bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5661: Akt/mTOR is an Essential Downstream Pathway of Platelet-Derived Growth Factor Signaling in Pulmonary Artery Smooth Muscle Cells
Authors: Ogawa Aiko , Firth Amy L , Yuan Jason X
Published: April 2024

Background: Platelet-derived growth factor (PDGF) is known to be a potent mitogen involved in the development and progression of pulmonary vascular remodeling. Recently, PDGF has attracted attention due to the inhibition of its receptor having a positive effect on hemodynamics, remodeling, and survival. Indeed, a PDGF receptor inhibitor is now undergoing clinical trials. It is, however, unclear which downstream pathway is important in pulmonary vascular remodeling. The Akt/mTOR pathway is central to many biological responses including cell growth, proliferation, survival, and metabolism. The involvement of the Akt/mTOR pathway in PDGF signaling was evaluated, in addition to a potential role in store-operated Ca
2+
entry (SOCE), which we previously reported to be important in cell proliferation and vascular remodeling in idiopathic and thromboembolic pulmonary hypertension.

Methods and Results: In human pulmonary artery smooth muscle cells (PASMC) and fibroblasts, PDGF transiently phosphorylated Akt, mTOR and p70S6K (24.8-, 3.6-, 8.1-fold increase in 15 minutes, P<0.01, 0.001, 0.01, respectively). Interestingly, PDGF failed to induce Akt phosphorylation in pulmonary artery endothelial cells. Chronic treatment with PDGF (10 ng/ml) significantly enhanced the increase in cytosolic Ca
2+
due to cyclopiazonic acid (10 microM)-induced Ca
2+
mobilization (from 0.75±0.02 to 0.88±0.03 arbitrary unit (a.u.), P<0.001) and SOCE (from 1.03±0.02 to 1.51±0.05 a.u., P<0.001). When treated with rapamycin (10 nM), an inhibitor of mTOR, or Akt inhibitor (1 microM), SOCE was significantly reduced (to 1.28±0.05, 0.60±0.02 a.u., respectively, P <0.001).

Conclusions: The data suggest that Akt/mTOR signaling is an important pathway in the regulation of SOCE in PASMC. A greater understanding of this pathway may enable the development of novel therapeutic approaches in patients with pulmonary vascular disease.
Abstract 5675: Local Gene Silencing of TLR4 Expression
in vivo
Improves Vein Graft Patency in Mice
Authors: Karper Jacco , de Vries Margreet , van den Brand Ben , Niessen Hans , Hoeffer Imo , Quax Paul
Published: April 2024

Introduction: Patency of vein grafts is limited by vascular remodelling processes such as neointima formation and accelerated atherosclerosis. Toll Like Receptor 4 (TLR4) is associated with neointima formation and atherosclerosis.

Hypothesis: We hypothesize that TLR4 can serve as a local therapeutic target against vein graft disease.

Methods: In human saphenous veins (huSV) immunohistochemical staining for TLR4 was performed on cross-sections of either directly harvested huSV (n=6),
in vitro
perfused huSV (n=6) and coronary graft huSV derived at autopsy (n=5). Mouse venous bypass grafting was performed in Balb/c mice (n=8) and in TLR4 deficient (C3H/Hej) mice (n=7) using a venous interposition model in the carotid artery. Five lentiviral TLR4 shRNAs were created and validated for local TLR4 gene silencing by measurement of murine TLR4/MD2 expression by FACS analysis and the best vector was selected for
in vivo
analysis. To test the effect of local TLR4 deficiency on vein graft disease virus was administered in pluronic gel and lubricated around vein grafts after vein grafting in APOE3*Leiden mice fed a high cholesterolemic diet.

Results: A marked induction of focal TLR4 expression was observed in HuSV, reperfused ex vivo with whole blood under pulsatile arterial pressure. Moreover, TLR4 was abundantly present in the neointima formed in autopsy grafts and preexisting intimal lesions in directly harvested huSV. Vein grafts in C3H/Hej mice had 48±12% less vessel wall thickening (p=0.04) than Balb/c controls, 28 days after surgery. Hypercholesterolemic APOE3*Leiden mice showed massive vein graft disease due to neointima formation and accelerated atherosclerosis after vein grafting. Local gene silencing of TLR4 led to a significant reduction of vessel wall thickening (38±10%, n=10, p=0.03) in the graft segment and superior graft patency (p=0.013) as compared to the control. In conclusion, the upregulation of TLR4 expression in huSV during remodelling, the reduced vein graft thickening in TLR deficient C3H/Hej mice and reduction of vessel wall thickening after local interfering in the TLR4 pathway indicate the potential of TLR4 as a local therapeutic target against vein graft disease to increase graft patency.
Abstract 5676: Free Fatty Acids Induce Dynamic Inflammatory Processes in Pancreatic Islets That Lead to
β
Cell Dysfunction
Authors: Eguchi Kosei , Manabe Ichiro , Shen Hua , Nagai Ryozo
Published: April 2024

