bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 399: End-stage Heart Failure Causes Severe Lung Fibrosis and Dysfunction
Authors: Liu Xiaohong , Wang Huan , Shang Ruru , Zhang Jin , Chen Yingjie
Published: June 2021

Chronic heart failure (CHF) causes trouble breathing in patients. We recently demonstrated that systolic pressure overload by transverse aortic constriction (TAC) causes severe left ventricular (LV) failure that is associated with massive lung fibrosis and lung vascular remodeling, and right ventricular (RV) dysfunction in mice. Here, we further studied the effect of CHF on lung structure and function in mice, and the effect of CHF on lung fibrosis in patients. We demonstrated that chronic TAC resulted in decrease of LV ejection fraction, and increases LV weight, lung weight, and RV weight, as well as their ratios to bodyweight. Interestingly, the development of LV failure is associated with a significant lung dysfunction as evidenced by a ~2-fold increase of lung resistance and a ~50% dramatic decrease of lung compliance
in vivo
. Lung compliance was also significantly reduced ~50% in lung isolated from CHF mice, indicating the decrease of lung compliance is due to the structure change of lung. The reduced lung compliance in CHF mice is significantly correlated with the decrease of LV ejection fraction, the increase of lung weight, and RV hypertrophy, suggesting the reduced lung compliance might contribute to the development of RV hypertrophy and failure. Histochemical analyses further demonstrated that CHF causes massive lung vascular, perivascular and interstitial fibrosis, as well as increase of lung myofibroblast proliferation. By using the chimeric mice created by transplantation of Bone Marrow Derived Cells (BMDCs) from GFP mice into wild type mice, we demonstrated that BMDCs contribute to the increased lung myofibroblasts and lung fibrosis. However, BMDCs don’t differentiate into lung smooth muscles cells in CHF mice. Moreover, we demonstrated that inhibition of lung inflammation by a cytokine therapy protocol is effective in attenuating TAC-induced lung fibrosis. Finally, we demonstrated that end-stage CHF causes increase of lung fibrosis in patients, and the increased lung fibrosis is associated with RV hypertrophy and dysfunction in patients. Together, our study demonstrated that end-stage CHF causes lung fibrosis and lung dysfunction, and inhibition of inflammation is effective in attenuating heart failure induced lung fibrosis.
Abstract 40: Interleukin-10 Deficiency Impairs Reparative Properties of Bone Marrow-derived Endothelial Progenitor Cell Exosomes Function in Ischemic Myocardium
Authors: Yue Yujia , Garikipati Venkata N , Tang Yan , Chen Zhongjian , Zhou Jibin , Nickoloff Emily , Wang Chunlin , Benedict Cindy , Gao Erhe , Khan Mohsin , Verma Suresh , Kishore Raj
Published: June 2021

Endothelial progenitor cell (EPC) based therapy promotes neovascularization in ischemic myocardium. Studies suggest that therapeutic effect of stem cells is largely due to paracrine mechanisms including stem cell-derived exosomes that are emerging as key paracrine mediators of stem cell functions. However, the autologous transplantation of EPCs in patients with systemic inflammation, which is a common symptom in patients with ischemic heart diseases, yield modest results suggesting the compromised cell function and altered exosome performance. We hypothesized that EPCs under inflammatory stress secrete dysfunctional exosomes with altered contents including packaged microRNAs, which eventually compromises exosomes repair ability in ischemic myocardium. Whether modulation of identified targets like specific microRNAs in the cargo of exosomes can rescue and/or enhance their reparative properties of dysfunctional exosomes is not known. We have previously shown in Interleukin-10 (IL-10) KO mice (model mimicking systemic inflammation) that loss of IL-10 impairs EPCs functions via miR-375. After cell expansion, we isolated exosomes from these two groups and compared their functions in terms of cell survival, proliferation, migration and angiogenic capacity
in vitro
and
in vivo
. We report that WT-EPC-exosomes (Exo) transplantation in the ischemic myocardium after MI significantly improved post-infarct repair, neovascularization and left ventricle functions. Our
in vitro
studies revealed that WT-EPC-Exo treatment enhanced endothelial cells proliferation and tube formation and inhibited apoptosis; whereas IL-10 KO-EPC-Exo exhibited opposite effects, suggesting that reparative capacity of WT-EPC-Exo is lost in exosomes derived from IL-10 KO-EPCs. Exosome miRNA profiling revealed a drastically different expression patterns with enrichment of inflammation induced microRNAs including significantly higher expression of miR-375. Interestingly, Knockdown of miR-375 in IL-10-EPCs decreased the expression level of miR-375 in their exosomes and partly rescued their angiogenic dysfunctions. Taken together, our studies suggest that modulation of miR-375 in IL-10 KO-EPC-Exo can rescue its phenotype and promote cardiac repair.
Abstract 406: Oxidative Stress Mediated Myocardial Lipid Dysfunction
Authors: Panchatcharam Manikandan , Chandra Mini , Fox Jonathan , Bhuiyan Shenuarin , Orr Wayne , Kevil Christopher , Alexander Jonathan Steven , Wu Joseph C , Escalante-Alcalde Diana , Miriyala Sumitra
Published: June 2021

