148. A Novel Microelectric Field Network-Gene Transfer Technique for Highly Efficient Therapeutic Gene Expression in Human Cardiac Stem Cells

Rationale: Cardiac stem cells (CSCs) are beneficial when administered to infarcted mouse or rat hearts. Though the mechanism of these benefits is unknown, CSC vitality likely plays a major role. Thus, investigating the factors governing CSC survival in the ischemic heart may lead to more effective therapeutic strategies. Our previous studies showed that misfolded proteins accumulate in the sarco/endoplasmic reticulum (SR/ER) of the ischemic heart. The transcription factor, ATF6, is a key component of the adaptive ER stress response because it induces genes that reduce the accumulation of misfolded proteins, improving myocyte survival during ischemic stress. While our lab has shown that, in cardiac myocytes, ATF6 is cardioprotective in the ischemic heart, neither the ER stress response nor ATF6 have been examined in CSCs. We hypothesize that ATF6 and the adaptive ER stress response are critical for optimal survival of CSCs. Objective/Methods: To gauge the relevance of the ER stress response in CSCs, we used MTT assays to compare the viabilities of mouse CSCs to neonatal rat ventricular myocytes (NRVM) subjected to treatments that mimic ischemic ER stress in the heart. We also assessed the effect of inhibiting ATF6 on both the ER stress response and CSC viability by using chemical inhibition of ATF6 activation or siRNA-mediated ATF6 knock down. Results: We found that, compared to NRVM, CSCs exhibited lower levels of adaptive ER stress response gene expression and decreased viability in response to ER stress. Thus, relative to NRVM, the adaptive ER stress response is not fully developed in CSCs. We also found that either chemical inhibition of ATF6 activation or ATF6 knock down decreased adaptive ER stress response gene expression. Strikingly, ATF6 inhibition or knockdown decreased CSC viability and cell number by as much as 70%. Conclusions: Thus, compared to cardiac myocytes, CSCs exhibit a reduced adaptive ER stress response and are more sensitive to ER stress, suggesting that enhancement of the ATF6-mediated adaptive ER stress response in CSCs may be a viable therapeutic approach for enhancing stem cell-mediated myocardial repair.

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Abstract 865: Gata-4 Is An Angiogenic Survival Factor Of The Infarcted Heart

Circulation ◽

10.1161/circ.116.suppl_16.ii_169 ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Olli Tenhunen ◽

Hanna Leskinen ◽

Raisa Serpi ◽

Jaana Rysä ◽

Harri Pennanen ◽

...

Keyword(s):

Heart Failure ◽

Stem Cells ◽

Gene Transfer ◽

Dna Binding ◽

Ventricular Remodeling ◽

Left Ventricular Remodeling ◽

Left Ventricular ◽

Therapeutic Interventions ◽

Cardiac Stem Cells

Recent data suggest that the cardiac-restricted transcription factor GATA-4 is an anti-apoptotic factor required for adaptive responses as well as a key regulator of hypertrophy and hypertrophy-associated genes in the heart. As a leading cause of chronic heart failure, reversal of post-infarction left ventricular remodeling represents an important target for therapeutic interventions. Here we studied the role of GATA-4 as a mediator of post-infarction remodeling. Rats were subjected to experimental myocardial infarction (MI) by ligating the left anterior descending coronary artery (LAD). Ligation of the LAD decreased the DNA binding activity of GATA-4 by 69 % at day 1 after MI (P<0.001, n=7– 8) as assessed by gel mobility shift assays. At 2 weeks the GATA-4 DNA binding was significantly upregulated (2.4-fold, P<0.05, n=7), and returned to baseline at 4 weeks. To determine the functional role of GATA-4, rats underwent LAD ligation followed by peri-infarct intramyocardial delivery of adenoviral vector expressing GATA-4. Hearts treated with the GATA-4 gene transfer exhibited significantly increased ejection fraction (58±5% vs. 38±3% in LacZ-treated control animals with MI, P<0.001, n=8 –9) and fractional shortening (28±3% vs. 16±1%, P<0.001, n=8 –9) 2 weeks after MI. Accordingly, the infarct size was significantly reduced (26±4% vs. 45±4%, P<0.01, n=8 –9). To determine the cardioprotective mechanisms of GATA-4, the number of cardiac stem cells, apoptotic cardiomyocytes and capillaries were assessed. The number of capillaries (59±4/field vs. 48±3/field, P<0.051, n=7– 8) and c-kit positive stem cells (13±5 cells vs. 4±2 cells, P<0.05, n=7– 8) were increased in GATA-4 treated hearts, and a tendency to decreased apoptosis was observed in TUNEL-stained histological sections. These results indicate that the reversal of reduced GATA-4 activity prevents adverse post-infarction remodeling through increased angiogenesis, recruitment of cardiac stem cells and anti-apoptosis. GATA-4-based gene transfer may represent a novel, efficient therapeutic approach for heart failure.

