skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Ultrasound-targeted microbubble destruction improves the low density lipoprotein receptor gene expression in HepG{sub 2} cells

Abstract

Ultrasound-targeted microbubble destruction had been employed in gene delivery and promised great potential. Liver has unique features that make it attractive for gene therapy. However, it poses formidable obstacles to hepatocyte-specific gene delivery. This study was designed to test the efficiency of therapeutic gene transfer and expression mediated by ultrasound/microbubble strategy in HepG{sub 2} cell line. Air-filled albumin microbubbles were prepared and mixed with plasmid DNA encoding low density lipoprotein receptor (LDLR) and green fluorescent protein. The mixture of the DNA and microbubbles was administer to cultured HepG{sub 2} cells under variable ultrasound conditions. Transfection rate of the transferred gene and cell viability were assessed by FACS analysis, confocal laser scanning microscopy, Western blot analysis and Trypan blue staining. The result demonstrated that microbubbles with ultrasound irradiation can significantly elevate exogenous LDLR gene expression and the expressed LDLRs were functional and active to uptake their ligands. We conclude that ultrasound-targeted microbubble destruction has the potential to promote safe and efficient LDLR gene transfer into hepatocytes. With further refinement, it may represent an effective nonviral avenue of gene therapy for liver-involved genetic diseases.

Authors:
 [1];  [2];  [2];  [3];  [3];  [3];  [3];  [1];  [2];  [2];  [4];  [5];  [5];  [5]
  1. Institute of Reproductive Medicine, Nanjing Medical University, Nanjing 210029 (China)
  2. (China)
  3. Atherosclerosis Research Center, Nanjing Medical University, Nanjing 210029 (China)
  4. Institute of Reproductive Medicine, Nanjing Medical University, Nanjing 210029 (China) and Atherosclerosis Research Center, Nanjing Medical University, Nanjing 210029 (China) and Provincial Laboratory of Human Functional Genomics, Nanjing Medical University, Nanjing 210029 (China). E-mail: lmfan@njmu.edu.cn
  5. Cardiovascular Division, Wuxi No.1 Hospital, Wuxi 214002 (China)
Publication Date:
OSTI Identifier:
20798932
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 343; Journal Issue: 2; Other Information: DOI: 10.1016/j.bbrc.2006.02.179; PII: S0006-291X(06)00480-3; Copyright (c) 2006 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ALBUMINS; GENE THERAPY; GENES; HEREDITARY DISEASES; IRRADIATION; LIGANDS; LIPOPROTEINS; LIVER; LIVER CELLS; MICROSCOPY; RECEPTORS; TRYPAN BLUE; UPTAKE

