bluuebunny/crossref_metadata_2025 huggingface dataset:
Severe Airflow Limitation After the Unifocalization Procedure
Authors: Schulze-Neick Ingram , Ho S. Yen , Bush Andrew , Rosenthal Mark , Franklin Rodney C. , Redington Andrew N. , Penny Daniel J.
Published: July 2021

Background
—While unifocalization techniques have improved the treatment options in patients with pulmonary atresia, ventricular septal defect (PA-VSD), and major aortopulmonary collaterals (MAPCAs), severe airflow limitation contributes to significant early postoperative morbidity and mortality. Although this has been attributed to bronchospasm, characteristically it is refractory to bronchodilators, suggesting that other mechanisms may play a role.

Methods and Results
—The clinical course and preoperative angiograms of patients who underwent unifocalization were reviewed. Patients who developed airflow limitation early after surgery underwent fiberoptic bronchoscopy. In addition, the anatomy of the MAPCAs was examined in 14 heart-lung blocks from patients with PA-VSD. Twenty-two procedures were performed in 16 children. Three developed marked airflow limitation early after surgery, necessitating prolonged high-pressure ventilation. Bronchoscopy demonstrated tracheobronchial epithelial necrosis in 2 and signs of tracheobronchial ischemia in the third. Two were successfully extubated after 15 and 16 days, but the third died after 57 days of ventilatory support. Review of the preoperative angiograms demonstrated an extensive peribronchial arterial supply arising from a MAPCA in 1 of the patients who developed severe airway necrosis after unifocalization. This was also obvious in a second patient, but the MAPCA was not included in the unifocalization. In 7 autopsy specimens, MAPCAs contributed to a peribronchial or peritracheal vascular network. Dissection of the distribution of these branches in 2 specimens revealed extensive intrapulmonary peribronchial anastomoses.

Conclusions
—Airflow limitation early after unifocalization is related to airway ischemia resulting from interruption of the tracheobronchial blood supply during mobilization of MAPCAs.
Congenital Mitral Stenosis With or Without Associated Defects
Authors: Serraf Alain , Zoghbi Joy , Belli Emré , Lacour-Gayet François , Aznag Hakim , Houyel Lucile , Lambert Virginie , Piot Dominique , Planché Claude
Published: July 2021

Background
—Congenital mitral stenosis (CMS) remains a surgical challenge, particularly when it is associated with other heart defects. As in other groups of heart defects, there is a trend toward early single-stage complete repair, but the optimal surgical approach remains unanswered.

Methods and Results
—This study was designed to analyze the evolution of surgical strategies in patients with CMS and associated defects through single-stage and staged repair. Between 1980 and 1999, 72 children were operated on for congenital heart defects, including CMS. Preoperative transmitral gradient was 12.6±7 mm Hg. Preoperatively, all the patients were NYHA class III to IV. Thirteen patients had an isolated CMS; in 59, it was associated with other heart defects, mainly ventricular septal defect (n=28) or multilevel left ventricular obstruction (n=41). In this group of patients, 33 had a staged approach, and 26 had a single-stage approach. Early mortality was 12.5% (9 patients). There were no deaths in the isolated CMS and single-stage repair groups. Logistic regression revealed that early mortality was influenced by association with left ventricular outflow tract obstruction (
P
<0.001) and by use of a staged approach (
P
<0.01). There was no late mortality in isolated CMS; there were 2 late deaths in the group of single-stage repair and 6 late deaths in the staged approach group (
P
<0.01). Reoperation was required in 24 patients, mainly for residual mitral valve dysfunction or residual left ventricular outflow tract obstruction. Including the reoperations, 10 patients received a prosthetic mitral valve. At 15 years after surgery, survival was 69.6±7.5%, freedom from reoperation was 70.8±6.3%, and freedom from mitral valve replacement was 69±6%.

Conclusions
—Surgery for isolated CMS gives excellent early and long-term results. In patients with associated heart defects, a single-stage operation seems superior to a staged approach. Mitral valve replacement in this category of patients should be reserved as a salvage procedure.
Vacuolar H
+
-ATPase Plays a Crucial Role in Growth and Phenotypic Modulation of Myofibroblasts in Cultured Human Saphenous Vein
Authors: Otani Hajime , Yamamura Tadashi , Nakao Yoshihisa , Hattori Reiji , Fujii Hirohumi , Ninomiya Hideki , Kido Masakuni , Kawaguchi Hideki , Osako Motohiko , Imamura Hiroji , Ohta Tetsuo , Ohkuma Shoji
Published: July 2021

Background
—The molecular mechanism of neointimal hyperplasia after vein graft surgery remains elusive. Vacuolar H
+
-ATPase (V-ATPase) is involved in intracellular trafficking and may play a crucial role in neointimal cell growth.

Methods and Results
—Cultured human saphenous vein segments developed neointimal formation within 10 days. Neointimal cells were positive for vimentin and α-smooth muscle actin but negative for desmin, which is indicative of myofibroblasts. Those myofibroblasts were found to have originated from periadventitial fibroblasts, which upregulated the expression of 16-kDa proteolipid of V-ATPase before proliferation and phenotypic modulation. Neointimal myofibroblast growth and survival were highly sensitive to inhibition of V-ATPase by bafilomycin A
1
(BA
1
), because the incorporation of [
3
H]thymidine into the myofibroblasts was significantly inhibited by nanomolar concentrations of BA
1
and apoptotic cell death was induced by a similar concentration range of BA
1
. In contrast, endothelial cells and differentiated smooth muscle cells were resistant to apoptosis by BA
1
.

