bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5211: Identification of a Direct Substrate of Thioredoxin 1 in Cardiac Myocytes
Authors: Oka Shinichi , Shao Dan , Li Hong , Sadhoshima Junichi
Published: April 2024

Thioredoxin 1 (Trx1), a 12 kD anti-oxidant, reduces oxidized proteins by transferring electrons from its conserved cysteine residues (Cys-32 and Cys-35) to its targets, a reaction termed the thiol-disulfide exchange reaction. Trx1 inhibits pathological hypertrophy in the heart, which is in part mediated by reduction of DnaJb5 and class II histone deacetylases. Thus, the goal in this study was to identify additional redox-sensitive targets of Trx1 in cardiac myocytes. When Trx1 reduces its target, Cys-32 in the catalytic center transiently forms an intermolecular disulfide bond with substrate proteins, which is in turn reduced by Cys-35. Thus, Trx1C35S, a Trx1 mutant, is expected to act as a substrate trapping mutant due to an incomplete dithiol-disulfide exchange reaction. To identify direct substrate proteins of Trx1 in cardiac myocytes, Flag-Trx1C35S-HA was expressed in cultured cardiac myocytes with adenovirus transduction. After myocytes were treated with H
2
O
2
, proteins trapped by Flag-Trx1C35S-HA were immunoprecipitated with anti-HA antibody. No protein was trapped when Flag-Trx1CC32/35SS-HA was used, suggesting that the trapping is Cys-32-dependent. Trapped substrates were separated by non-reducing SDS-PAGE and then subjected to mass spectrometry. We identified rat peroxiredoxin 1 (Prdx1), a Trx1-dependent peroxidase, as a major protein trapped by Flag-Trx1C35S-HA in hydrogen peroxide treated cardiac myocytes. Overexpression of Trx1 inhibited H
2
O
2
-induced oxidation of Prdx1 in cells
in vitro
(Oxidized/Reduced form of Prdx1: control 1.58, Trx1 overexpression 0.7), confirming that Prdx1 is reduced by Trx1. In order to identify
in vivo
targets of Trx1, transgenic mice (Tg) with cardiac specific expression of Flag-Trx1C35S-HA were generated. Heart homogenates obtained from Tg-Flag-Trx1C35S-HA mice were subjected to immunoprecipitation with anti-HA antibody and separated by non-reducing SDS-PAGE. Subsequent immunoblot analyses showed that Prdx1 was among the proteins trapped by Flag-Trx1C35S-HA
in vivo
. Thus, using a new Trx1 substrate trapping assay, we identified Prdx1 as a major direct substrate of Trx1 in cardiac myocytes both
in vitro
and
in vivo
.
Abstract 5217: Endothelial Vasomotor Dysfunction in the Brachial Artery Predicts Development of Early Stage Renal Dysfunction in Patients With Type 2 Diabetes Mellitus
Authors: Hirano Mitsumasa , Nakamura Takamitsu , Kitta Yoshinobu , Takishima Isao , Ino Yuri , Kobayashi Tsuyoshi , Makino Aritaka , Sano Keita , Saito Yukio , Kodama Yasushi , Obata Jun-ei , Kawabata Ken-ichi , Kugiyama Kiyotaka
Published: April 2024

Endothelial dysfunction in renal vasculature is implicated in the pathogenesis of renal failure in type 2 diabetes mellitus (DM). Endothelial dysfunction occurs in systemic vasculature of DM patients, and there may be a relationship between endothelial dysfunction in the brachial artery and renal vasculature. This study examined whether endothelial vasomotor dysfunction in the brachial artery predicts early renal dysfunction in DM patients.

Methods:
Flow-mediated endothelium-dependent dilation (FMD, % increase in artery diameter from baseline) in the brachial artery was measured in 275 consecutive DM patients with normal renal function at enrollment (serum creatinine levels [sCr] <1.0 mg/dl, urinary albumin excretion [UAE] < 25 mg/day and estimated glomerular filtration rate [eGFR] ≥ 60 mL/min/1.73 m
2
at baseline). They were prospectively followed for 1.5 years. The end point was development of early stage renal dysfunction, defined as occurrence of one or more of the following events: sCr ≥ 1.2 mg/dL, UAE ≥ 30 mg/day and eGFR < 60 mL/min/1.73 m
2
.

Results:
During the follow-up period, early stage renal dysfunction developed in 63 (46.7%) patients with impaired FMD (< 5.2%, 50
th
percentile of the distribution) and in 17 (12.1 %) patients with the preserved FMD (≥ 5.2%) (p < 0.0001). Using multivariate logistic analysis, the impaired FMD was an independent predictor of the development of early stage renal dysfunction (OR; 5.1, 95%CI; 2.62 - 9.68, p < 0.0001) after adjustment with covariates, including hypertension, age, use of anti-diabetic drugs and CRP levels. Using a
c
-statistic, the predictive value of impaired FMD was significantly incremental over that of the conventional known risks including hypertension, age, use of anti-diabetic drug and CRP levels for progression of renal dysfunction (area under the ROC curve; 0.88 and 0.72, respectively, p = 0.01). Dilator response of the brachial artery to nitrates had no significant predictive value.

Conclusions:
Endothelial vasomotor dysfunction in the brachial artery is an independent predictor of development of early renal dysfunction in DM patients. The measurement of FMD is useful for identifying patients with type 2 DM at risk of developing future renal dysfunction.
Abstract 5253: Human Microvascular Dysfunction Induced by AL Amyloid Light Chain Proteins is Attenuated by Tetrahydrobiopterin
Authors: Migrino Raymond Q , Truran Seth , Gutterman David D , Schlundt Brittany , Bright Megan , Hari Parameswaran
Published: April 2024

The mechanisms of injury in light chain (LC) amyloidosis (AL) remain unknown. We aim to measure endothelial/non-endothelial dilation and nitric oxide (NO) production in non-AL human coronary and adipose arterioles post exposure to LC±tetrahydrobiopterin (BH4).