Citation Formats

Guo Dongping, Atherosclerosis Research Center, Nanjing Medical University, Nanjing 210029, Provincial Laboratory of Human Functional Genomics, Nanjing Medical University, Nanjing 210029, Li Xiaoyu, Sun, Ping, Tang Yibo, Chen Xiuying, Chen Qi, Atherosclerosis Research Center, Nanjing Medical University, Nanjing 210029, Provincial Laboratory of Human Functional Genomics, Nanjing Medical University, Nanjing 210029, Fan Leming, Zang Bin, Shao Lizheng, and Li Xiaorong. Ultrasound-targeted microbubble destruction improves the low density lipoprotein receptor gene expression in HepG{sub 2} cells. United States: N. p., 2006. Web.
Guo Dongping, Atherosclerosis Research Center, Nanjing Medical University, Nanjing 210029, Provincial Laboratory of Human Functional Genomics, Nanjing Medical University, Nanjing 210029, Li Xiaoyu, Sun, Ping, Tang Yibo, Chen Xiuying, Chen Qi, Atherosclerosis Research Center, Nanjing Medical University, Nanjing 210029, Provincial Laboratory of Human Functional Genomics, Nanjing Medical University, Nanjing 210029, Fan Leming, Zang Bin, Shao Lizheng, & Li Xiaorong. Ultrasound-targeted microbubble destruction improves the low density lipoprotein receptor gene expression in HepG{sub 2} cells. United States.
Guo Dongping, Atherosclerosis Research Center, Nanjing Medical University, Nanjing 210029, Provincial Laboratory of Human Functional Genomics, Nanjing Medical University, Nanjing 210029, Li Xiaoyu, Sun, Ping, Tang Yibo, Chen Xiuying, Chen Qi, Atherosclerosis Research Center, Nanjing Medical University, Nanjing 210029, Provincial Laboratory of Human Functional Genomics, Nanjing Medical University, Nanjing 210029, Fan Leming, Zang Bin, Shao Lizheng, and Li Xiaorong. Fri . "Ultrasound-targeted microbubble destruction improves the low density lipoprotein receptor gene expression in HepG{sub 2} cells". United States. doi:.
@article{osti_20798932,
title = {Ultrasound-targeted microbubble destruction improves the low density lipoprotein receptor gene expression in HepG{sub 2} cells},
author = {Guo Dongping and Atherosclerosis Research Center, Nanjing Medical University, Nanjing 210029 and Provincial Laboratory of Human Functional Genomics, Nanjing Medical University, Nanjing 210029 and Li Xiaoyu and Sun, Ping and Tang Yibo and Chen Xiuying and Chen Qi and Atherosclerosis Research Center, Nanjing Medical University, Nanjing 210029 and Provincial Laboratory of Human Functional Genomics, Nanjing Medical University, Nanjing 210029 and Fan Leming and Zang Bin and Shao Lizheng and Li Xiaorong},
abstractNote = {Ultrasound-targeted microbubble destruction had been employed in gene delivery and promised great potential. Liver has unique features that make it attractive for gene therapy. However, it poses formidable obstacles to hepatocyte-specific gene delivery. This study was designed to test the efficiency of therapeutic gene transfer and expression mediated by ultrasound/microbubble strategy in HepG{sub 2} cell line. Air-filled albumin microbubbles were prepared and mixed with plasmid DNA encoding low density lipoprotein receptor (LDLR) and green fluorescent protein. The mixture of the DNA and microbubbles was administer to cultured HepG{sub 2} cells under variable ultrasound conditions. Transfection rate of the transferred gene and cell viability were assessed by FACS analysis, confocal laser scanning microscopy, Western blot analysis and Trypan blue staining. The result demonstrated that microbubbles with ultrasound irradiation can significantly elevate exogenous LDLR gene expression and the expressed LDLRs were functional and active to uptake their ligands. We conclude that ultrasound-targeted microbubble destruction has the potential to promote safe and efficient LDLR gene transfer into hepatocytes. With further refinement, it may represent an effective nonviral avenue of gene therapy for liver-involved genetic diseases.},
doi = {},
journal = {Biochemical and Biophysical Research Communications},
number = 2,
volume = 343,
place = {United States},
year = {Fri May 05 00:00:00 EDT 2006},
month = {Fri May 05 00:00:00 EDT 2006}
}
  • Recently GLP-1 was found to have cardioprotective effects independent of those attributable to tight glycemic control. Methods and results: We employed ultrasound targeted microbubble destruction (UTMD) to deliver piggybac transposon plasmids encoding the GLP-1 gene with a nuclear localizing signal to rat hearts with adriamycin cardiomyopathy. After a single UTMD treatment, overexpression of transgenic GLP-1 was found in nuclei of rat heart cells with evidence that transfected cardiac cells had undergone proliferation. UTMD-GLP-1 gene therapy restored LV mass, fractional shortening index, and LV posterior wall diameter to nearly normal. Nuclear overexpression of GLP-1 by inducing phosphorylation of FoxO1-S256 and translocationmore » of FoxO1 from the nucleus to the cytoplasm significantly inactivated FoxO1 and activated the expression of cyclin D1 in nuclei of cardiac muscle cells. Reversal of adriamycin cardiomyopathy appeared to be mediated by dedifferentiation and proliferation of nuclear FoxO1-positive cardiac muscle cells with evidence of embryonic stem cell markers (OCT4, Nanog, SOX2 and c-kit), cardiac early differentiation markers (NKX2.5 and ISL-1) and cellular proliferation markers (BrdU and PHH3) after UTMD with GLP-1 gene therapy. Conclusions: Intranuclear myocardial delivery of the GLP-1gene can reverse established adriamycin cardiomyopathy by stimulating myocardial regeneration. - Highlights: • The activation of nuclear FoxO1 in cardiac muscle cells associated with adriamycin cardiomyopathy. • Myocardial nuclear GLP-1 stimulates myocardial regeneration and reverses adriamycin cardiomyopathy. • The process of myocardial regeneration associated with dedifferentiation and proliferation.« less