Conclusions
—These results suggest that V-ATPase plays a crucial role in growth and phenotypic modulation of myofibroblasts that contributes to neointimal formation in cultured human saphenous vein.
Gene Therapy for Myocardial Protection
Authors: Jayakumar Jay , Suzuki Ken , Khan Mak , Smolenski Ryszard T. , Farrell Aldo , Latif Najma , Raisky Olivier , Abunasra Haitham , Sammut Ivan A. , Murtuza Bari , Amrani Mohamed , Yacoub Magdi H.
Published: July 2021

Background
—Heat shock protein 70 (HSP70) gene transfection has been shown to enhance myocardial tolerance after normothermic ischemia-reperfusion. We investigated the effect of HSP70 gene transfection on mechanical and endothelial function in a protocol mimicking clinical heart preservation.

Methods and Results
—Rat hearts were infused ex vivo with Hemagglutinating Virus of Japan–liposome complex containing HSP70 gene (HSP, n=8) or no gene (CON, n=8), and heterotopically transplanted into recipient rats. Four days after surgery, transfected hearts were perfused on a Langendorff apparatus for 45 minutes, arrested with St Thomas’ No. 1 cardioplegia for 4 hours at 4°C, and reperfused for 1 hour. Mechanical and endothelial function was studied before and after ischemia. Creatine kinase was measured in reperfusion effluent. Hearts underwent Western blotting and immunohistochemistry to confirm HSP70 overexpression. Postischemic recovery of mechanical function (% preischemic±SEM) was greater in HSP versus CON: Left ventricular developed pressure recovery was 76.7±3.9% versus 60.5±3.1% (
P
<0.05); dP/dtmax recovery was 79.4±4.9% versus 56.2±3.2% (
P
<0.05); dP/dtmin recovery was 74.8±4.6% versus 57.3±3.6% (
P
<0.05). Creatine kinase release was attenuated in HSP versus CON: 0.22±0.02 versus 0.32±0.04 IU/min/g wet wt. (
P
<0.05). Recovery of coronary flow was greater in HSP versus CON: 76.5±3.8% versus 59.2±3.2% (
P
<0.05). Recovery of coronary response to 5-hydroxytryptamine (5×10
−
5
mol/L) was 55.6±4.7% versus 23.9±3.2% (
P
<0.05); recovery of coronary response to glyceryltrinitrate (15 mg/L) was not different between HSP and CON: 87.4±6.9% versus 84.3±5.8% (NS).

Conclusions
—In a clinically relevant donor heart preservation protocol, HSP70 gene transfection protects both mechanical and endothelial function.
Development of a Novel Method for Cell Transplantation Through the Coronary Artery
Authors: Suzuki Ken , Brand Nigel J. , Smolenski Ryszard T. , Jayakumar Jay , Murtuza Bari , Yacoub Magdi H.
Published: July 2021

Background
—Cell transplantation is a promising strategy to treat end-stage heart failure. At present, a popular method to deliver cells into the heart is direct intramuscular injection. This method, however, may not be efficient in spreading cells globally into the myocardium. We have developed a novel method for cell transplantation using intracoronary infusion.

Methods and Results
—An L6 rat skeletal muscle cell line expressing β-galactosidase (β-gal) was generated by gene transfection and clonal selection. These cells (10
6
in 1 mL medium) were infused into explanted rat hearts through the coronary artery, followed by heterotopic heart transplantation into the abdomen of recipients. Control hearts were infused with cell-free medium. According to β-gal activity measurements, ≈5×10
5
grafted cells per heart existed on day 3, increasing to 5×10
6
on day 28 in the cell-transplanted hearts. At day 28, discrete loci positively stained for β-gal were observed throughout the cardiac layers of both left and right coronary territories. Some of them differentiated into β-gal–positive multinucleated myotubes that aligned with the cardiac fiber axis and integrated into the native myocardium, whereas others formed colonies consisting of undifferentiated myoblasts. Connexin 43, a cardiac gap junction protein, was expressed between grafted cells and native cardiomyocytes. No reduction in cardiac function was observed in a Langendorff perfusion system.

Conclusions
—We have developed a unique method for efficient cell transplantation based on intracoronary infusion. This method, potentially applicable in the clinical setting during cardiac surgery, could be useful to globally supply cells to the heart.
Angiotensin II type 1 receptor A1166C polymorphism in relation to incident cardiovascular events, hypertension, and blood pressure control in elderly Caucasians and African Americans
Authors: Hindorff Lucia A. , Heckbert Susan R. , Tracy Russ , Tang Zhonghua , Psaty Bruce M. , Kronmal Richard A. , Siscovick David S. , Nazar-Stewart Valle
Published: July 2021

P17

Objective: The C allele of the angiotensin II type 1 receptor (AT1R) A1166C polymorphism has been associated in several studies with increased risks of hypertension (HTN) and myocardial infarction (MI), and with enhanced blood pressure (BP) response to ACE inhibitors but not calcium antagonists. Most of these studies were small, and none included large numbers of African Americans. We examined these associations in the Cardiovascular Health Study, an observational cohort study of risk factors for coronary disease and stroke in men and women ≥ 65 years of age. Methods: All 771 self-identified African Americans plus 1333 randomly-selected Caucasians were genotyped for the AT1R polymorphism. BP, medication use, and HTN were assessed annually. Incident MI/coronary death (CHD, n = 125 in whites; n = 60 in blacks), incident total cardiovascular events (CVD, n = 226 in whites; n = 101 in blacks), incident congestive heart failure (CHF, n = 146 in whites; n = 55 in blacks), and incident HTN (n = 184 in whites; n = 85 in blacks) were identified during up to 8 years of follow-up. Results: Genotype frequency distributions differed between whites (AA 49%, AC 41%, CC 10%) and blacks (85%, 15%, 1%, p < 0.0005). Age, gender or HTN at baseline did not differ by genotype in either group. In both whites and blacks, genotype was not associated with incident CHD (HR for AC/CC vs. AA, 95% CI in whites = 1.0, 0.7-1.5; blacks, HR = 0.7, 0.3-1.5), incident total CVD events (whites, HR = 1.0, 0.8-1.3; blacks, HR = 0.9, 0.5-1.6), incident CHF (whites, HR = 1.1, 0.8-1.5; blacks, 0.6, 0.3-1.4) or newly-identified HTN (whites, HR = 0.8, 0.6-1.1; blacks, HR = 1.1, 0.6-1.9). Among treated hypertensives, the association of genotype with controlled BP (<140/<90) did not differ between users of calcium channel blockers and users of ACE inhibitors in either race. Conclusion: In this large population-based elderly cohort, the AT1R A1166C polymorphism was not associated with prevalent or newly-identified HTN, incident CVD events, or BP response to antihypertensive agents in either Caucasians or African Americans.
Gene-environment interaction in the regulation of HDL: The hepatic lipase gene promoter polymorpism modulates the physical activity associated increase in HDL
Authors: Hokanson John E , Hamman Richard F , Eckel Robert H , Kamboh M Ilyas
Published: July 2021

