bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 866: Secreted Frizzled Related Protein 2 Modulates Post Myocardial Infarct Remodeling by Inhibiting Collagen Maturation through the Inhibition of Bone Morphogenetic Protein 1 Activity
Authors: He Wei , Zhang Lunan , Pratt Richard E , Dzau Victor J
Published: July 2021

Myocardial infarction and post-infarction remodeling with heart failure are the major cause of mortality and morbidity in the United States. We recently reported that intracardiac implantation of genetically engineered mesenchymal stem cell (MSC) overexpressing the Akt gene dramatically reduced the infarct size and restored cardiac functions in rodent hearts after coronary artery ligation. Further, we identified Secreted Frizzled Related Protein 2 (sfrp2) as a key factor released by Akt-MSC mediating myocardial survival and repair. However, the underlying mechanism remains elusive. Bone Morphogenetic Protein1 (BMP1)/Tolloid (TLD)-like metalloproteinases belong to a subgroup of astacin family and play key roles in the regulation of extracelluar matrix (ECM) formation and cardiac fibrosis. These proteases have procollagen C-proteinase (PCP) activities which are responsible for the cleavage of C-propeptides from procollagen precursors to produce mature collagen fibrils. In this report, we showed that three days following myocardial infarction in rats, both BMP1 protein expression and activity were upregulated in the infarcted left ventricle. Interestingly, we found recombinant sfrp2 could inhibit BMP1 activity in MI tissue samples as measured by an in vitro PCP activity assay. Furthermore, using purified recombinant proteins, we demonstrated that sfrp2, but not sfrp1 or sfrp3, inhibited BMP-1 activity in vitro. Moreover, purified sfrp2 could physically interact with BMP1 protein as shown by the co-immunoprecipitation assay. To provide further evidence that sfrp2 can interfere with collagen processing, we demonstrated that exogenously added sfrp2 interfered with procollagen processing in primary cultures of cardiac fibroblast culture medium. Similar results were obtained when these cells were transiently transfected with sfrp2 expressing plasmids. In summary, our data suggest that one of the molecular mechanisms underlying the cardioprotective and repair effects of sfrp2 protein on myocardial infarction is through the inhibition of BMP-1 activity. Therefore, sfrp2 has the potential clinical application as a novel anti-fibrotic reagent for the modulation of cardiac remodeling after acute myocardial infarction.
Abstract 879: The Chemokine CXCL10 Is Involved In The Development Of Atherosclerotic Plaque Vulnerability
Authors: Segers Dolf , de Crom Rini , Leenen Pieter , Pasterkamp Gerard , Krams Rob
Published: July 2021

Introduction: Atherosclerosis is an inflammatory disease in which monocyte/macrophages and Th1 cells are crucial cell types. The CXC-chemokine CXCL10 might play a pivotal role in its pathogenesis as it mediates chemoattraction of these cells. We previously found that high CXCL10 levels in human carotid plaques are associated with a vulnerable phenotype. Moreover, we identified CXCL10 as a possible biomarker for vulnerability in a mouse model for vulnerable plaque. The aim of the current study was to evaluate if CXCL10 has a critical role in the development of plaque vulnerability.

Methods: Atherosclerosis-susceptible female ApoE -/-mice on high fat, high cholesterol diet were instrumented with a perivascular shear stress-altering plastic cylinder to induce vulnerable plaque. In the first three weeks following surgery, mice were injected 3 times/week with a bioactivity-neutralizing CXCL10 antibody. After 9 weeks, plaques were harvested and sections were evaluated for macrophages (CD68), immune activation (MHC class II), smooth muscle cells (a-actin), lipids (Oil Red O) and collagen (picrosirius red).

Results: Lesion sizes were equal in both the intervention group and the control group. Also lipid accumulation was comparable in both groups. Despite unaffected macrophage content (30.1 vs 28.7% p = 0.73), we did find a 2-fold reduction of MHC class-II expression (4.9 vs 14.5% p = 0.011) in the antibody-treated group. This decline in activation of the immune system coincided with an increase in the presence of smooth muscle cells (12.6 vs 4.2% p = 0.034) and an increase in collagen content (9.7 vs 21.8% p = 0.014).

Conclusion: This study confirms a pivotal role of CXCL10 in the development of vulnerable plaque. Inhibiting CXCL10 resulted in an increase of plaque stability by decreasing immune activation and increasing smooth muscle cell and collagen content. Accordingly, CXCL10 is a potential marker for both diagnostic and therapeutic intervention.
Abstract 915: Deletion of
JNK2
Prevents Hypercholesterolemia-Induced Oxidative Stress and Endothelial Dysfunction
Authors: Osto Elena , Matter Christian M , Kuroedov Alexey , Camici Giovanni , Malinski Tadeusz , Bachschmid Markus , Stallmach Thomas , Boren Jan , Iliceto Sabino , Luscher Thomas F , Cosentino Francesco
Published: July 2021

Background
Hypercholesterolemia-induced endothelial dysfunction (ED) is a major trigger for atherosclerosis. The c-Jun-N-terminal kinases (JNKs) belong to the mitogen-activated protein kinase family. Genetic deletion of
JNK2
has been shown to decrease atheroma formation. The present study was designed to investigate whether hypercholesterolemic
JNK2
knockout (
JNK2
−/−
) mice are protected from oxidative stress-induced ED.

