bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4882: Inhibitor Kappa B Kinase Regulates Endothelial Nitric Oxide Synthase Activity and Nitric Oxide Generation
Authors: Mohan Sumathy , Mohan Natarajan , Roman Linda , Humayun Saima , Konopinski Ryszard
Published: April 2024

The involvement of heat shock protein 90 (Hsp-90) in the regulation of eNOS activity is known. We recently demonstrated the increased interaction of Hsp-90 with IKK
β
in vascular endothelial cells under conditions of high glucose. However, whether this binding of IKK
β
to Hsp-90 will interfere with the binding of Hsp-90 to eNOS is not clear and the region on Hsp-90 to which IKK
β
binds is unknown. In this study, we examined whether the interaction of Hsp-90 with eNOS is compromised by competitive binding of IKK
β
. Our attempts to demonstrate competition between eNOS and IKK
β
for Hsp-90 using purified proteins confirmed that elevated levels of IKK
β
can dissociate eNOS-Hsp-90 complex. Hsp-90 and eNOS with increasing concentrations of purified Gst-IKK
β
were incubated for 2h at 4°C. Immunoprecipitation of this complex with anti-eNOS antibody followed by Immunoblotting using anti-Hsp-90 antibody demonstrated reduced amounts of Hsp-90 associated to eNOS as concentration of IKK
β
increases (Fig 1A

). The supernatants were re-precipitated with anti-GST antibody and the immune complex was analyzed for the presence of Hsp-90. Immunoblot showed the presence of Hsp-90 with higher concentration of IKK
β
(1B). Further, we investigated whether IKK
β
can inhibit eNOS enzyme activity by dissociating Hsp-90-eNOS complex. Nitric oxide formation was measured at 23°C using the hemoglobin capture assay. Addition of IKK
β
significantly blocked eNOS activity (Fig 2

). Also,
In vitro
kinase assay performed using bovine aortic endothelial cell lysates and purified proteins demonstrated possible phosphorylation of Hsp-90 by IKK
β
kinase. These results show the significance of IKK
β
in regulating eNOS activity.

Figure 1

Figure 2
Abstract 4883: Insulin-like Growth Factor Binding Protein-3 (IGFBP3) Modulates Vascular Tone via Scavenging Receptor-B1 (SRB1)-Dependent Nitric Oxide Release
Authors: Jarajapu Yagna P , Kielczewski Jennifer L , Baxter Robert C , Grant Maria B
Published: April 2024

Purpose: We recently reported the vascular protective effect of IGFBP3 in experimental models of ischemic vascular injury involving increased homing of circulating endothelial progenitor cells and activation of nitric oxide synthase (eNOS). The vascular protective potential of a molecule is determined in part by its ability to facilitate perfusion to the ischemic areas by dilation of the resident vasculature. Therefore, we hypothesized that IGFBP3 regulates vascular tone by causing vasodilation and thereby, enhances the perfusion of vascular bed.

Methods: We evaluated the effect of intraluminal IGFBP3 on the pressure- and agonist-induced constrictions in rat posterior cerebral arteries (PCAs) and compared with that of HDL, a known activator of SRB1. NO release and endothelial intracellular Ca2+ ([Ca2+]i) were evaluated by DAF-FM and Fura-2 fluorescence microscopy, respectively, in intact arteries and human microvascular endothelial cells (HMVECs). Signaling pathways mediating the effects of IGFBP3 were delineated in intact arteries and HMVECs. Activation of eNOS by IGFBP3 was evaluated by Western blotting of ser1177 phosphorylated eNOS in HMVECs. IGF-independent effect was confirmed by using a non-IGF-binding mutant of IGFBP3.

Results: Intraluminal IGFBP3 (100ng/ml) or HDL (1mg/ml) caused significant decrease in pressure- (40, 70 and 100 mmHg) and serotonin-induced constrictions of PCAs compared to the vehicle-control. These effects were blocked by L-NAME or SRB1-function blocking antibody (SRB1-ab). NO release was stimulated in PCAs by intraluminal IGFBP3 and HDL with no change in the [Ca2+]i. NO release by IGFBP3, with no change in the [Ca2+]i, in HMVECs was blocked by SRB1-ab, PP2 (cSrc inhibitor), LY294002 (PI3-kinase inhibitor) and triciribin (akt inhibitor) not by KN-92 (CamKII inhibitor). Non-IGF-binding mutant and wild type IGFBP3 showed similar effect in stimulating NO release in HMVECs. Phosphorylation of eNOS at ser1177 was increased in a time-dependent manner by IGFBP3 that was blocked by SRB1-ab.

Conclusions: These results suggest that IGFBP3 modulates vascular tone by NO release via SRB1-dependent cSrc/PI3-kinase/akt pathway and this response may significantly contribute to the vascular protective effect of IGFBP3.
Abstract 4889: Coupling Factor 6 Enhances the Action of Angiotensin II and Causes Insulin Resistance in Mice
Authors: Osanai Tomohiro , Tanaka Makoto , Echizen Takashi , Ashitate Toshiro , Izumiyama Kei , Sagara Shigeki , Tomita Hirofumi , Okumura Ken
Published: April 2024

