bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1930: 3D Volumetric Carotid Plaque Imaging by Cardiovascular MRI Solves the Mystery of the ‘Percent Stenosis Paradox’
Authors: Biederman Robert W , Williams Ronald B , Grant Saundra B , Rayarao Geetha , Yamrozik June A , Rathi Vikas K , Vido Diane A , Neff David R , Doyle Mark
Published: July 2021

Morphologic characteristics of plaque composition have been proposed to explain the apparent paradox of improved clinical events despite no or minimal reduction in % stenosis with statins. CMR can distinguish underlying features that determine plaque ‘vulnerability’. We hypothesize that in statin-naive pts with high-grade carotid artery stenosis, there will be a high degree of correlation in the relationship between the ‘unstable’ lipid pool and ‘stable’ fibrous plaque by 3D CMR, yet may be independent of 2D %stenosis. Representing 530-two mm contiguous CMR (1.5T GE)
in vivo
slices of advanced (mean 61±24% stenosis) carotid disease, 26 complete bilateral human (age: 66±14yrs) plaques were analyzed for 3D volumetric extent of vascular wall: lipid pool, fibrous cap, matrix and minima/maxima of each. All were related to fasting lipid levels relative to %stenosis via QPlaque (Medis, The Netherlands). Plaque morphology determined by T1, T2/PD CMR. In all, 25/26
in vivo
plaques were imaged. Mean resolution: 1x1x2mm. The mg/dL range of LDL-C was 63–186, HDL-C: 28–59 and TG: 81–213. Lipid pool represented 15±4% while fibrous plaque represented 5±15% of total vessel wall. Total Cholesterol (Chol
T
) and LDL-C were inversely related to minimum vessel wall thickness (r=−0.5 and −0.6, respectively, p<0.05, for both) while only Chol
T
was related to fibrous cap (r=0.6, p<0.01). The Chol
T
/LDL-C ratio was highly related to minimum fibrous plaque thickness (r=0.8, p<0.001). The 3D lipid pool was the only fraction highly correlated (>0.6) with triglycerides (r=0.6, p<0.01). A linear regression relating fibrous cap: vessel wall ratio to non-HDL cholesterol and Chol
T
was highly correlated (r=0.6, 0.7, respectively, p<0.01 for both) but was independent of
in vivo %
stenosis (r=0.1). Relating %stenosis to
any
lipid fraction or ratio showed no relationship. Percent stenosis provides relatively little information about vulnerability of
de novo
, statin-naive carotid plaques. As most current imaging studies concentrate on plaque stenosis, a more appropriate focus on plaque composition provides a more robust quantifiable volumetric metric and may be more indicative of the underlying pathology by high-resolution 3D CMR.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 1952: ABCA1 Deficency Diminishes Dysfunctional HDL And Inhibits Early Atherosclerotic Lesion Development In SR-BI Knockout Mice
Authors: Zhao Ying , Out Ruud , Ye Dan , Van Berkel Theo J , Van Eck Miranda
Published: July 2021

Objective:
ABCA1 and SR-BI play an essential role in reverse cholesterol transport (RCT) from macrophages in the arterial wall to the liver. The aim of this study was to investigate the effect of combined total-body ABCA1 and SR-BI deficiency on lipoprotein metabolism and atherosclerosis.

Methods & Results:
ABCA1/SR-BI double knockout (dKO), ABCA1 KO (AKO), SR-BI KO (SKO), and wildtype (WT) littermates were fed an atherogenic diet for 10 weeks to induce atherosclerosis. Under these conditions, SR-BI deficiency leads to largely increased serum cholesterol (C) levels (SKO: 470±68 mg/dL
vs
WT: 181±9 mg/dL, p<0.001), due to an impaired removal of HDL-C and VLDL-C, while hampered HDL production in AKO mice resulted in cholesterol levels of only 23±4 mg/dL (p<0.0001). In dKO mice, HDL-C was similarly low as in single AKO mice. Total serum cholesterol levels, however, were 3.8-fold (p<0.001) higher, due to increased VLDL-C levels. Interestingly, more extreme foam cell accumulation was observed in the peritoneal cavity of dKO mice ((30±6%, p<0.01), as compared to WT (1.7±0.2%), SKO (2.5±0.3%), and AKO (5±2%) mice. Combined deletion of ABCA1 and SR-BI thus induces macrophage foam cell formation
in vivo
. The accumulation of HDL-C and VLDL-C in SKO mice dramatically induced atherosclerotic lesion formation (206±16x10
3
μm
2
vs
12±2x10
3
μm
2
for WT mice, p<0.001)), while lesions were only 33±12x10
3
μm
2
(p<0.05) in AKO and 51±19x10
3
μm
2
(p<0.01) in dKO mice with low HDL-C levels. Thus, despite enhanced foam cell formation in dKO mice, ABCA1 deletion reduces the atherosclerosis susceptibility of SKO mice. Next, the serum atherogenicity was tested by incubation of RAM cells with 3% serum of different types of mice. Serum from SKO mice induced more extreme foam cell formation (89±2%, p<0.001), compared to serum from WT (31±3%), AKO (2.3±0.1%), and dKO (3.1±0.5%) mice. Interestingly, this effect highly correlated with both HDL-C (r=0.98, p<0.0001) and VLDL/LDL-C levels (r=0.96, p<0.0001).