Lipid phosphate phosphatases-3 control the conversion of bioactive lipid phosphates to their dephosphorylated counterparts. Oxidative stress transactivates microRNA-92a, which is a negative regulator of LPP3. We found that LPP3 expression was markedly downregulated in ischemic regions after ischemia/reperfusion (I/R) injury. We observed a similar trend in the myocardium from patients samples with acute MI at 24h. Our in vitro studies indicate that overexpression of LPP3 protects the cardiomyocyte against reactive oxygen species (ROS)-induced cardiac injury and knockout of LPP3 gene in the myocardium increases cardiac dysfunction and mortality. Using XF24 Seahorse analyzer we determined the effect of ROS on respiration in pluripotent stem cell-derived cardiomyocytes (iPSC-CM). Adding Phorbol 12-myristate 13-acetate (PMA) to these cells immediately increased oxygen consumption as compared to LPP3 overexpressed cells. This apparent increase in respiration was reversible by oligomycin, which blocks ATP synthase. The rate of oxygen consumption per cell was significantly lower in stimulated compared to LPP3 overexpressed iPSC-CM. The most noticeable difference in the O
2
consumption was found in the presence of carbonilcyanide p-trifluromethoxyphenylhydrazone (FCCP). FCCP is an inner membrane pore opener which resets the proton gradient between mitochondrial matrix and interspace, resulting in continuous transport of protons and consuming O
2
at the maximum potential. Remarkably, while the FCCP treatment increased O
2
consumption in LPP3 overexpressed cells (P<0.05), the treatment showed no effect on the O
2
consumption in the PMA stimulated alone. The result indicated that the low basal oxidative phosphorylation activity in stimulated cells was due to unusually low oxidative phosphorylation potential. To explore the free radical regulation of LPP3 overexpressed cells, superoxide anion was measured using dihydroethidium, a fluorescent cholesterol analog. The levels of superoxide radicals in PMA treated cells were consistently and significantly higher than the levels in LPP3 overexpressed cells (P<0.05). In turn, the radicals can be removed by adding MitoTEMPO (a specific scavenger of mitochondrial superoxide).
Abstract 419: Conserved Age-associated Cytoskeletal Remodeling Improves Cardiac Function and Lifespan
Authors: Kaushik Gaurav , Spenlehauer Alice , Sessions Ayla O , Trujillo Adriana S , Fuhrmann Alexander , Fu Zongming , Venkatraman Vidya , Pohl Danielle , Tuler Jeremy , Wang Mingyi , Lakatta Edward G , Ocorr Karen , Bodmer Rolf , Bernstein Sanford I , Van Eyk Jennifer E , Cammarato Anthony , Engler Adam J
Published: June 2021

The human heart is capable of functioning for decades despite minimal cell turnover or regeneration, suggesting that molecular alterations help sustain heart function with age. However, identification of compensatory remodeling events in the aging heart remains elusive. We present the cardiac proteomes of young and old rhesus monkeys and rats, from which we show that certain age-associated remodeling events within the cardiomyocyte cytoskeleton are highly conserved and beneficial rather than deleterious. Targeted transcriptomic analysis in Drosophila confirmed conservation and implicated vinculin as a unique molecular regulator of cardiac function during aging. Cardiac-restricted vinculin overexpression reinforced the cortical cytoskeleton and enhanced myofilament organization, leading to improved contractility and hemodynamic stress tolerance in healthy and myosindeficient fly hearts. Moreover, cardiac-specific vinculin overexpression increased median life span by more than 150% in flies and preserved their physical activity. A broad array of potential therapeutic targets and regulators of age-associated modifications, specifically for vinculin, are presented and suggest altered metabolism as a system mechanism for lifespan extension. These findings suggest that the heart has molecular mechanisms to sustain performance and promote longevity, which may be assisted by therapeutic intervention to ameliorate the decline of function in aging patient hearts.
Abstract 424: Effector T Cells Induce Cardiac Fibroblasts Transition to Myofibroblasts & Contribute to Pressure Overload Induced Cardiac Fibrosis
Authors: Nevers Tania A , Salvador Ane , Velazquez Francisco , Aronovitz Mark , Blanton Robert
Published: June 2021

Background:
Cardiac fibrogenesis is a major pathogenic factor that occurs in heart failure (HF) and results in contractile dysfunction and ventricular dilation. Recently, we showed that T cell deficient mice (TCRα
-/-
) do not develop cardiac fibrosis (CF) and have preserved cardiac function in the thoracic aortic constriction (TAC) mouse model of pressure overload (PO). Specifically, CD4
+
T cells are activated in the cardiac draining lymph nodes and infiltrate the LV, where the Th1 and Th17 effector T cell signature transcription factors are significantly upregulated as compared with control mice. However, the T cell subsets involved and the mechanisms by which they contribute to CF and pathogenesis of non-ischemic HF remains to be determined. Thus, we hypothesize that heart infiltrated effector T cells perpetuate the fibrotic response by regulating the differentiation and activation of extracellular matrix-producing cardiac myofibroblasts.

Methods and Results:
Naïve or effector T cells differentiated
in vitro
or isolated from mice undergoing TAC or Sham surgery were co-cultured with adult C57BL/6 cardiac fibroblasts (CFB). In contrast with naïve T cells, effector T cells and PO activated T cells strongly adhered to CFB and mediated fibroblast to myofibroblasts transition as depicted by immunofluorescence expression of SMAα. Effector T cell supernatants only slightly mediated this transition, indicating that effector T cells direct contact with CFB, rather than cytokine release is required to mediate CFB transformation. Adoptive transfer of effector, but not naïve T cells, into TCRα
-/-
recipient mice in the onset of TAC resulted in T cells infiltration into the left ventricle and increased CF.

Conclusions:
Our data indicate that CD4+ effector T cells directly interact with CFB to induce CF in response to PO induced CF. Future studies will determine the adhesion mechanisms regulating this crosstalk and evaluate the pro-fibrotic mechanisms induced and whether this is a T effector cell specific subset. These results will provide an attractive tool to counteract the inflammatory/fibrotic process as an alternative option for the treatment of CF in non- ischemic HF.
Abstract 427: Tissue Inhibitor of Matrix Metalloproteinase-3 (TIMP3) Provides Beneficial Effects Post-MI by Promoting Angiogenesis
Authors: Takawale Abhijit R , Zhang Pu , Basu Ratnadeep , Azad Abul , Farhan Maikel , Wang Xiuhua , Murray Allan , Kassiri Zamaneh
Published: June 2021

Introduction:
Myocardial infarction (MI) results in loss of cardiomyocytes, adverse extracellular matrix (ECM) remodelling, leading to left ventricular (LV) dilation and dysfunction. Tissue inhibitor of metalloproteinase (TIMPs) are MMP inhibitors, main regulators of ECM integrity. TIMPs can also regulate other aspects of myocardial remodeling such as hypertrophy, fibrosis and inflammation. TIMP3 levels are reduced in the peri-infarct zone within 24 hours post-MI in mice.

