bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1592: Electrical Stimulation of LV Fibroblasts Causes Frequency-Dependent Modulation of Matrix Metalloproteinase Release and Increase in Tissue Inhibitors of Metalloproteinases
Authors: Mukherjee Rupak , Sheats Nina J , Bouges Shenikqua , Chang Eileen I , Stroud Robert E , McDermott Paul J , Spinale Francis G
Published: July 2021

LV fibroblasts (LVMFs), which form a cellular netwrork throughout the myocardium including electrical connectivity to myocytes, are a primary cell type that contributes to remodfeling of the extracellular matrix through the synthesis of matrix metalloproteinases (MMP) and tissue inhibitors of the MMPs (TIMPs). While cytokines and other signaling molecules can induce MMP and TIMP synthesis and release from LVMFs, whether and to what degree LVMFs respond to changes in electrical stimulation with respect to modulation of MMP/TIMP release remains unknown. Porcine LVMFs were grown to confluence, serum deprived for 24 hours, and then randomized to electrical stimulation (6x10
5
cells/well, 130V 5ms pulses) for 24 hours at 1, 2, and 4 Hz (n=6 wells/frequency). Unstimulated cells (0 Hz) served as controls. Electrical stimulation had no effect on LVMF morphology. Cell media and pellets were collected for levels of MMP-2, MMP-9 (zymography), MT1-MMP, and TIMP-4 (immunoblotting). Changes in MMP and TIMP levels with stimulation are reported as a percentage of unstimulated values (TABLE

). MMP-2, MMP-9, and TIMP-1 levels were higher than unstimulated levels at 2 and 4 Hz. MT1-MMP levels were lower than unstimulated levels at 1 and 2 Hz. Ratios for each of the MMPs to TIMP-1 were computed (TABLE

). While the MMP-2/TIMP-1 and MMP-9/TIMP-1 ratios remained similar to unstimulated values, the MT1-MMP/TIMP-1 ratio was lower than unstimulated values at 1 and 2 Hz. The unique findings of this study are two-fold. First,
in vitro
electrical stimulation of LVMFs, which are generally considered to be electrically inert, resulted in demonstrable changes in the release of specific MMP and TIMP species. Second, the reduced MT1-MMP to TIMP-1 ratio suggests electrical stimulation of LVMFs caused a shift in the stoichiometric balance between MMPs and TIMPs that may favor accumulation of extracellular matrix components.
Abstract 1604: Improved Cardiac Function after Expression of the Human Elastin Gene in the Infarcted Myocardium
Authors: Li Shu-Hong , Sun Zhuo , Guo Lily , Han Mihan , Weisel Richard D , Li Ren-Ke
Published: July 2021

Rationale:
After a myocardial infarction, necrosed cardiomyocytes are replaced by fibrotic tissue. Subsequent thinning and expansion of this non-elastic scar contributes to cardiac dilatation and congestive heart failure. We attempted to restore the elastic properties of the scar by implanting the infarct area with cells expressing elastin.

Methods:
Full-length elastin cDNA was generated by PCR using the human lung cDNA library, and then transfected (in adenoviral vector) into rat bone marrow stromal cells (BMSCs). Western blot and immuno-staining confirmed expression of elastin protein. Seven days after coronary artery ligation, BMSCs transfected with genes for either adeno-elastin (BMSC+Elastin) or adeno-green fluorescence protein (BMSC), or media (control group) were injected into the infarct area of adult rats (n=8/group).

Results:
Over 8 weeks following cell or media implantation, cardiac function (fractional shortening by echocardiography), ventricular volumes, and load independent indices of cardiac function (end systolic and diastolic volumes, preload recruitable stroke work and end systolic elastance by pressure-volume catheter), were all significantly better preserved in both BMSC groups (p<0.05 vs. media), with further improvements in the BMSC+Elastin group (p<0.05 vs. BMSC group). Biochemical analysis detected over-expression of recombinant elastin, and histological examination revealed that collagen fiber length and diameter were preserved in the scar tissue. Myocardial birefringence determined by polarized light (linear retardance of the tissue structure) was significantly highest in the infarct area of the BMSC+Elastin group (p<0.05 compared to BMSC and media groups). Elastin over-expression by cell-based gene therapy restored the elastic matrix structure in the scar tissue, stabilized the infarct and prevented ventricular dilatation, producing the smallest (p<0.05) scar size and surface area in the BMSC+Elastin group.

Conclusions:
Over-expression of elastin in transplanted BMSCs contributed to the alignment and organization of an elastic extracellular matrix structure, which prevented cardiac dilatation and dysfunction. Elastin plays a key role in the remodeling of the extracellular matrix.
Abstract 1623: Reactive Oxygen Species increases Intracellular Calcium, alters the Electrophysiological Properties and Synaptic Transmission in Intrinsic Cardiac Ganglion Neurons:
in vitro
Authors: Dyavanapalli Jhansi , Rimmer Katrina , Harper Alexander A
Published: July 2021