Metabolic syndrome is a major risk factor of atherosclerotic cardiovascular disease and previous studies have suggested the pivotal involvement of inflammation in its development. Serum levels of free fatty acids are increased in obese subjects and have been implicated in pathogenesis of both cardiovascular and metabolic diseases. We found that palmitate, the major free fatty acid in blood, when administered to mice markedly exacerbated neointima formation. Interestingly, a continuous infusion of palmitate induced
β
cell dysfunction. Because neointima formation is an inflammatory process, we addressed if inflammation plays a role in the palmitate-induced islet pathology. In the islets of mice infused with palmitate, the reduction of mRNA of
β
cell functional markers was accompanied by induction of chemokines including MCP-1 and MIP-1
α
, and inflammatory cytokines including IL-1
β
and TNF-
α
. Flow cytometric analysis showed that palmitate infusion induced rapid accumulation of CD11b
hi
F4/80
+
Ly-6C
+
M1 macrophages in the islets. When macrophages were depleted in mice, the macrophage accumulation was suppressed and the induction of cytokines and chemokines, and
β
cell dysfunction induced by palmitate was ameliorated, indicating the causative involvement of macrophages in
β
cell dysfunction. We further analyzed the signaling mechanisms that controls response to palmitate and found that Toll-like receptor 4 (TLR4) signaling mediates chemokine production in
β
cells. As expected, palmitate-induced M1 macrophage recruitment and
β
cell dysfunction were markedly suppressed in TLR4 knockout (KO) and Myd88 KO mice. Finally, to confirm the relevance of these phenomena, the islets of db/db and db/+ mice were analyzed. In the islets of db/db mice,
β
cell dysfunction was accompanied by induction of cytokines and chemokines, and accumulation of CD11b
hi
F4/80
+
Ly-6C
+
M1 macrophages were observed. Taken together, results of the present study demonstrate that palmitate induces inflammatory processes in pancreatic islet by recruiting Ly-6C
+
inflammatory monocytes/macrophages, which leads to
β
cell dysfunction. In conclusion, chronic inflammation induced by free fatty acids appears to play important roles in both cardiovascular and metabolic diseases.
Abstract 5679: The Critical Role of p90 Ribosomal S6 Kinase (p90RSK) in Endothelial eNOS Expression is Mediated by ERK5 Transactivation via Its SUMOylation and Phosphorylation
Authors: tu LE Nhat , Lee Hakjoo , Heo Kyung-Sun , Woo Chang-Hoon , Nigro Patrizia , McClain Carolyn , Spangenberg Thomas , Thomas Tamlyn , Berk Bradford , Fujiwara Keigi , Abe Jun-Ichi
Published: April 2024

Emerging evidence shows the reduction of vascular reactivity in diabetic patients. We have reported that advanced glycated end products and reactive oxygen species inhibit laminar steady flow (s-flow) mediated-eNOS expression in endothelial cells (EC) via ERK5-SUMOylation, which subsequently inhibits ERK5 and KLF2 transactivation. However, it remains unknown how ERK5-SUMOylation is regulated in EC. In this study we investigated the involvement of redox sensitive kinase p90RSK on ERK5 activation and eNOS induction. Inhibition of ERK5 transactivation by H
2
O
2
was reversed by dominant negative p90RSK (21±7%-increase compared with H
2
O
2
treated cells, p<0.05), indicating that this inhibition is p90RSK-dependent. Transduction of an adenovirus vector expressing DN-p90RSK (Ad-DN-p90RSK) abolished H
2
O
2
-mediated inhibition of s-flow-mediated eNOS expression. Ad-DN-p90RSK significantly inhibited H
2
O
2
-mediated ERK5-SUMOylation, suggesting that p90RSK inhibits ERK5 by increasing ERK5-SUMOylation. To elucidate how p90RSK increases ERK5-SUMOylation, we investigated the effect of de-SUMOylation enzyme SENP2, which contains potential p90RSK phosphorylation sites. Depletion of SENP2 by SENP2 siRNA increased ERK5-SUMOylation. Over-expression of SENP2 inhibited ERK5-SUMOylation in a dose-dependent manner, suggesting that ERK5 is a substrate for SENP2. An
in vitro
kinase assay revealed that active p90RSK phosphorylated SENP2, suggesting SENP2 as a novel substrate of p90RSK. In addition, H
2
O
2
induced SENP2 nuclear export, which was inhibited by Ad-DN-p90RSK. These data suggest that p90RSK inactivates SENP2 by phosphorylating it, thus increasing ERK5-SUMOylation and decreasing ERK5/KLF2 transactivation and eNOS expression. En face confocal microscopy of normal mouse aorta showed p90RSK localization in the perinuclear region in EC. Interestingly, while p-p90RSK exhibited nuclear localization in s-flow areas, p-p90RSK localized in the cytoplasm of EC in disturbed flow (d-flow) areas which are prone to atherosclerosis. ApoE
−/−
mice revealed increased p90RSK activation in d-flow areas and early atherosclerotic lesions, also supporting the patho-physiological role of the p90RSK-SENP2 axis on EC dysfunction.

This research has received full or partial funding support from the American Heart Association, National Center.
Abstract 5681: GPR30 is Required for the Pro-apoptotic Effects of Estrogen in Vascular Smooth Muscle Cells
Authors: Gros Robert , Ding Qingming , Chorazcyzewski Jozef , Feldman Ross D
Published: April 2024

Rapid and non-genomic effects of estradiol (EST) may be mediated through the G-Protein-coupled receptor dubbed GPR30 receptor. The present studies tested the hypothesis that GPR30 expression, which we demonstrate is readily detectable in freshly isolated vascular tissue but is decreasingly detectable in cultured VSMCs, mediates the effects of EST to alter ERK phosphorylation status and apoptosis. In freshly isolated aortic tissue, EST-stimulated ERK phosphorylation whereas in cultured VSMCs, EST inhibits ERK phosphorylation in a dose-dependent manner. However, gene transfer of GPR30 resulted in EST-mediated stimulation of ERK phosphorylation (138±4%, n=10), which is in contrast to the effects of EST in GFP-expressing primary culture of VSMCs (68±3%, n=14). EST-mediated stimulation of ERK subsequent to heterologous GPR30 expression was pertussis toxin-sensitive and PI3 kinase-dependent; under these conditions, EST also inhibited PKA activation. In contrast, in the absence of GPR30 expression in cultured VSMC, EST stimulated PKA activity and inhibited ERK phosphorylation. We also assessed the impact of GPR30 expression on EST-mediated regulation of VSMCs apoptosis. In cultured VSMC transduced with GFP vector (control conditions), EST (10 nM) mediated a decrease in VSMC apoptosis (80±2%, n=7), an effect that was marginally but significantly enhanced by gene transfer of ER
α
(71±3%, n=7). The anti-apoptotic effect of EST was attenuated by co-incubation with the PKA inhibitor Rp-cAMP. However, PI3 kinase inhibition did not affect estradiol’s anti-apoptotic actions. In contrast, gene transfer of GPR30 into the VSMC resulted in enhancement of apoptotic rates following EST treatment (169±2%, n=7), and this GPR30-mediated pro-apoptotic effect of EST was attenuated by the PI3 kinase inhibitor, but not by Rp-cAMP. Thus, the effect of EST on vascular smooth muscle cell apoptosis is likely dependent on the balance between ER-mediated PKA activation and GPR30-mediated PKA inhibition and PI3 kinase activation. Taken together, we postulate that modulation of GPR30 expression and/or activity may be an important determinant of the effects of estradiol in the vasculature.
Abstract 5686: Stromal Cell-Derived Factor 1 (SDF-1) Signaling Regulates Platelet Migration
Authors: Kraemer Bjoern , Koch Miriam , Bigalke Boris , Daub Karin , Urban Bejamin , Schwanitz Laura , Gawaz Meinrad , Lindemann Stephan
Published: April 2024