Hypothesis:
Replenishment of TIMP3 post-MI limit infarct expansion, and attenuate LV dilation and dysfunction.

Methods:
MI was induced in adult male wildtype (C57BL/6) mice by ligation of the left anterior descending artery. Adenoviral constructs expressing human TIMP3 (Ad- hTIMP3) or no-TIMP (Ad-Null, control) were injected in the peri-infarct zone (5.4x10
7
pfu, 5 injections/heart). Cardiac function was assessed by echocardiography. Cardiomyocyte density (WGA/DAPI staining), vascular density (Fluo-lectin injection, CD31 IHC), ECM composition (PSR staining) were assessed at 3 and 7 days post-MI. In vitro, angiogenic potency of TIMP3 (rTIMP3) was assessed using the 3D fibrin gel-based angiogenesis assay using primary human vascular (HUVECs) and coronary artery endothelial cells (HCAECs), and co-IP between TIMP3 and VEGFR2.

Results:
Ad-TIMP3 injections significantly improved LV function and reduced LV dilation as compared to Ad-Null group post-MI. Infarct size was markedly reduced with TIMP3 injections and more viable myocytes were preserved in the infarct zone at 1wk post-MI. Ad-TIMP3-MI group showed a higher density of endothelial cells and increased coronary density in the infarct and peri-infarct regions compared to the Ad-null group. This suggested that Ad-TIMP3 promotes angiogenesis in the infarcted myocardium. In vitro studies confirmed that rTIMP3 promoted angiogenesis/sprouting in human endothelial cells up to100ng/ml. However at higher concentrations (>1ug/ml), rTIMP3 exerted anti-angiogenic effects by binding to VEGFR2. This function of rTIMP3 appears to be through an MMP-inhibitory mechanism.

Conclusion:
The novel pro-angiogenic function of TIMP3 post-MI could provide additional beneficial effects in post-MI treatment.
Abstract 438: Hydrogen Sulfide Promotes Mitochondrial Biogenesis Through the Regulation of AMP-activated Protein Kinase
Authors: Shimizu Yuuki , Polavarapu Rohini , Calvert John
Published: June 2021

Background:
Hydrogen sulfide (H
2
S) possesses numerous cellular actions that account for its cardioprotective effects. A mechanism of particular interest is its effects on the mitochondria. At low concentrations, H
2
S donates electrons to the electron transport chain, whereas at high concentrations it inhibits mitochondrial respiration. H
2
S therapy improves mitochondrial function and prevents the loss of mitochondria following the onset of myocardial ischemia. However, it is not known if these improvements are associated with simply a reduction in injury or if mitochondrial biogenesis is involved. Therefore, the purpose of this study was to determine if H
2
S regulates/induces mitochondrial biogenesis in the heart.

Methods and Results:
C57BL/6J mice (8 weeks of age) were given an orally active H
2
S donor (SG-1002; 20 mg/kg/day) in their chow for 4 weeks. For these studies we focused our analysis on an AMPK-PGC1α signaling cascade. SG-1002 significantly increased the phosphorylation of AMPK, the serine phosphorylation of PGC1α, and increased the nuclear localization of PGC1α. This was associated with an increase in the gene expression of PGC1α target genes associated with mitochondrial biogenesis, an increase in mitochondrial to nuclear DNA ratios and an increase in citrate synthase activity. SG-1002 failed to elicit these changes in AMPK deficient mice. Therefore, we sought to determine how SG-1002 activated AMPK. SG-1002 did not alter the phosphorylation of LKB1, an upstream kinase of AMPK, and did not alter the levels of AMP (activator of AMPK). SG-1002 did not alter the expression of protein phosphatase 2A (PP2A; dephosphorylates AMPK), but it did significantly decrease the activity of PP2A). This decrease was accompanied by an increase in the sulfhydration of PP2A, suggesting that this modification is inhibitory.

Conclusion:
These data suggest that H
2
S augments mitochondrial biogenesis in the heart via an AMPK-PGC1α signaling cascade. This is important because mitochondrial abnormalities are associated with a number of disease states (diabetes and heart failure) where H
2
S levels are decreased. Therefore, strategies aimed at increasing H
2
S levels could potentially induce the generation of new, healthy mitochondria.
Abstract 446: Estrogen and Estrogen Receptor Protect the Female Heart From Ischemia/reperfusion Injury by Increasing the Activity of Mitochondrial p38β and MnSOD
Authors: Luo Tao , Liu Han , Kim Jin K
Published: June 2021

Background:
We have previously demonstrated a novel
in-vitro
mechanism of how 17β-estradiol (E2) protects cultured neonatal cardiomyocytes from H/R by identifying a functionally active mitochondrial pool of p38β and E2-driven upregulation of manganese superoxide (MnSOD) activity via p38β. This protective mechanism leads to E2-mediated suppression of reactive oxygen species and attenuates apoptosis. However, little is known about these cytoprotective actions of E2 in the heart after myocardial I/R
in vivo
. The aim of this study is to determine whether the cardioprotective effects of E2 involve the interaction of mitochondrial p38β and MnSOD in the heart
in vivo
.

