bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1747: Therapeutic Potential of Mesenchymal Stem Cells Derived from Three Different Layers of Human Fetal Membrane in Hindlimb Ischemia Model
Authors: Harada Kazuhiko , Ohnishi Shunsuke , Yamahara Kenichi , Sada Masaharu , Ishikane Shin , Mishima Kenichi , Iwasaki Katsunori , Fujiwara Michihiro , Kangawa Kenji , Kitamura Soichiro , Nagaya Noritoshi , Ikeda Tomoaki
Published: July 2021

The fetal membrane (FM), which is reported to contain mesenchymal stam cells (MSC), has been regarded as an ideal source for regenerative medicine, although it is normally discarded after birth. Little information is available regarding the biological difference and therapeutic potential of MSC obtained from different layers of FM including amnion and chorion. We assessed the hypothesis that human FM-derived MSC (FM-MSC) might have therapeutic effects on hindlimb ischemia. FM was obtained following cesarean section and vaginal delivery of healthy donor mothers after obtaining informed consent. We mechanically and enzymatically separated human FM into three layers; amnion and inner and outer layers of chorion, and isolated dish-adherent cells from each layer of FM. These FM-derived cells showed similar surface antigen expression and pluripotency compared to bone marrow (BM)-derived MSC. Because cultured inner chorion-derived MSC (IC-MSC) secreted large amounts of growth factors including IGF-1(13.6 ± 0.08 ng/106cells/24hrs, p < 0.001 vs. amnion or outer chorion-derived MSC (AM-MSC or OC-MSC)) and HGF (7.94 ± 0.67 ng/106cells/24hrs, p < 0.001 vs. AM-MSC or OC-MSC) compared to AM-MSC or OC-MSC, we chose to use IC-MSC for further in vivo experiment. Hindlimb ischemia was induced in 6-week-old male NOD/SCID mice, which were divided into two groups:

IC-MSC transplanted group (1 × 10
6
cells/50 μl PBS, n = 9), and

control group (50 μl PBS, n = 10).

IC-MSC was injected into ischemic hindlimb muscle at five different points. Laser Doppler perfusion analyses showed that the ischemic/nonischemic blood flow ratio was significantly increased in IC-MSC group compared to control group (0.468 ± 0.093 vs. 0.227 ± 0.045 respectively, p < 0.01). Transplantation of FM-MSC showed significant neovascularization in murine hindlimb ischemia model. FM-MSC might have a potential to be an ideal source for treatment of ischemic cardiovascular disease.
Abstract 1765: Immunosuppressive Potential of Ibp - A Novel Inhibitor of the Calcineurin/NF-AT Cascade
Authors: Gebhardt Claudia , Burkard Natalie , Czolbe Martin , Williams Tatjana , Panther Franziska , Ritter Oliver
Published: July 2021

Background:
The calcineurin (Cn)/NF-AT signaling cascade plays a crucial role during T-cell activation and development of myocardial hypertrophy. We previously demonstrated that, in addition to NF-AT, Cn is translocated to the nucleus. We also developed a synthetic peptide (IBP) which inhibited the nuclear import of Cn without affecting calcineurin phosphatase activity. The antihypertrophic effect of IBP on the rat myocardium was shown
in vivo
and
in vitro
. Here we extended our studies on a potential use of IBP in the prevention of transplant rejection.

Methods and results:
Coimmunoprecipitation experiments revealed that the synthetic IBP peptide, which is identical to the nuclear localization sequence of calcineurin, disrupts importin/calcineurin interaction. We further demonstrated that inhibition of the importin/calcineurin interaction by the small competitive peptide (IBP) is sufficient to inactivate the calcineurin/NF-AT signaling cascade. Inhibitory effects of IBP on T-cell activation were analyzed by [H
3
]-thymidine incorporation (7135 ± 2503 vs. 2957 ± 1161 [%]) in activated T-cells
in vitro
(dentritic cells were used as antigen stimulation). To verify immunosuppressive effect of IBP
in vitro
, heterotopic heart transplantations were performed on rats. Following transplantation rats were treated with IBP (i.p. 130mg/kg/d) or peanut oil as control (i.p. 1 ml peanut oil/d) for 14 days. Time until transplant rejection was significantly prolonged in IBP treated animals compared to control group (15.2 ± 1.5 day vs. 4 ± 2.8 day; n = 8; p < 0.05). Immunohistochemical studies from transplanted hearts demonstrated that the synthetic calcineurin inhibitory peptide IBP prevented calcineurin nuclear translocation in infiltrating T cells in the donor hearts.

Conclusions:
The synthetic inhibitory peptide IBP disrupts the calcineurin/NF-AT cascade by inhibition of Cn nuclear import. This suppresses T-cell activation
in vitro
and
in vivo,
thereby revealing a potential use of IBP as novel agent to protect donor organs from rejection.
Abstract 1769: Caveolin-1 Regulates TGF-Beta Signaling in Cardiac Remodeling
Authors: Miyasato Shelley , Bollt Oana , Pike Joyce , Hashimoto Ann , Shohet Ralph V , Le Saux Claude Jourdan
Published: July 2021