Conclusion:
Total-body ABCA1 deficiency inhibits early atherosclerotic lesion development in SR-BI KO mice, which might be attributable to reduced circulating levels of pro-atherogenic HDL. Furthermore, both SR-BI and ABCA1 are essential for maintaining HDL-mediated RCT.
Abstract 3377: Modulation of Vessel Stability by the ELR-negative Chemokine IP-10
Authors: Bodnar Richard J , Yates Cecelia C , Du Xiaoping , Wells Alan
Published: July 2021

Angiogenesis plays a critical role in tissue development and wound repair. Regulation of the vascular tree is critical to organogenesis and neo-organogenesis. The initial vigorous vessel proliferation and arborization that occurs during tissue development and wound repair must be pruned to form mature tissue. The signals that trigger this vascular regression are still ill-defined. Mitotic endothelial cells express the CXC receptor 3 (CXCR3), which binds the ELR-negative chemokines IP-10 (CXCL10), platelet factor 4 (PF4, CXCL4), IP-9/I-TAC (CXCL11), and MIG (CXCL9), with IP-9 and IP-10 being expressed during the later remodeling phases of wound repair when vascular involution occurs. We hypothesized that not only does this signaling system limit angiogenesis, but that CXCR3 signaling also compromises endothelial cell tube integrity contributing to regression. Treatment of newly formed tubes/cords on Matrigel (in vitro) or vessels in a subcutaneous Matrigel plug (in vivo) with IP-10 in either presence or absence of VEGF caused a dissociation of the tubes and subsequent vascular involution. This followed CXCR3 induced cleavage of β3 integrin in endothelial cells. CXCR3 triggering of mu-calpain activity caused cleavage of the cytoplasmic tail of human β3 integrins at the calpain cleavage sites NPLY747 and TSTF754. IP-10 stimulation was also found to activate caspase 3, blockage of which prevented cell death but not tube dissociation. Thus, CXCR3 activation in newly formed vessels results in endothelial dissociation from matrix, followed by anoikis. This is the first direct evidence for an extracellular signaling mechanism through CXCR3 causing the dissociation of newly formed blood vessels.

This study was supported by funds from VA Medical Research Program and National Institute of General Medical Sciences (NIH, USA).
Abstract 3385: Cardiac Stem Cell Survival During DNA Damage Is Regulated By Modulation Of Histone Acetyl Transferase p300
Authors: Jain Sumit , Rodrigues Claudia , Yuan Huijun , Liu Jing , Webster Keith A , Bishopric Nanette H
Published: July 2021

Background: Many sources of oxidative stress, including ischemia/reperfusion and chemotherapy, can induce DNA damage in the heart, leading to loss of cardiomyocytes and development of heart failure. Cardiac stem cells (CSCs) may serve to regenerate injured myocardium. However, ischemic injury has been associated with senescence and apoptosis in CSCs, potentially limiting their contribution to tissue repair. The histone acetyltransferase p300 has been shown to be an important regulator of genome stability and is involved in cell survival in response to DNA damage. We hypothesized that p300 could play a role in CSC response to oxidant stress.

Methods: c-kit+, sca-1+ CSCs were isolated and cloned by serial dilution from adult mouse hearts. CSC clones were stably transfected with lentivirus vectors carrying shRNAmir against p300 or a non-silencing RNA (NS) and maintained under puromycin selection prior to treatment. CSCs were exposed to doxorubicin (DOX), a DNA-damaging agent, for defined intervals. Levels of p300 were monitored by Western analysis and expression of DNA damage-regulated genes was assessed using multiplex realtime QPCR. CSCs were treated with 1 uM DOX and survival was assessed over a period of 72h.

Results: Levels of p300 increased rapidly in CSCs within 4h after exposure to doxorubicin and were sustained for at least 24h. CSCs expressing p300 shRNAmir (p300 KD) contained ~20% of the amount of p300 in CSCs transfected with the NS sequence (NS CSCs). QPCR analysis of DNA repair and cell cycle control genes indicated that 44 were significantly expressed in CSCs; 13 of these were up-regulated by p300 loss, and 5 were down-regulated, indicative of a DNA damage response to p300 loss.

Both cell proliferation rates and progression through the cell cycle were slower in p300 KD vs. NS CSCs. Surprisingly, following 48h treatment with DOX, p300 KD CSCs had 32% improved survival vs. NS CSCs.

Conclusions: Under acute genotoxic stress, p300 accumulates in CSCs. Reducing p300 levels induces the expression of genes involved in the DNA damage response, probably as a compensatory response, and slows CSC proliferation. Loss or inhibition of p300 is likely to cause multifactorial growth arrest in CSCs and thereby reduce their vulnerability to genotoxic agents.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 3426: Myostatin Deletion Preserves Cardiac Function in Senescent Mice
Authors: Morissette Michael R , Stricker Janelle C , Rosenzweig Anthony
Published: July 2021

Myostatin (MSTN) is a well-known negative regulator of skeletal muscle mass, and MSTN inhibition is being considered as therapy for multiple conditions associated with muscle wasting, including sarcopenia of aging. We have previously shown that MSTN inhibits phenylephrine-induced cardiomyocyte hypertrophy, however whether MSTN has a physiological role in regulating cardiac hypertrophy or function at baseline or with aging remains unclear. To determine if MSTN is dynamically regulated with aging, we performed QRT-PCR on hearts from male wild-type (WT) senescent mice (24 months old (mos)) and rats (32 mos). MSTN mRNA levels were increased in old versus young (4 mos) hearts (2.5- and 4-fold respectively, p<0.05). To study the functional significance of MSTN in aging, we maintained germline MSTN-knockout mice (MSTN
−/−
) and their WT littermates for 24 –27 months. We found no difference in heart weight of aged male MSTN
−/−
compared to WT mice (162.5±17.0 (n=4) vs 153.2±4.2 (n=4) mg, p=0.51), which would argue against an inhibitory role for MSTN in age-related increases in cardiac mass. We also performed echocardiography on unanesthetized senescent MSTN
−/−
and WT mice. MSTN
−/−
mice had better fractional shortening (58.1±2.0 (n=7) vs 49.4±1.2 (n=8) %, p=0.002) and smaller LV end-diastolic diameter (3.41±0.19 vs 2.71±0.14 mm, p=0.012) compared to WT. The decreased cardiac function seen in aged WT mice was associated with increased cardiac fibrosis on Masson-Trichrome stained sections. Western blot analysis also demonstrated a 3.3-fold increase in phospholamban phosphorylation in MSTN
−/−
hearts (p<0.05), compared to WT, while no differences in SERCA2a or calsequestrin protein levels were seen. We conclude that MSTN increases in the heart with aging, and that genetic deletion of MSTN results in improved cardiac function without a difference in heart mass in senescent mice. Decreased cardiac fibrosis and increased inhibition (phosphorylation) of phospholamban likely contribute to the better cardiac function seen in senescent MSTN
−/−
mice. These results suggest that inhibiting MSTN for sarcopenia in the elderly may also benefit cardiac function and could represent a novel therapeutic approach for ameliorating cardiac dysfunction and/or fibrosis.