The lower values of intracellular pH are commonly observed in the hypertensive states including a high renin state. It is also reported that intracellular acidosis is related to the pathogenesis of diabetes by inducing insulin resistance in the skeletal muscle. We recently showed that the molecular rotary motor F
1
F
o
complex, ATP synthase, is present at the plasma membrane, and that coupling factor 6 (CF6) forces the backward rotation of F
o
after binding to F
1
, resulting in cellular acidosis (Hypertension, 2005). Using CF6-overexpressing transgenic (TG) mice and vascular smooth muscle cells (VSMC), we tested the hypothesis that CF6 enhances the action of angiotensin II (AngII) and causes insulin resistance. In TG mice, the introduced gene of human CF6 was expressed in overall tissues, and upregulated by 2 times. In the mesenteric arterioles isolated from TG mice, Ang II-induced vasoconstriction was enhanced compared with WT mice (p<0.05). Blockade of CF6 with its specific antibody remarkably attenuated the Ang II-induced vasoconstriction by 85±5% in TG mice and by 79±5% in WT mice, thereby eliminating the difference in contraction. In CF6-overexpressing (spontaneously hypertensive rats, SHR) VSMC, Ang II (10
−7
M)-induced increase in the spike phase of [Ca
2+
]
i
was higher compared with control (Wistar Kyoto rats) VSMC. In VSMC with a high affinity CF6 receptor (SHR), Ang II (10
−7
M)-induced increase in the spike phase of [Ca
2+
]
i
was enhanced by the presence of CF6 at 10
−7
M. All were blocked by PP1 at 50
μ
M, a c-Src inhibitor. When the mice were fed with high sucrose diet (20%) for 8 weeks, the TG mice exhibited insulin resistance: In glucose-tolerance test, the plasma glucose level was higher until 1.5 hours (p < 0.05, two-way ANOVA), and insulin level at 1 hour was higher in TG mice (1111 ± 118 vs 400 ± 54 pg/ml, p < 0.05). The TG mice exhibited no change in tyrosine phosphorylation of
β
subunit of insulin receptor but the decreases in the protein expression of insulin receptor substrate-1 by 64 ± 9%, phosphatidylinositol-3 kinase activity, Akt phosporylation, and the plasma membrane GLUT-4 protein by 69 ± 11% in the skeletal muscle (n = 5, all p < 0.05). These indicate that CF6 enhances the action of AngII by c-Src signaling and thereby causes diet-induced insulin resistance in mice.
Abstract 4891: Angiotensin II-activated Caspase 3-dependent Apoptosis in the RVLM Contributes to Sympathoexcitaion Through the Activation of Ras/MAPK/ERK Pathway
Authors: Kishi Takuya , Hirooka Yoshitaka , Sunagawa Kenji
Published: April 2024

Background:
The rostal ventrolateral medulla (RVLM) regulates sympathetic nerve activity (SNA). Angiotensin II-derived superoxide anion in the RVLM induces the pressor response via the activation of p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-regulated kinase (ERK). Small G protein Ras mediates caspase 3-dependent apoptosis through the activation of p38 MAPK and ERK. We hypothesized that angiotensin II/angiotensin II type 1 receptor (AT
1
R)-activated caspase 3-dependent apoptosis in the RVLM contributes to sympathoexcitation through the activation of Ras/p38 MAPK/ERK pathway in stroke-prone spontaneously hypertensive rats (SHRSP).

Methods and Results:
Telemetrically measured mean blood pressure (MBP) and heart rate (HR) under a conscious state, and SNA assessed by urinary norepinephrine excretion, were significantly higher in SHRSP than in Wistar-Kyoto (WKY) rats. Activities of Ras, p38 MAPK, ERK, and caspase 3 in the RVLM were significantly higher in SHRSP than in WKY (Ras; 1.7 ± 0.2 U vs 1.3 ± 0.2 U, p38 MAPK; 1.8 ± 0.1 U vs 1.2 ± 0.3 U, ERK; 2.1 ± 0.3 U vs 1.4 ± 0.3 U, caspase 3; 1.7 ± 0.1 U vs 1.1 ± 0.2 U, n = 5 for each,
P
< 0.01). Mitochondrial apoptotic proteins Bax and Bad in the RVLM were also significantly increased in SHRSP (Bax; + 38 ± 2%, Bad; + 43 ± 3%, n = 5 for each,
P
< 0.01). In SHRSP, intracerebroventricular (ICV) infusion of Ras inhibitor, Z-DEVD-FMK, significantly reduced MBP, HR, and SNA (MBP; −38 ± 4mmHg, HR; −44 ± 18bpm, SNA; −0.8 ± 0.2
μ
g/day, n = 5 for each,
P
< 0.01) and inhibited activities of Ras/p38 MAPK/ERK, Bax, Bad, and caspase 3 in the RVLM (Ras; −36 ± 3%, p38 MAPK; −27 ± 3%, ERK; −24 ± 2%, Bax; −21 ± 3%, Bad; −28 ± 5%, caspase 3; −39 ± 6%, n = 5 for each,
P
< 0.01). ICV of caspase 3 inhibitor also decreased MBP, HR, and SNA in SHRSP. ICV of AT
1
R blocker, candesartan, in SHRSP decreased MBP, HR, and SNA (MBP; −34 ± 5mmHg, HR; −52 ± 14bpm, SNA; −0.9 ± 0.2
μ
g/day, n = 5 for each,
P
< 0.01) and reduced activities of Ras/p38 MAPK/ERK, Bax, Bad, and caspase 3 in the RVLM.

Conclusion:
Angiotensin II/AT
1
R-activated caspase 3-dependent apoptosis in the RVLM increases SNA through the activation of Ras/p38 MAPK/ERK pathway in SHRSP. This mechanism of sympathoexcitation may play a crucial role in the pathogenesis of hypertension.
Abstract 4892: Olmesartan Potentiates the Vasorelaxation and Anti-hypertensive Effect of Angiotensin(1–7)
Authors: Santos Robson A , Savergnini Silvia Q , Lautner Roberto Q , Braga Aline N , Castro Carlos H
Published: April 2024

