bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4902: Renal Sinus Fat Influences the Number of Medications Needed to Regulate Blood Pressure in Patients With Hypertension
Authors: Chughtai Haroon L , Morgan Tim M , Ding Jingzhong , Brinkley Tina E , Rocco Michael , Hamilton Craig A , Hundley W. G
Published: April 2024

Introduction:
A potential consequence of increasing intraperitoneal (IP) fat is the accumulation of fat in the renal sinus (RS). Compression of low pressure vessels (renal veins, lymphatics), by RS fat may result in activation of the renin-angiotensin system (RAS) and promote more extensive use of antihypertensive therapy.

Hypothesis:
RS fat would be associated with IP fat and number of medications for blood pressure control.

Methods:
Two hundred five participants aged 55 to 85 years, without renal artery stenosis, underwent magnetic resonance imaging measures of RS and abdominal fat (subcutaneous + IP + retroperitoneal). Data were analyzed for possible associations of RS fat with renal function, BP, and the number of prescribed antihypertensives.

Results:
Age averaged 69 ± 9 years; 50% were women. RS fat averaged 5 ± 3 cm
3
(up to 16 cm3). RS fat correlated with retroperitoneal and IP fat volumes, and cardiac risk factors (hypertension, diabetes, and coronary artery disease) (r = 0.49, 0.42, and r = 0.18, p = 0.001, 0.001, and 0.009 respectively); it did not correlate with subcutaneous fat (r = −0.08, p = 0.23). RS fat also showed a positive association with number of antihypertensive medications, renal size, creatinine, pulse pressure, and an inverse correlation with serum potassium, a marker of RAS activation (r = 0.21, 0.44, 0.23, 0.13, and 0.16; r = −0.18, p = 0.01) [Table

]. These associations of RS fat persisted after adjustment for age, gender, height, diabetes, coronary artery disease, serum lipids, and IP fat (p = 0.01 for all). Importantly, after adjusting IP fat to RS fat in our regression model, RS fat remained associated with the number of prescribed anti-hypertensive medications (
B
= 0.27, p = 0.01), while IP fat did not.

Conclusions:
Higher volumes of renal sinus fat are associated with indirect evidence of the activation of the RAS and the number of medications used to treat hypertension. These data suggest a possible mechanism for central obesity to promote hypertension.

Table:

Correlations of different variables using renal sinus fat as a dependant variable
Abstract 4920: Endothelial-Targeted BMPR2 Loss-of-Function Mutations Cause Increased Apoptosis and Pulmonary Arterial Hypertension
Authors: Yelle Dominique , Deng Yupu , West James , Stewart Duncan J
Published: April 2024

Introduction: Pulmonary arterial hypertension (PAH) is a rare and fatal disease caused by excessive remodelling of small pulmonary arterioles. Heterozygous loss-of-function mutations in the bone morphogenetic protein receptor 2 (BMPR2) have recently been implicated in patients with familial and idiopathic PAH. However, how mutations in this ubiquitously expressed receptor result in a specific abnormality of the lung microcirculation is unknown. We hypothesized that mutations in BMPR2 lead to PAH by increasing the susceptibility of ECs to apoptosis, particularly within fragile pulmonary arterioles. Aims: To examine the effect of endothelial targeted overexpression of a BMPR2 deletional mutation on EC apoptosis, pulmonary hemodynamics and arteriolar remodelling.

Methods: We developed an endothelial-specific binary transgenic (BT) mouse model in which the driver mice express the tetracycline transactivator under the control of the endothelial-restricted V-cadherin promoter and the responder mice harbour a BMPR2 transgene containing an arginine-to-stop mutation in the receptor’s C-terminal domain driven by a tetracycline responsive promoter. Pulmonary artery pressure was evaluated by measuring right ventricular systolic pressure (RVSP). Fluorescent microangiography (FMA) was performed to visualize the 3D architecture of the pulmonary microcirculation.

Results: RVSP was significantly increased in BT mice compared to non binary controls (p < 0.05; n = 9), associated with increased muscularization of small pulmonary arterioles detected by
α
-smooth muscle actin immunostaining. In addition, FMA revealed decreased perfusion and distal pre-capillary discontinuity in BT mice. TUNEL staining also demonstrated a significant increase in the number of apoptotic cells in BT mice compared to non binary controls (5.8 ± 1.1, 1.0 ± 0.7 positive cells/HPF, respectively; p < 0.05; n = 4).

Conclusions: These data suggest that BMPR2 mutations increase the susceptibility to apoptosis, possibly leading to EC loss and pre-capillary arteriolar discontinuity. This new model will help elucidate the pathophysiological events leading to the development of PAH and provide a unique tool to evaluate novel potential treatments for this disease.
Abstract 4929: Injection of Human Neural Stem Cell Line in a Mouse Hind Limb Ischemia Model Promotes Muscle Neovascularisation
Authors: Katare Rajesh , Miljan Erik , Corteling Randolph , Hicks Caroline , Emanueli Costanza , Sinden John , Madeddu Paolo
Published: April 2024

Objective:
Recently, the human conditionally immortalized neural stem cell (hNSC) line, CTX0E03, has received approval from the UK MHRA for treatment, by direct intracerebral implantation, of stable stroke disability in elderly patients. We report here for the first time that intramuscular injection of the CTX0E03 hNSC line into a mouse model of hind-limb ischemia results in a repeatable significant, dose-dependent therapeutic improvement in blood flow.

Methods:
Unilateral limb ischemia was induced in immunodeficient (FoxnCD1
nu/nu
) mice. Suspensions of CTX0E03 cells were administered by injection into the ischemic limb adductor muscle. Five different doses of hNSCs were tested in two experiments (3 million; 1 million; 300,000 in Exp 1 and 300,000, 30,000 and 3,000 in Exp 2), with vehicle injections used as a control in both experiments (n=10/group). Laser Doppler measurements of the limbs were taken at Days 3, 7, 14 and 21. Capillary and arteriole density and absolute blood flow measurements (0.02
μ
m fluorescent microspheres) were done on Day 21.

