bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5322: Donepezil, an Acetylcholinesterase Inhibitor, Attenuates Atherogenesis in Apolipoprotein E Knockout Mice Through Antioxidative and Anti-inflammatory Effects
Authors: Inanaga Keita , Ichiki Toshihiro , Takeda Kotaro , Sunagawa Kenji
Published: April 2024

Objective Donepezil, a reversible acetylcholinesterase inhibitor, improves cognitive function of Alzheimer’s disease. Vagus nerve stimulation is known to attenuate inflammatory responses in mice with lipopolysaccharide-induced sepsis, which is designated cholinergic anti-inflammatory system. We investigated whether the pharmacological stimulation of cholinergic system by donepezil affects atherogenesis in apolipoprotein (Apo) E-knockout (KO) mice.

Methods and Results Male ApoE-KO mice (10-week-old) were fed a high fat diet (HFD) and received infusion of angiotensin (Ang) II (1.9 mg/kg/day). Oral administration of donepezil (5 mg/kg/day) or infusion of physostigmine (2mg/kg/day), another acetylcholinesterase inhibitor, for 4 weeks did not change hemodynamics or cholesterol level, but significantly reduced Oil Red O-positive area of en face aorta (Control 3.8±0.4%, AngII + HFD 13.6±2.2%, AngII + HFD and donepezil 6.9±0.9%*, AngII + HFD and physostigmine 8.2±0.6%*, *P<0.05 vs AngII + HFD) and macrophage infiltration into the aortic root (p<0.05). Donepezil also decreased monocyte chemoattractant protein (MCP)-1-positive area by 45% (p<0.05) in the aortic sinus. Donepezil administration also attenuated atherogenesis in ApoE-KO mice fed a HFD for 8 weeks without AngII infusion. Quantitative RT-PCR revealed that administration of donepezil to ApoE-KO mice treated with AngII and a HFD significantly suppressed expression of MCP-1 and tumor necrosis factor-alpha 52% and 70% reduction, respectively, P<0.05) in the aorta. Donepezil significantly suppressed NADPH oxidase activity (43% reduction, P<0.05) and production of reactive oxygen species in the aorta. Preincubation with acetylcholine completely inhibited AngII-induced NADPH oxidase 1 expression in cultured vascular smooth muscle cells (P<0.05).

Conclusion The present study revealed a novel anti-atherosclerotic effect of pharmacological stimulation of cholinergic system by donepezil. Donepezil may increase acetylcholine availability and downregulate NADPH oxidase followed by suppression of inflammatory cytokine production and atherogenesis. Cholinergic stimulation may be a novel therapeutic strategy for atherosclerotic cardiovascular diseases.
Abstract 5445: Tissue Kallikrein Deficiency Impairs Circulating Proangiogenic Cells Invasive Capacity
Authors: Spinetti Gaia , Fortunato Orazio , Cordella Daniela , Alhenc-Gelas Francois , Ritcher Christine , Vincent Marie-Pascale , Tonolo Giancarlo , Cherchi Sara , Emanueli Costanza , Madeddu Paolo R
Published: April 2024

Background: Circulating proangiogenic cells (PACs) express the serine protease tissue kallikrein (K1), which favours invasion through metalloproteinases activation.

Aim: To determine whether inherited or disease-induced K1 deficiency alters PACs migration and proangiogenic capacity, and whether K1-deficient PACs transduction with wild type or polymorphic variant (R53H) K1 can rescue functional defects.

Methods & Results: Wild type (WT, n=8) or K1-knockout mice (K1KO, n=7) lineage negative (Lin-) bone marrow mononuclear cells (BMMNC) were used in migration assays or differentiated into PACs. K1KO Lin-BMMNC showed defective migration to SDF-1
α
compared to WT (fold increase vs vehicle 2.2, p=N.S and 5.8, p<0.05 vs vehicle and K1KO, respectively, n=3). Similarly impaired was K1KO PACs migration (n=3, p<0.05 vs WT). Further, K1KO PACs were unable to promote endothelial cell (EC) networking in vitro (p<0.05). Adenovirus-mediated K1 transduction rescued both defects in K1KO PACs (n=3, p<0.05 vs Ad.Null). Next, we studied type 2 diabetic patients and age-matched healthy controls circulating PACs (n<6 per group). Diabetic PACs showed reduced migration and proangiogenic potential (−1.8 and 2.0 fold vs controls, p<0.05) and lower K1 protein (−56.6 %, p<0.05), but normal K1 mRNA abundance, pointing at a post-transcriptional defect. K1 overexpression increased healthy PACs migration and EC networking (p<0.01 vs Ad.Null), but could not revert diabetic PACs liabilities (p±N.S. vs Ad.Null). We then verified if K1 increases PACs invasive capacity. K1-PACs invaded matrix more efficiently (2.5-fold increase, p<0.01 vs Null-PACs). In contrast, the polymorphic variant R53H, which encodes for an enzymatic defective K1 form, did not improve invasion. Moreover, K1-PACs failed to invade the matrix when regular collagen was substituted with glycated one.

Conclusions: Inherited or diabetes-induced K1 deficit hampers PACs invasive and proangiogenic capacities, with K1 gene therapy being able to restore those functions except in diabetes. Diabetes could interfere with K1 enzymatic activity and make extracellular matrix impenetrable to PACs. Alternatively, additional migratory machinery deficit may overwhelm benefits of K1 overexpression.
Abstract 5482: Unexpected Role for Connexin37 in Circulating FVIII and Coagulation
Authors: Burnier Laurent , Pfenniger Anna , Taffet Steven , Delmar Mario , Fontana Pierre , Angelillo-Scherrer Anne , Kwak Brenda R
Published: April 2024

