Adeno-associated virus (AAV) vectors, derived from a non-pathogenic parvovirus, are considered to be an appropriate gene transfer vehicle for both dividing and non-dividing cells. In the present ...study, we investigated whether the rat heart could be efficiently transduced with AAV vectors. Rat cardiac myocytes (CM) were infected with AAV-lacZ vector containingβ-galactosidase (β-gal) genein vitro, and the expression ofβ-gal in CM was evaluated by X-gal staining andβ-gal ELISA. With increasing multiplicities of infection (MOI), more than 60% of CM were stained positively with X-gal, and theβ-gal expression increased to 31.1±4.6 ng/mg protein in a MOI-dependent manner (MOI: 104to 106particles/cell). Theβ-gal expression was also increased in an incubation period-dependent manner (1–24 h).β-gal expression was maximal at day 3 and then gradually decreased with time. However,β-gal expression at day 14 was almost the same level as that at day 1 (45.5±5.9v55.2±6.2 ng/mg protein). Excised rat right ventricular papillary muscles were also infected with AAV-lacZex vivo. When theβ-gal expression was evaluated by X-gal staining, more than 80% of CM in the papillary muscles were stained positively, indicating efficient gene transfer into CM using AAV vectors. These findings suggest that AAV vectors are promising for cardiac gene therapy.
The adhesive interaction of monocytes and vascular smooth muscle cells (VSMCs) has been suggested to be a regulatory signal in the cellular activation that is involved in the pathogenesis of ...atherosclerosis. We investigated the effects of monocyte-VSMC interaction on inducible nitric oxide (NO) synthase expression.
NO production by the cultured cells was determined by measuring the nitrite content of the culture media using the Griess reagent. The expression of inducible NO synthase protein was assayed by Western blotting.
Interleukin-1 beta (IL-1 beta) induced nitrite production by VSMCs in a time-dependent manner. The addition of the mouse monocyte cell line J774 to IL-1 beta-stimulated VSMCs further increased nitrite production in a monocyte number-dependent manner. Enhanced nitrite production by coculture was accompanied by increased inducible NO synthase protein accumulation. Addition of tumor necrosis factor-alpha (TNF-alpha) also enhanced IL-1 beta-induced nitrite production by VSMCs, but TNF-alpha showed no effect in the presence of monocytes. Coculture of monocytes and VSMCs in the presence of IL-1 beta secreted substantial amounts of TNF-alpha. The production of nitrite by coculture was markedly inhibited by an anti-TNF-alpha antibody.
The present study revealed that direct cell-to-cell interaction between monocytes and VSMCs enhances NO production, suggesting an important role for their interaction in the pathogenesis of atherosclerosis.
A study was conducted to determine whether sympathetic nerve activity, one of the main regulators of blood pressure, is involved in high blood pressure in the night-time and morning. Twenty-seven ...untreated hypertensive subjects, in whom hypertension was diagnosed by ambulatory blood pressure (ABP) measurement, who showed a 24h systolic ABP value over 140mmHg and/or 24h diastolic ABP over 90 mmHg were recruited. They also showed a night-time systolic ABP value of over 130mmHg and/or a night-time diastolic ABP of over 80mmHg. They were divided into two groups: "dippers (D)" whose night-time ambulatory blood pressure fell by more than 10% of the day-time blood pressure, and "nondippers (ND)" in whom this phenomenon was absent. We examined the effect of a long-acting α1-blocker (doxazosin) on diurnal blood pressure variation in these subjects with essential hypertension. Baseline casual blood pressure and 24h systolic ABP were not significantly different between the two groups. However, both night-time and morning ABP in ND were higher than those in D. Administration of doxazosin (mean 73±13 (SE) d) significantly decreased casual blood pressure, and 24h, day-time, nighttime and morning systolic ABP in the whole cohort. When subjects were divided into D and ND, the day-time and morning systolic ABP decreased significantly after doxazosin treatment in both groups, whereas the night-time systolic ABP decreased significantly only in ND but not in D. These results suggest that sympathetic nerve activity involved in elevating blood pressure during the night may differ between D and ND. (Hypertens Res 1995; 18: 125-130)
Echocardiography showed left ventricular hypertrophy with ventricular septum thickness (VST) of 15 mm and left ventricular posterior wall thickness (LVPWT) of 12 mm. Left ventricular mass index ...(LVMI) and relative wall thickness (RWT) were 229 g/m2 and 0.68, respectively.