Results:
The three highest doses of CTX0E03 cells markedly increased the recovery of blood flow to the ischemic limb as measured by Doppler (Figure A

), adductor muscle blood flow (Figure B

) and capillary and arteriole density. The two lower doses, 30,000 and 3,000 cells produced no significant improvements. Histological analysis demonstrated CTX0E03 cell survival at Day 21 in some, but not all, of the implanted muscles.

Conclusion:
CTX0E03 cell therapy significantly improved clinically relevant aspects of hind limb ischemia in nude mice dose dependently by enhancing neovascularization.
Abstract 4938: Agtrl-1/Apelin Binding Specifically Enhances Myocardial Ischemia Driven Neovascularization and Left Ventricular Function by Attracting EPCs
Authors: Tempel Dennie , Herpers Robert J , van Deel Elza D , de Boer Martine , Bos Frank , van de Kamp Esther H , Haasdijk Remco A , Schulte-Merker Stefan , Duckers Henricus J
Published: April 2024

The molecular mechanisms that govern the formation of new blood vessels following ischemia remain largely unknown. In order to identify new genes involved in blood vessel formation, we performed a genome-wide microarray screen using the affymetrix platform in mice and zebrafish. Gene expression profiles of isolated Flk1+ angioblasts during murine and piscine embryonic development were compared to those of Flk1− cells. Angiotensin receptor 2 like 1 (Agtrl-1) is one of the genes that was identified. In adult C57bl/6 mice Agtrl-1 is highly expressed in mature endothelial cells and was also detected in cKit+/Flk1+/Lin- cells, but not in Sca+/Flk1+/Lin- cells. Remarkably, the number of cKit+/Flk1+/Agtrl-1 cells was increased in the ischemic area after myocardial ischemia (P<0.001), but not after hind limb ischemia. Apelin protein expression, the endogenous ligand of Agtrl-1, was also augmented by 3-fold in the infarct region of the myocardium (P<0.01) and systemic infusion of Apelin increased the recruitment cKit+/Flk1+ EPCs to the circulation by two-fold (P<0.03). Injection of Apelin into the ischemic myocardium resulted in increased homing to the injection site of this specific population and a two-fold increase in neovascularization of the ischemic tissue (P<0.01), consequently improving cardiac function, left ventricular dimensions and diminished scar formation. These findings indicate for the first time, that apelin in response to myocardial ischemia could function as a specific chemoattractant for Agtrl-1+ EPCs, stimulating migration into the infarcted area and subsequently promoting neovascularisation and myocardial repair.
Abstract 4944: Bayesian Analysis of TRITON-TIMI 38: Reassessing Prasugrel versus Clopidogrel in the Treatment of Patients With Acute Coronary Syndrome
Authors: Lang Christopher , Arora Rohit
Published: April 2024

Background: The application of Bayesian analytic techniques can provide clinically relevant information regarding efficacy of an intervention. Given the recent controversy surrounding prasgurel, as reported in the TRITON-TIMI 38 study, we sought to re-examine both the frequentist and Bayesian statistical methods which evaluated this study.

Methods: Magnitude of therapeutic benefit and a sensitivity analysis were performed, using posterior probabilities (Pr), in relation to probability of benefit versus harm, as well as probabilities of greater than 20% benefit (Pr>20%). The analysis was conducted with experiential probabilities obtained from the TRITON-TIMI 38 data, and a null prior probability with a log mean OR of 0, and a standard deviation of 2.

Results: Based on the study results, the Pr for the primary endpoint of death from cardiovascular causes, nonfatal MI, or nonfatal stroke is 99.9%. Calculated probability of at least a 20% benefit is 66.5%. This value is reasonably high enough to infer a clinically important benefit. All cause mortality was also found to have a clinically relevant benefit at Pr>20% of 99.1%. Analysis does indicate a clinically significant probability of harm pertaining to Non-CABG major and major plus minor bleeding, with a probability of at least 20% harm of 73.6 and 82.4% respectively.

Conclusions: On re-evaluation, the Bayesian analysis suggests the clinical relevance of prasugrel as a beneficial treatment in patients with ACS, which serves to support the implications of the conventional statistics reported in TRITON-TIMI 38. Of note, the clinically relevant benefit is juxtaposed against the harm related to bleeding complications.
Abstract 4948: Decrease in Poor Responders on Switching From Clopidogrel to Prasugrel: Results From the SWitching Anti Platelet (SWAP) Study
Authors: Saucedo Jorge F , Angiolillo Dominick J , DeRaad Roger , Frelinger Andrew L , Gurbel Paul A , Costigan Timothy M , Jakubowski Joseph A , Ojeh Clement K , Effron Mark B
Published: April 2024

Background:
The SWAP study, a prospective double-blind study in patients with a recent history of acute coronary syndrome (ACS), demonstrated that switching from a stable maintenance dose (MD) of clopidogrel (Clop) 75 mg/day to prasugrel (Pras) 10 mg/day, with or without a 60 mg loading dose (LD), resulted in significantly lower platelet aggregation by one week. This analysis reports on a secondary objective of the SWAP study evaluating the effect of switching from Clop to Pras on thienopyridine poor response rates.

Methods:
Poor response to Pras and Clop was evaluated in 100 patients (Placebo LD/Clop 75mg/day, n=33; Placebo LD/Pras 10mg/day, n=36; Pras 60 mg LD/Pras 10mg/day, n=31). Poor responders were defined as patients with maximum platelet aggregation (MPA) >65% to 20
μ
M ADP assessed by light transmittance aggregometry performed prior to randomization and at 2 hr, 24 hrs, and 7 days following start of study drug.

Results:
Prior to randomization, the Clop poor response rate was 27%. Poor responder rates in each group at 2 hrs, 24 hrs, and 7 days post study drug initiation are shown in the Figure

. At 2 hrs and 24 hrs, there were no poor responders in patients receiving a LD of Pras. At the end of the 7-day MD period, the poor responder rate in the combined Pras groups (with and without LD) was significantly lower compared with Clop (5% vs. 21%; p=0.014).