In cardiac fibrosis, fibrous tissue replaces healthy contractile tissue. The regulation of these processes is controlled in large part by transforming growth factor-β (TGF-β). Caveolin-1 (cav1) regulates TGF-β signaling by either sequestering the TGF-β receptor complex or enhancing its degradation. Thus, cav1 may prevent TGF-β directed fibrosis. To investigate the role of cav1 in cardiac remodeling, we performed left ventricular cryoinjury in Cav1-deficient (Cav1−/−) mice and wild-type controls. Ventricular function was followed by echocardiography, and 3, 14, and 30 days after surgery, cardiac RNA and protein were analyzed for inflammatory responses, connective tissue and TGF-β signaling related proteins. Cryoinjured WT presented reduced cav1 expression. Concurrently, evidence of activation of TGF-β signaling was measured as shown by increase of Smad2 phosphorylation. Moreover Cav1−/− cryoinjured hearts had enhanced Smad2 phosphorylation. Collagen gene expression was transiently upregulated in cryoinjured WT mice 3 days post surgery (2.5-fold) and this elevation persisted in Cav1−/− hearts (3.5-fold at 14 days). The level of collagenases (mmp-8 and -13) expression was dramatically increased in the 3-day cryoinjured WT but not in Cav1−/− mice. As a result, augmented collagen deposition, resulting from increased collagen expression and reduced degradation by collagenases, was observed by Masson’s trichrome and picrosirius staining in injured Cav1−/− hearts. WT mice had a transient decline in fractional shortening (FS) but function returned to baseline by 30 days post-injury. In contrast, cryoinjured Cav1−/− mice had a significant lower % FS after 30 days compared to baseline or to cryoinjured WT (67.4 ± 9.6, 76 ± 11, 76.9 ± 5.5, respectively). Moreover Cav1−/− mice presented an altered inflammatory response following cryoinjury. Reduced macrophage infiltrates and IL-6 level of expression were also measured in cryoinjured Cav1−/− mice. These data indicate that in absence of caveolae, TGF-β signaling is enhanced, and this leads to a disordered inflammatory response and suboptimal cardiac remodeling that may impair left ventricular function.
Abstract 1778: Estrogen Protects Cardiomyocytes from Apoptosis by Suppressing p38alpha-mediated p53 activation and by Downregulating a Negative Feedback from p53 on p38beta
Authors: Liu Han , Pedram Ali , Levin Ellis R , Kim Jin K
Published: July 2021

To investigate the molecular mechanisms of estrogen-mediated protection of cardiomyocytes in hypoxic stress. Estrogen is anti-apoptotic and promotes survival of cardiomyocytes under ischemia-related stress. We have previously shown that estrogen protects cardiomyocytes exposed to simulated ischemia-reperfusion (I/R) by differentially regulating pro-apoptotic p38alpha and pro-survival p38beta. However, little is known about how E2 modulation of the kinases alters the apoptotic signaling in cardiomyocytes. An attractive downstream target is p53, a well-known mediator of apoptosis and a substrate of p38alpha. We propose that the cytoprotective actions of estrogen involve regulation of p53 via p38. Cultured neonatal rat cardiomyocytes underwent hypoxia followed by reoxygenation (H/R) to simulate I/R. The extent of apoptosis was determined by examining annexin V-positive cells under fluorescent microscopy. The whole cell lysate was collected after H/R, and the protein of interest was immunoprecipitated and immunoblotted. The p38 activity was determined by immunoprecipitating the protein and performing in-vitro kinase assays on the substrate, ATF2. In our model, p53 played a significant role in H/R-induced myocyte death: Inhibition of p53 (by pifithrin-alpha or by siRNA) significantly reduced the number of apoptotic cells by half. Phosphorylation of p53 (p-p53) at serine 15 increased after H/R, while E2 effectively inhibited this form of p53 activation. Use of a specific agonist for each estrogen receptor isoform (ERalpha or ERbeta) demonstrated that both isoforms participate in inhibition of p-p53. E2 attenuated transcription of apoptosis-specific targets of p53,
puma
and
noxa
, as determined by RT PCR. In addition, inhibition of p53 augmented the p38beta activity, suggesting a baseline repression of this pro-survival kinase by p53 that may be reversed by E2. Our results demonstrate that during H/R stress, cardiomyocytes undergo p53-dependent apoptosis following phosphorylation of p53 by p38alpha. E2 protects cardiomyocytes by inhibiting p38alpha-p53 signaling, leading to pro-survival p38beta activation.
Abstract 1780: Caspase-independent Apoptosis Is Activated In The Late Stage Of Heart Failure In Mice Overexpressing Crma
In Vivo
Authors: Bae Soochan , Siu Parco , Choudhury Sangita , Ke Qingen , Koh Young Y , Kang Peter M
Published: July 2021

Caspase inhibition is thought to be an effective strategy for inhibiting apoptosis in heart. Recent data, however, suggest that caspase-independent apoptosis might be activated when caspases are inhibited. Here, we examined whether caspase-independent apoptosis involving apoptosis inducing factor (AIF) is important in the development of heart failure in the setting of caspase inhibition
in vivo.

Method:

We created transgenic mice with cardiac specific overexpression of cytokine response modifier A (CrmA), a known inhibitor of caspases, using αMHC promoter (CrmA Tg mice). A single dose of doxorubicin (DOX; 20 mg/kg) was injected
i.p.
into male mice to generate acute cardiomyopathy. A quantitative analysis of cardiac apoptosis was performed using TUNEL assay.

Results:

Caspase activity was completely inhibited in CrmA Tg mice hearts 6 days after DOX injection, whereas the WT hearts showed significant caspase activation. We also observed significantly improved survival in CrmA Tg compared to WT mice (81% vs 38%, p<0.05) 6 days after DOX injection. However, at 12 days, CrmA Tg and WT mice had a similar rate of cardiac apoptosis, and the survival benefit did not persist. Further analysis revealed that CrmA Tg but not WT mice hearts were characterized by a significant release of AIF from the mitochondria at 12 days. This evidence of AIF-induced apoptosis suggests a late activation of AIF in the setting of caspase inhibition. CrmA Tg mice were also pre-treated with 4-AN (4-amino-1,8-napthalimide, 3 mg/kg), a potent inhibitor of PARP, and examined 12 days after DOX injection. PARP is an important regulator of AIF-induced apoptosis, and PARP inhibition significantly improved DOX-impaired cardiac function (+91%, p<0.01) and survival compared to CrmA Tg mice without 4-AN (93% vs. 13%, p<0.05). In addition 4-AN pretreatment resulted in a significant inhibition of PARP activity, blockade of AIF released from mitochondria, and less apoptosis compared to CrmA Tg without 4-AN.