The gap junction protein connexin37 (Cx37) plays an important role in cell-cell communication in the vasculature. Cx37 is expressed in endothelial cells, platelets and megakaryocytes. We have recently shown that Cx37 limits thrombus propensity by permitting intercellular signaling between aggregating platelets. Here, we have performed high throughput phage display to identify potential binding partners for the regulatory intracellular C-terminus of Cx37 (Cx37CT). We retrieved 2 consensus binding motifs for Cx37CT: WHK…[K,R]XP… and FH-K…[K,R]XXP…. Sequence alignment against the NCBI protein database indicated 66% homology of one the selected peptides with FVIII B-domain. We performed cross-linking reactions using BS3 and confirmed that an 11-mer peptide of the FVIII B-domain sequence linked to recombinant Cx37CT. In vitro binding of this peptide to Cx37CT was also confirmed by surface plasmon resonance. The dissociation constant of FVIII B-domain peptides to Cx37CT was ~20 uM. Other peptide sequences, designed upstream or downstream of the FVIII B-domain sequence, showed very low or no affinity for Cx37CT. Finally, in vivo studies revealed that thrombin generation in platelet-poor plasma from Cx37−/− mice (endogenous thrombin potential: 634±11 nM min, mean±SEM) was increased compared to Cx37+/+ mice (427±12, P<0.001). Moreover, partial activated thromboplastin time (aPTT) was shorter in Cx37−/− (39.7±1.5 s) than in Cx37+/+ mice (45.9±1.8, P=0.03), whereas prothrombin time was comparable. The shorter aPTT in Cx37−/− mice correlated with higher circulating FVIII activity (46.0±0.7 vs. 53.5±2.7 s for Cx37+/+, P=0.03). Overall, our data show for the first time a functional interaction between FVIII and Cx37. This interaction may be relevant for the control of FVIII secretion and, thereby, in the regulation of levels of FVIII circulating in blood. In addition, these results may open new perspectives to improve the efficiency of recombinant FVIII manufacturing.
Abstract 5492: Risk of Major Adverse Cardiovascular Events in Patients on Clopidogrel and Proton Pump Inhibitors: A Meta-analysis
Authors: Singh Mukesh , Singh Sarabjeet , Molnar Janos , Arora Rohit
Published: April 2024

Background: The anti-platelet properties of clopidogrel are thought to be activated by cytochrome P450 2C19. This isoenzyme is potently inhibited by the most commonly prescribed proton pump inhibitors (PPIs) and therefore may interfere with clopidogrel activation and its anti-platelet effects. However, recently published studies have yielded mixed results, leaving the question unanswered. Hence a meta-analysis was performed to evaluate the potential impact of PPIs on the effectiveness of clopidogrel to prevent major adverse cardiovascular events (MACE).

Methods: A systematic review of literature revealed 5 studies involving 29749 patients. End points analyzed were major adverse cardiovascular events (acute myocardial infarction, repeat revascularization, death due to cardiac causes. Combined relative risks (RR) across all the studies and the 95% confidence intervals were computed using the Mantel-Haenszel fixed-effect model. A two-sided alpha error < 0.05 was considered to be statistically significant.

Results: There were no significant differences in patient demographics between both groups. Compared with patients on clopidogrel without PPI, the risk of MACE was significantly higher in patients taking clopidogrel with PPI (RR: 1.65, CI: 1.55 – 1.76; p=0.000).

Conclusions: The present meta-analysis suggests that combinig PPI with clopidogrel increases risk of MACE and it may be a class effect.
Abstract 5503: Sonothrombolysis in a Rabbit Model of Acute Insoluble Ischemic Stroke
Authors: Flores Rene , Lowery John D , Hennings Leah J , Brown Aliza T , Skinner Robert D , Culp William C
Published: April 2024

Introduction:
Evidence suggests that low-frequency ultrasound (US) improves tissue perfusion in the rabbit ischemic limb and enhances myocardial perfusion even without removing the arterial obstruction. We evaluated US-facilitated thrombolysis, or sonothrombolysis, with microbubbles (MB) and tissue plasminogen activator (tPA) for decreases in infarct volume in rabbits with insoluble middle cerebral artery (MCA) occlusions.

Hypothesis:
Sonothrombolysis with MB or tPA is effective at reducing infarcts without lysis of arterial obstruction.

Methods:
New Zealand White rabbits (n = 34; 5.2±0.07 kg) received angiography and three embolic spheres (diameter = 700 to 900
μ
m) were injected into the internal carotid artery occluding the MCA. Rabbits were randomly assigned to one of four groups. Control (n = 10) rabbits were embolized but received no US, tPA, or MB. Other groups were tPA (n = 7) without US, tPA+US (n = 9), and MB+US (n = 8). Treatment was initiated one hour following angiographic verification of occlusion. Rabbits with US received pulsed wave US (1 MHz at 0.8 W/cm
2
) for 1 hour and rabbits with tPA received intravenous tPA (0.9 mg/kg) over 1 hour. Rabbits with MB received intravenous MB (0.16 mg/kg) over 30 minutes. Rabbits were sacrificed at 24 hours and infarct volume was determined using vital stains on brain sections.

Results:
Percent infarct volume averaged 3.4±0.9%, 2.0±1.1%, 1.8±1.0%, and 0.92±0.9% for control, tPA, MB+US, and tPA+US rabbits, respectively; infarct volume tended (P = 0.066) to be decreased for rabbits treated with tPA+US compared to controls. Infarct volume did not differ (P > 0.26) between controls and rabbits treated with tPA and MB+US.

Conclusions:
Combined treatment with tPA and US shows a trend towards decreasing brain infarct volume in rabbits with permanent MCA occlusion, but not to the extent found in muscle or myocardium at lower frequencies.
Abstract 5519: Enhanced Age-related Endothelial Dysfunction in Genetic Deletion of JunD
Authors: Osto Elena , Coppolino Giuseppe , Mateescu Bogdan , Akhmedov Alexander , Malinski Tad , Perna Enrico , Mechta-Grigoriou Fatima , Matter Christian , Luescher Thomas F , Cosentino Francesco
Published: April 2024

JunD is a transcription factor that regulates genes involved in antioxidant defense. This study aimed to investigate whether JunD deficient mice (
JunD
−/−
) are more prone to age-related, oxidative stress-mediated, endothelial dysfunction in comparison to age-matched wild type (WT) mice.