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Blood Serum Stimulates p38-Mediated Proliferation and Changes in Global Gene Expression of Adult Human Cardiac Stem Cells

Cells ◽

10.3390/cells9061472 ◽

2020 ◽

Vol 9 (6) ◽

pp. 1472 ◽

Cited By ~ 1

Author(s):

Anna L. Höving ◽

Kazuko E. Schmidt ◽

Madlen Merten ◽

Jassin Hamidi ◽

Ann-Katrin Rott ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

P38 Mapk ◽

Regulatory Networks ◽

Human Blood Plasma ◽

Cardiac Stem Cells ◽

Dependent Manner ◽

Protective Effects ◽

Adult Human ◽

Age And Sex

During aging, senescent cells accumulate in various tissues accompanied by decreased regenerative capacities of quiescent stem cells, resulting in deteriorated organ function and overall degeneration. In this regard, the adult human heart with a generally low regenerative potential is of extreme interest as a target for rejuvenating strategies with blood borne factors that might be able to activate endogenous stem cell populations. Here, we investigated for the first time the effects of human blood plasma and serum on adult human cardiac stem cells (hCSCs) and showed significantly increased proliferation capacities and metabolism accompanied by a significant decrease of senescent cells, demonstrating a beneficial serum-mediated effect that seemed to be independent of age and sex. However, RNA-seq analysis of serum-treated hCSCs revealed profound effects on gene expression depending on the age and sex of the plasma donor. We further successfully identified key pathways that are affected by serum treatment with p38-MAPK playing a regulatory role in protection from senescence and in the promotion of proliferation in a serum-dependent manner. Inhibition of p38-MAPK resulted in a decline of these serum-mediated beneficial effects on hCSCs in terms of decreased proliferation and accelerated senescence. In summary, we provide new insights in the regulatory networks behind serum-mediated protective effects on adult human cardiac stem cells.

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Developing Lentiviral Vectors for Gene Therapy of Wiskott-Aldrich Syndrome.

Blood ◽

10.1182/blood.v104.11.3170.3170 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3170-3170

Author(s):

Marguerite V. Evans-Galea ◽

Matthew M. Wielgosz ◽

Ted S. Strom ◽

Hideki Hanawa ◽

John M. Cunningham ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Stem Cell ◽