Methods:
Urine LC were purified (5 AL subjects, 57±11 years). Adipose (n=19) and coronary (n=25) arterioles from discarded fat and atrial appendage from non-AL patients post surgery were cannulated, pressurized (60 mm Hg) and dilation to acetylcholine (adipose:10
−9
–10
−4
M) or bradykinin (coronary:10
−10
–10
−5
M) and papaverine (both:10
−4
M) were measured before and 1 hour post exposure to LC (20
μ
g/mL) or LC+BH4 (100
μ
M). Separately, NO was measured using 4,5-diaminofluorescein diacetate fluorescence in arterioles exposed to LC, LC+BH4 and vehicle.

Results:
Adipose arterioles had impaired dilation to acetylcholine (10
−4
M:47.4±8.2 versus 82.9±3.5% control, p=0.002) and papaverine (83.8±4.3 vs. 94.5±1.6% control, p=0.007) post-LC suggesting endothelial and non-endothelial dysfunction. Post LC, coronary dilation to bradykinin (10
−5
M:63.4±6.3 versus 84.8±3.1% control, p=0.004) but not papaverine was reduced signifying endothelial dysfunction. BH4 pretreatment restored adipose and coronary microvascular function (p=NS vs. control). NO production was reduced by LC but restored by BH4 (see figure

).

Conclusions:
Brief exposure to AL light chains impaired microvascular function and reduced NO production in non-AL human adipose and coronary arterioles; both were restored by tetrahydrobiopterin. This novel mechanism of injury may be important in AL pathophysiology and a potential therapeutic target.

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 5263: Renin Angiotensin System Plays a Crucial Role in the Development of Myocardial Ischemia/Reperfusion Injury With a Condition of Renal Failure in Mice
Authors: Suzuki Jun-ichi , Ogawa Masahito , Hirata Yasunobu , Nagai Ryozo , Isobe Mitsuaki
Published: April 2024

Background: The clinical relationship between the heart and renal diseases has been clarified. Although renin angiotensin system (RAS) mediates the development of cardio-renal syndrome, little is known about the etiology obtained from experimental models with myocardial ischemia and renal failure.

Methods: To test the hypothesis that RAS plays a pivotal role in cardio-renal syndrome, we developed a novel murine cardio-renal disease model using both the 5/6 subtotal nephrectomy (NTX) and the myocardial ischemia/reperfusion injury (I/R) techniques. Four weeks after the subtotal nephrectomy, the left coronary artery was occluded for 60 minutes followed by 24 hour reperfusion.

Results: Serum creatinine levels on day 28 were higher in the NTX group (0.28±0.04 mg/dl, n=8) compared to those in the non-NTX group (0.13±0.01 mg/dl, n=5, p<0.05). The infarction area/area at risk ratio significantly increased in NTX+I/R mice (50.7±5.9 %, n=7) compared to the non-NTX+I/R mice (20.8±3.1 %, n=8, p<0.05), while ischemic area/whole myocardial area was comparable between the two groups. The NTX+I/R hearts had significantly larger left ventricular diameters compared with that of the non-NTX+I/R mice. The number of infiltrating cells was enhanced by the NTX+I/R, while they were limited in non-NTX+I/R. Interstitial fibrosis also increased in NTX+I/R, while non-NTX+I/R suppressed this change. The hearts isolated from NTX+I/R mice increased the mRNA expression ratios (the NTX-IR group per the native group) of angiotensin-converting enzyme (1.57±1.26) and angiotensin II receptor type 1 levels (1.21±0.07) measured by quantitative RT-PCR.

Conclusion: RAS deeply involves the development of cardio-renal syndrome and it may have a clinical usefulness in the treatment of myocardial ischemia with renal dysfunction.
Abstract 5268: A Critical Role of Bradykinin in the Murine Model of Contrast Media-Induced Nephropathy
Authors: Suzuki Jun-ichi , Ogawa Masahito , Hirata Yasunobu , Nagai Ryozo , Isobe Mitsuaki
Published: April 2024

Background: Iodinated contrast media (CM) has been broadly used for diagnostic procedures; CM-induced nephropathy (CIN) affects morbidity and mortality of patients. Although CIN is known to be mediated by several factors, including the renin-angiotensin system (RAS), little is known about the evidence obtained from experimental models. Thus, we developed a murine CIN model to evaluate the mechanism.

Methods: We performed 5/6 subtotal nephrectomy (NTX) and administered CM (iopamidol, 500
μ
l/25 g mice) intravenously into the mice 4 weeks after NTX. We administered an angiotensin converting enzyme inhibitor (ACEI, imidapril, 1 mg/kg/day, n=6), an angiotensin II receptor blocker (ARB, TA606, 1 mg/kg/day, n=4), or ACEI plus a bradykinin B2 receptor antagonist (Hoe-140, 0.25 mg/kg, n=5) into the mice daily.

Results: Serum creatinine levels on day 28 were significantly elevated in the NTX group (0.26±0.01 mg/dl) compared to those in the non-NTX group (0.13±0.01 mg/dl). A day after CM injection, creatinine levels were significantly elevated in the non-treated group (0.41±0.01 mg/dl). While ACEI treatment significantly suppressed the increase in creatinine levels (0.34±0.04 mg/dl, p<0.05 vs. CIN group), ARB treatment did not suppress (0.47±0.06 mg/dl). A bradykinin antagoninst Hoe-140 negated (0.43±0.07 mg/dl, p<0.05 vs. CIN+ACEI) ACEI’s ability to suppress renal damage. We examined renal AT1R, COX-2, MCP-1 and MMP-9 mRNA levels using quantitative RT-PCR. AT1R mRNA levels were enhanced in the NTX and CIN groups compared to those in the native group. COX-2, MCP-1 and MMP-9 mRNA levels were enhanced in the CIN+ACEI group compared to the native and CIN groups. ACEI+Hoe-140 treatment suppressed the mRNA expression. Both treatments did not reduce their blood pressure compared to those of the vehicle treatment, statistically.

