Hydrogen gas attenuates embryonic gene expression and prevents left ventricular remodeling induced by intermittent hypoxia in cardiomyopathic hamsters.

The prevalence of sleep apnea is very high in patients with heart failure (HF). The aims of this study were to investigate the influence of intermittent hypoxia (IH) on the failing heart and to evaluate the antioxidant effect of hydrogen gas. Normal male Syrian hamsters (n = 22) and cardiomyopathic (CM) hamsters (n = 33) were exposed to IH (repeated cycles of 1.5 min of 5% oxygen and 5 min of 21% oxygen for 8 h during the daytime) or normoxia for 14 days. Hydrogen gas (3.05 vol/100 vol) was inhaled by some CM hamsters during hypoxia. IH increased the ratio of early diastolic mitral inflow velocity to mitral annulus velocity (E/e', 21.8 vs. 16.9) but did not affect the LV ejection fraction (EF) in normal Syrian hamsters. However, IH increased E/e' (29.4 vs. 21.5) and significantly decreased the EF (37.2 vs. 47.2%) in CM hamsters. IH also increased the cardiomyocyte cross-sectional area (672 vs. 443 µm(2)) and interstitial fibrosis (29.9 vs. 9.6%), along with elevation of oxidative stress and superoxide production in the left ventricular (LV) myocardium. Furthermore, IH significantly increased the expression of brain natriuretic peptide, ß-myosin heavy chain, c-fos, and c-jun mRNA in CM hamsters. Hydrogen gas inhalation significantly decreased both oxidative stress and embryonic gene expression, thus preserving cardiac function in CM hamsters. In conclusion, IH accelerated LV remodeling in CM hamsters, at least partly by increasing oxidative stress in the failing heart. These findings might explain the poor prognosis of patients with HF and sleep apnea.

Identification of a novel aldose reductase-like gene upregulated in the failing heart of cardiomyopathic hamster.

Cardiomyopathy (CM) is degenerative disease of myocardium which leads to severe cardiac failure. Although many causative genes for CM have been identified, molecular pathogenesis of CM is not fully understood. In this study, we searched for a novel pathway recruited in the development of CM by using BIO14.6 hamster as an animal model for human CM. We screened upregulated genes in the left ventricle by differential display technique and searched for a gene which had never been linked to CM. We identified a novel gene overexpressed in BIO14.6 hamster ventricles, which was considered to be a new member of aldo-keto reductase (AKR) superfamily. The cloned cDNA encoded a 316 amino acid polypeptide with calculated molecular mass of 35,804, which showed high amino acid sequence similarities to aldose reductase and its relative: 69.6% to AKR1B1 (human aldose reductase), 68.4% to AKR1B3 (mouse aldose reductase), and 85.8% to AKR1B7 (mouse vas deferens protein). The upregulation of this aldose reductase-like gene in BIO14.6 hamster ventricles (6.3 ± 0.8-fold) seemed to be influenced by the overexpression of activator protein-1 present there. With the fact that AKR1B1, AKR1B3, and AKR1B7 have synthetic activities of prostaglandin F2α, the aldose reductase-like protein could cause cardiac hypertrophy through production of prostaglandin F2α whose precursor and receptor were abundant in BIO14.6 hamster ventricles. Aldose reductase and its related proteins would give a new clue to dissect the pathogenesis of CM including oxidative stress and cardiac hypertrophy, and to develop a new drug for the treatment of CM.

Acid regulation of NaDC-1 requires a functional endothelin B receptor.

Metabolically generated acid is the major physiological stimulus for increasing proximal tubule citrate reabsorption, which leads to a decrease in citrate excretion. The activity of the Na-citrate cotransporter, NaDC-1, is increased in vivo by acid ingestion and in vitro by an acidic pH medium. In opossum kidney cells the acid stimulatory effect and the ability of endothelin-1 (ET-1) to stimulate NaDC-1 activity are both blocked by the endothelin B (ET(B)) receptor antagonist, BQ788. Acid feeding had no effect on brush border membrane NaDC-1 activity in mice in which ET(B) receptor expression was knocked out, whereas a stimulatory effect was found in wild-type mice. Using ET(A)/ET(B) chimeric and ET(B) C-terminal tail truncated constructs, ET-1 stimulation of NaDC-1 required a receptor C-terminal tail from either ET(A) or ET(B). The ET-1 effect was greatest when either the ET(B) transmembrane domain and C-terminal tail were present or the ET(B) C-terminal tail was linked to the ET(A) transmembrane domain. This effect was smaller when the ET(B) transmembrane domain was linked to the ET(A) C-terminal tail. Thus, the acid-activated pathway mediating stimulation of NaDC-1 activity requires a functional ET(B) receptor in vivo and in vitro, as does acid stimulation of NHE3 activity. Since increased NaDC-1 and NHE3 activities constitute part of the proximal tubule adaptation to an acid load, these studies indicate that there are similarities in the signaling pathway mediating these responses.