Methods and Results
Male
JNK2
−/−
and corresponding wild-type (WT) mice (8 weeks old) were fed either a high cholesterol diet (HCD, 1.25% total cholesterol) or normal chow (controls) for 14 weeks. WT mice fed a HCD showed a 2-fold increase in JNK phosphorylation as assessed by Western blotting (n=4 – 6, p<0.05 vs. controls). In parallel, endothelium-dependent relaxations to acetylcholine (Ach, 10
−9
-10
−6
mol/L) were impaired in WT mice exposed to a HCD as compared to WT controls (n=4 – 6 in each groups, p<0.05). In contrast,
JNK2
−/−
mice did not exhibit hypercholesterolemia-induced ED (96±5% max relaxation to Ach, n=4 – 6, p<0.05 vs. WT HCD). Endothelium-independent relaxations to sodium nitroprusside (10
−10
-10
−5
mol/L) were identical in all groups. Nitric oxide release, measured by nanosensors in single aortic endothelial cells, was decreased in WT but not in
JNK2
−/−
HCD mice (121 vs. 253 nmol/L in WT and
JNK2
−/−
HCD mice, respectively, n=4 – 6, p<0.05). Indeed, WT HCD mice displayed a marked increase in peroxynitrite (ONOO
−
) generation, nitrotyrosine expression,and lipid peroxidation (n=4 – 6 in each groups, p<0.05 vs. WT and
JNK2
−/−
control as well as
JNK2
−/−
HCD mice). Deletion of the
JNK2
gene prevented this hypercholesterolemia-induced increase in oxidative stress. Furthermore, expression of antioxidant defense systems such as manganese and extracellular superoxide dismutase as well as heme-oxygenase activity was significantly decreased in WT, but not in
JNK2
−/−
HCD mice (n=4 – 6, p<0.05).

Conclusions
These data suggest that genetic deletion of JNK2 protects against hypercholesterolemia-induced oxidative stress and endothelial dysfunction. Thus, inhibition of JNK2 may represent a novel therapeutic target to prevent initiation of atherogenesis.
Abstract 916: Klotho-protein Regulates Transient Receptor Potential Canonical-1-mediated Ca(2+)-influx In Endothelial Cells And Prevents Cell Death And Disruption Of Endothelial Integrity Induced By Ca(2+)-overload
Authors: Okigaki Mitsuhiko , Kusaba Tetsurou , Honsho Shoken , Matsui Akihiro , Matsunaga Shinsaku , Katsume Asako , Kishita Eigo , Matsubara Hiroaki
Published: July 2021

Introduction
Klotho-deficient mice have extensive vascular calcification. However, the mechanism involved has not been elucidated. We analyzed the endothelial cells of Klotho-deficient mice (Klotho-ECs) and found a novel functional role for the Klotho-protein in the maintenance of vascular homeostasis.

Methods and Results
Primary-cultured endothelial cells from the aorta of wild-type- (WT) and Klotho- deficient mice were loaded with fura-2 and stimulated with VEGF. In WT-ECs, intracellular Ca(2+) concentration ([Ca(2+)]i) elevated to the peak level, and it decreased to the basal level in +5 minutes. Whereas, in Klotho-ECs, after similar peak level to WT-ECs, the elevation of [Ca(2+)]i was sustained at ~35% of the peak level for more than 10 minutes. In Ca(2+) free medium, this sustained elevation was abolished. Immunostaining revealed that, in Klotho-ECs, VEGF-induced co-internalization of the Ca(2+) channel; TRPC-1, with VEGF-receptor-2 (VEGFR-2) was abolished, thereby, at 30 minutes after VEGF-stimulation, expression levels of TRPC-1 in the plasmamembrane was 2.2-fold higher than in WT-ECs. Klotho-protein constitutively bound to VEGFR-2 and TRPC-1, strengthened their association, and promoted their co-internalization. VEGF-induced activation of Rac-1 and reorganization of F-actin, which are involved in internalization, were impaired in Klotho-ECs, and restored by the replacement of Klotho-protein. VEGF-induced phosphorylation of VEGFR-2 and its downstream signaling molecules, including Src, Akt, PLCγ, and Caveolin1, was attenuated. As the biological consequence of sustained increase in [Ca(2+)]i, activation of calpain and caspase-3 followed by apoptosis was promoted in Klotho-ECs in vivo and vitro. Also, expression of VE-cadherin in the plasmamembrane was reduced and intimal hyperpermeability was observed in the aorta of Klotho-deficient mice, which presumably lead to exudation of the Ca(2+)/phosphor -rich serum into the vessel walls, followed by extensive vascular calcification.

Conclusion
Thus, Klotho-protein regulates ligand-induced Ca(2+) influx and subsequent biological functions involved in vascular calcification.
Abstract 946: Angiotensin II Type 1 Receptor Partially Mediates Stretch-Induced Potentiation of the Slow Component of Delayed Rectifier Potassium Current in Guinea Pig Atrium
Authors: Zankov Dimitar P , Toyoda Futoshi , Omatsu-Kanbe Mariko , Matsuura Hiroshi , Horie Minoru
Published: July 2021

Introduction:
Mechano-sensitive signal transduction in atrial myocytes is closely related to the development of atrial fibrillation (AF). A repolarizing conductance in the heart stimulated by stretch or swelling of myocytes is the slow component of the delayed rectifier K
+
current (
I
Ks
). Modulation of
I
Ks
appears to be linked to pathogenesis of AF because in hereditary cases, gain-of-function mutations in
I
Ks
channel genes have been detected. Moreover, membrane stretch activates AT
1
receptor even without angiotensin II.

Hypothesis:
We assessed the hypothesis that activation of AT
1
receptor mediates swelling-induced increase of
I
Ks
in atrium.

Methods:
Whole-cell patch-clamp method was used to record
I
Ks
and action potentials in guinea pig atrial myocytes.

Results:
Exposure of atrial myocytes to 70% hyposmotic solution (210 mOsmol/L, HS) enhanced
I
Ks
by 84.1± 8.4% (
n
= 12), abbreviated atrial action potential at 90% repolarization (APD
90
) by 16.8± 1.3%, and depolarized resting potential by 4.9±0.7 mV (
n
= 9). Selective blockade of AT
1
receptor with 1 or 5 μmol/L candesartan and 5 μmol/L olmesartan significantly attenuated the HS-induced
I
Ks
increase to 48.0±4.1% (
n
= 10), 47.2±2.7% (
n
= 9), and 59.4±5.5% (
n
= 9), respectively. Pretreatment with 5 μmol/L olmesartan also significantly reduced the HS-evoked shortening of APD
90
to 12.4± 1.4% (
n
= 10). Tyrosine kinase inhibitors tyrphostins A23 and A25 (20 μmol/L) reduced the HS-induced
I
Ks
enhancement to 42.0± 9.5 (
n
= 13) and 52.7±4.7% (
n
= 12), respectively, whereas their inactive analogue A1 had little effect. Block of tyrosine phosphatases by 500 μmol/L orthovanadate further promoted the potentiation of
I
Ks
by HS to 124.6± 10.6% (
n
= 8) and markedly decreased the recovery rate of
I
Ks
after withdrawal of HS. Pharmacological suppression of G proteins, phospholipase C, and protein kinase C did not appreciably affect the modulation of
I
Ks
.