Conclusion:

In the setting of caspase inhibition, apoptosis can be induced through caspase-independent mechanisms, predominately via AIF, in a model of heart failure
in vivo
. The concurrent inhibition of AIF activation provides a more effective and complete inhibition of apoptosis.
Abstract 1830: Protein Quality Control Inadequacy Activates NFAT Signaling in Cardiomyocytes
Authors: Kumarapeli Asangi R , Tang Mingxin , Zheng Hanqiao , Horak Kathleen M , Li Manixiang , Molkentin Jeffery D , Wang Xuejun
Published: July 2021

Protein Quality Control (PQC) assists proper (re)folding of nascent polypeptides, keeps normal mature proteins from denaturing or misfolding, and timely removes terminally misfolded and irreparable damaged proteins in the cell. PQC is enforced by collaboration between chaperones and targeted proteolysis. The latter is primarily mediated by the ubiquitin-proteasome system (UPS) while a large family of chaperones are heat shock proteins (HSPs), including small HSPs.
α
B-crystallin (CryAB) is the most abundant small HSP in cardiomyocytes. Signs of severe PQC inadequacy, including abnormal protein aggregation, reduced CryAB protein, and functional insufficiency of the proteasome, were all observed in failing human hearts, supporting a novel hypothesis that PQC inadequacy contributes to the genesis of congestive heart failure (CHF). It is well established that the calcineurin-NFAT pathway regulates the expression of a significant subset of genes associated with cardiac remodeling and failure. We present here several lines of new genetic and pharmacological evidence that PQC inadequacy suffices to activate NFAT signaling in both cultured neonatal rat cardiomyocytes (NRCMs) and intact mouse hearts. NFAT translocation from cytoplasm to the nucleus is critical to its activation. We observed in cultured NRCMs that CryAB overexpression (o/e) significantly reduced adrenergic agonists-induced NFAT nuclear translocation and myocyte hypertrophy while pharmacological inhibition of the proteasome induced significant calcineurin-dependent NFAT nuclear translocation. Using a NFAT-luciferase reporter mouse model, we detected that NFAT transactivation was increased by a factor of 3 in a mouse cardiomyopathy model that was created by transgenic o/e of a misfolded desmin protein and displayed severe PQC inadequacy. Similarly, we found that NFAT activities in the heart of CryAB/HSPB2 null mice were significantly increased. Consistently, these mice developed cardiac hypertrophy and malfunction at the baseline and displayed exacerbated cardiac hypertrophy and malfunction upon transverse aortic constriction (TAC). Finally, cardiac-specific CryAB o/e significantly attenuated NFAT activation and cardiac hypertrophic responses induced by TAC.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 1841: Stromal Cell Derived Factor-1 Increases Capillary Density But Does Not Contribute To Cardiomyocyte Replacement After Experimental Myocardial Infarction
Authors: Loffredo Francesco , Segers Vincent F , MacGillivray Catherine , Lee Richard T
Published: July 2021

Background. The mechanisms by which regenerative therapies improve cardiac function are incompletely defined. Numerous laboratories have demonstrated that the stem cell chemoattractant Stromal cell derived factor-1 (SDF-1) improves cardiac function after myocardial infarction (MI). This study used a genetic fate-mapping approach to ask the question: Is the beneficial effect of SDF-1 delivery due to cardiomyocyte regeneration, increased capillary density, or both?

Methods. We used a genetic fate-mapping system that allows “pulse-chase” studies of cardiomyocyte turnover in mice. We crossed an inducible cardiomyocyte-specific Mer-Cre-Mer transgenic mouse with Z/EG reporter mice. Using a high-efficiency 4-OH-tamoxifen protocol, we then induced Cre recombination and expression of GFP in cardiomyocytes only. SDF-1(S4V), a protease-resistant form of SDF-1, was delivered with nanofibers after coronary ligation.

Results. The percentage of pulse-labeled GFP+ cardiomyocytes was 81±4% (n=6) in sham-operated mice, as anticipated with this system. GFP+ cardiomyocytes were 60±5% and 69±4% (n=11) respectively at MI border and MI remote areas of the control nanofiber group, consistent with our previous study and suggesting a significant stem/precursor cell contribution to cardiomyocyte replacement after injury (P<0.0001). GFP+ cardiomyocytes were 65±9% and 76±6% (n=10) respectively at MI border and MI remote areas of the SDF-1(S4V)-RAD group (p=ns vs nanofiber control group), indicating no significantly increase in cardiomyocyte refreshment attributable to SDF-1. However, capillary density increased from 204.7 ± 10.1/mm2 in the nanofiber control group to 308.9 ± 21.9/mm2 in SDF-1(S4V)-RAD + nanofiber group (p =0.0003).

Conclusion. Using genetic “pulse-chase” fate mapping, these data indicate that the stem cell chemoattractant SDF-1 does not induce measurable adult mammalian cardiomyocyte replacement by stem/precursor cells following injury. However, SDF-1 significantly increases myocardial capillary density, suggesting that increased angiogenesis but not cardiogenesis is responsible for the beneficial effects of SDF-1 on cardiac function.
Abstract 1875: L-type Calcium Channel C-terminus Suppresses L-type Calcium Channel Transcription in Cardiomyocytes
Authors: Schroder Elizabeth A , Byse Miranda J , Satin Jonathan
Published: July 2021