Expression and function of ephrin-B1 and its cognate receptor EphB2 in human atherosclerosis: from an aspect of chemotaxis.

Although several cytokines and chemokines have been demonstrated to play pivotal roles in the pathophysiological conditions of atherosclerosis, few findings exist regarding the expression and function of cytokine-modulating molecules such as ephrin-Bs and their cognate receptors, EphBs, in human atherosclerosis. Therefore, in the present study, we screened novel genes modulating atherogenesis by cDNA array and quantitatively determined them by real-time RT (reverse transcription)-PCR in human carotid atherosclerotic plaques. Ephrin-B1 and EphB2, key regulators of embryogenesis, were significantly up-regulated in plaques compared with those in adjacent control tissues [ephrin-B1, 0.638+/-0.106 compared with 0.831+/-0.152, or 130% (P<0.05); EphB2, 1.296+/-0.281 compared with 2.233+/-0.506, or 172% (P<0.05)]. Immunohistological analysis demonstrated that both ephrin-B1 and EphB2 were expressed in macrophages and T-lymphocytes in plaques as well as in monocytes, T-lymphocytes and arterial endothelial cells isolated from healthy adults. Interestingly, the extracellular domains of ephrin-B1 and EphB2, the expression of which were both enhanced in stimulated THP-1 cells, significantly inhibited spontaneous (22.5 and 27.6% respectively; P<0.01) and MCP-1 (monocyte chemoattractant protein-1)-dependent (29.7 and 22.6% respectively; P<0.01) migration of monocytes. In conclusion, these results demonstrate that ephrin-B1 and EphB2 are overexpressed in atherosclerotic tissue and might locally regulate cell migration, possibly through modulating cytokine-related chemotaxic activity; however, the functional role of these molecules in atherogenesis should be investigated further.

Altered expression balance of matrix metalloproteinases and their inhibitors in human carotid plaque disruption: results of quantitative tissue analysis using real-time RT-PCR method.

BACKGROUND: The balance between degradation and synthesis of extracellular matrix determines its content in atherosclerotic tissue. To examine the role of expression balance of matrix metalloproteinases (MMPs) to their inhibitors, tissue inhibitors of metalloproteinases (TIMPs) and tissue factor pathway inhibitor-2 (TFPI-2) in the development and disruption of atherosclerotic plaque, these gene expressions in human carotid plaque were quantitatively determined by real-time reverse transcription (RT)-polymerase chain reaction (PCR) method. METHODS: Total RNA for cDNA synthesis was extracted from tissues in 24 patients with carotid endarterectomy. The amounts of cDNAs for MMP-1, -2, -3 and -9, TFPI-2 and TIMP-1, -2 and -3 were determined by real-time RT-PCR method, and normalized with glutaraldehyde 3-dehydrogenase. RESULTS: In plaques, the expression MMP-1 (1.53+/-0.25, mean+/-S.E.M.), MMP-3 (1.99+/-0.59) and MMP-9 (2.00+/-0.51) was augmented compared to those in the adjacent control regions (0.60+/-0.16, 0.46+/-0.18 and 0.58+/-0.21, respectively, p<0.05). The expression of TFPI-2 was lower in plaques (0.32+/-0.08) than in controls (0.94+/-0.23, p<0.01). Although the expression of TIMP-1 was higher in plaques (1.28+/-0.23) than in controls (0.81+/-0.10, p<0.05), the indices of MMP-1/TIMP-1, MMP-3/TIMP-3 and MMP-9/TIMP-1 were still significantly higher in plaques. Interestingly, MMP-9 and the resulting MMP-9/TIMP-1 balance in plaques with disruption were significantly higher (3.36+/-1.52 and 1.66+/-0.12, n=11) than those in non-disrupted plaques (1.11+/-0.52 and 0.76+/-0.12, n=13, p<0.05). CONCLUSION: With the decreased expression of TFPI-2, upregulation of MMPs in atherosclerotic plaque was disproportional to that of TIMPs, suggesting that imbalanced degradation and synthesis of extracellular matrix persists in advanced lesions, particularly in plaques with disruption.