Platelets have been regarded as static cells that do not move once they adhere to a matrix. In contrast to the current opinion, we found that platelets were mobile, able to migrate over a surface, and transmigrated through a transwell membrane and endothelium toward a source of SDF-1(See Sketch 1 and Image 2 below). Platelet migration was regulated by SDF-1 signaling, which involves Phosphoinositol 3-Kinase (PI3-Kinase) and downstream SGK1 (Serum- and Glucocorticoid regulated Kinase-1). SGK1 is a central element in ion channel homeostasis that is crucial for cytoskeletal rearrangement. Thus, the regulation of platelet migration describes an entirely novel functional area of SGK1.
In vivo
transmigration experiments in mice using intravital microscopy showed platelet invasion into post-ischemic vascular areas, but significantly decreased invasion by murine SGK1
−/−
platelets (see photomicrographs, Image 3 and Table 4). Corresponding
in vitro
transmigration experiments revealed that SGK1
−/−
platelets show significantly decreased response to SDF-1 and migratory activity (Table 4). The potential of platelets to migrate may substantially redefine the role of platelets in the pathophysiology of vascular inflammation, subsequent atherosclerotic degeneration and vascular regeneration.
Abstract 5687: Identification of HGF as a Novel Vascular ADAM Metalloprotease Substrate by Phage Display
Authors: Higuchi Sadaharu , Kimura Keita , Takaguri Akira , Hinoki Akinari , Eguchi Kunie , Frank Gerald D , Eguchi Satoru
Published: April 2024

Activation of a metalloprotease, ADAM17, and subsequent EGF receptor transactivation via proteolytic cleavage of its pro-ligand has been implicated in vascular remodeling induced by angiotensin II (AngII). The known substrates of ADAM17 include proHB-EGF and proTNF-alpha. However, whether other unidentified ADAM17 substrates are involved in AngII-induced vascular remodeling remains unanswered. To search for such substrates, we made a rat aortic cDNA phage library that has a hexa-histidine tag. After Ni-NTA agarose binding, the library phage was incubated with recombinant ADAM17 and only the cleaved phages were rescued and amplified (bio-panning). Upon repeating the panning 3 times, we obtained about 4000 phage colonies which potentially displayed ADAM17 substrates. From 128 colonies, we identified 71 candidate proteins in 88 readable sequences. Among these proteins, eleven proteins were known as ADAM17 substrates including (amphiregulin, Notch etc.) while six proteins were other protease substrates indicating strong reliability and provability of the technique. Among the novel substrate candidates, we focused on hepatocyte growth factor (HGF), a multifunctional growth factor with a proteolytic mechanism of activation. Upon transfection with a shedding assay vector encoding proHGF with its N terminus fused with ALP, the ALP activity in the condition medium was significantly reduced in ADAM17-null MEF cells compared to the control MEF cells. Moreover, the reduction was rescued with adenovirus vector encoding ADAM17. In rat VSMCs transfected with the assay vector, AngII markedly enhanced the ALP activity in the conditioned medium that was completely blocked with a MMP/ADAM inhibitor, BiPS. Also, AngII stimulated secretion of HGF-alpha in VSMCs. Recombinant HGF markedly stimulated ERK1/2 activity via the Met receptor in VSMCs. It also stimulated proliferation of VSMCs. In conclusion, we have established a novel phage display system for identification of a protease substrate with high probability. HGF was identified as a novel ADAM17 substrate potentially involved in vascular remodeling induced by AngII.

This research has received full or partial funding support from the American Heart Association, National Center.
Abstract 5688: HMGB1 is a Novel Substrate of Dypeptidyl Peptidase IV
Authors: Marchetti Carlo , De Cristofaro Raimondo , Mocini David , Olivieri Fabiola , Marra Maurizio , Bonfigli Anna Rita , Limana Federica , Capogrossi Maurizio C. , Germani Antonia
Published: April 2024

Introduction:
High Mobility Group Box 1 (HMGB1) is a cytokine with a key role in tissue regeneration and angiogenesis. HMGB1 levels are reduced in diabetic skin and topical application of HMGB1 to skin wounds of diabetic mice enhanced vessel density and accelerated wound healing, suggesting that diabetes may affect endogenous HMGB1 functions. CD26 is a protease which selectively removes the N-terminal X-Pro or X-Ala containing motifs from a number of chemokines modulating their activity. High levels of CD26 have been detected in plasma of type II diabetic patients and its inhibition has proved effective in the treatment of diabetes. Since HMGB1 contains potential CD26 cleavage sites we determined whether HMGB1 may be a substrates for CD26 and, eventually, whether CD26-mediated cleavage may alter its biologic activity.

Methods and results:
Recombinant HMGB1 (5 uM) was incubated overnight with CD26 (2.2 uM). RP-HPLC analysis on Aquapore C18 column yielded a peptide with a molecular weigh lower than 2000 kDa. The CD26-mediated deletion of the N-terminal region of HMGB1 was confirmed by western blot analysis: time-dependent disappearance of the molecular weigh band corresponding to HMGB1 was detected with an anti-HMGB1 antibody which recognizes its first 16 N-terminal amino-acids of HMGB1. It was previously found that HMGB1 promoted endothelial cell migration through the activation of c-Jun N-terminal kinase (JNK) and extracellular regulated kinase (ERK) signaling pathways. To evaluate whether CD26 altered HMGB1 functions, endothelial cells were induced to migrate in presence of HMGB1 and CD26. HMGB1-mediated chemotactic activity was reduced by CD26 in a dose-dependent manner. Similarly, CD26 abolished HMGB1-induced JNK and ERK activation. Since diabetes is associated with the increased CD26 activity, both CD26 activity and HMGB1 levels were measured in serum of diabetic patients. Although HMGB1 serum levels did not significantly differ between diabetic and normoglycemic patients, increased N-terminal deleted HMGB1 was detected in serum from diabetic patients.