P08

HDL-C is an important protective factor for coronary heart disease. Physical activity raises HDL-C, although the effect varies among individuals. The hepatic lipase (HL) promoter polymorphism (HL-514C→T) is associated with HDL-C. This study addresses the question: is the relationship between physical activity and HDL-C altered by an individual’s HL promoter polymorphism genotype? Non-diabetic subjects from the San Luis Valley Diabetes Study provided a 7 day physical activity recall from which metabolic equivalents (METS) were calculated. Anthropomorphic measures and fasting blood for lipid and DNA analysis were also collected. Women were excluded if postmenopausal or taking estrogen, resulting in 248 Hispanic and 363 non-Hispanic White (NHW) subjects. The frequency of the HL-514T allele was 0.18 in NHW compared to 0.47 in Hispanics (p<0.001). Women had higher HDL-C than men (52±13 vs. 45±11, p<0.001). The HL-514T allele was associated with higher HDL-C in men (46±12 vs. 44±11 for HL-514CC, p<0.03), but not in women (51±13 vs. 52±13 for the HL-514CC, p=0.5). METS were positively related to HDL-C in both men and women(r=0.18, p=0.001; and r=0.20, p<0.01, respectively). Multivariate analysis showed a statistically significant interaction between METS and the HL-514 allele on HDL-C (p<0.001). That is, subjects with the HL-514T allele showed less of an effect of METS on HDL-C compared to HL-514CC (slope=0.01, p=0.08 vs. slope=0.03, p=0.005, respectively), after adjusting for age, sex, ethnicity, BMI, waist/hip ratio, and log triglyceride. BMI was also independently related to HDL-C (p<0.0001). Thus, in this free living rural community, the beneficial effects of physical activity on HDL-C are modulated by the hepatic lipase gene promoter polymorphism. Individuals with the HL-514T allele are more resistant to exercise associated higher HDL-C. The allele-specific effects of physical activity on HDL-C were independent of body fat and fat distribution. This suggests a genotype-specific targeted approach of exercise intervention to raise HDL and reduce coronary disease risk may be warranted.
Cholesterol Screening in Children and Family History of Coronary Heart Disease.
Authors: Muratova Viktorina N , Islam Syed S , Spangler Emily C , Demerath Ellen W , Neal William A
Published: July 2021

P94

Background: Selective blood cholesterol screening of children based upon National Cholesterol Education Program (NCEP) guidelines of family history of premature cardiovascular disease (CVD) or parental hypercholesterolemia is inadequate in a population with high prevalence of coronary heart disease (CHD), low levels of cholesterol screening, low socio-economic status (SES) and diminished access to preventive health care. We hypothesize that universal cholesterol screening of pre-pubertal school children may be effective in identifying children and their parents with abnormal lipid levels in this high risk rural population. Fifth grade school children from seven rural Appalachian counties participated in a school based cholesterol screening program. Data on family history of premature CHD, anthropometric and blood pressure measurements, tobacco smoke exposure, dietary history and physical activity levels were collected at the time of screening. Seven hundred and nine 5

th

grade

students ( mean age 10.8 years) participated in the program. One hundred seventy four (24.5%) were considered presumptively dyslipidemic after non-fasting finger- stick (FS) cholesterol screening. Thirty six percent of these dyslipidemic children had a fasting lipid profile done. Dyslipidemia was confirmed in 37(59%) of these children. FS cholesterol levels were significantly correlated with fasting TC (r=0.80 p < 0.0001). Among confirmed dyslipidemic children, family history was not a good predictor of dyslipidemia (sensitivity 21.6%). Seventy nine parents of dyslipidemic children participated in fasting lipid profile assessment. Fifty two parents (67%) were dyslipidemic, most of them (79%) did not have a family history of premature CHD or hypercholesterolemia. FS cholesterol levels were also correlated with fasting TC of fathers (r=0.46 p=0.01), and mothers (r=0.32 p=0.02). Conclusion: Significant correlation exists between non-fasting FS cholesterol levels of children and subsequent fasting lipid profile of children and their parents. Family history has low sensitivity in predicting children with elevated serum cholesterol concentrations.
Serum Homocysteine levels are independently associated with mortality in Primary and Secondary Prevention patients derived from a high-risk clinic population.
Authors: Acevedo Monica , Pearce Gregory L. , Sprecher Dennis L.
Published: July 2021