Calcium homeostasis is critical for cardiac myocyte function and must be tightly regulated. In this study we test the hypothesis that a mobile carboxyl-terminal cleavage product (CCt) of the cardiac L-type calcium channel (Ca
V
1.2) auto-regulates expression. Our ongoing
in vivo
studies showed that persistent Ca
V
1.2 block induced an up-regulation of Ca
V
1.2 mRNA, protein and I
Ca,L
. In the present study we test the hypothesis that CCt contributes to transcriptional signaling associated with cellular hypertrophy. We created eGFP-CCt fusion constructs to explore CCt regulation of the Ca
V
1.2 promoter, and to report sub-cellular localization of CCt in cardiac myocytes. Developing cardiac myocytes were cultured in 10% FBS. Over-expression of native, full-length CCt causes a 34 ± 8% decrease of Ca
V
1.2 promoter activity, and truncated CCt caused 80 ± 3% decrease (n = 12). Wild-type CCt distributes into cytosol and nucleus, with a greater presence in the nucleus than cytosol. A deletion mutant of CCt was also tested. Truncated CCt has a greater relative affinity for the nucleus than wild-type CCt. This is consistent with increased suppression of Ca
V
1.2 promoter activity by truncated CCt. Chromatin immunoprecipitation (ChIP) assay showed that CCt interacts with the Ca
V
1.2 promoter in adult ventricular cardiac myocytes at promoter modules containing NKX2.5/MEF2, C/EBp, and a cis regulatory module identified
in silico
. We next explored whether fetal cardiac myocyte Ca
V
1.2 was regulated by serum
in vitro
. We tested ANF promoter activity as a positive control, and measured serum-response, versus serum-withdrawal on Ca
V
1.2 promoter, mRNA, protein, and L-type current (I
Ca,L
) from fetal mouse ventricular myocytes. Serum increased ANF promoter activity and cell size. Serum withdrawal increased Ca
V
1.2 promoter activity, Ca
V
1.2 protein, and I
Ca,L
. Moreover, serum withdrawal decreased the relative nuclear localization of CCt. A combination of promoter deletion mutant analysis, and the response of promoter mutants to serum withdrawal support the conclusion that CCt, a proteolytic fragment of Ca
V
1.2 auto-regulates Ca
V
1.2 expression in cardiac myocytes. These data support the novel mechanism that a mobile segment of Ca
V
1.2 links Ca handling to nuclear signaling.
Abstract 1923: NPC1L1 Haplotype (1735C-25342A-27677T) is Related with Cholesterol Absorption from Intestine
Authors: Maeda Tomomi , Kusano Jun , Fujita Mineko , Yasuda Daijiro , Takeoka Yoko , Yoshimura Nakayuki , Ishikawa Toshio , Honda Akira , Matsuzaki Yasushi , Kinoshita Makoto , Teramoto Tamio
Published: July 2021

Background:
NPC1L1 is the intestinal sterol transporter that is a target molecule of ezetimibe. It has been reported that the LDL response to ezetimibe treatment is significantly different among NPC1L1 haplotypes. We hypothesized that common variants in NPC1L1 would affect cholesterol absorption from intestine and cholesterol synthesis in liver.

Methods:
Genomic DNAs were extracted from 140 healthy Japanese volunteers with informed consent. Three single nucleotide polymorphisms (SNPs) (1735C>G, 25342A>C, and 27677T>C) were determined by PCR-RFLP method. Plasma concentration of lathosterol (a marker of cholesterol synthesis), and sitosterol, campesterol (markers of cholesterol absorption) were determined by LC-MS/MS for 79 subjects whose plasma was available.

Results:
NPC1L1 genotype frequencies of these SNPs of Japanese were as follows; 1735C/C:0.35, 1735C/G:0.464, 1735G/G:0.186; 25342A/A:0.969, 25342A/C:0.031, 25342C/C:0; 27677T/T:0.969, 27677T/ C:0.031, 27677 C/C:0. There were no significant differences in serum lipids concentration among three groups of 1735C/C, C/G, and G/G. Major haplotype of NPC1L1 in Japanese is 1735C-25342A-27677T, whose frequency is 0.573. Plasma concentration of campesterol of those who have at least one haplotype of 1735C-25342A-27677T is lower than those who dose not have 1735C-25342A-27677T, significantly (4.67±2.15 vs. 6.08±4.08 (p<0.05)).

Conclusion:
NPC1L1 genotype frequencies are very different in Japanese populations from those reported in Caucasians. Those who have NPC1L1 haplotype 1735C-25342A-27677T had lower plasma campesterol concentration, which may indicate that cholesterol absorption is reduced in individuals with NPC1L1 haplotype 1735C-25342A-27677T. This may be the reason why ezetimibe is effective to lower LDL-cholesterol in individuals who lack this haplotype.
Abstract 3387: Hypoxia-induced Vascular Repair by Progenitor Cells Requires the Nadph Oxidase Nox2
Authors: Schröder Katrin , Kohen Andreas , Aicher Alexandra , Brandes Ralf P
Published: July 2021

Endothelial progenitor cells (EPC) promote vascular repair but the impact of systemic hypoxia on EPC function is unclear. Hypoxia is a strong stimulus for angiogenesis and affects reactive-oxygen species (ROS) production and signalling. We studied the role of hypoxia for EPC mobilization and vascular repair and the contribution of the NADPH oxidase Nox2, the most important source of ROS in endothelial cells, in this process. Murine EPCs were defined as linage negative and sca-1/flk-1 positive and measured by FACS from whole blood. Vascular repair was induced by the carotid artery wire injury model. Hypoxia (10%, 96hours) induced mobilisation of EPC and improved vascular repair after wire injury in wild-type mice but not in NADPH oxidase knockout mice (Nox2y/−). Hypoxia induces the formation of erythropoietin (EPO), which might contribute to EPC mobilization. EPO production in response to hypoxia however was enhanced in Nox2y/− as compared to WT mice. Importantly, EPO, which induced pronounced EPC mobilization in WT mice, failed to do so in Nox2−/− mice. These data suggest that EPO signalling is defective in Nox2y/− mice. Indeed, in EPCs from WT-mice, EPO induced ROS production and proliferation and both responses were missing in EPCs from Nox2y/− mice. Transplantation of bone marrow from Nox2y/− mice into WT mice resulted in an attenuated mobilization in response to hypoxia and EPO, whereas the transplantation of WT bone marrow into Nox2y/− mice restored the normal phenotype . This demonstrates that a failure of bone marrow stem cells to generate ROS in response to EPO underly the mobilization defect. We conclude that Nox2-derived ROS are required for EPO-signalling in stem cells and that hypoxia-induced EPC mobilisation and vascular repair requires the NADPH oxidase Nox2.
Abstract 3417: Unselected Human Cardiosphere-derived Cells are Functionally Superior to c-Kit- or CD90-Purified Cardiosphere-derived Cells
Authors: Smith Rachel Ruckdeschel , Chimenti Isotta , Marbán Eduardo
Published: July 2021