Conclusions:
CD26 cleaves HMGB1 and, via this mechanism, it inhibits its functions. These findings suggest that CD26 may account for the inhibition of HMGB1 effects found in diabetes.
Abstract 5753: An Axon-guiding Molecule Semaphorin3E is Critically Involved in Impaired Angiogenesis of Diabetes
Authors: Moriya Junji , Minamino Tohru , Tateno Kaoru , Okada Sho , Shimizu Ippei , Nojima Aika , Yokoyama Masataka , Komuro Issei
Published: April 2024

The axon-guiding molecules known as semaphorins and their specific receptors (plexins) regulate the vascular pattern and play an important role in development of the vascular network during embryogenesis. However, it remains unclear whether these molecules are involved in postnatal angiogenesis. Here we report that semaphorin3E (Sema3E), one of the class 3 semaphorins, and its specific receptor plexinD1 inhibit angiogenesis in adults. Sema3E inhibited cell growth and tube formation by suppressing the vascular endothelial growth factor (VEGF) signaling pathway. Expression of Sema3E and plexinD1 was markedly up-regulated in ischemic limbs of mice, and inhibition of this pathway by introduction of the plexinD1-Fc gene or disruption of Sema3E led to significant improvement of revascularization. We also found that Sema3E-deficient mice showed better blood recovery and a larger vessel area in their ischemic limbs than wild-type mice, suggesting that Sema3E/plexinD1 negatively regulate postnatal angiogenesis. Next, we identified a putative binding element of p53, the tumor suppressor protein, within the promotor region of the Sema3E gene, and found that hypoxia up-regulated Sema3E expression by activating p53 in endothelial cells. Consistent with our in vitro data, up-regulation of Sema3E expression was abolished in the ischemic limbs of p53-deficient mice, suggesting that an increase of p53 promotes Sema3E expression in ischemic tissue and thus inhibits blood flow recovery. It has been reported that angiogenic response to ischemia is attenuated in diabetes. We found that expression of p53 was increased in diabetic mice and this increase was further enhanced by ischemia. Likewise, expression levels of Sema3E were significantly higher in diabetic mice than in control mice. Consequently, blood flow recovery after VEGF treatment was impaired in diabetic mice compared with VEGF-treated control mice. These changes were effectively reversed by additional introduction of the plexinD1-Fc gene. These results indicate that Sema3E negatively regulates postnatal angiogenesis and suggest that inhibition of Sema3E would be a novel strategy for therapeutic angiogenesis, especially when VEGF treatment is ineffective, such as in the diabetic state.
Abstract 5757: RAGE in Bone Marrow Derived Cells Blunts Restoration of Blood Flow in Mice Subjected to Femoral Artery Ligation
Authors: Shen Xiaoping , Song Fei , Rosario Rosa , Morales Alfredo , Zou Yu Shan , Schmidt Ann Marie , Yan Shi Fang
Published: April 2024

Receptor for Advanced Glycation Endproducts (RAGE) plays central roles in the injury response to global hypoxia and ischemia/reperfusion (I/R) injury. Our previous data revealed that diabetic (D) and non-diabetic (ND) homozygous RAGE null mice subjected to hind limb ischemia displayed improvement in angiogenesis and restoration of blood flow. Here, we tested the hypothesis that endogenous RAGE expression in bone marrow (BM)-derived cells may contribute to impaired angiogenesis and blunted restoration of blood flow after femoral artery (FA) ligation. Wild type (WT) mice were rendered diabetic (D) with streptozotocin (stz); non-diabetic (ND) cohorts were treated with buffer alone.
N
D or D WT mice were used as recipient mice in BM transplantation. Rosa
+/−
transgenic mice containing a lacZ reporter gene were used as an indicator of transplantation of RAGE
+/+
or RAGE
−/−
BM, therefore, the chimerical mice containing RAGE
+/+
/Rosa
+/−
or RAGE
−/−
/Rosa
+/−
were generated as donor strains for BM transplantation. Immunostaining with anti-
β
-galactosidase IgG demonstrated that BM transplantation was successful from donors to recipients by localization of the expression of LacZ gene in the muscle tissues. FA ligation was performed on 13-week old ND or D WT recipient mice. Both ND and D WT recipient mice with transplantation of RAGE
−/−
/Rosa
+/−
BM displayed significantly improved blood flow ratios (injured/contralateral leg) by laser Doppler analysis on day 28 after FA ligation compared to ND or D WT recipient mice with transplantation of RAGE
+/+
/Rosa
+/−
BM (Table

). Our data suggested that RAGE expression in the bone marrow contributes to impairment in blood flow recovery after acute arterial injury, irrespective of the state of glycemia.

Table.

Doppler data of Blood Flow Ratios (BRF, %)
Abstract 5765: 9p21 Genetic Variant and Benefit From Intensive Statin Therapy After an Acute Coronary Syndrome
Authors: Mega Jessica L , Iakoubova Olga A , Devlin James J , Qin Jie , Cannon Christopher P , Braunwald Eugene , Sabatine Marc S
Published: April 2024

Background
Genetic variation at 9p21 has been associated with a greater risk of MI. The relationship of this locus to risk of recurrent coronary events in patients with ACS and to benefit from intensive statin therapy is unknown.

Methods
We studied 2029 pts in PROVE IT-TIMI 22, a trial of intensive (atorvastatin 80 mg/d) vs moderate (pravastatin 40 mg/d) statin therapy after recent ACS. Cox regression was used to assess risk of recurrent coronary events associated with 9p21 rs1333049 “C” allele adjusting for clinical risk factors and to test for an interaction between statin therapy and rs1333049, as well as
KIF6
719Arg, a gene variant previously shown to be associated with clinical benefit of statins.