Methods.
Thoracic aortic rings from young (3 months old), middle aged (6 months old) and old (22 months old) male
JunD
−/−
and WT mice were suspended for isometric tension recording. Endothelium-dependent relaxation to acetylcholine (Ach, 10
−9
–10
−6
mol/L) was assessed after submaximal contraction with norepinephrine (10
−6
mol/L). Calcium ionophore stimulated nitric oxide (NO), superoxide anion (O
2
−
) and peroxynitrite (ONOO
−
) were measured with electrochemical nanosensors placed near the surface (5±2
μ
m) of a single endothelial cell.

Results.
The age-associated impairment of endothelium-dependent relaxations to Ach (Ach, 10
−9
–10
−6
mol/L) was significantly enhanced in
JunD
−/−
as compared to age-matched WT. Maximal relaxations were 55±5 vs 78±4% at 6 months and 39±3 vs 50±2% at 22 months for JunD−/− and WT mice, respectively (n=6 – 8, p<0.05 vs age-matched group). Endothelium-independent relaxations to sodium nitroprusside (10
−10
–10
−5
mol/L) did not differ in
JunD
−/−
and WT of different age groups (n=6 – 8, p<NS). Age-induced decrease of NO production was higher in
JunD
−/−
as compared with WT (475±32 vs 350±28 nmol/L and 358±26 vs 220±23 nmol/L for 6 and 22 months old WT and
JunD
−/−
, respectively; n=3–5, p<0.05 vs age-matched group). O
2
−
and ONOO
−
generation increased with age in WT and more significantly in
JunD
−/−
mice (O
2
−
, 67±6 vs 103±8 nmol/L and 116±9 vs 210±16 nmol/L; ONOO
−
, 224±17 vs 319±22 nmol/L and 313±21 vs 492±29 nmol/L for 6 and 22 months old WT and
JunD
−/−
, respectively; n= 3–5, p<0.05 vs age-matched group). eNOS and MnSOD protein expression was downregulated in
JunD
−/−
mice as compared with WT controls (n=3–5, p<0.05 vs age-matched group). Relaxations to Ach in
JunD
−/−
mice were restored by free radical scavengers superoxide dismutase (SOD) (150 U/ml) and catalase (1200 U/ml).

Conclusion
. Our results indicate that JunD protects against vascular oxidative stress providing new insights into the pathophysiology of age-associated endothelial dysfunction.
Abstract 5558: Inflammasome Activation in Cardiac Fibroblasts is Essential for Myocardial Ischemia-reperfusion Injury
Authors: Kawaguchi Masanori , Takahashi Masafumi , Hata Takeki , Kashima Yuichiro , Takahashi Yasuko , Sagara Junji , Taniguchi Shun’ichiro , Ikeda Uichi
Published: April 2024

Background:
Inflammatory responses play a key role in the pathophysiology of myocardial ischemia-reperfusion (I/R) injury. We previously investigated the role of ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), an adaptor protein that forms inflammasome whose activation leads to caspase-1-dependent interleukin (IL)-1
β
maturation, in myocardial I/R injury and found that ASC deficiency prevented IL-1
β
secretion and inflammatory cell infiltration, resulting in improving myocardial injury and remodeling. In this study, we investigated the mechanisms by which I/R initiates activation of inflammasome in the heart.

Methods and Results:
To examine what kind of cells are responsible for inflammasome activation in the heart, primary cardiomyocytes (CM) and cardiac fibroblasts (CF) from the wild-type mice were prepared. Although ASC was clearly expressed in both CM and CF, IL-1
β
secretion was detected in only CF after stimulation with relatively high dose of lypopolysaccharide (LPS: 100 ng/mL). The intracellular pro-IL-1
β
accumulation in CF was similar between wild-type (WT) and ASC deficient (ASC
−/−
) mice; however, IL-1
β
processing and secretion were significantly inhibited in ASC
−/−
-CF, compared with that in WT-CF (p<0.01). Hypoxia/reoxygenation (H/R: 6 h of H following 18 h R) dramatically induced IL-1
β
secretion in CF after low dose (10 ng/mL) LPS priming. To determine the mechanisms by H/R might activate inflammasome, CF were treated with ATP (10 mM), HMGB1 (10 mg/mL), and K+ ionophore nigericin (1mM) after LPS priming. Marked IL-1
β
secretion was detected only in nigericin-treated CF. Further, H/R-induced IL-1
β
secretion was significantly inhibited by the treatment with antioxidant N-acetyl-L-cysteine (NAC: 10 mM), K
+
channel blockers (glimepiride 10 mM, glibenclamide 10 mM, TEA 10 mM) or high extracellular K
+
(130 mM), indicating that inflammasome activation in response to H/R might be mediated through K+ efflux and ROS production.

Conclusion:
These findings demonstrate that initial activation of inflammasome in response to I/R occurs in CF, but not in CM, in the heart, and suggest that inflammasome in the CF could be a potential target for the treatment of myocardial I/R injury.
Abstract 5569: Neointimal Hyperplasia After Mechanical Endovascular Injury is Augmented Through Angiotensin II-mediated Regional Vascular Inflammation via Activation of the JNK Pathway in Angiotensin-converting Enzyme 2-deficient Mice
Authors: Sahara Makoto , Sata Masataka , Morita Toshihiro , Hirata Yasunobu , Nagai Ryozo
Published: April 2024

Background:
Angiotensin-converting enzyme 2 (ACE2) is a negative regulator of the renin-angiotensin system, although its role on vascular inflammation and atherosclerosis remains unclear.