Conclusion: ACEI treatment is useful for the prevention of CM-induced nephropathycompared to that of ARB treatment in the murine model of CIN because bradykinin pathway is critical in regulating CIN development.
Abstract 5282: RAPL, a New Mediator of Angiogenesis, Promotes Integrin Endocytosis in Endothelial Cells
Authors: Carmona Guillaume , Dimmeler Stefanie , Chavakis Emmanouil
Published: April 2024

The molecular mechanisms regulating angiogenesis are not entirely understood. Integrins are critical mediators of angiogenesis. In our previous work, we identified the small GTPase Rap1 as a mediator of integrin affinity in endothelial cells (EC) and angiogenesis. RAPL is an effector protein of Rap1 promoting integrin activation and adhesion in lymphocytes. However, the role of RAPL for integrin regulation in EC and angiogenesis is unclear. Silencing of RAPL with siRNA significantly decreased angiogenic sprouting of HUVEC in a 3-dimensional spheroidal system (83 % inhibition) and blocked VEGF-induced invasion (68 % inhibition) in comparison to scrambled transfected EC. However, while silencing of Rap1 reduced HUVEC adhesion, silencing of RAPL unexpectedly significantly increased adhesion suggesting distinct roles for RAPL and Rap1 in integrin regulation in EC. In line with these results, silencing of RAPL significantly increased surface expression of
β
1-integrin as assessed by FACS, while not affecting
β
1-integrin mRNA and total protein levels in HUVEC. It is conceivable that the enhanced surface expression of
β
1-integrins upon silencing of RAPL is caused by a redistribution of the integrin from an intracellular pool to the cell surface. Therefore, we studied the role of RAPL for the endocytosis (internalization) of
β
1-integrins in HUVEC. Strikingly, silencing of RAPL blocked
β
1-integrin endocytosis by 43±6 % as determined by confocal microscopy. Additionally, silencing of RAPL reduced internalization of
β
1-integrin as assessed by a FACS-based endocytosis assay. Interestingly, RAPL co-immunoprecipitated with GEP100, a guanine nucleotide exchange factor (GEF) for Arf6, which mediates
β
1-integrin internalization in HeLa cells. Furthermore, silencing of GEP100 significantly impaired angiogenic sprouting of HUVEC (55 % inhibition) and VEGF-induced invasion. In conclusion, our results demonstrate for the first time that RAPL mediates, in association with GEP100,
β
1-integrin internalization thereby promoting angiogenic sprouting and migration. Taken together, our data identify a new angiogenic signaling pathway and shed light on the relevance of
β
1-integrin internalization for angiogenic functions such as sprouting and migration.
Abstract 5300: Targeting Ongoing Arterial Thrombosis With Platelet-Specific Microparticles
Authors: Ruggeri Zaverio M , Orje Jennifer N , Mitragorti Samir , Smith Jeffrey
Published: April 2024

In the arterial circulation with rapid blood flow, initial platelet adhesion at sites of vascular injury depends on the interaction between the amino terminal domain of platelet glycoprotein Ib (GPIb-NH2) and the A1 domain of von Willebrand Factor (VWF-A1) immobilized onto the vessel wall. Moreover, platelet aggregation responsible for subsequent thrombus growth is mediated by binding of soluble plasma VWF to GPIb on the platelet membrane. Thus, microparticles (MPs) presenting on their surface GPIb-NH2 or VWF-A1 should be capable of homing to sites of ongoing thrombus formation by mimicking platelets either without or with bound VWF, respectively. To test this hypothesis, we coated fluorescent MPs with recombinant VWF-A1 (residues 445–733 of the mature human VWF subunit) or GPIb-NH2 (residues -2–290). Both proteins were expressed as dimers in the attempt to mimic the corresponding native functional conformations. When added to a washed human red cell suspension and perfused over the appropriate substrate (i.e. MPs coated with VWF-A1 over surface-immobilized GPIb-NH2; or vice versa), both kinds of MPs adhered in equal number at shear rate as high as 10,000 s-1. This response was markedly decreased when MPs were coated with VWF-A1 carrying the loss-of-function mutation G561S. In vivo, derivatized MPs were used in mouse models of thrombosis in the carotid and femoral arteries and in the femoral vein in which a thrombogenic lesion was induce by superfusion of ferric chloride or with a pulsed nitrogen laser. Upon injection during the early stages of platelet response to injury, MPs coated with either VWF-A1 or GPIb-NH2 specifically adhered to the lesion, indicating that both GPIb and VWF were present on the surface of the forming thrombus. In contrast, MPs coated with VWF-A1 were remarkably more efficient than those coated with GPIb-NH2 in targeting nearly occluding thrombi, indicating that the surface of the latter presents GPIb (platelets) but not accessible VWF. These findings show that MPs derivatized with specific adhesive domains may be used to target areas of vascular injury with developing thrombi for diagnostic or therapeutic purposes, as well as to interrogate thrombus composition.
Abstract 5308: Role of Vascular Endothelial Growth Factor-A in the Development of Mice CaCl
2
Induced Abdominal Aortic Aneurysm
Authors: Kaneko Hidehiro , Anzai Toshihisa , Takahashi Toshiyuki , Maekawa Yuichiro , Shimoda Masayuki , Yoshimura Koichi , Aoki Hiroki , Shimizu Hideyuki , Okada Yasunori , Yozu Ryohei , Yoshikawa Tsutomu , Ogawa Satoshi
Published: April 2024

Background:
Abdominal aortic aneurysm (AAA) is histologically characterized by increased angiogenesis, chronic inflammation, and extracellular matrix degradation. We sought to elucidate the role of vascular endothelial growth factor (VEGF)-A, which is a potent angiogenic and proinflammatory factor, in the development of AAA.

