bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1106:
In Vivo
Molecular Imaging for Cardiac Regeneration
Authors: Wang Jingxiong , Rabinovich Brian , Zhang Sui , Shpall Elizabeth , Gelovani Juri G , Willerson James T , Yeh Edward T
Published: July 2021

Introduction
We have showed that the transplanted human peripheral blood CD34
+
cells can transform into smooth muscle, endothelial, and myocardial cells in infarcted hearts in mice. However, the fate of the transplanted cells
in vivo
is unknown.

Hypothesis
Molecular imaging is of substantial value for non-invasively tracking the fate of transplanted stem cells and for developing stem cell transplantation toward a clinically accepted therapy.

Objectives
To develop and validate molecular imaging techniques for reliably tracking the fate of transplanted stem cells in ischemic hearts
in vivo
and to follow the tempo-spatial dynamics of their transformation and function.

Methods
Using SCID mice transplanted with the CD34
+
cells after myocardial infarction as a model, the CD34
+
cells were transduced with 6 lentivectors bearing a fusion reporter gene driven by 6 different promoters. The reporter protein consists of herpes simplex virus thymidine kinase, green fluorescence protein, and luciferase, allowing us to monitor
in vivo
the fate of transplanted cells with bioluminescence imaging (BLI) and positron emission tomography (PET). In order to assess cardiac function, the mice were scanned with a 7.0-T MRI.

Results
The virus containing human ubiquitin promoter produced stable transduction efficiency 30%. Thereafter, 10
6
transduced CD34
+
cells were injected into peri-infarct area per mouse heart. At day 7, the BLI signals in hearts were 1.4 ± 0.12x10
5
photons(p)/s/cm
2
/sr (n=6). At day 14, the PET signals were 0.05 ± 0.001 injection dose (ID) %/g. Both signals increased progressively to week 4, then remained in plateaus of 1.8 ± 0.15x10
5
p/s/cm
2
/sr and 1.38 ± 0.002 ID%/g, respectively until week 6, thereafter declining gradually, but were still as high as 9 ± 0.11x10
4
p/s/cm
2
/sr and 0.92 ± 0.001 ID%/g at week 16, indicating the transplanted CD34
+
cells survive and function in the host. Co-register MRI/PET showed the concordance of anatomic and functional consequences after the transplantation of the CD34
+
cells.

Conclusion
Our results demonstrate the feasibility of using molecular imaging to reliably track human CD34
+
cell transplanted into infarcted hearts in mice, thus identifying PET/MRI imaging as a means to trace stem cell transplants in injured hearts.
Abstract 1115: Impaired Angiogenic Potential of Cardiac Progenitor Cells in Mice with a Cardiomyocyte-Restricted Knock Out of STAT3
Authors: Fischer Philipp , Missol-Kolka Ewa , Zschemisch Nils-Holger , Templin Christian , Drexler Helmut , Hilfiker-Kleiner Denise
Published: July 2021

Mice with a cardiomyocyte-restricted knock out of STAT3 (KO: alpha-MHC-Cre
tg/+;
STAT3
flox/flox
) show a continuous decrease of the cardiac capillary density and develop heart failure beyond the age of 9 months. We sought to determine the paracrine influence of cardiomyocyte STAT3 on the endothelial differentiation potential of cardiac progenitor cells (CPC) of the adult mouse heart. Sca-1
+
CPC were isolated from male mice hearts by MACS separation. STAT3 was entirely deleted in cardiomyocytes of KO mice, while CPC from KO showed normal expression of STAT3 (confirmed by PCR and Western blot). No difference in the total number of CPC per heart was observed between wildtype (WT: STAT3
flox/flox
) and KO mice. FACS analysis revealed a reduced number of endothelial progenitor cells (as defined by coexpression of Sca-1, CD31 and CD38, −25%, P<0.05) among CPC from KO compared to CPC from WT. The differentiation potential of CPC from WT and KO was analyzed during in vitro culture on fibronectin-coated plates. After 4 weeks of culture RT-PCR for CD31 and immunohistochemistry (IHC) for endothelial cell (EC) marker tie2 and isolectin B4 was performed. CPC from WT showed markedly more efficient EC differentiation and tube formation compared to CPC from KO (p<0.01). In contrast, adipocyte differentiation was enhanced in CPC from KO (p<0.05, oil red staining and RT-PCR). Proliferation capacity of CPC from KO was reduced by 33% (p<0.01) as compared to CPC from WT. Microarray results of freshly isolated CPC were consistent with the differences in EC and adipocyte differentiation (i.e. prostaglandin E receptor 3 up 2.3-fold in CPC from WT, Lipocalin-2 up 2.7-fold in CPC from KO). We did not observe cardiomyocyte differentiation (IHC for alpha-sarcomeric actinin; RT-PCR for Nkx 2.5, alpha-MHC, or alpha-skeletal actin) of CPC from both genotypes, neither in vitro by addition of oxytocin, 5-AZA, DMSO, nor following intramyocardial injection of CPC in vivo.

Conclusion: STAT3-dependent paracrine mediators released from cardiomyocytes are determinants of differentiation and vasculogenic properties of new EC derived from cardiac progenitor cells. The identification of these factors may offer new approaches to enforce the endogenous vasculogenic repair potential of the adult heart.
Abstract 1123: Critical Role Of Adipose Senescence In Insulin Resistance And Vascular Dysfunction
Authors: Orimo Masayuki , Minamino Tohru , Miyauchi Hideyuki , Tateno Kaoru , Okada Sho , Moriya Junji , Miura Kentaro , Shimizu Ippei , Nojima Aika , Komuro Issei
Published: July 2021

