bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5130: Leptin Increases Perihematomal Inflammation in Experimental Hemorrhagic Stroke
Authors: Kim Chi K , Ryu Wi-Sun , Kim Beom J , Lee Seung-Hoon , Yoon Byung-Woo
Published: April 2024

Background
: Leptin, one of the most alleged adipokines, exerts its tissue-protective effect in ischemic injuries by reducing apoptosis through extracellular signal-regulated kinase pathway. In this context, it may be speculated that leptin also confer beneficial effect on hemorrhagic events. However, few studies have elaborated on the association between exogenous leptin and tissue damage in experimental hemorrhagic stroke model.

Methods
: After inducing intracerebral hemorrhage in mouse brain via the sterotaxic infusion of collagenase, 4mg/kg or 8mg/kg of recombinant leptin or vehicle (phosphate buffered saline) was administered intraperitoneally. Brain water content, reflecting peri-hematomal edema, and hemorrhage volume were measured at 72 hours after inducing hemorrhage. Inflammatory cells expressing myeloperoxidase (MPO) or OX6 were counted at 48 hours, and western blotting for Akt, pAkt, STAT3, pSTAT3, ERK and pERK was performed at 24 hours.

Results
: Compared to the vehicle injected mice, exogenous leptin increased brain water contents (80.07±0.29% in vehicle group, 80.74±0.32% in 4mg/kg leptin, and 81.48±0.18% in 8mg/kg leptin; rho=0.525 and p<0.01). Based on this result, all the following experiments were performed with 8mg/kg of leptin. Leptin injection also augmented infiltration of MPO-stained cells (210±8 cells/mm
3
in leptin group and 133±8 cells/mm
3
in vehicle group; p<0.05) and OX6-stained cells (129±13 cells/mm
3
in leptin group and 88±10 cell/mm
3
in vehicle group; p<0.05). Mean hemorrhage volume was also increased in 8mg/kg leptin group (26.83±3.22 mm
3
in leptin group and 17.98±2.73 mm
3
in vehicle group; p<0.05). The expression level of phosphorylated STAT3 was significantly elevated in mice with 8mg/kg leptin injection (p<0.05), however, pAkt and pERK did not augmented by induced hyperleptinemia.

Conclusion
: Our study documented that peri-hematomal inflammation and hemorrhage volume were increased by exogenous leptin via STAT3 pathway. These results may indicate the potentially detrimental role of leptin on hemorrhagic stroke via augmentation of inflammatory response after tissue damage.
Abstract 5154: Isolated Nocturnal Hypertension is Associated With Pre-clinical Vascular Pathology in People of African Descent
Authors: Maseko Muzi J , Woodiwiss Agela J , Norton Gavin R
Published: April 2024

Recent large clinical studies indicate that an inability to decrease blood pressure (BP) at night is associated with excessive cardiovascular morbidity and mortality. In a randomly selected population sample of subjects (40% hypertensive, 60% overweight and obese, and 8% diabetic), in whom 458 had high quality 24-hour ambulatory BP monitoring and target organ assessments, we determined whether isolated nocturnal hypertension was associated with renal and vascular target organ changes. Pre-clinical renal target organ changes were assessed from urinary microalbumin-to-creatinine ratios (ACR) determined from 24-hour urine samples, and pre-clinical vascular pathology was assessed from carotid-femoral pulse wave velocity (PWV) determined using applanation tonometry. software. In the study sample 7.6% of participants had isolated nocturnal hypertension. Compared to the normomotensives, the isolated nocturnal hypertensives had a higher BMI (31 versus 27). In multivariate regression analysis adjusting for multiple confounders, participants with increased 24-hour BP values had a greater PWV (p<0.0001) and an increased urinary ACR (p<0.0001) as compared to participants with a normal 24-hour, day and night BP. Similarly, participants with isolated nocturnal hypertension, had an increased PWV as compared to participants with a normal 24-hour, day and night BP (p<0.0005). However, participants with isolated nocturnal hypertension had similar urinary ACR values as compared to participants with a normal 24-hour, day and night BP (p=0.62). There was also a strong relationship between nocturnal hypertension and both diabetes (p<0.017) and waist to hip ratio (p<0.038). Isolated nocturnal hypertension is associated with waist to hip ratio, diabetes and pre-clinical vascular but not renal target organ changes in a population sample of African ancestry. As pre-clinical vascular pathology, diabetes and increased waist to hip ratio are independent predictors of cardiovascular outcomes, these data suggest that outcome studies are required to determine whether isolated nocturnal hypertension is a clinical entity that requires therapeutic intervention and lifestyle changes.
Abstract 5195: Forced Angiogenesis in Adventitia Promotes Plaque Formation in Abdominal Aorta of Apolipoprotein E-deficient Mice
Authors: Tanaka Kimie , Nagata Daisuke , Hirata Yasunobu , Tabata Yasuhiko , Nagai Ryozo , Sata Masataka
Published: April 2024

Background:
Accumulating evidence suggests that exaggerated formation of vasa vasorum plays an important role in the pathogenesis of atherosclerotic plaque progression. It was reported that enhanced angiogenesis in adventitia promoted lesion progression after mechanical vascular injuries. However, it remains unclear whether augmented angiogenesis in adventitia could promote hyperlipidemia-induced atherosclerotic lesion formation.

Methods:
We investigated whether local administration of slow-releasing form (30 days) of basic fibroblast growth factor (bFGF) promotes plaque progression with enhanced perivascular angiogenesis. bFGF (100
μ
g/body) incorporated in acid gelatin hydrogel microspheres (AGHMs) (bFGF+AGHM group, n=4), AGHMs (AGHM group, n=7), or PBS (control group, n=9) was administrated into periaortic areas of retroperitoneal space of 10 to 11-week-old male ApoE-deficient mice. The mice were fed on western type diet. The abdominal aorta was harvested with perivascular soft tissues at 13 weeks after the operation. Staining with
Lycopersicon Esculentum
(Tomato) lectin was performed to visualize micro-vessels at 5 different sections from each aorta.

