C - from 57±2 to 68±2 (all values in mg/dl, p<0.001); in males TC was changed from 100±4 to 127±5 (p<0.001), HDL-C - from 73.5±2.6 to 84±3.5 (p<0.03). This effect of hABCA1 was found only on normal C57Bl background. When Tie2hABCA1 was transferred to ABC1-K/O or apoE-K/O backgrounds no effect of hABCA1 on plasma lipids was revealed. Experiments on cultivated in Transwells polarized lung endothelial cells isolated from Tie2hABCA1 mice on ABCA1-K/O background revealed that Tie2hABCA1 was able to promote cholesterol efflux to apoA1 directed mostly to apical side of the cells. Expression of Tie2hABCA1 in C57Bl mice protected them against diet-induced atherosclerosis. After 6 months on Cocoa Butter Diet (TD90221) control females had 6.5±0.8% of en face aorta lesions, whereas transgenic animals had 4±0.5% of the lesions (p<0.02). HDL-C after 6 months on diet was higher in transgenic vs control females (56±3 vs 40.5±3.5, p<0.003). There was no protection if hABCA1 was on ABCA1-K/O or apoE -K/O backgrounds. In summary, the results support a role of endothelial ABCA1 expression in the generation of HDL and in the protection of diet induced atherosclerosis.
Abstract 5489: Treatment of ApoE-Deficient Mice with Statin Inhibits the Oxidative Stress-Dependent Lysosomal Protease Cathepsin Activation System: Implication for Plaque Stability
Authors: Cheng Xian Wu , Kuzuya Masafumi , Okumura Kenji , Nakamura Kae , Inoue Aiko , Sasaki Takeshi , Song Haizhen , Takeshita Kyosuke , Kim Weon , Shi Guo-Ping , Murohara Toyoaki
Published: July 2021

Lysosomal protease cathepsin (Cat) overexpressed in atherosclerotic lesions has a strong proteolytic activity. Given that reactive oxygen species (ROS) are also implicated in protease activation in atherogenesis, we examined a possible role of the ROS-induced cathepsin activation system in neointima formation and atherosclerotic plaque in stability. Murine vascular injury-induced neointima formation was examined by use of a carotid artery ligation model, which demonstrated that cathepsin K deficiency impaired neointima formation and that cystatin C deficiency enhanced it (9-week-old males; n = 18 for each group,
P<
0.01). To examine statin-mediated pleiotropic actions on neointima formation and plaque stability, a mouse plaque disruption model (a combination of ligation and cuff placement treatment) was applied to apoE-deficient mice (9-week-old males). The apoE-deficient mice (n = 24–30 for each group) maintained on a normal diet were treated with (1) vehicle (0.5% carboxymethyl cellulose), pitavastatin (1 mg/kg/d), pitavastatin (10 mg/kg/d) for 10 days (before and after cuff treatment; all given by oral gavage every day). Pitavastatin suppressed not only macrophage infiltration but also levels of NADPH oxidase component mRNAs (p22
phox
and gp91
phox
) and O2-production in the atherosclerotic lesions. Moreover, pitavastatin increased the collagen fraction and reduced media elastic lamina fragmentation concomitant with decreases in the levels of cathepsin K mRNA and protein. Among these beneficial effects, high doses of pitavastatin further reduced intraplaque hemorrhage. The pitavastatin-mediated vascular-protective effects were mimicked by cathepsin K deficiency in apoE-deficient mice. Pitavastatin lowered the serum levels of cathepsin K but not plasma lipid profile. Pitavastatin inhibited H
2
O
2
-induced Cat K expression and activity in cultured smooth muscle cells and macrophages. Cathepsin K deficiency suppressed vascular injury-induced neointima formation. The data suggested that short-term administration of statin attenuates neointima formation and atherosclerotic plaque stability at least in part by inhibiting the oxidative stress-dependent Cat activation system.
Abstract 5494: ADAMTS-7 is Critical for Vascular Smooth Muscle Cell Migration and Neointima Formation in Injured Rat Carotid Artery
Authors: Kong Wei , Wang Li , Bai Xue , Liu Bo , Zhu Yi , Wang Xian
Published: July 2021