Anerobic metabolism generates reactive oxygen species (ROS) as by-products and this is increased during ischemia. ROS have been shown to interact with and impair the functioning of membrane proteins such as ion channels and transporters and cause disturbances in intracellular Ca
2+
homeostasis. We have investigated the effects of ROS upon [Ca
2+
]
i
, the intrinsic electrophysiological characteristics and synaptic transmission in neurons of the ICG regulating the sinoatrial node. The hypothesis is that ROS-induced damage increases [Ca
2+
]
i
and attenuates synaptic transmission in ICG. used a whole-mount adult rat ICG preparation, in vitro (rats were killed by stunning and cervical dislocation, according to current UK Home Office guidelines). Intracellular recordings were made using sharp glass microelectrodes filled with Oregon Green 488 BAPTA-1, allowing simultaneous recording of electrical properties and measurement of [Ca
2+
]
i
. Signals resulting from [Ca
2+
]
i
changes were expressed as the ratio of fluorescence changes over baseline fluorescence, (f-fo)/fo . The ROS-donors hydrogen peroxide (H
2
O
2
, 1 mM) and tert-butyl hydroperoxide (
t
-BHP, 1mM) had broadly similar actions on postganglionic somata, both producing membrane potential hyperpolarization (from −48.4 mV ± 6.5 S.D control to −69.9 mV ± 7.9 H
2
O
2
, n=7; and from −51.9 mV ± 7.1 control to −59.2 mV ± 7.9
t
-BHP, n=17, p<0.001). Considering the somatic action potential (AP),
t
-BHP decreased the rate of rise (from 159 V/sec ± 79 control to 117 V/sec ± 53, n=6, p<0.05), but did not alter the rate of fall and increased AP duration (measured at 0 mV) from 0.7ms (± 0.2) control to 1.1ms (± 0.3, n=5, p<0.05). In addition,
t
-BHP reduced the AP afterhyper-polarization (AHP) amplitude (from 15.5 mV ± 5.2 control to 9.9 mV ± 4.5, n=10, p<0.001) but had no impact on AP overshoot or AHP duration.
t
-BHP and H
2
O
2
markedly increased resting [Ca
2+
]
i
to 1.59 (± 0.03, n=8,p<0.001) and 2.36 (± 0.04, n=3, p<0.05) respectively, of control values (~ 60 nM ). H
2
O
2
blocked synaptic transmission in 3 /4 neurons. In contrast,
t
-BHP had no significant action on synaptic transmission. Together, these data demonstrate that ROS alters the excitability of ICG neurons attenuating parasympathetic control of the heart during ischemia/ reperfusion.
Abstract 1633: RhoA and Rho Kinase Activation in Human Pulmonary Hypertension - Role of 5-HT Signaling
Authors: Guilluy Christophe , Eddahibi Saadia , Agard Christian , Savale Laurent , Fadel Elie , Adnot Serge , Pacaud Pierre , Loirand Gervaise
Published: July 2021

Background- The complex and multifactorial pathogenesis of pulmonary hypertension (PH) involves constriction, remodeling, and in situ thrombosis of pulmonary vessels. Both serotonin (5-HT) and Rho kinase signaling may contribute to these alterations. Here, we investigated possible links between the 5-HT transporter (5-HTT) and RhoA/Rho kinase pathways, as well as their involvement in the progression of human and experimental PH.

Methods and Results- Lungs, platelets, and quiescent cultured pulmonary-artery smooth-muscle cells (PA-SMCs) from patients with idiopathic PH (iPH) were characterized by marked elevations in RhoA and Rho kinase activity and by a strong increase in serotonin binding to RhoA, compared to controls. The 5-HTT inhibitor fluoxetine and the type 2 transglutaminase inhibitor monodansylcadaverin prevented 5-HT-induced RhoA serotonylation and RhoA/Rho kinase activation, as well as 5-HT-induced proliferation of PA-SMCs from iPH patients, that was also inhibited by the Rho kinase inhibitor fasudil. Increased Rho kinase activity, RhoA activation, and RhoA serotonylation were also observed in lungs from SM22–5-HTT+ mice, which overexpress 5-HTT in smooth muscle and spontaneously develop PH. Treatment of SM22–5-HTT+ mice with either fasudil or fluoxetine limited both PH progression and RhoA/Rho kinase activation.

Conclusions- RhoA and Rho kinase activities are increased in iPH, as a result of transglutaminase-mediated transamidation of RhoA by 5-HT internalized via 5-HTT. Direct involvement of this pathway in 5-HT-mediated PA-SMC proliferation and platelet activation during PH progression identify RhoA/Rho kinase signaling as a promising target for new treatments against PH.
Abstract 1637: Difference of Pulmonary Arterial Microvasculature in Patients with Pulmonary Hypertension Using Scanning Electron Microscope
Authors: Miura Aya , Nakamura Kazufumi , Kusano Kengo F , Matsubara Hiromi , Miura Daiji , Miura Ryuji , Ogawa Aiko , Ikeda Tetsuya , Fujio Hideki , Miyaji Katsumasa , Nishii Nobuhiro , Morita Hiroshi , Oka Takefumi , Ohtsuka Aiji , Date Hiroshi , Ohe Tohru
Published: July 2021

Background and Objectives: Pulmonary vascular diseases (pulmonary arterial hypertension: PAH, pulmonary veno-occlusive disease: PVOD and pulmonary capillary hemangiomatosis: PCH) are well-known diseases that cause pulmonary hypertension (PH). However, the differences of structural characteristics of pulmonary microvasculature in these diseases have not been elucidated enough. We examined the morphological differences among these diseases by methacrylate resin (Mercox) perfusion using scanning electron microscope.

Methods and Results: Ten lung samples were 8 patients with PH and 2 control subjects without PH. These were obtained at lung transplantations or autopsies. Microvascular corrosion replicas were made by Mercox perfusion from the pulmonary artery. Pathological diagnosis revealed 5 PAH, 2 PVOD and 1 PCH in patients with PH. In PAH samples, small arteries or arterioles showed twisted and tortuous courses, but such vessels were not observed in samples from control, PVOD and PCH. Binary branching was observed in control, but not in PVOD and PCH. In PVOD and PCH, several branching was commonly observed. Moreover, the loss of capillary network and the disruption of arterioles were more severe in samples from PVOD and PCH patients than in samples from PAH patients.

Conclusion: Pulmonary microvascular morphology is seriously different among the type of underlying diseases in PH patients.
Abstract 1684: Toll-like Receptor 4 Deficiency Attenuates Angiotensin II-induced Atherosclerosis and Abdominal Aortic Aneurysms via a MyD88-dependent Mechanism
Authors: Owens A. Phillip , Howatt Deborah A , Daugherty Alan
Published: July 2021

Objective:

We previously demonstrated that angiotensin II (AngII) infusion into myeloid differentiation factor 88 deficient mice (MyD88−/−) resulted in a profound reduction of atherosclerosis and abdominal aortic aneurysms (AAAs) in apoE−/− mice. Furthermore, AngII directly regulated toll-like receptor (TLR) mRNA in cell types associated with these diseases. The objective of this study was to determine the specific TLR responsible for the MyD88 mediated reduction in vascular pathology.