Results
: Larger lesions were observed only in the bFGF+AGHMs group ( bFGF+AGHM: 3.5×10
4
±1.2 × 10
4
μ
m
2
, AGHM:0.8 × 10
4
±0.7×10
4
μ
m
2
, control 0
μ
m
2
, p<0.005). Plaque formation was associated with increased neovascularization in the adventitia. When the aortic section were divided into 16 radial areas, there was a relation between the number of capillaries and the plaque size (r=0.77, p<0.001). In time course study, proliferation of vasa vasorum and accumulation of Mac3-positive macrophages were observed prior to atherosclerotic lesion formation.

Conclusions:
Local administration of bFGF in adventita induces development of vasa vasorum, which potentially accelerates plaque lesion progression in ApoE-deficient mice.
Abstract 5199: Oral Administration of an Active Form Vitamin D
3
Decreases Atherosclerosis in Mice via Modulating Immune Cell Phenotypes and Functions
Authors: Takeda Masafumi , Yamashita Tomoya , Shinohara Masakazu , Sasaki Naoto , Nakajima Kenji , Kita Tomoyuki , Hirata Ken-ichi
Published: April 2024

Background and Objectives:
Recent several clinical studies demonstrated the low level of plasma vitamin D
3
would be a risk of cardiovascular events. An active form vitamin D
3
not only regulates bone metabolisms but also has an immunosuppressive and anti-inflammatory actions which target dendritic cells (DCs) and T lymphocytes. Vitamin D
3
has been reported to inhibit the maturation of DCs and to induce regulatory T cells (Tregs) in experimental autoimmune disease models. We hypothesized an active form vitamin D
3
(calcitriol), could prevent atherosclerosis through changing immune responses.

Method and Results
: Six-week-apolipoprotein E knockout mice on a standard diet were orally administered 200 ng calcitriol dissolved in carboxymethyl cellulose (n=12) or carboxymethyl cellulose alone (n=15; control) twice a week for 12 weeks and atherosclerosis was assessed at 18 weeks old. Calcitriol administration decreased atherosclerotic lesion 39.1 % at aortic sinus compared to control group (mean plaque area: 0.18±0.05×10
6
vs. 0.27±0.07×10
6
μ
m
2
, p<0.01) without lowering cholesterol. The drug was also shown to reduce the infiltration of inflammatory cells to the atherosclerotic lesions by immunohistochemistry. To evaluate the induction of Tregs and immature state of DCs (iDC) in small intestine and other organs, we examined the ratio of Tregs and iDC by flow cytometry in mesenteric lymph nodes (MLNs) and spleens. Calcitriol markedly increased CD4
+
CD25
+
FoxP3
+
Tregs in MLNs by 17.1% and spleens by 23.4% (p<0.01), and further reduced the mature state (CD11c
+
CD80
+
CD86
+
) DCs in MLNs by 10.6% (p=0.02) compared to control. RT-PCR demonstrated mRNA expressions of CD80 and interferon-gamma were markedly decreased in MLNs and spleens in calcitriol-treated group, respectively.

Conclusion
: An active form vitamin D
3
could prevent the development of atherosclerosis via changing the function or differentiation of DCs and T lymphocytes. At least, we demonstrated calcitriol increased the number of Tregs and reduced the mature state DCs in the present study, though further studies are required to examine the role of iDC and Tregs in this process. This treatment could be a potential immunotherapy for preventing atherosclerosis and must contribute to the clinics.
Abstract 5203: Arterial Stiffness and Urinary Albumin Excretion in Hypertension: The Emerging Role of the Soluble Receptor for Advanced Glycation End Products
Authors: Dimitriadis Kyriakos , Tsioufis Costas , Thomopoulos Costas , Andrikou Ioannis , Tatsis Hraklis , Kalovidouris Nikolaos , Tzamou Vanessa , Tsiamis Eleftherios , Stefanadis Christodoulos
Published: April 2024

Introduction:
Current evidence suggests that the soluble receptor for advanced glycation end products (sRAGE) participates in the development of vascular disease, while albuminuria and arterial stiffening are associated with atherosclerosis progression.

Hypothesis:
Urinary albumin excretion, expressed as the albumin to creatinine ratio (ACR), may be related to sRAGE and arterial stiffness in essential hypertensives.

Methods:
Our population consisted of 80 newly diagnosed untreated non-diabetic patients with stage I to II essential hypertension [48 men, mean age=52 years, office blood pressure (BP)=145/93 mmHg]. According to the ACR values determined as the mean of two non-consecutive morning spot urine samples, patients were divided into microalbuminurics (n=27) (mean ACR=30 –300 mg/g) and normoalbuminurics (n=53) (mean ACR<30 mg/g). Moreover, arterial stiffness was evaluated on the basis of carotid to femoral pulse wave velocity (PWV), by means of a computerized method (Complior SP).

Results:
Microalbuminurics compared to normoalbuminurics were older (53±6 vs 49±5 years, p<0.05), had higher 24-h systolic BP (140±13 vs 132±12 mmHg, p=0.001), while did not differ regarding sex and metabolic profile (p=NS). Moreover, microalbuminurics compared to normoalbuminurics exhibited higher levels of PWV (8.9±1.8 vs 7.9±1.5 m/sec, p<0.0001), whereas had lower sRAGE (1011±458 vs 1503±931 pg/ml, p=0.003), independently of confounders. In the total population, ACR was positively related to 24-h systolic BP (r=0.514, p<0.0001) and PWV (r=0.588, p<0.0001), whereas it was negatively correlated with sRAGE (r=−0.275, p=0.018). Regarding sRAGE, it was associated with body mass index (r=−0.241, p=0.003), 24-h pulse pressure (r=−0.372, p=0.001) and PWV (r=−0.403, p<0.0001). Multiple regression analysis revealed that age, 24-h systolic BP, PWV and sRAGE were the independent predictors of ACR (
R

2

=0.49, p<0.0001).