Synthesis and activity of the enzymatic equivalent of the sodium pump, Na,K-ATPase, are regulated by thyroid hormone in responsive tissues. The purpose of this study was to determine whether ...triiodothyronine (T3) regulates the level of the messenger RNA (mRNA) coding for Na,K-ATPase alpha- and beta-subunits in the heart. The expression of Na,K-ATPase mRNAs in in vitro myocardial cells was directly assayed by Northern and slot blot hybridization using Na,K-ATPase alpha- and beta-isoform-specific cDNA probes. Exposure of cultured neonatal rat cardiocytes to 10(-8) M T3 resulted in 1) threefold to fourfold increase in alpha 1- and beta 1-mRNA accumulation, with a maximum elevation at 48 hours, 2) sevenfold increase in alpha 2-mRNA accumulation with a peak elevation at 72 hours, and 3) transient threefold increase in alpha 3-mRNA within the first 24 hours followed by a deinduction thereafter. The increase in alpha 1-mRNA accumulation by T3 occurred over the physiological T3 concentration range with an EC50 of 5 x 10(-10) M. This was associated with a twofold increase in alpha 1-subunit protein accumulation and an increase in Na,K-ATPase transport activity. The half-life of alpha 1-mRNA analyzed by actinomycin D chase was less than 3 hours and was not affected by T3. Transfection experiments with the luciferase reporter gene revealed that thyroid hormone response sequences are located within the 5'-flanking regions of each alpha-isoform gene.
Cytokine induction of intercellular adhesion molecule-1 (ICAM-1) on cardiac myocytes may be a critical step in cardiac inflammation associated with acute myocardial infarction and myocarditis. The ...aim of this study was to investigate the involvement of monocyte chemoattractant protein-1 (MCP-1), a homologue of mouse JE, in the neutrophil-myocyte adhesion in vitro.
MCP-1/JE and ICAM-1 mRNA expression in cultured neonatal rat cardiac myocytes was evaluated by northern blot analysis. ICAM-1 molecule content on myocytes was determined by ELISA. For adherence assay, myocytes and neutrophils were co-incubated and the number of bounded neutrophils was counted.
MCP-1/JE transcripts were not clearly observed in cultured neonatal rat cardiac myocytes; however, its transcripts were clearly detected by exposure to interleukin 1 alpha (100 U.ml-1), lipopolysaccharide (1 microgram.ml-1), or hypoxia (95% N2 + 5% CO2). In ELISA analysis, the expression of ICAM-1 molecules on cardiac myocytes was significantly stimulated by MCP-1 in a dose dependent manner, and the effect of MCP-1 was observed as early as at 6 h. In northern blot analysis, ICAM-1 mRNA expression was constitutively observed in myocytes, and the expression was markedly stimulated by exposure to MCP-1 with a peak elevation at 2 h. In adherence assay, MCP-1 stimulated the adhesion of rat neutrophils to rat cardiac myocytes, and this effect of MCP-1 was inhibited by an anti-ICAM-1 MAb.
These results suggest that cardiac myocytes produce MCP-1, which could in turn promote the adhesion of neutrophils to myocytes via ICAM-1 expression, suggesting the involvement of MCP-1 in cardiac inflammation associated with acute myocardial infarction and myocarditis.
We analyzed the expression of mouse DMAHPISix5 (the myotonic dystrophy-associated homeodomain protein gene) during embryogenesis and in various tissues by northern blotting. Expression was observed ...as early as embryonic day 7 (E7) and continued to E17. Abundant expression was observed in neonatal heart and skeletal muscle with potential links to the pheno-type of myotonic dystrophy. The transcription initiation sites of the gene were analyzed in mouse E11 and E15 embryos and in adult skeletal and heart muscle. Three major transcription initiation sites were identified, the proximal site was specific to the early E11 embryo, while the other two were common among the heart and skeletal muscle and E11 and E15 embryos. All transcription initiation sites were downstream of the corresponding CTG repeat locus of the mouse gene (−1195), excluding a possible inclusion of the CUG repeat sequence in mRNA leading to abnormal splicing or to translation of aberrant protein. For analysis of the regulatory elements in the promoter region, we used P19 embryonal carcinoma cells which abundantly express mouse DMAHPISix5. Multiple positive and negative elements were identified in the promoter region. All positive elements were Sp1/Sp3 binding sites and one of the negative elements was a novel factor binding site. The transcription initiation sites and regulatory elements are conserved between human and mouse DMAHP.
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