Yoshihide Mitani

Prolonged Administration of l-Arginine Ameliorates Chronic Pulmonary Hypertension and Pulmonary Vascular Remodeling in Rats

BACKGROUND Endothelium-dependent nitric oxide-mediated vasodilation is impaired in rats with pulmonary hypertension (PH) induced by chronic hypoxia or by monocrotaline injection. We therefore investigated whether the prolonged administration of the nitric oxide precursor L-arginine would alleviate PH in both rat models. METHODS AND RESULTS Fifty-nine rats were exposed to hypobaric hypoxia (380 mm Hg, 10 days) or room air and injected intraperitoneally with L-arginine (500 mg/kg), D-arginine (500 mg/kg), or saline once daily from day -3 to day 10. An additional 38 rats injected subcutaneously with monocrotaline (60 mg/kg) or saline were treated similarly with L-arginine or saline from day -3 to day 17. At the end of the experiment, awake mean pulmonary arterial pressure was determined. The heart was dissected to weigh the right ventricle, and the lungs were obtained for vascular morphometric analysis. Hypoxic rats developed PH (30.8+/-0.7 versus 19.2+/-0.4 mm Hg in controls; P<.05) and right ventricular hypertrophy. Their pulmonary arterial wall thickness and the proportion of muscular arteries in the peripheral arteries increased. L-Arginine but not D-arginine reduced PH (24.8+/-0.7 mm Hg; P<.05), right ventricular hypertrophy, and pulmonary vascular disease. Monocrotaline rats developed PH (34.9+/-2.1 versus 18.8+/-1.2 mm Hg in controls; P<.05), right ventricular hypertrophy, and pulmonary vascular disease. Again, L-arginine reduced PH (24.3+/-1.7 mm Hg; P<.05), right ventricular hypertrophy, and pulmonary vascular disease. CONCLUSIONS We conclude that L-arginine ameliorated the changes associated with PH in rats, perhaps by modifying the endogenous nitric oxide production.

Elevated Levels of High-Sensitivity C-Reactive Protein and Serum Amyloid-A Late After Kawasaki Disease: Association Between Inflammation and Late Coronary Sequelae in Kawasaki Disease

Background—Coronary sequelae that persist after Kawasaki disease (KD) have been associated with obstructive changes of the lesions and coronary vascular events in adolescents and young adults. However, little is known about the association between sequelae late after KD and inflammatory markers, which are potential mediators and markers for atherogenesis. Methods and Results—Cross-sectional study was performed to test the hypothesis that coronary sequelae are associated with elevated levels of inflammatory markers in patients late after KD (mean time interval after the onset, 10 years, 10 months). Levels of high-sensitivity C-reactive protein (CRP), serum amyloid-A (SAA), interleukin-6, and soluble intercellular adhesion molecule-1 were measured in the 4 groups (n=80): the referent group (n=15) and KD subgroups with normal coronary arteries from the onset (n=27); with regressed aneurysms (n=18); and with coronary artery lesions, such as persistent aneurysms, stenosis, and occlusion (n=20). CRP levels were significantly elevated in a KD subgroup with coronary artery lesions compared with the referent or other KD subgroups, as analyzed by ANOVA and ANCOVA after adjustment for a confounding factor body mass index. Levels of CRP, SAA, and interleukin-6 were positively correlated. Stepwise regression and logistic regression analyses support the association between the persistence of coronary artery lesions and the levels of CRP and SAA. Conclusions—Results demonstrate that the persistence of coronary lesions late after KD was independently associated with levels of CRP and SAA, suggesting that inflammation may be a novel functional aspect of coronary artery diseases late after KD.

Nitric oxide reduces vascular smooth muscle cell elastase activity through cGMP-mediated suppression of ERK phosphorylation and AML1B nuclear partitioning

Nitric oxide (NO) reduces the severity of pulmonary vascular disease in rats as do elastase inhibitors. We therefore hypothesized that NO inhibits elastase by suppressing mitogen‐activated protein kinases that trans‐activate AML1B, a transcription factor for elastase. We used cultured pulmonary artery smooth muscle cells in which serum‐treated elastin (STE) induces a > threefold increase in elastase activity as evaluated by solubilization of [3H]‐elastin. NO donors (SNAP and DETA NONO‐ate) inhibited elastase in a dose‐dependent manner as did a cGMP mimetic (8‐pCPT‐cGMP). SNAP inhibition of elastase was reversed by coadministration of a cGMP‐PKG inhibitor (Rp‐8‐pCPT‐cGMP). The STE‐induced increase in phospho‐ERK was suppressed by NO donors and the cGMP mimetic, and reversed by cGMP‐PKG inhibitor, as was expression of AML1B and DNA binding in nuclear extracts. A concomitant increase in p38 phosphorylation was also inhibited by SNAP, but whereas MEK inhibitor (PD98059) suppressed elastase and AML1B‐DNA binding, a p38 inhibitor (SB202190) did not. Our study uniquely links NO with inhibition of elastase‐dependent matrix remodeling in vascular disease by suggesting a cGMP‐PKG‐related mechanism suppressing ERK‐mediated partitioning of AML1B in nuclear extracts.—Mitani, Y., Zaidi, S. H. E., Dufourcq, P., Thompson, K., Rabinovitch, M. Nitric oxide reduces vascular smooth muscle cell elastase activity through cGMP‐mediated suppression of ERK phosphorylation and AML1B nuclear partitioning. FASEB J. 14, 805–814 (2000)