Results
Among those treated with moderate statin therapy, the adj HR for CHD/MI over 2 yrs was 1.35 (95% CI 0.80 –2.28) for carriers (75% of pts) vs non-carriers. Benefit from intensive vs moderate statin therapy was significantly greater among rs1333049 “C” allele carriers than non-carriers (Fig

). In a multilocus model, both rs1333049 and
KIF6
variants modified statin therapy (P
interaction
0.035 & 0.029), and a particularly large benefit of intensive statin therapy was seen in carriers of both variants (HR 0.32, 95% CI 0.17– 0.59, P<0.0001).

Conclusion
The rs1333049 variant at 9p21 is associated with a trend towards increased risk of recurrent CHD/MI in patients who receive moderate statin therapy following an ACS, consistent with the risk seen for MI in population studies. Carriers of the rs1333049 “C” and the
KIF6
719Arg variants appear to receive significantly greater benefit from intensive versus moderate statin therapy. Further study of these pharmacogenetic interactions is warranted.
Abstract 5768: The Effect of Ticagrelor on Cardiopulmonary Function in Patients With Stable Coronary Artery Disease
Authors: Storey Robert F , Bliden Kevin , Patil Shankar B , Karunakaran Arun , Ecob Rosemary , Butler Kathleen , Teng Renli , Gurbel Paul
Published: April 2024

Background:
A higher incidence of dyspnea has been observed in patients treated with ticagrelor (T) versus clopidogrel (C). As part of a multicenter, randomized, double-blind, parallel-group study (the ONSET-OFFSET study) in patients with stable coronary artery disease (CAD), we investigated whether T treatment is associated with any change in cardiopulmonary function.

Methods:
A total of 123 stable aspirin-treated CAD patients were randomized to T (180 mg load then 90 mg bid [n=57]), C (600 mg load then 75 mg qd [n=54]), or placebo (n=12) for 6 weeks. Echocardiography, N-terminal pro-brain natriuretic peptide (NT-proBNP) measurements, and comprehensive pulmonary function tests were performed prior to (baseline) and 6 weeks after drug administration and/or following development of dyspnea. ANCOVA was used to analyze cardiopulmonary function changes from baseline.

Results:
The incidence of new-onset dyspnea following randomization was 24.6%, 5.6%, and 0% in the T, C, and placebo groups, respectively. The majority of instances were mild and/or lasted less than 24 h, although 3 patients on T versus 0 on C or placebo discontinued due to dyspnea. Seven of 16 and 8/16 patients experiencing dyspnea did so within 24 h and 1 week, respectively, after drug administration. In all treatment groups, there were no significant changes between baseline and 6 weeks in any of the cardiopulmonary function parameters (Table

). Subgroup analysis of patients experiencing dyspnea also showed no significant changes in these parameters. C
max
and AUC
0-inf
were similar between T-treated patients who experienced dyspnea and those who did not.

Conclusions:
Ticagrelor treatment had no effect on cardiopulmonary function in stable CAD patients. Dyspnea occurred with a higher frequency with ticagrelor versus clopidogrel but was not accompanied by any significant change in cardiopulmonary function.
Abstract 5799: Overexpression of Integrin Beta5 Enhances the Paracrine Angiogenic Properties of Early Outgrowth Endothelial Progenitor Cells via Src Kinase-Mediated Activation of STAT3
Authors: Leifheit Maren , Conrad Gaby , Heida Nana-Maria , Schroeter Marco , Hasenfuss Gerd , Konstantinides Stavros , Schaefer Katrin
Published: April 2024

Integrin alphavbeta5 is expressed on endothelial (progenitor) cells and has been implicated in angiogenesis. We have previously shown that stimulation of endothelial progenitor cells (EPC) with leptin upregulates alphavbeta5 integrin expression and enhances their angiogenic and reparative properties. The aim of the present study was to determine the intracellular mechanisms mediating the angiogenic effects of integrin alphavbeta5 in endothelial progenitor cells. For this purpose, EPC isolated from human venous blood and cultivated under endothelial conditions were transiently transfected with the full-length cDNA of human integrin beta 5 (EPC-ITGB5) or empty vector control (EPC-control), and analyzed 48 hours later (on day 7). Integrin beta5 overexpression was confirmed using flow cytometry (149±12%; P=0.0001 vs. EPC-control), Western blot (138±10%; P<0.01) and PCR analysis (135±13%; P<0.05). Moreover, it significantly enhanced the angiogenic capacities of EPC in vitro (spheroid and matrigel angiogenesis assay) and stimulated new vessel formation in vivo (chorioallantoic membrane assay and murine hindlimb ischemia model). Western blot and immunoprecipitation experiments revealed that overexpression of ITGB5 resulted in elevated integrin alphavbeta5 phosphorylation, followed by activation of Src kinase and signal transducer and activator of transcription 3 (STAT3). The specificity of ITGB5-dependent Src kinase and STAT3 phosphor-ylation was confirmed using function-blocking antibodies. Analysis of STAT3 target gene transcription revealed elevated mRNA and protein expression of the angiogenic chemokines IL8 and MCP1 in EPC-ITGB5, and significantly elevated IL8 and MCP1 protein levels in the conditioned medium (CdM) from EPC-ITGB5 were confirmed using quantitative ELISA. Moreover, CdM from EPC-ITGB5 enhanced the sprouting of co-incubated human umbilical vein endothelial cells (HUVEC) in a STAT3-dependent manner. Taken together, our findings suggest an important role for the cytoplasmic protein kinase Src in linking alphavbeta5 integrin to STAT3-dependent gene expression and angiogenesis. This pathway deserves further investigation as a possible means for (therapeutically) enhancing the paracrine potency of EPC.
Abstract 5805: Adenosine Down-Regulates Soluble Vascular Endothelial Growth Factor Receptor-1
Authors: Léonard Frédérique , Ernens Isabelle , Vausort Mélanie , Rolland-Turner Magali , Devaux Yvan , Wagner Daniel R
Published: April 2024

Background: The cardioprotective nucleoside adenosine acts through 4 types of receptors: A1, A2a, A2b, A3. An emerging concept is that adenosine is a major factor driving angiogenesis, thus contributing to the repair of the ischemic heart. Angiogenesis is controlled by a plethora of vasculogenic factors, among which Vascular Endothelial Growth Factor (VEGF) and its receptor VEGFR1 (or Flt-1). A soluble form of VEGFR1 (sFlt-1) traps circulating VEGF, thereby preventing its binding to VEGF receptors. Both VEGF and sFlt-1 are induced by Hypoxia Inducible Factor 1
α
. The aim of the present study was to characterize the effect of adenosine on the production sFlt-1.