Cardiosphere-derived cells (CDCs), a naturally heterogeneous mixture of cell sub-populations, were grown from percutaneous endomyocardial adult human biopsy specimens (n=6). c-Kit
+
and CD90
+
CDCs were selected using magnetic-activated cell sorting with excellent purity as determined by flow cytometry. Immunostaining revealed that ~30% of c-Kit
+
CDCs expressed Nkx2.5, ~100% of CD90
+
CDCs expressed procollagen type I, and ~100% of both sub-populations expressed CD105. When placed in co-culture with neonatal myocytes and fibroblasts, c-Kit
+
CDCs expressed cardiac troponin I, while CD90
+
CDCs expressed vimentin. In order to assess the therapeutic potential of purified CDCs, acute myocardial infarcts (MIs) were created in immunodeficient mice and c-Kit
+
(n=16), CD90
+
(n=14), or CD105
+
(n=3) CDCs were injected into the border zone. Echocardiograms were performed 3 weeks post-MI to measure left ventricular ejection fraction (LVEF). CD105-injected mice were comparable to an historical control group of mixed CDC-injected mice (LVEF = 41.3±2.9% CD105 vs. 42.8±10.4% CDC [n=11], p=0.60), indicating that the sorting process did not itself impair the therapeutic potential of CDCs. c-Kit- and CD90-injected mice were indistinguishable from one another (LVEF=31.7±8.2% c-Kit vs. 32.1±11.8% CD90, p=0.92), and both groups were significantly outperformed by the CD105-injected mice (p=0.01 and p=0.03, respectively). All groups were then compared to two other historical control groups, mice treated with normal human dermal fibroblasts (NHDFs [n=7]) and mice treated with phosphate-buffered saline (PBS [n=11]). c-Kit-injected mice did significantly outperform both NHDF- (p=0.04) and PBS-injected mice (p=0.03), while more variability in the CD90-injected group resulted in nearly significant comparisons with the NHDF (p=0.08) and PBS groups (p=0.08). While the therapeutic mechanisms of action of these two distinct sub-populations are undoubtedly different, both offer similar global functional benefits in the setting of acute MI. We conclude that the spontaneously-emerging unselected CDC population serves as a therapeutic cell cocktail, and that no functional advantage is conferred by the extra step of sorting for c-Kit
+
or CD90
+
sub-populations.
Abstract 3461: Inhibition of the p38 MAP Kinase in Vivo Improves Number and Functional Activity of Progenitor Cells and Reduces Atherosclerotic Disease Progression
Authors: Seeger Florian , Sedding Daniel , Langheinrich Alexander C , Rasper Tina , Zeiher Andreas M , Dimmeler Stefanie
Published: July 2021

Initial trials suggest that bone marrow-derived mononuclear cells (BMC) and ex vivo expanded endothelial progenitor cells (EPC) augment neovascularisation in patients after myocardial infarction. However, patients with cardiovascular risk factors show reduced number and impaired functional activity of EPC and increased activity of the p38 MAP kinase. Therefore, we examined the effect of p38 MAP kinase inhibition on progenitor cells in an in vivo model of metabolic syndrome and atherosclerosis. Consistent with the impairment of EPC by risk factors for coronary artery disease in humans, Lepr
db
mice exhibited a significantly lower number of EPC (sca-1
+
/flk-1
+
cells) and an increased number of inflammatory cells (Gr1
+
/CD45
+
cells) compared to their wild type littermates (0.05±0.07% vs. 1.6±1.1%, and 48.2±11% vs. 23.4±5.9%, respectively). Treatment of Lepr
db
mice with the p38 inhibitor SB203580 significantly increased the number of sca-1
+
/flk-1
+
cells (29±7 -fold increase) and lowered the number of GR1
+
/CD45
+
cells (69±11% of Lepr
db
mice). Moreover, both, reduced EPC colony forming activity (60±6 % of wt), and impaired BMC invasion capacity (58±13% of wt) were significantly improved in Lepr
db
mice by p38 inhibition. Finally, treatment of ApoE
−/−
mice with the SB203580 for 4 months reduced atheromatous lesion size by 51±3% (p<0.05). This study provides first evidence that p38 MAP kinase inhibition with SB203580 improves the impaired progenitor cell number and functional activity in an animal model of metabolic syndrome. Improvement of progenitor cell function is associated with reduction of atheroscle-rotic disease progression in ApoE
−/−
mice.
Abstract 3467: Fibulin-2 Enhances Angiotensin-II-Induced Transforming Growth Factor (TGF)-β Activation in Promoting Cardiac Remodeling in the Mouse Model I
n Vivo
Authors: Zhang Hangxiang , Dong Hailong , Wu Jing , Chu Mon-Li , Tsuda Takeshi
Published: July 2021

Background:
Angiotensin-II (Ang-II) is a potent neurohormone responsible for progression of cardiac remodeling in which TGF-β serves as a principal downstream mediator. In our previous study, genetic deletion of fibulin-2 attenuated progression of ventricular dysfunction after experimental myocardial infarction (MI). Because Ang-II plays a central role in post-MI ventricular remodeling, we tested the hypothesis that fibulin-2 modulates Ang-II-induced cardiac remodeling.

Methods:
Subpressor dosage of Ang-II (0.2 μg/kg/min) was infused over 4 weeks by mini-osmotic-pump in age matched wild-type (WT), heterozygous, and fibulin-2 null (Fbln2
−/−
) adult male mice. Sham mice received normal saline.