Methods and Results:
We obtained human aortic wall samples (n=10) during surgical repair of AAA. Autopsy samples from normal aortic wall were served as control (n=6). Immunohistochemistry showed increased CD31 positive vessels, VEGF-A expression, and CD68 positive macrophage infiltration in the aortic wall of AAA compared with control samples. Next, we induced AAA in mice by periaortic application of CaCl
2
. Mice were treated with intraperitoneal injection of soluble VEGF-A receptor-1 (sFlt-1) 1mg/g (FLT, n=8) or PBS 1
μ
l (CON, n=8) on alternative day and sacrificed 28 days after the operation. Blood pressure, heart rate, and body weight on day 28 were comparable between FLT and CON. Treatment with sFlt-1 significantly reduced aneurismal size (1.7±0.9 vs. 1.3±0.2 mm, p< 0.01) in association with lowering serum VEGF-A level (p< 0.01). Elastica von Gieson staining showed that wavy morphology of the elastic lamellae was destroyed in CON, but was preserved in FLT. Administration of sFlt-1 reduced zymographic active form of matrix metalloproteinase (MMP)-9 abundance and decreased MMP-9 positive cell count in the aortic wall compared with CON (both p<0.05). Immunohistochemistry showed significantly fewer Mac-3 positive monocytes/macrophages, CD31 and VEGF-A positive cells in periaortic tissues in FLT compared with CON (all p<0.05). Aortic wall mRNA expression of MCP-1, TNF-alpha, and TNF-alpha converting enzyme (TACE) in FLT was lower than that in CON (all p< 0.05). Immunoblotting showed decreased expression of p-JNK in FLT compared with CON (p< 0.05).

Conclusions:
VEGF-A was overexpressed in arterial wall of human AAA and experimental model of AAA. Treatment with sFlt-1 in mice prevented AAA development, in association with reduced neoangiogenesis, MMPs activity, infiltration of inflammatory cells and JNK activity, and preserved ECM structure. These findings suggest a crucial role of VEGF-A in the development of AAA.
Abstract 5309: Perforin-Independent Granzyme B-Mediated Extracellular Matrix Protein Cleavage Contributes to Abdominal Aortic Aneurysm
Authors: Ang Lisa S , Boivin Wendy A , Williams Sarah J , Chamberlain Ciara M , Zhao Hongyan , Allard Michael F , McManus Bruce M , Granville David J
Published: April 2024

Background:

Granzyme B (GrB) is a pro-apoptotic serine protease present in atherosclerotic human vessels, where its expression corresponds to increased disease severity and plaque instability. In addition to its perforin-dependent cytotoxic role, GrB accumulates extracellularly and contributes to the degradation of extracellular matrix (ECM) proteins.

Hypothesis:

GrB contributes to abdominal aortic aneurysm (AAA) through the cleavage of medial and/or adventitial ECM proteins.

Methods

: Human AAA and thoracic aortic aneurysm (TAA) samples were assessed for the presence of granzymes, immune cell markers, and ECM. Apolipoprotein E (apoE)−/− × GrB−/− double knockout (GDKO) and apoE−/− × perforin−/− DKO (PDKO) mice were generated to assess the functional intracellular and extracellular roles of GrB, respectively, in AAA. To induce AAA, mice were implanted with osmotic mini pumps containing angiotensin II (AII, 1000 ng/kg/min) or saline for 28 days. To further assess extracellular GrB activity, a novel serpin inhibitor of GrB was injected 3h prior to pump implantation. Survival, degree of AAA, ECM composition and GrB localization were evaluated.
In vitro
protease assays were performed to identify novel extracellular GrB substrates that may contribute to AAA.

Results:

While GrB was not present in healthy human aorta, GrB was abundantly expressed in the vessel wall and thrombus of aneurysmal tissue. AII-treated GDKO mice but not PDKO mice had increased 28-day survival (92.86%; n=14 vs. 56.25%, n=16) when compared to apoE-KO mice (53.33%; n=15). While AAA rupture was observed frequently in apoE-KO (46.7%; n=15) and PDKO (43.3%; n=16) mice, rupture was rarely observed in GDKO (7.1%; n=14) mice. Reduced degradation of medial fibrillin-1 and adventitial decorin and collagen was observed in the blood vessels of GDKO and serpin-treated mice versus apoE-KO mice. GrB localized to macrophages, mast cells and areas of medial disruption and adventitial weakening. Protease assays demonstrated GrB-cleavage of fibrillin-1 and decorin along with other ECM components.

Conclusions:

GrB contributes to the loss of vessel wall integrity and AAA through the cleavage of medial and adventitial ECM. Inhibition of GrB attenuates medial disruption and aortic rupture in mice.
Abstract 5336: Presence of Ischemia and Non-Sustained Ventricular Tachycardia is Strongly Associated With the Risk of Cardiovascular Outcomes: Observations From the MERLIN-TIMI 36 Trial
Authors: Harkness James R , Morrow David A , Braunwald Eugene , Qin Jie , Belardinelli Luiz , Wang Whedy , Karwatowska-Prokopczuk Ewa , Lopez-Sendon Jose , Bode Christophe , Scirica Benjamin M
Published: April 2024

Background: Among pts with NSTEACS, recurrent ischemia and ventricular arrhythmias detected on continuous ECG (cECG) remain common clinical events with uncertain clinical significance.

Methods: We determined the risk associated with the presence of ischemia (> 1mm ST depression lasting > 1min) and NSVT (> 4beats) alone or combined on 7-day cEKG in 6355 pts with NSTEACS. Pts were categorized into 4 groups:

No ischemia or NSVT,

NSVT alone,

Ischemia alone or

Both ischemia and NSVT.

Median clinical follow-up was 348 days.

Results: A total of 43.6% pts had no VT or ischemia; 31.2% had NSVT alone; 18.5% had ischemia alone; and 6.8% had both NSVT and ischemia. Pts with either NSVT or ischemia on cECG were at increased risk of CV outcomes (Table

). In a MV analysis, recurrent ischemia alone increased the risk of all CV outcomes while NSVT alone increased the risk of CVD, SCD, and subsequent heart failure. Pts with both ischemia and NSVT were at high-risk for CVD (10.1 v 3.0%, p<0.001), SCD (7.8 v 0.9%, p<0.001), recurrent MI (15.4 v 6.2%, p<0.001) and heart failure (13.0 v 3.0%, p<0.001) compared to pts with neither. In pts with both ischemia and NSVT, 47.1% of SCD occurred within 90d (Figure

).

Conclusion: In more than 6300 pts with NSTEACS, the presence of either ischemia or NSVT alone or particularly in combination was independently associated with poor CV outcomes, including a particularly high risk for early SCD. These findings raise the question whether such pts should be considered for ICD implantation.