  • Five mutations in the low density lipoprotein (LDL) receptor (R) gene account for approximately 83% of cases of heterozygous familial hypercholesterolemia (hFH) in French Canadians in Quebec. The two most prevalent mutations are a >10kb deletion (10kb) of the promoter region resulting in a null allele (60.5% of cases) and a trp{sub 66}{r_arrow}gly missense mutation in exon 3 (ex3) resulting in a binding-defective R (11.7%). We have compared the phenotypic expression of these two mutations in 427 10kb hFH patients, 239 women (age 37.5 {plus_minus} 14.2 years) and 188 men (33.7 {plus_minus} 11.7) and 69 ex3 hFH patients, 42 womenmore » (40.6 {plus_minus} 14.3) and 27 men (36.8 {plus_minus}13.2). All data were analyzed separately for women and men. Tendon xanthomas were more prevalent in the 10kb (women 63%, men 68%) than in the ex3 patients (48%,48%). Total and LDL cholesterol were significantly higher in the 10kb patients with than without xanthomas but similar in ex3 patients. There were no significant differences in plasma lipoprotein concentrations between 10kb and ex3 patients with coronary artery disease (CAD) or between 10kb and ex3 patients without CAD. Among men with CAD, those with 10kb were significantly younger than those with ex3 (39.6 {plus_minus} 9.8, n=93 and 46.4 {plus_minus} 7.0, n=9, respectively). In both sexes, high plasma lipoprotein concentrations conferred an increased risk of CAD in 10kb but not in ex3 patients. Thus, as in homozygotes (previous study), the >10kb deletion is associated with more severe expression of FH than is the exon 3 mutation, although the plasma lipoprotein concentrations are not significantly different between the 10kb and ex3 heterozygotes. Since in homozygotes plasma cholesterol levels in 10kb are 60% higher than in ex3 patients, these observations suggest that the expression of the normal LDL-R allele compensates for the lack of a second allele in 10kb heterozygotes.« less
  • Approximately 60% of familial hypercholesterolemia (FH) in French Canadians is due to a > 10 kb deletion of the promoter region of the gene encoding the low density lipoprotein (LDL) receptor (LDL-R), allowing determination of the influence of a single LDL-R allele on phenotypic expression of FH. Normal allele haplotypes of approximately 250 heterozygotes were determined with 7 RFLPs. In vitro maximal LDL-R activity of blood lymphocytes from a subset of approximately 150 heterozygotes, measured by immunocytofluorometry, was significantly higher (20 to 30%) in subjects with LDL-R normal allele haplotype G (n=11), and O (n=7) compared to the most frequentmore » haplotype F (n=43), while no differences were observed among F, E (n=11), and the 2 other most prevalent haplotypes (n=43). LDL-R mRNA in these lymphocytes was significantly elevated 2.3-, 1.7-, and 1.8- fold, in G, O, and E, respectively, compared to F, while no significant differences were apparent between F and the other two most frequent haplotyes. Large interindividual variability in lymphocyte LDL-R mRNA levels and activity was observed even among subjects with the same LDL-R normal allele haplotype. However, maximally induced lymphocyte LDL-R mRNA levels correlated poorly with levels measured in freshly isolated cells (n=14). Relative to haplotype F (n=47 women (W), 39 men (M)), mean plasma LDL cholesterol levels adjusted for age and apolipoprotein E genotype were 5-10% lower in men and women with haplotypes G (n=16 W, 12 M) and O (n=8 W, 6 M), and 20% lower in 7 W with haplotype E. These results suggest that (1) normal LDL-R allele haplotype G and O may contain sequence variations which confer relatively high gene expression and (2) environmental and genetic influences other than the LDL-R gene contribute substantially to variability in LDL-R expression and plasma LDL cholesterol levels in French Canadian FH heterozygotes.« less
  • PCSK9 is a secreted protein that degrades low density lipoprotein receptors (LDLRs) in liver by binding to the epidermal growth factor-like repeat A (EGF-A) domain of the LDLR. It is not known whether PCSK9 causes degradation of LDLRs within the secretory pathway or following secretion and reuptake via endocytosis. Here we show that a mutation in the LDLR EGF-A domain associated with familial hypercholesterolemia, H306Y, results in increased sensitivity to exogenous PCSK9-mediated cellular degradation because of enhanced PCSK9 binding affinity. The crystal structure of the PCSK9-EGF-A(H306Y) complex shows that Tyr-306 forms a hydrogen bond with Asp-374 in PCSK9 at neutralmore » pH, which strengthens the interaction with PCSK9. To block secreted PCSK9 activity, LDLR (H306Y) subfragments were added to the medium of HepG2 cells stably overexpressing wild-type PCSK9 or gain-of-function PCSK9 mutants associated with hypercholesterolemia (D374Y or S127R). These subfragments blocked secreted PCSK9 binding to cell surface LDLRs and resulted in the recovery of LDLR levels to those of control cells. We conclude that PCSK9 acts primarily as a secreted factor to cause LDLR degradation. These studies support the concept that pharmacological inhibition of the PCSK9-LDLR interaction extracellularly will increase hepatic LDLR expression and lower plasma low density lipoprotein levels.« less
  • The very low density lipoprotein receptor (VLDL-r) is a cell-surface molecule specialized for the internalization of multiple diverse ligands, including apolipoprotein E (apoE)-containing lipoprotein particles, via clathrin-coated pits. Its structure is similar to the low-density lipoprotein receptor (LDL-r), although the two have substantially different systemic distributions and regulatory pathways. The present work examines the distribution of VLDL-r in the central nervous system (CNS) and in relation to senile plaques in Alzheimer disease (AD). VLDL-r is present on resting and activated microglia, particularly those associated with senile plaques (SPs). VLDL-r immunoreactivity is also found in cortical neurons. Two exons of VLDL-rmore » mRNA are differentially spliced in the mature receptor mRNA. One set of splice forms gives rise to receptors containing (or lacking) an extracellular O-linked glycosylation domain near the transmembrane portion of the molecule. The other set of splice forms appears to be brain-specific, and is responsible for the presence or absence of one of the cysteine-rich repeat regions in the binding region of the molecule. Ratios of the receptor variants generated from these splice forms do not differ substantially across different cortical areas or in AD. We hypothesize that VLDL-r might contribute to metabolism of apoE and apoE/A{beta} complexes in the brain. Further characterization of apoE receptors in Alzheimer brain may help lay the groundwork for understanding the role of apoE in the CNS and in the pathophysiology of AD. 43 refs., 5 figs.« less