bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 631 ADAM12 Is An Important Regulator Of Myocyte Apoptosis Induced By β-adrenergic Receptor Stimulation
Authors: Xi Hang , Rafiq Khadija , Hanscom Marie , Seqqat Rachid , Malinin Nikolay L , Griswold-prenner Irene , Sabri Abdelkarim
Published: July 2021

ADAM (A Disintegrin And Metalloprotease)12 is a member of a family of cell surface proteins with protease and cell-binding activities. Recent work showed ADAM12 up-regulation in human heart failure. However, the activation mechanisms of ADAM12 in the heart are obscure. We hypothesized that β-adrenergic receptors (AR) stimulation regulates ADAM12 activation in neonatal rat ventricular myocytes (NRVMs) in-vitro and after injection of isoproterenol (ISO) in-vivo. Wistar rats received a single injection of ISO (5 mg/kg) and were sacrificed 6, 24 and 72 hrs later. In comparison with controls, left ventricular function was impaired in rats 24 hrs after ISO injection and started to improve at 72 hrs. The fraction of myocytes undergoing apoptosis peaked 24 hrs after ISO injection and declined thereafter. ADAM12 protein was reduced in hearts from ISO treated animals at 6 hrs, pointing to a possible increase in ADAM12 proteolytic activity. However, both ADAM12 expression and activation were significantly up-regulated at 24 and 72 hrs after ISO injection. We therefore assessed whether ADAM12 activation was involved in myocyte apoptosis secondary to excess exposure of catecholamine. Acute stimulation with ISO (10 μM, 30 min to 3 hrs) induced accumulation of ADAM12 N-terminal cleavage product in conditioned medium, demonstrating activation of the ADAM metalloprotease activity. However, chronic stimulation with ISO for 24 hrs and 48 hrs significantly increased both ADAM12 expression and secretion. This ISO-induced ADAM12 expression/activation was mediated through β
1
-AR stimulation and was dependent on intracellular calcium elevation and protein kinase C activation. Adenoviral expression of an ADAM12 protease-deficient mutant (ADAM12DeltaMP) blocks β-AR-induced myocyte apoptosis, while transduction of NRVMs with adenovirus harboring ADAM12 significantly increased myocyte apoptosis. These data suggest that ADAM12 is a regulator of myocyte apoptosis induced by β-AR in NRVMs and may play an important autocrine role in mediating the effects of β-AR on myocardial remodeling.
Abstract 638: A Novel Mutation In The
KCR1
Gene May Influence Susceptibility To The Acquired Long QT Syndrome
Authors: Hayashi Kenshi , Ino Hidekazu , Fujino Noboru , Uchiyama Katsuharu , Masuta Eiichi , Funada Akira , Sakamoto Yuichirou , Tsubokawa Toshinari , Yasuda Toshihiko , Kanaya Honin , Kim Min Young , Kupershmidt Sabina , Higashida Haruhiro , Yamagishi Masakazu
Published: July 2021

Introduction
: Acquired QT interval prolongation and torsade de pointes can be caused by exposure to certain drugs, electrolyte imbalance, and myocardial ischemia. Acquired LQTS is more prevalent than the congenital LQTS and represents a significant problem for public health. Previous reports showed that K
+
channel regulator 1 (KCR1), an α glucosyltransferase, diminishes HERG drug sensitivity , and that a
KCR1
polymorphism, Ile447Val, is associated with protection against drug-induced LQTS in humans. However, few studies address the effects of genetic changes in
KCR1
and we screened the coding region of
KCR1
in Japanese patients with the acquired LQTS.

Hypothesis
: We hypothesized that mutations or polymorphisms in
KCR1
may influence the development of acquired LQTS.

Methods:
The genomic DNA of 70 unrelated patients with congenital LQTS, 41 unrelated patients with acquired LQTS, and 130 unrelated control subjects was screened by PCR-SSCP, PCR-RFLP, and direct sequencing. Appropriate sequence changes were introduced into subcloned KCR1 cDNA using site-directed mutagenesis, and HERG plus WT or mutant KCR1 cDNA was transiently transfected into CHO-K1 cells. HERG current was recorded using whole cell patch-clamp techniques and drug block of HERG current was assessed.

Results
: We identified a novel mutation in
KCR1
, Glu33Asp in a Japanese acquired LQTS patient. Similar to what we reported previously, WT KCR1 diminished the susceptibility of HERG current to dofetilide (50 nM). After 15 min of continuous pulsing, 74±7% current remained in the presence of WT KCR1 vs 54±5% in the absence of KCR1. In contrast, 48±6% of HERG current remained in the presence of Glu33Asp KCR1 which was comparable to that of HERG alone. Significantly, we found that unlike WT KCR1, Glu33Asp KCR1 is no longer able to complement the genetic defect in α glucosyltransferase activity in yeast strain YG649, suggesting that enzymatic function of Glu33Asp KCR1 is compromised. We found no evidence for the Ile447Val KCR1 in Japanese LQTS patients and control subjects, which is present at an allele frequency of 7% allele frequency in a control population of the United States.

Conclusion:
These data suggest that the Glu33Asp
KCR1
genetic change may predispose to the development of acquired LQTS.
Abstract 678: Administration of Intravenous High Density Lipoprotein Leads to Acute Changes in Human Atherosclerotic Plaque In Vivo
Authors: Shaw James A , Blombery Peter , Lyon Stuart , Sviridov Dimitri , Bobik Alex , Kanellakis Peter , Dart Anthony M
Published: July 2021

Introduction
High density lipoprotein (HDL) is an inverse predictor of cardiovascular events. Recent studies have shown a reduction in plaque volume and change in plaque ultrasound characteristics after 4 infusions of reconstituted HDL (r HDL CSL-111) given weekly over 1 month. Whether rHDL infusion leads to acute changes in plaque composition in vivo is not known.

Methods
Patients with symptom limiting claudication planned for percutaneous superficial femoral artery (sfa) revascularisation were randomised in a double blind manner to either placebo or IV r HDL (80mg/Kg given over 4 hrs). Prior to the treatment bloods were taken for measurement of lipid levels and inflammatory markers. 5–7 days following the infusion patients returned, repeat bloods were taken for lipids and inflammatory markers and revascularisation was performed including atherectomy (Foxhollow CA) to excise plaque from the sfa.