Vascular smooth muscle cell phenotypes in primary pulmonary hypertension.

Primary pulmonary hypertension (PPH) is associated with specific structural alterations, including cellular intimal thickening, intimal fibrosis, and plexiform lesions. To determine the phenotypes of smooth muscle cells (SMCs) in such lesions, the authors conducted an immunohistochemical analysis of lung tissues from two patients with PPH, using two antimuscle actin antibodies, HHF35 and CGA7, and two anti-SMC myosin heavy chain markers, anti-SM1 and anti-SM2 antibodies and related antibodies. Cells that stained positive (+) with HHF35, CGA7, anti-SM1, and anti-SM2 were considered to be SMCs of a mature state. Conversely, those that stained positive with HHF35 and anti-SM1, but weakly positive (+/-) or negative (-) with CGA7 and anti-SM2, were considered to be SMCs exhibiting an immature state. Cellular intimal thickening was composed of SMCs of an immature phenotype (HHF35+, CGA7+/-, SM1+, SM2+/-), accompanied by the expression of fibronectin and the presence of macrophages; intimal fibrosis contained mature SMCs (HHF35+, CGA7+, SM1+, SM2+); and plexiform lesion consisted of proliferative endothelial cells (von Willebrand factor-positive cells, proliferating cell nuclear antigen-positive cells) and underlying immature SMCs (HHF35+, CGA7-, SM1+, SM2-) associated with fibronectin expression and macrophage infiltration. These findings suggest that smooth muscle cells with specific phenotypes may contribute to the development of specific vascular lesions in primary pulmonary hypertension.

In Vivo Plaque Composition and Morphology in Coronary Artery Lesions in Adolescents and Young Adults Long After Kawasaki Disease. A Virtual Histology–Intravascular Ultrasound Study

Background— Coronary artery lesions (CALs) late after Kawasaki disease were characterized by endothelial dysfunction and low-grade inflammation, surrogate markers for atherosclerosis. We tested the hypothesis that CALs in patients long after Kawasaki disease are accompanied by atheroma-like features, as assessed by virtual histology–intravascular ultrasound, a new method to assess coronary plaque composition and morphology in vivo. Methods and Results— Virtual histology–intravascular ultrasound was performed in 13 Japanese Kawasaki disease patients (median age, 18.3 years; interquartile range, 16.9 to 23.3 years) an interval after Kawasaki disease (median, 15.9 years; interquartile range, 14.3 to 21.9 years). We investigated 6 sites with localized stenosis, 15 sites with an aneurysm, 29 sites with a regressed aneurysm, and 50 sites with a normal coronary segment. Plaque components were categorized into 4 parts: fibrous, fibrofatty, necrotic core, and dense calcium areas. Qualitatively, the normal segment had no or trivial intravascular ultrasound–visible plaque area, whereas the CAL exhibited a heterogeneous plaque area with the 4 components in different amounts and proportions. Quantitatively, a combined group of CALs had a higher absolute value of fibrous, dense calcium, and necrotic core areas than the normal segment. In further analyses of 3 subtypes of CALs, localized stenosis, an advanced lesion, exhibited higher absolute and relative values of dense calcium and necrotic core areas and a lower relative value of the fibrous area than regressed and persistent aneurysms. Conclusion— The present limited but initial virtual histology–intravascular ultrasound findings give new insight into the potential role of atherogenesis in the evolution of CALs in adolescents and young adults long after Kawasaki disease and therefore warrant further investigation.

Ultrafiltration of the priming blood before cardiopulmonary bypass attenuates inflammatory response and improves postoperative clinical course in pediatric patients.