Methods and Results:

MyD88 mice were bred onto an LDLr−/− background. Deficiency in this hyperlipidemic strain caused similar decreases in AngII-induced atherosclerosis and aneurysm to those we previously noted in apoE−/− mice. Male TLR4+/+ (n = 14) or −/− (n = 19), on an LDLr−/− background, were fed a fat-enriched diet (21% milk fat, 0.15% cholesterol) and infused with AngII (1,000ng/kg/min) for 28 days. TLR4−/− mice had significantly attenuated systolic blood pressure from TLR4+/+ mice both prior to and during AngII infusion (P < .01). However, AngII did increase systolic blood pressure similarly in both groups (+/+: pre-infusion 142 ± 2, post-infusion 169 ± 3 mmHg; −/−: pre-infusion 130 ± 1, post-infusion 158 ± 3 mmHg; P < .001). Neither TLR4 genotype nor AngII infusions had significantly different effects on total plasma cholesterol concentrations or lipoprotein-cholesterol distributions. TLR4 deficiency dramatically decreased AngII-induced atherosclerotic lesion areas in both the aortic arch (50% decrease, P < .004), and thoracic aorta (66% decrease, P < .001). TLR4 deficiency decreased the diameter of the suprarenal abdominal aortic region from 2.31 ± 0.3 to 1.2 ± 0.06 mm (P < 0.001) and the incidence of AAAs from 93% to 26% (P < 0.001), versus control animals. Conversely, TLR2 deficiency reduced AngII-induced atherosclerosis in LDLr−/− mice, but had no significant effect on AAA formation.

Conclusion:

TLR4 deficiency attenuated both AngII-induced atherosclerosis and AAAs, in LDLr−/− mice, in a manner similar to the effects of MyD88 deficiency. TLR2 deficiency decreased AngII-induced atherosclerosis, but had no effect on AAAs. These data are consistent with TLR4 being the major receptor for MyD88-induced effects on AngII-induced AAAs.

This research has received full or partial funding support from the American Heart Association, AHA Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).
Abstract 1692: The Impact of Hypertriglyceridemia on Clinical Outcome of Patients Subjected to Percutaneous Coronary Intervention in the Presence of High and Low High-Density Lipoprotein Cholesterol
Authors: Torguson Rebecca , Kaneshige Kimberly , Xue Zhenyi , Okubagzi Petros , Pichard Augusto , Suddath William , Kent Kenneth , Satler Lowell , Waksman Ron , Brewer H. Bryan
Published: July 2021

Background:
High-density lipoprotein cholesterol (HDLc) is known to have protective cardiovascular effects. Elevated triglycerides (TRI) are often targeted with therapeutic agents. This study aimed to investigate the effects of TRI in the presence of high and low levels of HDLc on clinical outcomes of patients undergoing contemporary percutaneous coronary intervention (PCI).

Methods:
We analyzed a series of 4391 patients who presented for elective contemporary PCI since 2003 and have completed 1-year clinical follow up. The study cohort comprised of 4 subgroups: high TRI with high HDLc, high TRI with low HDLc, normal TRI with high HDLc, and normal TRI with low HDLc. High TRI was defined as ≥ 200 mg/dl, normal TRI as < 200 mg/dl, high HDLc was defined as ≥ 40mg/dl for males and ≥ 45mg/dl for females, and low HDLc as < 40mg/dl for males and < 45mg/dl for females. The assessed endpoints, death, Q-wave myocardial infarction (Q wave MI), target vessel revascularization (TVR), and a composite of major adverse cardiac events (MACE) were then compared between groups.

Results
Approximately 98.1% of the overall population is on statin therapy. As expected, patients with high HDLc have significantly better outcomes as compared to those with low HDLc and normal TRI; however, the high TRI low HDLc group demonstrated similar outcomes to patients with high HDLc and normal TRI. Additionally, among the group with high TRI, there were no significant differences in 1-year MACE outcomes between tertiles of non-HDL cholesterol. (Table

)

Conclusion:
In patients undergoing PCI with normal triglycerides low HDLc was associated with increased MACE and death at 1 year when compared to high HDLc patients. In contrast, a similar incidence of death and MACE was observed in hypertriglycer-idemic patients with low HDLc and high HDLc levels. Therapies targeted to elevate HDLc among patients with normal TRI may play a critical role in the prevention of recurrent cardiac events post PCI.

Table:

1 Year Outcomes
Abstract 1703: Involvement of Adam17 in Neointima Formation after Vascular Injury; Potential Role of Tyrosine Phosphorylation on its Activity
Authors: Hinoki Akinari , Higuchi Sadaharu , kimura Keita , Eguchi Kunie , Autieri Michael V , Sommerville Laura J , Frank Gerald D , Eguchi Satoru
Published: July 2021

Angiotensin II (AngII) and its G protein-coupled receptor, AngII type-1 receptor (AT
1
), play critical roles in mediating cardiovascular diseases such as hypertension and atherosclerosis. It is widely believed that AngII promotes these diseases by inducing vascular remodeling that involves hypertrophy and hyperplasia of vascular smooth muscle cells (VSMCs). We and others have shown that the EGF receptor transactivation pathway is the key signaling mechanism by which AngII induces VSMC growth. Recently, we have further reported that production of HB-EGF via a metalloprotease, ADAM17, leads to EGF receptor transactivation by AngII in vitro. Here, we hypothesised that tyrosine phosphorylation of ADAM17 is essential for ADAM17 activation by the AT
1
receptor in VSMCs, and that ADAM17 is a critical target of intervention for prevention of vascular remodeling in vivo. Our result revealed that not only a forced expression of dominant-negative (dn) ADAM17-E406A by adenovirus but also the tyrosine phosphorylation incapable ADAM17-Y702F mutant markedly inhibited AngII-induced EGF receptor transactivation compared to the stimulated control in vector-infected VSMCs. The neointimal cells appeared 14 days after balloon injury are strongly positive for ADAM17 immunostaining. Moreover, we observed marked inhibition of intimal hyperplasia in dnADAM17 adenovirus treated carotid artery after the balloon injury. We also confirmed marked expression of dnADAM17 in the artery after the virus infection. Taken together, these results suggest the involvement of ADAM17 in neointima formation. ADAM17 may be activated at the lesion by an upstream tyrosine kinase in response to AngII and other hormonal factors.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 1727: Apolipoprotein E (ApoE) Induces an Anti-inflammatory Phenotype in Macrophages
Authors: Baitsch Daniel , Telgmann Ralph , Varga Georg , Muller-Tidow Carsten , Bot Martine , Nofer Jerzy Roch
Published: July 2021