Conclusions:
In hypertension, microalbuminuria is accompanied by pronounced arterial stiffening and diffuse vascular dysfunction as reflected by lower sRAGE levels. Moreover, the association of sRAGE with ACR and PWV, underscores the catalytic role of this receptor in the progression of the hypertensive atherosclerotic disease.
Abstract 5219: Reduced Number of CD34+ Circulating Progenitor Cells Predict Long-term Cardiovascular Outcome in Chronic Kidney Disease Patients With Hemodialysis
Authors: Tanaka Miho , Ishii Hideki , Aoyama Toru , Kamoi Daisuke , Kawamura Yoshihiro , Kumada Yoshitaka , Takahashi Hiroshi , Maruyama Syuichi , matsuo Seiichi , Murohara Toyoaki
Published: April 2024

Background:
Patients with chronic kidney disease(CKD) on hemodialysis (HD) are the highest risk population for cardiovascular (CV) disease, and endothelial dysfunction is regarded as an important cause of CV risk in this population. Circulating endothelial progenitor cells (EPCs) are believed to be involved in maintenance of endothelial integrity, and are close proximity to CD34+ circulating progenitor cells (CPCs). We prospectively examined whether reduced number of CD34+ CPCs would predict long-term CV outcome in HD patients.

Methods:
The number of CD34+ CPCs was determined with the use of flow-cytometry in blood sample before dialysis session in 216 outer HD patients. To avoid the influence of body fluid overload, the ratio of CD34+ CPCs to peripheral mononuclear cells (CD34+/PMNCs) were calculated. The patients were divided into tertiles according to CD34+/PMNCs levels; tertile 1 (T1): <0.012, T2: 0.013–0.022 and T3: >0.023 (n=72 each), and were prospectively followed for 4 years.

Results:
During 42±10months, 67 CV events and 35 deaths (21 CV deaths) occurred. Four-year event-free survival were 54%, 68% and 82% for CV events (p=0.0013), 78%, 92% and 98% for CV death (p=0.0009) and 73%, 84% and 93% for all-cause death (p=0.0058) in T1, T2 and T3, respectively. On Cox multivariate analysis, reduced CD34+CPCs levels were independent predictor for CV events (HR 2.40, 95%CI 1.25–4.62, p=0.028 for T1 vs. T3), for CV death (HR 5.46, 95%CI 1.18–25.26, p=0.029 for T1 vs. T3) and for all-cause death (HR 3.72, 95%CI 1.37–10.09, p=0.030 for T1 vs. T3), respectively.

Conclusion:
These data suggest that reduced number of CD34+ CPCs predicts long-term CVoutcome in CKD patients with HD.
Abstract 5226: Endothelial Nox4 NADPH Oxidase Enhances Vasodilatation via Hydrogen Peroxide-induced Hyperpolarization and Reduces Blood Pressure
Authors: Wang Minshu , Ray Robin , Murdoch Colin , Zhang Min , Walker Simon , Brewer Alison , Shah Ajay
Published: April 2024

Introduction NADPH oxidases (Noxs) are reactive oxygen species-generating enzymes implicated in cardiovascular disease. Nox4 is the most abundantly expressed isoform in endothelial cells but its function is unclear. We investigated the role of endothelial Nox4 on vascular function
in vivo
.

Methods and Results We generated transgenic mice with endothelium-specific overexpression of Nox4 (Nox4TG) and studied the effects on endothelial function (aortic rings ex vivo) and blood pressure (BP, telemetry). Nox4 protein levels were 2-fold higher in Nox4TG aorta compared to wild-type littermates (WT), with no changes in expression of other Nox isoforms. Nox4TG had enhanced relaxation to acetylcholine (ACh) than WT (−log EC50 7.76±0.07 vs. 7.20±0.05; n=12; p<0.001) but similar relaxation to sodium nitroprusside. The ACh response in Nox4TG and WT was identical in the presence of catalase (1500U/ml) but remained greater in Nox4TG in the presence of inhibitors of nitric oxide synthesis (L-NMMA, 100
μ
M; −log EC50 7.44±0.22 vs. 6.93±0.22; n=6; p<0.01), soluble guanylate cyclase (ODQ, 5
μ
M; −log EC50 6.92±0.21 vs. 6.78±0.30; n=6; p<0.001) or protein kinase G (KT5823, 2
μ
M; −log EC50 7.77±0.11 vs. 7.27±0.12; n=6; p<0.001). However, the difference between Nox4TG and WT was abolished in the presence of high extracellular potassium (30mM). Nox4TG also had significantly lower BP than WT (mean BP 102.5±1.8 vs. 109.5±2.0mmHg; n=10; P<0.05), which was abolished after chronic treatment with N-acetylcysteine or an SOD/catalase mimetic, EUK-8. Plasma nitrite/nitrate levels and aortic levels of phosphorylated VASP were identical and acute iv treatment with L-NMMA (10mg/kg) increased BP to a similar extent in Nox4TG and WT. The hypertensive response to chronic 14-day angiotensin II infusion (1.1mg/kg/d) was lower in Nox4TG compared to WT (mean BP 116.7±4.7 vs. 129.4±3.5mmHg; n=10; P<0.05).