Methods and Results:
Left coronary ligation for 45 min and reperfusion for 24 hr was used to produce myocardial I/R injury in ovariectomized female mice with or without E2 pellet (0.1 mg/pellet). TTC staining showed that the infarct size was approximately 30% of the left ventricle at risk in untreated mice after I/R. The infarct size was reduced by more than half in the E2-treated group. Western blotting showed that the phosphorylation of p38β (p-p38β) in the heart homogenate was upregulated by E2, compared to the untreated mice, while the total p38β protein level was not altered. Mitochondrial p-p38β isolated from the hearts decreased after I/R, while E2 administration attenuated this effect. The total protein level of MnSOD decreased after I/R, regardless of E2 treatment. However, the MnSOD activity was normalized by E2 treatment, suggesting that E2 specifically increased the enzymatically active subset of MnSOD. Physical interaction between mitochondrial p38β and MnSOD was found by using co-immunoprecipitation from the hearts of the studied animals. Estrogen receptor (ER) α, β and double knockout female mice exhibited larger infarct size after I/R injury than that of WT mice while no significant difference was observed between the knockout mice. Loss of both ERs led to reduced activity of mitochondrial p38β and MnSOD at the baseline and after myocardial I/R.

Conclusion:
This work offers
in-vivo
evidence to our previously detailed mechanistic data that the protective effects of estrogen and ER against cardiac I/R injury involve the regulation of MnSOD activity via mitochondrial p38β.
Abstract 450: Unveiling Human Cardiac Stem Cells Role in Myocardial Ischemia-reperfusion Injury
Authors: Sebastião Maria J , Serra Margarida , Pereira Rute , Brito Catarina , Palacios Itziar , Gomes-Alves Patrícia , Alves Paula M
Published: June 2021

After an Acute Myocardial Infarction (AMI), Ischemia-Reperfusion (I/R) injury is responsible for a critical decrease in the number of viable cardiomyocytes (hCMs). Human adult myocardium harbors a population of endogenous cardiac stem cells (hCSCs) that is activated upon I/R injury, contributing to myocardial repair through the establishment of an auto/paracrine molecular crosstalk between hCSCs and hCMs in stress. Our work aims at setting up the first
in vitro
human I/R injury model in order to decipher the role of hCSCs and correspondent cross talk between hCSCs and hCMs upon AMI using proteomic tools. Human CSCs, hCMs cultures and co-cultures were established using human donor derived CSCs (c-kit
+
, CD45
-
) and hCMs derived from human induced pluripotent stem cells at different maturation stages (hiPSC-CMs). Ischemia was mimicked by substituting growth media by Ischemia Mimetic Solution (including nutrient depletion, lactate accumulation, acidosis and hyperosmosis) and placing the cells at 0% O
2
for 5 hours. In the reperfusion step, cells were placed back in their physiological culture conditions (3% O
2
). The effect of I/R injury on growth factor secretion, cells’ viability, as well as on hCSC proliferation was accessed in both mono- and co-culture systems. In addition, hCSCs total proteome analysis was performed at different timepoints (before injury, after ischemia and after 1h and 16h of reoxygenation) by LC-MS. Important features of I/R injury were successfully captured in our model, namely hCSC proliferation activation upon insult, the increase in HGF secretion during the first hour after reoxygenation, and the protective role of hCSCs on hiPSC-CMs. The maturation stage of hiPSC-CM showed to be of high relevance in the response to injury. More than 2000 proteins were identified in hCSCs per experimental time point; proteins associated with mitochondrial dysfunction and oxidative stress response were identified in hCSCs exposed to injury. This system will allow further understanding on the molecular landscape of the myocardium during AMI, namely regarding hCSC regenerative response and hCM survival. The knowledge gained in this work will potentiate the development of novel therapies for myocardium regeneration.
Abstract 453: The G Protein-coupled Estrogen Receptor GPER/GPR30 is a Potential Therapeutic Target for Cardiovascular Inflammation
Authors: Flores Roxana , Nizet Victor , Insel Paul A , Corriden Ross
Published: June 2021

Abstract: Neutrophil extracellular traps (NETs), DNA-based structures that play an important role in pathogen killing/capture, are also thought to contribute to inflammatory disease and have been implicated in thrombosis. Pharmacological agents that can modulate the formation of extracellular traps are as-yet unknown. We hypothesized that targeting G protein-coupled receptors (GPCRs) may provide such agents and thereby could aid in treating inflammation in the cardiovascular system. Here, we investigated the role of the G protein-coupled estrogen receptor (GPER/GPR30), which has recently been shown to play a role in innate immune function (Cabas et al.
J Immunol
. 191: 4628-39 [2013]). Quantitative PCR revealed that GPER is highly expressed in human neutrophils isolated from healthy male and female donors. Fluorescent quantification of extracellular DNA using PicoGreen revealed that both selective (G-36) and general (raloxifene) antagonists of GPER, but not selective antagonists for the nuclear estrogen receptors ERa (MPP) or ERb (PHTPP) inhibited phorbol myristate acetate (PMA)-induced NET production by human neutrophils. These results were verified using high throughput imaging of fixed, SytoxGreen-stained neutrophils. The activity of GPER antagonists appeared to be independent of Erk1/2 signaling and reactive oxygen species (ROS) generation, pathways that are mediators of NET production. Immunocytochemistry revealed that G-36 treatment of neutrophils raises their concentration of cAMP, which can inhibit production of NETs. Taken together, our data suggest that GPER may be a novel therapeutic target for modulating NET production and inflammation in the cardiovascular system.
Abstract 456: Early Doxorubicin Exposure Upregulates P-STAT3 Expression in Adult Murine Model
Authors: Harris Tanoya L , Aune Gregory J
Published: June 2021

Background:
Anthracyclines such as doxorubicin (DOX) are a key component of chemotherapy treatments and also have cardiotoxic effects that can manifest decades after initial exposure. Long term survivors have a 15 times greater risk of heart failure than the general population. Previously, our lab has shown an increase in serum leptin in DOX treated animals 15 months after exposure. Altered p-STAT3 activation has been shown in patient and mouse models of heart failure and is downstream of leptin receptor activation. Our overall aim was to evaluate p- STAT3 levels in mice hearts of aged mice previously exposed to doxorubicin during development.