Cellular senescence is originally described as the finite replicative lifespan of human somatic cells in culture. As a consequence of semi-conservative DNA replication, the extreme terminals of the chromosomes are not duplicated completely, resulting in successive shortening of telomeres with each cell division. Telomerase is a ribonucleoprotein that adds telomeres to the ends of chromosomes. Critically short telomeres are thought to trigger DNA damage response, thereby inducing cellular senescence. Accumulating evidence has suggested that senescent cells promote aging phenotypes or age-related pathologies. Here we show that adipose senescence is critically involved in the regulation of insulin resistance that underlies age-associated cardiovascular disease. The later generation of telomerase-deficient mice with short telomeres exhibited insulin resistance and vascular dysfunction when fed on a high-calorie diet. Adipose tissue of these mice revealed senescence-like phenotypes such as an increase in neutral β galactosidase activity and upregulation of p53 and pro-inflammatory cytokines. Serum levels of pro-inflammatory cytokines were markedly elevated in telomerase-deficient mice and treatment of these mice with a neutralizing antibody against TNF-α significantly improved insulin and glucose intolerance. Removal of senescent adipose tissue reduced serum levels of pro-inflammatory cytokines and thereby improved insulin resistance in telomerase-deficient mice. Conversely, implantation of senescent adipose tissue to wild-type mice impaired insulin sensitivity and glucose tolerance in recipients. Introduction of telomere dysfunction to young adipose tissue markedly upregulated p53 expression and increased the production of pro-inflammatory cytokines. Inhibition of p53 activity significantly improved senescence-like phenotypes of adipose tissue, insulin resistance, and vascular dysfunction in telomerase-deficient mice. These results disclose a novel mechanism of insulin resistance and suggest that adipose senescence is a potential therapeutic target for the treatment of diabetes and diabetic vasculopathy.
Abstract 1127: Natural-Killer T Cells are Involved in the Development of Glucose Intolerance and Microalbuminuria in Diet-Induced Obese Mice
Authors: Ohmura Kazue , Ishimori Naoki , Ohmura Yoshinori , Fujii Satoshi , Andoh Yasuhiro , Tsutsui Hiroyuki
Published: July 2021

Introduction:
Natural killer T (NKT) cells are a unique subset of T lymphocytes, which recognize glycolipids and integrate inflammation producing pro-inflammatory cytokines on activation. We have demonstrated that NKT cells are present in atherosclerotic lesions and play a pivotal role in atherogenesis. NKT cells also reside in visceral adipose tissues, however, their pathogenic role has not been studied in metabolic derangements.

Methods and Results:
To determine whether NKT cells are involved in the development of metabolic phenotypes, male β
2
microglobulin knockout (KO) mice, which lack T cells and NKT cells, and C57BL/6J (WT) mice were fed an western diet (WD) containing 21% fat and 0.15% cholesterol or a chow diet for 13 weeks. In both KO (n=14) and WT (n=10) mice fed WD, visceral adipose-tissue weight, adipose cell size, and plasma leptin and non-HDL cholesterol levels were significantly increased compared to those fed a chow (
p<
0.05) but did not differ between KO and WT mice. However, in KO mice fed WD, impaired glucose tolerance (plasma glucose 15 minutes after intraperitoneal glucose injection, 228±7 vs 343±31 mg/dl,
p<
0.001) and microalbuminuria (urine albumin/creatinin ratio, 10±1 vs 24±2 μg/mg,
p<
0.0001) were significantly ameliorated, but not in WT mice. Plasma TNF-α was significantly lower in KO mice than in WT mice (29±6 vs 85±40 pg/ml,
p<
0.05). Immunohistochemical staining of visceral adipose tissues revealed that F4/80 (+) macrophages in KO mice were markedly reduced to 34% of those in WT mice (
p<
0.0001). To further confirm that NKT cells can aggravate metabolic derangements, WT mice received α-galactosylceramide (0.1μg/g), which specifically activates NKT cells, (αgC, n=5) or saline (PBS, n=5) injection after 13 weeks of feeding WD. In αgC mice, impaired glucose tolerance (347±9 vs 308±13 mg/dl,
p<
0.05) and microalbuminuria (35±8 vs 17±1 μg/mg,
p<
0.05) were significantly exacerbated as compared to PBS mice.

Conclusions:
NKT cells enhance chronic inflammation in visceral adipose tissues and contribute to the development of metabolic derangements in diet-induced obese mice. NKT cells may be novel therapeutic targets in the prevention of metabolic syndrome.
Abstract 1135: Hydrogen Peroxide Plays a Crucial Role in Coronary Microvascular Vasodilation with Increased Oxidative Stress in Right Ventricles of Pulmonary Hypertensive Rats
Authors: Kajiya Masahito , Inai Yousuke , Morimoto Taro , Iwasaki Tatsuo , Endo Kousuke , Mohri Satoshi , Yada Toyotaka , Naruse Keiji , Ohe Tohru , Kajiya Fumihiko
Published: July 2021

Background:
Hydrogen peroxide (H
2
O
2
) is a well recognized candidate for EDHF. The aim of this study was to examine H
2
O
2
-EDHF function in coronary arterial microvessels under conditions of increased oxidative stress in RV of pulmonary hypertension (PH).

Methods:
Monocrotaline (60 mg/kg SC) was administrated in 5 week-age male Sprague-Dawley rats to induce PH (PH; n=52). Saline was injected in control rats (n=47). After 3 weeks, RV coronary arterioles (CA; 30 –100 μm) and small arteries (CSA; 100 –200 μm) were visualized
in vivo
using our intravital videomicroscopy. After cyclooxigenase blockade, Acetylcholine (ACh; 2 μg/kg/min IV) induced vasodilation was evaluated under three conditions; ACh alone, N
ω
nitro-L-arginine methyl ester (L-NAME), L-NAME+catalase. Differences of percentage vasodilation between ACh alone and L-NAME, and between L-NAME and L-NAME+catalase were used as relative indices for the contributions of NO and H
2
O
2
-EDHF, respectively. Malondialdehyde (MDA) in plasma was measured with colormetric assay. Superoxide production in RV was determined by lucigenin-derived chemiluminescence.

