bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5515: Mitochondrial Membrane Hyperpolarization and Reduced Mitochondrial Mass Characterize the Arteriolar Endothelium and Mononuclear Cells of Humans With Type 2 Diabetes Mellitus-
in vivo
and
in vitro
Assessment of Endothelial Function
Authors: Widlansky Michael E , Vita Joseph A , Kizhakekuttu Tinoy J , Wang Jingli , Arthur Emily I , Gutterman David D
Published: April 2024

Background:
Endothelial dysfunction and inflammation are central to the vascular pathology of Type 2 diabetes (DM).
In vitro
data suggest excessive mitochondrial reactive oxygen species production contributes to endothelial dysfunction in part due to pathological hyperpolarization of mitochondrial membrane potential (ΔΨ
m
). ΔΨ
m
hyperpolarization in leukocytes is also associated with activation of the inflammatory response. Mitochondrial mass and morphology are also central to mitochondrial function. The relevance of these mechanisms to the human vasculature in DM is unknown.

Methods:
Blood mononuclear cells were isolated from 30 DM patients and 32 non-diabetics. Subcutaneous arterioles were harvested by gluteal adipose tissue biopsy in a subset (15 DM, 17 controls). ΔΨ
m
was measured in cells and arterioles using the membrane potential-sensitive fluorescent dye JC-1. Mitochondrial mass was measured using the cardiolipin-specific fluorophore nonyl acridine orange. Endothelium dependent vasodilation was assessed using video microscopy (arterioles) and ultrasonography (brachial artery). Arteriolar vasoreactivity was also determined in the setting of low-dose (100 nM) CCCP to reduce ΔΨ
m
magnitude.

Results:
ΔΨ
m
magnitude of the arteriolar endothelium and mononuclear cells were significantly greater in DM patients [P=0.01 (endothelium), P < 0.0001 (mononuclear cells)]. Mitochondrial mass was significantly lower in DM mononuclear cells (P=0.004), and in DM endothelium following age adjustment (P=0.003). Endothelium dependent vasodilation was impaired in DM in the brachial artery and subcutaneous arterioles (both P<0.001). CCCP treatment of the DM arteriolar endothelium reversed microvascular endothelial dysfunction (P<0.001 vs. untreated).

Conclusions:
ΔΨ
m
is hyperpolarized and mitochondrial mass is lower in the arteriolar endothelium and mononuclear cells of DM patients with endothelial dysfunction. Pharmacological normalization of ΔΨ
m
reverses microvascular endothelial dysfunction in DM. These findings are consistent with experimental studies suggesting a link between mitochondria and vascular disease. Interventions that restore mitochondrial membrane potential and mass in DM may have therapeutic potential.
Abstract 5538: Apoptosis Signal-Regulating Kinase (ASK) 2 Deficient Mice Have Salt-Resistant Hypertension With Cardiac Hypertrophy
Authors: Kataoka Keiichiro , Nakamura Taishi , Fukuda Masaya , Nako Hisato , Dong Yi fei , Liu Rei , Tokutomi Yoshiko , Ogawa Hisao , Mitsuyama Shokei
Published: April 2024

Background:
Apoptosis signal-regulating kinase (ASK) 2 is a MAP3K that is highly related to ASK1. ASK2 is activated in response to oxidative stress. There have been no reports about the cardiovascular functions of ASK2. Herein, we show the cardiovascular phenotypes of ASK2 deficient mice (ASK2KO).

Methods and Results:

ASK2KO had higher blood pressures than wild type mice (WT) (122±1 vs. 104±0.4 mmHg, p<0.01). Urinary catecholamine excretion in ASK2KO was significantly increased compared to WT, indicating that hypertension in ASK2KO is associated with enhanced sympathetic nerve activity. Histological examinations revealed that myocardial fibrosis was increased in ASK2KO compared to WT. The number of macrophages and superoxide levels were greater in ASK2KO than in WT.

ASK2KO were administered either olmesartan or hydralazine in order to determine if myocardial fibrosis in ASK2KO is associated with the renin-angiotensin system (RAS). Although blood pressures of both groups were decreased to the same levels as normal WT levels, only the olmesartan group had both decreased myocardial fibrosis and inflammatory cell infiltration; this indicates that cardiac hypertrophy in ASK2KO is associated with RAS.

ASK2KO and WT were fed a high salt diet (8% NaCl) to examine whether hypertension in ASK2KO is salt-sensitive or not.

Blood pressures of ASK2KO were significantly decreased by salt loading, although those of WT were not. Increased cardiac fibrosis and inflammatory cell infiltration observed in normal diet ASK2KO were also significantly decreased by salt loading. This shows that hypertension and cardiac hypertrophy in ASK2KO are salt resistant. Salt loading increased ASK2 mRNA in the heart tissues of WT, and it enhanced the inflammatory responses, oxidative stress and TGFβ expression in WT hearts, but these effects were not observed in ASK2KO, which indicates that ASK2 is involved in salt induced cardiac injury.

Conclusions:

ASK2KO have salt-resistant hypertension with cardiac hypertrophy. ASK2 plays an important role in blood pressure regulation and cardiac hypertrophy in association with RAS.

ASK2 plays a pivotal role in salt-induced cardiac injuries in association with oxidative stress and inflammatory responses.
Abstract 5541: Erythropoietin Receptor Signaling Exacerbates Neointimal Hyperplasia in the Rat Carotid Artery After Balloon Injury
Authors: Tsujimoto Akiko , Takemura Genzou , Okubo Munenori , Kanamori Hiromitsu , Ogino Atsushi , Kawamura Itta , Kawaguchi Tomonori , Takeyama Toshiaki , Watanabe Takatomo , Goto Kazuko , Kawasaki Masanori , Fujiwara Takako , Fujiwara Hisayoshi , MInatoguchi Shinya
Published: April 2024