Apolipoprotein E (apoE) exerts anti-atherogenic effects by promoting cholesterol efflux and hepatic lipoprotein clearance. However, apoE retains protective effects even under experimental settings, in which its influence on plasma cholesterol is negligible suggesting that this lipoprotein inhibits atherosclerosis independently from cholesterol transport. To gain further insight into mechanisms underlying apoE-mediated atheroprotection, we investigated its effect on the functional phenotype of RAW 264.7 macrophages overexpressing either of two apoE receptors: ApoER2/LRP8 or VLDL-R. Incubation of ApoER2/LRP8- or VLDL-R-expressing macrophages with apoE downregulated markers of pro-inflammatory M1 functional phenotype (expression and activity of iNOS, production of IL-12), whereas markers of anti-inflammatory M2 phenotype (expression and activity of arginase-I, production of IL-1RA) were upregulated. In addition, macrophage responses typical for M1 phenotype (migration, generation of reactive oxygen species, antibody-dependent cell cytotoxicity) were suppressed in ApoER2/LRP8- or VLDL-R-expressing cells in the presence of apoE. Finally, apoE prevented LPS- and IFN-γ-induced activation of ApoER2/LRP8- or VLDL-R-expressing macrophages as documented by reduced production of IL-12, TNF-α and MCP-1, reduced expression and activity of iNOS and COX2, and reduced activation and/or phosphorylation of NF-κB, IκB and STAT1. The modulatory effects of apoE on macrophage phenotype were inhibited by SB220025, a p38MAP kinase inhibitor, and PP1A, a tyrosine kinase inhibitor. Accordingly, apoE induced tyrosine kinase-dependent activation of p38MAP kinase in ApoER2/LRP8- or VLDL-R-expressing macrophages. Under
in vivo
conditions, apoE
−/−
mice transplanted with apoE-producing wild-type bone marrow presented with increased plasma IL-1RA levels. In addition, peritoneal macrophages from transplanted animals demonstrated enhanced M2 phenotype (increased IL-1RA production and CD206 expression). We conclude that apoE signalling over ApoER2/LRP8 or VLDL-R promotes macrophage conversion from pro-inflammatory M1 to anti-inflammatory M2 phenotype. This effect may represent a novel anti-atherogenic activity of apoE.
Abstract 1737: FoxO4 is an Atheroprotective Factor
Authors: Zhang Qing-Jun , Zhou Wen , Liu Zhi-Ping
Published: July 2021

Atherosclerosis is a chronic inflammatory process of arterial wall as the result of interactions between genetic and environmental factors. The pathological change of atherosclerosis involves interactions of endothelial cells, smooth muscle cells and infiltrating immune cells including macrophages and T cells. We have found that one of the family members of FoxO transcription factor, FoxO4, plays important roles in the regulation of phenotype switch of smooth muscle cells, matrix remodeling, and immune cell recruitment. Based on these observations, we hypothesize that FoxO4 may regulate atherogenesis through one or more aforementioned processes. To test this hypothesis, we generated
ApoE/Foxo4
double knockout mice (DKO). The
ApoE/Foxo4
DKO mice and
ApoE
single knockout littermates (SKO) were fed with high fat diet for 8 weeks to induce atherosclerosis. At the end of the 8 weeks, the aortas and hearts were dissected out and plasmas were collected. The aortic plaque was stained with Oil Red O
en face
and the percentage of plaque area to aortic face area was determined. Results show that there was no difference of plasma cholesterol level between DKO mice and control group (2300 ± 414mg/L in
ApoE
group vs 2667 ± 344mg/L in
ApoE/Foxo4
DKO group, p = 0.28). No body weight difference was observed between these two groups of mice neither before nor after high fat diet.
ApoE/Foxo4
DKO mice developed significantly more atheroscle-rotic plaques than that of
ApoE
SKO littermate controls (6.28 ± 1.40 in
ApoE
SKO group vs 9.47 ± 2.59 in
ApoE/Foxo4
DKO group, p = 0.0016), suggesting that FoxO4 is an atheroprotective factor. We are currently investigating the molecular mechanism(s) by which FoxO4 regulates atherogenesis.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 1743: Stem/Progenitor Markers Sca-1 versus C-kit and CD31 Determine Angiogenic Potential of Mouse Bone Marrow-derived Endothelial Progenitor Cells
Authors: Tanaka Toshikazu D , Ii Masaaki , Sekiguchi Haruki , Jujo Kentaro , Misener Sol , Losordo Douglas W
Published: July 2021

Background:
Endothelial progenitor cells (EPCs) have been shown to have angiogenic potential contributing to neovascularization. However, the definition of EPC, including surface marker expression of EPCs promoting vasculo-/angiogenesis in ischemic tissue, remains uncertain. We hypothesized that stem/progenitor (c-kit vs. sca-1) and endothelial cell (EC) markers (CD31) may identify cells with enhanced EPC potential.