Methods and Results:
Male ACE2-deficient (ACE2
−

/y

) and wild-type (ACE2
+

/y

) mice underwent wire-mediated endovascular injury in femoral artery. After 4 weeks, the injured artery in ACE2
−

/y

mice exhibited marked neointimal hyperplasia compared with that in ACE2
+

/y

mice. The intima/media ratio in the vascular lesion was 1.97±0.31 in ACE2
+

/y

mice and 2.78±0.49 in ACE2
−

/y

mice (
P
<0.05). There were no significant differences in blood pressure between both mice. In histology, the accumulation of macrophages in lesions was significantly augmented in ACE2
−

/y

mice. ACE2 protein expression in vascular cells in wild-type mice was detected by immunohistochemistry and western blotting, and the concentration of angiotensin II (Ang-II) in the injured artery was higher in ACE2
−

/y

mice than in ACE2
+

/y

mice. In addition, in the injured artery, quantitative mRNA expressions of vascular inflammatory molecules such as VCAM-1, MCP-1, TNF
α
, and MMP9 were increased more markedly in ACE2
−

/y

mice. In contrast, an ARB, olmesartan, attenuated the lesion formation and mRNA expressions of these molecules in the injured artery in ACE2
−

/y

mice. Next, we analyzed phosphorylation status of ERK and JNK in aortic vascular smooth muscle cells (VSMCs), which were derived from ACE2
+

/y

or ACE2
−

/y

mice and were stimulated with Ang-II. Although Ang-II increased ERK and JNK phosphorylation in VSMCs of both mice, the increase was greater in VSMCs of ACE2
−

/y

mice. Moreover, we evaluated which signaling pathway mediated Ang-II-induced upregulation of VCAM-1 and MMP9 in VSMCs. Although an ERK inhibitor PD98059 had no impact on Ang-II-induced upregulation of VCAM-1 and MMP9 mRNA expressions, a JNK inhibitor SP600125 significantly attenuated them, suggesting the central role of the JNK pathway in transducing the impact of Ang-II on vascular inflammation.

Conclusions:
ACE2 deletion induces marked neointimal formation in femoral artery after vascular injury through Ang-II-mediated regional vascular inflammation via activation of the JNK pathway. ACE2 may confer protection against vascular diseases.
Abstract 5575: Ca2+/Redox-Sensitive Tyrosine Kinase PYK2 Promotes Atherogenesis Through OxLDL/ROS/p21-Dependent Premature Senescence in Endothelium
Authors: Katsume Asako , Okigaki Mitsuhiko , Matsui Akihiro , ji shen Che , Yamaguchi Shinihiro , Kishita Eigo , Yamada Hiroyuki , Matsubara Hiroaki
Published: April 2024

Background: Calcium/redox-sensitive tyrosine kinase PYK2 plays crucial roles in cell migration. Analysis of PYK2-deficient macrophages has been reported that their directional migration was impaired due to dysfunction of PI3K, small GTPase Rho, and Ca (2+)-mobilization. Since monocyte/macrophage plays crucial role in atherogenesis, we newly generated the PYK2-knock-out mice and investigated the role of PYK2 in atherogenesis.

Methods: PYK2−/− mice (P-KO) were crossbred with ApoE−/− mice (E-KO), and PYK2−/−/ApoE−/− double knockout (D-KO) mice were established. Four-week-old mice were fed with high-fat-diet for 8 weeks, and the thoracic aorta and aortic root were histologically analyzed. Then we studied the mechanisms of their difference in atherogenesis.

Results: At the 8th week, atherosclerotic area of thoracic aorta in D-KO was less than E-KO (~54%, P<0.01, n=15, respectively). Numbers of infiltrating macrophage in D-KO mice was also reduced (~52%, P<0.01, n=15). We also verified that no significant difference in atherosclerotic area between E-KO recipient with D-KO bone marrow and D-KO recipient with E-KO bone marrow (n=6). At the 7th day, in E-KO aorta, we detected the senescence-associated-beta-gal staining area, however in D-KO aorta there is no area stained with SA-beta-gal. The expression of p21 was significantly increased in E-KO aorta at the 7th day, but in D-KO aorta there is no increase in p21 expression. In primary-cultured endothelial cells (ECs), Lysophosphatidylcholine (LPC) induced p21 expression in WT was significantly higher than that in P-KO. And we studied that these results were caused by ROS and JNK dependent pathway. The stained area with SA-beta-gal and the expression of p21 was impaired by intra peritoneal injection with JNK inhibitor (SA600125). The stained area with 8OHDG in E-KO was higher than that in D-KO, and the number of the stained endothelial cells with DCF in WT was much higher than that in P-KO. The expression of VCAM-1 and MCP-1 in E-KO was significantly increased than that in D-KO.

Conclusion: PYK2 promotes atherogenesis by inducing ROS/p21-dependent premature senescence and cytokine induction in endothelium
Abstract 5599: Secreted Frizzled Protein 2, a Cardioprotective Gene is Upregulated in Mesenchymal Stem Cells Following Engraftment in the Injured Myocardium
Authors: Kavanagh Claire , Duffy Garry P , Mashayekhi Kaveh , O’Brien Timothy , Barry Frank
Published: April 2024

A variety of stem cells including Mesenchymal Stem Cells (MSCs) have been shown to have therapeutic potential in the treatment of myocardial damage. While the mechanism of action still remains unknown, MSCs have been shown to produce and secrete a broad variety of cytokines, chemokines and growth factors that may potentially be involved in cardiac repair. This has been confirmed by reports that human MSCs can exert favourable cardiac effects after myocardial infarction (MI) without persistence, engraftment, transdifferentation or cell fusion. Thus, the mechanisms responsible for their functional benefits appear to be attributable to paracrine effects. Secreted frizzled protein 2 (SFRP2) is expressed in MSCs following Akt gene transfer and has been shown to have paracrine protective effects. The objective of this study was to determine the effect of the ischemic environment on the engrafted MSC in order to understand the host - stem cell interaction. Using laser microdissection (LMD), the engrafted MSCs were retrieved from the injured myocardium 1 week post MI. To determine if SFRP2 was upregulated in our retrieved MSC population, RT PCR analysis of the retrieved MSCs Vs control MSCs was performed. SFRP2 was found to be 5,700 fold upregulated in MSCs exposed to the infarcted myocardium compared to control MSCs. Subsequent in vitro cardioprotection assays identified SFRP2 as a potent protector of cardiomyocytes during hypoxia treatment. Mitochondrial activity increased by 52% when neonatal rat cardiomyocytes were treated with SFRP2 recombinant protein during hypoxia treatment when compared to hypoxia alone. Caspase activity was also decreased in cardiomyocytes treated with SFRP2 during hypoxia. Cells also showed a healthy morphology and maintained beating after exposure to hypoxia. These data suggest that the injured host can induce MSCs to express cytoprotective factors such as SFRP2 and identify the host - stem cell interaction as an important determinant of the therapeutic effect of stem cell administration.
Abstract 5602: The Transcription Factor Islet-1: A Novel Gene Target for Future Cardiac Repair?
Authors: Barzelay Aya , Hochhauser Edith , Keren Gad , George Jacob
Published: April 2024