Treatment with erythropoietin (EPO) was reported to accelerate reendothelialization, accompanying endothelial progenitor cell (EPC) induction, to result in suppression of neointimal hyperplasia in the carotid artery after balloon injury. We injured the right carotid artery of male SD rats (200 to 250 g) with an oversized balloon. Treatment with recombinant human EPO (1000 IU/kg/day) or asialoerythropoietin (AEPO, 1000 IU/kg/day), a nonerythrogenic derivative of EPO, was started at 1 week after injury when neointimal proliferates and continued for 3 days. The rats were examined 2 weeks after the surgery. The hemoglobin level was significantly increased in the EPO-treated rats but was unchanged in the saline control and AEPO-treated groups. FACS analysis revealed significantly increased circulating EPCs in the EPO-treated group but not in the AEPO-treated group. Reendothelialization was accelerated in both EPO- and AEPO-treated groups. However, the area of neointima was significantly greater in both treatment groups compared with the control group and accordingly the area of neointimal to medial area ratio (%) was significantly greater in the EPO- (150+/−26%) and AEPO-treated (157+/−15%) groups compared with the control group (132+/−7%). Alpha-smooth muscle actin-positive cell population was also greater in both treatment groups. The incidence of proliferating cells labeled with Ki-67 antigen was significantly greater not only in the endothelium but also in the neointima of the EPO- and AEPO-treated groups compared with the control group. In conclusion, EPO receptor signaling can proliferate not only endothelial cells but also intimal smooth muscle-like cells independently of EPC induction, which might have aggravated neointimal hyperplasia when EPO receptor was stimulated at the phase of intimal proliferation after injury. These findings caution an easy use of EPO in patients after vascular intervention.
Abstract 5546: Krüppel-like Factor 2 (KLF)2 Inhibits Macrophage Pro-inflammatory Activation
Authors: Hampole Ashwin V , Mahabeleshwar Ganapati H , Sharma Nikunj , Jain Mukesh K
Published: April 2024

Background: Inflammation contributes to a broad spectrum of human disease states including cardiovascular disease. Macrophages are critical regulators of the innate inflammatory response. KLF2 is a member of the Krüppel-like family of zinc-finger transcription factors implicated in cellular development and differentiation. Previous studies from our laboratory indicate that KLF2 inhibits pro-inflammatory activation of monocytic cell lines. However, the role of KLF2 in macrophage activation and function of primary cells remains unknown.

Methods and Results: To gain insights regarding the role of KLF2 in macrophage biology, both gain-of-function and loss-of-function studies were undertaken. For gain-of-function analyses, RAW264.7 cells were infected with empty control virus (Ad-EV) and KLF2 virus (Ad-K2) for 24 hours followed by assessment of key inflammatory targets by quantitative PCR and ELISA. Our studies revealed a marked reduction in both mRNA expression and protein secretion of CD40L, MCP-1, IL-1
β
, and TNF
α
. To assess the effect of KLF2 deficiency, we harvested primary peritoneal macrophages from KLF2
+/+
and KLF2
+/−
mice. These cells were cultured for 24 hours, stimulated with LPS (50 ng/ml) overnight, and then assessed for target genes. As expected, the expression of all 4 targets was increased in KLF2
+/+
; however, this induction was markedly enhanced in KLF2
+/−
macrophages. Finally, to gain insights regarding the effect of KLF2 deficiency on macrophage function, migration (Transwell) and invasion (Matrigel) assays were performed. We find that KLF2 deficiency augments both macrophage migration (KLF2
+/+
: 256±8.8% versus KLF2
+/−
: 380±9.7%, P<5×10
−6
) and invasion (KLF2
+/+
: 248±10.6% versus KLF2
+/−
: 329±10.2%, P<1×10
−4
) following LPS activation. Finally, our mechanistic studies suggest that the anti-inflammatory effects of KLF2 are mediated, at least in part, by its ability to inhibit the NF-kB pathway.

Conclusion: Our results indicate that KLF2 is novel regulator of macrophage activation. As such, targeting of KLF2 function may be beneficial in preventing a wide spectrum of inflammatory disease such as atherosclerosis.
Abstract 5552: Notch-peroxisome Proliferator Activated Receptor Gamma 2 Pathway Regulates Transendothelial Fatty Acid Transport via Induction of Fatty Acid Binding Protein 4 and Fatty Acid Translocase in Capillary Endothelial Cells in Heart
Authors: Goto Kosaku , Iso Tatsuya , Suga Toshihiro , Matsui Hiroki , Arai Masashi , Gonzalez Frank J , Kurabayashi Masahiko
Published: April 2024

Notch signaling, an evolutionarily conserved pathway for cell fate determination, is essential for normal vascular development and maintenance as well as other multiple aspects of vascular function. We have reported that fatty acid binding protein 4 (FABP4), also known as aP2, is a potential candidate of Notch target genes in capillary endothelial cells (EC) of heart, skeletal muscle and adipose tissue. Given that FABP4 is well known as an adipocyte-specific marker and downstream of PPAR-
γ
, we hypothesized that Notch signaling may regulate several genes abundant in adipocyte via induction of peroxisome proliferator activated receptor-
γ
(PPAR-
γ
). Here we show that Notch signaling induces several adipocyte-related genes in capillary EC via induction of PPAR-
γ
2, an isoform known to play a major role in adipocyte differentiation. Overexpression of Notch intracellular domain (NICD), an activated form of Notch, in human cardiac microvessel endothelial cells (HCMEC) induced expression of FABP4, lipoprotein lipase (LPL), but not fatty acid translocase (FAT/CD36). The induction was accompanied by expression of PPAR-
γ
2, but not PPAR-
γ
1. Administration of synthetic PPAR-
γ
ligands, thiazolidindione (TZD), markedly enhanced expression of these genes including even FAT/CD36. Their induction was mediated by PPAR-
γ
2 because their expression by NICD plus TZD was abolished by administration of GW9662, a PPAR-
γ
inhibitor. Of importance, the gene induction by NICD and the enhancement by TZD were not observed in other cells such as human umbilical vein EC and 3T3L1 fibroblast/adipocyte, suggesting capillary EC-specific effect of Notch-PPAR-
γ
pathway. The gene induction was observed in HCMEC when co-cultured with cells expressing Dll4, a Notch ligand, in the presence of TZD. Consistent with these findings, these genes including a Notch receptor Notch1, Dll4, FABP4 and FAT/CD36 were co-localized in capillary EC. Given that long chain fatty acids are central source of energy metabolism in heart, skekletal muscle and adipocyte, adipocyte-related genes such as FABP4 and FAT/CD36 induced by Notch-PPAR pathway in capillary EC may play a crucial role in fatty acid transport system through endothelial layer to supply adequate fatty acids to the fatty acid-burning organs.
Abstract 5557: Lack of Interleukin-1 Receptor Antagonist in Inflammatory Cells Causes Spontaneous Femoral Artery Aneurysms in Mice
Authors: Isoda Kikuo , Niida Tomiharu , Kondo Harumi , Arai Yuko , Hayase Taichirou , Kujiraoka Takehiko , Kitagaki Manabu , Ishigami Norio , Adachi Takeshi , Ohsuzu Fumitaka
Published: April 2024