Conclusions:
In patients with recent ACS, switching from Clop to Pras MD is associated with reduced poor response rates (or frequency of high platelet reactivity), while Pras LD overcomes any poor response seen with Clop.
Abstract 4959: Imaging of Atherosclerotic Lesions in Apoe−/− Mice by Targeting of CD40
Authors: Grafe Michael , Dietrich Thore , Stibenz Dietger , Klohs Jan , Lutgens Esther , Schulz Petra , Groetzinger Carsten , Bourayou Riad , Fleck Eckart , Graf Kristof
Published: April 2024

We investigated whether in-vivo imaging of the CD40 receptor can be used for identification of inflammatory atherosclerotic lesions.

Methods: Monoclonal antibodies against CD40 (CD40ab), IgGA2 (Coab) and Fab2 fragments (CD40Fab) were coupled to the near-infrared fluorescence (NIRF) dyes Cy5′5 or Cy7. Apolipoprotein E deficient mice (apoE−/−) and apoE−/−CD40−/− deficient mice (apoE−/−CD40−/−) were fed with high cholesterol diet for 8 –12 month. Antibody conjugates were injected into the tail vein and mice were investigated 6 to 48 hours after injection by conventional NIRF imaging, fluorescence microscopy of the aortic arch and in-vivo 3D fluorescence molecular tomography (FMT). Sudan staining and histological analysis were performed after imaging procedures.

Results: Application of Cy5′5-labeled CD40ab resulted in accumulation of fluorescence signal intensity mainly in Sudan-positive areas of the thoracic aorta, which was also seen with the Coab at high concentrations. Fluorescence increased slightly after 24 hours compared to 6 hours after injection. In vivo imaging was not feasible using conjugated antibodies due to circulating intravasal fluorescence (>48h). Significant background fluorescence was detected after application of CD40ab and Coab in all mice, probably due to FC receptor-dependent binding. Application of Cy7-CD40Fab induced strong in-vivo fluorescence detected by FMT in projection to the aortic arch and carotid arteries of apoE−/− (n=4), but not in apoE−/−CD40−/− mice (n=4). No signal was observed in mice injected with unlabelled Cy7. Ex-vivo imaging of the thoracic aorta confirmed a Cy7 signal in atherosclerotic lesions of apoE−/−, but not in apoE−/−CD40−/− after application of CD40Fab. Data were also confirmed by microscopic fluorescence imaging of the aortic wall and atherosclerotic lesions.

Conclusion: In vivo detection of CD40 in atherosclerotic plaques was feasible using antibody fragments but not with full anti-CD40 antibodies. The approach is applicable for detection of vascular inflammation, since CD40 is strongly associated with inflammatory lesion formation.
Abstract 5007: Variability in the Response to Aspirin
Authors: Grosser Tilo , Fries Susanne , Lawson John , FitzGerald Garret
Published: April 2024

Low dose aspirin is efficacious in the secondary prevention of stroke and myocardial infarction. It has been suggested that a syndrome of “aspirin resistance” constrains its efficacy. Despite this concern, no clear definition of aspirin resistance or a segregation of its elements has emerged and estimates of its frequency have varied up to 50% of aspirin users. We aimed to determine the incidence of an internally consistent, stable and specific “aspirin resistant” phenotype. Healthy volunteers (n=314; targeted enrollment n=400) were screened for their platelet aggregation response before and 4 or 8 hours after a single oral dose of 325 mg enteric coated aspirin testing four stimuli of platelet activation including arachidonic acid (AA). Individuals who showed a less than 60% inhibition upon AA stimulation underwent repeat testing. Individuals who failed to respond to aspirin twice were exposed to low dose aspirin (81 mg) and clopidogrel (75 mg) for one week each in a cross over design. Inhibition of the cyclooxygenases was assessed in ex vivo whole blood assays and by mass spectrometric quantitation of urinary prostaglandin metabolites. On the first occasion, 102 (32.5%) subjects responded with a reduction of the AA induced aggregation by less than 60%. However, only 23 subjects (7.3%) remained non-responsive when aspirin was added to the assay in vitro. Six individuals (1.9%) remained non-responsive when tested a second time. Currently, only one individual remains non-responsive upon exposure to low dose aspirin (81 mg daily for one week) on the third replicate measurement. There was a striking inverse relationship between “exposure” and “non-responsiveness”: Both, prolonging the post dosing interval and adding increasing concentrations of aspirin in vitro reduced the number of non-responsive individuals. Variable pharmacokinetics caused a high frequency of apparent non-responsiveness to a single dose of 325 mg enteric coated aspirin. The majority of individuals responded to aspirin upon repeated exposure, extension the post dosing interval or addition of aspirin in vitro. The incidence of internally consistent, stable and specific “aspirin resistance” - which might be explained by genetic causes - is lower than 1% in healthy individuals.
Abstract 5024: Diagnostic and Prognostic Value of High Sensitive Troponin T in Patients With Acute Coronary Syndromes
Authors: Weber Michael W , Nef Holger , Moellmann Helge , Mayer Sarah , Liebetrau Christoph , Woelken Mariella , Hamm Christian W
Published: April 2024

Background: Cardiac troponins are the gold standard for diagnosis and risk stratification of patients with ACS. As a threshold for troponins, the serum concentration which is identical to the 99
th
percentile of a healthy population has been defined. However, this threshold has to measurable with sufficient analytical precision (CV<10%). In the past, troponin assays were not able to measure a troponin concentration at the 99th percentile with sufficient precision. However newly developed highsensitive troponin assays will enable measurement at the 99th percentile with the required precision. The aim of the present study is to evaluate the diagnostic and prognostic value of a new hs TnT assay in comparison to the established TnT assay.