Results
10 patients (9 male) average age 65 ± 10 yrs (mean ± sd) were recruited. 7 had a history of documented coronary artery disease and all patients were on aspirin and 8 on statins. 6 of the patients received r HDL and 4 placebo. There was a significant increase in Ankle brachial index in both groups after atherectomy (0.57 ± 0.22 to 0.92 ± 0.12 (p < 0.01) in HDL gp, 0.58 ± 0.1 to 0.93 ± 0.05 (p < 0.01) in placebo gp.) There were no significant differences in LDL, HDL or CRP after the infusion in either group. In the SFA plaque there was significantly less Vascular Cell Adhesion Molecule 1(VCAM1) staining (23 ± 8.7% vs 41.8 ± 2.7% p < 0.05) and evidence of oxygen free radicals (31.5 ± 12.0 vs 90.3 ± 24.3 p < 0.05 (arbitrary units)) in the HDL treated subjects compared to placebo. The amount of lipid in the plaque tended to be lower in the HDL group.

Conclusion
Intravenous infusion of 1 dose of reconstituted HDL leads to acute changes in plaque with a reduction in measures of inflammation and oxidised free radicals in subjects with peripheral vascular disease. These changes may explain the presumed cardioprotective effects of HDL.
Abstract 738: Role of the Pulmonary Veins in Experimental Paradigms of Remodeling- Basic Science Associated Atrial Fibrillation
Authors: Sakabe Masao , Lemola Kristina , Chiba Katsuyoshi , Katsouras Grigorios , Shiroshita-Takeshita Akiko , Nattel Stanley
Published: July 2021

Background
: Pulmonary vein (PV) activity is crucial in some forms of clinical atrial fibrillation (AF), particularly paroxysmal lone AF. The precise importance of PVs in AF associated with structural heart disease is less clear. Some evidence suggests that PVs may be important for AF in experimentally-remodeled atrial substrates, e.g. atrial-tachycardia remodeling (ATR) and congestive heart failure (CHF)-induced structural remodeling, but contradictory findings also exist. This study assessed the role of PVs in canine remodeling-associated AF by evaluating the changes induced by PV-LA disconnection via encircling epicardial ablation.

Methods:
AF was induced before and after complete isolation of all PVs in dogs with:

ATR induced by atrial tachypacing (400 bpm x 1 wk; with AV block and 80 bpm ventricular pacing to control ventricular rate, n=5); and

CHF induced by ventricular tachypacing (VTP, 240 bpm x 2 wks, n=7).

Electrophysiological measurements and AF mapping with 240 unipolar atrial electro-grams in both atria and all PVs were also obtained before and after PV isolation.

Results
ATR reduced atrial ERPs and ERP rate adaptation. The shortest AF cycle length (AFCL) in each ATR dog was recorded from PVs (mean 84±3 ms), but the mean AFCL in each of the PVs (range 97–103 ms) was not different from the mean AFCL at LA sites (98 –103 ms). PV ablation did not affect AF duration in ATR dogs (mean 284±265 pre- vs. 304±225 s post-ablation, P=NS), nor did it significantly alter RA or LA AFCL. In CHF dogs, ERPs and ERP rate adaptation were preserved. The shortest AFCL was recorded in PVs in 2 dogs (108,112 ms) and in the left side of Bachmann’s bundle in 3 (97–117 ms). PV ablation failed to alter AF duration in CHF dogs (mean 778±203 pre- vs. 644±206 s post-ablation), and increased AFCL in RA and LA slightly (10 –15%) but not significantly.

Conclusions
: Uncoupling of the PVs from the LA fails to significantly affect the AF substrate in 2 clinically-relevant animal paradigms of AF-promoting atrial remodeling. These findings suggest that in the presence of a favorable atrial substrate for AF the PVs are not needed for AF maintenance, and are consistent with clinical observations that complete PV isolation may not be essential for effective ablation therapy of all groups of AF patients.
Abstract 182: Dosing Time of Angiotensin Converting Enzyme Inhibitor is Important for Protecting Organ Damage in Hypertension
Authors: Tsujino Takeshi , Matsumoto Mika , Naito Yoshiro , Chikako Yoshida , Wakabayashi Kana , Sakoda Tsuyoshi , Ohyanagi Mitsumasa , Masuyama Tohru
Published: July 2021

Introduction: There is a difference in the pattern of the diurnal variations of the aortic and cardiac mRNA expression of renin-angiotensin system between spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats. Angiotensin II-dependent upregulation of gene expression was enhanced only in the daytime in the aorta of SHR.

Hypothesis: Dosing time of angiotensin converting enzyme inhibitor may influence its organ protective effect in genetic hypertension.

Methods: 10-week-old stroke prone spontaneously hypertensive rats (SHR-SP) were kept in a room in which a 12-h light/12-h dark cycle was maintained [lights on at 7 a.m.; Zeitgeber time (ZT) 0]. They were divided into three groups:

the control group(C group: n=28)was given vehicle

the morning group (M group: n=23) was given captopril (30mg/kg) at ZT0

the evening group (E group: n=23) was given captopril (30mg/kg) at ZT12.

After 1 week of treatment, ACE activities of heart and kidney were measured 6 hours after drug-administration (n=8 for each group). After 7 weeks of treatment, urinary protein excretion, creatinine clearance, and heart weight/body weight ratio (HW/BW, mg/g) were examined. Five rats for each group underwent long-term implantation of a device that telemetrically monitors blood pressure (BP).

Results: Mean BP elevation and premature death were equally prevented in the M group and E group compared to the C group. BP was lower in the day and higher in the night in the M group (day time BP vs. night time BP: 145.9 ± 10.3 vs. 163.7 ± 19.2 mmHg, p<0.01), while BP was stable throughout a day in the E group (day time BP vs. night time BP: 152.2 ± 14.2 vs. 153.5 ± 14.7 mmHg, n.s.). After 1 week of treatment, cardiac and renal ACE activities were lower in the M group than in the E group (cardiac ACE activities: 0.639 ± 0.130 vs. 0.842 ± 0.085 nmol/mg tissue/hr, p<0.01; renal ACE activities: 0.402 ± 0.145 vs. 0.728 ± 0.165 nmol/mg tissue/hr, p<0.001). After 7 weeks of treatment, creatinine clearance was higher and HW/BW was lower in the M group than in the E group (2.93± 0.57 vs. 2.01± 0.43, p<0.005; 4.33± 0.19 vs. 4.63 ± 0.36 g/kg, p<0.05).