Results:
RV systolic pressure was significantly increased in PH (75±13 vs. 31±3 mmHg; p<0.05). In PH, Ach-induced vasodilation was significantly reduced in both CA and CSA compared with control (CA; 5.2±1.5% vs. 10.8±3.5%, CSA; 3.8±1.0% vs. 6.0±1.9%, both p<0.05). Inhibition of vasodilation by L-NAME in PH was markedly decreased for both CA (from 4.0% to 1.0%) and CSA (from 3.6% to 1.0%) (both p<0.05). Suppression of vasodilation by the addition of catalase in PH was decreased for CA (from 5.2% to 3.1%, p<0.05), but maintained or even increasing tendency for CSA (from 1.7% to 2.0%, NS). Notably, the relative contribution of H
2
O
2
-EDHF in PH was greatly increased for both CA (from 48% to 60%) and CSA (from 28% to 53%) (both p<0.05). Plasma MDA and RV superoxide in PH were markedly higher than those in control (MDA; 1.63±0.53 vs. 0.93±0.11 μm, superoxide; 290±52 vs 158±25 cpm/mg, both p<0.05). Collectively, increased oxidative stress may augment H
2
O
2
produciton and contribute to the robust EDHF function.

Conclusion:
Under conditions of increased oxidative stress in PH, H
2
O
2
-EDHF plays a crucial role against NO dysfunction in RV coronary microcirculation.
Abstract 1163: Engineering Biomaterials For Vascular Differentiation And Regeneration
Authors: Gerecht Sharon , Vunjak-Novakovic Gordana , Langer Robert
Published: July 2021

The goal of our studies was to utilize engineered biomaterials to understand the mechanisms underlying microenvironment regulatory cascades during vascular differentiation and regeneration. We found that the ECM polysaccharide hyaluronic acid (HA) is a developmentally relevant material for the growth of human embryonic stem cels (hESCs). Human ESCs encapsulated in HA hydrogels in conditioned media, maintained their undifferentiated state and could be further initiate vasculogenesis within the same system by supplementation with VEGF. In a continuous study we have developed a method for introducing pores into photocurable bioelastomer while maintaining mechanical properties. Biocompatibility studies demonstrated the ability to encapsulate, grow and differentiated cells
in vitro
, while subcutaneous transplantation revealed inflammatory response similar to other biocompatible materials. In addition, these
In vivo
experiments showed that the porous bioelastomer promotes ingrowth and integration with the host circulation, suggesting that porous bioelastomer may be utilized for tissue engineering applications. Mammalian cells respond to their substrates by complex changes in gene expression profiles, morphology, proliferation and migration. We report that the nanotopography of the substrate can be used to control various cellular responses of hESCs and human endothelial progenitor cell (hEPC). Poly(dimethylsiloxane) (PDMS) films were replica-molded on passivated silicon wafers to yield line-grating (600 nm ridges with 600 nm spacing and 600 ± 150 nm feature height), coated with fibronectin or collagen, and seeded with single-cell suspensions. These nanopattered PDMS substrates induced hESC alignment and elongation, mediated the organization of cytoskeletal components, and reduced proliferation. The addition of actin disrupting agents attenuated the alignment and proliferative effects of nanotopography. Human EPCs cultured on nanotopographic substrates elongated, and aligned with the structures, while their migration was also enhanced. Long-term cultures led to the formation of band-like structures of hEPCs as their proliferation was reduced, and the addition of matrigel led to the formation of ordered tube structures.
Abstract 1166: In-vivo Serial Assessment of Aortic Aneurysm Formation in a Hyperlipidimic Mouse Model
Authors: Turner Gregory , Olzinski Alan , Bernard Roberta , Aravindhan Karpagam , Karr Heather , Willette Robert , Gough Peter , Jucker Beat
Published: July 2021

Introduction
Abdominal aortic aneurysms (AAA) can be consistently produced in hyperlipidimic mice by continuous infusion of Angiotensin-II (Ang-II). MRI allows for AAA visualization
in-vivo
with sufficient spatial resolution to assess changes in aneurysm size and morphology. Bright-blood contrast allows for detection of medial breaks, an index of AAA severity, and USPIO contrast agent uptake allows for detection of macrophage activity in AAA. For this study we used MRI to characterize AAA development in a murine model and examined the effect of the broad-based MMP inhibitor, doxycycline, on AAA progression over a 4 week period.

Methods
Osmotic pumps were implanted subcutaneously in ApoE−/− mice for infusion of Ang-II (1000 ng/kg/min) over 4 weeks. Animals were given 30 mg/kg/day doxycycline or vehicle (n=15 per group) via drinking water. Weekly scans were performed using a 9.4T MRI scanner. In addition, uptake of USPIO (Combidex, 1000 μmol/kg) in AAA was measured.

Results
MRI revealed two distinct features of AAA development: remodeling of the adventitia which compresses the aortic lumen (Fig 1B

) and a break in the medial wall characterized by bright-blood signal within the AAA (Fig 1C

). Higher incidence of AAA formation in the vehicle vs. doxycycline group was detected along with a significantly larger (P<.01) AAA area. For mice given USPIO, MRI revealed signal loss in the periphery and shoulder region of the AAA which corresponded to macrophage rich regions as determined by IHC.

Conclusion
These results demonstrate MRI’s potential for non-invasively and temporally assessing AAA development and pharmacological intervention in this pre-clinical cardiovascular disease model.
Abstract 1167: A Path To Vascular Disease Through The Biological Clock
Authors: Rudic R. Daniel , Anea Ciprian B , Stepp David W , Simkins G. Bryan , Reed Guy , Fulton David J
Published: July 2021