Migration of vascular smooth muscle cells (VSMCs) plays an essential role during vascular development, in response to vascular injury and during atherogenesis. Extensive studies have implicated the importance of extracellular matrix (ECM)-degrading proteinases during VSMCs migration. ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs), a recently described family of proteinases, is capable of degrading vascular ECM proteins. However, the relevance of ADAMTS family members in cardiovascular disease is poorly understood. In this study, we sought to determine whether ADAMTS-7 is involved in VSMC migration and neointima formation in response to vascular injury. Denudation of rat carotid arteries with a balloon catheter led to an initial decrease of ADAMTS-7 protein level in injured compared with sham-operated arteries within the first 24 hours, followed by a subsequent increase during the 4 to 14 days after injury. In primary VSMCs, the pro-inflammatory cytokine TNF-α increased ADAMTS-7 mRNA level through transcriptional factors nuclear factor-kappa B and AP-1. VSMCs infected with ADAMTS-7 adenovirus (Ad-ADAMTS-7) greatly accelerated their migration and invasion
in vitro
. Conversely, suppression of ADAMTS-7 expression by small interfering RNA (siRNA) markedly retarded VSMC movement by 50% than that with scramble siRNA. At 7 days after injury, the neointimal area of the vascular wall was sixfold greater in Ad-ADAMTS-7-infected than that in Ad-GFP-infected vessels (3.10±0.88 vs. 0.52±0.28 ×10
4
μm
2
, n=8 per group,
P
<0.05). By contrast, perivascular administration of ADAMTS-7 siRNA, but not scramble siRNA to injured arteries resulted in prolonged ADAMTS-7 silencing and diminished neointimal thickening without affecting medial areas. This inhibitory effect was sustained up to 14 days after injury. As well, ADAMTS-7 mediated degradation of the vascular ECM cartilage oligomeric matrix protein (COMP) in injured vessels, which might facilitate VSMC migration and neointimal thickening. ADAMTS-7 directs VSMC migration and neointima formation and therefore may serve as a novel therapeutic target for vascular restenosis and atherogenesis.
Abstract 5500: Nanoparticle Inhalation Impairs Endothelium-dependent Vasoreactivity in Coronary Arterioles
Authors: LeBlanc Amanda J , Hu Yanduan , Muller-Delp Judy M , Chen B. T , Frazer D. , Castranova V. , Nurkiewicz Timothy R
Published: July 2021

Epidemiological studies have shown that exposure to particulate matter (PM) is associated with an increased risk for myocardial infarction (MI). We have recently shown that systemic microvascular function is significantly impaired after inhalation exposure to PM and nanoparticles. However, it remains unclear how endothelial function in the coronary microcirculation responds to such insults and if impaired vasoreactivity as a result of exposure contributes to the increased risk for MI. Male Sprague-Dawley rats were exposed to filtered air (control), fine TiO
2
particles (primary particle diameter, ~1μm), or TiO
2
nanoparticles (primary particle diameter, ~21 nm) via inhalation at concentrations relevant to ambient air pollution (Fine TiO
2
, ~ 8–90 μg measured pulmonary deposition; nano-TiO
2
, ~ 4–38 μg measured pulmonary deposition). Coronary arterioles (≤ 150 μm in diameter) were isolated from the left anterior descending artery distribution and responses to flow (FID), acetylcholine (ACh), endothelin and dea-NONO-ate were assessed. Exposure to either fine TiO
2
or nano-TiO
2
impaired endothelium-dependent FID (Control 78±8%, Fine TiO
2
62±5%, Nano-TiO
2
60±8%). Similarly, endothelium-dependent vasodilation to ACh tended to be lower in coronary arterioles from rats exposed to fine TiO
2
and nano-TiO
2
compared to arterioles from control rats, but this did not reach statistical significance. Interestingly, vascular smooth muscle nitric oxide (NO) sensitivity (dea-NONO-ate) was decreased by exposure to nano-TiO
2
, but not fine TiO
2
(Nano-TiO
2
36±8%, Fine TiO
2
69±5%). Furthermore, nano-TiO
2
exposure diminished vasoconstrictor responses to endothelin, whereas fine TiO
2
exposure had no effect on endothelin reactivity (Nano-TiO
2
65±6%, Fine TiO
2
80±4%). These results suggest that nanoparticle exposure causes significantly more microvascular dysfunction than exposure to fine TiO
2
. It is probable that such disturbances in coronary microvascular function contribute to the cardiac events associated with particle pollution exposure.