Sudden Cardiac Death

1-yr Clinical Outcomes(%) by NSVT or Ischemia detected on cECG monitoring
Abstract 5337: Morphological Characteristics of the Culprit Lesion Correlate With the Angiographic Flow Grade Following Thrombolysis in Myocardial Infarction. An Optical Coherence Tomography Study
Authors: Karanasos Antonis , Toutouzas Konstantinos , Drakopoulou Maria , Synetos Andreas , Riga Maria , Tsiamis Eleftherios , Klettas Dimitrios , Tentolouris Costas , Stefanadi Elli , Stefanadis Christodoulos
Published: April 2024

Background: Intravenous thrombolysis fails to restore blood flow in a significant percentage of patients with ST-elevation myocardial infarction (STEMI). It remains unknown, whether certain morphological characteristics of the culprit lesion (CL) can predict the success of thrombolysis. We hypothesized that the thickness of the fibrous cap and the incidence of plaque rupture at the CL, as measured by Optical Coherence Tomography (OCT), may correlate with the outcome of thrombolysis.

Methods: We prospectively enrolled 55 consecutive patients with STEMI that were treated with thrombolysis 6 hours from symptom onset. All patients underwent coronary angiography within 24 hours from thrombolysis. We estimated TIMI flow grade and the corrected TIMI frame count (CTFC) for patients with TIMI flow II or III. In target vessels with TIMI flow grade ≤1 aspiration thrombectomy was performed. We then acquired OCT images from the CL. We measured the fibrous cap thickness (FCT) at the thinnest part overlying a lipid pool, the lipid content (in quadrants) and the incidence of plaque rupture, which was defined as the presence of fibrous-cap discontinuity.

Results: Of the total study population, 15 patients had TIMI flow 0 or I, 15 had TIMI flow II, and 25 had TIMI flow III. The number of lipid quadrants was greater in patients with TIMI flow 0-I than in patients with TIMI flow II and III (median 4, (range 1) vs. median 3 (range 2) vs. median 2 (range 1), p<0.001). The culprit lesion of the patients with TIMI flow 0 or I was more often characterized by a ruptured plaque compared to those with TIMI flow II and III (n=13 (86.67%) for TIMI 0–1, n=8 (53.33%) for TIMI II and n=6 (24%) for TIMI III, p<0.001). For patients with TIMI 0 and I, minimum FCT was 41±7
μ
m, for patients with TIMI II 55±23
μ
m and for those with TIMI III 87±26
μ
m (p<0.001). In patients with TIMI flow II or III, a correlation was found between CTFC and the minimal cap thickness (r=−0.423, p=0.006).

Conclusion: In patients with STEMI, TIMI flow III following thrombolysis is associated with greater fibrous cap thickness, reduced lipid content and reduced incidence of plaque rupture in the CL. This study suggests that specific morphological features of the CL correlate with the flow grade following thrombolytic therapy in patients with STEMI.
Abstract 5438: Directed Differentiation of Endothelial Cells From Coronary Artery Disease (CAD) Patients-derived iPS Cells
Authors: Kim Sinae , Park Changwon , Han Ji Woong , Kim Sung-Whan , Kim Hyongbum , Choi Yong Jin , Lee Eugine , Patel Riyaz , Park In-Hyun , Daley George Q. , Quyyumi Arshed , Yoon Young-sup
Published: April 2024

Background.
We recently generated Induced pluripotent stem (iPS) cells generated from peripheral blood mononuclear cells of coronary artery disease (CAD) patients. In this study, we sought to differentiate these iPS cells into endothelial cells (ECs), and to confirm their vasculogenic potential in vivo using multiple animal models.

Methods and Results.
In these experiments, iPS cells were spontaneously differentiated through embryoid body (EB) formation and were sorted for KDR using FACS. Next, we further cultured these KDR
+
cells for 21 days and sorted for KDR and CD31. These KDR
+
CD31
+
cells showed a higher rate (~70%) of double positivity for UEA1 lectin and ac-LDL uptake. The KDR
+
CD31
+
cells expressed endothelial specific proteins such as VE-cadherin and von Willebrand factor (vWF) under immunohistochemical examination, formed tubes when cultured in Matrigel, and expressed EC-specific genes (
KDR, CD31, CD34, eNOS, VE-cadherin, vWF
) as shown by qRT-PCR analysis. In this culture condition, the EC phenotype was maintained > 2 months. Next, to verify their EC characteristics in vivo and provide bona fide evidence of EC commitment of iPS cells, Matrigel plug assay and a hindlimb ischemia model were employed. First, we injected a mixture of Matrigel and the KDR
+
CD31
+
cells labeled with a red fluorescent dye, DiI, into the backs of athymic mice subcutaneously. Two months later, the mice were perfused with an endothelial cell marker, isolectin B4 (ILB4) to stain functional endothelium. The injected cells gave rise to vascular structures and were stained positive for ILB4. Second, we surgically induced hindlimb ischemia in nude mice and injected the KDR
+
CD31
+
cells labeled with DiI into the hindlimb muscles immediately after the surgery. After sacrificing the mice nine days later, muscles were harvested and the tissues were subjected to immunohistochemistry with ILB4. Injected KDR
+
CD31
+
cells expressed ILB4 and were incorporated into vascular structures, suggesting vasculogenesis.