Application of real-time RT-PCR to quantifying gene expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human abdominal aortic aneurysm.

BACKGROUND: The relative expression levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), key regulators in remodeling of extracellular matrix, are considered to play a pivotal role in the development of abdominal aortic aneurysm (AAA). However, few data exist regarding quantitative assessment of their expression in clinical settings. METHODS: In 22 patients with AAA who underwent graft replacement, tissue samples of the AAA and non-dilated aorta were obtained. Using a real-time RT-PCR method that enabled quantitative measurement of mRNA levels in small tissue samples, we determined gene expression levels of MMPs and TIMPs relative to that of glutaraldehyde 3-phosphate dehydrogenase in each sample. RESULTS: The expression levels of the MMP-1 and -3 genes were significantly augmented in AAA compared with non-dilated regions (4.48 +/- 2.01 versus 0.26 +/- 0.12, P < 0.01 and 1.89 +/- 1.00 versus 5.01 +/- 0.97, P < 0.05, respectively). Although genes for TIMP-1, -2 and -3 tended to be upregulated in AAA, relative expression levels of MMP-1 to TIMP-1, MMP-1 to TIMP-2, MMP-1 to TIMP-3, and MMP-3 to TIMP-2 were still higher in AAA than in non-dilated regions (1.12 +/- 0.63 versus 0.10 +/- 0.03, 4.13 +/- 1.12 versus 0.43 +/- 0.11, 1.61 +/- 0.59 versus 0.14 +/- 0.03, and 7.81 +/- 1.60 versus 2.56 +/- 0.76, respectively, P < 0.05). CONCLUSION: These results demonstrate that the present real-time RT-PCR method is reliable for the determination of mRNA levels in small samples of vascular tissue and that disproportional expression of both MMP-1 and MMP-3 relative to TIMPs relates pathologically to the evolution of AAA.

Molecular pathogenesis of severe cardiomyopathy in the TO-2 hamster.

BACKGROUND: Cardiomyopathy (CM) is a life-threatening disease with progressive degeneration of cardiac muscle. From a representative animal model of CM, the BIO14.6 hamster, arose the TO-2 strain manifesting a severe, dilated form of CM. Previous studies demonstrated that both strains have an identical genomic deletion disrupting the delta-sarcoglycan gene to cause CM. OBJECTIVE: To elucidate an additional pathogenesis for cardiac dilation in the TO-2 hamster. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was used to assess the expression levels of genes for cardiac hypertrophy, such as beta-myosin heavy chain and preproendothelin-1. The involvement of apoptosis in CM was tested using terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labelling (TUNEL) and DNA ladder assays. The progression of cardiac degeneration was visualized using specific histological staining techniques. Echocardiography was performed to estimate the wall thickness and the movement of a living hamster heart. RESULTS: RT-PCR showed the expression of the genes involved in cardiac hypertrophy to be higher in TO-2 hamsters than in BIO14.6 hamsters, contary to what the thin myocardium may suggest. DNA ladder and TUNEL assays showed no significant difference in apoptosis in the hearts of either strain. In contrast, the infiltrate of inflammatory cells was prominent in the myocardium of TO-2 hamsters. In addition to dilation of the cardiac chamber, echocardiography also revealed disharmonized contraction in TO-2 hearts without apparent arrhythmias. CONCLUSIONS: Impairment of compensatory cardiac hypertrophy or involvement of apoptosis is less likely to be an additional pathogenesis in the TO-2 hamster. The present data suggest that augmented necrosis is the principal cause of severe CM in the TO-2 hamster. Further analysis of the molecular pathogenesis of TO-2 would help to disclose the final common pathways for the manifestation of CM.

Expression and Function of Ephrin-B1 and Its Cognate Receptor EphB2 in Human Abdominal Aortic Aneurysm.