Results:
There was no blood pressure change throughout Ang-II treatment. WT developed significant left ventricular (LV) hypertrophy by Ang-II, whereas Ang-II-treated Fbln2
−/−
mice showed no noticeable hypertrophy compared with sham: LV/body weight ratio (WT 4.83±0.18 vs. Fbln2
−/−
4.01± 0.12 mg/g,
p
< 0.05) and LV posterior wall thickness by echocardiogram (WT 0.76± 0.03 vs. Fbln2
−/−
0.71± 0.02 mm,
p
< 0.05). Atrial natriuretic peptide (ANP) mRNA expression was significantly increased in Ang-II-treated WT compared with sham, but not in Ang-II-treated Flbn2
−/−
. Ang-II also induced significant up-regulation in fibulin-2, Collagen I, Collagen III, and MMP-2 mRNA level in WT, but not in Fbln2
−/−
. Both TGF-β1 mRNA and protein expression were significantly up-regulated in Ang-II-treated WT, but were unchanged in Ang-II-treated Fbln2
−/−
compared with sham. Activation of TGF-β downstream signaling proteins, phosphorylated forms of Smad2, TGF-β-activated kinase 1 (TAK1), and p38MAPK, were all significantly increased in Ang-II-treated WT, as opposed to no increase in Ang-II-treated Fbln2
−/−
compared with sham. Heterozygous mice showed intermediate increase in LV hypertrophy, matrix protein synthesis, and activation of TGF-β downstream signaling pathways between WT and Fbln2
−/−
.

Conclusions:
Our data suggest that fibulin-2 enhances Ang-II-induced myocardial hypertrophy via up-regulation of TGF-β and its downstream signaling pathways in dose-dependent fashion and that fibulin-2 is required for Ang-II-induced TGF-β activation.

This research has received full or partial funding support from the American Heart Association, AHA Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).
Abstract 3486: Albuminuria is Associated with Angiographically Determined Coronary Atherosclerosis
Authors: Rein Philipp , Saely Christoph H , Vonbank Alexander , Beer Stefan , Drexel Heinz
Published: July 2021

Introduction
: In spite of the overwhelming evidence linking albuminuria to cardiovascular events, data on its relationship to atherosclerosis are scarce and conflicting. It is still unclear whether albuminuria is associated with a particular phase of the atherothrombotic process.

Hypothesis:
We assessed the hypothesis that albuminuria is associated with angiographically determined coronary atherosclerosis.

Methods:
The urinary albumin-to-creatinine ratio (ACR) was determined and the eGFR was calculated by the quadratic Mayo Clinic equation in a large population of 907 consecutive patients undergoing coronary angiography for the evaluation of CAD.

Results:
From our patients, 297 had an eGFR <90ml/min/1.73m
2
, and 212 had albuminuria (ACR ≥30 mg/g). When compared to subjects with both normal eGFR and normal urinary albumin excretion (n = 494), the prevalence of significant coronary stenoses (i.e. stenoses with lumen narrowing ≥50%) was significantly higher in patients with normal eGFR and albuminuria (n = 116) and in those with decreased eGFR and albuminuria (n = 96), but similar in those (n = 201) who had decreased eGFR but not albuminuria (52.8 vs. 65.5%, p = 0.013; 52.8 vs. 65.6%, p = 0.021; and 52.8 vs. 47.3%.; p = 0.183, respectively). Concordantly, in logistic regression analysis the ACR but not the eGFR predicted significant coronary stenoses after multivariate adjustment, with standardized adjusted odds ratios (OR) of 1.27 [95% CI 1.03 – 1.58]; p = 0.027 and 1.05 [0.89 – 1.28]; p = 0.61, respectively. The association between the ACR and significant coronary stenoses remained significant after further adjustment for eGFR (OR = 1.30 [1.04 – 1.62]; p = 0.021).

Conclusions:
In conclusion, albuminuria is strongly associated with angiographically determined coronary atherosclerosis, independent of conventional cardiovascular risk factors and of the eGFR.
Abstract 3542: Role of Mitochondrial Oxidative Stress in Angiotensin II Mediated Hypertension
Authors: Dikalova Anna E , Bikineyeva Alfiya T , Harrison David G , Dikalov Sergey I
Published: July 2021

Oxidative stress is strongly implicated in the pathogenesis of hypertension; however, the role of mitochondrial oxidative stress is not clear. We have investigated the role of mitochondria in endothelial dysfunction and hypetention using cultured bovine endothelial cells (BAECs) and mice infused with angiotensin II (AngII). Production of O
2
•−
was measured by dihydroethidium and HPLC. Nitric oxide and H
2
O
2
were detected by ESR. Sytolic blood presure was measured in mice infused with saline, AngII (0.7mg/kg/day), AngII plus mitochondria targeted antioxidant mitoTEMPO or non-targeted antioxidant TEMPOL (50 μg/kg/day). AngII significantly increased mitochondrial H
2
O
2
. MitoTEMPO blocked AngII-induced oxidative stress and restored NO
•
production in BAECs and mouse aorta. Supplementation of BAECs with malonate, inhibitor of complex II, or rotenone abolished AngII induced oxidative stress. Interestingly, inhibition of mitochondrial oxidative stress decreased the activity of NADPH oxidases. Co-infusion of mitoTEMPO with AngII significantly attenuated the increase in the blood pressure by 30-mm Hg (Fig. 1

), while i.p. injection to hypertensive mice decreased the blood presure by 20-mm Hg. We suggest that mitoTEMPO can be used as an effective inhibitor of the oxidative stress induced by Ang II as well as an antihypertensive agent and, thereby, can be effective in ameliorating hypertension-related nephrosclerosis, diabetic nephropathy, or atherosclerosis. Figure 1. (A) Attenuation of hypertensive effect of Ang II in C57Blk/6 mice by co-infusion of mitoTEMPO. (B) Dose dependent effect of mitoTEMPO (50, 150 and 500 μg/kg/day) after 14-days of Ang II and drug administration.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 3570: A Critical and Novel Role of Nogo (Neurite Outgrowth Inhibitor) in Pulmonary Arterial Hypertension
Authors: Sutendra Gopinath , Bonnet Sebastien , Wright Paulette , Dromparis Peter , Haromy Alois , Sessa William , Michelakis Evangelos
Published: July 2021