In conclusion.
iPS cells derived from CAD patients can generate functionally competent ECs in vivo, Together the present study suggest that endothelially differentiated cells from iPS cells can be used therapeutically in an autologous manner to treat tissue ischemia.
Abstract 5444: Cbx3 Identified by Nuclear Proteomics Plays a Key Role in Stem Cell Differentiation Into Smooth Muscle Cells in vitro and in vivo
Authors: Xiao Qingzhong , Wang Gang , Bai Xue , Yin Xiaoke , Mayr Manuel , Wang Xian , Xu Qingbo
Published: April 2024

Our previous studies demonstrated that Sca-1+ progenitors derived from embryonic stem cells (ESCs) and adventitial tissues of adult apoE−/− mice can differentiate into smooth muscle cells (SMCs) that participated in the neointimal formation. However, the molecular mechanism of stem cell differentiation into SMCs is not fully elucidated. To identify potential signal transducers mediating SMC differentiation, nuclear proteins were harvested from undifferentiated mouse ESCs and differentiating ESC at different time points, and subjected to proteomic analysis by nanoflow liquid chromatography tandem mass spectrometry. We found that the majority of upregulated nuclear proteins during SMC differentiation were involved in chromatin remodeling, cell proliferation, and RNA processing processes. Among those upregulated nuclear proteins, we further focused on chromobox protein homolog 3 (Cbx3) because Cbx3 functions as activator and/or repressor within both heterochromatin and euchromatin. Knockdown of Cbx3 in the differentiating stem cells resulted in down-regulation of smooth muscle markers including smooth muscle actin, SM22, calponin and smooth muscle myosin heavy chain, while enforced expression of this gene enhanced SMC differentiation in a dose-dependent manner. CHIP assay suggests that Cbx3 mediated SMC differentiation through regulation of smooth muscle specific transcription factor, serum response factor (SRF), and coactivatior myocardin. Furthermore, we also demonstrated that the transcription factor, Dia1, functioned as a bridge protein between Cbx3 and SRF, through which Cbx3 modulates SRF activation, mediates ultimately SMC differentiation. Interestingly, in situ hybridization and immunohistochemical staining indicates that Cbx3 localized at smooth muscle system during development in different stages of chicken embryos. Importantly, in vivo knockdown of Cbx3 in the vessel wall significant increased wire-injury-induced neointima formation in mice. Our findings demonstrated for the first time that Cbx3 has a crucial role in SMC differentiation and possesses a protective function in vessel injury-induced neointima formation, providing novel information on potential therapeutic targets for vascular diseases.
Abstract 5464: The Cell Proliferative Effect of LRP6 Mutation is Mediated by Increased PDGF Dependent Cyclin D1 Expression
Authors: Keramati Ali R , Liu Wenzhong , Nottoli Timothy , Faramarzi Saeed , Tellides George , Mani Arya
Published: April 2024

We recently identified a disease causing mutation (R611C) at a highly conserved residue of the LDL receptor like protein 6 (LRP6) in a family with early onset coronary artery disease (CAD), and metabolic syndrome. This finding signifies the effect of a single gene defect in LRP6 on development of atherosclerosis and underscores emerging evidence implicating effect of altered Wnt signaling on atherosclerosis. The exact mechanisms linking LRP6 mutation with atherosclerosis are not well understood. mRNA expression analysis in cells from LRP6 mutation carriers has demonstrated that LRP6 mutation has a heterogeneous effect on Wnt signaling. Strikingly, cyclin D1, a target of Wnt signaling and a cell cycle activator is expressed at significantly higher levels in the mutant compared to the wildtype cells. Smooth muscle cell proliferation is a major component in the process of atherosclerosis development. We therefore dissected Wnt signaling and examined cell proliferation in aortic smooth muscle cell (SMC) cultures isolated from LRP6
R593C
knock-in and LRP6 +/− knockout mice. The proliferation rate of LRP6
R593C
SMCs was highest and those of the wildtype littermates were the lowest among the three, indicating that both haploinsufficiency as well as impaired LRP6 function result in cell cycle activation. GSK3
β
is an intermediate peptide of the Wnt signaling pathway that inhibits cyclin D1expression. PDGF inactivates GSK3
β
and promotes cellular proliferation by increasing the expression levels of cyclin D1. Our experiments showed that GSK3
β
is at baseline more activated in cells expressing the mutant vector compared to the wildtype cells. However, after stimulation with PDGF, GSK3
β
activation in the mutant cells diminishes to levels significantly lower than wildtype cells. This effect was mediated by higher expression levels of PDGF receptor in the mutant compared to the wildtype cells. Our result signifies the important role of LRP6 and its interaction with PDGF in the process of smooth cell proliferation and identifies LRP6 and GSK3
β
as potential targets for both prevention and pharmacotherapy of atherosclerosis.
Abstract 5481: Heparanase Regulates the Thrombotic Potential of Vascular Injury and Stent Placement
Authors: Baker Aaron , Gibson William , Kolachalama Vijaya B , Golumb Mordachai , Indolfi Laura , Edelman Elazer R
Published: April 2024

Background:
Heparanase cleaves heparan sulfate proteoglycans (HSPGs), key components of the extracellular matrix and critical endothelial mediators of hemostasis.

Methods:
Mice transgenic for human heparanase were used to evaluate the influence of heparanase overexpression on thrombosis in an ex-vivo and in-vivo model. To evaluate thrombotic potential, a laser induced thrombosis model was used. Arterial injury was also performed prior to measuring thrombosis to examine the effects of light and heavy injury on thrombosis in the heparanase transgenic mice. A novel ex-vivo murine assay of stent thrombosis was performed in which mouse aortas were stented into a flow loop perfused with mouse blood.

Results:
In the absence of injury there was no difference in the time for formation of thrombosis in the wildtype (WT) and heparanase transgenic (HPA Tg) mice. However, in the presence of light injury or heavy injury thrombosis in HPA Tg mice occurred significantly faster than in WT mice (63.0 minutes vs. 41.8 minutes, p=.034). The prothrombotic phenotype of HPA Tg mice was masked when heparin was administered prior to injury (68.5 minutes vs. 69.2 minutes p=NS). Blood from HPA Tg mice increased stent thrombosis threefold versus WT in flow loops as measured by optical density (2.81 vs. 1.0 optical density, p=.023) and increased adherent platelets were found in electron microscopy.

Conclusions:
Increased arterial expression of heparanase leads to increased thrombosis in the injured artery. In particular, endovascular stenting induces injury and flow disturbance that exacerbated the thrombotic potential of blood with increased heparanase levels.

Figure 1.