Cardiovascular disease is the leading cause of death for both men and women in the United States and the world. There is a profound pattern in the time of day at which the death occurs; it is in the morning, when the endothelium is most vulnerable and blood pressure surges, that stroke and heart attack most frequently happen. Though the biological clock rhythmically oscillates in blood vessels, evidence of a direct function in the vasculature for Bmal1, an essential clock component, is lacking. We have found that chronic flow interruption in arteries of mice with targeted disruption of Bmal1 gene (Bmal1-KO) causes pathological vascular remodeling which worsens to thrombosis with ageing. In addition, naïve aortic arteries of Bmal1-KO mice exhibit endothelial dysfunction, which is improved by relief of oxidant stress. Akt signalling, a pathway critical to vascular function, was significantly attenuated in arteries of Bmal1-KO mice. Co-transfection of cultured cells with the transcription factors Bmal-Npas2 or Bmal1-Clock induced a robust activation of the Akt promoter, providing evidence of a direct link between the circadian clock and Akt pathways. Our data reveals a new role for the biological clock in the control of endothelial signalling and vascular remodeling which may be of significant impact in the progression of vascular disease.
Abstract 1169: Critical Role of Leukocyte ROCK1 in Mediating Neointimal Formation Following Vascular Injury
Authors: Noma Kensuke , Rikitake Yoshiyuki , Oyama Naotsugu , Ahl Daniella , Liu Ping-Yen , Wang Hongwei , Sawada Naoki , Okamoto Ryuji , Hiroi Yukio , Liao James K
Published: July 2021

Background
- Rho kinase (ROCK) has been implicated in vascular inflammation, remodeling, and atherosclerosis. However, the tissue- and isoform-specific roles of ROCKs in mediating these processes are not known.

Methods and Results
- To determine the role of ROCKs following vascular injury, we ligated the left common carotid artery in
WT, ROCK1
+/−
, and ROCK2
+/−
mice. Four weeks after carotid ligation, the neointimal areas were decreased in
ROCK1
+/−
(6,900 ± 3,800 μm
2
,
P
< 0.05), but not in
ROCK2
+/−
mice (31,200 ± 8,500 μm
2
,
P
= NS), compared to that of
WT
mice (26,800 ± 5,600 μm
2
). This correlated with substantial decreases in vascular ROCK activity, neointimal proliferation, proinflammatory adhesion molecules, and leukocyte infiltration in
ROCK1
+/−
mice. Indeed, neutrophil recruitment into the peritoneal cavity after intraperitoneal injection of thioglycollate was reduced in
ROCK1
+/−
mice (7.0 ± 0.3 x 10
6
/ml,
P <
0.01) compared to that in
WT
mice (13.9 ± 0.8 x 10
6
/ml), U937-LAM monocyte accumulation on TNFα-stimulated endothelial cells (EC) from
ROCK1
+/−
mice was 2.5-fold lower than that from
WT
mice, and thrombin-induced increases in ROCK activity and ICAM-1 and VCAM-1 expressions were impaired in EC from
ROCK1
+/−
mice. Furthermore, in vascular smooth muscle cells from
ROCK1
+/−
mice, PDGF-induced increases in ROCK activity and migration, but not proliferation, were reduced. To determine the role of leukocyte ROCK1 in this process, we transplanted the bone marrow (BM) from donor
WT
into recipient irradiated
WT
mice, from
WT
into irradiated
ROCK1
+/−
mice, and from
ROCK1
+/−
into irradiated
WT
mice. Transplantation with BM from
ROCK1
+/−
into
WT
mice resulted in reduced neointimal formation and leukocyte infiltration, while transplantation with BM from
WT
into
ROCK1
+/−
mice had no substantial effect on them.

Conclusion
- These findings indicate that ROCK1, but not ROCK2, mediates leukocyte recruitment and neointimal formation following vascular injury and suggest that leukocyte ROCK1 may be an important therapeutic target in vascular inflammatory and proliferative diseases.
Abstract 1190: The Complement Component C5a is Present in Human Unstable Coronary Lesions in Vivo and Induces the Expression of MMP-9 in Human Macrophages via Nf-kappaB Activation In Vitro
Authors: Speidl Walter S , Kastl Stefan P , Hutter Randolp , Kaun Christoph , Bauriedel Gerhard , Fuster Valentin , Maurer Gerald , Badimon Juan J , Wojta Johann
Published: July 2021

Background. The complement component C5a formed during activation of the complement cascade exerts chemotactic and proinflammatory effects. We have recently shown that serum levels of C5a predict cardiovascular risk in patients with advanced atherosclerosis. However, it is not known whether C5a may play an active role in the onset of acute coronary events.

Methods and Results. Immunohistochemical analysis of human coronary plaques obtained by atherectomy demonstrated the presence of C5a in macrophage rich areas and the necrotic core. Unstable plaques (n=9) showed significantly higher staining for C5a than stable coronary lesions (n=10; p<0.05). Double-labeling studies demonstrated colocalization of C5a and MMP-9. When human monocyte derived macrophages were stimulated in vitro with rhC5a, MMP-9 mRNA levels were increase 6-fold. In addition, MMP-9 antigen as detected by ELISA and MMP-9 activity as detected by a specific activity assay significantly increased after stimulation with rhC5a. Nuclear shift assay revealed that rhC5a stimulated NF-κB DNA binding and the NF-κB inhibitor 6-amino-4-phenoxyphenethylaminoquinazoline blocked the up-regulation of MMP-9 by rhC5a.

Conclusion. C5a is increased in unstable coronary plaques, colocalizes with MMP-9 and induces the expression of MMP-9 in human macrophages via NF-κB activation. Taken this observations together, activation of the complement cascade and formation of C5a may be involved in the onset of acute coronary events.
Abstract 1214: Rational Design of a Potent Bipartite Inhibitor of Nuclear Factor of Activated T Cells (NFAT), a Key Regulator of Cardiovascular Hyperplasia
Authors: Yu Haixiang , Bot Ilze , Sliedregt Karen , Xu Xingfu , van der Marel Gijs A , Overkleeft Herman S , van Berkel Theo J , Biessen Erik A
Published: July 2021

Purpose:
Targeted intervention in calcineurin/NFAT signaling is an effective strategy in the treatment of vascular smooth muscle cell (vSMC) hyperplasia and restenosis. We have recently shown that VIVIT, a novel peptide inhibitor of NFAT, is more selective and less toxic than cyclosporine A (CsA) and holds promise in antirestenotic therapy. However, its suboptimal pharmacological features preclude direct clinical use. In this study, we sought to overcome these setbacks by stepwise optimization of VIVIT.