Conclusions Nox4TG had significantly enhanced ACh-induced vasodilatation compared to WT as a result of H
2
O
2
-induced hyperpolarization. Nox4TG also had a lower BP, which was not attributable to altered nitric oxide bioactivity but was normalized by chronic antioxidant treatment. These results suggest that endothelial Nox4 has potentially beneficial effects on vascular tone and BP.
Abstract 5257: Inhibition of Myosin Light Chain Phosphatase by Rho Kinase (ROCK) Modulates Arteriolar Tone and Constriction of Coronary Arterioles
Authors: Lu Guangrong , Hein Travis W , Xu Xin , Kuo Lih
Published: April 2024

ROCK has been suggested to regulate smooth muscle contraction of conduit artery via multiple pathways including direct myosin light chain (MLC) phosphorylation or inhibition of MLC phosphatase (MLCP) activity. Although recent studies have implicated the involvement of ROCK in pathogenesis of cardiovascular disease, the role and expression of ROCK isoforms (ROCK1 and ROCK2) and their signaling pathway for vasomotor regulation in resistance arterioles remain unclear. Using an isolated and pressurized vessel approach, we found that the resting tone and the endothelin-1 (ET-1)-induced constriction of porcine coronary arterioles (60±4
μ
m resting diameter) were preferentially inhibited, in a dose-dependent manner, by a potent ROCK2 inhibitor H-1152 compared to a non-selective ROCK antagonist Y-27632. MLCP inhibitor calyculin A reversed the inhibitory effects of H-1152 and Y-27632, but ROCK inhibition failed to prevent or reverse calyculin A-induced arteriolar constriction. These results indicate that ROCK is involved in the development of basal tone and vasoconstriction to ET-1 and that MLCP is a major target of ROCK. Immunoblotting and immunohistochemical data showed that ROCK2 expression is 2-fold higher than ROCK1 in coronary arterioles with preferential media-layer distribution. Both ROCK1 and ROCK2 were also expressed in cultured porcine coronary arteriolar smooth muscle cells (PCASMC) with ROCK2 as the major isoform. In cultured PCASMC, MLC phosphorylation (ser19) elicited by ET-1, thromboxane analogue (U46619) and 80 mM KCl was sensitive to H-1152, supporting the involvement of ROCK2 in regulating coronary arteriolar tone via MLC phosphorylation. Consistent with our functional data, calyculin A increased MLC phosphorylation (ser19) in PCASMC, but ROCK2 inhibition (by H-1152) failed to reverse such effect. We conclude that ROCK2 is the major isoform mediating the development of basal tone and vasoconstrictor activity in coronary resistance arterioles primarily via regulation of MLCP activity.
Abstract 5262: Progressive Decline in C-Type Natriuretic Peptide With Aging is Associated With Cardiorenal Fibrosis, Myocardial Dysfunction and Proteinuria
Authors: Sangaralingham S. Jeson , Martin Fernando L , Ichiki Tomoko , McKie Paul M , Mohammed Selma F , Harders Gerald E , Oehler Elise A , Chen Horng H , Redfield Margaret M , Burnett John C
Published: April 2024

Background:
C-type natriuretic peptide (CNP) is of endothelial cell origin and represents the most potent anti-fibrotic peptide of the natriuretic peptide family and is important for extracellular matrix homeostasis including mediating bone growth in the young. Cardiorenal aging is associated with altered cardiac and renal structure and function that may contribute to the development of heart and kidney failure, particularly in the elderly. The impact of aging on circulating CNP and associated changes in cardiorenal structure and function are undefined.

Hypothesis:
We hypothesized that a decrease in endogenous plasma CNP occurs with aging and is associated with an increase in cardiorenal fibrosis and altered left ventricular (LV) and renal structure and function.

Methods:
Studies were performed in 2, 11 and 20 month old male Fischer rats (n = 8 –10/group). Echo was used to assess LV structure and function. Blood pressure (BP), urinary protein excretion, plasma B-type natriuretic peptide (BNP) and CNP were measured. Kidneys and LV were harvested for gross and histology analysis. Mean±SE, *p<0.05.

Results:
Aging from 2 to 20 months was associated with progressive reductions in plasma CNP (30±3 to 21±1* to 9±1*pg/ml). Significant and progressive cardiac and renal fibrosis were observed with aging (LV: 9±0.3 to 15±0.7* to 21±1*% and kidney: 1±0.3 to 8±2* to 17±1* %). In contrast to CNP, plasma BNP was modestly increased from the 2 to 20 month old group (21±2 to 26±1*pg/ml). The reduction in CNP and increase in BNP, the latter a biomarker for LV dysfunction, were associated with decreased EF (88±1 to 80±1*%) and increased LVEDd (6.61±0.09 to 7.48±0.09*mm). BP and urinary protein excretion were mildly elevated with aging (91±1 to 101±3* mmHg and 8±0.3 to 20±3* mg/day, respectively).

Conclusion:
We report for the first time that aging is associated with a progressive decline in circulating CNP and progressive cardiorenal fibrosis, systolic dysfunction and proteinuria. Further studies are warranted to explore the hypothesis that a mechanism of cardiorenal aging with altered cardiorenal structure and function may include a decrease in the bioavailability of CNP.
Abstract 5269: Asymmetric Dimethylarginine Triggers Vasculo-Renal Interaction in CKD Mice
Authors: Kajimoto Hidemi , Kai Hisashi , Yasuoka Suguru , Anegawa Takahiro , Aoki Hiroki , Imaizumi Tsutomu
Published: April 2024

Background: Patients with chronic kidney disease (CKD) also suffer increased morbidity and mortality from cardiovascular diseases (CVD). Endothelial dysfunction is a major factor underlying the pathogenesis of CVD. Asymmetric dimethylarginine (ADMA), an endogenous endothelial nitric oxide synthase (eNOS) inhibitor, is elevated in CKD. We hypothesized that ADMA has a role in endothelial dysfunction in CKD.

Methods and Results: We created a CKD model by performing 5/6 nephrectomy (Nx) in mice. Blood and 24-hour urine were collected for measurements of renal function and ADMA, and NOx levels at 4 weeks after Nx. Nx induced renal failure (serum blood urea nitrogen, controls: 26.9±1.6 mg/dL, Nx mice: 61.0±3.8 mg/dL, p <0.001; serum creatinine, controls: 0.19±0.01 mg/dl, Nx mice: 0.31±0.03 mg/dL, p <0.01) without changing blood pressure. The serum ADMA level was higher (p <0.01) in Nx mice (1.20±0.05 nmol/mL) than in controls (1.00±0.05 nmol/mL). Urinary NOx was lower in Nx mice (controls: 2153±189 nmol/L, Nx mice: 706±335 nmol/L, p <0.05). Isometric tension study using aortic rings showed that endothelium-mediated relaxation induced by acetylcholine was impaired in Nx mice. Furthermore, pretreatment with exogenously administered ADMA at a serum concentration comparable to that in Nx mice reduced acetylcholine-induced relaxation in the aorta of controls. Western blotting showed the phosphorylated eNOS level of the aorta was lower in Nx mice than in controls, whereas eNOS levels were similar. The expression level of dimethylarginine dimethylaminohydrolase-1 (DDAH-1), a degradation enzyme of ADMA, was reduced in the kidney, but not in the aorta of Nx mice. In an ex vivo experiment using cultured human umbilical vein endothelial cells, ADMA treatment with a concentration corresponding to the serum level in Nx mice reduced the eNOS phosphorylation level.