Conclusions: Morning administration of captopril was more effective than evening one in terms of the strength of ACE inhibition and organ protection in SHR-SP.
Abstract 796: Reduction of Cardiac Calsequestrin Increases Sarcoplasmic Reticulum Ca
2+
-Leak Independent of Luminal Ca
2+
and Triggers Ventricular Arrhythmias in Mice
Authors: Chopra Nagesh , Kannankeril Prince J , Yang Tao , Hlaing Thinn , Holinstat Izabela A , Ettensohn Kristen , Pfeifer Karl , Akin Brandy , Jones Larry R , Franzini-Armstrong Clara , Knollmann Bjorn C
Published: July 2021

Deletion of cardiac calsequestrin (Casq2) causes VT in mice akin to
CASQ2
mutations. However, other sarcoplasmic reticulum (SR) proteins (triadin-1/junctin) are also reduced in homozygous (
Casq2
&
minus;/−
) mice and may contribute to their arrhythmia phenotype. Furthermore, it is unknown if Casq2 regulates SR Ca
2+
-release directly or indirectly by buffering SR luminal Ca
2+
. To address both questions, we examined heterozygous (
Casq2

+/
−

) mice with a 25% reduction in Casq2, but no decrease in other SR proteins.

Results:

Casq2

+/
−

mice (n=35) challenged with isoproterenol displayed 3-fold higher rates of ventricular ectopy than
Casq2
+/+
mice (n=31; p<0.05). Programmed stimulation induced significantly more VT in
Casq2

+
/
−

mice. Field-stimulated Ca
2+
transients, cell shortening, L-type Ca
2+
current and SR volume were not different between the 2 groups. However, in presence of isoproterenol, SR Ca
2+
leak was significantly higher in
Casq2

+
/
−

myocytes (
Casq2
+/−
0.18±0.02 F
ratio
vs Casq2
+/+
0.11±0.01 F
ratio
, n=57, 60; p=0.01), resulting in a significantly higher rate of spontaneous SR Ca
2+
-releases/triggered beats (
Casq2
+/−
0.38±0.07
vs Casq2
+/+
0.12±0.06, n=47, 34; p=0.007). Luminal SR Ca
2+
was not significantly different between the 2 groups. However, on plotting SR Ca
2+
leak as a function of luminal SR Ca
2+,
leak remained significantly higher in
Casq2
+/−
myocytes (figure

).

Conclusion:

Even modest reductions in Casq2 increase SR Ca
2+
leak and cause VT susceptibility under stress, which could explain increased risk for VT in
CASQ2
+/−
mutations. The underlying mechanism is likely the direct modulation of SR Ca
2+
-release by Casq2 rather than decreased Ca
2+
buffering in the SR lumen.
Abstract 798: Ca
2+
Sparks are Modulated by the Mitochondrial Energy State
Authors: Zhou Lufang , Aon Miguel A , O’Rourke Brian
Published: July 2021

Mitochondria produce reactive oxygen species (ROS) but are also a target of oxidative stress that can induce rapid oscillations of mitochondrial inner membrane potential (ΔΨ
m
). The proximity of mitochondria to SR Ca
2+
release sites suggests that the mitochondrial energy state will determine the local redox and energetic status of the Ca
2+
handling subsystem. Because ryanodine receptors, SERCA, and L-type Ca
2+
channels are all sensitive to ATP, Mg
2+
and thiol oxidation, we investigated whether laser flash-triggered mitochondrial ΔΨ
m
oscillations influence resting Ca
2+
spark frequency in fluo-4 + TMRE-loaded cardiomyocytes using two-photon microscopy to monitorΔΨ
m
, cytosolic [Ca
2+
], and [NADH]. The correlation between resting Ca
2+
spark frequency and ΔΨ
m
was analyzed over several cycles of ΔΨ
m
oscillation in each cell. Notably, Ca
2+
spark frequency increased with ΔΨ
m
depolarization and decreased withΔΨ
m
repolarization, indicative of close coupling between energetics and Ca
2+
cycling (See Figure

). Moreover, spontaneous Ca
2+
waves were often observed when mitochondria were depolarized but not whenΔΨ
m
recovered. There were no significant effects on Ca
2+
spark durations or rise times. In conclusion, the results show that stress-induced mitochondrial depolarization (involving NADH oxidation, ROS release, and depletion of glutathione) leads to increased Ca
2+
spark frequency, most likely as a result of modification of the local redox status near the dyad. These findings provide important insights into the connection between metabolism and Ca
2+
cycling in the heart with relevance to the mechanism of contractile and electrical dysfunction in cardiac disease states.
Abstract 805: Differential Regulation Of Cytokine-induced Alternative Splicing Of The TF Gene By Serine/Arginine Rich Protein Kinases
Authors: Rauch Ursula , Eisenreich Andreas , Poller Wolfgang , Schultheiss Heinz-Peter
Published: July 2021

Background: Higher eukaryotes control gene expression and increase protein diversity by alternative splicing of pre-mRNA. The Cdc2-like kinase (Clk) family, DNA topoisomerase I (DNA topo I) or Akt kinase are involved in splicing control by regulating the phosphorylation state of serine/arginine rich (SR) proteins. We recently showed that alternatively spliced human tissue factor (asHTF), a soluble isoform of tissue factor (TF), the primary initiator of coagulation, is expressed in HUVECs in response to inflammatory cytokines. This study investigated the role of Clks, DNA topo I and the PI3K-Pathway in regulation of TF-splicing in TNF-α induced HUVECs.

Methods: HUVECs were incubated with inhibitors of Clks, DNA-topo I or PI3K and were then stimulated with TNF-α. The SR protein phosphorylation state was determined 2 min post induction. The full length (fl) TF and asHTF mRNA were assessed 60 min post induction by Real-Time PCR. Proteins were measured 5 and 8 hours after stimulation by Western blots and the cell thrombogenicity was analyzed via a chromogenic assay.