Methods and results: From April 2003 until April 2005 all consecutive patients (n=1023, 30% females, median age 65 years) admitted because of an ACS within the last 48 hours were included. Diagnosis was myocardial infarction in 857 (84%) patients. Clinical follow up after 6 months was available. During this period 72 (7.0%) patients died. Conventional TnT was measured on admission and at day 1 (Elecsys®, Roche, Germany). A TnT > 0.03 ng/ml was considered positive. High sensitive troponin T (hsTnT) was measured on admission (Elecsys®, Roche, Germany). For this assay, a threshold of 0.012 ng/ml, which is the 99
th
percentile and identical with the 10% CV, has been applied. On admission 694 (68%) of the patients were TnT positive, yielding a sensitivity of 81% for the diagnosis of MI. In comparison, 879 (86%) of the patients were hsTnT positive on admission yielding a sensitivity of 96%. The AUC of the ROC curves for TnT and hsTnT for the diagnosis of MI were comparable (0.929 for TnT and 0.954 for hsTnT). Among the 329 patients who were TnT negative on admission, in 133 patients MI could have been diagnosed correctly by hsTnT (sensitivity of 82%, specificity of 67%). In a multivariate Cox regression model, hsTnT was an independent predictor for mortality. In this model, conventional TnT lost its predictive value.

Conclusion: A new hs TnT assay enables early diagnosis of MI with a higher sensitivity as conventional TnT assays. Furthermore, hsTnT levels provide stronger prognostic information for mortality within 6 months as conventional TnT levels.
Abstract 5025: Diagnostic Value of Serial Changes in Low-Positive Extra-Sensitive Troponin I Levels for Acute Myocardial Infarction
Authors: Chugh Atul R , Elmore Julius B , Kuznetsov Sofya , Loughran John H , Flaherty Michael P , Platt Melissa A , Coleman Royce D , Miller James J , Dawn Buddhadeb
Published: April 2024

Background:
The use of high sensitivity troponin I (TnI-ES) assays results in higher rates of low-positive (LP-TnI-ES) results. Diagnostic value of serial changes in LP-TnI-ES levels remains unclear.

Methods:
Results of serial TnI-ES tests (VITROS ECi, Ortho-Clinical Diagnostics) were obtained from July 2008 to March 2009. Demographic and clinical data were recorded. Positive (>0.034 ng/ml [the 99
th
percentile]) results were divided into low (0.035– 0.100 ng/ml), intermediate (0.101– 0.300 ng/ml), and high (>0.300 ng/ml) categories. Serial LP-TnI-ES results were tracked in hospitalized patients for progression to a higher category, regression to normal, or no significant change. Time interval for such changes was calculated. ANOVA and Pearson’s chi-Square tests were used to analyze association with outcome variables.

Results:
TnI-ES was performed in a total of 4090 unique patients, of whom 1134 (27.7%) had at least one positive TnI-ES value. At least one LP-TnI-ES value was noted in 818 (73.5%; 59% male, 37% African American; age 68±11 years) of these 1134 pts. Patients diagnosed with myocardial infarction (MI) comprised 7.3% of the LP-TnI-ES group. Predictors of MI by univariate analysis included higher initial LP-TnI-ES level (
P
=0.002), progression from lower or regression from higher values (
P
<0.001), higher GFR (
P
=0.05), lower creatinine (
P
=0.05), and prior history of MI (
P
<0.001); while age, gender, diabetes, and race were not significant. Results from a multivariate regression model are shown in Table 1

. Progression of LP-TnI-ES to high in less than 12 hours was most predictive of MI (mean LP-TnI-ES change: 10.646±27.054; mean time to change: 6:24±3:01;
P
<0.001).

Conclusions:
In patients with a low-positive TnI-ES result, serial changes and higher initial LP-TnI-ES levels, prior history of MI, and higher GFR were predictive of MI. These variables may be utilized to construct a clinical algorithm to aid decision-making in patients with LP-TnI-ES in the ER.

Table 1-

Results of Multivariate Logistic Regression Model
Abstract 5042: Interferon-gamma Induced Adipose Inflammation Linked to the Impairment of Vascular Function in Type 2 Diabetic Mice
Authors: Zhang Hanrui , Zhang Cuihua
Published: April 2024

Interferon-gamma (IFN) is the hallmark cytokine of Th1 cells and plays a major role in immuno-mediated inflammation in type 1 diabetes. However, the role of IFN in initiating adipose inflammation and subsequent vascular dysfunction in type 2 diabetes remains to be determined. Accordingly, we hypothesized that IFN induces inflammatory cell infiltration and chemoattractant gene expression in mesenteric adipose tissue (MAT) and impairs vascular function of small mesenteric arteries (MA) of type 2 diabetic mice. To test this, we studied MAT inflammation, and vascular function of MA in control mice (m Lepr
db
), diabetic mice (Lepr
db
), m Lepr
db
treated with IFN, Lepr
db
treated with anti-IFN or anti-monocyte chemoattractant protein (MCP)-1. Acetylcholine (ACh)-induced vasodilation was impaired in Lepr
db
vs. m Lepr
db
, but sodium nitroprusside (SNP)-induced vasodilation was comparable. Both anti-IFN and anti-MCP-1 improved endothelial function in Lepr
db
, while IFN induced impaired vascular function in m Lepr
db
. Mesenteric bed weight and MAT adipocyte size were greatly elevated (P<0.05) in Lepr
db
vs. m Lepr
db
. Immunohistochemical staining results revealed that CD3-positve T-lymphocytes, F4/80 and Mac-3 positive macrophage infiltration into MAT was markedly increased in Lepr
db
and IFN-treated m Lepr
db
. It showed reduced MAT macrophages infiltration in Lepr
db
treated with anti-IFN vs. Lepr
db
. Interestingly, Mac-3 positive macrophage infiltration into the adventitia of small mesenteric arteries was significantly increased in Lepr
db
and IFN-treated m Lepr
db
, suggesting the possible crosstalk between adipose inflammation and vascular dysfunction. Western blotting results showed that anti-IFN decreased MAT IFN and MCP-1 protein expression in Lepr
db
, while IFN and MCP-1 expression was elevated in IFN-treated m Lepr
db
. Anti-MCP-1 treatment only decreased MCP-1 expression in Lepr
db
without affecting IFN levels, indicating that IFN may act as an initiator of MAT inflammation and chemoattractant gene expression. These results suggest a role for IFN in the regulation of visceral adipose inflammatory response and vascular dysfunction in type 2 diabetes.