T Cell ◽

Gene Transfer ◽

Hela Cells ◽

Lentiviral Vectors ◽

Proximal Promoter ◽

Wiskott Aldrich Syndrome ◽

Wasp Gene

Abstract The Wiskott-Aldrich syndrome (WAS) is a rare X-linked recessive disorder caused by mutations in the WASP gene. WASP is an effector protein in the actin polymerization pathway. Characterized by immunodeficiency, micro-thrombocytopenia and eczema, untreated WAS patients may also suffer hemorrhages, recurring infections and potential malignancies. WAS can be cured by bone marrow (BM) transplant but because many patients lack a suitable donor, stem cell-targeted gene transfer is being developed as an alternative therapeutic approach. We have demonstrated correction of the T-cell proliferation defect in Wasp− mice using MSCV oncoretroviral vectors (Blood102:3108, 2003). However, our competitive repopulation studies in mice with both wild-type (WT) and gene-corrected Wasp− BM, demonstrated only a modest selective advantage for gene modified lymphocytes. Correction of the lymphocytopenia was observed only in animals having high proportions of transduced cells. In addition, variability in the level of gene expression among gene-corrected cells was associated with only partial correction of the T-cell cytokine secretory defects. Thus, the efficiency of stem cell-targeted gene transfer as well as the level and consistency of gene expression are likely to be key factors that determine success in any clinical application of gene transfer for the treatment of WAS. In attempting to improve vector design, we have used lentiviral vectors because of their greater efficiency in transducing repopulating stem cells as we recently demonstrated in a non-human primate model (Blood103:4062, 2004). The WASP gene is regulated by two promoter regions. The proximal promoter lies immediately adjacent to the translation start site with the distal promoter found 6 kb upstream, followed by an alternate first exon. We have developed a series of third generation, self-inactivating lentiviral vectors containing the MSCV, proximal or distal WASP promoters driving GFP in the reverse transcriptional orientation. The WASP promoters were active in lymphocytes but not HeLa cells in vitro. However, expression was low in lymphocytes and granulocytes in mice transplanted with genetically modified stem cells. In an effort to abrogate any position-effect variegation and enhance expression, we generated a new series of vectors with the transcriptional unit in the forward orientation that also contained the woodchuck post-transcriptional regulatory element, the chicken beta-globin 5′ DNase I hypersensitive site 4 (I) and human beta-interferon scaffold attachment region (S) insulator elements. Either GFP or murine Wasp cDNA were included for expression analysis. Despite the complexity of the SI-containing vectors, titers of concentrated preparations ranged from 9x106 TU/ml to 5x107 TU/ml and enabled transduction of both cell lines and murine hematopoietic stem cells. Low-level GFP expression from the proximal promoter was detected in HeLa cells with higher expression found in lymphocytes (NALM6 and Jurkat cells). Expression of GFP under the control of the proximal WASP promoter was detected in vivo in multiple hematopoietic lineages in mice transplanted with transduced stem cells. Future efforts will focus on further characterization and optimization of vector design with the goal of achieving consistent, high level expression.

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Local Gene Delivery System by Bubble Liposomes and Ultrasound Exposure into Joint Synovium

Journal of Drug Delivery ◽

10.1155/2011/203986 ◽

2011 ◽

Vol 2011 ◽

pp. 1-7 ◽

Cited By ~ 13

Author(s):

Yoichi Negishi ◽

Yuka Tsunoda ◽

Yoko Endo-Takahashi ◽

Yusuke Oda ◽

Ryo Suzuki ◽

...

Keyword(s):

Gene Expression ◽

Gene Transfer ◽

Gene Delivery ◽

Delivery System ◽

Imaging System ◽

Effective Means ◽

Gene Delivery System ◽

Highly Efficient ◽

Efficient Gene

Recently, we have developed novel polyethylene glycol modified liposomes (bubble liposomes; BL) entrapping an ultrasound (US) imaging gas, which can work as a gene delivery tool with US exposure. In this study, we investigated the usefulness of US-mediated gene transfer systems with BL into synoviocytes in vitro and joint synovium in vivo. Highly efficient gene transfer could be achieved in the cultured primary synoviocytes transfected with the combination of BL and US exposure, compared to treatment with plasmid DNA (pDNA) alone, pDNA plus BL, or pDNA plus US. When BL was injected into the knee joints of mice, and US exposure was applied transcutaneously to the injection site, highly efficient gene expression could be observed in the knee joint transfected with the combination of BL and US exposure, compared to treatment with pDNA alone, pDNA plus BL, or pDNA plus US. The localized and prolonged gene expression was also shown by an in vivo luciferase imaging system. Thus, this local gene delivery system into joint synovium using the combination of BL and US exposure may be an effective means for gene therapy in joint disorders.

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