It has been described that Olmesartan increases plasma angiotensin-(1–7) [Ang-(1–7)] through increase in ACE2 expression. Here we evaluated the effect of acute treatment with Olmesartan on the anti-hypertensive and vasorelaxing effects of Ang-(1–7). Vascular activity of Ang-(1–7) was evaluated in intact aorta rings of Wistar rats, pre-contracted with phenylephrine (10
−7
M). Olmesartan or Losartan were added to the bath at a concentration of 10
−7
M, 30 minutes before adding phenylephrine and performing a dose-response curve of Ang-(1–7) (10
−10
to 10
−6
M). To evaluate the acute effect of Olmesartan on the anti-hypertensive action of Ang-(1–7), experiments were performed in male SHR (14 –16 week-old). Twenty-four hours before the experiments, catheters were implanted into the abdominal aorta for arterial pressure and heart rate (HR) measurements and the femoral vein for intravenous infusions. Different doses of Ang-(1–7) (0.003, 0.03, 0.3 and 3nmol/Kg/min) or saline were infused (30 minutes each), before and after one hour administration of Olmesartan or Losartan (1mg/Kg) by gavage. Acute treatment with Olmesartan augmented the vasodilator effect of Ang-(1–7) in endothelium-intact aortic rings (E
max
: 28.28 ± 2.94 vs 14.94 ± 1.37% in vehicle-treated rings). The same treatment with Losartan did not change the Ang-(1–7)-induced vasorelaxation (E
max
: 11.08 ± 0.98). Acute infusion of Ang-(1–7) produced a dose-dependent decrease in MAP in conscious SHR. The maximum change during infusion was observed at the highest dose (3 nmol/Kg/min, Δ: −16.6 ± 3.8 mmHg). The anti-hypertensive effect of Ang-(1–7) was markedly enhanced after Olmesartan administration (maximum change: −35.4 ± 5.4 mmHg, at the dose of 3nmol/Kg/min). Losartan treatment produced only a slight increase in the Ang-(1–7) anti-hypertensive effect (Δ: −9.9 ± 6.3 mmHg). Vehicle administration did not change the anti-hypertensive effect of Ang-(1–7). At the doses used, acute administration of both AT1 blockers alone did not change blood pressure. These data show that Olmesartan but not Losartan potentiated the vasorelaxation and anti-hypertensive effects of Ang-(1–7). The absence of effect of the other AT1 blocker Losartan, suggest that this is not a class effect.
Abstract 4898: Genetic Polymorphism on Type 2 Receptor of Angiotensin II Affects Systemic Inflammatory Process in Hypertensive Individuals
Authors: Tousoulis Dimitris , Koumallos Nikolaos , Antoniades Charalambos , Miliou Antigoni , Antonopoulos Alexis , Mentzikof Dimitris , Marinou Kyriakoula , Bakogiannis Constantinos , Stougiannos Pavlos , Kallikazarou Eftihia , Tsioufis Costas , Stefanadis Christodoulos
Published: April 2024

Introduction: Evidence suggests that there is a balance between angiotensin II effects on proatherogenic constitutive type 1, and antiatherogenic inducible type 2 (AT2R) receptors. The AT2R gene is located on chromosome X, and the biological effect of a newly described polymorphism (A1675G) in this gene is unclear. Aim: We examined the impact of A1675G polymorphism on AT2R, on the risk for coronary atherosclerosis, and its effect on the expression of proatherogenic inflammatory molecules.

Methods: The study population consisted of 437 males: 155 with coronary artery disease (111 with hypertension) and 282 healthy age-matched controls (121 with hypertension). The presence of A1675G polymorphism on AT2R gene (located in chromosome X) was determined by PCR. Serum levels of C-reactive protein, fibrinogen, interleukin-6 (IL-6) and soluble vascular cell adhesion molecule-1 (sVCAM-1) were measured in all the participants.

Results: The frequency of the A allele was similar among healthy individuals (41.8% 118/282) and CAD patients (47.7%, 74/155, p = NS). However the presence of the A allele was more frequent in hypertensives with CAD (55%, 59/107) than in hypertensives without CAD (35.8%, 43/120, p <0.01). Importantly, the A allele was associated with increased risk for CAD among hypertensive individuals (OR: 2.201[95%CI: 1.291–3.752], p = 0.004), an effect which was not seen among normotensive subjects (p = NS). Importantly, the presence of the A allele was also associated with significantly higher levels of CRP (mean[25th–95th percentile]: A:3.52[1.98 – 6.08] vs G:1.18[0.66 –1.71] mg/dL, p = 0.0001), fibrinogen (A:407[347–513] vs G:369[320 – 416] mg/dl, p = 0.001), IL-6 (A:1.55[3.63–5.40] vs G:0.99[0.51–2.46] pg/ml, p = 0.002), and sVCAM-1 (A:702[648 –925] vs G:621[476 –799] ng/ml, p = 0.03).

Conclusions: Genetic polymorphism A1675G on AT2R affects systemic inflammatory mechanisms, since the presence of the A allele is associated with higher levels of CRP, fibrinogen, IL-6 and sVCAM-1. In addition, the A allele is associated with elevated cardiovascular risk among hypertensive individuals.
Abstract 4899: Improved Blood Pressure Control and Lack of Gender Bias in a Large Veterans Affairs Population
Authors: Fletcher Ross D , Papademetriou Vasilios , Amdur Richard L , Singh Sephen , McManus Christopher , Jones Ronald E
Published: April 2024

Introduction:
Despite the proven benefits, blood pressure (BP) control rates remain low. Gender bias has emerged in several aspects of treatment of cardiovascular disease. Thus, we assessed the effect of interventions to control blood pressure in men and women in the Department of Veteran Affairs.

Methods:
From January of 2001 to December 2007, we assessed BP control in 460,278 men and 17,821 women patients from 15 VA hospitals in the United States. Medical interventions to control BP included use of electronic medical records with automated reminders, medical regimen adjustments, and frequent visits to both specialty and nurse run clinics.

Results
: Control rates (BP ≤ 140/90) over time are shown (Figure

). In Jan 2001 control rates were: 43.8% for men and 47.0% for women (p < 0.001) and in June 2001 were 51.8% for men and 54.2% for women (p < 0.001). After 7 years of follow up, control rates improved in both genders as follows: Jan 2007, 67.3% for men and 67.6 for women (p = NS), June 2007, 72.9% for men and 72.9% for women (p = NS, p < 0.001 for seasonal differences). Overall during the follow up period while there was a 23.1% increase in control rates for men and 19.2% for women (p = 0.007), the final control rate was the same.

Conclusions:

High rates of BP control can be achieved in both men and women.

Although early on more women than men were controlled, the control rates were identical at the end of the follow up period.

No gender bias was detected for blood pressure treatment and control in this cohort.

Seasonal BP variation was seen in both genders throughout the follow up period.
Abstract 4908: Athero-protective Effects of Immunization With apoB-100 Related Peptide Vaccine in apoE−/− Mice is Associated With Enhanced CD8 Regulatory T Cell Response
Authors: Zhao Xiaoning , Chyu Kuang-Yuh , Dimayuga Paul C , Yano Juliana , Li Xiaojun , Trinidad Portia , Zhou Jianchang , Lio Wai Man , Kirzner Jonathan , Chan Lai F , Cercek Bojan , Shah Prediman K
Published: April 2024

Background:
We previously reported that immunization of apoE−/− mice with human apoB-100 related peptide P210 (KTTKQ SFDLS VKAQY KKNKH) resulted in a 60% reduction in atherosclerosis by a regulatory T-cell (Treg) response elicited by the vaccine. In this study, we investigated the mechanisms involved in this regulatory response.