Conclusion:
These results indicate involvement of AT
1
receptor and tyrosine kinases in the hyposmotic potentiation of atrial
I
Ks
. It is likely that in the clinical settings, AT
1
receptor antagonists can attenuate the stretch-induced
I
Ks
increase and action potential shortening and thereby exert an acute antiarrhythmic action on stretch-related atrial arrhythmias such as AF.
Abstract 948: An Allosteric Mechanism for Drug Block of a Cardiac Potassium Channel
Authors: Yang Tao , Smith Jarrod A , Meiler Jens , Wingo Tammy L , Kanki Hideaki , Leake Brenda , Sanders Charles R , Balser Jeffrey R , Roden Dan M
Published: July 2021

Background: A common mechanism for block of ion channels is current inhibition by drug binding to residues - notably in S6 - within the permeating pore. Open state block is commonly observed.

Methods and Results: In this study, we used a combination of alanine/cysteine mutagenesis, patch clamp, and homology modeling to identify a novel allosteric mechanism for drug block of the human cardiac potassium channel KCNQ1 (the α-subunit encoding IKs). The initial mutagenesis experiments studied a total of 45 amino acid residues in the S6 segment (30 residues), and the proximal C-terminus (15 residues) of KCNQ1. Residues required for drug block were identified as alanine/cysteine mutants displaying <50% block of expressed current (n=4 – 6 each) upon exposure to drug concentrations inhibiting wild-type current by ≥90% (quinidine 100 μm, clofilium 30 μm and L-7 10 μm). Among the 30 residues in S6, there were five (F332, F335, S338, A341 and G348) displaying such reduced block and in the proximal C-terminus, 5/15 were less sensitive, including F351 which was near-totally resistant (current decrease = 4±3%). Homology modeling of KCNQ1 based on the Kv1.2 structure unexpectedly suggested that F351 faces away from the permeating pore, arguing against pore block as the mechanism for drug inhibition of KCNQ1 current. In the open channel model, F351 lines a pocket that also includes L251 and V254 in S4–S5. The model allows drug access to this pocket, and alanine substitutions at both sites yielded potassium currents that were resistant to drug block (↓34±4% for L251A and ↓40±5% for V254A).

Conclusion: These data strongly support a model in which open state block of this channel occurs not via binding to a site in the pore but rather by a novel allosteric mechanism, drug access to a side pocket generated in the open channel configuration and lined by S6 and S4/S5 residues.
Abstract 950: Direct Evidence for an Important Role of Agonist-independent Constitutive I
K,ACh
Channel Activity in Atrial Tachycardia Remodeling
Authors: Voigt Niels , Maguy Ange , Yeh Yung-Hsin , Qi Xiao-Yan , Ravens Ursula , Dobrev Dobromir , Nattel Stanley
Published: July 2021

Background:
Although atrial tachycardia (AT) appears to promote agonist-independent constitutively active I
K,ACh
that increases susceptibility to AF, direct demonstration of dysregulated I
K,ACh
channel function is lacking. We studied AT effects on single I
K,ACh
channel activity in dog atria.

Methods:
I
K,ACh
channel activity was recorded with cell-attached patch clamp in isolated atrial myocytes of control (CTL) and AT (7 days, 400 min
−1
) dogs.

Results
: AT prolonged inducible AF duration from 44±22 to 413±167 s; N=9 dogs/gp, P<0.001. In the absence of cholinergic stimulation, single-channel openings with typical I
K,ACh
conductance and rectification were observed in CTL and AT (Figure

). AT produced prominent agonist-independent I
K,ACh
activity due to 7-fold increased opening frequency (f
o
) and 10-fold increased open probability (P
o
) vs CTL (P<0.01 for each), but unaltered open time and single channel conductance. With maximum I
K,ACh
activation (10 μm carbachol, CCh), f
o
was 38% lower, open time constant 25% higher, and P
o
and unitary conductance unchanged for AT vs CTL. The selective Kir3 blocker tertiapin (100 nM) reduced f
o
and P
o
by 48% and 51% (P<0.05 each) without altering other channel properties, confirming the identity of
I
K,ACh.

Conclusions
: AT produces prominent agonist-independent constitutive single-channel I
K,ACh
activity, providing a molecular basis for previously-observed AT-enhanced macroscopic I
K,ACh
, as well as associated AP-shortening and tertiapin-suppressible AF promotion. These results suggest an important role for constitutively active I
K,ACh
channels in AT-remodeling and support their interest as a potential novel AF-therapy target.
Abstract 200: Beta-Myosin Heavy Chain Induction after Pressure Overload Is in a Minor Sub-population of Myocytes and Requires the Alpha-1A-Adrenergic Receptor
Authors: Lopez Javier E , Myagmar Bat-Erdene , Swigart Philip , Bigos Marty , Rodrigo Manoj , Simpson Paul
Published: July 2021

Background: Induction of the fetal hypertrophic gene beta-myosin heavy chain (MHC) is a signature feature of pressure overload, and is thought to occur in most hypertrophied myocytes. Beta-MHC mRNA is not induced after transverse aortic constriction (TAC) in a double knockout (KO) model of the alpha-1A and alpha-1B-adrenergic receptor (AR) subtypes, but it is unknown whether the A or B or both are required.

Hypothesis: We tested the hypothesis that native beta-MHC protein induction is in a sub-population of myocytes and requires only a single alpha-1 subtype.

Methods: TAC was done in wild type (WT) and alpha-1 KO male mice ages 12–14 weeks. Beta-MHC protein was measured in isolated adult myocytes by a novel flow cytometry approach, with antibodies validated for beta-MHC and total sarcomeric MHC.