The LIM-homeobox transcription factor isl1 plays a crucial role during heart embryogenesis. Embryonic isl1+ precursors give rise to over two-thirds of the heart and to its subsequent lineages: cardiac muscle, smooth muscle and endothelium. Interestingly, a subset of Isl1+ progenitors remains embedded in the postnatal heart. In the current study, we investigated whether isl1 is expressed in
adult
mesenchymal stem cells (MSCs), and after experimental myocardial infarction (MI). Additionally, we examined whether isl1 overexpression in MSCs could promote their vasculogenic properties, and whether intramyocardial gene transfer of naked DNA encoding isl1 could promote a functional recovery after MI. We used the transgenic mice
isl1/cre/Z/EG
, in order to detect isl1 expression in MSCs of adult mice, complemented by qRT-PCR and immunostaining for isl1 detection in rat- MSCs. Four weeks after MI induction, isl1 expression was assessed in bone marrow and spleen cells by qRT-PCR and immunostaining. Isl1 was retrovirally transduced to MSCs. Endothelial markers were examined by FACS and tube formation capacity was assessed on matrigel. Furthermore, intramyocardial injection of plasmid encoding isl1 to mice after ligation of the LAD was performed. We report for the first time, the identification of isl1+ progenitors in
adult
bone marrow. The number of isl1+ progenitors increased after
in vitro
cell culture, and also in the splenocytes after acute experimental myocardial infarction. Isl1 overexpression in MSCs promoted their differentiation towards endothelium. Finally, isl1 gene transfer to the peri-infarct region after MI induction resulted in partial salvage of ventricular function evident by echocardiography. Thus, the isl1 gene may stand as an attractive target for future cell based therapy for regenerative myocardial dysfunction.
Abstract 5645: Novel Cross-Talk Between Oxidized LDL, Angiotensin II and Oxidative Stress After AT1 Blockade and AT1a/AT1b Double Knockout
Authors: Morawietz Henning , Catar Rusan A , Goettsch Claudia , Taye Ashraf , Muller Gregor , Lehmann Susann , Ziegler Christian G , Bornstein Stefan R , Krug Alexander W , Walther Thomas
Published: April 2024

Oxidized low-density-lipoprotein (oxLDL) and angiotensin II (Ang II) can increase formation of reactive oxygen species in endothelial cells. However, the potential cross-talk between both risk factors, the impact of Ang II receptor 1 (AT
1
) blockade on lipoprotein-induced oxidative stress and endothelial function and of AT
1a
/AT
1b
double knockout on oxidative stress is not well understood. First, we studied the impact of native and oxidized LDL (nLDL, oxLDL) on Ang II receptor expression and formation of reactive oxygen species in primary cultures of human umbilical artery endothelial cells (HUAEC). Native and oxLDL (max. 100
μ
g/mL) induced after 1 h AT
1
(3.3- and 3.7-fold) and AT
2
(1.8- and 2.8-fold) receptor mRNA expression in HUAEC. Increased AT
1
and AT
2
mRNA and protein expression could be observed for up to 24 h. Both lipoproteins increased intracellular AT
1
receptor immunofluorescence in HUAEC. Native LDL and oxLDL activated MEK/ERK and p38 MAPK pathways (ELISA, Western blot) in HUAEC. OxLDL induced oxLDL receptor LOX-1 expression in HUAEC (RT-PCR, Western blot). Induction of both Ang II receptors and of LOX-1 by oxLDL was reduced by AT
1
receptor antagonist candesartan. OxLDL induced in contrast to native LDL superoxide anion formation in HUAEC (chemiluminescence). AT
1
receptor blockade prevented lipoprotein-induced oxidative stress in HUAEC like SOD. Furthermore, AT
1
blockade prevented impaired endothelial function (increase of log EC
50
values and impaired max. relaxation by oxLDL in phenylephrine-preconstricted vessels). In aortic rings of wild-type mice, oxLDL-induced vascular superoxide anion formation was reduced by AT
1
blockade. Finally, deletion of AT
1
receptor subtypes 1a and 1b in AT
1a
/AT
1b
double knockout mice resulted in the aorta in significant downregulation of protein expression of LOX-1, Nox subunits Nox2, p47phox and p22phox, compared with wild-type mice. In conclusion, augmented vascular oxidative stress and endothelial dysfunction in response to lipoproteins involves induction of the AT
1
receptor. Double knockout of AT
1
receptors reduce LOX-1 and NAD(P)H oxidase expression. Our data suggest a cross-talk between oxidized LDL, angiotensin II and oxidative stress after AT
1
blockade and AT
1a
/AT
1b
double knockout.
Abstract 5662: Reductions in Atrophic E3 Ubiquitin Ligase Expression in Pulmonary Arteries Correlate With the Development of Pulmonary Hypertension
Authors: Paffett Michael L , Lund Amie K , Campen Matthew J
Published: April 2024

Vascular remodeling in pulmonary hypertensive syndromes is driven by increased activity of nuclear factor of activated T-cells (NFAT) in smooth muscle cells. Although much is known about the regulation of NFAT activity by calcineurin, very little is known about vascular smooth muscle atrophy F-box ubiquitin E3 ligase expression, which targets calcineurin for proteosomal degradation and inhibits pathological hypertrophy. Therefore, we hypothesized the ubiquitin E3 ligases muscle atrophy F-box (MAFbx) and muscle ring finger (MuRF-1) expression would decline during the development of pulmonary hypertension. To investigate this hypothesis, rats were given a single injection of MCT (50 mg/kg i.p.) and indices of pulmonary hypertension (RVSP and RV/LVS) were measured at 0, 7, 14, 28 and 42 days post MCT injection (n=7–10/group). Left and right pulmonary arteries were harvested from each rat at the conclusion of these measures and mRNA expression was assessed by real-time multiplex PCR analysis for MAFbx and MuRF-1. Consistent with our hypothesis, pulmonary vascular smooth muscle MAFbx and MuRF-1 expression were attenuated from 14d to 42d time points compared to 0d when normalized to b-actin mRNA expression, which correlated negatively with RVSP and right ventricular hypertrophy. Interestingly, while treatment with sildenafil from days 28 – 42 reduced RVSP and RV/LVS, MAFbx expression was further compromised. These results suggest that the E3 ubiquitin ligases MAFbx and MuRF-1 may play a role in the development of MCT-induced pulmonary hypertension.
Abstract 5682: Novel Role of Copper Transporters CTR1 and ATP7A in PDGF-induced Vascular Smooth Muscle Cell Migration and Vascular Remodeling
Authors: Ashino Takashi , Urao Norifumi , Oshikawa Jin , Wang Huan , Huo Yuqing , McKinney Ronal D , Ushio-Fukai Masuko , Fukai Tohru
Published: April 2024