Background:
Arterial aneurysms represent a complex degenerative disorder involving chronic arterial wall inflammation and destructive remodeling of structural connective tissue. The relative balance of pro- and anti-inflammatory cytokines likely controls inflammation in the aneurysm wall. The interleukin (IL)-1 receptor antagonist (IL-1Ra) negatively regulates IL-1 signaling. However, the role of IL-1Ra in aneurysm is poorly understood.

Methods and Results:
IL-1Ra-deficient (IL-1Ra−/−) mice (backcrossed 8 generations to the B6 background) and wild-type (WT) mice did not differ with regard to systolic arterial pressure and plasma lipid levels. However, IL-1Ra−/− mice had spontaneously developed femoral artery aneurysms after 8-month-old, and both maximal vessel area and the intimal + medial area of femoral arteries in IL-1Ra−/− mice were significantly larger compared with WT mice at 9 months of age. Real-time PCR of 9-month-old IL-1Ra−/− mice revealed significantly increased mRNA levels of IL-1
β
(6.6-fold, P<0.05), tumor necrosis factor (TNF)-
α
(12.4-fold, P<0.05) and matrix metaroproteinase-9 (6.0-fold, P<0.01) compared with WT mice. Histological analysis revealed numerous inflammatory cells around the femoral artery aneurysm in IL-1Ra−/− mice, and elastin staining showed destruction of the internal and external elastic lamina in IL-1Ra−/− mice. Immunostaining revealed many macrophages and some CD4-positive T cells in the adventitia. Then we studied the function of macrophage and T cell. After LPS (1
μ
g/ml) stimulation, IL-1Ra deficient macrophages produced much higher levels of TNF-
α
than those from WT mice. Furthermore TNF-
α
production of T cells from IL-1Ra−/− mice stimulated with anti-CD3 antibody was also higher than those from WT mice. Finally, we performed bone marrow (BM) cell transplantation. We detected femoral artery aneurysms with many inflammatory cells in the adventitia in several WT mice receiving BM cells from IL-1Ra−/− (44%) but not WT mice (0%).

Conclusions:
This study demonstrates that IL-1Ra-deficiency in inflammatory cells disrupts immune system homeostasis, resulting in the development of autoimmunity and inflammation. Then the inflammation causes spontaneous femoral artery aneurysms in B6 mice.
Abstract 5601: Podoplanin Expressing Cells Isolated From Bone Marrow and Peripheral Blood Function as Lymphatic Endothelial Progenitor Cells
Authors: Lee Ji Yoon , Park Changwon , Lee Eugine , Kim Hyongbum , Yoon Young-sup
Published: April 2024

Background: Previously, we demonstrated that podoplanin expressing (pod+) cells can be culture-isolated from bone marrow (BM) mononuclear cells (MNCs) and that these cells have a potential to differentiate into lymphatic endothelial cells (LECs). In this study, we investigated whether pod+ cells exist in BM and peripheral blood (PB), and they can function as lymphatic endothelial progenitor cells (LEPCs).

Methods and Results. First we performed FACS analysis and found that pod+ cells are present in BM and PB less than 0.5% in C57/BL6 mice. Thus, next to investigate whether the number of pod+ cells would be increased in the BM or PB under conditions which promote lymphatic vessel growth, mice were injected with B16-F1 melanoma cells into the back skins. FACS analysis conducted 7 days later showed that the number of pod+ cells in BM and PB were about 15 fold increased in mice with melanoma growth (normal vs tumor, BM: 0.2±0.5% vs. 3.2±1.0%, PB: 0.4±0.3% vs. 6.7±2.0%, P < 0.01). FACS analysis further revealed that the BM pod+ cells derived from the tumor-bearing mice exhibited high levels of VEGFR-3+ and c-KIT+, but not LYVE-1+ cells, but PB pod+ cells derived from the same mice displayed VEGFR-3+ and LYVE-1+, but not c-KIT+ cells. These data suggest that more undifferentiated LEPC phenotypes exist in BM and undergo differentiation during mobilization into PB. Next, to determine the lymphvasculogenic potential of the isolated BM pod+ cells in vivo, we injected the pod+ cells (Dil-labeled or GFP mouse derived) isolated from BM of tumor-bearing mice into skin and ear wound models. Immunohistochemistry showed that the injected cells were incorporated into lymphatic vessels and expressed LEC-specific markers, suggesting LEC differentiation. We also found that these pod+ cells express CD45 in BM but lost CD45 expression after incorporation into lymphatic vessels, suggesting that pod+ cells are hematopoietic in origin but lost hematopoietic properties during differentiation into LEC. In

Conclusion. This study for the first time demonstrated that pod+ cells are present in BM and PB, are augmented under lymphatic vascular growth conditions, and contributed to lymphvasculogenesis, suggesting a role of pod+ cells as LEPCs.
Abstract 5622: Effectiveness of Hybrid Therapy With Warfarin and Statins to Reduce the Incidence of Ischemic Stroke in High-risk Patients With Atrial Fibrillation
Authors: Hama Yoshiyuki , Ueda Marehiko , Takano Hiroyuki , Hiranuma Yasunori , Hori Yasuhiko , Miyazaki Akira , Yamanouchi Masato , Kanda Junji , Kuwabara Yoichi , Komuro Issei
Published: April 2024

Background HMG-CoA reductase inhibitor statins have been recently used to prevent ischemic stroke in patients with coronary artery disease. However, whether or not such statins truly reduce the risk of ischemic stroke in patients with atrial fibrillation (AF) remains to be clearly elucidated.