Results: In WT mice, the fraction of myocytes expressing beta-MHC was 3% in shams (SE =1, n =4 hearts), and increased to 26% of myocytes at 1–3 weeks after TAC (SE =4, n =8, p< 0.01 vs. sham). Myocytes expressing beta-MHC were predominantly large cells (see Figure

). In alpha-1A KO mice (AKO), beta-MHC was induced in only 6% of myocytes (SE =2, n =3, p<0.05 vs. WT TAC, p =NS vs. sham), or in only 15% as many myocytes as in WT hearts. Western blotting and quantitative RT-PCR confirmed reduced beta-MHC induction in alpha-1A KO myocytes.

Conclusion: Beta-MHC induction after pressure overload is in only a minor sub-population of cardiac myocytes, contrary to common models of fetal hypertrophic gene induction. Furthermore, beta-MHC induction requires a single alpha-1-AR subtype, the alpha-1A, which cannot be compensated by other hypertrophic receptors.
Abstract 977: Mesenchymal Stem Cell Therapy Prevents Ischemia/Reperfusion Injury and Promotes Tissue Regeneration
Authors: Manning Eddie , Salgar Shashikumar , Hernandez Mireya , Mateu Dania , Vazquez-Padron Roberto , Li Sen , Mathew James , Pham Si
Published: July 2021

Ischemic heart disease, especially myocardial infarction, is an important cause of heart failure. However, therapeutic potential of mesenchymal stem cells (MSC) for myocardial or pulmonary ischemia reperfusion (IR) injury is not well studied. Interleukin-10 (IL-10) is an anti-inflammatory cytokine. In this study MSC and IL-10 engineered MSC were tested for their ability to prevent IR injury and promote tissue repair.

Methods:
Bone marrow cells from Lewis rat were cultured in plastic tissue culture flasks for 72 hours; cells in suspension were discarded and the adherent cells (MSC) were expanded. MSC were transduced with
r
vIL-10-retrovirus and selected on neomycin (1mg/ml for 7 days). Experimental design: following 120 minutes of left lung ischemia, Group A, rats received vIL10 transduced MSC (∼15 x 10
6
; i.v.); Group B, rats received empty vector engineered MSC; Group C, received MSC; and Group D, received saline. Group E received no ischemia or MSC. Left lung ischemia was performed by clamping left pulmonary artery/vein/bronchus at end inspiration. At 24h, 72 h, & 7 days following ischemia, blood was collected from left and right pulmonary veins, separately.

Results
: Mean blood oxygenation (PaO2/FIO2 ratio, mmHg) at day 7 was significantly (P<0.05) reduced following IR injury in Group B (161± 77) and Group D (181± 64), compared to MSC-vIL10 (404± 32; Group A) & MSC (294±103;Group C) treated animals. In Group E, PaO2/FIO2 was 440± 42mm Hg. Significant improvement in lung oxygenation with MSC and vIL-10 therapy was observed as early as 3 days. MSC produced vIL-10 (∼5ng/ml)
ex vivo
. HP of lungs demonstrated reduced PMN cells with MSC therapy. Inflammatory mediators IL-1a, MCP-1, MIP-1a, and IL1-b protein levels were markedly increased in the injured lung; their analysis in MSC treated groups are underway. I.V. injected
Tg
GFP+MSC trafficked to the lung & other tissues. VEGF & HGF mRNA in MSC was 66% & 43% of β-actin, respectively.
Ex vivo
expanded MSC were CD34-, CD45+ (5%), CD29+ (92%), CD80-, CD86+ (10%), CD90+ (∼90%), MHC Class I
low
& MHC Class II-.

Conclusions:
Autologous MSC therapy prevented IR injury and served as an excellent vehicle to deliver cytokine. MSC therapy to prevent IR injury and enhance tissue regeneration in cardiopulmonary diseases seems promising.
Abstract 205: Dysfunction of the 19S Proteosome Contributes to Adverse Cardiac Remodeling in Mice With Cardiac Restricted Overexpression of Tumor Necrosis Factor
Authors: Wang Wei , Szalai Gabor , Sivasubramanian Natarajan , Mann Douglas L
Published: July 2021

We have shown previously that mice with cardiac restricted overexression of tumor necrosis factor (sTNF mice) develop progressive cardiac remodeling. Given that the ubiquitin/ proteasome system regulates protein turnover in eukaryotic cells and is thereby a major factor in controlling cell cycle, apoptosis, signal transduction pathways, which are important in cardiac remodeling, the
purpose
of this study was to assess the function of 19S, 20S and 26S proteasome in sTNF mice and littermate control mice.

Results
: Activity of the 26S proteasome was significantly suppressed in the hearts of the sTNF mice compared with the wild type littermates, and was detectable as early as 4-weeks of age. To explore the mechanism for the dysfunction of the 26S proteosome, we examined the activity of the 19S and 20S subunits. We observed cleavage of two subunits of the 19S regulatory complex of the proteasome, namely S1 and S10b, in the hearts of the sTNF mice. S1 is involved in the linking of the lid and the base of the 19S complex, while S10b is one of the six ATPase subunits in the base of the 19S complex. Interestingly, we observed that the expression of six subunits of the 20S core complex, β2, β2i, β3, β4, β5, and β7 were up-regulated in the hearts of the sTNF mice compared with the wild type littermate control mice. Despite the higher expression of the six β subunits in the 20S complex, we observed no difference in the 20S proteasome activity in hearts between the sTNF mice and the wild type littermate controls.

Conclusion
: Taken together, these results suggest that cleavage of S1 and S10b might contribute to the low 26S proteasome activity in the hearts of the sTNF mice. Given that the 26S proteasome is responsible for degrading pro-apoptotic pathways, as well as turning off pro-fibrotic signaling (TGF-beta), these results suggest that dysfunction of the 26S proteasome might be one of the mechanisms underlying left ventricular remodeling in the setting of sustained inflammation.
Abstract 1007: Platelet Glycoprotein VI (GPVI) Deletion Reduces Myocardial Infarction in Mice
Authors: Li Haiquan , Takizawa Hisao , Gong Xiaoqi , Concepcion Alice , Kambayashi Junichi , Tandon Narendra , Liu Yongge
Published: July 2021