0015

Background: Elevated total homocysteine levels (tHCY) have been implicated as an independent risk factor for mortality in patients with known and no known CAD. Few data are available that compare tHCY as a predictor of mortality in both groups. Objectives: In an ongoing, high-risk cardiovascular Prevention Clinic we sought to determine whether tHCY showed evidence of short-term mortality independently of traditional risk factors in primary and secondary prevention patients. Methods: Short term mortality (mean follow-up 26 ± 15 months) was assessed in 2441 clinic patients (1103 primary and 1338 secondary prevention). Cox models were used to assess the unadjusted relationships between tHCY (quartiles: < 9.4, 9.5-11.7, 11.8-14.7, >14.7 μmol/L) and survival time with hazard ratios (HR) used to estimate mortality risk. Framingham global risk scores were calculated for each patient in order to adjust for traditional risk factors (age, gender, LDL-C, HDL-C, blood pressure, diabetes and smoking). Results: The mean age of the overall group was 56 ± 12 years, 34% were women and 62 patients died during the follow-up (18 for primary and 44 for secondary prevention). Serum tHCY quartile showed a relationship to mortality for primary and secondary prevention patients (HR 3.02 and 1.69 respectively) with and without adjustment for traditional risk factors and creatinine (

Table). Conclusions: Serum homocysteine levels are independently associated with mortality in primary and secondary Prevention patients derived from the same high-risk clinic population.

Table 1.
Coronary Artery Calcification is Inversely Related to Bone Mineral Density in Young Women:
Authors: Schreiner Pamela J , Lewis Cora E , Detrano Robert C , Hulley Stephen B
Published: July 2021

0003

Coronary artery calcification (CAC), a marker of subclinical atherosclerosis, is related to low bone mineral density (BMD) in postmenopausal women, potentially due to age or declining estrogen levels. Little is known about this association in premenopausal women. During the Year 10 CARDIA Study, BMD was measured by dual energy X-ray absorptiometry at the hip (pelvis, femoral neck, intertrochanteric (IT) region, Ward’s triangle) and the lumbar spine in a subgroup; CAC was measured in a different subgroup by electron beam tomography within the same year. A calcium score was calculated as the product of an area of potential calcification covered by 6 adjacent pixels and the peak CT number within that region; a calcium score >0 indicates the presence of any CAC. Data for both measures were available for 75 women, ages 29-41 years (mean age 35 years). CAC was present in 14.7% of these young women. Mean weight was 75.2±19.1 kg, range 48.6 to 124.4 kg. Mean areal BMD was consistent with the age and reproductive status of these women: 1.18, 0.89, 1.15, and 0.83 g/cm

2

for the pelvis, femoral neck, IT region and Ward’s triangle, respectively; and 1.09 g/cm

2

for the lumbar spine. Cross-sectional logistic regression was performed to determine the crude association between BMD and CAC. BMD was not related to CAC at any site. Weight, however, was strongly associated with CAC, with a prevalence odds ratio (pOR) of 2.53 (95% CI 1.43, 4.49) for a 1-sd increment in weight. When weight was considered as a confounder to the association between BMD and CAC, BMD in the IT region was inversely related to CAC, with a 1-sd (0.17 g/cm

2

) decrement in the IT region BMD associated with a 233% increase in the odds of CAC (pOR=3.33, 95% CI 1.08, 10.4). This association was not materially affected by adjustment for age, ethnicity, or height. Nearly identical results were seen for CARDIA men, with an inverse association between IT region BMD and CAC after adjustment for weight. These data suggest that in a population-based group of young women with low prevalence of either osteopenia or osteoporosis, lower BMD may be a marker of subclinical atherosclerosis, comparable to findings in postmenopausal women.
The Association of Physical Activity with Obesity Development during Adolescence:
Authors: Kimm Sue Y. S. , Glynn Nancy W. , Barton Bruce A. , Kronsberg Shari S. , Schreiber George B. , Daniels Stephen R. , Obarzanek Eva
Published: July 2021

0006

The longitudinal data from NGHS show that physical activity (PA) fell by 35% in daily and 83% in weekly levels during adolescence, with greater decline for black (B) than white (W) girls. During these 10 years, the prevalence of obesity in NGHS doubled with higher rates in B girls than in W. The aim of this report is to examine the longitudinal association of PA and obesity development in 1213 B and 1166 W NGHS girls with annual followup from ages 9-10 to 18-19 years. Activity was assessed with a 3-day diary (AD) in tandem with a food diary and a habitual (past year) activity questionnaire (HAQ). Obesity was defined as ≥85

th

percentile of the sum of skinfolds (at triceps, subscapular and suprailiac sites) using age-specific cutpoints. Longitudinal analysis (GEE) was done with the likelihood (odds ratio, OR) of obesity as the outcome measure. Predictor variables included race, visit, age of menarche, average daily energy intake, and activity (as average daily activity, AD score, in one model and average weekly activity, HAQ score, in another). Visit (age) and race were significant predictors (1.51 OR for black race) of obesity. Age of menarche, energy intake, and HAQ scores were significantly (p<0.001 for all) and inversely associated with obesity. There was no significant interaction between race and HAQ, p=0.27. However, interaction between visit and HAQ was significant (p=0.01), indicating lower OR with higher HAQ from age 15 onward. The above analyses were repeated for AD in place of HAQ. Daily activity was not significantly (p=0.37) associated with obesity risk. We conclude that habitual, rather than short-term (AD), activity exerted a significant protective effect on the risk of developing obesity during adolescence. This finding is consistent with our previously reported lack of association between daily PA and obesity development in the NGHS cohort. Habitual PA during mid- to late adolescence seems to confer a greater protective effect on obesity risk. Our study confirms the role of PA in the marked increase in obesity prevalence during adolescence. Hence, obesity prevention needs to aim at fostering habitual activity, especially for black girls.
Vascular dysfunction after repair of coarctation of the aorta
Authors: de Divitiis Marcello , Pilla Carlo , Kattenhorn Mia , Zadinello Mariutzka , Donald Ann , Leeson Paul , Wallace Sharon , Redington Andrew , Deanfield John E.
Published: July 2021

Background

Patients with repaired coarctation are at increased risk of hypertension and cardiovascular disease despite successful repair. We studied the function of conduit arteries in upper and lower limbs of patients late after successful coarctation repair and its relation to age at surgery.

