Chun Xue

Induction of Nitric Oxide Synthase in the Human Cardiac Allograft Is Associated With Contractile Dysfunction of the Left Ventricle

BACKGROUND The mechanisms underlying cardiac contractile dysfunction after transplantation remain poorly defined. Previous work has revealed that inducible nitric oxide synthase (iNOS) is expressed in the rat heterotopic cardiac allograft during rejection; resultant overproduction of nitric oxide (NO) might cause cardiac contractile dysfunction via the negative inotropic and cytotoxic actions of NO. In this investigation, we tested the hypothesis that induction of iNOS may occur and be associated with cardiac allograft contractile dysfunction in humans. METHODS AND RESULTS We prospectively studied 16 patients in the first year after cardiac transplantation at the time of serial surveillance endomyocardial biopsy. Clinical data, the results of biopsy histology, and echocardiographic and Doppler evaluation of left ventricular systolic and diastolic function were recorded. Total RNA was extracted from biopsy specimens, and mRNA for beta-actin, detected by reverse transcription-polymerase chain reaction (RT-PCR) using human specific primers, was used as a constitutive gene control; iNOS mRNA was similarly detected by RT-PCR using human specific primers. iNOS protein was detected in biopsy frozen sections by immunofluorescence. Myocardial cGMP was measured by radioimmunoassay, and serum nitrogen oxide levels (NOx = NO2 + NO3) were measured by chemiluminescence. iNOS mRNA was detected in allograft myocardium at some point in each patient and in 59 of 123 biopsies (48%) overall. In individual patients, iNOS mRNA expression was episodic and time dependent; the frequency of expression was highest during the first 180 days after transplant (P = .0006). iNOS protein associated with iNOS mRNA was detected by immunofluorescence in cardiac myocytes. iNOS mRNA expression was not related to the ISHLT histological grade of rejection or to serum levels of NOx but was associated with increased levels of myocardial cGMP (P = .01) and with both systolic (P = .024) and diastolic (P = .006) left ventricular contractile dysfunction measured by echocardiography and Doppler. CONCLUSIONS These data support a relation between iNOS mRNA expression and contractile dysfunction in the human cardiac allograft.

Upregulation of Nitric Oxide Synthase Correlates Temporally With Onset of Pulmonary Vascular Remodeling in the Hypoxic Rat

Alterations in nitric oxide signaling have been hypothesized to have an etiologic role in the development of hypoxic pulmonary hypertension. However, changes in the expression of nitric oxide synthase (NOS) in hypoxic lungs remains controversial. In this study, we used (1) Northern and Western analyses to measure NOS mRNA and protein expressions, (2) lung histology together with measurements of lung and heart weights to monitor pulmonary vascular remodeling, and (3) immunohistochemistry to localize NOS proteins. The data demonstrated that endothelial NOS mRNA and protein were upregulated over 1 to 7 days of hypoxia that temporally correlated with and preceded the vascular remodeling that occurred in the course of the development of hypoxic pulmonary hypertension. Hypoxia also induced brain NOS in bronchial epithelium and inducible NOS in vascular smooth muscle but did not affect inducible NOS expression in macrophages or basal guanylyl cyclase activity in the lung. These findings showed that upregulation of endothelial NOS was tightly correlated with the vascular remodeling induced by hypoxia, suggesting a role for nitric oxide in the development of pulmonary hypertension.

Localization of endothelial NOS at the basal microtubule membrane in ciliated epithelium of rat lung.

Nitric oxide (NO), an important cell messenger molecule, is formed endogenously in the lung airway. Three individual genes of NO synthase (NOS), which represent brain NOS (bNOS), inducible NOS (iNOS), and endothelial NOS (eNOS), have been reported in the cultured lung epithelium. Although studies in vivo showed that bNOS and iNOS were expressed and localized in the cytoplasm of bronchial epithelium, the expression and localization of eNOS remains to be determined. Therefore, we employed an eNOS monoclonal antibody whose immunospecificity was tested by both Western blot and preadsorption immunohistochemistry to immunostain rat lungs from fetus to adult. The results showed that eNOS immunoreactivity began to appear in the lung epithelium within 2 hr after birth. Six hours later (8 hr after birth), the NOS immunoreaction was concentrated near the surface of the ciliated epithelial cells. This staining pattern appeared in lungs at Day 1, Week 1, Week 2, and in adult rats. By electron microscopy, eNOS immunoreactivity was confirmed within ciliated epithelium and was shown to be associated with the basal microtubule membrane of the cilia. Nonciliated cells were not stained. Type II epithelial cells also contain eNOS immunoreactivity, which is primarily associated with rough endoplasmic reticulum, and free ribosomes. However, macrophages in the lungs lacked eNOS immunoreactivity. This study demonstrated that eNOS was postnatally expressed in rat bronchial ciliated epithelium. The localization of eNOS at the basal membrane of ciliary microtubules suggests that eNOS may be involved in the function of epithelial cilia, consistent with previous physiological studies.