Fibrillar collagen, exposed by damage of vascular endothelium, is the most thrombogenic component of the subendothelium, which, in addition to supporting platelet adhesion, is also a potent platelet activator. Platelet glycoprotein VI (GPVI) plays a major role in collagen-induced platelet activation, leading to thrombus formation. Inhibition of GPVI function may be a novel anti-platelet therapy with minimal bleeding risk. Recent evidence in FcRγ-chain-knockout (KO) mice, which also lack GPVI, shows attenuated myocardial infarction after ischemia and reperfusion. Unlike GPVI, which is exclusively expressed in platelets and megakaryocytes, FcRγ-chain also exists in white blood cells and immune cells. Thus, it is unclear whether GPVI deletion is cardioprotective. In this study, we compared myocardial infarction in GPVI-KO and age-matched wild-type (WT) mice. In parallel FcRγ-chain-KO mice were compared with age-matched WT mice. A major branch of left coronary artery was ligated for 30 min followed by reperfusion for 24 hrs. Myocardial infarction was measured by tetrozolium staining. Hemodynamics were not statistically different among the groups. Risk zone sizes were also similar between WT and GPVI-KO mice (0.020 ± 0.004 cm
3
and 0.022 ± 0.005 cm
3
, respectively) (Mean ± SD). However, percentage infarction of the risk zone was significantly smaller in GPVI-KO mice (averaged 22 ± 8 % vs. 45 ± 18 % in WT). Comparable protection was observed in FcRγ-chain-KO mice (12 ± 6 % vs. 46 ± 13 % in WT). Significant exposure of vascular collagen in the ischemia/reperfusion region of myocardium was revealed by FITC-labeled recombinant GPVI protein, injected prior to ischemia, indicating endothelium injury. Furthermore, there was significantly less expression of P-selectin, a marker of activated platelets, in the myocardium from GPVI-KO and FcRγ-KO mice when compared with WT mice. It is concluded that deletion of GPVI reduces myocardial infarction induced by ischemia and reperfusion, likely due to the attenuation of the platelet-collagen interaction. With its known anti-thrombotic effect and minimal risk of bleeding in animals, anti-GPVI therapy may offer a promising novel treatment for cardiovascular diseases.
Abstract 1033: Simultaneous Clot-targeted Factor Xa Inhibition And Selective Blockade Of Activated GPIIb/IIIa On Platelets Results In Delayed But Potent Antithrombotic Effects Without Bleeding Time Prolongation.
Authors: Hagemeyer Christoph E , Eisenhardt Steffen U , Bassler Nicole , Stoll Patrick , Schwarz Meike , Chen Yung C , Ahrens Ingo , Bode Christoph , Peter Karlheinz
Published: July 2021

Background: We generated phage-display-derived anti-GPIIb/IIIa single-chain antibodies (e.g. scFv SCE5) that specifically bind to the activated GPIIb/IIIa only and thus specifically block activated platelets only. ScFv SCE5 demonstrates strong antithrombotic potency, comparable to the conformation-unspecific blockers tirofiban and eptifibatide. In contrast bleeding times were not prolonged with scFv SCE5. Here we now use the possibility to add effector molecules using molecular biology methods. The highly potent anticoagulant TAP (tick anticoagulant peptide), which is a direct factor Xa (fXa) inhibitor, was used as an effector molecule.

Methods and Results: We genetically fused the activation-specific scFv with TAP, expressed the constructs in E.coli and purified the 39 kDa protein via its Histag binding to Nickel beads. Specific binding of the fusion molecules MA2/SCE5-TAP and strong inhibition of fibrinogen binding was proven in flow cytometry; anti-fXa activity was demonstrated in chromogenic assays. In vivo anticoagulative efficiency was determined by Doppler-flow in a ferric chloride-induced carotid artery thrombosis model in mice. Prolongation in occlusion time with SCE5-TAP was significantly stronger compared to SCE5 alone, recombinant TAP, non-binding mut-scFv-TAP as well as the clinical used drugs enoxaparine and eptifibatide. In contrast to the other anticoagulants tested, bleeding time was not prolonged by SCE5-TAP. Flow experiments studying platelet adhesion on collagen revealed a possible mechanism for the unique finding of a fully normal bleeding time: LIBS exposure on adhering platelets and as such the anticoagulative targeting potency of SCE5-TAP was delayed until considerable layers of platelets were deposited.

Conclusions: The combination of activation-specific GPIIb/IIIa blockade and fXa inhibition in one clot-targeted molecule further improves in-vivo antithrombotic efficiency without causing any bleeding time prolongation. The delay of the observed targeting effect may allow a sealing of injuries with platelet layers but may be in time for the prevention of occlusive platelet aggregates. The described blockers represent a new type of highly selective drugs that warrant further clinical development.
Abstract 1045: E-ntpdase-1/Cd39 Confers Anti-inflammatory And Immunomodulatory Properties To Circulating Membrane-derived Microparticles
Authors: Banz Yara , Beldi Guido , Wu Yan , Usheva Anny , Robson Simon C
Published: July 2021

Background: Microparticles (MPs, <1.5ÂμM) are circulating, membrane-derived micro-vesicles shed from cells and platelets under physiological conditions as well as following stress, such as post-myocardial infarction. MPs have been implicated in inflammation, coagulation and vascular function. CD39, the prototype nucleoside triphosphate diphosphohydrolase (NTP-Dase1) within the vasculature modulates extracellular purinergic signalling through ATP and ADP hydrolysis, conferring anti-inflammatory, anti-thrombotic properties to the intravascular space. The aim of this study was to determine if CD39 is present on MPs and whether this impacts MP phenotype and function.

Methods: The proteome, absolute numbers as well as cellular origin of circulating MPs from wild type (wt) and cd39-null C57Bl6 mice were compared by 2D-gel electrophoresis, western blot and flow cytometry. Isolated MPs were incubated with endothelial cells (EC) to analyze their intrinsic pro-inflammatory potential.

Results: CD39 can be demonstrated and has ecto-enzymatic activity on circulating MPs from wt mice. 2D-gel analysis revealed that cd39-null MPs express lower levels of talin (component of focal adhesion) and ATP synthase, when compared to wild type MPs. Mutant mice have increased numbers of circulating MPs, with significantly higher fractions derived from platelets and endothelium as compared to wt mice (p<0.05). The presence of CD39 on wt MPs significantly attenuates inflammatory responses following incubation with EC as compared to cd39-null MPs (reduced IL-6 and TNF-alpha secretion, ICAM-1 and VCAM-1 expression, vWF release and NF-kappa B activation, p<0.05).

