Shuyuan Chen

Echocardiographic Destruction of Albumin Microbubbles Directs Gene Delivery to the Myocardium

BACKGROUND The noninvasive, tissue-specific delivery of therapeutic agents to the heart would be a valuable clinical tool. This study addressed the hypothesis that albumin-coated microbubbles could be used to effectively deliver an adenoviral transgene to rat myocardium by ultrasound-mediated microbubble destruction. METHODS AND RESULTS Recombinant adenovirus containing beta-galactosidase and driven by a constitutive promoter was attached to the surface of albumin-coated, perfluoropropane-filled microbubbles. These bubbles were infused into the jugular vein of rats with or without simultaneous echocardiography. Additional controls included ultrasound of microbubbles that did not contain virus, virus alone, and virus plus ultrasound. One group underwent ultrasound-mediated destruction of microbubbles followed by adenovirus infusion. Rats were killed after 4 days and examined for beta-galactosidase expression. The hearts of all rats that underwent ultrasound-mediated destruction of microbubbles containing virus showed nuclear staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside substrate, indicating expression of the transgene. None of the control animals showed myocardial expression of the beta-galactosidase transgene. By quantitative analysis, beta-galactosidase activity was 10-fold higher in the treated group than in controls (P<0.0001). CONCLUSIONS Ultrasound-mediated destruction of albumin-coated microbubbles is a promising method for the delivery of bioactive agents to the heart.

Ultrasound-Targeted Microbubble Destruction Can Repeatedly Direct Highly Specific Plasmid Expression to the Heart

Background—Noninvasive, tissue-specific delivery of therapeutic agents would be a valuable clinical tool. We have previously shown that ultrasound-targeted microbubble destruction can direct expression of an adenoviral reporter to the heart. The present study shows that this method can be applied to selectively deliver plasmid vectors to the heart. Methods and Results—We used albumin and lipid microbubbles containing plasmids with a luciferase transgene to target the heart in rats. After 4 days, organs were harvested and analyzed for reporter gene expression. In a second set of experiments, the hearts of rats treated with plasmids were harvested at various time points during a 4-week period. Both luciferase activity and mRNA concentrations were measured. Luciferase transfection with plasmids showed highly specific gene expression in the heart, with hardly any activity in control organs. Time course evaluation showed high transgene expression in the first 4 days, with a rapid decline thereafter. Repeated treatment produced a second peak of transgene expression with similar decay. Conclusions—Ultrasound-mediated destruction of microbubbles directs plasmid transgene expression to the heart with much greater specificity than viral vectors and can be regulated by repeated treatments. This noninvasive technique is a promising method for cardiac gene therapy.

Optimization of ultrasound parameters for cardiac gene delivery of adenoviral or plasmid deoxyribonucleic acid by Ultrasound-Targeted microbubble destruction

OBJECTIVES This study was undertaken to optimize echocardiographic parameters for successful gene delivery to the heart and to extend the method from adenoviral to plasmid deoxyribonucleic acid (DNA). BACKGROUND We have previously shown that ultrasound-targeted microbubble destruction can direct tissue expression of adenoviral transgenes to the heart. The optimal echocardiographic parameters for this technique have not been reported. METHODS Adenoviral or plasmid DNA encoding the luciferase reporter gene was incorporated into liposome microbubbles and infused intravenously into anesthetized rats. We systematically evaluated the effects of ultrasound parameters known to influence microbubble destruction, including electrocardiogram (ECG) triggering, ultrasound frequency, mode of ultrasound, and mechanical index, on gene expression in rat myocardium four days after treatment. In addition, gene expression in heart, liver, and skeletal muscle were compared between adenoviral and plasmid DNA. RESULTS Optimal ultrasound parameters for this technique include low-transmission frequency (1.3 MHz), maximal mechanical index, and ECG triggering to allow complete filling of the myocardial capillary bed by microbubbles. No difference was seen between ultraharmonics and power Doppler mode. Using adenoviral DNA, optimal ultrasound parameters yielded myocardial luciferase activity on the order of 104 relative light units/mg protein/min but with even higher liver activity. Plasmid DNA was expressed in rat myocardium at similar levels but without detectable liver expression. CONCLUSIONS Ultrasound-targeted microbubble destruction can be used to deliver adenoviral or plasmid DNA to the myocardium. This technique holds great promise in applying the rapidly expanding repertoire of gene therapies being developed for cardiac disease.

DNA-loaded albumin microbubbles enhance ultrasound-mediated transfection in vitro.