ABSTRACT The priming solution using in cardiopulmonary bypass (CPB) for infants undergoing cardiac surgery includes considerable amounts of stored blood. Our objective was to test the hypothesis that ultrafiltration (UF) of the stored blood before CPB reduces the unfavorable effects of stored blood and the production of inflammatory cytokines. Fifty pediatric patients with congenital heart defects took part in this study. The patients were randomly divided into two groups: the UF (27 pediatric patients who received UF) and control (23 pediatric patients who did not receive UF) groups. UF was performed with a polysulphone ultrafiltrator before CPB. Blood samples were collected immediately before, during, and 1 h after CPB. The levels of cytokines (TNF‐&agr;, IL‐1&bgr;, IL‐8), NH3, and bradykinin were determined. The serum concentrations of NH3 and bradykinin decreased significantly after UF. Compared with the control group, the UF group had significantly lower cytokine production. Water balance in UF group was better than that of control group. The UF group received significantly less inotropic support and shorter duration of ventilator support and ICU stay. We conclude that removal of bradykinin and a decrease in the levels of NH3, potassium, and pH play a significant role in reducing water retention and postoperative lung injury. UF of the blood used to prime the circuit for CPB is a safe and efficient method for use in open heart surgery in small pediatric patients.

The ex utero intrapartum treatment (EXIT) procedure in giant fetal neck masses.

Giant fetal neck masses can cause airway obstructions with potential poor fetal prognosis after delivery. The relationship between the fetal neck mass and airway structure can be defined prenatally with ultrasound and magnetic resonance imaging (MRI). The ex utero intrapartum treatment (EXIT) procedure is an available technique to obtain a fetal airway while feto-maternal circulation is preserved. We present a case in which prenatally a giant fetal neck mass was diagnosed on ultrasound and MRI, and a successful EXIT procedure could be performed.

Correlation of inhaled nitric-oxide induced reduction of pulmonary artery pressure and vascular changes

The purpose of the present study was to determine the relationship between hypertensive pulmonary vascular remodelling and the changes in mean pulmonary artery pressure (mPAP) during low-dose nitric oxide (NO) inhalation. Rats were exposed to chronic hypobaric hypoxia (air at 50.5 kPa (380 mmHg), 10% oxygen, for 5–29 days) to induce chronic pulmonary hypertension (PH) with pulmonary vascular structural changes. After the chronic hypoxic exposure, the rats had an indwelling pulmonary artery catheter inserted and changes in mPAP with NO were correlated to morphometrical analysis of pulmonary vascular changes. All concentrations of inhaled NO (0.1–2.0 parts per million) reduced mPAP with a similar per cent reduction from baseline mPAP in PH rats, while no changes were observed in control rats. During NO inhalation in PH rats, the absolute value of the decrease in mPAP, but not per cent reduction in mPAP, significantly correlated with baseline mPAP, the percentage of muscularised arteries at the alveolar wall level and at the alveolar duct level, and the per cent medial wall thickness of muscularised arteries. In the chronic hypoxic pulmonary hypertension model, the severity of pulmonary vascular remodelling did not alter the reactivity of the pulmonary arteries to nitric oxide and might, in part, determine the magnitude of nitric-oxide induced absolute reduction in mean pulmonary artery pressure.

Genetic mutation analysis in Japanese patients with non-syndromic congenital heart disease.

Congenital heart disease (CHD) is the most common birth defect occurring in humans and some transcriptional factors have been identified as causative. However, additional mutation analysis of these genes is necessary to develop effective diagnostic and medical treatment methods. We conducted sequence analysis of the coding regions of NKX2.5, GATA4, TBX1, TBX5, TBX20, CFC1 and ZIC3 in 111 Japanese patients with non-syndromic CHD and 9 of their relatives. All patient samples were also analyzed by multiplex ligation-dependent probe amplification using probes involved in chromosome deletion related to CHD. Five novel variations of TBX5, GATA4 and TBX20 were detected in 6 of the patients, whereas none were found in 200 controls. The TBX5 variation p.Pro108Thr, located in the T-box domain, was identified in a patient with tricuspid atresia, an exon–intron boundary variation of GATA4 (IVS4+5G>A) was detected in a Tetralogy of Fallot patient and an 8p23 microdeletion was detected in one patient with atrioventricular septal defect and psychomotor delay. A total of seven non-synonymous polymorphisms were found in the patients and controls. Accumulation of novel variations of genes involving the cardiac development may be required for better understanding of CHD.

Pediatric Cardiac Remodeling After Cardiac Resynchronization Therapy

A 1.8-year-old male required a conventional DDD pacemaker for an atrioventricular block after congenital heart surgery. Five years later, heart failure due to left ventricular (LV) dyssynchrony progressed and we performed cardiac resynchronization therapy (CRT). Long-term echocardiographic follow-up showed that LV shortening fraction had improved within the first year after CRT, and LV end diastolic dimension had decreased after the first year. During LV remodeling (1–24 months after CRT), the QRS duration shortened without a change in the JT and Tpeak-end interval. The New York Heart Association class improved from III to I during the 2.3-year follow-up.