Methods and Results:
Mononuclear cells (MNCs) were isolated from mouse bones, and Lin+ cells were depleted by magnetic cell sorting. Lin- cells were further sorted with the following markers (% of total MNCs) by FACS: c-kit+ (1.87%), sca-1+ (0.6%), c-kit+ /CD31+ (1.1%) and sca-1+ /CD31+ (0.28%). Non-sorted MNCs were used as a control. To examine EC phenotype in culture, cells were labeled with DiI and co-cultured with mature ECs (human microvascular endothelial cells: HMVECs). The percent incorporation of DiI labeled cells into HMVEC tube structures 12 hours after co-culture and BS1-lectin positivity/acLDL uptake were: sca-1+ /CD31+ cells (87 ± 2%) > c-kit+ /CD31+ (79 ± 8%) > sca-1+ (62 ± 8%) > c-kit+ (59 ± 5%) > MNC (50 ± 3%
)
. Next, we examined homing capacity of these cells to ischemic myocardium using a mouse myocardial infarction (MI) model. DiI-labeled cells (5x10
4
, IV) were injected to splenectomized mice 3 days after MI, and the hearts were excised 24 hours after the cell injection for histological analysis. Interestingly, the number of recruited/retained DiI-labeled-cells in the MI hearts exactly replicated the findings of the in vitro tube formation assay (cells/HPF): sca-1+ /CD31+ (108 ± 26) > c-kit+ /CD31+ (77 ± 16) > sca-1+ (71 ± 14) > c-kit+ (67 ± 1) > MNCs (48 ±
6)
, suggesting that sca-1+ /CD31+ cells might have great functional activities as endothelial precursors.

Conclusions:
Both stem/progenitor marker Sca-1 and EC marker CD31 expressing EPCs exhibited high potential angiogenic capacity with EC phenotypic features compared with c-kit expressing cells. Our data suggest that Sca-1+ /CD31+ cells may represent EPC-rich cell population, and Sca-1/CD31 could be useful markers to enrich for cells with EPC potential. Ongoing studies will determine the in vivo characteristics of these cells for ischemic tissue repair.
Abstract 1766: Diet-Induced Obesity Causes Inflammation by Activating Toll-Like Receptor 4 Signaling and Downregulates AMPK in Heart
Authors: Ko Hwi Jin , Jung Dae Young , Ma Zhexi , Kim Jason K
Published: July 2021

Increasing evidence implicates the role of inflammation in diabetes and complications. Macrophages are shown to infiltrate adipose tissue in obesity, and inflammatory cytokines alter glucose metabolism in peripheral organs. Male C57BL/6 mice were fed high-fat diet (HFD; 55% fat by calories) or chow diet for 6 weeks, and heart samples were taken for analysis (n = 5~7). Chronic HFD increased whole body fat mass, measured by
1
H-MRS, by 3-fold, and elevated plasma IL-6 and TNF-α levels by 40%. Diet-induced obesity caused inflammation in heart and increased macrophage-specific CD68 levels by 5-fold (Fig. 1) (*
P
< 0.05 vs Chow). Diet-induced cardiac inflammation was associated with significant increases in toll-like receptor 4 (TLR4) and MyD88 levels in heart (Fig. 2). HFD also increased cardiomyocyte SOCS3 levels by more than 3-fold (Fig. 3). Myocardial glucose metabolism was measured using intravenous injection of 2-[
14
C]deoxyglucose in awake mice (n = 6). Chronic HFD reduced myocardial glucose uptake by 50%, and this was associated with significant reductions in total GLUT4 and GLUT1 protein levels. Further, Thr
172
phosphorylation of AMPK, a critical regulator of energy balance, was markedly reduced in heart following HFD (Fig. 4). These results demonstrate that diet-induced obesity causes macrophage infiltration and inflammation in heart by increasing TLR4 signaling in cardiomyocytes. Similar to the effects of inflammation on peripheral glucose metabolism, diet-induced cardiac inflammation reduced myocardial glucose metabolism by downregulating AMPK and GLUT protein levels. Thus, our findings underscore an important role of inflammation in diabetic heart.
Abstract 1817: Chronic Treatment with Resveratrol Restores Serca 2a Expression and Improves Cardiac Function of Diabetic Mice
Authors: Gupta Madhu , Sulaiman Mohamed , Periasamy Muthu , Griffin Andrew J , Gupta Mahesh P
Published: July 2021

Background:
Diabetic cardiomyopathy is characterized by impaired cardiac contractility. An important contributor to diastolic dysfunction in the diabetic heart is the reduced activity of sarcoplasmic reticulum calcium pump (SERCA2a). Over expression of SERCA2a alone in transgenic mice protected diabetic hearts from severe contractile dysfunction. Resveratrol (RSV) is a phytoalexin, present in dietary supplements and found naturally in red grape skin and red wine. It shows a multitude of cardio protective effects including anti-hypertrophic, anti-oxidant, anti-ischemic and antiarrythmic. Its role in diabetic cardiomyopathy has not been evaluated.

Objectives:
We tested the hypothesis that chronic treatment with RSV improves contractile function of the diabetic heart by enhancing SERCA2a expression involving transcriptional regulatory mechanisms.

Methods and Results:
Adult CD 1 mice were injected with streptozotocin (150 mg / kg i.p.). Mice with more than 400 mg /dL blood glucose levels were considered diabetic, vehicle injected animals served as controls. Half of the diabetic animals were fed RSV enriched diet (0.067%). After three months, by echocardiography diabetic animals showed increased LVESD (38%), increased LVEDD (22%), and reduced fractional shortening (46%). This was accompanied by reduced SERCA2a and SIRT1 protein expression as well as a 3-fold decline in SERCA2a mRNA as assessed by real-time PCR. RSV treatment improved LV function and SERCA2a mRNA to almost normal levels. In primary cultures of cardiomyocytes, transient transfection analysis of SERCA2a promoter / CAT reporter construct (consisting of 1.5 Kb SERCA2a promoter region) revealed a 4-fold decline in SERCA2a promoter activity when cells were grown in high glucose (33 mM) for 5 days as compared to cells grown in normal glucose (5.5 mM) for a comparable period of time. The decline in reporter expression was prevented by pretreatment with RSV ( 0.5 uM).