Ultrasound (US), with or without microbubbles, enhances gene transfer in cultured cells, but the effect is modest. We tested if attaching DNA to albumin microbubbles during bubble synthesis could enhance gene expression. Plasmid DNA was loaded on the albumin shell over a range of concentrations (500 to 10,000 microg/mL). Optimal gene expression occurred with loading doses of 4000 microg DNA/mL (4k-loaded bubbles). These microbubbles had diameters of 2.4 +/- 0.7 microm and carried 40 pg DNA/microbubble. DNA-loaded microbubbles had optimal transfection at higher delivered doses of DNA than unloaded bubbles mixed with plasmid. The 4k-loaded bubbles demonstrated a fivefold (p = 0.0003) increase in luciferase reporter expression over that with unloaded bubbles. Similarly, transfection efficiency was better for 4k-loaded microbubbles than unloaded microbubbles (41 +/- 3% vs. 9 +/- 3%, p < 0.0001). DNA loading of microbubbles enhances gene expression and transfection efficiency in US-targeted transfection in vitro and may represent an improved avenue for therapeutic gene delivery in vivo.

Targeting of VEGF-mediated angiogenesis to rat myocardium using ultrasonic destruction of microbubbles

Myocardial angiogenesis mediated by human vascular endothelial growth factor 165 (hVEGF165) cDNA was promoted in rat myocardium using an in vivo-targeted gene delivery system known as ultrasound-targeted microbubble destruction (UTMD). Microbubbles carrying plasmids encoding hVEGF165, or control solutions were infused intravenously during ultrasonic destruction of the microbubbles within the myocardium. Biochemical and histological assessment of gene expression and angiogenesis were performed 5, 10, and 30 days after UTMD. UTMD-treated myocardium contained hVEGF165 protein and mRNA. The myocardium of UTMD-treated animals showed hypercellular foci associated with hVEGF165 expression and endothelial cell markers. Capillary density in UTMD-treated rats increased 18% at 5 days and 33% at 10 days, returning to control levels at 30 days (P<0.0001). Similarly, arteriolar density increased 22% at 5 days, 86% at 10 days, and 31% at 30 days (P<0.0001). Thus, noninvasive delivery of hVEGF165 to rat myocardium by UTMD resulted in significant increases in myocardial capillary and arteriolar density.

Adipogenic capacity and the susceptibility to type 2 diabetes and metabolic syndrome

To determine whether adipocyte storage capacity influences the onset and severity of type 2 diabetes and other components of the metabolic syndrome, we made normal and db/db mice resistant to obesity by overexpressing leptin receptor-b on the aP2-Lepr-b promoter. On a 4% diet, these mice have no phenotype, but on a 60% fat diet, they resist diet-induced obesity because constitutive adipocyte-specific overexpression of Lepr-b prevents obesity via the antilipogenic autocrine/paracrine action of leptin on adipocytes. After 8 months on the same 60% fat diet, body fat of transgenic mice was 70% below WT controls. Cardiac and liver fat was elevated in the transgenics, and their hyperinsulinemia was more marked, suggesting greater insulin resistance. The aP2-Lepr-b transgene also prevented obesity in db/db mice; at 10 weeks of age their body fat was half that of the db/db mice. This lack of obesity was attributable to reduced expression of sterol regulatory element binding protein-1c and its target lipogenic enzymes in adipose tissue and a 6-fold increase in Pref-1 mRNA. Severe diabetes was present in transgenics at 4 weeks of age, 10 weeks before db/db controls. Echocardiographic evidence of cardiomyopathy appeared at 10 weeks, weeks before the db/db mice. Histologically, loss of β cells and myocardial fibrosis was present in the transgenic group at least 6 weeks before the db/db mice. These results suggest that the expression level of genes that regulate the adipogenic response to overnutrition profoundly influences the age of onset and severity of diet-induced type 2 diabetes and co-morbidities.

Bioeffects of myocardial contrast microbubble destruction by echocardiography.