Methods and Results:
In the first stage stepwise truncation and alanine scan studies were performed to establish the critical importance of the HPVIVIT motif for NFAT inhibition. In order to create a non-toxic bipartite compound that simultaneously targets two recently identified NFAT docking sites on calcineurin, the HPVIVIT motif was conjugated to a maleimide derivative via a series of flexible spacers of 8–28 Å. One of the conjugates, maleimide conjugated HPVIVIT or MCV, was 10,000 more potent in inhibiting NFAT (IC50=1.9 nM) than the parent peptide. MCV was able to abrogate ionomycin induced nuclear translocation of NFAT1-GFP at 100 nM and this effect persisted for >36h. Moreover, it profoundly inhibited platelet-derived growth factor-BB stimulated vSMC proliferation at concentrations as low as 10 nM (P=0.04; ~80%). MCV (100 nM) selectively disrupted calcineurin NFAT interaction in a GST pull-down assay but did not affect that of other binding partners (AKAP-79/Cain). It was at least equally potent as CsA in inhibiting ionomycin induced NFAT dephosphorylation in COS-1 cells. Unlike CsA however, MCV appeared to be non-toxic and did not impair the activity of calcineurin phosphatase. Moreover, in contrast to CsA MCV did not affect mitogen-activated protein kinase activity after PMA stimulation in COS-1 cells at concentrations up to 10 μM nor did it influence NFκB activity. Finally, CsA prevented nuclear accumulation of calcineurin, which remained unaffected by MCV1 (1 μM).

Conclusion
: The potency, chemical features and superior selectivity of the bipartite NFAT inhibitor MCV render this compound an excellent lead in the treatment of inflammatory disorders implicating NFAT activation, including myocardiac hypertrophy and restenosis.
Abstract 1221: Targeted Deletion of ROCK2 in Cardiomyocytes Prevents Angiotensin II-Induced Cardiac Hypertrophy
Authors: Okamoto Ryuji , Noma Kensuke , Sawada Naoki , Hiroi Yukio , Liu Ping-Yen , Liao James K
Published: July 2021

Background
- Previous studies have shown that Rho kinase (ROCK) inhibitors prevent the development of cardiac hypertrophy. Because ROCK inhibitors inhibit both ROCK isoforms, ROCK1 and ROCK2, the isoform-specific role of ROCK cannot be elucidated from these studies. Hence, a genetic approach with targeted deletion of ROCK in cardiomyocytes provides the best opportunity towards understanding the role of ROCK isoforms in the development of cardiac hypertrophy. Previous studies showed that ROCK1 KO mice develop cardiac hypertrophy to angiotensin II infusion similar to WT mice, but do not develop cardiac fibrosis. However, the role of ROCK2 in the development of cardiac hypertrophy remains to be determined.

Methods and Results
- Mice deficient in cardiomyocyte-specific ROCK2 (c-ROCK2
−/−
) were generated by crossing mice with loxP-flanked ROCK2 allele with transgenic mice expressing a Cre protein under the control of the cardiomyocyte-specific alpha-myosin heavy chain promoter. The ROCK2 expression levels in the c-ROCK2
−/−
mice heart was decreased to less than 30% compared with wild-type mice (ROCK2
+/+
mice) in the whole heart. Heart rate, blood pressures and cardiac systolic function were normal in c-ROCK2
−/−
mice. Ang II (400ng/kg/min) or vehicle was subcutaneously infused into c-ROCK2
−/−
and ROCK2
+/+
male mice (each group; n=10) for 28 days. Ang II-induced cardiac hypertrophy assessed by an increase in heart weight, left ventricular mass, myocyte cross-sectional area and cardiac hypertrophy-related genes expressions were attenuated in c-ROCK2
−/−
mice compared with ROCK2
+/+
mice. The basal activity of extracellular signal-regulated kinase (ERK) were similar in hearts between two groups but the activation of ERK was attenuated and the activity was downregulated earlier in c-ROCK2
−/−
than in ROCK2
+/+
mice. The activity of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK) and Akt activity were similar between two groups.

Conclusions
- These results indicate that ROCK2 is necessary for Ang II-induced cardiac hypertrophy. The mechanism, in part, involves the activation of ERK by ROCK2. Thus, selective ROCK2 inhibitors may be beneficial for preventing cardiac hypertrophy.
Abstract 1228: Decreased Mitochondrial Biogenesis and Increased Pro-oxidant Activity Are Associated with Oxidative Impairment of Complex II in the Postischemic Heart
Authors: Chen Jingfeng , Chen Chwen-Lih , Palmer Brian , Zweier Jay L , Chen Yeong-Renn
Published: July 2021

Increased O
2
·−
generated in mitochondria and NO is a key mechanism of cell death in the postischemic injury. Succinate ubiquinone reductase (SQR or Complex II) is a crucial segment of electron transport chain. Oxidative impairment of SQR has been observed in the postischemic myocardium. When rats are subjected to a 30 min coronary ligation, followed by a 24 h reperfusion, two polypeptides in tissue homogenate were found to react with the antibody against nuclear-encoded 29 kDa iron-sulfur protein (ISP) of SQR. One at 29 kDa corresponded to the mature form localized in mitochondria was present in normal amounts. The other with higher molecular weight 32 kDa corresponded to the precursor localized in cytosol was marked deficient. Similar results were observed when isolated heart was subjected to global ischemia (30min) and reperfusion (1h). Taken together, these results implicate mitochondria myopathy with an abnormality of biogenesis in the postischemic heart. The SQR mRNA level analyzed by quantitative real-time PCR was decreased 51%. Both mRNA and protein expressions of transcription coactivator PGC-1α were decreased in postischemic models 44% and 38% respectively, indicating a defect in the regulation and coordination of mitochondrial biogenesis. The mRNA levels of downstream transcription factors and antioxidant enzymes including NRF, TFAM, MnSOD, and UCP were similarly decreased; indicating that defect in mitochondrial biogenesis augments the oxidative stress in the postischemic heart. In addition, peroxynitrite formation was detected in the mitochondria of postischemic myocyte. The SQR isolated from myocardium was further used to study the interaction of NO with SQR
in vitro
. Under the conditions of enzyme turnover, SQR obtained after NO (25 fold excess) pretreatment gained catalytic function to generate hydroxyl radical detected by EPR spin trapping using DEPMPO. In the presence of catalase and iron chelator, the EPR signal of associated with DEPMPO/
·
OH was completely abolished, suggesting the involvement of iron-H
2
O
2
dependent Fenton reaction. Direct EPR measurement at 77 °K indicated the formation of nonheme iron-NO complex, implying electron leakage to O
2
had occurred at the ISP of SQR and excess NO predisposed SQR to pro-oxidant activity.
Abstract 1229: Molecular Mechanisms of Angiotensin II-Mediated Mitochondrial Dysfunction: Linking Mitochondrial Oxidative Damage and Vascular Endothelial Dysfunction
Authors: Doughan Abdulrahman , Harrison David , Dikalov Sergey
Published: July 2021