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 5054: Bone Marrow MyD88 Contributes to Ischemic Myocardial Infarction and Modulates Neutrophil Function and Chemokine Receptor Expression
Authors: Feng Yan , Zou Lin , Si Ri , Nagasaka Yasuko , Shen Shiqian , Chao Wei
Published: April 2024

INTRODUCTION:
MyD88 is an adaptor protein critical for innate immune signaling and plays a pivotal role in host defense against infection, but its role in
non-infectious
myocardial inflammation and injury after ischemia-reperfusion (I/R) is unclear. Our previous studies in MyD88
−/−
(KO) mice have demonstrated that MyD88 is essential for I/R-induced myocardial injury. The current study was to delineate the specific contribution of
cardiac
and
bone marrow (BM)-derived
MyD88 in mediating ischemic cardiac injury and to explore the underlying mechanisms.

METHODS:
Chimeric models were generated by lethal irradiation and BM reconstruction. For I/R model, the coronary artery was ligated for 60 min and followed by reperfusion. At the end of reperfusion, the hearts were examined for ischemic and infarct sizes. To assess neutrophil mobility function, thioglycollate was injected intra-peritoneally. Six hours later, peritoneal cells were counted. Neutrophils were labeled with anti-Ly-6C and counted by flow cytometry. CXCR2 expression, which is critical for neutrophil mobility function, was measured by flow cytometry.

RESULTS:
There was a significant decrease in infarct sizes in KO compared to WT (22±1% vs. 37±2%,
p
<0.001). Compared to WT→WT control (Donor→Recipient), KO→WT mice, which lacked MyD88 in BM cells but maintained intact MyD88 signaling in the heart, had smaller MI sizes (
p
< 0.05). Surprisingly, infarct sizes of WT→WT mice were also significantly reduced compared to WT (
p
< 0.05). To determine if MyD88 deficiency inhibits neutrophil function that contributes the observed cardiac benefit, we examined the neutrophil mobility and CXCR2 expression. Indeed, neutrophil mobility was dramatically decreased in KO mice compared to WT mice (
p
< 0.01). Neutrophil mobilization also decreased in WT→WT and KO→WT chimeric mice (
p
< 0.01). Moreover, there was a significant decrease in CXCR2 expression on the peritoneal neutrophils of KO and KO→WT mice (
p
< 0.01), but not in WT→WT mice.

CONCLUSIONS:
Taken together, these findings suggest that MyD88 signaling, most likely that of BM-derived neutrophils plays a critical role in mediating myocardial I/R injury. MyD88 may mediate I/R injury partly by modulating CXCR2 expression and thus neutrophil function.

This research has received full or partial funding support from the American Heart Association, Founders Affiliate (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont).
Abstract 5065: Statins Modulate the Expression of Kruppel-Like Factor 2 in T Lymphocytes and Inhibit T Cell Responses
Authors: Bu De-xiu , Garcia-Cardena Guillermo , Tarrio Margarite , Maganto-Garcia Elena , Grabie Nir , Lichtman Andrew H
Published: April 2024

Kruppel-Like Factor 2 (KLF2) is a zinc -finger transcription factor, essential for maintenance of T cell quiescence and migration. Statins upregulate KLF2 expression in endothelial cells, but direct effects of statins on T cells are not characterized. We hypothesized that immunomodu-latory effects of statins are related partially to their influence on T cell KLF2 expression. Therefore, we characterized KLF2 expression pattern during different phases of T cell activation, examined the effects of Statins on KLF2 expression in T cells, and determined if Statins modulated pathologic T cell responses in a KLF2-dependent manner. Quantitative RT-PCR analyses indicated that KLF2 expression is relatively high in naïve mouse CD4+ and CD8+ T cells, but drops rapidly after TCR and CD28 co-stimulation and increases again as effector T cell are rested for several days. Interestingly, we also found that KLF2 is upregulated in CD8+ T cells that are functionally suppressed by FOXP3+ regulatory T cells. Effector T cells treated with four different lipophilic Statins (10 micromolar, 18h) enhanced expression of KLF2 and sphingosine 1 phosphate receptor (S1P1) and down-regulated interferon gamma expression. Statins also diminished T cell proliferation in response to TCR stimulation. Similar patterns of KLF2 expression and of Statin modulation of KLF2 were detected in human T cells, by qRT-PCR and Western blot analyses. Lovastatin (20mg/kg, i.p) treatment of C57B6 mice enhanced T cell KLF2 expression and induced resistance to T cell reactivation ex vivo. In a mouse model of myocarditis induced by adoptive transfer of heart-antigen-specific CTL, we found that simvastatin treatment of transferred T cells or retroviral-mediated over expression of KLF2 in the transferred T cells had similar effects; both reduced severity of myocarditis, down-regulated inflammatory gene expression in the hearts, and reduced cardiac draining lymph node responses. We conclude that both Statins and KLF2 modulate T cell activation and effector responses and that Statins upregulate KLF2 in T cells. Therefore, modulation of KLF2 by Statins may have therapeutic significance for cardiovascular diseases such as myocarditis and atherosclerosis.
Abstract 5067: Interleukin-7 is an Active Participant in Atherogenesis by Promoting Monocyte/macrophage Arrest on Endothelial Cells
Authors: Li Rongying , Paul Antoni , Ko Kerry W.S. , Sheldon Michael , Rich Benjamin E. , Chan Lawrence , Oka Kazuhiro
Published: April 2024