Methods and Results:
Male apoE−/− mice were immunized at 6, 9 and 11 weeks of age with P210 conjugated to cBSA, and alum as adjuvant, at a dose of 0.1 mg/mouse and euthanized at 13 weeks of age, or fed atherogenic diet until euthanasia at 25 weeks of age. At 13 weeks, FACS analysis of regional LN showed significantly reduced CD11c + CD86 + dendritic cells (DC) in the p210 group (Table

), suggesting an immune-regulatory response. Treg profiling showed significantly increased CD8 + CD25 + Treg in the peripheral blood (PBMC) of P210 group (Table

) without changes in the CD4+CD25+ and CD8+CD28- Treg. CD11c+ DCs in atherosclerotic lesions at 25 weeks were reduced in P210 (2.9± 1.7%, n = 12; p<0.05) compared with cBSA or PBS group (3.9± 1.6%, n = 7 and 4.5± 1.2%, n = 18; respectively) with concomitant reduction of macrophages in P210 group compared with cBSA and PBS (12.4± 2.5% vs. 15.4± 3.1% and 16.1± 2.9%, respectively; p<0.05).

Conclusion:
Our results indicate that P210 vaccination resulted in a CD8+CD25+ Treg response mediated by decreased DC co-stimulatory signaling, leading to a reduction of plaque DC and macrophage content, and attenuated plaque growth, suggesting a novel role of CD8+ Treg in reducing atherosclerosis.
Abstract 4909: Nox2-Containing NADPH Oxidase Deficiency Protects Against Age-Dependent Inhibition of Neovascularization
Authors: Turgeon Julie , Haddad Paola , Dussault Sylvie , Groleau Jessika , Maingrette Fritz , Perez Gemma , Rivard Alain
Published: April 2024

Background:
We have previously shown that aging is associated with an impaired angiogenic response following ischemia. Because aging leads to an increased generation of reactive oxygen species (ROS), here we investigated the role of Nox2-containing NADPH oxidase ROS generation for the modulation of neovascularization by aging.

Methods:
Hindlimb ischemia was surgically induced by femoral artery removal in young (5–7 weeks) and older (9 months) Nox2
−/−
and control (Nox2
+/+
) mice.

Results:
We found that blood flow recovery is significantly reduced in older compared to younger mice at day 21 after surgery (Doppler flow ratios: 0.51± 0.05 vs. 0.72± 0.05; p<0.05). At the microvascular level, capillary density was also significantly reduced in ischemic muscles of older animals (53.4± 6.3 capillaries/field vs. 82.5± 4.9 capillaries/field; p<0.001). This was associated with a significant increase of oxidative stress levels (nitrotyrosine immunostaining) in ischemic tissues of older animals. Importantly, we found that Nox2 deficiency reduces oxidative stress levels in ischemic tissues and restores blood flow recuperation in older animals. Endothelial progenitor cells (EPCs) have been shown to have an important role for postnatal neovascularization. Here we found that the functional activities of EPCs (adhesion to HUVECs, migration) are significantly impaired in old compared to young mice. However, Nox2 deficiency rescues EPCs functional activities in older animals. We also demonstrate an age-dependent pathological increase of oxidative stress levels (DCF-DA, DHE) in EPCs that is not present in Nox2-deficient animals.

Conclusion:
Nox2-containing NADPH oxidase deficiency protects against age-dependent impairment of neovascularization. Potential mechanisms include reduced ROS generation in ischemic tissues and preserved angiogenic activities of EPCs.
Abstract 4916: Hepatocyte Growth Factor Mediates Proangiogenic Effects by Activating the NADPH Oxidase Nox2
Authors: Schröder Katrin , Schütz Susanne , Bätz Sina , Wandzioch Katalin , Rennecke Stephan , Brandes Ralf P
Published: April 2024

Hepatocyte growth factor (HGF) is a potent pro-angiogenic stimulus. We determined the effect of HGF on the mobilization of endothelial progenitor cells (EPCs) and endothelial aortic ring outgrowth as a model for ex vivo angiogenesis. As growth factor signaling frequently involves reactive oxygen species (ROS), we analyzed the effects of HGF on ROS formation and identified their enzymatic sources and the contribution of ROS to HGF signaling. In vivo application of HGF in mice induced the mobilization of EPCs (identified by FACS analysis as sca-1+, flk-1+, lineage negative mononuclear cells). Ex vivo, HGF stimulated endothelial cell outgrowth and increased the H2O2 formation of EPCs and the peroxynitrite formation in HUVEC as determined by peroxidase-catalyzed amplex red oxidation and DCHF-DA oxidation, respectively. This difference in ROS formation between EPCs and HUVECs was a consequence of the low expression of endothelial NO synthase (eNOS) in EPCs: In HUVECs, HGF increased the phosphorylation of eNOS at Ser1177, which increases the catalytic activity of the enzyme. As NADPH oxidases of the Nox family are important sources of ROS, their contribution to the signaling of HGF was determined. Down-regulation or genetic deletion of Nox2 prevented the HGF-stimulated ROS formation. As compared to wild type controls, the HGF-induced endothelial outgrowth was significantly reduced in aortic rings from Nox2 deficient (Nox2y/-) mice. Moreover, in vivo, the HGF-induced EPC mobilization was absent in Nox2y/- mice. Accordingly, HGF induced colony forming unit (CFU) formation in EPCs of WT but not Nox2y/- mice. ROS formation was essential for HGF-induced signaling: In HUVEC, HGF stimulated the phosphorylation of Jak2 and STAT3 and these effects were absent after depletion of the NADPH oxidase Nox2. We conclude that HGF-signaling critically involves the NADPH oxidase Nox2 and that ROS-formation by this enzyme contributes to the pro-angiogenic effects of HGF by stimulating EPC mobilization and endothelial activity.
Abstract 4921: Endothelial-targeted Expression of a Conditional Fas-fKBP Chimera in Transgenic Mice Establishes the Role of Lung Microvascular Apoptosis in Pulmonary Hypertension
Authors: Jiang Jin-Yi , Jurasz Paul , Courtman David W , Deng Yupu , Yang Anli , Zhang Qiuwang , Turksen Kursad , Sinclair Tammy , Stewart Duncan J
Published: April 2024

Introduction: Pulmonary arterial hypertension (PAH) is a lethal disorder characterized by pulmonary arteriolar remodeling and obliteration. Although its pathogenesis is still unclear, endothelial cell (EC) apoptosis has been suggested to be an initiating event in PAH, possibly leading directly to the degeneration of fragile precapillary arterioles. This study was to create a transgenic model of tritrable EC apoptosis to test the hypothesis that this is sufficient to induce the PAH phenotype.