Nogo
was first identified as an inhibitor of neuronal axonal regeneration. Recently,
Nogo-B
was implicated in the proliferative and anti-apoptotic remodeling in systemic arteries; reduced
Nogo-B
expression was seen in remodeled mouse femoral arteries following injury. Pulmonary arterial hypertension (PAH) is also characterized by proliferative/anti-apoptotic remodeling in pulmonary arteries (PA), sparing systemic vessels. PAH PA smooth muscle cells (PASMC) are characterized by mitochondrial hyperpolarization (increased ΔΨm), decreased production of reactive oxygen species (ROS) (suppressing mitochondria-dependent apoptosis), down-regulation of Kv1.5 and activation of the transcription factor NFAT (promoting contraction and proliferation). We found that in contrast to systemic vessels,
Nogo-B
expression is significantly increased in vivo and in vitro in PAs and PASMCs from patients (n=6) and mice (n=42) with PAH, compared to normals. We hypothesized that
Nogo
is involved in the pathogenesis of PAH
. Nogo
−/−
mice (n=7) had a normal phenotype and, in contrast to
Nogo
+/+
, did not develop chronic hypoxia (CH)-induced PAH assessed invasively (catheterization, RV/LV+Septum) and non-invasively (pulmonary artery acceleration time and treadmill performance) (n=7, Table

). CH-
Nogo
+/+
PASMC had the expected increase in ΔΨm (measured by TMRM), decreased ROS (MitoSOX), increased [Ca
++
]
i
(FLUO3), decreased Kv1.5 (immunohistochemistry) and NFAT activation (nuclear translocation). None of these changes occurred in CH-
Nogo
−/−
PASMC while all were induced in normoxic
Nogo
+/+
PASMC by adenoviral over-expression of
Nogo-B
. Heterozygote CH-
Nogo
+/−
(n=7) values were between
Nogo
−/−
and
Nogo
+/+
suggesting a gene dose-dependent effect.
Nogo
is over-expressed in human and rodent PAH and induces critical features of the PAH phenotype.
Nogo
targeting might represent a novel and selective therapeutic strategy for PAH.

Table
Abstract 3600: HspA12B Promotes Angiogenesis through Suppressing AKAP12 and Up-Regulating VEGF Pathway
Authors: Steagall Rebecca J , Hua Fang , Thirunazukarasu Mahesh , Zhan Lijun , Li Chuanfu , Maulik Nilanjana , Han Zhihua
Published: July 2021

We have previously shown that HspA12B, a member of HspA70 family subfamily 12, is a novel angiogenesis regulator that is preferentially expressed in endothelial cells (ECs) and required for angiogenesis
in vitro
. The mechanism by which HspA12B regulates angiogenesis, however, is unknown. In this study we identified AKAP12/SSeCKS as a HSPA12B-interacting protein through a yeast two-hybrid screening and confirmed the interaction by co-immunoprecipitation and co-localization. We observed that HspA12B negatively regulated the expression of AKAP12/SSeCKS, a cancer metastasis repressor that inhibits VEGF expression and angiogen-esis. In HUVEC, HspA12B knockdown increased AKAP12 levels, decreased VEGF by more than 75%, and down-regulated Akt and pAkt; whereas HspA12B over expression decreased AKAP12 and more than doubled VEGF levels. We further identified a 32-AA domain in AKAP12 that was capable of interacting with HspA12B. Overexpression of this 32-AA domain in HUVEC disrupted the HspA12B-AKAP12 interaction and decreased VEGF expression by more than 70%, suggesting the importance of HspA12B-AKAP12 interaction in regulating VEGF. We also observed that HspA12B expression was increased more than 2 folds in ECs by hypoxia or shearing stress, and induced in ischemic rat heart. Inhibition of HspA12B abolished hypoxia-induced tubule formation. Adeno-HspA12B promoted angiogenesis in DIVAA assay. We concluded that this is the first evidence that HspA12B promotes angiogenesis through regulating VEGF by way of suppressing AKAP12. Our finding is the first example of an EC-specific molecular chaperone acting as the regulator of angiogenesis.
Abstract 3620: Cellular Pcsk9 Autocatalytic Activity Measured in Hepatocytes
Authors: Kourimate Sanae , Lalanne Florent , Costet Philippe
Published: July 2021

Proprotein convertase subtilisin kexin type 9 (PCSK9) is a natural inhibitor of the Low density lipoprotein receptor (LDLr). Its autocatalytic cleavage is necessary to its processing within the cell and the targeting of the LDLr. Therefore identifying molecules that would modulate this activity is an important challenge. The possibility of measuring this activity has been debated. Using whole hepatocytes lysates and optimized conditions, we showed that the cleavage of a fluorogenic peptide corresponding to proPCSK9 cleavage site is specific to this proprotein convertase. Indeed, the fluorogenic activity was virtually absent in primary hepatocytes from PCSK9 knockout mice compared with wild type hepatocytes. In human immortalized hepatocytes (IHH), a reduction of PCSK9 expression by 40 to 60% using SiRNA technology reproducibly decreased PCSK9 activity in a range of similar magnitude. Maldi-Toff analysis of the cell lysate following the the experiment showed that the fluorogenic peptide is cleaved at the right site LVFAQ152. In IHH cells exposed to the Liver X agonist T0901317 1 microM or fenofibric acid 250microM, or to both drugs concomitantly for 48h, PCSK9 protein content was respectively increased by 60% (p<0.5), decreased by 70% (p<0.01) or decreased by 30% (p<0.01). We observed similar variations of the fluorogenic activity of these cell lysates. We also observed that PCSK9 autocatalytic activity was increased in cells accumulating PCSK9 upon exposure to pravastatin 10microM for 48h, and that the repressive effect of fenofibric acid 250microM overcame the induction by pravastatin, in agreement with our previous works on PCSK9 transcriptional regulation. We also analysed the catalytic activity of PCSK9 gain of function variants. Altogether, these results show that PCSK9 endogenous autocatalytic activity can be measured in cellulo. Futhermore, the catalytic activity reflects PCSK9 protein abundance. We suggest that this test could also be used to identify postraductional modulators of PCSK9 activity.
Abstract 3651: Caloric Restriction Stimulates Ischemia-induced Angiogenesis through an eNOS-dependent Pathway
Authors: Shibata Rei , Kondo Megumi , Shimano Masayuki , Kondo Kazuhisa , Walsh Kenneth , Ouchi Noriyuki , Murohara Toyoaki
Published: July 2021