This research has received full or partial funding support from the American Heart Association, AHA Founders Affiliate (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont).
Abstract 3460: Molecular Characterization, Safety and Feasibility of Induced Pluripotent Stem (iPS) Cell Derived Cardiomyocytes for Heart Regenerative Therapy
Authors: Ohno Yohei , Yuasa Shinsuke , Onizuka Takeshi , Egashira Toru , Shimoji Kenichiro , Yoon Sung Han , Arai Takahide , Endo Jin , Kageyama Toshimi , Chen Hao , Tanaka Tomofumi , Hattori Fumiyuki , Ogawa Satoshi , Yamanaka Shinya , Fukuda Keiichi
Published: July 2021

[Background]
We recently reported that mouse and human iPS cells could be generated from somatic cells by gene transfer of Oct3/4, Sox2, c-Myc and Klf4. Although the morphology, growth characteristics and pluripotency of iPS cells are believed to be similar to those of ES cells, there are several versions such as Fbx-iPS, Nanog-iPS cells, and it remains unknown whether what type of iPS cell could be appropriate for the cardiovascular regeneration therapy. This study investigated the cardiomyocyte differentiation potential of mouse iPS cells, molecular characterization of iPS-induced cardiomyocytes, and the safety and feasibility of their transplantation into the heart.

[Methods and Results]

We used several lines of Fbx-iPS and Nanog-iPS cells and induced cardiomyocyte differentiation using the hanging drop culture method. Mouse ES cells were used as control. Both Fbx-iPS and Nanog-iPS cells could differentiate into cardiomyocytes, and the incidence of beating embryoid bodies was similar to that of ES cells.

RT-PCR analyses and immunohistochemistry revealed that both iPS cells expressed all the cardiomyocyte marker genes including Nkx2.5, GATA4, MEF2C, myosin light chain-2v, α-myosin heavy chain, atrial natriuretic peptide, and α-sarcomeric actinin, and they showed normal structures.

Electron microscopic study revealed that iPS cell derived cardiomyocytes had the typical morphological cardiomyocyte structure, which is difficult to distinguish from that of ES cell derived cells.

Electrical physiological study showed that iPS cell derived cardiomyocytes had sinus node-like or fetal ventricular type action potentials.

Purified iPS cell derived cardiomyocytes could be successfully transplanted into murine hearts and the engrafted cells survived and were integrated into host myocardium.

Nanog-iPS cell derived cells showed no severe complication after transplantation, however Fbx-iPS cell derived cells showed teratoma in the host tissue. Fbx-iPS cells expressed extrinsic c-myc after differentiation.

[Conclusions]
The iPS cells proved to be an attractive cell source for the regeneration of cardiomyocytes, however safety and careful characterization is necessary before clinical application.
Abstract 3491: Endo-Epicardial Dissociation of Electrical Activity Increases During the Development of the Substrate for Atrial Fibrillation in the Goat
Authors: Eckstein Jens , Maesen Bart , Verheule Sander , Allessie Maurits , Schotten Ulrich
Published: July 2021

Background: The high incidence of transmural conduction of fibrillation waves (breakthroughs) in a complex substrate for atrial fibrillation (AF) implies the presence of electrical dissociation between the subepicardial layer (Epi) and the endocardial bundle network (Endo). The presence of this Endo/Epi dissociation (EED) in remodeled atria and its role in the progressive stabilization of AF over time has not been studied yet.

Methods: We developed a mapping tool for synchronous Endo/Epi mapping (spatial resolution 1.6mm) with 90 exactly opposing electrode pairs (open chest experiment). We included 3 groups of goats: C = Control (acute AF induced by 50Hz burst pacing, n=7), 3wk = 3weeks AF (n=7) and 6mo = 6months AF (n=7). Dissociated activity was postulated when either activation times differed by more than 12ms vertically or 8ms horizontally (indicating a local conduction velocity < 20cm/s) or local direction of propagation between Endo and Epi differed by more than 90 degrees. To monitor AF stability, repetitive in-vivo cardioversion experiments with class 1C drugs were performed in 6 of the 6mo goats at 2,6,10 and 14wk AF.

Results: Applying the time criterion, EED increased from 15±4% (C) to 22±11% (3wk) and 35±13% (6mo, p=0.002 vs. C). Also the differences in the direction of propagation significantly contributed to EED. Using the combined criterion, EED increased from 38±5% (C) to 46±10% (3wk) and 53±11% (6mo, p=0.007 vs. C). Dissociation within the epicardial and the endocardial layer (time criterion) increased to a comparable extent (19±8% vs. 27±14% vs. 37± 7%, p<0.001 C vs. 6mo). Mean Endo/Epi activation time differences were close to 0ms in all three groups (−1.0±15ms vs. −0.8±16ms vs. −0.3±20ms), ruling out preferential conduction from Endo to Epi or vice versa. Success rate of cardioversion experiments decreased from 83% (2w) to 33% (6wk) to 16% (10wk) to 0% (14wk) indicating increasing stability of AF over time.

Conclusion: During AF, pronounced EED occurs. EED (like dissociation within Endo and Epi) increases over time, contributing to the progressive stabilization of AF. Enhanced EED might explain the high incidence of transmural conduction (breakthroughs) in a complex substrate for AF.
Abstract 3501: Calmodulin Kinase II Activation of Mutant Ryanodine Channels Promotes Ectopic Activity and Atrial Fibrillation
Authors: Chelu Mihail G , Sood Subeena , Wang Sufen , Sarma Satyam , van Oort Ralph J , Skapura Darlene G , Anderson Mark E , Dobrev Dobromir , Valderrabano Miguel , Wehrens Xander H
Published: July 2021