Conclusions and Implications:
Our results indicate that in diabetic hearts, resveratrol improves cardiac function by enhancing SERCA2a expression. This effect is mediated at the transcription level possibly involving SIRT1 activiity.
Abstract 1831: Phosphodiesterase-5 Inhibition Prevents Cardiac Hypertrophy, Independently of the Calcineurin Pathway
Authors: Hsu Steven , Nagayama Takahiro , Koitabashi Norimichi , Zhou Liye , Zhang Manling , Bedja Djahida , Gabrielson Kathleen , Kass David A , Takimoto Eiki
Published: July 2021

Cyclic GMP and its downstream kinase protein kinase G (PKG) negatively regulate cardiac hypertrophy. To date the only documented target of this cascade is the serine-threonine phosphatase calcineurin (Cn), whose activation is central to the development of pathologic cardiac hypertrophy. Recently, we reported that phosphodiesterase 5 (PDE5) inhibition (sildenafil, SIL) activates myocardial PKG and prevents pressure-overload induced hypertrophy by suppressing multiple cascades including Cn. To test the centrality of Cn signaling to the
in vivo
anti-hypertrophic effects of SIL, we subjected mice deficient in the Cn-A
β
subunit (CnA
β
−/−
) to severe trans-aortic constriction (TAC) with or without SIL (100mg/kg/day, p.o.) for 3-wks. TAC induced less hypertrophy that was more concentric in CnA
β
−/−
vs WT-controls (50% vs 100% increase in heart mass/tibia length, p<0.03). SIL completely blocked the hypertrophic response and fully normalized fetal gene re-expression (e.g ANP, BNP and
β
MHC) in CnA
β
−/−
TAC hearts, while it inhibited LVH by 60% and suppressed ANP and
β
MHC in WT-TAC hearts. SIL improved cardiac systolic and diastolic function (pressure-volume analysis) in CnA
β
−/−
TAC hearts much as in WT-TAC hearts. In CnA
β
−/−
TAC hearts, phosphorylated calcium calmodulin kinase II (CaMK II) increased 10-fold versus only a 2-fold rise in WT-TAC, whereas Akt and glycogen synthase kinase 3
β
(GSK3
β
) activation were comparable between groups. Extracellular response kinase (ERK) 1/2 was activated with TAC in WT hearts only. Importantly, SIL stimulated myocardial PKG and markedly inhibited the activation of CaMKII, Akt and GSK3
β
similarly in both groups exposed to TAC. Thus, Cn is not required for the anti-hypertrophic effects of SIL. Though TAC-induced hypertrophy is less in CnA
β
−/−
mice, SIL remains effective in suppressing the residual response by targeting alternative cascades such as CaMK II. These findings suggest that SIL acts either on multiple pathways concurrently, or at a node proximal to these pathways likely at or near the sarcolemmal membrane.
Abstract 1844: Temporal engraftment and functional outcome of Bone Marrow Derived Multipotent Progenitor cell transplantation (MPC) in a porcine model of postinfarction Left Ventricular (LV) Remodeling
Authors: Jameel Mohammad N , Wang Xiaohong , Li Qinglu , Mansoor Abdul , Xiong Qiang , Swingen Cory , Zhang Jianyi
Published: July 2021

This study compares the short term and long term functional outcomes of bone marrow derived multipotent progenitor cells (MPC) transplantation. In the short term study, the LAD was occluded distal to the 2
nd
diagonal branch with PTCA balloon dilatation for 60 minutes (IR, 1 month follow-up) and MPC were delivered through a transarterial catheter, while in the long term study, the coronary artery was occluded with ligation (OCCL, 4 months follow-up). In both IR and OCCL cases, intra-myocardial injection of 50 million LacZ labeled MPC (Cell) was performed in the periscar region with 5 equal injections immediately after myocardial infarction, while in control animals (CONT) saline was injected. The myocardial bioenergetics (P-31 MRS) and LV function (MRI) were followed-up monthly (Table

). MPC transplantation resulted in improvement of ejection fraction in the OCCL study, which persists for 4 months. In the IR study, although the EF was not significantly different between the two groups at 7days postinfarction, Cell induced a significant improvement of EF at week 4 (Table

). The myocardial phosphorylation potential that is reflected by the PCr/ATP ratio was significantly higher in CELL treated group of both studies, which persists during high cardiac workstates secondary to catecholamine stimulation. Although the engraftment was low (less than 0.35%), a small portion of the engrafted cells were found co-staining positive for troponin T and/or N-cadherin suggesting differentiation to myogenic cells. The infarct size was similar between the groups at 10 days. However, the infarct size was significantly reduced at 4 months in the Cell group that was not observed in the CONT. These data demonstrate that MPC transplantation associated improvement in ventricular function and myocardial energetics persists for 4 months follow-up. The beneficial effects are likely caused by cell transplantation induced mobilization and differentiation of endogenous cardiac progenitors.

This research has received full or partial funding support from the American Heart Association, AHA Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).

Improvement in Ejection Fraction after MPC transplantation
Abstract 1847: Modulation of Cardiac Remodeling with Acellular Matrix Emulsion is Associated with Myofibroblast Proliferation and Angiogenesis via Recruiting C-Kit Positive Cells after Myocardial Infarction
Authors: Zhao Zhi-Qing , Puskas John D , Xu Di , Wang Ning-Ping , Guyton Robert A , Vinten-Johansen Jakob , Matheny Robert
Published: July 2021