Methods and Results

Flow-mediated dilatation (FMD) and the dilatation after sublingual nitroglycerin (NTG, 25 μg) were measured by using high-resolution ultrasound in the brachial artery in 64 coarctation patients (44 males and 20 females, aged 19±10 years; median age at operation 4 months) and 45 control subjects (28 males and 17 females, aged 19±10 years) and in the posterior tibial artery in 37 patients and 22 control subjects. Arterial stiffness was determined by pulse-wave velocity (PWV) of the brachioradial and femoral-dorsalis pedis tracts. Patients, compared with control subjects, had lower brachial FMD (7.16±3.4% versus 8.62±2.3%, respectively;
P
=0.02) and NTG (11.46±4.3% versus 13.21±4.6%, respectively;
P
=0.046) and higher brachioradial PWV (9.17±3.1 versus 8.06±1.9 m/s, respectively;
P
=0.05). In contrast, posterior tibial FMD, NTG, and lower limb PWV were comparable. Age (months) at the time of repair was related to brachioradial PWV (
r
=0.42,
P
=0.002) but not to brachial FMD or NTG.

Conclusions

Patients with repaired aortic coarctation have impaired conduit artery function, with abnormal responses to flow and NTG, and increased vascular stiffness confined to the upper part of the body. Early repair is associated with preserved elastic properties of conduit arteries, but reduced reactivity remains.
Cell Transplantation for the Treatment of Acute Myocardial Infarction Using Vascular Endothelial Growth Factor–Expressing Skeletal Myoblasts
Authors: Suzuki Ken , Murtuza Bari , Smolenski Ryszard T. , Sammut Ivan A. , Suzuki Noriko , Kaneda Yasufumi , Yacoub Magdi H.
Published: July 2021

Background

Vascular endothelial growth factor (VEGF) is a promising reagent for inducing myocardial angiogenesis. Skeletal myoblast transplantation has been shown to improve cardiac function in chronic heart failure models by regenerating muscle. We hypothesized that transplantation of VEGF-expressing myoblasts could effectively treat acute myocardial infarction by providing VEGF-induced cardioprotection through vasodilatation in the early phase, followed by angiogenesis effects in salvaging ischemic host myocardium combined with the functional benefits of newly formed, skeletal myoblast-derived muscle in the later phase.

Methods and Results

Primary rat skeletal myoblasts were transfected with the human VEGF
165
gene using hemagglutinating virus of Japan-liposome with >95% transfection efficiency. Four million of these myoblasts (VEGF group), control-transfected myoblasts (control group), or medium only (medium group) was injected into syngeneic rat hearts 1 hour after left coronary artery occlusion. Myocardial VEGF-expression increased for 2 weeks in the VEGF group, resulting in enhanced angiogenesis without the formation of tumors. Grafted myoblasts had differentiated into multinucleated myotubes within host myocardium. Infarct size (33.3±1.4%, 38.1±1.4%, and 43.7±1.6% for VEGF, control, and medium groups, respectively;
P
=0.0005) was significantly reduced with VEGF treatment, and cardiac function improved in the VEGF group (maximum dP/dt: 4072.0±93.6, 3772.5±101.1, and 3482.5±90.6 mm Hg/s in the 3 groups, respectively;
P
=0.0011; minimum dP/dt: −504.2±68.5, −2311.3±57.0, and −2124.0±57.9 mm Hg/s, respectively;
P
=0.0008).

Conclusions

This combined strategy of cell transplantation with gene therapy could be of importance for the treatment of acute myocardial infarction.
Pathogenesis of Mitral Regurgitation in Tachycardia-Induced Cardiomyopathy
Authors: Timek Tomasz A. , Dagum Paul , Lai David T. , Liang David , Daughters George T. , Ingels Neil B. , Miller D. Craig
Published: July 2021

Background

Dilated cardiomyopathy is often associated with mitral regurgitation (MR), or so-called functional MR, the mechanism of which continues to be debated. We studied the valvular and ventricular 3D geometric perturbations associated with MR in an ovine model of tachycardia-induced cardiomyopathy (TIC).

Methods and Results

Nine sheep underwent myocardial marker implantation in the left ventricle (LV), mitral annulus, and mitral leaflets. After 5 to 8 days, the animals were studied with biplane videofluoroscopy (baseline), and mitral competence was assessed by transesophageal echocardiography. Rapid ventricular pacing (180 to 230 bpm) was subsequently initiated for 15±6 days until the development of TIC and MR, whereupon biplane videofluoroscopy and transesophageal echocardiography studies were repeated. LV volume was calculated from the epicardial marker array. Valve closure time was defined as the time after end diastole when the distance between leaflet edge markers reached its minimal plateau. TIC resulted in increased LV end-diastolic volume (
P
=0.001) and LV end-systolic volume (
P
=0.0001) and greater LV sphericity (
P
=0.02). MR increased significantly (grade 0.2±0.3 versus 2.2±0.9,
P
=0.0001), as did mitral annulus area (817±146 versus 1100±161 mm
2
,
P
=0.0001) and mitral annulus septal-lateral diameter (28.2±3.5 versus 35.1±2.6 mm,
P
=0.0001). Time of valve closure (70±18 versus 87±14 ms,
P
=0.23) and angular displacement of both the anterior (29±5° versus 27±3°,
P
=0.3) and posterior (55±15° versus 44±11°,
P
=0.13) leaflet edges relative to the mitral annulus after valve closure did not change, but leaflet edge separation after closure increased (5.2±0.9 versus 6.8±1.2 mm,
P
=0.019).