Methods We performed a retrospective cohort study at 4 Japanese centers. The study sample consisted of a cohort of 1152 patients (mean age, 67±10 years; men, 64 %; 513 paroxysmal AF) who had documented AF on electrocardiograms before August 2002 and who also visited our hospitals after September 2002. The statin group consisted of 179 patients (16%). A cox proportional hazards analysis was performed to control for baseline differences. We assessed the incidence of ischemic stroke.

Results During a follow-up of 61±23 months, 151 patients experienced ischemic stroke. Since the statin group had more risk factors for stroke, such as a history of congestive heart failure, hypertension and diabetes mellitus, than the non-statin group, the statin group had a higher CHADS2 score (congestive heart failure, hypertension, age, diabetes, stroke [doubled]) than the non-statin group (statin group 1.93±1.15 vs. non-statin group 1.42±1.19, p<0.001). The incidence of ischemic stroke was similar in both groups (statin group 12.0% vs. non-statin group, 17.5%, P = 0.08). For patients with a CHADS2 score ranging from 2– 6 with concomitant warfarin use, however, ischemic stroke was significantly less frequent in the statin group (statin group 11.7% vs. non-statin group, 26.0%, P = 0.045) although the incidence of major bleeding was similar in both groups (statin group 14.4% vs. non-statin group, 21.1%, P = 0.508). After adjustment for differences in baseline clinical variables, statin use was significantly associated with the incidence of ischemic stroke (hazard ratio[HR], 0.43; 95% confidence interval[CI], 0.23 to 0.83; P = 0.012),

Conclusions In comparison to warfarin alone, the hybrid therapy with warfarin and statin was therefore suggested to be more effective in high-risk patients with atrial fibrillation.
Abstract 5646: Endothelial Intrinsic NF-
κ
B Signaling Regulates iNOS And eNOS Expression
in vivo
in Endotoxemic Mice
Authors: Ye Xiaobing , Song Dongmei , Liu Shu Fang
Published: April 2024

We have previously demonstrated that endothelial NF-
κ
B blockade mitigates septic shock and septic vascular dysfunction (
Circulation
118: S570, 2008). Since endothelial and inducible nitric oxide synthase (iNOS and eNOS) are known to be involved in vascular dysfunction, this study examined the contribution of endothelial intrinsic NF-
κ
B signaling in LPS induced alterations in iNOS and eNOS expression. Wild type (WT) and transgenic mice (TG) that conditionally overexpress a mutant I-
κ
B
α
selectively on endothelium were injected with saline (1 ml/kg, i.p.) or
E Coli
LPS (10 mg/kg, i.p.). Aortic levels of iNOS and eNOS proteins were determined by Western blot. The iNOS and eNOS bands were quantified using densitometry and expressed as arbitrary optical density units. Plasma and aortic levels of nitrite/nitrate, and aortic level of TNF-
α
were measured using commercial kits. Aortic level of iNOS protein (O.D units) for WT-Con, TG-Con, WT-LPS and TG-LPS groups were 0±0, 0±0, 935±107 and 257±58 (p < 0.01, WT-LPS vs other 3 groups). Plasma and aortic levels of nitrite/nitrate, for WT-Con, TG-Con, WT-LPS and TG-LPS groups, were 4.4±0.7, 3.9±0.4, 19.3±3.4 and 6.9±1.3
μ
M in plasma, and 5.8±1.9, 5.8±0.9, 40.2±3.9 and 19.9±2.0 nmole/mg protein in aorta (p < 0.01, WT-LPS vs other 3 groups). Aortic level of eNOS protein (O.D units) for WT-Con, TG-Con, WT-LPS and TG-LPS groups were 896±54, 799±61, 586±46 and 838±49 (p < 0.05, WT-LPS vs other 3 groups). Down-regulation of eNOS expression was associated with increased aortic TNF-
α
expression. Aortic levels of TNF-
α
(pg/mg protein) for WT-Con, TG-Con, WT-LPS and TG-LPS groups were 68±26, 23±12, 753±132 and 369±54 (p < 0.01, WT-LPS vs other 3 groups). IHC staining of aortic sections showed that TG-LPS mice had a significantly reduction in LPS-induced iNOS or TNF-
α
expression in both endothelial and smooth muscle cells, indicating a paracrine interaction between the two cells. These results suggest blockade of endothelial NF-
κ
B pathway mitigates septic vascular dysfunction by inhibiting vascular iNOS expression and by preventing eNOS down-regulation.

(Supported by NIH GM063907)
Abstract 5658: Immune Reconstitution Prevents Autoimmune Inflammation and Right Ventricular Remodeling in Immunodeficiency-Associated Pulmonary Hypertension
Authors: Tamosiuniene Rasa , Long Carlin , Ambler Kelly , Sung Yon , Gera Lajos , Wang Lijuan , Nicolls Mark
Published: April 2024

Pulmonary arterial hypertension (PH) is a frequently lethal condition commonly associated with autoimmune conditions. Because autoimmunity is closely linked to immunodeficiency, we utilize athymic (AT) nude rats lacking normal T cells to demonstrate how PH is accentuated by a lack of normal regulatory T cell activity. The purpose of this study was to determine whether immune reconstitution (IR) prevents autoimmune inflammation (AI), right ventricular (RV) remodeling and PH using inbred AT rats reconstituted with euthymic spleen cells.

Methods and Results: Inbred WAG AT nude rats were given a single injection of vascular endothelial growth factor receptor (VEGFR) antagonist SU5416 (20mg/kg sc), AT+SU group. Control AT was treated with vehicle only (DMSO). In adoptive transfer IR experiments, AT rats received unfractionated 20×10
6
euthymic rat spleen cells, CD4+T cells, CD8+T cells, CD4+CD25+T cells isolated with magnetic cell sorting before or after SU5416 administration. 2D-Echo, pulmonary hemodynamics and RV/LV+S ratio were assessed at 10d and 21d. Echo revealed decreased RV fractional shortening (p<0.05) and PA acceleration time (p<0.005) in AT+SU compared with IR and controls. IR prevented increase of RVSP compared to AT+SU (P<0.05) and of RV/LV+S (P<0.001). RV fetal cardiac gene expression (a-MHC, b-MHC, SERCA, BNP, SkACT, ANP) was consistent with pathologic stress pattern injury pattern and was significantly attenuated by IR. Furthermore, IR reduced expression of Th1, TH2 cytokines and pulmonary infiltration of macrophages, mast cells, B cells, IgG deposition and up-regulated lung BMPR2, FOXP3+, c-kit+, HOX11+ expression with increased c-kit+ and BMPR2 expression being noted in the pulmonary vascular endothelium.