Conclusions: Elevated levels of circulating ADMA, probably resulting from decreased DDAH-1 expression in the kidney, appear to impair endothelial function by inhibiting eNOS activity in CKD. ADMA may thus have a triggering role in the vasculo-renal interaction in CKD.
Abstract 5312: Deletion of Microsomal Prostaglandin E
2
Synthase-1 Modulates the Response to Vascular Injury in Mice
Authors: Wang Miao , Tamby Mathieu C , Ihida-Stansbury Kaori , Yu Zhou , Cheng Yan , Jones Peter L , FitzGerald Garret A
Published: April 2024

Microsomal (m) prostaglandin (PG) E
2
synthase(S)-1 catalyzes the isomerization of the cyclooxygenase (COX) product, PGH
2
, into PGE
2
. Previous studies in mice suggest that targeting of mPGES-1 may less likely cause hypertension or thrombosis than COX-2 selective inhibitors
in vivo
. Indeed, deletion of mPGES-1 retards atherogenesis and angiotensin II induced aortic aneurysm formation. To address the role of mPGES-1 in the response to vascular injury, the effect of enzyme deletion on neointimal formation four weeks after wire injury of the femoral artery was assessed. mPGES-1 suppressed urinary excretion of the major metabolite of PGE
2
, while augmented that of prostacyclin (PGI
2
), a prostanoid that restrains vascular proliferation after the injury. Both maximal neointimal area and vascular stenosis were reduced significantly in mPGES-1 knock out mice compared to wild type controls (65.6±5.7 vs 37.7±5.1×10
3
pixel area and 70.5±13.4% vs 47.7±17.4%, respectively; in both cases, p < 0.01). Neointimal Tenascin C, a promoter of vascular smooth muscle cell (VSMC) proliferation is upregulated in response to vascular injury, and this was suppressed by mPGES-1 deletion. VSMCs isolated from mPGES-1 deficient mice exhibited delayed attachment and less coordinate migratory morphology when cultured on fibrillar type I collagen. Deletion of mPGES-1 impairs VSMC mobility and attenuates neointimal formation in response to vascular injury. Inhibition of mPGES-1 has therapeutic potential for the prevention of restenosis.

This research has received full or partial funding support from the American Heart Association, National Center.
Abstract 5323: Inhibition of NADPH Oxidase is Not Sufficient for Protection Against Age-associated Changes in Atherosclerosis, Arterial Phenotype and Cardiac Function
Authors: Vendrov Aleksandr E , Madamanchi Nageswara R , Molnar Kimberly C , Runge Marschall S
Published: April 2024

The incidence of cardiovascular diseases (CVD) increases with age. Increased inflammation and ROS levels were implicated in this process. To examine the interplay of aging, ROS and CVD, we performed vascular phenotyping of young (4 months) and aged (16 months) apoE−/− and apoE−/−/p47phox−/− mice fed a normal or Western diet. As we previously reported, young apoE−/−/p47phox−/− mice had significantly less atherosclerosis compared to apoE−/− mice. However, a significant increase in atherosclerosis was observed in aged apoE−/−/p47phox−/− compared to young apoE−/−/p47phox−/− mice, regardless of the diet. Significant decrease in aortic compliance was observed in aged apoE−/− and apoE−/−/p47phox−/− compared to the respective young mice regardless of the diet. Left ventricle (LV) ejection fraction was significantly decreased in the aged mice while LV end diastolic volume, LV mass and posterior wall thickness were significantly increased compared with young mice of the same genotype. Aortic sections from aged apoE−/− and apoE−/−/p47phox−/− mice showed increased collagen, decreased elastin content and focal calcification. Increased total and mitochondrial ROS production was also observed in aortic sections from aged apoE−/− and apoE−/−/p47phox−/− mice compared to those in young mice of the same genotype. Aortic vascular smooth muscle cells (VSMC) from aged wild-type and p47phox−/− mice had higher levels of total and mitochondrial ROS compared to VSMC from respective young mice. ROS levels were higher in aged p47phox−/− VSMC. Expression of Nox1 and Nox4 subunits was significantly increased in aged vs young wild-type VSMC while only a modest increase was observed in aged p47phox−/− animals as detected by RT-PCR and histochemistry. Expression of uncoupling protein 2 and PPAR
γ
coactivator 1 was significantly increased in aged wild-type but decreased in aged p47phox−/− VSMC compared to the respective young VSMC indicating higher mitochondrial membrane potential and increased mitochondrial dysfunction in p47phox−/− cells. Together, these data suggest that suppression of NADPH oxidase activity is not sufficient for protection from age-associated changes in VSMC and cardiovasculature and from atherosclerosis in the presence of hypercholesterolemia.
Abstract 5324: Skeletal Muscle-specific Overexpression of Insulin-like Growth Factor I Suppresses Angiotensin II-induced Oxidative Stress and Prevents Skeletal Muscle Wasting
Authors: Sukhanov Sergiy , Semprun-Prieto Laura , Yoshida Tadashi , Delafontaine Patrick
Published: April 2024