Platelet-derived growth factor (PDGF) stimulates vascular smooth muscle cell (VSMC) migration primary through the PDGF receptor-beta (PDGFR), which contributes to vascular remodeling in vivo. Previous study shows that copper chelation inhibits neointimal formation in response to injury. However, underlying mechanisms remain unknown. Intracellular copper levels are tightly regulated by the copper importer CTR1 which localizes at plasma membrane (PM) and the copper exporter ATP7A which traffics between trans-Golgi network (TGN) to PM. Here we show that both CTR1 and ATP7A are expressed in cultured VSMC. Co-immunoprecipitation assay shows that ATP7A binds to PDGFR basally and PDGF stimulation rapidly promotes their association within 5 min, which is prevented by knockdown of CTR1 with siRNA (85%). Proteomic analysis in caveolae/lipid raft (C/LR) fraction reveals that ATP7A is localized in C/LR where PDGFR is found. PDGF stimulation promotes recruitment of ATP7A to C/LR fraction, which is inhibited by CTR1 siRNA (95%). Furthermore, inductively coupled plasma mass spectrometry (ICP-MS) shows that C/LR contains high amount of copper (139.7 (C/LR) vs. 17.9 (non-C/LR) ppb/mg proteins). In response to wound injury or PDGF stimulation, ATP7A translocates from TGN to PM at the leading edge where it colocalizes with PDGFR and the LR marker cholera toxin subunit B in actively migrating VSMCs, which is prevented by CTR1 siRNA or C/LR disruptor methyl-beta cyclodextrin. Note that this ATP7A translocation is associated with decrease in copper levels in C/LR, suggesting increase in copper efflux in C/LR during active VSMC migration. Functionally, PDGF-induced VSMC migration is significantly inhibited by knockdown of CTR1 or ATP7A with siRNAs (84% and 68%, respectively) as well as treatment with cell permeable copper chelator TTM (50.2%) and cell impermeable copper chelator BCS (39.1%). Moreover, ATP7A expression is dramatically increased in the neointima in a mouse wire injury model. In summary, CTR1-dependent translocation of the copper exporter ATP7A to C/LR and its binding to PDGFR plays an important role in PDGF-induced VSMC migration, which may contributes to vascular remodeling in response to injury and development of atherosclerosis.

This research has received full or partial funding support from the American Heart Association, Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).
Abstract 5756: The MicroRNA-17–92 Cluster Exhibits a Cell Intrinsic Anti-angiogenic Function in Endothelial Cells
Authors: Döbele Carmen , Bonauer Angelika , Fischer Ariane , Reiss Yvonne , Zeiher Andreas M , Dimmeler Stefanie
Published: April 2024

MicroRNAs (miRs) are endogenously expressed small noncoding RNA molecules that are able to regulate gene expression on the posttranscriptional level by binding to the mRNAs of their target genes, thus inducing inhibition of translation or mRNA degradation. The miR-17–92 cluster (encoding miR-17, -18a, -19a, -20a, -19b and miR-92a) was shown to augment tumor angiogenesis if overexpressed in tumor cells. Whereas miR-92a was recently identified as negative regulator of vessel growth, the individual functions of the other members of the miR-17–92 cluster in endothelial cells is less clear and only a combination of miR-17, -18a and -20a was shown to partially rescue the defective angiogenesis under conditions of Dicer deficiency. To investigate the involvement of the individual members of the miR-17–92 cluster in angiogenesis, human umbilical vein endothelial cells (HUVECs) were transfected with precursor molecules for miR-17, -18a, -19a and -20a. Overexpression of the different members significantly inhibited 3D spheroid sprouting (Pre-17:10±3%,-18a: 28±5%,-19a: 45±4%,-20a: 33±8% of control) and impaired the assembly of capillary networks in vitro. Conversely, inhibition of miR-17 (142±15% of control), miR-18a (152±6% of control) and miR-20a (153±9% of control) augmented spheroid sprout formation. Next, we addressed the in vivo relevance by using antagomirs to block the individual miRs. Antagomir-17 (which also blocks miR-20a) significantly increased the number of lectin-positive vessels in matrigel plugs (190±26% of control), whereas antagomirs, which specifically affect miR-18a and miR-19a were less effective. Antagomir-17 additionally improved blood flow after hind limb ischemia (217±24% of control) and increased tumor growth (140±14% of control). To identify direct targets of miR-17, we combined microarray gene expression analysis with target prediction. Among others, Janus kinase 1 (Jak1) was significantly downregulated on mRNA (38±5% of control) and protein (41±14% of control) level by miR-17. In summary, we show that the individual members of the miR-17–92 clusters exhibit a cell intrinsic anti-angiogenic activity in endothelial cells. Inhibition of miR-17 efficiently increased neovascularization and tumor growth in vivo.
Abstract 5777: SIRT1 Inhibits VSMC Proliferation and Migration Underlying Neointima Formation Following Vascular Injury
Authors: Li Li , Zhang Hui-Na , Chen Hou-Zao , Gao Peng , Zhang Qing-Jun , Zhang Ran , Lv Xiang , Liu De-Pei , Liang Chih-Chuan
Published: April 2024