Degradation of native extracellular matrix (ECM) has been associated with maladaptive cardiac remodeling after infarction. This study tested the hypothesis that preservation of ECM by injecting a xenogegeic acellular matrix emulsion in infarct myocardium promotes myofibroblast proliferation and angiogenesis by recruiting host c-kit positive cells. Sixty-four rats were subjected to 45 minutes regional ischemia followed by 3, 7, 21 and 42 days of reperfusion. Histological examination was performed by immunohistological staining and cardiac function was analyzed using echocardiography. ECM emulsion (30–50
μ
l) was injected into the area at risk myocardium after reperfusion and localization of the emulsion was confirmed with Masson Trichome staining. At 7 days of reperfusion, the population of c-kit positive cells within the emulsion area increased significantly relative to the control (32±0.6* vs. 15±3/1000 nuclei), consistent with significantly enhanced expression of 31 kDa stem cell factor detected by Western blotting. Along with this change, myofibroblasts accumulated in the emulsion region to a significant extent compared to the control (59±8* vs. 30±3/HPF). Strong immunoreactivity of VEGF was observed in the emulsion area and angiogenesis was significantly enhanced relative to the control, evidenced by increased density of
α
-smooth muscle actin-positive vessels (70±10* vs. 20±4/HPF) and vWF-positive vessels (95±14* vs. 34±8/HPF), respectively. At 42 days of reperfusion, echocardiography showed improvements in end-systolic volume (0.3±0.1* vs. 0.6±0.3 ml)), fractional shortening (33±5* vs. 24±6%) and ejection fraction (67±6* vs. 53±10%) in the emulsion group. The wall thickness of the infarcted middle anterior septum in the emulsion group was also significantly greater than that in the Control (0.19±0.02* vs. 0.15±0.02cm). Intramyocardial injection of an acellular extracellular matrix emulsion into the ischemic/reperfused myocardium attenuates maladaptive cardiac remodeling and preserves cardiac function, potentially mediated by enhanced myofibroblast proliferation and angiogenesis via recruiting c-kit positive cells. * p<0.05 emulsion vs. control.
Abstract 1878: Beneficial Electrophysiological Effects of bi-Ventricular Pacing for Cardiac Resynchronization Therapy in a Canine Model of Dyssynchronous Heart Failure
Authors: Aiba Takeshi , Barth Andreas S , Liu Ting , Chakir Khalid , Hesketh Geoffrey G , Dimaano Veronica L , Abraham Theodore P , O’Rourke Brian , Akar Fadi G , Kass David A , Tomaselli Gordon F
Published: July 2021

Background

: Cardiac resynchronization therapy (CRT) using bi-ventricular (bi-V) pacing improves cardiac function and mortality in patients with heart failure and dyssynchronous contraction (DHF). However, the mechanisms by which bi-V pacing restores DHF-induced electrophysiological remodeling remain controversial. We test the hypothesis that bi-V pacing has enhanced beneficial effects on DHF beyond resynchronization of contraction.

Methods and Results

: Adult dogs underwent RV pacing (190–200 bpm) for 6 weeks (DHF: n = 6), or 3-week DHF followed by 3 weeks of resynchronization by either bi-V pacing (n = 6) or RA pacing (A-pace: n = 5) at the same pacing rate. Myocytes were isolated from LV anterior (ANT) and lateral (LTR) walls in non-failing (NF), DHF, bi-V and A-pace dogs. Whole cell patch clamp was performed to measure action potential (AP), transient outward (I
to
), inward rectifier (I
K1
) and delayed rectifier K
+
currents (I
K
). L-type Ca
2+
current (I
Ca,L
) and [Ca
2+
]
i
transients (CaT) were measured in the absence (baseline) and presence of isoproterenol (ISO) (35°C). The QRS duration was wider in bi-V than in A-pace, the other ECG and hemodynamic parameters were not statistically different between Bi-V and A-pace dogs. Bi-V abbreviated DHF-induced prolongation of APD in the LTR but not ANT cells and diminished the regional gradient of APD, whereas A-pace less abbreviated APD in the LTR cells and still remained the gradient. Bi-V partially restored the DHF-induced down regulation of I
K1
and I
K
but not I
to
, whereas A-pace restored only I
K1
but not I
K
and I
to
. Furthermore, bi-V fully restored the DHF-induced reduction of I
Ca,L
at baseline and its ISO response, whereas A-pace did not restore I
Ca,L
at baseline but restored its ISO responsiveness. The CaT amplitude at baseline and in ISO were partially restored by both bi-V and A-pace but the restoration was more complete with bi-V compared to A-pace.

Conclusion

: Although both bi-V and A-pace resynchronize the LV contraction, DHF-induced downregulation of K
+
currents, Ca
2+
current and handling were less completely restored by A-pace compared with bi-V pace, suggesting bi-V pacing not only resynchronizes LV contraction but improves regional heterogeneity of repolarization and Ca
2+
homeostasis in DHF.
Abstract 1891: Human Vascular Progenitor Cells Form Conductive Coronary Artery in Dogs with Critical Coronary Artery Stenosis
Authors: Monaco Francesco Lo , Ojaimi Caroline , Dora Massimo , Bearzi Claudia , Gatti Alessandro , Kajstura Jan , Leri Annarosa , Anversa Piero , Hintze Thomas H
Published: July 2021

To determine whether the human heart possesses a coronary vascular progenitor cell (VPC) that regulates the growth of endothelial cells (ECs), smooth muscle cells (SMCs), and vasculogenesis, we have identified vascular niches composed of c-kit-positive KDR-positive VPCs within the coronary circulation. VPCs are connected by functional gap junctions to ECs, SMCs and fibroblasts which operate as supporting cells. Isolated VPCs were self-renewing and clonogenic and differentiated into ECs and SMCs. Subsequently, we tested whether human VPCs possess the ability to create conductive coronary arteries in immunosuppressed dogs with critical coronary stenosis. The presence of a critical stenosis was documented by the absence of reactive hyperemia after the release of a 15 sec occlusion of the LAD. VPCs infected with a lentiviral vector carrying EGFP were then injected above, laterally and below the site of constriction. At 10 days, the lack of reactive hyperemia persisted while at 30 days there was a slow return of CBF possibly mediated by the formation of coronary vessels. Thus, myocardial perfusion was determined; gold-labeled microspheres were injected with the LAD open to establish baseline CBF while lutetium-labeled microspheres were administered after LAD occlusion. Multiple sections from the region proximal to the region distal to the occlusion were processed to evaluate CBF distribution. Every other section was collected for histology. CBF at baseline was similar in all sections but after LAD occlusion there was a doubling in CBF in the fully dilated coronary circulation distal to the stenotic vessel. Histologically, new coronary arteries, 0.8–1.5 mm in diameter, were detected in proximity of the stenotic vessel together with resistance arterioles and capillaries, pointing to vessel regeneration as the mechanism of enhanced CBF in the ischemic myocardium. ECs and SMCs within the vessel wall were all EGFP-positive; the human origin of these cells was confirmed by the detection of human DNA sequences with an
Alu
probe. In conclusion, the human heart possesses a coronary VPC that can be isolated and expanded in vitro for subsequent autologous transplantation in patients with coronary artery disease and the formation of a biological bypass.
Abstract 1904: Myocardial Postconditioning Against Ischemia and Reperfusion Injury by Near Infrared Electromagnetic Radiation
Authors: Pratt Phillip F , Bienengraeber Martin , Weihrauch Dorothee , Kersten Judy R , Warltier David C
Published: July 2021