Methods: Primary blood monocytes obtained from healthy volunteers and patients with acute myocardial infarction (MI) were differentiated to macrophages in vitro. Treatments included: adenosine (10
μ
M),
CGS21680
(A2a receptor agonist, 0.1–10
μ
M),
SCH58261
(A2a receptor antagonist, 0.1–10
μ
M), and/or LPS (100 ng/mL). ELISA, immuno-blotting and TransAM™ assay were used to measure expression levels and activation of sFlt-1 and HIF-1
α
. In vitro formation of microtubules by human coronary artery endothelial cells (HCAEC) was used to evaluate angiogenesis.

Results: LPS induced sFlt-1 production by macrophages from healthy volunteers and adenosine inhibited this induction (43% decrease, P=0.006, n=12). This inhibition was replicated by the A2a agonist and reversed by the A2a antagonist. The formation of a network of microtubules by HCAEC was enhanced in the presence of conditioned medium from adenosine-treated macrophages and impaired by exogenous sFlt-1. Adenosine inhibited the LPS-induced nuclear translocation of HIF-1
α
, thereby preventing its activation. The effect of adenosine was replicated in macrophages isolated from acute MI patients.

Conclusion: We show here for the first time that adenosine down-regulates the production of sFlt-1. This mechanism involves the adenosine A2a receptor and the transcriptional activator HIF-1
α
. Our data suggest that this effect of adenosine may play a significant role in angiogenesis.
Abstract 5807: Psychological Stress Impairs Ischemia-induced Neovascularization
Authors: Maingrette Fritz , Turgeon Julie , Haddad Paola , Groleau Jessika , Perez Gemma , Dussault Sylvie , Rivard Alain
Published: April 2024

Background:
Psychological stress (PS) is a major risk factor for cardiovascular diseases but the underlying mechanisms are still largely unknown. Here we tested the hypothesis that PS might decrease neovascularization in response to ischemia.

Methods and results:
Balb/c mice were subjected to restraint stress by placing them in a well ventilated 50 mL conical centrifuge tube for 20 min each day for 14 days. Control mice were left unperturbed in their home cage with free access to food and water. After two weeks of this stress protocol, hindlimb ischemia was surgically provoked by femoral artery removal and hindlimb perfusion was then evaluated by Laser Doppler. Mice exposed to PS showed a significantly lower rate of blood flow recovery compared to control mice at day 14 (0.60±0.04 vs 0.78±0.05, p<0.05) and day 21 (0.61±0.07 vs 0.80±0.07, p<0.05) and had a significantly lower capillary density in ischemic muscles (38±1 vs 74±3 capillaries/mm
2
, p<0.001), as assessed by CD31 immunostaining. Endothelial progenitor cells (EPCs) have been shown to have an important role for postnatal neovascularization. We found that the number of peripheral EPCs was significantly decreased in PS mice compared to control mice and that EPCs from PS mice showed a significant impairment of their angiogenic activities (adhesion, migration, integration into tubules). Moreover, oxidative stress levels in EPCs isolated from mice exposed to PS were significantly increased as determined by DCF-DA and DHE immunostainings.

Conclusions:
PS is associated with a reduced neovascularization following ischemia. The potential mechanisms involved include increased oxidative stress, decreased EPCs number and impairment of their functional activities.
Abstract 5808: Endothelial Cell Polyploidization Induces an Atherogenic Phenotype and is Prevented by Activating the Nampt-SIRT1 Axis
Authors: Borradaile Nica M , Pickering J. Geoffrey
Published: April 2024

Polyploid endothelial cells (ECs) are found in aged and atherosclerotic arteries. However, whether increased chromosome content has an impact on EC function is unknown. To interrogate the process by which ECs become polyploid we tracked cellular DNA content by flow cytometry in human aortic ECs as they replicated over 130 days. Strikingly, we observed that almost all ECs (99%) became tetraploid as they approached replicative senescence. Furthermore, the accumulation of tetraploid ECs was accelerated when cells were concurrently subjected to the oxidative stress of high glucose. To determine the impact of tetraploidy on EC function, independent of replicative senescence, we induced tetraploidy using the spindle poison, nocodazole. After a 24 h recovery, 70% of the EC population was tetraploid with no change in senescence-associated
β
-galactosidase activity. High density microarray expression analyses of tetraploid ECs revealed an atherogenic phenotype characterized by a cell cycle arrest profile (decreased
CCNE2/A2
,
RBL1
,
BUB1B
, increased
CDKN1A
) and increased expression of genes involved in inflammation (
IL32
,
TNFRSF21/10C
,
PTGS1
) and extracellular matrix remodeling (
COL5A1
,
FN1
,
MMP10/14
). Interestingly, aging-induced polyploidization was prevented by modest (1.6-fold) overexpression of the NAD
+
-regenerating enzyme, nicotinamide phosphoribosyltransferase (Nampt). The protection from polyploidization conferred by Nampt was not associated with enhanced PARP-1 or SIRT2 activity but with increased SIRT1 activity, which reduced cellular ROS and the associated oxidative stress stimulus for polyploidization. We conclude that human aortic ECs are prone to chromosome duplication that, in and of itself, can induce an atherogenic EC phenotype. Moreover, the emergence of tetraploid ECs can be prevented by optimizing the Nampt-SIRT1 axis. Strategies to maintain EC NAD
+
levels may represent a novel approach to mitigate the potentially deleterious accumulation of polyploid ECs associated with vascular disease.
Abstract 5821: The Effect of Spironolactone on Biomarkers of Inflammation and Extracellular Remodelling in Early Chronic Kidney Disease
Authors: Edwards Nicola C , Kirkwood Helen , Cramb Robert , Hutchinson Colin A , Steeds Richard P , Ferro Charles J , Townend John N
Published: April 2024