Atherosclerosis is a chronic inflammatory disease, but can regress. Despite the importance of atherosclerotic lesion regression, its mechanisms are not well understood. We induced regression of advanced atherosclerosis in
Apoe
−/−
mice and studied potential mechanisms. A single i.v. injection of helper-dependent adenoviral vector expressing human apoE3 (HDAd-gE3) into
Apoe
−/−
mice resulted in a 28±4% reduction of the advanced atherosclerosis (n= 7 vs. baseline n=8, p=0.012) after 41 weeks. Using this system, we analyzed gene expression profiles in the aorta at day 10, when plasma cholesterol was normalized. Pathway and ontology analyses revealed multiple signaling pathways influenced by the treatment; however, after correction for multiple testing we unexpectedly found that interleukin-7 (IL-7) was significantly downregulated. IL-7 is a nonredundant cytokine for B- and T-cell development. Although several studies have suggested that IL-7 is associated with cardiovascular disease, IL-7 has not been implicated as having a direct role in atherogenesis. IL-7 positive areas in the aorta were also reduced in the HDAd-gE3 group by 53% (n=5– 6/group, p<0.01) after 41 weeks of treatment. IL-7 could be a marker for lesion regression or reduced inflammation, or be directly involved in atherosclerosis. To test this hypothesis, we analyzed the effects of IL-7 on expression of adhesion molecules and chemokines in human aortic endothelial cells (HAECs). IL-7 upregulated adhesion molecules and chemokines which include VCAM-1, ICAM-1, E-selectin and monocyte chemotactic protein-1. This upregulation was time- and dose-dependent and promoted monocyte adhesion to HAECs. Moreover, the increase of plasma IL-7 levels in
Apoe
−/−
mice by HDAd-IL-7 enhanced homing of monocyte/macrophage to the lesions by 2-fold (n=6/group, p<0.01) without increasing plasma cholesterol. In contrast, transplantation of bone marrow cells isolated from
Il-7
−/−
mice into
Ldlr
−/−
mice reduced macrophage positive areas by 26% after 20 weeks compared with those isolated from wild type mice (n=6/group, p<0.05). These results suggest that IL-7 plays a direct role in atherosclerosis by promoting monocyte/macrophage homing to atherosclerotic lesions.
Abstract 5075: Beneficial Effects of Combination of ACE Inhibitor and Rho-kinase Inhibitor on Tubulointerstitial Fibrosis Induced by Unilateral Ureteral Obstruction
Authors: Nishikimi Toshio , Takeda Yosuke , Ishimura Kimihiko , Tadokoro Kazuyoshi , Iemura-Inaba Chikako , Akimoto Kazumi , Ishimitsu Toshihiko , Matsuoka Hiroaki
Published: April 2024

Objects: We recently reported that long-term Rho-kinase inhibition attenuates glomerulosclerosis in severe hypertension, salt-induced hypertension, and malignant hypertension rat model. In addition, ACE inhibitor is well known to prevent renal fibrosis. This study was design to compare the effects of ACE inhibitor (imidapril), Rho-kinase inhibitor (fasudil), and their combination on renal interstitial fibrosis induced by unilateral ureteral obstruction (UUO). We also attempted to elucidate the mechanism involved.

Methods: Imidapril (50mg/L), fasudil (1g/L), or their combination was given in drinking water to mouse (C57BL/6, each group: n =8), and their effects were compared on renal interstitial fibrosis induced by UUO. We measured histological findings, immunohistological findings, oxidative stress, and various mRNA expressions in the kidney by UUO.

Results: Eleven days after UUO, wild type kidney was characterized increased fibrotic area (Masson’s trichrome stain positive area), dihydroethidium (DHE) positive area,
α
-SMA positive area, F4/80 positive area, and increased various mRNA expressions. Fasudil and imidapril similarly improved fibrotic area (−23%, −15%), DHE positive area (−13%, −11%),
α
-SMA positive area (−22%, −15%), F4/80 positive area (−42%, −34%), and various mRNA expressions (TGF-
β
: −20%, −18%; CollagenI: −37%, −19%; CollagenIII: −36%, −26%; MCP-1: −22%, −34%, TNF-
α
: −7%, −13%), most of which were significant (P< 0.05). Combination of imidapril and fasudil further improved fibrotic area (−52%), DHE positive area (−26%),
α
-SMA positive area (−33%), F4/80 positive area (−62%), and various mRNA expressions (TGF-
β
:−48%; CollagenI:−65%; CollagenIII:−59%; MCP-1:−54%, TNF-
α
: −33%) (all P <0.05 vs monotherapy).

Conclusion: Compared with either agent alone, the combination of an ACE inhibitor and a Rho-kinase inhibitor is more effective in the prevention of renal interstitial fibrosis due to inhibition of TGF-
β
/collagen, migration of monocyte/macrophage, differentiation of fibroblast, inflammation, and oxidative stress pathway. Combination of an ACE inhibitor and a Rho-kinase inhibitor may be a possible new therapeutic strategy for chronic kidney disease.
Abstract 5080: Role of Mineralocorticoid Receptor/Rho/Rho-kinase Pathway in Obesity-Related Renal Injury
Authors: Tokuyama Hirobumi , Wakino Shu , Tatematsu Satoru , Yoshioka Kyoko , Hasegawa Kazuhiro , Hara Yoshikazu , Minakuchi Hitoshi , Sueyasu Keiko , Homma Koichiro , Washida Naoki , Hayashi Koichi , Itoh Hiroshi
Published: April 2024