Results: TNF-α inceased the mRNA expression of asHTF and flTF in HUVECs. The Clk-inhibitor completely inhibited the TNF-α induced expression of asHTF and reduced flTF by 30 %. Inhibition of DNA topo I increased asHTF expression and reduced the flTF expression. Inhibition of the PI3K/Akt-pathway had no effect on TF mRNA expression. Reduced Clk-inhibition the TF activity by 50 % whereas DNA topo I inhibition significantly decreased the procoagulant TF activity 8 hours post TNF-α induction. The Clk- and DNA-topo I-inhibitors altered the SR-protein phosphorylation pattern post TNF-α-induction. Additionally resulted inhibition of Clks in the generation of a third TF mRNA-splice variant, TF-A.

Conclusion: Selective inhibition of Clks or DNA topo I leads to alterations of SR-protein phosphorylation and affects the differential expression of TF isoforms, thereby modulating the thrombogenicity of HUVECs. The inhibition of Clks contributes to the generation of a third TF splice variant. The inhibition of these kinases gives new insights into the regulation of the TF gene splicing process, which may result in new therapeutic strategies for modulating cellular thrombogenicity.
Abstract 806: Protein Disulfide Isomerase (PDI) signals Tissue Factor (TF)-dependent Fibrin formation in vivo
Authors: von Bruehl Marie-Luise , Grahl Lenka , Konrad Ildiko , Lorenz Michael , Schulz Christian , Reinhardt Christoph , Manukyan Davit , Altmann Berid , Dlugai Silke , Mackman Nigel , Engelmann Bernd , Massberg Steffen
Published: July 2021

Background: Pathological fibrin formation is a predominant cause for arterial thrombosis, triggering myocardial infarction and stroke. Tissue factor (TF), which is expressed by vascular endothelium and cell-derived microparticles, is a central trigger of intravascular fibrin generation. In intact blood vessels, TF is only minimally active, and the coagulation system is down-regulated. In contrast, TF is rapidly stimulated following endothelial disruption. However, the molecular mechanisms that trigger fibrin formation via regulation of TF activation are largely undefined.

Methods and Results: Here, we have identified a novel pathway that leads to TF activation in vitro and contributes to intravascular coagulation in vivo. We show that the protein disulfide isomerase (PDI) induces disulfide formation of the Cys186/Cys209 pair located in the extracellular domain of TF. In vitro, PDI-mediated disulfide formation led to profound activation of TF. In vivo, we found that PDI is not present in the intact vessel wall, but becomes abundantly expressed following vessel damage. We show that injured smooth muscle cells and adherent/activated platelets are the major cellular sources of PDI at sites of vascular injury. We then assessed fibrin formation following injury of the common carotid artery using intravital microscopy. We observed that PDI-blockade strongly reduced fibrin formation by approximately 45– 60%. Likewise, intravenous infusion of PDI significantly enhanced TF-dependent fibrin formation. Together, this implies that PDI initiates coagulation in vitro and also during arterial thrombosis in vivo. Notably, PDI did not act on platelets to trigger intravascular coagulation. Correspondingly, PDI blockade also inhibited fibrin formation, when platelet adhesion was prevented using a function-blocking anti-GPVI antibody.

Conclusion: These findings reveal for the first time in vivo that the thiol isomerase PDI is a major molecular trigger of blood coagulation, which is exposed after vessel injury and converts TF into its functionally active form. Hence, PDI represents a promising novel target for the treatment of pathologic fibrin formation in patients with thrombotic disorders.
Abstract 811: Long-term Patency of a Small-diameter Vascular Graft Made of Fibroin, a Silk-based Biodegradable Material
Authors: Enomoto Soichiro , Sata Masataka , Sumi Makoto , Asakura Tetsuo , Nagai Ryozo
Published: July 2021

Background: There is an increasing need for vascular grafts in the field of surgical revascularization. However, smaller vascular grafts made from synthetic biomaterials, particularly smaller than 5 mm in diameter, are associated with high incidence of thrombosis. Fibroin is a biodegradable protein derived from silk. Fibroin provides anti-thrombotic surface and serves as a scaffold for various cell types in tissue-engineering. In this study, we evaluate the potential of fibroin to generate artificial vascular prosthesis for small arteries.

Methods and Results: A small artificial vessel with three layers was woven from fibroin thread. Fibroin-based grafts (1.5 mm in diameter, 10 mm in length) were implanted into the abdominal aorta of male 10 to 14-week-old Sprague Dawley rats by end-to-end anastomosis. Polytetrafluoroethylene (PTFE)-based grafts were used as control. Patency of fibroin grafts at 1 year after implantation was significantly higher than that of PTFE grafts (85.1% vs. 30%, p<0.01). Endothelial cells and smooth muscle cells migrated into the fibroin graft early after implantation and organized endothelial and medial layers, as determined by anti-CD31 or anti-α-smooth muscle actin immunostaining. Vasa vasorum was also formed in adventitia. Sirius-red staining revealed that the content of collagen in fibroin grafts significantly increased at 3 months after implantation (1613.5 ± 246.3%, p<0.01) with decrease in fibroin fiber content (70.6 ± 4.6%, p<0.05). To investigate the origin of the cells of neointima and media, we performed bone marrow transplantation from GFP rats to wild-type rats. GFP-positive cells substantially contributed to organization of endothelium and smooth muscle layers in the fibroin grafts that had been implanted into the bone marrow chimeric rats.