Methods: The Fas-Induced Apoptosis (FIA) construct contains the Fas cytoplasmic death domain fused with 2 copies of the FK506-binding peptide (FKBP) domain. This construct was targeted to ECs (i.e. EFIA) under the control of the Tie2 promoter and apoptosis was induced by a small molecule dimerizing agent, AP20187, which binds tightly to the FKBP domain. After
in vitro
validation of its activity, the EFIA gene construct was microinjected into fertilized oocytes in CD1 mice. Apoptosis was assessed by TUNEL staining and right ventricular systolic pressure (RVSP) and hypertrophy (RV/LV+septal weight) were determined with and without i.p. injection of AP20187.

Results: EFIA transgenic mice appeared normal, however, administration of the AP20187 at 10 mg/kg/day for 2 weeks resulted in an increase in RVSP, which was greater in homozygous vs. heterozygous EFIA mice (37 ± 7 and 31 ± 7 mm Hg, respectively) compared WT mice either with or without AP20187 (26 ± 2 and 23 ± 2, respectively; p = 0.02). As well, there was a significant increase in right ventricular remodeling in homozygous EFIA mice treated with AP20187 compared to WT mice (RV/LV+ septal wt: 0.366 ±0.132 vs. 0.244 ± 0.048, respectively). AP20187 treatment also induced apoptosis in the peripheral lung (0.22 ± 0.05%) as well as in the kidney (9 ± 2%), which was significantly correlated with increases in RVSP. Of noted, increased lung cell proliferation, as evidenced by PCNA staining, was seen only in AP20187-treated EFIA mice.

Conclusions: These results provide the first evidence that EC apoptosis is sufficient to produce PAH. This was characterized in EFIA mice by both increased peripheral lung apoptosis and reactive cellular proliferation, thus representing a new tool to further explore the pathogenesis of the lethal disease.
Abstract 4931: RGDS-Epitope Presenting Peptide Amphiphile Nanofibers Enhance Therapeutic Potency of Cell-based Strategies in Ischemic Tissue
Authors: Tongers Joern , Webber Matthew G , Renault Marie-Ange , Roncalli Jerome G , Jujo Kentaro , Wu Xintong , Klyachko Ekaterina , Thorne Tina , Stupp Samuel I , Losordo Douglas W
Published: April 2024

Cell-based therapies have emerged as a promising option in ischemic tissue repair. Impaired viability and retention of injected cells in the ischemic target zone and reduced biopotency of autologous cells from pts with advanced disease remain significant challenges. Advances in bionanotechnology have led to development of biomaterials that may offer solutions to overcome limitations and to enhance the efficacy of cell therapy by modulating the microenvironment. Interaction with fibronectin (FN) has been shown to effect cell survival, adhesion and motility. We developed a self-assembling peptide amphiphile (PA) nanofiber that presents the binding domain epitope of FN, Arg-Gly-Asp-Ser (RGDS). Incubation of BM-derived pro-angiogenic cells (BMPAC) with this RGDS-PA (0.2w%) reduced H
2
O
2
-induced apoptosis (0.22±0.02 TUNEL
+
/TUNEL
−
cells) compared to cells without RGDS-PA (0.60±0.07, n=5–10, P<0.01). In addition, RGDS-PA increased proliferation (MTS assay; RGDS-PA 174±30% vs. 100% control, n=7–12, P<0.01), adhesion to uncoated (185±95 vs. 59±25, P<0.05) and vitronectin (201±84 vs. 53±26, P<0.05) surfaces. Tube formation (matrigel assay) and migration (Boyden chamber), endpoints that integrate several cell functions, were enhanced and impaired respectively. Local injection of RGDS-PA combined with a
sub-therapeutic
dose of BMPAC (10
5
) (R+B) vs. BMPAC alone (B) 3 days after surgical hind-limb ischemia (HLI) in 8-week-old, male FVB/N mice, improved blood flow recovery (laser Doppler, ischemic/non-ischemic limb ratio: R+B, 0.67±0.06; B, 0.55±0.04), limb salvage (score: R+B, 3.4±0.3; B 2.9±0.4; n=8 –9/group, P<0.05), and motor function of the ischemic limb (score: R+B, 3.6±0.2; B, 2.9±0.2; n=8 –9/group, P<0.05) at day 28. Viablity tracking with
β
-actin-luc
+
cells revealed higher bioluminescent signals, reflecting more viable cells at day 7 (R+B, 692±141%; B, 132±50%; n=7–9/group, p<0.01). These findings correspond with incr CD31
+
capillaries in the ischemic limb muscle at day 28 (Tx: 56±2/hpf; R+B: 91±5/hpf; B: 72±2/hpf; n=5–9/group, p<0.001).

Conclusion
: RGDS-PA nanofibers enhance functionality of BMPAC and thereby augment potency of cell-based therapies in ischemic tissue repair.

This research has received full or partial funding support from the American Heart Association, Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).
Abstract 4933: A 17B-estradiol Conjugated Chitosan-phosphorylcholine Polymer Supports Survival and Amplification of Endothelial Progenitor Cells (EPCs)
Authors: Tardif Kim , Cloutier Isabelle , Miao Zheime , Lemieux Caroline , Winnik Françoise , Tanguay Jean-François
Published: April 2024

Introduction The mobilization of bone marrow derived cells (BMDC) including endothelial progenitors cells (EPCs) accelerates the reendothelialization process. Therefore, methodologies to favour EPC mobilization and amplification at the lesion site are needed. 17B-estradiol (E2) was shown to favour vascular healing through mechanisms involving EPCs. Chitosan (CH), a biodegradable polymer when combined to phosphorylcholine (PC) groups demonstrates an enhanced biocompatibility within a physiological environment and represents an attractive tool for tissue engineering. Hypothesis E2 conjugated CH-PC polymer improves survival, amplification and functionality of bone marrow derived EPC.