Introduction Early stage chronic kidney disease (CKD) is associated with major abnormalities of arterial and ventricular stiffness and function and with a high rate of adverse cardiovascular events. We have previously demonstrated that in this high risk group, significant reductions in LV mass and markers of arterial stiffness occurred after 40 weeks of treatment with the aldosterone antagonist, spironolactone. We hypothesized that serological markers of collagen turnover, inflammation and ventricular stress might also be improved with this treatment.

Methods 112 patients participating in the double blind randomized controlled trial investigating the effect of spironolactone in early CKD had serum biomarkers of

collagen turnover; amino-terminal propeptide of procollagen type III (PNIIIP),

inflammation; high sensitivity C-Reactive Protein (hsCRP), and

ventricular stress- N-terminal Pro Beta- Natriuretic Peptide (BNP) measured. Patients had stage 2 and 3 CKD, were on ACE Inhibitor or ARB therapy, with controlled hypertension (<130/85mmHg) and no history of cardiovascular disease or diabetes. Samples were measured at baseline and after 40 weeks of blinded treatment with either spironolactone 25mg once a day or matching placebo.

Results PNIIIP levels were unchanged with spironolactone but increased significantly in the placebo group (0.06
μ
g/L±0.70 vs 0.46
μ
g/L±0.82, p<0.01). There was no difference in median hsCRP levels (0.01mg/L (IQR 1.63) vs. 0.08mg/L (IQR 2.76), p =NS). Spironolactone resulted in significant reduction in median BNP levels (−0.4pg/L (IQR 70.9) vs. 9.3pg/L (IQR 54.7), p<0.01). All baseline biomarkers were positively correlated; PNIIIP with hsCRP (
r
= 0.24, p<0.05) PNIIIP and BNP (
r
= 0.27, p<0.01), hsCRP and BNP (
r
= 0.39, p<0.01).

Conclusion Treatment with spironolactone prevents deterioration of or improves biomarkers of collagen synthesis and of ventricular stress in CKD. These data support the hypothesis that deleterious effects of aldosterone on extracellular remodeling can be attenuated by spironolactone in CKD as has been observed in clinical outcome studies of heart failure. The association of all biomarkers suggests a complicated interplay of collagen turnover, inflammation and myocyte stretch.
Abstract 16644: TGF-β/Smad3 Activation in the Diabetic Mouse Heart Induces Fibrotic Cardiac Remodeling and Diastolic Dysfunction While Protecting From Excessive Matrix Degradation and Preventing the Development of Systolic Dysfunction
Authors: Biernacka Anna , Gonzalez-Quesada Carlos , Tian Qi , Li Na , Dobaczewski Marcin , Wang Xiao-Fan , Frangogiannis Nikolaos G
Published: April 2024

Hypertrophy, fibrosis and diastolic dysfunction are the main characteristics of diabetic cardiomyopathy. Our study examines the role of the pro-fibrotic TGF-β/Smad3 pathway in the diabetic heart using leptin resistant db/db mice. db/db mice developed diabetes and severe obesity and had marked elevation in circulating leptin levels. Despite exhibiting central leptin resistance (resulting in hyperphagia), db/db mice expressed a functional short-form leptin receptor in the myocardium. db/db hearts had concentric hypertrophy and diastolic dysfunction; systolic function was preserved. Collagen deposition was increased in db/db hearts; fibrotic changes were preceded by cytokine and chemokine induction and by activation of Smad3 signaling. In vitro, db/db cardiac fibroblasts exhibited marked upregulation of cytokines, chemokines and MMPs upon leptin stimulation indicating full responsiveness to the actions of leptin. In order to examine the role of Smad3 in morphology and function of the db/db heart we generated db/db Smad3 mutant mice. db/db Smad3 -/- mice exhibited high early mortality; the causes of early death are under investigation. In contrast, db/db Smad3 +/- heterozygotes (het) had similar survival curves, comparable weight gain and fat content with db/db Smad3+/+ animals. However, het mice had significant ventricular dilation (LVEDV, db/db: 82.9+4.7 vs. het: 125.6+6.4; p<0.01) accompanied by systolic dysfunction (FS, db/db: 42.8+2.9 vs. het: 29.1+2.7; p<0.01). Diastolic dysfunction, assessed through P:V loop analysis, was attenuated in het animals. In contrast, non-obese het mice had comparable chamber dimensions and function with WT Smad3 +/+ animals. The dilative remodeling, systolic dysfunction and attenuated diastolic dysfunction observed in obese diabetic het mice were associated with reduced collagen deposition, and increased MMP activity. Cardiac function and geometry in the diabetic heart are dependent on a balance between leptin and Smad3-mediated actions. Attenuation of Smad3 signaling in db/db mice prevents fibrosis and diastolic dysfunction, but also allows unopposed pro-inflammatory leptin-mediated effects resulting in impaired matrix preservation, chamber dilation and systolic dysfunction.
Abstract 16658: Greater Post-Aspirin Residual Platelet Aggregation is Associated With Increased Risk of Incident Acute Coronary Syndromes in Healthy Individuals
Authors: Qayyum Rehan , Kral Brian G , Yanek Lisa R , Vaidya Dhananjay , Nyquist Paul A , Mathias Rasika , Becker Lewis C , Becker Diane M
Published: April 2024

Background:
Platelet aggregation plays an essential role in the pathogenesis of acute coronary syndrome (ACS). Aspirin (ASA), as an inhibitor of the cyclooxygenase-1 (COX-1) and thromboxane-dependent platelet activation pathways, is the mainstay of primary chemoprophylaxis. However, studies have demonstrated that persons still experience an ACS event while on ASA, suggesting that variation in indirect COX-1 pathways may contribute to clinical outcomes. We determined the extent to which an increased risk of incident ACS would be observed in healthy individuals with residual platelet aggregation in an important indirect COX-1 pathway following ASA.