Some individuals with mutations in the cardiac ryanodine receptor gene (RyR2) develop stress-induced atrial fibrillation (AF), but the underlying mechanisms are unknown. This study explored the molecular basis of AF susceptibility in mice heterozygous for the human arrhythmia-linked RyR2 mutation R176Q (RQ mice). AF inducibility was assessed with atrial burst pacing in WT (n=34), RQ mice (n=43) and RQ mice crossed with transgenic mice expressing the CaMKII-inhibitory peptide AC3I (RQxAC3I, n=6) or the inactive analog AC3C (RQxAC3C, n=9). Dual voltage (RH237) and calcium (Rhod-2 AM) optical mapping was applied to monitor simultaneous Ca2+ and membrane-voltage changes. Ca2+ fluorescence (Fluo-4 AM) was used to study SR Ca2+ release. Expression/phosphorylation of CaMKII and RyR2 were determined by immunostaining in mice and human atrial biopsies from sinus rhythm (SR) (n=7) and chronic AF (n=7) patients. Atrial burst pacing-induced AF was more common in RQ (55%) than in WT mice (11%*; *P<0.05) and was associated with ~50% higher CaMKII-dependent RyR2 phopshorylation (Ser2814 site). Expression and phosphorylation (Thr287) of CaMKIId were increased in AF patients by 63%* and 40%*, respectively, and CaMKII-phopshorylation of RyR2 at Ser2814 was 80%* higher in AF than in SR patients. Genetic inhibition of CaMKII reduced AF incidence in RQxAC3I (16%*), but not in RQxAC3C mice (62%). Optical mapping revealed more atrial burst pacing-induced ectopic beats (and re-entry) in RQ than WT atria (296 vs.19* ectopic beats, 120 ms cycle length) that were depressed by CaMKII inhibiton with KN-93 (1 μM). [Ca2+]i release by 10 mM local caffeine puffs (an index of SR Ca2+ content) were similar in RQ and WT mice (4.59±0.42 vs 4.04±0.41 [a.u.], respectively), whereas SR Ca2+ leak defined as tetracaine (5 μM)-sensitive decrease in diastolic [Ca2+]i was higher in RQ than in WT mice (% of SR Ca2+ content: 10.2±0.9 vs 7.7±0.8*). CaMKII inhibition with KN-93 suppressed SR Ca2+ leak. High rate-associated CaMKII activation promotes AF inducibility in mice with a stress-induced arrhythmia-linked R176Q mutation in RyR2. Inhibition of CaMKII may be a viable drug target for effective AF therapy.

This research has received full or partial funding support from the American Heart Association, AHA South Central Affiliate (Arkansas, New Mexico, Oklahoma & Texas).
Abstract 3559: Insulin Receptor Substrates (IRS) Signaling are Essential Regulators of Mitochondrial Function and Cardiomyocyte Survival
Authors: Riehle Christian , Wayment Benjamin , Pires Karla M , Moreira Annie B , Theobald Heather , Dong Xiaocheng , White Morris F , Litwin Sheldon E , Abel E. Dale
Published: July 2021

It has recently been reported that impaired PI3K signaling leads to cardiac mitochondrial dysfunction and that cardiac insulin receptor deficiency impairs cardiac growth. Signaling from insulin and insulin-like growth factor 1 receptors to PI3K is mediated by insulin receptor substrates (IRS) 1 and 2. Our objective was to determine the contribution of IRS isoforms on cardiac structure, mitochondrial and contractile function. For this purpose we generated mice with cardiomyocyte-specific deletion of IRS1 (CIRS1KO) or IRS2 (CIRS2KO) or IRS1 + IRS2 (CIRS12KO). While the single KOs had normal survival, CIRS12KO mice died from heart failure within the first eleven weeks of life. At the age of 8 weeks, heart weight-to-tibia length ratios, HW/TL were reduced by 11% (p<0.05) in CIRS1KO versus WT. ADP-stimulated mitochondrial oxygen consumption (V-ADP) was decreased by 22% (p<0.05) and ATP synthesis by 16% (p<0.05) in saponin-permeabilized cardiac fibers using pyruvate as substrate but not with palmitoyl carnitine (PC) or glutamate. In contrast, HW/TL was unchanged in CIRS2KO at the age of 8 weeks and was increased at the age of 14 weeks (+7%, p<0.05). Similarly, ADP-stimulated respirations were reduced in CIRS2KO at both ages by −21% and −16% respectively, (p < 0.05) only with pyruvate as substrate. In contrast, CIRS12KO developed dilated cardiomyopathy at the age of 4 weeks (LVDs + 18.9%, LVPWd −12.7%, LVPWs −21.4%, FS −36.7%, EF −26.3%, p<0.05). Histology revealed myofibrillar loss and disarray and increased fibrosis. Using stereology, cardiomyocyte nuclei per area was reduced by 27% and cardiomyocyte cross-sectional area was increased by 33% (both, p<0.05). Using pyruvate, PC or glutamate as substrates, V-ADP and ATP-synthesis rates were reduced by 33– 41%. Transcriptional analysis revealed a coordinate downregulation (by 40 – 60%) of PDK4 and PDH subunits, FAO and OXPHOS genes that were associated with a 35% reduction in expression of PGC-1β. At the protein level, expression of the complex II subunit, succinate-ubiquinol oxidoreductase was reduced by −51% (p<0.01). These data identify a critical role for IRS-mediated signaling in the regulation mitochondrial gene and protein expression, mitochondrial function and cardiomyocyte survival.
Abstract 3563: Postconditioning Inhibits Mitochondrial Permeability Transition Pore Opening Independently of any Modification of the Oxidative Phosphorylation and of the Inner Membrane Potential at Reperfusion
Authors: Paillard Mélanie , Gomez Ludovic , Augeul Lionel , Loufouat Joseph , Ovize Michel
Published: July 2021

Mitochondrial permeability transition pore (mPTP) inhibition plays a crucial role in postconditioning (PostC). We sought to determine whether oxidative phosphorylation and mitochondrial membrane potential (ΔΨ
m
), which both modulate mPTP opening, are involved in the inhibition of mPTP opening in the postconditioned heart. Anesthetized rabbits underwent 30 minutes of ischemia followed by 10 minutes of reperfusion. At the onset of reperfusion, they received either no intervention (Control, C), 4 cycles of 1 min ischemia followed by 1 min reperfusion (PostC), or an IV injection of 5mg/kg of the powerful inhibitor of mPTP opening, i.e. cyclosporine A (CsA). Sham rabbits underwent no ischemic insult throughout the 40 minute experiment. At the end of the 10 minute reperfusion period, the myocardial area at risk was excised, and mitochondria were isolated by differential centrifugations. Calcium retention capacity, an index of mPTP inhibition (CRC: nmol Ca
2+
/mg prot) and ΔΨ
m
(at state 4: % of FCCP-evoked maximum) were assessed by spectrofluorimetry in isolated subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria. Oxidative phosphorylation (at states 3 and 4: nmol O
2
/min/mg) was assessed using a Clark-type electrode (RCR: state 3 / state 4). As expected, PostC and CsA treatments improved CRC when compared to the C group. Control, PostC and CsA mitochondria exhibited a comparable significant dissipation of ΔΨ
m
, together with a comparable significant decrease of RCR in both SSM and IFM. In all three groups, this latter effect was related to a concomitant significant decrease in state 3 and to an increase in state 4 respiration. These data suggest that during the early minutes of reperfusion, postconditioning inhibits mPTP opening, independent of any specific modification of the oxidative phosphorylation or of ΔΨ
m
.