Disclaimer: The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the National Institute for Occupational Safety and Health.
Abstract 5501: Reduced Coronary Reserve as a Mechanism in Beta-Adrenergic Receptor Mediated Cardiomyopathy and its Rescue by Adenylyl Cyclase Type 5 Knockout
Authors: Gao Shumin , Yan Lin , Hong Chull , Zhao Xin , Chen Li , Shen You-Tang , Vatner Stephen F , Vatner Dorothy E
Published: July 2021

Reduced coronary reserve (CR) is a cardinal feature of human cardiomyopathy, but it has not been studied extensively in mouse models of cardiomyopathy, potentially because of difficulties inherent to measuring CR in such a small heart. The first goal of this study was to determine if reduced CR is a potential mechanism involved in a transgenic (Tg) mouse model of cardiomyopathy, induced by cardiac overexpression of beta
2
-adrenergic receptors (beta
2
-AR Tg). Older beta
2
-AR Tg mice (16–17 months, n=7) developed cardiomyopathy, as reflected by decreased LV ejection fraction (LVEF, 50±7%), and increased fibrosis (5.2±0.4%) and apoptosis (3.8±0.6%). CR was obtained by measuring LV coronary blood flow at baseline and with adenosine (160 microg/kg/min) induced hyperemia using high resolution ultrasound (Vevo 770). CR was significantly decreased in beta
2
-AR Tg (1.8±0.2) compared with wild type (WT) (3.0±0.3, n=6). The next goal was to determine if the rescue of the beta
2
-AR Tg cardiomyopathy resulted in a rescue of reduced CR. Since adenylyl cyclase type 5 knockout (AC5 KO) mice are protected against catecholamine stress and the development of heart failure (HF), and exhibit enhanced CR (3.7±0.4, n=5), we hypothesized that mating these mice with beta
2
-AR Tg might rescue the cardiomyopathy. Older (16–17 months, n=4) bigenic mice (beta
2
-AR Tg x AC5 KO) demonstrated a rescue of cardiomyopathy, as reflected by normalized LVEF (71±1%) and levels of apoptosis (0.11±0.01%) and fibrosis (1.09±0.33%), similar to WT. In these mice, CR was also normalized (3.4±0.6). Reduced CR in beta
2
-AR Tg cardiomyopathy is similar to that observed in large mammalian models, and in patients with cardiomyopathy. Thus, this mechanism is important to consider in mouse models of cardiomyopathy, in general, and also in particular for the mechanism of the rescue of the beta
2
-AR Tg cardiomyopathy by AC5 KO.
Abstract 5508: PSGL-1-expressing CD4 T Cells Induce Endothelial Injury in Perimenopausal Women with Low Estrogen Levels
Authors: Gomita Keiko , Sato Kayoko , Kitamura Kazutaka , Yoshida Masayuki , Hagiwara Nobuhisa
Published: July 2021

Background:
Atherosclerosis develops rapidly in postmenopausal women, and plaque erosion induces acute coronary syndromes (ACS) in perimenopausal women (PMW). However, the causal relationship between low estrogen levels and the mechanism of plaque erosion in PMW is not fully resolved. We examined whether PWM have historical T cell dysfunction related to adhesion molecules, and whether they experience accelerated plaque erosion as a result.