Congestive heart failure (CHF) is a leading cause of cardiovascular mortality and morbidity and is associated with elevated circulating levels of angiotensin II (ANG) and muscle wasting (cachexia), which is an important predictor of poor outcome. We have shown that ANG infusion in FVB mice reduces skeletal muscle IGF-1 expression, increases oxidative stress and induces skeletal muscle wasting. We hypothesized that skeletal muscle-specific IGF-1 overexpression would decrease ANG-induced oxidative stress and muscle wasting. Mice overexpressing IGF-1 under control of the skeletal muscle-specific type II myosin light chain promoter (MLC) and control mice (FVB) were infused with 1 ug/kg ANG or vehicle (SHM) and were pair-fed for 7 days. ANG decreased skeletal muscle weight in FVB (ANG/SHM, gastrocnemius: 99.8±3.6/122.3±4.2 mg, P< 0.01; tibialis anterior: 32.0±1.3/41.3±1.3 mg, P< 0.05; quadriceps: 117.6±6.1/160.6±6.7 mg, P< 0.05), but not in MLC. ANG induced a leftward shift in myofiber size distribution and decreased cross-sectional area (CSA) of type I and type II myofibers in FVB (ANG/SHM, gastrocnemius, type I, 443±22/528±28 um2; type II, 620±40/835±44 um2) and this effect was completely inhibited in MLC as detected by immunostaining with isoform-specific antibodies. Oxidative stress was assessed in muscle homogenates using superoxide-sensitive dye L-012. ANG increased superoxides in FVB by 67% (228±25 vs. 149±21 RFU/mg protein, p<0.005) but did not significantly increase superoxides in MLC (376±99 vs. 298±81 RFU/mg protein, p=NS). 250 uM Apocynin (a specific NADPH oxidase inhibitor) suppressed ANG-induced superoxides in FVB (91±5% decrease vs. DMSO control). Analysis of NADPH oxidase subunit expression indicated that ANG upregulated NOX4 mRNA expression in skeletal muscle and this effect was greater in FVB (3.6-fold increase, p<0.05) compared to MLC (2.1-fold increase, p=NS, RT-PCR). Thus, IGF-1 overexpression suppresses oxidative stress and efficiently blocks skeletal muscle wasting, possibly via inhibition of ANG-induced NOX4 upregulation and NADPH oxidase-dependent superoxide formation. IGF-1 inhibition of skeletal muscle NADPH oxidase activity could provide a novel approach for the treatment of cachexia.
Abstract 5424: Hypoxia Induces the Histone Demethylase Jmjd3 and Epigenetically Reprograms Endothelial Progenitor Cells
Authors: Ohtani Kisho , Koyanagi Masamichi , Boeckel Jes-Niels , Zeiher Andreas , Dimmeler Stefanie
Published: April 2024

Endothelial progenitor cells (EPCs) contribute to vascular repair and postnatal neovascularisation. Cell therapy using EPCs improved neovascularisation and functional recovery after ischemia. However, it has been extensively debated whether EPCs can differentiate into endothelial cells and the mechanisms of EPC differentiation are poorly understood. Endothelial differentiation requires the expression of endothelial marker genes, which are silenced by chromatin dependent repressive mechanisms such as histone H3 lysine 27 trimethylation (H3K27me3) in non-endothelial cells. We observed that induction of hind limb ischemia increased the expression of Jmjd3, which was shown to demethylate H3K27me3 thereby removing repressive histone marks. Therefore, we examined whether EPC might be reprogrammed by hypoxic/ischemic injury through epigenetic pathways. We report here that Jmjd3 was induced when EPCs were exposed to hypoxia for more than 24 hours. Hypoxia additionally stimulated the nuclear translocation of Jmjd3 in EPC as shown by immunostaining (12.0±3.2% in normoxia, 68.5±3.6% in hypoxia, Jmjd3 positive nuclei/whole nuclei). Increased expression and nuclear translocation of Jmjd3 after hypoxic stimulation was associated with a reduction of global H3K27m3. Additionally, chromatin immunoprecipitation showed that the promoter of eNOS and KDR, which is transcriptionally silenced by high levels of H3K27me3 in early EPC under normoxic conditions, is activated by hypoxia. Hypoxia induced a down-regulation of H3K27me3 (0.045±0.017% input in normoxia and 0.024±0.010% input in hypoxia at the eNOS promoter, 0.197±0.067% input in normoxia and 0.126±0.030% input in hypoxia at the KDR promoter) and increased the ratio of active versus repressive chromatin marks at the eNOS and KDR promoter. Consistently, eNOS and KDR mRNA levels in EPC were increased after hypoxia. In summary, these data demonstrate that hypoxia-induced up-regulation and translocation of the histone demethylase Jmjd3 is associated with a reduction of repressive histone marks at the promoter of endothelial genes. These findings indicate that hypoxia may epigenetically modulate endothelial commitment.
Abstract 5429: Bone Marrow-derived Cells Contribute to Vascular Inflammation but Do Not Differentiate Into Smooth Muscle Cell Lineages
Authors: Iwata Hiroshi , Manabe Ichiro , Fujiu Katsuhito , Takeda Norifumi , Eguchi Kosei , Sata Masataka , Nagai Ryozo
Published: April 2024

It has been proposed that bone marrow-derived cells infiltrate the neointima, where they differentiate into smooth muscle cells (SMCs). This is based on the observation that those cells express SM
α
-actin; however, technical limitations have hindered clear identification of the lineages of bone marrow-derived “SMC-like” cells. Using a specific antibody against the definitive SMC-lineage marker SM myosin heavy chain (SM-MHC) and mouse lines in which reporter genes were driven by regulatory programs for either SM-MHC or SM
α
-actin, we demonstrated that while some bone marrow-derived cells express SM
α
-actin in the wire injury-induced neointima, those cells did not express SM-MHC, even 30 weeks after injury. Likewise, no SM-MHC+ bone marrow-derived cells were found in vascular lesions in apolipoprotein E−/− mice or in a heart transplantation vasculopathy model. Instead, the majority of bone marrow-derived SM
α
-actin+ cells expressed monocyte/macrophage markers, and expression of inflammation-related genes was significantly higher in neointimal subregions rich in bone marrow-derived SM
α
-actin+ cells than in other regions. In addition, flow cytometric analysis showed that bone marrow-derived SM
α
-actin+ cells were also CD115+ CD11b+ F4/80+ Ly6c+ CD11c−, which is the surface phenotype of inflammatory monocytes. It thus appears that bone marrow-derived cells are of monocyte/macrophage lineage and acquire a phenotype in which SMC early differentiation markers are expressed. Although these cells are involved in remodeling at active lesions in the severely injured vessel wall, it is very unlikely they acquire the definitive SMC lineage.
Abstract 5451: Coronary Spatial Distribution of Non-ST Elevation Myocardial Infarction is Non-Uniform and Clustered in the Proximal Portion of the Coronary Tree
Authors: Khan M Faisal , Candia Susana C , Jeon Cathy , Wang John C , Kuntz Richard , Mauri Laura
Published: April 2024