Caloric restriction (CR) can extend longevity and modulate the features of obesity-related metabolic and vascular diseases. However, little is known about the role of CR in the process of ischemia-induced vascular remodeling. Here, we investigated whether CR regulates angiogenic responses in vivo employing the hindlimb model of ischemia-induced angiogenesis. Wild-type (WT), endothelial nitric oxide synthase deficient (eNOS-KO) or adiponectin deficient (APN-KO) mice were randomly divided into 2 groups that were fed either ad libitum (AL) or CR (65% of the diet consumption of AL). Four weeks later, mice were subjected to unilateral hindlimb surgery. Serum adiponectin levels were measured by ELISA. The expression and activation of eNOS were assessed by Western blot analysis. Body weight of WT mice fed CR (CR-WT) was decreased by 26% compared to WT mice fed AL (AL-WT). Angiogenic repair of ischemic hindlimb was accelerated in CR-WT compared with AL-WT as evaluated by laser Doppler blood flow (p<0.05) and capillary density analyses (p<0.05). In WT mice, CR significantly enhanced ischemia-induced increase in eNOS activation of muscle tissues and increased serum adiponectin levels. In eNOS-KO mice, CR significantly increased serum adiponectin levels but did not enhance limb perfusion after surgery. In APN-KO mice, ischemia induction did not increase eNOS activation in muscle tissues, and CR had no effects on blood flow recovery and eNOS activation in ischemic limb. Our observation indicates that CR can promote angiogenesis in response to tissue ischemia through its ability to increase eNOS activation that is possibly mediated by adiponectin.
Abstract 3654: Semaphorin3E is a Novel Angiogenic Regulator
Authors: Moriya Junji , Minamino Tohru , Tateno Kaoru , Orimo Masayuki , Miyauchi Hideyuki , Okada Sho , Shimizu Ippei , Nojima Aika , Yokoyama Masataka , Komuro Issei
Published: July 2021

Semaphorin3E (sema3E) and its specific receptor plexinD1 are known to regulate the patterning of vessels during embryogenesis. However, it remains unclear whether these molecules are involved in postnatal angiogenesis. To elucidate the role of sema3E/plexinD1, we performed in vitro assay using human umbilical vein endothelial cells (HUVECs). Treatment with vascular endothelial growth factor (VEGF) increased proliferation and tube formation and this increase was significantly inhibited by sema3E. Moreover, treatment with the plexinD1-Fc fusion protein antagonized this anti-angiogenic activity of sema3E. Western blot analyses revealed that sema3E suppressed VEGF-induced phosphorylation of VEGFR2, suggesting that sema3E negatively regulates angiogenesis by inhibiting the VEGF signaling pathway. Expression of sema3E and plexinD1 was markedly upregulated in ischemic limbs. Immunohistochemistry showed that sema3E was expressed by the arterioles, myocytes, and capillary endothelial cells in ischemic tissue. Introduction of the plexinD1-Fc gene into ischemic limbs led to significant improvement of blood flow recovery and an increase in the number of CD31-positive cells. It has been reported that other members of the sema3 family are transcriptionally regulated by p53, a tumor suppressor protein that inhibits neovascularization in tumors. Consistent with these reports, forced expression of p53 was found to upregulate sema3E expression in HUVECs. We also found that the expression of p53 was markedly increased in ischemic limbs and that this increase was further enhanced in ischemic tissues of diabetic mice. Consequently, expression of sema3E was significantly higher in ischemic limbs of diabetic mice than in control mice, and the blood flow recovery after ischemia was strongly impaired in these mice even though treated with VEGF. In contrast, treatment with both VEGF and PlexinD1-Fc markedly improved blood flow recovery in diabetic mice. These results indicate that sema3E/plexinD1 negatively regulates postnatal angiogenesis under the regulation of p53 and suggest that inhibition of sema3E would be a novel strategy for therapeutic angiogenesis, especially when VEGF treatment is ineffective.
Abstract 3707: The Multiplier Effect of Telmisartan and Fenofibrate in Patients with Metabolic Syndrome
Authors: Maruyama Katsuya , Sutani Yasuo , Abe Yoshiteru , Motohiro Masayuki , Katoh Shuji
Published: July 2021

Background:
Telmisartan, a partial agonist of PPARγ, improve insulin resistance with increase adiponectin and especially high molecular weight adiponectin (HMW adipo) in patients with metabolic syndrome. Fenofibrate, an antihyperlipidemic drug, can function as an agonist of PPARα and is increased adiponectin receptors. In this study, we examined that telmisartan with fenofibrate influences multiplier effect of insulin resistance and anti-arteriosclerosis action in patients with metabolic syndrome.

Methods:
25 patients with metabolic syndrome took telmisartan (40mg/day) for 6 months (stage 1), and they were measured HOMA-R, high sensitive CRP (hsCRP), adiponectin, HMW adipo, TNFα, both visceral fat (VF) area and subcutaneous fat (SF) area by CT scan and mean intima-media thickness (M-IMT) of common carotid artery. After stage 1 they took telmisartan (40mg/day) together with fenofibrate (100mg/day) for 6 months (stage 2), and they were measured them again.

Results:
We compared these parameters of stage 1 and stage 2. HOMA-R, hsCRP, and TNFα were decreased for both stage 1 and stage 2. HOMA-R especially was significant (from 3.97 to 3.09; p<0.05) for stage 2. Both adiponectin and HMW adipo were increased for stage 1, but they were decreased for stage 2. Both VF area and SF area were decreased between stage1 and stage2. SF area especially was significant (from 194.9 to 184.3 cm2; p<0.05) for stage 2. M-IMT of common carotid artery significantly was decreased (from 0.97 to 0.84 mm; p<0.01) between stage 1 and stage 2.

Conclusion:
Our findings indicate that although telmisartan with fenofibrate are decreased both fat area (especially SF area) and secretion of adiponectin, they influences multiplier effect of insulin resistance and anti-arteriosclerosis action through the improvement of adiponectin sensitivity.