Summarized data:
Abstract 3593: Wild Type Human Tissue Kallikrein, but Not its Common Polymorphic Variant R53H, Promotes Arteriogenesis in Normoperfused and Ischemic Rodent Models.
Authors: Stone Oliver A , Emanueli Costanza , Harper Steven J , Giudicelli Christine , Alhenc-Gelas Francois , Sala-Newby Graciella , Bates David O , Madeddu Paolo R
Published: July 2021

Therapeutic angiogenesis via administration of recombinant growth factors or vector-mediated overexpression has been largely unsuccessful in part due to the inadequacy of a single growth factor to induce all aspects of vascular growth. We have previously shown that human tissue kallikrein (hK1) promotes angiogenesis by kinin-mediated activation of the Akt-eNOS pathway, independently of VEGF. To further characterise the cellular mechanisms of hK1 induced neovascularisation, we delivered adenovirus encoding the wild type kallikrein gene (
Ad.KLK1
, 5x10
6
or 5x10
5
pfu) or a polymorphic variant (
Ad.R53H-KLK1
), which is 50% less potent as a kinin generating enzyme, or control virus (
Ad.eGFP
) in the rat mesenteric assay, a non-ischemic model of angiogenesis. In separate experiments,
Ad.KLK1
was injected into the mesentery of rats given the bradykinin-2-receptor (B2R) antagonist, Icatibant (Subc 300nmol/kg/day). Intravital and confocal imaging was used to examine vessel morphology and histology.
Ad.KLK1
increased the functional vessel area (445±76 vs 32±4% in
Ad.eGFP,
p<0.01), conduit vessel density (193±12 vs 63±7mm
−2
, p<0.001) and average vessel diameter (15±1 vs 12.6±1μm, p<0.001).
Ad.KLK1
-induced neovasculature showed an increased pericyte (58±5% vs 31±5, p<0.05) and smooth muscle cell coverage (4±1 vs 0%, p<0.001). In addition, we found that B2R co-localises with pericytes in the mesentery. Following Icatibant,
Ad.KLK1
stimulated the formation of a network of small-size vessels, without pericyte or smooth muscle cell coverage and surrounded by spots of micro-hemorrhaging.
Ad.R53H-KLK1
was less efficient than
Ad.KLK1
in promoting angiogenesis and recruiting vascular smooth muscle cells. In a model of limb ischemia, disruption of the kallikrein gene resulted in dysfunctional reparative arteriogenesis and delayed hemodynamic recovery, which were both corrected by local
Ad.KLK1
but not by
Ad.R53H-KLK1.
These data provide a mechanistic explanation for the robust arteriogenic effect of
Ad.KLK1
, indicating a key role for the kinin B2R. We also demonstrate for the first time that a single nucleotide polymorphism in the
KLK1
gene, present in 5–7% of the Caucasian population, leads to a reduced neovascularisation
in-vivo
. All data mean±SEM, n=6
Abstract 3608: High Mobility Group A1 Protein - A New Regulator for Peroxisome Proliferator-Activated Receptor Gamma Transcriptional Repression in Human Aortic Smooth Muscle Cells
Authors: Bloch Mandy , Foryst-Ludwig Anna , Unger Thomas , Kintscher Ulrich
Published: July 2021

The study aimed to identify new nuclear cofactors for PPARgamma (peroxisome proliferator-activated receptor gamma)-dependent gene transcription in human aortic smooth muscle cells (HASMC) in order to develop new PPARgamma-ligands with improved clinical safety in the absence of deleterious cardiovascular side effects. Using an Oligo GEArray® Human Nuclear Receptors and Coregulators Microarray for gene expression profiling, we identified the transcriptional regulator and chromatin modifying High Mobility Group (HMG) A1 protein highly expressed in unstimulated HASMC. PPARgamma-dependent gene regulation was studied by analysis of PMA-induced MMP-9 (matrix metalloproteinase 9) expression ± pioglitazone (pio 10μM). PMA (50ng/ml) stimulated MMP-9 mRNA expression by 46.3±22.3-fold (p<0.05 vs. vehicle) which was markedly blocked by pio (10μM: 17.4±4.8-fold vs. PMA alone p<0.05). Pio also blocked PMA-induced MMP-9 promoter activity by 45% in transactivation assays in HEK293 using a pGL3-MMP-9 2.2 kb construct. To evaluate the role of HMGA1, gene silencing experiments with siRNA for HMGA1 were performed (91 % in HASMC and 80.2% in HEK293 reduction of HMGA1 protein expression). HMGA1 siRNA completely abolished PPARgamma-mediated MMP9-mRNA repression (control siRNA: pio-mediated MMP-9 regulation vs. PMA alone: −66.8 % in HASMC and −59.3% in HEK293 p<0.01; HMGA1 siRNA: pio-mediated MMP-9 regulation vs. PMA alone: +10.7 % in HASMC and +14.7% in HEK293 vs. PMA alone; p=n.s.). Knockdown of HMGA1 expression reverse trans-repression of MMP9 by PPARgamma in HASMCs. By using ChIP assay we could demonstrate that pio-induced PPARgamma activation leads to a potent recruitment of PPARgamma (3.0 fold vs.1.15 fold PMA alone) and HMGA1 complexes (1.24 fold vs. 0.0 fold PMA alone) to the MMP9 promoter in HASMC. In consonance with reduced promoter activity, RNA-Polymerase II was removed from the MMP9 promoter by pio (0.08 fold vs 1.04 fold PMA alone). In conclusion, HMGA1 is required for PPARgamma-mediated repression of MMP-9 gene transcription. Ligand-induced HMGA1-PPARgamma interactions might be an important determinant for ligand-specific anti-atherosclerotic actions.
Abstract 3614: Adiponectin Inhibits Vascular Inflammation and Rapid Progression of Atherosclerosis Induced by Angiotensin II
Authors: Liu Joey , Becerra Diana , Boyadjian Rima , Kim Jason , Tangirala Rajendra K
Published: July 2021