Vascular SMC (VSMC) proliferation and migration are crucial events involved in the pathophys-iology of vascular diseases. SIRT1, a class III histone deacetylase (HDAC), has been implicated in controlling angiogenesis. However, the role of SIRT1 during the response of VSMCs to injury remains unknown. To study the role of SIRT1 in the neointima formation, we generated transgenic mice expressing the human SIRT1 from a minimal
SM22α
promoter. We demonstrated that in a mouse carotid artery ligation model, SMC-specific overexpression of SIRT1 substantially reduced neointima formation. SIRT1 overexpression inhibited VSMC proliferation and migration, and induced cell cycle arrest at the G1/S transition. Accordingly, overexpression of SIRT1 decreased cyclin D1 and MMP-9 expression at both mRNA and protein level, whereas knockdown of SIRT1 by RNAi resulted in the reverse effect on their protein expression. Moreover, decreased cyclin D1 and MMP-9 expression/activity were observed in injured carotid arteries of SMC-SIRT1 Tg mice. In addition, two components of activator protein-1(AP-1), c-Fos and c-Jun, interacting with and being deacetylated by SIRT1, were involved in the downregulation of cyclin D1 and MMP-9 expression. These findings demonstrate the inhibitive effect of SIRT1 on neointima formation after vascular injury and suggest a potential pharmacological target for prevention of vascular diseases.
Abstract 5802: Ischemia-induced Angiogenesis is Impaired in Aminopeptidase-A deficient Mice with Decreased Stability of HIF-1
α
Authors: Kubota Ryuji , Numaguchi Yasushi , Ishii Masakazu , Naruse Keiji , Okumura Kenji , Murohara Toyoaki
Published: April 2024

Background:
Aminopeptidase A (APA) is a membrane-bound metalloprotease degreading bioactive peptidase by cleaving from N-terminal amino acids with several functions in angiogenesis. To assess whether APA regulates ischemia-induced angiogenesis, we explored the mechanism by which APA affects angiogenesis under hypoxia conditions and evaluated the process of perfusion recovery in the hindlimb ischemia model of APA knock-out mice (APAKO).

Methods and Results:
Western blotting analysis and immunostaining of endothelial cells (ECs) from mouse aorta revealed that the accumulation HIF-1
α
protein and nuclear translocation in response to hypoxic challenge (1%O
2
, 12h) were blunted in APAKO (HIF-1
α
/
β
-Actin ratio, C57BL/6J (WT) vs APAKO: 0.987±0.069 vs 0.467±0.063, mean±SE, p=0.018). Although the levels of E3 ubiquitin ligase Siah2 and prolyl-hydroxylase 3 (PHD3), both upstream regulators of HIF-1
α
, did not alter under hypoxia (1%O
2
, 6h), proteasome inhibitor MG-132 restored the decreased accumulation of HIF-1
α
in ECs from APAKO similar to WT. Tube formation assay under hypoxia showed ECs of APAKO formed significantly less endothelial network (Tube length, mm/field, WT vs APAKO: 35.2±3.4 vs 27.6±5.0, p=0.024), and this was partly reversed by VEGF (50ng/mL) treatment (31.2±4.8, p=0.048), but not by soluble APA peptide supplementation (26.6±4.3, p=NS). APAKO and WT mice were subjected to unilateral hindlimb ischemia. Laser Doppler perfusion imaging demonstrated decreased blood flow recovery in APAKO, (Ischemic/Non ischemic limb, WT vs APAKO: 0.80±0.07 vs 0.70±0.04, at 4 weeks, p=0.046). Tissue capillary density at 2 weeks was significantly decreased in APAKO (ECs/Muscle Ratio, WT vs APAKO: 1.57±0.15 vs 1.25±0.12; p=0.039). FACS analysis of endothelial progenitor cells revealed no significant difference between in APAKO and WT mice, suggesting that vasculogenesis was minimally involved in the reparative process from ischemia in APAKO mice.

Conclusion:
Ischemia-induced angiogenesis is impaired in APAKO mice due to decreased stability of HIF-1
α
with upregulation of proteasomal degradation under hypoxic conditions and subsequent shortage of growth factor secretion. APA would be a functional target of ischemia-induced angiogenesis.
Abstract 5817: A Proatherogenic Role for HMGB1 in Atherosclerosis
Authors: Bobik Alex , Kanellakis Peter , Agrotis Alex , Koulis Christine , Ahrens Ingo , Peter Karlheinz , Mori Shuji , Takahashi Nideo K , Liu Keyue , Nishibori Masahiro
Published: April 2024

HMGB1 is a highly conserved nuclear protein that modulates chromatin structure. It is exported from the nucleus into the cytoplasm and secreted by immune cells and is also released by necrotic cells. In vitro, HMGB1 is a chemoattractant for inflammatory and smooth muscle cells and stimulates cytokine secretion by macrophages. Since it is highly expressed in human atherosclerotic lesions we tested the hypothesis that extracellular HMGB1 may be proatherogenic, augmenting development of atherosclerotic lesions in ApoE−/− mice. Initially we confirmed that like human atherosclerotic lesions, lesions in ApoE−/− mice also express HMGB1. To determine its significance for atherosclerosis, ApoE−/− mice were injected i.v. twice weekly with a HMGB1 monoclonal neutralising antibody and fed a high fat diet over 8 weeks. Treatment with the antibody attenuated atherosclerosis by 55%. Macrophage accumulation was reduced by 43% whilst expression of VCAM-1 and MCP-1 were reduced by 48 and 72% respectively. The total (CD11c+) dendritic cell population was reduced by 65% and mature (CD83+) dendritic cells were reduced by nearly 60% suggesting a major effect on the dendritic cell population. Treatment also reduced CD4+ T-cell numbers by 50% and attenuated smooth muscle cell accumulation within developing lesions but did not affect endothelial cells. To determine whether HMGB1 differentially affected the CD4+ T-cell Th1 and Th2 populations we measured Tim3 and Tim1 mRNA levels. Tim 1, a marker for Th2 cells was not detectable in lesions of control ApoE−/− mice or mice treated with the neutralising antibody, but Tim3 mRNA was reduced by 55% with the neutralising antibody. Since in vitro HMGB1 stimulates secretion of proinflammatory cytokines by macrophages we also examined whether proinflammatory cytokine expression was affected in lesions. mRNAs encoding TNF-alpha and IL-1 beta were unchanged but IFN-alpha and IL-6 were reduced by nearly 50% by the neutralising antibody. Also, mRNA encoding ABCA1 was unaffected but mRNA encoding FABP4 was elevated 5-fold. We conclude that HMGB1 contributes to atherosclerosis development by augmenting intima development, stimulating dendritic cell migration, thereby increasing CD4+ Th1T-cell accumulation as well as IFN-alpha and IL-6.
Abstract 5818: Uncovering the Role of Circulating Soluble Receptor for Advanced Glycation End Products in Hypertension: Relationships With Albuminuria
Authors: Tsioufis Costas , Dimitriadis Kyriakos , Syrseloudis Dimitris , Thomopoulos Costas , Mazaraki Anastasia , Miliou Antigoni , Sideris Skevos , Stefanadi Elli , Tousoulis Dimitris , Stefanadis Christodoulos
Published: April 2024