Methods:
C57/BL6J mice were injected weekly intraperitoneally with saline or 1 mg/kg, 3 mg/kg/ and 5mg/kg/ week of the DOX from 2 weeks to 6 weeks of age. At 7 weeks one cohort of mice were harvested. At 15 months a second cohort was harvested. Hearts were cut in half and one half paraffin sectioned. From the other half, heart lysates were prepared. P-STAT3 and STAT3 expression was evaluated by Immunohistochemistry and Western blots.

Results and Conclusions:
Western blot confirms a dose-dependent upregulation of p-STAT3 expression in DOX treated mice hearts at 80 weeks old compared to controls (1.13 VS. 0.04, p-STAT3/STAT3 ratio). In addition, p-STAT3 is increased by immunohistochemistry. This indicates that early exposure to DOX may produce changes in the heart that continue throughout life. These findings suggest that gene targets of activated p-STAT3 may drive the gradual progression to heart failure in patients exposed to anthracyclines during childhood. Future studies, may elucidate therapeutic targets to slow the progression of heart failure in these patients.
Abstract 50: DNA Methylation Signatures in Blood Distinguish Patients at Risk of Post-operative Atrial Fibrillation Following Cardiopulmonary Bypass
Authors: Monte Emma , Fischer Matthew A , Wisniewski Nicholas , Scovotti Jennifer , Howard-Quijano Kimberly , Kimball Todd , Hofer Ira , Wang Yibin , Pellegrini Matteo , Mahajan Aman , Vondriska Thomas
Published: June 2021

Post-operative atrial fibrillation (AF) is the most common complication for cardiac surgery, with an incidence of 27-40% and is associated with increased 30-day mortality. Several models exist to predict post-op AF using clinical data, however none have been widely implemented, and all have room for improvement (average area under ROC curve is ~0.70). We tested whether DNA methylation, assayed from pre-operative blood draws, could add predictive value.

We enrolled a cohort of 55 adult patients undergoing cardiopulmonary bypass, with a 36% incidence of post-op AF. Reduced representative bisulfite sequencing provided single-base resolution for an average of 3 million CpGs with 10x sequencing coverage per sample. We first tested whether these CpGs were correlated with AF. Of the 809,569 CpGs that passed thresholds in all samples, 16,774 were significantly correlated with AF (corrected p-value <=0.01 using methylKit in R). We used the R package Glmnet to perform logistic regression on the 16,774 CpGs in 40 of the 55 patients using 5-fold cross validation to build a model and selecting the 381 CpGs which were included in 95% of the 5,000 models. Our assumption being that these 381 CpGs were predictive no matter the subset of patients selected in the randomly generated groups employed during cross validation. Using these 381 CpGs as inputs we built a final model that ultimately incorporated 51 CpGs. None of the 51 CpGs were significantly correlated with age (corrected p-value <=0.01), ruling out age as a confounding factor. We tested our model on the remaining 15 patients. Our model correctly predicted 5 of the 5 AF cases, and 9 of the 10 AF-free cases. Validation of our model in a larger cohort is ongoing, and future directions will incorporate our epigenomic signature with readily available clinical risk factors to further optimize the model.
Abstract 56: Growth Hormone Releasing Hormone Improves Sarcomere Relaxation and Calcium Decline in Cardiomyocyte From a Mouse Model of Heart Failure With Preserved Ejection Fraction
Authors: Dulce Raul A , Kanashiro-Takeuchi Rosemeire M , Takeuchi Lauro M , Balkan Wayne , Cai Renzhi , Schally Andrew V , Hare Joshua M
Published: June 2021

Heart failure with preserved ejection fraction (HFpEF) is characterized by impaired relaxation, ventricular stiffening and fibrosis. Growth hormone releasing hormone (GHRH) agonists reduce fibrosis in rat and swine models of ischemic myocardial injury. However, their effect on cardiomyocytes is not known. We hypothesized that activation of GHRH receptor signaling improves impaired cardiomyocyte relaxation in a mouse model of HFpEF. C57BL6N mice (n=4-5) were implanted with a mini-osmotic pump to deliver angiotensin-II (Ang-II: 0.8 mg/kg/day) for 4 weeks and received daily injections of GHRH-Agonist (GHRH-A [MR-409]: 100 μg/kg) or vehicle (DMSO+propylene-glycol). Cardiomyocytes were isolated and calcium and sarcomere shortening assessed. Ang-II-treated cardiomyocytes exhibited reduced sarcomere length, indicating an inability to completely relax, despite lower resting calcium. These cardiomyocytes also exhibited impaired ability to contract with no changes in calcium transient amplitude, deficient relaxation and delayed calcium decay. MR-409 treatment restored resting calcium and resting sarcomere length; improved sarcomere shortening and completely abrogated Ang-II-induced delay in calcium decline and relaxation (see figure 1). Our findings demonstrate that chronic administration of Ang-II mediates structural and functional changes consistent with HFpEF and suggest that activation of the GHRH receptor signaling pathways prevents HFpEF-associated cardiomyocyte performance alterations.
Abstract 81: Absence of Macrophage microRNA-155 Affects Cardiac Metabolic and Inflammatory Remodeling Following Diabesotension and Protects Against Development of Heart Failure
Authors: Rech Monika , Wouters Kristiaan , Lecomte Julie , Bitsch Nicole , Heymans Stephane , van Bilsen Marc , Schroen Blanche
Published: June 2021

The co-existence of diabetes, obesity, hypertension - diabesotension - is a major burden in western societies and often culminates into heart failure (HF). Diabesotension is associated with a chronic status of low-grade inflammation, involving macrophage infiltration into adipose and cardiac tissues, local activation of inflammatory pathways and concomitant insulin-resistance. We showed that the microRNA miR-155 is a powerful modulator of hypertension-induced cardiac inflammation and HF. Here we hypothesize that absence of miR-155 in macrophages protects against adipose tissue and cardiac inflammation during diabesotension and thereby prevents insulin resistance and cardiac dysfunction.