Electron microscopy of stents exposed to WT and HPA Tg blood.
Abstract 5507: Metabolic Regulation in Coronary Vascular Tone: Various Vasodilative Substances in Myocardial Ischemia
Authors: Kaneshiro Takashi , Saitoh Shuichi , Machii Hirohumi , Yamaguchi Osamu , Ishibashi Toshiyuki , Takeishi Yasuchika
Published: April 2024

Metabolic regulation plays an important role in modifying coronary vascular tone. We hypothesized that H
2
O
2
, purinergic components, and angiotensin, produced by cardiac myocytes control coronary vascular tone in proportion to ischemia. To test this hypothesis, we measured changes in the diameter of isolated, pressurized coronary arterioles (65±5
μ
m, n=42) in response to supernatant collected from isolated paced (400 bpm) cardiac myocytes in rat. Changes in diameter of arterioles were determined under control conditions following treatment of the isolated arterioles with A1, A2 antagonist, 8-PSPT (50
μ
M), P2Y1 antagonist, MRS-2179 (100
μ
M), or angiotensin II type-1 receptor antagonist, olmesartan (1
μ
M). In a supernatant from 120 min pacing myocytes, increase of lactate level and decrease of %O
2
concentration were observed compared to unpacing (112±8.2 mg/dl vs. 2±0.4 mg/dl, 2±1% vs. 48±5%, P<0.01, respectively). A supernatant (500
μ
l to a 2 ml bath) from myocytes paced for 20, 60 and 120 min caused a graded vasodilation(14.1±0.4 %, 20.2±1.6 %, 53.8±6.2 %, respectively). After a 20 min pacing, catalase (10, 000 U) eliminated vasodilation (0.9±0.8 %, P<0.01). However, 8-PSPT, MRS-2179 or olmesartan did not affect the vasodilative property. Following a 60 min pacing, catalase converted vasodilation to vasoconstriction (−15.1±1.0%, P<0.01). This converted vasoconstriction was completely eliminated with olmesartan (−0.6±0.4%, P<0.01). 8-PSPT or MRS-2179 did not affect the vasoactive property. Upon a 120 min pacing, catalase partially reduced vasodilation (37.2±3.8 %, P<0.05). The remaining vasodilation was converted to vasoconstriction with 8-PSPT + MRS-2179 (−23.4±3.0 %, P<0.01), and this vasoconstriction was completely eliminated with olmesartan (−0.5±0.9%, P<0.01). The SOD activity with an ESR spectrometer attenuated 16.2±1.4 %, and H
2
O
2
concentration in myocytes supernatant were decreased in 120 min pacing compared to those in 60 min pacing (22±0.8 to 31±1.4
μ
M, P<0.05). ADP level in myocytes increased in 120 min pacing [8.2±0.8
μ
mol/g, P<0.01 vs. unpacing (not detected)]. These results suggest that main vasodilative metabolic modulators released from myocytes replaced from H
2
O
2
to purinergic components in proportion to ischemia.
Abstract 5508: Angiotensin Type-2 Receptor-mediated Coronary Arteriolar Dilation is Enhanced in Rats With Type-2 Diabetes Mellitus: A Role of Enhanced Nitric Oxide Mechansim via the Overexpression of Bradykinin B
2
Receptor
Authors: Oikawa Koichi , Kushibiki Motoi , Yamada Masahiro , Osanai Tomohiro , Okumura Ken
Published: April 2024

Angiotensin type-2 (AT
2
) receptor is said to function as a counterregulator against angiotensin type-1 (AT
1
) receptor, and its expression was shown to be increased in various pathological states. However, the role of AT
2
receptor in type-2 diabetes mellitus (DM) is unclear. We examined AT
2
receptor-mediated coronary arteriolar dilation comparatively in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a type-2 DM model, and control Long-Evans Tokushima Otsuka (LETO) rats. At the age of 40 weeks, the coronary arterioles were isolated, cannulated, pressurized with 60 mmHg, and preconstricted with 40 mM potassium chloride. In the presence of valsartan (10
−4
M), each of angiotensin II (10
−11
to 10
−6
M) and a selective AT
2
receptor agonist CGP-42112A (CGP, 10
−11
to 10
−6
M) dilated the coronary arterioles in a dose-dependent manner, and the dilator responses were greater in OLETF than in LETO rats (both p<0.05). An AT
2
receptor antagonist PD 123319 (10
−4
M) and N
G
-nitro-L-arginine methyl ester (10
−4
M) completely abolished the dilator effects of angiotensin II and CGP. Bradykinin B
2
receptor antagonist HOE-140 significantly attenuated the vasodilator effects of CGP and bradykinin, but did not alter the effect of acetylcholine. Reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated that AT
1
receptor mRNA level in the coronary arterioles was upregulated by 1.44±0.14 times in OLETF compared with that in LETO (p<0.05). In contrast, AT
2
receptor mRNA level did not differ between LETO and OLETF. Bradykinin B
2
receptor mRNA level was upregulated by 2.56±0.38 times in OLETF compared with that in LETO (p<0.05). In conclusion, in the presence of AT
1
receptor blocker, angiotensin II causes vasodilation in the rat coronary arterioles by a nitric oxide mechanism. This AT
2
receptor-mediated vasodilation is enhanced in rats with type-2 DM, which can be explained, at least in part, by the overexpression of bradykinin B
2
receptor in DM rats.
Abstract 5512: Vascular Rhexis: A Potentially Modifiable Contributor to Negative Remodeling After Myocardial Infarction
Authors: Zaman Akm Tarikuz , French Christopher J , Sobel Burton E
Published: April 2024