Mitochondrial (mito) dysfunction is a prominent feature of most cardiovascular diseases including hypertension, diabetes, and heart failure. Angiotensin II (Ang II) is an important stimulus for atherogenesis and hypertension; however, its effects on mito function remain unknown. We hypothesized that Ang II may induce mito oxidative damage that in turn may decrease endothelial nitric oxide (NO
•
) and promote vascular oxidative stress. Mitochondria isolated from control and Ang II (200 nM, 4 hours) treated bovine aortic endothelial cells II were investigated. Electron Spin Resonance was used to study the effect of Ang II on mito ROS production, mito respiration, glutathione content and endothelial NO
•
. Mitochondrial membrane potential was examined using fluorescence microscopy. Endothelial O
2−
•
formation was quantified using an HPLC-based dihydroethidium assay. Mitochondrial H
2
O
2
and endothelial O
2−
•
production increased significantly after treatment of endothelial cells with Ang II. This increase was blocked by preincubation of intact cells with apocynin (NADPH oxidase inhibitor), uric acid (scavenger of peroxynitrite), chelerythrine (PKC inhibitor), N (G)-nitro-L-arginine methyl ester (L-NAME) (nitric oxide synthase inhibitor), 5-hydroxydecanoate (5-HD, mitochondrial ATP-sensitive potassium channels inhibitor) or glibenclamide. Depletion of p22phox subunit of NADPH oxidase with siRNA led to significant (P<0.001) reduction in Ang II-mediated mito ROS production. Angiotensin II decreased mito glutathione content, and mito respiratory control ratio. These responses were attenuated by apocynin, 5-HD and glibenclamide. In addition, 5-HD prevented the Ang II-induced decrease in endothelial NO
•
(73% in Ang II-treated cells and 94% in Ang II plus 5-HD vs. 100% in control; P < 0.05) and mito membrane potential. These events were not associated with activation of caspase-3. Angiotensin II induces mito dysfunction via a pathway that depends on PKC, the NADPH oxidase and formation of peroxynitrite. Furthermore, mito dysfunction in response to Ang II modulates endothelial NO
•
and O
2−
•
generation, which may in turn have ramifications for the development of endothelial dysfunction.
Abstract 229: The Novel Angiogenic Cytokine Secretoneurin promotes Angiogenesis, Arteriogenesis and Vasculogenesis in the Hind-Limb Ischemia Model
Authors: Schgoer Wilfried , Egger Margot , Peer Arno , Jeschke Johannes , Tancevski Ivan , Ritsch Andreas , Piza-Katzer Hildegunde , Patsch Josef R , Schratzberger Peter , Kirchmair Rudolf
Published: July 2021

Introduction

- Secretoneurin (SN) represents a sensory, inflammatory neuropeptide which was recently demonstrated to act as an angiogenic and vasculogenic cytokine in vitro and in vivo. The present study was conducted to test the hypothesis that SN may be implicated in reparative angiogenesis. Furthermore, we challenged the healing potential of SN applied as a newly generated SN gene therapy vector in the setting of limb ischemia.

Methods and Results

- We cloned the human SN coding sequence into the pAAV plasmid containing a cytomegalovirus enhancer/promoter sequence. Bioactivity of recombinant SN was shown by proliferative and chemotactic activity on endothelial cells in vitro. Unilateral limb ischemia was induced in C57/bl mice by femoral artery resection. By Real Time PCR, Western Blotting, SN-specific RIA and Immunhistochemistry, we documented that SN is up-regulated in ischemic muscles. Next, we tested whether SN gene therapy may exert curative effects in this ischemia model. Injection of the SN plasmid into ischemic adductor muscles increased capillary (0.67 vs. 0.35, n = 24, p = 0.02) and arteriole (0.16 vs. 0.8, n = 24, p = 0.04) density, reduced endothelial cell apoptosis, and accelerated perfusion recovery as shown by Laser Doppler Perfusion Index (LDPI ratio ischemic/control leg after 28 days of ischemia 1.1 vs. 0.7, n = 24, p < 0.01) in comparson to pAAV-GFP (green-fluorescence protein) treated mice. Furthermore, SN gene therapy significantly reduced toe necrosis of ischemic limbs compared to control animals (26% vs. 50%, n = 24, p < 0.05). In bone marrow transplantation models, increased vascularity of ischemic hind-limbs after SN gene therapy was shown to be mediated, at least in part, by enhanced recruitment of bone marrow-derived endothelial progenitor cells.

