Gene Transfer into Cultured Cardiac Myocytes Mediated by Retrovirus

BACKGROUND: Transplantation of cardiac myocytes (CMs) into the injured heart emerges as a potential alternative for the treatment of heart failure. Genetic modification of CMs could enhance and/or modify its therapeutic effects. The characteristics of retroviral gene delivery, which is most commonly used in human trial, has been minimally studied in CMs due to its low efficiency in non-dividing cells. In this study, using newly developed high-titer retrovirus, we evaluated 1) the efficiency of gene transfer into CMs, 2) whether S phase during infection is necessary for the transduction, and 3) characteristics of gene delivery to mononucleated vs binucleated CMs. METHODS: Enriched CMs were cultured from the ventricles of 1 day-old rat hearts. The cells were transduced by MFG-nls-LacZ retroviruses (5x107 IU/ml) in the presence or absence of polybrene. 3H-thymidine was added to label cells in S phase. The cells were stained for b-galactosidase activity and then immunostained using MF20Ab to identify CMs. The cells were subsequently processed for in vitro autoradiography. RESULTS: 1)With 3 rounds of infection, 5.9% of total cultured cells were LacZ-positive. The efficiency of transduction reached upto 7.4% in the presence of polybrene 8 microgram/ml. 2)Nuclear morphology of LacZ-positive CMs was pleomorphic from mononucleated to multinucleated, mostly binucleated. 3)Among mononucleated CMs, 17% of cells were labelled with thymidine. Transduction efficiency (TDE) of thymidine-positive and -negative mononucleated CMs were 37.9% and 3.1%, respectively. Among binucleated cells, 28.9% of cells were labelled with thymidine. TDE of thymidine-positive and -negative binucleated CMs were 75.4% and 13.6%, respectively. 4)In total, TDE of binucleated cells were 3.5 times compared to the one of mononucleated cells (31.5% vs 9.0%). CONCLUSION: TDE of CMs using high-titer retrovirus is relatively low. S phase of cells during retroviral infection is not mandatory for the retroviral transduction. Binucleated CMs are susceptible to retroviral gene delivery and their TDE is higher than the one of mononucleated CMs.

The Effects of Aldosterone and Cytokines IL-1beta, TNF-alpha on the Expression of Angiotensin Converting Enzyme Gene in Vascular Smooth Muscle Cells

BACKGROUND: It has been suggested that all components of the renin-angiotensin-aldosterone system (RAAS) are present in the vascular wall and that the vascular RAAS modulates vascular tone and vascular hypertrophy. One of the catalytic step in the RAAS cascade is the local conversion of angiotensin I to angiotensin II (Ang II) by angiotensin converting enzyme (ACE). One of the major sources of ACE in the vasculature is vascular smooth muscle cells (VSMC). Here, we provide insight into the intrinsic mechanisms by which the components of RAAS regulate gene expression of ACE in cultured smooth muscle cells of the rat and we also investigated the effects of cytokines on ACE mRNA. METHODS: RNA was extracted from the primary cultured VSMCs. We analyzed the expression levels of ACE by competitive reverse transcription-PCR using recombinant RNA as an internal standard. RESULTS: 1) ACE mRNA level was increased markedly by aldosterone in a dose- and time-dependent manner, indicating that there exists positive feedback mechanism within RAAS. 2) The induction of ACE mRNA by aldosterone was inhibited by spironolactone. 3) Aldosterone-stimulated expression of ACE was also inhibited by Ang II, which shows that Ang II acts as a negative regulator of the expression of ACE in RAAS cascade. 4) Interleukin-1beta or TNF-alpha did not induce ACE mRNA expression. 5) However, mixture of interleukin-1betaand TNF-alpha(CytoMix) significantly increased the expression of ACE. It was also shown that CytoMix increased aldosterone-stimulated ACE mRNA expression in an additative manner. CONCLUSION: These results indicate that the expression of ACE in smooth muscle cells is modulated by the components of RAAS and cytokines. The intrinsic positive and negative feedback controls of RAAS would play an important role in the pathogenesis of vascular diseases.

Development of Quantitative Reverse Transcription-Polymerase Chain Reaction for the Mesurement of Angiotensin Converting Enzyme mRNA

BACKGROUND: The analysis of ACE gene expression in vital to study the role of angiotensin conveting enzyme(ACE) in the pathogenesis of cardiovascular disease. Traditionally, levels of individual mRNA expression have been analyzed by semiquantitative Northern blotting, which requires a large quantity of tissue. Therefore, gene expression of a little biopsy specimen from the human heart or atherectomy specimen from the blood vessel cannot be measured easily. Reverse transcription-polymerase chain reaction(RT-PCR) is very effective, sensitive and rapid method of detecting the method of quantitative RT-PCR(QRT-PCR) using recombinant RNA template as internal standard to measure the expression of ACE. METHOD: Recombinant RNA(rcRNA) was designed to yield PCR product which differs in size by about 200bp from that of the target RNA. Initially, spacer gene, which was composed of ACE sense primer, antisense primer, T7 promotor and poly(dT) tail with glutathione transferase(GSTM) gene of 180bp in the middle, was constructed. Then, standard rcRNA was obtained by in vitro transcription. Target RNA was mixed with rcRNA and amplified by PCR, togather with P-dCTP. PCR products were analyzed by gel electrophoresis. For quantitation, either gel was cut and radioactivity was counted or gel was dried and exposed to X-ray film and density was measured using image densitometer. We carried out semiquantitative RT-PCR to study the modulation of ACE expression in vascular smooth muscle cell(VSMC) by dexamethasone and basis FGF(bFGF). RESULT: The size difference of PCR products from the standard RNA and the extracted target RNA was matched as designed. By using QRT-PCR, there was 1.7*10(8) ACE mRNA molecules in 1 ng of rat lung total RNA. bFGF and dexamethasone upregulated ACE mRNA expression in cultured VSMC. CONCLUSION: These results suggest that RT-PCR using rcRNA as internal standard is a very useful method for quantitation or semiquantitation of ACE mRNA from a small amount of tissue or cultured cells. Expression of ACE in VSMC can be modulated by various stimuli such as basic FGF and dexamethasone. QRT-PCR could be widely used in the studies of expression of specific human genes.