Male C57Bl/6J mice received a bone marrow transplantation of miR-155 WT (n=30) or miR-155 KO (n=30) donors with same genetic background. High fat (HFD; n=22/gr) or chow diet (n=8/gr) was given for 20 weeks. An HFD subgroup was subjected to pressure overload by angiotensin II (1.5mg/kg/d) for 4 weeks (HFD+PO; n=12/gr). Glycaemia, glucose tolerance test, cardiac MRI were performed after 16 and/or 20 weeks. Organs were stored for histology, RNA and FACS analysis.

After 16 weeks of HFD, body weight (+11% WT; +14% KO; p<0.05) and glycaemia (+16% WT; +18% KO; p<0.05) increased compared to chow diet animals in both genotypes, confirming the status of obesity and pre-diabetes. Absence of macrophage miR-155 protected HFD-mice from glucose intolerance (at T15: WT 25.08; KO 18.15 mmol/L; p<0.05), suggesting that adipose tissue macrophages might contribute to insulin resistance. In addition, HFD+PO caused mild systolic dysfunction in WT but not in miR-155 KO animals compared to control (ejection fraction: -19% WT, p<0.05; -7% KO, n.s.). While cardiac interstitial fibrosis was not affected by absence of macrophage miR-155, cardiac CD45-positive leukocyte infiltration upon HFD+PO was abrogated. HFD+PO-induced cardiac expression of two PPAR-dependent metabolic genes, angiopoietin-like 4 and uncoupling protein 3, is repressed in the absence of macrophage miR-155.

Our data suggest that macrophage miR-155 plays a role in the development of insulin resistance and cardiac dysfunction, possibly by affecting local immune cell function and metabolic programming.
Abstract 96: Simvastatin Prevents TGF-β1-induced SMAD2/3 Phosphorylation Involved in Ventricular Fibrosis: Role of Protein Phosphatases
Authors: Rizvi Farhan , Siddiqui Ramail , DeFranco Alessandra , Holmuhamedov Alisher , Xu Hao , Emelyanova Larisa , Ross Gracious , Shi Yang , Holmuhamedov Ekhson , Tajik A. Jamil , Jahangir Arshad
Published: June 2021

Background:
Ventricular fibrosis leads to progressive cardiac dysfunction and heart failure (HF). Statins are reported to reduce cardiac fibrosis through the cholesterol-independent pathway, but mechanisms remain elusive. We hypothesize simvastatin reduced TGF-β1-induced ventricular fibrosis through activation of SMAD protein phosphatase Mg
2+
/Mn
2+
-1A (PPM1A), -2A (PP2A).

Methods:
In the absence and presence of TGF-β1 (5ng) with or without simvastatin (1μM), the rate of fibroblast proliferation (doubling time), myofibroblast differentiation (ICC), α-SMA mRNA (RT-PCR) and protein expression (Western blot) and the release of collagen synthesis markers, pro-collagen type I C-terminal peptide (PICP) and pro-collagen type III N-terminal peptide (PIIINP), in the media (ELISA) were determined along with protein interaction between SMAD2/3 and PPM1A or PP2A (Co-IP) and SMAD2/3 phosphorylation (Western blot).

Results:
Simvastatin reduced the effect of TGF-β1 on hVF proliferation by 47% (50000 to 26500), p<0.01; myofibroblast differentiated population from 48% (avg 48/100) to 11% (avg 11/100), p<0.01; expression of α-SMA mRNA by 76%, p<0.01; and protein by 60%, p<0.05. Simvastatin also decreased release of PICP by 66%, p<0.01, and PIIINP by 83%, p<0.01, into the media. Time-dependent increases in SMAD2/3 phosphorylation were reduced by simvastatin through activation of protein phosphatases PPM1A and PP2A by interacting with SMAD2/3.

Conclusion:
Involvement of PPM1A and PP2A in the anti-fibrotic effect of simvastatin reveals novel signaling mediators that may be selectively targeted for prevention of myocardial injury-induced ventricular fibrosis and HF.
Abstract 97: Ca
2+
Influx Via

I
CRAC

Channels is Involved in Increased Secretion/Deposition of Collagen by Human Failing Heart Ventricular Fibroblasts
Authors: Ross Gracious R. , Emelyanova Larisa , Rizvi Farhan , Shi Yang , Holmuhamedov Ekhson L. , Werner Paul , Tajik A. Jamil , Jahangir Arshad
Published: June 2021

Introduction:
Excessive cardiac fibrosis (CF) underlies mechanical and electrical dysfunction in heart failure (HF). However, the underlying mechanisms of increased CF are unclear. Recently, we reported increased Orai1 expression and

I
CRAC

(calcium release-activated channels) -Ca
2+
influx in failing heart ventricular fibroblasts (hVF). Therefore, we studied the magnitude of collagen secretion/deposition by hVF from failing hearts and the involvement of Ca
2+
influx through

I
CRAC

channels in this process.

Hypothesis:
We tested the hypothesis that collagen secretion/deposition by hVFs from failing hearts will be high and sensitive to inhibition of

I
CRAC

channels, compared to normal hearts.

Methods:
Primary cultures of hVF obtained from HF patients undergoing LV assist device implantation (n=9) were compared to non-diseased hVF from trauma victims (n=4). The degree of fibrosis was analyzed by histology of LV tissue sections (5μm) with Masson’s trichrome staining. Collagen secretion/deposition after 48 h culture of hVFs from failing or normal hearts in the presence or absence of

I
CRAC

inhibitor, YM-58483(10 μM), was assayed calorimetrically using Sircol™ kit. Data were analyzed by unpaired Student’s t-test.

Results:
Histology of the failing heart LV sections with Masson’s trichome staining showed severe fibrosis compared to control sections. The percentage of positive staining area to total area, measured using custom-developed ImageJ macro, was significantly (p<0.05) higher in the failing heart (4.5±0.2%) compared to that of normal (0.1± 0.01%). The secretion/deposition of soluble collagen was significantly (p<0.05) higher in failing heart hVF (86± 7.6 μg/10
6
cells) than normal heart hVF (58.4±8.8 μg/10
6
cells). Incubation of fibroblasts with the

I
CRAC

inhibitor YM58486 (10 μM) for 48 h eliminated the increase in collagen secretion of HF fibroblasts (55.8± 7 μg/10
6
cells vs 66.8± 6 μg/10
6
cells, p=0.27).