Methods: A system of mixed BMDC isolated from C57BL/6 female mice was cultured on fibronectin (control matrix), CH-PC-E2 or CH-PC alone or with soluble E2. BMDC proliferation, survival and organization were evaluated over 14 days. Proportion of mesenchymal cells (CD44+) and hematopoietic cells (CD117+) was determined by flow cytometry analysis. Cell migration through a CH-PC, CH-PC-E2 or fibronectin microfilm was assessed in a transwell system where E2 was used as the chemoattractant.

Results: BMDC adhesion and organization were comparable on the CH-PC, CH-PC-E2 and fibronectin matrixes over tested times. Soluble E2 or conjugated to CH-PC did not affect cell growth in standard proliferation assay. However, in specific population analysis, CH-PC-E2 markedly increased the proportion of hematopoietic cells (7,7±1,2 % vs CH-PC 2,4±0,65% and fibronectin 0,98±0,2%, p<0.05) and EPCs (2,31±0,3% vs CH-PC 0,2±0,05% and fibronectin 0,03±0,01, p<0.05). At the opposite, it decreased mesenchymal fraction (82,4±5,2% vs CH-PC 94,7±1,2% and fibronectin 89,4±3,0%, p<0.05). Moreover, even if all matrixes preserved the heterogeneous morphology of cell populations, only the CH-PC based polymers triggered spontaneous tubules formation suggesting a proangiogenic potential. Finally, the E2-induced migratory activity of BMDC was reduced by half but not abrogated on CH-based matrixes.

Conclusion: E2 conjugated CH-PC promotes EPC amplification and is a promising vehicle for cell delivery at the vascular lesion to increase healing process.
Abstract 4942: Identification of Insulin Receptor Substrate Family Proteins Mediating PAI-1 Production in Cardiac and Adipose Tissues From IRS-1 Null Mice
Authors: Furumoto Tomoo , Fujii Satoshi , Tsutsui Hiroyuki
Published: April 2024

Obesity is associated with dysfunctional insulin receptor substrate (IRS)-1 pathway and compensatory hyperinsulinemia. Insulin can stimulate production of plasminogen activator inhibitor (PAI)-1, the physiologic inhibitor of fibrinolysis, in adipocytes. Because hypofibrinolysis by PAI-1 confers cardiovascular risk, the role of IRS-1-mediated signaling in PAI-1 expression was elucidated in IRS-1 knockout (KO) mice. PAI-1 mRNA was increased by 10-fold in adipose tissue from IRS-1 KO (P<0.001). Abdominal adipocyes isolated from IRS-1 KO released 11-fold more PAI-1 to the conditioned media (P<0.001), indicating increased PAI-1 production in IRS-1 KO. IRS-2 was barely detectable in abdominal adipose tissue of IRS-1 KO. In contrast, IRS-3 was increased by 60% in IRS-1 KO, suggesting that IRS-3-mediated pathway was compensating for the lack of IRS-1 in adipose tissue. PAI-1 mRNA was increased in IRS-1 heart and perivascular fibrosis was persistently augmented, suggesting that PAI-1 can contribute to coronary arterial remodeling. In IRS-1 KO, cardiac expressions of IRS-2 and p85, a substrate of IRS-2, were increased by 2.7- and 2.4-fold over control and active JNK1 was increased by 1.36-fold and active JNK2 was increased by 2.18-fold, suggesting the presence of compensatory IRS-2-mediated pathway. Thus, impaired IRS-1 mediated signaling can be compensated by diverse alternative pathways in IRS-1 KO. These alternative pathways may contribute to persistent PAI-1 expression and hypofibrinlysis in the setting of hyperinsulinemia in obesity.
Abstract 5008: Effect of Clopidogrel Discontinuation on Platelet Function
Authors: Lordkipanidze Marie , Diodati Jean G , Schampaert Erick , Palisaitis Donald A , Sia Edmond , Pharand Chantal
Published: April 2024

Objective:
Clopidogrel discontinuation is associated with increased incidence of ischemic events, which may signal a pharmacological rebound. We evaluated the impact of clopidogrel discontinuation on platelet function in CAD patients.

Methods:
Platelet aggregation was measured by VerifyNow
®
P2Y
12
and by light transmission aggregometry (LTA) in response to ADP 1, 2, 3, 5, 10, and 20
μ
M, in 37 CAD patients scheduled to discontinue clopidogrel treatment (aspirin treated patients clinically stable 1 year after clopidogrel initiation), and 37 CAD patients not taking clopidogrel. Platelet function was assessed the day before clopidogrel cessation and 1, 3, 7, 14, 21, 28 days after.

Results:
Subjects were on average 65±10 years old, and presented classic CAD risk factors similarly treated between groups. Subjects discontinuing clopidogrel were more likely to have undergone stenting. VerifyNow
®
P2Y
12
did not detect any rebound platelet activity, but ADP-induced LTA showed significant increase in platelet aggregation, particularly at low ADP levels, suggestive of platelet hypersensitivity (Fig

). Rebound platelet activity was detectable at 7 days after clopidogrel cessation, and remained higher than in controls 28 days of discontinuation. No clinical events were seen at 28 days in either group.

Conclusion:
Platelet sensitization to ADP as a consequence of chronic clopidogrel administration may partially explain the recrudescence of ischemic events following clopidogrel discontinuation in otherwise stable CAD patients. Further research into the pathophysiological processes of platelet function recovery is required.

Figure:

Platelet aggregation as a function of time
Abstract 5029: Carotid Plaque Regression Following 6-Month Statin Therapy Assessed by 3T MRI: Comparison With Ultrasound Intima-Media Thickness
Authors: Bowers Mark , LaDisa John F , Harmann Leanne , Prost Robert , Doppalapudi Anil , Migrino Raymond Q
Published: April 2024

Statin’s role in

short-term

plaque regression is unknown. 3T MRI has superior plaque volume (PV) measurement capacity compared to ultrasound intima-media thickness (IMT). We tested the hypothesis that carotid plaque progression is attenuated by 6 month statin therapy and this can be detected by MRI PV but not IMT.