Adiponectin, an adipocytokine derived from adipose tissue, has anti-inflammatory and antiatherogenic properties and low plasma adiponectin levels are associated with the metabolic syndrome and cardiovascular disease. However, it is unknown whether elevated plasma adiponectin levels provide atheroprotection against angiotensin II (AngII)-induced vascular inflammation and accelerated atherosclerosis. We investigated whether adenoviral adiponectin expression provides protection against AngII-induced inflammation and atherosclerosis. Thirteen week-old low-density lipoprotein receptor-deficient (LDLR
−/−
) mice were fed Western diet and infused with Ang-II. Mice were injected with 5 X 10
8
particles of adenovirus expressing either GFP (Ad-GFP, n=16/group) or mouse adiponectin (Ad-APN, n=16/group). Eight weeks after injection, plasma adiponectin levels in Ad-APN-injected mice were increased 12-fold compared to control Ad-GFP mice (306.4 mg/dL vs 25.3 mg/dL). Elevation of plasma adiponectin levels led to a significant increase in plasma HDL levels (23.6% increase vs. control), whereas the plasma cholesterol and triglyceride levels were unchanged. Quantification of atherosclerosis in the entire aortas by
en face
method, revealed a significant inhibition of atherosclerosis in Ad-APN mice (45% reduction, p<0.01 vs. Ad-GFP mice). Furthermore, adiponectin expression substantially inhibited the mRNA expression of inflammatory and atherogenic genes (ICAM-1, osteopontin, MCP-1 and CCR2) in the vessel wall. Interestingly, adiponectin also increased the mRNA expression of critical reverse cholesterol transport genes, ABCA1 and ABCG1, in the aorta. These results demonstrate that increasing plasma adiponectin levels may be an effective therapeutic strategy to inhibit angiotensin-II induced vascular inflammation and rapid progression of atherosclerosis.
Abstract 3622: Oxidized LDL-derived Reactive Aldehydes Induce ER Stress and Trigger the Unfolded Protein Response: Implications for Atherosclerotic Lesion Formation
Authors: Vladykovskaya Elena N , Haberzettl Petra , Barski Oleg A , Sithu Srinivas D , D’Souza Stanley E , Bhatnagar Aruni , Srivastava Sanjay
Published: July 2021

All stages of atherosclerotic lesion formation are associated with the accumulation of proteins modified by aldehydes that are generated from the oxidation of membrane phospholipids and lipoproteins. Although aldehyde-adducted proteins elicit strong immunogenic responses, the role of lipid peroxidation products in promoting and sustaining atherogenesis remains unclear. To examine aldehyde-induced protein modification and to assess its contribution to vascular stress, vascular smooth muscle and endothelial cells (EC) were exposed to 4-hydroxy-trans-2-nonenal (HNE), one of the most abundant and reactive oxidized lipid-derived aldehyde (LDA). Exposure to HNE (25 μM, 2h) resulted in extensive modification of cellular proteins in both cell types. Majority of these proteins, identified by MALDI/MS analysis, were ER-resident protein chaperones that assist in protein folding. Confocal imaging of HNE treated EC showed that the modified proteins were associated with the ER. Exposure to HNE led to the splicing of the bZIP transcription factor, XBP-1, a characteristic feature of the alarm phase of ER stress, and an increase in the phosphorylation of eIF2α, which regulates the expression of multiple ER-stress dependent transcription factors. These transcription factors, ATF3, ATF4, and CHOP were also strongly (2–5-fold) induced by HNE. Treatment with HNE was associated with a 2–5 fold increase in the expression of ER chaperones, GRP78 and HERP, indicating that HNE triggers the adaptive phase of the response, which increases the protein folding capacity of the ER. Pretreatment with the chemical chaperone, phenylbutyric acid (PBA), to facilitate protein folding, completely abolished HNE-induced increase in the expression of ATF3. EC treated with HNE enhanced monocyte adhesion, which was attenuated by PBA, indicating that early stages of lesion formation may be ER-stress dependent. In intermediate arterial lesion of 20 week old apoE-null mice, HNE-adducted proteins were localized to areas immunopositive for ATF3, ATF4, and phospho-PERK. These results suggest that protein modifications by LDA induce ER stress and the alarm and adaptive responses to this stress may be significant determinants of lesion progression and stability.
Abstract 3636: The Effects of Arginase II Overexpression on Endothelial Function in Transgenic Mouse Model
Authors: Vaisman Boris L , Wood Katherine C , Demosky Stephen J , Knapper Catherine L , Chin-Dusting Jaye , Morris Sidney M , Gladwin Mark T , Remaley Alan T
Published: July 2021