Conclusion:
LV fibrosis is severe in HF patients. The magnitude of pro-fibrotic collagen secretion/deposition by hVF from HF patients is significantly high but sensitive to

I
CRAC

inhibition. Selective targeting of fibroblast

I
CRAC

may be a potential therapeutic tool to mitigate progression of CF and HF.
Abstract 104: Navigated Brain Stimulation for Upper Limb Recovery After Stroke: A Randomized Sham Controlled Clinical Trial of Low Frequency r-TMS to Non-injured Hemisphere Combined With Upper Limb Rehabilitation
Authors: Harvey Richard L , Liu Charles , Edwards Dylan , Dunning Kari , Fregni Felipe , Stein Joel , Laine Jarmo
Published: June 2021

Introduction:
Upper limb function following stroke is limited, with only 50% regaining some function and less than 20% regaining normal function. Repetitive TMS has promise as an adjunct to upper limb therapy after stroke. We aimed to determine if navigated brain stimulation (NBS) with a low-frequency (1 Hz) protocol to non-injured hemisphere combined with upper limb rehabilitation would improve arm motor function better than rehabilitation alone.

Method:
We enrolled 199 patients with hemiplegia from ischemic or hemorrhagic stroke within 3-12 m post ictus. Randomization was in a 2:1 ratio to NBS with 18 sessions of rehabilitation over 6 weeks, or to sham NBS with therapy. The primary end-point was rate of achieving a 5-point improvement on the upper limb Fugl-Myer (ULFM) score at 6 m post-treatment and safety. Secondary outcomes included post-treatment, 1 m, 3 m and 6 m change on ULFM, action research arm test (ARAT), and EQ-5D-3L health questionnaire.

Results:
Of 199 subjects enrolled, 167 completed treatment and follow-up due to early stoppage of data collection after interim futility analysis. All subjects improved significantly on each outcome measure at each point of follow up, including 6 m post-treatment ULFM: treatment (8.1±7.4, p<0.001) and sham (8.5±8.7, p<0.001). In the ITT analysis, there was no difference on achievement of 5 points on ULFM at 6 m post-treatment: 67% treatment vs. 65% sham (chi-sq 1.105, p=0.76). Repeated measures ANCOVA group*time interaction showed no significant difference between groups for ULFM (p=0.87), ARAT (p=0.80) and the EQ 5D (p=0.96). There were no study or device related serious adverse events and no difference in SAE’s between groups.

Conclusion:
NBS can be safely used in the clinical setting. Clinically important gains were observed in both study arms suggesting no additional effect of 1 Hz NBS in stroke subjects within 3-12 m post ictus. The lack of NBS effects may be explained by the large effect size (ceiling effect) or potentially that the sham coil unintentionally induced cortical currents. Further analysis of the sham method and also secondary subgroup analyses will provide further insights and generate novel hypothesis to be confirmed in future NBS trials.
Abstract 105: Early But Not Too Early, High Intensity Physical Exercise Rehabilitation May be Beneficial: A Pilot Clinical Study
Authors: Tong Yanna , Ding Jamie Y , Sadowsky Mordechai G , Du Huishan , Geng Xiaokun , Ding Yuchuan
Published: June 2021

Introduction:
Very early mobilization (VEM) is thought to be beneficial for stroke-unit care, but many key factors, such as initiation time and intensity, have not been optimized. We attempted to determine an optimal mobilization plan with well-defined initiation time and intensity in clinical practice.

Methods:
We conducted a randomized controlled trial with blinded assessment at follow-up. Patients with ischemic stroke, first or recurrent, admitted to the stroke unit within 24 h of stroke onset who met physiological criteria were randomly assigned (1:1:1) to 3 groups: Routine Mobilization (RM) received a lower dose of out-of-bed mobilization (<1.5h/d) 24-48 h post-stroke, Very Early and Intensive Mobilization (VEIM) received a higher dose of out-of-bed mobilization (≥3h/d) within 24 h, and Early and Intensive Mobilization (EIM) received a higher dose of out-of-bed mobilization (≥3h/d) 24-48 h post-stroke. Out-of-bed mobilization involves sitting, standing, and walking with or without assistance. The intervention period lasted 10-14 days. All the patients received standard medical therapy according to their different conditions. Patient function was evaluated with the modified Rankin Scale (mRS) score at discharge, 3 months after, and 6 months after. The primary target was a favorable outcome at follow-up, defined as an mRS score of 0-2. We used the Friedman test and the chi-squared test on a fourfold table for significance.

Results:
A total of 150 patients were recruited, and 120 (80%) finished the training and follow-up assessment with 30 patients dropping out (10 in RM, 12 in VEIM and 8 in EIM). Patients receiving VEIM had lower odds of a favorable outcome at both 3 and 6 months, although the difference did not reach the p = 0.05 level of significance. In the VEIM group, only 42.1% of patients had a favorable outcome 6 months after discharge, as opposed to 55.0% of RM patients and 61.9% of EIM patients.

Conclusions:
The higher dose mobilization protocol started within 24 h post-stroke was not associated with an increase of favorable outcomes at both 3 and 6 months post-discharge, but rather with a trend toward poorer outcomes. However, the higher dose mobilization protocol started within 48 h post-stroke may lead to a better outcome.
Abstract 106: High Red Blood Cell Components of Retrieved Clot Related With Recanalization During Intraarterial Thrombectomy
Authors: SHIN JONG WOOK , Jeong Hye Seon , Song Hee-Jung , Kwon Hyon-Jo , Song Kyu Sang , Kim Jei
Published: June 2021

Background:
Retrieved clots obtained during intraarterial thrombectomy (IAT) had variable histologic composition containing red (RBC) and white blood cells (WBC), congregated fibrin/platelets. We analyzed histologic composition differences of the retrieved clots by stroke subtypes and recanalization status of the occluded vessels after IAT.