Conclusions

MR in TIC resulted from decreased leaflet coaptation secondary to annular dilatation in the septal-lateral direction. These data support the use of annular reduction procedures, such as rigid, complete ring annuloplasty, to address functional MR in patients with dilated cardiomyopathy.
Abstract 559: Diacylglycerol Kinase-ζ Rescues Gαq-induced Heart Failure
Authors: Niizeki Takeshi , Takeishi Yasuchika , Koyama Yo , Kitahara Tatsuro , Suzuki Satoshi , Mende Ulrike , Kubota Isao
Published: July 2021

Background:
It has been reported that the expression of a constitutively active mutant of the G protein αq subunit in the hearts of transgenic mice (Gαq-TG) induces cardiac hypertrophy and lethal heart failure. Thus, the Gαq protein-coupled receptor (GPCR) signaling pathway, which includes diacylglycerol (DAG) and protein kinase C (PKC), plays a critical role in the development of cardiac hypertrophy and heart failure. DAG kinase (DGK) catalyzes DAG and controls cellular DAG levels, and thus may act as a negative regulator of GPCR signaling. In this study, we tested the hypothesis that DGKζ rescues Gαq-TG mice from developing heart failure.

Methods and Results:
We generated double transgenic mice (Gαq/DGKζ-TG) with cardiac-specific overexpression of both DGKζ and constitutive active Gαq by crossing Gαq-TG mice with transgenic mice with cardiac-specific overexpression of DGKζ (DGKζ-TG), and the pathophysiological consequences were analyzed. DGKζ inhibited cardiac hypertrophy and progression to heart failure in Gαq-TG mice (Table

). DGKζ prevented dilatation of left ventricular dimension and reduction of left ventricular fractional shortening in Gαq-TG mice. Markedly increased left ventricular end-diastolic pressure in Gαq-TG mice was normalized in Gαq/DGKζ-TG mice. Increases in heart weight/body weight ratio and cardiomyocyte cross sectional area were attenuated in Gαq/DGKζ-TG mice. Translocation of PKC α and ε isoforms, activation of JNK and p38 MAPK induced by Gαq were attenuated by DGKζ. DGKζ reduced fibrotic changes and concomitant upregulation of fibrosis-related genes such as collagen type I and type III induced by Gαq. DGKζ improved the survival rate of Gαq-TG mice.

Conclusions:
These results demonstrate the first evidence that DGKζ prevents cardiac dysfunction and heart failure by activated Gαq without detectable adverse effects in
in vivo
hearts and suggest that DGKζ represents a novel therapeutic target for cardiac hypertrophy and heart failure.

Summary of the results
Abstract 562: Improvement In Left Ventricular Function Due To A Reduction Of Myocardial Fibrosis Following Myocardial Infarction Using A Purpose Designed Novel Anti Fibrotic Drug FT-011
Authors: Elsik Maros , Zhang Yuan , Wang Bing , Kompa Andrew , Gilbert Richard , Krum Henry , Kelly Darren
Published: July 2021

Introduction:
Following myocardial infarction (MI), pathological deposition of excess collagen adversely effects cardiac function. We evaluated the role of a novel anti-fibrotic drug FT-011 (Fibrotech, Australia) in preventing fibrosis and preserving ventricular function.

Methods
: To first test for anti-fibrotic effects, isolated rat neonatal cardiac fibroblasts were stimulated with angiotensin II (AII)(0.1 μmol/L) or TGF-ß(10ng/ml) and treated with FT-011 (30–200 μmol/L) alone or in combination with the angiotensin II antagonist irbesartan (Irb)(10
−5
-10
−7
mmol/l). Effects on collagen synthesis were assessed by
3
H-proline incorporation. Subsequently, ten week old Sprague Dawley rats underwent LAD artery ligation to induce MI (or sham procedure), then treatment for 4 weeks with FT-011(200 mg/kg/d) or vehicle, starting 1 week post surgery. Left ventricular (LV) function was assessed by echocardiography (day 2 and 35 post-surgery) and cardiac catheterization (day 35). Total collagen (TC) deposition (by picrosirius red staining) and collagen subtypes I and III (C-I, C-III, by immunohistochemistry) were assessed.

Results:
A dose dependent reduction of collagen synthesis with FT-011 in response to both AII or TGF-ß stimulation (p<0.01), and a further reduction with FT-011±Irb was observed in vitro. In vivo, MIs were of comparable size. Treatment with FT-011 significantly reduced TC, C-I and C-III (p<0.05) in the myocardium, resulting in improved LV systolic function, normalization of diastolic function parameters (Table

) and improvement in heart failure with a reduction in lung/body weight ratio with FT-011 (p<0.05).

Table:

35 day data [mean (SEM)].

Conclusion:
Post MI treatment with FT-011 improved heart function by reducing pathological remodeling of the ventricle. This appears to be occurring at least in part via a direct anti-fibrotic effect of the drug.
Abstract 567: Ivabradine Improved Cardiac Function, Fibrosis And Hyperexcitability In Rat Post-myocardial Infarction Severe Heart Failure.
Authors: Milliez Paul , Nehme Johnny , Robidel Estelle , Rodriguez Camille , Samuel JaneLise , Delcayre Claude
Published: July 2021

The selective sinus node I

f

current inhibitor Ivabradine (Iva) reduces heart rate (HR) without any other hemodynamic effects. We investigated in a rat model of post-MI severe heart failure, the effects of a chronic Iva administration on LV function, structure and electrical remodelling.

Methods
. MI was induced by coronary artery ligation in adult rats: echocardiography and Holter ECG were performed 2 months (m) after MI i.e. before randomization (n=12/group) into MI and MI+Iva (10 mg/kg/d) groups, and 3 m later. In ventricles, collagen was quantified after Sirius red staining of section and ACE and AT1-receptor mRNA expression (normalized to GAPDH) was assayed by RT-PCR.