Methods

: 15 subjects (68±3 years, 4F) with carotid atherosclerosis had carotid MRI (4 channel coil, resolution 0.3– 0.6×0.3– 0.6×2mm) and IMT assessed at baseline and 6 months post-statin treatment. T1, T2, proton density and time of flight images were obtained±12 mm from flow divider. PV and components were measured with Plaqueview software. Same day mean posterior IMT were measured by standard method. Observers were blinded to subject/scan order.

Results

: LDL changed from 90±7 to 78±7 mg/dL (p=NS). MRI PV decreased (1166±83 to 1084±102 mm
3
, p=0.007). Mean IMT was unchanged (1.14±0.07 vs. 1.14±0.07 mm, p=NS). Change in necrotic and hemorrhage volume correlated with MRI PV change (R=0.8, 0.7, all p<0.001), but not IMT change. In 8 subjects, plaque spatial distribution (6 quadrants) was analyzed (see figure

). Greatest thickness was seen opposite the flow divider (241–300°, p<0.01), a region not accessed by US IMT.

Conclusions

: In carotid disease, there was a measurable reduction in plaque burden following

short-term

statin treatment using 3T MRI that was not seen with US IMT. The heterogenous spatial distribution of plaque supports use of volumetric measurement for serial studies. 3T MRI may be useful to assess treatment efficacy, reduce number of subjects needed and be a better imaging tool to assess effects of novel therapies for carotid atherosclerosis in clinical trials.
Abstract 5030: Sex Differences of Carotid Atherosclerotic Plaque: In vivo 3T MRI Study
Authors: Ota Hideki , Reeves Mathew J , Zhu David C , Yuan Chun , DeMarco J K
Published: April 2024

Background:
Carotid endarterectomy (CEA) to prevent stroke is less beneficial for women compared to men in asymptomatic patients. Sex differences in plaque characteristics may help explain why women receive less benefit from CEA. Numerous plaque features are associated with increased stroke risk such as thin/ruptured fibrous cap, larger lipid-rich/necrotic core (LRNC) and hemorrhage. We tested the hypothesis that within an asymptomatic carotid stenosis population, men have more of these high-risk carotid plaque features than women as detected by
in vivo
3T MRI.

Methods:
One hundred twenty consecutive patients (men, 64, women, 56, age 45– 86) with >=50% asymptomatic carotid stenosis measured by duplex ultrasound were included. Two independent and blinded reviewers interpreted multi-contrast MR images of the more stenotic carotid. Presence of thin/ruptured fibrous cap and plaque components (LRNC, hemorrhage and calcium), and % component volume, if present, were documented. Multivariate logistic and linear regression analyses were used to quantify the association between sex and individual plaque characteristics adjusting for other potential confounders: age, statin use, smoking, history of hyperlipidemia, hypertension, coronary artery disease and diabetes.

Results:
The prevalence of plaque features and % volume components in men versus women were, respectively: thin/ruptured fibrous cap (47% vs 20%, p<0.01), LRNC (72% vs 48%, p=0.01), hemorrhage (31% vs 17%, p=0.11) and calcium (84% vs 89%, p=0.45); mean % volume of LRNC (5.7% vs 2.4%, p<0.01), hemorrhage (2.2% vs 0.3%, p=0.03) and calcium (4.6% vs 4.1%, p=0.58). Only statin use was found to be a significant confounder and included in the multivariate models. The adjusted odds ratio (aOR) were significantly greater for men (vs women) for presence of thin/ruptured fibrous cap (aOR, 3.5, 95%CI= 1.5, 8.0), and LRNC (aOR 3.6, 95%CI= 1.6, 8.1). Plaque volumes were also significantly larger in men for % LRNC (3.7%, 95%CI= 1.6, 5.7%), and % hemorrhage volume (1.9%, 95% CI=0.5, 3.2%).

Conclusion:
In patients with asymptomatic >=50% carotid stenosis, men tended to have higher-risk plaque features than women. These findings indicate a possible reason for why CEA is less effective in asymptomatic women.

This research has received full or partial funding support from the American Heart Association, Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).
Abstract 5068: Interaction Between CD36 and Oxidized LDL Induces Loss of Cell Polarity and Inhibits Macrophage Migration: A Mechanism of Macrophage Trapping
Authors: Park Young M , Drazba Judith A , Febbraio Maria , Silverstein Roy L
Published: April 2024

Trapping of lipid-laden macrophages in the arterial intima is a critical but reversible step in atherosclerosis. Previous studies from other laboratories showed that regressed atherosclerotic lesions were characterized by emigration of lipid-laden macrophages. CD36, a class B scavenger receptor expressed on macrophages is a pathogenic factor for foam cell formation and atherosclerosis. We recently showed that oxidized LDL (oxLDL) modulates macrophage cytoskeletal signaling via CD36, inhibiting migration and thus potentially contributing to intimal trapping. We now hypothesize that oxLDL-CD36 inhibition of migration is the result of specific intracellular signals that regulate cell polarity.

Methods/Results:
Live cell imaging of murine peritoneal macrophages showed that oxLDL, but not native LDL, induced loss of cell polarity. Macrophages from
cd36 null
mice did not show this effect. Similarly, macrophages from mice null for Vav, a guanine nucleotide exchange factor recently shown to be a downstream effector of CD36 signaling, did not show oxLDL-induced loss of polarity. Macrophage migration velocity and dynamic movement of macrophage membrane were decreased by oxLDL in wild type macrophages by 85% and 86% respectively, but not in
cd36 null
or
vav null
macrophages. These findings appear to be related to activity of myosin II, a recently suggested cell polarity determinant. Western blots for phosphorylated myosin regulatory light chain (MRLC) showed that oxLDL, but not native LDL, induced de-phosphorylation of MRLC by 80%. Enzyme linked immunosorbent assays for GTP-bound Rac, a regulator of MRLC phosphorylation, showed that macrophage Rac-GTPase activity was increased 2.6 fold by oxLDL. These findings were not observed in macrophages from
cd36 null
or
vav null
mice. 6-thio-GTP, a Rac-GTPase inhibitor that inhibits binding of Vav to Rac1 abrogated the effect of oxLDL on MRLC dephosphorylation.