Near-infrared electromagnetic radiation (NIR) has been reported to stimulate biochemical processes within cells and tissue, but whether NIR is capable of acutely protecting myocardium against ischemia+reperfusion injury
in vivo
is unknown. We tested the hypothesis that NIR (670 nm, 30 nm bandwith, 50 mW/cm
2
) exposure immediately before and during early reperfusion protects rabbit myocardium against infarction that is dependent upon reactive oxygen species (ROS), nitric oxide synthase (NOS), mitochondrial K
ATP
(mito K
ATP
) channels and inhibition of mitochondrial permeability transition pore (mPTP) opening. Rabbits subjected to a 30 min left anterior descending coronary artery occlusion and reperfusion received no irradiation (control) or continuous NIR (beginning 3 min before and ending 7 min after reperfusion) in the presence or absence of ROS scavengers [N-acetylcysteine (NAC; 150 mg/kg), or tempol (30 mg/kg)], nonselective NOS inhibitor L-nitro-arginine methyl ester (L-NAME; 10 mg/kg), selective mitoK
ATP
channel antagonist 5-hydroxydecanoate (5-HD; 10 mg/kg) or mPTP opener atractyloside (5 mg/kg). Dihydroethidium and diaminofluroscein immunofluorescence were used to detect ROS and NO production, respectively, in additional rabbits with or without NIR exposure. NIR reduced infarct size (24±4% of the area at risk; triphenyltetrazolium chloride staining) compared with control (46±3%). NAC, tempol, L-NAME, 5-HD, and atractyloside alone did not affect infarct size, but these drugs abolished the cardioprotective effect of NIR. NIR increased ROS production independent of ischemia and reperfusion, and this effect was blocked by NAC. NIR also enhanced NO generation during early reperfusion. In addition, NIR increased ATP synthesis in isolated rabbit mitochondria. Thus, NIR postconditions myocardium against infarction concomitant with ROS and NO production. MitoK
ATP
channels and mPTP mediate cardioprotection by NIR.
Abstract 1924: Carriers of Loss-of-Function Mutations in ABCA1 Display Pancreatic Beta Cell Dysfunction
Authors: Vergeer Menno , Brunham Liam R , Koetsveld Joris , Kruit Janine K , Verchere C B , Kastelein John J , Hayden Michael R , Stroes Erik S
Published: July 2021

Background
The ATP Binding Cassette transporter A1 (ABCA1) transports free cholesterol to nascent high-density lipoproteins (HDL) and maintains plasma HDL levels. In mice, ABCA1 is essential in regulating intracellular cholesterol homeostasis and insulin secretion in the β cell. The role of ABCA1 in human glucose metabolism is unclear.

Objective and methods
To assess the effects of ABCA1 dysfunction on glucose homeostasis in humans
, we matched heterozygous carriers of disruptive mutations in ABCA1 and non-carriers for age, gender and BMI and performed oral glucose tolerance tests (OGTT; 9 vs. 8 respectively) and hyperglycemic clamping experiments (6 vs. 6).

Results
Carriers had lower HDL-C levels than non-carriers (0.58 ± 0.3 vs. 1.46 ± 0.4 mmol/L, p=0.001) but LDL-C did not differ (3.4 ± 1.0 vs. 2.8 ± 0.8 mmol/L, p=0.21). Fasting plasma glucose was not different (5.2 ± 1.5 vs. 5.0 ± 0.4 mmol/L). Glucose curves after OGTT were significantly higher in carriers than in non-carriers (genotype * time interaction, p=0.005; plasma glucose at t=60 min 9.0 ± 3.0 mmol/L vs. 6.0 ± 1.4 mmol/L respectively, p=0.02). During hyperglycemic clamps, carriers showed a lower first phase insulin and C-peptide response than non-carriers (genotype * time interaction, p<0.05 and p<0.01 respectively; insulin at t=5 min 164±118 vs. 352 ±141 pmol/L, p<0.05; C-peptide at t=5 min 1033 ± 628 vs. 1942 ± 723 pmol/L, p<0.05) but no difference in insulin sensitivity index (0.0216 ± 0.012 mg kg
−1
. min
−1
. pM
−1
for carriers and 0.0197 ± 0.005 mg kg
−1
. min
−1
. pM
−1
for non-carriers; p=0.73). Disposition index - a measure of β cell function, adjusted for insulin sensitivity - was lower in carriers than in non-carriers (1037 ± 610 vs. 2718 ± 1524; p<0.05). Non-carriers responded to an arginine stimulus with an increase in C-peptide levels (from 3558 ± 1240 pM to 6817 ± 1665 pM; p<0.005), whereas in carriers this increase did not reach statistical significance (from 3727 ± 1843 pM to 5480 ± 1757 pM; p=0.12).

Conclusion
Carriers of loss-of-function mutations in ABCA1 show impaired insulin secretion without insulin resistance, resulting in glucose intolerance. Our data confirm previous studies in mice and provide evidence for a role of ABCA1 in β cell dysfunction and the pathophysiology of diabetes mellitus in man.