Results
: 2m-post MI, all rats displayed severe decreased LVEF, and increased LVEDP compared to sham-operated (28%±3 vs 66%±5 and 32±2 vs 10±1 mmHg, respectively; p<0.05). In 5m-post-MI, LVEF and LVEDP were stabilized with Iva (31±1% and 24±2 mmHg), while they were worsened in MI groups (21±3% and 38±2mmHg). Blood pressure, body and heart weights were similar in MI and MI+Iva. Iva reduced HR (RR: 201±5 vs 179±3 ms in MI; p<0.05) and ventricular premature beats (514±152 vs 4717±1363/day for MI; p<0.05), and improved HR variability (SDRR: 5±1.5 vs 3.9± 0.6 for MI; p<0.05). There were no effects of Iva on duration (ms) of PR (45±3 vs 44±3), QRS (51±3 vs 46±3) and QT (106±7 vs 99±6, for MI and MI+Iva, respectively). The increased ventricular collagen in MI (4.0±0.1 vs 0.8±0.2 % in sham; p<0.05) was markedly reduced in MI+Iva (1.8±0.1%, p<0.0001 vs MI). The increases in ventricular gene expression of ACE and AT-1 receptor in MI rats (2.5 and 6 folds versus sham operated, respectively, p<0.05) were completely blunted by Iva treatment.

Conclusion:
Iva treatment of the post-MI heart failure:
1-
markedly reduced HR and ventricular excitability and without harmful effects on conduction;
2-
prevented worsening of LV dysfunction and remodelling, and
3-
induced a downregulation of cardiac RAAS transcripts. Thus, through its negative chronotropic effects, Iva allowed the maintenance of cardiac function below the threshold for a tissular RAAS stimulation even in severe post-MI cardiac failure. Such beneficial effects of Iva on cardiac remodelling open new perspectives for the treatment of severe heart failure
Abstract 579: De-differentiation of Somatic Fibroblasts by Mouse Embryonic Stem Cell-free Extracts: Multi-lineage Re-differentiation and Therapeutic Efficacy of Re-programmed Cells in Mouse Models of Hind limb and Acute Myocardial Ischemia
Authors: Rajasingh Johnson , Lambers Erin M , Bord Evelyn , Hamada Hiromichi , Goukassian Ilona , Qin Gangjian , Losordo Douglas W , Kishore Raj
Published: July 2021

Background: De-differentiation of adult somatic cells into multipotent progenitor cells might provide an attractive, oocyte-independent alternate source for therapeutic cloning to generate pluripotent, autologous stem cells for regenerative medicine. We tested the hypothesis that exposure of reversibly permeabilized NIH3T3 fibroblasts to mouse embryonic stem cell (mES) extracts may provide regulatory molecules required for epigenetic changes leading to re-programming of terminally differentiated somatic cells and that such reprogrammed cells may display multi-lineage differentiation.

Methods and Results: Streptolysin O-permeabilized NIH3T3 cells were exposed to mESC extracts in ATP-regenerating system. Plasma membrane was resealed by culturing in medium 2mM CaCl2. We report that upon exposure to mES extracts 3T3 cells undergo dedifferentiation as evident from morphological changes, induction of mES specific and the loss of lamin A/C, a specific marker of the soma. At epigenetic level, mES extract treatment induced de-methylation of Oct4 promoter and histone3 hyperacetylation at lysine 9 residue, as evident from restriction enzyme mapping, bisulphite genomic sequencing and chromatin immuno-precipitation experiments. Under defined culture conditions in vitro, reprogrammed 3T3 cells can be differentiated into neuronal, adipocyte, cardiomyocyte (CMC) and endothelial cells (EC). Moreover, transplantation of labeled reprogrammed 3T3 cells into mouse hind limb ischemia and acute myocardial infarction models resulted in the gain of physiological functions and showed evidence of acquiring the expression of muscle, EC and CMC specific proteins, in vivo. Moreover, when injected in SCID mice, reprogrammed cells formed teratomas.

Conclusion: Taken together our biochemical, molecular and functional data provide a novel and oocyte- independent approach for the generation of functional autologous stem like cells from terminally differentiated somatic cells which may potentially be of therapeutic application in regenerative medicine.
Abstract 580: The Production of a Bio-Engineered Endothelial Intima From Cultured Cells Using Whole Cardiac Cadaveric Extracellular Matrix.
Authors: Kren Stefan , Caron Gabriel , Taylor Doris A
Published: July 2021