Conclusion: A small-diameter fibroin-based vascular graft has significantly higher patency than a PTFE-based graft. Bone marrow-derived cells contribute to vascular remodeling after graft implantation. Fibroin might be a promising material to engineer vascular prosthesis for small arteries.
Abstract 825: Impaired ABCA-1 Mediated Cholesterol Efflux In Hypoxic Macrophages
Authors: Mick Stephanie L , Parathath Sajesh , Maitin Vatsala , Feig Jonathan E , Grauer Lisa , Joaquin Victor , Habiel David , Fisher Edward A
Published: July 2021

Atherosclerotic plaques contain large numbers of metabolically active cells that can be deprived of normal oxygen (0
2
) tension, with levels documented below 10mm Hg (~1% O
2
) in rabbit and human plaques (vs. normal tissue; 20 –70mmHg, 2.5%–9% O
2
). Hypoxia has been assumed, but not documented in mouse plaques. Hypoxia stabilizes transcription factor HIF-1α, the master regulator of the cellular response to hypoxia and also involved in reduction-oxidation and inflammatory pathways. Atherosclerotic plaques of apoE knockout mice were immunostained and areas + for HIF-1α as well as two downstream targets (VEGF and Glut-1) were present in increased levels in deeper plaque regions. We next investigated the effect of hypoxia on macrophage function
in vitro
by incubating murine J774 macrophages in 1% 0
2
in DMEM containing 15% FBS for 24 h and found a 30% increase in cellular total cholesterol (TC) associated with hypoxia. In cells loaded by cyclodextrin-cholesterol, a ~400% increase in TC was observed in hypoxic relative to normoxic cells. Assays using apoAI as a cholesterol acceptor showed that in 1% 0
2
there was a ~90% decrease in ABCA-1 mediated efflux relative to normoxia. In spite of this, ABCA-1 mRNA was increased ~2.5 fold in hypoxic vs. normoxic macrophages. ABCA-1 protein levels by Western analysis remained equal in the 2 conditions. However, by immunofluorescence and confocal microscopy, in sharp distinction to the plasma membrane and cytoplasmic distribution seen in normoxic cells, ABCA-1 localized mainly in a juxtanuclear location in hypoxic macrophages, suggesting impaired trafficking to the membrane.

Conclusions:

HIF-1α is present in murine plaques, suggesting that they have true hypoxic areas;

Hypoxia
in vitro
promotes macrophage cholesterol accumulation concurrent with ABCA-1 dysfunction; and

Hypoxia redistributes ABCA1 from the plasma membrane to the juxtanuclear region.
Abstract 831: Functional Analysis of S127 and D374 Residues of PCSK9: Distinct Hot Spots for Gain of Function
Authors: Fisher Timothy S , Pandit Shilpa , Wisniewski Douglas , Santoro Joseph C , Cubbon Rose M , Ha Sookhee , Cummings Richard T , Wright Samuel D , Sparrow Carl P , Sitlani Ayesha
Published: July 2021

Mutations within proprotein convertase subtilisin/kexin type 9 (PCSK9) are associated with dominant forms of familial hypercholesterolemia. Recent studies have shown that PCSK9 directly binds the LDL receptor (LDLR) and that addition of PCSK9 to cells promotes degradation of LDLR. Mutations associated with hypercholesterolemia (S127R and D374Y) bind the LDLR stronger and are more potent in decreasing LDL-uptake. Based on these data, we sought to better understand the mechanism by which mutations at both the S127 and D374 residues of PCSK9 affect PCSK9 function. A limited vertical scanning mutagenesis was done, resulting in the following mutants: S127(R, A, D, K, L, T) and D374(Y, A, E, F, K, L). Expression of several S127 alleles in HEK293 cells resulted in less efficient processing of PCSK9, with S127L severely defective in processing. In contrast, all D374 alleles tested had no effect on PCSK9 processing. In a cell-based functional assay of PCSK9 activity, both S127R and S127K proteins were significantly more potent (2– 4 fold) in decreasing LDL-uptake than wild-type PCSK9, while all other S127 mutants were similar to wild-type. Each D374 mutant tested was more potent than wild-type PCSK9 in reducing LDL-uptake (D374Y, F > A, L > E, K > wild-type). To determine if the potencies of S127 and D374 mutations in lowering LDL-uptake correlated with their ability to interact with the LDLR, each mutant was tested in a PCSK9-LDLR time-resolved FRET assay. Results indicate that potency in the cell-based LDL-uptake assay correlates with PCSK9’s binding affinity for the LDLR. Combination of S127R and D374Y was also found to have an additive effect in enhancing PCSK9’s ability to reduce LDL-uptake. Molecular modeling indicates that mutations at S127 and D374 which enhance PCSK9 function stabilize or destabilize the protein, respectively. In conclusion, these results suggest a model in which mutations at S127 and D374 residues modulate PCSK9’s ability to regulate LDLR function through distinct mechanisms.
Abstract 860: Phenotypic Characterization Of Murine And Human Cardiac-resident Progenitor Cells Isolated On Basis Of Aldehyde-dehydrogenase Activity
Authors: Spicher Albert , Meinhardt Andrea , Roehrich Marc-Estienne , Vassalli Giuseppe
Published: July 2021

Identification of stem cells based on hematopoietic stem cell (HSC) surface markers, such as stem cell antigen-1 (Sca-1) and the c-kit receptor, has limited specificity. High aldehyde-dehydrogenase (ALDH) activity is a general cellular property of stem cells shared by HSC, neural, and intestinal stem cells. The presence of cells with high ALDH activity in the adult heart has not been investigated.

Methods: Cells were isolated from adult mouse hearts, and from atrial appendage samples from humans with ischemic or valvular heart disease. Myocyte-depleted mouse Sca-1+, and lineage (Lin)-negative/c-kit+ human heart cells were purified with immunomagnetic beads. ALDH-high cells were identified using a specific fluorescent substrate, and sorted by FACS. Cell surface marker analysis was performed by flow cytometry.

Results: Myocyte-depleted mouse heart cells contained 4.8+/−3.2% ALDH-high/SSC-low and 32.6+/−1.6% Sca-1+ cells. ALDH-high cells were Lin-negative, Sca-1+ CD34+ CD105+ CD106+, contained small CD44+ (27%) and CD45+ (15%) subpopulations, and were essentially negative for c-kit (2%), CD29, CD31, CD133 and Flk-1. After several passages in culture, ~20% of ALDH-high cells remained ALDH-high. Myocyte-depleted human atrial cells contained variable numbers of ALDH-high cells ranging from 0.5% to 11%, and 4% Lin-negative/c-kit+ cells. ALDH-high cells were CD29+ CD105+, contained a small c-kit+ subpopulation (5%), and were negative for CD31, CD45 and CD133. After 5 passages in culture, the majority of ALDH-high cells remained ALDH-high.