Introduction:
We hypothesized that plaque rupture leading to NSTEMI is a non-uniform process with variable risk for different anatomic locations within each coronary artery. It is not known whether this distribution is similar to that in STEMI, where we have previously observed proximal clustering of lesions.

Methods:
We used quantitative angiographic analysis (QCA) to compare the spatial location of lesions in NSTEMI to STEMI in consecutive patients undergoing coronary angiography at the Brigham and Women’s Hospital over a 24-month period for NSTEMI and STEMI (N= 200 and 208 respectively). The segment lengths of the culprit vessel and its side branches were measured. The Wilcoxon rank sum test was used to compare the distribution of culprit lesions in NSTEMI vs. STEMI.

Results:
NSTEMI patients were older and had a higher prevalence of prior infarction, diabetes mellitus, hypertension, dyslipidemia, and renal insufficiency. Despite these differences, lesions in both NSTEMI and STEMI were clustered in the proximal third of each of the coronary arteries. There was no significant difference in the spatial distribution of the lesions in NSTEMI as compared to STEMI in the LAD, p=0.36 (Fig 1a & b

), LCx, p=0.13 or RCA, p=0.22.)

Conclusion:
We conclude that NSTEMI plaque ruptures are not uniformly distributed but proximally clustered similar to STEMI lesions. This suggests that the anatomic zones at risk for acute thrombosis are independent of the type of acute coronary syndrome and that the proximal segments are at highest risk.

Figure 1a & b.

LAD Absolute Distance Analysis and Cumulative Frequency Distribution.
50% of NSTEMI lesions occurred within the first 35mm of the vessel (proximal 1/3
rd
Abstract 5465: SIRT1 Decreases Lox-1 Mediated Foam Cell Formation in Atherogenesis
Authors: Stein M A , Lohmann Christine , Schäfer Nicola , Hofmann Janin , Rohrer Lucia , Breitenstein Alexander , Besler Christian , Becher Burkhard , Hottiger Michael O , McBurney Michael W , Luscher Thomas F , Matter Christian M
Published: April 2024

Background - Atherosclerosis is a chronic inflammatory disease that results from interaction between oxLDL, activated endothelial cells, monocyte-derived macrophages, T cells, and the arterial wall. Infiltrating macrophages become foam cells upon uptake of oxidized low-density lipoproteins (oxLDL) via scavenger receptors such as lectin-like oxLDL receptor-1 (Lox-1). SIRT1 is a class III deacetylase that senses caloric restriction, improves insulin secretion in islet cells, and reduces accumulation of fatty acids in white adipose. The relevance of endogenous SIRT1 on atherosclerosis remains unknown.

Methods - To investigate the role of SIRT1 in these cellular and molecular processes, we compared hypercholesterolemic ApoE−/− SIRT1+/+ and ApoE−/− SIRT1+/− mice that were kept on a high-cholesterol diet for 12 weeks. Peritoneal-elicited macrophages, human aortic endothelial cells (HAEC) and RAW macrophages were used to verify our in vivo findings. Bone-marrow transplantations were performed to assess the relevance of macrophage-derived SIRT1 on atherogenesis.

Results - Our plaque analyses reveal that SIRT1 prevents atherogenesis without affecting total plasma cholesterol or lipid subfractions. ApoE−/− SIRT1+/− mice show increased features of plaque vulnerability with thinner fibrous caps and enhanced plaque inflammation (increased number of macrophages and T cells as well as induced expression of adhesion molecules and inflammatory cytokines). Furthermore, peritoneal-elicited macrophages from heterozygous mice accumulate increased levels of oxLDL. In vitro, we show that the atheroprotective effects of SIRT1 are mediated via NF-
κ
B inhibition: in HAEC SIRT1 inhibits NF-
κ
B signaling and diminishes expression of adhesion molecules; in macrophages the inhibitory effect reduces expression of Lox-1. Bone-marrow transplantations demonstrate that macrophage.-derived SIRT1 is crucial for preventing disease progression.

Conclusions - Our data show that SIRT1 prevents atherosclerotic plaque formation. The availability of specific SIRT1-activating drugs suggests that pharmacological activation of SIRT1 may become an attractive anti-atherogenic strategy.
Abstract 5472: Nox4 is a Novel Inducible Source of Intracellular Reactive Oxygen Species in Human Monocyte-Derived Macrophages and Mediates Oxidized LDL-induced Macrophage Death
Authors: Lee Chi Fung , Qiao Mu , Schroeder Katrin , Asmis Reto
Published: April 2024