Background; Bioengineered solutions to failing cardiac tissue have been difficult to achieve due partially to adverse interactions between circulating blood and the engineered surface. The aim of this study was to determine if by using naturally-derived ECM and cultured endothelial cells, a bioengineered whole-heart vascular intima could be generated. The matrix substrate for organ culture was produced by a perfusion-based detergent decelluarization of cadaveric rat heart. This process maintained ECM protein integrity as indicated by a glycosaminoglycan assay, with ~ equivalent amounts present relative to cadaveric rat heart. Its acellular nature was confirmed by loss of > 96% DNA (p = 0.001) compared to normal rat heart. In vitro infusion of aqueous dye or Mercox resin suggested a complete arterial tree, with structural preservation of vascular conduits. In vivo perfusability of the ECM was demonstrated by heterotopic transplantation with anticoagulation (n=4) into RNU rats for 7 days. Recellularization of the vascular tree was attempted by In Vitro Langendorff perfusion of 2 x 107 rat aortal endothelial cells (ECs) followed by a 7d incubation with escalating pulsatile flow in a 3D bioreactor. CellTracker Green assessed EC viability and permitted visualization of engrafted cells by fluorescent microscopy. Vessels of different diameters contained “patches” of confluent endothelium with complete circumferential lining of many of the matrix conduits. ECs lining both chamber walls and trabeculae were also observed. Nuclear staining showed 537.8 +/− 67.6 ECs / mm2 on endocardial surfaces, as well as 311.7 +/− 61.8 ECs / mm2 in vessels. To enhance the delivery of cells into the ventricular walls, a microcanulization of the brachiocephalic artery with sustained aortal perfusion was undertaken. This technique diverted more cells to the vasculature and more broadly distributed the cells in each area resulting in a lower cell density; 199.8 +/− 25.0 ECs / mm2 in vessels vs 125.8 +/− 43.4 ECs / mm2 on endocardial surfaces. In conclusion, these data suggest that by using detergent prepared acellular ECM of a whole organ, generation of a complete endothelial lining of vascular structures may be possible.
Abstract 587: Ablation of AngiotensinIV Receptor Attenuates Hypofibrinolysis and Inhibits Neointimal Formation in Murine Vascular Injury Model
Authors: Numaguchi Yasushi , Kubota Ryuji , Ishii Masakazu , Ma Xiuyang , Nagasaka Tetsuro , Mizutani Shigehiko , Murohara Toyoaki
Published: July 2021

Reduced fibrinolytic activity is associated with adverse cardiovascular events. Recently, it has been shown that angiotensin (Ang) IV receptor (AT4R) regulated fibrinolyasis via plasminogen activator inhibitor-1 (PAI-1) activation. However, it remains unknown how much this Ang IV-AT4R signaling affects the neointimal formation in the reparative process after vascular injury. To investigate if AngIV would inhibit fibrinolysis and promote neointimal formation, we evaluated the degree of proliferation of vascular smooth muscle cells and compared with the role of AT1R using AT1R and AT4R knockout mice (
AT1R

−
/
−

and
AT4R

−
/
−

, respectively).

[Methods and Results]
AT1R

−
/
−

and
AT4R

−
/
−

showed normal hemostasis patterns including bleeding time if compared with wild type mice (C57BL6/J; WT). To assess fibrinolysis in an acute thrombosis model, renal glomerular fibrin deposition was counted after lipopolysaccharide injection (0.2mg/kg, i.v., n=5). Fibrin deposition was reduced in
AT4R

−
/
−

as compared to WT and
AT1R

−
/
−

at 4 hrs (12±3% of total glomeruli, 27±4% and 25±4%, p=0.012 vs. WT). The levels of plasma active PAI-1 antigen measured by ELISA were also reduced in the order of
AT4R

−
/
−

,
AT1R

−
/
−

and WT (4.2±0.8*, 8.0±1 and 14±3 ng/mL, *p<0.001vs. WT). For a vascular injury model, neointimal formation was observed over 7 days after carotid artery ligation and cuff placement. In the carotid arteries of WT and
AT1R
−/−
, significant hyperplasia of adventitial tissues and neointimal formation were observed after vascular injury, while those of
AT4R
−/−
were minimally affected (n=6 each) in accordance with greater lumen diameters. In this model, genetic ablation of AT4R, but not AT1R, showed beneficial effects in vascular remodeling.

[Conclusions]
In murine vascular injury model, disruption of AngIV-AT4R signaling leads to accelerated fibrinolysis and decreased neointimal formation, suggesting that AT4R is a novel therapeutic target against cardiovascular disease.

Morphometric Analyses of the Sections from Murine Carotid Arteries
Abstract 588: Sequential Roles For Myosin-X In Bmp6-dependent Filopodial Extension, Migration, And Transport Of Bmp Receptors
Authors: Pi Xinchun , Ren Rongqin , Kelley Russell , Patterson Cam
Published: July 2021

Endothelial cell migration is a key step during angiogenesis and its dysregulation contributes to aberrant neovascularization. The bone morphogenetic proteins (BMP) are potent stimulators of cell migration and angiogenesis. However, the signaling pathways necessary for migration induced by BMPs are still poorly understood. Using microarray analyses, we found that Myosin X (Myo10), which is known to regulate filopodial integrity, is potently upregulated by BMP2 or BMP6. RT-PCR and western blotting analysis indicate more than 10-fold increase of Myo10 mRNA and protein levels. In endothelial cells, BMP6-induced Myo10 localizes in filopodia and filopodia number increases from 20±11 to 54±17 per cell as determined by scanning electron microscopy. This finding is consistent with our observation that increased filopodial extensions are induced by overexpression of Myo10. Likewise, cellular alignment induced by BMP6 is Myo10-dependent. Using Dunn chamber assay, MEC transfected with control siRNA and pretreated with BMP6 for 4 hours before application of the BMP6 gradient demonstrated increased alignment towards the BMP6 gradient (from 32±10 to 95±5 cells). However, cells transfected with Myo10 siRNA failed to align in the proper direction (from 28±7 to 27±9 cells). Boyden chamber assays indicate that the knockdown of Myo10 with specific small interfering RNA (siRNA) inhibits BMP6-directed cell migration (from 53±5 to 4±2 cells per field), surporting the model that Myo10 located in filopodia plays a key role in endothelial migration directed by BMP6. Surprisingly, we found that Myo10 and BMP6 receptor ALK6 co-localized in a BMP6-dependent fashion. ALK6 was translocated into filopodia after BMP6 stimulation, and both ALK6 and Myo10 possessed intrafilopodial motility. Additionally, Myo10 was required for BMP6-dependent Smad activation, indicating that-in addition to its role in filopodial assembly-Myo10 also participates in a requisite amplification loop for BMP signaling. Our data indicate that Myo10 plays a critical role in the guidance of endothelial migration towards BMP6 gradients via the regulation of filopodial function and amplification of BMP signals.