We previously noticed in studies of plasminogen activator inhibitor type-1 (PAI-1) knockout (PKO) mice that infarction was often hemorrhagic. Thus, we hypothesized that the intensified fibrinolysis was unmasking a loss of integrity of coronary vessels (vascular rhexis) potentially caused in part by inflammation. To test this hypothesis, in the present study we induced myocardial infarction (MI) in PKO and control C57BL6 mice by ligation of the left coronary artery for 48 hr, harvested the hearts for assessment of infarct size (measured based on depletion of left ventricular [LV] creatinine kinase activity), and quantified hemorrhage by assaying with non-cross reacting ELISAs hemoglobin content corrected for myoglobin content in LV myocardium. Normal mouse LV myocardium contains approximately 40
μ
l blood/g. PKO mice (n = 9) had marked intramyocardial hemorrhage in infarct zones (432±27 SEM
μ
l blood/g). Vascular rhexis was evident in C57BL6 controls as well (n = 5) as judged from the presence of 51±3
μ
l blood/g LV 48 hr after coronary occlusion despite less blood in the infarct zone within 4 hr after coronary occlusion (10±5
μ
l blood/g, n = 13). Intramyocardial hemorrhage in PKO mice was significantly reduced (302±37
μ
l blood/g, p < 0.01 vs control PKO) by treatment with cyclophosphamide (n = 12) before coronary occlusion sufficient to induce profound leukopenia and by treatment with aprotinin (n = 9) after coronary occlusion to inhibit plasmin (220±40
μ
l blood/g, p < 0.01 vs control PKO). Treatment with the combination of both (n = 4) virtually eliminated hemorrhage (49±5
μ
l blood/g, p < 0.01 vs control PKO). Treatment did not modify infarct size (PKO: 46±2% of LV mass; PKO with cycloposphamide: 45±1%; PKO with aprotinin: 43±2%). Thus, rupture of coronary vessels occurs within 48 hr after the onset of coronary occlusion leading to hemorrhage that may exacerbate late, negative ventricular remodeling; cyclophosphamide reduces vascular rhexis by attenuating inflammation; and aprotinin reduces hemorrhage associated with vascular rhexis unmasked by intensified fibrinolysis by inhibiting plasmin.
Abstract 5521: Rosuvastatin Prevents Ischemia and Reperfusion-Induced Endothelial Dysfunction by a COX-2 Dependent Mechanism
Authors: Liuni Andrew , Luca Mary Clare , Gori Tommaso , Parker John D
Published: April 2024

BACKGROUND:
Animal studies have demonstrated that rosuvastatin (RSV) can limit the damage induced by ischemia-reperfusion (IR) in the cardiac, cerebral and mesenteric circulation. It remains unknown whether this beneficial effect of RSV can also be demonstrated in humans, and by what mechanism this may occur.

METHODS AND RESULTS:
Twenty healthy volunteers were randomized to a single dose of oral RSV (40 mg) or placebo. 24 hours later, endothelium-dependent, flow-mediated dilation (FMD) of the radial artery was measured before and after IR (15 minutes of ischemia at the level of the brachial artery followed by 15 minutes of reperfusion). There was no difference in pre-IR FMD between groups (P=NS). In the placebo group, FMD was significantly blunted after IR (FMD pre-IR: 6.4+/−0.4%, post-IR: 1.1+/−1.2%, P<0.01). This impairment in FMD with IR was prevented with RSV (Table

; FMD before IR: 7.5+/−1.0%; after IR: 6.2+/−1.2%,
P
=NS compared with RSV before IR; P<0.05 vs. placebo). In a separate protocol, 18 healthy volunteers received the cyclooxygenase-2 (COX-2) inhibitor celecoxib (200mg BID p.o.) for 5 days. On day 4, subjects were randomized to a single dose of RSV (40mg) or placebo and 24 hours later underwent the same protocol as above. Pretreatment with celecoxib completely abolished RSV’s protective effect (Table

; FMD pre-IR: 8.0±2.2%, post-IR: 1.4±2.0%, P<0.001 before versus after IR, P=NS vs. placebo). IR did not modify resting arterial diameter, baseline blood flow or peak hyperemic blood flow during FMD in either protocol (Table

; P=NS for all).

CONCLUSIONS:
The present findings demonstrate, for the first time in humans, the ability of RSV to pharmacologically prevent the development of IR-induced endothelial dysfunction. In addition, these data demonstrate the importance of COX-2 in the manifestation of this preconditioned phenotype, evidence that also provides potential mechanistic insight into the observed cardiovascular toxicity with COX-2 inhibitors.
Abstract 5522: Improvement of Angiotensin-II-Induced Vascular Dysfunction and Oxidative Stress by the Organic Nitrate Pentaerithrityl Tetranitrate Depends on Heme Oxygenase-1-Induction
Authors: Wenzel Philip , Schuhmacher Swenja , Schulz Eberhard , Oelze Matthias , Kamuf Jens , Gori Tommaso , Hortmann Marcus , Bhatnagar Aruni , Daiber Andreas , Münzel Thomas
Published: April 2024

Objective: Pentaerithrityl tetranitrate (PETN)-treatment is devoid of nitrate tolerance which has been attributed to the induction of the antioxidant enzyme heme oxygenase-1 (HO-1). With the present study we tested, whether chronic treatment with PETN can improve angiotensin-II (AT-II)-induced vascular oxidative stress and dysfunction.

Methods and Results: In contrast to isosorbide mononitrate (ISMN, 75mg/kg/d/7d), co-treatment with PETN (15mg/kg/d/7d) improved impaired endothelial and smooth muscle function and normalized vascular and cardiac ROS production (mitochondria, NADPH oxidase activity and uncoupled endothelial nitric oxide synthase [eNOS]) as assessed by dihydroethidine (DHE)-staining, lucigenin-enhanced chemiluminescence and quantitative HPLC of DHE-oxidation products in AT-II (1mg/kg/d/7d) treated rats. The antioxidant features of PETN were recapulated in spontaneously hypertensive rats. In addition to increase in HO-1 protein expression, PETN but not ISMN augmented aortic protein levels of the tetrahydrobiopterin-synthesizing enzymes GTP-cyclohydrolase-I and dihydrofolate reductase in AT-II-treated rats, thereby preventing eNOS uncoupling. In AT-II-treated (0.1mg/kg/d/7d) HO-1+/− mice, PETN failed to be antioxidant, whereas HO-1 induction by hemin (25mg/kg) mimicked the PETN effect in AT-II-treated HO-1+/+ mice.

Conclusions: PETN improves AT-II-induced vascular oxidative stress and dysfunction via induction of HO-1. Thus, PETN is the first organic nitrate to be beneficial in an experimental model of arterial hypertension.