Background: Microbubble destruction during contrast echocardiography is known to cause capillary leaks and red blood cell extravasation in skeletal muscle. This study evaluated the biological effects of microbubble destruction on cardiac muscle. Methods: Contrast echocardiography was performed in 36 rats randomized to receive either Definity or Optison at a mechanical index (MI) of 1.6, 1.2, or 0.8. Myocardial bioeffects were assessed by measuring left ventricular (LV) size and fractional area shortening and histopathology. In addition, blood samples for troponin T were drawn at baseline, postinfusion (30 minutes), day 1, day 4, and day 7. LV size and function were measured at baseline and immediately prior to euthanasia on day 7, after which the heart was removed and sectioned for histopathology. Results: There was no statistical difference in LV size or function regardless of the contrast agent or MI, nor was there any histopathological evidence of myocardial damage. However, troponin T increased over time (F = 3.77, P = 0.012), peaking at 30 minutes and returning to normal by day 4. The difference between Definity and Optison was not statistically significant. However, troponin T values were higher at a higher MI (F = 5.01, P = 0.012). Of 12 rats imaged at a MI of 1.6, 9 (75%) had elevated troponin T as compared to 4 (33%) of 12 at a MI of 1.2. None of the 12 rats imaged at a MI of 0.8 had an elevated troponin T at any time point. Conclusions: Microbubble destruction at high acoustic power (MI 1.6) can cause mild troponin T elevations that are not associated with LV dysfunction or histopathological evidence of myocardial damage.

Regeneration of pancreatic islets in vivo by ultrasound-targeted gene therapy

This study uses a novel approach to gene therapy in which plasmid DNA is targeted to the pancreas in vivo using ultrasound-targeted microbubble destruction (UTMD) to achieve islet regeneration. Intravenous microbubbles carrying plasmids are destroyed within the pancreatic microcirculation by ultrasound, achieving local gene expression that is further targeted to β-cells by a modified rat insulin promoter (RIP3.1). A series of genes implicated in endocrine development were delivered to rats 2 days after streptozotocin-induced diabetes. The genes, PAX4, Nkx2.2, Nkx6.1, Ngn3 and Mafa, produced α-cell hyperplasia, but no significant improvement in β-cell mass or blood glucose level 30 days after UTMD. In contrast, RIP3.1-NeuroD1 promoted islet regeneration from surviving β-cells, with normalization of glucose, insulin and C-peptide levels at 30 days. In a longer-term experiment, four of six rats had a return of diabetes at 90 days, accompanied by β-cell apoptosis on Tunel staining. Pretreatment with the JNK inhibitor SP600125 successfully blocked β-cell apoptosis and resulted in restoration of β-cell mass and normalization of blood glucose level for up to 90 days. This technique allows in vivo islet regeneration, restoration of β-cell mass and normalization of blood sugar, insulin and C-peptide in rats without viruses.

Reversal of streptozotocin-induced diabetes in rats by gene therapy with betacellulin and pancreatic duodenal homeobox-1

Ultrasound-targeted microbubble destruction (UTMD) was used to direct betacellulin (BTC) and pancreatic duodenal homeobox-1 (PDX1) to rat pancreas 48 h after islet destruction by streptozotocin (STZ). Sprague–Dawley rats were rendered diabetic by STZ injection. Controls included normal rats, STZ only without UTMD, and UTMD with DsRed reporter gene. Blood glucose increased dramatically in all rats 48 h after STZ, and continued to rise after UTMD with BTC alone. Blood glucose declined from day 3 to day 10 after UTMD with PDX1, but remained elevated (261±8 mg/dl). However, in rats treated with both BTC and PDX1, blood glucose remained below 200 mg/dl throughout day 10. This was accompanied by normalization of blood insulin and C-peptide. Histology demonstrated islet-like clusters of glucagon-staining cells in the rats treated with BTC and PDX1, but these clusters disappeared by 30 days after UTMD treatment. Although regeneration of insulin-producing islets was not seen, diabetes was reversed for up to 15 days after a single UTMD treatment by ectopic insulin production by pancreatic acinar cells. These cells co-expressed amylase and insulin and demonstrated several β-cell markers by reverse transcription-PCR. Gene therapy by UTMD can reverse diabetes in vivo in adult rats by restoring pancreatic insulin production.

Transient overexpression of cyclin D2/CDK4/GLP1 genes induces proliferation and differentiation of adult pancreatic progenitors and mediates islet regeneration

The molecular mechanism of β-cell regeneration remains poorly understood. Cyclin D2/CDK4 expresses in normal β cells and maintains adult β-cell growth. We hypothesized that gene therapy with cyclin D2/CDK4/GLP-1 plasmids targeted to the pancreas of STZ-treated rats by ultrasound-targeted microbubble destruction (UTMD) would force cell cycle re-entry of residual G0-phase islet cells into G1/S phase to regenerate β cells. A single UTMD treatment induced β-cell regeneration with reversal of diabetes for 6 mo without evidence of toxicity. We observed that this β-cell regeneration was not mediated by self-replication of pre-existing β cells. Instead, cyclin D2/CDK4/GLP-1 initiated robust proliferation of adult pancreatic progenitor cells that exist within islets and terminally differentiate to mature islets with β cells and α cells.