Conclusions: CD39 confers anti-inflammatory and immunomodulatory properties upon circulating MPs. These results highlight the protective potential of CD39; a finding that may be exploited therapeutically, with the prospective of administering soluble CD39 (apyrase) to the site of intravascular injury to ameliorate inflammation, as occurs for instance in acute coronary syndromes.
Abstract 1090: Goal Attainment for LDL-C, HDL-C, Triglycerides, and All Lipids in U.S. Adults: National Health and Nutrition Examination Survey 2003–2004
Authors: Ghandehari Heli , Kamal-Bahl Sachin , Wong Nathan D
Published: July 2021

Background:
Recent U.S. population data are lacking regarding the achievement of recommended lipid levels according to demographic factors and comorbidities. We evaluated the extent of LDL-C, HDL-C, triglyceride, and overall lipid goal attainment in U.S. adults.

Design, Setting, and Participants:
Data analysis from adults aged ≥20 who had fasted for ≥8 hours (n=2987 representing 132.7 million) in the National Health and Nutrition Examination Survey 2003–2004, a representative cross-sectional survey of the non-institutionalized civilian U.S. population.

Main Outcome Measures:
The proportion of adults at recommended levels for LDL-C, HDL-C, triglycerides, and all lipids were examined by gender, age, ethnicity and the presence of cardiovascular disease (CVD), diabetes (DM) or metabolic syndrome (MetS). Goals were <100 mg/dl for LDL-C for DM, CVD, or 2+ risk factors and >20% 10-year risk for coronary heart disease (CHD), <130 mg/dl if 2+ risk factors and ≤20% 10-year risk for CHD, or <160 mg/dl if <2 risk factors; ≥40 mg/dl (male) or ≥50 mg/dl (female) for HDL-C; and <150 mg/dl for triglycerides.

Results:
The proportion at goal for LDL-C was 74.5%, HDL-C 73.2%, triglycerides 73.6%, and 47.5% for all lipids. Control of LDL-C and triglycerides was greater in females, and HDL-C greater in males. Mexican-Americans and persons with CVD, DM, or MetS were least likely to be at recommended levels (see Table

). Table

. Proportion (%) of US adults 2003–2004 at recommended levels for LDL-C, HDL-C, triglycerides, and all lipids

Conclusions:
While three-fourths of U.S. adults are at recommended levels for LDL-C, HDL-C and triglycerides individually, less than half are at goal for all lipids. For persons with DM or CVD, only a third are at goal for LDL-C and only a sixth are at goal for all lipids. Improved treatment efforts to optimize goal attainment of all lipids are required.
Abstract 1094: Inflammation Results In Increased Allele-specific Apo(a) Levels In African Americans: Potential Explanation For Inter-ethnic Difference?
Authors: Erdembileg Anuurad , Chiem Alan , Tracy Russell P , Pearson Thomas A , Berglund Lars
Published: July 2021

Background:
The role of inflammation in the pathogenesis of cardiovascular disease (CVD) is well recognized. Lipoprotein(a), Lp(a), is an independent CVD risk factor where plasma levels are largely determined by apo(a) gene size. Allele-specific apo(a) levels are higher among African Americans (AA) compared to Caucasians (C), and the most pronounced inter-ethnic difference is seen for medium-sized apo(a) alleles. This difference remains unexplained. The apo(a) gene contains response elements for inflammatory factors. However, the effect of inflammation on Lp(a) level, particularly on allele-specific apo(a) levels is unknown.

Objective:
We investigated the effect of inflammation measured by two acute-phase reactants (CRP and fibrinogen) on allele-specific apo(a) levels in AA and C.

Methods:
We determined Lp(a) levels, apo(a) sizes, allele-specific apo(a) levels, fibrinogen and CRP levels in 167 AA and 259 C.

Results:
Lp(a) levels were increased among AA with higher vs. lower CRP (>3 mg/dl) or fibrinogen (>340 mg/dl) levels (143 vs. 108 nmol/l,
P
=0.009, and 146 vs. 105 nmol/l,
P
=0.002, respectively). We next analyzed allele-specific apo(a) levels for different apo(a) sizes. No differences in allele-specific apo(a) levels across CRP (>3 vs. ≤3 mg/dl) or fibrinogen (>340 vs. ≤340 mg/dl) groups were seen among AA or C for small apo(a) sizes (<22 K4). Allele specific apo(a) levels for medium apo(a) sizes (23–30 K4) were significantly higher among AA with high levels of CRP or fibrinogen compared to those with low levels (88 vs. 67 nmol/l,
P
=0.014 and 91 vs. 59 nmol/l,
P<
0.0001, respectively). In contrast, there were no significant differences in allele-specific apo(a) levels across CRP or fibrinogen groups among C with medium sized apo(a) alleles.

Conclusion:
Increased levels of CRP or fibrinogen are associated with higher allele-specific medium-sized apo(a) levels in AA, but not in C. Our results implicate that a pro-inflammatory stimulus may result in a selective increase in Lp(a) levels among AA carrying medium-sized apo(a). These findings provide a potential explanation for differences in Lp(a) levels between African Americans and Caucasians and suggest that inflammation-associated events may contribute to this inter-ethnic difference.
Abstract 1114: Deregulation of MicroRNAs in the Heart of c-kit-Defective Mice
Authors: Hosoda Toru , Yasuzawa-Amano Saori , Amano Katsuya , Boni Alessandro , Urbanek Konrad , Delucchi Francesca , Arcarese Michael L , Miqueo Arantxa Gonzalez , Siggins Robert W , Misao Yu , LeCapitaine Nicole J , Mosna Federico , Sonnenblick Edmund H , Kajstura Jan , Bolli Roberto , Anversa Piero , Leri Annarosa
Published: July 2021