Both aldosterone and Rho-kinase are demonstrated independently to play an important role in renal injury and impaired insulin signalling. We therefore examined whether aldosterone/Rho/Rho-kinase pathway contributed to obesity-associated nephropathy. C57BL/6J mice were fed a high fat (HFD) or low fat diet (LFD) and mice on HFD were treated with a mineralocorticoid receptor (MR) antagonist, eplerenone. The mice on HFD developed obesity, and hyperglycemia compared with those on LFD, but manifested similar levels of systolic blood pressure. In HFD-fed mice, glomerular hypercellularity and increased mesangial matrix were noted (66.7±3.0 vs. 32.3±2.5 nuclei/gcs, p<0.01), which paralleled the increase in albuminuria (HFD, 0.13±0.04mg/gCr, P<0.01 vs LFD, 0.02±0.01mg/gCr). The treatment with eplerenone alleviated the histological changes and albuminuria (0.04±0.01mg/gCr, P<0.01 vs HFD) without alterations in systemic blood pressure. Adipose tissue weight and adipocyte size of HFD-fed mice were increased, which were ameliorated by eplerenone. Furthermore, enhanced Rho-kinase activity was noted in kidneys (2.0-fold, p<0.05) and adipose tissues (1.8-fold, p<0.05) from HFD-fed mice, as well as increased expressions of MCP-1, TNF-
α
and PDGF-B. All of these changes were attenuated by eplerenone. In HFD-fed mice, MR protein levels in the nuclear fraction were increased in kidneys (2.3-fold, p<0.05) and adipose tissues (2.0-fold, p<0.05) without elevation in serum aldosterone levels (LFD, 278±14mg/dl, HFD, 297±12mg/dl), and Sgk1, an effector of aldosterone, was upregulated (kidneys; 5.5-fold, p<0.01, adipose tissues; 2.2-fold, p<0.01). Finally, in mesangial cells, stimulation with aldosterone increased Rho-kinase activity, and pre-incubation with eplerenone prevented the aldosterone-induced activation of Rho-kinase. In conclusion, excess fat intake causes obesity and renal injury in C57BL/6J mice, and these changes are mediated by enhanced MR/Rho/Rho-kinase pathway and subsequent inflammatory process. Furthermore, both MR activation and the subsequent Rho-kinase stimulation are likely to participate in the development of obesity-associated nephropathy without elevation in serum aldosterone levels.
Abstract 5081: Circulating Microparticles as Potential Biomarkers for Primary Intracerebral Hemorrhage Stroke
Authors: Chen Shuzhen , Zhang Dongsheng , Zhang Wenfeng , Wang Jinju , Wang Yigang , Chen Yanfang
Published: April 2024

Introduction:
Primary intracerebral hemorrhage (ICH) stroke is a serious complication of uncontrolled hypertension. Recent clinical studies have shown that circulating microparticles (MPs) from endothelia (EMPs), platelets (PMPs) and endothelial progenitor cells (EPC-MPs) are potential biomarkers for cardiovascular diseases. However, the role of circulating MPs in ICH stroke is unknown.

Methods and Results:
The human renin and angiotensinogen transgenic (R+A+) and the control (R−A−) mice (male, 8 –10 weeks) were fed with normal diet or high salt diet (8% NaCl, HSD) plus nitric oxide synthase inhibitor L-NAME (120 mg/kg/day) in drinking water for two weeks. Mean arterial pressure (MAP) was measured using telemetric probes. Circulating MPs were identified by membrane markers, CD62E (for EMPs), CD62P (for PMPs), and CD34 and VEGFR2 (for EPC-MPs) using flow cytometry. The severity of ICH was determined by histological analysis of total bleeding area. The lumen/wall in the middle cerebral artery (MCA) was histologically measured. Results (Table

and Figure

) showed:

The R+A+ mice were hypertensive and had higher basal level of circulating PMPs and EMPs with no difference in EPC-MPs;

After fed with HSD and L-NAME, R+A+ mice had severe hypertension and 50% of them developed ISH stroke within two weeks;

There was a further increase of EMPs and a decrease of EPC-MPs in R+A+ mice with ICH stroke;

There were strong correlations of EMPs (r=0.44~0.61) and EPC-MPs (r=−0.52~0.78) with MAP, MCA lumen/wall and total area of bleeding sites.

Conclusion:
Circulating EPC-MPs and EMPs could serve as useful biomarkers for uncontrolled hypertension, MCA remodeling and ICH stroke.

Table.

Blood pressure, circulating MPs and MCA lumen/wall in R+A+ and R−A− mice fed with normal diet or HSD and L-NAME for two weeks

Fig.

Hemorrhages in the brain (A, D: brainstem: B, E: cerebellum; C,F: basal ganglia) and MCA remodeling, A–C; R+A+ mice fed with HSD+L-NAME; D–F: R−A− mice fed with HSD+L-NAME; G: R−A− mice fed with normal diet, H: R−A− mice fed with HSD+L-NAME; I: R+A+ mice fed with HSD+L-NAME
Abstract 5083: Klotho Gene Delivery Delays and Attenuates Spontaneous Hypertension
Authors: Sun Zhongjie
Published: April 2024

Background

-
Klotho is a recently-discovered anti-aging gene. Knockout of klotho gene shortens the lifespan while over-expression of klotho gene extends lifespan of mice. The prevalence of hypertension increases with age. Therefore, hypertension and related cardiovascular diseases are aging disorders. The expression of klotho was decreased significantly in SHRs. The purpose of this experiment was to test our hypothesis that klotho gene delivery attenuates spontaneous hypertension.

Methods and Results-

Recombinant adeno-associated virus (AAV-2) carrying mouse klotho full-length cDNA (AAV.mKL) was constructed for
in vivo
expression of klotho. Four groups of male SHR (4 weeks) (8 rats/group) and 1 group of age-matched WKY rats (5 rats/group) were used. Blood pressure (BP) and body weight (BW) were measured twice in all animals before gene delivery in SHR. Briefly, the 4 groups of SHR received IV injection of AAV.mKL, AAV.LacZ, AAV.GFP, and PBS, respectively. The WKY group received PBS as a control. Following gene delivery, BP and BW was monitored weekly. BPs of SHR groups treated with AAV.LacZ, AAV.GFP, or PBS were significantly higher than that of the WKY group treated with PBS. AAV.mKL delayed and significantly attenuated the elevation of BP without affecting the body weight gain in SHRs. One single dose of AAV.mKL attenuated spontaneous hypertension for up to 12 weeks (length of the study). The systolic BP was 140±3.6, 174±4, and 126±2.8 mmHg for SHR-mKL, SHR-LacZ, and WKY-PBS groups, respectively. AAV.mKL also significantly increased hypotensive responses to acetylcholine, suggesting that klotho gene delivery improved endothelial function. AAV.mKL significantly decreased NADPH oxidase activity and superoxide production in mesenteric resistance arteries. Klotho gene delivery abolished renal damage in SHRs.