Methods and Results:
Fresh CD4 T cells were isolated from 62 PMW (mean age 54.0±5.0 years) and 69 women with regular menstruation cycles (NC, mean age 33.0±8.0 years). Estrogen levels were lower in PMW than in NC (P<0.0001). Measurements of DAPI-binding nuclear protein fragmentations showed that CD4 T cells from PMW induced significant human umbilical vein endothelial cells (HUVEC) apoptosis compared to CD4 T cells from NC (P<0.002). Furthermore, CD4 T cells from PMW had high T cell activation marker CD69 (P<0.001) and expressed strong p-selectin glycoprotein ligand-1(PSGL-1) and integrinβ
2
but not L-selectin and integrin
α
M by FACS (P<0.0001, P<0.04, respectively). Estrogen levels correlated negatively with PSGL-1 (R=0.448, P<0.02) and integrinβ
2
(R=0.442, P<0.03), and negatively with CD4 T cell-induced HUVEC apoptosis (R=0.524, P<0.03). Rolling and adhesion of CD4 T cells to activated HUVEC under laminar flow showed strong correlation with HUVEC apoptosis (R=0.717, P<0.03 and R=0.562, P<0.03, respectively). Anti-PSGL-1 Ab inhibited both CD4 T cell-rolling and CD4 T cell-adhesion to activated HUVEC (P<0.05, P<0.009, respectively), but anti-CD18 Ab only affected cell-adhesion (P<0.006). Finally, we included anti-PSGL-1 Ab and anti-CD18 Ab in the apoptosis assay due to investigate whether the CD4 T cell-mediated apoptosis of endothelial cells was dependent on PSGL-1. Anti PSGL-1Ab treatment reduced CD4 T cell-induced apoptosis of endothelial cells (P=0.001) while anti integlinβ
2
Ab did not.

Conclusions:
From these results, we concluded that PSGL-1-expressing CD4 T cells in PMW with low estrogen levels is a factor that contributes to the acceleration of atherosclerosis and plaque erosion.
Abstract 5521: Activation of Farnesoid X Receptor (FXR) Reduces Atherosclerosis in LDLRKO and apoEKO Mice
Authors: Hartman Helen B , Harnish Douglas C , Evans Mark J
Published: July 2021

The nuclear hormone receptors farnesoid X Receptor (FXR) and small heterodimer partner (SHP) regulate bile acid synthesis and cholesterol homeostasis in a complex fashion. In regard to atherosclerosis, FXR repression of bile acid synthesis has been proposed to be either detrimental due to reduced elimination of cholesterol or beneficial due to decreased cholesterol absorption. FXRKO mice have failed to resolved this question, with atherosclerotic lesions in FXRKO mice either enhanced (apoE, FXRdKO mice) or reduced (LDLR, FXRdKO mice). Here we demonstrate that the synthetic ligand FXR-450 blocked Western diet-mediated increases in VLDL and LDL cholesterol in both male or female LDLRKO and male or female apoEKO mice. Correspondingly, FXR-450 strongly reduced lesion development in female and male ApoEKO mice fed a Western diet. To determine if FXR-450 reduction of lipids and atherosclerotic lesion size is solely mediated by induction of SHP and repression of bile acid synthesis, LDLRKO/SHPKO and ApoEKO/SHPKO were similarly analyzed. In female LDLRKO/SHPKO or female apoEKO/SHPKO, FXR-450 no longer reduced VLDL or LDL cholesterol. Surprisingly, FXR-450 still reduced VLDL and LDL cholesterol in male LDLRKO/SHPKO and male apoEKO/SHPKO. Further FXR-450 significantly reduced lesion development in male apoEKO/SHPKO mice. These results demonstrate activation of FXR has highly beneficial effects on plasma lipids and lesion formation in multiple mouse atherosclerosis models. Further, while induction of SHP appears to be the predominant protective mechanism of FXR activation in female mice, additional mechanisms may play a role, particularly in male mice.
Abstract 5535: In Vivo Activation Of Myocardial At2 Receptor Expression By Endogenous No And Pentaerythritol Tetranitrate
Authors: Dao Thao-Vi V , Balz Vera , Oppermann Marc , Suvorava Tatsiana , Kojda Georg
Published: July 2021