Conclusions

-These results suggest that the novel angiogenic cytokine Secretoneurin is up-regulated by ischemia in skeletal muscle cells. Furthermore, results from gene therapy in this ischemia model suggest that Secretoneurin represent a promising new substance for therapeutic angiogenesis.
Abstract 1240: Sodium Channel Splice Variants Seen in Human Heart Failure Result in Dominant Negative Suppression of the Wild-Type Current
Authors: Shang Lijuan L , Sanyal Shamarendra , Dudley Samuel C
Published: July 2021

Patients with heart failure have increased risk of death and reduced Na
+
channels. We have identified a possible mechanism whereby Na
+
channels are decreased in human heart failure. We have observed that heart failure is associated with increases of 14.2 fold and 3.8 fold in two (C and D) of three cardiac Na
+
channel mRNA splice variants. These variants encode for channels that are truncated before the domain IV pore-forming loop and are predicted to be non-functional. We tested this prediction by expressing the truncated forms in HEK cells. The truncated SCN5A cDNAs labeled either with IRES (internal ribosome entry site)-mediated GFP or fused GFP were transitively transfected into HEK cells or a HEK cell line stably expressing the native human SCN5A. HEK cell lines stably expressing the truncation variants were created by selection for three weeks with geneticin after transfection. Neither of the two truncations generated current when transfected alone. When transfected in a cell line stably expressing wild-type Na
+
channels, the presence of the truncations resulted in 54.6% (±8.5, p<0.01, N=14) and 56.0% (±8.9, p<0.01, N=10) reductions in peak current with the C and D variants respectively when compared to the wild-type alone. The reduction of wild-type current in the presence of the truncations showed variant dose-dependency when varying the amount of vector encoding for the splice variants. Laser fluorescent scanning for N-terminal GFP labeled Na
+
channels confirmed a reduced number of wild-type channels in the membrane in the presence of mRNA for the two truncations. In conclusion, truncated Na
+
channel mRNA splice variants are increased in human heart failure. These truncations have a dominant negative effect on the wild-type allele. This may help explain the reduced Na
+
current and increased arrhythmic risk in heart failure.
Abstract 1257: Autophagy Plays Both Protective and Detrimental Roles in Post Myocardial Infarction Hearts
Authors: Takagi Hiromitsu , Kyoi Shiori , Liu Huasheng , Hsu Chiao-Po , Gao Shumin , Yan Lin , Levine Beth , Vatner Dorothy E , Vatner Stephen F , Sadoshima Junichi
Published: July 2021

Autophagy is an important mechanism of protein degradation involving formation of double membrane vesicles termed autophagosomes and lysosomal degradation. Autophagy is activated in the heart both under basal conditions and in response to stress. During acute ischemia, autophagy is protective, but during reperfusion, it may promote cell death, suggesting that the function of autophagy is context-dependent. In order to elucidate the role of autophagy in other pathological conditions in the heart, we examined whether autophagy is activated after myocardial infarction (MI) in vivo. On Day 2, autophagosome formation, as evaluated by GFP-LC3 dots, was significantly increased in the border zone compared with the remote area (25 vs 2 dots/field, p<0.05, n=5). The functional significance of autophagy was evaluated using
beclin 1
+/− mice, in which autophagy is significantly attenuated due to reduced expression of Beclin 1, a critical component of the autophagic machinery. Control (WT) and
beclin 1
+/− mice (3 months old) were subjected to permanent coronary artery ligation. Kaplan Meier survival analyses indicated that the rate of mortality was significantly greater in WT (4 out of 13) than in
beclin 1
+/− (0 out of 11) mice (p<0.05) at early stages (0 –11 days), whereas there was no significant difference in mortality at later stages (11–29 days), suggesting that inhibition of autophagy is protective during early stages of MI. After 4 weeks,
beclin 1
+/− mice exhibited larger LV myocyte cross sectional areas in remote areas (254 vs 220 μm
2
, p<0.005) with greater posterior wall thickness (1.24 vs 1.11 mm, p<0.05) and increased levels of cardiac myocyte apoptosis in the scar area (7.6 vs 2.8/mm
2
) and the adjacent area (1.2 vs 0.6/mm
2
) compared with WT mice. These results suggest that suppression of autophagy at late stages of MI promotes apoptosis and hypertrophy, and may be detrimental. In summary, autophagy is induced in the heart after MI and exhibits time-dependent functions. Although autophagy is detrimental at early (within 11 days) stages of MI, it may inhibit cardiac remodeling, and, thus, is protective during chronic stages.
Abstract 232: Vascular Dysfunction Induced By High Pressure Levels Is Mediated By Rac-1
Authors: Vecchione Carmine , Gentile Maria Teresa , Di Pardo Alba , Bettarini Umberto , Lembo Giuseppe
Published: July 2021

Oxidative stress plays a crucial role in the vascular dysfunction present in hypertension. The molecular mechanism involved are not completely elucidated. One of the principal source of oxidative stress at vascular level is the NADPH oxidase enzyme which needs Rac-1 to be activated. Aim of our study was to evaluate the role of Rac-1 in vascular dysfunction induced by high pressure levels. To test this hypothesis, carotid of mice were placed on pressure system and subjected to increasing levels of pressure (100 and 180 mmHg). In particular, after a period of perfusion (1h) with 100 mmHg vessels were subjected to 180 mmHg for three hours. At the end of perfusion period the vascular reactivity was tested. Our results demonstrate that vessels perfused with 180mmHg (n=8) showed an impaired vasorelaxation to acetylcholine as compared to vessels perfused with 100 mmHg (n=7) (% max vasorelaxation: 47±3 vs 88±7, p<0.01). Vasorelaxation to nitroglycerin was comparable between vessels perfused respectively at 100 and 180 mmHg. Some vessels were used to evaluate oxidative stress (n=5) by dihydroethidium and Rac-1 activity (n=8) by Rac-1/PAK complex. Our results demonstrate that high pressure levels induced an increased oxidative stress and more interesting an increased Rac-1 activity as compared to vessels perfused with 100 mmHg. Thus, to clarify the role of Rac-1 in high pressure evoked oxidative stress and vascular dysfunction, some vessels (n=6) were infected with adenoviral vector containing Rac-1 dominant negative. In this experimental setting vessels perfused at 180 mmHg showed an oxidative stress and endothelial vasorelaxation comparable to that observed at 100 mmHg (% max vasorelaxation: 79±6 vs 85±7, n.s.), thus demonstrating a key role of Rac-1 in oxidative stress and endothelial dysfunction evoked by high pressure levels. Thus, Rac-1 could represent the target of novel therapeutic strategies for reducing vascular damage in hypertension.
Abstract 1337: Attenuation of the Leptin-Induced Hypertrophy by NF-kB Inhibition Requires Activation of AMPK in Neonatal Cardiomyocytes
Authors: Rajapurohitam Venkatesh , Javadov Sabzali , Purdham Daniel M , Karmazyn Morris
Published: July 2021