Conclusions: This study demonstrates that T cell deficiency is permissive for PH development and that IR prevents hemodynamic RV remodeling, attenuates AI and limits PH. Interestingly, that PH prevention is associated with increased regenerative HOX11+, C-kit stem cell populations.
Abstract 5685: TGF-beta Signaling and Kruppel-like Factor 10 Regulate Bone Marrow-derived EPC Differentiation, Function, and Neovascularization
Authors: Wara Akm , Foo ShiYin , Icli Basak , Cao Zhuoxiao , Esen Fehim , Subramaniam Malayannan , Spelsberg Thomas C , Rosenzweig Anthony , Feinberg Mark W
Published: April 2024

Background: Endothelial progenitor cells (EPCs) may originate from the bone marrow and are capable of contributing to cardiovascular regeneration. However, the identities of these bone marrow cells and the signaling pathways that regulate their differentiation into functional EPCs remain poorly understood. Hematopoiesis consists of a cascade by which hematopoietic stem cells (HSCs) give rise to either a common myeloid progenitor (CMPs) or common lymphoid progenitor (CLPs). The CMP cells can give rise to either granulocyte-monocyte progenitors (GMPs) or megakaryocyte-erythrocyte progenitors (MEPs).

Methods and Results: Herein, we identify that CMPs and GMPs preferentially differentiate into CD34+KDR+ EPCs as measured by FACS, uptake of DiIacLDL and U.lex Lectin, matrigel studies, and endothelial markers. Co-culture studies in matrigel demonstrate increased network formation and incorporation when HUVECs were co-incubated with CMPs and GMPs only. Treatment of CMPs or GMPs with IFN-g blocked EPC differentiation, whereas treatment with TGF-b1 potently induced EPC differentiation. Conversely, EPC formation was markedly impaired using blocking antibodies or TGF-b1 deficient CMPs or GMPs, whereas IFN-g deficient mice have increased EPC differentiation. Functionally, EPCs derived from CMPs and GMPs more potently adhered to fibronectin-coated plates, migrated faster in transwell Boyden chambers, and released VEGF nearly ~2 to 2.5-fold higher than EPCs from MEPs and HSCs. Finally, we identified that a TGF-b1-responsive Kruppel-like transcription factor, KLF10, as robustly expressed ~60-fold in EPCs derived from CMPs and GMPs, compared to hematopoietic progenitors lacking EPC markers. Consistently, KLF10−/− mice have marked defects in EPC differentiation (−23.6% vs. WT, P<0.002), TGF-b1 responsiveness (completedly abolished), and impaired blood flow recovery in response to hindlimb ischemia (−23.8% vs. WT, P<0.008), an effect associated with reduced levels of circulating EPCs (−28.2% vs. WT, P<0.05).

Conclusion: Among bone marrow progenitors, a hierarchy exists for EPC differentiation. Targeting KLF10 may alter EPC differentiation from bone marrow-derived CMPs and GMPs and influence cardiovascular repair.
Abstract 5758: The Anti-Angiogenic Activity of rPAI-1
23
Promotes Vasa Vasorum Regression in Atherogenic Mice Through a Plasmin-Dependent Mechanism
Authors: Kehoe Mary Mulligan
Published: April 2024

Introduction
: Angiogenic vasa vasorum is considered the conduit for nutrient supplies to atherosclerotic plaque and its inhibition is associated with reduced plaque progression. We reported that the anti-angiogenic activity of rPAI-1
23
, a truncated PAI-1 protein, stimulates a highly significant decrease in vasa vasorum vessel area and length, plaque area and plaque cholesterol in the descending aorta in atherogenic mice. High levels of FGF-2 associated with adventitial vascular structures of saline treated mice appear to stimulate tube formation and vessel guidance, which is inhibited by rPAI-1
23
.

Objective
: In this study we investigated the mechanism of angiogenic vasa vasorum regression in atherogenic mice treated with rPAI-1
23
.

Methods and results
: Plasma from female LDLR
−/−
/ApoB-100 mice fed Paigen’s diet without cholate for 20 weeks while receiving rPAI-1
23
treatment (n = 21) for the last six weeks was compared to control mice treated with saline (n= 16). The rPAI-1
23
treated mice have significantly elevated plasmin, MMP-3 and MMP-2 activities accompanied by increased fibrinogen breakdown. Protease activities correspond with basement membrane breakdown visualized in confocal microscopy images of nidogen-probed adventitia. Immunoprecipitation of descending aorta adventitial protein pulls down a fibrinogen• FGF-2 complex in both treatment groups, but FGF-2 and fibrinogen levels are significantly less in rPAI-1
23
treated mice. In vitro studies show that FGF-2• fibrinogen complexes increase endothelial cell permeability and tube formation by 3- and 4-fold, respectively; rPAI-1
23
inhibits both angiogenic processes.