There are many vascular signaling interactions between the vascular endothelial NO system and the renin-angiotensin-system. We hypothesized that endothelial NO might impact on the expression of angiotensin (AT) type 1 (AT1) and type 2 (AT2) receptors. We generated mice with an endothelial-specific overexpression of endothelial NO
•
-synthase (eNOS) using the Tie-2 promotor. Two of these lines were characterized by eNOS-Western blot analyses and blood pressure measurements in comparison to transgene negative controls. In addition, C57Bl/6 mice were fed with either 6 or 60 mg pentaerythritol tetranitrate (PETN) for 4 weeks and characterized as well. Analysis of line 1 of transgenic eNOS mice (1-eNOS) showed a 2.3±0.15 fold higher aortic expression of eNOS and a reduction of blood pressure to 109.6±2.0 mmHg (P<0.01, n=4–6). Line 2 of transgenic eNOS mice (2-eNOS) showed a 3.3±0.3 fold higher aortic expression of eNOS and a reduction of blood pressure to 105.0±3.0 mmHg (n=6, P<0.01). Treatment of 2-eNOS with the NOS-inhibitor L-nitroarginine (L-NAME) for 30 days inhibited the difference in blood pressure suggesting that this effect is due to increased bioavailability of endogenous nitric oxide (NO). In lungs and left ventricular myocardium of 2-eNOS the expression of AT1 receptors was similar to the controls (121.6±14.77%, and 100.1±12.43%, respectively, n=8, P>0.05). In striking contrast, the expression of AT2 receptors was increased by endothelial overexpression of eNOS in a gene-dose-dependent manner in the myocardium. In 1-eNOS the increase was 134.3±10.66% (P<0.05) and in 2-eNOS the increase was 177.7±20.93% (P<0.05). In lung tissue the increase of AT2 receptor expression in line 2 amounted to 139.3±13.73% (P<0.05) and to 131.8±.7 in line 1 of eNOS transgenic mice suggesting a vascular rather than a cardiomyocyte effect. Furthermore, preliminary experiments showed a significant increase of AT2 receptor expression in myocardial tissue of PETN-fed mice and a significant decrease in myocardial tissue of L-NAME-fed mice (respectively, n=4, P<0.05). These results show that NO can upregulate vascular AT2 receptor expression in the lung and in the myocardium in-vivo. This newly discovered regulation might contribute to vasoprotective effects of NO.
Abstract 5542: Role of Scavenger Receptor BI in Sepsis
Authors: Guo Ling , Smart Eric , Li Xiangan
Published: July 2021

Scavenger receptor BI (SR-BI) is a well-established HDL receptor that regulates plasma HDL cholesterol levels by selectively uptake cholesterol ester from HDL. We recently reported a novel function of SR-BI namely protection against endotoxin-induced animal death. Using a lipopolysaccharides (LPS) induced endotoxic animal model, we found that LPS challenge induced 90% fatality of SR-BI null mice while all the wild type littermates survived, indicating that SR-BI plays a pivotal role in protection against LPS toxicity in vivo. In the current studies, we determined whether SR-BI plays a protective role in sepsis using an established septic animal model (cecal ligation and puncture, CLP). We demonstrate that CLP treatment induced 100% fatality of SR-BI null mice (n=20) whereas only 21% fatality of wild type littermates (n=27) and 36% fatality of heterozygous littermates (n=30). Moreover, SR-BI null mice exhibited exaggerated and prolonged inflammatory response as shown by elevated proinflammatory cytokine levels in serum upon CLP treatment compared to wild type littermates. Further studies revealed that the plasma LPS levels were significantly higher in SR-BI null mice compared to wild type control mice, indicating that SR-BI is required for LPS clearance in sepsis. Our findings clearly demonstrate that SR-BI is a critical protective modulator in sepsis in mice, which may reveal a novel target for the intervention of sepsis.

(Supported by grant from American Heart Association 0530241N)

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 5546: Anti-Inflammatory Effects of Nicotinic Acid: Suppression of Fractalkine, RANTES, and MCP-1 and Upregulation of Adiponectin in 3T3-L1 Adipocytes
Authors: Digby Janet E , Dyar Oliver J , McNeill Eileen , Greaves David R , Choudhury Robin P
Published: July 2021

Introduction: The atheroprotective effects of nicotinic acid (NA) may be mediated through well-defined effects on plasma lipoproteins. However, a major site of action for nicotinic acid is adipose tissue, via the G-protein coupled receptor, GPR109A. Since, adipose tissue is an active secretory organ that contributes both positively and negatively to the systemic inflammatory process associated with cardiovascular disease, we hypothesized that: NA would act directly upon adipocytes to alter the expression of pro-inflammatory chemokines, and the anti-inflammatory adipokine adiponectin.