Endothelium is a major regulator of local vascular homeostasis. Its normal function is crucial for prevention of the development of atherosclerosis, hypertension and other cardiovascular disorders. Reduced nitric oxide (NO) bioavailability is one of the earliest and most important markers of endothelial dysfunction. L-arginine is the substrate for nitric oxide synthases (NOS). Arginase (Arg) can compete with eNOS for L-arginine and thus may play a role in endothelial dysfunction. To further investigate the role of ArgII in endothelial function and in atherosclerosis we generated transgenic mice with human ArgII (hArgII) gene under control of endothelial-specific Tie2 promoter. Expression of hArgII was measured by RT-PCR in eight tissues of transgenic males and compared with the level of mouse ArgII (mArgII) expression in kidneys of normal C57Bl mice, which was taken as 100%. hArgII was expressed at very high levels in all tissues, especially in aorta (2700%), heart (3500%), kidney (1600%), lung (9860%) and muscle (2000%). Arginase activity was elevated 4.6 – 62 fold in all tissues except liver. Lung endothelial cells isolated from hArgII transgenic mice had 4.4-fold greater of arginase activity than whole lung. Resident peritoneal macrophages from hArgII transgenic and normal mice had similar levels of arginase activity, confirming endothelial specificity of the Tie2 promoter. Overexpression of hArgII neither led to significant changes in plasma level of arginine, citrulline, NOHA, ADMA, SDMA and ornithine, nor to changes in plasma lipid levels. However, ArgII overexpression on apoE-knockout background was accompanied by a 10% increase in plasma total cholesterol (p<0.05). hArgII transgenic mice also had blood pressure (mean arterial and diastolic) that averaged 17% higher than controls. Aortic ring segments from hArgII transgenic mice, precontracted with phenylephrine (10
−6
M), exhibited decreased endothelium-dependent relaxation to increasing concentrations of acetylcholine (10
−9
to 10
−3.5
M), indicating endothelial dysfunction secondary to NO insufficiency. These results show that the Tie2hArgII transgenic mouse is a new model for investigating the role of ArgII in endothelial function and in atherosclerosis.
Abstract 3640: Mitochondrial Uncoupling Protein 2 Facilitates Endothelial Cell Growth and Angiogenesis via Reduction of Mitochondrial Superoxide
Authors: Shimasaki Yukio , Chen Kai , Keaney John F
Published: July 2021

Background:
Growing evidence suggests that mitochondrial function contributes to cell phenotype. One important component of mitochondrial function is the membrane potential that is controlled, in part, by uncoupling proteins (UCPs). Based on our previous data, the UCP2 is predominantly expressed in cultured endothelial cells. Therefore, we sought to examine the role of UCP2 in endothelial cell growth and angiogenesis.

Methods and Results:
Murine lung endothelial cells (MLECs) were isolated from UCP2-null and wild-type mice. UCP2-null cells were found less proliferative than wild-type cells (P<0.02, UCP2-null cells vs. wild-type cells, n=4). This defect of UCP2-null cells was rescued by UCP2 adenovirus transfection (19% increase, p<0.02 vs. LacZ adenovirus treated cells, n=3), and also rescued by transfection with manganese superoxide dismutase (MnSOD) adenovirus (53% increase, P<0.002 vs. LacZ adenovirus treated cells, n=3). We found a reciprocal relation such as no UCP2 expression and higher mitochondrial superoxide level in the MLECs (P<0.005, UCP2-null cells vs. wild-type cells, n=3), suggesting that mitochondrial superoxide may regulate endothelial cell growth. Then, we prepared murine aortic rings from UCP2-null and wild-type mice and embedded in rat tail collagen gel. The sprouting angiogenesis of UCP2-null explants was significantly less than wild-type explants (P<0.02, UCP2-null explants vs. wild-type explants, n=3– 4). Furthermore, MLECs from MnSOD-heterozygous mice showed less proliferation with lower expression of UCP2 protein and higher mitochondrial superoxide level compared to the MLECs from wild-type littermates (P<0.02, MnSOD-heterozygous cells vs. wild-type cells, n=4 – 8). We also observed less sprouting angiogenesis in MnSOD-heterozygous aortic explants than wild-type aortic explants (P<0.05, MnSOD-heterozygous explants vs. wild-type explants, n=3– 6).

Conclusions:
These data indicate that mitochondrial superoxide controls endothelial cell proliferation and angiogenesis, suggesting that mitochondrial metabolism modulates the endothelial cell growth and angiogenesis.
Abstract 3642: Local Therapeutic Angiogenesis Is Limited by Microvascular Clearance
Authors: Le Kha , Hwang Chao-Wei , Tzafriri A. Rami , Lovich Mark , Edelman Elazer
Published: July 2021

Local growth factor (GF) delivery is an attractive mode of vascular regenerative therapy for ischemic tissues. Yet, local release of proangiogenic GFs does not produce a sustained angiogenic response in clinical trials. We hypothesize that angiogenic therapies are self-regulated by dynamically altering tissue transport characteristics. Induced neocapillaries increase drug clearance rates, limiting tissue drug levels and induced vessel growth. We examined the interdependence of FGF distribution and angiogenesis in the rabbit heart
in vivo
(Fig

).
35
S-FGF1 penetrated 0.6 mm into the myocardium within 48 hr of release from epicardial controlled release patches. Drug release remained constant and angiogenesis was achieved but peaked at day 8 and regressed by day 31, as neocapillary clearance induced rapid increase of GF washout. An ex-vivo perfused heart model supported this interpretation. FGF2 distributions followed linear diffusion with low diffusivity (0.02 μm
2
/s) and low tissue penetration in the absence of coronary flow. Flow restoration enhanced clearance, contracting FGF2 penetration −46%. Findings were explained by a diffusion-clearance model, with a clearance rate constant K = 1.15×10
−4
sec
−1
. The validated model of myocardial GF transport was coupled to a model angiogenic response to provide an integrated computational framework for evaluating local angiogenic therapy (insert). Induced angiogenesis is a double-edged sword. Early release of angiogenic compounds stimulates neo-vascularization but the very efficacy of these compounds enhances their clearance and abrogates their pharmacologic benefit.
Abstract 3648: A Dual Role for Granulocyte-Colony Stimulating Factor in Promotion of Coronary Collateral Growth and Production of Cardiomyocyte Reactive Oxygen Species
Authors: Carrão Ana C , Chilian Wiliam M , Yun June , Kolz Chris , Buschmann Ivo
Published: July 2021