Nox2/gp91
phox
is the large subunit of the integral membrane component of the phagocytic NADPH oxidase (Nox) complex and was believed to be the sole Nox isoform present in monocytes and macrophages. Previously, we reported that increased intracellular ROS formation is required for OxLDL-induced cell death in human monocyte-derived macrophage (HMDM), but the source of these intracellular ROS was unknown (Asmis, Circ.Res. 2003). We now identified Nox4, not Nox2, as the source of OxLDL-induced ROS generation. Nox4 mRNA was detected in human blood monocytes and HMDM. Using a novel rabbit monoclonal antibody that detects Nox4 as a single 66 kDa band, we localized Nox4 to the ER, the nucleus and focal adhesion sites of HMDM, suggesting that Nox4 may play a role in macrophage redox signaling. After stimulation of HMDM with OxLDL, but not native LDL, Nox4 mRNA and protein expression were up-regulated in a dose- and time-dependent manner. Nox4 protein expression was maximal after 6 h and coincided with both increased p22 expression and increased intracellular ROS formation, suggesting that the Nox4/p22 complex is responsible for OxLDL-induced ROS formation in HMDM. Inhibition of MEK1 and ERK1/2 phosphorylation blocked both the induction of Nox4 expression and intracellular ROS formation induced by OxLDL, and protected HMDM from OxLDL-mediated cytotoxicity. In contrast, inhibitors of p38 MAPK or JNK affect neither Nox4 induction nor ROS formation, and showed no cytoprotection. Knockdown of Nox4 with adenovirus-delivered siRNA decreased OxLDL-induced ROS formation and protected macrophages from OxLDL-induced cell injury. In summary, we identified Nox4 as a novel, inducible source of intracellular ROS in human monocytes and macrophages. Nox4 is likely to play important roles in redox signaling and macrophage function. However, if overinduced Nox4-derived ROS promote macrophage death, a mechansim which may have important implications for the pathogenesis of atherosclerosis.

This research has received full or partial funding support from the American Heart Association, South Central Affiliate (Arkansas, New Mexico, Oklahoma & Texas).
Abstract 5486: High Density Lipoprotein (HDL) From Healthy Subjects, but Not From Patients With Coronary Artery Disease, Exerts Anti-thrombotic Effects on Human Endothelial Cells
Authors: Holy Erik W , Besler Christian , Landmesser Ulf , Luscher Thomas F , Tanner Felix C
Published: April 2024

Background: Arterial thrombus formation is determined by the balance between pro-thrombotic mediators such as tissue factor (TF) and plasminogen activator inhibitor type 1 (PAI-1), and anti-thrombotic factors like tissue factor pathway inhibitor (TFPI) or tissue plasminogen activator (tPA). Native HDL from healthy subjects (HS) has anti-thrombotic properties; however, it remains unknown whether this is the case for HDL from patients with stable coronary disease (CAD) or acute coronary syndrome (ACS).

Methods: HDL was isolated by sequential ultracentrifugation from HS and patients with CAD and ACD. The effects of HDL (50
μ
g/ml) on TF, TFPI, and PAI-1 expression in human endothelial cells were determined by Western blot analysis; tPA release was measured by ELISA.

Results: HDL from HS impaired thrombin-induced TF expression (−45±5%, p<0.05, n=16) and activity (−33±8%, p<0.05, n±7); in contrast, HDL from CAD and ACS patients did not (p=NS, n=12 and n=8). Similarly, HDL from HS increased TFPI expression by 2-fold (p<0.01, n=8), while HDL from CAD and ACS patients had no effect (p=NS). HDL from HS enhanced tPA release (+26±3%; p=0.05, n=8); in contrast, HDL from CAD and ACS patients did not (p=NS, n=6). Furthermore, HDL from HS did not affect PAI-1 expression, while HDL from CAD patients enhanced PAI-1 expression by 62% (p<0.05 vs. healthy, n=12) and HDL from ACS patients by 2 fold (p<0.05 vs. control and p<0.05 vs. healthy, n=8). Pretreatment with the inhibitor of NO formation, L-NAME (100
μ
M), abolished the anti-thrombotic effects of HDL from HS on TF, TFPI, and tPA expression. The exogenous nitric oxide donor, DETANO, mimicked the effects of HDL from HS on TF, TFPI, and tPA.

Conclusion: This study demonstrates that HDL from healthy subjects exerts anti-thrombotic effects on endothelial cells. In contrast, HDL from CAD and ACS patients loses these antithrombotic properties and instead enhances PAI-1 expression, thereby becoming pro-thrombotic. This observation might be highly relevant for HDL-targeted therapies.
Abstract 5496: Antiplatelet Effects and Bleeding Risk of DZ-697b, a Novel, Oral Antiplatelet Agent, versus Clopidogrel: Results of a Phase-I Study
Authors: Zafar Mohammad U , Ibanéz Borja , Choi Brian G , Vorchheimer David A , Pinéro Antonio , Vila Peter M , Speidl Walter S , Jin Xioping , Sorokin Igor , Sharma Raman K , Badimon Juan J
Published: April 2024

INTRODUCTION
: DZ-697b is a new orally active antiplatelet agent that inhibits collagen and ristocetin-mediated platelet aggregation. It does not require metabolization for the generation of its active compound and has a safer profile than clopidogrel in pre-clinical studies. The aim of this study was to investigate the platelet inhibitory effects and the bleeding risk of 3 doses of DZ-697b compared to 300mg clopidogrel.

METHODS
: In a 4-treatment, 3-period, crossover design, 20 healthy subjects (31±7 years, 85% male) were randomized to a single oral dose of DZ-697b 60mg, 120mg and 360mg, and clopidogrel 300mg (n=15 in each treatment). Antiplatelet effects were assessed by measuring changes from pre-dose to 6 hours post-dose in platelet-thrombus size at high- and low-flow rates in the Badimon chamber, and platelet adhesion at high- and low-shear rates using Diamed Impact-R platelet function assay. Bleeding risk was assessed using surgicutt bleeding time procedure, measured at predose and 6 hours postdose.

RESULTS
: DZ-697b caused dose-dependent reductions in thrombus size and platelet adhesion (Table

). Antiplatelet effects of DZ-697b 120mg were closest to clopidogrel 300mg. Bleeding times with all DZ-697b doses were significantly shorter than clopidogrel.

CONCLUSIONS
: The oral agent DZ-697b shows potent, dose-dependent, antiplatelet properties. Bleeding time prolongations with even the highest tested dose of DZ-697b is significantly shorter than 300mg clopidogrel. Further studies are warranted.