MicroRNAs (miRNAs) are a family of non-coding RNAs that regulate gene expression by repressing mRNA function at the post-transcriptional level. Recently, the involvement of miRNAs in the modulation of cardiac function has been described in vivo in the developing and adult heart. Whether miRNAs play an important role in the control of the growth and differentiation of c-kit-positive cardiac progenitor cells (CPCs) is currently unknown. Therefore, two transgenic mouse models characterized by defects in the c-kit receptor or its ligand, stem cell factor (SCF), were studied in combination with wild-type littermates. The first one has a spontaneous point mutation of the c-kit receptor in one allele (W
V
) coupled with an amino acid deletion in the other allele (W); and the second has a complete deletion of the SCF gene locus in one allele (Sl) and deletion of the membrane-bound ligand in the other (Sl
d
). The ventricular myocardium of transgenic and non-transgenic mice was analyzed with respect to miRNA expression by microarray. We have identified three miRNAs that showed an extremely high level of expression in the heart of transgenic and non-transgenic animals: miR-150, miR-451 and miR-805. These miRNAs have not been described previously in the myocardium and were consistently upregulated in the heart of wild-type mice; a 1.4 –1.8-fold difference was found with respect to transgenic animals. The major difference was detected for miR-150. So far, this miRNA was considered to be restricted to hematopoietic progenitors. Surveying the predicted targets of miR-150, we identified a cluster of genes involved in cell proliferation that were expected to be upregulated in W/W
V
and Sl/Sl
d
mice: Akt3, ELK1, FOXP1, Pim-1, IGF-1, Myb and PA2G4. Consistently, W/W
V
mice showed a two-fold or larger mRNA and protein levels for genes implicated in cell cycle progression. A similar analysis was not possible for miR-451 and miR-805 since the predicted targets of these miRNAs remain to be recognized. In conclusion, a novel miRNA critical for hematopoiesis has been identified in the heart and this miRNA is downregulated when defects of c-kit signaling are present.
Abstract 1128: The Leukocyte Integrin Mac-1 Promotes Acute Cardiac Allograft Rejection
Authors: Shimizu Koichi , Libby Peter , Sakuma Masashi , Wang Yunmei , Mitchell Richard N , Simon Daniel I
Published: July 2021

Background: In allograft rejection, donor endothelial cells (EC) are the principal early targets of host leukocytes and alloantibodies. Although the leukocyte integrin Mac-1(αmβ2, CD11b/ CD18) facilitates leukocyte-leukocyte as well as leukocyte-EC interactions, its roles in allograft survival and vasculopathy remains unclear. This study used murine cardiac transplantation to test the hypothesis that Mac-1 deficiency in the recipient reduces accumulation of graft infiltrating cells and attenuates cardiac allograft rejection.

Results: In total-allomismatched murine cardiac allografts (BALB/c donor hearts and C57BL/6 recipients), survival averaged 13.8 ± 2.3 days (n=6) for allografts in Mac-1-deficient (Mac-1−/−) recipients compared to 8.3 ± 1.3 days (n = 18, p<0.0001) in wild-type (WT) recipients. At 7 d post-transplantation, allografts in Mac-1−/− recipients had significantly lower parenchymal rejection (PR) scores (2.3 ± 0.4) than WT recipients (3.0 ± 0.4, p<0.01). Allograft accumulation of neutrophils, T cells, and macrophages (Mϕ) significantly diminished in Mac-1−/− compared to WT recipients. To determine the cell type involved in acute graft dysfunction, we performed adoptive transfer experiments. Adoptive transfer of WT, but not Mac-1−/−, Mϕ exacerbated parenchymal rejection of 7-day allografts placed into Mac-1−/− recipients (PR score: 2.3 ± 0.4 for WT vs. 1.5 ± 0.5 for Mac1−/− Mϕ; p < 0.05), and significantly reduced graft survival (8.0 ± 1.4 vs. 13.5 ± 2.6 days, p<0.01). Neither WT nor Mac-1−/− neutrophil adoptive transfer affected graft survival in Mac-1−/− recipients. Mac-1−/− Mϕ co-cultured with allo-EC (BALB/c) expressed significantly lower levels of the co-stimulatory molecules CD40 and CD80, and mixed leukocyte reaction using allo-EC-primed Mac-1−/− Mϕ showed significantly lower antigen-presenting function than WT Mϕ. Despite attenuating acute rejection, recipient Mac-1-deficiency did not prevent graft arterial disease.

Conclusion: These studies demonstrate that Mac-1 contributes to acute allograft rejection by modulating macrophage recruitment, antigen presentation, and T cell priming. These observations establish a molecular target for modulating recipient responses to prolong graft survival.
Abstract 1131: PARP1 is required for Adhesion Molecule Expression in Atherogenesis
Authors: von Tobias Lukowicz , Hassa Paul O , Lohmann Christine , Borén Jan , Braunersreuther Vincent , Mach François , Odermatt Bernhard , Gersbach Monika , Camici Giovanni G , Tanner Felix C , Hottiger Michael O , Luescher Thomas F , Matter Christian M
Published: July 2021

Objective
- Atherosclerosis is a chronic inflammatory disease resulting from the interaction between modified lipoproteins, activated endothelial cells, macrophages, T-cells, and elements of the arterial wall. However, the key mediators linking recruitment of inflammatory cells to atherogenesis remain poorly defined. Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear enzyme that plays a role in acute inflammatory responses of cells and organs.

Methods and Results
- After 12 weeks of high-cholesterol diet, plaque formation in male
apolipoprotein E-
deficient mice was decreased by chronic inhibition of enzymatic PARP activity or genetic deletion of
PARP1
by 46 or 51 %, respectively (
P <
0.05, n ≥ 9). Pharmacological PARP inhibition using PJ34 or
PARP
1 deletion reduced PARP activity and diminished expression of inducible nitric oxide synthase (iNOS), vascular cell adhesion molecule-1 (VCAM-1), P-, and E-selectin (see Figure

). Furthermore, chronic PARP inhibition reduced plaque macrophage (CD68) and T-cell infiltration (CD3), increased fibrous cap thickness, and decreased necrotic core size and cell death (
P <
0.05, n ≥ 6).