Methods:
1507 apparently healthy individuals with a family history of premature coronary artery disease were enrolled for 2-week therapy with of ASA 81 mg/day. At the end of second week, whole blood impedance aggregometry after collagen-mediated (5ug/mL) platelet aggregation was used to measure maximal platelet aggregation. ACS events during follow-up were adjudicated from medical records by 3 independent reviewers.

Results:
At baseline, the mean (SD) age was 46.3 (11.5) years; 57% female; 37% African American; 23% current smokers, 34% hypertensive, and 8% diabetes. During follow-up (mean 5[1.8] years), there were 26 ACS events. Post-ASA residual collagen-mediated platelet aggregation was greater in participants who had an ACS compared with those without ACS (28.5 ohms vs. 24.0 ohms, p<0.001). In an age, sex, race, and cardiovascular risk factors (LDL cholesterol, current smoking, diabetes, and hypertension) adjusted model, one ohm difference in residual collagen-mediated platelet aggregation was strongly associated with a 7% increase in the hazard of an ACS event (HR = 1.07, 95%CI = 1.02 to 1.11, p = 0.002).

Conclusions:
Healthy subjects with greater residual collagen-mediated platelet aggregation in an indirect COX-1 pathway after ASA therapy are at significantly higher risk of ACS than subjects with lower aggregation. Platelet aggregation pathways not directly inhibited by ASA may play an important role in the pathogenesis of ACS.
Abstract 16730: Enhancing the Cardiomyogenic Potential of Human Mesenchymal Stem Cells by Modulation of miR Expression
Authors: Cervio Elisabetta , Pisano Federica , Danieli Patrizia , Gnecchi Massimiliano
Published: April 2024

Introduction:
it has been shown that mesenchymal stem cells (MSC) can differentiate into cardiomyocytes (CMC) both in vitro and in vivo. However, the efficiency of cardiac differentiation is very limited. Recently, it has been suggested that miR1, 133 and 499 are involved in cardiac development and stem cell differentiation. The role of these miR in MSC differentiation is currently unknown. Accordingly, we tested if the overexpression of miR1, 133 and 499 may favour the differentiation of MSC into CMC.

Methods:
miR1, 133, and 499 were transiently overexpressed, one by one or in different combinations, in human MSC. Cardiac differentiation was evaluated by measuring the expression of the cardiac specific markers GATA4, Nkx2.5, Tbx5, MEF2c, MLC2v, Cx43, cTnT and α-MHC. Western Blot and immunocytochemistry (ICC) for cTnT and Cx43 were also performed.

Results:
miR499 alone increased the expression of the early cardiac genes GATA4, Tbx5, MLC2v. In particular, miR499 exerted more pronounced effects compared with miR1 and miR133. Concomitant overexpression of miR499 and miR133 further increased the expression of Tbx5 and MEF2c. Western Blot analysis showed that miR499 increased the expression of late cardiac genes when overexpressed alone or in combination with miR133. Finally, ICC staining confirmed that miR499 in association with miR133 increase Cx43 and cTnT protein expression. Finally, the expression of cardiac specific protein α-MHC was more pronounced in MSC overexpressing miR499 alone or in combination with miR133.

Conclusions:
concomitant overexpression of miR499 and miR133 successfully commit AMSC into CMC. Our data suggests that miR modulation can represent a promising approach to improve the efficiency of cardioregnerative therapy with multipotent stem cells.
Abstract 16739: Regional Variation in the Relationship of Infarct Size to Left Ventricular Ejection Fraction: Implications for Risk Stratification for Sudden Cardiac Death
Authors: Aquino Alejandro , Goldberger Jeffrey J , Wu Edwin , Carr Maria L , Bakhos Lara , Izquierdo Gomez Maria M , Benefield Brandon , Kadish Alan H , Lee Daniel C
Published: April 2024

Introduction:
In patients with coronary artery disease (CAD), guidelines for primary prevention of sudden cardiac death (SCD) use left ventricular ejection fraction (LVEF) ≤35%. The substrate for SCD is myocardial infarction (MI); indeed, MI size expressed as percent of LV mass (IM%) measured by cardiac magnetic resonance imaging (CMR) has been shown to be a better predictor of inducible ventricular tachycardia (VT) on electrophysiologic study than LVEF, with a cutoff of > 10% providing the highest sensitivity.

Purpose:
To examine the influence of MI location on the relationship between IM% and LVEF.

Methods:
We analyzed cine and late gadolinium enhanced (LGE) CMR images for 412 patients with CAD (81% male, age 62.3 ± 11.4) and a history of MI from the DEfibrillators To REduce Risk by MagnetIc ResoNance Imaging Evaluation Trial (DETERMINE trial,
www.clinicaltrials.gov
). LVEF and IM% were quantified (Medis QMass MR 7.2). We performed segmental visual scoring of cine (from 0 = normal to 4 = dyskinesis) and LGE images (from 0 = none to 4 = 76-100%). Each of the 17 segments was assigned to a coronary artery territory based on American Heart Association segmentation recommendations. MI location was calculated as the territory with the highest LGE score: left anterior descending (LAD), left circumflex (LCX), and right coronary artery (RCA).

Results:
Overall, 206/412 (50%) of patients had IM% > 10% while maintaining LVEF > 35%. The distribution of MI location was LAD (205), LCX (84), and RCA (123). The impact of increasing IM% on LVEF differed based on MI location (figure). The expected IM% required to reduce LVEF to 35% for a LCX MI was 19%, for LAD was 26%, and for RCA was 34%.

Conclusions:
As a marker of MI burden, LVEF is insensitive and is strongly influenced by MI location. As it best represents the substrate for SCD and is related to both mortality and inducible VT, further efforts are required to investigate IM% as a predictor of SCD, independent of LVEF.