We examined the expression of ephrin-B1 and its cognate receptor EphB2, key regulators of angiogenesis and embryogenesis, in human abdominal aortic aneurysm (AAA) and analyzed their functional roles in cell migration. From 10 patients (9 males and 1 female; age, 68.5 ± 2.4) who underwent vascular surgery for AAA, we obtained AAA and adjacent control tissues. Using real-time RT-PCR, we analyzed expression of ephrin-B1 and EphB2. We also histologically localized these molecules in AAA tissues. Finally, effects of ephrin-B1 and EphB2 on inflammatory cell chemotaxis were examined by cell migration assay. Expression levels of ephrin-B1 (0.410 ± 0.046 versus 1.198 ± 0.252, P = 0.027) and EphB2 (0.764 ± 0.212 versus 1.272 ± 0.137, P = 0.594) were higher in AAA than normal control. Both ephrin-B1 and EphB2 were expressed in macrophages, T lymphocytes, and endothelial cells within AAA. In chemotaxis assay, ephrin-B1 and EphB2 inhibited mononuclear-cell chemotaxis induced by stromal derived factor-1 down to 54.7 ± 12.7% (P = 0.01) and 50.7 ± 13.1% (P = 0.01), respectively. These data suggest that ephrin-B1 and EphB2 might be functional in human adult inflammatory cells and involved in the pathogenesis of AAA, although specific roles of these molecules should further be sought.

A consensus sequence in the endothelin-B receptor second intracellular loop is required for NHE3 activation by endothelin-1.

Endothelin-1 (ET-1) increases the activity of Na(+)/H(+) exchanger 3 (NHE3), the major proximal tubule apical membrane Na(+)/H(+) antiporter. This effect is seen in opossum kidney (OKP) cells expressing the endothelin-B (ET(B)) and not in cells expressing the endothelin-A (ET(A)) receptor. However, ET-1 causes similar patterns of protein tyrosine phosphorylation, adenylyl cyclase inhibition, and increases in cell [Ca(2+)] in ET(A)- and ET(B)-expressing OKP cells, implying that an additional mechanism is required for NHE3 stimulation by the ET(B) receptor. The present studies used ET(A) and ET(B) receptor chimeras and site-directed mutagenesis to identify the ET receptor domains that mediate ET-1 regulation of NHE3 activity. We found that binding of ET-1 to the ET(A) receptor inhibits NHE3 activity, an effect for which the COOH-terminal tail is necessary and sufficient. ET-1 stimulation of NHE3 activity requires the COOH-terminal tail and the second intracellular loop of the ET(B) receptor. Within the second intracellular loop, a consensus sequence was identified, KXXXVPKXXXV, that is required for ET-1 stimulation of NHE3 activity. This sequence suggests binding of a homodimeric protein that mediates NHE3 stimulation.

Increased gene expression of collagen Types I and III is inhibited by beta-receptor blockade in patients with dilated cardiomyopathy.

AIMS: To elucidate the cellular mechanisms of cardioprotection of beta-blockers in patients with heart failure, we investigated the effects of beta-blockers on collagen synthesis in patients with dilated cardiomyopathy (DCM). METHODS AND RESULTS: We examined the gene expression before and 4 months after the administration of a beta-blocker in 17 DCM patients. The messenger ribonucleic acid expression of collagen Types I and III (Col I and III) and transforming growth factor-beta(1) (TGF-beta(1)) of right ventricular tissues obtained by the endomyocardial biopsy were assessed by quantitative reverse transcriptase-polymerase chain reaction. Cardiac sympathetic nerve activity was assessed by the washout rate (WR) of (123)I-metaiodobenzylguanidine from the heart. Left ventricular ejection fraction (21 +/- 7 vs. 35 +/- 9%) and WR (53+/-14 vs. 42 +/- 13%) improved significantly. Before the beta-blocker treatment, the expressions of both Col I (r = 0.560, P = 0.041) and Col III (r = 0.630, P = 0.008) genes were correlated with WR. The expression levels of both Col I (1.08 +/- 0.72 vs. 0.65 +/- 0.26, P = 0.024) and Col III (2.06 +/- 1.81 vs. 1.05 +/- 0.74, P = 0.018) were reduced by a beta-blocker. Changes in TGF-beta(1) correlated with those in WR (r = 0.606, P = 0.002). CONCLUSION: beta-Blockers are considered to inhibit the expression of collagen-related genes in DCM, which seems to be mediated by TGF-beta(1).

Sustained upregulation of inflammatory chemokine and its receptor in aneurysmal and occlusive atherosclerotic disease: results form tissue analysis with cDNA macroarray and real-time reverse transcriptional polymerase chain reaction methods.