Conclusion-

AAV delivery of mouse klotho gene delayed and attenuated hypertension in SHR. The antihypertensive effect of klotho may be mediated by inhibition of NADPH oxidases. Klotho deficiency may play a role in the development of spontaneous hypertension.

This research has received full or partial funding support from the American Heart Association, Greater Southeast Affiliate (Alabama, Florida, Georgia, Louisiana, Mississippi, Puerto Rico & Tennessee).
Abstract 5106: C-c Chemokine Receptor 2 Inhibitor Ameliorates Diet-induced Insulin Resistance and Hepatic Steatosis in Mice
Authors: Tamura Yukinori , Murayama Toshinori , Minami Manabu , Nishikaze Yuki , Kita Toru , Yokode Masayuki , Arai Hidenori
Published: April 2024

Visceral obesity and insulin resistance are common underlying factors of the metabolic syndrome (MetS), which is associated with a chronic low-grade inflammatory state and cardiovascular disease. C-C chemokine receptor-2 (CCR2), known as a receptor for MCP-1, plays a role in monocyte/macrophage (M
φ
) recruitment and M
φ
-dependent inflammatory response. Recently, adipose tissue inflammation by M
φ
infiltration through MCP-1/CCR2 is considered to play a pivotal role in the development of visceral obesity and insulin resistance. However, the effect of postnatal inhibition of CCR2 on the development of diet-induced obesity (DIO) and insulin resistance remains unclear. To further explore the role of CCR2 in the development of visceral obesity and its associated metabolic disorders, we studied the effect of pharmacological inhibition of CCR2 before the onset of DIO in C57BL/6 mice. To study the effect of CCR2 inhibition, C57BL/6 mice were fed a high fat and high sugar diet with or without propagermanium (Pro, dose of 5mg and 50mg/kg/day), as a CCR2 inhibitor for 12 weeks from 6 weeks of age. At the end of study the gene expression of F4/80 and CD11c was reduced by 50%, while that of IL-10 and MGL-1 was increased by 2-fold in white adipose tissue (WAT), indicating that Pro suppressed adipose tissue M
φ
accumulation and shifted the infiltration of pro-inflammatory M1 M
φ
to anti-inflammatory M2 M
φ
. These data are consistent with a marked reduction of pro-inflammatory cytokine, TNF-
α
by Pro by 50% in WAT and liver. Diet-induced hypoadiponectinemia and hyperleptinemia were also improved by Pro treatment. Further, Pro treatment ameliorated insulin sensitivity and glucose tolerance, resulting in a marked reduction of plasma glucose and insulin level. Finally, Pro treatment decreased hepatic triglyceride contents by 45% in DIO mice. In conclusion, Pro improved obesity and its associated metabolic disorders, such as insulin resistance and hepatic steatosis by suppressing infiltration of pro-inflammatory M1 M
φ
into WAT. Our data also implicate that CCR2 contributes to DIO and type 2 diabetes by accelerating adipose tissue inflammation, and that Pro may be a beneficial drug for the treatment of such metabolic disorders by blocking CCR2-mediated inflammation.
Abstract 5109: LKB1 in Vascular Endothelial Cells is Essential for Revascularization in a Mouse Ischemic Hind-limb Model
Authors: Ohashi Koji , Ouchi Noriyuki , Walsh Kenneth
Published: April 2024

Recent reports have indicated that LKB1 is a regulator of AMP-activated protein kinase (AMPK) signaling in many tissues including liver, skeletal muscle and cardiac myocytes. In vascular endothelial cells, AMPK signaling is essential for cell function and angiogenic responses to tissue ischemia. Homozygous LKB1 deficiency in mice leads to embryonic lethality associated with abnormal vascular development. Here, we investigated whether heterozygous LKB1 deficiency in endothelial cells has a role in regulating revascularization to tissue ischemia in hind limb ischemic mouse model. We also explored the role of AMPK signaling downstream of LKB1 in vascular endothelial cells. Homozygous vascular endothelial cell deletion of LKB1, using the conditional loxP gene regulation system crossed with Tie2-Cre transgenic mice, led to an embryonic lethal phenotype. Thus, we produced endothelial specific reduced LKB1 model,
Lkb1

flox/

+
;Tie2

Tg/

+
(heterozygous-KO) mice. LKB1 expression level was decreased to half in the isolated endothelial cells from heterozygous-KO mice compared with those from
Lkb1

flox/

+
;Tie2
+/+
(control) mice, but mice were normal in viability, body weight and vascular density. Revascularization of hind limb following ischemic surgery was significantly impaired in heterozygous-KO mice compared with control mice as evaluated by laser Doppler Flow method and capillary density analyses at day 14 and 28 after surgery. In cultured endothelial cells, reduction of LKB1, using both siRNA and adenovirus expressing non-functional kinase dead LKB1 protein, attenuated differentiation into network structures that was accompanied by diminished phosphorylation levels of AMPK (Thr-172) and acetyl-CoA carboxylase (ACC) (Ser-79). Conversely, LKB1 over-expression by adenovirus (Ad-LKB1) augmented differentiation into network structure with elevated phosphorylation levels of AMPK, ACC and eNOS (Ser-1177). The augmented network structure induced by Ad-LKB1 was abrogated by the co-transduction of a dominant negative mutant of AMPK. Taken together, our observations suggest that LKB1-AMPK axis in endothelial cells plays a pivotal role in the revascularization response to tissue ischemia.