Developmental expression and localization of the catalytic subunit of protein phosphatase 2A in rat lung

Protein phosphatase type‐2A (PP2A) is a highly conserved serine/threonine phosphatase known to play a key role in cell proliferation and differentiation in vitro, but the role of PP2A in mammalian embryogenesis remains unexplored. No particular information exists as to the tissue or cell specific expression of PP2A or the relevance of PP2A expression to mammalian development in vivo. To examine expression of PP2A during mammalian lung development, we studied fetal rats from day 14 of gestation (the lung bud is formed on day 12 of gestation) to parturition. Western analysis with a specific PP2A catalytic subunit antibody identified a single 36 kDa protein, with protein levels two‐fold higher in the 17 and 19 day embryonic lung as compared to the adult. With in situ hybridization and immunohistochemistry, both mRNA and protein for PP2A were localized equally to the epithelial lining of the embryonic lung airway and the surrounding mesenchyme in the 14 day embryonic lung. With maturation of the lung, PP2A becomes highly expressed in respiratory epithelium. The highest level of expression was in the earliest developing airways with columnar epithelium (the pseudoglandular stage, 15–18 days of gestation). There was a decrease in expression with the transformation to cuboidal epithelium by day 20 of gestation. This was most noticeable in the developing bronchial epithelium of the 19 and 20 day gestation lungs where only an occasional cell continues to express PP2A. Mesenchymal hybridization was most obvious in early endothelial cells of forming vascular channels at 17–19 days of gestation. PP2A respiratory epithelial expression mimicked the centrifugal development of the respiratory tree where the highest expression was in the peripheral columnar epithelium (15–18 days gestation) with only an occasional central bronchiolar cell continuing to express PP2A at 19 and 20 days gestation. Endothelial hybridization decreased with muscularization of large pulmonary arteries with low levels of expression detected in bronchial or vascular smooth muscle. In the newborn lung PP2A expression was decreased, but detectable in alveolar epithelium and vascular endothelium. In summary; 1) PP2A mRNA and protein exhibit cell specific expression during rat lung development; 2) PP2A is highly expressed in the respiratory epithelium of the fetal rat lung and is temporally related to the maturation of the bronchial epithelium; 3) and the PP2A subunit is highly expressed in early vascular endothelium, but not smooth muscle of the rat lung. Dev. Dyn. 1998;211:1–10.

Expression and Mapping of Protein Phosphatase 2A α in the Developing Rat Heart

Protein phosphatase 2A (PP2A) is a second messenger involved in cell cycle regulation, cell transformation, and cell fate determination. We previously identified a gene encoding the α catalytic subunit of PP2A in the embryonic rat heart, but its role in cardiac morphogenesis was unknown. In this study, we examined the developmental expression of PP2Aα mRNA and protein in the heart using Northern and Western analysis, in situ hybridization, and immumohistochemical staining. We found two major PP2Aα transcripts in the rat heart (1.8 and 2.4 kb), at all stages examined. By Western blotting, PP2Aα protein levels were twice as high in the embryonic rat heart compared with the adult. In situ hybridization on embryonic d 12 showed that PP2Aα mRNA was expressed in the heart, brain, tail, and limb buds. Cardiac PP2Aα expression was regionally restricted to the atrium, ventricle, and truncus arteriosus. PP2Aα expression did not extend into the more distal aortic sac or aortic arches. Cross-sectional hybridization revealed PP2Aα mRNA in the epicardium, pericardium, and endothelium. Later in development, mRNA expression was also detected at high levels in mesenchymal cells populating the endocardial cushions and in myocardium. At term, PP2Aα was highly expressed in endothelial cells, but not in the underlying myocardium. PP2Aα protein had a similar distribution at all embryonic stages examined. These results show that there is transcriptional, translational, and cell-specific regulation of PP2Aα during heart development. We speculate on the role of PP2Aα-mediated dephosphorylation in cardiac morphogenesis and suggest a number of possible molecular targets.

Increased Pulmonary Blood Flow as a Regulator of Pulmonary Vascular Remodeling: Role of eNOS ♦ 105

To better understand the mechanisms whereby pulmonary blood flow regulates pulmonary vascular bed growth in congenital heart lesions, 42 day old Sprague-Dawley rats (n = 24) had creation of an aortocaval shunt to increase pulmonary blood flow for six weeks. The shunt resulted in a significant increase in heart (2.89 ± 0.4 vs 6.1 ± 0.9, P ≤ 0.05) and lung (1.93 ± 0.5 vs 4.6 ± 1.4, P ≤ 0.05) to body weight (sham vs shunt, mean ± standard deviation) without significantly altering pulmonary (16 ± 3 vs 19 ± 4, NS) or carotid (125 ± 5 vs 130 ± 6, NS) blood pressures (mean mm Hg ± standard deviation). Histologic examination of the lungs revealed significant thickening of the pulmonary arterial medial wall relative to the vessel diameter (35% ± 7.2 vs 57% ± 11.8, P ≤ 0.05, sham vs shunt, mean ± standard deviation) and increased muscularization of very small arteries (≤ 80μM) as evidenced by α-actin smooth muscle staining. Proliferating cell nuclear antigen staining and bromo-deoxyuridine labeling of proliferating cells demonstrated that the thickened arterial wall was a result of medial hypertrophy and not proliferation. To determine the role of nitric oxide (NO) in the regulation of the arteriolar hypertrophy or the affect of hypertrophy on NO expression in the pulmonary vasculature, endothelial NO synthase (eNOS) gene and protein expression were determined in lungs from shunt and sham operated animals. Western and northern analysis demonstrated that eNOS protein and mRNA levels were not altered in the shunt lungs. In addition NAPDH diaphorase staining was negative for increased NOS in the hypertrophied pulmonary vasculature of the shunt animals. Therefore increased pulmonary flow without increased pressure resulted in pulmonary artery medial hypertrophy and not hyperplasia. We speculate that the pulmonary vascular remodeling resulting from increased pulmonary blood flow alone is not mediated by eNOS.