Methods and Results: 3T3-L1 pre-adipocytes were differentiated into adipocytes using insulin and dexamethasone (10
−7
M) for 10 days. Adipocytes were incubated with NA (10
−6
M to 10
−3
M) under basal conditions, and/or with TNFα (1.0 ng/ml for 4 hours) to provoke inflammation. Quantitative real-time PCR for mRNA showed increased fractalkine (CX3CL1) (9-fold ± 3.3:
P
< 0.01), monocyte chemoattractant protein-1 (MCP-1) (24-fold ± 1.2:
P
< 0.001), and RANTES (CCL5) (500-fold ± 55: P < 0.001) in response to TNFα treatment (n=6). The addition of nicotinic acid (10
−4
M) to TNFα treated adipocytes attenuated expression of CXCL3 (50 ±12%: P < 0.01); MCP-1 (50 ± 6%:
P
< 0.01) and CCL5 (70 ± 3%:
P
< 0.01). By contrast, NA, (10
−6
M to 10
−3
M) increased in a dose-dependent manner mRNA of the atheroprotective hormone adiponectin (3- to 5-fold n=6,
P
<0.01 after 4 hours) and protein expression (2-fold) after 24 hours (n=4,
P
< 0.05).

Conclusions:

NA directly suppresses pro-atherogenic chemokines and promotes secretion of atheroprotective adiponectin by adipocytes.

This is the first report of fracktalkine expression by adipose tissue.

Since adipose tissue has the potential to contribute to both systemic and local (perivascular) inflammation associated with atherosclerosis our results suggest a new “pleiotropic” role for NA.
Abstract 5553: Hypercholesterolemia Induced Overexpression of Caveolin-1 and LOX-1 Downregulates HO-1/HSP-90 Mediated e-NOS Activation: A Novel Therapeutic Strategy for the Improvement of Left Ventricular Function
Authors: Penumathsa Suresh Varma , Koneru Srikanth , Maulik Gautam , Maulik Nilanjana
Published: July 2021

Hypercholesterolemia (HC) related decrease in eNOS phosphorylation & endothelial dysfunction may account for impaired angiogenesis and subsequent increased ventricular remodeling. Over expression of Caveolin-1 (Cav-1) and Lectin-like oxidized LDL receptor (LOX-1) has been demonstrated during HC but the mechanism needs to be elucidated. To investigate this we randomized the rats into control (normal diet) and HC (5% high cholesterol diet for 8 weeks). The cholesterol, triglycerides & LDL levels were increased & the HDL levels decreased in HC compared to control. After the experimental diet period the rats were subjected to Left Anterior Descending Artery (LAD) ligation. We evaluated the expression of Cav-1, LOX-1, Heme Oxygenase (HO-1), HSP-90, phospho(p)-Akt & p-eNOS in HC and control. Significant increase in Cav-1, LOX-1 (2, 1.8 fold) & decrease in HO-1, HSP-90, p-Akt, p-eNOS & VEGF (0.5, 0.6, 0.4, 0.5 & 0.6 fold) was observed in HC compared to control. The LV functional reserve was evaluated by measuring the ejection fraction (EF), fractional shortening (FS) & LV internal diameter (LVID) after 30 days of LAD ligation. Significant increase in LVID ( 8.6 vs 7) and decrease in EF (39 vs 53%), FS (20 vs 28%), LVID (2 vs 2.7mm) as well as capillary (1888 vs 2424) & arteriolar density (1.5 vs 2.5) counts/mm2 was observed in HC compared to control. Earlier we have reported that over expression of HO-1 mediates eNOS activation & cardioprotection in MI model. To investigate the mechanism involved in HO-1 mediated cardioprotection we generated cardiac specific HO-1 over expressed transgenic (Tg) mice. Immunohistochemical analysis of HO-1 Tg mice has clearly demonstrated decreased Cav-1-eNOS interaction. Immunoblot analysis has shown to decrease Cav-1 (0.6 fold) & increased HSP-90, p-Akt, p-eNOS & VEGF expression (1.5, 1.6, 1.4 & 1.5 fold) as compared to control. These findings demonstrated that HO-1 over expression regulates HSP-90 & Cav-1 for eNOS activation. In conclusion, we demonstrate a novel mechanism of HO-1/HSP-90 mediated Cav-1-eNOS regulation leading to increased neovascularization & reduced ventricular remodeling during HC for the regression of clinical complications which would be crucial for cardiovascular drug therapy.