Introduction:
The soluble receptor for advanced glycation end-products (sRAGE) participates in the development and acceleration of atherosclerosis, while albuminuria is an established marker of target organ damage.

Hypothesis:
Urinary albumin excretion, expressed as the albumin to creatinine ratio (ACR), may be related to sRAGE in essential hypertensives.

Methods:
Our population consisted of 80 newly diagnosed untreated non-diabetics with stage I to II essential hypertension [48 men, mean age=52 years, office blood pressure (BP)=145/93 mmHg]. According to the ACR values determined as the mean of two non-consecutive morning spot urine samples, the study population was divided into microalbuminurics (n=27) (mean ACR=30–300 mg/g) and normoalbuminurics (n=53) (mean ACR<30 mg/g). Moreover, in all patients venous blood sampling was performed for estimation sRAGE concentrations.

Results:
Microalbuminurics compared to normoalbuminurics were older (53±6 vs 49±5 years, p<0.05), had higher 24-h systolic BP (140±13 vs 132±12 mmHg, p=0.001), while did not differ regarding sex and metabolic profile (p=NS). Moreover, microalbuminurics compared to normoalbuminurics exhibited lower levels of sRAGE (1011±458 vs 1503±931 pg/ml, p<0.003). In the total population, ACR was positively related to age (r=0.345, p=0.004), body mass index (r=0.217, p<0.003) and 24-h systolic BP (r=0.514, p<0.0001), whereas it was negatively correlated with sRAGE (r= −0.275, p=0.018). Regarding sRAGE, it was associated with body mass index (r= −0.241, p=0.003), waist to hip ratio (r= −0.463, p<0.0001) and 24-h pulse pressure (r= −0.372, p=0.001). Multiple regression analysis revealed that age, 24-h systolic BP and sRAGE were the independent predictors of ACR (
R

2

=0.47, p<0.0001). Furthermore, analysis of covariance showed that sRAGE values were significantly different between groups even after adjustment for confounding factors (p<0.05).

Conclusions:
In essential hypertension, microalbuminuria is accompanied by attenuated levels of sRAGE, reflecting pronounced vascular dysfunction. Moreover, the close association of sRAGE with ACR, suggests active involvement of sRAGE in atherosclerotic target organ damage progression in non-diabetic essential hypertensives.
Abstract 5819: Long-term Existence of the Autoantibodies Against the Second Extracellular Loop of
β
1-Adrenoceptor Induces Kidney Injury in Rats
Authors: Liu Jingyi , Zuo Lin , Wang Ke , Ma Jihua , Bao Haijun , Tian Jue , Yan Li , Liu Huirong
Published: April 2024

Background:
The effects of antibodies against the second extracellular loop (EC
II
) of
β
1
-adrenoceptors (
β
1
-AA) have been extensive investigated in cardiovascular disease. Our previous study has demonstrated that peritoneal exudate had been induced after long-term active immunization with synthetic peptides corresponding to human
β
1
-AR-EC
II
in rats. We hypothesis that
β
1
-AA may also bind with
β
1
-AR in kidney and display pathophysiological roles.

Methods:
Immunization models were set up using either the peptides corresponding to the EC
II
of
β
1
-adrenoceptors or
β
1
-AA in Rats; the long-term effects of anti-
β
1
-AR-EC
II
on kidney structure and function were observed.

Results:
Compared with the vehicle group, blood urea nitrogen (BUN), creatinine (CR), and uric acid (UA) increased, and the BUN-to-CR ratio decreased markedly at the 8
th
week after immunization with peptides (BUN: 8.9±0.95 mM
vs
6.7±0.35 mM,
P
<0.05; CR: 70.0±3.85 mM
vs
40.9±1.38 mM,
P
<0.01; UA: 235.6±16.43 mM
vs
89.5±9.80 mM,
P
<0.01; BUN/CR: 0.11±0.008
vs
0.16±0.008,
P
<0.01). At the 28
th
week after immunization, BUN and CR were still higher than those in the vehicle group (
P
<0.05,
P
<0.01); BUN/CR was also lower than the vehicle group (
P
<0.05). HE and Masson’s staining revealed some mononuclear cell infiltration around the renal glomerulus, and collagen fibers accumulation around the renal tubules in 9 rats. To exclude the possible toxic role of antigen peptides to kidney in active immunization, passive immunization model by injection of purified
β
1
-AA was used to confirm the above results. BUN, CR, UA significantly increased, and BUN/CR decreased as comparing with the negative sera group at 24
th
week after immunization (BUN: 9.25±0.43 mM
vs
6.56±0.28 mM; CR: 68.8±8.05 mM
vs
41.8±1.39 mM; UA: 166.5±21.14 mM
vs
86.3±12.35 mM; BUN/CR: 0.14±0.009
vs
0.16±0.006; all
P
<0.01). The morphological changes after passive immunization by HE or Masson’s staining is consistent with the results of active immunization.

Conclusion:
Anti-
β
1
-AA could lead impairment of kidney function and structure, suggesting that anti-
β
1
-AA may have a broad spectrum of organ injury effect in addition to its reported harmful effects in cardiovascular system.