Conclusions
- Our data provide pharmacologic and genetic evidence that endogenous
PARP1
is required for the development of atherosclerosis
in vivo
by increasing adhesion molecules due to endothelial activation, enhancing inflammation, and inducing features of plaque vulnerability. Thus, inhibition of PARP1 may represent a promising novel therapeutic target in atherosclerosis.
Abstract 1132: Antinflammatory role of macrophage Cyclooxygenase 2 derived prostaglandins in vivo
Authors: Hui Yiqun , Wang Dairong , Yu Zhou , Ricciotti Emanuela , Cheng Yan , FitzGerald Garret A
Published: July 2021

Nonsteroidal anti-inflammatory drugs, including those selective for cyclooxygenase (COX) - 2 relieve pain and inflammation. Thus, suppression of COX-2 derived proinflammatory products is a cardinal feature of NSAID efficacy. The use of conventional COX-2 knockout mice to explore this biology is constrained by the multiple roles of COX-2 in development and early postnatal life. To circumvent these limitations we generated mice with tissue-specific COX-2 deletion in macrophages (COX-2
Mac−/−
) confirmed by absence of LPS inducible mRNA and protein and maximal suppression of evoked formation of thromboxane (Tx), prostacyclin (PGI
2
) and prostaglandin E
2
by macrophages ex vivo. Deletion of macrophage COX-2 depressed LPS evoked systemic generation of these prostanoids by 35 – 40%. Surprisingly, deletion of macrophage COX-2 amplified the hypotensive response and blunted the thermal response to systemic LPS and shortened survival. Exposure of peritoneal macrophages to LPS ex vivo revealed a pro-inflammatory cytokine profile in the COX-2
Mac−/−
cells; higher levels of tumor necrosis factor - α and interleukin 6, but a reduction in interleukin 10. Products of COX-2 in macrophages can subserve both pro- and antinflammatory roles in vivo. Cell selective deletion of COX-2 permits delineation of cellular contributions to indices of systemic prostanoid biosynthesis and elucidation of prostanoid biology in complex multicellular disease processes, such as pertain in atherosclerosis.
Abstract 1143: Imaging of Vasa Vasorum in Atherosclerotic Plaque with
99m
Tc-labeled single chain-VEGF
Authors: Ohshima Satoru , Fujimoto Shinichiro , Tsimikas Sotirios , Kolodgie Frank D , Virmani Renu , Levashova Zoya , Backer Joseph M , Blankenberg Francis G , Petrov Artiom , Narula Jagat
Published: July 2021

Introduction:
Adventitial vasa vasorum proliferation and neointimal neovascularization are associated with intraplaque hemorrhage, expansion of necrotic core and hence plaque vulnerability. Increased expression of VEGF and its receptors accompany neoangiogenic process. We used
99m
Tc -labeled single chain VEGF (TcV) for developing potentially noninvasive imaging modality in experimentally induced aortic atherosclerotic lesion.

Methods :
Noninva-sive radionuclide imaging was performed with TcV (6.85 ±0. 27 mCi) in 6 NZW rabbits receiving high cholesterol diet (0.2% cholesterol, 4% fat) for one year and compared with 3 control rabbits receiving normal rabbit chow. Four hours after intravenous administration of TcV, micro SPECT/microCT imaging was performed for in vivo localization of tracer activity. Aortas were then explanted, and gamma counted for determination of % injected dose per gram (%ID/g). The aortas were then submitted for histopathologic characterization.

Results :
The uptake in thoracic aorta was clearly visualized non-invasively by TcV in vivo imaging in 4 of 5 rabbits in hypercholesterolemic rabbits, but not in the control animals. The %ID/g of each parts of aorta in hypercholesterolemic rabbits (Arch : 0.036 ± 0.020 %, Thoracic : 0.026 ± 0.012 %, Abd : 0.019 ± 0.009 %) was about 2.5-fold higher than that in control group (Arch : 0.014 ± 0.004 %, Thoracic : 0.009 ± 0.003 %, Abd : 0.009 ± 0.003 %) (figure a

). Ex vivo images of each group are shown as figure b

.

Conclusions :
This preliminary study suggests a potentially novel strategy for non-invasive imaging of neoangiogenesis in atherosclerotic plaque and may allow identification of unstable plaques.
Abstract 1162: Proteomic Characterization of the Secretome of Peripheral Blood-derived Smooth Muscle Progenitors
Authors: Simper David , Mayr Ursula , Benbow Ulrike , Newby Andrew , Xu Qingbo , Mayr Manuel
Published: July 2021

Background. Recent studies on circulating progenitor cells of the myeloid lineage have led to a new appreciation of their secretome, as paracrine factors may support tissue regeneration.

Methods and Results. Using a mass-spectrometry-based proteomic approach complemented by protein separation using two-dimensional gel electrophoresis, we compared the conditioned medium of plasma-derived smooth muscle progenitors (SPCs) and human aortic smooth muscle cells (SMCs). In total, over 150 individual protein features were identified. The majority of proteins were common to both cell lines, but higher amounts of the interstitial collagenase MMP-1, and of inflammatory cytokines, i.e. IL-8 and MCP-1, were found in the conditioned medium of SMCs compared to SPCs, which was subsequently verified by multiplex cytokine assays. ELISA measurements also confirmed that the concentrations of MMP-1 in the conditioned medium of SMCs exceeded the levels in the supernatant of SPCs. Although MMP-1 activity was partially antagonized in SMCs by a coordinated increase in TIMP-1, MMP-1 activity remained lower in SPCs. As a functional consequence, SMCs shedded more N-cadherin, an established MMP target, from their cell surface and showed a stronger invasive capacity. In contrast, SPCs produced more collagen, fibronectin and biglycan, but less perlecan than SMCs. A proteomic comparison between SPCs and endothelial progenitor cells (EPCs) revealed that the production of extracellular matrix proteins and the attenuated secretion of inflammatory cytokines was a specific characteristic of SPCs. EPCs secreted high levels of cathepsins, predominantly cathepsin D, B, Z and L, which were not detected in the conditioned medium of SPCs.

Conclusion. The present study constitutes the first comprehensive assessment of different circulating progenitors of the myeloid lineage demonstrating that apart from their potential physical contribution to lesion formation, SPCs might contribute to plaque stability by limiting inflammatory reactions and contributing to extracellular matrix deposition.