Conclusion
: In conclusion, rPAI-1
23
promotes vasa vasorum regression in atherogenic female LDLR
−/−
/ApoB-100 mice through a plasmin-dependent mechanism that negatively regulates the fibrinogenic and angiogenic pathways. Enhanced plasmin activity, in response to rPAI-1
23
, stimulates activation of MMPs known to degrade basement membrane and fibrinogen, thus leading to vessel collapse. Fibrinogen degradation inhibits the angiogenic potential of the fibrinogen•FGF-2 complex.
Abstract 5770: The Platelet Activity After Clopidogrel Termination (PACT) Study
Authors: Frelinger Andrew L , Barnard Marc R , Fox Marsha L , Michelson Alan D
Published: April 2024

“Rebound” platelet hyperreactivity upon discontinuation of clopidogrel has been proposed to lead to increased thrombotic risk including late stent thrombosis. However the hypothesis that discontinuation of clopidogrel results in platelet hyperreactivity has never been rigorously tested. To test this, we performed a prospective, randomized, double-blind, placebo-controlled, crossover study: the Platelet Activity after Clopidogrel Termination (PACT) study. Platelet reactivity was measured before, during clopidogrel 75 mg or placebo daily for 14 days, and on days 1, 4, 8, 11, 15 and 45 after discontinuation of clopidogrel or placebo in 15 healthy subjects. All subjects received aspirin 81 mg daily. Platelet reactivity was assessed by platelet surface activated GPIIb-IIIa and surface P-selectin, measured by whole blood flow cytometry, in response to ADP 0.5, 1, and 20
μ
M, thrombin receptor activating peptide (TRAP) 1 and 20
μ
M, and both collagen 5
μ
g/mL and epinephrine 5
μ
M, and by light transmission platelet aggregation with ADP 2.5, 5, and 20
μ
M, TRAP 2 and 20
μ
M, and collagen 6
μ
g/mL. Immature platelet fraction (IPF) was measured in the Sysmex XE-2100. At no time point after discontinuation of clopidogrel was platelet reactivity, as determined by all assay end points, or the IPF significantly greater than after discontinuation of placebo. The figure

shows representative results. In conclusion, this double-blind, placebo-controlled, crossover study demonstrates that discontinuation of clopidogrel does not result in “rebound” platelet hyperreactivity -as determined by multiple time points, assays, agonists, and agonist concentrations.

Fig.

ADP 1 μM-stimulated platelet surface activated GPIIb-IIIa before, during, and after treatment with placebo

or clopidogrel

.
Abstract 5786: Prostaglandin E
2
and EP3 Activation Potentiate Human Platelet Responsiveness
Authors: Rocca Bianca , Petrucci Giovanna , Patrono Carlo
Published: April 2024

Activated human platelets synthesize prostaglandin (PG) E
2
, although at much lower rates than thromboxane A
2
. PGE
2
binds different receptors (EP1–4). EP3-deleted mice displayed lower platelet response to sub-threshold concentrations of agonists and less susceptibility to experimental thrombosis than wild type mice. However, the role of PGE
2
in human platelet function remains poorly characterized. We investigated the effect of PGE
2
and EP-agonists on human platelet responsiveness to other agonists. Platelets were incubated with increasing concentrations of PGE
2
or different EP agonists, and stimulated by aggregating doses of adenosine diphosphate (ADP), collagen or arachidonic acid (AA). Aggregation was measured by the optical method. EP expression was studied by immunohistochemistry in platelets and bone marrow megakaryocytes. PGE
2
in the microM range (1–200 microM) dose-dependently inhibited 10microM ADP-induced aggregation. However nanoM PGE
2
concentrations (2–200 nanoM), dose-dependently increased the slope (velocity) of the secondary wave of 8 microM ADP-induced platelet aggregation, with an EC
50
of 25±6 nanoM (n=15), without affecting maximal aggregation. At lower (4 microM) ADP concentration, PGE
2
reverted the reversible aggregation, with an EC
50
of 29±10 nanoM (n=6). In
in vitro
aspirinated platelets, 200 nanoM PGE
2
in part counteracted the effect of aspirin in abolishing the secondary wave of 8 microM ADP-induced aggregation. In addition, PGE
2
dose-dependently abolished shape change caused by 10 microg/ml collagen and 50 microg/ml AA, with an IC
50
of 157±4 nanoM. The EP3 agonist, 11-deoxy-16,16-dimetyl PGE
2
, mimicked the PGE
2
effect on the secondary wave of ADP-induced aggregation, with an EC
50
58±1 nanoM (n=10), while the EP2 agonist butaprost was ineffective. The EP3 agonist at microM concentrations, dose-dependently induced spontaneous platelet aggregation with an EC
50
of 5.5±1 microM. Immunostains revealed EP3 expression in human platelets and megakaryocytes. We conclude that PGE
2
-EP3 interaction potentiates human platelet response to physiological concentrations of common agonists. EP3 might represent a novel pharmacological target for inhibiting platelet responsiveness in atherothrombotic diseases.
Abstract 5814: Vascular Lectin-Like-OxLDL Scavenger Receptor (LOX-1) Mediates Oxidative Stress, Endothelin-1, and Matrix Metalloproteinase Expression in the Vasculature of Vehicular Engine Emissions-Exposed Mice
Authors: Lund Amie K , Lucero JoAnn , Mathews Nadine , Harman Melissa , Lucas Selita’ , Campen Matthew J
Published: April 2024

A positive correlation between exposure to traffic-related air pollution and increased rates of cardiovascular morbidity and mortality has been well documented; however, underlying mechanisms have not yet been elucidated. We have reported that vascular oxidative stress levels are significantly elevated in atherosclerotic apolipoprotein KO (ApoE−/−) mice exposed to vehicular emissions, which appears to mediate increases in vascular matrix metalloproteinases (MMPs) and endothelin-1 (ET-1), expression of which are associated with progression of atherosclerosis. Since ROS production promotes formation of oxidized LDL (oxLDL), we investigated whether the effects of oxLDL on the vasculature, in response to exposure to vehicular emissions, are mediated via the lectin-like-ox-LDL receptor (LOX-1). To test this hypothesis, 10 wk old male ApoE−/− mice on a high fat diet received either mouse IgG (control, 16
μ
g protein/ml, 0.1 ml/mouse, i.p.) or the neutralizing antibodies to LOX-1 (anti-mouse LOX-1/SR-E1 antibody: 16
μ
g protein/ml, 0.1 ml/mouse, i.p.) every other day. Mice were randomly assigned to inhalational exposure of either filtered-air (FA: n=12 anti-LOX-1, n=12 IgG) or a mixed exposure of 250
μ
g PM/m
3
diesel exhaust + 50
μ
g PM/m
3
gasoline engine exhaust (n=12 anti-LOX-1, n=12 IgG) for 6 hr/day for 7 days. Analysis showed significant elevations in oxLDL and aorta oxidative stress from mice exposed to vehicular exhaust compared to FA. Anti-LOX-1 treatment ameliorated the increase in circulating oxLDL, as well as vascular lipid peroxidation in vehicular exhaust-exposed animals. Aorta LOX-1, ET-1, MMP-9 and tissue inhibitor of MMPs, TIMP-1 and -2 mRNA expression, as well as MMP-9 protein and activity, were also observed to be significantly elevated with exposure to mixed vehicular exhaust, of which LOX-1, ET-1 and MMP-9 were attenuated through LOX-1 Ab treatment. In agreement with LOX-1 mRNA findings, LOX-1 protein and soluble LOX-1 (sLOX-1) plasma levels were also found increased in exposure animals. Such findings indicate that effects of ROS (and oxLDL), as well as expression of vascular factors associated with progression of atherosclerosis (ET-1 and MMPs), induced by vehicular exhaust exposure, are likely mediated through the LOX-1 receptor.
Abstract 16698: Induction of Mesp1 by Brachyury(t) Generates the Common Multipotent Cardiovascular Stem Cell
Authors: David Robert , Jarsch Veronica , Schwarz Florian , Franz Wolfgang-Michael
Published: April 2024