Conclusion:
OxLDL interaction with CD36 inhibited macrophage migration and caused loss of cell polarity. The mechanism by which this occurs is via Vav-dependent activation of Rac-GTPase and dephosphorylation of MRLC. Activation of this pathway in the vessel wall may induce trapping of macrophages in the arterial intima and promote atherosclerosis.
Abstract 5091: Long-Term Clinical Outcome of Therapeutic Angiogenesis by Autologous Transplantation of Bone Marrow Mononuclear Cells for Patients With Chronic Limb Ischemia - Results of the TACT Trial, Prognosis, Efficacy and Safety
Authors: Matoba Satoaki , Matsubara Hiroaki , Imaizumi Tsutomu , Murohara Toyoaki , Saito Yoshihiko , Fukushima Masanori
Published: April 2024

Therapeutic Angiogenesis by Cell Transplantation (TACT) study is the first randomized controlled angiogenic cell therapy by intramuscular injection of autologous bone marrow mononuclear cells (BM-MNCs) in the patients with critical limb ischemia. The feasibility of TACT was reported in the Lancet (2002).

Objective and Results

The present study was designed to assess the 3-year safety and clinical outcomes of this angiogenic cell therapy by investigating the mortality and leg amputation-free interval as primary endpoints. Three year overall survival rates were 80% in patients with peripheral artery disease (PAD) (11 died in 74 patients) and 100% (no death) in 41 patients with thromboangiitis obliterans (TAO, Buerger’s disease). Three-year amputation-free rate was 60% in PAD and 91% in TAO patients (Fig

). These mortality and amputation-free rate were lower than the historical data from the conventional therapies. The multi-variate analysis revealed that the severity of rest pain and repeated experience of bypass surgery are the prognostic factors negatively affecting amputation-free interval. The significant improvement in the rest pain, ulcer size and walking distance was maintained during at least 2 year after the therapy.

Conclusions

The present study demonstrates that the angiogenic cell therapy using BM-MNCs has an ability to induce a long-term efficient increase in the regional perfusion in ischemic limbs, leading to extension of amputation-free interval. The safety and efficacy are superior to the conventional revascularization therapies. The severity of rest pain and the history of bypass surgery negatively influence the responsiveness to this cell therapy.
Abstract 5093: Atorvastatin Reduces Macrophage Accumulation in the Atherosclerotic Plaque: A Comparison versus Non-statin Based Regimen in Patients Undergoing Carotid Endarterectomy
Authors: Puato Massimo , Faggin Elisabetta , Rattazzi Marcello , Cipollone Francesco , Zambon Alberto , Grego Franco , Mezzetti Andrea , Pauletto Paolo
Published: April 2024

Several studies suggested that statins may exert anti-atherosclerotic effects beyond cholesterol lowering. No definite data on humans are available confirming this hypothesis. To compare the effect of high dose
vs
low dose of atorvastatin
vs
non-statin based treatment (cholestyramine plus sistosterol) on carotid plaque composition, we studied 60 hypercholesterolemic patients (total cholesterol, TC, 225–295 mg/dl) eligible for carotid endarterectomy. Three months prior surgery patients were randomized into 3 groups (n=20) receiving atorvastatin 10 mg/day (AT-10), or atorvastatin 80 mg/day (AT-80), or cholestyramine 8 g/day plus sitosterol 2.5 g/day (C-S). Blood samples were collected to assess lipid profile and inflammatory markers levels. Analysis of carotid plaque cell composition and lipid content was performed on endarterectomy specimens. The AT-80 group showed the highest reduction in TC and LDL cholesterol (LDL-C) levels. A comparable TC and LDL-C level was achieved in the AT-10 and C-S groups. The three regimens did not significantly influence the levels of inflammatory markers (including hs-PCR). A lower content in macrophages was observed in the AT-10 group plaques compared to the C-S group. The macrophage content was further on reduced in the AT-80 group plaques. No difference in lymphocyte number was observed among treatments while the content of smooth muscle cells was higher in the AT- 80 group. The plaque lipid content was similar in the 3 groups. In conclusion, dspite a comparable reduction in cholesterol levels, atorvastatin treatment was superior to a non-statin based regimen in blunting macrophage accumulation inside carotid atherosclerotic plaque.
Abstract 5094: Effect of Celiprolol on Prevention of Cardiovascular Events in Vascular Ehlers-Danlos Syndrome
Authors: Ong Kim-Thanh , Perdu Jérôme , Plauchu Henri , De Backer Julie , De Paepe Anne , Emmerich Joseph , Jeunemaitre Xavier , Germain Dominique , Collignon Patrick , Georgesco Gabriella , Bozec Erwan , Hulot Jean-Sébastien , Laurent Stéphane , Boutouyrie Pierre
Published: April 2024

Background: Vascular Ehlers-Danlos syndrome (vEDS) is a rare severe genetic disease which results from mutations in the gene encoding type III procollagen (COL3A1), characterized by vascular and/or hollow organic ruptures. No treatment is yet validated. We tested the ability of celiprolol, a beta1-adrenoceptor antagonist with a beta2-adrenoceptor agonist action, for preventing the complications of vEDS in a prospective, randomized, open, blinded endpoints trial.

Methods: Fifty three previously untreated vEDS patients were randomized to a 5-year treatment with either celiprolol (n=25) or no treatment (n=28). The two groups were matched for demographic, medical historic and clinical characteristics. Celiprolol was up-titrated from 100 to 400 mg by steps of 100 mg every 6 months. The primary end-point was an arterial event (rupture or dissection, fatal or not) occurring during follow-up. Secondary endpoints were intestinal or uterine rupture or major clinical events, related to vEDS, judged by the event committee.

Results: Mean duration of follow-up was 47 (±15) months. The study was ended prematurely by the safety monitoring board since significant differences were reached between two groups. The primary endpoint was reached by 5 patients (20%) in the celiprolol group and by 14 patients (50%) in the control group (hazard ratio, 0.36; 95% CI, 0.15 to 0.88; P=0.04). Primary plus secondary endpoints occurred in 6 patients (24%) in the celiprolol group and in 17 patient (61%) in the control group (hazard ratio, 0.31; 95% CI, 0.14 to 0.71; P=0.0097).

Conclusions: Celiprolol effectively reduced both vascular complications and organic ruptures in vEDS patients.