BACKGROUND: Although cytokines are known to be pivotal in the development of atherosclerotic diseases, few data exist regarding their expressions in the established stages such as aneurysmal or occlusive lesions. Therefore, in the present study the gene expression levels of cytokine-related substances in abdominal aortic aneurysm (AAA) and carotid artery stenosis (CAS) were determined using cDNA macroarray and real-time reverse transcriptase polymerase chain reaction (RT-PCR) methods. METHODS AND RESULTS: Tissue samples were obtained from 31 patients with AAA and 24 with CAS. The array-specific [33P]-labeled cDNA probe mixture synthesized from 2.5 microg total RNA with gene-specific primers was hybridized with nylon membranes containing 375 cDNA clones. Densitometric analysis confirmed differences in expression (>5-fold) for 97 of the cytokine-related gene products between AAA and adjacent control tissue. Among these, simultaneous upregulation was found in the expression of interleukin (IL)-8 (9-fold) and its receptor, CXCR-2 (11-fold). Thus, the expressions of IL-8 and CXCR-2 were further quantified by real-time RT-PCR. The expression of both the genes was significantly upregulated in both AAA and CAS compared with control regions as followed: IL-8=0.53+/-0.16 vs 0.11+/-0.04 (p<0.01); CXCR-2=2.04+/-0.75 vs 0.29+/-0.10 (p<0.01) in AAA, and IL-8=1.35 +/-0.25 vs 0.60+/-0.16; CXCR-2=2.00 +/-0.51 vs 0.58+/-0.21 (p<0.05) in CAS. Under these conditions, the gene expressions of monocyte chemotactic protein-1 and its receptor, CCR-2, were not significantly different in the control and diseased regions of both AAA and CAS. CONCLUSIONS: Sustained upregulation of IL-8 and CXCR-2 was observed in both AAA and CAS, suggesting the inflammatory process is still active in established dilated and occlusive atherosclerotic diseases. Whether upregulation of this system could be protective or not protective for disease development requires further study.

Electrical and ionic abnormalities in the heart of cardiomyopathic hamsters: in quest of a new paradigm for cardiac failure and lethal arrhythmia.

Cardiomyopathy is primary degenerative disease of myocardium, which leads to cardiac failure and lethal arrhythmia. An appropriate model animal of a particular disease is, in general, greatly helpful for better understanding of its pathogenesis. In 1962, a naturally occurring mutant line of Syrian hamster named BIO1.50 was reported, which inherited cardiomyopathy and muscular dystrophy as autosomal recessive mode with 100% penetrance. To date, several sublines of cardiomyopathic hamsters (CM hamsters) have been derived. The genomic deletion of delta-sarcoglycan, a member of dystrophin-associated proteins, was demonstrated to be the common genetic cause of CM hamsters in 1997. Over the past 40 years, hundreds of papers have been published on the pathophysiological aspects of CM hamsters. The aim of this paper is to annotate every one of the CM hamsters with its historical background and then summarize the previous findings on CM hamsters with special focus on electrical and ionic properties. This review article is expected to serve as a basis to build up a new paradigm for the pathogenesis of cardiac failure and severe arrhythmia.

Changes in myocardial gene expression associated with beta-blocker therapy in patients with chronic heart failure.

BACKGROUND: The left ventricular functional recovery by beta-blocker therapy is now attributed to time-dependent biologic effects on cardiomyocytes. METHODS AND RESULTS: To elucidate the cellular mechanism of these biologic effects, we treated 9 patients with dilated cardiomyopathy for 4 months with beta-blockers and examined the gene expressions linked to an improvement of left ventricular ejection fraction (EF). Gene expressions of the biopsied right ventricular endomyocardium were assessed by real-time reverse transcription-polymerase chain reaction. A decrease in beta-myosin heavy chain (1.23+/-0.49 versus 0.86+/-0.45, P<.05) was observed 4 months after the administration of beta-blockers. The expression levels of both sarcoplasmic reticulum Ca(2+) ATPase (SERCA) (0.80+/-0.28 versus 1.39+/-0.44, P<.01) and phospholamban (PLB) (0.49+/-0.08 versus 0.88+/-0.34, P<.05) increased, whereas the expression levels of Na(+)-Ca(2+) exchanger (NCX), beta-adrenoreceptor kinase 1, and ryanodine receptor 2 were unchanged. The SERCA/NCX ratio (0.68+/-0.14 versus 0.96+/-0.33, P<.05) also increased. The increase in SERCA mRNA expression correlated with the degree of changes in EF (%deltaEF) (r=0.679, P<.05), and none of changes in these genes expression correlated with changes in the plasma brain natriuretic peptide concentration. CONCLUSIONS: The functional recovery resulting from beta-blockers may be associated with the restoration of the unfavorable gene expression that controls Ca(2+) handlings in the failing heart.