Conclusions: Adult mouse and human hearts contain significant numbers of cells with high ALDH activity, a general cellular property that stem cells possess in different organs, and express stem cell markers (Sca-1 and CD34 in the mouse). The immunophenotype of cardiac-resident ALDH-high cells differs from that previously described for bone marrow ALDH-high HSC, and suggests that this cell population may be enriched in mesenchymal progenitors. Analysis of lineage differentiation potential of ALDH-high cells is in progress. ALDH activity provides a new, practical approach to purifying cardiac-resident progenitor cells.
Abstract 876: Combined Inhibition Of Ccl2, Cx3cr1 And Ccr5 Abrogates Ly6chi And Ly6clo Monocytosis And Almost Abolishes Atherosclerosis In Hypercholesterolemic Mice
Authors: Potteaux Stephane , Combadiere Christophe , Pezard Adeline , Riou Stephanie , Simon Tabassome , Rodero Mathieu , Esposito Bruno , Merval Regine , Proudfoot Amanda , Lehoux Stephanie , Tedgui Alain , Mallat Ziad
Published: July 2021

Monocytes are critical mediators of atherogenesis. Specialized chemokine/chemokine receptor pathways orchestrate the infiltration of specific monocyte subsets within atherosclerotic arteries. Interruption of individual chemokine pathways including CCL2/CCR2, CCL5/CCR5, CX3CL1/CX3CR1, CXCL8/CXCR2 or CXCL10/CXCR3, leads to significant but only partial inhibition of lesion development, whereas deficiency in other signals such as CXCL16 or CCR1 accelerates atherosclerosis. Evidence that particular chemokine pathways may cooperate to promote monocyte recruitment into atherosclerotic arteries is still lacking. Here, we show that in addition to the current prevailing paradigm accounting for the role of chemokines as modulators of monocyte trafficking between the blood and the vessel wall, chemokine-mediated signals critically determine the frequency of monocytes in the blood and the bone marrow under both non-inflammatory and atherosclerotic conditions. Particularly, CCL2, CX3CR1 and CCR5-dependent signals differentially alter CD11b+ Ly6G- 7/4hi (also known as Ly6Chi) and CD11b + Ly6G- 7/4lo (Ly6Clo) monocytosis. Combined inhibition of these pathways in hypercholesterolemic, atherosclerosis susceptible apolipoprotein E-deficient mice leads to additive reduction in bone marrow and circulating monocytes, associated with a marked and additive 90% reduction in atherosclerosis. Lesion size highly correlates with the number of circulating monocytes. Thus, signals mediated through CCL2, CX3CR1 and CCR5 critically determine the frequency of circulating monocyte subsets and thereby account for almost all macrophage accumulation into atherosclerotic arteries.
Abstract 198: Endothelin-1 and Phenylephrine Activate Global Protein Kinase D Similarly in Adult Cardiac Myocytes, but Differ Greatly in Nuclear versus Sarcolemmal Targeting
Authors: Bossuyt Julie , Martin Jody L , Hench Karl , Kunkel Maya T , Newton Alexandra C , Robia Seth L , Bers Donald M
Published: July 2021

Endothelin-1(ET-1) and phenylephrine (PE) both activate hypertrophic signaling in cardiac myocytes via nuclear export of class II histone deacetylase (HDAC), and protein kinase D (PKD) is critical in mediating this for HDAC5. However, we now find that in adult ventricular myocytes PE-induced HDAC5 nuclear export is more critically PKD-dependent than for ET-1. Here, we use novel FRET-based PKD activity reporter (DKAR) and GFP-tagged PKD (both in adenoviral vectors) expressed in adult rabbit cardiac myocytes, along with confocal and TIRF microcsopy to localize ET-1
vs
. PE-induced PKD activation and translocation. On-line PKD activity (sensed by DKAR FRET) was similarly activated by 100 nM ET-1, 10 μM PE or 300 nM PDBu (4.6 ± 0.4, 5.9 ± 0.7 or 5.6 ± 0.6 units) in adult myocytes overexpressing PKD. PKD-GFP expressed in myocytes is relatively uniform and cytosolic at rest (slightly higher at Z-lines), and largely non-nuclear (F
nuc
/F
cyto
0.5 ± 0.03). ET-1 caused rapid sustained PKD recruitment to the plasma membrane (46 ± 3% increase) and also at Z-lines, but only slow and modest PKD nuclear import (12 ± 8% rise in F
nuc
/F
cyto
). In contrast, PE more modestly increased membrane and Z-line recruitment (12 ± 0.9%), but caused more dramatic nuclear translocation (47 ± 4% rise in F
nuc
/F
cyto
). PDBu caused both membrane (120 ± 15% increase) and nuclear localization of PKD (44 ± 4% increase in F
nuc
/F
cyto
). Interestingly, SR Ca depletion by thapsigargin caused an ET-1-like effect, whereas strophantidin did not elicit PKD mobilization. We conclude that ET-1 and PE differ in adult myocyte PKD activation, not in extent, but in cellular localization or translocation. These results are consistent with a more prominent nuclear action of PKD in response to PE vs. ET-1, and with the more critical role of PKD in PE-induced HDAC5 nuclear export.
Abstract 919: Suppression of Abdominal Aortic Aneurysms in the Angiotensin II-infused ApoE Deficient Mice by Treatment with Chymase Inhibitor
Authors: Inoue Nao , Muramatsu Michiko , Jin Denan , Takai Shinji , Hayashi Tetsuya , Katayama Hiroshi , Tamai Hiroshi , Kitaura Yasushi , Miyazaki Mizuo
Published: July 2021