Aims:
Our recent work demonstrated that common cardiovascular progenitor cells are characterized and induced by the expression of the transcription factor MesP1 in vertebrate embryos and murine embryonic stem cells. As the proliferative potential of stem cell derived cardiomyocytes is limited, it is crucial to understand how MesP1 expression is mediated, in order to achieve reasonable and reliable yields for novel stem cell based therapeutic options. As potential upstream regulators of MesP1 we therefore analysed Eomes and Brachyury(T), which had been controversely discussed to be crucial for cardiovasculogenic lineage formation.

Methods and results:
Wild-type and transgenic murine embryonic stem cell lines, mRNA-analyses, embryoid body formation, and cell sorting revealed that the MesP1 positive population emerges from the Brachyury(T) positive fraction.
In situ
hybridizations using wild-type mouse embryos confirmed that Brachyury(T) colocalises with MesP1
in vivo
. Likewise, shRNA based loss of Brachyury(T) causes a dramatic decrease of MesP1 expression accompanied by reduced cardiac markers in differentiating embryonic stem cells, which is reflected
in vivo
via
in situs
using Brachyury(T) knock-out embryos where MesP1 mRNA is greatly abolished. We finally defined a 3.4 kb proximal MesP1-promoter fragment which is directly bound and activated by Brachyury(T) via a T responsive element as shown via Bandshift, Chromatin Immuneprecipitation and Reporter Assays.

Conclusion:
Our work contributes to the understanding of the earliest cardiovasculogenic events and may become an important prerequisite for cell therapy, tissue engineering and pharmacological testing in the culture dish using pluripotent stem cell derived as well as directly reprogrammed cardiovascular cell types.
Abstract 16804: Long-Term Differential Effects of Amlodipine and Atenolol based Antihypertensive Therapy on Left Ventricular Structure and Function: An ASCOT Sub-Study
Authors: Barron Anthony J , Tapp Robyn J , Sharp Andrew , Baksi Arun J , Hughes Alun D , Mayet Jamil ,   ASCOT Investigators
Published: April 2024

Background:
Both left ventricular hypertrophy (LVH) and impairment of LV diastolic function have been shown to be important predictors of morbidity and mortality in hypertension. Anti-hypertensive treatment can cause regression of LVH and improve LV diastolic function. However little is known about whether these changes are sustained with long term treatment.

Methods:
Six-hundred and sixty-nine patients (mean age 62 years, 78.9% male) in the ASCOT study who were randomised to either an amlodipine-based regimen (Amlod), or an atenolol-based antihypertensive regimen (Aten) had LV structure and diastolic function assessed by tissue Doppler echocardiography. Measurements were made a median time of 1.48 (Phase 1) and 3.49 (Phase 2) years after randomisation.

Results:
Blood pressure was lower in Amlod both at phase 1 (141/80 vs 146/83) and phase 2 (138/79 vs 143/82).Left ventricular mass index had significantly improved between Phase 1 and Phase 2 assessment in both treatment groups (Amlod: 119.6 to 116.6 g/m
2
, p=0.007, Aten: 121.8 to 117.2 g/m
2
, p<0.001).At the initial Phase 1 assessment LV diastolic function was better in Amlod compared with Aten (e' 8.5cm/s versus 7.7cm/s, p>0.001; E/e' 7.6 versus 8.1, p=0.008). With a further 2 years of antihypertensive therapy with good BP control there was a small, significant drop in e' but no change in E:e'with Amlod (e' Phase 2 8.1cm/s, versus phase 1 p<0.001; E/e' Phase 2 7.8, versus phase 1 p=0.29); in contrast in Aten there was no change in e' but a small, significant rise in E:e' (e' Phase 2 7.7cm/s, versus phase 1 p=0.59; E/e' Phase 2 8.4, versus phase 1 p=0.02).By the end of the phase 2, after correction for age, gender and systolic blood pressure, e' in Amlod remained significantly higher than aten (p<0.01).

Conclusions:
Patients with hypertension and good BP control had further regression of LV mass between 1.5 and 3.5 years of antihypertensive therapy with both Amlod and Aten. Those receiving Amlod had better echocardiographic indices of diastolic function at both time points when compared with Aten. Appropriate antihypertensive therapy may lead to early improvement of diastolic function and allow maintenance of a more normal pattern at longer follow-up with improvement in left ventricular mass.
Character Count: 1926
Abstract 16815: Cholesterol Efflux Capcity in Genetic Forms of HDL Deficiency
Authors: Alrasheed Sara
Published: April 2024

Objective:
High-density lipoprotein cholesterol (HDL-C) deficiency is the most frequent lipoprotein abnormality in patients with premature coronary artery disease. In order to determine the functionality of HDL particles in genetically defined HDL deficiency (<5th percentile), we employed a cell-based assay to examine ABCA1-dependent cholesterol efflux capacity in apoB-depleted plasma isolated from 19 probands of French Canadian descent. First degree relatives with normal HDL levels served as controls.