Method:
We analyzed histologic and angiographic findings of 69 retrieved clots obtained from 26 acute ischemic stroke patients (large-artery atherosclerosis, 6; cardioembolism, 15; undetermined etiology, 5 patients), who received IAT. The individual clot was measured positive pixels of RBC, WBC, and congregated-fibrin/platelet components on Hematoxylin-eosin stained clot using semiautomated quantitative analysis (Aperioscanscope, ver. 12.2, Leica Biosystems). Differences of the three components were compared by the stroke subtypes and by achievement of recanalization status defined as 2 or 3 of the Arterial Occlusive Lesion Scale. Finally, relationship of the recanalization status with clot composition and other risk factors was evaluated using multivariate analysis.

Result:
RBC component was higher in subtype of cardioembolism (36.04%) than large-artery atherosclerosis (18.18%) and undetermined etiology (24.26%, p=0.019, Kruskal-Wallis test). Instead, congregated-fibrin/platelet component was higher in large-artery atherosclerosis (78.87%) than cardioembolism (59.95%) and undetermined etiology (72.28%, p=0.012). On comparison by the recanalization status, RBC component was higher in retrieved clots achieved recanalization (34.65%) than no-recanalization (13.98%, p=0.003, Mann-Whitney test) of the occluded vessel. Congregated-fibrin/platelet component was lower in the recanalized (61.81%) than the no-recanalized clot (81.63%, p=0.003). On multivariate analysis, RBC component only showed statistical significance as a factor related with recanalization status of the occluded vessels.

Conclusion:
The present study showed that the high RBC and low fibrin/platelet composition of retrieved clot related with cardioembolic stroke. The RBC-rich clot was related with achievement of recanalization after IAT rather than the platelet/fibrin-rich clot.
Abstract 120: Radiocarbon Birth Dating of Cerebral Arteriovenous Malformations
Authors: Cooke Daniel L , Halberkann Meike , Matzenauer Christian , Kim Helen , Lawton Michael , Beseoglu Kerim , Dreier Rita , Buchholz Bruce , Macdonald R. Loch , Etminan Nima
Published: June 2021

Introduction:
The chronological development of brain arteriovenous malformations (AVM) is controversial. Atmospheric nuclear testing until 1963 produced a global bomb-pulse of
14
C and thus a chronometer of molecular synthesis in the entire biosphere from 1955 to the present. We isolated and dated a durable, representative structural protein from human cerebral arteries (CA) and brain AVMs.

Method:
Samples from cadaveric CAs or from brain AVMs from patients undergoing surgical resection were processed to ultra- purify elastin. The age of purified elastin extracted from cerebral arteries or from AVMs was measured using
14
C bomb-pulse technique. We modified a
14
C biological incorporation equation model using variable AVM formation dates and elastin formation and degradation rates.

Results:
A total of 62 CAs from individuals with ages ranging from 1 month to 100 years and 30 unruptured or ruptured AVMs were collected. All CAs and nearly all AVMs yielded ultra-purified elastin in sufficient amount for further
14
C birth dating. Mean age of elastin was 13.4± 11.2 years (n = 34), whereas elastin derived from AVMs was less than 5 years old (n = 15, p = 0.002). Based on
14
C content relative subject birthdate and date of tissue collection, best-fit modeling of CA and AVM elastin demonstrated turnover rates less than 2% and 25 - 50%, respectively. Amongst AVMs, those with associated aneurysms had younger elastin (p=0.007). Larger AVMs tended to have younger elastin (p=0.086) and AVM rupture was not associated with elastin age (p=0.358).

Conclusions:
Our data provides the first chronological evidence for the longevity of elastin in CAs, as evident based on the inclusion of bomb carbon in elastin from subjects who reached maturity prior to 1955. Importantly it also demonstrates elastin isolated from brain AVMs is contemporary and likely immature suggesting a non-congenital origin and/or rapid vascular turnover.
Abstract 122: Transit Time on Digital Subtraction Angiography Correlates with Cerebral Arteriovenous Malformation Flow Measured by Quantitative Magnetic Resonance Angiography
Authors: Brunozzi Denise , Hussein Ahmed E , Shakur Sophia F , Linninger Andreas , Hsu Chih-Yang , Charbel Fady T , Alaraj Ali
Published: June 2021

Introduction:
Digital subtraction angiography (DSA) currently provides angioarchitectural features of cerebral arteriovenous malformations (AVMs) but its role in the hemodynamic evaluation of AVMs is poorly understood. Here, we assess transit time (TT) of contrast on DSA relative to AVM flow measured using quantitative magnetic resonance angiography (QMRA).

Methods:
Patients seen at our institution between 2007 and 2014 with a cerebral AVM and DSA and QMRA obtained prior to any treatment were retrospectively reviewed. TT on DSA was defined as time needed for contrast to change image intensity from 10%-100%, 100%-10%, and 25%-25%. TT was correlated to AVM total flow and angio-architectural features.

Results:
33 patients (mean age 34.8 years) were included. 7 patients presented with hemorrhage. Mean AVM volume was 13.61 mL (range 0.28-84.2 mL). Higher total flow significantly correlated with shorter TT100%-10% and TT25%-25% (P=0.04, P=0.03, respectively). Total flow was also significantly related to the ratio sum of draining vein diameters/TT (TT10%-100% P=0.04, TT100%-10% P=0.01, TT25%-25% P=0.02). Presence of venous stenosis (P=0.01) and arterial ectasia (P=0.03) correlated significantly with TT100%-10%. TT was not significantly related to hemorrhagic presentation, deep drainage, single draining vein, or Spetzler-Martin grade.

Conclusions:
TT and the ratio sum of draining vein diameters/TT correlated significantly with AVM total flow measured using QMRA, and so TT along with venous diameters determined from DSA may be used as a surrogate for AVM flow.