Chymase promotes not only angiotensin II production but also matrix metalloproteinase (MMP)-9 activation, which have a critical role on development of abdominal aortic aneurysms (AAAs). The purpose of this study is to examine the effects of chymase inhibitor, NK3201, on the MMP-9 activity and development of AAA in the angiotensin II-induced apolipoprotein E (apoE)-deficient mice. Method: Angiotensin II (1000ng/kg/min) (vehicle group) or saline (control group) were infused into 16-week-old male apoE-deficient mice for 4 weeks. To examine the effect of chymase inhibition for AAA, we administered NK3201 (30mg/kg/day) to angiotensin II-infused group (NK3201-treated group) for the same period. At the end of angiotensin II infusion, we measured the diameters of suprarenal and infrarenal aorta. AAA severities were scored using the suprarenal aortic diameter/infrarenal aortic diameter ratio and presence of thrombus formation, i.e. under 2.0 was 0, from 2.0 to 2.5 was 1, from 2.5 to 3.0 was 2, over 3.0 was 3, and presence of thrombus was 4. We also determined the chymase and MMP-9 activities using total aorta. Results: The scores that reflected the progression and severity of AAA were increased in vehicle group compared with control group ( 2.35±0.30 vs. 0.27±0.12, p<0.01). This progression was inhibited in NK3201-treated group compared with vehicle group (1.13±0.35, p<0.05 vs. vehicle group). Chymase activity was significantly increased in vehicle group compared with control group. MMP-9 activity was also increased in vehicle group, however it was decreased significantly in NK3201-treated group.Discussion: We demonstrated that chymase inhibition could reduce AAA progression through inhibition of MMP-9 in angiotensin II-induced apoE-deficient mice. Chymase inhibitor might be a novel strategy for preventing AAAs.
Abstract 923: Dissociation Between Superoxide Accumulation And Nitroglycerin Induced Tolerance
Authors: Addanki Vamsi K , Tsou Pei-Suen , Fung Ho-Leung
Published: July 2021

We hypothesize that superoxide (SO) accumulation is not a critical causative factor in inducing nitroglycerin (NTG) tolerance. Using p47phox−/− and gp91−/− mice vs. their respective wild-type (WT) controls, we showed that aorta from mice null of these critical NADPH oxidase subunits exhibited similar vascular tolerance after NTG dosing (20 mg/kg sc, tid for 3 days), as indicated by their ex vivo pEC
50
and cyclic guanosine monophosphate (cGMP, pmol/mg protein) accumulation upon NTG challenge. In vitro aorta SO production (cpm/mg dry weight) was enhanced by NTG incubation both in p47phox null and WT mice (Table 1

). Pre-exposure of isolated mice aorta to 100 microM NTG for 1 hr resulted in vascular tolerance toward NTG and increased SO accumulation. Oxypurinol (Oxy, 1mM) reduced SO but failed to attenuate vascular tolerance (Table 2

). In LLC-PK1 cells, pre-exposure to NTG (1 microM for 4 hours) resulted in increased SO accumulation and reduced cGMP response to 3.16 microM NTG vs. vehicle control. Exposure to 1 microM angiotensin II increased SO but did not reduce cGMP response. Taken together, these results indicate that in vivo vascular NTG tolerance in mice does not require the presence of the p47phox and gp91phox subunits of NADPH oxidase, and that increased SO accumulation may be a consequence, rather than a cause, of NTG tolerance.

Table 1

Table 2
Abstract 963: Metabolic Syndrome is Characterized by Ventriculo-Arterial Mismatch with Disproportionate Increase of End-systolic Ventricular Stiffness
Authors: Melenovsky Vojtech , Chantler Paul D , Majidi Farzad , Ferucci Luigi , Kass David A , Lakatta Edward G , Najjar Samer S
Published: July 2021

Objective:
Ventricular end-systolic stiffness (Ees) reflects the contractile behavior of the left ventricle, geometry and mass. In subjects without heart disease, Ees is matched to arterial load (arterial elastance, Ea) so that ventriculo-vascular coupling is preserved and cardiac performance optimized. However, it remains unknown whether metabolic syndrome (MS) affects the coupling ratio (Ees/Ea).

Methods
: Ea and Ees were non-invasively estimated by single-beat methods based on resting echocardiography and blood pressure data from 176 participants (age 64±13 y, 51% females) in the Baltimore Longitudinal Study of Aging. Presence (+) or absence (−) of MS and the MS score were defined by ATP III-NCEP.

Results:
MS+ subjects had higher Ees/Ea ratio than MS - (2.1 vs. 1.9, p=0.02) due to higher Ees (3.1 vs. 2.6 mmHg/ml, p=0.002), even if adjusted to body size. Vascular load (Ea, 1.5 vs. 1.4 mmHg/ml, p=0.07), LV mass, age, gender were similar (p>0.1) in both groups, but pulse pressure and relative LV wall thickness were higher in MS+. Ees/Ea and Ees correlated with MS score (r=0.18 and 0.27, p=0.02 and <0.001) and plasma leptin level (r=0.31 and 0.20, p<0.001 and 0.02). From individual MS components, Ees/Ea was most affected by waist circumferrence. Ees and Ees/Ea predicted attained peak VO
2
(r= − 0.33 and − 0.29, p<0.001), being lower in MS+ (19±6 vs. 25±6 ml/kg/min, p<0.001).

Conclusions:
In MS, ventricular end-systolic stiffening rises beyond that predicted by arterial load. Leptin and other endocrine factors related to central obesity may be implicated in disproportionate increase of Ees. Abnormal coupling and low systolic reserve likely contribute to exercise intolerance in MS.
Abstract 973: A Novel Anti-Inflammatory Therapeutic Approach for Pulmonary Arterial Hypertension: Blockade of NF-kB by Nano-DDS of NF-kB decoy to the lung ameliorates monocrotaline-induced PAH
Authors: Kimura Satoshi , Egashira Kensuke , Ling Chen , Tsujimoto Hiroyuki , Hara Kaori , Kawashima Yoshiaki , Tominaga Ryuji , Sunagawa Kenji
Published: July 2021

Background:
Inflammatory mechanisms are implicated in the pathogenesis of pulmonary arterial hypertension (PAH), and thus can be a promising therapeutic target of the devastating disease. Nuclear factor-κB (NF-κB) is a redox-sensitive transcription factor that controls expression of important inflammatory cytokines. However, no prior study addressed the role of NF-κB in PAH. We have developed nanotechnology-based drug delivery system (Nano-DDS) to the lung using intratracheal injection of bioabsorbable polymeric PLGA nanoparticle (NP, a mean diameter of 50 nm) for clinical application.

Hypothesis:
Blockade of NF-κB by Nano-DDS of NF-κB decoy to the lung ameliorates monocrotaline (MCT)-induced PAH.