Methods and Results:
We determined the ability of apoB-depleted plasma to promote cholesterol efflux from [
3
H] cholesterol-labeled, cAMP-stimulated J774 macrophages. Cases consisted of 16 ABCA1 heterozygotes, and three APOA1 E136X heterozygotes. We obtained evidence that ApoB-depleted plasma isolated from subjects with genetic forms of HDL deficiency had significantly reduced levels of ABCA1-mediated cholesterol efflux as compared to sibling controls (72.33 %). Conversely, when normalized to apoA-I levels, the efflux capacity of apoB-depleted plasma from subjects was attenuated as compared to sibling controls.

Linear regression was used to determine which biochemical measurement (apoA-I concentration, HDL-C level and HDL subfractions quantified by 2DPAGGE) best explained the cholesterol efflux results. A strong, positive correlation was found between cholesterol efflux measurements and apoA-I levels, HDL-C levels, alpha-2 HDL levels, and alpha-3 HDL levels (all p< 0.05). However, HDL-C levels only explained approximately 27% of the variability in cellular cholesterol efflux, suggesting that efflux capacity may help better understand HDL functionality.

Conclusion:
While HDL obtained from subjects with ABCA1 or APOA1 gene defects had decreased cellular cholesterol efflux compared with sibling controls, this difference was markedly attenuated when corrected for apoA-I mass, suggesting that such particles remain functional in cholesterol efflux through ABCA1.
Abstract 16838: Genetics of Insulin Response to Fenofibrate in the Goldn Study
Authors: Irvin Ryan , Wood Alexis C , Vaughan Laura Kelly , Kabagambe Edmond , Sha Jin , Tiwari Hemant , Borecki Ingrid , Ordovas Jose , Arnett Donna
Published: April 2024

Background:
Fenofibrate is an effective treatment for hypertriglyceridemia but also improves cholesterol levels, markers of inflammation, and insulin resistance. Fenofibrate binds to the transcription factor peroxisome proliferator-activated receptor-α (PPARα) which regulates expression of a number of genes. How fenofibrate improves measures of insulin resistance is not completely understood. We sought to determine whether there are genes that influence the effect of fenofibrate on measures of insulin resistance.

Methods:
We performed a genome wide association study (GWAS) of change in fasting insulin after fenofibrate treatment in the Genetics of Lipid Lowering and Diet Network (GOLDN) study. We calculated change in fasting insulin concentration among 750 GOLDN participants without diabetes that were treated with fenofibrate (160 mg/day) for 3 weeks. Genotyping was done with Affymetrix Human SNP Array 6.0. Of one million SNPs genotyped, 718, 542 met the genotype quality control criteria and were tested for association with change in fasting insulin. Genetic variants were modeled using multivariable linear regression models adjusting for age, sex, center, and family relationship.

Results:
The three most statistically significant loci were near inositol 1,4,5-triphosphate receptor, type 2 (
ITPR2
, rs16930147,
P
=2.6*10
-7
), monooxygenase DBH-like 1 isoform 2 (
MOXD1
, rs7751860,
P
=1.3*10
-6
) and chromatin modifying protein 4C (
CHMP4C
, rs11996762,
P
=1.4*10
-6
).
ITPR2
has been associated with waist-to-hip ratio and is highly expressed in human adipocytes. MOXD1 has been shown to be upregulated in the livers of transgenic mice with reduced bioavailability of insulin. Finally, CHMP4C has been implicated in p53 regulation of the endosomal compartment in response to stress where the endosome plays a major role in intracellular cholesterol metabolism.

Conclusions:
The results show novel loci that may play a role in insulin response to fenofibrate treatment, and can shed light on the pathways underlying this treatment benefit which can help reduce cardiovascular disease risk in treated patients.
Abstract 16840: AMP-Activated Protein Kinase Prevents Myocardial Injury During Ischemia-Reperfusion by Suppressing the c-Jun Terminal Kinase Pathway and Inhibiting Mitochondrial Transition Permeability Pore Opening
Authors: Zaha Vlad G , Qi Dake , Palmeri Monica , Young Lawrence H
Published: April 2024

AMP-activated kinase (AMPK) is a stress responsive kinase that coordinates energy conservation, activating catabolic pathways and inhibiting fuel storage and protein synthesis. In the heart, functional AMPK is essential to myocardial survival and functional recovery following ischemia-reperfusion. The mechanisms by which intrinsic AMPK activation protects against ischemic injury are not well understood. We investigated the role of AMPK in modulating mitogen activated protein kinase pathway and mitochondrial integrity during ischemia-reperfusion. Hearts from wild type (WT) and kinase dead AMPK (KD) mice were perfused with 7mM glucose, 0.4 mM oleate, 1% BSA and 10µU/mL insulin in vitro and subjected to 15 min of global ischemia and 10 min reperfusion. KD hearts had similar baseline contractile function, but markedly decreased recovery of left ventricular developed pressure (LVDP) after ischemia-reperfusion compared to WT (4.6±1.4 vs. 26.4±5.2% of baseline, n=9 and 11, p<0.05). Mitochondria isolated from non-ischemic KD and WT hearts had similar calcium induced, cyclosporin-A inhibitable mitochondrial permeability transition pore (mPTP) opening (29.3±1.9 vs. 23.2±1.6µM calcium concentration at mPTP opening, n=4 per group). However, following ischemia-reperfusion mPTP opening was triggered by significantly less calcium in KD mitochondria compared to WT (9.0±0.6 vs. 12.6±1.3µM calcium, n=5 and 6, respectively, p<0.05), indicating higher susceptibility of KD mitochondria to mPTP opening. KD hearts had increased phosphorylation of mitogen activated protein kinase kinase 4 (MKK4) and downstream c-Jun terminal kinase (JNK) during early reperfusion compared to WT hearts (p<0.05). Pharmacologic inhibition of JNK during ischemia-reperfusion with SP600125 (10µM) resulted in improved recovery selectively in KD hearts (4.6±1.4 vs. 15.3±6.5%, of baseline, n=4 and 5, respectively, p<0.05), without changes in WT. JNK inhibition also eliminated the increased susceptibility of mitochondria from KD hearts to mPTP opening. In conclusion, intrinsic AMPK activation prevents cardiac injury during ischemia-reperfusion, suppressing activation of signaling through the MKK4-JNK pathway and inhibiting mitochondrial transition pore opening.