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic cytokine that promotes proliferation and differentiation of neutrophil progenitors. Because G-CSF ameliorates myocar-dial ischemic injury, we projected this effect would also translate into stimulating myocardial adaptations to ischemia. Accordingly, we hypothesized that G-CSF stimulates coronary collateral growth (CCG) in a rat model of repetitive episodic ischemia (RI): 40 sec LAD occlusion every 20 min for 2h20min, 3 times/day for 5 days. CCG was deduced from collateral-dependent flow (flow to LAD region during occlusion [neutron activated microspheres]) and expressed as the increase in the ratio between collateral-dependent and normal zone flows from the initial measurement to that after 5 days of RI. Following RI, G-CSF (100 microg/Kg/day) increased CCG (P<0.01) (0.47 +− 0.15) versus vehicle (0.14 +− 0.06). Surprisingly, G-CSF treatment without RI increased CCG (0.57 +− 0.18, P<0.01 vs vehicle) equal to G-CSF +RI. Because redox signaling is critical for CCG and neutrophils are a rich source of NADPH oxidase and reactive oxygen species (ROS), we hypothesized that G-CSF stimulates production of ROS. We evaluated ROS by dihydroethidine (DHE) fluorescence (LV injection, 60 microg/kg, during two episodes of ischemia). DHE fluorescence was double in G-CSF+RI vs vehicle+RI (P<0.01), and even higher in G-CSF without RI (P<0.01). Interestingly, the DHE signal did not co-localize with myeloperoxidase (immunostaining, neutrophil marker) but appeared in cardiac myocytes. To unequivocally determine if G-CSF stimulates ROS production in cardiac myocytes, we studied isolated cardiac myocytes and found the cytokine stimulates ROS. In addition to affecting neutrophils, G-CSF directly targets cardiac myocytes to produce ROS. In conclusion, G-CSF stimulates production of ROS by cardiac myocytes, which likely plays a pivotal role in adaptations of the heart to ischemia including growth of the coronary collateral circulation.
Abstract 3657: Cell-Specific Delivery and Monitoring of Antiangiogenic Therapy in Experimental Aortic Valve Disease with Theranostic Integrin-Targeted 19F Nanobeacons
Authors: Waters Emily A , Chen Junjie , Allen John S , Zhang Huiying , Fuhrhop Ralph , Lanza Gregory M , Wickline Samuel A
Published: July 2021

Although angiogenesis plays a critical role in the development of atherosclerosis, its participation in early valvular disease remains speculative. To delineate the presence of angiogenesis and its potential responsiveness to medical therapy in experimental valve disease, we used unique signaling (
19
F), background-free molecular MR nanobeacons as simultaneous
diagnostic
and
therapeutic
(theranostic) tools to quantify valvular angiogenesis in 24 cholesterol-fed NZW rabbits (0.25% cholesterol, 6 mo.) and its response to nanoparticle-borne antiangiogenic therapy with an aminopeptidase-2 inhibitor, fumagillin. Perfluorocarbon nanobeacons (~250 nm) targeted to neovascular α
v
β
3
integrins and loaded with 0.2% fumagillin were given i.v. at 1 mL/kg for 4 weekly doses (n=8; 30 μg/kg fumagillin/dose). Controls included treatment with nontargeted drug-bearing nanoparticles (n=7) and saline (n=9). MR spectroscopy (470 MHz) for detection of
19
F signal from bound nanoparticles was performed after valve excision. Immunocytochemistry confirmed angiogenesis in all valves. In the targeted drug treatment group, both 50% fewer vessels and 50% lower
19
F signal were observed vs nontargeted or saline groups (p<0.01, p<0.001), confirming efficacy of the theranostic agent in limited dosing (Figure 1

). Across all animals, cholesterol levels exerted a strong linear effect on the extent of angiogenesis (r=0.52; p<0.01). We speculate that targeted therapy of angiogenesis with theranostic nanoparticles could offer an alternative or conjunctive approach for rational medical management of valvular disease.

Figure 1:

(Left) Least squares means of
19
F signal from the saline (S), targeted fumagillin (TF), and untargeted fumagillin (UF) treated groups, normalized to the saline control. Note 50% reduction of
19
F signal in the group treated with targeted fumagillin nanoparticles compared to the untreated and untargeted controls. (Right) LS means of vessel count on stained valve sections. Note 50% fewer vessels in targeted fumagillin group corresponding to reduction in
19
F signal. (* p<0.01, ** p<0.001)
Abstract 3664: Mitochondrial p27 Is Required For Endothelial Energy Metabolism And Cell Migration
Authors: Spyridopoulos Ioakim , Altschmied Joachim , Andres Vicente , Haendeler Judith
Published: July 2021

The migratory capacity of endothelial cells (EC) is a key prerequisite for the growth of new blood vessels during neovascularisation of ischemic and/or tumor tissues. Recently, mitochondria have been identified as signalling organelles in EC, which may contribute to migratory capacity. Therefore, the aim of this study was to investigate whether a molecular link exists between mitochondrial energy metabolism and migratory capacity of EC. To determine whether mitochondria are required for migratory capacity, we incubated EC with 50 ng/ml ethidiume bromide (EtBr) in the presence of uridine to deplete mitochondrial DNA (mtDNA) and, thereby, destroy active mitochondria. EC, which were depleted of mtDNA, did not migrate in a scratch wound assay (− EtBr: 22 +/− 3 migrated cells; + EtBr: 0.15 +/− 0.1 migrated cells). Since protein translocation is a major mechanism for modulating the functional capacity of the mitochondria, we hypothesized that induction of migration by the promigratory stimulus vascular endothelial growth factor (VEGF) leads to translocation of proteins to mitochondria. Recent studies demonstrated that a non-nuclear pool of p27 exerts biological activities including increase of migratory capacity independent of its cell cycle inhibitory activity. Stimulation of EC with VEGF induced a 2.5 +/− 0.4-fold increase in mitochondrially localized p27 protein. Reducing p27 expression by two different siRNAs completely abrogated VEGF-induced migration of EC (98 +/− 2 % inhibition). Next, we cloned p27 into a mitochondrial targeted vector (p27mito) to exclusively overexpress p27 in the mitochondria. Overexpression of p27mito stimulated mitochondrial ATP biosynthesis (3.24 +/− 1.06-fold empty vector), increased mitochondrial membrane potential and enhanced migratory capacity of EC to a similar extent as compared to VEGF. Cells overexpressing p27mito did not demonstrate further increased migratory capacity in response to VEGF. Finally, only p27mito but not nuclear-targeted p27 overexpression rescued the impaired migratory capacity after siRNA-mediated genetic ablation of endogenous p27 expression. These data demonstrate a novel role of mitochondrial p27 as a molecular link between cellular energy metabolism and migratory capacity of EC.