Introduction:
Leptin, a 16 kDa product of the obesity gene, has been shown to be elevated in patients with heart failure. Recently we demonstrated that leptin induces hypertrophy in cultured cardiomyocytes, however the molecular mechanisms underlying the hypertrophic effect of leptin remain to be elucidated. In the present study we investigated whether nuclear factor-kB (NF-kB) and adenosine monophosphate-activated kinase (AMPK) are key mediators in leptin-induced cardiac hypertrophy.

Methods and Results:
Leptin (3.1 nM) treatment for 24 hrs increased cell surface area by 40% (P<0.002) as measured by Sigma scan software. The increase in cell surface area was completely blocked by NF-kB activation inhibitor (1μM) and AICAR (1 mM), the AMPK activator (P<0.001). Incubation with leptin for 24 hrs had no effect on PRKA-α (α-subunit of AMPK) but upregulated the PRKAβ2 (β-subunit of AMPK) gene by 85% (P<0.04) as determined by real-time PCR. In contrast, AICAR treatment increased the PRKA-α by 256% (P<0.02) but not the PRKA-β gene. The leptin-dependent 90% increase in α-skeletal actin, a hypertrophic marker gene was inhibited by AICAR (P<0.01). Western blot analysis revealed that leptin treatment for 5 min increased the phosphorylation of IkBs, a group of cytoplasmic NF-kB inhibitors by 51% (P<0.03). Phosphorylation triggers their degradation and allows NF-kB to translocate into the nucleus. Leptin-induced IkB-α phosphorylation was associated with significant activation of NF-kB p65 by phosphorylation at Ser536 which is important for translocation into the nucleus. Inhibition of NF-kB induced upregulation of P-Thr172-AMPK but not P-Ser485 AMPK levels. Cglitazone (5 μM), a potent and selective PPAR-γ ligand, or AICAR treatment for 30 min significantly upregulated the expression of P-Thr172-AMPK while leptin had no effect. In addition, AICAR treatment for 24 hrs increased the expression of PPAR-γ by 450% (P<0.006), FATCD36 by 35% (P<0.03) and GLUT1 by 220% (P<0.03).

Conclusion
: Attenuation of leptin-induced hypertrophy by NF-kB inhibition is mediated by AMPK activation. The anti-hypertrophic effect of AMPK activation is mediated through upregulation of PPAR-γ, FATCD36 and GLUT1 genes expression responsible for maintaining energy homeostasis.
Abstract 1352: Gender Differences In Cardiac Repolarization In Transgenic Long QT Syndrome 2 (LQT2) Rabbits
Authors: Odening Katja E , Hajjiri Mohammad , Brunner Michael , Lorvidhaya Peem , Schofield Lorraine , Peng Xuwen , Zehender Manfred , Kirk Malcolm , Koren Gideon
Published: July 2021

Introduction:
Adult women with LQT2 are at higher risk for clinical events and sudden cardiac death (SCD) than men. We have created transgenic rabbits over-expressing pore mutants of the human KvLQT1 (LQT1) and HERG (LQT2) selectively in the heart. We report the gender differences in cardiac repolarization, incidence of polymorphic VT and SCD in these cohorts.

Methods:
Adult female and male LQT1, LQT2, and littermate controls (ages 5 to 33 mo were similar in f/m, LQT2 were younger due to higher mortality) underwent telemetric ECG monitoring, surface ECG and
in vivo
electrophysiological studies under general anaesthesia with isoflurane (2–5%).

Results:
Monitoring data showed a steeper QT/RR slope in female (0.745 ± 0.05, n=4) than in male (0.513 ± 0.06, n=9; p<0.05) LQT2 rabbits. No significant gender differences were observed in QT/RR slopes in either LQT1 or WT rabbits. QT-, QTpeak-index and Tp-e were significantly longer in female than in male LQT2 rabbits (females, n=6: QT: 129.2% ± 3.9; QTp: 105.9% ± 2.1; Tp-e: 42.9ms ± 4.1 vs. males, n=8: QT: 117.5% ± 4.3; p<0.05; QTp: 93.5% ± 5.6, p<0.05; Tp-e: 29.5ms ± 2.9, p<0.02). EP studies revealed significantly longer atrial (AERP) and ventricular (VERP) refractory periods in LQT2 females compared to males (females, n=4: AERP: 143.3 ± 5.8 ms; VERP: 212.5 ± 22.2 ms vs. males, n=8: AERP: 102.5 ± 7.7 ms, p<0.01; VERP: 178.1 ± 7.8 ms, p<0.05). AERP and VERP were significantly longer in LQT2 females than in LQT1 females (LQT1 females, n=7: AERP: 101.4 ± 7.7 ms, p<0.01, VERP: 156.9 ± 6.2, p<0.001), whereas in male LQT rabbits this genotype difference was only found in VERP but not in AERP. Survival was significantly shorter in female LQT2 rabbits compared to LQT1 or WT controls, with 4 sudden deaths among 10 LQT2, 1 among 19 WT and no SCD in 13 LQT1 females (p<0.02). No gender difference was observed in mortality. All cases of SCD occurred after sexual maturation and two LQT2 females died during lactation. Monitoring revealed the cause of SCD was polymorphic VT.

Conclusions:
Monitoring of LQT rabbits reveal gender differences in LQT2 rabbits. QT/RR slope is steeper in female than in male adult LQT2 rabbits. AERP and VERP are longer in LQT2 females than in males. Finally, in LQT2 females, SCD was associated with lactation, but not pregnancy.