Methods and Results:
[Immunohistochemical studies in human samples from autopsy] Immunohistochemical study using an antibody against activated form (α-p65) showed that activation of NF-κB was noted in the alveolar macrophage and pulmonary vasculature with medial hypertrophy from lung cross-sections from patients with severe PAH. These pathologic changes were associated with increased staining of IL-6 and MCP-1. [Experimental animal studies] Single intratracheal injection of NP incorporated with fluorescence labeled NF-κB decoy in rats resulted in sustained intracellular delivery into macrophages and pulmonary vasculature until 7 days. After MCT injection, rats were divided into no treatment (No Tx) group, and those received single intratracheal decoy alone (50 μg), blank NPs alone, or decoy NP (n=6 – 8, each). No Tx group developed significant PAH, pulmonary arterial remodeling, and increased infiltration of macrophages 3 weeks after MCT injection. These pathologic changes were ameliorated by treatment with decoy NP, but not with decoy only or blank NP only. Increased activity of NF-κB in alveolar macrophages in No Tx group was attenuated by intratracheal decoy NP, but not by decoy only or blank NP only.

Conclusions:
We have developed a novel Nano-DDS of NF-κB decoy into the lung using bioabsorbable NP, and demonstrated its therapeutic benefit associated with anti-inflammatory effects in MCT-induced PAH. This study provides an innovative future direction of less invasive and dependable Nano-DDS for patients with severe PAH.
Abstract 125: Proteome Analysis of a Regulatory Pathway of Pathologic Vascular Injury as Mediated by KLF5 and its Transcriptional Complexes
Authors: Aizawa Kenichi , Suzuki Toru , Mastumura Takayoshi , Kada Nanae , Sawaki Daigo , Zhan Hong , Nagai Ryozo
Published: July 2021

Background:
Transcription factor Krüppel-like factor 5 (KLF5) is a key element linking external stress and cardiovascular remodeling by up-regulating platelet derived growth factor (PDGF)-A chain gene activity. However, the underlying mechanisms remain to be elucidated. The unambiguous and comprehensive identification of interacting proteins is crucial for understanding these mechanisms. In the present study, we identified interacting factors of KLF5 by proteomic analysis and characterized their regulation in the vascular pathogenic response.

Methods&Results:
Double-stranded oligonucleotide containing the binding sequence for KLF5 in the PDGF-A promoter was synthesized and attached to metal beads, to which cell nuclear extract was applied. SDS-PAGE visualized specific bands to the sequence, which were subjected to in-gel digestion and peptide mass fingerprinting by MALDI-TOF/MS spectrometry. Factors that are known to be important in the DNA damage/repair pathway were successively identified. We therefore examined the involvement of the complex in vascular pathologies. Double-strand break as determined by immunohistochemistry using γ-H2AX antibody, a marker of activation of the double-stranded DNA damage/repair response, was observed in pathogenically stimulated vascular endothelial cells (HUVEC) and neointimal tissues in rat carotid artery balloon injury model. Further, KLF5 was shown to mediate the response on γ-H2AX as shown by co-immunoprecipitation and confocal microscopy.

Discussion:
We show a hitherto unknown regulatory mechanism by DNA double-strand break/repair system involving KLF5 in the vascular pathogenic response. Our findings might provide a clue to understanding the initiation of pathological cell proliferation observed in atherosclerosis or restenosis after coronary intervention. This new pathway might also be a tempting target for therapeutic intervention aimed at modulating the activity of KLF5 upon PDGF-A chain and its associated pathologies in the cardiovascular system.
Abstract 1008: A Protective Role for Cardiac Specific Muscle Ring Finger-1 (MuRF1) in Ischemia/Reperfusion Injury In Vivo
Authors: Willis Monte S , Rojas Mauricio , Lockyer Pamela , Hampton Thomas G , Li Luge , Meredith Dane , Patterson Cam
Published: July 2021

We previously identified a critical role for MuRF1 in suppressing pathologic cardiac hypertrophy. To extend these observations to other pathologic processes, we tested the role of MuRF1 in cardiac ischemia reperfusion (I/R) injury. We challenged MuRF1 transgenic (Tg) mice to I/R injury both ex situ and in vivo. First, we examined isolated MuRF1 Tg and age-matched sibling wild-type (WT) hearts after global ischemia (15 min) followed by reperfusion (20 min) in a Langendorff apparatus. Baseline function of MuRF1 Tg hearts did not significantly differ from WT hearts (mean left ventricular developed pressure (LVDP) 88.5 +/− 18 vs. 82.5 +/− 6.7, respectively; n = 4/group). Mean LVDP of hearts from MuRF1 Tg mice after reperfusion was 76.0 +/− 22.9% of baseline function compared to 27.2 +/− 13.3% in WT hearts (N = 5/group, P< 0.05)). To confirm that MuRF1 is cardioprotective in vivo, we subjected MuRF1 Tg and WT mice to a 30 minute ligation of the left anterior descending coronary artery, followed by 24 hours reperfusion. Mice underwent conscious echocardiography at baseline and after 24 hours; cardiac function was further interrogated by Millar pressure volume catheterization at 24 hours. Additionally, hearts underwent a histological evaluation of area at risk and infarct size. By echocardiography, a ~7% decrease in fractional shortening was identified in MuRF1 Tg mice after 24 hours reperfusion compared to baseline. This was in striking contrast to WT mice, which exhibited ~48% decrease in fractional shortening. Steady state catheterization measurements showed a significantly higher ejection fraction in MuRF1 Tg compared to WT mice after I/R injury (81.6 ± 2.3% vs. 49.0 +/− 4.0%, P < 0.05). Contractility reflected by +dP/dt max was better preserved in MuRF1 Tg compared to WT mice after I/R injury (12,614 +/− 776 vs. 7,448 +/−752, N = 3–12/group, P < 0.05). Histologically, the area of infarct in MuRF1 Tg mice was significantly smaller (10.0 +/− 0.8%) than in WT mice (25.5 +/− 2.5%, N = 4/group, P < 0.05). We demonstrate here for the first time that cardiac MuRF1 expression preserves function after I/R injury in vivo. Since MuRF1 is known to interact with metabolic and structural targets, this model will allow us to identify mechanisms by which MuRF1 modifies cardiac pathophysiology.